Detailed analysis of inflammatory and neuromodulatory cytokine secretion from human NT2 astrocytes using multiplex bead array

Astrocytes are a very important cell type in the brain fulfilling roles in both neuroimmunology and neurotransmission. We have conducted the most comprehensive analysis of secreted cytokines conducted to date (astrocytes of any source) to determine whether astrocytes derived from the human Ntera2 (NT2) cell-line are a good model of human primary astrocytes. We have compared the secretion of cytokines from NT2 astrocytes with those produced in astrocyte enriched human brain cultures and additional cytokines implicated in brain injury or known to be expressed in the human brain. The concentration of cytokines was measured in astrocyte conditioned media using multiplex bead array (MBA), where 18 cytokines were measured simultaneously. Resting NT2 astrocytes produced low levels (～1-30 pg/ml) of MIP1α, IL-6 and GM-CSF and higher levels of MCP-1, IP-10 and IL-8 (1-11 ng/ml) under non-inflammatory conditions. All of these in addition to IL-1β, TNFα, and IL-13, were increased by pro-inflammatory activation (TNFα or IL-1β stimulation). In contrast, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, LTα, and IFNγ were not detected in astrocyte conditioned media under any of the culture conditions tested. NT2 astrocytes were unresponsive to IL-2 and the adenyl cyclase agonist, forskolin. Interestingly, IFNγ stimulation selectively increased IP-10 secretion only. As astrocytes stimulated with IL-1β or TNFα produced several chemokines in the ng/ml range, we next assessed the chemoattractant properties of these cells. Conditioned media from TNFα-stimulated astrocytes significantly chemoattracted leukocytes from human blood. This study provides the most comprehensive analysis of cytokine production by human astrocytes thus far, and shows that NT2 astrocytes are highly responsive to pro-inflammatory mediators including TNFα and IL-1β, producing cytokines and chemokines capable of attracting leukocytes from human blood. We conclude that in the absence of adult human primary astrocytes that NT2-astrocytes may provide a valuable alternative to study the immunological behaviour of human astrocytes.     

Temperature-dependent calcium-induced calcium release via InsP3 receptors in mouse olfactory ensheathing glial cells

Cooling can induce Ca(2+) signaling via activation of temperature-sensitive ion channels such as TRPM8, TRPA1 and ryanodine receptor channels. Here we have studied the mechanism of cooling-evoked Ca(2+) signaling in mouse olfactory ensheathing cells (OECs), a specialized type of glial cells in the olfactory nerve layer of the olfactory bulb. Reducing the temperature from above 30°C to 28°C and below triggered Ca(2+) transients that persisted in the absence of external Ca(2+), but were suppressed after Ca(2+) store depletion by cyclopiazonic acid. Cooling-evoked Ca(2+) transients were present in mice deficient of TRPM8 and TRPA1, and were not inhibited by ryanodine receptor antagonists. Inhibition of InsP(3) receptors with 2-APB and caffeine entirely blocked cooling-evoked Ca(2+) transients. Moderate Ca(2+) increases, as evoked by flash photolysis of NP-EGTA (caged Ca(2+)) and cyclopiazonic acid, triggered InsP(3) receptor-mediated Ca(2+) release at 22°C, but not at 31°C. The results suggest that InsP(3) receptors mediate Ca(2+)-induced Ca(2+) release in OECs, and that this Ca(2+) release is temperature-sensitive and can be suppressed at temperatures above 28°C.     

Molecular basis of Colorado potato beetle adaptation to potato plant defence at the level of digestive cysteine proteinases

Potato synthesises high levels of proteinase inhibitors in response to insect attack. This can adversely affect protein digestion in the insects, leading to reduced growth, delayed development and lowered fecundity. Colorado potato beetle overcomes this defence mechanism by changing the composition of its digestive proteinases. The induced cysteine proteinases in the adapted gut sustain a normal rate of protein hydrolysis either by inactivating the inhibitors by cleavage or by insensitivity to the inhibitors as a result of high Kis. In this study cDNA clones of cysteine proteinases in adapted guts were isolated by nested PCR on the basis of N-terminal sequences previously determined for purified enzymes (Gruden et al., 2003). The cysteine proteinase cDNAs can be classified into three groups: intestains A, B and C. The amino acid identity is more than 91% within and 35-62% between the groups. They share 43-50% identity to mammalian cathepsins S, L, K, H, J and cathepsin-like enzymes from different arthropods. Homology modelling predicts that intestains A, B and C follow the general fold of papain-like proteinases. Intestains from each group, however, differ in some specific structural characteristics in the S1 and S2 binding sites that could influence enzyme-inhibitor interaction and thus, provide different mechanisms of resistance to inhibitors for the different enzymes. Gene expression analysis revealed that the intestains A and C, but not B, are induced twofold by potato plants with high levels of proteinase inhibitors.     

