The affinity of EBNA1 for its origin of DNA synthesis is a determinant of the origin's replicative efficiency

Epstein-Barr virus (EBV) replicates its genome as a licensed plasmid in latently infected cells. Although replication of this plasmid is essential for EBV latent infection, its synthesis still fails for 16% of the templates in S phase. In order to understand these failures, we sought to determine whether the affinity of the initiator protein (EBNA1) for its binding sites in the origin affects the efficiency of plasmid replication. We have answered this question by using several engineered origins modeled upon the arrangement of EBNA1-binding sites found in DS, the major plasmid origin of EBV. The human TRF2 protein also binds to half-sites in DS and increases EBNA1's affinity for its own sites; we therefore also tested origin efficiency in the presence or absence of these sites. We have found that if TRF2-half-binding sites are present, the efficiency of supporting the initiation of DNA synthesis and of establishing a plasmid bearing that origin directly correlates with the affinity of EBNA1 for that origin. Moreover, the presence of TRF2-half-binding sites also increases the average level of EBNA1 and ORC2 bound to those origins in vivo, as measured by chromatin immunoprecipitation. Lastly, we have created an origin of DNA synthesis from high-affinity EBNA1-binding sites and TRF2-half-binding sites that functions severalfold more efficiently than does DS. This finding indicates that EBV has selected a submaximally efficient origin of DNA synthesis for the latent phase of its life cycle. This enhanced origin could be used practically in human gene vectors to improve their efficiency in therapy and basic research.     

钠盐和氯盐胁迫下胡杨木质部汁液ABA、离子浓度和叶片气体交换的变化

研究了渗透胁迫和盐胁迫下一年生胡杨(Populus euphratica Oliv.)幼苗的木质部汁液脱落酸(ABA)、离子浓度及叶片气体交换的变化.PEG 6000 (溶液渗透势 -0.24 MPa)、50 mmol/L含钠离子的盐溶液 (NaNO3∶NaHCO3∶NaH2PO4=5∶4∶1, pH 6.8, 渗透势 -0.24 MPa)和50 mmol/L含氯离子的盐溶液 (KCl∶NH4Cl=1∶1, 渗透势 -0.24 MPa) 3种处理都显著降低了苗木的净光合速率(Pn)和蒸腾速率(TRN),但盐处理植株的TRN高于PEG处理的苗木.木质部汁液ABA的浓度在PEG处理后1 h达到峰值,之后开始下降,降到对照水平后又逐渐回升.盐处理苗木的ABA也是在处理开始后就迅速升高,但之后ABA水平明显高于PEG处理的植株.结果显示,渗透胁迫和离子胁迫都能提高胡杨木质部汁液ABA的浓度: 盐处理开始后ABA的迅速升高主要是渗透胁迫的作用,而此后离子胁迫(Na+和Cl-)对ABA水平的提高具有重要作用.钠盐处理对胡杨净光合速率和蒸腾速率的抑制作用高于氯盐处理,其木质部汁液中较高水平的ABA和盐离子(Na+和Cl-)是可能的原因.钠盐处理苗木的盐离子(Na+和Cl-)水平高于氯盐处理,主要是由以下两方面的原因所致: (1)细胞膜上的Ca2+被Na+所取代, 增加了膜的透性; (2)胡杨根细胞液泡对Na+的区隔化能力较弱(与区隔Cl-相比).另外,盐胁迫下胡杨能保持对营养元素K+、Ca2+和Mg2+的吸收,这也是其抗盐性强的重要原因.     

Degradation of lignin and lignin related compounds by protoplasts isolated from Fomes annosus

Protoplasts from a lignolytic fungus Fomes annosus were prepared through enzymatic hydrolysis of mycelium utilizing Novozym, a wall lytic enzyme preparation. Isolated protoplasts and living mycelium were compared in their ability to degrade ¹⁴C-labelled lignin related phenols and dehydropolymers of labelled coniferyl alcohol (synthetic lignin). The amounts of ¹⁴CO₂ released from O¹⁴CH₃-groups, ¹⁴C-2-side chains and ¹⁴C-rings by protoplasts was in the same range as those for intact mycelium. The methoxyl groups of synthetic lignin were more rapidly metabolized by protoplasts than by mycelium. When calculated in dpm of released ¹⁴CO₂ per mg protein the decomposition of ¹⁴C-labelled synthetic lignin and lignin-related monomers in a hyphae-free system of protoplasts was considerable higher than that obtained by the intact mycelium. The presence of intact hyphae is thus not necessary for lignin degradation to occur.Non-common-abbreviations used DHP Dehydropolymer of coniferyl alcohol - LS lignosulfonates prepared from DHP     

Regulation of proteolytic enzymes in axenically grown myxamoebae of Physarum polycephalum

