The human cytomegalovirus-specific UL1 gene encodes a late-phase glycoprotein incorporated in the virion envelope

We have investigated the previously uncharacterized human cytomegalovirus (HCMV) UL1 open reading frame (ORF), a member of the rapidly evolving HCMV RL11 family. UL1 is HCMV specific; the absence of UL1 in chimpanzee cytomegalovirus (CCMV) and sequence analysis studies suggest that UL1 may have originated by the duplication of an ancestor gene from the RL11-TRL cluster (TRL11, TRL12, and TRL13). Sequence similarity searches against human immunoglobulin (Ig)-containing proteins revealed that HCMV pUL1 shows significant similarity to the cellular carcinoembryonic antigen-related (CEA) protein family N-terminal Ig domain, which is responsible for CEA ligand recognition. Northern blot analysis revealed that UL1 is transcribed during the late phase of the viral replication cycle in both fibroblast-adapted and endotheliotropic strains of HCMV. We characterized the protein encoded by hemagglutinin (HA)-tagged UL1 in the AD169-derived HB5 background. UL1 is expressed as a 224-amino-acid type I transmembrane glycoprotein which becomes detectable at 48 h postinfection. In infected human fibroblasts, pUL1 colocalized at the cytoplasmic site of virion assembly and secondary envelopment together with TGN-46, a marker for the trans-Golgi network, and viral structural proteins, including the envelope glycoprotein gB and the tegument phosphoprotein pp28. Furthermore, analyses of highly purified AD169 UL1-HA epitope-tagged virions revealed that pUL1 is a novel constituent of the HCMV envelope. Importantly, the deletion of UL1 in HCMV TB40/E resulted in reduced growth in a cell type-specific manner, suggesting that pUL1 may be implicated in regulating HCMV cell tropism.     

Activated platelets and monocytes generate four hydroxyphosphatidylethanolamines via lipoxygenase

12/15-Lipoxygenase (LOX) mediates immune-regulatory activities not accounted for by its known free acid eicosanoids, suggesting that additional lipids may be generated by activated cells. To characterize novel LOX-derived lipids, a lipidomic approach was utilized. Ionophore-activated interleukin-4-treated human peripheral monocytes generated up to 10-fold more esterified 15-hydroxyeicosatetraenoic acid (15-HETE) than free in a phosphatidylinositol 3-kinase- and protein kinase C-sensitive manner. Precursor scanning electrospray ionization/tandem spectroscopy for m/z 319 (HETE, [M-H](-)) showed 4 ions at m/z 738, 764, 766, and 782 that were identified using tandem spectroscopy and MS3 as specific diacyl and plasmalogen 15-HETE phosphatidylethanolamines. Using H (18)(2)O water, the compounds were shown to form by direct oxidation of endogenous phosphatidylethanolamine (PE) by 15-LOX, with PE being the preferred phospholipid pool containing 15-HETE. Similarly, human platelets generated 4 analogous PE lipids that contained 12-HETE and increased significantly in response to ionophore, collagen, or convulxin. These products were retained in the cells, in contrast to free acids, which are primarily secreted. Precursor scanning of platelet extracts for the major platelet-derived prostanoid, thromboxane B2 (m/z 369.2), did not reveal PE esters, indicating that this modification is restricted to the LOX pathway. In summary, we show formation of PE-esterified HETEs in immune cells that may contribute to LOX signaling in inflammation.     

Cellular repressor of E1A-stimulated genes is a bona fide lysosomal protein which undergoes proteolytic maturation during its biosynthesis

Cellular repressor of E1A-stimulated genes (CREG) has been reported to be a secretory glycoprotein implicated in cellular growth and differentiation. We now show that CREG is predominantly localized within intracellular compartments. Intracellular CREG was found to lack an N-terminal peptide present in the secreted form of the protein. In contrast to normal cells, CREG is largely secreted by fibroblasts missing both mannose 6-phosphate receptors. This is not observed in cells lacking only one of them. Mass spectrometric analysis of recombinant CREG revealed that the protein contains phosphorylated oligosaccharides at either of its two N-glycosylation sites. Cellular CREG was found to cosediment with lysosomal markers upon subcellular fractionation by density-gradient centrifugation. In fibroblasts expressing a CREG-GFP fusion construct, the heterologous protein was detected in compartments containing lysosomal proteins. Immunolocalization of endogenous CREG confirmed that intracellular CREG is localized in lysosomes. Proteolytic processing of intracellular CREG involves the action of lysosomal cysteine proteinases. These results establish that CREG is a lysosomal protein that undergoes proteolytic maturation in the course of its biosynthesis, carries the mannose 6-phosphate recognition marker and depends on the interaction with mannose 6-phosphate receptors for efficient delivery to lysosomes.     

