The Use of Background Signal in the Transformation of cDNA-Microarray Measurements

As the application field of microarrays grows, so does the need for appropriate statistical tools to analyse the signal intensity measurements. Normalisation procedures are required to make the signals from different channels and arrays comparable. One objective, which is also the focus of this report, is to remove the curvature seen on plots of the log ratio versus the mean log intensity values of two channels.A number of methods already exist that are based on the assumption of a shift between the measurements of the two channels. In this article, we explore the use of background measurements to estimate and correct for the shift. We compare our proposal with some well known methods by applying them to microarrays from two studies. These two studies investigate the effect of vegetable diets on the gene expression in colon and lung tissue of mice.Graphical illustrations and a robust summary statistic show that all transformations are an improvement to the raw data. Overall, the best results are obtained with our proposed transformation that takes the background measurements into account.     

Tissue-specific expression of ferritin H regulates cellular iron homoeostasis in vivo

Ferritin is a ubiquitously distributed iron-binding protein. Cell culture studies have demonstrated that ferritin plays a role in maintenance of iron homoeostasis and in the protection against cytokine- and oxidant-induced stress. To test whether FerH (ferritin H) can regulate tissue iron homoeostasis in vivo, we prepared transgenic mice that conditionally express FerH and EGFP (enhanced green fluorescent protein) from a bicistronic tetracycline-inducible promoter. Two transgenic models were explored. In the first, the FerH and EGFP transgenes were controlled by the tTA(CMV) (Tet-OFF) (where tTA and CMV are tet transactivator protein and cytomegalovirus respectively). In skeletal muscle of mice bearing the FerH/EGFP and tTA(CMV) transgenes, FerH expression was increased 6.0+/-1.1-fold (mean+/-S.D.) compared with controls. In the second model, the FerH/EGFP transgenes were controlled by an optimized Tet-ON transactivator, rtTA2(S)-S2(LAP) (where rtTA is reverse tTA and LAP is liver activator protein), resulting in expression predominantly in the kidney and liver. In mice expressing these transgenes, doxycycline induced FerH in the kidney by 14.2+/-4.8-fold (mean+/-S.D.). Notably, increases in ferritin in overexpressers versus control littermates were accompanied by an elevation of IRP (iron regulatory protein) activity of 2.3+/-0.9-fold (mean+/-S.D.), concurrent with a 4.5+/-2.1-fold (mean+/-S.D.) increase in transferrin receptor, indicating that overexpression of FerH is sufficient to elicit a phenotype of iron depletion. These results demonstrate that FerH not only responds to changes in tissue iron (its classic role), but can actively regulate overall tissue iron balance.     

Palynological diversity and major evolutionary trends in Cyperaceae

Pollen and orbicule morphology of 84 species, representing 52 genera from all tribes and subfamilies are investigated, in order to assess the systematic value of palynological data and to determine palynological evolutionary trends in Cyperaceae. A total of 90% of the species are examined for the first time with scanning electron microscopy. Pollen grains of Cyperaceae are oblate spheroidal to perprolate in shape, inaperturate to polyporate with opercula or pontopercula on pori or colpi. We distinguished seven different sexine ornamentation patterns. Orbicules occur in all species investigated. Pollen morphological variation within Cyperaceae is considerable and includes dispersal unit; number, location and degree of differentiation of apertural zones; and sexine ornamentation patterns. In subfamily Mapanioideae both tribes can be characterized by palynological synapomorphies. However, in subfamily Cyperoideae, the observed pattern of variation does not fit the most recent molecular phylogeny due to high levels of homoplasy and polymorphism in major pollen characters.     

