A TSG101/MDM2 regulatory loop modulates MDM2 degradation and MDM2/p53 feedback control

The p53 tumor suppressor protein and the MDM2 oncoprotein form a feedback-control loop that up-regulates cellular MDM2 production, blocks p53 activity, and promotes p53 decay. tsg101 was discovered as a gene whose deficiency results in neoplastic transformation of NIH 3T3 cells and the ability to generate metastatic tumors in nude mice. Its protein product contains a domain, Ubc, characteristic of the catalytic domain of ubiquitin conjugase (E2) enzymes but lacking an active-site cysteine crucial for ubiquitin conjugase activity. Here we report that TSG101 participates with MDM2 in an autoregulatory loop that modulates the cellular levels of both proteins, and also of p53, by affecting protein decay. We show that the Ubc domain of TSG101 interferes with ubiquitination of MDM2, that TSG101 inhibits MDM2 decay and elevates its steady-state level, and that these events are associated with down-regulation of p53 protein. Conversely, pulse–chase and Western blot experiments in wild-type and mutant fibroblasts indicate that elevation of MDM2 by overexpression of wild-type p53, by amplification of the endogenous MDM2 gene, or by transfection of MDM2-expressing constructs promotes TSG101 loss, which we show occurs by 26S proteasome-dependent decay. Our results identify TSG101 as both a regulator of, and target of, MDM2/p53 circuitry.

Thermostable and site-specific DNA binding of the gene product ORF56 from the Sulfolobus islandicus plasmid pRN1, a putative archael plasmid copy control protein

There is still a lack of information on the specific characteristics of DNA-binding proteins from hyperthermophiles. Here we report on the product of the gene orf56 from plasmid pRN1 of the acidophilic and thermophilic archaeon Sulfolobus islandicus. orf56 has not been characterised yet but low sequence similarily to several eubacterial plasmid-encoded genes suggests that this 6.5 kDa protein is a sequence-specific DNA-binding protein. The DNA-binding properties of ORF56, expressed in Escherichia coli, have been investigated by EMSA experiments and by fluorescence anisotropy measurements. Recombinant ORF56 binds to double-stranded DNA, specifically to an inverted repeat located within the promoter of orf56. Binding to this site could down-regulate transcription of the orf56 gene and also of the overlapping orf904 gene, encoding the putative initiator protein of plasmid replication. By gel filtration and chemical crosslinking we have shown that ORF56 is a dimeric protein. Stoichiometric fluorescence anisotropy titrations further indicate that ORF56 binds as a tetramer to the inverted repeat of its target binding site. CD spectroscopy points to a significant increase in ordered secondary structure of ORF56 upon binding DNA. ORF56 binds without apparent cooperativity to its target DNA with a dissociation constant in the nanomolar range. Quantitative analysis of binding isotherms performed at various salt concentrations and at different temperatures indicates that approximately seven ions are released upon complex formation and that complex formation is accompanied by a change in heat capacity of –6.2 kJ/mol. Furthermore, recombinant ORF56 proved to be highly thermostable and is able to bind DNA up to 85°C.

Ribozyme minigene-mediated RAD51 down-regulation increases radiosensitivity of human prostate cancer cells

The strand transferase RAD51 is a component of the homologous recombination repair pathway. To examine the contribution of RAD51 to the genotoxic effects of ionising radiation, we have used a novel ribozyme strategy. A reporter gene vector was constructed so that expression of an inserted synthetic double-stranded ribozyme-encoding oligonucleotide would be under the control of the cytomegalovirus immediate-early gene enhancer/promoter system. The prostate tumour cell line LNCaP was transfected with this vector or a control vector, and a neomycin resistance gene on the vector was used to create geneticin-resistant stable cell lines. Three stable cell lines were shown by western blot analysis to have significant down-regulation of RAD51 to 20–50% of the levels expressed in control cell lines. All three cell lines had a similar increased sensitivity to γ-irradiation by 70 and 40%, respectively, compared to normal and empty vector-transfected cells, corresponding to dose-modifying factors of ∼2.0 and 1.5 in the mid-range of the dose-response curves. The amount of RAD51 protein in transfected cell lines was shown to strongly correlate with the α parameter obtained from fitted survival curves. These results highlight the importance of RAD51 in cellular responses to radiation and are the first to indicate the potential use of RAD51-targeted ribozyme minigenes in tumour radiosensitisation.

