The CaMV 35S promoter has a weak expression activity in dark grown tissues of moss Physcomitrella patens.

The constitutive Cauliflower Mosaic Virus 35S promoter (CaMV 35S) is widely used as a tool to express recombinant proteins in plants, but with different success. We previously showed that the expression of an F-actin marker, GFP-talin, in Physcomitrella patens using the CaMV 35S promoter failed to homogenously label moss tissues. Here, we show a significant diminution of the GFP fluorescence in dark grown old moss cells and complete lack of labelling in newly differentiated cells. Furthermore, we demonstrate that stable moss lines harbouring a resistance cassette driven by the CaMV 35S are unable to grow in darkness in the presence of the antibiotic. In contrast to the CaMV 35S, the heat inducible promoter, hsp17.3B showed uniform expression pattern in all cells and tissues following a mild heat shock.

Time-resolved lanthanide luminescence for lab-on-a-chip detection of biomarkers on cancerous tissues.

PDMS-based microfluidic devices combined with lanthanide-based immunocomplexes have been successfully tested for the multiplex detection of biomarkers on cancerous tissues, revealing an enhanced sensitivity compared to classical organic dyes.

Dense granular bodies: a novel nucleoplasmic structure in hibernating dormice.

Dense granular bodies (DGB) are particular structural constituents observed in cell nuclei of different tissues-liver, pancreas, brown adipose tissue, adrenal cortex-of hibernating dormice. They appear as strongly electron-dense clusters of closely packed granules, with thin fibrils spreading out at their periphery. DGB always occur in the nucleoplasm, sometimes making contact with other nuclear structural constituents typical of the hibernating state, such as coiled bodies, amorphous bodies and nucleoplasmic fibrils. DGB are present only during deep hibernation and rapidly disappear upon arousal from hibernation. Cytochemical and immunocytochemical analyses showed that DGB contain ribonucleoproteins and several nucleoplasmic RNA processing factors, suggesting that DGB can represent accumulation sites of splicing factors which are provided to splicing sites when normal metabolic activity is rapidly restored during arousal.

Microarray and RNAi Analysis of P450s in Anopheles gambiae Male and Female Steroidogenic Tissues: CYP307A1 Is Required for Ecdysteroid Synthesis.

In insects, the steroid hormone 20-hydroxyecdysone (20E) coordinates major developmental transitions. While the first and the final steps of 20E biosynthesis are characterized, the pathway from 7-dehydrocholesterol to 5β-ketodiol, commonly referred as the "black box", remains hypothetical and whether there are still unidentified enzymes is unknown. The black box would include some oxidative steps, which are believed to be mediated by P450 enzymes. To identify new enzyme(s) involved in steroid synthesis, we analyzed by small-scale microarray the expression of all the genes encoding P450 enzymes of the malaria mosquito Anopheles gambiae in active steroidogenic organs of adults, ovaries from blood-fed females and male reproductive tracts, compared to inactive steroidogenic organs, ovaries from non-blood-fed females. Some genes encoding P450 enzymes were specifically overexpressed in female ovaries after a blood-meal or in male reproductive tracts but only three genes were found to be overexpressed in active steroidogenic organs of both females and males: cyp307a1, cyp4g16 and cyp6n1. Among these genes, only cyp307a1 has an expression pattern similar to other mosquito steroidogenic genes. Moreover, loss-of-function by transient RNAi targeting cyp307a1 disrupted ecdysteroid production demonstrating that this gene is required for ecdysteroid biosynthesis in Anopheles gambiae.

Lower thigh subcutaneous and higher visceral abdominal adipose tissue content both contribute to insulin resistance.

