Bacillus methanolicus pyruvate carboxylase and homoserine dehydrogenase I and II and their roles for L-lysine production from methanol at 50 degrees C.

PubMed

Brautaset, Trygve; Jakobsen, Øyvind M; Degnes, Kristin F; Netzer, Roman; Naerdal, Ingemar; Krog, Anne; Dillingham, Rick; Flickinger, Michael C; Ellingsen, Trond E

2010-07-01

We here present the pyc gene encoding pyruvate carboxylase (PC), and the hom-1 and hom-2 genes encoding two active homoserine dehydrogenase (HD) proteins, in methylotrophic Bacillus methanolicus MGA3. In general, both PC and HD are regarded as key targets for improving bacterial L-lysine production; PC plays a role in precursor oxaloacetate (OAA) supply while HD controls an important branch point in the L-lysine biosynthetic pathway. The hom-1 and hom-2 genes were strongly repressed by L-threonine and L-methionine, respectively. Wild-type MGA3 cells secreted 0.4 g/l L-lysine and 59 g/l L-glutamate under optimised fed batch methanol fermentation. The hom-1 mutant M168-20 constructed herein secreted 11 g/l L-lysine and 69 g/l of L-glutamate, while a sixfold higher L-lysine overproduction (65 g/l) of the previously constructed classical B. methanolicus mutant NOA2#13A52-8A66 was accompanied with reduced L-glutamate production (28 g/l) and threefold elevated pyc transcription level. Overproduction of PC and its mutant enzyme P455S in M168-20 had no positive effect on the volumetric L-lysine yield and the L-lysine yield on methanol, and caused significantly reduced volumetric L-glutamate yield and L: -glutamate yield on methanol. Our results demonstrated that hom-1 represents one key target for achieving L-lysine overproduction, PC activity plays an important role in controlling L-glutamate production from methanol, and that OAA precursor supply is not a major bottleneck for L-lysine overproduction by B. methanolicus.

Vaccination with plasmid DNA encoding a mutant toxic shock syndrome toxin-1 ameliorates toxin-induced lethal shock in mice.

PubMed

Feng, Mao-Hui; Cui, Jing-Chun; Nakane, Akio; Hu, Dong-Liang

2013-09-01

Staphylococcal toxic shock syndrome toxin-1 (TSST-1), a superantigenic toxin produced by Staphylococcus (S.) aureus, is a major cause of septic shock and toxic shock syndrome. To investigate whether vaccination with a plasmid DNA encoding a non-toxic mutant TSST-1 (mTSST-1) can protect mice against wild-type TSST-1-induced lethal shock, the mice were intranasally immunized with the plasmid DNA (named pcDNA-mTSST-1) plus a mucosal adjuvant, a non-toxic mutant labile toxin (mLT). After the immunization, the mice were challenged with TSST-1 and lipopolysaccharide (LPS). The survival rate of mice immunized with pcDNA-mTSST-1 plus mLT was higher than that of the control mice immunized with PBS alone, mLT alone, pcDNA-mTSST-1 alone, or a parent plasmid plus mLT. The titers of interferon-γ (IFN-γ) in the sera of mice immunized with pcDNA-mTSST-1 plus mLT were significantly lower than those of the mLT control mice. Immunization with pcDNA-mTSST-1 plus mLT increased the serum levels of TSST-1-specific antibodies, especially immunoglobulin G1 (IgG1) and IgG2a subclasses. Furthermore, the sera obtained from mice immunized with pcDNA-mTSST-1 plus mLT significantly inhibited the TSST-1-induced secretion of IFN-γ and tumor necrosis factor-α (TNF-α) in murine spleen cells in vitro. These results indicate that immunization with pcDNA-mTSST-1 plus mLT provides protection against the lethal toxic shock of mice induced by wild-type TSST-1. The protective effect could be due to TSST-1-specific neutralizing antibodies as well as the inhibition of IFN-γ and TNF-α secretions. Since TSST-1 is commonly released by invasive S. aureus, the pcDNA-mTSST-1 should be useful in preventing toxin-induced shock resulting from S. aureus infection.

Cellular Prion Protein Combined with Galectin-3 and -6 Affects the Infectivity Titer of an Endogenous Retrovirus Assayed in Hippocampal Neuronal Cells

PubMed Central

Shin, Hae-Young; Goto, Joy J.; Carp, Richard I.; Choi, Eun-Kyoung; Kim, Yong-Sun

2016-01-01

Prion diseases are infectious and fatal neurodegenerative diseases which require the cellular prion protein, PrPC, for development of diseases. The current study shows that the PrPC augments infectivity and plaque formation of a mouse endogenous retrovirus, MuLV. We have established four neuronal cell lines expressing mouse PrPC, PrP+/+; two express wild type PrPC (MoPrPwild) and the other two express mutant PrPC (MoPrPmut). Infection of neuronal cells from various PrP+/+ and PrP-/- (MoPrPKO) lines with MuLV yielded at least three times as many plaques in PrP+/+ than in PrP-/-. Furthermore, among the four PrP+/+ lines, one mutant line, P101L, had at least 2.5 times as many plaques as the other three PrP+/+ lines. Plaques in P101L were four times larger than those in other PrP+/+ lines. Colocalization of PrP and CAgag was seen in MuLV-infected PrP+/+ cells. In the PrP-MuLV interaction, the involvement of galectin-3 and -6 was observed by immunoprecipitation with antibody to PrPC. These results suggest that PrPC combined with galectin-3 and -6 can act as a receptor for MuLV. P101L, the disease form of mutant PrPC results suggest the genetic mutant form of PrPC may be more susceptible to viral infection. PMID:27936017

Spontaneous Generation of Infectious Prion Disease in Transgenic Mice

PubMed Central

Castilla, Joaquín; Pintado, Belén; Gutiérrez-Adan, Alfonso; Andréoletti, Olivier; Aguilar-Calvo, Patricia; Arroba, Ana-Isabel; Parra-Arrondo, Beatriz; Ferrer, Isidro; Manzanares, Jorge; Espinosa, Juan-Carlos

2013-01-01

We generated transgenic mice expressing bovine cellular prion protein (PrPC) with a leucine substitution at codon 113 (113L). This protein is homologous to human protein with mutation 102L, and its genetic link with Gerstmann–Sträussler–Scheinker syndrome has been established. This mutation in bovine PrPC causes a fully penetrant, lethal, spongiform encephalopathy. This genetic disease was transmitted by intracerebral inoculation of brain homogenate from ill mice expressing mutant bovine PrP to mice expressing wild-type bovine PrP, which indicated de novo generation of infectious prions. Our findings demonstrate that a single amino acid change in the PrPC sequence can induce spontaneous generation of an infectious prion disease that differs from all others identified in hosts expressing the same PrPC sequence. These observations support the view that a variety of infectious prion strains might spontaneously emerge in hosts displaying random genetic PrPC mutations. PMID:24274622

Revisiting PC1/3 Mutants: Dominant-Negative Effect of Endoplasmic Reticulum-Retained Mutants.

PubMed

Blanco, Elias H; Ramos-Molina, Bruno; Lindberg, Iris

2015-10-01

Prohormone convertase 1/3 (PC1/3), encoded by the gene PCSK1, is critical for peptide hormone synthesis. An increasing number of studies have shown that inactivating mutations in PCSK1 are correlated with endocrine pathologies ranging from intestinal dysfunction to morbid obesity, whereas the common nonsynonymous polymorphisms rs6232 (N221D) and rs6234-rs6235 (Q665E-S690T) are highly associated with obesity risk. In this report, we revisited the biochemical and cellular properties of PC1/3 variants in the context of a wild-type PC1/3 background instead of the S357G hypermorph background used for all previous studies. In the wild-type background the PC1/3 N221D variant exhibited 30% lower enzymatic activity in a fluorogenic assay than wild-type PC1/3; this inhibition was greater than that detected in an equivalent experiment using the PC1/3 S357G background. A PC1/3 variant with the linked carboxyl-terminal polymorphisms Q665E-S690T did not show this difference. We also analyzed the biochemical properties of 2 PC1/3 mutants, G209R and G593R, which are retained in the endoplasmic reticulum (ER), and studied their effects on wild-type PC1/3. The expression of ER-retained mutants induced ER stress markers and also resulted in dominant-negative blockade of wild-type PC1/3 prodomain cleavage and decreased expression of wild-type PC1/3, suggesting facilitation of the entry of wild-type protein to a degradative proteasomal pathway. Dominant-negative effects of PC1/3 mutations on the expression and maturation of wild-type protein, with consequential effects on PC1/3 availability, add a new element which must be considered in population and clinical studies of this gene.

The single-strand DNA binding activity of human PC4 preventsmutagenesis and killing by oxidative DNA damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Jen-Yeu; Sarker, Altaf Hossain; Cooper, Priscilla K.

Human positive cofactor 4 (PC4) is a transcriptional coactivator with a highly conserved single-strand DNA (ssDNA) binding domain of unknown function. We identified PC4 as a suppressor of the oxidative mutator phenotype of the Escherichia coli fpg mutY mutant and demonstrate that this suppression requires its ssDNA binding activity. Yeast mutants lacking their PC4 ortholog Sub1 are sensitive to hydrogen peroxide and exhibit spontaneous and peroxide induced hypermutability. PC4 expression suppresses the peroxide sensitivity of the yeast sub l{Delta} mutant, suggesting that the human protein has a similar function. A role for yeast and human proteins in DNA repair ismore » suggested by the demonstration that Sub1 acts in a peroxide-resistance pathway involving Rad2 and by the physical interaction of PC4 with the human Rad2 homolog XPG. We show XPG recruits PC4 to a bubble-containing DNA substrate with resulting displacement of XPG and formation of a PC4-DNA complex. We discuss the possible requirement for PC4 in either global or transcription-coupled repair of oxidative DNA damage to mediate the release of XPG bound to its substrate.« less

A New Mouse Allele of Glutamate Receptor Delta 2 with Cerebellar Atrophy and Progressive Ataxia

PubMed Central

Miyoshi, Yuka; Yoshioka, Yoshichika; Suzuki, Kinuko; Miyazaki, Taisuke; Koura, Minako; Saigoh, Kazumasa; Kajimura, Naoko; Monobe, Yoko; Kusunoki, Susumu; Matsuda, Junichiro; Watanabe, Masahiko; Hayasaka, Naoto

2014-01-01

Spinocerebellar degenerations (SCDs) are a large class of sporadic or hereditary neurodegenerative disorders characterized by progressive motion defects and degenerative changes in the cerebellum and other parts of the CNS. Here we report the identification and establishment from a C57BL/6J mouse colony of a novel mouse line developing spontaneous progressive ataxia, which we refer to as ts3. Frequency of the phenotypic expression was consistent with an autosomal recessive Mendelian trait of inheritance, suggesting that a single gene mutation is responsible for the ataxic phenotype of this line. The onset of ataxia was observed at about three weeks of age, which slowly progressed until the hind limbs became entirely paralyzed in many cases. Micro-MRI study revealed significant cerebellar atrophy in all the ataxic mice, although individual variations were observed. Detailed histological analyses demonstrated significant atrophy of the anterior folia with reduced granule cells (GC) and abnormal morphology of cerebellar Purkinje cells (PC). Study by ultra-high voltage electron microscopy (UHVEM) further indicated aberrant morphology of PC dendrites and their spines, suggesting both morphological and functional abnormalities of the PC in the mutants. Immunohistochemical studies also revealed defects in parallel fiber (PF)–PC synapse formation and abnormal distal extension of climbing fibers (CF). Based on the phenotypic similarities of the ts3 mutant with other known ataxic mutants, we performed immunohistological analyses and found that expression levels of two genes and their products, glutamate receptor delta2 (grid2) and its ligand, cerebellin1 (Cbln1), are significantly reduced or undetectable. Finally, we sequenced the candidate genes and detected a large deletion in the coding region of the grid2 gene. Our present study suggests that ts3 is a new allele of the grid2 gene, which causes similar but different phenotypes as compared to other grid2 mutants. PMID:25250835

Underlying mechanisms of cyclic peptide inhibitors interrupting the interaction of CK2α/CK2β: comparative molecular dynamics simulation studies.

PubMed

Zhou, Yue; Zhang, Na; Chen, Wenjuan; Zhao, Lijiao; Zhong, Rugang

2016-04-07

Protein-protein interactions (PPIs) are fundamental to all biological processes. Recently, the CK2β-derived cyclic peptide Pc has been demonstrated to efficiently antagonize the CK2α/CK2β interaction and strongly affect the phosphorylation of CK2β-dependent CK2 substrate specificity. The binding affinity of Pc to CK2α is destroyed to different extents by two single-point mutations of Tyr188 to Ala (Y188A) and Phe190 to Ala (F190A), which exert negative effects on the inhibitory activity (IC50) of Pc against the CK2α/CK2β interaction from 3.0 μM to 54.0 μM and ≫100 μM, respectively. However, the structural influences of Y188A and F190A mutations on the CK2α-Pc complex remain unclear. In this study, comparative molecular dynamics (MD) simulations, principal component analysis (PCA), domain cross-correlation map (DCCM) analysis and energy calculations were performed on wild type (WT), Y188A mutant, and F190A mutant systems. The results revealed that ordered communications between hydrophobic and polar interactions were essential for CK2α-Pc binding in the WT system. In addition to the loss of the hydrogen bond between Gln36 of CK2α and Gly189 of Pc in the two mutants, the improper recognition mechanisms occurred through different pathways. These pathways included the weakened hydrophobic interactions in the Y188A mutant as well as decreased polar and hydrophobic interactions in the F190A mutant. The energy analysis results qualitatively elucidated the instability of the two mutants and energetic contributions of the key residues. This study not only revealed the structural mechanisms for the decreased binding affinity of Y188A and F190A mutant CK2α-Pc complexes, but also provided valuable clues for the rational design of CK2α/CK2β subunit interaction inhibitors with high affinity and specificity.

Phototrophic cultivation of NaCl-tolerant mutant of Spirulina platensis for enhanced C-phycocyanin production under optimized culture conditions and its dynamic modeling.

PubMed

Gupta, Apurva; Mohan, Devendra; Saxena, Rishi Kumar; Singh, Surendra

2018-02-01

Commercial cultivation of Spirulina sp. is highly popular due to the presence of high amount of C-phycocyanin (C-PC) and other valuable chemicals like carotenoids and γ-linolenic acid. In this study, the pH and the concentrations of nitrogen and carbon source were manipulated to achieve improved cell growth and C-PC production in NaCl-tolerant mutant of Spirulina platensis. In this study, highest C-PC (147 mg · L -1 ) and biomass (2.83 g · L -1 ) production was achieved when a NaCl-tolerant mutant of S. platensis was cultivated in a nitrate and bicarbonate sufficient medium (40 and 60 mM, respectively) at pH 9.0 under phototrophic conditions. Kinetic study of wildtype S. platensis and its NaCl-tolerant mutant was also done to determine optimum nitrate concentrations for maximum growth and C-PC production. Kinetic parameter of inhibition (Haldane model) was fitted to the relationship between specific growth rate and substrate concentration obtained from the growth curves. Results showed that the maximum specific growth rate (μ max ) for NaCl-tolerant mutant increased by 17.94% as compared to its wildtype counterpart, with a slight increase in half-saturation constant (K s ), indicating that this strain could grow well at high concentration of NaNO 3 . C-PC production rate (C max ) in mutant cells increased by 12.2% at almost half the value of K s as compared to its wildtype counterpart. Moreover, the inhibition constant (K i ) value was 207.85% higher in NaCl-tolerant mutant as compared to its wildtype strain, suggesting its ability to produce C-PC even at high concentrations of NaNO 3 . © 2017 Phycological Society of America.

A defect in the inflammation-primed macrophage-activation cascade in osteopetrotic rats.

PubMed

Yamamoto, N; Lindsay, D D; Naraparaju, V R; Ireland, R A; Popoff, S N

1994-05-15

Macrophages were activated by administration of lysophosphatidylcholine (lyso-Pc) or dodecylglycerol (DDG) to wild-type rats but not in osteopetrotic (op) mutant rats. In vitro treatment of wild-type rat peritoneal cells with lyso-Pc or DDG efficiently activated macrophages whereas treatment of op mutant rat peritoneal cells with lyso-Pc or DDG did not activate macrophages. The inflammation-primed macrophage activation cascade in rats requires participation of B lymphocytes and vitamin D binding protein (DBP). Lyso-Pc-inducible beta-galactosidase of wild-type rat B lymphocytes can convert DBP to the macrophage-activating factor (MAF), whereas B lymphocytes of the op mutant rats were shown to be deficient in lyso-Pc-inducible beta-galactosidase. DBP is conserved among mammalian species. Treatment of human DBP (Gc1 protein) with commercial glycosidases yields an extremely high titrated MAF as assayed on mouse and rat macrophages. Because the enzymatically generated MAF (GcMAF) bypasses the role of lymphocytes in macrophage activation, the op mutant rat macrophages were efficiently activated by administration of a small quantity (100 pg/rat) of GcMAF. Likewise, in vitro treatment of op rat peritoneal cells with as little as 40 pg GcMAF/ml activated macrophages.

Cadmium-sensitive, cad1 mutants of Arabidopsis thaliana are phytochelatin deficient.

PubMed Central

Howden, R; Goldsbrough, P B; Andersen, C R; Cobbett, C S

1995-01-01

An allelic series of cad1, cadmium-sensitive mutants of Arabidopsis thaliana, was isolated. These mutants were sensitive to cadmium to different extents and were deficient in their ability to form cadmium-peptide complexes as detected by gel-filtration chromatography. Each mutant was deficient in its ability to accumulate phytochelatins (PCs) as detected by high-performance liquid chromatography and the amount of PCs accumulated by each mutant correlated with its degree of sensitivity to cadmium. The mutants had wild-type levels of glutathione, the substrate for PC biosynthesis, and in vitro assays demonstrated that each of the mutants was deficient in PC synthase activity. These results demonstrate conclusively the importance of PCs for cadmium tolerance in plants. PMID:7770517

Phytochelatin Synthesis Promotes Leaf Zn Accumulation of Arabidopsis thaliana Plants Grown in Soil with Adequate Zn Supply and is Essential for Survival on Zn-Contaminated Soil.

PubMed

Kühnlenz, Tanja; Hofmann, Christian; Uraguchi, Shimpei; Schmidt, Holger; Schempp, Stefanie; Weber, Michael; Lahner, Brett; Salt, David E; Clemens, Stephan

2016-11-01

Phytochelatin (PC) synthesis is essential for the detoxification of non-essential metals such as cadmium (Cd). In vitro experiments with Arabidopsis thaliana seedlings had indicated a contribution to zinc (Zn) tolerance as well. We addressed the physiological role of PC synthesis in Zn homeostasis of plants under more natural conditions. Growth responses, PC accumulation and leaf ionomes of wild-type and AtPCS1 mutant plants cultivated in different soils representing adequate Zn supply, Zn deficiency and Zn excess were analyzed. Growth on Zn-contaminated soil triggers PC synthesis and is strongly impaired in PC-deficient mutants. In fact, the contribution of AtPCS1 to tolerating Zn excess is comparable with that of the major Zn tolerance factor MTP1. For plants supplied with a normal level of Zn, a significant reduction in leaf Zn accumulation of AtPCS1 mutants was detected. In contrast, AtPCS1 mutants grown under Zn-limited conditions showed wild-type levels of Zn accumulation, suggesting the operation of distinct Zn translocation pathways. Contrasting phenotypes of the tested AtPCS1 mutant alleles upon growth in Zn- or Cd-contaminated soil indicated differential activation of PC synthesis by these metals. Experiments with truncated versions identified a part of the AtPCS1 protein required for the activation by Zn but not by Cd. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

The Legionella pneumophila Dot/Icm-secreted Effector PlcC/CegC1 Together with PlcA and PlcB Promotes Virulence and Belongs to a Novel Zinc Metallophospholipase C Family Present in Bacteria and Fungi*

PubMed Central

Aurass, Philipp; Schlegel, Maren; Metwally, Omar; Harding, Clare R.; Schroeder, Gunnar N.; Frankel, Gad; Flieger, Antje

2013-01-01

Legionella pneumophila is a water-borne bacterium that causes pneumonia in humans. PlcA and PlcB are two previously defined L. pneumophila proteins with homology to the phosphatidylcholine-specific phospholipase C (PC-PLC) of Pseudomonas fluorescens. Additionally, we found that Lpg0012 shows similarity to PLCs and has been shown to be a Dot/Icm-injected effector, CegC1, which is designated here as PlcC. It remained unclear, however, whether these L. pneumophila proteins exhibit PLC activity. PlcC expressed in Escherichia coli hydrolyzed a broad phospholipid spectrum, including PC, phosphatidylglycerol (PG), and phosphatidylinositol. The addition of Zn2+ ions activated, whereas EDTA inhibited, PlcC-derived PLC activity. Protein homology search revealed that the three Legionella enzymes and P. fluorescens PC-PLC share conserved domains also present in uncharacterized fungal proteins. Fifteen conserved amino acids were essential for enzyme activity as identified via PlcC mutagenesis. Analysis of defined L. pneumophila knock-out mutants indicated Lsp-dependent export of PG-hydrolyzing PLC activity. PlcA and PlcB exhibited PG-specific activity and contain a predicted Sec signal sequence. In line with the reported requirement of host cell contact for Dot/Icm-dependent effector translocation, PlcC showed cell-associated PC-specific PLC activity after bacterial growth in broth. A PLC triple mutant, but not single or double mutants, exhibited reduced host killing in a Galleria mellonella infection model, highlighting the importance of the three PLCs in pathogenesis. In summary, we describe here a novel Zn2+-dependent PLC family present in Legionella, Pseudomonas, and fungi with broad substrate preference and function in virulence. PMID:23457299

The Legionella pneumophila Dot/Icm-secreted effector PlcC/CegC1 together with PlcA and PlcB promotes virulence and belongs to a novel zinc metallophospholipase C family present in bacteria and fungi.

PubMed

Aurass, Philipp; Schlegel, Maren; Metwally, Omar; Harding, Clare R; Schroeder, Gunnar N; Frankel, Gad; Flieger, Antje

2013-04-19

Legionella pneumophila is a water-borne bacterium that causes pneumonia in humans. PlcA and PlcB are two previously defined L. pneumophila proteins with homology to the phosphatidylcholine-specific phospholipase C (PC-PLC) of Pseudomonas fluorescens. Additionally, we found that Lpg0012 shows similarity to PLCs and has been shown to be a Dot/Icm-injected effector, CegC1, which is designated here as PlcC. It remained unclear, however, whether these L. pneumophila proteins exhibit PLC activity. PlcC expressed in Escherichia coli hydrolyzed a broad phospholipid spectrum, including PC, phosphatidylglycerol (PG), and phosphatidylinositol. The addition of Zn(2+) ions activated, whereas EDTA inhibited, PlcC-derived PLC activity. Protein homology search revealed that the three Legionella enzymes and P. fluorescens PC-PLC share conserved domains also present in uncharacterized fungal proteins. Fifteen conserved amino acids were essential for enzyme activity as identified via PlcC mutagenesis. Analysis of defined L. pneumophila knock-out mutants indicated Lsp-dependent export of PG-hydrolyzing PLC activity. PlcA and PlcB exhibited PG-specific activity and contain a predicted Sec signal sequence. In line with the reported requirement of host cell contact for Dot/Icm-dependent effector translocation, PlcC showed cell-associated PC-specific PLC activity after bacterial growth in broth. A PLC triple mutant, but not single or double mutants, exhibited reduced host killing in a Galleria mellonella infection model, highlighting the importance of the three PLCs in pathogenesis. In summary, we describe here a novel Zn(2+)-dependent PLC family present in Legionella, Pseudomonas, and fungi with broad substrate preference and function in virulence.

An Arabidopsis mutant showing reduced feedback inhibition of photosynthesis.

PubMed

Van Oosten, J J; Gerbaud, A; Huijser, C; Dijkwel, P P; Chua, N H; Smeekens, S C

1997-11-01

Many plant genes are responsive to sugars but the mechanisms used by plants to sense sugars are unknown. A genetic approach has been used in Arabidopsis to identify genes involved in perception and transduction of sugar signals. For this purpose, an in vivo reporter system was established consisting of the light- and sugar-regulated plastocyanin promoter, fused to the luciferase coding sequence (PC-LUC construct). At the seedling stage, expression of the PC-LUC gene is repressed by sucrose, and a number of sucrose-uncoupled (sun) mutants were selected in which sucrose is unable to repress the activity of the PC promoter. Three mutants have been characterized in more detail. The sugar analog 2-deoxy-D-glucose (2DG) was used to repress whole plant photosynthesis, PC-LUC gene expression and total ribulose-1,5-bisphosphate activity. It was found that the sun6 mutation makes plants unresponsive to these 2DG-induced effects. Moreover, unlike wild-type plants, sun6 mutants are insensitive to elevated levels of glucose in the growth medium. These findings suggest that the SUN6 gene is active in a hexose-activated signal transduction pathway.

Alteration of cell wall xylan acetylation triggers defense responses that counterbalance the immune deficiencies of plants impaired in the β-subunit of the heterotrimeric G-protein.

PubMed

Escudero, Viviana; Jordá, Lucía; Sopeña-Torres, Sara; Mélida, Hugo; Miedes, Eva; Muñoz-Barrios, Antonio; Swami, Sanjay; Alexander, Danny; McKee, Lauren S; Sánchez-Vallet, Andrea; Bulone, Vincent; Jones, Alan M; Molina, Antonio

2017-11-01

Arabidopsis heterotrimeric G-protein complex modulates pathogen-associated molecular pattern-triggered immunity (PTI) and disease resistance responses to different types of pathogens. It also plays a role in plant cell wall integrity as mutants impaired in the Gβ- (agb1-2) or Gγ-subunits have an altered wall composition compared with wild-type plants. Here we performed a mutant screen to identify suppressors of agb1-2 (sgb) that restore susceptibility to pathogens to wild-type levels. Out of the four sgb mutants (sgb10-sgb13) identified, sgb11 is a new mutant allele of ESKIMO1 (ESK1), which encodes a plant-specific polysaccharide O-acetyltransferase involved in xylan acetylation. Null alleles (sgb11/esk1-7) of ESK1 restore to wild-type levels the enhanced susceptibility of agb1-2 to the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM), but not to the bacterium Pseudomonas syringae pv. tomato DC3000 or to the oomycete Hyaloperonospora arabidopsidis. The enhanced resistance to PcBMM of the agb1-2 esk1-7 double mutant was not the result of the re-activation of deficient PTI responses in agb1-2. Alteration of cell wall xylan acetylation caused by ESK1 impairment was accompanied by an enhanced accumulation of abscisic acid, the constitutive expression of genes encoding antibiotic peptides and enzymes involved in the biosynthesis of tryptophan-derived metabolites, and the accumulation of disease resistance-related secondary metabolites and different osmolites. These esk1-mediated responses counterbalance the defective PTI and PcBMM susceptibility of agb1-2 plants, and explain the enhanced drought resistance of esk1 plants. These results suggest that a deficient PTI-mediated resistance is partially compensated by the activation of specific cell-wall-triggered immune responses. © 2017 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

Autism-associated R451C mutation in neuroligin3 leads to activation of the unfolded protein response in a PC12 Tet-On inducible system

PubMed Central

Ulbrich, Lisa; Favaloro, Flores Lietta; Trobiani, Laura; Marchetti, Valentina; Patel, Vruti; Pascucci, Tiziana; Comoletti, Davide; Marciniak, Stefan J.; De Jaco, Antonella

2015-01-01

Several forms of monogenic heritable autism spectrum disorders are associated with mutations in the neuroligin genes. The autism-linked substitution R451C in neuroligin3 induces local misfolding of its extracellular domain, causing partial retention in the ER (endoplasmic reticulum) of expressing cells. We have generated a PC12 Tet-On cell model system with inducible expression of wild-type or R451C neuroligin3 to investigate whether there is activation of the UPR (unfolded protein response) as a result of misfolded protein retention. As a positive control for protein misfolding, we also expressed the mutant G221R neuroligin3, which is known to be completely retained within the ER. Our data show that overexpression of either R451C or G221R mutant proteins leads to the activation of all three signalling branches of the UPR downstream of the stress sensors ATF6 (activating transcription factor 6), IRE1 (inositol-requiring enzyme 1) and PERK [PKR (dsRNA-dependent protein kinase)-like endoplasmic reticulum kinase]. Each branch displayed different activation profiles that partially correlated with the degree of misfolding caused by each mutation. We also show that up-regulation of BiP (immunoglobulin heavy-chain-binding protein) and CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein] was induced by both mutant proteins but not by wild-type neuroligin3, both in proliferative cells and cells differentiated to a neuron-like phenotype. Collectively, our data show that mutant R451C neuroligin3 activates the UPR in a novel cell model system, suggesting that this cellular response may have a role in monogenic forms of autism characterized by misfolding mutations. PMID:26621873

Dopamine D2 receptor signaling modulates mutant ataxin-1 S776 phosphorylation and aggregation.

PubMed

Hearst, Scoty M; Lopez, Mariper E; Shao, Qingmei; Liu, Yong; Vig, Parminder J S

2010-08-01

Spinocerebellar ataxia 1 (SCA1) is a dominantly inherited neurodegenerative disease associated with progressive ataxia resulting from the loss of cerebellar Purkinje cells (PCs) and neurons in the brainstem. In PCs of SCA1 transgenic mice, the disease causing ataxin-1 protein mediates the formation of S100B containing cytoplasmic vacuoles and further self-aggregates to form intranuclear inclusions. The exact function of the ataxin-1 protein is not fully understood. However, the aggregation and neurotoxicity of the mutant ataxin-1 protein is dependent on the phosphorylation at serine 776 (S776). Although protein kinase A (PKA) has been implicated as the S776 kinase, the mechanism of PKA/ataxin-1 regulation in SCA1 is still not clear. We propose that a dopamine D(2) receptor (D2R)/S100B pathway may be involved in modulating PKA activity in PCs. Using a D2R/S100B HEK stable cell line transiently transfected with GFP-ataxin-1[82Q], we demonstrate that stimulation of the D2R/S100B pathway caused a reduction in mutant ataxin-1 S776 phosphorylation and ataxin-1 aggregation. Activation of PKA by forskolin resulted in an enhanced S776 phosphorylation and increased ataxin-1 nuclear aggregation, which was suppressed by treatment with D2R agonist bromocriptine and PKA inhibitor H89. Furthermore, treating SCA1 transgenic PC slice cultures with forskolin induced neurodegenerative morphological abnormalities in PC dendrites consistent with those observed in vivo. Taken together our data support a mechanism where PKA dependent mutant ataxin-1 phosphorylation and aggregation can be regulated by D2R/S100B signaling.

Dopamine D2 Receptor Signaling Modulates Mutant Ataxin-1 S776 Phosphorylation and Aggregation

PubMed Central

Hearst, SM; Lopez, ME; Shao, Q; Liu, Y; Vig, PJS

2010-01-01

Spinocerebellar ataxia 1 (SCA1) is a dominantly inherited neurodegenerative disease associated with progressive ataxia resulting from the loss of cerebellar Purkinje cells (PCs) and neurons in the brainstem. In PCs of SCA1 transgenic (Tg) mice, the disease causing ataxin-1 protein mediates the formation of S100B containing cytoplasmic vacuoles and further self-aggregates to form intranuclear inclusions. The exact function of the ataxin-1 protein is not fully understood. However, the aggregation and neurotoxicity of the mutant ataxin-1 protein is dependent on the phosphorylation at serine 776 (S776). Although protein kinase A (PKA) has been implicated as the S776 kinase, the mechanism of PKA/ataxin-1 regulation in SCA1 is still not clear. We propose that a dopamine D2 receptor (D2R)/S100B pathway may be involved in modulating PKA activity in PCs. Using a D2R/S100B HEK stable cell line transiently transfected with GFP-ataxin-1[82Q], we demonstrate that stimulation of the D2R/S100B pathway caused a reduction in mutant ataxin-1 S776 phosphorylation and ataxin-1 aggregation. Activation of PKA by forskolin resulted in an enhanced S776 phosphorylation and increased ataxin-1 nuclear aggregation, which was suppressed by treatment with D2R agonist bromocriptine and PKA inhibitor H89. Furthermore, treating SCA1 Tg PC slice cultures with forskolin induced neurodegenerative morphological abnormalities in PC dendrites consistent with those observed in vivo. Taken together our data support a mechanism where PKA dependent mutant ataxin-1 phosphorylation and aggregation can be regulated by D2R/S100B signaling. PMID:20477910

Down-regulation of polycystin in lymphatic malformations: possible role in the proliferation of lymphatic endothelial cells.

PubMed

Ren, Jian-Gang; Xia, Hou-Fu; Yang, Jie-Gang; Zhu, Jun-Yi; Zhang, Wei; Chen, Gang; Zhao, Ji-Hong; Sun, Yan-Fang; Zhao, Yi-Fang

2017-07-01

Lymphatic malformations (LMs) are composed of aberrant lymphatic vessels and regarded as benign growths of the lymphatic system. Recent studies have demonstrated that the mutant embryos of PKD1 and PKD2, encoding polycystin-1 (PC-1) and polycystin-2 (PC-2), respectively, result in aberrant lymphatic vessels similar to those observed in LMs. In this study, for the first time, we investigated PC-1 and PC-2 expression and assessed their roles in the development of LMs. Our results demonstrated that PC-1 and PC-2 gene and protein expressions were obviously decreased in LMs compared with normal skin tissues. In addition, the expression of phosphorylated ERK but not total ERK was up-regulated in LMs and negatively correlated with the expression of PC-1 and PC-2. Moreover, up-regulation of Ki67 was detected in LMs and positively correlated with ERK phosphorylation levels. Furthermore, cluster analysis better reflected close correlation between these signals. All of the above results provided strong evidence suggesting that the hyperactivation of the ERK pathway may be caused by down-regulation of PC-1 and PC-2 in LMs, contributing to increased proliferation of lymphatic endothelial cells in LMs. Our present study sheds light on novel potential mechanisms involved in LMs and may help to explore novel treatments for LMs. Copyright © 2017 Elsevier Inc. All rights reserved.

