Thrombopoietin complements G(i)- but not G(q)-dependent pathways for integrin {alpha}(IIb){beta}(3) activation and platelet aggregation

Binding of thrombopoietin (TPO) to the cMpl receptor on human platelets potentiates aggregation induced by a number of agonists, including ADP. In this work, we found that TPO was able to restore ADP-induced platelet aggregation upon blockade of the G(q)-coupled P2Y1 purinergic receptor but not upon inhibition of the G(i)-coupled P2Y12 receptor. Moreover, TPO triggered platelet aggregation upon co-stimulation of G(z) by epinephrine but not upon co-stimulation of G(q) by the thromboxane analogue U46619. Platelet aggregation induced by TPO and G(i) stimulation was biphasic, and cyclooxygenase inhibitors prevented the second but not the first phase. In contrast to ADP, TPO was unable to induce integrin alpha(IIb)beta(3) activation, as evaluated by binding of both fibrinogen and PAC-1 monoclonal antibody. However, ADP-induced activation of integrin alpha(IIb)beta(3) was blocked by antagonists of the G(q)-coupled P2Y1 receptor but was completely restored by the simultaneous co-stimulation of cMpl receptor by TPO. Inside-out activation of integrin alpha(IIb)beta(3) induced by TPO and G(i) stimulation occurred independently of thromboxane A(2) production and was not mediated by protein kinase C, MAP kinases, or Rho-dependent kinase. Importantly, TPO and G(i) activation of integrin alpha(IIb)beta(3) was suppressed by wortmannin and Ly294002, suggesting a critical regulation by phosphatidylinositol 3-kinase. We found that TPO did not activate phospholipase C in human platelets and was unable to restore ADP-induced phospholipase C activation upon blockade of the G(q)-coupled P2Y1 receptor. TPO induced a rapid and sustained activation of the small GTPase Rap1B through a pathway dependent on phosphatidylinositol 3-kinase. In ADP-stimulated platelets, Rap1B activation was reduced, although not abolished, upon blockade of the P2Y1 receptor. However, accumulation of GTP-bound Rap1B in platelets activated by co-stimulation of cMpl and P2Y12 receptor was identical to that induced by the simultaneous ligation of P2Y1 and P2Y12 receptor by ADP. These results indicate that TPO can integrate G(i), but not G(q), stimulation and can efficiently support integrin alpha(IIb)beta(3) activation platelet aggregation by an alternative signaling pathway independent of phospholipase C but involving the phosphatidylinositol 3-kinase and the small GTPase Rap1B.     

Identification, cloning and functional characterization of a novel dermonecrotic toxin (phospholipase D) from brown spider (Loxosceles intermedia) venom

Brown spider bites are associated with lesions including dermonecrosis, gravitational spreading and a massive inflammatory response, along with systemic problems that may include hematological disturbances and renal failure. The mechanisms by which the venom exerts its noxious effects are currently under investigation. It is known that the venom contains a major toxin (dermonecrotic toxin, biochemically a phospholipase D) that can experimentally induce dermonecrosis, inflammatory response, animal mortality and platelet aggregation. Herein, we describe cloning, heterologous expression, purification and functionality of a novel isoform of the 33 kDa dermonecrotic toxin. Circular dichroism analysis evidenced correct folding for the toxin. The recombinant toxin was recognized by whole venom serum antibodies and by a specific antibody to a previously described dermonecrotic toxin. The identified toxin was found to display phospholipase activity and dermonecrotic properties. Additionally, the toxin caused a massive inflammatory response in rabbit skin dermis, evoked platelet aggregation, increased vascular permeability, caused edema and death in mice. These characteristics in combination with functional studies for other dermonecrotic toxins illustrate that a family of dermonecrotic toxins exists, and includes a novel member with high activity that may be useful for future structural and functional studies.     

Cloning of the <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. FG1 dehalogenase genes and construction of hybrid pathways in <Emphasis Type="Italic">Pseudomonas putida</Emphasis> strains

An Arthrobacter strain, able to utilize 4-chlorobenzoic acid as the sole carbon and energy source, was isolated and characterized. The first step of the catabolic pathway was found to proceed via a hydrolytic dehalogenation that leads to the formation of 4-hydroxybenzoic acid. The dehalogenase encoding genes (fcb) were sequenced and found highly homologous to and organized as those of other 4-chlorobenzoic acid degrading Arthrobacter strains. The fcb genes were cloned and successfully expressed in the heterologous host Pseudomonas putida PaW340 and P. putida KT2442 upper TOL, which acquired the ability to grow on 4-chlorobenzoic acid and 4-chlorotoluene, respectively. The cloned dehalogenase displayed a high specificity for para-substituted haloaromatics with affinity Cl > Br > I > F, in the order.     

