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Sample records for a53t-alpha-synuclein overexpression impairs

  1. Age-dependent effects of A53T alpha-synuclein on behavior and dopaminergic function.

    PubMed

    Oaks, Adam W; Frankfurt, Maya; Finkelstein, David I; Sidhu, Anita

    2013-01-01

    Expression of A53T mutant human alpha-synuclein under the mouse prion promoter is among the most successful transgenic models of Parkinson's disease. Accumulation of A53T alpha-synuclein causes adult mice to develop severe motor impairment resulting in early death at 8-12 months of age. In younger, pre-symptomatic animals, altered motor activity and anxiety-like behaviors have also been reported. These behavioral changes, which precede severe neuropathology, may stem from non-pathological functions of alpha-synuclein, including modulation of monoamine neurotransmission. Our analysis over the adult life-span of motor activity, anxiety-like, and depressive-like behaviors identifies perturbations both before and after the onset of disease. Young A53T mice had increased distribution of the dopamine transporter (DAT) to the membrane that was associated with increased striatal re-uptake function. DAT function decreased with aging, and was associated with neurochemical alterations that included increased expression of beta-synuclein and gamma synuclein. Prior to normalization of dopamine uptake, transient activation of Tau kinases and hyperphosphorylation of Tau in the striatum were also observed. Aged A53T mice had reduced neuron counts in the substantia nigra pars compacta, yet striatal medium spiny neuron dendritic spine density was largely maintained. These findings highlight the involvement of the synuclein family of proteins and phosphorylation of Tau in the response to dopaminergic dysfunction of the nigrostriatal pathway.

  2. Astrocytic expression of Parkinson's disease-related A53T alpha-synuclein causes neurodegeneration in mice.

    PubMed

    Gu, Xing-Long; Long, Cai-Xia; Sun, Lixin; Xie, Chengsong; Lin, Xian; Cai, Huaibin

    2010-04-21

    Parkinson's disease (PD) is the most common movement disorder. While neuronal deposition of alpha-synuclein serves as a pathological hallmark of PD and Dementia with Lewy Bodies, alpha-synuclein-positive protein aggregates are also present in astrocytes. The pathological consequence of astrocytic accumulation of alpha-synuclein, however, is unclear. Here we show that PD-related A53T mutant alpha-synuclein, when selectively expressed in astrocytes, induced rapidly progressed paralysis in mice. Increasing accumulation of alpha-synuclein aggregates was found in presymptomatic and symptomatic mouse brains and correlated with the expansion of reactive astrogliosis. The normal function of astrocytes was compromised as evidenced by cerebral microhemorrhage and down-regulation of astrocytic glutamate transporters, which also led to increased inflammatory responses and microglial activation. Interestingly, the activation of microglia was mainly detected in the midbrain, brainstem and spinal cord, where a significant loss of dopaminergic and motor neurons was observed. Consistent with the activation of microglia, the expression level of cyclooxygenase 1 (COX-1) was significantly up-regulated in the brain of symptomatic mice and in cultured microglia treated with conditioned medium derived from astrocytes over-expressing A53T alpha-synuclein. Consequently, the suppression of COX-1 activities extended the survival of mutant mice, suggesting that excess inflammatory responses elicited by reactive astrocytes may contribute to the degeneration of neurons. Our findings demonstrate a critical involvement of astrocytic alpha-synuclein in initiating the non-cell autonomous killing of neurons, suggesting the viability of reactive astrocytes and microglia as potential therapeutic targets for PD and other neurodegenerative diseases.

  3. Proteinase K-resistant alpha-synuclein is deposited in presynapses in human Lewy body disease and A53T alpha-synuclein transgenic mice.

    PubMed

    Tanji, Kunikazu; Mori, Fumiaki; Mimura, Junsei; Itoh, Ken; Kakita, Akiyoshi; Takahashi, Hitoshi; Wakabayashi, Koichi

    2010-08-01

    Abnormally modified alpha-synuclein is a pathological hallmark of Parkinson's disease and the other alpha-synucleinopathies. Since proteinase K (PK) treatment is known to enhance the immunoreactivity of abnormal alpha-synuclein, we immunohistochemically examined the brain of transgenic (Tg) mice expressing human mutant A53T alpha-synuclein using this retrieval method. PK treatment abolished the immunoreactivity of alpha-synuclein in abnormal inclusions as well as of endogenous alpha-synuclein in Tg mice, whereas PK-resistant alpha-synuclein was found in the presynaptic nerve terminals, especially in the hippocampus and temporal cortex. In human Lewy body disease, PK-resistant alpha-synuclein was deposited in Lewy bodies and Lewy neurites, as well as in the presynapses in distinct brain regions, including the hippocampus, temporal cortex and substantia nigra. Biochemical analysis revealed that PK-resistant alpha-synuclein was detected in the presynaptic fraction in Tg mice and human Lewy body disease. Although PK-resistant alpha-synuclein was found in the presynapse in Tg mice even at 1 week of age, it was not phosphorylated until at least 8 months of age. Moreover, PK-resistant alpha-synuclein in the presynapse was not phosphorylated in human Lewy body disease. These findings suggest that phosphorylation is not necessary to cause the conversion of soluble form to PK-resistant alpha-synuclein. Considering that native alpha-synuclein is a soluble protein localized to the presynaptic terminals, our findings suggest that PK-resistant alpha-synuclein may disturb the neurotransmission in alpha-synucleinopathies.

  4. rAAV2/7 vector-mediated overexpression of alpha-synuclein in mouse substantia nigra induces protein aggregation and progressive dose-dependent neurodegeneration

    PubMed Central

    2013-01-01

    Background Alpha-synuclein is a key protein implicated in the pathogenesis of Parkinson's disease (PD). It is the main component of the Lewy bodies, a cardinal neuropathological feature in the disease. In addition, whole locus multiplications and point mutations in the gene coding for alpha-synuclein lead to autosomal dominant monogenic PD. Over the past decade, research on PD has impelled the development of new animal models based on alpha-synuclein. In this context, transgenic mouse lines have failed to reproduce several hallmarks of PD, especially the strong and progressive dopaminergic neurodegeneration over time that occurs in the patients. In contrast, viral vector-based models in rats and non-human primates display prominent, although highly variable, nigral dopaminergic neuron loss. However, the few studies available on viral vector-mediated overexpression of alpha-synuclein in mice report a weak neurodegenerative process and no clear Lewy body-like pathology. To address this issue, we performed a comprehensive comparative study of alpha-synuclein overexpression by means of recombinant adeno-associated viral vectors serotype 2/7 (rAAV2/7) at different doses in adult mouse substantia nigra. Results We noted a significant and dose-dependent alpha-synucleinopathy over time upon nigral viral vector-mediated alpha-synuclein overexpression. We obtained a strong, progressive and dose-dependent loss of dopaminergic neurons in the substantia nigra, reaching a maximum of 82% after 8 weeks. This effect correlated with a reduction in tyrosine hydroxylase immunoreactivity in the striatum. Moreover, behavioural analysis revealed significant motor impairments from 12 weeks after injection on. In addition, we detected the presence of alpha-synuclein-positive aggregates in the remaining surviving neurons. When comparing wild-type to mutant A53T alpha-synuclein at the same vector dose, both induced a similar degree of cell death. These data were supported by a biochemical

  5. rAAV2/7 vector-mediated overexpression of alpha-synuclein in mouse substantia nigra induces protein aggregation and progressive dose-dependent neurodegeneration.

    PubMed

    Oliveras-Salvá, Marusela; Van der Perren, Anke; Casadei, Nicolas; Stroobants, Stijn; Nuber, Silke; D'Hooge, Rudi; Van den Haute, Chris; Baekelandt, Veerle

    2013-11-25

    Alpha-synuclein is a key protein implicated in the pathogenesis of Parkinson's disease (PD). It is the main component of the Lewy bodies, a cardinal neuropathological feature in the disease. In addition, whole locus multiplications and point mutations in the gene coding for alpha-synuclein lead to autosomal dominant monogenic PD. Over the past decade, research on PD has impelled the development of new animal models based on alpha-synuclein. In this context, transgenic mouse lines have failed to reproduce several hallmarks of PD, especially the strong and progressive dopaminergic neurodegeneration over time that occurs in the patients. In contrast, viral vector-based models in rats and non-human primates display prominent, although highly variable, nigral dopaminergic neuron loss. However, the few studies available on viral vector-mediated overexpression of alpha-synuclein in mice report a weak neurodegenerative process and no clear Lewy body-like pathology. To address this issue, we performed a comprehensive comparative study of alpha-synuclein overexpression by means of recombinant adeno-associated viral vectors serotype 2/7 (rAAV2/7) at different doses in adult mouse substantia nigra. We noted a significant and dose-dependent alpha-synucleinopathy over time upon nigral viral vector-mediated alpha-synuclein overexpression. We obtained a strong, progressive and dose-dependent loss of dopaminergic neurons in the substantia nigra, reaching a maximum of 82% after 8 weeks. This effect correlated with a reduction in tyrosine hydroxylase immunoreactivity in the striatum. Moreover, behavioural analysis revealed significant motor impairments from 12 weeks after injection on. In addition, we detected the presence of alpha-synuclein-positive aggregates in the remaining surviving neurons. When comparing wild-type to mutant A53T alpha-synuclein at the same vector dose, both induced a similar degree of cell death. These data were supported by a biochemical analysis that showed

  6. Overexpression of Lamin B Receptor Results in Impaired Skin Differentiation.

    PubMed

    Sola Carvajal, Agustín; McKenna, Tomás; Wallén Arzt, Emelie; Eriksson, Maria

    2015-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare segmental progeroid disorder commonly caused by a point mutation in the LMNA gene that results in the increased activation of an intra-exonic splice site and the production of a truncated lamin A protein, named progerin. In our previous work, induced murine epidermal expression of this specific HGPS LMNA mutation showed impaired keratinocyte differentiation and upregulated lamin B receptor (LBR) expression in suprabasal keratinocytes. Here, we have developed a novel transgenic animal model with induced overexpression of LBR in the interfollicular epidermis. LBR overexpression resulted in epidermal hypoplasia, along with the downregulation and mislocalization of keratin 10, suggesting impaired keratinocyte differentiation. Increased LBR expression in basal and suprabasal cells did not coincide with increased proliferation. Similar to our previous report of HGPS mice, analyses of γH2AX, a marker of DNA double-strand breaks, revealed an increased number of keratinocytes with multiple foci in LBR-overexpressing mice compared with wild-type mice. In addition, suprabasal LBR-positive cells showed densely condensed and peripherally localized chromatin. Our results show a moderate skin differentiation phenotype, which indicates that upregulation of LBR is not the sole contributor to the HGPS phenotype.

  7. Overexpression of Lamin B Receptor Results in Impaired Skin Differentiation

    PubMed Central

    Sola Carvajal, Agustín; McKenna, Tomás; Wallén Arzt, Emelie; Eriksson, Maria

    2015-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare segmental progeroid disorder commonly caused by a point mutation in the LMNA gene that results in the increased activation of an intra-exonic splice site and the production of a truncated lamin A protein, named progerin. In our previous work, induced murine epidermal expression of this specific HGPS LMNA mutation showed impaired keratinocyte differentiation and upregulated lamin B receptor (LBR) expression in suprabasal keratinocytes. Here, we have developed a novel transgenic animal model with induced overexpression of LBR in the interfollicular epidermis. LBR overexpression resulted in epidermal hypoplasia, along with the downregulation and mislocalization of keratin 10, suggesting impaired keratinocyte differentiation. Increased LBR expression in basal and suprabasal cells did not coincide with increased proliferation. Similar to our previous report of HGPS mice, analyses of γH2AX, a marker of DNA double-strand breaks, revealed an increased number of keratinocytes with multiple foci in LBR-overexpressing mice compared with wild-type mice. In addition, suprabasal LBR-positive cells showed densely condensed and peripherally localized chromatin. Our results show a moderate skin differentiation phenotype, which indicates that upregulation of LBR is not the sole contributor to the HGPS phenotype. PMID:26053873

  8. Skeletal overexpression of gremlin impairs bone formation and causes osteopenia.

    PubMed

    Gazzerro, Elisabetta; Pereira, Renata C; Jorgetti, Vanda; Olson, Sarah; Economides, Aris N; Canalis, Ernesto

    2005-02-01

    Skeletal cells synthesize bone morphogenetic proteins (BMPs) and BMP antagonists. Gremlin, a BMP antagonist, is expressed in osteoblasts and opposes BMP effects on osteoblastic differentiation and function in vitro. However, its effects in vivo are not known. To investigate the actions of gremlin on bone remodeling in vivo, we generated transgenic mice overexpressing gremlin under the control of the osteocalcin promoter. Gremlin transgenics exhibited bone fractures and reduced bone mineral density by 20-30%, compared with controls. Static and dynamic histomorphometry of femurs revealed that gremlin overexpression caused reduced trabecular bone volume and the appearance of woven bone. Polarized light microscopy revealed disorganized collagen bundles at the endosteal cortical surface. Gremlin transgenic mice displayed a 70% decrease in the number of osteoblasts/trabecular area and reduced mineral apposition and bone formation rates. In vivo bromodeoxyuridine labeling and marrow stromal cell cultures demonstrated an inhibitory effect of gremlin on osteoblastic cell replication, but no change on apoptosis was detected. Marrow stromal cells from gremlin transgenics displayed a reduced response to BMP on phosphorylated mothers against decapentaplegic 1/5/8 phosphorylation and reduced free cytosolic beta-catenin levels. In conclusion, transgenic mice overexpressing gremlin in the bone microenvironment have decreased osteoblast number and function leading to osteopenia and spontaneous fractures.

  9. Overexpression of alpha2C-adrenoceptors impairs water maze navigation.

    PubMed

    Björklund, M; Sirviö, J; Riekkinen, M; Sallinen, J; Scheinin, M; Riekkinen, P

    2000-01-01

    We investigated the role of overexpression of alpha2C-adrenoceptors in water maze navigation in mice transgenically manipulated to have a threefold overexpression of the alpha2C-adrenoreceptors. Alpha2C-adrenoreceptors overexpressing mice swam more in the peripheral annulus of the pool and did not find the hidden escape platform as well as the wild type control mice. A subtype-nonselective alpha2-adrenoreceptor antagonist, atipamezole (ATI, 1000 microg/kg, s.c.), fully reversed the deficit in platform finding and search strategy in overexpressing mice. Noradrenaline depletion (-95%) induced by N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) did not impair platform finding of wild type or overexpressing mice. The DSP-4 lesion slightly increased swimming in the peripheral annulus in wild type mice, but not in overexpressing mice. The DSP-4 lesion produced a dissociable effect on the action of atipamezole to improve platform finding and search strategy in overexpressing mice: atipamezole did not alleviate the platform finding deficit in DSP-4 lesioned overexpressing mice, but normalized their abnormal search strategy. These results suggest that the abnormal search pattern and deficit in the accuracy of platform finding are mediated by constitutive activity of overexpressed alpha2C-adrenoreceptors.

  10. DNA Adenine Methyltransferase (Dam) Overexpression Impairs Photorhabdus luminescens Motility and Virulence.

    PubMed

    Payelleville, Amaury; Lanois, Anne; Gislard, Marie; Dubois, Emeric; Roche, David; Cruveiller, Stéphane; Givaudan, Alain; Brillard, Julien

    2017-01-01

    Dam, the most described bacterial DNA-methyltransferase, is widespread in gamma-proteobacteria. Dam DNA methylation can play a role in various genes expression and is involved in pathogenicity of several bacterial species. The purpose of this study was to determine the role played by the dam ortholog identified in the entomopathogenic bacterium Photorhabdus luminescens. Complementation assays of an Escherichia coli dam mutant showed the restoration of the DNA methylation state of the parental strain. Overexpression of dam in P. luminescens did not impair growth ability in vitro. In contrast, compared to a control strain harboring an empty plasmid, a significant decrease in motility was observed in the dam-overexpressing strain. A transcriptome analysis revealed the differential expression of 208 genes between the two strains. In particular, the downregulation of flagellar genes was observed in the dam-overexpressing strain. In the closely related bacterium Xenorhabdus nematophila, dam overexpression also impaired motility. In addition, the dam-overexpressing P. luminescens strain showed a delayed virulence compared to that of the control strain after injection in larvae of the lepidopteran Spodoptera littoralis. These results reveal that Dam plays a major role during P. luminescens insect infection.

  11. Catalase overexpression does not impair extensor digitorum longus muscle function in normal mice.

    PubMed

    Liu, Mingju; Yue, Yongping; Li, Dejia; Duan, Dongsheng

    2007-12-01

    Catalase is a major antioxidant enzyme. Increasing catalase expression represents a promising avenue to improve muscle function in certain physiological conditions and in some muscle diseases. We hypothesized that catalase overexpression should not impair normal muscle contraction. We delivered a hemagglutinin (HA)-tagged human catalase gene to normal mouse muscle by an adeno-associated viral vector (AAV). Western blot and immunostaining revealed efficient expression of HA-tagged catalase. Enzymatic assay demonstrated an approximately threefold increase in catalase activity in AAV-infected muscles. Catalase overexpression impaired neither twitch nor tetanic tension in the extensor digitorum longus (EDL) muscle. Furthermore, EDL fatigue response was not altered. Taken together, we have developed a novel AAV vector to enhance catalase expression. Lack of apparent toxicity in normal muscle strongly supports further exploration of this vector to reduce oxidative stress-induced muscle damage.

  12. p130Cas over-expression impairs mammary branching morphogenesis in response to estrogen and EGF.

    PubMed

    Camacho Leal, Maria del Pilar; Pincini, Alessandra; Tornillo, Giusy; Fiorito, Elisa; Bisaro, Brigitte; Di Luca, Elisa; Turco, Emilia; Defilippi, Paola; Cabodi, Sara

    2012-01-01

    p130Cas adaptor protein regulates basic processes such as cell cycle control, survival and migration. p130Cas over-expression has been related to mammary gland transformation, however the in vivo consequences of p130Cas over-expression during mammary gland morphogenesis are not known. In ex vivo mammary explants from MMTV-p130Cas transgenic mice, we show that p130Cas impairs the functional interplay between Epidermal Growth Factor Receptor (EGFR) and Estrogen Receptor (ER) during mammary gland development. Indeed, we demonstrate that p130Cas over-expression upon the concomitant stimulation with EGF and estrogen (E2) severely impairs mammary morphogenesis giving rise to enlarged multicellular spherical structures with altered architecture and absence of the central lumen. These filled acinar structures are characterized by increased cell survival and proliferation and by a strong activation of Erk1/2 MAPKs and Akt. Interestingly, antagonizing the ER activity is sufficient to re-establish branching morphogenesis and normal Erk1/2 MAPK activity. Overall, these results indicate that high levels of p130Cas expression profoundly affect mammary morphogenesis by altering epithelial architecture, survival and unbalancing Erk1/2 MAPKs activation in response to growth factors and hormones. These results suggest that alteration of morphogenetic pathways due to p130Cas over-expression might prime mammary epithelium to tumorigenesis.

  13. Brain-derived neurotrophic factor (BDNF) overexpression in the forebrain results in learning and memory impairments.

    PubMed

    Cunha, Carla; Angelucci, Andrea; D'Antoni, Angela; Dobrossy, Mate D; Dunnett, Stephen B; Berardi, Nicoletta; Brambilla, Riccardo

    2009-03-01

    In this study we analyzed the effect on behavior of a chronic exposure to brain-derived neurotrophic factor (BDNF), by analysing a mouse line overexpressing BDNF under the alphaCaMKII promoter, which drives the transgene expression exclusively to principal neurons of the forebrain. BDNF transgenic mice and their WT littermates were examined with a battery of behavioral tests, in order to evaluate motor coordination, learning, short and long-term memory formation. Our results demonstrate that chronic BDNF overexpression in the central nervous system (CNS) causes learning deficits and short-term memory impairments, both in spatial and instrumental learning tasks. This observation suggests that a widespread increase in BDNF in forebrain networks may result in adverse effects on learning and memory formation.

  14. Select Overexpression of Homer1a in Dorsal Hippocampus Impairs Spatial Working Memory

    PubMed Central

    Celikel, Tansu; Zivkovic, Aleksandar; Resnik, Evgeny; Hasan, Mazahir T.; Licznerski, Pawel; Osten, Pavel; Rozov, Andrej; Seeburg, Peter H.; Schwarz, Martin K.

    2007-01-01

    Long Homer proteins forge assemblies of signaling components involved in glutamate receptor signaling in postsynaptic excitatory neurons, including those underlying synaptic transmission and plasticity. The short immediate-early gene (IEG) Homer1a can dynamically uncouple these physical associations by functional competition with long Homer isoforms. To examine the consequences of Homer1a-mediated “uncoupling” for synaptic plasticity and behavior, we generated forebrain-specific tetracycline (tet) controlled expression of Venus-tagged Homer1a (H1aV) in mice. We report that sustained overexpression of H1aV impaired spatial working but not reference memory. Most notably, a similar impairment was observed when H1aV expression was restricted to the dorsal hippocampus (HP), which identifies this structure as the principal cortical area for spatial working memory. Interestingly, H1aV overexpression also abolished maintenance of CA3-CA1 long-term potentiation (LTP). These impairments, generated by sustained high Homer1a levels, identify a requirement for long Homer forms in synaptic plasticity and temporal encoding of spatial memory. PMID:18982121

  15. AP-1 overexpression impairs corticosteroid inhibition of collagen production by fibroblasts isolated from asthmatic subjects.

    PubMed

    Jacques, Eric; Semlali, Abdelhabib; Boulet, Louis Philippe; Chakir, Jamila

    2010-08-01

    Asthma is characterized by airway remodeling associated with an increase in the deposition of ECM proteins such as type I collagen. These components are mainly produced by fibroblasts. Inhaled corticosteroids are considered the cornerstone of asthma therapy. Despite substantial evidence as to the anti-inflammatory action of corticosteroids, their effect on controlling ECM protein deposition in the airways is not completely understood. This study determined the effect of dexamethasone (Dex) on collagen production by bronchial fibroblasts derived from asthmatic and healthy subjects. Expression of procollagen mRNA in fibroblasts from asthmatics and normal controls was determined by quantitative PCR. Regulation of the procollagen-alpha(1)I promoter was evaluated by transient transfections. Transforming growth factor-beta (TGF-beta) protein expression was determined by ELISA. Protein expression of glucocorticoid receptor (GR) and interaction with activator protein-1 (AP-1), a collagen regulatory transcription factor, was assessed by Western blots, coimmunoprecipitations, and EMSA. AP-1 overexpression was performed by transient transfection using c-Fos/c-Jun expression plasmids. Dex significantly downregulated procollagen production and promoter activity in normal fibroblasts but had no effect on asthmatic fibroblasts. AP-1 and GR interaction increased after Dex stimulation in asthmatic fibroblasts. AP-1 overexpression in control fibroblasts abrogated collagen gene response to Dex. These results show that Dex failed to reduce collagen production in fibroblasts from asthmatic subjects. This impaired response may be related to AP-1 overexpression in these cells.

  16. Leucine-rich α2-glycoprotein overexpression in the brain contributes to memory impairment.

    PubMed

    Akiba, Chihiro; Nakajima, Madoka; Miyajima, Masakazu; Ogino, Ikuko; Miura, Masami; Inoue, Ritsuko; Nakamura, Eri; Kanai, Fumio; Tada, Norihiro; Kunichika, Miyuki; Yoshida, Mitsutaka; Nishimura, Kinya; Kondo, Akihide; Sugano, Hidenori; Arai, Hajime

    2017-08-24

    We previously reported increase in leucine-rich α2-glycoprotein (LRG) concentration in cerebrospinal fluid is associated with cognitive decline in humans. To investigate relationship between LRG expression in the brain and memory impairment, we analyzed transgenic mice overexpressing LRG in the brain (LRG-Tg) focusing on hippocampus. Immunostaining and Western blotting revealed age-related increase in LRG expression in hippocampal neurons in 8-, 24-, and 48-week-old controls and LRG-Tg. Y-maze and Morris water maze tests indicated retained spatial memory in 8- and 24-week-old LRG-Tg, while deteriorated in 48-week-old LRG-Tg compared with age-matched controls. Field excitatory postsynaptic potentials declined with age in LRG-Tg compared with controls at 8, 24, and 48 weeks. Paired-pulse ratio decreased with age in LRG-Tg, while increased in controls. As a result, long-term potentiation was retained in 8- and 24-week-old LRG-Tg, whereas diminished in 48-week-old LRG-Tg compared with age-matched controls. Electron microscopy observations revealed fewer synaptic vesicles and junctions in LRG-Tg compared with age-matched controls, which became significant with age. Hippocampal LRG overexpression contributes to synaptic dysfunction, which leads to memory impairment with advance of age. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  17. MicroRNA-126 overexpression rescues diabetes-induced impairment in efferocytosis of apoptotic cardiomyocytes

    PubMed Central

    Suresh Babu, Sahana; Thandavarayan, Rajarajan A.; Joladarashi, Darukeshwara; Jeyabal, Prince; Krishnamurthy, Shashirekha; Bhimaraj, Arvind; Youker, Keith A.; Krishnamurthy, Prasanna

    2016-01-01

    Efferocytosis, a process of clearance of apoptotic cells by phagocytes, is essential for successful resolution of inflammation and maintenance of tissue homeostasis. Diabetes compromises the function of macrophages leading to adverse inflammatory response during wound healing, myocardial injury, atherosclerosis and autoimmune disorders. However, the effect of diabetes on macrophage-mediated efferocytosis of apoptotic cardiomyocytes (ACM) and the molecular mechanisms involved are not understood so far. In the present study we found that invitro efferocytosis of ACM was impaired in macrophages from db/db (diabetic) mice. Macrophages exposed to high glucose (HG) decreases microRNA-126 (miR-126) expression with a corresponding increase in ADAM9 expression. Dual-luciferase reporter assay confirms that ADAM9 3′UTR contains miR-126 target site. ADAM9 inhibition reduces HG-induced proteolytic cleavage of Mer tyrosine receptor kinase (MerTK, a proto-oncogene that plays a critical role in phagocytosis), resulting in shedding of soluble-Mer (sMER) and loss of MERTK function. Over-expression of miR-126 attenuates HG-induced impairment of efferocytosis. Furthermore, human diabetic hearts show lower miR-126 expression with a corresponding increase in ADAM9 expression vs. normal counterparts. These data suggests that diabetes impairs efferocytosis of ACM and that strategies to enhance efferocytosis might attenuate diabetes-induced impairment in inflammation resolution and cardiac repair after injury. PMID:27827458

  18. β-Cell-Specific Mafk Overexpression Impairs Pancreatic Endocrine Cell Development

    PubMed Central

    Abdellatif, Ahmed M.; Oishi, Hisashi; Itagaki, Takahiro; Jung, Yunshin; Shawki, Hossam H.; Okita, Yukari; Hasegawa, Yoshikazu; Suzuki, Hiroyuki; El-Morsy, Salah E.; El-Sayed, Mesbah A.; Shoaib, Mahmoud B.; Sugiyama, Fumihiro; Takahashi, Satoru

    2016-01-01

    The MAF family transcription factors are homologs of v-Maf, the oncogenic component of the avian retrovirus AS42. They are subdivided into 2 groups, small and large MAF proteins, according to their structure, function, and molecular size. MAFK is a member of the small MAF family and acts as a dominant negative form of large MAFs. In previous research we generated transgenic mice that overexpress MAFK in order to suppress the function of large MAF proteins in pancreatic β-cells. These mice developed hyperglycemia in adulthood due to impairment of glucose-stimulated insulin secretion. The aim of the current study is to examine the effects of β-cell-specific Mafk overexpression in endocrine cell development. The developing islets of Mafk-transgenic embryos appeared to be disorganized with an inversion of total numbers of insulin+ and glucagon+ cells due to reduced β-cell proliferation. Gene expression analysis by quantitative RT-PCR revealed decreased levels of β-cell-related genes whose expressions are known to be controlled by large MAF proteins. Additionally, these changes were accompanied with a significant increase in key β-cell transcription factors likely due to compensatory mechanisms that might have been activated in response to the β-cell loss. Finally, microarray comparison of gene expression profiles between wild-type and transgenic pancreata revealed alteration of some uncharacterized genes including Pcbd1, Fam132a, Cryba2, and Npy, which might play important roles during pancreatic endocrine development. Taken together, these results suggest that Mafk overexpression impairs endocrine development through a regulation of numerous β-cell-related genes. The microarray analysis provided a unique data set of differentially expressed genes that might contribute to a better understanding of the molecular basis that governs the development and function of endocrine pancreas. PMID:26901059

  19. Transglutaminase 2 overexpression induces depressive-like behavior and impaired TrkB signaling in mice

    PubMed Central

    Pandya, Chirayu D; Hoda, Nasrul; Crider, Amanda; Peter, Diya; Kutiyanawalla, Ammar; Kumar, Sanjiv; Ahmed, Anthony O; Turecki, Gustavo; Hernandez, Caterina M; Terry, Alvin V

    2016-01-01

    Serotonin (5-HT) and brain derived neurotrophic factor (BDNF) are two signaling molecules that play important regulatory roles in the development and plasticity of neural circuits that are known to be altered in depression. However, the mechanism by which 5-HT regulates BDNF signaling is unknown. In the present study, we found that 5-HT treatment increases BDNF receptor, TrkB (tropomyosin related kinase B) levels in mouse primary cortical neurons via a Rac1 (RAS-related C3 botulinum toxin substrate 1)-dependent mechanism. Significant increases in the levels of transglutaminase 2 (TG2, which is implicated in transamidation of 5-HT to Rac1) are observed in the mouse prefrontal cortex (PFC) following chronic exposure to stress. We also found that TG2 levels are increased in the postmortem PFC of depressed suicide subjects relative to matched controls. Moreover, in mice, neuronal overexpression of TG2 resulted in the atrophy of neurons and reduced levels of TrkB in the PFC as well as a depressive-like phenotype. Overexpression of TG2 in mouse cortical neurons reduced TrkB levels as a result of impaired endocytosis of TrkB. TG2 inhibition by either a viral particle or pharmacological approach attenuated behavioral deficits caused by chronic unpredictable stress. Moreover, the overexpression of TrkB in the mouse PFC ameliorated the depressive-like phenotype of TG2 overexpressed mice. Taken together, these postmortem and preclinical findings identify TG2 as a critical mediator of the altered TrkB expression and depressive-like behaviors associated with chronic exposure to stress and suggest that TG2 may represent a novel therapeutic target in depression. PMID:27620841

  20. Flightless I over-expression impairs skin barrier development, function and recovery following skin blistering.

    PubMed

    Kopecki, Zlatko; Yang, Gink N; Arkell, Ruth M; Jackson, Jessica E; Melville, Elizabeth; Iwata, Hiroaki; Ludwig, Ralf J; Zillikens, Detlef; Murrell, Dedee F; Cowin, Allison J

    2014-04-01

    Development of an intact epidermis is critical for maintaining the integrity of the skin. Patients with epidermolysis bullosa (EB) experience multiple erosions, which breach the epidermal barrier and lead to increased microbial colocalization of wounds, infections and sepsis. The cytoskeletal protein Flightless I (Flii) is a known regulator of both development and wound healing. Using Flii(+/-), WT and Flii(Tg/Tg) mice, we investigated the effect of altering Flii levels in embryos and adult mice on the development of the epidermal barrier and, consequently, how this affects the integrity of the skin in EB. Flii over-expression resulted in delayed formation of the epidermal barrier in embryos and decreased expression of tight junction (TJ) proteins Claudin-1 and ZO-2. Increased intercellular space and transepidermal water loss was observed in Flii(Tg)(/Tg) adult mouse skin, while Flii(Tg/Tg) keratinocytes showed altered TJ protein localization and reduced transepithelial resistance. Flii is increased in the blistered skin of patients with EB, and over-expression of Flii in experimental EBA showed impaired Claudin-1 and -4 TJ protein expression and delayed recovery of functional barrier post-blistering. Immunoprecipitation confirmed Flii associated with TJ proteins and in vivo actin assays showed that the effect of Flii on actin polymerization underpinned the impaired barrier function observed in Flii(Tg/Tg) mice. These results therefore demonstrate an important role for Flii in the development and regulation of the epidermal barrier, which may contribute to the impaired healing and skin fragility of EB patients. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  1. Muscle-specific Drp1 overexpression impairs skeletal muscle growth via translational attenuation

    PubMed Central

    Touvier, T; De Palma, C; Rigamonti, E; Scagliola, A; Incerti, E; Mazelin, L; Thomas, J-L; D'Antonio, M; Politi, L; Schaeffer, L; Clementi, E; Brunelli, S

    2015-01-01

    Mitochondrial fission and fusion are essential processes in the maintenance of the skeletal muscle function. The contribution of these processes to muscle development has not been properly investigated in vivo because of the early lethality of the models generated so far. To define the role of mitochondrial fission in muscle development and repair, we have generated a transgenic mouse line that overexpresses the fission-inducing protein Drp1 specifically in skeletal muscle. These mice displayed a drastic impairment in postnatal muscle growth, with reorganisation of the mitochondrial network and reduction of mtDNA quantity, without the deficiency of mitochondrial bioenergetics. Importantly we found that Drp1 overexpression activates the stress-induced PKR/eIF2α/Fgf21 pathway thus leading to an attenuated protein synthesis and downregulation of the growth hormone pathway. These results reveal for the first time how mitochondrial network dynamics influence muscle growth and shed light on aspects of muscle physiology relevant in human muscle pathologies. PMID:25719247

  2. Nigericin-induced impairment of autophagic flux in neuronal cells is inhibited by overexpression of Bak.

    PubMed

    Lim, Junghyun; Lee, Yunsu; Kim, Hyun-Wook; Rhyu, Im Joo; Oh, Myung Sook; Youdim, Moussa B H; Yue, Zhenyu; Oh, Young J

    2012-07-06

    Bak is a prototypic pro-apoptotic Bcl-2 family protein expressed in a wide variety of tissues and cells. Recent studies have revealed that Bcl-2 family proteins regulate apoptosis as well as autophagy. To investigate whether and how Bak exerts a regulatory role on autophagy-related events, we treated independent cell lines, including MN9D neuronal cells, with nigericin, a K(+)/H(+) ionophore. Treatment of MN9D cells with nigericin led to an increase of LC3-II and p62 levels with concomitant activation of caspase. Ultrastructural examination revealed accumulation of autophagic vacuoles and swollen vacuoles in nigericin-treated cells. We further found that the LC3-II accumulated as a consequence of impaired autophagic flux and the disrupted degradation of LC3-II in nigericin-treated cells. In this cell death paradigm, both transient and stable overexpression of various forms of Bak exerted a protective role, whereas it did not inhibit the extent of nigericin-mediated activation of caspase-3. Subsequent biochemical and electron microscopic studies revealed that overexpressed Bak maintained autophagic flux and reduced the area occupied by swollen vacuoles in nigericin-treated cells. Similar results were obtained in nigericin-treated non-neuronal cells and another proton ionophore-induced cell death paradigm. Taken together, our study indicates that a protective role for Bak during ionophore-induced cell death may be closely associated with its regulatory effect on maintenance of autophagic flux and vacuole homeostasis.

  3. Overexpression of mineralocorticoid and transdominant glucocorticoid receptor blocks the impairing effects of glucocorticoids on memory.

    PubMed

    Ferguson, Deveroux; Sapolsky, Robert

    2008-01-01

    It is well established that mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) influence hippocampal-dependent spatial memory. MRs are saturated in the presence of low corticosterone (CORT) levels; consequently receptor protein levels play a rate-limiting role in regulating the positive effects of MR-mediated gene transcription. In this study, viral vector-mediated transgene expression was used to simultaneously manipulate both MR and GR signaling. This approach allowed us to investigate the effects of spatially restricted overexpression of MR and a negative transdominant GR (TD) in the dentate gyrus (DG) subfield of the hippocampus, on short term and long term spatial memory in animals overexpressing one copy of MR or TD, two copies of MR ("MR/MR"), or one copy of each ("MR/TD"). Expression of transgenes did not influence the acquisition (learning) phase of the Morris water maze task. However, we found an overall enhancing effect of MR/MR expression on short term memory performance. In addition, rats expressing TD and MR/TD blocked the high CORT-induced impairments on long term spatial memory retrieval. These findings illustrate the potential beneficial effects of increasing MR signaling or decreasing GR signaling to enhance specific aspects of cognitive function.

  4. Overexpression of a proton-coupled vacuolar glucose exporter impairs freezing tolerance and seed germination.

    PubMed

    Klemens, Patrick A W; Patzke, Kathrin; Trentmann, Oliver; Poschet, Gernot; Büttner, Michael; Schulz, Alexander; Marten, Irene; Hedrich, Rainer; Neuhaus, H Ekkehard

    2014-04-01

    Arabidopsis vacuoles harbor, besides sugar transporter of the TMT-type, an early response to dehydration like 6 (ERDL6) protein involved in glucose export into the cytosol. However, the mode of transport of ERDL6 and the plant's feedback to overexpression of its activity on essential properties such as, for example, seed germination or freezing tolerance, remain unexplored. Using patch-clamp studies on vacuoles expressing AtERDL6 we demonstrated directly that this carrier operates as a proton-driven glucose exporter. Overexpression of BvIMP, the closest sugar beet (Beta vulgaris) homolog to AtERDL6, in Arabidopsis leads surprisingly to impaired seed germination under both conditions, sugar application and low environmental temperatures, but not under standard conditions. Upon cold treatment, BvIMP overexpressor plants accumulated lower quantities of monosaccharides than the wild-type, a response in line with the reduced frost tolerance of the transgenic Arabidopsis plants, and the fact that cold temperatures inhibits BvIMP transcription in sugar beet leaves. With these findings we show that the tight control of vacuolar sugar import and export is a key requisite for cold tolerance and seed germination of plants.

  5. Nigericin-induced Impairment of Autophagic Flux in Neuronal Cells Is Inhibited by Overexpression of Bak*

    PubMed Central

    Lim, Junghyun; Lee, Yunsu; Kim, Hyun-Wook; Rhyu, Im Joo; Oh, Myung Sook; Youdim, Moussa B. H.; Yue, Zhenyu; Oh, Young J.

    2012-01-01

    Bak is a prototypic pro-apoptotic Bcl-2 family protein expressed in a wide variety of tissues and cells. Recent studies have revealed that Bcl-2 family proteins regulate apoptosis as well as autophagy. To investigate whether and how Bak exerts a regulatory role on autophagy-related events, we treated independent cell lines, including MN9D neuronal cells, with nigericin, a K+/H+ ionophore. Treatment of MN9D cells with nigericin led to an increase of LC3-II and p62 levels with concomitant activation of caspase. Ultrastructural examination revealed accumulation of autophagic vacuoles and swollen vacuoles in nigericin-treated cells. We further found that the LC3-II accumulated as a consequence of impaired autophagic flux and the disrupted degradation of LC3-II in nigericin-treated cells. In this cell death paradigm, both transient and stable overexpression of various forms of Bak exerted a protective role, whereas it did not inhibit the extent of nigericin-mediated activation of caspase-3. Subsequent biochemical and electron microscopic studies revealed that overexpressed Bak maintained autophagic flux and reduced the area occupied by swollen vacuoles in nigericin-treated cells. Similar results were obtained in nigericin-treated non-neuronal cells and another proton ionophore-induced cell death paradigm. Taken together, our study indicates that a protective role for Bak during ionophore-induced cell death may be closely associated with its regulatory effect on maintenance of autophagic flux and vacuole homeostasis. PMID:22493436

  6. Overexpression of SIRT1 in Mouse Forebrain Impairs Lipid/Glucose Metabolism and Motor Function

    PubMed Central

    Wu, Dongmei; Qiu, Yifu; Gao, Xiang; Yuan, Xiao-Bing; Zhai, Qiwei

    2011-01-01

    SIRT1 plays crucial roles in glucose and lipid metabolism, and has various functions in different tissues including brain. The brain-specific SIRT1 knockout mice display defects in somatotropic signaling, memory and synaptic plasticity. And the female mice without SIRT1 in POMC neuron are more sensitive to diet-induced obesity. Here we created transgenic mice overexpressing SIRT1 in striatum and hippocampus under the control of CaMKIIα promoter. These mice, especially females, exhibited increased fat accumulation accompanied by significant upregulation of adipogenic genes in white adipose tissue. Glucose tolerance of the mice was also impaired with decreased Glut4 mRNA levels in muscle. Moreover, the SIRT1 overexpressing mice showed decreased energy expenditure, and concomitantly mitochondria-related genes were decreased in muscle. In addition, these mice showed unusual spontaneous physical activity pattern, decreased activity in open field and rotarod performance. Further studies demonstrated that SIRT1 deacetylated IRS-2, and upregulated phosphorylation level of IRS-2 and ERK1/2 in striatum. Meanwhile, the neurotransmitter signaling in striatum and the expression of endocrine hormones in hypothalamus and serum T3, T4 levels were altered. Taken together, our findings demonstrate that SIRT1 in forebrain regulates lipid/glucose metabolism and motor function. PMID:21738790

  7. Cerebralcare Granule® attenuates cognitive impairment in rats continuously overexpressing microRNA-30e

    PubMed Central

    XU, YONG; LIU, ZHIFEN; SONG, XI; ZHANG, KERANG; LI, XINGRONG; LI, JIANHONG; YAN, XU; LI, YUAN; XIE, ZHONGCHEN; ZHANG, HUI

    2015-01-01

    Previous studies have demonstrated that dysregulation of micro (mi)RNAs is associated with the etiology of various neuropsychiatric disorders, including depression and schizophrenia. Cerebralcare Granule® (CG) is a Chinese herbal medicine, which has been reported to have an ameliorative effect on brain injury by attenuating blood-brain barrier disruption and improving hippocampal neural function. The present study aimed to evaluate the cognitive behavior of rats continuously overexpressing miRNA-30e (lenti-miRNA-30e), prior to and following the administration of CG. In addition, the mechanisms underlying the ameliorative effects of CG were investigated. The cognitive ability of the rats was assessed using an open-field test and a Morris water maze spatial reference/working memory test. A terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to detect neuronal apoptosis in the dentate gyrus of the hippocampus. Immunohistochemical analysis and western blotting were conducted to detect the expression levels of B-cell lymphoma 2 (BCL-2) and ubiquitin-conjugating enzyme 9 (UBC9), in order to examine neuronal apoptosis. The lenti-miRNA-30e rats exhibited increased signs of anxiety, depression, hyperactivity and schizophrenia, which resulted in a severe impairment in cognitive ability. Furthermore, in the dentate gyrus of these rats, the expression levels of BCL-2 and UBC9 were reduced and apoptosis was increased. The administration of CG alleviated cognitive impairment, enhanced the expression levels of BCL-2 and UBC9, and reduced apoptosis in the dentate gyrus in the lenti-miRNA-30e rats. No significant differences were detected in behavioral indicators between the lenti-miRNA-30e rats treated with CG and the normal controls. These findings suggested that CG exerts a potent therapeutic effect, conferred by its ability to enhance the expression levels of BCL-2 and UBC9, which inhibits the apoptotic process in neuronal cells. Therefore, CG may be

  8. C/EBPalpha inactivation in FAK-overexpressed HL-60 cells impairs cell differentiation.

    PubMed

    Hashimoto, Ken-ichiro; Sonoda, Yoshiko; Yamakado, Masakazu; Funakoshi-Tago, Megumi; Yoshida, Naomi; Rokudai, Akiko; Aizu-Yokota, Eriko; Kasahara, Tadashi

    2006-07-01

    We previously demonstrated that focal adhesion kinase (FAK)-overexpressed (HL-60/FAK) cells have marked resistance against various apoptotic stimuli such as oxidative stress, ionizing radiation and TNF-receptor-induced ligand (TRAIL) compared with vector-transfected (HL-60/Vect) cells. Here, we show that HL-60/FAK cells are highly resistant to all-trans retinoic acid (ATRA)-induced differentiation, whereas original HL-60 or HL-60/Vect cells are sensitive. Treatment with ATRA at 1 muM for 5 days markedly inhibited the proliferation and increased the expression of differentiation markers (CD38, CD11b) in HL-60/Vect cells, but showed no such effect in HL-60/FAK cells. Electrophoretic mobility shift assay (EMSA) using an oligonucleotide for the c/EBP consensus binding sequence showed that c/EBPalpha was activated in ATRA-treated HL-60/Vect cells but not in HL-60/FAK cells, indicating that c/EBPalpha activation by ATRA was impaired in HL-60/FAK cells. In addition, the association of retinoblastoma protein (pRb) and c/EBPalpha after treatment with ATRA was seen in HL-60/Vect cells but not in HL-60/FAK cells. Further, hyperphosphorylation of pRb was observed in HL-60/FAK cells. Finally, the introduction of FAK siRNA into HL-60/FAK cells resulted in the recovery of sensitivity to ATRA-induced differentiation, confirming that the inhibition of HL-60/FAK differentiation resulted from both the induction of pRb hyperphosphorylation and the inhibition of association of pRb and c/EBPalpha.

  9. Overexpression of NYGGF4 (PID1) induces mitochondrial impairment in 3T3-L1 adipocytes.

    PubMed

    Zhao, Yaping; Zhang, Chunmei; Chen, Xiaohui; Gao, Chunlin; Ji, Chenbo; Chen, Fukun; Zhu, Chun; Zhu, Jingai; Wang, Jialin; Qian, Lingmei; Guo, Xirong

    2010-07-01

    NYGGF4 is a recently discovered gene that is involved in obesity-associated insulin resistance. The exact mechanism by which NYGGF4 induces insulin resistance has not yet been fully elucidated. In this study, we demonstrated that the overexpression of NYGGF4 in 3T3-L1 adipocytes decreased mitochondrial mass, mitochondrial DNA, and intracellular ATP synthesis. In addition, NYGGF4 overexpression also led to an imbalance of the mitochondrial dynamics and excess intracellular ROS production. Collectively, our results indicated that the overexpression of NYGGF4 caused mitochondrial dysfunction in adipocytes, which might be responsible for the development of NYGGF4-induced insulin resistance.

  10. HMGA1 overexpression in adipose tissue impairs adipogenesis and prevents diet-induced obesity and insulin resistance.

    PubMed

    Arce-Cerezo, Altamira; García, Miquel; Rodríguez-Nuevo, Aida; Crosa-Bonell, Mireia; Enguix, Natalia; Peró, Albert; Muñoz, Sergio; Roca, Carles; Ramos, David; Franckhauser, Sylvie; Elias, Ivet; Ferre, Tura; Pujol, Anna; Ruberte, Jesús; Villena, Josep A; Bosch, Fàtima; Riu, Efrén

    2015-09-28

    High-Mobility-Group-A1 (HMGA1) proteins are non-histone proteins that regulate chromatin structure and gene expression during embryogenesis, tumourigenesis and immune responses. In vitro studies suggest that HMGA1 proteins may be required to regulate adipogenesis. To examine the role of HMGA1 in vivo, we generated transgenic mice overexpressing HMGA1 in adipose tissues. HMGA1 transgenic mice showed a marked reduction in white and brown adipose tissue mass that was associated with downregulation of genes involved in adipogenesis and concomitant upregulation of preadipocyte markers. Reduced adipogenesis and decreased fat mass were not associated with altered glucose homeostasis since HMGA1 transgenic mice fed a regular-chow diet exhibited normal glucose tolerance and insulin sensitivity. However, when fed a high-fat diet, overexpression of HMGA1 resulted in decreased body-weight gain, reduced fat mass, but improved insulin sensitivity and glucose tolerance. Although HMGA1 transgenic mice exhibited impaired glucose uptake in adipose tissue due to impaired adipogenesis, the increased glucose uptake observed in skeletal muscle may account for the improved glucose homeostasis. Our results indicate that HMGA1 plays an important function in the regulation of white and brown adipogenesis in vivo and suggests that impaired adipocyte differentiation and decreased fat mass is not always associated with impaired whole-body glucose homeostasis.

  11. HMGA1 overexpression in adipose tissue impairs adipogenesis and prevents diet-induced obesity and insulin resistance

    PubMed Central

    Arce-Cerezo, Altamira; García, Miquel; Rodríguez-Nuevo, Aida; Crosa-Bonell, Mireia; Enguix, Natalia; Peró, Albert; Muñoz, Sergio; Roca, Carles; Ramos, David; Franckhauser, Sylvie; Elias, Ivet; Ferre, Tura; Pujol, Anna; Ruberte, Jesús; Villena, Josep A.; Bosch, Fàtima; Riu, Efrén

    2015-01-01

    High-Mobility-Group-A1 (HMGA1) proteins are non-histone proteins that regulate chromatin structure and gene expression during embryogenesis, tumourigenesis and immune responses. In vitro studies suggest that HMGA1 proteins may be required to regulate adipogenesis. To examine the role of HMGA1 in vivo, we generated transgenic mice overexpressing HMGA1 in adipose tissues. HMGA1 transgenic mice showed a marked reduction in white and brown adipose tissue mass that was associated with downregulation of genes involved in adipogenesis and concomitant upregulation of preadipocyte markers. Reduced adipogenesis and decreased fat mass were not associated with altered glucose homeostasis since HMGA1 transgenic mice fed a regular-chow diet exhibited normal glucose tolerance and insulin sensitivity. However, when fed a high-fat diet, overexpression of HMGA1 resulted in decreased body-weight gain, reduced fat mass, but improved insulin sensitivity and glucose tolerance. Although HMGA1 transgenic mice exhibited impaired glucose uptake in adipose tissue due to impaired adipogenesis, the increased glucose uptake observed in skeletal muscle may account for the improved glucose homeostasis. Our results indicate that HMGA1 plays an important function in the regulation of white and brown adipogenesis in vivo and suggests that impaired adipocyte differentiation and decreased fat mass is not always associated with impaired whole-body glucose homeostasis. PMID:26411793

  12. MMP-8 overexpression and persistence of neutrophils relate to stress-impaired healing and poor collagen architecture in mice

    PubMed Central

    Gajendrareddy, Praveen K.; Engeland, Christopher G.; Junges, Roger; Horan, Michael P.; Rojas, Isolde G.; Marucha, Phillip T.

    2013-01-01

    Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) are critical for tissue remodeling during wound repair. Psychological stress has been found to impair wound healing in humans and animals. The objective of this study was to assess MMP and TIMP gene expression during stress-impaired healing. Female SKH-1 mice (n = 299) were divided into control and stress groups (13 h restraint/day for 3 days prior to and 5 days post-wounding). Two 3.5 mm cutaneous full-thickness wounds were placed on the dorsum of each mouse and wound measurements were performed daily. RTPCR for gene expression of MMP-2, MMP-8, MMP-9, TIMP-1 and TIMP-2 was performed at days 1, 3 and 5. Immunohistochemical analyses of the healed wounds were performed at days 15 and 28. As expected, wounds healed more slowly in restraint-stressed mice compared to controls. Stressed mice exhibited MMP-8 overexpression and lower TIMP-1 levels during healing, and poorer collagen organization once healed. MMP-8 overexpression may have stemmed from a higher level of neutrophils, observed in wound tissue on days 3 and 5. These findings implicate higher neutrophil numbers, MMP-8 overexpression, and TIMP-1 under-expression, as mechanisms that may compromise wound outcomes such as scarring under conditions of stress. PMID:23103444

  13. Amphiregulin impairs apoptosis-stimulating protein 2 of p53 overexpression-induced apoptosis in hepatoma cells.

    PubMed

    Liu, Kai; Lin, Dongdong; Ouyang, Yabo; Pang, Lijun; Guo, Xianghua; Wang, Shanshan; Zang, Yunjin; Chen, Dexi

    2017-03-01

    Overexpression of apoptosis-stimulating protein 2 of p53 (ASPP2) induces apoptotic cell death in hepatoma cells (e.g. HepG2 cells) by enhancing the transactivation activity of p53, but long-term ASPP2 overexpression fails to induce more apoptosis since activation of the epidermal growth factor/epidermal growth factor receptor/SOS1 pathway impairs the pro-apoptotic role of ASPP2. In this study, in recombinant adenovirus-ASPP2-infected HepG2 cells, ASPP2 overexpression induces amphiregulin expression in a p53-dependent manner. Although amphiregulin initially contributes to ASPP2-induced apoptosis, it eventually impairs the pro-apoptotic function of ASPP2 by activating the epidermal growth factor/epidermal growth factor receptor/SOS1 pathway, leading to apoptosis resistance. Moreover, blocking soluble amphiregulin with a neutralizing antibody also significantly increased apoptotic cell death of HepG2 cells due to treatment with methyl methanesulfonate, cisplatin, or a recombinant p53 adenovirus, suggesting that the function of amphiregulin involved in inhibiting apoptosis may be a common mechanism by which hepatoma cells escape from stimulus-induced apoptosis. Thus, our data elucidate an apoptosis-evasion mechanism in hepatocellular carcinoma and have potential implications for hepatocellular carcinoma therapy.

  14. Obesity-induced overexpression of miR-802 impairs glucose metabolism through silencing of Hnf1b.

    PubMed

    Kornfeld, Jan-Wilhelm; Baitzel, Catherina; Könner, A Christine; Nicholls, Hayley T; Vogt, Merly C; Herrmanns, Karolin; Scheja, Ludger; Haumaitre, Cécile; Wolf, Anna M; Knippschild, Uwe; Seibler, Jost; Cereghini, Silvia; Heeren, Joerg; Stoffel, Markus; Brüning, Jens C

    2013-02-07

    Insulin resistance represents a hallmark during the development of type 2 diabetes mellitus and in the pathogenesis of obesity-associated disturbances of glucose and lipid metabolism. MicroRNA (miRNA)-dependent post-transcriptional gene silencing has been recognized recently to control gene expression in disease development and progression, including that of insulin-resistant type 2 diabetes. The deregulation of miRNAs miR-143 (ref. 4), miR-181 (ref. 5), and miR-103 and miR-107 (ref. 6) alters hepatic insulin sensitivity. Here we report that the expression of miR-802 is increased in the liver of two obese mouse models and obese human subjects. Inducible transgenic overexpression of miR-802 in mice causes impaired glucose tolerance and attenuates insulin sensitivity, whereas reduction of miR-802 expression improves glucose tolerance and insulin action. We identify Hnf1b (also known as Tcf2) as a target of miR-802-dependent silencing, and show that short hairpin RNA (shRNA)-mediated reduction of Hnf1b in liver causes glucose intolerance, impairs insulin signalling and promotes hepatic gluconeogenesis. In turn, hepatic overexpression of Hnf1b improves insulin sensitivity in Lepr(db/db) mice. Thus, this study defines a critical role for deregulated expression of miR-802 in the development of obesity-associated impairment of glucose metabolism through targeting of Hnf1b, and assigns Hnf1b an unexpected role in the control of hepatic insulin sensitivity.

  15. Histone Deacetylase 5 Is Overexpressed in Scleroderma Endothelial Cells and Impairs Angiogenesis via Repression of Proangiogenic Factors.

    PubMed

    Tsou, Pei-Suen; Wren, Jonathan D; Amin, M Asif; Schiopu, Elena; Fox, David A; Khanna, Dinesh; Sawalha, Amr H

    2016-12-01

    Vascular dysfunction represents a disease-initiating event in systemic sclerosis (SSc; scleroderma). Results of recent studies suggest that epigenetic dysregulation impairs normal angiogenesis and can result in abnormal patterns of blood vessel growth. Histone deacetylases (HDACs) control endothelial cell (EC) proliferation and regulate EC migration. Specifically, HDAC-5 appears to be antiangiogenic. This study was undertaken to test whether HDAC-5 contributes to impaired angiogenesis in SSc by repressing proangiogenic factors in ECs. Dermal ECs were isolated from patients with diffuse cutaneous SSc and healthy controls. Angiogenesis was assessed using an in vitro Matrigel tube formation assay. An assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed to assess and localize the genome-wide effects of HDAC5 knockdown on chromatin accessibility. The expression of HDAC5 was significantly increased in ECs from patients with SSc compared to healthy control ECs. Silencing of HDAC5 in SSc ECs restored normal angiogenesis. HDAC5 knockdown followed by ATAC-seq assay in SSc ECs identified key HDAC5-regulated genes involved in angiogenesis and fibrosis, such as CYR61, PVRL2, and FSTL1. Simultaneous knockdown of HDAC5 in conjunction with either CYR61, PVRL2, or FSTL1 inhibited angiogenesis in SSc ECs. Conversely, overexpression of these genes individually led to an increase in tube formation as assessed by Matrigel assay, suggesting that these genes play functional roles in the impairment of angiogenesis in SSc. Several novel HDAC5-regulated target genes associated with impaired angiogenesis were identified in SSc ECs by ATAC-seq. The results of this study provide a potential link between epigenetic regulation and impaired angiogenesis in SSc, and identify a novel mechanism for the dysregulated angiogenesis that characterizes this disease. © 2016, American College of Rheumatology.

  16. Mice overexpressing p40 in lungs have reduced leucocyte influx and slightly impaired resistance during tuberculosis

    PubMed Central

    Leemans, Jaklien C; Wieland, Catharina W; Florquin, Sandrine; van der Poll, Tom; Vervoordeldonk, Margriet J B M

    2006-01-01

    Interleukin (IL)-12 (p70) is a heterodimeric cytokine composed of p40 and p35, that plays a major role in the protective immune response to Mycobacterium tuberculosis. To define the role of p40 in lungs during pulmonary M. tuberculosis infection we generated transgenic (Tg) mice overexpressing p40 under control of the surfactant protein C promoter. Tg mice expressed the transgene in their lungs, yet demonstrated elevated pulmonary p40 protein levels. After infection, Tg mice displayed higher pulmonary p40 and p70 levels than wild type mice. Interferon-γ concentrations were similar in uninfected and infected Tg and wild type mice, arguing against agonistic effects of p40. Tg mice demonstrated reduced recruitment of macrophages, lymphocytes and neutrophils to the lungs early after infection. This was accompanied by reduced pulmonary tumour necrosis factor-α, macrophage inflammatory protein (MIP)-2 and MIP-1 α levels. This suggests that elevated p40 concentrations inhibited the chemotactic effects of p70 on leucocytes. Furthermore, Tg mice displayed slightly higher pulmonary mycobacterial outgrowth late in the infection than wild type mice. Taken together, we demonstrate that constitutive overexpression of p40 in lungs negatively influences IL-12-mediated leucocyte migration and protection against lung tuberculosis. This suggests a novel antagonistic role for p40 homodimers in regulating the chemotactic bioactivity of IL-12 after pulmonary mycobacterial infection. PMID:16476061

  17. Overexpression of Homer1a in the basal and lateral amygdala impairs fear conditioning and induces an autism-like social impairment.

    PubMed

    Banerjee, Anwesha; Luong, Jonathan A; Ho, Anthony; Saib, Aeshah O; Ploski, Jonathan E

    2016-01-01

    Autism spectrum disorders (ASDs) represent a heterogeneous group of disorders with a wide range of behavioral impairments including social and communication deficits. Apart from these core symptoms, a significant number of ASD individuals display higher levels of anxiety, and some studies indicate that a subset of ASD individuals have a reduced ability to be fear conditioned. Deciphering the molecular basis of ASD has been considerably challenging and it currently remains poorly understood. In this study we examined the molecular basis of autism-like impairments in an environmentally induced animal model of ASD, where pregnant rats are exposed to the known teratogen, valproic acid (VPA), on day 12.5 of gestation and the subsequent progeny exhibit ASD-like symptoms. We focused our analysis on the basal and lateral nucleus of the amygdala (BLA), a region of the brain found to be associated with ASD pathology. We performed whole genome gene expression analysis on the BLA using DNA microarrays to examine differences in gene expression within the amygdala of VPA-exposed animals. We validated one VPA-dysregulated candidate gene (Homer1a) using both quantitative PCR (qRT-PCR) and western blot. Finally, we overexpressed Homer1a within the basal and lateral amygdala of naïve animals utilizing adeno-associated viruses (AAV) and subsequently examined these animals in a battery of behavioral tests associated with ASD, including auditory fear conditioning, social interaction and open field. Our microarray data indicated that Homer1a was one of the genes which exhibited a significant upregulation within the amygdala. We observed an increase in Homer1a messenger RNA (mRNA) and protein in multiple cohorts of VPA-exposed animals indicating that dysregulation of Homer1a levels might underlie some of the symptoms exhibited by VPA-exposed animals. To test this hypothesis, we overexpressed Homer1a within BLA neurons utilizing a viral-mediated approach and found that overexpression of

  18. SK3 Channel Overexpression in Mice Causes Hippocampal Shrinkage Associated with Cognitive Impairments.

    PubMed

    Martin, Sabine; Lazzarini, Marcio; Dullin, Christian; Balakrishnan, Saju; Gomes, Felipe V; Ninkovic, Milena; El Hady, Ahmed; Pardo, Luis A; Stühmer, Walter; Del-Bel, Elaine

    2017-03-01

    The dysfunction of the small-conductance calcium-activated K(+) channel SK3 has been described as one of the factors responsible for the progress of psychoneurological diseases, but the molecular basis of this is largely unknown. This report reveals through use of immunohistochemistry and computational tomography that long-term increased expression of the SK3 small-conductance calcium-activated potassium channel (SK3-T/T) in mice induces a notable bilateral reduction of the hippocampal area (more than 50 %). Histological analysis showed that SK3-T/T mice have cellular disarrangements and neuron discontinuities in the hippocampal formation CA1 and CA3 neuronal layer. SK3 overexpression resulted in cognitive loss as determined by the object recognition test. Electrophysiological examination of hippocampal slices revealed that SK3 channel overexpression induced deficiency of long-term potentiation in hippocampal microcircuits. In association with these results, there were changes at the mRNA levels of some genes involved in Alzheimer's disease and/or linked to schizophrenia, epilepsy, and autism. Taken together, these features suggest that augmenting the function of SK3 ion channel in mice may present a unique opportunity to investigate the neural basis of central nervous system dysfunctions associated with schizophrenia, Alzheimer's disease, or other neuropsychiatric/neurodegenerative disorders in this model system. As a more detailed understanding of the role of the SK3 channel in brain disorders is limited by the lack of specific SK3 antagonists and agonists, the results observed in this study are of significant interest; they suggest a new approach for the development of neuroprotective strategies in neuropsychiatric/neurodegenerative diseases with SK3 representing a potential drug target.

  19. Lentiviral-Mediated Overexpression of the 18 kDa Translocator Protein (TSPO) in the Hippocampal Dentate Gyrus Ameliorates LPS-Induced Cognitive Impairment in Mice

    PubMed Central

    Wang, Wei; Zhang, Liming; Zhang, Xiaoying; Xue, Rui; Li, Lei; Zhao, Weixing; Fu, Qiang; Mi, Weidong; Li, Yunfeng

    2016-01-01

    The 18 kDa translocator protein (TSPO) is involved in the immune/inflammatory response. However, the exact role that TSPO plays in neuroinflammation-induced cognitive impairment is still elusive. The purpose of our present study was to investigate the effects of lentiviral-mediated hippocampal overexpression of the TSPO in a mouse model of LPS-induced cognitive impairment. We established a mouse cognitive impairment model using systematic daily administration of lipopolysaccharide (LPS) (0.5 mg/kg). Microinjection of the dentate gyrus of the mouse with lentiviral vectors, which contained a cDNA targeting TSPO (Lv-TSPO), resulted in a significant increase in TSPO expression and allopregnanolone production. Mice treated with LPS showed cognitive deficits in the novel object recognition test and the Morris water maze test that could be ameliorated by TSPO overexpression. In addition, TSPO overexpression reversed LPS-induced microglial activation and accumulation of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α. Moreover, TSPO overexpression attenuated the LPS-induced impairment of hippocampal neurogenesis. Our results suggest that local overexpression of TSPO in the hippocampal dentate gyrus alleviated LPS-induced cognitive deficits, and its effects might be mediated by the attenuation of inflammatory cytokines, inhibition of microglial activation, and promotion of neurogenesis. PMID:27803668

  20. Overexpression of SlRBZ Results in Chlorosis and Dwarfism through Impairing Chlorophyll, Carotenoid, and Gibberellin Biosynthesis in Tomato

    PubMed Central

    Fan, Mingqin; Gao, Shenghua; Ren, Junling; Yang, Qihong; Li, Hanxia; Yang, Changxian; Ye, Zhibiao

    2016-01-01

    ZFPs play important roles in many biological processes, including plant development, stress response, and phytohormone response. RanBP2-type zinc finger transcription factors have been characterized in animals and humans. However, their functions remain largely unknown in plants. In this study, we identified a RanBP2-type zinc finger protein gene (SlRBZ) in tomato. SlRBZ was constitutively expressed in roots, stems, leaves, flowers, and fruits. The SlRBZ-GFP fused protein was localized in the nucleus. Overexpression of SlRBZ resulted in chlorosis and dwarf phenotypes in tomato. Determination of physiological index showed that chlorophyll, carotenoid, and GAs contents were evidently decreased in transgenic plants. Furthermore, the qRT-PCR and RNA-Seq analyses demonstrated that the transcription of the genes involved in these biosynthesis pathways obviously decreased in SlRBZ-OE plants. In addition, ultrastructural observation by transmission electron microscopy indicated that plastids could not develop into mature chloroplasts with normal chloroplast membrane and thylakoid membrane system in SlRBZ-OE plants. The results suggest that overexpression of SlRBZ may impair the biosynthesis of chlorophyll, carotenoid, and gibberellin through blocking chloroplast development, resulting in chlorosis and dwarfism in tomato. PMID:27446137

  1. Overexpression of EphB2 in hippocampus rescues impaired NMDA receptors trafficking and cognitive dysfunction in Alzheimer model.

    PubMed

    Hu, Rui; Wei, Pan; Jin, Lu; Zheng, Teng; Chen, Wen-Yu; Liu, Xiao-Ya; Shi, Xiao-Dong; Hao, Jing-Ru; Sun, Nan; Gao, Can

    2017-03-30

    Alzheimer's disease (AD) is a progressive neurodegenerative disease, which affects more and more people. But there is still no effective treatment for preventing or reversing the progression of the disease. Soluble amyloid-beta (Aβ) oligomers, also known as Aβ-derived diffusible ligands (ADDLs) play an important role in AD. Synaptic activity and cognition critically depend on the function of glutamate receptors. Targeting N-methyl-D-aspartic acid (NMDA) receptors trafficking and its regulation is a new strategy for AD early treatment. EphB2 is a key regulator of synaptic localization of NMDA receptors. Aβ oligomers could bind to the fibronectin repeats domain of EphB2 and trigger EphB2 degradation in the proteasome. Here we identified that overexpression of EphB2 with lentiviral vectors in dorsal hippocampus improved impaired memory deficits and anxiety or depression-like behaviors in APPswe/PS1-dE9 (APP/PS1) transgenic mice. Phosphorylation and surface expression of GluN2B-containing NMDA receptors were also improved. Overexpression of EphB2 also rescued the ADDLs-induced depletion of the expression of EphB2 and GluN2B-containing NMDA receptors trafficking in cultured hippocampal neurons. These results suggest that improving the decreased expression of EphB2 and subsequent GluN2B-containing NMDA receptors trafficking in hippocampus may be a promising strategy for AD treatment.

  2. Overexpression of SlRBZ Results in Chlorosis and Dwarfism through Impairing Chlorophyll, Carotenoid, and Gibberellin Biosynthesis in Tomato.

    PubMed

    Fan, Mingqin; Gao, Shenghua; Ren, Junling; Yang, Qihong; Li, Hanxia; Yang, Changxian; Ye, Zhibiao

    2016-01-01

    ZFPs play important roles in many biological processes, including plant development, stress response, and phytohormone response. RanBP2-type zinc finger transcription factors have been characterized in animals and humans. However, their functions remain largely unknown in plants. In this study, we identified a RanBP2-type zinc finger protein gene (SlRBZ) in tomato. SlRBZ was constitutively expressed in roots, stems, leaves, flowers, and fruits. The SlRBZ-GFP fused protein was localized in the nucleus. Overexpression of SlRBZ resulted in chlorosis and dwarf phenotypes in tomato. Determination of physiological index showed that chlorophyll, carotenoid, and GAs contents were evidently decreased in transgenic plants. Furthermore, the qRT-PCR and RNA-Seq analyses demonstrated that the transcription of the genes involved in these biosynthesis pathways obviously decreased in SlRBZ-OE plants. In addition, ultrastructural observation by transmission electron microscopy indicated that plastids could not develop into mature chloroplasts with normal chloroplast membrane and thylakoid membrane system in SlRBZ-OE plants. The results suggest that overexpression of SlRBZ may impair the biosynthesis of chlorophyll, carotenoid, and gibberellin through blocking chloroplast development, resulting in chlorosis and dwarfism in tomato.

  3. Overexpression of CB2 cannabinoid receptors decreased vulnerability to anxiety and impaired anxiolytic action of alprazolam in mice.

    PubMed

    García-Gutiérrez, María S; Manzanares, Jorge

    2011-01-01

    Mice overexpressing CB2r (CB2xP) were exposed to open field (OF), light-dark box (LDB) and elevated plus maze (EPM) tests. Corticotropin-releasing factor (CRF) and pro-opiomelanocortin (POMC) mRNA were measured in paraventricular (PVN) and arcuate (ARC) nuclei of the hypothalamus after 30 minutes of restraint stress (RS). Anxiolytic effects of alprazolam (45 or 70 µg/kg, ip) were evaluated. GABA(A)α(2) and GABA(A)γ(2) mRNA were measured in the hippocampus (HIPP) and amygdala (AMY) of CB2xP and wild type (WT) mice. No differences were observed in the total distance travelled by CB2xP and WT mice in OF. Central and peripheral distances travelled significantly increased and decreased in CB2xP mice. Overexpression of CB2r reduced anxiety-like behaviours in LDB and EPM. In WT mice, RS increased CRF (82%) and POMC (42%) mRNA in the PVN and ARC nuclei, respectively. In CB2xP mice, RS also increased POMC (22%) mRNA in the ARC nucleus, but had no effect on CRF mRNA in the PVN nucleus. Administration of alprazolam was without effect in CB2xP mice. An increase of GABA(A)α(2) and GABA(A)γ(2) mRNA in the hippocampus and amygdala of CB2xP mice was observed. Our findings revealed that increased expression of CB2r significantly reduced anxiogenic-related behaviours, modified the response to stress and impaired the action of anxiolytic drugs.

  4. Intracellular Aβ pathology and early cognitive impairments in a transgenic rat overexpressing human amyloid precursor protein: a multidimensional study.

    PubMed

    Iulita, M Florencia; Allard, Simon; Richter, Luise; Munter, Lisa-Marie; Ducatenzeiler, Adriana; Weise, Christoph; Do Carmo, Sonia; Klein, William L; Multhaup, Gerhard; Cuello, A Claudio

    2014-06-05

    Numerous studies have implicated the abnormal accumulation of intraneuronal amyloid-β (Aβ) as an important contributor to Alzheimer's disease (AD) pathology, capable of triggering neuroinflammation, tau hyperphosphorylation and cognitive deficits. However, the occurrence and pathological relevance of intracellular Aβ remain a matter of controversial debate. In this study, we have used a multidimensional approach including high-magnification and super-resolution microscopy, cerebro-spinal fluid (CSF) mass spectrometry analysis and ELISA to investigate the Aβ pathology and its associated cognitive impairments, in a novel transgenic rat model overexpressing human APP. Our microscopy studies with quantitative co-localization analysis revealed the presence of intraneuronal Aβ in transgenic rats, with an immunological signal that was clearly distinguished from that of the amyloid precursor protein (APP) and its C-terminal fragments (CTFs). The early intraneuronal pathology was accompanied by a significant elevation of soluble Aβ42 peptides that paralleled the presence and progression of early cognitive deficits, several months prior to amyloid plaque deposition. Aβ38, Aβ39, Aβ40 and Aβ42 peptides were detected in the rat CSF by MALDI-MS analysis even at the plaque-free stages; suggesting that a combination of intracellular and soluble extracellular Aβ may be responsible for impairing cognition at early time points. Taken together, our results demonstrate that the intraneuronal development of AD-like amyloid pathology includes a mixture of molecular species (Aβ, APP and CTFs) of which a considerable component is Aβ; and that the early presence of these species within neurons has deleterious effects in the CNS, even before the development of full-blown AD-like pathology.

  5. Synaptic transmission is impaired prior to plaque formation in amyloid precursor protein-overexpressing mice without altering behaviorally-correlated sharp wave-ripple complexes.

    PubMed

    Hermann, D; Both, M; Ebert, U; Gross, G; Schoemaker, H; Draguhn, A; Wicke, K; Nimmrich, V

    2009-09-15

    One of the hallmarks of Alzheimer's disease is the accumulation of amyloid plaques in brains of affected patients. Several recent studies provided evidence that soluble oligomer forms of amyloid-beta (Abeta) rather than plaques determine cognitive decline. In vitro studies using artificial Abeta oligomer preparations suggest that such pathophysiology is caused by a specific impairment of synaptic function. We examined whether synaptic deficits occur before deposition of insoluble fibrillar Abeta by analyzing brain slices taken from young Tg2576 mice overexpressing mutant amyloid precursor protein. Excitatory synaptic transmission in the hippocampal CA1 region was strongly impaired before plaque development, suggesting a dissociation of an early synaptic impairment, probably caused by soluble oligomeric amyloid-beta, from subsequent plaque formation. At higher age neurotransmission was also decreased in wild type mice, paralleling a cognitive decline of normal aged animals. Memory formation in rats is accompanied by distinct hippocampal network oscillations. It has recently been shown that hippocampal gamma oscillations, a network correlate of exploratory behavior, are impaired in amyloid precursor protein (APP)-overexpressing mice. We determined whether sharp wave-ripple complexes, which contribute to memory consolidation during slow wave-sleep, are modified in Tg2576 mice. Interestingly, neither sharp waves nor superimposed ripples were changed at pre-plaque or plaque stages. During aging, however, there was a strong reduction of sharp wave frequency and ripple energy in wild type and APP-overexpressing animals. This indicates that the reported changes in network oscillations following APP-overexpression are specific for gamma oscillations, whereas aging has a more general effect on network properties. Taken together our data suggest that non-fibrillar forms of Abeta--possibly Abeta oligomers--specifically interfere with synaptic function in Tg2576, but do not

  6. Emotional memory impairments induced by AAV-mediated overexpression of human α-synuclein in dopaminergic neurons of the ventral tegmental area.

    PubMed

    Alvarsson, A; Caudal, D; Björklund, A; Svenningsson, P

    2016-01-01

    Parkinson's disease (PD) is associated with extensive degeneration of dopaminergic neurons originating in the substantia nigra pars compacta, but neuronal loss is also found in the ventral tegmental area (VTA). The VTA projects to areas involved in cognitive and emotional processes, including hippocampus, amygdala, nucleus accumbens and prefrontal cortex, and has thus been proposed to play a role in emotional memory impairments in PD. Since the formation of α-synuclein inclusions throughout the central nervous system is a pathological hallmark of PD, we studied the progressive effects of α-synuclein overexpression in the VTA on motor functions, emotional behaviour and emotional memory. Adeno-associated viral (AAV) vectors encoding either human α-synuclein or green fluorescent protein (GFP) were injected stereotactically into the VTA, and behaviour was monitored 3 and 8 weeks following AAV injection. At week 8, there was a 22% reduction of TH+ neurons in the VTA. We demonstrate that α-synuclein overexpression in dopaminergic neurons of the VTA induced mild motor deficits that appeared 3 weeks following AAV-α-synuclein injection and were aggravated at week 8. No depressive- or anxiety-like behaviours were found. To address emotional memory, we used the passive avoidance test, a one-trial associative learning paradigm based on contextual conditioning which requires minimal training. Interestingly, emotional memory impairments were found in α-synuclein overexpressing animals at week 8. These findings indicate that α-synuclein overexpression induces progressive memory impairments likely caused by a loss of function of mesolimbic dopaminergic projections. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. [Effect of Moxibustion on Learning-memory Ability and Hippocampal Amyloid beta Protein Overexpression in Mild Cognitive Impairment Rats].

    PubMed

    Zhu, Cai-feng; Sun, Jian-jian; Han, Wei; Yang, Jun

    2016-04-01

    To observe the effect of moxibustion of "Baihui" (GV 20), etc. on learning-memory ability, hip- pocampal amyloid beta (AP) protein expression and immune activity in mild cognitive impairment (MCI) rats, so as to reveal its mechanism underlying improving cognitive impairment. A total of 48 SD rats were randomly divided into normal, model, moxibustion, and medication groups (n = 12 in each group). The MCI model was established by intraperitoneal injection of 2 mL mixture solution containing D-galactose (120 mg - kg- - d-) and Sodium Nitrite (90 mg x kg(-1) x d(-1)), once daily for 40 days. Moxibustion (separated by Radix Aconiti Praeparata cake) was applied to "Baihui" (GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14) for 20 min, once daily for 2 weeks, with one day's rest between two weeks. The rats of the medication group were given with Nimodipine (2 mg x kg(-1) x d(-1), t.i.d.) by lavage for 2 weeks (except Sundays). The learning-memory ability was detected by Morris maze water swimming tasks. The expression level of hippocampal AP protein was detected by immunohistochemistry, and those of hippocampal presenilin-1 (PS-1) mRNA and cleaving enzyme (BACE-1) mRNA were detected by real time-PCR, and serum IL-6 level was assayed by ELISA. Following modeling, the average escape latency of location navigation tests of Morris maze water swimming tests, the expression levels of hippocampal Abeta protein, PS-1 mRNA and BACE-1 mRNA, and serum IL-6 content were significantly increased in the model group( P<0.01) , while the target-platform crossing times and the percentage of target-quadrant swimming duration of spacial probe trials were remarkably decreased in the model group (P<0.01). After moxibustion, the increased escape latency, hippocampal AP protein, PS-i mHNA and BACE-1 mRNA ex- pression and serum IL-6 content, and the decreased target-platform crossing times and the percentage of target-quadrant swim- ming duration were reversed in both moxibustion and medication

  8. Overexpression of the ped/pea-15 gene causes diabetes by impairing glucose-stimulated insulin secretion in addition to insulin action.

    PubMed

    Vigliotta, Giovanni; Miele, Claudia; Santopietro, Stefania; Portella, Giuseppe; Perfetti, Anna; Maitan, Maria Alessandra; Cassese, Angela; Oriente, Francesco; Trencia, Alessandra; Fiory, Francesca; Romano, Chiara; Tiveron, Cecilia; Tatangelo, Laura; Troncone, Giancarlo; Formisano, Pietro; Beguinot, Francesco

    2004-06-01

    Overexpression of the ped/pea-15 gene is a common feature of type 2 diabetes. In the present work, we show that transgenic mice ubiquitously overexpressing ped/pea-15 exhibited mildly elevated random-fed blood glucose levels and decreased glucose tolerance. Treatment with a 60% fat diet led ped/pea-15 transgenic mice to develop diabetes. Consistent with insulin resistance in these mice, insulin administration reduced glucose levels by only 35% after 45 min, compared to 70% in control mice. In vivo, insulin-stimulated glucose uptake was decreased by almost 50% in fat and muscle tissues of the ped/pea-15 transgenic mice, accompanied by protein kinase Calpha activation and block of insulin induction of protein kinase Czeta. These changes persisted in isolated adipocytes from the transgenic mice and were rescued by the protein kinase C inhibitor bisindolylmaleimide. In addition to insulin resistance, ped/pea-15 transgenic mice showed a 70% reduction in insulin response to glucose loading. Stable overexpression of ped/pea-15 in the glucose-responsive MIN6 beta-cell line also caused protein kinase Calpha activation and a marked decline in glucose-stimulated insulin secretion. Antisense block of endogenous ped/pea-15 increased glucose sensitivity by 2.5-fold in these cells. Thus, in vivo, overexpression of ped/pea-15 may lead to diabetes by impairing insulin secretion in addition to insulin action.

  9. A mouse model for MeCP2 duplication syndrome: MeCP2 overexpression impairs learning and memory and synaptic transmission.

    PubMed

    Na, Elisa S; Nelson, Erika D; Adachi, Megumi; Autry, Anita E; Mahgoub, Melissa A; Kavalali, Ege T; Monteggia, Lisa M

    2012-02-29

    Rett syndrome and MECP2 duplication syndrome are neurodevelopmental disorders that arise from loss-of-function and gain-of-function alterations in methyl-CpG binding protein 2 (MeCP2) expression, respectively. Although there have been studies examining MeCP2 loss of function in animal models, there is limited information on MeCP2 overexpression in animal models. Here, we characterize a mouse line with MeCP2 overexpression restricted to neurons (Tau-Mecp2). This MeCP2 overexpression line shows motor coordination deficits, heightened anxiety, and impairments in learning and memory that are accompanied by deficits in long-term potentiation and short-term synaptic plasticity. Whole-cell voltage-clamp recordings of cultured hippocampal neurons from Tau-Mecp2 mice reveal augmented frequency of miniature EPSCs with no change in miniature IPSCs, indicating that overexpression of MeCP2 selectively impacts excitatory synapse function. Moreover, we show that alterations in transcriptional repression mechanisms underlie the synaptic phenotypes in hippocampal neurons from the Tau-Mecp2 mice. These results demonstrate that the Tau-Mecp2 mouse line recapitulates many key phenotypes of MECP2 duplication syndrome and support the use of these mice to further study this devastating disorder.

  10. Selective overexpression of Toll-like receptor-4 in skeletal muscle impairs metabolic adaptation to high-fat feeding

    PubMed Central

    McMillan, Ryan P.; Wu, Yaru; Voelker, Kevin; Fundaro, Gabrielle; Kavanaugh, John; Stevens, Joseph R.; Shabrokh, Elika; Ali, Mostafa; Harvey, Mordecai; Anderson, Angela S.; Boutagy, Nabil E.; Mynatt, Randall L.; Frisard, Madlyn I.

    2015-01-01

    Toll-like receptor-4 (TLR-4) is elevated in skeletal muscle of obese humans, and data from our laboratory have shown that activation of TLR-4 in skeletal muscle via LPS results in decreased fatty acid oxidation (FAO). The purpose of this study was to determine whether overexpression of TLR-4 in skeletal muscle alters mitochondrial function and whole body metabolism in the context of a chow and high-fat diet. C57BL/6J mice (males, 6–8 mo of age) with skeletal muscle-specific overexpression of the TLR-4 (mTLR-4) gene were created and used for this study. Isolated mitochondria and whole muscle homogenates from rodent skeletal muscle (gastrocnemius and quadriceps) were investigated. TLR-4 overexpression resulted in a significant reduction in FAO in muscle homogenates; however, mitochondrial respiration and reactive oxygen species (ROS) production did not appear to be affected on a standard chow diet. To determine the role of TLR-4 overexpression in skeletal muscle in response to high-fat feeding, mTLR-4 mice and WT control mice were fed low- and high-fat diets for 16 wk. The high-fat diet significantly decreased FAO in mTLR-4 mice, which was observed in concert with elevated body weight and fat, greater glucose intolerance, and increase in production of ROS and cellular oxidative damage compared with WT littermates. These findings suggest that TLR-4 plays an important role in the metabolic response in skeletal muscle to high-fat feeding. PMID:26084695

  11. Selective overexpression of Toll-like receptor-4 in skeletal muscle impairs metabolic adaptation to high-fat feeding.

