Characterization of a Listeria monocytogenes Ca(2+) pump: a SERCA-type ATPase with only one Ca(2+)-binding site

We have characterized a putative Ca(2+)-ATPase from the pathogenic bacterium Listeria monocytogenes with the locus tag lmo0841. The purified and detergent-solubilized protein, which we have named Listeria monocytogenes Ca(2+)-ATPase 1 (LMCA1), performs a Ca(2+)-dependent ATP hydrolysis and actively transports Ca(2+) after reconstitution in dioleoylphosphatidyl-choline vesicles. Despite a high sequence similarity to the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) and plasma membrane Ca(2+)-ATPase (PMCA), LMCA1 exhibits important biochemical differences such as a low Ca(2+) affinity (K(0.5) ～80 μm) and a high pH optimum (pH ～9). Mutational studies indicate that the unusually high pH optimum can be partially ascribed to the presence of an arginine residue (Arg-795), corresponding in sequence alignments to the Glu-908 position at Ca(2+) binding site I of rabbit SERCA1a, but probably with an exposed position in LMCA1. The arginine is characteristic of a large group of putative bacterial Ca(2+)-ATPases. Moreover, we demonstrate that H(+) is countertransported with a transport stoichiometry of 1 Ca(2+) out and 1 H(+) in per ATP hydrolyzed. The ATPase may serve an important function by removing Ca(2+) from the microorganism in environmental conditions when e.g. stressed by high Ca(2+) and alkaline pH.     

Structural Basis for Recognizing Phosphoarginine and Evolving Residue-Specific Protein Phosphatases in Gram-Positive Bacteria

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The relationships between Late Ordovician sea-level changes and faunal turnover in western Baltica: Geochemical evidence of oxic and dysoxic bottom-water conditions

The late Sandbian to the early Katian (Late Ordovician) in southeastern Norway is dominated by marine mudstones that contain an exceptional spectrum of macrofauna and trace element geochemistry, recording both abrupt and gradual faunal changes during major sea-level and environmental shifts. This study investigates the relationships between sea-level changes and their influence on the source of clastic material, oxygen levels in the bottom water and faunal changes, with special emphasis on the brachiopod fauna. Trace element ratios indicate that nearly stable upper dysoxic bottom-water conditions prevailed in the northwestern part of the Baltoscandian Sea. The exception is during the major shallowing in the earliest Katian, when there was an abrupt shift to oxic conditions as the sea bottom came within a normal storm wave base. Nonetheless, the pre-shallowing epibenthic fauna (though not the shelly endobenthic) in this area was rich and diverse and two major immigration phases of new brachiopod taxa are seen well before the shallowing. This indicates that the immigration of new taxa was not the result of an increase in oxygen content in the bottom water. More brachiopod genera stayed during the abrupt shallowing and increased oxic level than did during the following gradual return to deeper, dysoxic environments. The major brachiopod immigration phases took place markedly earlier in this northwestern part of the Baltoscandian Sea than in the central part (Eastern Baltic) and possibly also the comparable faunal turnover in Laurentia. The following disappearance of taxa during an early Katian transgressive event coincided with the faunal turnover in the shallow-water environments of the East Baltic.The depositional history was consistent within the investigated mudstone-dominated offshore facies of the Oslo Region, which are comparable to the Central Baltoscandian Confacies Belt of Baltoscandia. The composition of the siliciclastic material which was deposited in the basin was nearly constant through the sequence, though the land area expanded to include ophiolites, expressed by Cr enrichment, coinciding with the major sea-level drop. The Cr enrichment indicates that, by that time at least, the ophiolite complexes north of the Oslo Region became subaerially exposed. This enrichment thus forms a potential geochemical marker horizon representing the basal Katian Stage.     

N‐linked glycosylation is an important parameter for optimal selection of cell lines producing biopharmaceutical human IgG

We studied the variations in N‐linked glycosylation of human IgG molecules derived from 105 different stable cell lines each expressing one of the six different antibodies. Antibody expression was based on glutamine synthetase selection technology in suspension growing CHO‐K1SV cells. The glycans detected on the Fc fragment were mainly of the core‐fucosylated complex type containing zero or one galactose and little to no sialic acid. The glycosylation was highly consistent for the same cell line when grown multiple times, indicating the robustness of the production and glycan analysis procedure. However, a twofold to threefold difference was observed in the level of galactosylation and/or non‐core‐fucosylation between the 105 different cell lines, suggesting clone‐to‐clone variation. These differences may change the Fc‐mediated effector functions by such antibodies. Large variation was also observed in the oligomannose‐5 glycan content, which, when present, may lead to undesired rapid clearance of the antibody in vivo. Statistically significant differences were noticed between the various glycan parameters for the six different antibodies, indicating that the variable domains and/or light chain isotype influence Fc glycosylation. The glycosylation altered when batch production in shaker was changed to fed‐batch production in bioreactor, but was consistent again when the process was scaled from 400 to 5,000 L. Taken together, the observed clone‐to‐clone glycosylation variation but batch‐to‐batch consistency provides a rationale for selection of optimal production cell lines for large‐scale manufacturing of biopharmaceutical human IgG. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Postembryonic development of Antygomonas incomitata (Kinorhyncha: Cyclorhagida)

Postembryonic development in the kinorhynch species Antygomonas incomitata was examined using scanning electron microscopy. The morphology of the six juvenile stages, J‐1 to J‐6, varies at numerous details, but they can also be distinguished by a few key characters. Juvenile stage 1 by its composition of only nine trunk segments; J‐2 by the combination of possessing 10 trunk segments, but no cuspidate spines on segment 9; J‐3 by the presence of cuspidate spines on segment 9, but only one pair of cuspidate spines on segment 8; J‐4 by the combination of 10 trunk segments only, but having two pairs of cuspidate spines on segment 8; J‐5 by possessing 11 trunk segments and same spine compositions as adults but is still maintaining postmarginal spiculae; J‐6 specimens closely resemble adults and are most easily identified by their reduced trunk lengths. New segments are formed in a growth zone in the anterior part of the terminal segment. The complete number of segments is reached in J‐5. Development of cuticular head and trunk structures are described through all postembryonic stages and following developmental patterns could be outlined: the mouth cone possesses outer oral styles from J‐1, but in J‐1 to J‐3, the styles alternate in size. Scalids of the introvert are added after each molt, and scalids appear earliest in the anterior rings, whereas scalids in more posterior rings are added in older postembryonic stages. The early J‐1 stage is poor in spines and sensory spots and both structures increase in number after each molt. The complete spine composition is reached in J‐4, whereas new sensory spots appear after all molts, inclusive the final one from J‐6 to adult. Sensory spots in the paraventral positions often appear as Type 3 sensory spots but are through development transformed to Type 2. This transformation happens earliest on the anterior segments. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.