Sequence elements in both subunits of the DNA fragmentation factor are essential for its nuclear transport

DNA cleavage is a biochemical hallmark of apoptosis. In humans, apoptotic DNA cleavage is executed by DNA fragmentation factor (DFF) 40. In proliferating cells DFF40 is expressed in the presence of its chaperone and inhibitor DFF45, which results in the formation of the DFF complex. Here, we present a systematic analysis of the nuclear import of the DFF complex. Our in vitro experiments demonstrate that the importin alpha/beta-heterodimer mediates the translocation of the DFF complex from the cytoplasm to the nucleus. Both DFF subunits interact directly with the importin alpha/beta-heterodimer. However, importin alpha/beta binds more tightly to the DFF complex compared with the individual subunits. Additionally, the isolated C-terminal regions of both DFF subunits together bind importin alpha/beta more strongly than the individual C termini. Our results from in vivo studies reveal that the C-terminal regions of both DFF subunits harbor nuclear localization signals. Furthermore, nuclear import of the DFF complex requires the C-terminal regions of both subunits. In more detail, one basic cluster in the C-terminal region of each subunit, DFF40 (RLKRK) and DFF45 (KRAR), is essential for nuclear accumulation of the DFF complex. Based on these findings two alternative models for the interaction of importin alpha/beta with the DFF complex are presented.     

Tissue strain amplification at the osteocyte lacuna: a microstructural finite element analysis

A parametric finite element model of an osteocyte lacuna was developed to predict the microstructural response of the lacuna to imposed macroscopic strains. The model is composed of an osteocyte lacuna, a region of perilacunar tissue, canaliculi, and the surrounding bone tissue. A total of 45 different simulations were modeled with varying canalicular diameters, perilacunar tissue material moduli, and perilacunar tissue thicknesses. Maximum strain increased with a decrease in perilacunar tissue modulus and decreased with an increase in perilacunar tissue modulus, regardless of the thickness of the perilacunar region. An increase in the predicted maximum strain was observed with an increase in canalicular diameter from 0.362 to 0.421 microm. In response to the macroscopic application of strain, canalicular diameters increased 0.8% to over 1.0% depending on the perilacunar tissue modulus. Strain magnification factors of over 3 were predicted. However, varying the size of the perilacunar tissue region had no effect on the predicted perilacunar tissue strain. These results indicate that the application of average macroscopic strains similar to strain levels measured in vivo can result in significantly greater perilacunar tissue strains and canaliculi deformations. A decrease in the perilacunar tissue modulus amplifies the perilacunar tissue strain and canaliculi deformation while an increase in the local perilacunar tissue modulus attenuates this effect.     

Human Cep192 is required for mitotic centrosome and spindle assembly

As cells enter mitosis, centrosomes dramatically increase in size and ability to nucleate microtubules. This process, termed centrosome maturation, is driven by the accumulation and activation of gamma-tubulin and other proteins that form the pericentriolar material on centrosomes during G2/prophase. Here, we show that the human centrosomal protein, Cep192 (centrosomal protein of 192 kDa), is an essential component of the maturation machinery. Specifically, we have found that siRNA depletion of Cep192 results in a complete loss of functional centrosomes in mitotic but not interphase cells. In mitotic cells lacking Cep192, microtubules become organized around chromosomes but rarely acquire stable bipolar configurations. These cells contain normal numbers of centrioles but cannot assemble gamma-tubulin, pericentrin, or other pericentriolar proteins into an organized PCM. Alternatively, overexpression of Cep192 results in the formation of multiple, extracentriolar foci of gamma-tubulin and pericentrin. Together, our findings support the hypothesis that Cep192 stimulates the formation of the scaffolding upon which gamma-tubulin ring complexes and other proteins involved in microtubule nucleation and spindle assembly become functional during mitosis.     

Arabidopsis: a model genus for speciation

What genetic and epigenetic changes underlie adaptation and divergence? Arabidopsis thaliana and its relatives are increasingly being employed to address such central questions of evolutionary biology. For example, comparative, genomic and classical genetic approaches are revealing mechanisms underlying processes relevant to speciation, including mating system evolution, the effects of ploidy and other chromosomal differences, and the roles that specific genes might play in Dobzhansky-Muller type incompatibilities. The considerable body of knowledge and resources available for A. thaliana and improvements in tools and technology applied to its close relatives are opening doors for combining experimental and comparative analyses to elucidate fundamental mechanisms of evolution.     

