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Background: Understanding of the pathophysiology of autism spectrum disorder (ASD) remains limited. Brain overgrowth has been hypothesized to be associated with the development of ASD. A derivative of amyloid-β precursor protein (APP), secreted APPα (sAPPα), has neuroproliferative effects and has been shown to be elevated in the plasma of persons with ASD compared to control subjects. Reduction in sAPPα holds promise as a novel molecular target of treatment in ASD. Research into the neurochemistry of ASD has repeatedly implicated excessive glutamatergic and deficient GABAergic neurotransmission in the disorder. With this in mind, acamprosate, a novel modulator of glutamate and GABA function, has been studied in ASD. No data is available on the impact of glutamate or GABA modulation on sAPPα function., Methods: Plasma APP derivative levels pre- and post-treatment with acamprosate were determined in two pilot studies involving youth with idiopathic and fragile X syndrome (FXS)-associated ASD. We additionally compared baseline APP derivative levels between youth with FXS-associated or idiopathic ASD., Results: Acamprosate use was associated with a significant reduction in plasma sAPP(total) and sAPPα levels but no change occurred in Aβ40 or Aβ42 levels in 15 youth with ASD (mean age: 11.1 years). Youth with FXS-associated ASD (n = 12) showed increased sAPPα processing compared to age-, gender- and IQ-match youth with idiopathic ASD (n = 11)., Conclusions: Plasma APP derivative analysis holds promise as a potential biomarker for use in ASD targeted treatment. Reduction in sAPP (total) and sAPPα may be a novel pharmacodynamic property of acamprosate. Future study is required to address limitations of the current study to determine if baseline APP derivative analysis may predict subgroups of persons with idiopathic or FXS-associated ASD who may respond best to acamprosate or to potentially other modulators of glutamate and/or GABA neurotransmission., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Alzheimer's disease (AD) is characterized by formation of neuritic plaque primarily composed of a small filamentous protein called amyloid-β peptide (Aβ). The rate-limiting step in the production of Aβ is the processing of Aβ precursor protein (APP) by β-site APP-cleaving enzyme (BACE1). Hence, BACE1 activity plausibly plays a rate-limiting role in the generation of potentially toxic Aβ within brain and the development of AD, thereby making it an interesting drug target. A phase II trial of the promising LY2886721 inhibitor of BACE1 was suspended in June 2013 by Eli Lilly and Co., due to possible liver toxicity. This outcome was apparently a surprise to the study's team, particularly since BACE1 knockout mice and mice treated with the drug did not show such liver toxicity. Lilly proposed that the problem was not due to LY2886721 anti-BACE1 activity. We offer an alternative hypothesis, whereby anti-BACE1 activity may induce apparent hepatotoxicity through inhibiting BACE1's processing of β-galactoside α-2,6-sialyltransferase I (STGal6 I). In knockout mice, paralogues, such as BACE2 or cathepsin D, could partially compensate. Furthermore, the short duration of animal studies and short lifespan of study animals could mask effects that would require several decades to accumulate in humans. Inhibition of hepatic BACE1 activity in middle-aged humans would produce effects not detectable in mice. We present a testable model to explain the off-target effects of LY2886721 and highlight more broadly that so-called off-target drug effects might actually represent off-site effects that are not necessarily off-target. Consideration of this concept in forthcoming drug design, screening, and testing programs may prevent such failures in the future., (Copyright © 2014 The Alzheimer's Association. All rights reserved.)

Late-onset Alzheimer's disease (LOAD) is the most common neurodegenerative disorder in older adults, affecting over 50% of those over age 85. Aging is the most important risk factor for the development of LOAD. Aging is associated with the decrease in the ability of cells to cope with cellular stress, especially protein aggregation. Here we describe how the process of aging affects pathways that control the processing and degradation of abnormal proteins including amyloid-β (Aβ). Genetic association studies in LOAD have successfully identified a large number of genetic variants involved in the development of the disease. However, there is a gap in understanding the interconnections between these pathomolecular events that prevent us from discovering therapeutic targets. We propose novel, pertinent links to elucidate how the biology of aging affects the sequence of events in the development of LOAD. Furthermore we analyze and synthesize the molecular-pathologic-clinical correlations of the aging process, involving the HSF1 and FOXO family pathways, Aβ metabolic pathway, and the different clinical stages of LOAD. Our new model postulates that the aging process would precede Aβ accumulation, and attenuation of HSF1 is an "upstream" event in the cascade that results in excess Aβ and synaptic dysfunction, which may lead to cognitive impairment and/or trigger "downstream" neurodegeneration and synaptic loss. Specific host factors, such as the activity of FOXO family pathways, would mediate the response to Aβ toxicity and the pace of progression toward the clinical manifestations of AD.

Autism is a neurodevelopmental disorder marked by social skills and communication deficits and interfering repetitive behavior. Intellectual disability often accompanies autism. In addition to behavioral deficits, autism is characterized by neuropathology and brain overgrowth. Increased intracranial volume often accompanies this brain growth. We have found that the Alzheimer's disease (AD) associated amyloid-β precursor protein (APP), especially its neuroprotective processing product, secreted APP α, is elevated in persons with autism. This has led to the "anabolic hypothesis" of autism etiology, in which neuronal overgrowth in the brain results in interneuronal misconnections that may underlie multiple autism symptoms. We review the contribution of research in brain volume and of APP to the anabolic hypothesis, and relate APP to other proteins and pathways that have already been directly associated with autism, such as fragile X mental retardation protein, Ras small GTPase/extracellular signal-regulated kinase, and phosphoinositide 3 kinase/protein kinase B/mammalian target of rapamycin. We also present additional evidence of magnetic resonance imaging intracranial measurements in favor of the anabolic hypothesis. Finally, since it appears that APP's involvement in autism is part of a multi-partner network, we extend this concept into the inherently interactive realm of epigenetics. We speculate that the underlying molecular abnormalities that influence APP's contribution to autism are epigenetic markers overlaid onto potentially vulnerable gene sequences due to environmental influence.

The goal of genome-wide association studies is to identify SNPs unique to disease. It usually involves a single sampling from subjects' lifetimes. While primary DNA sequence variation influences gene-expression levels, expression is also influenced by epigenetics, including the 'somatic epitype' (G(SE)), an epigenotype acquired postnatally. While genes are inherited, and novel polymorphisms do not routinely appear, G(SE) is fluid. Furthermore, G(SE) could respond to environmental factors (such as heavy metals) and to differences in exercise, maternal care and dietary supplements - all of which postnatally modify oxidation or methylation of DNA, leading to altered gene expression. Change in epigenetic status may be critical for the development of many diseases. We propose a 'longitudinal epigenome-wide association study', wherein G(SE) are measured at multiple time points along with subjects' histories. This Longitudinal epigenome-wide association study, based on the 'dynamic' somatic epitype over the 'static' genotype, merits further investigation.