Prion Propagation in Cells Expressing PrP Glycosylation Mutants ▿

PubMed Central

Salamat, Muhammad K.; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

2011-01-01

Infection by prions involves conversion of a host-encoded cell surface protein (PrPC) to a disease-related isoform (PrPSc). PrPC carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrPC glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrPSc and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrPSc, while PrPC with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrPC, were able to form infectious PrPSc. Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection. PMID:21248032

Regulation of Polycystin-1 Function by Calmodulin Binding

PubMed Central

Doerr, Nicholas; Wang, Yidi; Kipp, Kevin R.; Liu, Guangyi; Benza, Jesse J.; Pletnev, Vladimir; Pavlov, Tengis S.; Staruschenko, Alexander; Mohieldin, Ashraf M.; Takahashi, Maki; Nauli, Surya M.; Weimbs, Thomas

2016-01-01

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common genetic disease that leads to progressive renal cyst growth and loss of renal function, and is caused by mutations in the genes encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. The PC1/PC2 complex localizes to primary cilia and can act as a flow-dependent calcium channel in addition to numerous other signaling functions. The exact functions of the polycystins, their regulation and the purpose of the PC1/PC2 channel are still poorly understood. PC1 is an integral membrane protein with a large extracytoplasmic N-terminal domain and a short, ~200 amino acid C-terminal cytoplasmic tail. Most proteins that interact with PC1 have been found to bind via the cytoplasmic tail. Here we report that the PC1 tail has homology to the regulatory domain of myosin heavy chain including a conserved calmodulin-binding motif. This motif binds to CaM in a calcium-dependent manner. Disruption of the CaM-binding motif in PC1 does not affect PC2 binding, cilia targeting, or signaling via heterotrimeric G-proteins or STAT3. However, disruption of CaM binding inhibits the PC1/PC2 calcium channel activity and the flow-dependent calcium response in kidney epithelial cells. Furthermore, expression of CaM-binding mutant PC1 disrupts cellular energy metabolism. These results suggest that critical functions of PC1 are regulated by its ability to sense cytosolic calcium levels via binding to CaM. PMID:27560828

Regulation of polycystin-1 ciliary trafficking by motifs at its C-terminus and polycystin-2 but not by cleavage at the GPS site

PubMed Central

Su, Xuefeng; Wu, Maoqing; Yao, Gang; El-Jouni, Wassim; Luo, Chong; Tabari, Azadeh; Zhou, Jing

2015-01-01

ABSTRACT Failure to localize membrane proteins to the primary cilium causes a group of diseases collectively named ciliopathies. Polycystin-1 (PC1, also known as PKD1) is a large ciliary membrane protein defective in autosomal dominant polycystic kidney disease (ADPKD). Here, we developed a large set of PC1 expression constructs and identified multiple sequences, including a coiled-coil motif in the C-terminal tail of PC1, regulating full-length PC1 trafficking to the primary cilium. Ciliary trafficking of wild-type and mutant PC1 depends on the dose of polycystin-2 (PC2, also known as PKD2), and the formation of a PC1–PC2 complex. Modulation of the ciliary trafficking module mediated by the VxP ciliary-targeting sequence and Arf4 and Asap1 does not affect the ciliary localization of full-length PC1. PC1 also promotes PC2 ciliary trafficking. PC2 mutations truncating its C-terminal tail but not those changing the VxP sequence to AxA or impairing the pore of the channel, leading to a dead channel, affect PC1 ciliary trafficking. Cleavage at the GPCR proteolytic site (GPS) of PC1 is not required for PC1 trafficking to cilia. We propose a mutually dependent model for the ciliary trafficking of PC1 and PC2, and that PC1 ciliary trafficking is regulated by multiple cis-acting elements. As all pathogenic PC1 mutations tested here are defective in ciliary trafficking, ciliary trafficking might serve as a functional read-out for ADPKD. PMID:26430213

Regulation of polycystin-1 ciliary trafficking by motifs at its C-terminus and polycystin-2 but not by cleavage at the GPS site.

PubMed

Su, Xuefeng; Wu, Maoqing; Yao, Gang; El-Jouni, Wassim; Luo, Chong; Tabari, Azadeh; Zhou, Jing

2015-11-15

Failure to localize membrane proteins to the primary cilium causes a group of diseases collectively named ciliopathies. Polycystin-1 (PC1, also known as PKD1) is a large ciliary membrane protein defective in autosomal dominant polycystic kidney disease (ADPKD). Here, we developed a large set of PC1 expression constructs and identified multiple sequences, including a coiled-coil motif in the C-terminal tail of PC1, regulating full-length PC1 trafficking to the primary cilium. Ciliary trafficking of wild-type and mutant PC1 depends on the dose of polycystin-2 (PC2, also known as PKD2), and the formation of a PC1-PC2 complex. Modulation of the ciliary trafficking module mediated by the VxP ciliary-targeting sequence and Arf4 and Asap1 does not affect the ciliary localization of full-length PC1. PC1 also promotes PC2 ciliary trafficking. PC2 mutations truncating its C-terminal tail but not those changing the VxP sequence to AxA or impairing the pore of the channel, leading to a dead channel, affect PC1 ciliary trafficking. Cleavage at the GPCR proteolytic site (GPS) of PC1 is not required for PC1 trafficking to cilia. We propose a mutually dependent model for the ciliary trafficking of PC1 and PC2, and that PC1 ciliary trafficking is regulated by multiple cis-acting elements. As all pathogenic PC1 mutations tested here are defective in ciliary trafficking, ciliary trafficking might serve as a functional read-out for ADPKD. © 2015. Published by The Company of Biologists Ltd.

Prion Protein Promotes Kidney Iron Uptake via Its Ferrireductase Activity*

PubMed Central

Haldar, Swati; Tripathi, Ajai; Qian, Juan; Beserra, Amber; Suda, Srinivas; McElwee, Matthew; Turner, Jerrold; Hopfer, Ulrich; Singh, Neena

2015-01-01

Brain iron-dyshomeostasis is an important cause of neurotoxicity in prion disorders, a group of neurodegenerative conditions associated with the conversion of prion protein (PrPC) from its normal conformation to an aggregated, PrP-scrapie (PrPSc) isoform. Alteration of iron homeostasis is believed to result from impaired function of PrPC in neuronal iron uptake via its ferrireductase activity. However, unequivocal evidence supporting the ferrireductase activity of PrPC is lacking. Kidney provides a relevant model for this evaluation because PrPC is expressed in the kidney, and ∼370 μg of iron are reabsorbed daily from the glomerular filtrate by kidney proximal tubule cells (PT), requiring ferrireductase activity. Here, we report that PrPC promotes the uptake of transferrin (Tf) and non-Tf-bound iron (NTBI) by the kidney in vivo and mainly NTBI by PT cells in vitro. Thus, uptake of 59Fe administered by gastric gavage, intravenously, or intraperitoneally was significantly lower in PrP-knock-out (PrP−/−) mouse kidney relative to PrP+/+ controls. Selective in vivo radiolabeling of plasma NTBI with 59Fe revealed similar results. Expression of exogenous PrPC in immortalized PT cells showed localization on the plasma membrane and intracellular vesicles and increased transepithelial transport of 59Fe-NTBI and to a smaller extent 59Fe-Tf from the apical to the basolateral domain. Notably, the ferrireductase-deficient mutant of PrP (PrPΔ51–89) lacked this activity. Furthermore, excess NTBI and hemin caused aggregation of PrPC to a detergent-insoluble form, limiting iron uptake. Together, these observations suggest that PrPC promotes retrieval of iron from the glomerular filtrate via its ferrireductase activity and modulates kidney iron metabolism. PMID:25572394

Brucella abortus Synthesizes Phosphatidylcholine from Choline Provided by the Host

PubMed Central

Comerci, Diego J.; Altabe, Silvia; de Mendoza, Diego; Ugalde, Rodolfo A.

2006-01-01

The Brucella cell envelope is characterized by the presence of phosphatidylcholine (PC), a common phospholipid in eukaryotes that is rare in prokaryotes. Studies on the composition of Brucella abortus 2308 phospholipids revealed that the synthesis of PC depends on the presence of choline in the culture medium, suggesting that the methylation biosynthetic pathway is not functional. Phospholipid composition of pmtA and pcs mutants indicated that in Brucella, PC synthesis occurs exclusively via the phosphatidylcholine synthase pathway. Transformation of Escherichia coli with an expression vector containing the B. abortus pcs homologue was sufficient for PC synthesis upon induction with IPTG (isopropyl-β-d-thiogalactopyranoside), while no PC formation was detected when bacteria were transformed with a vector containing pmtA. These findings imply that Brucella depends on choline provided by the host cell to form PC. We could not detect any obvious associated phenotype in the PC-deficient strain under vegetative or intracellular growth conditions in macrophages. However, the pcs mutant strain displays a reproducible virulence defect in mice, which suggests that PC is necessary to sustain a chronic infection process. PMID:16484204

Regulation of phospholipid synthesis in phosphatidylserine synthase-deficient (chol) mutants of Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Letts, V.A.; Henry, S.A.

1985-08-01

Saccharomyces cerevisiae mutants, chol, are deficient in the synthesis of the phospholipid phosphatidylserine owing to lowered activity of the membrane-associated enzyme phosphatidylserine synthase. These mutants are auxotrophic for ethanolamine or choline and, in the absence of these supplements, cannot synthesize phosphatidylethanolamine or phosphatidylcholine (PC). The authors exploited these characteristics of the chol mutants to examine the regulation of phospholipid metabolism in S. cerevisiae. Macromolecular synthesis and phospholipid metabolism were examined in chol cells starved for ethanolamine. Coupled to the decline in PC biosynthesis was a simultaneous decrease in the overall rate of phospholipid synthesis. In particular, the rate of synthesismore » of phosphatidylinositol decreased in parallel with the decline in PC biosynthesis. However, under conditions of ethanolamine deprivation in chol cells, the cytoplasmic enzyme inositol-1-phosphate synthase could not be repressed by exogenous inositol, and the endogenous synthesis of the phospholipid precursor inositol appeared to be elevated. The implications of these findings with respect to the coordinated regulation of phospholipid synthesis are discussed.« less

PCSK1 Variants and Human Obesity.

PubMed

Ramos-Molina, B; Martin, M G; Lindberg, I

2016-01-01

PCSK1, encoding prohormone convertase 1/3 (PC1/3), was one of the first genes linked to monogenic early-onset obesity. PC1/3 is a protease involved in the biosynthetic processing of a variety of neuropeptides and prohormones in endocrine tissues. PC1/3 activity is essential for the activating cleavage of many peptide hormone precursors implicated in the regulation of food ingestion, glucose homeostasis, and energy homeostasis, for example, proopiomelanocortin, proinsulin, proglucagon, and proghrelin. A large number of genome-wide association studies in a variety of different populations have now firmly established a link between three PCSK1 polymorphisms frequent in the population and increased risk of obesity. Human subjects with PC1/3 deficiency, a rare autosomal-recessive disorder caused by the presence of loss-of-function mutations in both alleles, are obese and display a complex set of endocrinopathies. Increasing numbers of genetic diagnoses of infants with persistent diarrhea has recently led to the finding of many novel PCSK1 mutations. PCSK1-deficient infants experience severe intestinal malabsorption during the first years of life, requiring controlled nutrition; these children then become hyperphagic, with associated obesity. The biochemical characterization of novel loss-of-function PCSK1 mutations has resulted in the discovery of new pathological mechanisms affecting the cell biology of the endocrine cell beyond simple loss of enzyme activity, for example, dominant-negative effects of certain mutants on wild-type PC1/3 protein, and activation of the cellular unfolded protein response by endoplasmic reticulum-retained mutants. A better understanding of these molecular and cellular pathologies may illuminate possible treatments for the complex endocrinopathy of PCSK1 deficiency, including obesity. Copyright © 2016 Elsevier Inc. All rights reserved.

Blocking phosphatidylcholine utilization in Pseudomonas aeruginosa, via mutagenesis of fatty acid, glycerol and choline degradation pathways, confirms the importance of this nutrient source in vivo.

PubMed

Sun, Zhenxin; Kang, Yun; Norris, Michael H; Troyer, Ryan M; Son, Mike S; Schweizer, Herbert P; Dow, Steven W; Hoang, Tung T

2014-01-01

Pseudomonas aeruginosa can grow to very high-cell-density (HCD) during infection of the cystic fibrosis (CF) lung. Phosphatidylcholine (PC), the major component of lung surfactant, has been hypothesized to support HCD growth of P. aeruginosa in vivo. The phosphorylcholine headgroup, a glycerol molecule, and two long-chain fatty acids (FAs) are released by enzymatic cleavage of PC by bacterial phospholipase C and lipases. Three different bacterial pathways, the choline, glycerol, and fatty acid degradation pathways, are then involved in the degradation of these PC components. Here, we identified five potential FA degradation (Fad) related fadBA-operons (fadBA1-5, each encoding 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA thiolase). Through mutagenesis and growth analyses, we showed that three (fadBA145) of the five fadBA-operons are dominant in medium-chain and long-chain Fad. The triple fadBA145 mutant also showed reduced ability to degrade PC in vitro. We have previously shown that by partially blocking Fad, via mutagenesis of fadBA5 and fadDs, we could significantly reduce the ability of P. aeruginosa to replicate on FA and PC in vitro, as well as in the mouse lung. However, no studies have assessed the ability of mutants, defective in choline and/or glycerol degradation in conjunction with Fad, to grow on PC or in vivo. Hence, we constructed additional mutants (ΔfadBA145ΔglpD, ΔfadBA145ΔbetAB, and ΔfadBA145ΔbetABΔglpD) significantly defective in the ability to degrade FA, choline, and glycerol and, therefore, PC. The analysis of these mutants in the BALB/c mouse lung infection model showed significant inability to utilize PC in vitro, resulted in decreased replication fitness and competitiveness in vivo compared to the complement strain, although there was little to no variation in typical virulence factor production (e.g., hemolysin, lipase, and protease levels). This further supports the hypothesis that lung surfactant PC serves as an important nutrient for P. aeruginosa during CF lung infection.

Identification of a β-lactamase inhibitory protein variant that is a potent inhibitor of Staphylococcus PC1 β-lactamase

PubMed Central

Yuan, Ji; Chow, Dar-Chone; Huang, Wanzhi; Palzkill, Timothy

2011-01-01

The β-lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A β-lactamases. Widespread resistance to β-lactam antibiotics currently limits treatment strategies for Staphylococcus infections. The goal of this study was to determine the binding affinity of BLIP for S. aureus PC1 β-lactamase and to identify mutants that alter binding affinity. The BLIP inhibition constant (Ki) for the PC1 β-lactamase was measured at 350 nM and isothermal titration calorimetry (ITC) experiments indicated a binding constant (Kd) of 380 nM. A total of 23 residue positions in BLIP that contact β-lactamase were randomized and phage display was used to sort the libraries for tight binders to immobilized PC1 β-lactamase. The BLIP K74G mutant was the dominant clone selected and it was found to inhibit the PC1 β-lactamase with a Ki of 42 nM while calorimetry indicated a Kd of 26 nM. Molecular modeling studies suggested BLIP binds weakly to the PC1 β-lactamase due to the presence of alanine at position 104 of PC1. This position is occupied by glutamate in the TEM-1 enzyme where it forms a salt bridge with BLIP residue Lys74 that is important for the stability of the complex. This hypothesis was confirmed by showing that the A104E PC1 enzyme binds BLIP with 15-fold greater affinity than wild type PC1 β-lactamase. Kinetic measurements indicated similar association rates for all complexes with the variation in affinity due to altered dissociation rate constants suggesting changes in short-range interactions are responsible for the altered binding properties of the mutants. PMID:21238457

Functional defect of variants in the adenosine triphosphate-binding sites of ABCB4 and their rescue by the cystic fibrosis transmembrane conductance regulator potentiator, ivacaftor (VX-770).

PubMed

Delaunay, Jean-Louis; Bruneau, Alix; Hoffmann, Brice; Durand-Schneider, Anne-Marie; Barbu, Véronique; Jacquemin, Emmanuel; Maurice, Michèle; Housset, Chantal; Callebaut, Isabelle; Aït-Slimane, Tounsia

2017-02-01

ABCB4 (MDR3) is an adenosine triphosphate (ATP)-binding cassette (ABC) transporter expressed at the canalicular membrane of hepatocytes, where it mediates phosphatidylcholine (PC) secretion. Variations in the ABCB4 gene are responsible for several biliary diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3), a rare disease that can be lethal in the absence of liver transplantation. In this study, we investigated the effect and potential rescue of ABCB4 missense variations that reside in the highly conserved motifs of ABC transporters, involved in ATP binding. Five disease-causing variations in these motifs have been identified in ABCB4 (G535D, G536R, S1076C, S1176L, and G1178S), three of which are homologous to the gating mutations of cystic fibrosis transmembrane conductance regulator (CFTR or ABCC7; i.e., G551D, S1251N, and G1349D), that were previously shown to be function defective and corrected by ivacaftor (VX-770; Kalydeco), a clinically approved CFTR potentiator. Three-dimensional structural modeling predicted that all five ABCB4 variants would disrupt critical interactions in the binding of ATP and thereby impair ATP-induced nucleotide-binding domain dimerization and ABCB4 function. This prediction was confirmed by expression in cell models, which showed that the ABCB4 mutants were normally processed and targeted to the plasma membrane, whereas their PC secretion activity was dramatically decreased. As also hypothesized on the basis of molecular modeling, PC secretion activity of the mutants was rescued by the CFTR potentiator, ivacaftor (VX-770). Disease-causing variations in the ATP-binding sites of ABCB4 cause defects in PC secretion, which can be rescued by ivacaftor. These results provide the first experimental evidence that ivacaftor is a potential therapy for selected patients who harbor mutations in the ATP-binding sites of ABCB4. (Hepatology 2017;65:560-570). © 2016 by the American Association for the Study of Liver Diseases.

Triple Inhibition of EGFR, Met, and VEGF Suppresses Regrowth of HGF-Triggered, Erlotinib-Resistant Lung Cancer Harboring an EGFR Mutation

PubMed Central

Nakade, Junya; Takeuchi, Shinji; Nakagawa, Takayuki; Ishikawa, Daisuke; Sano, Takako; Nanjo, Shigeki; Yamada, Tadaaki; Ebi, Hiromichi; Zhao, Lu; Yasumoto, Kazuo; Matsumoto, Kunio; Yonekura, Kazuhiko

2014-01-01

Introduction: Met activation by gene amplification and its ligand, hepatocyte growth factor (HGF), imparts resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant lung cancer. We recently reported that Met activation by HGF stimulates the production of vascular endothelial growth factor (VEGF) and facilitates angiogenesis, which indicates that HGF induces EGFR-TKI resistance and angiogenesis. This study aimed to determine the effect of triple inhibition of EGFR, Met, and angiogenesis on HGF-triggered EGFR-TKI resistance in EGFR-mutant lung cancer. Methods: Three clinically approved drugs, erlotinib (an EGFR inhibitor), crizotinib (an inhibitor of anaplastic lymphoma kinase and Met), and bevacizumab (anti-VEGF antibody), and TAS-115, a novel dual TKI for Met and VEGF receptor 2, were used in this study. EGFR-mutant lung cancer cell lines PC-9, HCC827, and HGF-gene–transfected PC-9 (PC-9/HGF) cells were examined. Results: Crizotinib and TAS-115 inhibited Met phosphorylation and reversed erlotinib resistance and VEGF production triggered by HGF in PC-9 and HCC827 cells in vitro. Bevacizumab and TAS-115 inhibited angiogenesis in PC-9/HGF tumors in vivo. Moreover, the triplet erlotinib, crizotinib, and bevacizumab, or the doublet erlotinib and TAS-115 successfully inhibited PC-9/HGF tumor growth and delayed tumor regrowth associated with sustained tumor vasculature inhibition even after cessation of the treatment. Conclusion: These results suggest that triple inhibition of EGFR, HGF/Met, and VEGF/VEGF receptor 2, by either a triplet of clinical drugs or TAS-115 combined with erlotinib, may be useful for controlling progression of EGFR-mutant lung cancer by reversing EGFR-TKI resistance and for inhibiting angiogenesis. PMID:24828661

BLISTER Regulates Polycomb-Target Genes, Represses Stress-Regulated Genes and Promotes Stress Responses in Arabidopsis thaliana.

PubMed

Kleinmanns, Julia A; Schatlowski, Nicole; Heckmann, David; Schubert, Daniel

2017-01-01

HIGHLIGHTS The PRC2 interacting protein BLISTER likely acts downstream of PRC2 to silence Polycomb target genes and is a key regulator of specific stress responses in Arabidopsis . Polycomb group (PcG) proteins are key epigenetic regulators of development. The highly conserved Polycomb repressive complex 2 (PRC2) represses thousands of target genes by trimethylating H3K27 (H3K27me3). Plant specific PcG components and functions are largely unknown, however, we previously identified the plant-specific protein BLISTER (BLI) as a PRC2 interactor. BLI regulates PcG target genes and promotes cold stress resistance. To further understand the function of BLI , we analyzed the transcriptional profile of bli-1 mutants. Approximately 40% of the up-regulated genes in bli are PcG target genes, however, bli-1 mutants did not show changes in H3K27me3 levels at all tested genes, indicating that BLI regulates PcG target genes downstream of or in parallel to PRC2. Interestingly, a significant number of BLI regulated H3K27me3 target genes is regulated by the stress hormone absciscic acid (ABA). We further reveal an overrepresentation of genes responding to abiotic stresses such as drought, high salinity, or heat stress among the up-regulated genes in bli mutants. Consistently, bli mutants showed reduced desiccation stress tolerance. We conclude that the PRC2 associated protein BLI is a key regulator of stress-responsive genes in Arabidopsis : it represses ABA-responsive PcG target genes, likely downstream of PRC2, and promotes resistance to several stresses such as cold and drought.

Integration host factor is necessary for lysogenization of Escherichia coli by bacteriophage P2.

PubMed

Saha, S; Haggård-Ljungquist, E; Nordström, K

1990-01-01

Whether infection by bacteriophage P2 results in lysogenization of the host or vegetative growth of the phage depends upon a race between transcription from the repressor promoter Pc and the early promoter Pe; transcription from these promoters is mutually exclusive, since the Pc repressor Cox is formed from the Pe transcript and the Pe repressor C from the Pc transcript. The involvement of integration host factor (IHF) in the lysogenization of Escherichia coli K12 by P2 was tested by comparing wild-type and IHF-deficient (himA and himD) mutants. No lysogenic clones were formed following infection of the mutant bacteria. A switch plasmid that contains Pc-C-cat and Pe-cox-kan was used to test the choice for expression of Pc versus Pe. In the wild-type K12 bacteria, 20% of the clones expressed Pe transcription and 80% Pc transcription, whereas all transformed IHF-defective clones expressed transcription from Pe only. The effects of IHF on the in vivo expression of the Pe and Pc promoters were only marginal. The IHF protein was found to bind upstream of the Pe promoter, where a potential ihf sequence is located.

Enhanced susceptibility of Prnp-deficient mice to kainate-induced seizures, neuronal apoptosis, and death: Role of AMPA/kainate receptors.

PubMed

Rangel, Alejandra; Burgaya, Ferran; Gavín, Rosalina; Soriano, Eduardo; Aguzzi, Adriano; Del Río, José A

2007-09-01

Normal physiologic functions of the cellular prion protein (PrPc) are still elusive. This GPI-anchored protein exerts many functions, including roles in neuron proliferation, neuroprotection or redox homeostasis. There are, however, conflicting data concerning its role in synaptic transmission. Although several studies report that PrPc participates in NMDA-mediated neurotransmission, parallel studies describe normal behavior of PrPc-mutant mice. Abnormal axon connections have been described in the dentate gyrus of the hippocampi of PrPc-deficient mice similar to those observed in epilepsy. A study indicates increased susceptibility to kainate (KA) in these mutant mice. We extend the observation of these studies by means of several histologic and biochemical analyses of KA-treated mice. PrPc-deficient mice showed increased sensitivity to KA-induced seizures in vivo and in vitro in organotypic slices. In addition, we show that this sensitivity is cell-specific because interference experiments to abolish PrPc expression increased susceptibility to KA in PrPc-expressing cells. We indicate a correlation of susceptibility to KA in cells lacking PrPc with the differential expression of GluR6 and GluR7 KA receptor subunits using real-time RT-PCR methods. These results indicate that PrPc exerts a neuroprotective role against KA-induced neurotoxicity, probably by regulating the expression of KA receptor subunits. (c) 2007 Wiley-Liss, Inc.

Loss of Activating EGFR Mutant Gene Contributes to Acquired Resistance to EGFR Tyrosine Kinase Inhibitors in Lung Cancer Cells

PubMed Central

Kubo, Takuya; Murakami, Yuichi; Kawahara, Akihiko; Azuma, Koichi; Abe, Hideyuki; Kage, Masayoshi; Yoshinaga, Aki; Tahira, Tomoko; Hayashi, Kenshi; Arao, Tokuzo; Nishio, Kazuto; Rosell, Rafael; Kuwano, Michihiko; Ono, Mayumi

2012-01-01

Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR) mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11–18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11–18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11–18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2) or BIBW2992 (pan-TKI of EGFR family proteins). Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance. PMID:22815900

A novel homozygous no-stop mutation in G6PC gene from a Chinese patient with glycogen storage disease type Ia.

PubMed

Gu, Lei-Lei; Li, Xin-Hua; Han, Yue; Zhang, Dong-Hua; Gong, Qi-Ming; Zhang, Xin-Xin

2014-02-25

Glycogen storage disease type Ia (GSD-Ia) is an autosomal recessive genetic disorder resulting in hypoglycemia, hepatomegaly and growth retardation. It is caused by mutations in the G6PC gene encoding Glucose-6-phosphatase. To date, over 80 mutations have been identified in the G6PC gene. Here we reported a novel mutation found in a Chinese patient with abnormal transaminases, hypoglycemia, hepatomegaly and short stature. Direct sequencing of the coding region and splicing-sites in the G6PC gene revealed a novel no-stop mutation, p.*358Yext*43, leading to a 43 amino-acid extension of G6Pase. The expression level of mutant G6Pase transcripts was only 7.8% relative to wild-type transcripts. This mutation was not found in 120 chromosomes from 60 unrelated healthy control subjects using direct sequencing, and was further confirmed by digestion with Rsa I restriction endonuclease. In conclusion, we revealed a novel no-stop mutation in this study which expands the spectrum of mutations in the G6PC gene. The molecular genetic analysis was indispensable to the diagnosis of GSD-Ia for the patient. Copyright © 2013 Elsevier B.V. All rights reserved.

Visually induced initiation of Drosophila innate courtship-like following pursuit is mediated by central excitatory state.

PubMed

Kohatsu, Soh; Yamamoto, Daisuke

2015-03-06

The courtship ritual of male Drosophila represents an innate behaviour that is initiated by female-derived sensory stimuli. Here we report that moving light spots can induce courtship-like following pursuit in tethered wild-type male flies provided the fly is primed by optogenetic stimulation of specific dsx-expressing neuronal clusters in the lateral protocerebrum (LPR). Namely, stimulation of the pC1 neuronal cluster initiates unilateral wing extension and vibration of both sides, whereas stimulation of the pC2l cluster initiates only contralateral wing displays. In addition, stimulation of pC2l but not pC1 neurons induced abdominal bending and proboscis extension. Ca(2+) imaging of the pC1 cluster revealed periodic Ca(2+) rises, each corresponding to a turn of the male fly during courtship. In contrast, group-reared fru mutant males exhibit light spot-induced courtship pursuit without optogenetic priming. Ca(2+) imaging revealed enhanced responses of LPR neurons to visual stimuli in the mutants, suggesting a neural correlate of the light spot-induced courtship behaviour.

Unsaturation of Very-Long-Chain Ceramides Protects Plant from Hypoxia-Induced Damages by Modulating Ethylene Signaling in Arabidopsis

PubMed Central

Yu, Lu-Jun; Huang, Li; Chen, Liang; Wang, Feng-Zhu; Xia, Fan-Nv; Zhu, Tian-Ren; Wu, Jian-Xin; Yin, Jian; Liao, Bin; Shi, Jianxin; Zhang, Jian-Hua; Aharoni, Asaph; Yao, Nan; Shu, Wensheng; Xiao, Shi

2015-01-01

Lipid remodeling is crucial for hypoxic tolerance in animals, whilst little is known about the hypoxia-induced lipid dynamics in plants. Here we performed a mass spectrometry-based analysis to survey the lipid profiles of Arabidopsis rosettes under various hypoxic conditions. We observed that hypoxia caused a significant increase in total amounts of phosphatidylserine, phosphatidic acid and oxidized lipids, but a decrease in phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Particularly, significant gains in the polyunsaturated species of PC, PE and phosphatidylinositol, and losses in their saturated and mono-unsaturated species were evident during hypoxia. Moreover, hypoxia led to a remarkable elevation of ceramides and hydroxyceramides. Disruption of ceramide synthases LOH1, LOH2 and LOH3 enhanced plant sensitivity to dark submergence, but displayed more resistance to submergence under light than wild type. Consistently, levels of unsaturated very-long-chain (VLC) ceramide species (22:1, 24:1 and 26:1) predominantly declined in the loh1, loh2 and loh3 mutants under dark submergence. In contrast, significant reduction of VLC ceramides in the loh1-1 loh3-1 knockdown double mutant and lacking of VLC unsaturated ceramides in the ads2 mutants impaired plant tolerance to both dark and light submergences. Evidence that C24:1-ceramide interacted with recombinant CTR1 protein and inhibited its kinase activity in vitro, enhanced ER-to-nucleus translocation of EIN2-GFP and stabilization of EIN3-GFP in vivo, suggests a role of ceramides in modulating CTR1-mediated ethylene signaling. The dark submergence-sensitive phenotypes of loh mutants were rescued by a ctr1-1 mutation. Thus, our findings demonstrate that unsaturation of VLC ceramides is a protective strategy for hypoxic tolerance in Arabidopsis. PMID:25822663

The Prion Protein Modulates A-type K+ Currents Mediated by Kv4.2 Complexes through Dipeptidyl Aminopeptidase-like Protein 6*

PubMed Central

Mercer, Robert C. C.; Ma, Li; Watts, Joel C.; Strome, Robert; Wohlgemuth, Serene; Yang, Jing; Cashman, Neil R.; Coulthart, Michael B.; Schmitt-Ulms, Gerold; Jhamandas, Jack H.; Westaway, David

2013-01-01

Widely expressed in the adult central nervous system, the cellular prion protein (PrPC) is implicated in a variety of processes, including neuronal excitability. Dipeptidyl aminopeptidase-like protein 6 (DPP6) was first identified as a PrPC interactor using in vivo formaldehyde cross-linking of wild type (WT) mouse brain. This finding was confirmed in three cell lines and, because DPP6 directs the functional assembly of K+ channels, we assessed the impact of WT and mutant PrPC upon Kv4.2-based cell surface macromolecular complexes. Whereas a Gerstmann-Sträussler-Scheinker disease version of PrP with eight extra octarepeats was a loss of function both for complex formation and for modulation of Kv4.2 channels, WT PrPC, in a DPP6-dependent manner, modulated Kv4.2 channel properties, causing an increase in peak amplitude, a rightward shift of the voltage-dependent steady-state inactivation curve, a slower inactivation, and a faster recovery from steady-state inactivation. Thus, the net impact of wt PrPC was one of enhancement, which plays a critical role in the down-regulation of neuronal membrane excitability and is associated with a decreased susceptibility to seizures. Insofar as previous work has established a requirement for WT PrPC in the Aβ-dependent modulation of excitability in cholinergic basal forebrain neurons, our findings implicate PrPC regulation of Kv4.2 channels as a mechanism contributing to the effects of oligomeric Aβ upon neuronal excitability and viability. PMID:24225951

Pharmacological chaperoning: a primer on mechanism and pharmacology.

PubMed

Leidenheimer, Nancy J; Ryder, Katelyn G

2014-05-01

Approximately forty percent of diseases are attributable to protein misfolding, including those for which genetic mutation produces misfolding mutants. Intriguingly, many of these mutants are not terminally misfolded since native-like folding, and subsequent trafficking to functional locations, can be induced by target-specific, small molecules variably termed pharmacological chaperones, pharmacoperones, or pharmacochaperones (PCs). PC targets include enzymes, receptors, transporters, and ion channels, revealing the breadth of proteins that can be engaged by ligand-assisted folding. The purpose of this review is to provide an integrated primer of the diverse mechanisms and pharmacology of PCs. In this regard, we examine the structural mechanisms that underlie PC rescue of misfolding mutants, including the ability of PCs to act as surrogates for defective intramolecular interactions and, at the intermolecular level, overcome oligomerization deficiencies and dominant negative effects, as well as influence the subunit stoichiometry of heteropentameric receptors. Not surprisingly, PC-mediated structural correction of misfolding mutants normalizes interactions with molecular chaperones that participate in protein quality control and forward-trafficking. A variety of small molecules have proven to be efficacious PCs and the advantages and disadvantages of employing orthostatic antagonists, active-site inhibitors, orthostatic agonists, and allosteric modulator PCs are considered. Also examined is the possibility that several therapeutic agents may have unrecognized activity as PCs, and this chaperoning activity may mediate/contribute to therapeutic action and/or account for adverse effects. Lastly, we explore evidence that pharmacological chaperoning exploits intrinsic ligand-assisted folding mechanisms. Given the widespread applicability of PC rescue of mutants associated with protein folding disorders, both in vitro and in vivo, the therapeutic potential of PCs is vast. This is most evident in the treatment of lysosomal storage disorders, cystic fibrosis, and nephrogenic diabetes insipidus, for which proof of principle in humans has been demonstrated. Copyright © 2014 Elsevier Ltd. All rights reserved.

Kicking against the PRCs – A Domesticated Transposase Antagonises Silencing Mediated by Polycomb Group Proteins and Is an Accessory Component of Polycomb Repressive Complex 2

PubMed Central

Perera, Pumi; Mora-García, Santiago; de Leau, Erica; Thornton, Harry; de Alves, Flavia Lima; Rapsilber, Juri; Yang, Suxin; James, Geo Velikkakam; Schneeberger, Korbinian; Finnegan, E. Jean; Turck, Franziska; Goodrich, Justin

2015-01-01

The Polycomb group (PcG) and trithorax group (trxG) genes play crucial roles in development by regulating expression of homeotic and other genes controlling cell fate. Both groups catalyse modifications of chromatin, particularly histone methylation, leading to epigenetic changes that affect gene activity. The trxG antagonizes the function of PcG genes by activating PcG target genes, and consequently trxG mutants suppress PcG mutant phenotypes. We previously identified the ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN1 (ALP1) gene as a genetic suppressor of mutants in the Arabidopsis PcG gene LIKE HETEROCHROMATIN PROTEIN1 (LHP1). Here, we show that ALP1 interacts genetically with several other PcG and trxG components and that it antagonizes PcG silencing. Transcriptional profiling reveals that when PcG activity is compromised numerous target genes are hyper-activated in seedlings and that in most cases this requires ALP1. Furthermore, when PcG activity is present ALP1 is needed for full activation of several floral homeotic genes that are repressed by the PcG. Strikingly, ALP1 does not encode a known chromatin protein but rather a protein related to PIF/Harbinger class transposases. Phylogenetic analysis indicates that ALP1 is broadly conserved in land plants and likely lost transposase activity and acquired a novel function during angiosperm evolution. Consistent with this, immunoprecipitation and mass spectrometry (IP-MS) show that ALP1 associates, in vivo, with core components of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a widely conserved PcG protein complex which functions as a H3K27me3 histone methyltransferase. Furthermore, in reciprocal pulldowns using the histone methyltransferase CURLY LEAF (CLF), we identify not only ALP1 and the core PRC2 components but also plant-specific accessory components including EMBRYONIC FLOWER 1 (EMF1), a transcriptional repressor previously associated with PRC1-like complexes. Taken together our data suggest that ALP1 inhibits PcG silencing by blocking the interaction of the core PRC2 with accessory components that promote its HMTase activity or its role in inhibiting transcription. ALP1 is the first example of a domesticated transposase acquiring a novel function as a PcG component. The antagonistic interaction of a modified transposase with the PcG machinery is novel and may have arisen as a means for the cognate transposon to evade host surveillance or for the host to exploit features of the transposition machinery beneficial for epigenetic regulation of gene activity. PMID:26642436

Iron-Regulated Phospholipase C Activity Contributes to the Cytolytic Activity and Virulence of Acinetobacter baumannii

PubMed Central

Fiester, Steven E.; Schmidt, Robert E.; Beckett, Amber C.; Ticak, Tomislav; Carrier, Mary V.; Ghosh, Rajarshi; Ohneck, Emily J.; Metz, Maeva L.; Sellin Jeffries, Marlo K.; Actis, Luis A.

