A non-catalytic role for inositol 1,3,4,5,6-pentakisphosphate 2-kinase in the synthesis of ribosomal RNA.

Fundamental to the life and destiny of every cell is the regulation of protein synthesis through ribosome biogenesis, which begins in the nucleolus with the production of ribosomal RNA (rRNA). Nucleolar organization is a highly dynamic and tightly regulated process; the structural factors that direct nucleolar assembly and disassembly are just as important in controlling rRNA synthesis as are the catalytic activities that synthesize rRNA. Here, we report that a signaling enzyme, inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP5K) is also a structural component in the nucleolus. We demonstrate that IP5K has functionally significant interactions with three proteins that regulate rRNA synthesis: protein kinase CK2, TCOF1 and upstream-binding-factor (UBF). Through molecular modeling and mutagenic studies, we identified an Arg-Lys-Lys tripeptide located on the surface of IP5K that mediates its association with UBF. Nucleolar IP5K spatial dynamics were sensitive to experimental procedures (serum starvation or addition of actinomycin D) that inhibited rRNA production. We show that IP5K makes stoichiometrically sensitive contributions to the architecture of the nucleoli in intact cells, thereby influencing the degree of rRNA synthesis. Our study adds significantly to the biological significance of IP5K; previously, it was the kinase activity of this protein that had attracted attention. Our demonstration that IP5K 'moonlights' as a molecular scaffold offers an unexpected new example of how the biological sophistication of higher organisms can arise from gene products acquiring multiple functions, rather than by an increase in gene number.

Discovery of InsP6-kinases as InsP6-dephosphorylating enzymes provides a new mechanism of cytosolic InsP6 degradation driven by the cellular ATP/ADP ratio.

InsP6 (inositol hexakisphosphate), the most abundant inositol phosphate in metazoa, is pyrophosphorylated to InsP7 [5PP-InsP5 (diphosphoinositol pentakisphosphate)] by cytosolic and nuclear IP6Ks (InsP6 kinases) and to 1PP-InsP5 by another InsP6/InsP7 kinase family. MINPP1 (multiple inositol-polyphosphate phosphatase 1), the only known InsP6 phosphatase, is localized in the ER (endoplasmic reticulum) and lysosome lumina. A mechanism of cytosolic InsP6 dephosphorylation has remained enigmatic so far. In the present study, we demonstrated that IP6Ks change their kinase activity towards InsP6 at a decreasing ATP/ADP ratio to an ADP phosphotransferase activity and dephosphorylate InsP6. Enantio-selective analysis revealed that Ins(2,3,4,5,6)P5 is the main InsP5 product of the IP6K reaction, whereas the exclusive product of MINPP1 activity is the enantiomer Ins(1,2,4,5,6)P5. Whereas lentiviral RNAi-based depletion of MINPP1 at falling cellular ATP/ADP ratios had no significant impact on Ins(2,3,4,5,6)P5 production, the use of the selective IP6K inhibitor TNP [N2-(m-trifluorobenzyl),N6-(p-nitrobenzyl)purine] abolished the production of this enatiomer in different types of cells. Furthermore, by analysis of rat tissue and human blood samples all (main and minor) dephosphorylation products of InsP6 were detected in vivo. In summary, we identified IP6Ks as novel nuclear and cytosolic InsP6- (and InsP5-) dephosphorylating enzymes whose activity is sensitively driven by a decrease in the cellular ATP/ADP ratio, thus suggesting a role for IP6Ks as cellular adenylate energy 'sensors'.

Tumour cells can employ extracellular Ins(1,2,3,4,5,6)P(6) and multiple inositol-polyphosphate phosphatase 1 (MINPP1) dephosphorylation to improve their proliferation.

