Membrane association induces a conformational change in the Ebola virus matrix protein

The matrix protein VP40 from Ebola virus is targeted to the plasma membrane, where it is thought to induce assembly and budding of virions through its association with the lipid bilayer. Ebola virus VP40 is expressed as a monomeric molecule in solution, consisting of two loosely associated domains. Here we show that a C-terminal truncation of seven residues destabilizes the monomeric closed conformation and induces spontaneous hexamerization in solution, as indicated by chemical cross-linking and electron microscopy. Three-dimensional reconstruction of electron microscopy images shows ring-like structures consisting of the N-terminal domain along with evidence for flexibly attached C-terminal domains. In vitro destabilization of the monomer by urea treatment results in similar hexameric molecules in solution. In addition, we demonstrate that membrane association of wild-type VP40 also induces the conformational switch from monomeric to hexameric molecules that may form the building blocks for initiation of virus assembly and budding. Such a conformational change induced by bilayer targeting may be a common feature of many viral matrix proteins and its potential inhibition may result in new anti-viral therapies.     

Genomic structure and fine mapping of the two human filamin gene paralogues FLNB and FLNC and comparative analysis of the filamin gene family

The genomic structure of the filamin gene paralogues FLNB and FLNC was determined and related to FLNA. FLNB consists of 45 exons and 44 introns and spans approximately 80 kb of genomic DNA. FLNC is divided into 48 exons and 47 introns and covers approximately 29.5 kb of genomic DNA. A previously unknown intron was found in FLNA. The comparison of all three filamin gene paralogues revealed a highly conserved exon-intron structure with significant differences in the exons 32 of all paralogues encoding the hinge I region, as well as the insertion of a novel exon 40A in FLNC only. Gene organization does not correlate with the domain structures of the respective proteins. To improve candidate gene cloning approaches, FLNB was precisely mapped at 3p14 in an interval of 0.81 cM between WI3771 and WI6691 and FLNC at 7q32 in an interval of 2.07 cM between D7S530 and D7S649.     

Structural and functional differences of two toxins from the scorpion Pandinus imperator

The Pandinotoxins, PiTX-K alpha and PiTX-K beta, are members of the Charybdotoxin family of scorpion toxins that can be used to characterize K+ channels. PiTX-K alpha differs from PiTX-K beta, another peptide from Pandinus imperator, by one residue (P10E). When the two toxins are compared in a physiological assay, the affinity of PiTX-K beta for voltage-gated, rapidly inactivating K+ channels in dorsal root ganglia (DRG) neurons is 800-fold lower than that of PiTX-K alpha (K alpha-IC50 = 8.0 nM versus K beta-IC50 = 6,500 nM). To understand this difference, the three-dimensional structure of PiTX-K beta was determined by nuclear magnetic resonance (NMR) spectroscopy and compared to that of PiTX-K alpha. This comparison shows that structural differences between the two toxins occur at a residue that is critical for blocking K+ channels (K27) as well as at the site of the natural mutation (P10E). In PiTX-K beta, the negatively charged carboxylate oxygen of E10 can approach the positive charge of K27 and presumably reduces the net positive charge in this region of the toxin. This is likely the reason why PiTX-K beta binds K+ channels from DRG neurons with a much lower affinity than does PiTX-K alpha.     

Enantioselective Uptake and Degradation of the Chiral Herbicide Dichlorprop [(RS)-2-(2,4-Dichlorophenoxy)propanoic acid] by Sphingomonas herbicidovorans MH

Sphingomonas herbicidovorans MH was able to completely degrade both enantiomers of the chiral herbicide dichlorprop [(RS)-2-(2,4-dichlorophenoxy)propanoic acid], with preferential degradation of the (S) enantiomer over the (R) enantiomer. These results are in agreement with the recently reported enantioselective degradation of mecoprop [(RS)-2-(4-chloro-2-methylphenoxy)propanoic acid] by this bacterium (C. Zipper, K. Nickel, W. Angst, and H.-P. E. Kohler, Appl. Environ. Microbiol. 62:4318–4322, 1996). Uptake of (R)-dichlorprop, (S)-dichlorprop, and 2,4-D (2,4-dichlorophenoxyacetic acid) was inducible. Initial uptake rates of cells grown on the respective substrate showed substrate saturation kinetics with apparent affinity constants (K_t) of 108, 93, and 117 μM and maximal velocities (V_max) of 19, 10, and 21 nmol min⁻¹ mg of protein⁻¹ for (R)-dichlorprop, (S)-dichlorprop, and 2,4-D, respectively. Transport of (R)-dichlorprop, (S)-dichlorprop, and 2,4-D was completely inhibited by various uncouplers and by nigericin but was only marginally inhibited by valinomycin and by the ATPase inhibitor N,N′-dicyclohexylcarbodiimine. Experiments on the substrate specificity of the putative transport systems revealed that (R)-dichlorprop uptake was inhibited by (R)-mecoprop but not by (S)-mecoprop, (S)-dichlorprop, or 2,4-D. On the other hand, the (S)-dichlorprop transport was inhibited by (S)-mecoprop but not by (R)-mecoprop, (R)-dichlorprop, or 2,4-D. These results provide evidence that the first step in the degradation of dichlorprop, mecoprop, and 2,4-D by S. herbicidovorans is active transport and that three inducible, proton gradient-driven uptake systems exist: one for (R)-dichlorprop and (R)-mecoprop, another for (S)-dichlorprop and (S)-mecoprop, and a third for 2,4-D.     

