Production of Highly Polarized Positrons Using Polarized Electrons at MeV Energies.

The Polarized Electrons for Polarized Positrons experiment at the injector of the Continuous Electron Beam Accelerator Facility has demonstrated for the first time the efficient transfer of polarization from electrons to positrons produced by the polarized bremsstrahlung radiation induced by a polarized electron beam in a high-Z target. Positron polarization up to 82% have been measured for an initial electron beam momentum of 8.19 MeV/c, limited only by the electron beam polarization. This technique extends polarized positron capabilities from GeV to MeV electron beams, and opens access to polarized positron beam physics to a wide community.

Gene expression analysis in serial liver fine needle aspirates.

No method with low morbidity presently exists for obtaining serial hepatic gene expression measurements in humans. While hepatic fine needle aspiration (FNA) has lower morbidity than core needle biopsy, applicability is limited due to blood contamination, which confounds quantification of gene expression changes. The aim of this study was to validate FNA for assessment of hepatic gene expression. Liver needle biopsies and FNA procedures were simultaneously performed on 17 patients with chronic hepatitis C virus infection with an additional FNA procedure 1 week later. Nine patients had mild/moderate fibrosis and eight advanced fibrosis. Gene expression profiling was performed using Affymetrix microarrays and TaqMan qPCR; pathway analysis was performed using Ingenuity. We developed a novel strategy that applies liver-enriched normalization genes to determine the percentage of liver in the FNA sample, which enables accurate gene expression measurements overcoming biases derived from blood contamination. We obtained almost identical gene expression results (ρ = 0.99, P < 0.0001) comparing needle biopsy and FNA samples for 21 preselected genes. Gene expression results were also validated in dogs. These data suggest that liver FNA is a reliable method for serial hepatic tissue sampling with potential utility for a variety of preclinical and clinical applications.

Widespread aneuploidy revealed by DNA microarray expression profiling.

Expression profiling using DNA microarrays holds great promise for a variety of research applications, including the systematic characterization of genes discovered by sequencing projects. To demonstrate the general usefulness of this approach, we recently obtained expression profiles for nearly 300 Saccharomyces cerevisiae deletion mutants. Approximately 8% of the mutants profiled exhibited chromosome-wide expression biases, leading to spurious correlations among profiles. Competitive hybridization of genomic DNA from the mutant strains and their isogenic parental wild-type strains showed they were aneuploid for whole chromosomes or chromosomal segments. Expression profile data published by several other laboratories also suggest the use of aneuploid strains. In five separate cases, the extra chromosome harboured a close homologue of the deleted gene; in two cases, a clear growth advantage for cells acquiring the extra chromosome was demonstrated. Our results have implications for interpreting whole-genome expression data, particularly from cells known to suffer genomic instability, such as malignant or immortalized cells.

Drug target validation and identification of secondary drug target effects using DNA microarrays.

We describe here a method for drug target validation and identification of secondary drug target effects based on genome-wide gene expression patterns. The method is demonstrated by several experiments, including treatment of yeast mutant strains defective in calcineurin, immunophilins or other genes with the immunosuppressants cyclosporin A or FK506. Presence or absence of the characteristic drug 'signature' pattern of altered gene expression in drug-treated cells with a mutation in the gene encoding a putative target established whether that target was required to generate the drug signature. Drug dependent effects were seen in 'targetless' cells, showing that FK506 affects additional pathways independent of calcineurin and the immunophilins. The described method permits the direct confirmation of drug targets and recognition of drug-dependent changes in gene expression that are modulated through pathways distinct from the drug's intended target. Such a method may prove useful in improving the efficiency of drug development programs.

Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles.

Genome-wide transcript profiling was used to monitor signal transduction during yeast pheromone response. Genetic manipulations allowed analysis of changes in gene expression underlying pheromone signaling, cell cycle control, and polarized morphogenesis. A two-dimensional hierarchical clustered matrix, covering 383 of the most highly regulated genes, was constructed from 46 diverse experimental conditions. Diagnostic subsets of coexpressed genes reflected signaling activity, cross talk, and overlap of multiple mitogen-activated protein kinase (MAPK) pathways. Analysis of the profiles specified by two different MAPKs-Fus3p and Kss1p-revealed functional overlap of the filamentous growth and mating responses. Global transcript analysis reflects biological responses associated with the activation and perturbation of signal transduction pathways.

Role of scaffolds in MAP kinase pathway specificity revealed by custom design of pathway-dedicated signaling proteins.