InsP(6) [Ins(1,2,3,4,5,6)P6; phytate] is the most abundant inositol phosphate in mammalian cells with cytosolic/nuclear concentrations of up to 50 µM. We noticed that InsP6 in culture medium at a concentration of ≤50 µM significantly stimulates H1299 tumour cell growth, whereas larger concentrations of InsP6 inhibit growth. A detailed study of the fate of 30 µM InsP6 added to H199 cells revealed a major fraction of InsP6 initially precipitates as cell-surface metal complexes, but becomes slowly re-solubilized by extracellular dephosphorylation first to InsP3 isomers and subsequently to free myo-inositol. The precipitated metal-InsP6 complex is endocytosed in a receptor-independent but intact-glycocalyx-dependent manner and appears in lysosomes, where it is immediately dephosphorylated to Ins(1,2,4,5,6)P5 and very slowly to free inositol. By RNA knockdown, we identified secreted and lysosome targeted MINPP1 (multiple inositol-polyphosphate phosphatase 1), the mammalian 3-phytase, to be essentially involved both in extracellular and in lysosomal InsP6 dephosphorylation. The results of the present study indicate that tumour cells employ this enzyme to utilize the micronutrients myo-inositol and metal-phosphate when encountering extracellular InsP6 and thus to enhance their growth potential.

Upward Lightning at Wind Turbines: Risk Assessment From Larger-Scale Meteorology.

Upward lightning (UL) has become a major threat to the growing number of wind turbines producing renewable electricity. It can be much more destructive than downward lightning due to the large charge transfer involved in the discharge process. Ground-truth lightning current measurements indicate that less than 50% of UL could be detected by lightning location systems (LLS). UL is expected to be the dominant lightning type during the cold season. However, current standards for assessing the risk of lightning at wind turbines mainly consider summer lightning, which is derived from LLS. This study assesses the risk of LLS-detectable and LLS-undetectable UL at wind turbines using direct UL measurements at instrumented towers. These are linked to meteorological data using random forests. The meteorological drivers for the absence/occurrence of UL are found from these models. In a second step, the results of the tower-trained models are extended to a larger study area (central and northern Germany). The tower-trained models for LLS-detectable lightning are independently verified at wind turbine sites in this area and found to reliably diagnose this type of UL. Risk maps based on cold season case study events show that high probabilities in the study area coincide with actual UL flashes. This lends credibility to the application of the model to all UL types, increasing both risk and affected areas.

Amplification of annual and diurnal cycles of alpine lightning.

The response of lightning to a changing climate is not fully understood. Historic trends of proxies known for fostering convective environments suggest an increase of lightning over large parts of Europe. Since lightning results from the interaction of processes on many scales, as many of these processes as possible must be considered for a comprehensive answer. Recent achievements of decade-long seamless lightning measurements and hourly reanalyses of atmospheric conditions including cloud micro-physics combined with flexible regression techniques have made a reliable reconstruction of cloud-to-ground lightning down to its seasonally varying diurnal cycle feasible. The European Eastern Alps and their surroundings are chosen as reconstruction region since this domain includes a large variety of land-cover, topographical and atmospheric circulation conditions. The most intense changes over the four decades from 1980 to 2019 occurred over the high Alps where lightning activity doubled in the 2010 s compared to the 1980 s. There, the lightning season reaches a higher maximum and starts one month earlier. Diurnally, the peak is up to 50% stronger with more lightning strikes in the afternoon and evening hours. Signals along the southern and northern alpine rim are similar but weaker whereas the flatlands surrounding the Alps have no significant trend.

Upward Lightning at the Gaisberg Tower: The Larger-Scale Meteorological Influence on the Triggering Mode and Flash Type.

