The structural and electrochemical consequences of hydrogenating copper N2S2 Schiff base macrocycles

A series of cis and trans tetradentate copper macrocyclic complexes, of ring size 14-16, that employ amine and thioether donor groups are reported. Apart from 5,6,15,16-bisbenzo-8,13-diaza-1,4-dithia-cyclohexadecane copper(I) (cis-[Cu(H₄N_buS_en)]⁺) all of the complexes are obtained in the copper(II) form. Crystallographic analysis shows that the copper(II) complexes all adopt a distorted planar geometry around the copper. In contrast, cis-[Cu(H₄N_buS_en)]⁺ is found to adopt a distorted tetrahedral geometry. The complexes were subjected to electrochemical analysis in water and acetonitrile. The effect of the solvent, positions of the donor atoms (cis/trans) on E^1/2 is discussed as is the comparison of the electrochemical behaviour of these complexes with their parent Schiff base macrocycles.     

Acquisition of Temozolomide Chemoresistance in Gliomas Leads to Remodeling of Mitochondrial Electron Transport Chain

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of high-grade gliomas. Acquired chemoresistance is a severe limitation to this therapy with more than 90% of recurrent gliomas showing no response to a second cycle of chemotherapy. Efforts to better understand the underlying mechanisms of acquired chemoresistance to TMZ and potential strategies to overcome chemoresistance are, therefore, critically needed. TMZ methylates nuclear DNA and induces cell death; however, the impact on mitochondria DNA (mtDNA) and mitochondrial bioenergetics is not known. Herein, we tested the hypothesis that TMZ-mediated alterations in mtDNA and respiratory function contribute to TMZ-dependent acquired chemoresistance. Using an in vitro model of TMZ-mediated acquired chemoresistance, we report 1) a decrease in mtDNA copy number and the presence of large heteroplasmic mtDNA deletions in TMZ-resistant glioma cells, 2) remodeling of the entire electron transport chain with significant decreases of complexes I and V and increases of complexes II/III and IV, and 3) pharmacologic and genetic manipulation of cytochrome c oxidase, which restores sensitivity to TMZ-dependent apoptosis in resistant glioma cells. Importantly, human primary and recurrent pairs of glioblastoma multiforme (GBM) biopsies as well as primary and TMZ-resistant GBM xenograft lines exhibit similar remodeling of the ETC. Overall these results suggest that TMZ-dependent acquired chemoresistance may be due to a mitochondrial adaptive response to TMZ genotoxic stress with a major contribution from cytochrome c oxidase. Thus, abrogation of this adaptive response may reverse chemoresistance and restore sensitivity to TMZ, providing a strategy for improved therapeutic outcomes in GBM patients.     

Regulatory volume increase (RVI) by in situ and isolated bovine articular chondrocytes

Metabolism of the matrix by chondrocytes is sensitive to alterations in cell volume that occur, for example, during static loading and osteoarthritis. The ability of chondrocytes to respond to changes in volume could be important, and this study was aimed at testing the hypothesis that chondrocytes can regulate their volume following cell shrinking by regulatory volume increase (RVI). We used single cell fluorescence imaging of in situ bovine articular chondrocytes, cells freshly isolated into 280 or 380 mOsm, or 2-D cultured chondrocytes loaded with calcein or fura-2, to investigate RVI and changes to [Ca2+]i during shrinkage. Following a 42% hyperosmotic challenge, chondrocytes rapidly shrunk, however, only approximately 6% of the in situ or freshly isolated chondrocytes demonstrated RVI. This contrasted with 2D-cultured chondrocytes where approximately 54% of the cells exhibited RVI. The rate of RVI was the same for all preparations. During the 'post-RVD/RVI protocol', approximately 60% of the in situ and freshly isolated chondrocytes demonstrated RVD, but only approximately 5% showed RVI. There was no relationship between [Ca2+]i and RVI either during hyperosmotic challenge, or during RVD suggesting that changes to [Ca2+]i were not required for RVI. Depolymerisation of the actin cytoskeleton by latrunculin, increased RVI by freshly isolated chondrocytes, in a bumetanide-sensitive manner. The results showed that in situ and freshly isolated articular chondrocytes have only limited RVI capacity. However, RVI was stimulated by treating freshly isolated chondrocytes with latrunculin B and following 2D culture of chondrocytes, suggesting that cytoskeletal integrity plays a role in regulating RVI activity which appears to be mediated principally by the Na+ - K+ -2Cl- cotransporter.     

