Henning Ulrich

University of São Paulo, San Paulo, São Paulo, Brazil

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Publications (108)307.03 Total impact

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    ABSTRACT: The N-methyl D-aspartate (NMDA) class of ionotropic glutamate receptors plays important roles in learning and memory as well as in a number of neurological disorders including Huntington's disease and cerebral ischemia. Here, we describe the isolation and characterization of a 2́ F-modified RNA aptamers directed against GluN2A-containing NMDA receptors. By adding a negative selection step towards the closely related AMPA and kainate receptors, the RNA aptamers specifically recognize NMDA receptors with dissociation constants in the nanomolar range. Electrophysiological characterization of these aptamers using rapid perfusion in outside-out patches reveals that they selectively inhibit the GluN2A containing subtype of NMDA receptors with little effect on the AMPA and kainate receptor subtypes. We also demonstrate that this RNA aptamer significantly reduces neurotoxicity in an in vitro model of cerebral ischemia. Given that the RNA based antagonist can be readily modified it can be used as a tool in targeted drug delivery or for imaging purposes in addition to having the potential use as a therapeutic intervention in disorders involving glutamate receptors.
    ACS Chemical Neuroscience 04/2014; · 3.87 Impact Factor
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    Henning Ulrich, Attila Tárnok
    Cytometry Part A 03/2014; 85(3):201-2. · 3.71 Impact Factor
  • H Ulrich, J Bocsi, T Glaser, A Tárnok
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    ABSTRACT: During brain development, a population of uniform embryonic cells migrates and differentiates into a large number of neural phenotypes - origin of the enormous complexity of the adult nervous system. Processes of cell proliferation, differentiation and programmed death of no longer required cells, do not occur only during embryogenesis, but are also maintained during adulthood and are affected in neurodegenerative and neuropsychiatric disease states. As neurogenesis is an endogenous response to brain injury, visible as proliferation (of to this moment silent stem or progenitor cells), its further stimulation can present a treatment strategy in addition to stem cell transfer for cell regeneration therapy. Concise techniques for studying such events in vitro and in vivo permit understanding of underlying mechanisms. Detection of subtle physiological alterations in brain cell proliferation and neurogenesis can be explored, that occur during environmental stimulation, exercise and ageing. Here, we have collected achievements in the field of basic research on applications of cytometry, including automated imaging for quantification of morphological or fluorescence-based parameters in cell cultures, towards imaging of three-dimensional brain architecture together with DNA content and proliferation data. Multi-parameter and more recently in vivo flow cytometry procedures, have been developed for quantification of phenotypic diversity and cell processes that occur during brain development as well as in adulthood, with importance for therapeutic approaches.
    Cell Proliferation 02/2014; 47(1):12-9. · 2.27 Impact Factor
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    ABSTRACT: Oligonucleotide aptamers are selected from libraries typically comprising up to 10(15) different sequences by an iterative process of binding, separation, amplification and purification, called SELEX. During this process, the diversity of the oligonucleotide pool decreases until, presumably, only sequences with highest binding affinities towards chosen targets remain. This selection technique is time-consuming, labor-intensive and expensive. Though well posed in principles, the SELEX procedure is noise sensitive, due to amplification of unspecific-binding sequences, and it is not surprising that aptamer selection is often not successful in practice. In view of that, a follow-up of the progress of selection during its course with simple yet reliable methods is necessary. In this paper, we describe five independent assays to estimate the sequence complexity of SELEX pools including qualitative restriction fragment length polymorphism analysis, melting curve analysis, quantitative fluorescence intensity measurements of bound ssDNA, real time PCR quantification and pool dissociation constant analysis during the progress of aptamer selection against streptavidin. Properties and features of each method are discussed and compared. Pool dissociation constant analysis and sequencing serve as reference methods.
