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Studies of childhood memory typically show that our earliest memories come from between three and four years of age. This finding is not universal, however. The age estimate varies across cultures and is affected by social influences. Research from the judgments and decision-making literature suggests that these estimates might also involve a judgment under uncertainty. Therefore, they might be susceptible to less social influences such as heuristics and biases. To investigate this possibility, we conducted two experiments that used anchoring paradigms to influence participants' estimates of their age during early autobiographical memories. In Experiment 1, participants answered either a high-anchor or a low-anchor question, and were warned that the anchor was uninformative; they went on to estimate their age during their earliest autobiographical memory. In Experiment 2, we replicated Experiment 1 and extended the design to examine additional early autobiographical memories. In both experiments, participants in the low-anchor condition gave earlier age estimates than those in the high-anchor condition. These results provide new insights into the methods used to investigate autobiographical memory. Moreover, they show that reports of early autobiographical memories can be influenced by a relatively light touch - a change to a single digit in a single question.

In creating a culture of service, can law firms engage transactional lawyers in pro bono work too? In this chapter, Daniel Greenberg argues that firms can, if their pro bono programs move beyond litigation and incorporate transactional projects that build on the skills and knowledge of what their transactional lawyers do. At Greenberg's firm, their pro bono program successfully recruits volunteer transactional lawyers to represent nonprofits just like they represent their corporate clients advising on real estate issues, drafting contract language, or addressing intellectual property concerns. –Eds. Any analysis of access to justice for Americans of average means must take note of the pro bono programs of major law firms. They are a huge potential source of advocacy for individual Americans of average means, and for the organizations that advocate for them. To maximize that potential, these firms will need to expand their services not by doing more of the same, but by rethinking the structure of their pro bono programs. This chapter argues that when a firm adopts a transactional pro bono model, thus transforming the scope of its services, not only will Americans of average means benefit, but the firm, its partners and associates will end up benefitting as well. Historically, as is still the case in most law firms today, pro bono work has been tied to the firm's litigation practice. In the 1950s, the NAACP Legal Defense Fund, Inc. used courts to jump-start the civil rights movement. In 1963, President John F. Kennedy enlisted fifty of the country's largest law firms to work for equal justice through litigation with the creation of The Lawyers Committee for Civil Rights under Law. Framing large issues in law reform terms, and using class action litigation as its tool, private lawyers attacked segregation, discriminatory education and election laws, and other civil rights violations in federal courts that were largely sympathetic to their cause. Simultaneously, new legal services arose with a focus on the rights of poor people. These programs collaborated with big law firms in their quest for housing rights, access to government benefits, and consumer protections. Later, advocates in the women's, anti-war, and environmental movements emulated this model.

Semantic dementia (SD) is a progressive neurological disorder characterized by temporal lobe atrophy and the gradual decline of semantic memory (Snowden, Goulding, & Neary, 1989). Individuals with SD also have impaired autobiographical event memory (AM), but the extent of this impairment has been controversial. Some investigations have found that older memories tend to be impoverished while those from more recent periods tend to be intact (Graham et al., 2003; Moss et al., 2003; Nestor et al., 2002; Piolino et al., 2003; Snowden, Griffiths, & Neary, 1996), which is the reverse of the Ribot gradient seen in Alzheimer’s disease (e. g. Piolino et al., 2003). Others have found that AM drops off precipitously for events beyond the last few years, suggesting that a step function better describes the impairment (Graham & Hodges, 1997; Hou, Miller, & Kramer, 2005). Under some testing conditions, the impairment is ungraded (Ivaniou et al., 2006) or entirely absent (McKinnon et al., 2006; Moss et al., 2003; Westmacott et al., 2001). Data on the temporal gradient in SD have been used to refine neuropsychological models of memory (Moscovitch et al., 2005), so this issue is of theoretical importance. Several factors could account for the differences across studies. First, some reports suggest that patients with mild SD have a graded impairment while those with moderate SD show a step function instead (Ivaniou et al., 2006, Matuszewski et al., 2009), meaning that the results of a particular investigation may depend on the severity of the disorder in the patient or patients tested. Moreover, in mild SD, additional retrieval support (i.e., detailed cues) can elevate performance to normal or near-normal levels (McKinnon et al., 2006; Moss et al., 2003), perhaps because retrieval support helps patients overcome strategic-retrieval deficits secondary to frontal pathology (Moss et al., 2003; Nestor et al., 2002). The choice of test modality can also influence the pattern of AM impairment. Most AM tests are essentially verbal; in the Galton-Crovitz test, for example, memories are cued with single keywords such as HORSE. SD patients have particular difficulty with verbal material, however, and it would be unsurprising to find that a patient cannot generate a detailed memory to the keyword HORSE if he or she no longer knows what horses are. Moreover, SD patients can relearn words (Snowden & Neary, 2002), and these words might be disproportionately linked to recent experiences (Moss et al., 2003). Therefore, the apparent temporal gradient found in cue-word studies might reflect the degradation and reacquisition of semantic knowledge rather than the integrity of AM. Two studies tried to circumvent these problems by using visual stimuli. Westmacott and colleagues (2001) cued AMs of an SD patient with family photographs. The patient seemed to perform well (there was no normal control group, but performance was at 85% or higher) and no temporal gradient was found. Graham and colleagues (2003), however, tested a patient using family photographs and found that he scored about 50% during the remote period. They did not test the recent period and therefore could not assess the temporal gradient. Although these studies suggested that visual cues might facilitate recall in SD, they did not completely resolve the question. The main limitation arises from fundamental differences between the visual and verbal cues that have been used to test AM in SD. The nouns used in Galton-Crovitz tasks are generally high-frequency and easily imageable, but not personally relevant. Family photographs, by contrast, are more personally relevant than a noun, and they contain additional cues (such as the people involved) that might help patients construct appropriate stories. Accordingly, existing studies suffer from a confound between cue modality and retrieval support. Other nonverbal cues have not been investigated. Odors, for example, can evoke autobiographical memories (Rubin, Groth, & Goldsmith, 1984), particularly memories that are older (Willander & Larson, 2006), more emotional (Herz & Cupchik, 1995; Rubin et al., 1984), and more detailed (Chu & Downes, 2002); additionally, odor-evoked memories have not been thought about or talked about as frequently (Rubin et al., 1984; Willander & Larsson, 2006). While odor identification is impaired in SD (Luzzi et al., 2007), an odor can trigger a memory even if a participant cannot identify it (Rubin et al., 1984). Thus, odors tend to elicit the very memories that SD patients find hardest to retrieve. We therefore tested a group of SD patients and controls with a Galton-Crovitz paradigm that used words, simple pictures, and odors as cues. We hypothesized that pictures and words would be equally effective, while odors would be superior; we also predicted a temporal gradient with superior recall for recent memories.

