Tetracapsuloides bryosalmonae infection affects the expression of genes involved in cellular signal transduction and iron metabolism in the kidney of the brown trout Salmo trutta

Tetracapsuloides bryosalmonae is an enigmatic endoparasite which causes proliferative kidney disease in various species of salmonids in Europe and North America. The life cycle of the European strain of T. bryosalmonae generally completes in an invertebrate host freshwater bryozoan and vertebrate host brown trout (Salmo trutta) Linnaeus, 1758. Little is known about the gene expression in the kidney of brown trout during the developmental stages of T. bryosalmonae. In the present study, quantitative real-time PCR was applied to quantify the target genes of interest in the kidney of brown trout at different time points of T. bryosalmonae development. PCR primers specific for target genes were designed and optimized, and their gene expression levels were quantified in the cDNA kidney samples using SYBR Green Supermix. Expression of Rab GDP dissociation inhibitor beta, integral membrane protein 2B, NADH dehydrogenase 1 beta subcomplex subunit 6, and 26S protease regulatory subunit S10B were upregulated significantly in infected brown trout, while the expression of the ferritin M middle subunit was downregulated significantly. These results suggest that host genes involved in cellular signal transduction, proteasomal activities, including membrane transporters and cellular iron storage, are differentially upregulated or downregulated in the kidney of brown trout during parasite development. The gene expression pattern of infected renal tissue may support the development of intraluminal sporogonic stages of T. bryosalmonae in the renal tubular lumen of brown trout which may facilitate the release of viable parasite spores to transmit to the invertebrate host bryozoan

The high-affinity binding site for tricyclic antidepressants resides in the outer vestibule of the serotonin transporter

The structure of the bacterial leucine transporter from Aquifex aeolicus (LeuT(Aa)) has been used as a model for mammalian Na+/Cl--dependent transporters, in particular the serotonin transporter (SERT). The crystal structure of LeuT(Aa) liganded to tricyclic antidepressants predicts simultaneous binding of inhibitor and substrate. This is incompatible with the mutually competitive inhibition of substrates and inhibitors of SERT. We explored the binding modes of tricyclic antidepressants by homology modeling and docking studies. Two approaches were used subsequently to differentiate between three clusters of potential docking poses: 1) a diagnostic SERTY95F mutation, which greatly reduced the affinity for [H-3] imipramine but did not affect substrate binding; 2) competition binding experiments in the presence and absence of carbamazepine (i.e., a tricyclic imipramine analog with a short side chain that competes with [3H] imipramine binding to SERT). Binding of releasers (para-chloroamphetamine, methylene-dioxy-methamphetamine/ecstasy) and of carbamazepine were mutually exclusive, but Dixon plots generated in the presence of carbamazepine yielded intersecting lines for serotonin, MPP+, paroxetine, and ibogaine. These observations are consistent with a model, in which 1) the tricyclic ring is docked into the outer vestibule and the dimethyl-aminopropyl side chain points to the substrate binding site; 2) binding of amphetamines creates a structural change in the inner and outer vestibule that precludes docking of the tricyclic ring; 3) simultaneous binding of ibogaine (which binds to the inward-facing conformation) and of carbamazepine is indicative of a second binding site in the inner vestibule, consistent with the pseudosymmetric fold of monoamine transporters. This may be the second low-affinity binding site for antidepressants

Role of Marine Pollutants in Impairment of DNA Integrity

Abstract In this article, we present an overview on the role of marine pollutants in impairment of DNA integrity in marine gastropods exposed to xenobiotics released from various sources into the coastal ecosystem. We provide an insight into the impact of various types of genotoxic compounds on the physiologic status of marine organisms, with specific focus on DNA integrity. DNA damage in marine gastropods was found to be highly correlated to the level of contaminants prevalent in the marine environment. DNA integrity in marine organisms was significantly affected upon exposure to genotoxicants such as polycyclic aromatic hydrocarbons (PAH), persistent organic pollutants, cadmium, lead, manganese, chromium, copper, etc. The prevalence of persistent xenobiotic contaminants in the marine environment coupled to their deleterious impact on marine organisms, exemplifies the measurement of DNA integrity as a prognostic tool for marine pollution assessment, control and policy-making

Do the binding sites of substrates and tricyclic antidepressants overlap on the human serotonin transporter?

