Science.gov

Sample records for abnormal protein accumulation

  1. The sequential development of abnormal prion protein accumulation in mice with Creutzfeldt-Jakob disease.

    PubMed Central

    Muramoto, T.; Kitamoto, T.; Tateishi, J.; Goto, I.

    1992-01-01

    The distribution and sequential development of prion protein (PrP) accumulation in the central nervous system (CNS) and non-neuronal organs of mice infected with Creutzfeldt-Jakob disease (CJD) were investigated immunohistochemically using a new pretreatment method that greatly enhanced the immunoreactivity of PrP. Prion protein accumulation in the CNS was first detected at 30 days after inoculation and then developed near the inoculation site or periventricular area, and later spread to the whole cerebrum and then to the pons. Its staining took some characteristic forms. Among non-neuronal organs, PrP accumulated in the follicular dendritic cells (FDCs) in spleen, lymph node, Peyer's patch, and thymus. FDCs staining appeared in spleen, lymph node, and Peyer's patch at 21 or 30 days after inoculation, and in thymus at 90 days. Germinal centers developed in the thymus of some CJD-infected mice. No PrP staining was detected in any examined organs of age-matched control mice. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:1376559

  2. The abnormal isoform of the prion protein accumulates in late-endosome-like organelles in scrapie-infected mouse brain.

    PubMed

    Arnold, J E; Tipler, C; Laszlo, L; Hope, J; Landon, M; Mayer, R J

    1995-08-01

    The prion encephalopathies are characterized by accumulation in the brain of the abnormal form PrPsc of a normal host gene product PrPc. The mechanism and site of formation of PrPsc from PrPc are currently unknown. In this study, ME7 scrapie-infected mouse brain was used to show, both biochemically and by double-labelled immunogold electron microscopy, that proteinase K-resistant PrPsc is enriched in subcellular structures which contain the cation-independent mannose 6-phosphate receptor, ubiquitin-protein conjugates, beta-glucuronidase, and cathepsin B, termed late endosome-like organelles. The glycosylinositol phospholipid membrane-anchored PrPc will enter such compartment for normal degradation and the organelles may therefore act as chambers for the conversion of PrPc into infectious PrPsc in this murine model of scrapie.

  3. PROTEIN L-ISOASPARTYL METHYLTRANSFERASE2 is differentially expressed in chickpea and enhances seed vigor and longevity by reducing abnormal isoaspartyl accumulation predominantly in seed nuclear proteins.

    PubMed

    Verma, Pooja; Kaur, Harmeet; Petla, Bhanu Prakash; Rao, Venkateswara; Saxena, Saurabh C; Majee, Manoj

    2013-03-01

    PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT) is a widely distributed protein-repairing enzyme that catalyzes the conversion of abnormal l-isoaspartyl residues in spontaneously damaged proteins to normal aspartyl residues. This enzyme is encoded by two divergent genes (PIMT1 and PIMT2) in plants, unlike many other organisms. While the biological role of PIMT1 has been elucidated, the role and significance of the PIMT2 gene in plants is not well defined. Here, we isolated the PIMT2 gene (CaPIMT2) from chickpea (Cicer arietinum), which exhibits a significant increase in isoaspartyl residues in seed proteins coupled with reduced germination vigor under artificial aging conditions. The CaPIMT2 gene is found to be highly divergent and encodes two possible isoforms (CaPIMT2 and CaPIMT2') differing by two amino acids in the region I catalytic domain through alternative splicing. Unlike CaPIMT1, both isoforms possess a unique 56-amino acid amino terminus and exhibit similar yet distinct enzymatic properties. Expression analysis revealed that CaPIMT2 is differentially regulated by stresses and abscisic acid. Confocal visualization of stably expressed green fluorescent protein-fused PIMT proteins and cell fractionation-immunoblot analysis revealed that apart from the plasma membrane, both CaPIMT2 isoforms localize predominantly in the nucleus, while CaPIMT1 localizes in the cytosol. Remarkably, CaPIMT2 enhances seed vigor and longevity by repairing abnormal isoaspartyl residues predominantly in nuclear proteins upon seed-specific expression in Arabidopsis (Arabidopsis thaliana), while CaPIMT1 enhances seed vigor and longevity by repairing such abnormal proteins mainly in the cytosolic fraction. Together, our data suggest that CaPIMT2 has most likely evolved through gene duplication, followed by subfunctionalization to specialize in repairing the nuclear proteome.

  4. Progressive accumulation of the abnormal conformer of the prion protein and spongiform encephalopathy in the obex of nonsymptomatic and symptomatic Rocky Mountain elk (Cervus elaphus nelsoni) with chronic wasting disease.

    PubMed

    Spraker, Terry R; Gidlewski, Thomas; Powers, Jenny G; Nichols, Tracy; Balachandran, Aru; Cummings, Bruce; Wild, Margaret A; VerCauteren, Kurt; O'Rourke, Katherine I

    2015-07-01

    The purpose of our study was to describe the progressive accumulation of the abnormal conformer of the prion protein (PrP(CWD)) and spongiform degeneration in a single section of brain stem in Rocky Mountain elk (Cervus elaphus nelsoni) with chronic wasting disease (CWD). A section of obex from 85 CWD-positive elk was scored using the presence and abundance of PrP(CWD) immunoreactivity and spongiform degeneration in 10 nuclear regions and the presence and abundance of PrP(CWD) in 10 axonal tracts, the subependymal area of the fourth ventricle, and the thin subpial astrocytic layer (glial limitans). Data was placed in a formula to generate an overall obex score. Data suggests that PrP(CWD) immunoreactivity and spongiform degeneration has a unique and relatively consistent pattern of progression throughout a section of obex. This scoring technique utilizing a single section of obex may prove useful in future work for estimating the presence and abundance of PrP(CWD) in peripheral tissues and the nervous system in elk with CWD.

  5. Progressive accumulation of the abnormal conformer of the prion protein and spongiform encephalopathy in the obex of nonsymptomatic and symptomatic Rocky Mountain elk (Cervus elaphus nelsoni) with chronic wasting disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chronic wasting disease (CWD), a transmissible spongiform encephalopathy, has been reported in captive and free-ranging cervids. An abnormal isoform of a prion protein (PrP-CWD) has been associated with CWD in Rocky Mountain elk (Cervus elaphus nelsoni) and this prion protein can be detected with i...

  6. A SENSITIVE IMMUNOFLUORESCENCE ASSAY FOR DETECTION OF P53 PROTEIN ACCUMULATION IN SPUTUM

    EPA Science Inventory

    p53 mutations are common genetic alterations in lung cancers and usually result in p53 protein accumulation in tumor cells. Sputum is noninvasive to collect and ideal for screening p53 abnormalities. This study was to determine the feasibility of detecting p53 protein accumulatio...

  7. Accumulation of organotins in seafood leads to reproductive tract abnormalities in female rats.

    PubMed

    Podratz, Priscila L; Merlo, Eduardo; Sena, Gabriela C; Morozesk, Mariana; Bonomo, Marina M; Matsumoto, Silvia T; da Costa, Mércia B; Zamprogno, Gabriela C; Brandão, Poliane A A; Carneiro, Maria T W D; Miguel, Emilio de C; Miranda-Alves, Leandro; Silva, Ian V; Graceli, Jones B

    2015-11-01

    Organotins (OTs) are environmental contaminants used as biocides in antifouling paints that have been shown to be endocrine disrupters. However, studies evaluating the effects of OTs accumulated in seafood (LNI) on reproductive health are particularly sparse. This study demonstrates that LNI leads to impairment in the reproductive tract of female rats, as the estrous cycle development, as well as for ovary and uterus morphology. Rats were treated with LNI, and their reproductive morphophysiology was assessed. Morphophysiological abnormalities, such as irregular estrous cycles, abnormal ovarian follicular development and ovarian collagen deposition, were observed in LNI rats. An increase in luminal epithelia and ERα expression was observed in the LNI uteri. Together, these data provide in vivo evidence that LNI are toxic for reproductive morphophysiology, which may be associated with risks to reproductive function.

  8. Insufficient Lymph Drainage Causes Abnormal Lipid Accumulation and Vein Wall Degeneration

    PubMed Central

    Tanaka, Hiroki; Yamamoto, Naoto; Suzuki, Minoru; Mano, Yuuki; Sano, Masaki; Zaima, Nobuhiro; Sasaki, Takeshi; Setou, Mitsutoshi

    2016-01-01

    Objective: Previously, we analyzed human varicose veins (VV) using imaging mass spectrometry (IMS) and detected the abnormal accumulation of lipid molecules in the walls of VV, possibly due to insufficient lipid drainage by the lymphatic vessels. In this study, we created an animal model of lymphatic insufficiency to investigate the effects of insufficient lymph drainage on vein walls. Methods: In rats, the lymphatic collecting vessels surrounding the femoral vein were ligated on one side (the model tissue), which caused the local retention of lymphatic fluid in the perivascular tissue. The equivalent contralateral tissue was used as a control. A histological study of the femoral vein and the surrounding perivascular tissue was conducted. IMS was used to analyze the distribution of lipid molecules in the perivascular tissue. Results: Fourteen days after the procedure, the lymphatic vessels in the model tissue were significantly dilated. Furthermore, IMS revealed that the composition of the lipid molecules in the perivascular regions of the model tissue had altered. Compared with the control tissue, the model tissue exhibited marked perivascular accumulation of lysophosphatidylcholine (1-acyl 16:0), phosphatidylcholine (16:0/20:4), and triglycerides (52:2). Interestingly, the walls of the femoral veins running through the model tissue were 3.4-fold thicker than those of the femoral veins running through the control tissue. The number of tumor necrosis factor α-positive adipocytes was increased in the perivascular regions of the model tissue. Conclusion: The findings of this study indicated that the accumulation of lymphatic fluid due to insufficient lymph drainage changes the structure of vein walls, and such changes might be associated with chronic venous insufficiency. (This is a translation of Jpn J Phlebol 2015; 26: 227–235.) PMID:28018498

  9. Intracellular accumulation and resistance to degradation of the Alzheimer amyloid A4/beta protein.

    PubMed Central

    Knauer, M F; Soreghan, B; Burdick, D; Kosmoski, J; Glabe, C G

    1992-01-01

    The A4 or beta protein is a peptide that constitutes the major protein component of senile plaques in Alzheimer disease. The A4/beta protein is derived from a larger, transmembrane amyloid precursor protein (APP). The putative abnormal processing events leading to amyloid accumulation are largely unknown. Here we report that a 42-residue synthetic peptide, beta 1-42, corresponding to one of the longer forms of the A4/beta protein, accumulates in cultured human skin fibroblasts and is stable for at least 3 days. The peptide appears to accumulate intracellularly, since it does not accumulate under conditions that prevent endocytosis and accumulation is correlated with the acquisition of resistance to removal by trypsin digestion. This intracellular accumulation is also correlated with the ability of the peptide to aggregate as determined by SDS/polyacrylamide gel electrophoresis. At low concentrations of the beta 1-42 peptide, which favor the nonaggregated state, no accumulation is observed. Shorter peptide analogs (28 or 39 residues) that are truncated at the C terminus, which lack the ability to aggregate in SDS gels, fail to accumulate. The accumulated intracellular beta 1-42 peptide is in an aggregated state and is contained in a dense organellar compartment that overlaps the distribution of late endosomes or secondary lysosomes. Immunofluorescence of the internalized peptide in permeabilized cells reveals that it is contained in granular deposits, consistent with localization in late endosomes or secondary lysosomes. Sequence analysis indicates that some of the internalized peptide is subject to N-terminal trimming. These results suggest that the aggregated A4/beta protein may be resistant to degradation and suggest that the A4/beta protein may arise, at least in part, by endosomal or lysosomal processing of APP. Our results also suggest that relatively nonspecific proteolysis may be sufficient to generate the A4/beta protein if this part of APP is selectively

  10. Selenocystamine improves protein accumulation in chloroplasts of eukaryotic green algae.

    PubMed

    Ferreira-Camargo, Livia S; Tran, Miller; Beld, Joris; Burkart, Michael D; Mayfield, Stephen P

    2015-12-01

    Eukaryotic green algae have become an increasingly popular platform for recombinant proteins production. In particular, Chlamydomonas reinhardtii, has garnered increased attention for having the necessary biochemical machinery to produce vaccines, human antibodies and next generation cancer targeting immunotoxins. While it has been shown that chloroplasts contain chaperones, peptidyl prolylisomerases and protein disulfide isomerases that facilitate these complex proteins folding and assembly, little has been done to determine which processes serve as rate-limiting steps for protein accumulation. In other expression systems, as Escherichia coli, Chinese hamster ovary cells, and insect cells, recombinant protein accumulation can be hampered by cell's inability to fold the target polypeptide into the native state, resulting in aggregation and degradation. To determine if chloroplasts' ability to oxidize proteins that require disulfide bonds into a stable conformation is a rate-limiting step of protein accumulation, three recombinant strains, each expressing a different recombinant protein, were analyzed. These recombinant proteins included fluorescent GFP, a reporter containing no disulfide bonds; Gaussia princeps luciferase, a luminescent reporter containing disulfide bonds; and an immunotoxin, an antibody-fusion protein containing disulfide bonds. Each strain was analyzed for its ability to accumulate proteins when supplemented with selenocystamine, a small molecule capable of catalyzing the formation of disulfide bonds. Selenocystamine supplementation led to an increase in luciferase and immunotoxin but not GFP accumulation. These results demonstrated that selenocystamine can increase the accumulation of proteins containing disulfide bonds and suggests that a rate-limiting step in chloroplast protein accumulation is the disulfide bonds formation in recombinant proteins native structure.

  11. CONCEPTUAL MODEL FOR ORIGIN OF ABNORMALLY PRESSURED GAS ACCUMULATIONS IN LOW-PERMEABILITY RESERVOIRS.

    USGS Publications Warehouse

    Law, B.E.; Dickinson, W.W.

    1985-01-01

    The paper suggests that overpressured and underpressured gas accumulations of this type have a common origin. In basins containing overpressured gas accumulations, rates of thermogenic gas accumulation exceed gas loss, causing fluid (gas) pressure to rise above the regional hydrostatic pressure. Free water in the larger pores is forced out of the gas generation zone into overlying and updip, normally pressured, water-bearing rocks. While other diagenetic processes continue, a pore network with very low permeability develops. As a result, gas accumulates in these low-permeability reservoirs at rates higher than it is lost. In basins containing underpressured gas accumulations, rates of gas generation and accumulation are less than gas loss. The basin-center gas accumulation persists, but because of changes in the basin dynamics, the overpressured accumulation evolves into an underpressured system.

  12. Positive effects of duckweed polycultures on starch and protein accumulation.

    PubMed

    Li, Yang; Zhang, Fantao; Daroch, Maurycy; Tang, Jie

    2016-10-01

    The effect of duckweed species composition (Lemna aequinoctialis 5505, Landoltia punctata 5506 and Spirodela polyrhiza 5507) in polyculture and monoculture on biomass and starch/protein content were investigated at different levels of temperature, light intensity, nitrogen and phosphorus concentrations. The three growth parameters significantly affect duckweed biomass accumulation. Different combinations of duckweed species greatly varied in starch/protein content. Although all the polycultures showed a median relative growth rate and the majority of the polycultures showed a median and starch/protein content as compared with their respective monocultures, some of the polycultures were found to promote the accumulation of starch/protein at different growth conditions. These findings indicated that proper combination of duckweed species could facilitate desirable biomass accumulation and improve biomass quality. The present study provides useful references for future large-scale duckweed cultivation.

  13. Positive effects of duckweed polycultures on starch and protein accumulation

    PubMed Central

    Li, Yang; Zhang, Fantao; Daroch, Maurycy; Tang, Jie

    2016-01-01

    The effect of duckweed species composition (Lemna aequinoctialis 5505, Landoltia punctata 5506 and Spirodela polyrhiza 5507) in polyculture and monoculture on biomass and starch/protein content were investigated at different levels of temperature, light intensity, nitrogen and phosphorus concentrations. The three growth parameters significantly affect duckweed biomass accumulation. Different combinations of duckweed species greatly varied in starch/protein content. Although all the polycultures showed a median relative growth rate and the majority of the polycultures showed a median and starch/protein content as compared with their respective monocultures, some of the polycultures were found to promote the accumulation of starch/protein at different growth conditions. These findings indicated that proper combination of duckweed species could facilitate desirable biomass accumulation and improve biomass quality. The present study provides useful references for future large-scale duckweed cultivation. PMID:27515418

  14. Membrane toxicity of abnormal prion protein in adrenal chromaffin cells of scrapie infected sheep.

    PubMed

    McGovern, Gillian; Jeffrey, Martin

    2013-01-01

    Transmissible spongiform encephalopathies (TSEs) or prion diseases are associated with accumulations of disease specific PrP (PrP(d)) in the central nervous system (CNS) and often the lymphoreticular system (LRS). Accumulations have additionally been recorded in other tissues including the peripheral nervous system and adrenal gland. Here we investigate the effect of sheep scrapie on the morphology and the accumulation of PrP(d) in the adrenal medulla of scrapie affected sheep using light and electron microscopy. Using immunogold electron microscopy, non-fibrillar forms of PrP(d) were shown to accumulate mainly in association with chromaffin cells, occasional nerve endings and macrophages. PrP(d) accumulation was associated with distinctive membrane changes of chromaffin cells including increased electron density, abnormal linearity and invaginations. Internalisation of PrP(d) from the chromaffin cell plasma membrane occurred in association with granule recycling following hormone exocytosis. PrP(d) accumulation and internalisation from membranes is similarly associated with perturbations of membrane structure and trafficking in CNS neurons and tingible body macrophages of the LRS. These data suggest that a major toxic effect of PrP(d) is at the level of plasma membranes. However, the precise nature of PrP(d)-membrane toxicity is tissue and cell specific suggesting that the normal protein may act as a multi-functional scaffolding molecule. We further suggest that the co-localisation of PrP(d) with exocytic granules of the hormone trafficking system may provide an additional source of infectivity in blood.

  15. GFP-like proteins stably accumulate in lysosomes.

    PubMed

    Katayama, Hiroyuki; Yamamoto, Akitsugu; Mizushima, Noboru; Yoshimori, Tamotsu; Miyawaki, Atsushi

    2008-01-01

    Green fluorescent protein (GFP) from the jellyfish Aequorea victoria, its GFP variants (Aequorea GFPs), and more recently the novel GFP-like proteins from Anthozoa have greatly advanced our technologies for fluorescently labeling cells, organelles, and proteins. It has been shown, however, that some GFP-like proteins have a tendency to oligomerize and aggregate. Transfection of GFP-like proteins into cultured mammalian cells results in bright punctate structures, which are thought to be cytosolic protein aggregates. In this study, we demonstrate that these structures are not cytosolic aggregates but lysosomes that have accumulated the GFP-like proteins. Our biochemical and immunocytochemical experiments have revealed that certain GFP-like proteins expressed in the cytosol enter lysosomes possibly by an autophagy-related mechanism, but retain their fluorescence because of resistance not only to acidity but also to lysosomal proteases.

  16. Sporadic inclusion-body myositis: A degenerative muscle disease associated with aging, impaired muscle protein homeostasis and abnormal mitophagy.

    PubMed

    Askanas, Valerie; Engel, W King; Nogalska, Anna

    2015-04-01

    Sporadic inclusion-body myositis (s-IBM) is the most common degenerative muscle disease in which aging appears to be a key risk factor. In this review we focus on several cellular molecular mechanisms responsible for multiprotein aggregation and accumulations within s-IBM muscle fibers, and their possible consequences. Those include mechanisms leading to: a) accumulation in the form of aggregates within the muscle fibers, of several proteins, including amyloid-β42 and its oligomers, and phosphorylated tau in the form of paired helical filaments, and we consider their putative detrimental influence; and b) protein misfolding and aggregation, including evidence of abnormal myoproteostasis, such as increased protein transcription, inadequate protein disposal, and abnormal posttranslational modifications of proteins. Pathogenic importance of our recently demonstrated abnormal mitophagy is also discussed. The intriguing phenotypic similarities between s-IBM muscle fibers and the brains of Alzheimer and Parkinson's disease patients, the two most common neurodegenerative diseases associated with aging, are also discussed. This article is part of a Special Issue entitled: Neuromuscular Diseases: Pathology and Molecular Pathogenesis.

  17. Protein repair L-isoaspartyl methyltransferase in plants. Phylogenetic distribution and the accumulation of substrate proteins in aged barley seeds.

    PubMed Central

    Mudgett, M B; Lowenson, J D; Clarke, S

    1997-01-01

    Protein L-isoaspartate (D-aspartate) O-methyltransferases (MTs; EC 2.1.1.77) can initiate the conversion of detrimental L-isoaspartyl residues in spontaneously damaged proteins to normal L-aspartyl residues. We detected this enzyme in 45 species from 23 families representing most of the divisions of the plant kingdom. MT activity is often localized in seeds, suggesting that it has a role in their maturation, quiescence, and germination. The relationship among MT activity, the accumulation of abnormal protein L-isoaspartyl residues, and seed viability was explored in barley (Hordeum vulgare cultivar Himalaya) seeds, which contain high levels of MT. Natural aging of barley seeds for 17 years resulted in a significant reduction in MT activity and in seed viability, coupled with increased levels of "unrepaired" L-isoaspartyl residues. In seeds heated to accelerate aging, we found no reduction of MT activity, but we did observe decreased seed viability and the accumulation of isoaspartyl residues. Among populations of accelerated aged seed, those possessing the highest levels of L-isoaspartyl-containing proteins had the lowest germination percentages. These results suggest that the MT present in seeds cannot efficiently repair all spontaneously damaged proteins containing altered aspartyl residues, and their accumulation during aging may contribute to the loss of seed viability. PMID:9414558

  18. Evolutionary origins of abnormally large shoot sodium accumulation in nonsaline environments within the Caryophyllales.

    PubMed

    White, Philip J; Bowen, Helen C; Broadley, Martin R; El-Serehy, Hamed A; Neugebauer, Konrad; Taylor, Anna; Thompson, Jacqueline A; Wright, Gladys

    2017-04-01

    The prevalence of sodium (Na)-'hyperaccumulator' species, which exhibit abnormally large shoot sodium concentrations ([Na]shoot ) when grown in nonsaline environments, was investigated among angiosperms in general and within the Caryophyllales order in particular. Shoot Na concentrations were determined in 334 angiosperm species, representing 35 orders, grown hydroponically in a nonsaline solution. Many Caryophyllales species exhibited abnormally large [Na]shoot when grown hydroponically in a nonsaline solution. The bimodal distribution of the log-normal [Na]shoot of species within the Caryophyllales suggested at least two distinct [Na]shoot phenotypes within this order. Mapping the trait of Na-hyperaccumulation onto the phylogenetic relationships between Caryophyllales families, and between subfamilies within the Amaranthaceae, suggested that the trait evolved several times within this order: in an ancestor of the Aizoaceae, but not the Phytolaccaceae or Nyctaginaceae, in ancestors of several lineages formerly classified as Chenopodiaceae, but not in the Amaranthaceae sensu stricto, and in ancestors of species within the Cactaceae, Portulacaceae, Plumbaginaceae, Tamaricaceae and Polygonaceae. In conclusion, a disproportionate number of Caryophyllales species behave as Na-hyperaccumulators, and multiple evolutionary origins of this trait can be identified within this order.

  19. Abnormal recruitment of extracellular matrix proteins by excess Notch3 ECD: a new pathomechanism in CADASIL.

    PubMed

    Monet-Leprêtre, Marie; Haddad, Iman; Baron-Menguy, Céline; Fouillot-Panchal, Maï; Riani, Meriem; Domenga-Denier, Valérie; Dussaule, Claire; Cognat, Emmanuel; Vinh, Joelle; Joutel, Anne

    2013-06-01

    -linked and soluble TIMP3 species. Moreover, reverse zymography assays show a significant elevation of TIMP3 activity in the brain vessels from mice and patients with CADASIL. Collectively, our findings lend support to a Notch3(ECD) cascade hypothesis in CADASIL disease pathology, which posits that aggregation/accumulation of Notch3(ECD) in the brain vessels is a central event, promoting the abnormal recruitment of functionally important extracellular matrix proteins that may ultimately cause multifactorial toxicity. Specifically, our results suggest a dysregulation of TIMP3 activity, which could contribute to mutant Notch3(ECD) toxicity by impairing extracellular matrix homeostasis in small vessels.

  20. The use of Diagnostic Imaging for Identifying Abnormal Gas Accumulations in Cetaceans and Pinnipeds

    PubMed Central

    Dennison, Sophie; Fahlman, Andreas; Moore, Michael

    2012-01-01

    Recent dogma suggested that marine mammals are not at risk of decompression sickness due to a number of evolutionary adaptations. Several proposed adaptations exist. Lung compression and alveolar collapse that terminate gas-exchange before a depth is reached where supersaturation is significant and bradycardia with peripheral vasoconstriction affecting the distribution, and dynamics of blood and tissue nitrogen levels. Published accounts of gas and fat emboli and dysbaric osteonecrosis in marine mammals and theoretical modeling have challenged this view-point, suggesting that decompression-like symptoms may occur under certain circumstances, contrary to common belief. Diagnostic imaging modalities are invaluable tools for the non-invasive examination of animals for evidence of gas and have been used to demonstrate the presence of incidental decompression-related renal gas accumulations in some stranded cetaceans. Diagnostic imaging has also contributed to the recognition of clinically significant gas accumulations in live and dead cetaceans and pinnipeds. Understanding the appropriate application and limitations of the available imaging modalities is important for accurate interpretation of results. The presence of gas may be asymptomatic and must be interpreted cautiously alongside all other available data including clinical examination, clinical laboratory testing, gas analysis, necropsy examination, and histology results. PMID:22685439

  1. The use of Diagnostic Imaging for Identifying Abnormal Gas Accumulations in Cetaceans and Pinnipeds.

    PubMed

    Dennison, Sophie; Fahlman, Andreas; Moore, Michael

    2012-01-01

    Recent dogma suggested that marine mammals are not at risk of decompression sickness due to a number of evolutionary adaptations. Several proposed adaptations exist. Lung compression and alveolar collapse that terminate gas-exchange before a depth is reached where supersaturation is significant and bradycardia with peripheral vasoconstriction affecting the distribution, and dynamics of blood and tissue nitrogen levels. Published accounts of gas and fat emboli and dysbaric osteonecrosis in marine mammals and theoretical modeling have challenged this view-point, suggesting that decompression-like symptoms may occur under certain circumstances, contrary to common belief. Diagnostic imaging modalities are invaluable tools for the non-invasive examination of animals for evidence of gas and have been used to demonstrate the presence of incidental decompression-related renal gas accumulations in some stranded cetaceans. Diagnostic imaging has also contributed to the recognition of clinically significant gas accumulations in live and dead cetaceans and pinnipeds. Understanding the appropriate application and limitations of the available imaging modalities is important for accurate interpretation of results. The presence of gas may be asymptomatic and must be interpreted cautiously alongside all other available data including clinical examination, clinical laboratory testing, gas analysis, necropsy examination, and histology results.

  2. Distribution of abnormal prion protein in a sheep affected with L-type bovine spongiform encephalopathy.

    PubMed

    Matsuura, Y; Iwamaru, Y; Masujin, K; Imamura, M; Mohri, S; Yokoyama, T; Okada, H

    2013-07-01

    To investigate the topographical distribution and patterns of deposition of immunolabelled abnormal prion protein (PrP(Sc)), interspecies transmission of atypical L-type bovine spongiform encephalopathy (BSE) to Cheviot ewes (ARQ/ARQ genotype) was performed. L-type BSE was successfully transmitted via the intracerebral route to a ewe, with an incubation period of 1,562 days. Minimal vacuolar change was detected in the basal ganglia, thalamus and brainstem, and PrP(Sc) accumulated throughout the brain. The L-type BSE-affected sheep was characterized by conspicuous fine particulate deposits in the neuropil, particulate and/or granular intraneuronal and intraglial deposits, and the absence of PrP(Sc) plaques or stellate deposits. In addition, immunohistochemical and western blot analyses revealed that PrP(Sc) accumulation was present in peripheral nervous tissues (including the trigeminal ganglia and dorsal root ganglion) and adrenal glands, but was absent in lymphoid tissues. These results suggest that L-type BSE has distinct and distinguishable characteristics as well as PrP(Sc) tissue tropism in sheep.

  3. Accumulation of protein carbonyls within cerebellar astrocytes in murine experimental autoimmune encephalomyelitis

    PubMed Central

    Zheng, Jianzheng; Bizzozero, Oscar A.

    2010-01-01

    Recent work from our laboratory has implicated protein carbonylation in the pathophysiology of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The present study was designed to determine the changes in protein carbonylation during the disease progression, and to identify the target cells and modified proteins in the cerebellum of EAE animals, prepared by active immunization of C57/BL6 mice with MOG35-55 peptide. In this model, protein carbonylation was maximal at the peak of the disease (acute phase) to decrease thereafter (chronic phase). Double immunofluorescence microscopy of affected cerebella showed that carbonyls accumulate in white matter astrocytes, and to a lesser extent in microglia/macrophages, both in the acute and chronic phase. Surprisingly, T cells, oligodendrocytes and neurons were barely stained. By 2D-oxyblot and mass spectrometry, β-actin, β-tubulin, GFAP and HSC-71 were identified as the major targets of carbonylation throughout disease. Using a pull-down/western blot method we found a significant increase in the proportion of carbonylated β-actin, β-tubulin and GFAP in the chronic phase but not in the acute phase. These results suggest that as disease progresses from the inflammatory to the neurodegenerative phase there may be an inappropriate removal of oxidized cytoskeletal proteins. Additionally, the extensive accumulation of carbonylated GFAP in the chronic phase of EAE may be responsible for the abnormal shape of astrocytes observed at this stage. PMID:20857508

  4. p53 gene alterations and protein accumulation in colorectal cancer

    PubMed Central

    Bertorelle, R; Esposito, G; Belluco, C; Bonaldi, L; Del Mistro, A; Nitti, D; Lise, M; Chieco-Bianchi, L

    1996-01-01

    Aim—To correlate immunohistochemical staining with single strand conformation polymorphism (SSCP) analysis of the p53 gene in colorectal cancer in order to understand how the findings provided by the two techniques complement each other in defining p53 functional status. Methods—Frozen tumour tissue from 94 patients with colorectal cancer was studied for p53 protein accumulation and gene mutations. Accumulation of p53 protein was detected by immunohistochemistry using PAb1801 and BP53-12-1 monoclonal antibodies. The findings were then compared with SSCP analysis of exons 5 to 8 of the p53 gene. All cases with a positive result by SSCP analysis were confirmed by sequencing. Results—Nuclear staining was observed in 51 (54.2%) cases. SSCP analysis of the DNA amplified by PCR revealed that the electrophoretic pattern had shifted in 30 cases; sequence analysis confirmed the occurrence of a mutation in 29 cases and of a polymorphism in one. In 27 cases both assays gave a positive result, and in 40 both were negative; therefore, concordance between PCR-SSCP and immunohistochemistry was seen in 72% of cases. Conclusion—The data indicate that positive immunostaining corresponds with the presence of a mutation in most, but not all, cases studied; other mechanisms could be responsible for stabilisation and accumulation of p53 protein in the nucleus. Nonsense mutations which do not confer stability on the protein will not be detected by immunohistochemistry and false negative results can also occur with SSCP analysis. Images PMID:16696056

  5. Extracellular cystatin SN and cathepsin B prevent cellular senescence by inhibiting abnormal glycogen accumulation.

    PubMed

    Oh, Sang-Seok; Park, Soojong; Lee, Ki-Won; Madhi, Hamadi; Park, Sae Gwang; Lee, Hee Gu; Cho, Yong-Yeon; Yoo, Jiyun; Dong Kim, Kwang

    2017-04-06

    Cystatin SN (CST1), a known inhibitor of cathepsin B (CatB), has important roles in tumor development. Paradoxically, CatB is a member of the cysteine cathepsin family that acts in cellular processes, such as tumor development and invasion. However, the relationship between CST1 and CatB, and their roles in tumor development are poorly understood. In this study, we observed that the knockdown of CST1 induced the activity of senescence-associated β-galactosidase, a marker of cellular senescence, and expression of senescence-associated secretory phenotype genes, including interleukin-6 and chemokine (C-C motif) ligand 20, in MDA-MB-231 and SW480 cancer cells. Furthermore, CST1 knockdown decreased extracellular CatB activity, and direct CatB inhibition, using specific inhibitors or shCatB, induced cellular senescence. Reconstitution of CST1 restored CatB activity and inhibited cellular senescence in CST1 knockdown cells. CST1 knockdown or CatB inhibition increased glycogen synthase (GS) kinase 3β phosphorylation at serine 9, resulting in the activation of GS and the induction of glycogen accumulation associated with cellular senescence. Importantly, CST1 knockdown suppressed cancer cell proliferation, soft agar colony growth and tumor growth in a xenograft model. These results indicate that CST1-mediated extracellular CatB activity enhances tumor development by preventing cellular senescence. Our findings suggest that antagonists of CST1 or inhibitors of CatB are potential anticancer agents.

  6. Manipulating corn germplasm to increase recombinant protein accumulation.

    PubMed

    Hood, Elizabeth E; Devaiah, Shivakumar P; Fake, Gina; Egelkrout, Erin; Teoh, Keat Thomas; Requesens, Deborah Vicuna; Hayden, Celine; Hood, Kendall R; Pappu, Kameshwari M; Carroll, Jennifer; Howard, John A

    2012-01-01

    Using plants as biofactories for industrial enzymes is a developing technology. The application of this technology to plant biomass conversion for biofuels and biobased products has potential for significantly lowering the cost of these products because of lower enzyme production costs. However, the concentration of the enzymes in plant tissue must be high to realize this goal. We describe the enhancement of the accumulation of cellulases in transgenic maize seed as a part of the process to lower the cost of these dominant enzymes for the bioconversion process. We have used breeding to move these genes into elite and high oil germplasm to enhance protein accumulation in grain. We have also explored processing of the grain to isolate the germ, which preferentially contains the enzymes, to further enhance recovery of enzyme on a dry weight basis of raw materials. The enzymes are active on microcrystalline cellulose to release glucose and cellobiose.

  7. Focal adhesion protein abnormalities in myelodysplastic mesenchymal stromal cells

    SciTech Connect

    Aanei, Carmen Mariana; Eloae, Florin Zugun; Flandrin-Gresta, Pascale; Tavernier, Emmanuelle; Carasevici, Eugen; Guyotat, Denis; Campos, Lydia

    2011-11-01

    Direct cell-cell contact between haematopoietic progenitor cells (HPCs) and their cellular microenvironment is essential to maintain 'stemness'. In cancer biology, focal adhesion (FA) proteins are involved in survival signal transduction in a wide variety of human tumours. To define the role of FA proteins in the haematopoietic microenvironment of myelodysplastic syndromes (MDS), CD73-positive mesenchymal stromal cells (MSCs) were immunostained for paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} and p130CAS, and analysed for reactivity, intensity and cellular localisation. Immunofluorescence microscopy allowed us to identify qualitative and quantitative differences, and subcellular localisation analysis revealed that in pathological MSCs, paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} formed nuclear molecular complexes. Increased expression of paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} and enhanced nuclear co-localisation of these proteins correlated with a consistent proliferative advantage in MSCs from patients with refractory anaemia with excess blasts (RAEB) and negatively impacted clonogenicity of HPCs. These results suggest that signalling via FA proteins could be implicated in HPC-MSC interactions. Further, because FAK is an HSP90{alpha}/{beta} client protein, these results suggest the utility of HSP90{alpha}/{beta} inhibition as a target for adjuvant therapy for myelodysplasia.

  8. Accumulation of guanylate binding proteins in patients treated with interferons.

    PubMed

    Cheng, Y S; Becker-Manley, M F; Rucker, R G; Borden, E C

    1988-06-01

    We have previously described an interferon (IFN)-induced protein with a molecular weight of 67,000. This protein has an affinity to guanylates and is thus called guanylate binding protein (GBP). The synthesis of GBP is inducible by IFNs in all human diploid fibroblast cell lines that we studied. To determine whether or not the GBP synthesis is IFN-inducible in humans as well as in cultured cells, we have studied the levels of GBP in the peripheral blood leukocytes (PBL) of patients treated with either type I or type II IFN. An increased GBP level was found the day immediately after treatment with either type of IFN, and the elevated GBP levels were maintained for at least 8 days. Among the patients studied, we found a higher level GBP accumulation (2.3x) in patients treated with IFN-beta than in those treated with IFN-gamma (1.6x). The increase of GBP in patients receiving IFN-gamma correlated with increases in class II histocompatibility antigens, HLA-DR and HLA-DQ in monocytes. Thus, the levels of GBP in peripheral blood leukocytes may be used as a parameter for the study of IFN responses in patients.

  9. Prion protein accumulation in lipid rafts of mouse aging brain.

    PubMed

    Agostini, Federica; Dotti, Carlos G; Pérez-Cañamás, Azucena; Ledesma, Maria Dolores; Benetti, Federico; Legname, Giuseppe

    2013-01-01

    The cellular form of the prion protein (PrP(C)) is a normal constituent of neuronal cell membranes. The protein misfolding causes rare neurodegenerative disorders known as transmissible spongiform encephalopathies or prion diseases. These maladies can be sporadic, genetic or infectious. Sporadic prion diseases are the most common form mainly affecting aging people. In this work, we investigate the biochemical environment in which sporadic prion diseases may develop, focusing our attention on the cell membrane of neurons in the aging brain. It is well established that with aging the ratio between the most abundant lipid components of rafts undergoes a major change: while cholesterol decreases, sphingomyelin content rises. Our results indicate that the aging process modifies the compartmentalization of PrP(C). In old mice, this change favors PrP(C) accumulation in detergent-resistant membranes, particularly in hippocampi. To confirm the relationship between lipid content changes and PrP(C) translocation into detergent-resistant membranes (DRMs), we looked at PrP(C) compartmentalization in hippocampi from acid sphingomyelinase (ASM) knockout (KO) mice and synaptosomes enriched in sphingomyelin. In the presence of high sphingomyelin content, we observed a significant increase of PrP(C) in DRMS. This process is not due to higher levels of total protein and it could, in turn, favor the onset of sporadic prion diseases during aging as it increases the PrP intermolecular contacts into lipid rafts. We observed that lowering sphingomyelin in scrapie-infected cells by using fumonisin B1 led to a 50% decrease in protease-resistant PrP formation. This may suggest an involvement of PrP lipid environment in prion formation and consequently it may play a role in the onset or development of sporadic forms of prion diseases.

  10. Prion Protein Accumulation in Lipid Rafts of Mouse Aging Brain

    PubMed Central

    Agostini, Federica; Dotti, Carlos G.; Pérez-Cañamás, Azucena; Ledesma, Maria Dolores; Benetti, Federico; Legname, Giuseppe

    2013-01-01

    The cellular form of the prion protein (PrPC) is a normal constituent of neuronal cell membranes. The protein misfolding causes rare neurodegenerative disorders known as transmissible spongiform encephalopathies or prion diseases. These maladies can be sporadic, genetic or infectious. Sporadic prion diseases are the most common form mainly affecting aging people. In this work, we investigate the biochemical environment in which sporadic prion diseases may develop, focusing our attention on the cell membrane of neurons in the aging brain. It is well established that with aging the ratio between the most abundant lipid components of rafts undergoes a major change: while cholesterol decreases, sphingomyelin content rises. Our results indicate that the aging process modifies the compartmentalization of PrPC. In old mice, this change favors PrPC accumulation in detergent-resistant membranes, particularly in hippocampi. To confirm the relationship between lipid content changes and PrPC translocation into detergent-resistant membranes (DRMs), we looked at PrPC compartmentalization in hippocampi from acid sphingomyelinase (ASM) knockout (KO) mice and synaptosomes enriched in sphingomyelin. In the presence of high sphingomyelin content, we observed a significant increase of PrPC in DRMS. This process is not due to higher levels of total protein and it could, in turn, favor the onset of sporadic prion diseases during aging as it increases the PrP intermolecular contacts into lipid rafts. We observed that lowering sphingomyelin in scrapie-infected cells by using fumonisin B1 led to a 50% decrease in protease-resistant PrP formation. This may suggest an involvement of PrP lipid environment in prion formation and consequently it may play a role in the onset or development of sporadic forms of prion diseases. PMID:24040215

  11. Accumulator

    NASA Technical Reports Server (NTRS)

    Fenwick, J. R.; Karigan, G. H. (Inventor)

    1977-01-01

    An accumulator particularly adapted for use in controlling the pressure of a stream of fluid in its liquid phase utilizing the fluid in its gaseous phase was designed. The accumulator is characterized by a shell defining a pressure chamber having an entry throat for a liquid and adapted to be connected in contiguous relation with a selected conduit having a stream of fluid flowing through the conduit in its liquid phase. A pressure and volume stabilization tube, including an array of pressure relief perforations is projected into the chamber with the perforations disposed adjacent to the entry throat for accommodating a discharge of the fluid in either gaseous or liquid phases, while a gas inlet and liquid to gas conversion system is provided, the chamber is connected with a source of the fluid for continuously pressuring the chamber for controlling the pressure of the stream of liquid.

  12. Disruption of Axonal Transport Perturbs Bone Morphogenetic Protein (BMP) - Signaling and Contributes to Synaptic Abnormalities in Two Neurodegenerative Diseases

    PubMed Central

    Kang, Min Jung; Hansen, Timothy J.; Mickiewicz, Monique; Kaczynski, Tadeusz J.; Fye, Samantha; Gunawardena, Shermali

    2014-01-01

    Formation of new synapses or maintenance of existing synapses requires the delivery of synaptic components from the soma to the nerve termini via axonal transport. One pathway that is important in synapse formation, maintenance and function of the Drosophila neuromuscular junction (NMJ) is the bone morphogenetic protein (BMP)-signaling pathway. Here we show that perturbations in axonal transport directly disrupt BMP signaling, as measured by its downstream signal, phospho Mad (p-Mad). We found that components of the BMP pathway genetically interact with both kinesin-1 and dynein motor proteins. Thick vein (TKV) vesicle motility was also perturbed by reductions in kinesin-1 or dynein motors. Interestingly, dynein mutations severely disrupted p-Mad signaling while kinesin-1 mutants showed a mild reduction in p-Mad signal intensity. Similar to mutants in components of the BMP pathway, both kinesin-1 and dynein motor protein mutants also showed synaptic morphological defects. Strikingly TKV motility and p-Mad signaling were disrupted in larvae expressing two human disease proteins; expansions of glutamine repeats (polyQ77) and human amyloid precursor protein (APP) with a familial Alzheimer's disease (AD) mutation (APPswe). Consistent with axonal transport defects, larvae expressing these disease proteins showed accumulations of synaptic proteins along axons and synaptic abnormalities. Taken together our results suggest that similar to the NGF-TrkA signaling endosome, a BMP signaling endosome that directly interacts with molecular motors likely exist. Thus problems in axonal transport occurs early, perturbs BMP signaling, and likely contributes to the synaptic abnormalities observed in these two diseases. PMID:25127478

  13. Proteomic identification of abnormally expressed proteins in early-stage placenta derived from cloned cat embryos.

    PubMed

    Bang, Jae-Il; Lee, Hyo-Sang; Deb, Gautam Kumar; Ha, A-Na; Kwon, Young-Sang; Cho, Seong-Keun; Kim, Byeong-Woo; Cho, Kyu-Woan; Kong, Il-Keun

    2013-01-15

    It is unknown whether gene expression in cloned placenta during pre- and postimplantation is associated with early pregnancy failure in the cat. In this study, protein expression patterns were examined in early-stage (21-day-old) domestic cat placentas of fetuses derived from AI (CP; N = 4) and cloned embryo transfer (CEP; N = 2). Differentially expressed proteins were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrometry (MS). A total of 21 proteins were aberrantly expressed (P < 0.05) by >1.5-fold in CEP compared with CP. Compared with CP, 12 proteins were upregulated in CEP (peptidyl-prolyl cis-trans isomerase A, annexin A2, protein DJ-1, adenylate kinase isoenzyme 1, protein disulfide-isomerase A3, actin cytoplasmic 1, serum albumin, protein disulfide-isomerase A6, and triosephosphate isomerase), and nine proteins were downregulated (triosephosphate isomerase; heterogeneous nuclear ribonucleoprotein H; tropomyosin alpha-4; triosephosphate isomerase 1; 60 kDa heat shock protein, mitochondrial; serum albumin; calumenin; keratin type 1; and prohibitin). The identities of the differentially expressed proteins were validated by peptide mass fingerprinting using matrix-assisted laser desorption/ionization-TOF/TOF MS/MS. The abnormally expressed proteins identified in this study might be associated with impaired development and dysfunction of CEP during early pregnancy. Abnormal protein expression might also induce fetal loss and contribute to failure to maintain pregnancy to term.

  14. Uptake and Degradation of Protease-Sensitive and -Resistant Forms of Abnormal Human Prion Protein Aggregates by Human Astrocytes

    PubMed Central

    Choi, Young Pyo; Head, Mark W.; Ironside, James W.; Priola, Suzette A.

    2015-01-01

    Sporadic Creutzfeldt-Jakob disease is the most common of the human prion diseases, a group of rare, transmissible, and fatal neurologic diseases associated with the accumulation of an abnormal form (PrPSc) of the host prion protein. In sporadic Creutzfeldt-Jakob disease, disease-associated PrPSc is present not only as an aggregated, protease-resistant form but also as an aggregated protease-sensitive form (sPrPSc). Although evidence suggests that sPrPSc may play a role in prion pathogenesis, little is known about how it interacts with cells during prion infection. Here, we show that protease-sensitive abnormal PrP aggregates derived from patients with sporadic Creutzfeldt-Jakob disease are taken up and degraded by immortalized human astrocytes similarly to abnormal PrP aggregates that are resistant to proteases. Our data suggest that relative proteinase K resistance does not significantly influence the astrocyte's ability to degrade PrPSc. Furthermore, the cell does not appear to distinguish between sPrPSc and protease-resistant PrPSc, suggesting that sPrPSc could contribute to prion infection. PMID:25280631

  15. [Abnormal Serum Total Protein Measurement by Lipoprotein-X in an Infant with Biliary Atresia].

    PubMed

    Futatsugi, Akiko; Hidaka, Eiko; Kubota, Noriko; Nishijima, Fumie; Yoshizawa, Katsumi; Ishimine, Nau; Sugano, Mitsutoshi; Hori, Atsushi; Hidaka, Hiroya

    2015-11-01

    Lipoprotein-X (LP-X) in cholestatic jaundice causes abnormal reaction in assays for low-density lipoprotein-cholesterol, but the effects on other test items are unknown. Here, we report an infant with biliary atresia showing abnormal reaction in total serum protein assay using the biuret method, and lipoprotein-X (LP-X) was then detected. In this 11-month-old female infant, jaundice was observed at 2 months old, and a diagnosis of biliary atresia was made. On biochemical tests at 12 months old, the total serum protein concentrations detected by the biuret method were very high, and the response curve and linearity of dilution were abnormal. LP-X was detected by agar electrophoresis. In addition and recovery experiments with normal serum fractionation of the patient's LP-X-rich lipoprotein fraction prepared by ultracentrifugation, normal γ-globulin fractionation showed an abnormal reaction by the biuret method. In infants with biliary atresia, we showed that the total serum protein assay by the biuret method was influenced by LP-X-rich lipoprotein, which may be caused by abnormal reaction of LP-X and γ-globulin. [Case Report].

  16. Reduced miR-200b and miR-200c expression contributes to abnormal hepatic lipid accumulation by stimulating JUN expression and activating the transcription of srebp1

    PubMed Central

    Sun, Libo; Lu, Yonggang; Dou, Lin; Huang, Xiuqing; Sun, Mingxiao; Pang, Cheng; Qu, Jing; Liu, Guanghui; Li, Jian

    2016-01-01

    Previous studies indicated that miR-200s participated in IL-6-induced hepatic insulin resistance. However, the role of miR-200s in hepatic lipid accumulation has not been elucidated. Here we found that miR-200b and miR-200c were reduced in the steatotic livers of mice fed a high-fat diet (HFD) and patients with nonalcoholic fatty liver disease. This down-regulation was accompanied by an increase in the expression of lipogenic proteins such as sterol regulatory element-binding protein 1 (SREBP1) and fatty acid synthase (FAS). The suppression of miR-200b and miR-200c in Hep1-6 and NCTC1469 hepatocytes enhanced intracellular triglyceride levels, which were associated with increased SREBP-1 and FAS protein levels. In contrast, the over-expression of miR-200b and miR-200c suppressed lipid accumulation and reduced the expression of SREBP1 and FAS in Hep1-6 and NCTC1469 cells transfected with miR-200b or miR-200c mimics. Importantly, the up-regulation of miR-200b and miR-200c could reverse oleic acid/palmitic acid-induced lipid accumulation in hepatocytes. A luciferase reporter assay identified that miR-200b and miR-200c could directly bind the 3′UTR of jun. JUN activated the transcription of srebp1 to increase lipid accumulation. The data also demonstrated that increased miR-200b and miR-200c expression might be associated with sitagliptin-reduced hepatic lipid accumulation in mice fed a HFD. These findings suggest, for the first time, that reduced miR-200b and miR-200c expression contributes to abnormal hepatic lipid accumulation by stimulating JUN expression and activating the transcription of srebp1. PMID:27166182

  17. Mutations in the SPTLC1 protein cause mitochondrial structural abnormalities and endoplasmic reticulum stress in lymphoblasts.

    PubMed

    Myers, Simon J; Malladi, Chandra S; Hyland, Ryan A; Bautista, Tara; Boadle, Ross; Robinson, Phillip J; Nicholson, Garth A

    2014-07-01

    Mutations in serine palmitoyltransferase long chain subunit 1 (SPTLC1) cause the typical length-dependent axonal degeneration hereditary sensory neuropathy type 1 (HSN1). Transmission electron microscopy studies on SPTLC1 mutant lymphoblasts derived from patients revealed specific structural abnormalities of mitochondria. Swollen mitochondria with abnormal cristae were clustered around the nucleus, with some mitochondria being wrapped in rough endoplasmic reticulum (ER) membranes. Total mitochondrial counts revealed a significant change in mitochondrial numbers between healthy and diseased lymphocytes but did not reveal any change in length to width ratios nor were there any changes to cellular function. However, there was a notable change in ER homeostasis, as assessed using key ER stress markers, BiP and ERO1-Lα, displaying reduced protein expression. The observations suggest that SPTLC1 mutations cause mitochondrial abnormalities and ER stress in HSN1 cells.

  18. Abnormal membrane protein methylation and merocyanine 540 fluorescence in sickle erythrocyte membranes.

    PubMed

    Manna, C; Hermanowicz, N; Ro, J Y; Neilan, B; Glushko, V; Kim, S

    1984-06-01

    Sickle cell erythrocytes exhibit reduced carboxyl methylation of membrane proteins compared to normal erythrocytes. This altered methylation in sickle membrane proteins is also observable when extracted membranes, both intact and alkali treated, were used as substrates for the homologous protein methylase II (S-adenosylmethionine:protein-carboxyl O-methyltransferase, EC. 2.1.1.24). However, when glycophorin A, one of the major methyl acceptors in both membranes, was extracted by lithium diiodosalicylate and used as the methyl acceptor, the proteins from both membranes were methylated equally, suggesting an involvement of membrane structure in membrane-bound protein methylation. Merocyanine 540 (MC-540), a fluorescent probe, was used to determine if the membranes differed in organization. Incubation of both normal and sickle erythrocytes membranes with MC-540 produced a marked increase in extrinsic fluorescence, reflecting a relatively nonpolar environment for the dye bound to the membranes. The fluorescence from sickle cell ghosts was only 87% as intense as that from normal ghosts, while the actual amount of MC-540 associated with sickle cell membranes was only 62% of normal. These data suggest that differences exist in the distribution of surface charges on these plasma membranes. These results are consistent with the hypothesis that abnormal levels of membrane protein methylation observed in sickle erythrocytes may be a result of abnormal membrane organization characteristic to sickle cell anemia.

  19. Gla-rich protein is a novel vitamin K-dependent protein present in serum that accumulates at sites of pathological calcifications.

    PubMed

    Viegas, Carla S B; Cavaco, Sofia; Neves, Pedro L; Ferreira, Ana; João, Alexandre; Williamson, Matthew K; Price, Paul A; Cancela, M Leonor; Simes, Dina C

    2009-12-01

    Mineralization of soft tissues is an abnormal process that occurs in any body tissue and can greatly increase morbidity and mortality. Vitamin K-dependent (VKD) proteins play a crucial role in these processes; matrix Gla protein is considered one of the most relevant physiological inhibitors of soft tissue calcification know to date. Several studies have suggested that other, still unknown, VKD proteins might also be involved in soft tissue calcification pathologies. We have recently identified in sturgeon a new VKD protein, Gla-rich protein (GRP), which contains the highest ratio between number of Gla residues and size of the mature protein so far identified. Although mainly expressed in cartilaginous tissues of sturgeon, in rat GRP is present in both cartilage and bone. We now show that GRP is a circulating protein that is also expressed and accumulated in soft tissues of rats and humans, including the skin and vascular system in which, when affected by pathological calcifications, GRP accumulates at high levels at sites of mineral deposition, indicating an association with calcification processes. The high number of Gla residues and consequent mineral binding affinity properties strongly suggest that GRP may directly influence mineral formation, thereby playing a role in processes involving connective tissue mineralization.

  20. Stimulation of adenosine A2A receptors reduces intracellular cholesterol accumulation and rescues mitochondrial abnormalities in human neural cell models of Niemann-Pick C1.

    PubMed

    Ferrante, A; De Nuccio, C; Pepponi, R; Visentin, S; Martire, A; Bernardo, A; Minghetti, L; Popoli, P

    2016-04-01

    Niemann Pick C 1 (NPC1) disease is an incurable, devastating lysosomal-lipid storage disorder characterized by hepatosplenomegaly, progressive neurological impairment and early death. Current treatments are very limited and the research of new therapeutic targets is thus mandatory. We recently showed that the stimulation of adenosine A2A receptors (A2ARs) rescues the abnormal phenotype of fibroblasts from NPC1 patients suggesting that A2AR agonists could represent a therapeutic option for this disease. However, since all NPC1 patients develop severe neurological symptoms which can be ascribed to the complex pathology occurring in both neurons and oligodendrocytes, in the present paper we tested the effects of the A2AR agonist CGS21680 in human neuronal and oligodendroglial NPC1 cell lines (i.e. neuroblastoma SH-SY5Y and oligodendroglial MO3.13 transiently transfected with NPC1 small interfering RNA). The down-regulation of the NPC1 protein effectively resulted in intracellular cholesterol accumulation and altered mitochondrial membrane potential. Both effects were significantly attenuated by CGS21680 (500 nM). The protective effects of CGS were prevented by the selective A2AR antagonist ZM241385 (500 nM). The involvement of calcium modulation was demonstrated by the ability of Bapta-AM (5-7 μM) in reverting the effect of CGS. The A2A-dependent activity was prevented by the PKA-inhibitor KT5720, thus showing the involvement of the cAMP/PKA signaling. These findings provide a clear in vitro proof of concept that A2AR agonists are promising potential drugs for NPC disease.

  1. Early Golgi abnormalities and neurodegeneration upon loss of presynaptic proteins Munc18-1, syntaxin-1 or SNAP-25.

    PubMed

    Santos, Tatiana C; Wierda, Keimpe; Broeke, Jurjen H; Toonen, Ruud F; Verhage, Matthijs

    2017-03-27

    The loss of presynaptic proteins Munc18-1, syntaxin-1 or SNAP-25 is known to produce cell death, but the underlying features have not been compared experimentally. Here, we investigated these features in cultured mouse CNS and dorsal root ganglion neurons. Side-by-side comparisons confirmed massive cell death, before synaptogenesis, within 1-4 days in vitro (DIV) upon loss of t-SNAREs (syntaxin-1, SNAP-25) or Munc18-1, but not v-SNAREs (synaptobrevins/VAMP1/2/3 using Tetanus Neurotoxin (TeNT), also in TI-VAMP/VAMP7 knock-out (KO) neurons). A condensed cis-Golgi was the first abnormality observed upon Munc18-1 or SNAP-25 loss within 3 DIV. This phenotype was distinct from the Golgi fragmentation observed in apoptosis. Cell death was too rapid after syntaxin-1 loss to study Golgi abnormalities. Syntaxin-1 and Munc18-1 depend on each other for normal cellular levels. We observed that endogenous syntaxin-1 accumulates at the Golgi of Munc18-1 KO neurons. However, expression of a non-neuronal Munc18 isoform that does not bind syntaxin-1, Munc18-3, in Munc18-1 KO neurons prevented cell death and restored normal cis-Golgi morphology, but not synaptic transmission or syntaxin-1 targeting. Finally, we observed that dorsal root ganglion neurons are the only Munc18-1 KO neurons that do not degenerate in vivo or in vitro In these neurons, cis-Golgi abnormalities were less severe, with no changes in Golgi shape. Together these data demonstrate that cell death upon Munc18-1, syntaxin-1 or SNAP-25 loss occurs via a degenerative pathway unrelated to the known synapse function of these proteins and involving early cis-Golgi abnormalities, distinct from apoptosis.SIGNIFICANCE STATEMENTThis study provides new insights in a neurodegeneration pathway triggered by the absence of specific proteins involved in synaptic transmission (syntaxin-1, Munc18-1, SNAP-25), while other proteins involved in the same molecular process (synaptobrevins, Munc13-1/2) do not cause degeneration. Massive

  2. Knockout of G protein β5 impairs brain development and causes multiple neurologic abnormalities in mice

    PubMed Central

    Zhang, Jian-Hua; Pandey, Mritunjay; Seigneur, Erica M.; Panicker, Leelamma M.; Koo, Lily; Schwartz, Owen M.; Chen, Weiping; Chen, Ching-Kang; Simonds, William F.

    2011-01-01

    Gβ5 is a divergent member of the signal-transducing G protein β subunit family encoded by GNB5 and expressed principally in brain and neuronal tissue. Among heterotrimeric Gβ isoforms, Gβ5 is unique in its ability to heterodimerize with members of the R7 subfamily of the regulator of G protein signaling (RGS) proteins that contain G protein-γ like domains. Previous studies employing Gnb5 knockout (KO) mice have shown that Gβ5 is an essential stabilizer of such RGS proteins and regulates the deactivation of retinal phototransduction and the proper functioning of retinal bipolar cells. However, little is known of the function of Gβ5 in the brain outside the visual system. We show here that mice lacking Gβ5 have a markedly abnormal neurologic phenotype that includes impaired development, tiptoe-walking, motor learning and coordination deficiencies, and hyperactivity. We further show that Gβ5-deficient mice have abnormalities of neuronal development in cerebellum and hippocampus. We find that the expression of both mRNA and protein from multiple neuronal genes is dysregulated in Gnb5 KO mice. Taken together with previous observations from Gnb5 KO mice, our findings suggest a model in which Gβ5 regulates dendritic arborization and/or synapse formation during development, in part by effects on gene expression. PMID:21883221

  3. Caterpillar attack triggers accumulation of the toxic maize protein RIP2.

    PubMed

    Chuang, Wen-Po; Herde, Marco; Ray, Swayamjit; Castano-Duque, Lina; Howe, Gregg A; Luthe, Dawn S

    2014-02-01

    Some plant-derived anti-herbivore defensive proteins are induced by insect feeding, resist digestion in the caterpillar gut and are eliminated in the frass. We have identified several maize proteins in fall armyworm (Spodoptera frugiperda) frass that potentially play a role in herbivore defense. Furthermore, the toxicity of one of these proteins, ribosome-inactivating protein 2 (RIP2), was assessed and factors regulating its accumulation were determined. To understand factors regulating RIP2 protein accumulation, maize (Zea mays) plants were infested with fall armyworm larvae or treated with exogenous hormones. The toxicity of recombinant RIP2 protein against fall armyworm was tested. The results show that RIP2 protein is synthesized as an inactive proenzyme that can be processed in the caterpillar gut. Also, caterpillar feeding, but not mechanical wounding, induced foliar RIP2 protein accumulation. Quantitative real-time PCR indicated that RIP2 transcripts were rapidly induced (1 h) and immunoblot analysis indicated that RIP2 protein accumulated soon after attack and was present in the leaf for up to 4 d after caterpillar removal. Several phytohormones, including methyl jasmonate, ethylene, and abscisic acid, regulated RIP2 protein expression. Furthermore, bioassays of purified recombinant RIP2 protein against fall armyworm significantly retarded caterpillar growth. We conclude that the toxic protein RIP2 is induced by caterpillar feeding and is one of a potential suite of proteins that defend maize against chewing herbivores.

  4. Melanin or a Melanin-Like Substance Interacts with the N-Terminal Portion of Prion Protein and Inhibits Abnormal Prion Protein Formation in Prion-Infected Cells.

    PubMed

    Hamanaka, Taichi; Nishizawa, Keiko; Sakasegawa, Yuji; Oguma, Ayumi; Teruya, Kenta; Kurahashi, Hiroshi; Hara, Hideyuki; Sakaguchi, Suehiro; Doh-Ura, Katsumi

    2017-03-15

    Prion diseases are progressive fatal neurodegenerative illnesses caused by the accumulation of transmissible abnormal prion protein (PrP). To find treatments for prion diseases, we searched for substances from natural resources that inhibit abnormal PrP formation in prion-infected cells. We found that high-molecular-weight components from insect cuticle extracts reduced abnormal PrP levels. The chemical nature of these components was consistent with that of melanin. In fact, synthetic melanin produced from tyrosine or 3-hydroxy-l-tyrosine inhibited abnormal PrP formation. Melanin did not modify cellular or cell surface PrP levels, nor did it modify lipid raft or cellular cholesterol levels. Neither did it enhance autophagy or lysosomal function. Melanin was capable of interacting with PrP at two N-terminal domains. Specifically, it strongly interacted with the PrP region of amino acids 23 to 50 including a positively charged amino acid cluster and weakly interacted with the PrP octarepeat peptide region of residues 51 to 90. However, the in vitro and in vivo data were inconsistent with those of prion-infected cells. Abnormal PrP formation in protein misfolding cyclic amplification was not inhibited by melanin. Survival after prion infection was not significantly altered in albino mice or exogenously melanin-injected mice compared with that of control mice. These data suggest that melanin, a main determinant of skin color, is not likely to modify prion disease pathogenesis, even though racial differences in the incidence of human prion diseases have been reported. Thus, the findings identify an interaction between melanin and the N terminus of PrP, but the pathophysiological roles of the PrP-melanin interaction remain unclear.IMPORTANCE The N-terminal region of PrP is reportedly important for neuroprotection, neurotoxicity, and abnormal PrP formation, as this region is bound by many factors, such as metal ions, lipids, nucleic acids, antiprion compounds, and several

  5. Identification and Kinetics of Accumulation of Proteins Induced by Ethylene in Bean Abscission Zones 1

    PubMed Central

    del Campillo, Elena; Lewis, Lowell N.

    1992-01-01

    A two-dimensional gel electrophoresis system that combines a cationic polyacrylamide gel electrophoresis at pH near neutrality with sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the spectrum of basic polypeptides that accumulate in bean (Phaseolus vulgaris) abscission zones after treatment with ethylene. Results showed that, as abscission progressed, at least seven basic proteins accumulated in the abscission zone prior to the accumulation of 9.5 cellulase. Six of the seven proteins correspond to pathogenesis-related (PR) proteins. Among them, two isoforms of β-1,3-glucanase and multiple isoforms of chitinase were identified. A 22 kilodalton polypeptide that accumulated to high levels was identified as a thaumatin-like protein by analysis of its N-terminal sequence (up to 20 amino acids) and its serological relationship with heterologous thaumatin antibodies. A 15 kilodalton polypeptide serologically related to PR P1 (p14) from tomato was identified as bean PR P1 (p14)-like protein. The kinetics of accumulation of glucanases, chitinases, thaumatin-like and PR P1 (p14)-like proteins during ethylene treatment were similar and they showed that PR proteins accumulated in abscission zones prior to the increase in 9.5 cellulase. Addition of indoleacetic acid, a potent inhibitor of abscission, reduced the accumulation of these proteins to a similar extent (60%). The synchronized accumulation of this set of PR proteins, early in the abscission process, may play a role in induced resistance to possible fungal attack after a plant part is shed. The seventh protein does not correspond to any previously characterized PR protein. This new 45 kilodalton polypeptide accumulated in abscission zones on exposure to ethylene concomitantly with the increase in 9.5 cellulase. Its N-terminal sequence (up to 15 amino acids) showed some homology with the amino terminal sequence of chitinase. Polyclonal antibodies against chitinase recognized the 45

  6. Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation.

    PubMed

    Alvarez, M Lucrecia; Topal, Emel; Martin, Federico; Cardineau, Guy A

    2010-01-01

    Improving foreign protein accumulation is crucial for enhancing the commercial success of plant-based production systems since product yields have a major influence on process economics. Cereal grain evolved to store large amounts of proteins in tightly organized aggregates. In maize, gamma-Zein is the major storage protein synthesized by the rough endoplasmic reticulum (ER) and stored in specialized organelles called protein bodies (PB). Zera (gamma-Zein ER-accumulating domain) is the N-terminal proline-rich domain of gamma-zein that is sufficient to induce the assembly of PB formation. Fusion of the Zera domain to proteins of interest results in assembly of dense PB-like, ER-derived organelles, containing high concentration of recombinant protein. Our main goal was to increase recombinant protein accumulation in plants in order to enhance the efficiency of orally-delivered plant-made vaccines. It is well known that oral vaccination requires substantially higher doses than parental formulations. As a part of a project to develop a plant-made plague vaccine, we expressed our model antigen, the Yersinia pestis F1-V antigen fusion protein, with and without a fused Zera domain. We demonstrated that Zera-F1-V protein accumulation was at least 3x higher than F1-V alone when expressed in three different host plant systems: Ncotiana benthamiana, Medicago sativa (alfalfa) and Nicotiana tabacum NT1 cells. We confirmed the feasibility of using Zera technology to induce protein body formation in non-seed tissues. Zera expression and accumulation did not affect plant development and growth. These results confirmed the potential exploitation of Zera technology to substantially increase the accumulation of value-added proteins in plants.

  7. Minos-insertion mutant of the Drosophila GBA gene homologue showed abnormal phenotypes of climbing ability, sleep and life span with accumulation of hydroxy-glucocerebroside.

    PubMed

    Kawasaki, Haruhisa; Suzuki, Takahiro; Ito, Kumpei; Takahara, Tsubasa; Goto-Inoue, Naoko; Setou, Mitsutoshi; Sakata, Kazuki; Ishida, Norio

    2017-03-07

    Gaucher's disease in humans is considered a deficiency of glucocerebrosidase (GlcCerase) that result in the accumulation of its substrate, glucocerebroside (GlcCer). Although mouse models of Gaucher's disease have been reported from several laboratories, these models are limited due to the perinatal lethality of GlcCerase gene. Here, we examined phenotypes of Drosophila melanogaster homologues genes of the human Gaucher's disease gene by using Minos insertion. One of two Minos insertion mutants to unknown function gene (CG31414) accumulates the hydroxy-GlcCer in whole body of Drosophila melanogaster. This mutant showed abnormal phenotypes of climbing ability and sleep, and short lifespan. These abnormal phenotypes are very similar to that of Gaucher's disease in human. In contrast, another Minos insertion mutant (CG31148) and its RNAi line did not show such severe phenotype as observed in CG31414 gene mutation. The data suggests that Drosophila CG31414 gene mutation might be useful for unraveling the molecular mechanism of Gaucher's disease.

  8. C-terminally mutated tubby protein accumulates in aggresomes

    PubMed Central

    Kim, Sunshin; Sung, Ho Jin; Lee, Ji Won; Kim, Yun Hee; Oh, Yong-Seok; Yoon, Kyong-Ah; Heo, Kyun; Suh, Pann-Ghill

    2017-01-01

    The tubby protein (Tub), a putative transcription factor, plays important roles in the maintenance and function of neuronal cells. A splicing defect-causing mutation in the 3′-end of the tubby gene, which is predicted to disrupt the carboxy-terminal region of the Tub protein, causes maturity-onset obesity, blindness, and deafness in mice. Although this pathological Tub mutation leads to a loss of function, the precise mechanism has not yet been investigated. Here, we found that the mutant Tub proteins were mostly localized to puncta found in the perinuclear region and that the C-terminus was important for its solubility. Immunocytochemical analysis revealed that puncta of mutant Tub co-localized with the aggresome. Moreover, whereas wild-type Tub was translocated to the nucleus by extracellular signaling, the mutant forms failed to undergo such translocation. Taken together, our results suggest that the malfunctions of the Tub mutant are caused by its misfolding and subsequent localization to aggresomes. PMID:27697107

  9. Diaphragm Abnormalities in Patients with End-Stage Heart Failure: NADPH Oxidase Upregulation and Protein Oxidation

    PubMed Central

    Ahn, Bumsoo; Coblentz, Philip D.; Beharry, Adam W.; Patel, Nikhil; Judge, Andrew R.; Moylan, Jennifer. S.; Hoopes, Charles W.; Bonnell, Mark R.; Ferreira, Leonardo F.

    2017-01-01

    Patients with heart failure (HF) have diaphragm abnormalities that contribute to disease morbidity and mortality. Studies in animals suggest that reactive oxygen species (ROS) cause diaphragm abnormalities in HF. However, the effects of HF on ROS sources, antioxidant enzymes, and protein oxidation in the diaphragm of humans is unknown. NAD(P)H oxidase, especially the Nox2 isoform, is an important source of ROS in the diaphragm. Our main hypothesis was that diaphragm from patients with HF have heightened Nox2 expression and p47phox phosphorylation (marker of enzyme activation) that is associated with elevated protein oxidation. We collected diaphragm biopsies from patients with HF and brain-dead organ donors (controls). Diaphragm mRNA levels of Nox2 subunits were increased 2.5–4.6-fold over controls (p < 0.05). Patients also had increased protein levels of Nox2 subunits (p47phox, p22phox, and p67phox) and total p47phox phosphorylation, while phospho-to-total p47phox levels were unchanged. The antioxidant enzyme catalase was increased in patients, whereas glutathione peroxidase and superoxide dismutases were unchanged. Among markers of protein oxidation, carbonyls were increased by ~40% (p < 0.05) and 4-hydroxynonenal and 3-nitrotyrosines were unchanged in patients with HF. Overall, our findings suggest that Nox2 is an important source of ROS in the diaphragm of patients with HF and increases in levels of antioxidant enzymes are not sufficient to maintain normal redox homeostasis. The net outcome is elevated diaphragm protein oxidation that has been shown to cause weakness in animals. PMID:28119629

  10. CCDC115 Deficiency Causes a Disorder of Golgi Homeostasis with Abnormal Protein Glycosylation

    PubMed Central

    Jansen, Jos C.; Cirak, Sebahattin; van Scherpenzeel, Monique; Timal, Sharita; Reunert, Janine; Rust, Stephan; Pérez, Belén; Vicogne, Dorothée; Krawitz, Peter; Wada, Yoshinao; Ashikov, Angel; Pérez-Cerdá, Celia; Medrano, Celia; Arnoldy, Andrea; Hoischen, Alexander; Huijben, Karin; Steenbergen, Gerry; Quelhas, Dulce; Diogo, Luisa; Rymen, Daisy; Jaeken, Jaak; Guffon, Nathalie; Cheillan, David; van den Heuvel, Lambertus P.; Maeda, Yusuke; Kaiser, Olaf; Schara, Ulrike; Gerner, Patrick; van den Boogert, Marjolein A.W.; Holleboom, Adriaan G.; Nassogne, Marie-Cécile; Sokal, Etienne; Salomon, Jody; van den Bogaart, Geert; Drenth, Joost P.H.; Huynen, Martijn A.; Veltman, Joris A.; Wevers, Ron A.; Morava, Eva; Matthijs, Gert; Foulquier, François; Marquardt, Thorsten; Lefeber, Dirk J.

    2016-01-01

    Disorders of Golgi homeostasis form an emerging group of genetic defects. The highly heterogeneous clinical spectrum is not explained by our current understanding of the underlying cell-biological processes in the Golgi. Therefore, uncovering genetic defects and annotating gene function are challenging. Exome sequencing in a family with three siblings affected by abnormal Golgi glycosylation revealed a homozygous missense mutation, c.92T>C (p.Leu31Ser), in coiled-coil domain containing 115 (CCDC115), the function of which is unknown. The same mutation was identified in three unrelated families, and in one family it was compound heterozygous in combination with a heterozygous deletion of CCDC115. An additional homozygous missense mutation, c.31G>T (p.Asp11Tyr), was found in a family with two affected siblings. All individuals displayed a storage-disease-like phenotype involving hepatosplenomegaly, which regressed with age, highly elevated bone-derived alkaline phosphatase, elevated aminotransferases, and elevated cholesterol, in combination with abnormal copper metabolism and neurological symptoms. Two individuals died of liver failure, and one individual was successfully treated by liver transplantation. Abnormal N- and mucin type O-glycosylation was found on serum proteins, and reduced metabolic labeling of sialic acids was found in fibroblasts, which was restored after complementation with wild-type CCDC115. PSI-BLAST homology detection revealed reciprocal homology with Vma22p, the yeast V-ATPase assembly factor located in the endoplasmic reticulum (ER). Human CCDC115 mainly localized to the ERGIC and to COPI vesicles, but not to the ER. These data, in combination with the phenotypic spectrum, which is distinct from that associated with defects in V-ATPase core subunits, suggest a more general role for CCDC115 in Golgi trafficking. Our study reveals CCDC115 deficiency as a disorder of Golgi homeostasis that can be readily identified via screening for abnormal

  11. ELLI-1, a novel germline protein, modulates RNAi activity and P-granule accumulation in Caenorhabditis elegans

    PubMed Central

    Andralojc, Karolina M.; Kelly, Ashley L.; Tanner, Paige C.

    2017-01-01

    Germ cells contain non-membrane bound cytoplasmic organelles that help maintain germline integrity. In C. elegans they are called P granules; without them, the germline undergoes partial masculinization and aberrant differentiation. One key P-granule component is the Argonaute CSR-1, a small-RNA binding protein that antagonizes accumulation of sperm-specific transcripts in developing oocytes and fine-tunes expression of proteins critical to early embryogenesis. Loss of CSR-1 complex components results in a very specific, enlarged P-granule phenotype. In a forward screen to identify mutants with abnormal P granules, ten alleles were recovered with a csr-1 P-granule phenotype, eight of which contain mutations in known components of the CSR-1 complex (csr-1, ego-1, ekl-1, and drh-3). The remaining two alleles are in a novel gene now called elli-1 (enlarged germline granules). ELLI-1 is first expressed in primordial germ cells during mid-embryogenesis, and continues to be expressed in the adult germline. While ELLI-1 forms cytoplasmic aggregates, they occasionally dock, but do not co-localize with P granules. Instead, the majority of ELLI-1 aggregates accumulate in the shared germline cytoplasm. In elli-1 mutants, several genes that promote RNAi and P-granule accumulation are upregulated, and embryonic lethality, sterility, and RNAi resistance in a hypomorphic drh-3 allele is enhanced, suggesting that ELLI-1 functions with CSR-1 to modulate RNAi activity, P-granule accumulation, and post-transcriptional expression in the germline. PMID:28182654

  12. Characterization of the Proteostasis Roles of Glycerol Accumulation, Protein Degradation and Protein Synthesis during Osmotic Stress in C. elegans

    PubMed Central

    Choung-Hee Lee, Elaine; Deonarine, Andrew; Strange, Kevin

    2012-01-01

    Exposure of C. elegans to hypertonic stress-induced water loss causes rapid and widespread cellular protein damage. Survival in hypertonic environments depends critically on the ability of worm cells to detect and degrade misfolded and aggregated proteins. Acclimation of C. elegans to mild hypertonic stress suppresses protein damage and increases survival under more extreme hypertonic conditions. Suppression of protein damage in acclimated worms could be due to 1) accumulation of the chemical chaperone glycerol, 2) upregulation of protein degradation activity, and/or 3) increases in molecular chaperoning capacity of the cell. Glycerol and other chemical chaperones are widely thought to protect proteins from hypertonicity-induced damage. However, protein damage is unaffected by gene mutations that inhibit glycerol accumulation or that cause dramatic constitutive elevation of glycerol levels. Pharmacological or RNAi inhibition of proteasome and lyosome function and measurements of cellular protein degradation activity demonstrated that upregulation of protein degradation mechanisms plays no role in acclimation. Thus, changes in molecular chaperone capacity must be responsible for suppressing protein damage in acclimated worms. Transcriptional changes in chaperone expression have not been detected in C. elegans exposed to hypertonic stress. However, acclimation to mild hypertonicity inhibits protein synthesis 50–70%, which is expected to increase chaperone availability for coping with damage to existing proteins. Consistent with this idea, we found that RNAi silencing of essential translational components or acute exposure to cycloheximide results in a 50–80% suppression of hypertonicity-induced aggregation of polyglutamine-YFP (Q35::YFP). Dietary changes that increase protein production also increase Q35::YFP aggregation 70–180%. Our results demonstrate directly for the first time that inhibition of protein translation protects extant proteins from damage

  13. Endometrial inflammation and abnormal expression of extracellular matrix proteins induced by Mycoplasma bovis in dairy cows.

    PubMed

    Guo, Mengyao; Wang, Guoqing; Lv, Tingting; Song, Xiaojing; Wang, Tiancheng; Xie, Guanghong; Cao, Yongguo; Zhang, Naisheng; Cao, Rongfeng

    2014-03-15

    Mycoplasma bovis infection can cause endometrial inflammation leading to infertility and involuntary culling in dairy cows. Because extracellular matrix (ECM) proteins affect the adherence of mycoplasma to eukaryotic cell surface, they may play a role in the pathogenesis of the bacteria. The objective of the present study was to evaluate the endometrial inflammatory response and ECM protein expression induced by M bovis. Endometrial concentrations of inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and mRNA and protein expression of collagen IV (CL-IV), fibronectin (FN), and laminin (LN) were evaluated 10, 20, and 30 days after M bovis intrauterine infusion in breed cows 18 days postpartum. The presence of the bacteria in the uterus was detected by nested polymerase chain reaction and denaturing gradient gel electrophoresis. Endometrial TNF-α, IL-1β, and IL-6 concentrations in the treatment group were greater (P < 0.05) than in the positive and negative control groups 20 and 30 days after infusion. Endometrial CL-IV, FN, and LN mRNA and protein expression increased (P < 0.01) 20 days after infusion in all groups. However, the increase was more pronounced in the treatment group and reactive expressions were greater (P < 0.05) than in the positive and negative control groups 10, 20, and 30 days after infusion. In conclusion, M bovis triggered endometrial inflammatory response and increased CL-IV, FN, and LN mRNA and protein expression. The abnormal expression of ECM these proteins may promote the pathogenic effects of M bovis that lead to endometrial tissue damage and infertility.

  14. HDAC6 controls major cell response pathways to cytotoxic accumulation of protein aggregates

    PubMed Central

    Boyault, Cyril; Zhang, Yu; Fritah, Sabrina; Caron, Cécile; Gilquin, Benoit; Kwon, So Hee; Garrido, Carmen; Yao, Tso-Pang; Vourc’h, Claire; Matthias, Patrick; Khochbin, Saadi

    2007-01-01

    A cellular defense mechanism counteracts the deleterious effects of misfolded protein accumulation by eliciting a stress response. The cytoplasmic deacetylase HDAC6 (histone deacetylase 6) was previously shown to be a key element in this response by coordinating the clearance of protein aggregates through aggresome formation and their autophagic degradation. Here, for the first time, we demonstrate that HDAC6 is involved in another crucial cell response to the accumulation of ubiquitinated protein aggregates, and unravel its molecular basis. Indeed, our data show that HDAC6 senses ubiquitinated cellular aggregates and consequently induces the expression of major cellular chaperones by triggering the dissociation of a repressive HDAC6/HSF1 (heat-shock factor 1)/HSP90 (heat-shock protein 90) complex and a subsequent HSF1 activation. HDAC6 therefore appears as a master regulator of the cell protective response to cytotoxic protein aggregate formation. PMID:17785525

  15. iTRAQ-based proteomic analysis of plasma reveals abnormalities in lipid metabolism proteins in chronic kidney disease-related atherosclerosis

    PubMed Central

    Luczak, Magdalena; Formanowicz, Dorota; Marczak, Łukasz; Suszyńska-Zajczyk, Joanna; Pawliczak, Elżbieta; Wanic-Kossowska, Maria; Stobiecki, Maciej

    2016-01-01

    Patients with chronic kidney disease (CKD) have a considerably higher risk of death due to cardiovascular causes. Using an iTRAQ MS/MS approach, we investigated the alterations in plasma protein accumulation in patients with CKD and classical cardiovascular disease (CVD) without CKD. The proteomic analysis led to the identification of 130 differentially expressed proteins among CVD and CKD patients and healthy volunteers. Bioinformatics analysis revealed that 29 differentially expressed proteins were involved in lipid metabolism and atherosclerosis, 20 of which were apolipoproteins and constituents of high-density lipoprotein (HDL) and low-density lipoprotein (LDL). Although dyslipidemia is common in CKD patients, we found that significant changes in apolipoproteins were not strictly associated with changes in plasma lipid levels. A lack of correlation between apoB and LDL concentration and an inverse relationship of some proteins with the HDL level were revealed. An increased level of apolipoprotein AIV, adiponectin, or apolipoprotein C, despite their anti-atherogenic properties, was not associated with a decrease in cardiovascular event risk in CKD patients. The presence of the distinctive pattern of apolipoproteins demonstrated in this study may suggest that lipid abnormalities in CKD are characterized by more qualitative abnormalities and may be related to HDL function rather than HDL deficiency. PMID:27600335

  16. Biogenesis of protein bodies during legumin accumulation in developing olive (Olea europaea L.) seed.

    PubMed

    Jimenez-Lopez, Jose C; Zienkiewicz, Agnieszka; Zienkiewicz, Krzysztof; Alché, Juan D; Rodríguez-García, Maria I

    2016-03-01

    Much of our current knowledge about seed development and differentiation regarding reserves synthesis and accumulation come from monocot (cereals) plants. Studies in dicotyledonous seeds differentiation are limited to a few species and in oleaginous species are even scarcer despite their agronomic and economic importance. We examined the changes accompanying the differentiation of olive endosperm and cotyledon with a focus on protein bodies (PBs) biogenesis during legumin protein synthesis and accumulation, with the aim of getting insights and a better understanding of the PBs' formation process. Cotyledon and endosperm undergo differentiation during seed development, where an asynchronous time-course of protein synthesis, accumulation, and differential PB formation patterns was found in both tissues. At the end of seed maturation, a broad population of PBs, particularly in cotyledon cells, was distinguishable in terms of number per cell and morphometric and cytochemical features. Olive seed development is a tissue-dependent process characterized by differential rates of legumin accumulation and PB formation in the main tissues integrating seed. One of the main features of the impressive differentiation process is the specific formation of a broad group of PBs, particularly in cotyledon cells, which might depend on selective accumulation and packaging of proteins and specific polypeptides into PBs. The nature and availability of the major components detected in the PBs of olive seed are key parameters in order to consider the potential use of this material as a suitable source of carbon and nitrogen for animal or even human use.