    Journal of pharmaceutical and biomedical analysis 01/2014; 91C:151-159. · 2.45 Impact Factor
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    ABSTRACT: Novel developmental functions have been attributed to the P2X7 receptor (P2X7R) including proliferation stimulation and neural differentiation. Mouse embryonic stem cells (ESC), induced with retinoic acid to neural differentiation, closely assemble processes occurring during neuroectodermal development of the early embryo. P2X7R expression together with the pluripotency marker Oct-4 was highest in undifferentiated ESC. In undifferentiated cells, the P2X7R agonist Bz-ATP accelerated cell cycle entry, which was blocked by the specific P2X7R inhibitor KN-62. ESC induced to neural differentiation with retinoic acid, reduced Oct-4 and P2X7R expression. P2X7R receptor-promoted intracellular calcium fluxes were obtained at lower Bz-ATP ligand concentrations in undifferentiated and in neural-differentiated cells compared to other studies. The presence of KN-62 led to increased number of cells expressing SSEA-1, Dcx and β3-tubulin, as well as the number of SSEA-1 and β3-tubulin-double-positive cells confirming that onset of neuroectodermal differentiation and neuronal fate determination depends on suppression of P2X7R activity. Moreover, an increase in the number of Ki-67 positive cells in conditions of P2X7R inhibition indicates rescue of progenitors into the cell cycle, augmenting the number of neuroblasts and consequently neurogenesis. In embryonic cells, P2X7R expression and activity is upregulated, maintaining proliferation, while upon induction to neural differentiation P2X7 receptor expression and activity needs to be suppressed.
    PLoS ONE 01/2014; 9(5):e96281. · 3.73 Impact Factor
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    ABSTRACT: Stem cells are known for their capacity to self-renew and differentiate into at least one specialized cell type. Mesenchymal stem cells (MSCs) were isolated initially from bone marrow but are now known to exist in all vascularized organ or tissue in adults. MSCs are particularly relevant for therapy due to their simplicity of isolation and cultivation. The International Society for Cellular Therapy (ISCT) has proposed a set of standards to define hMSCs for laboratory investigations and preclinical studies: adherence to plastic in standard culture conditions; in vitro differentiation into osteoblasts, adipocytes, and chondroblasts; specific surface antigen expression in which ≥95% of the cells express the antigens recognized by CD105, CD73, and CD90, with the same cells lacking (≤2% positive) the antigens CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR. In this review we will take an historical overview of how umbilical cord blood, bone marrow, adipose-derived, placental and amniotic fluid, and menstrual blood stem cells, the major sources of human MSC, can be obtained, identified and how they are being used in clinical trials to cure and treat a very broad range of conditions, including heart, hepatic, and neurodegenerative diseases. An overview of protocols for differentiation into hepatocytes, cardiomyocytes, neuronal, adipose, chondrocytes, and osteoblast cells are highlighted. We also discuss a new source of stem cells, induced pluripotent stem cells (iPS cells) and some pathways, which are common to MSCs in maintaining their pluripotent state. © 2013 International Society for Advancement of Cytometry
    Cytometry Part A 10/2013; · 3.71 Impact Factor
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    ABSTRACT: Bradykinin-potentiating peptides from Bothrops jararaca (Bj) discovered in the early 1960s, were the first natural inhibitors of the angiotensin-converting enzyme (ACE). These peptides belong to a large family of snake venom proline-rich oligopeptides (PROs). One of these peptides, Bj-PRO-9a, was essential for defining ACE as effective drug target and development of captopril, an active site-directed inhibitor of ACE used worldwide for the treatment of human arterial hypertension. Recent experimental evidences demonstrated that cardiovascular effects exerted by different Bj-PROs are due to distinct mechanisms besides of ACE inhibition. In the present work, we have investigated the cardiovascular actions of four Bj-PROs, namely Bj-PRO-9a, -11e, -12b e -13a. Bj-PRO-9a acts upon ACE and BK activities to promote blood pressure reduction. Although the others Bj-PROs are also able to inhibit the ACE activity and to potentiate the BK effects, our results indicate that antihypertensive effect evoked by them involve new mechanisms. Bj-PRO-11e and Bj-PRO-12b involves induction of [Ca(2+)]i transients by so far unknown receptor proteins. Moreover, we have suggested argininosuccinate synthetase and M3 muscarinic receptor as targets for cardiovascular effects elicited by Bj-PRO-13a. In summary, the herein reported results provide evidence that Bj-PRO-mediated effects are not restricted to ACE inhibition or potentiation of BK-induced effects and suggest different actions for each peptide for promoting arterial pressure reduction. The present study reveals the complexity of the effects exerted by Bj-PROs for cardiovascular control, opening avenues for the better understanding of blood pressure regulation and for the development of novel therapeutic approaches.
    Peptides 08/2013; · 2.52 Impact Factor
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    ABSTRACT: Throughout the development of the central nervous system, neural crest cells and the primary neural stem cells originate several non-neuronal and neuronal cell types. Undifferentiated stem cells exist in the adult brain, mainly in the dentate gyrus of the hippocampus and in the subventricular zone of the lateral ventricles, and can produce new neurons, participating in brain plasticity and tissue regeneration. Neurogenesis in the embryonic and adult brain occurs under the control of intrinsic and extrinsic factors. However, the mechanisms, by which cell cycle components control neural stem cell proliferation and consequently neurogenesis, still lack further investigation. We discuss here recent knowledge obtained on cell cycle components as key regulators of neural stem and progenitor cell proliferation and differentiation in the embryonic and adult brain.