Regulation of Riboflavin Biosynthesis in Bacillus subtilis Is Affected by the Activity of the Flavokinase/Flavin Adenine Dinucleotide Synthetase Encoded by ribC

This work shows that the ribC wild-type gene product has both flavokinase and flavin adenine dinucleotide synthetase (FAD-synthetase) activities. RibC plays an essential role in the flavin metabolism of Bacillus subtilis, as growth of a ribC deletion mutant strain was dependent on exogenous supply of FMN and the presence of a heterologous FAD-synthetase gene in its chromosome. Upon cultivation with growth-limiting amounts of FMN, this ribC deletion mutant strain overproduced riboflavin, while with elevated amounts of FMN in the culture medium, no riboflavin overproduction was observed. In a B. subtilis ribC820 mutant strain, the corresponding ribC820 gene product has reduced flavokinase/FAD-synthetase activity. In this strain, riboflavin overproduction was also repressed by exogenous FMN but not by riboflavin. Thus, flavin nucleotides, but not riboflavin, have an effector function for regulation of riboflavin biosynthesis in B. subtilis, and RibC seemingly is not directly involved in the riboflavin regulatory system. The mutation ribC820 leads to deregulation of riboflavin biosynthesis in B. subtilis, most likely by preventing the accumulation of the effector molecule FMN or FAD.     

Eye Movement Deficits Are Consistent with a Staging Model of pTDP-43 Pathology in Amyotrophic Lateral Sclerosis

Background

The neuropathological process underlying amyotrophic lateral sclerosis (ALS) can be traced as a four-stage progression scheme of sequential corticofugal axonal spread. The examination of eye movement control gains deep insights into brain network pathology and provides the opportunity to detect both disturbance of the brainstem oculomotor circuitry as well as executive deficits of oculomotor function associated with higher brain networks.

Objective

To study systematically oculomotor characteristics in ALS and its underlying network pathology in order to determine whether eye movement deterioration can be categorized within a staging system of oculomotor decline that corresponds to the neuropathological model.

Methods

Sixty-eight ALS patients and 31 controls underwent video-oculographic, clinical and neuropsychological assessments.

Results

Oculomotor examinations revealed increased anti- and delayed saccades’ errors, gaze-palsy and a cerebellary type of smooth pursuit disturbance. The oculomotor disturbances occurred in a sequential manner: Stage 1, only executive control of eye movements was affected. Stage 2 indicates disturbed executive control plus ‘genuine’ oculomotor dysfunctions such as gaze-paly. We found high correlations (p<0.001) between the oculomotor stages and both, the clinical presentation as assessed by the ALS Functional Rating Scale (ALSFRS) score, and cognitive scores from the Edinburgh Cognitive and Behavioral ALS Screen (ECAS).

Conclusions

Dysfunction of eye movement control in ALS can be characterized by a two-staged sequential pattern comprising executive deficits in Stage 1 and additional impaired infratentorial oculomotor control pathways in Stage 2. This pattern parallels the neuropathological staging of ALS and may serve as a technical marker of the neuropathological spreading.     

Streptomonospora algeriensis sp. nov., a halophilic actinomycete isolated from soil in Algeria

A halophilic actinomycete strain, designated H27^T, was isolated from a soil sample collected from a hypersaline habitat in Djelfa Province (North-Central Algeria), and then investigated using a polyphasic taxonomic approach. The strain was observed to produce poor aerial mycelium, which formed short chains of oval to cylindrical-shaped spores at maturity, and non fragmented substrate mycelium. The optimum NaCl concentration for growth was found to be 10–15 % (w/v) and the optimum growth temperature and pH were found to be 28–37 °C and 6–7, respectively. The diagnostic diamino acid in the cell-wall peptidoglycan was identified as meso-diaminopimelic acid. The predominant menaquinones of strain H27^T were identified as MK-11 (H₄) and MK-10 (H₆). The major fatty acids were found to be iso-C_16:0, anteiso-C_17:0, 10 methyl C_17:0 and 10 methyl C_16:0. The diagnostic phospholipids detected were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and phosphatidylinositol. The chemotaxonomic properties of strain H27^T are consistent with those shared by members of the genus Streptomonospora. 16S rRNA gene sequence analysis indicated that strain H27^T is most closely related to Streptomonospora alba DSM 44588^T (98.8 %) and Streptomonospora flavalba DSM 45155^T (98.7 %) whereas the DNA–DNA relatedness values between strain H27^T and the two type strains were 17.1 and 57.9 %, respectively. Based on the combined genotypic and phenotypic evidence, it is proposed that strain H27^T should be classified as representative of a novel species, for which the name Streptomonospora algeriensis sp. nov. is proposed. The type strain is H27^T (=DSM 45604^T =CCUG 63369^T =MTCC 11563^T).     