  17. Perivascular Accumulation of β-Sheet-Rich Proteins in Offspring Brain following Maternal Exposure to Carbon Black Nanoparticles

    PubMed Central

    Onoda, Atsuto; Kawasaki, Takayasu; Tsukiyama, Koichi; Takeda, Ken; Umezawa, Masakazu

    2017-01-01

    Environmental stimulation during brain development is an important risk factor for the development of neurodegenerative disease. Clinical evidence indicates that prenatal exposure to particulate air pollutants leads to diffuse damage to the neurovascular unit in the developing brain and accelerates neurodegeneration. Maternal exposure to carbon black nanoparticles (CB-NPs), used as a model for particulate air pollution, induces long-lasting diffuse perivascular abnormalities. We aimed to comprehensively characterize the perivascular abnormalities related to maternal NPs exposure using Fourier transform infrared microspectroscopy (in situ FT-IR) and classical staining analysis. Pregnant ICR mice were intranasally treated with a CB-NPs suspension (95 μg/kg at a time) on gestational days 5 and 9. Brains were collected 6 weeks after birth and sliced to prepare 10-μm-thick serial sections. Reflective spectra of in situ FT-IR were acquired using lattice measurements (x-axis: 7, y-axis: 7, 30-μm apertures) around a centered blood vessel. We also performed mapping analysis of protein secondary structures. Serial sections were stained with using periodic acid-Schiff or immunofluorescence to examine the phenotypes of the perivascular areas. Peaks of amide I bands in spectra from perivascular areas were shifted by maternal NPs exposure. However, there were two types of peak-shift in one mouse in the exposure group. Some vessels had a large peak-shift and others had a small peak-shift. In situ FT-IR combined with traditional staining revealed that the large peak-shift was induced around blood vessel adjacent to astrocytes with glial fibrillary acidic protein and aquaporin-4 over-expression and perivascular macrophages (PVMs) with enlarged lysosome granules. Furthermore, protein secondary structural analysis indicated that maternal NPs exposure led to increases in β-sheet content and decreases in α-helix content in areas that are mostly close to the centered blood vessel

  18. Growth of plasmodium falciparum in human erythrocytes containing abnormal membrane proteins

    SciTech Connect

    Schulman, S. City Univ. of New York, NY ); Roth, E.F. Jr.; Cheng, B.; Rybicki, A.C.; Sussman, I.I.; Wong, M.; Nagel, R.L.; Schwartz, R.S. ); Wang, W. ); Ranney, H.M. )

    1990-09-01

    To evaluate the role of erythrocyte (RBC) membrane proteins in the invasion and maturation of Plasmodium falciparum, the authors have studied, in culture, abnormal RBCs containing quantitative or qualitative membrane protein defects. These defects included hereditary spherocytosis (HS) due to decreases in the content of spectrin (HS(Sp{sup +})), hereditary elliptocytosis (HE) due to protein 4.1 deficiency (HE(4.1{sup 0})), HE due to a spectrin {alpha}I domain structural variant that results in increased content of spectrin dimers (HE(Sp{alpha}{sup I/65})), and band 3 structural variants. Parasite invasion, measured by the initial uptake of ({sup 3}H)hypoxanthine 18 hr after inoculation with merozoites, was normal in all of the pathologic RBCs. In contrast, RBCs from six HS(Sp{sup +}) subjects showed marked growth inhibition that became apparent after the first or second growth cycle. The extent of decreased parasite growth in HS(Sp{sup +}) RBCs closely correlated with the extent of RBC spectrin deficiency. Homogeneous subpopulations of dense HS RBCs exhibited decreased parasite growth to the same extent as did HS whole blood. RBCs from four HE subjects showed marked parasite growth and development.

  19. Genetic suppression of neurodegeneration and neurotransmitter release abnormalities caused by expanded full-length huntingtin accumulating in the cytoplasm.

    PubMed Central

    Romero, Eliana; Cha, Guang-Ho; Verstreken, Patrik; Ly, Cindy V.; Hughes, Robert; Bellen, Hugo J.; Botas, Juan

    2008-01-01

    Summary Huntington's Disease (HD) is a dominantly inherited neurodegenerative disorder caused by expansion of a translated CAG repeat in the N-terminus of the huntingtin protein. Here we describe the generation and characterization of a novel full-length HD Drosophila model to reveal a previously unknown disease mechanism that occurs early in the course of pathogenesis, before expanded huntingtin is cleaved and imported into the nucleus in detectable amounts. We find that expanded full-length huntingtin (128QhttFL) leads to behavioral, neurodegenerative, and electrophysiological phenotypes. These phenotypes are caused by a Ca2+-dependent increase in neurotransmitter release efficiency in 128QhttFL animals. Partial loss of function in synaptic transmission (Syntaxin, Snap, Rop) and voltage-gated Ca2+ channel genes suppresses both the electrophysiological and the neurodegenerative phenotypes. Thus, our data indicate that increased neurotransmission is at the root of neuronal degeneration caused by expanded full-length huntingtin during early stages of pathogenesis. PMID:18184562

  20. Chicken antibody against a restrictive epitope of prion protein distinguishes normal and abnormal prion proteins.

    PubMed

    Miyamoto, Kazuyoshi; Kimura, Sota; Nakamura, Naoto; Yokoyama, Takashi; Horiuchi, Hiroyuki; Furusawa, Shuichi; Matsuda, Haruo

    2007-10-01

    Recently, we reported the application of a recombinant chicken IgY monoclonal antibody, Ab3-15, against mammalian prion protein (PrP), for the diagnosis of bovine spongiform encephalopathy in cattle. In this study, we have characterized a soluble, single-chain variable fragment (scFv) form of this antibody, sphAb3-15 using brain homogenates from mice. This sphAb3-15 antibody recognized denatured forms of both PrP(C) and PrP(Sc), and PrP(Sc) after PK-treatment, on Western blotting. In sandwich ELISAs, on dot blots and by immunoprecipitation, sphAb3-15 efficiently bound to PrP from normal brain homogenates, but weakly bound PrP from scrapie-infected brain homogenates. These results suggest that sphAb3-15 selectively recognizes PrP(C) under native conditions and that the epitope recognized by sphAb3-15 may undergo conformational changes during the conversion of PrP(C) into PrP(Sc).

  1. IFT proteins accumulate during cell division and localize to the cleavage furrow in Chlamydomonas.

    PubMed

    Wood, Christopher R; Wang, Zhaohui; Diener, Dennis; Zones, James Matt; Rosenbaum, Joel; Umen, James G

    2012-01-01

    Intraflagellar transport (IFT) proteins are well established as conserved mediators of flagellum/cilium assembly and disassembly. However, data has begun to accumulate in support of IFT protein involvement in other processes elsewhere in the cell. Here, we used synchronous cultures of Chlamydomonas to investigate the temporal patterns of accumulation and localization of IFT proteins during the cell cycle. Their mRNAs showed periodic expression that peaked during S and M phase (S/M). Unlike most proteins that are synthesized continuously during G1 phase, IFT27 and IFT46 levels were found to increase only during S/M phase. During cell division, IFT27, IFT46, IFT72, and IFT139 re-localized from the flagella and basal bodies to the cleavage furrow. IFT27 was further shown to be associated with membrane vesicles in this region. This localization pattern suggests a role for IFT in cell division.

  2. Calmodulin-binding protein CBP60g functions as a negative regulator in Arabidopsis anthocyanin accumulation

    PubMed Central

    Zou, Bo; Wan, Dongli; Li, Ruili; Han, Xiaomin; Li, Guojing; Wang, Ruigang

    2017-01-01

    Anthocyanins, a kind of flavonoid, normally accumulate in the flowers and fruits and make them colorful. Anthocyanin accumulation is regulated via the different temporal and spatial expression of anthocyanin regulatory and biosynthetic genes. CBP60g, a calmodulin binding protein, has previously been shown to have a role in pathogen resistance, drought tolerance and ABA sensitivity. In this study, we found that CBP60g repressed anthocyanin accumulation induced by drought, sucrose and kinetin. The expression pattern of CBP60g was in accordance with the anthocyanin accumulation tissues. Real-time qPCR analysis revealed that the anthocyanin biosynthetic genes CHS, CHI and DFR, as well as two members of MBW complex, PAP1, a MYB transcription factor, and TT8, a bHLH transcription factor, were down regulated by CBP60g. PMID:28253311

  3. Accumulation of Group 3 Late Embryogenesis Abundant Proteins in Zea mays Embryos 1

    PubMed Central

    Thomann, Estela B.; Sollinger, John; White, Constance; Rivin, Carol J.

    1992-01-01

    Several different types of proteins that are modulated by abscisic acid (ABA) accumulate in developing embryos of maize (Zea mays L.). Some of these proteins are specific to the developing seed, such as the storage globulin, GLB1, whereas others are involved in general responses to water deficit. Here we describe a maize protein family of this second type, a Group 3 late embryogenesis abundant (MLG3). Like other proteins of this class, MLG3 polypeptides are ABA-responsive. They are found in maturing seeds and in dehydrating plant tissues. Antigenically related proteins are found in other cereals. To distinguish the regulation of developmentally programmed ABA responses from those that are environmentally induced, we compared the ontological pattern and accumulation requirements of MLG3 polypeptides with those we previously described for GLB1. GLB1 accumulation begins early in the maturation phase and specifically requires high levels of ABA and the participation of the Viviparous-1 (Vp1) gene product. Vp1 is required for other ABA-modulated events in maize seed development as well. In experiments using vp1 mutants and mutants deficient in ABA synthesis (vp5 mutation), we show that MLG3 accumulation also is dependent upon ABA, but it shows striking differences from GLB1. MLG3 accumulates much later in embryogenesis, coincident with the onset of dehydration. In contrast to GLB1, MLG3 proteins can be induced by de novo ABA synthesis in response to culturing in high osmoticum. Unlike GLB1, MLG3 has no specific requirement for the Vp1 gene product. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6Figure 7Figure 8 PMID:16668930

  4. Abnormal expression of FLI1 protein is an adverse prognostic factor in acute myeloid leukemia

    PubMed Central

    Qiu, Yi Hua; Zhang, Nianxiang; Singh, Neera; Faderl, Stefan; Ferrajoli, Alessandra; York, Heather; Qutub, Amina A.; Coombes, Kevin R.; Watson, Dennis K.

    2011-01-01

    Friend leukemia virus integration 1 (FLI1), an Ets transcription factor family member, is linked to acute myelogenous leukemia (AML) by chromosomal events at the FLI1 locus, but the biologic impact of FLI1 expression on AML is unknown. FLI1 protein expression was measured in 511 newly diagnosed AML patients. Expression was similar in peripheral blood (PB) and BM and higher at diagnosis than at relapse (P = .02). Compared with normal CD34+ cells, expression in AML was above or below normal in 32% and 5% of patients, respectively. Levels were negatively correlated with an antecedent hematologic disorder (P = .002) but not with age or cytogenetics. Mutated NPM1 (P = .0007) or FLT3-ITD (P < .02) had higher expression. FLI1 levels were negatively correlated with 10 of 195 proteins associated with proliferation and stromal interaction, and positively correlated (R > 0.3) with 19 others. The FLI1 level was not predictive of remission attainment, but patients with low or high FLI1 expression had shorter remission duration (22.6 and 40.3 vs 51.1 weeks, respectively; P = .01) and overall survival (45.2 and 35.4 vs 59.4 weeks, respectively; P = .03). High FLI1 levels were adverse in univariate and multivariate analysis. FLI1 expression is frequently abnormal and prognostically adverse in AML. FLI1 and/or its response genes may be therapeutically targetable to interfere with AML cell biology. PMID:21917756

  5. Proteolysis of abnormal prion protein with a thermostable protease from Thermococcus kodakarensis KOD1.

    PubMed

    Koga, Yuichi; Tanaka, Shun-ichi; Sakudo, Akikazu; Tobiume, Minoru; Aranishi, Mutsuo; Hirata, Azumi; Takano, Kazufumi; Ikuta, Kazuyoshi; Kanaya, Shigenori

    2014-03-01

    The abnormal prion protein (scrapie-associated prion protein, PrP(Sc)) is considered to be included in the group of infectious agents of transmissible spongiform encephalopathies. Since PrP(Sc) is highly resistant to normal sterilization procedures, the decontamination of PrP(Sc) is a significant public health issue. In the present study, a hyperthermostable protease, Tk-subtilisin, was used to degrade PrP(Sc). Although PrP(Sc) is known to be resistant toward proteolytic enzymes, Tk-subtilisin was able to degrade PrP(Sc) under extreme conditions. The level of PrP(Sc) in brain homogenates was found to decrease significantly in vitro following Tk-subtilisin treatment at 100 °C, whereas some protease-resistant fractions remain after proteinase K treatment. Rather small amounts of Tk-subtilisin (0.3 U) were required to degrade PrP(Sc) at 100 °C and pH 8.0. In addition, Tk-subtilisin was observed to degrade PrP(Sc) in the presence of sodium dodecyl sulfate or other industrial surfactants. Although several proteases degrading PrP(Sc) have been reported, practical decontamination procedures using enzymes are not available. This report aims to provide basic information for the practical use of a proteolytic enzyme for PrP(Sc) degradation.

  6. ATP6AP1 deficiency causes an immunodeficiency with hepatopathy, cognitive impairment and abnormal protein glycosylation

    PubMed Central

    Jansen, Eric J. R.; Timal, Sharita; Ryan, Margret; Ashikov, Angel; van Scherpenzeel, Monique; Graham, Laurie A.; Mandel, Hanna; Hoischen, Alexander; Iancu, Theodore C.; Raymond, Kimiyo; Steenbergen, Gerry; Gilissen, Christian; Huijben, Karin; van Bakel, Nick H. M.; Maeda, Yusuke; Rodenburg, Richard J.; Adamowicz, Maciej; Crushell, Ellen; Koenen, Hans; Adams, Darius; Vodopiutz, Julia; Greber-Platzer, Susanne; Müller, Thomas; Dueckers, Gregor; Morava, Eva; Sykut-Cegielska, Jolanta; Martens, Gerard J. M.; Wevers, Ron A.; Niehues, Tim; Huynen, Martijn A.; Veltman, Joris A.; Stevens, Tom H.; Lefeber, Dirk J.

    2016-01-01

    The V-ATPase is the main regulator of intra-organellar acidification. Assembly of this complex has extensively been studied in yeast, while limited knowledge exists for man. We identified 11 male patients with hemizygous missense mutations in ATP6AP1, encoding accessory protein Ac45 of the V-ATPase. Homology detection at the level of sequence profiles indicated Ac45 as the long-sought human homologue of yeast V-ATPase assembly factor Voa1. Processed wild-type Ac45, but not its disease mutants, restored V-ATPase-dependent growth in Voa1 mutant yeast. Patients display an immunodeficiency phenotype associated with hypogammaglobulinemia, hepatopathy and a spectrum of neurocognitive abnormalities. Ac45 in human brain is present as the common, processed ∼40-kDa form, while liver shows a 62-kDa intact protein, and B-cells a 50-kDa isoform. Our work unmasks Ac45 as the functional ortholog of yeast V-ATPase assembly factor Voa1 and reveals a novel link of tissue-specific V-ATPase assembly with immunoglobulin production and cognitive function. PMID:27231034

  7. Prognostic value of serum tumor abnormal protein in gastric cancer patients

    PubMed Central

    LAN, FENG; ZHU, MING; QI, QIUFENG; ZHANG, YAPING; LIU, YONGPING

    2016-01-01

    Aberrant glycosylation of protein occurs in nearly all types of cancers and has been confirmed to be associated with tumor progression, metastasis and the survival rate of patients. The present study aimed to explore the prognostic value of tumor abnormal protein (TAP) in gastric cancer patients. TAP was detected in the blood of 42 gastric cancer patients and 56 healthy volunteers by using the TAP testing kit. Univariate and multivariate Cox regression analysis were performed to evaluate the prognostic value of TAP. In total, 64.3% of gastric cancer patients were positive for TAP, and TAP was significantly correlated with poor prognosis [progression-free survival (PFS), 4.2 vs. 12.6 months; P=0.043]. TAP [hazard ratio (HR), 64.487; P<0.01), differentiation (HR, 17.279; P<0.01) and TNM stage (HR, 45.480; P<0.01) were found to be independent predictive factors for PFS. Furthermore, Kaplan-Meier curves indicated that TAP is associated with a reduced PFS in gastric cancer patients. The results of the present study therefore indicated that the TAP test has significant prognostic value for gastric cancer patients. PMID:27330802

  8. ATP6AP1 deficiency causes an immunodeficiency with hepatopathy, cognitive impairment and abnormal protein glycosylation.

    PubMed

    Jansen, Eric J R; Timal, Sharita; Ryan, Margret; Ashikov, Angel; van Scherpenzeel, Monique; Graham, Laurie A; Mandel, Hanna; Hoischen, Alexander; Iancu, Theodore C; Raymond, Kimiyo; Steenbergen, Gerry; Gilissen, Christian; Huijben, Karin; van Bakel, Nick H M; Maeda, Yusuke; Rodenburg, Richard J; Adamowicz, Maciej; Crushell, Ellen; Koenen, Hans; Adams, Darius; Vodopiutz, Julia; Greber-Platzer, Susanne; Müller, Thomas; Dueckers, Gregor; Morava, Eva; Sykut-Cegielska, Jolanta; Martens, Gerard J M; Wevers, Ron A; Niehues, Tim; Huynen, Martijn A; Veltman, Joris A; Stevens, Tom H; Lefeber, Dirk J

    2016-05-27

    The V-ATPase is the main regulator of intra-organellar acidification. Assembly of this complex has extensively been studied in yeast, while limited knowledge exists for man. We identified 11 male patients with hemizygous missense mutations in ATP6AP1, encoding accessory protein Ac45 of the V-ATPase. Homology detection at the level of sequence profiles indicated Ac45 as the long-sought human homologue of yeast V-ATPase assembly factor Voa1. Processed wild-type Ac45, but not its disease mutants, restored V-ATPase-dependent growth in Voa1 mutant yeast. Patients display an immunodeficiency phenotype associated with hypogammaglobulinemia, hepatopathy and a spectrum of neurocognitive abnormalities. Ac45 in human brain is present as the common, processed ∼40-kDa form, while liver shows a 62-kDa intact protein, and B-cells a 50-kDa isoform. Our work unmasks Ac45 as the functional ortholog of yeast V-ATPase assembly factor Voa1 and reveals a novel link of tissue-specific V-ATPase assembly with immunoglobulin production and cognitive function.

  9. Abnormal expression of FLI1 protein is an adverse prognostic factor in acute myeloid leukemia.

    PubMed

    Kornblau, Steven M; Qiu, Yi Hua; Zhang, Nianxiang; Singh, Neera; Faderl, Stefan; Ferrajoli, Alessandra; York, Heather; Qutub, Amina A; Coombes, Kevin R; Watson, Dennis K

    2011-11-17

    Friend leukemia virus integration 1 (FLI1), an Ets transcription factor family member, is linked to acute myelogenous leukemia (AML) by chromosomal events at the FLI1 locus, but the biologic impact of FLI1 expression on AML is unknown. FLI1 protein expression was measured in 511 newly diagnosed AML patients. Expression was similar in peripheral blood (PB) and BM and higher at diagnosis than at relapse (P = .02). Compared with normal CD34(+) cells, expression in AML was above or below normal in 32% and 5% of patients, respectively. Levels were negatively correlated with an antecedent hematologic disorder (P = .002) but not with age or cytogenetics. Mutated NPM1 (P = .0007) or FLT3-ITD (P < .02) had higher expression. FLI1 levels were negatively correlated with 10 of 195 proteins associated with proliferation and stromal interaction, and positively correlated (R > 0.3) with 19 others. The FLI1 level was not predictive of remission attainment, but patients with low or high FLI1 expression had shorter remission duration (22.6 and 40.3 vs 51.1 weeks, respectively; P = .01) and overall survival (45.2 and 35.4 vs 59.4 weeks, respectively; P = .03). High FLI1 levels were adverse in univariate and multivariate analysis. FLI1 expression is frequently abnormal and prognostically adverse in AML. FLI1 and/or its response genes may be therapeutically targetable to interfere with AML cell biology.

  10. Stable accumulation of seed storage proteins containing vaccine peptides in transgenic soybean seeds.

    PubMed

    Maruyama, Nobuyuki; Fujiwara, Keigo; Yokoyama, Kazunori; Cabanos, Cerrone; Hasegawa, Hisakazu; Takagi, Kyoko; Nishizawa, Keito; Uki, Yuriko; Kawarabayashi, Takeshi; Shouji, Mikio; Ishimoto, Masao; Terakawa, Teruhiko

    2014-10-01

    There has been a significant increase in the use of transgenic plants for the large-scale production of pharmaceuticals and industrial proteins. Here, we report the stable accumulation of seed storage proteins containing disease vaccine peptides in transgenic soybean seeds. To synthesize vaccine peptides in soybean seeds, we used seed storage proteins as a carrier and a soybean breeding line lacking major seed storage proteins as a host. Vaccine peptides were inserted into the flexible disordered regions in the A1aB1b subunit three-dimensional structure. The A1aB1b subunit containing vaccine peptides in the disordered regions were sorted to the protein storage vacuoles where vaccine peptides are partially cleaved by proteases. In contrast, the endoplasmic reticulum (ER)-retention type of the A1aB1b subunit containing vaccine peptides accumulated in compartments that originated from the ER as an intact pro-form. These results indicate that the ER may be an organelle suitable for the stable accumulation of bioactive peptides using seed storage proteins as carriers.

  11. ATM is a cytoplasmic protein in mouse brain required to prevent lysosomal accumulation

    PubMed Central

    Barlow, Carrolee; Ribaut-Barassin, Catherine; Zwingman, Theresa A.; Pope, Amber J.; Brown, Kevin D.; Owens, Jennie W.; Larson, Denise; Harrington, Elizabeth A.; Haeberle, Anne-Marie; Mariani, Jean; Eckhaus, Michael; Herrup, Karl; Bailly, Yannick; Wynshaw-Boris, Anthony

    2000-01-01

    We previously generated a mouse model with a mutation in the murine Atm gene that recapitulates many aspects of the childhood neurodegenerative disease ataxia-telangiectasia. Atm-deficient (Atm−/−) mice show neurological defects detected by motor function tests including the rota-rod, open-field tests and hind-paw footprint analysis. However, no gross histological abnormalities have been observed consistently in the cerebellum of any line of Atm−/− mice analyzed in most laboratories. Therefore, it may be that the neurologic dysfunction found in these animals is associated with predegenerative lesions. We performed a detailed analysis of the cerebellar morphology in two independently generated lines of Atm−/− mice to determine whether there was evidence of neuronal abnormality. We found a significant increase in the number of lysosomes in Atm−/− mice in the absence of any detectable signs of neuronal degeneration or other ultrastructural anomalies. In addition, we found that the ATM protein is predominantly cytoplasmic in Purkinje cells and other neurons, in contrast to the nuclear localization of ATM protein observed in cultured cells. The cytoplasmic localization of ATM in Purkinje cells is similar to that found in human cerebellum. These findings suggest that ATM may be important as a cytoplasmic protein in neurons and that its absence leads to abnormalities of cytoplasmic organelles reflected as an increase in lysosomal numbers. PMID:10639172

  12. Abnormal Intracellular Accumulation and Extracellular Aβ Deposition in Idiopathic and Dup15q11.2-q13 Autism Spectrum Disorders

    PubMed Central

    Wegiel, Jerzy; Frackowiak, Janusz; Mazur-Kolecka, Bozena; Schanen, N. Carolyn; Cook, Edwin H.; Sigman, Marian; Brown, W. Ted; Kuchna, Izabela; Wegiel, Jarek; Nowicki, Krzysztof; Imaki, Humi; Ma, Shuang Yong; Chauhan, Abha; Chauhan, Ved; Miller, David L.; Mehta, Pankaj D.; Flory, Michael; Cohen, Ira L.; London, Eric; Reisberg, Barry; de Leon, Mony J.; Wisniewski, Thomas

    2012-01-01

    Background It has been shown that amyloid ß (Aβ), a product of proteolytic cleavage of the amyloid β precursor protein (APP), accumulates in neuronal cytoplasm in non-affected individuals in a cell type–specific amount. Methodology/Principal Findings In the present study, we found that the percentage of amyloid-positive neurons increases in subjects diagnosed with idiopathic autism and subjects diagnosed with duplication 15q11.2-q13 (dup15) and autism spectrum disorder (ASD). In spite of interindividual differences within each examined group, levels of intraneuronal Aβ load were significantly greater in the dup(15) autism group than in either the control or the idiopathic autism group in 11 of 12 examined regions (p<0.0001 for all comparisons; Kruskall-Wallis test). In eight regions, intraneuronal Aβ load differed significantly between idiopathic autism and control groups (p<0.0001). The intraneuronal Aβ was mainly N-terminally truncated. Increased intraneuronal accumulation of Aβ17–40/42 in children and adults suggests a life-long enhancement of APP processing with α-secretase in autistic subjects. Aβ accumulation in neuronal endosomes, autophagic vacuoles, Lamp1-positive lysosomes and lipofuscin, as revealed by confocal microscopy, indicates that products of enhanced α-secretase processing accumulate in organelles involved in proteolysis and storage of metabolic remnants. Diffuse plaques containing Aβ1–40/42 detected in three subjects with ASD, 39 to 52 years of age, suggest that there is an age-associated risk of alterations of APP processing with an intraneuronal accumulation of a short form of Aβ and an extracellular deposition of full-length Aβ in nonfibrillar plaques. Conclusions/Significance The higher prevalence of excessive Aβ accumulation in neurons in individuals with early onset of intractable seizures, and with a high risk of sudden unexpected death in epilepsy in autistic subjects with dup(15) compared to subjects with idiopathic

  13. Mutations in nucleolar proteins lead to nucleolar accumulation of polyA+ RNA in Saccharomyces cerevisiae.

    PubMed Central

    Kadowaki, T; Schneiter, R; Hitomi, M; Tartakoff, A M

    1995-01-01

    Synthesis of mRNA and rRNA occur in the chromatin-rich nucleoplasm and the nucleolus, respectively. Nevertheless, we here report that a Saccharomyces cerevisiae gene, MTR3, previously implicated in mRNA transport, codes for a novel essential 28-kDa nucleolar protein. Moreover, in mtr3-1 the accumulated polyA+ RNA actually colocalizes with nucleolar antigens, the nucleolus becomes somewhat disorganized, and rRNA synthesis and processing are inhibited. A strain with a ts conditional mutation in RNA polymerase I also shows nucleolar accumulation of polyA+ RNA, whereas strains with mutations in the nucleolar protein Nop1p do not. Thus, in several mutant backgrounds, when mRNA cannot be exported i concentrates in the nucleolus. mRNA may normally encounter nucleolar components before export and proteins such as Mtr3p may be critical for export of both mRNA and ribosomal subunits. Images PMID:8534909

  14. Complex proteinopathy with accumulations of prion protein, hyperphosphorylated tau, α-synuclein and ubiquitin in experimental bovine spongiform encephalopathy of monkeys.

    PubMed

    Piccardo, Pedro; Cervenak, Juraj; Bu, Ming; Miller, Lindsay; Asher, David M

    2014-07-01

    Proteins aggregate in several slowly progressive neurodegenerative diseases called 'proteinopathies'. Studies with cell cultures and transgenic mice overexpressing mutated proteins suggested that aggregates of one protein induced misfolding and aggregation of other proteins as well - a possible common mechanism for some neurodegenerative diseases. However, most proteinopathies are 'sporadic', without gene mutation or overexpression. Thus, proteinopathies in WT animals genetically close to humans might be informative. Squirrel monkeys infected with the classical bovine spongiform encephalopathy agent developed an encephalopathy resembling variant Creutzfeldt-Jakob disease with accumulations not only of abnormal prion protein (PrP(TSE)), but also three other proteins: hyperphosphorylated tau (p-tau), α-synuclein and ubiquitin; β-amyloid protein (Aβ) did not accumulate. Severity of brain lesions correlated with spongiform degeneration. No amyloid was detected. These results suggested that PrP(TSE) enhanced formation of p-tau and aggregation of α-synuclein and ubiquitin, but not Aβ, providing a new experimental model for neurodegenerative diseases associated with complex proteinopathies.

  15. Altered neuronal mitochondrial coenzyme A synthesis in neurodegeneration with brain iron accumulation caused by abnormal processing, stability, and catalytic activity of mutant pantothenate kinase 2.

    PubMed

    Kotzbauer, Paul T; Truax, Adam C; Trojanowski, John Q; Lee, Virginia M-Y

    2005-01-19

    Mutations in the pantothenate kinase 2 (PANK2) gene have been identified in patients with neurodegeneration with brain iron accumulation (NBIA; formerly Hallervorden-Spatz disease). However, the mechanisms by which these mutations cause neurodegeneration are unclear, especially given the existence of multiple pantothenate kinase genes in humans and multiple PanK2 transcripts with potentially different subcellular localizations. We demonstrate that PanK2 protein is localized to mitochondria of neurons in human brain, distinguishing it from other pantothenate kinases that do not possess mitochondrial-targeting sequences. PanK2 protein translated from the most 5' start site is sequentially cleaved at two sites by the mitochondrial processing peptidase, generating a long-lived 48 kDa mature protein identical to that found in human brain extracts. The mature protein catalyzes the initial step in coenzyme A (CoA) synthesis but displays feedback inhibition in response to species of acyl CoA rather than CoA itself. Some, but not all disease-associated point mutations result in significantly reduced catalytic activity. The most common mutation, G521R, results in marked instability of the intermediate PanK2 isoform and reduced production of the mature isoform. These results suggest that NBIA is caused by altered neuronal mitochondrial lipid metabolism caused by mutations disrupting PanK2 protein levels and catalytic activity.

  16. Loss of Clcc1 Results in ER Stress, Misfolded Protein Accumulation, and Neurodegeneration

    PubMed Central

    Jia, Yichang; Jucius, Thomas J.; Cook, Susan A.

    2015-01-01

    Folding of transmembrane and secretory proteins occurs in the lumen of the endoplasmic reticulum (ER) before transportation to the cell surface and is monitored by the unfolded protein response (UPR) signaling pathway. The accumulation of unfolded proteins in the ER activates the UPR that restores ER homeostasis by regulating gene expression that leads to an increase in the protein-folding capacity of the ER and a decrease in the ER protein-folding load. However, prolonged UPR activity has been associated with cell death in multiple pathological conditions, including neurodegeneration. Here, we report a spontaneous recessive mouse mutation that causes progressive cerebellar granule cell death and peripheral motor axon degeneration. By positional cloning, we identify the mutation in this strain as a retrotransposon insertion in the Clcc1 gene, which encodes a putative chloride channel localized to the ER. Furthermore, we demonstrate that the C3H/HeSnJ inbred strain has late onset cerebellar degeneration due to this mutation. Interestingly, acute knockdown of Clcc1 expression in cultured cells increases sensitivity to ER stress. In agreement, GRP78, the major HSP70 family chaperone in the ER, is upregulated in Clcc1-deficient granule cells in vivo, and ubiquitinated proteins accumulate in these neurons before their degeneration. These data suggest that disruption of chloride homeostasis in the ER disrupts the protein-folding capacity of the ER, leading to eventual neuron death. PMID:25698737

  17. Identification of Ubiquilin, a Novel Presenilin Interactor That Increases Presenilin Protein Accumulation

    PubMed Central

    Mah, Alex L.; Perry, George; Smith, Mark A.; Monteiro, Mervyn J.

    2000-01-01

    Mutations in the highly homologous presenilin genes encoding presenilin-1 and presenilin-2 (PS1 and PS2) are linked to early-onset Alzheimer's disease (AD). However, apart from a role in early development, neither the normal function of the presenilins nor the mechanisms by which mutant proteins cause AD are well understood. We describe here the properties of a novel human interactor of the presenilins named ubiquilin. Yeast two-hybrid (Y2H) interaction, glutathione S-transferase pull-down experiments, and colocalization of the proteins expressed in vivo, together with coimmunoprecipitation and cell fractionation studies, provide compelling evidence that ubiquilin interacts with both PS1 and PS2. Ubiquilin is noteworthy since it contains multiple ubiquitin-related domains typically thought to be involved in targeting proteins for degradation. However, we show that ubiquilin promotes presenilin protein accumulation. Pulse-labeling experiments indicate that ubiquilin facilitates increased presenilin synthesis without substantially changing presenilin protein half-life. Immunohistochemistry of human brain tissue with ubiquilin-specific antibodies revealed prominent staining of neurons. Moreover, the anti-ubiquilin antibodies robustly stained neurofibrillary tangles and Lewy bodies in AD and Parkinson's disease affected brains, respectively. Our results indicate that ubiquilin may be an important modulator of presenilin protein accumulation and that ubiquilin protein is associated with neuropathological neurofibrillary tangles and Lewy body inclusions in diseased brain. PMID:11076969

  18. Two Outer Membrane Proteins Contribute to Caulobacter crescentus Cellular Fitness by Preventing Intracellular S-Layer Protein Accumulation

    DOE PAGES

    Overton, K. Wesley; Park, Dan M.; Yung, Mimi C.; ...

    2016-09-23

    Surface layers, or S-layers, are two-dimensional protein arrays that form the outermost layer of many bacteria and archaea. They serve several functions, including physical protection of the cell from environmental threats. The high abundance of S-layer proteins necessitates a highly efficient export mechanism to transport the S-layer protein from the cytoplasm to the cell exterior.Caulobacter crescentusis unique in that it has two homologous, seemingly redundant outer membrane proteins, RsaFaand RsaFb, which together with other components form a type I protein translocation pathway for S-layer export. These proteins have homology toEscherichia coliTolC, the outer membrane channel of multidrug efflux pumps. Heremore » we provide evidence that, unlike TolC, RsaFaand RsaFbare not involved in either the maintenance of membrane stability or the active export of antimicrobial compounds. Rather, RsaFaand RsaFbare required to prevent intracellular accumulation and aggregation of the S-layer protein RsaA; deletion of RsaFaand RsaFbled to a general growth defect and lowered cellular fitness. Using Western blotting, transmission electron microscopy, and transcriptome sequencing (RNA-seq), we show that loss of both RsaFaand RsaFbled to accumulation of insoluble RsaA in the cytoplasm, which in turn caused upregulation of a number of genes involved in protein misfolding and degradation pathways. These findings provide new insight into the requirement for RsaFaand RsaFbin cellular fitness and tolerance to antimicrobial agents and further our understanding of the S-layer export mechanism on both the transcriptional and translational levels inC. crescentus. IMPORTANCEDecreased growth rate and reduced cell fitness are common side effects of protein production in overexpression systems. Inclusion bodies typically form inside the cell, largely due to a lack of sufficient export machinery to transport the overexpressed proteins to the extracellular environment. This phenomenon can

  19. Evidence for degradation of abnormal prion protein in tissues from sheep with scrapie during composting

    PubMed Central

    Huang, Hongsheng; Spencer, J. Lloyd; Soutyrine, Andrei; Guan, Jeiwen; Rendulich, Jasmine; Balachandran, Aru

    2007-01-01

    This study investigated whether the abnormal prion protein (PrPSc) in tissues from sheep with scrapie would be destroyed by composting. Tissues from sheep naturally infected with scrapie were placed within fiberglass mesh bags and buried in compost piles for 108 d in experiment 1 or 148 d in experiment 2. The temperature in the compost piles rose quickly; it was above 60°C for about 2 wk and then slowly declined to the ambient temperature. Before composting, PrPSc was detected in all the tissues by Western blotting. In experiment 1, PrPSc was not detected after composting in the tissue remnants or the surrounding sawdust. In experiment 2, 1 of 5 specimens tested negative after composting, whereas PrPSc was detected in the other 4 bags, though in reduced amounts compared with those before composting. Tissue weights were reduced during composting. Analysis of the tissue remnants for microbial 16S ribosomal DNA demonstrated that there were more diverse microbes involved in experiment 1 than in experiment 2 and that the guanine and cytosine content of the microbial 16S DNA was higher in the specimens of experiment 1 than in those of experiment 2, which suggests greater dominance of thermophilic microbes in experiment 1. These results indicate that composting may have value as a means for degrading PrPSc in carcasses and other wastes. PMID:17193880

  20. Positive Lysosomal Modulation As a Unique Strategy to Treat Age-Related Protein Accumulation Diseases

    PubMed Central

    Wisniewski, Meagan L.; Butler, David

    2012-01-01

    Abstract Lysosomes are involved in degrading and recycling cellular ingredients, and their disruption with age may contribute to amyloidogenesis, paired helical filaments (PHFs), and α-synuclein and mutant huntingtin aggregation. Lysosomal cathepsins are upregulated by accumulating proteins and more so by the modulator Z-Phe-Ala-diazomethylketone (PADK). Such positive modulators of the lysosomal system have been studied in the well-characterized hippocampal slice model of protein accumulation that exhibits the pathogenic cascade of tau aggregation, tubulin breakdown, microtubule destabilization, transport failure, and synaptic decline. Active cathepsins were upregulated by PADK; Rab proteins were modified as well, indicating enhanced trafficking, whereas lysosome-associated membrane protein and proteasome markers were unchanged. Lysosomal modulation reduced the pre-existing PHF deposits, restored tubulin structure and transport, and recovered synaptic components. Further proof-of-principle studies used Alzheimer disease mouse models. It was recently reported that systemic PADK administration caused dramatic increases in cathepsin B protein and activity levels, whereas neprilysin, insulin-degrading enzyme, α-secretase, and β-secretase were unaffected by PADK. In the transgenic models, PADK treatment resulted in clearance of intracellular amyloid beta (Aβ) peptide and concomitant reduction of extracellular deposits. Production of the less pathogenic Aβ1–38 peptide corresponded with decreased levels of Aβ1–42, supporting the lysosome's antiamyloidogenic role through intracellular truncation. Amelioration of synaptic and behavioral deficits also indicates a neuroprotective function of the lysosomal system, identifying lysosomal modulation as an avenue for disease-modifying therapies. From the in vitro and in vivo findings, unique lysosomal modulators represent a minimally invasive, pharmacologically controlled strategy against protein accumulation disorders

  1. MTHFSD and DDX58 are novel RNA-binding proteins abnormally regulated in amyotrophic lateral sclerosis.

    PubMed

    MacNair, Laura; Xiao, Shangxi; Miletic, Denise; Ghani, Mahdi; Julien, Jean-Pierre; Keith, Julia; Zinman, Lorne; Rogaeva, Ekaterina; Robertson, Janice

    2016-01-01

    Tar DNA-binding protein 43 (TDP-43) is an RNA-binding protein normally localized to the nucleus of cells, where it elicits functions related to RNA metabolism such as transcriptional regulation and alternative splicing. In amyotrophic lateral sclerosis, TDP-43 is mislocalized from the nucleus to the cytoplasm of diseased motor neurons, forming ubiquitinated inclusions. Although mutations in the gene encoding TDP-43, TARDBP, are found in amyotrophic lateral sclerosis, these are rare. However, TDP-43 pathology is common to over 95% of amyotrophic lateral sclerosis cases, suggesting that abnormalities of TDP-43 play an active role in disease pathogenesis. It is our hypothesis that a loss of TDP-43 from the nucleus of affected motor neurons in amyotrophic lateral sclerosis will lead to changes in RNA processing and expression. Identifying these changes could uncover molecular pathways that underpin motor neuron degeneration. Here we have used translating ribosome affinity purification coupled with microarray analysis to identify the mRNAs being actively translated in motor neurons of mutant TDP-43(A315T) mice compared to age-matched non-transgenic littermates. No significant changes were found at 5 months (presymptomatic) of age, but at 10 months (symptomatic) the translational profile revealed significant changes in genes involved in RNA metabolic process, immune response and cell cycle regulation. Of 28 differentially expressed genes, seven had a ≥ 2-fold change; four were validated by immunofluorescence labelling of motor neurons in TDP-43(A315T) mice, and two of these were confirmed by immunohistochemistry in amyotrophic lateral sclerosis cases. Both of these identified genes, DDX58 and MTHFSD, are RNA-binding proteins, and we show that TDP-43 binds to their respective mRNAs and we identify MTHFSD as a novel component of stress granules. This discovery-based approach has for the first time revealed translational changes in motor neurons of a TDP-43 mouse model

  2. Poly(ADP-ribosyl)ation enhances H-RAS protein stability and causes abnormal cell cycle progression in human TK6 lymphoblastoid cells treated with hydroquinone.

    PubMed

    Liu, Linhua; Ling, Xiaoxuan; Tang, Huanwen; Chen, Jialong; Wen, Qiaosheng; Zou, Fei

    2015-08-05

    Hydroquinone (HQ), one of the most important benzene-derived metabolites, can induce aberrant cell cycle progression; however, the mechanism of this induction remains unclear. Poly(ADP-ribosyl)ation (PARylation), which is catalysed primarily by poly(ADP-ribose) polymerase-1 (PARP-1), participates in various biological processes, including cell cycle control. The results of the present study show an accumulation in G1 phase versus S phase of TK6 human lymphoblast cells treated with HQ for 48h compared with PBS-treated cells; after 72h of HQ treatment, the cells transitioned from G1 arrest to S phase arrest. We examined the expression of six genes related to the cell cycle or leukaemia to further explore the reason for this phenomenon. Among these genes, H-RAS was found to be associated with this phenomenon because its mRNA and protein expression decreased at 48h and increased at 72h. Experiments for PARP activity induction and inhibition revealed that the observed PARylation was positively associated with H-RAS expression. Moreover, in cells treated with HQ in conjunction with PARP-1 knockdown, expression of the H-RAS protein decreased and the number of cells in G1 phase increased. The degree of poly(ADP-ribosyl) modification of the H-RAS protein increased in cells treated with HQ for 72h, further supporting that changes in PARylation contributed to the rapid alteration of H-RAS protein expression, followed by abnormal progression of the cell cycle. Co-immunoprecipitation (co-IP) assays were employed to determine whether protein complexes were formed by PARP-1 and H-RAS proteins, and the direct interaction between these proteins indicated that PARylation regulated H-RAS expression. As detected by confocal microscopy, the H-RAS protein was found in the nucleus and cytoplasm. To our knowledge, this study is the first to reveal that H-RAS protein can be modified by PARylation.

  3. Accumulation of plant small heat-stress proteins in storage organs.

    PubMed

    Lubaretz, Olga; Zur Nieden, Uta

    2002-06-01

    Plant small heat-stress proteins (sHSPs) have been shown to be expressed not only after exposure to elevated temperatures, but also at particular developmental stages such as embryogenesis, microsporogenesis, and fruit maturation. This paper presents new data on the occurrence of sHSPs in vegetative tissues, their tissue-specific distribution, and cellular localization. We have found sHSPs in 1-year-old twigs of Acer platanoides L. and Sambucus nigra L. and in the liana Aristolochia macrophylla Lamk. exclusively in the winter months. In tendrils of Aristolochia, sHSPs were localized in vascular cambium cells. After budding, in spring, these proteins were no longer present. Furthermore, accumulation of sHSPs was demonstrated in tubers and bulbs of Allium cepa L., Amaryllis ( Hippeastrum hybridum hort.), Crocus albiflorus L., Hyacinthus orientalis L., Narcissus pseudonarcissus L., Tulipa gesneriana L., and Solanum tuberosum L. (potato). In potato tubers and bulb scales of Narcissus the stress proteins were localized in the central vacuoles of storage parenchyma cells. In order to obtain more information on a possible functional correlation between storage proteins and sHSPs, the accumulation of both types of protein in tobacco seeds during seed ripening and germination was monitored. The expression of sHSPs and globulins started simultaneously at about the 17th day after anthesis. During seed germination the sHSPs disappeared in parallel with the storage proteins. Furthermore, in embryos of transgenic tobacco plants, which do not contain any protein bodies or storage proteins, no sHSPs were found. Thus, the occurrence of sHSPs in perennial plant storage organs seems to be associated with the presence of storage proteins.

  4. Influence of host chloroplast proteins on Tobacco mosaic virus accumulation and intercellular movement.

    PubMed

    Bhat, Sumana; Folimonova, Svetlana Y; Cole, Anthony B; Ballard, Kimberly D; Lei, Zhentian; Watson, Bonnie S; Sumner, Lloyd W; Nelson, Richard S

    2013-01-01

    Tobacco mosaic virus (TMV) forms dense cytoplasmic bodies containing replication-associated proteins (virus replication complexes [VRCs]) upon infection. To identify host proteins that interact with individual viral components of VRCs or VRCs in toto, we isolated viral replicase- and VRC-enriched fractions from TMV-infected Nicotiana tabacum plants. Two host proteins in enriched fractions, ATP-synthase γ-subunit (AtpC) and Rubisco activase (RCA) were identified by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry or liquid chromatography-tandem mass spectrometry. Through pull-down analysis, RCA bound predominantly to the region between the methyltransferase and helicase domains of the TMV replicase. Tobamovirus, but not Cucumber mosaic virus or Potato virus X, infection of N. tabacum plants resulted in 50% reductions in Rca and AtpC messenger RNA levels. To investigate the role of these host proteins in TMV accumulation and plant defense, we used a Tobacco rattle virus vector to silence these genes in Nicotiana benthamiana plants prior to challenge with TMV expressing green fluorescent protein. TMV-induced fluorescent lesions on Rca- or AtpC-silenced leaves were, respectively, similar or twice the size of those on leaves expressing these genes. Silencing Rca and AtpC did not influence the spread of Tomato bushy stunt virus and Potato virus X. In AtpC- and Rca-silenced leaves TMV accumulation and pathogenicity were greatly enhanced, suggesting a role of both host-encoded proteins in a defense response against TMV. In addition, silencing these host genes altered the phenotype of the TMV infection foci and VRCs, yielding foci with concentric fluorescent rings and dramatically more but smaller VRCs. The concentric rings occurred through renewed virus accumulation internal to the infection front.

  5. Paired helical filaments from Alzheimer disease brain induce intracellular accumulation of Tau protein in aggresomes.

    PubMed

    Santa-Maria, Ismael; Varghese, Merina; Ksiezak-Reding, Hanna; Dzhun, Anastasiya; Wang, Jun; Pasinetti, Giulio M

    2012-06-08

    Abnormal folding of tau protein leads to the generation of paired helical filaments (PHFs) and neurofibrillary tangles, a key neuropathological feature in Alzheimer disease and tauopathies. A specific anatomical pattern of pathological changes developing in the brain suggests that once tau pathology is initiated it propagates between neighboring neuronal cells, possibly spreading along the axonal network. We studied whether PHFs released from degenerating neurons could be taken up by surrounding cells and promote spreading of tau pathology. Neuronal and non-neuronal cells overexpressing green fluorescent protein-tagged tau (GFP-Tau) were treated with isolated fractions of human Alzheimer disease-derived PHFs for 24 h. We found that cells internalized PHFs through an endocytic mechanism and developed intracellular GFP-Tau aggregates with attributes of aggresomes. This was particularly evident by the perinuclear localization of aggregates and redistribution of the vimentin intermediate filament network and retrograde motor protein dynein. Furthermore, the content of Sarkosyl-insoluble tau, a measure of abnormal tau aggregation, increased 3-fold in PHF-treated cells. An exosome-related mechanism did not appear to be involved in the release of GFP-Tau from untreated cells. The evidence that cells can internalize PHFs, leading to formation of aggresome-like bodies, opens new therapeutic avenues to prevent propagation and spreading of tau pathology.

  6. Paired Helical Filaments from Alzheimer Disease Brain Induce Intracellular Accumulation of Tau Protein in Aggresomes*

    PubMed Central

    Santa-Maria, Ismael; Varghese, Merina; Ksiȩżak-Reding, Hanna; Dzhun, Anastasiya; Wang, Jun; Pasinetti, Giulio M.

    2012-01-01

    Abnormal folding of tau protein leads to the generation of paired helical filaments (PHFs) and neurofibrillary tangles, a key neuropathological feature in Alzheimer disease and tauopathies. A specific anatomical pattern of pathological changes developing in the brain suggests that once tau pathology is initiated it propagates between neighboring neuronal cells, possibly spreading along the axonal network. We studied whether PHFs released from degenerating neurons could be taken up by surrounding cells and promote spreading of tau pathology. Neuronal and non-neuronal cells overexpressing green fluorescent protein-tagged tau (GFP-Tau) were treated with isolated fractions of human Alzheimer disease-derived PHFs for 24 h. We found that cells internalized PHFs through an endocytic mechanism and developed intracellular GFP-Tau aggregates with attributes of aggresomes. This was particularly evident by the perinuclear localization of aggregates and redistribution of the vimentin intermediate filament network and retrograde motor protein dynein. Furthermore, the content of Sarkosyl-insoluble tau, a measure of abnormal tau aggregation, increased 3-fold in PHF-treated cells. An exosome-related mechanism did not appear to be involved in the release of GFP-Tau from untreated cells. The evidence that cells can internalize PHFs, leading to formation of aggresome-like bodies, opens new therapeutic avenues to prevent propagation and spreading of tau pathology. PMID:22496370

  7. The Cytoplasmic Zinc Finger Protein ZPR1 Accumulates in the Nucleolus of Proliferating Cells

    PubMed Central

    Galcheva-Gargova, Zoya; Gangwani, Laxman; Konstantinov, Konstantin N.; Mikrut, Monique; Theroux, Steven J.; Enoch, Tamar; Davis, Roger J.

    1998-01-01

    The zinc finger protein ZPR1 translocates from the cytoplasm to the nucleus after treatment of cells with mitogens. The function of nuclear ZPR1 has not been defined. Here we demonstrate that ZPR1 accumulates in the nucleolus of proliferating cells. The role of ZPR1 was examined using a gene disruption strategy. Cells lacking ZPR1 are not viable. Biochemical analysis demonstrated that the loss of ZPR1 caused disruption of nucleolar function, including preribosomal RNA expression. These data establish ZPR1 as an essential protein that is required for normal nucleolar function in proliferating cells. PMID:9763455

  8. Mitochondrial iron accumulation exacerbates hepatic toxicity caused by hepatitis C virus core protein

    SciTech Connect

    Sekine, Shuichi; Ito, Konomi; Watanabe, Haruna; Nakano, Takafumi; Moriya, Kyoji; Shintani, Yoshizumi; Fujie, Hajime; Tsutsumi, Takeya; Miyoshi, Hideyuki; Fujinaga, Hidetake; Shinzawa, Seiko; Koike, Kazuhiko; Horie, Toshiharu

    2015-02-01

    Patients with long-lasting hepatitis C virus (HCV) infection are at major risk of hepatocellular carcinoma (HCC). Iron accumulation in the livers of these patients is thought to exacerbate conditions of oxidative stress. Transgenic mice that express the HCV core protein develop HCC after the steatosis stage and produce an excess of hepatic reactive oxygen species (ROS). The overproduction of ROS in the liver is the net result of HCV core protein-induced dysfunction of the mitochondrial respiratory chain. This study examined the impact of ferric nitrilacetic acid (Fe-NTA)-mediated iron overload on mitochondrial damage and ROS production in HCV core protein-expressing HepG2 (human HCC) cells (Hep39b cells). A decrease in mitochondrial membrane potential and ROS production were observed following Fe-NTA treatment. After continuous exposure to Fe-NTA for six days, cell toxicity was observed in Hep39b cells, but not in mock (vector-transfected) HepG2 cells. Moreover, mitochondrial iron ({sup 59}Fe) uptake was increased in the livers of HCV core protein-expressing transgenic mice. This increase in mitochondrial iron uptake was inhibited by Ru360, a mitochondrial Ca{sup 2+} uniporter inhibitor. Furthermore, the Fe-NTA-induced augmentation of mitochondrial dysfunction, ROS production, and cell toxicity were also inhibited by Ru360 in Hep39b cells. Taken together, these results indicate that Ca{sup 2+} uniporter-mediated mitochondrial accumulation of iron exacerbates hepatocyte toxicity caused by the HCV core protein. - Highlights: • Iron accumulation in the livers of patients with hepatitis C virus (HCV) infection is thought to exacerbate oxidative stress. • The impact of iron overload on mitochondrial damage and ROS production in HCV core protein-expressing cells were examined. • Mitochondrial iron uptake was increased in the livers of HCV core protein-expressing transgenic mice. • Ca{sup 2+} uniporter-mediated mitochondrial accumulation of iron exacerbates

  9. Mutational analysis of PVX TGBp3 links subcellular accumulation and protein turnover

    SciTech Connect

    Ju, H.-J.; Ye, C.-M.; Verchot-Lubicz, Jeanmarie

    2008-05-25

    Potato virus X (PVX) TGBp3 is required for virus cell-to-cell transport, has an N-terminal transmembrane domain, and a C-terminal cytosolic domain. In the absence of virus infection TGBp3:GFP is seen in the cortical and perinuclear ER. In PVX infected cells the TGBp3:GFP fusion is also seen in the nucleoplasm indicating that events during PVX infection trigger entry into the nucleus. Mutational analysis failed to identify a nuclear targeting domain. Mutations inhibiting TGBp3 association with the ER and inhibiting virus movement did not block TGBp3:GFP in the nucleoplasm. A mutation disrupting the N-terminal transmembrane domain of TGBp3 caused the fusion to accumulate in the nucleus indicating that nuclear import is regulated by ER interactions. Tunicamycin, an ER-stress inducing chemical, caused lower levels of GFP and TGBp3:GFP to accumulate in virus infected protoplasts. MG115 and MG132 were used to demonstrate that wild-type and mutant TGBp3:GFP fusions were degraded by the 26S proteasome. These observations are consistent with an ER-associated protein degradation (ERAD) pathway suggesting that PVX TGBp3, similar to aberrant ER proteins, is translocate to the cytoplasm for degradation. Nuclear accumulation of mutant and wild-type TGBp3:GFP is independent of other PVX proteins and may be another feature of an ERAD pathway.

  10. Clinical significance of the appearance of abnormal protein band in patients with multiple myeloma.

    PubMed

    Jo, Jae-Cheol; Yoon, Dok Hyun; Kim, Shin; Lee, Kyoungmin; Kang, Eun Hee; Jang, Seongsoo; Park, Chan-Jeoung; Chi, Hyun-Sook; Huh, Jooryung; Park, Chan-Sik; Suh, Cheolwon

    2014-03-01

    Multiple myeloma (MM) is characterized by clonal expansion of malignant bone marrow cells producing a unique monoclonal immunoglobulin. The appearance of abnormal protein band (APB) in MM has been reported during follow-up. We aimed to evaluate the clinical characteristics and outcomes of patients with APB in a single center cohort. A total of 377 consecutive MM patients were treated at the Asan Medical Center between January 2002 and December 2012. We compared clinical characteristics and survival outcome between those with and without APB. Of the 377 patients, 34 (9 %) experienced APB. They comprised 18.2 % (27/148) of patients treated with autologous stem cell transplantation (ASCT) and 3.1 % (7/229) of those not receiving ASCT. APB occurred after a median of 7.9 months (range, 2.2-95.7 months) from diagnosis. Immunoglobulin isotypes at diagnosis were as follows: IgG (n = 10), IgA (n = 8), IgD (n = 5), free κ (n = 4), and free λ (n = 7). Nine patients experienced a second APB. With a median follow-up of 54.1 months, the median overall survival (OS) has not been reached in patients with APB and was 38.3 months in patients without (P < 0.001). Multivariate analysis indicated that the development of APB was a significant favorable prognostic factor for OS (hazard ratio 0.21; 95 % confidence interval 0.08-0.52). Serum β₂-microglobulin, albumin, creatinine, and ASCT were also independent prognostic factors for OS. Further investigation is required to establish the mechanisms underlying APB in MM.

  11. Nuclear poly(A)-binding protein aggregates misplace a pre-mRNA outside of SC35 speckle causing its abnormal splicing

    PubMed Central

    Klein, Pierre; Oloko, Martine; Roth, Fanny; Montel, Valérie; Malerba, Alberto; Jarmin, Susan; Gidaro, Teresa; Popplewell, Linda; Perie, Sophie; Lacau St Guily, Jean; de la Grange, Pierre; Antoniou, Michael N.; Dickson, George; Butler-Browne, Gillian; Bastide, Bruno; Mouly, Vincent; Trollet, Capucine

    2016-01-01

    A short abnormal polyalanine expansion in the polyadenylate-binding protein nuclear-1 (PABPN1) protein causes oculopharyngeal muscular dystrophy (OPMD). Mutated PABPN1 proteins accumulate as insoluble intranuclear aggregates in muscles of OPMD patients. While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice have been established, the molecular mechanisms which trigger pathological defects in OPMD and the role of aggregates remain to be determined. Using exon array, for the first time we have identified several splicing defects in OPMD. In particular, we have demonstrated a defect in the splicing regulation of the muscle-specific Troponin T3 (TNNT3) mutually exclusive exons 16 and 17 in OPMD samples compared to controls. This splicing defect is directly linked to the SC35 (SRSF2) splicing factor and to the presence of nuclear aggregates. As reported here, PABPN1 aggregates are able to trap TNNT3 pre-mRNA, driving it outside nuclear speckles, leading to an altered SC35-mediated splicing. This results in a decreased calcium sensitivity of muscle fibers, which could in turn plays a role in muscle pathology. We thus report a novel mechanism of alternative splicing deregulation that may play a role in various other diseases with nuclear inclusions or foci containing an RNA binding protein. PMID:27507886

  12. Reproductive Aging Drives Protein Accumulation in the Uterus and Limits Lifespan in C. elegans

    PubMed Central

    Zimmerman, Stephanie M.; Hinkson, Izumi V.; Elias, Joshua E.; Kim, Stuart K.

    2015-01-01

    Aging in Caenorhabditis elegans is characterized by widespread physiological and molecular changes, but the mechanisms that determine the rate at which these changes occur are not well understood. In this study, we identify a novel link between reproductive aging and somatic aging in C. elegans. By measuring global age-related changes in the proteome, we identify a previously uncharacterized group of secreted proteins in the adult uterus that dramatically increase in abundance with age. This accumulation is blunted in animals with an extended reproductive period and accelerated in sterile animals lacking a germline. Uterine proteins are not removed in old post-reproductive animals or in young vulvaless worms, indicating that egg-laying is necessary for their rapid removal in wild-type young animals. Together, these results suggest that age-induced infertility contributes to extracellular protein accumulation in the uterus with age. Finally, we show that knocking down multiple age-increased proteins simultaneously extends lifespan. These results provide a mechanistic example of how the cessation of reproduction contributes to detrimental changes in the soma, and demonstrate how the timing of reproductive decline can influence the rate of aging. PMID:26656270

  13. Correlation between protein accumulation profiles and conventional toxicological findings using a model antiandrogenic compound, flutamide.

    PubMed

    Friry-Santini, Claire; Rouquié, David; Kennel, Philippe; Tinwell, Helen; Benahmed, Mohamed; Bars, Rémi

    2007-05-01

    In conventional rodent toxicity studies the characterization of the adverse effects of a chemical relies primarily on gravimetric, and histopathological data. The aim of this study was to evaluate if the use of two-dimensional gel electrophoresis could generate protein accumulation profiles, which were in accordance with conventional toxicological findings by investigating a model antiandrogen, flutamide (FM), whose toxic effects, as measured using standard approaches, are well characterized. Male Sprague-Dawley rats were orally exposed to FM (0, 6, 30, and 150 mg/kg/day) for 28 days. The expected inhibition of androgen-dependent tissue stimulation, increased luteinizing hormone and testosterone plasma levels, and Leydig cell hyperplasia were observed. Changes in testicular protein accumulation profiles were evaluated in rats exposed to 150 mg/kg/day FM. Several proteins involved in steroidogenesis (e.g., StAR, ApoE, Hmgcs1, Idi1), cell cycle, and cancer (e.g., Ddx1, Hspd1) were modulated by FM, and these data provided molecular evidence for the hormonal and testicular histopathology changes recorded. Changes in proteins associated with spermatogenesis were also recorded, and these are discussed within the context of the testicular phenotype observed following FM treatment (i.e., normal spermatogenesis but Leydig cell hyperplasia). Overall, our data indicate that the combination of conventional toxicology measurements with omic observations has the potential to improve our global understanding of the toxicity of a compound.

  14. ESCRTs regulate amyloid precursor protein sorting in multivesicular bodies and intracellular amyloid-β accumulation.

    PubMed

    Edgar, James R; Willén, Katarina; Gouras, Gunnar K; Futter, Clare E

    2015-07-15

    Intracellular amyloid-β (Aβ) accumulation is a key feature of early Alzheimer's disease and precedes the appearance of Aβ in extracellular plaques. Aβ is generated through proteolytic processing of amyloid precursor protein (APP), but the intracellular site of Aβ production is unclear. APP has been localized to multivesicular bodies (MVBs) where sorting of APP onto intraluminal vesicles (ILVs) could promote amyloidogenic processing, or reduce Aβ production or accumulation by sorting APP and processing products to lysosomes for degradation. Here, we show that APP localizes to the ILVs of a subset of MVBs that also traffic EGF receptor (EGFR), and that it is delivered to lysosomes for degradation. Depletion of the endosomal sorting complexes required for transport (ESCRT) components, Hrs (also known as Hgs) or Tsg101, inhibited targeting of APP to ILVs and the subsequent delivery to lysosomes, and led to increased intracellular Aβ accumulation. This was accompanied by dramatically decreased Aβ secretion. Thus, the early ESCRT machinery has a dual role in limiting intracellular Aβ accumulation through targeting of APP and processing products to the lysosome for degradation, and promoting Aβ secretion.

  15. mTORC1 Coordinates Protein Synthesis and Immunoproteasome Formation via PRAS40 to Prevent Accumulation of Protein Stress.

    PubMed

    Yun, Young Sung; Kim, Kwan Hyun; Tschida, Barbara; Sachs, Zohar; Noble-Orcutt, Klara E; Moriarity, Branden S; Ai, Teng; Ding, Rui; Williams, Jessica; Chen, Liqiang; Largaespada, David; Kim, Do-Hyung

    2016-02-18

    Reduction of translational fidelity often occurs in cells with high rates of protein synthesis, generating defective ribosomal products. If not removed, such aberrant proteins can be a major source of cellular stress causing human diseases. Here, we demonstrate that mTORC1 promotes the formation of immunoproteasomes for efficient turnover of defective proteins and cell survival. mTORC1 sequesters precursors of immunoproteasome β subunits via PRAS40. When activated, mTORC1 phosphorylates PRAS40 to enhance protein synthesis and simultaneously to facilitate the assembly of the β subunits for forming immunoproteasomes. Consequently, the PRAS40 phosphorylations play crucial roles in clearing aberrant proteins that accumulate due to mTORC1 activation. Mutations of RAS, PTEN, and TSC1, which cause mTORC1 hyperactivation, enhance immunoproteasome formation in cells and tissues. Those mutations increase cellular dependence on immunoproteasomes for stress response and survival. These results define a mechanism by which mTORC1 couples elevated protein synthesis with immunoproteasome biogenesis to protect cells against protein stress.

  16. Ice-binding proteins that accumulate on different ice crystal planes produce distinct thermal hysteresis dynamics

    PubMed Central

    Drori, Ran; Celik, Yeliz; Davies, Peter L.; Braslavsky, Ido

    2014-01-01

    Ice-binding proteins that aid the survival of freeze-avoiding, cold-adapted organisms by inhibiting the growth of endogenous ice crystals are called antifreeze proteins (AFPs). The binding of AFPs to ice causes a separation between the melting point and the freezing point of the ice crystal (thermal hysteresis, TH). TH produced by hyperactive AFPs is an order of magnitude higher than that produced by a typical fish AFP. The basis for this difference in activity remains unclear. Here, we have compared the time dependence of TH activity for both hyperactive and moderately active AFPs using a custom-made nanolitre osmometer and a novel microfluidics system. We found that the TH activities of hyperactive AFPs were time-dependent, and that the TH activity of a moderate AFP was almost insensitive to time. Fluorescence microscopy measurement revealed that despite their higher TH activity, hyperactive AFPs from two insects (moth and beetle) took far longer to accumulate on the ice surface than did a moderately active fish AFP. An ice-binding protein from a bacterium that functions as an ice adhesin rather than as an antifreeze had intermediate TH properties. Nevertheless, the accumulation of this ice adhesion protein and the two hyperactive AFPs on the basal plane of ice is distinct and extensive, but not detectable for moderately active AFPs. Basal ice plane binding is the distinguishing feature of antifreeze hyperactivity, which is not strictly needed in fish that require only approximately 1°C of TH. Here, we found a correlation between the accumulation kinetics of the hyperactive AFP at the basal plane and the time sensitivity of the measured TH. PMID:25008081

  17. Transgenic rice seeds accumulating recombinant hypoallergenic birch pollen allergen Bet v 1 generate giant protein bodies.

    PubMed

    Wang, Shuyi; Takahashi, Hideyuki; Kajiura, Hiroyuki; Kawakatsu, Taiji; Fujiyama, Kazuhito; Takaiwa, Fumio

    2013-06-01

    A versatile hypoallergenic allergen derivative against multiple allergens is an ideal tolerogen for allergen-specific immunotherapy. Such a tolerogen should exhibit high efficacy, without side effects, when administered at high doses and should be applicable to several allergens. Tree pollen chimera 7 (TPC7), a hypoallergenic Bet v 1 tolerogen against birch pollen allergy, was previously selected by DNA shuffling of 14 types of Fagales tree pollen allergens. In this study, transgenic rice seed accumulating TPC7 was generated as an oral vaccine against birch pollen allergy by expressing this protein as a secretory protein using the N-terminal signal peptide and the C-terminal KDEL tag under the control of an endosperm-specific glutelin promoter. The highest level of TPC7 accumulation was approximately 207 µg grain(-1). Recombinant TPC7 is a glycoprotein with high mannose-type N-glycan, but without β1,2-xylose or α1,3-fucose, suggesting that TPC7 is retained in the endoplasmic reticulum (ER). TPC7 is deposited as a novel, giant spherical ER-derived protein body, >20 µm in diameter, which is referred to as the TPC7 body. Removal of the KDEL retention signal or mutation of a cysteine residue resulted in an alteration of TPC7 body morphology, and deletion of the signal peptide prevented the accumulation of TPC7 in rice seeds. Therefore, the novel TPC7 bodies may have formed aggregates within the ER lumen, primarily due to the intrinsic physicochemical properties of the protein.

  18. Ice-binding proteins that accumulate on different ice crystal planes produce distinct thermal hysteresis dynamics.

    PubMed

    Drori, Ran; Celik, Yeliz; Davies, Peter L; Braslavsky, Ido

    2014-09-06

    Ice-binding proteins that aid the survival of freeze-avoiding, cold-adapted organisms by inhibiting the growth of endogenous ice crystals are called antifreeze proteins (AFPs). The binding of AFPs to ice causes a separation between the melting point and the freezing point of the ice crystal (thermal hysteresis, TH). TH produced by hyperactive AFPs is an order of magnitude higher than that produced by a typical fish AFP. The basis for this difference in activity remains unclear. Here, we have compared the time dependence of TH activity for both hyperactive and moderately active AFPs using a custom-made nanolitre osmometer and a novel microfluidics system. We found that the TH activities of hyperactive AFPs were time-dependent, and that the TH activity of a moderate AFP was almost insensitive to time. Fluorescence microscopy measurement revealed that despite their higher TH activity, hyperactive AFPs from two insects (moth and beetle) took far longer to accumulate on the ice surface than did a moderately active fish AFP. An ice-binding protein from a bacterium that functions as an ice adhesin rather than as an antifreeze had intermediate TH properties. Nevertheless, the accumulation of this ice adhesion protein and the two hyperactive AFPs on the basal plane of ice is distinct and extensive, but not detectable for moderately active AFPs. Basal ice plane binding is the distinguishing feature of antifreeze hyperactivity, which is not strictly needed in fish that require only approximately 1°C of TH. Here, we found a correlation between the accumulation kinetics of the hyperactive AFP at the basal plane and the time sensitivity of the measured TH.

  19. UCH-L1 induces podocyte hypertrophy in membranous nephropathy by protein accumulation.

    PubMed

    Lohmann, Frithjof; Sachs, Marlies; Meyer, Tobias N; Sievert, Henning; Lindenmeyer, Maja T; Wiech, Thorsten; Cohen, Clemens D; Balabanov, Stefan; Stahl, R A K; Meyer-Schwesinger, Catherine

    2014-07-01

    Podocytes are terminally differentiated cells of the glomerular filtration barrier that react with hypertrophy in the course of injury such as in membranous nephropathy (MGN). The neuronal deubiquitinase ubiquitin C-terminal hydrolase L1 (UCH-L1) is expressed and activated in podocytes of human and rodent MGN. UCH-L1 regulates the mono-ubiquitin pool and induces accumulation of poly-ubiquitinated proteins in affected podocytes. Here, we investigated the role of UCH-L1 in podocyte hypertrophy and in the homeostasis of the hypertrophy associated "model protein" p27(Kip1). A better understanding of the basic mechanisms leading to podocyte hypertrophy is crucial for the development of specific therapies in MGN. In human and rat MGN, hypertrophic podocytes exhibited a simultaneous up-regulation of UCH-L1 and of cytoplasmic p27(Kip1) content. Functionally, inhibition of UCH-L1 activity and knockdown or inhibition of UCH-L1 attenuated podocyte hypertrophy by decreasing the total protein content in isolated glomeruli and in cultured podocytes. In contrast, UCH-L1 levels and activity increased podocyte hypertrophy and total protein content in culture, specifically of cytoplasmic p27(Kip1). UCH-L1 enhanced cytoplasmic p27(Kip1) levels by nuclear export and decreased poly-ubiquitination and proteasomal degradation of p27(Kip1). In parallel, UCH-L1 increased podocyte turnover, migration and cytoskeletal rearrangement, which are associated with known oncogenic functions of cytoplasmic p27(Kip1) in cancer. We propose that UCH-L1 induces podocyte hypertrophy in MGN by increasing the total protein content through altered degradation and accumulation of proteins such as p27(Kip1) in the cytoplasm of podocytes. Modification of both UCH-L1 activity and levels could be a new therapeutic avenue to podocyte hypertrophy in MGN.

  20. Selective Estrogen Receptor Modulators Suppress Hif1α Protein Accumulation in Mouse Osteoclasts

    PubMed Central

    Iwasaki, Ryotaro; Kobayashi, Tami; Watanabe, Ryuichi; Oike, Takatsugu; Toyama, Yoshiaki; Matsumoto, Morio; Nakamura, Masaya; Kawana, Hiromasa; Nakagawa, Taneaki; Miyamoto, Takeshi

    2016-01-01

    Anti-bone resorptive drugs such as bisphosphonates, the anti-RANKL antibody (denosumab), or selective estrogen receptor modulators (SERMs) have been developed to treat osteoporosis. Mechanisms underlying activity of bisphosphonates or denosumab in this context are understood, while it is less clear how SERMs like tamoxifen, raloxifene, or bazedoxifene inhibit bone resorption. Recently, accumulation of hypoxia inducible factor 1 alpha (Hif1α) in osteoclasts was shown to be suppressed by estrogen in normal cells. In addition, osteoclast activation and decreased bone mass seen in estrogen-deficient conditions was found to require Hif1α. Here, we used western blot analysis of cultured osteoclast precursor cells to show that tamoxifen, raloxifene, or bazedoxifene all suppress Hif1α protein accumulation. The effects of each SERM on osteoclast differentiation differed in vitro. Our results suggest that interventions such as the SERMs evaluated here could be useful to inhibit Hif1α and osteoclast activity under estrogen-deficient conditions. PMID:27802325

  1. Proteomic profiling of maize opaque endosperm mutants reveals selective accumulation of lysine-enriched proteins

    PubMed Central

    Morton, Kyla J.; Jia, Shangang; Zhang, Chi; Holding, David R.

    2016-01-01

    Reduced prolamin (zein) accumulation and defective endoplasmic reticulum (ER) body formation occurs in maize opaque endosperm mutants opaque2 (o2), floury2 (fl2), defective endosperm*B30 (DeB30), and Mucronate (Mc), whereas other opaque mutants such as opaque1 (o1) and floury1 (fl1) are normal in these regards. This suggests that other factors contribute to kernel texture. A liquid chromatography approach coupled with tandem mass spectrometry (LC-MS/MS) proteomics was used to compare non-zein proteins of nearly isogenic opaque endosperm mutants. In total, 2762 proteins were identified that were enriched for biological processes such as protein transport and folding, amino acid biosynthesis, and proteolysis. Principal component analysis and pathway enrichment suggested that the mutants partitioned into three groups: (i) Mc, DeB30, fl2 and o2; (ii) o1; and (iii) fl1. Indicator species analysis revealed mutant-specific proteins, and highlighted ER secretory pathway components that were enriched in selected groups of mutants. The most significantly changed proteins were related to stress or defense and zein partitioning into the soluble fraction for Mc, DeB30, o1, and fl1 specifically. In silico dissection of the most significantly changed proteins revealed novel qualitative changes in lysine abundance contributing to the overall lysine increase and the nutritional rebalancing of the o2 and fl2 endosperm. PMID:26712829

  2. Two Outer Membrane Proteins Contribute to Caulobacter crescentus Cellular Fitness by Preventing Intracellular S-Layer Protein Accumulation

    SciTech Connect

    Overton, K. Wesley; Park, Dan M.; Yung, Mimi C.; Dohnalkova, Alice C.; Smit, John; Jiao, Yongqin

    2016-09-23

    Surface layers, or S-layers, are two-dimensional protein arrays that form the outermost layer of many bacteria and archaea. They serve several functions, including physical protection of the cell from environmental threats. The high abundance of S-layer proteins necessitates a highly efficient export mechanism to transport the S-layer protein from the cytoplasm to the cell exterior.Caulobacter crescentusis unique in that it has two homologous, seemingly redundant outer membrane proteins, RsaFaand RsaFb, which together with other components form a type I protein translocation pathway for S-layer export. These proteins have homology toEscherichia coliTolC, the outer membrane channel of multidrug efflux pumps. Here we provide evidence that, unlike TolC, RsaFaand RsaFbare not involved in either the maintenance of membrane stability or the active export of antimicrobial compounds. Rather, RsaFaand RsaFbare required to prevent intracellular accumulation and aggregation of the S-layer protein RsaA; deletion of RsaFaand RsaFbled to a general growth defect and lowered cellular fitness. Using Western blotting, transmission electron microscopy, and transcriptome sequencing (RNA-seq), we show that loss of both RsaFaand RsaFbled to accumulation of insoluble RsaA in the cytoplasm, which in turn caused upregulation of a number of genes involved in protein misfolding and degradation pathways. These findings provide new insight into the requirement for RsaFaand RsaFbin cellular fitness and tolerance to antimicrobial agents and further our understanding of the S-layer export mechanism on both the transcriptional and translational levels inC. crescentus.

  3. Two Outer Membrane Proteins Contribute to Caulobacter crescentus Cellular Fitness by Preventing Intracellular S-Layer Protein Accumulation

    SciTech Connect

    Overton, K. Wesley; Park, Dan M.; Yung, Mimi C.; Dohnalkova, Alice C.; Smit, John; Jiao, Yongqin; Parales, R. E.

    2016-09-23

    ABSTRACT

    Surface layers, or S-layers, are two-dimensional protein arrays that form the outermost layer of many bacteria and archaea. They serve several functions, including physical protection of the cell from environmental threats. The high abundance of S-layer proteins necessitates a highly efficient export mechanism to transport the S-layer protein from the cytoplasm to the cell exterior.Caulobacter crescentusis unique in that it has two homologous, seemingly redundant outer membrane proteins, RsaFaand RsaFb, which together with other components form a type I protein translocation pathway for S-layer export. These proteins have homology toEscherichia coliTolC, the outer membrane channel of multidrug efflux pumps. Here we provide evidence that, unlike TolC, RsaFaand RsaFbare not involved in either the maintenance of membrane stability or the active export of antimicrobial compounds. Rather, RsaFaand RsaFbare required to prevent intracellular accumulation and aggregation of the S-layer protein RsaA; deletion of RsaFaand RsaFbled to a general growth defect and lowered cellular fitness. Using Western blotting, transmission electron microscopy, and transcriptome sequencing (RNA-seq), we show that loss of both RsaFaand RsaFbled to accumulation of insoluble RsaA in the cytoplasm, which in turn caused upregulation of a number of genes involved in protein misfolding and degradation pathways. These findings provide new insight into the requirement for RsaFaand RsaFbin cellular fitness and tolerance to antimicrobial agents and further our understanding of the S-layer export mechanism on both the transcriptional and translational levels inC. crescentus

  4. The abnormal phosphorylation of tau protein at Ser-202 in Alzheimer disease recapitulates phosphorylation during development.

    PubMed

    Goedert, M; Jakes, R; Crowther, R A; Six, J; Lübke, U; Vandermeeren, M; Cras, P; Trojanowski, J Q; Lee, V M

    1993-06-01

    Tau is a neuronal phosphoprotein whose expression is developmentally regulated. A single tau isoform is expressed in fetal human brain but six isoforms are expressed in adult brain, with the fetal isoform corresponding to the shortest of the adult isoforms. Phosphorylation of tau is also developmentally regulated, as fetal tau is phosphorylated at more sites than adult tau. In Alzheimer disease, the six adult tau isoforms become abnormally phosphorylated and form the paired helical filament, the major fibrous component of the characteristic neurofibrillary lesions. We show here that Ser-202 (in the numbering of the longest human brain tau isoform) is a phosphorylation site that distinguishes fetal from adult tau and we identify it as one of the abnormal phosphorylation sites in Alzheimer disease. The abnormal phosphorylation of tau at Ser-202 in Alzheimer disease thus recapitulates normal phosphorylation during development.

  5. [Ocular diseases caused by accumulation of proteins with post-translational modifications].

    PubMed

    Kaji, Yuichi

    2009-03-01

    Long-term duration of lifestyle-related diseases including diabetes induces various ocular diseases. For this reason, the development of lifestyle-related ocular diseases is closely related to the aging process. In the present study, we tried to reveal the molecular mechanism of lifestyle-related ocular diseases, especially diabetic complications of the eyes, in relation to aging. To unify the molecular mechanisms of diabetic complications and aging changes of the eyes, we focused on two kinds of nonenzymatic post-translational modification products: advanced glycation end products (AGEs) and D-amino acids. We found that the accumulation of proteins rich in AGEs and D-amino acids plays a central role in the development of both diabetic complications and such changes of the eyes as diabetic retinopathy, diabetic keratopathy, pinguecula, spheroidal degeneration of the cornea, and drusen. In addition, decreased function in AGE-modified and D-amino acid-containing proteins is a factor in the development of diabetic complications and aging changes in eyes. In this way, posttranslational changes in molecules and amino acids are important contributing factors in the development of diabetic complications and aging changes in eyes. In conclusion, accumulation of AGE-modified and D-amino acid-containing proteins is the molecular mechanism of both diabetic complications and the aging changes in eyes.

  6. Uniform accumulation of recombinant miraculin protein in transgenic tomato fruit using a fruit-ripening-specific E8 promoter.

    PubMed

    Hirai, Tadayoshi; Kim, You-Wang; Kato, Kazuhisa; Hiwasa-Tanase, Kyoko; Ezura, Hiroshi

    2011-12-01

    The E8 promoter, a tomato fruit-ripening-specific promoter, and the CaMV 35S promoter, a constitutive promoter, were used to express the miraculin gene encoding the taste-modifying protein in tomato. The accumulation of miraculin protein and mRNA was compared among transgenic tomatoes expressing the miraculin gene driven by these promoters. Recombinant miraculin protein predominantly accumulated in transgenic tomato lines using the E8 promoter (E8-MIR) only at the red fruit stage. The accumulations were almost uniform among all fruit tissues. When the 35S promoter (35S-MIR) was used, miraculin accumulation in the exocarp was much higher than in other tissues, indicating that the miraculin accumulation pattern can be regulated by using different types of promoters. We also discuss the potential of the E8-MIR lines for practical use.

