Exploring the functional role of the bacterial protein SeqA and co--?occurring genome stability systems in TnsE--?mediated Tn7 transposition using the E. coli model system

Transposon Tn7, is unique among transposons for both its target specificity and selectivity. One of its target selection pathways, mediated by the protein TnsE, has been found to allow Tn7 to preferentially transpose into mobile plasmids despite there only composing approximately 1% of the bacterial genome. Prior research in the Peters laboratory revealed both a physical interaction between TnsE and the bacterial protein SeqA as well as a functional role of the TnsE--?SeqA interaction in TnsE--?mediated transposition. Based on this work a model was developed, whereby SeqA occludes TnsE’s access to the replicating chromosome. An unrelated study from another group found that the genes yfbV, metJ, holD, mutH, and matP co--?occurred with seqA in genomes with an ancestral dam gene. In this project, an in vivo Tn7 transposition assay was used to investigate the role of the SeqA--?TnsE interaction in the TnsE pathway of Tn7 transposition. The same transposition assay was also used on the co--?occuring set of bacterial proteins in order to identify a possible association with their gene products and TnsE--?mediated transposition. The Tn7 transposition events were then mapped using two complementary assays. The results suggest that in the absence of SeqA, TnsE--?mediated Tn7 transposition increases significantly and that a much higher percentage of the insertions occur into the chromosome in a DseqA background, thus supporting our model. The results for the other proteins were much less clear, with the only significant change in TnsE--?mediated transposition frequency occurring in the DmetJ mutant, where no successful Tn7 transpositions were observed within the detection of our assay

Depolarization of the intergeniculate leaflet neurons by serotonin : in vitro study

The intergeniculate leaflet of the thalamus (IGL) is a part of the mammalian biological clock which integrates photic and non-photic information and conveys it to the master biological clock - suprachiasmatic nuclei (SCN). One of the non-photic cues is delivered by the serotoninergic projection from dorsal raphe nucleus. In vitro electrophysiological recordings were performed from single IGL neurons using whole-cell patch clamp technique. We investigated the influence of serotonin (serotonin creatinine sulfate complex, 5HT) on 'spontaneous' neuronal activity in this structure. In most of recorded cells, 5-HT caused significant increases in firing rate. In majority of cases the effect was resynaptic. However, in some cases we observed postsynaptic depolarization. To our knowledge, depolarizing influence of 5HT on the single neurons in the IGL has been shown here for the first time

Charakterystyka napięciowo-zależnych zmian zaangażowanych w generowanie aktywności erupcyjnej neuronów listka ciała kolankowatego bocznego : badania in vitro patch clamp

