Iron alters glutamate secretion by regulating cytosolic aconitase activity

Glutamate has many important physiological functions, including its role as a neurotransmitter in the retina and the central nervous system. We have made the novel observations that retinal pigment epithelial cells underlying and intimately interacting with the retina secrete glutamate and that this secretion is significantly affected by iron. In addition, iron increased secretion of glutamate in cultured lens and neuronal cells, indicating that this may be a common mechanism for the regulation of glutamate production in many cell types. The activity of the iron-dependent enzyme cytosolic aconitase (c-aconitase) is increased by iron. The conversion of citrate to isocitrate by c-aconitase is the first step in a three-step process leading to glutamate formation. In the present study, iron increased c-aconitase activity, and this increase was associated with an increase in glutamate secretion. Inhibition of c-aconitase by oxalomalate decreased glutamate secretion and completely inhibited the iron-induced increase in glutamate secretion. Derangements in both glutamate secretion and iron metabolism have been noted in neurological diseases and retinal degeneration. Our results are the first to provide a functional link between these two physiologically important substances by demonstrating a significant role for iron in the regulation of glutamate production and secretion in mammalian cells resulting from iron regulation of aconitase activity. Glutamatergic systems are found in many nonneuronal tissues. We provide the first evidence that, in addition to secreting glutamate, retinal pigment epithelial cells express the vesicular glutamate transporter VGLUT1 and that regulated vesicular release of glutamate from these cells can be inhibited by riluzole. retinal pigment epithelial cells; lens epithelial cells     

Plasmodium falciparum 2-Cys peroxiredoxin reacts with plasmoredoxin and peroxynitrite

Thioredoxin peroxidase 1 (TPx1) of the malarial parasite Plasmodium falciparum is a 2-Cys peroxiredoxin involved in the detoxification of reactive oxygen species and - as shown here - of reactive nitrogen species. As novel electron acceptor of reduced TPx1, we characterised peroxynitrite; the rate constant for ONOO- reduction by the enzyme (1 x 10(6) M(-1) s(-1) at pH 7.4 and 37 degrees C) was determined by stopped-flow measurements. As reducing substrate of TPx1, we identified - aside from thioredoxin - plasmoredoxin; this 22-kDa protein occurs only in malarial parasites. When studying the potential roles of Cys74 and Cys170 of Tpx1 in catalysis, as well as in oligomerisation behaviour, we found that replacement of Cys74 by Ala influenced neither the dimerisation nor enzymatic activity of TPx1. In the C170A mutant, however, the kcat/Km for reduced Trx as a substrate was shown to be approximately 50-fold lower and, in contrast to the wild-type enzyme, covalently linked dimers were not formed. For the catalytic cycle of TPx1, we conclude that oxidation of the peroxidatic Cys50 by the oxidising substrate is followed by the formation of an intermolecular disulfide bond between Cys50 and Cys170' of the second subunit, which is then attacked by an external electron donor such as thioredoxin or plasmoredoxin.     

Experimentally derived estimates of growth by juvenile Tachypleus tridentatus and Carcinoscorpius rotundicauda (Xiphosura) from nursery beaches in Hong Kong

Juvenile Tachypleus tridentatus and Carcinoscorpius rotundicauda with prosomal widths of between 17.1 and 91.1 mm were obtained from their nursery beaches in Hong Kong. They were kept in the laboratory and fed a mixture of squid, prawn and fish. Prosomal width and wet weight were measured weekly to obtain growth data. Over half of the individuals molted during a five and a half month captivity period. After every ecdysis, prosomal width and wet weight of T. tridentatus grew by averages of 24.2% and 71.5% over pre-molt measurements, respectively. Similar values were obtained for C. rotundicauda, i.e. 24.0% and 77.3%, respectively. Three T. tridentatus with prosomal widths of between 26.5 and 35.0 mm molted twice between 89 and 149 days, leading to an average growth rate of 0.1 mm·day⁻¹ and 0.04 g·day⁻¹ in terms of prosomal width and wet weight, respectively. A positive growth allometry (b coefficient=2.97) was identified, indicating that weight gain for T. tridentatus, and possibly C. rotundicauda, was faster than prosomal width growth after each ecdysis. The effect of temperature on growth was also determined by comparing the percentages of T. tridentatus which molted at ∼28 °C and ∼18 °C. Fifty percent of individuals molted at the former, but only 10% at the latter. This study indicates that Hong Kong horseshoe crabs take a shorter time to reach sexual maturity, as compared with conspecifics in Japan, because they can molt more frequently at higher sediment/water temperatures (∼28 °C) if food is available.     

