Molecular and behavioural abnormalities in the FUS‐tg mice mimic frontotemporal lobar degeneration: Effects of old and new anti‐inflammatory therapies

Genetic mutations in FUS, a DNA/RNA‐binding protein, are associated with inherited forms of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). A novel transgenic FUS[1‐359]‐tg mouse line recapitulates core hallmarks of human ALS in the spinal cord, including neuroinflammation and neurodegeneration, ensuing muscle atrophy and paralysis, as well as brain pathomorphological signs of FTLD. However, a question whether FUS[1‐359]‐tg mouse displays behavioural and brain pro‐inflammatory changes characteristic for the FTLD syndrome was not addressed. Here, we studied emotional, social and cognitive behaviours, brain markers of inflammation and plasticity of pre‐symptomatic FUS[1‐359]‐tg male mice, a potential FTLD model. These animals displayed aberrant behaviours and altered brain expression of inflammatory markers and related pathways that are reminiscent to the FTLD‐like syndrome. FTLD‐related behavioural and molecular Journal of Cellular and Molecular Medicine features were studied in the pre‐symptomatic FUS[1‐359]‐tg mice that received standard or new ALS treatments, which have been reported to counteract the ALS‐like syndrome in the mutants. We used anti‐ALS drug riluzole (8 mg/kg/d), or anti‐inflammatory drug, a selective blocker of cyclooxygenase‐2 (celecoxib, 30 mg/kg/d) for 3 weeks, or a single intracerebroventricular (i.c.v.) infusion of human stem cells (Neuro‐Cells, 500 000‐CD34⁺), which showed anti‐inflammatory properties. Signs of elevated anxiety, depressive‐like behaviour, cognitive deficits and abnormal social behaviour were less marked in FUS‐tg–treated animals. Applied treatments have normalized protein expression of interleukin‐1β (IL‐1β) in the prefrontal cortex and the hippocampus, and of Iba‐1 and GSK‐3β in the hippocampus. Thus, the pre‐symptomatic FUS[1‐359]‐tg mice demonstrate FTLD‐like abnormalities that are attenuated by standard and new ALS treatments, including Neuro‐Cell preparation.     

Chloroplast Genome of Native Silene latifolia subsp. alba from Fennoscandia Shows High Level of Differences from Invasive White Campion

Silene latifolia is an herbaceous plant with great invasive potential. Spread along trade routes from Europe to almost all continents, white campion became particularly widespread in North America. We sequenced the chloroplast genome of S. latifolia subsp. alba from a native range in southeast Fennoscandia. The chloroplast genome of native S. latifolia subsp. alba forms a 151,747-bp circle, has two inverted repeat regions (25,993 bp each), large single copy (82,708 bp), and small single copy (17,106 bp) regions. It contains 77 protein-coding genes, 30 tRNA genes, and four rRNA genes. SSRs and long DNA repeats were identified. Comparison of a newly sequenced plastome of S. latifolia subsp. alba with plastomes of invasive specimens of species from North America and Japan revealed a high level of single nucleotide polymorphisms (SNPs) among them. A total of 214 SNPs were found, among which 110 were identified in intergenic spacers, 74 in exons, and 30 in introns. Intraspecific shifts in inverted repeat boundaries were identified. Our research suggests that high polymorphic regions may be potential molecular markers for population studies and that high intraspecific genetic polymorphism may contribute to a species’ invasive success.

     

Does Interaction between the Motor and Regulatory Domains of the Myosin Head Occur during ATPase Cycle? Evidence from Thermal Unfolding Studies on Myosin Subfragment 1

Myosin head (myosin subfragment 1, S1) consists of two major structural domains, the motor (or catalytic) domain and the regulatory domain. Functioning of the myosin head as a molecular motor is believed to involve a rotation of the regulatory domain (lever arm) relative to the motor domain during the ATPase cycle. According to predictions, this rotation can be accompanied by an interaction between the motor domain and the C-terminus of the essential light chain (ELC) associated with the regulatory domain. To check this assumption, we applied differential scanning calorimetry (DSC) combined with temperature dependences of fluorescence to study changes in thermal unfolding and the domain structure of S1, which occur upon formation of the ternary complexes S1-ADP-AlF₄ ^- and S1-ADP-BeF_x that mimic S1 ATPase intermediate states S1**-ADP-P_i and S1*-ATP, respectively. To identify the thermal transitions on the DSC profiles (i.e. to assign them to the structural domains of S1), we compared the DSC data with temperature-induced changes in fluorescence of either tryptophan residues, located only in the motor domain, or recombinant ELC mutants (light chain 1 isoform), which were first fluorescently labeled at different positions in their C-terminal half and then introduced into the S1 regulatory domain. We show that formation of the ternary complexes S1-ADP-AlF₄ ^- and S1-ADP-BeF_x significantly stabilizes not only the motor domain, but also the regulatory domain of the S1 molecule implying interdomain interaction via ELC. This is consistent with the previously proposed concepts and also adds some new interesting details to the molecular mechanism of the myosin ATPase cycle.     

Regulation of the tetrapyrrole biosynthetic pathway leading to heme and chlorophyll in plants and cyanobacteria

Photosynthetic organisms synthesize chlorophylls, hemes, and bilin pigments via a common tetrapyrrole biosynthetic pathway. This review summarizes current knowledge about the regulation of this pathway in plants, algae, and cyanobacteria. Particular emphasis is placed on the regulation of glutamate-1-semialdehyde formation and on the channelling of protoporphyrin IX into the heme and chlorophyll branches. The potential role of chlorophyll molecules that are not bound to photosynthetic pigment-protein complexes ('free chlorophylls') or of other Mg-containing porphyrins in regulation of tetrapyrrole synthesis is also discussed.     