Mzabimyces algeriensis gen. nov., sp. nov., a halophilic filamentous actinobacterium isolated from a Saharan soil,and proposal of Mzabimycetaceae fam. nov.

Three halophilic mycelium-forming actinobacteria, strains H195^T, H150 and H151, were isolated from a Saharan soil sample collected from Béni-isguen in the Mzab region (Ghardaïa, South of Algeria) and subjected to a polyphasic taxonomic characterisation. These strains were observed to show an aerial mycelium differentiated into coccoid spore chains and fragmented substrate mycelium. Comparative analysis of the 16S rRNA gene sequences revealed that the highest sequence similarities were to Saccharopolyspora qijiaojingensis YIM 91168^T (92.02 % to H195^T). Phylogenetic analyses showed that the strains H195^T, H150 and H151 represent a distinct phylogenetic lineage. The cell-wall hydrolysate was found to contain meso-diaminopimelic acid, and the diagnostic whole-cell sugars were identified as arabinose and galactose. The major cellular fatty acids were identified as iso-C_15:0, iso-C_16:0, iso-C_17:0 and anteiso-C_17:0. The diagnostic phospholipid detected was phosphatidylcholine and MK-9 (H₄) was found to be the predominant menaquinone. The genomic DNA G+C content of strain H195^T was 68.2 mol%. On the basis of its phenotypic features and phylogenetic position, we propose that strain H195^T represents a novel genus and species, Mzabimyces algeriensis gen. nov., sp. nov., within a new family, Mzabimycetaceae fam. nov. The type strain of M. algeriensis is strain H195^T (=DSM 46680^T = MTCC 12101^T).     

Nocardia vulneris sp. nov., isolated from wounds of human patients in North America

Nocardia species are ubiquitous in the environment with an increasing number of species isolated from clinical sources. From 2005 to 2009, eight isolates (W9042, W9247, W9290, W9319, W9846, W9851^T, W9865, and W9908) were obtained from eight patients from three states in the United States and Canada; all were from males ranging in age from 47 to 81 years old; and all were obtained from finger (n = 5) or leg (n = 3) wounds. Isolates were characterized by polyphasic analysis using molecular, phenotypic, morphologic and chemotaxonomic methods. Sequence analysis of 16S rRNA gene sequences showed the eight isolates are 100 % identical to each other and belong in the genus Nocardia. The nearest phylogenetically related neighbours were found to be the type strains for Nocardia altamirensis (99.33 % sequence similarity), Nocardia brasiliensis (99.37 %), Nocardia iowensis (98.95 %) and Nocardia tenerifensis (98.44 %). The G+C content of isolate W9851^T was determined to be 68.4 mol %. The DNA–DNA relatedness between strain W9851^T and the N. brasiliensis type strain was 72.8 % and 65.8 % when measured in the laboratory and in silico from genome sequences, respectively, and 95.6 % ANI. Whole-cell peptidoglycan was found to contain meso-diaminopimelic acid; MK-8-(H₄)_ω-cyc was identified as the major menaquinone; the major fatty acids were identified as C_16:0, 10 Me C_18:0, and C_{18:1 w9c}, the predominant phospholipids were found to include diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides; whole-cell sugars detected were arabinose and galactose; and mycolic acids ranging from 38 to 60 carbon atoms were found to be present. These chemotaxonomic analyses are consistent with assignment of the isolates to the genus Nocardia. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectra of the clinical isolates showed genus and species level profiles that were different from other Nocardia species. All isolates were resistant to ciprofloxacin, clarithromycin and imipenem but were susceptible to amikacin, amoxicillin/clavulanate, linezolid and trimethoprim/sulfamethoxazole. The results of our polyphasic analysis suggest the new isolates obtained from wound infections represent a novel species within the genus Nocardia, for which the name Nocardia vulneris sp. nov. is proposed, with strain W9851^T (= DSM 45737^T = CCUG 62683^T = NBRC 108936^T) as the type strain.     

Inhibition of oxidative stress-induced amyloid β formation in NT2 neurons by culture filtrate of a strain of Streptomyces antibioticus

Actinomycetes isolated from Iran soil habitats were tested for the capacity to produce compounds which can protect neurons from cell death generated by oxidative stress in NT2 neurons. Confirmation of our initial hit was accomplished via the determination of amyloid β level using the enzyme-linked immunosorbent assay test. The most interesting amyloid β formation inhibitor discovered in our study was a secondary metabolite which was produced by strain HM45. This bioactive strain was identified as a strain of Streptomyces antibioticus DSM 40234 using polyphasic approach. The strain HM45 was deposited in Deutsche Sammlung von Mikroorganismen und Zellkulturen as S. antibioticus DSM 41955 and University of Tehran Microorganisms Sollection as S. antibioticus UTMC 00105. This work is the first report on efficiency of an actinomycete metabolite in prohibition of neurons death caused by amyloid β formation.