Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-modified lentiviral vector.

One of the major hurdles for the development of gene therapy for Fanconi anemia (FA) is the increased sensitivity of FA stem cells to free radical-induced DNA damage during ex vivo culture and manipulation. To minimize this damage, we have developed a brief transduction procedure for lentivirus vector-mediated transduction of hematopoietic progenitor cells from patients with Fanconi anemia complementation group A (FANCA). The lentiviral vector FancA-sW contains the phosphoglycerate kinase promoter, the FANCA cDNA, and a synthetic, safety-modified woodchuck post transcriptional regulatory element (sW). Bone marrow mononuclear cells or purified CD34(+) cells from patients with FANCA were transduced in an overnight culture on recombinant fibronectin peptide CH-296, in low (5%) oxygen, with the reducing agent, N-acetyl-L-cysteine (NAC), and a combination of growth factors, granulocyte colony-stimulating factor (G-CSF), Flt3 ligand, stem cell factor, and thrombopoietin. Transduced cells plated in methylcellulose in hypoxia with NAC showed increased colony formation compared with 21% oxygen without NAC (P<0.03), showed increased resistance to mitomycin C compared with green fluorescent protein (GFP) vector-transduced controls (P<0.007), and increased survival. Thus, combining short transduction and reducing oxidative stress may enhance the viability and engraftment of gene-corrected cells in patients with FANCA.

Classification of oral streptococci by two-dimensional gel electrophoresis with direct activity stain for glycosyltransferases.

Thirty eight strains of oral streptococci were divided into six types by two-dimensional gel electrophoresis (2-DE) followed by glycosyltransferase (GLT) activity stain: Type 1, Streptococcus mutans; Type 2, Streptococcus rattus; Type 3, Streptococcus sobrinus and Streptococcus downei; Type 4, Streptococcus cricetus; Type 5, Streptococcus salivarius; and Type 6, Streptococcus sanguis, Streptococcus oralis and Streptococcus gordonii. In Types 1, 2 and 5, two glucosyltransferases synthesizing water-insoluble (GTF-I) and water-soluble glucans (GTF-S) and a fructosyltransferase (FTF) were detected. In Types 3 and 4, GTF-I and two GTF-Ss were detected. Only one GTF-S was detected in Type 6. The 2-DE patterns for these six types were clearly distinguished from each other based on the kind, number and location of GLTs in gel.

Verbal and design fluency in patients with frontal lobe lesions.

The ability to generate items belonging to categories in verbal fluency tasks has been attributed to frontal cortex. Nonverbal fluency (e.g., design fluency) has been assessed separately and found to rely on the right hemisphere or right frontal cortex. The current study assessed both verbal and nonverbal fluency in a single group of patients with focal, frontal lobe lesions and age- and education-matched control participants. In the verbal fluency task, participants generated items belonging to both letter cues (F, A, and S) and category cues (animals and boys' names). In the design fluency task, participants generated novel designs by connecting dot arrays with 4 straight lines. A switching condition was included in both verbal and design fluency tasks and required participants to switch back and forth between different sets (e.g., between naming fruits and furniture). As a group, patients with frontal lobe lesions were impaired, compared to control participants, on both verbal and design fluency tasks. Patients with left frontal lesions performed worse than patients with right frontal lesions on the verbal fluency task, but the 2 groups performed comparably on the design fluency task. Both patients and control participants were impacted similarly by the switching conditions. These results suggest that verbal fluency is more dependent on left frontal cortex, while nonverbal fluency tasks, such as design fluency, recruit both right and left frontal processes.

Dextran acceptor reaction of Streptococcus sobrinus glucosyltransferase GTF-I as revealed by using uniformly 13C-labeled sucrose.