Alzheimer's disease (AD) is the leading cause of dementia in the elderly. Extraneuronal plaque comprising mostly the amyloid β peptide and intraneuronal tangles of hyperphosphorylated microtubule-associated τ protein (τ, gene MAPT) are typical of AD. Misfolded τ is also implicated in Parkinson's disease and frontotemporal dementia. We aim to understand the regulation of the human MAPT promoter by mapping its functional domains. We subcloned a 4868 base pair (bp) fragment from human BAC RPCI-11 100C5. Sequence analysis revealed an H2 haplotype MAPT promoter, 5'-UTR, and intronal fragment. Database analysis of the fragment showed 50%-75% homology with mouse and >90% with rhesus monkey. Comparison with human H1 sequences revealed differences that crossed predicted transcription factor sites. DNA-protein interaction studies by electrophoretic mobility shift assay suggested hypoxia response and an active specificity protein 1 (SP1) site in the 5'-untranslated region. Transfection of a series of MAPT promoter deletions revealed unique functional domains. The distal-most had different activities in neuronal vs. non-neuronal cells. We have cloned, sequenced, and functionally characterized a 4868bp fragment of the human MAPT 5'-flanking region, including the core promoter region (-302/+4), neurospecific domains (-4364/-1992 and +293/+504, relative to +1 TSS), and a hypoxia-inducible element (+60/+84). Our work extended functional analysis of the MAPT sequence further upstream, and explores cell-type specificity of MAPT promoter activity. Finally, we provided direct comparison of likely transcription factor binding sites, which are useful to understand differences between H1/H2 pathogenic associations., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Late onset Alzheimer's disease (LOAD) is a non-familial, progressive neurodegenerative disease and the most prominent form of dementia in the elderly. Accumulating evidence suggests that LOAD not only results from the combined effects of variation in a number of genes and environmental factors, but also from epigenetic abnormalities such as histone modifications or DNA methylation. In comparison to monogenic diseases, LOAD exhibits numerous anomalies that suggest an epigenetic component in disease etiology. Evidence against a monogenic course and for an epigenetic component include: 1) the dominance of sporadic cases over familial ones and the low estimated concordance rates for monozygotic twins; 2) gender specific susceptibility and course of disease; 3) parent-of-origin effects, and late age of onset; 4) brain chromatin abnormalities, non-Mendelian inheritance patterns, and atypical levels of folate and homocysteine; and 5) monoallelic expression patterns of susceptibility genes [1]. The epigenome is particularly susceptible to deregulation during early embryonic and neonatal periods and thus disturbances during these periods can have latent lasting effects. The Latent Early-life Associated Regulation (LEARn) model attempts to explain these consequences from a brain specific point of view. In the present review we present the evidence that support the role of epigenetics in the development of AD and explore the potential pathways and mechanisms that may be involved.

Alzheimer's disease (AD) is a leading cause of aging related dementia and has been extensively studied by several groups around the world. A general consensus, based on neuropathology, genetics and cellular and animal models, is that the 4 kDa amyloid β protein (Aβ) triggers a toxic cascade that induces microtubule-associated protein τ (MAPT) hyperphosphorylation and deposition. Together, these lesions lead to neuronal dysfunction and neurodegeneration, modeled in animals, that ultimately causes dementia. Genetic studies show that a simple duplication of the Aβ precursor (APP) gene, as occurs in Down syndrome (trisomy 21), with a 1.5-fold increase in expression, can cause dementia with the complete AD associated neuropathology. The most fully characterized form of AD is early onset familial AD (FAD). Unfortunately, by far the most common form of AD is late onset AD (LOAD). FAD has well-identified autosomally dominant genetic causes, absent in LOAD. It is reasonable to hypothesize that environmental influences play a much stronger role in etiology of LOAD than of FAD. Since AD pathology in LOAD closely resembles FAD with accumulation of both Aβ and MAPT, it is likely that the environmental factors foster accumulation of these proteins in a manner similar to FAD mutations. Therefore, it is important to identify environmentally driven changes that "phenocopy" FAD in order to find ways to prevent LOAD. Epigenetic changes in expression are complex but stable determinants of many complex traits. Some aspects are regulated by prenatal and early post-natal development, others punctuate specific periods of maturation, and still others occur throughout life, mediating predictable changes that take place during various developmental stages. Environmental agents such as mercury, lead, and pesticides can disrupt the natural epigenetic program and lead to developmental deficits, mental retardation, feminization, and other complex syndromes. In this review we discuss latent early- life associated regulation (LEARn), where apparently temporary changes, induced by environmental agents, become latent and present themselves again at maturity or senescence to increase production of Aβ that may cause AD. The model provides us with a novel direction for identifying potentially harmful agents that may induce neurodegeneration and dementia later in life and provides hope that we may be able to prevent age-related neurodegenerative disease by "detoxifying" our environment.

Diabetes, cardiovascular disease, hypertension, and other disorders have been unified within the metabolic syndrome. Recently, it has been proposed that Alzheimer's disease (AD) and other degenerative, age-related neurological disorders may also be etiologically linked to the metabolic syndrome in a metabolic-cognitive syndrome. We review current evidence in the field for this unification. In addition, we describe how the latent early-life associated regulation (LEARn) model provides specific mechanisms to predict genetic targets for both metabolic disorders, e.g., diabetes, and neurodegenerative disorders, e.g., AD. The LEARn model is based on environmental induction of latent epigenetic misregulation, which develops into disease upon suffering additional environmental insults. We review structural differences between gene sequences that are and are not susceptible to LEARn misregulation. In addition to suggesting research targets such as the IDE and SORCS1 genes, which are implicated in both AD and diabetes, LEARn suggests specific mechanisms for pre-disease remediation, based on nutritional adjustment of aberrant DNA methylation and oxidation. The possibility of a single metabolic-cognitive disorder opens up the possibility of unified preventative treatments that reduce monetary and social costs of disease. LEARn suggests specific, testable pathways within the large theory.

Amyloid-β peptide (Aβ) plaque in the brain is the primary (post mortem) diagnostic criterion of Alzheimer's disease (AD). The physiological role(s) of Aβ are poorly understood. We have previously determined an Aβ interacting domain (AβID) in the promoters of AD-associated genes (Maloney and Lahiri, 2011. Gene. 15,doi:10.1016/j.gene.2011.06.004. epub ahead of print.). This AβID interacts in a DNA sequence-specific manner with Aβ. We now demonstrate novel Aβ activity as a possible transcription factor. Herein, we detected Aβ-chromatin interaction in cell culture by ChIP assay. We observed that human neuroblastoma (SK-N-SH) cells treated with FITC conjugated Aβ1-40 localized Aβ to the nucleus in the presence of H2O2-mediated oxidative stress. Furthermore, primary rat fetal cerebrocortical cultures were transfected with APP and BACE1 promoter-luciferase fusions, and rat PC12 cultures were transfected with polymorphic APP promoter-CAT fusion clones. Transfected cells were treated with different Aβ peptides and/or H2O2. Aβ treatment of cell cultures produced a DNA sequence-specific response in cells transfected with polymorphic APP clones. Our results suggest the Aβ peptide may regulate its own production through feedback on its precursor protein and BACE1, leading to amyloidogenesis in AD., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Deposition of extracellular plaques, primarily consisting of amyloid β peptide (Aβ), in the brain is the confirmatory diagnostic of Alzheimer's disease (AD); however, the physiological and pathological role of Aβ is not fully understood. Herein, we demonstrate novel Aβ activity as a putative transcription factor upon AD-associated genes. We used oligomers from 5'-flanking regions of the apolipoprotein E (APOE), Aβ-precursor protein (APP) and β-amyloid site cleaving enzyme-1 (BACE1) genes for electrophoretic mobility shift assay (EMSA) with different fragments of the Aβ peptide. Our results suggest that Aβ bound to an Aβ-interacting domain (AβID) with a consensus of "KGGRKTGGGG". This peptide-DNA interaction was sequence specific, and mutation of the first "G" of the decamer's terminal "GGGG" eliminated peptide-DNA interaction. Furthermore, the cytotoxic Aβ25-35 fragment had greatest DNA affinity. Such specificity of binding suggests that the AβID is worth of further investigation as a site wherein the Aβ peptide may act as a transcription factor., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Noroviruses are members of the family Caliciviridae, and cause a highly communicable gastroenteritis in humans. We explored the potential to develop a plant-based vaccine against Narita 104 virus, a Genogroup II Norovirus. In stably transgenic potato, we obtained very poor expression of Narita 104 virus capsid protein (NaVCP) despite the use of a strong constitutive promoter (dual enhancer 35S) driving the native coding sequence. We identified potentially detrimental sequence motifs that could mediate aberrant mRNA processing via spurious polyadenylation signals. Northern blots and RT-PCR analysis of total RNA revealed truncated transcripts that suggested premature polyadenylation. Site-directed mutagenesis to remove one potential polyadenylation near-upstream element resulted in an increased expression of NaVCP when transiently expressed in leaves of Nicotiana benthamiana. Further, cloning of the truncated cDNAs from transgenic NaVCP potato plants and transiently transfected N. benthamiana allowed us to identify at least ten different truncated transcripts resulting from premature polyadenylation of full length NaVCP transcripts. Comparative studies using real time PCR analysis from cDNA samples revealed lower accumulation of full length transcripts of NaVCP as compared to those from a gene encoding Norwalk Virus capsid protein (a related Genogroup I Norovirus) in transiently transfected plants. These findings provide evidence for impaired expression of NaVCP in transgenic plants mediated by spurious polyadenylation signals, and demonstrate the need to scrupulously search for potential polyadenylation signals in order to improve transgene expression in plants.

