Publications by authors named "Cord-Michael Becker"

62 Publications

A proline-rich motif in the large intracellular loop of the glycine receptor α1 subunit interacts with the Pleckstrin homology domain of collybistin.

J Adv Res 2021 03 8;29:95-106. Epub 2020 Oct 8.

Department of Biochemistry, German University in Cairo, Egypt.

Introduction: The inhibitory glycine receptor (GlyR), a mediator of fast synaptic inhibition, is located and held at neuronal synapses through the anchoring proteins gephyrin and collybistin. Stable localization of neurotransmitter receptors is essential for synaptic function. In case of GlyRs, only beta subunits were known until now to mediate synaptic anchoring.

Objectives: We identified a poly-proline II helix (PPII) in position 365-373 of the intra-cellular TM3-4 loop of the human GlyRα1 subunit as a novel potential synaptic anchoring site. The potential role of the PPII helix as synaptic anchoring site was tested.

Methods: Glycine receptors and collybistin variants were generated and recombinantly expressed in HEK293 cells and cultured neurons. Receptor function was assessed using patch-clamp electrophysiology, protein-protein interaction was studied using co-immuno-precipitation and pulldown experiments.

Results: Recombinantly expressed collybistin bound to isolated GlyRα1 TM3-4 loops in GST-pulldown assays. When the five proline residues P365A, P366A, P367A, P369A, P373A (GlyRα1) located in the GlyRα1-PPII helix were replaced by alanines, the PPII secondary structure was disrupted. Recombinant GlyRα1 mutant subunits displayed normal cell surface expression and wildtype-like ion channel function, but binding to collybistin was abolished. The GlyRα1-collybistin interaction was independently confirmed by o-immunoprecipitation assays using full-length GlyRα1 subunits. Surprisingly, the interaction was not mediated by the SH3 domain of collybistin, but by its Pleckstrin homology (PH) domain. The mutation GlyRα1, identified in a hyperekplexia patient, is also disrupting the PPII helix, and caused reduced collybistin binding.

Conclusion: Our data suggest a novel interaction between α1 GlyR subunits and collybistin, which is physiologically relevant in vitro and in vivo and may contribute to postsynaptic anchoring of glycine receptors.
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http://dx.doi.org/10.1016/j.jare.2020.09.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8020344PMC
March 2021

PKA and PKC Modulators Affect Ion Channel Function and Internalization of Recombinant Alpha1 and Alpha1-Beta Glycine Receptors.

Front Mol Neurosci 2018 14;11:154. Epub 2018 May 14.

Department of Biochemistry, German University in Cairo, New Cairo, Egypt.

Glycine receptors (GlyRs) are important mediators of fast inhibitory neurotransmission in the mammalian central nervous system. Their function is controlled by multiple cellular mechanisms, including intracellular regulatory processes. Modulation of GlyR function by protein kinases has been reported for many cell types, involving different techniques, and often yielding contradictory results. Here, we studied the effects of protein kinase C (PKC) and cAMP-dependent protein kinase A (PKA) on glycine induced currents in HEK293 cells expressing human homomeric α1 and heteromeric α1-β GlyRs using whole-cell patch clamp techniques as well as internalization assays. In whole-cell patch-clamp measurements, modulators were applied in the intracellular buffer at concentrations between 0.1 μM and 0.5 μM. EC of glycine increased upon application of the protein kinase activators Forskolin and phorbol-12-myristate-13-acetate (PMA) but decreased in the presence of the PKC inhibitor Staurosporine aglycon and the PKA inhibitor H-89. Desensitization of recombinant α1 receptors was significantly increased in the presence of Forskolin. Staurosporine aglycon, on the other hand decreased desensitization of heteromeric α1-β GlyRs. The time course of receptor activation was determined for homomeric α1 receptors and revealed two simultaneous effects: cells showed a decrease of EC after 3-6 min of establishing whole-cell configuration. This effect was independent of protein kinase modulators. All modulators of PKA and PKC, however, produced an additional shift of EC, which overlay and eventually exceeded the cells intrinsic variation of EC. The effect of kinase activators was abolished if the corresponding inhibitors were co-applied, consistent with PKA and PKC directly mediating the modulation of GlyR function. Direct effects of PKA- and PKC-modulators on receptor expression on transfected HEK cells were monitored within 15 min of drug application, showing a significant increase of receptor internalization with PKA and PKC activators, while the corresponding inhibitors had no significant effect on receptor surface expression or internalization. Our results confirm the observation that phosphorylation via PKA and PKC has a direct effect on the GlyR ion channel complex and plays an important role in the fine-tuning of glycinergic signaling.
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http://dx.doi.org/10.3389/fnmol.2018.00154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5961436PMC
May 2018

Loss of glycine receptors containing the α3 subunit compromises auditory nerve activity, but not outer hair cell function.

Hear Res 2016 07 18;337:25-34. Epub 2016 May 18.

Department of Otorhinolaryngology, Saarland University Medical Center, Homburg/Saar, Germany.

Inhibitory glycine receptors containing the α3 subunit (GlyRα3) regulate sensory information processing in the CNS and retina. In previous work, we demonstrated the presence of postsynaptic GlyRα3 immunoreactivity at efferent synapses of the medial and lateral olivocochlear bundle in the organ of Corti; however, the role of these α3-GlyRs in auditory signalling has remained elusive. The present study analyzes distortion-product otoacoustic emissions (DPOAEs) and auditory brainstem responses (ABRs) of knockout mice with a targeted inactivation of the Glra3 gene (Glra3(-/-)) and their wildtype littermates (Glra3(+/+)) before and seven days after acoustic trauma (AT; 4-16 kHz, 120 dB SPL, 1 h). Before AT, DPOAE thresholds were slightly, but significantly lower, and DPOAE amplitudes were slightly larger in Glra3(-/-) as compared to Glra3(+/+) mice. While click- and f-ABR thresholds were similar in both genotypes before AT, threshold-normalized click-ABR wave I amplitudes were smaller in Glra3(-/-) mice as compared to their wildtype littermates. Following AT, both the decrement of ABR wave I amplitudes and the delay of wave I latencies were more pronounced in Glra3(-/-) than Glra3(+/+) mice. Accordingly, correlation between early click-evoked ABR signals (0-2.5 ms from stimulus onset) before and after AT was significantly reduced for Glra3(-/-) as compared to Glra3(+/+) mice. In summary, these results show that loss of α3-GlyRs compromises suprathreshold auditory nerve activity, but not outer hair cell function.
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http://dx.doi.org/10.1016/j.heares.2016.05.004DOI Listing
July 2016

Posttranslational modification and mutation of histidine 50 trigger alpha synuclein aggregation and toxicity.