Upward lightning is rarer than downward lightning and requires tall (100+ m) structures to initiate. It may be either self-initiated or triggered by other lightning discharges. While conventional lightning location systems (LLSs) detect most of the upward lightning flashes superimposed by pulses or return strokes, they miss a specific flash type that consists only of a continuous current. Globally, only few specially instrumented towers can record this flash type. The proliferation of wind turbines in combination with damages from upward lightning necessitates an improved understanding under which conditions self-initiated upward lightning and the continuous-current-only subtype occur. This study uses a random forest machine learning model to find the larger-scale meteorological conditions favoring the occurrence of the different phenomena. It combines ground truth lightning current measurements at the specially instrumented tower at Gaisberg mountain in Austria with variables from larger-scale meteorological reanalysis data (ERA5). These variables reliably explain whether upward lightning is self-initiated or triggered by other lightning discharges. The most important variable is the height of the -10°C isotherm above the tall structure: the closer it is, the higher is the probability of self-initiated upward lightning. For the different flash types, this study finds a relationship to the larger-scale electrification conditions and the LLS-detected lightning situation in the vicinity. Lower amounts of supercooled liquid water, solid, and liquid differently sized particles and no LLS-detected lightning events nearby favor the continuous-current-only subtype compared to the other subtypes, which preferentially occur with LLS-detected lightning events within 3 km from the Gaisberg Tower.

Frequency and predictors of poststroke epilepsy after mechanical thrombectomy for large vessel occlusion stroke: results from a multicenter cohort study.

BACKGROUND: Poststroke epilepsy (PSE) represents an important complication of stroke. Data regarding the frequency and predictors of PSE in patients with large-vessel occlusion stroke receiving mechanical thrombectomy (MT) are scarce. Furthermore, information on acute and preexisting lesion characteristics on brain MRI has not yet been systematically considered in risk prediction of PSE. This study thus aims to assess PSE risk after acute ischemic stroke treated with MT, based on clinical and MRI features. METHODS: In this multicenter study from two tertiary stroke centers, we included consecutive acute ischemic stroke patients who had received MT for acute intracranial large vessel occlusion (LVO) between 2011 and 2017, in whom post-interventional brain MRI and long term-follow-up data were available. Infarct size, affected cerebrovascular territory, hemorrhagic complications and chronic cerebrovascular disease features were assessed on MRI (blinded to clinical information). The primary outcome was the occurrence of PSE (> 7 days after stroke onset) assessed by systematic follow-up via phone interview or electronic records. RESULTS: Our final study cohort comprised 348 thrombectomy patients (median age: 67 years, 45% women) with a median long-term follow-up of 78 months (range 0-125). 32 patients (9%) developed PSE after a median of 477 days (range 9-2577 days). In univariable analyses, larger postinterventional infarct size, infarct location in the parietal, frontal or temporal lobes and cerebral microbleeds were associated with PSE. Multivariable Cox regression analysis confirmed larger infarct size (HR 3.49; 95% CI 1.67-7.30) and presence of cerebral microbleeds (HR 2.56; 95% CI 1.18-5.56) as independent predictors of PSE. CONCLUSION: In our study, patients with large vessel occlusion stroke receiving MT had a 9% prevalence of PSE over a median follow-up period of 6.5 years. Besides larger infarct size, presence of cerebral microbleeds on brain MRI predicted PSE occurrence.

Human inositol 1,4,5-trisphosphate 3-kinase isoform B (IP3KB) is a nucleocytoplasmic shuttling protein specifically enriched at cortical actin filaments and at invaginations of the nuclear envelope.

Recent studies have shown that inositol 1,4,5-trisphosphate 3-kinase isoform B (IP3KB) possesses important roles in the development of immune cells. IP3KB can be targeted to multiple cellular compartments, among them nuclear localization and binding in close proximity to the plasma membrane. The B isoform is the only IP3K that is almost ubiquitously expressed in mammalian cells. Detailed mechanisms of its targeting regulation will be important in understanding the role of Ins(1,4,5)P(3) phosphorylation on subcellular calcium signaling and compartment-specific initiation of pathways leading to regulatory active higher phosphorylated inositol phosphates. Here, we identified an exportin 1-dependent nuclear export signal ((134)LQRELQNVQV) and characterized the amino acids responsible for nuclear localization of IP3KB ((129)RKLR). These two targeting domains regulate the amount of nuclear IP3KB in cells. We also demonstrated that the localization of IP3KB at the plasma membrane is due to its binding to cortical actin structures. Intriguingly, all three of these targeting activities reside in one small polypeptide segment (amino acids 104-165), which acts as a multitargeting domain (MTD). Finally, a hitherto unknown subnuclear localization of IP3KB could be demonstrated in rapidly growing H1299 cells. IP3KB is specifically enriched at nuclear invaginations extending perpendicular between the apical and basal surface of the nucleus of these flat cells. Such nuclear invaginations are known to be involved in Ins(1,4,5)P(3)-mediated Ca(2+) signaling of the nucleus. Our findings indicate that IP3KB not only regulates cytoplasmic Ca(2+) signals by phosphorylation of subplasmalemmal and cytoplasmic Ins(1,4,5)P(3) but may also be involved in modulating nuclear Ca(2+) signals generated from these nuclear envelope invaginations.