Purpose: To describe the frequency, nature, trends, predictors, and outcomes of chemotherapy-related hospitalizations (CRHs) among a nonselected population of cancer patients treated at a community cancer center, and to explore the feasibility of implementing continuous quality improvement methodologies in routine oncology practice., Methods: We conducted a prospective cohort study of consecutive adult cancer patients who received chemotherapy at a community cancer center January 2003 to December 2006. Demographic, comorbidity, diagnosis, treatment, and laboratory data were collected via medical record abstraction. Hospitalizations were classified as chemotherapy related or unrelated by a multidisciplinary panel. Patients who experienced CRHs were compared with those who did not. Using a randomly sampled subset of cases and controls, we built a logistic regression model to identify independent predictors of CRH., Results: Of 2,068 chemotherapy recipients, 179 (8.7%) experienced 262 CRHs. Most hospitalizations were not chemotherapy related (73.7%). The mean monthly rate of CRH was 1.5%, the median length of stay was 5 days, the most common type of CRH was gastrointestinal (46.1%) followed by infectious (31.4%), and 0.9% of chemotherapy recipients had a fatal CRH. Significant predictors of CRH included having a comorbidity score of 3-4 versus 0 and having a higher creatinine level., Conclusions: Although the vast majority of chemotherapy recipients did not experience a CRH, these events were, unfortunately, not without serious consequences. Care should be taken when offering chemotherapy to patients with multiple comorbid conditions. Systematic efforts to monitor toxicity can lead directly to improvements in quality of care.

Alzheimer's disease (AD) currently has over 6 million victims in the USA, alone. The recently FDA approved drugs for AD only provide mild, transient relief for symptoms without addressing underlying mechanisms to a significant extent. Basic understanding of the activities of the amyloid beta peptide (Abeta) and associated proteins such as beta-site APP-cleaving enzyme 1 (BACE1) is necessary to develop effective medical responses to AD. Recently (Exper. Neurol. 2010. 221, 18-25), Tabaton et al. have presented a model of both non-pathological and pathological Abeta activities and suggest potential therapeutic pathways based on their proposed framework of Abeta acting as the signal that induces a kinase cascade, ultimately stimulating transcription factors that upregulate genes such as BACE1. We respond by presenting evidence of Abeta's other activities, including protection against metal-induced reactive oxidizing species (ROS), modification of cholesterol transport, and potential activity as a transcription factor in its own right. We touch upon clinical implications of each of these functions and highlight the currently unexplored implications of our suggested novel function of Abeta as a transcription factor. Abeta appears to be a highly multi-functional peptide, and any or all of the pathways it engages in is a likely candidate for antiAD drug development., (Copyright 2010 Elsevier Inc. All rights reserved.)

The neurodegenerative disorder Alzheimer's disease (AD) is the 6th leading cause of death in the USA. In addition to neurological and psychiatric symptoms, AD is characterized by deficiencies in S-adenylmethionine (SAM), vitamin B12, and folate. Deficiency in these nutrients has been shown to result in gene promoter methylation with upregulation of AD-associated genes. While some cases of AD are due to specific mutations in genes such as presenilin 1 (PSEN) and the amyloid-beta peptide precursor protein (APP), these familial AD (FAD) cases account for a minority of cases. The majority of genetic contribution consists of risk factors with incomplete penetrance. Several environmental risk factors, such as cholesterol and diet, head trauma, and reduced levels of exercise, have also been determined for AD. Nevertheless, the majority of risk for AD appears to be established early in life. We propose to explain this via the LEARn (Latent Early-life Associated Regulation) model. LEARn-AD (LAD) would be a "two-hit" disorder, wherein the first hit would occur due to environmental stress within the regulatory sequences of AD-associated genes, maintained by epigenetic changes such as in DNA methylation. This hit would most likely come in early childhood. The second hit could consist of further stress, such as head trauma, poor mid-life diet, or even general changes in expression of genes that occur later in life independent of any pathogenesis. Given that the primary risk for LAD would be maintained by DNA (hypo)methylation, we propose that long-term nutritional remediation based on the LEARn model, or LEARn-based nutritional gain (LEARnING), beginning early in life, would significantly reduce risk for AD late in life., (2010 Elsevier Inc. All rights reserved.)

Variations in levels of apolipoprotein E (ApoE) have been tied to the risk and progression of Alzheimer's disease (AD). Our group has previously compared and contrasted the promoters of the mouse and human ApoE gene (APOE) promoter sequences and found notable similarities and significant differences that suggest the importance of the APOE promoter's role in the human disease. We examine here three specific single-nucleotide polymorphisms within the human APOE promoter region, specifically at -491 (A/T), -427 (T/C), and at -219 (G/T) upstream from the +1 transcription start site. The -219 and -491 polymorphic variations have significant association with instance of AD, and -491AA has significant risk even when stratified for the APOEepsilon4 allele. We also show significant effects on reporter gene expression in neuronal cell cultures, and, notably, these effects are modified by species origin of the cells. The -491 and -219 polymorphisms may have an interactive effect in addition to any independent activity. DNA-protein interactions differ between each polymorphic state. We propose SP1 and GATA as candidates for regulatory control of the -491 and -219 polymorphic sites. This work's significance lies in drawing connection among APOE promoter polymorphisms' associations with AD to functional promoter activity differences and specific changes in DNA-protein interactions in cell culture-based assays. Taken together, these results suggest that APOE expression levels are a risk factor for AD irrespective of APOEepsilon4 allele status., ((c) 2009 Wiley-Liss, Inc.)

Obese patients in the ICU present unique challenges to the health care team and specific challenges to nurses. This article reviews the science and art of resource use for obese patients in the ICU. Staff nurses and advanced practice nurses can make important contributions in evaluating optimal resource use and improving outcomes in this population of vulnerable patients.