Single amino acids in the lumenal loop domain influence the stability of the major light-harvesting chlorophyll a/b complex

The major light-harvesting complex of photosystem II (LHCIIb) is one of the most abundant integral membrane proteins. It greatly enhances the efficiency of photosynthesis in green plants by binding a large number of accessory pigments that absorb light energy and conduct it toward the photosynthetic reaction centers. Most of these pigments are associated with the three transmembrane and one amphiphilic alpha helices of the protein. Less is known about the significance of the loop domains connecting the alpha helices for pigment binding. Therefore, we randomly exchanged single amino acids in the lumenal loop domain of the bacterially expressed apoprotein Lhcb1 and then reconstituted the mutant protein with pigments in vitro. The resulting collection of mutated recombinant LHCIIb versions was screened by using a 96-well-format plate-based procedure described previously [Heinemann, B., and Paulsen, H. (1999) Biochemistry 38, 14088-14093], enabling us to test several thousand mutants for their ability to form stable pigment-protein complexes in vitro. At least one-third of the positions in the loop domain turned out to be sensitive targets; i.e., their exchange abolished formation of LHCIIb in vitro. This confirms our earlier notion that the LHCIIb loop domains contribute more specifically to complex formation and/or stabilization than by merely connecting the alpha helices. Among the target sites, glycines and hydrophilic amino acids are more prominently represented than hydrophobic ones. Specifically, the exchange of any of the three acidic amino acids in the lumenal loop abolishes reconstitution of stable pigment-protein complexes, suggesting that ionic interactions with other protein domains are important for correct protein folding or complex stabilization. One hydrophobic amino acid, tryptophan in position 97, has been hit repeatedly in independent mutation experiments. From the LHCIIb structure and previous mutational analyses, we propose a stabilizing interaction between this amino acid and F195 near the C-proximal end of the third transmembrane helix.     

Functional properties of proteins from the coelomic fluid of the wounded sea star Asterias rubens (L)

Impact on viability and adhesion of three protein fractions, separated by size, from the coelomic fluid of wounded Asterias rubens′, was tested on autologous coelomocytes. In addition antimicrobial property of the protein fractions was tested on the Gram-negative bacterium Vibrio parahaemolyticus. All fractions promoted viability and the larger proteins facilitated adhesion of the coelomocytes. The strongest antimicrobial effect was caused by the fraction with the smallest proteins.     

ATM and ATR promote Mre11 dependent restart of collapsed replication forks and prevent accumulation of DNA breaks

Ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia Rad3-related (ATR) and the Mre11/Rad50/Nbs1 complex ensure genome stability in response to DNA damage. However, their essential role in DNA metabolism remains unknown. Here we show that ATM and ATR prevent accumulation of DNA double-strand breaks (DSBs) during chromosomal replication. Replicating chromosomes accumulate DSBs in Xenopus laevis egg extracts depleted of ATM and ATR. Addition of ATM and ATR proteins to depleted extracts prevents DSB accumulation by promoting restart of collapsed replication forks that arise during DNA replication. We show that collapsed forks maintain MCM complex but lose Pol epsilon, and that Pol epsilon reloading requires ATM and ATR. Replication fork restart is abolished in Mre11 depleted extracts and is restored by supplementation with recombinant human Mre11/Rad50/Nbs1 complex. Using a novel fluorescence resonance energy transfer-based technique, we demonstrate that ATM and ATR induce Mre11/Rad50/Nbs1 complex redistribution to restarting forks. This study provides direct biochemical evidence that ATM and ATR prevent accumulation of chromosomal abnormalities by promoting Mre11/Rad50/Nbs1 dependent recovery of collapsed replication forks.     

