Biomedical subjects

Elena E Pohl

Publications and source records attributed to Elena E Pohl.

8 recordsLinked to original sources

PubMed2006

A new automated technique for the reconstitution of hydrophobic proteins into planar bilayer membranes. Studies of human recombinant uncoupling protein 1.

Electrophysiological characterisation of the vast number of annotated channel and transport proteins in the postgenomic era would be greatly facilitated by the introduction of rapid and robust methods for the functional incorporation of membrane proteins into defined lipid bilayers. Here, we describe an automated technique for reconstitution of membrane proteins into lipid bilayer membranes, which substantially reduces both the reconstitution time and the amount of protein required for the membrane formation. The method allows the investigation of single protein channels as well as insertion of multiple copies (approximately 10(7)) into a single bilayer. Despite a comparatively large membrane area (up to 300 microm diameter), the high stability of the membrane permits the application of transmembrane voltages up to 300 mV. This feature is especially important for studies of inner membrane mitochondrial proteins, since they act at potentials up to approximately 200 mV under physiological conditions. It is a combination of these advantages that enables the detailed investigation of the minuscule single protein conductances typical for proton transporters. We have applied the new technique for the reconstitution and electrophysiological characterisation of human recombinant uncoupling protein 1, hUCP1, that has been overexpressed in E. coli and purified from inclusion bodies. We demonstrate that hUCP1 activity in the presence of fatty acids is comparable to the activity of UCP1 isolated from brown adipose tissue.

Carrier Proteins↗

PubMed2006

Macrophage/microglia activation factor expression is restricted to lesion-associated microglial cells after brain trauma.

After traumatic brain lesion, microglial cells are rapidly activated, migrate toward the sites of injury, and cause secondary damage that accounts for most of the loss of brain function. In the present study, we have characterized a new macrophage/microglia activation factor (MAF). Using the monocytic cell line U937, we were able to demonstrate that MAF is upregulated after TPA-induced differentiation into macrophages. We have generated a specific antibody against MAF. In BV-2 microglial cells, MAF is partially co-localized with IB4, a classical microglial marker. In addition, we have analyzed the in vivo expression patterns of MAF after entorhinal cortex lesion. We were able to show a substantial upregulation of MAF on selected CD11b(+) and IB4(+) macrophages/microglial cells in the deafferented hippocampus and in the perilesional region, while no MAF expression was detectable on the contralateral side. Confocal microscopy revealed a lysosome-like expression pattern in BV-2 cells, as well as in ECL-associated macrophages/microglial cells in vivo. Furthermore, we were able to demonstrate that U937 cells with downregulated MAF converted slower and to a significantly reduced extent to the macrophageal phenotype after TPA treatment. In addition, MAF downregulation in BV-2 microglial cells substantially reduced the phagocytotic uptake of dextran beads. Our data indicate that MAF is expressed in selected macrophages/microglial cells around the lesion and in the degenerating hippocampus after ECL. Furthermore, MAF expression in monocytic cells seems to play a functional role in the differentiation to a phagocytosing phenotype and may be, at least partially, required for phagocytotic activity, specifically in lesioned tissue after brain trauma.

Animals↗

PubMed2005

NMDA antagonist inhibits the extracellular signal-regulated kinase pathway and suppresses cancer growth.

Glutamate antagonists limit the growth of human cancers in vitro. The mechanism of anticancer action of NMDA antagonists is not known, however. In this article, we report that the NMDA antagonist dizocilpine inhibits the extracellular signal-regulated kinase 1/2 pathway, an intracellular signaling cascade that is activated by growth factors and controls the proliferation of cancer cells. Dizocilpine reduces the phosphorylation of cAMP-responsive element binding protein, suppresses the expression of cyclin D1, up-regulates the cell cycle regulators and tumor suppressor proteins p21 and p53, and increases the number of lung adenocarcinoma cells in the G(2) and S phases of the cell cycle. Silencing of the tumor suppressor protein p21 abolishes antiproliferative action of dizocilpine. Consistent with inhibition of the extracellular signal-regulated kinase 1/2-signaling cascade, dizocilpine reverses the stimulation of proliferation induced by epidermal, insulin, and basic fibroblast growth factors in lung adenocarcinoma cells. Furthermore, dizocilpine prolongs the survival of mice with metastatic lung adenocarcinoma and slows the growth of neuroblastoma and rhabdomyosarcoma in mice. These findings reveal the mechanism of antiproliferative action of dizocilpine and indicate that it may be useful in the therapy of human cancers.

Antineoplastic Agents↗

PubMed2004

In vivo detection limits of magnetically labeled embryonic stem cells in the rat brain using high-field (17.6 T) magnetic resonance imaging.

