Ultrastructural localization of adenosine triphosphatase activity in HeLa cells at various stages of the cell cycle

Summary HeLa cells in a monolayer culture were synchronized to S, G₂ and mitotic phases by use of excess (2.5 mM) deoxythymidine double-block technique. The localizations of Ca⁺⁺-activated adenosine triphosphatase (ATPase) at different phases of the cell cycle were studied using light and electron-microscopic histochemical techniques, and microphotometric comparisons of the densities of reaction products. Enzyme reaction product was always localized in the endoplasmic reticulum, nuclear membrane, mitochondria and Golgi apparatus, but there were qualitative and quantitative differences related to the phases of the cell cycle. In S phase the activity was mainly concentrated in a perinuclear area of the cytoplasm whereas in G₂ and mitosis the activity was scattered throughout the cell. The total activity per cell was maximal in G₂, was less in S phase and least in mitosis. Activity in the mitochondria and endoplasmic reticulum was distinctly less in mitosis than in other phases of the cell cycle. The mitochondrial ATPase differed from the ATPase at other sites in ion dependence and sensitivity to oligomycin. The results suggest that there may be several distinct ATPases in proliferating cells.     

The effect of membrane stabilizers on phytochrome-controlled anthocyanin biosynthesis in Brassica oleracea

The promotion of anthocyanin synthesis in red-cabbage seedlings by 5 min exposure to R light is inhibited by subsequent application of CaCl₂. The stimulation of dark synthesis of anthocyanin by n-PrOH and by kinetin is also reduced by Ca²⁺ and by cholesterol, both of which are well known to stabilize cell membranes. By contrast, EDTA, which chelates Ca²⁺, promotes dark synthesis of anthocyanin. Assay of native Ca²⁺ extractable from seedlings immersed in EDTA demonstrates that R light exposure promotes a highly significant increase in extractable Ca²⁺. It is suggested that the molecular configuration of the phytochrome molecule affects the ability of a membrane to bind Ca²⁺ and that this in turn affects the permeability to substrates which are required for anthocyanin biosynthesis.     

Tumor ablation with irreversible electroporation

We report the first successful use of irreversible electroporation for the minimally invasive treatment of aggressive cutaneous tumors implanted in mice. Irreversible electroporation is a newly developed non-thermal tissue ablation technique in which certain short duration electrical fields are used to permanently permeabilize the cell membrane, presumably through the formation of nanoscale defects in the cell membrane. Mathematical models of the electrical and thermal fields that develop during the application of the pulses were used to design an efficient treatment protocol with minimal heating of the tissue. Tumor regression was confirmed by histological studies which also revealed that it occurred as a direct result of irreversible cell membrane permeabilization. Parametric studies show that the successful outcome of the procedure is related to the applied electric field strength, the total pulse duration as well as the temporal mode of delivery of the pulses. Our best results were obtained using plate electrodes to deliver across the tumor 80 pulses of 100 micros at 0.3 Hz with an electrical field magnitude of 2500 V/cm. These conditions induced complete regression in 12 out of 13 treated tumors, (92%), in the absence of tissue heating. Irreversible electroporation is thus a new effective modality for non-thermal tumor ablation.     

Allometric growth ratios are independent of Igf2 gene dosage during development

In the mouse, allelic dosage of the paternally expressed gene coding for insulin-like growth factor II (Igf2), from null to bi-allelic, results in dose-dependent growth, an effect which appears to be fully established during a discrete period of embryogenesis that then persists throughout life. Here, we specifically quantify the influence of Igf2 allelic dosage on the proportionality of regional embryonic growth rather than overall growth. Remarkably, preservation of allometric growth ratios between head and body regions were observed throughout development, irrespective of the range of overall growth phenotype (60-130% of wild type). Evaluation of log-log plots suggests that each allele of Igf2 expressed corresponds to the equivalent of 2-4 days of relative growth. Igf2 is predominantly expressed in extra-embryonic mesoderm (E7.5-E8.25), 24 h before alterations in cell number are known to occur in embryos with disruption of the paternally expressed allele. We hypothesized that the preservation of proportionality may result from modification of extra-embryonic development and subsequent alteration of systemic nutritional supply. Morphological analyses of chorio-allantoic and placental development between E9 and E9.5 appeared Igf2 independent. This suggests either an intrinsic but systemic Igf2-dependent activity within the embryo or a more complex developmental mechanism accounts for the proportional phenotype. Allelic IGF2 expression is subject to stochastic variation in humans, with 10% of the population estimated to be functionally bi-allelic. Evaluation of allometric growth of normal and pathological human embryos, suggest intra-uterine growth phenotypes associated with altered IGF2 imprinting are also likely to be proportionate.     

