Phosphorylated and unphosphorylated forms of human single-stranded DNA-binding protein are equally active in simian virus 40 DNA replication and in nucleotide excision repair.

The trimeric human single-stranded DNA-binding protein (HSSB; also called RP-A) plays an essential role in DNA replication, nucleotide excision repair, and homologous DNA recombination. The p34 subunit of HSSB is phosphorylated at the G1/S boundary of the cell cycle or upon exposure of cells to DNA damage-inducing agents including ionizing and UV radiation. We have previously shown that the phosphorylation of p34 is catalyzed by both cyclin-dependent kinase-cyclin A complex and DNA-dependent protein kinase. In this study, we investigated the effect of phosphorylation of p34 by these kinases on the replication and repair function of HSSB. We observed no significant difference with the unphosphorylated and phosphorylated forms of HSSB in the simian virus 40 DNA replication or nucleotide excision repair systems reconstituted with purified proteins. The phosphorylation status of the p34 subunit of HSSB was unchanged during the reactions. We suggest that the phosphorylated HSSB has no direct effect on the basic mechanism of DNA replication and nucleotide excision repair reactions in vitro, although we cannot exclude a role of p34 phosphorylation in modulating HSSB function in vivo through a yet poorly understood control pathway in the cellular response to DNA damage and replication.

Inactivation of ethanol-inducible cytochrome P450 and other microsomal P450 isozymes by trans-4-hydroxy-2-nonenal, a major product of membrane lipid peroxidation.

Of the microsomal P450 cytochromes, the ethanol-inducible isoform, P450 2E1, is believed to be predominant in leading to oxidative damage, including the generation of radical species that contribute to lipid peroxidation, and in the reductive beta-scission of lipid hydroperoxides to give hydrocarbons and aldehydes. In the present study, the sensitivity of a series of P450s to trans-4-hydroxy-2-nonenal (HNE), a known toxic product of membrane lipid peroxidation, was determined. After incubation of a purified cytochrome with HNE, the other components of the reconstituted system (NADPH-cytochrome P450 reductase, phosphatidylcholine, and NADPH) were added, and the rate of oxygenation of 1-phenylethanol to yield acetophenone was assayed. Inactivation occurs in a time-dependent and HNE concentration-dependent manner, with P450s 2E1 and 1A1 being the most sensitive, followed by isoforms 1A2, 3A6, and 2B4. At an HNE concentration of 0.24 microM, which was close to the micromolar concentration of the enzyme, four of the isoforms were significantly inhibited, but not P450 2B4. In other experiments, the reductase was shown to be only relatively weakly inactivated by HNE. P450s 2E1 and 2B4 in microsomal membranes from animals induced with acetone or phenobarbital, respectively, are as readily inhibited as the purified forms. Evidence was obtained that the P450 heme is apparently not altered and the sulfur ligand is not displaced, that substrate protects against HNE, and that the inactivation is reversed upon dialysis. Higher levels of reductase or substrate do not restore the activity of inhibited P450 in the catalytic assay. Our results suggest that the observed inhibition of the various P450s is of sufficient magnitude to cause significant changes in the metabolism of foreign compounds such as drugs and chemical carcinogens by the P450 oxygenase system at HNE concentrations that occur in biological membranes. In view of the known activities of P450 2E1 in generating lipid hydroperoxides and in their beta-scission, its inhibition by this product of membrane peroxidation may provide a negative regulatory function.

A chloroplast homologue of the signal recognition particle subunit SRP54 is involved in the posttranslational integration of a protein into thylakoid membranes.

The mechanisms involved in the integration of proteins into the thylakoid membrane are largely unknown. However, many of the steps of this process for the light-harvesting chlorophyll a/b protein (LHCP) have been described and reconstituted in vitro. LHCP is synthesized as a precursor in the cytosol and posttranslationally imported into chloroplasts. Upon translocation across the envelope membranes, the N-terminal transit peptide is cleaved, and the apoprotein is assembled into a soluble "transit complex" and then integrated into the thylakoid membrane via three transmembrane helices. Here we show that 54CP, a chloroplast homologue of the 54-kDa subunit of the mammalian signal recognition particle (SRP54), is essential for transit complex formation, is present in the complex, and is required for LHCP integration into the thylakoid membrane. Our data indicate that 54CP functions posttranslationally as a molecular chaperone and potentially pilots LHCP to the thylakoids. These results demonstrate that one of several pathways for protein routing to the thylakoids is homologous to the SRP pathway and point to a common evolutionary origin for the protein transport systems of the endoplasmic reticulum and the thylakoid membrane.

