E4 Gene Research Articles

482. Suppression of the Leaky Expression of Adenovirus Genes Following Transduction with a Replication-Incompetent Adenovirus Vector by Incorporating microRNA-Targeted Sequences into the 3'-Untranslated Region of the E2A, E4, or pIX Gene

482. Suppression of the Leaky Expression of Adenovirus Genes Following Transduction with a Replication-Incompetent Adenovirus Vector by Incorporating microRNA-Targeted Sequences into the 3'-Untranslated Region of the E2A, E4, or pIX Gene

Detection of the human papillomavirus 58 physical state using the amplification of papillomavirus oncogene transcripts assay

HPV 58 is detected commonly in cervical cancer in East Asian countries. To evaluate the HPV 58 physical state, the amplification of papillomavirus oncogene transcripts (APOT) and hybridisation assays were established. Episome- and integrate-derived transcripts were confirmed by direct sequencing. Twenty-nine HPV 58 positive samples from various cervical lesions were used. The results showed that the episome-derived transcripts were recognised as two major specific amplified products (1040 and 714bp). Two splice donor sites were mapped to the 5′ splice site of the E1 gene on SD898 and SD899 and spliced to the 3′ acceptor site of the E4 gene on SA3353, SA3356 and SA3365. The episome-derived transcripts were found 100% in normal cervical epithelia and low-grade lesions (9/9 cases) while the integrate-derived transcripts were detected in 13.3% of high-grade lesions (2/15 cases) and in 20% of carcinomas (1/5 cases). HPV 58 integration sites were found on chromosomes 4q21, 12q24 and 18q12. Using the established APOT assay, the results revealed not only novel information on the HPV 58 transcription patterns of episomal transcripts, but also integration site. The APOT assay is a reliable and useful tool for the detection of the HPV 58 physical state and its oncogene expression.

Genetic Variation in Soybean at the Maturity Locus E4 Is Involved in Adaptation to Long Days at High Latitudes

Soybean (Glycine max) cultivars adapted to high latitudes have a weakened or absent sensitivity to photoperiod. The purposes of this study were to determine the molecular basis for photoperiod insensitivity in various soybean accessions, focusing on the sequence diversity of the E4 (GmphyA2) gene, which encodes a phytochrome A (phyA) protein, and its homoeolog (GmphyA1), and to disclose the evolutionary consequences of two phyA homoeologs after gene duplication. We detected four new single-base deletions in the exons of E4, all of which result in prematurely truncated proteins. A survey of 191 cultivated accessions sourced from various regions of East Asia with allele-specific molecular markers reliably determined that the accessions with dysfunctional alleles were limited to small geographical regions, suggesting the alleles’ recent and independent origins from functional E4 alleles. Comparison of nucleotide diversity values revealed lower nucleotide diversity at non-synonymous sites in GmphyA1 than in E4, although both have accumulated mutations at almost the same rate in synonymous and non-coding regions. Natural mutations have repeatedly generated loss-of-function alleles at the E4 locus, and these have accumulated in local populations. The E4 locus is a key player in the adaptation of soybean to high-latitude environments under diverse cropping systems.

Adenoviral E4 Gene Stimulates Secretion of Pigmental Epithelium Derived Factor (PEDF) that Maintains Long-term Survival of Human Glomerulus-derived Endothelial Cells

Renal glomerular endothelial cells are specialized cells with an important role in physiological filtration and glomerular disease. However, maintenance of human primary endothelial cells requires stimulation with serum and growth factors that often results in modification of the cells properties. Previously, expression of early adenovirus region E4 was shown to help maintaining long-term survival of human endothelial cells in serum free media without addition of growth factors. In the current study, we showed that media conditioned with human epithelial cells stably transfected with Ad E4 region also supported survival of human glomerulus-derived endothelial cells in serum-free media. Mass-spectrometry analysis of the conditioned media identified pigmental epithelium derived factor (PEDF) as a major component of the conditioned media. PEDF expression in 293-E4 cells was validated by RT-PCR, Western blot and ELISA analysis. PEDF expression was detected in mouse glomeruli. Supplementation with recombinant PEDF supported survival of primary endothelial cells and the cells transformed with SV40 large T antigen in serum-free media, and extended the life-span of both cell cultures. PEDF did not inhibit FGF-2 stimulated growth and tubulogenesis of endothelial cells. Thus we demonstrated that adenoviral E4 region stimulated expression and secretion of PEDF by human renal epithelial cells that acted as a survival factor for glomerulus-derived endothelial cells.