    Stem cell reviews 07/2013; · 5.08 Impact Factor
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    ABSTRACT: The kinin-B2 receptor (B2BKR) activated by its endogenous ligand bradykinin participates in various metabolic processes including control of arterial pressure and inflammation. Recently, functions for this receptor in brain development and protection against glutamate-provoked excitotoxicity have been proposed. Here, we report neuroprotective properties for bradykinin against organophosphate poisoning using acute hippocampal slices as an in vitro model. Following slice perfusion for 10 min with diisopropylfluorophosphate (DFP) to initiate the noxious stimulus, responses of pyramidal neurons upon an electric impulse were reduced to less than 30% of control amplitudes. Effects on synaptic-elicited population spikes were reverted when preparations had been exposed to bradykinin 30 min after challenging with DFP. Accordingly, bradykinin-induced population spike recovery was abolished by HOE-140, a B2BKR antagonist. However, the kinin-B1 receptor (B1BKR) agonist Lys-des-Arg(9)-bradykinin, inducing phosphorylation of MEK/MAPK and cell death, abolished bradykinin-mediated neuroprotection, an effect, which was reverted by the ERK inhibitor PD98059. In agreement with pivotal B1BKR functions in this process, antagonism of endogenous B1BKR activity alone was enough for restoring population spike activity. On the other hand pralidoxime, an oxime, reactivating AChE after organophosphate poisoning, induced population spike recovery after DFP exposure in the presence of bradykinin and Lys-des-Arg(9)-bradykinin. Lys-des-Arg(9)-bradykinin did not revert protection exerted by pralidoxime, however when instead bradykinin and Ly-des-Arg(9)-bradykinin were superfused together, recovery of population spikes diminished. These findings again confirm the neuroprotective feature of bradykinin, which is, diminished by its endogenous metabolites, stimulating the B1BKR, providing a novel understanding of physiological roles of these receptors.
    Neuroscience 06/2013; · 3.12 Impact Factor
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  • Tina Smuc, Il-Young Ahn, Henning Ulrich
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    ABSTRACT: Aptamers are small nucleic acid molecules capable of binding to a wide range of target molecules with high affinity and specificity. They have been developed and widely used not only as research tools, but also as biosensors, specific antagonists, and diagnostic markers and as protein purification platform for many pharmaceutical and clinical applications. Here, in this paper we will explore biochemical aspects of aptamer-target interactions and show why aptamers rival antibodies in target recognition and purification procedures. This review will focus on strategies of using aptamers as affinity ligands for molecules of therapeutic and pharmaceutical interest including applications in chromatography and capillary electrophoresis for protein and small molecule purification. Moreover, we will also discuss aptamers whose binding parameters can be controlled on demand for diagnostic approaches and used as sensitive receptors in biosensorics. Aptamers have opened up exciting fields in basic and applied research of pharmaceutical and biotechnological interest.
    Journal of pharmaceutical and biomedical analysis 03/2013; 81-82C:210-217. · 2.45 Impact Factor
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    ABSTRACT: Enhanced activity of the sympatho-adrenal axis and augmented circulating catecholamines has been implicated in the development of hypertension. Release of catecholamine from stimulated adrenal medulla chromaffin cells has been shown to be higher and longer in spontaneously hypertensive rats (SHRs), compared with normotensive Wistar rats (NWRs). Whether differences in the functional expression of voltage-dependent calcium channels (VDCCs) of the L-, N-, or P/Q subtypes may contribute to such distinct secretory behaviour, is unknown. We therefore approached here this study in voltage-clamped NWR and SHR chromaffin cells, using 10mM Ba(2+) as charge carrier (IBa) and selective blockers of each channel type. We found that compared with NWR cells, SHR chromaffin cells exhibited the following differences: (1) 30% diminution of the IBa fraction carried by L channels; (2) a doubling of the IBa fraction carried by P/Q channels; (3) more visible current modulation by ATP that could be linked to a 10-fold higher mRNA levels for purinergic receptors of the P2Y2 subtype; and (3) a higher contribution of PQ channels to the transients of the cytosolic calcium concentrations ([Ca(2+)]c) generated by K(+), compared with L channels. These results may contribute to the better understanding of the greater calcium signalling and exocytotic responses of SHR compared with NWR chromaffin cells, found in three previous reports from our laboratories.