    PubMed

    McMillan, Ryan P; Wu, Yaru; Voelker, Kevin; Fundaro, Gabrielle; Kavanaugh, John; Stevens, Joseph R; Shabrokh, Elika; Ali, Mostafa; Harvey, Mordecai; Anderson, Angela S; Boutagy, Nabil E; Mynatt, Randall L; Frisard, Madlyn I; Hulver, Matthew W

    2015-08-01

    Toll-like receptor-4 (TLR-4) is elevated in skeletal muscle of obese humans, and data from our laboratory have shown that activation of TLR-4 in skeletal muscle via LPS results in decreased fatty acid oxidation (FAO). The purpose of this study was to determine whether overexpression of TLR-4 in skeletal muscle alters mitochondrial function and whole body metabolism in the context of a chow and high-fat diet. C57BL/6J mice (males, 6-8 mo of age) with skeletal muscle-specific overexpression of the TLR-4 (mTLR-4) gene were created and used for this study. Isolated mitochondria and whole muscle homogenates from rodent skeletal muscle (gastrocnemius and quadriceps) were investigated. TLR-4 overexpression resulted in a significant reduction in FAO in muscle homogenates; however, mitochondrial respiration and reactive oxygen species (ROS) production did not appear to be affected on a standard chow diet. To determine the role of TLR-4 overexpression in skeletal muscle in response to high-fat feeding, mTLR-4 mice and WT control mice were fed low- and high-fat diets for 16 wk. The high-fat diet significantly decreased FAO in mTLR-4 mice, which was observed in concert with elevated body weight and fat, greater glucose intolerance, and increase in production of ROS and cellular oxidative damage compared with WT littermates. These findings suggest that TLR-4 plays an important role in the metabolic response in skeletal muscle to high-fat feeding. Copyright © 2015 the American Physiological Society.

  12. SET overexpression decreases cell detoxification efficiency: ALDH2 and GSTP1 are downregulated, DDR is impaired and DNA damage accumulates.

    PubMed

    Almeida, Luciana O; Goto, Renata N; Pestana, Cezar R; Uyemura, Sérgio A; Gutkind, Silvio; Curti, Carlos; Leopoldino, Andréia M

    2012-12-01

    Alcohol and tobacco consumption are risk factors for head and neck squamous cell carcinoma (HNSCC). Aldehyde dehydrogenase 2 (ALDH2) and glutathione S-transferase pi 1 (GSTP1) are important enzymes for cellular detoxification and low efficiencies are implicated in cancer. We assessed the potential role of SET protein overexpression, a histone acetylation modulator accumulated in HNSCC, in gene regulation and protein activity of ALDH2 and GSTP1. SET was knocked down in HN13, HN12 and Cal27, and overexpressed in HEK293 cells; ethanol and cisplatin were the chemical agents. Cells with SET overexpression (HEK293/SET, HN13 and HN12) showed lower ALDH2 and GSTP1 mRNA levels and trichostatin A increased them (real-time PCR). Ethanol upregulated GSTP1 and ALDH2 mRNAs, whereas cisplatin upregulated GSTP1 in HEK293 cells. SET-chromatin binding revealed SET interaction with ALDH2 and GSTP1 promoters, specifically via SET NAP domain; ethanol and cisplatin abolished SET binding. ALDH2 and GSTP1 efficiency was assessed by enzymatic and comet assay. A lower ALDH2 activity was associated with greater DNA damage (tail intensity) in HEK293/SET compared with HEK293 cells, whereas HN13/siSET showed ALDH2 activity higher than HN13 cells. HN13/siSET cells showed increased tail intensity. Cisplatin-induced DNA damage response showed negative relationship between SET overexpression and BRCA2 recruitment. SET downregulated repair genes ATM, BRCA1 and CHEK2 and upregulated TP53. Cisplatin-induced cell-cycle arrest occurred in G(0) /G(1) and S in HEK293 cells, whereas HEK293/SET showed G(2) /M stalling. Overall, cisplatin was more cytotoxic for HN13 than HN13/siSET cells. Our data suggest a role for SET in cellular detoxification, DNA damage response and genome integrity.

  13. FGFR2 is overexpressed in myxoid liposarcoma and inhibition of FGFR signaling impairs tumor growth in vitro

    PubMed Central

    Künstlinger, Helen; Fassunke, Jana; Schildhaus, Hans-Ulrich; Brors, Benedikt; Heydt, Carina; Ihle, Michaela Angelika; Mechtersheimer, Gunhild; Wardelmann, Eva; Büttner, Reinhard; Merkelbach-Bruse, Sabine

    2015-01-01

    Myxoid liposarcomas account for more than one third of liposarcomas and about 10% of all adult soft tissue sarcomas. The tumors are characterized by specific chromosomal translocations leading to the chimeric oncogenes FUS-DDIT3 or EWS1R-DDIT3. The encoded fusion proteins act as aberrant transcription factors. Therefore, we implemented comparative expression analyses using whole-genome microarrays in tumor and fat tissue samples. We aimed at identifying differentially expressed genes which may serve as diagnostic or prognostic biomarkers or as therapeutic targets. Microarray analyses revealed overexpression of FGFR2 and other members of the FGF/FGFR family. Overexpression of FGFR2 was validated by qPCR, immunohistochemistry and western blot analysis in primary tumor samples. Treatment of the myxoid liposarcoma cell lines MLS 402 and MLS 1765 with the FGFR inhibitors PD173074, TKI258 (dovitinib) and BGJ398 as well as specific siRNAs reduced cell proliferation, induced apoptosis and delayed cell migration. Combination of FGFR inhibitors with trabectedin further increased the effect. Our study demonstrates overexpression of FGFR2 and a functional role of FGFR signaling in myxoid liposarcoma. As FGFR inhibition showed effects on proliferation and cell migration and induced apoptosis in vitro, our data indicate the potential use of FGFR inhibitors as a targeted therapy for these tumors. PMID:26036639

  14. FGFR2 is overexpressed in myxoid liposarcoma and inhibition of FGFR signaling impairs tumor growth in vitro.

    PubMed

    Künstlinger, Helen; Fassunke, Jana; Schildhaus, Hans-Ulrich; Brors, Benedikt; Heydt, Carina; Ihle, Michaela Angelika; Mechtersheimer, Gunhild; Wardelmann, Eva; Büttner, Reinhard; Merkelbach-Bruse, Sabine

    2015-08-21

    Myxoid liposarcomas account for more than one third of liposarcomas and about 10% of all adult soft tissue sarcomas. The tumors are characterized by specific chromosomal translocations leading to the chimeric oncogenes FUS-DDIT3 or EWS1R-DDIT3. The encoded fusion proteins act as aberrant transcription factors. Therefore, we implemented comparative expression analyses using whole-genome microarrays in tumor and fat tissue samples. We aimed at identifying differentially expressed genes which may serve as diagnostic or prognostic biomarkers or as therapeutic targets. Microarray analyses revealed overexpression of FGFR2 and other members of the FGF/FGFR family. Overexpression of FGFR2 was validated by qPCR, immunohistochemistry and western blot analysis in primary tumor samples. Treatment of the myxoid liposarcoma cell lines MLS 402 and MLS 1765 with the FGFR inhibitors PD173074, TKI258 (dovitinib) and BGJ398 as well as specific siRNAs reduced cell proliferation, induced apoptosis and delayed cell migration. Combination of FGFR inhibitors with trabectedin further increased the effect. Our study demonstrates overexpression of FGFR2 and a functional role of FGFR signaling in myxoid liposarcoma. As FGFR inhibition showed effects on proliferation and cell migration and induced apoptosis in vitro, our data indicate the potential use of FGFR inhibitors as a targeted therapy for these tumors.

  15. Reverse of age-dependent memory impairment and mitochondrial DNA damage in microglia by an overexpression of human mitochondrial transcription factor a in mice.

    PubMed

    Hayashi, Yoshinori; Yoshida, Masayoshi; Yamato, Mayumi; Ide, Tomomi; Wu, Zhou; Ochi-Shindou, Mayumi; Kanki, Tomotake; Kang, Dongchon; Sunagawa, Kenji; Tsutsui, Hiroyuki; Nakanishi, Hiroshi

    2008-08-20

    Mitochondrial DNA (mtDNA) is highly susceptible to injury induced by reactive oxygen species (ROS). During aging, mutations of mtDNA accumulate to induce dysfunction of the respiratory chain, resulting in the enhanced ROS production. Therefore, age-dependent memory impairment may result from oxidative stress derived from the respiratory chain. Mitochondrial transcription factor A (TFAM) is now known to have roles not only in the replication of mtDNA but also its maintenance. We herein report that an overexpression of TFAM in HeLa cells significantly inhibited rotenone-induced mitochondrial ROS generation and the subsequent NF-kappaB (nuclear factor-kappaB) nuclear translocation. Furthermore, TFAM transgenic (TG) mice exhibited a prominent amelioration of an age-dependent accumulation of lipid peroxidation products and a decline in the activities of complexes I and IV in the brain. In the aged TG mice, deficits of the motor learning memory, the working memory, and the hippocampal long-term potentiation (LTP) were also significantly improved. The expression level of interleukin-1beta (IL-1beta) and mtDNA damages, which were predominantly found in microglia, significantly decreased in the aged TG mice. The IL-1beta amount markedly increased in the brain of the TG mice after treatment with lipopolysaccharide (LPS), whereas its mean amount was significantly lower than that of the LPS-treated aged wild-type mice. At the same time, an increased mtDNA damage in microglia and an impaired hippocampal LTP were also observed in the LPS-treated aged TG mice. Together, an overexpression of TFAM is therefore considered to ameliorate age-dependent impairment of the brain functions through the prevention of oxidative stress and mitochondrial dysfunctions in microglia.

  16. Impaired repression at a vasopressin promoter polymorphism underlies overexpression of vasopressin in a rat model of trait anxiety.

    PubMed

    Murgatroyd, Chris; Wigger, Alexandra; Frank, Elisabeth; Singewald, Nicolas; Bunck, Mirjam; Holsboer, Florian; Landgraf, Rainer; Spengler, Dietmar

    2004-09-01

    Two inbred rat lines have been developed that show either high (HAB) or low (LAB) anxiety-related behavior. The behavioral phenotype correlates with arginine vasopressin (AVP) expression at the level of the hypothalamic paraventricular nucleus (PVN), but not supraoptic nucleus, with HAB animals overexpressing the neuropeptide in both magnocellular and parvocellular subdivisions of the PVN. We detected a number of single nucleotide polymorphisms (SNPs) in the AVP locus that differ between the HAB and LAB animals, two of which were embedded in cis-regulatory elements. The HAB-specific allele of the AVP gene promoter occurs in 1.5% of outbred Wistar rats and is more transcriptionally active in vivo, as revealed by allele-specific transcription studies in cross-mated HAB/LAB F1 animals. Interestingly, one specific SNP [A(-1276)G] conferred reduced binding of the transcriptional repressor CArG binding factor A (CBF-A) in the HAB allele, the consequent differential regulation of the AVP promoter resulting in an overexpression of AVP in vitro and in vivo. Furthermore, CBF-A is highly coexpressed in AVP-containing neurons of the PVN supporting an important role for regulation of AVP gene expression in vivo. Taken together, our results demonstrate a role for an AVP gene polymorphism and CBF-A in elevated AVP expression in the PVN of HAB rats likely to contribute to their behavioral and neuroendocrine phenotype.

  17. Altered intracellular distribution of PrPC and impairment of proteasome activity in tau overexpressing cortical neurons.

    PubMed

    Canu, Nadia; Filesi, Ilaria; Pristerà, Andrea; Ciotti, Maria Teresa; Biocca, Silvia

    2011-01-01

    The microtubule associated protein tau plays a crucial role in Alzheimer's disease and in many neurodegenerative disorders collectively known as tauopathies. Recently, tau pathology has been also documented in prion diseases although the possible molecular events linking these two proteins are still unknown. We have investigated the fate of normal cellular prion protein (PrP(C)) in primary cortical neurons overexpressing tau protein. We found that overexpression of tau reduces PrP(C) expression at the cell surface and causes its accumulation and aggregation in the cell body but does not affect its maturation and glycosylation. Trapped PrP(C) forms detergent-insoluble aggregates, mainly composed of un-glycosylated and mono-glycosylated forms of prion protein. Interestingly, co-transfection of tau gene in cortical neurons with a proteasome activity reporter, consisting of a short peptide degron fused to the carboxyl-terminus of green fluorescent protein (GFP-CL1), results in down-regulation of the proteasome system, suggesting a possible mechanism that contributes to intracellular PrP(C) accumulation. These findings open a new perspective for the possible crosstalk between tau and prion proteins in the pathogenesis of tau induced-neurodegeneration.

  18. Specific epiblast loss and hypoblast impairment in cattle embryos sensitized to survival signalling by ubiquitous overexpression of the proapoptotic gene BAD.

    PubMed

    van Leeuwen, Jessica; Berg, Debra K; Smith, Craig S; Wells, David N; Pfeffer, Peter L

    2014-01-01

    Early embryonic lethality is common, particularly in dairy cattle. We made cattle embryos more sensitive to environmental stressors by raising the threshold of embryo survival signaling required to overcome the deleterious effects of overexpressing the proapoptotic protein BAD. Two primary fibroblast cell lines expressing BAD and exhibiting increased sensitivity to stress-induced apoptosis were used to generate transgenic Day 13/14 BAD embryos. Transgenic embryos were normal in terms of retrieval rates, average embryo length or expression levels of the trophectoderm marker ASCL2. However both lines of BAD-tg embryos lost the embryonic disc and thus the entire epiblast lineage at significantly greater frequencies than either co-transferrred IVP controls or LacZ-tg embryos. Embryos without epiblast still contained the second ICM-derived lineage, the hypopblast, albeit frequently in an impaired state, as shown by reduced expression of the hypoblast markers GATA4 and FIBRONECTIN. This indicates a gradient of sensitivity (epiblast > hypoblast > TE) to BAD overexpression. We postulate that the greater sensitivity of specifically the epiblast lineage that we have seen in our transgenic model, reflects an inherent greater susceptibility of this lineage to environmental stress and may underlie the epiblast-specific death seen in phantom pregnancies.

  19. Over-expression of Runx1 transcription factor impairs the development of thymocytes from the double-negative to double-positive stages.

    PubMed

    Wong, Won F; Nakazato, Megumi; Watanabe, Toshio; Kohu, Kazuyoshi; Ogata, Takehiro; Yoshida, Naomi; Sotomaru, Yusuke; Ito, Mamoru; Araki, Kimi; Telfer, Janice; Fukumoto, Manabu; Suzuki, Daisuke; Sato, Takehito; Hozumi, Katsuto; Habu, Sonoko; Satake, Masanobu

    2010-06-01

    Runx1 transcription factor is highly expressed at a CD4/CD8-double-negative (DN) stage of thymocyte development but is down-regulated when cells proceed to the double-positive (DP) stage. In the present study, we examined whether the down-regulation of Runx1 is necessary for thymocyte differentiation from the DN to DP stage. When Runx1 was artificially over-expressed in thymocytes by Lck-driven Cre, the DN3 population was unaffected, as exemplified by proper pre-T-cell receptor expression, whereas the DN4 population was perturbed as shown by the decrease in the CD27(hi) sub-fraction. In parallel, the growth rate of DN4 cells was reduced by half, as measured by bromodeoxyuridine incorporation. These events impaired the transition of DN4 cells to the DP stage, resulting in the drastic reduction of the number of DP thymocytes. The Runx1 gene has two promoters, a proximal and a distal promoter; and, in thymocytes, endogenous Runx1 was mainly transcribed from the distal promoter. Interestingly, only distal, but not proximal, Runx1 over-expression exhibited an inhibitory effect on thymocyte differentiation, suggesting that the distal Runx1 protein may fulfil a unique function. Our collective results indicate that production of the distal Runx1 protein must be adequately down-regulated for thymocytes to transit from the DN to the DP stage, a critical step in the massive expansion of the T-cell lineage.

  20. Impairment of Mitochondria in Adult Mouse Brain Overexpressing Predominantly Full-Length, N-Terminally Acetylated Human α-Synuclein

    PubMed Central

    Sarafian, Theodore A.; Ryan, Christopher M.; Souda, Puneet; Masliah, Eliezer; Kar, Upendra K.; Vinters, Harry V.; Mathern, Gary W.; Faull, Kym F.; Whitelegge, Julian P.; Watson, Joseph B.

    2013-01-01

    While most forms of Parkinson’s Disease (PD) are sporadic in nature, a small percentage of PD have genetic causes as first described for dominant, single base pair changes as well as duplication and triplication in the α-synuclein gene. The α-synuclein gene encodes a 140 amino acid residue protein that interacts with a variety of organelles including synaptic vesicles, lysosomes, endoplasmic reticulum/Golgi vesicles and, reported more recently, mitochondria. Here we examined the structural and functional interactions of human α-synuclein with brain mitochondria obtained from an early, pre-manifest mouse model for PD over-expressing human α-synuclein (ASOTg). The membrane potential in ASOTg brain mitochondria was decreased relative to wildtype (WT) mitochondria, while reactive oxygen species (ROS) were elevated in ASOTg brain mitochondria. No selective interaction of human α-synuclein with mitochondrial electron transport complexes cI-cV was detected. Monomeric human α-synuclein plus carboxyl terminally truncated forms were the predominant isoforms detected in ASOTg brain mitochondria by 2-dimensional PAGE (Native/SDS) and immunoblotting. Oligomers or fibrils were not detected with amyloid conformational antibodies. Mass spectrometry of human α-synuclein in both ASOTg brain mitochondria and homogenates from surgically resected human cortex demonstrated that the protein was full-length and postranslationally modified by N-terminal acetylation. Overall the study showed that accumulation of full-length, N-terminally acetylated human α-synuclein was sufficient to disrupt brain mitochondrial function in adult mice. PMID:23667637

  1. Selective Impairment of TH17-Differentiation and Protection against Autoimmune Arthritis after Overexpression of BCL2A1 in T Lymphocytes

    PubMed Central

    Iglesias, Marcos; Augustin, Juan Jesús; Alvarez, Pilar; Santiuste, Inés; Postigo, Jorge; Merino, Jesús; Merino, Ramón

    2016-01-01

    The inhibition of apoptotic cell death in T cells through the dysregulated expression of BCL2 family members has been associated with the protection against the development of different autoimmune diseases. However, multiple mechanisms were proposed to be responsible for such protective effect. The purpose of this study was to explore the effect of the T-cell overexpression of BCL2A1, an anti-apoptotic BCL2 family member without an effect on cell cycle progression, in the development of collagen-induced arthritis. Our results demonstrated an attenuated development of arthritis in these transgenic mice. The protective effect was unrelated to the suppressive activity of regulatory T cells but it was associated with a defective activation of p38 mitogen-activated protein kinase in CD4+ cells after in vitro TCR stimulation. In addition, the in vitro and in vivo TH17 differentiation were impaired in BCL2A1 transgenic mice. Taken together, we demonstrated here a previously unknown role for BCL2A1 controlling the activation of CD4+ cells and their differentiation into pathogenic proinflammatory TH17 cells and identified BCL2A1 as a potential target in the control of autoimmune/inflammatory diseases. PMID:27433938

  2. Impaired natriuretic response to high-NaCl diet plus aldosterone infusion in mice overexpressing human CD39, an ectonucleotidase (NTPDase1)

    PubMed Central

    Zhang, Yue; Robson, Simon C.; Morris, Kaiya L.; Heiney, Kristina M.; Dwyer, Karen M.; Ecelbarger, Carolyn M.

    2015-01-01

    Extracellular nucleotides acting through P2 receptors facilitate natriuresis. To define how purinergic mechanisms are involved in sodium homeostasis, we used transgenic (TG) mice that globally overexpress human CD39 (hCD39, NTPDase1), an ectonucleotidase that hydrolyzes extracellular ATP/ADP to AMP, resulting in an altered extracellular purine profile. On a high-sodium diet (HSD, 3.5% Na+), urine volume and serum sodium were significantly higher in TG mice but sodium excretion was unaltered. Furthermore, TG mice showed an attenuated fall in urine aldosterone with HSD. Western blot analysis revealed significantly lower densities (∼40%) of the β-subunit of the epithelial sodium channel (ENaC) in medulla, and the major band (85-kDa) of γ-ENaC in TG mice cortex. To evaluate aldosterone-independent differences, in a second experiment, aldosterone was clamped by osmotic minipump at 20 μg/day, and mice were fed either an HSD or a low-sodium diet (LSD, 0.03% Na+). Here, no differences in urine volume or osmolality, or serum aldosterone were found, but TG mice showed a modest, yet significant impairment in late natriuresis (days 3 and 4). Several major sodium transporters or channel subunits were differentially expressed between the genotypes. HSD caused a downregulation of Na-Cl cotransporter (NCC) in both genotypes; and had higher cortical levels of NCC, Na-K-ATPase (α-1 subunit), and α- and γ-ENaC. The Na-K-2Cl cotransporter (NKCC2) was downregulated by HSD in wild-type mice, but it increased in TG mice. In summary, our data support the concept that extracellular nucleotides facilitate natriuresis; they also reveal an aldosterone-independent downregulation of major renal sodium transporters and channel subunits by purinergic signaling. PMID:25877509

  3. TREM2 Overexpression has No Improvement on Neuropathology and Cognitive Impairment in Aging APPswe/PS1dE9 Mice.

    PubMed

    Jiang, Teng; Wan, Yu; Zhang, Ying-Dong; Zhou, Jun-Shan; Gao, Qing; Zhu, Xi-Chen; Shi, Jian-Quan; Lu, Huan; Tan, Lan; Yu, Jin-Tai

    2017-03-01

    Previously, we showed that overexpression of triggering receptor expressed on myeloid cells 2 (TREM2), a microglia-specific immune receptor, in the brain of a middle-aged (7 months old) APPswe/PS1dE9 mice could ameliorate Alzheimer's disease (AD)-related neuropathology by enhancement of microglial amyloid-β (Aβ) phagocytosis. Since AD is an age-related neurodegenerative disorder, it is critical to assess the efficacy of TREM2 overexpression in aging animals with an advanced disease stage. In vivo, we employed a lentiviral strategy to overexpress TREM2 in the brain of aging (18 months old) APPswe/PS1dE9 mice, and observed its efficacy on AD-related neuropathology and cognitive functions. Afterwards, we directly isolated microglia from middle-aged and aging APPswe/PS1dE9 mice and determined effects of TREM2 overexpression on microglial Aβ phagocytosis and Aβ-binding receptors expression in vitro. In aging APPswe/PS1dE9 mice, TREM2 overexpression has no beneficial effect on AD-related neuropathology and spatial cognitive functions. Of note, in vitro experiments showed a significant reduction of Aβ phagocytosis in microglia from aging APPswe/PS1dE9 mice, possibly attributing to the declined expression of Aβ-binding receptors. Meanwhile, this phagocytic deficit in microglia from aging APPswe/PS1dE9 mice cannot be rescued by TREM2 overexpression. Taken together, our study shows that TREM2 overexpression fails to provide neuroprotection in aging APPswe/PS1dE9 mice, possibly attributing to deficits in microglial Aβ phagocytosis at the late-stage of disease progression. These findings indicate that TREM2-mediated protection in AD is at least partially dependent on the reservation of microglial phagocytic functions, emphasizing the importance of early therapeutic interventions for this devastating disease.

  4. YIH1 is an actin-binding protein that inhibits protein kinase GCN2 and impairs general amino acid control when overexpressed.

    PubMed

    Sattlegger, Evelyn; Swanson, Mark J; Ashcraft, Emily A; Jennings, Jennifer L; Fekete, Richard A; Link, Andrew J; Hinnebusch, Alan G

    2004-07-16

    The general amino acid control (GAAC) enables yeast cells to overcome amino acid deprivation by activation of the alpha subunit of translation initiation factor 2 (eIF2alpha) kinase GCN2 and consequent induction of GCN4, a transcriptional activator of amino acid biosynthetic genes. Binding of GCN2 to GCN1 is required for stimulation of GCN2 kinase activity by uncharged tRNA in starved cells. Here we show that YIH1, when overexpressed, dampens the GAAC response (Gcn- phenotype) by suppressing eIF2alpha phosphorylation by GCN2. The overexpressed YIH1 binds GCN1 and reduces GCN1-GCN2 complex formation, and, consistent with this, the Gcn- phenotype produced by YIH1 overexpression is suppressed by GCN2 overexpression. YIH1 interacts with the same GCN1 fragment that binds GCN2, and this YIH1-GCN1 interaction requires Arg-2259 in GCN1 in vitro and in full-length GCN1 in vivo, as found for GCN2-GCN1 interaction. However, deletion of YIH1 does not increase eIF2alpha phosphorylation or derepress the GAAC, suggesting that YIH1 at native levels is not a general inhibitor of GCN2 activity. We discovered that YIH1 normally resides in a complex with monomeric actin, rather than GCN1, and that a genetic reduction in actin levels decreases the GAAC response. This Gcn- phenotype was partially suppressed by deletion of YIH1, consistent with YIH1-mediated inhibition of GCN2 in actin-deficient cells. We suggest that YIH1 resides in a YIH1-actin complex and may be released for inhibition of GCN2 and stimulation of protein synthesis under specialized conditions or in a restricted cellular compartment in which YIH1 is displaced from monomeric actin.

  5. Overexpressed neuroglobin raises threshold for nitric oxide-induced impairment of mitochondrial respiratory activities and stress signaling in primary cortical neurons

    PubMed Central

    Singh, Shilpee; Zhuo, Ming; Gorgun, Murat; Englander, Ella W.

    2013-01-01

    Surges of nitric oxide compromise mitochondrial respiration primarily by competitive inhibition of oxygen binding to cytochrome c oxidase (complex IV) and are particularly injurious in neurons, which rely on oxidative phosphorylation for all their energy needs. Here, we show that transgenic overexpression of the neuronal globin protein, neuroglobin, helps diminish protein nitration, preserve mitochondrial function and sustain ATP content of primary cortical neurons challenged by extended nitric oxide exposure. Specifically, in transgenic neurons, elevated neuroglobin curtailed nitric oxide-induced alterations in mitochondrial oxygen consumption rates, including baseline oxygen consumption, consumption coupled with ATP synthesis, proton leak and spare respiratory capacity. Concomitantly, activation of genes involved in sensing and responding to oxidative/nitrosative stress, including the early-immediate c-Fos gene and the phase II antioxidant enzyme, heme oxygenase-1, was diminished in neuroglobin-overexpressing compared to wild-type neurons. Taken together, these differences reflect a lesser insult produced by similar concentrations of nitric oxide in neuroglobin-overexpressing compared to wild-type neurons, suggesting that abundant neuroglobin buffers nitric oxide and raises the threshold of nitric oxide-mediated injury in neurons. PMID:23587847

  6. The over-expression of the β2 catalytic subunit of the proteasome decreases homologous recombination and impairs DNA double-strand break repair in human cells.

    PubMed

    Collavoli, Anita; Comelli, Laura; Cervelli, Tiziana; Galli, Alvaro

    2011-01-01

    By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR) when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB). This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p.

  7. The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells

    PubMed Central

    Collavoli, Anita; Comelli, Laura; Cervelli, Tiziana; Galli, Alvaro

    2011-01-01

    By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR) when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB). This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p. PMID:21660142

  8. Overexpression of the calpain-specific inhibitor calpastatin reduces human alpha-Synuclein processing, aggregation and synaptic impairment in [A30P]αSyn transgenic mice

    PubMed Central

    Diepenbroek, Meike; Casadei, Nicolas; Esmer, Hakan; Saido, Takaomi C.; Takano, Jiro; Kahle, Philipp J.; Nixon, Ralph A; Rao, Mala V.; Melki, Ronald; Pieri, Laura; Helling, Stefan; Marcus, Katrin; Krueger, Rejko; Masliah, Eliezer; Riess, Olaf; Nuber, Silke

    2014-01-01

    Lewy bodies, a pathological hallmark of Parkinson's disease (PD), contain aggregated alpha-synuclein (αSyn), which is found in several modified forms and can be discovered phosphorylated, ubiquitinated and truncated. Aggregation-prone truncated species of αSyn caused by aberrant cleavage of this fibrillogenic protein are hypothesized to participate in its sequestration into inclusions subsequently leading to synaptic dysfunction and neuronal death. Here, we investigated the role of calpain cleavage of αSyn in vivo by generating two opposing mouse models. We crossed into human [A30P]αSyn transgenic (i) mice deficient for calpastatin, a calpain-specific inhibitor, thus enhancing calpain activity (SynCAST(−)) and (ii) mice overexpressing human calpastatin leading to reduced calpain activity (SynCAST(+)). As anticipated, a reduced calpain activity led to a decreased number of αSyn-positive aggregates, whereas loss of calpastatin led to increased truncation of αSyn in SynCAST(−). Furthermore, overexpression of calpastatin decreased astrogliosis and the calpain-dependent degradation of synaptic proteins, potentially ameliorating the observed neuropathology in [A30P]αSyn and SynCAST(+) mice. Overall, our data further support a crucial role of calpains, particularly of calpain 1, in the pathogenesis of PD and in disease-associated aggregation of αSyn, indicating a therapeutic potential of calpain inhibition in PD. PMID:24619358

  9. Receptor-interacting protein 140 overexpression impairs cardiac mitochondrial function and accelerates the transition to heart failure in chronically infarcted rats.

    PubMed

    Chen, YanFang; Chen, ShaoRui; Yue, ZhongBao; Zhang, YiQiang; Zhou, ChangHua; Cao, WeiWei; Chen, Xi; Zhang, LuanKun; Liu, PeiQing

    2017-02-01

    Heart failure (HF) is associated with myocardial energy metabolic abnormality. Receptor-interacting protein 140 (RIP140) is an important transcriptional cofactor for maintaining energy balance in high-oxygen consumption tissues. However, the role of RIP140 in the pathologic processes of HF remains to be elucidated. In this study, we investigated the role of RIP140 in mitochondrial and cardiac functions in rodent hearts under myocardial infarction (MI) stress. MI was created by a permanent ligation of left anterior descending coronary artery and exogenous expression of RIP140 by adenovirus (Ad) vector delivery. Four weeks after MI or Ad-RIP140 treatment, cardiac function was assessed by echocardiographic and hemodynamics analyses, and the mitochondrial function was determined by mitochondrial genes expression, biogenesis, and respiration rates. In Ad-RIP140 or MI group, a subset of metabolic genes changed, accompanied with slight reductions in mitochondrial biogenesis and respiration rates but no change in adenosine triphosphate (ATP) content. Cardiac malfunction was compensated. However, under MI stress, rats overexpressing RIP140 exhibited greater repressions in mitochondrial genes, state 3 respiration rates, respiration control ratio, and ATP content and had further deteriorated cardiac malfunction. In conclusion, RIP140 overexpression leads to comparable cardiac function as resulted from MI, but RIP140 aggravates metabolic repression, mitochondrial malfunction, and further accelerates the transition to HF in response to MI stress.

  10. miR-143 Overexpression Impairs Growth of Human Colon Carcinoma Xenografts in Mice with Induction of Apoptosis and Inhibition of Proliferation

    PubMed Central

    Borralho, Pedro M.; Simões, André E. S.; Gomes, Sofia E.; Lima, Raquel T.; Carvalho, Tânia; Ferreira, Duarte M. S.; Vasconcelos, Maria H.; Castro, Rui E.; Rodrigues, Cecília M. P.

    2011-01-01

    Background MicroRNAs (miRNAs) are aberrantly expressed in human cancer and involved in the (dys)regulation of cell survival, proliferation, differentiation and death. Specifically, miRNA-143 (miR-143) is down-regulated in human colon cancer. In the present study, we evaluated the role of miR-143 overexpression on the growth of human colon carcinoma cells xenografted in nude mice (immunodeficient mouse strain: N: NIH(s) II-nu/nu). Methodology/Principal Findings HCT116 cells with stable miR-143 overexpression (Over-143) and control (Empty) cells were subcutaneously injected into the flanks of nude mice, and tumor growth was evaluated over time. Tumors arose ∼ 14 days after tumor cell implantation, and the experiment was ended at 40 days after implantation. miR-143 was confirmed to be significantly overexpressed in Over-143 versus Empty xenografts, by TaqMan® Real-time PCR (p<0.05). Importantly, Over-143 xenografts displayed slower tumor growth compared to Empty xenografts from 23 until 40 days in vivo (p<0.05), with final volumes of 928±338 and 2512±387 mm3, respectively. Evaluation of apoptotic proteins showed that Over-143 versus Empty xenografts displayed reduced Bcl-2 levels, and increased caspase-3 activation and PARP cleavage (p<0.05). In addition, the incidence of apoptotic tumor cells, assessed by TUNEL, was increased in Over-143 versus Empty xenografts (p<0.01). Finally, Over-143 versus Empty xenografts displayed significantly reduced NF-κB activation and ERK5 levels and activation (p<0.05), as well as reduced proliferative index, evaluated by Ki-67 immunohistochemistry (p<0.01). Conclusions Our results suggest that reduced tumor volume in Over-143 versus Empty xenografts may result from increased apoptosis and decreased proliferation induced by miR-143. This reinforces the relevance of miR-143 in colon cancer, indicating an important role in the control of in vivo tumor progression, and suggesting that miR-143 may constitute a putative novel

  11. Perturbation of auxin homeostasis by overexpression of wild-type IAA15 results in impaired stem cell differentiation and gravitropism in roots.

    PubMed

    Yan, Da-Wei; Wang, Jing; Yuan, Ting-Ting; Hong, Li-Wei; Gao, Xiang; Lu, Ying-Tang

    2013-01-01

    Aux/IAAs interact with auxin response factors (ARFs) to repress their transcriptional activity in the auxin signaling pathway. Previous studies have focused on gain-of-function mutations of domain II and little is known about whether the expression level of wild-type Aux/IAAs can modulate auxin homeostasis. Here we examined the perturbation of auxin homeostasis by ectopic expression of wild-type IAA15. Root gravitropism and stem cell differentiation were also analyzed. The transgenic lines were less sensitive to exogenous auxin and exhibited low-auxin phenotypes including failures in gravity response and defects in stem cell differentiation. Overexpression lines also showed an increase in auxin concentration and reduced polar auxin transport. These results demonstrate that an alteration in the expression of wild-type IAA15 can disrupt auxin homeostasis.

  12. Combined Effects of Cadmium and UVB Radiation on Sea Urchin Embryos: Skeleton Impairment Parallels p38 MAPK Activation and Stress Genes Overexpression.

    PubMed

    Bonaventura, Rosa; Russo, Roberta; Zito, Francesca; Matranga, Valeria

    2015-05-18

    Human and natural activities release many pollutants in the marine environment. The mixture of pollutants can affect many organisms concurrently. We used Paracentrotus lividus as a model to analyze the effects on signal transduction pathways and stress gene expression in embryos exposed continuously to double stress, i.e., cadmium (Cd) from fertilization and UVB at cleavage (Cd/UVB-embryos). By microscopical inspection, we evaluated embryonic morphology after 72 h of development. Tissue-specific markers were used to assess mesoderm differentiation by immunofluorescence. We analyzed p38MAPK, ERK1/2, and JNK activation by Western blot and mRNA profiles of Pl-MT, Pl-14-3-3epsilon, and Pl-jun genes by real-time quantitative polymerase chain reaction (qPCR) and the localization of their transcripts by whole mount in situ hybridization (WMISH). We found that the Cd/UVB combined exposure induced morphological malformations in 76% of pluteus embryos, mainly affecting the development of the skeleton, including the normal branching of skeletal roads. In Cd/UVB-embryos, p38MAPK was activated 1 h after UVB exposure and a remarkable overexpression of the Pl-MT, Pl-14.3.3epsilon, and Pl-jun genes 24 h after UVB exposure. Pl-MT and Pl-14.3.3epsilon mRNAs were misexpressed as they were localized in a position different from that observed in wild-type embryos, i.e., the intestine. On the contrary, Pl-jun mRNA has remained localized in the skeletogenic cells despite their displacement in exposed embryos. In conclusion, Cd/UVB exposure affected skeletal patterning producing alternative morphologies in which p38MAPK activation and Pl-MT, Pl-14.3.3epsilon, and Pl-jun gene overexpression seem linked to a protective role against the stress response induced by Cd/UVB.

  13. Overexpression of FAM83H-AS1 indicates poor patient survival and knockdown impairs cell proliferation and invasion via MET/EGFR signaling in lung cancer

    PubMed Central

    Zhang, Jie; Feng, Shumei; Su, Wenmei; Bai, Shengbin; Xiao, Lei; Wang, Lihui; Thomas, Dafydd G.; Lin, Jules; Reddy, Rishindra M.; Carrott, Philip W.; Lynch, William R.; Chang, Andrew C.; Beer, David G.; Guo, You-min; Chen, Guoan

    2017-01-01

    Whole transcriptome analyses of next generation RNA sequencing (RNA-Seq) data from human cancer samples reveled thousands of uncharacterized non-coding RNAs including long non-coding RNA (lncRNA). Recent studies indicated that lncRNAs are emerging as crucial regulators in cancer processes and potentially useful as biomarkers for cancer diagnosis and prognosis. To delineate dysregulated lncRNAs in lung cancer, we analyzed RNA-Seq data from 461 lung adenocarcinomas (LUAD) and 156 normal lung tissues. FAM83H-AS1, one of the top dysregulated lncRNAs, was found to be overexpressed in tumors relative to normal lung and significantly associated with worse patient survival in LUAD. We verified this diagnostic/prognostic potential in an independent cohort of LUAD by qRT-PCR. Cell proliferation, migration and invasion were decreased after FAM83H-AS1 knockdown using siRNAs in lung cancer cells. Flow cytometry analysis indicated the cell cycle was arrested at the G2 phase after FAM83H-AS1 knockdown. Mechanistically, we found that MET/EGFR signaling was regulated by FAM83H-AS1. Our study indicated that FAM83H-AS1 plays an important role in lung tumor progression and may be potentially used as diagnostic/prognostic marker. Further characterization of this lncRNA may provide a novel therapeutic target impacting MET/EGFR signaling. PMID:28198463

  14. Targeted overexpression of Dkk1 in osteoblasts reduces bone mass but does not impair the anabolic response to intermittent PTH treatment in mice.

    PubMed

    Yao, Gang-Qing; Wu, Jian-Jun; Troiano, Nancy; Insogna, Karl

    2011-03-01

    Parathyroid hormone (PTH) is a potent anabolic agent, but the cellular mechanisms by which it increases bone mass are not fully understood. Dickkopf 1 (Dkk1) is an endogenous inhibitor of Wnt signaling and suppresses bone formation in vivo. We sought to determine if Dkk1 and anabolic PTH treatment interact in regulating bone mass. PTH treatment of primary murine osteoblasts for 24 h reduced Dkk1 expression by 90% as quantified by real-time PCR, whereas PTH treatment in vivo reduced Dkk1 expression by 30% when given as a single daily subcutaneous dose. To directly determine whether Dkk1 modulates the anabolic response of PTH in vivo, we engineered transgenic (TG) mice expressing murine Dkk1 under the control of the 2.3-kb rat collagen alpha-1 promoter. TG mice had significantly reduced bone mass, which was accompanied by reduced histomorphometric parameters of bone formation (reduced OV/TV, ObS/OS, and NOb/TAR). Treatment of TG mice and wild-type (WT) littermates with 95 ng/g body weight of human (1-34) PTH daily for 34 days resulted in comparable increases in bone mass at all skeletal sites. Histomorphometric analyses indicated that PTH treatment increased the numbers of both osteoblasts and osteoclasts in WT mice but only increased the numbers of osteoblasts in TG mice. We conclude that overexpression of Dkk1 does not attenuate the anabolic response to PTH in vivo.

  15. Intra-hippocampal transplantation of neural precursor cells with transgenic over-expression of IL-1 receptor antagonist rescues memory and neurogenesis impairments in an Alzheimer's disease model.

    PubMed

    Ben-Menachem-Zidon, Ofra; Ben Menachem-Zidon, Ofra; Ben-Menahem, Yair; Ben-Hur, Tamir; Yirmiya, Raz

    2014-01-01

    Ample evidence implicates neuroinflammatory processes in the etiology and progression of Alzheimer's disease (AD). To assess the specific role of the pro-inflammatory cytokine interleukin-1 (IL-1) in AD we examined the effects of intra-hippocampal transplantation of neural precursor cells (NPCs) with transgenic over-expression of IL-1 receptor antagonist (IL-1raTG) on memory functioning and neurogenesis in a murine model of AD (Tg2576 mice). WT NPCs- or sham-transplanted Tg2576 mice, as well as naive Tg2576 and WT mice served as controls. To assess the net effect of IL-1 blockade (not in the context of NPCs transplantation), we also examined the effects of chronic (4 weeks) intra-cerebroventricular (i.c.v.) administration of IL-1ra. We report that 12-month-old Tg2576 mice exhibited increased mRNA expression of hippocampal IL-1β, along with severe disturbances in hippocampal-dependent contextual and spatial memory as well as in neurogenesis. Transplantation of IL-1raTG NPCs 1 month before the neurobehavioral testing completely rescued these disturbances and significantly increased the number of endogenous hippocampal cells expressing the plasticity-related molecule BDNF. Similar, but less-robust effects were also produced by transplantation of WT NPCs and by i.c.v. IL-1ra administration. NPCs transplantation produced alterations in hippocampal plaque formation and microglial status, which were not clearly correlated with the cognitive effects of this procedure. The results indicate that elevated levels of hippocampal IL-1 are causally related to some AD-associated memory disturbances, and provide the first example for the potential use of genetically manipulated NPCs with anti-inflammatory properties in the treatment of AD.

  16. Overexpression of TFAM protects 3T3-L1 adipocytes from NYGGF4 (PID1) overexpression-induced insulin resistance and mitochondrial dysfunction.

    PubMed

    Shi, Chun-Mei; Xu, Guang-Feng; Yang, Lei; Fu, Zi-Yi; Chen, Ling; Fu, Hai-Long; Shen, Ya-Hui; Zhu, Lu; Ji, Chen-Bo; Guo, Xi-Rong

    2013-07-01

    NYGGF4, also known as phosphotyrosine interaction domain containing 1(PID1), is a recently discovered gene which is involved in obesity-related insulin resistance (IR) and mitochondrial dysfunction. We aimed to further elucidate the effects and mechanisms underlying NYGGF4-induced IR by investigating the effect of overexpressing mitochondrial transcription factor A (TFAM), which is essential for mitochondrial DNA transcription and replication, on NYGGF4-induced IR and mitochondrial abnormalities in 3T3-L1 adipocytes. Overexpression of TFAM increased the mitochondrial copy number and ATP content in both control 3T3-L1 adipocytes and NYGGF4-overexpressing adipocytes. Reactive oxygen species (ROS) production was enhanced in NYGGF4-overexpressing adipocytes and reduced in TFAM-overexpressing adipocytes; co-overexpression of TFAM significantly attenuated ROS production in NYGGF4-overexpressing adipocytes. However, overexpression of TFAM did not affect the mitochondrial transmembrane potential (ΔΨm) in control 3T3-L1 adipocytes or NYGGF4-overexpressing adipocytes. In addition, co-overexpression of TFAM-enhanced insulin-stimulated glucose uptake by increasing Glucose transporter type 4 (GLUT4) translocation to the PM in NYGGF4-overexpressing adipocytes. Overexpression of NYGGF4 significantly inhibited tyrosine phosphorylation of Insulin receptor substrate 1 (IRS-1) and serine phosphorylation of Akt, whereas overexpression of TFAM strongly induced phosphorylation of IRS-1 and Akt in NYGGF4-overexpressing adipocytes. This study demonstrates that NYGGF4 plays a role in IR by impairing mitochondrial function, and that overexpression of TFAM can restore mitochondrial function to normal levels in NYGGF4-overexpressing adipocytes via activation of the IRS-1/PI3K/Akt signaling pathway.

  17. Decreased proliferation kinetics of mouse myoblasts overexpressing FRG1.

    PubMed

    Chen, Steven C; Frett, Ellie; Marx, Joseph; Bosnakovski, Darko; Reed, Xylena; Kyba, Michael; Kennedy, Brian K

    2011-01-01

    Although recent publications have linked the molecular events driving facioscapulohumeral muscular dystrophy (FSHD) to expression of the double homeobox transcription factor DUX4, overexpression of FRG1 has been proposed as one alternative causal agent as mice overexpressing FRG1 present with muscular dystrophy. Here, we characterize proliferative defects in two independent myoblast lines overexpressing FRG1. Myoblasts isolated from thigh muscle of FRG1 transgenic mice, an affected dystrophic muscle, exhibit delayed proliferation as measured by decreased clone size, whereas myoblasts isolated from the unaffected diaphragm muscle proliferated normally. To confirm the observation that overexpression of FRG1 could impair myoblast proliferation, we examined C2C12 myoblasts with inducible overexpression of FRG1, finding increased doubling time and G1-phase cells in mass culture after induction of FRG1 and decreased levels of pRb phosphorylation. We propose that depressed myoblast proliferation may contribute to the pathology of mice overexpressing FRG1 and may play a part in FSHD.

  18. Reduced viability of neuronal cells after overexpression of protein histidine phosphatase.

    PubMed

    Krieglstein, Josef; Lehmann, Martina; Mäurer, Anette; Gudermann, Thomas; Pinkenburg, Olaf; Wieland, Thomas; Litterscheid, Sarah; Klumpp, Susanne

    2008-11-01

    Protein histidine phosphatase (PHP) has just recently been discovered in eukaryotes and ATP-citrate lyase (ACL) was shown to be one of its substrates. Since ACL is crucial for cellular energy and fat metabolism we made an attempt to study the influence of PHP on cell viability. Using an adenoviral vector PHP was overexpressed in SN56 cholinergic murine neuroblastoma cells and in primary cultures of hippocampal neurons obtained from embryonic rats (E18). Overexpression of PHP in these cells caused a decrease in ACL activity and consequently impaired viability. To be sure that the reduced cellular viability was achieved by overexpression of PHP we also downregulated ACL in SN56 cells using RNAi-technology. Downregulation of ACL was harmful to the cells similar to what was observed upon overexpression of PHP. Taken together, it is concluded that overexpression of PHP results in increased dephosphorylation with concomitant inactivation of ACL, thus finally leading to cell damage.

  19. Vldlr overexpression causes hyperactivity in rats

    PubMed Central

    2012-01-01

    Background Reelin regulates neuronal positioning in cortical brain structures and neuronal migration via binding to the lipoprotein receptors Vldlr and Lrp8. Reeler mutant mice display severe brain morphological defects and behavioral abnormalities. Several reports have implicated reelin signaling in the etiology of neurodevelopmental and psychiatric disorders, including autism, schizophrenia, bipolar disorder, and depression. Moreover, it has been reported that VLDLR mRNA levels are increased in the post-mortem brain of autistic patients. Methods We generated transgenic (Tg) rats overexpressing Vldlr, and examined their histological and behavioral features. Results Spontaneous locomotor activity was significantly increased in Tg rats, without detectable changes in brain histology. Additionally, Tg rats tended to show performance deficits in the radial maze task, suggesting that their spatial working memory was slightly impaired. Thus, Vldlr levels may be involved in determining locomotor activity and memory function. Conclusions Unlike reeler mice, patients with neurodevelopmental or psychiatric disorders do not show striking neuroanatomical aberrations. Therefore, it is notable, from a clinical point of view, that we observed behavioral phenotypes in Vldlr-Tg rats in the absence of neuroanatomical abnormalities. PMID:23110844

  20. Vldlr overexpression causes hyperactivity in rats.

    PubMed

    Iwata, Keiko; Izumo, Nobuo; Matsuzaki, Hideo; Manabe, Takayuki; Ishibashi, Yukiko; Ichitani, Yukio; Yamada, Kazuo; Thanseem, Ismail; Anitha, Ayyappan; Vasu, Mahesh Mundalil; Shimmura, Chie; Wakuda, Tomoyasu; Kameno, Yosuke; Takahashi, Taro; Iwata, Yasuhide; Suzuki, Katsuaki; Nakamura, Kazuhiko; Mori, Norio

    2012-10-30

    Reelin regulates neuronal positioning in cortical brain structures and neuronal migration via binding to the lipoprotein receptors Vldlr and Lrp8. Reeler mutant mice display severe brain morphological defects and behavioral abnormalities. Several reports have implicated reelin signaling in the etiology of neurodevelopmental and psychiatric disorders, including autism, schizophrenia, bipolar disorder, and depression. Moreover, it has been reported that VLDLR mRNA levels are increased in the post-mortem brain of autistic patients. We generated transgenic (Tg) rats overexpressing Vldlr, and examined their histological and behavioral features. Spontaneous locomotor activity was significantly increased in Tg rats, without detectable changes in brain histology. Additionally, Tg rats tended to show performance deficits in the radial maze task, suggesting that their spatial working memory was slightly impaired. Thus, Vldlr levels may be involved in determining locomotor activity and memory function. Unlike reeler mice, patients with neurodevelopmental or psychiatric disorders do not show striking neuroanatomical aberrations. Therefore, it is notable, from a clinical point of view, that we observed behavioral phenotypes in Vldlr-Tg rats in the absence of neuroanatomical abnormalities.

  1. Phospholamban Overexpression in Transgenic Rabbits

    PubMed Central

    Pattison, J. Scott; Waggoner, Jason R.; James, Jeanne; Martin, Lisa; Gulick, James; Osinska, Hanna; Klevitsky, Raisa; Kranias, Evangelia G.; Robbins, Jeffrey

    2008-01-01

    There has been considerable interest in pursuing phospholamban as a putative therapeutic target for overcoming depressed calcium handling in human heart failure. Studies predominantly done in mice have shown that phospholamban is a key regulator of sarcoplasmic reticulum calcium cycling and cardiac function. However, mice differ significantly from humans in how they regulate calcium, whereas rabbits better recapitulate human cardiac function and calcium handling. To investigate phospholamban’s role in the rabbit heart, transgenic rabbits that overexpressed wild-type phospholamban in the ventricular cardiomyocytes and slow-twitch skeletal muscles were generated. Rabbits expressing high levels of phospholamban were not viable due to severe skeletal muscle wasting, the onset of cardiac pathology and early death. A viable transgenic line exhibited a 30% increase in PLN protein levels in the heart. These animals showed isolated foci of cardiac pathology, but cardiac function as well as the response to β-adrenergic stimulation were normal. SR-calcium uptake measurements showed that the transgenic hearts had the expected reduced affinity for calcium. The data show that phospholamban-overexpressing transgenic rabbits differ markedly in phenotype from analogous transgenic mice in that rabbits are quite sensitive to alterations in phospholamban levels. Exceeding a relatively narrow window of phospholamban expression results in significant morbidity and early death. PMID:17882530

  2. Axonal Transport Rates In Vivo Are Unaffected by Tau Deletion or Overexpression in Mice

    PubMed Central

    Yuan, Aidong; Kumar, Asok; Peterhoff, Corrinne; Duff, Karen; Nixon, Ralph A.

    2010-01-01

    Elevated tau expression has been proposed as a possible basis for impaired axonal transport in Alzheimer’s disease. To address this hypothesis, we analyzed the movement of pulse radiolabeled proteins in vivo along retinal ganglion cell (RGC) axons of mice that lack tau or overexpress human tau isoforms. Here, we show that the global axonal transport rates of slow and fast transport cargoes in axons are not significantly impaired when tau expression is eliminated or increased. In addition, markers of slow transport (neurofilament light subunit) and fast transport (snap25) do not accumulate in retinas and are distributed normally along optic axons in mice that lack or overexpress tau. Finally, ultrastructural analyses revealed no abnormal accumulations of vesicular organelles or neurofilaments in RGC perikarya or axons in mice overexpressing or lacking tau. These results suggest that tau is not essential for axonal transport and that transport rates in vivo are not significantly affected by substantial fluctuations in tau expression. PMID:18272688

  3. Emotional and learning behaviour in mice overexpressing heat shock protein 70.

    PubMed

    Ammon-Treiber, Susanne; Grecksch, Gisela; Angelidis, Charalampos; Vezyraki, Patra; Höllt, Volker; Becker, Axel

    2008-09-01

    The effects of inducible heat shock protein 70 (HSP70) on emotional and learning behaviour as well as hippocampal long-term potentiation was investigated in transgenic HSP70 overexpressing mice. In active two-way avoidance learning (shuttle box) as well as spatial 8-arm radial maze learning, the HSP70 overexpressing mice showed diminished learning performance. In several tests there was no indication of differences in anxiety behaviour between transgenic mice and wild-type mice. This suggests that impairment in learning behaviour is unrelated to the learning task and motivational aspects of behaviour. To investigate the neurophysiological correlate of learning, long-term potentiation experiments were performed. In transversal hippocampal slices, an enhanced amplitude of the population spike was found in HSP70 overexpressing mice. It was hypothesised that enhanced potentiation in conjunction with potentiation effects due to learning led to learning impairment.

  4. Working Memory Deficits, Increased Anxiety-Like Traits, and Seizure Susceptibility in BDNF Overexpressing Mice

    ERIC Educational Resources Information Center

    Papaleo, Francesco; Silverman, Jill L.; Aney, Jordan; Tian, Qingjun; Barkan, Charlotte L.; Chadman, Kathryn K.; Crawley, Jacqueline N.

    2011-01-01

    BDNF regulates components of cognitive processes and has been implicated in psychiatric disorders. Here we report that genetic overexpression of the BDNF mature isoform (BDNF-tg) in female mice impaired working memory functions while sparing components of fear conditioning. BDNF-tg mice also displayed reduced breeding efficiency, higher…

  5. Working Memory Deficits, Increased Anxiety-Like Traits, and Seizure Susceptibility in BDNF Overexpressing Mice

    ERIC Educational Resources Information Center

    Papaleo, Francesco; Silverman, Jill L.; Aney, Jordan; Tian, Qingjun; Barkan, Charlotte L.; Chadman, Kathryn K.; Crawley, Jacqueline N.

    2011-01-01

    BDNF regulates components of cognitive processes and has been implicated in psychiatric disorders. Here we report that genetic overexpression of the BDNF mature isoform (BDNF-tg) in female mice impaired working memory functions while sparing components of fear conditioning. BDNF-tg mice also displayed reduced breeding efficiency, higher…

  6. NUCKS overexpression in breast cancer

    PubMed Central

    Drosos, Yiannis; Kouloukoussa, Mirsini; Østvold, Anne Carine; Grundt, Kirsten; Goutas, Nikos; Vlachodimitropoulos, Dimitrios; Havaki, Sophia; Kollia, Panagoula; Kittas, Christos; Marinos, Evangelos; Aleporou-Marinou, Vassiliki

    2009-01-01

    Background NUCKS (Nuclear, Casein Kinase and Cyclin-dependent Kinase Substrate) is a nuclear, DNA-binding and highly phosphorylated protein. A number of reports show that NUCKS is highly expressed on the level of mRNA in several human cancers, including breast cancer. In this work, NUCKS expression on both RNA and protein levels was studied in breast tissue biopsies consisted of invasive carcinomas, intraductal proliferative lesions, benign epithelial proliferations and fibroadenomas, as well as in primary cultures derived from the above biopsies. Specifically, in order to evaluate the level of NUCKS protein in correlation with the histopathological features of breast disease, immunohistochemistry was employed on paraffin sections of breast biopsies of the above types. In addition, NUCKS expression was studied by means of Reverse Transcription PCR (RT-PCR), real-time PCR (qRT-PCR) and Western immunoblot analyses in the primary cell cultures developed from the same biopsies. Results The immunohistochemical Results showed intense NUCKS staining mostly in grade I and II breast carcinomas compared to normal tissues. Furthermore, NUCKS was moderate expressed in benign epithelial proliferations, such as adenosis and sclerosing adenosis, and highly expressed in intraductal lesions, specifically in ductal carcinomas in situ (DCIS). It is worth noting that all the fibroadenoma tissues examined were negative for NUCKS staining. RT-PCR and qRT-PCR showed an increase of NUCKS expression in cells derived from primary cultures of proliferative lesions and cancerous tissues compared to the ones derived from normal breast tissues and fibroadenomas. This increase was also confirmed by Western immunoblot analysis. Although NUCKS is a cell cycle related protein, its expression does not correlate with Ki67 expression, neither in tissue sections nor in primary cell cultures. Conclusion The results show overexpression of the NUCKS protein in a number of non malignant breast lesions and

  7. Taste - impaired

    MedlinePlus

    ... longer. Causes of impaired taste include: Bell's palsy Common cold Flu and other viral infections Nasal infection, nasal ... your diet. For taste problems due to the common cold or flu, normal taste should return when the ...

  8. Impaired Driving

    MedlinePlus

    Impaired driving is dangerous. It's the cause of more than half of all car crashes. It means operating a ... texting Having a medical condition which affects your driving For your safety and the safety of others, ...

  9. Impaired Driving

    MedlinePlus

    ... people were killed in alcohol-impaired driving crashes, accounting for nearly one-third (31%) of all traffic- ... promotion efforts into practice that influence economic, organizational, policy, and school/community action. 13,14 Using community- ...

  10. Physical Impairment

    NASA Astrophysics Data System (ADS)

    Trewin, Shari

    Many health conditions can lead to physical impairments that impact computer and Web access. Musculoskeletal conditions such as arthritis and cumulative trauma disorders can make movement stiff and painful. Movement disorders such as tremor, Parkinsonism and dystonia affect the ability to control movement, or to prevent unwanted movements. Often, the same underlying health condition also has sensory or cognitive effects. People with dexterity impairments may use a standard keyboard and mouse, or any of a wide range of alternative input mechanisms. Examples are given of the diverse ways that specific dexterity impairments and input mechanisms affect the fundamental actions of Web browsing. As the Web becomes increasingly sophisticated, and physically demanding, new access features at the Web browser and page level will be necessary.

  11. Hearing Impairment

    MedlinePlus

    ... may include inserting an object such as a cotton swab too far into the ear, a sudden explosion or other loud noise, a sudden change in air pressure, a head injury, or repeated ear infections. Sensorineural hearing impairment results from problems with or damage ...

  12. Mechanism of action and therapeutic efficacy of Aurora kinase B inhibition in MYC overexpressing medulloblastoma.

    PubMed

    Diaz, Roberto Jose; Golbourn, Brian; Faria, Claudia; Picard, Daniel; Shih, David; Raynaud, Denis; Leadly, Michael; MacKenzie, Danielle; Bryant, Melissa; Bebenek, Matthew; Smith, Christian A; Taylor, Michael D; Huang, Annie; Rutka, James T

    2015-02-20

    Medulloblastoma comprises four molecular subgroups of which Group 3 medulloblastoma is characterized by MYC amplification and MYC overexpression. Lymphoma cells expressing high levels of MYC are susceptible to apoptosis following treatment with inhibitors of mitosis. One of the key regulatory kinases involved in multiple stages of mitosis is Aurora kinase B. We hypothesized that medulloblastoma cells that overexpress MYC would be uniquely sensitized to the apoptotic effects of Aurora B inhibition. The specific inhibition of Aurora kinase B was achieved in MYC- overexpressing medulloblastoma cells with AZD1152-HQPA. MYC overexpression sensitized medulloblastoma cells to cell death upon Aurora B inhibition. This process was found to be independent of endoreplication. Using both flank and intracranial cerebellar xenografts we demonstrate that tumors formed from MYC-overexpressing medulloblastoma cells show a response to Aurora B inhibition including growth impairment and apoptosis induction. Lastly, we show the distribution of AZD1152-HQPA within the mouse brain and the ability to inhibit intracranial tumor growth and prolong survival in mice bearing tumors formed from MYC-overexpressing medulloblastoma cells. Our results suggest the potential for therapeutic application of Aurora kinase B inhibitors in the treatment of Group 3 medulloblastoma.

  13. Mechanism of action and therapeutic efficacy of Aurora kinase B inhibition in MYC overexpressing medulloblastoma

    PubMed Central

    Faria, Claudia; Picard, Daniel; Shih, David; Raynaud, Denis; Leadly, Michael; MacKenzie, Danielle; Bryant, Melissa; Bebenek, Matthew; Smith, Christian A.; Taylor, Michael D.; Huang, Annie; Rutka, James T.

    2015-01-01

    Medulloblastoma comprises four molecular subgroups of which Group 3 medulloblastoma is characterized by MYC amplification and MYC overexpression. Lymphoma cells expressing high levels of MYC are susceptible to apoptosis following treatment with inhibitors of mitosis. One of the key regulatory kinases involved in multiple stages of mitosis is Aurora kinase B. We hypothesized that medulloblastoma cells that overexpress MYC would be uniquely sensitized to the apoptotic effects of Aurora B inhibition. The specific inhibition of Aurora kinase B was achieved in MYC-overexpressing medulloblastoma cells with AZD1152-HQPA. MYC overexpression sensitized medulloblastoma cells to cell death upon Aurora B inhibition. This process was found to be independent of endoreplication. Using both flank and intracranial cerebellar xenografts we demonstrate that tumors formed from MYC-overexpressing medulloblastoma cells show a response to Aurora B inhibition including growth impairment and apoptosis induction. Lastly, we show the distribution of AZD1152-HQPA within the mouse brain and the ability to inhibit intracranial tumor growth and prolong survival in mice bearing tumors formed from MYC-overexpressing medulloblastoma cells. Our results suggest the potential for therapeutic application of Aurora kinase B inhibitors in the treatment of Group 3 medulloblastoma. PMID:25739120

  14. Tuning Escherichia coli for membrane protein overexpression.

    PubMed

    Wagner, Samuel; Klepsch, Mirjam M; Schlegel, Susan; Appel, Ansgar; Draheim, Roger; Tarry, Michael; Högbom, Martin; van Wijk, Klaas J; Slotboom, Dirk J; Persson, Jan O; de Gier, Jan-Willem

    2008-09-23

    A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used "Walker strains" C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.

  15. Tuning Escherichia coli for membrane protein overexpression

    PubMed Central

    Wagner, Samuel; Klepsch, Mirjam M.; Schlegel, Susan; Appel, Ansgar; Draheim, Roger; Tarry, Michael; Högbom, Martin; van Wijk, Klaas J.; Slotboom, Dirk J.; Persson, Jan O.; de Gier, Jan-Willem

    2008-01-01

    A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used “Walker strains” C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications. PMID:18796603

  16. Thidoredxin-2 overexpression fails to rescue chronic high calorie diet induced hippocampal dysfunction.

    PubMed

    Liu, Yong; Yang, Ying; Dong, Hui; Cutler, Roy G; Strong, Randy; Mattson, Mark P

    2016-01-01

    A high calorie diet (HCD) can impair hippocampal synaptic plasticity and cognitive function in animal models. Mitochondrial thioredoxin 2 (TRX-2) is critical for maintaining intracellular redox status, but whether it can protect against HCD-induced impairment of synaptic plasticity is unknown. We found that levels of TRX-2 are reduced in the hippocampus of wild type mice maintained for 8 months on a HCD, and that the mice on the HCD exhibit impaired hippocampal synaptic plasticity (long-term potentiation at CA1 synapses) and cognitive function (novel object recognition). Transgenic mice overexpressing human TRX-2 (hTRX-2) exhibit increased resistance to diquat-induced oxidative stress in peripheral tissues. However, neither the HCD nor hTRX-2 overexpression affected levels of lipid peroxidation products (F2 isoprostanes) in the hippocampus, and hTRX-2 transgenic mice were not protected against the adverse effects of the HCD on hippocampal synaptic plasticity and cognitive function. Our findings indicate that TRX-2 overexpression does not mitigate adverse effects of a HCD on synaptic plasticity, and also suggest that oxidative stress may not be a pivotal factor in the impairment of synaptic plasticity and cognitive function caused by HCDs.

  17. Craniosynostosis in transgenic mice overexpressing Nell-1

    PubMed Central

    Zhang, Xinli; Kuroda, Shun’ichi; Carpenter, Dale; Nishimura, Ichiro; Soo, Chia; Moats, Rex; Iida, Keisuke; Wisner, Eric; Hu, Fei-Ya; Miao, Steve; Beanes, Steve; Dang, Catherine; Vastardis, Heleni; Longaker, Michael; Tanizawa, Katsuyuki; Kanayama, Norihiro; Saito, Naoaki; Ting, Kang

    2002-01-01

    Previously, we reported NELL-1 as a novel molecule overexpressed during premature cranial suture closure in patients with craniosynostosis (CS), one of the most common congenital craniofacial deformities. Here we describe the creation and analysis of transgenic mice overexpressing Nell-1. Nell-1 transgenic animals exhibited CS-like phenotypes that ranged from simple to compound synostoses. Histologically, the osteogenic fronts of abnormally closing/closed sutures in these animals revealed calvarial overgrowth and overlap along with increased osteoblast differentiation and reduced cell proliferation. Furthermore, anomalies were restricted to calvarial bone, despite generalized, non-tissue-specific overexpression of Nell-1. In vitro, Nell-1 overexpression accelerated calvarial osteoblast differentiation and mineralization under normal culture conditions. Moreover, Nell-1 overexpression in osteoblasts was sufficient to promote alkaline phosphatase expression and micronodule formation. Conversely, downregulation of Nell-1 inhibited osteoblast differentiation in vitro. In summary, Nell-1 overexpression induced calvarial overgrowth resulting in premature suture closure in a rodent model. Nell-1, therefore, has a novel role in CS development, perhaps as part of a complex chain of events resulting in premature suture closure. On a cellular level, Nell-1 expression may modulate and be both sufficient and required for osteoblast differentiation. PMID:12235118

  18. Overexpression of Catalase Diminishes Oxidative Cysteine Modifications of Cardiac Proteins.

    PubMed

    Yao, Chunxiang; Behring, Jessica B; Shao, Di; Sverdlov, Aaron L; Whelan, Stephen A; Elezaby, Aly; Yin, Xiaoyan; Siwik, Deborah A; Seta, Francesca; Costello, Catherine E; Cohen, Richard A; Matsui, Reiko; Colucci, Wilson S; McComb, Mark E; Bachschmid, Markus M

    2015-01-01

    Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H2O2), react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential cysteine thiolates. We previously found that cardiac-specific overexpression of catalase (Cat), an enzyme that detoxifies excess H2O2, protected from oxidative stress and delayed cardiac aging in mice. Using redox proteomics and systems biology, we sought to identify the cysteines that could play a key role in cardiac disease and aging. With a 'Tandem Mass Tag' (TMT) labeling strategy and mass spectrometry, we investigated differential reversible cysteine oxidation in the cardiac proteome of wild type and Cat transgenic (Tg) mice. Reversible cysteine oxidation was measured as thiol occupancy, the ratio of total available versus reversibly oxidized cysteine thiols. Catalase overexpression globally decreased thiol occupancy by ≥1.3 fold in 82 proteins, including numerous mitochondrial and contractile proteins. Systems biology analysis assigned the majority of proteins with differentially modified thiols in Cat Tg mice to pathways of aging and cardiac disease, including cellular stress response, proteostasis, and apoptosis. In addition, Cat Tg mice exhibited diminished protein glutathione adducts and decreased H2O2 production from mitochondrial complex I and II, suggesting improved function of cardiac mitochondria. In conclusion, our data suggest that catalase may alleviate cardiac disease and aging by moderating global protein cysteine thiol oxidation.

  19. Overexpression of VOZ2 confers biotic stress tolerance but decreases abiotic stress resistance in Arabidopsis

    PubMed Central

    Nakai, Yusuke; Fujiwara, Sumire; Kubo, Yasuyuki; Sato, Masa H.

    2013-01-01

    VOZ (vascular plant one zinc-finger protein) is a plant specific one-zinc finger type transcriptional activator, which is highly conserved through land plant evolution. We have previously shown that loss-of-function mutations in VOZ1 and VOZ2 showed increased cold and drought stress tolerances whereas decreased biotic stress resistance in Arabidopsis. Here, we demonstrate that transgenic plants overexpressing VOZ2 impairs freezing and drought stress tolerances but increases resistance to a fungal pathogen, Colletoricum higginsianum. Consistent with changes in the tolerance to biotic and abiotic stresses, the expression of marker genes for these stresses is significantly altered compared with those of the wild-type plant. These results indicate that a overexpression of VOZ2 confers biotic stress tolerance but impairs abiotic stress tolerances in Arabidopsis. PMID:23299334

  20. Overexpression of VOZ2 confers biotic stress tolerance but decreases abiotic stress resistance in Arabidopsis.

    PubMed

    Nakai, Yusuke; Fujiwara, Sumire; Kubo, Yasuyuki; Sato, Masa H

    2013-03-01

    VOZ (vascular plant one zinc-finger protein) is a plant specific one-zinc finger type transcriptional activator, which is highly conserved through land plant evolution. We have previously shown that loss-of-function mutations in VOZ1 and VOZ2 showed increased cold and drought stress tolerances whereas decreased biotic stress resistance in Arabidopsis. Here, we demonstrate that transgenic plants overexpressing VOZ2 impairs freezing and drought stress tolerances but increases resistance to a fungal pathogen, Colletoricum higginsianum. Consistent with changes in the tolerance to biotic and abiotic stresses, the expression of marker genes for these stresses is significantly altered compared with those of the wild-type plant. These results indicate that a overexpression of VOZ2 confers biotic stress tolerance but impairs abiotic stress tolerances in Arabidopsis.

  1. Comparative Plasmodium gene overexpression reveals distinct perturbation of sporozoite transmission by profilin

    PubMed Central

    Sato, Yuko; Hliscs, Marion; Dunst, Josefine; Goosmann, Christian; Brinkmann, Volker; Montagna, Georgina N.; Matuschewski, Kai

    2016-01-01

    Plasmodium relies on actin-based motility to migrate from the site of infection and invade target cells. Using a substrate-dependent gliding locomotion, sporozoites are able to move at fast speed (1–3 μm/s). This motility relies on a minimal set of actin regulatory proteins and occurs in the absence of detectable filamentous actin (F-actin). Here we report an overexpression strategy to investigate whether perturbations of F-actin steady-state levels affect gliding locomotion and host invasion. We selected two vital Plasmodium berghei G-actin–binding proteins, C-CAP and profilin, in combination with three stage-specific promoters and mapped the phenotypes afforded by overexpression in all three extracellular motile stages. We show that in merozoites and ookinetes, additional expression does not impair life cycle progression. In marked contrast, overexpression of C-CAP and profilin in sporozoites impairs circular gliding motility and salivary gland invasion. The propensity for productive motility correlates with actin accumulation at the parasite tip, as revealed by combinations of an actin-stabilizing drug and transgenic parasites. Strong expression of profilin, but not C-CAP, resulted in complete life cycle arrest. Comparative overexpression is an alternative experimental genetic strategy to study essential genes and reveals effects of regulatory imbalances that are not uncovered from deletion-mutant phenotyping. PMID:27226484

  2. Fetal PGC-1α Overexpression Programs Adult Pancreatic β-Cell Dysfunction

    PubMed Central

    Valtat, Bérengère; Riveline, Jean-Pierre; Zhang, Ping; Singh-Estivalet, Amrit; Armanet, Mathieu; Venteclef, Nicolas; Besseiche, Adrien; Kelly, Daniel P.; Tronche, François; Ferré, Pascal; Gautier, Jean-François; Bréant, Bernadette; Blondeau, Bertrand

    2013-01-01

    Adult β-cell dysfunction, a hallmark of type 2 diabetes, can be programmed by adverse fetal environment. We have shown that fetal glucocorticoids (GCs) participate in this programming through inhibition of β-cell development. Here we have investigated the molecular mechanisms underlying this regulation. We showed that GCs stimulate the expression of peroxisome proliferator–activated receptor-γ coactivator-1α (PGC-1α), a coregulator of the GCs receptor (GR), and that the overexpression of PGC-1α represses genes important for β-cell development and function. More precisely, PGC-1α inhibited the expression of the key β-cell transcription factor pancreatic duodenal homeobox 1 (Pdx1). This repression required the GR and was mediated through binding of a GR/PGC-1α complex to the Pdx1 promoter. To explore PGC-1α function, we generated mice with inducible β-cell PGC-1α overexpression. Mice overexpressing PGC-1α exhibited at adult age impaired glucose tolerance associated with reduced insulin secretion, decreased β-cell mass, and β-cell hypotrophy. Interestingly, PGC-1α expression in fetal life only was sufficient to impair adult β-cell function whereas β-cell PGC-1α overexpression from adult age had no consequence on β-cell function. Altogether, our results demonstrate that the GR and PGC-1α participate in the fetal programming of adult β-cell function through inhibition of Pdx1 expression. PMID:23274887

  3. Overexpression of dilp2 causes nutrient-dependent semi-lethality in Drosophila

    PubMed Central

    Sato-Miyata, Yukiko; Muramatsu, Keigo; Funakoshi, Masabumi; Tsuda, Manabu; Aigaki, Toshiro

    2014-01-01

    Insulin/insulin-like growth factor (IGF) plays an important role as a systemic regulator of metabolism in multicellular organisms. Hyperinsulinemia, a high level of blood insulin, is often associated with impaired physiological conditions such as hypoglycemia, insulin resistance, and diabetes. However, due to the complex pathophysiology of hyperinsulinemia, the causative role of excess insulin/IGF signaling has remained elusive. To investigate the biological effects of a high level of insulin in metabolic homeostasis and physiology, we generated flies overexpressing Drosophila insulin-like peptide 2 (Dilp2), which has the highest potential of promoting tissue growth among the Ilp genes in Drosophila. In this model, a UAS-Dilp2 transgene was overexpressed under control of sd-Gal4 that drives expression predominantly in developing imaginal wing discs. Overexpression of Dilp2 caused semi-lethality, which was partially suppressed by mutations in the insulin receptor (InR) or Akt1, suggesting that dilp2-induced semi-lethality is mediated by the PI3K/Akt1 signaling. We found that dilp2-overexpressing flies exhibited intensive autophagy in fat body cells. Interestingly, the dilp2-induced autophagy as well as the semi-lethality was partially rescued by increasing the protein content relative to glucose in the media. Our results suggest that excess insulin/IGF signaling impairs the physiology of animals, which can be ameliorated by controlling the nutritional balance between proteins and carbohydrates, at least in flies. PMID:24795642

  4. Mice with cardiac overexpression of PPARγ have impaired repolarization and spontaneous fatal ventricular arrhythmias (Morrow, PPARγ overexpression induces fatal arrhythmias)

    PubMed Central

    Morrow, John P.; Katchman, Alexander; Son, Ni-Huiping; Trent, Chad M.; Khan, Raffay; Shiomi, Takayuki; Huang, Haiyan; Amin, Vaibhav; Lader, Joshua M.; Vasquez, Carolina; Morley, Gregory E.; D'Armiento, Jeanine; Homma, Shunichi; Goldberg, Ira J.; Marx, Steven O.

    2011-01-01

    Background Diabetes and obesity, which confer an increased risk of sudden cardiac death, are associated with cardiomyocyte lipid accumulation and altered cardiac electrical properties, manifested by prolongation of the QRS duration and QT interval. It is difficult to distinguish the contribution of cardiomyocyte lipid accumulation versus the contribution of global metabolic defects to the increased incidence of sudden death and electrical abnormalities. Methods and Results In order to study the effects of metabolic abnormalities on arrhythmias without the complex systemic effects of diabetes and obesity, we studied cardiac-specific transgenic mice expressing PPARγ1 via the cardiac α-myosin heavy-chain promoter. The PPARγ-transgenic mice develop abnormal accumulation of intracellular lipids and die as young adults, prior to a significant reduction in systolic function. Using implantable ECG telemeters, we found that these mice have prolongation of the QRS and QT intervals, and spontaneous ventricular arrhythmias, including polymorphic ventricular tachycardia and ventricular fibrillation. Isolated cardiomyocytes demonstrated prolonged action potential duration caused by reduced expression and function of the potassium channels responsible for repolarization. Short-term exposure to pioglitazone, a PPARγ agonist, had no effect on mortality or rhythm in WT mice, but further exacerbated the arrhythmic phenotype and increased the mortality in the PPARγ TG mice. Conclusions Our findings support an important link between PPARγ activation, cardiomyocyte lipid accumulation, ion channel remodeling and increased cardiac mortality. PMID:22124376

  5. All Vision Impairment

    MedlinePlus

    ... USAJobs Home > Statistics and Data > All Vision Impairment All Vision Impairment Vision Impairment Defined Vision impairment is ... being blind by the U.S. definition.) The category “All Vision Impairment” includes both low vision and blindness. ...

  6. Overexpression of Glucocorticoid Receptor β Enhances Myogenesis and Reduces Catabolic Gene Expression

    PubMed Central

    Hinds, Terry D.; Peck, Bailey; Shek, Evan; Stroup, Steven; Hinson, Jennifer; Arthur, Susan; Marino, Joseph S.

    2016-01-01

    Unlike the glucocorticoid receptor α (GRα), GR β (GRβ) has a truncated ligand-binding domain that prevents glucocorticoid binding, implicating GRα as the mediator of glucocorticoid-induced skeletal muscle loss. Because GRβ causes glucocorticoid resistance, targeting GRβ may be beneficial in impairing muscle loss as a result of GRα activity. The purpose of this study was to determine how the overexpression of GRβ affects myotube formation and dexamethasone (Dex) responsiveness. We measured GR isoform expression in C2C12 muscle cells in response to Dex and insulin, and through four days of myotube formation. Next, lentiviral-mediated overexpression of GRβ in C2C12 was performed, and these cells were characterized for cell fusion and myotube formation, as well as sensitivity to Dex via the expression of ubiquitin ligases. GRβ overexpression increased mRNA levels of muscle regulatory factors and enhanced proliferation in myoblasts. GRβ overexpressing myotubes had an increased fusion index. Myotubes overexpressing GRβ had lower forkhead box O3 (Foxo3a) mRNA levels and a blunted muscle atrophy F-box/Atrogen-1 (MAFbx) and muscle ring finger 1 (MuRF1) response to Dex. We showed that GRβ may serve as a pharmacological target for skeletal muscle growth and protection from glucocorticoid-induced catabolic signaling. Increasing GRβ levels in skeletal muscle may cause a state of glucocorticoid resistance, stabilizing muscle mass during exposure to high doses of glucocorticoids. PMID:26875982

  7. Long-term in vivo Resistin Overexpression Induces Myocardial Dysfunction and Remodeling in Rats

    PubMed Central

    Chemaly, Elie R.; Hadri, Lahouaria; Zhang, Shihong; Kim, Maengjo; Kohlbrenner, Erik; Sheng, Jipo; Liang, Lifan; Chen, Jiqiu; K-Raman, Purushothaman; Hajjar, Roger J.; Lebeche, Djamel

    2011-01-01

    We have previously reported that resistin induces hypertrophy and impairs contractility in isolated rat cardiomyocytes. To examine the long-term cardiovascular effects of resistin, we induced in vivo overexpression of resistin using adeno-associated virus serotype 9 injected by tail vein in rats and compared to control animals. Ten weeks after viral injection, overexpression of resistin was associated with increased ratio of left ventricular (LV) weight/body weight increased end-systolic LV volume and significant decrease in LV contractility, measured by the end-systolic pressure volume relationship slope in LV pressure volume loops, compared to controls. At the molecular level, mRNA expression of ANF and β-MHC, and protein levels of phospholamban were increased in the resistin group without a change in the level of SERCA2a protein expression. Increased fibrosis by histology, associated with increased mRNA levels of collagen, fibronectin and connective tissue growth factor were observed in the resistin-overexpressing hearts. Resistin overexpression was also associated with increased apoptosis in vivo, along with an apoptotic molecular phenotype in vivo and in vitro. Resistin-overexpressing LV tissue had higher levels of TNF-α receptor 1 and iNOS, and reduced levels of eNOS. Cardiomyocytes overexpressing resistin in vitro produced larger amounts of TNFα in the medium, had increased phosphorylation of IκBα and displayed increased intracellular reactive oxygen species (ROS) content with increased expression and activity of ROS-producing NADPH oxidases compared to controls. Long-term resistin overexpression is associated with a complex phenotype of oxidative stress, inflammation, fibrosis, apoptosis and myocardial remodeling and dysfunction in rats. This phenotype recapitulates key features of diabetic cardiomyopathy. PMID:21549710

  8. CREB Overexpression Ameliorates Age-related Behavioral and Biophysical Deficits

    NASA Astrophysics Data System (ADS)

    Yu, Xiao-Wen

    Age-related cognitive deficits are observed in both humans and animals. Yet, the molecular mechanisms underlying these deficits are not yet fully elucidated. In aged animals, a decrease in intrinsic excitability of pyramidal neurons from the CA1 sub-region of hippocampus is believed to contribute to age-related cognitive impairments, but the molecular mechanism(s) that modulate both these factors has yet to be identified. Increasing activity of the transcription factor cAMP response element-binding protein (CREB) in young adult rodents has been shown to facilitate cognition, and increase intrinsic excitability of their neurons. However, how CREB changes with age, and how that impacts cognition in aged animals, is not clear. Therefore, we first systematically characterized age- and training-related changes in CREB levels in dorsal hippocampus. At a remote time point after undergoing behavioral training, levels of total CREB and activated CREB (phosphorylated at S133, pCREB) were measured in both young and aged rats. We found that pCREB, but not total CREB was significantly reduced in dorsal CA1 of aged rats. Importantly, levels of pCREB were found to be positively correlated with short-term spatial memory in both young and aged rats i.e. higher pCREB in dorsal CA1 was associated with better spatial memory. These findings indicate that an age-related deficit in CREB activity may contribute to the development of age-related cognitive deficits. However, it was still unclear if increasing CREB activity would be sufficient to ameliorate age-related cognitive, and biophysical deficits. To address this question, we virally overexpressed CREB in CA1, where we found the age-related deficit. Young and aged rats received control or CREB virus, and underwent water maze training. While control aged animals exhibited deficits in long-term spatial memory, aged animals with CREB overexpression performed at levels comparable to young animals. Concurrently, aged neurons

  9. Hand1 overexpression inhibits medulloblastoma metastasis

    SciTech Connect

    Asuthkar, Swapna; Guda, Maheedhara R.; Martin, Sarah E.; Antony, Reuben; Fernandez, Karen; Lin, Julian; Tsung, Andrew J.; Velpula, Kiran K.

