Identification of genes that are associated with DNA repeats in prokaryotes

Using in silico analysis we studied a novel family of repetitive DNA sequences that is present among both domains of the prokaryotes (Archaea and Bacteria), but absent from eukaryotes or viruses. This family is characterized by direct repeats, varying in size from 21 to 37 bp, interspaced by similarly sized non-repetitive sequences. To appreciate their characteri-stic structure, we will refer to this family as the clustered regularly interspaced short palindromic repeats (CRISPR). In most species with two or more CRISPR loci, these loci were flanked on one side by a common leader sequence of 300-500 b. The direct repeats and the leader sequences were conserved within a species, but dissimilar between species. The presence of multiple chromosomal CRISPR loci suggests that CRISPRs are mobile elements. Four CRISPR-associated (cas) genes were identified in CRISPR-containing prokaryotes that were absent from CRISPR-negative prokaryotes. The cas genes were invariably located adjacent to a CRISPR locus, indicating that the cas genes and CRISPR loci have a functional relationship. The cas3 gene showed motifs characteristic for helicases of the superfamily 2, and the cas4 gene showed motifs of the RecB family of exonucleases, suggesting that these genes are involved in DNA metabolism or gene expression. The spatial coherence of CRISPR and cas genes may stimulate new research on the genesis and biological role of these repeats and genes.     

Novel Subtype of Peroxisomal Acyl-CoA Oxidase Deficiency and Bifunctional Enzyme Deficiency with Detectable Enzyme Protein: Identification by Means of Complementation Analysis

We describe four infants with a novel subtype of an isolated deficiency of one of the peroxisomal β-oxidation enzymes with detectable enzyme protein. The patients showed characteristic clinical and biochemical abnormalities, including hypotonia, psychomotor retardation, hepatomegaly, typical facial appearance, accumulation of very-long-chain fatty acids, and decreased lignoceric acid oxidation. However, β-oxidation enzyme proteins were detected by immunoblot analyses, and large peroxisomes were identified by immunofluorescence staining. In order to identify the underlying defect in these patients, complementation analysis was introduced using fibroblasts from these patients and patients with an established deficiency of either acyl-CoA oxidase or bifunctional enzyme, as identified by immunoblotting. In the complementing combinations, fused cells showed increased lignoceric acid oxidation, resistance against 1-pyrene dodecanoic acid/UV selection, and normalization of the size and the distribution of peroxisomes. The results indicate that two patients with a more severe clinical course were suffering from bifunctional enzyme deficiency and that the other two infants, who were siblings and had a less severe clinical presentation, were the first patients with acyl-CoA oxidase deficiency with detectable enzyme protein.     

Regulation of CTP: phosphocholine cytidylyltransferase activity by the physical properties of lipid membranes: an important role for stored curvature strain energy

CTP:Phosphocholine cytidylyltransferase (CT) catalyzes the key step in phosphatidylcholine (PC) synthesis. CT is activated by binding to certain lipid membranes. The membrane binding affinity of CT can vary from micromolar to millimolar K(d), depending on the lipid composition of the target membrane. Class II CT activators like diacylglycerols and unsaturated phosphatidylethanolamines (PE) favor inverted lipid phase formation. The mechanism(s) governing CT's association with class II lipid membranes and subsequent activation are relatively unknown. We measured CT activation by vesicles composed of PC and one of three unsaturated PEs, dioleoylglycerol (DOG), or cholesterol. For each lipid system, we estimated the stored curvature strain energy of the monolayer when confined to a relatively flat bilayer. CT binding and activation correlate very well with the curvature strain energy of several chemically distinct class II lipid systems, with the exception of those containing cholesterol, in which CT activation was less than the increase in curvature strain. CT activation by membranes containing DOG was reversed by inclusion of specific lysolipids, which reduce curvature strain energy. LysoPC, which has a larger positive curvature than lysoPE, produced greater inhibition of CT activation. Stored curvature strain energy is thus an important determinant of CT activation. Membrane interfacial polarity was investigated using a membrane-anchored fluorescent probe. Decreases in quenching of this interfacial probe by doxyl-PCs in class II membranes suggest the probe adopts a more superficial membrane location. This may reflect an increased surface hydrophobicity of class II lipid membranes, implying a role for surface dehydration in CT's interactions with membranes containing class II lipids. Cholesterol, a poor activator of CT, did not affect the positioning of the polarity-sensitive probe, suggesting that one reason for its ineffectiveness is an inability to enhance surface hydrophobicity.     

Identification of oxidative stress and Toll-like receptor 4 signaling as a key pathway of acute lung injury

Multiple lung pathogens such as chemical agents, H5N1 avian flu, or SARS cause high lethality due to acute respiratory distress syndrome. Here we report that Toll-like receptor 4 (TLR4) mutant mice display natural resistance to acid-induced acute lung injury (ALI). We show that TLR4-TRIF-TRAF6 signaling is a key disease pathway that controls the severity of ALI. The oxidized phospholipid (OxPL) OxPAPC was identified to induce lung injury and cytokine production by lung macrophages via TLR4-TRIF. We observed OxPL production in the lungs of humans and animals infected with SARS, Anthrax, or H5N1. Pulmonary challenge with an inactivated H5N1 avian influenza virus rapidly induces ALI and OxPL formation in mice. Loss of TLR4 or TRIF expression protects mice from H5N1-induced ALI. Moreover, deletion of ncf1, which controls ROS production, improves the severity of H5N1-mediated ALI. Our data identify oxidative stress and innate immunity as key lung injury pathways that control the severity of ALI.     

Cu,Zn Superoxide Dismutase Maturation and Activity Are Regulated by COMMD1

The maturation and activation of the anti-oxidant Cu,Zn superoxide dismutase (SOD1) are highly regulated processes that require several post-translational modifications. The maturation of SOD1 is initiated by incorporation of zinc and copper ions followed by disulfide oxidation leading to the formation of enzymatically active homodimers. Our present data indicate that homodimer formation is a regulated final step in SOD1 maturation and implicate the recently characterized copper homeostasis protein COMMD1 in this process. COMMD1 interacts with SOD1, and this interaction requires CCS-mediated copper incorporation into SOD1. COMMD1 does not regulate disulfide oxidation of SOD1 but reduces the level of SOD1 homodimers. RNAi-mediated knockdown of COMMD1 expression results in a significant induction of SOD1 activity and a consequent decrease in superoxide anion concentrations, whereas overexpression of COMMD1 exerts exactly the opposite effects. Here, we identify COMMD1 as a novel protein regulating SOD1 activation and associate COMMD1 function with the production of free radicals.