Aberrant expression and biological significance of Sox2, an embryonic stem cell transcriptional factor, in ALK-positive anaplastic large cell lymphoma.

Sox2 (sex-determining region Y-Box) is one of the master transcriptional factors that are important in maintaining the pluripotency of embryonic stem cells (ESCs). In line with this function, Sox2 expression is largely restricted to ESCs and somatic stem cells. We report that Sox2 is expressed in cell lines and tumor samples derived from ALK-positive anaplastic large cell lymphoma (ALK(+)ALCL), for which the normal cellular counterpart is believed to be mature T-cells. The expression of Sox2 in ALK(+)ALCL can be attributed to nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), the oncogenic fusion protein carrying a central pathogenetic role in these tumors. By confocal microscopy, Sox2 protein was detectable in virtually all cells in ALK(+)ALCL cell lines. However, the transcriptional activity of Sox2, as assessed using a Sox2-responsive reporter construct, was detectable only in a small proportion of cells. Importantly, downregulation of Sox2 using short interfering RNA in isolated Sox2(active) cells, but not Sox2(inactive) cells, resulted in a significant decrease in cell growth, invasiveness and tumorigenicity. To conclude, ALK(+)ALCL represents the first example of a hematologic malignancy that aberrantly expresses Sox2, which represents a novel mechanism by which NPM-ALK mediates tumorigenesis. We also found that the transcriptional activity and oncogenic effects of Sox2 can be heterogeneous in cancer cells.

A study of the reproducibility and etiology of diffusion anisotropy differences in developmental stuttering: a potential role for impaired myelination.

Several diffusion tensor imaging (DTI) studies have reported fractional anisotropy (FA) reductions within the left perisylvian white matter (WM) of persistent developmental stutterers (PSs). However, these studies have not reached the same conclusions in regard to the presence, spatial distribution (focal/diffuse), and directionality (elevated/reduced) of FA differences outside of the left perisylvian region. In addition, supplemental DTI measures (axial and radial diffusivities, diffusion trace) have yet to be utilized to examine the potential etiology of these FA reductions. Therefore, the present study sought to reexamine earlier findings through a sex- and age-controlled replication analysis and then to extend these findings with the aforementioned non-FA measures. The replication analysis showed that robust FA reductions in PSs were largely focal, left hemispheric, and within late-myelinating associative and commissural fibers (division III of the left superior longitudinal fasciculus, callosal body, forceps minor of the corpus callosum). Additional DTI measures revealed that these FA reductions were attributable to an increase in diffusion perpendicular to the affected fiber tracts (elevated radial diffusivity). These findings suggest a hypothesis that will be testable in future studies: that myelogenesis may be abnormal in PSs within left-hemispheric fiber tracts that begin a prolonged course of myelination in the first postnatal year.

The Shb signalling scaffold binds to and regulates constitutive signals from the Epstein-Barr virus LMP2A membrane protein.

The Epstein-Barr virus latency-associated membrane protein LMP2A has been shown to activate the survival kinase Akt in epithelial and B cells in a phosphoinositide 3-kinase-dependent fashion. In this study, we demonstrate that the signalling scaffold Shb associates through SH2 and PTB domain interactions with phosphorylated tyrosine motifs in the LMP2A N-terminal tail. Additionally, we show that mutation of tyrosines in these motifs as well as shRNA-mediated downregulation of Shb leads to a loss of constitutive Akt-activation in LMP2A-expressing cells. Furthermore, utilization by Shb of the LMP2A ITAM motif regulates stability of the Syk tyrosine kinase in LMP2A-expressing cells. Our data set the precedent for viral utilization of the Shb signalling scaffold and implicate Shb as a regulator of LMP2A-dependent Akt activation.

Hypophonia in Parkinson's disease: neural correlates of voice treatment revealed by PET.