Mol Neurodegener 2015 Mar 11;10. Epub 2015 Mar 11.

Department of Molecular Neurology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 91054, Erlangen, Germany.

Background: Aggregation and aggregation-mediated formation of toxic alpha synuclein (aSyn) species have been linked to the pathogenesis of sporadic and monogenic Parkinson's disease (PD). A novel H50Q mutation of aSyn, resulting in the substitution of histidine by glutamine, has recently been identified in PD patients. We have previously shown that the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) induces the formation of HNE-aSyn adducts, thereby promoting aSyn oligomerization and increasing its extracellular toxicity to human dopaminergic neurons. Intriguingly, we identified histidine 50 (H50) of aSyn as one of the HNE modification target residues. These converging lines of evidence support the hypothesis that changes in H50 via posttranslational modification (PTM) and mutation trigger the formation of aggregated, toxic aSyn species, which interfere with cellular homeostasis. In the present study, we aim to elucidate 1) the role of H50 in HNE-mediated aSyn aggregation and toxicity, and 2) the impact of H50 mutation on aSyn pathology. Besides the PD-related H50Q, we analyze a PD-unrelated control mutation, in which H50 is replaced by an arginine residue (H50R).

Results: Analysis of HNE-treated aSyn revealed that H50 is the most susceptible residue of aSyn to HNE modification and is crucial for HNE-mediated aSyn oligomerization. Overexpression of aSyn with substituted H50 in H4 neuroglioma cells reduced HNE-induced cell damage, indicating a pivotal role of H50 in HNE modification-induced aSyn toxicity. Furthermore, we showed in vitro that H50Q/R mutations substantially increase the formation of high density and fibrillar aSyn species, and potentiate the oligomerization propensity of aSyn in the presence of a nitrating agent. Cell-based experiments also revealed that overexpression of H50Q aSyn in H4 cells promotes aSyn oligomerization. Importantly, overexpression of both H50Q/R aSyn mutants in H4 cells significantly increased cell death when compared to wild type aSyn. This increase in cell death was further exacerbated by the application of H2O2.

Conclusion: A dual approach addressing alterations of H50 showed that either H50 PTM or mutation trigger aSyn aggregation and toxicity, suggesting an important role of aSyn H50 in the pathogenesis of both sporadic and monogenic PD.
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http://dx.doi.org/10.1186/s13024-015-0004-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4365527PMC
March 2015

Disturbed neuronal ER-Golgi sorting of unassembled glycine receptors suggests altered subcellular processing is a cause of human hyperekplexia.

J Neurosci 2015 Jan;35(1):422-37

Institute for Clinical Neurobiology, Julius-Maximilians-University of Würzburg, 97078 Würzburg, Germany,

Recent studies on the pathogenic mechanisms of recessive hyperekplexia indicate disturbances in glycine receptor (GlyR) α1 biogenesis. Here, we examine the properties of a range of novel glycine receptor mutants identified in human hyperekplexia patients using expression in transfected cell lines and primary neurons. All of the novel mutants localized in the large extracellular domain of the GlyR α1 have reduced cell surface expression with a high proportion of receptors being retained in the ER, although there is forward trafficking of glycosylated subpopulations into the ER-Golgi intermediate compartment and cis-Golgi compartment. CD spectroscopy revealed that the mutant receptors have proportions of secondary structural elements similar to wild-type receptors. Two mutants in loop B (G160R, T162M) were functional, but none of those in loop D/β2-3 were. One nonfunctional truncated mutant (R316X) could be rescued by coexpression with the lacking C-terminal domain. We conclude that a proportion of GlyR α1 mutants can be transported to the plasma membrane but do not necessarily form functional ion channels. We suggest that loop D/β2-3 is an important determinant for GlyR trafficking and functionality, whereas alterations to loop B alter agonist potencies, indicating that residues here are critical elements in ligand binding.
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http://dx.doi.org/10.1523/JNEUROSCI.1509-14.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4287157PMC
January 2015

Single expressed glycine receptor domains reconstitute functional ion channels without subunit-specific desensitization behavior.

J Biol Chem 2014 Oct 20;289(42):29135-47. Epub 2014 Aug 20.

Institute of Biochemistry, Emil Fischer Center, Friedrich-Alexander University Erlangen-Nürnberg, Fahrstrasse 17, 91054 Erlangen, Germany, the Institute for Clinical Neurobiology, University of Würzburg, Versbacherstrasse 5, 97078 Würzburg, Germany, and

Cys loop receptors are pentameric arrangements of independent subunits that assemble into functional ion channels. Each subunit shows a domain architecture. Functional ion channels can be reconstituted even from independent, nonfunctional subunit domains, as shown previously for GlyRα1 receptors. Here, we demonstrate that this reconstitution is not restricted to α1 but can be transferred to other members of the Cys loop receptor family. A nonfunctional GlyR subunit, truncated at the intracellular TM3-4 loop by a premature stop codon, can be complemented by co-expression of the missing tail portion of the receptor. Compared with α1 subunits, rescue by domain complementation was less efficient when GlyRα3 or the GABAA/C subunit ρ1 was used. If truncation disrupted an alternative splicing cassette within the intracellular TM3-4 loop of α3 subunits, which also regulates receptor desensitization, functional rescue was not possible. When α3 receptors were restored by complementation using domains with and without the spliced insert, no difference in desensitization was found. In contrast, desensitization properties could even be transferred between α1/α3 receptor chimeras harboring or lacking the α3 splice cassette proving that functional rescue depends on the integrity of the alternative splicing cassette in α3. Thus, an intact α3 splicing cassette in the TM3-4 loop environment is indispensable for functional rescue, and the quality of receptor restoration can be assessed from desensitization properties.
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http://dx.doi.org/10.1074/jbc.M114.559138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4200267PMC
October 2014

Peptide labeling with photoactivatable trifunctional cadaverine derivative and identification of interacting partners by biotin transfer.

