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.     

PEATmoss (Physcomitrella Expression Atlas Tool): a unified gene expression atlas for the model plant Physcomitrella patens

Physcomitrella patens is a bryophyte model plant that is often used to study plant evolution and development. Its resources are of great importance for comparative genomics and evo‐devo approaches. However, expression data from Physcomitrella patens were so far generated using different gene annotation versions and three different platforms: CombiMatrix and NimbleGen expression microarrays and RNA sequencing. The currently available P. patens expression data are distributed across three tools with different visualization methods to access the data. Here, we introduce an interactive expression atlas, Physcomitrella Expression Atlas Tool (PEATmoss), that unifies publicly available expression data for P. patens and provides multiple visualization methods to query the data in a single web‐based tool. Moreover, PEATmoss includes 35 expression experiments not previously available in any other expression atlas. To facilitate gene expression queries across different gene annotation versions, and to access P. patens annotations and related resources, a lookup database and web tool linked to PEATmoss was implemented. PEATmoss can be accessed at https://peatmoss.online.uni-marburg.de     

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.     

Poor lysosomal membrane integrity in proximal tubule cells of haptoglobin 2-2 genotype mice with diabetes mellitus

The haptoglobin (Hp) genotype is a major determinant of progression of nephropathy in individuals with diabetes mellitus (DM). The major function of the Hp protein is to bind and modulate the fate of extracorpuscular hemoglobin and its iron cargo. We have previously demonstrated an interaction between the Hp genotype and the DM on the accumulation of iron in renal proximal tubule cells. The primary objective of this study was to determine the intracellular localization of this iron in the proximal tubule cell and to assess its potential toxicity. Transmission electron microscopy demonstrated a marked accumulation of electron-dense deposits in the lysosomes of proximal tubules cells in Hp 2-2 DM mice. Energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy were used to perform elemental analysis of these deposits and demonstrated that these deposits were iron rich. These deposits were associated with lysosomal membrane lipid peroxidation and loss of lysosomal membrane integrity. Vitamin E administration to Hp 2-2 DM mice resulted in a significant decrease in both intralysosomal iron-induced oxidation and lysosomal destabilization. Iron-induced renal tubular injury may play a major role in the development of diabetic nephropathy and may be a target for slowing the progression of renal disease.     

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.