Transmembrane peptides as inhibitors of ErbB receptor signaling

Receptor tyrosine kinases have a single transmembrane (TM) segment that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor. However, mutations within some of these receptors, and recent studies with the epidermal growth factor (EGF) and ErbB2 receptors have indicated that interactions between TM domains do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimerization and activation. One consequence of the importance of these interactions is that short hydrophobic peptides corresponding to these domains should act as specific inhibitors. To test this hypothesis, we constructed expression vectors encoding short fusion peptides encompassing native or mutated TM domains of the EGF, ErbB2, and insulin receptors. In human cell lines overexpressing the wild-type EGF receptor or ErbB2, we observed that the peptides are expressed at the cell surface and that they inhibit specifically the autophosphorylation and signaling pathway of their cognate receptor. Identical results were obtained with peptides chemically synthesized. Mechanism of action involves inhibition of dimerization of the receptors as shown by the lack of effects of mutant nondimerizing sequences, completed by density centrifugation and covalent cross-linking experiments. Our findings stress the role of TM domain interactions in ErbB receptor function, and possibly for other single-spanning membrane proteins.     

Hidden in plain sight: subtle effects of the 8-oxoguanine lesion on the structure, dynamics, and thermodynamics of a 15-base pair oligodeoxynucleotide duplex

The base lesion 8-oxoguanine is formed readily by oxidation of DNA, potentially leading to G → T transversion mutations. Despite the apparent similarity of 8-oxoguanine-cytosine base pairs to normal guanine-cytosine base pairs, cellular base excision repair systems effectively recognize the lesion base. Here we apply several techniques to examine a single 8-oxoguanine lesion at the center of a nonpalindromic 15-mer duplex oligonucleotide in an effort to determine what, if anything, distinguishes an 8-oxoguanine-cytosine (8oxoG-C) base pair from a normal base pair. The lesion duplex is globally almost indistinguishable from the unmodified parent duplex using circular dichroism spectroscopy and ultraviolet melting thermodynamics. The DNA mismatch-detecting photocleavage agent Rh(bpy)(2)chrysi(3+) cleaves only weakly and nonspecifically, revealing that the 8oxoG-C pair is locally stable at the level of the individual base pairs. Nuclear magnetic resonance spectra are also consistent with a well-conserved B-form duplex structure. In the two-dimensional nuclear Overhauser effect spectra, base-sugar and imino-imino cross-peaks are strikingly similar between parent and lesion duplexes. Changes in chemical shift due to the 8oxoG lesion are localized to its complementary cytosine and to the 2-3 bp immediately flanking the lesion on the lesion strand. Residues further removed from the lesion are shown to be unperturbed by its presence. Notably, imino exchange experiments indicate that the 8-oxoguanine-cytosine pair is strong and stable, with an apparent equilibrium constant for opening equal to that of other internal guanine-cytosine base pairs, on the order of 10(-6). This collection of experiments shows that the 8-oxoguanine-cytosine base pair is incredibly stable and similar to the native pair.     

Nonsense-mediated mRNA decay impacts MSI-driven carcinogenesis and anti-tumor immunity in colorectal cancers