Synthesis and biological actions of diphosphoinositol phosphates (inositol pyrophosphates), regulators of cell homeostasis.

Diphosphoinositol phosphates are a subclass of inositol phosphates possessing one or two high energy diphosphate groups instead of phosphoester substituents of the myo-inositol. Here we describe the enzymes responsible for their synthesis and degradation and how these may be regulated. Formation of diphosphoinositol phosphates in yeast and mammals is driven by an increase of the cellular energy charge, a lack of inorganic phosphate, and in mammals by osmotic or heat stress and in some cases by receptor mediated signaling. Known cellular actions are an improvement of the cell homeostasis by a reduction of the energy charge, increased phosphate uptake, improvement of mitochondrial performance, and an increase of insulin secretion in mammals. The underlying molecular mechanisms of action are far from being clarified but an increasing body of knowledge about molecular details has highlighted their complex participation in many cellular systems and metabolic processes.

Nucleocytoplasmic shuttling of human inositol phosphate multikinase is influenced by CK2 phosphorylation.

Human inositol phosphate multikinase (IPMK) is a multifunctional protein in cellular signal transduction, namely, a multispecific inositol phosphate kinase, phosphatidylinositol 3-kinase, and a scaffold within the mTOR-raptor complex. To fulfill these nuclear and cytoplasmic functions, intracellular targeting of IPMK needs to be regulated. We show here that IPMK, which has been considered to be a preferentially nuclear protein, is a nucleocytoplasmic shuttling protein, whose nuclear export is mediated by classical nuclear export receptor CRM1. We identified a functional nuclear export signal (NES) additionally to its previously described nuclear import signal (NLS). Furthermore, we describe a mechanism by which the activity of the IPMK-NLS is controlled. Protein kinase CK2 binds endogenous IPMK and phosphorylates it at serine 284. Interestingly, this phosphorylation can decrease nuclear localization of IPMK cell type specifically. A controlled nuclear import of IPMK may direct its actions either toward nuclear inositol phosphate (InsPx) metabolism or cytoplasmic actions on InsPx, phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2], as well as mTOR-raptor.

A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells.

Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner.

Essential role of microRNA-155 in the pathogenesis of autoimmune arthritis in mice.

OBJECTIVE: MicroRNAs (miRNA) are a new class of regulatory elements. Altered expression of miRNA has been demonstrated in the inflamed joints of patients with rheumatoid arthritis (RA). The aim of this study was to examine the role of miRNA in the pathogenesis of autoimmune arthritis, using 2 murine models. METHODS: Collagen-induced arthritis (CIA) and K/BxN serum-transfer arthritis were induced in wild-type (WT) and miR-155-deficient (miR-155(-/-) ) mice. The severity of arthritis was determined clinically and histologically. Anticollagen antibodies and cytokines were measured by enzyme-linked immunosorbent assay. The cellular composition of the draining lymph nodes after induction of CIA was measured by flow cytometry. RESULTS: The miR-155(-/-) mice did not develop CIA. Deficiency in miR-155 prevented the generation of pathogenic autoreactive B and T cells, since anticollagen antibodies and the expression levels of antigen-specific T cells were strongly reduced in miR-155(-/-) mice. Moreover, Th17 polarization of miR-155(-/-) mouse T cells was impaired, as shown by a significant decrease in the levels of interleukin-17 (IL-17) and IL-22. In the K/BxN serum-transfer arthritis model, which only depends on innate effector mechanisms, miR-155(-/-) mice showed significantly reduced local bone destruction, attributed to reduced generation of osteoclasts, although the severity of joint inflammation was similar to that in WT mice. CONCLUSION: These results demonstrate that miR-155 is essentially involved in the adaptive and innate immune reactions leading to autoimmune arthritis, and therefore miR-155 might provide a novel target for the treatment of patients with RA.