Background: The importance of genetic variation to the etiology of neuropsychiatric disorders is well established and is currently being examined for diagnosis and treatment. The most popular method of obtaining material for genotype analysis, high-yielding DNA extraction from blood, has several limitations, including invasiveness, need for skilled individuals to collect material, and requirement for cold storage. Saliva sampling is noninvasive and trained personnel are less necessary, but it still requires a relatively high level of subject compliance. Buccal mucosa cells sampling is almost completely noninvasive, reducing compliance issues significantly. Samples collected have been shown to produce usable DNA after shipment through conventional mail. The DNA produced by rapid elution of these swabs in chaotropic buffers is, however, of limited quality and low purity., Objective: Our aim was to develop a rapid, economical, and environmentally safe method for extraction of high-quality genomic DNA, which can be used to determine clinically important genotypes from trace quantity samples and which has sufficient yield for multiple assays., Methods: We developed a method of extracting high-quality genomic DNA from buccal swab, which we termed the 'rapid method for swab' (RMS). We compared RMS with two established procedures, specifically the original rapid method and the commercially available Buccal Amp method. We assessed the generated genomic DNAs by their (i) quality, (ii) quantity, (iii) restriction enzyme digestibility, and (iv) PCR-based genotyping in addition to time, cost, and environmental impact of the procedures., Main Results: DNA generated by RMS was of higher purity than that by Buccal Amp. RMS is nonenzymatic and does not use strong chaotropic salts or extreme pH. We also showed the suitability of RMS-DNA for LA/LG genotyping as generated by PCR using 7-deaza-dGTP., Conclusion: The RMS procedure is novel, efficient, safe, and yields sufficient material for multiple genotyping analyses. The RMS produces DNA of high quality from a single human buccal swab. RMS is a noninvasive technique and particularly suitable for children and older individuals and in field collection settings.

Alzheimer's disease (AD) is characterized by plaques of amyloid-beta (Abeta) peptide, cleaved from amyloid-beta protein precursor (AbetaPP). Our hypothesis is that lifespan profiles of AD-associated mRNA and protein levels in monkeys would differ from mice and that differential lifespan expression profiles would be useful to understand human AD pathogenesis. We compared profiles of AbetaPP mRNA, AbetaPP protein, and Abeta levels in rodents and primates. We also tracked a transcriptional regulator of the AbetaPP gene, specificity protein 1 (SP1), and the beta amyloid precursor cleaving enzyme (BACE1). In mice, AbetaPP and SP1 mRNA and their protein products were elevated late in life; Abeta levels declined in old age. In monkeys, SP1, AbetaPP, and BACE1 mRNA declined in old age, while protein products and Abeta levels rose. Proteolytic processing in both species did not match production of Abeta. In primates, AbetaPP and SP1 mRNA levels coordinate, but an inverse relationship exists with corresponding protein products as well as Abeta levels. Comparison of human DNA and mRNA sequences to monkey and mouse counterparts revealed structural features that may explain differences in transcriptional and translational processing. These findings are important for selecting appropriate models for AD and other age-related diseases.

Alzheimer's disease (AD), the most common form of dementia among the elderly, manifests mostly late in adult life. However, it is presently unclear when the disease process starts and how long the pathobiochemical processes take to develop. Our goal is to address the timing and nature of triggers that lead to AD. To explain the etiology of AD, we have recently proposed a "Latent Early-life Associated Regulation" (LEARn) model, which postulates a latent expression of specific genes triggered at the developmental stage. This model integrates both the neuropathological features (e.g., amyloid-loaded plaques and tau-laden tangles) and environmental factors (e.g., diet, metal exposure, and hormones) associated with the disease. Environmental agents perturb gene regulation in a long-term fashion, beginning at early developmental stages, but these perturbations do not have pathological results until significantly later in life. The LEARn model operates through the regulatory region (promoter) of the gene and by affecting the methylation status within the promoter of specific genes.

Background: Serotonin transporter promoter (5-HTTLPR) genotype appears to increase risk for depression in the context of stressful life events. However, the effects of this genotype on measures of stress sensitivity are poorly understood. Therefore, this study examined whether 5-HTTLPR genotype was associated with negative information processing biases in early childhood., Method: Thirty-nine unselected seven-year-old children completed a negative mood induction procedure and a Self-Referent Encoding Task designed to measure positive and negative schematic processing. Children were also genotyped for the 5-HTTLPR gene., Results: Children who were homozygous for the short allele of the 5-HTTLPR gene showed greater negative schematic processing following a negative mood prime than those with other genotypes. 5-HTTLPR genotype was not significantly associated with positive schematic processing., Limitations: The sample size for this study was small. We did not analyze more recently reported variants of the 5-HTTLPR long alleles., Conclusions: 5-HTTLPR genotype is associated with negative information processing styles following a negative mood prime in a non-clinical sample of young children. Such cognitive styles are thought to be activated in response to stressful life events, leading to depressive symptoms; thus, cognitive styles may index the "stress-sensitivity" conferred by this genotype.

Cyclin-dependent kinase 5 (cdk5) has been implicated in Alzheimer's disease (AD) pathogenesis. Here, we demonstrate that overexpression of p25, an activator of cdk5, led to increased levels of BACE1 mRNA and protein in vitro and in vivo. A p25/cdk5 responsive region containing multiple sites for signal transducer and activator of transcription (STAT1/3) was identified in the BACE1 promoter. STAT3 interacts with the BACE1 promoter, and p25-overexpressing mice had elevated levels of pSTAT3 and BACE1, whereas cdk5-deficient mice had reduced levels. Furthermore, mice with a targeted mutation in the STAT3 cdk5 responsive site had lower levels of BACE1. Increased BACE levels in p25 overexpressing mice correlated with enhanced amyloidogenic processing that could be reversed by a cdk5 inhibitor. These data demonstrate a pathway by which p25/cdk5 increases the amyloidogenic processing of APP through STAT3-mediated transcriptional control of BACE1 that could have implications for AD pathogenesis.

Alzheimer's disease is characterized by amyloid-beta peptide (Abeta)-loaded plaques in the brain. Abeta is a cleavage fragment of amyloid-beta protein precursor (APP) and over production of APP may lead to amyloidogenesis. The regulatory region of the APP gene contains consensus sites recognized by the transcription factor, specificity protein 1 (SP1), which has been shown to be required for the regulation of APP and Abeta. To understand the role of SP1 in APP biogenesis, herein we have characterized the relative distribution and localization of SP1, APP, and Abeta in various brain regions of rodent and primate models using immunohistochemistry. We observed that overall distribution and cellular localization of SP1, APP, and Abeta are similar and neuronal in origin. Their distribution is abundant in various layers of neocortex, but restricted to the Purkinje cell layer of the cerebellum, and the pyramidal cell layer of hippocampus. These findings suggest that overproduction of Abeta in vivo may be associated with transcriptional pathways involving SP1 and the APP gene.

The sporadic nature of Alzheimer's disease (AD) argues for an environmental link that may drive AD pathogenesis; however, the triggering factors and the period of their action are unknown. Recent studies in rodents have shown that exposure to lead (Pb) during brain development predetermined the expression and regulation of the amyloid precursor protein (APP) and its amyloidogenic beta-amyloid (Abeta) product in old age. Here, we report that the expression of AD-related genes [APP, BACE1 (beta-site APP cleaving enzyme 1)] as well as their transcriptional regulator (Sp1) were elevated in aged (23-year-old) monkeys exposed to Pb as infants. Furthermore, developmental exposure to Pb altered the levels, characteristics, and intracellular distribution of Abeta staining and amyloid plaques in the frontal association cortex. These latent effects were accompanied by a decrease in DNA methyltransferase activity and higher levels of oxidative damage to DNA, indicating that epigenetic imprinting in early life influenced the expression of AD-related genes and promoted DNA damage and pathogenesis. These data suggest that AD pathogenesis is influenced by early life exposures and argue for both an environmental trigger and a developmental origin of AD.