Anal Biochem 2014 Jul 13;456:14-21. Epub 2014 Apr 13.

Institut für Biochemie, Emil Fischer Zentrum, Friedrich Alexander Universität Erlangen-Nürnberg, 91054 Erlangen, Germany. Electronic address:

A new photoactivatable trifunctional cross-linker, cBED (cadaverine-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3'-dithiopropionate), was synthesized by chemical conversion of sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3'-dithiopropionate) with cadaverine. This cross-linker was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. cBED is based on sulfo-SBED that has a photoactivatable azido group, a cleavable disulfide bond for label transfer methods, and a biotin moiety for highly sensitive biotin/avidin detection. By ultraviolet (UV) light, the azido group is converted to a reactive nitrene, transforming transient bindings of interacting structures to covalent bonds. In contrast to the sulfo-N-hydroxysuccinimide (sulfo-NHS) moiety of sulfo-SBED, which attaches quite unspecifically to amino groups, cBED includes a cadaverine moiety that can be attached by transglutaminase more specifically to certain glutamine residues. For instance, thymosin β4 can be labeled with cBED using tissue transglutaminase. By high-resolution HPLC/ESI-MS (electrospray ionization-mass spectrometry) and tandem MS (MS/MS) of the trypsin digest, it was established that glutamine residues at positions 23 and 36 were labeled, whereas Q39 showed no reactivity. The covalent binding of cBED to thymosin β4 did not influence its G-actin sequestering activity, and the complex could be used to identify new interaction partners. Therefore, cBED can be used to better understand the multifunctional role of thymosin β4 as well as of other proteins and peptides.
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http://dx.doi.org/10.1016/j.ab.2014.04.003DOI Listing
July 2014

Oxidative stress-induced posttranslational modifications of alpha-synuclein: specific modification of alpha-synuclein by 4-hydroxy-2-nonenal increases dopaminergic toxicity.

Mol Cell Neurosci 2013 May 28;54:71-83. Epub 2013 Jan 28.

Institute of Biochemistry (Emil-Fischer-Center), Friedrich-Alexander-University of Erlangen Nürnberg, 91054 Erlangen, Germany.

Aggregation and neurotoxicity of misfolded alpha-synuclein (αSyn) are crucial mechanisms for progressive dopaminergic neurodegeneration associated with Parkinson's disease (PD). Posttranslational modifications (PTMs) of αSyn caused by oxidative stress, including modification by 4-hydroxy-2-nonenal (HNE-αSyn), nitration (n-αSyn), and oxidation (o-αSyn), have been implicated to promote oligomerization of αSyn. However, it is yet unclear if these PTMs lead to different types of oligomeric intermediates. Moreover, little is known about which PTM-derived αSyn species exerts toxicity to dopaminergic cells. In this study, we directly compared aggregation characteristics of HNE-αSyn, n-αSyn, and o-αSyn. Generally, all of them promoted αSyn oligomerization. Particularly, HNE-αSyn and n-αSyn were more prone to forming oligomers than unmodified αSyn. Moreover, these PTMs prevented the formation of amyloid-like fibrils, although HNE-αSyn and o-αSyn were able to generate protofibrillar structures. The cellular effects associated with distinct PTMs were studied by exposing modified αSyn to dopaminergic Lund human mesencephalic (LUHMES) neurons. The cellular toxicity of HNE-αSyn was significantly higher than other PTM species. Furthermore, we tested the toxicity of HNE-αSyn in dopaminergic LUHMES cells and other cell types with low tyrosine hydroxylase (TH) expression, and additionally analyzed the loss of TH-immunoreactive cells in HNE-αSyn-treated LUHMES cells. We observed a selective toxicity of HNE-αSyn to neurons with higher TH expression. Further mechanistic studies showed that HNE-modification apparently increased the interaction of extracellular αSyn with neurons. Moreover, exposure of differentiated LUHMES cells to HNE-αSyn triggered the production of intracellular reactive oxygen species, preceding neuronal cell death. Antioxidant treatment effectively protected cells from the damage triggered by HNE-αSyn. Our findings suggest a specific pathological effect of HNE-αSyn on dopaminergic neurons.
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http://dx.doi.org/10.1016/j.mcn.2013.01.004DOI Listing
May 2013

Oxidative stress-induced posttranslational modifications of human hemoglobin in erythrocytes.

Arch Biochem Biophys 2013 Jan 29;529(1):34-44. Epub 2012 Nov 29.

Institut für Biochemie, Emil-Fischer-Zentrum, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.

Posttranslational modifications (PTMs) have been reported in hemoglobin (Hb) treated with ROS/RNS in cell-free experiments. However, little is known about oxidative PTMs of Hb occurring within the erythrocytes. The aim of this study is to characterize the patterns of Hb PTMs in erythrocytes under oxidative stress. Using mass spectrometry, we investigated specifically methionine/tryptophan oxidation, tyrosine nitration, and the modification via 4-hydroxynonenal (HNE), a product of lipid-peroxidation, on Hb. We demonstrated that the treatment with H(2)O(2)/nitrite induced higher levels of Hb oxidation/nitration in purified Hb preparations than in unpurified hemolysates and erythrocytes, indicating that ROS/RNS are primarily removed by antioxidative mechanisms. We further studied Hb from erythrocytes exposed to γ-irradiation. An irradiation of 30-100 Gy triggered a remarkable increase of intracellular ROS. However, 30 Gy did not induce apparent changes in Hb oxidation/nitration and hemolysis, while Hb oxidation/nitration and hemolysis were significantly enhanced by 100 Gy, suggesting that Hb oxidation/nitration are the consequence of overwhelmed antioxidative mechanisms after oxidative attack and reflect the severity of the oxidative damage of erythrocytes. Although irradiation was known to induce lipid-peroxidation, we could not detect HNE-Hb adducts in irradiated erythrocytes. Analyzing PTM patterns suggests Hb nitration as a more suitable indicator of the oxidative damage of erythrocytes.
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http://dx.doi.org/10.1016/j.abb.2012.11.002DOI Listing
January 2013

Inhibitory glycine receptors: an update.