It is well known that visceral adipose tissue (VAT) is associated with insulin resistance (IR). Considerable debate remains concerning the potential positive effect of thigh subcutaneous adipose tissue (TSAT). Our objective was to observe whether VAT and TSAT are opposite, synergistic or additive for both peripheral and hepatic IR. Fifty-two volunteers (21 male/31 female) between 30 and 75 years old were recruited from the general population. All subjects were sedentary overweight or obese (mean BMI 33.0 ± 3.4 kg/m(2)). Insulin sensitivity was determined by a 4-h hyperinsulinemic-euglycemic clamp with stable isotope tracer dilution. Total body fat and lean body mass were determined by dual X-ray absorptiometry. Abdominal and mid-thigh adiposity was determined by computed tomography. VAT was negatively associated with peripheral insulin sensitivity, while TSAT, in contrast, was positively associated with peripheral insulin sensitivity. Subjects with a combination of low VAT and high TSAT had the highest insulin sensitivity, subjects with a combination of high VAT and low TSAT were the most insulin resistant. These associations remained significant after adjusting for age and gender. These data confirm that visceral excess abdominal adiposity is associated with IR across a range of middle-age to older men and women, and further suggest that higher thigh subcutaneous fat is favorably associated with better insulin sensitivity. This strongly suggests that these two distinct fat distribution phenotypes should both be considered in IR as important determinants of cardiometabolic risk.

Stimulation by leptin of 3H GDP binding to brown adipose tissue of fasted but not fed rats.

OBJECTIVES: To assess the effects of intracerebroventricular (i.c.v.) leptin administration on rats fed ad libitum or fasted on 3H GDP binding to brown adipose tissue (BAT). SUBJECTS: Groups of 5-6 ten-week-old male Wistar rats. EXPERIMENTAL DESIGN: An i.c.v. cannula was inserted and unilateral denervation of interscapular brown adipose tissue (BAT) was performed 5 d before each study. Thereafter, leptin was infused i.c.v. during 72 h while rats were fed ad libitum or fasted. Vehicle-infused, pair-fed or fasted rats were used as controls. MEASUREMENTS: 3H GDP binding to innervated and denervated BAT mitochondria. RESULTS: 3H GDP binding to innervated or denervated BAT of rats fed ab libitum compared to vehicle-infused, pair-fed rats was not increased by i.c.v. leptin. 3H GDP binding was lower in fasted than in fed rats, and the difference was larger in innervated than denervated BAT. I.c.v. leptin increased 3H GDP binding by 30% in innervated, and by 51% in denervated BAT (P < 0.05) in fasted rats. CONCLUSIONS: I.c.v. leptin does not increase 3H GDP binding to BAT of rats fed ad libitum compared to pair-fed (food-restricted) rats. In contrast, i.c.v. leptin produces a mild stimulation of 3H GDP binding to BAT of fasted rats. This effect is not mediated by the sympathetic nervous system, because it is observed in both innervated and denervated BAT. These results are compatible with the concept that, in fasting rats, the decrease in leptin secretion contributes to the reduction in 3H GDP binding to BAT mitochondria.

Precision of a new tool to measure visceral adipose tissue (VAT) using dual-energy X-Ray absorptiometry (DXA).

OBJECTIVE: A new tool to quantify visceral adipose tissue (VAT) over the android region of a total body dual-energy x-ray absorptiometry (DXA) scan has recently been reported. The measurement, CoreScan, is currently available on Lunar iDXA densitometers. The purpose of the study was to determine the precision of the CoreScan VAT measurement, which is critical for understanding the utility of this measure in longitudinal trials. DESIGN AND METHODS: VAT precision was characterized in both an anthropomorphic imaging phantom (measured on 10 Lunar iDXA systems) and a clinical population consisting of obese women (n = 32). RESULTS: The intrascanner precision for the VAT phantom across 9 quantities of VAT mass (0-1,800 g) ranged from 28.4 to 38.0 g. The interscanner precision ranged from 24.7 to 38.4 g. There was no statistical dependence on the quantity of VAT for either the inter- or intrascanner precision result (p = 0.670). Combining inter- and intrascanner precision yielded a total phantom precision estimate of 47.6 g for VAT mass, which corresponds to a 4.8% coefficient of variance (CV) for a 1 kg VAT mass. Our clinical population, who completed replicate total body scans with repositioning between scans, showed a precision of 56.8 g on an average VAT mass of 1110.4 g. This corresponds to a 5.1% CV. Hence, the in vivo precision result was similar to the phantom precision result. CONCLUSIONS: The study suggests that CoreScan has a relatively low precision error in both phantoms and obese women and therefore may be a useful addition to clinical trials where interventions are targeted towards changes in visceral adiposity.

A validated assay for measuring doxorubicin in biological fluids and tissues in an isolated lung perfusion model: matrix effect and heparin interference strongly influence doxorubicin measurements.