A sucrose glucosyltransferase GTF-I from cariogenic Streptococcus sobrinus transferred the uniformly 13C-labeled glucosyl residue ([U-(13)C]Glc) from [U-(13)C]sucrose to exogenous dextran T500 at the non-reducing-end, mostly by alpha-(1-->6) linkages and partially by alpha-(1-->3) linkages, as revealed by the 13C-(13)C NMR coupling pattern. With increasing amounts of [U-(13)C]sucrose, transfer of [U-(13)C]Glc to the alpha-(1-->3)-linked chain became predominant without increase in the number of chains. The transfer of [U-(13)C]Glc to an isomaltopentaose acceptor occurred similarly to its transfer to T500. alpha-(1-->3)-branches in the [U-(13)C]dextran, specifically synthesized from [U-(13)C]sucrose by a Streptococcus bovis dextransucrase, were not formed by GTF-I, as judged by the observation that a newly-formed alpha-1,3,6-branched [U-(13)C]Glc was not detected, which could have been formed by transferring the unlabeled Glc from sucrose to the internal alpha-(1-->6)-linked [U-(13)C]Glc at C-3. The 13C-(13)C one-bond coupling constants (1J) were also recorded for the C-1--C-6 bond of the internal alpha-(1-->6)-linked [U-(13)C]Glc and of the non-reducing-end [U-(13)C]Glc.

The Ponzo illusion and the perception of orientation.

A new theory, called the tilt constancy theory, claims that the Ponzo illusion is caused by the misperception of orientation induced by local visual cues. The theory relates the Ponzo illusion-along with the Zöllner, Poggendorff, Wündt-Hering, and cafe wall illusions-to the mechanisms that enable us to perceive stable orientations despite changes in retinal orientation or body orientation. In Experiment 1, the magnitude of the misperception of orientation was compared with the magnitude of the Ponzo illusion. In Experiment 2, predictions of the tilt constancy theory were compared with accounts based on (1) low spatial frequencies in the image, (2) memory comparisons (pool-and-store model), and (3) relative sizejudgments. In Experiment 3, predictions of the tilt constancy theory were tested against predictions of the assimilation theory of Pressey and his colleagues. In the final experiment, the orientation account was compared with theories based on linear perspective and inappropriate size constancy. The results support the tilt constancy theory.

Disruption of 3-phosphoinositide-dependent kinase 1 (PDK1) signaling by the anti-tumorigenic and anti-proliferative agent n-alpha-tosyl-l-phenylalanyl chloromethyl ketone.

The anti-tumorigenic and anti-proliferative effects of N-alpha-tosyl-l-phenylalanyl chloromethyl ketone (TPCK) have been known for more than three decades. Yet little is known about the discrete cellular targets of TPCK controlling these effects. Previous work from our laboratory showed TPCK, like the immunosuppressant rapamycin, to be a potent inhibitor of the 70-kilodalton ribosomal S6 kinase 1 (S6K1), which mediates events involved in cell growth and proliferation. We show here that rapamycin and TPCK display distinct inhibitory mechanisms on S6K1 as a rapamycin-resistant form of S6K1 was TPCK-sensitive. Additionally, we show that TPCK inhibited the activation of the related kinase and proto-oncogene Akt. Upstream regulators of S6K1 and Akt include phosphoinositide 3-kinase (PI 3-K) and 3-phosphoinositide-dependent kinase 1 (PDK1). Whereas TPCK had no effect on either mitogen-regulated PI 3-K activity or total cellular PDK1 activity, TPCK prevented phosphorylation of the PDK1 regulatory sites in S6K1 and Akt. Furthermore, whereas both PDK1 and the mitogen-activated protein kinase (MAPK) are required for full activation of the 90-kilodalton ribosomal S6 kinase (RSK), TPCK inhibited RSK activation without inhibiting MAPK activation. Consistent with the capacity of RSK and Akt to mediate a cell survival signal, in part through phosphorylation of the pro-apoptotic protein BAD, TPCK reduced BAD phosphorylation and led to cell death in interleukin-3-dependent 32D cells. Finally, in agreement with results seen in embryonic stem cells lacking PDK1, protein kinase A activation was not inhibited by TPCK showing TPCK specificity for mitogen-regulated PDK1 signaling. TPCK inhibition of PDK1 signaling thus disables central kinase cascades governing diverse cellular processes including proliferation and survival and provides an explanation for its striking biological effects.

Source memory enhancement for emotional words.