Improved comparative proteome analysis based on two-dimensional gel electrophoresis

The purpose of this study was to test the extent to which differences in spot intensity can be reliably recognized between two groups of two-dimensional electrophoresis gels (pH 4-7, visualized with ruthenium fluorescent stain) each loaded with different amounts of protein from rat brain (power analysis). Initial experiments yielded only unsatisfactory results: 546 spots were matched from two groups of 6 gels each loaded with 200 microg and 250 microg protein, respectively. Only 72 spots were higher (p<0.05), while 58 spots were significantly lower in the 250-microg group. The construction of new apparatuses that allowed the simultaneous processing of 24 gels throughout all steps between rehydration and staining procedure considerably lowered the between-gel variation. This resulted in the detection of significant differences in spot intensities in 77-90% of all matched spots on gel groups with a 25% difference in protein load. This applied both when protein from 24 biological replicates was loaded onto two groups of 12 gels and when two pooled tissue samples were each loaded onto 6 gels. At a difference of 50% in protein load, more than 90% of all spots differed significantly between two experimental groups.     

Characterization of the hematopoietic target cells of AEV, MC29 and AMV avian leukemia viruses

The hematopoietic target cells of the three prototype strains of replication defective avian leukemia viruses (DLVs) were studied, using a newly developed, quantitative in vitro transformation assay. Our results show that the target cells of avian erythroblastosis virus (AEV) belong to the erythroid lineage while those of myelocytomatosis virus 29 (MC29) and avian myeloblastosis virus (AMV) belong to the myeloid lineage. As judged from suicide experiments using BUdR incorporation and irradiation with visible light, a higher proportion of AEV- and AMV-target cells are in cycle than MC29-target cells. Using differentiation specific antisera directed against cell surface antigens, we could demonstrate that the target cells of AEV express erythroblast-specific antigen(s) and less intensively erythrocyte-specific antigen(s), while those of MC29 and AMV express myeloblast-specific antigen(s). In addition, MC29-target cells express macrophage-specific antigen(s). None of the AEV-target cells are adherent or phagocytic, while a small proportion of the AMV-target cells are adherent and about half of the MC29-target cells are both adherent and phagocytic. Our results support the concept that DLVs specifically transform certain types of committed erythroid and myeloid progenitor cells. The target cells of AEV and AMV appear to resemble the respective transformed cells in their state of differentiation, whereas those of MC29 appear to be more immature than the corresponding transformed cells.     

Selbstreinigung und Ciliatenbesiedlung in saurem Milieu (Modellversuche)

It is well known that acid precipitation originating from air pollution may increase the acidity of lakes and rivers. In the present experimental study the effects of acidification on the population dynamics of bacteria and protozoa associated with the decomposition of peptone have been investigated.

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Institut für landwirtschaftliche Zoologie und Bienenkunde der Universität Bonn     

Cytomegalovirus subverts macrophage identity

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Water clusters in the reaction centre of Rhodobacter sphaeroides

The structure of the reaction centre from Rhodobacter sphaeroides has been refined up to 2.4 Å resolution. Several clusters of firmly bound water molecules were found proximal to the primary and secondary quinones. They represent putative pathways for proton transfer.     

Die Hemmung der Carotinoidsynthese im Stickstoffmangel

Summary The ability to synthesize carotenoids during normal or strongly reduced nitrogen supply was tested in a higher plant (Hordeum vulgare L.) and in a green alga (Scenedesmus obliquus). Though carotenoids are nitrogenfree, their synthesis is strongly inhibited during nitrogen deficiency. The formation of secondary carotenoids, which apparently proceeds at the expense of the primary carotenoid components, occurs in Scenedesmus but not in Hordeum plants. It is concluded that the dimerisation of geranly-geranyl-pyrophosphate is the limiting step in the carotenoid synthesis during nitrogen deficiency. The synthesis of prenyl chains is less affected by nitrogen deficiency.