Characterization of the trans Watson-Crick GU base pair located in the catalytic core of the antigenomic HDV ribozyme

The HDV ribozyme's folding pathway is, by far, the most complex folding pathway elucidated to date for a small ribozyme. It includes 6 different steps that have been shown to occur before the chemical cleavage. It is likely that other steps remain to be discovered. One of the most critical of these unknown steps is the formation of the trans Watson-Crick GU base pair within loop III. The U(23) and G(28) nucleotides that form this base pair are perfectly conserved in all natural variants of the HDV ribozyme, and therefore are considered as being part of the signature of HDV-like ribozymes. Both the formation and the transformation of this base pair have been studied mainly by crystal structure and by molecular dynamic simulations. In order to obtain physical support for the formation of this base pair in solution, a set of experiments, including direct mutagenesis, the site-specific substitution of chemical groups, kinetic studies, chemical probing and magnesium-induced cleavage, were performed with the specific goal of characterizing this trans Watson-Crick GU base pair in an antigenomic HDV ribozyme. Both U(23) and G(28) can be substituted for nucleotides that likely preserve some of the H-bond interactions present before and after the cleavage step. The formation of the more stable trans Watson-Crick base pair is shown to be a post-cleavage event, while a possibly weaker trans Watson-Crick/Hoogsteen interaction seems to form before the cleavage step. The formation of this unusually stable post-cleavage base pair may act as a driving force on the chemical cleavage by favouring the formation of a more stable ground state of the product-ribozyme complex. To our knowledge, this represents the first demonstration of a potential stabilising role of a post-cleavage conformational switch event in a ribozyme-catalyzed reaction.     

Shrub cover in northern Nunavik: can herbivores limit shrub expansion?

Recent climate changes have increased the primary productivity of many Arctic and subarctic regions. Erected shrub has been shown to increase in abundance over the last decades in northern regions in response to warmer climate. At the same time, caribou herds are declining throughout the circumboreal regions. Based on observation of heavy browsing on shrubs at Deception Bay (Nunavik, Canada), we hypothesized that the densification of shrubs observed in nearby locations did not occur at our study site despite of observed warming because of a recent peak of the Rivière-aux-Feuilles caribou herd. To assess shrub cover changes, we compared a 1972 mosaic of aerial photos to a 2010 satellite image over a 5 km² area, divided into 56 grids of 100 30 m × 30 m cells. Most cells (n = 4,502) did not show any changes in the cover of shrubs but those who did were as likely to increase as to decrease. The relative cover of shrubs in cells who changed was not higher in 2010 (6.1 ± 0.2 %) than in 1972 (7.3 ± 0.4 %). More than 70 % of birch and willow had more than 50 % of their shoot browsed, suggesting that caribou may limit shrub expansion at this site. We cannot rule out that abiotic factors also contribute to the inertia in shrub cover. Increases in shrub abundance reported in Nunavik and elsewhere were located closer to the tree line or in discontinuous permafrost, whereas our site is characterized by herbaceous arctic tundra, continuous permafrost and relatively low annual precipitation.     

Identification of Mutations in TMEM5 and ISPD as a Cause of Severe Cobblestone Lissencephaly

Cobblestone lissencephaly is a peculiar brain malformation with characteristic radiological anomalies. It is defined as cortical dysplasia that results when neuroglial overmigration into the arachnoid space forms an extracortical layer that produces agyria and/or a “cobblestone” brain surface and ventricular enlargement. Cobblestone lissencephaly is pathognomonic of a continuum of autosomal-recessive diseases characterized by cerebral, ocular, and muscular deficits. These include Walker-Warburg syndrome, muscle-eye-brain disease, and Fukuyama muscular dystrophy. Mutations in POMT1, POMT2, POMGNT1, LARGE, FKTN, and FKRP identified these diseases as alpha-dystroglycanopathies. Our exhaustive screening of these six genes, in a cohort of 90 fetal cases, led to the identification of a mutation in only 53% of the families, suggesting that other genes might also be involved. We therefore decided to perform a genome-wide study in two multiplex families. This allowed us to identify two additional genes: TMEM5 and ISPD. Because TMEM has a glycosyltransferase domain and ISPD has an isoprenoid synthase domain characteristic of nucleotide diP-sugar transferases, these two proteins are thought to be involved in the glycosylation of dystroglycan. Further screening of 40 families with cobblestone lissencephaly identified nonsense and frameshift mutations in another four unrelated cases for each gene, increasing the mutational rate to 64% in our cohort. All these cases displayed a severe phenotype of cobblestone lissencephaly A. TMEM5 mutations were frequently associated with gonadal dysgenesis and neural tube defects, and ISPD mutations were frequently associated with brain vascular anomalies.     

Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay

Quantitation of plasma lipopolysaccharides (LPSs) might be used to document Gram-negative bacterial infection. In the present work, LPS-derived 3-hydroxymyristate was extracted from plasma samples with an organic solvent, separated by reversed phase HPLC, and quantitated by MS/MS. This mass assay was combined with the limulus amebocyte lysate (LAL) bioassay to monitor neutralization of LPS activity in biological samples. The described HPLC/MS/MS method is a reliable, practical, accurate, and sensitive tool to quantitate LPS. The combination of the LAL and HPLC/MS/MS analyses provided new evidence for the intrinsic capacity of plasma lipoproteins and phospholipid transfer protein to neutralize the activity of LPS. In a subset of patients with systemic inflammatory response syndrome, with documented infection but with a negative plasma LAL test, significant amounts of LPS were measured by the HPLC/MS/MS method. Patients with the highest plasma LPS concentration were more severely ill. HPLC/MS/MS is a relevant method to quantitate endotoxin in a sample, to assess the efficacy of LPS neutralization, and to evaluate the proinflammatory potential of LPS in vivo.     

Changes in cell-wall polysaccharide composition of developingNitella internodes

Changes in the uronide, neutral-polysacharide, and cellulose composition of the cell wall ofNitella axillaris Braun were followed throughout development of the internodes and correlated with changes in growth rate. As the cells increased in length from 4 to 80 mm during development, the relative growth rate decreased. Cell wall thickness, as measured by wall density, increased in direct proportion to diameter, indicating that cell-wall stress did not change during elogation. Cell-wall analyses were adapted to allow determination of the composition of the wall of single cells. The total amounts of uronides, neutral sugars and cellulose all increased during development. However, as the growth rate decreased, the relative proportions of uronides and neutral sugars, expressed as percent of the dry weight of the wall, decreased, while the proportion of cellulose increased. The neutral sugars liberated upon hydrolysis ofNitella walls are qualitatively similar to those found in hydrolysates of higher plant cell walls: glucose, xylose, mannose, galactose, arabinose fucose and rhamnose. Only the percentage of galactose was found to increase in walls of mature cells, while the percentage of all other sugars decreased. The rate of apposition (g of wall material deposited per unit wall surface area per hour) of neutral polysaccharides decreased rapidly with decreasing growth rate during the early stages of development. The rate of apposition of uronides decreased more steadily throughout development, while that of cellulose, after an early decline, remained constant until dropping off at the end of the elongation period. These correlations between decreasing growth rate and decreasing rate of apposition of neutral sugars and uronides indicate that synthesis of these cell-wall components could be involved in the regulation of the rate of cell elongation inNitella.     

Influenza Vaccine Manufacturing: Effect of Inactivation,Splitting and Site of Manufacturing. Comparison of Influenza Vaccine Production Processes

The aim of this study was to evaluate the impact of different inactivation and splitting procedures on influenza vaccine product composition, stability and recovery to support transfer of process technology. Four split and two whole inactivated virus (WIV) influenza vaccine bulks were produced and compared with respect to release criteria, stability of the bulk and haemagglutinin recovery. One clarified harvest of influenza H3N2 A/Uruguay virus prepared on 25.000 fertilized eggs was divided equally over six downstream processes. The main unit operation for purification was sucrose gradient zonal ultracentrifugation. The inactivation of the virus was performed with either formaldehyde in phosphate buffer or with beta-propiolactone in citrate buffer. For splitting of the viral products in presence of Tween^®, either Triton^™ X-100 or di-ethyl-ether was used. Removal of ether was established by centrifugation and evaporation, whereas removal of Triton-X100 was performed by hydrophobic interaction chromatography. All products were sterile filtered and subjected to a 5 months real time stability study. In all processes, major product losses were measured after sterile filtration; with larger losses for split virus than for WIV. The beta-propiolactone inactivation on average resulted in higher recoveries compared to processes using formaldehyde inactivation. Especially ether split formaldehyde product showed low recovery and least stability over a period of five months.     

Functional and structural aspects of poplar cytosolic and plastidial type a methionine sulfoxide reductases

The genome of Populus trichocarpa contains five methionine sulfoxide reductase A genes. Here, both cytosolic (cMsrA) and plastidial (pMsrA) poplar MsrAs were analyzed. The two recombinant enzymes are active in the reduction of methionine sulfoxide with either dithiothreitol or poplar thioredoxin as a reductant. In both enzymes, five cysteines, at positions 46, 81, 100, 196, and 202, are conserved. Biochemical and enzymatic analyses of the cysteine-mutated MsrAs support a catalytic mechanism involving three cysteines at positions 46, 196, and 202. Cys(46) is the catalytic cysteine, and the two C-terminal cysteines, Cys(196) and Cys(202), are implicated in the thioredoxin-dependent recycling mechanism. Inspection of the pMsrA x-ray three-dimensional structure, which has been determined in this study, strongly suggests that contrary to bacterial and Bos taurus MsrAs, which also contain three essential Cys, the last C-terminal Cys(202), but not Cys(196), is the first recycling cysteine that forms a disulfide bond with the catalytic Cys(46). Then Cys(202) forms a disulfide bond with the second recycling cysteine Cys(196) that is preferentially reduced by thioredoxin. In agreement with this assumption, Cys(202) is located closer to Cys(46) compared with Cys(196) and is included in a (202)CYG(204) signature specific for most plant MsrAs. The tyrosine residue corresponds to the one described to be involved in substrate binding in bacterial and B. taurus MsrAs. In these MsrAs, the tyrosine residue belongs to a similar signature as found in plant MsrAs but with the first C-terminal cysteine instead of the last C-terminal cysteine.     