OBJECTIVE: To investigate the neural correlates of hypophonia in individuals with idiopathic PD (IPD) before and after voice treatment with the Lee Silverman Voice Treatment method (VT) using (15)O-H(2)O PET. METHODS: Regional cerebral blood flow (rCBF) changes associated with overt speech-motor tasks relative to the resting state were measured in the IPD subjects before and after VT, and in a group of healthy control volunteers. RESULTS: Behavioral measures of voice loudness significantly improved following treatment. Before VT, patients had strong speech-related activations in motor and premotor cortex (M1-mouth, supplementary motor cortex [SMA], and inferior lateral premotor cortex [ILPm]), which were significantly reduced post-VT. Similar to the post-treatment session, premotor activations were absent (SMA) or below statistical threshold (M1-mouth) in the healthy control group. In addition, following VT treatment, significant right-sided activations were present in anterior insular cortex, caudate head, putamen, and dorsolateral prefrontal cortex (DLPFC). Finally, the VT-induced neural changes were not present with transient experimenter-cued increases of loudness in VT-untreated patients. CONCLUSIONS: Effective improvement of IPD hypophonia following voice treatment with VT was accompanied by a reduction of cortical motor-premotor activations, resembling the functional pattern observed in healthy volunteers and suggesting normalization, and additional recruitment of right anterior insula, caudate head, putamen, and DLPFC. This treatment-dependent functional reorganization suggests a shift from an abnormally effortful (premotor cortex) to a more automatic (basal ganglia, anterior insula) implementation of speech-motor actions.

Brain imaging studies of developmental stuttering.

UNLABELLED: This paper reviews recent brain imaging research on stuttering against a background of studies that the writer and colleagues have been conducting at the University of Texas Health Science Center in San Antonio. The paper begins by reviewing some pertinent background to recent neuroimaging investigations of developmental stuttering. It then outlines the findings from four brain imaging studies that the San Antonio group has conducted using H2(15)O positron emission tomography (PET). Finally, some of the principal findings that are emerging across brain imaging studies of stuttering are reviewed, while also highlighting--and attempting to resolve--some apparent across-study inconsistencies among the findings. Research on stuttering using magnetoencephalogaphy (MEG) and transcranial magnetic stimulation (TMS) is also considered. The findings increasingly point to a failure of normal temporal lobe activation during speech that may either contribute to (or is the result of) a breakdown in the sequencing of processing among premotor regions implicated in phonologic planning. LEARNING OUTCOMES: As a result of this activity, the participant will become familiar with some recent neurophysiological correlates of stuttering and what they suggest about the nature of this disorder.

The modification of speech naturalness during rhythmic stimulation treatment of stuttering.

This study investigated the modification of speech naturalness during stuttering treatment. It systematically replicated an earlier study (Ingham & Onslow, 1985) that demonstrated that unnatural-sounding stutter-free speech could be shaped into more natural-sounding stutter-free speech by using regular feedback of speech-naturalness ratings during speaking tasks. In the present study, the some procedure was used with three persons who stutter-2 adolescent girls and 1 adult man-during rhythmic stimulation conditions. The two adolescent participants spoke only English, but Spanish was the first and English the second language (ESL) of the adult participant. For the 2 adolescents, it was demonstrated that their unnatural-sounding rhythmic speech could be shaped to levels found among normally fluent speakers without losing the fluency-inducing benefits of rhythmic speech. The findings indicate that speech-naturalness feedback may be a powerful procedure for overcoming a problematic aspect of rhythmic speech treatments of stuttering. However, it was not possible to deliver reliable speech-naturalness feedback to the adult ESL speaker, who also displayed a strong dialect. The study highlights the need to find strategies to improve interjudge agreement when using speech naturalness ratings with speakers who display a strong dialect.

The Gab1 docking protein links the b cell antigen receptor to the phosphatidylinositol 3-kinase/Akt signaling pathway and to the SHP2 tyrosine phosphatase.

