CD133/prominin-1 is a potential therapeutic target for antibody-drug conjugates in hepatocellular and gastric cancers.

CD133/prominin-1 is a pentaspan transmembrane glycoprotein overexpressed in various solid tumours including colorectal and glioblastomas. CD133 was found here to be highly expressed in >or=50% of pancreatic, gastric and intrahepatic cholangiocarcinomas. Quantitative flow cytometric analysis showed that a panel of established hepatocellular, pancreatic and gastric cancer cell lines expressed CD133 at levels higher than normal epithelial cells or bone marrow progenitor cells. A murine anti-human CD133 antibody (AC133) conjugated to a potent cytotoxic drug, monomethyl auristatin F (MMAF), effectively inhibited the growth of Hep3B hepatocellular and KATO III gastric cancer cells in vitro with IC(50) values of 2-7 ng ml(-1). MMAF induced apoptosis in the cancer cells as measured by caspase activation. The anti-CD133-drug conjugate (AC133-vcMMAF) was shown to internalise and colocalised with the lysosomal marker CD107a in the sensitive cell lines. In contrast, in the resistant cell line Su.86.86, the conjugate internalised and colocalised with the caveolae marker, Cav-1. Addition of ammonium chloride, an inhibitor of lysosomal trafficking and processing, suppressed the cytotoxic effect of AC133-vcMMAF in both Hep3B and KATO III. Anti-CD133-drug conjugate treatment resulted in significant delay of Hep3B tumour growth in SCID mice. Anti-CD133 antibody-drug conjugates warrant further evaluation as a therapeutic strategy to eradicate CD133+ tumours.

Eye activity correlates of workload during a visuospatial memory task.

Changes in six measures of eye activity were assessed as a function of task workload in a target identification memory task. Eleven participants completed four 2-hr blocks of a mock anti-air-warfare task, in which they were required to examine and remember target classifications (friend/enemy) for subsequent prosecution (fire upon/allow to pass), while targets moved steadily toward two centrally located ship icons. Target density served as the task workload variable; between one and nine targets were simultaneously present on the display. For each participant, moving estimates of blink frequency and duration, fixation frequency and dwell time, saccadic extent, and mean pupil diameter, integrated over periods of 10 to 20 s, demonstrated systematic changes as a function of target density. Nonlinear regression analyses found blink frequency, fixation frequency, and pupil diameter to be the most predictive variables relating eye activity to target density. Participant-specific artificial neural network models, developed through training on two or three sessions and subsequently tested on a different session from the same participant, correlated well with actual target density levels (mean R = 0.66). Results indicate that moving mean estimation and artificial neural network techniques enable information from multiple eye measures to be combined to produce reliable near-real-time indicators of workload in some visuospatial tasks. Potential applications include the monitoring of visual activity of system opetators for indications of visual workload and scanning efficiency.

Insight into the tumor suppressor function of CBP through the viral oncoprotein tax.

CREB binding protein (CBP) is a cellular coactivator protein that regulates essentially all known pathways of gene expression. The transcriptional coactivator properties of CBP are utilized by at least 25 different transcription factors representing nearly all known classes of DNA binding proteins. Once bound to their target genes, these transcription factors are believed to tether CBP to the promoter, leading to activated transcription. CBP functions to stimulate transcription through direct recruitment of the general transcription machinery as well as acetylation of both histone and transcription factor substrates. Recent observations indicate that a critical dosage of CBP is required for normal development and tumor suppression, and that perturbations in CBP concentrations may disrupt cellular homeostasis. Furthermore, there is accumulating evidence that CBP deregulation plays a direct role in hematopoietic malignancies. However, the molecular events linking CBP deregulation and malignant transformation are unclear. Further insight into the function of CBP, and its role as a tumor suppressor, can be gained through recent studies of the human T-cell leukemia virus, type I (HTLV-I) Tax oncoprotein. Tax is known to utilize CBP to stimulate transcription from the viral promoter. However, recent data suggest that as a consequence of the Tax-CBP interaction, many cellular transcription factor pathways may be deregulated. Tax disruption of CBP function may play a key role in transformation of the HTLV-I-infected cell. Thus, Tax derailment of CBP may lend important information about the tumor suppressor properties of CBP and serve as a model for the role of CBP in hematopoietic malignancies.