2016-01-01

Acinetobacter baumannii is an opportunistic Gram-negative pathogen that causes a wide range of infections including pneumonia, septicemia, necrotizing fasciitis and severe wound and urinary tract infections. Analysis of A. baumannii representative strains grown in Chelex 100-treated medium for hemolytic activity demonstrated that this pathogen is increasingly hemolytic to sheep, human and horse erythrocytes, which interestingly contain increasing amounts of phosphatidylcholine in their membranes. Bioinformatic, genetic and functional analyses of 19 A. baumannii isolates showed that the genomes of each strain contained two phosphatidylcholine-specific phospholipase C (PC-PLC) genes, which were named plc1 and plc2. Accordingly, all of these strains were significantly hemolytic to horse erythrocytes and their culture supernatants tested positive for PC-PLC activity. Further analyses showed that the transcriptional expression of plc1 and plc2 and the production of phospholipase and thus hemolytic activity increased when bacteria were cultured under iron-chelation as compared to iron-rich conditions. Testing of the A. baumannii ATCC 19606T plc1::aph-FRT and plc2::aph isogenic insertion derivatives showed that these mutants had a significantly reduced PC-PLC activity as compared to the parental strain, while testing of plc1::ermAM/plc2::aph demonstrated that this double PC-PLC isogenic mutant expressed significantly reduced cytolytic and hemolytic activity. Interestingly, only plc1 was shown to contribute significantly to A. baumannii virulence using the Galleria mellonella infection model. Taken together, our data demonstrate that both PLC1 and PLC2, which have diverged from a common ancestor, play a concerted role in hemolytic and cytolytic activities; although PLC1 seems to play a more critical role in the virulence of A. baumannii when tested in an invertebrate model. These activities would provide access to intracellular iron stores this pathogen could use during growth in the infected host. PMID:27875572

Stuxnet Facilitates the Degradation of Polycomb Protein during Development.

PubMed

Du, Juan; Zhang, Junzheng; He, Tao; Li, Yajuan; Su, Ying; Tie, Feng; Liu, Min; Harte, Peter J; Zhu, Alan Jian

2016-06-20

Polycomb-group (PcG) proteins function to ensure correct deployment of developmental programs by epigenetically repressing target gene expression. Despite the importance, few studies have been focused on the regulation of PcG activity itself. Here, we report a Drosophila gene, stuxnet (stx), that controls Pc protein stability. We find that heightened stx activity leads to homeotic transformation, reduced Pc activity, and de-repression of PcG targets. Conversely, stx mutants, which can be rescued by decreased Pc expression, display developmental defects resembling hyperactivation of Pc. Our biochemical analyses provide a mechanistic basis for the interaction between stx and Pc; Stx facilitates Pc degradation in the proteasome, independent of ubiquitin modification. Furthermore, this mode of regulation is conserved in vertebrates. Mouse stx promotes degradation of Cbx4, an orthologous Pc protein, in vertebrate cells and induces homeotic transformation in Drosophila. Our results highlight an evolutionarily conserved mechanism of regulated protein degradation on PcG homeostasis and epigenetic activity. Copyright © 2016 Elsevier Inc. All rights reserved.

Sigma-1 receptor agonist PRE084 is protective against mutant huntingtin-induced cell degeneration: involvement of calpastatin and the NF-κB pathway

PubMed Central

Hyrskyluoto, A; Pulli, I; Törnqvist, K; Huu Ho, T; Korhonen, L; Lindholm, D

2013-01-01

Alterations in mitochondria and increased oxidative stress are associated with the disease progression in Huntington's disease (HD). Endoplasmic reticulum (ER) stress and oxidative damage are linked through the close communication between the ER and mitochondria. Sigma-1 receptor (Sig-1R) is a chaperone protein in the ER that is involved in ER stress regulation, but little is known about its role in HD or the mechanisms for cell protection. Here we show that the Sig-1R agonist, PRE084 increases cell survival and counteracts the deleterious effects caused by N-terminal mutant huntingtin proteins in neuronal PC6.3 cells. Particularly, PRE084 increased the levels of cellular antioxidants by activating the NF-κB pathway that is compromised by the expression of mutant huntingtin proteins. These results show that the Sig-1R agonist has beneficial effects in models of HD and that compounds affecting the Sig-1R may be promising targets for future drug development in HD. PMID:23703391

Cell-surface prion protein interacts with glycosaminoglycans.

PubMed Central

Pan, Tao; Wong, Boon-Seng; Liu, Tong; Li, Ruliang; Petersen, Robert B; Sy, Man-Sun

2002-01-01

We used ELISA and flow cytometry to study the binding of prion protein PrP to glycosaminoglycans (GAGs). We found that recombinant human PrP (rPrP) binds GAGs including chondroitin sulphate A, chondroitin sulphate B, hyaluronic acid, and heparin. rPrP binding to GAGs occurs via the N-terminus, a region known to bind divalent cations. Additionally, rPrP binding to GAGs is enhanced in the presence of Cu2+ and Zn2+, but not Ca2+ and Mn2+. rPrP binds heparin strongest, and the binding is inhibited by certain heparin analogues, including heparin disaccharide and sulphate-containing monosaccharides, but not by acetylated heparin. Full-length normal cellular prion protein (PrPC), but not N-terminally truncated PrPC species, from human brain bind GAGs in a similar Cu2+/Zn2+-enhanced fashion. We found that GAGs specifically bind to a synthetic peptide corresponding to amino acid residues 23-35 in the N-terminus of rPrP. We further demonstrated that while both wild-type PrPC and an octapeptide-repeat-deleted mutant PrP produced by transfected cells bound heparin at the cell surface, the PrP N-terminal deletion mutant and non-transfectant control failed to bind heparin. Binding of heparin to wild-type PrPC on the cell surface results in a reduction of the level of cell-surface PrPC. These results provide strong evidence that PrPC is a surface receptor for GAGs. PMID:12186633

Saccharomyces Cerevisiae Cho2 Mutants Are Deficient in Phospholipid Methylation and Cross-Pathway Regulation of Inositol Synthesis

PubMed Central

Summers, E. F.; Letts, V. A.; McGraw, P.; Henry, S. A.

1988-01-01

Five allelic Saccharomyces cerevisiae mutants deficient in the methylation of phosphatidylethanolamine (PE) have been isolated, using two different screening techniques. Biochemical analysis suggested that these mutants define a locus, designated CHO2, that may encode a methyltransferase. Membranes of cho2 mutant cells grown in defined medium contain approximately 10% phosphatidylcholine (PC) and 40-50% PE as compared to wild-type levels of 40-45% PC and 15-20% PE. In spite of this greatly altered phospholipid composition, cho2 mutant cells are viable in defined medium and are not auxotrophic for choline or other phospholipid precursors such as monomethylethanolamine (MME). However, analysis of yeast strains carrying more than one mutation affecting phospholipid biosynthesis indicated that some level of methylated phospholipid is essential for viability. The cho2 locus was shown by tetrad analysis to be unlinked to other loci affecting phospholipid synthesis. Interestingly, cho2 mutants and other mutant strains that produce reduced levels of methylated phospholipids are unable to properly repress synthesis of the cytoplasmic enzyme inositol-1-phosphate synthase. This enzyme was previously shown to be regulated at the level of mRNA abundance in response to inositol and choline in the growth medium. We cloned the CHO2 gene on a 3.6-kb genomic DNA fragment and created a null allele of cho2 by disrupting the CHO2 gene in vivo. The cho2 disruptant, like all other cho2 mutants, is viable, exhibits altered regulation of inositol biosynthesis and is not auxotrophic for choline or MME. PMID:3066687

Onjisaponin B derived from Radix Polygalae enhances autophagy and accelerates the degradation of mutant α-synuclein and huntingtin in PC-12 cells.

PubMed

Wu, An-Guo; Wong, Vincent Kam-Wai; Xu, Su-Wei; Chan, Wai-Kit; Ng, Choi-In; Liu, Liang; Law, Betty Yuen-Kwan

2013-11-15

Emerging evidence indicates important protective roles being played by autophagy in neurodegenerative disorders through clearance of aggregate-prone or mutant proteins. In the current study, we aimed to identify autophagy inducers from Chinese medicinal herbs as a potential neuroprotective agent that enhances the clearance of mutant huntingtin and α-synuclein in PC-12 cells. Through intensive screening using the green fluorescent protein-light chain 3 (GFP-LC3) autophagy detection platform, we found that the ethanol extracts of Radix Polygalae (Yuan Zhi) were capable of inducing autophagy. Further investigation showed that among three single components derived from Radix Polygalae--i.e., polygalacic acid, senegenin and onjisaponin B--onjisaponin B was able to induce autophagy and accelerate both the removal of mutant huntingtin and A53T α-synuclein, which are highly associated with Huntington disease and Parkinson disease, respectively. Our study further demonstrated that onjisaponin B induces autophagy via the AMPK-mTOR signaling pathway. Therefore, findings in the current study provide detailed insights into the protective mechanism of a novel autophagy inducer, which is valuable for further investigation as a new candidate agent for modulating neurodegenerative disorders through the reduction of toxicity and clearance of mutant proteins in the cellular level.

Onjisaponin B Derived from Radix Polygalae Enhances Autophagy and Accelerates the Degradation of Mutant α-Synuclein and Huntingtin in PC-12 Cells

PubMed Central

Wu, An-Guo; Wong, Vincent Kam-Wai; Xu, Su-Wei; Chan, Wai-Kit; Ng, Choi-In; Liu, Liang; Law, Betty Yuen-Kwan

2013-01-01

Emerging evidence indicates important protective roles being played by autophagy in neurodegenerative disorders through clearance of aggregate-prone or mutant proteins. In the current study, we aimed to identify autophagy inducers from Chinese medicinal herbs as a potential neuroprotective agent that enhances the clearance of mutant huntingtin and α-synuclein in PC-12 cells. Through intensive screening using the green fluorescent protein-light chain 3 (GFP-LC3) autophagy detection platform, we found that the ethanol extracts of Radix Polygalae (Yuan Zhi) were capable of inducing autophagy. Further investigation showed that among three single components derived from Radix Polygalae—i.e., polygalacic acid, senegenin and onjisaponin B—onjisaponin B was able to induce autophagy and accelerate both the removal of mutant huntingtin and A53T α-synuclein, which are highly associated with Huntington disease and Parkinson disease, respectively. Our study further demonstrated that onjisaponin B induces autophagy via the AMPK-mTOR signaling pathway. Therefore, findings in the current study provide detailed insights into the protective mechanism of a novel autophagy inducer, which is valuable for further investigation as a new candidate agent for modulating neurodegenerative disorders through the reduction of toxicity and clearance of mutant proteins in the cellular level. PMID:24248062

X-ray structures of the Pseudomonas cichorii D-tagatose 3-epimerase mutant form C66S recognizing deoxy sugars as substrates.

PubMed

Yoshida, Hiromi; Yoshihara, Akihide; Ishii, Tomohiko; Izumori, Ken; Kamitori, Shigehiro

2016-12-01

Pseudomonas cichorii D-tagatose 3-epimerase (PcDTE), which has a broad substrate specificity, efficiently catalyzes the epimerization of not only D-tagatose to D-sorbose but also D-fructose to D-psicose (D-allulose) and also recognizes the deoxy sugars as substrates. In an attempt to elucidate the substrate recognition and catalytic reaction mechanisms of PcDTE for deoxy sugars, the X-ray structures of the PcDTE mutant form with the replacement of Cys66 by Ser (PcDTE_C66S) in complexes with deoxy sugars were determined. These X-ray structures showed that substrate recognition by the enzyme at the 1-, 2-, and 3-positions is responsible for enzymatic activity and that substrate-enzyme interactions at the 4-, 5-, and 6-positions are not essential for the catalytic reaction of the enzyme leading to the broad substrate specificity of PcDTE. They also showed that the epimerization site of 1-deoxy 3-keto D-galactitol is shifted from C3 to C4 and that 1-deoxy sugars may bind to the catalytic site in the inhibitor-binding mode. The hydrophobic groove that acts as an accessible surface for substrate binding is formed through the dimerization of PcDTE. In PcDTE_C66S/deoxy sugar complex structures, bound ligand molecules in both the linear and ring forms were detected in the hydrophobic groove, while bound ligand molecules in the catalytic site were in the linear form. This result suggests that the sugar-ring opening of a substrate may occur in the hydrophobic groove and also that the narrow channel of the passageway to the catalytic site allows a substrate in the linear form to pass through.

Common nonsynonymous variants in PCSK1 confer risk of obesity.

PubMed

Benzinou, Michael; Creemers, John W M; Choquet, Helene; Lobbens, Stephane; Dina, Christian; Durand, Emmanuelle; Guerardel, Audrey; Boutin, Philippe; Jouret, Beatrice; Heude, Barbara; Balkau, Beverley; Tichet, Jean; Marre, Michel; Potoczna, Natascha; Horber, Fritz; Le Stunff, Catherine; Czernichow, Sebastien; Sandbaek, Annelli; Lauritzen, Torsten; Borch-Johnsen, Knut; Andersen, Gitte; Kiess, Wieland; Körner, Antje; Kovacs, Peter; Jacobson, Peter; Carlsson, Lena M S; Walley, Andrew J; Jørgensen, Torben; Hansen, Torben; Pedersen, Oluf; Meyre, David; Froguel, Philippe

2008-08-01

Mutations in PCSK1 cause monogenic obesity. To assess the contribution of PCSK1 to polygenic obesity risk, we genotyped tag SNPs in a total of 13,659 individuals of European ancestry from eight independent case-control or family-based cohorts. The nonsynonymous variants rs6232, encoding N221D, and rs6234-rs6235, encoding the Q665E-S690T pair, were consistently associated with obesity in adults and children (P = 7.27 x 10(-8) and P = 2.31 x 10(-12), respectively). Functional analysis showed a significant impairment of the N221D-mutant PC1/3 protein catalytic activity.

Chimeras of the native form or achondroplasia mutant (G375C) of human fibroblast growth factor receptor 3 induce ligand-dependent differentiation of PC12 cells.

PubMed Central

Thompson, L M; Raffioni, S; Wasmuth, J J; Bradshaw, R A

1997-01-01

Mutations in the gene for human fibroblast growth factor receptor 3 (hFGFR3) cause a variety of skeletal dysplasias, including the most common genetic form of dwarfism, achondroplasia (ACH). Evidence indicates that these phenotypes are not due to simple haploinsufficiency of FGFR3 but are more likely related to a role in negatively regulating skeletal growth. The effects of one of these mutations on FGFR3 signaling were examined by constructing chimeric receptors composed of the extracellular domain of human platelet-derived growth factor receptor beta (hPDGFR beta) and the transmembrane and intracellular domains of hFGFR3 or of an ACH (G375C) mutant. Following stable transfection in PC12 cells, which lack platelet-derived growth factor (PDGF) receptors, all clonal cell lines, with either type of chimera, showed strong neurite outgrowth in the presence of PDGF but not in its absence. Antiphosphotyrosine immunoblots showed ligand-dependent autophosphorylation, and both receptor types stimulated strong phosphorylation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase, an event associated with the differentiative response of these cells. In addition, ligand-dependent phosphorylation of phospholipase Cgamma and Shc was also observed. All of these responses were comparable to those observed from ligand activation, such as by nerve growth factor, of the native PC12 cells used to prepare the stable transfectants. The cells with the chimera bearing the ACH mutation were more rapidly responsive to ligand with less sustained MAPK activation, indicative of a preactivated or primed condition and consistent with the view that these mutations weaken ligand control of FGFR3 function. However, the full effect of the mutation likely depends in part on structural features of the extracellular domain. Although FGFR3 has been suggested to act as a negative regulator of long-bone growth in chrondrocytes, it produces differentiative signals similar to those of FGFR1, to which only positive effects have been ascribed, in PC12 cells. Therefore, its regulatory effects on bone growth likely result from cellular contexts and not the induction of a unique FGFR3 signaling pathway. PMID:9199352

Cytoplasm-predominant Pten associates with increased region-specific brain tyrosine hydroxylase and dopamine D2 receptors in mouse model with autistic traits.

PubMed

He, Xin; Thacker, Stetson; Romigh, Todd; Yu, Qi; Frazier, Thomas W; Eng, Charis

2015-01-01

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders characterized by impairment in social communication/interaction and inflexible/repetitive behavior. Several lines of evidence support genetic factors as a predominant cause of ASD. Among those autism susceptibility genes that have been identified, the PTEN tumor suppressor gene, initially identified as predisposing to Cowden heritable cancer syndrome, was found to be mutated in a subset of ASD patients with extreme macrocephaly. However, the ASD-relevant molecular mechanism mediating the effect of PTEN mutations remains elusive. We developed a Pten knock-in murine model to study the effects of Pten germline mutations, specifically altering subcellular localization, in ASD. Proteins were isolated from the hemispheres of the male littermates, and Western blots were performed to determine protein expression levels of tyrosine hydroxylase (TH). Immunohistochemical stains were carried out to validate the localization of TH and dopamine D2 receptors (D2R). PC12 cells ectopically expressing either wild-type or missense mutant PTEN were then compared for the differences in TH expression. Mice carrying Pten mutations have high TH and D2R in the striatum and prefrontal cortex. They also have increased phosphorylation of cAMP response element-binding protein (CREB) and TH. Mechanistically, PTEN downregulates TH production in PC12 cells via inhibiting the phosphoinositide 3-kinase (PI3K)/CREB signaling pathway, while PTEN reduces TH phosphorylation via suppressing MAPK pathway. Unlike wild-type PTEN but similar to the mouse knock-in mutant Pten, three naturally occurring missense mutations of PTEN that we previously identified in ASD patients, H93R, F241S, and D252G, were not able to suppress TH when overexpressed in PC12 cells. In addition, two other PTEN missense mutations, C124S (pan phosphatase dead) and G129E (lipid phosphatase dead), failed to suppress TH when ectopically expressed in PC12 cells. Our data reveal a non-canonical PTEN-TH pathway in the brain that may work as a core regulator of dopamine signaling, which when dysfunctional is pathogenic in ASD.

Site-directed mutagenesis of GH10 xylanase A from Penicillium canescens for determining factors affecting the enzyme thermostability.

PubMed

Denisenko, Yury A; Gusakov, Alexander V; Rozhkova, Aleksandra M; Osipov, Dmitry O; Zorov, Ivan N; Matys, Veronika Yu; Uporov, Igor V; Sinitsyn, Arkady P

2017-11-01

In order to investigate factors affecting the thermostability of GH10 xylanase A from Penicillium canescens (PcXylA) and to obtain its more stable variant, the wild-type (wt) enzyme and its mutant forms, carrying single amino acid substitutions, were cloned and expressed in Penicillium verruculosum B1-537 (niaD-) auxotrophic strain under the control of the cbh1 gene promoter. The recombinant PcXylA-wt and I6V, I6L, L18F, N77D, Y125R, H191R, S246P, A293P mutants were successfully expressed and purified for characterization. The mutations did not affect the enzyme specific activity against xylan from wheat as well as its pH-optimum of activity. One mutant (L18F) displayed a higher thermostability relative to the wild-type enzyme; its half-life time at 50-60°C was 2-2.5-fold longer than that for the PcXylA-wt, and the melting temperature was 60.0 and 56.1°C, respectively. Most of other mutations led to decrease in the enzyme thermostability. This study, together with data of other researchers, suggests that multiple mutations should be introduced into GH10 xylanases in order to dramatically improve their stability. Copyright © 2017 Elsevier B.V. All rights reserved.

Therapeutic siRNAs for dominant genetic skin diseases including pachyonychia congenita

PubMed Central

Leachman, Sancy A.; Hickerson, Robyn P.; Hull, Peter R.; Smith, Frances J. D.; Milstone, Leonard M.; Lane, E. Birgitte; Bale, Sherri J.; Roop, Dennis R.; McLean, W. H. Irwin; Kaspar, Roger L.

2008-01-01

The field of science and medicine has experienced a flood of data and technology associated with the human genome project. Over 10,000 human diseases have been genetically defined, but little progress has been made with respect to the clinical application of this knowledge. A notable exception to this exists for pachyonychia congenita (PC), a rare, dominant negative keratin disorder. The establishment of a non-profit organization, PC Project, has led to an unprecedented coalescence of patients, scientists, and physicians with a unified vision of developing novel therapeutics for PC. Utilizing the technological by-products of the human genome project, such as RNA interference (RNAi) and quantitative RT-PCR (qRT-PCR), physicians and scientists have collaborated to create a candidate siRNA therapeutic that selectively inhibits a mutant allele of KRT6A, the most commonly affected PC keratin. In vitro investigation of this siRNA demonstrates potent inhibition of the mutant allele and reversal of the cellular aggregation phenotype. In parallel, an allele-specific quantitative real time RT-PCR assay has been developed and validated on patient callus samples in preparation for clinical trials. If clinical efficacy is ultimately demonstrated, this “first-in-skin” siRNA may herald a paradigm shift in the treatment of dominant negative genetic disorders. PMID:18495438

Therapeutic siRNAs for dominant genetic skin disorders including pachyonychia congenita.

PubMed

Leachman, Sancy A; Hickerson, Robyn P; Hull, Peter R; Smith, Frances J D; Milstone, Leonard M; Lane, E Birgitte; Bale, Sherri J; Roop, Dennis R; McLean, W H Irwin; Kaspar, Roger L

2008-09-01

The field of science and medicine has experienced a flood of data and technology associated with the human genome project. Over 10,000 human diseases have been genetically defined, but little progress has been made with respect to the clinical application of this knowledge. A notable exception to this exists for pachyonychia congenita (PC), a rare, dominant-negative keratin disorder. The establishment of a non-profit organization, PC Project, has led to an unprecedented coalescence of patients, scientists, and physicians with a unified vision of developing novel therapeutics for PC. Utilizing the technological by-products of the human genome project, such as RNA interference (RNAi) and quantitative RT-PCR (qRT-PCR), physicians and scientists have collaborated to create a candidate siRNA therapeutic that selectively inhibits a mutant allele of KRT6A, the most commonly affected PC keratin. In vitro investigation of this siRNA demonstrates potent inhibition of the mutant allele and reversal of the cellular aggregation phenotype. In parallel, an allele-specific quantitative real-time RT-PCR assay has been developed and validated on patient callus samples in preparation for clinical trials. If clinical efficacy is ultimately demonstrated, this "first-in-skin" siRNA may herald a paradigm shift in the treatment of dominant-negative genetic disorders.

MLH1 as a direct target of MiR-155 and a potential predictor of favorable prognosis in pancreatic cancer.

PubMed

Liu, Wen-Jing; Zhao, Yu-Pei; Zhang, Tai-Ping; Zhou, Li; Cui, Quan-Cai; Zhou, Wei-Xun; You, Lei; Chen, Ge; Shu, Hong

2013-08-01

The regulation of Mut L homologue 1 (MLH1) expression by microRNA (miR)-155 and its prognostic significance in pancreatic cancer (PC) remain to be elucidated. This study aimed to address the issues. MiR-155 mimics and inhibitor were transfected to PC cell lines, Panc-1 and Capan-1. Expression of MLH1 was subsequently evaluated. Then, luciferase activity was detected after miR-155 mimics and pRL-TK plasmids containing wild-type and mutant 3'UTRs of MLH1 mRNA were co-transfected. Finally, immunohistochemical staining for MLH1 was performed in PC samples. Transfection of miR-155 mimics and inhibitor led to reversely altered protein expressions of miR-155 and MLH1, whereas the corresponding mRNA expressions were similar. A significant decrease in luciferase activity in the cells transfected with the wild-type pRL-TK plasmid was shown in contrast to those transfected with the mutant one. In addition, MLH1 was less expressed in tumor than in para-tumor tissues of PC. Extensive MLH1 expression was significantly associated with favorable differentiation and less lymph node metastasis. MLH1 expression was found to be a prognosticator in univariate analysis, and being of marginally significant impact in multivariate test. MLH1 might serve as a direct target of miR-155 and a potential prognosis predictor in PC.

[Role of phosphorylation of MARCKS-PSD in the secretion of MUC5AC induced by cold temperatures in human airway epithelial cells].

PubMed

Li, Minchao; Perelman, Juliy M; Zhou, Xiangdong

2012-05-01

To construct phosphorylation sites domain (PSD) mutant of myristoylated alaninerich C kinase substrate (MARCKS) and explore the role of transient receptor potential melastatin 8 cation channels (TRPM8) and MARCKS in cold-induced synthesis and exocytosis of mucin (MUC) 5AC. Human placental cDNA was used as a template to amplify the full coding region of MARCKS cDNA by PCR. Ser159, Ser 163, Ser 167, Ser 170 in the PSD were mutated to aspartic acids by an overlap PCR method. The resultant PSD mutant cDNA and the wild-type MARCKS cDNA were each subcloned into a mammalian expression vector pcDNA3.0. Recombinant constructs were confirmed by restriction enzyme digestion analysis and DNA sequencing. In intervention experiments, cells were pretreated with the TRPM8 channel antagonist BCTC and transfected with MARCKS-PSD mutant cDNA, and thereafter cold stimulation was applied. The levels of MUC5AC were measured by immunofluorescence and ELISA to clarify the roles of TRPM8 and PSD mutant on the synthesis and secretion of MUC5AC induced by cold, respectively. Restriction enzyme digestion analysis and DNA sequencing revealed that the pcDNA3.0- MARCKS and pcDNA3.0-MARCKS-PSD mutants were successfully constructed. The levels of intracellular and secreted MUC5AC of cold treated group were significantly higher than those of control group (P<0.05). BCTC attenuated the cold-induced synthesis and secretion of MUC5AC when compared with cold treated group (P<0.05). Transfection of 16HBE cells with the MARCKS-PSD mutant cDNA resulted in significant inhibition of mucin secretion in response to cold, and significantly higher level of intracellular MUC5AC than that of control group (P<0.01), whereas transfection with the vector DNA or the wild-type MARCKS cDNA had no effect on the mucin synthesis and secretion in response to cold (P>0.05). TRPM8 and phosphorylation of MARCKS-PSD mediates the cold-induced exocytosis of MUC5AC by airway epithelial cells.

Lgr4 Protein Deficiency Induces Ataxia-like Phenotype in Mice and Impairs Long Term Depression at Cerebellar Parallel Fiber-Purkinje Cell Synapses*

PubMed Central

Guan, Xin; Duan, Yanhong; Zeng, Qingwen; Pan, Hongjie; Qian, Yu; Li, Dali; Cao, Xiaohua; Liu, Mingyao

2014-01-01

Cerebellar dysfunction causes ataxia characterized by loss of balance and coordination. Until now, the molecular and neuronal mechanisms of several types of inherited cerebellar ataxia have not been completely clarified. Here, we report that leucine-rich G protein-coupled receptor 4 (Lgr4/Gpr48) is highly expressed in Purkinje cells (PCs) in the cerebellum. Deficiency of Lgr4 leads to an ataxia-like phenotype in mice. Histologically, no obvious morphological changes were observed in the cerebellum of Lgr4 mutant mice. However, the number of PCs was slightly but significantly reduced in Lgr4−/− mice. In addition, in vitro electrophysiological analysis showed an impaired long term depression (LTD) at parallel fiber-PC (PF-PC) synapses in Lgr4−/− mice. Consistently, immunostaining experiments showed that the level of phosphorylated cAMP-responsive element-binding protein (Creb) was significantly decreased in Lgr4−/− PCs. Furthermore, treatment with forskolin, an adenylyl cyclase agonist, rescued phospho-Creb in PCs and reversed the impairment in PF-PC LTD in Lgr4−/− cerebellar slices, indicating that Lgr4 is an upstream regulator of Creb signaling, which is underlying PF-PC LTD. Together, our findings demonstrate for first time an important role for Lgr4 in motor coordination and cerebellar synaptic plasticity and provide a potential therapeutic target for certain types of inherited cerebellar ataxia. PMID:25063812

SNF1-Related Protein Kinases Type 2 Are Involved in Plant Responses to Cadmium Stress1[C][W

PubMed Central

Kulik, Anna; Anielska-Mazur, Anna; Bucholc, Maria; Koen, Emmanuel; Szymańska, Katarzyna; Żmieńko, Agnieszka; Krzywińska, Ewa; Wawer, Izabela; McLoughlin, Fionn; Ruszkowski, Dariusz; Figlerowicz, Marek; Testerink, Christa; Skłodowska, Aleksandra; Wendehenne, David; Dobrowolska, Grażyna

2012-01-01

Cadmium ions are notorious environmental pollutants. To adapt to cadmium-induced deleterious effects plants have developed sophisticated defense mechanisms. However, the signaling pathways underlying the plant response to cadmium are still elusive. Our data demonstrate that SnRK2s (for SNF1-related protein kinase2) are transiently activated during cadmium exposure and are involved in the regulation of plant response to this stress. Analysis of tobacco (Nicotiana tabacum) Osmotic Stress-Activated Protein Kinase activity in tobacco Bright Yellow 2 cells indicates that reactive oxygen species (ROS) and nitric oxide, produced mainly via an l-arginine-dependent process, contribute to the kinase activation in response to cadmium. SnRK2.4 is the closest homolog of tobacco Osmotic Stress-Activated Protein Kinase in Arabidopsis (Arabidopsis thaliana). Comparative analysis of seedling growth of snrk2.4 knockout mutants versus wild-type Arabidopsis suggests that SnRK2.4 is involved in the inhibition of root growth triggered by cadmium; the mutants were more tolerant to the stress. Measurements of the level of three major species of phytochelatins (PCs) in roots of plants exposed to Cd2+ showed a similar (PC2, PC4) or lower (PC3) concentration in snrk2.4 mutants in comparison to wild-type plants. These results indicate that the enhanced tolerance of the mutants does not result from a difference in the PCs level. Additionally, we have analyzed ROS accumulation in roots subjected to Cd2+ treatment. Our data show significantly lower Cd2+-induced ROS accumulation in the mutants’ roots. Concluding, the obtained results indicate that SnRK2s play a role in the regulation of plant tolerance to cadmium, most probably by controlling ROS accumulation triggered by cadmium ions. PMID:22885934

Arsenic tolerance in Arabidopsis is mediated by two ABCC-type phytochelatin transporters

PubMed Central

Song, Won-Yong; Park, Jiyoung; Mendoza-Cózatl, David G.; Suter-Grotemeyer, Marianne; Shim, Donghwan; Hörtensteiner, Stefan; Geisler, Markus; Weder, Barbara; Rea, Philip A.; Rentsch, Doris; Schroeder, Julian I.; Lee, Youngsook; Martinoia, Enrico

2010-01-01

Arsenic is an extremely toxic metalloid causing serious health problems. In Southeast Asia, aquifers providing drinking and agricultural water for tens of millions of people are contaminated with arsenic. To reduce nutritional arsenic intake through the consumption of contaminated plants, identification of the mechanisms for arsenic accumulation and detoxification in plants is a prerequisite. Phytochelatins (PCs) are glutathione-derived peptides that chelate heavy metals and metalloids such as arsenic, thereby functioning as the first step in their detoxification. Plant vacuoles act as final detoxification stores for heavy metals and arsenic. The essential PC–metal(loid) transporters that sequester toxic metal(loid)s in plant vacuoles have long been sought but remain unidentified in plants. Here we show that in the absence of two ABCC-type transporters, AtABCC1 and AtABCC2, Arabidopsis thaliana is extremely sensitive to arsenic and arsenic-based herbicides. Heterologous expression of these ABCC transporters in phytochelatin-producing Saccharomyces cerevisiae enhanced arsenic tolerance and accumulation. Furthermore, membrane vesicles isolated from these yeasts exhibited a pronounced arsenite [As(III)]–PC2 transport activity. Vacuoles isolated from atabcc1 atabcc2 double knockout plants exhibited a very low residual As(III)–PC2 transport activity, and interestingly, less PC was produced in mutant plants when exposed to arsenic. Overexpression of AtPCS1 and AtABCC1 resulted in plants exhibiting increased arsenic tolerance. Our findings demonstrate that AtABCC1 and AtABCC2 are the long-sought and major vacuolar PC transporters. Modulation of vacuolar PC transporters in other plants may allow engineering of plants suited either for phytoremediation or reduced accumulation of arsenic in edible organs. PMID:21078981

Glycoform-independent prion conversion by highly efficient, cell-based, protein misfolding cyclic amplification

PubMed Central

Moudjou, Mohammed; Chapuis, Jérôme; Mekrouti, Mériem; Reine, Fabienne; Herzog, Laetitia; Sibille, Pierre; Laude, Hubert; Vilette, Didier; Andréoletti, Olivier; Rezaei, Human; Dron, Michel; Béringue, Vincent

2016-01-01

Prions are formed of misfolded assemblies (PrPSc) of the variably N-glycosylated cellular prion protein (PrPC). In infected species, prions replicate by seeding the conversion and polymerization of host PrPC. Distinct prion strains can be recognized, exhibiting defined PrPSc biochemical properties such as the glycotype and specific biological traits. While strain information is encoded within the conformation of PrPSc assemblies, the storage of the structural information and the molecular requirements for self-perpetuation remain uncertain. Here, we investigated the specific role of PrPC glycosylation status. First, we developed an efficient protein misfolding cyclic amplification method using cells expressing the PrPC species of interest as substrate. Applying the technique to PrPC glycosylation mutants expressing cells revealed that neither PrPC nor PrPSc glycoform stoichiometry was instrumental to PrPSc formation and strainness perpetuation. Our study supports the view that strain properties, including PrPSc glycotype are enciphered within PrPSc structural backbone, not in the attached glycans. PMID:27384922

Mitochondrially-targeted bacterial phosphatidylethanolamine methyltransferase sustained phosphatidylcholine synthesis of a Saccharomyces cerevisiae Δpem1 Δpem2 double mutant without exogenous choline supply.

PubMed

Kobayashi, Shingo; Mizuike, Aya; Horiuchi, Hiroyuki; Fukuda, Ryouichi; Ohta, Akinori

2014-09-01

In eukaryotic cells, phospholipids are synthesized exclusively in the defined organelles specific for each phospholipid species. To explain the reason for this compartmental specificity in the case of phosphatidylcholine (PC) synthesis, we constructed and characterized a Saccharomyces cerevisiae strain that lacked endogenous phosphatidylethanolamine (PE) methyltransferases but had a recombinant PE methyltransferase from Acetobacter aceti, which was fused with a mitochondrial targeting signal from yeast Pet100p and a 3×HA epitope tag. This fusion protein, which we named as mitopmt, was determined to be localized to the mitochondria by fluorescence microscopy and subcellular fractionation. The expression of mitopmt suppressed the choline auxotrophy of a double deletion mutant of PEM1 and PEM2 (pem1Δpem2Δ) and enabled it to synthesize PC in the absence of choline. This growth suppression was observed even if the Kennedy pathway was inactivated by the repression of PCT1 encoding CTP:phosphocholine cytidylyltransferase, suggesting that PC synthesized in the mitochondria is distributed to other organelles without going through the salvage pathway. The pem1Δpem2Δ strain deleted for PSD1 encoding the mitochondrial phosphatidylserine decarboxylase was able to grow because of the expression of mitopmt in the presence of ethanolamine, implying that PE from other organelles, probably from the ER, was converted to PC by mitopmt. These results suggest that PC could move out of the mitochondria, and raise the possibility that its movement is not under strict directional limitations. Copyright © 2014 Elsevier B.V. All rights reserved.

Procyanidins from apples (Malus pumila Mill.) extend the lifespan of Caenorhabditis elegans.

PubMed

Sunagawa, Tadahiro; Shimizu, Takahiko; Kanda, Tomomasa; Tagashira, Motoyuki; Sami, Manabu; Shirasawa, Takuji

2011-01-01

Apple polyphenols (AP) mainly consist of procyanidins (PC), which are composed of (-)-epicatechins and (+)-catechins. In order to investigate the antiageing effects of PC, we measured the lifespan of CAENORHABDITIS ELEGANS worms treated with PC. Treatment with 65 µg/mL PC extended the mean lifespan of wild-type N2 and FEM-1 worms by 12.1 % and 8.4 %, respectively, i.e., to a similar extent as resveratrol. In addition, treatment with 100 µg/mL AP also significantly prolonged the mean lifespan of the same worms by 12.0 % and 5.3 %, respectively, i.e., to a similar extent as PC. In contrast, treatment with (-)-epicatechin did not extend the lifespan of the worms. PC did not modify the growth, food intake, or fecundity of C. elegans. Treatment with PC did not extend the lifespan of MEV-1 worms, which show excessive oxidative stress, indicating that PC had no antioxidant ability in the MEV-1 mutant. Moreover, treatment with PC had no effect on the longevity of SIR-2.1 worms, which lack the activity of SIR-2, a member of the sirtuin family of NAD (+)-dependent protein deacetylases. These results indicated that PC has SIR-2.1-dependent antiageing effects on C. elegans. © Georg Thieme Verlag KG Stuttgart · New York.

