Molecular dynamics of FMRP and other RNA-binding proteins in MEG-01 differentiation: the role of mRNP complexes in non-neuronal development.

Asymmetrically differentiating cells are formed with the aid of RNA-binding proteins (RBPs), which can bind, stabilize, regulate, and transport target mRNAs. The loss of RBPs in neurons may lead to severe neurodevelopmental diseases such as the Fragile X Syndrome with the absence of the Fragile X Mental Retardation Protein (FMRP). Because the latter is ubiquitous and shares many similarities with other RBPs involved in the development of peripheral cells, we suggest that FMRP would have a role in the differentiation of all tissues where it is expressed. A MEG-01 differentiation model was, therefore, established to study the global developmental functions of FMRP. PMA induction of MEG-01 cells causes important morphological changes driven by cytoskeletal dynamics. Cytoskeleton change and colocalization analyses were performed by confocal microscopy and sucrose gradient fractionation. Total cellular protein content and de novo synthesis were also analyzed. Microtubular transport mediates the displacement of FMRP and other RBP-containing mRNP complexes towards regions of the cell in development. De novo protein synthesis decreases significantly upon differentiation and total protein content composition is altered. Because those results are comparable with those obtained in neurons, the absence of FMRP would have significant consequences in cells everywhere in the body. The latter should be further investigated to give a better understanding of the systemic implications of imbalances of FMRP and other functionally similar RBPs.

Dual-wavelength digital holography for 3D particle image velocimetry: experimental validation.

A multi-exposure digital in-line hologram of a particle field is recorded by two successive pulses of different wavelengths. During the reconstruction step, each recording can be independently analyzed by selecting a given wavelength. This procedure enables avoiding the superimposition of particle images that may be close to each other.

Quantitative measurement of FMRP in blood platelets as a new screening test for fragile X syndrome.

The fragile X syndrome usually results from CGG repeats expansion and methylation of the FMR1 gene leading to the absence of expression of its encoded protein, fragile X mental retardation protein (FMRP). Therefore, its diagnosis is traditionally based on the detection of these molecular alterations. As an alternative, FMRP-based screening methods have been proposed over the years. Most of them are based on immunohistochemistry analyses applied to a restricted number of lymphocytes (100) or hair roots (10-20) with limited diagnosis potential. In this study, we describe a truly quantitative approach using a new model, the blood platelet, which can be recovered easily with very high purity (99.9%). FMRP levels in platelets were first measured in a control population (n = 124) and reference values were established. FMRP measurements were also performed in confirmed fragile X subjects. Receiver operating characteristic curve analysis has shown that our test can easily discriminate fragile X males and females from controls (area under curve, AUC = 0.948). Cognitive functions were also assessed in these individuals using age-specific Wechsler Intelligence Scales for Children and the Vineland Adaptive Behavior Scales. A proportional relationship between FMRP levels, intelligence quotient and adaptive behavior was observed among fragile X individuals, suggesting that our test would be able to detect fragile X cases and may predict cognitive functions.

Monte Carlo Aggregation Code (MCAC) Part 2: Application to soot agglomeration, highlighting the importance of primary particles.

During the agglomeration of nanoparticles and in particular, soot, a change in both the flow regime (from free molecular to near continuum) as well as the change of agglomeration regime (from ballistic to diffusive) is expected. However, these effects are rarely taken into account in numerical simulations of particle agglomeration and yet, they are suspected to have an important impact on the agglomeration kinetics, particle morphologies, and size distributions. This work intends to study these properties by using the Monte Carlo Aggregation Code (MCAC) presented in the preceding work (part 1), focusing on the physical impacts of varying the particle volume fraction and monomers size and polydispersity. The results show an important sensitivity of the kinetics of agglomeration, coagulation homogeneity, and agglomerate morphology to the size of monomers. First, for smaller monomer diameters, the agglomeration kinetic is enhanced and agglomerates are characterized by larger fractal dimensions. Second, for large monomer diameters, fractal dimensions down to 1.67 can be found being smaller than the classical 1.78 for Diffusion Limited Cluster Agglomeration (DLCA) mechanism. One important conclusion is that variation in time of both regimes has to be considered for a more accurate simulation of the agglomerate size distribution and morphology.

Impact of hemodialysis on the level of high-sensitivity cardiac troponins T in patients with end-stage renal disease.