Doxorubicin is an antineoplasic agent active against sarcoma pulmonary metastasis, but its clinical use is hampered by its myelotoxicity and its cumulative cardiotoxicity, when administered systemically. This limitation may be circumvented using the isolated lung perfusion (ILP) approach, wherein a therapeutic agent is infused locoregionally after vascular isolation of the lung. The influence of the mode of infusion (anterograde (AG): through the pulmonary artery (PA); retrograde (RG): through the pulmonary vein (PV)) on doxorubicin pharmacokinetics and lung distribution was unknown. Therefore, a simple, rapid and sensitive high-performance liquid chromatography method has been developed to quantify doxorubicin in four different biological matrices (infusion effluent, serum, tissues with low or high levels of doxorubicin). The related compound daunorubicin was used as internal standard (I.S.). Following a single-step protein precipitation of 500 microl samples with 250 microl acetone and 50 microl zinc sulfate 70% aqueous solution, the obtained supernatant was evaporated to dryness at 60 degrees C for exactly 45 min under a stream of nitrogen and the solid residue was solubilized in 200 microl of purified water. A 100 microl-volume was subjected to HPLC analysis onto a Nucleosil 100-5 microm C18 AB column equipped with a guard column (Nucleosil 100-5 microm C(6)H(5) (phenyl) end-capped) using a gradient elution of acetonitrile and 1-heptanesulfonic acid 0.2% pH 4: 15/85 at 0 min-->50/50 at 20 min-->100/0 at 22 min-->15/85 at 24 min-->15/85 at 26 min, delivered at 1 ml/min. The analytes were detected by fluorescence detection with excitation and emission wavelength set at 480 and 550 nm, respectively. The calibration curves were linear over the range of 2-1000 ng/ml for effluent and plasma matrices, and 0.1 microg/g-750 microg/g for tissues matrices. The method is precise with inter-day and intra-day relative standard deviation within 0.5 and 6.7% and accurate with inter-day and intra-day deviations between -5.4 and +7.7%. The in vitro stability in all matrices and in processed samples has been studied at -80 degrees C for 1 month, and at 4 degrees C for 48 h, respectively. During initial studies, heparin used as anticoagulant was found to profoundly influence the measurements of doxorubicin in effluents collected from animals under ILP. Moreover, the strong matrix effect observed with tissues samples indicate that it is mandatory to prepare doxorubicin calibration standard samples in biological matrices which would reflect at best the composition of samples to be analyzed. This method was successfully applied in animal studies for the analysis of effluent, serum and tissue samples collected from pigs and rats undergoing ILP.

Diabetes mellitus, a microRNA-related disease?

Diabetes mellitus is a complex disease resulting in altered glucose homeostasis. In both type 1 and type 2 diabetes mellitus, pancreatic β cells cannot secrete appropriate amounts of insulin to regulate blood glucose level. Moreover, in type 2 diabetes mellitus, altered insulin secretion is combined with a resistance of insulin-target tissues, mainly liver, adipose tissue, and skeletal muscle. Both environmental and genetic factors are known to contribute to the development of the disease. Growing evidence indicates that microRNAs (miRNAs), a class of small noncoding RNA molecules, are involved in the pathogenesis of diabetes. miRNAs function as translational repressors and are emerging as important regulators of key biological processes. Here, we review recent studies reporting changes in miRNA expression in tissues isolated from different diabetic animal models. We also describe the role of several miRNAs in pancreatic β cells and insulin-target tissues. Finally, we discuss the possible use of miRNAs as blood biomarkers to prevent diabetes development and as tools for gene-based therapy to treat both type 1 and type 2 diabetes mellitus.

Extra microchromosome mosaicism in amniotic cells confirmed in fetal tissues.

Mosaicism for an extra microchromosome was discovered in amniotic cell cultures of a 39-year-old woman. Using G, Q, C bands and silver staining, it was concluded that the extra chromosome was bisatellited. Parents' karyotype was normal. Parents elected for termination of the pregnancy. The presence of the extra microchromosome was confirmed in various tissues of the aborted fetus. The literature on the subject is briefly reviewed.

Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting.