Tulving’s (1972) theory of memory draws a distinction between general knowledge (semantic memory) and memory for events (episodic memory). Neuropsychological studies have generally examined each type of memory in isolation, but theorists have long argued that these two forms of memory are interdependent. Here we review several lines of neuropsychological research that have explored the interdependence of episodic and semantic memory. The studies show that these forms of memory can affect each other both at encoding and at retrieval. We suggest that theories of memory should be revised to account for all of the interdependencies between episodic and semantic memory; they should also incorporate forms of memory that do not fit neatly into either category. (JINS, 2010, 16, 748–753.)

Memory tasks are often classified as semantic or episodic, but recent research shows that these types of memory are highly interactive. Category fluency, for example, is generally considered to reflect retrieval from semantic memory, but behavioral evidence suggests that episodic memory is also involved: participants frequently draw on autobiographical experiences while generating exemplars of certain categories. Neuroimaging studies accordingly have reported increased medial temporal lobe (MTL) activation during exemplar generation. Studies of fluency in MTL amnesics have yielded mixed results but were not designed to determine the precise contributions of episodic memory. We addressed this issue by asking MTL amnesics and controls to generate exemplars of three types of categories. One type tended to elicit autobiographical and spatial retrieval strategies (AS). Another type elicited strategies that were autobiographical but nonspatial (AN). The third type elicited neither autobiographical nor spatial strategies (N). Amnesic patients and control participants generated exemplars for eight categories of each type. Patients were impaired on all category types but were more impaired on AS and AN categories. After covarying for phonemic fluency (total FAS score), the N category impairment was not significant, but the impairment on AS and AN categories remained. The same results were obtained for patients with lesions restricted to the MTL and those with more extensive lesions. We conclude that patients' episodic memory impairment hindered their performance on this putatively semantic task. This interaction between episodic and semantic memory might partially account for fluency deficits seen in aging, mild cognitive impairment, and Alzheimer's disease.

We sought to map the time course of autobiographical memory retrieval, including brain regions that mediate phenomenological experiences of reliving and emotional intensity. Participants recalled personal memories to auditory word cues during event-related functional magnetic resonance imaging (fMRI). Participants pressed a button when a memory was accessed, maintained and elaborated the memory, and then gave subjective ratings of emotion and reliving. A novel fMRI approach based on timing differences capitalized on the protracted reconstructive process of autobiographical memory to segregate brain areas contributing to initial access and later elaboration and maintenance of episodic memories. The initial period engaged hippocampal, retrosplenial, and medial and right prefrontal activity, whereas the later period recruited visual, precuneus, and left prefrontal activity. Emotional intensity ratings were correlated with activity in several regions, including the amygdala and the hippocampus during the initial period. Reliving ratings were correlated with activity in visual cortex and ventromedial and inferior prefrontal regions during the later period. Frontopolar cortex was the only brain region sensitive to emotional intensity across both periods. Results were confirmed by time-locked averages of the fMRI signal. The findings indicate dynamic recruitment of emotion-, memory-, and sensory-related brain regions during remembering and their dissociable contributions to phenomenological features of the memories.

Major predictors of Alzheimer's disease (AD) include apolipoprotein E (APOE)-epsilon4, hippocampal atrophy on magnetic resonance imaging (MRI), and memory dysfunction prior to diagnosis. We examined 159 normal elderly subjects with MRI and the California Verbal Learning Test (CVLT); 84 returned for longitudinal follow-up 5 years later. Analyses at baseline revealed significant variance in hippocampal volume accounted for by cerebral volume and age but not by APOE isoform. However, interactions involving APOE isoform and laterality were observed. As hypothesized, an APOE x time interaction was revealed for CVLT long-delay free recall: APOE-epsilon3/4 subjects had significantly poorer performance than APOE-epsilon3/3 subjects at follow-up. Forward stepwise multiple regression analysis predicting follow-up long-delay free recall selected baseline recall, followed by number of APOE-epsilon4 alleles, followed by left-hippocampal volume. Age and sex did not enter into the model. We conclude that APOE-epsilon4 predicts longitudinal memory decline in healthy controls and that MRI morphometry of hippocampus adds slightly to predictive value.

BACKGROUND Although protease-activated receptors (PARs) have been described to play a role in different malignancies, their expression and biological activity in prostate cancer are mostly unknown. METHODS PAR expression in radical prostatectomy specimens was investigated by immunohistochemistry (IHC, 40 patients) and RT-PCR. Their role in LNCaP prostate cancer cell migration and Rac1/Cdc42 signaling was assessed with Boyden chamber analysis and Western blot, respectively. RESULTS PAR mRNA expression was higher in cancer, and protein expression was increased in PAR-1 (45%), PAR-2 (42%), and PAR-4 (68%), compared to normal glands. Increased PAR-1 (periglandular stroma) was associated with higher rates of biochemical recurrence (median follow-up, 5 years; P = 0.006). LNCaP migration was enhanced twofold and Rac1/Cdc42 signaling was activated by stimulation of PAR-1 and PAR-2. CONCLUSIONS PARs are overexpressed in prostate cancer and may serve as potential predictors of recurrence. The data suggest potential role of PARs in autocrine and paracrine mechanisms of prostate cancer. Prostate 67: 743–756, 2007. © 2007 Wiley-Liss, Inc.

Functional MRI was used to investigate the role of medial temporal lobe and inferior frontal lobe regions in autobiographical recall. Prior to scanning, participants generated cue words for 50 autobiographical memories and rated their phenomenological properties using our autobiographical memory questionnaire (AMQ). During scanning, the cue words were presented and participants pressed a button when they retrieved the associated memory. The autobiographical retrieval task was interleaved in an event-related design with a semantic retrieval task (category generation). Region-of-interest analyses showed greater activation of the amygdala, hippocampus, and right inferior frontal gyrus during autobiographical retrieval relative to semantic retrieval. In addition, the left inferior frontal gyrus showed a more prolonged duration of activation in the semantic retrieval condition. A targeted correlational analysis revealed pronounced functional connectivity among the amygdala, hippocampus, and right inferior frontal gyrus during autobiographical retrieval but not during semantic retrieval. These results support theories of autobiographical memory that hypothesize co-activation of frontotemporal areas during recollection of episodes from the personal past.