B cell antigen receptor (BCR) signaling causes tyrosine phosphorylation of the Gab1 docking protein. This allows phosphatidylinositol 3-kinase (PI3K) and the SHP2 tyrosine phosphatase to bind to Gab1. In this report, we tested the hypothesis that Gab1 acts as an amplifier of PI3K- and SHP2-dependent signaling in B lymphocytes. By overexpressing Gab1 in the WEHI-231 B cell line, we found that Gab1 can potentiate BCR-induced phosphorylation of Akt, a PI3K-dependent response. Gab1 expression also increased BCR-induced tyrosine phosphorylation of SHP2 as well as the binding of Grb2 to SHP2. We show that the pleckstrin homology (PH) domain of Gab1 is required for BCR-induced phosphorylation of Gab1 and for Gab1 participation in BCR signaling. Moreover, using confocal microscopy, we show that BCR ligation can induce the translocation of Gab1 from the cytosol to the plasma membrane and that this requires the Gab1 PH domain as well as PI3K activity. These findings are consistent with a model in which the binding of the Gab1 PH domain to PI3K-derived lipids brings Gab1 to the plasma membrane, where it can be tyrosine-phosphorylated and then act as an amplifier of BCR signaling.

Evaluation of a stuttering treatment based on reduction of short phonation intervals.

This paper reports the results of an efficacy study of a stuttering treatment program known as Modifying Phonation Intervals (MPI), which trains stuttering speakers to reduce the frequency of relatively short phonation intervals (PIs) during connected speech across speaking tasks and situations. Five young adult male stuttering speakers were treated in this computer-based program that systematically trains speakers to reduce selected short PIs found to functionally control stuttering. The treatment process was evaluated using multiple-baseline designs. Treatment was largely self-managed and based on a performance-contingent schedule of within-clinic speaking tasks (Establishment), beyond-clinic speaking tasks (Transfer), and systematic decreases in assessment occasions (Maintenance). Assessments were made at regular intervals before, during, and after treatment. All speakers achieved stutter-free and natural-sounding speech during within- and beyond-clinic speaking tasks at the completion of Maintenance. All were tested 12 months after completion of Maintenance, and all maintained the results. The findings from this study suggest that this procedure may make a significant contribution to stuttering treatment practice.

Is overt stuttered speech a prerequisite for the neural activations associated with chronic developmental stuttering?

Four adult right-handed chronic stutterers and four age-matched controls completed H(2)(15)O PET scans involving overt and imagined oral reading tasks. During overt stuttered speech prominent activations occurred in SMA (medial), BA 46 (right), anterior insula (bilateral), and cerebellum (bilateral) plus deactivations in right A2 (BA 21/22). These activations and deactivations also occurred when the same stutterers imagined they were stuttering. Some parietal regions were significantly activated during imagined stuttering, but not during overt stuttering. Most regional activations changed in the same direction when overt stuttering ceased (during chorus reading) and when subjects imagined that they were not stuttering (also during chorus reading). Controls displayed fewer similarities between regional activations and deactivations during actual and imagined oral reading. Thus overt stuttering appears not to be a prerequisite for the prominent regional activations and deactivations associated with stuttering.

Targets of B-cell antigen receptor signaling: the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase-3 signaling pathway and the Rap1 GTPase.

In this review, we discuss the role of phosphatidylinositol 3-kinase (PI3K) and Rap 1 in B-cell receptor (BCR) signaling. PI3K produces lipids that recruit pleckstrin homology domain-containing proteins to the plasma membrane. Akt is a kinase that the BCR activates in this manner. Akt phosphorylates several transcription factors as well as proteins that regulate apoptosis and protein synthesis. Akt also regulates glycogen synthase kinase-3, a kinase whose substrates include the nuclear factor of activated T cells (NF-AT)cl and beta-catenin transcriptional activators. In addition to Akt, PI3K-derived lipids also regulate the activity and localization of other targets of BCR signaling. Thus, a key event in BCR signaling is the recruitment of PI3K to the plasma membrane where its substrates are located. This is mediated by binding of the Src homology (SH) 2 domains in PI3K to phosphotyrosine-containing sequences on membrane-associated docking proteins. The docking proteins that the BCR uses to recruit PI3K include CD19, Cbl, Gab1, and perhaps Gab2. We have shown that Gab1 colocalizes PI3K with SH2 domain-containing inositol phosphatase (SHIP) and SHP2, two enzymes that regulate PI3K-dependent signaling. In contrast to PI3K, little is known about the Rap1 GTPase. We showed that the BCR activates Rap1 via phospholipase C-dependent production of diacylglycerol. Since Rap1 is thought to regulate cell adhesion and cell polarity, it may be involved in B-cell migration.