ELANE mutant-specific activation of different UPR pathways in congenital neutropenia.

PubMed

Nustede, Rainer; Klimiankou, Maksim; Klimenkova, Olga; Kuznetsova, Inna; Zeidler, Cornelia; Welte, Karl; Skokowa, Julia

2016-01-01

A number of studies have demonstrated induction of the unfolded protein response (UPR) in patients with severe congenital neutropenia (CN) harbouring mutations of ELANE, encoding neutrophil elastase. Why UPR is not activated in patients with cyclic neutropenia (CyN) carrying the same ELANE mutations is unclear. We evaluated the effects of ELANE mutants on UPR induction in myeloid cells from CN and CyN patients, and analysed whether additional CN-specific defects contribute to the differences in UPR induction between CN and CyN patients harbouring identical ELANE mutations. We investigated CN-specific p.C71R and p.V174_C181del (NP_001963.1) and CN/CyN-shared p.S126L (NP_001963.1) ELANE mutants. We found that transduction of haematopoietic cells with p.C71R, but not with p.V174_C181del or p.S126L ELANE mutants induced expression of ATF6, and the ATF6 target genes PPP1R15A, DDIT3 and HSPA5. Recently, we found that levels of secretory leucocyte protease inhibitor (SLPI), a natural ELANE inhibitor, are diminished in myeloid cells from CN patients, but not CyN patients. Combined knockdown of SLPI by shRNA and transduction of ELANE p.S126L in myeloid cells led to elevated levels of ATF6, PPP1R15A and HSPA5 RNA, suggesting that normal levels of SLPI in CyN patients might protect them from the UPR induced by mutant ELANE. In summary, different ELANE mutants have different effects on UPR activation, and SLPI regulates the extent of ELANE-triggered UPR. © 2015 John Wiley & Sons Ltd.

Choline Uptake in Agrobacterium tumefaciens by the High-Affinity ChoXWV Transporter▿

PubMed Central

Aktas, Meriyem; Jost, Kathinka A.; Fritz, Christiane; Narberhaus, Franz

2011-01-01

Agrobacterium tumefaciens is a facultative phytopathogen that causes crown gall disease. For successful plant transformation A. tumefaciens requires the membrane lipid phosphatidylcholine (PC), which is produced via the methylation and the PC synthase (Pcs) pathways. The latter route is dependent on choline. Although choline uptake has been demonstrated in A. tumefaciens, the responsible transporter(s) remained elusive. In this study, we identified the first choline transport system in A. tumefaciens. The ABC-type choline transporter is encoded by the chromosomally located choXWV operon (ChoX, binding protein; ChoW, permease; and ChoV, ATPase). The Cho system is not critical for growth and PC synthesis. However, [14C]choline uptake is severely reduced in A. tumefaciens choX mutants. Recombinant ChoX is able to bind choline with high affinity (equilibrium dissociation constant [KD] of ≈2 μM). Since other quaternary amines are bound by ChoX with much lower affinities (acetylcholine, KD of ≈80 μM; betaine, KD of ≈470 μM), the ChoXWV system functions as a high-affinity transporter with a preference for choline. Two tryptophan residues (W40 and W87) located in the predicted ligand-binding pocket are essential for choline binding. The structural model of ChoX built on Sinorhizobium meliloti ChoX resembles the typical structure of substrate binding proteins with a so-called “Venus flytrap mechanism” of substrate binding. PMID:21803998

Modulation of Bacillus thuringiensis Phosphatidylinositol-Specific Phospholipase C Activity by Mutations in the Putative Dimerization Interface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, X.; Shao, C; Zhang, X

2009-01-01

Cleavage of phosphatidylinositol (PI) to inositol 1,2-(cyclic)-phosphate (cIP) and cIP hydrolysis to inositol 1-phosphate by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C are activated by the enzyme binding to phosphatidylcholine (PC) surfaces. Part of this reflects improved binding of the protein to interfaces. However, crystallographic analysis of an interfacially impaired phosphatidylinositol-specific phospholipase (W47A/W242A) suggested protein dimerization might occur on the membrane. In the W47A/W242A dimer, four tyrosine residues from one monomer interact with the same tyrosine cluster of the other, forming a tight dimer interface close to the membrane binding regions. We have constructed mutant proteins in which two or more ofmore » these tyrosine residues have been replaced with serine. Phospholipid binding and enzymatic activity of these mutants have been examined to assess the importance of these residues to enzyme function. Replacing two tyrosines had small effects on enzyme activity. However, removal of three or four tyrosine residues weakened PC binding and reduced PI cleavage by the enzyme as well as PC activation of cIP hydrolysis. Crystal structures of Y247S/Y251S in the absence and presence of myo-inositol as well as Y246S/Y247S/Y248S/Y251S indicate that both mutant proteins crystallized as monomers, were very similar to one another, and had no change in the active site region. Kinetic assays, lipid binding, and structural results indicate that either (i) a specific PC binding site, critical for vesicle activities and cIP activation, has been impaired, or (ii) the reduced dimerization potential for Y246S/Y247S/Y248S and Y246S/Y247S/Y248S/Y251S is responsible for their reduced catalytic activity in all assay systems.« less

Regulation of GABAA and Glutamate Receptor Expression, Synaptic Facilitation and Long-Term Potentiation in the Hippocampus of Prion Mutant Mice

PubMed Central

Rangel, Alejandra; Madroñal, Noelia; Massó, Agnès Gruart i.; Gavín, Rosalina; Llorens, Franc; Sumoy, Lauro; Torres, Juan María; Delgado-García, José María; Río, José Antonio Del

2009-01-01

Background Prionopathies are characterized by spongiform brain degeneration, myoclonia, dementia, and periodic electroencephalographic (EEG) disturbances. The hallmark of prioniopathies is the presence of an abnormal conformational isoform (PrPsc) of the natural cellular prion protein (PrPc) encoded by the Prnp gene. Although several roles have been attributed to PrPc, its putative functions in neuronal excitability are unknown. Although early studies of the behavior of Prnp knockout mice described minor changes, later studies report altered behavior. To date, most functional PrPc studies on synaptic plasticity have been performed in vitro. To our knowledge, only one electrophysiological study has been performed in vivo in anesthetized mice, by Curtis and coworkers. They reported no significant differences in paired-pulse facilitation or LTP in the CA1 region after Schaffer collateral/commissural pathway stimulation. Methodology/Principal Findings Here we explore the role of PrPc expression in neurotransmission and neural excitability using wild-type, Prnp −/− and PrPc-overexpressing mice (Tg20 strain). By correlating histopathology with electrophysiology in living behaving mice, we demonstrate that both Prnp −/− mice but, more relevantly Tg20 mice show increased susceptibility to KA, leading to significant cell death in the hippocampus. This finding correlates with enhanced synaptic facilitation in paired-pulse experiments and hippocampal LTP in living behaving mutant mice. Gene expression profiling using Illumina™ microarrays and Ingenuity pathways analysis showed that 129 genes involved in canonical pathways such as Ubiquitination or Neurotransmission were co-regulated in Prnp −/− and Tg20 mice. Lastly, RT-qPCR of neurotransmission-related genes indicated that subunits of GABAA and AMPA-kainate receptors are co-regulated in both Prnp −/− and Tg20 mice. Conclusions/Significance Present results demonstrate that PrPc is necessary for the proper homeostatic functioning of hippocampal circuits, because of its relationships with GABAA and AMPA-Kainate neurotransmission. New PrPc functions have recently been described, which point to PrPc as a target for putative therapies in Alzheimer's disease. However, our results indicate that a “gain of function” strategy in Alzheimer's disease, or a “loss of function” in prionopathies, may impair PrPc function, with devastating effects. In conclusion, we believe that present data should be taken into account in the development of future therapies. PMID:19855845

HSPB1 facilitates ERK-mediated phosphorylation and degradation of BIM to attenuate endoplasmic reticulum stress-induced apoptosis

PubMed Central

Kennedy, Donna; Mnich, Katarzyna; Oommen, Deepu; Chakravarthy, Reka; Almeida-Souza, Leonardo; Krols, Michiel; Saveljeva, Svetlana; Doyle, Karen; Gupta, Sanjeev; Timmerman, Vincent; Janssens, Sophie; Gorman, Adrienne M; Samali, Afshin

2017-01-01

BIM, a pro-apoptotic BH3-only protein, is a key regulator of the intrinsic (or mitochondrial) apoptosis pathway. Here, we show that BIM induction by endoplasmic reticulum (ER) stress is suppressed in rat PC12 cells overexpressing heat shock protein B1 (HSPB1 or HSP27) and that this is due to enhanced proteasomal degradation of BIM. HSPB1 and BIM form a complex that immunoprecipitates with p-ERK1/2. We found that HSPB1-mediated proteasomal degradation of BIM is dependent on MEK-ERK signaling. Other studies have shown that several missense mutations in HSPB1 cause the peripheral neuropathy, Charcot-Marie-Tooth (CMT) disease, which is associated with nerve degeneration. Here we show that cells overexpressing CMT-related HSPB1 mutants exhibited increased susceptibility to ER stress-induced cell death and high levels of BIM. These findings identify a novel function for HSPB1 as a negative regulator of BIM protein stability leading to protection against ER stress-induced apoptosis, a function that is absent in CMT-associated HSPB1 mutants. PMID:29048431

HSPB1 facilitates ERK-mediated phosphorylation and degradation of BIM to attenuate endoplasmic reticulum stress-induced apoptosis.

PubMed

Kennedy, Donna; Mnich, Katarzyna; Oommen, Deepu; Chakravarthy, Reka; Almeida-Souza, Leonardo; Krols, Michiel; Saveljeva, Svetlana; Doyle, Karen; Gupta, Sanjeev; Timmerman, Vincent; Janssens, Sophie; Gorman, Adrienne M; Samali, Afshin

2017-08-31

BIM, a pro-apoptotic BH3-only protein, is a key regulator of the intrinsic (or mitochondrial) apoptosis pathway. Here, we show that BIM induction by endoplasmic reticulum (ER) stress is suppressed in rat PC12 cells overexpressing heat shock protein B1 (HSPB1 or HSP27) and that this is due to enhanced proteasomal degradation of BIM. HSPB1 and BIM form a complex that immunoprecipitates with p-ERK1/2. We found that HSPB1-mediated proteasomal degradation of BIM is dependent on MEK-ERK signaling. Other studies have shown that several missense mutations in HSPB1 cause the peripheral neuropathy, Charcot-Marie-Tooth (CMT) disease, which is associated with nerve degeneration. Here we show that cells overexpressing CMT-related HSPB1 mutants exhibited increased susceptibility to ER stress-induced cell death and high levels of BIM. These findings identify a novel function for HSPB1 as a negative regulator of BIM protein stability leading to protection against ER stress-induced apoptosis, a function that is absent in CMT-associated HSPB1 mutants.

A Mutation within the Extended X Loop Abolished Substrate-induced ATPase Activity of the Human Liver ATP-binding Cassette (ABC) Transporter MDR3*

PubMed Central

Kluth, Marianne; Stindt, Jan; Dröge, Carola; Linnemann, Doris; Kubitz, Ralf; Schmitt, Lutz

2015-01-01

The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain. PMID:25533467

The clinical presentation and genotype of protein C deficiency with double mutations of the protein C gene.

PubMed

Inoue, Hirofumi; Terachi, Shin-Ichi; Uchiumi, Takeshi; Sato, Tetsuji; Urata, Michiyo; Ishimura, Masataka; Koga, Yui; Hotta, Taeko; Hara, Toshiro; Kang, Dongchon; Ohga, Shouichi

2017-07-01

Severe protein C (PC) deficiency is a rare heritable thrombophilia leading to thromboembolic events during the neonatal period. It remains unclear how individuals with complete PC gene (PROC) defects develop or escape neonatal stroke or purpura fulminans (PF). We studied the onset of disease and the genotype of 22 PC-deficient patients with double mutations in PROC based on our cohort (n = 12) and the previous reports (n = 10) in Japan. Twenty-two patients in 20 unrelated families had 4 homozygous and 18 compound heterozygous mutations. Sixteen newborns presented with PF (n = 11, 69%), intracranial thromboembolism and hemorrhage (n = 13, 81%), or both (n = 8, 50%), with most showing a plasma PC activity of <10%. Six others first developed overt thromboembolism when they were over 15 years of age, showing a median PC activity of 31% (range: 19-52%). Fifteen of the 22 patients (68%) had the five major mutations (G423VfsX82, V339M, R211W, M406I, and F181V) or two others (E68K and K193del) that have been reported in Japan. Three of the six late-onset cases, but none of the 16 neonatal cases, had the K193del mutation, which has been reported to be the most common variant of Chinese thrombophilia. A novel mutation of A309V was determined in a family of two patients with late onset. The genotype of double-PROC mutants might show less diversity than heterozygous mutants in terms of the timing of the onset of thrombophilia (newborn onset or late onset). © 2017 Wiley Periodicals, Inc.

Improved antimicrobial activity of Pediococcus acidilactici against Salmonella Gallinarum by UV mutagenesis and genome shuffling.

PubMed

Han, Geon Goo; Song, Ahn Ah; Kim, Eun Bae; Yoon, Seong-Hyun; Bok, Jin-Duck; Cho, Chong-Su; Kil, Dong Yong; Kang, Sang-Kee; Choi, Yun-Jaie

2017-07-01

Pediococcus acidilactici is a widely used probiotic, and Salmonella enterica serovar Gallinarum (SG) is a significant pathogen in the poultry industry. In this study, we improved the antimicrobial activity of P. acidilactici against SG using UV mutation and genome shuffling (GS). To improve antimicrobial activity against SG, UV mutagenesis was performed against wild-type P. acidilactici (WT), and five mutants showed improved antimicrobial activity. To further improve antimicrobial activity, GS was performed on five UV mutants. Following GS, four mutants showed improved antimicrobial activity compared with the UV mutants and WT. The antimicrobial activity of GS1 was highest among the mutants; however, the activity was reduced when the culture supernatant was treated with proteinase K, suggesting that the improved antimicrobial activity is due to a proteinous substance such as bacteriocin. To validate the activity of GS1 in vivo, we designed multi-species probiotics and performed broiler feeding experiments. Groups consisted of no treatment (NC), avilamycin-treated (PC), probiotic group 1 containing WT (T1), and probiotic group 2 containing GS1 (T2). In broiler feeding experiments, coliform bacteria were significantly reduced in T2 compared with NC, PC, and T1. The cecal microbiota was modulated and pathogenic bacteria were reduced by GS1 oral administration. In this study, GS1 showed improved antimicrobial activity against SG in vitro and reduced pathogenic bacteria in a broiler feeding experiment. These results suggest that GS1 can serve as an efficient probiotic, as an alternative to antibiotics in the poultry industry.

Phosphatidylethanolamine Synthesis Is Required for Optimal Virulence of Brucella abortus▿

PubMed Central

Bukata, Lucas; Altabe, Silvia; de Mendoza, Diego; Ugalde, Rodolfo A.; Comerci, Diego J.

2008-01-01

The Brucella cell envelope contains the zwitterionic phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Synthesis of PC occurs exclusively via the PC synthase pathway, implying that the pathogen depends on the choline synthesized by the host cell to form PC. Notably, PC is necessary to sustain a chronic infection process, which suggests that the membrane lipid content is relevant for Brucella virulence. In this study we investigated the first step of PE biosynthesis in B. abortus, which is catalyzed by phosphatidylserine synthase (PssA). Disruption of pssA abrogated the synthesis of PE without affecting the growth in rich complex medium. In minimal medium, however, the mutant required choline supplementation for growth, suggesting that at least PE or PC is necessary for Brucella viability. The absence of PE altered cell surface properties, but most importantly, it impaired several virulence traits of B. abortus, such as intracellular survival in both macrophages and HeLa cells, the maturation of the replicative Brucella-containing vacuole, and mouse colonization. These results suggest that membrane phospholipid composition is critical for the interaction of B. abortus with the host cell. PMID:18931122

Endothelial expression of selectins during endotoxin preconditioning.

PubMed

Bauer, P; Welbourne, T; Shigematsu, T; Russell, J; Granger, D N

2000-12-01

Although bacterial endotoxins [lipopolysaccharide (LPS)] can confer tissue resistance to subsequent inflammatory insults, the mechanisms that underlie this LPS-preconditioning (LPS-PC) response remain poorly defined. The dual-radiolabeled monoclonal antibody technique was used to examine whether LPS-PC alters the upregulation (protein) of E- and P-selectins after subsequent LPS challenge. In the gut of wild-type (C57BL/6J) mice, LPS-PC was associated with a reduction in E- (66%) and P-selectin (33%) expression. A similar reduction in E-selectin expression was observed in mutant mice that were genetically deficient in either the endothelial or inducible isoform of nitric oxide synthase or that overexpressed the human gene for Cu/Zn superoxide dismutase. Severe combined immunodeficient mice, genetically devoid of lymphocytes, did exhibit partial inhibition of the LPS-PC response. We conclude that 1) LPS-PC can be demonstrated for E- and P-selectins in some vascular beds (e.g., gut), 2) the mechanism(s) underlying this blunted selectin response does not include a major role for either nitric oxide and superoxide, and 3) circulating lymphocytes may contribute to the LPS-PC response.

Tunicate cytostatic factor TC14-3 induces a polycomb group gene and histone modification through Ca2+ binding and protein dimerization

PubMed Central

2012-01-01

Background As many invertebrate species have multipotent cells that undergo cell growth and differentiation during regeneration and budding, many unique and interesting homeostatic factors are expected to exist in those animals. However, our understanding of such factors and global mechanisms remains very poor. Single zooids of the tunicate, Polyandrocarpa misakiensis, can give off as many as 40 buds during the life span. Bud development proceeds by means of transdifferentiation of very limited number of cells and tissues. TC14-3 is one of several different but closely related polypeptides isolated from P. misakiensis. It acts as a cytostatic factor that regulates proliferation, adhesion, and differentiation of multipotent cells, although the molecular mechanism remains uncertain. The Polycomb group (PcG) genes are involved in epigenetic control of genomic activity in mammals. In invertebrates except Drosophila, PcG and histone methylation have not been studied so extensively, and genome-wide gene regulation is poorly understood. Results When Phe65 of TC14-3 was mutated to an acidic amino acid, the resultant mutant protein failed to dimerize. The replacement of Thr69 with Arg69 made dimers unstable. When Glu106 was changed to Gly106, the resultant mutant protein completely lost Ca2+ binding. All these mutant proteins lacked cytostatic activity, indicating the requirement of protein dimerization and calcium for the activity. Polyandrocarpa Eed, a component of PcG, is highly expressed during budding, like TC14-3. When wild-type and mutant TC14-3s were applied in vivo and in vitro to Polyandrocarpa cells, only wild-type TC14-3 could induce Eed without affecting histone methyltransferase gene expression. Eed-expressing cells underwent trimethylation of histone H3 lysine27. PmEed knockdown by RNA interference rescued cultured cells from the growth-inhibitory effects of TC14-3. Conclusion These results show that in P. misakiensis, the cytostatic activity of TC14-3 is mediated by PmEed and resultant histone modification, and that the gene expression requires both the protein dimerization and Ca2+-binding of TC14-3. This system consisting of a humoral factor, PcG, and histone methylation would contribute to the homeostatic regulation of cell growth and terminal differentiation of invertebrate multipotent cells. PMID:22296827

Host plant-dependent phenotypic reversion of Ralstonia solanacearum from non-pathogenic to pathogenic forms via alterations in the phcA gene.

PubMed

Poussier, Stéphane; Thoquet, Philippe; Trigalet-Demery, Danièle; Barthet, Séverine; Meyer, Damien; Arlat, Matthieu; Trigalet, André

2003-08-01

Ralstonia solanacearum is a plant pathogenic bacterium that undergoes a spontaneous phenotypic conversion (PC) from a wild-type pathogenic to a non-pathogenic form. PC is often associated with mutations in phcA, which is a key virulence regulatory gene. Until now, reversion to the wild-type pathogenic form has not been observed for PC variants and the biological significance of PC has been questioned. In this study, we characterized various alterations in phcA (eight IS element insertions, three tandem duplications, seven deletions and a base substitution) in 19 PC mutants from the model strain GMI1000. In five of these variants, reversion to the pathogenic form was observed in planta, while no reversion was ever noticed in vitro whatever culture media used. However, reversion was observed for a 64 bp tandem duplication in vitro in the presence of tomato root exudate. This is the first report showing a complete cycle of phenotypic conversion/reversion in a plant pathogenic bacterium.

The metabolic enhancer piracetam ameliorates the impairment of mitochondrial function and neurite outgrowth induced by beta-amyloid peptide.

PubMed

Kurz, C; Ungerer, I; Lipka, U; Kirr, S; Schütt, T; Eckert, A; Leuner, K; Müller, W E

2010-05-01

beta-Amyloid peptide (Abeta) is implicated in the pathogenesis of Alzheimer's disease by initiating a cascade of events from mitochondrial dysfunction to neuronal death. The metabolic enhancer piracetam has been shown to improve mitochondrial dysfunction following brain aging and experimentally induced oxidative stress. We used cell lines (PC12 and HEK cells) and murine dissociated brain cells. The protective effects of piracetam in vitro and ex vivo on Abeta-induced impairment of mitochondrial function (as mitochondrial membrane potential and ATP production), on secretion of soluble Abeta and on neurite outgrowth in PC12 cells were investigated. Piracetam improves mitochondrial function of PC12 cells and acutely dissociated brain cells from young NMRI mice following exposure to extracellular Abeta(1-42). Similar protective effects against Abeta(1-42) were observed in dissociated brain cells from aged NMRI mice, or mice transgenic for mutant human amyloid precursor protein (APP) treated with piracetam for 14 days. Soluble Abeta load was markedly diminished in the brain of those animals after treatment with piracetam. Abeta production by HEK cells stably transfected with mutant human APP was elevated by oxidative stress and this was reduced by piracetam. Impairment of neuritogenesis is an important consequence of Abeta-induced mitochondrial dysfunction and Abeta-induced reduction of neurite growth in PC12 cells was substantially improved by piracetam. Our findings strongly support the concept of improving mitochondrial function as an approach to ameliorate the detrimental effects of Abeta on brain function.

Trastuzumab emtansine delays and overcomes resistance to the third-generation EGFR-TKI osimertinib in NSCLC EGFR mutated cell lines.

PubMed

La Monica, Silvia; Cretella, Daniele; Bonelli, Mara; Fumarola, Claudia; Cavazzoni, Andrea; Digiacomo, Graziana; Flammini, Lisa; Barocelli, Elisabetta; Minari, Roberta; Naldi, Nadia; Petronini, Pier Giorgio; Tiseo, Marcello; Alfieri, Roberta

2017-12-04

Osimertinib is a third-generation EGFR-TKI with a high selective potency against T790M-mutant NSCLC patients. Considering that osimertinib can lead to enhanced HER-2 expression on cell surface and HER-2 overexpression is a mechanism of resistance to osimertinib, this study was addressed to investigate the potential of combining osimertinib with trastuzumab emtansine (T-DM1) in order to improve the efficacy of osimertinib and delay or overcome resistance in NSCLC cell lines with EGFR activating mutation and with T790M mutation or HER-2 amplification. The effects of osimertinib combined with T-DM1 on cell proliferation, cell cycle, cell death, antibody-dependent cell-mediated cytotoxicity (ADCC), and acquisition of osimertinib resistance was investigated in PC9, PC9-T790M and H1975 cell lines. The potential of overcoming osimertinib resistance with T-DM1 was tested in a PC9/HER2c1 xenograft model. T-DM1 exerted an additive effect when combined with osimertinib in terms of inhibition of cell proliferation, cell death and ADCC induction in PC9, PC9-T790M and H1975 cell lines. Combining osimertinib and T-DM1 using different schedules in long-term growth experiments revealed that the appearance of osimertinib-resistance was prevented in PC9-T790M and delayed in H1975 cells when the two drugs were given together. By contrast, when osimertinib was followed by T-DM1 an antagonistic effect was observed on cell proliferation, cell death and resistance acquisition. In xenograft models, we demonstrated that HER-2 amplification was associated with osimertinib-resistance and that T-DM1 co-administration is a potential strategy to overcome this resistance. Our data suggest that concomitant treatment with osimertinib and T-DM1 may be a promising therapeutic strategy for EGFR-mutant NSCLC.

Congenital Insensitivity to Pain: Novel SCN9A Missense and In-Frame Deletion Mutations

PubMed Central

Cox, James J; Sheynin, Jony; Shorer, Zamir; Reimann, Frank; Nicholas, Adeline K; Zubovic, Lorena; Baralle, Marco; Wraige, Elizabeth; Manor, Esther; Levy, Jacov; Woods, C Geoffery; Parvari, Ruti

2010-01-01

SCN9A encodes the voltage-gated sodium channel Nav1.7, a protein highly expressed in pain-sensing neurons. Mutations in SCN9A cause three human pain disorders: bi-allelic loss of function mutations result in Channelopathy-associated Insensitivity to Pain (CIP), whereas activating mutations cause severe episodic pain in Paroxysmal Extreme Pain Disorder (PEPD) and Primary Erythermalgia (PE). To date, all mutations in SCN9A that cause a complete inability to experience pain are protein truncating and presumably lead to no protein being produced. Here, we describe the identification and functional characterization of two novel non-truncating mutations in families with CIP: a homozygously-inherited missense mutation found in a consanguineous Israeli Bedouin family (Nav1.7-R896Q) and a five amino acid in-frame deletion found in a sporadic compound heterozygote (Nav1.7-ΔR1370-L1374). Both of these mutations map to the pore region of the Nav1.7 sodium channel. Using transient transfection of PC12 cells we found a significant reduction in membrane localization of the mutant protein compared to the wild type. Furthermore, voltage clamp experiments of mutant-transfected HEK293 cells show a complete loss of function of the sodium channel, consistent with the absence of pain phenotype. In summary, this study has identified critical amino acids needed for the normal subcellular localization and function of Nav1.7. © 2010 Wiley-Liss, Inc. PMID:20635406

Congenital insensitivity to pain: novel SCN9A missense and in-frame deletion mutations.

PubMed

Cox, James J; Sheynin, Jony; Shorer, Zamir; Reimann, Frank; Nicholas, Adeline K; Zubovic, Lorena; Baralle, Marco; Wraige, Elizabeth; Manor, Esther; Levy, Jacov; Woods, C Geoffery; Parvari, Ruti

2010-09-01

SCN9Aencodes the voltage-gated sodium channel Na(v)1.7, a protein highly expressed in pain-sensing neurons. Mutations in SCN9A cause three human pain disorders: bi-allelic loss of function mutations result in Channelopathy-associated Insensitivity to Pain (CIP), whereas activating mutations cause severe episodic pain in Paroxysmal Extreme Pain Disorder (PEPD) and Primary Erythermalgia (PE). To date, all mutations in SCN9A that cause a complete inability to experience pain are protein truncating and presumably lead to no protein being produced. Here, we describe the identification and functional characterization of two novel non-truncating mutations in families with CIP: a homozygously-inherited missense mutation found in a consanguineous Israeli Bedouin family (Na(v)1.7-R896Q) and a five amino acid in-frame deletion found in a sporadic compound heterozygote (Na(v)1.7-DeltaR1370-L1374). Both of these mutations map to the pore region of the Na(v)1.7 sodium channel. Using transient transfection of PC12 cells we found a significant reduction in membrane localization of the mutant protein compared to the wild type. Furthermore, voltage clamp experiments of mutant-transfected HEK293 cells show a complete loss of function of the sodium channel, consistent with the absence of pain phenotype. In summary, this study has identified critical amino acids needed for the normal subcellular localization and function of Na(v)1.7. Copyright 2010 Wiley-Liss, Inc.

Lipid Rafts and Clathrin Cooperate in the Internalization of PrPC in Epithelial FRT Cells

PubMed Central

Casanova, Philippe; Puri, Claudia; Paladino, Simona; Tivodar, Simona S.; Campana, Vincenza; Tacchetti, Carlo; Zurzolo, Chiara

2009-01-01

Background The cellular prion protein (PrPC) plays a key role in the pathogenesis of Transmissible Spongiform Encephalopathies in which the protein undergoes post-translational conversion to the infectious form (PrPSc). Although endocytosis appears to be required for this conversion, the mechanism of PrPC internalization is still debated, as caveolae/raft- and clathrin-dependent processes have all been reported to be involved. Methodology/Principal Findings We have investigated the mechanism of PrPC endocytosis in Fischer Rat Thyroid (FRT) cells, which lack caveolin-1 (cav-1) and caveolae, and in FRT/cav-1 cells which form functional caveolae. We show that PrPC internalization requires activated Cdc-42 and is sensitive to cholesterol depletion but not to cav-1 expression suggesting a role for rafts but not for caveolae in PrPC endocytosis. PrPC internalization is also affected by knock down of clathrin and by the expression of dominant negative Eps15 and Dynamin 2 mutants, indicating the involvement of a clathrin-dependent pathway. Notably, PrPC co-immunoprecipitates with clathrin and remains associated with detergent-insoluble microdomains during internalization thus indicating that PrPC can enter the cell via multiple pathways and that rafts and clathrin cooperate in its internalization. Conclusions/Significance These findings are of particular interest if we consider that the internalization route/s undertaken by PrPC can be crucial for the ability of different prion strains to infect and to replicate in different cell lines. PMID:19503793

Detection of Cell Wall Chemical Variation in Zea Mays Mutants Using Near-Infrared Spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buyck, N.; Thomas, S.

Corn stover is regarded as the prime candidate feedstock material for commercial biomass conversion in the United States. Variations in chemical composition of Zea mays cell walls can affect biomass conversion process yields and economics. Mutant lines were constructed by activating a Mu transposon system. The cell wall chemical composition of 48 mutant families was characterized using near-infrared (NIR) spectroscopy. NIR data were analyzed using a multivariate statistical analysis technique called Principal Component Analysis (PCA). PCA of the NIR data from 349 maize leaf samples reveals 57 individuals as outliers on one or more of six Principal Components (PCs) atmore » the 95% confidence interval. Of these, 19 individuals from 16 families are outliers on either PC3 (9% of the variation) or PC6 (1% of the variation), the two PCs that contain information about cell wall polymers. Those individuals for which altered cell wall chemistry is confirmed with wet chemical analysis will then be subjected to fermentation analysis to determine whether or not biomass conversion process kinetics, yields and/or economics are significantly affected. Those mutants that provide indications for a decrease in process cost will be pursued further to identify the gene(s) responsible for the observed changes in cell wall composition and associated changes in process economics. These genes will eventually be incorporated into maize breeding programs directed at the development of a truly dual use crop.« less

Familial CJD Associated PrP Mutants within Transmembrane Region Induced Ctm-PrP Retention in ER and Triggered Apoptosis by ER Stress in SH-SY5Y Cells

PubMed Central

Wang, Xin; Shi, Qi; Xu, Kun; Gao, Chen; Chen, Cao; Li, Xiao-Li; Wang, Gui-Rong; Tian, Chan; Han, Jun; Dong, Xiao-Ping

2011-01-01

Background Genetic prion diseases are linked to point and inserted mutations in the prion protein (PrP) gene that are presumed to favor conversion of the cellular isoform of PrP (PrPC) to the pathogenic one (PrPSc). The pathogenic mechanisms and the subcellular sites of the conversion are not completely understood. Here we introduce several PRNP gene mutations (such as, PrP-KDEL, PrP-3AV, PrP-A117V, PrP-G114V, PrP-P102L and PrP-E200K) into the cultured cells in order to explore the pathogenic mechanism of familial prion disease. Methodology/Principal Findings To address the roles of aberrant retention of PrP in endoplasmic reticulum (ER), the recombinant plasmids expressing full-length human PrP tailed with an ER signal peptide at the COOH-terminal (PrP-KDEL) and PrP with three amino acids exchange in transmembrane region (PrP-3AV) were constructed. In the preparations of transient transfections, 18-kD COOH-terminal proteolytic resistant fragments (Ctm-PrP) were detected in the cells expressing PrP-KDEL and PrP-3AV. Analyses of the cell viabilities in the presences of tunicamycin and brefeldin A revealed that expressions of PrP-KDEL and PrP-3AV sensitized the transfected cells to ER stress stimuli. Western blots and RT-PCR identified the clear alternations of ER stress associated events in the cells expressing PrP-KDEL and PrP-3AV that induced ER mediated apoptosis by CHOP and capase-12 apoptosis pathway. Moreover, several familial CJD related PrP mutants were transiently introduced into the cultured cells. Only the mutants within the transmembrane region (G114V and A117V) induced the formation of Ctm-PrP and caused the ER stress, while the mutants outside the transmembrane region (P102L and E200K) failed. Conclusions/Significance The data indicate that the retention of PrP in ER through formation of Ctm-PrP results in ER stress and cell apoptosis. The cytopathic activities caused by different familial CJD associated PrP mutants may vary, among them the mutants within the transmembrane region undergo an ER-stress mediated cell apoptosis. PMID:21298055

The association of GSK3 beta with E2F1 facilitates nerve growth factor-induced neural cell differentiation.

PubMed

Zhou, Fangfang; Zhang, Long; Wang, Aijun; Song, Bo; Gong, Kai; Zhang, Lihai; Hu, Min; Zhang, Xiufang; Zhao, Nanming; Gong, Yandao

2008-05-23

It is widely acknowledged that E2F1 and GSK3beta are both involved in the process of cell differentiation. However, the relationship between E2F1 and GSK3beta in cell differentiation has yet to be discovered. Here, we provide evidence that in the differentiation of PC12 cells induced by nerve growth factor (NGF), GSK3beta was increased at both the mRNA and protein levels, whereas E2F1 at these two levels was decreased. Both wild-type GSK3beta and its kinase-defective mutant GSK3beta KM can inhibit E2F1 by promoting its ubiquitination through physical interaction. In addition, the colocalization of GSK3beta and E2F1 and their subcellular distribution, regulated by NGF, were observed in the process of PC12 differentiation. At the tissue level, GSK3beta colocalized and interacted with E2F1 in mouse hippocampus. Furthermore, GSK3beta facilitated neurite outgrowth by rescuing the promoter activities of Cdk inhibitors p21 and p15 from the inhibition caused by E2F1. To summarize, our findings suggest that GSK3beta can promote the ubiquitination of E2F1 via physical interaction and thus inhibit its transcription activity in a kinase activity independent manner, which plays an important role in the NGF-induced PC12 differentiation.

Instability of buried hydration sites increases protein subdomains fluctuations in the human prion protein by the pathogenic mutation T188R

NASA Astrophysics Data System (ADS)

Tomobe, Katsufumi; Yamamoto, Eiji; Akimoto, Takuma; Yasui, Masato; Yasuoka, Kenji

2016-05-01

The conformational change from the cellular prion protein (PrPc) to scrapie prion protein (PrPsc) is a key process in prion diseases. The prion protein has buried water molecules which significantly contribute to the stability of the protein; however, there has been no report investigating the influence on the buried hydration sites by a pathogenic mutation not adjacent to the buried hydration sites. Here, we perform molecular dynamics simulations of wild type (WT) PrPc and pathogenic point mutant T188R to investigate conformational changes and the buried hydration sites. In WT-PrPc, four buried hydration sites are identified by residence time and rotational relaxation analysis. However, there are no stable buried hydration sites in one of T188R simulations, which indicates that T188R sometimes makes the buried hydration sites fragile. We also find that fluctuations of subdomains S1-H1-S2 and H1-H2 increase in T188R when the buried hydration sites become unstable. Since the side chain of arginine which is replaced from threonine in T188R is larger than of threonine, the side chain cannot be embedded in the protein, which is one of the causes of the instability of subdomains. These results show correlations between the buried hydration sites and the mutation which is far from them, and provide a possible explanation for the instability by mutation.