The influence of emotional stimuli on source memory was investigated by using emotionally valenced words. The words were colored blue or yellow (Experiment 1) or surrounded by a blue or yellow frame (Experiment 2). Participants were asked to associate the words with the colors. In both experiments, emotionally valenced words elicited enhanced free recall compared with nonvalenced words; however, recognition memory was not affected. Source memory for the associated color was also enhanced for emotional words, suggesting that even memory for contextual information is benefited by emotional stimuli. This effect was not due to the ease of semantic clustering of emotional words because semantically related words were not associated with enhanced source memory, despite enhanced recall (Experiment 3). It is suggested that enhancement resulted from facilitated arousal or attention, which may act to increase organization processes important for source memory.

Differential effects of cue dependency on item and source memory.

The authors investigated the cue dependency of source and item memory. Individuals listened to words spoken by a man or a woman and later determined whether a test word was previously presented by a man or by a woman, or whether it was a new word. Cue dependent effects were assessed by presenting test words with (a) the same voice (match condition) that originally presented the word, (b) a different but familiar voice (mismatch condition), (c) a novel test voice (novel condition), and (d) no test voice (control condition). Compared with the control condition, source recollection was facilitated in matching-context conditions, disrupted in mismatching-context conditions, and not affected in novel test conditions. By contrast, item recognition was not affected by the match-mismatch manipulation but was significantly worse in novel test voice conditions. The authors propose an associative source interference view to account for the voice match-mismatch effects observed in source recollection.

Toward a cognitive neuroscience of metacognition.

The relationship between metacognition and executive control is explored. According to an analysis by Fernandez-Duque, Baird, and Posner (this issue), metacognitive regulation involves attention, conflict resolution, error correction, inhibitory control, and emotional regulation. These aspects of metacognition are presumed to be mediated by a neural circuit involving midfrontal brain regions. An evaluation of the proposal by Fernandez-Duque et al. is made, and it is suggested that there is considerable convergence of issues associated with metacognition, executive control, working memory, and frontal lobe function. By integrating these domains and issues, significant progress could be made toward a cognitive neuroscience of metacognition.

Nigerooligosaccharide acceptor reaction of Streptococcus sobrinus glucosyltransferase GTF-I.

Nigerose and nigerooligosaccharides served as acceptors for a glucosyltransferase GTF-I from cariogenic Streptococcus sobrinus to give a series of homologous acceptor products. The soluble oligosaccharides (dp 5-9) strongly activated the acceptor reaction, resulting in the accumulation of water-insoluble (1-->3)-alpha-D-glucan. The enzyme transferred the labeled glucosyl residue from D-[U-13C]sucrose to the 3-hydroxyl group at the non-reducing end of the (1-->3)-alpha-D-oligosaccharides, as unequivocally shown by NMR 13C-13C coupling patterns. The values of the 13C-13C one-bond coupling constant (1J) are also presented for the C-1-C-6 of the 13C-labeled alpha-(1-->3)-linked glucosyl residue and of the non-reducing-end residue.

Rsk1 mediates a MEK-MAP kinase cell survival signal.

BACKGROUND: Growth factors activate an array of cell survival signaling pathways. Mitogen-activated protein (MAP) kinases transduce signals emanating from their upstream activators MAP kinase kinases (MEKs). The MEK-MAP kinase signaling cassette is a key regulatory pathway promoting cell survival. The downstream effectors of the mammalian MEK-MAP kinase cell survival signal have not been previously described. RESULTS: We identify here a pro-survival role for the serine/threonine kinase Rsk1, a downstream target of the MEK-MAP kinase signaling pathway. In cells that are dependent on interleukin-3 (IL-3) for survival, pharmacological inhibition of MEKs antagonized the IL-3 survival signal. In the absence of IL-3, a kinase-dead Rsk1 mutant eliminated the survival effect afforded by activated MEK. Conversely, a novel constitutively active Rsk1 allele restored the MEK-MAP kinase survival signal. Experiments in vitro and in vivo demonstrated that Rsk1 directly phosphorylated the pro-apoptotic protein Bad at the serine residues that, when phosphorylated, abrogate Bad's pro-apoptotic function. Constitutively active Rsk1 caused constitutive Bad phosphorylation and protection from Bad-modulated cell death. Kinase-inactive Rsk1 mutants antagonize Bad phosphorylation. Bad mutations that prevented phosphorylation by Rsk1 also inhibited Rsk1-mediated cell survival. CONCLUSIONS: These data support a model in which Rsk1 transduces the mammalian MEK-MAP kinase signal in part by phosphorylating Bad.