The cultivation of Physarum polycephalum amoebae in two media with different protein contents revealed a regulation of aminopeptidases and proteases depending on the albumin content of the medium: in growing amoebae and plasmodia the aminopeptidases have similar isoenzyme patterns and relative activities against nitroanilides. One alanine and four leucine aminopeptidase isoenzymes were found within the slightly acid pH range. During growth amoebae secrete—different from plasmodia—leucine aminopeptidase into the medium with low protein content. In an albumin-rich medium additional alanine aminopeptidase activity was found. Out of nine plasmodial proteases four were found in amoebae too. Only one band (pI 3.6) was present in the protein-poor medium. No protease activity could be detected in the proteinrich medium.Abbreviations BSA bovine serum albumin - SD semi-defined (medium with low protein content; Table 1) The investigation formed a part of the Ph.D. thesis of A. Haars, Göttingen, 1976     

Cell cycle regulation of chromatin at an origin of DNA replication

Selection and licensing of mammalian DNA replication origins may be regulated by epigenetic changes in chromatin structure. The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) uses the cellular licensing machinery to regulate replication during latent infection of human cells. We found that the minimal replicator sequence of OriP, referred to as the dyad symmetry (DS), is flanked by nucleosomes. These nucleosomes were subject to cell cycle-dependent chromatin remodeling and histone modifications. Restriction enzyme accessibility assay indicated that the DS-bounded nucleosomes were remodeled in late G1. Remarkably, histone H3 acetylation of DS-bounded nucleosomes decreased during late G1, coinciding with nucleosome remodeling and MCM3 loading, and preceding the onset of DNA replication. The ATP-dependent chromatin-remodeling factor SNF2h was also recruited to DS in late G1, and formed a stable complex with HDAC2 at DS. siRNA depletion of SNF2h reduced G1-specific nucleosome remodeling, histone deacetylation, and MCM3 loading at DS. We conclude that an SNF2h-HDAC1/2 complex coordinates G1-specific chromatin remodeling and histone deacetylation with the DNA replication initiation process at OriP.     

Cholesterol absorption inhibitor Ezetimibe blocks uptake of oxidized LDL in human macrophages

Ezetimibe belongs to a group of selective and very effective 2-azetidione cholesterol absorption inhibitors which act on the level of cholesterol entry into enterocytes. Recent data indicated that the drug prevents the formation of a heterocomplex consisting of annexin-2 and caveolin-l and leads to specific inhibition of an NPCILI-dependent cholesterol uptake pathway required for uptake of micellar cholesterol into enterocytes. Earlier studies have shown that caveolin-l and annexin-2 are also expressed in human macro-phages and we show in this study that human macrophages express NPC1L1. Moreover in human macrophages, Ezetimibe(SCH58235) and its analogue, SCH354909, are bound to specific cell surface receptors followed by endocytosis via the classical endocytic pathway. SCH58235 had no effect on uptake and/or processing of acetylated LDL (Ac-LDL). In contrast, the compound inhibited uptake of oxidized LDL (Ox-LDL) by -50% in a dose-dependent manner. SCH58235 blocked the lipid-induced induction of LXR/RXR target genes ABCAI, ABCGI, and apolipoprotein E distinctively more effectively in macrophages loaded with Ox-LDL than in those loaded with Ac-LDL. Based on these findings, we presume that the caveolin-l-, annexin-2-, and NPClLI-dependent cholesterol uptake system that is operating in enterocytes may also contribute to class B scavenger receptor-dependent uptake of Ox-LDL in human monocyte-derived macrophages.     

Über ein bakteriolytisches Enzym vonArchangium violaceum (Myxobacteriales)

Zusammenfassung In der Nährlösung von Kulturen vonArchangium violaceum konnten folgende Enzymaktivitäten nachgewiesen werden: bakteriolytische, proteolytische und gegen Hefe lytische. Die Abgabe dieser Enzyme war bei den untersuchten Stämmen unterschiedlich. Drei verschiedene Typen ließen sich unterscheiden, solche die Hefe und Bakterien auflösten, solche die nur Bakterien lysierten und lytisch inaktive. Die Aktivität des bakteriolytischen Enzyms ist von der jeweiligen Wachstumsphase abhängig, beim Eintritt in das Stadium mit geringerer Wachstumsgeschwindigkeit sinkt die Aktivität. Die Enzymproduktion ist in weitem Bereich unabhängig vom pH-Wert der Nährlösung.Archangium violaceum ist in der Lage, den pH-Wert des Nährmediums in beide Richtungen bis zu pH±0,4 zu verändern.

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Studies of a bacteriolytic enzyme of Archangium violaceum (Myxobacteriales)I. Enzyme activities in vivo under different conditions

Summary Three different types of enzyme activities could be shown in the medium ofArchangium violaceum cultures: a bacteriolytic one, one which lyses yeasts and proteolytic ones. The production of these enzymes varies among the strains examined. We found three types of them: one lysing yeasts as well as bacteria, one active only against bacteria and one that was lytically inactive. The bacteriolytic activity is dependent on the phase of growth and decreases when the culture reaches the stage of slower growth. The enzyme production is within a wide range independent of the pH of the medium.Archangium violaceum is able to alter the pH of the medium in both directions up to ±0.4.