Molecular and phylogenetic analysis of the HPV 16 E4 gene in cervical lesions from women in Greece

The HPV16 E1(∧)E4 protein is thought to contribute to the release of newly formed viral particles from infected epithelia. In order to investigate amino acid mutations in the HPV16 E1(∧)E4 protein, the complete E4 ORF was amplified by PCR in 27 HPV16-positive cervical samples, and the amplicons were cloned. Fifteen nucleic acid variations were identified in the E4 ORF, including seven silent nucleic acid mutations. In addition, nine amino acid mutations (A7V, A7P, L16I, D45E, L59I, L59T, Q66P, S72F, H75Q) were detected in the E1(∧)E4 protein, and these were associated with the severity of cervical malignancy. A maximum-likelihood phylogenetic tree was constructed based on the E4 ORF, and nucleotide sequence analysis of the E4, E6 and E7 genes from the same samples was conducted in order to determine the phylogenetic origin of the cloned sequences from the amplified HPV16 E4. Based on the nucleotide sequence and phylogenetic analysis it was revealed that even though E4 ORF constitutes a small polymorphic portion of the viral genome (288 bp), it could provide valuable information about the origins of the HPV16 genome. In addition, molecular evolutionary analysis of the E4 coding region revealed that neutral selection is dominant in the overlapping region of the E4 and E2 ORFs.

381. Development of a Novel Adenovirus Vector Carrying microRNA-Targeted Sequences in the 3'-Untranslated Region of the pIX, E2A, or E4 Gene for Suppression of the Leaky Expression of Adenovirus Genes

381. Development of a Novel Adenovirus Vector Carrying microRNA-Targeted Sequences in the 3'-Untranslated Region of the pIX, E2A, or E4 Gene for Suppression of the Leaky Expression of Adenovirus Genes

Characterization and functional analysis of methionine sulfoxide reductase A gene family in tomato

In this study, four methionine sulfoxide reductase A (MSRA) genes, SlMSRA2-5, were identified in Micro-Tom (Solanum lycopersicum cv.'Micro-Tom') based on the tomato database. Exon/intron distribution assays indicated that the gene sequence of SlMSRA2, SlMSRA3, and SlMSRA4 contained two exons and one intron in the tomato genome, while the coding sequence of SlMSRA5 was interrupted by three introns. Southern blot analysis revealed that the members of the MSRA family existed as low copies in the tomato genome. Although SlMSRA2 and E4 gene (one of ethylene-inducible gene) share a similarity of 93.4% in terms of their amino acid sequence, organ-specific expression revealed that gene expression were remarkably different based on Northern blot analysis. In addition, SlMSRA3 and SlMSRA4 transcripts were abundant in leaves while expression of SlMSRA5 was weak in most of organs examined. The expression pattern of SlMSRAs induced in response to hormones and stresses suggested that the tomato MSRA genes might be involved in resistance against pathogens and various abiotic stresses. In addition, kinetics parameters suggested that recombinant SlMSRA4 protein exhibited a highest affinity for dabsyl-labeled methionine sulfoxide and its catalytic efficiency (K (cat) /K (m) ) was threefold and 6.5-fold higher than that of SlMSRA2 and SlMSRA3. However, the activity of purified SlMSRA5 was not detected in the DTT-dependent reducing system.

