Emendation of the Genus Auritidibacter Yassin et al. 2011 and Auritidibacter ignavus Yassin et al. 2011 based on features observed from Canadian and Swiss clinical isolates and whole-genome sequencing analysis.

Auritidibacter ignavus is a Gram-stain-positive bacillus derived from otorrhea. Four strains derived from ear discharges in Canada and Switzerland, with features consistent with but distinguishable from Auritidibacter ignavus IMMIB L-1656T (accession number FN554542) by 16S rRNA gene sequencing (97.5 % similarity), were thought to represent a novel species of the genus Auritidibacter. Auritidibacter ignavus DSM 45359T (=IMMIB L-1656T) was acquired to compare with Canadian and Swiss strains by whole-genome sequencing (WGS). Unexpectedly, those isolates were observed to be consistent with A. ignavus DSM 45359T by WGS (ANIb scores >98 %), MALDI-TOF (Bruker), cellular fatty acid analysis and biochemically (some differences were observed). A nearly full 16S rRNA gene sequence could not be readily prepared from A. ignavus DSM 45359T, even after multiple attempts. A 16S rRNA gene chimeric consensus sequence created from the genome assembly of A. ignavus DSM 45359T had only 97.5 % similarity to that of A. ignavus IMMIB L-1656T, implying that 16S rRNA sequence accession number FN554542 could not be replicated. We concluded that our isolates of members of the genus Auritidibacter were consistent with A. ignavus DSM 45359T, did not represent a novel species, and that the sequence corresponding to FN554542 was not reproducible. By WGS, A. ignavus DSM 45359T had genome of 2.53×106 bp with a DNA G+C content of 59.34%, while genomes of Canadian and Swiss isolates ranged from 2.47 to 2.59×106 bp with DNA G+C contents of 59.3-59.52 %. A. ignavus NML 100628 (=NCTC 14178=LMG 30897) did not demonstrate a rodcoccus cycle. Emendation of Auritidibacter ignavus was proposed based on these results.

Hepatitis B Virus Genotype G forms core-like particles with unique structural properties.

We have determined the structure of the core capsid of an unusual variant of hepatitis B virus, genotype G (HBV/G) at 14Å resolution, using cryo-electron microscopy. The structure reveals surface features not present in the prototype HBV/A genotype. HBV/G is novel in that it has a unique 36-bp insertion downstream of the core gene start codon. This results in a twelve amino acid insertion at the N-terminal end of the core protein, and two stop codons in the precore region that prevent the expression of HBeAg. HBV/G replication in patients is associated with co-infection with another genotype of HBV, suggesting that HBV/G may have reduced replication efficiency in vivo. We localized the N-terminal insertion in HBV/G and show that it forms two additional masses on the core surface adjacent to each of the dimer-spikes and have modelled the structure of the additional residues within this density. We show that the position of the insertion would not interfere with translocation of nucleic acids through the pores to the core interior compartment. However, the insertion may partially obscure several residues on the core surface that are known to play a role in envelopment and secretion of virions, or that could affect structural rearrangements that may trigger envelopment after DNA second-strand synthesis.

Quaternary structure of the insulin-insulin receptor complex.

The three-dimensional (3D) structure of the intrinsically dimeric insulin receptor bound to its ligand, insulin, was determined by electron cryomicroscopy. Gold-labeled insulin served to locate the insulin-binding domain. The 3D structure was then fitted with available known high-resolution domain substructures to obtain a detailed contiguous model for this heterotetrameric transmembrane receptor. The 3D reconstruction indicates that the two alpha subunits jointly participate in insulin binding and that the kinase domains in the two beta subunits are in a juxtaposition that permits autophosphorylation of tyrosine residues in the first step of insulin receptor activation.

The creation of stable cell lines expressing Ebola virus glycoproteins and the matrix protein VP40 and generating Ebola virus-like particles utilizing an ecdysone inducible mammalian expression system.

