Structure and microtubule-nucleation activity of isolated Drosophila embryo centrosomes characterized by whole mount scanning and transmission electron microscopy

Experimental approaches in Drosophila melanogaster over the last 20 years have played a fundamental role in elucidating the function, structure and molecular composition of the centrosome. However, quantitative data on the structure and function of the Drosophila centrosome are still lacking. This study uses, for the first time, whole mount electron microscopy in combination with negative staining on isolated centrosomes from the early Drosophila embryos to analyze its dimensions, structure and capacity to nucleate microtubules in vitro. We show that these organelles are on average 0.75 μm in diameter and have abundant pericentriolar material which often appears fibrillar and with bulbous protrusions. Corresponding to the abundant pericentriolar material, extensive microtubule nucleation occurs. Quantification of the number of microtubules nucleated showed that 50–300 active nucleation sites are present. We examined via electron microscopy immunogold labeling the distribution of γ-tubulin, CNN, Asp and the MPM-2 epitopes that are phosphorylated through Polo and the Cdk1 kinase. The distribution of these proteins is homogeneous, with the MPM-2 epitopes exhibiting the highest density. In contrast, centrosomal subdomains are identified using a centriole marker to relate centrosome size to the centriole number by electron microscopy. In conclusion, we present a clear-cut technique assaying and quantifying the microtubule nucleation capacity and antigen distribution complementing molecular studies on centrosome protein complexes, cell organelle assembly and protein composition

Interplay of inequivalent atomic positions in resonant x-ray diffraction of Fe3BO6

'Forbidden' Bragg reflections of iron orthoborate Fe3BO6 were studied theoretically and experimentally in the vicinity of the iron K edge. Their energy spectra are explained as resulting from the interference of x-rays scattered from two inequivalent crystallographic sites occupied by iron ions. This particular structure property gives rise to complex azimuthal dependences of the reflection intensities in the pre-edge region as they result from the interplay of site specific dipole-quadrupole and quadrupole-quadrupole resonant scattering. Also evidenced is an isotropic character of the absorption spectrum. Self-absorption correction to the diffraction data, as well as possible contributions of thermal vibrations and magnetic order, are discussed. Particular care is given to extracting clean spectra from the data, and it is demonstrated that excellent results can be obtained even from measurements that appear corrupted by several effects such as poor crystal quality and multiple scattering.Comment: 12 page

Polarization Dependence of Anomalous X-ray Scattering in Orbital Ordered Manganites

In order to determine types of the orbital ordering in manganites, we study theoretically the polarization dependence of the anomalous X-ray scattering which is caused by the anisotropy of the scattering factor. The general formulae of the scattering intensity in the experimental optical system is derived and the atomic scattering factor is calculated in the microscopic electronic model. By using the results, the X-ray scattering intensity in several types of the orbital ordering is numerically calculated as a function of azimuthal and analyzer angles.Comment: 9 pages, 7 figure

Low-level APC mutational mosaicism is the underlying cause in a substantial fraction of unexplained colorectal adenomatous polyposis cases

BACKGROUND: In 30-50% of patients with colorectal adenomatous polyposis, no germline mutation in the known genes APC, causing familial adenomatous polyposis, MUTYH, causing MUTYH-associated polyposis, or POLE or POLD1, causing polymerase-proofreading-associated polyposis can be identified, although a hereditary aetiology is likely. This study aimed to explore the impact of APC mutational mosaicism in unexplained polyposis. METHODS: To comprehensively screen for somatic low-level APC mosaicism, high-coverage next-generation sequencing of the APC gene was performed using DNA from leucocytes and a total of 53 colorectal tumours from 20 unrelated patients with unexplained sporadic adenomatous polyposis. APC mosaicism was assumed if the same loss-of-function APC mutation was present in ≥2 anatomically separated colorectal adenomas/carcinomas per patient. All mutations were validated using diverse methods. RESULTS: In 25% (5/20) of patients, somatic mosaicism of a pathogenic APC mutation was identified as underlying cause of the disease. In 2/5 cases, the mosaic level in leucocyte DNA was slightly below the sensitivity threshold of Sanger sequencing; while in 3/5 cases, the allelic fraction was either very low (0.1-1%) or no mutations were detectable. The majority of mosaic mutations were located outside the somatic mutation cluster region of the gene. CONCLUSIONS: The present data indicate a high prevalence of pathogenic mosaic APC mutations below the detection thresholds of routine diagnostics in adenomatous polyposis, even if high-coverage sequencing of leucocyte DNA alone is taken into account. This has important implications for both routine work-up and strategies to identify new causative genes in this patient group

Interference with Activator Protein-2 transcription factors leads to induction of apoptosis and an increase in chemo- and radiation-sensitivity in breast cancer cells