    European journal of pharmacology 03/2013; · 2.59 Impact Factor
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    Talita Glaser, Rodrigo R Resende, Henning Ulrich
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    ABSTRACT: Purinergic receptors participate, in almost every cell type, in controlling metabolic activities and many physiological functions including signal transmission, proliferation and differentiation. While most of P2Y receptors induce transient elevations of intracellular calcium concentration by activation of intracellular calcium pools and forward these signals as waves which can also be transmitted into neighboring cells, P2X receptors produce calcium spikes which also include activation of voltage-operating calcium channels. P2Y and P2X receptors induce calcium transients that activate transcription factors responsible for the progress of differentiation through mediators including calmodulin and calcineurin. Expression of P2X2 as well as of P2X7 receptors increases in differentiating neurons and glial cells, respectively. Gene expression silencing assays indicate that these receptors are important for the progress of differentiation and neuronal or glial fate determination. Metabotropic receptors, mostly P2Y1 and P2Y2 subtypes, act on embryonic cells or cells at the neural progenitor stage by inducing proliferation as well as by regulation of neural differentiation through NFAT translocation. The scope of this review is to discuss the roles of purinergic receptor-induced calcium spike and wave activity and its codification in neurodevelopmental and neurodifferentiation processes.
    Cell Communication and Signaling 02/2013; 11(1):12. · 5.09 Impact Factor
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    ABSTRACT: (-)-Carvone is an antinociceptive monoterpene found as the main active constituent of essential oils obtained from plants of the genus Mentha. Here, we have investigated the pharmacology of this monoterpene in dorsal root ganglia (DRG) neurons and TRPV1-expressing HEK293 cells. (-)-carvone at pharmacological active concentrations did not reveal significant cytotoxicity to the cells used in this study, as investigated by neutral red and propidium iodide flow cytometry assays. In calcium imaging experiments 1 mM (-)-carvone increased the cytosolic calcium levels in DRG neurons from 120.6 ± 5.0 nM (basal) to 310.7 ± 23.1 nM (P < 0.05). These effects were completely abolished when neurons were preincubated with calcium-free bath solution or ruthenium-red (5 µM) and capsazepine (10 µM), suggesting the possibility of TRPV1 channel-activation by (-)-carvone. Activity of (-)-carvone on TRPV1 channels was further investigated in HEK293 cells expressing recombinant human TRPV1 channels revealing dose-dependent calcium transients with an EC(50) of 1.3 ± 0.2 mM (Hill coefficient = 2.5). In conclusion, we show for the first time the ability of (-)-carvone to induce increases in cytosolic calcium concentration through TRPV1 activation. © 2013 International Society for Advancement of Cytometry.
    Cytometry Part A 01/2013; · 3.71 Impact Factor
  • Vera S Donnenberg, Henning Ulrich
    Cytometry Part A 01/2013; 83(1):8-10. · 3.71 Impact Factor
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    ABSTRACT: Worldwide the entire human population is at risk of infectious diseases of which a high degree is caused by pathogenic protozoans, worms, bacteria, and virus infections. Moreover the current medications against pathogenic agents are losing their efficacy due to increasing and even further spreading drug resistance. Therefore, there is an urgent need to discover novel diagnostic as well as therapeutic tools against infectious agents. In view of that, the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) represents a powerful technology to target selectively pathogenic factors as well as entire bacteria or viruses. SELEX uses a large combinatorial oligonucleic acid library (DNA or RNA) which is processed a by high-flux in vitro screen of iterative cycles. The selected ligands, termed aptamers, are characterized by high specificity and affinity to their target molecule, which are already exploited in diagnostic and therapeutic applications. In this minireview we will discuss the current status of the SELEX technique applied on bacterial and viral pathogens.
    BioMed research international. 01/2013; 2013:731516.
