Longitudinal MR assessment of hypoxic ischemic injury in the immature rat brain.

Extremely preterm infants commonly show brain injury with long-term structural and functional consequences. Three-day-old (P3) rat pups share some similarities in terms of cerebral development with the very preterm infant (born at 24-28 weeks of gestation). The aim of this study was to assess longitudinally the cerebral structural and metabolic changes resulting from a moderate neonatal hypoxic ischemic injury in the P3 rat pup using high-field (9.4 T) MRI and localized (1) H magnetic resonance spectroscopy techniques. The rats were scanned longitudinally at P3, P4, P11, and P25. Volumetric measurements showed that the percentage of cortical loss in the long term correlated with size of damage 6 h after hypoxia-ischemia, male pups being more affected than female. The neurochemical profiles revealed an acute decrease of most of metabolite concentrations and an increase in lactate 24 h after hypoxia-ischemia, followed by a recovery phase leading to minor metabolic changes at P25 in spite of an abnormal brain development. Further, the increase of lactate concentration at P4 correlated with the cortical loss at P25, giving insight into the early prediction of long-term cerebral alterations following a moderate hypoxia-ischemia insult that could be of interest in clinical practice.

Intrauterine devices and endometrial cancer risk : a pooled analysis of the Epidemiology of Endometrial Cancer Consortium

Intrauterine devices (IUDs), long-acting and reversible contraceptives, induce a number of immunological and biochemical changes in the uterine environment that could affect endometrial cancer (EC) risk. We addressed this relationship through a pooled analysis of data collected in the Epidemiology of Endometrial Cancer Consortium. We combined individual-level data from 4 cohort and 14 case-control studies, in total 8,801 EC cases and 15,357 controls. Using multivariable logistic regression, we estimated pooled odds ratios (pooled-ORs) and 95% confidence intervals (CIs) for EC risk associated with ever use, type of device, ages at first and last use, duration of use and time since last use, stratified by study and adjusted for confounders. Ever use of IUDs was inversely related to EC risk (pooled-OR = 0.81, 95% CI = 0.74-0.90). Compared with never use, reduced risk of EC was observed for inert IUDs (pooled-OR = 0.69, 95% CI = 0.58-0.82), older age at first use (≥35 years pooled-OR = 0.53, 95% CI = 0.43-0.67), older age at last use (≥45 years pooled-OR = 0.60, 95% CI = 0.50-0.72), longer duration of use (≥10 years pooled-OR = 0.61, 95% CI = 0.52-0.71) and recent use (within 1 year of study entry pooled-OR = 0.39, 95% CI = 0.30-0.49). Future studies are needed to assess the respective roles of detection biases and biologic effects related to foreign body responses in the endometrium, heavier bleeding (and increased clearance of carcinogenic cells) and localized hormonal changes.

Chromatin domain boundaries delimited by a histone-binding protein in yeast.

When located next to chromosomal elements such as telomeres, genes can be subjected to epigenetic silencing. In yeast, this is mediated by the propagation of the SIR proteins from telomeres toward more centromeric regions. Particular transcription factors can protect downstream genes from silencing when tethered between the gene and the telomere, and they may thus act as chromatin domain boundaries. Here we have studied one such transcription factor, CTF-1, that binds directly histone H3. A deletion mutagenesis localized the barrier activity to the CTF-1 histone-binding domain. A saturating point mutagenesis of this domain identified several amino acid substitutions that similarly inhibited the boundary and histone binding activities. Chromatin immunoprecipitation experiments indicated that the barrier protein efficiently prevents the spreading of SIR proteins, and that it separates domains of hypoacetylated and hyperacetylated histones. Together, these results suggest a mechanism by which proteins such as CTF-1 may interact directly with histone H3 to prevent the propagation of a silent chromatin structure, thereby defining boundaries of permissive and silent chromatin domains.

Ab initio absorption spectrum of NiO combining molecular dynamics with the embedded cluster approach in a discrete reaction field

We developed a procedure that combines three complementary computational methodologies to improve the theoretical description of the electronic structure of nickel oxide. The starting point is a Car-Parrinello molecular dynamics simulation to incorporate vibrorotational degrees of freedom into the material model. By means ofcomplete active space self-consistent field second-order perturbation theory (CASPT2) calculations on embedded clusters extracted from the resulting trajectory, we describe localized spectroscopic phenomena on NiO with an efficient treatment of electron correlation. The inclusion of thermal motion into the theoretical description allowsus to study electronic transitions that, otherwise, would be dipole forbidden in the ideal structure and results in a natural reproduction of the band broadening. Moreover, we improved the embedded cluster model by incorporating self-consistently at the complete active space self-consistent field (CASSCF) level a discrete (or direct) reaction field (DRF) in the cluster surroundings. The DRF approach offers an efficient treatment ofelectric response effects of the crystalline embedding to the electronic transitions localized in the cluster. We offer accurate theoretical estimates of the absorption spectrum and the density of states around the Fermi level of NiO, and a comprehensive explanation of the source of the broadening and the relaxation of the charge transferstates due to the adaptation of the environment

Selective distribution of lactate dehydrogenase isoenzymes in neurons and astrocytes of human brain.