Brain correlates of stuttering and syllable production. A PET performance-correlation analysis.

To distinguish the neural systems of normal speech from those of stuttering, PET images of brain blood flow were probed (correlated voxel-wise) with per-trial speech-behaviour scores obtained during PET imaging. Two cohorts were studied: 10 right-handed men who stuttered and 10 right-handed, age- and sex-matched non-stuttering controls. Ninety PET blood flow images were obtained in each cohort (nine per subject as three trials of each of three conditions) from which r-value statistical parametric images (SPI¿r¿) were computed. Brain correlates of stutter rate and syllable rate showed striking differences in both laterality and sign (i.e. positive or negative correlations). Stutter-rate correlates, both positive and negative, were strongly lateralized to the right cerebral and left cerebellar hemispheres. Syllable correlates in both cohorts were bilateral, with a bias towards the left cerebral and right cerebellar hemispheres, in keeping with the left-cerebral dominance for language and motor skills typical of right-handed subjects. For both stutters and syllables, the brain regions that were correlated positively were those of speech production: the mouth representation in the primary motor cortex; the supplementary motor area; the inferior lateral premotor cortex (Broca's area); the anterior insula; and the cerebellum. The principal difference between syllable-rate and stutter-rate positive correlates was hemispheric laterality. A notable exception to this rule was that cerebellar positive correlates for syllable rate were far more extensive in the stuttering cohort than in the control cohort, which suggests a specific role for the cerebellum in enabling fluent utterances in persons who stutter. Stutters were negatively correlated with right-cerebral regions (superior and middle temporal gyrus) associated with auditory perception and processing, regions which were positively correlated with syllables in both the stuttering and control cohorts. These findings support long-held theories that the brain correlates of stuttering are the speech-motor regions of the non-dominant (right) cerebral hemisphere, and extend this theory to include the non-dominant (left) cerebellar hemisphere. The present findings also indicate a specific role of the cerebellum in the fluent utterances of persons who stutter. Support is also offered for theories that implicate auditory processing problems in stuttering.

Tyrosine phosphorylation of shc in response to B cell antigen receptor engagement depends on the SHIP inositol phosphatase.

Tyrosine phosphorylation of Shc in response to B cell Ag receptor (BCR) engagement creates binding sites for the Src homology 2 (SH2) domain of Grb2. This facilitates the recruitment of both Grb2. Sos complexes and Grb2. SHIP complexes to the plasma membrane where Sos can activate Ras and SH2 domain-containing inositol phosphatase (SHIP) can dephosphorylate phosphatidylinositol 3,4,5-trisphosphate. Given the importance of Shc phosphorylation, we investigated the mechanism by which the BCR stimulates this response. We found that both the SH2 domain and phosphotyrosine-binding (PTB) domain of Shc are important for BCR-induced tyrosine phosphorylation of Shc and the subsequent binding of Grb2 to Shc. The unexpected finding that the PTB domain of Shc is required for Shc phosphorylation was investigated further. Because the major ligand for the Shc PTB domain is SHIP, we asked whether the interaction of Shc with SHIP was required for BCR-induced tyrosine phosphorylation of Shc. Using SHIP-deficient DT40 cells, we show that SHIP is necessary for the BCR to induce significant levels of Shc tyrosine phosphorylation. BCR-induced tyrosine phosphorylation of Shc could be restored in the these cells by expressing wild-type SHIP but not by expressing a mutant form of SHIP that cannot bind to Shc. This suggests that BCR-induced tyrosine phosphorylation of Shc may depend on the binding of SHIP to the Shc PTB domain. Thus, we have described a novel role for SHIP in BCR signaling, promoting the tyrosine phosphorylation of Shc.