  7. GUN1 Controls Accumulation of the Plastid Ribosomal Protein S1 at the Protein Level and Interacts with Proteins Involved in Plastid Protein Homeostasis1

    PubMed Central

    Pesaresi, Paolo; Rossi, Fabio; Guljamow, Arthur; Sommer, Frederik; Mühlhaus, Timo; Schroda, Michael; Masiero, Simona; Rothbart, Maxi; Hedtke, Boris

    2016-01-01

    Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the plant cell. Such intracellular responses are associated with signals that originate in chloroplasts and convey information on their physiological status to the nucleus, which leads to large-scale changes in gene expression (retrograde signaling). A screen designed to identify components of retrograde signaling resulted in the discovery of the so-called genomes uncoupled (gun) mutants. Genetic evidence suggests that the chloroplast protein GUN1 integrates signals derived from perturbations in plastid redox state, plastid gene expression, and tetrapyrrole biosynthesis (TPB) in Arabidopsis (Arabidopsis thaliana) seedlings, exerting biogenic control of chloroplast functions. However, the molecular mechanism by which GUN1 integrates retrograde signaling in the chloroplast is unclear. Here we show that GUN1 also operates in adult plants, contributing to operational control of chloroplasts. The gun1 mutation genetically interacts with mutations of genes for the chloroplast ribosomal proteins S1 (PRPS1) and L11. Analysis of gun1 prps1 lines indicates that GUN1 controls PRPS1 accumulation at the protein level. The GUN1 protein physically interacts with proteins involved in chloroplast protein homeostasis based on coimmunoprecipitation experiments. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments suggest that GUN1 might transiently interact with several TPB enzymes, including Mg-chelatase subunit D (CHLD) and two other TPB enzymes known to activate retrograde signaling. Moreover, the association of PRPS1 and CHLD with protein complexes is modulated by GUN1. These findings allow us to speculate that retrograde signaling might involve GUN1-dependent formation of protein complexes. PMID:26823545

  8. GUN1 Controls Accumulation of the Plastid Ribosomal Protein S1 at the Protein Level and Interacts with Proteins Involved in Plastid Protein Homeostasis.

    PubMed

    Tadini, Luca; Pesaresi, Paolo; Kleine, Tatjana; Rossi, Fabio; Guljamow, Arthur; Sommer, Frederik; Mühlhaus, Timo; Schroda, Michael; Masiero, Simona; Pribil, Mathias; Rothbart, Maxi; Hedtke, Boris; Grimm, Bernhard; Leister, Dario

    2016-03-01

    Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the plant cell. Such intracellular responses are associated with signals that originate in chloroplasts and convey information on their physiological status to the nucleus, which leads to large-scale changes in gene expression (retrograde signaling). A screen designed to identify components of retrograde signaling resulted in the discovery of the so-called genomes uncoupled (gun) mutants. Genetic evidence suggests that the chloroplast protein GUN1 integrates signals derived from perturbations in plastid redox state, plastid gene expression, and tetrapyrrole biosynthesis (TPB) in Arabidopsis (Arabidopsis thaliana) seedlings, exerting biogenic control of chloroplast functions. However, the molecular mechanism by which GUN1 integrates retrograde signaling in the chloroplast is unclear. Here we show that GUN1 also operates in adult plants, contributing to operational control of chloroplasts. The gun1 mutation genetically interacts with mutations of genes for the chloroplast ribosomal proteins S1 (PRPS1) and L11. Analysis of gun1 prps1 lines indicates that GUN1 controls PRPS1 accumulation at the protein level. The GUN1 protein physically interacts with proteins involved in chloroplast protein homeostasis based on coimmunoprecipitation experiments. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments suggest that GUN1 might transiently interact with several TPB enzymes, including Mg-chelatase subunit D (CHLD) and two other TPB enzymes known to activate retrograde signaling. Moreover, the association of PRPS1 and CHLD with protein complexes is modulated by GUN1. These findings allow us to speculate that retrograde signaling might involve GUN1-dependent formation of protein complexes.

  9. Proinflammatory S100 proteins regulate the accumulation of myeloid-derived suppressor cells.

    PubMed

    Sinha, Pratima; Okoro, Chinonyerem; Foell, Dirk; Freeze, Hudson H; Ostrand-Rosenberg, Suzanne; Srikrishna, Geetha

    2008-10-01

    Chronic inflammation is a complex process that promotes carcinogenesis and tumor progression; however, the mechanisms by which specific inflammatory mediators contribute to tumor growth remain unclear. We and others recently demonstrated that the inflammatory mediators IL-1beta, IL-6, and PGE(2) induce accumulation of myeloid-derived suppressor cells (MDSC) in tumor-bearing individuals. MDSC impair tumor immunity and thereby facilitate carcinogenesis and tumor progression by inhibiting T and NK cell activation, and by polarizing immunity toward a tumor-promoting type 2 phenotype. We now show that this population of immature myeloid cells induced by a given tumor share a common phenotype regardless of their in vivo location (bone marrow, spleen, blood, or tumor site), and that Gr1(high)CD11b(high)F4/80(-)CD80(+)IL4Ralpha(+/-)Arginase(+) MDSC are induced by the proinflammatory proteins S100A8/A9. S100A8/A9 proteins bind to carboxylated N-glycans expressed on the receptor for advanced glycation end-products and other cell surface glycoprotein receptors on MDSC, signal through the NF-kappaB pathway, and promote MDSC migration. MDSC also synthesize and secrete S100A8/A9 proteins that accumulate in the serum of tumor-bearing mice, and in vivo blocking of S100A8/A9 binding to MDSC using an anti-carboxylated glycan Ab reduces MDSC levels in blood and secondary lymphoid organs in mice with metastatic disease. Therefore, the S100 family of inflammatory mediators serves as an autocrine feedback loop that sustains accumulation of MDSC. Since S100A8/A9 activation of MDSC is through the NF-kappaB signaling pathway, drugs that target this pathway may reduce MDSC levels and be useful therapeutic agents in conjunction with active immunotherapy in cancer patients.

  10. An early nodulin-like protein accumulates in the sieve element plasma membrane of Arabidopsis.

    PubMed

    Khan, Junaid A; Wang, Qi; Sjölund, Richard D; Schulz, Alexander; Thompson, Gary A

    2007-04-01

    Membrane proteins within the sieve element-companion cell complex have essential roles in the physiological functioning of the phloem. The monoclonal antibody line RS6, selected from hybridomas raised against sieve elements isolated from California shield leaf (Streptanthus tortuosus; Brassicaceae) tissue cultures, recognizes an antigen in the Arabidopsis (Arabidopsis thaliana) ecotype Columbia that is associated specifically with the plasma membrane of sieve elements, but not companion cells, and accumulates at the earliest stages of sieve element differentiation. The identity of the RS6 antigen was revealed by reverse transcription-PCR of Arabidopsis leaf RNA using degenerate primers to be an early nodulin (ENOD)-like protein that is encoded by the expressed gene At3g20570. Arabidopsis ENOD-like proteins are encoded by a multigene family composed of several types of structurally related phytocyanins that have a similar overall domain structure of an amino-terminal signal peptide, plastocyanin-like copper-binding domain, proline/serine-rich domain, and carboxy-terminal hydrophobic domain. The amino- and carboxy-terminal domains of the 21.5-kD sieve element-specific ENOD are posttranslationally cleaved from the precursor protein, resulting in a mature peptide of approximately 15 kD that is attached to the sieve element plasma membrane via a carboxy-terminal glycosylphosphatidylinositol membrane anchor. Many of the Arabidopsis ENOD-like proteins accumulate in gametophytic tissues, whereas in both floral and vegetative tissues, the sieve element-specific ENOD is expressed only within the phloem. Members of the ENOD subfamily of the cupredoxin superfamily do not appear to bind copper and have unknown functions. Phenotypic analysis of homozygous T-DNA insertion mutants for the gene At3g20570 shows minimal alteration in vegetative growth but a significant reduction in the overall reproductive potential.

  11. Virus Induction of Heat Shock Protein 70 Reflects a General Response to Protein Accumulation in the Plant Cytosol1

    PubMed Central

    Aparicio, Frederic; Thomas, Carole L.; Lederer, Carsten; Niu, Yan; Wang, Daowen; Maule, Andrew J.

    2005-01-01

    Different cytoplasmically replicating RNA viruses were shown to induce a specific subset of heat-inducible heat shock protein 70 (HSP70) genes in Arabidopsis (Arabidopsis thaliana). To identify the inducing principle, a promoter∷reporter system was developed for the facile analysis of differentially responding Arabidopsis HSP70 genes, by infiltration into Nicotiana benthamiana leaves. Through transient expression of individual viral cistrons or through deletion analysis of a viral replicon, we were unable to identify a unique inducer of HSP70. However, there was a positive correlation between the translatability of the test construct and the differential induction of HSP70. Since these data implied a lack of specificity in the induction process, we also expressed a random series of cytosolically targeted Arabidopsis genes and showed that these also differentially induced HSP70. Through a comparison of different promoter∷reporter constructs and through measurements of the steady-state levels of the individual proteins, it appeared that the HSP70 response reflected the ability of the cytosol to sense individual properties of particular proteins when expressed at high levels. This phenomenon is reminiscent of the unfolded protein response observed when the induced accumulation of proteins in the endoplasmic reticulum also induces a specific suite of chaperones. PMID:15805473

  12. Accumulation of the inner nuclear envelope protein Sun1 is pathogenic in progeric and dystrophic laminopathies.

    PubMed

    Chen, Chia-Yen; Chi, Ya-Hui; Mutalif, Rafidah Abdul; Starost, Matthew F; Myers, Timothy G; Anderson, Stasia A; Stewart, Colin L; Jeang, Kuan-Teh

    2012-04-27

    Human LMNA gene mutations result in laminopathies that include Emery-Dreifuss muscular dystrophy (AD-EDMD) and Hutchinson-Gilford progeria, the premature aging syndrome (HGPS). The Lmna null (Lmna(-/-)) and progeroid LmnaΔ9 mutant mice are models for AD-EDMD and HGPS, respectively. Both animals develop severe tissue pathologies with abbreviated life spans. Like HGPS cells, Lmna(-/-) and LmnaΔ9 fibroblasts have typically misshapen nuclei. Unexpectedly, Lmna(-/-) or LmnaΔ9 mice that are also deficient for the inner nuclear membrane protein Sun1 show markedly reduced tissue pathologies and enhanced longevity. Concordantly, reduction of SUN1 overaccumulation in LMNA mutant fibroblasts and in cells derived from HGPS patients corrected nuclear defects and cellular senescence. Collectively, these findings implicate Sun1 protein accumulation as a common pathogenic event in Lmna(-/-), LmnaΔ9, and HGPS disorders.

  13. Evaluation of heat shock protein (HSP-60) induction on accumulation of carbohydrate in Isochrysis galbana

    SciTech Connect

    Olsen, H.; Wolfe, M.; Tell, J.; Tjeerdema, R.

    1995-12-31

    Primary levels of the marine food chain may play an important role in the fate of petroleum hydrocarbons in both chemically dispersed and un-dispersed oil spills. HSP-60 proteins, members of the chaperonin family of stress proteins, are induced in response to a wide variety of environmental agents, including UV light, heavy metals, and xenobiotics. Increased production and storage of carbohydrate in I. galbana has been associated with aging and stress. Thus, HSP-60 and carbohydrate storage were selected as sublethal endpoints of exposure to the primary producer, I. galbana, a golden brown, unicellular algae, and a significant component of the marine phytoplankton community. The authors have found that I. galbana cultures exposed to water-accommodated fractions (WAF) of Prudhoe Bay Crude Oil (PBCO), and PBCO/dispersant preparations efficiently induce HSP-60. Studies indicated that WAF produced a dose-related response in I. galbana, which increased as a function of time. Dispersant alone showed the greatest induction, while combined WAF-dispersant showed less induction, suggesting a possible competition between crude oil and algae for dispersant interaction. In addition, they have demonstrated that I. galbana accumulates carbohydrates in response to exposure to WAF and PBCO/dispersant preparations and therefore represents another index of stress in this organism. They were interested in determining if induction of stress proteins and HSP60 in particular represented an adaptive-mechanism, allowing this algae to better cope with exposure to petroleum hydrocarbons released in the marine environment during an oil spill. In an effort to determine if stress protein induction serves as a protective adaptive response to exposure to petroleum hydrocarbons they examined the effect of heat shock induction on the accumulation of carbohydrates by these organisms in response to exposure to WAF and dispersed oil preparations.

  14. Differential accumulation of transcripts encoding protein kinase homologs in greening pea seedlings.

    PubMed Central

    Lin, X; Feng, X H; Watson, J C

    1991-01-01

    Degenerate oligonucleotides, corresponding to conserved regions within the catalytic domain of known protein-serine/threonine kinases, were used as primers for the polymerase chain reaction to amplify cDNA synthesized from poly(A)+ RNA purified from the apical buds of 7-day-old pea seedlings. Five partial cDNAs were obtained and designated PsPK1 through PsPK5 (for Pisum sativum protein kinase) in order of decreasing length. The deduced amino acid sequences show that each member of the PsPK series is different in length, and, although their sequences are quite similar overall, each has a unique sequence. Moreover, each member of the PsPK series has structural features typical of members of the protein-serine/threonine kinase family of protein kinases. All are equally similar to cyclic nucleotide-dependent protein kinase and protein kinase C, suggesting that the pea homologs may be involved in signal transduction. DNA gel blots show that each PsPK cDNA is likely to be encoded by a single gene within the pea genome. RNA blot analyses show that the PsPK transcripts accumulate differentially during greening of etiolated seedlings. PsPK3 and PsPK5 transcripts show a large and rapid decline during deetiolation. In contrast, the level of PsPK4 RNA increases steadily during deetiolation whereas PsPK1 and PsPK2 transcripts show little change during the greening period. Thus light regulates changes in the levels of transcripts encoding putative protein kinases in plants. Images PMID:1714582

  15. Expression of EGFP and NPTII protein is not associated with organ abnormalities in deceased transgenic cloned cattle.

    PubMed

    Liu, Yan; Wu, Qian; Cui, Huiting; Li, Qinghe; Zhao, Yiqiang; Luo, Juan; Liu, Qiuyue; Sun, Xiuzhu; Tang, Bo; Zhang, Lei; Dai, Yunping; Li, Ning

    2008-12-01

    Both enhanced green fluorescence protein (EGFP) and neomycin phosphotransferase type II enzyme (NPTII) are widely used in transgenic studies, but their side effects have not been extensively investigated. In this study, we evaluated the expression profiles of the two marker genes and the relationship between their expression and organ abnormalities. Eight transgenic cloned cattle were studied, four harboring both EGFP and NPTII, and four harboring only the NPTII gene. Four age-matched cloned cattle were used as controls. EGFP and NPTII expression were measured and detected by Q-PCR, Western blot, ELISA, and RIA in heart, liver, and lungs, and the values ranged from 0.3 to 5 microg/g. The expression profiles exhibited differential or mosaic pattern between the organs, the pathologic symptoms of which were identified, but were similar to those of age-matched cloned cattle. All data indicated that the expression of EGFP and NPTII is not associated with organ abnormalities in transgenic cloned cattle.

  16. Protein storage vacuoles of Brassica napus zygotic embryos accumulate a BURP domain protein and perturbation of its production distorts the PSV.

    PubMed

    Teerawanichpan, Prapapan; Xia, Qun; Caldwell, Sarah J; Datla, Raju; Selvaraj, Gopalan

    2009-11-01

    BNM2is a prototypical member of the enigmatic BURP domain protein family whose members contain the signature FX6-7GX10-28PX25-31CX11-12X2SX45-56CHX10 CHX25-29CHX2TX15-16PX5CH in the C-terminus. This protein family occurs only in plants, and the cognate genes vary very widely in their expression contexts in vegetative and reproductive tissues. None of theBURP family members has been assigned any biochemical function. BNM2 was originally discovered as a gene expressed in microspore derived embryos (MDE) of Brassica napus but we found that MDE do not contain the corresponding protein. We show that BNM2 protein production is confined to the seeds and localized to the protein storage vacuoles (PSV) even though the transcript is found in vegetative parts and floral buds as well. In developing seeds, transcript accumulation precedes protein appearance by more than 18 days. RNA accumulation peaks at approximately 20 days post anthesis (DPA) whereas protein accumulation reaches its maximum at approximately 40 DPA. Transgenic expression of BNM2 does not abrogate this regulation to yield ectopic protein production or to alter the temporal aspect ofBNM2 accumulation. Overexpression ofBNM2 led to spatial distortion of storage protein accumulation within PSV and to some morphological alterations of PSVs. However, the overall storage protein content was not altered.

  17. The accumulation of lipids and proteins during red blood cell storage: the roles of leucoreduction and experimental filtration

    PubMed Central

    Silliman, Christopher C.; Burke, Timothy; Kelher, Marguerite R.

    2017-01-01

    Pre-storage leucoreduction has been universally adopted in most developed countries in Asia, Europe and the Americas. It decreases febrile transfusion reactions, alloimmunisation to HLA antigens, cytomegalovirus exposure, the accumulation of a number of pro-inflammatory mediators in the supernatant, including the accumulation of platelet-and leucocyte-derived proteins and metabolites during routine storage. This review will highlight the lipids and proteins, biological response modifiers (BRMs) that accumulate, their clinical effects in transfused hosts, and methods of mitigation. PMID:28263170

  18. Two chitinase-like proteins abundantly accumulated in latex of mulberry show insecticidal activity

    PubMed Central

    2010-01-01

    Background Plant latex is the cytoplasm of highly specialized cells known as laticifers, and is thought to have a critical role in defense against herbivorous insects. Proteins abundantly accumulated in latex might therefore be involved in the defense system. Results We purified latex abundant protein a and b (LA-a and LA-b) from mulberry (Morus sp.) and analyzed their properties. LA-a and LA-b have molecular masses of approximately 50 and 46 kDa, respectively, and are abundant in the soluble fraction of latex. Western blotting analysis suggested that they share sequence similarity with each other. The sequences of LA-a and LA-b, as determined by Edman degradation, showed chitin-binding domains of plant chitinases at the N termini. These proteins showed small but significant chitinase and chitosanase activities. Lectin RCA120 indicated that, unlike common plant chitinases, LA-a and LA-b are glycosylated. LA-a and LA-b showed insecticidal activities when fed to larvae of the model insect Drosophila melanogaster. Conclusions Our results suggest that the two LA proteins have a crucial role in defense against herbivorous insects, possibly by hydrolyzing their chitin. PMID:20109180

  19. Heat shock proteins in porcine ovary: synthesis, accumulation and regulation by stress and hormones.

    PubMed

    Sirotkin, Alexander V; Bauer, Miroslav

    2011-07-01

    The present studies aimed to understand the interrelationships between stress, hormones and heat shock proteins (HSPs) in the ovary. We examined (1) whether HSP70.2, HSP72 and HSP105/110 can be produced and accumulated in porcine ovarian tissue, (2) whether these HSPs could be indicators of stress, i.e. whether two kinds of stress (high temperatures and malnutrition/serum deprivation) can affect them, and (3) whether some hormonal regulators of ovarian functions (insulin-like growth factor (IGF)-I, leptin and follicle-stimulating hormone (FSH)) can affect these HSPs and response of ovaries to HSP-related stress. We analysed the expression of HSP70.2, HSP72 and HSP105/110 mRNA (by using real-time reverse transcriptase polymerase chain reaction) in porcine ovarian granulosa cells, as well as the accumulation of HSP70 protein (by using sodium dodecyl sulphate polyacrylamide gel electrophoresis-Western) in either whole ovarian follicles and granulose cells cultured at normal (37.5°C) or high (41.5°C) temperature, with and without serum and with and without IGF-I, leptin and FSH. Expression of mRNA for HSP70.2, HSP72 and HSP105/110 in ovarian granulosa cells and accumulation of HSP70 protein in whole ovarian follicles and granulosa cells were demonstrated. In all the groups, addition of either IGF-I, leptin and FSH reduced the expression of HSP70.2, HSP72 and HSP105/110 mRNA. Both high temperature, serum deprivation and their combination resulted in increase in mRNAs for all three analysed HSPs. Additions of either IGF-I, leptin and FSH prevented the stimulatory effect of both high temperature and serum deprivation on the transcription of HSP70.2, HSP72 and HSP105/110. In contrast, high temperature reduced accumulation of peptide HSP70 in both ovarian follicles and granulosa cell. Serum deprivation promoted accumulation of HSP70 in granulosa cells, but not in ovarian follicles. Addition of IGF-I, leptin and FSH was able to alter accumulation of HSP70 in both follicles

  20. Using transgenic modulation of protein synthesis and accumulation to probe protein signaling networks in Arabidopsis thaliana

    PubMed Central

    Warnasooriya, Sankalpi N

    2011-01-01

    Deployment of new model species in the plant biology community requires the development and/or improvement of numerous genetic tools. Sequencing of the Arabidopsis thaliana genome opened up a new challenge of assigning biological function to each gene. As many genes exhibit spatiotemporal or other conditional regulation of biological processes, probing for gene function necessitates applications that can be geared toward temporal, spatial and quantitative functional analysis in vivo. The continuing quest to establish new platforms to examine plant gene function has resulted in the availability of numerous genomic and proteomic tools. Classical and more recent genome-wide experimental approaches include conventional mutagenesis, tagged DNA insertional mutagenesis, ectopic expression of transgenes, activation tagging, RNA interference and two-component transactivation systems. The utilization of these molecular tools has resulted in conclusive evidence for the existence of many genes, and expanded knowledge on gene structure and function. This review covers several molecular tools that have become increasingly useful in basic plant research. We discuss their advantages and limitations for probing cellular protein function while emphasizing the contributions made to lay the fundamental groundwork for genetic manipulation of crops using plant biotechnology. PMID:21862868

  1. Congenital Abnormalities

    MedlinePlus

    ... Listen Español Text Size Email Print Share Congenital Abnormalities Page Content Article Body About 3% to 4% ... of congenital abnormalities earlier. 5 Categories of Congenital Abnormalities Chromosome Abnormalities Chromosomes are structures that carry genetic ...

  2. Hypoxia induces p53 accumulation in the S-phase and accumulation of hypophosphorylated retinoblastoma protein in all cell cycle phases of human melanoma cells.

    PubMed Central

    Danielsen, T.; Hvidsten, M.; Stokke, T.; Solberg, K.; Rofstad, E. K.

    1998-01-01

    Hypoxia has been shown to induce accumulation of p53 and of hypophosphorylated retinoblastoma protein (pRb) in tumour cells. In this study, the cell cycle dependence of p53 accumulation and pRb hypophosphorylation in four human melanoma cell lines that are wild type for p53 was investigated using two-parameter flow cytometry measurements of p53 or pRb protein content and DNA content. The hypoxia-induced increase in p53 protein was higher in S-phase than in G1 and G2 phases in all cell lines. The accumulation of p53 in S-phase during hypoxia was not related to hypoxia-induced apoptosis or substantial cell cycle specific cell inactivation during the first 24 h of reoxygenation. pRb was hypophosphorylated in all cell cycle phases by hypoxia treatment. The results did not support a direct link between p53 and pRb during hypoxia because p53 was induced in a cell cycle-specific manner, whereas no cell cycle-dependent differences in pRb hypophosphorylation were detected. Only a fraction of the cell populations (0.60+/-0.10) showed hypophosphorylated pRb. Thus, pRb is probably not the only mediator of the hypoxia-induced cell cycle block seen in all cells and all cell cycle phases. Moreover, the cell cycle-dependent induction of p53 by hypoxia suggests that the primary function of p53 accumulation during hypoxia is other than to arrest the cells. Images Figure 4 Figure 7 PMID:9862563

  3. Glycosylation inhibition reduces cholesterol accumulation in NPC1 protein-deficient cells.

    PubMed

    Li, Jian; Deffieu, Maika S; Lee, Peter L; Saha, Piyali; Pfeffer, Suzanne R

    2015-12-01

    Lysosomes are lined with a glycocalyx that protects the limiting membrane from the action of degradative enzymes. We tested the hypothesis that Niemann-Pick type C 1 (NPC1) protein aids the transfer of low density lipoprotein-derived cholesterol across this glycocalyx. A prediction of this model is that cells will be less dependent upon NPC1 if their glycocalyx is decreased in density. Lysosome cholesterol content was significantly lower after treatment of NPC1-deficient human fibroblasts with benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside, an inhibitor of O-linked glycosylation. Direct biochemical measurement of cholesterol showed that lysosomes purified from NPC1-deficient fibroblasts contained at least 30% less cholesterol when O-linked glycosylation was blocked. As an independent means to modify protein glycosylation, we used Chinese hamster ovary ldl-D cells defective in UDP-Gal/UDP-GalNAc 4-epimerase in which N- and O-linked glycosylation can be controlled. CRISPR generated, NPC1-deficient ldl-D cells supplemented with galactose accumulated more cholesterol than those in which sugar addition was blocked. In the absence of galactose supplementation, NPC1-deficient ldl-D cells also transported more cholesterol from lysosomes to the endoplasmic reticulum, as monitored by an increase in cholesteryl [(14)C]-oleate levels. These experiments support a model in which NPC1 protein functions to transfer cholesterol past a lysosomal glycocalyx.

  4. Human 14-3-3 gamma protein results in abnormal cell proliferation in the developing eye of Drosophila melanogaster

    PubMed Central

    Hong, Sophia W; Qi, Wenqing; Brabant, Marc; Bosco, Giovanni; Martinez, Jesse D

    2008-01-01

    Background 14-3-3 proteins are a family of adaptor proteins that participate in a wide variety of cellular processes. Recent evidence indicates that the expression levels of these proteins are elevated in some human tumors providing circumstantial evidence for their involvement in human cancers. However, the mechanism through which these proteins act in tumorigenesis is uncertain. Results To determine whether elevated levels of 14-3-3 proteins may perturb cell growth we overexpressed human 14-3-3 gamma (h14-3-3 gamma) in Drosophila larvae using the heat shock promoter or the GMR-Gal4 driver and then examined the effect that this had on cell proliferation in the eye imaginal discs of third instar larvae. We found that induction of h14-3-3 gamma resulted in the abnormal appearance of replicating cells in the differentiating proneural photoreceptor cells of eye imaginal discs where h14-3-3 gamma was driven by the heat shock promoter. Similarly, we found that driving h14-3-3 gamma expression specifically in developing eye discs with the GMR-Gal4 driver resulted in increased numbers of replicative cells following the morphogenetic furrow. Interestingly, we found that the effects of overexpressing h1433 gamma on eye development were increased in a genetic background where String (cdc25) function was compromised. Conclusion Taken together our results indicate that h14-3-3 gamma can promote abnormal cell proliferation and may act through Cdc25. This has important implications for 14-3-3 gamma as an oncogene as it suggests that elevated levels of 14-3-3 may confer a growth advantage to cells that overexpress it. PMID:18194556

  5. LDL Receptor-Related Protein-1 (LRP1) Regulates Cholesterol Accumulation in Macrophages

    PubMed Central

    Lillis, Anna P.; Muratoglu, Selen Catania; Au, Dianaly T.; Migliorini, Mary; Lee, Mi-Jeong; Fried, Susan K.; Mikhailenko, Irina; Strickland, Dudley K.

    2015-01-01

    Within the circulation, cholesterol is transported by lipoprotein particles and is taken up by cells when these particles associate with cellular receptors. In macrophages, excessive lipoprotein particle uptake leads to foam cell formation, which is an early event in the development of atherosclerosis. Currently, mechanisms responsible for foam cell formation are incompletely understood. To date, several macrophage receptors have been identified that contribute to the uptake of modified forms of lipoproteins leading to foam cell formation, but the contribution of the LDL receptor-related protein 1 (LRP1) to this process is not known. To investigate the role of LRP1 in cholesterol accumulation in macrophages, we generated mice with a selective deletion of LRP1 in macrophages on an LDL receptor (LDLR)-deficient background (macLRP1-/-). After feeding mice a high fat diet for 11 weeks, peritoneal macrophages isolated from Lrp+/+ mice contained significantly higher levels of total cholesterol than those from macLRP1-/- mice. Further analysis revealed that this was due to increased levels of cholesterol esters. Interestingly, macLRP1-/- mice displayed elevated plasma cholesterol and triglyceride levels resulting from accumulation of large, triglyceride-rich lipoprotein particles in the circulation. This increase did not result from an increase in hepatic VLDL biosynthesis, but rather results from a defect in catabolism of triglyceride-rich lipoprotein particles in macLRP1-/- mice. These studies reveal an important in vivo contribution of macrophage LRP1 to cholesterol homeostasis. PMID:26061292

  6. 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.

  7. Chromothripsis in healthy individuals affects multiple protein-coding genes and can result in severe congenital abnormalities in offspring.

    PubMed

    de Pagter, Mirjam S; van Roosmalen, Markus J; Baas, Annette F; Renkens, Ivo; Duran, Karen J; van Binsbergen, Ellen; Tavakoli-Yaraki, Masoumeh; Hochstenbach, Ron; van der Veken, Lars T; Cuppen, Edwin; Kloosterman, Wigard P

    2015-04-02

    Chromothripsis represents an extreme class of complex chromosome rearrangements (CCRs) with major effects on chromosomal architecture. Although recent studies have associated chromothripsis with congenital abnormalities, the incidence and pathogenic effects of this phenomenon require further investigation. Here, we analyzed the genomes of three families in which chromothripsis rearrangements were transmitted from a mother to her child. The chromothripsis in the mothers resulted in completely balanced rearrangements involving 8-23 breakpoint junctions across three to five chromosomes. Two mothers did not show any phenotypic abnormalities, although 3-13 protein-coding genes were affected by breakpoints. Unbalanced but stable transmission of a subset of the derivative chromosomes caused apparently de novo complex copy-number changes in two children. This resulted in gene-dosage changes, which are probably responsible for the severe congenital phenotypes of these two children. In contrast, the third child, who has a severe congenital disease, harbored all three chromothripsis chromosomes from his healthy mother, but one of the chromosomes acquired de novo rearrangements leading to copy-number changes. These results show that the human genome can tolerate extreme reshuffling of chromosomal architecture, including breakage of multiple protein-coding genes, without noticeable phenotypic effects. The presence of chromothripsis in healthy individuals affects reproduction and is expected to substantially increase the risk of miscarriages, abortions, and severe congenital disease.

  8. The HIV matrix protein p17 induces hepatic lipid accumulation via modulation of nuclear receptor transcriptoma

    PubMed Central

    Renga, Barbara; Francisci, Daniela; Carino, Adriana; Marchianò, Silvia; Cipriani, Sabrina; Chiara Monti, Maria; Del Sordo, Rachele; Schiaroli, Elisabetta; Distrutti, Eleonora; Baldelli, Franco; Fiorucci, Stefano

    2015-01-01

    Liver disease is the second most common cause of mortality in HIV-infected persons. Exactly how HIV infection per se affects liver disease progression is unknown. Here we have investigated mRNA expression of 49 nuclear hormone receptors (NRs) and 35 transcriptional coregulators in HepG2 cells upon stimulation with the HIV matrix protein p17. This viral protein regulated mRNA expression of some NRs among which LXRα and its transcriptional co-activator MED1 were highly induced at mRNA level. Dissection of p17 downstream intracellular pathway demonstrated that p17 mediated activation of Jak/STAT signaling is responsible for the promoter dependent activation of LXR. The treatment of both HepG2 as well as primary hepatocytes with HIV p17 results in the transcriptional activation of LXR target genes (SREBP1c and FAS) and lipid accumulation. These effects are lost in HepG2 cells pre-incubated with a serum from HIV positive person who underwent a vaccination with a p17 peptide as well as in HepG2 cells pre-incubated with the natural LXR antagonist gymnestrogenin. These results suggest that HIV p17 affects NRs and their related signal transduction thus contributing to the progression of liver disease in HIV infected patients. PMID:26469385

  9. The HIV matrix protein p17 induces hepatic lipid accumulation via modulation of nuclear receptor transcriptoma.

    PubMed

    Renga, Barbara; Francisci, Daniela; Carino, Adriana; Marchianò, Silvia; Cipriani, Sabrina; Chiara Monti, Maria; Del Sordo, Rachele; Schiaroli, Elisabetta; Distrutti, Eleonora; Baldelli, Franco; Fiorucci, Stefano

    2015-10-15

    Liver disease is the second most common cause of mortality in HIV-infected persons. Exactly how HIV infection per se affects liver disease progression is unknown. Here we have investigated mRNA expression of 49 nuclear hormone receptors (NRs) and 35 transcriptional coregulators in HepG2 cells upon stimulation with the HIV matrix protein p17. This viral protein regulated mRNA expression of some NRs among which LXRα and its transcriptional co-activator MED1 were highly induced at mRNA level. Dissection of p17 downstream intracellular pathway demonstrated that p17 mediated activation of Jak/STAT signaling is responsible for the promoter dependent activation of LXR. The treatment of both HepG2 as well as primary hepatocytes with HIV p17 results in the transcriptional activation of LXR target genes (SREBP1c and FAS) and lipid accumulation. These effects are lost in HepG2 cells pre-incubated with a serum from HIV positive person who underwent a vaccination with a p17 peptide as well as in HepG2 cells pre-incubated with the natural LXR antagonist gymnestrogenin. These results suggest that HIV p17 affects NRs and their related signal transduction thus contributing to the progression of liver disease in HIV infected patients.

  10. Ki67 antigen contributes to the timely accumulation of protein phosphatase 1γ on anaphase chromosomes.

    PubMed

    Takagi, Masatoshi; Nishiyama, Yuko; Taguchi, Atsuko; Imamoto, Naoko

    2014-08-15

    Ki67 is a protein widely used as cell-proliferation marker, with its cellular functions being hardly unveiled. In this paper, we present the direct interaction between Ki67 and PP1γ, a protein phosphatase showing characteristic accumulation on anaphase chromosomes via the canonical PP1-binding motif within Ki67. In cells depleted of Ki67, PP1γ is targeted to anaphase chromosomes less efficiently. Additionally, overexpression of Ki67, but not a mutant form without the ability to bind PP1γ, induced ectopic localization of PP1γ οn metaphase chromosomes. These observations demonstrate that Ki67 is one factor that defines the cellular behavior of PP1γ in anaphase. To explore the specific roles of the subset of PP1γ recruited on chromosome via its interaction with Ki67 (PP1γ-Ki67), endogenous Ki67 was replaced with a Ki67 mutant deficient in its ability to interact with PP1γ. Although no obvious defects in the progression of mitosis were observed, the timing of dephosphorylation of the mutant Ki67 in anaphase was delayed, indicating that Ki67 itself is one of the substrates of PP1γ-Ki67.

  11. Nonpeptidic Lysosomal Modulators Derived from Z-Phe-Ala-Diazomethylketone for Treating Protein Accumulation Diseases

    PubMed Central

    2012-01-01

    Lysosomes are involved in protein turnover and removing misfolded species, and their enzymes have the potential to offset the defect in proteolytic clearance that contributes to the age-related dementia Alzheimer's disease (AD). The weak cathepsin B and L inhibitor Z-Phe-Ala-diazomethylketone (PADK) enhances lysosomal cathepsin levels at low concentrations, thereby eliciting protective clearance of PHF-τ and Aβ42 in the hippocampus and other brain regions. Here, a class of positive modulators is established with compounds decoupled from the cathepsin inhibitory properties. We utilized PADK as a departure point to develop nonpeptidic structures with the hydroxyethyl isostere. The first-in-class modulators SD1002 and SD1003 exhibit improved levels of cathepsin up-regulation but almost complete removal of cathepsin inhibitory properties as compared to PADK. Isomers of the lead compound SD1002 were synthesized, and the modulatory activity was determined to be stereoselective. In addition, the lead compound was tested in transgenic mice with results indicating protection against AD-type protein accumulation pathology. PMID:24900408

  12. Effect of progerin on the accumulation of oxidized proteins in fibroblasts from Hutchinson Gilford progeria patients.

    PubMed

    Viteri, Gabriela; Chung, Youn Wook; Stadtman, Earl R

    2010-01-01

    The mutation responsible for Hutchinson Gilford Progeria Syndrome (HGPS) causes abnormal nuclear morphology. Previous studies show that free radicals and reactive oxygen species play major roles in the etiology and/or progression of neurodegenerative diseases and aging. This study compares oxidative stress responses between progeric and normal fibroblasts. Our data revealed higher ROS levels in HGPS cells compared to age-matched controls. In response to oxidative challenge, progeric cells showed increased mRNA levels for mitochondrial superoxide dismutase (SOD) and SOD protein content. However, this did not prevent a drop in the ATP content of progeria fibroblasts. Previous studies have shown that declines in human fibroblast ATP levels interfere with programmed cell death and promote necrotic inflammation. Notably, in our investigations the ATP content of progeria fibroblasts was only approximately 50% of that found in healthy controls. Furthermore, HGPS fibroblast analysis revealed a decrease in total caspase-like proteasome activity and in the levels of two active proteolytic complex subunits (beta(5) and beta(7)). A number of studies indicate that the molecular mechanisms causing accelerated aging in progeric patients also occur in healthy cells of older individuals. Thus, the results of this study may also help explain some of the cellular changes that accompany normal aging.

  13. Hepcidin Suppresses Brain Iron Accumulation by Downregulating Iron Transport Proteins in Iron-Overloaded Rats.

    PubMed

    Du, Fang; Qian, Zhong-Ming; Luo, Qianqian; Yung, Wing-Ho; Ke, Ya

    2015-08-01

    Iron accumulates progressively in the brain with age, and iron-induced oxidative stress has been considered as one of the initial causes for Alzheimer's disease (AD) and Parkinson's disease (PD). Based on the role of hepcidin in peripheral organs and its expression in the brain, we hypothesized that this peptide has a role to reduce iron in the brain and hence has the potential to prevent or delay brain iron accumulation in iron-associated neurodegenerative disorders. Here, we investigated the effects of hepcidin expression adenovirus (ad-hepcidin) and hepcidin peptide on brain iron contents, iron transport across the brain-blood barrier, iron uptake and release, and also the expression of transferrin receptor-1 (TfR1), divalent metal transporter 1 (DMT1), and ferroportin 1 (Fpn1) in cultured microvascular endothelial cells and neurons. We demonstrated that hepcidin significantly reduced brain iron in iron-overloaded rats and suppressed transport of transferrin-bound iron (Tf-Fe) from the periphery into the brain. Also, the peptide significantly inhibited expression of TfR1, DMT1, and Fpn1 as well as reduced Tf-Fe and non-transferrin-bound iron uptake and iron release in cultured microvascular endothelial cells and neurons, while downregulation of hepcidin with hepcidin siRNA retrovirus generated opposite results. We concluded that, under iron-overload, hepcidin functions to reduce iron in the brain by downregulating iron transport proteins. Upregulation of brain hepcidin by ad-hepcidin emerges as a new pharmacological treatment and prevention for iron-associated neurodegenerative disorders.

  14. Modeling Grain Nitrogen Accumulation and Protein Composition to Understand the Sink/Source Regulations of Nitrogen Remobilization for Wheat

    PubMed Central

    Martre, Pierre; Porter, John R.; Jamieson, Peter D.; Triboï, Eugène

    2003-01-01

    A functional explanation for the regulation of grain nitrogen (N) accumulation in cereal by environmental and genetic factors remains elusive. Here, new mechanistic hypotheses of grain N accumulation are proposed and tested for wheat (Triticum aestivum). First, we tested experimentally the hypothesis that grain N accumulation is mostly source regulated. Four contrasting cultivars, in terms of their grain N concentrations and yield potentials, were grown with non-limiting N supply. Grain number per ear was reduced by removing the top part of the ear at anthesis. Reduction in grain number gave a significant increase in N content per grain for all cultivars, showing that grain N accumulation was source regulated. However, on a per ear basis, cultivars with a high grain number fully compensated their N accumulation for reduced grain number at anthesis. Cultivars with a lower grain number did not compensate completely, and grain N per ear was decreased by 16%. Second, new mechanistic hypotheses of the origins of grain N source regulation and its response to environment were tested by simulation. The hypotheses were: (a) The regulation by N sources of grain N accumulation applies only for the storage proteins (i.e. gliadin and glutenin fractions); (b) accumulation of structural and metabolic proteins (i.e. albumin-globulin and amphiphilic fractions) is sink-regulated; and (c) N partitioning between gliadins and glutenins is constant during grain development and unmodified by growing conditions. Comparison of experimental and simulation results of the accumulation of grain protein fractions under wide ranges of N fertilization, temperatures, and irrigation supported these hypotheses. PMID:14630962

  15. No Obvious Abnormality in Mice Deficient in Receptor Protein Tyrosine Phosphatase β

    PubMed Central

    Harroch, S.; Palmeri, M.; Rosenbluth, J.; Custer, A.; Okigaki, M.; Shrager, P.; Blum, M.; Buxbaum, J. D.; Schlessinger, J.

    2000-01-01

    The development of neurons and glia is governed by a multitude of extracellular signals that control protein tyrosine phosphorylation, a process regulated by the action of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Receptor PTPβ (RPTPβ; also known as PTPζ) is expressed predominantly in the nervous system and exhibits structural features common to cell adhesion proteins, suggesting that this phosphatase participates in cell-cell communication. It has been proposed that the three isoforms of RPTPβ play a role in regulation of neuronal migration, neurite outgrowth, and gliogenesis. To investigate the biological functions of this PTP, we have generated mice deficient in RPTPβ. RPTPβ-deficient mice are viable, are fertile, and showed no gross anatomical alterations in the nervous system or other organs. In contrast to results of in vitro experiments, our study demonstrates that RPTPβ is not essential for neurite outgrowth and node formation in mice. The ultrastructure of nerves of the central nervous system in RPTPβ-deficient mice suggests a fragility of myelin. However, conduction velocity was not altered in RPTPβ-deficient mice. The normal development of neurons and glia in RPTPβ-deficient mice demonstrates that RPTPβ function is not necessary for these processes in vivo or that loss of RPTPβ can be compensated for by other PTPs expressed in the nervous system. PMID:11003666

  16. Inhibition of protein kinase C phosphorylation of hepatitis B virus capsids inhibits virion formation and causes intracellular capsid accumulation.

    PubMed

    Wittkop, Linda; Schwarz, Alexandra; Cassany, Aurelia; Grün-Bernhard, Stefanie; Delaleau, Mildred; Rabe, Birgit; Cazenave, Christian; Gerlich, Wolfram; Glebe, Dieter; Kann, Michael

    2010-07-01

    Capsids of hepatitis B virus and other hepadnaviruses contain a cellular protein kinase, which phosphorylates the capsid protein. Some phosphorylation sites are shown to be essential for distinct steps of viral replication as pregenome packaging or plus strand DNA synthesis. Although different protein kinases have been reported to phosphorylate the capsid protein, varying experimental approaches do not allow direct comparison. Furthermore, the activity of a specific protein kinase has not yet been correlated to steps in the hepadnaviral life cycle. In this study we show that capsids from various sources encapsidate active protein kinase Calpha, irrespective of hepatitis B virus genotype and host cell. Treatment of a virion expressing cell line with a pseudosubstrate inhibitor showed that inhibition of protein kinase C phosphorylation did not affect genome maturation but resulted in capsid accumulation and inhibited virion release to the medium. Our results imply that different protein kinases have distinct functions within the hepadnaviral life cycle.

  17. Storage Protein Accumulation in the Absence of the Vacuolar Processing Enzyme Family of Cysteine ProteasesW⃞

    PubMed Central

    Gruis, Darren; Schulze, Jan; Jung, Rudolf

    2004-01-01

    The role(s) of specific proteases in seed protein processing is only vaguely understood; indeed, the overall role of processing in stable protein deposition has been the subject of more speculation than direct investigation. Seed-type members of the vacuolar processing enzyme (VPE) family were hypothesized to perform a unique function in seed protein processing, but we demonstrated previously that Asn-specific protein processing in developing Arabidopsis seeds occurs independently of this VPE activity. Here, we describe the unexpected expression of vegetative-type VPEs in developing seeds and test the role(s) of all VPEs in seed storage protein accumulation by systematically stacking knockout mutant alleles of all four members (αVPE, βVPE, γVPE, and δVPE) of the VPE gene family in Arabidopsis. The complete removal of VPE function in the αvpe βvpe γvpe δvpe quadruple mutant resulted in a total shift of storage protein accumulation from wild-type processed polypeptides to a finite number of prominent alternatively processed polypeptides cleaved at sites other than the conserved Asn residues targeted by VPE. Although alternatively proteolyzed legumin-type globulin polypeptides largely accumulated as intrasubunit disulfide-linked polypeptides with apparent molecular masses similar to those of VPE-processed legumin polypeptides, they showed markedly altered solubility and protein assembly characteristics. Instead of forming 11S hexamers, alternatively processed legumin polypeptides were deposited primarily as 9S complexes. However, despite the impact on seed protein processing, plants devoid of all known functional VPE genes appeared unchanged with regard to protein content in mature seeds, relative mobilization rates of protein reserves during germination, and vegetative growth. These findings indicate that VPE-mediated Asn-specific proteolytic processing, and the physiochemical property changes attributed to this specific processing step, are not required for

  18. A RAS oncogene imparts growth factor independence to myeloid cells that abnormally regulate protein kinase C: a nonautocrine transformation pathway.

    PubMed

    Boswell, H S; Nahreini, T S; Burgess, G S; Srivastava, A; Gabig, T G; Inhorn, L; Srour, E F; Harrington, M A

    1990-06-01

    The factor-dependent cell line FDC-P1 has been utilized as a model of interleukin 3 (IL-3)-dependent myeloid cell proliferation. However, it has been recently observed that active phorbol esters (e.g., phorbol 12-myristate 13-acetate) may entirely replace IL-3 to promote its proliferation. These observations reveal abnormal regulation of protein kinase C (pkC) (absence of downregulation or overexpression). This property allowed a test of the hypothesis that the T24 RAS (codon 12) oncogene acts by constitutive and persistent pkC activation, driving proliferation. FDC-P1 cells were transfected by electroporation with the T24 RAS-containing vector pAL 8, or with a control vector pSVX Zip Neo, and neomycin-resistant clones were selected. Multiple RAS-transfectant clones were categorized for their growth factor requirement and incorporation of the 6.6-kb human mutant H-RAS genome. IL-3-independent clones had incorporated multiple (more than two) copies of the entire 6.6-kb RAS genome. The incorporation of multiple 6.6-kb RAS genomes was correlated with high-level p21 RAS expression. No evidence for autostimulatory growth factor production by clones containing the RAS oncogene was observed. Thus, acquisition of growth factor independence in myeloid cells by abundant expression of a RAS oncogene is linked, in part, to abnormal regulation of pkC, which acts as a collaborating oncogene.

  19. Protein and glycoprotein abnormalities in platelets from human Chediak-Higashi syndrome: polyacrylamide gel electrophoretic study of platelets from five patients.

    PubMed

    Ledezma, E; Apitz-Castro, R

    1985-10-01

    Polyacrylamide electrophoretic analysis of proteins and Tritium-labelled glycoproteins of the platelets from five patients with Chediak-Higashi Syndrome shows the existence of marked quantitative differences when compared to normal platelets. While the glycoprotein abnormalities are solely related to the plasma membrane, some of the abnormalities detected in the Coomasie blue pattern are probably representative of defects related to the dense bodies and the alpha-granules. Some of the abnormalities found may, in part, explain the variability of aggregatory responses described in these patients, as well as the marked tendency towards desaggregation exhibited by platelets from humans with the Chediak-Higashi Syndrome.

  20. Proteolysis of chloroplast proteins is responsible for accumulation of free amino acids in dark-treated tea (Camellia sinensis) leaves.

    PubMed

    Chen, Yiyong; Fu, Xiumin; Mei, Xin; Zhou, Ying; Cheng, Sihua; Zeng, Lanting; Dong, Fang; Yang, Ziyin

    2017-03-22

    Shade management (dark treatment) on tea (Camellia sinensis) plants is a common approach to improve free amino acids in raw materials of tea leaves. However, the reason for amino acid accumulation in dark-treated tea leaves is still unknown. In the present study, dark treatment significantly increased content of free amino acids and reduced content of soluble proteins in tea leaves. Quantitative proteomics analysis showed that most enzymes involved in biosyntheses of amino acids were down-accumulated by dark treatment. Chloroplast numbers reduced in dark-treated leaves and the content of soluble proteins reduced in the chloroplasts isolated from dark-treated leaves compared to control. These suggest that proteolysis of chloroplast proteins contributed to amino acid accumulation in dark-treated leaves. Two chloroplasts proteases, ATP-dependent Clp protease proteolytic subunit 3 and protease Do-like 2, were up-accumulated in dark-treated leaves. This study firstly elucidated the mechanism of accumulation of amino acids in dark-treated tea leaves.

  1. Apolipoprotein A-I Helsinki promotes intracellular acyl-CoA cholesterol acyltransferase (ACAT) protein accumulation.

    PubMed

    Toledo, Juan D; Garda, Horacio A; Cabaleiro, Laura V; Cuellar, Angela; Pellon-Maison, Magali; Gonzalez-Baro, Maria R; Gonzalez, Marina C

    2013-05-01

    Reverse cholesterol transport is a process of high antiatherogenic relevance in which apolipoprotein AI (apoA-I) plays an important role. The interaction of apoA-I with peripheral cells produces through mechanisms that are still poorly understood the mobilization of intracellular cholesterol depots toward plasma membrane. In macrophages, these mechanisms seem to be related to the modulation of the activity of acyl-CoA cholesterol acyltransferase (ACAT), the enzyme responsible for the intracellular cholesterol ester biosynthesis that is stored in lipid droplets. The activation of ACAT and the accumulation of lipid droplets play a key role in the transformation of macrophages into foam cells, leading to the formation of atheroma or atherosclerotic plaque. ApoA-I Helsinki (or ∆K107) is a natural apoA-I variant with a lysine deletion in the central protein region, carriers of which have increased atherosclerosis risk. We herein show that treatment of cultured RAW macrophages or CHOK1 cells with ∆K107, but not with wild-type apoA-I or a variant containing a similar deletion at the C-terminal region (∆K226), lead to a marked increase (more than 10 times) in the intracellular ACAT1 protein level as detected by western blot analysis. However, we could only detect a slight increase in cholesteryl ester produced by ∆K107 mainly when Chol loading was supplied by low-density lipoprotein (LDL). Although a similar choline-phospholipid efflux is evoked by these apoA-I variants, the change in phosphatidylcholine/sphyngomyelin distribution produced by wild-type apoA-I is not observed with either ∆K107 or ∆K226.

  2. Arabidopsis SEIPIN Proteins Modulate Triacylglycerol Accumulation and Influence Lipid Droplet Proliferation[OPEN

    PubMed Central

    2015-01-01

    The lipodystrophy protein SEIPIN is important for lipid droplet (LD) biogenesis in human and yeast cells. In contrast with the single SEIPIN genes in humans and yeast, there are three SEIPIN homologs in Arabidopsis thaliana, designated SEIPIN1, SEIPIN2, and SEIPIN3. Essentially nothing is known about the functions of SEIPIN homologs in plants. Here, a yeast (Saccharomyces cerevisiae) SEIPIN deletion mutant strain and a plant (Nicotiana benthamiana) transient expression system were used to test the ability of Arabidopsis SEIPINs to influence LD morphology. In both species, expression of SEIPIN1 promoted accumulation of large-sized lipid droplets, while expression of SEIPIN2 and especially SEIPIN3 promoted small LDs. Arabidopsis SEIPINs increased triacylglycerol levels and altered composition. In tobacco, endoplasmic reticulum (ER)-localized SEIPINs reorganized the normal, reticulated ER structure into discrete ER domains that colocalized with LDs. N-terminal deletions and swapping experiments of SEIPIN1 and 3 revealed that this region of SEIPIN determines LD size. Ectopic overexpression of SEIPIN1 in Arabidopsis resulted in increased numbers of large LDs in leaves, as well as in seeds, and increased seed oil content by up to 10% over wild-type seeds. By contrast, RNAi suppression of SEIPIN1 resulted in smaller seeds and, as a consequence, a reduction in the amount of oil per seed compared with the wild type. Overall, our results indicate that Arabidopsis SEIPINs are part of a conserved LD biogenesis machinery in eukaryotes and that in plants these proteins may have evolved specialized roles in the storage of neutral lipids by differentially modulating the number and sizes of lipid droplets. PMID:26362606

  3. Nanoscale Drug Delivery Platforms Overcome Platinum-Based Resistance in Cancer Cells Due to Abnormal Membrane Protein Trafficking

    PubMed Central

    Xue, Xue; Hall, Matthew D.; Zhang, Qiang; Wang, Paul C.; Gottesman, Michael M.; Liang, Xing-Jie

    2014-01-01

    The development of cellular resistance to platinum-based chemotherapies is often associated with reduced intracellular platinum concentrations. In some models, this reduction is due to abnormal membrane protein trafficking, resulting in reduced uptake by transporters at the cell surface. Given the central role of platinum drugs in the clinic, it is critical to overcome cisplatin resistance by bypassing the plasma membrane barrier to significantly increase the intracellular cisplatin concentration enough to inhibit the proliferation of cisplatin-resistant cells. Therefore, rational design of appropriate nanoscale drug delivery platforms (nDDPs) loaded with cisplatin or other platinum analogs as payloads is a possible strategy to solve this problem. This review will focus on the known mechanism of membrane trafficking in cisplatin-resistant cells, and the development and employment of nDDPs to improve cell uptake of cisplatin. PMID:24219825

  4. Nanoscale drug delivery platforms overcome platinum-based resistance in cancer cells due to abnormal membrane protein trafficking.

    PubMed

    Xue, Xue; Hall, Matthew D; Zhang, Qiang; Wang, Paul C; Gottesman, Michael M; Liang, Xing-Jie

    2013-12-23

    The development of cellular resistance to platinum-based chemotherapies is often associated with reduced intracellular platinum concentrations. In some models, this reduction is due to abnormal membrane protein trafficking, resulting in reduced uptake by transporters at the cell surface. Given the central role of platinum drugs in the clinic, it is critical to overcome cisplatin resistance by bypassing the plasma membrane barrier to significantly increase the intracellular cisplatin concentration enough to inhibit the proliferation of cisplatin-resistant cells. Therefore, rational design of appropriate nanoscale drug delivery platforms (nDDPs) loaded with cisplatin or other platinum analogues as payloads is a possible strategy to solve this problem. This review will focus on the known mechanism of membrane trafficking in cisplatin-resistant cells and the development and employment of nDDPs to improve cell uptake of cisplatin.

  5. The effect of protein supplementation on lactate accumulation during submaximal and maximal exercise.

    PubMed

    Vukovich, M D; Sharp, R L; King, D S; Kershishnik, K

    1992-12-01

    Eleven subjects performed a graded exercise test after 1 week of protein supplementation (PRO) or glucose polymer placebo (CON), randomly assigned in a double blind fashion. The exercise consisted of 3-min graded exercise bouts separated by 10 min of active recovery at zero pedal resistance. Subjects then performed a 30-sec Wingate test (WIN) to assess performance during supramaximal exercise. Blood samples were obtained in the last 15 sec of each exercise and recovery period. PRO resulted in a decrease in blood lactate following 120% VO2max and WIN, an increase in blood alanine at all time points, and lower postexercise muscle lactate and glycogen. Resting muscle GPT activity was 47% higher during the PRO trial. Mean power output during the WIN did not differ between PRO and CON. The WIN fatigue index was not significantly different between PRO and CON. The increased alanine may reflect increased transamination of pyruvate, thereby reducing the accumulation of lactate, which in turn had a marginal effect on performance during supramaximal exercise.

  6. Induced changes in chloroplast protein accumulation during heat bleaching in Euglena gracilis

    SciTech Connect

    Ortiz, W.; Wilson, C.J. )

    1988-02-01

    When growing cultures of light-grown Euglena gracilis Z are exposed to slightly elevated temperatures (33{degree}C) there is a time-dependent decrease in chlorophyll (bleaching) and a gradual transformation of chloroplasts into rudimentary plastids. A study was undertaken whose primary objective was to document major changes in polypeptide composition in the stroma and in thylakoids of cells that have been exposed to the bleaching temperature for up to 57 hours. A novel polypeptide of about 60,000 to 63,000 M{sub r} whose function is presently unknown, accumulates in the stroma and in thylakoids in response to growth at the bleaching temperature. The levels of the large and small subunit of ribuolosebisphosphate carboxylase, on the other hand, decrease to very low levels at about 33 hours and remain very low for the duration of the temperature treatment. Of two polypeptides associated with the light-harvesting chlorophyll-protein complex of photosystem II (28,000 and 24,500 M{sub r}) only the level of the smaller polypeptide decreases at the elevated temperature. The levels of 28,000 M{sub r} species remain virtually unchanged throughout the temperature treatment period.

  7. Accumulation of non-outer segment proteins in the outer segment underlies photoreceptor degeneration in Bardet–Biedl syndrome

    PubMed Central

    Datta, Poppy; Allamargot, Chantal; Hudson, Joseph S.; Andersen, Emily K.; Bhattarai, Sajag; Drack, Arlene V.; Sheffield, Val C.; Seo, Seongjin

    2015-01-01

    Compartmentalization and polarized protein trafficking are essential for many cellular functions. The photoreceptor outer segment (OS) is a sensory compartment specialized for phototransduction, and it shares many features with primary cilia. As expected, mutations disrupting protein trafficking to cilia often disrupt protein trafficking to the OS and cause photoreceptor degeneration. Bardet–Biedl syndrome (BBS) is one of the ciliopathies associated with defective ciliary trafficking and photoreceptor degeneration. However, precise roles of BBS proteins in photoreceptor cells and the underlying mechanisms of photoreceptor degeneration in BBS are not well understood. Here, we show that accumulation of non-OS proteins in the OS underlies photoreceptor degeneration in BBS. Using a newly developed BBS mouse model [Leucine zipper transcription factor-like 1 (Lztfl1)/Bbs17 mutant], isolated OSs, and quantitative proteomics, we determined 138 proteins that are enriched more than threefold in BBS mutant OS. In contrast, only eight proteins showed a more than threefold reduction. We found striking accumulation of Stx3 and Stxbp1/Munc18-1 and loss of polarized localization of Prom1 within the Lztfl1 and Bbs1 mutant OS. Ultrastructural analysis revealed that large vesicles are formed in the BBS OS, disrupting the lamellar structure of the OS. Our findings suggest that accumulation (and consequent sequestration) of non-OS proteins in the OS is likely the primary cause of photoreceptor degeneration in BBS. Our data also suggest that a major function of BBS proteins in photoreceptors is to transport proteins from the OS to the cell body or to prevent entry of non-OS proteins into the OS. PMID:26216965

  8. Familial abnormalities of thyroxine binding proteins: some problems of recognition and interpretation.

    PubMed Central

    Neild, J E; Byfield, P G; Lalloz, M R; Tait, D; Marigold, J H; Croft, D N; Slavin, B M

    1985-01-01

    A three generation family study was carried out after inappropriate treatment with radioactive iodine of a 50 year old woman with a raised serum total thyroxine concentration and free thyroxine index. Subsequent investigations showed that she and five members of her family had raised thyroxine binding globulin concentrations. Free thyroxine and free triiodothyronine concentrations were normal. Problems encountered in the recognition of this thyroxine binding protein disorder are discussed. Clinicians and clinical biochemists should be aware of these pitfalls and thus avoid further incorrect treatment on the basis of biochemical findings, even though free hormone estimations are now becoming readily available. PMID:3919066

  9. Heterogeneity of the Abnormal Prion Protein (PrPSc) of the Chandler Scrapie Strain

    PubMed Central

    Kasai, Kazuo; Iwamaru, Yoshifumi; Masujin, Kentaro; Imamura, Morikazu; Mohri, Shirou; Yokoyama, Takashi

    2013-01-01

    The pathological prion protein, PrPSc, displays various sizes of aggregates. In this study, we investigated the conformation, aggregation stability and proteinase K (PK)-sensitivity of small and large PrPSc aggregates of mouse-adapted prion strains. We showed that small PrPSc aggregates, previously thought to be PK-sensitive, are resistant to PK digestion. Furthermore, we showed that small PrPSc aggregates of the Chandler scrapie strain have greater resistance to PK digestion and aggregation-denaturation than large PrPSc aggregates of this strain. We conclude that this strain consists of heterogeneous PrPSc. PMID:25436883

  10. Abnormal lipopolysaccharide binding protein as marker of gastrointestinal inflammation in Parkinson disease

    PubMed Central

    Pal, Gian D.; Shaikh, Maliha; Forsyth, Christopher B.; Ouyang, Bichun; Keshavarzian, Ali; Shannon, Kathleen M.

    2015-01-01

    Objective: An inflammation-driven model of PD has been proposed based on the endotoxin lipopolysaccaride (LPS), a potential source of inflammation in the gastrointestinal system linked to neurotoxicity. Systemic exposure to bacterial endotoxin (LPS) can be determined by measuring plasma LPS binding protein (LBP). We aimed to evaluate whether lipopolysaccharide binding protein (LBP) can be used to distinguish PD subjects from control subjects and to assess whether LBP levels correlate with PD disease severity. Methods: We measured plasma LBP (ng/ml) using an ELISA kit in 94 PD subjects of various stages and 97 control subjects. Disease severity was assessed using the UPDRS and Hoehn and Yahr staging. The LBP level between the PD and control groups was compared using analysis of covariance. Spearman correlation was used to explore the relationship between LBP level and disease severity. Results: The mean LBP level in PD subjects (n = 94) was significantly different from control subjects (n = 95, p = 0.018). In PD subjects, we did not find a correlation between mean LBP level and disease severity. Conclusions: Our data suggests that LBP is one GI biomarker related to LPS induced neurotoxicity. However, there was significant variability in LBP levels within the PD and control groups, limiting its utility as a stand-alone biomarker. This study supports the role of LPS induced neurotoxicity in PD and further exploration of this pathway may be useful in developing sensitive and specific biomarkers for PD. PMID:26388718

  11. PMPCA mutations cause abnormal mitochondrial protein processing in patients with non-progressive cerebellar ataxia

    PubMed Central

    Jobling, Rebekah K.; Assoum, Mirna; Gakh, Oleksandr; Blaser, Susan; Raiman, Julian A.; Mignot, Cyril; Roze, Emmanuel; Dürr, Alexandra; Brice, Alexis; Lévy, Nicolas; Prasad, Chitra; Paton, Tara; Paterson, Andrew D.; Roslin, Nicole M.; Marshall, Christian R.; Desvignes, Jean-Pierre; Roëckel-Trevisiol, Nathalie; Scherer, Stephen W.; Rouleau, Guy A.; Mégarbané, André; Isaya, Grazia

    2015-01-01

    Non-progressive cerebellar ataxias are a rare group of disorders that comprise approximately 10% of static infantile encephalopathies. We report the identification of mutations in PMPCA in 17 patients from four families affected with cerebellar ataxia, including the large Lebanese family previously described with autosomal recessive cerebellar ataxia and short stature of Norman type and localized to chromosome 9q34 (OMIM #213200). All patients present with non-progressive cerebellar ataxia, and the majority have intellectual disability of variable severity. PMPCA encodes α-MPP, the alpha subunit of mitochondrial processing peptidase, the primary enzyme responsible for the maturation of the vast majority of nuclear-encoded mitochondrial proteins, which is necessary for life at the cellular level. Analysis of lymphoblastoid cells and fibroblasts from patients homozygous for the PMPCA p.Ala377Thr mutation and carriers demonstrate that the mutation impacts both the level of the alpha subunit encoded by PMPCA and the function of mitochondrial processing peptidase. In particular, this mutation impacts the maturation process of frataxin, the protein which is depleted in Friedreich ataxia. This study represents the first time that defects in PMPCA and mitochondrial processing peptidase have been described in association with a disease phenotype in humans. PMID:25808372

  12. Epilepsy, Behavioral Abnormalities, and Physiological Comorbidities in Syntaxin-Binding Protein 1 (STXBP1) Mutant Zebrafish

    PubMed Central

    Grone, Brian P.; Marchese, Maria; Hamling, Kyla R.; Kumar, Maneesh G.; Krasniak, Christopher S.; Sicca, Federico; Santorelli, Filippo M.; Patel, Manisha; Baraban, Scott C.

    2016-01-01

    Mutations in the synaptic machinery gene syntaxin-binding protein 1, STXBP1 (also known as MUNC18-1), are linked to childhood epilepsies and other neurodevelopmental disorders. Zebrafish STXBP1 homologs (stxbp1a and stxbp1b) have highly conserved sequence and are prominently expressed in the larval zebrafish brain. To understand the functions of stxbp1a and stxbp1b, we generated loss-of-function mutations using CRISPR/Cas9 gene editing and studied brain electrical activity, behavior, development, heart physiology, metabolism, and survival in larval zebrafish. Homozygous stxbp1a mutants exhibited a profound lack of movement, low electrical brain activity, low heart rate, decreased glucose and mitochondrial metabolism, and early fatality compared to controls. On the other hand, homozygous stxbp1b mutants had spontaneous electrographic seizures, and reduced locomotor activity response to a movement-inducing “dark-flash” visual stimulus, despite showing normal metabolism, heart rate, survival, and baseline locomotor activity. Our findings in these newly generated mutant lines of zebrafish suggest that zebrafish recapitulate clinical phenotypes associated with human syntaxin-binding protein 1 mutations. PMID:26963117

  13. Abnormal phosphoinositide metabolism and protein phosphorylation in platelets from a patient with the grey platelet syndrome.

    PubMed

    Rendu, F; Marche, P; Hovig, T; Maclouf, J; Lebret, M; Tenza, D; Levy-Toledano, S; Caen, J P

    1987-10-01

    Washed platelets isolated from one patient suffering from the inherited grey platelet syndrome were studied during thrombin-induced activation. The agonist-induced changes in (i) morphology, (ii) typical functional cell responses, (iii) membrane phospholipid metabolism and protein phosphorylation were studied and compared with the changes obtained with normal platelets. The morphology of the platelets as visualized by electron microscopy confirmed the almost total absence of intracellular alpha-granules and marked vacuolization. During thrombin stimulation the morphological changes were clearly delayed as compared to normal platelets, the granule centralization and aggregation occurred only 15 s after thrombin addition instead of 5 s in normal platelets. After 15 s, however, even though no alpha-granules were observed, a ring-like structure occurred centrally, indicating that they are not a prerequisite for this reaction. The whole release reaction, i.e. liberation of [14C]serotonin from dense granules and beta-N-acetylglucosaminidase activity from lysosomes, and the thromboxane synthesis were delayed and remained lower than in normal platelets. No thrombin-induced phosphatidyl 4,5-bisphosphate breakdown was measurable on 32P-prelabelled platelets although [32P]phosphatidate formation occurred normally. Phosphorylation time courses of myosin light chain (P20) and of protein P43 (mol wt 43,000) markedly differed from those of controls, being less than half of the normal during the first 15 s and remaining subnormal even after complete aggregation. These results suggest that in platelets devoid of alpha-granules a deficient transmembrane signalling system is likely responsible for the impaired physiological responses.

  14. Abnormal spermatogenesis and male infertility in testicular zinc finger protein Zfp318-knockout mice.

    PubMed

    Ishizuka, Masamichi; Ohtsuka, Eri; Inoue, Atsuto; Odaka, Mirei; Ohshima, Hirotaka; Tamura, Norihisa; Yoshida, Kaoru; Sako, Norihisa; Baba, Tadashi; Kashiwabara, Shin-Ichi; Okabe, Masaru; Noguchi, Junko; Hagiwara, Hiromi

    2016-09-01

    Zfp318, a mouse gene with a Cys2/His2 zinc finger motif, is mainly expressed in germ cells in the testis. It encodes two alternative transcripts, which regulate androgen receptor-mediated transcriptional activation or repression by overexpression of them. However, the role of Zfp318 is still obscure in vivo, especially in spermatogenesis. To elucidate the role of Zfp318 during gamete production, we established a knockout mouse line. Zfp318-null male mice exhibited infertility, whereas Zfp318-null female mice displayed normal fertility. ZFP318 was expressed during multiple stages of spermatogenesis, from spermatocytes to round spermatids. The nuclei of secondary spermatocytes showed high levels of expression. Histological analysis and quantitative analysis of DNA content showed decreased numbers of both spermatids in the seminiferous tubules and mature spermatozoa in the epididymides of Zfp318-null mice. These results suggest that Zfp318 is expressed as a functional protein in testicular germ cells and plays an important role in meiosis during spermatogenesis.

  15. Zinc and/or cadmium accumulation in Gynura pseudochina (L.) DC. studied in vitro and the effect on crude protein

    NASA Astrophysics Data System (ADS)

    Panitlertumpai, Natthawoot; Nakbanpote, Woranan; Sangdee, Aphidech; Thumanu, Kanjana; Nakai, Izumi; Hokura, Akiko

    2013-03-01

    Gynura pseudochina (L.) DC. is a zinc (Zn)/cadmium (Cd) hyperaccumulative plant. The aim of this study was to examine the tolerance of G. pseudochina (L.) DC. for Zn and/or Cd accumulation and protein expression. An in vitro tissue culture system was used to control the environment and effects of the microorganisms. Treatments with higher Zn and Cd concentrations increased chlorosis and the accumulation of metals in the root and shoot. Cd treatment at low levels induced the growth of the plant, and the translocation factor was high. A dual treatment with Cd and Zn decreased the metals' toxicity and demonstrated the plant's proclivity to accumulate Cd. The SDS-PAGE and FT-IR analyses showed the effect of the metals' toxicity on protein expression and secondary structure. Moreover, using XAFS techniques, it was demonstrated that treatment with a high Zn concentration (100 mg l-1) resulted in tetrahedral coordination with mixed S/O ligation in the protein extract as compared with Znsbnd O coordination in the protein extract from the control plant cultured in the presence of trace levels of Zn (0.04 mg l-1). This research suggested that G. pseudochina (L.) DC. had properties to tolerate a high Zn and Cd concentration, related to the sulphur proteins.

  16. Accumulation of Ku80 proteins at DNA double-strand breaks in living cells

    SciTech Connect

    Koike, Manabu Koike, Aki

    2008-03-10

    Ku plays a key role in multiple nuclear processes, e.g., DNA double-strand break (DSB) repair. The regulation mechanism of the localizations of Ku70 and Ku80 plays a key role in regulating the multiple functions of Ku. Although numerous biochemical studies in vitro have elucidated the DNA binding mechanism of Ku, no accumulation mechanisms of Ku70 and Ku80 at DSBs have been clarified in detail in vivo. In this study, we examined the accumulation mechanism of Ku80 at DSBs in living cells. EGFP-Ku80 accumulation at DSBs began immediately after irradiation. On the other hand, our data show that Ku70 alone, which has DNA binding activity independent of Ku80, cannot accumulate at the DSBs, whereas Ku70 bound to Ku80 can. The deletion of the C-terminal DNA-PKcs-binding domain and the mutation at the SUMOylation site of Ku80 had no effect on Ku80 accumulation. Unexpectedly, N-terminal deletion mutants of Ku80 fully lost their accumulation activity, although the mutants retained their Ku70 binding activity. Altogether, these data demonstrate that Ku80 is essential for Ku70 accumulation at DSBs. Furthermore, three domains of Ku80, i.e., the N-terminal {alpha}/{beta}, the DNA-binding, and Ku70-binding domains, seem to necessary for the accumulation at or recognition of DSBs in the early stage after irradiation.

  17. How Integrated Management Strategies Promote Protein Quality of Cotton Embryos: High Levels of Soil Available N, N Assimilation and Protein Accumulation Rate

    PubMed Central

    Yang, HongKun; Meng, YaLi; Chen, BingLin; Zhang, XingYue; Wang, YouHua; Zhao, WenQing; Zhou, ZhiGuo

    2016-01-01

    Cottonseed is widely used as a source of ruminant feed and for industrial purposes. Therefore, there is a tremendous need to improve the nutritional value of cotton embryos. In this study, a conventional management (CM) and two integrated cotton management strategies (IMS1, IMS2) were performed at two soil fertility levels to study the relationships among soil N, N assimilation, embryonic protein accumulation and protein quality. The levels of proteins, essential amino acids, and semi-essential amino acids, especially those of glutamate, lysine, and methionine, were higher in IMS1 and IMS2 embryos than in CM embryos. These changes were significantly positively correlated with the soil-available N content, glutamine synthetase activity and peak value of protein accumulation rate and were negatively correlated with the free amino acid level. These results illustrated that integrated management strategies, especially the rates and timing of N application, raise the level of soil available N, which is beneficial for N assimilation in developing cotton embryos. The protein content was limited by the rate of protein accumulation rather than by the free amino acid content. The combination of target yield fertilization, a growth-driven N application schedule, a high plant density and the seedling raising with bio-organic fertilizer can substantially improve protein quality in cotton embryos, especially at a soil with low soil organic matter and total nitrogen. PMID:27532007

  18. How Integrated Management Strategies Promote Protein Quality of Cotton Embryos: High Levels of Soil Available N, N Assimilation and Protein Accumulation Rate.

    PubMed

    Yang, HongKun; Meng, YaLi; Chen, BingLin; Zhang, XingYue; Wang, YouHua; Zhao, WenQing; Zhou, ZhiGuo

    2016-01-01

    Cottonseed is widely used as a source of ruminant feed and for industrial purposes. Therefore, there is a tremendous need to improve the nutritional value of cotton embryos. In this study, a conventional management (CM) and two integrated cotton management strategies (IMS1, IMS2) were performed at two soil fertility levels to study the relationships among soil N, N assimilation, embryonic protein accumulation and protein quality. The levels of proteins, essential amino acids, and semi-essential amino acids, especially those of glutamate, lysine, and methionine, were higher in IMS1 and IMS2 embryos than in CM embryos. These changes were significantly positively correlated with the soil-available N content, glutamine synthetase activity and peak value of protein accumulation rate and were negatively correlated with the free amino acid level. These results illustrated that integrated management strategies, especially the rates and timing of N application, raise the level of soil available N, which is beneficial for N assimilation in developing cotton embryos. The protein content was limited by the rate of protein accumulation rather than by the free amino acid content. The combination of target yield fertilization, a growth-driven N application schedule, a high plant density and the seedling raising with bio-organic fertilizer can substantially improve protein quality in cotton embryos, especially at a soil with low soil organic matter and total nitrogen.

  19. Endothelial cell protein C receptor-mediated redistribution and tissue-level accumulation of factor VIIa

    PubMed Central

    Clark, C A; Vatsyayan, R; Hedner, U; Esmon, C T; Pendurthi, U R; Rao, L V M

    2012-01-01

    Background Recent studies show that activated factor VII (FVIIa) binds to the endothelial cell protein C receptor (EPCR) on the vascular endothelium; however, the importance of this interaction in hemostasis or pathophysiology is unknown. Objective The aim of the present study was to investigate the role of the FVIIa interaction with EPCR on the endothelium in mediating FVIIa transport from the circulation to extravascular tissues. Methods Wild-type, EPCR-deficient or ECPR-over-expressing mice were injected with human recombinant (r)FVIIa (120 μg kg−1 body weight) via the tail vein. At varying time intervals after rFVIIa administration, blood and various tissues were collected to measure FVIIa antigen and activity levels. Tissue sections were analyzed by immunohistochemistry for FVIIa and EPCR. Results The data reveal that, after intravenous (i.v.) injection, rFVIIa rapidly disappears from the blood and associates with the endothelium in an EPCR-dependent manner. Immunohistochemical analyses revealed that the association of FVIIa with the endothelium was maximal at 30 min and thereafter progressively declined. The FVIIa association with the endothelium was undetectable at time points exceeding 24 h post-FVIIa administration. The levels of rFVIIa accumulated in tissue correlate with expression levels of EPCR in mice and FVIIa associated with tissues remained functionally active for periods of at least 7 days. Conclusions The observation that an EPCR-dependent association of FVIIa with the endothelium is most pronounced soon after rFVIIa administration and subsequently declines temporally, combined with the retention of functionally active FVIIa in tissue homogenates for extended periods, indicates that FVIIa binding to EPCR on the endothelium facilitates the transport of FVIIa from circulation to extravascular tissues where TF resides. PMID:22950420

  20. Protein Kinase Cβ Modulates Ligand-induced Cell Surface Death Receptor Accumulation

    PubMed Central

    Meng, Xue Wei; Heldebrant, Michael P.; Flatten, Karen S.; Loegering, David A.; Dai, Haiming; Schneider, Paula A.; Gomez, Timothy S.; Peterson, Kevin L.; Trushin, Sergey A.; Hess, Allan D.; Smith, B. Douglas; Karp, Judith E.; Billadeau, Daniel D.; Kaufmann, Scott H.

    2010-01-01

    Although treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) is known to protect a subset of cells from induction of apoptosis by death ligands such as Fas ligand and tumor necrosis factor-α-related apoptosis-inducing ligand, the mechanism of this protection is unknown. This study demonstrated that protection in short term apoptosis assays and long term proliferation assays was maximal when Jurkat or HL-60 human leukemia cells were treated with 2–5 nm PMA. Immunoblotting demonstrated that multiple PKC isoforms, including PKCα, PKCβ, PKCϵ, and PKCθ, translocated from the cytosol to a membrane-bound fraction at these PMA concentrations. When the ability of short hairpin RNA (shRNA) constructs that specifically down-regulated each of these isoforms was examined, PKCβ shRNA uniquely reversed PMA-induced protection against cell death. The PKCβ-selective small molecule inhibitor enzastaurin had a similar effect. Although mass spectrometry suggested that Fas is phosphorylated on a number of serines and threonines, mutation of these sites individually or collectively had no effect on Fas-mediated death signaling or PMA protection. Further experiments demonstrated that PMA diminished ligand-induced cell surface accumulation of Fas and DR5, and PKCβ shRNA or enzastaurin reversed this effect. Moreover, enzastaurin sensitized a variety of human tumor cell lines and clinical acute myelogenous leukemia isolates, which express abundant PKCβ, to tumor necrosis factor-α related apoptosis-inducing ligand-induced death in the absence of PMA. Collectively, these results identify a specific PKC isoform that modulates death receptor-mediated cytotoxicity as well as a small molecule inhibitor that mitigates the inhibitory effects of PKC activation on ligand-induced death receptor trafficking and cell death. PMID:19887445

  1. Abnormal Sensory Protein Expression and Urothelial Dysfunction in Ketamine-Related Cystitis in Humans

    PubMed Central

    2016-01-01

    Purpose The aim of this study was to analyze patterns of sensory protein expression and urothelial dysfunction in ketamine-related cystitis (KC) in humans. Methods Biopsies of bladder mucosa were performed in 29 KC patients during cystoscopy. Then specimens were analyzed for tryptase, zonula occludens-1 (ZO-1), E-cadherin, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) with immunofluorescence staining and quantification. In addition, 10 healthy control bladder specimens were analyzed and compared with the KC specimens. Another 16 whole bladder specimens obtained from partial cystectomy were also analyzed for the muscarinic receptors M2 and M3, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), β-3 adrenergic receptors (β3-ARs), and the P2X3 receptor by western blotting. In addition, 3 normal control bladder specimens were analyzed and compared with the KC specimens. Results The KC bladder mucosa revealed significantly less expression of ZO-1 and E-cadherin, and greater expression of TUNEL and tryptase activity than the control samples. The expression of M3 and β3-AR in the KC specimens was significantly greater than in the controls. The expression of iNOS, eNOS, M2, and P2X3 was not significantly different between the KC and control specimens. Conclusions The bladder tissue of KC patients revealed significant urothelial dysfunction, which was associated with mast-cell mediated inflammation, increased urothelial cell apoptosis, and increased expression of the M3 and β3-AR. PMID:27706016

  2. Metformin reduces lipid accumulation in macrophages by inhibiting FOXO1-mediated transcription of fatty acid-binding protein 4

    SciTech Connect

    Song, Jun; Ren, Pingping; Zhang, Lin; Wang, Xing Li; Chen, Li; Shen, Ying H.

    2010-02-26

    Objective: The accumulation of lipids in macrophages contributes to the development of atherosclerosis. Strategies to reduce lipid accumulation in macrophages may have therapeutic potential for preventing and treating atherosclerosis and cardiovascular complications. The antidiabetic drug metformin has been reported to reduce lipid accumulation in adipocytes. In this study, we examined the effects of metformin on lipid accumulation in macrophages and investigated the mechanisms involved. Methods and results: We observed that metformin significantly reduced palmitic acid (PA)-induced intracellular lipid accumulation in macrophages. Metformin promoted the expression of carnitine palmitoyltransferase I (CPT-1), while reduced the expression of fatty acid-binding protein 4 (FABP4) which was involved in PA-induced lipid accumulation. Quantitative real-time PCR showed that metformin regulates FABP4 expression at the transcriptional level. We identified forkhead transcription factor FOXO1 as a positive regulator of FABP4 expression. Inhibiting FOXO1 expression with FOXO1 siRNA significantly reduced basal and PA-induced FABP4 expression. Overexpression of wild-type FOXO1 and constitutively active FOXO1 significantly increased FABP4 expression, whereas dominant negative FOXO1 dramatically decreased FABP4 expression. Metformin reduced FABP4 expression by promoting FOXO1 nuclear exclusion and subsequently inhibiting its activity. Conclusions: Taken together, these results suggest that metformin reduces lipid accumulation in macrophages by repressing FOXO1-mediated FABP4 transcription. Thus, metformin may have a protective effect against lipid accumulation in macrophages and may serve as a therapeutic agent for preventing and treating atherosclerosis in metabolic syndrome.

  3. Blocking protein farnesylation improves nuclear shape abnormalities in keratinocytes of mice expressing the prelamin A variant in Hutchinson-Gilford progeria syndrome.