In vertebrates, the interconversion of lactate and pyruvate is catalyzed by the enzyme lactate dehydrogenase. Two distinct subunits combine to form the five tetrameric isoenzymes of lactate dehydrogenase. The LDH-5 subunit (muscle type) has higher maximal velocity (Vmax) and is present in glycolytic tissues, favoring the formation of lactate from pyruvate. The LDH-1 subunit (heart type) is inhibited by pyruvate and therefore preferentially drives the reaction toward the production of pyruvate. There is mounting evidence indicating that during activation the brain resorts to the transient glycolytic processing of glucose. Indeed, transient lactate formation during physiological stimulation has been shown by 1H-magnetic resonance spectroscopy. However, since whole-brain arteriovenous studies under basal conditions indicate a virtually complete oxidation of glucose, the vast proportion of the lactate transiently formed during activation is likely to be oxidized. These in vivo data suggest that lactate may be formed in certain cells and oxidized in others. We therefore set out to determine whether the two isoforms of lactate dehydrogenase are localized to selective cell types in the human brain. We report here the production and characterization of two rat antisera, specific for the LDH-5 and LDH-1 subunits of lactate dehydrogenase, respectively. Immunohistochemical, immunodot, and western-blot analyses show that these antisera specifically recognize their homologous antigens. Immunohistochemistry on 10 control cases demonstrated a differential cellular distribution between both subunits in the hippocampus and occipital cortex: neurons are exclusively stained with the anti-LDH1 subunit while astrocytes are stained by both antibodies. These observations support the notion of a regulated lactate flux between astrocytes and neurons.

Compartmentalization in membrane rafts defines a pool of N-cadherin associated with catenins and not engaged in cell-cell junctions in melanoma cells.

Melanoma progression is associated with changes in adhesion receptor expression, in particular upregulation of N-cadherin which promotes melanoma cell survival and invasion. Plasma membrane lipid rafts contribute to the compartmentalization of signaling complexes thereby regulating their function, but how they may affect the properties of adhesion molecules remains elusive. In this study, we addressed the question whether lipid rafts in melanoma cells may contribute to the compartmentalization of N-cadherin. We show that a fraction of N-cadherin in a complex with catenins is associated with cholesterol/sphingolipid-rich membrane microdomains in aggressive melanoma cells in vitro and experimental melanomas in vivo. Partitioning of N-cadherin in membrane rafts is not modulated by growth factors and signaling pathways relevant to melanoma progression, is not necessary for cell-cell junctions' establishment or maintenance, and is not affected by cell-cell junctions' and actin cytoskeleton disruption. These results reveal that two independent pools of N-cadherin exist on melanoma cell surface: one pool is independent of lipid rafts and is engaged in cell-cell junctions, while a second pool is localized in membrane rafts and does not participate in cell-cell adhesions. Targeting to membrane rafts may represent a previously unrecognized mechanism regulating N-cadherin function in melanoma cells.

Cancers du sein : comment associer l'hormonothérapie et la radiothérapie en situation adjuvante [How to combine hormonotherapy and radiation treatment in adjuvant breast cancer?]

Combined radiation and hormone therapies have become common clinical practice in recent years for locally-advanced prostate cancers. The use of such concomitant therapy in the treatment of breast disease has been infrequently reported in the literature, but seems justified given the common hormonal dependence of breast cancer and the potential synergistic effect of these two treatment modalities. As adjuvant therapy, two strategies are used in daily clinical practice: upfront aromatase inhibitors or sequentially after a variable delay of tamoxifen. These molecules may, thus, interact with radiotherapy. Retrospectives studies recently published did not show any differences in terms of locoregional recurrences between concurrent or sequential radiohormonotherapy. Lung and skin fibroses due to concurrent treatment are still under debate. Nevertheless, late side effects appeared to be increased by such a treatment, particularly in hypersensitive patients identified at risk by the lymphocyte predictive test. Concurrent radiohormonotherapy should, thus, be delivered cautiously at least for these patients. This article details the potent advantages and risks of concurrent use of adjuvant hormonotherapy and radiotherapy in localized breast cancers.