Stem cell transplantation is a promising therapeutic approach for several neurological disorders. However, it has yet to fulfill its high expectations, partially due to the lack of a reliable noninvasive method for monitoring the biodistribution of the grafted stem cells in vivo. We have used high-resolution magnetic resonance imaging (MRI) at 17.6 T, combined with efficient magnetic labeling of the stem cells with iron oxide nanoparticles, in order to assess the in vivo detection limit in small animal models. Injection of different concentrations of magnetically labeled stem cells in gel phantoms led to significant reductions in image intensity from small cellular clusters of less than 10 cells. To determine the detection limit in vivo, various numbers of both labeled and unlabeled cells were injected stereotactically into the striatum of rats. Significant hypointense signal changes were observed for 100 labeled cells. After injection of approximately 20 labeled cells, signal reduction at the injection site was observed but could not be assigned unambiguously to the cells. Our results show that high-field MRI allows tracking of a minimal number of cells in vivo, well below the number used in previous studies, opening the possibility of gaining new insights into cell migration and differentiation.

Animals↗

PubMed2004

Membrane destabilization by ricin.

Ricin is a promising candidate for the treatment of cancer because it can be selectively targeted to tumor cells via linkage to monoclonal antibodies. Biochemical evidence suggests that escape of ricin or its ribosome-inactivating subunit from an intracellular compartment is mediated by retrograde transport to the endoplasmic reticulum and subsequent direction into the ER-associated degradation pathway. Alternatively, lipase activity of ricin may facilitate leakage from endocytic vesicles. We have observed ricin-mediated release of macromolecular dyes from lipid vesicles that mimic the composition of endosomal membranes. Release of small molecules occurs to the same extent, suggesting an all-or-none mechanism due to bilayer destabilization. The level of accompanying membrane fusion depends on vesicle composition. Since it takes 24 h of incubation before the first traces of lysolipids are detectable by matrix-assisted laser desorption/ionization mass spectrometry, membrane destabilization is not due to the lipase activity of ricin.

Diffusion↗

PubMed2004

Direct impact of T cells on neurons revealed by two-photon microscopy in living brain tissue.

Encephalitogenic T cells invade the brain during neuroinflammation such as multiple sclerosis (MS), inducing damage to myelin sheaths and oligodendrocytes. Only recently, neuronal structures were reported to be a crucial target in the disease. Here, two-photon microscopy using ion-sensitive dyes revealed that within the complex cellular network of living brain tissue, proteolipid protein (PLP)-specific T cells and T cells recognizing the nonmurine antigen ovalbumin (OVA) directly and independently of the major histocompatibility complex (MHC) contact neurons in which they induce calcium oscillations. T cell contact finally resulted in a lethal increase in neuronal calcium levels. This could be prevented by blocking both perforin and glutamate receptors. For the first time, our data provide direct insight into the activity of T cells in the living brain and their detrimental impact on neurons.

Animals↗

PubMed2003

Transport kinetics of uncoupling proteins. Analysis of UCP1 reconstituted in planar lipid bilayers.

According to alternative hypotheses, mitochondrial uncoupling protein 1 (UCP1) is either a proton channel ("buffering model") or a fatty acid anion carrier ("fatty acid cycling"). Transport across the proton channel along a chain of hydrogen bonds (Grotthus mechanism) may include fatty acid carboxyl groups or occur in the absence of fatty acids. In this work, we demonstrate that planar bilayers reconstituted with UCP1 exhibit an increase in membrane conductivity exclusively in the presence of fatty acids. Hence, we can exclude the hypothesis considering a preexisting H+ channel in UCP1, which does not require fatty acid for function. The augmented conductivity is nearly completely blocked by ATP. Direct application of transmembrane voltage and precise current measurements allowed determination of ATP-sensitive conductances at 0 and 150 mV as 11.5 and 54.3 pS, respectively, by reconstituting nearly 3 x 10(5) copies of UCP1. The proton conductivity measurements carried out in presence of a pH gradient (0.4 units) allowed estimation of proton turnover numbers per UCP1 molecule. The observed transport rate of 14 s-1 is compatible both with carrier and channel nature of UCP1.

Adenosine Triphosphate↗

PubMed2002

Origin of membrane dipole potential: contribution of the phospholipid fatty acid chains.

The large intrinsic membrane dipole potential, phi(d), is important for protein insertion and functioning as well as for ion transport across natural and model membranes. However, the origin of phi(d) is controversial. From experiments carried out with lipid monolayers, a significant dependence on the fatty acid chain length is suggested, whereas in experiments with lipid bilayers, the contribution of additional -CH(2)-groups seems negligibly small compared with that of the phospholipid carbonyl groups and lipid-bound water molecules. To compare the impact of the -CH(2)-groups of dipalmitoylphosphatidylcholine (DPPC) near and far from the glycerol backbone, we have varied the structure of DPPC by incorporation of sulfur atoms in place of methylene groups in different positions of the fatty acid chain. The phi(d) of symmetric lipid bilayers containing one heteroatom was obtained from the charge relaxation of oppositely charged hydrophobic ions. We have found that the substitution for a S-atom of a -CH(2)-group decreases phi(d). The effect (deltaphi(d) = -22.6 mV) is most pronounced for S-atoms near the lipid head group while a S-atom substitution in the C(13)- or C(14)-position of the hydrocarbon chain does not effect the bilayer dipole potential. Most probably deltaphi(d) does not originate from an altered dipole potential of the acyl chain containing an heteroatom but is mediated by the disruption of chain packing, leading to a decreased density of lipid dipoles in the membrane.

1,2-Dipalmitoylphosphatidylcholine↗