Effects of time-interval judgement training on real-time measurement of stuttering.

The purpose of this study was to investigate whether a previously developed interval-based training program could improve judges' stuttering event judgments. Two groups of judges made real-time stuttering event judgments (computer-mouse button presses) in 3 to 6 trials before the response-contingent judgment training program and in another 3 to 6 trials after training, for recordings of 9 adults who stuttered. Their judgments were analyzed in terms of number of stuttering events, duration of stuttering, and 5-s intervals of speech that could be categorized as judged (or not judged) to contain stuttering. Results showed (a) changes in the amount of stuttering identified by the judges; (b) improved correspondence between the judges' identifications of stuttering events and interval-based standards previously developed from judgments made by experienced, authoritative judges; (c) improved correspondence between interval-based analyses of the judges' stuttering judgments and the previously developed standards; (d) improved intrajudge agreement; (e) improved interjudge agreement; and (f) convergence between the 2 judge groups, for samples and speakers used during training tasks and also for other speakers. Some implications of these findings for developing standardized procedures for the real-time measurement of stuttering are discussed.

The Gab1 protein is a docking site for multiple proteins involved in signaling by the B cell antigen receptor.

Gab1 is a member of the docking/scaffolding protein family which includes IRS-1, IRS-2, c-Cbl, p130(cas), and p62(dok). These proteins contain a variety of protein-protein interaction motifs including multiple tyrosine residues that when phosphorylated can act as binding sites for Src homology 2 (SH2) domain-containing signaling proteins. We show in the RAMOS human B cell line that Gab1 is tyrosine-phosphorylated in response to B cell antigen receptor (BCR) engagement. Moreover, tyrosine phosphorylation of Gab1 correlated with the binding of several SH2-containing signaling proteins to Gab1 including Shc, Grb2, phosphatidylinositol 3-kinase, and the SHP-2 tyrosine phosphatase. Far Western analysis showed that the SH2 domains of Shc, SHP-2, and the p85 subunit of phosphatidylinositol 3-kinase could bind directly to tyrosine-phosphorylated Gab1 isolated from activated RAMOS cells. In contrast, the Grb2 SH2 domain did not bind directly to Gab1 but instead to the Shc and SHP-2 associated with Gab1. We also show that Gab1 is present in the membrane-enriched particulate fraction of RAMOS cells and that Gab1/signaling protein complexes are found in this fraction after BCR engagement. Thus, tyrosine-phosphorylated Gab1 may recruit cytosolic signaling proteins to cellular membranes where they can act on membrane-bound targets. This may be a critical step in the activation of multiple BCR signaling pathways.

Activation of the Rap1 GTPase by the B cell antigen receptor.

The B cell antigen receptor (BCR) activates Ras, a GTPase that promotes cell proliferation by activating the Raf-1/MEK/ERK signaling module and other signaling enzymes. In its active GTP-bound form, the Rap1 GTPase may act as a negative regulator of Ras-mediated signaling by sequestering Ras effectors (e.g., Raf-1) and preventing their activation. In this report, we show that BCR engagement activates Rap1 and that this is dependent on production of diacylglycerol (DAG) by phospholipase C-gamma. Activation of Rap1 by the BCR was greatly reduced in phospholipase C-gamma-deficient B cells, whereas both a synthetic DAG and phorbol dibutyrate could activate Rap1 in B cells. We had previously shown that C3G, an activator of Rap1, associates with the Crk adaptor proteins in B cells and that BCR engagement causes Crk to bind to the Cas and Cbl docking proteins. However, the DAG-dependent pathway by which the BCR activates Rap1 apparently does not involve Crk signaling complexes since phorbol dibutyrate could activate Rap1 without inducing the formation of these complexes. Thus, the BCR activates Rap1 via a novel DAG-dependent pathway.

Children recovered from stuttering without formal treatment: perceptual assessment of speech normalcy.

