3D modeling of ribosomal RNA using cryo-electron microscopy density maps

Ribosomes are macromolecular protein-RNA complexes translating mRNA into protein. To date, crystal structures are available for the bacterial 30S and archaeal 50S subunits, as well as the complete bacterial 70S ribosomes. Eukaryotic ribosomes are much more complex in terms of ribosomal RNA and proteins. However, to date high-resolution crystal structures of eukaryotic ribosomes or ribosomal subunits are lacking. In order to build reliable models for the eukaryotic rRNA, we developed an approach for large scale homology and de novo modeling of RNA and subsequent exible tting into high-resolution cryo-EM density maps. Using this approach we built a model of the T. aestivum and the S. cerevisiae ribosome based on available cryo-EM maps at 5.5 Å and 6.1 Å resolution, respectively. The model comprises of 98% of the eukaryotic rRNA including all 21 RNA expansion segments (ES) and structurally six variable regions. Further, we were able to localize 74/80 (92.5%) of the ribosomal proteins. The model reveals unique ES-ES and r-protein-ES interactions, providing new insight into the structure and evolution of the eukaryotic ribosome. Moreover, the model was used for analyzing functional ribosomal complexes, i.e. the characterization of dierent nascent polypeptide chains within the ribosomal tunnel, intermediates of protein translocation as well as mRNA quality control

Experimentelle Coxsackievirus B3-Infektion primärer muriner und humaner CD19+ B-Lymphozyten

Coxsackievirus B3 (CVB3), ein typischer Vertreter der Picornaviridae, ist nicht nur in der Lage Zielorgane wie Herz und Pankreas zu infizieren, woraus akute/chronische Krankheitsbilder wie z. B. Myokarditis oder Pankreatitis entstehen, sondern kann auch in verschiedenen Zellen des Immunsystems detektiert werden. In der vorliegenden Arbeit konnte erstmals gezeigt werden, dass CVB3 aktiv in primären murinen und humanen CD19+ B-Zellen repliziert. Dabei ist die virale Infektiösität für diese Lymphozyten in Anwesenheit virusspezifischer Antikörper deutlich gesteigert. Untersuchungen zum zugrunde liegenden Mechanismus ergaben, dass der Eintritt von CVB3 in murine CD19+ B-Zellen über diese Antikörper scheinbar nicht durch zelluläre Fc-Rezeptoren vermittelt wird, sondern eher abhängig von einzelnen Komplement-Komponenten sein könnte. Eine Stimulation der B-Zellen z. B. durch Lipopolysaccharid führte ebenfalls zur Steigerung der Empfänglichkeit dieser Immunzellen. In Übereinstimmung mit den In vitro-Daten konnte auch nach einer Infektion von BALB/c-Mäusen mit CVB3 nachgewiesen werden, dass CD19+ B-Lymphozyten des Milzgewebes infiziert werden, wobei jedoch 3 d p. i. nur 0,6-1 % der Zellen virus-positiven sind. Der Einsatz zweier unterschiedlicher Pan-Caspase-Inhibitoren ergab weiterhin, dass die aktive Virusreplikation in primären murinen und humanen CD19+ B-Zellen sowie Raji-Zellen mit apoptotischen Prozessen assoziiert ist. Dieser Zusammenhang konnte nachfolgend auch durch eine In vivo-Applikation beider Substanzen im Zuge einer CVB3-Infektion immunkompetenter BALB/c-Mäuse bestätigt werden. Dabei hatte diese Behandlung eine Senkung der Viruslast in Herz- und Milzgewebe und eine Reduktion des Anteils infizierter CD19+ B-Zellen zur Folge

Nat Struct Mol Biol

As translation proceeds, the nascent polypeptide chain passes through a tunnel in the large ribosomal subunit. Although this ribosomal exit tunnel was once thought only to be a passive conduit for the growing nascent chain, accumulating evidence suggests that it may in fact play a more active role in regulating translation and initial protein folding events. Here we have determined single-particle cryo-electron microscopy reconstructions of eukaryotic 80S ribosomes containing nascent chains with high alpha-helical propensity located within the exit tunnel. The maps enable direct visualization of density for helices as well as allowing the sites of interaction with the tunnel wall components to be elucidated. In particular regions of the tunnel, the nascent chain adopts distinct conformations and establishes specific contacts with tunnel components, both ribosomal RNA and proteins, that have been previously implicated in nascent chain-ribosome interaction

Basal cytokeratins and their relationship to the cellular origin and functional classification of breast cancer