Phosphorylation on Ser-279 and Ser-282 of connexin43 regulates endocytosis and gap junction assembly in pancreatic cancer cells

PubMed Central

Johnson, Kristen E.; Mitra, Shalini; Katoch, Parul; Kelsey, Linda S.; Johnson, Keith R.; Mehta, Parmender P.

2013-01-01

The molecular mechanisms regulating the assembly of connexins (Cxs) into gap junctions are poorly understood. Using human pancreatic tumor cell lines BxPC3 and Capan-1, which express Cx26 and Cx43, we show that, upon arrival at the cell surface, the assembly of Cx43 is impaired. Connexin43 fails to assemble, because it is internalized by clathrin-mediated endocytosis. Assembly is restored upon expressing a sorting-motif mutant of Cx43, which does not interact with the AP2 complex, and by expressing mutants that cannot be phosphorylated on Ser-279 and Ser-282. The mutants restore assembly by preventing clathrin-mediated endocytosis of Cx43. Our results also document that the sorting-motif mutant is assembled into gap junctions in cells in which the expression of endogenous Cx43 has been knocked down. Remarkably, Cx43 mutants that cannot be phosphorylated on Ser-279 or Ser-282 are assembled into gap junctions only when connexons are composed of Cx43 forms that can be phosphorylated on these serines and forms in which phosphorylation on these serines is abolished. Based on the subcellular fate of Cx43 in single and contacting cells, our results document that the endocytic itinerary of Cx43 is altered upon cell–cell contact, which causes Cx43 to traffic by EEA1-negative endosomes en route to lysosomes. Our results further show that gap-junctional plaques formed of a sorting motif–deficient mutant of Cx43, which is unable to be internalized by the clathrin-mediated pathway, are predominantly endocytosed in the form of annular junctions. Thus the differential phosphorylation of Cx43 on Ser-279 and Ser-282 is fine-tuned to control Cx43’s endocytosis and assembly into gap junctions. PMID:23363606

Interaction of KMP-11 with Phospholipid Membranes and Its Implications in Leishmaniasis: Effects of Single Tryptophan Mutations and Cholesterol.

PubMed

Sannigrahi, Achinta; Maity, Pabitra; Karmakar, Sanat; Chattopadhyay, Krishnananda

2017-03-02

KMP-11 is a small protein that is believed to control the overall bilayer pressure of the Leishmania parasite. Recent results have suggested that membrane binding and the presence of cholesterol affect the efficacy of Leishmanial infection, in which KMP-11 plays an important role. Nevertheless, there exists no systematic study of membrane interaction with KMP-11 either in the absence or presence of cholesterol. In this article, we investigated the interaction between KMP-11 and phospholipid membranes using an unsaturated (PC 18:1; 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)) and saturated (PC 12:0; 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)) lipid as membrane mimics. Additionally, we studied the effect of cholesterol on the protein-membrane interaction. Steady-state as well as time-resolved fluorescence spectroscopy, isothermal titration calorimetry (ITC), and ζ-potential measurements were used for the determination of the binding constants for the wild-type (WT) and single-site tryptophan mutants. Single-site tryptophan mutants were designed to make sure that the tryptophan residues sample different surface exposures in different mutants. In the absence of cholesterol, the membrane-binding affinities of the partially exposed and buried tryptophan mutants (Y5W and Y48W, respectively) were found to be greater than those of the WT protein. In the presence of cholesterol, the binding constants of the WT and Y48W mutant were found to decrease with an increase in cholesterol concentration. This was in contrast to that in the Y5W and F77W mutants, in which the binding constants increased on adding cholesterol. The present study highlights the interplay among the conformational architecture of a protein, its interaction with the membrane, and membrane composition in modulating the survival of a Leishmania parasite inside host macrophages.

Polycystin-2-dependent control of cardiomyocyte autophagy.

PubMed

Criollo, Alfredo; Altamirano, Francisco; Pedrozo, Zully; Schiattarella, Gabriele G; Li, Dan L; Rivera-Mejías, Pablo; Sotomayor-Flores, Cristian; Parra, Valentina; Villalobos, Elisa; Battiprolu, Pavan K; Jiang, Nan; May, Herman I; Morselli, Eugenia; Somlo, Stefan; de Smedt, Humbert; Gillette, Thomas G; Lavandero, Sergio; Hill, Joseph A

2018-05-01

Considerable evidence points to critical roles of intracellular Ca 2+ homeostasis in the modulation and control of autophagic activity. Yet, underlying molecular mechanisms remain unknown. Mutations in the gene (pkd2) encoding polycystin-2 (PC2) are associated with autosomal dominant polycystic kidney disease (ADPKD), the most common inherited nephropathy. PC2 has been associated with impaired Ca 2+ handling in cardiomyocytes and indirect evidence suggests that this protein may be involved in autophagic control. Here, we investigated the role for PC2 as an essential regulator of Ca 2+ homeostasis and autophagy. Activation of autophagic flux triggered by mTOR inhibition either pharmacologically (rapamycin) or by means of nutrient depletion was suppressed in cells depleted of PC2. Moreover, cardiomyocyte-specific PC2 knockout mice (αMhc-cre;Pkd2 F/F mice) manifested impaired autophagic flux in the setting of nutrient deprivation. Stress-induced autophagy was blunted by intracellular Ca 2+ chelation using BAPTA-AM, whereas removal of extracellular Ca 2+ had no effect, pointing to a role of intracellular Ca 2+ homeostasis in stress-induced cardiomyocyte autophagy. To determine the link between stress-induced autophagy and PC2-induced Ca 2+ mobilization, we over-expressed either wild-type PC2 (WT) or a Ca 2+ -channel deficient PC2 mutant (PC2-D509V). PC2 over-expression increased autophagic flux, whereas PC2-D509V expression did not. Importantly, autophagy induction triggered by PC2 over-expression was attenuated by BAPTA-AM, supporting a model of PC2-dependent control of autophagy through intracellular Ca 2+ . Furthermore, PC2 ablation was associated with impaired Ca 2+ handling in cardiomyocytes marked by partial depletion of sarcoplasmic reticulum Ca 2+ stores. Finally, we provide evidence that Ca 2+ -mediated autophagy elicited by PC2 is a mechanism conserved across multiple cell types. Together, this study unveils PC2 as a novel regulator of autophagy acting through control of intracellular Ca 2+ homeostasis. Copyright © 2018. Published by Elsevier Ltd.

HSAN1 mutations in serine palmitoyltransferase reveal a close structure-function-phenotype relationship.

PubMed

Bode, Heiko; Bourquin, Florence; Suriyanarayanan, Saranya; Wei, Yu; Alecu, Irina; Othman, Alaa; Von Eckardstein, Arnold; Hornemann, Thorsten

2016-03-01

Hereditary sensory and autonomic neuropathy type 1 (HSAN1) is a rare autosomal dominant inherited peripheral neuropathy caused by mutations in the SPTLC1 and SPTLC2 subunits of serine palmitoyltransferase (SPT). The mutations induce a permanent shift in the substrate preference from L-serine to L-alanine, which results in the pathological formation of atypical and neurotoxic 1-deoxy-sphingolipids (1-deoxySL). Here we compared the enzymatic properties of 11 SPTLC1 and six SPTLC2 mutants using a uniform isotope labelling approach. In total, eight SPT mutants (STPLC1p.C133W, p.C133Y, p.S331F, p.S331Y and SPTLC2p.A182P, p.G382V, p.S384F, p.I504F) were associated with increased 1-deoxySL synthesis. Despite earlier reports, canonical activity with l-serine was not reduced in any of the investigated SPT mutants. Three variants (SPTLC1p.S331F/Y and SPTLC2p.I505Y) showed an increased canonical activity and increased formation of C20 sphingoid bases. These three mutations are associated with an exceptionally severe HSAN1 phenotype, and increased C20 sphingosine levels were also confirmed in plasma of patients. A principal component analysis of the analysed sphingoid bases clustered the mutations into three separate entities. Each cluster was related to a distinct clinical outcome (no, mild and severe HSAN1 phenotype). A homology model based on the protein structure of the prokaryotic SPT recapitulated the same grouping on a structural level. Mutations associated with the mild form clustered around the active site, whereas mutations associated with the severe form were located on the surface of the protein. In conclusion, we showed that HSAN1 mutations in SPT have distinct biochemical properties, which allowed for the prediction of the clinical symptoms on the basis of the plasma sphingoid base profile. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Effect of lysophosphatidylcholine on the filtration coefficient in intact dog lungs

NASA Technical Reports Server (NTRS)

Butler, B. D.; Davies, I.; Drake, R. E.

1989-01-01

Lysophosphatidylcholine (lyso-Pc) is a lysophospholipid normally found in low concentrations in the lung. At high concentrations lyso-Pc, instilled into the airways, causes pulmonary edema. The hypothesis was tested that the edema caused by lyso-Pc was due to an increase in pulmonary microvascular membrane permeability. In 11 anesthetized dogs, the left lower lobes (LLL) were continuously weighed while lyso-Pc (20 mM) was instilled into the LLL airways. After 30 min, the microvascular membrane fluid filtration coefficient (Kf) was determined from the relationship between the rate of LLL weight gain and the pulmonary microvascular pressure. Kf was not significantly different between the lyso-Pc-treated lobes vs control lobes. The data do not support the hypothesis that lyso-Pc, instilled into the airways, causes an increase in pulmonary microvascular permeability.

A mutation within the extended X loop abolished substrate-induced ATPase activity of the human liver ATP-binding cassette (ABC) transporter MDR3.

PubMed

Kluth, Marianne; Stindt, Jan; Dröge, Carola; Linnemann, Doris; Kubitz, Ralf; Schmitt, Lutz

2015-02-20

The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Mutations in the G6PC3 gene cause Dursun syndrome.

PubMed

Banka, Siddharth; Newman, William G; Ozgül, R Koksal; Dursun, Ali

2010-10-01

Dursun syndrome is a triad of familial primary pulmonary hypertension, leucopenia, and atrial septal defect. Here we demonstrate that mutations in G6PC3 cause Dursun syndrome. Mutations in G6PC3 are known to also cause severe congenital neutropenia type 4. Identification of the genetic basis of Dursun syndrome expands the pre-existing knowledge about the phenotypic effects of mutations in G6PC3. We propose that Dursun syndrome should now be considered as a subset of severe congenital neutropenia type 4 with pulmonary hypertension as an important clinical feature. Copyright © 2010 Wiley-Liss, Inc.

Roles of phospholipid methyltransferases in pycnidia development, stress tolerance and secondary metabolism in the taxol-producing fungus Pestalotiopsis microspore.

PubMed

Akhberdi, Oren; Zhang, Qian; Wang, Haichuan; Li, Yingying; Chen, Longfei; Wang, Dan; Yu, Xi; Wei, Dongsheng; Zhu, Xudong

2018-05-01

Phosphatidylcholine (PC) is an important membrane component of the eukaryotic cell. In yeast fungi, two phospholipid methyltransferases catalyze consecutive steps of methylation in the formation of phosphatidylcholine from phosphatidylethanolamine. However, roles of phospholipid methyltransferases in filamentous fungi remains less investigated. We report here the characterization of two genes, choA and choC, that putatively encoded phospholipid methyltransferases in the taxol-producing fungus Pestalotiopsis microspora. Deletion of choC resulted in defects in PC production, vegetative growth and development of asexual structure. The mutant strains exhibited multiple morphological abnormalities, e.g. swollen hyphal tips and enhanced hyphal branching, and even mycelial autolysis. Some novel roles for the genes were also revealed, for instance, the deletion of either choC or choA impaired the development of pycnidia and conidia, the cell wall integrity. The mutant strains displayed a hypersensitivity to stress conditions, e.g. osmotic stress, cold and metal ions. The osmotic hypersensitivity indicates a crosstalk of PC pathways to other signaling pathways, such as the HOG pathway. Still more, choA, but not choC, was required for the production of secondary metabolites, e.g. pestalotiollide B, suggesting distinct roles of the two genes. This work would contribute to better understanding the function of phospholipid methyltransferases in fungi. Copyright © 2018 Elsevier GmbH. All rights reserved.

Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2

PubMed Central

Furuta, Machi; Yano, Hideki; Zhou, An; Rouillé, Yves; Holst, Jens J.; Carroll, Raymond; Ravazzola, Mariella; Orci, Lelio; Furuta, Hiroto; Steiner, Donald F.

1997-01-01

The prohormone convertase SPC2 (PC2) participates in the processing of proinsulin, proglucagon, and a variety of other neuroendocrine precursors, acting either alone or in conjunction with the structurally related dense-core granule convertase SPC3 (PC3/PC1). We have generated a strain of mice lacking active SPC2 by introducing the neomycin resistance gene (Neor) into the third exon of the mSPC2 gene. This gene insertion results in the synthesis of an exon 3-deleted form of SPC2 that does not undergo autoactivation and is not secreted. The homozygous mutant mice appear to be normal at birth. However, they exhibit a small decrease in rate of growth. They also have chronic fasting hypoglycemia and a reduced rise in blood glucose levels during an intraperitoneal glucose tolerance test, which is consistent with a deficiency of circulating glucagon. The processing of proglucagon, prosomatostatin, and proinsulin in the alpha, delta, and beta cells, respectively, of the pancreatic islets is severely impaired. The islets in mutant mice at 3 months of age show marked hyperplasia of alpha and delta cells and a relative diminution of beta cells. SPC2-defective mice offer many possibilities for further delineating neuroendocrine precursor processing mechanisms and for exploring more fully the physiological roles of many neuropeptides and peptide hormones. PMID:9192619

Molecular and Functional Characterization of Broccoli EMBRYONIC FLOWER 2 Genes

PubMed Central

Chen, Long-Fang O.; Lin, Chun-Hung; Lai, Ying-Mi; Huang, Jia-Yuan; Sung, Zinmay Renee

2012-01-01

Polycomb group (PcG) proteins regulate major developmental processes in Arabidopsis. EMBRYONIC FLOWER 2 (EMF2), the VEFS domain-containing PcG gene, regulates diverse genetic pathways and is required for vegetative development and plant survival. Despite widespread EMF2-like sequences in plants, little is known about their function other than in Arabidopsis and rice. To study the role of EMF2 in broccoli (Brassica oleracea var. italica cv. Elegance) development, we identified two broccoli EMF2 (BoEMF2) genes with sequence homology to and a similar gene expression pattern to that in Arabidopsis (AtEMF2). Reducing their expression in broccoli resulted in aberrant phenotypes and gene expression patterns. BoEMF2 regulates genes involved in diverse developmental and stress programs similar to AtEMF2 in Arabidopsis. However, BoEMF2 differs from AtEMF2 in the regulation of flower organ identity, cell proliferation and elongation, and death-related genes, which may explain the distinct phenotypes. The expression of BoEMF2.1 in the Arabidopsis emf2 mutant (Rescued emf2) partially rescued the mutant phenotype and restored the gene expression pattern to that of the wild type. Many EMF2-mediated molecular and developmental functions are conserved in broccoli and Arabidopsis. Furthermore, the restored gene expression pattern in Rescued emf2 provides insights into the molecular basis of PcG-mediated growth and development. PMID:22537758

The design and delivery of a PKA inhibitory polypeptide to treat SCA1.

PubMed

Hearst, Scoty M; Shao, Qingmei; Lopez, Mariper; Raucher, Drazen; Vig, Parminder J S

2014-10-01

Spinocerebellar ataxia-1 (SCA1) is a neurodegenerative disease that primarily targets Purkinje cells (PCs) of the cerebellum. The exact mechanism of PC degeneration is unknown, however, it is widely believed that mutant ataxin-1 becomes toxic because of the phosphorylation of its serine 776 (S776) residue by cAMP-dependent protein kinase A (PKA). Therefore, to directly modulate mutant ATXN1 S776 phosphorylation and aggregation, we designed a therapeutic polypeptide to inhibit PKA. This polypeptide comprised of a thermally responsive elastin-like peptide (ELP) carrier, which increases peptide half-life, a PKA inhibitory peptide (PKI), and a cell-penetrating peptide (Synb1). We observed that our therapeutic polypeptide, Synb1-ELP-PKI, inhibited PKA activity at concentrations similar to the PKI peptide. Additionally, Synb1-ELP-PKI significantly suppressed mutant ATXN1 S776 phosphorylation and intranuclear inclusion formation in cell culture. Further, Synb1-ELP-PKI treatment improved SCA1 PC morphology in cerebellar slice cultures. Furthermore, the Synb1-ELP peptide carrier crossed the blood-brain barrier and localized to the cerebellum via the i.p. or intranasal route. Here, we show the intranasal delivery of ELP-based peptides to the brain as a novel delivery strategy. We also demonstrate that our therapeutic polypeptide has a great potential to target the neurotoxic S776 phosphorylation pathway in the SCA1 disease. © 2014 International Society for Neurochemistry.

HAL-2 Promotes Homologous Pairing during Caenorhabditis elegans Meiosis by Antagonizing Inhibitory Effects of Synaptonemal Complex Precursors

PubMed Central

Zhang, Weibin; Miley, Natasha; Zastrow, Michael S.; MacQueen, Amy J.; Sato, Aya; Nabeshima, Kentaro; Martinez-Perez, Enrique; Mlynarczyk-Evans, Susanna; Carlton, Peter M.; Villeneuve, Anne M.

2012-01-01

During meiosis, chromosomes align with their homologous pairing partners and stabilize this alignment through assembly of the synaptonemal complex (SC). Since the SC assembles cooperatively yet is indifferent to homology, pairing and SC assembly must be tightly coordinated. We identify HAL-2 as a key mediator in this coordination, showing that HAL-2 promotes pairing largely by preventing detrimental effects of SC precursors (SYP proteins). hal-2 mutants fail to establish pairing and lack multiple markers of chromosome movement mediated by pairing centers (PCs), chromosome sites that link chromosomes to cytoplasmic microtubules through nuclear envelope-spanning complexes. Moreover, SYP proteins load inappropriately along individual unpaired chromosomes in hal-2 mutants, and markers of PC-dependent movement and function are restored in hal-2; syp double mutants. These and other data indicate that SYP proteins can impede pairing and that HAL-2 promotes pairing predominantly but not exclusively by counteracting this inhibition, thereby enabling activation and regulation of PC function. HAL-2 concentrates in the germ cell nucleoplasm and colocalizes with SYP proteins in nuclear aggregates when SC assembly is prevented. We propose that HAL-2 functions to shepherd SYP proteins prior to licensing of SC assembly, preventing untimely interactions between SC precursors and chromosomes and allowing sufficient accumulation of precursors for rapid cooperative assembly upon homology verification. PMID:22912597

HAL-2 promotes homologous pairing during Caenorhabditis elegans meiosis by antagonizing inhibitory effects of synaptonemal complex precursors.

PubMed

Zhang, Weibin; Miley, Natasha; Zastrow, Michael S; MacQueen, Amy J; Sato, Aya; Nabeshima, Kentaro; Martinez-Perez, Enrique; Mlynarczyk-Evans, Susanna; Carlton, Peter M; Villeneuve, Anne M

2012-01-01

During meiosis, chromosomes align with their homologous pairing partners and stabilize this alignment through assembly of the synaptonemal complex (SC). Since the SC assembles cooperatively yet is indifferent to homology, pairing and SC assembly must be tightly coordinated. We identify HAL-2 as a key mediator in this coordination, showing that HAL-2 promotes pairing largely by preventing detrimental effects of SC precursors (SYP proteins). hal-2 mutants fail to establish pairing and lack multiple markers of chromosome movement mediated by pairing centers (PCs), chromosome sites that link chromosomes to cytoplasmic microtubules through nuclear envelope-spanning complexes. Moreover, SYP proteins load inappropriately along individual unpaired chromosomes in hal-2 mutants, and markers of PC-dependent movement and function are restored in hal-2; syp double mutants. These and other data indicate that SYP proteins can impede pairing and that HAL-2 promotes pairing predominantly but not exclusively by counteracting this inhibition, thereby enabling activation and regulation of PC function. HAL-2 concentrates in the germ cell nucleoplasm and colocalizes with SYP proteins in nuclear aggregates when SC assembly is prevented. We propose that HAL-2 functions to shepherd SYP proteins prior to licensing of SC assembly, preventing untimely interactions between SC precursors and chromosomes and allowing sufficient accumulation of precursors for rapid cooperative assembly upon homology verification.

Complex folding and misfolding effects of deer-specific amino acid substitutions in the β2-α2 loop of murine prion protein

NASA Astrophysics Data System (ADS)

Agarwal, Sonya; Döring, Kristina; Gierusz, Leszek A.; Iyer, Pooja; Lane, Fiona M.; Graham, James F.; Goldmann, Wilfred; Pinheiro, Teresa J. T.; Gill, Andrew C.

2015-10-01

The β2-α2 loop of PrPC is a key modulator of disease-associated prion protein misfolding. Amino acids that differentiate mouse (Ser169, Asn173) and deer (Asn169, Thr173) PrPC appear to confer dramatically different structural properties in this region and it has been suggested that amino acid sequences associated with structural rigidity of the loop also confer susceptibility to prion disease. Using mouse recombinant PrP, we show that mutating residue 173 from Asn to Thr alters protein stability and misfolding only subtly, whilst changing Ser to Asn at codon 169 causes instability in the protein, promotes oligomer formation and dramatically potentiates fibril formation. The doubly mutated protein exhibits more complex folding and misfolding behaviour than either single mutant, suggestive of differential effects of the β2-α2 loop sequence on both protein stability and on specific misfolding pathways. Molecular dynamics simulation of protein structure suggests a key role for the solvent accessibility of Tyr168 in promoting molecular interactions that may lead to prion protein misfolding. Thus, we conclude that ‘rigidity’ in the β2-α2 loop region of the normal conformer of PrP has less effect on misfolding than other sequence-related effects in this region.

Anti-Epidermal Growth Factor Vaccine Antibodies Enhance the Efficacy of Tyrosine Kinase Inhibitors and Delay the Emergence of Resistance in EGFR Mutant Lung Cancer Cells.

PubMed

Codony-Servat, Jordi; García-Roman, Silvia; Molina-Vila, Miguel Ángel; Bertran-Alamillo, Jordi; Giménez-Capitán, Ana; Viteri, Santiago; Cardona, Andrés F; d'Hondt, Erik; Karachaliou, Niki; Rosell, Rafael

2018-05-08

Mutations in EGFR correlate with impaired response to immune checkpoint inhibitors and the development of novel immunotherapeutic approaches for EGFR mutant non-small cell lung cancer (NSCLC) is of particular interest. Immunization against EGF has demonstrated efficacy in a phase III trial including unselected NSCLC patients, but little was known about the mechanisms involved in the effects of the anti-EGF antibodies generated by vaccination (anti-EGF VacAbs) or their activity in tumor cells with EGFR mutations. The EGFR-mutant, NSCLC cell lines H1975 and PC9, together with several gefitinib and osimertinib-resistant cells derived from PC9, were treated with anti-EGF VacAbs and/or EGFR tyrosine kinase inhibitors (TKIs). Cell viability was analyzed by proliferation assays, cell cycle by fluorescence-activated cell sorting analysis and levels of RNA and proteins by quantitative retro-transcription PCR and Western blotting. Anti-EGF VacAbs generated in rabbits suppressed EGF-induced cell proliferation and cycle progression and inhibited downstream EGFR signaling in EGFR-mutant cells. Sera from patients immunized with an EGF vaccine were also able to block activation of EGFR effectors. In combination, the anti-EGF VacAbs significantly enhanced the antitumor activity of all TKIs tested, suppressed Erk1/2 phosphorylation, blocked the activation of signal transducer and activator of transcription 3 (STAT3) and downregulated the expression of AXL. Finally, anti-EGF VacAbs significantly delayed the emergence in vitro of EGFR TKI resistant clones. EGFR-mutant patients can derive benefit from immunization against EGF, particularly if combined with EGFR TKIs. A Phase I trial of an EGF vaccine in combination with afatinib has been initiated. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Identification and Characterization of a High-Affinity Choline Uptake System of Brucella abortus

PubMed Central

Herrmann, Claudia K.; Bukata, Lucas; Melli, Luciano; Marchesini, M. Ines; Caramelo, Julio J.

2013-01-01

Phosphatidylcholine (PC), a common phospholipid of the eukaryotic cell membrane, is present in the cell envelope of the intracellular pathogen Brucella abortus, the etiological agent of bovine brucellosis. In this pathogen, the biosynthesis of PC proceeds mainly through the phosphatidylcholine synthase pathway; hence, it relies on the presence of choline in the milieu. These observations imply that B. abortus encodes an as-yet-unknown choline uptake system. Taking advantage of the requirement of choline uptake for PC synthesis, we devised a method that allowed us to identify a homologue of ChoX, the high-affinity periplasmic binding protein of the ABC transporter ChoXWV. Disruption of the choX gene completely abrogated PC synthesis at low choline concentrations in the medium, thus indicating that it is a high-affinity transporter needed for PC synthesis via the PC synthase (PCS) pathway. However, the synthesis of PC was restored when the mutant was incubated in media with higher choline concentrations, suggesting the presence of an alternative low-affinity choline uptake activity. By means of a fluorescence-based equilibrium-binding assay and using the kinetics of radiolabeled choline uptake, we show that ChoX binds choline with an extremely high affinity, and we also demonstrate that its activity is inhibited by increasing choline concentrations. Cell infection assays indicate that ChoX activity is required during the first phase of B. abortus intracellular traffic, suggesting that choline concentrations in the early and intermediate Brucella-containing vacuoles are limited. Altogether, these results suggest that choline transport and PC synthesis are strictly regulated in B. abortus. PMID:23161032

Enhanced Single-Step Bioproduction of the Simvastatin Precursor Monacolin J in an Industrial Strain of Aspergillus terreus by Employing the Evolved Lovastatin Hydrolase.

PubMed

Liang, Bo; Huang, Xuenian; Teng, Yun; Liang, Yajing; Yang, Yong; Zheng, Linghui; Lu, Xuefeng

2018-06-01

Biosynthesis of simvastatin, the active pharmaceutical ingredient of cholesterol-lowering drug Zocor, has drawn increasing global attention in recent years. Although single-step in vivo production of monacolin J, the intermediate biosynthetic precursor of simvastatin, has been realized by utilizing lovastatin hydrolase (PcEST) in our previous study, about 5% of residual lovastatin is still a problem for industrial production and quality control. In order to improve conversion efficiency and reduce lovastatin residues, modification of PcEST is carried out through directed evolution and a novel two-step high-throughput screening method. The mutant Q140L shows 18-fold improved whole-cell activity as compared to the wild-type, and one fold enhanced catalytic efficiency and 3 °C increased T 50 10 over the wild-type are observed by characterizing the purified protein. Finally, the engineered A. terreus strain overexpressing Q140L mutant exhibited the increased conversion efficiency and the reduced lovastatin residues by comparing with A. terreus strain overexpressing the wild-type PcEST, where almost 100% of the produced lovastatin is hydrolyzed to monacolin J. Therefore, this improved microbial cell factory can realize single-step bioproduction of monacolin J in a more efficient way, providing an attractive and eco-friendly substitute over the existing chemical synthetic routes of monacolin J and promoting complete bioproduction of simvastatin at industrial scale. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

INCURVATA2 Encodes the Catalytic Subunit of DNA Polymerase α and Interacts with Genes Involved in Chromatin-Mediated Cellular Memory in Arabidopsis thaliana

PubMed Central

Barrero, José María; González-Bayón, Rebeca; del Pozo, Juan Carlos; Ponce, María Rosa; Micol, José Luis

2007-01-01

Cell type–specific gene expression patterns are maintained by the stable inheritance of transcriptional states through mitosis, requiring the action of multiprotein complexes that remodel chromatin structure. Genetic and molecular interactions between chromatin remodeling factors and components of the DNA replication machinery have been identified in Schizosaccharomyces pombe, indicating that some epigenetic marks are replicated simultaneously to DNA with the participation of the DNA replication complexes. This model of epigenetic inheritance might be extended to the plant kingdom, as we report here with the positional cloning and characterization of INCURVATA2 (ICU2), which encodes the putative catalytic subunit of the DNA polymerase α of Arabidopsis thaliana. The strong icu2-2 and icu2-3 insertional alleles caused fully penetrant zygotic lethality when homozygous and incompletely penetrant gametophytic lethality, probably because of loss of DNA polymerase activity. The weak icu2-1 allele carried a point mutation and caused early flowering, leaf incurvature, and homeotic transformations of sepals into carpels and of petals into stamens. Further genetic analyses indicated that ICU2 interacts with TERMINAL FLOWER2, the ortholog of HETEROCHROMATIN PROTEIN1 of animals and yeasts, and with the Polycomb group (PcG) gene CURLY LEAF. Another PcG gene, EMBRYONIC FLOWER2, was found to be epistatic to ICU2. Quantitative RT-PCR analyses indicated that a number of regulatory genes were derepressed in the icu2-1 mutant, including genes associated with flowering time, floral meristem, and floral organ identity. PMID:17873092

An HPLC-ICP-MS technique for determination of cadmium-phytochelatins in genetically modified Arabidopsis thaliana.

PubMed

Sadi, Baki B M; Vonderheide, Anne P; Gong, Ji-Ming; Schroeder, Julian I; Shann, Jodi R; Caruso, Joseph A

2008-01-01

A reversed-phase high-performance liquid chromatographic technique was developed to separate cadmium-phytochelatin complexes (Cd-PC2, Cd-PC3, and Cd-PC4) of interest in the plant Arapidopsis thaliana. High-performance liquid chromatography (HPLC) was coupled to an inductively coupled plasma mass spectrometric (ICP-MS) system with some modification to the interface. This was done in order to sustain the plasma with optimum sensitivity for cadmium detection in the presence of the high methanol loads used in the gradient elution of the reversed-phase separation. The detection limits were found to be 91.8 ngl(-1), 77.2 ngl(-1) and 49.2 ngl(-1) for Cd-PC2, Cd-PC3, and Cd-PC4 respectively. The regression coefficients (r2) for Cd-PC2 to Cd-PC4 detection ranged from 0.998 to 0.999. The method was then used to investigate the occurrence and effect of cadmium-phytochelatin complexes in wild-type Arabidopsis and a phytochelatin-deficient mutant cad1-3 that had been genetically modified to ectopically express the wheat TaPCS1 phytochelatin synthase enzyme. The primary complex found in both wild-type and transgenic plants was Cd-PC2. In both lines, higher levels of Cd-PC2 were found in shoots than in roots, showing that phytochelatin synthases contribute to the accumulation of cadmium in shoots, in the Cd-PC2 form. Genetic modification did, however, impact the overall accumulation of Cd. Transgenic plants contained almost two times more cadmium in the form of Cd-PC2 in their roots than did the corresponding wild-type plants. Similarly, the shoot samples of the modified species also contained more (by 1.6 times) cadmium in the form of Cd-PC2 than the wild type. The enhanced role of PC2 in the transgenic Arabidopsis correlates with data showing long-distance transport of Cd in transgenic plants. Targeted transgenic expression of non-native phytochelatin synthases may contribute to improving the efficiency of plants for phytoremediation.

Signaling Pathways that Mediate Neurotoxin-Induced Death of Dopamine Neurons

DTIC Science & Technology

2008-11-01

lessons from the analysis of mutant mice. J. Cell Biol. 157, 441–453. Reimertz C., Kogel D., Rami A., Chittenden T. and Prehn J. H. (2003) Gene expression...Bauerbach E, Poppe M, Krieglstein J, Prehn JH (2002) p75 neurotrophin receptor is required for constitutive and NGF-induced survival signalling in PC12

Depletion of pro-oncogenic RUNX2 enhances gemcitabine (GEM) sensitivity of p53-mutated pancreatic cancer Panc-1 cells through the induction of pro-apoptotic TAp63.

PubMed

Ozaki, Toshinori; Nakamura, Mizuyo; Ogata, Takehiro; Sang, Meijie; Yoda, Hiroyuki; Hiraoka, Kiriko; Sang, Meixiang; Shimozato, Osamu

2016-11-01

Recently, we have described that siRNA-mediated silencing of runt-related transcription factor 2 (RUNX2) improves anti-cancer drug gemcitabine (GEM) sensitivity of p53-deficient human pancreatic cancer AsPC-1 cells through the augmentation of p53 family TAp63-dependent cell death pathway. In this manuscript, we have extended our study to p53-mutated human pancreatic cancer Panc-1 cells. According to our present results, knockdown of mutant p53 alone had a marginal effect on GEM-mediated cell death of Panc-1 cells. We then sought to deplete RUNX2 using siRNA in Panc-1 cells and examined its effect on GEM sensitivity. Under our experimental conditions, RUNX2 knockdown caused a significant enhancement of GEM sensitivity of Panc-1 cells. Notably, GEM-mediated induction of TAp63 but not of TAp73 was further stimulated in RUNX2-depleted Panc-1 cells, indicating that, like AsPC-1 cells, TAp63 might play a pivotal role in the regulation of GEM sensitivity of Panc-1 cells. Consistent with this notion, forced expression of TAp63α in Panc-1 cells promoted cell cycle arrest and/or cell death, and massively increased luciferase activities driven by TAp63-target gene promoters such as p21WAF1 and NOXA. In addition, immunoprecipitation experiments indicated that RUNX2 forms a complex with TAp63 in Panc-1 cells. Taken together, our current observations strongly suggest that depletion of RUNX2 enhances the cytotoxic effect of GEM on p53-mutated Panc-1 cells through the stimulation of TAp63-dependent cell death pathway even in the presence of a large amount of pro-oncogenic mutant p53, and might provide an attractive strategy to treat pancreatic cancer patients with p53 mutations.