Two routes of chlorophyllide synthesis that are differentially regulated by light in barley (Hordeum vulgare L.).

NADPH-protochlorophyllide oxidoreductase (POR; EC 1.6.99.1) catalyzes the only known light-dependent step in chlorophyll synthesis of higher plants, the reduction of protochlorophyllide (Pchlide) to chlorophyllide. In barley, two distinct immunoreactive POR proteins were identified. In contrast to the light-sensitive POR enzyme studied thus far (POR-A), levels of the second POR protein remained constant in seedlings during the transition from dark growth to the light and in green plants. The existence of a second POR-related protein was verified by isolating and sequencing cDNAs that encode a second POR polypeptide (POR-B) with an amino acid sequence identity of 75% to the POR-A. In the presence of NADPH and Pchlide, the in vitro-synthesized POR-A and POR-B proteins could be reconstituted to ternary enzymatically active complexes that reduced Pchlide to chlorophyllide only after illumination. Even though the in vitro activities of the two enzymes were similar, the expression of their genes during the light-induced transformation of etiolated to green seedlings was distinct. While the POR-A mRNA rapidly declined during illumination of dark-grown seedlings and soon disappeared, POR-B mRNA remained at an approximately constant level in dark-grown and green seedlings. Thus these results suggest that chlorophyll synthesis is controlled by two light-dependent POR enzymes, one that is active only transiently in etiolated seedlings at the beginning of illumination and the other that also operates in green plants.

The 62- and 80-kDa subunits of transcription factor IIH mediate the interaction with Epstein-Barr virus nuclear protein 2.

EBNA 2 (Epstein-Barr virus nuclear antigen 2) is an acidic transactivator essential for EBV transformation of B lymphocytes. We show that EBNA 2 directly interacts with general transcription factor IIH. Glutathione S-transferase (GST)-EBNA 2 acidic domain fusion protein depleted transcription factor IIH activity from a TFIIH nuclear fraction. The p89 (ERCC3), p80 (ERCC2), and p62 subunits of TFIIH were among the proteins retained by GST-EBNA 2. Eluates from the GST-EBNA 2 beads reconstituted activity in a TFIIH-dependent in vitro transcription assay. The p62 and p80 subunits of TFIIH independently bound to GST-EBNA 2, whereas the p34 subunit of TFIIH only bound in the presence of p62. A Trp-->Thr mutation in the EBNA 2 acidic domain abolishes EBNA 2 transactivation in vivo and greatly compromised EBNA 2 association with TFIIH activity and with the p62 and p80 subunits, providing a link between EBNA 2 transactivation and these interactions. Antibodies directed against the p62 subunit of TFIIH coimmunoprecipitated EBNA 2 from EBV-transformed B lymphocytes, indicating that EBNA 2 associates with TFIIH in vivo.

Tratamento de soro de queijo no ASBR: influência da estratégia de alimentação

Avaliou-se o desempenho do reator anaeróbio em batelada seqüencial com biomassa imobilizada (ASBBR) no tratamento de soro de queijo quanto submetido a diferentes estratégias de alimentação e cargas orgânicas volumétricas (COV). O reator operou com agitação mecânica através de impelidor do tipo hélice na rotação de 500 rpm. Um volume de 2 litros foi alimentado por ciclo com 1 litro de volume residual, totalizando 3 litros. O substrato utilizado foi soro de queijo desidratado reconstituído. Suplementou-se o sistema com NaHCO3 na razão de 50% NaHCO3/DQO. Foram testadas as seguintes COVs: 2, 4, 8 e 12 gDQO/l.d. Para ciclos de 8 horas e em cada COV, três estratégias de alimentação foram testadas: (a) operação em batelada com ciclo de 8 horas, (b) batelada alimentada de 2 horas (c) batelada alimentada de 4 horas. Na COV de 2 gDQO/l.d, a conversão de matéria orgânica como DQO em amostras filtradas foi de 92, 96 e 91% para as estratégias de alimentação (a), (b) e (c), respectivamente. Para a COV de 4 gDQO/l.d, o desempenho foi de 94, 97 e 93%, respectivamente. Para a COV de 8 gDQO/l.d houve redução nas eficiências de conversão a 83, 85 e 86%, respectivamente. O aumento da COV para 12 gDQO/l.d, resultou na redução em eficiências de 72, 73 e 81%, respectivamente. Os perfis durante os ciclos da concentração de ácidos voláteis totais mostraram que, apesar do aumento gradual com o tempo de enchimento aumentando, nenhuma diferença significativa foi detectada em termos dos seus valores máximos. Foi observada a redução de ácido propiônico como conseqüência do aumento do tempo de enchimento. Assim, para COV de 2 e 4 gDQO/l.d, a estratégia de alimentação (b) proporcionou maiores eficiências de conversão e estabilidade operacional, enquanto que este comportamento foi observado na estratégia de alimentação (c) para os valores de COV de 8 e 12 gDQO/l.d.