Prolonged deprivation of food induces dramatic changes in mammalian metabolism, including the release of large amounts of fatty acids from the adipose tissue, followed by their oxidation in the liver. The nuclear receptor known as peroxisome proliferator-activated receptor alpha (PPARalpha) was found to play a role in regulating mitochondrial and peroxisomal fatty acid oxidation, suggesting that PPARalpha may be involved in the transcriptional response to fasting. To investigate this possibility, PPARalpha-null mice were subjected to a high fat diet or to fasting, and their responses were compared with those of wild-type mice. PPARalpha-null mice chronically fed a high fat diet showed a massive accumulation of lipid in their livers. A similar phenotype was noted in PPARalpha-null mice fasted for 24 hours, who also displayed severe hypoglycemia, hypoketonemia, hypothermia, and elevated plasma free fatty acid levels, indicating a dramatic inhibition of fatty acid uptake and oxidation. It is shown that to accommodate the increased requirement for hepatic fatty acid oxidation, PPARalpha mRNA is induced during fasting in wild-type mice. The data indicate that PPARalpha plays a pivotal role in the management of energy stores during fasting. By modulating gene expression, PPARalpha stimulates hepatic fatty acid oxidation to supply substrates that can be metabolized by other tissues.

Vitamin E, Vitamin A, and carotenoids in male common lizard tissues

Vitamin E, vitamin A, and carotenoids are essential micronutrients for animals because of their antioxidant and immunostimulant functions and their implications for growth, development, and reproduction. In contrast to mammals and birds, information about their occurrence and distribution is generally lacking in reptiles, constraining our understanding of the use of these micronutrients. Using high-performance liquid chromatography, we determined the concentrations of vitamin E, vitamin A, and carotenoids in plasma, storage sites (liver and abdominal fat bodies), and in the colored ventral skin of male Common Lizards, Lacerta vivipara. All tissues shared a similar micronutrient profile, except the liver, which also showed traces of vitamin A(1). The main vitamin E compound present was a-tocopherol followed by lower concentrations of gamma-(beta-)tocopherol. Vitamin A(2) was the main vitamin A compound and it showed the highest concentration in the liver, where vitamin A(2) esters and traces of vitamin A(1) were found. Lutein was the main carotenoid, and it formed esters in the liver and the ventral skin. Zeaxanthin and low concentrations of beta-carotene were also present. The liver was the main storage site for carotenoid and vitamin A, whereas hepatic vitamin E concentrations resembled those present in abdominal Fat bodies. Compared with abdominal fat bodies, the ventral skin contained lower concentrations of vitamin A and vitamin E, but similar concentrations of carotenoicls. These results suggest that important differences exist in micronutrient presence, concentration, and distribution among tissues of lizards and other taxa such as birds and mammals.

Resistance to diet-induced obesity and associated metabolic perturbations in haploinsufficient monocarboxylate transporter 1 mice

The monocarboxylate transporter 1 (MCT1 or SLC16A1) is a carrier of short-chain fatty acids, ketone bodies, and lactate in several tissues. Genetically modified C57BL/6J mice were produced by targeted disruption of the mct1 gene in order to understand the role of this transporter in energy homeostasis. Null mutation was embryonically lethal, but MCT1 (+/-) mice developed normally. However, when fed high fat diet (HFD), MCT1 (+/-) mice displayed resistance to development of diet-induced obesity (24.8% lower body weight after 16 weeks of HFD), as well as less insulin resistance and no hepatic steatosis as compared to littermate MCT1 (+/+) mice used as controls. Body composition analysis revealed that reduced weight gain in MCT1 (+/-) mice was due to decreased fat accumulation (50.0% less after 9 months of HFD) notably in liver and white adipose tissue. This phenotype was associated with reduced food intake under HFD (12.3% less over 10 weeks) and decreased intestinal energy absorption (9.6% higher stool energy content). Indirect calorimetry measurements showed ∼ 15% increase in O2 consumption and CO2 production during the resting phase, without any changes in physical activity. Determination of plasma concentrations for various metabolites and hormones did not reveal significant changes in lactate and ketone bodies levels between the two genotypes, but both insulin and leptin levels, which were elevated in MCT1 (+/+) mice when fed HFD, were reduced in MCT1 (+/-) mice under HFD. Interestingly, the enhancement in expression of several genes involved in lipid metabolism in the liver of MCT1 (+/+) mice under high fat diet was prevented in the liver of MCT1 (+/-) mice under the same diet, thus likely contributing to the observed phenotype. These findings uncover the critical role of MCT1 in the regulation of energy balance when animals are exposed to an obesogenic diet.