J Biol Chem 2012 Nov 4;287(48):40216-23. Epub 2012 Oct 4.

Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia.

Strychnine-sensitive glycine receptors (GlyRs) mediate synaptic inhibition in the spinal cord, brainstem, and other regions of the mammalian central nervous system. In this minireview, we summarize our current view of the structure, ligand-binding sites, and chloride channel of these receptors and discuss recently emerging functions of distinct GlyR isoforms. GlyRs not only regulate the excitability of motor and afferent sensory neurons, including pain fibers, but also are involved in the processing of visual and auditory signals. Hence, GlyRs constitute promising targets for the development of therapeutically useful compounds.
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http://dx.doi.org/10.1074/jbc.R112.408229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3504737PMC
November 2012

The importance of TM3-4 loop subdomains for functional reconstitution of glycine receptors by independent domains.

J Biol Chem 2012 Nov 20;287(46):39205-15. Epub 2012 Sep 20.

Institute of Biochemistry, Emil Fischer Center, Friedrich-Alexander University Erlangen-Nuernberg, Fahrstrasse 17, 91054 Erlangen, Germany.

Truncated glycine receptors that have been found in human patients suffering from the neuromotor disorder hyperekplexia or in spontaneous mouse models resulted in non-functional ion channels. Rescue of function experiments with the lacking protein portion expressed as a separate independent domain demonstrated restoration of glycine receptor functionality in vitro. This construct harbored most of the TM3-4 loop, TM4, and the C terminus and was required for concomitant transport of the truncated α1 and the complementation domain from the endoplasmic reticulum toward the cell surface, thereby enabling complex formation of functional glycine receptors. Here, the complementation domain was stepwise truncated from its N terminus in the TM3-4 loop. Truncation of more than 49 amino acids led again to loss of functionality in the receptor complex expressed from two independent domain constructs. We identified residues 357-418 in the intracellular TM3-4 loop as being required for reconstitution of functional glycine-gated channels. All complementation constructs showed cell surface protein expression and correct orientation according to glycine receptor topology. Moreover, we demonstrated that the truncations did not result in a decreased protein-protein interaction between both glycine receptor domains. Rather, deletions of more than 49 amino acids abolished conformational changes necessary for ion channel opening. When the TM3-4 loop subdomain harboring residues 357-418 was expressed as a third independent construct together with the truncated N-terminal and C-terminal glycine receptor domains, functionality of the glycine receptor was again restored. Thus, residues 357-418 represent an important determinant in the process of conformational rearrangements following ligand binding resulting in channel opening.
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http://dx.doi.org/10.1074/jbc.M112.376053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3493960PMC
November 2012

A retroelement modifies pre-mRNA splicing: the murine Glrb(spa) allele is a splicing signal polymorphism amplified by long interspersed nuclear element insertion.

J Biol Chem 2012 Sep 10;287(37):31185-94. Epub 2012 Jul 10.

Institut für Biochemie, Emil Fischer Zentrum, Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.

The glycine receptor-deficient mutant mouse spastic carries a full-length long interspersed nuclear element (LINE1) retrotransposon in intron 6 of the glycine receptor β subunit gene, Glrb(spa). The mutation arose in the C57BL/6J strain and is associated with skipping of exon 6 or a combination of the exons 5 and 6, thus resulting in a translational frameshift within the coding regions of the GlyR β subunit. The effect of the Glrb(spa) LINE1 insertion on pre-mRNA splicing was studied using a minigene approach. Sequence comparison as well as motif prediction and mutational analysis revealed that in addition to the LINE1 insertion the inactivation of an exonic splicing enhancer (ESE) within exon 6 is required for skipping of exon 6. Reconstitution of the ESE by substitution of a single residue was sufficient to prevent exon skipping. In addition to the ESE, two regions within the 5' and 3' UTR of the LINE1 were shown to be critical determinants for exon skipping, indicating that LINE1 acts as efficient modifier of subtle endogenous splicing phenotypes. Thus, the spastic allele of the murine glycine receptor β subunit gene is a two-hit mutation, where the hypomorphic alteration in an ESE is amplified by the insertion of a LINE1 element in the adjacent intron. Conversely, the LINE1 effect on splicing may be modulated by individual polymorphisms, depending on the insertional environment within the host genome.
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http://dx.doi.org/10.1074/jbc.M112.375691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3438950PMC
September 2012

Functional implications of novel human acid sphingomyelinase splice variants.

PLoS One 2012 27;7(4):e35467. Epub 2012 Apr 27.

Department of Psychiatry and Psychotherapy, University Hospital, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany.

Background: Acid sphingomyelinase (ASM) hydrolyses sphingomyelin and generates the lipid messenger ceramide, which mediates a variety of stress-related cellular processes. The pathological effects of dysregulated ASM activity are evident in several human diseases and indicate an important functional role for ASM regulation. We investigated alternative splicing as a possible mechanism for regulating cellular ASM activity.

Methodology/principal Findings: We identified three novel ASM splice variants in human cells, termed ASM-5, -6 and -7, which lack portions of the catalytic- and/or carboxy-terminal domains in comparison to full-length ASM-1. Differential expression patterns in primary blood cells indicated that ASM splicing might be subject to regulatory processes. The newly identified ASM splice variants were catalytically inactive in biochemical in vitro assays, but they decreased the relative cellular ceramide content in overexpression studies and exerted a dominant-negative effect on ASM activity in physiological cell models.

Conclusions/significance: These findings indicate that alternative splicing of ASM is of functional significance for the cellular stress response, possibly representing a mechanism for maintaining constant levels of cellular ASM enzyme activity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035467PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338701PMC
September 2012

Modulation of TTX-sensitive voltage-dependent Na+ channels by β-bungarotoxin in rat cerebellar neurons.

BMC Neurosci 2012 Mar 29;13:36. Epub 2012 Mar 29.

Experimental Ophthalmology, Eye Hospital, University Medical Center Regensburg, Franz-Josef-Strauss Allee 11, 93053 Regensburg, Germany.

Background: The modulation of voltage-dependent Na+ channels by lipid metabolites such as arachidonic acid or eicosanoids plays a role in physiological functions as well as in degenerative diseases. So far TTX-resistant channels were found mainly to be regulated by lipid metabolites.