Halofuginone inhibits Smad3 phosphorylation via the PI3K/Akt and MAPK/ERK pathways in muscle cells: Effect on myotube fusion

Halofuginone, a novel inhibitor of Smad3 phosphorylation, has been shown to inhibit muscle fibrosis and to improve cardiac and skeletal muscle functions in the mdx mouse model of Duchenne muscular dystrophy. Here, we demonstrate that halofuginone promotes the phosphorylation of Akt and mitogen-activated protein kinase (MAPK) family members in a C2 muscle cell line and in primary myoblasts derived from wild-type and mdx mice diaphragms. Halofuginone enhanced the association of phosphorylated Akt and MAPK/extracellular signal-regulated protein kinase (ERK) with the non-phosphorylated form of Smad3, accompanied by a reduction in Smad3 phosphorylation levels. This reduction was reversed by inhibitors of the phosphoinositide 3′-kinase/Akt (PI3K/Akt) and MAPK/ERK pathways, suggesting their specific role in mediating halofuginone's inhibitory effect on Smad3 phosphorylation. Halofuginone enhanced Akt, MAPK/ERK and p38 MAPK phosphorylation and inhibited Smad3 phosphorylation in myotubes, all of which are crucial for myotube fusion. In addition, halofuginone increased the association Akt and MAPK/ERK with Smad3. As a consequence, halofuginone promoted myotube fusion, as reflected by an increased percentage of C2 and mdx myotubes containing high numbers of nuclei, and this was reversed by specific inhibitors of the PI3K and MAPK/ERK pathways. Together, the data suggest a role, either direct or via inhibition of Smad3 phosphorylation, for Akt or MAPK/ERK in halofuginone-enhanced myotube fusion, a feature which is crucial to improving muscle function in muscular dystrophies.     

Application of small interfering RNAs modified by unlocked nucleic acid (UNA) to inhibit the heart-pathogenic coxsackievirus B3

This study describes the first application of unlocked nucleic acid (UNA)-modified small interfering RNAs (siRNAs) directed against a medically relevant target, the coxsackievirus B3. We systematically analyzed the impact of different siRNA modification patterns and observed good compatibility of the introduction of UNA with the maintenance of high antiviral activity. Additionally, the polarity of an siRNA was successfully reversed by modulating the relative stability of the termini with locked nucleic acid (LNA) and UNA as shown in a reporter assay. The potency of the reversed siRNA against the full-length target was, however, too low to inhibit the infectious virus. Altogether, combined modification of siRNAs with LNA und UNA provides a promising approach to alter and improve properties of an siRNA.     

Intracellular Cyclic AMP Inhibits Constitutive and Phorbol Ester-Stimulated Secretory Cleavage of Amyloid Precursor Protein

Abstract: α-Secretase cleaves the full-length Alzheimer's amyloid precursor protein (APP) within the amyloid β peptide sequence, thus precluding amyloid formation. The resultant soluble truncated APP is constitutively secreted. This nonamyloidogenic processing of APP is increased on stimulation of the phospholipase C/protein kinase C pathway by phorbol esters. Here we used C6 cells transfected with APP₇₅₁ to examine whether the α-secretase cleavage is regulated by the adenylate cyclase signal transduction pathway. Forskolin, an activator of adenylate cyclase, inhibited both the constitutive and phorbol ester-stimulated secretion of nexin II (NXII), the secreted product of the α-secretase cleavage of APP₇₅₁. At 1 µ M , forskolin inhibited secretion of NXII by ∼50% without affecting either the intracellular levels of total APP or the secretion of secretory alkaline phosphatase. In contrast, 1,9-dideoxyforskolin, an inactive analogue of forskolin, did not affect secretion of NXII. These results indicated that forskolin specifically inhibited the α-secretase cleavage of APP₇₅₁. Forskolin treatment increased the intracellular concentration of cyclic AMP (cAMP), suggesting that the forskolin effects on APP cleavage may be mediated by cAMP. In support of this suggestion, both dibutyryl cAMP, a cAMP analogue, and isoproterenol, an activator of adenylate cyclase, also inhibited secretion of NXII. These data indicate that forskolin inhibition of the nonamyloidogenic cleavage of APP is mediated by the second messenger cAMP, which together with the protein kinase C signal transduction pathway modulates the secretory cleavage of APP.     

