Presynaptic Plasticity as a Hallmark of Rat Stress Susceptibility and Antidepressant Response

Two main questions are important for understanding and treating affective disorders: why are certain individuals susceptible or resilient to stress, and what are the features of treatment response and resistance? To address these questions, we used a chronic mild stress (CMS) rat model of depression. When exposed to stress, a fraction of rats develops anhedonic-like behavior, a core symptom of major depression, while another subgroup of rats is resilient to CMS. Furthermore, the anhedonic-like state is reversed in about half the animals in response to chronic escitalopram treatment (responders), while the remaining animals are resistant (non-responder animals). Electrophysiology in hippocampal brain slices was used to identify a synaptic hallmark characterizing these groups of animals. Presynaptic properties were investigated at GABAergic synapses onto single dentate gyrus granule cells. Stress-susceptible rats displayed a reduced probability of GABA release judged by an altered paired-pulse ratio of evoked inhibitory postsynaptic currents (IPSCs) (1.48 ± 0.25) compared with control (0.81 ± 0.05) and stress-resilient rats (0.78 ± 0.03). Spontaneous IPSCs (sIPSCs) occurred less frequently in stress-susceptible rats compared with control and resilient rats. Finally, a subset of stress-susceptible rats responding to selective serotonin reuptake inhibitor (SSRI) treatment showed a normalization of the paired-pulse ratio (0.73 ± 0.06) whereas non-responder rats showed no normalization (1.2 ± 0.2). No changes in the number of parvalbumin-positive interneurons were observed. Thus, we provide evidence for a distinct GABAergic synaptopathy which associates closely with stress-susceptibility and treatment-resistance in an animal model of depression.     

RNA modifications by oxidation: A novel disease mechanism?

The past decade has provided exciting insights into a novel class of central (small) RNA molecules intimately involved in gene regulation. Only a small percentage of our DNA is translated into proteins by mRNA, yet 80% or more of the DNA is transcribed into RNA, and this RNA has been found to encompass various classes of novel regulatory RNAs, including, e.g., microRNAs. It is well known that DNA is constantly oxidized and repaired by complex genome maintenance mechanisms. Analogously, RNA also undergoes significant oxidation, and there are now convincing data suggesting that oxidation, and the consequent loss of integrity of RNA, is a mechanism for disease development. Oxidized RNA is found in a large variety of diseases, and interest has been especially devoted to degenerative brain diseases such as Alzheimer disease, in which up to 50-70% of specific mRNA molecules are reported oxidized, whereas other RNA molecules show virtually no oxidation. The iron-storage disease hemochromatosis exhibits the most prominent general increase in RNA oxidation ever observed. Oxidation of RNA primarily leads to strand breaks and to oxidative base modifications. Oxidized mRNA is recognized by the ribosomes, but the oxidation results in ribosomal stalling and dysfunction, followed by decreased levels of functional protein as well as the production of truncated proteins that do not undergo proper folding and may result in protein aggregation within the cell. Ribosomal dysfunction may also signal apoptosis by p53-independent pathways. There are very few reports on interventions that reduce RNA oxidation, one interesting observation being a reduction in RNA oxidation by ingestion of raw olive oil. High urinary excretion of 8-oxo-guanosine, a biomarker for RNA oxidation, is highly predictive of death in newly diagnosed type 2 diabetics; this demonstrates the clinical relevance of RNA oxidation. Taken collectively the available data suggest that RNA oxidation is a contributing factor in several diseases such as diabetes, hemochromatosis, heart failure, and β-cell destruction. The mechanism involves free iron and hydrogen peroxide from mitochondrial dysfunction that together lead to RNA oxidation that in turn gives rise to truncated proteins that may cause aggregation. Thus RNA oxidation may well be an important novel contributing mechanism for several diseases.     

A rhodamine-labeled citalopram analogue as a high-affinity fluorescent probe for the serotonin transporter

A novel fluorescent ligand was synthesized as a high-affinity, high specificity probe for visualizing the serotonin transporter (SERT). The rhodamine fluorophore was extended from an aniline substitution on the 5-position of the dihydroisobenzofuran ring of citalopram (2, 1-(3-(dimethylamino)propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile), using an ethylamino linker. The resulting rhodamine-labeled ligand 8 inhibited [³H]5-HT uptake in COS-7 cells (K_i = 225 nM) with similar potency to the tropane-based JHC 1-064 (1), but with higher specificity towards the SERT relative to the transporters for dopamine and norepinephrine. Visualization of the SERT with compound 8 was demonstrated by confocal microscopy in HEK293 cells stably expressing EGFP–SERT.     

Mechanisms Preserving Insulin Action during High Dietary Fat Intake

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Stimulus responsive elastin biopolymers: Applications in medicine and biotechnology

Elastin-like polypeptides (ELPs) are artificial polypeptides, derived from Val-Pro-Gly-Xaa-Gly (VPGXG) pentapeptide repeats found in human tropoelastin, that reversibly coacervate above a critical temperature. Genetically encodable ELPs are monodisperse, stimuli responsive, and biocompatible, properties that make them attractive for drug delivery and tissue engineering. The potential of ELPs to self-assemble into nanostructures in response to environmental triggers is another interesting feature of these polypeptides that promises to lead to a host of new applications.     

