Microstructural investigation of Au ion-irradiated Eu-doped LaPO4 ceramics and single crystals

Ceramics and single crystals of LaPO4 monazite doped with Eu(III) were irradiated with 14 MeV Au5+ ions at three different fluences. Changes to crystallinity, local coordination environments, and topography were probed using grazing-incidence X-ray diffraction (GIXRD), vertical scanning interferometry (VSI), scanning electron microscopy (SEM), Raman, and luminescence spectroscopy. GIXRD data of the ceramics revealed fluence dependent amorphization. A similar level of amorphization was detected for samples irradiated with 5 × 1013 ions/cm2 and 1 × 1014 ions/cm2, whereas the sample irradiated with the highest fluence of 1 × 1015 ions/cm2 appeared slightly less amorphous. VSI showed clear swelling of entire grains at the highest ion fluence, while more localized damage to grain boundaries was detected for ceramic samples irradiated at the lowest fluence. Single crystal specimens showed no pronounced topography changes following irradiation. SEM images of the ceramic irradiated at the highest fluence showed topological features indicative of grain surface melting. Raman and luminescence data showed a different degree of disorder in polycrystalline vs. single crystal samples. While changes to PO4 vibrational modes were observed in the ceramics, changes were more subtle or not present in the single crystals. The opposite was observed when probing the local Ln-O environment using Eu(III) luminescence, where the larger changes in terms of an elongation of the Eu-O (or La-O) bond and an increasing relative disorder with increasing fluence were observed only for the single crystals. The dissimilar trends observed in irradiated single crystals and ceramics indicate that grain boundary chemistry likely plays a significant role in the radiation response

Synthesis and biological evaluation of novel MB327 analogs as resensitizers for desensitized nicotinic acetylcholine receptors after intoxication with nerve agents

Poisoning with organophosphorus compounds, which can lead to a cholinergic crisis due to the inhibition of acetylcholinesterase and the subsequent accumulation of acetylcholine (ACh) in the synaptic cleft, is a serious problem for which treatment options are currently insufficient. Our approach to broadening the therapeutic spectrum is to use agents that interact directly with desensitized nicotinic acetylcholine receptors (nAChRs) in order to induce functional recovery after ACh overstimulation. Although MB327, one of the most prominent compounds investigated in this context, has already shown positive properties in terms of muscle force recovery, this compound is not suitable for use as a therapeutic agent due to its insufficient potency. By means of in silico studies based on our recently presented allosteric binding pocket at the nAChR, i.e. the MB327-PAM-1 binding site, three promising MB327 analogs with a 4-aminopyridinium ion partial structure (PTM0056, PTM0062, and PTM0063) were identified. In this study, we present the synthesis and biological evaluation of a series of new analogs of the aforementioned compounds with a 4-aminopyridinium ion partial structure (PTM0064-PTM0072), as well as hydroxy-substituted analogs of MB327 (PTMD90–0012 and PTMD90–0015) designed to substitute entropically unfavorable water clusters identified during molecular dynamics simulations. The compounds were characterized in terms of their binding affinity towards the aforementioned binding site by applying the UNC0642 MS Binding Assays and in terms of their muscle force reactivation in rat diaphragm myography. More potent compounds were identified compared to MB327, as some of them showed a higher affinity towards MB327-PAM-1 and also a higher recovery of neuromuscular transmission at lower compound concentrations. To improve the treatment of organophosphate poisoning, direct targeting of nAChRs with appropriate compounds is a key step, and this study is an important contribution to this research

Natriuretic Peptide Expression and Function in GH3 Somatolactotropes and Feline Somatotrope Pituitary Tumours.

Patients harbouring mutations in genes encoding C-type natriuretic peptide (CNP; NPPC) or its receptor guanylyl cyclase B (GC-B, NPR2) suffer from severe growth phenotypes; loss-of-function mutations cause achondroplasia, whereas gain-of-function mutations cause skeletal overgrowth. Although most of the effects of CNP/GC-B on growth are mediated directly on bone, evidence suggests the natriuretic peptides may also affect anterior pituitary control of growth. Our previous studies described the expression of NPPC and NPR2 in a range of human pituitary tumours, normal human pituitary, and normal fetal human pituitary. However, the natriuretic peptide system in somatotropes has not been extensively explored. Here, we examine the expression and function of the CNP/GC-B system in rat GH3 somatolactotrope cell line and pituitary tumours from a cohort of feline hypersomatotropism (HST; acromegaly) patients. Using multiplex RT-qPCR, all three natriuretic peptides and their receptors were detected in GH3 cells. The expression of Nppc was significantly enhanced following treatment with either 100 nM TRH or 10 µM forskolin, yet only Npr1 expression was sensitive to forskolin stimulation; the effects of forskolin and TRH on Nppc expression were PKA- and MAPK-dependent, respectively. CNP stimulation of GH3 somatolactotropes significantly inhibited Esr1, Insr and Lepr expression, but dramatically enhanced cFos expression at the same time point. Oestrogen treatment significantly enhanced expression of Nppa, Nppc, Npr1, and Npr2 in GH3 somatolactotropes, but inhibited CNP-stimulated cGMP accumulation. Finally, transcripts for all three natriuretic peptides and receptors were expressed in feline pituitary tumours from patients with HST. NPPC expression was negatively correlated with pituitary tumour volume and SSTR5 expression, but positively correlated with D2R and GHR expression. Collectively, these data provide mechanisms that control expression and function of CNP in somatolactotrope cells, and identify putative transcriptional targets for CNP action in somatotropes

Natriuretic Peptide Expression and Function in GH3 Somatolactotropes and Feline Somatotrope Pituitary Tumours.

