N',2-diphenylquinoline-4-carbohydrazide based NK3 receptor antagonists

A new class of potent NK3R antagonists based on the N',2-diphenylquinoline-4-carbohydrazide core is described. In an ex vivo assay in gerbil, the lead compound 2g occupies receptors within the CNS following oral dosing (Occ(90) 30 mg/kg po; plasma Occ(90) 0.95 microM) and has good selectivity and promising PK properties.     

A Conserved Arginine-Rich Motif within the Hypervariable N-Domain of Drosophila Centromeric Histone H3 (CenH3CID) Mediates BubR1 Recruitment

Background

Centromere identity is determined epigenetically by deposition of CenH3, a centromere-specific histone H3 variant that dictates kinetochore assembly. The molecular basis of the contribution of CenH3 to centromere/kinetochore functions is, however, incompletely understood, as its interactions with the rest of centromere/kinetochore components remain largely uncharacterised at the molecular/structural level.

Principal Findings

Here, we report on the contribution of Drosophila CenH3^CID to recruitment of BubR1, a conserved kinetochore protein that is a core component of the spindle attachment checkpoint (SAC). This interaction is mediated by the N-terminal domain of CenH3^CID (NCenH3^CID), as tethering NCenH3^CID to an ectopic reporter construct results in BubR1 recruitment and BubR1-dependent silencing of the reporter gene. Here, we also show that this interaction depends on a short arginine (R)-rich motif and that, most remarkably, it appears to be evolutionarily conserved, as tethering constructs carrying the highly divergent NCenH3 of budding yeast and human also induce silencing of the reporter. Interestingly, though NCenH3 shows an exceedingly low degree of conservation, the presence of R-rich motives is a common feature of NCenH3 from distant species. Finally, our results also indicate that two other conserved sequence motives within NCenH3^CID might also be involved in interactions with kinetochore components.

Conclusions

These results unveil an unexpected contribution of the hypervariable N-domain of CenH3 to recruitment of kinetochore components, identifying simple R-rich motives within it as evolutionary conserved structural determinants involved in BubR1 recruitment.     

Cell Adhesion and Proliferation on Sulfonated and Non-Modified Chitosan Films

Three types of chitosan-based films have been prepared and evaluated: a non-modified chitosan film bearing cationizable aliphatic amines and two films made of N-sulfopropyl chitosan derivatives bearing both aliphatic amines and negative sulfonate groups at different ratios. Cell adhesion and proliferation on chitosan films of C2C12 pre-myoblastic cells and B16 cells as tumoral model have been tested. A differential cell behavior has been observed on chitosan films due to their different surface modification. B16 cells have shown lower vinculin expression when cultured on sulfonated chitosan films. This study shows how the interaction among cells and material surface can be modulated by physicochemical characteristics of the biomaterial surface, altering tumoral cell adhesion and proliferation processes.     

Proteomic analysis of Arabidopsis protein S-nitrosylation in response to inoculation with <Emphasis Type="Italic">Pseudomonas syringae</Emphasis>

Nitric oxide (NO) is a key signaling molecule in plants, being its biological effects mainly mediated through S-nitrosylation of cysteine thiols. Using the biotin switch method combined with mass spectrometry analysis we have identified 127 targets of S-nitrosylation in Arabidopsis cell suspension cultures and leaves challenged with virulent and avirulent isolates of Pseudomonas syringae pv. tomato. The NO targets are proteins associated with carbon, nitrogen, and sulpfur metabolism, photosynthesis, the cytoskeleton, stress-, pathogen- and redox-related and signaling proteins. Some proteins were previously identified in plants and mammals, while others (63%) represent novel targets of S-nitrosylation. Our data suggest that NO might be orchestrating the whole plant physiology, presumably through covalent modification of proteins.     