Lipoprotein lipase and leptin are accumulated in different secretory compartments in rat adipocytes.

Adipose cells produce and secrete several physiologically important proteins, such as lipoprotein lipase (LPL), leptin, adipsin, Acrp30, etc. However, secretory pathways in adipocytes have not been characterized, and vesicular carriers responsible for the accumulation and transport of secreted proteins have not been identified. We have compared the intracellular localization of two proteins secreted from adipose cells: leptin and LPL. Adipocytes accumulate large amounts of both proteins, suggesting that neither of them is targeted to the constitutive secretory pathway. By means of velocity centrifugation in sucrose gradients, equilibrium density centrifugation in iodixanol gradients, and immunofluorescence confocal microscopy, we determined that LPL and leptin were localized in different membrane structures. LPL was found mainly in the endoplasmic reticulum with a small pool being present in low density membrane vesicles that may represent a secretory compartment in adipose cells. Virtually all intracellular leptin was localized in these low density secretory vesicles. Insulin-sensitive Glut4 vesicles did not contain either LPL or leptin. Thus, secretion from adipose cells is controlled both at the exit from the endoplasmic reticulum as well as at the level of "downstream" secretory vesicles.

On the cellular site of two-pore channel TPC1 action in the Poaceae.

The slow vacuolar (SV) channel has been characterized in different dicots by patch-clamp recordings. This channel represents the major cation conductance of the largest organelle in most plant cells. Studies with the tpc1-2 mutant of the model dicot plant Arabidopsis thaliana identified the SV channel as the product of the TPC1 gene. By contrast, research on rice and wheat TPC1 suggested that the monocot gene encodes a plasma membrane calcium-permeable channel. To explore the site of action of grass TPC1 channels, we expressed OsTPC1 from rice (Oryza sativa) and TaTPC1 from wheat (Triticum aestivum) in the background of the Arabidopsis tpc1-2 mutant. Cross-species tpc1 complementation and patch-clamping of vacuoles using Arabidopsis and rice tpc1 null mutants documented that both monocot TPC1 genes were capable of rescuing the SV channel deficit. Vacuoles from wild-type rice but not the tpc1 loss-of-function mutant harbor SV channels exhibiting the hallmark properties of dicot TPC1/SV channels. When expressed in human embryonic kidney (HEK293) cells OsTPC1 was targeted to Lysotracker-Red-positive organelles. The finding that the rice TPC1, just like those from the model plant Arabidopsis and even animal cells, is localized and active in lyso-vacuolar membranes associates this cation channel species with endomembrane function.

Interaction of cationic ultrasmall superparamagnetic iron oxide nanoparticles with human melanoma cells.

Ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) are currently under development for the intracellular delivery of therapeutics. However, the mechanisms of cellular uptake and the cellular reaction to this uptake, independent of therapeutics, are not well defined. The interactions of biocompatible cationic aminoUSPIONs with human cells was studied in 2D and 3D cultures using biochemical and electron microscopy techniques. AminoUSPIONs were internalized by human melanoma cells in 2D and 3D cultures. Uptake was clathrin mediated and the particles localized in lysosomes, inducing activation of the lysosomal cathepsin D and decreasing the expression of the transferrin receptor in human melanoma cells and/or skin fibroblasts. AminoUSPIONs deeply invaded 3D spheroids of human melanoma cells. Thus, aminoUSPIONs can invade tumors and their uptake by human cells induces cell reaction.

Tightness of random knotting.

Long polymers in solution frequently adopt knotted configurations. To understand the physical properties of knotted polymers, it is important to find out whether the knots formed at thermodynamic equilibrium are spread over the whole polymer chain or rather are localized as tight knots. We present here a method to analyze the knottedness of short linear portions of simulated random chains. Using this method, we observe that knot-determining domains are usually very tight, so that, for example, the preferred size of the trefoil-determining portions of knotted polymer chains corresponds to just seven freely jointed segments.

Morphologic and electroretinographic phenotype of SR-BI knockout mice after a long-term atherogenic diet.