Recent publications have classified breast cancers on the basis of expression of cytokeratin-5 and -17 at the RNA and protein levels, and demonstrated the importance of these markers in defining sporadic tumours with bad prognosis and an association with BRCA1-related breast cancers. These important observations using different technology platforms produce a new functional classification of breast carcinoma. However, it is important in developing hypotheses about the pathogenesis of this tumour type to review the nomenclature that is being used to emphasize potential confusion between terminology that defines clinical subgroups and markers of cell lineage. This article reviews the lineages in the normal breast in relation to what have become known as the 'basal-like' carcinomas

Human Cardiac-Derived Adherent Proliferating Cells Reduce Murine Acute Coxsackievirus B3-Induced Myocarditis

BACKGROUND: Under conventional heart failure therapy, inflammatory cardiomyopathy typically has a progressive course, indicating a need for alternative therapeutic strategies to improve long-term outcomes. We recently isolated and identified novel cardiac-derived cells from human cardiac biopsies: cardiac-derived adherent proliferating cells (CAPs). They have similarities with mesenchymal stromal cells, which are known for their anti-apoptotic and immunomodulatory properties. We explored whether CAPs application could be a novel strategy to improve acute Coxsackievirus B3 (CVB3)-induced myocarditis. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the safety of our approach, we first analyzed the expression of the coxsackie- and adenovirus receptor (CAR) and the co-receptor CD55 on CAPs, which are both required for effective CVB3 infectivity. We could demonstrate that CAPs only minimally express both receptors, which translates to minimal CVB3 copy numbers, and without viral particle release after CVB3 infection. Co-culture of CAPs with CVB3-infected HL-1 cardiomyocytes resulted in a reduction of CVB3-induced HL-1 apoptosis and viral progeny release. In addition, CAPs reduced CD4 and CD8 T cell proliferation. All CAPs-mediated protective effects were nitric oxide- and interleukin-10-dependent and required interferon-γ. In an acute murine model of CVB3-induced myocarditis, application of CAPs led to a decrease of cardiac apoptosis, cardiac CVB3 viral load and improved left ventricular contractility parameters. This was associated with a decline in cardiac mononuclear cell activity, an increase in T regulatory cells and T cell apoptosis, and an increase in left ventricular interleukin-10 and interferon-γ mRNA expression. CONCLUSIONS: We conclude that CAPs are a unique type of cardiac-derived cells and promising tools to improve acute CVB3-induced myocarditis

The key hypoxia regulated gene CAIX is upregulated in basal-like breast tumours and is associated with resistance to chemotherapy

Basal-like tumours account for 15% of invasive breast carcinomas and are associated with a poorer prognosis and resistance to therapy. We hypothesised that this aggressive phenotype is because of an intrinsically elevated hypoxic response. Microarrayed tumours from 188 patients were stained for hypoxia-inducible factor (HIF)-1α, prolyl hydroxylase (PHD)1, PHD2, PHD3 and factor inhibiting HIF (FIH)-1, and carbonic anhydrase (CA) IX stained in 456 breast tumours. Tumour subtypes were correlated with standard clincopathological parameters as well as hypoxic markers. Out of 456 tumours 62 (14%) tumours were basal-like. These tumours were positively correlated with high tumour grade (P<0.001) and were associated with a significantly worse disease-free survival compared with luminal tumours (P<0.001). Fifty percent of basal-like tumours expressed HIF-1α, and more than half expressed at least one of the PHD enzymes and FIH-1. Basal-like tumours were nine times more likely to be associated with CAIX expression (P<0.001) in a multivariate analysis. Carbonic anhydrase IX expression was positively correlated with tumour size (P=0.005), tumour grade (P<0.001) and oestrogen receptor (ER) negativity (P<0.001). Patients with any CAIX-positive breast tumour phenotype and in the basal tumour group had a significantly worse prognosis than CAIX-negative tumours when treated with chemotherapy (P<0.001 and P=0.03, respectively). The association between basal phenotype and CAIX suggests that the more aggressive behaviour of these tumours is partly due to an enhanced hypoxic response. Further, the association with chemoresistance in CAIX-positive breast tumours and basal-like tumours in particular raises the possibility that targeted therapy against HIF pathway or downstream genes such as CAs may be an approach to investigate for these patients

Inhibitory Effects of Prior Low-dose X-irradiation on Ischemia-reperfusion Injury in Mouse Paw