Ebola virus is a filovirus that causes hemorrhagic fever in humans and is associated with case fatality rates of up to 90%. The lack of therapeutic interventions in combination with the threat of weaponizing this organism has enhanced research investigations. The expression of key viral proteins and the production of virus-like particles in mammalian systems are often pursued for characterization and functional studies. Common practice is to express these proteins through transient transfection of mammalian cells. Unfortunately the transfection reagents are expensive and the process is time consuming and labour intensive. This work describes utilizing an ecdysone inducible mammalian expression system to create stable cell lines that express the Ebola virus transmembrane glycoprotein (GP), the soluble glycoprotein (sGP) and the matrix protein (VP40) individually as well as GP and VP40 simultaneously (for the production of virus like particles). These products were the same as those expressed by the transient system, by Western blot analysis and electron microscopy. The inducible system proved to be an improvement of the current technology by enhancing the cost effectiveness and simplifying the process.

First detection and confirmation of spring viraemia of carp virus in common carp, Cyprinus carpio L., from Hamilton Harbour, Lake Ontario, Canada.

In June 2006, 150 wild common carp were sampled from Hamilton Harbour, Lake Ontario, Canada. Tissue pools consisting of kidney, spleen and encephalon were screened for viruses as a condition facilitating the export of live carp to France. Cytopathic effect (CPE), indicative of a viral infection, became evident after 8 days of incubation at 15 degrees C. Eighteen of 30 tissue pools (five fish per pool) eventually demonstrated viral CPE. The viral pathogen was initially cultured and isolated on the epithelioma papulosum cyprini cell line and subsequently shown to produce CPE in the fathead minnow and bluegill fin cell lines. Electron microscopy demonstrated the virus to be a rhabdovirus. Reverse transcriptase-polymerase chain reaction assay and nucleotide sequence analysis identified the isolate as spring viraemia of carp virus (SVCV). Phylogenetic analysis of a 533 bp region of the glycoprotein gene grouped the Canadian isolate in SVCV genogroup Ia together with isolates from Asia and the USA. Sequence comparisons revealed the Hamilton Harbour, Lake Ontario isolate to be most similar to an isolate obtained from common carp in the Calumet Sag Channel in Illinois in 2003 (98.9% nucleotide identity). This is the first report of the detection of SVCV in Canada.

Structures of small subunit ribosomal RNAs in situ from Escherichia coli and Thermomyces lanuginosus.

Small ribosomal subunits from the prokaryote Escherichia coli and the eukaryote Thermomyces lanuginosus were imaged electron spectroscopically, and single particle analysis used to yield three-dimensional reconstructions of the net phosphorus distribution representing the nucleic acid (RNA) backbone. This direct approach showed both ribosomal RNAs to have a three domain structure and other characteristic morphological features. The eukaryotic small ribosomal subunit had a prominent bill present in the head domain, while the prokaryotic subunit had a small vestigial bill. Both ribosomal subunits contained a thick 'collar' central domain which correlates to the site of the evolutionarily conserved ribosomal RNA core, and the location of the majority of ribosomal RNA bases that have been implicated in translation. The reconstruction of the prokaryotic subunit had a prominent protrusion extending from the collar, forming a channel approximately 1.5 nm wide and potentially representing a 'bridge' to the large subunit in the intact monosome. The basal domain of the prokaryotic ribosomal subunit was protein free. In this region of the eukaryotic subunit, there were two basal lobes composed of ribosomal RNA, consistent with previous hypotheses that this is a site for the 'non-conserved core' ribosomal RNA.

Does the eukaryotic large ribosomal subunit have a channel passing through it?

The large ribosomal subunit of the thermophilic fungus Thermomyces lanuginosus was treated with 2.96 M NH4Cl to remove specific complements of ribosomal proteins, and the core particles thereby derived were imaged by bright field transmission electron microscopy, and recurring views computed by single particle electron image analysis. A new characteristic projection was elucidated which showed a large depression or channel passing through the subunit. Such a channel has been perceived in the prokaryotic large ribosomal subunit under certain conditions and has been postulated to be the exit pathway for the nascent polypeptide chain, but its existence has not hitherto been demonstrated in eukaryotes.

Ribosomal proteins of Thermomyces lanuginosus--characterisation by two-dimensional gel electrophoresis and differential disassembly.