Inositol 1,4,5-trisphosphate 3-kinase-A is a new cell motility-promoting protein that increases the metastatic potential of tumor cells by two functional activities.

Cellular migration is an essential prerequisite for metastatic dissemination of cancer cells. This study demonstrates that the neuron/testis-specific F-actin-targeted inositol 1,4,5-trisphosphate 3-kinase-A (ITPKA) is ectopically expressed in different human tumor cell lines and during tumor progression in the metastatic tumor model Balb-neuT. High expression of ITPKA increases invasive migration in vitro and metastasis in a xenograft SCID mouse model. Mechanistic studies show that ITPKA promotes migration of tumor cells by two different mechanisms as follows: growth factor independently high levels of ITPKA induce the formation of large cellular protrusions by directly modulating the actin cytoskeleton. The F-actin binding activity of ITPKA stabilizes and bundles actin filaments and thus increases the levels of cellular F-actin. In growth factor-stimulated cells, the catalytically active domain enhances basal ITPKA-induced migration by activating store-operated calcium entry through production of inositol 1,3,4,5-tetrakisphosphate and subsequent inhibition of inositol phosphate 5-phosphatase. These two functional activities of ITPKA stimulating tumor cell migration place the enzyme among the potential targets of anti-metastatic therapy.

Functional role of inositol-1,4,5-trisphosphate-3-kinase-A for motility of malignant transformed cells.

Cell migration is one of the hallmarks of metastatic disease and thus identification of migration promoting proteins is crucial for the understanding of metastasis formation. Here we show that the neuron-specific, F-actin bundling inositol-1,4,5-trisphosphate-3-kinase-A (ITPKA) is ectopically expressed in tumor cells and critically involved in migration. Down-regulation of ITPKA expression in transformed cell-lines with ectopic expression of ITPKA significantly decreased migration and the number of linear and branched cell protrusion. Conversely, up-regulation of ITPKA in tumor cell lines with low endogenous ITPKA expression increased migration and formation of cell processes. In vitro, ITPKA alone induced the formation of linear actin filaments, whereas ITPKA mediated formation of branched protrusions seems to result from interaction between ITPKA and the F-actin cross-linking protein filamin C. Based on these actin-modulating and migration-promoting effects of ITPKA we examined its expression in clinical samples of different tumor entities, starting with the analysis of multiple tumor tissue arrays. As in lung adenocarcinoma specimens, the highest ITPKA expression rate was found, this tumor entity was examined in more detail. ITPKA was expressed early in adenocarcinoma progression (pN0) and was largely maintained in invasive and metastatic tumor cell populations (pN1/2, lymph node metastases). Together with our result that high expression of ITPKA increases motility of tumor cells we conclude that the observed expression of ITPKA early in tumor development increases the metastatic potential of lung adenocarcinoma cells. Therefore, we suggest that ITPKA may be a promising therapeutic molecular target for anti metastatic therapy of lung cancer.

Ins(1,4,5)P3 3-kinase-A overexpression induces cytoskeletal reorganization via a kinase-independent mechanism.