An ionic model for rhythmic activity in small clusters of embryonic chick ventricular cells

We recorded transmembrane potential in whole cell recording mode from small clusters (2-4 cells) of spontaneously beating 7-day embryonic chick ventricular cells after 1-3 days in culture and investigated effects of the blockers D-600, diltiazem, almokalant, and Ba2+. Electrical activity in small clusters is very different from that in reaggregates of several hundred embryonic chick ventricular cells, e.g., TTX-sensitive fast upstrokes in reaggregates vs. TTX-insensitive slow upstrokes in small clusters (maximum upstroke velocity approximately 100 V/s vs. approximately 10 V/s). On the basis of our voltage- and current-clamp results and data from the literature, we formulated a Hodgkin-Huxley-type ionic model for the electrical activity in these small clusters. The model contains a Ca2+ current (ICa), three K+ currents (IKs, IKr, and IK1), a background current, and a seal-leak current. ICa generates the slow upstroke, whereas IKs, IKr, and IK1 contribute to repolarization. All the currents contribute to spontaneous diastolic depolarization, e.g., removal of the seal-leak current increases the interbeat interval from 392 to 535 ms. The model replicates the spontaneous activity in the clusters as well as the experimental results of application of blockers. Bifurcation analysis and simulations with the model predict that annihilation and single-pulse triggering should occur with partial block of ICa. Embryonic chick ventricular cells have been used as an experimental model to investigate various aspects of spontaneous beating of cardiac cells, e.g., mutual synchronization, regularity of beating, and spontaneous initiation and termination of reentrant rhythms; our model allows investigation of these topics through numerical simulation.     

Syndecan-4 Is a Major Syndecan in Primary Human Endothelial Cells In Vitro,Modulated by Inflammatory Stimuli and Involved in Wound Healing

Syndecans are important cell surface proteoglycans with many functions; yet, they have not been studied to a very large extent in primary human endothelial cells. The purpose of this study was to investigate syndecan-4 expression in cultured human umbilical vein endothelial cells (HUVECs) and assess its role in inflammatory reactions and experimental wound healing. qRT-PCR analysis revealed that syndecan-3 and syndecan-4 were highly expressed in HUVECs, whereas the expression of syndecan-1 and -2 was low. HUVECs were cultured with the inflammatory mediators lipopolysaccharide (LPS) and interleukin 1β (IL-1β). As a result, syndecan-4 expression showed a rapid and strong increase. Syndecan-1 and -2 expressions decreased, whereas syndecan-3 was unaffected. Knockdown of syndecan-4 using siRNA resulted in changes in cellular morphology and focal adhesion sites, delayed wound healing and tube formation, and increased secretion of the pro-inflammatory and angiogenic chemokine, CXCL8. These data suggest functions for syndecan-4 in inflammatory reactions, wound healing and angiogenesis in primary human endothelial cells.     

A magnetic adsorbent-based process for semi-continuous PEGylation of proteins

A semi-continuous magnetic particle-based process for the controlled attachment of PEG (PEGylation) to proteins is described for the first time. Trypsin and 2 kDa mono-activated PEG were used to systematically develop the steps in the process. Proof of concept was shown in a microfluidics system to minimize reagent consumption. Two streams containing (i) 1.2 g/L trypsin and (ii) 4 g/L magnetic adsorbents derivatized with the reversible affinity ligand benzamidine were pumped into a pipe reactor. At the exit, a third solution of activated PEG (0-40 g/L) was introduced and the solutions immediately fed into a second reactor. Upon exiting, the mixture was combined in a third reactor with a fourth stream of free amine groups to stop the reaction (50 mM lysine). The mixture continued into a high-gradient magnetic separator where magnetic supports, with PEGylated trypsin still attached, were captured and washing and elution steps were subsequently carried out. Analysis of the conjugates (with SDS-PAGE & LC-MS) showed that the extent of PEGylation could be controlled by varying the reaction time or PEG concentration. Furthermore, the PEG-conjugates had higher enzyme activity compared to PEGylation of non-immobilized trypsin.     

Temperature Effects on Kinetic Parameters and Substrate Affinity of Cel7A Cellobiohydrolases

We measured hydrolytic rates of four purified cellulases in small increments of temperature (10–50 °C) and substrate loads (0–100 g/liter) and analyzed the data by a steady state kinetic model that accounts for the processive mechanism. We used wild type cellobiohydrolases (Cel7A) from mesophilic Hypocrea jecorina and thermophilic Rasamsonia emersonii and two variants of these enzymes designed to elucidate the role of the carbohydrate binding module (CBM). We consistently found that the maximal rate increased strongly with temperature, whereas the affinity for the insoluble substrate decreased, and as a result, the effect of temperature depended strongly on the substrate load. Thus, temperature had little or no effect on the hydrolytic rate in dilute substrate suspensions, whereas strong temperature activation (Q₁₀ values up to 2.6) was observed at saturating substrate loads. The CBM had a dual effect on the activity. On one hand, it diminished the tendency of heat-induced desorption, but on the other hand, it had a pronounced negative effect on the maximal rate, which was 2-fold larger in variants without CBM throughout the investigated temperature range. We conclude that although the CBM is beneficial for affinity it slows down the catalytic process. Cel7A from the thermophilic organism was moderately more activated by temperature than the mesophilic analog. This is in accord with general theories on enzyme temperature adaptation and possibly relevant information for the selection of technical cellulases.