INTRODUCTION The intergeniculate leaflet of the thalamus (IGL) is one of the two major neu-ronal structures of the mammalian biological clock. IGL neurons in rats, investi-gated in vivo, show a characteristic isoperiodic infra-slow oscillatory pattern (ISO) of activity. The mean period of this activity is about 120 s. The function of oscillatory activity, commonly observed in other biological clock structures, could be connected with the secretion of neuropeptides. Molecular study of the bases of bursting activity evoked in vitro, can help to better understand the mechanism of these oscillations. It was shown that the key element in this phe-nomenon is the expression of HCN family nonselective cationic channels and voltage-dependent T-type calcium channels. The generated h-current (the base of voltage sag) and t-current (causing low threshold spikes; LTS) are the topic of many studies revealing the mechanism of rhythmic neuronal activity. MATERIALS AND METHODS Based on our results of a patch clamp study, we have proposed the division of IGL cells into groups (using cluster analysis) based on different amplitudes of voltage components evoked by the h-current and t-current. The location of the investigated neurons chosen for further analysis was confirmed by immunohisto-chemical staining and confocal microscopy. RESULTS AND CONCLUSIONS The IGL neurons were classified into four groups showing different amplitudes of voltage sag and LTS. This classification obtained during experiments conducted at in vitro conditions, may provide information about the oscillatory na-ture of the neuron observed in the in vivo study.WSTĘP Listek ciała kolankowatego bocznego wzgórza (intergeniculate leaflet – IGL) jest jedną z dwóch głównych neuronalnych struktur zegara biologicznego ssaków. U szczura, w badaniach in vivo, neurony IGL wykazują charakterystyczny izoperiodyczny wzór infrawolnych oscylacji (infra-slow oscillation – ISO) generowania potencjałów czynnościowych, o okresie około 120 s. Przypuszcza się, że taka aktywność neuronalna, będąca cechą wspólną struktur zegara biologicznego, może ułatwiać sekrecję neuropeptydów. Badania molekularnych podstaw aktywności erupcyjnej, która może być wywołana w warunkach in vitro, przybliżają nas do poznania mechanizmu powstawania tych oscylacji. Wykazano, że kluczowym elementem, w generowaniu tego rodzaju oscylacji, jest obecność nieselektywnych kanałów kationowych z rodziny HCN oraz napięciowozależnych kanałów wapniowych typu-T. Przepływający przez nie prąd-h (powodujący sag napięciowy) oraz prąd-t (manifestujący się występowaniem niskoprogowych potencjałów wapniowych (low threshold spike – LTS) to obiekty wielu badań, dotyczących rytmicznej aktywności komórek nerwowych. MATERIAŁY I METODY Opierajac się na wynikach własnych badań, metodą elektrofizjologicznej rejestracji aktywności pojedynczych neuronów – patch clamp – autorzy zaproponowali podział neuronów IGL (na podstawie analizy klastrów), w zależności od amplitudy zmian napięcia, wywołanych prądem-h i prądem-t. Lokalizacja analizowanych neuronów została potwierdzona dzięki barwieniom immunohistochemicznym i mikroskopii konfokalnej. WYNIKI I WNIOSKI Otrzymano cztery grupy neuronów IGL, różniących się amplitudą sagu napięciowego oraz LTS. Przeprowadzony podczas badań in vitro podział może dostarczyć informacji na temat zdolności komórek do generowania aktywności oscylacyjnej w warunkach in vivo

Proglucagon signalling in the rat dorsomedial hypothalamus - physiology and high-fat diet-mediated alterations

A relatively new pharmacological target in obesity treatment has been the preproglucagon (PPG) signalling, predominantly with glucagon-like peptide (GLP) 1 receptor agonists. As far as the PPG role within the digestive system is well recognised, its actions in the brain remain understudied. Here, we investigated PPG signalling in the Dorsomedial Hypothalamus (DMH), a structure involved in feeding regulation and metabolism, using in situ hybridisation, electrophysiology, and immunohistochemistry. Our experiments were performed on animals fed both control, and high-fat diet (HFD), uncovering HFD-mediated alterations. First, sensitivity to exendin-4 (Exn4, a GLP1R agonist) was shown to increase under HFD, with a higher number of responsive neurons. The amplitude of the response to both Exn4 and oxyntomodulin (Oxm) was also altered, diminishing its relationship with the cells' spontaneous firing rate. Not only neuronal sensitivity, but also GLP1 presence, and therefore possibly release, was influenced by HFD. Immunofluorescent labelling of the GLP1 showed changes in its density depending on the metabolic state (fasted/fed), but this effect was eliminated by HFD feeding. Interestingly, these dietary differences were absent after a period of restricted feeding, allowing for an anticipation of the alternating metabolic states, which suggests possible prevention of such outcome

Cryo-EM structures of the human Elongator complex at work

tRNA modifications affect ribosomal elongation speed and co-translational folding dynamics. The Elongator complex is responsible for introducing 5-carboxymethyl at wobble uridine bases (cm5U34) in eukaryotic tRNAs. However, the structure and function of human Elongator remain poorly understood. In this study, we present a series of cryo-EM structures of human ELP123 in complex with tRNA and cofactors at four different stages of the reaction. The structures at resolutions of up to 2.9 Å together with complementary functional analyses reveal the molecular mechanism of the modification reaction. Our results show that tRNA binding exposes a universally conserved uridine at position 33 (U33), which triggers acetyl-CoA hydrolysis. We identify a series of conserved residues that are crucial for the radical-based acetylation of U34 and profile the molecular effects of patient-derived mutations. Together, we provide the high-resolution view of human Elongator and reveal its detailed mechanism of action