    2016-08-19

    Medulloblastoma (MB) is the most frequent malignant pediatric brain tumor. Current treatment includes surgery, radiation and chemotherapy. However, ongoing treatment in patients is further classified according to the presence or absence of metastasis. Since metastatic medulloblastoma are refractory to current treatments, there is need to identify novel biomarkers that could be used to reduce metastatic potential, and more importantly be targeted therapeutically. Previously, we showed that ionizing radiation-induced uPAR overexpression is associated with increased accumulation of β-catenin in the nucleus. We further demonstrated that uPAR protein act as cytoplasmic sequestration factor for a novel basic helix-loop-helix transcription factor, Hand1. Among the histological subtypes classical and desmoplastic subtypes account for the majority while large cell/anaplastic variant is most commonly associated with metastatic disease. In this present study using immunohistochemical approach and patient data mining for the first time, we demonstrated that Hand1 expression is observed to be downregulated in all the subtypes of medulloblastoma. Previously we showed that Hand1 overexpression regulated medulloblastoma angiogenesis and here we investigated the role of Hand1 in the context of Epithelial-Mesenchymal Transition (EMT). Moreover, UW228 and D283 cells overexpressing Hand1 demonstrated decreased-expression of mesenchymal markers (N-cadherin, β-catenin and SOX2); metastatic marker (SMA); and increased expression of epithelial marker (E-cadherin). Strikingly, human pluripotent stem cell antibody array showed that Hand1 overexpression resulted in substantial decrease in pluripotency markers (Nanog, Oct3/4, Otx2, Flk1) suggesting that Hand1 expression may be essential to attenuate the EMT and our findings underscore a novel role for Hand1 in medulloblastoma metastasis. - Highlights: • Hand1 expression is downregulated in Medulloblastoma. • Hand1 over expression reduce

  10. Cortical Visual Impairment

    MedlinePlus

    ... Frequently Asked Questions Español Condiciones Chinese Conditions Cortical Visual Impairment En Español Read in Chinese What is cortical visual impairment? Cortical visual impairment (CVI) is a decreased ...

  11. Virus-induced over-expression of protein phosphatase 2A inhibits insulin signalling in chronic hepatitis C.

    PubMed

    Bernsmeier, Christine; Duong, Francois H T; Christen, Verena; Pugnale, Paolo; Negro, Francesco; Terracciano, Luigi; Heim, Markus H

    2008-09-01

    Hepatitis C virus (HCV) infection disturbs glucose and lipid metabolism contributing to the development of liver steatosis, insulin resistance and type 2 diabetes mellitus. On the other hand, insulin resistance and steatosis have been found to be associated with increased rates of fibrosis progression and lower rates of response to interferon therapy in chronic hepatitis C (CHC). The molecular mechanisms contributing to insulin resistance in CHC are not well understood. We have shown previously that protein phosphatase 2A (PP2A) is over-expressed in biopsies from patients with CHC. In this study, we tested if PP2A over-expression leads to insulin resistance. We studied insulin signalling in cell lines that allow the regulated over-expression of HCV proteins and of the PP2A catalytic subunit (PP2Ac). Insulin signalling and PP2Ac expression were also studied in HCV transgenic mice and in liver biopsies from patients with CHC. Over-expression of PP2Ac in cells inhibited insulin signalling by dephosphorylation of PKB/Akt. PP2Ac over-expression and impaired insulin signalling were found in the liver of HCV transgenic mice and in liver biopsies of patients with CHC. HCV-induced over-expression of PP2A in the liver contributes to the pathogenesis of insulin resistance in patients with CHC.

  12. Overexpression of facioscapulohumeral muscular dystrophy region gene 1 causes primary defects in myogenic stem cells.

    PubMed

    Xynos, Alexandros; Neguembor, Maria Victoria; Caccia, Roberta; Licastro, Danilo; Nonis, Alessandro; Di Serio, Clelia; Stupka, Elia; Gabellini, Davide

    2013-05-15

    Overexpression of facioscapulohumeral muscular dystrophy region gene 1 (FRG1) in mice, frogs and worms leads to muscular and vascular abnormalities. Nevertheless, the mechanism that follows FRG1 overexpression and finally leads to muscular defects is currently unknown. Here, we show that the earliest phenotype displayed by mice overexpressing FRG1 is a postnatal muscle-growth defect. Long before the development of muscular dystrophy, FRG1 mice also exhibit a muscle regeneration impairment. Ex vivo and in vivo experiments revealed that FRG1 overexpression causes myogenic stem cell activation and proliferative, clonogenic and differentiation defects. A comparative gene expression profiling of muscles from young pre-dystrophic wild-type and FRG1 mice identified differentially expressed genes in several gene categories and networks that could explain the emerging tissue and myogenic stem cell defects. Overall, our study provides new insights into the pathways regulated by FRG1 and suggests that muscle stem cell defects could contribute to the pathology of FRG1 mice.

  13. Frequent Nek1 overexpression in human gliomas

    SciTech Connect

    Zhu, Jun; Cai, Yu; Liu, Pin; Zhao, Weiguo

    2016-08-05

    Never in mitosis A (NIMA)-related kinase 1 (Nek1) regulates cell cycle progression to mitosis. Its expression and potential functions in human gliomas have not been studied. Here, our immunohistochemistry (IHC) assay and Western blot assay results showed that Nek1 expression was significantly upregulated in fresh and paraffin-embedded human glioma tissues. Its level in normal brain tissues was low. Nek1 overexpression in human gliomas was correlated with the proliferation marker (Ki-67), tumor grade, Karnofsky performance scale (KPS) and more importantly, patients’ poor survival. Further studies showed that Nek1 expression level was also increased in multiple human glioma cell lines (U251-MG, U87-MG, U118, H4 and U373). Significantly, siRNA-mediated knockdown of Nek1 inhibited glioma cell (U87-MG/U251-MG) growth. Nek1 siRNA also sensitized U87-MG/U251-MG cells to temozolomide (TMZ), causing a profound apoptosis induction and growth inhibition. The current study indicates Nek1 might be a novel and valuable oncotarget of glioma, it is important for glioma cell growth and TMZ-resistance. - Highlights: • Nek1 is upregulated in multiple human glioma tissues and cell lines. • Nek1 overexpression correlates with glioma grades and patients’ KPS score. • Nek1 overexpression correlates with patients’ poor overall survival. • siRNA knockdown of Nek1 inhibits glioma cell growth. • siRNA knockdown of Nek1 sensitizes human glioma cells to temozolomide.

  14. Comparative Plasmodium gene overexpression reveals distinct perturbation of sporozoite transmission by profilin.

    PubMed

    Sato, Yuko; Hliscs, Marion; Dunst, Josefine; Goosmann, Christian; Brinkmann, Volker; Montagna, Georgina N; Matuschewski, Kai

    2016-07-15

    Plasmodium relies on actin-based motility to migrate from the site of infection and invade target cells. Using a substrate-dependent gliding locomotion, sporozoites are able to move at fast speed (1-3 μm/s). This motility relies on a minimal set of actin regulatory proteins and occurs in the absence of detectable filamentous actin (F-actin). Here we report an overexpression strategy to investigate whether perturbations of F-actin steady-state levels affect gliding locomotion and host invasion. We selected two vital Plasmodium berghei G-actin-binding proteins, C-CAP and profilin, in combination with three stage-specific promoters and mapped the phenotypes afforded by overexpression in all three extracellular motile stages. We show that in merozoites and ookinetes, additional expression does not impair life cycle progression. In marked contrast, overexpression of C-CAP and profilin in sporozoites impairs circular gliding motility and salivary gland invasion. The propensity for productive motility correlates with actin accumulation at the parasite tip, as revealed by combinations of an actin-stabilizing drug and transgenic parasites. Strong expression of profilin, but not C-CAP, resulted in complete life cycle arrest. Comparative overexpression is an alternative experimental genetic strategy to study essential genes and reveals effects of regulatory imbalances that are not uncovered from deletion-mutant phenotyping. © 2016 Sato et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  15. Nuclear overexpression of the overexpressed in lung cancer 1 predicts worse prognosis in gastric adenocarcinoma.

    PubMed

    Wang, Jue; Shen, Hongchang; Fu, Guobin; Zhao, Dandan; Wang, Weibo

    2017-02-07

    We have performed this retrospective study to elucidate whether elevated expression of the overexpressed in lung cancer 1 (OLC1) was related to the clinicopathological parameters and prognosis of patients with gastric adenocarcinoma. Additionally, different effects of various subcellular OLC1 expression on gastric adeno-carcinogenesis were focused on in our study. Both overall and subcellular expression of OLC1 was evaluated by immunohistochemistry(IHC) via tissue microarrays from total 393 samples. The Kaplan-Meier method and Cox's proportional hazard model were exerted to further explore the correlation between OLC1 and prognosis. Total overexpression of OLC1 was significantly associated with stage (P = 0.004) and differentiation (P = 0.009), and only the strong total expression could predict a poor prognosis (HR = 1.31, P = 0.04). There were significant associations found between nuclear overexpression and tumor invasion depth(P = 0.002), lymph node (P < 0.001), stage (P = 0.004), differentiation (P < 0.001) and smoking history (P = 0.045). Furthermore, over-expressed nuclear OLC1 protein could be an independent risk factor for gastric adenocarcinoma (univariate: HR = 1.43, P = 0.003; multivariate: HR = 1.39, P = 0.011). In general, both total and nuclear overexpression of OLC1 could be the signs of gastric adeno-carcinogenesis, which might be served as the biomarkers for diagnosis at an early stage, even at the onset of tumorigenesis. Rather than the total expression, nuclear overexpression of OLC1 was correlated with most clinicopathological parameters and could predict a poor overall survival as an independent factor for prognosis, which made it a more effective and sensitive biomarker for gastric adenocarcinoma.

  16. Effect of overexpression of PPARgamma on the healing process of corneal alkali burn in mice.

    PubMed

    Saika, Shizuya; Yamanaka, Osamu; Okada, Yuka; Miyamoto, Takeshi; Kitano, Ai; Flanders, Kathleen C; Ohnishi, Yoshitaka; Nakajima, Yuji; Kao, Winston W-Y; Ikeda, Kazuo

    2007-07-01

    Wound healing involves both local cells and inflammatory cells. Alkali burn of ocular surface tissue is a serious clinical problem often leading to permanent visual impairment resulting from ulceration, scarring and neovascularization during healing. Behaviors of corneal cells and inflammatory cells are orchestrated by growth factor signaling networks that have not been fully uncovered. Here we showed that adenoviral gene introduction of peroxisome proliferator-activated receptor-gamma (PPARgamma) inhibits activation of ocular fibroblasts and macrophages in vitro and also induced anti-inflammatory and anti-fibrogenic responses in an alkali-burned mouse cornea. PPARgamma overexpression suppressed upregulation of inflammation/scarring-related growth factors and matrix metalloproteinases (MMPs) in macrophages. It also suppressed expression of such growth factors and collagen Ialpha2 and myofibroblast generation upon exposure to TGFbeta1. Exogenous PPARgamma did not alter phosphorylation of Smad2, but inhibited its nuclear translocation. PPARgamma overexpression enhanced proliferation of corneal epithelial cells, but not of fibroblasts in vitro. Epithelial cell expression of MMP-2/-9 and TGFbeta1 and its migration were suppressed by PPARgamma overexpression. In vivo experiments showed that PPARgamma gene introduction suppressed monocytes/macrophages invasion and suppressed the generation of myofibroblasts, as well as upregulation of cytokines/growth factors and MMPs in a healing cornea. In vivo re-epitheliazation with basement membrane reconstruction in the healing, burned, cornea was accelerated by PPARgamma-Ad expression, although PPARgamma overexpression was considered to be unfavorable for cell migration. Together, these data suggest that overexpression of PPARgamma may represent an effective new strategy for treatment of ocular surface burns.

  17. PGC-1β regulates HER2-overexpressing breast cancer cells proliferation by metabolic and redox pathways.

    PubMed

    Victorino, Vanessa Jacob; Barroso, W A; Assunção, A K M; Cury, V; Jeremias, I C; Petroni, R; Chausse, B; Ariga, S K; Herrera, A C S A; Panis, C; Lima, T M; Souza, H P

    2016-05-01

    Breast cancer is a prevalent neoplastic disease among women worldwide which treatments still present several side effects and resistance. Considering that cancer cells present derangements in their energetic homeostasis, and that peroxisome proliferator-activated receptor- gamma coactivator 1 (PGC-1) is crucial for cellular metabolism and redox signaling, the main objective of this study was to investigate whether there is a relationship between PGC-1 expression, the proliferation of breast cancer cells and the mechanisms involved. We initially assessed PGC-1β expression in complementary DNA (cDNA) from breast tumor of patients bearing luminal A, luminal B, and HER2-overexpressed and triple negative tumors. Our data showed that PGC-1β expression is increased in patients bearing HER2-overexpressing tumors as compared to others subtypes. Using quantitative PCR and immunoblotting, we showed that breast cancer cells with HER2-amplification (SKBR-3) have greater expression of PGC-1β as compared to a non-tumorous breast cell (MCF-10A) and higher proliferation rate. PGC-1β expression was knocked down with short interfering RNA in HER2-overexpressing cells, and cells decreased proliferation. In these PGC-1β-inhibited cells, we found increased citrate synthase activity and no marked changes in mitochondrial respiration. Glycolytic pathway was decreased, characterized by lower intracellular lactate levels. In addition, after PGC-1β knockdown, SKBR-3 cells showed increased reactive oxygen species production, no changes in antioxidant activity, and decreased expression of ERRα, a modulator of metabolism. In conclusion, we show an association of HER2-overexpression and PGC-1β. PGC-1β knockdown impairs HER2-overexpressing cells proliferation acting on ERRα signaling, metabolism, and redox balance.

  18. Overexpression of multisubunit replication factors in yeast.

    PubMed

    Burgers, P M

    1999-07-01

    Facile genetic and biochemical manipulation coupled with rapid cell growth and low cost of growth media has established the yeast Saccharomyces cerevisiae as a versatile workhorse. This article describes the use of yeast expression systems for the overproduction of complex multipolypeptide replication factors. The regulated overexpression of these factors in yeast provides for a readily accessible and inexpensive source of these factors in large quantities. The methodology is illustrated with the five-subunit replication factor C. Whole-cell extracts are prepared by blending yeast cells with glass beads or frozen yeast with dry ice. Procedures are described that maximize the yield of these factors while minimizing proteolytic degradation.

  19. Visual impairment in the hearing impaired students.

    PubMed

    Gogate, Parikshit; Rishikeshi, Nikhil; Mehata, Reshma; Ranade, Satish; Kharat, Jitesh; Deshpande, Madan

    2009-01-01

    Ocular problems are more common in children with hearing problems than in normal children. Neglected visual impairment could aggravate educational and social disability. To detect and treat visual impairment, if any, in hearing-impaired children. Observational, clinical case series of hearing-impaired children in schools providing special education. Hearing-impaired children in selected schools underwent detailed visual acuity testing, refraction, external ocular examination and fundoscopy. Ocular motility testing was also performed. Teachers were sensitized and trained to help in the assessment of visual acuity using Snellen's E charts. Refractive errors and squint were treated as per standard practice. Excel software was used for data entry and SSPS for analysis. The study involved 901 hearing-impaired students between four and 21 years of age, from 14 special education schools. A quarter of them (216/901, 24%) had ocular problems. Refractive errors were the most common morbidity 167(18.5%), but only 10 children were using appropriate spectacle correction at presentation. Fifty children had visual acuity less than 20/80 at presentation; after providing refractive correction, this number reduced to three children, all of whom were provided low-vision aids. Other common conditions included strabismus in 12 (1.3%) children, and retinal pigmentary dystrophy in five (0.6%) children. Ocular problems are common in hearing-impaired children. Screening for ocular problems should be made mandatory in hearing-impaired children, as they use their visual sense to compensate for the poor auditory sense.

  20. Tumorigenesis promotes Mdm4-S overexpression

    PubMed Central

    Pant, Vinod; Larsson, Connie A.; Aryal, Neeraj; Xiong, Shunbin; You, M. James; Quintas-Cardama, Alfonso; Lozano, Guillermina

    2017-01-01

    Disruption of the p53 tumor suppressor pathway is a primary cause of tumorigenesis. In addition to mutation of the p53 gene itself, overexpression of major negative regulators of p53, MDM2 and MDM4, also act as drivers for tumor development. Recent studies suggest that expression of splice variants of Mdm2 and Mdm4 may be similarly involved in tumor development. In particular, multiple studies show that expression of a splice variant of MDM4, MDM4-S correlates with tumor aggressiveness and can be used as a prognostic marker in different tumor types. However, in the absence of prospective studies, it is not clear whether expression of MDM4-S in itself is oncogenic or is simply an outcome of tumorigenesis. Here we have examined the role of Mdm4-S in tumor development in a transgenic mouse model. Our results suggest that splicing of Mdm4 does not promote tumor development and does not cooperate with other oncogenic insults to alter tumor latency or aggressiveness. We conclude that Mdm4-S overexpression is a consequence of splicing defects in tumor cells rather than a cause of tumor evolution. PMID:28460439

  1. Tumorigenesis promotes Mdm4-S overexpression.

    PubMed

    Pant, Vinod; Larsson, Connie A; Aryal, Neeraj; Xiong, Shunbin; You, M James; Quintas-Cardama, Alfonso; Lozano, Guillermina

    2017-04-18

    Disruption of the p53 tumor suppressor pathway is a primary cause of tumorigenesis. In addition to mutation of the p53 gene itself, overexpression of major negative regulators of p53, MDM2 and MDM4, also act as drivers for tumor development. Recent studies suggest that expression of splice variants of Mdm2 and Mdm4 may be similarly involved in tumor development. In particular, multiple studies show that expression of a splice variant of MDM4, MDM4-S correlates with tumor aggressiveness and can be used as a prognostic marker in different tumor types. However, in the absence of prospective studies, it is not clear whether expression of MDM4-S in itself is oncogenic or is simply an outcome of tumorigenesis. Here we have examined the role of Mdm4-S in tumor development in a transgenic mouse model. Our results suggest that splicing of Mdm4 does not promote tumor development and does not cooperate with other oncogenic insults to alter tumor latency or aggressiveness. We conclude that Mdm4-S overexpression is a consequence of splicing defects in tumor cells rather than a cause of tumor evolution.

  2. Nucleophosmin is overexpressed in thyroid tumors

    SciTech Connect

    Pianta, Annalisa; Puppin, Cinzia; Franzoni, Alessandra; Fabbro, Dora; Di Loreto, Carla; Bulotta, Stefania; Deganuto, Marta; Paron, Igor; Tell, Gianluca; Puxeddu, Efisio; Filetti, Sebastiano; Russo, Diego; Damante, Giuseppe

    2010-07-02

    Nucleophosmin (NPM) is a protein that contributes to several cell functions. Depending on the context, it can act as an oncogene or tumor suppressor. No data are available on NPM expression in thyroid cells. In this work, we analyzed both NPM mRNA and protein levels in a series of human thyroid tumor tissues and cell lines. By using immunohistochemistry, NPM overexpression was detected in papillary, follicular, undifferentiated thyroid cancer, and also in follicular benign adenomas, indicating it as an early event during thyroid tumorigenesis. In contrast, various levels of NPM mRNA levels as detected by quantitative RT-PCR were observed in tumor tissues, suggesting a dissociation between protein and transcript expression. The same behavior was observed in the normal thyroid FRTL5 cell lines. In these cells, a positive correlation between NPM protein levels, but not mRNA, and proliferation state was detected. By using thyroid tumor cell lines, we demonstrated that such a post-mRNA regulation may depend on NPM binding to p-Akt, whose levels were found to be increased in the tumor cells, in parallel with reduction of PTEN. In conclusion, our present data demonstrate for the first time that nucleophosmin is overexpressed in thyroid tumors, as an early event of thyroid tumorigenesis. It seems as a result of a dysregulation occurring at protein and not transcriptional level related to an increase of p-Akt levels of transformed thyrocytes.

  3. Overexpression of UCP1 in tobacco induces mitochondrial biogenesis and amplifies a broad stress response

    PubMed Central

    2014-01-01

    Overexpression of UCP1 in the mitochondrial inner membrane induced increased uncoupling respiration, decreased ROS accumulation under abiotic stresses, and diminished cellular ATP content. These events may have triggered the expression of mitochondrial and stress-responsive genes in a coordinated manner. Because these metabolic alterations did not impair plant growth and development, UCP1 overexpression can potentially be used to create crops better adapted to abiotic stress conditions. PMID:24886177

  4. Overexpression of UCP1 in tobacco induces mitochondrial biogenesis and amplifies a broad stress response.

    PubMed

    Barreto, Pedro; Okura, Vagner Katsumi; Neshich, Izabella Agostinho Pena; Maia, Ivan de Godoy; Arruda, Paulo

    2014-05-28

    mitochondrial inner membrane induced increased uncoupling respiration, decreased ROS accumulation under abiotic stresses, and diminished cellular ATP content. These events may have triggered the expression of mitochondrial and stress-responsive genes in a coordinated manner. Because these metabolic alterations did not impair plant growth and development, UCP1 overexpression can potentially be used to create crops better adapted to abiotic stress conditions.

  5. VEGF overexpression in clinically localized prostate tumors and neuropilin-1 overexpression in metastatic forms.

    PubMed

    Latil, A; Bièche, I; Pesche, S; Valéri, A; Fournier, G; Cussenot, O; Lidereau, R

    2000-03-20

    Studies comparing tumor neovascularity with pathological findings suggest that angiogenesis contributes to the pathogenesis of prostate cancer. We have examined 42 primary sporadic prostate tumors at different clinical stages, together with 3 prostate cancer cell lines (DU145, PC3 and LNCaP), for expression of VEGF and the gene encoding the recently identified VEGF165 isoform-specific receptor neuropilin-1, by using a quantitative reverse transcription (RT)-PCR method. We also evaluated the VEGF transcription pattern. Upregulation of VEGF and neuropilin-1 was observed in 12 and 14 tumors, respectively. The VEGF165 isoform was slighly overrepresented in tumors that overexpressed VEGF. VEGF overexpression correlated with stage II disease (p < 0.05); neuropilin-1 overexpression correlated with advanced disease (p < 0. 01) and a high Gleason grade (p < 0.02). Our observations suggest that VEGF expression could be used as a prognostic marker in early-stage prostate tumors, whereas neuropilin-1 overexpression might be a marker of aggressiveness. Copyright 2000 Wiley-Liss, Inc.

  6. The influence of follistatin on mechanical properties of bone tissue in growing mice with overexpression of follistatin.

    PubMed

    Gajos-Michniewicz, Anna; Pawlowska, Elzbieta; Ochedalski, Tomasz; Piastowska-Ciesielska, Agnieszka

    2012-07-01

    Mechanical competence of bones is mainly associated with tissue quality that depends on proper bone metabolism processes. An imbalance in the regulation of bone metabolism leads to pathological changes in bone tissue leading to susceptibility to bone fractures and bone deterioration processes. Bone metabolism is regulated to a large extent by the members of the transforming growth factor-β superfamily, i.e., activins and bone morphogenetic proteins. However, their function is regulated by a single-chain protein called follistatin (FS). The aim of this study was to test the hypothesis that overexpression of FS in growing mice results in impairments in bone morphology and mechanical properties. Moreover, we wanted to investigate how geometrical, structural and material properties of bone tissue change with age. The experiment was performed on growing C57BL/6 TgNK14-mFst/6J mice, overexpressing FS (F mice) versus C57BL/6J mice used as controls (C mice). To establish how overexpression of FS influences bone tissue quality, we studied mice femurs to determine geometrical, structural and material properties of the skeleton. To determine mechanical resistance of bone tissue, femurs were loaded to failure in a three-point bending test. Obtained results indicated that overexpression of FS negatively influences bone metabolism. It was found that mutation results with a significant decrease of all measured biomechanical strength variables in F mice in comparison to C mice. Overexpression of FS leads to decreased quality of skeleton, increasing susceptibility to bone fractures.

  7. Impaired Waters and TMDLs

    EPA Pesticide Factsheets

    The 303(d) program provides guidance and technical resources to assist States in submitting lists of impaired waterbodies and the development of Total Maximum Daily Loads of the pollutant causing the impairment.

  8. Impaired Driving - Multiple Languages

    MedlinePlus

    ... Are Here: Home → Multiple Languages → All Health Topics → Impaired Driving URL of this page: https://medlineplus.gov/languages/ ... V W XYZ List of All Topics All Impaired Driving - Multiple Languages To use the sharing features on ...

  9. Adapting for Impaired Patrons.

    ERIC Educational Resources Information Center

    Schuyler, Michael

    1999-01-01

    Describes how a library, with an MCI Corporation grant, approached the process of setting up computers for the visually impaired. Discusses preparations, which included hiring a visually-impaired user as a consultant and contacting the VIP (Visually Impaired Persons) group; equipment; problems with the graphical user interface; and training.…

  10. Overexpression of membrane proteins using Pichia pastoris.

    PubMed

    Bornert, Olivier; Alkhalfioui, Fatima; Logez, Christel; Wagner, Renaud

    2012-02-01

    Among the small number of expression systems validated for the mass production of eukaryotic membrane proteins (EMPs), the methylotrophic yeast Pichia pastoris stands as one of the most efficient hosts. This system has been used to produce crystallization-grade proteins for a variety of EMPs, from which high-resolution 3D structures have been determined. This unit describes a set of guidelines and instructions to overexpress membrane proteins using the P. pastoris system. Using a G protein-coupled receptor (GPCR) as a model EMP, these protocols illustrate the necessary steps, starting with the design of the DNA sequence to be expressed, through the preparation and analysis of samples containing the corresponding membrane protein of interest. In addition, recommendations are given on a series of experimental parameters that can be optimized to substantially improve the amount and/or the functionality of the expressed EMPs.

  11. Transgenic Mice Overexpressing Renin Exhibit Glucose Intolerance and Diet-Genotype Interactions

    PubMed Central

    Fletcher, Sarah J.; Kalupahana, Nishan S.; Soltani-Bejnood, Morvarid; Kim, Jung Han; Saxton, Arnold M.; Wasserman, David H.; De Taeye, Bart; Voy, Brynn H.; Quignard-Boulange, Annie; Moustaid-Moussa, Naima

    2013-01-01

    Numerous animal and clinical investigations have pointed to a potential role of the renin-angiotensin system (RAS) in the development of insulin resistance and diabetes in conditions of expanded fat mass. However, the mechanisms underlying this association remain unclear. We used a transgenic mouse model overexpressing renin in the liver (RenTgMK) to examine the effects of chronic activation of RAS on adiposity and insulin sensitivity. Hepatic overexpression of renin resulted in constitutively elevated plasma angiotensin II (four- to six-fold increase vs. wild-type, WT). Surprisingly, RenTgMK mice developed glucose intolerance despite low levels of adiposity and insulinemia. The transgenics also had lower plasma triglyceride levels. Glucose intolerance in transgenic mice fed a low-fat diet was comparable to that observed in high-fat fed WT mice. These studies demonstrate that overexpression of renin and associated hyperangiotensinemia impair glucose tolerance in a diet-dependent manner and further support a consistent role of RAS in the pathogenesis of diabetes and insulin resistance, independent of changes in fat mass. PMID:23308073

  12. BAX gene over-expression via nucleofection to induce apoptosis in human lens epithelial cells.

    PubMed

    Fang, Yanwen; Mo, Xiaofen; Luo, Yi; Lu, Yi

    2012-09-01

    Despite significant advances in cataract surgery techniques, posterior capsule opacification (PCO) remains a common complication. In PCO, remaining epithelial cells cloud the lens capsule and impair postoperative vision. This in vitro study was designed to investigate the potential of a gene-based approach, specifically over-expression of the proapoptotic BAX gene, to prevent PCO. Human lens epithelial cells (HLECs) were transfected by nucleofection with a plasmid encoding a fusion protein of green fluorescent protein and human BAX. The expression levels of BAX and its antiapoptotic counterpart BCL2 were determined by realtime reverse transcription polymerase chain reaction, Western blotting and immunofluorescence. BAX over-expression-induced cell death was analyzed by fluorescence-activated cell sorting using the Annexin V antibody. Fluorescence microscopy and transmission electron microscopy were used to assess changes in morphology and ultrastructure. Differential expression of the downstream apoptosis-related factor, caspase 3, was detected by Western blotting. Nucleofection efficiency was high (nearly 80%). BAX-transfected HLECs showed remarkably enhanced BAX gene expression and BAX:BCL2 ratio, but relatively little change in endogenous BCL2 expression. BAX over-expression also led to significant cytotoxicity, induction of apoptosis-related characteristics and activation of caspase 3. In conclusion, our results indicate that BAX gene over-expression can trigger cell death in HLECs via an apoptotic pathway. Thus, BAX may be a promising candidate for human gene therapy to treat PCO.

  13. Arabidopsis thaliana transgenics overexpressing IBR3 show enhanced susceptibility to the bacterium Pseudomonas syringae.

    PubMed

    Huang, T-Y; Desclos-Theveniau, M; Chien, C-T; Zimmerli, L

    2013-09-01

    The gene, indole-3-butyric acid (IBA)-RESPONSE (IBR) 3, is thought to participate in peroxisomal β-oxidation of IBA to indole-3-acetic acid. Here we show that IBR3 may also play a role in Arabidopsis thaliana defence response to microbial pathogens. IBR3 is up-regulated during infection by virulent Pseudomonas syringae pv. tomato (Pst) DC3000 bacteria. Although mutant ibr3-4 did not show a pathogen phenotype, lines overexpressing IBR3 demonstrated enhanced susceptibility to Pst DC3000. Increased susceptibility phenotypes of IBR3 overexpressors were correlated with defective SA defence signalling and impairment of pattern-triggered immunity (PTI) activation. Notably, reactive oxygen species production was reduced in IBR3 overexpressors after treatment with the microbe-associated molecular patterns flg22 and efl26. Later PTI responses, such as accumulation of FRK1 transcripts and callose deposition were also reduced in transgenics overexpressing IBR3 after inoculation with the Type III secretion system deficient bacterial mutant Pst DC3000 hrcC or treatment with flg22 or elf26. Importantly, overexpression of IBR3 did not affect indole-3-acetic acid content or auxin-responsive gene expression. These results suggest a novel role for IBR3 in A. thaliana defence response against bacterial pathogens. © 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.

  14. Influenza A induces dysfunctional immunity and death in MeCP2-overexpressing mice

    PubMed Central

    Cronk, James C.; Herz, Jasmin; Kim, Taeg S.; Louveau, Antoine; Moser, Emily K.; Smirnov, Igor; Tung, Kenneth S.; Braciale, Thomas J.

    2017-01-01

    Loss of function or overexpression of methyl-CpG-binding protein 2 (MeCP2) results in the severe neurodevelopmental disorders Rett syndrome and MeCP2 duplication syndrome, respectively. MeCP2 plays a critical role in neuronal function and the function of cells throughout the body. It has been previously demonstrated that MeCP2 regulates T cell function and macrophage response to multiple stimuli, and that immune-mediated rescue imparts significant benefit in Mecp2-null mice. Unlike Rett syndrome, MeCP2 duplication syndrome results in chronic, severe respiratory infections, which represent a significant cause of patient morbidity and mortality. Here, we demonstrate that MeCP2Tg3 mice, which overexpress MeCP2 at levels 3- to 5-fold higher than normal, are hypersensitive to influenza A/PR/8/34 infection. Prior to death, MeCP2Tg3 mice experienced a host of complications during infection, including neutrophilia, increased cytokine production, excessive corticosterone levels, defective adaptive immunity, and vascular pathology characterized by impaired perfusion and pulmonary hemorrhage. Importantly, we found that radioresistant cells are essential to infection-related death after bone marrow transplantation. In all, these results demonstrate that influenza A infection in MeCP2Tg3 mice results in pathology affecting both immune and nonhematopoietic cells, suggesting that failure to effectively respond and clear viral respiratory infection has a complex, multicompartment etiology in the context of MeCP2 overexpression. PMID:28138553

  15. CREB overexpression in dorsal CA1 ameliorates long-term memory deficits in aged rats

    PubMed Central

    Yu, Xiao-Wen; Curlik, Daniel M; Oh, M Matthew; Yin, Jerry CP; Disterhoft, John F

    2017-01-01

    The molecular mechanisms underlying age-related cognitive deficits are not yet fully elucidated. In aged animals, a decrease in the intrinsic excitability of CA1 pyramidal neurons is believed to contribute to age-related cognitive impairments. Increasing activity of the transcription factor cAMP response element-binding protein (CREB) in young adult rodents facilitates cognition, and increases intrinsic excitability. However, it has yet to be tested if increasing CREB expression also ameliorates age-related behavioral and biophysical deficits. To test this hypothesis, we virally overexpressed CREB in CA1 of dorsal hippocampus. Rats received CREB or control virus, before undergoing water maze training. CREB overexpression in aged animals ameliorated the long-term memory deficits observed in control animals. Concurrently, cells overexpressing CREB in aged animals had reduced post-burst afterhyperpolarizations, indicative of increased intrinsic excitability. These results identify CREB modulation as a potential therapy to treat age-related cognitive decline. DOI: http://dx.doi.org/10.7554/eLife.19358.001 PMID:28051768

  16. Calpastatin overexpression limits calpain-mediated proteolysis and behavioral deficits following traumatic brain injury

    PubMed Central

    Schoch, Kathleen M.; Evans, Heather N.; Brelsfoard, Jennifer M.; Madathil, Sindhu K.; Takano, Jiro; Saido, Takaomi C.; Saatman, Kathryn E.

    2012-01-01

    Traumatic brain injury (TBI) results in abrupt, initial cell damage leading to delayed neuronal death. The calcium-activated proteases, calpains, are known to contribute to this secondary neurodegenerative cascade. Although the specific inhibitor of calpains, calpastatin, is present within neurons, normal levels of calpastatin are unable to fully prevent the damaging proteolytic activity of calpains after injury. In this study, increased calpastatin expression was achieved using transgenic mice that overexpress the human calpastatin (hCAST) construct under control of a calcium-calmodulin dependent kinase II α promoter. Naïve hCAST transgenic mice exhibited enhanced neuronal calpastatin expression and significantly reduced protease activity. Acute calpain-mediated spectrin proteolysis in the cortex and hippocampus induced by controlled cortical impact brain injury was significantly attenuated in calpastatin overexpressing mice. Aspects of posttraumatic motor and cognitive behavioral deficits were also lessened in hCAST transgenic mice compared to their wildtype littermates. However, volumetric analyses of neocortical contusion revealed no histological neuroprotection at either acute or long-term time points. Partial hippocampal neuroprotection observed at a moderate injury severity was lost after severe TBI. This study underscores the effectiveness of calpastatin overexpression in reducing calpain-mediated proteolysis and behavioral impairment after TBI, supporting the therapeutic potential for calpain inhibition. In addition, the reduction in spectrin proteolysis without accompanied neocortical neuroprotection suggests the involvement of other factors that are critical for neuronal survival after contusion brain injury. PMID:22572592

  17. Targeted Overexpression of EZH2 in the Mammary Gland Disrupts Ductal Morphogenesis and Causes Epithelial Hyperplasia

    PubMed Central

    Li, Xin; Gonzalez, Maria E.; Toy, Katherine; Filzen, Tracey; Merajver, Sofia D.; Kleer, Celina G.

    2009-01-01

    The Polycomb group protein enhancer of zeste homolog 2 (EZH2), which has roles during development of numerous tissues, is a critical regulator of cell type identity. Overexpression of EZH2 has been detected in invasive breast carcinoma tissue samples and is observed in human breast tissue samples of morphologically normal lobules up to 12 years before the development of breast cancer. The function of EZH2 during preneoplastic progression in the mammary gland is unknown. To investigate the role of EZH2 in the mammary gland, we targeted the expression of EZH2 to mammary epithelial cells using the mouse mammary tumor virus long terminal repeat. EZH2 overexpression resulted in aberrant terminal end bud architecture. By the age of 4 months, 100% of female mouse mammary tumor virus-EZH2 virgin mice developed intraductal epithelial hyperplasia resembling the human counterpart accompanied by premature differentiation of ductal epithelial cells and up-regulation of the luminal marker GATA-3. In addition, remodeling of the mammary gland after parturition was impaired and EZH2 overexpression caused delayed involution. Mechanistically, we found that EZH2 physically interacts with β-catenin, inducing β-catenin nuclear accumulation in mammary epithelial cells and activating Wnt/β-catenin signaling. The biological significance of these data to human hyperplasias is demonstrated by EZH2 up-regulation and colocalization with β-catenin in human intraductal epithelial hyperplasia, the earliest histologically identifiable precursor of breast carcinoma. PMID:19661437

  18. Bone Morphogenetic Protein-4 Inhibitor Gremlin Is Overexpressed in Idiopathic Pulmonary Fibrosis

    PubMed Central

    Koli, Katri; Myllärniemi, Marjukka; Vuorinen, Kirsi; Salmenkivi, Kaisa; Ryynänen, Merja J.; Kinnula, Vuokko L.; Keski-Oja, Jorma

    2006-01-01

    Idiopathic pulmonary fibrosis (IPF), ie, usual interstitial pneumonia in histopathology, is a disease characterized by tissue destruction and active areas of fibroproliferation in the lung. Gremlin (Drm), a member of the cysteine knot family of bone morphogenetic protein (BMP) inhibitors, functions to antagonize BMP-4-mediated signals during lung development. We describe here consistent overexpression of gremlin in the lung interstitium of IPF patients. Quantitative real-time reverse transcriptase-polymerase chain reaction analyses revealed considerably higher levels of gremlin mRNA in lung biopsies from IPF patients, the highest level being 35-fold higher compared to controls. Lung fibroblasts isolated from IPF patients also expressed elevated levels of gremlin, which was associated with impaired responsiveness to endogenous and exogenous BMP-4. Transforming growth factor-β-induced epithelial-to-mesenchymal transition of A549 lung epithelial cells in culture was also associated with induction of gremlin mRNA expression. In addition, A549 cells transfected to overexpress gremlin were more susceptible to transforming growth factor-β-induced epithelial-to-mesenchymal transition. Gremlin-mediated inhibition of BMP-4 signaling pathways is likely to enhance the fibrotic response and reduce epithelial regeneration in the lung. The overexpression of this developmental gene in IPF may be a key event in the persistence of myofibroblasts in the lung interstitium and provides a potential target for therapeutic intervention. PMID:16816361

  19. Bone morphogenetic protein-4 inhibitor gremlin is overexpressed in idiopathic pulmonary fibrosis.

    PubMed

    Koli, Katri; Myllärniemi, Marjukka; Vuorinen, Kirsi; Salmenkivi, Kaisa; Ryynänen, Merja J; Kinnula, Vuokko L; Keski-Oja, Jorma

    2006-07-01

    Idiopathic pulmonary fibrosis (IPF), ie, usual interstitial pneumonia in histopathology, is a disease characterized by tissue destruction and active areas of fibroproliferation in the lung. Gremlin (Drm), a member of the cysteine knot family of bone morphogenetic protein (BMP) inhibitors, functions to antagonize BMP-4-mediated signals during lung development. We describe here consistent overexpression of gremlin in the lung interstitium of IPF patients. Quantitative real-time reverse transcriptase-polymerase chain reaction analyses revealed considerably higher levels of gremlin mRNA in lung biopsies from IPF patients, the highest level being 35-fold higher compared to controls. Lung fibroblasts isolated from IPF patients also expressed elevated levels of gremlin, which was associated with impaired responsiveness to endogenous and exogenous BMP-4. Transforming growth factor-beta-induced epithelial-to-mesenchymal transition of A549 lung epithelial cells in culture was also associated with induction of gremlin mRNA expression. In addition, A549 cells transfected to overexpress gremlin were more susceptible to transforming growth factor-beta-induced epithelial-to-mesenchymal transition. Gremlin-mediated inhibition of BMP-4 signaling pathways is likely to enhance the fibrotic response and reduce epithelial regeneration in the lung. The overexpression of this developmental gene in IPF may be a key event in the persistence of myofibroblasts in the lung interstitium and provides a potential target for therapeutic intervention.

  20. MARCKS protein overexpression in inflammatory breast cancer

    PubMed Central

    Manai, Maroua; Lopez, Marc; Eghozzi, Radhia; Ayadi, Sinda; Lamine, Olfa Ben; Manai, Mohamed; Rahal, Khaled; Charafe-Jauffret, Emmanuelle; Jacquemier, Jocelyne; Viens, Patrice; Birnbaum, Daniel; Boussen, Hamouda; Chaffanet, Max; Bertucci, François

    2017-01-01

    Background Inflammatory breast cancer (IBC) is the most aggressive form of locally-advanced breast cancer. Identification of new therapeutic targets is crucial. We previously reported MARCKS mRNA overexpression in IBC in the largest transcriptomics study reported to date. Here, we compared MARCKS protein expression in IBC and non-IBC samples, and searched for correlations between protein expression and clinicopathological features. Results Tumor samples showed heterogeneity with respect to MARCKS staining: 18% were scored as MARCKS-positive (stained cells ≥ 1%) and 82% as MARCKS-negative. MARCKS expression was more frequent in IBC (36%) than in non-IBC (11%; p = 1.4E−09), independently from molecular subtypes and other clinicopathological variables. We found a positive correlation between protein and mRNA expression in the 148/502 samples previously analyzed for MARCKS mRNA expression. MARCKS protein expression was associated with other poor-prognosis features in the whole series of samples such as clinical axillary lymph node or metastatic extension, high pathological grade, ER-negativity, PR-negativity, HER2-positivity, and triple-negative and HER2+ statutes. In IBC, MARCKS expression was the sole tested variable associated with poor MFS. Materials and Methods We retrospectively analyzed MARCKS protein expression by immunohistochemistry in 502 tumors, including 133 IBC and 369 non-IBC, from Tunisian and French patients. All samples were pre-therapeutic clinical samples. We searched for correlations between MARCKS expression and clinicopathological features including the IBC versus non-IBC phenotype and metastasis-free survival (MFS). Conclusions MARCKS overexpression might in part explain the poor prognosis of IBC. As an oncogene associated with poor MFS, MARCKS might represent a new potential therapeutic target in IBC. PMID:28009981

  1. Targeted anticancer therapy: overexpressed receptors and nanotechnology.

    PubMed

    Akhtar, Mohd Javed; Ahamed, Maqusood; Alhadlaq, Hisham A; Alrokayan, Salman A; Kumar, Sudhir

    2014-09-25

    Targeted delivery of anticancer drugs to cancer cells and tissues is a promising field due to its potential to spare unaffected cells and tissues, but it has been a major challenge to achieve success in these therapeutic approaches. Several innovative approaches to targeted drug delivery have been devised based on available knowledge in cancer biology and on technological advancements. To achieve the desired selectivity of drug delivery, nanotechnology has enabled researchers to design nanoparticles (NPs) to incorporate anticancer drugs and act as nanocarriers. Recently, many receptor molecules known to be overexpressed in cancer have been explored as docking sites for the targeting of anticancer drugs. In principle, anticancer drugs can be concentrated specifically in cancer cells and tissues by conjugating drug-containing nanocarriers with ligands against these receptors. Several mechanisms can be employed to induce triggered drug release in response to either endogenous trigger or exogenous trigger so that the anticancer drug is only released upon reaching and preferentially accumulating in the tumor tissue. This review focuses on overexpressed receptors exploited in targeting drugs to cancerous tissues and the tumor microenvironment. We briefly evaluate the structure and function of these receptor molecules, emphasizing the elegant mechanisms by which certain characteristics of cancer can be exploited in cancer treatment. After this discussion of receptors, we review their respective ligands and then the anticancer drugs delivered by nanotechnology in preclinical models of cancer. Ligand-functionalized nanocarriers have delivered significantly higher amounts of anticancer drugs in many in vitro and in vivo models of cancer compared to cancer models lacking such receptors or drug carrying nanocarriers devoid of ligand. This increased concentration of anticancer drug in the tumor site enabled by nanotechnology could have a major impact on the efficiency of cancer

  2. Overexpression of Colligin 2 in Glioma Vasculature is Associated with Overexpression of Heat Shock Factor 2.

    PubMed

    Mustafa, Dana A M; Sieuwerts, Anieta M; Zheng, Ping Pin; Kros, Johan M

    2010-10-20

    In previous studies we found expression of the protein colligin 2 (heat shock protein 47 (HSP47), SERPINH1) in glioma neovasculature while not in normal brain tissue. Generally, the regulation of heat shock gene expression in eukaryotes is mediated by heat shock factors (HSF). In mammals, three heat shock transcription factors, HSF-1, -2, and -4, have been isolated. Here we investigated the relation between the expression of colligin 2 and these heat shock factors at the mRNA level using real-time reverse transcriptase PCR (qRT-PCR) in different grades of astrocytic tumorigenesis, viz., low-grade glioma and glioblastoma. Endometrium samples, representing physiological angiogenesis, were included as controls. Since colligin 2 is a chaperon for collagens, the gene expression of collagen I (COL1A1) was also investigated. The blood vessel density of the samples was monitored by expression of the endothelial marker CD31 (PECAM1). Because NG2-immunopositive pericytic cells are involved in glioma neovascularization, the expression of NG2 (CSPG4) was also measured.We demonstrate overexpression of HSF2 in both stages of glial tumorigenesis (reaching significance only in low-grade glioma) and also minor elevated levels of HSF1 as compared to normal brain. There were no differences in expression of HSF4 between low-grade glioma and normal brain while HSF4 was downregulated in glioblastoma. In the endometrium samples, none of the HSFs were upregulated. In the low-grade gliomas SERPINH appeared to be slightly overexpressed with a parallel 4-fold upregulation of COL1A1, while in glioblastoma there was over 5-fold overexpression of SERPINH1 and more than 150-fold overexpression of COL1A1. In both the lowgrade gliomas and the glioblastomas overexpression of CSPG4 was found and overexpression of PECAM1 was only found in the latter. Our data suggest that the upregulated expression of colligin 2 in glioma is accompanied by upregulation of COL1A1, CSPG4, HSF2 and to a lesser extent

  3. Prion protein and Aβ-related synaptic toxicity impairment

    PubMed Central

    Calella, Anna Maria; Farinelli, Mélissa; Nuvolone, Mario; Mirante, Osvaldo; Moos, Rita; Falsig, Jeppe; Mansuy, Isabelle M; Aguzzi, Adriano

    2010-01-01

    Alzheimer's disease (AD), the most common neurodegenerative disorder, goes along with extracellular amyloid-β (Aβ) deposits. The cognitive decline observed during AD progression correlates with damaged spines, dendrites and synapses in hippocampus and cortex. Numerous studies have shown that Aβ oligomers, both synthetic and derived from cultures and AD brains, potently impair synaptic structure and functions. The cellular prion protein (PrPC) was proposed to mediate this effect. We report that ablation or overexpression of PrPC had no effect on the impairment of hippocampal synaptic plasticity in a transgenic model of AD. These findings challenge the role of PrPC as a mediator of Aβ toxicity. PMID:20665634

  4. Memory Impairment in Children with Language Impairment

    ERIC Educational Resources Information Center

    Baird, Gillian; Dworzynski, Katharina; Slonims, Vicky; Simonoff, Emily

    2010-01-01

    Aim: The aim of this study was to assess whether any memory impairment co-occurring with language impairment is global, affecting both verbal and visual domains, or domain specific. Method: Visual and verbal memory, learning, and processing speed were assessed in children aged 6 years to 16 years 11 months (mean 9y 9m, SD 2y 6mo) with current,…

  5. Visual impairment in the hearing impaired students

    PubMed Central

    Gogate, Parikshit; Rishikeshi, Nikhil; Mehata, Reshma; Ranade, Satish; Kharat, Jitesh; Deshpande, Madan

    2009-01-01

    Background: Ocular problems are more common in children with hearing problems than in normal children. Neglected visual impairment could aggravate educational and social disability. Aim: To detect and treat visual impairment, if any, in hearing-impaired children. Setting and Design: Observational, clinical case series of hearing-impaired children in schools providing special education. Materials and Methods: Hearing-impaired children in selected schools underwent detailed visual acuity testing, refraction, external ocular examination and fundoscopy. Ocular motility testing was also performed. Teachers were sensitized and trained to help in the assessment of visual acuity using Snellen's E charts. Refractive errors and squint were treated as per standard practice. Statistical Analysis: Excel software was used for data entry and SSPS for analysis. Results: The study involved 901 hearing-impaired students between four and 21 years of age, from 14 special education schools. A quarter of them (216/901, 24%) had ocular problems. Refractive errors were the most common morbidity 167(18.5%), but only 10 children were using appropriate spectacle correction at presentation. Fifty children had visual acuity less than 20/80 at presentation; after providing refractive correction, this number reduced to three children, all of whom were provided low-vision aids. Other common conditions included strabismus in 12 (1.3%) children, and retinal pigmentary dystrophy in five (0.6%) children. Conclusion: Ocular problems are common in hearing-impaired children. Screening for ocular problems should be made mandatory in hearing-impaired children, as they use their visual sense to compensate for the poor auditory sense. PMID:19861747

  6. Memory Impairment in Children with Language Impairment

    ERIC Educational Resources Information Center

    Baird, Gillian; Dworzynski, Katharina; Slonims, Vicky; Simonoff, Emily

    2010-01-01

    Aim: The aim of this study was to assess whether any memory impairment co-occurring with language impairment is global, affecting both verbal and visual domains, or domain specific. Method: Visual and verbal memory, learning, and processing speed were assessed in children aged 6 years to 16 years 11 months (mean 9y 9m, SD 2y 6mo) with current,…

  7. Changes in brain cholinergic markers and spatial learning in old galanin-overexpressing mice.

    PubMed

    Pirondi, S; D'Intino, G; Gusciglio, M; Massella, A; Giardino, L; Kuteeva, E; Ogren, S-O; Hökfelt, T; Calzà, L

    2007-03-23

    The cholinergic forebrain system is involved in learning and memory, and its age-dependent decline correlates with a decrease in cognitive performance. Since the neuropeptide galanin participates in cholinergic neuron regulation, we have studied 19- to 23-month-old male mice overexpressing galanin under the platelet-derived growth factor B promoter (GalOE) and wild-type (WT) littermates by monitoring behavioral, neurochemical and morphological/histochemical parameters. In the Morris water maze test, old transgenic animals showed a significant impairment in escape latency in the hidden platform test compared to age-matched WT animals. The morphological/histochemical studies revealed that cholinergic neurons in the basal forebrain display a slight, age- but not genotype-related, alteration in choline acetyltransferase- (ChAT) immunoreactivity. The neurochemical studies showed an age-related decline in ChAT activity in the cerebral cortex of all mice, whereas in the hippocampal formation this effect was seen in GalOE but not WT animals. Expression of BDNF mRNA in the hippocampal formation, as evaluated by RT-PCR, was reduced in old animals; no age- or genotype-induced variations in NGF mRNA expression were observed. These data suggest that galanin overexpression further accentuates the age-related decline of the cholinergic system activity in male mice, resulting in impairment of water maze performance in old animals.

  8. Transgenic overexpression of ADAM12 suppresses muscle regeneration and aggravates dystrophy in aged mdx mice.

    PubMed

    Jørgensen, Louise Helskov; Jensen, Charlotte Harken; Wewer, Ulla M; Schrøder, Henrik Daa

    2007-11-01

    Muscular dystrophies are characterized by insufficient restoration and gradual replacement of the skeletal muscle by fat and connective tissue. ADAM12 has previously been shown to alleviate the pathology of young dystrophin-deficient mdx mice, a model for Duchenne muscular dystrophy. The observed effect of ADAM12 was suggested to be mediated via a membrane-stabilizing up-regulation of utrophin, alpha7B integrin, and dystroglycans. Ectopic ADAM12 expression in normal mouse skeletal muscle also improved regeneration after freeze injury, presumably by the same mechanism. Hence, it was suggested that ADAM12 could be a candidate for nonreplacement gene therapy of Duchenne muscular dystrophy. We therefore evaluated the long-term effect of ADAM12 overexpression in muscle. Surprisingly, we observed loss of skeletal muscle and accelerated fibrosis and adipogenesis in 1-year-old mdx mice transgenically overexpressing ADAM12 (ADAM12(+)/mdx mice), even though their utrophin levels were mildly elevated compared with age-matched controls. Thus, membrane stabilization was not sufficient to provide protection during prolonged disease. Consequently, we reinvestigated skeletal muscle regeneration in ADAM12 transgenic mice (ADAM12(+)) after a knife cut lesion and observed that the regeneration process was significantly impaired. ADAM12 seemed to inhibit the satellite cell response and delay myoblast differentiation. These results discourage long-term therapeutic use of ADAM12. They also point to impaired regeneration as a possible factor in development of muscular dystrophy.

  9. Lethal lung hypoplasia and vascular defects in mice with conditional Foxf1 overexpression

    PubMed Central

    Dharmadhikari, Avinash V.; Sun, Jenny J.; Gogolewski, Krzysztof; Carofino, Brandi L.; Ustiyan, Vladimir; Hill, Misty; Majewski, Tadeusz; Szafranski, Przemyslaw; Justice, Monica J.; Ray, Russell S.; Dickinson, Mary E.; Kalinichenko, Vladimir V.; Gambin, Anna

    2016-01-01

    ABSTRACT FOXF1 heterozygous point mutations and genomic deletions have been reported in newborns with the neonatally lethal lung developmental disorder, alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). However, no gain-of-function mutations in FOXF1 have been identified yet in any human disease conditions. To study the effects of FOXF1 overexpression in lung development, we generated a Foxf1 overexpression mouse model by knocking-in a Cre-inducible Foxf1 allele into the ROSA26 (R26) locus. The mice were phenotyped using micro-computed tomography (micro-CT), head-out plethysmography, ChIP-seq and transcriptome analyses, immunohistochemistry, and lung histopathology. Thirty-five percent of heterozygous R26-Lox-Stop-Lox (LSL)-Foxf1 embryonic day (E)15.5 embryos exhibit subcutaneous edema, hemorrhages and die perinatally when bred to Tie2-cre mice, which targets Foxf1 overexpression to endothelial and hematopoietic cells. Histopathological and micro-CT evaluations revealed that R26Foxf1; Tie2-cre embryos have immature lungs with a diminished vascular network. Neonates exhibited respiratory deficits verified by detailed plethysmography studies. ChIP-seq and transcriptome analyses in E18.5 lungs identified Sox11, Ghr, Ednrb, and Slit2 as potential downstream targets of FOXF1. Our study shows that overexpression of the highly dosage-sensitive Foxf1 impairs lung development and causes vascular abnormalities. This has important clinical implications when considering potential gene therapy approaches to treat disorders of FOXF1 abnormal dosage, such as ACDMPV. PMID:27638768

  10. IL10R2 Overexpression Promotes IL22/STAT3 Signaling in Colorectal Carcinogenesis.

    PubMed

    Khare, Vineeta; Paul, Gregor; Movadat, Oliver; Frick, Adrian; Jambrich, Manuela; Krnjic, Anita; Marian, Brigitte; Wrba, Friedrich; Gasche, Christoph

    2015-11-01

    The mucosal immune response in the setting of intestinal inflammation contributes to colorectal cancer. IL10 signaling has a central role in gut homeostasis and is impaired in inflammatory bowel disease (IBD). Out of two IL10 receptor subunits, IL10R1 and IL10R2, the latter is shared among the IL10 family of cytokines and activates STAT signaling. STAT3 is oncogenic in colorectal cancer; however, knowledge about IL10 signaling upstream of STAT3 in colorectal cancer is lacking. Here, expression of IL10 signaling genes was examined in matched pairs from normal and tumor tissue from colorectal cancer patients showing overexpression (mRNA, protein) of IL10R2 and STAT3 but not IL10R1. IL10R2 overexpression was related to microsatellite stability. Transient overexpression of IL10R2 in HT29 cells increased proliferation upon ligand activation (IL10 and IL22). IL22, and not IL10, phosphorylated STAT3 along with increased phosphorylation of AKT and ERK. A significantly higher expression of IL22R1 and IL10R2 was also confirmed in a separate cohort of colorectal cancer samples. IL22 expression was elevated in gut mucosa from patients with IBD and colitis-associated cancer, which also exhibited increased expression of IL22R1 but not its coreceptor IL10R2. Overall, these data indicate that overexpression of IL10R2 and STAT3 contributes to colorectal carcinogenesis in microsatellite-stable tumors through IL22/STAT3 signaling.

  11. PTEN loss is associated with a poor response to trastuzumab in HER2-overexpressing gastroesophageal adenocarcinoma.

    PubMed

    Deguchi, Yasunori; Okabe, Hiroshi; Oshima, Nobu; Hisamori, Shigeo; Minamiguchi, Sachiko; Muto, Manabu; Sakai, Yoshiharu

    2017-05-01

    Although trastuzumab improves the outcome of patients with human epidermal growth factor receptor 2 (HER2)-overexpressing gastric or gastroesophageal junction adenocarcinoma (collectively referred to as "gastroesophageal adenocarcinoma"; GEA), no clinical response is observed in a substantial population of patients. A predictive biomarker of trastuzumab response is required. The aim of this study was to evaluate whether the hyperactivation of the downstream phosphatidylinositol 3-kinase pathway, due to phosphatase and tensin homolog (PTEN) loss or PIK3CA mutations, could provide trastuzumab resistance in GEA. Expression of HER2 and PTEN, and PIK3CA gene mutations were screened in 264 surgically resected GEA specimens. The effects of PTEN knockdown on the response to trastuzumab on cell viability, HER2 downstream signaling, apoptosis, and cell cycle were evaluated in HER2-overexpressing NCI-N87 gastric adenocarcinoma and OE19 esophageal adenocarcinoma cell lines. Inhibition of xenograft tumor growth by trastuzumab was investigated in OE19 cells with or without PTEN knockdown. The PTEN expression and objective response were analyzed in 23 GEA patients who received trastuzumab-based therapy. PTEN loss was identified in 34.5 % of HER2-overexpressing GEA patients, whereas PIK3CA mutations were rare (5.6 %). Trastuzumab-mediated growth suppression, apoptosis, and G1 cell cycle arrest were inhibited by PTEN knockdown through Akt activation in NCI-N87 and OE19 cells. PTEN knockdown impaired the antiproliferative effect of trastuzumab in OE19 xenograft models. A clinical response was observed in 50 % of PTEN-positive tumors (9 of 18) but in no tumors with PTEN loss (none of 5). PTEN loss was frequently found in HER2-overexpressing tumors, and was associated with a poor response to trastuzumab-based therapy in patients with GEA.

  12. Lysyl oxidase overexpression accelerates cardiac remodeling and aggravates angiotensin II-induced hypertrophy.

    PubMed

    Galán, María; Varona, Saray; Guadall, Anna; Orriols, Mar; Navas, Miquel; Aguiló, Silvia; de Diego, Alicia; Navarro, María A; García-Dorado, David; Rodríguez-Sinovas, Antonio; Martínez-González, José; Rodriguez, Cristina

    2017-09-01

    Lysyl oxidase (LOX) controls matrix remodeling, a key process that underlies cardiovascular diseases and heart failure; however, a lack of suitable animal models has limited our knowledge with regard to the contribution of LOX to cardiac dysfunction. Here, we assessed the impact of LOX overexpression on ventricular function and cardiac hypertrophy in a transgenic LOX (TgLOX) mouse model with a strong cardiac expression of human LOX. TgLOX mice exhibited high expression of the transgene in cardiomyocytes and cardiofibroblasts, which are associated with enhanced LOX activity and H2O2 production and with cardiofibroblast reprogramming. LOX overexpression promoted an age-associated concentric remodeling of the left ventricle and impaired diastolic function. Furthermore, LOX transgenesis aggravated angiotensin II (Ang II)-induced cardiac hypertrophy and dysfunction, which triggered a greater fibrotic response that was characterized by stronger collagen deposition and cross-linking and high expression of fibrotic markers. In addition, LOX transgenesis increased the Ang II-induced myocardial inflammatory infiltrate, exacerbated expression of proinflammatory markers, and decreased that of cardioprotective factors. Mechanistically, LOX overexpression enhanced oxidative stress and potentiated the Ang II-mediated cardiac activation of p38 MAPK while reducing AMPK activation. Our findings suggest that LOX induces an age-dependent disturbance of diastolic function and aggravates Ang II-induced hypertrophy, which provides novel insights into the role of LOX in cardiac performance.-Galán, M., Varona, S., Guadall, A., Orriols, M., Navas, M., Aguiló, S., de Diego, A., Navarro, M. A., García-Dorado, D., Rodríguez-Sinovas, A., Martínez-González, J., Rodriguez, C. Lysyl oxidase overexpression accelerates cardiac remodeling and aggravates angiotensin II-induced hypertrophy. © FASEB.

  13. Depressive-like phenotype induced by AAV-mediated overexpression of human α-synuclein in midbrain dopaminergic neurons.

    PubMed

    Caudal, D; Alvarsson, A; Björklund, A; Svenningsson, P

    2015-11-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by a progressive loss of nigral dopaminergic neurons and by the presence of aggregates containing α-synuclein called Lewy bodies. Viral vector-induced overexpression of α-synuclein in dopaminergic neurons represents a model of PD which recapitulates disease progression better than commonly used neurotoxin models. Previous studies using this model have reported motor and cognitive impairments, whereas depression, mood and anxiety phenotypes are less described. To investigate these psychiatric phenotypes, Sprague-Dawley rats received bilateral injections of a recombinant adeno-associated virus (AAV) vector expressing human α-synuclein or GFP into the substantia nigra pars compacta. Behavior was assessed at two timepoints: 3 and 8 weeks post-injection. We report that nigral α-synuclein overexpression led to a pronounced nigral dopaminergic cell loss accompanied by a smaller cell loss in the ventral tegmental area, and to a decreased striatal density of dopaminergic fibers. The AAV-α-synuclein group exhibited modest, but significant motor impairments 8 weeks after vector administration. The AAV-α-synuclein group displayed depressive-like behavior in the forced swim test after 3 weeks, and reduced sucrose preference at week 8. At both timepoints, overexpression of α-synuclein was linked to a hyperactive hypothalamic-pituitary-adrenal (HPA) axis regulation of corticosterone. The depressive-like phenotype was also correlated with decreased nigral brain-derived neurotrophic factor and spinophilin levels, and with decreased striatal levels of the activity-regulated cytoskeleton-associated protein. This study demonstrates that AAV-mediated α-synuclein overexpression in dopamine neurons is not only useful to model motor impairments of PD, but also depression. This study also provides evidence that depression in experimental Parkinsonism is correlated to dysregulation of the HPA axis and to

  14. Overexpression of phosphodiesterase-4 subtypes involved in surgery-induced neuroinflammation and cognitive dysfunction in mice.

    PubMed

    Wang, Wei; Zhang, Xiao-Ying; Feng, Ze-Guo; Wang, Dong-Xin; Zhang, Hao; Sui, Bo; Zhang, Yong-Yi; Zhao, Wei-Xing; Fu, Qiang; Xu, Zhi-Peng; Mi, Wei-Dong

    2017-02-21

    Postoperative cognitive dysfunction (POCD) is characterized by cognitive impairments in patients after surgery. Hippocampal neuroinflammation induced by surgery is highly associated with POCD. Phosphodiesterase-4 (PDE4) is an enzyme that specifically hydrolyses cyclic adenosine monophosphate (cAMP), which plays an important role during neuroinflammation and the process of learning and memory. However, the role of PDE4 in the development of POCD remains unclear. Male 14-month-old C57BL/6 mice received carotid artery exposure to mimic POCD. First, we evaluated cognitive performance by a Morris water maze (MWM) and fear conditioning system (FCS) test after surgery. The expression of PDE4 subtypes, pro-inflammatory cytokines, p-CREB and PSD95 as well as cAMP levels were investigated. Then, we used rolipram, a PDE4 inhibitor, to block the effects of PDE4. The cognitive performance of the mice and the expression of PDE4 subtypes, pro-inflammatory cytokines, p-CREB and PSD95 as well as cAMP levels were examined again. Mice displayed learning and memory impairment, overexpression of PDE4B and PDE4D, elevation of pro-inflammatory cytokines, and reduction in the expression of p-CREB, PSD95 and cAMP levels after surgery. The expression of PDE4B and PDE4D in the hippocampus decreased following blocking of PDE4 by rolipram. Meanwhile, rolipram attenuated the cognitive impairment and the elevation of pro-inflammatory cytokines induced by surgery. Moreover, rolipram reversed the reduction of p-CREB and PSD95. These results indicate that PDE4 subtype overexpression may be involved in the development of surgery-induced cognitive dysfunction in mice.

  15. Creatine Kinase-Overexpression Improves Myocardial Energetics, Contractile Dysfunction and Survival in Murine Doxorubicin Cardiotoxicity

    PubMed Central

    Gupta, Ashish; Rohlfsen, Cory; Leppo, Michelle K.; Chacko, Vadappuram P.; Wang, Yibin; Steenbergen, Charles; Weiss, Robert G.

    2013-01-01

    Doxorubicin (DOX) is a commonly used life-saving antineoplastic agent that also causes dose-dependent cardiotoxicity. Because ATP is absolutely required to sustain normal cardiac contractile function and because impaired ATP synthesis through creatine kinase (CK), the primary myocardial energy reserve reaction, may contribute to contractile dysfunction in heart failure, we hypothesized that impaired CK energy metabolism contributes to DOX-induced cardiotoxicity. We therefore overexpressed the myofibrillar isoform of CK (CK-M) in the heart and determined the energetic, contractile and survival effects of CK-M following weekly DOX (5mg/kg) administration using in vivo31P MRS and 1H MRI. In control animals, in vivo cardiac energetics were reduced at 7 weeks of DOX protocol and this was followed by a mild but significant reduction in left ventricular ejection fraction (EF) at 8 weeks of DOX, as compared to baseline. At baseline, CK-M overexpression (CK-M-OE) increased rates of ATP synthesis through cardiac CK (CK flux) but did not affect contractile function. Following DOX however, CK-M-OE hearts had better preservation of creatine phosphate and higher CK flux and higher EF as compared to control DOX hearts. Survival after DOX administration was significantly better in CK-M-OE than in control animals (p<0.02). Thus CK-M-OE attenuates the early decline in myocardial high-energy phosphates and contractile function caused by chronic DOX administration and increases survival. These findings suggest that CK impairment plays an energetic and functional role in this DOX-cardiotoxicity model and suggests that metabolic strategies, particularly those targeting CK, offer an appealing new strategy for limiting DOX-associated cardiotoxicity. PMID:24098344

  16. [Effects of NYGGF4 gene over-expression on the insulin sensitivity and secretory function of adipocytes].

    PubMed

    Zhang, Chun-Mei; Qiu, Jie; Chen, Xiao-Hui; Wang, Bin; Zhang, Min; Guo, Xi-Rong

    2009-10-01

    To study the effect of a new obesity-related gene NYGGF4 on the insulin sensitivity and secretory function of adipocytes. 3T3-L1 preadipocytes transfected with either an empty expression vector (pcDNA3.1; control group) or an NYGGF4 expression vector (NYGGF4-pcDNA3.1) were cultured in vitro and differentiated into the matured adipocytes with the standard insulin plus dexamethasone plus 3-isobutyl-methylxanthine (MDI) induction cocktail. 2-deoxy-D-[3H] glucose uptake was determined by liquid scintillation counting. Western blot was performed to detect the protein content and translocation of glucose transporter 4 (GLUT4). The supernatant concentrations of TNF-alpha, IL-6, adiponectin and resistin were measured using ELISA. NYGGF4 over-expression in 3T3-L1 adipocytes reduced insulin-stimulated glucose uptake. NYGGF4 over-expression impaired insulin-stimulated GLUT4 translocation without affecting the total protein content of GLUT4. The concentrations of TNF-alpha, IL-6, adiponectin and resistin in the culture medium of 3T3-L1 transfected with NYGGF4 were not significantly different from those in the control group. NYGGF4 over-expression impairs the insulin sensitivity of 3T3-L1 adipocytes through decreasing GLUT4 translocation and had no effects on the secretory function of adipocytes.

  17. Phenotypic analysis of mutant and overexpressing strains of lipid metabolism genes in Saccharomyces cerevisiae: implication in growth at low temperatures.

    PubMed

    López-Malo, María; Chiva, Rosana; Rozes, Nicolas; Guillamon, José Manuel

    2013-03-01

    The growing demand for wines with a more pronounced aromatic profile calls for low temperature alcoholic fermentations (10-15°C). However, there are certain drawbacks to low temperature fermentations such as reduced growth rate, long lag phase and sluggish or stuck fermentations. The lipid metabolism of Saccharomyces cerevisiae plays a central role in low temperature adaptation. The aim of this study was to detect lipid metabolism genes involved in cold adaptation. To do so, we analyzed the growth of knockouts in phospholipids, sterols and sphingolipids, from the EUROSCARF collection S. cerevisiae BY4742 strain at low and optimal temperatures. Growth rate of these knockouts, compared with the control, enabled us to identify the genes involved, which were also deleted or overexpressed in a derivative haploid of a commercial wine strain. We identified genes involved in the phospholipid (PSD1 and OPI3), sterol (ERG3 and IDI1) and sphingolipid (LCB3) pathways, whose deletion strongly impaired growth at low temperature and whose overexpression reduced generation or division time by almost half. Our study also reveals many phenotypic differences between the laboratory strain and the commercial wine yeast strain, showing the importance of constructing mutant and overexpressing strains in both genetic backgrounds. The phenotypic differences in the mutant and overexpressing strains were correlated with changes in their lipid composition. Copyright © 2013. Published by Elsevier B.V.

  18. Education for the Hearing Impaired (Auditorily Impaired).

    ERIC Educational Resources Information Center

    World Federation of the Deaf, Rome (Italy).

    Education for the hearing impaired is discussed in nine conference papers. J. N. Howarth describes "The Education of Deaf Children in Schools for Hearing Pupils in the United Kingdom" and A.I.Dyachkov of the U.S.S.R. outlines Didactical Principles of Educating the Deaf in the Light of their Rehabilitation Goal." Seven papers from…

  19. Development or Impairment?

    ERIC Educational Resources Information Center

    Hakansson, Gisela

    2010-01-01

    Joanne Paradis' Keynote Article on bilingualism and specific language impairment (SLI) is an impressive overview of research in language acquisition and language impairment. Studying different populations is crucial both for theorizing about language acquisition mechanisms, and for practical purposes of diagnosing and supporting children with…

  20. Development or Impairment?

    ERIC Educational Resources Information Center

    Hakansson, Gisela

    2010-01-01

    Joanne Paradis' Keynote Article on bilingualism and specific language impairment (SLI) is an impressive overview of research in language acquisition and language impairment. Studying different populations is crucial both for theorizing about language acquisition mechanisms, and for practical purposes of diagnosing and supporting children with…

  1. GFAP expression and social deficits in transgenic mice overexpressing human sAPPα

    PubMed Central

    Bailey, Antoinette R; Hou, Huayan; Song, Min; Obregon, Demian F; Portis, Samantha; Barger, Steven; Shytle, Doug; Stock, Saundra; Mori, Takashi; Sanberg, Paul G; Murphy, Tanya; Tan, Jun

    2013-01-01

    Autistic individuals display impaired social interactions and language, and restricted, stereotyped behaviors. Elevated levels of secreted amyloid precursor protein-alpha (sAPPα), the product of α-secretase cleavage of APP, are found in the plasma of some individuals with autism. The sAPPα protein is neurotrophic and neuroprotective and recently showed a correlation to glial differentiation in human neural stem cells (NSCs) via the IL-6 pathway. Considering evidence of gliosis in postmortem autistic brains, we hypothesized that subsets of patients with autism would exhibit elevations in CNS sAPPα and mice generated to mimic this observation would display markers suggestive of gliosis and autism-like behavior. Elevations in sAPPα levels were observed in brains of autistic patients compared to controls. Transgenic mice engineered to overexpress human sAPPα (TgsAPPα mice) displayed hypoactivity, impaired sociability, increased brain glial fibrillary acidic protein (GFAP) expression, and altered Notch1 and IL-6 levels. NSCs isolated from TgsAPPα mice, and those derived from wild-type mice treated with sAPPα, displayed suppressed β-tubulin III and elevated GFAP expression. These results suggest that elevations in brain sAPPα levels are observed in subsets of individuals with autism and TgsAPPα mice display signs suggestive of gliosis and behavioral impairment. PMID:23840007

  2. GFAP expression and social deficits in transgenic mice overexpressing human sAPPα.

    PubMed

    Bailey, Antoinette R; Hou, Huayan; Song, Min; Obregon, Demian F; Portis, Samantha; Barger, Steven; Shytle, Doug; Stock, Saundra; Mori, Takashi; Sanberg, Paul G; Murphy, Tanya; Tan, Jun

    2013-09-01

    Autistic individuals display impaired social interactions and language, and restricted, stereotyped behaviors. Elevated levels of secreted amyloid precursor protein-alpha (sAPPα), the product of α-secretase cleavage of APP, are found in the plasma of some individuals with autism. The sAPPα protein is neurotrophic and neuroprotective and recently showed a correlation to glial differentiation in human neural stem cells (NSCs) via the IL-6 pathway. Considering evidence of gliosis in postmortem autistic brains, we hypothesized that subsets of patients with autism would exhibit elevations in CNS sAPPα and mice generated to mimic this observation would display markers suggestive of gliosis and autism-like behavior. Elevations in sAPPα levels were observed in brains of autistic patients compared to controls. Transgenic mice engineered to overexpress human sAPPα (TgsAPPα mice) displayed hypoactivity, impaired sociability, increased brain glial fibrillary acidic protein (GFAP) expression, and altered Notch1 and IL-6 levels. NSCs isolated from TgsAPPα mice, and those derived from wild-type mice treated with sAPPα, displayed suppressed β-tubulin III and elevated GFAP expression. These results suggest that elevations in brain sAPPα levels are observed in subsets of individuals with autism and TgsAPPα mice display signs suggestive of gliosis and behavioral impairment. Copyright © 2013 Wiley Periodicals, Inc.

  3. Glial dysfunction causes age-related memory impairment in Drosophila.

    PubMed

    Yamazaki, Daisuke; Horiuchi, Junjiro; Ueno, Kohei; Ueno, Taro; Saeki, Shinjiro; Matsuno, Motomi; Naganos, Shintaro; Miyashita, Tomoyuki; Hirano, Yukinori; Nishikawa, Hiroyuki; Taoka, Masato; Yamauchi, Yoshio; Isobe, Toshiaki; Honda, Yoshiko; Kodama, Tohru; Masuda, Tomoko; Saitoe, Minoru

    2014-11-19

    Several aging phenotypes, including age-related memory impairment (AMI), are thought to be caused by cumulative oxidative damage. In Drosophila, age-related impairments in 1 hr memory can be suppressed by reducing activity of protein kinase A (PKA). However, the mechanism for this effect has been unclear. Here we show that decreasing PKA suppresses AMI by reducing activity of pyruvate carboxylase (PC), a glial metabolic enzyme whose amounts increase upon aging. Increased PC activity causes AMI through a mechanism independent of oxidative damage. Instead, increased PC activity is associated with decreases in D-serine, a glia-derived neuromodulator that regulates NMDA receptor activity. D-serine feeding suppresses both AMI and memory impairment caused by glial overexpression of dPC, indicating that an oxidative stress-independent dysregulation of glial modulation of neuronal activity contributes to AMI in Drosophila.

  4. Vascular Cognitive Impairment.

    PubMed

    Dichgans, Martin; Leys, Didier

    2017-02-03

    Cerebrovascular disease typically manifests with stroke, cognitive impairment, or both. Vascular cognitive impairment refers to all forms of cognitive disorder associated with cerebrovascular disease, regardless of the specific mechanisms involved. It encompasses the full range of cognitive deficits from mild cognitive impairment to dementia. In principle, any of the multiple causes of clinical stroke can cause vascular cognitive impairment. Recent work further highlights a role of microinfarcts, microhemorrhages, strategic white matter tracts, loss of microstructural tissue integrity, and secondary neurodegeneration. Vascular brain injury results in loss of structural and functional connectivity and, hence, compromise of functional networks within the brain. Vascular cognitive impairment is common both after stroke and in stroke-free individuals presenting to dementia clinics, and vascular pathology frequently coexists with neurodegenerative pathology, resulting in mixed forms of mild cognitive impairment or dementia. Vascular dementia is now recognized as the second most common form of dementia after Alzheimer's disease, and there is increasing awareness that targeting vascular risk may help to prevent dementia, even of the Alzheimer type. Recent advances in neuroimaging, neuropathology, epidemiology, and genetics have led to a deeper understanding of how vascular disease affects cognition. These new findings provide an opportunity for the present reappraisal of vascular cognitive impairment. We further briefly address current therapeutic concepts.

  5. Phenotypic effects of membrane protein overexpression in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Melén, Karin; Blomberg, Anders; von Heijne, Gunnar

    2006-07-01

    Large-scale protein overexpression phenotype screens provide an important complement to the more common gene knockout screens. Here, we have targeted the so far poorly understood Saccharomyces cerevisiae membrane proteome and report growth phenotypes for a strain collection overexpressing 600 C-terminally tagged integral membrane proteins grown both under normal and three different stress conditions. Although overexpression of most membrane proteins reduce the growth rate in synthetic defined medium, we identify a large number of proteins that, when overexpressed, confer specific resistance to various stress conditions. Our data suggest that regulation of glycosylphosphatidylinositol anchor biosynthesis and the Na+/K+ homeostasis system constitute major downstream targets of the yeast PKA/RAS pathway and point to a possible connection between the early secretory pathway and the cells' response to oxidative stress. We also have quantified the expression levels for >550 membrane proteins, facilitating the choice of well expressing proteins for future functional and structural studies. caffeine | paraquat | salt tolerance | yeast

  6. Overexpression of vsr in Escherichia coli is mutagenic.

    PubMed

    Doiron, K M; Viau, S; Koutroumanis, M; Cupples, C G

    1996-07-01

    Overexpression of vsr in Escherichia coli stimulates transition and frameshift mutations. The pattern of mutations suggests that mutagenesis is due to saturation or inactivation of dam-directed mismatch repair.

  7. [Overexpression of FKS1 to improve yeast autolysis-stress].

    PubMed

    Li, Jia; Wang, Jinjing; Li, Qi

    2015-09-01

    With the development of high gravity brewing, yeast cells are exposed to multiple brewing-associated stresses, such as increased osmotic pressure, enhanced alcohol concentration and nutritional imbalance. These will speed up yeast autolysis, which seriously influence beer flavor and quality. To increase yeast anti-autolytic ability, FKS1 overexpression strain was constructed by 18S rDNA. The concentration of β-1,3-glucan of overexpression strain was 62% higher than that of wild type strain. Meantime, FKS1 overexpression strain increased anti-stress ability at 8% ethanol, 0.4 mol/L NaCl and starvation stress. Under simulated autolysis, FKS1 showed good anti-autolytic ability by slower autolysis. These results confirms the potential of FKS1 overexpression to tackle yeast autolysis in high-gravity brewing.

  8. Impairment in Non-Word Repetition: A Marker for Language Impairment or Reading Impairment?

    ERIC Educational Resources Information Center

    Baird, Gillian; Slonims, Vicky; Simonoff, Emily; Dworzynski, Katharina

    2011-01-01

    Aim: A deficit in non-word repetition (NWR), a measure of short-term phonological memory proposed as a marker for language impairment, is found not only in language impairment but also in reading impairment. We evaluated the strength of association between language impairment and reading impairment in children with current, past, and no language…

  9. Impairment in Non-Word Repetition: A Marker for Language Impairment or Reading Impairment?

    ERIC Educational Resources Information Center

    Baird, Gillian; Slonims, Vicky; Simonoff, Emily; Dworzynski, Katharina

    2011-01-01

    Aim: A deficit in non-word repetition (NWR), a measure of short-term phonological memory proposed as a marker for language impairment, is found not only in language impairment but also in reading impairment. We evaluated the strength of association between language impairment and reading impairment in children with current, past, and no language…

  10. Hearing or speech impairment - resources

    MedlinePlus

    Resources - hearing or speech impairment ... The following organizations are good resources for information on hearing impairment or speech impairment: Alexander Graham Bell Association for the Deaf and Hard of Hearing -- www.agbell. ...

  11. Diaphragm assessment in mice overexpressing phospholamban in slow-twitch type I muscle fibers.

    PubMed

    Fajardo, Val Andrew; Smith, Ian Curtis; Bombardier, Eric; Chambers, Paige J; Quadrilatero, Joe; Tupling, Allan Russell

    2016-06-01

    Phospholamban (PLN) and sarcolipin (SLN) are small inhibitory proteins that regulate the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pump. Previous work from our laboratory revealed that in the soleus and gluteus minimus muscles of mice overexpressing PLN (Pln (OE)), SERCA function was impaired, dynamin 2 (3-5 fold) and SLN (7-9 fold) were upregulated, and features of human centronuclear myopathy (CNM) were observed. Here, we performed structural and functional experiments to evaluate whether the diaphragm muscles of the Pln (OE) mouse would exhibit CNM pathology and muscle weakness. Diaphragm muscles from Pln (OE) and WT mice were subjected to histological/histochemical/immunofluorescent staining, Ca(2+)-ATPase and Ca(2+) uptake assays, Western blotting, and in vitro electrical stimulation. Our results demonstrate that PLN overexpression reduced SERCA's apparent affinity for Ca(2+) but did not reduce maximal SERCA activity or rates of Ca(2+) uptake. SLN was upregulated 2.5-fold, whereas no changes in dynamin 2 expression were found. With respect to CNM, we did not observe type I fiber predominance, central nuclei, or central aggregation of oxidative activity in diaphragm, although type I fiber hypotrophy was present. Furthermore, in vitro contractility assessment of Pln (OE) diaphragm strips revealed no reductions in force-generating capacity, maximal rates of relaxation or force development, but did indicate that ½ relaxation time was prolonged. Therefore, the effects of PLN overexpression on skeletal muscle phenotype differ between diaphragm and the postural soleus and gluteus minimus muscles. Our findings here point to differences in SLN expression and type I fiber distribution as potential contributing factors.

  12. FHL1 reduces dystrophy in transgenic mice overexpressing FSHD muscular dystrophy region gene 1 (FRG1).

    PubMed

    Feeney, Sandra J; McGrath, Meagan J; Sriratana, Absorn; Gehrig, Stefan M; Lynch, Gordon S; D'Arcy, Colleen E; Price, John T; McLean, Catriona A; Tupler, Rossella; Mitchell, Christina A

    2015-01-01

    Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease with no effective treatment. The genetic cause of FSHD is complex and the primary pathogenic insult underlying the muscle disease is unknown. Several disease candidate genes have been proposed including DUX4 and FRG1. Expression analysis studies of FSHD report the deregulation of genes which mediate myoblast differentiation and fusion. Transgenic mice overexpressing FRG1 recapitulate the FSHD muscular dystrophy phenotype. Our current study selectively examines how increased expression of FRG1 may contribute to myoblast differentiation defects. We generated stable C2C12 cell lines overexpressing FRG1, which exhibited a myoblast fusion defect upon differentiation. To determine if myoblast fusion defects contribute to the FRG1 mouse dystrophic phenotype, this strain was crossed with skeletal muscle specific FHL1-transgenic mice. We previously reported that FHL1 promotes myoblast fusion in vitro and FHL1-transgenic mice develop skeletal muscle hypertrophy. In the current study, FRG1 mice overexpressing FHL1 showed an improvement in the dystrophic phenotype, including a reduced spinal kyphosis, increased muscle mass and myofiber size, and decreased muscle fibrosis. FHL1 expression in FRG1 mice, did not alter satellite cell number or activation, but enhanced myoblast fusion. Primary myoblasts isolated from FRG1 mice showed a myoblast fusion defect that was rescued by FHL1 expression. Therefore, increased FRG1 expression may contribute to a muscular dystrophy phenotype resembling FSHD by impairing myoblast fusion, a defect that can be rescued by enhanced myoblast fusion via expression of FHL1.