Combined eye activity measures accurately estimate changes in sustained visual task performance.

Five concurrent eye activity measures were used to model fatigue-related changes in performance during a visual compensatory tracking task. Nine participants demonstrated considerable variations in performance level during two 53-min testing sessions in which continuous video-based eye activity measures were obtained. Using a trackball, participants were required to maneuver a target disk (destabilized by pseudorandom wind forces) within the center of an annulus on a CRT display. Mean tracking performance as a function of time across 18 sessions demonstrated a monotonic increase in error from 0 to 11 min, and a performance plateau thereafter. Individual performance fluctuated widely around this trend - with an average root mean square (RMS) error of 2.3 disk radii. For each participant, moving estimates of blink duration and frequency, fixation dwell time and frequency, and mean pupil diameter were analyzed using non-linear regression and artificial neural network techniques. Individual models were derived using eye and performance data from one session and cross-validated on data from a second session run on a different day. A general regression model (based only on fixation dwell time and frequency) trained on data from both sessions from all participants produced a correlation of estimated to actual tracking performance of R=0.68 and an RMS error of 1.55 (S. D.=0.26) disk radii. Individual non-linear regression models containing a general linear model term produced the cross-session correlations of estimated to actual tracking performance of R=0.67. Individualized neural network models derived from the data of both experimental sessions produced the lowest RMS error (mean=1.23 disk radii, S.D.=0.13) and highest correlation (R=0.82) between eye activity-based estimates and actual tracking performance. Results suggest that information from multiple eye measures may be combined to produce accurate individualized real-time estimates of sub-minute scale performance changes during sustained tasks.

Binding of p53 to the KIX domain of CREB binding protein. A potential link to human T-cell leukemia virus, type I-associated leukemogenesis.

The pleiotropic cellular coactivator CREB binding protein (CBP) plays a critical role in supporting p53-dependent tumor suppressor functions. p53 has been shown to directly interact with a carboxyl-terminal region of CBP for recruitment of the coactivator to p53-responsive genes. In this report, we identify the KIX domain as a new p53 contact point on CBP. We show that both recombinant and endogenous forms of p53 specifically interact with KIX. We demonstrate that the activation domain of p53 participates in KIX binding and provide evidence showing that this interaction is critical for p53 transactivation function. The human T-cell leukemia virus, type-I-encoded oncoprotein Tax is a well established repressor of p53 transcription function. Like p53, Tax also binds to KIX. The finding that both transcription factors bind to a common region of CBP suggests that coactivator competition may account for the observed repression. We demonstrate reciprocal repression between Tax and p53 in transient transfection assays, supporting the idea of intracellular coactivator competition. We biochemically confirm coactivator competition by directly showing that both transcription factors bind to KIX in a mutually exclusive fashion. These data provide molecular evidence for the observed intracellular competition and suggest that Tax inhibits p53 function by abrogating a novel p53-KIX interaction. Thus, Tax competition for the p53-KIX complex may be a pivotal event in the human T-cell leukemia virus, type I transformation pathway.

Binding of the human T-cell leukemia virus Tax protein to the coactivator CBP interferes with CBP-mediated transcriptional control.