Feedback Control and Diurnal Regulation of Gibberellin 20-Oxidase Transcript Levels in Potato1

Tuber formation in potato (Solanum tuberosum) is promoted by short photoperiods and is inhibited by gibberellins (GAs). Endogenous levels of GA1 were shown to decrease in stolons and leaves of potato plants induced to tuberize, which suggests that photoperiodic regulation of GA biosynthesis may play a role in tuber induction. We report the isolation of three potato cDNA clones (StGA20ox1–3) encoding GA 20-oxidase, a key regulatory enzyme in the GA-biosynthetic pathway. Using northern analysis, we detected a differential pattern of tissue-specific expression of the mRNAs corresponding to these clones. StGA20ox mRNAs were also very abundant in leaves of the potato ga1 mutant, which is blocked in the 13-hydroxylation step, and were strongly down-regulated by gibberellic acid, suggesting a feedback regulation of these genes. In plants grown in short-day (inductive) conditions, levels of the StGA20ox transcripts in leaves fluctuated during a 24-h period, with a peak of accumulation observed about 4 h after the lights were turned off. Interruption of the night with a 30-min “night break” of light (noninductive conditions) did not have a marked effect on the levels of accumulation of the three GA 20-oxidase mRNAs during the day, but it induced a second peak of expression of StGA20ox1 and StGA20ox3 transcripts late in the night. This observation, together with the finding that StGA20ox1 mRNA is expressed at high levels in leaves, suggests that night-break induction of this gene might play a role in the control of tuberization by regulating endogenous levels of GAs in response to daylength conditions.

Down-regulation of RFL, the FLO/LFY homolog of rice, accompanied with panicle branch initiation

FLORICAULA (FLO) of Antirrhinum and LEAFY (FLY) of Arabidopsis regulate the formation of floral meristems. To examine whether same mechanisms control floral development in distantly related species such as grasses, we isolated RFL, FLO-LFY homolog of rice, and examined its expression and function. Northern analysis showed that RFL is expressed predominantly in very young panicle but not in mature florets, mature leaves, or roots. In situ hybridization revealed that RFL RNA was expressed in epidermal cells in young leaves at vegetative growth stage. After the transition to reproductive stage, RFL RNA was detected in all layers of very young panicle including the apical meristem, but absent in the incipient primary branches. As development of branches proceeds, RFL RNA accumulation localized in the developing branches except for the apical meristems of the branches and secondary branch primordia. Expression pattern of RFL raised a possibility that, unlike FLO and LFY, RFL might be involved in panicle branching. Transgenic Arabidopsis plants constitutively expressing RFL from the cauliflower mosaic virus 35S promoter were produced to test whether 35S-RFL would cause similar phenotype as observed in 35S-LFY plants. In 35S-RFL plants, transformation of inflorescence meristem to floral meristem was rarely observed. Instead, development of cotyledons, rosette leaves, petals, and stamens was severely affected, demonstrating that RFL function is distinct from that of LFY. Our results suggest that mechanisms controlling floral development in rice might be diverged from that of Arabidopsis and Antirrhinum.

Muscarinic acetylcholine receptor down-regulation limits the extent of inhibition of cell cycle progression in Chinese hamster ovary cells.

Cellular desensitization is believed to be important for growth control but direct evidence is lacking. In the current study we compared effects of wild-type and down-regulation-resistant mutant m3 muscarinic receptors on Chinese hamster ovary (CHO-K1) cell desensitization, proliferation, and transformation. We found that down-regulation of m3 muscarinic acetylcholine receptors was the principal mechanism of desensitization of receptor-activated inositol phosphate phospholipid hydrolysis in these cells. Activation of wild-type and mutant receptors inhibited anchorage-independent growth as assayed by colony formation in agar. However, the potency for inhibition of anchorage-independent growth was greater for cells expressing the mutant receptor. Activation of either receptor also initially inhibited anchorage-dependent cell proliferation in randomly growing populations. Rates of DNA synthesis and cell division were profoundly reduced by carbachol in cells expressing either receptor at early time points. Analysis of cell cycle parameters indicated that cell cycle progression was inhibited at transitions from G1 to S and G2/M to G1 phases. However, mutant receptor effects on anchorage-dependent growth were sustained, whereas wild-type receptor effects were transient. Thus, receptor down-regulation restored cell cycle progression. In contrast, activation of either receptor blocked entry into the cell cycle from quiescence, and this response was not reduced by receptor down-regulation. Therefore, activation of m3 muscarinic acetylcholine receptors inhibited CHO cell anchorage-dependent and -independent growth. In anchored cells carbachol inhibited the cell cycle at three distinct points. Inhibitions at two of these points were eliminated by wild-type receptor down-regulation while the other was not. These results directly demonstrate that desensitization mechanisms can act as principal determinants of cellular growth responses.