Synthesis, liposomal preparation, and in vitro toxicity of two novel dodecaborate cluster lipids for boron neutron capture therapy

A new class of lipids, containing the closo-dodecaborate cluster, has been synthesized. Two lipids, S-(N, N-(2-dimyristoyloxyethyl)acetamido)thioundecahydro-closo-dodecaborate (2-) (B-6-14) and S-(N, N-(2-dipalmitoyloxyethyl)acetamido)thioundecahydro-closo-dodecaborate (2-) (B-6-16) are described. Both of them have a double-tailed lipophilic part and a headgroup carrying two negative charges. Differential scanning calorimetry shows that B-6-14 and B-6-16 bilayers have main phase transition temperatures of 18.8 and 37.9 degrees C, respectively. Above the transition temperature of 18.8 degrees C, B-6-14 can form liposomal vesicles, representing the first boron-containing lipid with this capability. Upon cooling below the transition temperature, stiff bilayers are formed. When incorporated into liposomal formulations with equimolar amounts of distearoyl phosphatidylcholine (DSPC) and cholesterol, stable liposomes are obtained. The zeta-potential measurements indicate that both B-6-14- and B-6-16-containing vesicles are negatively charged, with the most negative potential described of any liposome so far. The liposomes are of high potential value as transporters of boron to tumor cells in treatments based on boron neutron capture therapy (BNCT). Liposomes prepared from B-6-14 were slightly less toxic in V79 Chinese hamster cells (IC50 5.6 mM) than unformulated Na2B12H11SH (IC50 3.9 mM), while liposomes prepared from B-6-16 were not toxic even at 30 mM.     

Molecular phylogeny of the Heterotrichea (Ciliophora, Postciliodesmatophora) based on small subunit rRNA gene sequences

A comprehensive molecular analysis of the phylogenetic relationships within the Heterotrichea including all described families is still lacking. For this reason, the complete nuclear small subunit (SSU) rDNA was sequenced from further representatives of the Blepharismidae and the Stentoridae. In addition, the SSU rDNA of a new, undescribed species of the genus Condylostomides (Condylostomatidae) was sequenced. The detailed phylogenetic analyses revealed a consistent branching pattern: while the terminal branches are generally well resolved, the basal relationships remain unsolved. Moreover, the data allow some conclusions about the macronuclear evolution within the genera Blepharisma, Stentor, and Spirostomum suggesting that a single, compact macronucleus represents the ancestral state.     

Suppression of influenza A virus nuclear antigen production and neuraminidase activity by a nutrient mixture containing ascorbic acid, green tea extract and amino acids

Influenza, one of the oldest and most common infections, poses a serious health problem causing significant morbidity and mortality, and imposing substantial economic costs. The efficacy of current drugs is limited and improved therapies are needed. A unique nutrient mixture (NM), containing ascorbic acid, green tea extract, lysine, proline, N-acetyl cysteine, selenium among other micronutrients, has been shown to exert anti-carcinogenic and anti-atherogenic activity both in vitro and in vivo. Many of the constituents of NM have been shown to have an inhibitory effect on replication of influenza virus and HIV. This prompted us to study the effect of NM on influenza A virus multiplication in infected cells and neuraminidase activity (NA) in virus particles. Addition of NM to Vero or MDCK cells post infection resulted in dose-dependent inhibition of viral nucleoprotein (NP) production in infected cells. NM-mediated inhibition of viral NP was selective and not due to cytotoxicity towards host cells. This antiviral effect was enhanced by pretreatment of virus with the nutrient mixture. Individual components of NM, namely ascorbic acid and green tea extract, also blocked viral NP production, conferring enhanced inhibition when tested in combination. Incubation of cell-free virus with NM resulted in dose-dependent inhibition of associated NA enzyme activity. In conclusion, the nutrient mixture exerts an antiviral effect against influenza A virus by lowering viral protein production in infected cells and diminishing viral enzymatic activity in cell-free particles.     

Apoptotic DNA fragmentation is not related to the phosphorylation state of histone H1

Changes in chromatin structure, histone phosphorylation and cleavage of DNA into nucleosome-size fragments are characteristic features of apoptosis. Since H1 histones bind to the site of DNA cleavage between nucleosomal cores, the question arises as to whether the state of H1 phosphorylation influences the rate of internucleosomal cleavage. Here, we tested the relation between DNA fragmentation and H1 phosphorylation both in cultured cells and in vitro. In Jurkat cells, hyperosmotic mannitol concentration resulted in apoptosis, including nucleosomal fragmentation, whereas apoptosis induction by increased NaCl concentration was not accompanied by DNA fragmentation. However, both treatments induced dephosphorylation of H1 histones. In contrast, treatment of Raji cells with alkylphosphocholine led to induction of apoptosis with internucleosomal fragmentation, albeit without notable histone H1 dephosphorylation. These results demonstrate that dephosphorylation of H1 histones is neither a prerequisite for nor a consequence of internucleosomal cleavage. Moreover, we observed with an in vitro assay that the known enhancing effect of H1 histones on the activity of the apoptosis-induced endonuclease DFF40 is independent of the subtype or the phosphorylation state of the linker histone.