In vivo gene expression in granulosa cells during pig terminal follicular development.

Ovarian antral follicular development is clearly dependent on pituitary gonadotrophins FSH and LH. Although the endocrine mechanism that controls ovarian folliculogenesis leading to ovulation is quite well understood, the detailed mechanisms and molecular determinants in the different follicular compartments remain to be clarified. The aim of this study was to identify the genes differentially expressed in pig granulosa cells along the terminal ovarian follicle growth, to gain a comprehensive view of these molecular mechanisms. First, we developed a specific micro-array using cDNAs from suppression subtractive hybridization libraries (345 contigs) obtained by comparison of three follicle size classes: small, medium and large antral healthy follicles. In a second step, a transcriptomic analysis using cDNA probes from these three follicle classes identified 79 differentially expressed transcripts along the terminal follicular growth and 26 predictive genes of size classes. The differential expression of 18 genes has been controlled using real-time PCR experiments validating the micro-array analysis. Finally, the integration of the data using Ingenuity Pathways Analysis identified five gene networks providing descriptive elements of the terminal follicular development. Specifically, we observed: (1) the down-expression of ribosomal protein genes, (2) the genes involved in lipid metabolism and (3) the down-expression of cell morphology and ion-binding genes. In conclusion, this study gives new insight into the gene expression during pig terminal follicular growth in vivo and suggested, in particular, a morphological change in pig granulosa cells accompanying terminal follicular growth.

Clustering time-series gene expression data using smoothing spline derivatives.

Microarray data acquired during time-course experiments allow the temporal variations in gene expression to be monitored. An original postprandial fasting experiment was conducted in the mouse and the expression of 200 genes was monitored with a dedicated macroarray at 11 time points between 0 and 72 hours of fasting. The aim of this study was to provide a relevant clustering of gene expression temporal profiles. This was achieved by focusing on the shapes of the curves rather than on the absolute level of expression. Actually, we combined spline smoothing and first derivative computation with hierarchical and partitioning clustering. A heuristic approach was proposed to tune the spline smoothing parameter using both statistical and biological considerations. Clusters are illustrated a posteriori through principal component analysis and heatmap visualization. Most results were found to be in agreement with the literature on the effects of fasting on the mouse liver and provide promising directions for future biological investigations.

Identification of QTLs for Ralstonia solanacearum race 3-phylotype II resistance in tomato.

Resistance against a Ralstonia solanacearum race 3-phylotype II strain JT516 was assessed in a F(2:3) and a population of inbred lines (RIL), both derived from a cross between L. esculentum cv. Hawaii 7996 (partially resistant) and L. pimpinellifolium WVa700 (susceptible). Resistance criteria used were the percentage of wilted plants to calculate the AUDPC value, and bacterial colonization scores in roots and stem (hypocotyl and epicotyl) assessed in two independent greenhouse experiments conducted during the cool and hot seasons in Réunion Island, France. Symptoms were more severe during the cool season trials. Heritability estimates in individual seasons ranged from 0.82 to 0.88, depending on resistance criterion. A set of 76 molecular markers was used for quantitative trait loci (QTL) mapping using the single- and composite- interval mapping methods, as well as ANOVA. Four QTLs, named Bwr- followed by a number indicating their map location, were identified. They explained from 3.2 to 29.8% of the phenotypic variation, depending on the resistance criterion and the season. A major QTL, Bwr-6, and a minor one, Bwr-3, were detected in each season for all resistance criteria. Both QTLs showed stronger effects in the hot season than in the cool one. Their role in resistance to R. solanacearum race 3-phylotype II was subsequently confirmed in the RIL population derived from the same cross. Two other QTLs, Bwr-4 and Bwr-8, with intermediate and minor effects, respectively, were only detected in the hot season, demonstrating that environmental factors may strongly influence the expression of resistance against the race 3-phylotype II strain JT516. These QTLs were compared with those detected in the RIL population against race 1-phylotype I strain JT519 as well as those detected in other previous studies in the same genetic background against other race 1-phylotype I and II strains. This comparison revealed the possible occurrence of some phylotype-specific resistance QTLs in Hawaii 7996.

Sample patterning on NMR surface microcoils.

