Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen

Binding of different regulatory subunits and methylation of the catalytic (C) subunit carboxy-terminal leucine 309 are two important mechanisms by which protein phosphatase 2A (PP2A) can be regulated. In this study, both genetic and biochemical approaches were used to investigate regulation of regulatory subunit binding by C subunit methylation. Monoclonal antibodies selectively recognizing unmethylated C subunit were used to quantitate the methylation status of wild-type and mutant C subunits. Analysis of 13 C subunit mutants showed that both carboxy-terminal and active site residues are important for maintaining methylation in vivo. Severe impairment of methylation invariably led to a dramatic decrease in Balpha subunit binding but not of striatin, SG2NA, or polyomavirus middle tumor antigen (MT) binding. In fact, most unmethylated C subunit mutants showed enhanced binding to striatin and SG2NA. Certain carboxy-terminal mutations decreased Balpha subunit binding without greatly affecting methylation, indicating that Balpha subunit binding is not required for a high steady-state level of C subunit methylation. Demethylation of PP2A in cell lysates with recombinant PP2A methylesterase greatly decreased the amount of C subunit that could be coimmunoprecipitated via the Balpha subunit but not the amount that could be coimmunoprecipitated with Aalpha subunit or MT. When C subunit methylation levels were greatly reduced in vivo, Balpha subunits were found complexed exclusively to methylated C subunits, whereas striatin and SG2NA in the same cells bound both methylated and unmethylated C subunits. Thus, C subunit methylation is critical for assembly of PP2A heterotrimers containing Balpha subunit but not for formation of heterotrimers containing MT, striatin, or SG2NA. These findings suggest that methylation may be able to selectively regulate the association of certain regulatory subunits with the A/C heterodimer.     

PELAGIC AND BENTHIC ENVIRONMENTAL CONTROLS ON THE SPATIAL DISTRIBUTION OF A VIABLE DIATOM PROPAGULE BANK ON THE SWEDISH WEST COAST1

The distribution of viable diatom resting stages in sediments on the Swedish west coast was assessed by the most probable number (MPN) culture technique. Multivariate analyses correlated benthic and pelagic environmental factors to the observed spatial variations in the size and taxonomic composition of the propagule bank. Viable diatom resting stages were plentiful (0.2–4.8 million cells·g ^{? 1} 1 Received 11 September 2001. Accepted 12 June 2002.
dry weight) and were dominated by the genera Skeletonema, Detonula, Chaetoceros, and Thalassiosira. Size of the propagule bank was primarily related to planktonic biomass (measured as chl a) and was highest in the Orust‐Tjörn fjord system. Species composition in this fjord system was dominated by D. confervacea (Cleve) Gran and T. nordenskioeldii Cleve in contrast to stations on the outer coast, which contained more cells of T. minima Gaarder, Asterionellopsis glacialis (Castracane) Round, and Leptocylindrus danicus Cleve. These taxonomic variations were principally influenced by deep water oxygen concentrations and water column stability. Benthic resting cells of S. costatum (Greville) Cleve were abundant all along the coast but showed reduced viability in low oxygen environments. Calculations based on MPN values estimated that resuspension of sediment could provide a sizable inoculum to the plankton, although the development of planktonic blooms will also depend on forces of hydrography and weather. Although benthic resting stages may not be absolutely necessary for survival of all diatoms, these cells may be important in determining species cycles, succession, and the spatial distribution of diatoms.     

