In Vitro Studies on the Putative Function of N-acetylaspartate as an Osmoregulator

Efflux and tissue content of N-acetylaspartate (NAA) and amino acids were evaluated from cultured and acutely prepared hippocampal slices in response to changes in osmolarity. The osmoregulator taurine, but not NAA, was lost from both types of slices after moderate reductions in extracellular osmolarity (−60 mOsm) for 10–48 h. Hypoosmotic shock (−166 mOsm) for 5 min resulted in unselective efflux of several amino acids from acutely prepared slices. Notably, the efflux of taurine, but not NAA, was prominent also after the shock. Efflux of NAA was markedly enhanced by NMDA and high K⁺, in particular after the stimulation period. The high K⁺-mediated efflux was decreased by high extracellular osmolarity and a NMDA-receptor antagonist. The results indicate that NAA efflux can be induced by a sudden non-physiological decrease in extracellular osmolarity but not by prolonged more moderate changes in osmolarity. The mechanisms behind the efflux of NAA by high K⁺ are complex and may involve both swelling and activation of NMDA-receptors.     

Inflammation-associated S100 proteins: new mechanisms that regulate function

This review focuses on new aspects of extracellular roles of the calgranulins. S100A8, S100A9 and S100A12 are constitutively expressed in neutrophils and induced in several cell types. The S100A8 and S100A9 genes are regulated by pro- and anti-inflammatory mediators and their functions may depend on cell type, mediators within a particular inflammatory milieu, receptors involved in their recognition and their post-translational modification. The S100A8 gene induction in macrophages is dependent on IL-10 and potentiated by immunosuppressive agents. S100A8 and S100A9 are oxidized by peroxide, hypochlorite and nitric oxide (NO). HOCl generates intra-chain sulfinamide bonds; stronger oxidation promotes cross-linked forms that are seen in human atheroma. S100A8 is >200-fold more sensitive to oxidative cross-linking than low-density lipoprotein and may reduce oxidative damage. S100A8 and S100A9 can be S-nitrosylated. S100A8–SNO suppresses mast cell activation and inflammation in the microcirculation and may act as an NO transporter to regulate vessel tone in inflammatory lesions. S100A12 activates mast cells and is a monocyte and mast cell chemoattractant; a G-protein-coupled mechanism may be involved. Structure–function studies are discussed in relation to conservation and divergence of functions in S100A8. S100A12 induces cytokines in mast cells, but not monocytes/macrophages. It forms complexes with Zn²⁺ and, by chelating Zn²⁺, S100A12 significantly inhibits MMPs. Zn²⁺ in S100A12 complexes co-localize with MMP-9 in foam cells in atheroma. In summary, S100A12 has pro-inflammatory properties that are likely to be stable in an oxidative environment, because it lacks Cys and Met residues. Conversely, S100A8 and S100A9 oxidation and S-nitrosylation may have important protective mechanisms in inflammation.     

Human Alzheimer’s disease synaptic <Emphasis Type=Italic>O</Emphasis>-GlcNAc site mapping and iTRAQ expression proteomics with ion trap mass spectrometry

Neuronal synaptic functional deficits are linked to impaired learning and memory in Alzheimer’s disease (AD). We recently demonstrated that O-GlcNAc, a novel cytosolic and nuclear carbohydrate post-translational modification, is enriched at neuronal synapses and positively regulates synaptic plasticity linked to learning and memory in mice. Reduced levels of O-GlcNAc have been observed in AD, suggesting a possible link to deficits in synaptic plasticity. Using lectin enrichment and mass spectrometry, we mapped several human cortical synaptic O-GlcNAc modification sites. Overlap in patterns of O-GlcNAcation between mouse and human appears to be high, as previously mapped mouse synaptic O-GlcNAc sites in Bassoon, Piccolo, and tubulin polymerization promoting protein p25 were identified in human. Novel O-GlcNAc modification sites were identified on Mek2 and RPN13/ADRM1. Mek2 is a signaling component of the Erk 1/2 pathway involved in synaptic plasticity. RPN13 is a component of the proteasomal degradation pathway. The potential interplay of phosphorylation with mapped O-GlcNAc sites, and possible implication of those sites in synaptic plasticity in normal versus AD states is discussed. iTRAQ is a powerful differential isotopic quantitative approach in proteomics. Pulsed Q dissociation (PQD) is a recently introduced fragmentation strategy that enables detection of low mass iTRAQ reporter ions in ion trap mass spectrometry. We optimized LTQ ion trap settings for PQD-based iTRAQ quantitation and demonstrated its utility in O-GlcNAc site mapping. Using iTRAQ, abnormal synaptic expression levels of several proteins previously implicated in AD pathology were observed in addition to novel changes in synaptic specific protein expression including Synapsin II.     

Vanadyl bisacetylacetonate protects β cells from palmitate-induced cell death through the unfolded protein response pathway

Endoplasmic reticulum (ER) stress induced by free fatty acids (FFA) is important to β-cell loss during the development of type 2 diabetes. To test whether vanadium compounds could influence ER stress and the responses in their mechanism of antidiabetic effects, we investigated the effects and the mechanism of vanadyl bisacetylacetonate [VO(acac)₂] on β cells upon treatment with palmitate, a typical saturated FFA. The experimental results showed that VO(acac)₂ could enhance FFA-induced signaling pathways of unfolded protein responses by upregulating the prosurvival chaperone immunoglobulin heavy-chain binding protein/78-kDa glucose-regulated protein and downregulating the expression of apoptotic C/EBP homologous protein, and consequently the reduction of insulin synthesis. VO(acac)₂ also ameliorated FFA-disturbed Ca²⁺ homeostasis in β cells. Overall, VO(acac)₂ enhanced stress adaption, thus protecting β cells from palmitate-induced apoptosis. This study provides some new insights into the mechanisms of antidiabetic vanadium compounds.     