BACKGROUND: Signal transduction pathways with shared components must be insulated from each other to avoid the inappropriate activation of multiple pathways by a single stimulus. Scaffold proteins are thought to contribute to this specificity by binding select substrates. RESULTS: We have studied the ability of scaffold proteins to influence signaling by the yeast kinase Ste11, a MAPKKK molecule that participates in three distinct MAP kinase pathways: mating, filamentation, and HOG. We used protein fusions to force Ste11 to associate preferentially with a subset of its possible binding partners in vivo, including Ste5, Ste7, and Pbs2. Signaling became confined to a particular pathway when Ste11 was covalently attached to these scaffolds or substrates. This pathway bias was conferred upon both stimulus-activated and constitutively active forms of Ste11. We also used membrane-targeted derivatives of the mating pathway scaffold, Ste5, to show that stimulus-independent signaling initiated by this scaffold remained pathway specific. Finally, we demonstrate that loss of pathway insulation has a negative physiological consequence, as nonspecific activation of both the HOG and mating pathways interfered with proper execution of the mating pathway. CONCLUSIONS: The signaling properties of these kinase fusions support a model in which scaffold proteins dictate substrate choice and promote pathway specificity by presenting preferred substrates in high local concentration. Furthermore, insulation is inherent to scaffold-mediated signaling and does not require that signaling be initiated by pathway-specific stimuli or activator proteins. Our results give insight into the mechanisms and physiological importance of pathway insulation and provide a foundation for the design of customized signaling proteins.

GCN1, a translational activator of GCN4 in Saccharomyces cerevisiae, is required for phosphorylation of eukaryotic translation initiation factor 2 by protein kinase GCN2.

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) by the protein kinase GCN2 mediates increased translation of the transcriptional activator GCN4 in amino acid-starved yeast cells. We show that this key phosphorylation event and the attendant translational induction of GCN4 are dependent on the product of a previously uncharacterized gene, GCN1. Inactivation of GCN1 did not affect the level of eIF-2 alpha phosphorylation when mammalian eIF-2 alpha kinases were expressed in yeast cells in place of GCN2, arguing against an involvement of GCN1 in dephosphorylation of eIF-2 alpha. In addition, while GCN1 is required in vivo for phosphorylation of eIF-2 alpha by GCN2, cell extracts from gcn1 delta strains contained wild-type levels of GCN2 eIF-2 alpha-kinase activity. On the basis of these results, we propose that GCN1 is not needed for GCN2 kinase activity per se but is required for in vivo activation of GCN2 in response to the starvation signal, uncharged tRNA. GCN1 encodes a protein of 297 kDa with an 88-kDa region that is highly similar in sequence to translation elongation factor 3 identified in several fungal species. This sequence similarity raises the possibility that GCN1 interacts with ribosomes or tRNA molecules and functions in conjunction with GCN2 in monitoring uncharged tRNA levels during the process of translation elongation.

Evidence that GCN1 and GCN20, translational regulators of GCN4, function on elongating ribosomes in activation of eIF2alpha kinase GCN2.

In the yeast Saccharomyces cerevisiae, phosphorylation of translation initiation factor eIF2 by protein kinase GCN2 leads to increased translation of the transcriptional activator GCN4 in amino acid-starved cells. The GCN1 and GCN20 proteins are components of a protein complex required for the stimulation of GCN2 kinase activity under starvation conditions. GCN20 is a member of the ATP-binding cassette (ABC) family, most of the members of which function as membrane-bound transporters, raising the possibility that the GCN1/GCN20 complex regulates GCN2 indirectly as an amino acid transporter. At odds with this idea, indirect immunofluorescence revealed cytoplasmic localization of GCN1 and no obvious association with plasma or vacuolar membranes. In addition, a fraction of GCN1 and GCN20 cosedimented with polysomes and 80S ribosomes, and the ribosome association of GCN20 was largely dependent on GCN1. The C-terminal 84% of GCN20 containing the ABCs was found to be dispensable for complex formation with GCN1 and for the stimulation of GCN2 kinase function. Because ABCs provide the energy-coupling mechanism for ABC transporters, these results also contradict the idea that GCN20 regulates GCN2 as an amino acid transporter. The N-terminal 15 to 25% of GCN20, which is critically required for its regulatory function, was found to interact with an internal segment of GCN1 similar in sequence to translation elongation factor 3 (EF3). Based on these findings, we propose that GCN1 performs an EF3-related function in facilitating the activation of GCN2 by uncharged tRNA on translating ribosomes. The physical interaction between GCN20 and the EF3-like domain in GCN1 could allow for modulation of GCN1 activity, and the ABC domains in GCN20 may be involved in this regulatory function. A human homolog of GCN1 has been identified, and the portion of this protein most highly conserved with yeast GCN1 has sequence similarity to EF3. Thus, similar mechanisms for the detection of uncharged tRNA on translating ribosomes may operate in yeast and human cells.

Transcriptional profiling shows that Gcn4p is a master regulator of gene expression during amino acid starvation in yeast.

