Analysis of Nuclear Export Sequence Regions of FUS-Related RNA-Binding Proteins in Essential Tremor

Background and Objective

Genes encoding RNA-binding proteins, including FUS and TDP43, play a central role in different neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Recently, a mutation located in the nuclear export signal (NES) of the FUS gene has been reported to cause an autosomal dominant form of familial Essential tremor.

Material and Methods

We sequenced the exons coding the NES domains of five RNA-binding proteins (TARDBP, hnRNPA2B1, hnRNPA1, TAF15 and EWSR1) that have been previously implicated in neurodegeneration in a series of 257 essential tremor (ET) cases and 376 healthy controls. We genotyped 404 additional ET subjects and 510 healthy controls to assess the frequency of the EWSR1 p.R471C substitution.

Results

We identified a rare EWSR1 p.R471C substitution, which is highly conserved, in a single subject with familial ET. The pathogenicity of this substitution remains equivocal, as DNA samples from relatives were not available and the genotyping of 404 additional ET subjects did not reveal any further carriers. No other variants were observed with significant allele frequency differences compared to controls in the NES coding regions.

Conclusions

The present study demonstrates that the NES domains of RNA-binding proteins are highly conserved. The role of the EWSR1 p.R471C substitution needs to be further evaluated in future studies.     

Immune mobilization of autologous blood progenitor cells: direct influence on the cellular subsets collected

Background aimsA phase I trial examined the ability of immunotherapy to mobilize progenitor and activated T cells.MethodsInterleukin (IL)-2 was administered subcutaneously for 11 days, with granulocyte (G)-colony-stimulating factor (CSF) (5 mcg/kg/day) and granulocyte–macrophage (GM)-CSF (7.5 mcg/kg/day) added for the last 5 days. Leukapheresis was initiated on day 11. Thirteen patients were treated (myeloma n = 11, non-Hodgkin's lymphoma n = 2).ResultsToxicities were minimal. IL-2 was stopped in two patients because of capillary leak (n = 1) and diarrhea (n = 1). Each patient required 2.5 leukaphereses (median; range 1–3) to collect 3.2 × 10⁶ CD34⁺ cells/kg (median; range 1.9–6.6 × 10⁶/kg). Immune mobilization increased the number of CD3⁺ CD8⁺ T cells (P = 0.002), CD56⁺ natural killer (NK) cells (P = 0.0001), CD8⁺ CD56⁺ T cells (P = 0.002) and CD4⁺ CD25⁺ cells (P = 0.0001) compared with cancer patients mobilized with G-CSF alone. There was increased lysis of myeloma cells after 7 days (P = 0.03) or 11 days (P = 0.02). The maximum tolerated dose of IL-2 was 1 × 10⁶ IU/m²/day.ConclusionsImmune mobilization is well tolerated with normal subsequent marrow engraftment. As cells within the graft influence lymphocyte recovery, an increased number of functional lymphocytes may result in more rapid immune reconstitution.     

Enzymatically stable 5' mRNA cap analogs: synthesis and binding studies with human DcpS decapping enzyme

Four novel 5' mRNA cap analogs have been synthesized with one of the pyrophosphate bridge oxygen atoms of the triphosphate linkage replaced with a methylene group. The analogs were prepared via reaction of nucleoside phosphor/phosphon-1-imidazolidates with nucleoside phosphate/phosphonate in the presence of ZnCl2. Three of the new cap analogs are completely resistant to degradation by human DcpS, the enzyme responsible for hydrolysis of free cap resulting from 3' to 5' cellular mRNA decay. One of the new analogs has very high affinity for binding to human DcpS. Two of these analogs are Anti Reverse Cap Analogs which ensures that they are incorporated into mRNA chains exclusively in the correct orientation. These new cap analogs should be useful in a variety of biochemical studies, in the analysis of the cellular function of decapping enzymes, and as a basis for further development of modified cap analogs as potential anti-cancer and anti-parasite drugs.     

Detection of the change point in oxygen uptake during an incremental exercise test using recursive residuals: relationship to the plasma lactate accumulation and blood acid base balance

