Isolation and functional characterization of a stable complex between photoactivated rhodopsin and the G protein, transducin

Transitory binding between photoactivated rhodopsin (Rho* or Meta II) and the G protein transducin (Gt-GDP) is the first step in the visual signaling cascade. Light causes photoisomerization of the 11-cis-retinylidene chromophore in rhodopsin (Rho) to all-trans-retinylidene, which induces conformational changes that allow Gt-GDP to dock onto the Rho* surface. GDP then dissociates from Gt, leaving a transient nucleotide-empty Rho*-Gt(e) complex before GTP becomes bound, and Gt-GTP then dissociates from Rho*. Further biochemical advances are required before structural studies of the various Rho*-Gt complexes can be initiated. Here, we describe the isolation of n-dodecyl-beta-maltoside solubilized, stable, functionally active, Rho*-Gt(e), Rho(e)*-Gt(e), and 9-cis-retinal/11-cis-retinal regenerated Rho-Gt(e) complexes by sucrose gradient centrifugation. In these complexes, Rho* spectrally remained in its Meta II state, and Gt(e) retained its ability to interact with GTPgammaS. Removal of all-trans-retinylidene from Rho*-Gt(e) had no effect on the stability of the Rho(e)*-Gt(e) complex. Moreover, opsin in the Rho(e)*-Gt(e) complex with an empty nucleotide-binding pocket in Gt and an empty retinoid-binding pocket in Rho was regenerated up to 75% without complex dissociation. These results indicate that once Rho* couples with Gt, the chromophore plays a minor role in stabilizing this complex. Moreover, in complexes regenerated with 9-cis-retinal/11-cis-retinal, Rho retains a conformation similar to Rho* that is stabilized by Gt(e) apo-protein.

Biochemical characterization of detergent-resistant membranes: a systematic approach

Lateral segregation of cholesterol- and sphingomyelin-rich rafts and glycerophospholipid-containing non-raft microdomains has been proposed to play a role in a variety of biological processes. The most compelling evidence for membrane segregation is based on the observation that extraction with non-ionic detergents leads to solubilization of a subset of membrane components only. However, one decade later, a large body of inconsistent detergent-extraction data is threatening the very concept of membrane segregation. We have assessed the validity of the existing paradigms and we show the following. (i) The localization of a membrane component within a particular fraction of a sucrose gradient cannot be taken as a yardstick for its solubility: a variable localization of the DRMs (detergent-resistant membranes) in sucrose gradients is the result of complex associations between the membrane skeleton and the lipid bilayer. (ii) DRMs of variable composition can be generated by using a single detergent, the increasing concentration of which gradually extracts one protein/lipid after another. Therefore any extraction pattern obtained by a single concentration experiment is bound to be 'investigator-specific'. It follows that comparison of DRMs obtained by different detergents in a single concentration experiment is prone to misinterpretations. (iii) Depletion of cholesterol has a graded effect on membrane solubility. (iv) Differences in detergent solubility of the members of the annexin protein family arise from their association with chemically different membrane compartments; however, these cannot be attributed to the 'brick-like' raft-building blocks of fixed size and chemical composition. Our findings demonstrate a need for critical re-evaluation of the accumulated detergent-extraction data.

Equine peripheral blood-derived progenitors in comparison to bone marrow-derived mesenchymal stem cells

Fibroblast-like cells isolated from peripheral blood of human, canine, guinea pig, and rat have been demonstrated to possess the capacity to differentiate into several mesenchymal lineages. The aim of this work was to investigate the possibility of isolating pluripotent precursor cells from equine peripheral blood and compare them with equine bone marrow-derived mesenchymal stem cells. Human mesenchymal stem cells (MSCs) were used as a control for cell multipotency assessment. Venous blood (n = 33) and bone marrow (n = 5) were obtained from adult horses. Mononuclear cells were obtained by Ficoll gradient centrifugation and cultured in monolayer, and adherent fibroblast-like cells were tested for their differentiation potential. Chondrogenic differentiation was performed in serum-free medium in pellet cultures as a three-dimensional model, whereas osteogenic and adipogenic differentiation were induced in monolayer culture. Evidence for differentiation was made via biochemical, histological, and reverse transcription-polymerase chain reaction evaluations. Fibroblast-like cells were observed on day 10 in 12 out of 33 samples and were allowed to proliferate until confluence. Equine peripheral blood-derived cells had osteogenic and adipogenic differentiation capacities comparable to cells derived from bone marrow. Both cell types showed a limited capacity to produce lipid droplets compared to human MSCs. This result may be due to the assay conditions, which are established for human MSCs from bone marrow and may not be optimal for equine progenitor cells. Bone marrow-derived equine and human MSCs could be induced to develop cartilage, whereas equine peripheral blood progenitors did not show any capacity to produce cartilage at the histological level. In conclusion, equine peripheral blood-derived fibroblast-like cells can differentiate into distinct mesenchymal lineages but have less multipotency than bone marrow-derived MSCs under the conditions used in this study.

Retinal differentiation of human bone marrow-derived stem cells by co-culture with retinal pigment epithelium in vitro.