    PubMed

    Wang, Yuexia; Ostlund, Cecilia; Worman, Howard J

    2010-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is an accelerated aging disorder caused by mutations in LMNA leading to expression of a truncated prelamin A variant termed progerin. Whereas a farnesylated polypeptide is normally removed from the carboxyl-terminus of prelamin A during endoproteolytic processing to lamin A, progerin lacks the cleavage site and remains farnesylated. Cultured cells from human subjects with HGPS and genetically modified mice expressing progerin have nuclear morphological abnormalities, which are reversed by inhibitors of protein farnesylation. In addition, treatment with protein farnesyltransferase inhibitors improves whole animal phenotypes in mouse models of HGPS. However, improvement in nuclear morphology in tissues after treatment of animals has not been demonstrated. We therefore treated transgenic mice that express progerin in epidermis with the protein farnesyltransferase inhibitor FTI-276 or a combination of pravastatin and zoledronate to determine if they reversed nuclear morphological abnormalities in tissue. Immunofluorescence microscopy and "blinded" electron microscopic analysis demonstrated that systemic administration of FTI-276 or pravastatin plus zoledronate significantly improved nuclear morphological abnormalities in keratinocytes of transgenic mice. These results show that pharmacological blockade of protein prenylation reverses nuclear morphological abnormalities that occur in HGPS in vivo. They further suggest that skin biopsy may be useful to determine if protein farnesylation inhibitors are exerting effects in subjects with HGPS in clinical trials.

  4. Fragile X-like behaviors and abnormal cortical dendritic spines in cytoplasmic FMR1-interacting protein 2-mutant mice.

    PubMed

    Han, Kihoon; Chen, Hogmei; Gennarino, Vincenzo A; Richman, Ronald; Lu, Hui-Chen; Zoghbi, Huda Y

    2015-04-01

    Silencing of fragile X mental retardation 1 (FMR1) gene and loss of fragile X mental retardation protein (FMRP) cause fragile X syndrome (FXS), a genetic disorder characterized by intellectual disability and autistic behaviors. FMRP is an mRNA-binding protein regulating neuronal translation of target mRNAs. Abnormalities in actin-rich dendritic spines are major neuronal features in FXS, but the molecular mechanism and identity of FMRP targets mediating this phenotype remain largely unknown. Cytoplasmic FMR1-interacting protein 2 (Cyfip2) was identified as an interactor of FMRP, and its mRNA is a highly ranked FMRP target in mouse brain. Importantly, Cyfip2 is a component of WAVE regulatory complex, a key regulator of actin cytoskeleton, suggesting that Cyfip2 could be implicated in the dendritic spine phenotype of FXS. Here, we generated and characterized Cyfip2-mutant (Cyfip2(+/-)) mice. We found that Cyfip2(+/-) mice exhibited behavioral phenotypes similar to Fmr1-null (Fmr1(-/y)) mice, an animal model of FXS. Synaptic plasticity and dendritic spines were normal in Cyfip2(+/-) hippocampus. However, dendritic spines were altered in Cyfip2(+/-) cortex, and the dendritic spine phenotype of Fmr1(-/y) cortex was aggravated in Fmr1(-/y); Cyfip2(+/-) double-mutant mice. In addition to the spine changes at basal state, metabotropic glutamate receptor (mGluR)-induced dendritic spine regulation was impaired in both Fmr1(-/y) and Cyfip2(+/-) cortical neurons. Mechanistically, mGluR activation induced mRNA translation-dependent increase of Cyfip2 in wild-type cortical neurons, but not in Fmr1(-/y) or Cyfip2(+/-) neurons. These results suggest that misregulation of Cyfip2 function and its mGluR-induced expression contribute to the neurobehavioral phenotypes of FXS.

  5. The molecular chaperone TRiC/CCT binds to the Trp-Asp 40 (WD40) repeat protein WDR68 and promotes its folding, protein kinase DYRK1A binding, and nuclear accumulation.

    PubMed

    Miyata, Yoshihiko; Shibata, Takeshi; Aoshima, Masato; Tsubata, Takuichi; Nishida, Eisuke

    2014-11-28

    Trp-Asp (WD) repeat protein 68 (WDR68) is an evolutionarily conserved WD40 repeat protein that binds to several proteins, including dual specificity tyrosine phosphorylation-regulated protein kinase (DYRK1A), MAPK/ERK kinase kinase 1 (MEKK1), and Cullin4-damage-specific DNA-binding protein 1 (CUL4-DDB1). WDR68 affects multiple and diverse physiological functions, such as controlling anthocyanin synthesis in plants, tissue growth in insects, and craniofacial development in vertebrates. However, the biochemical basis and the regulatory mechanism of WDR68 activity remain largely unknown. To better understand the cellular function of WDR68, here we have isolated and identified cellular WDR68 binding partners using a phosphoproteomic approach. More than 200 cellular proteins with wide varieties of biochemical functions were identified as WDR68-binding protein candidates. Eight T-complex protein 1 (TCP1) subunits comprising the molecular chaperone TCP1 ring complex/chaperonin-containing TCP1 (TRiC/CCT) were identified as major WDR68-binding proteins, and phosphorylation sites in both WDR68 and TRiC/CCT were identified. Co-immunoprecipitation experiments confirmed the binding between TRiC/CCT and WDR68. Computer-aided structural analysis suggested that WDR68 forms a seven-bladed β-propeller ring. Experiments with a series of deletion mutants in combination with the structural modeling showed that three of the seven β-propeller blades of WDR68 are essential and sufficient for TRiC/CCT binding. Knockdown of cellular TRiC/CCT by siRNA caused an abnormal WDR68 structure and led to reduction of its DYRK1A-binding activity. Concomitantly, nuclear accumulation of WDR68 was suppressed by the knockdown of TRiC/CCT, and WDR68 formed cellular aggregates when overexpressed in the TRiC/CCT-deficient cells. Altogether, our results demonstrate that the molecular chaperone TRiC/CCT is essential for correct protein folding, DYRK1A binding, and nuclear accumulation of WDR68.

  6. Low expression of secreted frizzled-related protein 2 and nuclear accumulation of β-catenin in aggressive nonfunctioning pituitary adenoma

    PubMed Central

    WU, YOUTU; BAI, JIWEI; HONG, LINCHUAN; LIU, CHUNHUI; YU, SHENGYUAN; YU, GUOQIANG; ZHANG, YAZHUO

    2016-01-01

    The identification of a specific molecular marker for aggressiveness of nonfunctioning pituitary adenomas (NFPAs) is urgently required in order to guide the clinical diagnosis and treatment of NFPAs. In the present study, low expression of secreted frizzled-related protein 2 (sFRP2) in NFPAs was demonstrated by reverse transcription-quantitative polymerase chain reaction, western blot and immunohistochemical analyses. The results confirmed an abnormal accumulation of free β-catenin in the nuclei of NFPAs, which is the core step for the activation of the Wnt canonical signaling pathway. Furthermore, cyclin D1 and c-Myc, the downstream proteins of the Wnt canonical signaling pathway, were overexpressed in aggressive NFPAs. These findings demonstrated the activation of the Wnt canonical signaling pathway in aggressive NFPAs. In addition, sFRP2 expression was observed to be inversely correlated to the aggressiveness of NFPAs. Therefore, sFRP2 may act as a tumor suppressor through modulation of the cellular cytosolic pool of β-catenin in NFPAs. Furthermore, the expression of sFRP2 may serve as a biomarker for NFPAs aggressiveness and prognosis. PMID:27347125

  7. The apple WD40 protein MdTTG1 interacts with bHLH but not MYB proteins to regulate anthocyanin accumulation.

    PubMed

    An, Xiu-Hong; Tian, Yi; Chen, Ke-Qin; Wang, Xiao-Fei; Hao, Yu-Jin

    2012-05-01

    The abundance of anthocyanins and proanthocyanins in apples is tightly regulated by three classes of regulatory factors, MYB, bHLH and WD40 proteins, only some of which have been previously identified. In this study, we identified an apple WD40 protein (MdTTG1) that promotes the accumulation of anthocyanins. The biosynthetic genes required downstream in the flavonoid pathway were up-regulated when MdTTG1 was over-expressed in Arabidopsis. Consistent with its role as a transcriptional regulator, an MdTTG1-GFP fusion protein was observed only in the nucleus. We assayed the expression patterns of this gene in different organs and found that they were positively correlated with anthocyanin accumulation in the apple. Yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that MdTTG1 interacted with bHLH transcription factors (TFs) but not MYB protein, whereas bHLH was known to interact with MYB in apples. However, based on a ChIP assay, MdTTG1 does not appear to bind to the promoter of the anthocyanin biosynthetic genes MdDFR and MdUFGT. Taken together, these results suggest that the apple WD40 protein MdTTG1 interacts with bHLH but not MYB proteins to regulate anthocyanin accumulation.

  8. Accumulation of p21 proteins at DNA damage sites independent of p53 and core NHEJ factors following irradiation

    SciTech Connect

    Koike, Manabu; Yutoku, Yasutomo; Koike, Aki

    2011-08-19

    Highlights: {yields} p21 accumulated rapidly at laser-irradiated sites via its C-terminal region. {yields} p21 colocalized with the DSB marker {gamma}-H2AX and the DSB sensor Ku80. {yields} Accumulation of p21 is dependent on PCNA, but not p53 and the NHEJ core factors. {yields} Accumulation activity of p21 was conserved among human and animal cells. {yields} p21 is a useful tool as a detection marker of DNA damaged sites. -- Abstract: The cyclin-dependent kinase (CDK) inhibitor p21 plays key roles in p53-dependent DNA-damage responses, i.e., cell cycle checkpoints, senescence, or apoptosis. p21 might also play a role in DNA repair. p21 foci arise at heavy-ion-irradiated DNA-double-strand break (DSB) sites, which are mainly repaired by nonhomologous DNA-end-joining (NHEJ). However, no mechanisms of p21 accumulation at double-strand break (DSB) sites have been clarified in detail. Recent works indicate that Ku70 and Ku80 are essential for the accumulation of other NHEJ core factors, e.g., DNA-PKcs, XRCC4 and XLF, and other DNA damage response factors, e.g., BRCA1. Here, we show that p21 foci arise at laser-irradiated sites in cells from various tissues from various species. The accumulation of EGFP-p21 was detected in not only normal cells, but also transformed or cancer cells. Our results also showed that EGFP-p21 accumulated rapidly at irradiated sites, and colocalized with the DSB marker {gamma}-H2AX and with the DSB sensor protein Ku80. On the other hand, the accumulation occurred in Ku70-, Ku80-, or DNA-PKcs-deficient cell lines and in human papillomavirus 18-positive cells, whereas the p21 mutant without the PCNA-binding region (EGFP-p21(1-146)) failed to accumulate at the irradiated sites. These findings suggest that the accumulation of p21, but not functional p53 and the NHEJ core factors, is dependent on PCNA. These findings also suggest that the accumulation activity of p21 at DNA damaged sites is conserved among human and animal cells, and p21 is a useful

  9. Renal mass reduction results in accumulation of lipids and dysregulation of lipid regulatory proteins in the remnant kidney.

    PubMed

    Kim, Hyun Ju; Moradi, Hamid; Yuan, Jun; Norris, Keith; Vaziri, Nosratola D

    2009-06-01

    A significant reduction of renal mass results in proteinuria, glomerulosclerosis, and tubulointerstitial injury, culminating in end-stage chronic renal failure (CRF). The accumulation of lipids in the kidney can cause renal disease. Uptake of oxidized lipoproteins via scavenger receptors, reabsorption of filtered protein-bound lipids via the megalin-cubilin complex, and increased glucose load per nephron can promote lipid accumulation in glomerular, tubular, and interstitial cells in CRF. Cellular lipid homeostasis is regulated by lipid influx, synthesis, catabolism, and efflux. We examined lipid-regulatory factors in the remnant kidney of rats 11 wk after nephrectomy (CRF) or sham operation. CRF resulted in azotemia, proteinuria, lipid accumulation in the kidney, upregulation of megalin, cubilin, mediators of lipid influx (scavenger receptor class A and lectin-like oxidized receptor-1), lipid efflux (liver X receptor alpha/beta and ATP-binding cassette transporter), and fatty acid biosynthesis (carbohydrate-response element binding protein, fatty acid synthase, and acetyl-CoA carboxylase). However, factors involved in cholesterol biosynthesis (sterol regulatory element binding protein, 3-hydroxy-3-methylglutaryl coenzyme A reductase, SCAP, Insig-1, and Insig-2) and fatty acid oxidation (peroxisome proliferator-activated receptor, acyl-CoA oxidase, and liver-type fatty acid binding protein) were reduced in the remnant kidney. Thus CRF results in heavy lipid accumulation in the remnant kidney, which is mediated by upregulation of pathways involved in tubular reabsorption of filtered protein-bound lipids, influx of oxidized lipoproteins and synthesis of fatty acids, and downregulation of pathways involved in fatty acid catabolism.

  10. Convergent Signaling Pathways Controlled by LRP1 (Receptor-related Protein 1) Cytoplasmic and Extracellular Domains Limit Cellular Cholesterol Accumulation.

    PubMed

    El Asmar, Zeina; Terrand, Jérome; Jenty, Marion; Host, Lionel; Mlih, Mohamed; Zerr, Aurélie; Justiniano, Hélène; Matz, Rachel L; Boudier, Christian; Scholler, Estelle; Garnier, Jean-Marie; Bertaccini, Diego; Thiersé, Danièle; Schaeffer, Christine; Van Dorsselaer, Alain; Herz, Joachim; Bruban, Véronique; Boucher, Philippe

    2016-03-04

    The low density lipoprotein receptor-related protein 1 (LRP1) is a ubiquitously expressed cell surface receptor that protects from intracellular cholesterol accumulation. However, the underlying mechanisms are unknown. Here we show that the extracellular (α) chain of LRP1 mediates TGFβ-induced enhancement of Wnt5a, which limits intracellular cholesterol accumulation by inhibiting cholesterol biosynthesis and by promoting cholesterol export. Moreover, we demonstrate that the cytoplasmic (β) chain of LRP1 suffices to limit cholesterol accumulation in LRP1(-/-) cells. Through binding of Erk2 to the second of its carboxyl-terminal NPXY motifs, LRP1 β-chain positively regulates the expression of ATP binding cassette transporter A1 (ABCA1) and of neutral cholesterol ester hydrolase (NCEH1). These results highlight the unexpected functions of LRP1 and the canonical Wnt5a pathway and new therapeutic potential in cholesterol-associated disorders including cardiovascular diseases.

  11. Alveolar abnormalities

    MedlinePlus

    ... page: //medlineplus.gov/ency/article/001093.htm Alveolar abnormalities To use the sharing features on this page, please enable JavaScript. Alveolar abnormalities are changes in the tiny air sacs in ...

  12. Nail abnormalities

    MedlinePlus

    Beau's lines; Fingernail abnormalities; Spoon nails; Onycholysis; Leukonychia; Koilonychia; Brittle nails ... 2012:chap 71. Zaiac MN, Walker A. Nail abnormalities associated with systemic pathologies. Clin Dermatol . 2013;31: ...

  13. Degradation of Ndd1 by APC/C(Cdh1) generates a feed forward loop that times mitotic protein accumulation.

    PubMed

    Sajman, Julia; Zenvirth, Drora; Nitzan, Mor; Margalit, Hanah; Simpson-Lavy, Kobi J; Reiss, Yuval; Cohen, Itamar; Ravid, Tommer; Brandeis, Michael

    2015-05-11

    Ndd1 activates the Mcm1-Fkh2 transcription factor to transcribe mitotic regulators. The anaphase-promoting complex/cyclosome activated by Cdh1 (APC/C(Cdh1)) mediates the degradation of proteins throughout G1. Here we show that the APC/C(Cdh1) ubiquitinates Ndd1 and mediates its degradation, and that APC/C(Cdh1) activity suppresses accumulation of Ndd1 targets. We confirm putative Ndd1 targets and identify novel ones, many of them APC/C(Cdh1) substrates. The APC/C(Cdh1) thus regulates these proteins in a dual manner—both pretranscriptionally and post-translationally, forming a multi-layered feedforward loop (FFL). We predict by mathematical modelling and verify experimentally that this FFL introduces a lag between APC/C(Cdh1) inactivation at the end of G1 and accumulation of genes transcribed by Ndd1 in G2. This regulation generates two classes of APC/C(Cdh1) substrates, early ones that accumulate in S and late ones that accumulate in G2. Our results show how the dual state APC/C(Cdh1) activity is converted into multiple outputs by interactions between its substrates.

  14. Consumption of sucrose from infancy increases the visceral fat accumulation, concentration of triglycerides, insulin and leptin, and generates abnormalities in the adrenal gland.

    PubMed

    Díaz-Aguila, Yadira; Castelán, Francisco; Cuevas, Estela; Zambrano, Elena; Martínez-Gómez, Margarita; Muñoz, Alvaro; Rodríguez-Antolín, Jorge; Nicolás-Toledo, Leticia

    2016-03-01

    Consumption of sugar-sweetened beverages promotes the development of metabolic syndrome (MetS) and type 2 diabetes mellitus in humans. One factor related to the appearance of MetS components is the dysfunction of the adrenal gland. In fact, the experimental generation of hyperglycemia has been associated with morphological and microvascular changes in the adrenal glands of rats. We hypothesized that high sucrose consumption from infancy promotes histological disruption of the adrenal glands associated with the appearance of metabolic syndrome indicators. Male Wistar rats were separated at weaning (21 days old) into two groups: free access to tap water (control group, C) or 30 % sucrose diluted in water (sugar-fed group). After 12 weeks, high sucrose consumption promoted an increase in visceral fat accumulation, adipose cell number, and insulin resistance. Also, a rise in the concentration of triglycerides, very low-density lipoprotein, insulin and leptin was observed. In control rats, a histomorphometric asymmetry between the right and left adrenal glands was found. In the sugar-fed group, sucrose consumption produced a major change in adrenal gland asymmetry. No changes in corticosterone serum level were observed in either group. Our results suggest that a high sucrose liquid-diet from early life alters the morphology of adrenocortical zones, leading to MetS indicators.

  15. Caspase-mediated cleavage of C53/LZAP protein causes abnormal microtubule bundling and rupture of the nuclear envelope.

    PubMed

    Wu, Jianchun; Jiang, Hai; Luo, Shouqing; Zhang, Mingsheng; Zhang, Yinghua; Sun, Fei; Huang, Shuang; Li, Honglin

    2013-05-01

    Apoptotic nucleus undergoes distinct morphological and biochemical changes including nuclear shrinkage, chromatin condensation and DNA fragmentation, which are attributed to caspase-mediated cleavage of several nuclear substrates such as lamins. As most of active caspases reside in the cytoplasm, disruption of the nuclear-cytoplasmic barrier is essential for caspases to reach their nuclear targets. The prevailing proposed mechanism is that the increase in the permeability of nuclear pores induced by caspases allows the caspases and other apoptotic factors to diffuse into the nucleus, thereby resulting in the nuclear destruction. Here, we report a novel observation that physical rupture of the nuclear envelope (NE) occurs in the early stage of apoptosis. We found that the NE rupture was caused by caspase-mediated cleavage of C53/LZAP, a protein that has been implicated in various signaling pathways, including NF-κB signaling and DNA damage response, as well as tumorigenesis and metastasis. We also demonstrated that C53/LZAP bound indirectly to the microtubule (MT), and expression of the C53/LZAP cleavage product caused abnormal MT bundling and NE rupture. Taken together, our findings suggest a novel role of C53/LZAP in the regulation of MT dynamics and NE structure during apoptotic cell death. Our study may provide an additional mechanism for disruption of the nuclear-cytoplasmic barrier during apoptosis.

  16. Caspase-mediated cleavage of C53/LZAP protein causes abnormal microtubule bundling and rupture of the nuclear envelope

    PubMed Central

    Wu, Jianchun; Jiang, Hai; Luo, Shouqing; Zhang, Mingsheng; Zhang, Yinghua; Sun, Fei; Huang, Shuang; Li, Honglin

    2013-01-01

    Apoptotic nucleus undergoes distinct morphological and biochemical changes including nuclear shrinkage, chromatin condensation and DNA fragmentation, which are attributed to caspase-mediated cleavage of several nuclear substrates such as lamins. As most of active caspases reside in the cytoplasm, disruption of the nuclear-cytoplasmic barrier is essential for caspases to reach their nuclear targets. The prevailing proposed mechanism is that the increase in the permeability of nuclear pores induced by caspases allows the caspases and other apoptotic factors to diffuse into the nucleus, thereby resulting in the nuclear destruction. Here, we report a novel observation that physical rupture of the nuclear envelope (NE) occurs in the early stage of apoptosis. We found that the NE rupture was caused by caspase-mediated cleavage of C53/LZAP, a protein that has been implicated in various signaling pathways, including NF-κB signaling and DNA damage response, as well as tumorigenesis and metastasis. We also demonstrated that C53/LZAP bound indirectly to the microtubule (MT), and expression of the C53/LZAP cleavage product caused abnormal MT bundling and NE rupture. Taken together, our findings suggest a novel role of C53/LZAP in the regulation of MT dynamics and NE structure during apoptotic cell death. Our study may provide an additional mechanism for disruption of the nuclear-cytoplasmic barrier during apoptosis. PMID:23478299

  17. Dysferlin and other non-red cell proteins accumulate in the red cell membrane of Diamond-Blackfan Anemia patients.

    PubMed

    Pesciotta, Esther N; Sriswasdi, Sira; Tang, Hsin-Yao; Speicher, David W; Mason, Philip J; Bessler, Monica

    2014-01-01

    Diamond Blackfan Anemia (DBA) is a congenital anemia usually caused by diverse mutations in ribosomal proteins. Although the genetics of DBA are well characterized, the mechanisms that lead to macrocytic anemia remain unclear. We systematically analyzed the proteomes of red blood cell membranes from multiple DBA patients to determine whether abnormalities in protein translation or erythropoiesis contribute to the observed macrocytosis or alterations in the mature red blood cell membrane. In depth proteome analysis of red cell membranes enabled highly reproducible identification and quantitative comparisons of 1100 or more proteins. These comparisons revealed clear differences between red cell membrane proteomes in DBA patients and healthy controls that were consistent across DBA patients with different ribosomal gene mutations. Proteins exhibiting changes in abundance included those known to be increased in DBA such as fetal hemoglobin and a number of proteins not normally found in mature red cell membranes, including proteins involved in the major histocompatibility complex class I pathway. Most striking was the presence of dysferlin in the red blood cell membranes of DBA patients but absent in healthy controls. Immunoblot validation using red cell membranes isolated from additional DBA patients and healthy controls confirmed a distinct membrane protein signature specific to patients with DBA.

  18. Endogenous salicylic acid levels correlate with accumulation of pathogenesis-related proteins and virus resistance in tobacco

    SciTech Connect

    Yalpani, N.; Shulaev, V.; Raskin, I. )

    1993-07-01

    Salicylic acid (SA) is hypothesized to be an endogenous regulator of local and systemic disease resistance and an inducer of pathogenesis-related (PR) proteins among plants. High levels of PR proteins have been observed in an uninoculated amphidiploid hybrid of Nicotiana glutinosa [times] N. debneyi, which is highly resistant to tobacco mosaic virus (TMV). Fluoresence, UV, and mass spectral analysis established that the levels of SA in healthy N. glutinosa [times] N. debneyi leaves were 30 times greater than in N. tabacum [open quotes]Xanthi-nc[close quotes] tobacco, which does not constitutively express PR proteins and is less resistant to TMV. Upon TMV-inoculation SA levels increased at least 70-fold leaves of Xanthi-nc but role only slightly in the hybrid. Phloem exudates of N. glutinosa [times] N. debneyi contained at least 500 times more SA than those of Xanthi-nc. SA treatment caused the appearance of PR-1 protein in Xanthi-nc but did not affect constitutively high levels of PR-1 protein in N. glutinosa [times] N. debneyi. In contrast to Xanthi-nc tobacco, TMV-inoculated N. glutinosa [times] N. debneyi kept at 32 C accumulated more than 0.5 [mu]g SA/g fresh weight, maintained high levels of PR proteins, and developed a hypersensitive response to TMV. PR proteins have previously been shown to accumulate in the lower leaves of healthy, flowering Xanthi-nc tobacco, which exhibited increased resistance to TMV. These developmentally induced increases in resistance and PR-1 proteins positively correlated with tissue levels of SA. These results affirm the regulatory role of SA in disease resistance and PR protein production. 31 refs., 9 figs., 1 tab.

  19. The Citrus transcription factor, CitERF13, regulates citric acid accumulation via a protein-protein interaction with the vacuolar proton pump, CitVHA-c4

    PubMed Central

    Li, Shao-jia; Yin, Xue-ren; Xie, Xiu-lan; Allan, Andrew C.; Ge, Hang; Shen, Shu-ling; Chen, Kun-song

    2016-01-01

    Organic acids are essential to fruit flavor. The vacuolar H+ transporting adenosine triphosphatase (V-ATPase) plays an important role in organic acid transport and accumulation. However, less is known of V-ATPase interacting proteins and their relationship with organic acid accumulation. The relationship between V-ATPase and citric acid was investigated, using the citrus tangerine varieties ‘Ordinary Ponkan (OPK)’ and an early maturing mutant ‘Zaoshu Ponkan (ZPK)’. Five V-ATPase genes (CitVHA) were predicted as important to citric acid accumulation. Among the genes, CitVHA-c4 was observed, using a yeast two-hybrid screen, to interact at the protein level with an ethylene response factor, CitERF13. This was verified using bimolecular fluorescence complementation assays. A similar interaction was also observed between Arabidopsis AtERF017 (a CitERF13 homolog) and AtVHA-c4 (a CitVHA-c4 homolog). A synergistic effect on citric acid levels was observed between V-ATPase proteins and interacting ERFs when analyzed using transient over-expression in tobacco and Arabidopsis mutants. Furthermore, the transcript abundance of CitERF13 was concomitant with CitVHA-c4. CitERF13 or AtERF017 over-expression leads to significant citric acid accumulation. This accumulation was abolished in an AtVHA-c4 mutant background. ERF-VHA interactions appear to be involved in citric acid accumulation, which was observed in both citrus and Arabidopsis. PMID:26837571

  20. Influence of Host Chloroplast Proteins on Tobacco mosaic virus Accumulation and Intercellular Movement1[C][W][OA

    PubMed Central

    Bhat, Sumana; Folimonova, Svetlana Y.; Cole, Anthony B.; Ballard, Kimberly D.; Lei, Zhentian; Watson, Bonnie S.; Sumner, Lloyd W.; Nelson, Richard S.

    2013-01-01

    Tobacco mosaic virus (TMV) forms dense cytoplasmic bodies containing replication-associated proteins (virus replication complexes [VRCs]) upon infection. To identify host proteins that interact with individual viral components of VRCs or VRCs in toto, we isolated viral replicase- and VRC-enriched fractions from TMV-infected Nicotiana tabacum plants. Two host proteins in enriched fractions, ATP-synthase γ-subunit (AtpC) and Rubisco activase (RCA) were identified by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry or liquid chromatography-tandem mass spectrometry. Through pull-down analysis, RCA bound predominantly to the region between the methyltransferase and helicase domains of the TMV replicase. Tobamovirus, but not Cucumber mosaic virus or Potato virus X, infection of N. tabacum plants resulted in 50% reductions in Rca and AtpC messenger RNA levels. To investigate the role of these host proteins in TMV accumulation and plant defense, we used a Tobacco rattle virus vector to silence these genes in Nicotiana benthamiana plants prior to challenge with TMV expressing green fluorescent protein. TMV-induced fluorescent lesions on Rca- or AtpC-silenced leaves were, respectively, similar or twice the size of those on leaves expressing these genes. Silencing Rca and AtpC did not influence the spread of Tomato bushy stunt virus and Potato virus X. In AtpC- and Rca-silenced leaves TMV accumulation and pathogenicity were greatly enhanced, suggesting a role of both host-encoded proteins in a defense response against TMV. In addition, silencing these host genes altered the phenotype of the TMV infection foci and VRCs, yielding foci with concentric fluorescent rings and dramatically more but smaller VRCs. The concentric rings occurred through renewed virus accumulation internal to the infection front. PMID:23096159

  1. Subcellular Clearance and Accumulation of Huntington Disease Protein: A Mini-Review

    PubMed Central

    Zhao, Ting; Hong, Yan; Li, Xiao-Jiang; Li, Shi-Hua

    2016-01-01

    Huntington’s disease (HD) is an autosomal dominant, progressive neurodegenerative disease caused by an expanded polyglutamine (polyQ) tract in the N-terminal region of mutant huntingtin (mHtt). As a result, mHtt forms aggregates that are abundant in the nuclei and processes of neuronal cells. Although the roles of mHtt aggregates are still debated, the formation of aggregates points to deficient clearance of mHtt in brain cells. Since the accumulation of mHtt is a prerequisite for its neurotoxicity, exploring the mechanisms for mHtt accumulation and clearance would advance our understanding of HD pathogenesis and help us develop treatments for HD. We know that the ubiquitin-proteasome system (UPS) and autophagy play important roles in clearing mHtt; however, how mHtt preferentially accumulates in neuronal nuclei and processes remains unclear. Studying the clearance of mHtt in neuronal cells is a challenge because neurons are morphologically and functionally polarized, which means the turnover of mHtt may be distinct in different cellular compartments. In this review, we discuss our current knowledge about the clearance and accumulation of mHtt and strategies examining mHtt clearance and accumulation in different subcellular regions. PMID:27147961

  2. Differential accumulation of photosynthetic proteins regulates diurnal photochemical adjustments of PSII in common fig (Ficus carica L.) leaves.

    PubMed

    Mlinarić, Selma; Antunović Dunić, Jasenka; Skendrović Babojelić, Martina; Cesar, Vera; Lepeduš, Hrvoje

    2017-02-01

    Molecular processes involved in photosystem II adaptation of woody species to diurnal changes in light and temperature conditions are still not well understood. Regarding this, here we investigated differences between young and mature leaves of common fig (Ficus carica L.) in photosynthetic performance as well as accumulation of the main photosynthetic proteins: light harvesting complex II, D1 protein and Rubisco large subunit. Investigated leaf types revealed different adjustment mechanisms to keep effective photosynthesis. Rather stable diurnal accumulation of light harvesting complex II in mature leaves enabled efficient excitation energy utilization (negative L-band) what triggered faster D1 protein degradation at high light. However, after photoinhibition, greater accumulation of D1 during the night enabled them faster recovery. So, the most photosynthetic parameters, as the maximum quantum yield for primary photochemistry, electron transport and overall photosynthetic efficiency in mature leaves successfully restored to their initial values at 1a.m. Reduced connectivity of light harvesting complexes II to its reaction centers (positive L-band) in young leaves increased dissipation of excess light causing less pressure to D1 and its slower degradation. Decreased electron transport in young leaves, due to reduced transfer beyond primary acceptor QA(-) most probably additionally induced degradation of Rubisco large subunit what consequently led to the stronger decrease of overall photosynthetic efficiency in young leaves at noon.

  3. A protein related to prokaryotic UMP kinases is involved in psaA/B transcript accumulation in Arabidopsis.

    PubMed

    Hein, Paul; Stöckel, Jana; Bennewitz, Stefan; Oelmüller, Ralf

    2009-03-01

    Dpt1 (defect in p saA/B transcript accumulation 1) is a novel photosystem (PS) I mutant in Arabidopsis. dpt1 mutants fail to grow photoautotrophically, and are impaired in the accumulation of psaA/B transcripts while the transcript levels for the remaining PSI subunits, for subunits of the PSII, the cyt-b ( 6 )/f-complex, and the ribulose-1,5-bisphosphate carboxylase are comparable to the wild type. In-organello run-on transcription assays demonstrate that the lower psaA/B transcript abundance in dpt1-1 is not caused by the inability to transcribe the psaA/psaB/rps14 operon. psaA/B transcripts in the mutant are associated with polyribosomes and translated. Thus, the mutation affects post-transcriptional processes specific for psaA/B. The dpt1 gene was isolated by map-based cloning. The protein is localized in the stroma of the chloroplast and exhibits striking similarities to UMP kinases of prokaryotic origin. Our results show that the nuclear encoded protein Dpt1 is essential for retaining photosynthetic activity in higher plant chloroplasts and involved in post-transcriptional steps of psaA/B transcript accumulation. We discuss that Dpt1 may be a bifunctional protein that couples the pyrimidine metabolism to the photosynthetic electron transport.

  4. The Thylakoid Membrane Protein CGL160 Supports CF1CF0 ATP Synthase Accumulation in Arabidopsis thaliana

    PubMed Central

    Fristedt, Rikard; Martins, Nádia Figueira; Strenkert, Daniela; Clarke, Cornelia A.; Suchoszek, Monika; Thiele, Wolfram; Schöttler, Mark Aurel; Merchant, Sabeeha S.

    2015-01-01

    The biogenesis of the major thylakoid protein complexes of the photosynthetic apparatus requires auxiliary proteins supporting individual assembly steps. Here, we identify a plant lineage specific gene, CGL160, whose homolog, atp1, co-occurs with ATP synthase subunits in an operon-like arrangement in many cyanobacteria. Arabidopsis thaliana T-DNA insertion mutants, which no longer accumulate the nucleus-encoded CGL160 protein, accumulate less than 25% of wild-type levels of the chloroplast ATP synthase. Severe cosmetic or growth phenotypes result under either short day or fluctuating light growth conditions, respectively, but this is ameliorated under long day constant light growth conditions where the growth, ATP synthase activity and photosynthetic electron transport of the mutants are less affected. Accumulation of other photosynthetic complexes is largely unaffected in cgl160 mutants, suggesting that CGL160 is a specific assembly or stability factor for the CF1CF0 complex. CGL160 is not found in the mature assembled complex but it does interact specifically with subunits of ATP synthase, predominantly those in the extrinsic CF1 sub-complex. We suggest therefore that it may facilitate the assembly of CF1 into the holocomplex. PMID:25835989

  5. Transgenic soya bean seeds accumulating β-carotene exhibit the collateral enhancements of oleate and protein content traits.

    PubMed

    Schmidt, Monica A; Parrott, Wayne A; Hildebrand, David F; Berg, R Howard; Cooksey, Amanda; Pendarvis, Ken; He, Yonghua; McCarthy, Fiona; Herman, Eliot M

    2015-05-01

    Transgenic soya bean (Glycine max) plants overexpressing a seed-specific bacterial phytoene synthase gene from Pantoea ananatis modified to target to plastids accumulated 845 μg β carotene g(-1) dry seed weight with a desirable 12:1 ratio of β to α. The β carotene accumulating seeds exhibited a shift in oil composition increasing oleic acid with a concomitant decrease in linoleic acid and an increase in seed protein content by at least 4% (w/w). Elevated β-carotene accumulating soya bean cotyledons contain 40% the amount of abscisic acid compared to nontransgenic cotyledons. Proteomic and nontargeted metabolomic analysis of the mid-maturation β-carotene cotyledons compared to the nontransgenic did not reveal any significant differences that would account for the altered phenotypes of both elevated oleate and protein content. Transcriptomic analysis, confirmed by RT-PCR, revealed a number of significant differences in ABA-responsive transcripton factor gene expression in the crtB transgenics compared to nontransgenic cotyledons of the same maturation stage. The altered seed composition traits seem to be attributed to altered ABA hormone levels varying transcription factor expression. The elevated β-carotene, oleic acid and protein traits in the β-carotene soya beans confer a substantial additive nutritional quality to soya beans.

  6. Overexpression of a stress-responsive U-box protein gene VaPUB affects the accumulation of resistance related proteins in Vitis vinifera 'Thompson Seedless'.

    PubMed

    Jiao, Li; Zhang, Yali; Lu, Jiang

    2017-03-01

    Many U-box proteins have been identified and characterized as important factors against environmental stresses such as chilling, heat, salinity and pathogen attack in plant. Our previous research reported the cloning of a novel U-box protein gene VaPUB from Vitis amurensis 'Zuoshanyi' grape and suggested a function of it in related to cold stress in the model plant Arabidopsis system. In this study, the role of VaPUB in response to biotic and abiotic stress was further analyzed in the homologous grapevine system by studying the transcript regulation and the protein accumulation in VaPUB transgenic vines. The expression analysis assay shown that VaPUB was significantly up-regulated 6 h after cold treatment and as early as 2 h post inoculation with Plasmopara viticola, a pathogen causing downy mildew disease in grapevine. Over-expressing VaPUB in V. Vinifera 'Thompson Seedless' affected the microstructure of leaves. The proteome assay shown that the accumulation of pathogenesis-related protein PR10 and many proteins involved in carbon and energy metabolism, oxidation reaction and protein metabolism were significantly altered in transgenic vines. In comparison with wild type plants, the expression level of PR10 family genes was significantly decreased in VaPUB transgenic vines under P. viticola treatment or cold stress. Results from this study showed that the U-box protein gene PUB quickly responded to both biotic stress and abiotic stress and significantly influenced the accumulation of resistance related proteins in grapevine.

  7. pFPL Vectors for High-Throughput Protein Localization in Fungi: Detecting Cytoplasmic Accumulation of Putative Effector Proteins.

    PubMed

    Gong, Xiaoyan; Hurtado, Oscar; Wang, Baohua; Wu, Congqing; Yi, Mihwa; Giraldo, Martha; Valent, Barbara; Goodin, Michael; Farman, Mark

    2015-02-01

    As part of a large-scale project whose goal was to identify candidate effector proteins in Magnaporthe oryzae, we developed a suite of vectors that facilitate high-throughput protein localization experiments in fungi. These vectors utilize Gateway recombinational cloning to place a gene's promoter and coding sequences upstream and in frame with enhanced cyan fluorescent protein, green fluorescent protein (GFP), monomeric red fluorescence protein (mRFP), and yellow fluorescent protein or a nucleus-targeted mCHERRY variant. The respective Gateway cassettes were incorporated into Agrobacterium-based plasmids to allow efficient fungal transformation using hygromycin or geneticin resistance selection. mRFP proved to be more sensitive than the GFP spectral variants for monitoring proteins secreted in planta; and extensive testing showed that Gateway-derived fusion proteins produced localization patterns identical to their "directly fused" counterparts. Use of plasmid for fungal protein localization (pFPL) vectors with two different selectable markers provided a convenient way to label fungal cells with different fluorescent proteins. We demonstrate the utility of the pFPL vectors for identifying candidate effector proteins and we highlight a number of important factors that must be taken into consideration when screening for proteins that are translocated across the host plasma membrane.

  8. A generalised module for the selective extracellular accumulation of recombinant proteins

    PubMed Central

    2012-01-01

    Background It is widely believed that laboratory strains of Escherichia coli, including those used for industrial production of proteins, do not secrete proteins to the extracellular milieu. Results Here, we report the development of a generalised module, based on an E. coli autotransporter secretion system, for the production of extracellular recombinant proteins. We demonstrate that a wide variety of structurally diverse proteins can be secreted as soluble proteins when linked to the autotransporter module. Yields were comparable to those achieved with other bacterial secretion systems. Conclusions The advantage of this module is that it relies on a relatively simple and easily manipulated secretion system, exhibits no apparent limitation to the size of the secreted protein and can deliver proteins to the extracellular environment at levels of purity and yields sufficient for many biotechnological applications. PMID:22640772

  9. Clusterin: full-length protein and one of its chains show opposing effects on cellular lipid accumulation

    PubMed Central

    Matukumalli, Suvarsha Rao; Tangirala, Ramakrishna; Rao, C. M.

    2017-01-01

    Proteins, made up of either single or multiple chains, are designed to carry out specific biological functions. We found an interesting example of a two-chain protein where administration of one of its chains leads to a diametrically opposite outcome than that reported for the full-length protein. Clusterin is a highly glycosylated protein consisting of two chains, α- and β-clusterin. We have investigated the conformational features, cellular localization, lipid accumulation, in vivo effects and histological changes upon administration of recombinant individual chains of clusterin. We demonstrate that recombinant α- and β-chains exhibit structural and functional differences and differ in their sub-cellular localization. Full-length clusterin is known to lower lipid levels. In contrast, we find that β-chain-treated cells accumulate 2-fold more lipid than controls. Interestingly, α-chain-treated cells do not show such increase. Rabbits injected with β-chain, but not α-chain, show ~40% increase in weight, with adipocyte hypertrophy, liver and kidney steatosis. Many, sometimes contrasting, roles are ascribed to clusterin in obesity, metabolic syndrome and related conditions. Our findings of differential localization and activities of individual chains of clusterin should help in understanding better the roles of clusterin in metabolism. PMID:28120874

  10. Casitas B-cell lymphoma (Cbl) proteins protect mammary epithelial cells from proteotoxicity of active c-Src accumulation

    PubMed Central

    Mukhopadhyay, Chandrani; Triplett, Aleata; Bargar, Tom; Heckman, Carol; Wagner, Kay-Uwe

    2016-01-01

    Casitas B-cell lymphoma (Cbl) family ubiquitin ligases negatively regulate tyrosine kinase-dependent signal transduction by promoting degradation of active kinases. We and others previously reported that loss of Cbl functions caused hyperproliferation in lymphoid and hematopoietic systems. Unexpectedly, Cbl deletion in Cbl-b–null, Cbl-c–null primary mouse mammary epithelial cells (MECs) (Cbl triple-deficiency) induced rapid cell death despite enhanced MAP kinase and AKT activation. Acute Cbl triple-deficiency elicited distinct transcriptional and biochemical responses with partial overlap with previously described cellular reactions to unfolded proteins and oxidative stress. Although the levels of reactive oxygen species were comparable, detergent-insoluble protein aggregates containing phosphorylated c-Src accumulated in Cbl triple-deficient MECs. Treatment with a broad-spectrum kinase inhibitor dasatinib blocked protein aggregate accumulation and restored in vitro organoid formation. This effect is most likely mediated through c-Src because Cbl triple-deficient MECs were able to form organoids upon shRNA-mediated c-Src knockdown. Taking these data together, the present study demonstrates that Cbl family proteins are required to protect MECs from proteotoxic stress-induced cell death by promoting turnover of active c-Src. PMID:27930322

  11. Two-Step Regulation of LAX PANICLE1 Protein Accumulation in Axillary Meristem Formation in Rice[W

    PubMed Central

    Oikawa, Tetsuo; Kyozuka, Junko

    2009-01-01

    Axillary meristem (AM) formation is an important determinant of plant architecture. In rice (Oryza sativa), LAX PANICLE1 (LAX1) function is required for the generation of AM throughout the plant's lifespan. Here, we show a close relationship between AM initiation and leaf development; specifically, the plastochron 4 (P4) stage of leaf development is crucial for the proliferation of meristematic cells. Coincident with this, LAX1 expression starts in the axils of leaves at P4 stage. LAX1 mRNA accumulates in two to three layers of cells in the boundary region between the initiating AM and the shoot apical meristem. In lax1 mutants, the proliferation of meristematic cells is initiated but fails to progress into the formation of AM. The difference in sites of LAX1 mRNA expression and its action suggests non-cell-autonomous characteristics of LAX1 function. We found that LAX1 protein is trafficked to AM in a stage- and direction-specific manner. Furthermore, we present evidence that LAX1 protein movement is required for the full function of LAX1. Thus, we propose that LAX1 protein accumulates transiently in the initiating AM at P4 stage by a strict regulation of mRNA expression and a subsequent control of protein trafficking. This two-step regulation is crucial to the establishment of the new AM. PMID:19346465

  12. HCV core protein induces hepatic lipid accumulation by activating SREBP1 and PPAR{gamma}

    SciTech Connect

    Kim, Kook Hwan; Hong, Sung Pyo; Kim, KyeongJin; Park, Min Jung; Kim, Kwang Jin; Cheong, JaeHun . E-mail: molecule85@pusan.ac.kr

    2007-04-20

    Hepatic steatosis is a common feature in patients with chronic hepatitis C virus (HCV) infection. HCV core protein plays an important role in the development of hepatic steatosis in HCV infection. Because SREBP1 (sterol regulatory element binding protein 1) and PPAR{gamma} (peroxisome proliferators-activated receptor {gamma}) are involved in the regulation of lipid metabolism of hepatocyte, we sought to determine whether HCV core protein may impair the expression and activity of SREBP1 and PPAR{gamma}. In this study, it was demonstrated that HCV core protein increases the gene expression of SREBP1 not only in Chang liver, Huh7, and HepG2 cells transiently transfected with HCV core protein expression plasmid, but also in Chang liver-core stable cells. Furthermore, HCV core protein enhanced the transcriptional activity of SREBP1. In addition, HCV core protein elevated PPAR{gamma} transcriptional activity. However, HCV core protein had no effect on PPAR{gamma} gene expression. Finally, we showed that HCV core protein stimulates the genes expression of lipogenic enzyme and fatty acid uptake associated protein. Therefore, our finding provides a new insight into the mechanism of hepatic steatosis by HCV infection.

  13. Accumulation of 8-oxo-2'-deoxyguanosine and increased expression of hMTH1 protein in brain tumors.

    PubMed Central

    Iida, T.; Furuta, A.; Kawashima, M.; Nishida, J.; Nakabeppu, Y.; Iwaki, T.

    2001-01-01

    Oxidative DNA damage generated by an attack of reactive oxygen species causes mutation or cell death that may lead to various diseases and may be related to initiation or progression of carcinogenesis. 8-Oxo-2'-deoxyguanosine (8-oxo-dG) is a major oxidative DNA damage product that can result in mutation, and hMTH1, human MutT homolog protein 1, has been identified as an enzyme that hydrolyzes 8-oxo-dGTP to the monophosphate, thus preventing accumulation of 8-oxo-dG in DNA. With immunohistochemical approaches, we investigated accumulation of 8-oxo-dG and expression of hMTH1 in brain tumor tissues obtained from surgical and autopsy cases, including 42 neuroepithelial tumors, 5 meningiomas, 2 metastatic brain tumors, and 1 schwannoma. 8-Oxo-dG accumulation and hMTH1 expression were increased in various brain tumors. Nuclei of brain tumor cells were immunoreactive for 8-oxo-dG in all cases. In most cases, both nuclei and cytoplasm of the tumor cells were immunoreactive for hMTH1. Both 8-oxo-dG accumulation and hMTH1 expression were most evident in high-grade gliomas, indicating that oxidative stress was high in these gliomas. Thus, the defense mechanism against such oxidative stress may be enhanced as well. These results suggest that oxidative stress may play a role in tumor progression. PMID:11296483

  14. Abnormal Expression of Urea Transporter Protein in a Rat Model of Hepatorenal Syndrome Induced by Succinylated Gelatin

    PubMed Central

    Song, Weiping; Qi, Xiaolong; Zhang, Wenhui; Zhao, C Yingying; Cao, Yan; Wang, Fei; Yang, Changqing

    2015-01-01

    Background Hepatorenal syndrome (HRS) is a serious complication of advanced chronic liver disease. Abdominal compartment syndrome (ACS) occurs with dysfunction of multiple organs when abdominal pressure increases. Here, we report on a novel model of ACS with ascites and a model of HRS in rats to observe the urea transporter protein (UT) expression in the 2 models. Material/Methods A liver cirrhosis model was induced by CCl4. After changes of liver histopathology were observed, rats were injected intraperitoneally with succinylated gelatin to establish a model of ACS and HRS. Then, changes in BUN, Cr, and renal histopathology were detected. Moreover, the UT in ACS and HRS were also quantified. Results The surfaces of liver in the cirrhotic group became coarse, with visible small nodules and became yellow and greasy. The normal structure of the hepatic lobules were destroyed, and hyperplasia of fibrotic tissue and pseudo-lobe was observed. The levels of BUN and Cr were significantly increased in rats suffering from ACS and HRS, respectively, compared to their control groups. In addition, the mRNA levels of UT-A2 and UT-A3 decreased in rats with HRS compared to cirrhotic rats. However, there was no significant difference between the mRNA levels of UT-A2, UT-A3, and UT-B in rats with ACS vs. normal rats. Conclusions It is feasible to model ACS in rats by injecting succinylated gelatin into the abdominal cavity. Increasing the intra-abdominal pressure by succinylated gelatin is also a novel approach for modeling HRS in cirrhotic rats. Compared with control rats, there is an abnormal mRNA expression of UT in ACS rats and HRS rats. PMID:26414230

  15. Essentiality Is a Strong Determinant of Protein Rates of Evolution during Mutation Accumulation Experiments in Escherichia coli.

    PubMed

    Alvarez-Ponce, David; Sabater-Muñoz, Beatriz; Toft, Christina; Ruiz-González, Mario X; Fares, Mario A

    2016-09-26

    The Neutral Theory of Molecular Evolution is considered the most powerful theory to understand the evolutionary behavior of proteins. One of the main predictions of this theory is that essential proteins should evolve slower than dispensable ones owing to increased selective constraints. Comparison of genomes of different species, however, has revealed only small differences between the rates of evolution of essential and nonessential proteins. In some analyses, these differences vanish once confounding factors are controlled for, whereas in other cases essentiality seems to have an independent, albeit small, effect. It has been argued that comparing relatively distant genomes may entail a number of limitations. For instance, many of the genes that are dispensable in controlled lab conditions may be essential in some of the conditions faced in nature. Moreover, essentiality can change during evolution, and rates of protein evolution are simultaneously shaped by a variety of factors, whose individual effects are difficult to isolate. Here, we conducted two parallel mutation accumulation experiments in Escherichia coli, during 5,500-5,750 generations, and compared the genomes at different points of the experiments. Our approach (a short-term experiment, under highly controlled conditions) enabled us to overcome many of the limitations of previous studies. We observed that essential proteins evolved substantially slower than nonessential ones during our experiments. Strikingly, rates of protein evolution were only moderately affected by expression level and protein length.

  16. Accumulation of the Drosophila Torso-like protein at the blastoderm plasma membrane suggests that it translocates from the eggshell.

    PubMed

    Mineo, Alessandro; Furriols, Marc; Casanova, Jordi

    2015-04-01

    The eggshell serves as a depository for proteins that play an important role in early embryonic development. In particular, the Drosophila eggshell is responsible for transferring asymmetries from the egg chamber to specify the regions at both ends of the embryo through the uneven activation of the Torso (Tor) receptor in its membrane. This process relies on the restricted expression of the gene torso-like (tsl) in subpopulations of follicle cells during oogenesis and its protein accumulation at both poles of the eggshell, but it is not known how this signal is transmitted to the embryo. Here, we show that Tsl accumulates at the embryonic plasma membrane, even in the absence of the Tor receptor. However, during oogenesis, we detected Tsl accumulation only at the eggshell. These results suggest that there is a two-step mechanism to transfer the asymmetric positional cues from the egg chamber into the early embryo: initial anchoring of Tsl at the eggshell as it is secreted, followed by its later translocation to the egg plasma membrane, where it enables Tor receptor activation. Translocation of anchored determinants from the eggshell might then regulate the spatial and temporal control of early embryonic developmental processes.

  17. A Sugar-Inducible Protein Kinase, VvSK1, Regulates Hexose Transport and Sugar Accumulation in Grapevine Cells

    PubMed Central

    Lecourieux, Fatma; Lecourieux, David; Vignault, Céline; Delrot, Serge

    2010-01-01

    In grapevine (Vitis vinifera), as in many crops, soluble sugar content is a major component of yield and economical value. This paper identifies and characterizes a Glycogen Synthase Kinase3 protein kinase, cloned from a cDNA library of grape Cabernet Sauvignon berries harvested at the ripening stage. This gene, called VvSK1, was mainly expressed in flowers, berries, and roots. In the berries, it was strongly expressed at postvéraison, when the berries accumulate glucose, fructose, and abscisic acid. In grapevine cell suspensions, VvSK1 transcript abundance is increased by sugars and abscisic acid. In transgenic grapevine cells overexpressing VvSK1, the expression of four monosaccharide transporters (VvHT3, VvHT4, VvHT5, and VvHT6) was up-regulated, the rate of glucose uptake was increased 3- to 5-fold, and the amount of glucose and sucrose accumulation was more than doubled, while the starch amount was not affected. This work provides, to our knowledge, the first example of the control of sugar uptake and accumulation by a sugar-inducible protein kinase. PMID:19923236

  18. Accumulation of stress protein 72 (HSP72) in muscle and spleen of goldfish taken into space

    NASA Astrophysics Data System (ADS)

    Ohnishi, T.; Tsuji, K.; Ohmura, T.; Matsumoto, H.; Wang, X.; Takahahsi, A.; Nagaoka, S.; Takabayashi, A.

    Using Western blot analysis, here, we report the levels of HSP72 in several organs from goldfish which were taken into space on the NASA space shuttle. A remarkable accumulation of HSP72 was detected in muscle and spleen of those fish taken into space as compared with controls. These results suggested that the HSP72 induction is a kind of stress response at the molecular level introduced by the space environment consisting of microgravity and/or cosmic radiation as stressors.

  19. Protein glycosylation and advanced glycosylated endproducts (AGEs) accumulation: an avian solution?

    PubMed

    Iqbal, M; Probert, L L; Alhumadi, N H; Klandorf, H

    1999-04-01

    The objectives of this study were to determine the effect of diet restriction (DR) and the crosslinking inhibitor, aminoguanidine (AG), on PMA-induced respiratory burst, concentrations of uric acid, and the rate of pentosidine accumulation in the skin (Ps) of naturally hyperglycemic broiler breeder hens. Female chicks (n = 450) were randomly assigned to four groups from 8 to 92 weeks after hatch: ad libitum (AL), diet restricted (DR), AL and DR groups supplemented with 400 ppm AG each (AL + AG and DR + AG). No consistent effects of treatments were observed on plasma concentrations of glucose. The accumulation of Ps in AL birds increased linearly with age (p < .001) and was significantly retarded in all treatment groups (p < .001). Ps in the AL + AG group was comparable to that in the DR or DR + AG groups. PMA-induced respiratory bursts in blood leukocytes were significantly retarded in DR or AG-supplemented (p < .0001) groups. Although there was a marginal increase in overall mean concentrations of plasma uric acid for the DR group, no consistent differences were observed on individual time points. It is concluded that the glycosylation process may not be the primary cause of glucose-derived crosslinks and that the accumulation of Ps can be retarded by DR and AG in broiler breeder hens.

  20. Electrical nerve stimulation and the relief of chronic pain through regulation of the accumulation of synaptic Arc protein.

    PubMed

    Liu, Yue-peng; Liu, Su

    2013-08-01

    Electrical nerve stimulation (ENS) is used in clinical settings for the treatment of chronic pain, but the mechanism underlying its effects remains unknown. ENS has been found to mimic neural activity, inducing the accumulation of Arc in synapses. Activity-dependent synaptic accumulation of Arc protein has been shown to reduce synaptic strength by promoting endocytosis of the AMPA receptors in the synaptic membrane. These receptors play a decisive role in central sensitization, which is one of the main mechanisms underlying chronic pain. It is here hypothesized that ENS induces Arc expression in synapses, where Arc promotes endocytosis of membrane AMPARs that are up-regulated during chronic pain. High frequency and high intensity are characteristics of ENS, which may be effective in the treatment of chronic pain. Stimulation-site of ENS may also influence the outcome of ENS.

  1. MdHIR proteins repress anthocyanin accumulation by interacting with the MdJAZ2 protein to inhibit its degradation in apples

    PubMed Central

    Chen, Ke-Qin; Zhao, Xian-Yan; An, Xiu-Hong; Tian, Yi; Liu, Dan-Dan; You, Chun-Xiang; Hao, Yu-Jin

    2017-01-01

    In higher plants, jasmonate ZIM-domain (JAZ) proteins negatively regulate the biosynthesis of anthocyanins by interacting with bHLH transcription factors. However, it is largely unknown if and how other regulators are involved in this process. In this study, the apple MdJAZ2 protein was characterized in regards to its function in the negative regulation of anthocyanin accumulation and peel coloration. MdJAZ2 was used as a bait to screen a cDNA library using the yeast two-hybrid method. The hypersensitive induced reaction (HIR) proteins, MdHIR2 and MdHIR4, were obtained from this yeast two-hybrid. The ZIM domain of MdJAZ2 and the PHB domain of the MdHIR proteins are necessary for their interactions. The interactions were further verified using an in vitro pull-down assay. Subsequently, immunoblotting assays demonstrated that MdHIR4 enhanced the stability of the MdJAZ2-GUS protein. Finally, a viral vector-based transformation method showed that MdHIR4 inhibited anthocyanin accumulation and fruit coloration in apple by modulating the expression of genes associated with anthocyanin biosynthesis. PMID:28317851

  2. Abnormal amounts of intracellular calcium regulatory proteins in SHRSP.Z-Lepr(fa)/IzmDmcr rats with metabolic syndrome and cardiac dysfunction.

    PubMed

    Kagota, Satomi; Maruyama, Kana; Tada, Yukari; Wakuda, Hirokazu; Nakamura, Kazuki; Kunitomo, Masaru; Shinozuka, Kazumasa

    2013-02-01

    Metabolic syndrome is known to increase the risk of abnormal cardiac structure and function, which are considered to contribute to increased incidence of cardiovascular disease and mortality. We previously demonstrated that ventricular hypertrophy and diastolic dysfunction occur in SHRSP.Z-Lepr(fa)/IzmDmcr (SHRSP fatty) rats with metabolic syndrome. The aim of this study was to investigate the possible mechanisms underlying abnormal heart function in SHRSP fatty rats. The amount of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) 2a, phospholamban (PLB) protein, and Ser(16)-phosphorylated PLB was decreased in cardiomyocytes from SHRSP fatty rats compared with those from control Wistar-Kyoto rats at 18 weeks of age, and the PLB-to-SERCA2a ratio was increased. Left ventricular developed pressure was unchanged, and coronary flow rate and maximum rate of left ventricular pressure decline (-dP/dt) was decreased in SHRSP fatty rats. Treatment with telmisartan reversed the abnormalities of PLB amount, coronary flow rate, and -dP/dt in SHRSP fatty rats. These results indicate that abnormal amounts of intracellular Ca(2+) regulatory proteins in cardiomyocytes, leading to reduced intracellular Ca(2+) reuptake into the sarcoplasmic reticulum, may play a role in the diastolic dysfunction in SHRSP fatty rats and that these effects are partially related to decreased coronary circulation. Telmisartan may be beneficial in protecting against disturbances in cardiac function associated with metabolic syndrome.

  3. β-Propeller protein-associated neurodegeneration: a new X-linked dominant disorder with brain iron accumulation.

    PubMed

    Hayflick, Susan J; Kruer, Michael C; Gregory, Allison; Haack, Tobias B; Kurian, Manju A; Houlden, Henry H; Anderson, James; Boddaert, Nathalie; Sanford, Lynn; Harik, Sami I; Dandu, Vasuki H; Nardocci, Nardo; Zorzi, Giovanna; Dunaway, Todd; Tarnopolsky, Mark; Skinner, Steven; Holden, Kenton R; Frucht, Steven; Hanspal, Era; Schrander-Stumpel, Connie; Mignot, Cyril; Héron, Delphine; Saunders, Dawn E; Kaminska, Margaret; Lin, Jean-Pierre; Lascelles, Karine; Cuno, Stephan M; Meyer, Esther; Garavaglia, Barbara; Bhatia, Kailash; de Silva, Rajith; Crisp, Sarah; Lunt, Peter; Carey, Martyn; Hardy, John; Meitinger, Thomas; Prokisch, Holger; Hogarth, Penelope

    2013-06-01

    Neurodegenerative disorders with high iron in the basal ganglia encompass an expanding collection of single gene disorders collectively known as neurodegeneration with brain iron accumulation. These disorders can largely be distinguished from one another by their associated clinical and neuroimaging features. The aim of this study was to define the phenotype that is associated with mutations in WDR45, a new causative gene for neurodegeneration with brain iron accumulation located on the X chromosome. The study subjects consisted of WDR45 mutation-positive individuals identified after screening a large international cohort of patients with idiopathic neurodegeneration with brain iron accumulation. Their records were reviewed, including longitudinal clinical, laboratory and imaging data. Twenty-three mutation-positive subjects were identified (20 females). The natural history of their disease was remarkably uniform: global developmental delay in childhood and further regression in early adulthood with progressive dystonia, parkinsonism and dementia. Common early comorbidities included seizures, spasticity and disordered sleep. The symptoms of parkinsonism improved with l-DOPA; however, nearly all patients experienced early motor fluctuations that quickly progressed to disabling dyskinesias, warranting discontinuation of l-DOPA. Brain magnetic resonance imaging showed iron in the substantia nigra and globus pallidus, with a 'halo' of T1 hyperintense signal in the substantia nigra. All patients harboured de novo mutations in WDR45, encoding a beta-propeller protein postulated to play a role in autophagy. Beta-propeller protein-associated neurodegeneration, the only X-linked disorder of neurodegeneration with brain iron accumulation, is associated with de novo mutations in WDR45 and is recognizable by a unique combination of clinical, natural history and neuroimaging features.

  4. Differential accumulation of a 24-kd dehydrin protein in wheat seedlings correlates with drought stress tolerance at grain filling.

    PubMed

    Lopez, C G; Banowetz, G; Peterson, C J; Kronstad, W E

    2001-01-01

    The Pacific Northwest (PNW), an important region for wheat production in the USA, is often subject to water deficits during sowing and grain filling. These deficits reduce the quality and yield of the crop. As a consequence, an important objective of breeding programs in the region is improving the genetic adaptation of wheat cultivars to drought stress. One response to dehydrative stresses is the accumulation of proteins called dehydrins, which are believed to protect membranes and macromolecules against denaturation. We characterized dehydrin accumulation in seedlings during drought stress and its correlation with stress tolerance during grain filling in seven wheat cultivars, 'Connie', 'Gene', 'TAM105', 'Rod', 'Hiller', 'Rhode', and 'Stephens'. A 24-kd dehydrin accumulated in seedlings under stress, but not in irrigated control plants. Connie, TAM105, and Gene started to accumulate dehydrins at the fourth day of stress, while the other cultivars showed dehydrins after twelve days of stress. This differential accumulation in seedlings was associated with stress tolerance at grain filling, characterized by a lower reduction in yield and in the rate of decrease in leaf water potential per day of stress. Connie, TAM105, Gene and Rod where the most tolerant cultivars. The results indicate that expression of this 24-kd dehydrin might serve as a rapid and non-destructive screening technique at the seedling stage. Even though the results are promising, selection experiments using a population segregating for stress tolerance are needed to test more conclusively whether this dehydrin can serve as a genetic marker for cultivars with tolerance to drought stress.

  5. Crystal Structure of Okadaic Acid Binding Protein 2.1: A Sponge Protein Implicated in Cytotoxin Accumulation.

    PubMed

    Ehara, Haruhiko; Makino, Marie; Kodama, Koichiro; Konoki, Keiichi; Ito, Takuhiro; Sekine, Shun-ichi; Fukuzawa, Seketsu; Yokoyama, Shigeyuki; Tachibana, Kazuo

    2015-07-06

    Okadaic acid (OA) is a marine polyether cytotoxin that was first isolated from the marine sponge Halichondria okadai. OA is a potent inhibitor of protein serine/threonine phosphatases (PP) 1 and 2A, and the structural basis of phosphatase inhibition has been well investigated. However, the role and mechanism of OA retention in the marine sponge have remained elusive. We have solved the crystal structure of okadaic acid binding protein 2.1 (OABP2.1) isolated from H. okadai; it has strong affinity for OA and limited sequence homology to other proteins. The structure revealed that OABP2.1 consists of two α-helical domains, with the OA molecule deeply buried inside the protein. In addition, the global fold of OABP2.1 was unexpectedly similar to that of aequorin, a jellyfish photoprotein. The presence of structural homologues suggested that, by using similar protein scaffolds, marine invertebrates have developed diverse survival systems adapted to their living environments.

  6. A protein kinase binds the C-terminal domain of the readthrough protein of Turnip yellows virus and regulates virus accumulation

    SciTech Connect

    Rodriguez-Medina, Caren; Boissinot, Sylvaine; Chapuis, Sophie; Gereige, Dalya; Rastegar, Maryam; Erdinger, Monique; Revers, Frédéric; Ziegler-Graff, Véronique; Brault, Véronique

    2015-12-15

    Turnip yellows virus (TuYV), a phloem-limited virus, encodes a 74 kDa protein known as the readthrough protein (RT) involved in virus movement. We show here that a TuYV mutant deleted of the C-terminal part of the RT protein (TuYV-∆RT{sub Cter}) was affected in long-distance trafficking in a host-specific manner. By using the C-terminal domain of the RT protein as a bait in a yeast two-hybrid screen of a phloem cDNA library from Arabidopsis thaliana we identified the calcineurin B-like protein-interacting protein kinase-7 (AtCIPK7). Transient expression of a GFP:CIPK7 fusion protein in virus-inoculated Nicotiana benthamiana leaves led to local increase of wild-type TuYV accumulation, but not that of TuYV-∆RT{sub Cter}. Surprisingly, elevated virus titer in inoculated leaves did not result in higher TuYV accumulation in systemic leaves, which indicates that virus long-distance movement was not affected. Since GFP:CIPK7 was localized in or near plasmodesmata, CIPK7 could negatively regulate TuYV export from infected cells. - Highlights: • The C-terminal domain of TuYV-RT is required for long-distance movement. • CIPK7 from Arabidopsis interacts with RT{sub Cter} in yeast and in plants. • CIPK7 overexpression increases virus titer locally but not virus systemic movement. • CIPK7 localizes to plasmodesmata. • CIPK7 could be a defense protein regulating virus export.

  7. Heterologous expression of AtClo1, a plant oil body protein, induces lipid accumulation in yeast.

    PubMed

    Froissard, Marine; D'andréa, Sabine; Boulard, Céline; Chardot, Thierry

    2009-05-01

    Proteomic approaches on lipid bodies have led to the identification of proteins associated with this compartment, showing that, rather than the inert fat depot, lipid droplets appear as complex dynamic organelles with roles in metabolism control and cell signaling. We focused our investigations on caleosin [Arabidopsis thaliana caleosin 1 (AtClo1)], a minor protein of the Arabidopsis thaliana seed lipid body. AtClo1 shares an original triblock structure, which confers to the protein the capacity to insert at the lipid body surface. In addition, AtClo1 possesses a calcium-binding domain. The study of plants deficient in caleosin revealed its involvement in storage lipid degradation during seed germination. Using Saccharomyces cerevisiae as a heterologous expression system, we investigated the potential role of AtClo1 in lipid body biogenesis and filling. The green fluorescent protein-tagged protein was correctly targeted to lipid bodies. We observed an increase in the number and size of lipid bodies. Moreover, transformed yeasts accumulated more fatty acids (+46.6%). We confirmed that this excess of fatty acids was due to overaccumulation of lipid body neutral lipids, triacylglycerols and steryl esters. We showed that the original intrinsic properties of AtClo1 protein were sufficient to generate a functional lipid body membrane and to promote overaccumulation of storage lipids in yeast oil bodies.

  8. Meiotic abnormalities

    SciTech Connect

    1993-12-31

    Chapter 19, describes meiotic abnormalities. These include nondisjunction of autosomes and sex chromosomes, genetic and environmental causes of nondisjunction, misdivision of the centromere, chromosomally abnormal human sperm, male infertility, parental age, and origin of diploid gametes. 57 refs., 2 figs., 1 tab.

  9. Stable Accumulation of Modified 2S Albumin Seed Storage Proteins with Higher Methionine Contents in Transgenic Plants 1

    PubMed Central

    De Clercq, Ann; Vandewiele, Martine; Van Damme, Jozef; Guerche, Philippe; Van Montagu, Marc; Vandekerckhove, Joël; Krebbers, Enno

    1990-01-01

    We present the results of two sets of experiments designed to express high methionine proteins in transgenic seeds in three different plant species. In the first approach, two chimeric genes were constructed in which parts of the Arabidopsis 2S albumin gene 1 (AT2S1) were fused at different positions to a Brazil nut 2S albumin cDNA clone. Brazil nut 2S albumin was found to accumulate stably in transgenic Arabidopsis, Brassica napus, and tobacco seeds. In the second approach, methionine-enriched AT2S1 genes were constructed by deleting sequences encoding a region of the protein which is not highly conserved among 2S albumins of different species and replacing them with methioninerich sequences. Introduction of the modified AT2S1 genes into three different plant species resulted in the accumulation of the methionine-enriched 2S albumins in all three species at levels reaching 1 to 2% of the total high salt-extractable seed protein. Images Figure 3 Figure 4 PMID:16667878

  10. A mutation in the CASQ1 gene causes a vacuolar myopathy with accumulation of sarcoplasmic reticulum protein aggregates.

    PubMed

    Rossi, Daniela; Vezzani, Bianca; Galli, Lucia; Paolini, Cecilia; Toniolo, Luana; Pierantozzi, Enrico; Spinozzi, Simone; Barone, Virginia; Pegoraro, Elena; Bello, Luca; Cenacchi, Giovanna; Vattemi, Gaetano; Tomelleri, Giuliano; Ricci, Giulia; Siciliano, Gabriele; Protasi, Feliciano; Reggiani, Carlo; Sorrentino, Vincenzo

    2014-10-01

    A missense mutation in the calsequestrin-1 gene (CASQ1) was found in a group of patients with a myopathy characterized by weakness, fatigue, and the presence of large vacuoles containing characteristic inclusions resulting from the aggregation of sarcoplasmic reticulum (SR) proteins. The mutation affects a conserved aspartic acid in position 244 (p.Asp244Gly) located in one of the high-affinity Ca(2+) -binding sites of CASQ1 and alters the kinetics of Ca(2+) release in muscle fibers. Expression of the mutated CASQ1 protein in COS-7 cells showed a markedly reduced ability in forming elongated polymers, whereas both in cultured myotubes and in in vivo mouse fibers induced the formation of electron-dense SR vacuoles containing aggregates of the mutant CASQ1 protein that resemble those observed in muscle biopsies of patients. Altogether, these results support the view that a single missense mutation in the CASQ1 gene causes the formation of abnormal SR vacuoles containing aggregates of CASQ1, and other SR proteins, results in altered Ca(2+) release in skeletal muscle fibers, and, hence, is responsible for the clinical phenotype observed in these patients.

  11. Angiopoietin Like Protein 2 (ANGPTL2) Promotes Adipose Tissue Macrophage and T lymphocyte Accumulation and Leads to Insulin Resistance

    PubMed Central

    Sasaki, Yusuke; Ohta, Masayuki; Desai, Dhruv; Figueiredo, Jose-Luiz; Whelan, Mary C.; Sugano, Tomohiro; Yamabi, Masaki; Yano, Wataru; Faits, Tyler; Yabusaki, Katsumi; Zhang, Hengmin; Mlynarchik, Andrew K.; Inoue, Keisuke; Mizuno, Ken; Aikawa, Masanori

    2015-01-01

    Objectives Angiopoietin-like protein 2 (ANGPTL2), a recently identified pro-inflammatory cytokine, is mainly secreted from the adipose tissue. This study aimed to explore the role of ANGPTL2 in adipose tissue inflammation and macrophage activation in a mouse model of diabetes. Methodology/Principal Findings Adenovirus mediated lacZ (Ad-LacZ) or human ANGPTL2 (Ad-ANGPTL2) was delivered via tail vein in diabetic db/db mice. Ad-ANGPTL2 treatment for 2 weeks impaired both glucose tolerance and insulin sensitivity as compared to Ad-LacZ treatment. Ad-ANGPTL2 treatment significantly induced pro-inflammatory gene expression in white adipose tissue. We also isolated stromal vascular fraction from epididymal fat pad and analyzed adipose tissue macrophage and T lymphocyte populations by flow cytometry. Ad-ANGPTL2 treated mice had more adipose tissue macrophages (F4/80+CD11b+) and a larger M1 macrophage subpopulation (F4/80+CD11b+CD11c+). Moreover, Ad-ANGPTL2 treatment increased a CD8-positive T cell population in adipose tissue, which preceded increased macrophage accumulation. Consistent with our in vivo results, recombinant human ANGPTL2 protein treatment increased mRNA levels of pro-inflammatory gene products and production of TNF-α protein in the human macrophage-like cell line THP-1. Furthermore, Ad-ANGPTL2 treatment induced lipid accumulation and increased fatty acid synthesis, lipid metabolism related gene expression in mouse liver. Conclusion ANGPTL2 treatment promotes macrophage accumulation and activation. These results suggest potential mechanisms for insulin resistance. PMID:26132105

  12. Responsibility of regulatory gene expression and repressed protein synthesis for triacylglycerol accumulation on sulfur-starvation in Chlamydomonas reinhardtii.

    PubMed

    Sato, Atsushi; Matsumura, Rie; Hoshino, Naomi; Tsuzuki, Mikio; Sato, Norihiro

    2014-01-01

    Triacylglycerol (TG) synthesis is induced for energy and carbon storage in algal cells under nitrogen(N)-starved conditions, and helps prevent reactive oxygen species (ROS) production through fatty acid synthesis that consumes excessive reducing power. Here, the regulatory mechanism for the TG content in sulfur(S)-starved cells of Chlamydomonas reinhardtii was examined, in comparison to that in N- or phosphorus(P)-starved cells. S- and N- starved cells exhibited markedly increased TG contents with up-regulation of mRNA levels of diacylglycerol acyltransferase (DGAT) genes. S-Starvation also induced expression of the genes for phosphatidate synthesis. In contrast, P-starved cells exhibited little alteration of the TG content with almost no induction of these genes. The results implied deficient nutrient-specific regulation of the TG content. An arg9 disruptant defective in arginine synthesis, even without nutritional deficiencies, exhibited an increased TG content upon removal of supplemented arginine, which repressed protein synthesis. Repression of protein synthesis thus seemed crucial for TG accumulation in S- or N- starved cells. Meanwhile, the results of inhibitor experiments involving cells inferred that TG accumulation during S-starvation is supported by photosynthesis and de novo fatty acid synthesis. During S-starvation, sac1 and snrk2.2 disruptants, which are defective in the response to the ambient S-status, accumulated TG at lower and higher levels, respectively, than the wild type. The sac1 and snrk2.2 disruptants showed no or much greater up-regulation of DGAT genes, respectively. In conclusion, TG synthesis would be activated in S-starved cells, through the diversion of metabolic carbon-flow from protein to TG synthesis, and simultaneously through up-regulation of the expression of a particular set of genes for TG synthesis at proper levels through the actions of SAC1 and SNRK2.2.

  13. Rapid and Highly Sensitive Detection of Variant Creutzfeldt - Jakob Disease Abnormal Prion Protein on Steel Surfaces by Protein Misfolding Cyclic Amplification: Application to Prion Decontamination Studies

    PubMed Central

    Belondrade, Maxime; Nicot, Simon; Béringue, Vincent; Coste, Joliette; Lehmann, Sylvain; Bougard, Daisy

    2016-01-01

    The prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the population remains uncertain, although it has been estimated that 1 in 2000 people in the United Kingdom are positive for abnormal prion protein (PrPTSE) by a recent survey of archived appendix tissues. The prominent lymphotropism of vCJD prions raises the possibility that some surgical procedures may be at risk of iatrogenic vCJD transmission in healthcare facilities. It is therefore vital that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated. A current limitation is the lack of a rapid model permissive to human prions. Here, we developed a prion detection assay based on protein misfolding cyclic amplification (PMCA) technology combined with stainless-steel wire surfaces as carriers of prions (Surf-PMCA). This assay allowed the specific detection of minute quantities (10−8 brain dilution) of either human vCJD or ovine scrapie PrPTSE adsorbed onto a single steel wire, within a two week timeframe. Using Surf-PMCA we evaluated the performance of several reference and commercially available prion-specific decontamination procedures. Surprisingly, we found the efficiency of several marketed reagents to remove human vCJD PrPTSE was lower than expected. Overall, our results demonstrate that Surf-PMCA can be used as a rapid and ultrasensitive assay for the detection of human vCJD PrPTSE adsorbed onto a metallic surface, therefore facilitating the development and validation of decontamination procedures against human prions. PMID:26800081

  14. Accumulation of isolevuglandin-modified protein in normal and fibrotic lung

    PubMed Central

    Mont, Stacey; Davies, Sean S.; Roberts second, L. Jackson; Mernaugh, Raymond L.; McDonald, W. Hayes; Segal, Brahm H.; Zackert, William; Kropski, Jonathan A.; Blackwell, Timothy S.; Sekhar, Konjeti R.; Galligan, James J.; Massion, Pierre P.; Marnett, Lawrence J.; Travis, Elizabeth L.; Freeman, Michael L.

    2016-01-01

    Protein lysine modification by γ-ketoaldehyde isomers derived from arachidonic acid, termed isolevuglandins (IsoLGs), is emerging as a mechanistic link between pathogenic reactive oxygen species and disease progression. However, the questions of whether covalent modification of proteins by IsoLGs are subject to genetic regulation and the identity of IsoLG-modified proteins remain unclear. Herein we show that Nrf2 and Nox2 are key regulators of IsoLG modification in pulmonary tissue and report on the identity of proteins analyzed by LC-MS following immunoaffinity purification of IsoLG-modified proteins. Gene ontology analysis revealed that proteins in numerous cellular pathways are susceptible to IsoLG modification. Although cells tolerate basal levels of modification, exceeding them induces apoptosis. We found prominent modification in a murine model of radiation-induced pulmonary fibrosis and in idiopathic pulmonary fibrosis, two diseases considered to be promoted by gene-regulated oxidant stress. Based on these results we hypothesize that IsoLG modification is a hitherto unrecognized sequelae that contributes to radiation-induced pulmonary injury and IPF. PMID:27118599

  15. Mandibular coronoid process in parathyroid hormone-related protein-deficient mice shows ectopic cartilage formation accompanied by abnormal bone modeling.

    PubMed

    Shibata, Shunichi; Suda, Naoto; Fukada, Kenji; Ohyama, Kimie; Yamashita, Yasuo; Hammond, Vicki E

    2003-07-01

    Parathyroid hormone-related protein (PTHrP) null mutant mice were analyzed to investigate an additional role for PTHrP in cell differentiation. We found ectopic cartilage formation in the mandibular coronoid process in newborn mice. While many previous studies involving PTHrP gene knockout mouse have shown that the cartilage in various regions becomes smaller, this is the first report showing an "increase" of cartilage volume. Investigations of mandibular growth using normal mice indicated that coronoid secondary cartilage never formed from E 15 to d 4, but small amount of cartilage temporally formed at d 7, and this also applies to PTHrP-wild type mice. Therefore, PTHrP deficiency consequently advanced the secondary cartilage formation, which is a novel role of PTHrP in chondrocyte differentiation. In situ hybridization of matrix proteins showed that this coronoid cartilage had characteristics of the lower hypertrophic cell zone usually present at the site of endochondral bone formation and/or "chondroid bone" occasionally found in distraction osteogenesis. In addition, the coronoid process in the PTHrP-deficient mouse also showed abnormal expansion of bone marrow and an increase in the number of multinucleated osteoclasts, an indication of abnormal bone modeling. These results indicate that PTHrP is involved in bone modeling as well as in chondrocyte differentiation. In situ hybridization of matrix protein mRNAs in the abnormal mandibular condylar cartilage revealed that this cartilage was proportionally smaller, supporting previous immunohistochemical results.

  16. Growth rate, protein accumulation, and catabolic enzyme activity of skeletal muscles of galliform birds.

    PubMed

    Shea, Russell E; Olson, John M; Ricklefs, Robert E

    2007-01-01

    We measured the mass and several potential indices of functional capacity of the leg and pectoral muscles through 21 d of age in chicks of three species of galliform birds and the domesticated turkey. The study was conducted to test the hypothesis that the growth rate of a tissue is inversely related to its capacity for mature function across species. We measured the proportion of protein and the activities of the catabolic enzymes citrate synthase (CS), pyruvate kinase (PK), and beta -hydroxy-acyl-CoA-dehydrogenase (HOAD) and estimated exponential growth rate (EGR) from growth increments. EGR was negatively related to proportion of protein, PK, and HOAD and positively related to CS activity. In a multiple regression, EGR was uniquely related only to proportion of protein; it was higher in pectoral muscles and increased in this order: wild turkeyprotein, but domestication and selection for rapid growth and large muscle size in turkeys resulted in substantially elevated growth rate. When the proportion of protein was normalized by its maximum value for each species and muscle type, the relationship between EGR and normalized protein did not differ significantly among species or muscle type. Thus, if we accept the proportion of protein relative to the mature level as an index of functional capacity--presumably representing the assembly of the contractile apparatus--then growth rate is consistently inversely related to a muscle's capacity for mature function, that is, force generation.

  17. Aging induces cardiac diastolic dysfunction, oxidative stress, accumulation of advanced glycation endproducts and protein modification.

    PubMed

    Li, Shi-Yan; Du, Min; Dolence, E Kurt; Fang, Cindy X; Mayer, Gabriele E; Ceylan-Isik, Asli F; LaCour, Karissa H; Yang, Xiaoping; Wilbert, Christopher J; Sreejayan, Nair; Ren, Jun

    2005-04-01

    Evidence suggests that aging, per se, is a major risk factor for cardiac dysfunction. Oxidative modification of cardiac proteins by non-enzymatic glycation, i.e. advanced glycation endproducts (AGEs), has been implicated as a causal factor in the aging process. This study was designed to examine the role of aging on cardiomyocyte contractile function, cardiac protein oxidation and oxidative modification. Mechanical properties were evaluated in ventricular myocytes from young (2-month) and aged (24-26-month) mice using a MyoCam system. The mechanical indices evaluated were peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocity of shortening/relengthening (+/- dL/dt). Oxidative stress and protein damage were evaluated by glutathione and glutathione disulfide (GSH/GSSG) ratio and protein carbonyl content, respectively. Activation of NAD(P)H oxidase was determined by immunoblotting. Aged myocytes displayed a larger cell cross-sectional area, prolonged TR90, and normal PS, +/- dL/dt and TPS compared with young myocytes. Aged myocytes were less tolerant of high stimulus frequency (from 0.1 to 5 Hz) compared with young myocytes. Oxidative stress and protein oxidative damage were both elevated in the aging group associated with significantly enhanced p47phox but not gp91phox expression. In addition, level of cardiac AGEs was approximately 2.5-fold higher in aged hearts than young ones determined by AGEs-ELISA. A group of proteins with a molecular range between 50 and 75 kDa with pI of 4-7 was distinctively modified in aged heart using one- or two-dimension SDS gel electrophoresis analysis. These data demonstrate cardiac diastolic dysfunction and reduced stress tolerance in aged cardiac myocytes, which may be associated with enhanced cardiac oxidative damage, level of AGEs and protein modification by AGEs.

