Probing the pharmacological properties of distinct subunit interfaces within heteromeric glycine receptors reveals a functional ββ agonist-binding site

Synaptic glycine receptors (GlyRs) are hetero-pentameric chloride channels composed of α and β subunits, which are activated by agonist binding at subunit interfaces. To examine the pharmacological properties of each potential agonist-binding site, we substituted residues of the GlyR α(1) subunit by the corresponding residues of the β subunit, as deduced from sequence alignment and homology modeling based on the recently published crystal structure of the glutamate-gated chloride channel GluCl. These exchange substitutions allowed us to reproduce the βα, αβ and ββ subunit interfaces present in synaptic heteromeric GlyRs by generating recombinant homomeric receptors. When the engineered α(1) GlyR mutants were expressed in Xenopus oocytes, all subunit interface combinations were found to form functional agonist-binding sites as revealed by voltage clamp recording. The ββ-binding site displayed the most distinct pharmacological profile towards a range of agonists and modulators tested, indicating that it might be selectively targeted to modulate the activity of synaptic GlyRs. The mutational approach described here should be generally applicable to heteromeric ligand-gated ion channels composed of homologous subunits and facilitate screening efforts aimed at targeting inter-subunit specific binding sites.     

Ammonium formation and assimilation in P(SARK)∷IPT tobacco transgenic plants under low N

Wild Type (WT) and transgenic tobacco plants expressing isopentenyltransferase (IPT), a gene encoding the enzyme regulating the rate-limiting step in cytokinins (CKs) synthesis, were grown under limited nitrogen (N) conditions. We analyzed nitrogen forms, nitrogen metabolism related-enzymes, amino acids and photorespiration related-enzymes in WT and P_SARK∷IPT tobacco plants. Our results indicate that the WT plants subjected to N deficiency displayed reduced nitrate (NO₃⁻) assimilation. However, an increase in the production of ammonium (NH₄⁺), by the degradation of proteins and photorespiration led to an increase in the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle in WT plants. In these plants, the amounts of amino acids decreased with N deficiency, although the relative amounts of glutamate and glutamine increased with N deficiency. Although the transgenic plants expressing P_SARK∷IPT and growing under suboptimal N conditions displayed a significant decline in the N forms in the leaf, they maintained the GS/GOGAT cycle at control levels. Our results suggest that, under N deficiency, CKs prevented the generation and assimilation of NH₄⁺ by increasing such processes as photorespiration, protein degradation, the GS/GOGAT cycle, and the formation of glutamine.     

The role of amino acid transporters in GSH synthesis in the blood-brain barrier and central nervous system

Glutathione (GSH) plays a critical role in protecting cells from oxidative stress and xenobiotics, as well as maintaining the thiol redox state, most notably in the central nervous system (CNS). GSH concentration and synthesis are highly regulated within the CNS and are limited by availability of the sulfhydryl amino acid (AA) l-cys, which is mainly transported from the blood, through the blood-brain barrier (BBB), and into neurons. Several antiporter transport systems (e.g., x(c)(-), x(-)(AG), and L) with clearly different luminal and abluminal distribution, Na(+), and pH dependency have been described in brain endothelial cells (BEC) of the BBB, as well as in neurons, astrocytes, microglia and oligodendrocytes from different brain structures. The purpose of this review is to summarize information regarding the different AA transport systems for l-cys and its oxidized form l-cys(2) in the CNS, such as expression and activity in blood-brain barrier endothelial cells, astrocytes and neurons and environmental factors that modulate transport kinetics.     