Apolipoprotein E (ApoE), encoded by the apolipoprotein E gene (APOE), plays an important role in the pathogenesis of Alzheimer's disease (AD). The APOE epsilon4 variant is strongly associated with AD. APOE promoter polymorphisms have also been reported to associate with higher AD risk. Mouse models of APOE expression have long been used to study the pathogenesis of AD. Elucidating the role of the APOE gene in AD requires understanding of how its regulation differs between mouse and human APOE genes, and how the differences influence AD risk. We compared the structure and function of both the human APOE gene promoter (hAPOEP) and mouse APOE gene promoter (mAPOEP) regions. Homology is less than 40% at 180 bp or more upstream of the two species' transcription start site (TSS, +1). Functional analysis revealed both similarities and important differences between the two sequences, significantly affected by human versus rodent cell line origin. We likewise probed nuclear extracts from several cell lines of different origins (astrocytic, glial, and neuronal) and mouse brain with specific hAPOEP and mAPOEP fragments. Each fragment shared DNA-protein interactions with the other but, notably, also bound distinct factors, demonstrated by gel shift and southwestern analyses. We determined possible identities for these distinct factors. These results suggest that regulation of mouse and human APOE genes may be sufficiently unique to justify the use of both the human APOE promoter sequence in transgenic rodent models and non-rodent AD models for studying factors involved in AD pathogenesis.

Objectives: Early-emerging, temperamental differences in fear-related traits may be a heritable vulnerability factor for anxiety disorders. Previous research indicates that the serotonin transporter promoter region polymorphism is a candidate gene for such traits., Methods: Associations between 5-HTTLPR genotype and indices of fearful child temperament, derived from maternal report and standardized laboratory observations, were examined in a community sample of 95 preschool-aged children., Results: Children with one or more long alleles of the 5-HTTLPR gene were rated as significantly more nervous during standardized laboratory tasks than children who were homozygous for the short alleles. Children homozygous for the short alleles were also rated as significantly shyer, by maternal report, than those with at least one copy of the long allele of the 5-HTTLPR gene., Conclusions: This study extends the literature linking the short alleles of the serotonin transporter promoter region polymorphism to fear and anxiety-related traits in early childhood and adulthood, and is one of very few studies to examine the molecular genetics of preschoolers' temperament using multiple measures of traits in a normative sample.

Alzheimer's disease (AD) is currently the most prominent form of dementia among the elderly. Although AD manifests in late adult life, it is not clear when the disease actually starts and how long the neuropathological processes take to develop AD. The major unresolved question is the timing and the nature of triggering leading to AD. Is it an early or developmental and/or late phenomenon and what are the factors that trigger the cascade of pathobiochemical processes? To explain the etiology of AD one should consider the neuropathological features, such as neuronal cell death, tau tangles, and amyloid plaque, and environmental factors associated with AD, such as diet, toxicological exposure, and hormonal factors. Current dominant theories of AD etiology are "protein-only", they attribute the cause of the disease directly to the activities of associated proteins once they have been produced; the major limitation is that protein aggregations occur "late in the game". Development and progression of AD has not been explained by protein-only models. In view of this limitation, we propose a "Latent Early-Life Associated Regulation" (LEARn) model, which postulates a latent expression of specific genes triggered at the developmental stage. According to this model, environmental agents (e.g., heavy metals), intrinsic factors (e.g., cytokines), and dietary factors (e.g., cholesterol) perturb gene regulation in a long-term fashion, beginning at early developmental stages; however, these perturbations do not have pathological results until significantly later in life. For example, such actions would perturb APP gene regulation at very early stage via its transcriptional machinery, leading to delayed overexpression of APP and subsequently of Abeta deposition. This model operates on the regulatory region (promoter) of the gene and by the effect of methylation at certain sites within the promoter of specific genes. Promoters tend to have both positive and negative regulatory elements, and promoter activity can be altered by changes in the primary DNA sequence and by epigenetic changes through mechanisms such as DNA methylation at CpG dinucleotides or oxidation of guanosine residues. The basis of the LEARn model is that environmental factors, including metals and dietary factors, operate by interfering the interaction of methylated CpG clusters with binding proteins, such as MeCP2 and SP1. The LEARn model may explain the etiology of AD and other neuropsychiatric and developmental disorders.

Major characteristics of Alzheimer's disease (AD) are synaptic loss, cholinergic dysfunction, and abnormal protein depositions in the brain. The amyloid beta-peptide (Abeta), a proteolytic fragment of amyloid beta precursor protein (APP), aggregates to form neuritic plaques and has a causative role in AD. A present focus of AD research is to develop safe Abeta-lowering drugs. A selective acetylcholinesterase inhibitor, phenserine, in current human trials lowers both APP and Abeta. Phenserine is dose-limited in animals by its cholinergic actions; its cholinergically inactive enantiomer, posiphen (+)-[phenserine], was assessed. In cultured human neuroblastoma cells, posiphen, like phenserine, dose- and time-dependently lowered APP and Abeta levels by reducing the APP synthesis rate. This action translated to an in vivo system. Posiphen administration to mice (7.5-75 mg/kg daily, 21 consecutive days) significantly decreased levels of total APP (tissue mass-adjusted) in a dose-dependent manner. Abeta40 and Abeta42 levels were significantly lowered by posiphen (> or =15 mg/kg) compared with controls. The activities of alpha-, beta-, and gamma-secretases were assessed in the same brain samples, and beta-secretase activity was significantly reduced. Posiphen, like phenserine, can lower Abeta via multiple mechanisms and represents an interesting drug candidate for AD treatment.

Major hallmarks of Alzheimer's disease (AD) include brain deposition of the amyloid-beta peptide (Abeta), which is proteolytically cleaved from a large Abeta precursor protein (APP) by beta and gamma- secretases. A transmembrane aspartyl protease, beta-APP cleaving enzyme (BACE1), has been recognized as the beta-secretase. We review the structure and function of the BACE1 protein, and of 4129 bp of the 5'-flanking region sequence of the BACE1 gene and its interaction with various transcription factors involved in cell signaling. The promoter region and 5'-untranslated region (UTR) contain multiple transcription factor binding sites, such as AP-1, CREB and MEF2. A 91 bp fragment is the shortest region with significant reporter gene activity and constitutes the minimal promoter element for BACE1. The BACE1 promoter contains six unique functional domains and three structural domains of increasing sequence complexity as the "ATG" start codon is approached. Notably, the BACE1 gene promoter contains basal regulatory elements, inducible features and sites for regulation by various important transcription factors. Herein, we also discuss and speculate how the interaction of these transcription factors with the BACE1 promoter can modulate synaptic plasticity, neuronal apoptosis and oxidative stress, which are pertinent to the pathogenesis and progression of AD.

Autism is characterized by restricted, repetitive behaviors and impairment in socialization and communication. Although no neuropathologic substrate underlying autism has been found, the findings of brain overgrowth via neuroimaging studies and increased levels of brain-derived neurotrophic factor (BDNF) in neuropathologic and blood studies favor an anabolic state. We examined acetylcholinesterase, plasma neuronal proteins, secreted beta-amyloid precursor protein (APP), and amyloid-beta 40 and amyloid-beta 42 peptides in children with and without autism. Children with severe autism and aggression expressed secreted beta-amyloid precursor protein at two or more times the levels of children without autism and up to four times more than children with mild autism. There was a trend for children with autism to show higher levels of secreted beta-amyloid precursor protein and nonamyloidogenic secreted beta-amyloid precursor protein and lower levels of amyloid-beta 40 compared with controls. This favors an increased alpha-secretase pathway in autism (anabolic), opposite to what is seen in Alzheimer disease. Additionally, a complex relationship between age, acetylcholinesterase, and plasma neuronal markers was found.