Amnesia typically results from trauma to the medial temporal regions that coordinate activation among the disparate areas of cortex that represent the information that make up autobiographical memories. We proposed that amnesia should also result from damage to these regions, particularly regions that subserve long-term visual memory [Rubin, D. C., & Greenberg, D. L. (1998). Visual memory-deficit amnesia: A distinct amnesic presentation and etiology. Proceedings of the National Academy of Sciences of the USA, 95, 5413-5416]. We previously found 11 such cases in the literature, and all 11 had amnesia. We now present a detailed investigation of one of these patients. M.S. suffers from long-term visual memory loss along with some semantic deficits; he also manifests a severe retrograde amnesia and moderate anterograde amnesia. The presentation of his amnesia differs from that of the typical medial-temporal or lateral-temporal amnesic; we suggest that his visual deficits may be contributing to his autobiographical amnesia.

A total of 30 undergraduates recalled the same 20 autobiographical memories at two sessions separated by 2 weeks. At each session they dated their memories and rated them on 18 properties commonly studied in autobiographical memory experiments. Individuals showed moderate stability in their ratings on the 18 scales (r approximately .5), with consistency of dating being much higher (r = .96). There was more stability in the individuals' average rating on each scale (r approximately .8), even when the averages were calculated on different memories in the different sessions. The results are consistent with a constructive view of autobiographical memory, in which stable individual differences in cognitive style are important.

Many people claim to remember how they heard about the terrorist attacks of September 11, 2001—including George W. Bush, the President of the United States at the time. On at least three occasions, the President was asked how he heard the news of the attacks. His answers contained substantial inconsistencies and provide a near-perfect example of a false flashbulb memory. Copyright © 2004 John Wiley & Sons, Ltd.

In three experiments, undergraduates rated autobiographical memories on scales derived from existing theories of memory. In multiple regression analyses, ratings of the degree to which subjects recollected (i.e., relived) their memories were predicted by visual imagery, auditory imagery, and emotions, whereas ratings of belief in the accuracy of their memories were predicted by knowledge of the setting. Recollection was predicted equally well in between- and within-subjects analyses, but belief consistently had smaller correlations and multiple regression predictions between subjects; individual differences in the cognitive scales that we measured could not account well for individual differences in belief. In contrast, measures of mood (Beck Depression Index) and dissociation (Dissociative Experience Scale) added predictive value for belief, but not for recollection. We also found that highly relived memories almost always had strong visual images and that remember/know judgments made on autobiographical memories were more closely related to belief than to recollection.

Experimental laboratories in psychology are a bit different from the typical hard-science lab; in fact, to the casual visitor, they look a lot like ordinary office space. Nevertheless, these labs generally need certain features to be viable, including dedicated research space, reliable access to participants, and equipment for participant testing. The precise needs vary from investigator to investigator, and this article does not attempt to cover them all; rather, it seeks to provide a sense of the key issues that arise when investigators, department chairs, and administrators set out to build a new psychology lab.

© 2014, Psychonomic Society, Inc. Visual imagery plays a fundamental role in autobiographical memory, but several aspects of this role remain unclear. We conducted three experiments to explore this relationship. In the first experiment, we examined the relation between the phenomenological properties of autobiographical memory and several measures of visual-imagery ability. We found no significant positive relation between imagery ability and autobiographical memory, except on a measure of cognitive style. In a second experiment, we examined the autobiographical memories of people with different cognitive styles—namely, visualizers and verbalizers. We found that, for both kinds of participant, visual imagery was correlated with the feeling that they were reliving their memories, but auditory imagery played a greater role in verbalizers. In a third experiment, we examined the memories of individuals who had a congenital absence of visual imagery. We found that they had a deficit of auditory imagery, as well; moreover, they were much less likely than controls to feel as though they were reliving their memories. The results support the idea that visual imagery plays a vital and irreplaceable role in autobiographical recall.

This study investigated the pollutant emission reduction and demand-side management potential of 16 photovoltaic (PV) systems installed across the US during 1993 and 1994. The US Environmental Protection Agency (EPA) and 11 electric power companies sponsored the project. This article presents results of analyses of each PV system's ability to offset power plant emissions of sulfur dioxide (SO2), nitrogen oxides (NOx), carbon dioxide (CO2) and particulates and to provide power during peak demand hours for the individual host buildings and peak load hours for the utility. The analyses indicate a very broad range in the systems' abilities to offset pollutant emissions, due to variation in the solar resource available and the emission rates of the participating utilities' load following generation plants. Each system's ability to reduce building peak demand was dependent on the correlation of that load to the available solar resource. Most systems operated in excess of 50% of their capacity during building peak load hours in the summer months, but well below that level during winter peak hours. Similarly, many systems operated above 50% of their capacity during utility peak load hours in the summer months, but at a very low level during winter peak hours.

We describe a form of amnesia, which we have called visual memory-deficit amnesia, that is caused by damage to areas of the visual system that store visual information. Because it is caused by a deficit in access to stored visual material and not by an impaired ability to encode or retrieve new material, it has the otherwise infrequent properties of a more severe retrograde than anterograde amnesia with no temporal gradient in the retrograde amnesia. Of the 11 cases of long-term visual memory loss found in the literature, all had amnesia extending beyond a loss of visual memory, often including a near total loss of pretraumatic episodic memory. Of the 6 cases in which both the severity of retrograde and anterograde amnesia and the temporal gradient of the retrograde amnesia were noted, 4 had a more severe retrograde amnesia with no temporal gradient and 2 had a less severe retrograde amnesia with a temporal gradient.