Deletion of Specific Immune-Modulatory Genes from Modified Vaccinia Virus Ankara-Based HIV Vaccines Engenders Improved Immunogenicity in Rhesus Macaques

Modified vaccinia virus Ankara (MVA) is a safe, attenuated orthopoxvirus that is being developed as a vaccine vector but has demonstrated limited immunogenicity in several early-phase clinical trials. Our objective was to rationally improve the immunogenicity of MVA-based HIV/AIDS vaccines via the targeted deletion of specific poxvirus immune-modulatory genes. Vaccines expressing codon-optimized HIV subtype C consensus Env and Gag antigens were generated from MVA vector backbones that (i) harbor simultaneous deletions of four viral immune-modulatory genes, encoding an interleukin-18 (IL-18) binding protein, an IL-1β receptor, a dominant negative Toll/IL-1 signaling adapter, and CC-chemokine binding protein (MVAΔ4-HIV); (ii) harbor a deletion of an additional (fifth) viral gene, encoding uracil-DNA glycosylase (MVAΔ5-HIV); or (iii) represent the parental MVA backbone as a control (MVA-HIV). We performed head-to-head comparisons of the cellular and humoral immune responses that were elicited by these vectors during homologous prime-boost immunization regimens utilizing either high-dose (2 × 10⁸ PFU) or low-dose (1 × 10⁷ PFU) intramuscular immunization of rhesus macaques. At all time points, a majority of the HIV-specific T cell responses, elicited by all vectors, were directed against Env, rather than Gag, determinants, as previously observed with other vector systems. Both modified vectors elicited up to 6-fold-higher frequencies of HIV-specific CD8 and CD4 T cell responses and up to 25-fold-higher titers of Env (gp120)-specific binding (nonneutralizing) antibody responses that were relatively transient in nature. While the correlates of protection against HIV infection remain incompletely defined, our results indicate that the rational deletion of specific genes from MVA vectors can positively alter their cellular and humoral immunogenicity profiles in nonhuman primates.     

A 91-year record of seasonal and interannual variability of diatoms from laminated sediments in Saanich Inlet, British Columbia

Diatom seasonal succession and interannual variability werestudied using laminated sediments from Saanich Inlet, BritishColumbia, for the years 1900–1991. Frozen sediment coresallowed fine-scale sampling of laminae for each year. Thus,three ‘seasons’ for each year were identified basedon species composition. Thalassiosira species were indicatorsof spring deposition. Skeletonema costatum was abundant in samplesfollowing Thalassiosira, probably deposited in late spring andsummer. Rhizosolenia sp. was most abundant in fall/winter samples.Diatom stratigraphies were related to sea surface temperature,salinity, sea level and the Pacific North American Index (PNA)using canonical correspondence analysis (CCA). CCA showed thatspecies of a particular season generally had optima for temperatureand salinity characteristic of that time. Interannual changesin diatom species composition and abundance were most prevalentin the decades 1920–1940, with the exception of S.costatumwhich showed cyclic changes in abundance. Skeletonema was moreabundant during periods of cool temperatures, while littoraldiatoms were more abundant during times of heavy winter rains.Sea level was an important variable in CCA and while its relationshipto diatoms is not clear, it may be related to variations innutrient supply to diatoms in surface waters.     

The Adenovirus E4orf4 Protein Induces G2/M Arrest and Cell Death by Blocking Protein Phosphatase 2A Activity Regulated by the B55 Subunit