Adenovirus E4orf3 Targets Transcriptional Intermediary Factor 1γ for Proteasome-Dependent Degradation during Infection

The ability of adenovirus early region proteins, E1B-55K and E4orf6, to usurp control of cellular ubiquitin ligases and target proteins for proteasome-dependent degradation during infection is well established. Here we show that the E4 gene product, E4orf3 can, independently of E1B-55K and E4orf6, target the transcriptional corepressor transcriptional intermediary factor 1γ (TIF1γ) for proteasome-mediated degradation during infection. Initial mass spectrometric studies identified TIF1 family members-TIF1α, TIF1β, and TIF1γ-as E1B-55K-binding proteins in both transformed and infected cells, but analyses revealed that, akin to TIF1α, TIF1γ is reorganized in an E4orf3-dependent manner to promyelocytic leukemia protein-containing nuclear tracks during infection. The use of a number of different adenovirus early region mutants identified the specific and sole requirement for E4orf3 in mediating TIF1γ degradation. Further analyses revealed that TIF1γ is targeted for degradation by a number of divergent human adenoviruses, suggesting that the ability of E4orf3 to regulate TIF1γ expression is evolutionarily conserved. We also determined that E4orf3 does not utilize the Cullin-based ubiquitin ligases, CRL2 and CRL5, or the TIF1α ubiquitin ligase in order to promote TIF1γ degradation. Further studies suggested that TIF1γ possesses antiviral activity and limits adenovirus early and late gene product expression during infection. Indeed, TIF1γ knockdown accelerates the adenovirus-mediated degradation of MRE11, while TIF1γ overexpression delays the adenovirus-mediated degradation of MRE11. Taken together, these studies have identified novel adenovirus targets and have established a new role for the E4orf3 protein during infection.

A comprehensive strategy for dementia from primary prevention to end‐stage management

A comprehensive strategy for dementia from primary prevention to end‐stage management

Shape changes in the CA3 and dentate regions of the hippocampus in individuals with mild cognitive impairment

Background: Shape and volume changes in the subfields in the hippocampus have been reported in MCI patients and have been shown to predict whether or not patients decline. Previous studies that reported shape changes in the CA1 region of the hippocampus did not segment the individual subfields of the hippocampus, thus these deflections could indeed be due to shape changes in the deeper subfields. More recent reports suggest that there may be shape and volume changes in the CA3 and dentate gyrus (DG) regions of the hippocampus that may differentiate healthy adults from those early in the course of AD or in some cases in asymptomatic carriers of the ApoE e4 gene. We sought to investigate the detailed profile of volume and shape changes in MCI patients (all CDR 0.5) using the OASIS (Open Access Series of Imaging Studies) freely available data set. Methods: We used an alternative approach to previous studies by combining traditional grayscale data with manual segmentations of the subfields. In this technique, we manually segmented hippocampal subfields on each individual scan and used a region-of-interest based diffeomorphic alignment technique (ROI-ANTS) that we recently developed in order to quantify each subfield’s shape changes. The method uses information from grayscale images but weights the transformations based on ROI segmentations, resulting in optimum cross-participant overlap. Results: We found significant shape changes in the deeper subfields (CA3/DG) in MCI patients compared to age and gender matched controls, suggesting that this is one of the first hippocampal regions that deteriorates in the course of AD and that previously studies likely underestimated the extent to which it was involved due to an inherent bias in the alignment techniques. Conclusions: Using a robust diffeomorphic technique and precise anatomical segmentations, we find evidence for shape changes in the CA3/DG that may precede other hippocampal changes very early in the course of AD. This is consistent with animal studies suggesting that loss of synaptic input into the DG early in AD may result in shape changes in this region. This can potentially be a viable biomarker for predicting AD onset.

Age-Dependent Effect of Apolipoprotein E4 on Functional Outcome after Controlled Cortical Impact in Mice

The apolipoprotein E4 (APOE4) gene leads to increased brain amyloid beta (Aβ) and poor outcome in adults with traumatic brain injury (TBI); however, its role in childhood TBI is controversial. We hypothesized that the transgenic expression of human APOE4 worsens the outcome after controlled cortical impact (CCI) in adult but not immature mice. Adult and immature APOE4 mice had worse motor outcome after CCI (P<0.001 versus wild type (WT)), but the Morris water maze performance was worse only in adult APOE4 mice (P=0.028 at 2 weeks, P=0.019 at 6 months versus WT), because immature APOE4 mice had performance similar to WT for up to 1 year after injury. Brain lesion size was similar in adult APOE4 mice but was decreased (P=0.029 versus WT) in injured immature APOE4 mice. Microgliosis was similar in all groups. Soluble brain Aβ(40) was increased at 48 hours after CCI in adult and immature APOE4 mice and in adult WT (P<0.05), and was dynamically regulated during the chronic period by APOE4 in adults but not immature mice. The data suggest age-dependent effects of APOE4 on cognitive outcome after TBI, and that therapies targeting APOE4 may be more effective in adults versus children with TBI.

