The Structure of a Rigorously Conserved RNA Element within the SARS Virus Genome

We have solved the three-dimensional crystal structure of the stem-loop II motif (s2m) RNA element of the SARS virus genome to 2.7-Å resolution. SARS and related coronaviruses and astroviruses all possess a motif at the 3′ end of their RNA genomes, called the s2m, whose pathogenic importance is inferred from its rigorous sequence conservation in an otherwise rapidly mutable RNA genome. We find that this extreme conservation is clearly explained by the requirement to form a highly structured RNA whose unique tertiary structure includes a sharp 90° kink of the helix axis and several novel longer-range tertiary interactions. The tertiary base interactions create a tunnel that runs perpendicular to the main helical axis whose interior is negatively charged and binds two magnesium ions. These unusual features likely form interaction surfaces with conserved host cell components or other reactive sites required for virus function. Based on its conservation in viral pathogen genomes and its absence in the human genome, we suggest that these unusual structural features in the s2m RNA element are attractive targets for the design of anti-viral therapeutic agents. Structural genomics has sought to deduce protein function based on three-dimensional homology. Here we have extended this approach to RNA by proposing potential functions for a rigorously conserved set of RNA tertiary structural interactions that occur within the SARS RNA genome itself. Based on tertiary structural comparisons, we propose the s2m RNA binds one or more proteins possessing an oligomer-binding-like fold, and we suggest a possible mechanism for SARS viral RNA hijacking of host protein synthesis, both based upon observed s2m RNA macromolecular mimicry of a relevant ribosomal RNA fold

Preparation of Group I Introns for Biochemical Studies and Crystallization Assays by Native Affinity Purification

The study of functional RNAs of various sizes and structures requires efficient methods for their synthesis and purification. Here, 23 group I intron variants ranging in length from 246 to 341 nucleotides—some containing exons—were subjected to a native purification technique previously applied only to shorter RNAs (<160 nucleotides). For the RNAs containing both exons, we adjusted the original purification protocol to allow for purification of radiolabeled molecules. The resulting RNAs were used in folding assays on native gel electrophoresis and in self-splicing assays. The intron-only RNAs were subjected to the regular native purification scheme, assayed for folding and employed in crystallization screens. All RNAs that contained a 3′ overhang of one nucleotide were efficiently cleaved off from the support and were at least 90% pure after the non-denaturing purification. A representative subset of these RNAs was shown to be folded and self-splicing after purification. Additionally, crystals were grown for a 286 nucleotide long variant of the Clostridium botulinum intron. These results demonstrate the suitability of the native affinity purification method for the preparation of group I introns. We hope these findings will stimulate a broader application of this strategy to the preparation of other large RNA molecules

The spectrum of thyroid dysfunction in an Australian hepatitis C population treated with combination Interferon-α2β and Ribavirin

BACKGROUND: The study aims to assess the pattern of thyroid response to combination Interferon-α2β (IFN-α) and Ribavirin (RBV) anti-viral therapy in an Australian hepatitis C cohort. These include the prevalence of thyroid dysfunction (TD) including hyperthyroidism and hypothyroidism and their possible predictors, the common overall pattern of thyroid function tests whilst receiving therapy and TD outcomes, and the correlation with HCV status outcome. METHODS: A retrospective analysis of all medical records was performed to assess thyroid function in Hepatitis C Virus (HCV) patients who were treated at the Hunter Area hepatitis C treatment center between 1995 and March 2004. The centre is part of the John Hunter hospital, a major tertiary referral centre in New South Wales, Australia. RESULTS: There were 272 cases available for review. The prevalence of TD is 6.7 percent and is made up predominantly of females (80 percent). There were 3 (1.1 percent) cases of hyperthyroidism with 2 (67 percent) females. Thirteen out of fifteen (80 percent) cases of hypothyroidism were females with the overall prevalence of 5.5 percent. The majority of hypothyroid patients still required Thyroxine supplement at the end of follow up. CONCLUSION: Ninety three percent of HCV treated patients have intact thyroid function at the end of treatment. The predominant TD is hypothyroidism. The predominant pattern of thyrotoxicosis (TTX) is that of thyroiditis although the number is small. Graves' like disease was not observed. People with pre-existing thyroid auto-antibodies should be closely monitored for thyroid dysfunction, particularly hypothyroidism