  13. FHL1 Reduces Dystrophy in Transgenic Mice Overexpressing FSHD Muscular Dystrophy Region Gene 1 (FRG1)

    PubMed Central

    Feeney, Sandra J.; McGrath, Meagan J.; Sriratana, Absorn; Gehrig, Stefan M.; Lynch, Gordon S.; D’Arcy, Colleen E.; Price, John T.; McLean, Catriona A.; Tupler, Rossella; Mitchell, Christina A.

    2015-01-01

    Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease with no effective treatment. The genetic cause of FSHD is complex and the primary pathogenic insult underlying the muscle disease is unknown. Several disease candidate genes have been proposed including DUX4 and FRG1. Expression analysis studies of FSHD report the deregulation of genes which mediate myoblast differentiation and fusion. Transgenic mice overexpressing FRG1 recapitulate the FSHD muscular dystrophy phenotype. Our current study selectively examines how increased expression of FRG1 may contribute to myoblast differentiation defects. We generated stable C2C12 cell lines overexpressing FRG1, which exhibited a myoblast fusion defect upon differentiation. To determine if myoblast fusion defects contribute to the FRG1 mouse dystrophic phenotype, this strain was crossed with skeletal muscle specific FHL1-transgenic mice. We previously reported that FHL1 promotes myoblast fusion in vitro and FHL1-transgenic mice develop skeletal muscle hypertrophy. In the current study, FRG1 mice overexpressing FHL1 showed an improvement in the dystrophic phenotype, including a reduced spinal kyphosis, increased muscle mass and myofiber size, and decreased muscle fibrosis. FHL1 expression in FRG1 mice, did not alter satellite cell number or activation, but enhanced myoblast fusion. Primary myoblasts isolated from FRG1 mice showed a myoblast fusion defect that was rescued by FHL1 expression. Therefore, increased FRG1 expression may contribute to a muscular dystrophy phenotype resembling FSHD by impairing myoblast fusion, a defect that can be rescued by enhanced myoblast fusion via expression of FHL1. PMID:25695429

  14. Neuroprotective effects of Bcl-2 overexpression on nerve cells of rats with acute cerebral infarction.

    PubMed

    Zhang, H R; Peng, J H; Zhu, G Y; Xu, R X

    2015-07-13

    We aimed to investigate the influence of lentiviral-mediated Bcl-2 overexpression in cerebral tissues of rats with acute cerebral infarction. Forty-five rats were randomly divided into sham, model, and treatment groups. The sham and model groups were administered a control lentiviral vector via the intracranial arteries 10 days before surgery, while the treatment group received lentivirus encoding a Bcl-2 overexpression vector. We induced cerebral artery infarction using a suture-occlusion method and analyzed the cerebral expression levels of apoptosis-related genes (caspase-3, Bax), total cerebral apoptosis, range of cerebral tissue infarction, and changes in nerve cell function after 72 h. The Bcl-2-encoding lentivirus was well expressed in rat cerebral tissues. The treatment group had significantly higher expression levels of Bcl-2 than the other two groups. After cerebral infarction, the model group had significantly increased expression levels of caspase-3 and Bax protein in cerebral tissues than the sham (P < 0.05). Expression of these apoptosis-related proteins in the treatment group was obviously lower than that in the model group (P < 0.05), but significantly higher than in the sham group (P < 0.05). Compared to sham, neuronal apoptosis levels and infarction range of cerebral tissues was increased in the model and treatment groups; however, these values in the treatment group were significantly lower than that in the model group (P < 0.05). Importantly, the treatment group had significantly decreased neurological impairment scores (P < 0.05). In conclusion, Bcl-2 over-expression can decrease neuronal apoptosis in rat cerebral tissue, and thus is neuroprotective after cerebral ischemia.

  15. A transgenic mouse model of neuroepithelial cell specific inducible overexpression of dopamine D1-receptor

    PubMed Central

    Fujimoto, Kumiko; Araki, Kiyomi; McCarthy, Deirdre M.; Sims, John R.; Ren, Jia-Qian; Zhang, Xuan; Bhide, Pradeep G.

    2010-01-01

    Dopamine and its receptors appear in the brain during early embryonic period suggesting a role for dopamine in brain development. In fact, dopamine receptor imbalance resulting from impaired physiological balance between D1- and D2-receptor activities can perturb brain development and lead to persisting changes in brain structure and function. Dopamine receptor imbalance can be produced experimentally using pharmacological or genetic methods. Pharmacological methods tend to activate or antagonize the receptors in all cell types. In the traditional gene knockout models the receptor imbalance occurs during development and also at maturity. Therefore, assaying the effects of dopamine imbalance on specific cell types (e.g. precursor versus postmitotic cells) or at specific periods of brain development (e.g. pre- or postnatal periods) is not feasible in these models. We describe a novel transgenic mouse model based on the tetracycline dependent inducible gene expression system in which dopamine D1-receptor transgene expression is induced selectively in neuroepithelial cells of the embryonic brain at experimenter-chosen intervals of brain development. In this model, doxycycline-induced expression of the transgene causes significant overexpression of the D1-receptor and significant reductions in the incorporation of the S-phase marker bromodeoxyuridine into neuroepithelial cells of the basal and dorsal telencephalon indicating marked effects on telencephalic neurogenesis. The D1-receptor overexpression occurs at higher levels in the medial ganglionic eminence than the lateral ganglionic eminence or cerebral wall. Moreover, although the transgene is induced selectively in the neuroepithelium, D1-receptor protein overexpression appears to persist in postmitotic cells. The mouse model can be modified for neuroepithelial cell-specific inducible expression of other transgenes or induction of the D1-receptor transgene in other cells in specific brain regions by crossbreeding

  16. Muscle-specific growth hormone receptor (GHR) overexpression induces hyperplasia but not hypertrophy in transgenic zebrafish.

    PubMed

    Figueiredo, Marcio Azevedo; Mareco, Edson A; Silva, Maeli Dal Pai; Marins, Luis Fernando

    2012-06-01

    Even though growth hormone (GH) transgenesis has demonstrated potential for improved growth of commercially important species, the hormone excess may result in undesired collateral effects. In this context, the aim of this work was to develop a new model of transgenic zebrafish (Danio rerio) characterized by a muscle-specific overexpression of the GH receptor (GHR) gene, evaluating the effect of transgenesis on growth, muscle structure and expression of growth-related genes. In on line of transgenic zebrafish overexpressing GHR in skeletal muscle, no significant difference in total weight in comparison to non-transgenics was observed. This can be explained by a significant reduction in expression of somatotrophic axis-related genes, in special insulin-like growth factor I (IGF-I). In the same sense, a significant increase in expression of the suppressors of cytokine signaling 1 and 3 (SOCS) was encountered in transgenics. Surprisingly, expression of genes coding for the main myogenic regulatory factors (MRFs) was higher in transgenic than non-transgenic zebrafish. Genes coding for muscle proteins did not follow the MRFs profile, showing a significant decrease in their expression. These results were corroborated by the histological analysis, where a hyperplasic muscle growth was observed in transgenics. In conclusion, our results demonstrated that GHR overexpression does not induce hypertrophic muscle growth in transgenic zebrafish probably because of SOCS impairment of the GHR/IGF-I pathway, culminating in IGF-I and muscle proteins decrease. Therefore, it seems that hypertrophy and hyperplasia follow two different routes for entire muscle growth, both of them triggered by GHR activation, but regulated by different mechanisms.

  17. Over-Expression of DSCR1 Protects against Post-Ischemic Neuronal Injury

    PubMed Central

    Corlett, Alicia; Broughton, Brad R. S.; Kim, Hyun Ah; Thundyil, John; Drummond, Grant R.; Arumugam, Thiruma V.; Pritchard, Melanie A.

    2012-01-01

    Background and Purpose The Down syndrome candidate region 1 (DSCR1) gene is located on human chromosome 21 and its protein is over-expressed in brains of Down syndrome individuals. DSCR1 can modulate the activity of calcineurin, a phosphatase abundant in the brain, but its influence on stroke outcome is not clear. We compared stroke outcome in wildtype (WT) and transgenic (DSCR1-TG) mice which over-express isoform 1 of human DSCR1. Methods Transient cerebral ischemia was produced by occlusion of the middle cerebral artery for 0.5 h. After 23.5 h reperfusion, we assessed neurological impairment, brain infarct and edema volume, leukocyte infiltration and markers of inflammation. Intrinsic resistance to apoptosis following glucose deprivation was also assessed in primary cultures of WT and DSCR1-TG neurons. Results In contrast to WT, DSCR1-TG mice had an improved neurological deficit score, greater grip strength, attenuated infarct volume and brain swelling, and lacked hippocampal lesions after stroke. Expression of mouse DSCR1-1, but not DSCR1-4, mRNA and protein was increased by ischemia in both WT and DSCR1-TG. Brain calcineurin activity was increased to a similar degree after ischemia in each genotype. DSCR1-TG mice had fewer infiltrating neutrophils and activated microglia compared with WT, in association with an attenuated upregulation of several pro-inflammatory genes. Neurons from DSCR1-TG mice were more resistant than WT neurons to apoptotic cell death following 24 h of glucose deprivation. Conclusions Over-expression of DSCR1 in mice improves outcome following stroke. Mechanisms underlying this protection may involve calcineurin-independent, anti-inflammatory and anti-apoptotic effects mediated by DSCR1 in neurons. PMID:23144708

  18. Overexpression of plastidial thioredoxins f and m differentially alters photosynthetic activity and response to oxidative stress in tobacco plants

    PubMed Central

    Rey, Pascal; Sanz-Barrio, Ruth; Innocenti, Gilles; Ksas, Brigitte; Courteille, Agathe; Rumeau, Dominique; Issakidis-Bourguet, Emmanuelle; Farran, Inmaculada

    2013-01-01

    Plants display a remarkable diversity of thioredoxins (Trxs), reductases controlling the thiol redox status of proteins. The physiological function of many of them remains elusive, particularly for plastidial Trxs f and m, which are presumed based on biochemical data to regulate photosynthetic reactions and carbon metabolism. Recent reports revealed that Trxs f and m participate in vivo in the control of starch metabolism and cyclic photosynthetic electron transfer around photosystem I, respectively. To further delineate their in planta function, we compared the photosynthetic characteristics, the level and/or activity of various Trx targets and the responses to oxidative stress in transplastomic tobacco plants overexpressing either Trx f or Trx m. We found that plants overexpressing Trx m specifically exhibit altered growth, reduced chlorophyll content, impaired photosynthetic linear electron transfer and decreased pools of glutathione and ascorbate. In both transplastomic lines, activities of two enzymes involved in carbon metabolism, NADP-malate dehydrogenase and NADP-glyceraldehyde-3-phosphate dehydrogenase are markedly and similarly altered. In contrast, plants overexpressing Trx m specifically display increased capacity for methionine sulfoxide reductases, enzymes repairing damaged proteins by regenerating methionine from oxidized methionine. Finally, we also observed that transplastomic plants exhibit distinct responses when exposed to oxidative stress conditions generated by methyl viologen or exposure to high light combined with low temperature, the plants overexpressing Trx m being notably more tolerant than Wt and those overexpressing Trx f. Altogether, these data indicate that Trxs f and m fulfill distinct physiological functions. They prompt us to propose that the m type is involved in key processes linking photosynthetic activity, redox homeostasis and antioxidant mechanisms in the chloroplast. PMID:24137166

  19. Overexpression of dystrophin in transgenic mdx mice eliminates dystrophic symptoms without toxicity.

    PubMed

    Cox, G A; Cole, N M; Matsumura, K; Phelps, S F; Hauschka, S D; Campbell, K P; Faulkner, J A; Chamberlain, J S

    1993-08-19

    Duchenne and Becker muscular dystrophy (DMD and BMD) are X-linked recessive diseases caused by defective expression of dystrophin. The mdx mouse, an animal model for DMD, has a mutation that eliminates expression of the 427K muscle and brain isoforms of dystrophin. Although these animals do not display overt muscle weakness or impaired movement, the diaphragm muscle of the mdx mouse is severely affected and shows progressive myofibre degeneration and fibrosis which closely resembles the human disease. Here we explore the feasibility of gene therapy for DMD by examining the potential of a full-length dystrophin transgene to correct dystrophic symptoms in mdx mice. We find that expression of dystrophin in muscles of transgenic mdx mice eliminates the morphological and immunohistological symptoms of muscular dystrophy. In addition, overexpression of dystrophin prevents the development of the abnormal mechanical properties associated with dystrophic muscle without causing deleterious side effects. Our results provide functional evidence for the feasibility of gene therapy for DMD.

  20. Overexpression of BMI-1 Promotes Cell Growth and Resistance to Cisplatin Treatment in Osteosarcoma

    PubMed Central

    Chen, Dafu; Hao, Dongsheng; Duan, Yuanhui; Qiu, Guixing; Wang, Yipeng

    2011-01-01

    Background BMI-1 is a member of the polycomb group of genes (PcGs), and it has been implicated in the development and progression of several malignancies, but its role in osteosarcoma remains to be elucidated. Methodology/Principal Findings In the present study, we found that BMI-1 was overexpressed in different types of osteosarcomas. Downregulation of BMI-1 by lentivirus mediated RNA interference (RNAi) significantly impaired cell viability and colony formation in vitro and tumorigenesis in vivo of osteosarcoma cells. BMI-1 knockdown sensitized cells to cisplatin-induced apoptosis through inhibition of PI3K/AKT pathway. Moreover, BMI-1-depletion-induced phenotype could be rescued by forced expression of BMI-1 wobble mutant which is resistant to inhibition by the small interfering RNA (siRNA). Conclusions/Significance These findings suggest a crucial role for BMI-1 in osteosarcoma pathogenesis. PMID:21311599

  1. Kids' Quest: Vision Impairment

    MedlinePlus

    ... most important job. Return to Steps World-Wide Web Search Kids Health: What is Vision Impairment What ... for the Blind (AFB) created the Braille Bug web site to teach sighted children about braille, and ...

  2. Speech impairment (adult)

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003204.htm Speech impairment (adult) To use the sharing features on ... 2017, A.D.A.M., Inc. Duplication for commercial use must be authorized in writing by ADAM ...

  3. Evaluation of hearing impairment.

    PubMed

    Zadeh, M H; Selesnick, S H

    2001-01-01

    Hearing impairment is among the most common medical condition presenting to health care professionals. Ear anatomy, physiology, and pathology resulting in hearing loss are discussed. A systematic approach to evaluation, diagnosis, and treatment is presented.

  4. DWARF overexpression induces alteration in phytohormone homeostasis, development, architecture and carotenoid accumulation in tomato.

    PubMed

    Li, Xiao-Jing; Chen, Xiao-Juan; Guo, Xie; Yin, Ling-Ling; Ahammed, Golam Jalal; Xu, Chang-Jie; Chen, Kun-Song; Liu, Chao-Chao; Xia, Xiao-Jian; Shi, Kai; Zhou, Jie; Zhou, Yan-Hong; Yu, Jing-Quan

    2016-03-01

    Brassinosteroids (BRs) play a critical role in plant growth, development and stress response; however, genetic evidence for the BR-mediated integrated regulation of plant growth still remains elusive in crop species. Here, we clarified the function of DWARF (DWF), the key BR biosynthetic gene in tomato, in the regulation of plant growth and architecture, phytohormone homeostasis and fruit development by comparing wild type, d^(im), a weak allele mutant impaired in DWF, and DWF-overexpressing plants in tomato. Results showed that increases in DWF transcripts and endogenous BR level resulted in improved germination, lateral root development, CO2 assimilation and eventually plant growth as characterized by slender and compact plant architecture. However, an increase in DWF transcript down-regulated the accumulation of gibberellin, which was associated with decreases in leaf size and thickness. BRs positively regulated lateral bud outgrowth, which was associated with decreased transcript of Aux/IAA3, and the ethylene-dependent petiole bending and fruit ripening. Notably, overexpression of DWF did not significantly alter fruit yield per plant; however, increases by 57.4% and 95.3% might be estimated in fruit yield per square metre in two transgenic lines due to their compact architecture. Significantly, BR level was positively related with the carotenoid accumulation in the fruits. Taken together, our results demonstrate that BRs are actively involved in the regulation of multiple developmental processes relating to agronomical important traits.

  5. Viral-mediated Ntf3 overexpression disrupts innervation and hearing in nondeafened guinea pig cochleae.

    PubMed

    Lee, Min Young; Kurioka, Takaomi; Nelson, Megan M; Prieskorn, Diane M; Swiderski, Donald L; Takada, Yohei; Beyer, Lisa A; Raphael, Yehoash

    2016-01-01

    Synaptopathy in the cochlea occurs when the connection between inner hair cells and the auditory nerve is disrupted, leading to impaired hearing and nerve degeneration. Experiments using transgenic mice have shown that overexpression of NT3 by supporting cells repairs synaptopathy caused by overstimulation. To accomplish such therapy in the clinical setting, it would be necessary to activate the neurotrophin receptor on auditory neurons by other means. Here we test the outcome of NT3 overexpression using viral-mediated gene transfer into the perilymph versus the endolymph of the normal guinea pig cochlea. We inoculated two different Ntf3 viral vectors, adenovirus (Adv) or adeno-associated virus (AAV) into the perilymph, to facilitate transgene expression in the mesothelial cells and cochlear duct epithelium, respectively. We assessed outcomes by comparing Auditory brainstem response (ABR) thresholds prior to that at baseline to thresholds at 1 and 3 weeks after inoculation, and then performed histologic evaluation of hair cells, nerve endings, and synaptic ribbons. We observed hearing threshold shifts as well as disorganization of peripheral nerve endings and disruption of synaptic connections between inner hair cells and peripheral nerve endings with both vectors. The data suggest that elevation of NT3 levels in the cochlear fluids can disrupt innervation and degrade hearing.

  6. Overexpression of Notch1 ectodomain in myeloid cells induces vascular malformations through a paracrine pathway.

    PubMed

    Li, Xiujie; Calvo, Ezequiel; Cool, Marc; Chrobak, Pavel; Kay, Denis G; Jolicoeur, Paul

    2007-01-01

    We previously reported that truncation of Notch1 (N1) by provirus insertion leads to overexpression of both the intracellular (N1(IC)) and the extracellular (N1(EC)) domains. We produced transgenic (Tg) mice expressing N1(EC) in T cells and in cells of the myeloid lineage under the regulation of the CD4 gene. These CD4C/N1(EC) Tg mice developed vascular disease, predominantly in the liver: superficial distorted vessels, cavernae, lower branching of parenchymal vessels, capillarized sinusoids, and aberrant smooth muscle/endothelial cell topography. The disease developed in lethally irradiated normal mice transplanted with Tg bone marrow or fetal liver cells as well as in Rag-/- Tg mice. In nude mice transplanted with fetal liver cells from (ROSA26 x CD4C/N1(EC)) F1 Tg mice, abnormal vessels were of recipient origin. Transplantation of Tg peritoneal macrophages into normal recipients also induced abnormal vessels. These Tg macrophages showed impaired functions, and their conditioned medium inhibited the proliferation of liver sinusoid endothelial cells in vitro. The Egr-1 gene and some of its targets (Jag1, FIII, FXIII-A, MCP-1, and MCP-5), previously implicated in hemangioma or vascular malformations, were overexpressed in Tg macrophages. These results show that myeloid cells can be reprogrammed by N1(EC) to induce vascular malformations through a paracrine pathway.

  7. Overexpressed of RAD51 suppresses recombination defects: a possible mechanism to reverse genomic instability

    SciTech Connect

    Schild, David; Wiese, Claudia

    2009-10-15

    RAD51, a key protein in the homologous recombinational DNA repair (HRR) pathway, is the major strand-transferase required for mitotic recombination. An important early step in HRR is the formation of single-stranded DNA (ss-DNA) coated by RPA (a ss-DNA binding protein). Displacement of RPA by RAD51 is highly regulated and facilitated by a number of different proteins known as the 'recombination mediators'. To assist these recombination mediators, a second group of proteins also is required and we are defining these proteins here as 'recombination co-mediators'. Defects in either recombination mediators or comediators, including BRCA1 and BRCA2, lead to impaired HRR that can genetically be complemented for (i.e. suppressed) by overexpression of RAD51. Defects in HRR have long been known to contribute to genomic instability leading to tumor development. Since genomic instability also slows cell growth, precancerous cells presumably require genomic restabilization to gain a growth advantage. RAD51 is overexpressed in many tumors, and therefore, we hypothesize that the complementing ability of elevated levels of RAD51 in tumors with initial HRR defects limits genomic instability during carcinogenic progression. Of particular interest, this model may also help explain the high frequency of TP53 mutations in human cancers, since wild-type p53 represses RAD51.

  8. Viral-mediated Ntf3 overexpression disrupts innervation and hearing in nondeafened guinea pig cochleae

    PubMed Central

    Lee, Min Young; Kurioka, Takaomi; Nelson, Megan M; Prieskorn, Diane M; Swiderski, Donald L; Takada, Yohei; Beyer, Lisa A; Raphael, Yehoash

    2016-01-01

    Synaptopathy in the cochlea occurs when the connection between inner hair cells and the auditory nerve is disrupted, leading to impaired hearing and nerve degeneration. Experiments using transgenic mice have shown that overexpression of NT3 by supporting cells repairs synaptopathy caused by overstimulation. To accomplish such therapy in the clinical setting, it would be necessary to activate the neurotrophin receptor on auditory neurons by other means. Here we test the outcome of NT3 overexpression using viral-mediated gene transfer into the perilymph versus the endolymph of the normal guinea pig cochlea. We inoculated two different Ntf3 viral vectors, adenovirus (Adv) or adeno-associated virus (AAV) into the perilymph, to facilitate transgene expression in the mesothelial cells and cochlear duct epithelium, respectively. We assessed outcomes by comparing Auditory brainstem response (ABR) thresholds prior to that at baseline to thresholds at 1 and 3 weeks after inoculation, and then performed histologic evaluation of hair cells, nerve endings, and synaptic ribbons. We observed hearing threshold shifts as well as disorganization of peripheral nerve endings and disruption of synaptic connections between inner hair cells and peripheral nerve endings with both vectors. The data suggest that elevation of NT3 levels in the cochlear fluids can disrupt innervation and degrade hearing. PMID:27525291

  9. Overexpression of RAD51 suppresses recombination defects: a possible mechanism to reverse genomic instability

    PubMed Central

    Schild, David; Wiese, Claudia

    2010-01-01

    RAD51, a key protein in the homologous recombinational DNA repair (HRR) pathway, is the major strand-transferase required for mitotic recombination. An important early step in HRR is the formation of single-stranded DNA (ss-DNA) coated by RPA (a ss-DNA-binding protein). Displacement of RPA by RAD51 is highly regulated and facilitated by a number of different proteins known as the ‘recombination mediators’. To assist these recombination mediators, a second group of proteins also is required and we are defining these proteins here as ‘recombination co-mediators’. Defects in either recombination mediators or co-mediators, including BRCA1 and BRCA2, lead to impaired HRR that can genetically be complemented for (i.e. suppressed) by overexpression of RAD51. Defects in HRR have long been known to contribute to genomic instability leading to tumor development. Since genomic instability also slows cell growth, precancerous cells presumably require genomic re-stabilization to gain a growth advantage. RAD51 is overexpressed in many tumors, and therefore, we hypothesize that the complementing ability of elevated levels of RAD51 in tumors with initial HRR defects limits genomic instability during carcinogenic progression. Of particular interest, this model may also help explain the high frequency of TP53 mutations in human cancers, since wild-type p53 represses RAD51 expression. PMID:19942681

  10. Adipose-derived stromal cells overexpressing vascular endothelial growth factor accelerate mouse excisional wound healing.

    PubMed

    Nauta, Allison; Seidel, Catharina; Deveza, Lorenzo; Montoro, Daniel; Grova, Monica; Ko, Sae Hee; Hyun, Jeong; Gurtner, Geoffrey C; Longaker, Michael T; Yang, Fan

    2013-02-01

    Angiogenesis is essential to wound repair, and vascular endothelial growth factor (VEGF) is a potent factor to stimulate angiogenesis. Here, we examine the potential of VEGF-overexpressing adipose-derived stromal cells (ASCs) for accelerating wound healing using nonviral, biodegradable polymeric vectors. Mouse ASCs were transfected with DNA plasmid encoding VEGF or green fluorescent protein (GFP) using biodegradable poly (β-amino) esters (PBAE). Cells transfected using Lipofectamine 2000, a commercially available transfection reagent, were included as controls. ASCs transfected using PBAEs showed enhanced transfection efficiency and 12-15-fold higher VEGF production compared with cells transfected using Lipofectamine 2000 (*P < 0.05). When transplanted into a mouse wild-type excisional wound model, VEGF-overexpressing ASCs led to significantly accelerated wound healing, with full wound closure observed at 8 days compared to 10-12 days in groups treated with ASCs alone or saline control (*P < 0.05). Histology and polarized microscopy showed increased collagen deposition and more mature collagen fibers in the dermis of wound beds treated using PBAE/VEGF-modified ASCs than ASCs alone. Our results demonstrate the efficacy of using nonviral-engineered ASCs to accelerate wound healing, which may provide an alternative therapy for treating many diseases in which wound healing is impaired.

  11. Mannose 6-Phosphate Receptor Is Reduced in -Synuclein Overexpressing Models of Parkinsons Disease

    PubMed Central

    Matrone, Carmela; Dzamko, Nicolas; Madsen, Peder; Nyegaard, Mette; Pohlmann, Regina; Søndergaard, Rikke V.; Lassen, Louise B.; Andresen, Thomas L.; Halliday, Glenda M.; Jensen, Poul Henning

    2016-01-01

    Increasing evidence points to defects in autophagy as a common denominator in most neurodegenerative conditions. Progressive functional decline in the autophagy-lysosomal pathway (ALP) occurs with age, and the consequent impairment in protein processing capacity has been associated with a higher risk of neurodegeneration. Defects in cathepsin D (CD) processing and α-synuclein degradation causing its accumulation in lysosomes are particularly relevant for the development of Parkinson's disease (PD). However, the mechanism by which alterations in CD maturation and α-synuclein degradation leads to autophagy defects in PD neurons is still uncertain. Here we demonstrate that MPR300 shuttling between endosomes and the trans Golgi network is altered in α-synuclein overexpressing neurons. Consequently, CD is not correctly trafficked to lysosomes and cannot be processed to generate its mature active form, leading to a reduced CD-mediated α-synuclein degradation and α-synuclein accumulation in neurons. MPR300 is downregulated in brain from α-synuclein overexpressing animal models and in PD patients with early diagnosis. These data indicate MPR300 as crucial player in the autophagy-lysosomal dysfunctions reported in PD and pinpoint MRP300 as a potential biomarker for PD. PMID:27509067

  12. Smad6/Smurf1 overexpression in cartilage delays chondrocyte hypertrophy and causes dwarfism with osteopenia

    PubMed Central

    Horiki, Mitsuru; Imamura, Takeshi; Okamoto, Mina; Hayashi, Makoto; Murai, Junko; Myoui, Akira; Ochi, Takahiro; Miyazono, Kohei; Yoshikawa, Hideki; Tsumaki, Noriyuki

    2004-01-01

    Biochemical experiments have shown that Smad6 and Smad ubiquitin regulatory factor 1 (Smurf1) block the signal transduction of bone morphogenetic proteins (BMPs). However, their in vivo functions are largely unknown. Here, we generated transgenic mice overexpressing Smad6 in chondrocytes. Smad6 transgenic mice showed postnatal dwarfism with osteopenia and inhibition of Smad1/5/8 phosphorylation in chondrocytes. Endochondral ossification during development in these mice was associated with almost normal chondrocyte proliferation, significantly delayed chondrocyte hypertrophy, and thin trabecular bone. The reduced population of hypertrophic chondrocytes after birth seemed to be related to impaired bone growth and formation. Organ culture of cartilage rudiments showed that chondrocyte hypertrophy induced by BMP2 was inhibited in cartilage prepared from Smad6 transgenic mice. We then generated transgenic mice overexpressing Smurf1 in chondrocytes. Abnormalities were undetectable in Smurf1 transgenic mice. Mating Smad6 and Smurf1 transgenic mice produced double-transgenic pups with more delayed endochondral ossification than Smad6 transgenic mice. These results provided evidence that Smurf1 supports Smad6 function in vivo. PMID:15123739

  13. Amniotic-fluid-derived mesenchymal stem cells overexpressing interleukin-1 receptor antagonist improve fulminant hepatic failure.

    PubMed

    Zheng, Yu-Bao; Zhang, Xiao-Hong; Huang, Zhan-Lian; Lin, Chao-Shuang; Lai, Jing; Gu, Yu-Rong; Lin, Bin-Liang; Xie, Dong-Ying; Xie, Shi-Bin; Peng, Liang; Gao, Zhi-Liang

    2012-01-01

    Uncontrolled hepatic immunoactivation is regarded as the primary pathological mechanism of fulminant hepatic failure (FHF). The major acute-phase mediators associated with FHF, including IL-1β, IL-6, and TNF-α, impair the regeneration of liver cells and stem cell grafts. Amniotic-fluid-derived mesenchymal stem cells (AF-MSCs) have the capacity, under specific conditions, to differentiate into hepatocytes. Interleukin-1-receptor antagonist (IL-1Ra) plays an anti-inflammatory and anti-apoptotic role in acute and chronic inflammation, and has been used in many experimental and clinical applications. In the present study, we implanted IL-1Ra-expressing AF-MSCs into injured liver via the portal vein, using D-galactosamine-induced FHF in a rat model. IL-1Ra expression, hepatic injury, liver regeneration, cytokines (IL-1β, IL-6), and animal survival were assessed after cell transplantation. Our results showed that AF-MSCs over-expressing IL-1Ra prevented liver failure and reduced mortality in rats with FHF. These animals also exhibited improved liver function and increased survival rates after injection with these cells. Using green fluorescent protein as a marker, we demonstrated that the engrafted cells and their progeny were incorporated into injured livers and produced albumin. This study suggests that AF-MSCs genetically modified to over-express IL-1Ra can be implanted into the injured liver to provide a novel therapeutic approach to the treatment of FHF.

  14. Chronic overexpression of PNPLA3I148M in mouse liver causes hepatic steatosis

    PubMed Central

    Li, John Zhong; Huang, Yongcheng; Karaman, Ruchan; Ivanova, Pavlina T.; Brown, H. Alex; Roddy, Thomas; Castro-Perez, Jose; Cohen, Jonathan C.; Hobbs, Helen H.

    2012-01-01

    A genetic variant in PNPLA3 (PNPLA3I148M), a triacylglycerol (TAG) hydrolase, is a major risk factor for nonalcoholic fatty liver disease (NAFLD); however, the mechanism underlying this association is not known. To develop an animal model of PNPLA3-induced fatty liver disease, we generated transgenic mice that overexpress similar amounts of wild-type PNPLA3 (PNPLA3WT) or mutant PNPLA3 (PNPLA3I148M) either in liver or adipose tissue. Overexpression of the transgenes in adipose tissue did not affect liver fat content. Expression of PNPLA3I148M, but not PNPLA3WT, in liver recapitulated the fatty liver phenotype as well as other metabolic features associated with this allele in humans. Metabolic studies provided evidence for 3 distinct alterations in hepatic TAG metabolism in PNPLA3I148M transgenic mice: increased formation of fatty acids and TAG, impaired hydrolysis of TAG, and relative depletion of TAG long-chain polyunsaturated fatty acids. These findings suggest that PNPLA3 plays a role in remodeling TAG in lipid droplets, as they accumulate in response to food intake, and that the increase in hepatic TAG levels associated with the I148M substitution results from multiple changes in hepatic TAG metabolism. The development of an animal model that recapitulates the metabolic phenotype of the allele in humans provides a new platform in which to elucidate the role of PNLPA3I148M in NAFLD. PMID:23023705

  15. Seeding and transgenic overexpression of alpha-synuclein triggers dendritic spine pathology in the neocortex.

    PubMed

    Blumenstock, Sonja; Rodrigues, Eva F; Peters, Finn; Blazquez-Llorca, Lidia; Schmidt, Felix; Giese, Armin; Herms, Jochen

    2017-05-01

    Although misfolded and aggregated α-synuclein (α-syn) is recognized in the disease progression of synucleinopathies, its role in the impairment of cortical circuitries and synaptic plasticity remains incompletely understood. We investigated how α-synuclein accumulation affects synaptic plasticity in the mouse somatosensory cortex using two distinct approaches. Long-term in vivo imaging of apical dendrites was performed in mice overexpressing wild-type human α-synuclein. Additionally, intracranial injection of preformed α-synuclein fibrils was performed to induce cortical α-syn pathology. We find that α-synuclein overexpressing mice show decreased spine density and abnormalities in spine dynamics in an age-dependent manner. We also provide evidence for the detrimental effects of seeded α-synuclein aggregates on dendritic architecture. We observed spine loss as well as dystrophic deformation of dendritic shafts in layer V pyramidal neurons. Our results provide a link to the pathophysiology underlying dementia associated with synucleinopathies and may enable the evaluation of potential drug candidates on dendritic spine pathology in vivo. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  16. Adipose-derived Stromal Cells Overexpressing Vascular Endothelial Growth Factor Accelerate Mouse Excisional Wound Healing

    PubMed Central

    Nauta, Allison; Seidel, Catharina; Deveza, Lorenzo; Montoro, Daniel; Grova, Monica; Ko, Sae Hee; Hyun, Jeong; Gurtner, Geoffrey C; Longaker, Michael T; Yang, Fan

    2013-01-01

    Angiogenesis is essential to wound repair, and vascular endothelial growth factor (VEGF) is a potent factor to stimulate angiogenesis. Here, we examine the potential of VEGF-overexpressing adipose-derived stromal cells (ASCs) for accelerating wound healing using nonviral, biodegradable polymeric vectors. Mouse ASCs were transfected with DNA plasmid encoding VEGF or green fluorescent protein (GFP) using biodegradable poly (β-amino) esters (PBAE). Cells transfected using Lipofectamine 2000, a commercially available transfection reagent, were included as controls. ASCs transfected using PBAEs showed enhanced transfection efficiency and 12–15-fold higher VEGF production compared with cells transfected using Lipofectamine 2000 (*P < 0.05). When transplanted into a mouse wild-type excisional wound model, VEGF-overexpressing ASCs led to significantly accelerated wound healing, with full wound closure observed at 8 days compared to 10–12 days in groups treated with ASCs alone or saline control (*P < 0.05). Histology and polarized microscopy showed increased collagen deposition and more mature collagen fibers in the dermis of wound beds treated using PBAE/VEGF-modified ASCs than ASCs alone. Our results demonstrate the efficacy of using nonviral-engineered ASCs to accelerate wound healing, which may provide an alternative therapy for treating many diseases in which wound healing is impaired. PMID:23164936

  17. Glutaminase C overexpression in the brain induces learning deficits, synaptic dysfunctions, and neuroinflammation in mice.

    PubMed

    Wang, Yi; Li, Yuju; Zhao, Runze; Wu, Beiqing; Lanoha, Blaise; Tong, Zenghan; Peer, Justin; Liu, Jianhui; Xiong, Huangui; Huang, Yunlong; Zheng, Jialin

    2017-06-15

    Glutaminolysis, a metabolic process that converts glutamine to glutamate, is particularly important for the central nervous system since glutamate is the major transmitter of excitatory synapses. Glutaminase is the mitochondrial enzyme that catalyzes the first step of glutaminolysis. Two genes encode at least four isoforms of glutaminase in humans. Gls1 gene encodes isoforms kidney-type glutaminase (KGA) and glutaminase C (GAC) through alternative splicing, whereas Gls2 gene encodes liver-type glutaminase isoforms. KGA and GAC have been associated with several neurological diseases. However, it remains unclear whether changes in their expressions can directly cause brain abnormalities. Using a transgenic approach, we generated mice that overexpressed GAC in the brain. The resulting transgenic mice had severe impairments in spatial and fear learning compared with littermate controls. The learning deficits were consistent with diminished hippocampal long-term potentiation in the hippocampal slices of the GAC transgenic mice. Furthermore, we found increases in astrocyte and microglia markers, inflammatory factors, and a decrease in synapse marker synaptophysin, suggesting neuroinflammation and synaptic changes in the GAC transgenic mouse brains. In conclusion, these findings provide the first evidence that GAC overexpression in the brain has deleterious effects on learning and synaptic integrity in vivo. Copyright © 2017. Published by Elsevier Inc.

  18. TROP2 overexpression promotes proliferation and invasion of lung adenocarcinoma cells

    SciTech Connect

    Li, Zanhua; Jiang, Xunsheng; Zhang, Wei

    2016-01-29

    Recent studies suggest that the human trophoblast cell-surface antigen TROP2 is highly expressed in a number of tumours and is correlated with poor prognosis. However, its role in non-small cell lung carcinoma (NSCLC) remains largely unknown. Here we examined TROP2 expression by immunohistochemistry in a series of 68 patients with adenocarcinoma (ADC). We found significantly elevated TROP2 expression in ADC tissues compared with normal lung tissues (P < 0.05), and TROP2 overexpression was significantly associated with TNM (tumour, node, metastasis) stage (P = 0.012), lymph node metastasis (P = 0.038), and histologic grade (P = 0.013). Kaplan–Meier survival analysis revealed that high TROP2 expression correlated with poor prognosis (P = 0.046). Multivariate analysis revealed that TROP2 expression was an independent prognostic marker for overall survival of ADC patients. Moreover, TROP2 overexpression enhanced cell proliferation, migration, and invasion in the NSCLC cell line A549, whereas knockdown of TROP2 induced apoptosis and impaired proliferation, migration, and invasion in the PC-9 cells. Altogether, our data suggest that TROP2 plays an important role in promoting ADC and may represent a novel prognostic biomarker and therapeutic target for the disease.

  19. Immunohistochemical COX-2 overexpression correlates with HER-2/neu overexpression in invasive breast carcinomas: a pilot study.

    PubMed

    Çiriş, Ibrahim Metin; Bozkurt, Kemal Kürşat; Başpinar, Sirin; Kapucuoğlu, Fatma Nilgün

    2011-03-15

    Cyclooxygenase-2 (COX-2) is a prostaglandin synthase that catalyzes the synthesis of prostaglandin G2 and H2. It has been shown that COX-2 plays an important role in tumorigenesis of different tumor types and it is thought to take part in breast carcinogenesis. In the present study, we aimed to investigate the relationship of immunohistochemical COX-2 expression with clinicopathological parameters, including HER-2/neu overexpression in invasive breast carcinoma (IBC). Our study population comprised 10 normal breasts, 25 ductal carcinomas in situ (DCIS), and 51 invasive breast carcinomas. Immunohistochemical overexpressions of COX-2 and HER-2/neu were investigated in sections of formalin-fixed, paraffin-embedded blocks by 3 observers. In normal breast, DCIS and IBC, the COX-2 overexpression rate was 0%, 84%, and 58.8%, respectively. In IBC, COX-2 overexpression had a significant relationship with HER-2/neu overexpression (p=0.026) and a high histological grade (p=0.026). COX-2 expression in both DCIS (n=25) and IBC (n=51) was significantly higher than in normal breast tissue (p<0.0001). In addition, the COX-2 expression rate was significantly higher in DCIS than in IBC (p=0.042). Our results indicated that COX-2 overexpression correlates with aggressive phenotypic features, such as HER-2/neu overexpression and high histological grade in IBC. Increased expression of COX-2 in both DCIS and IBC in comparison to normal breast could indicate a role in breast carcinogenesis. COX-2 overexpression may provide a clinically useful biomarker for estimating tumor aggressiveness.

  20. Over-expression of microRNA-1 causes arrhythmia by disturbing intracellular trafficking system

    PubMed Central

    Su, Xiaomin; Liang, Haihai; Wang, He; Chen, Guizhi; Jiang, Hua; Wu, Qiuxia; Liu, Tianyi; Liu, Qiushuang; Yu, Tong; Gu, Yunyan; Yang, Baofeng; Shan, Hongli

    2017-01-01

    Dysregulation of intracellular trafficking system plays a fundamental role in the progression of cardiovascular disease. Up-regulation of miR-1 contributes to arrhythmia, we sought to elucidate whether intracellular trafficking contributes to miR-1-driven arrhythmia. By performing microarray analyses of the transcriptome in the cardiomyocytes-specific over-expression of microRNA-1 (miR-1 Tg) mice and the WT mice, we found that these differentially expressed genes in miR-1 Tg mice were significantly enrichment with the trafficking-related biological processes, such as regulation of calcium ion transport. Also, the qRT-PCR and western blot results validated that Stx6, Braf, Ube3a, Mapk8ip3, Ap1s1, Ccz1 and Gja1, which are the trafficking-related genes, were significantly down-regulated in the miR-1 Tg mice. Moreover, we found that Stx6 was decreased in the heart of mice after myocardial infarction and in the hypoxic cardiomyocytes, and further confirmed that Stx6 is a target of miR-1. Meanwhile, knockdown of Stx6 in cardiomyocytes resulted in the impairments of PLM and L-type calcium channel, which leads to the increased resting ([Ca2+]i). On the contrary, overexpression of Stx6 attenuated the impairments of miR-1 or hypoxia on PLM and L-type calcium channel. Thus, our studies reveals that trafficking-related gene Stx6 may regulate intracellular calcium and is involved in the occurrence of cardiac arrhythmia, which provides new insights in that miR-1 participates in arrhythmia by regulating the trafficking-related genes and pathway. PMID:28397788

  1. Calpastatin Overexpression Preserves Cognitive Function Following Seizures, While Maintaining Post-Injury Neurogenesis

    PubMed Central

    Machado, Vanessa M.; Lourenço, Ana Sofia; Florindo, Cláudia; Fernandes, Raquel; Carvalho, Caetana M.; Araújo, Inês M.

    2017-01-01

    In the adult mammalian brain, new neurons continue to be produced throughout life in two main regions in the brain, the subgranular zone (SGZ) in the hippocampus and the subventricular zone in the walls of the lateral ventricles. Neural stem cells (NSCs) proliferate in these niches, and migrate as neuroblasts, to further differentiate in locations where new neurons are needed, either in normal or pathological conditions. However, the endogenous attempt of brain repair is not very efficient. Calpains are proteases known to be involved in neuronal damage and in cell proliferation, migration and differentiation of several cell types, though their effects on neurogenesis are not well known. Previous work by our group has shown that the absence of calpastatin (CAST), the endogenous inhibitor of calpains, impairs early stages of neurogenesis. Since the hippocampus is highly associated with learning and memory, we aimed to evaluate whether calpain inhibition would help improve cognitive recovery after lesion and efficiency of post-injury neurogenesis in this region. For that purpose, we used the kainic acid (KA) model of seizure-induced hippocampal lesion and mice overexpressing CAST. Selected cognitive tests were performed on the 3rd and 8th week after KA-induced lesion, and cell proliferation, migration and differentiation in the dentate gyrus (DG) of the hippocampus of adult mice were analyzed using specific markers. Cognitive recovery was evaluated by testing the animals for recognition, spatial and associative learning and memory. Cognitive function was preserved by CAST overexpression following seizures, while modulation of post-injury neurogenesis was similar to wild type (WT) mice. Calpain inhibition could still be potentially able to prevent the impairment in the formation of new neurons, given that the levels of calpain activity could be reduced under a certain threshold and other harmful effects from the pathological environment could also be controlled. PMID

  2. Conditional corticotropin-releasing hormone overexpression in the mouse forebrain enhances rapid eye movement sleep

    PubMed Central

    Kimura, M; Müller-Preuss, P; Lu, A; Wiesner, E; Flachskamm, C; Wurst, W; Holsboer, F; Deussing, J M

    2009-01-01

    Impaired sleep and enhanced stress hormone secretion are the hallmarks of stress-related disorders, including major depression. The central neuropeptide, corticotropin-releasing hormone (CRH), is a key hormone that regulates humoral and behavioral adaptation to stress. Its prolonged hypersecretion is believed to play a key role in the development and course of depressive symptoms, and is associated with sleep impairment. To investigate the specific effects of central CRH overexpression on sleep, we used conditional mouse mutants that overexpress CRH in the entire central nervous system (CRH-COE-Nes) or only in the forebrain, including limbic structures (CRH-COE-Cam). Compared with wild-type or control mice during baseline, both homozygous CRH-COE-Nes and -Cam mice showed constantly increased rapid eye movement (REM) sleep, whereas slightly suppressed non-REM sleep was detected only in CRH-COE-Nes mice during the light period. In response to 6-h sleep deprivation, elevated levels of REM sleep also became evident in heterozygous CRH-COE-Nes and -Cam mice during recovery, which was reversed by treatment with a CRH receptor type 1 (CRHR1) antagonist in heterozygous and homozygous CRH-COE-Nes mice. The peripheral stress hormone levels were not elevated at baseline, and even after sleep deprivation they were indistinguishable across genotypes. As the stress axis was not altered, sleep changes, in particular enhanced REM sleep, occurring in these models are most likely induced by the forebrain CRH through the activation of CRHR1. CRH hypersecretion in the forebrain seems to drive REM sleep, supporting the notion that enhanced REM sleep may serve as biomarker for clinical conditions associated with enhanced CRH secretion. PMID:19455148

  3. Conditional corticotropin-releasing hormone overexpression in the mouse forebrain enhances rapid eye movement sleep.

    PubMed

    Kimura, M; Müller-Preuss, P; Lu, A; Wiesner, E; Flachskamm, C; Wurst, W; Holsboer, F; Deussing, J M

    2010-02-01

    Impaired sleep and enhanced stress hormone secretion are the hallmarks of stress-related disorders, including major depression. The central neuropeptide, corticotropin-releasing hormone (CRH), is a key hormone that regulates humoral and behavioral adaptation to stress. Its prolonged hypersecretion is believed to play a key role in the development and course of depressive symptoms, and is associated with sleep impairment. To investigate the specific effects of central CRH overexpression on sleep, we used conditional mouse mutants that overexpress CRH in the entire central nervous system (CRH-COE-Nes) or only in the forebrain, including limbic structures (CRH-COE-Cam). Compared with wild-type or control mice during baseline, both homozygous CRH-COE-Nes and -Cam mice showed constantly increased rapid eye movement (REM) sleep, whereas slightly suppressed non-REM sleep was detected only in CRH-COE-Nes mice during the light period. In response to 6-h sleep deprivation, elevated levels of REM sleep also became evident in heterozygous CRH-COE-Nes and -Cam mice during recovery, which was reversed by treatment with a CRH receptor type 1 (CRHR1) antagonist in heterozygous and homozygous CRH-COE-Nes mice. The peripheral stress hormone levels were not elevated at baseline, and even after sleep deprivation they were indistinguishable across genotypes. As the stress axis was not altered, sleep changes, in particular enhanced REM sleep, occurring in these models are most likely induced by the forebrain CRH through the activation of CRHR1. CRH hypersecretion in the forebrain seems to drive REM sleep, supporting the notion that enhanced REM sleep may serve as biomarker for clinical conditions associated with enhanced CRH secretion.

  4. Profiling posttraumatic functional impairment.

    PubMed

    Norman, Sonya B; Stein, Murray B; Davidson, Jonathan R T

    2007-01-01

    Many individuals who have been exposed to psychological trauma suffer from impaired functioning, regardless of whether they have PTSD. Our purpose was to identify a subset of PTSD symptoms linked to functional impairment to a) improve the likelihood that individuals with posttraumatic impairment receive treatment, and b) offer a method to assess cost-burden of trauma history in epidemiological studies. We examined patterns of trauma-related symptoms in two independent community surveys (N=1002 and 630). Rank ordering of symptoms and their associations with impairment guided construction of an impairment-related profile in the first data set. The profile was then tested in the second data set. The derived symptom profile, consisting of intense recollections and/or emotional symptoms upon exposure to reminders, plus one or more of numbing/detachment, avoidance, sleep problems, concentration problems, or hypervigilance, detected the majority (88% and 74%) of persons with posttraumatic functional impairment. The symptom profile can help identify traumatized individuals who may benefit from treatment but do not necessarily meet criteria for PTSD.

  5. Defective renal water handling in transgenic mice over-expressing human CD39/NTPDase1

    PubMed Central

    Zhang, Yue; Morris, Kaiya L.; Sparrow, Shannon K.; Dwyer, Karen M.; Enjyoji, Keiichi; Robson, Simon C.

    2012-01-01

    Ectonucleoside triphosphate diphosphohydrolase-1 hydrolyzes extracellular ATP and ADP to AMP. Previously, we showed that CD39 is expressed at several sites within the kidney and thus may impact the availability of type 2 purinergic receptor (P2-R) ligands. Because P2-Rs appear to regulate urinary concentrating ability, we have evaluated renal water handling in transgenic mice (TG) globally overexpressing hCD39. Under basal conditions, TG mice exhibited significantly impaired urinary concentration and decreased protein abundance of AQP2 in the kidney compared with wild-type (WT) mice. Urinary excretion of total nitrates/nitrites was significantly higher in TG mice, but the excretion of AVP or PGE2 was equivalent to control WT mice. There were no significant differences in electrolyte-free water clearance or fractional excretion of sodium. Under stable hydrated conditions (gelled diet feeding), the differences between the WT and TG mice were negated, but the decrease in urine osmolality persisted. When water deprived, TG mice failed to adequately concentrate urine and exhibited impaired AVP responses. However, the increases in urinary osmolalities in response to subacute dDAVP or chronic AVP treatment were similar in TG and WT mice. These observations suggest that TG mice have impaired urinary concentrating ability despite normal AVP levels. We also note impaired AVP release in response to water deprivation but that TG kidneys are responsive to exogenous dDAVP or AVP. We infer that heightened nucleotide scavenging by increased levels of CD39 altered the release of endogenous AVP in response to dehydration. We propose that ectonucleotidases and modulated purinergic signaling impact urinary concentration and indicate potential utility of targeted therapy for the treatment of water balance disorders. PMID:22622462

  6. Physiological Response to Membrane Protein Overexpression in E. coli*

    PubMed Central

    Gubellini, Francesca; Verdon, Grégory; Karpowich, Nathan K.; Luff, Jon D.; Boël, Grégory; Gauthier, Nils; Handelman, Samuel K.; Ades, Sarah E.; Hunt, John F.

    2011-01-01

    Overexpression represents a principal bottleneck in structural and functional studies of integral membrane proteins (IMPs). Although E. coli remains the leading organism for convenient and economical protein overexpression, many IMPs exhibit toxicity on induction in this host and give low yields of properly folded protein. Different mechanisms related to membrane biogenesis and IMP folding have been proposed to contribute to these problems, but there is limited understanding of the physical and physiological constraints on IMP overexpression and folding in vivo. Therefore, we used a variety of genetic, genomic, and microscopy techniques to characterize the physiological responses of Escherichia coli MG1655 cells to overexpression of a set of soluble proteins and IMPs, including constructs exhibiting different levels of toxicity and producing different levels of properly folded versus misfolded product on induction. Genetic marker studies coupled with transcriptomic results indicate only minor perturbations in many of the physiological systems implicated in previous studies of IMP biogenesis. Overexpression of either IMPs or soluble proteins tends to block execution of the standard stationary-phase transcriptional program, although these effects are consistently stronger for the IMPs included in our study. However, these perturbations are not an impediment to successful protein overexpression. We present evidence that, at least for the target proteins included in our study, there is no inherent obstacle to IMP overexpression in E. coli at moderate levels suitable for structural studies and that the biochemical and conformational properties of the proteins themselves are the major obstacles to success. Toxicity associated with target protein activity produces selective pressure leading to preferential growth of cells harboring expression-reducing and inactivating mutations, which can produce chemical heterogeneity in the target protein population, potentially

  7. Fibroblast-specific upregulation of Flightless I impairs wound healing.

    PubMed

    Turner, Christopher T; Waters, James M; Jackson, Jessica E; Arkell, Ruth M; Cowin, Allison J

    2015-09-01

    The cytoskeletal protein Flightless (Flii) is a negative regulator of wound healing. Upregulation of Flii is associated with impaired migration, proliferation and adhesion of both fibroblasts and keratinocytes. Importantly, Flii translocates from the cytoplasm to the nucleus in response to wounding in fibroblasts but not keratinocytes. This cell-specific nuclear translocation of Flii suggests that Flii may directly regulate gene expression in fibroblasts, providing one potential mechanism of action for Flii in the wound healing response. To determine whether the tissue-specific upregulation of Flii in fibroblasts was important for the observed inhibitory effects of Flii on wound healing, an inducible fibroblast-specific Flii overexpressing mouse model was generated. The inducible ROSA26 system allowed the overexpression of Flii in a temporal and tissue-specific manner in response to tamoxifen treatment. Wound healing in the inducible mice was impaired, with wounds at day 7 postwounding significantly larger than those from non-inducible controls. There was also reduced collagen maturation, increased myofibroblast infiltration and elevated inflammation. The impaired healing response was similar in magnitude to that observed in mice with non-tissue-specific upregulation of Flii suggesting that fibroblast-derived Flii may have an important role in the wound healing response. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Targeted overexpression of leptin to keratinocytes in transgenic mice results in lack of skin phenotype but induction of early leptin resistance.

    PubMed

    Rico, Laura; Del Rio, Marcela; Bravo, Ana; Ramirez, Angel; Jorcano, José L; Page, M Angustias; Larcher, Fernando

    2005-10-01

    The epidermis has a great potential as a bioreactor to produce proteins with systemic action. However, the consequences of ectopic epidermal protein overexpression need to be carefully addressed to avoid both local and systemic adverse effects. Thus, the long-term effects of leptin on skin physiology have not been studied, and the metabolic consequences of sustained keratinocyte-derived leptin overexpression are unknown. Herein we describe that very high serum leptin levels can be achieved from a cutaneous source in transgenic mice in which leptin cDNA overexpression was driven by the keratin K5 gene regulatory sequences. Histopathological analysis including the study of skin differentiation and proliferation markers in these transgenic mice revealed that keratinocyte-derived leptin overexpression appears not to have any impact on cutaneous homeostasis. Although young K5-leptin transgenic mice showed remarkable thinness and high glucose metabolism as shown in other leptin transgenic mouse models, a marked leptin insensitivity become apparent as early as 3-4 months of age as demonstrated by increased weight gain and insulin resistance development. Other signs of leptin/insulin resistance included increased bone mass, organomegaly, and wound healing impairment. In addition, to provide evidence for the lack of untoward effects of leptin on epidermis, this transgenic mouse helps us to establish the safe ranges of keratinocyte-derived leptin overexpression and may be useful as a model to study leptin resistance.

  9. Neuronal-specific overexpression of a mutant valosin-containing protein associated with IBMPFD promotes aberrant ubiquitin and TDP-43 accumulation and cognitive dysfunction in transgenic mice.

    PubMed

    Rodriguez-Ortiz, Carlos J; Hoshino, Hitomi; Cheng, David; Liu-Yescevitz, Liqun; Blurton-Jones, Mathew; Wolozin, Benjamin; LaFerla, Frank M; Kitazawa, Masashi

    2013-08-01

    Mutations in valosin-containing protein (VCP) cause a rare, autosomal dominant disease called inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD). One-third of patients with IBMPFD develop frontotemporal dementia, characterized by an extensive neurodegeneration in the frontal and temporal lobes. Neuropathologic hallmarks include nuclear and cytosolic inclusions positive to ubiquitin and transactive response DNA-binding protein 43 (TDP-43) in neurons and glial activation in affected regions. However, the pathogenic mechanisms by which mutant VCP triggers neurodegeneration remain unknown. Herein, we generated a mouse model selectively overexpressing a human mutant VCP in neurons to study pathogenic mechanisms of mutant VCP-mediated neurodegeneration and cognitive impairment. The overexpression of VCPA232E mutation in forebrain regions produced significant progressive impairments of cognitive function, including deficits in spatial memory, object recognition, and fear conditioning. Although overexpressed or endogenous VCP did not seem to focally aggregate inside neurons, TDP-43 and ubiquitin accumulated with age in transgenic mouse brains. TDP-43 was also found to co-localize with stress granules in the cytosolic compartment. Together with the appearance of high-molecular-weight TDP-43 in cytosolic fractions, these findings demonstrate the mislocalization and accumulation of abnormal TDP-43 in the cytosol of transgenic mice, which likely lead to an increase in cellular stress and cognitive impairment. Taken together, these results highlight an important pathologic link between VCP and cognition.

  10. Targeted Overexpression of Amelotin Disrupts the Microstructure of Dental Enamel

    PubMed Central

    Lacruz, Rodrigo S.; Nakayama, Yohei; Holcroft, James; Nguyen, Van; Somogyi-Ganss, Eszter; Snead, Malcolm L.; White, Shane N.; Paine, Michael L.; Ganss, Bernhard

    2012-01-01

    We have previously identified amelotin (AMTN) as a novel protein expressed predominantly during the late stages of dental enamel formation, but its role during amelogenesis remains to be determined. In this study we generated transgenic mice that produce AMTN under the amelogenin (Amel) gene promoter to study the effect of AMTN overexpression on enamel formation in vivo. The specific overexpression of AMTN in secretory stage ameloblasts was confirmed by Western blot and immunohistochemistry. The gross histological appearance of ameloblasts or supporting cellular structures as well as the expression of the enamel proteins amelogenin (AMEL) and ameloblastin (AMBN) was not altered by AMTN overexpression, suggesting that protein production, processing and secretion occurred normally in transgenic mice. The expression of Odontogenic, Ameloblast-Associated (ODAM) was slightly increased in secretory stage ameloblasts of transgenic animals. The enamel in AMTN-overexpressing mice was much thinner and displayed a highly irregular surface structure compared to wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. Tomes' process, the hallmark of secretory stage ameloblasts, did not form in transgenic mice. Collectively our data demonstrate that the overexpression of amelotin has a profound effect on enamel structure by disrupting the formation of Tomes' process and the orderly growth of enamel prisms. PMID:22539960

  11. Targeted overexpression of amelotin disrupts the microstructure of dental enamel.

    PubMed

    Lacruz, Rodrigo S; Nakayama, Yohei; Holcroft, James; Nguyen, Van; Somogyi-Ganss, Eszter; Snead, Malcolm L; White, Shane N; Paine, Michael L; Ganss, Bernhard

    2012-01-01

    We have previously identified amelotin (AMTN) as a novel protein expressed predominantly during the late stages of dental enamel formation, but its role during amelogenesis remains to be determined. In this study we generated transgenic mice that produce AMTN under the amelogenin (Amel) gene promoter to study the effect of AMTN overexpression on enamel formation in vivo. The specific overexpression of AMTN in secretory stage ameloblasts was confirmed by Western blot and immunohistochemistry. The gross histological appearance of ameloblasts or supporting cellular structures as well as the expression of the enamel proteins amelogenin (AMEL) and ameloblastin (AMBN) was not altered by AMTN overexpression, suggesting that protein production, processing and secretion occurred normally in transgenic mice. The expression of Odontogenic, Ameloblast-Associated (ODAM) was slightly increased in secretory stage ameloblasts of transgenic animals. The enamel in AMTN-overexpressing mice was much thinner and displayed a highly irregular surface structure compared to wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. Tomes' process, the hallmark of secretory stage ameloblasts, did not form in transgenic mice. Collectively our data demonstrate that the overexpression of amelotin has a profound effect on enamel structure by disrupting the formation of Tomes' process and the orderly growth of enamel prisms.

  12. RECK overexpression reduces invasive ability in ameloblastoma cells.

    PubMed

    Liang, Qi-xiang; Liang, Yan-can; Xu, Zhi-ying; Chen, Wei-liang; Xie, Hong-liang; Zhang, Bin

    2014-09-01

    Ameloblastoma is a frequent odontogenic neoplasm characterized by local invasiveness and high risk of recurrence. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a tumor suppressor that inhibits metastasis and angiogenesis. The aim of this study was to investigate effects of RECK overexpression on invasive potential in ameloblastoma cells. Lentiviral vectors containing human RECK gene were created and subsequently stably transfected into immortalized ameloblastoma cell line hTERT(+) -AM. Functional characteristics of hTERT(+) -AM cells with stable RECK overexpression included proliferation, migration, invasion, and regulation of matrix metalloproteinases (MMP)-2, MMP-9 measured by zymography or commercially available assays. The stable and higher expression of RECK mRNA and protein (P < 0.01) was detected in RECK-transfected hTERT(+) -AM cells. RECK overexpression caused a decrease in migration and invasion (P < 0.01) for hTERT(+) -AM cells and a decrease in activity of MMP-2, MMP-9 (P < 0.01). Proliferation was not affected by RECK overexpression (P > 0.05). Overexpression of RECK gene significantly inhibited cell invasive ability of hTERT(+) -AM cells, suggesting RECK may be a new target for ameloblastoma treatment. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Temporal Effects of Catalase Overexpression on Healing Following Myocardial Infarction

    PubMed Central

    Pendergrass, Karl D.; Varghese, Susan T.; Maiellaro-Rafferty, Kathryn; Brown, Milton E.; Taylor, W. Robert; Davis, Michael E.

    2011-01-01

    Background Reactive oxygen species, such as hydrogen peroxide (H2O2), contribute to progression of dysfunction following myocardial infarction (MI). However, chronic overexpression studies do not agree with acute protein delivery studies. The purpose of the present study was to assess the temporal role of cardiomyocyte-derived H2O2 scavenging on cardiac function after infarction using an inducible system. Methods and Results We developed a tamoxifen-inducible, cardiomyocyte-specific catalase overexpressing mouse. Catalase overexpression was induced either 5 days pre or post-MI. Mice exhibited a 3-fold increase in cardiac catalase activity that was associated with a significant decrease in H2O2 levels at both 7 and 21 days. However, cardiac function improved only at the later time point. Pro-inflammatory and fibrotic genes were acutely upregulated after MI, but catalase overexpression abolished the increase, despite no acute change in function. This led to reduced overall scar formation, with lower levels of Collagen 1A and increased contractile Collagen 3A expression at 21 days. Conclusions In contrast to prior studies, there were no acute functional improvements with physiological catalase overexpression prior to MI. Scavenging of H2O2 however, reduced pro-inflammatory cytokines and altered cardiac collagen isoforms, associated with an improvement in cardiac function after 21 days. Our results suggest that sustained H2O2 levels, rather than acute levels immediately following MI, may be critical in directing remodeling and cardiac function at later time points. PMID:20971939

  14. Genetic basis for p53 overexpression in human breast cancer.

    PubMed Central

    Davidoff, A M; Humphrey, P A; Iglehart, J D; Marks, J R

    1991-01-01

    Overexpression of an activated form of the p53 protein may be involved in neoplastic transformation. We found widespread overexpression of p53 by immunohistochemical staining in 11 (22%) of 49 primary invasive human breast cancers. Northern blot analysis showed that this overexpression was not due to an increase in the steady-state level of p53 mRNA. The p53 gene was directly sequenced in 7 of these tumors with elevated levels of the protein and, in each case, a mutation that altered the coding sequence for p53 was found in a highly conserved region of the gene. Whereas 4 of these tumors contained only a mutant p53 allele, the other 3 tumors exhibited coding sequences from both a mutant and a wild-type allele. p53 mutations have previously been correlated with allelic loss of part of chromosome 17p that contains the p53 locus. Examination of all 49 breast tumors revealed a 61% frequency of deletion at or near the p53 locus. However, the presence of allelic deletion did not correlate with overexpression of the protein. Six tumors that were deleted but did not express high levels of the protein were sequenced and all retained a wild-type p53 allele. In this series of human breast cancers, overexpression of the p53 protein, not allelic loss on chromosome 17p, was always associated with mutation of the p53 gene. Images PMID:2052583

  15. PGM2 overexpression improves anaerobic galactose fermentation in Saccharomyces cerevisiae

    PubMed Central

    2010-01-01

    Background In Saccharomyces cerevisiae galactose is initially metabolized through the Leloir pathway after which glucose 6-phosphate enters glycolysis. Galactose is controlled both by glucose repression and by galactose induction. The gene PGM2 encodes the last enzyme of the Leloir pathway, phosphoglucomutase 2 (Pgm2p), which catalyses the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Overexpression of PGM2 has previously been shown to enhance aerobic growth of S. cerevisiae in galactose medium. Results In the present study we show that overexpression of PGM2 under control of the HXT7'promoter from an integrative plasmid increased the PGM activity 5 to 6 times, which significantly reduced the lag phase of glucose-pregrown cells in an anaerobic galactose culture. PGM2 overexpression also increased the anaerobic specific growth rate whereas ethanol production was less influenced. When PGM2 was overexpressed from a multicopy plasmid instead, the PGM activity increased almost 32 times. However, this increase of PGM activity did not further improve aerobic galactose fermentation as compared to the strain carrying PGM2 on the integrative plasmid. Conclusion PGM2 overexpression in S. cerevisiae from an integrative plasmid is sufficient to reduce the lag phase and to enhance the growth rate in anaerobic galactose fermentation, which results in an overall decrease in fermentation duration. This observation is of particular importance for the future development of stable industrial strains with enhanced PGM activity. PMID:20507616

  16. Phytochrome a overexpression inhibits hypocotyl elongation in transgenic Arabidopsis.

    PubMed Central

    Boylan, M T; Quail, P H

    1991-01-01

    To develop a model plant system for efficient functional analysis of mutagenized phytochrome polypeptides, we have overexpressed oat phytochrome A in Arabidopsis thaliana. R1 seedlings from selfed primary transformants segregated for hypocotyl length, when grown in the light, with a ratio of 3 short to 1 of normal length. When homozygous lines were established from these two size classes, accumulation of immunologically detectable oat phytochrome cosegregated with the short-hypocotyl trait. The short-hypocotyl seedlings contained substantially more spectrally active phytochrome than their normal-sized siblings, indicating that the introduced oat protein was photoreversible. The short-hypocotyl phenotype was strictly light-dependent, since no morphological effects of phytochrome overexpression could be seen in etiolated seedlings. Overexpression of only the chromophore-bearing, N-terminal domain of phytochrome A did not induce short hypocotyls in light-grown seedlings, indicating that additional sequence is essential for photoreceptor function. Similarly, overexpression of a full-length sequence mutated at the chromophore attachment site had no effect on phenotype, indicating the absence of any detectable dominant negative effect of the chromophoreless polypeptide on the activity of endogenous Arabidopsis phytochrome. Thus, the readily scorable short-hypocotyl phenotype of Arabidopsis seedlings overexpressing phytochrome A provides a simple visual assay for rapidly monitoring the biological activity of mutagenized phytochrome A polypeptides. Images PMID:11607244

  17. Clinical significance of Her2/neu overexpression in urothelial carcinomas.

    PubMed

    Alexa, Aurora; Baderca, Flavia; Zăhoi, Delia Elena; Lighezan, Rodica; Izvernariu, D; Raica, M

    2010-01-01

    HER2/neu is a defective transmembrane tyrosine kinase receptor, homologue to the epidermal growth factor receptor, showing overexpression in a large variety of tumor cells. There are no studies published so far regarding HER2/neu overexpression and sensitivity of the urothelial tumors of the urinary bladder to anti-HER2/neu therapy. There are a relatively high number of articles in the literature referring to HER2/neu expression in urothelial tumors of the urinary bladder, but only two of them had investigated HER2/neu expression in patients with urothelial tumors of the upper urinary tract. We have studied HER2/neu overexpression in 59 patients with urothelial carcinomas of the urinary tract by immunohistochemistry. Normal urothelium and the elements of the neighboring renal parenchyma were negative. Out of the 59 cases of urothelial carcinomas, 38 were negative (0 and +1) and 21 were positive: eight were moderately and 13 were intensely positive (+2 and +3). The percentage of positive cases was 35.59%. The negative cases were mostly well-differentiated, G1 tumors, no matter the T-tumor stage. Most of the cases were diagnosed as papillary or, rarely, infiltrative. There is no correlation between HER2/neu overexpression and the tumor stage. The same was true for the lymph node status. The expression intensity, however, was significantly correlated with the differentiation grade. Overexpression was most likely present in tumors with high differentiation grade (p<0.05).

  18. Modification of photosynthetic regulation in tomato overexpressing glutathione peroxidase.

    PubMed

    Herbette, Stephane; Menn, Aline Le; Rousselle, Patrick; Ameglio, Thierry; Faltin, Zehava; Branlard, Gérard; Eshdat, Yuval; Julien, Jean-Louis; Drevet, Joël R; Roeckel-Drevet, Patricia

    2005-06-20

    To investigate the function of glutathione peroxidase (GPX) in plants, we produced transgenic tomato plants overexpressing an eukaryotic selenium-independent GPX (GPX5). We show here that total GPX activity was increased by 50% in transgenic plants, when compared to control plants transformed with the binary vector without the insert (PZP111). A preliminary two-dimensional electrophoretic protein analysis of the GPX overexpressing plants showed notably a decrease in the accumulation of proteins identified as rubisco small subunit 1 and fructose-1,6-bisphosphate aldolase, two proteins involved in photosynthesis. These observations, together with the fact that in standard culture conditions, GPX-overexpressing plants were not phenotypically distinct from control plants prompted us to challenge the plants with a chilling treatment that is known to affect photosynthesis activity. We found that upon chilling treatment with low light level, photosynthesis was not affected in GPX-overexpressing plants while it was in control plants, as revealed by chlorophyll fluorescence parameters and fructose-1,6-biphosphatase activity. These results suggest that overexpression of a selenium-independent GPX in tomato plants modifies specifically gene expression and leads to modifications of photosynthetic regulation processes.

  19. Overexpression of hypoxia-inducible factor 1 alpha improves immunomodulation by dental mesenchymal stem cells.

    PubMed

    Martinez, Victor G; Ontoria-Oviedo, Imelda; Ricardo, Carolina P; Harding, Sian E; Sacedon, Rosa; Varas, Alberto; Zapata, Agustin; Sepulveda, Pilar; Vicente, Angeles

    2017-09-29

    Human dental mesenchymal stem cells (MSCs) are considered as highly accessible and attractive MSCs for use in regenerative medicine, yet some of their features are not as well characterized as other MSCs. Hypoxia-preconditioning and hypoxia-inducible factor 1 (HIF-1) alpha overexpression significantly improves MSC therapeutics, but the mechanisms involved are not fully understood. In the present study, we characterize immunomodulatory properties of dental MSCs and determine changes in their ability to modulate adaptive and innate immune populations after HIF-1 alpha overexpression. Human dental MSCs were stably transduced with green fluorescent protein (GFP-MSCs) or GFP-HIF-1 alpha lentivirus vectors (HIF-MSCs). A hypoxic-like metabolic profile was confirmed by mitochondrial and glycolysis stress test. Capacity of HIF-MSCs to modulate T-cell activation, dendritic cell differentiation, monocyte migration, and polarizations towards macrophages and natural killer (NK) cell lytic activity was assessed by a number of functional assays in co-cultures. The expression of relevant factors were determined by polymerase chain reaction (PCR) analysis and enzyme-linked immunosorbent assay (ELISA). While HIF-1 alpha overexpression did not modify the inhibition of T-cell activation by MSCs, HIF-MSCs impaired dendritic cell differentiation more efficiently. In addition, HIF-MSCs showed a tendency to induce higher attraction of monocytes, which differentiate into suppressor macrophages, and exhibited enhanced resistance to NK cell-mediated lysis, which supports the improved therapeutic capacity of HIF-MSCs. HIF-MSCs also displayed a pro-angiogenic profile characterized by increased expression of CXCL12/SDF1 and CCL5/RANTES and complete loss of CXCL10/IP10 transcription. Immunomodulation and expression of trophic factors by dental MSCs make them perfect candidates for cell therapy. Overexpression of HIF-1 alpha enhances these features and increases their resistance to allogenic NK

  20. Mainstreaming the Hearing Impaired Student.

    ERIC Educational Resources Information Center

    Szelazkiewicz, Sara

    This review of the literature on mainstreaming students with hearing impairments begins by defining "hearing impairment,""deafness," and "hard of hearing". The paper briefly considers causes of hearing impairments and types of hearing impairments. Suggestions for preparing a class for a student with hearing loss includes having a visible class…

  1. Trainable Mentally Impaired/Severely Multiply Impaired/Autistic Impaired/Severely Mentally Impaired. Product Evaluation Report 1989-1990.

    ERIC Educational Resources Information Center

    Claus, Richard N.; And Others

    The evaluation report describes special education services provided to trainable mentally impaired (TMI), autistic impaired (AI), severely multiply impaired (SXI), and severely mentally impaired (SMI) students at and through the Melvin G. Millet Learning Center (Bridgeport, Michigan). The eight program components are described individually and…

  2. Combinatorial Method for Overexpression of Membrane Proteins in Escherichia coli*

    PubMed Central

    Leviatan, Shani; Sawada, Keisuke; Moriyama, Yoshinori; Nelson, Nathan

    2010-01-01

    Membrane proteins constitute 20–30% of all proteins encoded by the genome of various organisms. Large amounts of purified proteins are required for activity and crystallization attempts. Thus, there is an unmet need for a heterologous membrane protein overexpression system for purification, crystallization, and activity determination. We developed a combinatorial method for overexpressing and purifying membrane proteins using Escherichia coli. This method utilizes short hydrophilic bacterial proteins, YaiN and YbeL, fused to the ends of the membrane proteins to serve as facilitating factors for expression and purification. Fourteen prokaryotic and mammalian membrane proteins were expressed using this system. Moderate to high expression was obtained for most proteins, and detergent solubilization combined with a short purification process produced stable, monodispersed membrane proteins. Five of the mammalian membrane proteins, overexpressed using our system, were reconstituted into liposomes and exhibited transport activity comparable with the native transporters. PMID:20525689

  3. Combinatorial method for overexpression of membrane proteins in Escherichia coli.

    PubMed

    Leviatan, Shani; Sawada, Keisuke; Moriyama, Yoshinori; Nelson, Nathan

    2010-07-30

    Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. Large amounts of purified proteins are required for activity and crystallization attempts. Thus, there is an unmet need for a heterologous membrane protein overexpression system for purification, crystallization, and activity determination. We developed a combinatorial method for overexpressing and purifying membrane proteins using Escherichia coli. This method utilizes short hydrophilic bacterial proteins, YaiN and YbeL, fused to the ends of the membrane proteins to serve as facilitating factors for expression and purification. Fourteen prokaryotic and mammalian membrane proteins were expressed using this system. Moderate to high expression was obtained for most proteins, and detergent solubilization combined with a short purification process produced stable, monodispersed membrane proteins. Five of the mammalian membrane proteins, overexpressed using our system, were reconstituted into liposomes and exhibited transport activity comparable with the native transporters.

  4. Biglycan Overexpression on Tooth Enamel Formation in Transgenic Mice

    PubMed Central

    Wen, Xin; Zou, YanMing; Luo, Wen; Goldberg, Michel; Moats, Rex; Conti, Peter S.; Snead, Malcolm L.; Paine, Michael L.

    2008-01-01

    Previously it was shown that the volume of forming enamel of molar teeth in biglycan-null mice was greater than in genetically matched wild-type mice. This phenotypic change appeared to result from an increase in amelogenin expression, implying that biglycan directly influences amelogenin synthesis. To determine whether biglycan over-expression resulted in decreased amelogenin expression, we engineered transgenic mice to over-express biglycan in the enamel organ epithelium. Biglycan over-expression did not significantly affect the amelogenin expression in incisor and molar teeth in 3-day transgenic mice. In the transgenic animals we observed that the immature and mature enamel appeared normal. These results suggested that increasing the biglycan expression, in the cells that synthesize the precursor protein matrix for enamel, has a negligible influence on amelogenesis. PMID:18727043

  5. Geminin overexpression induces mammary tumors via suppressing cytokinesis.

    PubMed

    Blanchard, Zannel; Malik, Rohit; Mullins, Nicole; Maric, Christine; Luk, Hugh; Horio, David; Hernandez, Brenda; Killeen, Jeffrey; Elshamy, Wael M

    2011-12-01

    Aneuploidy plays an important role in the development of cancer. Here, we uncovered an oncogenic role for geminin in mitotic cells. In addition to chromatin, tyrosine phosphorylated geminin also localizes to centrosome, spindle, cleavage furrow and midbody during mitosis. Geminin binding to Aurora B prevents its binding to INCENP, and thus activation leading to lack of histone H3-(serine 10) phosphorylation, chromosome condensation failure, aborted cytokinesis and the formation of aneuploid, drug resistance cells. Geminin overexpressing human mammary epithelial cells form aneuploid, aggressive tumors in SCID mice. Geminin is overexpressed in more than half of all breast cancers analyzed. The current study reveals that geminin is a genuine oncogene that promotes cytokinesis failure and production of aneuploid, aggressive breast tumors when overexpressed and thus a worthy therapeutic target (oncotarget) for aggressive breast cancer.

  6. Social communication impairments: pragmatics.

    PubMed

    Russell, Robert L

    2007-06-01

    Social communication or pragmatic impairments are characterized and illustrated as involving inappropriate or ineffective use of language and gesture in social contexts. Three clinical vignettes illustrate different pragmatic impairments and the wealth of diagnostic information that can be garnered from observation of a child's social communication behavior. Definitions of, and developmental milestones in, domains of pragmatic competence are provided. Several screening instruments are suggested for use in assessing pragmatic competence within the time-frame of a pediatric examination. Frequent comorbid psychiatric conditions are described and a sample of current neurobiologic research is briefly summarized.

  7. Targeted overexpression of mitochondrial catalase protects against cancer chemotherapy-induced skeletal muscle dysfunction

    PubMed Central

    Lark, Daniel S.; Reese, Lauren R.; Torres, Maria J.; Ryan, Terence E.; Lin, Chien-Te; Cathey, Brook L.; Neufer, P. Darrell

    2016-01-01

    The loss of strength in combination with constant fatigue is a burden on cancer patients undergoing chemotherapy. Doxorubicin, a standard chemotherapy drug used in the clinic, causes skeletal muscle dysfunction and increases mitochondrial H2O2. We hypothesized that the combined effect of cancer and chemotherapy in an immunocompetent breast cancer mouse model (E0771) would compromise skeletal muscle mitochondrial respiratory function, leading to an increase in H2O2-emitting potential and impaired muscle function. Here, we demonstrate that cancer chemotherapy decreases mitochondrial respiratory capacity supported with complex I (pyruvate/glutamate/malate) and complex II (succinate) substrates. Mitochondrial H2O2-emitting potential was altered in skeletal muscle, and global protein oxidation was elevated with cancer chemotherapy. Muscle contractile function was impaired following exposure to cancer chemotherapy. Genetically engineering the overexpression of catalase in mitochondria of muscle attenuated mitochondrial H2O2 emission and protein oxidation, preserving mitochondrial and whole muscle function despite cancer chemotherapy. These findings suggest mitochondrial oxidants as a mediator of cancer chemotherapy-induced skeletal muscle dysfunction. PMID:27329802

  8. Targeted overexpression of mitochondrial catalase protects against cancer chemotherapy-induced skeletal muscle dysfunction.

    PubMed

    Gilliam, Laura A A; Lark, Daniel S; Reese, Lauren R; Torres, Maria J; Ryan, Terence E; Lin, Chien-Te; Cathey, Brook L; Neufer, P Darrell

    2016-08-01

    The loss of strength in combination with constant fatigue is a burden on cancer patients undergoing chemotherapy. Doxorubicin, a standard chemotherapy drug used in the clinic, causes skeletal muscle dysfunction and increases mitochondrial H2O2 We hypothesized that the combined effect of cancer and chemotherapy in an immunocompetent breast cancer mouse model (E0771) would compromise skeletal muscle mitochondrial respiratory function, leading to an increase in H2O2-emitting potential and impaired muscle function. Here, we demonstrate that cancer chemotherapy decreases mitochondrial respiratory capacity supported with complex I (pyruvate/glutamate/malate) and complex II (succinate) substrates. Mitochondrial H2O2-emitting potential was altered in skeletal muscle, and global protein oxidation was elevated with cancer chemotherapy. Muscle contractile function was impaired following exposure to cancer chemotherapy. Genetically engineering the overexpression of catalase in mitochondria of muscle attenuated mitochondrial H2O2 emission and protein oxidation, preserving mitochondrial and whole muscle function despite cancer chemotherapy. These findings suggest mitochondrial oxidants as a mediator of cancer chemotherapy-induced skeletal muscle dysfunction. Copyright © 2016 the American Physiological Society.

  9. Transgenic sugarcane overexpressing CaneCPI-1 negatively affects the growth and development of the sugarcane weevil Sphenophorus levis.

    PubMed

    Schneider, Vanessa Karine; Soares-Costa, Andrea; Chakravarthi, Mohan; Ribeiro, Carolina; Chabregas, Sabrina Moutinho; Falco, Maria Cristina; Henrique-Silva, Flavio

    2017-01-01

    Transgenic sugarcane expressing CaneCPI-1 exhibits resistance to Sphenophorus levis larvae. Transgenic plants have widely been used to improve resistance against insect attack. Sugarcane is an economically important crop; however, great losses are caused by insect attack. Sphenophorus levis is a sugarcane weevil that digs tunnels in the stem base, leading to the destruction of the crop. This insect is controlled inefficiently by chemical insecticides. Transgenic plants expressing peptidase inhibitors represent an important strategy for impairing insect growth and development. Knowledge of the major peptidase group present in the insect gut is critical when choosing the most effective inhibitor. S. levis larvae use cysteine peptidases as their major digestive enzymes, primarily cathepsin L-like activity. In this study, we developed transgenic sugarcane plants that overexpress sugarcane cysteine peptidase inhibitor 1 (CaneCPI-1) and assessed their potential through feeding bioassays with S. levis larvae. Cystatin overexpression in the transgenic plants was evaluated using semi-quantitative RT-PCR, RT-qPCR, and immunoblot assays. A 50% reduction of the average weight was observed in larvae that fed on transgenic plants in comparison to larvae that fed on non-transgenic plants. In addition, transgenic sugarcane exhibited less damage caused by larval attack than the controls. Our results suggest that the overexpression of CaneCPI-1 in sugarcane is a promising strategy for improving resistance against this insect.

  10. Inducible Conditional Vascular-Specific Overexpression of Peroxisome Proliferator-Activated Receptor Beta/Delta Leads to Rapid Cardiac Hypertrophy.

    PubMed

    Wagner, Kay-Dietrich; Vukolic, Ana; Baudouy, Delphine; Michiels, Jean-François; Wagner, Nicole

    2016-01-01

    Peroxisome proliferator-activated receptors are nuclear receptors which function as ligand-activated transcription factors. Among them, peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) is highly expressed in the heart and thought to have cardioprotective functions due to its beneficial effects in metabolic syndrome. As we already showed that PPARβ/δ activation resulted in an enhanced cardiac angiogenesis and growth without impairment of heart function, we were interested to determine the effects of a specific activation of PPARβ/δ in the vasculature on cardiac performance under normal and in chronic ischemic heart disease conditions. We analyzed the effects of a specific PPARβ/δ overexpression in endothelial cells on the heart using an inducible conditional vascular-specific mouse model. We demonstrate that vessel-specific overexpression of PPARβ/δ induces rapid cardiac angiogenesis and growth with an increase in cardiomyocyte size. Upon myocardial infarction, vascular overexpression of PPARβ/δ, despite the enhanced cardiac vessel formation, does not protect against chronic ischemic injury. Our results suggest that the proper balance of PPARβ/δ activation in the different cardiac cell types is required to obtain beneficial effects on the outcome in chronic ischemic heart disease.