Depletion of pro-oncogenic RUNX2 enhances gemcitabine (GEM) sensitivity of p53-mutated pancreatic cancer Panc-1 cells through the induction of pro-apoptotic TAp63

PubMed Central

Ozaki, Toshinori; Nakamura, Mizuyo; Ogata, Takehiro; Sang, Meijie; Yoda, Hiroyuki; Hiraoka, Kiriko; Sang, Meixiang; Shimozato, Osamu

2016-01-01

Recently, we have described that siRNA-mediated silencing of runt-related transcription factor 2 (RUNX2) improves anti-cancer drug gemcitabine (GEM) sensitivity of p53-deficient human pancreatic cancer AsPC-1 cells through the augmentation of p53 family TAp63-dependent cell death pathway. In this manuscript, we have extended our study to p53-mutated human pancreatic cancer Panc-1 cells. According to our present results, knockdown of mutant p53 alone had a marginal effect on GEM-mediated cell death of Panc-1 cells. We then sought to deplete RUNX2 using siRNA in Panc-1 cells and examined its effect on GEM sensitivity. Under our experimental conditions, RUNX2 knockdown caused a significant enhancement of GEM sensitivity of Panc-1 cells. Notably, GEM-mediated induction of TAp63 but not of TAp73 was further stimulated in RUNX2-depleted Panc-1 cells, indicating that, like AsPC-1 cells, TAp63 might play a pivotal role in the regulation of GEM sensitivity of Panc-1 cells. Consistent with this notion, forced expression of TAp63α in Panc-1 cells promoted cell cycle arrest and/or cell death, and massively increased luciferase activities driven by TAp63-target gene promoters such as p21WAF1 and NOXA. In addition, immunoprecipitation experiments indicated that RUNX2 forms a complex with TAp63 in Panc-1 cells. Taken together, our current observations strongly suggest that depletion of RUNX2 enhances the cytotoxic effect of GEM on p53-mutated Panc-1 cells through the stimulation of TAp63-dependent cell death pathway even in the presence of a large amount of pro-oncogenic mutant p53, and might provide an attractive strategy to treat pancreatic cancer patients with p53 mutations. PMID:27713122

Loss of MyD88 alters neuroinflammatory response and attenuates early Purkinje cell loss in a spinocerebellar ataxia type 6 mouse model

PubMed Central

Aikawa, Tomonori; Mogushi, Kaoru; Iijima-Tsutsui, Kumiko; Ishikawa, Kinya; Sakurai, Miyano; Tanaka, Hiroshi; Mizusawa, Hidehiro; Watase, Kei

2015-01-01

Spinocerebellar ataxia type 6 (SCA6) is dominantly inherited neurodegenerative disease, caused by an expansion of CAG repeat encoding a polyglutamine (PolyQ) tract in the Cav2.1 voltage-gated calcium channel. Its key pathological features include selective degeneration of the cerebellar Purkinje cells (PCs), a common target for PolyQ-induced toxicity in various SCAs. Mutant Cav2.1 confers toxicity primarily through a toxic gain-of-function mechanism; however, its molecular basis remains elusive. Here, we studied the cerebellar gene expression patterns of young Sca6-MPI118Q/118Q knockin (KI) mice, which expressed mutant Cav2.1 from an endogenous locus and recapitulated many phenotypic features of human SCA6. Transcriptional signatures in the MPI118Q/118Q mice were distinct from those in the Sca1154Q/2Q mice, a faithful SCA1 KI mouse model. Temporal expression profiles of the candidate genes revealed that the up-regulation of genes associated with microglial activation was initiated before PC degeneration and was augmented as the disease progressed. Histological analysis of the MPI118Q/118Q cerebellum showed the predominance of M1-like pro-inflammatory microglia and it was concomitant with elevated expression levels of tumor necrosis factor, interleukin-6, Toll-like receptor (TLR) 2 and 7. Genetic ablation of MyD88, a major adaptor protein conveying TLR signaling, altered expression patterns of M1/M2 microglial phenotypic markers in the MPI118Q/118Q cerebellum. More importantly, it ameliorated PC loss and partially rescued motor impairments in the early disease phase. These results suggest that early neuroinflammatory response may play an important role in the pathogenesis of SCA6 and its modulation could pave the way for slowing the disease progression during the early stage of the disease. PMID:26034136

Inhibition of prohormone convertase 1 (PC1) expression in cholecystokinin (CCK) expressing At-T20 cells decreased cellular content and secretion of CCK and caused a shift in molecular forms of CCK secreted.

PubMed

Beinfeld, Margery C; Vishnuvardhan, Daesety; Blum, Alissa; Reynolds, Nicole; Fannous, Sanya; Kitagawa, Kouki; Marchand, James E

2006-04-01

Two different RNAi methods were used to inhibit the expression of prohormone convertase 1 (PC1) in At-T20 cells. Transient transfection of double stranded RNA and stable expression of a vector expressing hairpin-loop RNA targeting PC1 reduced cholecystokinin (CCK) secretion from At-T20 cells. PC1 mRNA and protein were also decreased in the vector transfected cells. This treatment caused a shift in the forms of cholecystokinin (CCK) secreted, decreasing CCK 22 and increasing CCK 8. Stable expression of RNAi effectively decreased PC1 expression. The observed decrease in CCK seen with these RNAi treatments further supports a role for PC1 in CCK processing in these cells.

Gene therapy in pancreatic cancer

PubMed Central

Liu, Si-Xue; Xia, Zhong-Sheng; Zhong, Ying-Qiang

2014-01-01

Pancreatic cancer (PC) is a highly lethal disease and notoriously difficult to treat. Only a small proportion of PC patients are eligible for surgical resection, whilst conventional chemoradiotherapy only has a modest effect with substantial toxicity. Gene therapy has become a new widely investigated therapeutic approach for PC. This article reviews the basic rationale, gene delivery methods, therapeutic targets and developments of laboratory research and clinical trials in gene therapy of PC by searching the literature published in English using the PubMed database and analyzing clinical trials registered on the Gene Therapy Clinical Trials Worldwide website (http://www. wiley.co.uk/genmed/ clinical). Viral vectors are main gene delivery tools in gene therapy of cancer, and especially, oncolytic virus shows brighter prospect due to its tumor-targeting property. Efficient therapeutic targets for gene therapy include tumor suppressor gene p53, mutant oncogene K-ras, anti-angiogenesis gene VEGFR, suicide gene HSK-TK, cytosine deaminase and cytochrome p450, multiple cytokine genes and so on. Combining different targets or combination strategies with traditional chemoradiotherapy may be a more effective approach to improve the efficacy of cancer gene therapy. Cancer gene therapy is not yet applied in clinical practice, but basic and clinical studies have demonstrated its safety and clinical benefits. Gene therapy will be a new and promising field for the treatment of PC. PMID:25309069

Molecular basis of autosomal dominant neurohypophyseal diabetes insipidus. Cellular toxicity caused by the accumulation of mutant vasopressin precursors within the endoplasmic reticulum.

PubMed Central

Ito, M; Jameson, J L; Ito, M

1997-01-01

Mutations in the arginine vasopressin (AVP) gene cause autosomal dominant familial neurohypophyseal diabetes insipidus (FNDI). The dominant inheritance pattern has been postulated to reflect neuronal toxicity of the mutant proteins, but the mechanism for such cytotoxicity is unknown. In this study, wild-type or several different mutant AVP genes were stably expressed in neuro2A neuroblastoma cells. When cells were treated with valproic acid to induce neuronal differentiation, each of the mutants caused reduced viability. Metabolic labeling revealed diminished intracellular trafficking of mutant AVP precursors and confirmed inefficient secretion of immunoreactive AVP. Immunofluorescence studies demonstrated marked accumulation of mutant AVP precursors within the endoplasmic reticulum. These studies suggest that the cellular toxicity in FNDI may be caused by the intracellular accumulation of mutant precursor proteins. PMID:9109434

Severe congenital neutropenia resulting from G6PC3 deficiency with increased neutrophil CXCR4 expression and myelokathexis.

PubMed

McDermott, David H; De Ravin, Suk See; Jun, Hyun Sik; Liu, Qian; Priel, Debra A Long; Noel, Pierre; Takemoto, Clifford M; Ojode, Teresa; Paul, Scott M; Dunsmore, Kimberly P; Hilligoss, Dianne; Marquesen, Martha; Ulrick, Jean; Kuhns, Douglas B; Chou, Janice Y; Malech, Harry L; Murphy, Philip M

2010-10-14

Mutations in more than 15 genes are now known to cause severe congenital neutropenia (SCN); however, the pathologic mechanisms of most genetic defects are not fully defined. Deficiency of G6PC3, a glucose-6-phosphatase, causes a rare multisystem syndrome with SCN first described in 2009. We identified a family with 2 children with homozygous G6PC3 G260R mutations, a loss of enzymatic function, and typical syndrome features with the exception that their bone marrow biopsy pathology revealed abundant neutrophils consistent with myelokathexis. This pathologic finding is a hallmark of another type of SCN, WHIM syndrome, which is caused by gain-of-function mutations in CXCR4, a chemokine receptor and known neutrophil bone marrow retention factor. We found markedly increased CXCR4 expression on neutrophils from both our G6PC3-deficient patients and G6pc3(-/-) mice. In both patients, granulocyte colony-stimulating factor treatment normalized CXCR4 expression and neutrophil counts. In G6pc3(-/-) mice, the specific CXCR4 antagonist AMD3100 rapidly reversed neutropenia. Thus, myelokathexis associated with abnormally high neutrophil CXCR4 expression may contribute to neutropenia in G6PC3 deficiency and responds well to granulocyte colony-stimulating factor.

The impact of intermittent versus continuous exposure to EGFR tyrosine kinase inhibitor on selection of EGFR T790M-mutant drug-resistant clones in a lung cancer cell line carrying activating EGFR mutation

PubMed Central

Lee, Youngjoo; Choi, Yu-Ra; Kim, Kyoung-Yeon; Shin, Dong Hoon

2016-01-01

Drug-resistant cell lines are essential tools for investigating the mechanisms of resistance to molecular-targeted anti-cancer drugs. However, little is known about how to establish clinically relevant drug-resistant cell lines. Our study examined the impact of a drug-free period on the establishment of a cell line with clinically relevant resistance to molecular-targeted drugs. We used PC9 cells, a lung cancer cell line carrying EGFR mutation, because this is a validated target for EGFR tyrosine kinase inhibitors (TKI). PC9 cells were intermittently or continuously exposed to increasing concentrations of gefitinib (0.01 μM to 1.0 μM) and the emergence of the most common acquired resistance mutation in EGFR, T790M, was determined. T790M was detected at a 25-fold lower drug concentration in cells continuously exposed to gefitinib (PC9/GRc) than in cells intermittently exposed to gefitinib (PC9/GRi) (0.04 μM vs 1.0 μM, respectively). The mutation frequencies at those drug concentrations were 19.8% and 8.0% in PC9/GRc and PC9/GRi cells, respectively. After drug-free culture for 8 weeks, resistance to gefitinib decreased in the PC9/GRi cells but not in the PC9/GRc cells. In the PC9/GRc cells, the frequency of the T790M mutation was consistently about 20% from 0.04 μM to 1.0 μM of gefitinib. In the PC9/GRc cells, the T790M mutation was detected in all single-cell clones, at frequencies ranging from 7.0% to 37.0%, with a median of 19.5% (95% confidence interval, 17.3%–20.9%). In conclusion, compared with intermittent drug exposure, continuous exposure might select better minor drug-resistant clones when creating cell lines resistant to molecular-targeted drugs. PMID:27270313

The impact of intermittent versus continuous exposure to EGFR tyrosine kinase inhibitor on selection of EGFR T790M-mutant drug-resistant clones in a lung cancer cell line carrying activating EGFR mutation.

PubMed

Lee, Youngjoo; Choi, Yu-Ra; Kim, Kyoung-Yeon; Shin, Dong Hoon

2016-07-12

Drug-resistant cell lines are essential tools for investigating the mechanisms of resistance to molecular-targeted anti-cancer drugs. However, little is known about how to establish clinically relevant drug-resistant cell lines. Our study examined the impact of a drug-free period on the establishment of a cell line with clinically relevant resistance to molecular-targeted drugs. We used PC9 cells, a lung cancer cell line carrying EGFR mutation, because this is a validated target for EGFR tyrosine kinase inhibitors (TKI). PC9 cells were intermittently or continuously exposed to increasing concentrations of gefitinib (0.01 μM to 1.0 μM) and the emergence of the most common acquired resistance mutation in EGFR, T790M, was determined. T790M was detected at a 25-fold lower drug concentration in cells continuously exposed to gefitinib (PC9/GRc) than in cells intermittently exposed to gefitinib (PC9/GRi) (0.04 μM vs 1.0 μM, respectively). The mutation frequencies at those drug concentrations were 19.8% and 8.0% in PC9/GRc and PC9/GRi cells, respectively. After drug-free culture for 8 weeks, resistance to gefitinib decreased in the PC9/GRi cells but not in the PC9/GRc cells. In the PC9/GRc cells, the frequency of the T790M mutation was consistently about 20% from 0.04 μM to 1.0 μM of gefitinib. In the PC9/GRc cells, the T790M mutation was detected in all single-cell clones, at frequencies ranging from 7.0% to 37.0%, with a median of 19.5% (95% confidence interval, 17.3%-20.9%). In conclusion, compared with intermittent drug exposure, continuous exposure might select better minor drug-resistant clones when creating cell lines resistant to molecular-targeted drugs.

The Mutation Breeding and Mutagenic Effect of Air Plasma on Penicillium Chrysogenum

NASA Astrophysics Data System (ADS)

Gui, Fang; Wang, Hui; Wang, Peng; Liu, Hui; Cai, Xiaochun; Hu, Yihua; Yuan, Chengling; Zheng, Zhiming

2012-04-01

Low temperature air plasma was used as the mutation tool for penicillin-producing strain Penicillium chrysogenum. The discharge conditions were RF power of 360 W, temperature of 40°C in a sealed chamber, and pressure of 10 Pa to 30 Pa. The result showed that the kinetics of the survival rate followed a typical saddle-shaped curve. Based on a statistic analysis, at the treating duration of 10 min, the positive mutation rate was as high as 37.5% while the negative mutation rate was low. The colonial morphology changed obviously when the plasma treating duration reached or exceeded 45 min. After both primary and secondary screening, a mutant designated as aPc051310 with high productivity of penicillin was obtained, and a strong mutagenic effect on P. chrysogenum was observed in the process. It was proved that after five generations, the mutant aPc051310 still exhibits a high productivity. All the results prove that the plasma mutation method could be developed as a convenient and effective tool to breed high-yield strains in the fermentation industry, while expanding the plasm application at the same time.

Nucleotide substitutions revealing specific functions of Polycomb group genes.

PubMed

Bajusz, Izabella; Sipos, László; Pirity, Melinda K

2015-04-01

POLYCOMB group (PCG) proteins belong to the family of epigenetic regulators of genes playing important roles in differentiation and development. Mutants of PcG genes were isolated first in the fruit fly, Drosophila melanogaster, resulting in spectacular segmental transformations due to the ectopic expression of homeotic genes. Homologs of Drosophila PcG genes were also identified in plants and in vertebrates and subsequent experiments revealed the general role of PCG proteins in the maintenance of the repressed state of chromatin through cell divisions. The past decades of gene targeting experiments have allowed us to make significant strides towards understanding how the network of PCG proteins influences multiple aspects of cellular fate determination during development. Being involved in the transmission of specific expression profiles of different cell lineages, PCG proteins were found to control wide spectra of unrelated epigenetic processes in vertebrates, such as stem cell plasticity and renewal, genomic imprinting and inactivation of X-chromosome. PCG proteins also affect regulation of metabolic genes being important for switching programs between pluripotency and differentiation. Insight into the precise roles of PCG proteins in normal physiological processes has emerged from studies employing cell culture-based systems and genetically modified animals. Here we summarize the findings obtained from PcG mutant fruit flies and mice generated to date with a focus on PRC1 and PRC2 members altered by nucleotide substitutions resulting in specific alleles. We also include a compilation of lessons learned from these models about the in vivo functions of this complex protein family. With multiple knockout lines, sophisticated approaches to study the consequences of peculiar missense point mutations, and insights from complementary gain-of-function systems in hand, we are now in a unique position to significantly advance our understanding of the molecular basis of in vivo functions of PcG proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

The metabolic enhancer piracetam ameliorates the impairment of mitochondrial function and neurite outgrowth induced by ß-amyloid peptide

PubMed Central

Kurz, C; Ungerer, I; Lipka, U; Kirr, S; Schütt, T; Eckert, A; Leuner, K; Müller, WE

2010-01-01

Background and purpose: β-Amyloid peptide (Aβ) is implicated in the pathogenesis of Alzheimer's disease by initiating a cascade of events from mitochondrial dysfunction to neuronal death. The metabolic enhancer piracetam has been shown to improve mitochondrial dysfunction following brain aging and experimentally induced oxidative stress. Experimental approach: We used cell lines (PC12 and HEK cells) and murine dissociated brain cells. The protective effects of piracetam in vitro and ex vivo on Aβ-induced impairment of mitochondrial function (as mitochondrial membrane potential and ATP production), on secretion of soluble Aβ and on neurite outgrowth in PC12 cells were investigated. Key results: Piracetam improves mitochondrial function of PC12 cells and acutely dissociated brain cells from young NMRI mice following exposure to extracellular Aβ1-42. Similar protective effects against Aβ1-42 were observed in dissociated brain cells from aged NMRI mice, or mice transgenic for mutant human amyloid precursor protein (APP) treated with piracetam for 14 days. Soluble Aβ load was markedly diminished in the brain of those animals after treatment with piracetam. Aβ production by HEK cells stably transfected with mutant human APP was elevated by oxidative stress and this was reduced by piracetam. Impairment of neuritogenesis is an important consequence of Aβ-induced mitochondrial dysfunction and Aβ-induced reduction of neurite growth in PC12 cells was substantially improved by piracetam. Conclusion and implications: Our findings strongly support the concept of improving mitochondrial function as an approach to ameliorate the detrimental effects of Aβ on brain function. This article is commented on by Moncada, pp. 217–219 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00706.x and to view related papers by Pravdic et al. and Puerta et al. visit http://dx.doi.org/10.1111/j.1476-5381.2010.00698.x and http://dx.doi.org/10.1111/j.1476-5381.2010.00663.x PMID:20218980

Autistic-like behaviour and cerebellar dysfunction in Purkinje cell Tsc1 mutant mice.

PubMed

Tsai, Peter T; Hull, Court; Chu, YunXiang; Greene-Colozzi, Emily; Sadowski, Abbey R; Leech, Jarrett M; Steinberg, Jason; Crawley, Jacqueline N; Regehr, Wade G; Sahin, Mustafa

2012-08-30

Autism spectrum disorders (ASDs) are highly prevalent neurodevelopmental disorders, but the underlying pathogenesis remains poorly understood. Recent studies have implicated the cerebellum in these disorders, with post-mortem studies in ASD patients showing cerebellar Purkinje cell (PC) loss, and isolated cerebellar injury has been associated with a higher incidence of ASDs. However, the extent of cerebellar contribution to the pathogenesis of ASDs remains unclear. Tuberous sclerosis complex (TSC) is a genetic disorder with high rates of comorbid ASDs that result from mutation of either TSC1 or TSC2, whose protein products dimerize and negatively regulate mammalian target of rapamycin (mTOR) signalling. TSC is an intriguing model to investigate the cerebellar contribution to the underlying pathogenesis of ASDs, as recent studies in TSC patients demonstrate cerebellar pathology and correlate cerebellar pathology with increased ASD symptomatology. Functional imaging also shows that TSC patients with ASDs display hypermetabolism in deep cerebellar structures, compared to TSC patients without ASDs. However, the roles of Tsc1 and the sequelae of Tsc1 dysfunction in the cerebellum have not been investigated so far. Here we show that both heterozygous and homozygous loss of Tsc1 in mouse cerebellar PCs results in autistic-like behaviours, including abnormal social interaction, repetitive behaviour and vocalizations, in addition to decreased PC excitability. Treatment of mutant mice with the mTOR inhibitor, rapamycin, prevented the pathological and behavioural deficits. These findings demonstrate new roles for Tsc1 in PC function and define a molecular basis for a cerebellar contribution to cognitive disorders such as autism.

Delineation of the Clinical, Molecular and Cellular Aspects of Novel JAM3 Mutations Underlying the Autosomal Recessive Hemorrhagic Destruction of the Brain, Subependymal Calcification and Congenital Cataracts

PubMed Central

Akawi, Nadia A.; Canpolat, Fuat E.; White, Susan M.; Quilis-Esquerra, Josep; Sanchez, Martin Morales; Gamundi, Maria José; Mochida, Ganeshwaran H.; Walsh, Christopher A.; Ali, Bassam R.; Al-Gazali, Lihadh

2014-01-01

We have recently shown that the hemorrhagic destruction of the brain, subependymal calcification and congenital cataracts is caused by biallelic mutations in the gene encoding junctional adhesion molecule 3 (JAM3) protein. Affected members from three new families underwent detailed clinical examination including imaging of the brain. Affected individuals presented with a distinctive phenotype comprising hemorrhagic destruction of the brain, subependymal calcification and congenital cataracts. All patients had a catastrophic clinical course resulting in death in 7 out of 10 affected individuals. Sequencing the coding exons of JAM3 revealed three novel homozygous mutations: c.2T>G (p.M1R), c.346G>A (p.E116K) and c.656G>A (p.C219Y). The p.M1R mutation affects the start codon and therefore is predicted to impair protein synthesis. Cellular studies showed that the p.C219Y mutation resulted in a significant retention of the mutated protein in the endoplasmic reticulum, suggesting a trafficking defect. The p.E116K mutant traffics normally to the plasma membrane as the wild type and may have lost its function due to the lack of interaction with an interacting partner. Our data further support the importance of JAM3 in the development and function of the vascular system and the brain. PMID:23255084

Repeated Administration of D-Amphetamine Induces Distinct Alterations in Behavior and Metabolite Levels in 129Sv and Bl6 Mouse Strains.

PubMed

Vanaveski, Taavi; Narvik, Jane; Innos, Jürgen; Philips, Mari-Anne; Ottas, Aigar; Plaas, Mario; Haring, Liina; Zilmer, Mihkel; Vasar, Eero

2018-01-01

The main goal of the study was to characterize the behavioral and metabolomic profiles of repeated administration (for 11 days) of d-amphetamine (AMPH, 3 mg/kg i. p.), indirect agonist of dopamine (DA), in widely used 129S6/SvEvTac (129Sv) and C57BL/6NTac (Bl6) mouse strains. Acute administration of AMPH (acute AMPH) induced significantly stronger motor stimulation in Bl6. However, repeated administration of AMPH (repeated AMPH) caused stronger motor sensitization in 129Sv compared acute AMPH. Body weight of 129Sv was reduced after repeated saline and AMPH, whereas no change occurred in Bl6. In the metabolomic study, acute AMPH induced an elevation of isoleucine and leucine, branched chain amino acids (BCAA), whereas the level of hexoses was reduced in Bl6. Both BCAAs and hexoses remained on level of acute AMPH after repeated AMPH in Bl6. Three biogenic amines [asymmetric dimethylarginine (ADMA), alpha-aminoadipic acid (alpha-AAA), kynurenine] were significantly reduced after repeated AMPH. Acute AMPH caused in 129Sv a significant reduction of valine, lysophosphatidylcholines (lysoPC a C16:0, lysoPC a C18:2, lysoPC a C20:4), phosphatidylcholine (PC) diacyls (PC aa C34:2, PC aa C36:2, PC aa C36:3, PC aa C36:4) and alkyl-acyls (PC ae C38:4, PC ae C40:4). However, repeated AMPH increased the levels of valine and isoleucine, long-chain acylcarnitines (C14, C14:1-OH, C16, C18:1), PC diacyls (PC aa C38:4, PC aa C38:6, PC aa C42:6), PC acyl-alkyls (PC ae C38:4, PC ae C40:4, PC ae C40:5, PC ae C40:6, PC ae C42:1, PC ae C42:3) and sphingolipids [SM(OH)C22:1, SM C24:0] compared to acute AMPH in 129Sv. Hexoses and kynurenine were reduced after repeated AMPH compared to saline in 129Sv. The established changes probably reflect a shift in energy metabolism toward lipid molecules in 129Sv because of reduced level of hexoses. Pooled data from both strains showed that the elevation of isoleucine and leucine was a prominent biomarker of AMPH-induced behavioral sensitization. Simultaneously a significant decline of hexoses, citrulline, ADMA, and kynurenine occurred. The reduced levels of kynurenine, ADMA, and citrulline likely reflect altered function of N-methyl-D-aspartate (NMDA) and NO systems caused by repeated AMPH. Altogether, 129Sv strain displays stronger sensitization toward AMPH and larger variance in metabolite levels than Bl6.

TDP-43 causes differential pathology in neuronal versus glial cells in the mouse brain

PubMed Central

Yan, Sen; Wang, Chuan-En; Wei, Wenjie; Gaertig, Marta A.; Lai, Liangxue; Li, Shihua; Li, Xiao-Jiang

2014-01-01

Mutations in TAR DNA-binding protein 43 (TDP-43) are associated with familial forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Although recent studies have revealed that mutant TDP-43 in neuronal and glial cells is toxic, how mutant TDP-43 causes primarily neuronal degeneration in an age-dependent manner remains unclear. Using adeno-associated virus (AAV) that expresses mutant TDP-43 (M337V) ubiquitously, we found that mutant TDP-43 accumulates preferentially in neuronal cells in the postnatal mouse brain. We then ubiquitously or selectively expressed mutant TDP-43 in neuronal and glial cells in the striatum of adult mouse brains via stereotaxic injection of AAV vectors and found that it also preferentially accumulates in neuronal cells. Expression of mutant TDP-43 in neurons in the striatum causes more severe degeneration, earlier death and more robust symptoms in mice than expression of mutant TDP-43 in glial cells; however, aging increases the expression of mutant TDP-43 in glial cells, and expression of mutant TDP-43 in older mice caused earlier onset of phenotypes and more severe neuropathology than that in younger mice. Although expression of mutant TDP-43 in glial cells via stereotaxic injection does not lead to robust neurological phenotypes, systemic inhibition of the proteasome activity via MG132 in postnatal mice could exacerbate glial TDP-43-mediated toxicity and cause mice to die earlier. Consistently, this inhibition increases the expression of mutant TDP-43 in glial cells in mouse brains. Thus, the differential accumulation of mutant TDP-43 in neuronal versus glial cells contributes to the preferential toxicity of mutant TDP-43 in neuronal cells and age-dependent pathology. PMID:24381309

TDP-43 causes differential pathology in neuronal versus glial cells in the mouse brain.

PubMed

Yan, Sen; Wang, Chuan-En; Wei, Wenjie; Gaertig, Marta A; Lai, Liangxue; Li, Shihua; Li, Xiao-Jiang

2014-05-15

Mutations in TAR DNA-binding protein 43 (TDP-43) are associated with familial forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Although recent studies have revealed that mutant TDP-43 in neuronal and glial cells is toxic, how mutant TDP-43 causes primarily neuronal degeneration in an age-dependent manner remains unclear. Using adeno-associated virus (AAV) that expresses mutant TDP-43 (M337V) ubiquitously, we found that mutant TDP-43 accumulates preferentially in neuronal cells in the postnatal mouse brain. We then ubiquitously or selectively expressed mutant TDP-43 in neuronal and glial cells in the striatum of adult mouse brains via stereotaxic injection of AAV vectors and found that it also preferentially accumulates in neuronal cells. Expression of mutant TDP-43 in neurons in the striatum causes more severe degeneration, earlier death and more robust symptoms in mice than expression of mutant TDP-43 in glial cells; however, aging increases the expression of mutant TDP-43 in glial cells, and expression of mutant TDP-43 in older mice caused earlier onset of phenotypes and more severe neuropathology than that in younger mice. Although expression of mutant TDP-43 in glial cells via stereotaxic injection does not lead to robust neurological phenotypes, systemic inhibition of the proteasome activity via MG132 in postnatal mice could exacerbate glial TDP-43-mediated toxicity and cause mice to die earlier. Consistently, this inhibition increases the expression of mutant TDP-43 in glial cells in mouse brains. Thus, the differential accumulation of mutant TDP-43 in neuronal versus glial cells contributes to the preferential toxicity of mutant TDP-43 in neuronal cells and age-dependent pathology.

Fluorinated Chaperone-β-Cyclodextrin Formulations for β-Glucocerebrosidase Activity Enhancement in Neuronopathic Gaucher Disease.

PubMed

García-Moreno, M Isabel; de la Mata, Mario; Sánchez-Fernández, Elena M; Benito, Juan M; Díaz-Quintana, Antonio; Fustero, Santos; Nanba, Eiji; Higaki, Katsumi; Sánchez-Alcázar, José A; García Fernández, José M; Ortiz Mellet, Carmen

2017-03-09

Amphiphilic glycomimetics encompassing a rigid, undistortable nortropane skeleton based on 1,6-anhydro-l-idonojirimycin and a polyfluorinated antenna, when formulated as the corresponding inclusion complexes with β-cyclodextrin (βCD), have been shown to behave as pharmacological chaperones (PCs) that efficiently rescue lysosomal β-glucocerebrosidase mutants associated with the neuronopathic variants of Gaucher disease (GD), including the highly refractory L444P/L444P and L444P/P415R single nucleotide polymorphs, in patient fibroblasts. The body of work here presented includes the design criteria for the PC prototype, the synthesis of a series of candidates, the characterization of the PC:βCD complexes, the determination of the selectivity profiles toward a panel of commercial and human lysosomal glycosidases, the evaluation of the chaperoning activity in type 1 (non-neuronopathic), type 2 (acute neuronopathic), and type 3 (adult neuronopathic) GD fibroblasts, the confirmation of the rescuing mechanism by immunolabeling, and the analysis of the PC:GCase binding mode by docking experiments.

Post mortem survey of peripheral dental caries in 510 Swedish horses.

PubMed

Gere, I; Dixon, P M

2010-05-01

Peripheral caries (PC) of equine teeth is a poorly described disorder that can cause serious clinical problems if it progresses. To assess the prevalence, sites and severity of PC in a population of Swedish horses. A post mortem study of 510 equine skulls was performed in 2 Swedish equine abattoirs. PC only affected the cheek teeth (CT) and was present in 6.1% (31/510) of skulls. It affected mainly the peripheral cementum, and 87% of PC in the 29 affected mature horses occurred in the 3 caudal CT (Triadan 09-11). Concurrent infundibular caries involving most maxillary CT (mean 9.7/skull) was present in 32% of skulls affected with PC. Trotting horses (mean age 8.1 years) believed to be on a high concentrate and silage diet were preferentially affected with PC in this population. Food was usually tightly adherent to the PC lesions and this feature may have promoted the progression of the disease. Significantly increased levels of diastemata were present in PC-affected horses, and periodontal disease was present in areas adjacent to some PC lesions. PC is a relatively common disorder of horses under certain management conditions that can progress to cause serious dental disorders, especially if concurrent, widespread infundibular caries is present. Equine clinicians should be aware of this significant dental disorder and research into its aetiopathogenesis, possible prevention and treatment are required.

The γ-secretase cleavage product of Polycystin-1 regulates TCF and CHOP-mediated transcriptional activation through a p300-dependent mechanism

PubMed Central

Merrick, David; Chapin, Hannah; Baggs, Julie E.; Yu, Zhiheng; Somlo, Stefan; Sun, Zhaoxia; Hogenesch, John B.; Caplan, Michael

2011-01-01

Summary Mutations in Pkd1, encoding polycystin-1 (PC1), cause Autosomal Dominant Polycystic Kidney Disease (ADPKD). We show that the carboxy-terminal tail (CTT) of PC1 is released by γ-secretase-mediated cleavage and regulates the Wnt and CHOP pathways by binding the transcription factors TCF and CHOP, disrupting their interaction with the common transcriptional co-activator p300. Loss of PC1 causes increased proliferation and apoptosis, while reintroducing PC1-CTT into cultured Pkd1 null cells reestablishes normal growth rate, suppresses apoptosis, and prevents cyst formation. Inhibition of γ-secretase activity impairs the ability of PC1 to suppress growth and apoptosis, and leads to cyst formation in cultured renal epithelial cells. Expression of the PC1-CTT is sufficient to rescue the dorsal body curvature phenotype in zebrafish embryos resulting from either γ-secretase inhibition or suppression of Pkd1 expression. Thus, γ-secretase-dependent release of the PC1-CTT creates a protein fragment whose expression is sufficient to suppress ADPKD-related phenotypes in vitro and in vivo. PMID:22178500

From molecular PDT damage to cellular PDT responses: attempts at bridging the gap on the role of Bcl-2

NASA Astrophysics Data System (ADS)

Usuda, Jitsuo; Xue, Liang-yan; Chiu, Song-mao; Azizuddin, Kashif; Morris, Rachel L.; Mulvihill, John; Oleinick, Nancy L.

2003-06-01

Expression of the anti-apoptotic proteins Bcl-2 and/or Bcl-xL is greatly elevated in many advanced cancers, especially those resistant to standard therapies, such as radiation or chemotherapy. It has been suggested that those two proteins would be attractive targets for the development of new cancer treatments. Photodynamic therapy (PDT) with photosensitizers that localize in or target mitochondria, such as the phthalocyanine Pc 4, specifically attack the anti-apoptotic protein Bcl-2, generating a variety of oxidized, complexed, and cleaved photoproducts. The closely related protein Bcl-xL is also a target of Pc 4-PDT. In a recent study employing transient transfection of an expression vector encoding deletion mutants of Bcl-2, we identified the membrane anchorage regions of the protein that are required to form the photosensitive target. In spite of the demonstrated photodamage to Bcl-2 (and Bcl-xL), how the photodamage translates into changes in the sensitivity of cells to PDT-induced apoptosis or other modes of cell death is not clear, and it also remains unclear how elevated amounts of anti-apoptotic proteins in tumors might make them more or less responsive to PDT. In the present study, we have studied the PDT response of MCF7 human breast cancer cells overexpressing wild-type Bcl-2 or certain deletion mutants either in a transient or stable mode. We show that cells expressing modestly elevated amounts (<10-fold increase) of Bcl-2 and in which the pro-apoptotic protein Bax is not upregulated do not differ from the parental cells with respect to PDT-induced cell killing. In contrast, cells expressing higher amounts (>50-fold increase) of Bcl-2 or certain mutants are made significantly more resistant to the induction of apoptosis and the loss of clonogenicity upon exposure to Pc 4-PDT. In the presence of high levels of Bcl-2, extensive photodamage requires higher PDT doses. We conclude that Pc 4-PDT targets Bcl-2 and Bcl-xL, eliminating one mechanism that protects the tumor cells from other types of therapy. However, it is possible that cells expressing very high levels of the anti-apoptotic proteins might still be resistant to PDT. The data suggest that PDT with a non-vascular-targeting photosensitizer might be effective in a combination treatment in which Bcl-2 and Bcl-xL are first photodamaged before delivery of a second agent.

Novel mutations of androgen receptor: a possible mechanism of bicalutamide withdrawal syndrome.

PubMed

Hara, Takahito; Miyazaki, Jun-ichi; Araki, Hideo; Yamaoka, Masuo; Kanzaki, Naoyuki; Kusaka, Masami; Miyamoto, Masaomi

2003-01-01

Most prostate cancers (PCs) become resistant to combined androgen blockade therapy with surgical or medical castration and antiandrogens after several years. Some of these refractory PCs regress after discontinuation of antiandrogen administration [antiandrogen withdrawal syndrome (AWS)]. Although the molecular mechanisms of the AWS are not fully understood because of the lack of suitable experimental models, one hypothesis of the mechanism is mutation of androgen receptor (AR). However, bicalutamide, which has become the most prevalent pure antiandrogen, does not work as an agonist for any mutant AR detected thus far in PC. To elucidate the mechanisms of the AWS, we established and characterized novel LNCaP cell sublines, LNCaP-cxDs, which were generated in vitro by culturing androgen-dependent LNCaP-FGC human PC cells in androgen-depleted medium with bicalutamide to mimic the combined androgen blockade therapy. LNCaP-FGC cells did not grow at first, but they started to grow after 6-13 weeks of culture. Bicalutamide stimulated LNCaP-cxD cell growth and increased prostate-specific antigen secretion from LNCaP-cxD cells both in vitro and in vivo. Sequencing of AR transcripts revealed that the AR in LNCaP-cxD cells harbors a novel mutation in codon 741, TGG (tryptophan) to TGT (cysteine; W741C), or in codon 741, TGG to TTG (leucine; W741L), in the ligand-binding domain. Transactivation assays showed that bicalutamide worked as an agonist for both W741C and W741L mutant ARs. Importantly, another antiandrogen, hydroxyflutamide, worked as an antagonist for these mutant ARs. In summary, we demonstrate for the first time that within only 6-13 weeks of in vitro exposure to bicalutamide, LNCaP-FGC cells, whose growth had initially been suppressed, came to use bicalutamide as an AR agonist via W741 AR mutation to survive. Our data strongly support the hypothesis that AR mutation is one possible mechanism of the AWS and suggest that flutamide might be effective as a second-line therapy for refractory PC previously treated with bicalutamide.