The goal of this study was to assess the in vitro differentiation capacity of human bone marrow-derived stem cells (hBMSCs) along retinal lineages. Mononuclear cells (MNC) were isolated from bone marrow (BM) and mobilized peripheral blood (mPB) using Ficoll-Paque density gradient centrifugation, and were sorted by magnetic-activated cell sorting (MACS) for specific stem cell subsets (CD34(+)CD38(+)/CD34(+)CD38(-)). These cells were then co-cultured on human retinal pigment epithelial cells (hRPE) for 7 days. The expression of stem cell, neural and retina-specific markers was examined by immunostaining, and the gene expression profiles were assessed after FACS separation of the co-cultured hBMSCs by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, in vitro functionality of the differentiated cells was analyzed by quantifying phagocytosis of CY5-labeled photoreceptor outer segments (POS). After 7 days of co-culture, hBMSCs adopted an elongated epithelial-like morphology and expressed RPE-specific markers, such as RPE65 and bestrophin. In addition, these differentiated cells were able to phagocytose OS, one of the main characteristics of native RPE cells. Our data demonstrated that human CD34(+)CD38(-) hBMSC may differentiate towards an RPE-like cell type in vitro and could become a new type of autologous donor cell for regenerative therapy in retinal degenerative diseases.

Quantitative isolation of mouse and human intestinal intraepithelial lymphocytes by elutriation centrifugation

Intestinal intraepithelial lymphocytes (IEL) are specialized subsets of T cells with distinct functional capacities. While some IEL subsets are circulating, others such as CD8alphaalpha TCRalphabeta IEL are believed to represent non-circulating resident T cell subsets [Sim, G.K., Intraepithelial lymphocytes and the immune system. Adv. Immunol., 1995. 58: 297-343.]. Current methods to obtain enriched preparations of intraepithelial lymphocytes are mostly based on Percoll density gradient or magnetic bead-based technologies [Lundqvist, C., et al., Isolation of functionally active intraepithelial lymphocytes and enterocytes from human small and large intestine. J. Immunol. Methods, 1992. 152(2): 253-263.]. However, these techniques are hampered by a generally low yield of isolated cells, and potential artifacts due to the interference of the isolation procedure with subsequent functional assays, in particular, when antibodies against cell surface markers are required. Here we describe a new method for obtaining relatively pure populations of intestinal IEL (55-75%) at a high yield (>85%) by elutriation centrifugation. This technique is equally suited for the isolation and enrichment of intraepithelial lymphocytes of both mouse and human origin. Time requirements for fractionating cell suspensions by elutriation centrifugation are comparable to Percoll-, or MACS-based isolation procedures. Hence, the substantially higher yield and the consistent robust enrichment for intraepithelial lymphocytes, together with the gentle treatment of the cells during elutriation that does not interfere with subsequent functional assays, are important aspects that are in favor of using this elegant technology to obtain unmanipulated, unbiased populations of intestinal intraepithelial lymphocytes, and, if desired, also of pure epithelial cells.

Diagnostic performance and accuracy of 3-D spoiled gradient-dual-echo MRI with water- and fat-signal separation in liver-fat quantification: comparison to liver biopsy

To prospectively evaluate a 3-dimensional spoiled gradient-dual-echo (3D SPGR-DE) magnetic resonance imaging (MRI) sequence for the qualitative and quantitative analysis of liver fat content (LFC) in patients with the suspicion of fatty liver disease using histopathology as the standard of reference.

Gradient estimation in dendritic reinforcement learning

We study synaptic plasticity in a complex neuronal cell model where NMDA-spikes can arise in certain dendritic zones. In the context of reinforcement learning, two kinds of plasticity rules are derived, zone reinforcement (ZR) and cell reinforcement (CR), which both optimize the expected reward by stochastic gradient ascent. For ZR, the synaptic plasticity response to the external reward signal is modulated exclusively by quantities which are local to the NMDA-spike initiation zone in which the synapse is situated. CR, in addition, uses nonlocal feedback from the soma of the cell, provided by mechanisms such as the backpropagating action potential. Simulation results show that, compared to ZR, the use of nonlocal feedback in CR can drastically enhance learning performance. We suggest that the availability of nonlocal feedback for learning is a key advantage of complex neurons over networks of simple point neurons, which have previously been found to be largely equivalent with regard to computational capability.

Intravenous iron treatment in pregnancy: comparison of high-dose ferric carboxymaltose vs. iron sucrose

Oral iron substitution has shown to be insufficient for treatment of severe iron deficiency anemia in pregnancy. Ferric carboxymaltose is a new intravenous (i.v.) iron formulation promising to be more effective and as safe as iron sucrose. We aimed to assess side effects and tolerance of ferric carboxymaltose compared to i.v. iron sucrose in pregnant women.

Safety and efficacy of high-dose intravenous iron carboxymaltose vs. iron sucrose for treatment of postpartum anemia

The purpose of this study is to compare the safety and efficacy of intravenous (IV) high-dose iron carboxymaltose (ICM) with iron sucrose (IS) for the treatment of postpartum anemia.