Results: We investigated the lipid-dependent modulation of TTX-sensitive (TTX-s) Na+ channels using β-bungarotoxin (β-BuTX, 10 pM), which has an intrinsic phospholipase-A2 activity, and indomethacin (10 μM), which blocks cyclooxygenase activity in primary cerebellar neurons. To investigate TTX-s Na+ channels, whole-currents were measured under K+-free conditions and blocked by 10 nM TTX. The currents resulting from calculating the difference of currents measured in the presence and the absence of TTX were used for further analysis. Application of indomethacin mainly changed the current kinetics but has only minor effects on voltage-dependence. In contrast β-BuTX increased the maximal current amplitude and shifted the voltage-dependent activation towards more negative potentials. The effects of β-BuTX were blocked by indomethacin. Analysis of lipid metabolites which accumulate by treatment with β-BuTX using MALDI-TOF MS showed an increase of cyclooxygenase reaction products in relation to arachidonic acid.

Conclusions: In summary, we conclude that TTX-sensitive Na+ channels can be directly modulated by cyclooxygenase reaction products leading to higher activity at less depolarized potentials and subsequent higher excitability of neurons. Since activation of cyclooxygenase is also involved in pathways leading to apoptotic cells death this could play a role in degenerative diseases of the CNS and highlights a possible protective effect of cyclooxygenase inhibition.
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http://dx.doi.org/10.1186/1471-2202-13-36DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338087PMC
March 2012

Developmental regulation of glycine receptors at efferent synapses of the murine cochlea.

Histochem Cell Biol 2011 Oct 18;136(4):387-98. Epub 2011 Aug 18.

Department of Otorhinolaryngology, Head and Neck Surgery, Erlangen University Hospital, Erlangen, Germany.

Efferent olivocochlear feedback innervation modulates the stream of auditory information from cochlea to brainstem by regulating auditory nerve activity and controlling the contribution of cochlear outer hair cells to basilar membrane motion. In our previous work, we gave a first description of glycine receptors (GlyRs) in the rat cochlea indicating a possible localization at efferent cochlear synapses. Here, we analyze the developmental regulation of GlyR transcripts and protein within the developing murine organ of Corti (postnatal days P0-P21). Using quantitative RT-PCR, GlyRα1 and α2 were identified as the predominant GlyRα subunit transcripts before the onset of hearing (
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http://dx.doi.org/10.1007/s00418-011-0855-6DOI Listing
October 2011

Intracellular glycation of nuclear DNA, mitochondrial DNA, and cytosolic proteins during senescence-like growth arrest.

DNA Cell Biol 2011 Sep 25;30(9):681-9. Epub 2011 May 25.

Department of Chemistry and Pharmacy, Food Chemistry, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany.

To investigate the accumulation of intracellular advanced glycation end products (AGEs), a method was established for the simultaneous analysis of glycation products of cytosolic proteins, nuclear DNA, and mitochondrial DNA (mtDNA). Nuclear DNA, mtDNA, and cytosolic proteins were simultaneously isolated from one cell lysate by differential centrifugation and combined mechanical and chemical cell disruption methods. The major DNA-AGE N(2)-carboxyethyl-2'-deoxyguanosine (CEdG) was quantified in nuclear DNA and mtDNA by ELISA, whereas the protein-AGEs N(ɛ)-(carboxymethyl)lysine (CML) and N(ɛ)-(carboxyethyl)lysine (CEL) were determined by western blot. The method was used to analyze NIH3T3 fibroblasts. In untreated cells, CEdG levels of mtDNA (14.84 ± 3.07 pg CEdG/μg mtDNA) were significantly higher compared with nuclear DNA (4.40 ± 0.64 pg CEdG/μg DNA; p < 0.001). Then, fibroblasts were analyzed after 7 days of senescence-like growth arrest. In senescent fibroblasts, the CEdG content of nuclear DNA significantly increased by 25%. However, the CEdG level of mtDNA significantly decreased to 52%; in parallel, an increase in mitochondrial mass and mtDNA was observed. Senescence did not lead to general accumulation of protein-AGEs, but two protein bands at 32 and 34 kDa showed a significant increase in the CML/CEL modification rate (208%, p < 0.001; 196%, p = 0.0016) in senescent fibroblasts compared with control cells.
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http://dx.doi.org/10.1089/dna.2011.1236DOI Listing
September 2011

Structural characterization of intracellular C-terminal domains of group III metabotropic glutamate receptors.

FEBS Lett 2011 Feb 8;585(3):511-6. Epub 2011 Jan 8.

Institut für Biochemie (Emil-Fischer-Zentrum), Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.

Metabotropic glutamate receptors (mGluRs) are regulated by interacting proteins that mostly bind to their intracellular C-termini. Here, we investigated if mGluR6, mGluR7a and mGluR8a C-termini form predefined binding surfaces or if they were rather unstructured. Limited tryptic digest of purified peptides argued against the formation of stable globular folds. Circular dichroism, (1)H NMR and (1)H(15)N HSQC spectra indicated the absence of rigid secondary structure elements. Furthermore, we localized short linear binding motifs in the unstructured receptor domains. Our data provide evidence that protein interactions of the analyzed mGluR C-termini are mediated rather by short linear motifs than by preformed folds.
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http://dx.doi.org/10.1016/j.febslet.2010.12.042DOI Listing
February 2011

Developmental profiling by mass spectrometry of phosphocholine containing phospholipids in the rat nervous system reveals temporo-spatial gradients.

J Neurochem 2010 Aug 28;114(4):1119-34. Epub 2010 May 28.

Emil-Fischer-Zentrum, Institut für Biochemie, Universität Erlangen-Nürnberg, Erlangen, Germany.