Aligned microcontact printing for patterning the sample in areas of homogeneous RF-field on the highly sensitive surface of planar NMR microprobes is presented. We experimentally demonstrate that sample patterning allows drastic improvement of the spin excitation uniformity. The NMR microprobes are designed for cell analysis and characterized using lipid vesicles as cell substitutes. Lipid vesicles are advantageous as composition and concentration of the confined solution are precisely controlled and because of their similarity to living cells. Using aligned microcontact printing, a monolayer of lipid vesicles is immobilized on the surface of the planar NMR microprobe in a patterned way. 1H NMR spectra and CPMG spin echoes of sucrose solution confined within the lipid vesicles are successfully recorded. Nutation curves of the sample structured in different patterns demonstrate the impact of patterning on the spin excitation uniformity. The total detection volumes are between 1 and 2 nL and derived with help of a theoretic model based on 3D finite element simulation. This model predicts the signal-to-noise ratio and the progression of the nutation curves.

Conformational rigidification via derivatization facilitates the determination of absolute configuration using chiroptical spectroscopy: a case study of the chiral alcohol endo-borneol.

We demonstrate that derivatization of the OH group of endo-borneol, 1, leads to conformational rigidification. Conformational analysis (CA) of 1 and its methyl, acetate, tert-butyl, and trimethylsilyl derivatives, 2-5, is carried out using ab initio density functional theory (DFT). The number of thermally accessible stable conformations is reduced from 3 in 1, to 2 in 2, and to 1 in 3-5. Comparison of IR and vibrational circular dichroism (VCD) spectra of 1 and 3-5, calculated using DFT, to experimental spectra unambiguously confirms the DFT CA. The determination of absolute configurations (ACs) of chiral molecules via analysis of chiroptical spectra using DFT methods increases in complexity and decreases in reliability as the number of populated conformations increases. Our results for endo-borneol support the conclusion that, in the case of chiral alcohols, derivatization can lead to substantial rigidification and, as a result, significantly facilitate the determination of ACs.

Markers associated with stalk number and suckering in sugarcane colocate with tillering and rhizomatousness QTLs in sorghum.

Two important factors influencing sugar yield, the primary focus of sugarcane plant breeding programs, are stalk number and suckering. Molecular markers linked to both of these traits are sought to assist in the identification of high sugar yield, high stalk number, low-suckering sugarcane clones. In this preliminary mapping study, 108 progeny from a biparental cross involving two elite Australian sugarcane clones were evaluated at two sites for two years for both stalk number and suckering. A total of 258 DNA markers, including both restriction fragment length polymorphisms (RFLPs) and radio-labelled amplified fragments (RAFs), were scored and evaluated using single-factor analysis. Sixteen (7 RFLPs and 9 RAFs) and 14 (6 RFLPs and 8 RAFs) markers were identified that were significantly associated (P < 0.01) with stalk number and suckering, respectively, across both years and sites. The seven and six RFLP markers associated with stalk number and suckering, respectively, were generated by eight different RFLP probes, of which seven had been mapped in sorghum and (or) sugarcane. Of significant interest was the observation that all seven RFLP probes could be shown to be located within or near QTLs associated with tillering and rhizomatousness in sorghum. This observation highlights the usefulness of comparative mapping between sorghum and sugarcane and suggests that the identification of useful markers for stalk number and suckering in sugarcane would be facilitated by focussing on sorghum QTLs associated with related traits.

Planar microcoil-based microfluidic NMR probes.

Microfabricated small-volume NMR probes consisting of electroplated planar microcoils integrated on a glass substrate with etched microfluidic channels are fabricated and tested. 1H NMR spectra are acquired at 300 MHz with three different probes having observed sample volumes of respectively 30, 120, and 470 nL. The achieved sensitivity enables acquisition of an 1H spectrum of 160 microg sucrose in D2O, corresponding to a proof-of-concept for on-chip NMR spectroscopy. Increase of mass-sensitivity with coil diameter reduction is demonstrated experimentally for planar microcoils. Models that enable quantitative prediction of the signal-to-noise ratio and of the influence of microfluidic channel geometry on spectral resolution are presented and successfully compared to the experimental data. The main factor presently limiting sensitivity for high-resolution applications is identified as being probe-induced static magnetic field distortions. Finally, based on the presented model and measured data, future performance of planar microcoil-based microfluidic NMR probes is extrapolated and discussed.