Human adenovirus E4orf4 protein is toxic in human tumor cells. Its interaction with the Bα subunit of protein phosphatase 2A (PP2A) is critical for cell killing; however, the effect of E4orf4 binding is not known. Bα is one of several mammalian B-type regulatory subunits that form PP2A holoenzymes with A and C subunits. Here we show that E4orf4 protein interacts uniquely with B55 family subunits and that cell killing increases with the level of E4orf4 expression. Evidence suggesting that Bα-specific PP2A activity, measured in vitro against phosphoprotein substrates, is reduced by E4orf4 binding was obtained, and two potential B55-specific PP2A substrates, 4E-BP1 and p70^S6K, were seen to be hypophosphorylated in vivo following expression of E4orf4. Furthermore, treatment of cells with low levels of the phosphatase inhibitor okadaic acid or coexpression of the PP2A inhibitor I₁^PP2A enhanced E4orf4-induced cell killing and G₂/M arrest significantly. These results suggested that E4orf4 toxicity results from the inhibition of B55-specific PP2A holoenzymes, an idea that was strengthened by an observed growth arrest resulting from treatment of H1299 cells with Bα-specific RNA interference. We believe that E4orf4 induces growth arrest resulting in cell death by reducing the global level of B55-specific PP2A activity, thus preventing the dephosphorylation of B55-specific PP2A substrates, including those involved in cell cycle progression.Our research group and others have shown that the 114-residue product of early region E4 of human adenoviruses, termed E4orf4, induces p53-independent cell death in human tumor cells (24, 25, 34-36, 55) and in Saccharomyces cerevisiae (23, 53). E4orf4 protein, which shares no obvious homology with other viral or cellular products, kills a wide range of human cancer cells but is believed to have reduced activity against normal human primary cells (6, 55, 56). Although in some cases E4orf4-expressing cells exhibit characteristics typical of apoptosis, including the presence of irregularly shaped and shrunken nuclei, cytoplasmic vacuolization, and membrane blebbing (24, 25, 50, 55), cell death may more typically be independent of caspase activation (24, 25, 30, 32, 50). With H1299 human non-small-cell lung carcinoma cells, death is characterized by rapid cell rounding, enlargement, release from the surface of culture plates, cell cycle arrest in G₂/M and possibly G₁, and eventually, after an extended period, loss of membrane integrity (30). Both cytoplasmic and nuclear pathways have been observed, the former involving interactions with c-Src family kinases, activation of calpain, and remodeling of the actin cytoskeleton (7, 24, 50, 51, 58). Little is known about the nuclear pathway, which may represent the predominant death-inducing process. Our current evidence suggests that H1299 cells die following prolonged irreversible cell cycle arrest leading to mitotic catastrophe and death by a necrosis-like process (30).E4orf4 is known to associate with the Bα regulatory subunit of protein phosphatase 2A (PP2A) (22, 34), and this interaction appears to be necessary for the majority of E4orf4 toxicity in both yeast (23, 53) and human tumor cells (34, 56). PP2A is an abundant serine-threonine phosphatase involved in regulation of metabolism, splicing, translation, morphogenesis, development, and cell cycle progression (15, 19, 27, 43, 59). PP2A holoenzymes exist as multiple heterotrimeric complexes composed of a catalytic C subunit, an A subunit that functions as a scaffold, and a B-type regulatory subunit. Two forms each of the A and C subunits exist in mammalian cells; however, more than 20 B-type subunits have been identified in three unique classes (B/B55, B′/B56, B″/PR72), plus striatin/SG2NA (sometimes called B‴) (10, 19, 26). Although one group has suggested that E4orf4 protein interacts with one or more members of the B′/B56 class (57), it is generally accepted that interaction with the Bα/B55 subunit (Cdc55 in yeast) is important for induction of cell death in both human tumor cells and yeast (53, 57). Interestingly, a recent report has also suggested that in yeast, growth suppression induced by E4orf4 is mediated only in part by the catalytic C subunit of PP2A (31).In the present report, we show that E4orf4 protein interacts uniquely with members of the B55 class of PP2A B-type subunits, and at sufficient concentrations, it appears to become toxic by reducing dephosphorylation of substrates of B55-containing PP2A holoenzymes. As cell death is preceded by cell cycle arrest, we believe that key substrates may include proteins required for cell cycle progression.     

Viability of phytoplankton resting stages in the sediments of a coastal Swedish fjord

Viable diatom and dinoflagellate resting stages were recovered from sediments in Koljö Fjord on the west coast of Sweden. To determine the maximum survival time of buried resting stages, samples from sediment depths down to 50?cm were incubated at temperatures of 3, 10 and 18?°C. Sediment cores were dated by ²¹⁰Pb and the age of samples containing viable resting stages was determined using the constant rate of supply model. Dilution cultures of surface sediments allowed semiquantitative estimates of the potential seed bank. Dinoflagellate cysts from species such as Diplopsalis sp., Gymnodinium nolleri, Oblea rotunda and Protoceratium reticulatum were viable down to 15?cm depth, or 37 years old. Spores and resting cells of the diatoms Chaetoceros spp., Detonula confervacea and Skeletonema costatum were viable to over 40?cm depth, and may have been buried for many decades. The seed bank of living resting stages in surficial sediments was found to be rich (c. 57000 diatom resting stages g^?1 wet weight and c. 200 dinoflagellate cysts g^?1 wet weight), and the percentage of viable resting stages was higher for spore- and cyst-forming species. The oxygen-deficient sediments in Koljö Fjord appear to be a natural conservator of cell viability, a condition not easily simulated in laboratory studies. These results are ecologically important since spores and cysts are a repository of genetic material able to repopulate waters if resuspended and exposed to suitable light, temperature and nutrients.     