Phospholipids are important components of the nervous system, in particular of neuronal and glial membranes. Ontogenesis of the nervous system is associated with fundamental alterations in lipid patterns. Here, matrix-assisted-laser-desorption/ionization time-of-flight mass spectrometry and electro-spray-ionization mass spectrometry were combined to analyze phosphatidylcholines and sphingomyelins, allowing an assessment of individual molecular species. Analysis in eight different regions of the nervous system during development of the Wistar rat, from embryonic day 14 to adulthood, produced informative patterns of developmental and regional changes in lipid contents. Phospholipids containing long chain fatty acyl residues exhibited a characteristic patterning, with dramatic increases in the caudal parts of the nervous system 2 weeks after birth. In contrast, relative contents of short chain phosphatidylcholines were low in the perinatal CNS, decreasing even further during development. The relative amounts of sphingomyelins carrying the fatty acid residues 18:0, 22:0, 24:0, and 24:1 increased developmentally in the caudal nervous system. The rostro-caudal gradient of long chain lipid accumulation is matched by expression gradients of myelin structural and regulatory genes, as evident from bioinformatic analysis. These observations characterize the accumulation of individual lipid classes in the nervous system as a highly regulated process, with structurally related lipids showing a similar temporo-spatial distribution and developmental patterning.
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http://dx.doi.org/10.1111/j.1471-4159.2010.06836.xDOI Listing
August 2010

DARPP-32 binds to tra2-beta1 and influences alternative splicing.

Biochim Biophys Acta 2010 May-Jun;1799(5-6):448-53. Epub 2010 Jan 13.

University of Erlangen-Nuremberg, Institute for Biochemistry, Fahrstrasse 17, 91054 Erlangen, Germany.

The majority of human genes undergo alternative splicing, which is frequently altered in response to physiological stimuli. DARPP-32 (dopamine and cAMP regulated phosphoprotein, 32kDa) is a component of PKA-dependent signaling pathways. Here we show that DARPP-32 binds directly to the splicing factor tra2-beta1 (transformer 2). DARPP-32 changes the usage of tra2-beta1 dependent alternative exons in a concentration-dependent manner, suggesting that the DARPP-32:tra2-beta1 interaction is a molecular link between signaling pathways and pre-mRNA processing.
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http://dx.doi.org/10.1016/j.bbagrm.2010.01.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3100204PMC
June 2010

Multifunctional basic motif in the glycine receptor intracellular domain induces subunit-specific sorting.

J Biol Chem 2010 Feb 3;285(6):3730-3739. Epub 2009 Dec 3.

From the Institut für Biochemie (Emil-Fischer-Zentrum), Universität Erlangen-Nürnberg, Erlangen 91054, Germany. Electronic address:

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated ion channel that mediates fast synaptic inhibition in the vertebrate central nervous system. As a member of the family of Cys-loop receptors, it assembles from five homologous subunits (GlyRalpha1-4 and -beta). Each subunit contains an extracellular ligand binding domain, four transmembrane domains (TM), and an intracellular domain, formed by the loop connecting TM3 and TM4 (TM3-4 loop). The TM3-4 loops of the subunits GlyRalpha1 and -alpha3 harbor a conserved basic motif, which is part of a potential nuclear localization signal. When tested for functionality by live cell imaging of green fluorescent protein and beta-galactosidase-tagged domain constructs, the TM3-4 loops of GlyRalpha1 and -alpha3, but not of GlyRalpha2 and -beta, exhibited nuclear sorting activity. Subunit specificity may be attributed to slight amino acid alterations in the basic motif. In yeast two-hybrid screening and GST pulldown assays, karyopherin alpha3 and alpha4 were found to interact with the TM3-4 loop, providing a molecular mechanism for the observed intracellular trafficking. These results indicate that the multifunctional basic motif of the TM3-4 loop is capable of mediating a karyopherin-dependent intracellular sorting of full-length GlyRs.
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http://dx.doi.org/10.1074/jbc.M109.030460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2823514PMC
February 2010

Mapping of disulfide bonds within the amino-terminal extracellular domain of the inhibitory glycine receptor.

J Biol Chem 2009 Dec 27;284(52):36128-36136. Epub 2009 Oct 27.

Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen-Nürnberg, Erlangen 91054. Electronic address:

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel and a member of the superfamily of cysteine loop (Cys-loop) neurotransmitter receptors, which also comprises the nicotinic acetylcholine receptor (nAChR). Within the extracellular domain (ECD), the eponymous Cys-loop harbors two conserved cysteines, assumed to be linked by a superfamily-specific disulfide bond. The GlyR ECD carries three additional cysteine residues, two are predicted to form a second, GlyR-specific bond. The configuration of none of the cysteines of GlyR, however, had been determined directly. Based on a crystal structure of the nAChRalpha1 ECD, we generated a model of the human GlyRalpha1 where close proximity of the respective cysteines was consistent with the formation of both the Cys-loop and the GlyR-specific disulfide bonds. To identify native disulfide bonds, the GlyRalpha1 ECD was heterologously expressed and refolded under oxidative conditions. By matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we detected tryptic fragments of the ECD indicative of disulfide bond formation for both pairs of cysteines, as proposed by modeling. The identity of tryptic fragments was confirmed using chemical modification of cysteine and lysine residues. As evident from circular dichroism spectroscopy, mutagenesis of single cysteines did not impair refolding of the ECD in vitro, whereas it led to partial or complete intracellular retention and consequently to a loss of function of full-length GlyR subunits in human embryonic kidney 293 cells. Our results indicate that the GlyR ECD forms both a Cys-loop and a GlyR-specific disulfide bond. In addition, cysteine residues appear to be important for protein maturation in vivo.
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http://dx.doi.org/10.1074/jbc.M109.043448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2794728PMC
December 2009

Assessment of heat treatment of dairy products by MALDI-TOF-MS.

Mol Nutr Food Res 2009 Dec;53(12):1487-95

Department of Chemistry and Pharmacy, Emil-Fischer-Center, University of Erlangen-Nuremberg, Germany.

The formation of the Amadori product from lactose (protein lactosylation) is a major parameter to evaluate the quality of processed milk. Here, MALDI-TOF-MS was used for the relative quantification of lactose-adducts in heated milk. Milk was heated at a temperature of 70, 80, and 100 degrees C between 0 and 300 min, diluted, and subjected directly to MALDI-TOF-MS. The lactosylation rate of alpha-lactalbumin increased with increasing reaction temperature and time. The results correlated well with established markers for heat treatment of milk (concentration of total soluble protein, soluble alpha-lactalbumin and beta-lactoglobulin at pH 4.6, and fluorescence of advanced Maillard products and soluble tryptophan index; r=0.969-0.997). The method was also applied to examine commercially available dairy products. In severely heated products, protein pre-purification by immobilized metal affinity chromatography improved spectra quality. Relative quantification of protein lactosylation by MALDI-TOF-MS proved to be a very fast and reliable method to monitor early Maillard reaction during milk processing.
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http://dx.doi.org/10.1002/mnfr.200900008DOI Listing
December 2009

Recessive hyperekplexia mutations of the glycine receptor alpha1 subunit affect cell surface integration and stability.