Current evidence suggests that young children who recover from stuttering are essentially stutter-free. However, there is no evidence to indicate if their speech is perceptually indistinguishable from normally fluent peers or whether they retain perceptually unusual speech. One important example of recovery from stuttering is children who have recovered without receiving formal treatment. An investigation was conducted to determine if the speech of these children is perceptually different from the speech of children who have never stuttered. Speakers consisted of 10 preschool and early school-age children documented as recovered from stuttering without benefit of formal treatment. In a series of studies they were compared with 10 children who had never stuttered. Three groups of judges-sophisticated, unsophisticated, and experienced-were separately asked, using videotaped speech samples of the children, to decide which samples were from children who used to stutter. Results revealed that the children who recovered from stuttering were perceptually indistinguishable from the normal controls. The same result was obtained regardless of whether the samples were presented in paired-stimulus or single-stimulus mode. Two of the groups of judges were also instructed to rate the speech naturalness of the speech samples. The speakers were not distinguished on this measure either. Methodological issues and the implications of the findings are discussed.

Identifying the authoritative judgments of stuttering: comparisons of self-judgments and observer judgments.

Reliable and accurate stuttering measurement depends on the existence of unambiguous descriptions or exemplars of stuttered and nonstuttered speech. The development of clinically meaningful and useful exemplars, in turn, requires determining whether persons who stutter judge the same speech to be stuttered that other observers judge to be stuttered. The purpose of these experiments, therefore, was to compare stuttering judgements from several sources: 15 adults who stutter, judging their own spontaneous speech; the same adults who stutter, judging each other's speech; and a panel of 10 authorities on stuttering research and treatment. Judgments were mode under several conditions, including self-judgments made while the speaker was talking and self- and other-judgements made from recordings in continuous and interval formats. Results showed substantial differences in stuttering judgments across speakers, judges, and judgment conditions, but across-task comparisons were complicated by low self-agreement for many judges. Some intervals were judged consistently by all judges to be Stuttered or Nonstuttered, across multiple conditions, but many other intervals were either not assigned replicable judgments or were consistently judged to be Nonstuttered by the speaker who had produced them but were not assigned consistent judgments by other judges. The implications of these findings for stuttering measurement are considered.

Effect of speech dialect on speech naturalness ratings: a systematic replication of Martin, Haroldson, and Triden (1984).

This study investigated the effect of speech dialect on listeners' speech naturalness ratings by systematically replicating Martin, Haroldson, and Triden's (1984) study using three groups of speaker samples. Two groups consisted of speakers with General American dialect--one with persons who stutter and the other with persons who do not stutter. The third group also consisted of speakers who do not stutter but who spoke non-General American dialect. The results showed that speech naturalness ratings distinguished among the three speaker groups. The variables that appeared to influence speech naturalness ratings were type of dialect, speech fluency, and speaking rate, though they differed across speaker groups. The findings also suggested that strength of speech dialect may be a scaleable dimension that judges can rate with acceptable levels of reliability. Dialect may also be an important factor that needs to be incorporated or controlled within systems designed to train speech naturalness ratings. It may also be an important factor in determining the extent to which stuttering treatment produces natural sounding speech.

Experimental investigation of the effects of frequency-altered auditory feedback on the speech of adults who stutter.

A series of single-subject experiments evaluated the effects of frequency-altered auditory feedback (FAF) on the speech performance of four adult males who stutter. Using alterations of plus or minus one octave, FAF was compared with normal auditory feedback (NAF) in oral reading and spontaneous speech with measurements made of stuttered intervals, stutter-free speech rate, and speech naturalness. The effects of extended FAF conditions on spontaneous speech were also evaluated for two subjects who demonstrated a positive response to FAF. Results showed no consistencies across subjects in responses to FAF: One subject showed no response, another produced an initial temporary response, a third showed a deterioration in speech quality with minimal reductions in stuttering, and a fourth displayed substantial and sustained improvements in speech performance. Some implications of these findings for current research and theory about the relationship between stuttering and FAF are discussed.