Associations of APOE gene polymorphisms with bone mineral density and fracture risk: a meta-analysis

To determine the association of the Apolipoprotein E (APOE) E4 gene polymorphism with bone mineral density (BMD) and fractures we conducted a meta-analysis of 17 reports. Despite lower trochanteric and lumbar BMD in APOE4 carriers, there is insufficient evidence to support a consistent association of APOE with bone health. APOE has been studied for its potential role in osteoporosis risk. It is hypothesized that genetic variation at APOE locus, known as E2, E3, and E4, may modulate BMD through its effects on lipoproteins and vitamin K transport. The purpose of this study was to determine the association of the APOE-E4 gene polymorphism with bone-related phenotypes. We conducted a meta-analysis that combined newly analyzed individual data from two community-based cohorts, the Framingham Offspring Study (N = 1,495) and the vitamin K clinical trial (N = 377), with 15 other eligible published reports. Bone phenotypes included BMD measurements of the hip (total hip and trochanteric and femoral neck sites) and lumbar spine (from the L2 to L4 vertebrae) and prevalence or incidence of vertebral, hip, and other fractures. In sex-pooled analyses, APOE4 carriers had a 0.018 g/cm(2) lower weighted mean trochanteric BMD than non carriers (p = 0.0002) with no evidence for between-study heterogeneity. A significant association was also detected with lumbar spine BMD (p = 0.006); however, inter-study heterogeneity was observed. Associations with lumbar spine and trochanteric BMD were observed predominantly in women and became less significant in meta-regression (p = 0.055 and 0.01, respectively). There were no consistent associations of APOE4 genotype with BMD at other skeletal sites or with fracture risk. Based on these findings, there is insufficient evidence to support a strong and consistent association of the APOE genotype with BMD and fracture incidence.

Failed Drug Trials Suggest Alzheimer's Shift

Failed Drug Trials Suggest Alzheimer's Shift

Esterase‐based resistance in the tobacco‐adapted form of the green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae) in the eastern United States

Organophosphates and carbamates represent alternative insecticides in managing the tobacco-adapted form of the green peach aphid (TGPA), Myzus persicae (Sulzer), a major pest of tobacco in the United States and around the world. General esterases that detoxify these insecticides were assessed in green, red, and orange morphs of field-collected M. persicae. A total of 136 aphid colonies were collected from 2004 though 2007 and screened for total esterase activity. The green morphs had lower esterase levels, with a mean of 77+/-6.6 nmol/min/mg protein, as compared to red (84+/-2.9 nmol/min/mg protein) and orange morphs (172+/-16.5 nmol/min/mg protein). Overall esterase activities, and those for the red and green morphs, were positively correlated with LC(50) values for acephate (organophosphate) and methomyl (carbamate) assessed in leaf-dip bioassays. Esterase genes responsible for higher esterase activities were diagnosed by gene amplification studies. Twenty-three of 24 colonies tested had either the E4 or FE4 gene amplified, both known to confer esterase-based resistance. Fifteen out of the 24 colonies tested had amplified E4 gene and four colonies had FE4 gene amplification. All orange morphs and one green morph had both E4 and FE4 genes amplified. This unique phenotype, where two esterase genes were amplified had an 865-bp band characteristic of the FE4 gene and an additional 381-bp band characteristic of a deleted upstream region of the E4 gene. Changes that occurred in esterase-based resistance in the TGPA over the past two decades and their implications on insecticide resistance management are discussed.