In the present study, effects of increased IP3K-A [Ins(1,4,5)P(3) 3-kinase-A] expression were analysed. H1299 cells overexpressing IP3K-A formed branching protrusions, and under three-dimensional culture conditions, they exhibited a motile fibroblast-like morphology. They lost the ability to form actin stress fibres and showed increased invasive migration in vitro. Furthermore, expression levels of the mesenchymal marker proteins vimentin and N-cadherin were increased. The enzymatic function of IP3K-A is to phosphorylate the calcium-mobilizing second messenger Ins(1,4,5)P(3) to (Ins(1,3,4,5)P(4). Accordingly, cells overexpressing IP3K-A showed reduced calcium release and altered concentrations of InsPs, with decreasing concentrations of Ins(1,4,5)P(3), InsP(6) and Ins(1,2,3,4,5)P(5), and increasing concentrations of Ins(1,3,4,5)P(4). However, IP3K-A-induced effects on cell morphology do not seem to be dependent on enzyme activity, since a protein devoid of enzyme activity also induced the formation of branching protrusions. Therefore we propose that the morphological changes induced by IP3K-A are mediated by non-enzymatic activities of the protein.

A novel 6-pyrophosphorylating IP6 kinase (IP6-6K) discovered in the protozoon Trichomonas vaginalis.

The parasitic protozoon Trichomonas vaginalis is the pathogen of trichomoniasis, the most common non-viral, sexually transmitted disease in humans. Inositol phosphates function in the pathomechanisms of a number of human pathogenic protozoa. Recent findings point to a role of inositol phosphates in T. vaginalis' adaption to oxygen exposure during change of host. Six inositol phosphate kinase genes (tvip6k1-4, tvipk1-2) were identified in the T. vaginalis genome by us all coding for proteins containing canonical sequence motifs of the major group of animal inositol phosphate kinases (PDKG, SSLL, DFG/A). When characterizing the purified protein product of tvip6k1, we discovered that the major activity of the highly active enzyme (˜2 µmol/min/mg) is a conversion of InsP6 to 6PP-InsP5 and not 5PP-InsP5 as by animal isoforms. Thus TvIP6K1 is a novel IP6-6K. The enzyme also converts Ins(1,3,4,5,6)P5 to products pyrophosphorylated both at 6- and 4-phosphate still having a free 5-hydroxyl. In addition, the enzyme has a minor selectivity to phosphorylate the 3-OH in Ins(1,2,4,5)P4 and Ins(1,2,4,5,6)P5. To present knowledge this novel enzyme is restricted to protozoa. Since its structure is predicted to be distinctly different from animal IP6K (IP6-5K) forms, TvIP6-6K may become a promising target to search for novel trichomoniasis specific drugs.

Inositol hexakisphosphate increases the size of platelet aggregates.

The inositol phosphates, InsP5 and InsP6, have recently been identified as binding partners of fibrinogen, which is critically involved in hemostasis by crosslinking activated platelets at sites of vascular injury. Here, we investigated the putative physiological role of this interaction and found that platelets increase their InsP6 concentration upon stimulation with the PLC-activating agonists thrombin, collagen I and ADP and present a fraction of it at the outer plasma membrane. Cone and plate analysis in whole blood revealed that InsP6 specifically increases platelet aggregate size. This effect is fibrinogen-dependent, since it is inhibited by an antibody that blocks fibrinogen binding to platelets. Furthermore, InsP6 has only an effect on aggregate size of washed platelets when fibrinogen is present, while it has no influence in presence of von Willebrand factor or collagen. By employing blind docking studies we predicted the binding site for InsP6 at the bundle between the γ and ß helical subunit of fibrinogen. Since InsP6 is unable to directly activate platelets and it did not exhibit an effect on thrombin formation or fibrin structure, our data indicate that InsP6 might be a hemostatic agent that is produced by platelets upon stimulation with PLC-activating agonists to promote platelet aggregation by supporting crosslinking of fibrinogen and activated platelets.

Intracellular localization of human Ins(1,3,4,5,6)P5 2-kinase.