Capns1, a new binding partner of RasGAP-SH3 domain in K-Ras(V12) oncogenic cells: modulation of cell survival and migration.

PubMed

Pamonsinlapatham, Perayot; Gril, Brunilde; Dufour, Sylvie; Hadj-Slimane, Réda; Gigoux, Véronique; Pethe, Stéphanie; L'hoste, Sébastien; Camonis, Jacques; Garbay, Christiane; Raynaud, Françoise; Vidal, Michel

2008-11-01

Ras GTPase-activating protein (RasGAP) is hypothesized to be an effector of oncogenic Ras stimulating numerous downstream cellular signaling cascades involved in survival, proliferation and motility. In this study, we identified calpain small subunit-1 (Capns1) as a new RasGAP-SH3 domain binding partner, using yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation assay and was found specific to cells expressing oncogenic K-Ras. We used confocal microscopy to analyze our stably transfected cell model producing mutant Ras (PC3Ras(V12)). Staining for RasGAP-SH3/Capns1 co-localization was two-fold stronger in the protrusions of Ras(V12) cells than in PC3 cells. RasGAP or Capns1 knockdown in PC3Ras(V12) cells induced a two- to three-fold increase in apoptosis. Capns1 gene silencing reduced the speed and increased the persistence of movement in PC3Ras(V12) cells. In contrast, RasGAP knockdown in PC3Ras(V12) cells increased cell migration. Knockdown of both proteins altered the speed and directionality of cell motility. Our findings suggest that RasGAP and Capns1 interaction in oncogenic Ras cells is involved in regulating migration and cell survival.

Interaction Between the Biotin Carboxyl Carrier Domain and the Biotin Carboxylase Domain in Pyruvate Carboxylase from Rhizobium etli†

PubMed Central

Lietzan, Adam D.; Menefee, Ann L.; Zeczycki, Tonya N.; Kumar, Sudhanshu; Attwood, Paul V.; Wallace, John C.; Cleland, W. Wallace; Maurice, Martin St.

2011-01-01

Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To effect catalysis, the tethered biotin of PC must gain access to active sites in both the biotin carboxylase domain and the carboxyl transferase domain. Previous studies have demonstrated that a mutation of threonine 882 to alanine in PC from Rhizobium etli renders the carboxyl transferase domain inactive and favors the positioning of biotin in the biotin carboxylase domain. We report the 2.4 Å resolution X-ray crystal structure of the Rhizobium etli PC T882A mutant which reveals the first high-resolution description of the domain interaction between the biotin carboxyl carrier protein domain and the biotin carboxylase domain. The overall quaternary arrangement of Rhizobium etli PC remains highly asymmetrical and is independent of the presence of allosteric activator. While biotin is observed in the biotin carboxylase domain, its access to the active site is precluded by the interaction between Arg353 and Glu248, revealing a mechanism for regulating carboxybiotin access to the BC domain active site. The binding location for the biotin carboxyl carrier protein domain demonstrates that tethered biotin cannot bind in the biotin carboxylase domain active site in the same orientation as free biotin, helping to explain the difference in catalysis observed between tethered biotin and free biotin substrates in biotin carboxylase enzymes. Electron density located in the biotin carboxylase domain active site is assigned to phosphonoacetate, offering a probable location for the putative carboxyphosphate intermediate formed during biotin carboxylation. The insights gained from the T882A Rhizobium etli PC crystal structure provide a new series of catalytic snapshots in PC and offer a revised perspective on catalysis in the biotin-dependent enzyme family. PMID:21958016

Three-Dimensional Constructive Interference in Steady State Sequences and Phase-Contrast Magnetic Resonance Imaging of Arrested Hydrocephalus.

PubMed

Elkafrawy, Fatma; Reda, Ihab; Elsirafy, Mohamed; Gawad, Mohamed Saied Abdel; Elnaggar, Alaa; Khalek Abdel Razek, Ahmed Abdel

2017-02-01

To evaluate the role of three-dimensional constructive interference in steady state (3D-CISS) sequences and phase-contrast magnetic resonance imaging (PC-MRI) in patients with arrested hydrocephalus. A prospective study of 20 patients with arrested hydrocephalus was carried out. All patients underwent PC-MRI and 3D-CISS for assessment of the aqueduct. Axial (through-plane), sagittal (in-plane) PC-MRI, and sagittal 3D-CISS were applied to assess the cerebral aqueduct and the spontaneous third ventriculostomy if present. Aqueductal patency was graded using 3D-CISS and PC-MRI. Quantitative analysis of flow through the aqueduct was performed using PC-MRI. The causes of obstruction were aqueductal obstruction in 75% (n = 15), third ventricular obstruction in 5% (n = 1), and fourth ventricular obstruction in 20% (n = 4). The cause of arrest of hydrocephalus was spontaneous third ventriculostomy in 65% (n = 13), endoscopic third ventriculostomy in 10% (n = 2), and ventriculoperitoneal shunt in 5% (n = 1), and no cause could be detected in 20% of patients (n = 4). There is a positive correlation (r = 0.80) and moderate agreement (κ = 0.509) of grading with PC-MRI and 3D-CISS sequences. The mean peak systolic velocity of cerebrospinal fluid was 1.86 ± 2.48 cm/second, the stroke volume was 6.43 ± 13.81 μL/cycle, and the mean flow was 0.21 ± 0.32 mL/minute. We concluded that 3D-CISS and PC-MRI are noninvasive sequences for diagnosis of the level and cause of arrested hydrocephalus. Copyright © 2016 Elsevier Inc. All rights reserved.

Characterization of necrosis-inducing NLP proteins in Phytophthora capsici

PubMed Central

2014-01-01

Background Effector proteins function not only as toxins to induce plant cell death, but also enable pathogens to suppress or evade plant defense responses. NLP-like proteins are considered to be effector proteins, and they have been isolated from bacteria, fungi, and oomycete plant pathogens. There is increasing evidence that NLPs have the ability to induce cell death and ethylene accumulation in plants. Results We evaluated the expression patterns of 11 targeted PcNLP genes by qRT-PCR at different time points after infection by P. capsici. Several PcNLP genes were strongly expressed at the early stages in the infection process, but the expression of other PcNLP genes gradually increased to a maximum at late stages of infection. The genes PcNLP2, PcNLP6 and PcNLP14 showed the highest expression levels during infection by P. capsici. The necrosis-inducing activity of all targeted PcNLP genes was evaluated using heterologous expression by PVX agroinfection of Capsicum annuum and Nicotiana benthamiana and by Western blot analysis. The members of the PcNLP family can induce chlorosis or necrosis during infection of pepper and tobacco leaves, but the chlorotic or necrotic response caused by PcNLP genes was stronger in pepper leaves than in tobacco leaves. Moreover, PcNLP2, PcNLP6, and PcNLP14 caused the largest chlorotic or necrotic areas in both host plants, indicating that these three genes contribute to strong virulence during infection by P. capsici. This was confirmed through functional evaluation of their silenced transformants. In addition, we further verified that four conserved residues are putatively active sites in PcNLP1 by site-directed mutagenesis. Conclusions Each targeted PcNLP gene affects cells or tissues differently depending upon the stage of infection. Most PcNLP genes could trigger necrotic or chlorotic responses when expressed in the host C. annuum and the non-host N. benthamiana. Individual PcNLP genes have different phytotoxic effects, and PcNLP2, PcNLP6, and PcNLP14 may play important roles in symptom development and may be crucial for virulence, necrosis-inducing activity, or cell death during infection by P. capsici. PMID:24886309

Characterization of necrosis-inducing NLP proteins in Phytophthora capsici.

PubMed

Feng, Bao-Zhen; Zhu, Xiao-Ping; Fu, Li; Lv, Rong-Fei; Storey, Dylan; Tooley, Paul; Zhang, Xiu-Guo

2014-05-08

Effector proteins function not only as toxins to induce plant cell death, but also enable pathogens to suppress or evade plant defense responses. NLP-like proteins are considered to be effector proteins, and they have been isolated from bacteria, fungi, and oomycete plant pathogens. There is increasing evidence that NLPs have the ability to induce cell death and ethylene accumulation in plants. We evaluated the expression patterns of 11 targeted PcNLP genes by qRT-PCR at different time points after infection by P. capsici. Several PcNLP genes were strongly expressed at the early stages in the infection process, but the expression of other PcNLP genes gradually increased to a maximum at late stages of infection. The genes PcNLP2, PcNLP6 and PcNLP14 showed the highest expression levels during infection by P. capsici. The necrosis-inducing activity of all targeted PcNLP genes was evaluated using heterologous expression by PVX agroinfection of Capsicum annuum and Nicotiana benthamiana and by Western blot analysis. The members of the PcNLP family can induce chlorosis or necrosis during infection of pepper and tobacco leaves, but the chlorotic or necrotic response caused by PcNLP genes was stronger in pepper leaves than in tobacco leaves. Moreover, PcNLP2, PcNLP6, and PcNLP14 caused the largest chlorotic or necrotic areas in both host plants, indicating that these three genes contribute to strong virulence during infection by P. capsici. This was confirmed through functional evaluation of their silenced transformants. In addition, we further verified that four conserved residues are putatively active sites in PcNLP1 by site-directed mutagenesis. Each targeted PcNLP gene affects cells or tissues differently depending upon the stage of infection. Most PcNLP genes could trigger necrotic or chlorotic responses when expressed in the host C. annuum and the non-host N. benthamiana. Individual PcNLP genes have different phytotoxic effects, and PcNLP2, PcNLP6, and PcNLP14 may play important roles in symptom development and may be crucial for virulence, necrosis-inducing activity, or cell death during infection by P. capsici.

Binding of 3,4,5,6-Tetrahydroxyazepanes to the Acid-[beta]-glucosidase Active Site: Implications for Pharmacological Chaperone Design for Gaucher Disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Orwig, Susan D.; Tan, Yun Lei; Grimster, Neil P.

2013-03-07

Pharmacologic chaperoning is a therapeutic strategy being developed to improve the cellular folding and trafficking defects associated with Gaucher disease, a lysosomal storage disorder caused by point mutations in the gene encoding acid-{beta}-glucosidase (GCase). In this approach, small molecules bind to and stabilize mutant folded or nearly folded GCase in the endoplasmic reticulum (ER), increasing the concentration of folded, functional GCase trafficked to the lysosome where the mutant enzyme can hydrolyze the accumulated substrate. To date, the pharmacologic chaperone (PC) candidates that have been investigated largely have been active site-directed inhibitors of GCase, usually containing five- or six-membered rings, suchmore » as modified azasugars. Here we show that a seven-membered, nitrogen-containing heterocycle (3,4,5,6-tetrahydroxyazepane) scaffold is also promising for generating PCs for GCase. Crystal structures reveal that the core azepane stabilizes GCase in a variation of its proposed active conformation, whereas binding of an analogue with an N-linked hydroxyethyl tail stabilizes GCase in a conformation in which the active site is covered, also utilizing a loop conformation not seen previously. Although both compounds preferentially stabilize GCase to thermal denaturation at pH 7.4, reflective of the pH in the ER, only the core azepane, which is a mid-micromolar competitive inhibitor, elicits a modest increase in enzyme activity for the neuronopathic G202R and the non-neuronopathic N370S mutant GCase in an intact cell assay. Our results emphasize the importance of the conformational variability of the GCase active site in the design of competitive inhibitors as PCs for Gaucher disease.« less

CDC14A phosphatase is essential for hearing and male fertility in mouse and human.

PubMed

Imtiaz, Ayesha; Belyantseva, Inna A; Beirl, Alisha J; Fenollar-Ferrer, Cristina; Bashir, Rasheeda; Bukhari, Ihtisham; Bouzid, Amal; Shaukat, Uzma; Azaiez, Hela; Booth, Kevin T; Kahrizi, Kimia; Najmabadi, Hossein; Maqsood, Azra; Wilson, Elizabeth A; Fitzgerald, Tracy S; Tlili, Abdelaziz; Olszewski, Rafal; Lund, Merete; Chaudhry, Taimur; Rehman, Atteeq U; Starost, Matthew F; Waryah, Ali M; Hoa, Michael; Dong, Lijin; Morell, Robert J; Smith, Richard J H; Riazuddin, Sheikh; Masmoudi, Saber; Kindt, Katie S; Naz, Sadaf; Friedman, Thomas B

2018-03-01

The Cell Division-Cycle-14 gene encodes a dual-specificity phosphatase necessary in yeast for exit from mitosis. Numerous disparate roles of vertebrate Cell Division-Cycle-14 (CDC14A) have been proposed largely based on studies of cultured cancer cells in vitro. The in vivo functions of vertebrate CDC14A are largely unknown. We generated and analyzed mutations of zebrafish and mouse CDC14A, developed a computational structural model of human CDC14A protein and report four novel truncating and three missense alleles of CDC14A in human families segregating progressive, moderate-to-profound deafness. In five of these families segregating pathogenic variants of CDC14A, deaf males are infertile, while deaf females are fertile. Several recessive mutations of mouse Cdc14a, including a CRISPR/Cas9-edited phosphatase-dead p.C278S substitution, result in substantial perinatal lethality, but survivors recapitulate the human phenotype of deafness and male infertility. CDC14A protein localizes to inner ear hair cell kinocilia, basal bodies and sound-transducing stereocilia. Auditory hair cells of postnatal Cdc14a mutants develop normally, but subsequently degenerate causing deafness. Kinocilia of germ-line mutants of mouse and zebrafish have normal lengths, which does not recapitulate the published cdc14aa knockdown morphant phenotype of short kinocilia. In mutant male mice, degeneration of seminiferous tubules and spermiation defects result in low sperm count, and abnormal sperm motility and morphology. These findings for the first time define a new monogenic syndrome of deafness and male infertility revealing an absolute requirement in vivo of vertebrate CDC14A phosphatase activity for hearing and male fertility.

Severe hypertriglyceridemia due to two novel loss-of-function lipoprotein lipase gene mutations (C310R/E396V) in a Chinese family associated with recurrent acute pancreatitis.

PubMed

Lun, Yu; Sun, Xiaofang; Wang, Ping; Chi, Jingwei; Hou, Xu; Wang, Yangang

2017-07-18

Lipoprotein lipase (LPL) is widely expressed in skeletal muscles, cardiac muscles as well as adipose tissue and involved in the catabolism of triglyceride. Herein we have systematically characterized two novel loss-of-function mutations in LPL from a Chinese family in which afflicted members were manifested by severe hypertriglyceridemia and recurrent pancreatitis. DNA sequencing revealed that the proband was a heterozygote carrying a novel c.T928C (p.C310R) mutation in exon 6 of the LPL gene. Another member of the family was detected to be a compound heterozygote who along with the c.T928C mutation also carried a novel missense mutation c.A1187T (p.E396V) in exon 8 of the LPL gene. Furthermore, COS-1 cells were transfected with lentiviruses containing the mutant LPL genes. While C310R markedly reduced the overall LPL protein level, COS-1 cells carrying E396V or double mutations contained similar overall LPL protein levels to the wild-type. The specific activity of the LPL mutants remained at comparable magnitude to the wild-type. However, few LPL were detected in the culture medium for the mutants, suggesting that both mutations caused aberrant triglyceride catabolism. More specifically, E396V and double mutations dampened the transport of LPL to the cell surface, while for the C310R mutation, reducing LPL protein level might be involved. By characterizing these two novel LPL mutations, this study has expanded our understanding on the pathogenesis of familial hypertriglyceridemia (FHTG).

Novel histone deacetylase inhibitor CG200745 induces clonogenic cell death by modulating acetylation of p53 in cancer cells.

PubMed

Oh, Eun-Taex; Park, Moon-Taek; Choi, Bo-Hwa; Ro, Seonggu; Choi, Eun-Kyung; Jeong, Seong-Yun; Park, Heon Joo

2012-04-01

Histone deacetylase (HDAC) plays an important role in cancer onset and progression. Therefore, inhibition of HDAC offers potential as an effective cancer treatment regimen. CG200745, (E)-N(1)-(3-(dimethylamino)propyl)-N(8)-hydroxy-2-((naphthalene-1-loxy)methyl)oct-2-enediamide, is a novel HDAC inhibitor presently undergoing a phase I clinical trial. Enhancement of p53 acetylation by HDAC inhibitors induces cell cycle arrest, differentiation, and apoptosis in cancer cells. The purpose of the present study was to investigate the role of p53 acetylation in the cancer cell death caused by CG200745. CG200745-induced clonogenic cell death was 2-fold greater in RKO cells expressing wild-type p53 than in p53-deficient RC10.1 cells. CG200745 treatment was also cytotoxic to PC-3 human prostate cancer cells, which express wild-type p53. CG200745 increased acetylation of p53 lysine residues K320, K373, and K382. CG200745 induced the accumulation of p53, promoted p53-dependent transactivation, and enhanced the expression of MDM2 and p21(Waf1/Cip1) proteins, which are encoded by p53 target genes. An examination of CG200745 effects on p53 acetylation using cells transfected with various p53 mutants showed that cells expressing p53 K382R mutants were significantly resistant to CG200745-induced clonogenic cell death compared with wild-type p53 cells. Moreover, p53 transactivation in response to CG200745 was suppressed in all cells carrying mutant forms of p53, especially K382R. Taken together, these results suggest that acetylation of p53 at K382 plays an important role in CG200745-induced p53 transactivation and clonogenic cell death.

ALS mutations in FUS cause neuronal dysfunction and death in Caenorhabditis elegans by a dominant gain-of-function mechanism

PubMed Central

Murakami, Tetsuro; Yang, Seung-Pil; Xie, Lin; Kawano, Taizo; Fu, Donald; Mukai, Asuka; Bohm, Christopher; Chen, Fusheng; Robertson, Janice; Suzuki, Hiroshi; Tartaglia, Gian Gaetano; Vendruscolo, Michele; Kaminski Schierle, Gabriele S.; Chan, Fiona T.S.; Moloney, Aileen; Crowther, Damian; Kaminski, Clemens F.; Zhen, Mei; St George-Hyslop, Peter

2012-01-01

It is unclear whether mutations in fused in sarcoma (FUS) cause familial amyotrophic lateral sclerosis via a loss-of-function effect due to titrating FUS from the nucleus or a gain-of-function effect from cytoplasmic overabundance. To investigate this question, we generated a series of independent Caenorhabditis elegans lines expressing mutant or wild-type (WT) human FUS. We show that mutant FUS, but not WT-FUS, causes cytoplasmic mislocalization associated with progressive motor dysfunction and reduced lifespan. The severity of the mutant phenotype in C. elegans was directly correlated with the severity of the illness caused by the same mutation in humans, arguing that this model closely replicates key features of the human illness. Importantly, the mutant phenotype could not be rescued by overexpression of WT-FUS, even though WT-FUS had physiological intracellular localization, and was not recruited to the cytoplasmic mutant FUS aggregates. Our data suggest that FUS mutants cause neuronal dysfunction by a dominant gain-of-function effect related either to neurotoxic aggregates of mutant FUS in the cytoplasm or to dysfunction in its RNA-binding functions. PMID:21949354

ALS mutations in FUS cause neuronal dysfunction and death in Caenorhabditis elegans by a dominant gain-of-function mechanism.

PubMed

Murakami, Tetsuro; Yang, Seung-Pil; Xie, Lin; Kawano, Taizo; Fu, Donald; Mukai, Asuka; Bohm, Christopher; Chen, Fusheng; Robertson, Janice; Suzuki, Hiroshi; Tartaglia, Gian Gaetano; Vendruscolo, Michele; Kaminski Schierle, Gabriele S; Chan, Fiona T S; Moloney, Aileen; Crowther, Damian; Kaminski, Clemens F; Zhen, Mei; St George-Hyslop, Peter

2012-01-01

It is unclear whether mutations in fused in sarcoma (FUS) cause familial amyotrophic lateral sclerosis via a loss-of-function effect due to titrating FUS from the nucleus or a gain-of-function effect from cytoplasmic overabundance. To investigate this question, we generated a series of independent Caenorhabditis elegans lines expressing mutant or wild-type (WT) human FUS. We show that mutant FUS, but not WT-FUS, causes cytoplasmic mislocalization associated with progressive motor dysfunction and reduced lifespan. The severity of the mutant phenotype in C. elegans was directly correlated with the severity of the illness caused by the same mutation in humans, arguing that this model closely replicates key features of the human illness. Importantly, the mutant phenotype could not be rescued by overexpression of WT-FUS, even though WT-FUS had physiological intracellular localization, and was not recruited to the cytoplasmic mutant FUS aggregates. Our data suggest that FUS mutants cause neuronal dysfunction by a dominant gain-of-function effect related either to neurotoxic aggregates of mutant FUS in the cytoplasm or to dysfunction in its RNA-binding functions.

[Toxicity of Pythium oligandrum broth to animal and its control effect on rot diseases caused by Penicillium italicum and Penicillium digitatum in orange fruit storage].

PubMed

Tan, Yan; Peng, Liangzhi; Yuan, Ling; Wang, Shaobo

2015-11-04

In order to develop a safe, nontoxic and efficient biological antistaling agent and to decrease the incidence of rot diseases caused by the Penicillium italicum and Penicillium digitatum in orange fruit storage. the present experiment was carried out with Pythium oligandrum broth (POB) produced by our self-isolated strain (P. oligandrum CQ2010) to study the toxicity to animal. Thereafter, mycelium growth and spore germination of both P. digitatum and P. italicum and control effect of rot disease in orange storage were compared after treated by liquid culture medium (control), POB, prochloraz (PC) , and PC + POB. Gastric lavage with large amount POB did not influence mouse weight. The animals also showed no abnormality in appearance, behaviors and pathology changes in heart, liver, kidney, lung and intestine. POB decreased the hyphal growth by 70.24% - 93.74% and spore germination by 44.91% - 87.82% (24 h after POB addition) of these two pathogenic fungi. Disease incidence of orange fruit following P. italicum inoculation changed in the sequence: CK > POB > PC > PC + POB and the control efficacy behaved otherwise. In commercial simulation storage, the disease incidence of orange fruit caused by P. digitatum and P. italicum was above 50% of the total. The fruit rot rate was 26.40% (CK), 15.03% (POB), 16.61% (PC) and 4.21% (PC + POB). There were no significant differences in fruit quality under different treatments. POB was safe to animal and could decrease rot disease incidence caused by P. italicum and P. digitatum in orange storage whereby producing a positive interaction with prochloraz and controlling rot diseases caused by these two fungi.

Expanding the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum towards styrylalanines.

PubMed

Bencze, László Csaba; Filip, Alina; Bánóczi, Gergely; Toşa, Monica Ioana; Irimie, Florin Dan; Gellért, Ákos; Poppe, László; Paizs, Csaba

2017-05-03

This study focuses on the expansion of the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) towards the l-enantiomers of racemic styrylalanines rac-1a-d - which are less studied and synthetically challenging unnatural amino acids - by reshaping the aromatic binding pocket of the active site of PcPAL by point mutations. Ammonia elimination from l-styrylalanine (l-1a) catalyzed by non-mutated PcPAL (wt-PcPAL) took place with a 777-fold lower k cat /K M value than the deamination of the natural substrate, l-Phe. Computer modeling of the reactions catalyzed by wt-PcPAL indicated an unproductive and two major catalytically active conformations and detrimental interactions between the aromatic moiety of l-styrylalanine, l-1a, and the phenyl ring of the residue F137 in the aromatic binding region of the active site. Replacing the residue F137 by smaller hydrophobic residues resulted in a small mutant library (F137X-PcPAL, X being V, A, and G), from which F137V-PcPAL could transform l-styrylalanine with comparable activity to that of the wt-PcPAL with l-Phe. Furthermore, F137V-PcPAL showed superior catalytic efficiency in the ammonia elimination reaction of several racemic styrylalanine derivatives (rac-1a-d) providing access to d-1a-d by kinetic resolution, even though the d-enantiomers proved to be reversible inhibitors. The enhanced catalytic efficiency of F137V-PcPAL towards racemic styrylalanines rac-1a-d could be rationalized by molecular modeling, indicating the more relaxed enzyme-substrate complexes and the promotion of conformations with higher catalytic activities as the main reasons. Unfortunately, ammonia addition onto the corresponding styrylacrylates 2a-d failed with both wt-PcPAL and F137V-PcPAL. The low equilibrium constant of the ammonia addition, the poor ligand binding affinities of 2a-d, and the non-productive binding states of the unsaturated ligands 2a-d within the active sites of either wt-PcPAL or F137V-PcPAL - as indicated by molecular modeling - might be responsible for the inactivity of the PcPAL variants in the reverse reaction. Modeling predicted that the F137V mutation is beneficial for the KRs of 4-fluoro-, 4-cyano- and 4-bromostyrylalanines, but non-effective for the KR process of 4-trifluoromethylstyrylalanine.

[Prenatal care and hospital maternal mortality in Tijuana, Baja California, Mexico].

PubMed

Gonzaga-Soriano, María Rode; Zonana-Nacach, Abraham; Anzaldo-Campos, María Cecilia; Olazarán-Gutiérrez, Asbeidi

2014-01-01

To describe the prenatal care (PC) received in women with maternal hospital deaths from 2005 to 2011 in Tijuana, Baja California, Mexico. Were reviewed the medical chars and registrations of the maternal deaths by the local Committees of Maternal Mortality. There were 44 maternal hospital deaths. Thirty (68%) women assisted to PC appointments during pregnancy, the average number of PC visits was 3.8 and 18 (41%) had an adequate PC (≥ 5 visits). Six (14%) women didn't know they were pregnant; 19 (43%), 21 (48%) y 4 (9%) maternal deaths were due to direct, indirect obstetric cause or non-obstetric causes. Eighteen (18%), 2 (4 %) and 34 (77%) of the maternal deaths occurred during pregnancy, delivery or puerperium. It is necessary pregnancy women have an early, periodic and systematic PC to identify opportunely risk factors associated with pregnancy complications.

Discovery and characterization of a novel irreversible EGFR mutants selective and potent kinase inhibitor CHMFL-EGFR-26 with a distinct binding mode

PubMed Central

Chen, Cheng; Yu, Kailin; Zou, Fengming; Wang, Wenchao; Wang, Wei; Wu, Jiaxin; Liu, Juan; Wang, Beilei; Wang, Li; Ren, Tao; Zhang, Shanchun; Yun, Cai-Hong; Liu, Jing; Liu, Qingsong

2017-01-01

EGFR T790M mutation accounts for about 40-55% drug resistance for the first generation EGFR kinase inhibitors in the NSCLC. Starting from ibrutinib, a highly potent irreversible BTK kinase inhibitor, which was also found to be moderately active to EGFR T790M mutant, we discovered a highly potent irreversible EGFR inhibitor CHMFL-EGFR-26, which is selectively potent against EGFR mutants including L858R, del19, and L858R/T790M. It displayed proper selectivity window between the EGFR mutants and the wide-type. CHMFL-EGFR-26 exhibited good selectivity profile among 468 kinases/mutants tested (S score (1)=0.02). In addition, X-ray crystallography revealed a distinct “DFG-in” and “cHelix-out” inactive binding mode between CHMFL-EGFR-26 and EGFR T790M protein. The compound showed highly potent anti-proliferative efficacy against EGFR mutant but not wide-type NSCLC cell lines through effective inhibition of the EGFR mediated signaling pathway, induction of apoptosis and arresting of cell cycle progression. CHMFL-EGFR-26 bore acceptable pharmacokinetic properties and demonstrated dose-dependent tumor growth suppression in the H1975 (EGFR L858R/T790M) and PC-9 (EGFR del19) inoculated xenograft mouse models. Currently CHMFL-EGFR-26 is undergoing extensive pre-clinical evaluation for the clinical trial purpose. PMID:28407693

Synergistic effect between erlotinib and MEK inhibitors in KRAS wildtype human pancreatic cancer cells

PubMed Central

Diep, Caroline H.; Munoz, Ruben M.; Choudhary, Ashish; Von Hoff, Daniel D.; Han, Haiyong

2011-01-01

Purpose The combination of gemcitabine plus erlotinib has shown a small but statistically significant survival advantage when compared to gemcitabine alone in patients with advanced pancreatic cancer. However, the overall survival rate with the erlotinib and gemcitabine combination is still low. In this study we sought to identify gene targets that, when inhibited, would enhance the activity of EGFR-targeted therapies in pancreatic cancer cells. Experimental Design A high-throughput RNAi screen was carried out to identify candidate genes. Selected gene hits were further confirmed and mechanisms of action were further investigated using various assays. Results Six gene hits from siRNA screening were confirmed to significantly sensitize BxPC-3 pancreatic cancer cells to erlotinib. One of the hits, MAPK1, was selected for further mechanistic studies. Combination treatments of erlotinib plus two MAP kinase kinase (MEK) inhibitors, RDEA119 and AZD6244, showed significant synergistic effect for both combinations (RDEA119-erlotinib and AZD6244-erlotinib) compared to the corresponding single drug treatments in pancreatic cancer cell lines with wild-type KRAS (BxPC-3 and Hs 700T) but not in cell lines with mutant KRAS (MIA PaCa-2 and PANC-1). The enhanced antitumor activity of the combination treatment was further verified in the BxPC-3 and MIA PaCa-2 mouse xenograft model. Examination of the MAPK signaling pathway by Western blotting indicated effective inhibition of the EGFR signaling by the drug combination in KRAS wildtype cells but not in KRAS mutant cells. Conclusions Overall, our results suggest that combination therapy of an EGFR and MEK inhibitors may have enhanced efficacy in patients with pancreatic cancer. PMID:21385921

Tumorigenesis of K-ras mutation in human endometrial carcinoma via upregulation of estrogen receptor.

PubMed

Tu, Zheng; Gui, Liming; Wang, Jianliu; Li, Xiaoping; Sun, Pengming; Wei, Lihui

2006-05-01

To investigate the tumorigenesis of mutant [12Asp]-K-ras in endometrial carcinoma and its relationship with ER. We constructed pcDI-[12Asp]K-ras4B by inserting full-length [12Asp]K-ras4B from human endometrial carcinoma Hec-1A cells, into pcDI vector. Cell proliferation of NIH3T3 after transfection with pcDI-[12Asp]K-ras4B was measured by MTT assay. The cell transformation was determined by colony formation and tumor nodule development. [12Asp]-K-ras4B-NIH3T3 cells were transfected with constitutively active pCMV-RafCAAX and dominant-negative pCMV-RafS621A. Cell growth was measured by MTT assay and [3H]thymidine incorporation. After transfected with pcDI-[12Asp]K-ras4B or pCMV-RafS621A, the cells were harvested for Western blot and reporter assay to determine the expression and transcriptional activity of ERalpha and ERbeta, respectively. [12Asp]-K-ras4B enhanced NIH3T3 cells proliferation after 48 h post-transfection (P < 0.05). More colonies were grown 10 days after incubating pcDI-[12Asp]-K-ras4B-NIH3T3 cells (13.48%) than pcDI-NIH3T3 (4.26%) or untreated NIH3T3 (2.33%). The pcDI-[12Asp]-K-ras4B-NIH3T3 cells injected to the nude mice Balb/C developed tumor nodules with poor-differentiated cells after 12 days. An increase of ERalpha and ERbeta was observed in pcDI-[12Asp]-K-ras4B-NIH3T3 cells. RafS621A downregulated ERalpha and ERbeta expression. Estrogen induced the ER transcriptional activity by 5-fold in pcDI-NIH3T3 cells, 13-fold in pcDI-[12Asp]K-ras4B-NIH3T3 and 19-fold in HEC-1A. RafS621A suppressed the ER transcriptional activity. K-ras mutation induces tumorigenesis in endometrium, and this malignant transformation involves Raf signaling pathway and ER.

Calcium-Mediated Apoptosis and Apoptotic Sensitization in Prostate Cancer

DTIC Science & Technology

2003-06-01

tyrosine phosphatase, PTP1B . To study their direct involvement in apoptosis and signaling, PC cells were transfected with dominant negative caspase 7...and inducible constructs of activated PTP1B B. Dominant negative caspase 7 suppressed activation of endogenous caspase 7 by calcium ionophore...supporting a role for its recruitment into the calcium initiated apoptotic process. Activated PTP1B expression (but not a phosphatase-dead mutant

Endoplasmic reticulum-associated degradation of the mouse PC1/3-N222D hypomorph and human PCSK1 mutations contributes to obesity.

PubMed

Stijnen, P; Brouwers, B; Dirkx, E; Ramos-Molina, B; Van Lommel, L; Schuit, F; Thorrez, L; Declercq, J; Creemers, J W M

2016-06-01

The proprotein convertase 1/3 (PC1/3), encoded by proprotein convertase subtilisin/kexin type 1 (PCSK1), cleaves and hence activates several orexigenic and anorexigenic proproteins. Congenital inactivation of PCSK1 leads to obesity in human but not in mice. However, a mouse model harboring the hypomorphic mutation N222D is obese. It is not clear why the mouse models differ in phenotype. Gene expression analysis was performed with pancreatic islets from Pcsk1(N222D/N222D) mice. Subsequently, biosynthesis, maturation, degradation and activity were studied in islets, pituitary, hypothalamus and cell lines. Coimmunoprecipitation of PC1/3-N222D and human PC1/3 variants associated with obesity with the endoplasmic reticulum (ER) chaperone BiP was studied in cell lines. Gene expression analysis of islets of Pcsk1(N222D/N222D) mice showed enrichment of gene sets related to the proteasome and the unfolded protein response. Steady-state levels of PC1/3-N222D and in particular the carboxy-terminally processed form were strongly reduced in islets, pituitary and hypothalamus. However, impairment of substrate cleavage was tissue dependent. Proinsulin processing was drastically reduced, while processing of proopiomelanocortin (POMC) to adrenocorticotropic hormone (ACTH) in pituitary was only mildly impaired. Growth hormone expression and IGF-1 levels were normal, indicating near-normal processing of hypothalamic proGHRH. PC1/3-N222D binds to BiP and is rapidly degraded by the proteasome. Analysis of human PC1/3 obesity-associated mutations showed increased binding to BiP and prolonged intracellular retention for all investigated mutations, in particular for PC1/3-T175M, PC1/3-G226R and PC1/3-G593R. This study demonstrates that the hypomorphic mutation in Pcsk1(N222D) mice has an effect on catalytic activity in pancreatic islets, pituitary and hypothalamus. Reduced substrate processing activity in Pcsk1(N222D/N222D) mice is due to enhanced degradation in addition to reduced catalytic activity of the mutant. PC1/3-N222D binds to BiP, suggesting impaired folding and reduced stability. Enhanced BiP binding is also observed in several human obesity-associated PC1/3 variants, suggesting a common mechanism.

The E3 ubiquitin ligase CHIP selectively regulates mutant epidermal growth factor receptor by ubiquitination and degradation.