Nonsense-mediated mRNA Decay (NMD) degrades mutant mRNAs containing premature termination codon (PTC-mRNAs). Here we evaluate the consequence of NMD activity in colorectal cancers (CRCs) showing microsatellite instability (MSI) whose progression is associated with the accumulation of PTC-mRNAs encoding immunogenic proteins due to frameshift mutations in coding repeat sequences. Inhibition of UPF1, one of the major NMD factors, was achieved by siRNA in the HCT116 MSI CRC cell line and the resulting changes in gene expression were studied using expression microarrays. The impact of NMD activity was also investigated in primary MSI CRCs by quantifying the expression of several mRNAs relative to their mutational status and to endogenous UPF1 and UPF2 expression. Host immunity developed against MSI cancer cells was appreciated by quantifying the number of CD3epsilon-positive tumor-infiltrating lymphocytes (TILs). UPF1 silencing led to the up-regulation of 1251 genes in HCT116, among which a proportion of them (i.e. 38%) significantly higher than expected by chance contained a coding microsatellite (P<2x10(-16)). In MSI primary CRCs, UPF1 was significantly over-expressed compared to normal adjacent mucosa (P<0.002). Our data provided evidence for differential decay of PTC-mRNAs compared to wild-type that was positively correlated to UPF1 endogenous expression level (P = 0.02). A negative effect of UPF1 and UPF2 expression on the host's anti-tumor response was observed (P<0.01). Overall, our results show that NMD deeply influences MSI-driven tumorigenesis at the molecular level and indicate a functional negative impact of this system on anti-tumor immunity whose intensity has been recurrently shown to be an independent factor of favorable outcome in CRCs.     

Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo

New sources of red blood cells (RBCs) would improve the transfusion capacity of blood centers. Our objective was to generate cells for transfusion by inducing a massive proliferation of hematopoietic stem and progenitor cells, followed by terminal erythroid differentiation. We describe here a procedure for amplifying hematopoietic stem cells (HSCs) from human cord blood (CB) by the sequential application of specific combinations of growth factors in a serum-free culture medium. The procedure allowed the ex vivo expansion of CD34+ progenitor and stem cells into a pure erythroid precursor population. When injected into nonobese diabetic, severe combined immunodeficient (NOD/SCID) mice, the erythroid cells were capable of proliferation and terminal differentiation into mature enucleated RBCs. The approach may eventually be useful in clinical transfusion applications.     

Modulation of the phase heterogeneity of aminoglycerophospholipid mixtures by sphingomyelin and monovalent cations: maintenance of the lamellar arrangement in the biological membranes

The phase behaviour of mixed molecular species of phosphatidylethanolamine, phosphatidylserine and sphingomyelin of biological origin were examined in aqueous co-dispersions using synchrotron X-ray diffraction. The co-dispersions of phospholipids studied were aimed to model the mixing of lipids populating the cytoplasmic and outer leaflets in the resting or scrambled activated cell membrane. Mixtures enriched with phosphatidylethanolamine and phosphatidylserine were characterized by a phase separation of non-lamellar phases (cubic and inverted hexagonal) with a lamellar gel phase comprising the most saturated molecular species. Inclusion of sphingomyelin in the mixture resulted in a suppression of the hexagonal-II phase in favour of lamellar phases at temperatures where a proportion of the phospholipid was fluid. The effect was also dependent on the total amount of sphingomyelin in ternary mixtures, and the lamellar phase dominated in mixtures containing more than 30 mol%, irrespective of the relative proportions of phosphatidylserine/sphingomyelin. A transition from gel to liquid-crystal phase was detected by wide-angle scattering during heating scans of ternary mixtures enriched in sphingomyelin and was shown by thermal cycling experiments to be coupled with a hexagonal-II phase to lamellar transition. In such samples there was evidence of a coexistence of non-lamellar phases with a lamellar gel phase. A transition of the gel phase to the fluid state on heating from 35 to 41 °C was evidenced by a progressive increase in the lamellar d-spacing. The presence of calcium enhanced the phase separation of a lamellar gel phase from a hexagonal-II phase in mixtures enriched in phosphatidylserine. This effect was counteracted by charge screening with 150 mM NaCl. The effect of sphingomyelin on stabilizing the lamellar phase is discussed in the context of an altered composition in the cytoplasmic/outer leaflets of the plasma membrane resulting from scrambling of the phospholipid distribution. The results suggest that a lamellar structure can be retained by the inward translocation of sphingomyelin in biological membranes. The presence of monovalent cations serves also to stabilize the bilayer in activated cells where a translocation of aminoglycerophospholipids and an influx of calcium occur simultaneously.Abbreviations PC phosphatidylcholine - PE phosphatidylethanolamine - PS phosphatidylserine - SAXS small-angle X-ray scattering - SM sphingomyelin - WAXS wide-angle X-ray scattering - XRD X-ray diffraction     

