How flexible are poly(para-phenyleneethynylene)s?
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Journal Article↗
Biomedical subjects
Herbert Zimmermann
Publications and source records attributed to Herbert Zimmermann.
Cloning, purification, and identification of the liver canalicular ecto-ATPase as NTPDase8.
Extracellular nucleotides regulate critical liver functions via the activation of specific transmembrane receptors. The hepatic levels of extracellular nucleotides, and therefore the related downstream signaling cascades, are modulated by cell-surface enzymes called ectonucleotidases, including nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), NTPDase2/CD39L1, and ecto-5'-nucleotidase/CD73. The goal of this study was to determine the molecular identity of the canalicular ecto-ATPase/ATPDase that we hypothesized to correspond to the recently cloned NTPDase8. Human and rat NTPDase8 cDNAs were cloned, and the genes were located on chromosome loci 9q34 and 3p13, respectively. The recombinant proteins, expressed in COS-7 and HEK293T cells, were biochemically characterized. NTPDase8 was also purified from rat liver by Triton X-100 solubilization, followed by DEAE, Affigel Blue, and concanavalin A chromatographies. Importantly, NTPDase8 was responsible for the major ectonucleotidase activity in liver. The ion requirement, apparent K(m) values, nucleotide hydrolysis profile, and preference as well as the resistance to azide were similar for recombinant NTPDase8s and both purified rat NTPDase8 and porcine canalicular ecto-ATPase/ATPDase. The partial NH(2)-terminal amino acid sequences of all NTPDase8s share high identity with the purified liver canalicular ecto-ATPase/ATPDase. Histochemical analysis showed high ectonucleotidase activities in bile canaliculi and large blood vessels of rat liver, in agreement with the immunolocalization of NTPDase1, 2, and 8 with antibodies developed for this study. No NTPDase3 expression could be detected in liver. In conclusion, NTPDase8 is the canalicular ecto-ATPase/ATPDase and is responsible for the main hepatic NTPDase activity. The canalicular localization of this enzyme suggests its involvement in the regulation of bile secretion and/or nucleoside salvage.
Adenosine Triphosphatases↗
Polyoxometalates--a new class of potent ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) inhibitors.
Polyoxotungstates were identified as potent inhibitors of NTPDases1, 2, and 3. The most potent compound was K(6)H(2)[TiW(11)CoO(40)], exhibiting K(i) values of 0.140 microM (NTPDase1), 0.910 microM (NTPDase2), and 0.563 microM (NTPDase3). One of the compounds, (NH(4))(18)[NaSb(9)W(21)O(86)], was selective for NTPDases2 and 3 versus NTPDase1. NTPDase inhibition might contribute to the described biological effects of polyoxometalates, including their anti-cancer activity.
Adenosine Triphosphatases↗
Mesomorphism in columnar phases studied by solid-state nuclear magnetic resonance.
In this paper, we present 13C and 1H NMR investigations of 2, 3, 6, 7, 10, 11-hexahexyl-thiotriphenylene (HHTT). The measurements were carried out under both static and magic-angle spinning conditions. The phase diagram of HHTT is K<-->H<-->D(hd)<-->I , where H is a helical phase and D(hd) is a columnar liquid crystal. The motivation was to characterize the molecular order and dynamics and to investigate differences at the molecular level between the two mesophases: H and D(hd). It is shown that D(hd) is a conventional columnar liquid crystal, where the molecular core undergoes fast rotation about the symmetry axis. The orientational order in this mesophase is lower and the temperature dependence of the order parameter is steeper than in other triphenylene-based compounds. On the other hand, in the helical phase the core, similarly to the solid phase, is essentially rigid. The difference between the solid and helical phases is mainly manifested in an increased mobility of the aliphatic chains observed in the latter phase. In addition, the sample exhibits thermal history effects, which are observed in the different behavior upon cooling and heating.
Journal Article↗
Phylloquinone and related radical anions studied by pulse electron nuclear double resonance spectroscopy at 34 GHz and density functional theory.
1H hyperfine (hf) coupling constants of semiquinone radical anions of 1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone, and 2-methyl-3-phytyl-1,4-naphthoquinone in frozen alcoholic solutions were measured using pulse Q-band electron nuclear double resonance spectroscopy. The resolved signals of the quinone protons as well as from hydrogen bond and solvent shell protons were analyzed and assigned. Both in-plane and out-of-plane hydrogen bonding with respect to the pi-plane of the radical is observed. Interactions with nonexchangeable protons from the surrounding matrix are detected and assigned to solvent protons above and below the quinone plane. Density functional theory was used to calculate spin Hamiltonian parameters of the radical anions. Solvent molecules of the first solvent shell that provide hydrogen bonds to the quinones were included in the geometry optimization. The conductor-like screening model was employed to introduce additional effects of the solvent cage. From a comparison of the experimental and calculated hf tensors it is concluded that four solvent molecules are coordinated via hydrogen bonds to the quinone oxygens. For all radicals very good agreement between experimental and calculated data is observed. The influence of different substituents on the spin density distribution and hydrogen bond geometries is discussed.
