Pharmacological and structural characterization of long-sarafotoxins, a new family of endothelin-like peptides: Role of the C-terminus extension

Long-sarafotoxins (l-SRTXs) have recently been identified in both the venom of Atractaspis microlepidota and that of Atractaspis irregularis. They are characterized by different C-terminus extensions that follow the invariant Trp21, which plays a crucial role in endothelin-receptor binding. We initially determined the toxicity and three-dimensional structures of two chemically synthesized l-SRTXs that have different C-terminus extensions, namely SRTX-m (24 aa, including extension "D-E-P") and SRTX-i3 (25 aa, including extension "V-N-R-N"). Both peptides were shown to be highly toxic in mice and displayed the cysteine-stabilized α-helical motif that characterizes endothelins and short-SRTXs, to which a longer C-terminus with variable flexibility is added. To discern the functional and pharmacological consequences of the supplementary amino acids, different chimerical as well as truncated forms of SRTX were designed and synthesized. Thus, we either removed the extra-C-terminal residues of SRTX-m or i3, or grafted the latter onto the C-terminal extremity of a short-SRTX (s-SRTX) (ie. SRTX-b). Our competitive binding assays where SRTXs competed for iodinated endothelin-1 binding to cloned ET(A) and ET(B) receptor subtypes over-expressed in CHO cells, revealed the essential role of the C-terminus extensions for ET-receptor recognition. Indeed, l-SRTXs displayed an affinity three to four orders of magnitude lower as compared to SRTX-b for the two receptor subtypes. Moreover, grafting the C-terminus extension to SRTX-b induced a drastic decrease in affinity, while its removal (truncated l-SRTXs) yielded an affinity for ET-receptors similar to that of s-SRTXs. Furthermore, we established by intracellular Ca(2+) measurements that l-SRTXs, as well as s-SRTXs, display agonistic activities. We thus confirmed in these functional assays the major difference in potency for these two SRTX families as well as the crucial role of the C-terminus extension in their various pharmacological profiles. Finally, one of the chimeric toxin synthesized in this study appears to be one of the most potent and selective ligand of the ET(B) receptor known to date.     

The effects of different adhesive agents on the shear bond strength of a self-adhesive resin cement

Purpose: To compare the clinical success of a self-adhesive resin cement used in combination with different adhesive bonding systems with that of a conventional dual-cure resin cement. Methods: The study was performed with 136 freshly extracted molars embedded in acrylic resin blocks and 136 IPS e.max Press discs. Teeth and discs were randomly divided into four equal groups and cemented together using either RelyX ARC (ARC), RelyX Unicem (Unicem), RelyX Unicem+Adper-Prompt L-pop (L-pop), or RelyX and Unicem+Total-etch (Total-etch). Shear bond strength measurements were obtained before and after thermocycling. Following bond testing, the surfaces of one sample per subgroup (thermocycled and non-thermocycled), were assessed using scanning electron microscopy (SEM). Results: Among the non-thermocylced subgroups, ARC exhibited the highest bond strength values, followed by Total Etch, Unicem and L-pop. ARC also exhibited the highest bond strength values among the thermocycled subgroups, followed by Unicem, Total-etch, and L-pop. SEM analysis clearly revealed the negative effects of thermo-cycling on the mechanical properties of adhesive agents. Conclusions: RelyX Unicem may be preferable in many cases because of its simplified application and reduced technique-sensitivity.     

Temporal competition between differentiation programs determines cell fate choice

Multipotent differentiation, where cells adopt one of several possible fates, occurs in diverse systems ranging from bacteria to mammals. This decision‐making process is driven by multiple differentiation programs that operate simultaneously in the cell. How these programs interact to govern cell fate choice is poorly understood. To investigate this issue, we simultaneously measured activities of the competing sporulation and competence programs in single Bacillus subtilis cells. This approach revealed that these competing differentiation programs progress independently without cross‐regulation before the decision point. Cells seem to arrive at a fate choice through differences in the relative timing between the two programs. To test this proposed dynamic mechanism, we altered the relative timing by engineering artificial cross‐regulation between the sporulation and competence circuits. Results suggest a simple model that does not require a checkpoint or intricate cross‐regulation before cellular decision‐making. Rather, cell fate choice appears to be the outcome of a ‘molecular race’ between differentiation programs that compete in time, providing a simple dynamic mechanism for decision‐making.     

Agmatine attenuates neuropathic pain in rats: possible mediation of nitric oxide and noradrenergic activity in the brainstem and cerebellum

Effect of agmatine (10-400 mg/kg) on neuropathic pain in a rat model produced by loose ligatures around the common sciatic nerve was studied. The involvement of possible alterations in nitric oxide (NO) levels [measured as its stable metabolites nitrate + nitrite] and in noradrenergic activity [measured as norepinephrine and 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) levels] in this effect was also investigated biochemically in the brainstem and cerebellum. Agmatine increased the neuropathic pain threshold at 300 and 400 mg/kg. There was almost a twofold increase in nitrate + nitrite levels in the brainstem and cerebellum of the rats with neuropathic pain and agmatine decreased the high nitrate + nitrite levels only in the brainstem at 300 mg/kg and both in the brainstem and cerebellum at 400 mg/kg. Ligation of sciatic nerve resulted in almost twofold increase in norepinephrine and MHPG levels only in the brainstem of the rats. Agmatine decreased MHPG levels at 300 and 400 mg/kg, however it decreased norepinephrine levels only at the higher dose. These findings indicate that agmatine decreases neuropathic pain, an effect which may involve the reduction of NO levels and noradrenergic activity in the brain.     

