Effect of Recombinant Tissue Plasminogen Activator and 120-kHz Ultrasound on Porcine Intracranial Thrombus Density.

Surgical intervention for the treatment of intracerebral hemorrhage (ICH) has been limited by inadequate lysis of the target thrombus. Adjuvant transcranial ultrasound exposure is hypothesized to improve thrombolysis, expedite hematoma evacuation and improve clinical outcomes. A juvenile porcine intracerebral hemorrhage model was established by direct infusion of autologous blood into the porcine white matter. Thrombi were either not treated (sham) or treated with recombinant tissue plasminogen activator alone (rt-PA only) or in combination with pulsed transcranial 120-kHz ultrasound (sonothrombolysis). After treatment, pigs were euthanized, the heads frozen and sectioned and the thrombi extracted. D-Dimer and thrombus density assays were used to assess degree of lysis. Both porcine and human D-dimer assays tested did not have sufficient sensitivity to detect porcine D-dimer. Thrombi treated with rt-PA with or without 120-kHz ultrasound had a significantly lower density compared with sham-treated thrombi. No enhancement of rt-PA-mediated thrombolysis was noted with the addition of 120-kHz ultrasound (sonothrombolysis). The thrombus density assay revealed thrombolytic efficacy caused by rt-PA in an in vivo juvenile porcine model of intracerebral hemorrhage. Transcranial sonothrombolysis did not enhance rt-PA-induced thrombolysis, likely because of the lack of exogenous cavitation nuclei.

Structural insight into the functional mechanism of Nep1/Emg1 N1-specific pseudouridine methyltransferase in ribosome biogenesis.

Nucleolar Essential Protein 1 (Nep1) is required for small subunit (SSU) ribosomal RNA (rRNA) maturation and is mutated in Bowen-Conradi Syndrome. Although yeast (Saccharomyces cerevisiae) Nep1 interacts with a consensus sequence found in three regions of SSU rRNA, the molecular details of the interaction are unknown. Nep1 is a SPOUT RNA methyltransferase, and can catalyze methylation at the N1 of pseudouridine. Nep1 is also involved in assembly of Rps19, an SSU ribosomal protein. Mutations in Nep1 that result in decreased methyl donor binding do not result in lethality, suggesting that enzymatic activity may not be required for function, and RNA binding may play a more important role. To study these interactions, the crystal structures of the scNep1 dimer and its complexes with RNA were determined. The results demonstrate that Nep1 recognizes its RNA site via base-specific interactions and stabilizes a stem-loop in the bound RNA. Furthermore, the RNA structure observed contradicts the predicted structures of the Nep1-binding sites within mature rRNA, suggesting that the Nep1 changes rRNA structure upon binding. Finally, a uridine base is bound in the active site of Nep1, positioned for a methyltransfer at the C5 position, supporting its role as an N1-specific pseudouridine methyltransferase.

Crystal structure of iodotyrosine deiodinase, a novel flavoprotein responsible for iodide salvage in thyroid glands.

The flavoprotein iodotyrosine deiodinase (IYD) salvages iodide from mono- and diiodotyrosine formed during the biosynthesis of the thyroid hormone thyroxine. Expression of a soluble domain of this membrane-bound enzyme provided sufficient material for crystallization and characterization by x-ray diffraction. The structures of IYD and two co-crystals containing substrates, mono- and diiodotyrosine, alternatively, were solved at resolutions of 2.0, 2.45, and 2.6 A, respectively. The structure of IYD is homologous to others in the NADH oxidase/flavin reductase superfamily, but the position of the active site lid in IYD defines a new subfamily within this group that includes BluB, an enzyme associated with vitamin B(12) biosynthesis. IYD and BluB also share key interactions involving their bound flavin mononucleotide that suggest a unique catalytic behavior within the superfamily. Substrate coordination to IYD induces formation of an additional helix and coil that act as an active site lid to shield the resulting substrate.flavin complex from solvent. This complex is stabilized by aromatic stacking and extensive hydrogen bonding between the substrate and flavin. The carbon-iodine bond of the substrate is positioned directly over the C-4a/N-5 region of the flavin to promote electron transfer. These structures now also provide a molecular basis for understanding thyroid disease based on mutations of IYD.