An analysis of signal detection and threshold models of source memory.

The authors analyzed source memory performance with an unequal-variance signal detection theory model and compared the findings with extant threshold (multinomial and dual-process) models. In 3 experiments, receiver operating characteristic (ROC) analyses of source discrimination revealed curvilinear functions, supporting the relative superiority of a continuous signal detection model when compared with a threshold model. This result has implications for both multinomial and dual-process models, both of which assume linear ROCs in their description of source memory performance.

Retrieval-induced forgetting in episodic memory.

Participants learned the locations of 12 stimuli that were uniquely colored but could be grouped by shape (4 circles, 4 triangles, 4 crosses). Following the study, a retrieval-practice phase required participants to recall the colors of a subset of the stimuli (i.e., 2 circles, 2 triangles) using shape and location as cues. In a final test, participants recalled the colors of all 12 stimuli. Compared with the control set of stimuli (i.e., 4 crosses), memory was facilitated for practiced items but impaired for related items, which were not practiced but shared the same shape group. Across experiments, retrieval-induced forgetting was observed for different perceptual groupings and for different cuing procedures. The effect, however, required retrieval of information during the interpolated phase. Providing extra presentations did not disrupt memory for related items.

Ribosomal S6 kinase 1 (RSK1) activation requires signals dependent on and independent of the MAP kinase ERK.

BACKGROUND: The rsk1 gene encodes the 90 kDa ribosomal S6 kinase 1 (RSK1) protein, which contains two kinase domains. RSK1, which is involved in regulating cell survival and proliferation, lies at the end of the signaling cascade mediated by the extracellular signal-regulated kinase (ERK) subfamily of mitogen-activated protein (MAP) kinases. ERK activation and subsequent phosphorylation of the RSK1 carboxy-terminal catalytic loop stimulates phosphotransferase activity in the RSK1 amino-terminal kinase domain. When activated, RSK1 phosphorylates both nuclear and cytoplasmic substrates through this amino-terminal catalytic domain. It is thought that stimulation of the ERK/MAP kinase pathway is sufficient for RSK1 activation, but how ERK phosphorylation activates the RSK1 amino-terminal kinase domain is not known. RESULTS: The individual isolated RSK1 kinase domains were found to be under regulatory control. In vitro kinase assays established that ERK phosphorylates RSK1 within the carboxy-terminal kinase domain, and the phosphoinositide-dependent kinase 1 (PDK1) phosphorylates RSK1 within the amino-terminal kinase domain. In transiently transfected HEK 293E cells, PDK1 alone stimulated phosphotransferase activity of an isolated RSK1 amino-terminal kinase domain. Nevertheless, activation of full-length RSK1 in the absence of serum required activation by both PDK1 and ERK. CONCLUSIONS: RSK1 is phosphorylated by PDK1 in the amino-terminal kinase-activation loop, and by ERK in the carboxy-terminal kinase-activation loop. Activation of phosphotransferase activity of full-length RSK1 in vivo requires both PDK1 and ERK. RSK1 activation is therefore regulated by both the mitogen-stimulated ERK/MAP kinase pathway and a PDK1-dependent pathway.

Monitoring item and source information: evidence for a negative generation effect in source memory.

Item memory and source memory were assessed in a task that simulated a social conversation. Participants generated answers to questions or read statements presented by one of three sources (faces on a computer screen). Positive generation effects were observed for item memory. That is, participants remembered topics of conversation better if they were asked questions about the topics than if they simply read statements about topics. However, a negative generation effect occurred for source memory. That is, remembering the source of some information was disrupted if participants were required to answer questions pertaining to that information. These findings support the notion that item and source memory are mediated, as least in part, by different processes during encoding.

Distinct mathematical behavior of apoptotic versus non-apoptotic tumor cell death.