The promoter for the gene coding for human protein C has been characterized as to nucleotide sequences that regulate the synthesis of mRNA. The major transcription start site was found 65 nucleotides upstream from the first intron/exon boundary along with two minor sites. Functional characterization of 1528 base pairs at the 5′-end of the gene was then carried out by chloramphenicol acetyltransferase reporter assays, protection from DNase I digestion, and electrophoretic mobility shift assays employing HepG2 and HeLa cells. One of the upstream regions (nucleotides −25 to +9) contained binding sites for at least two different transcription factors, including a hepatic nuclear factor 1-binding site (−10 to +9) and two overlapping and oppositely oriented hepatic nuclear factor 3-binding sites (−25 to −11). A second major region (PCE1) (+12 to +30) appeared to be a unique, liver-specific regulatory sequence. An Sp1-binding site in exon I (+58 to +65) was also recognized by cotransfection experiments with an Sp1 expression plasmid. Specific mutations in these promoter elements reduced transcriptional activity and abolished the binding of hepatic nuclear proteins. Finally, a strong silencer element (PCS1) (between −162 and −82) and two possible liver-specific enhancer regions (PCE2 and PCE3), which interact coordinately with the promoter elements, were also found (between −1462 and −162).

Publisher Summary Implicit memory or nondeclarative memory does not involve conscious or deliberate retrieval. Although the different types of implicit memory have different behavioral characteristics and depend on different neural systems, they all appear to be intact in amnesic patients, and are thus independent of the brain structures damaged in amnesia. Implicit memory has been a focus in experimental psychology for more than two decades. More recently, implicit memory has become an important concept in studies of clinical populations. There are multiple forms of implicit memory that differ not only in terms of behavioral properties but also the brain systems that support them. The existence of multiple memory systems and processes reveal the immense capacity for plasticity and adaptation in the human brain. Two forms of implicit memory are studied such as priming (perceptual priming, conceptual priming, contamination from explicit memory, priming in amnesia, alzheimer's disease, frontotemporal lobar dementia, and neural basis of priming) and procedural memory. Procedural memory generally refers to memory for knowing how to do something. The knowledge in procedural memories is not accessible to awareness, but rather is manifested only through performance of a task. Like priming one is not necessarily aware of one's procedural knowledge. Unlike priming, which can occur after a single presentation of a stimulus, procedural memories are built up gradually and incrementally with practice.

We sought to examine the relations between age, gender and brain volumes in an elderly population; we also sought to examine ways of measuring these relations. Three sets of analyses were used: correlational analyses, in which correlations between independent variables and brain volumes were calculated without correction for intracranial volume (ICV); covariational analyses, in which ICV was used as a covariate in regression equations; and ratio analyses, in which the dependent variable was the ratio of brain volume to ICV. These analyses yielded similar results, except that (as expected) adjusting for ICV altered estimates of gender differences. Analyses of age showed decreases in left caudate, putamen, and right hippocampus and an increase in CSF, a result generally in accord with previous findings. However, we also found a significant decrease of white-matter volumes and no significant decrease in total gray-matter volumes. Correlational analyses showed that men did not always have larger volumes despite their larger head size; women generally had larger volumes after adjusting for ICV. We found no age-gender interactions.

Numerous studies have shown gender differences in the brain volumes of elderly adults. Some evidence shows that higher estrogen levels may be neuroprotective, suggesting that hormone therapy (HT) may in part be responsible for these gender differences; however, few studies have examined the relation between HT and brain volumes. Brain volumes of caudate, putamen, hippocampus, gray matter, white matter, white-matter lesions, and cerebrospinal fluid were measured on magnetic resonance imaging scans. A comprehensive neuropsychological battery was administered. Women were separated into two groups based on HT use, and we used multiple regression analyses to compare these groups with one another and with men. Results of brain-volume measurements showed that HT users had significantly less gray matter and more cerebrospinal fluid than nonusers. Results of the neuropsychological testing showed that HT users performed better on the Shipley Vocabulary Test than males did.

This special issue of Cortex focuses on the relative contribution of different neural networks to memory and the interaction of 'core' memory processes with other cognitive processes. In this article, we examine both. Specifically, we identify cognitive processes other than encoding and retrieval that are thought to be involved in memory; we then examine the consequences of damage to brain regions that support these processes. This approach forces a consideration of the roles of brain regions outside of the frontal, medial-temporal, and diencephalic regions that form a central part of neurobiological theories of memory. Certain kinds of damage to visual cortex or lateral temporal cortex produced impairments of visual imagery or semantic memory; these patterns of impairment are associated with a unique pattern of amnesia that was distinctly different from the pattern associated with medial-temporal trauma. On the other hand, damage to language regions, auditory cortex, or parietal cortex produced impairments of language, auditory imagery, or spatial imagery; however, these impairments were not associated with amnesia. Therefore, a full model of autobiographical memory must consider cognitive processes that are not generally considered 'core processes,' as well as the brain regions upon which these processes depend.

Thrombin and trypsin induce cell signaling through a subclass of G-protein-coupled receptors called the protease-activated receptors (PARs). In many cells, PAR signaling results in the activation of RhoA and other members of the Rho family of small GTPases which are involved in cytoskeletal reorganization. The expression of PARs and their role in the activation of Rho GTPases in prostate cancer cells are not clearly known. FACS analysis demonstrated that the androgen-dependent LNCaP cells express PAR1, PAR2, and PAR4 but not PAR3. Stimulation with thrombin and trypsin resulted in the rapid activation of RhoA in a dose-dependent manner with an EC(50) of 1.0 and 5 nM, respectively. Activation of RhoA was enhanced by, but not dependent on, the presence of 1 nM dihydrotestosterone. Inhibition of the proteolytic properties of thrombin by hirudin and trypsin by diisopropyl fluorophosphate abolished the observed RhoA activation. Stimulation with 150 microM PAR-activating peptides TFFLRN (PAR1), SLIGKV (PAR2), and AYPGKF (PAR4) demonstrated that PAR1 and PAR2 mediated protease-activated RhoA signaling. Fluorescent microscopy studies showed that LNCaP cells treated with either thrombin (10 nM) or trypsin (10 nM) developed an increased number of filopodia, stress fibers, and focal adhesions relative to untreated cells. These observations represent the first report of PAR signaling in prostate cancer cells as well as the ability of PAR2 to mediate RhoA activation. Since the activation of RhoA is important for cytoskeletal reorganization, we postulate that PAR-mediated RhoA activation may be a major signaling pathway in the biology of prostate cancer.