The HTLV-I oncoprotein Tax is required for high level viral transcription and is strongly linked to HTLV-I-associated malignant transformation. Tax stimulates HTLV-I transcription through high affinity binding to the KIX domain of CBP, a pleiotropic coactivator. Several cellular proteins, including c-jun, also bind to KIX and utilize CBP as a coactivator. To test whether Tax binding to KIX may disable cellular CBP function, we examined the potential interplay between Tax and c-jun for binding to KIX. We show that Tax represses the transcription function of c-jun in vivo and demonstrate that both transcription factors bind to an overlapping minimal region of KIX in vitro. c-jun binding to KIX is displaced by Tax, indicating that their binding is mutually exclusive and providing a molecular basis for the observed repression. The competition between Tax and cellular transcription factors for CBP represents a novel pathway for HTLV-I dependent deregulation of gene expression, and may have significant implications for cellular homeostasis and transformation in the HTLV-I infected T-cell.

Anchoring of CREB binding protein to the human T-cell leukemia virus type 1 promoter: a molecular mechanism of Tax transactivation.

The human T-cell leukemia virus type 1 (HTLV-1)-encoded Tax protein activates viral transcription through interaction with the cellular transcription factor CREB (cyclic AMP response element [CRE] binding protein). Although Tax stabilizes the binding of CREB to the Tax-responsive viral CREs in the HTLV-1 promoter, the precise molecular mechanism by which Tax mediates strong transcriptional activation through CREB remains unclear. In this report, we show that Tax promotes high-affinity binding of the KIX domain of CREB binding protein (CBP) to CREB-viral CRE complexes, increasing the stability of KIX in these nucleoprotein complexes by up to 4.4 kcal/mol. Comparable KIX binding affinities were measured for both phosphorylated and unphosphorylated forms of CREB, and in all cases high-affinity binding was dependent upon both Tax and the viral CRE. Tax also promoted association of KIX to a truncated form of CREB containing only the 73-amino-acid basic leucine zipper (bZIP) domain, indicating that the entire amino-terminal CBP-interacting domain of CREB is nonessential in the presence of Tax. Functional studies upheld the binding studies, as expression of the bZIP domain of CREB was sufficient to support Tax transactivation of HTLV-1 transcription in vivo. Finally, we show that transfection of a KIX expression plasmid, which lacks activation properties, inhibited Tax transactivation in vivo. This suggests that KIX occupies the CBP binding site on Tax, and therefore CBP is likely a cofactor in mediating Tax stimulation of HTLV-1 transcription. Together, these data support a model in which Tax anchors CBP to the HTLV-1 promoter, with strong transcriptional activation resulting from the CBP-associated activities of nucleosome remodeling and recruitment of the general transcription machinery.

Spatial frequency-dependent asymmetry of visual evoked potential amplitudes.

The extent to which pattern reversal evoked potential amplitudes are distributed symmetrically over the scalp was investigated as a function of stimulus spatial frequency. Nine right-handed male subjects viewed sinusoidal grating stimuli of 4.0 and 0.5 c/deg phase reversed every 900 msec. A visual half-field configuration enabled selective stimulation of the right- or left-hemisphere visual cortex. Evoked responses were recorded from the 2 cm above the inion (Oz) and at 7 and 13 cm lateral to Oz. Analyses of normalized evoked response amplitudes showed a significant asymmetry for the 4.0 c/deg stimulus; right-hemisphere amplitudes declined as a function of distance from the midline, while left-hemisphere amplitudes were greatest at the 7 cm recording site. No hemispheric differences were observed for the 0.5 c/deg stimulus; amplitudes for both hemispheres declined as a function of distance from the midline. The data are discussed in terms of hemispheric differences in morphology and functional asymmetries at early levels of sensory processing.

Redundant use of luminance and flashing with shape and color as highlighting codes in symbolic displays.