Determination of protein-derived epitopes by mass spectrometry

Mass spectrometry has evolved as a technique suitable for the characterization of peptides and proteins beyond their linear sequence. The advantages of mass spectrometric sample analysis are high sensitivity, high mass accuracy, rapid analysis time and low sample consumption. In epitope mapping, the molecular structure of an antigen (the epitope or antigenic determinant) that interacts with the paratope (recognition surface) of the antibody is identified. To obtain information on linear, conformational and/or discontinuous epitopes, various approaches have been developed in conjunction with mass spectrometry. These methods include limited proteolysis and epitope footprinting, epitope excision and epitope extraction for linear epitopes and probing the surface accessibility of residues by differential chemical modifications of specific amino acid side chains or by differential hydrogen/deuterium exchange of the protein backbone amides for conformational and discontinuous epitopes. Epitope mapping by mass spectrometry is applicable in basic biochemical research and, with increasing robustness, should soon find its implementation in routine clinical diagnosis.     

Potential application for mesenchymal stem cells in the treatment of cardiovascular diseases

Stem cells isolated from various sources have been shown to vary in their differentiation capacity or pluripotentiality. Two groups of stem cells, embryonic and adult stem cells, may be capable of differentiating into any desired tissue or cell type, which offers hope for the development of therapeutic applications for a large number of disorders. However, major limitations with the use of embryonic stem cells for human disease have led researchers to focus on adult stem cells as therapeutic agents. Investigators have begun to examine postnatal sources of pluripotent stem cells, such as bone marrow stroma or adipose tissue, as sources of mesenchymal stem cells. The following review focuses on recent research on the use of stem cells for the treatment of cardiovascular and pulmonary diseases and the future application of mesenchymal stem cells for the treatment of a variety of cardiovascular disorders.     

Keratinocyte galvanotaxis in combined DC and AC electric fields supports an electromechanical transduction sensing mechanism

Sedentary keratinocytes at the edge of a skin wound migrate into the wound, guided by the generation of an endogenous electric field (EF) generated by the collapse of the transepithelial potential. The center of the wound quickly becomes more negative than the surrounding tissue and remains the cathode of the endogenous EF until the wound is completely re‐epithelialized. This endogenous guidance cue can be studied in vitro. When placed in a direct current (DC) EF of physiological strength, 100 V/m, keratinocytes migrate directionally toward the cathode in a process known as galvanotaxis. Although a number of membrane‐bound (e.g., epidermal growth factor receptor (EGFR), integrins) and cytosolic proteins (cAMP, ERK, PI3K) are known to play a role in the downstream signaling mechanisms underpinning galvanotaxis, the initial sensing mechanism for this response is not understood. To investigate the EF sensor, we studied the migration of keratinocytes in a DC EF of 100 V/m, alternating current (AC) EFs of 40 V/m at either 1.6 or 160 Hz, and combinations of DC and AC EFs. In the AC EFs alone, keratinocytes migrated randomly. The 1.6 Hz AC EF combined with the DC EF suppressed the direction of migration but had no effect on speed. In contrast, the 160 Hz AC EF combined with the DC EF did not affect the direction of migration but increased the migration speed compared to the DC EF alone. These results can be understood in terms of an electromechanical transduction model, but not an electrodiffusion/osmosis or a voltage‐gated channel model. Bioelectromagnetics 34:85–94, 2013. © 2012 Wiley Periodicals, Inc.     