121 Influence of Benthic vs. Pelagic Seeding on Coastal Diatom Bloom Development

Diatom blooms may be initiated by cells that have survived in the plankton or germinated resting stages from the sediments. However, it is not well understood how these different inocula contribute to bloom development. We followed diatom community development in twenty‐liter microcosms given different inocula. Surface sediment and phytoplankton were collected in Gullmar Fjord, Sweden. Replicate microcosms were then dosed with local sediment and/or plankton and incubated in situ in Gullmar Fjord. We also followed the concurrent development of the phytoplankton community in the fjord. Experiments run in both spring and fall 2002 showed that bloom development in the microcosms was significantly faster when seeded by planktonic cells. However, addition of sediment may have stimulated planktonic growth and also provided additional propagules. The type of inoculum used strongly influenced the diatom composition in the microcosms. Sediment additions, through germination of resting stages, resulted in communities dominated by Detonula confervacea and Thalassiosira minima in spring, and Skeletonema costatum in fall. Planktonic inocula resulted in blooms of T. nordenskioeldii and Chaetoceros debilis in spring, and S. costatum and several Chaetoceros spp. in fall. Microcosms dosed with both plankton and sediment showed a mixed species assemblage. Comparisons between the microcosms and fjord phytoplankton suggest an important role for benthic seeding of diatom blooms.     

THE TYCHOPELAGIC DIATOM,PARALIA SULCATA,AS PALEOINDICATOR SPECIES IN COASTAL MARINE ENVIRONMENTS

Paralia sulcata is a diatom commonly found in both the plankton and benthos of coastal environments. This species is heavily silicified and, thus preserves well in sedimentary records making it a potentially useful paleoindicator species. However, its tychopelagic nature and its association with a wide range of environmental conditions have made detailed paleoecological interpretations complicated. High‐resolution sediment records from coastal fjords in both Canada and Sweden show variations in the abundance and morphology of P. sulcata that provide evidence of changes in benthic habitat distribution and surface water properties in the fjords on timescales of decades to centuries. These studies suggest that P. sulcata can be an important paleoindicator species when interpretations are made in the context of its complex ecology.     

COMPARISON OF THREE COMMON MOLECULAR TOOLS FOR DISTINGUISHING AMONG GEOGRAPHICALLY SEPARATED CLONES OF THE DIATOM SKELETONEMA MARINOI SARNO ET ZINGONE (BACILLARIOPHYCEAE)1

Skeletonema marinoi Sarno et Zingone is a planktonic marine diatom with a widespread geographic distribution. Different populations of this species may show distinct genetic signatures. We have evaluated the utility of three common molecular methods for distinguishing clones of S. marinoi from different geographic regions. Clonal cultures were isolated from the Canadian west coast, south west Portugal, and the east and west coasts of Sweden. All strains originated from resting stages in sediment. More than 90% of the individually isolated chains grew to densities suitable for DNA extraction. Genetic signatures of clones from each sample location were assessed by sequencing variable domains (D1–D3) of the nuclear large subunit (LSU) rRNA gene and internal transcriber spacer (ITS) (ITS‐1, 5.8S and ITS‐2) regions, and also by analysis of randomly amplified polymorphic DNA patterns. Analysis of molecular variance showed that strains from the four geographic areas were significantly separated by all three methods but that differences among European samples were best resolved by ITS 2 sequences.