  18. Abnormal accumulation of trace metals by plants

    SciTech Connect

    Reeves, R.D.; Brooks, R.R.; Baker, A.J.M.

    1996-12-31

    The article describes the hyperaccumulation of metals by plants. Ranges for low, normal, high, and hyperaccumulating uptake are established. A partial list of hyperaccumulator species and their localities is included. Studies are reviewed and summarized for zinc, cadmium and lead, nickel, cobalt and copper, selenium, and cadmium and manganese hyperaccumulation.

  19. Accumulation of mutant huntingtin fragments in aggresome-like inclusion bodies as a result of insufficient protein degradation.

    PubMed

    Waelter, S; Boeddrich, A; Lurz, R; Scherzinger, E; Lueder, G; Lehrach, H; Wanker, E E

    2001-05-01

    The huntingtin exon 1 proteins with a polyglutamine repeat in the pathological range (51 or 83 glutamines), but not with a polyglutamine tract in the normal range (20 glutamines), form aggresome-like perinuclear inclusions in human 293 Tet-Off cells. These structures contain aggregated, ubiquitinated huntingtin exon 1 protein with a characteristic fibrillar morphology. Inclusion bodies with truncated huntingtin protein are formed at centrosomes and are surrounded by vimentin filaments. Inhibition of proteasome activity resulted in a twofold increase in the amount of ubiquitinated, SDS-resistant aggregates, indicating that inclusion bodies accumulate when the capacity of the ubiquitin-proteasome system to degrade aggregation-prone huntingtin protein is exhausted. Immunofluorescence and electron microscopy with immunogold labeling revealed that the 20S, 19S, and 11S subunits of the 26S proteasome, the molecular chaperones BiP/GRP78, Hsp70, and Hsp40, as well as the RNA-binding protein TIA-1, the potential chaperone 14-3-3, and alpha-synuclein colocalize with the perinuclear inclusions. In 293 Tet-Off cells, inclusion body formation also resulted in cell toxicity and dramatic ultrastructural changes such as indentations and disruption of the nuclear envelope. Concentration of mitochondria around the inclusions and cytoplasmic vacuolation were also observed. Together these findings support the hypothesis that the ATP-dependent ubiquitin-proteasome system is a potential target for therapeutic interventions in glutamine repeat disorders.

  20. Ophthalmic acid accumulation in an Escherichia coli mutant lacking the conserved pyridoxal 5'-phosphate-binding protein YggS.

    PubMed

    Ito, Tomokazu; Yamauchi, Ayako; Hemmi, Hisashi; Yoshimura, Tohru

    2016-12-01

    Escherichia coli YggS is a highly conserved pyridoxal 5'-phosphate (PLP)-binding protein whose biochemical function is currently unknown. A previous study with a yggS-deficient E. coli strain (ΔyggS) demonstrated that YggS controls l-Ile- and l-Val-metabolism by modulating 2-ketobutyrate (2-KB), l-2-aminobutyrate (l-2-AB), and/or coenzyme A (CoA) availability in a PLP-dependent fashion. In this study, we found that ΔyggS accumulates an unknown metabolite as judged by amino acid analyses. LC/MS and MS/MS analyses of the compound with propyl chloroformate derivatization, and co-chromatography analysis identified this compound as γ-l-glutamyl-l-2-aminobutyryl-glycine (ophthalmic acid), a glutathione (GSH) analogue in which the l-Cys moiety is replaced by l-2-AB. We also determine the metabolic consequence of the yggS mutation. Absence of YggS initially increases l-2-AB availability, and then causes ophthalmic acid accumulation and CoA limitation in the cell. The expression of a γ-glutamylcysteine synthetase and a glutathione synthetase in a ΔyggS background causes high-level accumulation of ophthalmic acid in the cells (∼1.2 nmol/mg cells) in a minimal synthetic medium. This opens the possibility of a first fermentative production of ophthalmic acid.

  1. Increase of hepatic fat accumulation by liver specific expression of Hepatitis B virus X protein in zebrafish.

    PubMed

    Shieh, Yun-Sheng; Chang, Yin-Shan; Hong, Jiann-Ruey; Chen, Li-Je; Jou, Luen-Kuang; Hsu, Chia-Chun; Her, Guor Mour

    2010-07-01

    The pathogenesis of fatty liver disease remains largely unknown. Here, we assessed the importance of hepatic fat accumulation on the progression of hepatitis in zebrafish by liver specific expression of Hepatitis B virus X protein (HBx). Transgenic zebrafish lines, GBXs, which selectively express the GBx transgene (GFP-fused HBx gene) in liver, were established. GBX Liver phenotypes were evaluated by histopathology and molecular analysis of fatty acid (FA) metabolism-related genes expression. Most GBXs (66-81%) displayed obvious emaciation starting at 4 months old. Over 99% of the emaciated GBXs developed hepatic steatosis or steatohepatitis, which in turn led to liver hypoplasia. The liver histology of GBXs displayed steatosis, lobular inflammation, and balloon degeneration, similar to non-alcoholic steatohepatitis (NASH). Oil red O stain detected the accumulation of fatty droplets in GBXs. RT-PCR and Q-rt-PCR analysis revealed that GBx induced hepatic steatosis had significant increases in the expression of lipogenic genes, C/EBP-alpha, SREBP1, ChREBP and PPAR-gamma, which then activate key enzymes of the de novo FA synthesis, ACC1, FAS, SCD1, AGAPT, PAP and DGAT2. In addition, the steatohepatitic GBX liver progressed to liver degeneration and exhibited significant differential gene expression in apoptosis and stress. The GBX models exhibited both the genetic and functional factors involved in lipid accumulation and steatosis-associated liver injury. In addition, GBXs with transmissible NASH-like phenotypes provide a promising model for studying liver disease.

  2. Synergistic effects of the SAPK/JNK and the proteasome pathway on glial fibrillary acidic protein (GFAP) accumulation in Alexander disease.

    PubMed

    Tang, Guomei; Xu, Zhiheng; Goldman, James E

    2006-12-15

    Protein aggregates in astrocytes that contain glial fibrillary acidic protein (GFAP), small heat shock proteins, and ubiquitinated proteins are termed Rosenthal fibers and characterize Alexander disease, a leukodystrophy caused by heterozygous mutations in GFAP. The mechanisms responsible for the massive accumulation of GFAP in Alexander disease remain unclear. In this study, we show that overexpression of both wild type and R239C mutant human GFAP led to cytoplasmic inclusions. GFAP accumulation also led to a decrease of proteasome activity and an activation of the MLK2-JNK pathway. In turn, the expression of activated mixed lineage kinases (MLKs) induced JNK activation and increased GFAP accumulation, whereas blocking the JNK pathway decreased GFAP accumulation. Activated MLK also inhibited proteasome function. A direct inhibition of proteasome function pharmacologically further activated JNK. Our data suggest a synergistic interplay between the proteasome and the SAPK/JNK pathway in the context of GFAP accumulation. Feedback interactions among GFAP accumulation, SAPK/JNK activation, and proteasomal hypofunction cooperate to produce further protein accumulation and cellular stress responses.

  3. Heat shock protein synthesis and trehalose accumulation are not required for induced thermotolerance in depressed Saccharomyces cerevisiae.

    PubMed

    Gross, C; Watson, K

    1996-03-27

    Intrinsic and heat shock induced thermotolerance of Saccharomyces cerevisiae was investigated in cells grown on glucose and acetate supplemented media. Heat shocked cells (37 degrees C/30 min), in either medium, exhibited induced synthesis of heat shock proteins (hsp) and trehalose. In all cases, with the notable exception of repressed cells of a relatively thermosensitive strain, heat shock acquisition of thermotolerance also occurred in the absence of protein synthesis and coincident decrease in trehalose accumulation. Results indicted that the marked increase in thermotolerance exhibited by non-fermenting (acetate) cells compared with fermenting (glucose) cells was not closely correlated with levels of hsp or trehalose. It was concluded that mechanisms for intrinsic and induced thermotolerance appear to be different and that growth on acetate endows cells with a biochemical predisposition, other than hsp or trehalose, that confers intrinsic tolerance, a factor which may be subject to heat induced modification.

  4. Activation of the unfolded protein response pathway causes ceramide accumulation in yeast and INS-1E insulinoma cells.

    PubMed

    Epstein, Sharon; Kirkpatrick, Clare L; Castillon, Guillaume A; Muñiz, Manuel; Riezman, Isabelle; David, Fabrice P A; Wollheim, Claes B; Riezman, Howard

    2012-03-01

    Sphingolipids are not only important components of membranes but also have functions in protein trafficking and intracellular signaling. The LCB1 gene encodes a subunit of the serine palmitoyltransferase, which is responsible for the first step of sphingolipid synthesis. Here, we show that activation of the unfolded protein response (UPR) can restore normal ceramide levels and viability in yeast cells with a conditional defect in LCB1. Dependence on UPR was demonstrated by showing the HAC1-dependence of the suppression. A similar induction of ceramides by UPR seems to take place in mammalian cells. In rat pancreatic INS-1E cells, UPR activation induces the transcription of the CerS6 gene, which encodes a ceramide synthase. This correlates with the specific accumulation of ceramide with a C16 fatty acyl chain upon UPR activation. Therefore, our study reveals a novel connection between UPR induction and ceramide synthesis that seems to be conserved between yeast and mammalian cells.

  5. Accumulation of the NON-YELLOW COLORING 1 protein of the chlorophyll cycle requires chlorophyll b in Arabidopsis thaliana.

    PubMed

    Jia, Ting; Ito, Hisashi; Hu, Xueyun; Tanaka, Ayumi

    2015-02-01

    Chlorophyll a and chlorophyll b are interconverted in the chlorophyll cycle. The initial step in the conversion of chlorophyll b to chlorophyll a is catalyzed by the chlorophyll b reductases NON-YELLOW COLORING 1 (NYC1) and NYC1-like (NOL), which convert chlorophyll b to 7-hydroxymethyl chlorophyll a. This step is also the first stage in the degradation of the light-harvesting chlorophyll a/b protein complex (LHC). In this study, we examined the effect of chlorophyll b on the level of NYC1. NYC1 mRNA and NYC1 protein were in low abundance in green leaves, but their levels increased in response to dark-induced senescence. When the level of chlorophyll b was enhanced by the introduction of a truncated chlorophyllide a oxygenase gene and the leaves were incubated in the dark, the amount of NYC1 was greatly increased compared with that of the wild type; however, the amount of NYC1 mRNA was the same as in the wild type. In contrast, NYC1 did not accumulate in the mutant without chlorophyll b, even though the NYC1 mRNA level was high after incubation in the dark. Quantification of the LHC protein showed no strong correlation between the levels of NYC1 and LHC proteins. However, the level of chlorophyll fluorescence of the dark adapted plant (Fo ) was closely related to the accumulation of NYC1, suggesting that the NYC1 level is related to the energetically uncoupled LHC. These results and previous reports on the degradation of chlorophyllide a oxygenase suggest that the a feedforward and feedback network is included in chlorophyll cycle.

  6. Overexpression of UV-DAMAGED DNA BINDING PROTEIN 1 links plant development and phytonutrient accumulation in high pigment-1 tomato.

    PubMed

    Azari, Raviv; Reuveni, Moshe; Evenor, Dalia; Nahon, Sahadia; Shlomo, Haviva; Chen, Lea; Levin, Ilan

    2010-08-01

    Fruits of tomato plants carrying the high pigment-1 mutations hp-1 and hp-1(w) are characterized by an increased number of plastids coupled with enhanced levels of functional metabolites. Unfortunately, hp-1 mutant plants are also typified by light-dependent retardation in seedling and whole-plant growth and development, which limits their cultivation. These mutations were mapped to the gene encoding UV-DAMAGED DNA BINDING PROTEIN 1 (DDB1) and, recently, fruit-specific RNA interference studies have demonstrated an increased number of plastids and enhanced carotenoid accumulation in the transgenic tomato fruits. However, whole-plant overexpression of DDB1, required to substantiate its effects on seedling and plant development and to couple them with fruit phenotypes, has heretofore been unsuccessful. In this study, five transgenic lines constitutively overexpressing normal DDB1 in hp-1 mutant plants were analysed. Eleven-day-old seedlings, representing these lines, displayed up to approximately 73- and approximately 221-fold overexpression of the gene in hypocotyls and cotyledons, respectively. This overexpression resulted in statistically significant reversion to the non-mutant developmental phenotypes, including more than a full quantitative reversion. This reversion of phenotypes was generally accompanied by correlated responses in chlorophyll accumulation and altered expression of selected light signalling genes: PHYTOCHROME A, CRYPTOCHROME 1, ELONGATED HYPOCOTYL 5, and the gene encoding CHLOROPHYLL A/B-BINDING PROTEIN 4. Cumulatively, these results provide the missing link between DDB1 and its effects on tomato plant development.

  7. The MLN4924 inhibitor exerts a neuroprotective effect against oxidative stress injury via Nrf2 protein accumulation.

    PubMed

    Andérica-Romero, Ana Cristina; Hernández-Damián, Jacqueline; Vázquez-Cervantes, Gustavo Ignacio; Torres, Ismael; Pedraza-Chaverri, José

    2016-08-01

    It was explored the cytoprotective and antioxidant effect of MLN4924, a specific inhibitor of Nedd8-activating enzyme (NAE), against hydrogen peroxide (H2O2)-induced damage in cerebellar granule neurons (CGNs). Primary cultures of CGNs were exposed to H2O2 after preincubation with MLN4924. The compounds were removed, and CGNs were incubated in culture medium for 24h in order to determine cell viability by 3-[4,5-dimethylthiazol-2-yl)]-2,5-diphenyl-tetrazolium bromide (MTT) and fluorescein diacetate (FDA) assays. It was demonstrated that MLN4924 remarkably attenuated H2O2-induced cell damage. Meanwhile reactive oxygen species (ROS) production was evaluated with the fluorescent probe dihydroethidium (DHE). Interestingly H2O2-induced ROS production was inhibited by pretreatment with MLN4924. MLN4924 treatment in CGNs resulted in nuclear factor E2-related factor 2 (Nrf2) protein accumulation. Intriguingly this effect was observed in the cytosolic and nuclear compartments of the CGNs. The cytoprotective effect of MLN4924 was associated with its ability to diminish ROS production induced by H2O2 and the accumulation of Nrf2 protein levels in the cytoplasm and nucleus of the CGNs.

  8. Small deletions in the potato leafroll virus readthrough protein affect particle morphology, aphid transmission, virus movement and accumulation.

    PubMed

    Peter, Kari A; Liang, Delin; Palukaitis, Peter; Gray, Stewart M

    2008-08-01

    Potato leafroll virus (PLRV) capsid comprises 180 coat protein (CP) subunits, with some percentage containing a readthrough domain (RTD) extension located on the particle's surface. The RTD N terminus is highly conserved in luteovirids and this study sought to identify biologically active sites within this region of the PLRV RTD. Fourteen three-amino-acid-deletion mutants were generated from a cloned infectious PLRV cDNA and delivered to plants by Agrobacterium inoculations. All mutant viruses accumulated locally in infiltrated tissues and expressed the readthrough protein (RTP) containing the CP and RTD sequences in plant tissues; however, when purified, only three mutant viruses incorporated the RTP into the virion. None of the mutant viruses were aphid transmissible, but the viruses persisted in aphids for a period sufficient to allow for virus transmission. Several mutant viruses were examined further for systemic infection in four host species. All mutant viruses, regardless of RTP incorporation, moved systemically in each host, although they accumulated at different rates in systemically infected tissues. The biological properties of the RTP are sensitive to modifications in both the RTD conserved and variable regions.

  9. Molecular characterization of dendritic cell-derived exosomes. Selective accumulation of the heat shock protein hsc73.

    PubMed

    Théry, C; Regnault, A; Garin, J; Wolfers, J; Zitvogel, L; Ricciardi-Castagnoli, P; Raposo, G; Amigorena, S

    1999-11-01

    Exosomes are membrane vesicles secreted by hematopoietic cells upon fusion of late multivesicular endosomes with the plasma membrane. Dendritic cell (DC)-derived exosomes induce potent antitumor immune responses in mice, resulting in the regression of established tumors (Zitvogel, L., A. Regnault, A. Lozier, J. Wolfers, C. Flament, D. Tenza, P. Ricciardi-Castagnoli, G. Raposo, and S. Amigorena. 1998. Nat. Med. 4:594-600). To unravel the molecular basis of exosome-induced immune stimulation, we now analyze the regulation of their production during DC maturation and characterize extensively their protein composition by peptide mass mapping. Exosomes contain several cytosolic proteins (including annexin II, heat shock cognate protein hsc73, and heteromeric G protein Gi2alpha), as well as different integral or peripherally associated membrane proteins (major histocompatibility complex class II, Mac-1 integrin, CD9, milk fat globule-EGF-factor VIII [MFG-E8]). MFG-E8, the major exosomal component, binds integrins expressed by DCs and macrophages, suggesting that it may be involved in exosome targeting to these professional antigen-presenting cells. Another exosome component is hsc73, a cytosolic heat shock protein (hsp) also present in DC endocytic compartments. hsc73 was shown to induce antitumor immune responses in vivo, and therefore could be involved in the exosome's potent antitumor effects. Finally, exosome production is downregulated upon DC maturation, indicating that in vivo, exosomes are produced by immature DCs in peripheral tissues. Thus, DC-derived exosomes accumulate a defined subset of cellular proteins reflecting their endosomal biogenesis and accounting for their biological function.

  10. Linkage of protein kinase C-beta activation and intracellular interleukin-2 accumulation in human naive CD4 T cells.

    PubMed Central

    Hassan, J; Rainsford, E; Reen, D J

    1997-01-01

    A critical role for protein kinase C (PKC) in signal transduction events has been well established. Moreover, studies of regulation in PKC levels suggest participation in mediating long-term cellular functions. Protein kinase C-beta (PKC-beta) has been reported to be involved in interleukin-2 (IL-2) synthesis in T lymphocytes. In this study, the role of PKC-beta in intracellular accumulation of IL-2 was investigated using specific inhibitors. Preincubation with two different PKC inhibitors, one specific for classical isotypes (alpha and beta I) Go6976, and one which inhibits both classical and non-classical isotypes, GF109203X, caused a complete block in cytoplasmic IL-2 accumulation when naive CD4 T cells were stimulated in the presence of CD2+CD28+phorbol myristate acetate (PMA). In contrast, preincubation with up to 1000 ng/ml of cyclosporin A (CsA) resulted in a reduction in the intracellular IL-2 detected, as observed by a decrease in the proportion of positive cells as well as a fall in the mean fluorescence intensity (MFI). CsA did not influence PKC-beta translocation. Flow cytometric assessments of PKC-beta and its isoforms beta I and beta II correlated with Western blotting analysis and these results were further supported by the use of PKC-beta-positive (HUT 78) and -negative (BW5147) T-cell lines. Using the specific inhibitors, Go6976 and GF109203X, the findings in this study suggest that activation and translocation of PKC-beta is critical for accumulation of intracellular IL-2. The influence of CsA in reducing but not blocking IL-2 synthesis is discussed. PMA-induced down-regulation of the CD4 antigen was observed in the presence of Go6976 and but not GF109203X, suggesting regulation by non-classical PKC isoforms. Images Figure 4 PMID:9497487

  11. Arabidopsis SEIPIN proteins modulate triacylglycerol accumulation and influence lipid droplet proliferation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The lipodystrophy protein SEIPIN is important for lipid droplet (LD) biogenesis in human and yeast cells. By contrast to the single SEIPIN genes in humans and yeast, there are three SEIPIN homologues in Arabidopsis thaliana, designated At-SEIPIN1, At-SEIPIN2 and At-SEIPIN3. Here, a yeast (Saccharomy...

  12. Protein Hydrolysates from Beta-Conglycinin Enriched Soybean Genotypes Inhibit Lipid Accumulation and Inflammation in Vitro

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Obesity is a worldwide health concern and a well recognized predictor of premature mortality associated with a state of chronic inflammation. The objective was to evaluate the effect of soy protein hydrolysates (SPH) produced from different soybean genotypes by alcalase (SAH) or simulated gastroint...

  13. Accumulation of maize chlorotic dwarf virus proteins in its plant host and leafhopper vector.

    PubMed

    Chaouch-Hamada, Rym; Redinbaugh, Margaret G; Gingery, Roy E; Willie, Kristen; Hogenhout, Saskia A

    2004-08-01

    The genome of Maize chlorotic dwarf virus (MCDV; genus Waikavirus; family Sequiviridae) consists of a monopartite positive-sense RNA genome encoding a single large polyprotein. Antibodies were produced to His-fusions of three undefined regions of the MCDV polyprotein: the N-terminus of the polyprotein (R78), a region between coat proteins (CPs) and the nucleotide-binding site (NBS) (R37), and a region between the NBS and a 3C-like protease (R69). The R78 antibodies react with proteins of 50 kDa (P50), 35 kDa (P35), and 25 kDa (P25) in virus preparations, and with P35 in plant extracts. In extracts of the leafhopper vector Graminella nigrifrons fed on MCDV-infected plants, the R78 antibodies reacted with P25 but not with P50 and P35. The R69 antibodies bound proteins of approximately 36 kDa (P36), 30 kDa (P30), and 26 kDa (P26) in virus preparations, and P36 and P26 in plant extracts. Antibodies to R37 reacted with a 26-kDa protein in purified virus preparations, but not in plant extracts. Neither the R69 nor the R37 antibodies bound any proteins in G. nigrifrons. Thus, in addition to the three CPs, cysteine protease and RNA-dependent RNA polymerase, the MCDV polyprotein is apparently post-transitionally cleaved into P50, P35, P25, P36, P30, and P26.

  14. Expression of the IRT1 metal transporter is controlled by metals at the levels of transcript and protein accumulation.

    PubMed

    Connolly, Erin L; Fett, Janette P; Guerinot, Mary Lou

    2002-06-01

    Iron, an essential nutrient, is not readily available to plants because of its low solubility. In addition, iron is toxic in excess, catalyzing the formation of hydroxyl radicals that can damage cellular constituents. Consequently, plants must carefully regulate iron uptake so that iron homeostasis is maintained. The Arabidopsis IRT1 gene is the major transporter responsible for high-affinity iron uptake from the soil. Here, we show that the steady state level of IRT1 mRNA was induced within 24 h after transfer of plants to iron-deficient conditions, with protein levels peaking 72 h after transfer. IRT1 mRNA and protein were undetectable 12 h after plants were shifted back to iron-sufficient conditions. Overexpression of IRT1 did not confer dominant gain-of-function enhancement of metal uptake. Analysis of 35S-IRT1 transgenic plants revealed that although IRT1 mRNA was expressed constitutively in these plants, IRT1 protein was present only in the roots when iron is limiting. Under these conditions, plants that overexpressed IRT1 accumulated higher levels of cadmium and zinc than wild-type plants, indicating that IRT1 is responsible for the uptake of these metals and that IRT1 protein levels are indeed increased in these plants. Our results suggest that the expression of IRT1 is controlled by two distinct mechanisms that provide an effective means of regulating metal transport in response to changing environmental conditions.

  15. Identification of differentially accumulated proteins associated with embryogenic and non-embryogenic calli in saffron (Crocus sativus L.)

    PubMed Central

    2012-01-01

    Background Somatic embryogenesis (SE) is a complex biological process that occurs under inductive conditions and causes fully differentiated cells to be reprogrammed to an embryo like state. In order to get a better insight about molecular basis of the SE in Crocus sativus L. and to characterize differentially accumulated proteins during the process, a proteomic study based on two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry has been carried out. Results We have compared proteome profiles of non-embryogenic and embryogenic calli with native corm explants. Total soluble proteins were phenol-extracted and loaded on 18 cm IPG strips for the first dimension and 11.5% sodium dodecyl sulfate-polyacrylamide gels for the second dimension. Fifty spots with more than 1.5-fold change in abundance were subjected to mass spectrometry analysis for further characterization. Among them 36 proteins could be identified, which are classified into defense and stress response, protein synthesis and processing, carbohydrate and energy metabolism, secondary metabolism, and nitrogen metabolism. Conclusion Our results showed that diverse cellular and molecular processes were affected during somatic to embryogenic transition. Differential proteomic analysis suggests a key role for ascorbate metabolism during early stage of SE, and points to the possible role of ascorbate-glutathione cycle in establishing somatic embryos. PMID:22243837

  16. Decreased mTOR signalling reduces mitochondrial ROS in brain via accumulation of the telomerase protein TERT within mitochondria

    PubMed Central

    Wan, Tengfei; Bell, Amy; Hill, Kirsten N.; von Zglinicki, Thomas; Saretzki, Gabriele

    2016-01-01

    Telomerase in its canonical function maintains telomeres in dividing cells. In addition, the telomerase protein TERT has non-telomeric functions such as shuttling to mitochondria resulting in a decreased oxidative stress, DNA damage and apoptosis. TERT protein persists in adult neurons and can co-localise to mitochondria under various stress conditions. We show here that TERT expression decreased in mouse brain during aging while release of reactive oxygen species (ROS) from the mitochondrial electron transport chain increased. Dietary restriction (DR) caused accumulation of TERT protein in mouse brain mitochondria correlating to decreased ROS release and improved learning and spatial short-term memory. Decreased mTOR signalling is a mediator of DR. Accordingly, feeding mice with rapamycin increased brain mitochondrial TERT and reduced ROS release. Importantly, the beneficial effects of rapamycin on mitochondrial function were absent in brains and fibroblasts from first generation TERT −/− mice, and when TERT shuttling was inhibited by the Src kinase inhibitor bosutinib. Taken together, our data suggests that the mTOR signalling pathway impinges on the mitochondrial localisation of TERT protein, which might in turn contribute to the protection of the brain by DR or rapamycin against age-associated mitochondrial ROS increase and cognitive decline. PMID:27777385

  17. Regulation of myofibrillar accumulation in chick muscle cultures - Evidence for the involvement of calcium and lysosomes in non-uniform turnover of contractile proteins

    NASA Technical Reports Server (NTRS)

    Silver, Geri; Etlinger, Joseph D.

    1985-01-01

    The effects of calcium on the synthesis and the degradation of individual myofibrillar proteins were investigated using primary chick-leg skeletal muscle cultures labeled with S-35-methionine (for protein accumulation experiments) or Ca(2+)-45 (for calcium efflux experiments). It was found that the turnover of individual contractile proteins is regulated nonuniformly by a calcium-dependent mechanism involving lysosomes. The results also indicate that contractile proteins are released from the myofibril before their breakdown to amino acids.

  18. Mitochondrial Matrix Ca2+ Accumulation Regulates Cytosolic NAD+/NADH Metabolism, Protein Acetylation, and Sirtuin Expression

    PubMed Central

    Marcu, Raluca; Wiczer, Brian M.; Neeley, Christopher K.

    2014-01-01

    Mitochondrial calcium uptake stimulates bioenergetics and drives energy production in metabolic tissue. It is unknown how a calcium-mediated acceleration in matrix bioenergetics would influence cellular metabolism in glycolytic cells that do not require mitochondria for ATP production. Using primary human endothelial cells (ECs), we discovered that repetitive cytosolic calcium signals (oscillations) chronically loaded into the mitochondrial matrix. Mitochondrial calcium loading in turn stimulated bioenergetics and a persistent elevation in NADH. Rather than serving as an impetus for mitochondrial ATP generation, matrix NADH rapidly transmitted to the cytosol to influence the activity and expression of cytosolic sirtuins, resulting in global changes in protein acetylation. In endothelial cells, the mitochondrion-driven reduction in both the cytosolic and mitochondrial NAD+/NADH ratio stimulated a compensatory increase in SIRT1 protein levels that had an anti-inflammatory effect. Our studies reveal the physiologic importance of mitochondrial bioenergetics in the metabolic regulation of sirtuins and cytosolic signaling cascades. PMID:24865966

  19. TAZ Protein Accumulation Is Negatively Regulated by YAP Abundance in Mammalian Cells*

    PubMed Central

    Finch-Edmondson, Megan L.; Strauss, Robyn P.; Passman, Adam M.; Sudol, Marius; Yeoh, George C.; Callus, Bernard A.

    2015-01-01

    The mammalian Hippo signaling pathway regulates cell growth and survival and is frequently dysregulated in cancer. YAP and TAZ are transcriptional coactivators that function as effectors of this signaling pathway. Aberrant YAP and TAZ activity is reported in several human cancers, and normally the expression and nuclear localization of these proteins is tightly regulated. We sought to establish whether a direct relationship exists between YAP and TAZ. Using knockdown and overexpression experiments we show YAP inversely regulates the abundance of TAZ protein by proteasomal degradation. Interestingly this phenomenon was uni-directional since TAZ expression did not affect YAP abundance. Structure/function analyses suggest that YAP-induced TAZ degradation is a consequence of YAP-targeted gene transcription involving TEAD factors. Subsequent investigation of known regulators of TAZ degradation using specific inhibitors revealed a role for heat shock protein 90 and glycogen synthase kinase 3 but not casein kinase 1 nor LATS in YAP-mediated TAZ loss. Importantly, this phenomenon is conserved from mouse to human; however, interestingly, different YAP isoforms varied in their ability to degrade TAZ. Since shRNA-mediated TAZ depletion in HeLa and D645 cells caused apoptotic cell death, we propose that isoform-specific YAP-mediated TAZ degradation may contribute to the contradicting roles reported for YAP overexpression. This study identifies a novel mechanism of TAZ regulation by YAP, which has significant implications for our understanding of Hippo pathway regulation, YAP-isoform specific signaling, and the role of these proteins in cell proliferation, apoptosis, and tumorigenesis. PMID:26432639

  20. GNG11 (G-protein γ subunit 11) suppresses cell growth with induction of reactive oxygen species and abnormal nuclear morphology in human SUSM-1 cells.

    PubMed

    Takauji, Yuki; Kudo, Ikuru; En, Atsuki; Matsuo, Ryo; Hossain, Mohammad; Nakabayashi, Kazuhiko; Miki, Kensuke; Fujii, Michihiko; Ayusawa, Dai

    2017-04-05

    Enforced expression of GNG11, G-protein γ subunit 11, induces cellular senescence in normal human diploid fibroblasts. We here examined the effect of the expression of GNG11 on the growth of immortalized human cell lines, and found that it suppressed the growth of SUSM-1 cells, but not of HeLa cells. We then compared these two cell lines to understand the molecular basis for the action of GNG11. We found that expression of GNG11 induced the generation of reactive oxygen species (ROS) and abnormal nuclear morphology in SUSM-1 cells but not in HeLa cells. Increased ROS generation by GNG11 would likely be caused by the down-regulation of the antioxidant enzymes in SUSM-1 cells. We also found that SUSM-1 cells, even under normal culture conditions, showed higher levels of ROS and higher incidence of abnormal nuclear morphology than HeLa cells, and that abnormal nuclear morphology was relevant to the increased ROS generation in SUSM-1 cells. Thus, SUSM-1 and HeLa cells showed differences in the regulation of ROS and nuclear morphology, which might account for their different responses to the expression of GNG11. Then, SUSM-1 cells may provide a unique system to study the regulatory relationship between ROS generation, nuclear morphology, and G-protein signaling.

  1. The three classes of wheat xylanase-inhibiting proteins accumulate in an analogous way during wheat ear development and germination.

    PubMed

    Croes, Evi; Gebruers, Kurt; Luyten, Nikkie; Delcour, Jan A; Courtin, Christophe M

    2009-08-15

    Wheat contains high levels of the three classes of xylanase inhibitors (XIs), Triticum aestivum xylanase inhibitor (TAXI), xylanase-inhibiting protein (XIP) and thaumatin-like xylanase inhibitor (TLXI). These proteins have been linked to plant defense. In this study, expression of XIs during wheat ear development and germination was examined using immunoblotting. The three types of XIs accumulated at high levels between the milky and the soft dough stages of ear development, and reached the highest levels at the hard kernel stage. From the hard kernel stage to harvest ripeness, a slight drop in inhibitor levels was observed, which was more marked for TAXI and TLXI than for XIP. During germination, the levels of the three types of XIs initially decreased, but XIs accumulated again after 1-2d, reaching maximum levels between 5 and 9d after imbibition. The levels of TAXI, XIP and TLXI in the seedlings then gradually and continuously declined as a function of time. 1D- and 2D-immunoblotting indicated that the three types of XIs occur in a wide variety of forms. This polymorphism is maintained throughout ear development and germination, although the proportions of the different (iso)forms vary with time. A differential temporal profile was observed for the unprocessed and processed forms of TAXI-type proteins. Finally, the occurrence of TAXI and XIP, but not TLXI, in roots and shoots of young seedlings was demonstrated. No XIs were detected in roots, leaves or stems at later stages of ear development. Overall, the three classes of XIs show remarkable similarities in their temporal distribution, indicating a related function within the wheat plant.

  2. Coat Protein Regulation by CK2, CPIP, HSP70, and CHIP Is Required for Potato Virus A Replication and Coat Protein Accumulation.

    PubMed

    Lõhmus, Andres; Hafrén, Anders; Mäkinen, Kristiina

    2017-02-01

    We demonstrate here that both coat protein (CP) phosphorylation by protein kinase CK2 and a chaperone system formed by two heat shock proteins, CP-interacting protein (CPIP) and heat shock protein 70 (HSP70), are essential for potato virus A (PVA; genus Potyvirus) replication and that all these host proteins have the capacity to contribute to the level of PVA CP accumulation. An E3 ubiquitin ligase called carboxyl terminus Hsc70-interacting protein (CHIP), which may participate in the CPIP-HSP70-mediated CP degradation, is also needed for robust PVA gene expression. Residue Thr(243) within the CK2 consensus sequence of PVA CP was found to be essential for viral replication and to regulate CP protein stability. Substitution of Thr(243) either with a phosphorylation-mimicking Asp (CP(ADA)) or with a phosphorylation-deficient Ala (CP(AAA)) residue in CP expressed from viral RNA limited PVA gene expression to the level of nonreplicating PVA. We found that both the CP(AAA) mutant and CK2 silencing inhibited, whereas CP(ADA) mutant and overexpression of CK2 increased, PVA translation. From our previous studies, we know that phosphorylation reduces the RNA binding capacity of PVA CP and an excess of CP fully blocks viral RNA translation. Together, these findings suggest that binding by nonphosphorylated PVA CP represses viral RNA translation, involving further CP phosphorylation and CPIP-HSP70 chaperone activities as prerequisites for PVA replication. We propose that this mechanism contributes to shifting potyvirus RNA from translation to replication.

  3. The FKB2 gene of Saccharomyces cerevisiae, encoding the immunosuppressant-binding protein FKBP-13, is regulated in response to accumulation of unfolded proteins in the endoplasmic reticulum.

    PubMed Central

    Partaledis, J A; Berlin, V

    1993-01-01

    The FKB2 gene of Saccharomyces cerevisiae encodes a homolog of mammalian FKBP-13, an FK506/rapamycin-binding protein that localizes to the lumen of the endoplasmic reticulum (ER). We have found that FKB2 mRNA levels increase in response to the accumulation of unfolded precursor proteins in the ER. FKB2 mRNA levels are elevated in cells blocked in N-glycosylation--i.e., in wild-type cells treated with tunicamycin and in the sec53-6 mutant grown at the nonpermissive temperature. Mutations that block other steps in secretion have no effect on FKB2 mRNA levels, indicating that increases in FKB2 mRNA are not the consequence of a general block in secretion. The increase in FKB2 mRNA in response to unfolded proteins in the ER is mediated through a 21-bp unfolded-protein response (UPR) element located in the 5' noncoding region of FKB2. UPR elements present in other ER chaperone genes, such as yeast KAR2 (BiP), mammalian GRP78 (BiP), and GRP94, function in an analogous manner to that in FKB2. As with KAR2, FKB2 mRNA levels are also elevated by heat shock. The similarities in the regulation of FKB2 and other ER chaperone genes suggest that FKBP-13 may play a role in protein trafficking in the ER. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7685904

  4. Arabidopsis cold shock domain protein 2 influences ABA accumulation in seed and negatively regulates germination.

    PubMed

    Sasaki, Kentaro; Kim, Myung-Hee; Kanno, Yuri; Seo, Mitsunori; Kamiya, Yuji; Imai, Ryozo

    2015-01-02

    The cold shock domain (CSD) is the most conserved nucleic acid binding domain and is distributed from bacteria to animals and plants. CSD proteins are RNA chaperones that destabilize RNA secondary structures to regulate stress tolerance and development. AtCSP2 is one of the four CSD proteins in Arabidopsis and is up-regulated in response to cold. Since AtCSP2 negatively regulates freezing tolerance, it was proposed to be a modulator of freezing tolerance during cold acclimation. Here, we examined the function of AtCSP2 in seed germination. We found that AtCSP2-overexpressing lines demonstrated retarded germination as compared with the wild type, with or without stress treatments. The ABA levels in AtCSP2-overexpressing seeds were higher than those in the wild type. In addition, overexpression of AtCSP2 reduced the expression of an ABA catabolic gene (CYP707A2) and gibberellin biosynthesis genes (GA20ox and GA3ox). These results suggest that AtCSP2 negatively regulates seed germination by controlling ABA and GA levels.

  5. A Nitrogen-Fixing Subunit Essential for Accumulating 4Fe-4S-Containing Photosystem I Core Proteins1[OPEN

    PubMed Central

    Nath, Krishna; Wessendorf, Ryan L.

    2016-01-01

    Nitrogen-fixation-subunit-U (NFU)-type proteins have been shown to be involved in the biogenesis of iron-sulfur clusters. We investigated the molecular function of a chloroplastic NFU-type iron-sulfur scaffold protein, NFU3, in Arabidopsis (Arabidopsis thaliana) using genetics approaches. Loss-of-function mutations in the NFU3 gene caused yellow pigmentation in leaves, reductions in plant size, leaf size, and growth rate, delay in flowering and seeding, and decreases in seed production. Biochemical and physiological analyses indicated that these defects are due to the substantial reductions in the abundances of 4Fe-4S-containing photosystem I (PSI) core subunits PsaA (where Psa stands for PSI), PsaB, and PsaC and a nearly complete loss of PSI activity. In addition to the substantial decreases in the amounts of PSI core proteins, the content of 3Fe-4S-containing ferredoxin-dependent glutamine oxoglutarate aminotransferases declined significantly in the nfu3 mutants. Furthermore, the absorption spectrum of the recombinant NFU3 protein showed features characteristic of 4Fe-4S and 3Fe-4S clusters, and the in vitro reconstitution experiment indicated an iron-sulfur scaffold function of NFU3. These data demonstrate that NFU3 is involved in the assembly and transfer of 4Fe-4S and 3Fe-4S clusters and that NFU3 is required for the accumulation of 4Fe-4S- and 3Fe-4S-containing proteins, especially 4Fe-4S-containing PSI core subunits, in the Arabidopsis chloroplast. PMID:27784767

  6. Proteins iodinated by the chloramine-T method appear to be degraded at an abnormally rapid rate after endocytosis

    SciTech Connect

    Opresko, L.; Wiley, H.S.; Wallace, R.A.

    1980-03-01

    Proteins labeled with either /sup 3/H by reductive methylation or /sup 125/I by the chloramine-T method were incubated with Xenopus laevis oocytes; the incorporation and acid precipitability of the proteins were then studied. The uptake rates of both specifically incorporated (vitellogenin) and nonspecifically incorporated proteins (bovine serum albumin and X. laevis serum proteins lacking albumins) were not influenced by the method of labeling. However, /sup 125/I-labeled proteins were apparently degraded at rates far exceeding their /sup 3/H-labeled counterparts, based on the generation of acid-soluble radioactivity. Apparent degradation rates observed for endocytotically incorporated proteins may vary depending on the method used to label the protein and caution should be exercised when interpreting results obtained with labeled, particularly chloramine-T labeled, proteins.

  7. Niemann-Pick Type C2 Protein Mediates Hepatic Stellate Cells Activation by Regulating Free Cholesterol Accumulation.

    PubMed

    Twu, Yuh-Ching; Lee, Tzong-Shyuan; Lin, Yun-Lian; Hsu, Shih-Ming; Wang, Yuan-Hsi; Liao, Chia-Yu; Wang, Chung-Kwe; Liang, Yu-Chih; Liao, Yi-Jen

    2016-07-13

    In chronic liver diseases, regardless of their etiology, the development of fibrosis is the first step toward the progression to cirrhosis, portal hypertension, and hepatocellular carcinoma. Hepatic stellate cells (HSCs) are the main profibrogenic cells that promote the pathogenesis of liver fibrosis, and so it is important to identify the molecules that regulate HSCs activation and liver fibrosis. Niemann-Pick type C2 (NPC2) protein plays an important role in the regulation of intracellular cholesterol homeostasis by directly binding with free cholesterol. However, the roles of NPC2 in HSCs activation and liver fibrosis have not been explored in detail. Since a high-cholesterol diet exacerbates liver fibrosis progression in both rodents and humans, we propose that the expression of NPC2 affects free cholesterol metabolism and regulates HSCs activation. In this study, we found that NPC2 is decreased in both thioacetamide- and carbon tetrachloride-induced liver fibrosis tissues. In addition, NPC2 is expressed in quiescent HSCs, but its activation status is down-regulated. Knockdown of NPC2 in HSC-T6 cells resulted in marked increases in transforming growth factor-β1 (TGF-β1)-induced collagen type 1 α1 (Col1a1), α-smooth muscle actin (α-SMA) expression, and Smad2 phosphorylation. In contrast, NPC2 overexpression decreased TGF-β1-induced HSCs activation. We further demonstrated that NPC2 deficiency significantly increased the accumulation of free cholesterol in HSCs, increasing Col1a1 and α-SMA expression and activating Smad2, and leading to sensitization of HSCs to TGF-β1 activation. In contrast, overexpression of NPC2 decreased U18666A-induced free cholesterol accumulation and inhibited the subsequent HSCs activation. In conclusion, our study has demonstrated that NPC2 plays an important role in HSCs activation by regulating the accumulation of free cholesterol. NPC2 overexpression may thus represent a new treatment strategy for liver fibrosis.

  8. Niemann-Pick Type C2 Protein Mediates Hepatic Stellate Cells Activation by Regulating Free Cholesterol Accumulation

    PubMed Central

    Twu, Yuh-Ching; Lee, Tzong-Shyuan; Lin, Yun-Lian; Hsu, Shih-Ming; Wang, Yuan-Hsi; Liao, Chia-Yu; Wang, Chung-Kwe; Liang, Yu-Chih; Liao, Yi-Jen

    2016-01-01

    In chronic liver diseases, regardless of their etiology, the development of fibrosis is the first step toward the progression to cirrhosis, portal hypertension, and hepatocellular carcinoma. Hepatic stellate cells (HSCs) are the main profibrogenic cells that promote the pathogenesis of liver fibrosis, and so it is important to identify the molecules that regulate HSCs activation and liver fibrosis. Niemann-Pick type C2 (NPC2) protein plays an important role in the regulation of intracellular cholesterol homeostasis by directly binding with free cholesterol. However, the roles of NPC2 in HSCs activation and liver fibrosis have not been explored in detail. Since a high-cholesterol diet exacerbates liver fibrosis progression in both rodents and humans, we propose that the expression of NPC2 affects free cholesterol metabolism and regulates HSCs activation. In this study, we found that NPC2 is decreased in both thioacetamide- and carbon tetrachloride-induced liver fibrosis tissues. In addition, NPC2 is expressed in quiescent HSCs, but its activation status is down-regulated. Knockdown of NPC2 in HSC-T6 cells resulted in marked increases in transforming growth factor-β1 (TGF-β1)-induced collagen type 1 α1 (Col1a1), α-smooth muscle actin (α-SMA) expression, and Smad2 phosphorylation. In contrast, NPC2 overexpression decreased TGF-β1-induced HSCs activation. We further demonstrated that NPC2 deficiency significantly increased the accumulation of free cholesterol in HSCs, increasing Col1a1 and α-SMA expression and activating Smad2, and leading to sensitization of HSCs to TGF-β1 activation. In contrast, overexpression of NPC2 decreased U18666A-induced free cholesterol accumulation and inhibited the subsequent HSCs activation. In conclusion, our study has demonstrated that NPC2 plays an important role in HSCs activation by regulating the accumulation of free cholesterol. NPC2 overexpression may thus represent a new treatment strategy for liver fibrosis. PMID:27420058

  9. A Caleosin-Like Protein with Peroxygenase Activity Mediates Aspergillus flavus Development, Aflatoxin Accumulation, and Seed Infection

    PubMed Central

    Almousally, Ibrahem; Shaban, Mouhnad; Blee, Elizabeth

    2015-01-01

    Caleosins are a small family of calcium-binding proteins endowed with peroxygenase activity in plants. Caleosin-like genes are present in fungi; however, their functions have not been reported yet. In this work, we identify a plant caleosin-like protein in Aspergillus flavus that is highly expressed during the early stages of spore germination. A recombinant purified 32-kDa caleosin-like protein supported peroxygenase activities, including co-oxidation reactions and reduction of polyunsaturated fatty acid hydroperoxides. Deletion of the caleosin gene prevented fungal development. Alternatively, silencing of the gene led to the increased accumulation of endogenous polyunsaturated fatty acid hydroperoxides and antioxidant activities but to a reduction of fungal growth and conidium formation. Two key genes of the aflatoxin biosynthesis pathway, aflR and aflD, were downregulated in the strains in which A. flavus PXG (AfPXG) was silenced, leading to reduced aflatoxin B1 production in vitro. Application of caleosin/peroxygenase-derived oxylipins restored the wild-type phenotype in the strains in which AfPXG was silenced. PXG-deficient A. flavus strains were severely compromised in their capacity to infect maize seeds and to produce aflatoxin. Our results uncover a new branch of the fungal oxylipin pathway and may lead to the development of novel targets for controlling fungal disease. PMID:26116672

  10. Testosterone modulates gene expression pathways regulating nutrient accumulation, glucose metabolism and protein turnover in mouse skeletal muscle.

    PubMed

    Haren, M T; Siddiqui, A M; Armbrecht, H J; Kevorkian, R T; Kim, M J; Haas, M J; Mazza, A; Kumar, Vijaya B; Green, M; Banks, W A; Morley, J E

    2011-02-01

    Testosterone regulates energy metabolism and skeletal muscle mass in males, but the molecular mechanisms are not fully understood. This study investigated the response of skeletal muscle to castration and testosterone replacement in 8-week-old male mice. Using microarray analyses of mRNA levels in gastrocnemius muscle, 91 genes were found to be negatively regulated by testosterone and 68 genes were positively regulated. The mRNA levels of the insulin signalling suppressor molecule Grb10 and the glycogen synthesis inhibitors, protein phosphatase inhibitor-1 and phosphorylase kinase-γ, were negatively regulated by testosterone. The insulin-sensitive glucose and amino acid transporters, Glut3 and SAT2, the lipodystrophy gene, Lpin1 and protein targeting to glycogen were positively regulated. These changes would be expected to increase nutrient availability and sensing within skeletal muscle, increase metabolic rate and carbohydrate utilization and promote glycogen accumulation. The observed positive regulation of atrogin-1 (Fbxo32) by testosterone could be explained by the phosphorylation of Akt and Foxo3a, as determined by Western blotting. Testosterone prevented the castration-induced increase in interleukin-1α, the decrease in interferon-γ and the atrophy of the levator ani muscle, which were all correlated with testosterone-regulated gene expression. These findings identify specific mechanisms by which testosterone may regulate skeletal muscle glucose and protein metabolism.

  11. Subcellular targeting is a key condition for high-level accumulation of cellulase protein in transgenic maize seed.

    PubMed

    Hood, Elizabeth E; Love, Robert; Lane, Jeff; Bray, Jeff; Clough, Richard; Pappu, Kamesh; Drees, Carol; Hood, Kendall R; Yoon, Sangwoong; Ahmad, Atta; Howard, John A

    2007-11-01

    Ethanol from lignocellulosic biomass is being pursued as an alternative to petroleum-based transportation fuels. To succeed in this endeavour, efficient digestion of cellulose into monomeric sugar streams is a key step. Current production systems for cellulase enzymes, i.e. fungi and bacteria, cannot meet the cost and huge volume requirements of this commodity-based industry. Transgenic maize (Zea mays L.) seed containing cellulase protein in embryo tissue, with protein localized to the endoplasmic reticulum, cell wall or vacuole, allows the recovery of commercial amounts of enzyme. E1 cellulase, an endo-beta-1,4-glucanase from Acidothermus cellulolyticus, was recovered at levels greater than 16% total soluble protein (TSP) in single seed. More significantly, cellobiohydrolase I (CBH I), an exocellulase from Trichoderma reesei, also accumulated to levels greater than 16% TSP in single seed, nearly 1000-fold higher than the expression in any other plant reported in the literature. The catalytic domain was the dominant form of E1 that was detected in the endoplasmic reticulum and vacuole, whereas CBH I holoenzyme was present in the cell wall. With one exception, individual transgenic events contained single inserts. Recovery of high levels of enzyme in T2 ears demonstrated that expression is likely to be stable over multiple generations. The enzymes were active in cleaving soluble substrate.

  12. Receptor protein kinase FERONIA controls leaf starch accumulation by interacting with glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Yang, Tao; Wang, Long; Li, Chiyu; Liu, Ying; Zhu, Sirui; Qi, Yinyao; Liu, Xuanming; Lin, Qinglu; Luan, Sheng; Yu, Feng

    2015-09-11

    Cell expansion is coordinated by several cues, but available energy is the major factor determining growth. Receptor protein kinase FERONIA (FER) is a master regulator of cell expansion, but the details of its control mechanisms are not clear. Here we show that FER interacts with cytosolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GAPC1 and GAPC2), that catalyzes a key reaction in glycolysis, which contributes to energy production. When there is an FER deficiency, there are corresponding decreases in the enzyme activity of GAPDH and increased amounts of starch. More importantly, gapc1/2 mutants mimic fer4 mutants. These data indicate that FER regulated starch content is an evolutionarily conserved function in plants that connects the cell expansion and energy metabolism pathways.

  13. AniA regulates reserve polymer accumulation and global protein expression in Rhizobium etli.

    PubMed

    Encarnación, Sergio; del Carmen Vargas, María; Dunn, Michael F; Dávalos, Araceli; Mendoza, Guillermo; Mora, Yolanda; Mora, Jaime

    2002-04-01

    Previously, it was reported that the oxidative capacity and ability to grow on carbon sources such as pyruvate and glucose were severely diminished in the Rhizobium etli phaC::OmegaSm(r)/Sp(r) mutant CAR1, which is unable to synthesize poly-beta-hydroxybutyric acid (PHB) (M. A. Cevallos, S. Encarnación, A. Leija, Y. Mora, and J. Mora, J. Bacteriol. 178:1646-1654, 1996). By random Tn5 mutagenesis of the phaC strain, we isolated the mutants VEM57 and VEM58, both of which contained single Tn5 insertions and had recovered the ability to grow on pyruvate or glucose. Nucleotide sequencing of the region surrounding the Tn5 insertions showed that they had interrupted an open reading frame designated aniA based on its high deduced amino acid sequence identity to the aniA gene product of Sinorhizobium meliloti. R. etli aniA was located adjacent to and divergently transcribed from genes encoding the PHB biosynthetic enzymes beta-ketothiolase (PhaA) and acetoacetyl coenzyme A reductase (PhaB). An aniA::Tn5 mutant (VEM5854) was constructed and found to synthesize only 40% of the wild type level of PHB. Both VEM58 and VEM5854 produced significantly more extracellular polysaccharide than the wild type. Organic acid excretion and levels of intracellular reduced nucleotides were lowered to wild-type levels in VEM58 and VEM5854, in contrast to those of strain CAR1, which were significantly elevated. Proteome analysis of VEM58 showed a drastic alteration of protein expression, including the absence of a protein identified as PhaB. We propose that the aniA gene product plays an important role in directing carbon flow in R. etli.

  14. AniA Regulates Reserve Polymer Accumulation and Global Protein Expression in Rhizobium etli

    PubMed Central

    Encarnación, Sergio; Vargas, María del Carmen; Dunn, Michael F.; Dávalos, Araceli; Mendoza, Guillermo; Mora, Yolanda; Mora, Jaime

    2002-01-01

    Previously, it was reported that the oxidative capacity and ability to grow on carbon sources such as pyruvate and glucose were severely diminished in the Rhizobium etli phaC::ΩSmr/Spr mutant CAR1, which is unable to synthesize poly-β-hydroxybutyric acid (PHB) (M. A. Cevallos, S. Encarnación, A. Leija, Y. Mora, and J. Mora, J. Bacteriol. 178:1646-1654, 1996). By random Tn5 mutagenesis of the phaC strain, we isolated the mutants VEM57 and VEM58, both of which contained single Tn5 insertions and had recovered the ability to grow on pyruvate or glucose. Nucleotide sequencing of the region surrounding the Tn5 insertions showed that they had interrupted an open reading frame designated aniA based on its high deduced amino acid sequence identity to the aniA gene product of Sinorhizobium meliloti. R. etli aniA was located adjacent to and divergently transcribed from genes encoding the PHB biosynthetic enzymes β-ketothiolase (PhaA) and acetoacetyl coenzyme A reductase (PhaB). An aniA::Tn5 mutant (VEM5854) was constructed and found to synthesize only 40% of the wild type level of PHB. Both VEM58 and VEM5854 produced significantly more extracellular polysaccharide than the wild type. Organic acid excretion and levels of intracellular reduced nucleotides were lowered to wild-type levels in VEM58 and VEM5854, in contrast to those of strain CAR1, which were significantly elevated. Proteome analysis of VEM58 showed a drastic alteration of protein expression, including the absence of a protein identified as PhaB. We propose that the aniA gene product plays an important role in directing carbon flow in R. etli. PMID:11914361

  15. Rapid and systemic accumulation of chloroplast mRNA-binding protein transcripts after flame stimulus in tomato

    NASA Technical Reports Server (NTRS)

    Vian, A.; Henry-Vian, C.; Davies, E.

    1999-01-01

    It has been shown that tomato (Lycopersicon esculentum) plants respond to flame wounding and electrical stimulation by a rapid (15 min) and systemic up-regulation of proteinase inhibitor (pin) genes. To find other genes having a similar expression pattern, we used subtractive cDNA screening between flamed and control plants to select clones up-regulated by flame wounding. We report the characterization of one of them, a chloroplast mRNA-binding protein encoded by a single gene and expressed preferentially in the leaves. Systemic gene expression in response to flaming in the youngest terminal leaf exhibited three distinct phases: a rapid and transient increase (5-15 min) in transcript accumulation, a decline to basal levels (15-45 min), and then a second, more prolonged increase (60-90 min). In contrast, after a mechanical wound the rapid, transient increase (5 min) was followed by a rapid decline to basal levels but no later, prolonged accumulation. In the petiole, the initial flame-wound-evoked transient increase (15 min) was followed by a continuous decline for 3 h. The nature of the wound signal(s) causing such rapid changes in transcript abundance is discussed in relation to electrical signaling, which has recently been implicated in plant responses to wounding.

  16. Antidepressants Accumulate in Lipid Rafts Independent of Monoamine Transporters to Modulate Redistribution of the G Protein, Gαs.

    PubMed

    Erb, Samuel J; Schappi, Jeffrey M; Rasenick, Mark M

    2016-09-16

    Depression is a significant public health problem for which currently available medications, if effective, require weeks to months of treatment before patients respond. Previous studies have shown that the G protein responsible for increasing cAMP (Gαs) is increasingly localized to lipid rafts in depressed subjects and that chronic antidepressant treatment translocates Gαs from lipid rafts. Translocation of Gαs, which shows delayed onset after chronic antidepressant treatment of rats or of C6 glioma cells, tracks with the delayed onset of therapeutic action of antidepressants. Because antidepressants appear to specifically modify Gαs localized to lipid rafts, we sought to determine whether structurally diverse antidepressants accumulate in lipid rafts. Sustained treatment of C6 glioma cells, which lack 5-hydroxytryptamine transporters, showed marked concentration of several antidepressants in raft fractions, as revealed by increased absorbance and by mass fingerprint. Closely related molecules without antidepressant activity did not concentrate in raft fractions. Thus, at least two classes of antidepressants accumulate in lipid rafts and effect translocation of Gαs to the non-raft membrane fraction, where it activates the cAMP-signaling cascade. Analysis of the structural determinants of raft localization may both help to explain the hysteresis of antidepressant action and lead to design and development of novel substrates for depression therapeutics.

  17. Detection of the abnormal isoform of the prion protein associated with chronic wasting disease in the optic pathways of the brain and retina of Rocky Mountain elk (Cervus elaphus nelsoni).

    PubMed

    Spraker, T R; O'Rourke, K I; Gidlewski, T; Powers, J G; Greenlee, J J; Wild, M A

    2010-05-01

    Eyes and nuclei of the visual pathways in the brain were examined in 30 Rocky Mountain elk (Cervus elaphus nelsoni) representing 3 genotypes of the prion protein gene PRNP (codon 132: MM, ML, or LL). Tissues were examined for the presence of the abnormal isoform of the prion protein associated with chronic wasting disease (PrP(CWD)). Nuclei and axonal tracts from a single section of brain stem at the level of the dorsal motor nucleus of the vagus nerve were scored for intensity and distribution of PrP(CWD) immunoreactivity and degree of spongiform degeneration. This obex scoring ranged from 0 (elk with no PrP(CWD) in the brain stem) to 10 (representing elk in terminal stage of disease). PrP(CWD) was detected in the retina of 16 of 18 (89%) elk with an obex score of > 7. PrP(CWD) was not detected in the retina of the 3 chronic wasting disease-negative elk and 9 elk with an obex score of < 6. PrP(CWD) was found in the nuclei of the visual pathways in the brain before it was found in the retina. Within the retina, PrP(CWD) was first found in the inner plexiform layer, followed by the outer plexiform layer. Intracytoplasmic accumulation of PrP(CWD) was found in a few neurons in the ganglion cell layer in the PRNP 132ML elk but was a prominent feature in the PRNP 132LL elk. Small aggregates of PrP(CWD) were present on the inner surface of the outer limiting membrane in PRNP 132LL elk but not in PRNP 132MM or 132ML elk. This study demonstrates PrP(CWD) accumulation in nuclei of the visual pathways of the brain, followed by PrP(CWD) in the retina.

  18. Accumulate repeat accumulate codes

    NASA Technical Reports Server (NTRS)

    Abbasfar, Aliazam; Divsalar, Dariush; Yao, Kung

    2004-01-01

    In this paper we propose an innovative channel coding scheme called 'Accumulate Repeat Accumulate codes' (ARA). This class of codes can be viewed as serial turbo-like codes, or as a subclass of Low Density Parity Check (LDPC) codes, thus belief propagation can be used for iterative decoding of ARA codes on a graph. The structure of encoder for this class can be viewed as precoded Repeat Accumulate (RA) code or as precoded Irregular Repeat Accumulate (IRA) code, where simply an accumulator is chosen as a precoder. Thus ARA codes have simple, and very fast encoder structure when they representing LDPC codes. Based on density evolution for LDPC codes through some examples for ARA codes, we show that for maximum variable node degree 5 a minimum bit SNR as low as 0.08 dB from channel capacity for rate 1/2 can be achieved as the block size goes to infinity. Thus based on fixed low maximum variable node degree, its threshold outperforms not only the RA and IRA codes but also the best known LDPC codes with the dame maximum node degree. Furthermore by puncturing the accumulators any desired high rate codes close to code rate 1 can be obtained with thresholds that stay close to the channel capacity thresholds uniformly. Iterative decoding simulation results are provided. The ARA codes also have projected graph or protograph representation that allows for high speed decoder implementation.

  19. Leukocyte abnormalities.

    PubMed

    Gabig, T G

    1980-07-01

    Certain qualitative abnormalities in neutrophils and blood monocytes are associated with frequent, severe, and recurrent bacterial infections leading to fatal sepsis, while other qualitative defects demonstrated in vitro may have few or no clinical sequelae. These qualitative defects are discussed in terms of the specific functions of locomotion, phagocytosis, degranulation, and bacterial killing.

  20. Prion neuropathology follows the accumulation of alternate prion protein isoforms after infective titre has peaked

    PubMed Central

    Sandberg, Malin K.; Al-Doujaily, Huda; Sharps, Bernadette; De Oliveira, Michael Wiggins; Schmidt, Christian; Richard-Londt, Angela; Lyall, Sarah; Linehan, Jacqueline M.; Brandner, Sebastian; Wadsworth, Jonathan D. F.; Clarke, Anthony R.; Collinge, John

    2014-01-01

    Prions are lethal infectious agents thought to consist of multi-chain forms (PrPSc) of misfolded cellular prion protein (PrPC). Prion propagation proceeds in two distinct mechanistic phases: an exponential phase 1, which rapidly reaches a fixed level of infectivity irrespective of PrPC expression level, and a plateau (phase 2), which continues until clinical onset with duration inversely proportional to PrPC expression level. We hypothesized that neurotoxicity relates to distinct neurotoxic species produced following a pathway switch when prion levels saturate. Here we show a linear increase of proteinase K-sensitive PrP isoforms distinct from classical PrPSc at a rate proportional to PrPC concentration, commencing at the phase transition and rising until clinical onset. The unaltered level of total PrP during phase 1, when prion infectivity increases a million-fold, indicates that prions comprise a small minority of total PrP. This is consistent with PrPC concentration not being rate limiting to exponential prion propagation and neurotoxicity relating to critical concentrations of alternate PrP isoforms whose production is PrPC concentration dependent. PMID:25005024

  1. The relationships among IGF-1, DNA content, and protein accumulation during skeletal muscle hypertrophy

    NASA Technical Reports Server (NTRS)

    Adams, G. R.; Haddad, F.

    1996-01-01

    Insulin-like growth factor-1 (IGF-1) is known to have anabolic effects on skeletal muscle cells. This study examined the time course of muscle hypertrophy and associated IGF-1 peptide and mRNA expression. Data were collected at 3, 7, 14, and 28 days after surgical removal of synergistic muscles of both normal and hypophysectomized (HX) animals. Overloading increased the plantaris (Plant) mass, myofiber size, and protein-to-body weight ratio in both groups (normal and HX; P < 0.05). Muscle IGF-1 peptide levels peaked at 3 (normal) and 7 (HX) days of overloading with maximum 4.1-fold (normal) and 6.2-fold (HX) increases. Increases in muscle IGF-1 preceded the hypertrophic response. Total DNA content of the overloaded Plant increased in both groups. There was a strong positive relationship between IGF-1 peptide and DNA content in the overloaded Plant from both groups. These results indicate that 1) the muscles from rats with both normal and severely depressed systemic levels of IGF-1 respond to functional overload with an increase in local IGF-1 expression and 2) this elevated IGF-1 may be contributing to the hypertrophy response, possibly via the mobilization of satellite cells to provide increases in muscle DNA.

  2. Phosphate availability regulates ethylene biosynthesis gene expression and protein accumulation in white clover (Trifolium repens L.) roots

    PubMed Central

    Roldan, Marissa; Islam, Afsana; Dinh, Phuong T.Y.; Leung, Susanna; McManus, Michael T.

    2016-01-01

    The expression and accumulation of members of the 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) and ACC oxidase (ACO) gene families was examined in white clover roots grown in either Pi (phosphate) sufficient or Pi-deprived defined media. The accumulation of one ACO isoform, TR-ACO1, was positively influenced after only 1 h of exposure to low Pi, and this was maintained over a 7-day time-course. Up-regulation of TR-ACS1, TR-ACS2 and TR-ACS3 transcript abundance was also observed within 1 h of exposure to low Pi in different tissue regions of the roots, followed by a second increase in abundance of TR-ACS2 after 5–7 days of exposure. An increase in transcript abundance of TR-ACO1 and TR-ACO3, but not TR-ACO2, was observed after 1 h of exposure to low Pi, with a second increase in TR-ACO1 transcripts occurring after 2–5 days. These initial increases of the TR-ACS and TR-ACO transcript abundance occurred before the induction of Trifolium repens PHOSPHATE TRANSPORTER 1 (TR-PT1), and the addition of sodium phosphite did not up-regulate TR-ACS1 expression over 24 h. In situ hybridization revealed some overlap of TR-ACO mRNA accumulation, with TR-ACO1 and TR-ACO2 in the root tip regions, and TR-ACO1 and TR-ACO3 mRNA predominantly in the lateral root primordia. TR-ACO1p-driven GFP expression showed that activation of the TR-ACO1 promoter was initiated within 24 h of exposure to low Pi (as determined by GFP protein accumulation). These results suggest that the regulation of ethylene biosynthesis in white clover roots is biphasic in response to low Pi supply. PMID:27737923

  3. Essential role of conserved DUF177A protein in plastid 23S rRNA accumulation and plant embryogenesis

    PubMed Central

    Yang, Jiani; Suzuki, Masaharu; McCarty, Donald R.

    2016-01-01

    DUF177 proteins are nearly universally conserved in bacteria and plants except the Chlorophyceae algae. Thus far, duf177 mutants in bacteria have not established a function. In contrast, duf177a mutants have embryo lethal phenotypes in maize and Arabidopsis. In maize inbred W22, duf177a mutant embryos arrest at an early transition stage, whereas the block is suppressed in the B73 inbred background, conditioning an albino seedling phenotype. Background-dependent embryo lethal phenotypes are characteristic of maize plastid gene expression mutants. Consistent with the plastid gene expression hypothesis, quantitative real-time PCR revealed a significant reduction of 23S rRNA in an Escherichia coli duf177 knockout. Plastid 23S rRNA contents of duf177a mutant tissues were also markedly reduced compared with the wild-type, whereas plastid 16S, 5S, and 4.5S rRNA contents were less affected, indicating that DUF177 is specifically required for accumulation of prokaryote-type 23S rRNA. An AtDUF177A–green fluorescent protein (GFP) transgene controlled by the native AtDUF177A promoter fully complemented the Arabidopsis atduf177a mutant. Transient expression of AtDUF177A–GFP in Nicotiana benthamiana leaves showed that the protein was localized in chloroplasts. The essential role of DUF177A in chloroplast–ribosome formation is reminiscent of IOJAP, another highly conserved ribosome-associated protein, suggesting that key mechanisms controlling ribosome formation in plastids evolved from non-essential pathways for regulation of the prokaryotic ribosome. PMID:27574185

  4. Abnormal pressure in hydrocarbon environments

    USGS Publications Warehouse

    Law, B.E.; Spencer, C.W.

    1998-01-01

    Abnormal pressures, pressures above or below hydrostatic pressures, occur on all continents in a wide range of geological conditions. According to a survey of published literature on abnormal pressures, compaction disequilibrium and hydrocarbon generation are the two most commonly cited causes of abnormally high pressure in petroleum provinces. In young (Tertiary) deltaic sequences, compaction disequilibrium is the dominant cause of abnormal pressure. In older (pre-Tertiary) lithified rocks, hydrocarbon generation, aquathermal expansion, and tectonics are most often cited as the causes of abnormal pressure. The association of abnormal pressures with hydrocarbon accumulations is statistically significant. Within abnormally pressured reservoirs, empirical evidence indicates that the bulk of economically recoverable oil and gas occurs in reservoirs with pressure gradients less than 0.75 psi/ft (17.4 kPa/m) and there is very little production potential from reservoirs that exceed 0.85 psi/ft (19.6 kPa/m). Abnormally pressured rocks are also commonly associated with unconventional gas accumulations where the pressuring phase is gas of either a thermal or microbial origin. In underpressured, thermally mature rocks, the affected reservoirs have most often experienced a significant cooling history and probably evolved from an originally overpressured system.

  5. Accumulated SET protein up-regulates and interacts with hnRNPK, increasing its binding to nucleic acids, the Bcl-xS repression, and cellular proliferation

    SciTech Connect

    Almeida, Luciana O.; Garcia, Cristiana B.; Matos-Silva, Flavia A.; Curti, Carlos; Leopoldino, Andréia M.

    2014-02-28

    Highlights: • hnRNPK is a new target of SET. • SET regulates hnRNPK. • SET and hnRNPK accumulation promotes tumorigenesis. • SET accumulation is a potential model to study genes regulated by SET-hnRNPK. - Abstract: SET and hnRNPK are proteins involved in gene expression and regulation of cellular signaling. We previously demonstrated that SET accumulates in head and neck squamous cell carcinoma (HNSCC); hnRNPK is a prognostic marker in cancer. Here, we postulate that SET and hnRNPK proteins interact to promote tumorigenesis. We performed studies in HEK293 and HNSCC (HN6, HN12, and HN13) cell lines with SET/hnRNPK overexpression and knockdown, respectively. We found that SET and/or hnRNPK protein accumulation increased cellular proliferation. SET accumulation up-regulated hnRNPK mRNA and total/phosphorylated protein, promoted hnRNPK nuclear location, and reduced Bcl-x mRNA levels. SET protein directly interacted with hnRNPK, increasing both its binding to nucleic acids and Bcl-xS repression. We propose that hnRNPK should be a new target of SET and that SET–hnRNPK interaction, in turn, has potential implications in cell survival and malignant transformation.

  6. Rice PROTEIN l-ISOASPARTYL METHYLTRANSFERASE isoforms differentially accumulate during seed maturation to restrict deleterious isoAsp and reactive oxygen species accumulation and are implicated in seed vigor and longevity.

    PubMed

    Petla, Bhanu Prakash; Kamble, Nitin Uttam; Kumar, Meenu; Verma, Pooja; Ghosh, Shraboni; Singh, Ajeet; Rao, Venkateswara; Salvi, Prafull; Kaur, Harmeet; Saxena, Saurabh Chandra; Majee, Manoj

    2016-07-01

    PROTEIN l-ISOASPARTYL O-METHYLTRANSFERASE (PIMT) is a protein-repairing enzyme involved in seed vigor and longevity. However, the regulation of PIMT isoforms during seed development and the mechanism of PIMT-mediated improvement of seed vigor and longevity are largely unknown. In this study in rice (Oryza sativa), we demonstrate the dynamics and correlation of isoaspartyl (isoAsp)-repairing demands and PIMT activity, and their implications, during seed development, germination and aging, through biochemical, molecular and genetic studies. Molecular and biochemical analyses revealed that rice possesses various biochemically active and inactive PIMT isoforms. Transcript and western blot analyses clearly showed the seed development stage and tissue-specific accumulation of active isoforms. Immunolocalization studies revealed distinct isoform expression in embryo and aleurone layers. Further analyses of transgenic lines for each OsPIMT isoform revealed a clear role in the restriction of deleterious isoAsp and age-induced reactive oxygen species (ROS) accumulation to improve seed vigor and longevity. Collectively, our data suggest that a PIMT-mediated, protein repair mechanism is initiated during seed development in rice, with each isoform playing a distinct, yet coordinated, role. Our results also raise the intriguing possibility that PIMT repairs antioxidative enzymes and proteins which restrict ROS accumulation, lipid peroxidation, etc. in seed, particularly during aging, thus contributing to seed vigor and longevity.

  7. Cytoplasmic Retention of Protein Phosphatase 2A Inhibitor 2 (I2PP2A) Induces Alzheimer-like Abnormal Hyperphosphorylation of Tau*

    PubMed Central

    Arif, Mohammad; Wei, Jianshe; Zhang, Qi; Liu, Fei; Basurto-Islas, Gustavo; Grundke-Iqbal, Inge; Iqbal, Khalid

    2014-01-01

    Abnormal hyperphosphorylation of Tau leads to the formation of neurofibrillary tangles, a hallmark of Alzheimer disease (AD), and related tauopathies. The phosphorylation of Tau is regulated by protein phosphatase 2A (PP2A), which in turn is modulated by endogenous inhibitor 2 (I2PP2A). In AD brain, I2PP2A is translocated from neuronal nucleus to cytoplasm, where it inhibits PP2A activity and promotes abnormal phosphorylation of Tau. Here we describe the identification of a potential nuclear localization signal (NLS) in the C-terminal region of I2PP2A containing a conserved basic motif, 179RKR181, which is sufficient for directing its nuclear localization. The current study further presents an inducible cell model (Tet-Off system) of AD-type abnormal hyperphosphorylation of Tau by expressing I2PP2A in which the NLS was inactivated by 179RKR181 → AAA along with 168KR169 → AA mutations. In this model, the mutant NLS (mNLS)-I2PP2A (I2PP2AAA-AAA) was retained in the cell cytoplasm, where it physically interacted with PP2A and inhibited its activity. Inhibition of PP2A was associated with the abnormal hyperphosphorylation of Tau, which resulted in microtubule network instability and neurite outgrowth impairment. Expression of mNLS-I2PP2A activated CAMKII and GSK-3β, which are Tau kinases regulated by PP2A. The immunoprecipitation experiments showed the direct interaction of I2PP2A with PP2A and GSK-3β but not with CAMKII. Thus, the cell model provides insights into the nature of the potential NLS and the mechanistic relationship between I2PP2A-induced inhibition of PP2A and hyperphosphorylation of Tau that can be utilized to develop drugs preventing Tau pathology. PMID:25128526

  8. Accumulation of wild-type p53 protein in astrocytomas is not mediated by MDM2 gene amplification

    SciTech Connect

    Rubio, M.P.; Louis, D.N. Harvard Medical School, Boston, MA )

    1993-05-01

    The authors have previously described ten cases of astrocytoma (three WHO grade II, four grade III and four grade IV) with seemingly contradictory results on immunohistochemical analysis of the p53 protein and molecular genetic analysis of the p53 gene. Fixed, embedded tissues from these cases were immunohistochemically positive with the PAb 1801 antibody, which supposedly implies the presence of mutant protein. These ten cases, however, did not have mutations in exons 5 through 8 of the p53 gene, the conserved regions in which almost all human mutations have been described. The authors suggested that these cases might either represent overexpression of wild-type p53 protein (since the PAb 1801 antibody reacts with both wild-type and mutant p53 protein) or mutations in less conserved regions of the gene. To investigate these possibilities further, they performed single strand conformational polymorphism analysis and DNA sequencing on p53 exons 4, 9 and 10 in the nine cases with available DNA, since rare mutations have been noted at these loci. None of the cases showed alterations, making it highly unlikely that these tumors harbor mutations in exons of the p53 gene. They also performed immunohistochemistry on frozen sections from seven available tumors, using the mutant-specific antibody PAb 240 in addition to PAb 1801. All tumors continued to show positive staining with PAb 1801, but only one tumor reacted with PAb 240. The results support the hypothesis that the accumulated p53 protein in most cases is wild-type. Because the product of the MDM2 oncogene can bind to wild-type p53 protein, and because MDM2 amplification has recently been demonstrated in human tumors, the authors evaluated MDM2 amplification in the nine astrocytomas with available DNA. Using slot blot analysis with a 96-base pair, PCR-generated probe to the first exon of the MDM2 gene, they were unable to show MDM2 gene amplification in these tumors or in other assayed astrocytomas.

  9. Fatty Acid Transport Protein-2 inhibitor Grassofermata/CB5 protects cells against lipid accumulation and toxicity

    PubMed Central

    Saini, Nipun; Black, Paul N.; Montefusco, David; DiRusso, Concetta C.

    2015-01-01

    The inhibition of the fatty acid uptake into non-adipose tissues provides an attractive target for prevention of lipotoxicity leading to obesity-associated non-alcoholic fatty liver disease and type 2 diabetes. Fatty acid transport proteins (FATPs) are bifunctional proteins involved in the uptake and activation of fatty acids by esterification with coenzyme A. Here we characterize Grassofermata/CB5, previously identified as a fatty acid uptake inhibitor directed against HsFATP2. The compound was effective in inhibiting the uptake of fatty acids in the low micro-molar range (IC50 8–11μM) and prevented palmitate-mediated lipid accumulation and cell death in cell lines that are models for intestines, liver, muscle and pancreas. In adipocytes, uptake inhibition was less effective (IC50 58μM). Inhibition was specific for long chain fatty acids and was ineffective toward medium chain fatty acids, which are transported by diffusion. Kinetic analysis of Grassofermata-dependent FA transport inhibition verified a non-competitive mechanism. By comparison with Grassofermata, several atypical antipsychotic drugs previously implicated as inhibitors of FA uptake were ineffectual. In mice Grassofermata decreased absorption of 13C-oleate demonstrating its potential as a therapeutic agent. PMID:26284975

  10. Combined nitrogen limitation and cadmium stress stimulate total carbohydrates, lipids, protein and amino acid accumulation in Chlorella vulgaris (Trebouxiophyceae).

    PubMed

    Chia, Mathias Ahii; Lombardi, Ana Teresa; da Graça Gama Melão, Maria; Parrish, Christopher C

    2015-03-01

    Metals have interactive effects on the uptake and metabolism of nutrients in microalgae. However, the effect of trace metal toxicity on amino acid composition of Chlorella vulgaris as a function of varying nitrogen concentrations is not known. In this research, C. vulgaris was used to investigate the influence of cadmium (10(-7) and 2.0×10(-8)molL(-1) Cd) under varying nitrogen (2.9×10(-6), 1.1×10(-5) and 1.1×10(-3)molL(-1)N) concentrations on its growth rate, biomass and biochemical composition. Total carbohydrates, total proteins, total lipids, as well as individual amino acid proportions were determined. The combination of Cd stress and N limitation significantly inhibited growth rate and cell density of C. vulgaris. However, increasing N limitation and Cd stress stimulated higher dry weight and chlorophyll a production per cell. Furthermore, biomolecules like total proteins, carbohydrates and lipids increased with increasing N limitation and Cd stress. Ketogenic and glucogenic amino acids were accumulated under the stress conditions investigated in the present study. Amino acids involved in metal chelation like proline, histidine and glutamine were significantly increased after exposure to combined Cd stress and N limitation. We conclude that N limitation and Cd stress affects the physiology of C. vulgaris by not only decreasing its growth but also stimulating biomolecule production.

  11. Intermediate filament protein accumulation in motor neurons derived from giant axonal neuropathy iPSCs rescued by restoration of gigaxonin

    PubMed Central

    Johnson-Kerner, Bethany L.; Ahmad, Faizzan S.; Diaz, Alejandro Garcia; Greene, John Palmer; Gray, Steven J.; Samulski, Richard Jude; Chung, Wendy K.; Van Coster, Rudy; Maertens, Paul; Noggle, Scott A.; Henderson, Christopher E.; Wichterle, Hynek

    2015-01-01

    Giant axonal neuropathy (GAN) is a progressive neurodegenerative disease caused by autosomal recessive mutations in the GAN gene resulting in a loss of a ubiquitously expressed protein, gigaxonin. Gene replacement therapy is a promising strategy for treatment of the disease; however, the effectiveness and safety of gigaxonin reintroduction have not been tested in human GAN nerve cells. Here we report the derivation of induced pluripotent stem cells (iPSCs) from three GAN patients with different GAN mutations. Motor neurons differentiated from GAN iPSCs exhibit accumulation of neurofilament (NF-L) and peripherin (PRPH) protein and formation of PRPH aggregates, the key pathological phenotypes observed in patients. Introduction of gigaxonin either using a lentiviral vector or as a stable transgene resulted in normalization of NEFL and PRPH levels in GAN neurons and disappearance of PRPH aggregates. Importantly, overexpression of gigaxonin had no adverse effect on survival of GAN neurons, supporting the feasibility of gene replacement therapy. Our findings demonstrate that GAN iPSCs provide a novel model for studying human GAN neuropathologies and for the development and testing of new therapies in relevant cell types. PMID:25398950

  12. Through Its Nonstructural Protein NS1, Parvovirus H-1 Induces Apoptosis via Accumulation of Reactive Oxygen Species▿ †

    PubMed Central

    Hristov, Georgi; Krämer, Melanie; Li, Junwei; El-Andaloussi, Nazim; Mora, Rodrigo; Daeffler, Laurent; Zentgraf, Hanswalter; Rommelaere, Jean; Marchini, Antonio

    2010-01-01

    The rat parvovirus H-1 (H-1PV) attracts high attention as an anticancer agent, because it is not pathogenic for humans and has oncotropic and oncosuppressive properties. The viral nonstructural NS1 protein is thought to mediate H-1PV cytotoxicity, but its exact contribution to this process remains undefined. In this study, we analyzed the effects of the H-1PV NS1 protein on human cell proliferation and cell viability. We show that NS1 expression is sufficient to induce the accumulation of cells in G2 phase, apoptosis via caspase 9 and 3 activation, and cell lysis. Similarly, cells infected with wild-type H-1PV arrest in G2 phase and undergo apoptosis. Furthermore, we also show that both expression of NS1 and H-1PV infection lead to higher levels of intracellular reactive oxygen species (ROS), associated with DNA double-strand breaks. Antioxidant treatment reduces ROS levels and strongly decreases NS1- and virus-induced DNA damage, cell cycle arrest, and apoptosis, indicating that NS1-induced ROS are important mediators of H-1PV cytotoxicity. PMID:20375165

  13. The actin-related protein hArp8 accumulates on the mitotic chromosomes and functions in chromosome alignment

    SciTech Connect

    Aoyama, Naoki; Oka, Asako; Kitayama, Kumiko; Kurumizaka, Hitoshi; Harata, Masahiko

    2008-02-15

    The actin family consists of conventional actin and various actin-related proteins (Arps). Some of these Arps are localized in the nucleus, and a fraction of each of these nuclear Arps is functionally involved in chromatin remodeling and histone acetyltransferase complexes. On the other hand, in mitotic cells, the localization and function of the nuclear Arps are largely unknown. Human Arp8 (hArp8), an ortholog of yeast nuclear Arp8, was recently found to be associated with the hINO80-chromatin remodeling complex along with hArp5. Here we report that hArp8, but not hArp5, accumulates on mitotic chromosomes. This is the first example where a member of the actin family is found to be associated with mitotic chromosomes. Expression of truncated hArp8 proteins and depletion of endogenous hArp8 by RNA interference caused misalignment of mitotic chromosomes, suggesting that chromosome-associated hArp8 has a role in chromosome behavior. In contrast, depletion of hIno80 and hArp5 did not cause misalignment of chromosomes, suggesting that the role of hArp8 at mitotic chromosomes is independent of the activity of hINO80 complexes. These findings provide the first insight into a novel function of actin family members in mitosis.