Oxidative damage to DNA has been associated with neurodegenerative diseases. Developmental exposure to lead (Pb) has been shown to elevate the Alzheimer's disease (AD) related beta-amyloid peptide (Abeta), which is known to generate reactive oxygen species in the aging brain. This study measures the lifetime cerebral 8-hydroxy-2'-deoxyguanosine (oxo8dG) levels and the activity of the DNA repair enzyme 8-oxoguanine DNA glycosylase (Ogg1) in rats developmentally exposed to Pb. Oxo8dG was transiently modulated early in life (Postnatal day 5), but was later elevated 20 months after exposure to Pb had ceased, while Ogg1 activity was not altered. Furthermore, an age-dependent loss in the inverse correlation between Ogg1 activity and oxo8dG accumulation was observed. The effect of Pb on oxo8dG levels did not occur if animals were exposed to Pb in old age. These increases in DNA damage occurred in the absence of any Pb-induced changes in copper/zinc-superoxide dismutase (SOD1), manganese-SOD (SOD2), and reduced-form glutathion (GSH). These data suggest that oxidative damage and neurodegeneration in the aging brain could be impacted by the developmental disturbances.

We describe a patient with Wegener's granulomatosis (WG) who developed neurological symptoms attributed to meningeal involvement. The diagnosis of WG was complicated by persistently negative antineutrophil cytoplasmic antibodies (ANCA) and lack of specificity in the histopathological findings from multiple anatomical sites. This rare neurological manifestation of WG was treated successfully with oral cyclophosphamide and the patient has continued remission for 3 years taking oral methotrexate.

The main characteristic of Alzheimer's disease (AD) is brain deposition of the beta-amyloid (Abeta) peptide, generated endoproteolytically from Abeta precursor protein (APP) by beta- and gamma-secretases. A transmembrane aspartyl protease, beta-APP-cleaving enzyme (BACE1), was identified as beta-secretase. Although BACE1 cleaves APP at the beta-secretase site, the role of its homolog, beta-secretase 2 (BACE2) is poorly understood. We report the mRNA expression profile, DNA sequence, and molecular characterization of the BACE2 gene, located on chromosome 21q22.3. The BACE2 gene expresses more strongly in peripheral tissues, although BACE2 mRNA is found in the majority of brain regions, including the postcentral gyrus and temporal lobe. Characterization of 2932 bp of the BACE2 5'-flanking region (GC content of 55%), reveals the absence of canonical CCAAT and TATA boxes within 1 kb of the transcription start site (TSS). The sequence lacks significant internal repeats and has a housekeeping gene structure. Two active regions of the BACE2 promoter determine its basal expression and cell-type specificity. The proximal region (-31/+238) likely determines general basal expression, and the distal region (-2618/-1513), cell-type specificity. Several putative transcription factor sites, particularly SP1, Oct-1, and HES-1, are predicted to be within 1 kb of the TSS. On either side of the proximal promoter region, two negative regulatory domains might reduce BACE2 expression under an induced condition. The BACE2 5'-flanking region is likely to be highly regulated and expressed in a tissue type-specific manner.

The beta-amyloid (Abeta) protein present in the neuritic plaques of Alzheimer's disease is cleaved from Abeta precursor protein (APP) by beta- and gamma-secretases. Following identification of beta-APP cleaving enzyme (BACE1) as the beta-secretase, a homologous beta-secretase 2 (BACE2) was described. Our goal is to characterize the regulatory region of the BACE genes. We compare functional domains within the BACE1 and BACE2 regulatory regions. Both BACE genes lack canonical TATAand CAAT boxes, but they contain distinguishing transcription start sites and transcription factor-binding sites. The BACE1 sequence contains more repetitive elements than does BACE2 (no elements). Regulatory domains do not overlap strongly between the two promoter regions. The BACE1upstream sequence contains both negative and positive domains, separated from the transcription seat by a long neutral domain. The corresponding BACE2sequence consists of a weakly positive domain directly upstream of a strongly positive domain, near a functionally active domain. DNA-protein interaction was corroborated by functional data. In primary rat cortical cultures, BACE1-driven reporter protein's expression was twice that of BACE2- driven reporter. The BACE2 gene promoter relatively reduced function in neuronal cells compared with BACE1. The BACE1 gene might operate through a single transcriptional control site. BACE2 operates through dual transcriptional control sites. Two (or more) regulatory pathways might control transcription in BACE2. Thus, BACE2 is partially suppressed in normal neuronal cells and likely to be a highly regulated gene expressed in a particularly tissue-specific fashion.

The amyloid-beta (Abeta) peptide, the proteolytic fragment of Abeta precursor protein (APP), aggregates and forms neuritic plaques, a major hallmark of Alzheimer's disease (AD). The limiting step in generating the Abeta peptide from APP is cleavage by the beta-secretase enzyme, BACE1. Regulation of the BACE1 gene is likely to play an important role in AD etiology and treatment. We therefore studied the activity of a 4.1-kb 5'-flanking region (-3765/+364, +1 being the transcription start site) of the BACE1 gene, both in 5'- and 3'-deletion series and through Northern blotting. We show that the BACE1 promoter has regulatory activity throughout the 4.1-kb length, both positive and negative, and that this activity can be quantitatively modeled according to promoter sequence length, with the specific model depending on the presence of negative regulatory elements as the 5'- most portion of the sequence. We also examined a previously identified 141-bp proximal fragment (+224/+364) of the BACE1 promoter and two constituent (91- and 50-bp) subfragments. We report that the 91-bp fragment (+224/+314) is the most likely seat of neuronal expression of the BACE1 gene and that it is the portion of the 141-bp fragment that accounts for observed DNA-protein interactions in brain extracts. The 50-bp fragment (+315/+364), which showed significant reporter gene activity from the empty vector, binds nuclear proteins in a cell type-specific manner and contains the AP2 site as shown by the electrophoretic mobility shift assay. Overall, the 141-bp fragment had no strong matches within GenBank, and the 91-bp fragment is predicted to have several potential stem-loop sites. Taken together, BACE1 gene promoter activity is differentially regulated, and the 91-bp fragment represents a novel promoter region for cell type-specific regulation. This fragment might be a useful target to regulate BACE1 expression leading to Abeta production and to understand the neuropathogenesis of AD.

Alzheimer's disease (AD) is characterized by formation of plaques of amyloid beta peptide (Abeta). Autosomally-inherited or "familial" AD had been demonstrated only in connection with coding sequence mutations. We characterized DNA-protein interaction and expression influence of two polymorphisms that occur in the promoter (C<-->T at -3829 and T<-->C at -1023, +1 transcription start site) of the Abeta precursor protein (APP) gene. We report distinct functional differences in reporter expression and in DNA-protein interaction for variant sequences in both -3829 and -1023 polymorphic regions. The -3829T variant has reduced DNA-protein interaction and reporter expression compared to -3829C, while -1023C has greater DNA-protein interaction and reporter expression than -1023T. Our predictions for likely transcription factors for loss of function (-3829T) are ADR1, MIG1, and PuF, and for gain of function (-1023C) are E12/E47, ITF-2, and RFX2. Characterization of the activity of a regulatory polymorphism of the APP gene points towards understanding mechanisms that likely underlie the majority of AD cases and may contribute to promoter-based drug design.