InsP6 is an intracellular signal with several proposed functions that is synthesized by IP5K [Ins(1,3,4,5,6)P5 2-kinase]. In the present study, we overexpressed EGFP (enhanced green fluorescent protein)-IP5K fusion proteins in NRK (normal rat kidney), COS7 and H1299 cells. The results indicate that there is spatial microheterogeneity in the intracellular localization of IP5K that could also be confirmed for the endogenous enzyme. This may facilitate changes in InsP6 levels at its sites of action. For example, overexpressed IP5K showed a structured organization within the nucleus. The kinase was preferentially localized in euchromatin and nucleoli, and co-localized with mRNA. In the cytoplasm, the overexpressed IP5K showed locally high concentrations in discrete foci. The latter were attributed to stress granules by using mRNA, PABP [poly(A)-binding protein] and TIAR (TIA-1-related protein) as markers. The incidence of stress granules, in which IP5K remained highly concentrated, was further increased by puromycin treatment. Using FRAP (fluorescence recovery after photobleaching) we established that IP5K was actively transported into the nucleus. By site-directed mutagenesis we identified a nuclear import signal and a peptide segment mediating the nuclear export of IP5K.

Radiosensitization of tumour cell lines by the polyphenol Gossypol results from depressed double-strand break repair and not from enhanced apoptosis.

PURPOSE: New drugs are needed to increase the efficiency of radiotherapy in order to improve the therapeutic outcome of tumour patients. In this respect, the polyphenol Gossypol might be of interest, because of its effect on apoptosis and DNA repair, which is either mediated directly or indirectly via the inositol phosphate metabolism. It was investigated, whether these effects result in enhanced radiosensitivity of tumour cells. MATERIAL AND METHODS: Tumour cell lines investigated: A549, FaDu, H1299, MCF7 and Du145. Cell cycle distribution was determined by FACS analysis, apoptosis was measured by DAPI staining and caspase3/7 activity. Double-strand breaks (DSB) were investigated via gammaH2AX-foci and cell survival by colony formation assay. The level of inositol phosphates was determined by HPLC, protein expression by Western blot. RESULTS: In A549 cells, Gossypol at concentrations 1microM strongly affects proliferation with only a modest arrest in the G1-phase, but with no increase in the fraction of apoptotic cells or the number of additional DSB. Additional DSB were only seen in FaDu cells, where Gossypol (2microM) was extremely toxic with a plating efficiency <0.002. When combined with irradiation, incubation with Gossypol (1-2microM) was found to result in an enhanced radiosensitivity with, however, a substantial variation. While there was a strong radiosensitization for FaDu and Du145 cells, there was an intermediate response for A549 cells, but almost no effect for H1299 and MCF7 cells. This sensitization was not caused from an elevated rate of apoptosis, but primarily resulted from reduced DSB repair capacity. The reduction in DSB repair could be ascribed neither to changes in the level of repair proteins relevant for non-homologous end-joining (Ku70, Ku80, DNA-PKcs) nor to changes in the level of higher phosphorylated inositols, whereby the latter were even found to be enhanced by Gossypol. CONCLUSIONS: For some tumour cell lines treatment with low concentrations of Gossypol can be used to inhibit DSB repair capacity and with that to increase the cellular radiosensitivity.

Spatio-temporal precipitation climatology over complex terrain using a censored additive regression model.

Flexible spatio-temporal models are widely used to create reliable and accurate estimates for precipitation climatologies. Most models are based on square root transformed monthly or annual means, where a normal distribution seems to be appropriate. This assumption becomes invalid on a daily time scale as the observations involve large fractions of zero observations and are limited to non-negative values. We develop a novel spatio-temporal model to estimate the full climatological distribution of precipitation on a daily time scale over complex terrain using a left-censored normal distribution. The results demonstrate that the new method is able to account for the non-normal distribution and the large fraction of zero observations. The new climatology provides the full climatological distribution on a very high spatial and temporal resolution, and is competitive with, or even outperforms existing methods, even for arbitrary locations.