Anions↗
Nucleotide signaling in nervous system development.
The development of the nervous system requires complex series of cellular programming and intercellular communication events that lead from the early neural induction to the formation of a highly structured central and peripheral nervous system. Neurogenesis continuously takes place also in select regions of the adult mammalian brain. During the past years, a multiplicity of cellular control mechanisms has been identified, ranging from differential transcriptional mediators to inducers or inhibitors of cell specification or neurite outgrowth. While the identification of transcription factors typical for the stage-specific progression has been a topic of key interest for many years, less is known concerning the potential multiplicity of relevant intercellular signaling pathways and the fine tuning of epigenetic gene regulation. Nucleotide receptors can induce a multiplicity of cellular signaling pathways and are involved in multiple molecular interactions, thus opening the possibility of cross talk between several signaling pathways, including growth factors, cytokines, and extracellular matrix components. An increasing number of studies provides evidence for a role of nucleotide signaling in nervous system development. This includes progenitor cell proliferation, cell migration, neuronal and glial cellular interaction and differentiation, and synaptic network formation.
Adenosine Triphosphate↗
Ecto-5'-nucleotidase (cd73)-dependent and -independent generation of adenosine participates in the mediation of tubuloglomerular feedback in vivo.
Tubuloglomerular feedback (TGF) describes a sequence of events linking salt concentrations in tubular fluid at the macula densa to the vascular tone of the afferent arteriole and thus to the glomerular filtration rate (GFR) of the same nephron. The signal transduction pathways of TGF remain incompletely understood, but both ATP release from macula densa cells and local formation of adenosine were suggested to be involved in the process. To test the role of extracellular formation of adenosine by ecto-5'-nucleotidase (cd73) in TGF, in regulation of GFR, and in tubular reabsorption, renal clearance and micropunture experiments were performed in cd73 wild-type (cd73(+/+)) and knockout mice (cd73(-/-)). The cd73(-/-) mice presented normal mean arterial blood pressure, but modestly lower whole kidney and single nephron GFR (SNGFR). Fractional reabsorption of Na(+) and K(+) up to the late proximal tubule, distal tubule, as well as urine were not significantly different between cd73(-/-) and cd73(+/+) mice. Lack of cd73 resulted in a diminished TGF response, as indicated by smaller changes of stop-flow pressure in response to increasing loop of Henle perfusion from 0 to 25 nl/min, smaller differences in SNGFR determined from paired proximal and distal tubular collections, and by smaller fractional changes of distal SNGFR in response to adding 6 nl/min of artificial tubular fluid to free-flowing proximal tubules. The TGF response in cd73(+/+) mice and the residual TGF response in cd73(-/-) mice were completely inhibited by adenosine A(1)-receptor blockade. The results suggest that extracellular formation of adenosine by ecto-5'-nucleotidase (cd73) is dispensable for normal fluid, Na(+), or K(+) reabsorption along the nephron, but contributes to the regulation of GFR. Adenosine generated by both ecto-5'-nucleotidase (cd73)-dependent and -independent mechanisms participates in the mediation of TGF in vivo.
5'-Nucleotidase↗
Adenosine produced via the CD73/ecto-5'-nucleotidase pathway has no impact on erythropoietin production but is associated with reduced kidney weight.
CD73/ecto-5'-nucleotidase, which catalyzes the conversion of adenosine monophosphate to adenosine, has been implicated in vascular homeostasis. The aim of the present study was to evaluate the role of CD73 in erythropoietin (EPO) production and to determine its influence on basal kidney perfusion using a CD73 knockout mutant recently generated by us. Of all organs investigated, kidneys showed the most prominent CD73 activity, preferentially located in peritubular fibroblasts of the renal cortex and the glomerular mesangium. In the absence of CD73, alkaline phosphatase remained unchanged, but tissue adenosine was reduced under control conditions (by 76%) and during normobaric hypoxia (by 72%). Despite the loss of CD73 activity, EPO mRNA and plasma protein concentrations were not different under basal conditions as well as after normobaric hypoxia (8% O2) and carbon monoxide (0.1% CO) inhalation (both for 4 h). Although there were no differences in blood pressure and urine flow volume, average weight of both kidneys was reduced by 21% in the knockout (wild type 7.17+/-1.18 mg g-1 body wt, CD73-/- 5.70+/-1.91 mg g-1 body wt). Measurement of renal plasma flow and glomerular filtration revealed no significant differences when related to respective kidney weights. We conclude that adenosine derived by the extracellular CD73 pathway has no impact on EPO production under basal conditions and after hypoxic challenge but may determine kidney weight.