Structures of the Signal Recognition Particle Receptor from the Archaeon Pyrococcus furiosus: Implications for the Targeting Step at the Membrane

In all organisms, a ribonucleoprotein called the signal recognition particle (SRP) and its receptor (SR) target nascent proteins from the ribosome to the translocon for secretion or membrane insertion. We present the first X-ray structures of an archeal FtsY, the receptor from the hyper-thermophile Pyrococcus furiosus (Pfu), in its free and GDP•magnesium-bound forms. The highly charged N-terminal domain of Pfu-FtsY is distinguished by a long N-terminal helix. The basic charges on the surface of this helix are likely to regulate interactions at the membrane. A peripheral GDP bound near a regulatory motif could indicate a site of interaction between the receptor and ribosomal or SRP RNAs. Small angle X-ray scattering and analytical ultracentrifugation indicate that the crystal structure of Pfu-FtsY correlates well with the average conformation in solution. Based on previous structures of two sub-complexes, we propose a model of the core of archeal and eukaryotic SRP•SR targeting complexes

Molecular pathogenesis of T lymphocyte-induced liver injury in alcoholic hepatitis

The development of alcohol-induced liver injury is, in part, a consequence of the immunological/inflammatory response that alcohol stimulates. The abnormalities of immune function in heavy drinkers have been documented well. Cytokines, especially TNF α, produced from macrophages/Kupffer cells, play a role in the induction of liver cell necrosis and apoptosis. TNF α can cause liver cell apoptosis through the TNF α receptor or Fas/CD95 which is expressed by liver cells. Furthermore, chronic ethanol consumption may damage the liver by inhibiting the hepatotrophic and hepatoprotective actions of TNF α and other cytokines. There exists an intrinsic lymphocyte population in the normal liver. Intrahepatic T lymphocytes consist of a heterogeneous population of cells that has many and varied functional characteristics in addition to classical T cell activity. The population of intrahepatic T lymphocytes may arise via a thymus-independent pathway. Our recent work has demonstrated the role of liver-associated T lymphocytes in the pathogenesis of alcohol related liver injury initiated by a variety of stimuli such as endotoxin (lipopolysaccharide, LPS) or concanavalin A (Con A). Our studies have, for the first time, suggested that alcohol consumption alone does not lead to the development of marked liver necrosis (at least in the rat), but rather that a second insult is required for this to occur. Liver-associated T lymphocytes in rats spontaneously secrete interleukin-1 α, interleukin-6 and TNF α in vitro culture. There is a significant decline in the amounts of interleukin-1 α and TNF α secreted in ethanol-consuming rats compared with non-ethanol consuming rats. The numbers of T cells, NK cells and Kupffer cells in liver perfusates remains stable over a prolonged period of ethanol consumption. However, following Con A injection, there was an inappropriate increase in the amounts of interleukin-6 and TNF α secreted in in vitro culture of liver-associated T lymphocytes and a significant increase in the percentage of CD⁴⁺ T cells and CD²⁵⁺ T cells in liver perfusates compared with non-ethanol consuming rats. It suggested that liver-associated T lymphocytes are involved in the inflammatory process associated with alcohol related liver injury through increased cytokine secretion (TNF α)

How to treat: non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) and the subcategory of nonalcoholic steatohepatitis (NASH) was first described less than three decades ago and is strongly linked to obesity and the metabolic syndrome. The histological appearances of NAFLD and NASH resemble alcoholic steatosis and steatohepatitis respectively and can progress to similar sequelae. NAFLD is now the most common cause of chronic liver disease worldwide and will have a major impact on health care over subsequent years. There is an accumulating body of evidence to show that patients with NAFLD can progress to cirrhosis, liver failure and hepatocellular carcinoma

Methadone Maintenance in Australia

tag=1 data=Methadone Maintenance in Australia. tag=2 data=Caplehorn, John R.M.%Batey, Robert G. tag=3 data=Journal of Drug Issues, Vol 22, no. 3, tag=6 data=^d ^m ^y1992 tag=8 data=DRUGS tag=9 data=METHADONE tag=15 data=JO