Determination of absolute configuration using ab initio calculation of optical rotation.

Ab initio Density Functional Theory (DFT) calculations of transparent spectral region, discrete frequency specific rotations were used to assign the absolute configurations (ACs) of: 1, 2H-naphtho[1,8-bc]thiophene 1-oxide; 2, m-F-phenyl glycidic acid methyl ester; 3, o-Br-phenyl glycidic acid methyl ester; 4, p-CH(3)-phenyl glycidic acid methyl ester; 5, 2-(1-hydroxyethyl)-chromen-4-one; and 6, 6-Br-2-(1-hydroxyethyl)-chromen-4-one. The ACs of 5 and 6 were previously determined via X-ray crystallography to be: 5, R(-)/S(+); 6, R(+)/S(-). The ACs obtained using [alpha](D) are the same for both 5 and 6: R(+)/S(-). We conclude that the previously reported AC of 5 is incorrect.

Long-range (1)H-(15)N heteronuclear shift correlation at natural abundance: a tool to study benzothiazole biodegradation by two rhodococcus strains.

The biodegradation of benzothiazole and 2-hydroxybenzothiazole by two strains of Rhodococcus was monitored by reversed phase high-pressure liquid chromatography and by (1)H nuclear magnetic resonance (NMR). Both xenobiotics were biotransformed into a hydroxylated derivative of 2-hydroxybenzothiazole by these two strains. The chemical structure of this metabolite was determined by a new NMR methodology: long-range (1)H-(15)N heteronuclear shift correlation without any previous (15)N enrichment of the compound. This powerful NMR tool allowed us to assign the metabolite structure to 2,6-dihydroxybenzothiazole.

Deoxysugars via microbial reduction of 5-acyl-isoxazolines: application to the synthesis of 3-deoxy-D-fructose and derivatives.

5-Acylisoxazolines 3a-d were obtained by 1,3-dipolar cycloaddition from acetoxymethyl vinyl ketone and nitro precursors. Compounds 3a-d were biotransformed by Aspergillus niger into a 1:1 mixture of stereomers of 5-dihydroxyethyl isoxazolines (+)-4a-d (anti) and (-)-5a-d (syn). Both stereomers were obtained in good yields and with high optical purities. Carbonyl reduction by Aspergillus niger produces alcohols of R-configuration thus giving an access to D-sugar analogues: Compound (+)-4d was converted to 3-deoxy-D-erythro-hexulose and several protected derivatives. Total synthesis of 3-deoxy-D-fructose-6-phosphate was also achieved in two steps and 64% overall yield from (+)-4d.

Microbial transformations of 2-substituted benzothiazoles.

The occurrence of benzothiazoles in the environment seems to be restricted to aquatic compartments and is mainly associated with the manufacture and use of the rubber additive 2-mercaptobenzothiazole (MBT) and its derivatives. Although data on benzothiazole biotransformations in natural environments at ppb and ppt levels are scarce, the unsubstituted benzothiazole (BT) and 2-hydroxybenzothiazole (OBT) are generally considered to be biodegradable, whereas 2-methylthiobenzothiazole is recalcitrant. The fungicide 2-thiocyanomethylthiobenzothiazole is assumed to be hydrolysed to MBT, which is then further methylated. At higher concentration levels, similar conclusions can generally be drawn. In addition, BT, MBT, 2-aminobenzothiazole and benzothiazole-2-sulphonate can be biodegraded, although side- and end-products may form. For BT and MBT, threshold concentration were reported above which inhibitory effects on biological treatment processes occur. Due to the limited availability of axenic bacterial cultures capable of benzothiazole mineralization, only the initial steps of the degradation pathways have been elucidated so far.

Argatroban and alteplase in patients with acute myocardial infarction: the ARGAMI Study.