Human subcutaneous adipose cells support complete differentiation but not self-renewal of hematopoietic progenitors

Adipose tissue is now considered as an endocrine organ implicated in energy regulation, inflammation and immune response, and as a source of multipotent cells with a broad range of differentiation capacities. Some of these cells are of a mesenchymal type which can -- like their bone marrow (BM) counterpart -- support hematopoiesis, since in a previous study we were able to reconstitute lethally irradiated mice by cells isolated from adipose tissue. In the present study, we established that cells derived from the stroma-vascular fraction of human subcutaneous fat pads support the complete differentiation of hematopoietic progenitors into myeloid and B lymphoid cells. However, these cells are unable to maintain the survival and self-renewal of hematopoietic stem cells. These features, similar to those of BM adipocytes, are the opposite of those of other cell types derived from mesenchymal progenitors such as BM myofibroblasts or osteoblasts. Because it is abundant and accessible, adipose tissue could be a convenient source of cells for the short-term reconstitution of hematopoiesis in man.     

Artifical transfer and morphological description of virus particles associated with superparasitism behaviour in a parasitoid wasp

In parasitoids, the adaptive significance of superparasitism (laying of egg(s) in already parasitized hosts) has been the subject of strong controversy. The current view is to interpret this behaviour as an adaptation to increased competition for hosts, because the supernumerary egg still has a chance to win possession for the host. However, we recently discovered that in the solitary parasitoid Leptopilina boulardi, superparasitism is rather caused by an unknown infectious element: stable non superparasitizing lineages (NS) are transformed into stable superparasitizing lineages (S) after eggs from both lineages have competed inside the same host (superparasitism). In this report, we investigate the nature and location of the causative agent. Involvement of bacteria is unlikely because antibiotic treatments do not affect wasp phenotype and because bacterial 16S ribosomal DNA was not detected using PCR. We report successful injection experiments showing that the causative agents are located in wasp poison gland and ovaries and are stably inherited. Electron microscopic studies demonstrate that long filamentous virus particles located in wasp oviducts are strongly associated with superparasitism behaviour, leading to reconsider the adaptive significance of this behaviour in parasitoids. Interestingly, parasitoids are often infected with similar viruses for which no phenotypic effect has been documented. This raises the possibility that they could induce the same behavioural manipulation.     

Transmembrane proteins are not required for early stages of nuclear envelope assembly

All identified membrane fusion proteins are transmembrane proteins. In the present study, we explored the post-mitotic reassembly of the NE (nuclear envelope). The proteins that drive membrane rearrangements in NE assembly remain unknown. To determine whether transmembrane proteins are prerequisite components of this fusion machinery, we have focused on nuclear reconstitution in a cell-free system. Mixing of soluble interphase cytosolic extract and MV (membrane vesicles) from amphibian eggs with chromatin results in the formation of functional nuclei. We replaced MV and cytosol with protein-free phosphatidylcholine LS (liposomes) that were pre-incubated with interphase cytosol. While later stages of NE assembly yielding functional nucleus did not proceed without integral proteins of MV, LS-associated cytosolic proteins were sufficient to reconstitute membrane targeting to the chromatin and GTP-dependent lipid mixing. Binding involved LS-associated A-type lamin, and fusion involved Ran GTPase. Thus in contrast with post-fusion stages, fusion initiation in NE assembly, like membrane remodelling in budding and fission, does not require transmembrane proteins.     

Novel chromophores and buried charges control color in mFruits

mFruits are second-generation monomeric red fluorescent proteins (mRFPs) that have improved brightness and photostability compared to the first-generation mRFP1. The emission and excitation maxima are distributed over the remarkably large ranges of about 550-650 and 540-590 nm, respectively; however, the variations in the spectra can be traced to a few key amino acids. Spectroscopic and atomic resolution crystallographic analyses of three representatives, mOrange, mStrawberry, and mCherry, reveal that different mechanisms operate to establish the excitation and emission maxima. Evidently, they all undergo the second oxidation step to produce an acylimine linkage in the polypeptide backbone. In comparison to the progenitor DsRed, direct covalent modification to this linkage (mOrange) and indirect modification of the chromophore environment (mStrawberry and mCherry) produce strong blue- and red-shifted variants. The blue shift of mOrange is induced by an unprecedented covalent modification of the protein backbone. The electron-density map indicates the formation of a third heterocycle, 2-hydroxy-dihydrooxazole, upon the reaction of Thr 66 Ogamma with the polypeptide backbone, which in turn reduces the conjugation of the carbonyl at position 65 with the rest of the chromophore. In mStrawberry and mCherry, the movement of charged Lys 70 and protonation of Glu 215 are proposed to modify the chromophore electron-density distribution, inducing the red shift. pH-dependent spectral shifts of mCherry and mStrawberry appear to result from the titration of Glu 215, although, for mStrawberry, partial cyclization of Thr 66 may contribute at high pH.