A novel zinc bis(thiosemicarbazone) complex for live cell imaging

Fluorescent zinc complexes have recently attracted a lot of interest owing to their vast applications in cellular imaging. We report the synthesis as well as physical, chemical and biological studies of a novel zinc glyoxalbis(4-methyl-4-phenyl-3-thiosemicarbazone), [Zn(GTSC)]₃, complex. As compared with the well-studied zinc biacetylbis(4-methyl-3-thiosemicarbazone), Zn(ATSM), complex, which was used as a reference, [Zn(GTSC)]₃ had 2.5-fold higher fluorescence. When cellular fluorescence was measured using flow cytometry, we observed that [Zn(GTSC)]₃ had 3.4-fold to 12-fold higher fluorescence than Zn(ATSM) in various cell lines (n = 9) of different tissue origin. Confocal fluorescence microscopy results showed that [Zn(GTSC)]₃ appeared to have a nuclear localization within 30 min of addition to MCF7 cells. Moreover, [Zn(GTSC)]₃ showed minimal cytotoxicity compared with Zn(ATSM), suggesting that [Zn(GTSC)]₃ may be less deleterious to cells when used as an imaging agent. Our data suggest that the novel [Zn(GTSC)]₃ complex can potentially serve as a biocompatible fluorescent imaging agent for live cells.     

Biodiversity and conservation of insects and other invertebrates

The thematic issue of Biodiversity and Conservation devoted to the biodiversity and conservation of insects and other invertebrates is introduced. The issue comprises 23 original research papers covering diverse habitats from forests to grasslands, ponds and rivers to coasts, and the tropics to boreal regions. Amongst the organisms discussed are ants, bees, beetles, butterflies, crabs, microgastropods, millipedes, spiders, and weevils. Some of the difficulties of conserving the most species-rich groups of eukaryotes, in the face of ignorance as to their identities and positions in ecological processes, are noted and the precautionary principle is seen as a pragmatic and responsible approach.     

Non-native and translocated fish species in Serbia and their impact on the native ichthyofauna

A total of 22 fish species have been introduced into the inland waters of Serbia, either intentionally or accidentally. This paper provides a summary of data concerning time and reason of introduction, mode of expansion, degree of acclimatization, impact on native fish and estimated area of recent distribution. Four of the non-native fish species currently occupy more than 51% of Serbian territory while 5 of them occupy between 21–50% of territory. This paper reviews impacts of introduced freshwater fish in Serbia based on collected data.     

Intestinal iron absorption during suckling in mammals

The maintenance of appropriate iron levels is important for mammalian health, particularly during the rapid growth period following birth. Too little iron can lead to irreversible damage to the developing central nervous system and too much iron at this point can have adverse long term consequences, possibly due to excessive free radical production. In order to maintain iron levels, intestinal iron absorption is very efficient in young mammals, such that almost all of the iron in breast milk is utilized. However this high level of absorption is unable to be down regulated in response to excess iron as it can be in adults, implying that different regulatory processes are involved during suckling. Various mechanisms have been proposed to explain this high absorption, including enhanced expression of the proteins involved in iron absorption in adults (particularly DMT1 and ferroportin), non-specific uptake via pinocytosis, and the uptake of lactoferrin bound iron by the lactoferrin receptor. However, at present the precise mechanism is unclear. It is possible that all of these components contribute to the high intestinal iron absorption seen during suckling, or a novel, as yet undescribed, mechanism could be involved. This review summarises the evidence for and against each of the mechanisms described above and highlights how little is known about iron homeostasis in this vital stage of development.     

<Emphasis Type=Italic>Pseudomonas</Emphasis> siderophores in the sputum of patients with cystic fibrosis

The lungs of patients with cystic fibrosis become chronically infected with the bacterium Pseudomonas aeruginosa, which heralds progressive lung damage and a decline in health. Iron is a crucial micronutrient for bacteria and its acquisition is a key factor in infection. P. aeruginosa can acquire this element by secreting pyoverdine and pyochelin, iron-chelating compounds (siderophores) that scavenge iron and deliver it to the bacteria. Siderophore-mediated iron uptake is generally considered a key factor in the ability of P. aeruginosa to cause infection. We have investigated the amounts of pyoverdine in 148 sputum samples from 36 cystic fibrosis patients (30 infected with P. aeruginosa and 6 as negative controls). Pyoverdine was present in 93 samples in concentrations between 0.30 and 51 μM (median 4.6 μM) and there was a strong association between the amount of pyoverdine and the number of P. aeruginosa present. However, pyoverdine was not present, or below the limits of detection (~0.3 μM), in 21 sputum samples that contained P. aeruginosa. Pyochelin was also absent, or below the limits of detection (~1 μM), in samples from P. aeruginosa-infected patients with little or no detectable pyoverdine. Our data show that pyoverdine is an important iron-scavenging molecule for P. aeruginosa in many cystic fibrosis patients, but other P. aeruginosa iron-uptake systems must be active in some patients to satisfy the bacterial need for iron.