Patients harbouring mutations in genes encoding C-type natriuretic peptide (CNP; NPPC) or its receptor guanylyl cyclase B (GC-B, NPR2) suffer from severe growth phenotypes; loss-of-function mutations cause achondroplasia, whereas gain-of-function mutations cause skeletal overgrowth. Although most of the effects of CNP/GC-B on growth are mediated directly on bone, evidence suggests the natriuretic peptides may also affect anterior pituitary control of growth. Our previous studies described the expression of NPPC and NPR2 in a range of human pituitary tumours, normal human pituitary, and normal fetal human pituitary. However, the natriuretic peptide system in somatotropes has not been extensively explored. Here, we examine the expression and function of the CNP/GC-B system in rat GH3 somatolactotrope cell line and pituitary tumours from a cohort of feline hypersomatotropism (HST; acromegaly) patients. Using multiplex RT-qPCR, all three natriuretic peptides and their receptors were detected in GH3 cells. The expression of Nppc was significantly enhanced following treatment with either 100 nM TRH or 10 µM forskolin, yet only Npr1 expression was sensitive to forskolin stimulation; the effects of forskolin and TRH on Nppc expression were PKA- and MAPK-dependent, respectively. CNP stimulation of GH3 somatolactotropes significantly inhibited Esr1, Insr and Lepr expression, but dramatically enhanced cFos expression at the same time point. Oestrogen treatment significantly enhanced expression of Nppa, Nppc, Npr1, and Npr2 in GH3 somatolactotropes, but inhibited CNP-stimulated cGMP accumulation. Finally, transcripts for all three natriuretic peptides and receptors were expressed in feline pituitary tumours from patients with HST. NPPC expression was negatively correlated with pituitary tumour volume and SSTR5 expression, but positively correlated with D2R and GHR expression. Collectively, these data provide mechanisms that control expression and function of CNP in somatolactotrope cells, and identify putative transcriptional targets for CNP action in somatotropes

Structural aspects of molybdenum-transhydroxylase from Pelobacter acidigallici and tungsten-acetylene hydratase from Pelobacter acetylenicus

P. acetylenicus is a strictly anaerobic and mesophilic bacterium that is able to grow on acetylene as single energy and carbon source. The first step in the metabolization of acetylene is the transformation of acetylene to acetaldehyde. This addition of water is catalyzed by the W/Fe-S dependent enzyme acetylene hydratase.Acetylene hydratase from P. acetylenicus was purified to homogeneity. It is a monomer with a molecular mass of the amino acid chain of 81.9 kDa. BLASTP searches revealed that the enzyme is highly similar to enzymes of the DMSO-reductase family. Acetylene hydratase is a thermostable enzyme with a temperature optimum between 50 and 55°C. It is a very stable enzyme when stored under exclusion of dioxygen in a nitrogen/hydrogen atmosphere at 6°C (Abt, 2001). Within three months, there was no detectable loss of acetylene hydratase activity from tungstate-grown P. acetylenicus. Although acetylene hydratase catalyzes no redox reaction, it contains one [4Fe-4S] center and one W-bisMGD as redox-cofactors.Crystals of the w-acetylene hydratase were obtained both in presence and in absence (N2: H2 = 94: 6 v/v) of dioxygen. Only the crystals grown in absence of dioxygen were able to diffract X-ray-radiation. Dithionite-reduced enzyme crystals obtained under exclusion of dioxygen, could be measured at the Deutsches Elektronensynchrotron (DESY) in Hamburg up to resolution better then 2,5Å. Also crystals were measured at the ESRF in Grenoble at the tungsten L-edge, however, this dataset could not be used to solve the three dimensional structure because of high mosaicity and decreasing of resolution. This crystal was not stably enough to stand the measurement in the synchrotron radiation.P. acidigallici is a strictly anaerobic bacterium that is able to live on gallic acid (3,4,5-trihydroxybenzoic acid), pyrogallol (1,2,3-trihydroxybenzene), phloroglucinol (1,3,5-trihydroxy-benzene), or 2,4,6-trihydroxybenzoic acid. A crucial step in the fermentation of decarboxylated gallic acid (pyrogallol) is the transhydroxylation of pyrogallol to phloroglucinol. This reaction is catalyzed by the Mo/Fe-S dependent enzyme transhydroxylase (pyrogallol:phloroglucinol hydroxyltransferase E.C. 1.97.1.2).Transhydroxylase from P. acidigallici is a heterodimer consisting of a large subunit (100.4 kDa) and a small subunit (31.3 kDa). This enzyme is closely related to enzymes of the DMSO-reductase family. Although the overall reaction of transhydroxylase is no redox reaction it contains different iron-sulfur centers and one Mo-bisMGD as redox-cofactors.12 of the 13 cysteines in the small β-subunit are highly conserved. Some of them are referred to the [4Fe-4S] ferredoxins. The 15 cysteines of the big subunit do not align with the cysteines of related iron-sulfur proteins. Therefore, it is unlikely that an iron-sulfur center is located in the large subunit. It is more likely that there are three [4Fe-4S] clusters located in the small subunit.Crystals of as isolated transhydroxylase were not able to diffract X-ray radiation. Crystallization of dithionite reduced transhydroxylase under exclusion of dioxygen led to crystals which diffracted to resolution limits higher than 2.5 Å with synchrotron radiation.These crystals were measured with synchrotron radiation and the three-dimensional structure of transhydroxylase was solved. Even structures of transhydroxylase in complex with pyrogallol and inhibitor (1,2,4-trihydroxybenzene) were solved at high resolutions up to 2.0 Å.These result led to a new possible reaction mechanism. Hereby the function of the molybdenum ion is to coordinate the pyrogallol at its C1 position. The amino acids Asp A174, His A144 and Tyr A404 near the active site seem to have catalytic function