Demersal trawl discards with spatial and bathymetric emphasis in the Turkish coast of the Aegean Sea

The aim of the present study is to investigate the spatial and bathymetric distribution of demersal trawl discards in the Turkish coast of the Aegean Sea. Trawl hauls were performed in the legal trawling areas of the Turkish coast of the Aegean Sea (Çanakkale, Foça, Karaburun, S??ac?k, Güllük) between 2010 and 2012 by commercial trawlers. Depth of the trawl hauls ranged from 30?m in the south to 450?m in the north. As a result of 311 trawl samples, 200 species belonging to eight taxonomic groups (Porifera, Cnidaria, Annelida, Arthropoda, Mollusca, Echinodermata, Tunicata, Chordata) were identified. The changes in species composition based on depth and region were determined to be statistically significant. It was found that species composition distinctly changed around 200?m. In a regional assessment it was determined that the Turkish coast of the Aegean Sea is divided into three sub-regions, i.e. Çanakkale, Foça-Karaburun-S??ac?k and Güllük. The results of similar studies conducted in the Mediterranean were investigated and compared with the findings of this study.     

Viable mice with compound mutations in the Wnt/Dvl pathway antagonists nkd1 and nkd2

Gradients of Wnt/beta-catenin signaling coordinate development and physiological homeostasis in metazoan animals. Proper embryonic development of the fruit fly Drosophila melanogaster requires the Naked cuticle (Nkd) protein to attenuate a gradient of Wnt/beta-catenin signaling across each segmental anlage. Nkd inhibits Wnt signaling by binding the intracellular protein Dishevelled (Dsh). Mice and humans have two nkd homologs, nkd1 and nkd2, whose encoded proteins can bind Dsh homologs (the Dvl proteins) and inhibit Wnt signaling. To determine whether nkd genes are necessary for murine development, we replaced nkd exons that encode Dvl-binding sequences with IRES-lacZ/neomycin cassettes. Mutants homozygous for each nkd(lacZ) allele are viable with slightly reduced mean litter sizes. Surprisingly, double-knockout mice are viable, with subtle alterations in cranial bone morphology that are reminiscent of mutation in another Wnt/beta-catenin antagonist, axin2. Our data show that nkd function in the mouse is dispensable for embryonic development.     

Deletion of Chromosomal Region 8p21 Confers Resistance to Bortezomib and Is Associated with Upregulated Decoy TRAIL Receptor Expression in Patients with Multiple Myeloma

Loss of the chromosomal region 8p21 negatively effects survival in patients with multiple myeloma (MM) that undergo autologous stem cell transplantation (ASCT). In this study, we aimed to identify the immunological and molecular consequences of del(8)(p21) with regards to treatment response and bortezomib resistance.In patients receiving bortezomib as a single first line agent without any high-dose therapy, we have observed that patients with del(8)(p21) responded poorly to bortezomib with 50% showing no response while patients without the deletion had a response rate of 90%. In vitro analysis revealed a higher resistance to bortezomib possibly due to an altered gene expression profile caused by del(8)(p21) including genes such as TRAIL-R4, CCDC25, RHOBTB2, PTK2B, SCARA3, MYC, BCL2 and TP53. Furthermore, while bortezomib sensitized MM cells without del(8)(p21) to TRAIL/APO2L mediated apoptosis, in cells with del(8)(p21) bortezomib failed to upregulate the pro-apoptotic death receptors TRAIL-R1 and TRAIL-R2 which are located on the 8p21 region. Also expressing higher levels of the decoy death receptor TRAIL-R4, these cells were largely resistant to TRAIL/APO2L mediated apoptosis.Corroborating the clinical outcome of the patients, our data provides a potential explanation regarding the poor response of MM patients with del(8)(p21) to bortezomib treatment. Furthermore, our clinical analysis suggests that including immunomodulatory agents such as Lenalidomide in the treatment regimen may help to overcome this negative effect, providing an alternative consideration in treatment planning of MM patients with del(8)(p21).     

The Irish Potato Famine Pathogen Phytophthora infestans Translocates the CRN8 Kinase into Host Plant Cells

Phytopathogenic oomycetes, such as Phytophthora infestans, secrete an arsenal of effector proteins that modulate plant innate immunity to enable infection. We describe CRN8, a host-translocated effector of P. infestans that has kinase activity in planta. CRN8 is a modular protein of the CRN effector family. The C-terminus of CRN8 localizes to the host nucleus and triggers cell death when the protein is expressed in planta. Cell death induction by CRN8 is dependent on its localization to the plant nucleus, which requires a functional nuclear localization signal (NLS). The C-terminal sequence of CRN8 has similarity to a serine/threonine RD kinase domain. We demonstrated that CRN8 is a functional RD kinase and that its auto-phosphorylation is dependent on an intact catalytic site. Co-immunoprecipitation experiments revealed that CRN8 forms a dimer or multimer. Heterologous expression of CRN8 in planta resulted in enhanced virulence by P. infestans. In contrast, in planta expression of the dominant-negative CRN8^R469A;D470A resulted in reduced P. infestans infection, further implicating CRN8 in virulence. Overall, our results indicate that similar to animal parasites, plant pathogens also translocate biochemically active kinase effectors inside host cells.     

J deflections on ECG in severe hypothermia and hypokalaemia: a case report

The J wave, also known as Osborn wave, is a deflection that can be observed on the surface ECG as a late delta wave, seen at the end of the QRS complex. In this case, a 75-year-old woman, after 1 day of continuous haemodialysis, showed a marked hypothermia (28.5°C) and severe hypokalaemia (1.7 mEq/l). Bradycardia was seen on the monitor and J waves were recognised on the ECG recording. After appropriate replacement of potassium and treatment of hypothermia, the J waves disappeared spontaneously.