  11. Inducible Conditional Vascular-Specific Overexpression of Peroxisome Proliferator-Activated Receptor Beta/Delta Leads to Rapid Cardiac Hypertrophy

    PubMed Central

    Wagner, Kay-Dietrich; Vukolic, Ana; Baudouy, Delphine; Michiels, Jean-François

    2016-01-01

    Peroxisome proliferator-activated receptors are nuclear receptors which function as ligand-activated transcription factors. Among them, peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) is highly expressed in the heart and thought to have cardioprotective functions due to its beneficial effects in metabolic syndrome. As we already showed that PPARβ/δ activation resulted in an enhanced cardiac angiogenesis and growth without impairment of heart function, we were interested to determine the effects of a specific activation of PPARβ/δ in the vasculature on cardiac performance under normal and in chronic ischemic heart disease conditions. We analyzed the effects of a specific PPARβ/δ overexpression in endothelial cells on the heart using an inducible conditional vascular-specific mouse model. We demonstrate that vessel-specific overexpression of PPARβ/δ induces rapid cardiac angiogenesis and growth with an increase in cardiomyocyte size. Upon myocardial infarction, vascular overexpression of PPARβ/δ, despite the enhanced cardiac vessel formation, does not protect against chronic ischemic injury. Our results suggest that the proper balance of PPARβ/δ activation in the different cardiac cell types is required to obtain beneficial effects on the outcome in chronic ischemic heart disease. PMID:27057154

  12. Overexpression of GTP cyclohydrolase 1 feedback regulatory protein is protective in a murine model of septic shock.

    PubMed

    Starr, Anna; Sand, Claire A; Heikal, Lamia; Kelly, Peter D; Spina, Domenico; Crabtree, Mark; Channon, Keith M; Leiper, James M; Nandi, Manasi

    2014-11-01

    Overproduction of nitric oxide (NO) by inducible NO synthase contributes toward refractory hypotension, impaired microvascular perfusion, and end-organ damage in septic shock patients. Tetrahydrobiopterin (BH4) is an essential NOS cofactor. GTP cyclohydrolase 1 (GCH1) is the rate-limiting enzyme for BH4 biosynthesis. Under inflammatory conditions, GCH1 activity and hence BH4 levels are increased, supporting pathological NOS activity. GCH1 activity can be controlled through allosteric interactions with GCH1 feedback regulatory protein (GFRP). We investigated whether overexpression of GFRP can regulate BH4 and NO production and attenuate cardiovascular dysfunction in sepsis. Sepsis was induced in mice conditionally overexpressing GFRP and wild-type littermates by cecal ligation and puncture. Blood pressure was monitored by radiotelemetry, and mesenteric blood flow was quantified by laser speckle contrast imaging. Blood biochemistry data were obtained using an iSTAT analyzer, and BH4 levels were measured in plasma and tissues by high-performance liquid chromatography. Increased BH4 and NO production and hypotension were observed in all mice, but the extents of these pathophysiological changes were attenuated in GFRP OE mice. Perturbations in blood biochemistry were similarly attenuated in GFRP OE compared with wild-type controls. These results suggest that GFRP overexpression regulates GCH1 activity during septic shock, which in turn limits BH4 bioavailability for iNOS. We conclude that the GCH1-GFRP axis is a critical regulator of BH4 and NO production and the cardiovascular derangements that occur in septic shock.

  13. PGC-1α overexpression exacerbates β-amyloid and tau deposition in a transgenic mouse model of Alzheimer's disease.

    PubMed

    Dumont, Magali; Stack, Cliona; Elipenahli, Ceyhan; Jainuddin, Shari; Launay, Nathalie; Gerges, Meri; Starkova, Natalia; Starkov, Anatoly A; Calingasan, Noel Y; Tampellini, Davide; Pujol, Aurora; Beal, M Flint

    2014-04-01

    The peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) interacts with various transcription factors involved in energy metabolism and in the regulation of mitochondrial biogenesis. PGC-1α mRNA levels are reduced in a number of neurodegenerative diseases and contribute to disease pathogenesis, since increased levels ameliorate behavioral defects and neuropathology of Huntington's disease, Parkinson's disease, and amyotrophic lateral sclerosis. PGC-1α and its downstream targets are reduced both in postmortem brain tissue of patients with Alzheimer's disease (AD) and in transgenic mouse models of AD. Therefore, we investigated whether increased expression of PGC-1α would exert beneficial effects in the Tg19959 transgenic mouse model of AD; Tg19959 mice express the human amyloid precursor gene (APP) with 2 familial AD mutations and develop increased β-amyloid levels, plaque deposition, and memory deficits by 2-3 mo of age. Rather than an improvement, the cross of the Tg19959 mice with mice overexpressing human PGC-1α exacerbated amyloid and tau accumulation. This was accompanied by an impairment of proteasome activity. PGC-1α overexpression induced mitochondrial abnormalities, neuronal cell death, and an exacerbation of behavioral hyperactivity in the Tg19959 mice. These findings show that PGC-1α overexpression exacerbates the neuropathological and behavioral deficits that occur in transgenic mice with mutations in APP that are associated with human AD.

  14. Persistent Overexpression of Phosphoglycerate Mutase, a Glycolytic Enzyme, Modifies Energy Metabolism and Reduces Stress Resistance of Heart in Mice

    PubMed Central

    Shioi, Tetsuo; Kato, Takao; Inuzuka, Yasutaka; Kawashima, Tsuneaki; Tamaki, Yodo; Kawamoto, Akira; Tanada, Yohei; Iwanaga, Yoshitaka; Narazaki, Michiko; Matsuda, Tetsuya; Adachi, Souichi; Soga, Tomoyoshi; Takemura, Genzou; Kondoh, Hiroshi; Kita, Toru; Kimura, Takeshi

    2013-01-01

    Background Heart failure is associated with changes in cardiac energy metabolism. Glucose metabolism in particular is thought to be important in the pathogenesis of heart failure. We examined the effects of persistent overexpression of phosphoglycerate mutase 2 (Pgam2), a glycolytic enzyme, on cardiac energy metabolism and function. Methods and Results Transgenic mice constitutively overexpressing Pgam2 in a heart-specific manner were generated, and cardiac energy metabolism and function were analyzed. Cardiac function at rest was normal. The uptake of analogs of glucose or fatty acids and the phosphocreatine/βATP ratio at rest were normal. A comprehensive metabolomic analysis revealed an increase in the levels of a few metabolites immediately upstream and downstream of Pgam2 in the glycolytic pathway, whereas the levels of metabolites in the initial few steps of glycolysis and lactate remained unchanged. The levels of metabolites in the tricarboxylic acid (TCA) cycle were altered. The capacity for respiration by isolated mitochondria in vitro was decreased, and that for the generation of reactive oxygen species (ROS) in vitro was increased. Impaired cardiac function was observed in response to dobutamine. Mice developed systolic dysfunction upon pressure overload. Conclusions Constitutive overexpression of Pgam2 modified energy metabolism and reduced stress resistance of heart in mice. PMID:23951293

  15. CD147 overexpression promotes tumorigenicity in Chinese hamster ovary cells.

    PubMed

    Yong, Yu-Le; Liao, Cheng-Gong; Wei, Ding; Chen, Zhi-Nan; Bian, Huijie

    2016-04-01

    CD147 overexpresses in many epithelium-originated tumors and plays an important role in tumor migration and invasion. Most studies aim at the role of CD147 in tumor progression using tumor cell models. However, the influence of abnormal overexpression of CD147 on neoplastic transformation of normal cells is unknown. Here, the role of CD147 in malignant phenotype transformation in CHO cells was investigated. Three CHO cell lines that stably overexpressed CD147 (CHO-CD147), EGFP-CD147 (CHO-EGFP-CD147), and EGFP (CHO-EGFP) were generated by transfection of plasmids containing human CD147, EGFP-human CD147, and EGFP genes into CHO cells. Cell migration and invasion were detected by wound healing and transwell matrix penetration assay. Trypan blue exclusion, MTT, cell cycle analysis, and BrdU cell proliferation assay were used to detect cell viability and cell proliferation. Annexin V-FITC analysis was performed to detect apoptosis. We found that CD147 overexpression promoted the migration and invasion of CHO cells. CD147 accelerated the G1 to S phase transition and enhanced the CHO cell proliferation. Overexpression of CD147 inhibited both early- and late-stages of apoptosis of CHO-CD147 cells, which is caused by serum deprivation. CHO-EGFP-CD147 cells showed an increased anchorage-independent growth compared with CHO-EGFP cells as detected by soft-agar colony formation assay. The tumors formed by CHO-CD147 cells in nude mice were larger and coupled with higher expression of proliferating cell nuclear antigen and Ki-67 than that of CHO cells. In conclusion, human CD147 overexpression induces malignant phenotype in CHO cells.

  16. Functional assay using lectin gene targeting technologies (over-expression).

    PubMed

    Nonaka, Motohiro; Kawasaki, Toshisuke

    2014-01-01

    Function of lectin depends on its amino acid sequence of carbohydrate-recognition domain (CRD), conformation, and extracellular/intracellular localization. Altering lectin gene expression by over-expression or knockdown is a powerful tool for analyzing its cellular function. Here, we describe a method of lectin gene over-expression, taking a C-type lectin, mannan-binding protein (MBP), as an example. Carbohydrate-binding ability of MBP, its subcellular localization, and functional co-localization with ligand glycoprotein are assayed comparing with an inactive mutant MBP.

  17. Excess of Yra1 RNA-Binding Factor Causes Transcription-Dependent Genome Instability, Replication Impairment and Telomere Shortening.

    PubMed

    Gavaldá, Sandra; Santos-Pereira, José M; García-Rubio, María L; Luna, Rosa; Aguilera, Andrés

    2016-04-01

    Yra1 is an essential nuclear factor of the evolutionarily conserved family of hnRNP-like export factors that when overexpressed impairs mRNA export and cell growth. To investigate further the relevance of proper Yra1 stoichiometry in the cell, we overexpressed Yra1 by transforming yeast cells with YRA1 intron-less constructs and analyzed its effect on gene expression and genome integrity. We found that YRA1 overexpression induces DNA damage and leads to a transcription-associated hyperrecombination phenotype that is mediated by RNA:DNA hybrids. In addition, it confers a genome-wide replication retardation as seen by reduced BrdU incorporation and accumulation of the Rrm3 helicase. In addition, YRA1 overexpression causes a cell senescence-like phenotype and telomere shortening. ChIP-chip analysis shows that overexpressed Yra1 is loaded to transcribed chromatin along the genome and to Y' telomeric regions, where Rrm3 is also accumulated, suggesting an impairment of telomere replication. Our work not only demonstrates that a proper stoichiometry of the Yra1 mRNA binding and export factor is required to maintain genome integrity and telomere homeostasis, but suggests that the cellular imbalance between transcribed RNA and specific RNA-binding factors may become a major cause of genome instability mediated by co-transcriptional replication impairment.

  18. Excess of Yra1 RNA-Binding Factor Causes Transcription-Dependent Genome Instability, Replication Impairment and Telomere Shortening

    PubMed Central

    Gavaldá, Sandra; Santos-Pereira, José M.; García-Rubio, María L.; Luna, Rosa; Aguilera, Andrés

    2016-01-01

    Yra1 is an essential nuclear factor of the evolutionarily conserved family of hnRNP-like export factors that when overexpressed impairs mRNA export and cell growth. To investigate further the relevance of proper Yra1 stoichiometry in the cell, we overexpressed Yra1 by transforming yeast cells with YRA1 intron-less constructs and analyzed its effect on gene expression and genome integrity. We found that YRA1 overexpression induces DNA damage and leads to a transcription-associated hyperrecombination phenotype that is mediated by RNA:DNA hybrids. In addition, it confers a genome-wide replication retardation as seen by reduced BrdU incorporation and accumulation of the Rrm3 helicase. In addition, YRA1 overexpression causes a cell senescence-like phenotype and telomere shortening. ChIP-chip analysis shows that overexpressed Yra1 is loaded to transcribed chromatin along the genome and to Y’ telomeric regions, where Rrm3 is also accumulated, suggesting an impairment of telomere replication. Our work not only demonstrates that a proper stoichiometry of the Yra1 mRNA binding and export factor is required to maintain genome integrity and telomere homeostasis, but suggests that the cellular imbalance between transcribed RNA and specific RNA-binding factors may become a major cause of genome instability mediated by co-transcriptional replication impairment. PMID:27035147

  19. PP2A methylation controls sensitivity and resistance to β-amyloid–induced cognitive and electrophysiological impairments

    PubMed Central

    Nicholls, Russell E.; Sontag, Jean-Marie; Zhang, Hong; Staniszewski, Agnieszka; Yan, Shijun; Kim, Carla Y.; Yim, Michael; Woodruff, Caitlin M.; Arning, Erland; Wasek, Brandi; Yin, Deqi; Bottiglieri, Teodoro; Sontag, Estelle; Kandel, Eric R.; Arancio, Ottavio

    2016-01-01

    Elevated levels of the β-amyloid peptide (Aβ) are thought to contribute to cognitive and behavioral impairments observed in Alzheimer’s disease (AD). Protein phosphatase 2A (PP2A) participates in multiple molecular pathways implicated in AD, and its expression and activity are reduced in postmortem brains of AD patients. PP2A is regulated by protein methylation, and impaired PP2A methylation is thought to contribute to increased AD risk in hyperhomocysteinemic individuals. To examine further the link between PP2A and AD, we generated transgenic mice that overexpress the PP2A methylesterase, protein phosphatase methylesterase-1 (PME-1), or the PP2A methyltransferase, leucine carboxyl methyltransferase-1 (LCMT-1), and examined the sensitivity of these animals to behavioral and electrophysiological impairments caused by exogenous Aβ exposure. We found that PME-1 overexpression enhanced these impairments, whereas LCMT-1 overexpression protected against Aβ-induced impairments. Neither transgene affected Aβ production or the electrophysiological response to low concentrations of Aβ, suggesting that these manipulations selectively affect the pathological response to elevated Aβ levels. Together these data identify a molecular mechanism linking PP2A to the development of AD-related cognitive impairments that might be therapeutically exploited to target selectively the pathological effects caused by elevated Aβ levels in AD patients. PMID:26951658

  20. Altered Tnnt3 characterizes selective weakness of fast fibers in mice overexpressing FSHD region gene 1 (FRG1).

    PubMed

    Sancisi, Valentina; Germinario, Elena; Esposito, Alessandra; Morini, Elisabetta; Peron, Samantha; Moggio, Maurizio; Tomelleri, Giuliano; Danieli-Betto, Daniela; Tupler, Rossella

    2014-01-15

    Facioscapulohumeral muscular dystrophy (FSHD), a common hereditary myopathy, is characterized by atrophy and weakness of selective muscle groups. FSHD is considered an autosomal dominant disease with incomplete penetrance and unpredictable variability of clinical expression within families. Mice overexpressing FRG1 (FSHD region gene 1), a candidate gene for this disease, develop a progressive myopathy with features of the human disorder. Here, we show that in FRG1-overexpressing mice, fast muscles, which are the most affected by the dystrophic process, display anomalous fast skeletal troponin T (fTnT) isoform, resulting from the aberrant splicing of the Tnnt3 mRNA that precedes the appearance of dystrophic signs. We determine that muscles of FRG1 mice develop less strength due to impaired contractile properties of fast-twitch fibers associated with an anomalous MyHC-actin ratio and a reduced sensitivity to Ca(2+). We demonstrate that the decrease of Ca(2+) sensitivity of fast-twitch fibers depends on the anomalous troponin complex and can be rescued by the substitution with the wild-type proteins. Finally, we find that the presence of aberrant splicing isoforms of TNNT3 characterizes dystrophic muscles in FSHD patients. Collectively, our results suggest that anomalous TNNT3 profile correlates with the muscle impairment in both humans and mice. On the basis of these results, we propose that aberrant fTnT represents a biological marker of muscle phenotype severity and disease progression.

  1. Anarthria impairs subvocal counting.

    PubMed

    Cubelli, R; Nichelli, P; Pentore, R

    1993-12-01

    We studied subvocal counting in two pure anarthric patients. Analysis showed that they performed definitively worse than normal subjects free to articulate subvocally and their scores were in the lower bounds of the performances of subjects suppressing articulation. These results suggest that subvocal counting is impaired after anarthria.

  2. Aids for visual impairment.

    PubMed Central

    Dudley, N J

    1990-01-01

    This article provides only a flavour of the type and range of aids available to the visually impaired person. Many other aids for leisure, learning, and daily living are illustrated in the RNIB equipment and games catalogue. Images FIG 1 FIG 3 FIG 4 FIG 5 FIG 6 FIG 7 FIG 8 FIG 9 FIG 10 PMID:2252929

  3. Emotionally Impaired Elementary Curriculum.

    ERIC Educational Resources Information Center

    Taulbee, Dianne R.; And Others

    A curriculum is presented for teaching emotionally impaired elementary students. The curriculum document describes program management techniques, strategies for developing and maintaining teacher-student relationships, and therapy/change systems. It outlines referral and eligibility procedures and exit criteria. It contains job descriptions for…

  4. Emotionally Impaired Elementary Curriculum.

    ERIC Educational Resources Information Center

    Taulbee, Dianne R.; And Others

    The Jackson County (Michigan) Intermediate School District curriculum for teaching emotionally impaired elementary students is presented. The curriculum document describes program management techniques, strategies for developing and maintaining teacher-student relationships, and therapy/change systems. It outlines referral and eligibility…

  5. Overexpression of protein kinase STK25 in mice exacerbates ectopic lipid accumulation, mitochondrial dysfunction and insulin resistance in skeletal muscle.

    PubMed

    Chursa, Urszula; Nuñez-Durán, Esther; Cansby, Emmelie; Amrutkar, Manoj; Sütt, Silva; Ståhlman, Marcus; Olsson, Britt-Marie; Borén, Jan; Johansson, Maria E; Bäckhed, Fredrik; Johansson, Bengt R; Sihlbom, Carina; Mahlapuu, Margit

    2017-03-01

    Understanding the molecular networks controlling ectopic lipid deposition and insulin responsiveness in skeletal muscle is essential for developing new strategies to treat type 2 diabetes. We recently identified serine/threonine protein kinase 25 (STK25) as a critical regulator of liver steatosis, hepatic lipid metabolism and whole body glucose and insulin homeostasis. Here, we assessed the role of STK25 in control of ectopic fat storage and insulin responsiveness in skeletal muscle. Skeletal muscle morphology was studied by histological examination, exercise performance and insulin sensitivity were assessed by treadmill running and euglycaemic-hyperinsulinaemic clamp, respectively, and muscle lipid metabolism was analysed by ex vivo assays in Stk25 transgenic and wild-type mice fed a high-fat diet. Lipid accumulation and mitochondrial function were also studied in rodent myoblasts overexpressing STK25. Global quantitative phosphoproteomics was performed in skeletal muscle of Stk25 transgenic and wild-type mice fed a high-fat diet to identify potential downstream mediators of STK25 action. We found that overexpression of STK25 in transgenic mice fed a high-fat diet increases intramyocellular lipid accumulation, impairs skeletal muscle mitochondrial function and sarcomeric ultrastructure, and induces perimysial and endomysial fibrosis, thereby reducing endurance exercise capacity and muscle insulin sensitivity. Furthermore, we observed enhanced lipid accumulation and impaired mitochondrial function in rodent myoblasts overexpressing STK25, demonstrating an autonomous action for STK25 within cells. Global phosphoproteomic analysis revealed alterations in the total abundance and phosphorylation status of different target proteins located predominantly to mitochondria and sarcomeric contractile elements in Stk25 transgenic vs wild-type muscle, respectively, providing a possible molecular mechanism for the observed phenotype. STK25 emerges as a new regulator of the

  6. Vestibular Impairment in Dementia

    PubMed Central

    Harun, Aisha; Oh, Esther S.; Bigelow, Robin T.; Studenski, Stephanie; Agrawal, Yuri

    2017-01-01

    Objective Recent studies suggest an association between vestibular and cognitive function. The goal of the study was to investigate whether vestibular function was impaired in individuals with mild cognitive impairment (MCI) and Alzheimer’s disease (AD) compared to cognitively normal individuals. Study Design Cross-sectional study. Setting Outpatient memory clinic and longitudinal observational study unit. Patients Older individuals ≥ 55 years with MCI or AD. Age, gender and education-matched normal controls were drawn from the Baltimore Longitudinal Study of Aging (BLSA). Intervention Saccular and utricular function was assessed with cervical and ocular vestibular-evoked myogenic potentials (c- and oVEMPs) respectively, and horizontal semicircular canal function was assessed with video head impulse testing. Main Outcome Measures Presence or absence of VEMP responses, VEMP amplitude and vestibular ocular reflex (VOR) gain were measured. Results Forty-seven individuals with cognitive impairment (MCI N=15 and AD N=32) underwent testing and were matched with 94 controls. In adjusted analyses, bilaterally absent cVEMPs were associated with an over three-fold odds of AD (OR 3.42, 95% CI 1.33–8.91, p=0.011). One microvolt increases in both cVEMP and oVEMP amplitudes were associated with decreased odds of AD (OR 0.28, 95% CI 0.09–0.93, p=0.038 and OR 0.92, 95% CI 0.85–0.99, p=0.036, respectively). There was no significant difference in VOR gain between the groups. Conclusions These findings confirm and extend emerging evidence of an association between vestibular dysfunction and cognitive impairment. Further investigation is needed to determine the causal direction for the link between peripheral vestibular loss and cognitive impairment. PMID:27466890

  7. Vestibular Impairment in Dementia.

    PubMed

    Harun, Aisha; Oh, Esther S; Bigelow, Robin T; Studenski, Stephanie; Agrawal, Yuri

    2016-09-01

    Recent studies suggest an association between vestibular and cognitive function. The goal of the study was to investigate whether vestibular function was impaired in individuals with mild cognitive impairment (MCI) and Alzheimer's disease (AD) compared with cognitively normal individuals. Cross-sectional study. Outpatient memory clinic and longitudinal observational study unit. Older individuals ≥55 years with MCI or AD. Age, sex, and education-matched normal controls were drawn from the Baltimore Longitudinal Study of Aging (BLSA). Saccular and utricular function was assessed with cervical and ocular vestibular-evoked myogenic potentials (c- and oVEMPs) respectively, and horizontal semicircular canal function was assessed with video head impulse testing. Presence or absence of VEMP responses, VEMP amplitude, and vestibular ocular reflex (VOR) gain were measured. Forty-seven individuals with cognitive impairment (MCI N = 15 and AD N = 32) underwent testing and were matched with 94 controls. In adjusted analyses, bilaterally absent cVEMPs were associated with an over three-fold odds of AD (OR 3.42, 95% CI 1.33-8.91, p = 0.011). One microvolt increases in both cVEMP and oVEMP amplitudes were associated with decreased odds of AD (OR 0.28, 95% CI 0.09-0.93, p = 0.038 and OR 0.92, 95% CI 0.85-0.99, p = 0.036, respectively). There was no significant difference in VOR gain between the groups. These findings confirm and extend emerging evidence of an association between vestibular dysfunction and cognitive impairment. Further investigation is needed to determine the causal direction for the link between peripheral vestibular loss and cognitive impairment.

  8. Sarcolipin deletion exacerbates soleus muscle atrophy and weakness in phospholamban overexpressing mice

    PubMed Central

    Fajardo, Val A.; Gamu, Daniel; Mitchell, Andrew; Bloemberg, Darin; Bombardier, Eric; Chambers, Paige J.; Bellissimo, Catherine; Quadrilatero, Joe; Tupling, A. Russell

    2017-01-01

    Sarcolipin (SLN) and phospholamban (PLN) are two small proteins that regulate the sarco(endo)plasmic reticulum Ca2+-ATPase pumps. In a recent study, we discovered that Pln overexpression (PlnOE) in slow-twitch type I skeletal muscle fibers drastically impaired SERCA function and caused a centronuclear myopathy-like phenotype, severe muscle atrophy and weakness, and an 8 to 9-fold upregulation of SLN protein in the soleus muscles. Here, we sought to determine the physiological role of SLN upregulation, and based on its role as a SERCA inhibitor, we hypothesized that it would represent a maladaptive response that contributes to the SERCA dysfunction and the overall myopathy observed in the PlnOE mice. To this end, we crossed Sln-null (SlnKO) mice with PlnOE mice to generate a PlnOE/SlnKO mouse colony and assessed SERCA function, CNM pathology, in vitro contractility, muscle mass, calcineurin signaling, daily activity and food intake, and proteolytic enzyme activity. Our results indicate that genetic deletion of Sln did not improve SERCA function nor rescue the CNM phenotype, but did result in exacerbated muscle atrophy and weakness, due to a failure to induce type II fiber compensatory hypertrophy and a reduction in total myofiber count. Mechanistically, our findings suggest that impaired calcineurin activation and resultant decreased expression of stabilin-2, and/or impaired autophagic signaling could be involved. Future studies should examine these possibilities. In conclusion, our study demonstrates the importance of SLN upregulation in combating muscle myopathy in the PlnOE mice, and since SLN is upregulated across several myopathies, our findings may reveal SLN as a novel and universal therapeutic target. PMID:28278204

  9. Sarcolipin deletion exacerbates soleus muscle atrophy and weakness in phospholamban overexpressing mice.

    PubMed

    Fajardo, Val A; Gamu, Daniel; Mitchell, Andrew; Bloemberg, Darin; Bombardier, Eric; Chambers, Paige J; Bellissimo, Catherine; Quadrilatero, Joe; Tupling, A Russell

    2017-01-01

    Sarcolipin (SLN) and phospholamban (PLN) are two small proteins that regulate the sarco(endo)plasmic reticulum Ca2+-ATPase pumps. In a recent study, we discovered that Pln overexpression (PlnOE) in slow-twitch type I skeletal muscle fibers drastically impaired SERCA function and caused a centronuclear myopathy-like phenotype, severe muscle atrophy and weakness, and an 8 to 9-fold upregulation of SLN protein in the soleus muscles. Here, we sought to determine the physiological role of SLN upregulation, and based on its role as a SERCA inhibitor, we hypothesized that it would represent a maladaptive response that contributes to the SERCA dysfunction and the overall myopathy observed in the PlnOE mice. To this end, we crossed Sln-null (SlnKO) mice with PlnOE mice to generate a PlnOE/SlnKO mouse colony and assessed SERCA function, CNM pathology, in vitro contractility, muscle mass, calcineurin signaling, daily activity and food intake, and proteolytic enzyme activity. Our results indicate that genetic deletion of Sln did not improve SERCA function nor rescue the CNM phenotype, but did result in exacerbated muscle atrophy and weakness, due to a failure to induce type II fiber compensatory hypertrophy and a reduction in total myofiber count. Mechanistically, our findings suggest that impaired calcineurin activation and resultant decreased expression of stabilin-2, and/or impaired autophagic signaling could be involved. Future studies should examine these possibilities. In conclusion, our study demonstrates the importance of SLN upregulation in combating muscle myopathy in the PlnOE mice, and since SLN is upregulated across several myopathies, our findings may reveal SLN as a novel and universal therapeutic target.

  10. PGC-1α overexpression exacerbates β-amyloid and tau deposition in a transgenic mouse model of Alzheimer's disease

    PubMed Central

    Dumont, Magali; Stack, Cliona; Elipenahli, Ceyhan; Jainuddin, Shari; Launay, Nathalie; Gerges, Meri; Starkova, Natalia; Starkov, Anatoly A.; Calingasan, Noel Y.; Tampellini, Davide; Pujol, Aurora; Beal, M. Flint

    2014-01-01

    The peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) interacts with various transcription factors involved in energy metabolism and in the regulation of mitochondrial biogenesis. PGC-1α mRNA levels are reduced in a number of neurodegenerative diseases and contribute to disease pathogenesis, since increased levels ameliorate behavioral defects and neuropathology of Huntington's disease, Parkinson's disease, and amyotrophic lateral sclerosis. PGC-1α and its downstream targets are reduced both in postmortem brain tissue of patients with Alzheimer's disease (AD) and in transgenic mouse models of AD. Therefore, we investigated whether increased expression of PGC-1α would exert beneficial effects in the Tg19959 transgenic mouse model of AD; Tg19959 mice express the human amyloid precursor gene (APP) with 2 familial AD mutations and develop increased β-amyloid levels, plaque deposition, and memory deficits by 2–3 mo of age. Rather than an improvement, the cross of the Tg19959 mice with mice overexpressing human PGC-1α exacerbated amyloid and tau accumulation. This was accompanied by an impairment of proteasome activity. PGC-1α overexpression induced mitochondrial abnormalities, neuronal cell death, and an exacerbation of behavioral hyperactivity in the Tg19959 mice. These findings show that PGC-1α overexpression exacerbates the neuropathological and behavioral deficits that occur in transgenic mice with mutations in APP that are associated with human AD.—Dumont, M., Stack, C., Elipenahli, C., Jainuddin, S., Launay, N., Gerges, M., Starkova, N., Starkov, A. A., Calingasan, N. Y., Tampellini, D., Pujol, A., Beal, M. F. PGC-1α overexpression exacerbates β-amyloid and tau deposition in a transgenic mouse model of Alzheimer's disease. PMID:24398293

  11. Over-expression of Follistatin-like 3 attenuates fat accumulation and improves insulin sensitivity in mice.

    PubMed

    Brandt, Claus; Hansen, Rasmus Hvass; Hansen, Jakob Bondo; Olsen, Caroline Holkmann; Galle, Pia; Mandrup-Poulsen, Thomas; Gehl, Julie; Pedersen, Bente Klarlund; Hojman, Pernille

    2015-02-01

    Follistatin-like 3 (fstl3), a natural inhibitor of members of the TGF-β family, increases during resistance training in human plasma. Fstl3 primarily binds myostatin and activin A, and thereby inhibits their functions. We hypothesize that blocking myostatin and activin A signalling through systemic fstl3 over-expression protects against diet-induced obesity and insulin resistance. Fstl3 was over-expressed by DNA electrotransfer in tibialis anterior, quadriceps and gastrocnemius muscles in female C57BL/C mice, and the mice were subsequently randomized to chow or high-fat feeding. Body weight, food intake, fat accumulation by MR scanning, and glucose, insulin and glucagon tolerance were evaluated, as was the response in body weight and metabolic parameters to 24h fasting. Effects of fstl3 on pancreatic insulin and glucagon content, and pancreatic islet morphology were determined. Fstl3 over-expression reduced fat accumulation during high-fat feeding by 16%, and liver fat by 50%, as determined by MRI. No changes in body weight were observed, while the weight of the transfected muscles increased by 10%. No transcriptional changes were found in the subcutaneous adipose tissue. Fstl3 mice displayed improved insulin sensitivity and muscle insulin signalling. In contrast, glucose tolerance was impaired in high-fat fed fstl3 mice, which was explained by increased hepatic glucagon sensitivity and glucose output, as well as a decrease in the pancreatic insulin/glucagon ratio. Accordingly, fstl3 transfection improved counter-regulation to 24h fasting. Fstl3 over-expression regulates insulin and glucagon sensitivities through increased muscular insulin action, as well as increased hepatic glucagon sensitivity and pancreatic glucagon content. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Kinase Activity of Overexpressed HipA Is Required for Growth Arrest and Multidrug Tolerance in Escherichia coli▿

    PubMed Central

    Correia, Frederick F.; D'Onofrio, Anthony; Rejtar, Tomas; Li, Lingyun; Karger, Barry L.; Makarova, Kira; Koonin, Eugene V.; Lewis, Kim

    2006-01-01

    Overexpression of the HipA protein of the HipBA toxin/antitoxin module leads to multidrug tolerance in Escherichia coli. HipA is a “toxin” that causes reversible dormancy, whereas HipB is an antitoxin that binds HipA and acts as a transcriptional repressor of the hipBA operon. Comparative sequence analysis shows that HipA is a member of the phosphatidylinositol 3/4-kinase superfamily. The kinase activity of HipA was examined. HipA was autophosphorylated in the presence of ATP in vitro, and the purified protein appeared to carry a single phosphate group on serine 150. Thus, HipA is a serine kinase that is at least partially phosphorylated in vivo. Overexpression of HipA caused inhibition of cell growth and increase in persister formation. Replacing conserved aspartate 309 in the conserved kinase active site or aspartate 332 in the Mg2+-binding site with glutamine produced mutant proteins that lost the ability to stop cellular growth upon overexpression. Replacing serine 150 with alanine yielded a similarly inactive protein. The mutant proteins were then examined for their ability to increase antibiotic tolerance. Cells overexpressing wild-type HipA were highly tolerant to cefotaxime, a cell wall synthesis inhibitor, to ofloxacin, a fluoroquinolone inhibitor of DNA gyrase, and to topoisomerase IV and were almost completely resistant to killing by mitomycin C, which forms DNA adducts. The mutant proteins did not protect cells from cefotaxime or ofloxacin and had an impaired ability to protect from mitomycin C. Taken together, these results suggest that the protein kinase activity of HipA is essential for persister formation. PMID:17041039

  13. Lysophosphatidylcholine acyltransferase 1 (LPCAT1) overexpression in human colorectal cancer.

    PubMed

    Mansilla, Francisco; da Costa, Kerry-Ann; Wang, Shuli; Kruhøffer, Mogens; Lewin, Tal M; Orntoft, Torben F; Coleman, Rosalind A; Birkenkamp-Demtröder, Karin

    2009-01-01

    The alteration of the choline metabolite profile is a well-established characteristic of cancer cells. In colorectal cancer (CRC), phosphatidylcholine is the most prominent phospholipid. In the present study, we report that lysophosphatidylcholine acyltransferase 1 (LPCAT1; NM_024830.3), the enzyme that converts lysophosphatidylcholine into phosphatidylcholine, was highly overexpressed in colorectal adenocarcinomas when compared to normal mucosas. Our microarray transcription profiling study showed a significant (p < 10(-8)) transcript overexpression in 168 colorectal adenocarcinomas when compared to ten normal mucosas. Immunohistochemical analysis of colon tumors with a polyclonal antibody to LPCAT1 confirmed the upregulation of the LPCAT1 protein. Overexpression of LPCAT1 in COS7 cells localized the protein to the endoplasmic reticulum and the mitochondria and increased LPCAT1 specific activity 38-fold. In cultured cells, overexpressed LPCAT1 enhanced the incorporation of [(14)C]palmitate into phosphatidylcholine. COS7 cells transfected with LPCAT1 showed no growth rate alteration, in contrast to the colon cancer cell line SW480, which significantly (p < 10(-5)) increased its growth rate by 17%. We conclude that LPCAT1 may contribute to total choline metabolite accumulation via phosphatidylcholine remodeling, thereby altering the CRC lipid profile, a characteristic of malignancy.

  14. Laboratory and field studies of guayule modified to overexpress HMGR

    USDA-ARS?s Scientific Manuscript database

    We report the genetic modification of guayule to overexpress the isoprenoid pathway enzyme HMGR. The rubber content of two-month old in vitro transformed plantlets showed a 65% increase in rubber over the control for one line (HMGR6), and lower resin for another (HMGR2). In field evaluations HMGR6...

  15. Abetalipoproteinemia induced by overexpression of ORP150 in mice.

    PubMed

    Kobayashi, Tomohiro; Iguchi, Taisen; Ohta, Yasuhiko

    2007-06-01

    ORP150 is an endoplasmic-resident, hypoxic stress-induced protein, but little is known about the effects of its systemic overexpression. We have produced a transgenic strain of mice that overexpress ORP150 (ORP-Tg mice). These mice exhibit severe growth retardation concomitant with vacuolar degeneration in the heart. To investigate the cause of the observed growth retardation in response to ORP150 overexpression, we conducted a clinical evaluation of the ORP-Tg mice. Blood analysis showed significantly lower concentrations of serum triglyceride, cholesterol, glucose and insulin. The triglyceride components that were reduced in ORP-Tg mice were localized mainly at the origin and in the pre-beta fraction on agarose gel electrophoresis, corresponding to chylomicrons and very low-density lipoproteins. A lipid-loading test of ORP-Tg mice revealed reduced triglyceride uptake, which mainly was due to suppressed uptake of very low-density lipoproteins. An intraperitoneal glucose tolerance test indicated that the ORP-Tg mice have a significantly higher rate of glucose degradation. These findings suggest that overexpression of ORP150 in mice leads to abetalipoproteinemia with alteration of glucose and lipid metabolism. These data could provide clues for a therapeutic target of dyslipidemia or diabetes.

  16. Macrophages overexpressing Aire induce CD4+Foxp3+ T cells.

    PubMed

    Sun, Jitong; Fu, Haiying; Wu, Jing; Zhu, Wufei; Li, Yi; Yang, Wei

    2013-01-01

    Aire plays an important role in central immune tolerance by regulating the transcription of thousands of genes. However, the role of Aire in the peripheral immune system is poorly understood. Regulatory T (Treg) cells are considered essential for the maintenance of peripheral tolerance, but the effect of Aire on Treg cells in the peripheral immune system is currently unknown. In this study, we investigated the effects of macrophages overexpressing Aire on CD4+Foxp3+ Treg cells by co-culturing Aire-overexpressing RAW264.7 cells or their supernatant with splenocytes. The results show that macrophages overexpressing Aire enhanced the expression of Foxp3 mRNA and induced different subsets of Treg cells in splenocytes through cell-cell contact or a co-culture supernatants. TGF-β is a key molecule in the increases of CD4+CD45RA+Foxp3hi T cell and activating Treg (aTreg) levels observed following cell‑supernatant co-culturing. Subsets of Treg cells were induced by Aire-overexpressing macrophages, and the manipulation of Treg cells by the targeting of Aire may provide a method for the treatment of inflammatory or autoimmune diseases.

  17. POMC overexpression in the ventral tegmental area ameliorates dietary obesity.

    PubMed

    Andino, Lourdes M; Ryder, Daniel J; Shapiro, Alexandra; Matheny, Michael K; Zhang, Yi; Judge, Melanie K; Cheng, K Y; Tümer, Nihal; Scarpace, Philip J

    2011-08-01

    The activation of proopiomelanocortin (POMC) neurons in different regions of the brain, including the arcuate nucleus of the hypothalamus (ARC) and the nucleus of the solitary tract curtails feeding and attenuates body weight. In this study, we compared the effects of delivery of a recombinant adeno-associated viral (rAAV) construct encoding POMC to the ARC with delivery to the ventral tegmental area (VTA). F344×Brown Norway rats were high-fat (HF) fed for 14 days after which self-complementary rAAV constructs expressing either green fluorescent protein or the POMC gene were injected using coordinates targeting either the VTA or the ARC. Corresponding increased POMC levels were found at the predicted injection sites and subsequent α-melanocyte-stimulating hormone levels were observed. Food intake and body weight were measured for 4 months. Although caloric intake was unaltered by POMC overexpression, weight gain was tempered with POMC overexpression in either the VTA or the ARC compared with controls. There were parallel decreases in adipose tissue reserves. In addition, levels of oxygen consumption and brown adipose tissue uncoupling protein 1 were significantly elevated with POMC treatment in the VTA. Interestingly, tyrosine hydroxylase levels were increased in both the ARC and VTA with POMC overexpression in either the ARC or the VTA. In conclusion, these data indicate a role for POMC overexpression within the VTA reward center to combat HF-induced obesity.

  18. Control of cellulose biosynthesis by overexpression of a transcription factor

    DOEpatents

    Han, Kyung-Hwan; Ko, Jae-Heung; Kim, Won-Chan; Kim; , Joo-Yeol

    2017-05-16

    The invention relates to the over-expression of a transcription factor selected from the group consisting of MYB46, HAM1, HAM2, MYB112, WRKY11, ERF6, and any combination thereof in a plant, which can modulate and thereby modulating the cellulose content of the plant.

  19. Moesin overexpression is a unique biomarker of adenomyosis.

    PubMed

    Ohara, Rena; Michikami, Hiroo; Nakamura, Yuko; Sakata, Akiko; Sakashita, Shingo; Satomi, Kaishi; Shiba-Ishii, Aya; Kano, Junko; Yoshikawa, Hiroyuki; Noguchi, Masayuki

    2014-03-01

    Adenomyosis is characterized by extension of endometrial glands and stromal cells into the myometrium. Here we proved that 'moesin' is a unique biomarker of adenomyosis. We selected two cases of adenomyosis that had been surgically resected and fixed with formalin. Proteins were extracted from the infiltrating adenomyosis lesions and normal endometrium by tissue microdissection. The extracted proteins were examined using a LC-MS/MS system and the expression profiles of each region were compared. Two hundred and sixty proteins were detected, among which 73 were expressed more in adenomyosis than in normal endometrium. Among these proteins, we focused on overexpression of moesin in adenomyosis. Expression of moesin estimated semiquantitatively using an immunohistochemistry score was higher in adenomyosis than in normal endometrium. In particular, moesin was significanly overexpressed in stromal cells of adenomyosis than in those of normal endometrium. Relative to normal endometrium, moesin was also overexpressed at the RNA level in 9 of 14 cases of adenomyosis and at the protein level in all 14 cases. We also detected activated (phosphorylated) moesin in adenomyosis lesions. The present findings suggest that moesin is characteristically overexpressed and activated in adenomyosis, and that moesin activation may be related to extension of adenomyosis in the myometrium.

  20. Tumor Necrosis Factor–α Overexpression in Lung Disease

    PubMed Central

    Lundblad, Lennart K. A.; Thompson-Figueroa, John; Leclair, Timothy; Sullivan, Michael J.; Poynter, Matthew E.; Irvin, Charles G.; Bates, Jason H. T.

    2005-01-01

    Rationale: Tumor necrosis factor α (TNF-α) has been implicated as a key cytokine in many inflammatory lung diseases. These effects are currently unclear, because a transgenic mouse overexpressing TNF-α in the lung has been shown in separate studies to produce elements of both emphysema and pulmonary fibrosis. Objectives: We sought to elucidate the phenotypic effects of TNF-α overexpression in a mouse model. Measurements: We established the phenotype by measuring lung impedance and thoracic gas volume, and using micro–computed tomography and histology. Main Results: We found that airways resistance in this mouse was not different to control mice, but that lung tissue dampening, elastance, and hysteresivity were significantly elevated. Major heterogeneous abnormalities of the parenchyma were also apparent in histologic sections and in micro–computed tomography images of the lung. These changes included airspace enlargement, loss of small airspaces, increased collagen, and thickened pleural septa. We also found significant increases in lung and chest cavity volumes in the TNF-α–overexpressing mice. Conclusions: We conclude that TNF-α overexpression causes pathologic changes consistent with both emphysema and pulmonary fibrosis combined with a general lung inflammation, and consequently does not model any single human disease. Our study thus confirms the pleiotropic effects of TNF-α, which has been implicated in multiple inflammatory disorders, and underscores the necessity of using a wide range of investigative techniques to link gene expression and phenotype in animal models of disease. PMID:15805183

  1. Prion protein and Abeta-related synaptic toxicity impairment.

    PubMed

    Calella, Anna Maria; Farinelli, Mélissa; Nuvolone, Mario; Mirante, Osvaldo; Moos, Rita; Falsig, Jeppe; Mansuy, Isabelle M; Aguzzi, Adriano

    2010-08-01

    Alzheimer's disease (AD), the most common neurodegenerative disorder, goes along with extracellular amyloid-beta (Abeta) deposits. The cognitive decline observed during AD progression correlates with damaged spines, dendrites and synapses in hippocampus and cortex. Numerous studies have shown that Abeta oligomers, both synthetic and derived from cultures and AD brains, potently impair synaptic structure and functions. The cellular prion protein (PrP(C)) was proposed to mediate this effect. We report that ablation or overexpression of PrP(C) had no effect on the impairment of hippocampal synaptic plasticity in a transgenic model of AD. These findings challenge the role of PrP(C) as a mediator of Abeta toxicity.

  2. SUN2 Overexpression Deforms Nuclear Shape and Inhibits HIV

    PubMed Central

    Amraoui, Sonia; di Nunzio, Francesca; Kieffer, Camille; Porrot, Françoise; Opp, Silvana; Diaz-Griffero, Felipe; Casartelli, Nicoletta

    2016-01-01

    ABSTRACT In a previous screen of putative interferon-stimulated genes, SUN2 was shown to inhibit HIV-1 infection in an uncharacterized manner. SUN2 is an inner nuclear membrane protein belonging to the linker of nucleoskeleton and cytoskeleton complex. We have analyzed here the role of SUN2 in HIV infection. We report that in contrast to what was initially thought, SUN2 is not induced by type I interferon, and that SUN2 silencing does not modulate HIV infection. However, SUN2 overexpression in cell lines and in primary monocyte-derived dendritic cells inhibits the replication of HIV but not murine leukemia virus or chikungunya virus. We identified HIV-1 and HIV-2 strains that are unaffected by SUN2, suggesting that the effect is specific to particular viral components or cofactors. Intriguingly, SUN2 overexpression induces a multilobular flower-like nuclear shape that does not impact cell viability and is similar to that of cells isolated from patients with HTLV-I-associated adult T-cell leukemia or with progeria. Nuclear shape changes and HIV inhibition both mapped to the nucleoplasmic domain of SUN2 that interacts with the nuclear lamina. This block to HIV replication occurs between reverse transcription and nuclear entry, and passaging experiments selected for a single-amino-acid change in capsid (CA) that leads to resistance to overexpressed SUN2. Furthermore, using chemical inhibition or silencing of cyclophilin A (CypA), as well as CA mutant viruses, we implicated CypA in the SUN2-imposed block to HIV infection. Our results demonstrate that SUN2 overexpression perturbs both nuclear shape and early events of HIV infection. IMPORTANCE Cells encode proteins that interfere with viral replication, a number of which have been identified in overexpression screens. SUN2 is a nuclear membrane protein that was shown to inhibit HIV infection in such a screen, but how it blocked HIV infection was not known. We show that SUN2 overexpression blocks the infection of certain

  3. DEK over-expression promotes mitotic defects and micronucleus formation.

    PubMed

    Matrka, Marie C; Hennigan, Robert F; Kappes, Ferdinand; DeLay, Monica L; Lambert, Paul F; Aronow, Bruce J; Wells, Susanne I

    2015-01-01

    The DEK gene encodes a nuclear protein that binds chromatin and is involved in various fundamental nuclear processes including transcription, RNA splicing, DNA replication and DNA repair. Several cancer types characteristically over-express DEK at the earliest stages of transformation. In order to explore relevant mechanisms whereby DEK supports oncogenicity, we utilized cancer databases to identify gene transcripts whose expression patterns are tightly correlated with that of DEK. We identified an enrichment of genes involved in mitosis and thus investigated the regulation and possible function of DEK in cell division. Immunofluorescence analyses revealed that DEK dissociates from DNA in early prophase and re-associates with DNA during telophase in human keratinocytes. Mitotic cell populations displayed a sharp reduction in DEK protein levels compared to the corresponding interphase population, suggesting DEK may be degraded or otherwise removed from the cell prior to mitosis. Interestingly, DEK overexpression stimulated its own aberrant association with chromatin throughout mitosis. Furthermore, DEK co-localized with anaphase bridges, chromosome fragments, and micronuclei, suggesting a specific association with mitotically defective chromosomes. We found that DEK over-expression in both non-transformed and transformed cells is sufficient to stimulate micronucleus formation. These data support a model wherein normal chromosomal clearance of DEK is required for maintenance of high fidelity cell division and chromosomal integrity. Therefore, the overexpression of DEK and its incomplete removal from mitotic chromosomes promotes genomic instability through the generation of genetically abnormal daughter cells. Consequently, DEK over-expression may be involved in the initial steps of developing oncogenic mutations in cells leading to cancer initiation.

  4. AAV2-mediated follistatin overexpression induces ovine primary myoblasts proliferation.

    PubMed

    Nazari, Mahmood; Salabi, Fatemeh; Zhang, Li; Zhao, Fuping; Wei, Caihong; Du, Lixin

    2014-10-21

    Follistatin (FST) has been shown to bind to some TGF-β family members and can function as a potent myostatin (MSTN) antagonist. Recent studies have revealed that over-expression of FST by adeno-associated viruses increases muscle growth in mice, humans and nonhuman primates. In the present study, to determine the effect of FST on ovine primary myoblast (OPM) proliferation, FST was over-expressed using an adeno-associated virus serotype 2 (AAV 2) vector. Western blot results showed that AAV induced the expression of FST protein in transduced OPM cells. Real-time quantitative PCR results indicated that over-expression of FST resulted in a dramatic increase in Akt I and CDK2 expression and a decrease in p21 expression. Moreover, cell cycle analysis confirmed that FST down-regulated p21, a CDK inhibitor, and increased the level of CDK2 expression in OPM cells. Hence, follistatin positively regulated the G1 to S progression. Our results showed that FST induced proliferation through a down-regulation of p21, as only the p21 expression level was down-regulated as a result of FST over-expression in myoblasts, whereas no change was observed in the level of p57 expression. These results expanded our understanding of the regulation mechanism of FST in ovine primary myoblasts. Our results provide the first evidence that the AAV viral system can be used for gene transfer in ovine myoblast cells. Moreover, the results showed that an AAV vector can successfully induce the expression of FST in OPM cells in vitro. These findings demonstrated that FST over-expression induces proliferation through a down-regulation of the p21 gene under proliferating conditions.

  5. Simultaneous overexpression of Oct4 and Nanog abrogates terminal myogenesis.

    PubMed

    Lang, Kuan Chih; Lin, I Hsuan; Teng, Han Fang; Huang, Yi Cheng; Li, Chung Leung; Tang, Kam Tsun; Chen, Shen Liang

    2009-07-01

    Oct4 and Nanog are two embryonic stem (ES) cell-specific transcription factors that play critical roles in the maintenance of ES cell pluripotency. In this study, we investigated the effects of Oct4 and Nanog expression on the differentiation state of myogenic cells, which is sustained by a strong positive feedback loop. Oct4 and Nanog, either independently or simultaneously, were overexpressed in C2C12 myoblasts, and the expression of myogenic lineage-specific genes and terminal differentiation was observed by RT-PCR. Overexpression of Oct4 in C2C12 cultures repressed, while exogenous Nanog did not significantly alter C2C12 terminal differentiation. The expression of Pax7 was reduced in all Oct4-overexpressing myoblasts, and we identified a major Oct4-binding site in the Pax7 promoter. Simultaneous expression of Oct4 and Nanog in myoblasts inhibited the formation of myotubes, concomitant with a reduction in the endogenous levels of hallmark myogenic markers. Furthermore, overexpression of Oct4 and Nanog induced the expression of their endogenous counterparts along with the expression of Sox2. Using mammalian two-hybrid assays, we confirmed that Oct4 functions as a transcriptional repressor whereas Nanog functions as a transcriptional activator during muscle terminal differentiation. Importantly, in nonobese diabetic (NOD) severe combined immunodeficiency (SCID) mice, the pluripotency of C2C12 cells was conferred by overexpression of Oct4 and Nanog. These results suggest that Oct4 in cooperation with Nanog strongly suppresses the myogenic differentiation program and promotes pluripotency in myoblasts.

  6. Grammatical Impairments in PPA

    PubMed Central

    Thompson, Cynthia K.; Mack, Jennifer E.

    2015-01-01

    Background Grammatical impairments are commonly observed in the agrammatic subtype of primary progressive aphasia (PPA-G), whereas grammatical processing is relatively preserved in logopenic (PPA-L) and semantic (PPA-S) subtypes. Aims We review research on grammatical deficits in PPA and associated neural mechanisms, with discussion focused on production and comprehension of four aspects of morphosyntactic structure: grammatical morphology, functional categories, verbs and verb argument structure, and complex syntactic structures. We also address assessment of grammatical deficits in PPA, with emphasis on behavioral tests of grammatical processing. Finally, we address research examining the effects of treatment for progressive grammatical impairments. Main Contribution PPA-G is associated with grammatical deficits that are evident across linguistic domains in both production and comprehension. PPA-G is associated with damage to regions including the left inferior frontal gyrus (IFG) and dorsal white matter tracts, which have been linked to impaired comprehension and production of complex sentences. Detailing grammatical deficits in PPA is important for estimating the trajectory of language decline and associated neuropathology. We, therefore, highlight several new assessment tools for examining different aspects of morphosyntactic processing in PPA. Conclusions Individuals with PPA-G present with agrammatic deficit patterns distinct from those associated with PPA-L and PPA-S, but similar to those seen in agrammatism resulting from stroke, and patterns of cortical atrophy and white matter changes associated with PPA-G have been identified. Methods for clinical evaluation of agrammatism, focusing on comprehension and production of grammatical morphology, functional categories, verbs and verb argument structure, and complex syntactic structures are recommended and tools for this are emerging in the literature. Further research is needed to investigate the real

  7. Syndecan-4 shedding impairs macrovascular angiogenesis in diabetes mellitus

    SciTech Connect

    Li, Ran; Xie, Jun; Wu, Han; Li, Guannan; Chen, Jianzhou; Chen, Qinhua; Wang, Lian; Xu, Biao

    2016-05-20

    Purpose: Syndecan-4 (synd4) is a ubiquitous heparan sulfate proteoglycan cell surface receptor that modulates cell proliferation, migration, mechanotransduction, and endocytosis. The extracellular domain of synd4 sheds heavily in acute inflammation, but the shedding of synd4 in chronic inflammation, such as diabetes mellitus (DM), is still undefined. We investigated the alterations of synd4 endothelial expression in DM and the influence of impaired synd4 signaling on angiogenesis in human umbilical vein endothelial cells (HUVECs), diabetic rats, synd4 null mice, and db/db mice. Material and methods: HUVECs were incubated with advanced glycation end products (AGEs). Western blot analysis was used to determine synd4 protein expression and ELISA was used to detect soluble synd4 fragments. The concentration of synd4 in the aortic endothelia of diabetic rats was detected by immunohistochemical staining. Aortic ring assays were performed to study the process of angiogenesis in the diabetic rats and in synd4 null and db/db mice. Recombinant adenoviruses containing the synd4 gene or null were constructed to enhance synd4 aortic expression in db/db mice. Results: Western blot analysis showed decreased expression of the synd4 extracellular domain in HUVECs, and ELISA detected increased soluble fragments of synd4 in the media. Synd4 endothelial expression in the aortas of diabetic rats was decreased. Aortic ring assay indicated impaired angiogenesis in synd4 null and db/db mice, which was partially reversed by synd4 overexpression in db/db mice. Conclusion: Synd4 shedding from vascular endothelial cells played an important role in the diabetes-related impairment of angiogenesis. -- Highlights: •Synd4 shedding from endothelial cells is accelerated under the stimulation of AGEs. •Extracellular domain of synd4 is diminished in the endothelium of DM rats. •Aortic rings of synd4 null mice showed impaired angiogenesis. •Overexpression of synd4 partly rescues macrovascular

  8. Chemistry for the Visually Impaired.

    ERIC Educational Resources Information Center

    Ratliff, Judy L.

    1997-01-01

    Discusses modifications to general education or introductory chemistry courses that allow visually impaired students to participate productively. Describes a strategy for teaching about elements and density, and the construction of a conductivity tester for visually impaired students. (JRH)

  9. Impaired Consciousness in Epilepsy

    PubMed Central

    Blumenfeld, Hal

    2013-01-01

    Consciousness is essential to normal human life. In epileptic seizures consciousness is often transiently lost making it impossible for the individual to experience or respond. This has huge consequences for safety, productivity, emotional health and quality of life. To prevent impaired consciousness in epilepsy it is necessary to understand the mechanisms leading to brain dysfunction during seizures. Normally the “consciousness system”—a specialized set of cortical-subcortical structures—maintains alertness, attention and awareness. Recent advances in neuroimaging, electrophysiology and prospective behavioral testing have shed new light on how epileptic seizures disrupt the consciousness system. Diverse seizure types including absence, generalized tonic-clonic and complex partial seizures converge on the same set of anatomical structures through different mechanisms to disrupt consciousness. Understanding these mechanisms may lead to improved treatment strategies to prevent impaired consciousness and improve quality of life in people with epilepsy. PMID:22898735

  10. Overexpression of alpha-synuclein at non-toxic levels increases dopaminergic cell death induced by copper exposure via modulation of protein degradation pathways.

    PubMed

    Anandhan, Annadurai; Rodriguez-Rocha, Humberto; Bohovych, Iryna; Griggs, Amy M; Zavala-Flores, Laura; Reyes-Reyes, Elsa M; Seravalli, Javier; Stanciu, Lia A; Lee, Jaekwon; Rochet, Jean-Christophe; Khalimonchuk, Oleh; Franco, Rodrigo

    2015-09-01

    Gene multiplications or point mutations in alpha (α)-synuclein are associated with familial and sporadic Parkinson's disease (PD). An increase in copper (Cu) levels has been reported in the cerebrospinal fluid and blood of PD patients, while occupational exposure to Cu has been suggested to augment the risk to develop PD. We aimed to elucidate the mechanisms by which α-synuclein and Cu regulate dopaminergic cell death. Short-term overexpression of wild type (WT) or mutant A53T α-synuclein had no toxic effect in human dopaminergic cells and primary midbrain cultures, but it exerted a synergistic effect on Cu-induced cell death. Cell death induced by Cu was potentiated by overexpression of the Cu transporter protein 1 (Ctr1) and depletion of intracellular glutathione (GSH) indicating that the toxic effects of Cu are linked to alterations in its intracellular homeostasis. Using the redox sensor roGFP, we demonstrated that Cu-induced oxidative stress was primarily localized in the cytosol and not in the mitochondria. However, α-synuclein overexpression had no effect on Cu-induced oxidative stress. WT or A53T α-synuclein overexpression exacerbated Cu toxicity in dopaminergic and yeast cells in the absence of α-synuclein aggregation. Cu increased autophagic flux and protein ubiquitination. Impairment of autophagy by overexpression of a dominant negative Atg5 form or inhibition of the ubiquitin/proteasome system (UPS) with MG132 enhanced Cu-induced cell death. However, only inhibition of the UPS stimulated the synergistic toxic effects of Cu and α-synuclein overexpression. Our results demonstrate that α-synuclein stimulates Cu toxicity in dopaminergic cells independent from its aggregation via modulation of protein degradation pathways.

  11. Overexpression of alpha-synuclein at non-toxic levels increases dopaminergic cell death induced by copper exposure via modulation of protein degradation pathways

    PubMed Central

    Anandhan, Annadurai; Rodriguez-Rocha, Humberto; Bohovych, Iryna; Griggs, Amy M.; Zavala-Flores, Laura; Reyes-Reyes, Elsa M.; Seravalli, Javier; Stanciu, Lia A.; Lee, Jaekwon; Rochet, Jean-Christophe; Khalimonchuk, Oleh; Franco, Rodrigo

    2014-01-01

    Gene multiplications or point mutations in alpha (α)-synuclein are associated with familial and sporadic Parkinson’s disease (PD). An increase in copper (Cu) levels has been reported in the cerebrospinal fluid and blood of PD patients, while occupational exposure to Cu has been suggested to augment the risk to develop PD. We aimed to elucidate the mechanisms by which α-synuclein and Cu regulate dopaminergic cell death. Short-term overexpression of WT or A53T α-synuclein had no toxic effect in human dopaminergic cells and primary midbrain cultures, but it exerted a synergistic effect on Cu-induced cell death. Cell death induced by Cu was potentiated by overexpression of the Cu transporter protein 1 (Ctr1) and depletion of intracellular glutathione (GSH) indicating that the toxic effects of Cu are linked to alterations in its intracellular homeostasis. Using the redox sensor roGFP, we demonstrated that Cu-induced oxidative stress was primarily localized in the cytosol and not in the mitochondria. However, α-synuclein overexpression had no effect on Cu-induced oxidative stress. WT or A53T α-synuclein overexpression exacerbated Cu toxicity in dopaminergic cells and yeast in the absence of α-synuclein aggregation. Cu increased autophagic flux and protein ubiquitination. Impairment of autophagy by overexpression of a dominant negative Atg5 form or inhibition of the ubiquitin/proteasome system (UPS) with MG132 enhanced Cu-induced cell death. However, only inhibition of the UPS stimulated the synergistic toxic effects of Cu and α-synuclein overexpression. Our results demonstrate that α-synuclein stimulates Cu toxicity in dopaminergic cells independent from its aggregation via modulation of protein degradation pathways. PMID:25497688

  12. Age-Related Sensory Impairments and Risk of Cognitive Impairment.

    PubMed

    Fischer, Mary E; Cruickshanks, Karen J; Schubert, Carla R; Pinto, Alex A; Carlsson, Cynthia M; Klein, Barbara E K; Klein, Ronald; Tweed, Ted S

    2016-10-01

    To evaluate the associations between sensory impairments and 10-year risk of cognitive impairment. The Epidemiology of Hearing Loss Study (EHLS), a longitudinal, population-based study of aging in the Beaver Dam, Wisconsin community. Baseline examinations were conducted in 1993 and follow-up examinations have been conducted every 5 years. General community. EHLS members without cognitive impairment at EHLS-2 (1998-2000). There were 1,884 participants (mean age 66.7) with complete EHLS-2 sensory data and follow-up information. Cognitive impairment was defined as a Mini-Mental State Examination score of <24 or history of dementia or Alzheimer's disease. Hearing impairment was a pure-tone average of hearing thresholds (0.5, 1, 2, 4 kHz) of >25 dB hearing level in either ear, visual impairment was a Pelli-Robson contrast sensitivity of <1.55 log units in the better eye, and olfactory impairment was a San Diego Odor Identification Test score of <6. Hearing, visual, and olfactory impairment were independently associated with cognitive impairment risk (hearing: hazard ratio (HR) = 1.90, 95% confidence interval (CI) = 1.11-3.26; vision: HR = 2.05, 95% CI = 1.24-3.38; olfaction: HR = 3.92, 95% CI = 2.45-6.26)). Nevertheless, 85% of participants with hearing impairment, 81% with visual impairment, and 76% with olfactory impairment did not develop cognitive impairment during follow-up. The relationship between sensory impairment and cognitive impairment was not unique to one sensory system, suggesting that sensorineural health may be a marker of brain aging. The development of a combined sensorineurocognitive measure may be useful in uncovering mechanisms of healthy brain aging. © 2016, Copyright the Authors Journal compilation © 2016, The American Geriatrics Society.

  13. NO counterbalances HO-1 overexpression-induced acceleration of hepatocyte proliferation in mice.

    PubMed

    Schuett, Harald; Eipel, Christian; Maletzki, Claudia; Menger, Michael D; Vollmar, Brigitte

    2007-06-01

    The trigger for liver regeneration, including shear stress, has been the subject of ongoing debate. Blood vessel-derived gaseous molecules carbon monoxide (CO) and nitric oxide (NO) regulate vascular tone and play an important role in liver regeneration. In heme oxygenase-1 (HO-1) transgenic mice, it has been shown that CO-mediated impairment of vasorelaxation is an NO-dependent event. We therefore studied liver regeneration in HO-1 overexpressing animals in dependency of NO availability. Mice were subjected to (2/3) hepatectomy and were treated with either cobalt protoporphyrin-IX for induction of CO-liberating HO-1, N(omega)-nitro-L-arginine methyl ester (L-NAME) for blockade of NO synthase (NOS) or both. Application of molsidomine in L-NAME treated animals served for resubstitution of NO. Vehicle-treated animals served as respective control animals. We examined 5-bromo-2'-deoxyuridine incorporation and proliferating cell nuclear antigen expression as well as HO-1 and NOS-2 protein levels. Intrahepatic red blood cell velocity and volumetric blood flow were evaluated by in vivo fluorescence microscopy as indicators for microvascular shear stress. Hepatic regeneration remained unaffected by L-NAME application for NOS blockade. However, NOS blockade in HO-1 induced animals caused increased 5-bromo-2'-deoxyuridine and proliferating cell nuclear antigen measures of liver regeneration. In parallel, these animals revealed increased velocities and volumetric blood flow in the terminal afferent vessels and postsinusoidal venules. These local hemodynamic changes including enhanced hepatocyte proliferation could be reversed by NO liberation via molsidomine. The present findings stress the role of NO to counterbalance vascular tone in HO-1 overexpressing animals for maintenance of adequate perfusion and salutary shear force within the hepatic microvasculature upon liver resection.

  14. Overexpression of glutathione peroxidase attenuates myocardial remodeling and preserves diastolic function in diabetic heart.

    PubMed

    Matsushima, Shouji; Kinugawa, Shintaro; Ide, Tomomi; Matsusaka, Hidenori; Inoue, Naoki; Ohta, Yukihiro; Yokota, Takashi; Sunagawa, Kenji; Tsutsui, Hiroyuki

    2006-11-01

    Oxidative stress plays an important role in the structural and functional abnormalities of diabetic heart. Glutathione peroxidase (GSHPx) is a critical antioxidant enzyme that removes H(2)O(2) in both the cytosol and mitochondia. We hypothesized that the overexpression of GSHPx gene could attenuate left ventricular (LV) remodeling in diabetes mellitus (DM). We induced DM by injection of streptozotocin (160 mg/kg ip) in male GSHPx transgenic mice (TG+DM) and nontransgenic wildtype littermates (WT+DM). GSHPx activity was higher in the hearts of TG mice compared with WT mice, with no significant changes in other antioxidant enzymes. LV thiobarbituric acid-reactive substances measured in TG+DM at 8 wk were significantly lower than those in WT+DM (58 +/- 3 vs. 71 +/- 5 nmol/g, P < 0.05). Heart rate and aortic blood pressure were comparable between groups. Systolic function was preserved normal in WT+DM and TG+DM mice. In contrast, diastolic function was impaired in WT+DM and was improved in TG+DM as assessed by the deceleration time of peak velocity of transmitral diastolic flow and the time needed for relaxation of 50% maximal LV pressure to baseline value (tau; 13.5 +/- 1.2 vs. 8.9 +/- 0.7 ms, P < 0.01). The TG+DM values were comparable with those of WT+Control (tau; 7.8 +/- 0.2 ms). Improvement of LV diastolic function was accompanied by the attenuation of myocyte hypertrophy, interstitial fibrosis, and apoptosis. Overexpression of GSHPx gene ameliorated LV remodeling and diastolic dysfunction in DM. Therapies designed to interfere with oxidative stress might be beneficial to prevent cardiac abnormalities in DM.

  15. Dopamine D2 receptor over-expression alters behavior and physiology in Drd2-EGFP mice

    PubMed Central

    Kramer, Paul F.; Christensen, Christine H.; Hazelwood, Lisa A.; Dobi, Alice; Bock, Roland; Sibley, David R.; Mateo, Yolanda; Alvarez, Veronica A.

    2011-01-01

    BAC transgenic mice expressing the fluorescent reporter protein EGFP under the control of the D1 and D2 dopamine receptor promoters (Drd1-EGFP and Drd2-EGFP) have been widely used to study striatal function and have contributed to our understanding of the physiological and pathological function of the basal ganglia. These tools were produced and promptly made available to address questions in a cell-specific manner that has transformed the way we frame hypotheses in neuroscience. However, these mice have not been fully characterized until now. We found that Drd2-EGFP mice display a ~40% increase in membrane expression of the dopamine D2 receptor (D2R) and a two-fold increase in D2R mRNA levels in the striatum when compared to wild-type and Drd1-EGFP mice D2R over-expression was accompanied by behavioral hypersensitivity to D2R-like agonists, as well as enhanced electrophysiological responses to D2R activation in midbrain dopaminergic neurons. DA transients evoked by stimulation in the nucleus accumbens showed slower clearance in Drd2-EGFP mice and cocaine actions on DA clearance were impaired in these mice. Thus, it was not surprising to find that Drd2-EGFP mice were hyperactive when exposed to a novel environment and locomotion was suppressed by acute cocaine administration. All together, this study demonstrates that Drd2-EGFP mice over-express D2R and have altered dopaminergic signaling that fundamentally differentiates them from wild-type and Drd1-EGFP mice. PMID:21209197

  16. Butalbital and driving impairment.

    PubMed

    Yeakel, Jillian K; Logan, Barry K

    2013-07-01

    Butalbital (Fiorinal(®)), used in the treatment of migraines and muscle pain, is the most commonly encountered barbiturate in impaired driving cases. It has central nervous system (CNS) depressant properties, including sedation, drowsiness, and feelings of intoxication, which can contribute to driving impairment. Twenty-six driving under the influence cases are reviewed including results from field sobriety tests and toxicology testing. Blood samples were screened using enzyme multiplied immunoassay technique immunoassay, and the presence of butalbital was confirmed and quantified using gas chromatography/mass spectrometry, gas chromatography with flame ionization detection, or gas chromatography nitrogen/phosphorus detection. Butalbital concentrations ranged from 1.0 to 30.2 mg/L, with a mean and median of 16.0 mg/L. General impairment indicators in these cases included horizontal and vertical nystagmus, lack of convergence, poor motor coordination, and balance and speech problems, which are common to CNS depressant intoxication, similar to that associated with alcohol. These findings indicate the importance of toxicological testing for butalbital in cases where CNS depressants are indicated. © 2013 American Academy of Forensic Sciences.

  17. Osteoblast-Specific Krm2 Overexpression and Lrp5 Deficiency Have Different Effects on Fracture Healing in Mice

    PubMed Central

    Liedert, Astrid; Röntgen, Viktoria; Schinke, Thorsten; Benisch, Peggy; Ebert, Regina; Jakob, Franz; Klein-Hitpass, Ludger; Lennerz, Jochen K.; Amling, Michael; Ignatius, Anita

    2014-01-01

    The canonical Wnt/β-catenin pathway plays a key role in the regulation of bone remodeling in mice and humans. Two transmembrane proteins that are involved in decreasing the activity of this pathway by binding to extracellular antagonists, such as Dickkopf 1 (Dkk1), are the low-density lipoprotein receptor related protein 5 (Lrp5) and Kremen 2 (Krm2). Lrp 5 deficiency (Lrp5−/−) as well as osteoblast-specific overexpression of Krm2 in mice (Col1a1-Krm2) result in severe osteoporosis occurring at young age. In this study, we analyzed the influence of Lrp5 deficiency and osteoblast-specific overexpression of Krm2 on fracture healing in mice using flexible and semi-rigid fracture fixation. We demonstrated that fracture healing was highly impaired in both mouse genotypes, but that impairment was more severe in Col1a1-Krm2 than in Lrp5−/− mice and particularly evident in mice in which the more flexible fixation was used. Bone formation was more reduced in Col1a1-Krm2 than in Lrp5−/− mice, whereas osteoclast number was similarly increased in both genotypes in comparison with wild-type mice. Using microarray analysis we identified reduced expression of genes mainly involved in osteogenesis that seemed to be responsible for the observed stronger impairment of healing in Col1a1-Krm2 mice. In line with these findings, we detected decreased expression of sphingomyelin phosphodiesterase 3 (Smpd3) and less active β-catenin in the calli of Col1a1-Krm2 mice. Since Krm2 seems to play a significant role in regulating bone formation during fracture healing, antagonizing KRM2 might be a therapeutic option to improve fracture healing under compromised conditions, such as osteoporosis. PMID:25061805

  18. Neurological Impairment: Nomenclature and Consequences.

    ERIC Educational Resources Information Center

    Spears, Catherine E.; Weber, Robert E.

    Neurological impairment as discussed includes a range of disabilities referred to as neurological impairment: minimal brain dysfunction/damage, developmental disability, perceptual handicap, learning disability, hyperkinetic behavioral syndrome, and others. Defined are causes of neurological impairment and methods of diagnosis. Symptoms…

  19. Assessment of Hearing Impaired Youth.

    ERIC Educational Resources Information Center

    Hicks, Doin E., Ed.; And Others

    1980-01-01

    The issue of Directions contains 11 articles on assessment of hearing impaired individuals. Entries have the following titles and authors: "Classroom Assessment Techniques for Hearing Impaired Students--A Literature Review" (B. McKee, M. Hausknecht); "Informal Assessment of Hearing Impaired Students In the Classroom" (B. Culhane, R. Hein);…

  20. Assessment of Hearing Impaired Youth.

    ERIC Educational Resources Information Center

    Hicks, Doin E., Ed.; And Others

    1980-01-01

    The issue of Directions contains 11 articles on assessment of hearing impaired individuals. Entries have the following titles and authors: "Classroom Assessment Techniques for Hearing Impaired Students--A Literature Review" (B. McKee, M. Hausknecht); "Informal Assessment of Hearing Impaired Students In the Classroom" (B. Culhane, R. Hein);…

  1. Involvement of pregnane X receptor in the impaired glucose utilization induced by atorvastatin in hepatocytes.

    PubMed

    Ling, Zhaoli; Shu, Nan; Xu, Ping; Wang, Fan; Zhong, Zeyu; Sun, Binbin; Li, Feng; Zhang, Mian; Zhao, Kaijing; Tang, Xiange; Wang, Zhongjian; Zhu, Liang; Liu, Li; Liu, Xiaodong

    2016-01-15

    Accumulating evidences demonstrated that statins impaired glucose utilization. This study was aimed to investigate whether PXR was involved in the atorvastatin-impaired glucose utilization. Rifampicin/PCN served as PXR activator control. Glucose utilization, glucose uptake, protein levels of GLUT2, GCK, PDK2, PEPCK1 and G6Pase in HepG2 cells were measured. PXR inhibitors, PXR overexpression and PXR siRNA were applied to verify the role of PXR in atorvastatin-impaired glucose utilization in cells. Hypercholesterolemia rats induced by high fat diet feeding, orally received atorvastatin (5 and 10 mg/kg), pravastatin (10 mg/kg) for 14 days, or intraperitoneally received PCN (35 mg/kg) for 4 days. Results showed that glucose utilization was markedly inhibited by atorvastatin, simvastatin, pitavastatin, lovastatin and rifampicin. Neither rosuvastatin nor pravastatin showed the similar effect. Atorvastatin and pravastatin were selected for the following study. Atorvastatin and rifampicin significantly inhibited glucose uptake and down-regulated GLUT2 and GCK expressions. Similarly, overexpressed PXR significantly down-regulated GLUT2 and GCK expressions and impaired glucose utilization. Ketoconazole and resveratrol attenuated the impaired glucose utilization by atorvastatin and rifampicin in both parental and overexpressed PXR cells. PXR knockdown significantly up-regulated GLUT2 and GCK proteins and abolished the decreased glucose consumption and uptake by atorvastatin and rifampicin. Animal experiments showed that atorvastatin and PCN significantly elicited postprandial hyperglycemia, leading to increase in glucose AUC. Expressions of GLUT2 and GCK in rat livers were markedly down-regulated by atorvastatin and PCN. In conclusion, atorvastatin impaired glucose utilization in hepatocytes via repressing GLUT2 and GCK expressions, which may be partly due to PXR activation.

  2. Peroxidase-Generated Apoplastic ROS Impair Cuticle Integrity and Contribute to DAMP-Elicited Defenses.

    PubMed

    Survila, Mantas; Davidsson, Pär R; Pennanen, Ville; Kariola, Tarja; Broberg, Martin; Sipari, Nina; Heino, Pekka; Palva, Erkki T

    2016-01-01

    Cuticular defects trigger a battery of reactions including enhanced reactive oxygen species (ROS) production and resistance to necrotrophic pathogens. However, the source of ROS generated by such impaired cuticles has remained elusive. Here, we report the characterization of Arabidopsis thaliana ohy1 mutant, a Peroxidase 57 (PER57) - overexpressing line that demonstrates enhanced defense responses that result both from increased accumulation of ROS and permeability of the leaf cuticle. The ohy1 mutant was identified in a screen of A. thaliana seedlings for oligogalacturonides (OGs) insensitive/hypersensitive mutants that exhibit altered growth retardation in response to exogenous OGs. Mutants impaired in OG sensitivity were analyzed for disease resistance/susceptibility to the necrotrophic phytopathogens Botrytis cinerea and Pectobacterium carotovorum. In the ohy1 line, the hypersensitivity to OGs was associated with resistance to the tested pathogens. This PER57 overexpressing line exhibited a significantly more permeable leaf cuticle than wild-type plants and this phenotype could be recapitulated by overexpressing other class III peroxidases. Such peroxidase overexpression was accompanied by the suppressed expression of cutin biosynthesis genes and the enhanced expression of genes associated with OG-signaling. Application of ABA completely removed ROS, restored the expression of genes associated with cuticle biosynthesis and led to decreased permeability of the leaf cuticle, and finally, abolished immunity to B. cinerea. Our work demonstrates that increased peroxidase activity increases permeability of the leaf cuticle. The loss of cuticle integrity primes plant defenses to necrotrophic pathogens via the activation of DAMP-responses.

  3. Peroxidase-Generated Apoplastic ROS Impair Cuticle Integrity and Contribute to DAMP-Elicited Defenses

    PubMed Central

    Survila, Mantas; Davidsson, Pär R.; Pennanen, Ville; Kariola, Tarja; Broberg, Martin; Sipari, Nina; Heino, Pekka; Palva, Erkki T.

    2016-01-01

    Cuticular defects trigger a battery of reactions including enhanced reactive oxygen species (ROS) production and resistance to necrotrophic pathogens. However, the source of ROS generated by such impaired cuticles has remained elusive. Here, we report the characterization of Arabidopsis thaliana ohy1 mutant, a Peroxidase 57 (PER57) – overexpressing line that demonstrates enhanced defense responses that result both from increased accumulation of ROS and permeability of the leaf cuticle. The ohy1 mutant was identified in a screen of A. thaliana seedlings for oligogalacturonides (OGs) insensitive/hypersensitive mutants that exhibit altered growth retardation in response to exogenous OGs. Mutants impaired in OG sensitivity were analyzed for disease resistance/susceptibility to the necrotrophic phytopathogens Botrytis cinerea and Pectobacterium carotovorum. In the ohy1 line, the hypersensitivity to OGs was associated with resistance to the tested pathogens. This PER57 overexpressing line exhibited a significantly more permeable leaf cuticle than wild-type plants and this phenotype could be recapitulated by overexpressing other class III peroxidases. Such peroxidase overexpression was accompanied by the suppressed expression of cutin biosynthesis genes and the enhanced expression of genes associated with OG-signaling. Application of ABA completely removed ROS, restored the expression of genes associated with cuticle biosynthesis and led to decreased permeability of the leaf cuticle, and finally, abolished immunity to B. cinerea. Our work demonstrates that increased peroxidase activity increases permeability of the leaf cuticle. The loss of cuticle integrity primes plant defenses to necrotrophic pathogens via the activation of DAMP-responses. PMID:28066496

  4. Cardiac-Specific Overexpression of HIF-1α Prevents Deterioration of Glycolytic Pathway and Cardiac Remodeling in Streptozotocin-Induced Diabetic Mice

    PubMed Central

    Xue, Wanli; Cai, Lu; Tan, Yi; Thistlethwaite, Patricia; Kang, Y. James; Li, Xiaokun; Feng, Wenke

    2010-01-01

    Defective glycolysis and angiogenesis in the heart of diabetic patients and in experimental diabetic animal models have been reported. The aim of this study was to determine whether overexpression of hypoxia-inducible factor (HIF)-1α protects from myocardial injury in diabetic mice by increasing myocardial glycolysis and angiogenesis. Cardiac-specific HIF-1α–overexpressing transgenic and age-matched wild-type control mice were treated with streptozotocin to induce diabetes. Changes in glucose transporters, glycolytic enzymes, angiogenic factors and cardiac morphology were examined in the hearts by real-time RT-PCR, Western blotting, enzymatic assay, and histological assays. HIF-1α overexpression elevated hexokinase II (HK-II) protein level and total HK activity in nondiabetic heart and prevented the decreases in HK-II mRNA, protein, and total HK activity in diabetic heart. In addition, the reduction of glucose transporter I, but not glucose transporter 4, was restored in HIF transgenic mouse heart along with a recovery of myocardium ATP production. HIF-1α overexpression also normalized diabetes-reduced vascular endothelial growth factor concentration along with a sustained myocardial capillary density and an inhibition of cardiomyocyte hypertrophy and cardiac fibrosis. Therefore, elevation of HIF-1α provides a cardiac protection from diabetic-induced impairment in glucose metabolism and angiogenesis via up-regulation of HIF-1 target genes. PMID:20566749

  5. The Angiogenic Secretome in VEGF overexpressing Breast Cancer Xenografts

    PubMed Central

    Dore-Savard, Louis; Lee, Esak; Kakkad, Samata; Popel, Aleksander S.; Bhujwalla, Zaver M.

    2016-01-01

    The plasticity of cancer cells and the fluidity of the tumor microenvironment continue to present major challenges in the comprehensive understanding of cancer that is essential to design effective treatments. The tumor interstitial fluid (TIF) encompasses the secretome and holds the key to several of the phenotypic characteristics of cancer. Difficulties in sampling this fluid have resulted in limited characterization of its components. Here we have sampled TIF from triple negative and estrogen receptor (ER)-positive human breast tumor xenografts with or without VEGF overexpression. Angiogenesis-related factors were characterized in the TIF and plasma, to understand the relationship between the TIF and plasma secretomes. Clear differences were observed between the TIF and plasma angiogenic secretomes in triple negative MDA-MB-231 breast cancer xenografts compared to ER-positive MCF-7 xenografts with or without VEGF overexpression that provide new insights into TIF components and the role of VEGF in modifying the angiogenic secretome. PMID:27995973

  6. Overexpression of esterase D in kidney from trisomy 13 fetuses.

    PubMed Central

    Loughna, S; Bennett, P; Gau, G; Nicolaides, K; Blunt, S; Moore, G

    1993-01-01

    Human trisomy 13 (Patau syndrome) occurs in approximately 1 in 5,000 live births. It is compatible with life, but prolonged survival is rare. Anomalies often involve the urogenital, cardiac, craniofacial, and central nervous systems. It is possible that these abnormalities may be due to the overexpression of developmentally important genes on chromosome 13. The expression of esterase D (localized to chromosome 13q14.11) has been investigated in both muscle and kidney from trisomy 13 fetuses and has been compared with normal age- and sex-matched fetal tissues, by using northern analysis. More than a twofold increase in expression of esterase D was found in the kidney of two trisomy 13 fetuses, with normal levels in a third. Overexpression was not seen in the muscle tissues from these fetuses. Images Figure 1 Figure 2 Figure 3 PMID:8213811

  7. Overexpression of ankyrin1 promotes pancreatic cancer cell growth

    PubMed Central

    Omura, Noriyuki; Mizuma, Masamichi; MacGregor, Anne; Hong, Seung-Mo; Ayars, Michael; Almario, Jose Alejandro; Borges, Michael; Kanda, Mitsuro; Li, Ang; Vincent, Audrey; Maitra, Anirban; Goggins, Michael

    2016-01-01

    The methylation status of a promoter influences gene expression and aberrant methylation during tumor development has important functional consequences for pancreatic and other cancers. Using methylated CpG island amplification and promoter microarrays, we identified ANK1 as hypomethylated in pancreatic cancers. Expression analysis determined ANK1 as commonly overexpressed in pancreatic cancers relative to normal pancreas. ANK1 was co-expressed with miR-486 in pancreatic cancer cells. Stable knockdown of ANK1 in the pancreatic cancer cell line AsPC1 led to changes in cell morphology, and decreases in colony formation. Stable knockdown of ANK1 also marked reduced the growth of tumors in athymic nude mice. Among patients undergoing pancreaticoduodenectomy, those with pancreatic cancers expressing ANK1 had a poorer prognosis than those without ANK1 expression. These findings indicate a role for ANK1 overexpression in mediating pancreatic cancer tumorigenicity. PMID:27144336

  8. Enhanced learning after genetic overexpression of a brain growth protein.

    PubMed

    Routtenberg, A; Cantallops, I; Zaffuto, S; Serrano, P; Namgung, U

    2000-06-20

    Ramón y Cajal proposed 100 years ago that memory formation requires the growth of nerve cell processes. One-half century later, Hebb suggested that growth of presynaptic axons and postsynaptic dendrites consequent to coactivity in these synaptic elements was essential for such information storage. In the past 25 years, candidate growth genes have been implicated in learning processes, but it has not been demonstrated that they in fact enhance them. Here, we show that genetic overexpression of the growth-associated protein GAP-43, the axonal protein kinase C substrate, dramatically enhanced learning and long-term potentiation in transgenic mice. If the overexpressed GAP-43 was mutated by a Ser --> Ala substitution to preclude its phosphorylation by protein kinase C, then no learning enhancement was found. These findings provide evidence that a growth-related gene regulates learning and memory and suggest an unheralded target, the GAP-43 phosphorylation site, for enhancing cognitive ability.