Multicentre validation study of nucleic acids extraction from FFPE tissues.

In most pathology laboratories worldwide, formalin-fixed paraffin embedded (FFPE) samples are the only tissue specimens available for routine diagnostics. Although commercial kits for diagnostic molecular pathology testing are becoming available, most of the current diagnostic tests are laboratory-based assays. Thus, there is a need for standardized procedures in molecular pathology, starting from the extraction of nucleic acids. To evaluate the current methods for extracting nucleic acids from FFPE tissues, 13 European laboratories, participating to the European FP6 program IMPACTS (www.impactsnetwork.eu), isolated nucleic acids from four diagnostic FFPE tissues using their routine methods, followed by quality assessment. The DNA-extraction protocols ranged from homemade protocols to commercial kits. Except for one homemade protocol, the majority gave comparable results in terms of the quality of the extracted DNA measured by the ability to amplify differently sized control gene fragments by PCR. For array-applications or tests that require an accurately determined DNA-input, we recommend using silica based adsorption columns for DNA recovery. For RNA extractions, the best results were obtained using chromatography column based commercial kits, which resulted in the highest quantity and best assayable RNA. Quality testing using RT-PCR gave successful amplification of 200 bp-250 bp PCR products from most tested tissues. Modifications of the proteinase-K digestion time led to better results, even when commercial kits were applied. The results of the study emphasize the need for quality control of the nucleic acid extracts with standardised methods to prevent false negative results and to allow data comparison among different diagnostic laboratories.

Immunohistochemical expression of endothelial markers CD31, CD34, von Willebrand Factor and Fli-1 in normal human tissues

Résumé du travail de thèse Introduction : Les différentes cellules endothéliales du lit vasculaire ont de nombreuses similitudes fonctionnelles et morphologiques. Cependant, elles présentent également une importante hétérogénéité structurelle et fonctionnelle qui peut avoir des implications notamment dans l'angiogenèse et le développement des maladies cardio-vasculaires. Peu d'études ont été publiées au sujet de l'expression et de la distribution des marqueurs endothéliaux dans les tissus humain normaux. Objectif : Nous avons étudié l'expression immunohistochimique des marqueurs endothéliaux CD31, CD34, vWF et Fli-1 dans les vaisseaux périphériques du rein, du poumon, de la rate, du foie, du cour et des gros vaisseaux ; incluant l'aorte, la veine cave inférieure, l'artère rénale ainsi que les artères et veines pulmonaires et fémorales. Matériel et méthodes : Les échantillons tissulaires ont été obtenus à partir de matériel d'autopsie et de biopsies. Le matériel a été fixé en formaline et inclus en paraffine. Les coupes de paraffine ont été colorées immunohistochimiquement avec CD31, CD34 et vWF. Les biopsies ont également été colorées immunohistochimiquement avec Fli-1, D2-40 et Lyve-1. Résultats : L'expression immunohistochimique de ces marqueurs est hétérogène dans les différents organes étudiés. Dans le rein, l'endothélium fenêtré des glomérules exprime fortement CD31 et CD34. Par contre, il n'exprime pas ou alors de manière faible et focale vWF. Dans le poumon, les capillaires alvéolaires expriment fortement CD31 et CD34 mais sont habituellement négatifs pour le vWF. L'expression de vWF augmente graduellement avec le calibre vasculaire dans le poumon. Les sinusoïdes de la rate expriment CD31 de manière diffuse mais ils n'expriment pas CD34. Les sinusoïdes du foie expriment CD31 de part et d'autre des lobules. Par contre, CD34 est exprimé seulement dans la région périportale. L'expression de Fli-1 dans les cellules endothéliales est ubiquitaire et ne varie pas suivant le type de vaisseau ou d'organe. Fli-1 est également exprimé dans d'autres types de cellules, essentiellement des lymphocytes. D2-40 est exprimé seulement dans l'endothélium des vaisseaux lymphatiques. L'expression de Lyve-1 dans ce matériel de routine était inconstante et non reproductible. Conclusion : Ces résultats indiquent que l'expression des marqueurs endothéliaux CD31, CD34 et vWF est hétérogène dans le lit vasculaire et qu'elle varie entre différents vaisseaux et différents compartiments anatomiques du même organe. D2-40 ne marque que les cellules endothéliales lymphatiques.