Identification of equine P-selectin glycoprotein ligand-1 (CD162)

P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is a dimeric, mucin-like, transmembrane glycoprotein constitutively expressed on leukocytes. A high baseline level of P-selectin expression in circulating equine platelets suggests a primed state toward inflammation and thrombosis via P-selectin/PSGL-1 adhesion. To investigate the potential role of equine P-selectin in these events, we first identified the cDNA sequence of equine PSGL-1 (ePSGL-1) using degenerate PCR and RACE-PCR and then compared the predicted sequence with that of human PSGL-1 (hPSGL-1). ePSGL-1 protein subunit is predicted to be 43 kDa and composed of 420 amino acids with a predicted 18-amino-acid signal sequence showing 78% homology to hPSGL-1. Previously published work has shown that binding of P-selectin requires sulfation of at least one of three tyrosines and O-glycosylation of one threonine in the N-terminus of human PSGL-1. However, the corresponding domain in ePSGL-1, spanning residues 19–43, contains only one tyrosine in the vicinity of two threonines at positions 25 and 41. ePSGL-1 contains 14 threonine/serine-rich decameric repeats as compared to hPSGL-1 which contains 14–16 threonine-rich decameric repeats. The transmembrane and cytoplasmic domains display 91% and 74% homology to corresponding human PSGL-1 domains, respectively. In summary, there is 71% homology in comparing the open reading frame (ORF) of ePSGL-1 with that of hPSGL-1. The greatest homologies between species exist in the transmembrane domain and cytoplasmic tail while substantial differences exist in the extracellular domain.The nucleotide sequence data reported in this article has been submitted to GenBank and assigned the accession number AY298766.     

M line-deficient titin causes cardiac lethality through impaired maturation of the sarcomere

Titin, the largest protein known to date, has been linked to sarcomere assembly and function through its elastic adaptor and signaling domains. Titin's M-line region contains a unique kinase domain that has been proposed to regulate sarcomere assembly via its substrate titin cap (T-cap). In this study, we use a titin M line-deficient mouse to show that the initial assembly of the sarcomere does not depend on titin's M-line region or the phosphorylation of T-cap by the titin kinase. Rather, titin's M-line region is required to form a continuous titin filament and to provide mechanical stability of the embryonic sarcomere. Even without titin integrating into the M band, sarcomeres show proper spacing and alignment of Z discs and M bands but fail to grow laterally and ultimately disassemble. The comparison of disassembly in the developing and mature knockout sarcomere suggests diverse functions for titin's M line in embryonic development and the adult heart that not only involve the differential expression of titin isoforms but also of titin-binding proteins.     

The effect of constant darkness and short light periods on the survival and physiological fitness of two phytoplankton species and their growth potential after re-illumination

We tested the survival potential and fitness of two different algae strains (the diatom Thalassiosira weissflogii and the cryptophyceae Rhodomonas sp.) under different growth conditions (complete darkness and short light intervals, simulating conditions in a deep mixed water column) at different temperatures, plus the effect of these conditions on the physiological fitness and growth after re-illumination was examined. Both species survived the experimental conditions without significant cell loss or physiological damage. Two different survival strategies were observed: (1) the diatom T. weissflogii immediately reduced its metabolic rate and stopped cell division. The effect on chlorophyll a (chl-a) content and photosynthetic capacity was negligible. At 10 °C, T. weissflogii used the short light windows to metabolize carbohydrates and growth. (2) The cryptophyte Rhodomonas sp. initially continued to grow after transfer into all trials. However, the cell number decreased after day 6. Carbohydrate and chl-a content went on to decrease dramatically (70 and 50%, respectively). After 3 days of re-illumination, T. weissflogii grew faster than of Rhodomonas sp.. The diatom seemed to benefit from better start conditions and would out-compete the cryptophyte during a spring bloom. Our results highlight that these algae groups have different strategies in dealing with darkness, which potentially endow diatoms with a competitive advantage in deep mixed waters and in the season of early spring.     

Interaction network of the ribosome assembly machinery from a eukaryotic thermophile

Ribosome biogenesis in eukaryotic cells is a highly dynamic and complex process innately linked to cell proliferation. The assembly of ribosomes is driven by a myriad of biogenesis factors that shape pre‐ribosomal particles by processing and folding the ribosomal RNA and incorporating ribosomal proteins. Biochemical approaches allowed the isolation and characterization of pre‐ribosomal particles from Saccharomyces cerevisiae, which lead to a spatiotemporal map of biogenesis intermediates along the path from the nucleolus to the cytoplasm. Here, we cloned almost the entire set (～180) of ribosome biogenesis factors from the thermophilic fungus Chaetomium thermophilum in order to perform an in‐depth analysis of their protein–protein interaction network as well as exploring the suitability of these thermostable proteins for structural studies. First, we performed a systematic screen, testing about 80 factors for crystallization and structure determination. Next, we performed a yeast 2‐hybrid analysis and tested about 32,000 binary combinations, which identified more than 1000 protein–protein contacts between the thermophilic ribosome assembly factors. To exemplary verify several of these interactions, we performed biochemical reconstitution with the focus on the interaction network between 90S pre‐ribosome factors forming the ctUTP‐A and ctUTP‐B modules, and the Brix‐domain containing assembly factors of the pre‐60S subunit. Our work provides a rich resource for biochemical reconstitution and structural analyses of the conserved ribosome assembly machinery from a eukaryotic thermophile.     

A cyclophilin-like peptidyl-prolyl cis/trans isomerase from Legionella pneumophila – characterization, molecular cloning and overexpression

Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires’disease). A major virulence factor, the Mip protein (FK506-binding protein, FKBP25mem), belongs to the enzyme family of peptidyl-prolyl cis/trans isomerases (PPIases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPiase at a level of enzyme activity comparable to that of FKBP25mem. The N-terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPIases. The Icy gene (Legionella cycophn) was cloned and sequenced. It encodes a putative 164-amino-acid protein with a molecular mass of 17 968 Da called L. pneumophila cyclophilin 18 (L. p. Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coll with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19-amino-acid polypeptide extension including a 6-histi-dine tag and an enterokinase cleavage site exhibits