The apolipoprotein E gene (APOE) plays an important role in the pathogenesis of Alzheimer's disease (AD), and amyloid plaque comprised mostly of the amyloid-beta peptide (A(beta)) is one of the major hallmarks of AD. However, the relationship between these two important molecules is poorly understood. We examined how A(beta) treatment affects APOE expression in cultured cells and tested the role of the transcription factor NF-(kappa)B in APOE gene regulation. To delineate NF-(kappa)B's role, we have characterized a 1098 nucleotide (nt) segment containing the 5'-flanking region of the human APOE gene (-1054/+44, +1 transcription start site). Sequence analysis of this region suggests the presence of two potential NF-(kappa)B elements. To demonstrate promoter activity, the region was cloned upstream of a promoterless luciferase (reporter) gene. This segment was able to drive expression of luciferase in transient transfections of human fetal glial cells. Promoter activity was stimulated twofold by A(beta)(1-40) (25 microM, 24 h) treatment. Pretreatment with double-stranded DNA decoy oligonucleotides against NF-(kappa)B (2 microM) reduced A(beta) stimulation. Deletion and mutagenetic analyses demonstrated that the distal NF-(kappa)B element was functional and showed a strong DNA-protein complex band in gel shift analysis, similar to that from control NF-(kappa)B consensus element. An anti-inflammatory and anti-NF-(kappa)B drug, sodium salicylate, significantly blocked A(beta)-induced APOE promoter function. Our data provide evidence that upregulation of APOE by A(beta) in astroglial cells is mediated by an NF-(kappa)B-element present in the 5'-flanking region of the APOE gene.

Alzheimer's disease is characterized by brain deposition of toxic amyloid beta-peptide (Abeta), generated from the Abeta precursor protein (APP). APP gene expression is regulated via the proximal promoter region (PPR; -46/-1 in the human sequence; +1 transcription start) and the 5'-untranslated region (5'-UTR; +1/+147). We have recently identified a unique CAGA sequence, "amyloid" (+83/+86) present only in the APP gene from amyloid plaque-forming species, absent in all APP-like-proteins' (APLP1 and APLP2) genes. To assay functional activity of PPR + UTR and 5'-UTR regions that either contain or lack the "amyloid" box, we tested nine constructs in transient transfection studies. We observed significantly high reporter gene activity with -46/144, -46/100, -46/54, and 54/144 constructs. The 54/100 fragment, which contains a transforming growth factor-beta/ "amyloid"/interleukin-1 acute box cassette, showed different activity depending on cell type. Electrophoretic mobility shift assay (EMSA) showed distinct DNA-nuclear protein interaction in all fragments, differing among both cell types and specific fragment. Reporter gene expression corroborates with the DNA-protein binding pattern. To directly examine the "amyloid" box, we generated oligomers for CAGA mutants or mutated adjacent nucleotides. EMSA results showed that altering "amyloid" or adjacent sequence alters specific DNA-nuclear protein interaction in both mutation- and cell-type-specific manners. Reporter gene assay reveals mutant-specific expression effects. Therefore, the -46/54 region appears to be essential for basal expression of the APP gene, the 54/100 and 100/144 regions may have tissue-specific activity, and the "amyloid" CAGA box plays a role in APP gene regulation.

The amyloid beta-protein (Abeta) deposited in brains of Alzheimer's disease (AD) patients is proteolytically derived from a large Abeta precursor protein (APP). APP gene expression patterns in the AD brain region indicate that abnormalities of gene regulation may be important in AD pathology. To understand the contribution of different cell types to APP gene expression, we studied it at four levels: promoter activity (by reporter gene assay of transfected cells), DNA-nuclear protein interaction (by electrophoretic mobility shift assay), RNA message and protein (by northern and western blotting, respectively). APP mRNA and protein expression levels were greater in neuroblastoma and PC12 cells than in glial or cervix epithelial cells. Relative activity among 12 different promoter regions and within single regions varied according to cell type/cell line. An upstream regulatory region containing a GATA-1 site is necessary for activity in PC12 and glial cells but not in neuroblastoma cells. DNA-protein interactions were examined in three distal and one proximal promoter elements in nuclear extracts belonging to neuronal and non-neuronal cells. The proximal promoter region is important for cell line-specific APP gene expression. Characterization of the APP regulatory region's interaction with cell type-specific nuclear factor(s) is important to understand tissue-specific expression of APP seen in AD subjects.

Potentially toxic amyloid beta-peptide (Abeta) in Alzheimer's disease (AD) is generated from a family of Abeta-containing precursor proteins (APP), which is regulated via the 5'-untranslated region (5'-UTR) of its mRNA. We analyzed 5'-UTRs of the APP superfamily, including amyloid plaque-forming and non-amyloid plaque-forming species, and of prions (27 different DNA sequences). A "CAGA" sequence proximal to the "ATG" start codon was present in a location unique to APP genes of amyloid plaque-forming species and absent in all other genes surveyed. This CAGA box is immediately upstream of an interleukin-1-responsive element (acute box). In addition, the proximal CAGA box is predicted to appear on a stem-loop structure in both human and guinea pig APP mRNA. This stem-loop is part of a predicted bulge-loop that encompasses a known iron regulatory element (IRE). Electrophoretic mobility shift with segments of the APP 5'-UTR showed that a region with the proximal CAGA sequence binds nuclear proteins, and this UTR fragment is active in a reporter gene functional assay. Thus, the 5'-UTR in the human APP but not those of APP-like proteins contains a specific region that may participate in APP regulation and may determine a more general model for amyloid generation as seen in AD. The 5'-UTR of human APP contains several interesting control elements, such as an acute box element, a CAGA box, an IRE, and a transforming growth factor-beta-responsive element, that could control APP expression and provide suitable and specific drug targets for AD.

The first step in the generation of the amyloid-beta peptide (Abeta) deposited in the brains of patients with Alzheimer's disease (AD) is the processing of the larger Abeta precursor protein (APP) by an integral membrane aspartyl protease named the beta-site APP-cleaving enzyme (BACE). We present the genomic organization of the BACE gene. BACE mRNAs are synthesized as nine exons and eight introns from a 30.6 kb region of chromosome 11q23.2-11q23.3. Regulation of BACE may play an important role in regulating the levels of Abeta produced and is therefore likely to play an important role in AD. Herein, we report the cloning and detailed analysis of 3765 nucleotides of the promoter region of BACE and 364 nucleotides of the 5' untranslated region of the BACE mRNA (5' UTR). Characteristic "CAAT" and "TATA" boxes are absent within 1.5 kb of the transcription start site (TSS). The promoter region and 5' UTR contain multiple transcription factor binding sites, such as activator protein (AP)1, AP2, cAMP response element binding protein (CREB), estrogen responsive element (ERE), glucocorticoid responsive element (GRE), "GC" box, nuclear factor (NF)-kappaB, signal transducer and activator of transcription (STAT)1, stimulating protein (SP)1, metal-regulatory elements, and possible Zeste binding sites. Limited interspecies similarity was observed between the human sequence and corresponding genomic DNA from the rat and mouse sequences, but several transcription factor-binding sites are conserved. Thus, the BACE gene contains basal regulatory elements, inducible features and sites for regulated activity by various transcription factors. These results identify the important regions for functional analysis of the binding domains and neuron-specific expression (1). Such a study will allow us to further examine the possible role of changes in the promoter of BACE in AD pathogenesis.