Age control and stratigraphy of a sediment profile from the Lomonosov Ridge, Arctic Ocean

The upper 200 m of the sediments recovered during IODP Leg 302, the Arctic Coring Expedition (ACEX), to the Lomonosov Ridge in the central Arctic Ocean consist almost exclusively of detrital material. The scarcity of biostratigraphic markers severely complicates the establishment of a reliable chronostratigraphic framework for these sediments, which contain the first continuous record of the Neogene environmental and climatic evolution of the Arctic region. Here we present profiles of cosmogenic 10Be together with the seawater-derived fraction of stable 9Be obtained from the ACEX cores. The down-core decrease of 10Be/9Be provides an average sedimentation rate of 14.5 ± 1 m/Ma for the uppermost 151 m of the ACEX record and allows the establishment of a chronostratigraphy for the past 12.3 Ma. The age-corrected 10Be concentrations and 10Be/9Be ratios suggest the existence of an essentially continuous sea ice cover over the past 12.3 Ma.

Down-core distribution of live and dead benthic foraminifera in deep sea sediments from the Weddell Sea and the Californian continental borderland

Five short cores sub-sampled from box cores from three sites in the eastern Weddell Sea off Antarctica and in the eastern Pacific off southern California, covering a range in water depth from 500 to 2000 m, were analysed for the down-core distribution of live (stained with Rose Bengal) and dead benthic foraminifera. In the California continental borderland, Planulina ariminensis, Rosalina columbiensis and Trochammina spp. live attached to agglutinated polychaetes tubes that rise above the sedimentwater interface. Bolivina spissa lives exclusively in or on the uppermost sediment. Stained specimens of Chilostomella ovoidea are found down to 6 cm within the sediment and specimens of Globobulimina pacifica down to a maximum of 8 cm. Delta13C values of live G. pacifica decrease with increasing depth from the sediment surface down to 7 cm core depth, indicating that this infaunal species utilizes13C-depleted carbon from pore waters. In the dead, predominantly calcareous benthic forminiferal assemblage, selective dissolution of small delicate tests in the upper sediment column causes a continuous variation in species proportions. In the eastern Weddell Sea, the calcareous Bulimina aculeata lives in a carbonate corrosive environment exclusively in or on the uppermost sediment. The arenaceous Cribrostomoides subglobosum, Recurvoides contortus and some Reophax species are frequently found within the top 4 cm of the sediment, whereas stained specimens of Haplophragmoides bradyi, Glomospira charoides and Cribrostomoides wiesneri occur in maximum abundance below the uppermost 1.5 cm. Species proportions in the dead, predominantly arenaceous, benthic foraminiferal assemblage change in three distinct steps. The first change is caused by calcite dissolution at the sediment-water interface, the second coincides with the lower boundary of intense bioturbation, and the third results from the geochemical shift from oxidizing to reducing conditions below a compacted ash layer.

Biological Atlas of the Arctic Seas 2000: Physical oceanography, hydrochemistry, meteorology, and plankton of the Barents and Kara Seas

Presented are physical and biological data for the region extending from the Barents Sea to the Kara Sea during 158 scientific cruises for the period 1913-1999. Maps with the temporal distribution of physical and biological variables of the Barents and Kara Seas are presented, with proposed quality control criteria for phytoplankton and zooplankton data. Changes in the plankton community structure between the 1930s, 1950s, and 1990s are discussed. Multiple tables of Arctic Seas phytoplankton and zooplankton species are presented, containing ecological and geographic characteristics for each species, and images of live cells for the dominant phytoplankton species.