Water mass-specificity of bacterial communities in the North Atlantic revealed by massively parallel sequencing

Bacterial assemblages from subsurface (100 m depth), meso- (200-1000 m depth) and bathy-pelagic (below 1000 m depth) zones at 10 stations along a North Atlantic Ocean transect from 60°N to 5°S were characterized using massively parallel pyrotag sequencing of the V6 region of the 16S rRNA gene (V6 pyrotags). In a dataset of more than 830,000 pyrotags, we identified 10,780 OTUs of which 52% were singletons. The singletons accounted for less than 2% of the OTU abundance, whereas the 100 and 1000 most abundant OTUs represented 80% and 96% respectively of all recovered OTUs. Non-metric Multi-Dimensional Scaling and Canonical Correspondence Analysis of all the OTUs excluding the singletons revealed a clear clustering of the bacterial communities according to the water masses. More than 80% of the 1000 most abundant OTUs corresponded to Proteobacteria of which 55% were Alphaproteobacteria, mostly composed of the SAR11 cluster. Gammaproteobacteria increased with depth and included a relatively large number of OTUs belonging to Alteromonadales and Oceanospirillales. The bathypelagic zone showed higher taxonomic evenness than the overlying waters, albeit bacterial diversity was remarkably variable. Both abundant and low-abundance OTUs were responsible for the distinct bacterial communities characterizing the major deep-water masses. Taken together, our results reveal that deep-water masses act as bio-oceanographic islands for bacterioplankton leading to water mass-specific bacterial communities in the deep waters of the Atlantic.     

In silico identification and characterization of putative differentially expressed genes involved in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) seed development

Two genotypes of common bean (Phaseolus vulgaris L.) were studied to determine the structural cause of seed abortion in this species. In the non-abortive control (wild-type, cultivar BAT93), the histological analysis revealed a classical pattern of seed development and showed coordinated differentiation of the embryo proper, suspensor, endosperm tissue and seed coat. In contrast, the ethyl methanesulfonate (EMS) mutant (cultivar BAT93) showed disruption in the normal seed development leading to embryo abortion. Aborted embryos from these degenerate seeds showed abnormalities in suspensor and cotyledons at the globular, heart, torpedo and cotyledon stages. Exploring the feasibility of incorporating the available online bioinformatics databases, we identified 22 genes revealing high homology with genes involved in Arabidopsis thaliana embryo development and expressed in common bean immature seeds. The expression patterns of these genes were confirmed by RT–PCR. All genes were highly expressed in seed tissues. To study the expression profiles of isolated genes during Phaseolus embryogenesis, six selected genes were examined by quantitative RT–PCR analysis on the developing embryos of wild-type and EMS mutant plants. All selected genes were expressed differentially at different stages of embryo development. These results could help to improve understanding of the mechanism of common bean embryogenesis.     

Generation of two modified mouse alleles of the Hic1 tumor suppressor gene

HIC1 (hypermethylated in cancer 1) is a tumor suppressor gene located on chromosome 17p13.3, a region frequently hypermethylated or deleted in human neoplasias. In mouse, Hic1 is essential for embryonic development and exerts an antitumor role in adult animals. Since Hic1-deficient mice die perinatally, we generated a conditional Hic1 null allele by flanking the Hic1-coding region by loxP sites. When crossed to animals expressing Cre recombinase in a cell-specific manner, the Hic1 conditional mice will provide new insights into the function of Hic1 in developing and mature tissues. Additionally, we used gene targeting to replace sequence-encoding amino acids 186-893 of Hic1 by citrine fluorescent protein cDNA. We demonstrate that the distribution of Hic1-citrine fusion polypeptide corresponds to the expression pattern of wild-type Hic1. Consequently, Hic1-citrine "reporter" mice can be used to monitor the activity of the Hic1 locus using citrine fluorescence.