The purpose of this study was to develop a method to determine the power output at which oxygen uptake (V˙O₂) during an incremental exercise test begins to rise non-linearly. A group of 26 healthy non-smoking men [mean age 22.1 (SD 1.4) years, body mass 73.6 (SD 7.4) kg, height 179.4 (SD 7.5) cm, maximal oxygen uptake (V˙O_2max) 3.726 (SD 0.363) l · min⁻¹], experienced in laboratory tests, were the subjects in this study. They performed an incremental exercise test on a cycle ergometer at a pedalling rate of 70 rev · min⁻¹. The test started at a power output of 30 W, followed by increases amounting to 30 W every 3 min. At 5 min prior to the first exercise intensity, at the end of each stage of exercise protocol, blood samples (1 ml each) were taken from an antecubital vein. The samples were analysed for plasma lactate concentration [La]_pl, partial pressure of O₂ and CO₂ and hydrogen ion concentration [H⁺]_b. The lactate threshold (LT) in this study was defined as the highest power output above which [La⁻]_pl showed a sustained increase of more than 0.5 mmol · l⁻¹ · step⁻¹. The V˙O₂ was measured breath-by-breath. In the analysis of the change point (CP) of V˙O₂ during the incremental exercise test, a two-phase model was assumed for the 3rd-min-data of each step of the test: X _i =at _i +b+ɛ _i for i=1,2,…,T, and E(X _i )>at _i +b for i =T+1,…,n, where X ₁, … , X _n are independent and ɛ _i ∼N(0,σ²). In the first phase, a linear relationship between V˙O₂ and power output was assumed, whereas in the second phase an additional increase in V˙O₂ above the values expected from the linear model was allowed. The power output at which the first phase ended was called the change point in oxygen uptake (CP-V˙O₂). The identification of the model consisted of two steps: testing for the existence of CP and estimating its location. Both procedures were based on suitably normalised recursive residuals. We showed that in 25 out of 26 subjects it was possible to determine the CP-O₂ as described in our model. The power output at CP-V˙O₂ amounted to 136.8 (SD 31.3) W. It was only 11 W – non significantly – higher than the power output corresponding to LT. The V˙O₂ at CP-V˙O₂ amounted to 1.828 (SD 0.356) l · min⁻¹ was [48.9 (SD 7.9)% V˙O_{2 max} ]. The [La⁻]_pl at CP-V˙O₂, amounting to 2.57 (SD 0.69) mmol · l⁻¹ was significantly elevated (P<0.01) above the resting level [1.85 (SD 0.46) mmol · l⁻¹], however the [H⁺]_b at CP-V˙O₂ amounting to 45.1 (SD 3.0) nmol · l⁻¹, was not significantly different from the values at rest which amounted to 44.14 (SD 2.79) nmol · l⁻¹. An increase of power output of 30 W above CP-V˙O₂ was accompanied by a significant increase in [H⁺]_b above the resting level (P=0.03). Accepted: 25 March 1998     

Determination of some cyanopeptides synthesized by Woronichinia naegeliana (Chroococcales,Cyanophyceae)

Nineteen compounds isolated from field samples of cyanobacteria Woronichinia naegeliana (Unger) Elenkin were identified. They fell into four classes of peptides: cyanopeptolins (cyanopeptolin B, cyanopeptolin C, cyanopeptolin D, cyanopeptolin 880, micropeptin 88D, micropeptin 478‐B, micropeptin SD999, micropeptin T2, planktopeptin BL1061), microginins (microginin 478, microginin 757, microginin 51A, microginin 91E, microginin FR3, microginin FR4), anabaenopeptins (oscillamide B) and possibly microcystins (trace amounts of microcystin‐LR) showing respectively fragment patterns in their electrospray ion source‐MS spectra. The molecular masses of the determined peptides range from 700 to 1100 Da. These results confirm the remarkable ability of cyanobacteria to synthesize a wide array of peptides.     

Functional characteristics of green alga Scenedesmus obliquus (Chlorophyceae): 276–6 wild type and its two photosystems deficient mutants cultured under photoautotrophic,mixotrophic and heterotrophic conditions

Functional features of Scenedesmus obliquus: wild type 276–6 strain (WT) and its two mutants reported as photosystem I‐deficient (mutant 56.80) and photosystem II‐deficient (mutant 57.80) were characterized. Algae were cultured aseptically under continuous light or in darkness on mineral bold basal medium (BBM), yeast extract‐enriched BBM and yeast extract to evaluate the physiology of algal cells under photoautotrophic, mixotrophic and heterotrophic conditions. Growth, superoxide dismutase activity and photosynthetic parameters, including polyphasic fluorescence rise during the first seconds of chlorophyll a illumination (OJIP), were analyzed to find relationships between the photosynthetic/respiratory activity of the cells, occurrence of oxidative stress and trophic conditions applied to PSs‐deficient algae. Despite the highest superoxide dismutase activity, indicating the presence of oxidative stress, mixotrophic conditions appeared to be optimal for S. obliquus WT and mutant strains kept in non‐aerated cultures. OJIP analysis indicated that in mutant 56.80 part of photosystem (PS) I was functional and in mutant 57.80 residual PS II activity was found.     

Methods of the site-selective solid phase synthesis of peptide-derived Amadori products

Two procedures of glycated peptides’ synthesis have been developed. The first method involves reductive alkylation of the ε-amino groups of lysine with 2,3:4,5-di-O-isopropylidene-β-d-arabino-hexos-2-ulo-2,6-pyranose in the presence of sodium cyanoborohydride on solid support. The second one uses a new fully protected lysine derivative, which is a building block designed for direct introduction of the glycated lysine moiety into a peptide, according to the standard solid phase synthesis protocol. The applicability of the proposed methods for the synthesis of peptide-derived Amadori products is discussed. The structure of the synthesized glycated peptides was confirmed by high-resolution mass spectrometry and enzymatic hydrolysis. Circular dichroism studies, performed in water solution, revealed that the formation of the Amadori rearrangement product in the lysine side chain does not influence significantly the conformational preferences of the peptides studied. However, when the solvent was changed to trifluoroethanol, the glycated peptides preferred β-turn conformation.     

The plasticity of fungal interactions

Their hyphal structure, the common events of hybridization and horizontal gene transfer, as well as intimate associations with prokaryotes (including endobiotic bacteria) and cooperation with eukaryotes have made fungi very flexible at the genetic, physiological, and ecological levels. It is manifested with the fungal ability to perfectly exploit existing nutrient sources and plastically fit into a changing environment. Although the links between fungi and other ecosystem components are rarely clearly visible and unambiguous, fungi can be ecosystem buffers playing a homeostatic role throughout global ecosystems, reacting to changes in various ways, not only by modifications of gene expression but also by nuclear status and “extended phenotype”. The goal of this review is to underline some ecological interactions involving fungi and other organisms and to indicate high fungal plasticity in terms of ontogenetic perspective.