Whole cell cryo-electron tomography reveals distinct disassembly intermediates of vaccinia virus

At each round of infection, viruses fall apart to release their genome for replication, and then reassemble into stable particles within the same host cell. For most viruses, the structural details that underlie these disassembly and assembly reactions are poorly understood. Cryo-electron tomography (cryo-ET), a unique method to investigate large and asymmetric structures at the near molecular resolution, was previously used to study the complex structure of vaccinia virus (VV). Here we study the disassembly of VV by cryo-ET on intact, rapidly frozen, mammalian cells, infected for up to 60 minutes. Binding to the cell surface induced distinct structural rearrangements of the core, such as a shape change, the rearrangement of its surface spikes and de-condensation of the viral DNA. We propose that the cell surface induced changes, in particular the decondensation of the viral genome, are a prerequisite for the subsequent release of the vaccinia DNA into the cytoplasm, which is followed by its cytoplasmic replication. Generally, this is the first study that employs whole cell cryo-ET to address structural details of pathogen-host cell interaction.     

Characterization of the NADH:ubiquinone oxidoreductase (complex I) in the trypanosomatid Phytomonas serpens (Kinetoplastida)

NADH dehydrogenase activity was characterized in the mitochondrial lysates of Phytomonas serpens, a trypanosomatid flagellate parasitizing plants. Two different high molecular weight NADH dehydrogenases were characterized by native PAGE and detected by direct in-gel activity staining. The association of NADH dehydrogenase activities with two distinct multisubunit complexes was revealed in the second dimension performed under denaturing conditions. One subunit present in both complexes cross-reacted with the antibody against the 39 kDa subunit of bovine complex I. Out of several subunits analyzed by MS, one contained a domain characteristic for the LYR family subunit of the NADH:ubiquinone oxidoreductases. Spectrophotometric measurement of the NADH:ubiquinone 10 and NADH:ferricyanide dehydrogenase activities revealed their different sensitivities to rotenone, piericidin, and diphenyl iodonium.     

Properties of pyranose dehydrogenase purified from the litter-degrading fungus Agaricus xanthoderma

We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose-methanol-choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi-covalent flavinylation in PDH, 9-S-cysteinyl, 8-alpha-N3-histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are D-glucose, D-galactose, L-arabinose, and D-xylose. As shown by in situ NMR analysis, D-glucose and D-galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino-bis-(3-ethylbenzthiazolin-6-sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (k(cat)/K(m)). The enzyme may play a role in lignocellulose degradation.     

Structural protein analysis of the polyvalent staphylococcal bacteriophage 812

Phage 812 is a polyvalent phage with a very broad host range in the genus Staphylococcus, which makes it a suitable candidate for phage therapy of staphylococcal infections. This proteomic study, combining the results of both 1-DE and 2-DE followed by PMF, led to the identification of 24 virion proteins. Twenty new proteins, not yet identified by proteome analysis of closely related staphylococcal phages K and G1 were identified using this approach. Fifteen proteins were assigned unambiguously to the head-tail genome module; the remaining nine proteins are encoded by genes of the left or right arms of the phage genome. As expected, the most abundant proteins in the electrophoretic patterns are the major capsid protein, the major tail sheath protein and proteins identical to ORF 50 and ORF 95 of phage K, although their function is only putative. Identification of these 20 new proteins contributes substantially to a detailed characterization of phage virions, knowledge of which is necessary for rational phage therapy.     

The platinum-olefin binding energy in series of (PH<Subscript>3</Subscript>)<Subscript>2</Subscript>Pt(olefin) complexes - a theoretical study

Theoretical investigation of Pt(0)-olefin organometallic complexes containing tertiary phosphine ligands was focused on the strength of platinum-olefin electronic interaction. DFT theoretical study of electronic effects in a substantial number of ethylene derivatives was evaluated in terms of the Pt-olefin binding energy using MP2 correlation theory. Organometallics bearing coordinated olefins with general formula (R¹R²C = CR³R⁴)Pt(PH₃)₂ [R = various substituents] had been selected, including olefins containing both electron-donor substituents as well as electron-withdrawing groups. The stability of the corresponding complexes increases with a strengthening electron-withdrawal ability of the olefin substituents. Figure Representation of (CH₂ = CHR)Pt(PPh₃)₂ and the stability chart     

The Effect of Dominance Rank on the Distribution of Different Types of Male–Infant–Male Interactions in Barbary Macaques (Macaca sylvanus)

International Journal of Primatology - In several cercopithecine species males exhibit a specific type of male–infant–male interaction during which two males briefly manipulate an...     