Introducing “best single template” models as reference baseline for the Continuous Automated Model Evaluation (CAMEO)

Critical blind assessment of structure prediction techniques is crucial for the scientific community to establish the state of the art, identify bottlenecks, and guide future developments. In Critical Assessment of Techniques in Structure Prediction (CASP), human experts assess the performance of participating methods in relation to the difficulty of the prediction task in a biennial experiment on approximately 100 targets. Yet, the development of automated computational modeling methods requires more frequent evaluation cycles and larger sets of data. The “Continuous Automated Model EvaluatiOn (CAMEO)” platform complements CASP by conducting fully automated blind prediction evaluations based on the weekly pre-release of sequences of those structures, which are going to be published in the next release of the Protein Data Bank (PDB). Each week, CAMEO publishes benchmarking results for predictions corresponding to a set of about 20 targets collected during a 4-day prediction window. CAMEO benchmarking data are generated consistently for all methods at the same point in time, enabling developers to cross-validate their method's performance, and referring to their results in publications. Many successful participants of CASP have used CAMEO—either by directly benchmarking their methods within the system or by comparing their own performance to CAMEO reference data. CAMEO offers a variety of scores reflecting different aspects of structure modeling, for example, binding site accuracy, homo-oligomer interface quality, or accuracy of local model confidence estimates. By introducing the \"bestSingleTemplate\" method based on structure superpositions as a reference for the accuracy of 3D modeling predictions, CAMEO facilitates objective comparison of techniques and fosters the development of advanced methods.     

Development of (Na+ -K+)-ATPase in rat hindbrain: increments in parallel with Na+-dependent phosphorylation and K+-pnitrophenylphosphatase.

Rat hindbrain NaI-enriched microsomal (Na⁺-K⁺)-ATPase activity, K⁺-pNPPase activity, and Na⁺-dependent steady-state phosphorylation levels all increase approx 10-fold relative to microsomal protein between 5 days prenatally and 60 days postnatally. These activities, as well as the mean wet weight of the hindbrain, are at half of their 60 day values shortly after the 10th postnatal day. For all ages, these hindbrain activities average over twice those found in the forebrain in a companion paper (Bertoni & Siegel , 1978). Increases during development in the amount of the related phosphorylatable polypeptide, estimated by densitometry of stained polyacrylamide gels containing fixed amounts of microsomal protein dissolved in SDS, are in agreement with increases in steady state levels of Na⁺-dependent phosphorylation. The fraction of total phosphorylation that is Na⁺-dependent rises steadily during development consistent with, but not obligatorily due to, a conversion of some of the previously Na⁺-independent portion. Mg2⁺-ATPase and Mg²⁺ -pNPPase activities and steady-state Na⁺,-independent phosphorylation levels do not increase in parallel during development. These observations add further support to the proposed partial reaction scheme for (Na⁺- K⁺)-ATPase. The major increments in (Na ⁺-K⁺)-ATPase occur simultaneously with the deposition of specialized plasma membranes, particularly in the molecular layer of the cerebellum, as described in previous studies of rat hind brain.     

Microcolony formation by single-cell Synechococcus strains as a fast response to UV radiation

UV radiation (UVR) has different effects on prokaryotic cells, such as, for instance, filamentation and aggregation in bacteria. Here we studied the effect of UVR on microcolony formation in two freshwater Synechococcus strains of different ribotypes (group B and group I) and phycobiliprotein compositions (phycoerythrin [PE] and phycocyanin [PC]). Each strain was photoacclimated at two light intensities, low light (LL) (10 μmol m⁻² s⁻¹) and moderate light (ML) (100 μmol m⁻² s⁻¹). The cultures were exposed for 6 days to treatments with UVR or without UVR. PE-rich Synechococcus acclimated to LL had a low carotenoid/chlorophyll a (car/chl) ratio but responded faster to UVR treatment, producing the highest percentages of microcolonies and of cells in microcolonies. Conversely, the same strain acclimated to ML, with a higher car/chl ratio, did not aggregate significantly. These results suggest that microcolony formation by PE-rich Synechococcus is induced by UVR if carotenoid levels are low. PC-rich Synechococcus formed a very low percentage of microcolonies in both acclimations even with low car/chl ratio. The different responses of the two Synechococcus strains to UVR depend on their pigment compositions. On the other hand, this study does not exclude that UVR-induced microcolony formation could also be related to specific ribotypes.     

The current management of mycosis fungoides and Sézary syndrome and the role of radiotherapy: Principles and indications

Aim

To evaluate the current treatment of mycosis fungoides (MF) and Sézary syndrome (SS) focusing on the role of radiotherapy (RT), its principles and indications, and the perspectives of the novel irradiation technologies.

Background

MF and SS are rare lymphoproliferative diseases whose incidence is increasing. For a long time RT has been used as a single modality or in integrated treatment programs for these diseases.

Materials and methods

The latest systematic reviews, primary studies and new diagnostic and treatment guidelines on MF and SS were analyzed. Clinical outcomes together with the technical aspects and the role of RT were also evaluated.

Results

New data are available on pathogenesis, diagnostic criteria, classification and staging procedures for MF and SS and several local and systemic therapies are proposed. Localized RT can cure “minimal stage” MF while total skin electron beam irradiation (TSEI) may cure initial-stage disease and may offer important symptom relief (itch, erythroderma) in a more advanced setting. Despite its efficacy, RT is not largely used, mainly because of some technical difficulties but new RT technologies may be proposed to treat large skin surfaces.

Conclusions

New treatment programs offer good results, with median survival of more than 12 years in early-stage MF, but the median survival of 2.5 years or less in advanced stages is still a challenge. RT remains an option for all stages with a good cost/effectiveness ratio in a curative or palliative setting. New RT technologies can overcome some technical problems of treating large skin surfaces.