Nach neuronaler Stimulation werden Neurotransmitter in den synaptischen Spalt freigesetzt. Dort aktivieren sie prä- und postsynaptische Rezeptoren, um das elektrische Signal weiterzuleiten und über Rückkopplung ihre eigen Freisetzung zu steuern. Die synatische Neurotransmission unterliegt einer strengen räumlichen und zeitlichen Regulation durch membranstänfige Transporter aus der NSS/SLC6Familie ( "neurotransmitter:sodium symporters" =solute carier 6). Diese Tranporter sind an der präsynaptischen Nervendigung exprimiert und vermitteln die rasche Rückaufnahme des freigestezten Neurotransmitters aus dem synaptischen Spalt. Erste Einblicke in ihre drei-dimensionale Struktur wurden durch die Kristallisation eines bakteriellen Orthologen gewonnen, nämlich LeuTAa ein Leucintransporter des thermophilen Bakteriums Aquifex aeolicus. Die erste Kristallstruktur zeigt eine Konformation, die amehseten dem Übergangszustand ("occluded state")zwischen der auswärts und der einwärts gerichteten Konformation von LeuTAa entspricht. Von großem Interesse waren die in der Folge gelösten Strukuren, die LeuTAa in einem Komplex mit den trizyclischen Antidepressiva (TCA's) Clomipramin, Desipramin, Imipramin und Amitriptylin zeigten. Diese ermöglichten es, Modellvorstellungen zu entwickeln, wie die menschlichen NSS-Vertreter, inbesondere der Serotonintransporter (SERT), durch Inhibitoren gehemmt werden. Allerdings ist die Hemmung von LeuTAa durch TCA's nicht-kompetitiv, während TCA's den Serotonintransporters (SERT) kompetitiv hemmen. Dieser Widerspruch stellt die Gültigket von LeutAa als Modell für SERT in Frage. Die vorliegenden Experimente gehen von der Arbeitshypothese aus, dass diese Diskrepanz unter der Annahme erklärt werden kann, dass TCA's im äußeren Vestibül binden und dass ihre Seitenkette in die Substratbindungsstelle reicht. Weil Serotonin gößer als Leucin ist, verhindert dies die gleichzeitige Bindung von Serotonin und TCA's. Diese Hypothese wurde durch zwei Ansätze geprüft: (i) Verdränungsexperimente mit Carbamazepin (einem Analogon vn Imipramin, das nur eine kurze aliphatische Seitenkette hat und das die Bindung von [3H]Imipramin an SERT kompetitiv hemmt), (ii) gerichteter Mutagenese, mit der ich eine diagnostische SERTY95F Mutation erzeugte. Die Entfernung der Hydroxylgruppe setzte erwartungsgemäß die Affinität von [3H]Imipramin herab, hatte aber keinen Einfluss auf Substratbindung und Translokation. Mit Dixon Plots ließ sich nachweisen,dass Carbamazepin gleichzeitig mit Serotonin, Paroxetine oder Ibogain an den Transporter gebunden sein konnten. Hingegen war die gelichzeitige Bindung von Amphetaminen, nämlich para-Chloroamphetamin (PCA) oder Methylen-Dioxi-Methamphetamine (MDMA = 'ecstasy'), und von Carbamazepine nicht möglich. Diese Beobachtungen sind daher mit dem folgendeb Modell vereinbar: (i) Der trizyclische Ring von TCA's liebt im äußeren Verstbül von SERT und die Dimethylaminopropyl-Seitenkette bestezt die Substratbindungsstelle. (ii) Die Bindung von Amphetaminen führt zu einer Konformationsänderung im inneren und äußeren Vestibül, die ihre gleichzeitige Besetzung durch den trizyclischen Ring verhindert. (iii) Die simultane Bindung von Ibogain (das an die einwärts-gerichtete Konformation bindet) und von Carbamazepin an SERT ist mit einer zweiten niederaffinen Bindungsstelle im inneren Vestibül vereinbar. Dies stimmt mit der Pseudosymmetrie von NSS Proteinen und mit Hinweisen für eine zweite niederaffine Bindungsstelle von Imipramin.Upon neuronal stimulation, neurotransmitters are released into the synaptic cleft where they activate both pre- and post-synaptic receptors for propagation of the electrical nerve impulse. Neurotransmission across a synapse is under tight spatio-temporal regulation by high-affinity membrane transport proteins of the neurotransmitter:sodium symporter (NSS)-family. NSS proteins are localized on the pre-synaptic nerve terminals. They mediate rapid retrieval of the released neurotransmitters from the synaptic cleft. The first insight into the three-dimensional structure of this class of proteins was obtained by crystallization of the prokaryotic NSS member, LeuTAa, isolated from the thermophilic bacterium, Aquifex aeolicus. The first structure revealed a conformation likely representing an intermediate (i.e., the occluded state) between the 'outward-' and 'inward-' facing conformations of LeuTAa. Subsequent crystal structures of LeuTAa bound to tricyclic antidepressants (TCAs) viz., clomipramine, desipramine, imipramine and amitriptyline afforded mehanistic insights into the inhibition of NSS proteins. A major limitaion was noted: the inhibition of LeuTAa by TCAs is non-competitive, but TCAs are competitive inhibitors of the human serotonin transporter (SERT). Hence there is an obvious discrepancy, which questions the validity of the LeuTAa-based model. My thesis is based on the working hypothesis that the discrepancy can be resolved by assuming that TCA's bind in the outer vestibule and that their side chain reaches into the substrate binding site. Because serotonin is larger, this precludes simultaneous binding of serotonin and TCA's. I adressed this discrepancy via two approaches: (i) competition binding experiments with carbamazepine (i.e., an imipramine analog with a short aliphatic side chain, which was verifed to compete with [3H]imipramine binding to SERT), (ii) site-directed mutagenesis where I generated a diagnostic SERTY95F mutation which greatly reduced the affinity for [3H]imipramine but did not affect substrate binding. Dixon plots revealed that carbamazepine bound simultaneously to SERT in the presence of serotonin, paroxetine or ibogaine. In contrast, the binding of amphetamines, viz. para-chloroamphetamine (PCA) or methylene-dioxy-methamphetamine (MDMA or, 'ecstasy') and of carbamazepine was mutually exclusive. My observations are consistent with the following model: (i) the tricyclic ring of TCAs resides in the outer vestibule of SERT and the dimethyl-aminopropyl side chain occupies the substrate binding pocket; (ii) binding of amphetamines to SERT creates a structural change in the inner and outer vestibules which precludes simultaneous occupancy of the tricyclic ring in the vestibules, (iii) simultaneous binding of ibogaine (which binds to the 'inward-facing' conformation) and of carbamazepine to SERT is indicative of a second low-affinity binding site in the inner vestibule, consistent with the pseudo-symmetric fold of NSS proteins.submitted by Subhodeep SarkerAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Med. Univ., Diss., 2010OeBB(VLID)188337