Three visual search experiments evaluated the benefits and distracting effects of using luminance and flashing to highlight subclasses of symbols coded by shape and color. Each of three general shape/color classes (circular/blue, diamond/red, square/yellow) was divided into three subclasses by presenting the upper half, lower half, or entire symbol. Increasing the luminance of a subclass by a factor of two did not result in a significant improvement in search performance. Flashing a subclass at a rate of 3 Hz resulted in a significantly shorter mean search time (48% improvement). Increasing the luminance of one subclass (by a factor of five) while simultaneously flashing another significantly improved search times by 31% and 43% respectively, compared with nonhighlighted search conditions. In each experiment, the search times for nonhighlighted target subclasses were not affected by the presence of brighter and flashing targets. The failure of the initial experiment to find a significant performance improvement caused by increasing symbol luminance suggested that a larger luminance increase was necessary for this code to be effective. The overall results suggest that using luminance and flashing to highlight subclasses of color- and shape-coded symbols can reduce search times for these subclasses without producing a distraction effect by way of a concomitant increase in the search times for unhighlighted symbols.

Moderate cold exposure shortens evoked potential latencies in humans.

Visual, auditory event-related potentials, and brainstem auditory-evoked responses were recorded in as many as six young male subjects in order to study the effects of moderate cold air exposure on central nervous system functioning. Evoked potentials were recorded during repeated 50-min exposures to air of 4 and 22 degrees C; these levels of exposure resulted in no change in rectal core temperature. Evoked potentials recorded during exposures to 4 degrees air displayed consistently shorter latencies compared to those recorded at 22 degrees, suggesting faster CNS processing of sensory stimuli in the cold. These results are consistent with recent investigations of cold-induced behavioral response changes which indicate that increased arousal may occur with moderate (nonhypothermic) cold exposure.

Adrenergic responses to cognitive activity in a cold environment.

Adrenergic responses during physical stress such as cold exposure have been reported to differ from those responses observed during cognitive activity. Both the separate and the combined effects of cold and cognitive activity on catecholamine activity were examined in six male subjects. Alterations in plasma epinephrine and norepinephrine showed different patterns as a function of exposure to a 4 degrees C cold environment, a cognitive performance assessment battery (PAB), and the two conditions combined. Plasma epinephrine was not altered by exposure to cold and only slightly increased by PAB performance when given at 23 degrees C. However, epinephrine was substantially elevated by exposure to combined cold and PAB. Heart rate changes paralleled observed changes in epinephrine. Norepinephrine release was predominantly increased by cold exposure and was not altered by PAB performance.

Selective attenuation in brightness for brief stimuli and at low intensities supports age-related transient channel losses.

A brightness estimation experiment was conducted on 10 old (ages 60 to 77) and 10 young (ages 22 to 27) volunteers. Participants were introduced to magnitude estimation by scaling the lengths of line stimuli, after which they dark adapted for 10 minutes. Stimuli for brightness estimation were presented binocularly via a free-viewing system and consisted of circular flashes of 2 degrees. Stimuli covered a 3 log unit range of luminance levels in 0.5 log unit steps, and 3 durations (10, 100, 1000 msec). Linear regression analysis yielded dual-branched functions with a low intensity segment which was significantly steeper in slope than the high intensity segment. The slope for the older group was significantly less steep than that of the younger observers only at the low intensity segment. Findings with respect to stimulus duration showed a significantly attenuated slope for the old as compared to the young group only at 10 msec. The results extend previous threshold results to suprathreshold levels, and are consistent with an hypothesis of a selective loss of transient channels with age.

Older observers have attenuated increment thresholds upon transient backgrounds.

Foveal increment thresholds were measured in young, middle-aged, and older observers. These thresholds, which involved the detection of a small test flash as a function of the intensity of a larger background adapting field (AF), were measured at the instant of onset of the AF (transient condition) and when the eye had been fully light adapted to the AF (steady-state condition). All stimuli were presented to the left eye in a free-viewing system through a 2 mm artificial pupil. For the steady-state condition for all age groups, the functions were similar, but for the transient condition, the slope for the older observers was significantly less steep than that for the younger observers. These findings are consistent with an hypothesis of a selective loss of transient (Y) channels in the aging visual system.