cDNA cloning and expression of a hamster α-thrombin receptor coupled to Ca2+ mobilization

AbstractThe serine protease α-thrombin (thrombin) potently stimulates G-protein-coupled signaling pathways and DNA synthesis in CCL39 hamster lung fibroblasts. To clone a thrombin receptor cDNA, selective amplification of mRNA sequences displaying homology to the transmembrane domains of G-protein-coupled receptor genes was performed by polymerase chain reaction. Using reverse transcribed poly(A)+ RNA from CCL39 cells and degenerate primers corresponding to conserved regions of several phospholipase C-coupled receptors, three novel putative receptor sequences were identified. One corresponds to an mRNA transcript of 3.4 kb in CCL39 cells and a relatively abundant cDNA. Microinjection of RNA transcribed in vitro from this cDNA in Xenopus oocytes leads to the expression of a functional thrombin receptor. The hamster thrombin receptor consists of 427 amino acid residues with 8 hydrophobic domains, including one at the extreme N-terminus that is likely to represent a signal peptide. A thrombin consensus cleavage site is present in the N-terminal extracellular region of the receptor sequence followed by a negatively charged cluster of residues present in a number of proteins that interact with the anion-binding exosite of thrombin

Presentation of infection in older patients—a prospective study

<div><p></p><p><i>Background.</i> Traditional wisdom suggests that infections in older patients have atypical presentation, including blunted febrile response. Data are scarce.</p><p><i>Design.</i> We analyzed data from a prospectively collected database on presentation of infection in 4,308 patients, and compared the presentation of older patients (≥ 75 years) versus adults (< 75 years).</p><p><i>Settings.</i> Single tertiary medical center.</p><p><i>Participants.</i> Patients admitted with suspected bacterial infection during 2002–2004 and 2010–2011.</p><p><i>Measurements.</i> We evaluated clinical presentation on day of admission, including vital signs and laboratory parameters.</p><p><i>Results.</i> No difference in fever values as a presenting sign of infection was found between older patients and adults (median fever 38.3°C, interquartile range [IQR] 37.4–39.0°C; and 38.4°C, IQR 37.3–39.0°C, respectively, <i>P</i> = 0.08). Median leukocyte count was significantly higher in older patients (median 11.60, IQR 8.30–15.72 in older patients; 10.84, 7.50–15.00 in adults, <i>P</i> < 0.001). Presentation with septic shock, acute renal failure, and reduced consciousness was significantly more common in older patients. These findings were also consistent in the subgroups of bacteremic patients and patients with microbiologically documented infection.</p><p><i>Conclusion.</i> Elevated fever and leukocytosis were found to be at least equally common in older patients compared to younger adults as part of the presentation of infection.</p></div

Bioinformatic and functional analysis of RNA secondary structure elements among different genera of human and animal caliciviruses

The mechanism and role of RNA structure elements in the replication and translation of Caliciviridae remains poorly understood. Several algorithmically independent methods were used to predict secondary structures within the Norovirus, Sapovirus, Vesivirus and Lagovirus genera. All showed profound suppression of synonymous site variability (SSSV) at genomic 5 ends and the start of the sub-genomic (sg) transcript, consistent with evolutionary constraints from underlying RNA structure. A newly developed thermodynamic scanning method predicted RNA folding mapping precisely to regions of SSSV and at the genomic 3 end. These regions contained several evolutionarily conserved RNA secondary structures, of variable size and positions. However, all caliciviruses contained 3 terminal hairpins, and stemloops in the anti-genomic strand invariably six bases upstream of the sg transcript, indicating putative roles as sg promoters. Using the murine norovirus (MNV) reverse-genetics system, disruption of 5 end stemloops produced 15- to 20-fold infectivity reductions, while disruption of the RNA structure in the sg promoter region and at the 3 end entirely destroyed replication ability. Restoration of infectivity by repair mutations in the sg promoter region confirmed a functional role for the RNA secondary structure, not the sequence. This study provides comprehensive bioinformatic resources for future functional studies of MNV and other caliciviruses