Establishment of a confluent monolayer model with human primary trophoblast cells: novel insights into placental glucose transport

STUDY HYPOTHESIS Using optimized conditions, primary trophoblast cells isolated from human term placenta can develop a confluent monolayer in vitro, which morphologically and functionally resembles the microvilli structure found in vivo. STUDY FINDING We report the successful establishment of a confluent human primary trophoblast monolayer using pre-coated polycarbonate inserts, where the integrity and functionality was validated by cell morphology, biophysical features, cellular marker expression and secretion, and asymmetric glucose transport. WHAT IS KNOWN ALREADY Human trophoblast cells form the initial barrier between maternal and fetal blood to regulate materno-fetal exchange processes. Although the method for isolating pure human cytotrophoblast cells was developed almost 30 years ago, a functional in vitro model with primary trophoblasts forming a confluent monolayer is still lacking. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Human term cytotrophoblasts were isolated by enzymatic digestion and density gradient separation. The purity of the primary cells was evaluated by flow cytometry using the trophoblast-specific marker cytokeratin 7, and vimentin as an indicator for potentially contaminating cells. We screened different coating matrices for high cell viability to optimize the growth conditions for primary trophoblasts on polycarbonate inserts. During culture, cell confluency and polarity were monitored daily by determining transepithelial electrical resistance (TEER) and permeability properties of florescent dyes. The time course of syncytia-related gene expression and hCG secretion during syncytialization were assessed by quantitative RT-PCR and enzyme-linked immunosorbent assay, respectively. The morphology of cultured trophoblasts after 5 days was determined by light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Membrane makers were visualized using confocal microscopy. Additionally, glucose transport studies were performed on the polarized trophoblasts in the same system. MAIN RESULTS AND THE ROLE OF CHANCE During 5-day culture, the highly pure trophoblasts were cultured on inserts coated with reconstituted basement membrane matrix . They exhibited a confluent polarized monolayer, with a modest TEER and a size-dependent apparent permeability coefficient (Papp) to fluorescently labeled compounds (MW ∼400-70 000 Da). The syncytialization progress was characterized by gradually increasing mRNA levels of fusogen genes and elevating hCG secretion. SEM analyses confirmed a confluent trophoblast layer with numerous microvilli, and TEM revealed a monolayer with tight junctions. Immunocytochemistry on the confluent trophoblasts showed positivity for the cell-cell adhesion molecule E-cadherin, the tight junction protein 1 (ZO-1) and the membrane proteins ATP-binding cassette transporter A1 (ABCA1) and glucose transporter 1 (GLUT1). Applying this model to study the bidirectional transport of a non-metabolizable glucose derivative indicated a carrier-mediated placental glucose transport mechanism with asymmetric kinetics. LIMITATIONS, REASONS FOR CAUTION The current study is only focused on primary trophoblast cells isolated from healthy placentas delivered at term. It remains to be evaluated whether this system can be extended to pathological trophoblasts isolated from diverse gestational diseases. WIDER IMPLICATIONS OF THE FINDINGS These findings confirmed the physiological properties of the newly developed human trophoblast barrier, which can be applied to study the exchange of endobiotics and xenobiotics between the maternal and fetal compartment, as well as intracellular metabolism, paracellular contributions and regulatory mechanisms influencing the vectorial transport of molecules. LARGE-SCALE DATA Not applicable. STUDY FUNDING AND COMPETING INTERESTS This study was supported by the Swiss National Center of Competence in Research, NCCR TransCure, University of Bern, Switzerland, and the Swiss National Science Foundation (grant no. 310030_149958, C.A.). All authors declare that their participation in the study did not involve factual or potential conflicts of interests.