J Neurochem 2009 Nov 1;111(3):837-47. Epub 2009 Sep 1.

Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen-Nürnberg, Erlangen, Germany.

The human neurological disorder hyperekplexia is frequently caused by recessive and dominant mutations of the glycine receptor alpha1 subunit gene, GLRA1. Dominant forms are mostly attributed to amino acid substitutions within the ion pore or adjacent loops, resulting in altered channel properties. Here, the biogenesis of glycine receptor alpha1 subunit mutants underlying recessive forms of hyperekplexia was analyzed following recombinant expression in HEK293 cells. The alpha1 mutant S231R resulted in a decrease of surface integrated protein, consistent with reduced maximal current values. Decreased maximal currents shown for the recessive alpha1 mutant I244N were associated with protein instability, rather than decreased surface integration. The recessive mutants R252H and R392H encode exchanges of arginine residues delineating the intracellular faces of transmembrane domains. After expression, the mutant R252H was virtually absent from the cell surface, consistent with non-functionality and the importance of the positive charge for membrane integration. Surface expression of R392H was highly reduced, resulting in residual chloride conductance. Independent of the site of the mutation within the alpha1 polypeptide, metabolic radiolabelling and pulse chase studies revealed a shorter half-life of the full-length alpha1 protein for all recessive mutants as compared to the wild-type. Treatment with the proteasome blocker, lactacystin, significantly increased the accumulation of alpha1 mutants in intracellular membranes. These observations indicated that the recessive alpha1 mutants are recognized by the endoplasmatic reticulum control system, and degraded via the proteasome pathway. Thus, the lack of glycinergic inhibition associated with recessive hyperekplexia may be attributed to sequestration of mutant subunits within the endoplasmatic reticulum quality control system.
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http://dx.doi.org/10.1111/j.1471-4159.2009.06372.xDOI Listing
November 2009

Analysis of lysozyme in cheese by immunocapture mass spectrometry.

J Chromatogr B Analyt Technol Biomed Life Sci 2010 Jan 7;878(2):201-6. Epub 2009 Aug 7.

Department of Chemistry and Pharmacy, Emil Fischer Center, University of Erlangen-Nuremberg, Germany.

The enzyme lysozyme is used as a preservative to prevent late blowing of ripened cheese, caused by Clostridium tyrobutyricum. Since the enzyme is extracted from hen egg white, lysozyme has to be declared on food product labels as a potential allergen. Here, a method is reported that combines immunocapture purification and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis for the detection of lysozyme in cheese samples. Cheese extracts were treated with magnetic particles coated with a monoclonal antibody directed against lysozyme. After immunocapture purification, lysozyme was detected by MALDI-TOF-MS. The limit of detection of the assay was about 5mg/kg lysozyme in cheese. The method reliably distinguished between cheese samples which had been produced with and without lysozyme. Thus, the novel assay allows the reliable, sensitive, and specific detection of lysozyme in a food matrix. The assay could be easily adapted to other target peptides and proteins in complex food matrices and, therefore, has a broad application potential, e.g. for the analysis of allergens.
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http://dx.doi.org/10.1016/j.jchromb.2009.07.040DOI Listing
January 2010

Novel regulatory site within the TM3-4 loop of human recombinant alpha3 glycine receptors determines channel gating and domain structure.

J Biol Chem 2009 Oct 6;284(42):28624-33. Epub 2009 Aug 6.

Institut für Biochemie, Emil-Fischer-Zentrum, Friedrich-Alexander-Universität Erlangen-Nürnberg, D-91054 Erlangen, Germany.

Glycine receptors are Cys loop ligand-gated ion channels that mediate fast inhibitory synaptic transmission in the mammalian central nervous system. The functionally distinct splice variants alpha3L and alpha3K of the human glycine receptor differ by a 15-amino acid insert within the long intracellular TM3-4 loop, a region of high intersubunit diversity. In a mutational study, effects of the insert on ion channel function and secondary structure of the TM3-4 loop were investigated. Whole cell current responses and protein surface expression data indicated that the major effect of mutations within the insert was on channel gating. Changes in channel gating correlated with the distribution of charged residues about the splice region. Analysis of complex molecular weight indicated that recombinant TM3-4 loops of alpha3L and alpha3K associated into oligomers of different stoichiometry. Secondary structure analysis suggested that the insert stabilized the overall fold of the large cytoplasmic domain of alpha3L subunits. The absence of the insert resulted in a channel that was still functional, but the TM3-4 cytoplasmic domain appeared not stably folded. Thus, our data identified the spliced insert within the large TM 3-4 loop of alpha3 Gly receptors as a novel regulatory motif that serves a 2-fold role: (i) the presence of the insert stabilizes the overall spatial structure of the domain, and (ii) the insert presents a control unit that regulates gating of the receptor ion channel.
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http://dx.doi.org/10.1074/jbc.M109.043174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781406PMC
October 2009

Functional complementation of Glra1(spd-ot), a glycine receptor subunit mutant, by independently expressed C-terminal domains.

J Neurosci 2009 Feb;29(8):2440-52

Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.

The oscillator mouse (Glra1(spd-ot)) carries a 9 bp microdeletion plus a 2 bp microinsertion in the glycine receptor alpha1 subunit gene, resulting in the absence of functional alpha1 polypeptides from the CNS and lethality 3 weeks after birth. Depending on differential use of two splice acceptor sites in exon 9 of the Glra1 gene, the mutant allele encodes either a truncated alpha1 subunit (spd(ot)-trc) or a polypeptide with a C-terminal missense sequence (spd(ot)-elg). During recombinant expression, both splice variants fail to form ion channels. In complementation studies, a tail construct, encoding the deleted C-terminal sequence, was coexpressed with both mutants. Coexpression with spd(ot)-trc produced glycine-gated ion channels. Rescue efficiency was increased by inclusion of the wild-type motif RRKRRH. In cultured spinal cord neurons from oscillator homozygotes, viral infection with recombinant C-terminal tail constructs resulted in appearance of endogenous alpha1 antigen. The functional rescue of alpha1 mutants by the C-terminal tail polypeptides argues for a modular subunit architecture of members of the Cys-loop receptor family.
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http://dx.doi.org/10.1523/JNEUROSCI.4400-08.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6666244PMC
February 2009

Identification and site-specific relative quantification of beta-lactoglobulin modifications in heated milk and dairy products.