  14. Transgenic tobacco plants expressing siRNA targeted against the Mungbean yellow mosaic virus transcriptional activator protein gene efficiently block the viral DNA accumulation.

    PubMed

    Shanmugapriya, Gnanasekaran; Das, Sudhanshu Sekhar; Veluthambi, Karuppannan

    2015-06-01

    Mungbean yellow mosaic virus (MYMV) is a bipartite begomovirus that infects many pulse crops such as blackgram, mungbean, mothbean, Frenchbean, and soybean. We tested the efficacy of the transgenically expressed intron-spliced hairpin RNA gene of the transcriptional activator protein (hpTrAP) in reducing MYMV DNA accumulation. Tobacco plants transformed with the MYMV hpTrAP gene accumulated 21-22 nt siRNA. Leaf discs of the transgenic plants, agroinoculated with the partial dimers of MYMV, displayed pronounced reduction in MYMV DNA accumulation. Thus, silencing of the TrAP gene, a suppressor of gene silencing, emerged as an effective strategy to control MYMV.

  15. Behavioral abnormalities in prion protein knockout mice and the potential relevance of PrPc for the cytoskeleton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cellular prion protein (PrPC) is a highly conserved protein, which is anchored to the outer surface of the plasma membrane. Even though its physiological function has already been investigated in different cell or mouse models where PrPC expression is either up-regulated or depleted, its exact p...

  16. Herpes simplex virus 1 induces cytoplasmic accumulation of TIA-1/TIAR and both synthesis and cytoplasmic accumulation of tristetraprolin, two cellular proteins that bind and destabilize AU-rich RNAs.

    PubMed

    Esclatine, Audrey; Taddeo, Brunella; Roizman, Bernard

    2004-08-01

    Herpes simplex virus 1 causes a shutoff of cellular protein synthesis through the degradation of RNA that is mediated by the virion host shutoff (Vhs) protein encoded by the U(L)41 gene. We reported elsewhere that the Vhs-dependent degradation of RNA is selective, and we identified RNAs containing AU-rich elements (AREs) that were upregulated after infection but degraded by deadenylation and progressive 3'-to-5' degradation. We also identified upregulated RNAs that were not subject to Vhs-dependent degradation (A. Esclatine, B. Taddeo, L. Evans, and B. Roizman, Proc. Natl. Acad. Sci. USA 101:3603-3608, 2004). Among the latter was the RNA encoding tristetraprolin, a protein that binds AREs and is known to be associated with the degradation of RNAs containing AREs. Prompted by this observation, we examined the status of the ARE binding proteins tristetraprolin and TIA-1/TIAR in infected cells. We report that tristetraprolin was made and accumulated in the cytoplasm of wild-type virus-infected human foreskin fibroblasts as early as 2 h and in HEp-2 cells as early as 6 h after infection. The amounts of tristetraprolin that accumulated in the cytoplasm of cells infected with a mutant virus lacking U(L)41 were significantly lower than those in wild-type virus-infected cells. The localization of tristetraprolin was not modified in cells infected with a mutant lacking the gene encoding infected cell protein 4 (ICP4). TIA-1 and TIAR are two other proteins that are associated with the regulation of ARE-containing RNAs and that normally reside in nuclei. In infected cells, they started to accumulate in the cytoplasm after 6 h of infection. In cells infected with the mutant virus lacking U(L)41, TIA-1/TIAR accumulated in the cytoplasm in granular structures reminiscent of stress granules in a significant percentage of the cells. In addition, an antibody to tristetraprolin coprecipitated the Vhs protein from lysates of cells late in infection. The results indicate that the Vhs

  17. p53 nuclear protein accumulation correlates with mutations in the p53 gene, tumor grade, and stage in bladder cancer.

    PubMed

    Esrig, D; Spruck, C H; Nichols, P W; Chaiwun, B; Steven, K; Groshen, S; Chen, S C; Skinner, D G; Jones, P A; Cote, R J

    1993-11-01

    Seventy-three transitional cell carcinomas of the bladder were analyzed by immunohistochemistry for p53 nuclear accumulation, and the results were compared to mutations detected in the p53 gene by single strand conformational polymorphism analysis (SSCP) and DNA sequence analysis. Immunohistochemical studies were performed on formalin-fixed, paraffin-embedded tissue sections. A highly significant association between the presence of p53 mutations and p53 nuclear reactivity as detected by immunohistochemistry was found (P = 0.0001). Of 32 tumors that demonstrated p53 mutations by SSCP, 27 (84%) showed p53 nuclear reactivity. Of the five cases that did not demonstrate p53 nuclear reactivity, four had mutations in exon 5. However, of 41 tumors with no evidence of p53 mutation by molecular analysis, 12 (29%) showed p53 immunoreactivity. This indicates that immunohistochemical methods may be more sensitive than SSCP in detecting p53 mutations or that discordant cases represent tumors with accumulation of wild type p53 protein, without mutations at the p53 locus. Of the 15 tumors that were found to have mutations at exon 8, 13 demonstrated high-intensity homogeneous p53 nuclear reactivity by immunohistochemistry, and all mutations located at codon 280 demonstrated high-intensity homogeneous immunoreactivity. However, three of three tumors with exon 6 mutations demonstrated low-level p53 immunoreactivity, and four of six tumors with mutations in exon 5 showed no detectable p53 nuclear reactivity. This indicates that the heterogeneity of immunoreactivity observed when analyzing p53 nuclear accumulation may be related to the site of the p53 gene mutation. Information on tumor grade, stage, lymph node status, disease-free interval, and overall survival were available in 54 patients who had undergone cystectomy. A significant association was observed between p53 alterations (detected by immunohistochemistry and SSCP) and histological tumor grade (P = 0.003) and stage (P = 0

  18. Pho85p, a cyclin-dependent protein kinase, and the Snf1p protein kinase act antagonistically to control glycogen accumulation in Saccharomyces cerevisiae.

    PubMed Central

    Huang, D; Farkas, I; Roach, P J

    1996-01-01

    In Saccharomyces cerevisiae, nutrient levels control multiple cellular processes. Cells lacking the SNF1 gene cannot express glucose-repressible genes and do not accumulate the storage polysaccharide glycogen. The impaired glycogen synthesis is due to maintenance of glycogen synthase in a hyperphosphorylated, inactive state. In a screen for second site suppressors of the glycogen storage defect of snf1 cells, we identified a mutant gene that restored glycogen accumulation and which was allelic with PHO85, which encodes a member of the cyclin-dependent kinase family. In cells with disrupted PHO85 genes, we observed hyperaccumulation of glycogen, activation of glycogen synthase, and impaired glycogen synthase kinase activity. In snf1 cells, glycogen synthase kinase activity was elevated. Partial purification of glycogen synthase kinase activity from yeast extracts resulted in the separation of two fractions by phenyl-Sepharose chromatography, both of which phosphorylated and inactivated glycogen synthase. The activity of one of these, GPK2, was inhibited by olomoucine, which potently inhibits cyclin-dependent protein kinases, and contained an approximately 36-kDa species that reacted with antibodies to Pho85p. Analysis of Ser-to-Ala mutations at the three potential Gsy2p phosphorylation sites in pho85 cells implicated Ser-654 and/or Thr-667 in PHO85 control of glycogen synthase. We propose that Pho85p is a physiological glycogen synthase kinase, possibly acting downstream of Snf1p. PMID:8754836

  19. Proteins iodinated by the chloramine-T method appear to be degraded at an abnormally rapid rate after endocytosis

    SciTech Connect

    Opresko, L.; Wiley, H.S.; Wallace, R.A.

    1980-03-01

    Proteins labeled with either /sup 3/H by reductive methylation or /sup 125/I by the chloramine-T method were incubated with Xenopus laevis oocytes; the incorporation and acid precipitability of the proteins were then studied. The uptake rates of both specifically incorporated (vitellogenin) and nonspecifically incorporated proteins (bovine serum albumin and X. laevis serum proteins lacking albumin) were not influenced by the method of labeling. However, /sup 125/I-labeled proteins were apparently degraded at rates far exceeding their /sup 3/H-labeled counterparts, based on the generation of acid-soluble radioactivity. Thus, after a 3-hr incubation, 3 to 5 times more /sup 125/I-labeled bovine serum albumin and X. laevis serum proteins lacking albumin were degraded than the corresponding /sup 3/H-labeled proteins (95% compared to 30% and 75% compared to 15%, respectively), whereas after a 24-hr incubation, the degradation of /sup 125/I-labeled vitellogenin was 15 times greater than that of (/sup 3/H)vitellogenin labeled in vivo (60% compared to 4%). Moreover, examination of the relative amounts of /sup 3/H-compared to /sup 125/I-labeled bovine serum albumin deposited into the exogenously derived yolk platelet compartment of the oocyte revealed 7 times more acid-precipitable /sup 3/H-labeled protein, indicating that the observed discrepancies were not due to reincorporation of the /sup 3/H-labeled ligands. Passage of dissolved oocytes previously exposed to /sup 125/I-labeled bovine serum albumin (chloramine-T method) over a column of Bio-Gel P-10 revealed some breakdown of bovine serum albumin to intermediate molecular weight components and the presence of a large amount (approx. = 90%) of labeled low molecular weight compounds, which analysis showed to be 72% free iodine.

  20. Heat sensitivity in a bentgrass variant. Failure to accumulate a chloroplast heat shock protein isoform implicated in heat tolerance.

    PubMed

    Wang, Dongfang; Luthe, Dawn S

    2003-09-01

    Two variants of creeping bentgrass (Agrostis stolonifera cv palustris), developed using tissue culture, have been used to determine the roles of chloroplast-localized small heat shock proteins (CP-sHSPs) in heat tolerance. Results from previous research indicate that the heat-tolerant variant expressed two additional CP-sHSP isoforms not expressed in the heat-sensitive variant, that accumulation of the additional CP-sHSP isoforms was genetically linked to thermotolerance, and that the presence of the additional isoforms in the heat-tolerant variant provided greater protection to photosystem II during heat stress. To determine the basis of the differential expression, we isolated the genes encoding the CP-sHSPs from both variants and characterized their structure and expression. Two genes, ApHsp26.2 and ApHsp26.7a, were isolated from the heat-tolerant variant, and three genes, ApHsp26.2m, ApHsp26.8, and ApHsp26.7b, were isolated from the heat-sensitive variant. The sequence of ApHsp26.2m from the heat-sensitive variant was identical to ApHsp26.2, except for a point mutation that generated a premature stop codon. Therefore, the protein product of ApHsp26.2m did not accumulate in the heat-sensitive line. Mass spectrometry analysis confirmed that ApHsp26.2 encoded for the CP-sHSP isoforms unique to the heat-tolerant variant. An identical mutation was detected in one of the three parental lines used to develop the creeping bentgrass variants. This suggests that ApHsp26.2m was inherited from this parent and did not arise from a mutation that occurred during tissue culture. The presence of two isoforms encoded by the same gene might be due to differential processing of the N-terminal amino acids during or after import into the chloroplast.

  1. Role for the A domain of unprocessed accumulation-associated protein (Aap) in the attachment phase of the Staphylococcus epidermidis biofilm phenotype.

    PubMed

    Conlon, Brian P; Geoghegan, Joan A; Waters, Elaine M; McCarthy, Hannah; Rowe, Sarah E; Davies, Julia R; Schaeffer, Carolyn R; Foster, Timothy J; Fey, Paul D; O'Gara, James P

    2014-12-01

    The polysaccharide intercellular adhesin or the cell wall-anchored accumulation-associated protein (Aap) mediates cellular accumulation during Staphylococcus epidermidis biofilm maturation. Mutation of sortase, which anchors up to 11 proteins (including Aap) to the cell wall, blocked biofilm development by the cerebrospinal fluid isolate CSF41498. Aap was implicated in this phenotype when Western blots and two-dimensional (2D) electrophoresis revealed increased levels of the protein in culture supernatants. Unexpectedly, reduced levels of primary attachment were associated with impaired biofilm formation by CSF41498 srtA and aap mutants. In contrast to previous studies, which implicated Aap proteolytic cleavage and, specifically, the Aap B domains in biofilm accumulation, the CSF41498 Aap protein was unprocessed. Furthermore, aap appeared to play a less important role in the biofilm phenotype of S. epidermidis 1457, in which the Aap protein is processed. Anti-Aap A-domain IgG inhibited primary attachment and biofilm formation in strain CSF41498 but not in strain 1457. The nucleotide sequences of the aap gene A-domain region and cleavage site in strains CSF41498 and 1457 were identical, implicating altered protease activity in the differential Aap processing results in the two strains. These data reveal a new role for the A domain of unprocessed Aap in the attachment phase of biofilm formation and suggest that extracellular protease activity can influence whether Aap contributes to the attachment or accumulation phases of the S. epidermidis biofilm phenotype.

  2. Proteomics Profiling Reveals Carbohydrate Metabolic Enzymes and 14-3-3 Proteins Play Important Roles for Starch Accumulation during Cassava Root Tuberization.

    PubMed

    Wang, Xuchu; Chang, Lili; Tong, Zheng; Wang, Dongyang; Yin, Qi; Wang, Dan; Jin, Xiang; Yang, Qian; Wang, Liming; Sun, Yong; Huang, Qixing; Guo, Anping; Peng, Ming

    2016-01-21

    Cassava is one of the most important root crops as a reliable source of food and carbohydrates. Carbohydrate metabolism and starch accumulation in cassava storage root is a cascade process that includes large amounts of proteins and cofactors. Here, comparative proteomics were conducted in cassava root at nine developmental stages. A total of 154 identified proteins were found to be differentially expressed during starch accumulation and root tuberization. Many enzymes involved in starch and sucrose metabolism were significantly up-regulated, and functional classification of the differentially expressed proteins demonstrated that the majority were binding-related enzymes. Many proteins were took part in carbohydrate metabolism to produce energy. Among them, three 14-3-3 isoforms were induced to be clearly phosphorylated during storage root enlargement. Overexpression of a cassava 14-3-3 gene in Arabidopsis thaliana confirmed that the older leaves of these transgenic plants contained higher sugar and starch contents than the wild-type leaves. The 14-3-3 proteins and their binding enzymes may play important roles in carbohydrate metabolism and starch accumulation during cassava root tuberization. These results not only deepened our understanding of the tuberous root proteome, but also uncovered new insights into carbohydrate metabolism and starch accumulation during cassava root enlargement.

  3. Proteomics Profiling Reveals Carbohydrate Metabolic Enzymes and 14-3-3 Proteins Play Important Roles for Starch Accumulation during Cassava Root Tuberization

    PubMed Central

    Wang, Xuchu; Chang, Lili; Tong, Zheng; Wang, Dongyang; Yin, Qi; Wang, Dan; Jin, Xiang; Yang, Qian; Wang, Liming; Sun, Yong; Huang, Qixing; Guo, Anping; Peng, Ming

    2016-01-01

    Cassava is one of the most important root crops as a reliable source of food and carbohydrates. Carbohydrate metabolism and starch accumulation in cassava storage root is a cascade process that includes large amounts of proteins and cofactors. Here, comparative proteomics were conducted in cassava root at nine developmental stages. A total of 154 identified proteins were found to be differentially expressed during starch accumulation and root tuberization. Many enzymes involved in starch and sucrose metabolism were significantly up-regulated, and functional classification of the differentially expressed proteins demonstrated that the majority were binding-related enzymes. Many proteins were took part in carbohydrate metabolism to produce energy. Among them, three 14-3-3 isoforms were induced to be clearly phosphorylated during storage root enlargement. Overexpression of a cassava 14-3-3 gene in Arabidopsis thaliana confirmed that the older leaves of these transgenic plants contained higher sugar and starch contents than the wild-type leaves. The 14-3-3 proteins and their binding enzymes may play important roles in carbohydrate metabolism and starch accumulation during cassava root tuberization. These results not only deepened our understanding of the tuberous root proteome, but also uncovered new insights into carbohydrate metabolism and starch accumulation during cassava root enlargement. PMID:26791570

  4. Diverse accumulation of several dehydrin-like proteins in cauliflower (Brassica oleracea var. botrytis), Arabidopsis thaliana and yellow lupin (Lupinus luteus) mitochondria under cold and heat stress

    PubMed Central

    2010-01-01

    Background Dehydrins represent hydrophilic proteins acting mainly during cell dehydration and stress response. Dehydrins are generally thermostable; however, the so-called dehydrin-like (dehydrin-related) proteins show variable thermolability. Both groups immunoreact with antibodies directed against the K-segment of dehydrins. Plant mitochondrial dehydrin-like proteins are poorly characterized. The purpose of this study was to extend previous reports on plant dehydrins by comparing the level of immunoprecipitated dehydrin-like proteins in cauliflower (Brassica oleracea var. botrytis), Arabidopsis thaliana and yellow lupin (Lupinus luteus) mitochondria under cold and heat stress. Results All the analyzed plant species showed constitutive accumulation of thermostable mitochondrial putative dehydrins ranging from 50 to 70 kDa. The mitochondrial dehydrin-like proteins observed in cauliflower and Arabidopsis ranged from 10 to 100 kDa and in lupin imbibed seeds and hypocotyls - from 20 to 90 kDa. Cold treatment increased mainly the accumulation of 10-100 kDa cauliflower and Arabidopsis dehydrin-like proteins, in the patterns different in cauliflower leaf and inflorescence mitochondria. However, in lupin mitochondria, cold affected mainly 25-50 kDa proteins and seemed to induce the appearance of some novel dehydrin-like proteins. The influence of frost stress on cauliflower leaf mitochondrial dehydrin- like proteins was less significant. The impact of heat stress was less significant in lupin and Arabidopsis than in cauliflower inflorescence mitochondria. Cauliflower mitochondrial dehydrin-like proteins are localized mostly in the mitochondrial matrix; it seems that some of them may interact with mitochondrial membranes. Conclusions All the results reveal an unexpectedly broad spectrum of dehydrin-like proteins accumulated during some abiotic stress in the mitochondria of the plant species analyzed. They display only limited similarity in size to those reported previously

  5. Developmental and hormonal regulation of the arabidopsis CER2 gene that codes for a nuclear-localized protein required for the normal accumulation of cuticular waxes.

    PubMed Central

    Xia, Y; Nikolau, B J; Schnable, P S

    1997-01-01

    The previously cloned CER2 gene is required for the normal accumulation of cuticular waxes and encodes a novel protein. Earlier reports suggested that the CER2 protein is either a membrane-bound component of the fatty acid elongase complex or a regulatory protein. Cell fractionation and immunoblot analyses using polyclonal antibodies raised against a chemically synthesized peptide with a sequence based on the predicted CER2 protein sequence have demonstrated that the 47-kD CER2 protein is soluble and nuclear localized. These results are consistent with CER2 being a regulatory protein. Detailed studies of plants harboring a CER2 promoter/GUS transgene (CER2-GUS), in combination with immunoblot analyses, revealed that CER2 is expressed and the CER2 protein accumulates in a variety of organs and cell types. Expression is highest early in the development of these organs and is epidermis specific in most tissues. In agreement with the activity of the CER2 promoter in hypocotyls, cuticular wax accumulates on this organ in a CER2-dependent fashion. In leaves CER2 expression is confined to the guard cells, trichomes, and petioles. However, application of the cytokinin 6-benzylaminopurine induces ectopic expression of CER2-GUS in all cell types of leaves that emerge following treatment. PMID:9390429

  6. Analysis of nonadditive protein accumulation in young primary roots of a maize (Zea mays L.) F(1)-hybrid compared to its parental inbred lines.

    PubMed

    Hoecker, Nadine; Lamkemeyer, Tobias; Sarholz, Barbara; Paschold, Anja; Fladerer, Claudia; Madlung, Johannes; Wurster, Karl; Stahl, Mark; Piepho, Hans-Peter; Nordheim, Alfred; Hochholdinger, Frank

    2008-09-01

    Heterosis describes the superior performance of heterozygous F(1)-hybrids compared to their homozygous parental inbred lines. Heterosis is already manifested during early maize (Zea mays L.) primary root development. In this study, the most abundant soluble proteins have been investigated before the phenotypic manifestation of heterosis in 3.5-day-old primary roots in the flint inbred line UH002, the dent inbred line UH301 and the corresponding hybrid UH301 x UH002. In CBB-stained 2-DE gels, 150 of 304 detected proteins (49%) were accumulated in a nonadditive fashion in the hybrid compared to the average of their parental inbred lines (Student's t-test: p < 0.05). Remarkably, expression of 51% (76/150) of the nonadditively accumulated proteins exceeded the high parent or was below the low parent. ESI-MS/MS identified 75 of the 76 proteins that belonged to these expression classes. The most abundant functional classes among the 75 proteins that were encoded by 60 different genes were metabolism (58%) and disease and defense (19%). Nonadditive protein accumulation in primary roots of maize hybrids might be associated with heterosis manifestation. Identification of these proteins could therefore contribute to the better understanding of the molecular basis of heterosis.

  7. Leucine restores murine hepatic triglyceride accumulation induced by a low-protein diet by suppressing autophagy and excessive endoplasmic reticulum stress.

    PubMed

    Yokota, Shin-Ichi; Ando, Midori; Aoyama, Shinya; Nakamura, Kawai; Shibata, Shigenobu

    2016-04-01

    Although it is known that a low-protein diet induces hepatic triglyceride (TG) accumulation in both rodents and humans, little is known about the underlying mechanism. In the present study, we modeled hepatic TG accumulation by inducing dietary protein deficiency in mice and aimed to determine whether certain amino acids could prevent low-protein diet-induced TG accumulation in the mouse liver. Mice fed a diet consisting of 3 % casein (3C diet) for 7 days showed hepatic TG accumulation with up-regulation of TG synthesis for the Acc gene and down-regulation of TG-rich lipoprotein secretion from hepatocytes for Mttp genes. Supplementing the 3 % casein diet with essential amino acids, branched-chain amino acids, or the single amino acid leucine rescued hepatic TG accumulation. In the livers of mice fed the 3 % casein diet, we observed a decrease in the levels of the autophagy substrate p62, an increase in the expression levels of the autophagy marker LC3-II, and an increase in the splicing of the endoplasmic reticulum (ER) stress-dependent Xbp1 gene. Leucine supplementation to the 3 % casein diet did not affect genes related to lipid metabolism, but inhibited the decrease in p62, the increase in LC3-II, and the increase in Xbp1 splicing levels in the liver. Our results suggest that ER stress responses and activated autophagy play critical roles in low-protein diet-induced hepatic TG accumulation in mice, and that leucine suppresses these two major protein degradation systems. This study contributes to understanding the mechanisms of hepatic disorders of lipid metabolism.

  8. Excessive reactive oxygen species induces apoptosis in fibroblasts: Role of mitochondrially accumulated hyaluronic acid binding protein 1 (HABP1/p32/gC1qR)

    SciTech Connect

    Chowdhury, Anindya Roy; Ghosh, Ilora Datta, Kasturi

    2008-02-01

    Constitutively expressed HABP1 in normal murine fibroblast cell line induces growth perturbation, morphological abnormalities alongwith initiation of apoptosis. Here, we demonstrate that though HABP1 accumulation started in mitochondria from 48 hr of growth, induction of apoptosis with the release of cytochrome c and apoptosome complex formation occurred only after 60 hr. This mitochondrial dysfunction was due to gradual increase in ROS generation in HABP1 overexpressing cells. Along with ROS generation, increased Ca{sup 2+} influx in mitochondria leading to drop in membrane potential was evident. Interestingly, upon expression of HABP1, the respiratory chain complex I was shown to be significantly inhibited. Electronmicrograph confirmed defective mitochondrial ultrastructure. The reduction in oxidant generation and drop in apoptotic cell population accomplished by disruption of HABP1 expression, corroborating the fact that excess ROS generation in HABP1 overexpressing cells leading to apoptosis was due to mitochondrial HABP1 accumulation.

  9. Final Report for CRADA Agreement , AL-C-2006-01 with Microsens Biotechnologies: Detection of the Abnormal Prion Protein in Blood by Improving the Extraction of this Protein

    SciTech Connect

    Schmerr, Mary Jo

    2009-03-31

    Several conditions were examined to optimize the extraction protocol using Seprion beads for the abnormal prion protein. Different combinations of water, hexafluro-2-propanol and formic acid were used. The results of these extraction protocols showed that the magnetic beads coated with Seprion reagents were subject to degradation, themselves, when the extraction conditions that would solubilize the abnormal prion protein were used. These compounds caused interference in the immunoassay for the abnormal prion protein and rendered these protocols incompatible with the assay systems. In an attempt to overcome this problem, another approach was then used. The coated beads were used as an integral part of the assay platform. After washing away denaturing agents, the beads with the 'captured' abnormal prion were incubated directly in the immunoassay, followed by analysis by the capillary electrophoresis. When a capillary electrophoresis electro-kinetic separation was attempted, the beads disturbed the analysis making it impossible to interpret. A pressure separation method was then developed for capillary electrophoresis analysis. When 20 samples, 5 of which were positive were analyzed, the assay identified 4 of the 5 positives and had no false positives. When a larger number of samples were analyzed the results were not as good - there were false positives and false negatives. It was then observed that the amount of beads that were loaded was dependent upon how long the beads were allowed to settle before loading them into the capillary. This resulted in unacceptable variations in the results and explained that when large numbers of samples were evaluated the results were not consistent. Because the technical difficulties with using the Seprion beads could not be overcome at this time, another approach is underway that is outside of the scope of this CRADA. No further agreements have been developed. Because the results were not favorable, no manuscripts were written nor

  10. Quercetin and allopurinol reduce liver thioredoxin-interacting protein to alleviate inflammation and lipid accumulation in diabetic rats

    PubMed Central

    Wang, Wei; Wang, Chuang; Ding, Xiao-Qin; Pan, Ying; Gu, Ting-Ting; Wang, Ming-Xing; Liu, Yang-Liu; Wang, Fu-Meng; Wang, Shui-Juan; Kong, Ling-Dong

    2013-01-01

    Background and Purpose Thioredoxin-interacting protein (TXNIP), a regulator of cellular oxidative stress, has been associated with activation of NOD-like receptor 3 (NLRP3) inflammasome, inflammation and lipid metabolism, suggesting it has a role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) in diabetes. In this study we investigated whether TXNIP is involved in type 1 diabetes-associated NAFLD and whether antioxidants, quercetin and allopurinol, alleviate NAFLD by targeting TXNIP. Experimental Approach Diabetes was induced in male Sprague-Dawley rats by a single i.p. injection of 55 mg·kg−1 streptozotocin. Quercetin and allopurinol were given p.o. to diabetic rats for 7 weeks. Hepatic function, oxidative stress, inflammation and lipid levels were determined. Rat BRL-3A and human HepG2 cells were exposed to high glucose (30 mM) in the presence and absence of antioxidants, TXNIP siRNA transfection or caspase-1 inhibitor, Ac-YVAD-CMK. Key Results Quercetin and allopurinol significantly inhibited the TXNIP overexpression, activation of NLRP3 inflammasome, down-regulation of PPARα and up-regulation of sterol regulatory element binding protein-1c (SREBP-1c), SREBP-2, fatty acid synthase and liver X receptor α, as well as elevation of ROS and IL-1β in diabetic rat liver. These effects were confirmed in hepatocytes in vitro and it was further shown that TXNIP down-regulation contributed to the suppression of NLRP3 inflammasome activation, inflammation and changes in PPARα and SREBPs. Conclusions and Implications Inhibition of hepatic TXNIP by quercetin and allopurinol contributes to the reduction in liver inflammation and lipid accumulation under hyperglycaemic conditions. The targeting of hepatic TXNIP by quercetin and allopurinol may have therapeutic implications for prevention of type 1 diabetes-associated NAFLD. PMID:23647015

  11. Fatty acid transport protein-2 inhibitor Grassofermata/CB5 protects cells against lipid accumulation and toxicity

    SciTech Connect

    Saini, Nipun; Black, Paul N.; Montefusco, David; DiRusso, Concetta C.

    2015-09-25

    The inhibition of the fatty acid uptake into non-adipose tissues provides an attractive target for prevention of lipotoxicity leading to obesity-associated non-alcoholic fatty liver disease and type 2 diabetes. Fatty acid transport proteins (FATPs) are bifunctional proteins involved in the uptake and activation of fatty acids by esterification with coenzyme A. Here we characterize Grassofermata/CB5, previously identified as a fatty acid uptake inhibitor directed against HsFATP2. The compound was effective in inhibiting the uptake of fatty acids in the low micro-molar range (IC{sub 50} 8–11 μM) and prevented palmitate-mediated lipid accumulation and cell death in cell lines that are models for intestines, liver, muscle and pancreas. In adipocytes, uptake inhibition was less effective (IC{sub 50} 58 μM). Inhibition was specific for long chain fatty acids and was ineffective toward medium chain fatty acids, which are transported by diffusion. Kinetic analysis of Grassofermata-dependent FA transport inhibition verified a non-competitive mechanism. By comparison with Grassofermata, several atypical antipsychotic drugs previously implicated as inhibitors of FA uptake were ineffectual. In mice Grassofermata decreased absorption of {sup 13}C-oleate demonstrating its potential as a therapeutic agent. - Highlights: • Grassofermata is a small compound inhibitor of FATP2. • Uptake inhibition is specific for long chain fatty acids. • Uptake kinetics shows low specificity for adipocytes compared to other cell types. • Inhibition is by a non-competitive mechanism. • Atypical antipsychotics do not inhibit FA uptake by comparison with Grassofermata.

  12. Ampakine CX516 ameliorates functional deficits in AMPA receptors in a hippocampal slice model of protein accumulation.

    PubMed

    Kanju, Patrick M; Parameshwaran, Kodeeswaran; Sims, Catrina; Bahr, Ben A; Shonesy, Brian C; Suppiramaniam, Vishnu

    2008-11-01

    AMPAkines are positive modulators of AMPA receptors, and previous work has shown that these compounds can facilitate synaptic plasticity and improve learning and memory in both animals and humans; thus, their role in the treatment of cognitive impairment is worthy of investigation. In this study, we have utilized an organotypic slice model in which chloroquine-induced lysosomal dysfunction produces many of the pathogenic attributes of Alzheimer's disease. Our previous work demonstrated that synaptic AMPA receptor function is impaired in hippocampal slice cultures exhibiting lysosomal dysfunction leading to protein accumulation. The present study investigated the effect of the AMPAkine CX516 on AMPAR-mediated synaptic transmission as well as the CX516 induced modification of single channel AMPA receptor properties in this organotypic slice-culture model. In whole cell recordings from CA1 pyramidal neurons in chloroquine-treated slices we observed a significant decrease in AMPAR-mediated mEPSC frequency and amplitude indicating synaptic dysfunction. Following application of CX516, these parameters returned to nearly normal levels. Similarly, we report chloroquine-induced impairment of AMPAR single channel properties (decreased probability of opening and mean open time), and significant recovery of these properties following CX516 administration. These results suggest that AMPA receptors may be potential pharmaceutical targets for the treatment of neurodegenerative diseases, and highlights AMPAkines, in particular, as possible therapeutic agents.

  13. The accumulation of misfolded proteins in the mitochondrial matrix is sensed by PINK1 to induce PARK2/Parkin-mediated mitophagy of polarized mitochondria.

    PubMed

    Jin, Seok Min; Youle, Richard J

    2013-11-01

    Defective mitochondria exert deleterious effects on host cells. To manage this risk, mitochondria display several lines of quality control mechanisms: mitochondria-specific chaperones and proteases protect against misfolded proteins at the molecular level, and fission/fusion and mitophagy segregate and eliminate damage at the organelle level. An increase in unfolded proteins in mitochondria activates a mitochondrial unfolded protein response (UPR(mt)) to increase chaperone production, while the mitochondrial kinase PINK1 and the E3 ubiquitin ligase PARK2/Parkin, whose mutations cause familial Parkinson disease, remove depolarized mitochondria through mitophagy. It is unclear, however, if there is a connection between those different levels of quality control (QC). Here, we show that the expression of unfolded proteins in the matrix causes the accumulation of PINK1 on energetically healthy mitochondria, resulting in mitochondrial translocation of PARK2, mitophagy and subsequent reduction of unfolded protein load. Also, PINK1 accumulation is greatly enhanced by the knockdown of the LONP1 protease. We suggest that the accumulation of unfolded proteins in mitochondria is a physiological trigger of mitophagy.

  14. The roles of protein and lipid in the accumulation and distribution of perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) in plants grown in biosolids-amended soils.

    PubMed

    Wen, Bei; Wu, Yali; Zhang, Hongna; Liu, Yu; Hu, Xiaoyu; Huang, Honglin; Zhang, Shuzhen

    2016-09-01

    The roles of protein and lipid in the accumulation and distribution of perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) in seven species of plants from biosolids-amended soils were investigated. The PFOS and PFOA root concentration factors (Croot/Csoil) ranged from 1.37 to 4.68 and 1.69 to 10.3 (ng/groot)/(ng/gsoil), respectively, while the translocation factors (Cshoot/Croot) ranged from 0.055 to 0.16 and 0.093 to 1.8 (ng/gshoot)/(ng/groot), respectively. The PFOS and PFOA accumulations in roots correlated positively with root protein contents (P < 0.05), while negatively with root lipid contents (P < 0.05). These suggested the promotion effects of protein and inhibition effects of lipid on root uptake. The translocation factors correlated positively with the ratios between protein contents in shoots to those in roots (P < 0.05), showing the importance of protein on PFOS and PFOA translocation. This study is the first to reveal the different roles of protein and lipid in the accumulation and distribution of PFOS and PFOA in plants.

  15. Temporal resolution of misfolded prion protein transport, accumulation, glial activation, and neuronal death in the retinas of mice inoculated with scrapie

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Currently, there is a lack of pathologic landmarks to describe the progression of prion disease in vivo. The goal of this work was to determine the temporal relationship between the transport of misfolded prion protein from the brain to the retina, the accumulation of PrPSc in the retina, the respon...

  16. Altered expression of an ankyrin-repeat protein results in leaf abnormalities, necrotic lesions, and the elaboration of a systemic signal.

    PubMed

    Wirdnam, Corina; Motoyama, Andrea; Arn-Bouldoires, Estelle; van Eeden, Sjoerd; Iglesias, Alejandro; Meins, Frederick

    2004-11-01

    The PR-like proteins, class I beta-1,3-glucanase (GLU I) and chitinase (CHN I), are induced as part of a stereotypic response that can provide protection against viral, bacterial, and fungal pathogens. We have identified two Nicotiana plumbaginifolia ankyrin-repeat proteins, designated Glucanohydrolase Binding Proteins (GBP) 1 and 2, that bind GLU I and CHN I both in vitro and when expressed in yeast cells. Sense as well as antisense transformants of tobacco carrying the GBP1 gene elaborated graft-transmissible acropetally moving signals that induced the downward curling of young leaves. This phenotype was associated with reduced starch, sucrose, and fructose accumulation; the formation of necrotic lesions; and, the induction of markers for the hypersensitive response. GBP1/2 are members of a conserved Plant- Specific Ankyrin- repeat (PANK) family that includes proteins implicated in carbohydrate allocation, reactive oxygen metabolism, hypersensitive cell death, rapid elicitor responses, virus pathogenesis, and auxin signaling. The similarity in phenotype of PANK transformants and transformants altered in carbohydrate metabolism leads us to propose that PANK family members are multifunctional proteins involved in linking plant defense responses and carbohydrate metabolism.

  17. A Novel Asp121Asn Mutation of Myelin Protein Zero Is Associated with Late-Onset Axonal Charcot-Marie-Tooth Disease, Hearing Loss and Pupil Abnormalities

    PubMed Central

    Duan, Xiaohui; Gu, Weihong; Hao, Ying; Wang, Renbin; Wen, Hong; Sun, Shaojie; Jiao, Jinsong; Fan, Dongsheng

    2016-01-01

    Myelin protein zero (MPZ) is a major component of compact myelin in peripheral nerves. Mutations in MPZ have been associated with different Charcot–Marie–Tooth disease (CMT) phenotypes (CMT1B, CMT2I/J, CMTDI), Dejerine–Sottas syndrome, and congenital hypomyelination neuropathy. Here, we report phenotypic variability in a four-generation Chinese family with the MPZ mutation Asp121Asn. Genetic testing was performed on nine family members and 200 controls. Clinical, electrophysiological and skeletal muscle MRI assessments were available for review in six family members. A novel heterozygous missense mutation, Asp121Asn, was observed in five affected members of the family. Unaffected relatives and 200 normal controls were without the mutation. Four of the affected members of the family displayed late-onset, predominantly axonal sensory and motor neuropathy, pupil abnormalities, and progressive sensorineural hearing loss. One young affected member presented with Argyll–Robertson pupils and diminished deep tendon reflexes in the lower limbs. The MPZ mutation Asp121Asn may be associated with late-onset axonal neuropathy, early onset hearing loss and pupil abnormalities. Our report expands the number and phenotypic spectrum of MPZ mutations. PMID:27774063

  18. Abnormal serum thyroid hormones concentration with healthy functional gland: a review on the metabolic role of thyroid hormones transporter proteins.

    PubMed

    Azad, Reza Mansourian

    2011-03-01

    Laboratory findings can definitely help the patients not to enter into status, where the damage might be happen due to a miss-diagnosis based on clinical assessment alone. The secondary disease accompanied with thyroid patients should also carefully check out due to the interference which some diseases can cause in the amount of serum thyroid hormone, particularly the free thyroxin. The dilemma over thyroid clinical diagnosis occur due to variation on serum thyroid hormone which initiated by other non-thyroidal disorders which can play an important roles in metabolic disorders of thyroid hormone due to the alteration which occur on the serum level of thyroid hormone transporter proteins. The majority of serum thyroid hormones of up to 95-99% are bound to the carrier proteins mainly to Thyroxin-Binding Globulins (TBG), some transthyretin already known as pre-albumin and albumin which are all synthesis in the liver and any modification which alter their production may alter the status of thyroid hormones. It seems TBG, transthyretin and albumin carries 75, 20, 5% of thyroid hormones within blood circulation, respectively. The dilemma facing the thyroid hormones following disruption of thyroid hormone transporter protein synthesis originate from this fact that any alteration of these protein contribute to the alteration of total thyroid and free serum thyroid hormones which are in fact the biologically active form of thyroid hormones. The subsequent of latter implication result in miss-understanding and miss-diagnosis of thyroid function tests, with possible wrongly thyroid clinical care, followed by undesired therapy of otherwise healthy thyroid.

  19. Abnormal centrosome amplification in cells through the targeting of Ran-binding protein-1 by the human T cell leukemia virus type-1 Tax oncoprotein.

    PubMed

    Peloponese, Jean-Marie; Haller, Kerstin; Miyazato, Akiko; Jeang, Kuan-Teh

    2005-12-27

    Human T cell leukemia virus type-1 (HTLV-1) is an oncogenic retrovirus etiologically causal of adult T cell leukemia. The virus encodes a Tax oncoprotein that functions in transcriptional regulation, cell cycle control, and transformation. Because adult T cell leukemia like many other human cancers is a disease of genomic instability with frequent gains and losses of chromosomes, to understand this disease it is important to comprehend how HTLV-1 engenders aneuploidy in host cells. In this regard, loss of cell cycle checkpoints permits tolerance of aneuploidy but does not explain how aneuploidy is created. We show here that HTLV-1 Tax causes abnormal centrosome fragmentation in the mitotic phase of the cell cycle. We report that Tax directly binds Ran and Ran-binding protein-1, locates to centrosomes/spindle poles, and causes supernumerary centrosomes.

  20. Disruption of the gene encoding the latent transforming growth factor-beta binding protein 4 (LTBP-4) causes abnormal lung development, cardiomyopathy, and colorectal cancer.

    PubMed

    Sterner-Kock, Anja; Thorey, Irmgard S; Koli, Katri; Wempe, Frank; Otte, Jürgen; Bangsow, Thorsten; Kuhlmeier, Katharina; Kirchner, Thomas; Jin, Shenchu; Keski-Oja, Jorma; von Melchner, Harald

    2002-09-01

    Transforming growth factor-betas (TGF-betas) are multifunctional growth factors that are secreted as inactive (latent) precursors in large protein complexes. These complexes include the latency-associated propeptide (LAP) and a latent transforming growth factor-beta binding protein (LTBP). Four isoforms of LTBPs (LTBP-1-LTBP-4) have been cloned and are believed to be structural components of connective tissue microfibrils and local regulators of TGF-beta tissue deposition and signaling. By using a gene trap strategy that selects for integrations into genes induced transiently during early mouse development, we have disrupted the mouse homolog of the human LTBP-4 gene. Mice homozygous for the disrupted allele develop severe pulmonary emphysema, cardiomyopathy, and colorectal cancer. These highly tissue-specific abnormalities are associated with profound defects in the elastic fiber structure and with a reduced deposition of TGF-beta in the extracellular space. As a consequence, epithelial cells have reduced levels of phosphorylated Smad2 proteins, overexpress c-myc, and undergo uncontrolled proliferation. This phenotype supports the predicted dual role of LTBP-4 as a structural component of the extracellular matrix and as a local regulator of TGF-beta tissue deposition and signaling.

  1. Abnormalities of Endocytosis, Phagocytosis, and Development Process in Dictyostelium Cells That Over-Express Acanthamoeba castellanii Metacaspase Protein

    PubMed Central

    SAHEB, Entsar; TRZYNA, Wendy; MARINGER, Katherine; BUSH, John

    2015-01-01

    Background: Acanthamoeba castellanii forms a resistant cyst that protects the parasite against the host’s immune response. Acanthamoeba Type-I metacaspase (Acmcp) is a caspase-like protein that has been found to be expressed during the encystations. Dictyostelium discoideum is an organism closely related to Acanthamoeba useful for studying the molecular function of this protozoan caspase-like protein. Methods: The full length of Acmcp and a mutated version of the same gene, which lacks the proline rich N-terminal region (Acmcp-dpr), were cloned into the pDneo2a-GFP vector separately. The pDneo2a-GFP-Acmcp and pDneo2a-GFPAcmcp-dpr were electro-transfected into wild type D. discoideum cells to create cell lines that over-expressed Acmcp or Acmcp-dpr. Results: Both cell lines that over-expressed Acmcp and Acmcp-dpr showed a significant increase in the fluid phase internalization and phagocytosis rate compared to the control cells. Additionally, the cells expressing the Acmcp-dpr mutant were unable to initiate early development and failed to aggregate or form fruiting bodies under starvation conditions, whereas Acmcp over-expressing cells showed the opposite phenomena. Quantitative cell death analysis provided additional support for these findings. Conclusion: Acmcp is involved in the processes of endocytosis and phagocytosis. In addition, the proline rich region in Acmcp is important for cellular development in Dictyostelium. Given its important role in the development process, metacaspase protein is proposed as a candidate drug target against infections caused by A. castellanii. PMID:26246819

  2. Effect of light quality on the accumulation of photosynthetic pigments, proteins and mycosporine-like amino acids in the red alga Porphyra leucosticta (Bangiales, Rhodophyta).

    PubMed

    Korbee, Nathalie; Figueroa, Félix L; Aguilera, José

    2005-08-01

    The effect of different light qualities (white, blue, green, yellow and red light) on photosynthesis, measured as chlorophyll fluorescence, and the accumulation of photosynthetic pigments, proteins and the UV-absorbing mycosporine-like amino acids (MAAs) was studied in the red alga Porphyra leucosticta. Blue light promoted the highest accumulation of nitrogen metabolism derived compounds i.e., MAAs, phycoerythrin and proteins in previously N-starved algae after seven days culture in ammonium enriched medium. Similar results were observed in the culture under white light. In contrast, the lowest photosynthetic capacity i.e., lowest electron transport rate and lowest photosynthetic efficiency as well as the growth rate were found under blue light, while higher values were found in red and white lights. Blue light favored the accumulation of the MAAs porphyra-334, palythine and asterina-330 in P. leucosticta. However, white, green, yellow and red lights favored the accumulation of shinorine. The increase of porphyra-334, palythine and asterina-330 occurred in blue light simultaneous to a decrease in shinorine. The accumulation of MAAs and other nitrogenous compounds in P. leucosticta under blue light could not be attributed to photosynthesis and the action of a non-photosynthetic blue light photoreceptor is suggested. A non-photosynthetic photoreceptor could be also involved in the MAAs interconversion pathways in P. leucosticta.

  3. Proteomic analysis reveals differential accumulation of small heat shock proteins and late embryogenesis abundant proteins between ABA-deficient mutant vp5 seeds and wild-type Vp5 seeds in maize

    PubMed Central

    Wu, Xiaolin; Gong, Fangping; Yang, Le; Hu, Xiuli; Tai, Fuju; Wang, Wei

    2014-01-01

    ABA is a major plant hormone that plays important roles during many phases of plant life cycle, including seed development, maturity and dormancy, and especially the acquisition of desiccation tolerance. Understanding of the molecular basis of ABA-mediated plant response to stress is of interest not only in basic research on plant adaptation but also in applied research on plant productivity. Maize mutant viviparous-5 (vp5), deficient in ABA biosynthesis in seeds, is a useful material for studying ABA-mediated response in maize. Due to carotenoid deficiency, vp5 endosperm is white, compared to yellow Vp5 endosperm. However, the background difference at proteome level between vp5 and Vp5 seeds is unclear. This study aimed to characterize proteome alterations of maize vp5 seeds and to identify ABA-dependent proteins during seed maturation. We compared the embryo and endosperm proteomes of vp5 and Vp5 seeds by gel-based proteomics. Up to 46 protein spots, most in embryos, were found to be differentially accumulated between vp5 and Vp5. The identified proteins included small heat shock proteins (sHSPs), late embryogenesis abundant (LEA) proteins, stress proteins, storage proteins and enzymes among others. However, EMB564, the most abundant LEA protein in maize embryo, accumulated in comparable levels between vp5 and Vp5 embryos, which contrasted to previously characterized, greatly lowered expression of emb564 mRNA in vp5 embryos. Moreover, LEA proteins and sHSPs displayed differential accumulations in vp5 embryos: six out of eight identified LEA proteins decreased while nine sHSPs increased in abundance. Finally, we discussed the possible causes of global proteome alterations, especially the observed differential accumulation of identified LEA proteins and sHSPs in vp5 embryos. The data derived from this study provides new insight into ABA-dependent proteins and ABA-mediated response during maize seed maturation. PMID:25653661

  4. Accumulated SET protein up-regulates and interacts with hnRNPK, increasing its binding to nucleic acids, the Bcl-xS repression, and cellular proliferation.

    PubMed

    Almeida, Luciana O; Garcia, Cristiana B; Matos-Silva, Flavia A; Curti, Carlos; Leopoldino, Andréia M

    2014-02-28

    SET and hnRNPK are proteins involved in gene expression and regulation of cellular signaling. We previously demonstrated that SET accumulates in head and neck squamous cell carcinoma (HNSCC); hnRNPK is a prognostic marker in cancer. Here, we postulate that SET and hnRNPK proteins interact to promote tumorigenesis. We performed studies in HEK293 and HNSCC (HN6, HN12, and HN13) cell lines with SET/hnRNPK overexpression and knockdown, respectively. We found that SET and/or hnRNPK protein accumulation increased cellular proliferation. SET accumulation up-regulated hnRNPK mRNA and total/phosphorylated protein, promoted hnRNPK nuclear location, and reduced Bcl-x mRNA levels. SET protein directly interacted with hnRNPK, increasing both its binding to nucleic acids and Bcl-xS repression. We propose that hnRNPK should be a new target of SET and that SET-hnRNPK interaction, in turn, has potential implications in cell survival and malignant transformation.

  5. meso-Dihydroguaiaretic acid inhibits hepatic lipid accumulation by activating AMP-activated protein kinase in human HepG2 cells.

    PubMed

    Lee, Myoung-Su; Kim, Kyung Jin; Kim, Daeyoung; Lee, Kyung-Eun; Hwang, Jae-Kwan

    2011-01-01

    Hepatic lipid accumulation is a major risk factor for dyslipidemia, nonalcoholic fatty liver disease, and insulin resistance. The present study was conducted to evaluate hypolipidemic effects of meso-dihydroguaiaretic acid (MDA), anti-oxidative and anti-inflammatory compound isolated from the Myristica fragrans HOUTT., by oil red O staining, reverse transcription-polymerase chain reaction (RT-PCR), and Western blot. MDA significantly inhibited insulin-induced hepatic lipid accumulation in a dose-dependent manner. The lipid-lowering effect of MDA was accompanied by increased expression of proteins involved in fatty acid oxidation and decreased expression of lipid synthetic proteins. In addition, MDA activated AMP-activated protein kinase (AMPK) as determined by phosphorylation of acetyl-CoA carboxylase (ACC), a downstream target of AMPK. The effects of MDA on lipogenic protein expression were suppressed by pretreatment with compound C, an AMPK inhibitor. Taken together, these findings show that MDA inhibits insulin-induced lipid accumulation in human HepG2 cells by suppressing expression of lipogenic proteins through AMPK signaling, suggesting a potent lipid-lowering agent.

  6. An oleosin-fusion protein driven by the CaMV35S promoter is accumulated in Arabidopsis (Brassicaceae) seeds and correctly targeted to oil bodies.

    PubMed

    Li, W; Li, L G; Sun, X F; Tang, K X

    2012-08-13

    Oleosin-fusion technology is used to express desired proteins. It was developed based on the properties of oleosin; the heterologous protein gene is fused to the oleosin gene and the fusion gene is driven by a seed-specific promoter. We replaced the seed specific promoter with the CaMV35S promoter to dive a gfp-oleosin fusion gene in transformed Arabidopsis. The heterologous oleosin-fusion protein was mainly accumulated in the transgenic Arabidopsis seeds and correctly targeted to oil bodies. This provides an alternate choice of promoter in oleosin-fusion technology.

  7. Zinc finger protein genes from Cucurbita pepo are promising tools for conferring non-Cucurbitaceae plants with ability to accumulate persistent organic pollutants.

    PubMed

    Inui, Hideyuki; Hirota, Matashi; Goto, Junya; Yoshihara, Ryouhei; Kodama, Noriko; Matsui, Tomomi; Yamazaki, Kiyoshi; Eun, Heesoo

    2015-03-01

    Some cultivars of cucumbers, melons, pumpkins, and zucchini, which are members of the Cucurbitaceae family, are uniquely subject to contamination by hydrophobic pollutants such as the organohalogen insecticides DDT. However, the molecular mechanisms for the accumulation of these pollutants in cucurbits have not been determined. Here, cDNA subtraction analysis of Cucurbita pepo cultivars that are low and high accumulators of hydrophobic contaminants revealed that a gene for zinc finger proteins (ZFPs) are preferentially expressed in high accumulators. The cloned CpZFP genes were classified into 2 types: (1) the PBG type, which were expressed in C. pepo cultivars Patty Green, Black Beauty, and Gold Rush, and (2) the BG type, which were expressed in Black Beauty and Gold Rush. Expression of these CpZFP genes in transgenic tobacco plants carrying an aryl hydrocarbon receptor-based inducible gene expression system significantly induced β-glucuronidase activity when the plants were treated with a polychlorinated biphenyl (PCB) compound, indicating that highly hydrophobic PCBs accumulated in the plants. In transgenic tobacco plants carrying CpZFPs, accumulation of dioxins and dioxin-like compounds increased in their aerial parts when they were cultivated in the dioxin-contaminated soil. In summary, we propose that addition of CpZFP genes is a promising tool for conferring noncucurbits with the ability to accumulate hydrophobic contaminants.

  8. The transient accumulation of the signaling state of photoactive yellow protein is controlled by the external pH.

    PubMed

    Borucki, Berthold; Joshi, Chandra P; Otto, Harald; Cusanovich, Michael A; Heyn, Maarten P

    2006-10-15

    The signaling state of the photoreceptor photoactive yellow protein is the long-lived intermediate I(2)'. The pH dependence of the equilibrium between the transient photocycle intermediates I(2) and I(2)' was investigated. The formation of I(2)' from I(2) is accompanied by a major conformational change. The kinetics and intermediates of the photocycle and of the photoreversal were measured by transient absorption spectroscopy from pH 4.6 to 8.4. Singular value decomposition (SVD) analysis of the data at pH 7 showed the presence of three spectrally distinguishable species: I(1), I(2), and I(2)'. Their spectra were determined using the extrapolated difference method. I(2) and I(2)' have electronic absorption spectra, with maxima at 370 +/- 5 and 350 +/- 5 nm, respectively. Formation of the signaling state is thus associated with a change in the environment of the protonated chromophore. The time courses of the I(1), I(2), and I(2)' intermediates were determined from the wavelength-dependent transient absorbance changes at each pH, assuming that their spectra are pH-independent. After the formation of I(2)' ( approximately 2 ms), these three intermediates are in equilibrium and decay together to the initial dark state. The equilibrium between I(2) and I(2)' is pH dependent with a pK(a) of 6.4 and with I(2)' the main species above this pK(a). Measurements of the pH dependence of the photoreversal kinetics with a second flash of 355 nm at a delay of 20 ms confirm this pK(a) value. I(2) and I(2)' are photoreversed with reversal times of approximately 55 micros and several hundred microseconds, respectively. The corresponding signal amplitudes are pH dependent with a pK(a) of approximately 6.1. Photoreversal from I(2)' dominates above the pK(a). The transient accumulation of I(2)', the active state of photoactive yellow protein, is thus controlled by the proton concentration. The rate constant k(3) for the recovery to the initial dark state also has a pK(a) of

  9. Amyloid-β Precursor Protein Modulates the Sorting of Testican-1 and Contributes to Its Accumulation in Brain Tissue and Cerebrospinal Fluid from Patients with Alzheimer Disease

    PubMed Central

    Barrera-Ocampo, Alvaro; Arlt, Sönke; Matschke, Jakob; Hartmann, Ursula; Puig, Berta; Ferrer, Isidre; Zürbig, Petra; Glatzel, Markus; Jahn, Holger

    2016-01-01

    The mechanisms leading to amyloid-β (Aβ) accumulation in sporadic Alzheimer disease (AD) are unknown but both increased production or impaired clearance likely contribute to aggregation. To understand the potential roles of the extracellular matrix proteoglycan Testican-1 in the pathophysiology of AD, we used samples from AD patients and controls and an in vitro approach. Protein expression analysis showed increased levels of Testican-1 in frontal and temporal cortex of AD patients; histological analysis showed that Testican-1 accumulates and co-aggregates with Aβ plaques in the frontal, temporal and entorhinal cortices of AD patients. Proteomic analysis identified 10 fragments of Testican-1 in cerebrospinal fluid (CSF) from AD patients. HEK293T cells expressing human wild type or mutant Aβ precursor protein (APP) were transfected with Testican-1. The co-expression of both proteins modified the sorting of Testican-1 into the endocytic pathway leading to its transient accumulation in Golgi, which seemed to affect APP processing, as indicated by reduced Aβ40 and Aβ42 levels in APP mutant cells. In conclusion, patient data reflect a clearance impairment that may favor Aβ accumulation in AD brains and our in vitro model supports the notion that the interaction between APP and Testican-1 may be a key step in the production and aggregation of Aβ species. PMID:27486134

  10. Insulin-like growth factor binding protein-6 transgenic mice: postnatal growth, brain development, and reproduction abnormalities.

    PubMed

    Bienvenu, Géraldine; Seurin, Danielle; Grellier, Pascale; Froment, Pascal; Baudrimont, Marielle; Monget, Philippe; Le Bouc, Yves; Babajko, Sylvie

    2004-05-01

    In biological fluids, IGFs bind to six distinct binding proteins (IGFBP-1 to -6). IGFBP-6 is of particular interest because it has been shown to inhibit proliferation in many cell types and to be synthesized in the central nervous system (CNS). It also has the strongest affinity for IGF-II among the IGFBPs. To study IGFBP-6 function in vivo, we established IGFBP-6 transgenic mice in which human IGFBP-6 (hIGFBP-6) cDNA is expressed under the control of the glial fibrillary acidic protein (GFAP) promoter. Northern and Western blot analysis revealed strong transgene expression in the CNS. With histological examination of the CNS, cerebellum size and weight proved to be reduced by about 25% and 35%, respectively, and there were smaller numbers of differentiated, GFAP-expressing astrocytes than in wild-type mice. Between birth and 1 month of age, transgenic mice had high levels of circulating hIGFBP-6 and reduced plasma IGF-I, and, as a result, body weight was significantly reduced. Reproductive physiology was also affected. Litter size was reduced by 27% when wild-type males were mated with 3-month-old transgenic females and by 66% when mated with 6-month-old transgenic females. Histological examination of ovaries of transgenic mice revealed a marked decrease in weight and in the number of corpora lutea, suggesting altered ovulation, and circulating LH levels were reduced by 50%. Our results indicate that this new model of transgenic mouse may prove to be a useful tool in elucidating the in vivo role of IGFBP-6 in the brain, especially in regard to hypothalamic control, and in reproductive physiology.

  11. NPC1 late endosomes contain elevated levels of non-esterified (‘free’) fatty acids and an abnormally glycosylated form of the NPC2 protein

    PubMed Central

    2005-01-01

    NPC (Niemann–Pick type C) disease is a rare lipidosis characterized by the accumulation of LDL (low-density lipoprotein)-derived non-esterified cholesterol in the E/L (endosomal/lysosomal) system. The gene products that are responsible for the two NPC complementation groups are distinct and dissimilar, yet their cellular and disease phenotypes are virtually indistinguishable. To investigate the relationship between NPC1 and NPC2 and their potential role in NPC disease pathogenesis, we have developed a method for the rapid and efficient isolation of late endocytic vesicles from mouse liver by magnetic chromatography. Late endosomes from Wt (wild-type) and NPC1 mice were found to differ not only in their cholesterol and sphingomyelin content, as expected, but also in their non-esterified (‘free’) fatty acid content, with NPC1 vesicles showing an approx. 7-fold increase in non-esterified fatty acid levels compared with Wt vesicles. Furthermore, we show that the NPC2 protein is in an incompletely deglycosylated form in NPC1 late endosomes by a mechanism that is specific to the NPC2 protein and not a global aberration of protein glycosylation/deglycosylation or trafficking, since NPC2 secreted from NPC1 cells is indistinguishable from that secreted from Wt cells. Also, a greater proportion of the normally soluble cellular NPC2 protein partitions with detergent-insoluble late endosomal internal membrane domains in NPC1 vesicles. In addition, we show that, although a small amount of the NPC2 protein associates with these membranes in Wt vesicles, this localization becomes much more pronounced in NPC1 vesicles. These results suggest that the function of the NPC2 protein may be compromised as well in NPC1 endosomes, which might explain the paradoxical phenotypic similarities of the two NPC disease complementation groups. PMID:15896196

  12. p38 Mitogen-activated protein kinase and c-Jun NH2-terminal protein kinase regulate the accumulation of a tight junction protein, ZO-1, in cell-cell contacts in HaCaT cells.

    PubMed

    Minakami, Masahiko; Kitagawa, Norio; Iida, Hiroshi; Anan, Hisashi; Inai, Tetsuichiro

    2015-02-01

    To investigate the involvement of stress-activated protein kinases, JNK and p38 MAPK, in the assembly of tight junctions in keratinocytes, we treated HaCaT cells with various combinations of SP600125 (an inhibitor of JNK), SB202190 (an inhibitor of p38 MAPK) and anisomycin (an activator of both JNK and p38 MAPK) and examined the localization of ZO-1, an undercoat constitutive protein of the tight junction. Short-term (8h) incubation with SP600125, SB202190 or anisomycin induced the accumulation of ZO-1 in the cell-cell contacts, with reduced ZO-1 staining in the cytoplasm, while only long-term (24h) incubation with SP600125 induced the accumulation of ZO-1. SP600125, SB202190 or SP600125 plus SB202190 treatment induced thin linear staining for ZO-1 in the cell-cell contacts. Anisomycin treatment induced thick and irregular linear staining for ZO-1, while anisomycin plus SP600125 treatment induced zipper-like staining for ZO-1. Anisomycin plus SB202190 treatment or anisomycin plus both SP600125 and SB202190 treatment for 8h failed to lead to the accumulation of ZO-1 in cell-cell contacts, but induced thin linear staining with several gaps 16 h after removal of these agents. These results suggest that the localization of ZO-1 in cell-cell contacts is differently regulated by activation and inhibition of JNK and/or p38 MAPK depending on the incubation period.

  13. Influence of high temperature during grain filling on the accumulation of storage proteins and grain quality in rice (Oryza sativa L.).

    PubMed

    Lin, Chin-Ju; Li, Chia-Yu; Lin, Shao-Kai; Yang, Fan-Hsuan; Huang, Ji-Jwo; Liu, Yun-Hua; Lur, Huu-Sheng

    2010-10-13

    The present study was performed to understand the effects of high temperature (HT) during filling on the expression of storage proteins and the quality of rice grains. HT (35/30 °C day/night) reduced the weight, amylose content, and flour gel consistency of grains. It increased the accumulation of all classes of storage proteins at early filling stage but decreased the accumulation of prolamins at maturation. For albumins, the expressions of cyclophilin 2, peroxiredoxin, and HSP16.9 were differentially enhanced by HT. For globulins, HT decreased the accumulation of globulin but increased that of glyoxalase I and peroxiredoxin. HT enhanced the transcription of genes for glutelins, prolamins, globulins, and protein disulfide isomerase at early filling stage but decreased the expression of these genes at a later stage. Low amounts of prolamins and globulins, as well as low pH value, were found in sound, immature, and dead kernels grown under HT. The relationships among HT, storage proteins, and grain quality are discussed.

  14. Enzymatic activity of a subtilisin homolog, Tk-SP, from Thermococcus kodakarensis in detergents and its ability to degrade the abnormal prion protein

    PubMed Central

    2013-01-01

    Background Tk-SP is a member of subtilisin-like serine proteases from a hyperthermophilic archaeon Thermococcus kodakarensis. It has been known that the hyper-stable protease, Tk-SP, could exhibit enzymatic activity even at high temperature and in the presence of chemical denaturants. In this work, the enzymatic activity of Tk-SP was measured in the presence of detergents and EDTA. In addition, we focused to demonstrate that Tk-SP could degrade the abnormal prion protein (PrPSc), a protease-resistant isoform of normal prion protein (PrPC). Results Tk-SP was observed to maintain its proteolytic activity with nonionic surfactants and EDTA at 80°C. We optimized the condition in which Tk-SP functions efficiently, and demonstrated that the enzyme is highly stable in the presence of 0.05% (w/v) nonionic surfactants and 0.01% (w/v) EDTA, retaining up to 80% of its activity. Additionally, we also found that Tk-SP can degrade PrPSc to a level undetectable by western-blot analysis. Conclusions Our results indicate that Tk-SP has a great potential for technological applications, such as thermo-stable detergent additives. In addition, it is also suggested that Tk-SP-containing detergents can be developed to decrease the secondary infection risks of transmissible spongiform encephalopathies (TSE). PMID:23448268

  15. Accumulate-Repeat-Accumulate-Accumulate-Codes

    NASA Technical Reports Server (NTRS)

    Divsalar, Dariush; Dolinar, Sam; Thorpe, Jeremy

    2004-01-01

    Inspired by recently proposed Accumulate-Repeat-Accumulate (ARA) codes [15], in this paper we propose a channel coding scheme called Accumulate-Repeat-Accumulate-Accumulate (ARAA) codes. These codes can be seen as serial turbo-like codes or as a subclass of Low Density Parity Check (LDPC) codes, and they have a projected graph or protograph representation; this allows for a high-speed iterative decoder implementation using belief propagation. An ARAA code can be viewed as a precoded Repeat-and-Accumulate (RA) code with puncturing in concatenation with another accumulator, where simply an accumulator is chosen as the precoder; thus ARAA codes have a very fast encoder structure. Using density evolution on their associated protographs, we find examples of rate-lJ2 ARAA codes with maximum variable node degree 4 for which a minimum bit-SNR as low as 0.21 dB from the channel capacity limit can be achieved as the block size goes to infinity. Such a low threshold cannot be achieved by RA or Irregular RA (IRA) or unstructured irregular LDPC codes with the same constraint on the maximum variable node degree. Furthermore by puncturing the accumulators we can construct families of higher rate ARAA codes with thresholds that stay close to their respective channel capacity thresholds uniformly. Iterative decoding simulation results show comparable performance with the best-known LDPC codes but with very low error floor even at moderate block sizes.

  16. Lipid and protein accumulation in developing seeds of three lupine species: Lupinus luteus L., Lupinus albus L., and Lupinus mutabilis Sweet

    PubMed Central

    Borek, Sławomir; Pukacka, Stanisława; Michalski, Krzysztof; Ratajczak, Lech

    2009-01-01

    A comparative study was carried out on the dynamics of lipid accumulation in developing seeds of three lupine species. Lupine seeds differ in lipid content; yellow lupine (Lupinus luteus L.) seeds contain about 6%, white lupine (Lupinus albus L.) 7–14%, and Andean lupine (Lupinus mutabilis Sweet) about 20% of lipids by dry mass. Cotyledons from developing seeds were isolated and cultured in vitro for 96 h on Heller medium with 60 mM sucrose (+S) or without sucrose (–S). Each medium was additionally enriched with 35 mM asparagine or 35 mM NaNO3. Asparagine caused an increase in protein accumulation and simultaneously decreased the lipid content, but nitrate increased accumulation of both protein and lipid. Experiments with [1-14C]acetate and [2-14C]acetate showed that the decrease in lipid accumulation in developing lupine seeds resulted from exhaustion of lipid precursors rather than from degradation or modification of the enzymatic apparatus. The carbon atom from the C-1 position of acetate was liberated mainly as CO2, whereas the carbon atom from the C-2 position was preferentially used in anabolic pathways. The dominant phospholipid in the investigated lupine seed storage organs was phosphatidylcholine. The main fatty acid in yellow lupine cotyledons was linoleic acid, in white lupine it was oleic acid, and in Andean lupine it was both linoleic and oleic acids. The relationship between stimulation of lipid and protein accumulation by nitrate in developing lupine cotyledons and enhanced carbon flux through glycolysis caused by the inorganic nitrogen form is discussed. PMID:19635747

  17. Lipid and protein accumulation in developing seeds of three lupine species: Lupinus luteus L., Lupinus albus L., and Lupinus mutabilis Sweet.

    PubMed

    Borek, Slawomir; Pukacka, Stanisława; Michalski, Krzysztof; Ratajczak, Lech

    2009-01-01

    A comparative study was carried out on the dynamics of lipid accumulation in developing seeds of three lupine species. Lupine seeds differ in lipid content; yellow lupine (Lupinus luteus L.) seeds contain about 6%, white lupine (Lupinus albus L.) 7-14%, and Andean lupine (Lupinus mutabilis Sweet) about 20% of lipids by dry mass. Cotyledons from developing seeds were isolated and cultured in vitro for 96 h on Heller medium with 60 mM sucrose (+S) or without sucrose (-S). Each medium was additionally enriched with 35 mM asparagine or 35 mM NaNO3. Asparagine caused an increase in protein accumulation and simultaneously decreased the lipid content, but nitrate increased accumulation of both protein and lipid. Experiments with [1-14C]acetate and [2-14C]acetate showed that the decrease in lipid accumulation in developing lupine seeds resulted from exhaustion of lipid precursors rather than from degradation or modification of the enzymatic apparatus. The carbon atom from the C-1 position of acetate was liberated mainly as CO2, whereas the carbon atom from the C-2 position was preferentially used in anabolic pathways. The dominant phospholipid in the investigated lupine seed storage organs was phosphatidylcholine. The main fatty acid in yellow lupine cotyledons was linoleic acid, in white lupine it was oleic acid, and in Andean lupine it was both linoleic and oleic acids. The relationship between stimulation of lipid and protein accumulation by nitrate in developing lupine cotyledons and enhanced carbon flux through glycolysis caused by the inorganic nitrogen form is discussed.

  18. The proteins encoded by the Drosophila Planar Polarity Effector genes inturned, fuzzy and fritz interact physically and can re-pattern the accumulation of “upstream” Planar Cell Polarity proteins

    PubMed Central

    Wang, Ying; Yan, Jie; Lee, Haeryun; Lu, Qiuheng; Adler, Paul N.

    2014-01-01

    The frizzled/starry night pathway regulates planar cell polarity in a wide variety of tissues in many types of animals. It was discovered and has been most intensively studied in the Drosophila wing where it controls the formation of the array of distally pointing hairs that cover the wing. The pathway does this by restricting the activation of the cytoskeleton to the distal edge of wing cells. This results in hairs initiating at the distal edge and growing in the distal direction. All of the proteins encoded by genes in the pathway accumulate asymmetrically in wing cells. The pathway is a hierarchy with the Planar Cell Polarity (PCP) genes (aka the core genes) functioning as a group upstream of the Planar Polarity Effector (PPE) genes which in turn function as a group upstream of multiple wing hairs. Upstream proteins, such as Frizzled accumulate on either the distal and/or proximal edges of wing cells. Downstream PPE proteins accumulate on the proximal edge under the instruction of the upstream proteins. A variety of types of data support this hierarchy, however, we have found that when over expressed the PPE proteins can alter both the subcellular location and level of accumulation of the upstream proteins. Thus, the epistatic relationship is context dependent. We further show that the PPE proteins interact physically and can modulate the accumulation of each other in wing cells. We also find that over expression of Frtz results in a marked delay in hair initiation suggesting that it has a separate role/activity in regulating the cytoskeleton that is not shared by other members of the group. PMID:25072625

  19. The proteins encoded by the Drosophila Planar Polarity Effector genes inturned, fuzzy and fritz interact physically and can re-pattern the accumulation of "upstream" Planar Cell Polarity proteins.

    PubMed

    Wang, Ying; Yan, Jie; Lee, Haeryun; Lu, Qiuheng; Adler, Paul N

    2014-10-01

    The frizzled/starry night pathway regulates planar cell polarity in a wide variety of tissues in many types of animals. It was discovered and has been most intensively studied in the Drosophila wing where it controls the formation of the array of distally pointing hairs that cover the wing. The pathway does this by restricting the activation of the cytoskeleton to the distal edge of wing cells. This results in hairs initiating at the distal edge and growing in the distal direction. All of the proteins encoded by genes in the pathway accumulate asymmetrically in wing cells. The pathway is a hierarchy with the Planar Cell Polarity (PCP) genes (aka the core genes) functioning as a group upstream of the Planar Polarity Effector (PPE) genes which in turn function as a group upstream of multiple wing hairs. Upstream proteins, such as Frizzled accumulate on either the distal and/or proximal edges of wing cells. Downstream PPE proteins accumulate on the proximal edge under the instruction of the upstream proteins. A variety of types of data support this hierarchy, however, we have found that when over expressed the PPE proteins can alter both the subcellular location and level of accumulation of the upstream proteins. Thus, the epistatic relationship is context dependent. We further show that the PPE proteins interact physically and can modulate the accumulation of each other in wing cells. We also find that over expression of Frtz results in a marked delay in hair initiation suggesting that it has a separate role/activity in regulating the cytoskeleton that is not shared by other members of the group.

  20. Involvement of de Novo Protein Synthesis, Protein Kinase, Extracellular Ca2+, and Lipoxygenase in Arachidonic Acid Induction of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Genes and Isoprenoid Accumulation in Potato (Solanum tuberosum L.).

    PubMed Central

    Choi, D.; Bostock, R. M.

    1994-01-01

    A series of inhibitors were tested to determine the participation of de novo protein synthesis, protein kinase activity, extracellular Ca2+, and lipoxygenase activity in arachidonic acid elicitation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene expression and sesquiterpene phytoalexin biosynthesis in potato (Solanum tuberosum L. cv Kennebec). Gene-specific probes were used to discriminate effects on the expression of two HMGR genes (hmg1 and hmg2) that respond differentially in tuber tissue following wounding or elicitor treatment. Inhibition of protein synthesis with cycloheximide completely blocked arachidonate-induced hypersensitive necrosis and browning, including HMGR gene induction and phytoalexin accumulation. This suggests that proteins necessary for coupling arachidonic acid reception to HMGR mRNA accumulation are either rapidly turned over or not present constitutively and are induced following elicitor treatment. Staurosporin, a potent inhibitor of protein kinases, and ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime]-tetraacetic acid, a Ca2+ chelator, inhibited arachidonate-induction of hmg2 gene expression and phytoalexin accumulation but did not inhibit the wound-induced expression of hmg1. However, staurosporin inhibited arachidonate's suppression of hmg1 gene expression. Eicosatetraynoic acid, a lipoxygenase inhibitor that suppresses elicitor-induced phytoalexin accumulation, also inhibited arachidonate's suppression of hmg1 and induction of hmg2. The results indicate that arachidonate's suppression of hmg1 and activation of hmg2 depend on a common intermediate or set of intermediates whose generation is sensitive to the inhibitors tested. PMID:12232162

  1. Proteome analysis reveals differential expression of proteins involved in triacylglycerol accumulation by Rhodococcus jostii RHA1 after addition of methyl viologen.

    PubMed

    Dávila Costa, José Sebastián; Silva, Roxana A; Leichert, Lars; Alvarez, Héctor M

    2017-03-01

    Rhodococcus jostii RHA1 is able to degrade toxic compounds and accumulate high amounts of triacylglycerols (TAG) upon nitrogen starvation. These NADPH-dependent processes are essential for the adaptation of rhodococci to fluctuating environmental conditions. In this study, we used an MS-based, label-free and quantitative proteomic approach to better understand the integral response of R. jostii RHA1 to the presence of methyl viologen (MV) in relation to the synthesis and accumulation of TAG. The addition of MV promoted a decrease of TAG accumulation in comparison to cells cultivated under nitrogen-limiting conditions in the absence of this pro-oxidant. Proteomic analyses revealed that the abundance of key proteins of fatty acid biosynthesis, the Kennedy pathway, glyceroneogenesis and methylmalonyl-CoA pathway, among others, decreased in the presence of MV. In contrast, some proteins involved in lipolysis and β-oxidation of fatty acids were upregulated. Some metabolic pathways linked to the synthesis of NADPH remained activated during oxidative stress as well as under nitrogen starvation conditions. Additionally, exposure to MV resulted in the activation of complete antioxidant machinery comprising superoxide dismutases, catalases, mycothiol biosynthesis, mycothione reductase and alkyl hydroperoxide reductases, among others. Our study suggests that oxidative stress response affects TAG accumulation under nitrogen-limiting conditions through programmed molecular mechanisms when both stresses occur simultaneously.

  2. The development of frost tolerance and DHN5 protein accumulation in barley (Hordeum vulgare) doubled haploid lines derived from Atlas 68 x Igri cross during cold acclimation.

    PubMed

    Kosová, Klára; Tom Prásil, Ilja; Prásilová, Pavla; Vítámvás, Pavel; Chrpová, Jana

    2010-03-15

    The dynamics of a long-term cold acclimation (CA) was studied in spring barley cultivar Atlas 68, winter barley cultivar Igri and a set of doubled haploid (DH) lines derived from an Atlas 68xIgri cross. The aim was to evaluate the effect of plant development on the ability to induce frost tolerance (FT) and to accumulate dehydrin 5 (DHN5) during CA. The plant developmental stage was evaluated by phenological development of the shoot apex and by determination of days to heading after a certain period of CA. FT was determined by direct frost tests. Plant winter survival was also determined. DHN5 was evaluated by densitometric analysis of protein gel blots. Cold led to the induction of increased FT and to the accumulation of DHN5 in both spring and winter lines. However, with the progression of CA, differences between the growth habits occurred as the winter lines were able to maintain increased FT and DHN5 levels for a significantly longer period of time than the spring lines. After vegetative/reproductive transition, a significant decrease in DHN5 accumulation was found in all lines; however, a discrepancy between the acquired FT level and DHN5 accumulation in vernalized winter barley plants was found. A correlation between DHN5 accumulation and plant winter survival was found when the studied lines were differentiated according to their developmental stage and DHN5 level. Possible explanations for these phenomena are provided.

  3. Accumulation of 52 kDa glycine rich protein in auxin-deprived strawberry fruits and its role in fruit growth. [Fragaria ananassa

    SciTech Connect

    Reddy, A.S.N.; Poovaiah, B.W.

    1987-04-01

    Growth of strawberry (Fragaria ananassa Duch) receptacles can be stopped at any stage by deachening the fruits and can be resumed by exogenous application of auxin. In their earlier studies they demonstrated auxin regulated polypeptide changes at different stages of strawberry fruit development. Removal of achenes from fruits to deprive auxin resulted in the accumulation of 52 KDa polypeptide. This polypeptide is associated with cell wall and its concentration is increased in a time-dependent manner in auxin deprived receptacles. Incorporation studies with (/sup 35/S) methionine showed the promotion of labelling of 52 kDa polypeptide in the auxin-deprived receptacles within 12 h after removal of the achenes. Amino acid analysis revealed that the 52 KDa polypeptide is rich in glycine. Their studies, with normal and mutant strawberry receptacles, indicate that the synthesis and accumulation of this glycine rich protein correlates with cessation of receptacle growth. These results suggest a role for the glycine rich protein in growth.

  4. A Novel Kleefstra Syndrome-associated Variant That Affects the Conserved TPLX Motif within the Ankyrin Repeat of EHMT1 Leads to Abnormal Protein Folding*

    PubMed Central

    Blackburn, Patrick R.; Tischer, Alexander; Zimmermann, Michael T.; Kemppainen, Jennifer L.; Sastry, Sujatha; Knight Johnson, Amy E.; Cousin, Margot A.; Boczek, Nicole J.; Oliver, Gavin; Misra, Vinod K.; Gavrilova, Ralitza H.; Lomberk, Gwen; Auton, Matthew; Urrutia, Raul; Klee, Eric W.

    2017-01-01

    Kleefstra syndrome (KS) (Mendelian Inheritance in Man (MIM) no. 610253), also known as 9q34 deletion syndrome, is an autosomal dominant disorder caused by haploinsufficiency of euchromatic histone methyltransferase-1 (EHMT1). The clinical phenotype of KS includes moderate to severe intellectual disability with absent speech, hypotonia, brachycephaly, congenital heart defects, and dysmorphic facial features with hypertelorism, synophrys, macroglossia, protruding tongue, and prognathism. Only a few cases of de novo missense mutations in EHMT1 giving rise to KS have been described. However, some EHMT1 variants have been described in individuals presenting with autism spectrum disorder or mild intellectual disability, suggesting that the phenotypic spectrum resulting from EHMT1 alterations may be quite broad. In this report, we describe two unrelated patients with complex medical histories consistent with KS in whom next generation sequencing identified the same novel c.2426C>T (p.P809L) missense variant in EHMT1. To examine the functional significance of this novel variant, we performed molecular dynamics simulations of the wild type and p.P809L variant, which predicted that the latter would have a propensity to misfold, leading to abnormal histone mark binding. Recombinant EHMT1 p.P809L was also studied using far UV circular dichroism spectroscopy and intrinsic protein fluorescence. These functional studies confirmed the model-based hypotheses and provided evidence for protein misfolding and aberrant target recognition as the underlying pathogenic mechanism for this novel KS-associated variant. This is the first report to suggest that missense variants in EHMT1 that lead to protein misfolding and disrupted histone mark binding can lead to KS. PMID:28057753

  5. The overgrown hematopoietic organs-31 tumor suppressor gene of Drosophila encodes an Importin-like protein accumulating in the nucleus at the onset of mitosis

    PubMed Central

    1995-01-01

    The tumor suppressor gene overgrown hematopoietic organs-31 (oho31) of Drosophila encodes a protein with extensive homology to the Importin protein of Xenopus (50% identity), the related yeast SRP1 protein, and the mammalian hSRP1 and RCH1 proteins. A strong reduction in the expression of oho31 by a P element inserted in the 5' untranslated region of the oho31 transcript or a complete inactivation of oho31 by imprecise P element excision leads to malignant development of the hematopoietic organs and the genital disc, as shown by their growth autonomy in transplantation assays. We have cloned the oho31 gene of Drosophila melanogaster and determined its nucleotide sequence. The gene encodes a phosphoprotein of 522 amino acids made of three domains: a central hydrophobic domain of eight repeats of 42-44 amino acids each, displaying similarity to the arm motif found in junctional and nucleopore complex proteins, and flanked by two hydrophilic NH2- and COOH-terminal domains. Immunostaining revealed that the OHO31 protein is supplied maternally and rapidly degraded during the first 13 nuclear divisions. Thereafter, the OHO31 protein is predominantly expressed, albeit at reduced levels, in proliferating tissues. During the interphase of early embryonic cell cycles, the OHO31 protein is present in the cytoplasm and massively accumulates in the nucleus at the onset of mitosis in late interphase and prophase. The nuclear import of OHO31 is, however, less pronounced during later developmental stages. These results suggest that, similar to Importin, OHO31 may act as a cytosolic factor in nuclear transport. Moreover, the cell cycle-dependent accumulation of OHO31 in the nucleus indicates that this protein may be required for critical nuclear reactions occurring at the onset of mitosis. PMID:7790349

  6. Accumulate-Repeat-Accumulate-Accumulate Codes

    NASA Technical Reports Server (NTRS)

    Divsalar, Dariush; Dolinar, Samuel; Thorpe, Jeremy

    2007-01-01

    Accumulate-repeat-accumulate-accumulate (ARAA) codes have been proposed, inspired by the recently proposed accumulate-repeat-accumulate (ARA) codes. These are error-correcting codes suitable for use in a variety of wireless data-communication systems that include noisy channels. ARAA codes can be regarded as serial turbolike codes or as a subclass of low-density parity-check (LDPC) codes, and, like ARA codes they have projected graph or protograph representations; these characteristics make it possible to design high-speed iterative decoders that utilize belief-propagation algorithms. The objective in proposing ARAA codes as a subclass of ARA codes was to enhance the error-floor performance of ARA codes while maintaining simple encoding structures and low maximum variable node degree.