AIM: The majority of patients with end-stage renal disease have chronically elevated concentrations of troponins, thus obscuring the diagnosis of myocardial infarction. We conducted a prospective study to examine the impact of hemodialysis on the level of high-sensitivity cardiac troponins T in asymptomatic patients with end-stage renal disease. METHODS: High-sensitivity cardiac troponins T were measured in 43 patients, before and after three dialysis sessions, over a one week period. RESULTS: Following dialysis, an average decrease of 7.6 pg/mL in high-sensitivity cardiac troponin T levels was observed which represents a 10.3% drop from baseline. Mutlivariable mixed linear regression models taking into account dialysis session (session 1, 2 or 3), sampling moment (before and after dialysis) and repeated measures on individuals revealed that the presence of coronary artery disease and elevated body mass index were associated with higher high-sensitivity cardiac troponin T levels when the other variables were held constant (CAD: ß [fixed effect estimate]=31.13 pg/mL, P=0.022, 95%CI 4.46-57.80; body mass index: ß=2.57 kg/m2, P=0.008, 95%CI 0.68-4.46). The significant fixed effect estimate for the interaction between gender and sampling moment indicated that the drop in high-sensitivity cardiac troponin T levels following dialysis was greater for women than for men (ß=5.75, P=0.049, 95% CI 0.02-11.47). When controlling for the variables mentioned above, this analysis confirmed that hemodialysis accounted for an 11.31 pg/mL decrease in high-sensitivity cardiac troponin T levels (P<0.001, 95%CI -15.99 - -6.62) and that the values were higher in the first dialysis session than in the third (P=0.007; 95%CI 1.62-9.79). Ten patients (23%) were found to have no decrease or an increase in troponin levels after hemodialysis. CONCLUSION: In stable asymptomatic patients with end-stage renal disease, we have shown that hemodialysis reduces the blood concentration of high-sensitivity cardiac troponins T by at least 10%. Further studies are needed to confirm these results and determine their prognostic significance.

Development of an automated computer-controlled islet isolation system.

Before clinical islet transplantation can become an effective and reliable treatment for type 1 diabetic patients, there must be significant improvements in the methods employed for the isolation of islets of Langerhans. We have developed an automated cell extraction system (ACES), which allows computer control of the isolation process. As well, it incorporates a novel method of recombining dissociated pancreatic tissue. Following initial system design and testing to determine the optimal system configuration, a series of 12 consecutive canine islet isolations were performed. Pancreases were perfused with collagenase via the duct and dissociated and recombined using either the standard Ricordi-based protocol (group 1, n = 6) or dissociated and recombined using the ACES system (group 2, n = 6). A total of 90.8 +/- 21 x 10(3) islet equivalents (IE) (mean +/- SEM) were recovered in group 1 vs. 99 +/- 14 x 10(3) IE in group 2 (p = NS, student unpaired t-test). Following Ficoll purification the recovery was 56.2 +/- 14 x 10(3) IE for group 1 vs. 54.7 +/- 11 x 10(3) IE for group 2 (p = NS). Viability was equivalent with an 8.6-fold increase in insulin secretion for group 1 and an 8.8-fold increase for group 2 when the islets were exposed to high glucose solution supplemented with IBMX (3-isobutyl-1-methylxanthine) during static incubation. In vivo function was equivalent following transplantation of 2000 IE under the kidney capsule of alloxan-induced diabetic nude mice with five of six and five of seven mice surviving long-term (> 50 days posttransplant) (groups 1 and 2, respectively). This data shows that an entirely automated pancreatic islet extraction system can result in effective canine islet recovery without compromising islet yields and viability. The ACES system has several advantages over the standard isolation protocol. These include: 1) computer control and monitoring over all phases of the isolation, 2) a single-use sterile disposable tubing set, and 3) a novel method of tissue recombination.

Metabolism of metolachlor by a microsomal fraction isolated from grain sorghum (Sorghum bicolor) shoots.

A microsomal fraction isolated from the shoots of 3- to 4-day-old, dark-grown, grain sorghum (Sorghum bicolor cv. Funk G 522 DR) seedlings was characterized. The preparations had a cytochrome P-450 content that varied from approximately 90 to 150 pmol P-450/mg protein with cytochrome P-420 varying from 0 to 3% of the P-450 content. Type I difference spectra were formed with cinnamic acid and metolachlor, and a type II spectrum was formed with tetcyclacis. In short-term assays with [14C]metolachlor as substrate, the preparations produced a single time-dependent product that separated on silica gel TLC plates developed in benzene/acetone (2:1, v/v). RF values for metolachlor and the metabolite were approximately 0.70 and 0.48, respectively. The microsomal reaction required NADPH and oxygen, and was inhibited by carbon monoxide, with the inhibition being partially reversed by actinic light. Compounds known to inhibit the activity of cytochrome P-450 monooxygenases (piperonyl butoxide, tetcyclacis, and tridiphane) also prevented formation of the metabolite. Identity of the metabolite was confirmed by TLC and positive ion thermospray LC/MS to be 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-hydroxy-1-methylethyl)acetamide . Hence, the reaction catalyzed by the sorghum microsomes involved O-demethylation of the methoxypropyl side chain of metolachlor.