J Agric Food Chem 2008 Jul 7;56(13):5165-71. Epub 2008 Jun 7.

Department of Chemistry and Pharmacy, Food Chemistry, Emil-Fischer-Center, University of Erlangen-Nuremberg, Schuhstrasse 19, 91052 Erlangen, Germany.

During milk processing, proteins can be severely modified by oxidation, condensation, and Maillard reaction, leading to changes in their nutritional and technological properties. In this study, major modifications of beta-lactoglobulin, formed during the heating and processing of milk, were screened by mass spectrometry. For this purpose, beta-lactoglobulin was isolated from the milk samples by gel electrophoresis and analyzed by matrix-assisted laser desorption/ionization mass spectrometry after in-gel digestion with endoproteinase AspN. In heated milk, lactulosyllysine was detected at lysine 47 and 138 or 141 as well as methionine sulfoxide at methionine 7, 24, and 145. All these modifications increased gradually when raw milk was heated for 20, 40, and 60 min at 120 degrees C. The major modifications were also relatively quantified in dairy products, such as raw, high-temperature, ultra-high-temperature, sterilized, and condensed milk as well as infant formulas. The highest contents of lactulosyllysine at Lys47 were detected in powdered infant formulas, whereas lactulosyllysine at Lys138/141 was predominant in condensed milk samples. Methionine sulfoxide at Met7 and Met24 showed a trend toward higher modification rates in more severely processed products.
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http://dx.doi.org/10.1021/jf800571jDOI Listing
July 2008

Analysis of the peptide profile of milk and its changes during thermal treatment and storage.

J Agric Food Chem 2008 May 18;56(9):2899-906. Epub 2008 Apr 18.

Department of Chemistry and Pharmacy, Emil-Fischer-Center, University of Erlangen-Nuremberg, Schuhstrasse 19, 91052 Erlangen, Germany.

In this study a new method was developed for analysis of the low molecular weight protein fraction of milk, allowing a simple and fast overview of the peptide profile of various milk samples. For this purpose, immobilized metal affinity chromatography (IMAC) was coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). By this technique, two major peptides in milk could be identified as fragments of alpha-s1-casein. During heat treatment of raw milk, five new peptides were generated, the origin of which could be assigned to the casein fraction. Storage experiments with extended shelf life milk at 4 degrees C did not show any changes in the peptide profile, whereas in ultra high temperature milk stored at room temperature, one peptide increased significantly, which was identified as the N-terminus of alpha-s1-casein. The peptide was assumed to be formed in an enzymatic reaction, which was confirmed in a storage experiment with sterilized milk. Analyses of different commercially available milk samples confirmed the results obtained with the heated and stored milk. Furthermore, differences in the peptide profiles of the samples, probably due to different cow breeds or lactation stages, were observed. These results establish IMAC prior to MALDI-TOF-MS as a valid tool for the rapid analysis of the peptide profile of milk.
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http://dx.doi.org/10.1021/jf073479oDOI Listing
May 2008

Dependency of intraocular pressure elevation and glaucomatous changes in DBA/2J and DBA/2J-Rj mice.

Invest Ophthalmol Vis Sci 2008 Feb;49(2):613-21

Institute of Anatomy II, University of Erlangen-Nuremberg, Erlangen, Germany.

Purpose: In this study parameters relevant for glaucoma in DBA/2J (D2J) mice were compared with those in age-matched DBA/2J-Rj (D2Rj) mice, to challenge the postulated role of D2J mice as a model for secondary high-tension glaucoma.

Methods: Genotyping for three known short nucleotide polymorphisms (SNPs) in the Tyrp1 gene and the Gpnmb gene by MALDI-TOF-MS and immunohistochemical staining for Gpnmb was performed in D2J and D2Rj mice. Twelve C57Bl/6 (B6), 8 D2Rj, and 11 D2J mice between 1 and 4 months of age were screened qualitatively and quantitatively for morphologic differences within the anterior eye segment. The IOP progression of 25 D2Rj and 18 D2J mice were investigated between 4 to 10.5 months after birth. At the end of this study, in 10 randomly selected individuals of each D2J and D2Rj cohort, correlation of IOP progression and optic nerve damage were determined in each eye.

Results: D2J and D2Rj strains were homozygous for both Tyrp 1 amino acid substitutions, so far only described in D2J mice. The Gpnmb(R150X) point mutation present in D2J mice was not detected in D2Rj. Accordingly, immunoreactivity (IR) for Gpnmb was present only in D2Rj and B6 eyes, but not in D2J. Compared with B6, both DBA/2 mice (D2) showed a significantly narrowed chamber angle caused by an anteriorly displaced ciliary body. IOP measurements showed an average IOP of approximately 14 mm Hg between age 4 and 7 months in D2Rj, which decreased to approximately 11 mm Hg in the period from 8 to 10.5 months. In D2J the average IOP showed a steady increase in the observed period from 4 to 10.5 months (from 8.65 to 15.58 mm Hg). Individuals with IOP peaks up to 30 mm Hg were detected in D2Rj, but none of these mice showed signs of an optic neuropathy after 10.5 months. In contrast, 30% of the investigated D2J mice at the age of 10.5 months showed a severe optic neuropathy. Individual data analyses, however, showed no significant correlation between elevated IOP and glaucomatous changes within the D2J population.

Conclusions: Individual correlations of IOP course with axon loss in the single eyes confirmed that in D2J mice, hypertension is not the only causative factor in glaucomatous optic neuropathy. For further investigations on the pathogenesis of glaucoma in D2J mice, the D2Rj strain without a Gpnmb(R150X) mutation and without glaucomatous changes, but with individual IOP elevation, can be used as an interstrain control for D2J.
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http://dx.doi.org/10.1167/iovs.07-0745DOI Listing
February 2008