ARGAMI was designed to assess safety and efficacy of argatroban compared with heparin as adjunctive treatment to alteplase in the treatment of patients with acute myocardial infarction. ARGAMI consisted of an open-dose finding study (35 patients) followed by a placebo-controlled study with double dummy technique and 2:1 (argatroban:heparin) randomization. An argatroban dosage of 100 microg/kg bolus plus 3 microg/kg/min infusion for 72 hours was selected for the randomized study in which 82 patients were allocated to argatroban and 45 to heparin (5000 U intravenous bolus, 1000 U/h infusion). Patency of the infarct-related artery (Thrombolysis in Myocardial Infarction [TIMI] grade 2 or 3 flow) after 90 minutes was obtained in 62 patients (76%) allocated to argatroban versus 37 patients (82%) allocated to heparin (p=ns). Angiograms after 24 hours and 5 to 10 days showed low reocclusion rates in both groups. Bleeding complications were observed in 16 patients allocated to argatroban (19.5%) and in 9 patients allocated to heparin (20.0%). One patient allocated to heparin suffered from hemorrhage stroke. Argatroban, given as adjunctive treatment to alteplase, is tolerated well in patients with acute myocardial infarction. Safety and efficacy of the combination alteplase and argatroban (with this dose regimen) are similar to those of alteplase and heparin.

Metabolic control of contractile performance in isolated perfused rat heart. Analysis of experimental data by reaction:diffusion mathematical model.

The intracellular mechanisms of regulation of energy fluxes and respiration in contracting heart cells were studied. For this, we investigated the workload dependencies of the rate of oxygen consumption and metabolic parameters in Langendorff-perfused isolated rat hearts.(31)P NMR spectroscopy was used to study the metabolic changes during transition from perfusion with glucose to that with pyruvate with and without active creatine kinase system. The experimental results showed that transition from perfusion with glucose to that with pyruvate increased the phosphocreatine content and stability of its level at increased workloads. Inhibition of creatine kinase reaction by 15-min infusion of iodoacetamide decreased the maximal developed tension and respiration rates by a factor of two.(31)P NMR data were analyzed by a mathematical model of compartmentalized energy transfer, which is independent from the restrictions of the classical concept of creatine kinase equilibrium. The analysis of experimental data by this model shows that metabolic stability-constant levels of phosphocreatine, ATP and inorganic phosphate-at increased energy fluxes is an inherent property of the compartmentalized system. This explains the observed substrate specificity by changes in mitochondrial membrane potential. The decreased maximal respiration rate and maximal work output of the heart with inhibited creatine kinase is well explained by the rise in myoplasmic ADP concentration. This activates the adenylate kinase reaction in the myofibrillar space and in the mitochondria to fulfil the energy transfer and signal transmission functions, usually performed by creatine kinase. The activity of this system, however, is not sufficient to maintain high enough energy fluxes. Therefore, there is a kinetic explanation for the decreased maximal respiration rate of the heart with inhibited creatine kinase: i.e. a kinetically induced switch from an efficient energy transfer pathway (PCr-CK system) to a non-efficient one (myokinase pathway) within the energy transfer network of the cell under conditions of low apparent affinity of mitochondria to ADP in vivo. This may result in a significant decrease in the thermodynamic affinity of compartmentalized ATPase systems and finally in heart failure.

Common degradative pathways of morpholine, thiomorpholine, and piperidine by Mycobacterium aurum MO1: evidence from (1)H-nuclear magnetic resonance and ionspray mass spectrometry performed directly on the incubation medium.

In order to see if the biodegradative pathways for morpholine and thiomorpholine during degradation by Mycobacterium aurum MO1 could be generalized to other heterocyclic compounds, the degradation of piperidine by this strain was investigated by performing (1)H-nuclear magnetic resonance directly with the incubation medium. Ionspray mass spectrometry, performed without purification of the samples, was also used to confirm the structure of some metabolites during morpholine and thiomorpholine degradation. The results obtained with these two techniques suggested a general pathway for degradation of nitrogen heterocyclic compounds by M. aurum MO1. The first step of the degradative pathway is cleavage of the C---N bond; this leads formation of an intermediary amino acid, which is followed by deamination and oxidation of this amino acid into a diacid. Except in the case of thiodiglycolate obtained from thiomorpholine degradation, the dicarboxylates are completely mineralized by the bacterial cells. A comparison with previously published data showed that this pathway could be a general pathway for degradation by other strains of members of the genus Mycobacterium.