One- and two-dimensional gel electrophoresis were employed to characterise the proteins derived from the ribosomes of the thermophilic fungus Thermomyces lanuginosus. Approximately 32 (29 basic and 3 acidic) and 45 (43 basic and 2 acidic) protein spots were resolved from Th. lanuginosus small and large ribosomal subunits, respectively. The molecular weight of the small subunit proteins ranged from 9,800-36,000 Da with a number average molecular weight of 20,300 Da. The molecular weight range for the large subunit proteins was 12,000-48,500 Da with a number average molecular weight of 25,900 Da. Most proteins appeared to be present in unimolar amounts. These data are comparable with but not identical to those from other eukaryotic ribosomes. The sensitivities of the ribosomal proteins to increasing concentrations of NH4Cl were also evaluated by two-dimensional gel electrophoresis. Most ribosomal proteins were gradually released over a wide range of salt concentrations but some were preferentially enriched in one or two salt conditions.

Three-dimensional structure of myelin basic protein. II. Molecular modeling and considerations of predicted structures in multiple sclerosis.

A computational model of myelin basic protein (MBP) has been constructed based on the premise of a phylogenetically conserved beta-sheet backbone and on electron microscopical three-dimensional reconstructions. Many residues subject to post-translational modification (phosphorylation, methylation, or conversion of arginines to citrullines) were located in loop regions and thus accessible to modifying enzymes. The triproline segment (residues 99-101) is fully exposed on the back surface of the protein in a long crossover connection between two parallel beta-strands. The proximity of this region to the underlying beta-sheet suggests that post-translational modifications here might have potential synergistic effects on the entire structure. Post-translational modifications that lead to a reduced surface charge could result first in a weakened attachment to the myelin membrane rather than in a gross conformational change of the protein itself. Such mechanisms could be operative in demyelinating diseases such as multiple sclerosis.

Mechanism of transmembrane signaling: insulin binding and the insulin receptor.

Transmembrane signaling via receptor tyrosine kinases generally requires oligomerization of receptor monomers, with the formation of ligand-induced dimers or higher multimers of the extracellular domains of the receptors. Such formations are expected to juxtapose the intracellular kinase domains at the correct distances and orientations for transphosphorylation. For receptors of the insulin receptor family that are constitutively dimeric, or those that form noncovalent dimers without ligands, the mechanism must be more complex. For these, the conformation must be changed by the ligand from one that prevents activation to one that is permissive for kinase phosphorylation. How the insulin ligand accomplishes this action has remained a puzzle since the discovery of the insulin receptor over 2 decades ago, primarily because membrane proteins in general have been refractory to structure determination by crystallography. However, high-resolution structural evidence on individual separate subdomains of the insulin receptor and of analogous proteins has been obtained. The recently solved quaternary structure of the complete dimeric insulin receptor in the presence of insulin has now served as the structural envelope into which such individual domains were fitted. The combined structure has provided answers on the details of insulin/receptor interactions in the binding site and on the mechanism of transmembrane signaling of this covalent dimer. The structure explains many observations on the behavior of the receptor, from greater or lesser binding of insulin and its variants, point and deletion mutants of the receptor, to antibody-binding patterns, and to the effects on basal and insulin-stimulated autophosphorylation under mild reducing conditions.

The in situ architecture of Escherichia coli ribosomal RNA derived by electron spectroscopic imaging and three-dimensional reconstruction.

The structures of the large and small ribosomal subunits of Escherichia coli were reconstructed using spectroscopic electron microscopy and quaternion-assisted angular reconstitution to resolutions of better than 4 nm. In addition, the distributions of phosphorus within these complexes were reconstructed. The three-dimensional reconstruction of the distribution of this atomic element is an extension of microanalysis (in two dimensions) for phosphorus identification and mapping, as a signature of the arrangement of the phosphate backbones of the constituent ribosomal RNAs. The results on both the phosphorus reconstructions and the total reconstructions (protein and ribosomal RNA) reveal several passageways through both subunits. The structures correspond favourably with other independent reconstructions of the whole E. coli ribosome from cryoelectron micrographs and their accompanying models of translation (Frank et al., Nature, 376, 441-444, 1995; Stark et al., Structure, 3, 815-821, 1995). The overall reconstructions in conjunction with the phosphorus (rRNA) distributions are the first to be achieved synchronously for this nucleoprotein complex.