PURPOSE: The presence or absence of a p53-dependent apoptosis response has previously been shown to greatly influence radiosensitivity in tumor cells. Here, we examine clonogenic survival curves for two genetically related oncogene transformed cell lines differing in the presence or absence of p53 and apoptosis. Solid tumor radiosensitivity patterns have been previously described for these lines. MATERIALS AND METHODS: Oncogene-transformed fibroblasts derived from E1A + Ras transfection of p53-wild-type or p53-null mouse embryonic fibroblasts were plated as single cells and irradiated at increasing radiation doses in single fractions from 1.5 to 11 Gy. Clonogenic cell survival assays were obtained. Survival data are fit to a linear-quadratic relationship: S = e(-alphaD-betaD2). Apoptosis was assessed and quantitated morphologically by staining with the fluorescent nuclear dye DAPI, by TUNEL assay for DNA fragmentation, and by measurement of apoptotic cysteine protease cleavage activity in cytosolic extracts. RESULTS: Whereas radiation triggers massive apoptosis in the presence of p53, it produces no measurable DNA fragmentation, apoptotic cysteine protease cleavage activity, or morphological changes of apoptosis in the cells lacking p53. These contrasting mechanisms of death display dramatically different quantitative behavior: log-survival of apoptotic cells is linearly proportional to dose (S = e(-alphaD)), whereas survival of non-apoptotic (p53 null) is linear-quadratic with a significant quadratic contribution. The surviving fraction at 2 Gy (SF-2) for p53-null cells was 70% verses 12% for p53-intact cells. CONCLUSIONS: In this system, apoptosis appears to exhibit a dominance of single-event which produces a very high alpha/beta ratio, and no significant shoulder; whereas non-apoptotic death in this system exhibits a comparatively small linear component, a low alpha/beta ratio, and a larger shoulder.

Oncogene-dependent regulation of caspase activation by p53 protein in a cell-free system.

The mechanism by which p53 modulates apoptosis in cancer therapy is incompletely understood. Here, cell-free extracts from irradiated tumor cells are described in which endogenous p53 protein is shown to participate in caspase activation. This apoptotic activity is also oncogene-dependent, but independent of transcription in general or the presence of Bax or cytochrome c. A general use for this system is as a cell-free screen for apoptosis modulators. In this way, profound effects of protein kinase A were identified and corroborated in vivo by the protection conferred by cAMP against diverse triggers of p53-dependent apoptosis. This system provides direct biochemical evidence that p53 protein can transduce apoptotic signals through protein-protein interactions and reveals a modulator kinase pathway capable of regulating p53-dependent caspase activation.

On the recollection of specific- and partial-source information.

Memory judgments can be based on information that is more or less specific with respect to the source of an item. The authors introduce a procedure and multinomial model for measuring specific- and partial-source information. In 2 experiments, participants heard words spoken by 4 different voices: 2 male voices and 2 female voices. During the test, participants were required to remember who spoke the test items (e.g., Male 1, Male 2, Female 1, Female 2, or new word). Participants often remembered information about the gender of the source (i.e., partial-source information) when they did not remember information that identified the source itself (i.e., specific-source information). Dividing attention during retrieval impaired participants' memory for specific-source information (i.e., voice information) but did not affect memory for partial-source information (i.e., gender information).

Mapping symbols to response modalities: interference effects on Stroop-like tasks.

Response compatibility effects were assessed with a Stroop-like task which involved arrow and word stimuli. The subjects were required to respond to one stimulus--an arrow (e.g.,-->) or a word (e.g., left)--and ignore the other. It was shown that response compatibility played a significant role in generating Stroop-like interference. Robust interference effects were observed when the subjects responded manually to word stimuli (ignoring irrelevant arrows) and when they responded vocally to arrow stimuli (ignoring irrelevant words). Smaller interference effects were observed under response-compatible conditions, namely, responding manually to arrows and vocally to words. In the second experiment, within-dimension displays (e.g., arrow-arrow or word-word displays) yielded a pattern of interference that did not interact with response modality. These findings indicate that both stimulus-response compatibility effects and target-distractor similarity are crucial for understanding Stroop-like interference.