Structure and functional analysis of the IGF-II/IGF2R interaction

Embryonic development and normal growth require exquisite control of insulin-like growth factors (IGFs). In mammals the extracellular region of the cation-independent mannose-6-phosphate receptor has gained an IGF-II-binding function and is termed type II IGF receptor (IGF2R). IGF2R sequesters IGF-II; imbalances occur in cancers and IGF2R is implicated in tumour suppression. We report crystal structures of IGF2R domains 11-12, 11-12-13-14 and domains 11-12-13/IGF-II complex. A distinctive juxtaposition of these domains provides the IGF-II-binding unit, with domain 11 directly interacting with IGF-II and domain 13 modulating binding site flexibility. Our complex shows that Phe19 and Leu53 of IGF-II lock into a hydrophobic pocket unique to domain 11 of mammalian IGF2Rs. Mutagenesis analyses confirm this IGF-II 'binding-hotspot', revealing that IGF-binding proteins and IGF2R have converged on the same high-affinity site.     

Tumor destruction using electrochemotherapy followed by CpG oligodeoxynucleotide injection induces distant tumor responses

PURPOSE: Electrochemotherapy (ECT) is an effective local therapy of human cutaneous cancers but has no effect on distant untreated tumors. We addressed whether tumor-associated antigens released after ECT could induce an efficient systemic immunity when associated with an appropriate immunoadjuvant. METHODS AND RESULTS: We first studied the nature of the cellular recruitment and the expression of various toll-like receptors (TLRs) in tumors treated by ECT. We found that ECT induced a massive recruitment of CD11c and CD11b positive cells in the tumors and a strong increase of TLR9 expression. We then tested antitumor effects of the combination: ECT followed by TLR-9 ligands, CpG oligodeoxynucleotides (CpG ODN), in three murine tumor models. We found that this combination triggered both potent local synergistic antitumor effects, on the ipsi-lateral ECT-treated tumor, and more interestingly, a systemic antitumor response on the contra-lateral untreated tumor, in the three models. The systemic protection was T-cell dependent as it was not observed in nude littermates. The combination induced tumor-specific T cell effectors in the tumor-draining lymph nodes and in the spleen which secreted significantly more gamma-interferon upon activation than with ECT or CpG ODN alone. CONCLUSIONS: Our data show that ECT and CpG ODN synergize and induce a significant increase of the local effect and a systemic T-dependent antitumor response. Such combination constitutes a potential innovative vaccination strategy using in situ tumor-associated antigens that could eventually be translated into the clinic.     

The effect of kinetin in relation to photocontrol of anthocyani biosynthesis in Brassica oleracea

Kinetin stimulates the synthesis of anthocyanin in dark-grown seedlings of red-cabbage. When applied for only 15 min its effect resembles that of 5 min R light and can be nullified by a subsequent exposure to 5 min FR. However, kinetin fails to stimulate PAL activity in the dark-grown seedlings. It is suggested that the effect of kinetin, like that of R light, may be to increase membrane permeability, allowing a pool of endogenous substrate to reach the site of anthocyanin biosynthesis.     

Taxonomy and distribution of fresh water Ostracoda from Socotra Island,Yemen

Eleven species belonging to ten genera of fresh water Ostracoda, have been identified from thirteen sediment samples collected from the bottom of permanent streams running along the N-NE part of Socotra Island. The fauna shows close affinity with the S-E African and S-E Asian assemblages, with interesting findings regarding the Palearctic Limnocythere stationis Vávra, 1891. The taxonomy and geographic distribution of the fauna are here discussed in detail.     

Quantification of the Heterogeneity of Prognostic Cellular Biomarkers in Ewing Sarcoma Using Automated Image and Random Survival Forest Analysis

Driven by genomic somatic variation, tumour tissues are typically heterogeneous, yet unbiased quantitative methods are rarely used to analyse heterogeneity at the protein level. Motivated by this problem, we developed automated image segmentation of images of multiple biomarkers in Ewing sarcoma to generate distributions of biomarkers between and within tumour cells. We further integrate high dimensional data with patient clinical outcomes utilising random survival forest (RSF) machine learning. Using material from cohorts of genetically diagnosed Ewing sarcoma with EWSR1 chromosomal translocations, confocal images of tissue microarrays were segmented with level sets and watershed algorithms. Each cell nucleus and cytoplasm were identified in relation to DAPI and CD99, respectively, and protein biomarkers (e.g. Ki67, pS6, Foxo3a, EGR1, MAPK) localised relative to nuclear and cytoplasmic regions of each cell in order to generate image feature distributions. The image distribution features were analysed with RSF in relation to known overall patient survival from three separate cohorts (185 informative cases). Variation in pre-analytical processing resulted in elimination of a high number of non-informative images that had poor DAPI localisation or biomarker preservation (67 cases, 36%). The distribution of image features for biomarkers in the remaining high quality material (118 cases, 10⁴ features per case) were analysed by RSF with feature selection, and performance assessed using internal cross-validation, rather than a separate validation cohort. A prognostic classifier for Ewing sarcoma with low cross-validation error rates (0.36) was comprised of multiple features, including the Ki67 proliferative marker and a sub-population of cells with low cytoplasmic/nuclear ratio of CD99. Through elimination of bias, the evaluation of high-dimensionality biomarker distribution within cell populations of a tumour using random forest analysis in quality controlled tumour material could be achieved. Such an automated and integrated methodology has potential application in the identification of prognostic classifiers based on tumour cell heterogeneity.