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    Cytometry Part A 01/2013; 83(1):1-4. · 3.71 Impact Factor
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    ABSTRACT: Alzheimer's disease (AD) is characterized by brain accumulation of the neurotoxic amyloid-β peptide (Aβ) and by loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs). Recent evidence indicates that memory loss and cognitive decline in AD correlate better with the amount of soluble Aβ than with the extent of amyloid plaque deposits in affected brains. Inhibition of nAChRs by soluble Aβ40 is suggested to contribute to early cholinergic dysfunction in AD. Using phage display screening, we have previously identified a heptapeptide, termed IQ, homologous to most nAChR subtypes, binding with nanomolar affinity to soluble Aβ40 and blocking Aβ-induced inhibition of carbamylcholine-induced currents in PC12 cells expressing α7 nAChRs. Using alanine scanning mutagenesis and whole-cell current recording, we have now defined the amino acids in IQ essential for reversal of Aβ40 inhibition of carbamylcholine-induced responses in PC12 cells, mediated by α7 subtypes and other endogenously expressed nAChRs. We further investigated the effects of soluble Aβ, IQ and analogues of IQ on α3β4 nAChRs recombinantly expressed in HEK293 cells. Results show that nanomolar concentrations of soluble Aβ40 potently inhibit the function of α3β4 nAChRs, and that subsequent addition of IQ or its analogues does not reverse this effect. However, co-application of IQ makes the inhibition of α3β4 nAChRs by Aβ40 reversible. These findings indicate that Aβ40 inhibits different subtypes of nAChRs by interacting with specific receptor domains homologous to the IQ peptide, suggesting that IQ may be a lead for novel drugs to block the inhibition of cholinergic function in AD.
    PLoS ONE 01/2013; 8(7):e67194. · 3.73 Impact Factor
  • Henning Ulrich, Carsten Wrenger
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    ABSTRACT: In recent years, the SELEX (Systematic Evolution of Ligands by EXponential enrichment) technology has established itself as a powerful tool in basic research with promising applications in diagnostics and therapeutics. Oligonucleotides with high-affinities to their targets, denominated as aptamers, are obtained from partially random oligonucleotide pools by reiterative in vitro selection cycles and screening for binding activity. The original technique allowing the identification of aptamers binding to soluble targets, has recently been extended in order to produce aptamers binding to complex targets including receptors and ion channels embedded in the plasma membrane as well as whole cell surfaces or parasite organisms. In addition to discussing the most recent developments with focus on possible diagnostic and therapeutic application, we provide a simple protocol which has been successfully used to select for RNA aptamers as allosteric modulators of nicotinic receptor activity.
    Methods in molecular biology (Clifton, N.J.) 01/2013; 986:17-39. · 1.29 Impact Factor
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    ABSTRACT: Conus venoms are rich sources of biologically active peptides that act specifically on ionic channels and metabotropic receptors present at the neuromuscular junction, efficiently paralyzing the prey. Each species of Conus may have 50 to 200 uncharacterized bioactive peptides with pharmacological interest. Conus regius is a vermivorous species that inhabits Northeastern Brazilian tropical waters. In this work, we characterized one peptide with activity on neuronal acetylcholine receptor (nAChR). Crude venom was purified by reverse-phase HPLC and selected fractions were screened and sequenced by mass spectrometry, MALDI-ToF, and ESI-Q-ToF, respectively. A new peptide was identified, bearing two disulfide bridges. The novel 2,701 Da peptide belongs to the cysteine framework I, corresponding to the cysteine pattern CC-C-C. The biological activity of the purified peptide was tested by intracranial injection in mice, and it was observed that high concentrations induced hyperactivity in the animals, whereas lower doses caused breathing difficulty. The activity of this peptide was assayed in patch-clamp experiments, on nAChR-rich cells, in whole-cell configuration. The peptide blocked slow rise-time neuronal receptors, probably α 3 β 4 and/or α 3 β 4 α 5 subtype. According to the nomenclature, the new peptide was designated as α -RgIB.
    International Journal of Peptides 01/2013; 2013:543028.

Publication Stats

955 Citations
307.03 Total Impact Points

Institutions

  • 2002–2014
    • University of São Paulo
      • Department of Biochemistry (IQ)
      San Paulo, São Paulo, Brazil
  • 2013
    • Centre of Excellence for Biosensors, Instrumentation and Process Control
      Solkan, Nova Gorica, Slovenia
  • 2012
    • Universidade Federal de São Paulo
      • Departamento de Neurologia e Neurocirurgia
      São Paulo, Estado de Sao Paulo, Brazil
  • 2011
    • University College London
      Londinium, England, United Kingdom
  • 2001–2009
    • University of Leipzig
      Leipzig, Saxony, Germany
  • 1996–2001
    • Universität Hamburg
      • Center for Molecular Neurobiology (ZMNH)
      Hamburg, Hamburg, Germany
  • 1998–2000
    • Cornell University
      • Department of Molecular Biology and Genetics
      New York City, NY, United States