5'-Nucleotidase↗
Synaptic vesicle proteins under conditions of rest and activation: analysis by 2-D difference gel electrophoresis.
Synaptic vesicles are organelles of the nerve terminal that secrete neurotransmitters by fusion with the presynaptic plasma membrane. Vesicle fusion is tightly controlled by depolarization of the plasma membrane and a set of proteins that may undergo post-translational modifications such as phosphorylation. In order to identify proteins that undergo modifications as a result of synaptic activation, we induced massive exocytosis and analysed the synaptic vesicle compartment by benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE and difference gel electrophoresis (DIGE) followed by MALDI-TOF-MS. We identified eight proteins that revealed significant changes in abundance following nerve terminal depolarization. Of these, six were increased and two were decreased in abundance. Three of these proteins were phosphorylated as detected by Western blot analysis. In addition, we identified an unknown synaptic vesicle protein whose abundance increased on synaptic activation. Our results demonstrate that depolarization of the presynaptic compartment induces changes in the abundance of synaptic vesicle proteins and post-translational protein modification.
Animals↗
Analysis of the synaptic vesicle proteome using three gel-based protein separation techniques.
Synaptic vesicles are key organelles in neurotransmission. Their functions are governed by a unique set of integral and peripherally associated proteins. To obtain a complete protein inventory, we immunoisolated synaptic vesicles from rat brain to high purity and performed a gel-based analysis of the synaptic vesicle proteome. Since the high hydrophobicity of integral membrane proteins hampers their resolution by gel electrophoretic techniques, we applied in parallel three different gel electrophoretic methods for protein separation prior to MS. Synaptic vesicle proteins were subjected to either 1-D SDS-PAGE along with nano-LC ESI-MS/MS or to the 2-D gel electrophoretic techniques benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE, and double SDS (dSDS)-PAGE in combination with MALDI-TOF-MS. We demonstrate that the combination of all three methods provides a comprehensive survey of the proteinaceous inventory of the synaptic vesicle membrane compartment. The identified synaptic vesicle proteins include transporters, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), synapsins, rab and rab-interacting proteins, additional guanine nucleotide triphosphate (GTP) binding proteins, cytoskeletal proteins, and proteins modulating synaptic vesicle exo- and endocytosis. In addition, we identified novel proteins of unknown function. Our results demonstrate that the parallel application of three different gel-based approaches in combination with mass spectrometry permits a comprehensive analysis of the synaptic vesicle proteome that is considerably more complex than previously anticipated.
Animals↗
Uracil nucleotides stimulate human neural precursor cell proliferation and dopaminergic differentiation: involvement of MEK/ERK signalling.
Isolation and propagation of neural stem cells derived from human brain tissue uniquely enables the study of human neurogenesis in vitro. In addition, ex vivo-expanded human neural stem/precursor cells (NPCs) may offer novel therapeutic strategies. We investigated the effects of extracellular nucleotides on the proliferation and differentiation of human mesencephalic neural stem/precursor cells (hmNPCs). When combined with the mitogens epidermal growth factor and fibroblast growth factor 2, UTP (1 microm) boosted proliferation of hmNPCs as shown by increased expression of the proliferation marker proliferating cell nuclear antigen (330%). UTP-induced proliferation was abrogated by the preferential P2Y receptor blocker pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). UTP also stimulated dopaminergic differentiation. Treatment with UTP (100 microm) increased the number of tyrosine hydroxylase (TH)-positive cells and TH protein by 267 and 319% respectively. UTP-stimulated dopaminergic differentiation of hmNPCs was blocked by the P2 receptor antagonists suramin (10 microm) and PPADS (100 microm). In addition, UDP (1 microm) enhanced TH protein expression by 194%. During differentiation, treatment with UTP stimulated the extracellular signal-regulated kinase (ERK) pathway. Both ERK1/2 phosphorylation and dopaminergic differentiation were inhibited by U0126, a selective ERK kinase inhibitor, as well as by suramin. When other P2 receptor agonists (ATP, ADP and adenosine 5'-O-(2-thiophosphate) (ADPbetaS); all 100 microm) were applied, both proliferation and dopaminergic differentiation of NPCs were compromised. We conclude that uracil nucleotides exert specific P2 receptor-mediated effects on midbrain-derived human NPCs, and may be used to enhance both proliferation and dopaminergic differentiation.