  9. Lack of Gαi2 leads to dilative cardiomyopathy and increased mortality in β1-adrenoceptor overexpressing mice

    PubMed Central

    Keller, Kirsten; Maass, Martina; Dizayee, Sara; Leiss, Veronika; Annala, Suvi; Köth, Jessica; Seemann, Wiebke K.; Müller-Ehmsen, Jochen; Mohr, Klaus; Nürnberg, Bernd; Engelhardt, Stefan; Herzig, Stefan; Birnbaumer, Lutz; Matthes, Jan

    2015-01-01

    Aims Inhibitory G (Gi) proteins have been proposed to be cardioprotective. We investigated effects of Gαi2 knockout on cardiac function and survival in a murine heart failure model of cardiac β1-adrenoceptor overexpression. Methods and results β1-transgenic mice lacking Gαi2 (β1-tg/Gαi2−/−) were compared with wild-type mice and littermates either overexpressing cardiac β1-adrenoceptors (β1-tg) or lacking Gαi2 (Gαi2−/−). At 300 days, mortality of mice only lacking Gαi2 was already higher compared with wild-type or β1-tg, but similar to β1-tg/Gαi2−/−, mice. Beyond 300 days, mortality of β1-tg/Gαi2−/− mice was enhanced compared with all other genotypes (mean survival time: 363 ± 21 days). At 300 days of age, echocardiography revealed similar cardiac function of wild-type, β1-tg, and Gαi2−/− mice, but significant impairment for β1-tg/Gαi2−/− mice (e.g. ejection fraction 14 ± 2 vs. 40 ± 4% in wild-type mice). Significantly increased ventricle-to-body weight ratio (0.71 ± 0.06 vs. 0.48 ± 0.02% in wild-type mice), left ventricular size (length 0.82 ± 0.04 vs. 0.66 ± 0.03 cm in wild types), and atrial natriuretic peptide and brain natriuretic peptide expression (mRNA: 2819 and 495% of wild-type mice, respectively) indicated hypertrophy. Gαi3 was significantly up-regulated in Gαi2 knockout mice (protein compared with wild type: 340 ± 90% in Gαi2−/− and 394 ± 80% in β1-tg/Gαi2−/−, respectively). Conclusions Gαi2 deficiency combined with cardiac β1-adrenoceptor overexpression strongly impaired survival and cardiac function. At 300 days of age, β1-adrenoceptor overexpression alone had not induced cardiac hypertrophy or dysfunction while there was overt cardiomyopathy in mice additionally lacking Gαi2. We propose an enhanced effect of increased β1-adrenergic drive by the lack of protection via Gαi2. Gαi3 up-regulation was not sufficient to compensate for Gαi2 deficiency, suggesting an isoform-specific or

  10. Nmdmc overexpression extends Drosophila lifespan and reduces levels of mitochondrial reactive oxygen species

    SciTech Connect

    Yu, Suyeun; Jang, Yeogil; Paik, Donggi; Lee, Eunil; Park, Joong-Jean

    2015-10-02

    NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) is a bifunctional enzyme involved in folate-dependent metabolism and highly expressed in rapidly proliferating cells. However, Nmdmc physiological roles remain unveiled. We found that ubiquitous Nmdmc overexpression enhanced Drosophila lifespan and stress resistance. Interestingly, Nmdmc overexpression in the fat body was sufficient to increase lifespan and tolerance against oxidative stress. In addition, these conditions coincided with significant decreases in the levels of mitochondrial ROS and Hsp22 as well as with a significant increase in the copy number of mitochondrial DNA. These results suggest that Nmdmc overexpression should be beneficial for mitochondrial homeostasis and increasing lifespan. - Highlights: • Ubiquitous Nmdmc overexpression enhanced lifespan and stress tolerance. • Nmdmc overexpression in the fat body extended longevity. • Fat body-specific Nmdmc overexpression increased oxidative stress resistance. • Nmdmc overexpression decreased Hsp22 transcript levels and ROS. • Nmdmc overexpression increased mitochondrial DNA copy number.

  11. GRK5 deficiency leads to susceptibility to intermittent hypoxia-induced cognitive impairment

    PubMed Central

    Singh, Prabhakar; Peng, Wei; Zhang, Qiang; Ding, XueFeng; Suo, William Z.

    2016-01-01

    Obstructive sleep apnea (OSA) leads to cognitive impairment in about 25% patients, though it remains elusive what makes one more susceptible than the other to be cognitively impaired. G protein-coupled receptor kinase-5 (GRK5) deficiency is recently found to render subjects more susceptible to cognitive impairment triggered by over-expression of Swedish mutant ß-amyloid precursor protein. This study is to determine whether GRK5 deficiency also renders subjects more susceptible to the OSA-triggered cognitive impairment. Both wild type (WT) and GRK5 knockout (KO) mice were placed in conditions absence and presence of intermittent hypoxia (IH) with 8%/21% O2 90-second cycle for 8 hours a day for a month, and then followed by behavioral assessments with battery of tasks. We found that the selected IH condition only induced marginally abnormal behavior (slightly elevated anxiety with most others unchanged) in the WT mice but it caused significantly more behavioral deficits in the KO mice, ranging from elevated anxiety, impaired balancing coordination, and impaired short-term spatial memory. These results suggest that GRK5 deficiency indeed makes the mice more susceptible to wide range of behavioral impairments, including cognitive impairments. PMID:26778781

  12. Facioscapulohumeral muscular dystrophy in mice overexpressing FRG1.

    PubMed

    Gabellini, Davide; D'Antona, Giuseppe; Moggio, Maurizio; Prelle, Alessandro; Zecca, Chiara; Adami, Raffaella; Angeletti, Barbara; Ciscato, Patrizia; Pellegrino, Maria Antonietta; Bottinelli, Roberto; Green, Michael R; Tupler, Rossella

    2006-02-23

    Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that is not due to a classical mutation within a protein-coding gene. Instead, almost all FSHD patients carry deletions of an integral number of tandem 3.3-kilobase repeat units, termed D4Z4, located on chromosome 4q35 (ref. 3). D4Z4 contains a transcriptional silencer whose deletion leads to inappropriate overexpression in FSHD skeletal muscle of 4q35 genes located upstream of D4Z4 (ref. 4). To identify the gene responsible for FSHD pathogenesis, we generated transgenic mice selectively overexpressing in skeletal muscle the 4q35 genes FRG1, FRG2 or ANT1. We find that FRG1 transgenic mice develop a muscular dystrophy with features characteristic of the human disease; by contrast, FRG2 and ANT1 transgenic mice seem normal. FRG1 is a nuclear protein and several lines of evidence suggest it is involved in pre-messenger RNA splicing. We find that in muscle of FRG1 transgenic mice and FSHD patients, specific pre-mRNAs undergo aberrant alternative splicing. Collectively, our results suggest that FSHD results from inappropriate overexpression of FRG1 in skeletal muscle, which leads to abnormal alternative splicing of specific pre-mRNAs.

  13. Overexpression of Lymphotoxin in T Cells Induces Fulminant Thymic Involution

    PubMed Central

    Heikenwalder, Mathias; Prinz, Marco; Zeller, Nicolas; Lang, Karl S.; Junt, Tobias; Rossi, Simona; Tumanov, Alexei; Schmidt, Hauke; Priller, Josef; Rülicke, Thomas; Macpherson, Andrew J.; Holländer, Georg A.; Nedospasov, Sergei A.; Aguzzi, Adriano

    2008-01-01

    Activated lymphocytes and lymphoid-tissue inducer cells express lymphotoxins (LTs), which are essential for the organogenesis and maintenance of lymphoreticular microenvironments. Here we describe that T-cell-restricted overexpression of LT induces fulminant thymic involution. This phenotype was prevented by ablation of the LT receptors tumor necrosis factor receptor (TNFR) 1 or LT beta receptor (LTβR), representing two non-redundant pathways. Multiple lines of transgenic Ltαβ and Ltα mice show such a phenotype, which was not observed on overexpression of LTβ alone. Reciprocal bone marrow transfers between LT-overexpressing and receptor-ablated mice show that involution was not due to a T cell-autonomous defect but was triggered by TNFR1 and LTβR signaling to radioresistant stromal cells. Thymic involution was partially prevented by the removal of one allele of LTβR but not of TNFR1, establishing a hierarchy in these signaling events. Infection with the lymphocytic choriomeningitis virus triggered a similar thymic pathology in wt, but not in Tnfr1−/− mice. These mice displayed elevated TNFα in both thymus and plasma, as well as increased LTs on both CD8+ and CD4−CD8− thymocytes. These findings suggest that enhanced T cell-derived LT expression helps to control the physiological size of the thymic stroma and accelerates its involution via TNFR1/LTβR signaling in pathological conditions and possibly also in normal aging. PMID:18483211

  14. Role of overexpressed CFA/I fimbriae in bacterial swimming

    NASA Astrophysics Data System (ADS)

    Cao, Ling; Suo, Zhiyong; Lim, Timothy; Jun, SangMu; Deliorman, Muhammedin; Riccardi, Carol; Kellerman, Laura; Avci, Recep; Yang, Xinghong

    2012-06-01

    Enterotoxigenic Escherichia coli CFA/I is a protective antigen and has been overexpressed in bacterial vectors, such as Salmonella Typhimurium H683, to generate vaccines. Effects that overexpressed CFA/I may engender on the bacterial host remain largely unexplored. To investigate, we constructed a high CFA/I expression strain, H683-pC2, and compared it to a low CFA/I expression strain, H683-pC, and to a non-CFA/I expression strain, H683-pY. The results showed that H683-pC2 was less able to migrate into semisolid agar (0.35%) than either H683-pC or H683-pY. Bacteria that migrated showed motility halo sizes of H683-pC2 < H683-pC < H683-pY. In the liquid culture media, H683-pC2 cells precipitated to the bottom of the tube, while those of H683-pY did not. In situ imaging revealed that H683-pC2 bacilli tended to auto-agglutinate within the semisolid agar, while H683-pY bacilli did not. When the cfaBE fimbrial fiber encoding genes were deleted from pC2, the new plasmid, pC2(-), significantly recovered bacterial swimming capability. Our study highlights the negative impact of overexpressed CFA/I fimbriae on bacterial swimming motility.

  15. Conditional overexpression of transgenes in megakaryocytes and platelets in vivo

    PubMed Central

    Nguyen, Hao G.; Yu, Guangyao; Makitalo, Maria; Yang, Dan; Xie, Hou-Xiang; Jones, Matthew R.; Ravid, Katya

    2005-01-01

    Megakaryocyte (MK)–specific transgene expression has proved valuable in studying thrombotic and hemostatic processes. Constitutive expression of genes, however, could result in altered phenotypes due to compensatory mechanisms or lethality. To circumvent these limitations, we used the tetracycline/doxycycline (Tet)–off system to conditionally over-express genes in megakaryocytes and platelets in vivo. We generated 3 transactivator transgenic lines expressing the Tet transactivator element (tTA), under the control of the MK-specific platelet factor 4 promoter (PF4-tTA-VP16). Responder lines were simultaneously generated, each with a bidirectional minimal cytomegalovirus (CMV)–tTA responsive promoter driving prokaryotic β-galactosidase gene, as a cellular reporter, and a gene of interest (in this case, the mitotic regulator Aurora-B). A transactivator founder line that strongly expressed PF4-driven tTA–viral protein 16 (VP16) was crossbred to a responder line. The homozygous double-transgenic mouse line exhibited doxycycline-dependent transgene overexpression in MKs and platelets. Using this line, platelets were conveniently indicated at sites of induced stress by β-galactosidase staining. In addition, we confirmed our earlier report on effects of constitutive expression of Aurora-B, indicating a tight regulation at protein level and a modest effect on MK ploidy. Hence, we generated a new line, PF4-tTA-VP16, that is available for conditionally overexpressing genes of interest in the MK/platelet lineage in vivo. PMID:15890684

  16. Neuroligin-1 Overexpression in Newborn Granule Cells In Vivo

    PubMed Central

    Schnell, Eric; Bensen, AeSoon L.; Washburn, Eric K.; Westbrook, Gary L.

    2012-01-01

    Adult-born dentate granule cells integrate into the hippocampal network, extend neurites and form synapses in otherwise mature tissue. Excitatory and inhibitory inputs innervate these new granule cells in a stereotyped, temporally segregated manner, which presents a unique opportunity to study synapse development in the adult brain. To examine the role of neuroligins as synapse-inducing molecules in vivo, we infected dividing neural precursors in adult mice with a retroviral construct that increased neuroligin-1 levels during granule cell differentiation. By 21 days post-mitosis, exogenous neuroligin-1 was expressed at the tips of dendritic spines and increased the number of dendritic spines. Neuroligin-1-overexpressing cells showed a selective increase in functional excitatory synapses and connection multiplicity by single afferent fibers, as well as an increase in the synaptic AMPA/NMDA receptor ratio. In contrast to its synapse-inducing ability in vitro, neuroligin-1 overexpression did not induce precocious synapse formation in adult-born neurons. However, the dendrites of neuroligin-1-overexpressing cells did have more thin protrusions during an early period of dendritic outgrowth, suggesting enhanced filopodium formation or stabilization. Our results indicate that neuroligin-1 expression selectively increases the degree, but not the onset, of excitatory synapse formation in adult-born neurons. PMID:23110172

  17. Overexpression of calreticulin sensitizes SERCA2a to oxidative stress.

    PubMed

    Ihara, Yoshito; Kageyama, Kan; Kondo, Takahito

    2005-04-22

    Calreticulin (CRT), a Ca(2+)-binding molecular chaperone in the endoplasmic reticulum, plays a vital role in cardiac physiology and pathology. Oxidative stress is a main cause of myocardiac disorder in the ischemic heart, but the function of CRT under oxidative stress is not fully understood. In this study, the effect of overexpression of CRT on sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) 2a under oxidative stress was examined using myocardiac H9c2 cells transfected with the CRT gene. The in vitro activity of SERCA2a and uptake of (45)Ca(2+) into isolated microsomes were suppressed by H(2)O(2) in CRT-overexpressing cells compared with controls. Moreover, SERCA2a protein was degraded via a proteasome-dependent pathway following the formation of a complex with CRT under the stress with H(2)O(2). Thus, we conclude that overexpression of CRT enhances the inactivation and degradation of SERCA2a in the cells under oxidative stress, suggesting some pathophysiological functions of CRT in Ca(2+) homeostasis of myocardiac disease.

  18. Subcellular protein overexpression to develop abiotic stress tolerant plants

    PubMed Central

    Nouri, Mohammad-Zaman; Komatsu, Setsuko

    2012-01-01

    Environmental stresses are major factors limiting growth and development of crops. Plants respond to the stresses through a wide range of reactions from morphological changes to alterations in the patterns of protein expression. Understanding the mechanisms involved in the stress response is the first step to develop abiotic stress tolerant crops. Proteomics is a powerful tool in evaluating regulated proteins in the cell under stress and it is an efficient technique in studying stress tolerant plants. Because of the nature of abiotic stress, intracellular compartments play a main role in the stress response. Subcellular proteins such as ion and water transporters, reactive oxygen species (ROS) scavengers, and the proteins related to signaling and transcriptional regulation are frequently reported as being involved in stress tolerance. Overexpression of stress-responsive protein through generation of transgenic plants is one the main practical approaches in production of tolerant plants. In this article, recent studies on transgenic plants overexpressing subcellular proteins are reviewed and the role of organelles and over-expressed proteins is classified. PMID:23346093

  19. Overexpression of follistatin in trout stimulates increased muscling.

    PubMed

    Medeiros, Erika F; Phelps, Michael P; Fuentes, Fernando D; Bradley, Terence M

    2009-07-01

    Deletion or inhibition of myostatin in mammals has been demonstrated to markedly increase muscle mass by hyperplasia, hypertrophy, or a combination of both. Despite a remarkably high degree of conservation with the mammalian protein, the function of myostatin remains unknown in fish, many species of which continue muscle growth throughout the lifecycle by hyperplasia. Transgenic rainbow trout (Oncorhynchus mykiss) overexpressing follistatin, one of the more efficacious antagonists of myostatin, were produced to investigate the effect of this protein on muscle development and growth. P(1) transgenics overexpressing follistatin in muscle tissue exhibited increased epaxial and hypaxial muscling similar to that observed in double-muscled cattle and myostatin null mice. The hypaxial muscling generated a phenotype reminiscent of well-developed rectus abdominus and intercostal muscles in humans and was dubbed "six pack." Body conformation of the transgenic animals was markedly altered, as measured by condition factor, and total muscle surface area increased. The increased muscling was due almost exclusively to hyperplasia as evidenced by a higher number of fibers per unit area and increases in the percentage of smaller fibers and the number of total fibers. In several individuals, asymmetrical muscling was observed, but no changes in mobility or behavior of follistatin fish were observed. The findings indicate that overexpression of follistatin in trout, a species with indeterminate growth rate, enhances muscle growth. It remains to be determined whether the double muscling in trout is due to inhibition of myostatin, other growth factors, or both.

  20. Role of overexpressed CFA/I fimbriae in bacterial swimming.

    PubMed

    Cao, Ling; Suo, Zhiyong; Lim, Timothy; Jun, Sangmu; Deliorman, Muhammedin; Riccardi, Carol; Kellerman, Laura; Avci, Recep; Yang, Xinghong

    2012-06-01

    Enterotoxigenic Escherichia coli CFA/I is a protective antigen and has been overexpressed in bacterial vectors, such as Salmonella Typhimurium H683, to generate vaccines. Effects that overexpressed CFA/I may engender on the bacterial host remain largely unexplored. To investigate, we constructed a high CFA/I expression strain, H683-pC2, and compared it to a low CFA/I expression strain, H683-pC, and to a non-CFA/I expression strain, H683-pY. The results showed that H683-pC2 was less able to migrate into semisolid agar (0.35%) than either H683-pC or H683-pY. Bacteria that migrated showed motility halo sizes of H683-pC2 < H683-pC < H683-pY. In the liquid culture media, H683-pC2 cells precipitated to the bottom of the tube, while those of H683-pY did not. In situ imaging revealed that H683-pC2 bacilli tended to auto-agglutinate within the semisolid agar, while H683-pY bacilli did not. When the cfaBE fimbrial fiber encoding genes were deleted from pC2, the new plasmid, pC2(-), significantly recovered bacterial swimming capability. Our study highlights the negative impact of overexpressed CFA/I fimbriae on bacterial swimming motility.

  1. PACSIN3 Overexpression Increases Adipocyte Glucose Transport through GLUT1

    PubMed Central

    Roach, William; Plomann, Markus

    2007-01-01

    PACSIN family members regulate intracellular vesicle trafficking via their ability to regulate cytoskeletal rearrangement. These processes are known to be involved in trafficking of GLUT1 and GLUT4 in adipocytes. In this study PACSIN3 was observed to be the only PACSIN isoform that increases in expression during 3T3-L1 adipocyte differentiation. Overexpression of PACSIN3 in 3T3-L1 adipocytes caused an elevation of glucose uptake. Subcellular fractionation revealed that PACSIN3 overexpression elevated GLUT1 plasma membrane localization without effecting GLUT4 distribution. In agreement with this result, examination of GLUT exofacial presentation at the cell surface by photoaffinity labeling revealed significantly increased GLUT1, but not GLUT4, after overexpression of PACSIN3. These results establish a role for PACSIN3 in regulating glucose uptake in adipocytes via its preferential participation in GLUT1 trafficking. They are consistent with the proposal, which is supported by a recent study, that GLUT1, but not GLUT4, is predominantly endocytosed via the coated pit pathway in unstimulated 3T3-L1 adipocytes. PMID:17320047

  2. Mild Cognitive Impairment.

    PubMed

    Sanford, Angela M

    2017-08-01

    Mild cognitive impairment (MCI) occurs along a continuum from normal cognition to dementia. A roadblock to earlier diagnosis and potential treatment is the lack of consistency with screening for MCI. Universal screening would be ideal, but is limited. Once a diagnosis of MCI is made, it is important for the clinician to evaluate for reversible causes. At present time, there are no pharmacologic treatments proven to slow or cure progression of MCI to dementia; nonetheless, there is evidence that lifestyle modifications including diet, exercise, and cognitive stimulation may be effective. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Cognitive impairment and pragmatics.

    PubMed

    Gutiérrez-Rexach, Javier; Schatz, Sara

    2016-01-01

    One of the most important ingredients of felicitous conversation exchanges is the adequate expression of illocutionary force and the achievement of perlocutionary effects, which can be considered essential to the functioning of pragmatic competence. The breakdown of illocutionary and perlocutionary functions is one of the most prominent external features of cognitive impairment in Alzheimer's Disease, with devastating psychological and social consequences for patients, their family and caregivers. The study of pragmatic functions is essential for a proper understanding of the linguistic and communicative aspects of Alzheimer's disease.

  4. Passengers of Impaired Drivers

    PubMed Central

    Romano, Eduardo; Kelley-Baker, Tara; Lacey, John

    2012-01-01

    Introduction The aims of this study are (a) to estimate the prevalence of passengers riding with alcohol-impaired drivers; (b) to investigate the role of demographic factors (age, gender, race/ethnicity, educational status) and relevant driving conditions (time of the day, trip origin, vehicle ownership) on shaping the likelihood of alcohol-impaired driving; (c) to identify and estimate the prevalence of passengers as alternative drivers (PADs); and (d) to examine the role that vehicle ownership plays in shaping the occurrence of PADs. Method Data came from a unique convenience sample of passengers obtained from the 2007 National Roadside Survey, a random sample of drivers from the 48 contiguous states. Results The prevalence of PADs in the targeted population (mostly weekend night vehicles) was higher with drivers at .00

  5. Fertility impairment in radiotherapy

    PubMed Central

    Kuźba-Kryszak, Tamara; Nowikiewicz, Tomasz; Żyromska, Agnieszka

    2016-01-01

    Infertility as a result of antineoplastic therapy is becoming a very important issue due to the growing incidence of neoplastic diseases. Routinely applied antineoplastic treatments and the illness itself lead to fertility disorders. Therapeutic methods used in antineoplastic treatment may cause fertility impairment or sterilization due to permanent damage to reproductive cells. The risk of sterilization depends on the patient's sex, age during therapy, type of neoplasm, radiation dose and treatment area. It is known that chemotherapy and radiotherapy can lead to fertility impairment and the combination of these two gives an additive effect. The aim of this article is to raise the issue of infertility in these patients. It is of growing importance due to the increase in the number of children and young adults who underwent radiotherapy in the past. The progress in antineoplastic therapy improves treatment results, but at the same time requires a deeper look at existential needs of the patient. Reproductive function is an integral element of self-esteem and should be taken into account during therapy planning. PMID:27647982

  6. Neuroprotective potential of pleiotrophin overexpression in the striatonigral pathway compared with overexpression in both the striatonigral and nigrostriatal pathways

    PubMed Central

    Gombash, SE; Manfredsson, FP; Mandel, RJ; Collier, TJ; Fischer, DL; Kemp, CJ; Kuhn, NM; Wohlgenant, SL; Fleming, SM; Sortwell, CE

    2015-01-01

    Intrastriatal injection of recombinant adeno-associated viral vector serotype 2/1 (rAAV2/1) to overexpress the neurotrophic factor pleiotrophin (PTN) provides neuroprotection for tyrosine hydroxylase immunoreactive (THir) neurons in the substantia nigra pars compacta (SNpc), increases THir neurite density in the striatum (ST) and reverses functional deficits in forepaw use following 6-hydroxydopamine (6-OHDA) toxic insult. Glial cell line-derived neurotrophic factor (GDNF) gene transfer studies suggest that optimal neuroprotection is dependent on the site of nigrostriatal overexpression. The present study was conducted to determine whether enhanced neuroprotection could be accomplished via simultaneous rAAV2/1 PTN injections into the ST and SN compared with ST injections alone. Rats were unilaterally injected in the ST alone or injected in both the ST and SN with rAAV2/1 expressing either PTN or control vector. Four weeks later, all rats received intrastriatal injections of 6-OHDA. Rats were euthanized 6 or 16 weeks relative to 6-OHDA injection. A novel selective total enumeration method to estimate nigral THir neuron survival was validated to maintain the accuracy of stereological assessment. Long-term nigrostriatal neuroprotection and functional benefits were only observed in rats in which rAAV2/1 PTN was injected into the ST alone. Results suggest that superior preservation of the nigrostriatal system is provided by PTN overexpression delivered to the ST and restricted to the ST and SN pars reticulata and is not improved with overexpression of PTN within SNpc neurons. PMID:24807806

  7. Neuroprotective potential of pleiotrophin overexpression in the striatonigral pathway compared with overexpression in both the striatonigral and nigrostriatal pathways.

    PubMed

    Gombash, S E; Manfredsson, F P; Mandel, R J; Collier, T J; Fischer, D L; Kemp, C J; Kuhn, N M; Wohlgenant, S L; Fleming, S M; Sortwell, C E

    2014-07-01

    Intrastriatal injection of recombinant adeno-associated viral vector serotype 2/1 (rAAV2/1) to overexpress the neurotrophic factor pleiotrophin (PTN) provides neuroprotection for tyrosine hydroxylase immunoreactive (THir) neurons in the substantia nigra pars compacta (SNpc), increases THir neurite density in the striatum (ST) and reverses functional deficits in forepaw use following 6-hydroxydopamine (6-OHDA) toxic insult. Glial cell line-derived neurotrophic factor (GDNF) gene transfer studies suggest that optimal neuroprotection is dependent on the site of nigrostriatal overexpression. The present study was conducted to determine whether enhanced neuroprotection could be accomplished via simultaneous rAAV2/1 PTN injections into the ST and SN compared with ST injections alone. Rats were unilaterally injected in the ST alone or injected in both the ST and SN with rAAV2/1 expressing either PTN or control vector. Four weeks later, all rats received intrastriatal injections of 6-OHDA. Rats were euthanized 6 or 16 weeks relative to 6-OHDA injection. A novel selective total enumeration method to estimate nigral THir neuron survival was validated to maintain the accuracy of stereological assessment. Long-term nigrostriatal neuroprotection and functional benefits were only observed in rats in which rAAV2/1 PTN was injected into the ST alone. Results suggest that superior preservation of the nigrostriatal system is provided by PTN overexpression delivered to the ST and restricted to the ST and SN pars reticulata and is not improved with overexpression of PTN within SNpc neurons.

  8. The metabolic syndrome in mice overexpressing neuropeptide Y in noradrenergic neurons.

    PubMed

    Ailanen, Liisa; Ruohonen, Suvi T; Vähätalo, Laura H; Tuomainen, Katja; Eerola, Kim; Salomäki-Myftari, Henriikka; Röyttä, Matias; Laiho, Asta; Ahotupa, Markku; Gylling, Helena; Savontaus, Eriika

    2017-07-01

    A gain-of-function polymorphism in human neuropeptide Y (NPY) gene (rs16139) associates with metabolic disorders and earlier onset of type 2 diabetes (T2D). Similarly, mice overexpressing NPY in noradrenergic neurons (OE-NPY(DBH)) display obesity and impaired glucose metabolism. In this study, the metabolic syndrome-like phenotype was characterized and mechanisms of impaired hepatic fatty acid, cholesterol and glucose metabolism in pre-obese (2-month-old) and obese (4-7-month-old) OE-NPY(DBH) mice were elucidated. Susceptibility to T2D was assessed by subjecting mice to high caloric diet combined with low-dose streptozotocin. Contribution of hepatic Y1-receptor to the phenotype was studied using chronic treatment with an Y1-receptor antagonist, BIBO3304. Obese OE-NPY(DBH) mice displayed hepatosteatosis and hypercholesterolemia preceded by decreased fatty acid oxidation and accelerated cholesterol synthesis. Hyperinsulinemia in early obese state inhibited pyruvate- and glucose-induced hyperglycemia, and deterioration of glucose metabolism of OE-NPY(DBH) mice developed with aging. Furthermore, streptozotocin induced T2D only in OE-NPY(DBH) mice. Hepatic inflammation was not morphologically visible, but upregulated hepatic anti-inflammatory pathways and increased 8-isoprostane combined with increased serum resistin and decreased interleukin 10 pointed to increased NPY-induced oxidative stress that may predispose OE-NPY(DBH) mice to insulin resistance. Chronic treatment with BIBO3304 did not improve the metabolic status of OE-NPY(DBH) mice. Instead, downregulation of beta-1-adrenoceptors suggests indirect actions of NPY via inhibition of sympathetic nervous system. In conclusion, changes in hepatic fatty acid, cholesterol and glucose metabolism favoring energy storage contribute to the development of NPY-induced metabolic syndrome, and the effect is likely mediated by changes in sympathetic nervous system activity. © 2017 Society for Endocrinology.

  9. Sry-Independent Overexpression of Sox9 Supports Spermatogenesis and Fertility in the Mouse.

    PubMed

    Ortega, Egle A; Ruthig, Victor A; Ward, Monika A

    2015-12-01

    The Y chromosome gene Sry is responsible for sex determination in mammals and initiates a cascade of events that direct differentiation of bipotential genital ridges toward male-specific fate. Sox9 is an autosomal gene and a primary downstream target of SRY. The activation of Sox9 in the absence of Sry is sufficient for initiation of male-specific sex determination. Sry-to-Sox9 replacement has mostly been studied in the context of sex determination during early embryogenesis. Here, we tested whether Sry-to-Sox9 replacement affects male fertility in adulthood. We examined males with the Y chromosome carrying a deletion removing the endogenous Sry, with testes determination driven either by the Sox9 (XY(Tdym1)Sox9) or the Sry (XY(Tdym1)Sry) transgenes as well as wild-type males (XY). XY(Tdym1)Sox9 males had reduced testes size, altered testes shape and vasculature, and increased incidence of defects in seminiferous epithelium underlying the coelomic blood vessel region when compared to XY(Tdym1)Sry and XY. There were no differences between XY(Tdym1)Sry and XY(Tdym1)Sox9 males in respect to sperm number, motility, morphology, and ability to fertilize oocytes in vitro, but for some parameters, transgenic males were impaired when compared to XY. In fecundity trials, XY(Tdym1)Sry, XY(Tdym1)Sox9, and XY males yielded similar average numbers of pups and litters. Overall, our findings support that males lacking the testis determinant Sry can be fertile and reinforce the notion that Sry does not play a role in mature gonads. Although transgenic Sox9 overexpression in the absence of Sry results in certain testicular abnormalities, it does not translate into fertility impairment.

  10. Overexpression cdc42 attenuates isoflurane-induced neurotoxicity in developmental brain of rats.

    PubMed

    Fang, Xi; Li, Shiyong; Han, Qiang; Zhao, Yilin; Gao, Jie; Yan, Jing; Luo, Ailin

    2017-08-26

    Nowadays many children receive operations with general anesthesia. Isoflurane is a commonly-used general anesthetic. Numbers of studies demonstrated that isoflurane induced neurotoxicity and neurobehavioral deficiency in young rats, however, the underlying mechanism remained unknown. Cell division cycle 42 (cdc42) played an important role in regulating synaptic vesicle trafficking and actin dynamics in neuron, which closely linked to synaptic plasticity and dendritic spine formation. Meanwhile, cdc42 also involved in many neurodegenerative diseases. However, whether cdc42 provided a protective role in isoflurane induced synaptogenesis dysfunction still unknown. As the upstream of cdc42, calcium/Calmodulin-dependent protein kinase II (CaMKII) interacts with ion channels such as VDCCs and N-methyl-d-aspartate receptors (NMDARs), which closely associated with neuroapoptosis and cognitive deficiency in developing brain. The phosphorylation of CaMKIIα at Thr 286 plays an important role in introduction and maintenance of long-term potentiation (LTP). Therefore, we investigated the effect of isoflurane on cdc42 and its upstream Calcium/Calmodulin-dependent protein kinase II (CaMKII) and its downstream p21 activated kinase 3 (PAK3), then determined whether CaMKIIα/cdc42/PAK3 signaling pathway was involved in neurotoxicity and cognitive deficiency induced by isoflurane. Our study found that isoflurane induced neurotoxicity and resulted in cognitive impairment in young rats through suppressed CaMKIIα/cdc42/PAK3 signaling pathway. Cdc42 over-expression could reverse neurotoxicity and improve cognitive impairment induced by isoflurane. Copyright © 2017. Published by Elsevier Inc.

  11. Inhibition of osteoclast differentiation by overexpression of NDRG2 in monocytes

    SciTech Connect

    Kang, Kyeongah; Nam, Sorim; Kim, Bomi; Lim, Ji Hyun; Yang, Young; Lee, Myeong-Sok; Lim, Jong-Seok

    2015-12-25

    N-Myc downstream-regulated gene 2 (NDRG2), a member of the NDRG family of differentiation-related genes, has been characterized as a regulator of dendritic cell differentiation from monocytes, CD34{sup +} progenitor cells, and myelomonocytic leukemic cells. In this study, we show that NDRG2 overexpression inhibits the differentiation of U937 cells into osteoclasts in response to stimulation with a combination of macrophage colony-stimulating factor (M-CSF) and soluble receptor activator of NF-κB ligand (RANKL). U937 cells stably expressing NDRG2 are unable to differentiate into multinucleated osteoclast-like cells and display reduced tartrate-resistant acid phosphatase (TRAP) activity and resorption pit formation. Furthermore, NDRG2 expression significantly suppresses the expression of genes that are crucial for the proliferation, survival, differentiation, and function of osteoclasts, including c-Fos, Atp6v0d2, RANK, and OSCAR. The activation of ERK1/2 and p38 is also inhibited by NDRG2 expression during osteoclastogenesis, and the inhibition of osteoclastogenesis by NDRG2 correlates with the down-regulation of the expression of the transcription factor PU.1. Taken together, our results suggest that the expression of NDRG2 potentially inhibits osteoclast differentiation and plays a role in modulating the signal transduction pathway responsible for osteoclastogenesis. - Highlights: • The expression of NDRG2 significantly impairs osteoclast differentiation. • PU.1 and p38 MAPK inhibitions by NDRG2 are critical for the inhibition of osteoclastogenesis. • Knockdown of NDRG2 rescues the ability of monocytes to differentiate into osteoclasts. • NDRG2 expression in BM and primary macrophages also impairs osteoclast differentiation. • This study implies the potential of NDRG2 expression in the inhibition of osteoclastogenesis.

  12. Overexpression of long noncoding RNA HOTTIP promotes tumor invasion and predicts poor prognosis in gastric cancer

    PubMed Central

    Ye, Heng; Liu, Kun; Qian, Keqing

    2016-01-01

    Purpose Long noncoding RNAs have been proved to play important roles in the tumorigenesis and development of human gastric cancer (GC). Our study aims to investigate the expression and function of Homeobox A transcript at the distal tip (HOTTIP) in GC. Methods HOTTIP expression was detected in GC tissues and cell lines by using quantitative reverse transcription polymerase chain reaction. Association between HOTTIP levels and clinicopathological factors and patient prognosis was also analyzed. MTT, flow cytometry, and transwell invasion and migration assays were used to investigate the role of HOTTIP in the regulation of biological behaviors of GC cells. Results HOTTIP expression was remarkably increased in GC tissues and cell lines compared with that in the normal control. Clinicopathologic analysis revealed that high HOTTIP expression correlated with larger tumor size, deeper invasion depth, positive lymph node metastasis, advanced TNM stage, and shorter overall survival. Multivariate regression analysis identified HOTTIP overexpression as an independent unfavorable prognostic factor in GC patients. Moreover, HOTTIP downregulation by si-HOTTIP transfection impaired GC cell proliferation, promoted cell apoptosis, and reduced cell invasion and migration. Conclusion These findings suggested that HOTTIP may contribute to GC initiation and progression, and would be not only a novel prognostic marker but also a potential therapeutic target for this disease. PMID:27103834

  13. Overexpression of c-Jun contributes to sorafenib resistance in human hepatoma cell lines

    PubMed Central

    Haga, Yuki; Nakamura, Masato; Nakamoto, Shingo; Sasaki, Reina; Takahashi, Koji; Wu, Shuang; Yokosuka, Osamu

    2017-01-01

    Background Despite recent advances in treatment strategies, it is still difficult to cure patients with hepatocellular carcinoma (HCC). Sorafenib is the only approved multiple kinase inhibitor for systemic chemotherapy in patients with advanced HCC. The majority of advanced HCC patients are resistant to sorafenib. The mechanisms of sorafenib resistance are still unknown. Methods The expression of molecules involved in the mitogen-activated protein kinase (MAPK) signaling pathway in human hepatoma cell lines was examined in the presence or absence of sorafenib. Apoptosis of human hepatoma cells treated with sorafenib was investigated, and the expression of Jun proto-oncogene (c-Jun) was measured. Results The expression and phosphorylation of c-Jun were enhanced in human hepatoma cell lines after treatment with sorafenib. Inhibiting c-Jun enhanced sorafenib-induced apoptosis. The overexpression of c-Jun impaired sorafenib-induced apoptosis. The expression of osteopontin, one of the established AP-1 target genes, was enhanced after treatment with sorafenib in human hepatoma cell lines. Conclusions The protein c-Jun plays a role in sorafenib resistance in human hepatoma cell lines. The modulation and phosphorylation of c-Jun could be a new therapeutic option for enhancing responsiveness to sorafenib. Modulating c-Jun may be useful for certain HCC patients with sorafenib resistance. PMID:28323861

  14. Overexpression of the alpha1B-adrenergic receptor causes apoptotic neurodegeneration: multiple system atrophy.

    PubMed

    Zuscik, M J; Sands, S; Ross, S A; Waugh, D J; Gaivin, R J; Morilak, D; Perez, D M

    2000-12-01

    Progress toward elucidating the function of alpha1B-adrenergic receptors (alpha1BARs) in the central nervous system has been constrained by a lack of agonists and antagonists with adequate alpha1B-specificity. We have obviated this constraint by generating transgenic mice engineered to overexpress either wild-type or constitutively active alpha1BARs in tissues that normally express the receptor, including the brain. All transgenic lines showed granulovacular neurodegeneration, beginning in alpha1B-expressing domains of the brain and progressing with age to encompass all areas. The degeneration was apoptotic and did not occur in non-transgenic mice. Correspondingly, transgenic mice showed an age-progressive hindlimb disorder that was parkinsonian-like, as demonstrated by rescue of the dysfunction by 3, 4-dihydroxyphenylalanine and considerable dopaminergic-neuronal degeneration in the substantia nigra. Transgenic mice also had a grand mal seizure disorder accompanied by a corresponding dysplasia and neurodegeneration of the cerebral cortex. Both behavioral phenotypes (locomotor impairment and seizure) could be partially rescued with the alpha1AR antagonist terazosin, indicating that alpha1AR signaling participated directly in the pathology. Our results indicate that overstimulation of alpha1BAR leads to apoptotic neurodegeneration with a corresponding multiple system atrophy indicative of Shy-Drager syndrome, a disease whose etiology is unknown.

  15. Cardiomyocyte-specific perilipin 5 overexpression leads to myocardial steatosis and modest cardiac dysfunction1[S

    PubMed Central

    Wang, Hong; Sreenivasan, Urmila; Gong, Da-Wei; O'Connell, Kelly A.; Dabkowski, Erinne R.; Hecker, Peter A.; Ionica, Nicoleta; Konig, Manige; Mahurkar, Anup; Sun, Yezhou; Stanley, William C.; Sztalryd, Carole

    2013-01-01

    Presence of ectopic lipid droplets (LDs) in cardiac muscle is associated to lipotoxicity and tissue dysfunction. However, presence of LDs in heart is also observed in physiological conditions, such as when cellular energy needs and energy production from mitochondria fatty acid β-oxidation are high (fasting). This suggests that development of tissue lipotoxicity and dysfunction is not simply due to the presence of LDs in cardiac muscle but due at least in part to alterations in LD function. To examine the function of cardiac LDs, we obtained transgenic mice with heart-specific perilipin 5 (Plin5) overexpression (MHC-Plin5), a member of the perilipin protein family. Hearts from MHC-Plin5 mice expressed at least 4-fold higher levels of plin5 and exhibited a 3.5-fold increase in triglyceride content versus nontransgenic littermates. Chronic cardiac excess of LDs was found to result in mild heart dysfunction with decreased expression of peroxisome proliferator-activated receptor (PPAR)α target genes, decreased mitochondria function, and left ventricular concentric hypertrophia. Lack of more severe heart function complications may have been prevented by a strong increased expression of oxidative-induced genes via NF-E2-related factor 2 antioxidative pathway. Perilipin 5 regulates the formation and stabilization of cardiac LDs, and it promotes cardiac steatosis without major heart function impairment. PMID:23345411

  16. Long-Term Retinal PEDF Overexpression Prevents Neovascularization in a Murine Adult Model of Retinopathy

    PubMed Central

    Ribera, Albert; Bosch, Assumpcio; Ramos, David; Ruberte, Jesus; Bosch, Fatima

    2012-01-01

    Neovascularization associated with diabetic retinopathy (DR) and other ocular disorders is a leading cause of visual impairment and adult-onset blindness. Currently available treatments are merely palliative and offer temporary solutions. Here, we tested the efficacy of antiangiogenic gene transfer in an animal model that mimics the chronic progression of human DR. Adeno-associated viral (AAV) vectors of serotype 2 coding for antiangiogenic Pigment Epithelium Derived Factor (PEDF) were injected in the vitreous of a 1.5 month-old transgenic model of retinopathy that develops progressive neovascularization. A single intravitreal injection led to long-term production of PEDF and to a striking inhibition of intravitreal neovascularization, normalization of retinal capillary density, and prevention of retinal detachment. This was parallel to a reduction in the intraocular levels of Vascular Endothelial Growth Factor (VEGF). Normalization of VEGF was consistent with a downregulation of downstream effectors of angiogenesis, such as the activity of Matrix Metalloproteinases (MMP) 2 and 9 and the content of Connective Tissue Growth Factor (CTGF). These results demonstrate long-term efficacy of AAV-mediated PEDF overexpression in counteracting retinal neovascularization in a relevant animal model, and provides evidence towards the use of this strategy to treat angiogenesis in DR and other chronic proliferative retinal disorders. PMID:22911805

  17. Green Tea Polyphenols Rescue of Brain Defects Induced by Overexpression of DYRK1A

    PubMed Central

    Guedj, Fayçal; Sébrié, Catherine; Rivals, Isabelle; Ledru, Aurelie; Paly, Evelyne; Bizot, Jean C.; Smith, Desmond; Rubin, Edward; Gillet, Brigitte; Arbones, Mariona; Delabar, Jean M.

    2009-01-01

    Individuals with partial HSA21 trisomies and mice with partial MMU16 trisomies containing an extra copy of the DYRK1A gene present various alterations in brain morphogenesis. They present also learning impairments modeling those encountered in Down syndrome. Previous MRI and histological analyses of a transgenic mice generated using a human YAC construct that contains five genes including DYRK1A reveal that DYRK1A is involved, during development, in the control of brain volume and cell density of specific brain regions. Gene dosage correction induces a rescue of the brain volume alterations. DYRK1A is also involved in the control of synaptic plasticity and memory consolidation. Increased gene dosage results in brain morphogenesis defects, low BDNF levels and mnemonic deficits in these mice. Epigallocatechin gallate (EGCG) — a member of a natural polyphenols family, found in great amount in green tea leaves — is a specific and safe DYRK1A inhibitor. We maintained control and transgenic mice overexpressing DYRK1A on two different polyphenol-based diets, from gestation to adulthood. The major features of the transgenic phenotype were rescued in these mice. PMID:19242551

  18. Excess coenzyme A reduces skeletal muscle performance and strength in mice overexpressing human PANK2.

    PubMed

    Corbin, Deborah R; Rehg, Jerold E; Shepherd, Danielle L; Stoilov, Peter; Percifield, Ryan J; Horner, Linda; Frase, Sharon; Zhang, Yong-Mei; Rock, Charles O; Hollander, John M; Jackowski, Suzanne; Leonardi, Roberta

    2017-02-03

    Coenzyme A (CoA) is a cofactor that is central to energy metabolism and CoA synthesis is controlled by the enzyme pantothenate kinase (PanK). A transgenic mouse strain expressing human PANK2 was derived to determine the physiological impact of PANK overexpression and elevated CoA levels. The Tg(PANK2) mice expressed high levels of the transgene in skeletal muscle and heart; however, CoA was substantially elevated only in skeletal muscle, possibly associated with the comparatively low endogenous levels of acetyl-CoA, a potent feedback inhibitor of PANK2. Tg(PANK2) mice were smaller, had less skeletal muscle mass and displayed significantly impaired exercise tolerance and grip strength. Skeletal myofibers were characterized by centralized nuclei and aberrant mitochondria. Both the content of fully assembled complex I of the electron transport chain and ATP levels were reduced, while markers of oxidative stress were elevated in Tg(PANK2) skeletal muscle. These abnormalities were not detected in the Tg(PANK2) heart muscle, with the exception of spotty loss of cristae organization in the mitochondria. The data demonstrate that excessively high CoA may be detrimental to skeletal muscle function.

  19. Apparent Behavioral Benefits of Tau Overexpression in P301L Tau Transgenic Mice

    PubMed Central

    Morgan, Dave; Munireddy, Sanjay; Alamed, Jennifer; DeLeon, Jason; Diamond, David M.; Bickford, Paula; Hutton, Michael; Lewis, Jada; McGowan, Eileen; Gordon, Marcia N.

    2010-01-01

    Transgenic mice expressing human tau containing the P301L tau mutation (JNPL3; tau mice) develop motor neuron loss, paralysis and death between 7 and 12 months. Surprisingly, at 5 and 7 months of age, tau transgenic mice were superior to other genotypes in the rotarod task, and had near perfect scores on the balance beam and coat hanger tests. One tau transgenic mouse was performing at a superior level in the rotarod one day prior to developing paralysis. Cognitive function was also normal in the tau mice evaluated in the radial arm water maze and the Y-maze tasks. We also crossed the tau transgenic mice with Tg2576 amyloid-β protein precursor (AβPP) transgenic mice. Although AβPP mice were deficient in the radial arm maze task, AβPP+tau mice were not impaired, implying a benefit of the tau transgene. Some mice were homozygous for the retinal degeneration mutation (rd/rd) and excluded from the genotype analysis. Only the water maze task discriminated the rd/rd mice from nontransgenic mice. In conclusion, it seems that the modest tau overexpression or the presence of mutant tau in the JNPL3 tau mice may provide some benefit with respect to motor and cognitive performance before the onset of paralysis. PMID:19096159

  20. Impairments in Skin Integrity.

    PubMed

    Murphree, Rose W

    2017-09-01

    Altered skin integrity increases the chance of infection, impaired mobility, and decreased function and may result in the loss of limb or, sometimes, life. Skin is affected by both intrinsic and extrinsic factors. Intrinsic factors can include altered nutritional status, vascular disease issues, and diabetes. Extrinsic factors include falls, accidents, pressure, immobility, and surgical procedures. Ensuring skin integrity in the elderly requires a team approach and includes the individual, caregivers, and clinicians. The twenty-first century clinician has several online, evidence-based tools to assist with optimal treatment plans. Understanding best practices in addressing skin integrity issues can promote positive outcomes with the elderly. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Category specific semantic impairments.

    PubMed

    Warrington, E K; Shallice, T

    1984-09-01

    We report a quantitative investigation of the visual identification and auditory comprehension deficits of 4 patients who had made a partial recovery from herpes simplex encephalitis. Clinical observations had suggested the selective impairment and selective preservation of certain categories of visual stimuli. In all 4 patients a significant discrepancy between their ability to identify inanimate objects and inability to identify living things and foods was demonstrated. In 2 patients it was possible to compare visual and verbal modalities and the same pattern of dissociation was observed in both. For 1 patient, comprehension of abstract words was significantly superior to comprehension of concrete words. Consistency of responses was recorded within a modality in contrast to a much lesser degree of consistency between modalities. We interpret our findings in terms of category specificity in the organization of meaning systems that are also modality specific semantic systems.

  2. Overexpression of Indian hedgehog partially rescues short stature homeobox 2-overexpression-associated congenital dysplasia of the temporomandibular joint in mice

    PubMed Central

    LI, XIHAI; LIANG, WENNA; YE, HONGZHI; WENG, XIAPING; LIU, FAYUAN; LIN, PINGDONG; LIU, XIANXIANG

    2015-01-01

    The role of short stature homeobox 2 (shox2) in the development and homeostasis of the temporomandibular joint (TMJ) has been well documented. Shox2 is known to be expressed in the progenitor cells and perichondrium of the developing condyle. A previous study by our group reported that overexpression of shox2 leads to congenital dysplasia of the TMJ via downregulation of the Indian hedgehog (Ihh) signaling pathway, which is essential for embryonic disc primordium formation and mandibular condylar growth. To determine whether overexpression of Ihh may rescue the overexpression of shox2 leading to congenital dysplasia of the TMJ, a mouse model in which Ihh and shox2 were overexpressed (Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice) was utilized to assess the consequences of this overexpression on TMJ development during post-natal life. The results showed that the developmental process and expression levels of runt-related transcription factor 2 and sex determining region Y-box 9 in the TMJ of the Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice were similar to those in wild-type mice. Overexpression of Ihh rescued shox2 overexpression-associated reduction of extracellular matrix components. However, overexpression of Ihh did not inhibit the shox2 overexpression-associated increase of matrix metalloproteinases (MMPs) MMP9, MMP13 and apoptosis in the TMJ. These combinatory cellular and molecular defects appeared to account for the observed congenital dysplasia of TMJ, suggesting that overexpression of Ihh partially rescued shox2 overexpression-associated congenital dysplasia of the TMJ in mice. PMID:26096903

  3. Cognitive Impairment and Tramadol Dependence.

    PubMed

    Bassiony, Medhat M; Youssef, Usama M; Hassan, Mervat S; Salah El-Deen, Ghada M; El-Gohari, Hayam; Abdelghani, Mohamed; Abdalla, Ahmed; Ibrahim, Dalia H

    2017-02-01

    Cognitive impairment is one of the consequences of substance abuse. Tramadol abuse is a public health problem in Egypt. The objective of this study was to estimate the prevalence and correlates of cognitive impairment among tramadol-abuse patients and control subjects. This study included 100 patients with tramadol abuse and 100 control subjects (matched for age, sex, and education) who were recruited from Zagazig University Hospital, Egypt. Patients were divided into 2 groups: patients who used tramadol only (tramadol-alone group) and patients who used tramadol and other substances (polysubstance group). The participants were interviewed using Montreal Cognitive Assessment test and had urine screening for drugs. Twenty-four percent of the cases used tramadol alone, whereas the remaining used tramadol and other substances, mainly cannabis (66%) and benzodiazepines (27%). Tramadol-abuse patients were about 3 times more likely to have cognitive impairment than control subjects (81% vs 28%). Tramadol-alone patients were more than 2 times more likely to have cognitive impairment than control subjects (67% vs 28%). Cognitive impairment was significantly associated with polysubstance abuse. There was no association between cognitive impairment and sociodemographic or clinical factors. Cognitive impairment occurs commonly among tramadol-abuse patients. Memory impairment is the most common cognitive domain to be affected. There is a significant association between cognitive impairment and polysubstance abuse.

  4. [Multilingualism and specific language impairment].

    PubMed

    Arkkila, Eva; Smolander, Sini; Laasonen, Marja

    2013-01-01

    Specific language impairment is one of the most common developmental disturbances in childhood. With the increase of the foreign language population group an increasing number of children assimilating several languages and causing concern in language development attend clinical examinations. Knowledge of factors underlying the specific language impairment and the specific impairment in general, special features of language development of those learning several languages, as well as the assessment and support of the linguistic skills of a multilingual child is essential. The risk of long-term problems and marginalization is high for children having specific language impairment.

  5. Steroidogenic acute regulatory protein (StAR) overexpression attenuates HFD-induced hepatic steatosis and insulin resistance.

    PubMed

    Qiu, Yanyan; Sui, Xianxian; Zhan, Yongkun; Xu, Chen; Li, Xiaobo; Ning, Yanxia; Zhi, Xiuling; Yin, Lianhua

    2017-04-01

    Non-alcoholic fatty liver disease (NAFLD) covers a wide spectrum of liver pathology. Intracellular lipid accumulation is the first step in the development and progression of NAFLD. Steroidogenic acute regulatory protein (StAR) plays an important role in the synthesis of bile acid and intracellular lipid homeostasis and cholesterol metabolism. We hypothesize that StAR is involved in non-alcoholic fatty liver disease (NAFLD) pathogenesis. The hypothesis was identified using free fatty acid (FFA)-overloaded NAFLD in vitro model and high-fat diet (HFD)-induced NAFLD mouse model transfected by recombinant adenovirus encoding StAR (StAR). StAR expression was also examined in pathology samples of patients with fatty liver by immunohistochemical staining. We found that the expression level of StAR was reduced in the livers obtained from fatty liver patients and NAFLD mice. Additionally, StAR overexpression decreased the levels of hepatic lipids and maintained the hepatic glucose homeostasis due to the activation of farnesoid x receptor (FXR). StAR overexpression attenuated the impairment of insulin signaling in fatty liver. This protective role of StAR was owing to a reduction of intracellular diacylglycerol levels and the phosphorylation of PKCε. Furthermore, FXR inactivation reversed the observed beneficial effects of StAR. The present study revealed that StAR overexpression can reduce hepatic lipid accumulation, regulate glucose metabolism and attenuate insulin resistance through a mechanism involving the activation of FXR. Our study suggests that StAR may be a potential therapeutic target for NAFLD. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Over-expression of human endosulfatase-1 exacerbates cadmium-induced injury to transformed human lung cells in vitro.

    PubMed

    Zhang, Huiying; Newman, Donna R; Bonner, James C; Sannes, Philip L

    2012-11-15

    Environmental exposure to cadmium is known to cause damage to alveolar epithelial cells of the lung, impair their capacity to repair, and result in permanent structural alterations. Cell surface heparan sulfate proteoglycans (HSPGs) can modulate cell responses to injury through their interactions with soluble effector molecules. These interactions are often sulfate specific, and the removal of sulfate groups from HS side chains could be expected to influence cellular injury, such as that caused by exposure to cadmium. The goal of this study was to define the role 6-O-sulfate plays in cellular responses to cadmium exposure in two pulmonary epithelial cancer cell lines (H292 and A549) and in normal human primary alveolar type II (hAT2) cells. Sulfate levels were modified by transduced transient over-expression of 6-O-endosulfatase (HSulf-1), a membrane-bound enzyme which specifically removes 6-O-sulfate groups from HSPG side chains. Results showed that cadmium decreased cell viability and activated apoptosis pathways at low concentrations in hAT2 cells but not in the cancer cells. HSulf-1 over-expression, on the contrary, decreased cell viability and activated apoptosis pathways in H292 and A549 cells but not in hAT2 cells. When combined with cadmium, HSulf-1 over-expression further decreased cell viability and exacerbated the activation of apoptosis pathways in the transformed cells but did not add to the toxicity in hAT2 cells. The finding that HSulf-1 sensitizes these cancer cells and intensifies the injury induced by cadmium suggests that 6-O-sulfate groups on HSPGs may play important roles in protection against certain environmental toxicants, such as heavy metals. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Over-expression of Human Endosulfatase-1 Exacerbates Cadmium-induced Injury to Transformed Human Lung Cells In Vitro

    PubMed Central

    Zhang, Huiying; Newman, Donna R.; Bonner, James C.; Sannes, Philip L.

    2012-01-01

    Environmental exposure to cadmium is known to cause damage to alveolar epithelial cells of the lung, impair their capacity to repair, and result in permanent structural alterations. Cell surface heparan sulfate proteoglycans (HSPGs) can modulate cell responses to injury through their interactions with soluble effector molecules. These interactions are often sulfate specific, and the removal of sulfate groups from HS side chains could be expected to influence cellular injury, such as that caused by exposure to cadmium. The goal of this study was to define the role 6-O-sulfate plays in cellular responses to cadmium exposure in two pulmonary epithelial cancer cell lines (H292 and A549) and in normal human primary alveolar type II (hAT2) cells. Sulfate levels were modified by transduced transient over-expression of 6-O-endosulfatase (HSulf-1), a membrane-bound enzyme which specifically removes 6-O-sulfate groups from HSPG side chains. Results showed that cadmium decreased cell viability and activated apoptosis pathways at low concentrations in hAT2 cells but not in the cancer cells. HSulf-1 over-expression, on the contrary, decreased cell viability and activated apoptosis pathways in H292 and A549 cells but not in hAT2 cells. When combined with cadmium, HSulf-1 over-expression further decreased cell viability and exacerbated the activation of apoptosis pathways in the transformed cells but did not add to the toxicity in hAT2 cells. The finding that HSulf-1 sensitizes these cancer cells and intensifies the injury induced by cadmium suggests that 6-O-sulfate groups on HSPGs may play important roles in protection against certain environmental toxicants, such as heavy metals. PMID:23000194

  8. Overexpression of GTP Cyclohydrolase 1 Feedback Regulatory Protein Is Protective in a Murine Model of Septic Shock

    PubMed Central

    Starr, Anna; Sand, Claire A.; Heikal, Lamia; Kelly, Peter D.; Spina, Domenico; Crabtree, Mark; Channon, Keith M.; Leiper, James M.; Nandi, Manasi

    2014-01-01

    ABSTRACT Overproduction of nitric oxide (NO) by inducible NO synthase contributes toward refractory hypotension, impaired microvascular perfusion, and end-organ damage in septic shock patients. Tetrahydrobiopterin (BH4) is an essential NOS cofactor. GTP cyclohydrolase 1 (GCH1) is the rate-limiting enzyme for BH4 biosynthesis. Under inflammatory conditions, GCH1 activity and hence BH4 levels are increased, supporting pathological NOS activity. GCH1 activity can be controlled through allosteric interactions with GCH1 feedback regulatory protein (GFRP). We investigated whether overexpression of GFRP can regulate BH4 and NO production and attenuate cardiovascular dysfunction in sepsis. Sepsis was induced in mice conditionally overexpressing GFRP and wild-type littermates by cecal ligation and puncture. Blood pressure was monitored by radiotelemetry, and mesenteric blood flow was quantified by laser speckle contrast imaging. Blood biochemistry data were obtained using an iSTAT analyzer, and BH4 levels were measured in plasma and tissues by high-performance liquid chromatography. Increased BH4 and NO production and hypotension were observed in all mice, but the extents of these pathophysiological changes were attenuated in GFRP OE mice. Perturbations in blood biochemistry were similarly attenuated in GFRP OE compared with wild-type controls. These results suggest that GFRP overexpression regulates GCH1 activity during septic shock, which in turn limits BH4 bioavailability for iNOS. We conclude that the GCH1-GFRP axis is a critical regulator of BH4 and NO production and the cardiovascular derangements that occur in septic shock. PMID:25046538

  9. fadD deletion and fadL overexpression in Escherichia coli increase hydroxy long-chain fatty acid productivity.

    PubMed

    Bae, Jin H; Park, Beom Gi; Jung, Eunok; Lee, Pyung-Gang; Kim, Byung-Gee

    2014-11-01

    A major problem of long-chain fatty acid (LCFA) hydroxylation using Escherichia coli is that FadD (long-chain fatty acyl-CoA synthetase), which is necessary for exogenous LCFA transport, also initiates cellular consumption of LCFA. In this study, an effective method to prevent the cellular consumption of LCFA without impairing its transport is proposed. The main idea is that a heterologous enzyme which consumes LCFA can replace FadD in LCFA transport. For the model heterologous enzyme, CYP153A from Marinobacter aquaeolei, which converts palmitic acid into ω-hydroxy palmitic acid, was expressed in E. coli. When fadD was deleted from an E. coli strain, CYP153A indeed maintained the ability to transport LCFA. A disadvantage of fadD deletion mutant is the fact that FadD deficiency downregulates the transcription of fadL (outer membrane LCFA transporter) via FadR (fatty acid metabolism regulator protein), was solved by fadL overexpression from a plasmid. In addition, the overexpression of fadL was able to offset catabolite repression on fadL, allowing glucose to be used as the primary carbon source. In conclusion, the strain with fadD deletion and fadL overexpression showed 5.5-fold increase in productivity compared to the wild-type strain, converting 2.6 g/L (10.0 mM) of palmitic acid into 2.4 g/L (8.8 mM) of ω-hydroxy palmitic acid in a shake flask. This simple genetic manipulation can be applied to any LCFA hydroxylation using E. coli.

  10. Impaired growth and development of Colorado potato beetle larvae on potato plants overexpressing the oryzacystatin II gene

    USDA-ARS?s Scientific Manuscript database

    Plant proteinase inhibitors are attractive tools for crop improvement and their heterologous expression can enhance insect resistance in transgenic plants. Oryzacystatins I and II (OCI and OCII) show potential in controlling pests that utilize cysteine proteinases for protein digestion. To evaluate ...

  11. Impaired ventilatory acclimatization to hypoxia in female mice overexpressing erythropoietin: unexpected deleterious effect of estradiol in carotid bodies.

    PubMed

    Gassmann, Max; Pfistner, Christine; Doan, Van Diep; Vogel, Johannes; Soliz, Jorge

    2010-12-01

    Apart from enhancing the production of red blood cells, erythropoietin (Epo) alters the ventilatory response when oxygen supply is reduced. We recently demonstrated that Epo's beneficial effect on the ventilatory response to acute hypoxia is sex dependent, with female mice being better able to cope with reduced oxygenation. In the present work, we hypothesized that ventilatory acclimatization to chronic hypoxia (VAH) in transgenic female mice (Tg6) harboring high levels of Epo in the brain and blood will also be improved compared with wild-type (WT) animals. Surprisingly, VAH was blunted in Tg6 female mice. To define whether this phenomenon had a central (brain stem respiratory centers) and/or peripheral (carotid bodies) origin, a bilateral transection of carotid sinus nerve (chemodenervation) was performed. This procedure allowed the analysis of the central response in the absence of carotid body information. Interestingly, chemodenervation restored the VAH in Tg6 mice, suggesting that carotid bodies were responsible for the blunted response. Coherently with this observation, the sensitivity to oxygen alteration in arterial blood (Dejour test) after chronic hypoxia was lower in transgenic carotid bodies compared with the WT control. As blunted VAH occurred in female but not male transgenic mice, the involvement of sex female steroids was obvious. Indeed, measurement of sexual female hormones revealed that the estradiol serum level was 4 times higher in transgenic mice Tg6 than in WT animals. While ovariectomy decreased VAH in WT females, this treatment restored VAH in Tg6 female mice. In line with this observation, injections of estradiol in ovariectomized Tg6 females dramatically reduced the VAH. We concluded that during chronic hypoxia, estradiol in carotid bodies suppresses the Epo-mediated elevation of ventilation. Considering the increased application of recombinant Epo for a variety of disorders, our data imply the need to take the patient's hormonal status into consideration.

  12. Characterization of cognitive deficits in rats overexpressing human alpha-synuclein in the ventral tegmental area and medial septum using recombinant adeno-associated viral vectors.

    PubMed

    Hall, Hélène; Jewett, Michael; Landeck, Natalie; Nilsson, Nathalie; Schagerlöf, Ulrika; Leanza, Giampiero; Kirik, Deniz

    2013-01-01

    Intraneuronal inclusions containing alpha-synuclein (a-syn) constitute one of the pathological hallmarks of Parkinson's disease (PD) and are accompanied by severe neurodegeneration of A9 dopaminergic neurons located in the substantia nigra. Although to a lesser extent, A10 dopaminergic neurons are also affected. Neurodegeneration of other neuronal populations, such as the cholinergic, serotonergic and noradrenergic cell groups, has also been documented in PD patients. Studies in human post-mortem PD brains and in rodent models suggest that deficits in cholinergic and dopaminergic systems may be associated with the cognitive impairment seen in this disease. Here, we investigated the consequences of targeted overexpression of a-syn in the mesocorticolimbic dopaminergic and septohippocampal cholinergic pathways. Rats were injected with recombinant adeno-associated viral vectors encoding for either human wild-type a-syn or green fluorescent protein (GFP) in the ventral tegmental area and the medial septum/vertical limb of the diagonal band of Broca, two regions rich in dopaminergic and cholinergic neurons, respectively. Histopathological analysis showed widespread insoluble a-syn positive inclusions in all major projections areas of the targeted nuclei, including the hippocampus, neocortex, nucleus accumbens and anteromedial striatum. In addition, the rats overexpressing human a-syn displayed an abnormal locomotor response to apomorphine injection and exhibited spatial learning and memory deficits in the Morris water maze task, in the absence of obvious spontaneous locomotor impairment. As losses in dopaminergic and cholinergic immunoreactivity in both the GFP and a-syn expressing animals were mild-to-moderate and did not differ from each other, the behavioral impairments seen in the a-syn overexpressing animals appear to be determined by the long term persisting neuropathology in the surviving neurons rather than by neurodegeneration.

  13. Characterization of Cognitive Deficits in Rats Overexpressing Human Alpha-Synuclein in the Ventral Tegmental Area and Medial Septum Using Recombinant Adeno-Associated Viral Vectors

    PubMed Central

    Hall, Hélène; Jewett, Michael; Landeck, Natalie; Nilsson, Nathalie; Schagerlöf, Ulrika; Leanza, Giampiero; Kirik, Deniz

    2013-01-01

    Intraneuronal inclusions containing alpha-synuclein (a-syn) constitute one of the pathological hallmarks of Parkinson's disease (PD) and are accompanied by severe neurodegeneration of A9 dopaminergic neurons located in the substantia nigra. Although to a lesser extent, A10 dopaminergic neurons are also affected. Neurodegeneration of other neuronal populations, such as the cholinergic, serotonergic and noradrenergic cell groups, has also been documented in PD patients. Studies in human post-mortem PD brains and in rodent models suggest that deficits in cholinergic and dopaminergic systems may be associated with the cognitive impairment seen in this disease. Here, we investigated the consequences of targeted overexpression of a-syn in the mesocorticolimbic dopaminergic and septohippocampal cholinergic pathways. Rats were injected with recombinant adeno-associated viral vectors encoding for either human wild-type a-syn or green fluorescent protein (GFP) in the ventral tegmental area and the medial septum/vertical limb of the diagonal band of Broca, two regions rich in dopaminergic and cholinergic neurons, respectively. Histopathological analysis showed widespread insoluble a-syn positive inclusions in all major projections areas of the targeted nuclei, including the hippocampus, neocortex, nucleus accumbens and anteromedial striatum. In addition, the rats overexpressing human a-syn displayed an abnormal locomotor response to apomorphine injection and exhibited spatial learning and memory deficits in the Morris water maze task, in the absence of obvious spontaneous locomotor impairment. As losses in dopaminergic and cholinergic immunoreactivity in both the GFP and a-syn expressing animals were mild-to-moderate and did not differ from each other, the behavioral impairments seen in the a-syn overexpressing animals appear to be determined by the long term persisting neuropathology in the surviving neurons rather than by neurodegeneration. PMID:23705016

  14. Overexpression of Protochlorophyllide Oxidoreductase C Regulates Oxidative Stress in Arabidopsis

    PubMed Central

    Pattanayak, Gopal K.; Tripathy, Baishnab C.

    2011-01-01

    Light absorbed by colored intermediates of chlorophyll biosynthesis is not utilized in photosynthesis; instead, it is transferred to molecular oxygen, generating singlet oxygen (1O2). As there is no enzymatic detoxification mechanism available in plants to destroy 1O2, its generation should be minimized. We manipulated the concentration of a major chlorophyll biosynthetic intermediate i.e., protochlorophyllide in Arabidopsis by overexpressing the light-inducible protochlorophyllide oxidoreductase C (PORC) that effectively phototransforms endogenous protochlorophyllide to chlorophyllide leading to minimal accumulation of the photosensitizer protochlorophyllide in light-grown plants. In PORC overexpressing (PORCx) plants exposed to high-light, the 1O2 generation and consequent malonedialdehyde production was minimal and the maximum quantum efficiency of photosystem II remained unaffected demonstrating that their photosynthetic apparatus and cellular organization were intact. Further, PORCx plants treated with 5-aminolevulinicacid when exposed to light, photo-converted over-accumulated protochlorophyllide to chlorophyllide, reduced the generation of 1O2 and malonedialdehyde production and reduced plasma membrane damage. So PORCx plants survived and bolted whereas, the 5-aminolevulinicacid-treated wild-type plants perished. Thus, overexpression of PORC could be biotechnologically exploited in crop plants for tolerance to 1O2-induced oxidative stress, paving the use of 5-aminolevulinicacid as a selective commercial light-activated biodegradable herbicide. Reduced protochlorophyllide content in PORCx plants released the protochlorophyllide-mediated feed-back inhibition of 5-aminolevulinicacid biosynthesis that resulted in higher 5-aminolevulinicacid production. Increase of 5-aminolevulinicacid synthesis upregulated the gene and protein expression of several downstream chlorophyll biosynthetic enzymes elucidating a regulatory net work of expression of genes involved in 5

  15. Impaired Verb Fluency: A Sign of Mild Cognitive Impairment

    ERIC Educational Resources Information Center

    Ostberg, Per; Fernaeus, Sven-Erik; Hellstrom, Ake; Bogdanovic, Nenad; Wahlund, Lars Olof

    2005-01-01

    We assessed verb fluency vs. noun and letter-based fluency in 199 subjects referred for cognitive complaints including Subjective Cognitive Impairment, Mild Cognitive Impairment, and Alzheimer's disease. ANCOVAs and factor analyses identified verb, noun, and letter-based fluency as distinct tasks. Verb fluency performance in Mild Cognitive…

  16. Impaired Verb Fluency: A Sign of Mild Cognitive Impairment

    ERIC Educational Resources Information Center

    Ostberg, Per; Fernaeus, Sven-Erik; Hellstrom, Ake; Bogdanovic, Nenad; Wahlund, Lars Olof

    2005-01-01

    We assessed verb fluency vs. noun and letter-based fluency in 199 subjects referred for cognitive complaints including Subjective Cognitive Impairment, Mild Cognitive Impairment, and Alzheimer's disease. ANCOVAs and factor analyses identified verb, noun, and letter-based fluency as distinct tasks. Verb fluency performance in Mild Cognitive…

  17. Overexpression of a LAM domain containing RNA-binding protein LARP1c induces precocious leaf senescence in Arabidopsis.

    PubMed

    Zhang, Bangyue; Jia, Jianheng; Yang, Min; Yan, Chunxia; Han, Yuzhen

    2012-10-01

    Leaf senescence is the final stage of leaf life history, and it can be regulated by multiple internal and external cues. La-related proteins (LARPs), which contain a well-conserved La motif (LAM) domain and normally a canonical RNA recognition motif (RRM) or noncanonical RRM-like motif, are widely present in eukaryotes. Six LARP genes (LARP1a-1c and LARP6a-6c) are present in Arabidopsis, but their biological functions have not been studied previously. In this study, we investigated the biological roles of LARP1c from the LARP1 family. Constitutive or inducible overexpression of LARP1c caused premature leaf senescence. Expression levels of several senescence-associated genes and defense-related genes were elevated upon overexpression of LARP1c. The LARP1c null mutant 1c-1 impaired ABA-, SA-, and MeJA-induced leaf senescence in detached leaves. Gene expression profiles of LARP1c showed age-dependent expression in rosette leaves. Taken together, our results suggest LARP1c is involved in regulation of leaf senescence.

  18. Overexpression of a LAM Domain Containing RNA-Binding Protein LARP1c Induces Precocious Leaf Senescence in Arabidopsis

    PubMed Central

    Zhang, Bangyue; Jia, Jianheng; Yang, Min; Yan, Chunxia; Han, Yuzhen

    2012-01-01

    Leaf senescence is the final stage of leaf life history, and it can be regulated by multiple internal and external cues. La-related proteins (LARPs), which contain a well-conserved La motif (LAM) domain and normally a canonical RNA recognition motif (RRM) or noncanonical RRM-like motif, are widely present in eukaryotes. Six LARP genes (LARP1a-1c and LARP6a-6c) are present in Arabidopsis, but their biological functions have not been studied previously. In this study, we investigated the biological roles of LARP1c from the LARP1 family. Constitutive or inducible overexpression of LARP1c caused premature leaf senescence. Expression levels of several senescence-associated genes and defense-related genes were elevated upon overexpression of LARP1c. The LARP1c null mutant 1c-1 impaired ABA-, SA-, and MeJA-induced leaf senescence in detached leaves. Gene expression profiles of LARP1c showed age-dependent expression in rosette leaves. Taken together, our results suggest LARP1c is involved in regulation of leaf senescence. PMID:22965746

  19. Enhancing stress tolerance by overexpression of a methionine sulfoxide reductase A (MsrA) gene in Pleurotus ostreatus.

    PubMed

    Yin, Chaomin; Zheng, Liesheng; Zhu, Jihong; Chen, Liguo; Ma, Aimin

    2015-04-01

    Proteins are subjected to modification by reactive oxygen species (ROS), and oxidation of specific amino acid residues can impair their biological functions. Methionine as a sulfur-containing amino acid is easily oxidized to methionine sulfoxide (MetSO). The modified methionine can be repaired by methionine sulfoxide reductase (Msr), an enzyme that reverses oxidation of methionine in proteins. In this study, a methionine sulfoxide reductase A (PoMsrA) gene from Pleurotus ostreatus was cloned and characterized. Furthermore, the function of PoMsrA gene was analyzed by overexpression in P. ostreatus via Agrobacterium-mediated transformation. Stable integration of the target gene into the genome of P. ostreatus was confirmed by PCR, fluorescence observation, and Southern blot hybridization. qRT-PCR analysis showed that PoMsrA was highly expressed in the stage of mature and young fruiting bodies as well as the osmotic stress condition of 0.3 M NaCl. Additionally, the transgenic strains with PoMsrA overexpression exhibited an enhanced tolerance to high temperature, high osmotic stress, and oxidative stress. This suggests that PoMsrA is an active player in the protection of the cellular proteins from oxidative stress damage.

  20. Enhanced Stress Response in 5-HT1AR Overexpressing Mice: Altered HPA Function and Hippocampal Long-Term Potentiation.

    PubMed

    Pilar-Cuéllar, Fuencisla; Vidal, Rebeca; Díaz, Álvaro; Garro-Martínez, Emilio; Linge, Raquel; Castro, Elena; Haberzettl, Robert; Fink, Heidrun; Bert, Bettina; Brosda, Jan; Romero, Beatriz; Crespo-Facorro, Benedicto; Pazos, Ángel

    2017-08-18

    Postsynaptic 5-HT1A receptors (5-HT1AR) play an important role in anxiety and stress, although their contribution is still controversial. Previous studies report that mice overexpressing postsynaptic 5-HT1ARs show no changes in basal anxiety, though the influence of stress conditions has not been addressed yet. In this study, we used this animal model to evaluate the role of 5-HT1ARs in anxiety response after pre-exposure to an acute stressor. Under basal conditions, 5-HT1AR overexpressing animals presented high corticosterone levels and a lower mineralocorticoid/glucocorticoid receptor ratio. After pre-exposure to a single stressor, they showed a high anxiety-like response, associated with a blunted increase in corticosterone levels and higher c-Fos activation in the prefrontal cortex. Moreover, these mice also presented a lack of downregulation of hippocampal long-term potentiation after stress exposure. Therefore, higher postsynaptic 5-HT1AR activation might predispose to a high anxious phenotype and an impaired stress coping behavior.

  1. Over-expression of inducible HSP70 chaperone suppresses neuropathology and improves motor function in SCA1 mice.

    PubMed

    Cummings, C J; Sun, Y; Opal, P; Antalffy, B; Mestril, R; Orr, H T; Dillmann, W H; Zoghbi, H Y

    2001-07-01

    Many neurodegenerative diseases are caused by gain-of-function mechanisms in which the disease-causing protein is altered, becomes toxic to the cell, and aggregates. Among these 'proteinopathies' are Alzheimer's and Parkinson's disease, prion disorders and polyglutamine diseases. Members of this latter group, also known as triplet repeat diseases, are caused by the expansion of unstable CAG repeats coding for glutamine within the respective proteins. Spinocerebellar ataxia type 1 (SCA1) is one such disease, characterized by loss of motor coordination due to the degeneration of cerebellar Purkinje cells and brain stem neurons. In SCA1 and several other polyglutamine diseases, the expanded protein aggregates into nuclear inclusions (NIs). Because these NIs accumulate molecular chaperones, ubiquitin and proteasomal subunits--all components of the cellular protein re-folding and degradation machinery--we hypothesized that protein misfolding and impaired protein clearance might underlie the pathogenesis of polyglutamine diseases. Over-expressing specific chaperones reduces protein aggregation in transfected cells and suppresses neurodegeneration in invertebrate animal models of polyglutamine disorders. To determine whether enhancing chaperone activity could mitigate the phenotype in a mammalian model, we crossbred SCA1 mice with mice over-expressing a molecular chaperone (inducible HSP70 or iHSP70). We found that high levels of HSP70 did indeed afford protection against neurodegeneration.

  2. Overexpression of the mitochondrial methyltransferase TFB1M in the mouse does not impact mitoribosomal methylation status or hearing.

    PubMed

    Lee, Seungmin; Rose, Simon; Metodiev, Metodi D; Becker, Lore; Vernaleken, Alexandra; Klopstock, Thomas; Gailus-Durner, Valerie; Fuchs, Helmut; Hrabě De Angelis, Martin; Douthwaite, Stephen; Larsson, Nils-Göran

    2015-12-20

    Mitochondrial dysfunction is a well-established cause of sensorineural deafness, but the pathophysiological events are poorly understood. Non-syndromic deafness and predisposition to aminoglycoside-induced deafness can be caused by specific mutations in the 12S rRNA gene of mtDNA and are thus maternally inherited traits. The pathophysiology induced by mtDNA mutations has traditionally been attributed to deficient oxidative phosphorylation, which causes energy crisis with functional impairment of multiple cellular processes. In contrast, it was recently reported that signaling induced by 'hypermethylation' of two conserved adenosines of 12S rRNA in the mitoribosome is of key pathophysiological importance in sensorineural deafness. In support for this concept, it was reported that overexpression of the essential mitochondrial methyltransferase TFB1M in the mouse was sufficient to induce mitoribosomal hypermethylation and deafness. At variance with this model, we show here that 12S rRNA is near fully methylated in vivo in the mouse and thus cannot be further methylated to any significant extent. Furthermore, bacterial artificial chromosome transgenic mice overexpressing TFB1M have no increase of 12S rRNA methylation levels and hear normally. We thus conclude that therapies directed against mitoribosomal methylation are unlikely to be beneficial to patients with sensorineural hearing loss or other types of mitochondrial disease. © The Author 2015. Published by Oxford University Press.

  3. Developmental molecular and functional cerebellar alterations induced by PCP4/PEP19 overexpression: implications for Down syndrome.

    PubMed

    Mouton-Liger, François; Sahún, Ignasi; Collin, Thibault; Lopes Pereira, Patricia; Masini, Debora; Thomas, Sophie; Paly, Evelyne; Luilier, Sabrina; Même, Sandra; Jouhault, Quentin; Bennaï, Soumia; Beloeil, Jean-Claude; Bizot, Jean-Charles; Hérault, Yann; Dierssen, Mara; Créau, Nicole

    2014-03-01

    PCP4/PEP19 is a modulator of Ca(2+)-CaM signaling. In the brain, it is expressed in a very specific pattern in postmitotic neurons. In particular, Pcp4 is highly expressed in the Purkinje cell, the sole output neuron of the cerebellum. PCP4, located on human chromosome 21, is present in three copies in individuals with Down syndrome (DS). In a previous study using a transgenic mouse model (TgPCP4) to evaluate the consequences of 3 copies of this gene, we found that PCP4 overexpression induces precocious neuronal differentiation during mouse embryogenesis. Here, we report combined analyses of the cerebellum at postnatal stages (P14 and adult) in which we identified age-related molecular, electrophysiological, and behavioral alterations in the TgPCP4 mouse. While Pcp4 overexpression at P14 induces an earlier neuronal maturation, at adult stage it induces increase in cerebellar CaMK2alpha and in cerebellar LTD, as well as learning impairments. We therefore propose that PCP4 contributes significantly to the development of Down syndrome phenotypes through molecular and functional changes. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. LRRK2 overexpression alters glutamatergic presynaptic plasticity, striatal dopamine tone, postsynaptic signal transduction, motor activity and memory.

    PubMed

    Beccano-Kelly, Dayne A; Volta, Mattia; Munsie, Lise N; Paschall, Sarah A; Tatarnikov, Igor; Co, Kimberley; Chou, Patrick; Cao, Li-Ping; Bergeron, Sabrina; Mitchell, Emma; Han, Heather; Melrose, Heather L; Tapia, Lucia; Raymond, Lynn A; Farrer, Matthew J; Milnerwood, Austen J

    2015-03-01

    Mutations in leucine-rich repeat kinase 2 (Lrrk2) are the most common genetic cause of Parkinson's disease (PD), a neurodegenerative disorder affecting 1-2% of those >65 years old. The neurophysiology of LRRK2 remains largely elusive, although protein loss suggests a role in glutamatergic synapse transmission and overexpression studies show altered dopamine release in aged mice. We show that glutamate transmission is unaltered onto striatal projection neurons (SPNs) of adult LRRK2 knockout mice and that adult animals exhibit no detectable cognitive or motor deficits. Basal synaptic transmission is also unaltered in SPNs of LRRK2 overexpressing mice, but they do exhibit clear alterations to D2-receptor-mediated short-term synaptic plasticity, behavioral hypoactivity and impaired recognition memory. These phenomena are associated with decreased striatal dopamine tone and abnormal dopamine- and cAMP-regulated phosphoprotein 32 kDa signal integration. The data suggest that LRRK2 acts at the nexus of dopamine and glutamate signaling in the adult striatum, where it regulates dopamine levels, presynaptic glutamate release via D2-dependent synaptic plasticity and dopamine-receptor signal transduction.