Differences in kainate receptor involvement in hippocampal mossy fibre long-term potentiation depending on slice orientation

Long-term potentiation (LTP) is a well-established experimental model used to investigate the synaptic basis of learning and memory. LTP at mossy fibre - CA3 synapses in the hippocampus is unusual because it is normally N-methyl-d-aspartate (NMDA) receptor-independent. Instead it seems that the trigger for mossy fibre LTP involves kainate receptors (KARs). Although it is generally accepted that pre-synaptic KARs play an essential role in frequency facilitation and LTP, their subunit composition remains a matter of significant controversy. We have reported previously that both frequency facilitation and LTP can be blocked by selective antagonism of GluK1 (formerly GluR5/Glu(K5))-containing KARs, but other groups have failed to reproduce this effect. Moreover, data from receptor knockout and mRNA expression studies argue against a major role of GluK1, supporting a more central role for GluK2 (formerly GluR6/Glu(K6)). A potential reason underlying the controversy in the pharmacological experiments may reside in differences in the preparations used. Here we show differences in pharmacological sensitivity of synaptic plasticity at mossy fibre - CA3 synapses depend critically on slice orientation. In transverse slices, LTP of fEPSPs was invariably resistant to GluK1-selective antagonists whereas in parasagittal slices LTP was consistently blocked by GluK1-selective antagonists. In addition, there were pronounced differences in the magnitude of frequency facilitation and the sensitivity to the mGlu2/3 receptor agonist DCG-IV. Using anterograde labelling of granule cells we show that slices of both orientations possess intact mossy fibres and both large and small presynaptic boutons. Transverse slices have denser fibre tracts but a smaller proportion of giant mossy fibre boutons. These results further demonstrate a considerable heterogeneity in the functional properties of the mossy fibre projection.     

Identification of congenital muscular dystonia 2 associated with an inherited GlyT2 defect in Belgian Blue cattle from the United Kingdom

Two newborn Belgian Blue calves from a farm in the United Kingdom exhibited lateral recumbency, low head carriage and transient muscle spasms following tactile or auditory stimulation. DNA sequence analysis indicated that both calves were homozygous for the recessive congenital muscular dystonia type 2 (CMD2) mutation (c.809T>C, p.Leu270Pro) in SLC6A5, encoding the neuronal glycine transporter GlyT2. Further testing of animals from the index farm and a sample of Belgian Blue sires revealed an unexpectedly high frequency of CMD2 carriers. This implies that linked quantitative trait loci may be influencing the prevalence of CMD2 in the estimated 55,000 Belgian Blue cattle in the United Kingdom. We have therefore developed new inexpensive tests for the CMD2 allele that can be used to confirm diagnosis, identify carriers and guide future breeding strategy, thus avoiding animal distress/premature death and minimizing the future economic impact of this disorder.     

The multiple dimensions of racial mixture in Rio de Janeiro,Brazil: from whitening to Brazilian negritude

Abstract

The notion that racial mixture is a central feature of Latin American societies has been interpreted in different, if not strictly opposite, ways. On the one hand, scholars have presented it as evidence of weaker racial boundaries. On the other, it has been denounced as an expression of the illusion of harmonic racial relations. Relying on 160 interviews with black Brazilians, we argue that the valorization of racial mixture is an important response to stigmatization, but one that has multiple dimensions and different consequences for the maintenance of racial boundaries. We map out these different dimensions – namely, ‘whitening’, ‘Brazilian negritude’, ‘national identification’ and ‘non-essentialist racialism’ – and discuss how these dimensions are combined in different ways by our interviewees according to various circumstances. Exploring these multiple dimensions, we question any simplistic understanding of racial mixture as the blessing or the curse of Latin American racial dynamics.     

Development of novel low-copy nuclear markers for Hieraciinae (Asteraceae) and their perspective for other tribes

? Premise of the study: The development of three low-copy nuclear markers for low taxonomic level phylogenies in Asteraceae with emphasis on the subtribe Hieraciinae is reported. ? Methods and Results: Marker candidates were selected by comparing a Lactuca complementary DNA (cDNA) library with public DNA sequence databases. Interspecific variation and phylogenetic signal of the selected genes were investigated for diploid taxa from the subtribe Hieraciinae and compared to a reference phylogeny. Their ability to cross-amplify was assessed for other Asteraceae tribes. All three markers had higher variation (2.1-4.5 times) than the internal transcribed spacer (ITS) in Hieraciinae. Cross-amplification was successful in at least seven other tribes of the Asteraceae. Only three cases indicating the presence of paralogs or pseudogenes were detected. ? Conclusions: The results demonstrate the potential of these markers for phylogeny reconstruction in the Hieraciinae as well as in other Asteraceae tribes, especially for very closely related species.     