Mutations in the relay loop region result in dominant-negative inhibition of myosin II function in Dictyostelium

Dominant-negative inhibition is a powerful genetic tool for the characterization of gene function in vivo, based on the specific impairment of a gene product by the coexpression of a mutant version of the same gene product. We describe the detailed characterization of two myosin constructs containing either point mutations F487A or F506G in the relay region. Dictyostelium cells transformed with F487A or F506G myosin are unable to undergo processes that require myosin II function, including fruiting-body formation, normal cytokinesis and growth in suspension. Our results show that the dominant-negative inhibition of myosin function is caused by disruption of the communication between active site and lever arm, which blocks motor activity completely, and perturbation of the communication between active site and actin-binding site, leading to an ~100-fold increase in the mutants' affinity for actin in the presence of ATP.     

A new approach to revealing point mutations in DNA analyzed by colorimetric detection

A new approach to detection of point mutations in an amplified DNA was developed. The approach is based on highly selective ligation (T4 DNA ligase) of a tandem of short oligonucleotides one of which contains the biotin group. The ligation product is formed only when the hybridization complex DNA/tandem is formed and the tandem is perfect. The hybridization complex DNA/(biotinylated ligation product) was separated from the biotinylated component of the tandem by UV-immobilization of the reaction mixture on a nylon membrane. The immobilized hybridization complex was detected colorimetrically by a streptavidin-alkaline phosphatase cojugate with a chromogenic substrate.     

Nonradical mechanism of (bi)sulfite reaction with DEPMPO: cautionary note for SO3*- radical spin trapping

Knowledge of the formation of radicals from sulfites, in vivo, is of interest in understanding the allergic and inflammatory responses to environmental sulfur dioxide exposure. Sulfite anion trioxide (SO(3)(-*)) radical formation was measured in mice, preloaded with the spin trap, 5-(diethoxy-phosphoryl)-5-methyl-pyrrolidine-N-oxide, (DEPMPO). Based on spin trapping NMR, a surprising quantity of reduced SO(3)(-*)-adduct was observed that did not depend on co-administration of oxidizing agents, suggesting a possible nonradical reaction between (bi)sulfite and DEPMPO. The products of the reversible nucleophylic addition of (bi)sulfite to the nitrone functional group were identified using (31)P-NMR, (1)H-NMR, and (13)C-NMR spectroscopy as cis- and trans- stereoisomers of hydroxylamine and confirmed by quantum chemical calculations. Oxidation of the hydroxylamines results in the formation of two corresponding cis- and trans-isomeric nitroxides, only one of which has been earlier described as the paramagnetic adduct of genuinely trapped SO(3)(-*) radical. The results demonstrate that SO(3)(-*) detection using nitrone spin traps such as DEPMPO and DMPO may involve nonradical addition reactions except in cases when the required controls unambiguously prove a radical mechanism.     

Small heat shock protein Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin

Previously, we have shown that the small heat shock protein with apparent molecular mass 27 kDa (Hsp27) does not affect the thermal unfolding of F-actin, but effectively prevents aggregation of thermally denatured F-actin [Pivovarova AV, Mikhailova VV, Chernik IS, Chebotareva NA, Levitsky DI & Gusev NB (2005) Biochem Biophys Res Commun331, 1548-1553], and supposed that Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin. In the present work, we applied dynamic light scattering, analytical ultracentrifugation and size exclusion chromatography to examine the properties of complexes formed by denatured actin with a recombinant human Hsp27 mutant (Hsp27-3D) mimicking the naturally occurring phosphorylation of this protein at Ser15, Ser78, and Ser82. Our results show that formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. All the methods show that the size of actin-Hsp27-3D complexes decreases with increasing Hsp27-3D concentration in the incubation mixture and that saturation occurs at approximately equimolar concentrations of Hsp27-3D and actin. Under these conditions, the complexes exhibit a hydrodynamic radius of approximately 16 nm, a sedimentation coefficient of 17-20 S, and a molecular mass of about 2 MDa. It is supposed that Hsp27-3D binds to denatured actin monomers or short oligomers dissociated from actin filaments upon heating and protects them from aggregation by forming relatively small and highly soluble complexes. This mechanism might explain how small heat shock proteins prevent aggregation of denatured actin and by this means protect the cytoskeleton and the whole cell from damage caused by accumulation of large insoluble aggregates under heat shock conditions.     

Phycobilin/chlorophyll excitation equilibration upon carotenoid-induced non-photochemical fluorescence quenching in phycobilisomes of the cyanobacterium Synechocystis sp. PCC 6803

To determine the mechanism of carotenoid-sensitized non-photochemical quenching in cyanobacteria, the kinetics of blue-light-induced quenching and fluorescence spectra were studied in the wild type and mutants of Synechocystis sp. PCC 6803 grown with or without iron. The blue-light-induced quenching was observed in the wild type as well as in mutants lacking PS II or IsiA confirming that neither IsiA nor PS II is required for carotenoid-triggered fluorescence quenching. Both fluorescence at 660 nm (originating from phycobilisomes) and at 681 nm (which, upon 440 nm excitation originates mostly from chlorophyll) was quenched. However, no blue-light-induced changes in the fluorescence yield were observed in the apcE⁻ mutant that lacks phycobilisome attachment. The results are interpreted to indicate that interaction of the Slr1963-associated carotenoid with - presumably - allophycocyanin in the phycobilisome core is responsible for non-photochemical energy quenching, and that excitations on chlorophyll in the thylakoid equilibrate sufficiently with excitations on allophycocyanin in wild type to contribute to quenching of chlorophyll fluorescence.