  7. Loss of Cathepsin B and L Leads to Lysosomal Dysfunction, NPC-Like Cholesterol Sequestration and Accumulation of the Key Alzheimer's Proteins

    PubMed Central

    Cermak, Stjepko; Kosicek, Marko; Mladenovic-Djordjevic, Aleksandra; Smiljanic, Kosara; Kanazir, Selma

    2016-01-01

    Proper function of lysosomes is particularly important in neurons, as they cannot dilute accumulated toxic molecules and aggregates by cell division. Thus, impairment of lysosomal function plays an important role in neuronal degeneration and in the pathogenesis of numerous neurodegenerative diseases. In this work we analyzed how inhibition and/or loss of the major lysosomal proteases, the cysteine cathepsins B and L (CtsB/L), affects lysosomal function, cholesterol metabolism and degradation of the key Alzheimer's disease (AD) proteins. Here, we show that cysteine CtsB/L, and not the aspartyl cathepsin D (CtsD), represent a major lysosomal protease(s) that control lysosomal function, intracellular cholesterol trafficking and AD-like amyloidogenic features. Intriguingly, accumulation of free cholesterol in late endosomes/lysosomes upon CtsB/L inhibition resembled a phenotype characteristic for the rare neurodegenerative disorder Niemann-Pick type C (NPC). CtsB/L inhibition and not the inhibition of CtsD led to lysosomal impairment assessed by decreased degradation of EGF receptor, enhanced LysoTracker staining and accumulation of several lysosomal proteins LC3II, NPC1 and NPC2. By measuring the levels of NPC1 and ABCA1, the two major cholesterol efflux proteins, we showed that CtsB/L inhibition or genetic depletion caused accumulation of the NPC1 in lysosomes and downregulation of ABCA1 protein levels and its expression. Furthermore, we revealed that CtsB/L are involved in degradation of the key Alzheimer’s proteins: amyloid-β peptides (Aβ) and C-terminal fragments of the amyloid precursor protein (APP) and in degradation of β-secretase (BACE1). Our results imply CtsB/L as major regulators of lysosomal function and demonstrate that CtsB/L may play an important role in intracellular cholesterol trafficking and in degradation of the key AD proteins. Our findings implicate that enhancing the activity or levels of CtsB/L could provide a promising and a common

  8. Proteomic Characterization of Differential Abundant Proteins Accumulated between Lower and Upper Epidermises of Fleshy Scales in Onion (Allium cepa L.) Bulbs

    PubMed Central

    Wu, Xiaolin

    2016-01-01

    The onion (Allium cepa L.) is widely planted worldwide as a valuable vegetable crop. The scales of an onion bulb are a modified type of leaf. The one-layer-cell epidermis of onion scales is commonly used as a model experimental material in botany and molecular biology. The lower epidermis (LE) and upper epidermis (UE) of onion scales display obvious differences in microscopic structure, cell differentiation and pigment synthesis; however, associated proteomic differences are unclear. LE and UE can be easily sampled as single-layer-cell tissues for comparative proteomic analysis. In this study, a proteomic approach based on 2-DE and mass spectrometry (MS) was applied to compare LE and UE of fleshy scales from yellow and red onions. We identified 47 differential abundant protein spots (representing 31 unique proteins) between LE and UE in red and yellow onions. These proteins are mainly involved in pigment synthesis, stress response, and cell division. Particularly, the differentially accumulated chalcone-flavanone isomerase and flavone O-methyltransferase 1-like in LE may result in the differences in the onion scale color between red and yellow onions. Moreover, stress-related proteins abundantly accumulated in both LE and UE. In addition, the differential accumulation of UDP-arabinopyranose mutase 1-like protein and β-1,3-glucanase in the LE may be related to the different cell sizes between LE and UE of the two types of onion. The data derived from this study provides new insight into the differences in differentiation and developmental processes between onion epidermises. This study may also make a contribution to onion breeding, such as improving resistances and changing colors. PMID:28036352

  9. Proteomic Characterization of Differential Abundant Proteins Accumulated between Lower and Upper Epidermises of Fleshy Scales in Onion (Allium cepa L.) Bulbs.

    PubMed

    Wu, Si; Ning, Fen; Wu, Xiaolin; Wang, Wei

    2016-01-01

    The onion (Allium cepa L.) is widely planted worldwide as a valuable vegetable crop. The scales of an onion bulb are a modified type of leaf. The one-layer-cell epidermis of onion scales is commonly used as a model experimental material in botany and molecular biology. The lower epidermis (LE) and upper epidermis (UE) of onion scales display obvious differences in microscopic structure, cell differentiation and pigment synthesis; however, associated proteomic differences are unclear. LE and UE can be easily sampled as single-layer-cell tissues for comparative proteomic analysis. In this study, a proteomic approach based on 2-DE and mass spectrometry (MS) was applied to compare LE and UE of fleshy scales from yellow and red onions. We identified 47 differential abundant protein spots (representing 31 unique proteins) between LE and UE in red and yellow onions. These proteins are mainly involved in pigment synthesis, stress response, and cell division. Particularly, the differentially accumulated chalcone-flavanone isomerase and flavone O-methyltransferase 1-like in LE may result in the differences in the onion scale color between red and yellow onions. Moreover, stress-related proteins abundantly accumulated in both LE and UE. In addition, the differential accumulation of UDP-arabinopyranose mutase 1-like protein and β-1,3-glucanase in the LE may be related to the different cell sizes between LE and UE of the two types of onion. The data derived from this study provides new insight into the differences in differentiation and developmental processes between onion epidermises. This study may also make a contribution to onion breeding, such as improving resistances and changing colors.

  10. Abnormal body iron distribution and erythropoiesis in a novel mouse model with inducible gain of iron regulatory protein (IRP)-1 function.

    PubMed

    Casarrubea, D; Viatte, L; Hallas, T; Vasanthakumar, A; Eisenstein, R S; Schümann, K; Hentze, M W; Galy, B

    2013-07-01

    Disorders of iron metabolism account for some of the most common human diseases. Cellular iron homeostasis is maintained by iron regulatory proteins (IRP)-1 and 2 through their binding to cis-regulatory iron-responsive elements (IREs) in target mRNAs. Mouse models with IRP deficiency have yielded valuable insights into iron biology, but the physiological consequences of gain of IRP function in mammalian organisms have remained unexplored. Here, we report the generation of a mouse line allowing conditional expression of a constitutively active IRP1 mutant (IRP1) using Cre/Lox technology. Systemic activation of the IRP1 transgene from the Rosa26 locus yields viable animals with gain of IRE-binding activity in all the organs analyzed. IRP1 activation alters the expression of IRP target genes and is accompanied by iron loading in the same organs. Furthermore, mice display macrocytic erythropenia with decreased hematocrit and hemoglobin levels as well as impaired erythroid differentiation. Thus, inappropriately high IRP1 activity causes disturbed body iron distribution and erythropoiesis. This new mouse model further highlights the importance of appropriate IRP regulation in central organs of iron metabolism. Moreover, it opens novel avenues to study diseases associated with abnormally high IRP1 activity, such as Parkinson's disease or Friedreich's ataxia.

  11. Mutations in Radial Spoke Head Protein Genes RSPH9 and RSPH4A Cause Primary Ciliary Dyskinesia with Central-Microtubular-Pair Abnormalities

    PubMed Central

    Castleman, Victoria H.; Romio, Leila; Chodhari, Rahul; Hirst, Robert A.; de Castro, Sandra C.P.; Parker, Keith A.; Ybot-Gonzalez, Patricia; Emes, Richard D.; Wilson, Stephen W.; Wallis, Colin; Johnson, Colin A.; Herrera, Rene J.; Rutman, Andrew; Dixon, Mellisa; Shoemark, Amelia; Bush, Andrew; Hogg, Claire; Gardiner, R. Mark; Reish, Orit; Greene, Nicholas D.E.; O'Callaghan, Christopher; Purton, Saul; Chung, Eddie M.K.; Mitchison, Hannah M.

    2009-01-01

    Primary ciliary dyskinesia (PCD) is a genetically heterogeneous inherited disorder arising from dysmotility of motile cilia and sperm. This is associated with a variety of ultrastructural defects of the cilia and sperm axoneme that affect movement, leading to clinical consequences on respiratory-tract mucociliary clearance and lung function, fertility, and left-right body-axis determination. We performed whole-genome SNP-based linkage analysis in seven consanguineous families with PCD and central-microtubular-pair abnormalities. This identified two loci, in two families with intermittent absence of the central-pair structure (chromosome 6p21.1, Zmax 6.7) and in five families with complete absence of the central pair (chromosome 6q22.1, Zmax 7.0). Mutations were subsequently identified in two positional candidate genes, RSPH9 on chromosome 6p21.1 and RSPH4A on chromosome 6q22.1. Haplotype analysis identified a common ancestral founder effect RSPH4A mutation present in UK-Pakistani pedigrees. Both RSPH9 and RSPH4A encode protein components of the axonemal radial spoke head. In situ hybridization of murine Rsph9 shows gene expression restricted to regions containing motile cilia. Investigation of the effect of knockdown or mutations of RSPH9 orthologs in zebrafish and Chlamydomonas indicate that radial spoke head proteins are important in maintaining normal movement in motile, “9+2”-structure cilia and flagella. This effect is rescued by reintroduction of gene expression for restoration of a normal beat pattern in zebrafish. Disturbance in function of these genes was not associated with defects in left-right axis determination in humans or zebrafish. PMID:19200523

  12. Nuclear accumulation of epidermal growth factor receptor and acceleration of G1/S stage by Epstein-Barr-encoded oncoprotein latent membrane protein 1

    SciTech Connect

    Tao Yongguang; Song Xing; Deng Xiyun; Xie Daxin; Lee, Leo M.; Liu Yiping; Li Wei; Li Lili; Deng Lin; Wu Qiao; Gong Jianping; Cao Ya . E-mail: ycao98@public.cs.hn.cn

    2005-02-15

    Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is considered to be the major oncogenic protein of EBV-encoded proteins and has always been the core of the oncogenic mechanism of EBV. Advanced studies on nuclear translocation of the epidermal growth factor receptor (EGFR) family have greatly improved our knowledge of the biological function of cell surface receptors. In this study, we used the Tet-on LMP1 HNE2 cell line as a cell model, which is a dual-stable LMP1-integrated nasopharyngeal carcinoma (NPC) cell line and the expression of LMP1 which could be regulated by the Tet system. We found that LMP1 could regulate the nuclear accumulation of EGFR in a dose-dependent manner quantitatively and qualitatively. We also demonstrated that the nuclear localization sequence of EGFR played some roles in the location of the protein within the nucleus under LMP1 regulation and EGFR in the nucleus could bind to the promoters of cyclinD1 and cyclinE, respectively. We further demonstrated that EGFR is involved in the acceleration of the G1/S phase transition by LMP1 through binding to cyclinD1 and cyclinE directly. These findings provided a novel view that the acceleration of LMP1 on the G1/S transition via the nuclear accumulation of EGFR was critical in the process of nasopharyngeal carcinoma.

  13. [Molecular abnormalities in lymphomas].

    PubMed

    Delsol, G

    2010-11-01

    Numerous molecular abnormalities have been described in lymphomas. They are of diagnostic and prognostic value and are taken into account for the WHO classification of these tumors. They also shed some light on the underlying molecular mechanisms involved in lymphomas. Overall, four types of molecular abnormalities are involved: mutations, translocations, amplifications and deletions of tumor suppressor genes. Several techniques are available to detect these molecular anomalies: conventional cytogenetic analysis, multicolor FISH, CGH array or gene expression profiling using DNA microarrays. In some lymphomas, genetic abnormalities are responsible for the expression of an abnormal protein (e.g. tyrosine-kinase, transcription factor) detectable by immunohistochemistry. In the present review, molecular abnormalities observed in the most frequent B, T or NK cell lymphomas are discussed. In the broad spectrum of diffuse large B-cell lymphomas microarray analysis shows mostly two subgroups of tumors, one with gene expression signature corresponding to germinal center B-cell-like (GCB: CD10+, BCL6 [B-Cell Lymphoma 6]+, centerine+, MUM1-) and a subgroup expressing an activated B-cell-like signature (ABC: CD10-, BCL6-, centerine-, MUM1+). Among other B-cell lymphomas with well characterized molecular abnormalies are follicular lymphoma (BCL2 deregulation), MALT lymphoma (Mucosa Associated Lymphoid Tissue) [API2-MALT1 (mucosa-associated-lymphoid-tissue-lymphoma-translocation-gene1) fusion protein or deregulation BCL10, MALT1, FOXP1. MALT1 transcription factors], mantle cell lymphoma (cycline D1 [CCND1] overexpression) and Burkitt lymphoma (c-Myc expression). Except for ALK (anaplastic lymphoma kinase)-positive anaplastic large cell lymphoma, well characterized molecular anomalies are rare in lymphomas developed from T or NK cells. Peripheral T cell lymphomas not otherwise specified are a heterogeneous group of tumors with frequent but not recurrent molecular abnormalities

  14. Correction of metabolic abnormalities in a rodent model of obesity, metabolic syndrome, and type 2 diabetes mellitus by inhibitors of hepatic protein kinase C-ι.

    PubMed

    Sajan, Mini P; Nimal, Sonali; Mastorides, Stephen; Acevedo-Duncan, Mildred; Kahn, C Ronald; Fields, Alan P; Braun, Ursula; Leitges, Michael; Farese, Robert V

    2012-04-01

    Excessive activity of hepatic atypical protein kinase (aPKC) is proposed to play a critical role in mediating lipid and carbohydrate abnormalities in obesity, the metabolic syndrome, and type 2 diabetes mellitus. In previous studies of rodent models of obesity and type 2 diabetes mellitus, adenoviral-mediated expression of kinase-inactive aPKC rapidly reversed or markedly improved most if not all metabolic abnormalities. Here, we examined effects of 2 newly developed small-molecule PKC-ι/λ inhibitors. We used the mouse model of heterozygous muscle-specific knockout of PKC-λ, in which partial deficiency of muscle PKC-λ impairs glucose transport in muscle and thereby causes glucose intolerance and hyperinsulinemia, which, via hepatic aPKC activation, leads to abdominal obesity, hepatosteatosis, hypertriglyceridemia, and hypercholesterolemia. One inhibitor, 1H-imidazole-4-carboxamide, 5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy)methyl]cyclopentyl-[1R-(1a,2b,3b,4a)], binds to the substrate-binding site of PKC-λ/ι, but not other PKCs. The other inhibitor, aurothiomalate, binds to cysteine residues in the PB1-binding domains of aPKC-λ/ι/ζ and inhibits scaffolding. Treatment with either inhibitor for 7 days inhibited aPKC, but not Akt, in liver and concomitantly improved insulin signaling to Akt and aPKC in muscle and adipocytes. Moreover, both inhibitors diminished excessive expression of hepatic, aPKC-dependent lipogenic, proinflammatory, and gluconeogenic factors; and this was accompanied by reversal or marked improvements in hyperglycemia, hyperinsulinemia, abdominal obesity, hepatosteatosis, hypertriglyceridemia, and hypercholesterolemia. Our findings highlight the pathogenetic importance of insulin signaling to hepatic PKC-ι in obesity, the metabolic syndrome, and type 2 diabetes mellitus and suggest that 1H-imidazole-4-carboxamide, 5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy)methyl]cyclopentyl-[1R-(1a,2b,3b,4a)] and aurothiomalate or similar agents that

  15. Proteins that accumulate with age in human skeletal-muscle aggregates contribute to declines in muscle mass and function in Caenorhabditis elegans

    PubMed Central

    Ayyadevara, Srinivas; Balasubramaniam, Meenakshisundaram; Suri, Pooja; Mackintosh, Samuel G.; Tackett, Alan J.; Sullivan, Dennis H.; Shmookler Reis, Robert J.; Dennis, Richard A.

    2016-01-01

    Protein aggregation increases with age in normal tissues, and with pathology and age in Alzheimer's hippocampus and mouse cardiac muscle. We now ask whether human skeletal muscle accumulates aggregates with age. Detergent-insoluble protein aggregates were isolated from vastus lateralis biopsies from 5 young (23–27 years of age) and 5 older (64–80 years) adults. Aggregates, quantified after gel electrophoresis, contain 2.1-fold more protein (P<0.0001) when isolated from older subjects relative to young. Of 515 proteins identified by liquid chromatography coupled to tandem mass spectrometry, 56 (11%) were significantly more abundant in older muscle, while 21 (4%) were depleted with age (each P<0.05). Orthologs to seven of these proteins were then targeted in C. elegans by RNA interference. Six of the seven knockdown treatments decreased protein aggregation (range 6–45%, P<0.01 to <0.0001) and increased muscle mass (range 1.5- to 1.85-fold, P<0.01 to <0.0001) in aged nematodes, and rescued mobility (range 1.4 to 1.65-fold, P≤0.0005 each) in a nematode amyloidopathy model. We conclude that specific aggregate proteins, discovered as differentially abundant in aging human muscle, have orthologs that contribute functionally to aggregation and age-associated muscle loss in nematodes, and thus can be considered potential drug targets for sarcopenia in humans. PMID:27992858

  16. Label-free Proteomic Reveals that Cowpea Severe Mosaic Virus Transiently Suppresses the Host Leaf Protein Accumulation During the Compatible Interaction with Cowpea (Vigna unguiculata [L.] Walp.).

    PubMed

    Paiva, Ana L S; Oliveira, Jose T A; de Souza, Gustavo A; Vasconcelos, Ilka M

    2016-12-02

    Viruses are important plant pathogens that threaten diverse crops worldwide. Diseases caused by Cowpea severe mosaic virus (CPSMV) have drawn attention because of the serious damages they cause to economically important crops including cowpea. This work was undertaken to quantify and identify the responsive proteins of a susceptible cowpea genotype infected with CPSMV, in comparison with mock-inoculated controls, using label-free quantitative proteomics and databanks, aiming at providing insights on the molecular basis of this compatible interaction. Cowpea leaves were mock- or CPSMV-inoculated and 2 and 6 days later proteins were extracted and analyzed. More than 3000 proteins were identified (data available via ProteomeXchange, identifier PXD005025) and 75 and 55 of them differentially accumulated in response to CPSMV, at 2 and 6 DAI, respectively. At 2 DAI, 76% of the proteins decreased in amount and 24% increased. However, at 6 DAI, 100% of the identified proteins increased. Thus, CPSMV transiently suppresses the synthesis of proteins involved particularly in the redox homeostasis, protein synthesis, defense, stress, RNA/DNA metabolism, signaling, and other functions, allowing viral invasion and spread in cowpea tissues.

  17. Nickel accumulation and its effect on biomass, protein content and antioxidative enzymes in roots and leaves of watercress (Nasturtium officinale R. Br.).

    PubMed

    Duman, Fatih; Ozturk, Fatma

    2010-01-01

    In order to understand its response towards nickel stress, watercress (Nasturtium officinale R. Br.) was exposed to nickel (1-25 mg/L) for 1, 3, 5 and 7 days. The accumulation and translocation of nickel were determined and the influence of nickel on biomass, protein content and enzymatic antioxidants was examined for both roots and leaves. It was determined that N. officinale could accumulate appreciable amounts of Ni in both roots and leaves. Nickel accumulated particularly in the roots of plants. Biomass increased at low nickel concentrations but certain measurable change was not found at high concentrations. Under stress conditions the antioxidant enzymes were up-regulated compared to control. An increase in protein content and enzyme activities was observed at moderate exposure conditions followed by a decline at both roots and leaves. The maximum enzyme activities were observed at different exposure conditions. Our results showed that N. officinale had the capacity to overcome nickel-induced stress especially at moderate nickel exposure. Therefore, N. officinale may be used as a phytoremediator in moderately polluted aquatic ecosystems.

  18. Endoplasmic reticulum-localized small heat shock protein that accumulates in mulberry tree (Morus bombycis Koidz.) during seasonal cold acclimation is responsive to abscisic acid.

    PubMed

    Ukaji, Norifumi; Kuwabara, Chikako; Kanno, Yuri; Seo, Mitsunori; Takezawa, Daisuke; Arakawa, Keita; Fujikawa, Seizo

    2010-04-01

    With seasonal changes, several proteins accumulate in the endoplasmic reticulum (ER)-enriched fraction in the bark of mulberry tree (Morus bombycis Koidz.). Results of partial amino acid sequence analysis in our previous study suggested that one of these proteins is the ER-localized small heat shock protein (sHSP), designated 20-kD winter-accumulating protein (WAP20). In the present study, molecular and biochemical properties of WAP20 were investigated in detail. The deduced amino acid sequence of the cDNA has the predicted signal sequence to the ER, retention signal to the ER and two consensus regions conserved in sHSPs. Recombinant WAP20 expressed in Escherichia coli also showed typical biochemical features of sHSPs, including the formation of a high-molecular-mass complex between 200 and 300 kD under native conditions, promotion of the renaturation of chemically denaturated citrate synthase and prevention of heat stress-induced aggregation of the enzyme. Transcript levels of WAP20 in the bark tissue were seasonally changed, showing high expression levels from mid-October to mid-December, and the transcript levels were additionally increased and decreased by cold treatment and warm treatment, respectively. WAP20 transcripts were detected abundantly in bark tissue rather than xylem and winter bud tissues during seasonal cold acclimation. The bark tissue specificity of WAP20 accumulation was also observed by exogenous application of phytohormone abscisic acid (ABA) in de-acclimated twigs, whereas WAP20 transcripts were increased in all of these tissues by heat shock treatment at 37 degrees C in summer twigs. The results suggest that ABA may be involved in the expression of the WAP20 gene in bark tissue of the mulberry tree during seasonal cold acclimation.

  19. Dauricine inhibits insulin-like growth factor-I-induced hypoxia inducible factor 1α protein accumulation and vascular endothelial growth factor expression in human breast cancer cells

    PubMed Central

    Tang, Xu-dong; Zhou, Xin; Zhou, Ke-yuan

    2009-01-01

    Aim: To investigate the effects of dauricine (Dau) on insulin-like growth factor-I (IGF-I)-induced hypoxia inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression in human breast cancer cells (MCF-7). Methods: Serum-starved MCF-7 cells were pretreated for 1 h with different concentrations of Dau, followed by incubation with IGF-I for 6 h. HIF-1α and VEGF protein expression levels were analyzed by Western blotting and ELISA, respectively. HIF-1α and VEGF mRNA levels were determined by real-time PCR. In vitro angiogenesis was observed via the human umbilical vein endothelial cell (HUVEC) tube formation assay. An in vitro invasion assay on HUVECs was performed. Results: Dau significantly inhibited IGF-I-induced HIF-1α protein expression but had no effect on HIF-1α mRNA expression. However, Dau remarkably suppressed VEGF expression at both protein and mRNA levels in response to IGF-I. Mechanistically, Dau suppressed IGF-I-induced HIF-1α and VEGF protein expression mainly by blocking the activation of PI-3K/AKT/mTOR signaling pathway. In addition, Dau reduced IGF-I-induced HIF-1α protein accumulation by inhibiting its synthesis as well as by promoting its degradation. Functionally, Dau inhibited angiogenesis in vitro. Moreover, Dau had a direct effect on IGF-I-induced invasion of HUVECs. Conclusion: Dau inhibits human breast cancer angiogenesis by suppressing HIF-1α protein accumulation and VEGF expression, which may provide a novel potential mechanism for the anticancer activities of Dau in human breast cancer. PMID:19349962

  20. Metal accumulation and differentially expressed proteins in gill of oyster (Crassostrea hongkongensis) exposed to long-term heavy metal-contaminated estuary.

    PubMed

    Luo, Lianzhong; Ke, Caihuan; Guo, Xiaoyu; Shi, Bo; Huang, Miaoqin

    2014-06-01

    Bio-accumulation and bio-transmission of toxic metals and the toxicological responses of organisms exposed to toxic metals have been focused, due to heavy metal contaminations have critically threatened the ecosystem and food security. However, still few investigations focused on the responses of certain organisms exposed to the long term and severe heavy metal contamination in specific environments. In present investigation, the Hong Kong oyster, Crassostrea hongkongensis were obtained from 3 sites which were contaminated by different concentrations of heavy metals (such as zinc, copper, manganese and lead etc.), respectively. Heavy metal concentrations in the sea water samples collected from the 3 sites and the dissected tissues of the oysters with blue visceral mass were determinated to estimate the metal contamination levels in environments and the bio-accumulation ratios of the heavy metals in the different tissues of oysters. Moreover, Proteomic methods were employed to analyze the differentially expressed proteins in the gills of oysters exposed to long-term heavy metal contaminations. Results indicated that the Jiulong River estuary has been severely contaminated by Cu, Zn and slightly with Cr, Ni, Mn, etc, moreover, Zn and Cu were the major metals accumulated by oysters to phenomenally high concentrations (more than 3.0% of Zn and about 2.0% of Cu against what the dry weight of tissues were accumulated), and Cr, Ni, Mn, etc were also significantly accumulated. The differentially expressed proteins in the gills of oysters exposed to heavy metals participate in several cell processes, such as metal binding, transporting and saving, oxidative-reduction balance maintaining, stress response, signal transduction, etc. Significantly up-regulated expression (about 10 folds) of an important metal binding protein, metallothionein (MT) and granular cells was observed in the gills of oysters exposed to long-term and severely heavy-metal-contaminated estuary, it

  1. A Fusion between Domains of the Human Bone Morphogenetic Protein-2 and Maize 27 kD γ-Zein Accumulates to High Levels in the Endoplasmic Reticulum without Forming Protein Bodies in Transgenic Tobacco

    PubMed Central

    Ceresoli, Valentina; Mainieri, Davide; Del Fabbro, Massimo; Weinstein, Roberto; Pedrazzini, Emanuela

    2016-01-01

    Human Bone Morphogenetic Protein-2 (hBMP2) is an osteoinductive agent physiologically involved in bone remodeling processes. A commercialized recombinant hBMP2 produced in mammalian cell lines is available in different clinical applications where bone regeneration is needed, but widespread use has been hindered due to an unfavorable cost/effective ratio. Protein bodies are very large insoluble protein polymers that originate within the endoplasmic reticulum by prolamine accumulation during the cereal seed development. The N-terminal domain of the maize prolamin 27 kD γ-zein is able to promote protein body biogenesis when fused to other proteins. To produce high yield of recombinant hBMP2 active domain (ad) in stably transformed tobacco plants we have fused it to the γ-zein domain. We show that this zein-hBMP2ad fusion is retained in the endoplasmic reticulum without forming insoluble protein bodies. The accumulation levels are above 1% of total soluble leaf proteins, indicating that it could be a rapid and suitable strategy to produce hBMP2ad at affordable costs. PMID:27047526

  2. Effect of lipopolysaccharide on protein accumulation by murine peritoneal macrophages: the correlation to activation for macrophage tumoricidal function

    SciTech Connect

    Tannenbaum, C.S.

    1987-01-01

    The protein synthetic patterns of tumoricidal murine peritoneal macrophage populations have been compared to those of non-tumoricidal populations utilizing two dimensional polyacrylamide gel electrophoresis (2D PAGE) of (/sup 35/S)-methionine-labeled proteins. While the protein synthetic patterns exhibited by resident, inflammatory and activated macrophages had numerous common features which distinguished them from the other normal non-macrophage cell types examined, unique proteins also distinguished each macrophage population from the others. Peritoneal macrophages elicited by treatment with heat killed Propionibacterium acnes, the live, attenuated Mycobacterium bovis strain BCG, Listeria monocytogenes and the protozoan flagellate Trypanosoma rhodesiense, all exhibited tumoricidal activity in 16h or 72h functional assays, and shared a common protein synthetic profile which differentiated them from the synthetic patterns characteristic of the non-tumoricidal resident and inflammatory macrophages.

  3. RSK-mediated nuclear accumulation of the cold-shock Y-box protein-1 controls proliferation of T cells and T-ALL blasts.

    PubMed

    Gieseler-Halbach, Steffi; Meltendorf, Stefan; Pierau, Mandy; Weinert, Soenke; Heidel, Florian H; Fischer, Thomas; Handschuh, Juliane; Braun-Dullaeus, Ruediger C; Schrappe, Martin; Lindquist, Jonathan A; Mertens, Peter R; Thomas, Ulrich; Brunner-Weinzierl, Monika C

    2017-02-01

    Deregulated proliferation is key to tumor progression. Although unrestricted proliferation of solid tumor cells correlates with the cold-shock protein Y-box (YB)-binding protein-1 accumulation in the nuclei, little is known about its expression and function in hematopoietic malignancies, such as T-cell acute lymphoblastic leukemia (T-ALL). Here we show that YB-1 protein is highly enriched in the nuclei of activated T cells and malignant human T-ALL cell lines but not in resting T cells. YB-1 S(102) mutations that either mimic (S102D) or prevent phosphorylation (S102N) led to accumulation of YB-1 in the nucleus of T cells or strictly excluded it, respectively. Inactivation of ribosomal S6 kinase (RSK) was sufficient to abrogate T-cell and T-ALL cell proliferation, suggesting that RSK mediates cell-cycle progression, possibly dependent on YB-1-phosphorylation. Indeed, phosphomimetic YB-1(S102D) enhanced proliferation implying that S(102) phosphorylation is a prerequisite for malignant T-cell proliferation. At initial diagnosis of T-ALL, YB-1 localization was significantly altered in the nuclei of tumor blasts derived from bone marrow or peripheral blood. Our data show deregulated YB-1 in the nucleus as a yet unreported characteristic of T-ALL blasts and may refine strategies to restrict progression of hematopoietic tumors.

  4. Proline-rich cell wall proteins accumulate in growing regions and phloem tissue in response to water deficit in common bean seedlings.

    PubMed

    Battaglia, Marina; Solórzano, Rosa M; Hernández, Magdalena; Cuéllar-Ortiz, Sonia; García-Gómez, Blanca; Márquez, Judith; Covarrubias, Alejandra A

    2007-04-01

    Plant cell walls undergo dynamic changes in response to different environmental stress conditions. In response to water deficit, two related proline-rich glycoproteins, called p33 and p36, accumulate in the soluble fraction of the cell walls in Phaseolus vulgaris (Covarrubias et al. in Plant Physiol 107:1119-1128, 1995). In this work, we show that p33 and p36 are able to form a 240 kDa oligomer, which is found in the cell wall soluble fraction. We present evidence indicating that the highest accumulation of these proteins in response to water deficit occurs in the growing regions of common bean seedlings, particularly in the phloem tissues. These proteins were detected in P. vulgaris cell suspension cultures, where the p33/p36 ratio was higher under hyperosmotic conditions than in bean seedlings subjected to the same treatment. The results support a role for these proteins during the plant cell response to changes in its water status, and suggest that cell wall modifications are induced in active growing cells of common bean in response to water limitation.

  5. Quantitative Proteomics-Based Reconstruction and Identification of Metabolic Pathways and Membrane Transport Proteins Related to Sugar Accumulation in Developing Fruits of Pear (Pyrus communis).

    PubMed

    Reuscher, Stefan; Fukao, Yoichiro; Morimoto, Reina; Otagaki, Shungo; Oikawa, Akira; Isuzugawa, Kanji; Shiratake, Katsuhiro

    2016-03-01

    During their 6 month development, pear (Pyrus communis) fruits undergo drastic changes in their morphology and their chemical composition. To gain a better understanding of the metabolic pathways and transport processes active during fruit development, we performed a time-course analysis using mass spectrometry (MS)-based protein identification and quantification of fruit flesh tissues. After pre-fractionation of the samples, 2,841 proteins were identified. A principal component analysis (PCA) separated the samples from seven developmental stages into three distinct clusters representing the early, mid and late developmental phase. Over-representation analysis of proteins characteristic of each developmental phase revealed both expected and novel biological processes relevant at each phase. A high abundance of aquaporins was detected in samples from fruits in the cell expansion stage. We were able quantitatively to reconstruct basic metabolic pathways such as the tricarboxylic acid (TCA) cycle, which indicates sufficient coverage to reconstruct other metabolic pathways. Most of the enzymes that presumably contribute to sugar accumulation in pear fruits could be identified. Our data indicate that invertases do not play a major role in the sugar conversions in developing pear fruits. Rather, sucrose might be broken down by sucrose synthases. Further focusing on sugar transporters, we identified several putative sugar transporters from diverse families which showed developmental regulation. In conclusion, our data set comprehensively describes the proteome of developing pear fruits and provides novel insights about sugar accumulation as well as candidate genes for key reactions and transport steps.

  6. The methionine-rich low-molecular-weight chloroplast heat-shock protein: evolutionary conservation and accumulation in relation to thermotolerance.

    PubMed

    Downs, C; Heckathorn, S; Bryan, J; Coleman, J

    1998-02-01

    The evolutionary conservation of the low-molecular-weight chloroplast-localized heat-shock protein (LMW chlpHsp) in vascular plants was examined using immunological methods. An antibody (Abmet) specific to the LMW chlpHsp was produced using a synthetic 28-residue peptide containing the most conserved elements of its unique "methionine-rich domain" as an antigen. This antibody detected a heat-inducible low-molecular-weight chloroplast protein in plants of six divergent Anthophyta species, including C3, C4, CAM, monocot, and dicot species. Abmet also detected a LMW chlpHsp in species from the Divisions Psilotophyta, Equisetophyta, Polypodiophyta, and Ginkgophyta. A preliminary examination of the relationship between accumulation of the LMW chlpHsp and habitat was also conducted. Seven Anthophyta species originating from both warm- and cool-temperature habitats were grown at 28C and then heat stressed at 40C. A positive qualitative relationship between the accumulation of the LMW chlpHsp and organismal thermotolerance in these species was observed; similar results were obtained separately with four nonAnthophyta species. The strong evolutionary conservation of this LMW Hsp and its localization to the chloroplast, and the correlation between production of this protein and plant thermotolerance, suggest that the LMW chlpHsp plays an important role in adaptation to heat stress.

  7. Chromatin structure in granulocytes. A link between tight compaction and accumulation of a heterochromatin-associated protein (MENT).

    PubMed

    Grigoryev, S A; Woodcock, C L

    1998-01-30

    To study the mechanism of heterochromatin formation in vertebrate cells, we isolated nuclei from chicken polymorphonuclear granulocytes and examined the chromatin organization. We found granulocyte chromatin to remain insoluble after nuclease digestion and to be resistant to swelling in low salt/high pH media. Both insolubility and resistance to swelling were lost after washing with 0.3 M NaCl, a procedure that released two abundant tissue-specific proteins from granulocyte nuclei. One of them (42 kDa) is identified as MENT, a protein previously shown to be associated with repressed chromatin from mature chicken erythrocytes. We show that MENT is immunolocalized in granulocyte heterochromatin, where it is one of the most abundant chromatin proteins ( approximately 2 molecules/200 base pairs of DNA). MENT is the first nuclear protein structurally related to the serine protease inhibitor family. The other abundant protein is similar to or identical with mim-1, a myeloid-specific protein that is known to be stored in cell granules and to associate with isolated nuclei. MENT (but not mim-1) binds chromatin and free DNA, and, at its physiological protein/DNA ratio, enhances compaction and the reversible Mg2+-dependent self-association of nucleosome arrays. MENT appears to promote the formation of heterochromatin by acting as a "glue" within and between chains of nucleosomes.

  8. Is alopecia areata an autoimmune-response against melanogenesis-related proteins, exposed by abnormal MHC class I expression in the anagen hair bulb?

    PubMed Central

    Paus, R.; Slominski, A.; Czarnetzki, B. M.

    1993-01-01

    The etiology of alopecia areata (AA), a putative autoimmune disease characterized by sudden hair loss, has remained obscure. It is not understood, how the characteristic inflammatory infiltrate that selectively attacks anagen hair follicles in AA is generated. We hypothesize that this reflects an unexplored form of autoimmunity, a cytotoxic T cell attack on rhythmically synthesized autoantigens normally sequestered by a lack or very low level of MHC class I (MHC I)-expression, and suggest the following mechanism of AA pathogenesis: Microtrauma, neurogenic inflammation, or microbial antigens cause a localized breakdown of MHC I-"negativity" in the proximal anagen hair bulb via proinflammatory cytokines. This exposes autoantigens derived from melanogenesis-related proteins (MRP-DP), which are only generated during anagen, and triggers two successive waves of autoimmune responses: CD8+ cytotoxic T cells initiate AA after recognizing MRP-DP abnormally presented by MHC I molecules on hair matrix melanocytes and/or keratinocytes; a secondary attack, carried by CD4+ T cells and antigen presenting cells, is then mounted against MHC class II--presented additional autoantigens exposed by damaged melanocytes and keratinocytes. The latter causes most of the follicular damage, and extrafollicular disease, and depends greatly on the immunogenetic background of affected individuals. This unifying hypothesis explains the clinical heterogeneity and all salient features of AA, and argues that only the unlikely coincidence of multiple predisposing events triggers AA. The suppression of MHC I--expression and synthesis of MRP in the hair bulb, and the "tolerization" of MRP-DP autoreactive CD8+ T cells may be promising strategies for treating AA. PMID:7716973

  9. The Dietary Approaches to Stop Hypertension eating plan affects C-reactive protein, coagulation abnormalities, and hepatic function tests among type 2 diabetic patients.

    PubMed

    Azadbakht, Leila; Surkan, Pamela J; Esmaillzadeh, Ahmad; Willett, Walter C

    2011-06-01

    Few studies exist regarding the effects of the Dietary Approaches to Stop Hypertension (DASH) diet on novel cardiovascular risk factors among type 2 diabetic patients. We evaluated the effects of the DASH eating pattern on C-reactive protein (CRP) level, coagulation abnormalities, and hepatic function tests in type 2 diabetic patients. In this randomized, crossover clinical trial, 31 type 2 diabetic patients consumed a control diet or the DASH diet for 8 wk. The DASH diet was rich in fruits, vegetables, whole grains, and low-fat dairy products and low in saturated fat, total fat, cholesterol, refined grains, and sweets, with a total of 2400 mg/d sodium. The control diet was a standard diet for diabetic patients. There was a 4-wk washout between the 2 trial phases. The main outcome measures were CRP level, coagulation indices, and hepatic function tests. The mean percent change for plasma CRP level was -26.9 ± 3.5% after the DASH diet period and -5.1 ± 3.8% after the control diet period (P = 0.02). Decreases in both alanine aminotransferase and aspartate aminotransferase levels were greater after consuming the DASH diet compared with the control diet (-14.8 ± 3.0% vs -6.6 ± 3.4%; P = 0.001; -29.4 ± 3.7% vs -5.9 ± 1.4%; P = 0.001, respectively). The decrease in the plasma fibrinogen level during the DASH diet period (-11.4 ± 3.6%) was greater than that during the control diet (0.5 ± 3.4%) (P = 0.03). Among diabetic patients, the DASH diet can play an important role in reducing inflammation, plasma levels of fibrinogen, and liver aminotransferases. Future longer term studies are recommended.

  10. Prognostic value of coexistence of abnormal expression of micro-RNA-200b and cyclic adenosine monophosphate-responsive element-binding protein 1 in human astrocytoma.

    PubMed

    Zhang, Jun-qing; Yao, Qing-he; Kuang, Yong-qin; Ma, Yuan; Yang, Li-bin; Huang, Hai-dong; Cheng, Jing-ming; Yang, Tao; Liu, En-yu; Liang, Liang; Fan, Ke-xia; Zhao, Kai; Xia, Xun; Gu, Jian-wen

    2014-10-01

    Our aim was to investigate the expression of micro-RNA-200b (miR-200b) and cAMP-responsive element-binding protein 1 (CREB-1) in astrocytoma and its efficacy for predicting outcome. Both miR-200b and CREB-1 messenger RNA expression was measured in 122 astrocytomas and 30 nonneoplastic brain specimens by quantitative real-time polymerase chain reaction. Expression of miR-200b was significantly lower in astrocytoma than in nonneoplastic brain (P < .001), whereas CREB-1 messenger RNA expression was significantly elevated in the tumors (P < .001). Both miR-200b down-regulation and CREB-1 up-regulation were significantly associated with advanced pathologic grade (P = .002 and P = .006, respectively). Low miR-200b expression correlated negatively with Karnofsky performance score (P = .03), and high CREB-1 expression correlated positively with mean tumor diameter (P = .03). By Kaplan-Meier analysis, low miR-200b, high CREB-1, and coexistence of abnormal miR-200b and CREB-1 expression (low miR-200b/high CREB-1) were predictive of shorter progression-free survival and overall survival in both grade III and grade IV astrocytoma. By multivariate analysis, only low miR-200b/high CREB-1 expression was an independent prognostic factor for poor prognosis in astrocytoma of advanced grade. Both miR-200b and CREB-1 may play important cooperative roles in the progression of human astrocytoma. The efficacy of miR-200b and CREB-1 together as a predictor of prognosis in astrocytoma patients is shown for the first time.

  11. Increased placental fatty acid transporter 6 and binding protein 3 expression and fetal liver lipid accumulation in a mouse model of obesity in pregnancy.

    PubMed

    Díaz, Paula; Harris, Jessica; Rosario, Fredrick J; Powell, Theresa L; Jansson, Thomas

    2015-12-15

    Obesity in pregnancy is associated with increased fetal growth and adiposity, which, in part, is determined by transplacental nutrient supply. Trophoblast uptake and intracellular trafficking of lipids are dependent on placental fatty acid transport proteins (FATP), translocase (FAT/CD36), and fatty acid binding proteins (FABP). We hypothesized that maternal obesity in mice leads to increased placental expression of FAT/CD36, FATPs, and FABPs, and lipid accumulation in the fetal liver. C57/BL6J female mice were fed either a control (C; n = 10) or an obesogenic (OB; n = 10) high-fat, high-sugar diet before mating and throughout pregnancy. At E18.5, placentas and fetal livers were collected. Trophoblast plasma membranes (TPM) were isolated from placental homogenates. Expression of FAT/CD36 and FATP (TPM) and FABP (homogenates) was determined by immunoblotting. Gene expression was assessed by RT-quantitative PCR. Sections of fetal livers were stained for Oil Red O, and lipid droplets were quantified. TPM protein expression of FAT/CD36, FATP 2, and FATP 4 was comparable between C and OB groups. Conversely, TPM FATP 6 expression was increased by 35% in OB compared with C placentas without changes in mRNA expression. FABPs 1, 3-5 and PPARγ were expressed in homogenates, and FABP 3 expression increased 27% in OB compared with C placentas; however, no changes were observed in mRNA expression. Lipid droplet accumulation was 10-fold higher in the livers of fetuses from OB compared with C group. We propose that increased lipid transport capacity in obese mice promotes transplacental fatty acid transport and contributes to excess lipid accumulation in the fetal liver.

  12. Silencing of the SNARE protein NAPA sensitizes cancer cells to cisplatin by inducing ERK1/2 signaling, synoviolin ubiquitination and p53 accumulation.

    PubMed

    Wu, Zchong-Zcho; Sun, Nian-Kang; Chien, Kun-Yi; Chao, Chuck C-K

    2011-12-01

    We found earlier that NAPA represents an anti-apoptotic protein that promotes resistance to cisplatin in cancer cells by inducing the degradation of the tumor suppressor p53. In the present study, we investigated the cellular mechanism underlying the degradation of p53 by NAPA. Knockdown of NAPA using short-hairpin RNA was shown to induce p53 accumulation and to sensitize HEK293 cells to cisplatin. On the other hand, this sensitization effect was not found in H1299 lung carcinoma cells which lack p53. Expression of exogenous p53 in H1299 cells was increased following knockdown of NAPA and these cells showed increased sensitivity to cisplatin-induced apoptosis. Notably, knockdown of NAPA induced the ubiquitination and degradation of the E3 ubiquitin ligase synoviolin and the accumulation of p53 in unstressed HEK293 cells. Conversely, NAPA overexpression decreased the ubiquitination and degradation of synoviolin, and reduced p53 protein level. Knockdown of NAPA disrupted the interaction between synoviolin and proteins that form the endoplasmic reticulum-associated degradation (ERAD) complex and in turn decreased the ability of this complex to ubiquitinate p53. In addition, knockdown of NAPA induced the activation of the MAPK kinases ERK, JNK and p38, but only inhibition of ERK reduced synoviolin ubiquitination and p53 accumulation. These results indicate that NAPA promotes resistance to cisplatin through synoviolin and the ERAD complex which together induce the degradation of p53 and thus prevent apoptosis. Based on these findings, we propose that the combination of cisplatin and knockdown of NAPA represents a novel and attractive strategy to eradicate p53-sensitive cancer cells.

  13. WEE1 accumulation and deregulation of S-phase proteins mediate MLN4924 potent inhibitory effect on Ewing sarcoma cells.

    PubMed

    Mackintosh, C; García-Domínguez, D J; Ordóñez, J L; Ginel-Picardo, A; Smith, P G; Sacristán, M P; de Álava, E

    2013-03-14

    Ewing sarcoma (ES) is an aggressive bone and soft tissue tumor of children and young adults in which finding effective new targeted therapies is imperative. Here, we report an in-depth preclinical study of the investigational cullin-RING ubiquitin ligase (CRL) inhibitor MLN4924 in ES, as we have recently demonstrated the implication of a CRL component in the ES pathogenesis. First, our results support a high sensitivity of ES cells to MLN4924 growth inhibition both in vitro (14 ES cell lines tested, median IC50=81 nM) and in tumor xenografts (tumor regression achieved with 60 mg/kg BID, subcutaneously, n=9). Second, we report a dual mechanism of action of MLN4924 in ES cells: while a wide range of MLN4924 concentrations (∼30-300 nM) trigger a G2 arrest that can only be rescued by WEE1 kinase inhibition or depletion, saturating doses of the drug (>300 nM) cause a delay in S-phase progression concomitant with unbalanced CDK2-Cyclin E and CDK2-Cyclin A relative levels (accumulation of the first and depletion of the latter). The aberrant presence of CDC6 in the nucleus at late S-phase cell cycle stage confirmed the loss of CDK2-Cyclin A-specific functions. Remarkably, other mechanisms explored (P27 accumulation and DNA damage signaling pathways) were found unable to explain MLN4924 effects, strengthening the specificity of our findings and suggesting the absence of functionality of some CRL substrates accumulated in response to MLN4924. This study renders a rationale for clinical trials and contributes molecular mechanisms for a better understanding of this promising antitumoral agent.

  14. Chromoplast-specific carotenoid-associated protein appears to be important for enhanced accumulation of carotenoids in hp1 tomato fruits.

    PubMed

    Kilambi, Himabindu Vasuki; Kumar, Rakesh; Sharma, Rameshwar; Sreelakshmi, Yellamaraju

    2013-04-01

    Tomato (Solanum lycopersicum) high-pigment mutants with lesions in diverse loci such as DNA Damage-Binding Protein1 (high pigment1 [hp1]), Deetiolated1 (hp2), Zeaxanthin Epoxidase (hp3), and Intense pigment (Ip; gene product unknown) exhibit increased accumulation of fruit carotenoids coupled with an increase in chloroplast number and size. However, little is known about the underlying mechanisms exaggerating the carotenoid accumulation and the chloroplast number in these mutants. A comparison of proteome profiles from the outer pericarp of hp1 mutant and wild-type (cv Ailsa Craig) fruits at different developmental stages revealed at least 72 differentially expressed proteins during ripening. Hierarchical clustering grouped these proteins into three clusters. We found an increased abundance of chromoplast-specific carotenoid-associated protein (CHRC) in hp1 fruits at red-ripe stage that is also reflected in its transcript level. Western blotting using CHRC polyclonal antibody from bell pepper (Capsicum annuum) revealed a 2-fold increase in the abundance of CHRC protein in the red-ripe stage of hp1 fruits compared with the wild type. CHRC levels in hp2 were found to be similar to that of hp1, whereas hp3 and Ip showed intermediate levels to those in hp1, hp2, and wild-type fruits. Both CHRC and carotenoids were present in the isolated plastoglobules. Overall, our results suggest that loss of function of DDB1, DET1, Zeaxanthin Epoxidase, and Ip up-regulates CHRC levels. Increase in CHRC levels may contribute to the enhanced carotenoid content in these high-pigment fruits by assisting in the sequestration and stabilization of carotenoids.

  15. Red-backed vole brain promotes highly efficient in vitro amplification of abnormal prion protein from macaque and human brains infected with variant Creutzfeldt-Jakob disease agent.

    USGS Publications Warehouse

    Nemecek, Julie; Nag, Nabanita; Carlson, Christina M.; Schneider, Jay R.; Heisey, Dennis M.; Johnson, Christopher J.; Asher, David M.; Gregori, Luisa

    2013-01-01

    Rapid antemortem tests to detect individuals with transmissible spongiform encephalopathies (TSE) would contribute to public health. We investigated a technique known as protein misfolding cyclic amplification (PMCA) to amplify abnormal prion protein (PrPTSE) from highly diluted variant Creutzfeldt-Jakob disease (vCJD)-infected human and macaque brain homogenates, seeking to improve the rapid detection of PrPTSE in tissues and blood. Macaque vCJD PrPTSE did not amplify using normal macaque brain homogenate as substrate (intraspecies PMCA). Next, we tested interspecies PMCA with normal brain homogenate of the southern red-backed vole (RBV), a close relative of the bank vole, seeded with macaque vCJD PrPTSE. The RBV has a natural polymorphism at residue 170 of the PrP-encoding gene (N/N, S/S, and S/N). We investigated the effect of this polymorphism on amplification of human and macaque vCJD PrPTSE. Meadow vole brain (170N/N PrP genotype) was also included in the panel of substrates tested. Both humans and macaques have the same 170S/S PrP genotype. Macaque PrPTSE was best amplified with RBV 170S/S brain, although 170N/N and 170S/N were also competent substrates, while meadow vole brain was a poor substrate. In contrast, human PrPTSE demonstrated a striking narrow selectivity for PMCA substrate and was successfully amplified only with RBV 170S/S brain. These observations suggest that macaque PrPTSE was more permissive than human PrPTSE in selecting the competent RBV substrate. RBV 170S/S brain was used to assess the sensitivity of PMCA with PrPTSE from brains of humans and macaques with vCJD. PrPTSE signals were reproducibly detected by Western blot in dilutions through 10-12 of vCJD-infected 10% brain homogenates. This is the first report showing PrPTSE from vCJD-infected human and macaque brains efficiently amplified with RBV brain as the substrate. Based on our estimates, PMCA showed a sensitivity that might be sufficient to detect PrPTSE in v

  16. Red-Backed Vole Brain Promotes Highly Efficient In Vitro Amplification of Abnormal Prion Protein from Macaque and Human Brains Infected with Variant Creutzfeldt-Jakob Disease Agent

    PubMed Central

    Nemecek, Julie; Nag, Nabanita; Carlson, Christina M.; Schneider, Jay R.; Heisey, Dennis M.; Johnson, Christopher J.; Asher, David M.; Gregori, Luisa

    2013-01-01

    Rapid antemortem tests to detect individuals with transmissible spongiform encephalopathies (TSE) would contribute to public health. We investigated a technique known as protein misfolding cyclic amplification (PMCA) to amplify abnormal prion protein (PrPTSE) from highly diluted variant Creutzfeldt-Jakob disease (vCJD)-infected human and macaque brain homogenates, seeking to improve the rapid detection of PrPTSE in tissues and blood. Macaque vCJD PrPTSE did not amplify using normal macaque brain homogenate as substrate (intraspecies PMCA). Next, we tested interspecies PMCA with normal brain homogenate of the southern red-backed vole (RBV), a close relative of the bank vole, seeded with macaque vCJD PrPTSE. The RBV has a natural polymorphism at residue 170 of the PrP-encoding gene (N/N, S/S, and S/N). We investigated the effect of this polymorphism on amplification of human and macaque vCJD PrPTSE. Meadow vole brain (170N/N PrP genotype) was also included in the panel of substrates tested. Both humans and macaques have the same 170S/S PrP genotype. Macaque PrPTSE was best amplified with RBV 170S/S brain, although 170N/N and 170S/N were also competent substrates, while meadow vole brain was a poor substrate. In contrast, human PrPTSE demonstrated a striking narrow selectivity for PMCA substrate and was successfully amplified only with RBV 170S/S brain. These observations suggest that macaque PrPTSE was more permissive than human PrPTSE in selecting the competent RBV substrate. RBV 170S/S brain was used to assess the sensitivity of PMCA with PrPTSE from brains of humans and macaques with vCJD. PrPTSE signals were reproducibly detected by Western blot in dilutions through 10-12 of vCJD-infected 10% brain homogenates. This is the first report showing PrPTSE from vCJD-infected human and macaque brains efficiently amplified with RBV brain as the substrate. Based on our estimates, PMCA showed a sensitivity that might be sufficient to detect PrPTSE in vCJD-infected human

  17. Heterologous expression of Paranosema (Antonospora) locustae hexokinase in lepidopteran, Sf9, cells is followed by accumulation of the microsporidian protein in insect cell nuclei.

    PubMed

    Timofeev, Sergey A; Senderskiy, Igor V; Tsarev, Alexander A; Tokarev, Yuri S; Dolgikh, Viacheslav V

    2017-02-01

    Paranosema (Nosema, Antonospora) locustae is the only microsporidium produced as a commercial product for biological control. Molecular mechanisms of the effects of this pathogen and other invertebrate microsporidia on host cells remain uncharacterized. Previously, we immunolocalized P. locustae hexokinase in nuclei of Locusta migratoria infected adipocytes. Here, the microsporidian protein was expressed in the yeast Pichia pastoris and in lepidopteran Sf9 cells. During heterologous expression, P. locustae hexokinase was accumulated in the nuclei of insect cells but not in yeast cell nuclei. This confirms nuclear localization of hexokinase secreted by microsporidia into infected host cells and suggests convenient model for its further study.

  18. Accumulation of PrP-Sc in hemal nodes of naturally and experimentally scrapie-infected sheep

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Classical scrapie is a naturally occurring fatal disease of sheep and goats which is caused by prions, a novel class of infectious agent. Infection is accompanied by accumulation of abnormal isoforms of the prion protein (PrP-Sc) in certain neural and lymphoid tissues. Hemal nodes, which are unique ...

  19. Loss of lysosomal membrane protein NCU-G1 in mice results in spontaneous liver fibrosis with accumulation of lipofuscin and iron in Kupffer cells

    PubMed Central

    Kong, Xiang Y.; Nesset, Cecilie Kasi; Damme, Markus; Løberg, Else-Marit; Lübke, Torben; Mæhlen, Jan; Andersson, Kristin B.; Lorenzo, Petra I.; Roos, Norbert; Thoresen, G. Hege; Rustan, Arild C.; Kase, Eili T.; Eskild, Winnie

    2014-01-01

    Human kidney predominant protein, NCU-G1, is a highly conserved protein with an unknown biological function. Initially described as a nuclear protein, it was later shown to be a bona fide lysosomal integral membrane protein. To gain insight into the physiological function of NCU-G1, mice with no detectable expression of this gene were created using a gene-trap strategy, and Ncu-g1gt/gt mice were successfully characterized. Lysosomal disorders are mainly caused by lack of or malfunctioning of proteins in the endosomal-lysosomal pathway. The clinical symptoms vary, but often include liver dysfunction. Persistent liver damage activates fibrogenesis and, if unremedied, eventually leads to liver fibrosis/cirrhosis and death. We demonstrate that the disruption of Ncu-g1 results in spontaneous liver fibrosis in mice as the predominant phenotype. Evidence for an increased rate of hepatic cell death, oxidative stress and active fibrogenesis were detected in Ncu-g1gt/gt liver. In addition to collagen deposition, microscopic examination of liver sections revealed accumulation of autofluorescent lipofuscin and iron in Ncu-g1gt/gt Kupffer cells. Because only a few transgenic mouse models have been identified with chronic liver injury and spontaneous liver fibrosis development, we propose that the Ncu-g1gt/gt mouse could be a valuable new tool in the development of novel treatments for the attenuation of fibrosis due to chronic liver damage. PMID:24487409

  20. Evolution of the capsid protein genes of foot-and-mouth disease virus: antigenic variation without accumulation of amino acid substitutions over six decades.

    PubMed Central

    Martínez, M A; Dopazo, J; Hernández, J; Mateu, M G; Sobrino, F; Domingo, E; Knowles, N J

    1992-01-01

    The genetic diversification of foot-and-mouth disease virus (FMDV) of serotype C over a 6-decade period was studied by comparing nucleotide sequences of the capsid protein-coding regions of viruses isolated in Europe, South America, and The Philippines. Phylogenetic trees were derived for VP1 and P1 (VP1, VP2, VP3, and VP4) RNAs by using the least-squares method. Confidence intervals of the derived phylogeny (significance levels of nodes and standard deviations of branch lengths) were placed by application of the bootstrap resampling method. These procedures defined six highly significant major evolutionary lineages and a complex network of sublines for the isolates from South America. In contrast, European isolates are considerably more homogeneous, probably because of the vaccine origin of several of them. The phylogenetic analysis suggests that FMDV CGC Ger/26 (one of the earliest FMDV isolates available) belonged to an evolutionary line which is now apparently extinct. Attempts to date the origin (ancestor) of the FMDVs analyzed met with considerable uncertainty, mainly owing to the stasis noted in European viruses. Remarkably, the evolution of the capsid genes of FMDV was essentially associated with linear accumulation of silent mutations but continuous accumulation of amino acid substitutions was not observed. Thus, the antigenic variation attained by FMDV type C over 6 decades was due to fluctuations among limited combinations of amino acid residues without net accumulation of amino acid replacements over time. PMID:1316467

  1. Glycogen synthase kinase-3beta (GSK3beta) negatively regulates PTTG1/human securin protein stability, and GSK3beta inactivation correlates with securin accumulation in breast tumors.

    PubMed

    Mora-Santos, Mar; Limón-Mortés, M Cristina; Giráldez, Servando; Herrero-Ruiz, Joaquín; Sáez, Carmen; Japón, Miguel Á; Tortolero, Maria; Romero, Francisco

    2011-08-26

    PTTG1, also known as securin, is an inactivating partner of separase, the major effector for chromosome segregation during mitosis. At the metaphase-to-anaphase transition, securin is targeted for proteasomal destruction by the anaphase-promoting complex or cyclosome, allowing activation of separase. In addition, securin is overexpressed in metastatic or genomically instable tumors, suggesting a relevant role for securin in tumor progression. Stability of securin is regulated by phosphorylation; some phosphorylated forms are degraded out of mitosis, by the action of the SKP1-CUL1-F-box protein (SCF) complex. The kinases targeting securin for proteolysis have not been identified, and mechanistic insight into the cause of securin accumulation in human cancers is lacking. Here, we demonstrate that glycogen synthase kinase-3β (GSK3β) phosphorylates securin to promote its proteolysis via SCF(βTrCP) E3 ubiquitin ligase. Importantly, a strong correlation between securin accumulation and GSK3β inactivation was observed in breast cancer tissues, indicating that GSK3β inactivation may account for securin accumulation in breast cancers.

  2. Pathogenesis of scrapie in ARQ/ARQ sheep after subcutaneous infection: effect of lymphadenectomy and immune cell subset changes in relation to prion protein accumulation.

    PubMed

    Chianini, F; Sisó, S; Ricci, E; Eaton, S L; Finlayson, J; Pang, Y; Hamilton, S; Steele, P; Reid, H W; Cantile, C; Sales, J; Jeffrey, M; Dagleish, M P; González, L

    2013-04-15

    It is well established that the infectious agent of scrapie can replicate in the lymphoreticular system (LRS). However, the effects of removal of LRS target tissues on the pathogenesis of the infection and the accumulation of disease-associated prion protein (PrP(d)) in LRS tissues on specific immune cell subsets are poorly understood aspects. To address these questions 16 ARQ/ARQ sheep were subcutaneously inoculated in the drainage area of the prefemoral lymph node with brain homogenate derived from Suffolk sheep naturally infected with scrapie. Fourteen sheep were then subjected to either early (14-17 days post-inoculation [dpi]) or late (175-201 dpi) lymphadenectomy and culled at preclinical or clinical stages of infection. Neither late nor even early lymphadenectomy prevented infection or had any effect on the accumulation of PrP(d) in the LRS or CNS suggesting a rapid organic dissemination of the infectious agent after inoculation. Lymph nodes from eight scrapie inoculated sheep selected on the basis of the amount of PrP(d) in their LRS tissues (negative, low or high) were examined for six different immune cell markers. The PrP(d) negative lymph nodes from two sheep with no evidence of scrapie infection showed lower numbers of cluster of determination (CD) 21 positive cells than PrP(d) positive nodes, irrespective of their location (hind leg or head). However, quantitative differences in the expression of this marker were not detected when comparing lymph nodes with low and high levels of PrP(d) accumulation, suggesting that proliferation of CD21 positive cells is related to scrapie infection, but not directly linked to the magnitude of PrP(d) accumulation. An additional observation of the study was that sheep that were methionin-threonine at codon 112 of the prion protein gene showed lower attack rates than methionine homozygotes (67% and 100%, respectively) and also generally lower levels of PrP(d) accumulation in the LRS and brain and increased survival

  3. Tamm-Horsfall protein in recurrent calcium kidney stone formers with positive family history: abnormalities in urinary excretion, molecular structure and function.

    PubMed

    Jaggi, Markus; Nakagawa, Yasushi; Zipperle, Ljerka; Hess, Bernhard

    2007-04-01

    Tamm-Horsfall protein (THP) powerfully inhibits calcium oxalate crystal aggregation, but structurally abnormal THPs from recurrent calcium stone formers may promote crystal aggregation. Therefore, increased urinary excretion of abnormal THP might be of relevance in nephrolithiasis. We studied 44 recurrent idiopathic calcium stone formers with a positive family history of stone disease (RCSF(fam)) and 34 age- and sex-matched healthy controls (C). Twenty-four-hour urinary THP excretion was measured by enzyme linked immunosorbent assay. Structural properties of individually purified THPs were obtained from analysis of elution patterns from a Sepharose 4B column. Sialic acid (SA) contents of native whole 24-h urines, crude salt precipitates of native urines and individually purified THPs were measured. THP function was studied by measuring inhibition of CaOx crystal aggregation in vitro (pH 5.7, 200 mM sodium chloride). Twenty-four-hour urine excretion of THP was higher in RCSF(fam) (44.0 +/- 4.0 mg/day) than in C (30.9 +/- 2.2 mg/day, P = 0.015). Upon salt precipitation and lyophilization, elution from a Sepharose 4B column revealed one major peak (peak A, cross-reacting with polyclonal anti-THP antibody) and a second minor peak (peak B, not cross-reacting). THPs from RCSF(fam) eluted later than those from C (P = 0.021), and maximum width of THP peaks was higher in RCSF(fam )than in C (P = 0.024). SA content was higher in specimens from RCSF(fam) than from C, in native 24-h urines (207.5 +/- 20.4 mg vs. 135.2 +/- 16.1 mg, P = 0.013) as well as in crude salt precipitates of 24-h urines (10.4 +/- 0.5 mg vs. 7.4 +/- 0.9 mg, P = 0.002) and in purified THPs (75.3 +/- 9.3 microg/mg vs. 48.8 +/- 9.8 microg/mg THP, P = 0.043). Finally, inhibition of calcium oxalate monohydrate crystal aggregation by 40 mg/L of THP was lower in RCSF(fam) (6.1 +/- 5.5%, range -62.0 to +84.2%) than in C (24.9 +/- 6.0%, range -39.8 to +82.7%), P = 0.022, and only 25 out of 44 (57%) THPs from RCSF

  4. Lipopolysaccharide-induced neuroinflammation leads to the accumulation of ubiquitinated proteins and increases susceptibility to neurodegeneration induced by proteasome inhibition in rat hippocampus

    PubMed Central

    2012-01-01

    Background Neuroinflammation and protein accumulation are characteristic hallmarks of both normal aging and age-related neurodegenerative diseases. However, the relationship between these factors in neurodegenerative processes is poorly understood. We have previously shown that proteasome inhibition produced higher neurodegeneration in aged than in young rats, suggesting that other additional age-related events could be involved in neurodegeneration. We evaluated the role of lipopolysaccharide (LPS)-induced neuroinflammation as a potential synergic risk factor for hippocampal neurodegeneration induced by proteasome inhibition. Methods Young male Wistar rats were injected with 1 μL of saline or LPS (5 mg/mL) into the hippocampus to evaluate the effect of LPS-induced neuroinflammation on protein homeostasis. The synergic effect of LPS and proteasome inhibition was analyzed in young rats that first received 1 μL of LPS and 24 h later 1 μL (5 mg/mL) of the proteasome inhibitor lactacystin. Animals were sacrificed at different times post-injection and hippocampi isolated and processed for gene expression analysis by real-time polymerase chain reaction; protein expression analysis by western blots; proteasome activity by fluorescence spectroscopy; immunofluorescence analysis by confocal microscopy; and degeneration assay by Fluoro-Jade B staining. Results LPS injection produced the accumulation of ubiquitinated proteins in hippocampal neurons, increased expression of the E2 ubiquitin-conjugating enzyme UB2L6, decreased proteasome activity and increased immunoproteasome content. However, LPS injection was not sufficient to produce neurodegeneration. The combination of neuroinflammation and proteasome inhibition leads to higher neuronal accumulation of ubiquitinated proteins, predominant expression of pro-apoptotic markers and increased neurodegeneration, when compared with LPS or lactacystin (LT) injection alone. Conclusions Our results identify neuroinflammation

  5. Effects of EPA and DHA on lipid droplet accumulation and mRNA abundance of PAT proteins in caprine monocytes.

    PubMed

    Lecchi, Cristina; Invernizzi, Guido; Agazzi, Alessandro; Modina, Silvia; Sartorelli, Paola; Savoini, Giovanni; Ceciliani, Fabrizio

    2013-04-01

    The present study investigated the in vitro effects on caprine monocytes of two ω-3 PUFAs, namely eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on lipid droplet formation, an emerging process of fundamental importance in innate immunity regulation. The mRNA abundance of PAT protein family (PLIN1, PLIN2 and PLIN3), involved in the formation and trafficking of the droplets, was also assessed. The effects of EPA and DHA on monocyte apoptosis were studied as well. The number of lipid droplets per cell was found to be dependent on both type and concentration of fatty acid. ω-3 PUFAs upregulated PLIN3 and PLIN2 gene expression, as well as apoptosis rate. The present findings suggest that PUFA might modify innate immune functions of goat monocytes by interfering with the formation of lipid droplets and by upregulating proteins belonging to PAT protein family.

  6. Tlr4-mutant mice are resistant to acute alcohol-induced sterol-regulatory element binding protein activation and hepatic lipid accumulation.

    PubMed

    Zhang, Zhi-Hui; Liu, Xiao-Qian; Zhang, Cheng; He, Wei; Wang, Hua; Chen, Yuan-Hua; Liu, Xiao-Jing; Chen, Xi; Xu, De-Xiang

    2016-09-15

    Previous studies demonstrated that acute alcohol intoxication caused hepatic lipid accumulation. The present study showed that acute alcohol intoxication caused hepatic lipid accumulation in Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic sterol-regulatory element binding protein (SREBP)-1, a transcription factor regulating fatty acid and triglyceride (TG) synthesis, was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic Fas, Acc, Scd-1 and Dgat-2, the key genes for fatty acid and TG synthesis, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Additional experiment showed that hepatic MyD88 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic NF-κB was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Moreover, hepatic GSH content was reduced and hepatic MDA level was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic CYP2E1 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic p67phox and gp91phox, two NADPH oxidase subunits, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin-trapping agent, protected against alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. In conclusion, Tlr4-mutant mice are resistant to acute alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation.

  7. Tlr4-mutant mice are resistant to acute alcohol-induced sterol-regulatory element binding protein activation and hepatic lipid accumulation

    PubMed Central

    Zhang, Zhi-Hui; Liu, Xiao-Qian; Zhang, Cheng; He, Wei; Wang, Hua; Chen, Yuan-Hua; Liu, Xiao-Jing; Chen, Xi; Xu, De-Xiang

    2016-01-01

    Previous studies demonstrated that acute alcohol intoxication caused hepatic lipid accumulation. The present study showed that acute alcohol intoxication caused hepatic lipid accumulation in Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic sterol-regulatory element binding protein (SREBP)-1, a transcription factor regulating fatty acid and triglyceride (TG) synthesis, was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic Fas, Acc, Scd-1 and Dgat-2, the key genes for fatty acid and TG synthesis, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Additional experiment showed that hepatic MyD88 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic NF-κB was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Moreover, hepatic GSH content was reduced and hepatic MDA level was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic CYP2E1 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic p67phox and gp91phox, two NADPH oxidase subunits, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin-trapping agent, protected against alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. In conclusion, Tlr4-mutant mice are resistant to acute alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. PMID:27627966

  8. Far-infrared radiation protects viability in a cell model of Spinocerebellar Ataxia by preventing polyQ protein accumulation and improving mitochondrial function

    PubMed Central

    Chang, Jui-Chih; Wu, Shey-Lin; Hoel, Fredrik; Cheng, Yu-Shan; Liu, Ko-Hung; Hsieh, Mingli; Hoel, August; Tronstad, Karl Johan; Yan, Kuo-Chia; Hsieh, Ching-Liang; Lin, Wei-Yong; Kuo, Shou-Jen; Su, Shih-Li; Liu, Chin-San

    2016-01-01

    Far infrared radiation (FIR) is currently investigated as a potential therapeutic strategy in various diseases though the mechanism is unknown. Presently, we tested if FIR mediates beneficial effects in a cell model of the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3). SCA3 is caused by a mutation leading to an abnormal polyglutamine expansion (PolyQ) in ataxin-3 protein. The consequent aggregation of mutant ataxin-3 results in disruption of vital cell functions. In this study, neuroblastoma cells (SK-N-SH) was transduced to express either non-pathogenic ataxin-3-26Q or pathogenic ataxin-3-78Q proteins. The cells expressing ataxin-3-78Q demonstrated decreased viability, and increased sensitivity to metabolic stress in the presence rotenone, an inhibitor of mitochondrial respiration. FIR exposure was found to protect against these effects. Moreover, FIR improved mitochondrial respiratory function, which was significantly compromised in ataxin-3-78Q and ataxin-3-26Q expressing cells. This was accompanied by decreased levels of mitochondrial fragmentation in FIR treated cells, as observed by fluorescence microscopy and protein expression analysis. Finally, the expression profile LC3-II, Beclin-1 and p62 suggested that FIR prevent the autophagy inhibiting effects observed in ataxin-3-78Q expressing cells. In summary, our results suggest that FIR have rescuing effects in cells expressing mutated pathogenic ataxin-3, through recovery of mitochondrial function and autophagy. PMID:27469193

  9. Free cholesterol accumulation in macrophage membranes activates Toll-like receptors and p38 mitogen-activated protein kinase and induces cathepsin K.

    PubMed

    Sun, Yu; Ishibashi, Minako; Seimon, Tracie; Lee, Mingsum; Sharma, Sudarshana M; Fitzgerald, Katherine A; Samokhin, Andriy O; Wang, Yibin; Sayers, Scott; Aikawa, Masanori; Jerome, W Gray; Ostrowski, Michael C; Bromme, Dieter; Libby, Peter; Tabas, Ira A; Welch, Carrie L; Tall, Alan R

    2009-02-27

    The molecular events linking lipid accumulation in atherosclerotic plaques to complications such as aneurysm formation and plaque disruption are poorly understood. BALB/c-Apoe(-/-) mice bearing a null mutation in the Npc1 gene display prominent medial erosion and atherothrombosis, whereas their macrophages accumulate free cholesterol in late endosomes and show increased cathepsin K (Ctsk) expression. We now show increased cathepsin K immunostaining and increased cysteinyl proteinase activity using near infrared fluorescence imaging over proximal aortas of Apoe(-/-), Npc1(-/-) mice. In mechanistic studies, cholesterol loading of macrophage plasma membranes (cyclodextrin-cholesterol) or endosomal system (AcLDL+U18666A or Npc1 null mutation) activated Toll-like receptor (TLR) signaling, leading to sustained phosphorylation of p38 mitogen-activated protein kinase and induction of p38 targets, including Ctsk, S100a8, Mmp8, and Mmp14. Studies in macrophages from knockout mice showed major roles for TLR4, following plasma membrane cholesterol loading, and for TLR3, after late endosomal loading. TLR signaling via p38 led to phosphorylation and activation of the transcription factor Microphthalmia transcription factor, acting at E-box elements in the Ctsk promoter. These studies suggest that free cholesterol enrichment of either plasma or endosomal membranes in macrophages leads to activation of signaling via various TLRs, prolonged p38 mitogen-activated protein kinase activation, and induction of Mmps, Ctsk, and S100a8, potentially contributing to plaque complications.

  10. Accumulation of Epstein-Barr virus (EBV) BMRF1 protein EA-D during latent EBV activation of Burkitt's lymphoma cell line Raji.

    PubMed

    Ohashi, Makoto; Horie, Kazutaka; Hoshikawa, Yoshiko; Nagata, Keiko; Osaki, Mistuhiko; Ito, Hisao; Sairenji, Takeshi

    2007-02-01

    As a new model to elucidate molecular mechanisms in Epstein-Barr virus (EBV) activation, we tested the tetracycline-inducible (Tet-On)/BZLF1-oriP plasmid system in Raji cells. Cells transfected with this Tet-On plasmid did not activate EBV by doxycycline and surprisingly EBV latency was disrupted with large amounts of BMRF1 protein (EA-D) being accumulated in the cells. Brilliant EA-D fluorescence was markedly condensed in small sized cells, intra-cellular vesicles, and extra-cellular particles. Scanning electron microscopy demonstrated the extra-cellular particles to be covered with a membrane. EA-D molecules of 58, 50, 48, and 44kDa were expressed in the cells. The high (58 and 50kDa) and low (48 and 44kDa) EA-D molecules appeared in the early and late stages, respectively. Low EA-D molecules were detected mostly in EA-D positive cells separated into the heaviest density layer of a discontinuous Percoll gradient. Such molecules could be created from high EA-D molecules by protein phosphatase treatment. The EA-D molecules that appeared similar were detected in EBV-activated P3HR-1 and Akata cells. Several hypotheses concerning the accumulation of EA-D molecules of various polymorphic forms and their phosphorylation/dephosphorylation in this model system are presented, with possible biological and clinical relevance.

  11. Methyl jasmonate induces ATP biosynthesis deficiency and accumulation of proteins related to secondary metabolism in Catharanthus roseus (L.) G. hairy roots.

    PubMed

    Ruiz-May, Eliel; De-la-Peña, Clelia; Galaz-Ávalos, Rosa M; Lei, Zhentian; Watson, Bonnie S; Sumner, Lloyd W; Loyola-Vargas, Víctor M

    2011-08-01

    Jasmonates are specific signal molecules in plants that are involved in a diverse set of physiological and developmental processes. However, methyl jasmonate (MeJA) has been shown to have a negative effect on root growth and, so far, the biochemical mechanism for this is unknown. Using Catharanthus roseus hairy roots, we were able to observe the effect of MeJA on growth inhibition, cell disorganization and cell death of the root cap. Hairy roots treated with MeJA induced the perturbation of mitochondrial membrane integrity and a diminution in ATP biosynthesis. Furthermore, several proteins were identified that were involved in energy and secondary metabolism; the changes in accumulation of these proteins were observed with 100 μM MeJA. In conclusion, our results suggest that a switch of the metabolic fate of hairy roots in response to MeJA could cause an increase in the accumulation of secondary metabolites. This is likely to have important consequences in the production of specific alkaloids important for the pharmaceutical industry.

  12. ERK Signaling Pathway Is Involved in HPV-16 E6 but not E7 Oncoprotein-Induced HIF-1α Protein Accumulation in NSCLC Cells.

    PubMed

    Liu, Fei; Lin, Bihua; Liu, Xin; Zhang, Wenzhang; Zhang, Erying; Hu, Liang; Ma, Yuefan; Li, Xiangyong; Tang, Xudong

    2016-01-01

    Extracellular signal-regulated kinase (ERK)1/2 signaling pathway plays a critical role in regulating tumor angiogenesis. Our previous studies have demonstrated that HPV-16 oncoproteins enhanced hypoxia-inducible factor-1α (HIF-1α) protein accumulation and vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) expression in non-small cell lung cancer (NSCLC) cells, thus contributing to angiogenesis. In this study, we further investigated the role of ERK1/2 signaling pathway in HPV-16 oncoprotein-induced HIF-1α, VEGF, and IL-8 expression and in vitro angiogenesis in NSCLC cells. Our results showed that HPV-16 E6 and HPV-16 E7 oncoproteins promoted the activation of ERK1/2 signaling pathway in A549 and NCI-H460 cells. Moreover, PD98059, a specific inhibitor of ERK1/2, blocked in vitro angiogenesis stimulated by HPV-16 E6 but not E7 oncoprotein. Additionally, HIF-1α protein accumulation and VEGF and IL-8 expression in NSCLC cells induced by HPV-16 E6 but not E7 oncoprotein were significantly inhibited by PD98059. Taken together, our results suggest that ERK1/2 signaling pathway is involved in HPV-16 E6 but not E7 oncoprotein-induced HIF-1α, VEGF, and IL-8 expression in NSCLC cells, leading to the enhanced angiogenesis in vitro.

  13. Accumulation-Associated Protein Enhances Staphylococcus epidermidis Biofilm Formation under Dynamic Conditions and Is Required for Infection in a Rat Catheter Model

    PubMed Central

    Schaeffer, Carolyn R.; Woods, Keith M.; Longo, G. Matt; Kiedrowski, Megan R.; Paharik, Alexandra E.; Büttner, Henning; Christner, Martin; Boissy, Robert J.; Horswill, Alexander R.; Rohde, Holger

    2014-01-01

    Biofilm formation is the primary virulence factor of Staphylococcus epidermidis. S. epidermidis biofilms preferentially form on abiotic surfaces and may contain multiple matrix components, including proteins such as accumulation-associated protein (Aap). Following proteolytic cleavage of the A domain, which has been shown to enhance binding to host cells, B domain homotypic interactions support cell accumulation and biofilm formation. To further define the contribution of Aap to biofilm formation and infection, we constructed an aap allelic replacement mutant and an icaADBC aap double mutant. When subjected to fluid shear, strains deficient in Aap production produced significantly less biofilm than Aap-positive strains. To examine the in vivo relevance of our findings, we modified our previously described rat jugular catheter model and validated the importance of immunosuppression and the presence of a foreign body to the establishment of infection. The use of our allelic replacement mutants in the model revealed a significant decrease in bacterial recovery from the catheter and the blood in the absence of Aap, regardless of the production of polysaccharide intercellular adhesin (PIA), a well-characterized, robust matrix molecule. Complementation of the aap mutant with full-length Aap (containing the A domain), but not the B domain alone, increased initial attachment to microtiter plates, as did in trans expression of the A domain in adhesion-deficient Staphylococcus carnosus. These results demonstrate Aap contributes to S. epidermidis infection, which may in part be due to A domain-mediated attachment to abiotic surfaces. PMID:25332125

  14. Defects in the Expression of Chloroplast Proteins Leads to H2O2 Accumulation and Activation of Cyclic Electron Flow around Photosystem I.

    PubMed

    Strand, Deserah D; Livingston, Aaron K; Satoh-Cruz, Mio; Koepke, Tyson; Enlow, Heather M; Fisher, Nicholas; Froehlich, John E; Cruz, Jeffrey A; Minhas, Deepika; Hixson, Kim K; Kohzuma, Kaori; Lipton, Mary; Dhingra, Amit; Kramer, David M

    2016-01-01

    We describe a new member of the class of mutants in Arabidopsis exhibiting high rates of cyclic electron flow around photosystem I (CEF), a light-driven process that produces ATP but not NADPH. High cyclic electron flow 2 (hcef2) shows strongly increased CEF activity through the NADPH dehydrogenase complex (NDH), accompanied by increases in thylakoid proton motive force (pmf), activation of the photoprotective qE response, and the accumulation of H2O2. Surprisingly, hcef2 was mapped to a non-sense mutation in the TADA1 (tRNA adenosine deaminase arginine) locus, coding for a plastid targeted tRNA editing enzyme required for efficient codon recognition. Comparison of protein content from representative thylakoid complexes, the cytochrome bf complex, and the ATP synthase, suggests that inefficient translation of hcef2 leads to compromised complex assembly or stability leading to alterations in stoichiometries of major thylakoid complexes as well as their constituent subunits. Altered subunit stoichiometries for photosystem I, ratios and properties of cytochrome bf hemes, and the decay kinetics of the flash-induced thylakoid electric field suggest that these defect lead to accumulation of H2O2 in hcef2, which we have previously shown leads to activation of NDH-related CEF. We observed similar increases in CEF, as well as increases in H2O2 accumulation, in other translation defective mutants. This suggests that loss of coordination in plastid protein levels lead to imbalances in photosynthetic energy balance that leads to an increase in CEF. These results taken together with a large body of previous observations, support a general model in which processes that lead to imbalances in chloroplast energetics result in the production of H2O2, which in turn activates CEF. This activation could be from either H2O2 acting as a redox signal, or by a secondary effect from H2O2 inducing a deficit in ATP.