The authors investigated the role of baseline hippocampal volume on later clinical emergence of dementia in a group of older, non-demented depressed individuals. Subjects were 115 depressed, non-demented participants in a mental health clinical research center. All subjects were screened for dementia and agreed to have a magnetic resonance imaging (MRI) brain scan at baseline. Subjects were clinically evaluated by geriatric psychiatrists quarterly for up to 5 years and received annual neuropsychological testing. Bivariate analyses examined age, gender, race, educational level, baseline depression severity, age at depression onset, baseline Mini-Mental State Exam (MMSE), left and right hippocampal volume, and total cerebral volume. Age, baseline MMSE, total cerebral volume, and having a small left hippocampal volume were associated with later dementia and were included in subsequent survival analysis. Small left hippocampal volume was significantly associated with later dementia (hazard ratio=2.762). Small left hippocampal size on neuroimaging may be a marker for dementia in depressed patients who have not yet met criteria for a clinical diagnosis of a dementing disorder.

Background: There is a growing literature on the importance of hippocampal volume in geriatric depression. Methods: We examined hippocampal volume in a group of elderly depressed patients and a group of elderly control subjects ( N = 66 geriatric depressed patients and 18 elderly nondepressed control subjects) recruited through Duke's Mental Health Clinical Research Center for the Study of Depression in the Elderly. The subjects received a standardized evaluation, including a magnetic resonance imaging scan of the brain. Patients had unipolar major depression and were free of comorbid major psychiatric illness and neurologic illness. Differences were assessed using t tests and linear regression modeling. Results: Accounting for the effects of age, gender, and total brain volume, depressed patients tended to have smaller right hippocampal volume ( p = .014) and left hippocampal volume ( p = .073). Among depressed patients, age of onset was negatively but not significantly related to right hippocampal volume ( p = .052) and to left hippocampal volume ( p = .062). We noted that among subjects with either right or left hippocampal volume of 3 mL or less, the vast majority were patients rather than control subjects. Conclusions: These results support a role for hippocampal dysfunction in depression, particularly in late-age onset depression. Longitudinal studies examining both depressive and cognitive outcomes are needed to clarify the relationships between the hippocampus, depression, and dementia.

Top of pageAbstract Recombinant activated factor VII (rFVIIa) has been used as an effective alternative treatment for hemophilia patients who have developed inhibitors. We have successfully modeled a nonviral gene therapy approach to deliver factor VII (FVII) in a hemophilia A mouse model to decrease the cost and frequency of rFVIIa infusions. An engineered murine FVII (mFVIIa) cDNA was inserted into our gene transfer vector to generate a liver-specific construct pBS- HCRHPI-mFVIIa-A encoding a modified protein, which can be cleaved by intracellular proteases of the furin family to secrete the activated form of mFVII. Partial correction of bleeding was achieved following nonviral gene transfer of this construct into hemophilia A mice. To increase the safety and efficacy of FVII gene therapy, we devised two approaches. The first was to use a mFVII wild-type cDNA (mFVII) encoding the zymogen FVII to reduce the potential thrombotic risk associated with prolonged, high level FVIIa expression. The second was to use a mutant FVIIa or FVII molecule (mFVIIa-DVQ or mFVII-DVQ) encoding a superactive FVII molecule with amino-acid substitutions (V158D/E296V/M298Q; Persson et al., 2003) to increase the efficacy of nonviral gene transfer of FVII. Fifty mg of the respective plasmids encoding wild-type or modified proteins including mFVII, mFVIIa, mFVII-DVQ, and mFVIIa-DVQ, were delivered into 4 groups of hemophilia A mice by a rapid, high volume (hydrodynamics-based) infusion method (n=8 mice/group). Phenotypic correction was measured via tail-clip assays. A 30% reduction in hemoglobin loss was observed in mice treated with mFVII, and a 50% reduction in mice treated with mFVIIa. Significant and long-term correction in PT using 1:60 dilution of mouse plasma was observed in both mFVII and mFVIIa treated mice, with clotting values consistently lower than wild-type and hemophilia A controls (wild-type: 26.4 s, hemophilia A: 25.4 s, mFVII: 23.6 s, mFVIIa: 23.3 s). APTT performed on plasma diluted 1:3 similarly reflects the results of the PT assay, with lowered clotting times for both groups of treated mice (wild-type: 60.2, hemophilia A: 121.3, mFVII: 102.8, mFVIIa: 100.9). Both plasma- based and platelet-based thrombin generation (TG) assays indicated that both mFVII and mFVIIa have a positive effect on the three measured parameters with a lowered lag phase, lowered peak time and elevated peak thrombin levels. In addition, all these effects lasted long-term in plasmid treated mice for at least six months (experimental duration). Preliminary experiments using our most recently built constructs mFVII-DVQ and mFVIIa-DVQ showed reversion to wild-type phenotype in plasmid-treated mice as indicated by significantly reduced hemoglobin loss via tail clipping and substantial reduction in APTT. These results clearly demonstrate that nonviral gene therapy of either zymogen FVII or activated FVII can rescue bleeding in hemophilia A mice, and mFVII-DVQ/mFVIIa- DVQ may potentially recover wild-type clotting activity in treated mice.

Promoter and silencer elements of the immediate 5' flanking region of the gene coding for human factor VII were identified and characterized. The major transcription start site, designated as +1, was determined by RACE (rapid amplification of cDNA ends) analysis of human liver cDNA and was found to be located 50 bp upstream from the translation start site. Two minor transcription start sites were found at bp +32 bp and +37. Progressive deletions of the 5' flanking region were fused to the chloramphenicol acetyltransferase reporter gene and transient expression in HepG2 and HeLa cells was measured. Two promoter elements that were essential for hepatocyte-specific transcription were identified. The first site, FVIIP1, located at bp -19 to +1, functioned independently of orientation or position and contributed about one-third of the promoter activity of the factor VII gene. Electrophoretic mobility-shift, competition, and anti-hepatocyte nuclear factor 4 (HNF4) antibody supershift experiments demonstrated that this site contained an HNF-4 binding element homologous to the promoters in the genes coding for factor IX and factor X. The second site, FVIIP2, located at bp -50 to -26, also functioned independent of orientation or position and contributed about two thirds of the promoter activity in the gene for factor VII. Functional assays with mutant sequences demonstrated that a 10-bp G + C-rich core sequence which shares 90% sequence identity with the prothrombin gene enhancer was essential for the function of the second site. Mobility-shift and competition assays suggested that this site also binds hepatic-specific factors as well as the transcription factor Sp1. Two silencer elements located upstream of the promoter region spanning bp -130 to -103 (FVIIS1 site) and bp -202 to -130 (FVIIS2) were also identified by reporter gene assays.