Starvation for amino acids induces Gcn4p, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. In an effort to identify all genes regulated by Gcn4p during amino acid starvation, we performed cDNA microarray analysis. Data from 21 pairs of hybridization experiments using two different strains derived from S288c revealed that more than 1,000 genes were induced, and a similar number were repressed, by a factor of 2 or more in response to histidine starvation imposed by 3-aminotriazole (3AT). Profiling of a gcn4Delta strain and a constitutively induced mutant showed that Gcn4p is required for the full induction by 3AT of at least 539 genes, termed Gcn4p targets. Genes in every amino acid biosynthetic pathway except cysteine and genes encoding amino acid precursors, vitamin biosynthetic enzymes, peroxisomal components, mitochondrial carrier proteins, and autophagy proteins were all identified as Gcn4p targets. Unexpectedly, genes involved in amino acid biosynthesis represent only a quarter of the Gcn4p target genes. Gcn4p also activates genes involved in glycogen homeostasis, and mutant analysis showed that Gcn4p suppresses glycogen levels in amino acid-starved cells. Numerous genes encoding protein kinases and transcription factors were identified as targets, suggesting that Gcn4p is a master regulator of gene expression. Interestingly, expression profiles for 3AT and the alkylating agent methyl methanesulfonate (MMS) overlapped extensively, and MMS induced GCN4 translation. Thus, the broad transcriptional response evoked by Gcn4p is produced by diverse stress conditions. Finally, profiling of a gcn4Delta mutant uncovered an alternative induction pathway operating at many Gcn4p target genes in histidine-starved cells.

Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer.

We describe a flexible system for gene expression profiling using arrays of tens of thousands of oligonucleotides synthesized in situ by an ink-jet printing method employing standard phosphoramidite chemistry. We have characterized the dependence of hybridization specificity and sensitivity on parameters including oligonucleotide length, hybridization stringency, sequence identity, sample abundance, and sample preparation method. We find that 60-mer oligonucleotides reliably detect transcript ratios at one copy per cell in complex biological samples, and that ink-jet arrays are compatible with several different sample amplification and labeling techniques. Furthermore, results using only a single carefully selected oligonucleotide per gene correlate closely with those obtained using complementary DNA (cDNA) arrays. Most of the genes for which measurements differ are members of gene families that can only be distinguished by oligonucleotides. Because different oligonucleotide sequences can be specified for each array, we anticipate that ink-jet oligonucleotide array technology will be useful in a wide variety of DNA microarray applications.

Context effects on saccade-related brain potentials to words during reading.

In two experiments saccade-related brain potentials (SRPs) to sentences were investigated under conditions approximating natural reading. Our aim was to look for electrophysiological (SRP) signs of sentence context on the processing of final words that were either congruent or incongruent with the meaning of the sentence. In Experiment 1 subjects indicated by a button-press whether or not the final word was congruent with the context, while in Experiment 2 they read silently without an overt decision. In Experiment 1, SRPs to incongruent words were more negative than SRPs to congruent words between 80-310 msec (from saccade offset). In Experiment 2, however, the inconcruent SRPs became more negative than the congruent SRPs only between 280-460 msec. These results suggest that in Experiment 1, during the processing of incongruent words the early sign of registering mismatch appears simultaneously with the analysis of the visual features of the word.

Saccade-related brain potentials in semantic categorization tasks.

Saccade-related brain potentials (SRPs) were recorded in word categorization tasks in which subjects had to perform a saccade in order to perceive the stimulus. For all three conditions representing different degrees of complexity of semantic categorization, the stimuli belonged to one of two categories which appeared with the respective probabilities of either 0.20 or 0.80. The late positivity (P4) of the SRPs to infrequent stimuli appeared systematically later as the complexity of stimulus evaluation increased: The easiest categorization was accompanied by a P4 at 400 msec, in the more complex condition it peaked at 600 msec, and in the most difficult semantic categorization the P4 peaked even later, at 680 msec. This shift in peak latency with increasing complexity of categorization is in agreement with the results for traditional ERPs (e.g. Kutas and Donchin, 1978). The possible overlap of the late components was investigated by applying Principal Component-Varimax Analysis to the SRPs.

Saccade-related brain potentials during reading correct and incorrect versions of proverbs.

Using a reading task, the present study investigated saccade-related brain potentials (SRPs) accompanying the perception of the final words of proverbs, i.e. of sentences where the context allows a strong anticipation of the final word. The sentences were presented one at a time on a TV monitor. The proverbs appeared either in their original form or with their final word changed to be incongruous with the sentence context. SRPs to the two types of final words were recorded from 4 scalp areas. The onset of the saccade leading to the final word was used to trigger the averaging of SRPs. Incongruent and congruent brain responses were also compared by means of difference waveforms. The results showed that a difference between SRPs to congruous vs incongruous final words of proverbs already appeared simultaneously with the SRP component indicating the analysis of the visual pattern of the word. This finding supports an interactive model of word perception.