PURPOSE: To evaluate functional and ultrastructural changes in the retina of scavenger receptor B1 (SR-BI) knockout (KO) mice consuming a high fat cholate (HFC) diet. METHODS: Three-month-old male KO and wild-type (WT) mice were fed an HFC diet for 30 weeks. After diet supplementation, plasma cholesterol levels and electroretinograms were analyzed. Neutral lipids were detected with oil red O, and immunohistochemistry was performed on cryostat ocular tissue sections. The retina, Bruch's membrane (BM), retinal pigment epithelium (RPE), and choriocapillaris (CC) were analyzed by transmission electron microscopy. RESULTS: Using the WT for reference, ultrastructural changes were recorded in HFC-fed SR-BI KO mice, including lipid inclusions, a patchy disorganization of the photoreceptor outer segment (POS) and the outer nuclear layer (ONL), and BM thickening with sparse sub-RPE deposits. Within the CC, there was abnormal disorganization of collagen fibers localized in ectopic sites with sparse and large vacuolization associated with infiltration of macrophages in the subretinal space, reflecting local inflammation. These lesions were associated with electroretinographic abnormalities, particularly increasing implicit time in a- and b-wave scotopic responses. Abnormal vascular endothelial growth factor (VEGF) staining was detected in the outer nuclear layer. CONCLUSIONS: HFC-fed SR-BI KO mice thus presented sub-RPE lipid-rich deposits and functional and morphologic alterations similar to some features observed in dry AMD. The findings lend further support to the hypothesis that atherosclerosis causes retinal and subretinal damage that increases susceptibility to some forms of AMD.

Differential transgene expression profiles from rAAV2/1 vectors using the tetON and CMV promoters in the rat brain.

The biodistribution of transgene expression in the CNS after localized stereotaxic vector delivery is an important issue for safety of gene therapy for neurological diseases. The cellular specificity of transgene expression from rAAV2/1 vectors using the tetON expression cassette in comparison with the CMV promoter was investigated in the rat nigrostriatal pathway. After intrastriatal injection, although GFP was mainly expressed into neurons with both vectors, the relative proportions of DARPP-32+ projection neurons and parvalbumin+ interneurons were respectively 13:1 and 2:1 for the CMV and tetON vectors. DARP32+ neurons projecting to the globus pallidus were strongly GFP+ with both vectors, whereas those projecting to the substantia nigra pars reticulata (SNpr) were efficiently labeled by the CMV but poorly by the tetON vector. Numerous GFP+ cells were evidenced in the subventricular zone with both vectors. However, in the olfactory bulb (OB), GFP+ neurons were observed with the CMV but not the tetON vector. We conclude that the absence of significant amounts of transgene product in distant regions (SN and OB) constitutes a safety advantage of the AAV2/1-tetON vector for striatal gene therapy. Midbrain injections resulted in selective GFP expression in tyrosine hydroxylase+ neurons by the tetON vector whereas with the CMV vector, GFP+ cells covered a widespread area of the midbrain. The biodistribution of GFP protein corresponded to that of the transcripts and not of the viral genomes. We conclude that the rAAV2/1-tetON vector constitutes an interesting tool for specific transgene expression in midbrain dopaminergic neurons.

A-kinase anchoring proteins: scaffolding proteins in the heart.

The pleiotropic cyclic nucleotide cAMP is the primary second messenger responsible for autonomic regulation of cardiac inotropy, chronotropy, and lusitropy. Under conditions of prolonged catecholaminergic stimulation, cAMP also contributes to the induction of both cardiac myocyte hypertrophy and apoptosis. The formation of localized, multiprotein complexes that contain different combinations of cAMP effectors and regulatory enzymes provides the architectural infrastructure for the specialization of the cAMP signaling network. Scaffolds that bind protein kinase A are called "A-kinase anchoring proteins" (AKAPs). In this review, we discuss recent advances in our understanding of how PKA is compartmentalized within the cardiac myocyte by AKAPs and how AKAP complexes modulate cardiac function in both health and disease.

Differential migration of in vivo primed B and T lymphocytes to lymphoid and non-lymphoid organs.

Our study describes tissue-specific migration of T and B cells during a localized anti-viral immune response. After mouse mammary tumor virus (MMTV) injection, B lymphocytes of the draining lymph node become infected and present a retroviral superantigen to CD4(+) T lymphocytes. Infected B cells receive superantigen-mediated help in a fashion comparable to classical immune responses. To investigate the fate of T and B lymphocytes that had interacted via cognate help in the same peripheral lymph node microenvironment we adoptively transferred them into naive recipients. Here we show that MMTV-infected B cells and superantigen-stimulated T cells were programmed to migrate to distinct sites of the body. Plasmablasts but not T cells migrated to the mammary gland and activated alpha4beta1 integrins were found to have a crucial role in the migration to the mammary gland. In contrast, T cells had a much higher affinity for secondary lymphoid organs and large intestine. This demonstrates that upon antigen-driven B and T lymphocyte interaction in the local draining lymph node a subset-specific homing program for B and T lymphocytes is induced.