<p>Abstract</p> <p>Background</p> <p>Activator Protein-2 (AP-2) transcription factors are critically involved in a variety of fundamental cellular processes such as proliferation, differentiation and apoptosis and have also been implicated in carcinogenesis. Expression of the family members AP-2α and AP-2γ is particularly well documented in malignancies of the female breast. Despite increasing evaluation of single AP-2 isoforms in mammary tumors the functional role of concerted expression of multiple AP-2 isoforms in breast cancer remains to be elucidated. AP-2 proteins can form homo- or heterodimers, and there is growing evidence that the net effect whether a cell will proliferate, undergo apoptosis or differentiate is partly dependent on the balance between different AP-2 isoforms.</p> <p>Methods</p> <p>We simultaneously interfered with all AP-2 isoforms expressed in ErbB-2-positive murine N202.1A breast cancer cells by conditionally over-expressing a dominant-negative AP-2 mutant.</p> <p>Results</p> <p>We show that interference with AP-2 protein function lead to reduced cell number, induced apoptosis and increased chemo- and radiation-sensitivity. Analysis of global gene expression changes upon interference with AP-2 proteins identified 139 modulated genes (90 up-regulated, 49 down-regulated) compared with control cells. Gene Ontology (GO) investigations for these genes revealed <it>Cell Death </it>and <it>Cell Adhesion and Migration </it>as the main functional categories including 25 and 12 genes, respectively. By using information obtained from Ingenuity Pathway Analysis Systems we were able to present proven or potential connections between AP-2 regulated genes involved in cell death and response to chemo- and radiation therapy, (i.e. <it>Ctgf, Nrp1</it>, <it>Tnfaip3, Gsta3</it>) and AP-2 and other main apoptosis players and to create a unique network.</p> <p>Conclusions</p> <p>Expression of AP-2 transcription factors in breast cancer cells supports proliferation and contributes to chemo- and radiation-resistance of tumor cells by impairing the ability to induce apoptosis. Therefore, interference with AP-2 function could increase the sensitivity of tumor cells towards therapeutic intervention.</p

Enthalpy of formation of ye’elimite and ternesite

Calcium sulfoaluminate clinkers containing ye’elimite (Ca4Al6O12(SO4)) and ternesite (Ca5(SiO4)2SO4) are being widely investigated as components of calcium sulfoaluminate cement clinkers. These may become low energy replacements for Portland cement. Conditional thermodynamic data for ye’elimite and ternesite (enthalpy of formation) have been determined experimentally using a combination of techniques: isothermal conduction calorimetry, X-ray powder diffraction and thermogravimetric analysis. The enthalpies of formation of ye’elimite and ternesite at 25 °C were determined to be − 8523 and − 5993 kJ mol−1, respectively

Aluminum-rich belite sulfoaluminate cements: clinkering and early age hydration

Belite sulfoaluminate (BSA) cements have been proposed as environmentally friendly building materials, as their production may release up to 35% less CO2 into the atmosphere when compared to ordinary Portland cements. Here, we discuss the laboratory production of three aluminum-rich BSA clinkers with nominal mineralogical compositions in the range C2S (50-60%), C4A3$(20- 30$, i.e. a value as close as possible to the nominal composition. Under these experimental conditions, three different BSA clinkers, nominally with 20, 30 and 30 wt% of C4A3$, had 19.6, 27.1 and 27.7 wt$ respectively, as determined by Rietveld analysis. We also studied the complex hydration process of BSA cements prepared by mixing BSA clinkers and gypsum. We present a methodology to establish the phase assemblage evolution of BSA cement pastes with time, including amorphous phases and free water. The methodology is based on Rietveld quantitative phase analysis of synchrotron and laboratory X-ray powder diffraction data coupled with chemical constraints. A parallel calorimetric study is also reported. It is shown that the b-C2S phase is more reactive in aluminum-rich BSA cements than in standard belite cements. On the other hand, C4A3$ reacts faster than the belite phases. The gypsum ratio in the cement is also shown to be an important factor in the phase evolution

Anomalous Features of EMT during Keratinocyte Transformation

During the evolution of epithelial cancers, cells often lose their characteristic features and acquire a mesenchymal phenotype, in a process known as epithelial-mesenchymal transition (EMT). In the present study we followed early stages of keratinocyte transformation by HPV16, and observed diverse cellular changes, associated with EMT. We compared primary keratinocytes with early and late passages of HF1 cells, a cell line of HPV16-transformed keratinocytes. We have previously shown that during the progression from the normal cells to early HF1 cells, immortalization is acquired, while in the progression to late HF1, cells become anchorage independent. We show here that during the transition from the normal state to late HF1 cells, there is a progressive reduction in cytokeratin expression, desmosome formation, adherens junctions and focal adhesions, ultimately leading to poorly adhesive phenotype, which is associated with anchorage-independence. Surprisingly, unlike “conventional EMT”, these changes are associated with reduced Rac1-dependent cell migration. We monitored reduced Rac1-dependent migration also in the cervical cancer cell line SiHa. Therefore we can conclude that up to the stage of tumor formation migratory activity is eliminated

Can hippocampal neurites and growth cones climb over obstacles?

Guidance molecules, such as Sema3A or Netrin-1, can induce growth cone (GC) repulsion or attraction in the presence of a flat surface, but very little is known of the action of guidance molecules in the presence of obstacles. Therefore we combined chemical and mechanical cues by applying a steady Netrin-1 stream to the GCs of dissociated hippocampal neurons plated on polydimethylsiloxane (PDMS) surfaces patterned with lines 2 \ub5m wide, with 4 \ub5m period and with a height varying from 100 to 600 nm. GC turning experiments performed 24 hours after plating showed that filopodia crawl over these lines within minutes. These filopodia do not show staining for the adhesion marker Paxillin. GCs and neurites crawl over lines 100 nm high, but less frequently and on a longer time scale over lines higher than 300 nm; neurites never crawl over lines 600 nm high. When neurons are grown for 3 days over patterned surfaces, also neurites can cross lines 300 nm and 600 nm high, grow parallel to and on top of these lines and express Paxillin. Axons - selectively stained with SMI 312 - do not differ from dendrites in their ability to cross these lines. Our results show that highly motile structures such as filopodia climb over high obstacle in response to chemical cues, but larger neuronal structures are less prompt and require hours or days to climb similar obstacles