Adenine Nucleotides↗
Extracellular nucleotide signaling in adult neural stem cells: synergism with growth factor-mediated cellular proliferation.
We have previously shown that the extracellular nucleoside triphosphate-hydrolyzing enzyme NTPDase2 is highly expressed in situ by stem/progenitor cells of the two neurogenic regions of the adult murine brain: the subventricular zone (type B cells) and the dentate gyrus of the hippocampus (residual radial glia). We explored the possibility that adult multipotent neural stem cells express nucleotide receptors and investigated their functional properties in vitro. Neurospheres cultured from the adult mouse SVZ in the presence of epidermal growth factor and fibroblast growth factor 2 expressed the ecto-nucleotidases NTPDase2 and the tissue non-specific isoform of alkaline phosphatase, hydrolyzing extracellular ATP to adenosine. ATP, ADP and, to a lesser extent, UTP evoked rapid Ca(2+) transients in neurospheres that were exclusively mediated by the metabotropic P2Y(1) and P2Y(2) nucleotide receptors. In addition, agonists of these receptors and low concentrations of adenosine augmented cell proliferation in the presence of growth factors. Neurosphere cell proliferation was attenuated after application of the P2Y(1)-receptor antagonist MRS2179 and in neurospheres from P2Y(1)-receptor knockout mice. In situ hybridization identified P2Y(1)-receptor mRNA in clusters of SVZ cells. Our results infer nucleotide receptor-mediated synergism that augments growth factor-mediated cell proliferation. Together with the in situ data, this supports the notion that extracellular nucleotides contribute to the control of adult neurogenesis.
Adenosine↗
Ectonucleotidases in the nervous system.
Extracellular nucleotides are hydrolysed by enzymes of the plasma membrane with an extracellularly oriented catalytic site (ectonucleotidases). Members of several families of ectonucleotidases can contribute to extracellular nucleotide hydrolysis. They have been characterized in molecular and functional terms. A major role of these enzymes is in the modulation of ligand availability at nucleotide and nucleoside receptors. The enzymes reveal a wide and partially overlapping tissue distribution. The diversity of the individual family members is considerable and it is still difficult to assign identified enzymes to the modulation of purinergic signalling pathways. In the brain, members of all ectonucleotidase-families are expressed. Proposed physiological functions include modulation of synaptic transmission, of the ATP-mediated propagation of glial Ca2+ waves, of microglial function, adult neurogenesis or the control of vascular tone, haemostasis and thromboregulation.
Animals↗
Plasmodium falciparum glutathione S-transferase--structural and mechanistic studies on ligand binding and enzyme inhibition.
Glutathione S-transferase of the malarial parasite Plasmodium falciparum (PfGST) represents a novel class of GST isoenzymes. Since the architecture of the PfGST substrate binding site differs significantly from its human counterparts and there is only this one isoenzyme present in the parasite, PfGST is considered a highly attractive target for antimalarial drug development. Here we report the mechanistic, kinetic, and structural characterization of PfGST as well as its interaction with different ligands. Our data indicate that in solution PfGST is present as a tetramer that dissociates into dimers in the presence of glutathione (GSH). Fluorescence spectroscopy shows that in the presence of GSH GST serves as ligandin for parasitotoxic ferriprotoporphyrin IX with a high- and a low-affinity binding site. This is supported by a clear uncompetitive inhibition type. Site-directed mutagenesis studies demonstrate that neither Cys 86 nor Cys 101 contribute to the peroxidase activity of the enzyme, which is thus performed GSH-dependently at the active site. Tyr 9 is responsible for the deprotonation of GSH and Lys 15, but also Gln 71 are involved in GSH binding. We furthermore report the 2.4 A resolution X-ray structure of PfGST cocrystallized with the inhibitor S-hexylglutathione. In comparison with a previously reported structure obtained by crystal soaking, differences occur at the C-terminal end of helix alpha4 and at the S-hexylmoiety of the inhibitor. We furthermore show that, in contrast to previous reports, the antimalarial drug artemisinin is not metabolized by PfGST.
Animals↗
A combined 17O RAPT and MQ-MAS NMR study of L-leucine.