Abstract 21 Background: Low bone mineral density (BMD) and increased risk of fracture are increasingly being identified in patients with hemophilia. Multiple clinical studies have shown that both children and adults with hemophilia have significantly decreased BMD, with up to 70% of adult patients affected. Decreased BMD in this population has previously been attributed to inactivity due to hemophilic arthropathy and comorbid conditions including infection with HIV and hepatitis C. The factor VIII (FVIII) knockout (KO) mouse model has been extensively characterized with respect to bleeding and the development of inhibitors. However, the skeletal status of these animals has not been described. Therefore, the purpose of this study is to examine the skeletal phenotype of FVIII deficient mice and measure serum markers of bone turnover and regulation. Methods: We compared the skeletal phenotype of 11 male FVIII KO mice and 8 matched wild-type (WT) controls at 20 weeks of age. BMD was measured using dual energy x-ray absorptiometry (DXA), bone geometry was examined by μCT and, after sacrifice, femoral breaking strength was measured by three-point bending until failure (ultimate force). In a second group of 24 mice (12 KO and 12 WT), serum was obtained under uniform conditions at 20 weeks of age via cardiac puncture. Serum levels of receptor activator of nuclear factor kappa-B ligand (RANK-L), osteoprotegerin (OPG), interleukin 1α (IL-1α) and osteocalcin were measured using commercially available ELISAs. Alkaline phosphatase (alk phos) activity was measured using a colorimetric assay. The RANK-L/OPG system regulates bone turnover, osteocalcin and alk phos are markers of bone turnover and IL-1α is an inflammatory cytokine. Results: No differences in body weight, length, percent fat, or femoral length were observed between FVIII KO and WT mice (data not shown). No spontaneous bleeding was observed in any animal. As shown in Table I, KO mice had significantly decreased BMD, cortical thickness and stiffness compared to WT mice. This resulted in decreased resistance to fracture as measured via ultimate force. The data presented here demonstrate that adult FVIII KO male mice exhibit lower cortical bone mass resulting in femora less resistant to fracture compared with WT controls. Because FVIII KO mice do not have spontaneous joint hemorrhages and exhibit normal behavior and activity levels, these data suggest that there is an underlying connection between the coagulation system and bone metabolism that results in lower BMD and bone strength. To understand why KO mice have decreased BMD, we measured serum levels of regulators of bone metabolism, markers of bone turnover and inflammatory cytokines. There was no difference in RANK-L, OPG or the RANK-L/OPG ratio between the KO and WT mice. In addition, there was no observed difference in osteocalcin and alk phos. However, WT animals had significantly higher levels of IL-1α expression (P = 0.008). All of the KO animals had undetectable IL-1α levels under physiologic conditions whereas the mean IL-1α level in WT mice was 32.2 pg/ml. Thrombin production is reduced in hemophilia and thrombin has previously been shown to stimulate inflammatory cytokine production. Therefore, these data suggest that thrombin may be necessary for IL-1α production under non-stress conditions and that lower levels of IL-1α may be detrimental to bone health. Although increased inflammatory cytokines are typically associated with decreased BMD, prior research has shown that physiologic levels of cytokines are necessary to maintain bone health. Conclusions: These data provide compelling evidence that the link between FVIII deficiency and impaired skeletal health is real and independent of differences in physical activity and other medical co-morbidities. In addition, the decreased levels of IL-1α observed in KO mice suggest that inflammatory cytokines are involved in this pathophysiology. Ongoing work in our laboratory is aimed at further exploring these connections. Disclosures: No relevant conflicts of interest to declare.

Background: Blood platelets undergo a carefully regulated change in shape to serve as the primary mediators of hemostasis and thrombosis. These processes manifest through platelet spreading and aggregation and are dependent on platelet actin cytoskeletal changes orchestrated by the Rho GTPase family member Rac1. To elucidate how Rac1 is regulated in platelets, we captured Rac1-interacting proteins from platelets and identified Rac1-associated proteins by mass spectrometry. Findings: Here, we demonstrate that Rac1 captures the Rac guanine nucleotide exchange factor P-Rex1 from platelet lysates. Western blotting experiments confirmed that P-Rex1 is expressed in platelets and associated with Rac1. To investigate the functional role of platelet P-Rex1, platelets from P-Rex1 -/- -deficient mice were treated with platelet agonists or exposed to platelet activating surfaces of fibrinogen, collagen and thrombin. Platelets from P-Rex1 -/- mice responded to platelet agonists and activating surfaces similarly to wild type platelets. Conclusions: These findings suggest that P-Rex1 is not required for Rac1-mediated platelet activation and that the GEF activities of P-Rex1 may be more specific to GPCR chemokine receptor mediated processes in immune cells and tumor cells.

Abstract 4015 Poster Board III-951 A major signaling pathway that regulates platelet shape change and reorganization of the cytoskeleton involves the Rho family of GTPases; CDC42, Rac1 and RhoA. These GTPases are converted from their inactive or GDP-loaded state to the active or GTP-loaded state by a class of enzymes called Guanine Exchange Factors (GEFs). GEFs are a family of multi-domain proteins that contain a GDP-GTP exchange domain (DH-PH) as well as other protein interacting domains that are regulated by the activation of receptors present on the platelet surface. We previously identified the presence of two homologous GEFs, Cool-1 and Cool-2, in human platelets. In nucleated cells, the activities and substrate specificities of the Cool GEFs are regulated by complex interactions with p21-activated kinases (PAK), Gb/g heterodimers, focal adhesion kinase, and the scaffolding proteins GIT-1 and GIT-2. The Cool GEFs are found at the sites of focal adhesions during spreading and migration in nucleated cells, however, little is known about the regulation or activity of Cool-1 or Cool-2 in platelets. Co-immunoprecipitation experiments were performed with Cool-1 and Cool-2 antibodies using lysates from resting and thrombin stimulated human platelets. We analyzed the precipitated proteins by Mass Spectroscopy and found a number of structural and scaffold proteins bound to the Cool GEFs in the thrombin activated lysates. These cytoskeletal proteins include talin, multimerin, tubulin, filamin A, actin and fibrinogen. Interestingly, a large number of Factor XIII also co-immunoprecipitated with Cool-1 and Cool-2 in the thrombin activated platelet lysates. Western analysis of the co-immunoprecipated platelet proteins from thrombin, TRAP1 and TRAP4 demonstrated that the association of factor XIII with the Cool GEFs is calcium dependent. Inhibition of transglutaminase activity and presence of RGD peptide did not affect the association of Factor XIII with Cool-1 or Cool-2. Factor XIII deficiency is commonly thought to result in bleeding due to the impaired crosslinking of fibrin. However, platelet factor XIII may also play an important role in cross linking platelet cytoskeleletal proteins such as actin and myosin. Consistent with that hypothesis is a recent report showing that platelet factor XIII deficiency results in decreased lamellapodia formation under static adhesive conditions. The association of Factor XIII with the Cool GEFs further supports investigating the potential for the transglutaminase to crosslink platelet cytoskeletal proteins. Disclosures: No relevant conflicts of interest to declare.