  15. Defects in the Expression of Chloroplast Proteins Leads to H2O2 Accumulation and Activation of Cyclic Electron Flow around Photosystem I

    PubMed Central

    Strand, Deserah D.; Livingston, Aaron K.; Satoh-Cruz, Mio; Koepke, Tyson; Enlow, Heather M.; Fisher, Nicholas; Froehlich, John E.; Cruz, Jeffrey A.; Minhas, Deepika; Hixson, Kim K.; Kohzuma, Kaori; Lipton, Mary; Dhingra, Amit; Kramer, David M.

    2017-01-01

    We describe a new member of the class of mutants in Arabidopsis exhibiting high rates of cyclic electron flow around photosystem I (CEF), a light-driven process that produces ATP but not NADPH. High cyclic electron flow 2 (hcef2) shows strongly increased CEF activity through the NADPH dehydrogenase complex (NDH), accompanied by increases in thylakoid proton motive force (pmf), activation of the photoprotective qE response, and the accumulation of H2O2. Surprisingly, hcef2 was mapped to a non-sense mutation in the TADA1 (tRNA adenosine deaminase arginine) locus, coding for a plastid targeted tRNA editing enzyme required for efficient codon recognition. Comparison of protein content from representative thylakoid complexes, the cytochrome bf complex, and the ATP synthase, suggests that inefficient translation of hcef2 leads to compromised complex assembly or stability leading to alterations in stoichiometries of major thylakoid complexes as well as their constituent subunits. Altered subunit stoichiometries for photosystem I, ratios and properties of cytochrome bf hemes, and the decay kinetics of the flash-induced thylakoid electric field suggest that these defect lead to accumulation of H2O2 in hcef2, which we have previously shown leads to activation of NDH-related CEF. We observed similar increases in CEF, as well as increases in H2O2 accumulation, in other translation defective mutants. This suggests that loss of coordination in plastid protein levels lead to imbalances in photosynthetic energy balance that leads to an increase in CEF. These results taken together with a large body of previous observations, support a general model in which processes that lead to imbalances in chloroplast energetics result in the production of H2O2, which in turn activates CEF. This activation could be from either H2O2 acting as a redox signal, or by a secondary effect from H2O2 inducing a deficit in ATP. PMID:28133462

  16. Modified bean seed protein phaseolin did not accumulate stably in transgenic tobacco seeds after methionine enhancement mutations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The major seed storage protein phaseolin of common bean (Phaseolus vulgaris L.) is deficient in methionine, an essential amino acid for human and animal health. To improve the nutritional quality of common bean, we designed methionine enhancement of phaseolin based on the three dimensional structure...

  17. Heat-induced accumulation of protein synthesis elongation factor 1A indicates an important role in heat tolerance in potato

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Heat stress substantially reduces crop productivity worldwide, and will become more severe due to global warming. Identification of proteins involved in heat stress response may help develop varieties for heat tolerance. Eukaryotic elongation factor 1A (eEF1A) is a cytosolic, multifunctional protei...

  18. Protein Profiles for Muscle Development and Intramuscular Fat Accumulation at Different Post-Hatching Ages in Chickens

    PubMed Central

    Liu, Ranran; Zhao, Guiping; Zheng, Maiqing; Cui, Huanxian; Li, Qinghe; Song, Jiao; Wang, Jie; Wen, Jie

    2016-01-01

    Muscle development and growth influences the efficiency of poultry meat production, and is closely related to deposition of intramuscular fat (IMF), which is crucial in meat quality. To clarify the molecular mechanisms underlying muscle development and IMF deposition in chickens, protein expression profiles were examined in the breast muscle of Beijing-You chickens at ages 1, 56, 98 and 140 days, using isobaric tags for relative and absolute quantification (iTRAQ). Two hundred and four of 494 proteins were expressed differentially. The expression profile at day 1 differed greatly from those at day 56, 98 and 140. KEGG pathway analysis of differential protein expression from pair-wise comparisons (day 1 vs. 56; 56 vs. 98; 98 vs. 140), showed that the fatty acid degradation pathway was more active during the stage from day 1 to 56 than at other periods. This was consistent with the change in IMF content, which was highest at day 1 and declined dramatically thereafter. When muscle growth was most rapid (days 56–98), pathways involved in muscle development were dominant, including hypertrophic cardiomyopathy, dilated cardiomyopathy, cardiac muscle contraction, tight junctions and focal adhesion. In contrast with hatchlings, the fatty acid degradation pathway was downregulated from day 98 to 140, which was consistent with the period for IMF deposition following rapid muscle growth. Changes in some key specific proteins, including fast skeletal muscle troponin T isoform, aldehyde dehydrogenase 1A1 and apolipoprotein A1, were verified by Western blotting, and could be potential biomarkers for IMF deposition in chickens. Protein–protein interaction networks showed that ribosome-related functional modules were clustered in all three stages. However, the functional module involved in the metabolic pathway was only clustered in the first stage (day 1 vs. 56). This study improves our understanding of the molecular mechanisms underlying muscle development and IMF deposition

  19. gar2 is a nucleolar protein from Schizosaccharomyces pombe required for 18S rRNA and 40S ribosomal subunit accumulation.

    PubMed Central

    Gulli, M P; Girard, J P; Zabetakis, D; Lapeyre, B; Melese, T; Caizergues-Ferrer, M

    1995-01-01

    Several nucleolar proteins, such as nucleolin, NOP1/fibrillarin, SSB1, NSR1 and GAR1 share a common glycine and arginine rich structural motif called the GAR domain. To identify novel nucleolar proteins from fission yeast we screened Schizosaccharomyces pombe genomic DNA libraries with a probe encompassing the GAR structural motif. Here we report the identification and characterization of a S.pombe gene coding for a novel nucleolar protein, designated gar2. The structure of the fission yeast gar2 is reminiscent of that of nucleolin from vertebrates and NSR1 from Saccharomyces cerevisiae. In addition, like these proteins, gar2 has a nucleolar localisation. The disruption of the gar2+ gene affects normal cell growth, leads to an accumulation of 35S pre-rRNA and a decrease of mature 18S rRNA steady state levels. Moreover, ribosomal profiles of the mutant show an increase of free 60S ribosomal subunits and an absence of free 40S ribosomal subunits. gar2 is able to rescue a S.cerevisiae mutant lacking NSR1, thus establishing gar2 as a functional homolog of NSR1. We propose that gar2 helps the assembly of pre-ribosomal particles containing 18S rRNA. Images PMID:7596817

  20. Effect of ethephon on protein degradation and the accumulation of pathogensis-related (PR) proteins in tomato leaf discs. [Lycopersicon esculentum

    SciTech Connect

    Vera, P.; Conejero, V. )

    1990-01-01

    The effect of ethephon (2-chloroetylphosphonic acid) on the degradation of proteins and on the induction of Lycopersicon esculentum pathogenesis-related (PR) proteins was studied in tomato leaf discs. The rate of ribulose, -1,5-bisphosphate carboxylase/oxygenase (Rubisco) degradation was maximal in discs after 48 hours of incubation with 1 millimolar ethephon, leading to complete disappearance of Rubisco after 96 hours. This effect was correlated with an increase in PR protein synthesis and the induction of the previously reported alkaline proteolytic enzyme PR-P69. In vivo pulse-chase experiments demonstrated that ethephon not only affected Rubisco content but that of many other {sup 35}S-labeled proteins as well, indicating that ethylene activates a general and nonspecific mechanism of protein degradation. This effect was partially inhibited in vivo by the action of pCMB, a selective inhibitor of cysteine-proteinases such as P69. These data reinforce the hypothesis that P69 and perhaps other PR proteins are involved in the mechanism of accelerated protein degradation activated by ethylene.

  1. Effect of NO/sub 2/ inhalation and vitamin C deficiency on protein and lipid accumulation in the lung

    SciTech Connect

    Selgrade, M.K.; Mole, M.L.; Miller, F.J.; Hatch, G.E.; Gardner, D.E.; Hu, P.C

    1981-12-01

    Vitamin C-deficient and normal guinea pigs were exposed to various concentrations of NO/sub 2/ or air, and lavage fluid was obtained and analyzed for protein and lipid content. Exposure of normal animals to 752, 1880, 5640, or 9400 ..mu..g NO/sub 2//m/sup 3/ (0.4, 1.0, 3.0, or 5.0 ppm) for 72 hr did not alter the protein or lipid content of lung lavage fluid. However, exposure of vitamin C-deficient animals to the same concentrations of NO/sub 2/ caused marked increases in lavage proteins and lipids at all but the 752 ..mu..g/m/sup 3/ (0.4 ppm) level. At 9400 ..mu..g NO/sub 2//m/sup 3/ (5.0 ppm), 50% of the exposed C-deficient animals died, and pathologic study of the lungs showed proteinaceous edema fluid in the alveoli. Lungs from air-exposed animals and normal animals exposed to NO/sub 2/ appeared healthy. No effects were seen at 752 ..mu..g NO/sub 2/ (0.4 ppm) in either normal or deficient animals even when the time of exposure was extended to 1 week. At 9400 ..mu..g NO/sub 2//m/sup 3/ (5 ppm) effects could be seen in vitamin C-deficient animals even when the exposure period was shortened to 3 hr. Assessment of protein and lipid content of lavage fluid provided a sensitive method for determining subtle changes in the lung following NO/sub 2/ exposure.

  2. Dickkopf-related protein 1 (Dkk1) regulates the accumulation and function of myeloid derived suppressor cells in cancer.

    PubMed

    D'Amico, Lucia; Mahajan, Sahil; Capietto, Aude-Hélène; Yang, Zhengfeng; Zamani, Ali; Ricci, Biancamaria; Bumpass, David B; Meyer, Melissa; Su, Xinming; Wang-Gillam, Andrea; Weilbaecher, Katherine; Stewart, Sheila A; DeNardo, David G; Faccio, Roberta

    2016-05-02

    Tumor-stroma interactions contribute to tumorigenesis. Tumor cells can educate the stroma at primary and distant sites to facilitate the recruitment of heterogeneous populations of immature myeloid cells, known as myeloid-derived suppressor cells (MDSCs). MDSCs suppress T cell responses and promote tumor proliferation. One outstanding question is how the local and distant stroma modulate MDSCs during tumor progression. Down-regulation of β-catenin is critical for MDSC accumulation and immune suppressive functions in mice and humans. Here, we demonstrate that stroma-derived Dickkopf-1 (Dkk1) targets β-catenin in MDSCs, thus exerting immune suppressive effects during tumor progression. Mice bearing extraskeletal tumors show significantly elevated levels of Dkk1 in bone microenvironment relative to tumor site. Strikingly, Dkk1 neutralization decreases tumor growth and MDSC numbers by rescuing β-catenin in these cells and restores T cell recruitment at the tumor site. Recombinant Dkk1 suppresses β-catenin target genes in MDSCs from mice and humans and anti-Dkk1 loses its antitumor effects in mice lacking β-catenin in myeloid cells or after depletion of MDSCs, demonstrating that Dkk1 directly targets MDSCs. Furthermore, we find a correlation between CD15(+) myeloid cells and Dkk1 in pancreatic cancer patients. We establish a novel immunomodulatory role for Dkk1 in regulating tumor-induced immune suppression via targeting β-catenin in MDSCs.

  3. Vestibular abnormalities in congenital disorders.

    PubMed

    Sando, I; Orita, Y; Miura, M; Balaban, C D

    2001-10-01

    This paper reviews the histopathologic features of vestibular abnormalities in congenital disorders affecting the inner ear, based upon a comprehensive literature survey and a review of cases in our temporal bone collection. The review proceeds in three systematic steps. First, we surveyed associated diseases with the major phenotypic features of congenital abnormalities of the inner ear (including the internal auditory canal and otic capsule). Second, the vestibular anomalies are examined specifically. Finally, the anomalies are discussed from a developmental perspective. Among vestibular anomalies, a hypoplastic endolymphatic duct and sac are observed most frequently. Anomalies of the semicircular canals are also often observed. From embryological and clinical viewpoints, many of these resemble the structural features from fetal stages and appear to be associated with vestibular dysfunction. It is expected that progress in genetic analysis and accumulation of temporal bone specimens with vestibular abnormalities in congenital diseases will provide crucial information not only for pathology of those diseases, but also for genetic factors that are responsible for the specific vestibular abnormalities.

  4. Lon-Mediated Proteolysis of the FeoC Protein Prevents Salmonella enterica from Accumulating the Fe(II) Transporter FeoB under High-Oxygen Conditions

    PubMed Central

    Kim, Hyunkeun; Lee, Hwiseop

    2014-01-01

    The Salmonella Feo system consists of the FeoA, FeoB, and FeoC proteins and mediates ferrous iron [Fe(II)] import. FeoB is an inner membrane protein that, along with contributions from two small hydrophilic proteins, FeoA and FeoC, transports Fe(II). We previously reported that FeoC binds to and protects the FeoB transporter from FtsH-mediated proteolysis. In the present study, we report proteolytic regulation of FeoC that occurs in an oxygen-dependent fashion. While relatively stable under low-oxygen conditions, FeoC was rapidly degraded by the Lon protease under high-oxygen conditions. The putative Fe-S cluster of FeoC seemed to function as an oxygen sensor to control FeoC stability, as evidenced by the finding that mutation of the putative Fe-S cluster-binding site greatly increased FeoC stability under high-oxygen conditions. Salmonella ectopically expressing the feoB and feoC genes was able to accumulate FeoB and FeoC only under low-oxygen conditions, suggesting that FeoC proteolysis prevents Salmonella from accumulating the FeoB transporter under high-oxygen conditions. Finally, we propose that Lon-mediated FeoC proteolysis followed by FtsH-mediated FeoB proteolysis helps Salmonella to avoid uncontrolled Fe(II) uptake during the radical environmental changes encountered when shifting from low-iron anaerobic conditions to high-iron aerobic conditions. PMID:25313398

  5. Acetylation of TUG protein promotes the accumulation of GLUT4 glucose transporters in an insulin-responsive intracellular compartment.

    PubMed

    Belman, Jonathan P; Bian, Rachel R; Habtemichael, Estifanos N; Li, Don T; Jurczak, Michael J; Alcázar-Román, Abel; McNally, Leah J; Shulman, Gerald I; Bogan, Jonathan S

    2015-02-13

    Insulin causes the exocytic translocation of GLUT4 glucose transporters to stimulate glucose uptake in fat and muscle. Previous results support a model in which TUG traps GLUT4 in intracellular, insulin-responsive vesicles termed GLUT4 storage vesicles (GSVs). Insulin triggers TUG cleavage to release the GSVs; GLUT4 then recycles through endosomes during ongoing insulin exposure. The TUG C terminus binds a GSV anchoring site comprising Golgin-160 and possibly other proteins. Here, we report that the TUG C terminus is acetylated. The TUG C-terminal peptide bound the Golgin-160-associated protein, ACBD3 (acyl-CoA-binding domain-containing 3), and acetylation reduced binding of TUG to ACBD3 but not to Golgin-160. Mutation of the acetylated residues impaired insulin-responsive GLUT4 trafficking in 3T3-L1 adipocytes. ACBD3 overexpression enhanced the translocation of GSV cargos, GLUT4 and insulin-regulated aminopeptidase (IRAP), and ACBD3 was required for intracellular retention of these cargos in unstimulated cells. Sirtuin 2 (SIRT2), a NAD(+)-dependent deacetylase, bound TUG and deacetylated the TUG peptide. SIRT2 overexpression reduced TUG acetylation and redistributed GLUT4 and IRAP to the plasma membrane in 3T3-L1 adipocytes. Mutation of the acetylated residues in TUG abrogated these effects. In mice, SIRT2 deletion increased TUG acetylation and proteolytic processing. During glucose tolerance tests, glucose disposal was enhanced in SIRT2 knock-out mice, compared with wild type controls, without any effect on insulin concentrations. Together, these data support a model in which TUG acetylation modulates its interaction with Golgi matrix proteins and is regulated by SIRT2. Moreover, acetylation of TUG enhances its function to trap GSVs within unstimulated cells and enhances insulin-stimulated glucose uptake.

  6. Urine - abnormal color

    MedlinePlus

    ... medlineplus.gov/ency/article/003139.htm Urine - abnormal color To use the sharing features on this page, please enable JavaScript. The usual color of urine is straw-yellow. Abnormally colored urine ...

  7. Tooth - abnormal colors

    MedlinePlus

    ... medlineplus.gov/ency/article/003065.htm Tooth - abnormal colors To use the sharing features on this page, please enable JavaScript. Abnormal tooth color is any color other than white to yellowish- ...

  8. Abnormal Head Position

    MedlinePlus

    ... cause. Can a longstanding head turn lead to any permanent problems? Yes, a significant abnormal head posture could cause permanent ... occipitocervical synostosis and unilateral hearing loss. Are there any ... postures? Yes. Abnormal head postures can usually be improved depending ...

  9. Skeletal limb abnormalities

    MedlinePlus

    ... medlineplus.gov/ency/article/003170.htm Skeletal limb abnormalities To use the sharing features on this page, please enable JavaScript. Skeletal limb abnormalities refers to a variety of bone structure problems ...

  10. Abnormal Uterine Bleeding FAQ

    MedlinePlus

    ... PROBLEMS Abnormal Uterine Bleeding • What is a normal menstrual cycle? • When is bleeding abnormal? • At what ages is ... treat abnormal bleeding? •Glossary What is a normal menstrual cycle? The normal length of the menstrual cycle is ...

  11. Salidroside stimulates the accumulation of HIF-1α protein resulted in the induction of EPO expression: a signaling via blocking the degradation pathway in kidney and liver cells.

    PubMed

    Zheng, Ken Yu-Zhong; Zhang, Zhen-Xia; Guo, Ava Jiang-Yang; Bi, Cathy Wen-Chuang; Zhu, Kevin Yue; Xu, Sherry Li; Zhan, Janis Ya-Xian; Lau, David Tai-Wei; Dong, Tina Ting-Xia; Choi, Roy Chi-Yan; Tsim, Karl Wah-Keung

    2012-03-15

    Rhodiolae Crenulatae Radix et Rhizoma (Rhodiola), the root and rhizome of Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba, has been used as a traditional Chinese medicine (TCM) to increase the body resistance to mountain sickness in preventing hypoxia; however, the functional ingredient responsible for this adaptogenic effect has not been revealed. Here, we have identified salidroside, a glycoside predominantly found in Rhodiola, is the chemical in providing such anti-hypoxia effect. Cultured human embryonic kidney fibroblast (HEK293T) and human hepatocellular carcinoma (HepG2) were used to reveal the mechanism of this hematopoietic function mediated by salidroside. The application of salidroside in cultures induced the expression of erythropoietin (EPO) mRNA from its transcription regulatory element hypoxia response element (HRE), located on EPO gene. The application of salidroside stimulated the accumulation of hypoxia-inducible factor-1α (HIF-1α) protein, but not HIF-2α protein: the salidroside-induced HIF-1α protein was via the reduction of HIF-1α degradation but not the mRNA induction. The increased HIF-1α could account for the activation of EPO gene. These results supported the notion that hematopoietic function of Rhodiola was triggered, at least partially, by salidroside.

  12. Accumulation and altered localization of telomere-associated protein TRF2 in immortally transformed and tumor-derived human breast cells

    SciTech Connect

    Nijjar, Tarlochan; Bassett, Ekaterina; Garbe, James; Takenaka, Yasuhiro; Stampfer, Martha R.; Gilley, David; Yaswen, Paul

    2004-12-23

    We have used cultured human mammary epithelial cells (HMEC) and breast tumor-derived lines to gain information on defects that occur during breast cancer progression. HMEC immortalized by a variety of agents (the chemical carcinogen benzo(a)pyrene, oncogenes c-myc and ZNF217, and/or dominant negative p53 genetic suppressor element GSE22) displayed marked up regulation (10-15 fold) of the telomere binding protein, TRF2. Up-regulation of TRF2 protein was apparently due to differences in post-transcriptional regulation, as mRNA levels remained comparable in finite life span and immortal HMEC. TRF2 protein was not up-regulated by the oncogenic agents alone in the absence of immortalization, nor by expression of exogenously introduced hTERT genes. We found TRF2 levels to be at least 2-fold higher than in control cells in 11/15 breast tumor cell lines, suggesting that elevated TRF2 levels are a frequent occurrence during the transformation of breast tumor cells in vivo. The dispersed distribution of TRF2 throughout the nuclei in some immortalized and tumor-derived cells indicated that not all the TRF2 was associated with telomeres in these cells. The process responsible for accumulation of TRF2 in immortalized HMEC and breast tumor-derived cell lines may promote tumorigenesis by contributing to the cells ability to maintain an indefinite life span.

  13. Zn deficiency in Brassica napus induces Mo and Mn accumulation associated with chloroplast proteins variation without Zn remobilization.

    PubMed

    Billard, Vincent; Maillard, Anne; Garnica, Maria; Cruz, Florence; Garcia-Mina, José-Maria; Yvin, Jean-Claude; Ourry, Alain; Etienne, Philippe

    2015-01-01

    The importance of zinc (Zn) has been of little concern in human nutrition despite a strong decrease of this element in crops since the rise of high yielding varieties. For better food quality, Zn biofortification can be used, but will be optimal only if mechanisms governing Zn management are better known. Using Zn deficiency, we are able to demonstrate that Zn is not remobilized in Brassica napus (B. napus). Thus, remobilization processes should not be targeted by biofortification strategies. This study also complemented previous work by investigating leaf responses to Zn deficiency, especially from proteomic and ionomic points of view, showing for example, an increase in Manganese (Mn) content and of the Mn-dependent protein, Oxygen Evolving Enhancer.

  14. Copper-Deficiency in Brassica napus Induces Copper Remobilization, Molybdenum Accumulation and Modification of the Expression of Chloroplastic Proteins

    PubMed Central

    Billard, Vincent; Ourry, Alain; Maillard, Anne; Garnica, Maria; Coquet, Laurent; Jouenne, Thierry; Cruz, Florence; Garcia-Mina, José-Maria; Yvin, Jean-Claude; Etienne, Philippe

    2014-01-01

    During the last 40 years, crop breeding has strongly increased yields but has had adverse effects on the content of micronutrients, such as Fe, Mg, Zn and Cu, in edible products despite their sufficient supply in most soils. This suggests that micronutrient remobilization to edible tissues has been negatively selected. As a consequence, the aim of this work was to quantify the remobilization of Cu in leaves of Brassica napus L. during Cu deficiency and to identify the main metabolic processes that were affected so that improvements can be achieved in the future. While Cu deficiency reduced oilseed rape growth by less than 19% compared to control plants, Cu content in old leaves decreased by 61.4%, thus demonstrating a remobilization process between leaves. Cu deficiency also triggered an increase in Cu transporter expression in roots (COPT2) and leaves (HMA1), and more surprisingly, the induction of the MOT1 gene encoding a molybdenum transporter associated with a strong increase in molybdenum (Mo) uptake. Proteomic analysis of leaves revealed 33 proteins differentially regulated by Cu deficiency, among which more than half were located in chloroplasts. Eleven differentially expressed proteins are known to require Cu for their synthesis and/or activity. Enzymes that were located directly upstream or downstream of Cu-dependent enzymes were also differentially expressed. The overall results are then discussed in relation to remobilization of Cu, the interaction between Mo and Cu that occurs through the synthesis pathway of Mo cofactor, and finally their putative regulation within the Calvin cycle and the chloroplastic electron transport chain. PMID:25333918

  15. Copper-deficiency in Brassica napus induces copper remobilization, molybdenum accumulation and modification of the expression of chloroplastic proteins.

    PubMed

    Billard, Vincent; Ourry, Alain; Maillard, Anne; Garnica, Maria; Coquet, Laurent; Jouenne, Thierry; Cruz, Florence; Garcia-Mina, José-Maria; Yvin, Jean-Claude; Etienne, Philippe

    2014-01-01

    During the last 40 years, crop breeding has strongly increased yields but has had adverse effects on the content of micronutrients, such as Fe, Mg, Zn and Cu, in edible products despite their sufficient supply in most soils. This suggests that micronutrient remobilization to edible tissues has been negatively selected. As a consequence, the aim of this work was to quantify the remobilization of Cu in leaves of Brassica napus L. during Cu deficiency and to identify the main metabolic processes that were affected so that improvements can be achieved in the future. While Cu deficiency reduced oilseed rape growth by less than 19% compared to control plants, Cu content in old leaves decreased by 61.4%, thus demonstrating a remobilization process between leaves. Cu deficiency also triggered an increase in Cu transporter expression in roots (COPT2) and leaves (HMA1), and more surprisingly, the induction of the MOT1 gene encoding a molybdenum transporter associated with a strong increase in molybdenum (Mo) uptake. Proteomic analysis of leaves revealed 33 proteins differentially regulated by Cu deficiency, among which more than half were located in chloroplasts. Eleven differentially expressed proteins are known to require Cu for their synthesis and/or activity. Enzymes that were located directly upstream or downstream of Cu-dependent enzymes were also differentially expressed. The overall results are then discussed in relation to remobilization of Cu, the interaction between Mo and Cu that occurs through the synthesis pathway of Mo cofactor, and finally their putative regulation within the Calvin cycle and the chloroplastic electron transport chain.

  16. Nuclear accumulation of Yes-Associated Protein (YAP) maintains the survival of doxorubicin-induced senescent cells by promoting survivin expression.

    PubMed

    Ma, Kai; Xu, Qing; Wang, Shuren; Zhang, Weina; Liu, Mei; Liang, Shufang; Zhu, Hongxia; Xu, Ningzhi

    2016-05-28

    Although chemotherapeutic drugs can induce senescence to prohibit further division of tumor cells, senescence could also promote tumorigenesis mainly through a senescence-associated secretory phenotype. Therefore, senescent tumor cells should be eliminated immediately to prevent drug resistance and recurrence. Here, we used a doxorubicin-induced senescence model to explore the mechanism underlying the survival of therapy-induced senescent cells. After low-dose doxorubicin treatment, tumor cells turned on a senescence program and became large and flattened, increasing their contact area with the extracellular matrix (ECM). Furthermore, Yes-associated protein (YAP) accumulated in the nucleus and YAP activity was increased in doxorubicin-induced senescent cells. Knockdown of YAP increased the sensitivity of cells to low-dose doxorubicin treatment, causing apoptosis rather than senescence. Moreover, the anti-apoptotic gene survivin, a YAP target gene, was overexpressed in senescent cells. Inhibition of survivin could lead to selective elimination of senescent cells through apoptosis. Our study indicates that nuclear accumulation of YAP could promote the survival of senescent cells by increasing survivin expression. Therefore, targeting YAP or survivin might be a new strategy for clearing senescent cancer cells during drug treatment.

  17. Data on amyloid precursor protein accumulation, spontaneous physical activity, and motor learning after traumatic brain injury in the triple-transgenic mouse model of Alzheimer׳s disease.

    PubMed

    Kishimoto, Yasushi; Shishido, Hajime; Sawanishi, Mayumi; Toyota, Yasunori; Ueno, Masaki; Kubota, Takashi; Kirino, Yutaka; Tamiya, Takashi; Kawai, Nobuyuki

    2016-12-01

    This data article contains supporting information regarding the research article entitled "Traumatic brain injury accelerates amyloid-β deposition and impairs spatial learning in the triple-transgenic mouse model of Alzheimer׳s disease" (H. Shishido, Y. Kishimoto, N. Kawai, Y. Toyota, M. Ueno, T. Kubota, Y. Kirino, T. Tamiya, 2016) [1]. Triple-transgenic (3×Tg)-Alzheimer׳s disease (AD) model mice exhibited significantly poorer spatial learning than sham-treated 3×Tg-AD mice 28 days after traumatic brain injury (TBI). Correspondingly, amyloid-β (Aβ) deposition within the hippocampus was significantly greater in 3×Tg-AD mice 28 days after TBI. However, data regarding the short-term and long-term influences of TBI on amyloid precursor protein (APP) accumulation in AD model mice remain limited. Furthermore, there is little data showing whether physical activity and motor learning are affected by TBI in AD model mice. Here, we provide immunocytochemistry data confirming that TBI induces significant increases in APP accumulation in 3×Tg-AD mice at both 7 days and 28 days after TBI. Furthermore, 3×Tg-AD model mice exhibit a reduced ability to acquire conditioned responses (CRs) during delay eyeblink conditioning compared to sham-treated 3×Tg-AD model mice 28 days after TBI. However, physical activity and motor performance are not significantly changed in TBI-treated 3×Tg-AD model mice.

  18. Ubiquitin Accumulation on Disease Associated Protein Aggregates Is Correlated with Nuclear Ubiquitin Depletion, Histone De-Ubiquitination and Impaired DNA Damage Response

    PubMed Central

    Ben Yehuda, Adi; Risheq, Marwa; Novoplansky, Ofra; Bersuker, Kirill; Kopito, Ron R.; Goldberg, Michal; Brandeis, Michael

    2017-01-01

    Deposition of ubiquitin conjugates on inclusion bodies composed of protein aggregates is a definitive cytopathological hallmark of neurodegenerative diseases. We show that accumulation of ubiquitin on polyQ IB, associated with Huntington’s disease, is correlated with extensive depletion of nuclear ubiquitin and histone de-ubiquitination. Histone ubiquitination plays major roles in chromatin regulation and DNA repair. Accordingly, we observe that cells expressing IB fail to respond to radiomimetic DNA damage, to induce gamma-H2AX phosphorylation and to recruit 53BP1 to damaged foci. Interestingly ubiquitin depletion, histone de-ubiquitination and impaired DNA damage response are not restricted to PolyQ aggregates and are associated with artificial aggregating luciferase mutants. The longevity of brain neurons depends on their capacity to respond to and repair extensive ongoing DNA damage. Impaired DNA damage response, even modest one, could thus lead to premature neuron aging and mortality. PMID:28052107

  19. Rare Earth Ion Mediated Fluorescence Accumulation on a Single Microbead: An Ultrasensitive Strategy for the Detection of Protein Kinase Activity at the Single-Cell Level.

    PubMed

    Zhang, Xiaobo; Liu, Chenghui; Wang, Honghong; Wang, Hui; Li, Zhengping

    2015-12-07

    A single microbead-based fluorescence imaging (SBFI) strategy that enables detection of protein kinase activity from single cell lysates is reported. We systematically investigated the ability of various rare earth (RE) ions, immobilized on the microbead, for specific capturing of kinase-induced phosphopeptides, and Dy(3+) was found to be the most prominent one. Through the efficient concentration of kinase-induced fluorescent phosphopeptides on a Dy(3+) -functionalized single microbead, kinase activity can be detected and quantified by reading the fluorescence on the microbead with a confocal fluorescence microscope. Owing to the extremely specific recognition of Dy(3+) towards phosphopeptides and the highly-concentrated fluorescence accumulation on only one microbead, ultrahigh sensitivity has been achieved for the SBFI strategy which allows direct kinase analysis at the single-cell level.

  20. Effects of Homologous Phosphoenolpyruvate-Carbohydrate Phosphotransferase System Proteins on Carbohydrate Uptake and Poly(3-Hydroxybutyrate) Accumulation in Ralstonia eutropha H16▿†

    PubMed Central

    Kaddor, Chlud; Steinbüchel, Alexander

    2011-01-01

    Seven gene loci encoding putative proteins of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS) were identified in the genome of Ralstonia eutropha H16 by in silico analysis. Except the N-acetylglucosamine-specific PEP-PTS, an additional complete PEP-PTS is lacking in strain H16. Based on these findings, we generated single and multiple deletion mutants defective mainly in the PEP-PTS genes to investigate their influence on carbon source utilization, growth behavior, and poly(3-hydroxybutyrate) (PHB) accumulation. As supposed, the H16 ΔfrcACB and H16 ΔnagFEC mutants exhibited no growth when cultivated on fructose and N-acetylglucosamine, respectively. Furthermore, a transposon mutant with a ptsM-ptsH insertion site did not grow on both carbon sources. The observed phenotype was not complemented, suggesting that it results from an interaction of genes or a polar effect caused by the Tn5::mob insertion. ptsM, ptsH, and ptsI single, double, and triple mutants stored much less PHB than the wild type (about 10 to 39% [wt/wt] of cell dry weight) and caused reduced PHB production in mutants lacking the H16_A2203, H16_A0384, frcACB, or nagFEC genes. In contrast, mutant H16 ΔH16_A0384 accumulated 11.5% (wt/wt) more PHB than the wild type when grown on gluconate and suppressed partially the negative effect of the ptsMHI deletion on PHB synthesis. Based on our experimental data, we discussed whether the PEP-PTS homologous proteins in R. eutropha H16 are exclusively involved in the complex sugar transport system or whether they are also involved in cellular regulatory functions of carbon and PHB metabolism. PMID:21478317

  1. Enhanced cadmium accumulation and tolerance in transgenic tobacco overexpressing rice metal tolerance protein gene OsMTP1 is promising for phytoremediation.

    PubMed

    Das, Natasha; Bhattacharya, Surajit; Maiti, Mrinal K

    2016-08-01

    One of the most grievous heavy metal pollutants in the environment is cadmium (Cd), which is not only responsible for the crop yield loss owing to its phytotoxicity, but also for the human health hazards as the toxic elements usually accumulate in the consumable parts of crop plants. In the present study, we aimed to isolate and functionally characterize the OsMTP1 gene from indica rice (Oryza sativa L. cv. IR64) to study its potential application for efficient phytoremediation of Cd. The 1257 bp coding DNA sequence (CDS) of OsMTP1 encodes a ∼46 kDa protein belonging to the cation diffusion facilitator (CDF) or metal tolerance/transport protein (MTP) family. The OsMTP1 transcript in rice plant was found to respond during external Cd stress. Heterologous expression of OsMTP1 in tobacco resulted in the reduction of Cd stress-induced phytotoxic effects, including growth inhibition, lipid peroxidation, and cell death. Compared to untransformed control, the transgenic tobacco plants showed enhanced vacuolar thiol content, indicating vacuolar localization of the sequestered Cd. The transgenic tobacco plants exhibited significantly higher biomass growth (2.2-2.8-folds) and hyperaccumulation of Cd (1.96-2.22-folds) compared to untransformed control under Cd exposure. The transgenic plants also showed moderate tolerance and accumulation of arsenic (As) upon exogenous As stress, signifying broad substrate specificity of OsMTP1. Together, findings of our research suggest that the transgenic tobacco plants overexpressing OsMTP1 with its hyperaccumulating activity and increased growth rate could be useful for future phytoremediation applications to clean up the Cd-contaminated soil.

  2. Accumulation of mutant alpha1-antitrypsin Z in the endoplasmic reticulum activates caspases-4 and -12, NFkappaB, and BAP31 but not the unfolded protein response.

    PubMed

    Hidvegi, Tunda; Schmidt, Bela Z; Hale, Pamela; Perlmutter, David H

    2005-11-25

    In alpha(1)-antitrypsin (alpha1AT) deficiency, a polymerogenic mutant form of the secretory glycoprotein alpha1AT, alpha1ATZ, is retained in the endoplasmic reticulum (ER) of liver cells. It is not yet known how this results in liver injury in a subgroup of deficient individuals and how the remainder of deficient individuals escapes liver disease. One possible explanation is that the "susceptible" subgroup is unable to mount the appropriate protective cellular responses. Here we examined the effect of mutant alpha1ATZ on several potential protective signaling pathways by using cell lines with inducible expression of mutant alpha1AT as well as liver from transgenic mice with liver-specific inducible expression of mutant alpha1AT. The results show that ER retention of polymerogenic mutant alpha1ATZ does not result in an unfolded protein response (UPR). The UPR can be induced in the presence of alpha1ATZ by tunicamycin excluding the possibility that the pathway has been disabled. In striking contrast, ER retention of nonpolymerogenic alpha1AT mutants does induce the UPR. These results indicate that the machinery responsible for activation of the UPR can distinguish the physical characteristics of proteins that accumulate in the ER in such a way that it can respond to misfolded but not relatively ordered polymeric structures. Accumulation of mutant alpha1ATZ does activate specific signaling pathways, including caspase-12 in mouse, caspase-4 in human, NFkappaB, and BAP31, a profile that was distinct from that activated by nonpolymerogenic alpha1AT mutants.

  3. Accumulate Repeat Accumulate Coded Modulation

    NASA Technical Reports Server (NTRS)

    Abbasfar, Aliazam; Divsalar, Dariush; Yao, Kung

    2004-01-01

    In this paper we propose an innovative coded modulation scheme called 'Accumulate Repeat Accumulate Coded Modulation' (ARA coded modulation). This class of codes can be viewed as serial turbo-like codes, or as a subclass of Low Density Parity Check (LDPC) codes that are combined with high level modulation. Thus at the decoder belief propagation can be used for iterative decoding of ARA coded modulation on a graph, provided a demapper transforms the received in-phase and quadrature samples to reliability of the bits.

  4. A Single B-repeat of Staphylococcus epidermidis accumulation-associated protein induces protective immune responses in an experimental biomaterial-associated infection mouse model.

    PubMed

    Yan, Lin; Zhang, Lei; Ma, Hongyan; Chiu, David; Bryers, James D

    2014-09-01

    Nosocomial infections are the fourth leading cause of morbidity and mortality in the United States, resulting in 2 million infections and ∼100,000 deaths each year. More than 60% of these infections are associated with some type of biomedical device. Staphylococcus epidermidis is a commensal bacterium of the human skin and is the most common nosocomial pathogen infecting implanted medical devices, especially those in the cardiovasculature. S. epidermidis antibiotic resistance and biofilm formation on inert surfaces make these infections hard to treat. Accumulation-associated protein (Aap), a cell wall-anchored protein of S. epidermidis, is considered one of the most important proteins involved in the formation of S. epidermidis biofilm. A small recombinant protein vaccine comprising a single B-repeat domain (Brpt1.0) of S. epidermidis RP62A Aap was developed, and the vaccine's efficacy was evaluated in vitro with a biofilm inhibition assay and in vivo in a murine model of biomaterial-associated infection. A high IgG antibody response against S. epidermidis RP62A was detected in the sera of the mice after two subcutaneous immunizations with Brpt1.0 coadministered with Freund's adjuvant. Sera from Brpt1.0-immunized mice inhibited in vitro S. epidermidis RP62A biofilm formation in a dose-dependent pattern. After receiving two immunizations, each mouse was surgically implanted with a porous scaffold disk containing 5 × 10(6) CFU of S. epidermidis RP62A. Weight changes, inflammatory markers, and histological assay results after challenge with S. epidermidis indicated that the mice immunized with Brpt1.0 exhibited significantly higher resistance to S. epidermidis RP62A implant infection than the control mice. Day 8 postchallenge, there was a significantly lower number of bacteria in scaffold sections and surrounding tissues and a lower residual inflammatory response to the infected scaffold disks for the Brpt1.0-immunized mice than for of the ovalbumin (Ova

  5. Extracellular accumulation of potently microbicidal bactericidal/permeability-increasing protein and p15s in an evolving sterile rabbit peritoneal inflammatory exudate.

    PubMed Central

    Weinrauch, Y; Foreman, A; Shu, C; Zarember, K; Levy, O; Elsbach, P; Weiss, J

    1995-01-01

    To what extent the host defense role of granule-associated antibacterial proteins and peptides of PMN includes extracellular action has not been established. To address this question, we have analyzed the antibacterial activity of cell-free (ascitic) fluid (AF) obtained from glycogen-induced sterile inflammatory rabbit peritoneal exudates in which > 95% of the accumulating cells are PMN. AF, but not plasma collected in parallel, exhibits potent activity toward serum-resistant Gram-negative and Gram-positive bacteria. Total and specific antibacterial activity of AF increases during the first 12 h after injection of glycogen in parallel with the influx of PMN. At maximum, > 99% of 10(7) encapsulated Escherichia coli and Staphylococcus aureus are killed in 30 min/ml of AF. Neutralizing antibodies against the bactericidal/permeability-increasing protein (BPI) of PMN abolishes activity of AF toward encapsulated E. coli but has no effect on activity vs staphylococci. However, BPI alone (approximately 1 microgram/ml in AF) can only account for < or = 20% of AF activity toward E. coli. AF also contains 15 kD PMN proteins (p15s) that act in synergy with BPI. Purified BPI and p15s, in amounts present in AF, reconstitute the growth-inhibitory activity of AF toward encapsulated E. coli. These findings show for the first time an extracellular function of endogenous BPI, providing, together with the p15s, a potent microbicidal system toward Gram-negative bacteria resistant to plasma-derived proteins and phagocytes in inflammatory exudates. Images PMID:7706499

  6. Quantitative Comparison of Dense-Core Amyloid Plaque Accumulation in Amyloid-β Precursor Protein Transgenic Mice

    PubMed Central

    Liu, Peng; Reichl, John H.; Rao, Eshaan R.; McNellis, Brittany M.; Huang, Eric S.; Hemmy, Laura S.; Forster, Colleen L.; Kuskowski, Michael A.; Borchelt, David R.; Vassar, Robert; Ashe, Karen H.; Zahs, Kathleen R.

    2016-01-01

    There exist several dozen lines of transgenic mice that express human amyloid-β precursor protein (AβPP) with Alzheimer’s disease (AD)-linked mutations. AβPP transgenic mouse lines differ in the types and amounts of Aβ that they generate and in their spatiotemporal patterns of expression of Aβ assemblies, providing a toolkit to study Aβ amyloidosis and the influence of Aβ aggregation on brain function. More complete quantitative descriptions of the types of Aβ assemblies present in transgenic mice and in humans during disease progression should add to our understanding of how Aβ toxicity in mice relates to the pathogenesis of AD. Here, we provide a direct quantitative comparison of amyloid plaque burdens and plaque sizes in four lines of AβPP transgenic mice. We measured the fraction of cortex and hippocampus occupied by dense-core plaques, visualized by staining with Thioflavin S, in mice from young adulthood through advanced age. We found that the plaque burdens among the transgenic lines varied by an order of magnitude: at 15 months of age, the oldest age studied, the median cortical plaque burden in 5XFAD mice was already ~4.5 times that of 21-month Tg2576 mice and ~15 times that of 21–24-month rTg9191 mice. Plaque-size distributions changed across the lifespan in a line- and region-dependent manner. We also compared the dense-core plaque burdens in the mice to those measured in a set of pathologically-confirmed AD cases from the Nun Study. Cortical plaque burdens in Tg2576, APPSwePS1ΔE9, and 5XFAD mice eventually far exceeded those measured in the human cohort. PMID:28059792

  7. Tualang Honey Protects against BPA-Induced Morphological Abnormalities and Disruption of ERα, ERβ, and C3 mRNA and Protein Expressions in the Uterus of Rats

    PubMed Central

    Mohamad Zaid, Siti Sarah; Kassim, Normadiah M.; Othman, Shatrah

    2015-01-01

    Bisphenol A (BPA) is an endocrine disrupting chemical (EDC) that can disrupt the normal functions of the reproductive system. The objective of the study is to investigate the potential protective effects of Tualang honey against BPA-induced uterine toxicity in pubertal rats. The rats were administered with BPA by oral gavage over a period of six weeks. Uterine toxicity in BPA-exposed rats was determined by the degree of the morphological abnormalities, increased lipid peroxidation, and dysregulated expression and distribution of ERα, ERβ, and C3 as compared to the control rats. Concurrent treatment of rats with BPA and Tualang honey significantly improved the uterine morphological abnormalities, reduced lipid peroxidation, and normalized ERα, ERβ, and C3 expressions and distribution. There were no abnormal changes observed in rats treated with Tualang honey alone, comparable with the control rats. In conclusion, Tualang honey has potential roles in protecting the uterus from BPA-induced toxicity, possibly accounted for by its phytochemical properties. PMID:26788107

  8. Effective charge measurements reveal selective and preferential accumulation of anions, but not cations, at the protein surface in dilute salt solutions

    PubMed Central

    Gokarn, Yatin R; Fesinmeyer, R Matthew; Saluja, Atul; Razinkov, Vladimir; Chase, Susan F; Laue, Thomas M; Brems, David N

    2011-01-01

    Specific-ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusive. Although Hofmeister-ion effects on proteins are observed at higher (>0.3M) salt concentrations, in dilute (<0.1M) salt solutions nonspecific electrostatic screening is considered to be dominant. Here, using effective charge (Q*) measurements of hen-egg white lysozyme (HEWL) as a direct and differential measure of ion-association, we experimentally show that anions selectively and preferentially accumulate at the protein surface even at low (<100 mM) salt concentrations. At a given ion normality (50 mN), the HEWL Q* was dependent on anion, but not cation (Li+, Na+, K+, Rb+, Cs+, GdnH+, and Ca2+), identity. The Q* decreased in the order F− > Cl− > Br− > NO3− ∼ I− > SCN− > ClO4− ≫ SO42−, demonstrating progressively greater binding of the monovalent anions to HEWL and also show that the SO42− anion, despite being strongly hydrated, interacts directly with the HEWL surface. Under our experimental conditions, we observe a remarkable asymmetry between anions and cations in their interactions with the HEWL surface. PMID:21432935

  9. Cell wall accumulation of fluorescent proteins derived from a trans-Golgi cisternal membrane marker and paramural bodies in interdigitated Arabidopsis leaf epidermal cells.

    PubMed

    Akita, Kae; Kobayashi, Megumi; Sato, Mayuko; Kutsuna, Natsumaro; Ueda, Takashi; Toyooka, Kiminori; Nagata, Noriko; Hasezawa, Seiichiro; Higaki, Takumi

    2017-01-01

    In most dicotyledonous plants, leaf epidermal pavement cells develop jigsaw puzzle-like shapes during cell expansion. The rapid growth and complicated cell shape of pavement cells is suggested to be achieved by targeted exocytosis that is coordinated with cytoskeletal rearrangement to provide plasma membrane and/or cell wall materials for lobe development during their morphogenesis. Therefore, visualization of membrane trafficking in leaf pavement cells should contribute an understanding of the mechanism of plant cell morphogenesis. To reveal membrane trafficking in pavement cells, we observed monomeric red fluorescent protein-tagged rat sialyl transferases, which are markers of trans-Golgi cisternal membranes, in the leaf epidermis of Arabidopsis thaliana. Quantitative fluorescence imaging techniques and immunoelectron microscopic observations revealed that accumulation of the red fluorescent protein occurred mostly in the curved regions of pavement cell borders and guard cell ends during leaf expansion. Transmission electron microscopy observations revealed that apoplastic vesicular membrane structures called paramural bodies were more frequent beneath the curved cell wall regions of interdigitated pavement cells and guard cell ends in young leaf epidermis. In addition, pharmacological studies showed that perturbations in membrane trafficking resulted in simple cell shapes. These results suggested possible heterogeneity of the curved regions of plasma membranes, implying a relationship with pavement cell morphogenesis.

  10. Thallium-201 accumulation in cerebral candidiasis: Unexpected finding on SPECT

    SciTech Connect

    Tonami, N.; Matsuda, H.; Ooba, H.; Yokoyama, K.; Hisada, K.; Ikeda, K.; Yamashita, J. )

    1990-06-01

    The authors present an unexpected finding of Tl-201 uptake in the intracerebral lesions due to candidiasis. SPECT demonstrated the extent of the lesions and a high target-to-background ratio. The regions where abnormal Tl-201 accumulation was seen were nearly consistent with CT scans of those enhanced by a contrast agent. After treatment, most of the abnormal Tl-201 accumulation disappeared.

  11. The polypyrimidine tract-binding protein affects coronavirus RNA accumulation levels and relocalizes viral RNAs to novel cytoplasmic domains different from replication-transcription sites.

    PubMed

    Sola, Isabel; Galán, Carmen; Mateos-Gómez, Pedro A; Palacio, Lorena; Zúñiga, Sonia; Cruz, Jazmina L; Almazán, Fernando; Enjuanes, Luis

    2011-05-01

    The coronavirus (CoV) discontinuous transcription mechanism is driven by long-distance RNA-RNA interactions between transcription-regulating sequences (TRSs) located at the 5' terminal leader (TRS-L) and also preceding each mRNA-coding sequence (TRS-B). The contribution of host cell proteins to CoV transcription needs additional information. Polypyrimidine tract-binding protein (PTB) was reproducibly identified in association with positive-sense RNAs of transmissible gastroenteritis coronavirus (TGEV) TRS-L and TRS-B by affinity chromatography and mass spectrometry. A temporal regulation of PTB cytoplasmic levels was observed during infection, with a significant increase from 7 to 16 h postinfection being inversely associated with a decrease in viral replication and transcription. Silencing the expression of PTB with small interfering RNA in two cell lines (Huh7 and HEK 293T) led to a significant increase of up to 4-fold in mRNA levels and virus titer, indicating a negative effect of PTB on CoV RNA accumulation. During CoV infection, PTB relocalized from the nucleus to novel cytoplasmic structures different from replication-transcription sites in which stress granule markers T-cell intracellular antigen-1 (TIA-1) and TIA-1-related protein (TIAR) colocalized. PTB was detected in these modified stress granules in TGEV-infected swine testis cells but not in stress granules induced by oxidative stress. Furthermore, viral genomic and subgenomic RNAs were detected in association with PTB and TIAR. These cytoplasmic ribonucleoprotein complexes might be involved in posttranscriptional regulation of virus gene expression.

  12. Structurally abnormal human autosomes

    SciTech Connect

    1993-12-31

    Chapter 25, discusses structurally abnormal human autosomes. This discussion includes: structurally abnormal chromosomes, chromosomal polymorphisms, pericentric inversions, paracentric inversions, deletions or partial monosomies, cri du chat (cat cry) syndrome, ring chromosomes, insertions, duplication or pure partial trisomy and mosaicism. 71 refs., 8 figs.

  13. Morphological abnormalities among lampreys

    USGS Publications Warehouse

    Manion, Patrick J.

    1967-01-01

    The experimental control of the sea lamprey (Petromyzon marinus) in the Great Lakes has required the collection of thousands of lampreys. Representatives of each life stage of the four species of the Lake Superior basin were examined for structural abnormalities. The most common aberration was the presence of additional tails. The accessory tails were always postanal and smaller than the normal tail. The point of origin varied; the extra tails occurred on dorsal, ventral, or lateral surfaces. Some of the extra tails were misshaped and curled, but others were normal in shape and pigment pattern. Other abnormalities in larval sea lampreys were malformed or twisted tails and bodies. The cause of the structural abnormalities is unknown. The presence of extra caudal fins could be genetically controlled, or be due to partial amputation or injury followed by abnormal regeneration. Few if any lampreys with structural abnormalities live to sexual maturity.

  14. High-level accumulation of recombinant miraculin protein in transgenic tomatoes expressing a synthetic miraculin gene with optimized codon usage terminated by the native miraculin terminator.

    PubMed

    Hiwasa-Tanase, Kyoko; Nyarubona, Mpanja; Hirai, Tadayoshi; Kato, Kazuhisa; Ichikawa, Takanari; Ezura, Hiroshi

    2011-01-01

    In our previous study, a transgenic tomato line that expressed the MIR gene under control of the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator (tNOS) produced the taste-modifying protein miraculin (MIR). However, the concentration of MIR in the tomatoes was lower than that in the MIR gene's native miracle fruit. To increase MIR production, the native MIR terminator (tMIR) was used and a synthetic gene encoding MIR protein (sMIR) was designed to optimize its codon usage for tomato. Four different combinations of these genes and terminators (MIR-tNOS, MIR-tMIR, sMIR-tNOS and sMIR-tMIR) were constructed and used for transformation. The average MIR concentrations in MIR-tNOS, MIR-tMIR, sMIR-tNOS and sMIR-tMIR fruits were 131, 197, 128 and 287 μg/g fresh weight, respectively. The MIR concentrations using tMIR were higher than those using tNOS. The highest MIR accumulation was detected in sMIR-tMIR fruits. On the other hand, the MIR concentration was largely unaffected by sMIR-tNOS. The expression levels of both MIR and sMIR mRNAs terminated by tMIR tended to be higher than those terminated by tNOS. Read-through mRNA transcripts terminated by tNOS were much longer than those terminated by tMIR. These results suggest that tMIR enhances mRNA expression and permits the multiplier effect of optimized codon usage.

  15. Skeletal muscle cells lacking the retinoblastoma protein display defects in muscle gene expression and accumulate in S and G2 phases of the cell cycle

    PubMed Central

    1996-01-01

    Viral oncoproteins that inactivate the retinoblastoma tumor suppressor protein (pRb) family both block skeletal muscle differentiation and promote cell cycle progression. To clarify the dependence of terminal differentiation on the presence of the different pRb-related proteins, we have studied myogenesis using isogenic primary fibroblasts derived from mouse embryos individually deficient for pRb, p107, or p130. When ectopically expressed in fibroblasts lacking pRb, MyoD induces an aberrant skeletal muscle differentiation program characterized by normal expression of early differentiation markers such as myogenin and p21, but attenuated expression of late differentiation markers such as myosin heavy chain (MHC). Similar defects in MHC expression were not observed in cells lacking either p107 or p130, indicating that the defect is specific to the loss of pRb. In contrast to wild-type, p107- deficient, or p130-deficient differentiated myocytes that are permanently withdrawn from the cell cycle, differentiated myocytes lacking pRb accumulate in S and G2 phases and express extremely high levels of cyclins A and B, cyclin-dependent kinase (Cdk2), and Cdc2, but fail to readily proceed to mitosis. Administration of caffeine, an agent that removes inhibitory phosphorylations on inactive Cdc2/cyclin B complexes, specifically induced mitotic catastrophe in pRb-deficient myocytes, consistent with the observation that the majority of pRb- deficient myocytes arrest in S and G2. Together, these findings indicate that pRb is required for the expression of late skeletal muscle differentiation markers and for the inhibition of DNA synthesis, but that a pRb-independent mechanism restricts entry of differentiated myocytes into mitosis. PMID:8896600

  16. Absence of an Orphan Mitochondrial Protein, C19orf12, Causes a Distinct Clinical Subtype of Neurodegeneration with Brain Iron Accumulation

    PubMed Central

    Hartig, Monika B.; Iuso, Arcangela; Haack, Tobias; Kmiec, Tomasz; Jurkiewicz, Elzbieta; Heim, Katharina; Roeber, Sigrun; Tarabin, Victoria; Dusi, Sabrina; Krajewska-Walasek, Malgorzata; Jozwiak, Sergiusz; Hempel, Maja; Winkelmann, Juliane; Elstner, Matthias; Oexle, Konrad; Klopstock, Thomas; Mueller-Felber, Wolfgang; Gasser, Thomas; Trenkwalder, Claudia; Tiranti, Valeria; Kretzschmar, Hans; Schmitz, Gerd; Strom, Tim M.; Meitinger, Thomas; Prokisch, Holger

    2011-01-01

    The disease classification neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of progressive neurodegenerative disorders characterized by brain iron deposits in the basal ganglia. For about half of the cases, the molecular basis is currently unknown. We used homozygosity mapping followed by candidate gene sequencing to identify a homozygous 11 bp deletion in the orphan gene C19orf12. Mutation screening of 23 ideopathic NBIA index cases revealed two mutated alleles in 18 of them, and one loss-of-function mutation is the most prevalent. We also identified compound heterozygous missense mutations in a case initially diagnosed with Parkinson disease at age 49. Psychiatric signs, optic atrophy, and motor axonal neuropathy were common findings. Compared to the most prevalent NBIA subtype, pantothenate kinase associated neurodegeneration (PKAN), individuals with two C19orf12 mutations were older at age of onset and the disease progressed more slowly. A polyclonal antibody against the predicted membrane spanning protein showed a mitochondrial localization. A histopathological examination in a single autopsy case detected Lewy bodies, tangles, spheroids, and tau pathology. The mitochondrial localization together with the immunohistopathological findings suggests a pathomechanistic overlap with common forms of neurodegenerative disorders. PMID:21981780

  17. Hyaluronan and Hyaluronan-Binding Proteins Accumulate in Both Human Type 1 Diabetic Islets and Lymphoid Tissues and Associate With Inflammatory Cells in Insulitis

    PubMed Central

    Bogdani, Marika; Johnson, Pamela Y.; Potter-Perigo, Susan; Nagy, Nadine; Day, Anthony J.; Bollyky, Paul L.

    2014-01-01

    Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that is present in pancreatic islets, but little is known about its involvement in the development of human type 1 diabetes (T1D). We have evaluated whether pancreatic islets and lymphoid tissues of T1D and nondiabetic organ donors differ in the amount and distribution of HA and HA-binding proteins (hyaladherins), such as inter-α-inhibitor (IαI), versican, and tumor necrosis factor–stimulated gene-6 (TSG-6). HA was dramatically increased both within the islet and outside the islet endocrine cells, juxtaposed to islet microvessels in T1D. In addition, HA was prominent surrounding immune cells in areas of insulitis. IαI and versican were present in HA-rich areas of islets, and both molecules accumulated in diabetic islets and regions exhibiting insulitis. TSG-6 was observed within the islet endocrine cells and in inflammatory infiltrates. These patterns were only observed in tissues from younger donors with disease duration of <10 years. Furthermore, HA and IαI amassed in follicular germinal centers and in T-cell areas in lymph nodes and spleens in T1D patients compared with control subjects. Our observations highlight potential roles for HA and hyaladherins in the pathogenesis of diabetes. PMID:24677718

  18. ER reorganization is remarkably induced in COS-7 cells accumulating transmembrane protein receptors not competent for export from the endoplasmic reticulum.

    PubMed

    D'Agostino, Massimo; Crespi, Arianna; Polishchuk, Elena; Generoso, Serena; Martire, Gianluca; Colombo, Sara Francesca; Bonatti, Stefano

    2014-11-01

    The newly synthesized mutant L501fsX533 Frizzled-4 form and the alpha3beta4 nicotinic acetylcholine receptor expressed in the absence of nicotine accumulate in the endoplasmic reticulum of COS-7 cells and induce the formation of large areas of smooth and highly convoluted cisternae. This results in a generalized block of the transport to the Golgi complex of newly synthesized proteins. Intriguingly, both effects happen peculiarly in COS-7 cells; HeLa, Huh-7, and HEK293 cells expressing the two receptors at similar level than COS-7 cells show normal ER and normal transport toward the plasma membrane. These results question the conclusion that a dominant-negative mechanism would explain the dominance of the mutant L501fsX533 Fz4 allele in the transmission of a form of Familial exudative vitreoretinopathy. Moreover, they indicate that the coordination of endoplasmic reticulum homeostasis in COS-7 cells is particularly error prone. This finding suggests that COS-7 cells may be extremely useful to study the molecular mechanisms regulating endoplasmic reticulum size and architecture.

  19. Reduced cytosolic carboxypeptidase 6 (CCP6) level leads to accumulation of serum polyglutamylated DNAJC7 protein: A potential biomarker for renal cell carcinoma early detection

    PubMed Central

    Li, Yi; Zhu, Xiaoxiao; Ding, Juan; Ren, Shuangchun; Zhao, Heping; Wu, Song; Tian, Yong; Wang, Guo-Qing

    2016-01-01

    Renal cell carcinoma (RCC) is frequently diagnosed at advanced stages of disease, although early diagnosis has much favorable prognosis. This study assessed aberrant expression of cytosolic carboxypeptidase 6 (CCP6) leading to accumulation of serum polyglutamylated DNAJC7 as a biomarker for early RCC detection. A total of 835 RCCs, 143 chronic nephritis, 170 kidney stones and 415 health controls were collected for qRT-PCR, immunohistochemistry and Western blot analysis of CCP6 expression and mass spectrometry of DNAJC7 and polyglutamylated DNAJC7. The data showed that CCP6 expression was significantly decreased in 30 RCC tissues and that mass spectrometric and pull-down analysis identified DNAJC7 as a substrate of CCP6 and showed upregulated polyglutamylated-DNAJC7 (polyE-DNAJC7) in sera of RCC patients. The electrochemiluminescence immunoassay of large-scale serum samples from multi-institutes further confirmed the remarkable increase of polyE-DNAJC7 in 805 RCCs compared to that of 385 healthy controls (p < 0.001), 128 patients with chronic nephritis (p < 0.001), and 153 with kidney stone (p < 0.001). Serum level of DNAJC7-polyE protein was also associated with advanced RCC stage and grade in 805 patients. The data from the current study for the first time demonstrated increased serum polyglutamylated DNAJC7 as a potential biomarker for RCC early detection and association with advanced tumor stages and grade, which provides support of further polyglutamylation research in RCC. PMID:26993597

  20. The Craterostigma plantagineum glycine-rich protein CpGRP1 interacts with a cell wall-associated protein kinase 1 (CpWAK1) and accumulates in leaf cell walls during dehydration.

    PubMed

    Giarola, Valentino; Krey, Stephanie; von den Driesch, Barbara; Bartels, Dorothea

    2016-04-01

    Craterostigma plantagineum tolerates extreme desiccation. Leaves of this plant shrink and extensively fold during dehydration and expand again during rehydration, preserving their structural integrity. Genes were analysed that may participate in the reversible folding mechanism. Analysis of transcripts abundantly expressed in desiccated leaves identified a gene putatively coding for an apoplastic glycine-rich protein (CpGRP1). We studied the expression, regulation and subcellular localization of CpGRP1 and its ability to interact with a cell wall-associated protein kinase (CpWAK1) to understand the role of CpGRP1 in the cell wall during dehydration. The CpGRP1 protein accumulates in the apoplast of desiccated leaves. Analysis of the promoter revealed that the gene expression is mainly regulated at the transcriptional level, is independent of abscisic acid (ABA) and involves a drought-responsive cis-element (DRE). CpGRP1 interacts with CpWAK1 which is down-regulated in response to dehydration. Our data suggest a role of the CpGRP1-CpWAK1 complex in dehydration-induced morphological changes in the cell wall during dehydration in C. plantagineum. Cell wall pectins and dehydration-induced pectin modifications are predicted to be involved in the activity of the CpGRP1-CpWAK1 complex.

  1. Differential responses to different light spectral ranges of violaxanthin de-epoxidation and accumulation of Cbr, an algal homologue of plant early light inducible proteins, in two strains of Dunaliella.

    PubMed

    Banet; Pick; Malkin; Zamir

    1999-11-01

    Unicellular green algae of the genus Dunaliella, similar to higher plants, respond to light stress by enhanced de-epoxidation of violaxanthin and accumulation of Cbr, a protein homologous to early light inducible proteins (Elips) in plants. These proteins belong to the superfamily of chlorophyll a/b binding proteins. Two Dunaliella strains, D. bardawil and D. salina, were compared for these two responses under light in the UVA, blue, green and red spectral ranges. In D. bardawil, the two stress responses were similarly induced under UVA, blue or red light and to a lesser extent under green light. In D. salina, a similar spectral range dependence was exhibited for violaxanthin de-epoxidation. However, Cbr accumulated only under UVA or blue light but not under green or red light. A strong synergistic effect of a low dose of blue light superimposed on red light resulted in Cbr accumulation. These results reveal strain-specific differences in spectral range requirements of the two light-stress responses. In the two strains, violaxanthin de-epoxidation is triggered under photosynthetically-active spectral ranges but at least in D. salina, Cbr accumulation appears to require a specific light signal additionally to a signal(s) generated by light stress.

  2. Phorbol esters induce intracellular accumulation of the anti-apoptotic protein PED/PEA-15 by preventing ubiquitinylation and proteasomal degradation.

    PubMed

    Perfetti, Anna; Oriente, Francesco; Iovino, Salvatore; Alberobello, A Teresa; Barbagallo, Alessia P M; Esposito, Iolanda; Fiory, Francesca; Teperino, Raffaele; Ungaro, Paola; Miele, Claudia; Formisano, Pietro; Beguinot, Francesco

    2007-03-23

    Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA)-15 is an anti-apoptotic protein whose expression is increased in several cancer cells and following experimental skin carcinogenesis. Exposure of untransfected C5N keratinocytes and transfected HEK293 cells to phorbol esters (12-O-tetradecanoylphorbol-13-acetate (TPA)) increased PED/PEA-15 cellular content and enhanced its phosphorylation at serine 116 in a time-dependent fashion. Ser-116 --> Gly (PED(S116G)) but not Ser-104 --> Gly (PED(S104G)) substitution almost completely abolished TPA regulation of PED/PEA-15 expression. TPA effect was also prevented by antisense inhibition of protein kinase C (PKC)-zeta and by the expression of a dominant-negative PKC-zeta mutant cDNA in HEK293 cells. Similar to long term TPA treatment, overexpression of wild-type PKC-zeta increased cellular content and phosphorylation of WT-PED/PEA-15 and PED(S104G) but not of PED(S116G). These events were accompanied by the activation of Ca2+-calmodulin kinase (CaMK) II and prevented by the CaMK blocker, KN-93. At variance, the proteasome inhibitor lactacystin mimicked TPA action on PED/PEA-15 intracellular accumulation and reverted the effects of PKC-zeta and CaMK inhibition. Moreover, we show that PED/PEA-15 bound ubiquitin in intact cells. PED/PEA-15 ubiquitinylation was reduced by TPA and PKC-zeta overexpression and increased by KN-93 and PKC-zeta block. Furthermore, in HEK293 cells expressing PED(S116G), TPA failed to prevent ubiquitin-dependent degradation of the protein. Accordingly, in the same cells, TPA-mediated protection from apoptosis was blunted. Taken together, our results indicate that TPA increases PED/PEA-15 expression at the post-translational level by inducing phosphorylation at serine 116 and preventing ubiquitinylation and proteosomal degradation.

  3. Overexpression of the Saccharomyces cerevisiae mannosylphosphodolichol synthase-encoding gene in Trichoderma reesei results in an increased level of protein secretion and abnormal cell ultrastructure.

    PubMed

    Kruszewska, J S; Butterweck, A H; Kurzatkowski, W; Migdalski, A; Kubicek, C P; Palamarczyk, G

    1999-06-01

    Production of extracellular proteins plays an important role in the physiology of Trichoderma reesei and has potential industrial application. To improve the efficiency of protein secretion, we overexpressed in T. reesei the DPM1 gene of Saccharomyces cerevisiae, encoding mannosylphosphodolichol (MPD) synthase, under homologous, constitutively acting expression signals. Four stable transformants, each with different copy numbers of tandemly integrated DPM1, exhibited roughly double the activity of MPD synthase in the respective endoplasmic reticulum membrane fraction. On a dry-weight basis, they secreted up to sevenfold-higher concentrations of extracellular proteins during growth on lactose, a carbon source promoting formation of cellulases. Northern blot analysis showed that the relative level of the transcript of cbh1, which encodes the major cellulase (cellobiohydrolase I [CBH I]), did not increase in the transformants. On the other hand, the amount of secreted CBH I and, in all but one of the transformants, intracellular CBH I was elevated. Our results suggest that posttranscriptional processes are responsible for the increase in CBH I production. The carbohydrate contents of the extracellular proteins were comparable in the wild type and in the transformants, and no hyperglycosylation was detected. Electron microscopy of the DPM1-amplified strains revealed amorphous structure of the cell wall and over three times as many mitochondria as in the control. Our data indicate that molecular manipulation of glycan biosynthesis in Trichoderma can result in improved protein secretion.

  4. "Jeopardy" in Abnormal Psychology.

    ERIC Educational Resources Information Center

    Keutzer, Carolin S.

    1993-01-01

    Describes the use of the board game, Jeopardy, in a college level abnormal psychology course. Finds increased student interaction and improved application of information. Reports generally favorable student evaluation of the technique. (CFR)

  5. Re-analysis of protein data reveals the germination pathway and up accumulation mechanism of cell wall hydrolases during the radicle protrusion step of seed germination in Podophyllum hexandrum- a high altitude plant

    PubMed Central

    Dogra, Vivek; Bagler, Ganesh; Sreenivasulu, Yelam

    2015-01-01

    Podophyllum hexandrum Royle is an important high-altitude plant of Himalayas with immense medicinal value. Earlier, it was reported that the cell wall hydrolases were up accumulated during radicle protrusion step of Podophyllum seed germination. In the present study, Podophyllum seed Germination protein interaction Network (PGN) was constructed by using the differentially accumulated protein (DAP) data set of Podophyllum during the radicle protrusion step of seed germination, with reference to Arabidopsis protein–protein interaction network (AtPIN). The developed PGN is comprised of a giant cluster with 1028 proteins having 10,519 interactions and a few small clusters with relevant gene ontological signatures. In this analysis, a germination pathway related cluster which is also central to the topology and information dynamics of PGN was obtained with a set of 60 key proteins. Among these, eight proteins which are known to be involved in signaling, metabolism, protein modification, cell wall modification, and cell cycle regulation processes were found commonly highlighted in both the proteomic and interactome analysis. The systems-level analysis of PGN identified the key proteins involved in radicle protrusion step of seed germination in Podophyllum. PMID:26579141

  6. An XPA gene splicing mutation resulting in trace protein expression in an elderly xeroderma pigmentosum group A patient without neurological abnormalities.

    PubMed

    Takahashi, Y; Endo, Y; Kusaka, A; Nakamaura, S; Nakazawa, Y; Ogi, T; Uryu, M; Tsuji, M; Furue, M; Moriwaki, S

    2016-09-07

    A certain relationship between XPA gene mutations and the severity of symptoms has been observed in patients with xeroderma pigmentosum group A (XP-A). Patients with mutations within the DNA-binding domain usually exhibit severe symptoms, whereas splicing mutations in the same domain sometimes cause very mild symptoms. This inconsistency can be explained by a small amount of functional XPA protein produced from normally spliced transcripts. We herein report the case of an adult Japanese XP-A patient with unusually mild symptoms. We identified a homozygous c.529G>A mutation in exon 4 of the XPA gene, which resulted in aberrant splicing with a 29-bp deletion in exon 4 causing a frameshift. Intact mRNA was observable, but a Western blot analysis failed to detect any normal XPA protein. We therefore evaluated the DNA repair capacity in normal cells in which the XPA expression was artificially diminished. The repair capacity was still present in cells with trace levels of the XPA protein. The repair capacity of the cells derived from our patient with mild symptoms was poor by comparison, but still significant compared to that of the cells derived from an XP-A patient with severe symptoms. These results provide strong evidence that a trace level of XPA protein can still exert a relatively strong repair capacity, resulting in only a mild phenotype. This article is protected by copyright. All rights reserved.

  7. Abnormalities of the erythrocyte membrane.

    PubMed

    Gallagher, Patrick G

    2013-12-01

    Primary abnormalities of the erythrocyte membrane are characterized by clinical, laboratory, and genetic heterogeneity. Among this group, hereditary spherocytosis patients are more likely to experience symptomatic anemia. Treatment of hereditary spherocytosis with splenectomy is curative in most patients. Growing recognition of the long-term risks of splenectomy has led to re-evaluation of the role of splenectomy. Management guidelines acknowledge these considerations and recommend discussion between health care providers, patient, and family. The hereditary elliptocytosis syndromes are the most common primary disorders of erythrocyte membrane proteins. However, most elliptocytosis patients are asymptomatic and do not require therapy.

  8. Congenital abnormalities of the goat.

    PubMed

    Basrur, P K

    1993-03-01

    Congenital abnormalities of genetic and environmental causes constitute a striking proportion of the afflictions seen in goats. These include a variety of malformations and metabolic diseases that could occur in all breeds but tend to exhibit predisposition in some breeds of goats. Genetic abnormalities for which the carrier state is detectable with the aid of enzymes and surface protein markers can be eliminated from goat populations, whereas common polygenic disorders including udder problems in does and gynecomastia in bucks are more difficult to eradicate because the mutant genes responsible for these traits generally do not declare themselves until inbreeding brings together a critical concentration of liability genes to create a crisis. A substantial reduction of common abnormalities in this species, such as intersexuality in dairy breeds, abortion in Angora breed, and arthritis in the Pygmy breed, will require a change in breeders' preference and selection practice. In making these changes, however, the beneficial traits will have to be balanced against the undesirable effects of the selected mutant genes (pleiotropy), which hold the key to success or failure of a breed under domestication.

  9. Protein damage and repair controlling seed vigor and longevity.

    PubMed

    Ogé, Laurent; Broyart, Caroline; Collet, Boris; Godin, Béatrice; Jallet, Denis; Bourdais, Gildas; Job, Dominique; Grappin, Philippe

    2011-01-01

    The formation of abnormal isoaspartyl residues derived from aspartyl or asparaginyl residues is a major source of spontaneous protein misfolding in cells. The repair enzyme protein L: -isoaspartyl methyltransferase (PIMT) counteracts such damage by catalyzing the conversion of abnormal isoaspartyl residues to their normal aspartyl forms. Thus, this enzyme contributes to the survival of many organisms, including plants. Analysis of the accumulation of isoaspartyl-containing proteins and its modulation by the PIMT repair pathway, using germination tests, immunodetection, enzymatic assays, and HPLC analysis, gives new insights in understanding controlling mechanisms of seed longevity and vigor.

  10. The accumulation of macrophages expressing myeloid-related protein 8 (MRP8) and MRP14 in the spleen of BALB/cA mice during infection with Plasmodium berghei.

    PubMed

    Mizobuchi, Haruka; Yamakoshi, Sachiko; Omachi, Satoko; Osada, Yasutaka; Sanjoba, Chizu; Goto, Yasuyuki; Matsumoto, Yoshitsugu

    2014-03-01

    Splenomegaly is one of the typical symptoms of malaria. However, the pathogenesis of splenic enlargement still remains unclear. Spleen is a major organ for clearance of malaria parasites, but excessive response to the parasites can lead to splenomegaly. Myeloid-related protein (MRP) 8 and MRP14 are expressed by myeloid cells and are regarded as marker proteins of an immature and inflammatory subtype of macrophage. Previous studies have demonstrated that accumulation of MRP8(+) and MRP14(+) macrophages is associated with the pathological changes associated with various inflammatory diseases. In order to elucidate whether MRP8(+) and MRP14(+) cells are also involved in splenomegaly during malaria, we investigated expression of MRP8 and MRP14 in the spleens of mice infected with Plasmodium berghei. The MRP8 and MRP14 levels in the serum were analyzed by western blot, which confirmed that these proteins were elevated during infection compared with uninfected controls. Enlargement of the spleen was prominent at 7days of infection, and histological analysis of the spleens demonstrated deposition of malaria pigments and accumulation of mononuclear cells. Immunohistochemical staining of the tissue revealed the accumulation of cells expressing MRP8 and MRP14. In addition, the locations of those cells overlapped with CD11b(+) cells in the red pulp. These results suggest that splenomegaly in malaria is partly due to the accumulation of MRP8(+) and MRP14(+) macrophages.

  11. Retinal abnormalities in β-thalassemia major

    PubMed Central

    Bhoiwala, Devang L.; Dunaief, Joshua L.

    2015-01-01

    Patients with beta (β)-thalassemia (β-TM: thalassemia major, β-TI: thalassemia intermedia) have a variety of complications that may affect all organs, including the eye. Ocular abnormalities include retinal pigment epithelium degeneration, angioid streaks, venous tortuosity, night blindness, visual field defects, decreased visual acuity, color vision abnormalities, and acute visual loss. Patients with β-TM are transfusion dependent and require iron chelation therapy (ICT) in order to survive. Retinal degeneration may result from either retinal iron accumulation from transfusion-induced iron overload or retinal toxicity induced by ICT. Some who were never treated with ICT exhibited retinopathy, and others receiving ICT had chelator-induced retinopathy. We will focus on retinal abnormalities present in individuals with β-TM viewed in light of new findings on the mechanisms and manifestations of retinal iron toxicity. PMID:26325202

  12. Abnormal oxidant sensitivity and beta-chain structure of spectrin in hereditary spherocytosis associated with defective spectrin-protein 4.1 binding.

    PubMed Central

    Becker, P S; Morrow, J S; Lux, S E

    1987-01-01

    Hereditary spherocytosis (HS) is an inherited disorder of erythrocyte shape associated with spectrin deficiency and hemolytic anemia. In a subset of patients with the autosomal dominant form of HS, spectrin displays a reduced capacity to bind protein 4.1 and, therefore, actin; both functions that are critical to the membrane skeleton. A specific structural defect has not been identified in the spectrin from these patients. Chymotryptic digestion of the isolated spectrin chains shows impaired cleavage of the distal peptide of the beta subunit, the beta IV domain. In previous work, we have shown that mild oxidation markedly diminishes the binding capacity of normal spectrin for protein 4.1. Here we observe that chemical reduction of freshly isolated, untreated HS spectrin dramatically improves its function. Thus, a primary structural defect in the beta subunit of spectrin in this subtype of HS may lead to oxidant sensitivity, and secondarily, to a functional defect in the binding of spectrin to protein 4.1 and actin. Images PMID:3611357

  13. Genetic abnormalities and microsatellite instability in colorectal cancer.

    PubMed

    Iniesta, P; de Juan, C; Caldés, T; Vega, F J; Massa, M J; Cerdán, F J; López, J A; Fernández, C; Sánchez, A; Torres, A J; Balibrea, J L; Benito, M

    1998-01-01

    Our purpose was to investigate different genetic abnormalities, such as K-ras mutations, p53 alterations, and c-myc RNA overexpression, as well as microsatellite instability in 63 colorectal tumors obtained from patients that had undergone surgery. K-ras point mutations were analyzed by PCR-RFLP technique, followed by sequencing; p53 protein accumulation by immunohistochemistry; p53 gene mutations in exons 5-9 were studied by the SSCP and sequencing techniques, and c-myc overexpression by Northern blot. Microsatellite instability was performed at chromosomes 2p, 3p, and 11p by a PCR-based technique. Our data indicate a trend toward a poorer prognosis in patients who had K-ras transversions; besides, we have obtained a prevalence of c-myc RNA overexpression and p53 exon 7 mutations in the latest stages of tumor progression. In conclusion, our findings suggest that the recognition of molecular abnormalities might be used in colorectal cancer as a prognostic indicator or to determine the metastatic potential of colorectal adenocarcinomas.

  14. Molecular characterization of double-stranded RNA segments encoding the major capsid proteins of a Palyam serogroup orbivirus that caused an epizootic of congenital abnormalities in cattle.

    PubMed

    Yamakawa, M; Furuuchi, S; Minobe, Y

    1999-01-01

    cDNA cloning of the double-stranded RNA genome of Chuzan virus, a member of the Palyam serogroup orbiviruses, was carried out and the complete nucleotide sequences of RNA segments 2, 3, 6 and 7, encoding the major capsid proteins VP2, VP3, VP5 and VP7, respectively, were determined. The individual segments had single open reading frames and short inverted repeats adjacent to the conserved terminal sequences. Comparative sequence analysis with other serogroups of the genus Orbivirus suggested that VP2 is the principal determinant of serotype specificity and the neutralizing antigen of the Palyam serogroup. VP5 is also considered to be associated with antigenic variability. Both VP3 and VP7 probably contain serogroup-specific epitopes. Phylogenetic profiles demonstrated that the Palyam serogroup virus is more closely related to African horsesickness virus than to bluetongue virus and epizootic haemorrhagic disease virus.

  15. Expression of the translocator protein of 18 kDa by microglia, macrophages and astrocytes based on immunohistochemical localization in abnormal human brain

    PubMed Central

    Cosenza-Nashat, Melissa; Zhao, Meng-Liang; Suh, Hyeon-Sook; Morgan, Janet; Natividad, Ryan; Morgello, Susan; Lee, Sunhee C.

    2009-01-01

    Aims Microglia are involved in neurodegeneration, are prime targets for anti-inflammatory therapy and are potential biomarkers of disease progression. For example, positron emission tomography (PET) imaging employing radioligands for the mitochondrial translocator protein of 18 kDa (TSPO, formerly known as the peripheral benzodiazepine receptor) is being scrutinized to detect neuroinflammation in various diseases. TSPO is presumably present in activated microglia, but may be present in other neural cells. Methods We sought to elucidate the protein expression in normal human CNS, several neurological diseases (HIV encephalitis, Alzheimer’s disease, multiple sclerosis and stroke) and SIV encephalitis by performing immunohistochemistry with two anti-TSPO antibodies. Results Although the overall parenchymal staining was minimal in normal brain, endothelial and smooth muscle cells, subpial glia, intravascular monocytes and ependymal cells were TSPO positive. In disease states, elevated TSPO was present in parenchymal microglia, macrophages and some hypertrophic astrocytes, but the distribution of TSPO varied depending on the disease, disease stage and proximity to the lesion or relation to infection. Staining with the two antibodies correlated well in white matter, but one antibody also stained cortical neurons. Quantitative analysis demonstrated a significant increase in TSPO in the white matter of HIV encephalitis compared to brains without encephalitis. TSPO expression was also increased in SIV encephalitis. Conclusions This report provides the first comprehensive immunohistochemical analysis of the expression of TSPO. The results are useful for informing the usage of PET as an imaging modality and have an impact on the potential use of TSPO as an anti-inflammatory pharmacological target. PMID:19077109

  16. Yellow mosaic symptom caused by the nuclear shuttle protein gene of mungbean yellow mosaic virus is associated with single-stranded DNA accumulation and mesophyll spread of the virus.

    PubMed

    Kuruba, B L; Buvani, A P; Veluthambi, K

    Mungbean yellow mosaic virus-[India:Vigna] (MYMV-[IN:Vig]), a blackgram isolate of MYMV, causes yellow mosaic disease in blackgram and mungbean. Two variable DNA-B components, KA22 and KA27, cause distinct symptoms in blackgram [V. mungo (L.) Hepper] with the same DNA-A component. KA22 + DNA-A-agroinoculated blackgram plants displayed yellow mosaic symptom and accumulated high levels of viral single-stranded (ss) DNA. KA27 + D