  5. Overexpression of pyruvate kinase M2 associates with aggressive clinicopathological features and unfavorable prognosis in oral squamous cell carcinoma

    PubMed Central

    Wang, Yanling; Zhang, Xiaomin; Zhang, Yuchao; Zhu, Yuming; Yuan, Chunping; Qi, Bin; Zhang, Wei; Wang, Dongmiao; Ding, Xu; Wu, Heming; Cheng, Jie

    2015-01-01

    Abnormal glucose metabolism mediated by pyruvate kinase M2 (PKM2) fuels cancer overgrowth and propagation. However, its expression and oncogenic roles in in oral squamous cell carcinoma (OSCC) remains incompletely known. Here, we aimed to investigate the expression of PKM2, its prognostic values and oncogenic functions using 7,12-dimethyl-1,2-bezan-tracene (DMBA)-induced hamster buccal pouch SCC model, primary OSCC specimens as well as in vitro cellular assays. We found that in DMBA-induced OSCC model, negative PKM2 expression was commonly observed in normal epithelial, while more PKM2 abundance was detected in hyperplasia, dysplasia and SCC. Overexpression of PKM2 in a major fraction of OSCC significantly associated with tumor size (P = 0.027), cervical node metastasis (P = 0.004) and clinical stages (P = 0.000). Patients with increased PKM2 had remarkably reduced overall and disease-free survival. Multivariate survival analysis further revealed that PKM served as a critical independent prognostic factor for patients' overall survival. Furthermore, impaired cell proliferation and migration, and reduced apoptosis were detected upon PKM2 knockdown in HN4 and HN12 cells. Taken together, our findings reveal that PKM2 is critically involved in OSCC initiation and progression probably by promoting cell proliferation and migration as well as reducing apoptosis. Its overexpression correlates with aggressive clinicopathological features and poor patients' outcome. PMID:25970228

  6. Overexpression of Galectin-7 in Mouse Epidermis Leads to Loss of Cell Junctions and Defective Skin Repair

    PubMed Central

    Dang, Tien; Deshayes, Frédérique; Delacour, Delphine; Pichard, Evelyne; Advedissian, Tamara; Sidhu, Sukhvinder S.; Viguier, Mireille; Magnaldo, Thierry; Poirier, Francoise

    2015-01-01

    Background The proteins of the galectin family are implicated in many cellular processes, including cell interactions, polarity, intracellular trafficking, and signal transduction. In human and mouse, galectin-7 is almost exclusively expressed in stratified epithelia, notably in the epidermis. Galectin-7 expression is also altered in several human tumors of epithelial origin. This study aimed at dissecting the consequences of galectin-7 overexpression on epidermis structure and functions in vivo. Methods We established transgenic mice specifically overexpressing galectin-7 in the basal epidermal keratinocytes and analyzed the consequences on untreated skin and after UVB irradiation or mechanical injury. Results The intercellular cohesion of the epidermis is impaired in transgenic animals, with gaps developing between adjacent keratinocytes, associated with loss of adherens junctions. The epidermal architecture is aberrant with perturbations in the multilayered cellular organisation of the tissue, and structural defects in the basement membrane. These transgenic animals displayed a reduced re-epithelialisation potential following superficial wound, due to a defective collective migration of keratinocytes. Finally, a single mild dose of UVB induced an abnormal apoptotic response in the transgenic epidermis. Conclusion These results indicate that an excess of galectin-7 leads to a destabilisation of adherens junctions associated with defects in epidermal repair. As this phenotype shares similarities with that of galectin-7 null mutant mice, we conclude that a critical level of this protein is required for maintaining proper epidermal homeostasis. This study brings new insight into the mode of action of galectins in normal and pathological situations. PMID:25741714

  7. Overexpression of galectin-7 in mouse epidermis leads to loss of cell junctions and defective skin repair.

    PubMed

    Gendronneau, Gaëlle; Sanii, Sadaf; Dang, Tien; Deshayes, Frédérique; Delacour, Delphine; Pichard, Evelyne; Advedissian, Tamara; Sidhu, Sukhvinder S; Viguier, Mireille; Magnaldo, Thierry; Poirier, Francoise

    2015-01-01

    The proteins of the galectin family are implicated in many cellular processes, including cell interactions, polarity, intracellular trafficking, and signal transduction. In human and mouse, galectin-7 is almost exclusively expressed in stratified epithelia, notably in the epidermis. Galectin-7 expression is also altered in several human tumors of epithelial origin. This study aimed at dissecting the consequences of galectin-7 overexpression on epidermis structure and functions in vivo. We established transgenic mice specifically overexpressing galectin-7 in the basal epidermal keratinocytes and analyzed the consequences on untreated skin and after UVB irradiation or mechanical injury. The intercellular cohesion of the epidermis is impaired in transgenic animals, with gaps developing between adjacent keratinocytes, associated with loss of adherens junctions. The epidermal architecture is aberrant with perturbations in the multilayered cellular organisation of the tissue, and structural defects in the basement membrane. These transgenic animals displayed a reduced re-epithelialisation potential following superficial wound, due to a defective collective migration of keratinocytes. Finally, a single mild dose of UVB induced an abnormal apoptotic response in the transgenic epidermis. These results indicate that an excess of galectin-7 leads to a destabilisation of adherens junctions associated with defects in epidermal repair. As this phenotype shares similarities with that of galectin-7 null mutant mice, we conclude that a critical level of this protein is required for maintaining proper epidermal homeostasis. This study brings new insight into the mode of action of galectins in normal and pathological situations.

  8. Overexpression of zeaxanthin epoxidase gene enhances the sensitivity of tomato PSII photoinhibition to high light and chilling stress.

    PubMed

    Wang, Ning; Fang, Wei; Han, Han; Sui, Na; Li, Bin; Meng, Qing-Wei

    2008-03-01

    A tomato (Lycopersicon esculentum Mill.) zeaxanthin epoxidase gene (LeZE) was isolated. The deduced amino acid sequence of LeZE showed high identities with zeaxanthin epoxidase in other plant species. Northern blot analysis showed that the mRNA accumulation of LeZE in the wild-type (WT) was not induced by light and temperature but regulated by the diurnal rhythm. The sense transgenic plants were obtained under the control of the cauliflower mosaic virus 35S promoter (35S-CaMV). Northern and western blot analysis confirmed that sense LeZE was transferred into the tomato genome and overexpressed. The ratio of (A + Z)/(V + A + Z) and the values of non-photochemical quenching were lower in transgenic plants than in WT plants under high light and chilling stress with low irradiance. The O(2) evolution rate and the maximal photochemical efficiency of PSII (Fv/Fm) in transgenic plants decreased more quickly during both stresses and recovered slower than that in WT under optimal conditions. These results suggested that overexpression of LeZE impaired the function of the xanthophyll cycle and aggravated PSII photoinhibition in tomato under high light and chilling stress.

  9. Overexpression of the mitochondrial methyltransferase TFB1M in the mouse does not impact mitoribosomal methylation status or hearing

    PubMed Central

    Lee, Seungmin; Rose, Simon; Metodiev, Metodi D.; Becker, Lore; Vernaleken, Alexandra; Klopstock, Thomas; Gailus-Durner, Valerie; Fuchs, Helmut; Hrabě De Angelis, Martin; Douthwaite, Stephen; Larsson, Nils-Göran

    2015-01-01

    Mitochondrial dysfunction is a well-established cause of sensorineural deafness, but the pathophysiological events are poorly understood. Non-syndromic deafness and predisposition to aminoglycoside-induced deafness can be caused by specific mutations in the 12S rRNA gene of mtDNA and are thus maternally inherited traits. The pathophysiology induced by mtDNA mutations has traditionally been attributed to deficient oxidative phosphorylation, which causes energy crisis with functional impairment of multiple cellular processes. In contrast, it was recently reported that signaling induced by ‘hypermethylation’ of two conserved adenosines of 12S rRNA in the mitoribosome is of key pathophysiological importance in sensorineural deafness. In support for this concept, it was reported that overexpression of the essential mitochondrial methyltransferase TFB1M in the mouse was sufficient to induce mitoribosomal hypermethylation and deafness. At variance with this model, we show here that 12S rRNA is near fully methylated in vivo in the mouse and thus cannot be further methylated to any significant extent. Furthermore, bacterial artificial chromosome transgenic mice overexpressing TFB1M have no increase of 12S rRNA methylation levels and hear normally. We thus conclude that therapies directed against mitoribosomal methylation are unlikely to be beneficial to patients with sensorineural hearing loss or other types of mitochondrial disease. PMID:26464487

  10. Prevalent overexpression of prolyl isomerase Pin1 in human cancers.

    PubMed

    Bao, Lere; Kimzey, Amy; Sauter, Guido; Sowadski, Janusz M; Lu, Kun Ping; Wang, Da-Gong

    2004-05-01

    Phosphorylation of proteins on serine or threonine residues preceding proline (pSer/Thr-Pro) is a major regulatory mechanism in cell proliferation and transformation. Interestingly, the pSer/Thr-Pro motifs in proteins exist in two distinct cis and trans conformations, whose conversion rate is normally reduced on phosphorylation, but is catalyzed specifically by the prolyl isomerase Pin1. Pin1 can catalytically induce conformational changes in proteins after phosphorylation, thereby having profound effects on catalytic activity, dephosphorylation, protein-protein interactions, subcellular location, and/or turnover of certain phosphorylated proteins. Recently, it has been shown that Pin1 is overexpressed in human breast cancer cell lines and cancer tissues and plays a critical role in the transformation of mammary epithelial cells by activating multiple oncogenic pathways. Furthermore, Pin1 expression is an excellent independent prognostic marker in prostate cancer. However, little is known about Pin1 expression in other human normal and cancerous tissues. In the present study, we quantified Pin1 expression in 2041 human tumor samples and 609 normal tissue samples as well as normal and transformed human cell lines. We found that Pin1 was usually expressed at very low levels in most normal tissues and its expression was normally associated with cell proliferation, with high Pin1 levels being found only in a few cell types. However, Pin1 was strikingly overexpressed in many different human cancers. Most tumors (38 of 60 tumor types) have Pin1 overexpression in more than 10% of the cases, as compared with the corresponding normal controls, which included prostate, lung, ovary, cervical, brain tumors, and melanoma. Consistent with these findings, Pin1 expression in human cancer cell lines was also higher than that in the normal cell lines examined. These results indicate that Pin1 overexpression is a prevalent and specific event in human cancers. Given previous findings

  11. Oxidative stress-induced overexpression of miR-25: the mechanism underlying the degeneration of melanocytes in vitiligo.

    PubMed

    Shi, Q; Zhang, W; Guo, S; Jian, Z; Li, S; Li, K; Ge, R; Dai, W; Wang, G; Gao, T; Li, C

    2016-03-01

    Oxidative stress has a critical role in the pathogenesis of vitiligo. However, the specific molecular mechanism involved in oxidative stress-induced melanocyte death is not well characterized. Given the powerful role of microRNAs (miRNAs) in the regulation of cell survival as well as the fact that the generation of miRNAs can be affected by oxidative stress, we hypothesized that miRNAs may participate in vitiligo pathogenesis by modulating the expression of vital genes in melanocytes. In the present study, we initially found that miR-25 was increased in both serum and lesion samples from vitiligo patients, and its serum level was correlated with the activity of vitiligo. Moreover, restoration of miR-25 promoted the H2O2-induced melanocyte destruction and led to the dysfunction of melanocytes. Further experiments proved that MITF, a master regulator in melanocyte survival and function, accounted for the miR-25-caused damaging impact on melanocytes. Notably, other than the direct role on melanocytes, we observed that miR-25 inhibited the production and secretion of SCF and bFGF from keratinocytes, thus impairing their paracrine protective effect on the survival of melanocytes under oxidative stress. At last, we verified that oxidative stress could induce the overexpression of miR-25 in both melanocytes and keratinocytes possibly by demethylating the promoter region of miR-25. Taken together, our study demonstrates that oxidative stress-induced overexpression of miR-25 in vitiligo has a crucial role in promoting the degeneration of melanocytes by not only suppressing MITF in melanocytes but also impairing the paracrine protective effect of keratinocytes. Therefore, it is worthy to investigate the possibility of miR-25 as a potential drug target for anti-oxidative therapy in vitiligo.

  12. Impact of transgenic overexpression of SH2-containing inositol 5'-phosphatase 2 on glucose metabolism and insulin signaling in mice.

    PubMed

    Kagawa, Syota; Soeda, Yoshiyuki; Ishihara, Hajime; Oya, Takeshi; Sasahara, Masakiyo; Yaguchi, Saori; Oshita, Ryo; Wada, Tsutomu; Tsuneki, Hiroshi; Sasaoka, Toshiyasu

    2008-02-01

    SH2-containing inositol 5'-phosphatase 2 (SHIP2) is a 5'-lipid phosphatase hydrolyzing the phosphatidylinositol (PI) 3-kinase product PI(3,4,5)P(3) to PI(3,4)P(2) in the regulation of insulin signaling, and is shown to be increased in peripheral tissues of diabetic C57BL/KSJ-db/db mice. To clarify the impact of SHIP2 in the pathogenesis of insulin resistance with type 2 diabetes, we generated transgenic mice overexpressing SHIP2. The body weight of transgenic mice increased by 5.0% (P < 0.05) compared with control wild-type littermates on a normal chow diet, but not on a high-fat diet. Glucose tolerance and insulin sensitivity were mildly but significantly impaired in the transgenic mice only when maintained on the normal chow diet, as shown by 1.2-fold increase in glucose area under the curve over control levels at 9 months old. Insulin-induced phosphorylation of Akt was decreased in the SHIP2-overexpressing fat, skeletal muscle, and liver. In addition, the expression of hepatic mRNAs for glucose-6-phosphatase and phosphoenolpyruvate carboxykinase was increased, that for sterol regulatory element-binding protein 1 was unchanged, and that for glucokinase was decreased. Consistently, hepatic glycogen content was reduced in the 9-month-old transgenic mice. Structure and insulin content were histologically normal in the pancreatic islets of transgenic mice. These results indicate that increased abundance of SHIP2 in vivo contributes, at least in part, to the impairment of glucose metabolism and insulin sensitivity on a normal chow diet, possibly by attenuating peripheral insulin signaling and by altering hepatic gene expression for glucose homeostasis.

  13. Overexpression of Hsp20 prevents endotoxin-induced myocardial dysfunction and apoptosis via inhibition of NF-kappaB activation.

    PubMed

    Wang, Xiaohong; Zingarelli, Basilia; O'Connor, Michael; Zhang, Pengyuan; Adeyemo, Adeola; Kranias, Evangelia G; Wang, Yigang; Fan, Guo-Chang

    2009-09-01

    The occurrence of cardiovascular dysfunction in sepsis is associated with a significantly increased mortality rate of 70% to 90% compared with 20% in septic patients without cardiovascular impairment. Thus, rectification or blockade of myocardial depressant factors should partly ameliorate sepsis progression. Heat shock protein 20 (Hsp20) has been shown to enhance myocardial contractile function and protect against doxorubicin-induced cardiotoxicity. To investigate the possible role of Hsp20 in sepsis-mediated cardiac injury, we first examined the expression profiles of five major Hsps in response to lipopolysaccharide (LPS) challenge, and observed that only the expression of Hsp20 was downregulated in LPS-treated myocardium, suggesting that this decrease might be one of the mechanisms contributing to LPS-induced cardiovascular defects. Further studies using loss-of-function and gain-of-function approaches in adult rat cardiomyocytes verified that reduced Hsp20 levels were indeed correlated with the impaired contractile function. In fact, overexpression of Hsp20 significantly enhanced cardiomyocyte contractility upon LPS treatment. Moreover, after administration of LPS (25 microg/g) in vivo, Hsp20 transgenic mice (10-fold overexpression) displayed: 1) an improvement in myocardial function; 2) reduced the degree of cardiac apoptosis; and 3) decreased NF-kappaB activity, accompanied with reduced myocardial cytokines IL-1beta and TNF-alpha production, compared to the LPS-treated non-transgenic littermate controls. Thus, the increases in Hsp20 levels can protect against LPS-induced cardiac apoptosis and dysfunction, associated with inhibition of NF-kappaB activity, suggesting that Hsp20 may be a new therapeutic agent for the treatment of sepsis.

  14. Overexpression of endothelial nitric oxide synthase improves endothelium-dependent vasodilation in arteries infused with helper-dependent adenovirus.

    PubMed

    Jiang, Bo; Du, Liang; Flynn, Rowan; Dronadula, Nagadhara; Zhang, Jingwan; Kim, Francis; Dichek, David

    2012-11-01

    Adenoviral vectors (Ad) are useful tools for in vivo gene transfer into endothelial cells. However, endothelium-dependent vasodilation is impaired after Ad infusion, and this impairment is not prevented by use of advanced-generation "helper-dependent" (HD) Ad that lack all viral genes. We hypothesized that endothelium-dependent vasodilation could be improved in Ad-infused arteries by overexpression of endothelial nitric oxide synthase (eNOS). We tested this hypothesis in hyperlipidemic, atherosclerosis-prone rabbits because HDAd will likely be used for treating and preventing atherosclerosis. Moreover, the consequences of eNOS overexpression might differ in normal and atherosclerosis-prone arteries and could include atherogenic effects, as reported in transgenic mice. We cloned rabbit eNOS and constructed an HDAd that expresses it. HDAdeNOS increased NO production by cultured endothelial cells and increased arterial eNOS mRNA in vivo by ∼10-fold. Compared to arteries infused with a control HDAd, HDAdeNOS-infused arteries of hyperlipidemic rabbits had significantly improved endothelium-dependent vasodilation, and similar responses to phenylephrine and nitroprusside. Moreover, infusion of HDAdeNOS had local atheroprotective effects including large, significant decreases in intimal lipid accumulation and arterial tumor necrosis factor (TNF)-α expression (p≤0.04 for both). HDAdeNOS infusion yields a durable (≥2 weeks) increase in arterial eNOS expression, improves vasomotor function, and reduces artery wall inflammation and lipid accumulation. Addition of an eNOS expression cassette improves the performance of HDAd, has no harmful effects, and may reduce atherosclerotic lesion growth.

  15. Overexpression of Endothelial Nitric Oxide Synthase Improves Endothelium-Dependent Vasodilation in Arteries Infused with Helper-Dependent Adenovirus

    PubMed Central

    Jiang, Bo; Du, Liang; Flynn, Rowan; Dronadula, Nagadhara; Zhang, Jingwan; Kim, Francis

    2012-01-01

    Abstract Adenoviral vectors (Ad) are useful tools for in vivo gene transfer into endothelial cells. However, endothelium-dependent vasodilation is impaired after Ad infusion, and this impairment is not prevented by use of advanced-generation “helper-dependent” (HD) Ad that lack all viral genes. We hypothesized that endothelium-dependent vasodilation could be improved in Ad-infused arteries by overexpression of endothelial nitric oxide synthase (eNOS). We tested this hypothesis in hyperlipidemic, atherosclerosis-prone rabbits because HDAd will likely be used for treating and preventing atherosclerosis. Moreover, the consequences of eNOS overexpression might differ in normal and atherosclerosis-prone arteries and could include atherogenic effects, as reported in transgenic mice. We cloned rabbit eNOS and constructed an HDAd that expresses it. HDAdeNOS increased NO production by cultured endothelial cells and increased arterial eNOS mRNA in vivo by ∼10-fold. Compared to arteries infused with a control HDAd, HDAdeNOS-infused arteries of hyperlipidemic rabbits had significantly improved endothelium-dependent vasodilation, and similar responses to phenylephrine and nitroprusside. Moreover, infusion of HDAdeNOS had local atheroprotective effects including large, significant decreases in intimal lipid accumulation and arterial tumor necrosis factor (TNF)-α expression (p≤0.04 for both). HDAdeNOS infusion yields a durable (≥2 weeks) increase in arterial eNOS expression, improves vasomotor function, and reduces artery wall inflammation and lipid accumulation. Addition of an eNOS expression cassette improves the performance of HDAd, has no harmful effects, and may reduce atherosclerotic lesion growth. PMID:22906141

  16. Oxidative stress-induced overexpression of miR-25: the mechanism underlying the degeneration of melanocytes in vitiligo

    PubMed Central

    Shi, Q; Zhang, W; Guo, S; Jian, Z; Li, S; Li, K; Ge, R; Dai, W; Wang, G; Gao, T; Li, C

    2016-01-01

    Oxidative stress has a critical role in the pathogenesis of vitiligo. However, the specific molecular mechanism involved in oxidative stress-induced melanocyte death is not well characterized. Given the powerful role of microRNAs (miRNAs) in the regulation of cell survival as well as the fact that the generation of miRNAs can be affected by oxidative stress, we hypothesized that miRNAs may participate in vitiligo pathogenesis by modulating the expression of vital genes in melanocytes. In the present study, we initially found that miR-25 was increased in both serum and lesion samples from vitiligo patients, and its serum level was correlated with the activity of vitiligo. Moreover, restoration of miR-25 promoted the H2O2-induced melanocyte destruction and led to the dysfunction of melanocytes. Further experiments proved that MITF, a master regulator in melanocyte survival and function, accounted for the miR-25-caused damaging impact on melanocytes. Notably, other than the direct role on melanocytes, we observed that miR-25 inhibited the production and secretion of SCF and bFGF from keratinocytes, thus impairing their paracrine protective effect on the survival of melanocytes under oxidative stress. At last, we verified that oxidative stress could induce the overexpression of miR-25 in both melanocytes and keratinocytes possibly by demethylating the promoter region of miR-25. Taken together, our study demonstrates that oxidative stress-induced overexpression of miR-25 in vitiligo has a crucial role in promoting the degeneration of melanocytes by not only suppressing MITF in melanocytes but also impairing the paracrine protective effect of keratinocytes. Therefore, it is worthy to investigate the possibility of miR-25 as a potential drug target for anti-oxidative therapy in vitiligo. PMID:26315342

  17. Paradoxical resistance to high-fat diet-induced obesity and altered macrophage polarization in mineralocorticoid receptor-overexpressing mice.

    PubMed

    Kuhn, Emmanuelle; Bourgeois, Christine; Keo, Vixra; Viengchareun, Say; Muscat, Adeline; Meduri, Geri; Le Menuet, Damien; Fève, Bruno; Lombès, Marc

    2014-01-01

    The mineralocorticoid receptor (MR) exerts proadipogenic and antithermogenic effects in vitro, yet its in vivo metabolic impact remains elusive. Wild type (WT) and transgenic (Tg) mice overexpressing human MR were subjected to standard chow (SC) or high-fat diet (HFD) for 16 wk. Tg mice had a lower body weight gain than WT animals and exhibited a relative resistance to HFD-induced obesity. This was associated with a decrease in fat mass, an increased population of smaller adipocytes, and an improved glucose tolerance compared with WT animals. Quantitative RT-PCR studies revealed decreased expression of PPARγ2, a master adipogenic gene, and of glucocorticoid receptor and 11β-hydroxysteroid dehydrogenase type 1, consistent with an impaired local glucocorticoid signaling in adipose tissues (AT). This paradoxical resistance to HFD-induced obesity was not related to an adipogenesis defect since differentiation capacity of Tg preadipocytes isolated from stroma-vascular fractions was unaltered, suggesting that other nonadipocyte factors might compromise AT development. Although AT macrophage infiltration was not different between genotypes, Tg mice exhibited a distinct macrophage polarization, as revealed by FACS analysis and CD11c/CD206 expression studies. We further demonstrated that Tg macrophage-conditioned medium partially impaired preadipocyte differentiation. Therefore, we propose that modification of M1/M2 polarization of hMR-overexpressing macrophages could account in part for the metabolic phenotype of Tg mice. Collectively, our results provide evidence that MR exerts a pivotal immunometabolic role by controlling adipocyte differentiation processes directly but also indirectly through macrophage polarization regulation. Our findings should be taken into account for the pharmacological treatment of metabolic disorders.

  18. Fibroblasts of Machado Joseph Disease patients reveal autophagy impairment

    PubMed Central

    Onofre, Isabel; Mendonça, Nuno; Lopes, Sara; Nobre, Rui; de Melo, Joana Barbosa; Carreira, Isabel Marques; Januário, Cristina; Gonçalves, António Freire; de Almeida, Luis Pereira

    2016-01-01

    Machado Joseph Disease (MJD) is the most frequent autosomal dominantly inherited cerebellar ataxia caused by the over-repetition of a CAG trinucleotide in the ATXN3 gene. This expansion translates into a polyglutamine tract within the ataxin-3 protein that confers a toxic gain-of-function to the mutant protein ataxin-3, contributing to protein misfolding and intracellular accumulation of aggregates and neuronal degeneration. Autophagy impairment has been shown to be one of the mechanisms that contribute for the MJD phenotype. Here we investigated whether this phenotype was present in patient-derived fibroblasts, a common somatic cell type used in the derivation of induced pluripotent stem cells and subsequent differentiation into neurons, for in vitro disease modeling. We generated and studied adult dermal fibroblasts from 5 MJD patients and 4 healthy individuals and we found that early passage MJD fibroblasts exhibited autophagy impairment with an underlying mechanism of decreased autophagosome production. The overexpression of beclin-1 on MJD fibroblasts reverted partially autophagy impairment by increasing the autophagic flux but failed to increase the levels of autophagosome production. Overall, our results provide a well-characterized MJD fibroblast resource for neurodegenerative disease research and contribute for the understanding of mutant ataxin-3 biology and its molecular consequences. PMID:27328712

  19. KCNQ channels regulate age-related memory impairment.

    PubMed

    Cavaliere, Sonia; Malik, Bilal R; Hodge, James J L

    2013-01-01

    In humans KCNQ2/3 heteromeric channels form an M-current that acts as a brake on neuronal excitability, with mutations causing a form of epilepsy. The M-current has been shown to be a key regulator of neuronal plasticity underlying associative memory and ethanol response in mammals. Previous work has shown that many of the molecules and plasticity mechanisms underlying changes in alcohol behaviour and addiction are shared with those of memory. We show that the single KCNQ channel in Drosophila (dKCNQ) when mutated show decrements in associative short- and long-term memory, with KCNQ function in the mushroom body α/βneurons being required for short-term memory. Ethanol disrupts memory in wildtype flies, but not in a KCNQ null mutant background suggesting KCNQ maybe a direct target of ethanol, the blockade of which interferes with the plasticity machinery required for memory formation. We show that as in humans, Drosophila display age-related memory impairment with the KCNQ mutant memory defect mimicking the effect of age on memory. Expression of KCNQ normally decreases in aging brains and KCNQ overexpression in the mushroom body neurons of KCNQ mutants restores age-related memory impairment. Therefore KCNQ is a central plasticity molecule that regulates age dependent memory impairment.

  20. Overexpression of calpastatin inhibits L8 myoblast fusion

    SciTech Connect

    Barnoy, Sivia; E-mail: sivia@post.tau.ac.il; Maki, Masatoshi; Kosower, Nechama S.

    2005-07-08

    The formation of skeletal muscle fibers involves cessation of myoblast division, myoblast alignment, and fusion to multinucleated myofibers. Calpain is one of the factors shown to be involved in myoblast fusion. Using L8 rat myoblasts, we found that calpain levels did not change significantly during myoblast differentiation, whereas calpastatin diminished prior to myoblast fusion and reappeared after fusion. The transient diminution in calpastatin allows the Ca{sup 2+}-promoted activation of calpain and calpain-induced membrane proteolysis, which is required for myoblast fusion. Here we show that calpastatin overexpression in L8 myoblasts does not inhibit cell proliferation and alignment, but prevents myoblast fusion and fusion-associated protein degradation. In addition, calpastatin appears to modulate myogenic gene expression, as indicated by the lack of myogenin (a transcription factor expressed in differentiating myoblasts) in myoblasts overexpressing calpastatin. These results suggest that, in addition to the role in membrane disorganization in the fusing myoblasts, the calpain-calpastatin system may also modulate the levels of factors required for myoblast differentiation.

  1. Tubular Overexpression of Angiopoietin-1 Attenuates Renal Fibrosis

    PubMed Central

    Lee, Heedoo; Kim, Yeawon; Liu, Tuoen; Guo, Qiusha; Geminiani, Julio J.; Austin, Paul F.; Chen, Ying Maggie

    2016-01-01

    Emerging evidence has highlighted the pivotal role of microvasculature injury in the development and progression of renal fibrosis. Angiopoietin-1 (Ang-1) is a secreted vascular growth factor that binds to the endothelial-specific Tie2 receptor. Ang-1/Tie2 signaling is critical for regulating blood vessel development and modulating vascular response after injury, but is dispensable in mature, quiescent vessels. Although dysregulation of vascular endothelial growth factor (VEGF) signaling has been well studied in renal pathologies, much less is known about the role of the Ang-1/Tie2 pathway in renal interstitial fibrosis. Previous studies have shown contradicting effects of overexpressing Ang-1 systemically on renal tubulointerstitial fibrosis when different engineered forms of Ang-1 are used. Here, we investigated the impact of site-directed expression of native Ang-1 on the renal fibrogenic process and peritubular capillary network by exploiting a conditional transgenic mouse system [Pax8-rtTA/(TetO)7 Ang-1] that allows increased tubular Ang-1 production in adult mice. Using a murine unilateral ureteral obstruction (UUO) fibrosis model, we demonstrate that targeted Ang-1 overexpression attenuates myofibroblast activation and interstitial collagen I accumulation, inhibits the upregulation of transforming growth factor β1 and subsequent phosphorylation of Smad 2/3, dampens renal inflammation, and stimulates the growth of peritubular capillaries in the obstructed kidney. Our results suggest that Ang-1 is a potential therapeutic agent for targeting microvasculature injury in renal fibrosis without compromising the physiologically normal vasculature in humans. PMID:27454431

  2. Overexpression of Pto activates defense responses and confers broad resistance.

    PubMed

    Tang, X; Xie, M; Kim, Y J; Zhou, J; Klessig, D F; Martin, G B

    1999-01-01

    The tomato disease resistance (R) gene Pto specifies race-specific resistance to the bacterial pathogen Pseudomonas syringae pv tomato carrying the avrPto gene. Pto encodes a serine/threonine protein kinase that is postulated to be activated by a physical interaction with the AvrPto protein. Here, we report that overexpression of Pto in tomato activates defense responses in the absence of the Pto-AvrPto interaction. Leaves of three transgenic tomato lines carrying the cauliflower mosaic virus 35S::Pto transgene exhibited microscopic cell death, salicylic acid accumulation, and increased expression of pathogenesis-related genes. Cell death in these plants was limited to palisade mesophyll cells and required light for induction. Mesophyll cells of 35S::Pto plants showed the accumulation of autofluorescent compounds, callose deposition, and lignification. When inoculated with P. s. tomato without avrPto, all three 35S::Pto lines displayed significant resistance and supported less bacterial growth than did nontransgenic lines. Similarly, the 35S::Pto lines also were more resistant to Xanthomonas campestris pv vesicatoria and Cladosporium fulvum. These results demonstrate that defense responses and general resistance can be activated by the overexpression of an R gene.

  3. Nitrate metabolism in tobacco leaves overexpressing Arabidopsis nitrite reductase.

    PubMed

    Davenport, Susie; Le Lay, Pascaline; Sanchez-Tamburrrino, Juan Pablo

    2015-12-01

    Primary nitrogen assimilation in plants includes the reduction of nitrite to ammonium in the chloroplasts by the enzyme nitrite reductase (NiR EC:1.7.7.1) or in the plastids of non-photosynthetic organs. Here we report on a study overexpressing the Arabidopsis thaliana NiR (AtNiR) gene in tobacco plants under the control of a constitutive promoter (CERV - Carnation Etched Ring Virus). The aim was to overexpress AtNiR in an attempt to alter the level of residual nitrite in the leaf which can act as precursor to the formation of nitrosamines. The impact of increasing the activity of AtNiR produced an increase in leaf protein and a stay-green phenotype in the primary transformed AtNiR population. Investigation of the T1 homozygous population demonstrated elevated nitrate reductase (NR) activity, reductions in leaf nitrite and nitrate and the amino acids proline, glutamine and glutamate. Chlorophyl content of the transgenic lines was increased, as evidenced by the stay-green phenotype. This reveals the importance of NiR in primary nitrogen assimilation and how modification of this key enzyme affects both the nitrogen and carbon metabolism of tobacco plants.

  4. Prothymosin α overexpression contributes to the development of pulmonary emphysema.

    PubMed

    Su, Bing-Hua; Tseng, Yau-Lin; Shieh, Gia-Shing; Chen, Yi-Cheng; Shiang, Ya-Chieh; Wu, Pensee; Li, Kuo-Jung; Yen, Te-Hsin; Shiau, Ai-Li; Wu, Chao-Liang

    2013-01-01

    Emphysema is one of the disease conditions that comprise chronic obstructive pulmonary disease. Prothymosin α transgenic mice exhibit an emphysema phenotype, but the pathophysiological role of prothymosin α in emphysema remains unclear. Here we show that prothymosin α contributes to the pathogenesis of emphysema by increasing acetylation of histones and nuclear factor-kappaB, particularly upon cigarette smoke exposure. We find a positive correlation between prothymosin α levels and the severity of emphysema in prothymosin α transgenic mice and emphysema patients. Prothymosin α overexpression increases susceptibility to cigarette smoke-induced emphysema, and cigarette smoke exposure further enhances prothymosin α expression. We show that prothymosin α inhibits the association of histone deacetylases with histones and nuclear factor-kappaB, and that prothymosin α overexpression increases expression of nuclear factor-kappaB-dependent matrix metalloproteinase 2 and matrix metalloproteinase 9, which are found in the lungs of patients with chronic obstructive pulmonary disease. These results demonstrate the clinical relevance of prothymosin α in regulating acetylation events during the pathogenesis of emphysema.

  5. Overexpression of host plant urease in transgenic silkworms.

    PubMed

    Jiang, Liang; Huang, Chunlin; Sun, Qiang; Guo, Huizhen; Peng, Zhengwen; Dang, Yinghui; Liu, Weiqiang; Xing, Dongxu; Xu, Guowen; Zhao, Ping; Xia, Qingyou

    2015-06-01

    Bombyx mori and mulberry constitute a model of insect-host plant interactions. Urease hydrolyzes urea to ammonia and is important for the nitrogen metabolism of silkworms because ammonia is assimilated into silk protein. Silkworms do not synthesize urease and acquire it from mulberry leaves. We synthesized the artificial DNA sequence ureas using the codon bias of B. mori to encode the signal peptide and mulberry urease protein. A transgenic vector that overexpresses ure-as under control of the silkworm midgut-specific P2 promoter was constructed. Transgenic silkworms were created via embryo microinjection. RT-PCR results showed that urease was expressed during the larval stage and qPCR revealed the expression only in the midgut of transgenic lines. Urea concentration in the midgut and hemolymph of transgenic silkworms was significantly lower than in a nontransgenic line when silkworms were fed an artificial diet. Analysis of the daily body weight and food conversion efficiency of the fourth and fifth instar larvae and economic characteristics indicated no differences between transgenic silkworms and the nontransgenic line. These results suggested that overexpression of host plant urease promoted nitrogen metabolism in silkworms.

  6. Prothymosin α overexpression contributes to the development of pulmonary emphysema

    PubMed Central

    Su, Bing-Hua; Tseng, Yau-Lin; Shieh, Gia-Shing; Chen, Yi-Cheng; Shiang, Ya-Chieh; Wu, Pensee; Li, Kuo-Jung; Yen, Te-Hsin; Shiau, Ai-Li; Wu, Chao-Liang

    2013-01-01

    Emphysema is one of the disease conditions that comprise chronic obstructive pulmonary disease. Prothymosin α transgenic mice exhibit an emphysema phenotype, but the pathophysiological role of prothymosin α in emphysema remains unclear. Here we show that prothymosin α contributes to the pathogenesis of emphysema by increasing acetylation of histones and nuclear factor-kappaB, particularly upon cigarette smoke exposure. We find a positive correlation between prothymosin α levels and the severity of emphysema in prothymosin α transgenic mice and emphysema patients. Prothymosin α overexpression increases susceptibility to cigarette smoke-induced emphysema, and cigarette smoke exposure further enhances prothymosin α expression. We show that prothymosin α inhibits the association of histone deacetylases with histones and nuclear factor-kappaB, and that prothymosin α overexpression increases expression of nuclear factor-kappaB-dependent matrix metalloproteinase 2 and matrix metalloproteinase 9, which are found in the lungs of patients with chronic obstructive pulmonary disease. These results demonstrate the clinical relevance of prothymosin α in regulating acetylation events during the pathogenesis of emphysema. PMID:23695700

  7. Overexpression of neurofilament H disrupts normal cell structure and function

    NASA Technical Reports Server (NTRS)

    Szebenyi, Gyorgyi; Smith, George M.; Li, Ping; Brady, Scott T.

    2002-01-01

    Studying exogenously expressed tagged proteins in live cells has become a standard technique for evaluating protein distribution and function. Typically, expression levels of experimentally introduced proteins are not regulated, and high levels are often preferred to facilitate detection. However, overexpression of many proteins leads to mislocalization and pathologies. Therefore, for normative studies, moderate levels of expression may be more suitable. To understand better the dynamics of intermediate filament formation, transport, and stability in a healthy, living cell, we inserted neurofilament heavy chain (NFH)-green fluorescent protein (GFP) fusion constructs in adenoviral vectors with tetracycline (tet)-regulated promoters. This system allows for turning on or off the synthesis of NFH-GFP at a selected time, for a defined period, in a dose-dependent manner. We used this inducible system for live cell imaging of changes in filament structure and cell shape, motility, and transport associated with increasing NFH-GFP expression. Cells with low to intermediate levels of NFH-GFP were structurally and functionally similar to neighboring, nonexpressing cells. In contrast, overexpression led to pathological alterations in both filament organization and cell function. Copyright 2002 Wiley-Liss, Inc.

  8. SNEV overexpression extends the life span of human endothelial cells

    SciTech Connect

    Voglauer, Regina; Chang, Martina Wei-Fen; Dampier, Brigitta; Wieser, Matthias; Baumann, Kristin; Sterovsky, Thomas; Schreiber, Martin; Katinger, Hermann; Grillari, Johannes . E-mail: j.grillari@iam.boku.ac.at

    2006-04-01

    In a recent screening for genes downregulated in replicatively senescent human umbilical vein endothelial cells (HUVECs), we have isolated the novel protein SNEV. Since then SNEV has proven as a multifaceted protein playing a role in pre-mRNA splicing, DNA repair, and the ubiquitin/proteosome system. Here, we report that SNEV mRNA decreases in various cell types during replicative senescence, and that it is increased in various immortalized cell lines, as well as in breast tumors, where SNEV transcript levels also correlate with the survival of breast cancer patients. Since these mRNA profiles suggested a role of SNEV in the regulation of cell proliferation, the effect of its overexpression was tested. Thereby, a significant extension of the cellular life span was observed, which was not caused by altered telomerase activity or telomere dynamics but rather by enhanced stress resistance. When SNEV overexpressing cells were treated with bleomycin or bleomycin combined with BSO, inducing DNA damage as well as reactive oxygen species, a significantly lower fraction of apoptotic cells was found in comparison to vector control cells. These data suggest that high levels of SNEV might extend the cellular life span by increasing the resistance to stress or by improving the DNA repair capacity of the cells.

  9. Suppression of Hepatocellular Carcinoma by Inhibition of Overexpressed Ornithine Aminotransferase.

    PubMed

    Zigmond, Ehud; Ben Ya'acov, Ami; Lee, Hyunbeom; Lichtenstein, Yoav; Shalev, Zvi; Smith, Yoav; Zolotarov, Lidya; Ziv, Ehud; Kalman, Rony; Le, Hoang V; Lu, Hejun; Silverman, Richard B; Ilan, Yaron

    2015-08-13

    Hepatocellular carcinoma is the second leading cause of cancer death worldwide. DNA microarray analysis identified the ornithine aminotransferase (OAT) gene as a prominent gene overexpressed in hepatocellular carcinoma (HCC) from Psammomys obesus. In vitro studies demonstrated inactivation of OAT by gabaculine (1), a neurotoxic natural product, which suppressed in vitro proliferation of two HCC cell lines. Alpha-fetoprotein (AFP) secretion, a biomarker for HCC, was suppressed by gabaculine in both cell lines, but not significantly. Because of the active site similarity between GABA aminotransferase (GABA-AT) and OAT, a library of 24 GABA-AT inhibitors was screened to identify a more selective inhibitor of OAT. (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidene)cyclopentane-1-carboxylic acid (2) was found to be an inactivator of OAT that only weakly inhibits GABA-AT, l-aspartate aminotransferase, and l-alanine aminotransferase. In vitro administration of 2 significantly suppressed AFP secretion in both Hep3B and HepG2 HCC cells; in vivo, 2 significantly suppressed AFP serum levels and tumor growth in HCC-harboring mice, even at 0.1 mg/kg. Overexpression of the OAT gene in HCC and the ability to block the growth of HCC by OAT inhibitors support the role of OAT as a potential therapeutic target to inhibit HCC growth. This is the first demonstration of suppression of HCC by an OAT inactivator.

  10. Overexpression of Pto activates defense responses and confers broad resistance.

    PubMed Central

    Tang, X; Xie, M; Kim, Y J; Zhou, J; Klessig, D F; Martin, G B

    1999-01-01

    The tomato disease resistance (R) gene Pto specifies race-specific resistance to the bacterial pathogen Pseudomonas syringae pv tomato carrying the avrPto gene. Pto encodes a serine/threonine protein kinase that is postulated to be activated by a physical interaction with the AvrPto protein. Here, we report that overexpression of Pto in tomato activates defense responses in the absence of the Pto-AvrPto interaction. Leaves of three transgenic tomato lines carrying the cauliflower mosaic virus 35S::Pto transgene exhibited microscopic cell death, salicylic acid accumulation, and increased expression of pathogenesis-related genes. Cell death in these plants was limited to palisade mesophyll cells and required light for induction. Mesophyll cells of 35S::Pto plants showed the accumulation of autofluorescent compounds, callose deposition, and lignification. When inoculated with P. s. tomato without avrPto, all three 35S::Pto lines displayed significant resistance and supported less bacterial growth than did nontransgenic lines. Similarly, the 35S::Pto lines also were more resistant to Xanthomonas campestris pv vesicatoria and Cladosporium fulvum. These results demonstrate that defense responses and general resistance can be activated by the overexpression of an R gene. PMID:9878629

  11. Overexpression of neurofilament H disrupts normal cell structure and function

    NASA Technical Reports Server (NTRS)

    Szebenyi, Gyorgyi; Smith, George M.; Li, Ping; Brady, Scott T.

    2002-01-01

    Studying exogenously expressed tagged proteins in live cells has become a standard technique for evaluating protein distribution and function. Typically, expression levels of experimentally introduced proteins are not regulated, and high levels are often preferred to facilitate detection. However, overexpression of many proteins leads to mislocalization and pathologies. Therefore, for normative studies, moderate levels of expression may be more suitable. To understand better the dynamics of intermediate filament formation, transport, and stability in a healthy, living cell, we inserted neurofilament heavy chain (NFH)-green fluorescent protein (GFP) fusion constructs in adenoviral vectors with tetracycline (tet)-regulated promoters. This system allows for turning on or off the synthesis of NFH-GFP at a selected time, for a defined period, in a dose-dependent manner. We used this inducible system for live cell imaging of changes in filament structure and cell shape, motility, and transport associated with increasing NFH-GFP expression. Cells with low to intermediate levels of NFH-GFP were structurally and functionally similar to neighboring, nonexpressing cells. In contrast, overexpression led to pathological alterations in both filament organization and cell function. Copyright 2002 Wiley-Liss, Inc.

  12. Over-expression of nicotinamide phosphoribosyltransferase in ovarian cancers.

    PubMed

    Shackelford, Rodney E; Bui, Marilyn M; Coppola, Domenico; Hakam, Ardeshir

    2010-06-12

    Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the rate-limiting step of nicotinamide adenine dinucleotide (NAD(+)) synthesis and is required for cell growth, survival, DNA replication and repair, and angiogenesis. Nampt expression increases gene expression which promotes cell survival and increases SirT1 activity, promoting angiogenesis, and it is increased in several human malignancies. Recently, others have shown that ovarian serous adenocarcinomas (OSAs) express high levels of activated Stat3. Since Nampt expression is increased by Stat3, we hypothesized that Nampt protein might be highly expressed in OSAs. Using tissue microarray (TMA) and the avidin-biotin complex immunohistochemical technique we examined Nampt expression in 47 samples of benign ovarian tissue and 49 samples of ovarian serous adenoacarcinomas. Our data show that Nampt protein expression is significantly increased in OSAs as compared to benign ovarian tissue (0.49+/-0.12 benign vs. 4.78+/-0.46 malignant; +/-standard error of the mean). This is the first report demonstrating Nampt overexpression in OSA, which may shed light on the pathogenesis of OSA. Further studies of the role of Nampt overexpresion in OSA may shed light on the prognosis and clinical course of OSA. Last, since an effective pharmacologic Nampt inhibitor is currently in clinical use, further studies of Nampt overexpression in OSA may be used in selecting patients for Nampt inhibitor therapy.

  13. Overexpression of Indian hedgehog partially rescues short stature homeobox 2-overexpression-associated congenital dysplasia of the temporomandibular joint in mice.

    PubMed

    Li, Xihai; Liang, Wenna; Ye, Hongzhi; Weng, Xiaping; Liu, Fayuan; Lin, Pingdong; Liu, Xianxiang

    2015-09-01

    The role of short stature homeobox 2 (shox2) in the development and homeostasis of the temporomandibular joint (TMJ) has been well documented. Shox2 is known to be expressed in the progenitor cells and perichondrium of the developing condyle. A previous study by our group reported that overexpression of shox2 leads to congenital dysplasia of the TMJ via downregulation of the Indian hedgehog (Ihh) signaling pathway, which is essential for embryonic disc primordium formation and mandibular condylar growth. To determine whether overexpression of Ihh may rescue the overexpression of shox2 leading to congenital dysplasia of the TMJ, a mouse model in which Ihh and shox2 were overexpressed (Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice) was utilized to assess the consequences of this overexpression on TMJ development during post-natal life. The results showed that the developmental process and expression levels of runt-related transcription factor 2 and sex determining region Y-box 9 in the TMJ of the Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice were similar to those in wild‑type mice. Overexpression of Ihh rescued shox2 overexpression-associated reduction of extracellular matrix components. However, overexpression of Ihh did not inhibit the shox2 overexpression-associated increase of matrix metalloproteinases (MMPs) MMP9, MMP13 and apoptosis in the TMJ. These combinatory cellular and molecular defects appeared to account for the observed congenital dysplasia of TMJ, suggesting that overexpression of Ihh partially rescued shox2 overexpression‑associated congenital dysplasia of the TMJ in mice.

  14. The visually impaired patient.

    PubMed

    Rosenberg, Eric A; Sperazza, Laura C

    2008-05-15

    Blindness or low vision affects more than 3 million Americans 40 years and older, and this number is projected to reach 5.5 million by 2020. In addition to treating a patient's vision loss and comorbid medical issues, physicians must be aware of the physical limitations and social issues associated with vision loss to optimize health and independent living for the visually impaired patient. In the United States, the four most prevalent etiologies of vision loss in persons 40 years and older are age-related macular degeneration, cataracts, glaucoma, and diabetic retinopathy. Exudative macular degeneration is treated with laser therapy, and progression of nonexudative macular degeneration in its advanced stages may be slowed with high-dose antioxidant and zinc regimens. The value of screening for glaucoma is uncertain; management of this condition relies on topical ocular medications. Cataract symptoms include decreased visual acuity, decreased color perception, decreased contrast sensitivity, and glare disability. Lifestyle and environmental interventions can improve function in patients with cataracts, but surgery is commonly performed if the condition worsens. Diabetic retinopathy responds to tight glucose control, and severe cases marked by macular edema are treated with laser photocoagulation. Vision-enhancing devices can help magnify objects, and nonoptical interventions include special filters and enhanced lighting.

  15. VOT and hearing impairment

    NASA Astrophysics Data System (ADS)

    Lane, Harlan; Perkell, Joseph

    2001-05-01

    When deafened adults recover some hearing after receiving a cochlear implant, numerous changes in their speech occur at both phonemic and suprasegmental levels. If a change toward normative values is observed for some phonemic parameter, it may be attributed to the restored hearing; however, it may be a by-product of a suprasegmental change. Consistent with results reported for speakers with normal hearing, Lane et al. [J. Acoust. Soc. Am. 98, 3096-3106 (1995)] observed in implant users that VOT varies approximately linearly with syllable duration. Therefore, in comparing pre- and postimplant measures of VOT in five speakers, each token's VOT was adjusted for the change in syllable duration of that token relative to the mean syllable duration in a baseline session (called VOTc). Preimplant, the deaf speakers characteristically uttered plosives with abnormally short VOTc. With some hearing restored, four of the five lengthened VOTc. Changes in voiced plosives' VOTc with restored hearing were correlated with changes in SPL. Some of the reliable VOTc increases that were not correlated with SPL may have been caused by auditory validation of an internal model for phoneme production. Recent studies of VOT in hearing-impaired speakers will be reviewed in this light. [Work supported by NIDCD, NIH.

  16. Local over-expression of prolactin in differentiating mouse mammary gland induces functional defects and benign lesions, but no carcinoma.

    PubMed

    Manhès, Caroline; Kayser, Christine; Bertheau, Philippe; Kelder, Bruce; Kopchick, John J; Kelly, Paul A; Touraine, Philippe; Goffin, Vincent

    2006-08-01

    altered morphology of the gland, led to impaired milk extrusion through the nipple. In summary, these data show that over-expression of autocrine hPRL in a differentiating mammary gland induces dramatic functional and morphological defects, but not carcinoma. This deserves further investigations on the emerging concept that autocrine PRL may have different effects on pathological development of the mammary gland depending on the differentiation state of the latter.

  17. Del-1 overexpression potentiates lung cancer cell proliferation and invasion

    SciTech Connect

    Lee, Seung-Hwan; Kim, Dong-Young; Jing, Feifeng; Kim, Hyesoon; Yun, Chae-Ok; Han, Deok-Jong; Choi, Eun Young

    2015-12-04

    Developmental endothelial locus-1 (Del-1) is an endogenous anti-inflammatory molecule that is highly expressed in the lung and the brain and limits leukocyte migration to these tissues. We previously reported that the expression of Del-1 is positively regulated by p53 in lung endothelial cells. Although several reports have implicated the altered expression of Del-1 gene in cancer patients, little is known about its role in tumor cells. We here investigated the effect of Del-1 on the features of human lung carcinoma cells. Del-1 mRNA was found to be significantly decreased in the human lung adenocarcinoma cell lines A549 (containing wild type of p53), H1299 (null for p53) and EKVX (mutant p53), compared to in human normal lung epithelial BEAS-2B cells and MRC-5 fibroblasts. The decrease of Del-1 expression was dependent on the p53 activity in the cell lines, but not on the expression of p53. Neither treatment with recombinant human Del-1 protein nor the introduction of adenovirus expressing Del-1 altered the expression of the apoptosis regulators BAX, PUMA and Bcl-2. Unexpectedly, the adenovirus-mediated overexpression of Del-1 gene into the lung carcinoma cell lines promoted proliferation and invasion of the lung carcinoma cells, as revealed by BrdU incorporation and transwell invasion assays, respectively. In addition, overexpression of the Del-1 gene enhanced features of epithelial–mesenchymal transition (EMT), such as increasing vimentin while decreasing E-cadherin in A549 cells, and increases in the level of Slug, an EMT-associated transcription regulator. Our findings demonstrated for the first time that there are deleterious effects of high levels of Del-1 in lung carcinoma cells, and suggest that Del-1 may be used as a diagnostic or prognostic marker for cancer progression, and as a novel therapeutic target for lung carcinoma. - Highlights: • Developmental Endothelial Locus-1 (Del-1) expression is downregulated in human lung cancer cells.

  18. Vascular cognitive impairment and dementia

    PubMed Central

    Gorelick, Philip B.; Counts, Scott E.; Nyenhuis, David

    2016-01-01

    Vascular contributions to cognitive impairment are receiving heightened attention as potentially modifiable factors for dementias of later life. These factors have now been linked not only to vascular cognitive disorders but also Alzheimer’s disease. In this chapter we review 3 related topics that address vascular contributions to cognitive impairment: 1. vascular pathogenesis and mechanisms; 2. neuropsychological and neuroimaging phenotypic manifestations of cerebrovascular disease; and 3. prospects for prevention of cognitive impairment of later life based on cardiovascular and stroke risk modification.1 PMID:26704177

  19. Neurocognitive impairments in hiv infection.

    PubMed

    Edwin, T; Nammalvar, N; Sabhesan, S; Ganesh, R; Devarajan, H

    1999-01-01

    Neuropsychological impairments punctuate the early neurological involvement among HIV-1 infected patients. Three groups of patients, twenty in each were selected. The first group consisted of seronegative local controls, the second being a group of asymptomatic seropositive patients and the third a group of seropositive symptomatic individuals. All these three groups were tested using standard neuropsychological tests. Results indicate that a broad spectrum of impairments occur in the seropositive patients and that the impairments of various functions occur at different phases of the illness. The importance of these findings in prediction of early neurological disturbance is highlighted and their significance in the total management and rehabilitation is discussed.

  20. Mammary gland tumor formation in transgenic mice overexpressing stromelysin-1

    SciTech Connect

    Sympson, Carolyn J; Bissell, Mina J; Werb, Zena

    1995-06-01

    An intact basement membrane (BM) is essential for the proper function, differentiation and morphology of many epithelial cells. The disruption or loss of this BM occurs during normal development as well as in the disease state. To examine the importance of BM during mammary gland development in vivo, we generated transgenic mice that inappropriately express autoactivating isoforms of the matrix metalloproteinase stromelysin-1. The mammary glands from these mice are both functionally and morphologically altered throughout development. We have now documented a dramatic incidence of breast tumors in several independent lines of these mice. These data suggest that overexpression of stromelysin-1 and disruption of the BM may be a key step in the multi-step process of breast cancer.

  1. RNAi and overexpression of genes in ovarian somatic cells.

    PubMed

    Saito, Kuniaki

    2014-01-01

    Emerging evidence indicates that PIWI proteins, in collaboration with PIWI-interacting RNAs (piRNAs), play a critical role in retrotransposon silencing in Drosophila gonadal somatic and germ-line cells. The recent establishment of female germ-line stem cells/ovarian somatic sheet and its derivative cell line, ovarian somatic cells (OSCs), allows researchers to study the molecular functions of several protein factors involved in the primary piRNA pathway in Drosophila. Although transgene expression is difficult to achieve in gonad-derived cell lines, transfection of both expression vectors and knockdown reagents is highly effective in OSCs. Here, I focus on techniques that knockdown or overexpress genes of interest in OSCs.

  2. Over-expression of secreted proteins from mammalian cell lines

    PubMed Central

    Dalton, Annamarie C; Barton, William A

    2014-01-01

    Secreted mammalian proteins require the development of robust protein over-expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large-scale growth of cells in a variety of formats. PMID:24510886

  3. Overexpressed ribosomal proteins suppress defective chaperonins in Saccharomyces cerevisiae.

    PubMed

    Kabir, M Anaul; Sherman, Fred

    2008-12-01

    The chaperonin Cct complex of the yeast Saccharomyces cerevisiae is composed of eight different subunits encoded by eight essential genes, CCT1-CCT8. This Cct complex is responsible for the folding of a number of proteins including actin and tubulin. We have isolated and characterized 22 multicopy suppressors of the temperature-sensitive allele, cct4-1, which encodes an altered protein with a G345D replacement that diminishes ATP hydrolysis. Fourteen of the suppressors encode ribosomal proteins, four have roles in ribosome biogenesis, two have phosphatase activities, one is involved in protein synthesis and one of the suppressors corresponded to Cct4p. Some of the suppressors also acted on certain cct1, cct2, cct3 and cct6 mutations. We suggest that certain overexpressed ribosomal and other proteins can act as weak chaperones, phenotypically alleviating the partial defects of mutationally altered Cct subunits.

  4. Mutant HSPB1 overexpression in neurons is sufficient to cause age-related motor neuronopathy in mice

    PubMed Central

    Srivastava, Amit K.; Renusch, Samantha R.; Naiman, Nicole E.; Gu, Shuping; Sneh, Amita; Arnold, W. David; Sahenk, Zarife; Kolb, Stephen J.

    2012-01-01

    The small heat shock protein HSPB1 is a multifunctional, α-crystallin-based protein that has been shown to be neuroprotective in animal models of motor neuron disease and peripheral nerve injury. Missense mutations in HSPB1 result in axonal Charcot-Marie-Tooth disease with minimal sensory involvement (CMT2F) and distal hereditary motor neuropathy type 2 (dHMN-II). These disorders are characterized by a selective loss of motor axons in peripheral nerve resulting in distal muscle weakness and often severe disability. To investigate the pathogenic mechanisms of HSPB1 mutations in motor neurons in vivo, we have developed and characterized transgenic PrP-HSPB1 and PrP-HSPB1(R136W) mice. These mice express the human HSPB1 protein throughout the nervous system including in axons of peripheral nerve. Although both mouse strains lacked obvious motor deficits, the PrP-HSPB1(R136W) mice developed an age-dependent motor axonopathy. Mutant mice showed axonal pathology in spinal cord and peripheral nerve with evidence of impaired neurofilament cytoskeleton, associated with organelle accumulation. Accompanying these findings, increases in the number of Schmidt-Lanterman incisures, as evidence of impaired axon-Schwann cell interactions, were present. These observations suggest that overexpression of HSPB1(R136W) in neurons is sufficient to cause pathological and electrophysiological changes in mice that are seen in patients with hereditary motor neuropathy. PMID:22521462

  5. Effect of deletion and overexpression of tryptophan metabolism genes on growth and fermentation capacity at low temperature in wine yeast.

    PubMed

    López-Malo, María; García-Rios, Estefani; Chiva, Rosana; Guillamon, José Manuel; Martí-Raga, María

    2014-01-01

    Low-temperature fermentations produce wines with greater aromatic complexity, but the success of these fermentations greatly depends on the adaptation of yeast cells to cold. Tryptophan has been previously reported to be a limiting amino acid during Saccharomyces cerevisiae growth at low temperature. The objective of this study was to determine the influence of the tryptophan metabolism on growth and fermentation performance during low-temperature wine fermentation. To this end, we constructed the deletion mutants of the TRP1 and TAT2 genes in a derivative haploid of a commercial wine strain, and the TAT2 gene was overexpressed in the prototroph and auxotroph (Δtrp1) backgrounds. Then we characterized growth and fermentation activity during wine fermentation at low and optimum temperatures. Our results partially support the role of this amino acid in cold yeast growth. Although deletion of TRP1 impaired amino acid uptake and the growth rate at low temperature in synthetic must, this growth impairment did not affect the fermentation rate. Deletion of TAT2 endorsed this strain with the highest nitrogen consumption capacity and the greatest fermentation activity at low temperature. Our results also evidenced reduced ammonium consumption in all the strains at low temperature.

  6. Overexpression of Arabidopsis NLP7 improves plant growth under both nitrogen-limiting and -sufficient conditions by enhancing nitrogen and carbon assimilation.

    PubMed

    Yu, Lin-Hui; Wu, Jie; Tang, Hui; Yuan, Yang; Wang, Shi-Mei; Wang, Yu-Ping; Zhu, Qi-Sheng; Li, Shi-Gui; Xiang, Cheng-Bin

    2016-06-13

    Nitrogen is essential for plant survival and growth. Excessive application of nitrogenous fertilizer has generated serious environment pollution and increased production cost in agriculture. To deal with this problem, tremendous efforts have been invested worldwide to increase the nitrogen use ability of crops. However, only limited success has been achieved to date. Here we report that NLP7 (NIN-LIKE PROTEIN 7) is a potential candidate to improve plant nitrogen use ability. When overexpressed in Arabidopsis, NLP7 increases plant biomass under both nitrogen-poor and -rich conditions with better-developed root system and reduced shoot/root ratio. NLP7-overexpressing plants show a significant increase in key nitrogen metabolites, nitrogen uptake, total nitrogen content, and expression levels of genes involved in nitrogen assimilation and signalling. More importantly, overexpression of NLP7 also enhances photosynthesis rate and carbon assimilation, whereas knockout of NLP7 impaired both nitrogen and carbon assimilation. In addition, NLP7 improves plant growth and nitrogen use in transgenic tobacco (Nicotiana tabacum). Our results demonstrate that NLP7 significantly improves plant growth under both nitrogen-poor and -rich conditions by coordinately enhancing nitrogen and carbon assimilation and sheds light on crop improvement.

  7. Overexpression of Arabidopsis NLP7 improves plant growth under both nitrogen-limiting and -sufficient conditions by enhancing nitrogen and carbon assimilation

    PubMed Central

    Yu, Lin-Hui; Wu, Jie; Tang, Hui; Yuan, Yang; Wang, Shi-Mei; Wang, Yu-Ping; Zhu, Qi-Sheng; Li, Shi-Gui; Xiang, Cheng-Bin

    2016-01-01

    Nitrogen is essential for plant survival and growth. Excessive application of nitrogenous fertilizer has generated serious environment pollution and increased production cost in agriculture. To deal with this problem, tremendous efforts have been invested worldwide to increase the nitrogen use ability of crops. However, only limited success has been achieved to date. Here we report that NLP7 (NIN-LIKE PROTEIN 7) is a potential candidate to improve plant nitrogen use ability. When overexpressed in Arabidopsis, NLP7 increases plant biomass under both nitrogen-poor and -rich conditions with better-developed root system and reduced shoot/root ratio. NLP7–overexpressing plants show a significant increase in key nitrogen metabolites, nitrogen uptake, total nitrogen content, and expression levels of genes involved in nitrogen assimilation and signalling. More importantly, overexpression of NLP7 also enhances photosynthesis rate and carbon assimilation, whereas knockout of NLP7 impaired both nitrogen and carbon assimilation. In addition, NLP7 improves plant growth and nitrogen use in transgenic tobacco (Nicotiana tabacum). Our results demonstrate that NLP7 significantly improves plant growth under both nitrogen-poor and -rich conditions by coordinately enhancing nitrogen and carbon assimilation and sheds light on crop improvement. PMID:27293103

  8. Skeletal muscle-specific overexpression of IGFBP-2 promotes a slower muscle phenotype in healthy but not dystrophic mdx mice and does not affect the dystrophic pathology.

    PubMed

    Swiderski, Kristy; Martins, Karen Janet Bernice; Chee, Annabel; Trieu, Jennifer; Naim, Timur; Gehrig, Stefan Martin; Baum, Dale Michael; Brenmoehl, Julia; Chau, Luong; Koopman, René; Gregorevic, Paul; Metzger, Friedrich; Hoeflich, Andreas; Lynch, Gordon Stuart

    The insulin-like growth factor binding proteins (IGFBPs) are thought to modulate cell size and homeostasis via IGF-I-dependent and -independent pathways. There is a considerable dearth of information regarding the function of IGFBPs in skeletal muscle, particularly their role in the pathophysiology of Duchenne muscular dystrophy (DMD). In this study we tested the hypothesis that intramuscular IGFBP-2 overexpression would ameliorate the pathology in mdx dystrophic mice. 4week old male C57Bl/10 and mdx mice received a single intramuscular injection of AAV6-empty or AAV6-IGFBP-2 vector into the tibialis anterior muscle. At 8weeks post-injection the effect of IGFBP-2 overexpression on the structure and function of the injected muscle was assessed. AAV6-mediated IGFBP-2 overexpression in the tibialis anterior (TA) muscles of 4-week-old C57BL/10 and mdx mice reduced the mass of injected muscle after 8weeks, inducing a slower muscle phenotype in C57BL/10 but not mdx mice. Analysis of inflammatory and fibrotic gene expression revealed no changes between control and IGFBP-2 injected muscles in dystrophic (mdx) mice. Together these results indicate that the IGFBP-2-induced promotion of a slower muscle phenotype is impaired in muscles of dystrophin-deficient mdx mice, which contributes to the inability of IGFBP-2 to ameliorate the dystrophic pathology. The findings implicate the dystrophin-glycoprotein complex (DGC) in the signaling required for this adaptation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Disruption of Inhibitory Function in the Ts65Dn Mouse Hippocampus Through Overexpression of GIRK2

    DTIC Science & Technology

    2007-10-24

    Inhibitory Function in the Ts65Dn Mouse Hippocampus Through Overexpression of GIRK2" {O\\LldC0( Date Date Dissertation and Abstract Approved: Name of...thesis manuscript entitled: "Disruption of inhibitory function in the Ts65Dn mouse hippocampus through overexpression of GIRK2" is appropriately...Disruption of inhibitory function in the Ts65Dn mouse hippocampus through overexpression of GIRK2 by Tyler K. Best Doctoral Dissertation

  10. Overexpression and potential roles of NRIP1 in psoriasis

    PubMed Central

    Luan, Chao; Chen, Xu; Hu, Yu; Hao, Zhimin; Osland, Jared M.; Chen, Xundi; Gerber, Skyler D.; Chen, Min; Gu, Heng; Yuan, Rong

    2016-01-01

    Nuclear receptor interacting protein 1 (NRIP1, also known as RIP140) is a co-regulator for various transcriptional factors and nuclear receptors, and has been shown to take part in many biological and pathological processes, such as regulating mammary gland development and inflammatory response. The aim of this study is to investigate the expression of NRIP1 and to explore its roles in the pathogenesis of psoriasis. Thirty active psoriasis patients and 16 healthy volunteers were enrolled for this study. qRT-PCR analyses found that both NRIP1 and RelA/p65 were elevated in psoriatic lesions compared to psoriatic non-lesions and normal controls, and also overexpressed in peripheral blood mononuclear cell (PBMCs) of psoriasis patients. Suppression of NRIP1 in HaCaT cells could significantly inhibit cell growth and induce apoptosis, and the suppression of NRIP1 in CD4+ T cells isolated from psoriasis patients could downregulate the expression of RelA/p65 and decrease the secretion of IL-17. Furthermore, in Nrip1 knockout mice, IMQ-induced inflammation of skin was delayed and the RelA/p65 expression in lesions was reduced. In conclusion, our data suggests that NRIP1 is overexpressed both in skin and PBMCs of psoriasis patients and may be involved in the abnormal proliferation and apoptosis of keratinocytes, as well as the immune reaction through the regulation of RelA/p65. Therefore, NRIP1 may be a potential therapeutic target for psoriasis. PMID:27708240

  11. Focal adhesion kinase overexpression and its impact on human osteosarcoma

    PubMed Central

    Chen, Yong; Yang, Aizhen; Chen, Hui; Zhang, Jian; Wu, Sujia; Shi, Xin; Wang, Chen; Sun, Xiaoliang

    2015-01-01

    Focal adhesion kinase (FAK) has been implicated in tumorigenesis in various malignancies. We sought to examine the expression patterns of FAK and the activated form, phosphorylated FAK (pFAK), in human osteosarcoma and to investigate the correlation of FAK expression with clinicopathologic parameters and prognosis. In addition, the functional consequence of manipulating the FAK protein level was investigated in human osteosarcoma cell lines. Immunohistochemical staining was used to detect FAK and pFAK in pathologic archived materials from 113 patients with primary osteosarcoma. Kaplan-Meier survival and Cox regression analyses were performed to evaluate the prognoses. The role of FAK in the cytological behavior of MG63 and 143B human osteosarcoma cell lines was studied via FAK protein knock down with siRNA. Cell proliferation, migration, invasiveness and apoptosis were assessed using the CCK8, Transwell and Annexin V/PI staining methods. Both FAK and pFAK were overexpressed in osteosarcoma. There were significant differences in overall survival between the FAK-/pFAK- and FAK+/pFAK- groups (P = 0.016), the FAK+/pFAK- and FAK+/pFAK+ groups (P = 0.012) and the FAK-/pFAK- and FAK+/pFAK+ groups (P < 0.001). There were similar differences in metastasis-free survival between groups. The Cox proportional hazards analysis showed that the FAK expression profile was an independent indicator of both overall and metastasis-free survival. siRNA-based knockdown of FAK not only dramatically reduced the migration and invasion of MG63 and 143B cells, but also had a distinct effect on osteosarcoma cell proliferation and apoptosis. These results collectively suggest that FAK overexpression and phosphorylation might predict more aggressive biologic behavior in osteosarcoma and may be an independent predictor of poor prognosis. PMID:26393679

  12. CD3-epsilon overexpressed in prothymocytes acts as an oncogene.

    PubMed Central

    Wang, B.; She, J.; Salio, M.; Allen, D.; Lacy, E.; Lonberg, N.; Terhorst, C.

    1997-01-01

    BACKGROUND: Upon engagement of the T cell receptor for antigen, its associated CD3 proteins recruit signal transduction molecules, which in turn regulate T lymphocyte proliferation, apoptosis, and thymocyte development. Because some signal transducing molecules recruited by CD3-epsilon, i.e., p56lck and p59fyn, are oncogenic and since we previously found that overexpression of CD3-epsilon transgenes causes a block in T lymphocyte and NK cell development, we tested the hypothesis that aberrant CD3-epsilon signaling leads both to abnormal T lymphocyte death and lymphomagenesis. MATERIALS AND METHODS: Ten independently derived transgenic mouse lines were generated with four different genomic CD3-epsilon constructs. Mice either homozygous or hemizygous for each transgene were analyzed for an arrest in T lymphocyte development and for the occurrence of T cell lymphomas. RESULTS: Aggressive clonal T cell lymphomas developed at very high frequencies in seven mouse lines with intermediate levels of copies of CD3-epsilon derived transgenes. However, these lymphomas were not found when high copy numbers of CD3-epsilon transgenes caused a complete block in early thymic development or when a transgene was used in which the exons coding for the CD3-epsilon protein were deleted. Analyses of a series of double mutant mice, tgCD3-epsilon x RAG-2null, indicated that lymphomagenesis was initiated in lineage-committed prothymocytes, i.e., before rearrangement of the T cell receptor genes. In addition, the transgene coding for the CD3-epsilon cytoplasmic domain and its transmembrane region induced a T cell differentiation signal in premalignant tgCD3-epsilon x RAG-2null mice. CONCLUSION: The nonenzymatic CD3-epsilon protein acted as a potent oncogene when overexpressed early in T lymphocyte development. Lymphomagenesis was dependent on signal transduction events initiated by the cytoplasmic domain of CD3-epsilon. Images FIG. 2 FIG. 4 FIG. 5 PMID:9132282

  13. MCM10 overexpression implicates adverse prognosis in urothelial carcinoma

    PubMed Central

    Li, Wei-Ming; Huang, Chun-Nung; Ke, Hung-Lung; Li, Ching-Chia; Wei, Yu-Ching; Yeh, Hsin-Chih; Chang, Lin-Li; Huang, Chun-Hsiung; Liang, Peir-In; Yeh, Bi-Wen; Chan, Ti-Chun; Li, Chien-Feng; Wu, Wen-Jeng

    2016-01-01

    Urothelial carcinoma (UC) occurs in the upper urinary tract (UTUC) and the urinary bladder (UBUC). The molecular pathogenesis of UC has not been fully elucidated. Through data mining of a published transcriptome of UBUC (GSE31684), we identified Minichromosome Maintenance Complex Component 2 (MCM2) and MCM10 as the two most significantly upregulated genes in UC progression among the MCM gene family, the key factors for the initiation of DNA replication. To validate the clinical significance of MCM2 and MCM10, immunohistochemistry, evaluated by H-score, was used in a pilot study of 50 UTUC and 50 UBUC samples. Only a high expression level of MCM10 predicted worse disease-specific survival (DSS) and inferior metastasis-free survival (MeFS) for both UTUC and UBUC. Correspondingly, evaluation of MCM10 mRNA expression in 36 UTUCs and 30 UBUCs showed significantly upregulated levels in high stage UC, suggesting its role in tumor progression. Evaluation of 340 UTUC and 296 UBUC tissue samples, respectively, demonstrated that high MCM10 immunoexpression was significantly associated with advanced primary tumors, nodal status, and the presence of vascular invasion in both groups of UCs. In multivariate Cox regression analyses, adjusted for standard clinicopathological features, MCM10 overexpression was independently associated with DSS (UTUC hazard ratio [HR]=2.401, P = 0.013; UBUC HR=4.323, P=0.001) and with MeFS (UTUC HR=3.294, P<0.001; UBUC HR=1.972, P=0.015). In vitro, knockdown of MCM10 gene significantly suppressed cell proliferation in both J82 and TCCSUP cells. In conclusion, MCM10 overexpression was associated with unfavorable clinicopathological characteristics and independent negative prognostic effects, justifying its potential theranostic value in UC. PMID:27780919

  14. Pentylenetetrazol-kindling in mice overexpressing heat shock protein 70.

    PubMed

    Ammon-Treiber, Susanne; Grecksch, Gisela; Angelidis, Charalampos; Vezyraki, Patra; Höllt, Volker; Becker, Axel

    2007-04-01

    Kindling induced by the convulsant pentylenetetrazol (PTZ) is an accepted model of primary generalized epilepsy. Because seizures represent a strong distressing stimulus, stress-induced proteins such as heat shock proteins might counteract the pathology of increased neuronal excitation. Therefore, the aim of the present study was to determine whether PTZ kindling outcome parameters are influenced by heat shock protein 70 (Hsp70) overexpression in Hsp70 transgenic mice as compared to the respective wild-type mice. Kindling was performed by nine intraperitoneal injections of PTZ (ED(16) for induction of clonic-tonic seizures, every 48 h); control animals received saline instead of PTZ. Seven days after the final injection, all mice received a PTZ challenge dose. Outcome parameters included evaluation of seizure stages and overall survival rates. In addition, histopathological findings such as cell number in hippocampal subfields CA1 and CA3 were determined. The onset of the highest convulsion stage was delayed in Hsp70 transgenic mice as compared to wild-type mice, and overall survival during kindling was improved in Hsp70 transgenic mice as compared to wild-type mice. In addition, a challenge dose after termination of kindling produced less severe seizures in Hsp70 transgenic mice than in wild-type mice. PTZ kindling did not result in significant subsequent neuronal cell loss in CA1 or CA3 neither in wild-type mice nor in the Hsp70 transgenic mice. The results of the present experiments clearly demonstrate that overexpression of Hsp70 exerts protective effects regarding seizure severity and overall survival during PTZ kindling. In addition, the decreased seizure severity in Hsp70 transgenic mice after a challenge dose suggests an interference of Hsp70 with the developmental component of kindling.

  15. Sarcolipin overexpression improves muscle energetics and reduces fatigue.

    PubMed

    Sopariwala, Danesh H; Pant, Meghna; Shaikh, Sana A; Goonasekera, Sanjeewa A; Molkentin, Jeffery D; Weisleder, Noah; Ma, Jianjie; Pan, Zui; Periasamy, Muthu

    2015-04-15

    Sarcolipin (SLN) is a regulator of sarcoendoplasmic reticulum calcium ATPase in skeletal muscle. Recent studies using SLN-null mice have identified SLN as a key player in muscle thermogenesis and metabolism. In this study, we exploited a SLN overexpression (Sln(OE)) mouse model to determine whether increased SLN level affected muscle contractile properties, exercise capacity/fatigue, and metabolic rate in whole animals and isolated muscle. We found that Sln(OE) mice are more resistant to fatigue and can run significantly longer distances than wild-type (WT). Studies with isolated extensor digitorum longus (EDL) muscles showed that Sln(OE) EDL produced higher twitch force than WT. The force-frequency curves were not different between WT and Sln(OE) EDLs, but at lower frequencies the pyruvate-induced potentiation of force was significantly higher in Sln(OE) EDL. SLN overexpression did not alter the twitch and force-frequency curve in isolated soleus muscle. However, during a 10-min fatigue protocol, both EDL and soleus from Sln(OE) mice fatigued significantly less than WT muscles. Interestingly, Sln(OE) muscles showed higher carnitine palmitoyl transferase-1 protein expression, which could enhance fatty acid metabolism. In addition, lactate dehydrogenase expression was higher in Sln(OE) EDL, suggesting increased glycolytic capacity. We also found an increase in store-operated calcium entry (SOCE) in isolated flexor digitorum brevis fibers of Sln(OE) compared with WT mice. These data allow us to conclude that increased SLN expression improves skeletal muscle performance during prolonged muscle activity by increasing SOCE and muscle energetics.

  16. Transgenic cloned sheep overexpressing ovine toll-like receptor 4.

    PubMed

    Deng, Shoulong; Li, Guiguan; Zhang, Jinlong; Zhang, Xiaosheng; Cui, Maosheng; Guo, Yong; Liu, Guoshi; Li, Guangpeng; Feng, Jianzhong; Lian, Zhengxing

    2013-07-01

    An ovine fetal fibroblast cell line highly expressing TLR4 was established by inserting TLR4 into a reconstructive p3S-LoxP plasmid. Transgenic sheep overexpressing TLR4 were produced by transferring TLR4-transfected fetal fibroblasts into metaphase (M)II-stage enucleated oocytes (using SCNT). Because reconstructed embryos derived from MII-stage enucleated oocytes matured in vivo using a delayed-activated method had a higher pregnancy rate (18.52%) than that from MII-stage enucleated oocytes matured in vitro, the former procedure was used. Nine TLR4-transgenic live births were confirmed using polymerase chain reaction and Southern blot analysis. Increased expression of TLR4 at mRNA and protein levels in ear tissues of transgenic lambs were verified using reverse transcription polymerase chain reaction and immunohistochemistry, respectively. More toll-like receptor 4 protein was expressed by peripheral blood monocytes and/or macrophages collected from 3-month-old TLR4-transgenic than nontransgenic lambs at 0, 1, and 4 hours after lipopolysaccharide stimulation. Furthermore, interferon-γ and tumor necrosis factor α secreted by monocytes and/or macrophages of TLR4-transgenic lambs were significantly higher at 1 hour. Therefore, lipopolysaccharide-induced inflammatory responses from monocytes and/or macrophages occurred sooner in TLR4-transgenic lambs, consistent with an enhanced host immune response. In conclusion, transgenic sheep overexpressing TLR4 are a primary model to investigate the role of transgenic animals in disease resistance and have potential for breeding sheep with disease resistance.

  17. Statins Reduce Melanoma Development and Metastasis through MICA Overexpression.

    PubMed

    Pich, Christine; Teiti, Iotefa; Rochaix, Philippe; Mariamé, Bernard; Couderc, Bettina; Favre, Gilles; Tilkin-Mariamé, Anne-Françoise

    2013-01-01

    Survival of melanoma patients after metastases detection remains short. Several clinical trials have shown moderate efficiency in improving patient survival, and the search for pharmacological agents to enhance the immune response and reduce melanoma metastases is still necessary. Statins block the mevalonate pathway, which leads to decreases in GTPase isoprenylation and activity, particularly those of the Ras superfamily. They are widely used as hypocholesterolemic agents in cardiovascular diseases and several studies have shown that they also have protective effects against cancers. Furthermore, we have previously demonstrated that treatment of melanoma cells with inhibitors of the mevalonate pathway, such as statins, favor the development of specific adaptive immune responses against these tumors. In the present study, we tested statin impact on the innate immune response against human metastatic melanoma cells. Our data shows that treatment of two human melanoma cell lines with statins induced a weak but significant increase of MHC class I Chain-related protein A (MICA) membrane expression. Peroxisome Proliferator-Activated Receptor gamma is involved in this statin-induced MICA overexpression, which is independent of Ras and Rho GTPase signaling pathways. Interestingly, this MICA overexpression makes melanoma cells more sensitive to in vitro lysis by NK cells. The impact of statin treatment on in vivo development of melanoma tumors and metastases was investigated in nude mice, because murine NK cells, which express NKG2D receptors, are able to recognize and kill human tumor cells expressing MICA. The results demonstrated that both local tumor growth and pulmonary metastases were strongly inhibited in nude mice injected with statin-treated melanoma cells. These results suggest that statins could be effective in melanoma immunotherapy treatments.

