Screening for sequence-specific RNA-BPs by comprehensive UV crosslinking

BACKGROUND: Specific cis-elements and the associated trans-acting factors have been implicated in the post-transcriptional regulation of gene expression. In the era of genome wide analyses identifying novel trans-acting factors and cis-regulatory elements is a step towards understanding coordinated gene expression. UV-crosslink analysis is a standard method used to identify RNA-binding proteins. Uridine is traditionally used to radiolabel substrate RNAs, however, proteins binding to cis-elments particularly uridine poor will be weakly or not detected. We evaluate here the possibility of using UV-crosslinking with RNA substrates radiolabeled with each of the four ribonucleotides as an approach for screening for novel sequence specific RNA-binding proteins. RESULTS: The radiolabeled RNA substrates were derived from the 3'UTRs of the cloned Eg and c-mos Xenopus laevis maternal mRNAs. Specific, but not identical, uv-crosslinking signals were obtained, some of which corresponded to already identified proteins. A signal for a novel 90 kDa protein was observed with the c-mos 3'UTR radiolabeled with both CTP and GTP but not with UTP. The binding site of the 90 kDa RNA-binding protein was localised to a 59-nucleotide portion of the c-mos 3'UTR. CONCLUSION: That the 90 kDa signal was detected with RNAs radiolabeled with CTP or GTP but not UTP illustrates the advantage of radiolabeling all four nucleotides in a UV-crosslink based screen. This method can be used for both long and short RNAs and does not require knowledge of the cis-acting sequence. It should be amenable to high throughput screening for RNA binding proteins

Acceleration, current amplification and emittance in MBE-4, an experimental beam induction linear accelerator for heavy ions

We report on the implementation of a second schedule of acceleration and current amplification in MBE-4. Control of the beam current within the bunch is improved over that in the first schedule by the addition of several small amplitude induction pulsers to compensate for acceleration errors and to control the ends of the bunch. Measurements of the longitudinal and transverse emittance are presented. 5 refs., 3 figs., 1 tab

Cytotoxic CD8+ T cell-neuron interactions: perforin-dependent electrical silencing precedes but is not causally linked to neuronal cell death

Cytotoxic CD8(+) T cells are considered important effector cells contributing to neuronal damage in inflammatory and degenerative CNS disorders. Using time-lapse video microscopy and two-photon imaging in combination with whole-cell patch-clamp recordings, we here show that major histocompatibility class I (MHC I)-restricted neuronal antigen presentation and T cell receptor specificity determine CD8(+) T-cell locomotion and neuronal damage in culture and hippocampal brain slices. Two separate functional consequences result from a direct cell-cell contact between antigen-presenting neurons and antigen-specific CD8(+) T cells. (1) An immediate impairment of electrical signaling in single neurons and neuronal networks occurs as a result of massive shunting of the membrane capacitance after insertion of channel-forming perforin (and probably activation of other transmembrane conductances), which is paralleled by an increase of intracellular Ca(2+) levels (within <10 min). (2) Antigen-dependent neuronal apoptosis may occur independently of perforin and members of the granzyme B cluster (within approximately 1 h), suggesting that extracellular effects can substitute for intracellular delivery of granzymes by perforin. Thus, electrical silencing is an immediate consequence of MHC I-restricted interaction of CD8(+) T cells with neurons. This mechanism is clearly perforin-dependent and precedes, but is not causally linked, to neuronal cell death

Excitotoxic neuronal cell death during an oligodendrocyte-directed CD8+ T cell attack in the CNS gray matter

Background: Neural-antigen reactive cytotoxic CD8+ T cells contribute to neuronal dysfunction and degeneration in a variety of inflammatory CNS disorders. Facing excess numbers of target cells, CNS-invading CD8+ T cells cause neuronal cell death either via confined release of cytotoxic effector molecules towards neurons, or via spillover of cytotoxic effector molecules from 'leaky’ immunological synapses and non-confined release by CD8+ T cells themselves during serial and simultaneous killing of oligodendrocytes or astrocytes. Methods: Wild-type and T cell receptor transgenic CD8+ T cells were stimulated in vitro, their activation status was assessed by flow cytometry, and supernatant glutamate levels were determined using an enzymatic assay. Expression regulation of molecules involved in vesicular glutamate release was examined by quantitative real-time PCR, and mechanisms of non-vesicular glutamate release were studied by pharmacological blocking experiments. The impact of CD8+ T cell-mediated glutamate liberation on neuronal viability was studied in acute brain slice preparations. Results: Following T cell receptor stimulation, CD8+ T cells acquire the molecular repertoire for vesicular glutamate release: (i) they upregulate expression of glutaminase required to generate glutamate via deamination of glutamine and (ii) they upregulate expression of vesicular proton-ATPase and vesicular glutamate transporters required for filling of vesicles with glutamate. Subsequently, CD8+ T cells release glutamate in a strictly stimulus-dependent manner. Upon repetitive T cell receptor stimulation, CD25high CD8+ T effector cells exhibit higher estimated single cell glutamate release rates than CD25low CD8+ T memory cells. Moreover, glutamate liberation by oligodendrocyte-reactive CD25high CD8+ T effector cells is capable of eliciting collateral excitotoxic cell death of neurons (despite glutamate re-uptake by glia cells and neurons) in intact CNS gray matter. Conclusion: Glutamate release may represent a crucial effector pathway of neural-antigen reactive CD8+ T cells, contributing to excitotoxicity in CNS inflammation.<br

Post-stroke inhibition of induced NADPH oxidase type 4 prevents oxidative stress and neurodegeneration

Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox4(-/-)) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox4(-/-) mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy

Significantly lower antigenicity of incobotulinumtoxin than abo- or onabotulinumtoxin

BACKGROUND: For many indications, BoNT/A is repetitively injected with the risk of developing neutralizing antibodies (NABs). Therefore, it is important to analyze whether there is a difference in antigenicity between the different licensed BoNT/A preparations. METHODS: In this cross-sectional study, the prevalence of NABs was tested by means of the sensitive mouse hemidiaphragm assay (MHDA) in 645 patients. Patients were split into those having exclusively been treated with the complex protein-free incoBoNT/A preparation (CF-MON group) and those having started BoNT/A therapy with a complex protein-containing BoNT/A preparation (CC-I group). This CC-I group was split into those patients who remained either on abo- or onaBoNT/A (CC-MON group) and those who had been treated with at least two BoNT/A preparations (CC-SWI group). To balance treatment duration, only CC-MON patients who did not start their BoNT/A therapy more than 10 years before recruitment (CC-MON-10 group) were further analyzed. The log-rank test was used to compare the prevalence of NABs in the CF-MON and CC-MON-10 group. RESULTS: In the CF-MON subgroup, no patient developed NABs. In the CC-I group, 84 patients were NAB-positive. NABs were found in 33.3% of those who switched preparations (CC-SWI) and in 5.9% of the CC-MON-10 group. Kaplan-Meier curves for remaining NAB-negative under continuous BoNT/A therapy were significantly different (p < 0.035) between the CF-MON and CC-MON-10 group. CONCLUSION: Frequent injections of a complex protein-containing BoNT/A preparation are associated with significantly higher risks of developing NABs than injections with the same frequency using the complex protein-free incoBoNT/A preparation