Interaction of fensulfothion and phorate with preemergence herbicides on soybean parasitic nematodes.

The herbicides alachlor, linuron, vernolate, and metribuzin were applied to plots treated with the nematicide fensulfothion or the insecticide phorate and planted to soybean in two locations in North Carolina. In 1976 treatment with fensulfothion + alachlor or vernolate, phorate + alachlor or metribuzin resulted in greater nematode population densities than no treatment, or treatment with fensulfothion alone, or phorate alone. In 1977 fensulfothion and phorate alone and in combination with the preemergence herbicides effectively controlled Tylenchorhynchus cIaytoni. Late season population resurgence of Heterodera glycines occurred in fensulfothion + alachlor treated plots. Correlation coefficients for H. glycines vs. yield were -0.48 (P = 0.05) and -0.46 (P = 0.05) for 30 and 68 d after planting, respectively.

Population Dynamics of Heterodera glycines and Soybean Response in Soils Treated with Selected Nematicides and Herbicides.

Two field experiments were conducted in two locations to determine the effects of the nematicides aldicarb, phenamiphos, and ethoprop and/or the herbicides alachlor, linuron, or metribuzin on the population dynamics of Heterodera glycines and soybean growth and yield. Population densities of H. glycines were greater, at some time during the growing season, in several treatments with alachlor alone and in combination with nematicides. Numbers of H. glycines at harvest were greater in plots treated with aldicarb than in those treated with ethoprop or phenamiphos. The numbers in aldicarb treated plots were generally reduced when plots also received a herbicide. Soybean yields were negatively correlated with numbers of H. glycines eggs and juveniles in early to mid season but positively correlated with late season population densities.

Differential effect of halothane and forskolin on platelet cytosolic Ca2+ mobilization and aggregation.

BACKGROUND: Previous works have suggested that the impairment of platelet aggregation by halothane was partly related to a stimulation of cyclic adenosine monophosphate (cAMP) production, to an inhibitory effect on Ca2+ signaling, or both. Intracellular Ca2+ measurements therefore were undertaken, first to determine the critical steps in the platelet CaZ+ signaling cascade most likely to be affected by halothane or by an increase in cAMP production, and second to establish if the effect of halothane involves aggregation-related biochemical pathways triggered by an increase in internal Ca2+. METHODS: Human washed platelets were treated with halothane or forskolin for 5 min before application of either platelet-activating factor, thrombin, U46619, or thapsigargin. The cytosolic Ca2+ concentration ([Ca2+]i) was measured with the fluorescent Ca2+ indicator fura-2. Nephelometric measurements were also performed to assay the aggregation process. RESULTS: Our results indicate that pretreating platelets with halothane leads to a partial impairment of the [Ca2+]i increase induced either by U46619, thrombin, or platelet-activating factor, but this had no significant effect on the [Ca2+]i response triggered by thapsigargin. In addition, our results show that halothane inhibits platelet aggregation triggered by U46619, but not by thapsigargin. Conversely, forskolin completely inhibited the [Ca2+]i response to U46619 and thapsigargin and prevented platelet aggregation induced by both agonists. CONCLUSIONS: These results suggest that halothane and cAMP exert their effects on platelet aggregation and Ca2+ signaling through different mechanisms, and that halothane cannot impair platelet aggregation independently of phospholipase C stimulation.

The fragile X mental retardation protein is associated with poly(A)+ mRNA in actively translating polyribosomes.

The fragile X syndrome results from a transcriptional silencing of the FMR1 gene and the absence of its encoded protein. FMRP is a cytoplasmic RNA-binding protein, whose specific cellular function is still unknown. We present evidence that virtually all detectable cytoplasmic FMRP in mouse NIH 3T3 and human HeLa cells is found strictly in association with mRNA in actively translating polyribosomes. Furthermore, FMRP released from polyribosomes is associated with ribonucleoprotein complexes with sedimentation coefficients of 60-70S and selection on oligo(dT)-cellulose reveals that this association is specific to poly(A)-containing mRNPs. This association with actively translating polyribosomes is not affected by alteration of translational processes induced by serum stimulation and starvation in NIH 3T3 cells, suggesting that FMR1 expression is not cell cycle regulated and that FMRP might have a house-keeping function. FXR2 protein, which is closely related to FMRP, is also detected associated with mRNPs in translating polyribosomes. The results strongly suggest that FMRP might be a mRNA chaperone interacting with mRNP complexes.