We have reported that low-dose, unlike high-dose, irradiation enhanced antioxidation function and reduced oxidative damage. On the other hand, ischemia-reperfusion injury is induced by reactive oxygen species. In this study, we examined the inhibitory effects of prior low-dose X-irradiation on ischemia-reperfusion injury in mouse paw. BALB/c mice were irradiated by sham or 0.5 Gy of X-ray. At 4 hrs after irradiation, the left hind leg was bound 10 times with a rubber ring for 0.5, 1, or 2 hrs and the paw thickness was measured. Results show that the paw swelling thickness by ischemia for 0.5 hr was lower than that for 2 hrs. At 1 hr after reperfusion from ischemia for 1 hr, superoxide dismutase activity in serum was increased in those mice which received 0.5 Gy irradiation and in the case of the ischemia for 0.5 or 1 hr, the paw swelling thicknesses were inhibited by 0.5 Gy irradiation. In addition, interstitial edema in those mice which received 0.5 Gy irradiation was less than that in the mice which underwent by sham irradiation. These findings suggest that the ischemia-reperfusion injury is inhibited by the enhancement of antioxidation function by 0.5 Gy irradiation

Lipid synthesis and secretion by primary cultures of rat mammary epithelial cells

Lipid synthesis and secretion was measured in primary rat mammary epithelial cells cultured on basement matrix in medium supplemented with lactogenic hormones. The cells grew and differentiated to form alveolar‐like structures reminiscent of lactating mammary gland. They synthesized abundant triacylglycerol, containing fatty acids characteristic of rat milk (C10:O‐C14:0), using 14C‐glucose, 14C‐oleic acid or 14C‐glycerol as precursors. Basal levels of triacylglycerol secretion were measured using 14C‐oleic acid labeling; 1.3±0.3% of the labeled cellular triacylglycerol was secreted into the medium in 24 hours. Secreted lipid droplets were surrounded by a bilayer membrane with an electron‐dense inner coat characteristic of fat globules secreted by the mammary gland. The rate of triglycerol secretion was increased to 998±98% of control (P&lt;0.01) by the addition of phorbol 12‐myristate 13‐acetate (PMA) in combination with staurosporine, a protein kinase inhibitcn. Several other protein kinase inhibitors, when combined with PMA, also markedly stimulated secretion. Effective protein kinase inhibitors included sphingosine (has diverse cellular effects including the inhibition of protein kinase C; 13‐fold increase in secretion), and KT5823 (a cGMP dependent protein kinase inhibitor; 5‐fold increase). KT5720 (a cAMP‐dependent protein kinase inhibitor) did not alter secretion. Kinase inhibitors were effective only in the presence of a phorbol ester. 4α‐phorbol‐12,13‐didecanoate, a phorbol ester which does not activate protein kinase C (PKC), could substitute for PMA. Lipid release was not mediated by disruption of cell‐cell tight junctions, as EGTA did not release lipid. Based on these observations we suggest that two signals are needed to enable or stimulate lipid secretion in cultured rat mammary epithelial cells: (1) inhibition of a protein kinase and (2) a PKC‐independent effect of phorbol ester. We have, for the first time, characterized a cell culture model suitable for studying lipid synthesis and secretion by mammary epithelial cells

Thiram inhibits angiogenesis and slows the development of experimental tumours in mice

Thiram-tetramethylthiuram disulphide – a chelator of heavy metals, inhibited DNA synthesis and induced apoptosis in cultured bovine capillary endothelial cells. Bovine capillary endothelial cells were 10–60-fold more sensitive to thiram than other cell types. These effects were prevented by addition of antioxidants, indicating involvement of reactive oxygen species. Exogenously added Cu2+ impeded specifically and almost completely the inhibitory effect of thiram for bovine capillary endothelial cells. Moreover, thiram had markedly inhibited human recombinant Cu/Zn superoxide dismutase enzymatic activity (85%) in vitro. Moreover, PC12-SOD cells with elevated Cu/Zn superoxide dismutase were less sensitive to thiram treatment than control cells. These data indicate that the effects of thiram are mediated by inhibition of Cu/Zn superoxide dismutase activity. Oral administration of thiram (13–30 μg mouse−1), inhibited angiogenesis in CD1 nude mice. Tumour development is known to largely depend on angiogenesis. We found that oral administration of thiram (30 μg) to mice caused significant inhibition of C6 glioma tumour development (60%) and marked reduction (by 3–5-fold) in metastatic growth of Lewis lung carcinoma. The data establish thiram as a potential inhibitor of angiogenesis and raise the possibility for its use as therapy in pathologies in which neovascularisation is involved, including neoplasia