The cortactin-binding domain of WIP is essential for podosome formation and extracellular matrix degradation by murine dendritic cells

In immature dendritic cells (DCs) podosomes form and turn over behind the leading edge of migrating cells. The Arp2/3 complex activator Wiskott-Aldrich Syndrome Protein (WASP) localises to the actin core of forming podosomes together with WASP-Interacting Protein (WIP). A second weaker Arp2/3 activator, cortactin, is also found at podosomes where it has been proposed to participate in matrix metalloproteinase (MMP) secretion. We have previously shown that WIP(-/-) DCs are unable to make podosomes. WIP binds to cortactin and in this report we address whether WIP regulates cortactin-mediated MMP activity. Using DCs derived from splenic murine precursors, we found that wild-type cells were able to localise MMPs at podosomes where matrix degradation takes place. In contrast, WIP(-/-) DCs remain able to synthesise MMPs but do not degrade the extracellular matrix. Infection of WIP KO DCs with lentivirus expressing WIP restored both podosome formation and their ability to degrade the extracellular matrix, implicating WIP-induced podosomes as foci of functional MMP location. When WIP KO DCs were infected with a mutant form of WIP lacking the cortactin-binding domain (WIPΔ110-170) DCs were only able to elaborate disorganised podosomes that were unable to support MMP-mediated matrix degradation. Taken together, these results suggest a role for WIP not only in WASP-mediated actin polymerisation and podosome formation, but also in cortactin-mediated extracellular matrix degradation by MMPs.     

Role of WASP in cell polarity and podosome dynamics of myeloid cells

The integrin-dependent migration of myeloid cells requires tight coordination between actin-based cell membrane protrusion and integrin-mediated adhesion to form a stable leading edge. Under this mode of migration, polarised myeloid cells including dendritic cells, macrophages and osteoclasts develop podosomes that sustain the extending leading edge. Podosome integrity and dynamics vary in response to changes in the physical and biochemical properties of the cell environment. In the current article we discuss the role of various factors in initiation and stability of podosomes and the roles of the Wiskott Aldrich Syndrome Protein (WASP) in this process. We discuss recent data indicating that in a cellular context WASP is crucial not only for localised actin polymerisation at the leading edge and in podosome cores but also for coordination of integrin clustering and activation during podosome formation and disassembly.     

Pilot clinical trial of type 1 dendritic cells loaded with autologous tumor lysates combined with GM-CSF, pegylated IFN, and cyclophosphamide for metastatic cancer patients

Twenty-four patients with metastatic cancer received two cycles of four daily immunizations with monocyte-derived dendritic cells (DC). DC were incubated with preheated autologous tumor lysate and subsequently with IFN-α, TNF-α, and polyinosinic:polycytidylic acid to attain type 1 maturation. One DC dose was delivered intranodally, under ultrasound control, and the rest intradermally in the opposite thigh. Cyclophosphamide (day -7), GM-CSF (days 1-4), and pegIFN alpha-2a (days 1 and 8) completed each treatment cycle. Pretreatment with cyclophosphamide decreased regulatory T cells to levels observed in healthy subjects both in terms of percentage and in absolute counts in peripheral blood. Treatment induced sustained elevations of IL-12 in serum that correlated with the output of IL-12p70 from cultured DC from each individual. NK activity in peripheral blood was increased and also correlated with the serum concentration of IL-12p70 in each patient. Circulating endothelial cells decreased in 17 of 18 patients, and circulating tumor cells markedly dropped in 6 of 19 cases. IFN-γ-ELISPOT responses to DC plus tumor lysate were observed in 4 of 11 evaluated cases. Tracing DC migration with [(111)In] scintigraphy showed that intranodal injections reached deeper lymphatic chains in 61% of patients, whereas with intradermal injections a small fraction of injected DC was almost constantly shown to reach draining inguinal lymph nodes. Five patients experienced disease stabilization, but no objective responses were documented. This combinatorial immunotherapy strategy is safe and feasible, and its immunobiological effects suggest potential activity in patients with minimal residual disease. A randomized trial exploring this hypothesis is currently ongoing.