Remote Control Electrodeposition: Patterning on Substrates without Direct Electrical Connections

Thesis (Ph.D.)--University of Washington, 2016-06

Slowing down a pine invasion despite uncertainty in demography and dispersal

1. Management decisions regarding invasive plants often have to be made quickly and in the face of fragmentary knowledge of their population dynamics. However, recommendations are commonly made on the basis of only a restricted set of parameters. Without addressing uncertainty and variability in model parameters we risk ineffective management, resulting in wasted resources and an escalating problem if early chances to control spread are missed. 2. Using available data for Pinus nigra in ungrazed and grazed grassland and shrubland in New Zealand, we parameterized a stage-structured spread model to calculate invasion wave speed, population growth rate and their sensitivities and elasticities to population parameters. Uncertainty distributions of parameters were used with the model to generate confidence intervals (CI) about the model predictions. 3. Ungrazed grassland environments were most vulnerable to invasion and the highest elasticities and sensitivities of invasion speed were to long-distance dispersal parameters. However, there was overlap between the elasticity and sensitivity CI on juvenile survival, seedling establishment and long-distance dispersal parameters, indicating overlap in their effects on invasion speed. 4. While elasticity of invasion speed to long-distance dispersal was highest in shrubland environments, there was overlap with the CI of elasticity to juvenile survival. In shrubland invasion speed was most sensitive to the probability of establishment, especially when establishment was low. In the grazed environment elasticity and sensitivity of invasion speed to the severity of grazing were consistently highest. Management recommendations based on elasticities and sensitivities depend on the vulnerability of the habitat. 5. Synthesis and applications. Despite considerable uncertainty in demography and dispersal, robust management recommendations emerged from the model. Proportional or absolute reductions in long-distance dispersal, juvenile survival and seedling establishment parameters have the potential to reduce wave speed substantially. Plantations of wind-dispersed invasive conifers should not be sited on exposed sites vulnerable to long-distance dispersal events, and trees in these sites should be removed. Invasion speed can also be reduced by removing seedlings, establishing competitive shrubs and grazing. Incorporating uncertainty into the modelling process increases our confidence in the wide applicability of the management strategies recommended here.

On the chaotic instability of a nonsliding liquid-filled top with a small spheroidal base via Melnikov-Holmes-Marsden integrals

Chaotic orientations of a top containing a fluid filled cavity are investigated analytically and numerically under small perturbations. The top spins and rolls in nonsliding contact with a rough horizontal plane and the fluid in the ellipsoidal shaped cavity is considered to be ideal and describable by finite degrees of freedom. A Hamiltonian structure is established to facilitate the application of Melnikov-Holmes-Marsden (MHM) integrals. In particular, chaotic motion of the liquid-filled top is identified to be arisen from the transversal intersections between the stable and unstable manifolds of an approximated, disturbed flow of the liquid-filled top via the MHM integrals. The developed analytical criteria are crosschecked with numerical simulations via the 4th Runge-Kutta algorithms with adaptive time steps.

Top teams and strategy in a UK university

This paper reports on the results of an in-depth study of how a top management team (TMT) puts strategy into practice in a UK university. A study of the top team in Warwick University was conducted to analyse how strategy was formulated and implemented. The results suggest that a combination of two broad theoretical lenses provide useful analytical insight. These are strategy as practice and strategy as process. The main elements of this university’s strategy result from an interplay of localized routines and patterns of action within an organizational context, which both produces and is a product of such actions. The TMT itself was found to be clearly identifiable and stable in composition. The team exhibited identifiable patterns of strategic thinking and acting. However, the role of organizational structure was also found to be a key influence on the actions and processes of the TMT with strong central control tendencies in the team being counterbalanced by devolved operational control to individual departments. The data also reveal inter-relationships between organizational structures and the TMT in four key areas: direction-setting, monitoring and control, the allocation of resources, and processes of interaction. The overall conclusion is that to understand how strategy is practised, analysis needs to focus on how patterns of action are associated with the characteristics of both the team and the wider organization. The nature and characteristics of these patterns can be related to how strategy is put into practice.