Fungi are involved in the effects of litter mixtures on consumption by shredders

1. Decomposition of litter mixtures in both terrestrial and aquatic ecosystems often shows non‐additive diversity effects on decomposition rate, generally interpreted in streams as a result of the feeding activity of macroinvertebrates. The extent to which fungal assemblages on mixed litter may influence consumption by macroinvertebrates remains unknown. 2. We assessed the effect of litter mixing on all possible three‐species combinations drawn from four tree species (Alnus glutinosa, Betula pendula, Juglans regia and Quercus robur) on both fungal assemblages and the rate of litter consumption by a common shredder, Gammarus fossarum. After a 9‐week inoculation in a stream, batches of leaf discs were taken from all leaf species within litter mixture combinations. Ergosterol, an indicator of fungal biomass, and the composition of fungal assemblages, assessed from the conidia released, were determined, and incubated litter offered to G. fossarum in a laboratory‐feeding experiment. 3. Mixing leaf litter species enhanced both the Simpson’s index of the fungal assemblage and the consumption of litter by G. fossarum, but had no clear effect on mycelial biomass. Specifically, consumption rates of J. regia were consistently higher for mixed‐species litter packs than for single‐species litter. In contrast, the consumption rates of B. pendula were not affected by litter mixing, because of the occurrence of both positive and negative litter‐mixing effects in different litter species combinations that counteracted each other. 4. In some litter combinations, the greater development of some fungal species (e.g. Clavariopsis aquatica) as shown by higher sporulation rates coincided with increased leaf consumption, which may have resulted from feeding preferences by G. fossarum for these fungi. 5. Where litter mixture effects on decomposition rate are mediated via shredder feeding, this could be due to indirect effects of the fungal assemblage.     

Glycine to glutamic acid misincorporation observed in a recombinant protein expressed by Escherichia coli cells

A novel amino acid misincorporation, in which the intended glycine (Gly) residues were replaced by a glutamic acid (Glu), was observed in a recombinant protein expressed by Escherichia coli. The misincorporation was identified by peptide mapping and liquid chromatography-tandem mass spectrometric analysis on proteolyzed peptides of the protein and verified using the corresponding synthetic peptides containing the misincorporated residues. Analysis of the distribution of the misincorporated residues and their codon usage shows strong correlation between this misincorporation and the use of rarely used codon within the E. coli expression system. Results in this study suggest that the usage of the rare codon GGA has resulted in a Glu for Gly misincorporation.     

片断化生境中滇桐传粉生物学和繁育系统

通过野外观察和人工授粉试验方法,对云南省文山州西畴县法斗分布区片断化生境中濒危植物滇桐(Craigia yunnanensis)的传粉生物学特征和繁育系统进行研究.结果表明:滇桐每个聚伞花序有2～9朵两性花,单花花期为3～4d,单花雌雄蕊在时空上有一定的隔离;杂交指数(OCI) ＞4,花粉-胚珠比(P/O)为1381±53;有效传粉昆虫为大头丽蝇(Chrysomyia megacephala);同株异花授粉结实率低;滇桐的繁育系统属于异交为主,部分自交亲和,传粉过程需要传粉者;自然状态下滇桐的座果率(56.67％±3.85％)和结籽率(6.26％±0.75％)均较低,而异株异花授粉均可显著提高座果率及结籽率(P＜0.01),这与当前片断化生境中传粉昆虫少、效率低下等有关,表明生境片断化正在影响滇桐植株的早期生殖成功.