Stepwise consolidation of glacigenic sediments of the Prydz Bay

A series of samples from the five sites drilled across the continental shelf and upper slope in Prydz Bay during ODP Leg 119 were consolidation tested in an oedometer. Preconsolidation stresses increase downcore at Sites 739 and 742 in a stepwise manner, and the steps are interpreted to represent periods of increased action of grounded glaciers covering the entire shelf. By the use of theoretical ice sheet surface profiles giving the range of possible ice thicknesses, sediment loading and subsequent erosion seem to be the most important factor for increasing the overconsolidation ratios, and a total glacial erosion exceeding 1 km is possible. Four separate steps in consolidation, here termed "load events" have been identified. The lowermost load event, 1, is correlated to the onset of glaciations reaching the shelf edge and an early period of extensive glaciations, starting in early Oligocene or possibly earlier. Glacial activity related to the buildup of ice in West Antarctica in the late Miocene is tentatively correlated to load event 2. Event 3 is the trace of relatively extensive glacial erosion probably in the Pliocene, whereas the upper step in preconsolidation stress, load event 4, results from the last glaciation reaching the shelf edge, possibly during the late Weichselian. Correlations to other data related to Antarctic glacial history are, however, hampered by the poor age control of the cored diamictites. Consolidation tests may provide a tool for finding the position for hiatuses and unconformities formed subglacially and obscured by subglacial reworking.

(Table 3) Foraminiferal 87Sr/86Sr ratios and ages of ODP Hole 162-986D sediments

Site 986 was drilled to 965 meters below seafloor (mbsf) on the western Svalbard margin to record the onset of glaciations and to date and document the glacial evolution in the Svalbard-Barents Sea region during the Pliocene-Pleistocene. In this paper, results of sedimentological analyses are discussed in light of seismic stratigraphy and new age determinations. The latter were difficult to obtain in the glacial deposits, and datums are sparse. Through combined paleomagnetic data, biostratigraphy, and Sr isotopes, however, an overall chronology for the main evolutionary steps is suggested. The cored sequence at Site 986 is younger than 2.6 Ma, and the lower 60 m of the section contains no evidence of a major glacial influence. An initial glaciation is interpreted to have occurred at ~2.3 Ma, resulting in increased sand deposition from debris flows at Site 986 and forming a prominent seismic reflector, R7. However, glaciers probably did not reach the shelf break until ~1.6-1.7 Ma (Reflector R6), after which the depositional environment was dominated by diamictic debris flows. A gradual change in source area from the Barents Sea to Svalbard is recorded primarily by changes in carbonate and smectite content, ~355 mbsf (Reflector R5), at an interpolated age of 1.4-1.5 Ma. During the last ~1 m.y., Site 986 has undergone more distal deposition as the main depocenters have shifted laterally. This has resulted in less frequent debris flows and more turbidites and hemipelagic deposits, with a slight fining upward of the cored sediments.

Pollen abundances, and temperature and precipitation reconstruction for the Eocene to Oligocene transition in northern high latitudes