Electrocortical signs of word categorization in saccade-related brain potentials and visual evoked potentials.

Visual evoked potentials (VEPs) elicited by foveal presentation of words were compared to brain potentials evoked by the same words in a condition where subjects had to make a saccadic eye movement in order to perceive the words (saccade-related brain potentials, SRPs). Subjects had to categorize the words responding with a button press to stimuli belonging to the target (infrequent, P = 0.2) category. The VEP and SRP waveforms showed divergences in the early (up to 250 ms) components, but a marked similarity between the late components. Principal Component Analysis also revealed the same relationship between the two types of brain responses. Peak latency of the late SRP components measured from saccade offset showed an apparent processing advantage over the corresponding late components of VEPs. The N3 component, indexing semantic processing of visual patterns, peaked between 310 and 375 ms in the SRPs, while in the VEPs it appeared between 410 and 470 ms. The P4 component, associated with final stimulus evaluation, showed a similar latency benefit in favour of SRPs (420-500 ms vs 530-590 ms in VEPs). The mean reaction time was 74 ms shorter in the eye movement condition (measured from saccade offset) than in the VEP condition (703 vs 777 ms). The question of what kind of processes may contribute to the differences in mean RTs and to the latencies of the late components between the two conditions are discussed. We suggest that the late components (P3, N3 and P4) of the VEP and the SRP, respectively, index identical brain processes.

Responses to irrelevant probes during task-induced negative and positive shifts.

The functional significance of task-induced negative and positive cortical shifts were tested with the probe-stimulus method. Both shifts were induced within the same experimental situation in three variants of a CNV paradigm, where a slow positive wave (a variant of P300) appeared following S2. In Experiment I and II, S2 called for making or withholding a motor response (go/no-go); in Experiment III, S2 informed the subject about the correctness of a previous guess. Irrelevant probe-stimuli were applied in conjunction with the task during the CNV, the post-S2 positivity and the intertrial interval (ITI). The probe-evoked vertex EPs were smaller during the post-S2 positivity as compared to the CNV and the ITI. This was true not only for the motor task but also for the guessing task, where the effect is unlikely to have been contaminated by motor potentials. This indicates that positive shifts have an inhibitory effect on the processing of irrelevant probe-stimuli and possibly on information-processing in general.

Visual information processing with brain potentials that accompany saccadic eye movements.

The relation between stimulus probability and the late components of the saccade-related brain (lambda) potentials (SRPs) was studied in three kinds of cognitive tasks: guessing, counting and word categorization. These tasks, traditionally used to study event-related potentials, were modified in such a way that subjects had to perform a saccadic eye movement in order to perceive the target stimulus. In guessing and counting tasks three kinds of target stimulus appeared with the probabilities of 17, 33 and 50 percent, respectively. In the semantic word categorization task the stimuli belonged to one of two categories: frequent (80 percent, female names) and infrequent (20 percent, male names). In all three tasks the late positive components (P300 and P4) had greater amplitudes of SRPs elicited by the infrequent stimuli than by the frequent ones. A Principal Component-Varimax Analysis of the SRP data revealed factors corresponding to (1) a positive Slow Wave, (2) the positive P300 and (3) the P4 components, respectively. In addition, the semantic categorization task was correlated with a late (negative) SRP component.

[Splints in birth-related brachial plexus injuries]. / Zur Schienenbehandlung bei geburtstraumatischen Läsionen des Plexus brachialis.

Most cases of obstetrical brachial plexus palsies are mild traction injuries which resolve under physical therapy within several weeks or months. Severe ruptures or avulsion injuries of the plexus can lead to lifelong impairment of the upper extremities. Hence, in severe brachial plexus injuries the indications for brachial plexus reconstruction should be evaluated, early. At the age of about 3 months, the infant should be presented in a centre specialised in obstetrical brachial plexus palsies. In almost all cases intensive physical therapy is performed. In addition, many patients require splinting in order to gain function as part of the conservative therapy or for postoperative fixation. Depending on the type of splint, different demands are made on design, material and strategy of adjustment. Many different natural and synthetic materials are available for orthopaedic constructions. Because of its good adjustment options, the use of low temperature thermoplastic is steadily increasing. This contribution presents an overview of our currently used splints, new technical developments in our experience with more than 200 patients with obstetrical brachial plexus palsy. We present our experience with the most common splints for the use in fixation after birth-related brachial plexus surgery, subscapularis release, trapezius muscle transfer and functional improvement of hands with a lack of wrist extension.