Pathological characteristics of Alzheimer's disease (AD) include amyloid-beta (Abeta) plaques. Abeta is derived from the Abeta peptide precursor protein (APP) by gamma- and beta-secretases, the latter known as beta-site APP-cleaving enzyme 1 (BACE1, or herein BACE). We have also described potentially important regions in the promoter of BACE, which may regulate its activity (1). Herein, we have functionally dissected the regulatory regions within the BACE promoter into areas containing positive and negative regulatory elements. The 4.1 kb promoter region (-3765/+364, +1 being the transcription start site [TSS]) includes positive regulatory element in the -2975 to -2062 region flanked on either side by negative regulatory elements at -3764/-2975 and -2062/-1056. This is separated from the minimal promoter and 5' UTR by a neutral region of roughly 700 base pairs (bp). A 91 bp fragment (224/314) is the shortest region with a significant reporter gene activity and constitutes the minimal promoter element for BACE. Neuronal preference of the promoter was apparent in the 141 bp fragment (224/364) that contained the 91 bp fragment. Gel shift results also suggest the strongest signal of DNA-protein interaction with the 91 bp fragment in neuronal nuclear extracts. This interaction was strongly blocked by an activator protein (AP)2 binding oligomer. Super gel shift assays suggest that both the AP2-binding oligomer and the 91 bp fragment interfered with each other's binding capacity. An AP2 sequence is predicted to occur within the 91 bp fragment. We also found stimulating protein (SP)1-binding sites in this region of the promoter. Thus, functional and gel shift analysis indicate that the 91 bp fragment containing the AP2 and SP1 binding sites constitutes the core promoter region of BACE. Changes in the activity of this region could play an important role in regulating BACE activity in neurons.

Alzheimer's disease is characterized by the accumulation of amyloid-beta peptide, which is cleaved from the amyloid-beta precursor protein (APP). Reduction in levels of the potentially toxic amyloid-beta has emerged as one of the most important therapeutic goals in Alzheimer's disease. Key targets for this goal are factors that affect the regulation of the APP gene. Recent in vivo and in vitro studies have illustrated the importance of copper in Alzheimer's disease neuropathogenesis and suggested a role for APP and amyloid-beta in copper homeostasis. We hypothesized that metals and in particular copper might alter APP gene expression. To test the hypothesis, we utilized human fibroblasts overexpressing the Menkes protein (MNK), a major mammalian copper efflux protein. MNK deletion fibroblasts have high intracellular copper, whereas MNK overexpressing fibroblasts have severely depleted intracellular copper. We demonstrate that copper depletion significantly reduced APP protein levels and down-regulated APP gene expression. Furthermore, APP promoter deletion constructs identified the copper-regulatory region between -490 and +104 of the APP gene promoter in both basal MNK overexpressing cells and in copper-chelated MNK deletion cells. Overall these data support the hypothesis that copper can regulate APP expression and further support a role for APP to function in copper homeostasis. Copper-regulated APP expression may also provide a potential therapeutic target in Alzheimer's disease.

Ptosis of the midfacial tissues with resultant deepening of the melolabial folds, vertical lengthening of the lower eyelid, and depression of the oral commissure are generally only slightly improved with traditional superficial musculoaponeurotic system (SMAS) suspension or rhytidectomy techniques. Subperiosteal, deep plane, and composite rhytidectomies have evolved in an attempt to rejuvenate these areas. This article reviews a series of patients who underwent an endoscopic subperiosteal face-lift either as an isolated procedure or in conjunction with an SMAS rhytidectomy. Although all patients showed good initial improvement, patients with thin faces and well-defined facial bone structure maintained the best long-term result. Complications were minimal, with no permanent facial nerve injuries observed. The endoscopic subperiosteal face-lift is a useful technique as an isolated procedure or in conjunction with facial liposuction or SMAS suspension rhytidectomy. Arch Facial Plast Surg. 2000;2:274-278

Background: As the number and methods of skin resurfacing procedures are increasing, there is a small number of patients that develop a prolonged inflammation during the postoperative period., Objective: We attempted to correlate risk factors for the development of prolonged postpeel erythema (PPPE) and inflammation. A treatment regimen will be described to eliminate permanent skin changes., Methods: A retrospective chart review is presented to define and correlate risk factors for the development of PPPE and a treatment protocol is described. The setting is a large multisurgeon aesthetic center. Two-hundred and thirty-six consecutive chemical phenol peels on 196 patients over a 2-year period were reviewed., Results: Eleven percent of patients developed PPPE. Allergy to tape was the only factor significantly correlated with PPPE. All patients had complete resolution of skin changes with appropriate treatment., Conclusion: A small population of patients undergoing skin resurfacing procedures will develop prolonged erythema. No major risk factor could be correlated with its development. A treatment plan was devised and was successful in all cases.

Lips are the central feature in the lower third of the face. When they are full and well defined, they impart a sense of youth, health, and attractiveness to the bearer. Thin, flat lips, on the other hand, imply fragility and senility. The characteristics of the lips responsible for these qualities are the shape of Cupid's bow, the relative length of the upper lip, and the projection or bulk of the lips. Esthetic guidelines are presented for each of these characteristics, which when understood help the surgeon formulate an operative plan. Lip augmentation techniques using autogenous and alloplastic materials are presented. For patients with a long upper lip, vermilion advancement and nasal base resection are discussed in detail. With a look to the future, a discussion of preliminary experience with carbon dioxide laser lip advancement concludes the article.

Although a wide range of peeling agents are available, few can provide the dramatic improvement that is seen with phenol-based peeling. The nonoccluded phenol-based technique described in this article has been used successfully for over 20 years. Due to the deeper degree of injury and more involved recovery period, the surgeon will be best served by not only reading about this technique, but also observing an experienced surgeon.

Avian infectious laryngotracheitis virus (ILTV), a herpesvirus, is a highly contagious pathogen that causes an upper respiratory tract infection in chickens. It is one of the major problems in the poultry industry worldwide. Current vaccines are not satisfactory due to the induction of latent infection. Here we describe a system for the construction of recombinant ILTV. A 4-kbp ILTV EcoRI DNA fragment was cloned into plasmid pUC13 and sequenced. Computer prediction revealed two potential open reading frames with 216 and 259 amino acid residues, respectively. The 259-residue polypeptide was serine-rich. The beta-galactosidase (beta-gal) gene of E. coli was cloned into the XhoI/Bg/II site of this DNA fragment, integrated into the ILTV genome via homologous recombination, expressed under the control of the immediate-early cytomegalovirus promoter, and caused the formation of blue plaques in the presence of X-gal. The insertion of a foreign gene into the ILTV genome and the successful expression of the incorporated gene demonstrated the potential for the construction of attenuated recombinant ILTV vaccines and the development of ILTV as vectors for polyvalent vaccines against avian upper respiratory tract infections.

Infectious laryngotracheitis virus (ILTV) is the causative agent of a highly contagious upper respiratory tract infection in chickens. At present, ILTV vaccines are not satisfactory because of development of a latent carrier status in vaccinated birds. Development of recombinant virus vaccines has been hampered by the limited information available on the molecular level and organization of this virus. We isolated 3 assembly intermediates, designated A, B, and C from ILTV-infected cells. Analysis of [3H]thymidine-and [35S]methionine-labeled particles, and electron microscopic studies indicated that particle A was the empty capsid, particle B was the procapsid containing scaffolding protein, and particle C was the DNA-filled capsid. The ILTV procapsids could only be found in the nucleus, which indicated that procapsids could not translocate through the nuclear membrane until they packaged the DNA. The DNA-filled capsids migrated through the nuclear membrane and obtained an envelope from the inner membrane of the nucleus. The enveloped particles then migrated through the lumen of the endoplasmic reticulum into vacuoles in the cytoplasm. Infective virions were isolated from within the infected cells, indicating that budding through the cytoplasmic membrane is not a necessary step in ILTV maturation. Abundant arrays composed of tubules about 45 to 50 nm wide were found in the cytoplasm of chicken embryonic liver cells about 30 to 38 hours after infection. Comparison of the assembly intermediates and the DNA packaging pathway of ILTV with that of bacteriophage pi 29 indicates that similarity exists.(ABSTRACT TRUNCATED AT 250 WORDS)