Electron spectroscopic imaging of encapsidated DNA in vaccinia virus.

We have used electron spectroscopic imaging to locate the phosphorus in vaccinia DNA in situ in unstained, ultrathin sections of virions. The phosphorus of the DNA backbone appeared to form a halo on the core periphery surrounding a phosphorus-impoverished central element. These results constrain models for how DNA could be packaged into mature vaccinia particles.

Cryoelectron microscopy of protein-lipid complexes of human myelin basic protein charge isomers differing in degree of citrullination.

Myelin basic protein (MBP) is considered to be essential for the maintenance of stability of the myelin sheath. Reduction in cationicity of MBP, especially due to conversion of positively charged arginine residues to uncharged citrulline (Cit), has been found to be associated with multiple sclerosis (MS). Here, the interactions of an anionic phosphatidylserine/monosialoganglioside-G(M1) (4:1, w:w) lipid monolayer with 18.5-kDa MBP preparations from age-matched adult humans without MS (no Cit residues), with chronic MS (6 Cit), and with acute Marburg-type MS (18 Cit) were studied by transmission and ultralow dose scanning transmission electron microscopy under cryogenic conditions. Immunogold labeling and single particle electron crystallography were used to define the nature of the complexes visualized. These electron microscopical analyses showed that the three different MBP charge isomers all formed uniformly sized and regularly shaped protein-lipid complexes with G(M1), probably as hexamers, but exhibited differential association with and organization of the lipid. The least cationic Marburg MBP-Cit(18) formed the most open protein-lipid complex. The data show a disturbance in lipid-MBP interactions at the ultrastructural level that is related to degree of citrullination, and which may be involved in myelin degeneration in multiple sclerosis.

Marburg's variant of multiple sclerosis correlates with a less compact structure of myelin basic protein.

Multiple sclerosis (MS) is an autoimmune disease in which the myelin sheath of the central nervous system is degraded, and the 18.5 kDa isoform of myelin basic protein (MBP) is reduced in cationicity. In a unique case of acute, fulminating MS (Marburg's variant), MBP is considerably less cationic than MBP from both normal, and chronic MS-afflicted individuals. This electron microscopical study has identified that, in vitro, the less cationic Marburg MBP isomer forms a more extended protein-lipid complex than MBP from healthy or chronic MS-afflicted individuals. This correlation implies that chemical modifications to MBP in vivo contribute directly to the structural instability of myelin, and subsequent autoantigenic presentation of this protein, observed in vivo in MS.

Three-dimensional structure of myelin basic protein. I. Reconstruction via angular reconstitution of randomly oriented single particles.

Myelin basic protein (MBP) plays an integral role in the structure and function of the myelin sheath. In humans and cattle, an 18.5-kDa isoform of MBP predominates and exists as a multitude of charge isomers resulting from extensive and varied post-translational modifications. We have purified the least modified isomer (named C1) of the 18.5-kDa isoform of MBP from fresh bovine brain and imaged this protein as negatively stained single particles adsorbed to a lipid monolayer. Under these conditions, MBP/C1 presented diverse projections whose relative orientations were determined using an iterative quaternion-assisted angular reconstitution scheme. In different buffers, one with a low salt and the other with a high salt concentration, the conformation of the protein was slightly different. In low salt buffer, the three-dimensional reconstruction, solved to a resolution of 4 nm, had an overall "C" shape of outer radius 5.5 nm, inner radius 3 nm, overall circumference 15 nm, and height 4.7 nm. The three-dimensional reconstruction of the protein in high salt buffer, solved to a resolution of 2.8 nm, was essentially the same in terms of overall dimensions but had a somewhat more compact architecture. These results are the first structures achieved directly for this unusual macromolecule, which plays a key role in the development of multiple sclerosis.