We report the application of rotor-assisted population transfer (RAPT) to measure the quadrupolar coupling constant (C(q)) for spin 5/2 nuclei. Results from numerical simulations are presented on the magnitude of enhancement factor as a function of frequency offsets, i.e. the RAPT profile. Experimental O17 RAPT profile is traced for the amino acid L-leucine. In addition, results from MQ-MAS experiments are incorporated to determine the quadrupolar asymmetry parameter (eta(q)). Unlike previous reports, the O17 NMR parameters for an amino acid, L-leucine, is reported at a relatively low field of 9.4 T.
Algorithms↗
Immunoisolation of two synaptic vesicle pools from synaptosomes: a proteomics analysis.
The nerve terminal proteome governs neurotransmitter release as well as the structural and functional dynamics of the presynaptic compartment. In order to further define specific presynaptic subproteomes we used subcellular fractionation and a monoclonal antibody against the synaptic vesicle protein SV2 for immunoaffinity purification of two major synaptosome-derived synaptic vesicle-containing fractions: one sedimenting at lower and one sedimenting at higher sucrose density. The less dense fraction contains free synaptic vesicles, the denser fraction synaptic vesicles as well as components of the presynaptic membrane compartment. These immunoisolated fractions were analyzed using the cationic benzyldimethyl-n-hexadecylammonium chloride (BAC) polyacrylamide gel system in the first and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Protein spots were subjected to analysis by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS). We identified 72 proteins in the free vesicle fraction and 81 proteins in the plasma membrane-containing denser fraction. Synaptic vesicles contain a considerably larger number of protein constituents than previously anticipated. The plasma membrane-containing fraction contains synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery and numerous other proteins potentially involved in regulating the functional and structural dynamics of the nerve terminal.
Animals↗
Utilizing ESEEM spectroscopy to locate the position of specific regions of membrane-active peptides within model membranes.
Membrane-active peptides participate in many cellular processes, and therefore knowledge of their mode of interaction with phospholipids is essential for understanding their biological function. Here we present a new methodology based on electron spin-echo envelope modulation to probe, at a relatively high resolution, the location of membrane-bound lytic peptides and to study their effect on the water concentration profile of the membrane. As a first example, we determined the location of the N-terminus of two membrane-active amphipathic peptides, the 26-mer bee venom melittin and a de novo designed 15-mer D,L-amino acid amphipathic peptide (5D-L9K6C), both of which are antimicrobial and bind and act similarly on negatively charged membranes. A nitroxide spin label was introduced to the N-terminus of the peptides and measurements were performed either in H2O solutions with deuterated model membranes or in D2O solutions with nondeuterated model membranes. The lipids used were dipalmitoyl phosphatidylcholine (DPPC) and phosphatidylglycerol (PG), (DPPC/PG (7:3 w/w)), egg phosphatidylcholine (PC) and PG (PC/PG (7:3 w/w)), and phosphatidylethanolamine (PE) and PG (PE/PG, 7:3w/w). The modulation induced by the 2H nuclei was determined and compared with a series of controls that produced a reference "ruler". Actual estimated distances were obtained from a quantitative analysis of the modulation depth based on a simple model of an electron spin situated at a certain distance from the bottom of a layer with homogeneously distributed deuterium nuclei. The N-terminus of both peptides was found to be in the solvent layer in both the DPPC/PG and PC/PG membranes. For PE/PG, a further displacement into the solvent was observed. The addition of the peptides was found to change the water distribution in the membrane, making it "flatter" and increasing the penetration depth into the hydrophobic region.
1,2-Dipalmitoylphosphatidylcholine↗
Rotational motion in the molecular crystals meta- and ortho-carborane studied by deuteron nuclear magnetic resonance.
Spin-lattice and spin-spin-relaxation times, one- and two-dimensional spectra as well as two- and four-time correlation functions were measured for the molecular crystals ortho- and meta-carborane using deuteron nuclear magnetic resonance. It is found that in their noncubic phases these crystals exhibit highly anisotropic motions. In order to allow for a quantitative description of the motional geometry of the carboranes several stochastic models are formulated. By comparison of the model calculations with the experimental results it is found that the dynamics of these quasi-icosahedrally shaped molecules is governed by a composite reorientation process. Here the molecules perform threefold jumps around a molecule-fixed axis which itself can be tilted in four different directions with respect to a crystal-fixed axis. The tilt angle increases significantly with increasing temperature. On the basis of measurements of four-time stimulated-echo functions, implications for dynamic heterogeneity also in comparison with that of supercooled liquids are discussed.
Anisotropy↗