Eine Gelbfleckigkeit an Leonurus cardiaca L., verursacht vom Ackerbohnenwelke-Virus (broad bean wilt virus) (Kurze Mitteilung)

Das Tabakmosaik‐Virus (tobacco mosaic tobamovirus, TMV) und ein aus dem Wasser der Havel isoliertes, isometrisches Virus (Havel river ?tombusvirus, HRV) werden bei Hydrokultivierung von den Wurzeln experimentell infizierter Versuchspflanzen (Petunia hybrida bzw. Nicotiana megalos‐iphon) in die Nährlösung abgegeben und bleiben über lange Zeit infektiös. Die als Substrat verwendeten Blähtonkugeln erwiesen sich als viruskontaminiert. Bei erneuter Verwendung zur Hydrokultur können dadurch in unterschiedlich hohem Grade Spontaninfektionen der gesunden nachgebauten Pflanzen auftreten. Für das HRV wurde damit erstmalig eine vektorlose Übertragung nachgewiesen.

Nach Desinfektion der Gefäße und des Blähtons konnten dagegen keine Spontaninfektionen festgestellt werden.

Im Gegensatz zum TMV und HRV konnte für das Gurkenmosaik‐Virus (cucumber mosaic cucumovirus, CMV) eine derartige Übertragungsweise nicht nachgewiesen werden.     

FAS-Dependent Cell Death in α-Synuclein Transgenic Oligodendrocyte Models of Multiple System Atrophy

Multiple system atrophy is a parkinsonian neurodegenerative disorder. It is cytopathologically characterized by accumulation of the protein p25α in cell bodies of oligodendrocytes followed by accumulation of aggregated α-synuclein in so-called glial cytoplasmic inclusions. p25α is a stimulator of α-synuclein aggregation, and coexpression of α-synuclein and p25α in the oligodendroglial OLN-t40-AS cell line causes α-synuclein aggregate-dependent toxicity. In this study, we investigated whether the FAS system is involved in α-synuclein aggregate dependent degeneration in oligodendrocytes and may play a role in multiple system atrophy. Using rat oligodendroglial OLN-t40-AS cells we demonstrate that the cytotoxicity caused by coexpressing α-synuclein and p25α relies on stimulation of the death domain receptor FAS and caspase-8 activation. Using primary oligodendrocytes derived from PLP-α-synuclein transgenic mice we demonstrate that they exist in a sensitized state expressing pro-apoptotic FAS receptor, which makes them sensitive to FAS ligand-mediated apoptosis. Immunoblot analysis shows an increase in FAS in brain extracts from multiple system atrophy cases. Immunohistochemical analysis demonstrated enhanced FAS expression in multiple system atrophy brains notably in oligodendrocytes harboring the earliest stages of glial cytoplasmic inclusion formation. Oligodendroglial FAS expression is an early hallmark of oligodendroglial pathology in multiple system atrophy that mechanistically may be coupled to α-synuclein dependent degeneration and thus represent a potential target for protective intervention.     

Assessing the Conformational Changes of pb5, the Receptor-binding Protein of Phage T5, upon Binding to Its Escherichia coli Receptor FhuA

Within tailed bacteriophages, interaction of the receptor-binding protein (RBP) with the target cell triggers viral DNA ejection into the host cytoplasm. In the case of phage T5, the RBP pb5 and the receptor FhuA, an outer membrane protein of Escherichia coli, have been identified. Here, we use small angle neutron scattering and electron microscopy to investigate the FhuA-pb5 complex. Specific deuteration of one of the partners allows the complete masking in small angle neutron scattering of the surfactant and unlabeled proteins when the complex is solubilized in the fluorinated surfactant F₆-DigluM. Thus, individual structures within a membrane protein complex can be described. The solution structure of FhuA agrees with its crystal structure; that of pb5 shows an elongated shape. Neither displays significant conformational changes upon interaction. The mechanism of signal transduction within phage T5 thus appears different from that of phages binding cell wall saccharides, for which structural information is available.