A major signaling pathway that regulates platelet shape change and reorganization of the cytoskeleton involves the Rho family of GTPases; CDC42 (fillapodia), Rac1 (lamellapodia) and RhoA (focal adhesions). These GTPases are converted from their inactive or GDP-loaded state to the active or GTP-loaded state by a class of enzymes called Guanine Exchange Factors (GEFs). GEFs are a family of multi-domain proteins that contain a GDP-GTP exchange domain (DH-PH) as well as other protein interacting domains that are regulated by the activation of receptors present on the platelet surface. We used an affinity binding technique followed by mass spectroscopy to identify novel Rho family binding GEFs in platelet lysates. Recombinant GST-Rho fusion proteins bound to agarose beads were prepared in the GTP, GDP and nucleotide-free states and incubated with clarified human platelet lysates. Platelet lysate proteins associated with the different GST-Rho preparations were eluted and run on SDS-PAGE. Gel slices were then cut out, trypsin digested and analyzed by Mass Spectroscopy. Platelet GEFs were identified by the presence of a DH-PH domain. Using this technique we found Cool-1 and Cool-2, two closely related GEFs that regulate Rac1 and CDC42 activation in nucleated cells. Cool-1 has not been previously described in platelets. Homodimeric Cool-1 specifically activates CDC42, whereas Cool-2 is capable of activating Rac1 as a homodimer or CDC42 as a monomer. The substrate specificity of Cool-2 is regulated by complex interactions with p21-activated kinases (PAK) and Gb/g proteins. In nucleated cells, Cool-1 and Cool-2 are localized to focal adhesions by the GPCR-kinase-interacting proteins 1 and 2 (GIT1 and GIT2). GIT binding to the Cool proteins has been shown to inhibit their GEF activity. Here we show that Cool-1 is present only in the membrane fraction of resting platelet lysates while Cool-2 is present in both the membrane and cytoplasmic fractions. Upon activation with thrombin, about half the membrane bound Cool-1 and all the membrane bound Cool- 2 migrate to the cytoplasm. Precipitation experiments in thrombin activated platelets with anti-GIT1 and anti-GIT2 antibodies show GIT-1 avidly binds Cool-2 whereas GIT2 binds a small amount of Cool-1. These data suggest a complex mechanism of CDC42 and Rac1 activation in platelets by the Cool proteins. Based on our findings and the current understanding of the regulation of Cool proteins in nucleated cells, we hypothesize that early after thrombin activation, the CDC42 and Rac1 GEF activities of Cool-1 and Cool-2 result in fillapodia (CDC42) and lamellapodia (Rac1) formation. As integrant mediated focal adhesions are formed, the GEF activities of Cool-1 and Cool-2 are inhibited by the scaffolding proteins GIT2 and GIT1, respectively. In this manner, Rac1 and CDC42 mediated fillapodia and lamellapodia formation may be regulated by the extent of integrin engagement and platelet spreading.

A major signaling pathway that regulates platelet shape change and reorganization of the cytoskeleton involves the Rho family of GTPases whose members include Rac, CDC42 and RhoA. These GTPases are converted from their inactive or GDP-loaded state to the active or GTP-loaded state by a class of enzymes called Guanine-nucleotide Exchange Factors (GEFs). GEFs are a family of multi-domain proteins that contain a GDP-GTP exchange domain (DH-PH) as well as other protein interacting domains that are regulated by the activation of receptors present on the platelet surface. We used an affinity binding technique followed by mass spectrometry analysis to identify novel Rac binding GEFs from platelet lysates. Recombinant GST-Rac fusion proteins bound to agarose beads were prepared in the GTP, GDP and nucleotide-free states and incubated with human platelet lysates. Platelet lysate proteins associated with the different GST-Rac preparations (GTP-bound, GDP-bound or nucleotide-free) were eluted and run on SDS-PAGE. Gel slices were then cut out, trypsin digested and analyzed by mass spectrometry. Platelet GEFs were identified by the presence of a DH-PH domain. Using this technique we identified three novel Rac-associated platelet GEFs including P-REX1, a G-βγ protein and phosphatidylinositol (3,4,5)-triphosphate regulated GEF previously found in neutrophils and neurons (Welch, H.C.E et al., Cell108;809–822, 2002). PREX-1 is a 196kDa peptide composed of 1659 amino that specifically activates Rac in neutrophils. In addition to the DH-PH exchange domain, PREX-1 also contains tandem PDZ and DEP domains as well as significant similarity over its C-terminal half to Inositol Polyphosphate 4-Phosphatase. Western analysis with anti-P-REX1 antibody (gift of H.C. Welch, Barbraham Institute, U.K.) showed that PREX-1 is present in purified whole platelet lysates. We have also shown that platelet PREX-1 specifically associates with GST-Rac by affinity pull-down experiments. Functional studies to characterize the exchange activity of platelet PREX-1 are ongoing. PREX-1 is the only known GEF that is directly regulated by G-βγ protein (Hill, K. et al., J Biol Chem280(6):4166–4173, 2005) and represents a novel pathway for platelet G protein coupled receptor signaling through Rac in human platelets.

Owen et al . (Brevia, 8 September 2006, p. 1402) claimed that a patient's brain activity revealed that she was consciously responding to commands despite being in a vegetative state. However, several alternative explanations were not eliminated. Specifically, the activity could reflect unconscious reactions to the last word in the command, not conscious decisions to respond. A refined experimental design could clarify these issues.