A profound global climate shift took place at the Eocene-Oligocene transition (~33.5 million years ago) when Cretaceous/early Palaeogene greenhouse conditions gave way to icehouse conditions (Zachos et al., 2001, doi:10.1126/science.1059412; Coxall et al., 2005, doi:10.1038/nature03135; Lear et al., 2008, doi:10.1130/G24584A.1). During this interval, changes in the Earth's orbit and a long-term drop in atmospheric carbon dioxide concentrations (Pagani et al., 2005, doi:10.1126/science.1110063; Pearson and Palmer, 2000, doi:10.1038/35021000; DeConto and Pollard, 2003, doi:10.1038/nature01290) resulted in both the growth of Antarctic ice sheets to approximately their modern size (Coxall et al., 2005, doi:10.1038/nature03135; Lear et al., 2008, doi:10.1130/G24584A.1) and the appearance of Northern Hemisphere glacial ice (Eldrett et al., 2007, doi:10.1038/nature05591; Moran et al., 2006, doi:10.1038/nature04800). However, palaeoclimatic studies of this interval are contradictory: although some analyses indicate no major climatic changes (Kohn et al., 2004, doi:10.1130/G20442.1; Grimes et al., 2005, doi:10.1130/G21019.1), others imply cooler temperatures (Zanazzi et al., 2007, doi:10.1038/nature05551), increased seasonality (Ivany et al., 2000, doi:10.1038/35038044; Terry, 2001, doi:10.1016/S0031-0182(00)00248-0) and/or aridity (Ivany et al., 2000, doi:10.1038/35038044; Terry, 2001, doi:10.1016/S0031-0182(00)00248-0; Sheldon et al., 2002, doi:10.1086/342865; Dupont-Nivet et al., 2007, doi:10.1038/nature05516). Climatic conditions in high northern latitudes over this interval are particularly poorly known. Here we present northern high-latitude terrestrial climate estimates for the Eocene to Oligocene interval, based on bioclimatic analysis of terrestrially derived spore and pollen assemblages preserved in marine sediments from the Norwegian-Greenland Sea. Our data indicate a cooling of ~5 °C in cold-month (winter) mean temperatures to 0-2 °C, and a concomitant increased seasonality before the Oi-1 glaciation event. These data indicate that a cooling component is indeed incorporated in the d18O isotope shift across the Eocene-Oligocene transition. However, the relatively warm summer temperatures at that time mean that continental ice on East Greenland was probably restricted to alpine outlet glaciers.

Distribution of foraminifera, pre-Pliocene foraminifera and macrofossil debris in sediment core CRP-2 (Table 1)

The stratigraphic distribution, assemblage content, paleoecology and age of foraminifera recovered in fourteen of sixteen samples from the 5.63 m thick CRP-2 (Lithostratigraphic Unit 2.2) are discussed. LSU 2.2 comprises four discrete lithologic beds. The upward sequence is informally referred to as the lower sand bed, diamicton bed, middle sand bed, and upper sand bed and it is surmised that these four units are closely related in time. The lower sand bed (~1.5m), which overlies lower Miocene sediments and from which it is separated by the Ross Sea Unconformity, contains traces of recycled Miocene diatoms but is otherwise barren of biogenic material. The diamicton bed (~2.42 m) contains 21 species of benthic foraminifera, with assemblages consistently dominated by Cassidulinoides porrectus, Ammoelphidiella antarctica, Rosalina cf. globularis, Cibicides refulgens, and Ehrenbergina glabra. The overlying middle sand bed (~1.9 m) contains 13 species. with C. porrectus and E. glabra dominant and A. antarctica less common than in the underlying diamicton bed. The upper sand bed (~0.46 m) contains four species and very few tests. The diamicton bed and middle sand bed assemblages are considered to be near in situ thanatocoenoses; and sediments interpreted as marine in origin but influenced by hyposaline waters and nearby ice. Planktic taxa are absent, perhaps indicating the presence of tidewater glaciers, sea ice and/or hyposaline surface waters. The small assemblage in the upper sand bed is more problematic and may be recycled. On the basis of foraminifera in the diamicton and middle sand beds. LSU 2.2 is assigned to the Pliocene. The overlying diamicton in LSU 2.1 contains abundant Quaternary foraminifera.

Paleotemperatures and paleosalinities from the Celtic Margin (Bay of Biscay)

During the late Quaternary, both external and internal forcings have driven major climatic shifts from glacial to interglacial conditions. Nonlinear climatic steps characterized the transitions leading to these extrema, with intermediate excursions particularly well xpressed in the dynamics of the Northern Hemisphere cryosphere. Here we document the impact of these dynamics on the north-eastern North Atlantic Ocean, focussing on the 35-10 ka interval. Sea-surface salinities have been reconstructed quantitatively based on two independent methods from core MD95-2002, recovered from the northern Bay of Biscay adjacent to the axis of the Manche paleoriver outlet and thus in connection with proximal European ice sheets and glaciers. Quantitative reconstructions deriving from dinocyst and planktonic foraminiferal analyses have been combined within a robust chronology to assess the amplitude and timing of hydrological changes in this region. Our study evidences strong pulsed freshwater discharges which may have impacted the North Atlantic Meridional Overturning Circulation.