The male and female perspective in the link between male infant care and mating behaviour in Barbary macaques

Infant care from adult males is unexpected in species with high paternity uncertainty. Still, males of several polygynandrous primates engage in frequent affiliative interactions with infants. Two non‐exclusive hypotheses link male infant care to male mating strategies. The paternal investment hypothesis views infant care as a male strategy to maximize the survival of sired offspring, while the mating effort hypothesis predicts that females reward males who cared for their infant by preferably mating with them. Both hypotheses predict a positive relationship between infant care and matings with a particular female. However, the paternal investment hypothesis predicts that increased matings come before infant care whereas the mating effort hypothesis predicts that infant care precedes an increase in matings. Both hypotheses are usually tested from the perspective of the proportion of matings and care that individual females engage in and receive, rather than from the perspective of the care and mating behaviour of individual males. We tested the relationships between care and mating from both female and male perspectives in Barbary macaques. Mating predicted subsequent care and care predicted subsequent mating when viewed from the male but not the female perspective. Males mainly cared for infants of their main mating partners, but infants were not mainly cared for by their likely father. Males mated more with the mothers of their favourite infants, but females did not mate more with the main caretakers of their infants. We suggest that females do not choose their mating partners based on previous infant care, increasing paternity confusion. Males might try to increase paternal investment by distributing the care according to their own instead of female mating history. Further, males pursue females for mating opportunities based on previous care.     

Estimation of ABCB1 concentration in plasma membrane

The interaction between ABCB1 transporter and its substrates takes place in cell membranes but the available data precludes quantitative analysis of the interaction between transporter and substrate molecules. Further, the amount of transporter is usually expressed as a number of ABCB1 molecules per cell. In contrast, the substrate concentration in cell membranes is estimated by determination of substrate-lipid partition coefficient, as examples. In this study, we demonstrate an approach, which enables us to estimate the concentration of ABCB1 molecules within plasma membranes. For this purpose, human leukemia K562 cells with varying expression levels of ABCB1 were used: drug selected K562/Dox and K562/HHT cells with very high transporter expression, and K562/DoxDR2, K562/DoxDR1, and K562/DoxDR05 cells with gradually decreased expression of ABCB1 derived from K562/Dox cells using RNA interference technology. First, we determined the absolute amount of ABCB1 in cell lysates using immunoblotting and recombinant ABCB1 as a standard. We then determined the relative portion of transporter residing in the plasma membrane using immunohistochemistry in nonpermeabilized and permeabilized cells. These results enabled us to estimate the concentration of ABCB1 in the plasma membrane in resistant cells. The ABCB1 concentrations in the plasma membrane of drug selected K562/Dox and K562/HHT cells containing the highest amount of transporter reached millimolar levels. Concentrations of ABCB1 in the plasma membrane of resistant K562/DoxDR2, K562/DoxDR1, and K562/DoxDR05 cells with lower transporter expression were proportionally decreased.     

Conformal radiotherapy of maxilla tumors

PURPOSE: To demonstrate a conventional and a new therapeutic method of 3D treatment planning in maxilla tumors, the process of 3D treatment planning and its significance and to compare these two methods. METHOD: We performed 2D and 3D treatment plans. The ADAC planning system was used in the 3D treatment planning. CT and MRI scans were taken on the target volume and on each scan we demarcated the target volume and the critical organs. The irregular fields were obtained by 3D graphic reconstruction provided by the treatment planning programme. RESULTS: Compared to the conventional treatment planning more favourable dose distribution was obtained within the target volume and the radiation burden of the critical organs was kept under their tolerance doses. CONCLUSION: In conformal 3D treatment planning the shape and size of the irradiated volume are in good conformity with those of the target volume. In this way the radiation burden of the critical organs and adjacent intact tissues can be reduced.     

Importance of 3D conformal percutan and brachytherapy treatment planning based on CT and MRI examinations in treatment of oral cavity tumors

AIM: The importance of 3D conformal percutan and brachytherapy treatment planning based on CT and MRI examinations in treatment of oral cavity tumors. Introducing of the planning procedure and the selection aspects. METHOD: We present the treatment planning based on CT and MRI slices of an oral cavity tumor. The percutan or interstitial boost follow the percutan irradiation of the involved regions and lymph nodes, regarding to the target volume and the critical organs. RESULT: Our ADAC 3D planning system gives us the possibility to add the first line and the boost treatment plans, to determine and compare the dose distribution within the planned target volume and the radiation load of the critical organs. CONCLUSION: The comparative 3D radiation planning system allows higher local dose escalation required for the effective radiation treatment of oral cavity tumors with maximal protection of the surrounding healthy tissues.