Chromosome 17 abnormalities and lack of TP53 mutations in paediatric central nervous system tumours

Central nervous system (CNS) tumours are the most common solid tumours in children. Cytogenetic and molecular genetic studies of these neoplasms have previously shown abnormalities of chromosome 17, implicating genes on this autosome in tumorigenesis. To identify mutations in the TP53 tumour suppressor gene (17p13.1), we have sequenced the five highly conserved regions of this gene in 29 mixed paediatric CNS tumors. No mutations were detected by this analysis. In order to identify other candidate disease loci on chromosome 17, we have carried out a detailed deletion mapping analysis using 16 polymorphic DNA markers on 19 of the above tumours and an additional four cases. Abnormalities of chromosome 17 occurred in nine cases (39%), six of which were primitive neuroectodermal tumour (PNET)-medulloblastomas. These findings suggest that it is unlikely that the TP53 gene is directly involved in the development of common paediatric brain tumours. This is in contrast to findings from adult brain and other tumour types. Moreover, the frequency of chromosome 17 aberrations, especially in PNET-medulloblastomas, suggests that other genes on this chromosome contribute to tumourigenesis.     

Screening of the Antarctic marine sponges (Porifera) as a source of bioactive compounds

Sponges (Porifera) currently represent one of the richest sources of natural products and account for almost half of the pharmacologically active compounds of marine origin. However, to date very little is known about the pharmacological potential of the sponges from polar regions. In this work we report on screening of ethanolic extracts from 24 Antarctic marine sponges for different biological activities. The extracts were tested for cytotoxic effects against normal and transformed cell lines, red blood cells, and algae, for modulation of the activities of selected physiologically important enzymes (acetylcholinesterase, butyrylcholinesterase, and α-amylase), and for inhibition of growth of pathogenic and ecologically relevant bacteria and fungi. An extract from Tedania (Tedaniopsis) oxeata was selectively cytotoxic against the cancer cell lines and showed growth inhibition of all of the tested ecologically relevant and potentially pathogenic fungal isolates. The sponge extracts from Isodictya erinacea and Kirkpatrickia variolosa inhibited the activities of the cholinesterase enzymes, while the sponge extracts from Isodictya lankesteri and Inflatella belli reduced the activity of α-amylase. Several sponge extracts inhibited the growth of multiresistant pathogenic bacterial isolates of different origins, including extended-spectrum beta-lactamase and carbapenem-resistant strains, while sponge extracts from K. variolosa and Myxilla (Myxilla) mollis were active against a human methicillin-resistant Staphylococcus aureus strain. We conclude that Antarctic marine sponges represent a valuable source of biologically active compounds with pharmacological potential.     

Give What You Get: Capuchin Monkeys (Cebus apella) and 4-Year-Old Children Pay Forward Positive and Negative Outcomes to Conspecifics

The breadth of human generosity is unparalleled in the natural world, and much research has explored the mechanisms underlying and motivating human prosocial behavior. Recent work has focused on the spread of prosocial behavior within groups through paying-it-forward, a case of human prosociality in which a recipient of generosity pays a good deed forward to a third individual, rather than back to the original source of generosity. While research shows that human adults do indeed pay forward generosity, little is known about the origins of this behavior. Here, we show that both capuchin monkeys (Cebus apella) and 4-year-old children pay forward positive and negative outcomes in an identical testing paradigm. These results suggest that a cognitively simple mechanism present early in phylogeny and ontogeny leads to paying forward positive, as well as negative, outcomes.     

A novel alkyne cholesterol to trace cellular cholesterol metabolism and localization

Cholesterol is an important lipid of mammalian cells and plays a fundamental role in many biological processes. Its concentration in the various cellular membranes differs and is tightly regulated. Here, we present a novel alkyne cholesterol analog suitable for tracing both cholesterol metabolism and localization. This probe can be detected by click chemistry employing various reporter azides. Alkyne cholesterol is accepted by cellular enzymes from different biological species (Brevibacterium, yeast, rat, human) and these enzymes include cholesterol oxidases, hydroxylases, and acyl transferases that generate the expected metabolites in in vitro and in vivo assays. Using fluorescence microscopy, we studied the distribution of cholesterol at subcellular resolution, detecting the lipid in the Golgi and at the plasma membrane, but also in the endoplasmic reticulum and mitochondria. In summary, alkyne cholesterol represents a versatile, sensitive, and easy-to-use tool for tracking cellular cholesterol metabolism and localization as it allows for manifold detection methods including mass spectrometry, thin-layer chromatography/fluorography, and fluorescence microscopy.