[Coronary atherectomy. An essential tool for specific indications]. / Athérectomie coronaire. Un outil indispensable pour des indications spécifiques.

Technological advances in the manufacturing of stents have extended the indications of angioplasty and considerably reduced the immediate complications, death and myocardial infarction. Nevertheless, intra-stent restenosis remains a problem and some complex lesions are still inaccessible. Atherectomy has not been shown to be effective in limiting restenosis but it has a primordial role in the treatment of lesions of bifurcation and could improve long-term results as a complement of angioplasty and stenting. Rotational atherectomy is still useful, even essential, for lesions which cannot be passed with the balloon and for calcified plaques of atheroma. A possible new indication may be the treatment of intra-stent stenosis. The indications of directional atherectomy are more limited, mainly non-calcified ostial stenosis and of bifurcations of large arteries. The association with stenting has given encouraging results which require confirmation. These techniques have a place in the in the angioplasty physician's arsenal even though they are reserved for specific anatomical situations.

Expression of markers of platelet activation and the interpatient variation in response to abciximab.

Our study concerns the biological effects of abciximab (c7E3 Fab, ReoPro), a powerful new antiplatelet drug that blocks glycoprotein (GP) IIb-IIIa complexes. Samples were examined from 6 patients with coronary artery disease who received a bolus of abciximab followed by a 10- microg/min infusion for at least 18 hours before percutaneous transluminal coronary angioplasty. Inhibition of ADP-induced PA was maximal for 4 patients but partial (79% and 53%) for 2 others during the infusion. Flow cytometry performed with monoclonal antibodies (PAC-1, AP-6, and F26) specific for the "activated" GP IIb-IIIa complex revealed large decreases in the expression of activation markers on platelets during therapy, but these decreases were less marked when inhibition of ADP-induced PA was incomplete. Residual aggregation was seen for all patients during the infusion when TRAP 14-mer peptide or thrombin was the stimulus. Unblocked GP IIb-IIIa complexes were detected on thrombin-stimulated platelets from the patients by immunoelectron microscopy performed using the monoclonal antibody AP-2. Unblocked GP IIb-IIIa complexes were also detected by flow cytometry when platelets preincubated for 1 hour in vitro with abciximab under saturating conditions were (1) incubated with TRAP 14-mer or (2) permeabilized with Triton X-100. In confirming interpatient variation in the platelet response to a standard dose of abciximab, our results also show that an uninhibited internal pool of GP IIb-IIIa complexes may mediate a residual response to strong agonists.

[AS heterozygote hemoglobinopathy and coronary risk]. / Hémoglobinopathie hétérozygote AS et risque coronaire.

There have been several reports of vaso-occlusive events and sudden death in subjects with sickle cell trait. However, the precise mechanism underlying these episodes remains unclear. The clinical observations have been supported by in vitro studies in which haemoglobin AS (Hb AS) red cells showed abnormalities of their filterability, probably related to gelling or polymerisation of the Hb AS. These in vitro studies and reports in the literature of sickle-cell hearts led the authors to investigate the possible association between AS subject and coronary risk. The results of coronary angiography in 9 patients with Hb AS, paired with respect to the usual cardiovascular risk factors, were compared with those of 18 AA subjects. The number of patients who underwent coronary bypass surgery for three-vessel disease was much greater in the AS subjects. However, the difference was not statistically significant. This tendency of AS subjects to develop thrombosis and coronary artery disease requires further study with larger numbers of patients.

Primary cardiac lymphoma in an immunocompetent woman.

We report a fatal primary cardiac non-Hodgkin's lymphoma in a 62 years old immunocompetent woman presenting with tamponade and complete atrioventricular block. CT-scan, echocardiography and autopsy examination showed a tumor largely infiltrating the heart without extracardiac involvement. A surgical biopsy revealed high grade B-cell non-Hodgkin's lymphoma with a misleading myelomonocytic CD68 (KPI) expression. Polymerase Chain Reaction analysis revealed a clonal rearrangement of the immunoglobulin heavy chain gene and confirmed the B-cell origin of the lymphoma. Our report also emphasizes the role of immunohistochemical and molecular techniques in the diagnosis.