Moderate Prenatal Alcohol Exposure Enhances GluN2B Containing NMDA Receptor Binding and Ifenprodil Sensitivity in Rat Agranular Insular Cortex

Prenatal exposure to alcohol affects the expression and function of glutamatergic neurotransmitter receptors in diverse brain regions. The present study was undertaken to fill a current gap in knowledge regarding the regional specificity of ethanol-related alterations in glutamatergic receptors in the frontal cortex. We quantified subregional expression and function of glutamatergic neurotransmitter receptors (AMPARs, NMDARs, GluN2B-containing NMDARs, mGluR1s, and mGluR5s) by radioligand binding in the agranular insular cortex (AID), lateral orbital area (LO), prelimbic cortex (PrL) and primary motor cortex (M1) of adult rats exposed to moderate levels of ethanol during prenatal development. Increased expression of GluN2B-containing NMDARs was observed in AID of ethanol-exposed rats compared to modest reductions in other regions. We subsequently performed slice electrophysiology measurements in a whole-cell patch-clamp preparation to quantify the sensitivity of evoked NMDAR-mediated excitatory postsynaptic currents (EPSCs) in layer II/III pyramidal neurons of AID to the GluN2B negative allosteric modulator ifenprodil. Consistent with increased GluN2B expression, ifenprodil caused a greater reduction in NMDAR-mediated EPSCs from prenatal alcohol-exposed rats than saccharin-exposed control animals. No alterations in AMPAR-mediated EPSCs or the ratio of AMPARs/NMDARs were observed. Together, these data indicate that moderate prenatal alcohol exposure has a significant and lasting impact on GluN2B-containing receptors in AID, which could help to explain ethanol-related alterations in learning and behaviors that depend on this region.     

Petunia plants escape from negative selection against a transgene by silencing the foreign DNA via methylation

Summary TransgenicPetunia hybrida clones harbouring the T-DNA gene2 ofAgrobacterium tumefaciens were used to test a strategy for the trapping of plant transposable elements. In thePetunia line used, floral variegation is due to the presence of the non-autonomous transposable elementdTph1 at theAn1 locus. The gene2 product converts the auxin precursor indole-3-acetamide and its analogue 1-naphthalene acetamide into the active auxins indole-3-acetic acid and 1-naphthalene acetic acid. Plant cells that express gene2 can use a low concentration of the precursors as auxins and become sensitive to the toxicity of high concentrations of these compounds. By selecting protoplast-derived microcalli or seedlings able to grow on medium with high precursor concentrations, variant plants were obtained in which gene2 was no longer expressed. Southern analysis, using gene2-specific probes, revealed that in one variant the T-DNA was deleted. For 30 other variants no alteration in gene2 structure was observed, indicating that transposable element insertion was not responsible for the inactivation of gene2. Analysis with restriction enzymes allowing discrimination between methylated or non-methylated DNA sequences showed that the inactivated gene2 sequences were methylated. Addition of the in vivo methylation inhibitor 5-azacytidine to the medium led to reactivation of gene2 expression in some of the variants. These observations demonstrated that reversible DNA methylation was the main cause of silencing of gene2 in this system.     

Cytoprotective Effect of Ferritin H in Renal Ischemia Reperfusion Injury

Oxidative stress is a major contributor to kidney injury following ischemia reperfusion. Ferritin, a highly conserved iron-binding protein, is a key protein in the maintenance of cellular iron homeostasis and protection from oxidative stress. Ferritin mitigates oxidant stress by sequestering iron and preventing its participation in reactions that generate reactive oxygen species. Ferritin is composed of two subunit types, ferritin H and ferritin L. Using an in vivo model that enables conditional tissue-specific doxycycline-inducible expression of ferritin H in the mouse kidney, we tested the hypothesis that an increased level of H-rich ferritin is renoprotective in ischemic acute renal failure. Prior to induction of ischemia, doxycycline increased ferritin H in the kidneys of the transgenic mice nearly 6.5-fold. Following reperfusion for 24 hours, induction of neutrophil gelatinous-associated lipocalin (NGAL, a urine marker of renal dysfunction) was reduced in the ferritin H overexpressers compared to controls. Histopathologic examination following ischemia reperfusion revealed that ferritin H overexpression increased intact nuclei in renal tubules, reduced the frequency of tubular profiles with luminal cast materials, and reduced activated caspase-3 in the kidney. In addition, generation of 4-hydroxy 2-nonenal protein adducts, a measurement of oxidant stress, was decreased in ischemia-reperfused kidneys of ferritin H overexpressers. These studies demonstrate that ferritin H can inhibit apoptotic cell death, enhance tubular epithelial viability, and preserve renal function by limiting oxidative stress following ischemia reperfusion injury.