  18. Sarcolipin overexpression improves muscle energetics and reduces fatigue

    PubMed Central

    Sopariwala, Danesh H.; Pant, Meghna; Shaikh, Sana A.; Goonasekera, Sanjeewa A.; Molkentin, Jeffery D.; Weisleder, Noah; Ma, Jianjie; Pan, Zui

    2015-01-01

    Sarcolipin (SLN) is a regulator of sarcoendoplasmic reticulum calcium ATPase in skeletal muscle. Recent studies using SLN-null mice have identified SLN as a key player in muscle thermogenesis and metabolism. In this study, we exploited a SLN overexpression (SlnOE) mouse model to determine whether increased SLN level affected muscle contractile properties, exercise capacity/fatigue, and metabolic rate in whole animals and isolated muscle. We found that SlnOE mice are more resistant to fatigue and can run significantly longer distances than wild-type (WT). Studies with isolated extensor digitorum longus (EDL) muscles showed that SlnOE EDL produced higher twitch force than WT. The force-frequency curves were not different between WT and SlnOE EDLs, but at lower frequencies the pyruvate-induced potentiation of force was significantly higher in SlnOE EDL. SLN overexpression did not alter the twitch and force-frequency curve in isolated soleus muscle. However, during a 10-min fatigue protocol, both EDL and soleus from SlnOE mice fatigued significantly less than WT muscles. Interestingly, SlnOE muscles showed higher carnitine palmitoyl transferase-1 protein expression, which could enhance fatty acid metabolism. In addition, lactate dehydrogenase expression was higher in SlnOE EDL, suggesting increased glycolytic capacity. We also found an increase in store-operated calcium entry (SOCE) in isolated flexor digitorum brevis fibers of SlnOE compared with WT mice. These data allow us to conclude that increased SLN expression improves skeletal muscle performance during prolonged muscle activity by increasing SOCE and muscle energetics. PMID:25701006

  19. Statins Reduce Melanoma Development and Metastasis through MICA Overexpression

    PubMed Central

    Pich, Christine; Teiti, Iotefa; Rochaix, Philippe; Mariamé, Bernard; Couderc, Bettina; Favre, Gilles; Tilkin-Mariamé, Anne-Françoise

    2013-01-01

    Survival of melanoma patients after metastases detection remains short. Several clinical trials have shown moderate efficiency in improving patient survival, and the search for pharmacological agents to enhance the immune response and reduce melanoma metastases is still necessary. Statins block the mevalonate pathway, which leads to decreases in GTPase isoprenylation and activity, particularly those of the Ras superfamily. They are widely used as hypocholesterolemic agents in cardiovascular diseases and several studies have shown that they also have protective effects against cancers. Furthermore, we have previously demonstrated that treatment of melanoma cells with inhibitors of the mevalonate pathway, such as statins, favor the development of specific adaptive immune responses against these tumors. In the present study, we tested statin impact on the innate immune response against human metastatic melanoma cells. Our data shows that treatment of two human melanoma cell lines with statins induced a weak but significant increase of MHC class I Chain-related protein A (MICA) membrane expression. Peroxisome Proliferator-Activated Receptor gamma is involved in this statin-induced MICA overexpression, which is independent of Ras and Rho GTPase signaling pathways. Interestingly, this MICA overexpression makes melanoma cells more sensitive to in vitro lysis by NK cells. The impact of statin treatment on in vivo development of melanoma tumors and metastases was investigated in nude mice, because murine NK cells, which express NKG2D receptors, are able to recognize and kill human tumor cells expressing MICA. The results demonstrated that both local tumor growth and pulmonary metastases were strongly inhibited in nude mice injected with statin-treated melanoma cells. These results suggest that statins could be effective in melanoma immunotherapy treatments. PMID:23493799

  20. Tubular Overexpression of Gremlin Induces Renal Damage Susceptibility in Mice

    PubMed Central

    Droguett, Alejandra; Krall, Paola; Burgos, M. Eugenia; Valderrama, Graciela; Carpio, Daniel; Ardiles, Leopoldo; Rodriguez-Diez, Raquel; Kerr, Bredford; Walz, Katherina; Ruiz-Ortega, Marta; Egido, Jesus; Mezzano, Sergio

    2014-01-01

    A growing number of patients are recognized worldwide to have chronic kidney disease. Glomerular and interstitial fibrosis are hallmarks of renal progression. However, fibrosis of the kidney remains an unresolved challenge, and its molecular mechanisms are still not fully understood. Gremlin is an embryogenic gene that has been shown to play a key role in nephrogenesis, and its expression is generally low in the normal adult kidney. However, gremlin expression is elevated in many human renal diseases, including diabetic nephropathy, pauci-immune glomerulonephritis and chronic allograft nephropathy. Several studies have proposed that gremlin may be involved in renal damage by acting as a downstream mediator of TGF-β. To examine the in vivo role of gremlin in kidney pathophysiology, we generated seven viable transgenic mouse lines expressing human gremlin (GREM1) specifically in renal proximal tubular epithelial cells under the control of an androgen-regulated promoter. These lines demonstrated 1.2- to 200-fold increased GREM1 expression. GREM1 transgenic mice presented a normal phenotype and were without proteinuria and renal function involvement. In response to the acute renal damage cause by folic acid nephrotoxicity, tubule-specific GREM1 transgenic mice developed increased proteinuria after 7 and 14 days compared with wild-type treated mice. At 14 days tubular lesions, such as dilatation, epithelium flattening and hyaline casts, with interstitial cell infiltration and mild fibrosis were significantly more prominent in transgenic mice than wild-type mice. Tubular GREM1 overexpression was correlated with the renal upregulation of profibrotic factors, such as TGF-β and αSMA, and with increased numbers of monocytes/macrophages and lymphocytes compared to wild-type mice. Taken together, our results suggest that GREM1-overexpressing mice have an increased susceptibility to renal damage, supporting the involvement of gremlin in renal damage progression. This

  1. Tubular overexpression of gremlin induces renal damage susceptibility in mice.

    PubMed

    Droguett, Alejandra; Krall, Paola; Burgos, M Eugenia; Valderrama, Graciela; Carpio, Daniel; Ardiles, Leopoldo; Rodriguez-Diez, Raquel; Kerr, Bredford; Walz, Katherina; Ruiz-Ortega, Marta; Egido, Jesus; Mezzano, Sergio

    2014-01-01

    A growing number of patients are recognized worldwide to have chronic kidney disease. Glomerular and interstitial fibrosis are hallmarks of renal progression. However, fibrosis of the kidney remains an unresolved challenge, and its molecular mechanisms are still not fully understood. Gremlin is an embryogenic gene that has been shown to play a key role in nephrogenesis, and its expression is generally low in the normal adult kidney. However, gremlin expression is elevated in many human renal diseases, including diabetic nephropathy, pauci-immune glomerulonephritis and chronic allograft nephropathy. Several studies have proposed that gremlin may be involved in renal damage by acting as a downstream mediator of TGF-β. To examine the in vivo role of gremlin in kidney pathophysiology, we generated seven viable transgenic mouse lines expressing human gremlin (GREM1) specifically in renal proximal tubular epithelial cells under the control of an androgen-regulated promoter. These lines demonstrated 1.2- to 200-fold increased GREM1 expression. GREM1 transgenic mice presented a normal phenotype and were without proteinuria and renal function involvement. In response to the acute renal damage cause by folic acid nephrotoxicity, tubule-specific GREM1 transgenic mice developed increased proteinuria after 7 and 14 days compared with wild-type treated mice. At 14 days tubular lesions, such as dilatation, epithelium flattening and hyaline casts, with interstitial cell infiltration and mild fibrosis were significantly more prominent in transgenic mice than wild-type mice. Tubular GREM1 overexpression was correlated with the renal upregulation of profibrotic factors, such as TGF-β and αSMA, and with increased numbers of monocytes/macrophages and lymphocytes compared to wild-type mice. Taken together, our results suggest that GREM1-overexpressing mice have an increased susceptibility to renal damage, supporting the involvement of gremlin in renal damage progression. This

  2. Specific Language Impairment in Families: Evidence for Co-Occurrence with Reading Impairments.

    ERIC Educational Resources Information Center

    Flax, Judy F.; Realpe-Bonilla, Teresa; Hirsch, Linda S.; Brzustowicz, Linda M.; Bartlett, Christopher W.; Tallal, Paula

    2003-01-01

    Two family aggregation studies involving 25 children (ages 5-10) with specific language impairment (SLI) report the occurrence and co-occurrence of oral language impairments and reading impairments. Results indicate that when language impairments occur within families of SLI probands, these impairments generally co-occur with reading impairments.…

  3. Autophagy impairment induces premature senescence in primary human fibroblasts.

    PubMed

    Kang, Hyun Tae; Lee, Ki Baek; Kim, Sung Young; Choi, Hae Ri; Park, Sang Chul

    2011-01-01

    Recent studies have demonstrated that activation of autophagy increases the lifespan of organisms from yeast to flies. In contrast to the lifespan extension effect in lower organisms, it has been reported that overexpression of unc-51-like kinase 3 (ULK3), the mammalian homolog of autophagy-specific gene 1 (ATG1), induces premature senescence in human fibroblasts. Therefore, we assessed whether the activation of autophagy would genuinely induce premature senescence in human cells. Depletion of ATG7, ATG12, or lysosomal-associated membrane protein 2 (Lamp2) by transfecting siRNA or infecting cells with a virus containing gene-specific shRNA resulted in a senescence-like state in two strains of primary human fibroblasts. Prematurely senescent cells induced by autophagy impairment exhibited the senescent phenotypes, similar to the replicatively senescent cells, such as increased senescence associated β-galactosidase (SA-β-gal) activity, reactive oxygen species (ROS) generation, and accumulation of lipofuscin. In addition, expression levels of ribosomal protein S6 kinase1 (S6K1), p-S6K1, p-S6, and eukaryotic translation initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) in the mammalian target of rapamycin (mTOR) pathway and beclin-1, ATG7, ATG12-ATG5 conjugate, and the sequestosome 1 (SQSTM1/p62) monomer in the autophagy pathway were decreased in both the replicatively and the autophagy impairment-induced prematurely senescent cells. Furthermore, it was found that ROS scavenging by N-acetylcysteine (NAC) and inhibition of p53 activation by pifithrin-α or knockdown of p53 using siRNA, respectively, delayed autophagy impairment-induced premature senescence and restored the expression levels of components in the mTOR and autophagy pathways. Taken together, we concluded that autophagy impairment induces premature senescence through a ROS- and p53-dependent manner in primary human fibroblasts.

  4. 303(d) Listed Impaired Waters

    EPA Pesticide Factsheets

    Geospatial data for 303(d) Impaired Waters are available as prepackaged national downloads or as GIS web and and data services. EPA provides geospatial data in the formats: GIS compatible shapefiles and geodatabases and ESRI and OGC web mapping.

  5. Genetic Counseling in Hearing Impairment.

    ERIC Educational Resources Information Center

    Fraser, George R.

    1979-01-01

    The problem of counseling is dealt with mainly in the context of severe hearing impairment, since moderate forms are often due to illness or other nongenetic factors and do not constitute a grave handicap. (DLS)

  6. Psychiatric Issues in Cognitive Impairment

    PubMed Central

    Aarsland, Dag; Taylor, John-Paul; Weintraub, Daniel

    2015-01-01

    Neuropsychiatric symptoms (NPS) such as depression, hallucinations and apathy commonly occur in Parkinson’s disease (PD) and have major clinical consequences including a negative impact on quality of life. This review discusses the epidemiology, clinical features, diagnostic procedures and treatment issues of NPS in PD and related disorders in the perspective of cognitive impairment, focusing on depression, anxiety, visual hallucinations, apathy, sleep disturbances, impulse control disorder and non-motor fluctuations. The majority of NPS are more common in PD patients with dementia, possibly related to shared underlying pathologies. Recent studies also suggest that NPS are associated with mild cognitive impairment in PD, in particular with the amnestic type. Accurate diagnosis of NPS is important but can be difficult, due to overlapping symptoms and similar appearance of symptoms of motor symptoms of parkinsonism, cognitive impairment, mood disorders and apathy. There are few systematic studies focusing on the management of NPS in PD with cognitive impairment. PMID:24757113

  7. APP overexpression in the absence of NPC1 exacerbates metabolism of amyloidogenic proteins of Alzheimer's disease

    PubMed Central

    Maulik, Mahua; Peake, Kyle; Chung, JiYun; Wang, Yanlin; Vance, Jean E.; Kar, Satyabrata

    2015-01-01

    Amyloid-β (Aβ) peptides originating from β-amyloid precursor protein (APP) are critical in Alzheimer's disease (AD). Cellular cholesterol levels/distribution can regulate production and clearance of Aβ peptides, albeit with contradictory outcomes. To better understand the relationship between cholesterol homeostasis and APP/Aβ metabolism, we have recently generated a bigenic ANPC mouse line overexpressing mutant human APP in the absence of Niemann-Pick type C-1 protein required for intracellular cholesterol transport. Using this unique bigenic ANPC mice and complementary stable N2a cells, we have examined the functional consequences of cellular cholesterol sequestration in the endosomal–lysosomal system, a major site of Aβ production, on APP/Aβ metabolism and its relation to neuronal viability. Levels of APP C-terminal fragments (α-CTF/β-CTF) and Aβ peptides, but not APP mRNA/protein or soluble APPα/APPβ, were increased in ANPC mouse brains and N2a-ANPC cells. These changes were accompanied by reduced clearance of peptides and an increased level/activity of γ-secretase, suggesting that accumulation of APP-CTFs is due to decreased turnover, whereas increased Aβ levels may result from a combination of increased production and decreased turnover. APP-CTFs and Aβ peptides were localized primarily in early-/late-endosomes and to some extent in lysosomes/autophagosomes. Cholesterol sequestration impaired endocytic-autophagic-lysosomal, but not proteasomal, clearance of APP-CTFs/Aβ peptides. Moreover, markers of oxidative stress were increased in vulnerable brain regions of ANPC mice and enhanced β-CTF/Aβ levels increased susceptibility of N2a-ANPC cells to H2O2-induced toxicity. Collectively, our results show that cellular cholesterol sequestration plays a key role in APP/Aβ metabolism and increasing neuronal vulnerability to oxidative stress in AD-related pathology. PMID:26433932

  8. Characterization of PTZ-Induced Seizure Susceptibility in a Down Syndrome Mouse Model That Overexpresses CSTB

    PubMed Central

    Brault, Véronique; Martin, Benoît; Costet, Nathalie; Bizot, Jean-Charles; Hérault, Yann

    2011-01-01

    Down syndrome (DS) is a complex genetic syndrome characterized by intellectual disability, dysmorphism and variable additional physiological traits. Current research progress has begun to decipher the neural mechanisms underlying cognitive impairment, leading to new therapeutic perspectives. Pentylenetetrazol (PTZ) has recently been found to have positive effects on learning and memory capacities of a DS mouse model and is foreseen to treat DS patients. But PTZ is also known to be a convulsant drug at higher dose and DS persons are more prone to epileptic seizures than the general population. This raises concerns over what long-term effects of treatment might be in the DS population. The cause of increased propensity for epilepsy in the DS population and which Hsa21 gene(s) are implicated remain unknown. Among Hsa21 candidate genes in epilepsy, CSTB, coding for the cystein protease inhibitor cystatin B, is involved in progressive myoclonus epilepsy and ataxia in both mice and human. Thus we aim to evaluate the effect of an increase in Cstb gene dosage on spontaneous epileptic activity and susceptibility to PTZ-induced seizure. To this end we generated a new mouse model trisomic for Cstb by homologous recombination. We verified that increasing copy number of Cstb from Trisomy (Ts) to Tetrasomy (Tt) was driving overexpression of the gene in the brain, we checked transgenic animals for presence of locomotor activity and electroencephalogram (EEG) abnormalities characteristic of myoclonic epilepsy and we tested if those animals were prone to PTZ-induced seizure. Overall, the results of the analysis shows that an increase in Cstb does not induce any spontaneous epileptic activity and neither increase or decrease the propensity of Ts and Tt mice to myoclonic seizures suggesting that Ctsb dosage should not interfere with PTZ-treatment. PMID:22140471

  9. Suppression of Non-Homologous End Joining Repair by Overexpression of HMGA2

    PubMed Central

    Li, Angela Y.J.; Boo, Lee Ming; Wang, Shih-Ya; Lin, H. Helen; Wang, Clay C.C.; Yen, Yun; Chen, Benjamin P.C.; Chen, David J.; Ann, David K.

    2009-01-01

    Understanding the molecular details associated with aberrant high mobility group A2 (HMGA2) gene expression is key to establishing the mechanism(s) underlying its oncogenic potential and impact on the development of therapeutic strategies. Here, we report the involvement of HMGA2 in impairing DNA-dependent protein kinase (DNA-PK) during the non-homologous end joining (NHEJ) process. We demonstrated that HMGA2-expressing cells displayed deficiency in overall and precise DNA end-joining repair and accumulated more endogenous DNA damage. Proper and timely activation of DNA-PK, consisting of Ku70, Ku80 and DNA-PKcs subunits, is essential for the repair of DNA double strand breaks (DSBs) generated endogenously or by exposure to genotoxins. In cells overexpressing HMGA2, accumulation of histone 2A variant X phosphorylation at Ser-139 (γ-H2AX) was associated with hyper-phosphorylation of DNA-PKcs at Thr-2609 and Ser-2056 before and after the induction of DSBs. Also, the steady-state complex of Ku and DNA ends was altered by HMGA2. Microirradiation and real-time imaging in living cells revealed that HMGA2 delayed the release of DNA-PKcs from DSB sites, similar to observations found in DNA-PKcs mutants. Moreover, HMGA2 alone was sufficient to induce chromosomal aberrations, a hallmark of deficiency in NHEJ-mediated DNA repair. In summary, a novel role for HMGA2 to interfere with NHEJ processes was uncovered, implicating HMGA2 in the promotion of genome instability and tumorigenesis. PMID:19549901

  10. Over-expression of a human chromosome 22q11.2 segment including TXNRD2, COMT and ARVCF developmentally affects incentive learning and working memory in mice.

    PubMed

    Suzuki, Go; Harper, Kathryn M; Hiramoto, Takeshi; Funke, Birgit; Lee, MoonSook; Kang, Gina; Buell, Mahalah; Geyer, Mark A; Kucherlapati, Raju; Morrow, Bernice; Männistö, Pekka T; Agatsuma, Soh; Hiroi, Noboru

    2009-10-15

    Duplication of human chromosome 22q11.2 is associated with elevated rates of mental retardation, autism and many other behavioral phenotypes. However, because duplications cover 1.5-6 Mb, the precise manner in which segments of 22q11.2 causally affect behavior is not known in humans. We have now determined the developmental impact of over-expression of an approximately 190 kb segment of human 22q11.2, which includes the genes TXNRD2, COMT and ARVCF, on behaviors in bacterial artificial chromosome (BAC) transgenic (TG) mice. BAC TG mice and wild-type (WT) mice were tested for their cognitive capacities, affect- and stress-related behaviors and motor activity at 1 and 2 months of age. An enzymatic assay determined the impact of BAC over-expression on the activity level of COMT. BAC TG mice approached a rewarded goal faster (i.e. incentive learning), but were impaired in delayed rewarded alternation during development. In contrast, BAC TG and WT mice were indistinguishable in rewarded alternation without delays, spontaneous alternation, prepulse inhibition, social interaction, anxiety-, stress- and fear-related behaviors and motor activity. Compared with WT mice, BAC TG mice had an approximately 2-fold higher level of COMT activity in the prefrontal cortex, striatum and hippocampus. These data suggest that over-expression of this 22q11.2 segment enhances incentive learning and impairs the prolonged maintenance of working memory, but has no apparent effect on working memory per se, affect- and stress-related behaviors or motor capacity. High copy numbers of this 22q11.2 segment might contribute to a highly selective set of phenotypes in learning and cognition during development.

  11. RAC1b overexpression correlates with poor prognosis in KRAS/BRAF WT metastatic colorectal cancer patients treated with first-line FOLFOX/XELOX chemotherapy.

    PubMed

    Alonso-Espinaco, Virginia; Cuatrecasas, Miriam; Alonso, Vicente; Escudero, Pilar; Marmol, Maribel; Horndler, Carlos; Ortego, Javier; Gallego, Rosa; Codony-Servat, Jordi; Garcia-Albeniz, Xabier; Jares, Pedro; Castells, Antoni; Lozano, Juan José; Rosell, Rafael; Maurel, Joan

    2014-07-01

    Chemotherapy is the principal treatment in metastatic colorectal cancer (mCRC) patients. RAC1b, a RAC1 spliced variant, is over-expressed in colorectal cancer (CRC), and impairs apoptosis by activation of nuclear-factor-KB. Since RAC1b has been associated with the BRAF(V600E) mutation, associated with poor prognosis in CRC, we evaluated the role of RAC1b expression as a predictor of chemotherapy efficacy in mCRC. We analysed KRAS and BRAF mutation, microsatellite instability and RAC1b expression in 157 mCRC patients treated with FOLFOX/XELOX in first-line therapy. KRAS mutations were detected in 46 patients (34%), 10 patients were BRAF mutant (7%) and 79 were WT for both, KRAS and BRAF (59%). RAC1b overexpression was found in 30 patients (19%). In the multivariate analysis, BRAF mutational status was a poor prognostic factor for overall survival (OS); hazard ratio (HR), 2.78 (95% confidence interval (CI), 1.35-5.72; p=0.0057). RAC1b overexpression was a poor survival factor for OS (HR, 2.35; 95% CI, 1.2-4.59; p=0.01) and progression-free survival (PFS) (HR, 2.4; 95% CI, 1.2-4.78; p=0.01) in KRAS/BRAF WT mCRC patients. RAC1b overexpression constitutes a marker of poor prognosis in KRAS/BRAF WT mCRC patients treated with first-line FOLFOX/XELOX therapy. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Cardiac-restricted Overexpression of TRAF3 Interacting Protein 2 (TRAF3IP2) Results in Spontaneous Development of Myocardial Hypertrophy, Fibrosis, and Dysfunction.

    PubMed

    Yariswamy, Manjunath; Yoshida, Tadashi; Valente, Anthony J; Kandikattu, Hemanth Kumar; Sakamuri, Siva S V P; Siddesha, Jalahalli M; Sukhanov, Sergiy; Saifudeen, Zubaida; Ma, Lixin; Siebenlist, Ulrich; Gardner, Jason D; Chandrasekar, Bysani

    2016-09-09

    TRAF3IP2 (TRAF3 interacting protein 2; previously known as CIKS or Act1) is a key intermediate in the normal inflammatory response and the pathogenesis of various autoimmune and inflammatory diseases. Induction of TRAF3IP2 activates IκB kinase (IKK)/NF-κB, JNK/AP-1, and c/EBPβ and stimulates the expression of various inflammatory mediators with negative myocardial inotropic effects. To investigate the role of TRAF3IP2 in heart disease, we generated a transgenic mouse model with cardiomyocyte-specific TRAF3IP2 overexpression (TRAF3IP2-Tg). Echocardiography, magnetic resonance imaging, and pressure-volume conductance catheterization revealed impaired cardiac function in 2-month-old male transgenic (Tg) mice as evidenced by decreased ejection fraction, stroke volume, cardiac output, and peak ejection rate. Moreover, the male Tg mice spontaneously developed myocardial hypertrophy (increased heart/body weight ratio, cardiomyocyte cross-sectional area, GATA4 induction, and fetal gene re-expression). Furthermore, TRAF3IP2 overexpression resulted in the activation of IKK/NF-κB, JNK/AP-1, c/EBPβ, and p38 MAPK and induction of proinflammatory cytokines, chemokines, and extracellular matrix proteins in the heart. Although myocardial hypertrophy decreased with age, cardiac fibrosis (increased number of myofibroblasts and enhanced expression and deposition of fibrillar collagens) increased progressively. Despite these adverse changes, TRAF3IP2 overexpression did not result in cell death at any time period. Interestingly, despite increased mRNA expression, TRAF3IP2 protein levels and activation of its downstream signaling intermediates remained unchanged in the hearts of female Tg mice. The female Tg mice also failed to develop myocardial hypertrophy. In summary, these results demonstrate that overexpression of TRAF3IP2 in male mice is sufficient to induce myocardial hypertrophy, cardiac fibrosis, and contractile dysfunction. © 2016 by The American Society for Biochemistry

  13. Overexpression of a Calcium-Dependent Protein Kinase Confers Salt and Drought Tolerance in Rice by Preventing Membrane Lipid Peroxidation1[C][W

    PubMed Central

    Campo, Sonia; Baldrich, Patricia; Messeguer, Joaquima; Lalanne, Eric; Coca, María; San Segundo, Blanca

    2014-01-01

    The OsCPK4 gene is a member of the complex gene family of calcium-dependent protein kinases in rice (Oryza sativa). Here, we report that OsCPK4 expression is induced by high salinity, drought, and the phytohormone abscisic acid. Moreover, a plasma membrane localization of OsCPK4 was observed by transient expression assays of green fluorescent protein-tagged OsCPK4 in onion (Allium cepa) epidermal cells. Overexpression of OsCPK4 in rice plants significantly enhances tolerance to salt and drought stress. Knockdown rice plants, however, are severely impaired in growth and development. Compared with control plants, OsCPK4 overexpressor plants exhibit stronger water-holding capability and reduced levels of membrane lipid peroxidation and electrolyte leakage under drought or salt stress conditions. Also, salt-treated OsCPK4 seedlings accumulate less Na+ in their roots. We carried out microarray analysis of transgenic rice overexpressing OsCPK4 and found that overexpression of OsCPK4 has a low impact on the rice transcriptome. Moreover, no genes were found to be commonly regulated by OsCPK4 in roots and leaves of rice plants. A significant number of genes involved in lipid metabolism and protection against oxidative stress appear to be up-regulated by OsCPK4 in roots of overexpressor plants. Meanwhile, OsCPK4 overexpression has no effect on the expression of well-characterized abiotic stress-associated transcriptional regulatory networks (i.e. ORYZA SATIVA DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN1 and ORYZA SATIVA No Apical Meristem, Arabidopsis Transcription Activation Factor1-2, Cup-Shaped Cotyledon6 genes) and LATE EMBRYOGENESIS ABUNDANT genes in their roots. Taken together, our data show that OsCPK4 functions as a positive regulator of the salt and drought stress responses in rice via the protection of cellular membranes from stress-induced oxidative damage. PMID:24784760

  14. Overexpression of the scaffold WD40 protein WRAP53β enhances the repair of and cell survival from DNA double-strand breaks

    PubMed Central

    Rassoolzadeh, H; Böhm, S; Hedström, E; Gad, H; Helleday, T; Henriksson, S; Farnebo, M

    2016-01-01

    Altered expression of the multifunctional protein WRAP53β (WD40 encoding RNA Antisense to p53), which targets repair factors to DNA double-strand breaks and factors involved in telomere elongation to Cajal bodies, is linked to carcinogenesis. While loss of WRAP53β function has been shown to disrupt processes regulated by this protein, the consequences of its overexpression remain unclear. Here we demonstrate that overexpression of WRAP53β disrupts the formation of and impairs the localization of coilin to Cajal bodies. At the same time, the function of this protein in the repair of DNA double-strand breaks is enhanced. Following irradiation, cells overexpressing WRAP53β exhibit more rapid clearance of phospho-histone H2AX (γH2AX), and more efficient homologous recombination and non-homologous end-joining, in association with fewer DNA breaks. Moreover, in these cells the ubiquitylation of damaged chromatin, which is known to facilitate the recruitment of repair factors and subsequent repair, is elevated. Knockdown of the ubiquitin ligase involved, ring-finger protein 8 (RNF8), which is recruited to DNA breaks by WRAP53β, attenuated this effect, suggesting that overexpression of WRAP53β leads to more rapid repair, as well as improved cell survival, by enhancing RNF8-mediated ubiquitylation at DNA breaks. Our present findings indicate that WRAP53β and RNF8 are rate-limiting factors in the repair of DNA double-strand breaks and raise the possibility that upregulation of WRAP53β may contribute to genomic stability in and survival of cancer cells. PMID:27310875

  15. Impaired procedural learning in language impairment: results from probabilistic categorization.

    PubMed

    Kemeny, Ferenc; Lukacs, Agnes

    2010-03-01

    The Weather Prediction (WP) Task is a classical task of probabilistic category learning generally used for examining the dissociation of procedural and declarative memory. The current study focuses on performance of children with language impairment (LI) and compares their performance to that of typically developing (TD) children and adults with the aim of testing the procedural deficit hypothesis of LI (PDH; Ullman & Pierpont, 2005), which states that language impairment is not a specific linguistic phenomenon, but results from the dysfunction of a more general cognitive system: the procedural system. To test the generality of the procedural impairment, we needed a task that is dissimilar from language in that it does not build on sequential information. Children with language impairment show deficient learning on the Weather Prediction Task, which already appears at the early stages of the task. These results, in line with the PDH, point to the deficit of the procedural system in language impairment going beyond the language system. Whether this deficit is selective to the procedural system or is complemented by deficits in the declarative system is the subject of future studies.

  16. Skeletal muscle damage and impaired regeneration due to LPL-mediated lipotoxicity

    PubMed Central

    Tamilarasan, K P; Temmel, H; Das, S K; Al Zoughbi, W; Schauer, S; Vesely, P W; Hoefler, G

    2012-01-01

    According to the concept of lipotoxicity, ectopic accumulation of lipids in non-adipose tissue induces pathological changes. The most prominent effects are seen in fatty liver disease, lipid cardiomyopathy, non-insulin-dependent diabetes mellitus, insulin resistance and skeletal muscle myopathy. We used the MCK(m)-hLPL mouse distinguished by skeletal and cardiac muscle-specific human lipoprotein lipase (hLPL) overexpression to investigate effects of lipid overload in skeletal muscle. We were intrigued to find that ectopic lipid accumulation induced proteasomal activity, apoptosis and skeletal muscle damage. In line with these findings we observed reduced Musculus gastrocnemius and Musculus quadriceps mass in transgenic animals, accompanied by severely impaired physical endurance. We suggest that muscle loss was aggravated by impaired muscle regeneration as evidenced by reduced cross-sectional area of regenerating myofibers after cardiotoxin-induced injury in MCK(m)-hLPL mice. Similarly, an almost complete loss of myogenic potential was observed in C2C12 murine myoblasts upon overexpression of LPL. Our findings directly link lipid overload to muscle damage, impaired regeneration and loss of performance. These findings support the concept of lipotoxicity and are a further step to explain pathological effects seen in muscle of obese patients, patients with the metabolic syndrome and patients with cancer-associated cachexia. PMID:22825472

  17. Impaired Pain Processing Correlates with Cognitive Impairment in Parkinson's Disease

    PubMed Central

    Okada, Akinori; Nakamura, Tomohiko; Suzuki, Junichiro; Suzuki, Masashi; Hirayama, Masaaki; Katsuno, Masahisa; Sobue, Gen

    2016-01-01

    Objective Pain and cognitive impairment are important clinical features in patients with Parkinson's disease (PD). Although pain processing is associated with the limbic system, which is also closely linked to the cognitive function, the association between pain and cognitive impairment in PD is still not well understood. The aim of the study was to investigate the association between pain processing and cognitive impairment in patients with PD. Methods Forty-three patients with PD and 22 healthy subjects were studied. Pain-related somatosensory evoked potentials (SEPs) were generated using a thin needle electrode to stimulate epidermal Aδ fibers. Cognitive impairment was evaluated using the Mini-Mental State Examination (MMSE), the Frontal Assessment Battery, and Japanese version of the Montreal Cognitive Assessment (MoCA-J), and their correlation with pain-related SEPs was investigated. Results The N1/P1 amplitude was significantly lower in PD patients than the controls. N1/P1 peak-to-peak amplitudes correlated with the MMSE (r=0.66, p<0.001) and MoCA-J scores (r=0.38, p<0.01) in patients with PD. These amplitudes also strongly correlated with the domains of attention and memory in the MMSE (attention, r=0.52, p<0.001; memory, r=0.40, p<0.01) and MoCA-J (attention, r=0.45, p<0.005; memory, r=0.48, p<0.001), but not in control subjects. Conclusion A good correlation was observed between the decreased amplitudes of pain-related SEPs and an impairment of attention and memory in patients with PD. Our results suggest that pathological abnormalities of the pain pathway are significantly linked to cognitive impairment in PD. PMID:27803403

  18. Over-expression of Multi-heme C-type Cytochromes

    SciTech Connect

    Shi, Liang; Lin, Chiann Tso; Markillie, Lye Meng; Squier, Thomas C.; Hooker, Brian S.

    2005-02-01

    ABSTRACT-Because they contain covalently attached hemes, c-type cytochromes, especially those with multi-heme, are difficult to over-express. The gram negative bacterium Shewanella oneidensis MR-1 has been successfully used for over-expression of multi-heme c-type cytochromes...

  19. Effects of HMGB-1 Overexpression on Cell-Cycle Progression in MCF-7 Cells

    PubMed Central

    Yoon, Sarah; Lee, Jin Young; Yoon, Byung-Koo; Bae, DukSoo

    2004-01-01

    High mobility group-1 (HMGB-1) enhances the DNA interactions and possesses a transcriptional activation potential for several families of sequence-specific transcriptional activators. In order to examine the effect of HMGB-1 on the cell cycle progression in MCF-7 cells, the HMGB-1 expression vector was transfected into synchronized MCF-7 cells, and the effect of HMGB-1 overexpression on the cell cycle was examined. The HMGB-1 protein level in the transfected cells increased 4.87-fold compared to the non-transfected cells. There were few changes in the cell cycle phase distribution after HMGB-1 overexpression in the MCF-7 cells. Following the estrogen treatment, the cell cycle progressed in both the HMGB-1 overexpressed MCF-7 and the mock-treated cells. However, a larger proportion of HMGB-1 overexpressing MCF-7 cells progressed to the either S or G2 phase than the mock-treated cells. The mRNA levels of the cell cycle regulators changed after being treated with estrogen in both the HMGB-1 overexpressing MCF-7 and the mock-treated cells, but the changes in the expression level of the cell cycle regulator genes were more prominent in the HMGB-1 overexpressing MCF-7 cells than in the mock-treated cells. In conclusion, HMGB-1 overexpression itself does not alter the MCF-7 cell cycle progression, but the addition of estrogen to the HMGB-1 overexpressing MCF-7 cells appears to accelerate the cell cycle progression. PMID:15201494

  20. STAT1 negatively regulates spatial memory formation and mediates the memory-impairing effect of Aβ.

    PubMed

    Hsu, Wei-Lun; Ma, Yun-Li; Hsieh, Ding-You; Liu, Yen-Chen; Lee, Eminy Hy

    2014-02-01

    Signal transducer and activator of transcription-1 (STAT1) has an important role in inflammation and the innate immune response, but its role in the central nervous system is less well understood. Here, we examined the role of STAT1 in spatial learning and memory, and assessed the involvement of STAT1 in mediating the memory-impairing effect of amyloid-beta (Aβ). We found that water maze training downregulated STAT1 expression in the rat hippocampal CA1 area, and spatial learning and memory function was enhanced in Stat1-knockout mice. Conversely, overexpression of STAT1 impaired water maze performance. STAT1 strongly upregulated the expression of the extracellular matrix protein laminin β1 (LB1), which also impaired water maze performance in rats. Furthermore, Aβ impaired spatial learning and memory in association with a dose-dependent increase in STAT1 and LB1 expression, but knockdown of STAT1 and LB1 both reversed this effect of Aβ. This Aβ-induced increase in STAT1 and LB1 expression was also associated with a decrease in the expression of the N-methyl-D-aspartate receptor (NMDAR) subunits, NR1, and NR2B. Overexpression of NR1 or NR2B or exogenous application of NMDA reversed Aβ-induced learning and memory deficits as well as Aβ-induced STAT1 and LB1 expression. Our results demonstrate that STAT1 negatively regulates spatial learning and memory through transcriptional regulation of LB1 expression. We also identified a novel mechanism for Aβ pathogenesis through STAT1 induction. Notably, impairment of spatial learning and memory by this STAT1-mediated mechanism is independent of cAMP responsive element-binding protein signaling.

  1. 20 CFR 416.923 - Multiple impairments.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false Multiple impairments. 416.923 Section 416.923... DISABLED Determining Disability and Blindness Evaluation of Disability § 416.923 Multiple impairments. In... the combined effect of all of your impairments without regard to whether any such impairment,...

  2. Gadobutrol in Renally Impaired Patients

    PubMed Central

    Michaely, Henrik J.; Aschauer, Manuela; Deutschmann, Hannes; Bongartz, Georg; Gutberlet, Matthias; Woitek, Ramona; Ertl-Wagner, Birgit; Kucharczyk, Walter; Hammerstingl, Renate; De Cobelli, Francesco; Rosenberg, Martin; Balzer, Thomas; Endrikat, Jan

    2017-01-01

    Objective The aim of this study was to assess the potential risk of gadobutrol-enhanced magnetic resonance imaging (MRI) in patients with moderate to severe renal impairment for the development of nephrogenic systemic fibrosis (NSF). Materials and Methods We performed a prospective, international, multicenter, open-label study in 55 centers. Patients with moderate to severe renal impairment scheduled for any gadobutrol-enhanced MRI were included. All patients received a single intravenous bolus injection of gadobutrol at a dose of 0.1 mmol/kg body weight. The primary target variable was the number of patients who develop NSF within a 2-year follow-up period. Results A total of 908 patients were enrolled, including 586 with moderate and 284 with severe renal impairment who are at highest risk for developing NSF. The mean time since renal disease diagnosis was 1.83 and 5.49 years in the moderate and severe renal impairment cohort, respectively. Overall, 184 patients (20.3%) underwent further contrast-enhanced MRI with other gadolinium-based contrast agents within the 2-year follow-up. No patient developed symptoms conclusive of NSF. Conclusions No safety concerns with gadobutrol in patients with moderate to severe renal impairment were identified. There were no NSF cases. PMID:27529464

  3. Rcan1 deficiency impairs neuronal migration and causes periventricular heterotopia.

    PubMed

    Li, Yang; Wang, Jie; Zhou, Yang; Li, Dan; Xiong, Zhi-Qi

    2015-01-14

    Periventricular heterotopia (PH) is a cortical malformation characterized by aggregation of neurons lining the lateral ventricles due to abnormal neuronal migration. The molecular mechanism underlying the pathogenesis of PH is unclear. Here we show that Regulators of calcineurin 1 (Rcan1), a Down syndrome-related gene, plays an important role in radial migration of rat cortical neurons. Downregulation of Rcan1 by expressing shRNA impaired neural progenitor proliferation and led to defects in radial migration and PH. Two isoforms of Rcan1 (Rcan1-1 and Rcan1-4) are expressed in the rat brain. Migration defects due to downregulation of Rcan1 could be prevented by shRNA-resistant expression of Rcan1-1 but not Rcan1-4. Furthermore, we found that Rcan1 knockdown significantly decreased the expression level of Flna, an F-actin cross-linking protein essential for cytoskeleton rearrangement and cell migration, mutation of which causes the most common form of bilateral PH in humans. Finally, overexpression of FLNA in Rcan1 knockdown neurons prevented migration abnormalities. Together, these findings demonstrate that Rcan1 acts upstream from Flna in regulating radial migration and suggest that impairment of Rcan1-Flna pathway may underlie PH pathogenesis.

  4. The effects of bufadienolides on HER2 overexpressing breast cancer cells.

    PubMed

    Wang, Tianjiao; Mu, Lin; Jin, Haifeng; Zhang, Peng; Wang, Yueyue; Ma, Xiaochi; Pan, Jinjin; Miao, Jian; Yuan, Yuhui

    2016-06-01

    HER2 is a proto-oncogene frequently amplified in human breast cancer, its overexpression is correlated with tamoxifen resistance and decreased recurrence-free survival. Arenobufagin and bufalin are homogeneous bufadienolides of cardiac glycosides agents. In this research, we studied the effects of arenobufagin and bufalin on cellular survival and proliferation of HER2 overexpressing breast cancer cells and the mechanism under the results including the direct effect on HER2 downstream pathways. Our results showed that arenobufagin and bufalin could significantly inhibit the proliferation and survival of HER2 overexpressing breast cancer cells, along with the declination of SRC-1, SRC-3, nuclear transcription factor E2F1, phosphorylated AKT, and ERK. And the combination of each bufadienolide in low dose with tamoxifen could significantly enhance the inhibitory effect of tamoxifen on HER2 overexpressing breast cancer cells. All above suggest that arenobufagin and bufalin may be potential therapy adjuvants for HER2 overexpressing breast cancer therapy.

  5. Overexpression of Human Bone Alkaline Phosphatase in Pichia Pastoris

    NASA Technical Reports Server (NTRS)

    Karr, Laurel; Malone, Christine, C.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    The Pichiapastoris expression system was utilized to produce functionally active human bone alkaline phosphatase in gram quantities. Bone alkaline phosphatase is a key enzyme in bone formation and biomineralization, yet important questions about its structural chemistry and interactions with other cellular enzymes in mineralizing tissues remain unanswered. A soluble form of human bone alkaline phosphatase was constructed by deletion of the 25 amino acid hydrophobic C-terminal region of the encoding cDNA and inserted into the X-33 Pichiapastoris strain. An overexpression system was developed in shake flasks and converted to large-scale fermentation. Alkaline phosphatase was secreted into the medium to a level of 32mgAL when cultured in shake flasks. Enzyme activity was 12U/mg measured by a spectrophotometric assay. Fermentation yielded 880mgAL with enzymatic activity of 968U/mg. Gel electrophoresis analysis indicates that greater than 50% of the total protein in the fermentation is alkaline phosphatase. A purification scheme has been developed using ammonium sulfate precipitation followed by hydrophobic interaction chromatography. We are currently screening crystallization conditions of the purified recombinant protein for subsequent X-ray diffraction analyses. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

  6. Overexpression of Human Bone Alkaline Phosphatase in Pichia Pastoris

    NASA Technical Reports Server (NTRS)

    Karr, Laurel; Malone, Christine, C.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    The Pichiapastoris expression system was utilized to produce functionally active human bone alkaline phosphatase in gram quantities. Bone alkaline phosphatase is a key enzyme in bone formation and biomineralization, yet important questions about its structural chemistry and interactions with other cellular enzymes in mineralizing tissues remain unanswered. A soluble form of human bone alkaline phosphatase was constructed by deletion of the 25 amino acid hydrophobic C-terminal region of the encoding cDNA and inserted into the X-33 Pichiapastoris strain. An overexpression system was developed in shake flasks and converted to large-scale fermentation. Alkaline phosphatase was secreted into the medium to a level of 32mgAL when cultured in shake flasks. Enzyme activity was 12U/mg measured by a spectrophotometric assay. Fermentation yielded 880mgAL with enzymatic activity of 968U/mg. Gel electrophoresis analysis indicates that greater than 50% of the total protein in the fermentation is alkaline phosphatase. A purification scheme has been developed using ammonium sulfate precipitation followed by hydrophobic interaction chromatography. We are currently screening crystallization conditions of the purified recombinant protein for subsequent X-ray diffraction analyses. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

  7. Cardiac Muscarinic Receptor Overexpression in Sudden Infant Death Syndrome

    PubMed Central

    Livolsi, Angelo; Niederhoffer, Nathalie; Dali-Youcef, Nassim; Rambaud, Caroline; Olexa, Catherine; Mokni, Walid; Gies, Jean-Pierre; Bousquet, Pascal

    2010-01-01

    Background Sudden infant death syndrome (SIDS) remains the leading cause of death among infants less than 1 year of age. Disturbed expression of some neurotransmitters and their receptors has been shown in the central nervous system of SIDS victims but no biological abnormality of the peripheral vago-cardiac system has been demonstrated to date. The present study aimed to seek vago-cardiac abnormalities in SIDS victims. The cardiac level of expression of muscarinic receptors, as well as acetylcholinesterase enzyme activity were investigated. Methodology/Principal Findings Left ventricular samples and blood samples were obtained from autopsies of SIDS and children deceased from non cardiac causes. Binding experiments performed with [3H]NMS, a selective muscarinic ligand, in cardiac membrane preparations showed that the density of cardiac muscarinic receptors was increased as shown by a more than doubled Bmax value in SIDS (n = 9 SIDS versus 8 controls). On average, the erythrocyte acetylcholinesterase enzyme activity was also significantly increased (n = 9 SIDS versus 11 controls). Conclusions In the present study, it has been shown for the first time that cardiac muscarinic receptor overexpression is associated with SIDS. The increase of acetylcholinesterase enzyme activity appears as a possible regulatory mechanism. PMID:20209124

  8. Overexpression and topology of bacterial oligosaccharyltransferase PglB

    SciTech Connect

    Li, Lei; Woodward, Robert; Ding, Yan; Liu, Xian-wei; Yi, Wen; Bhatt, Veer S.; Chen, Min; Zhang, Lian-wen; Wang, Peng George

    2010-04-16

    Campylobacter jejuni contains a post-translational N-glycosylation system in which a STT3 homologue, PglB, functions as the oligosaccharyltransferase. Herein, we established a method for obtaining relatively large quantities of homogenous PglB proteins. PglB was overexpressed in Escherichia coli C43(DE3) at a level of 1 mg/L cell cultures. The activity of purified PglB was verified using a chemically synthesized sugar donor: N-acetylgalactosamine-diphospho-undecaprenyl (GalNAc-PP-Und) and a synthesized peptide acceptor. The result confirms that PglB is solely responsible for the oligosaccharyltransferase activity and complements the finding that PglB exhibits relaxed sugar substrate specificity. In addition, we performed the topology mapping of PglB using the PhoA/LacZ fusion method. The topological model shows that PglB possesses 11 transmembrane segments and two relatively large periplasmic regions other than the C-terminal domain, which is consistent with the proposal of the common N{sub cyt}-C{sub peri} topology with 11 transmembrane segments for the STT3 family proteins.

  9. The small co-chaperone p23 overexpressing transgenic mouse

    PubMed Central

    Zhang, Junli; Spilman, Patricia; Chen, Sylvia; Gorostiza, Olivia; Matalis, Alex; Niazi, Kayvan; Bredesen, Dale E.; Rao, Rammohan V.

    2012-01-01

    Studies from multiple laboratories have identified the roles of several ER stress-induced cell death modulators and effectors. Earlier, we described the role of p23 a small co-chaperone protein in preventing ER stress-induced cell death. p23 is cleaved by caspases at D142 to yield p19 (a 19kD product) during ER stress-induced cell death. Mutation of the caspase cleavage site not only blocks formation of the 19kD product but also attenuates the cell death process triggered by various ER stressors. Thus, uncleavable p23 (p23D142N) emerges as a reasonable candidate to test for potential inhibition of neurodegenerative disease phenotype that features misfolded proteins and ER stress. In the present work we report the generation of transgenic mouse lines that overexpress wild-type p23 or uncleavable p23 under the control of a ROSA promoter. These mice should prove useful for studying the role of p23 and/or uncleavable p23 in cellular stress-induced cell death. PMID:23022695

  10. The small co-chaperone p23 overexpressing transgenic mouse.

    PubMed

    Zhang, Junli; Spilman, Patricia; Chen, Sylvia; Gorostiza, Olivia; Matalis, Alex; Niazi, Kayvan; Bredesen, Dale E; Rao, Rammohan V

    2013-01-30

    Studies from multiple laboratories have identified the roles of several ER stress-induced cell death modulators and effectors. Earlier, we described the role of p23 a small co-chaperone protein in preventing ER stress-induced cell death. p23 is cleaved by caspases at D142 to yield p19 (a 19 kDa product) during ER stress-induced cell death. Mutation of the caspase cleavage site not only blocks formation of the 19 kDa product but also attenuates the cell death process triggered by various ER stressors. Thus, uncleavable p23 (p23D142N) emerges as a reasonable candidate to test for potential inhibition of neurodegenerative disease phenotype that features misfolded proteins and ER stress. In the present work we report the generation of transgenic mouse lines that overexpress wild-type p23 or uncleavable p23 under the control of a ROSA promoter. These mice should prove useful for studying the role of p23 and/or uncleavable p23 in cellular stress-induced cell death. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Overexpression of microRNA-99a Attenuates Cardiac Hypertrophy.

    PubMed

    Li, Qiaoling; Xie, Jun; Wang, Bingjian; Li, Ran; Bai, Jian; Ding, Liang; Gu, Rong; Wang, Lian; Xu, Biao

    2016-01-01

    Pathological cardiomyocyte hypertrophy is associated with significantly increased risk of heart failure, one of the leading medical causes of mortality worldwide. MicroRNAs are known to be involved in pathological cardiac remodeling. However, whether miR-99a participates in the signaling cascade leading to cardiac hypertrophy is unknown. To evaluate the role of miR-99a in cardiac hypertrophy, we assessed the expression of miR-99a in hypertrophic cardiomyocytes induced by isoprenaline (ISO)/angiotensin-II (Ang II) and in mice model of cardiac hypertrophy induced by transverse aortic constriction (TAC). Expression of miR-99a was evaluated in these hypertrophic cells and hearts. We also found that miR-99a expression was highly correlated with cardiac function of mice with heart failure (8 weeks after TAC surgery). Overexpression of miR-99a attenuated cardiac hypertrophy in TAC mice and cellular hypertrophy in stimuli treated cardiomyocytes through down-regulation of expression of mammalian target of rapamycin (mTOR). These results indicate that miR-99a negatively regulates physiological hypertrophy through mTOR signaling pathway, which may provide a new therapeutic approach for pressure-overload heart failure.

  12. Transgenic overexpression of ribonucleotide reductase improves cardiac performance

    PubMed Central

    Nowakowski, Sarah G.; Kolwicz, Stephen C.; Korte, Frederick Steven; Luo, Zhaoxiong; Robinson-Hamm, Jacqueline N.; Page, Jennifer L.; Brozovich, Frank; Weiss, Robert S.; Tian, Rong; Murry, Charles E.; Regnier, Michael

    2013-01-01

    We previously demonstrated that cardiac myosin can use 2-deoxy-ATP (dATP) as an energy substrate, that it enhances contraction and relaxation with minimal effect on calcium-handling properties in vitro, and that contractile enhancement occurs with only minor elevation of cellular [dATP]. Here, we report the effect of chronically enhanced dATP concentration on cardiac function using a transgenic mouse that overexpresses the enzyme ribonucleotide reductase (TgRR), which catalyzes the rate-limiting step in de novo deoxyribonucleotide biosynthesis. Hearts from TgRR mice had elevated left ventricular systolic function compared with wild-type (WT) mice, both in vivo and in vitro, without signs of hypertrophy or altered diastolic function. Isolated cardiomyocytes from TgRR mice had enhanced contraction and relaxation, with no change in Ca2+ transients, suggesting targeted improvement of myofilament function. TgRR hearts had normal ATP and only slightly decreased phosphocreatine levels by 31P NMR spectroscopy, and they maintained rate responsiveness to dobutamine challenge. These data demonstrate long-term (at least 5-mo) elevation of cardiac [dATP] results in sustained elevation of basal left ventricular performance, with maintained β-adrenergic responsiveness and energetic reserves. Combined with results from previous studies, we conclude that this occurs primarily via enhanced myofilament activation and contraction, with similar or faster ability to relax. The data are sufficiently compelling to consider elevated cardiac [dATP] as a therapeutic option to treat systolic dysfunction. PMID:23530224

  13. Over-Expression of Meteorin Drives Gliogenesis Following Striatal Injury

    PubMed Central

    Wright, Jordan L.; Ermine, Charlotte M.; Jørgensen, Jesper R.; Parish, Clare L.; Thompson, Lachlan H.

    2016-01-01

    A number of studies have shown that damage to brain structures adjacent to neurogenic regions can result in migration of new neurons from neurogenic zones into the damaged tissue. The number of differentiated neurons that survive is low, however, and this has led to the idea that the introduction of extrinsic signaling factors, particularly neurotrophic proteins, may augment the neurogenic response to a level that would be therapeutically relevant. Here we report on the impact of the relatively newly described neurotrophic factor, Meteorin, when over-expressed in the striatum following excitotoxic injury. Birth-dating studies using bromo-deoxy-uridine (BrdU) showed that Meteorin did not enhance injury-induced striatal neurogenesis but significantly increased the proportion of new cells with astroglial and oligodendroglial features. As a basis for comparison we found under the same conditions, glial derived neurotrophic factor significantly enhanced neurogenesis but did not effect gliogenesis. The results highlight the specificity of action of different neurotrophic factors in modulating the proliferative response to injury. Meteorin may be an interesting candidate in pathological settings involving damage to white matter, for example after stroke or neonatal brain injury. PMID:27458346

  14. Overexpression of Rad51C splice variants in colorectal tumors.

    PubMed

    Kalvala, Arjun; Gao, Li; Aguila, Brittany; Reese, Tyler; Otterson, Gregory A; Villalona-Calero, Miguel A; Duan, Wenrui

    2015-04-20

    Functional alterations in Rad51C are the cause of the Fanconi anemia complementation group O (FANCO) gene disorder. We have identified novel splice variants of Rad51C mRNA in colorectal tumors and cells. The alternatively spliced transcript variants are formed either without exon-7 (variant 1), without exon 6 and 7 (variant 2) or without exon 7 and 8 (variant 3). Real time PCR analysis of nine pair-matched colorectal tumors and non-tumors showed that variant 1 was overexpressed in tumors compared to matched non-tumors. Among 38 colorectal tumor RNA samples analyzed, 18 contained variant 1, 12 contained variant 2, 14 contained variant 3, and eight expressed full length Rad51C exclusively. Bisulfite DNA sequencing showed promoter methylation of Rad51C in tumor cells. 5-azacytidine treatment of LS-174T cells caused a 14 fold increase in variant 1, a 4.8 fold increase for variant 3 and 3.4 fold for variant 2 compared to 2.5 fold increase in WT. Expression of Rad51C variants is associated with FANCD2 foci positive colorectal tumors and is associated with microsatellite stability in those tumors. Further investigation is needed to elucidate differential function of the Rad51C variants to evaluate potential effects in drug resistance and DNA repair.

  15. Overexpression of microRNA-99a Attenuates Cardiac Hypertrophy

    PubMed Central

    Li, Ran; Bai, Jian; Ding, Liang; Gu, Rong; Wang, Lian; Xu, Biao

    2016-01-01

    Pathological cardiomyocyte hypertrophy is associated with significantly increased risk of heart failure, one of the leading medical causes of mortality worldwide. MicroRNAs are known to be involved in pathological cardiac remodeling. However, whether miR-99a participates in the signaling cascade leading to cardiac hypertrophy is unknown. To evaluate the role of miR-99a in cardiac hypertrophy, we assessed the expression of miR-99a in hypertrophic cardiomyocytes induced by isoprenaline (ISO)/angiotensin-II (Ang II) and in mice model of cardiac hypertrophy induced by transverse aortic constriction (TAC). Expression of miR-99a was evaluated in these hypertrophic cells and hearts. We also found that miR-99a expression was highly correlated with cardiac function of mice with heart failure (8 weeks after TAC surgery). Overexpression of miR-99a attenuated cardiac hypertrophy in TAC mice and cellular hypertrophy in stimuli treated cardiomyocytes through down-regulation of expression of mammalian target of rapamycin (mTOR). These results indicate that miR-99a negatively regulates physiological hypertrophy through mTOR signaling pathway, which may provide a new therapeutic approach for pressure-overload heart failure. PMID:26914935

  16. Reduced antimony accumulation in ARM58-overexpressing Leishmania infantum.

    PubMed

    Schäfer, Carola; Tejera Nevado, Paloma; Zander, Dorothea; Clos, Joachim

    2014-01-01

    Antimony-based drugs are still the mainstay of chemotherapy against Leishmania infections in many countries where the parasites are endemic. The efficacy of antimonials has been compromised by increasing numbers of resistant infections, the basis of which is not fully understood and likely involves multiple factors. By using a functional cloning strategy, we recently identified a novel antimony resistance marker, ARM58, from the parasite Leishmania braziliensis that protects the parasites against antimony-based antileishmanial compounds. Here we show that the Leishmania infantum homologue also confers resistance against antimony but not against other antileishmanial drugs and that its function depends critically on one of four conserved domains of unknown function. This critical domain requires at least two hydrophobic amino acids and is predicted to form a transmembrane structure. Overexpression of ARM58 in antimony-exposed parasites reduces the intracellular Sb accumulation by over 70%, indicating a role for ARM58 in Sb extrusion pathways, but without involvement of energy-dependent transporter proteins.

  17. Reduced Antimony Accumulation in ARM58-Overexpressing Leishmania infantum

    PubMed Central

    Schäfer, Carola; Tejera Nevado, Paloma; Zander, Dorothea

    2014-01-01

    Antimony-based drugs are still the mainstay of chemotherapy against Leishmania infections in many countries where the parasites are endemic. The efficacy of antimonials has been compromised by increasing numbers of resistant infections, the basis of which is not fully understood and likely involves multiple factors. By using a functional cloning strategy, we recently identified a novel antimony resistance marker, ARM58, from the parasite Leishmania braziliensis that protects the parasites against antimony-based antileishmanial compounds. Here we show that the Leishmania infantum homologue also confers resistance against antimony but not against other antileishmanial drugs and that its function depends critically on one of four conserved domains of unknown function. This critical domain requires at least two hydrophobic amino acids and is predicted to form a transmembrane structure. Overexpression of ARM58 in antimony-exposed parasites reduces the intracellular Sb accumulation by over 70%, indicating a role for ARM58 in Sb extrusion pathways, but without involvement of energy-dependent transporter proteins. PMID:24366738

  18. Generation of transgenic mice for conditional overexpression of Sox9

    PubMed Central

    Kim, Youngwoo; Murao, Hiroki; Yamamoto, Koji; Deng, Jian Min; Behringer, Richard R.; Nakamura, Takashi; Akiyama, Haruhiko

    2014-01-01

    Sox9 belongs to the family of Sry-related high-mobility group box transcription factors controlling cell fate, cell proliferation and differentiation in various tissues, including cartilage, testis, the central nervous system, kidney, and gastrointestine. Mice conditionally lacking Sox9 have revealed essential roles for Sox9 in these tissues. To gain further insight into the role of Sox9 in different tissues and at different stages of development, we have generated a transgenic mouse line to express Sox9 in a Cre recombinase-dependent manner. The construct contained the human cytomegalovirus enhancer and chicken β-actin promoter, and a floxed monomeric red fluorescence protein 1 (mRFP1) cassette to direct ubiquitous expression of mRFP1. Cre expression removed the mRFP1 gene, allowing expression of Sox9 and enhanced green fluorescent protein. Expectedly, overexpression of Sox9 in chondrocytes using Col2a1-Cre mice suppressed chondrocyte hypertrophy, and delayed terminal differentiation and subsequent ossification. Misexpression of Sox9 in hypertrophic chondrocytes using Col10a1-Cre knock-in mice also delayed terminal differentiation. This Sox9 conditional transgenic mouse line will be a valuable tool to uncover tissue-specific and developmental stage-specific functions of Sox9. PMID:20676705

  19. Overexpression of SOD2 increases salt tolerance of Arabidopsis.

    PubMed

    Gao, Xiuhua; Ren, Zhonghai; Zhao, Yanxiu; Zhang, Hui

    2003-12-01

    The yeast (Schizosaccharomyces pombe) SOD2 (Sodium2) gene was introduced into Arabidopsis under the control of the cauliflower mosaic virus 35S promoter. Transformants were selected for their ability to grow on medium containing kanamycin. Southern- and northern-blot analyses confirmed that SOD2 was transferred into the Arabidopsis genome. There were no obvious morphological or developmental differences between the transgenic and wild-type (wt) plants. Several transgenic homozygous lines and wt plants (control) were evaluated for salt tolerance and gene expression. Overexpression of SOD2 in Arabidopsis improved seed germination and seedling salt tolerance. Analysis of Na+ and K+ contents of the symplast and apoplast in the parenchyma cells of the root cortex and mesophyll cells in the spongy tissue of the leaf showed that transgenic lines accumulated less Na+ and more K+ in the symplast than the wt plants did. The photosynthetic rate and the fresh weight of the transgenic lines were distinctly higher than that of wt plants after NaCl treatment. Results from different tests indicated that the expression of the SOD2 gene promoted a higher level of salt tolerance in vivo in transgenic Arabidopsis plants.

  20. Overexpression and topology of bacterial oligosaccharyltransferase PglB.

    PubMed

    Li, Lei; Woodward, Robert; Ding, Yan; Liu, Xian-wei; Yi, Wen; Bhatt, Veer S; Chen, Min; Zhang, Lian-wen; Wang, Peng George

    2010-04-16

    Campylobacter jejuni contains a post-translational N-glycosylation system in which a STT3 homologue, PglB, functions as the oligosaccharyltransferase. Herein, we established a method for obtaining relatively large quantities of homogenous PglB proteins. PglB was overexpressed in Escherichia coli C43(DE3) at a level of 1 mg/L cell cultures. The activity of purified PglB was verified using a chemically synthesized sugar donor: N-acetylgalactosamine-diphospho-undecaprenyl (GalNAc-PP-Und) and a synthesized peptide acceptor. The result confirms that PglB is solely responsible for the oligosaccharyltransferase activity and complements the finding that PglB exhibits relaxed sugar substrate specificity. In addition, we performed the topology mapping of PglB using the PhoA/LacZ fusion method. The topological model shows that PglB possesses 11 transmembrane segments and two relatively large periplasmic regions other than the C-terminal domain, which is consistent with the proposal of the common N(cyt)-C(peri) topology with 11 transmembrane segments for the STT3 family proteins. Crown Copyright 2010. Published by Elsevier Inc. All rights reserved.

  1. Overexpression of calmodulin in pancreatic beta cells induces diabetic nephropathy.

    PubMed

    Yuzawa, Yukio; Niki, Ichiro; Kosugi, Tomoki; Maruyama, Shoichi; Yoshida, Futoshi; Takeda, Motohiro; Tagawa, Yoshiaki; Kaneko, Yukiko; Kimura, Toshihide; Kato, Noritoshi; Yamamoto, Jyunichiro; Sato, Waichi; Nakagawa, Takahiko; Matsuo, Seiichi

    2008-09-01

    Recently, endothelial dysfunction induced by an uncoupling of vascular endothelial growth factor (VEGF) and nitric oxide has been implicated in the pathogenesis of diabetic nephropathy (DN). Investigating the pathogenesis of DN has been limited, however, because of the lack of animal models that mimic the human disease. In this report, pancreatic beta cell-specific calmodulin-overexpressing transgenic (CaMTg) mice, a potential new model of DN, are characterized with particular emphasis on VEGF and related molecules. CaMTg mice developed hyperglycemia at 3 wk and persistent proteinuria by 3 mo. Morphometric analysis showed considerable increases in the glomerular and mesangial areas with deposition of type IV collagen. Moreover, the pathologic hallmarks of human DN (mesangiolysis, Kimmelstiel-Wilson-like nodular lesions, exudative lesions, and hyalinosis of afferent and efferent arteries with neovascularization) were observed. In addition, increased VEGF expression was associated with an increased number of peritubular capillaries. Expression of endothelial nitric oxidase synthase was reduced and that of VEGF was markedly elevated in CaMTg mice kidney compared with nontransgenic mice. No differences in VEGF receptor-1 or VEGF receptor-2 expression were observed between CaMTg mice and nontransgenic kidneys. In summary, CaMTg mice develop most of the distinguishing lesions of human DN, and the elevated VEGF expression in the setting of diminished endothelial nitric oxide synthase expression may lead to endothelial proliferation and dysfunction. This model may prove useful in the study of the pathogenesis and treatment of DN.

  2. [Overexpression of Aspergillus candidus lactase and analysis of enzymatic properties].

    PubMed

    Zhang, Wei; Fan, Yun-liu; Yao, Bin

    2005-04-01

    The lactase gene lacb' from Aspergillus candidus was fused behind alpha-factor signal sequence in the Pichia pastoris expression vector pPIC9, then integrated into the genome of P. pastoris by recombination events. The P. pastoris recombinants for lactase overexpression were screened by enzyme activity analysis and SDS-PAGE. The lactase expressed in P. pastoris was glycosylated protein with an apparent molecular weight of 130 kD, while the deglycosylated lactase treated with Endo H had an apparent molecular weight of about 110 kD. The expression level of secreted lactase protein in recombinant P. pastoris was 6 mg/mL with enzymatic activity of 3600 U/mL in the 5 L fermenter, which was the highest among that of all kinds of recombinant strains reported now. The optimal pH and optimal temperature of the lactase are 5.2 and 60 degrees C. The Vmax, Km, and specific activity of the lactase are 3.3 micromol/min, 1.7 mmol/L and 706.5 +/- 2.6 U/mg, respectively. Compare to the lactase from Aspergillus oryzae ATCC 20423, the expressed lactase from A. candidus have better enzymatic properties including the high thermostability, high specific activity and wide pH range for enzyme reaction.

  3. Overexpression of SOS genes in ciprofloxacin resistant Escherichia coli mutants.

    PubMed

    Pourahmad Jaktaji, Razieh; Pasand, Shirin

    2016-01-15

    Fluoroquinolones are important antibiotics for the treatment of urinary tract infections caused by Escherichia coli. Mutational studies have shown that ciprofloxacin, a member of fluoroquinolones induces SOS response and mutagenesis in pathogenic bacteria which in turn develop antibiotic resistance. However, inhibition of SOS response can increase recombination activity which in turn leads to genetic variation. The aim of this study was to measure 5 SOS genes expressions in nine E. coli mutants with different MICs for ciprofloxacin following exposure to ciprofloxacin. Gene expression was assessed by quantitative real time PCR. Gene alteration assessment was conducted by PCR amplification and DNA sequencing. Results showed that the expression of recA was increased in 5 mutants. This overexpression is not related to gene alteration, and enhances the expression of polB and umuCD genes encoding nonmutagenic and mutagenic polymerases, respectively. The direct relationship between the level of SOS expression and the level of resistance to ciprofloxacin was also indicated. It was concluded that novel therapeutic strategy that inhibits RecA activity would enhance the efficiency of common antibiotics against pathogenic bacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Changes in Gene Expression Associated with FTO Overexpression in Mice

    PubMed Central

    Kramer, Holger B.; McMurray, Fiona; Lafond, Mathilde; Boutens, Lily; Cox, Roger; Ashcroft, Frances M.

    2014-01-01

    Single nucleotide polymorphisms in the first intron of the fat-mass-and-obesity-related gene FTO are associated with increased body weight and adiposity. Increased expression of FTO is likely underlying this obesity phenotype, as mice with two additional copies of Fto (FTO-4 mice) exhibit increased adiposity and are hyperphagic. FTO is a demethylase of single stranded DNA and RNA, and one of its targets is the m6A modification in RNA, which might play a role in the regulation of gene expression. In this study, we aimed to examine the changes in gene expression that occur in FTO-4 mice in order to gain more insight into the underlying mechanisms by which FTO influences body weight and adiposity. Our results indicate an upregulation of anabolic pathways and a downregulation of catabolic pathways in FTO-4 mice. Interestingly, although genes involved in methylation were differentially regulated in skeletal muscle of FTO-4 mice, no effect of FTO overexpression on m6A methylation of total mRNA was detected. PMID:24842286

  5. MMSET is overexpressed in cancers: Link with tumor aggressiveness

    SciTech Connect

    Kassambara, Alboukadel; Klein, Bernard Moreaux, Jerome

    2009-02-20

    MMSET is expressed ubiquitously in early development and its deletion is associated with the malformation syndrome called Wolf-Hirschhorn syndrome. It is involved in the t(4; 14) (p16; q32) chromosomal translocation, which is the second most common translocation in multiple myeloma (MM) and is associated with the worst prognosis. MMSET expression has been shown to promote cellular adhesion, clonogenic growth and tumorigenicity in multiple myeloma. MMSET expression has been recently shown to increase with ascending tumor proliferation activity in glioblastoma multiforme. These data demonstrate that MMSET could be implicated in tumor emergence and/or progression. Therefore, we compared the expression of MMSET in 40 human tumor types - brain, epithelial, lymphoid - to that of their normal tissue counterparts using publicly available gene expression data, including the Oncomine Cancer Microarray database. We found significant overexpression of MMSET in 15 cancers compared to their normal counterparts. Furthermore MMSET is associated with tumor aggressiveness or prognosis in many types of these aforementioned cancers. Taken together, these data suggest that MMSET potentially acts as a pathogenic agent in many cancers. The identification of the targets of MMSET and their role in cell growth and survival will be key to understand how MMSET is associated with tumor development.

  6. Lymphopoiesis in transgenic mice over-expressing Artemis.

    PubMed

    Rivera-Munoz, P; Abramowski, V; Jacquot, S; André, P; Charrier, S; Lipson-Ruffert, K; Fischer, A; Galy, A; Cavazzana, M; de Villartay, J-P

    2016-02-01

    Artemis is a factor of the non-homologous end joining pathway involved in DNA double-strand break repair that has a critical role in V(D)J recombination. Mutations in DCLRE1C/ARTEMIS gene result in radiosensitive severe combined immunodeficiency in humans owing to a lack of mature T and B cells. Given the known drawbacks of allogeneic hematopoietic stem cell transplantation (HSCT), gene therapy appears as a promising alternative for these patients. However, the safety of an unregulated expression of Artemis has to be established. We developed a transgenic mouse model expressing human Artemis under the control of the strong CMV early enhancer/chicken beta actin promoter through knock-in at the ROSA26 locus to analyze this issue. Transgenic mice present a normal development, maturation and function of T and B cells with no signs of lymphopoietic malignancies for up to 15 months. These results suggest that the over-expression of Artemis in mice (up to 40 times) has no deleterious effects in early and mature lymphoid cells and support the safety of gene therapy as a possible curative treatment for Artemis-deficient patients.

  7. Overexpression of catalase targeted to mitochondria attenuates murine cardiac aging

    PubMed Central

    Dai, Dao-Fu; Santana, Luis F.; Vermulst, Marc; Tomazela, Daniela M.; Emond, M.J.; MacCoss, Michael J.; Gollahon, Katherine; Martin, George M.; Loeb, Lawrence A.; Ladiges, Warren C.; Rabinovitch, Peter S.

    2010-01-01

    Background: Age is a major risk for cardiovascular diseases. Although mitochondrial reactive oxygen species (ROS) have been proposed as one of the causes of aging, their role in cardiac aging remains unclear. We have previously shown that overexpression of catalase targeted to mitochondria (mCAT) prolongs murine median lifespan by 17-21%. Methods and Results: We used echocardiography to study cardiac function in aging cohorts of wild type (WT) and mCAT mice. Changes found in WT mice recapitulate human aging: age-dependent increases in left ventricular mass index (LVMI) and left atrial dimension, worsening of the myocardial performance index (MPI), and a decline in diastolic function. Cardiac aging in mice is accompanied by accumulation of mitochondrial protein oxidation, increased mitochondrial DNA mutations and deletions and mitochondrial biogenesis, increased ventricular fibrosis, enlarged myocardial fiber size, decreased cardiac SERCA2 protein and activation of the calcineurin-NFAT pathway. All of these age-related changes were significantly attenuated in mCAT mice. Analysis of survival of 130 mice demonstrated that echocardiographic cardiac aging risk scores were significant predictors of mortality. The estimated attributable risk to mortality for these two parameters was 55%. Conclusion: This study shows that cardiac aging in the mouse closely recapitulates human aging and demonstrates the critical role of mitochondrial ROS in cardiac aging and the impact of cardiac aging on survival. These findings also support the potential application of mitochondrial antioxidants in ROS-related cardiovascular diseases. PMID:19451351

  8. Endothelium-targeted overexpression of heat shock protein 27 ameliorates blood–brain barrier disruption after ischemic brain injury

    PubMed Central

    Jiang, Xiaoyan; Zhang, Lili; Pu, Hongjian; Hu, Xiaoming; Zhang, Wenting; Cai, Wei; Gao, Yanqin; Leak, Rehana K.; Keep, Richard F.; Bennett, Michael V. L.; Chen, Jun

    2017-01-01

    The damage borne by the endothelial cells (ECs) forming the blood–brain barrier (BBB) during ischemic stroke and other neurological conditions disrupts the structure and function of the neurovascular unit and contributes to poor patient outcomes. We recently reported that structural aberrations in brain microvascular ECs—namely, uncontrolled actin polymerization and subsequent disassembly of junctional proteins, are a possible cause of the early onset BBB breach that arises within 30–60 min of reperfusion after transient focal ischemia. Here, we investigated the role of heat shock protein 27 (HSP27) as a direct inhibitor of actin polymerization and protectant against BBB disruption after ischemia/reperfusion (I/R). Using in vivo and in vitro models, we found that targeted overexpression of HSP27 specifically within ECs—but not within neurons—ameliorated BBB impairment 1–24 h after I/R. Mechanistically, HSP27 suppressed I/R-induced aberrant actin polymerization, stress fiber formation, and junctional protein translocation in brain microvascular ECs, independent of its protective actions against cell death. By preserving BBB integrity after I/R, EC-targeted HSP27 overexpression attenuated the infiltration of potentially destructive neutrophils and macrophages into brain parenchyma, thereby improving long-term stroke outcome. Notably, early poststroke administration of HSP27 attached to a cell-penetrating transduction domain (TAT-HSP27) rapidly elevated HSP27 levels in brain microvessels and ameliorated I/R-induced BBB disruption and subsequent neurological deficits. Thus, the present study demonstrates that HSP27 can function at the EC level to preserve BBB integrity after I/R brain injury. HSP27 may be a therapeutic agent for ischemic stroke and other neurological conditions involving BBB breakdown. PMID:28137866

  9. Hepatic overexpression of SIRT1 in mice attenuates endoplasmic reticulum stress and insulin resistance in the liver

    PubMed Central

    Li, Yu; Xu, Shanqin; Giles, Amber; Nakamura, Kazuto; Lee, Jong Woo; Hou, Xiuyun; Donmez, Gizem; Li, Ji; Luo, Zhijun; Walsh, Kenneth; Guarente, Leonard; Zang, Mengwei

    2011-01-01

    Endoplasmic reticulum (ER) stress has been implicated in the pathophysiology of human type 2 diabetes (T2DM). Although SIRT1 has a therapeutic effect on metabolic deterioration in T2DM, the precise mechanisms by which SIRT1 improves insulin resistance remain unclear. Here, we demonstrate that adenovirus-mediated overexpression of SIRT1 in the liver of diet-induced insulin-resistant low-density lipoprotein receptor-deficient mice and of genetically obese ob/ob mice attenuates hepatic steatosis and ameliorates systemic insulin resistance. These beneficial effects were associated with decreased mammalian target of rapamycin complex 1 (mTORC1) activity, inhibited the unfolded protein response (UPR), and enhanced insulin receptor signaling in the liver, leading to decreased hepatic gluconeogenesis and improved glucose tolerance. The tunicamycin-induced splicing of X-box binding protein-1 and expression of GRP78 and CHOP were reduced by resveratrol in cultured cells in a SIRT1-dependent manner. Conversely, SIRT1-deficient mouse embryonic fibroblasts challenged with tunicamycin exhibited markedly increased mTORC1 activity and impaired ER homeostasi and insulin signaling. These effects were abolished by mTORC1 inhibition by rapamycin in human HepG2 cells. These studies indicate that SIRT1 serves as a negative regulator of UPR signaling in T2DM and that SIRT1 attenuates hepatic steatosis, ameliorates insulin resistance, and restores glucose homeostasis, largely through the inhibition of mTORC1 and ER stress.—Li, Y., Xu, S., Giles, A., Nakamura, K., Lee, J. W., Hou, X., Donmez, G., Li, J., Luo, Z., Walsh, K., Guarente, L., Zang, M. Hepatic overexpression of SIRT1 in mice attenuates endoplasmic reticulum stress and insulin resistance in the liver. PMID:21321189

  10. Overexpression of the transporters AtZIP1 and AtMTP1 in cassava changes zinc accumulation and partitioning

    PubMed Central

    Gaitán-Solís, Eliana; Taylor, Nigel J.; Siritunga, Dimuth; Stevens, William; Schachtman, Daniel P.

    2015-01-01

    Zinc deficiency in humans is a serious problem worldwide with an estimated one third of populations at risk for insufficient zinc in diet, which leads to impairment of cognitive abilities and immune system function. The goal of this research was to increase the bioavailable zinc in the edible portion of cassava roots to improve the overall zinc nutrition of populations that rely on cassava as a dietary staple. To increase zinc concentrations, two Arabidopsis thaliana genes coding for ZIP1 and MTP1 were overexpressed with a tuber-specific or constitutive promoter. Eighteen transgenic events from four constructs, out of a total of 73 events generated, showed significantly higher zinc concentrations in the edible portion of the storage root compared to the non-transgenic controls. The zinc content in the transgenic lines ranged from 4 to 73 mg/kg dry weight (DW) as compared to the non-transgenic control which contained 8 mg/kg. Striking changes in whole plant phenotype such as smaller plant size and chlorotic leaves were observed in transgenic lines that over accumulated zinc. In a confined field trial five transgenic events grown for 12 months showed a range of zinc concentrations from 18 to 217 mg/kg DW. Although the overexpression of zinc transporters was successful in increasing the zinc concentrations in 25% of the transgenic lines generated, it also resulted in a decrease in plant and tuber size and overall yield due to what appears to be zinc deficiency in the aerial parts of the plant. PMID:26217349

  11. Modeling Parkinson's disease pathology by combination of fibril seeds and α-synuclein overexpression in the rat brain.

    PubMed

    Thakur, Poonam; Breger, Ludivine S; Lundblad, Martin; Wan, Oi Wan; Mattsson, Bengt; Luk, Kelvin C; Lee, Virginia M Y; Trojanowski, John Q; Björklund, Anders

    2017-09-26

    Although a causative role of α-synuclein (α-syn) is well established in Parkinson's disease pathogenesis, available animal models of synucleinopathy do not replicate the full range of cellular and behavioral changes characteristic of the human disease. This study was designed to generate a more faithful model of Parkinson's disease by injecting human α-syn fibril seeds into the rat substantia nigra (SN), in combination with adenoassociated virus (AAV)-mediated overexpression of human α-syn, at levels that, by themselves, are unable to induce acute dopamine (DA) neurodegeneration. We show that the ability of human α-syn fibrils to trigger Lewy-like α-synuclein pathology in the affected DA neurons is dramatically enhanced in the presence of elevated levels of human α-syn. This synucleinopathy was fully developed already 10 days after fibril injection, accompanied by progressive degeneration of dopaminergic neurons in SN, neuritic swelling, reduced striatal DA release, and impaired motor behavior. Moreover, a prominent inflammatory response involving both activation of resident microglia and infiltration of CD4(+) and CD8(+) T lymphocytes was observed. Hypertrophic microglia were found to enclose or engulf cells and processes containing Lewy-like α-syn aggregates. α-Syn aggregates were also observed inside these cells, suggesting transfer of phosphorylated α-syn from the affected nigral neurons. The nigral pathology triggered by fibrils in combination with AAV-mediated overexpression of α-syn reproduced many of the cardinal features of the human disease. The short time span and the distinct sequence of pathological and degenerative changes make this combined approach attractive as an experimental model for the assessment of neuroprotective and disease-modifying strategies.

  12. Reg3α Overexpression Protects Pancreatic β Cells from Cytokine-Induced Damage and Improves Islet Transplant Outcome

    PubMed Central

    Ding, Ying; Xu, Yuemei; Shuai, Xuanyu; Shi, Xuhui; Chen, Xiang; Huang, Wenbin; Liu, Yun; Liang, Xiubin; Zhang, Zhihong; Su, Dongming

    2014-01-01

    The process of islet transplantation for treating type 1 diabetes has been limited by the high level of graft failure. This may be overcome by locally delivering trophic factors to enhance engraftment. Regenerating islet-derived protein 3α (Reg3α) is a pancreatic secretory protein which functions as an antimicrobial peptide in control of inflammation and cell proliferation. In this study, to investigate whether Reg3α could improve islet engraftment, a marginal mass of syngeneic islets pretransduced with adenoviruses expressing Reg3α or control EGFP were transplanted under the renal capsule of streptozotocin-induced diabetic mice. Mice receiving islets with elevated Reg3α production exhibited significantly lower blood glucose levels (9.057 ± 0.59 mmol/L versus 13.48 ± 0.35 mmol/L, P < 0.05) and improved glucose-stimulated insulin secretion (1.80 ± 0.17 ng/mL versus 1.16 ± 0.16 ng/mL, P < 0.05) compared with the control group. The decline of apoptotic events (0.57% ± 0.15% versus 1.06% ± 0.07%, P < 0.05) and increased β-cell proliferation (0.70% ± 0.10% versus 0.36% ± 0.14%, P < 0.05) were confirmed in islet grafts overexpressing Reg3α by morphometric analysis. Further experiments showed that Reg3α production dramatically protected cultured islets and pancreatic β cells from cytokine-induced apoptosis and the impairment of glucose-stimulated insulin secretion. Moreover, exposure to cytokines led to the activation of MAPKs in pancreatic β cells, which was reversed by Reg3α overexpression in contrast to control group. These results strongly suggest that Reg3α could enhance islet engraftments through its cytoprotective effect and advance the therapeutic efficacy of islet transplantation. PMID:25826674

  13. Sevoflurane Inhalation Accelerates the Long-Term Memory Consolidation via Small GTPase Overexpression in the Hippocampus of Mice in Adolescence.

    PubMed

    Nakamura, Emi; Kinoshita, Hiroyuki; Feng, Guo-Gang; Hayashi, Hisaki; Satomoto, Maiko; Sato, Motohiko; Fujiwara, Yoshihiro

    2016-01-01

    Sevoflurane exposure impairs the long-term memory in neonates. Whether the exposure to animals in adolescence affects the memory, however, has been unclear. A small hydrolase enzyme of guanosine triphosphate (GTPase) rac1 plays a role in the F-actin dynamics related to the synaptic plasticity, as well as superoxide production via reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. The current study was designed to examine whether sevoflurane exposure to mice in early adolescence modifies the long-term learning ability concomitantly with the changes in F-actin constitution as well as superoxide production in the hippocampus according to the levels of rac1 protein expression. Four-week-old mice were subjected to the evaluation of long-term learning ability for three days. On day one, each mouse was allowed to enter a dark chamber for five min to acclimatization. On day two, the procedure was repeated with the addition of an electric shock as soon as a mouse entered the dark chamber. All mice subsequently inhaled 2 L/min air with (Sevoflurane group) and without (Control group) 2.5% sevoflurane for three hours. On day three, each mouse was placed on the platform and retention time, which is the latency to enter the dark chamber, was examined. The brain removed after the behavior test, was used for analyses of immunofluorescence, Western immunoblotting and intracellular levels of superoxide. Sevoflurane exposure significantly prolonged retention time, indicating the enhanced long-term memory. Sevoflurane inhalation augmented F-actin constitution coexisting with the rac1 protein overexpression in the hippocampus whereas it did not alter the levels of superoxide. Sevoflurane exposure to 4-week-old mice accelerates the long-term memory concomitantly with the enhanced F-actin constitution coexisting with the small GTPase rac1 overexpression in the hippocampus. These results suggest that sevoflurane inhalation may amplify long-term memory

  14. Overexpression of tumor necrosis factor-α in the lungs alters immune response, matrix remodeling, and repair and maintenance pathways.

    PubMed

    Thomson, Errol M; Williams, Andrew; Yauk, Carole L; Vincent, Renaud

    2012-04-01

    Increased production of tumor necrosis factor (TNF)-α and matrix metalloproteinases (MMPs) is a feature of inflammatory lung diseases, including emphysema and fibrosis, but the divergent pathological characteristics that result indicate involvement of other processes in disease pathogenesis. Transgenic mice overexpressing TNF-α in type II alveolar epithelial cells under the control of the surfactant protein (SP)-C promoter develop pulmonary inflammation and emphysema but are resistant to induction of fibrosis by administration of bleomycin or transforming growth factor-β. To study the molecular mechanisms underlying the development of this phenotype, we used a microarray approach to characterize the pulmonary transcriptome of SP-C/TNF-α mice and wild-type littermates. Four-month-old SP-C/TNF-α mice displayed pronounced pulmonary inflammation, airspace enlargement, increased MMP-2 and MMP-9 levels, and altered expression of 2332 probes. The functional assessment of genes with increased expression revealed enrichment of inflammatory/immune responses and proteases, whereas genes involved in protease inhibition, angiogenesis, cross-linking of basement membrane proteins, and myofibroblast differentiation were predominantly decreased. Comparison with multiple lung disease models identified a set of genes unique to the SP-C/TNF-α model and revealed that lack of extracellular matrix production distinguished SP-C/TNF-α mice from fibrosis models. Activation of inflammatory and proteolytic pathways and disruption of maintenance and repair processes are central features of emphysema in this TNF-overexpression model. Impairment of myofibroblast differentiation and extracellular matrix production may underlie resistance to induction of fibrosis.

  15. Early and progressive microstructural brain changes in mice overexpressing human α-Synuclein detected by diffusion kurtosis imaging.

    PubMed

    Khairnar, Amit; Ruda-Kucerova, Jana; Szabó, Nikoletta; Drazanova, Eva; Arab, Anas; Hutter-Paier, Birgit; Neddens, Joerg; Latta, Peter; Starcuk, Zenon; Rektorova, Irena

    2017-03-01

    Diffusion kurtosis imaging (DKI) is sensitive in detecting α-Synuclein (α-Syn) accumulation-associated microstructural changes at late stages of the pathology in α-Syn overexpressing TNWT-61 mice. The aim of this study was to perform DKI in young TNWT-61 mice when α-Syn starts to accumulate and to compare the imaging results with an analysis of motor and memory impairment and α-Syn levels. Three-month-old (3mo) and six-month-old (6mo) mice underwent DKI scanning using the Bruker Avance 9.4T magnetic resonance imaging system. Region of interest (ROI) analyses were performed in the gray matter; tract-based spatial statistics (TBSS) analyses were performed in the white matter. In the same mice, α-Syn expression was evaluated using quantitative immunofluorescence. Mean kurtosis (MK) was the best differentiator between TNWT-61 mice and wildtype (WT) mice. We found increases in MK in 3mo TNWT-61 mice in the striatum and thalamus but not in the substantia nigra (SN), hippocampus, or sensorimotor cortex, even though the immunoreactivity of human α-Syn was similar or even higher in the latter regions. Increases in MK in the SN were detected in 6mo mice. These findings indicate that α-Syn accumulation-associated changes may start in areas wi