Effect of rapamycin on rat aortic ring vasomotion.

Rapamycin (RAPA) is an antifungal antibiotic with interesting new immunosuppressive properties. We evaluated RAPA's effects in vitro on basal and stimulated tension of isolated intact or denuded rat aortic rings. Rings were prepared in an organ chamber and contracted with 40 mM KCl (reference 100%). Some rings were treated with either RAPA's polysorbate/polyethylene glycol-based (PEG) vehicle (0.8% vol/vol) or with different concentrations of RAPA (10, 100, and 1,000 ng/ml) diluted in PEG; untreated rings were used as controls. Variation in tension with time (2 h) and the dose-response to thromboxane A2 analogue (U46619) and phenylephrine (PE) were measured in controls and treated rings. PEG potentiated the increase in basal tension in rings with endothelium after 2-h treatment (44.66 +/- 3.59 vs. 14.82 +/- 2.43% for controls, p < 0.05, n = 10). RAPA antagonized the contraction induced by its own vehicle dose dependently. At 1,000 ng/ml, RAPA caused relaxation of intact rings below the control level (4.29 +/- 2.20 vs. 14.82 +/- 2.43%, p < 0.05, n = 10), but not in rings without endothelium. RAPA did not modify the response to PE or U46619 in rings with endothelium. RAPA relaxed the vessels by an endothelium-dependent mechanism, and this effect can be modulated by its vasoconstrictive PEG vehicle.

Comparison of amino acid sequences deduced from a cDNA fragment obtained from infectious pancreatic necrosis virus (IPNV) strains of different serotypes.

Infectious pancreatic necrosis is an important viral disease of salmonid fish reared in hatchery. Its etiological agent, IPNV, showed a high degree of antigenic heterogeneity. Up to 10 serotypes and 2 serogroups were proposed. Yet, very little is known about genomic variations among viruses of different origin. In order to investigate these variations, a 310-bp cDNA fragment was prepared from 17 IPNV strains by reverse transcription of the viral genome and amplification by the polymerase chain reaction. These fragments were then cloned and sequenced. Comparison of the 17 sequences obtained with 3 previously published ones, at the amino acid level, showed that serologically related viruses are highly homologous (over 96% homology) but some strains which were reported to belong to different serotypes also appeared closely related. Thus, only three major groups, clearly distinct from each other, could be formed. Apart from this, a search for the exact cleavage site of the unprocessed polyprotein of IPNV was done since the amplified fragment used for sequencing was localized at the junction between two polypeptides of the virus, pVP2 and NS. No obvious sequence or dipeptide appeared conserved in all birnaviruses.

Expression of a soybean cytochrome P450 monooxygenase cDNA in yeast and tobacco enhances the metabolism of phenylurea herbicides.

A strategy based on the random isolation and screening of soybean cDNAs encoding cytochrome P450 monooxygenases (P450s) was used in an attempt to identify P450 isozymes involved in herbicide metabolism. Nine full-length (or near-full-length) P450 cDNAs representing eight distinct P450 families were isolated by using PCR-based technologies. Five of the soybean P450 cDNAs were expressed successfully in yeast, and microsomal fractions generated from these strains were tested for their potential to catalyze the metabolism of 10 herbicides and 1 insecticide. In vitro enzyme assays showed that the gene product of one heterologously expressed P450 cDNA (CYP71A10) specifically catalyzed the metabolism of phenylurea herbicides, converting four herbicides of this class (fluometuron, linuron, chlortoluron, and diuron) into more polar compounds. Analyses of the metabolites suggest that the CYP71A10 encoded enzyme functions primarily as an N-demethylase with regard to fluometuron, linuron, and diuron, and as a ring-methyl hydroxylase when chlortoluron is the substrate. In vivo assays using excised leaves demonstrated that all four herbicides were more readily metabolized in CYP71A10-transformed tobacco compared with control plants. For linuron and chlortoluron, CYP71A10-mediated herbicide metabolism resulted in significantly enhanced tolerance to these compounds in the transgenic plants.

Characterization of initial disturbances in a liquid jet by rainbow sizing.

The development of initial disturbances is relevant to the understanding of atomization processes in which droplets are generated by the breakup of a liquid jet. We theoretically and experimentally demonstrate that such disturbances can be characterized by rainbow sizing. More specifically, for a liquid jet with a diameter of 600 mum, disturbances in the range from 10 nm to 0.2 mum are accessible.