A major problem for clinical treatment of hemophilia A using factor replacement therapy is the high frequency formation of inhibitory antibodies against factor VIII. This problem is also predicted to occur following strategies currently aimed at targeted genetic correction of this disease. Recombinant activated factor VII (rFVIIa, NovoSeven®, Novo Nordisk, Bagsvaerd, Denmark) has been successfully used as an effective alternative treatment for hemophilia patients who have developed inhibitors. In order to decrease the cost and fluctuation of FVIIa levels associated with frequent infusions of rFVIIa, nonviral gene transfer of factor VII (FVII) was attempted in a hemophilia A mouse model. In addition, to investigate the potential thrombotic risks associated with prolonged, high level of FVIIa expression following gene transfer, we compared the effects of gene transfer vectors encoding zymogen FVII with an engineered secreted FVIIa in hemophilia A mice. We inserted murine FVII (mFVII) cDNA into a liver-specific vector developed recently in our lab to generate pBS-HCRHPI-mFVII-A. The mFVII cDNA sequence was then modified by site-directed mutagenesis to insert a protease cleavage site in between Arg152-Ile153. The resulting construct of murine FVIIa (mFVIIa) encodes a modified protein, which can be cleaved by intracellular proteases of the furin family to secrete the activated form of mFVII. Fifty mg of either mFVII or mFVIIa the plasmid was delivered into hemophilia A mouse liver by a rapid, high volume (hydrodynamics-based) infusion method (n=8 mice/group). Phenotypic correction was evaluated using tail-clip assays by measuring the hemoglobin levels in the collected blood. This assay indicated that vectors carrying either mFVII or mFVIIa can partially correct clotting following gene transfer. Significant correction in PT and APTT was observed in mFVII or mFVIIa-treated mice. Plasma-based and platelet-based thrombin generation (TG) assays indicated that either mFVII or mFVIIa significantly shorten peak time and peak thrombin levels of TG. In addition, these effects lasted long-term in plasmid treated mice for at least three months (experimental duration). We also constructed vectors carrying human FVII (hFVII) or FVIIa (hFVIIa) to test their efficacy for correcting hemophilia A. Constructs produced supra-physiological levels (800–1200ng/ml) of hFVII or hFVIIa assayed by ELISA at days 1 and 4 post-injection. However, the levels of hFVII and hFVIIa dropped precipitously at day 14 post-injection shown by both ELISA and Western blot, most likely due to the formation of species-specific antibodies against hFVII. These results clearly demonstrate that nonviral gene therapy of either zymogene FVII or activated FVII can rescue bleeding in hemophilia A mice.

A major problem for clinical treatment of hemophilia A using factor replacement therapy is the high frequency formation of inhibitory antibodies against factor VIII. This problem is also predicted to occur following strategies currently aimed at targeted genetic correction of this disease. Recombinant activated factor VII (rFVIIa) has been successfully used as an effective alternative treatment for hemophilia patients who developed inhibitors. In order to decrease the cost and fluctuation of FVIIa levels associated with frequent infusions of rFVIIa, nonviral gene transfer of factor VII (FVII) was attempted in a hemophilia A mouse model. To investigate the potential thrombotic risks associated with prolonged, high level of FVIIa expression following gene transfer, we compared the effects of gene transfer vectors encoding zymogen FVII and an engineered secreted FVIIa in hemophilia A mice. We have first inserted human FVII cDNA into a liver-specific vector developed recently in our lab to make pBS-HCRHPI-hFVIIA. The hFVII cDNA sequence was then modified by site-directed mutagenesis to insert a protease cleavage site in between Arg152-Ile153. The resulting construct, pBS-HCRHPI-hFVIIaA encodes a modified protein which can be cleaved by intracellular proteases of the furin family to secrete the activated form of FVII. Fifty mg of the plasmids were delivered into the hemophilia A mouse livers by a rapid, high volume (hydrodynamics-based) infusion method (n=8 mice/group). Both constructs produced supra-physiological levels (800-1200ng/ml) of hFVII or hFVIIa at days 1 and 4 post plasmid administration. Phenotypic correction was evaluated using bleeding assays (normal mice |[sim]|2-5mins, hemophilia A mice >30mins). The hemophilia A mice treated with pBS-HCRHPI-FVIIA had reduced bleeding times of 20-25 mins, although no significant reduction in clotting times by both prothrombin time (PT) and activated partial thromboplastin time (APTT) assays was observed. The normal mice treated with pBS-HCRHPI-FVIIaA had bleeding times of 5-10 mins with more significant reduction in clotting times by PT, and minor reduction by APTT. These results indicate that the evaluation of PT and APTT does not completely reflect the ability to achieve hemostasis in vivo upon injury under these experimental conditions. The levels of hFVII and hFVIIa dropped precipitously at day 14 post treatment possibly due to formation of species-specific antibodies against hFVII. Since interaction of human FVII with murine tissue factor is much weaker than with the human tissue factor, we next constructed vectors to transfer murine FVII or FVIIa to test their efficacy for correcting hemophilia A. Preliminary results in a tail-clip bleeding assay indicated that vectors carrying either mFVII or mFVIIa can partially correct clotting following gene transfer. Various assays including bleeding assays, PT, APTT, a plasma-based thrombin generation (TG) assay and a platelet-based TG assay with more treated animals will be used to assess their full function in rescuing bleeding.

Functional neuroimaging studies of episodic memory retrieval generally measure brain activity while participants remember items encountered in the laboratory (“controlled laboratory condition”) or events from their own life (“open autobiographical condition”). Differences in activation between these conditions may reflect differences in retrieval processes, memory remoteness, emotional content, retrieval success, self-referential processing, visual/spatial memory, and recollection. To clarify the nature of these differences, a functional MRI study was conducted using a novel “photo paradigm,” which allows greater control over the autobiographical condition, including a measure of retrieval accuracy. Undergraduate students took photos in specified campus locations (“controlled autobiographical condition”), viewed in the laboratory similar photos taken by other participants (controlled laboratory condition), and were then scanned while recognizing the two kinds of photos. Both conditions activated a common episodic memory network that included medial temporal and prefrontal regions. Compared with the controlled laboratory condition, the controlled autobiographical condition elicited greater activity in regions associated with self-referential processing (medial prefrontal cortex), visual/ spatial memory (visual and parahippocampal regions), and recollection (hippocampus). The photo paradigm provides a way of investigating the functional neuroanatomy of real-life episodic memory under rigorous experimental control.