Estrone sulfate (E1S), a prognosis marker for tumor aggressiveness in prostate cancer (PCa)

Seeking insight into the possible role of estrogens in prostate cancer (PCa) evolution, we assayed serum E₂, estrone (E₁), and estrone sulfate (E₁S) in 349 PCa and 100 benign prostatic hyperplasia (BPH) patients, and in 208 control subjects in the same age range (50–74 years).

E₁ (pmol/L ± S.D.) and E₁S (nmol/L ± S.D.) in the PCa and BPH patients (respectively 126.1 ± 66.1 and 2.82 ± 1.78, and 127.8 ± 56.4 and 2.78 ± 2.12) were significantly higher than in the controls (113.8 ± 47.6 and 2.11 ± 0.96). E₂ was not significantly different among the PCa, BPH, and control groups. These assays were also carried out in PCa patients after partition by prognosis (PSA, Gleason score (GS), histological stage, and surgical margins (SM)). Significantly higher E₁S levels were found in PCa with: PSA > 10 ng/L (3.05 ± 1.92) versus PSA ≤ 10 ng/mL (2.60 ± 1.55), stage pT3-T4 (2.99 ± 1.80) versus pT2 (2.58 ± 1.58), and positive (3.26 ± 1.95) versus negative margins (2.52 ± 1.48). E₁ was higher in poor- than in better-prognosis PCa. E₂ was significantly higher in PCa with GS ≥ 4 + 3 (109.5 ± 43.8) versus GS ≤ 3 + 4 (100.6 ± 36.5) and increased significantly when GS increased from 3 + 3 to 4 + 4. Estrogens, especially E₁S appeared to be possible markers of PCa progression.

Attempting to identify potential sources of E₂ in PCa according to prognosis, as well as in BPH, we found a significant correlation coefficient between E₁S and E₂ (0.266–0.347) in poor-prognosis PCa and no correlation in BPH (0.026) and better-prognosis PCa (0.013–0.104).

It is as though during progression of PCa from good to poor prognosis there were a shift in the E₁ to E₂ metabolic pathway from predominantly oxidative to predominantly reductive.     

Na movements and their oscillations during fertilization and the cell cycle in sea urchin eggs

We have analysed in detail the Na⁺ content and Na⁺ influx during fertilization and first divisions of the sea urchin egg (Paracentrotus lividus) using a filtration technique devised to eliminate rapidly contamination by the Na⁺ of external sea water. In the first 5 min following fertilization the egg fills up with Na⁺ (+ 30%). Thereafter Na⁺ is extruded and the Na⁺ content stabilizes at about 60% of the unfertilized egg level by the second cleavage (2 h). The initial increase in Na⁺ content is due to a large increase in Na⁺ influx already detected at 20 sec. The Na⁺ influx reaches its maximum at 1 min and its minimum at 5 min. H⁺ excretion follows the same kinetics. A second increase in Na⁺ influx is noted 5–10 min after fertilization; it reaches its maximum at prophase metaphase (30 min) and its minimum during cleavage (60 min). These oscillations in Na⁺ influx were observed for the first three divisions. Fertilization also immediately stimulates the Na⁺ efflux which remains elevated throughout the cell cycle and is responsible for the depletion of the Na⁺ content of the embryos. Activation of the eggs by weak amine bases (5 mM NH₄Cl) which bypasses the early cortical reaction produces only a depletion in the Na⁺ content of the egg similar to that produced by fertilization. NH₄Cl also increases the Na⁺ influx soon after fertilization, although no transient variations are noted.     

Alport syndrome: a genetic study of 31 families

Thirty one families with Alport syndrome including 3 families with associated syndromes were studied. The location of the COL4A5 gene, responsible for the Alport syndrome, was determined by linkage analysis with eight probes of the Xq arm and by a radiation hybrid panel. Concordant data indicated the localization of the Alport gene between DXS17 and DXS11. Four deletions and one single base mutation of the COL4A5 gene were detected. Homogeneity tests failed to show any evidence of genetic heterogeneity superimposed on clinical heterogeneity for ophthalmic signs and end-stage renal disease age.