Genetically Altering Aβ Distribution from the Brain to the Vasculature Ameliorates Tau Pathology

The inheritance of the epsilon4 allele of the apolipoprotein E (apoE) gene is the major genetic risk factor for developing late-onset Alzheimer disease. In transgenic mice overexpressing amyloid precursor protein (APP), replacing the endogenous mouse apoE gene with the human apolipoprotein E4 (apoE4) gene alters the distribution of amyloid-beta (Abeta) deposits from the brain parenchyma to the vasculature. However, the effects of this distribution on the onset and progression of tau pathology remain to be established. To address this issue, we used a genetic approach to replace the endogenous apoE gene with the human apoE4 allele in the 3xTg-AD mice. We showed that changing Abeta distribution from the parenchyma to the vasculature drastically reduces the tau pathology. The 3xTg-AD mice expressing the human apoE4 gene were virtually depleted of any somatodendritic tau deposits. These data strongly suggest that the somatodendritic tau accumulation is dependent on the parenchyma Abeta deposits.

World War II Vets Study Still Providing MH Data

World War II Vets Study Still Providing MH Data

Good News, Bad News on Alzheimer's Prevention

Good News, Bad News on Alzheimer's Prevention

IC-P3-202: Longitudinal association between the apolipoprotein E4 gene polymorphism and cerebral ventricular dilatation measured from serial magnetic resonance imaging in subjects participating in ADNI

IC-P3-202: Longitudinal association between the apolipoprotein E4 gene polymorphism and cerebral ventricular dilatation measured from serial magnetic resonance imaging in subjects participating in ADNI

The APOE4 genotype alters the response of microglia and macrophages to 17β-estradiol

The apolipoprotein E4 ( APOE4) gene is a well-known risk factor for Alzheimer's disease (AD) and other neurological disorders. Post-menopausal women with AD who express at least one APOE4 gene have more severe neuropathology and worsened cognitive scores than their non-expressing counterparts. Since 17β-estradiol down-regulates inflammation as part of its neuroprotective role, we examined the effect of 17β-estradiol on the response of microglia to immune activation as a function of APOE genotype. Our data show that the anti-inflammatory activity of 17β-estradiol is significantly reduced in APOE4 targeted replacement mice compared to APOE3 mice. A significant interaction between APOE genotype and the response to 17β-estradiol was observed for NO and cytokine production by immune activated microglia. The genotype specific effect was not restricted to brain macrophages since peritoneal macrophages from APOE4 ovariectomized mice also demonstrated a significant difference in 17β-estradiol responsiveness. ERβ protein levels in APOE4 microglia were higher than APOE3 microglia, suggesting a difference in post-translational protein regulation in the presence of the APOE4 gene. Overall, our data indicate that the APOE genotype may be a critical component in assessing the effectiveness of 17β-estradiol's action and may impact the neuroprotective role of 17β-estradiol and of hormone replacement therapy on brain function when the APOE4 gene is expressed.

Replication-deficient Adenovirus Induces Host Topoisomerase I Activity: Implications for Adenovirus-mediated Gene Expression

Replication-deficient adenoviruses are useful vectors for the transfer of therapeutic transgenes to malignant and non-malignant tissues. Yet their clinical application is limited by the potential toxicity of viral infection and the transient nature of transgene expression. Although transgene expression from adenovirus vectors is initially higher than expression of transgenes transduced by other viral or non-viral vectors, it is often insufficient to generate a significant therapeutic effect. We addressed this issue by searching for DNA-targeted viral-induced host responses potentially restricting transgene expression. Nuclear protein extracts from livers of rats systemically infected with replication-deficient adenovirus exhibited enhanced topoisomerase I activity compared with extracts from uninfected animals. Consequently, the inhibition of topoisomerase I by the anti-cancer drug topotecan greatly enhanced transgene expression in adenovirus-infected hepatic cells, colon cancer and prostate cancer cell cultures, mouse liver, human ex vivo tumor specimens, and mouse tumor in vivo. The enhancement could not be ascribed to non-specific genotoxic stress, cell death, or cell-cycle perturbation. These findings are significant for gene therapy as they reveal novel aspects of the host anti-adenovirus response and set the stage for the development of a rational molecular-pharmacological approach to increase the effectiveness, and safety, of adenovirus-mediated cancer therapeutics.