PubMed

Chung, Chaeuk; Yoo, Geon; Kim, Tackhoon; Lee, Dahye; Lee, Choong-Sik; Cha, Hye Rim; Park, Yeon Hee; Moon, Jae Young; Jung, Sung Soo; Kim, Ju Ock; Lee, Jae Cheol; Kim, Sun Young; Park, Hee Sun; Park, Myoungrin; Park, Dong Il; Lim, Dae-Sik; Jang, Kang Won; Lee, Jeong Eun

2016-10-14

Somatic mutation in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) is a decisive factor for the therapeutic response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in lung adenocarcinoma. The stability of mutant EGFR is maintained by various regulators, including heat shock protein 90 (Hsp90). The C terminus of Hsc70-interacting protein (CHIP) is a Hsp70/Hsp90 co-chaperone and exhibits E3 ubiquitin ligase activity. The high-affinity Hsp90-CHIP complex recognizes and selectively regulates their client proteins. CHIP also works with its own E3 ligase activity independently of Hsp70/Hsp90. Here, we investigated the role of CHIP in regulating EGFR in lung adenocarcinoma and also evaluated the specificity of CHIP's effects on mutant EGFR. In HEK 293T cells transfected with either WT EGFR or EGFR mutants, the overexpression of CHIP selectively decreased the expression of certain EGFR mutants (G719S, L747_E749del A750P and L858R) but not WT EGFR. In a pull-down assay, CHIP selectively interacted with EGFR mutants and simultaneously induced their ubiquitination and proteasomal degradation. The expressions of mutant EGFR in PC9 and H1975 were diminished by CHIP, while the expression of WT EGFR in A549 was nearly not affected. In addition, CHIP overexpression inhibited cell proliferation and xenograft's tumor growth of EGFR mutant cell lines, but not WT EGFR cell lines. EGFR mutant specific ubiquitination by CHIP may provide a crucial regulating mechanism for EGFR in lung adenocarcinoma. Our results suggest that CHIP can be novel therapeutic target for overcoming the EGFR TKI resistance. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of an N-terminal glycogen synthase kinase 3 phosphorylation site which regulates the functional localisation of polycystin-2 in vivo and in vitro

PubMed Central

Streets, Andrew J; Moon, David J; Kane, Michelle E; Obara, Tomoko; Ong, Albert CM

2008-01-01

PKD2 is mutated in 15% of patients with autosomal dominant polycystic kidney disease (ADPKD). Polycystin-2 (PC2), the PKD2 protein, is a nonselective Ca2+-permeable cation channel which may function at the cell surface and ER. Nevertheless, the factors that regulate the dynamic translocation of PC2 between the ER and other compartments are not well understood. Constitutive phosphorylation of PC2 at a single C-terminal site (Ser812) has been previously reported. Since we were unable to abolish phospholabelling of PC2 in HEK293 cells by site-directed mutagenesis of Ser812 or all 5 predicted phosphorylation sites in the C-terminus, we hypothesised that PC2 could also be phosphorylated at the N-terminus. In this paper, we report the identification of a new phosphorylation site for PC2 within its N-terminal domain (Ser76) and demonstrate that this residue is phosphorylated by glycogen synthase kinase 3 (GSK-3). The consensus recognition sequence for GSK-3 (Ser76/Ser80) is evolutionarily conserved down to lower vertebrates. In the presence of specific GSK-3 inhibitors, the lateral plasma membrane pool of endogenous PC2 redistributes into an intracellular compartment in MDCK cells without a change in primary cilia localization. Finally, co-injection of wild-type but not a S76A/S80A mutant PKD2 capped mRNA could rescue the cystic phenotype induced by an antisense morpholino oligonucleotide to pkd2 in zebrafish pronephric kidney. We conclude that surface localization of PC2 is regulated by phosphorylation at a unique GSK-3 site in its N-terminal domain in vivo and in vitro. This site is functionally significant for the maintenance of normal glomerular and tubular morphology. PMID:16551655

PcFKH1, a novel regulatory factor from the forkhead family, controls the biosynthesis of penicillin in Penicillium chrysogenum.

PubMed

Domínguez-Santos, Rebeca; García-Estrada, Carlos; Kosalková, Katarina; Prieto, Carlos; Santamarta, Irene; Martín, Juan-Francisco

2015-08-01

Penicillin biosynthesis in Penicillium chrysogenum (re-identified as Penicillium rubens) is a good example of a biological process subjected to complex global regulatory networks and serves as a model to study fungal secondary metabolism. The winged-helix family of transcription factors recently described, which includes the forkhead type of proteins, is a key type of regulatory proteins involved in this process. In yeasts and humans, forkhead transcription factors are involved in different processes (cell cycle regulation, cell death control, pre-mRNA processing and morphogenesis); one member of this family of proteins has been identified in the P. chrysogenum genome (Pc18g00430). In this work, we have characterized this novel transcription factor (named PcFKH1) by generating knock-down mutants and overexpression strains. Results clearly indicate that PcFKH1 positively controls antibiotic biosynthesis through the specific interaction with the promoter region of the penDE gene, thus regulating penDE mRNA levels. PcFKH1 also binds to the pcbC promoter, but with low affinity. In addition, it also controls other ancillary genes of the penicillin biosynthetic process, such as phlA (encoding phenylacetyl CoA ligase) and ppt (encoding phosphopantetheinyl transferase). PcFKH1 also plays a role in conidiation and spore pigmentation, but it does not seem to be involved in hyphal morphology or cell division in the improved laboratory reference strain Wisconsin 54-1255. A genome-wide analysis of processes putatively coregulated by PcFKH1 and PcRFX1 (another winged-helix transcription factor) in P. chrysogenum provided evidence of the global effect of these transcription factors in P. chrysogenum metabolism. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Novel mutations in the TSPAN12 gene in Chinese patients with familial exudative vitreoretinopathy

PubMed Central

Xu, Yu; Huang, Lulin; Li, Jing; Zhang, Qi; Fei, Ping; Zhu, Xiong; Tai, Zhengfu; Ma, Shi; Gong, Bo; Li, Yun; Zang, Weizhou; Zhu, Xianjun; Zhao, Peiquan

2014-01-01

Purpose Familial exudative vitreoretinopathy (FEVR) is a group of inherited blinding eye diseases characterized by defects in the development of the retinal vessels. Recent studies have identified genetic variants in tetraspanin 12 (TSPAN12) as a cause of FEVR. The purpose of this study was to identify novel TSPAN12 mutations in Chinese patients with FEVR and to describe the associated phenotypes. Methods Mutation screening was performed by directly sequencing PCR products of genomic DNA with primers designed to amplify the seven coding exons and adjacent intronic regions of the FEVR-causing gene TSPAN12. Clinical phenotypes of the patients with TSPAN12 mutations were documented. Wild-type and mutant TSPAN12 proteins were assayed for the Norrin-β-catenin signaling pathway with luciferase reporter assays. Results Three novel heterozygous mutations in TSPAN12 were identified: c.566G>A (p.C189Y), c.177delC (p.Y59fsX67), and c.C254T (p.T85M). All three mutations involved highly conserved residues and were not present in 200 normal individuals. Ocular phenotypes included increased ramification of the peripheral retinal vessels, a peripheral avascular zone, inferotemporal dragging of the optic disc and macula, and retinal folds. The probands showed relatively severe retinopathy, whereas the other family members were often asymptomatic. In SuperTopFlash (STF) cell line transfection studies, C189Y, Y59fsX67, and T85M mutants failed to induce luciferase reporter activity in response to Norrin. Conclusions We found three novel TSPAN12 mutations in Chinese patients with autosomal dominant FEVR, and suggest that TSPAN12 mutations cause FEVR. The phenotypes associated with the TSPAN12 mutations showed extensive variation in disease severity among members of the same family, which implied the complexity of FEVR mutations and phenotypes. PMID:25352738

The Orphan Disease Networks

PubMed Central

Zhang, Minlu; Zhu, Cheng; Jacomy, Alexis; Lu, Long J.; Jegga, Anil G.

2011-01-01

The low prevalence rate of orphan diseases (OD) requires special combined efforts to improve diagnosis, prevention, and discovery of novel therapeutic strategies. To identify and investigate relationships based on shared genes or shared functional features, we have conducted a bioinformatic-based global analysis of all orphan diseases with known disease-causing mutant genes. Starting with a bipartite network of known OD and OD-causing mutant genes and using the human protein interactome, we first construct and topologically analyze three networks: the orphan disease network, the orphan disease-causing mutant gene network, and the orphan disease-causing mutant gene interactome. Our results demonstrate that in contrast to the common disease-causing mutant genes that are predominantly nonessential, a majority of orphan disease-causing mutant genes are essential. In confirmation of this finding, we found that OD-causing mutant genes are topologically important in the protein interactome and are ubiquitously expressed. Additionally, functional enrichment analysis of those genes in which mutations cause ODs shows that a majority result in premature death or are lethal in the orthologous mouse gene knockout models. To address the limitations of traditional gene-based disease networks, we also construct and analyze OD networks on the basis of shared enriched features (biological processes, cellular components, pathways, phenotypes, and literature citations). Analyzing these functionally-linked OD networks, we identified several additional OD-OD relations that are both phenotypically similar and phenotypically diverse. Surprisingly, we observed that the wiring of the gene-based and other feature-based OD networks are largely different; this suggests that the relationship between ODs cannot be fully captured by the gene-based network alone. PMID:21664998

Congenital Proprotein Convertase 1/3 Deficiency Causes Malabsorptive Diarrhea and other Endocrinopathies in a Pediatric Cohort

PubMed Central

Martín, Martín G.; Lindberg, Iris; Solorzano-Vargas, R. Sergio; Wang, Jiafang; Avitzur, Yaron; Bandsma, Robert; Sokollik, Christiane; Lawrence, Sarah; Pickett, Lindsay A.; Chen, Zijun; Egritas, Odul; Dalgic, Buket; Albornoz, Valeria; de Ridder, Lissy; Hulst, Jessie; Gok, Faysal; Aydoğan, Ayşen; Al-Hussaini, Abdulrahman; Gok, Deniz Engin; Yourshaw, Michael; Wu, S. Vincent; Cortina, Galen; Stanford, Sara; Georgia, Senta

2013-01-01

Background & Aims Proprotein convertase 1/3 (PC1/3) deficiency, an autosomal recessive disorder caused by rare mutations in the PCSK1 gene, has been associated with obesity, severe malabsorptive diarrhea, and certain endocrine abnormalities. Common variants in PCSK1 have also been associated with obesity in heterozygotes in several population studies. PC1/3 is an endoprotease that processes many prohormones expressed in endocrine and neuronal cells. We investigated clinical and molecular features of PC1/3 deficiency. Methods We studied the clinical features of 13 children with PC1/3 deficiency and performed sequence analysis of PCSK1. We measured enzymatic activity of recombinant PC1/3 proteins. Results We identified a pattern of endocrinopathies that develop in an age-dependent manner. Eight of the mutations had severe biochemical consequences in vitro. Neonates had severe malabsorptive diarrhea and failure to thrive, required prolonged parenteral nutrition support, and had high mortality. Additional endocrine abnormalities developed as the disease progressed, including diabetes insipidus, growth hormone deficiency, primary hypogonadism, adrenal insufficiency, and hypothyroidism. We identified growth hormone deficiency, central diabetes insipidus, and male hypogonadism as new features of PCSK1 insufficiency. Interestingly, despite early growth abnormalities, moderate obesity, associated with severe polyphagia, generally appears. Conclusion In a study of 13 children with PC1/3 deficiency caused by disruption of PCSK1, failure of enteroendocrine cells to produce functional hormones resulted in generalized malabsorption. These findings indicate that PC1/3 is involved in processing of one or more enteric hormones that are required for nutrient absorption. PMID:23562752

[Regulation of [12Asp]K-ras4B on transcriptional activity of estrogen receptor in endometrial carcinoma HEC-1A cell lines].

PubMed

Gui, Li-ming; Wei, Li-hui; Xu, Ming-xu; Wang, Jian-liu; Zhong, Ying-cheng; Li, Xiao-ping; Tu, Zheng; Sun, Peng-ming; Ma, Da-long

2004-01-01

To investigate the effect of mutant-type [(12)Asp]K-ras4B gene on the expression of estrogen receptor (ER) alpha and beta and their transcriptional activity as a transcription factor in endometrial carcinoma HEC-1A cell line. (1) Effect of [(12)Asp]K-ras4B on the expression of ER alpha and beta were determined using Western blot assay. (2) Eukaryotic expression plasmid pGL3-luciferase-ERE containing luciferase report gene and estrogen receptor element (ERE) was constructed, and co-transfected into NIH3T3 and HEC-1A cell lines with pEGFP-N1 to examine the effect of [(12)Asp]K-ras4B on ER transcription that is regulated by estradiol. In addition, they were transfected into pSV5-HER0 (containing full length wide type ERalpha cDNA) and pCMV-rafS621A (inhibiting raf kinase) plasmids to test the effect of [(12)Asp]K-ras4B/raf signal pathway on transcriptional activity of ER proteins. (1) Protein level of ERs expressed in pcDI transfected control cells was low while it was increased for 3.6-fold (97 +/- 25, 349 +/- 67, P < 0.01) and 1.9-fold (128 +/- 37, 349 +/- 30, P < 0.05) in ERalpha and ERbeta, respectively, in pcDI-[(12)Asp]K-ras4B NIH3T3 cells after transfection. (2) In pcDI-[(12)Asp]K-ras4B NIH3T3 cells, the ratios for ERalpha and and ERbeta levels before transfection of rafS621A plasmids to that after the transfection, were 2.4:1 (724 +/- 45, 310 +/- 46, P < 0.05) and 1.8:1 (493 +/- 20, 284 +/- 20, P < 0.01), respectively; In HEC-1A cells, these ratios were 2.1:1 (566 +/- 22, 279 +/- 30, P < 0.01) and 2.4:1 (405 +/- 33, 165 +/- 15, P < 0.01), respectively. (3) In low serum (2%) culture condition, estradiol (E(2)) stimulated luciferase activity with an increase of 13-fold (130 +/- 42, 1681 +/- 242, P < 0.01) in pcDI-[(12)Asp] K-ras4B NIH3T3 cells, 19-fold (141 +/- 39, 2644 +/- 331, P < 0.001) in HEC-1A cells, respectively, when compared with those in the absence of E(2). (4) In pSV5-HER0 transfected pcDI-[(12)Asp] K-ras4B NIH3T3 cells and HEC-1A cells, compared to the untransfected cells, the ER transcriptional activity in the transfected cells increased markedly. The luciferase activity was increased for 8-fold (1048 +/- 91, 8099 +/- 452, P < 0.01) and 6-fold (2148 +/- 259, 12,705 +/- 2670, P < 0.001), respectively. rafS621A mutant had suppressive effects on luciferase activities in HEC-1A cells and pcDI-[(12)Asp]K-ras4B NIH3T3 cells. The ratio of luciferase activities in pcDI-[(12)Asp]K-ras4B NIH3T3 and HEC-1A cells, before and after transfection was 7.8:1 (1184 +/- 168, 152 +/- 27, P < 0.05) and 6.4:1 (1949 +/- 212, 304 +/- 60, P < 0.01), respectively. (1) [(12)Asp]K-ras4B can enhance the expression of ERalpha and beta proteins. This may be correlated with [(12)Asp]K-ras4B/raf signaling pathway. (2) The effect of mutant-type [(12)Asp]K-ras4B gene on ERs transcriptional activity in HEC-1A cells appears to need E(2).

Influence of Comorbidity on the Risk of Mortality in Men With Unfavorable-Risk Prostate Cancer Undergoing High-Dose Radiation Therapy Alone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huynh, Mai Anh, E-mail: mahuynh@lroc.harvard.edu; Chen, Ming-Hui; Wu, Jing

Purpose: To explore whether a subgroup of men with unfavorable-risk prostate cancer (PC) exists in whom high-dose radiation therapy (RT) alone is sufficient to avoid excess PC death due to competing risk from cardiometabolic comorbidity. Methods and Materials: This was a cohort study of 7399 men in whom comorbidity (including congestive heart failure, diabetes mellitus, or myocardial infarction) was assessed and recorded with T1-3NxM0 PC treated with brachytherapy with or without neoadjuvant RT, October 1997 to May 2013 at a single providing institution. Cox and competing risks regression analyses were used to assess whether men with unfavorable–intermediate/high-risk versus favorable–intermediate/low-risk PC weremore » at increased risk of PC-specific, all-cause, or other-cause mortality (PCSM, ACM, OCM), adjusting for number of comorbidities, age at and year of brachytherapy, RT use, and an RT treatment propensity score. Results: After a median follow-up of 7.7 years, 935 men died: 80 of PC and 855 of other causes. Among men with no comorbidity, PCSM risk (adjusted hazard ratio [AHR] 2.74 [95% confidence interval (CI) 1.49-5.06], P=.001) and ACM risk (AHR 1.30 [95% CI 1.07-1.58], P=.007) were significantly increased in men with unfavorable–intermediate/high-risk PC versus favorable–intermediate/low-risk PC, with no difference in OCM (P=.07). Although PCSM risk was increased in men with 1 comorbidity (AHR 2.87 [95% CI 1.11-7.40], P=.029), ACM risk was not (AHR 1.03 [95% CI 0.78-1.36], P=.84). Neither PCSM risk (AHR 4.39 [95% CI 0.37-51.98], P=.24) or ACM risk (AHR 1.43 [95% CI 0.83-2.45], P=.20) was increased in men with 2 comorbidities. Conclusions: To minimize death from PC, high-dose RT alone may be sufficient treatment in men with 2 or more cardiometabolic comorbidities and unfavorable–intermediate- and high-risk PC.« less

Multicentre study highlighting clinical relevance of new high-throughput methodologies in molecular epidemiology of Pneumocystis jirovecii pneumonia.

PubMed

Esteves, F; de Sousa, B; Calderón, E J; Huang, L; Badura, R; Maltez, F; Bassat, Q; de Armas, Y; Antunes, F; Matos, O

2016-06-01

Pneumocystis jirovecii causes severe interstitial pneumonia (PcP) in immunosuppressed patients. This multicentre study assessed the distribution frequencies of epidemiologically relevant genetic markers of P. jirovecii in different geographic populations from Portugal, the USA, Spain, Cuba and Mozambique, and the relationship between the molecular data and the geographical and clinical information, based on a multifactorial approach. The high-throughput typing strategy for P. jirovecii characterization consisted of DNA pooling using quantitative real-time PCR followed by multiplex-PCR/single base extension. The frequencies of relevant P. jirovecii single nucleotide polymorphisms (mt85, SOD110, SOD215, DHFR312, DHPS165 and DHPS171) encoded at four loci were estimated in ten DNA pooled samples representing a total of 182 individual samples. Putative multilocus genotypes of P. jirovecii were shown to be clustered due to geographic differences but were also dependent on clinical characteristics of the populations studied. The haplotype DHFR312T/SOD110C/SOD215T was associated with severe AIDS-related PcP and high P. jirovecii burdens. The frequencies of this genetic variant of P. jirovecii were significantly higher in patients with AIDS-related PcP from Portugal and the USA than in the colonized patients from Portugal, and Spain, and children infected with P. jirovecii from Cuba or Mozambique, highlighting the importance of this haplotype, apparently associated with the severity of the disease and specific clinical groups. Patients from the USA and Mozambique showed higher rates of DHPS mutants, which may suggest the circulation of P. jirovecii organisms potentially related with trimethoprim-sulfamethoxazole resistance in those geographical regions. This report assessed the worldwide distribution of P. jirovecii haplotypes and their epidemiological impact in distinct geographic and clinical populations. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. All rights reserved.

Bactericidal catechins damage the lipid bilayer.

PubMed

Ikigai, H; Nakae, T; Hara, Y; Shimamura, T

1993-04-08

The mode of antibacterial action of, the green tea (Camellia sinensis) extracts, (-)-epigallocatechin gallate (EGCg) and (-)-epicatechin (EC) was investigated. Strong bactericidal EGCg caused leakage of 5,6-carboxyfluorescein from phosphatidylcholine liposomes (PC), but EC with very weak bactericidal activity caused little damage to the membrane. Phosphatidylserine and dicetyl phosphate partially protected the membrane from EGCg-mediated damage when reconstituted into the liposome membrane with PC. EGCg, but not EC, caused strong aggregation and NPN-fluorescence quenching of PC-liposomes and these actions were markedly lowered in the presence of negatively charged lipids. These results show that bactericidal catechins primarily act on and damage bacterial membranes. The observation that Gram-negative bacteria are more resistant to bactericidal catechins than Gram-positive bacteria can be explained to some extent by the presence of negatively charged lipopolysaccharide.

Regulation of STATs by polycystin-1 and their role in polycystic kidney disease.

PubMed

Weimbs, Thomas; Olsan, Erin E; Talbot, Jeffrey J

2013-04-01

Autosomal-dominant polycystic kidney disease (ADPKD) is a common genetic disease caused by mutations in the gene coding for polycystin-1 (PC1). PC1 can regulate STAT transcription factors by a novel, dual mechanism. STAT3 and STAT6 are aberrantly activated in renal cysts. Genetic and pharmacological approaches to inhibit STAT3 or STAT6 have led to promising results in ADPKD mouse models. Here, we review current findings that lead to a model of PC1 as a key regulator of STAT signaling in renal tubule cells. We discuss how PC1 may orchestrate appropriate epithelial responses to renal injury, and how this system may lead to aberrant STAT activation in ADPKD thereby causing inappropriate activation of tissue repair programs that culminate in renal cyst growth and fibrosis.

Prostate-Specific Antigen (PSA)–Based Population Screening for Prostate Cancer: An Evidence-Based Analysis

PubMed Central

Pron, G

2015-01-01

Background Prostate cancer (PC) is the most commonly diagnosed non-cutaneous cancer in men and their second or third leading cause of cancer death. Prostate-specific antigen (PSA) testing for PC has been in common practice for more than 20 years. Objectives A systematic review of the scientific literature was conducted to determine the effectiveness of PSA-based population screening programs for PC to inform policy decisions in a publicly funded health care system. Data Sources A systematic review of bibliographic databases was performed for systematic reviews or randomized controlled trials (RCT) of PSA-based population screening programs for PC. Review Methods A broad search strategy was employed to identify studies reporting on key outcomes of PC mortality and all-cause mortality. Results The search identified 5 systematic reviews and 6 RCTs. None of the systematic reviews found a statistically significant reduction in relative risk (RR) of PC mortality or overall mortality with PSA-based screening. PC mortality reductions were found to vary by country, by screening program, and by age of men at study entry. The European Randomized Study of Screening for Prostate Cancer found a statistically significant reduction in RR in PC mortality at 11-year follow-up (0.79; 95% CI, 0.67–0.92), although the absolute risk reduction was small (1.0/10,000 person-years). However, the primary treatment for PCs differed significantly between countries and between trial arms. The American Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) found a statistically non-significant increase in RR for PC mortality with 13-year follow-up (1.09; 95% CI, 0.87–1.36). The degree of opportunistic screening in the control arm of the PLCO trial, however, was high. None of the RCTs found a reduction in all-cause mortality and all found a statistically significant increase in the detection of mainly low-risk, organ-confined PCs in the screening arm. Conclusions There was no evidence of a PC mortality reduction in the American PLCO trial, which investigated a screening program in a setting where opportunistic screening was already common practice. Given that opportunistic PSA screening practices in Canada are similar, it is unlikely that the introduction of a formal PSA screening program would reduce PC mortality. PMID:26366236

Prostate-Specific Antigen (PSA)-Based Population Screening for Prostate Cancer: An Evidence-Based Analysis.

PubMed

Pron, G

2015-01-01

Prostate cancer (PC) is the most commonly diagnosed non-cutaneous cancer in men and their second or third leading cause of cancer death. Prostate-specific antigen (PSA) testing for PC has been in common practice for more than 20 years. A systematic review of the scientific literature was conducted to determine the effectiveness of PSA-based population screening programs for PC to inform policy decisions in a publicly funded health care system. A systematic review of bibliographic databases was performed for systematic reviews or randomized controlled trials (RCT) of PSA-based population screening programs for PC. A broad search strategy was employed to identify studies reporting on key outcomes of PC mortality and all-cause mortality. The search identified 5 systematic reviews and 6 RCTs. None of the systematic reviews found a statistically significant reduction in relative risk (RR) of PC mortality or overall mortality with PSA-based screening. PC mortality reductions were found to vary by country, by screening program, and by age of men at study entry. The European Randomized Study of Screening for Prostate Cancer found a statistically significant reduction in RR in PC mortality at 11-year follow-up (0.79; 95% CI, 0.67-0.92), although the absolute risk reduction was small (1.0/10,000 person-years). However, the primary treatment for PCs differed significantly between countries and between trial arms. The American Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) found a statistically non-significant increase in RR for PC mortality with 13-year follow-up (1.09; 95% CI, 0.87-1.36). The degree of opportunistic screening in the control arm of the PLCO trial, however, was high. None of the RCTs found a reduction in all-cause mortality and all found a statistically significant increase in the detection of mainly low-risk, organ-confined PCs in the screening arm. There was no evidence of a PC mortality reduction in the American PLCO trial, which investigated a screening program in a setting where opportunistic screening was already common practice. Given that opportunistic PSA screening practices in Canada are similar, it is unlikely that the introduction of a formal PSA screening program would reduce PC mortality.

Congenital proprotein convertase 1/3 deficiency causes malabsorptive diarrhea and other endocrinopathies in a pediatric cohort.

PubMed

Martín, Martín G; Lindberg, Iris; Solorzano-Vargas, R Sergio; Wang, Jiafang; Avitzur, Yaron; Bandsma, Robert; Sokollik, Christiane; Lawrence, Sarah; Pickett, Lindsay A; Chen, Zijun; Egritas, Odul; Dalgic, Buket; Albornoz, Valeria; de Ridder, Lissy; Hulst, Jessie; Gok, Faysal; Aydoğan, Ayşen; Al-Hussaini, Abdulrahman; Gok, Deniz Engin; Yourshaw, Michael; Wu, S Vincent; Cortina, Galen; Stanford, Sara; Georgia, Senta

2013-07-01

Proprotein convertase 1/3 (PC1/3) deficiency, an autosomal-recessive disorder caused by rare mutations in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene, has been associated with obesity, severe malabsorptive diarrhea, and certain endocrine abnormalities. Common variants in PCSK1 also have been associated with obesity in heterozygotes in several population-based studies. PC1/3 is an endoprotease that processes many prohormones expressed in endocrine and neuronal cells. We investigated clinical and molecular features of PC1/3 deficiency. We studied the clinical features of 13 children with PC1/3 deficiency and performed sequence analysis of PCSK1. We measured enzymatic activity of recombinant PC1/3 proteins. We identified a pattern of endocrinopathies that develop in an age-dependent manner. Eight of the mutations had severe biochemical consequences in vitro. Neonates had severe malabsorptive diarrhea and failure to thrive, required prolonged parenteral nutrition support, and had high mortality. Additional endocrine abnormalities developed as the disease progressed, including diabetes insipidus, growth hormone deficiency, primary hypogonadism, adrenal insufficiency, and hypothyroidism. We identified growth hormone deficiency, central diabetes insipidus, and male hypogonadism as new features of PCSK1 insufficiency. Interestingly, despite early growth abnormalities, moderate obesity, associated with severe polyphagia, generally appears. In a study of 13 children with PC1/3 deficiency caused by disruption of PCSK1, failure of enteroendocrine cells to produce functional hormones resulted in generalized malabsorption. These findings indicate that PC1/3 is involved in the processing of one or more enteric hormones that are required for nutrient absorption. Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.

A novel mutation in FRMD7 causing X-linked idiopathic congenital nystagmus in a large family

PubMed Central

He, Xiang; Gu, Feng; Wang, Yujing; Yan, Jinting; Zhang, Meng; Huang, Shangzhi

2008-01-01

Purpose To identify the gene responsible for causing an X-linked idiopathic congenital nystagmus (XLICN) in a six-generation Chinese family. Methods Forty-nine members of an XLICN family were recruited and examined after obtaining informed consent. Affected male individuals were genotyped with microsatellite markers around the FRMD7 locus. Mutations were comprehensively screened by direct sequencing using gene specific primers. An X-inactivation pattern was investigated by X chromosome methylation analysis. Results The patients showed phenotypes consistent with XLICN. Genotype analysis showed that male affected individuals in the family shared a common haplotype with the selected markers. Sequencing FRMD7 revealed a G>T transversion (c.812G>T) in exon 9, which caused a conservative substitution of Cys to Phe at codon 271 (p.C271F). This mutation co-segregated with all affected individuals and was present in the obligate, non-penetrant female carriers. However, the mutation was not observed in unaffected familial males or 400 control males. Females with the mutant gene could be affected or carrier and they shared the same inactivated X chromosome harboring the mutation in blood cells, which showed there is no clear causal link between X-inactivation pattern and phenotype. Conclusions We identified a novel mutation in FRMD7 and confirmed the role of this mutation in the pathogenesis of X-linked congenital nystagmus. PMID:18246032

[Studies of immunomodulation caused by selenium-enriched phycocyanin].

PubMed

Egorova, E A; Gmoshinskiĭ, I V; Zorin, S N; Mazo, V K

2006-01-01

An influense was studied in rats of selenium enriched phycocyanin (Se-PC) from food microalgae Spirulina on anaphylactic reaction severity and circulating antibody response against model allergen--hen's egg white ovalbumin. Se-PC was introduced into diet in form of protein isolate precipitated with ammonia sulphate. Se-PC dosage made up to 450 mcg per rat daily that corresponded to 5 mcg of selenium. There were no differences revealed between experimental and control group that received standard diet in severity of anaphylactic reaction. Nevertheless rats receiving Se-PC demonstrated significantly increased specific IgG response. The probable immunomodulating properties of Se-PC included into food are discussed.

[Grape seed extract induces morphological changes of prostate cancer PC-3 cells].

PubMed

Shang, Xue-Jun; Yin, Hong-Lin; Ge, Jing-Ping; Sun, Yi; Teng, Wen-Hui; Huang, Yu-Feng

2008-12-01

To observe the morphological changes of prostate cancer PC-3 cells induced by grape seed extract (GSE). PC-3 cells were incubated with different concentrations of GSE (100, 200 and 300 microg/ml) for 24, 48 and 72 hours, and then observed for morphological changes by invert microscopy, HE staining and transmission electron microscopy. The incubated PC-3 cells appeared round, small, wrinkled and broken under the invert microscope and exhibited the classical morphological characteristics of cell death under the electron microscope, including cell atrophy, increased vacuoles, crumpled nuclear membrane, and chromosome aggregation. GSE can cause morphological changes and induce necrosis and apoptosis of PC-3 cells.

Expression of CALR mutants causes mpl-dependent thrombocytosis in zebrafish.

PubMed

Lim, K-H; Chang, Y-C; Chiang, Y-H; Lin, H-C; Chang, C-Y; Lin, C-S; Huang, L; Wang, W-T; Gon-Shen Chen, C; Chou, W-C; Kuo, Y-Y

2016-10-07

CALR mutations are identified in about 30% of JAK2/MPL-unmutated myeloproliferative neoplasms (MPNs) including essential thrombocythemia (ET) and primary myelofibrosis. Although the molecular pathogenesis of CALR mutations leading to MPNs has been studied using in vitro cell lines models, how mutant CALR may affect developmental hematopoiesis remains unknown. Here we took advantage of the zebrafish model to examine the effects of mutant CALR on early hematopoiesis and model human CALR-mutated MPNs. We identified three zebrafish genes orthologous to human CALR, referred to as calr, calr3a and calr3b. The expression of CALR-del52 and CALR-ins5 mutants caused an increase in the hematopoietic stem/progenitor cells followed by thrombocytosis without affecting normal angiogenesis. The expression of CALR mutants also perturbed early developmental hematopoiesis in zebrafish. Importantly, morpholino knockdown of mpl but not epor or csf3r could significantly attenuate the effects of mutant CALR. Furthermore, the expression of mutant CALR caused jak-stat signaling activation in zebrafish that could be blocked by JAK inhibitors (ruxolitinib and fedratinib). These findings showed that mutant CALR activates jak-stat signaling through an mpl-dependent mechanism to mediate pathogenic thrombopoiesis in zebrafish, and illustrated that the signaling machinery related to mutant CALR tumorigenesis are conserved between human and zebrafish.

Differential effects of lesion mimic mutants in barley on disease development by facultative pathogens

PubMed Central

McGrann, Graham R. D.; Steed, , Andrew; Burt, Christopher; Nicholson, Paul; Brown, James K. M.

2015-01-01

Lesion mimic mutants display spontaneous necrotic spots and chlorotic leaves as a result of mis-regulated cell death programmes. Typically these mutants have increased resistance to biotrophic pathogens but their response to facultative fungi that cause necrotrophic diseases is less well studied. The effect of altered cell death regulation on the development of disease caused by Ramularia collo-cygni, Fusarium culmorum and Oculimacula yallundae was explored using a collection of barley necrotic (nec) lesion mimic mutants. nec8 mutants displayed lower levels of all three diseases compared to nec9 mutants, which had increased R. collo-cygni but decreased F. culmorum disease symptoms. nec1 mutants reduced disease development caused by both R. collo-cygni and F. culmorum. The severity of the nec1-induced lesion mimic phenotype and F. culmorum symptom development was reduced by mutation of the negative cell death regulator MLO. The significant reduction in R. collo-cygni symptoms caused by nec1 was completely abolished in the presence of the mlo-5 allele and both symptoms and fungal biomass were greater than in the wild-type. These results indicate that physiological pathways involved in regulation of cell death interact with one another in their effects on different fungal pathogens. PMID:25873675

In vitro selection of resistance in haemophilus influenzae by 4 quinolones and 5 beta-lactams.

PubMed

Clark, Catherine; Kosowska, Klaudia; Bozdogan, Bülent; Credito, Kim; Dewasse, Bonifacio; McGhee, Pamela; Jacobs, Michael R; Appelbaum, Peter C

2004-05-01

We tested abilities of ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, amoxicillin, amoxicillin/clavulanate, cefixime, cefpodoxime, and cefdinir to select resistant mutants in 5 beta-lactamase positive and 5 beta-lactamase negative Haemophilus influenzae strains by single and multistep methodology. In multistep tests, amoxicillin, amoxicillin/clavulanate and cefpodoxime exposure did not cause >4-fold minimum inhibitory concentration (MIC) increase after 50 days. One mutant selected by cefdinir had one amino acid substitution (Gly490Glu) in PBP3 and became resistant to cefdinir. Cefixime exposure caused 8-fold MIC-increase in 1 strain with TEM but the mutant remained cefixime susceptible and had no alteration in PBP3 or TEM. Among 10 strains tested, ciprofloxacin, moxifloxacin, gatifloxacin, levofloxacin caused >4-fold MIC increase in 6, 6, 5, and 2 strain, respectively. Despite the increases in quinolone MICs, none of the mutants became resistant to quinolones by established criteria. Quinolone selected mutants had quindone resistance-determining region (QRDR) alterations in GyrA, GyrB, ParC, ParE. Four quinolone mutants had no QRDR alterations. Among beta-lactams cefdinir and cefixime selected one mutant each with higher MICs however amoxicillin, amoxicillin/clavulanate, and cefpodoxime exposure did not select resistant mutants.

Peak Pc Prediction in Conjunction Analysis: Conjunction Assessment Risk Analysis. Pc Behavior Prediction Models

NASA Technical Reports Server (NTRS)

Vallejo, J.J.; Hejduk, M.D.; Stamey, J. D.

2015-01-01

Satellite conjunction risk typically evaluated through the probability of collision (Pc). Considers both conjunction geometry and uncertainties in both state estimates. Conjunction events initially discovered through Joint Space Operations Center (JSpOC) screenings, usually seven days before Time of Closest Approach (TCA). However, JSpOC continues to track objects and issue conjunction updates. Changes in state estimate and reduced propagation time cause Pc to change as event develops. These changes a combination of potentially predictable development and unpredictable changes in state estimate covariance. Operationally useful datum: the peak Pc. If it can reasonably be inferred that the peak Pc value has passed, then risk assessment can be conducted against this peak value. If this value is below remediation level, then event intensity can be relaxed. Can the peak Pc location be reasonably predicted?

Polycystin-1 C-terminal Cleavage Is Modulated by Polycystin-2 Expression*

PubMed Central

Bertuccio, Claudia A.; Chapin, Hannah C.; Cai, Yiqiang; Mistry, Kavita; Chauvet, Veronique; Somlo, Stefan; Caplan, Michael J.

2009-01-01

Autosomal dominant polycystic kidney disease is caused by mutations in the genes encoding polycystin-1 (PC-1) and polycystin-2 (PC-2). PC-1 cleavage releases its cytoplasmic C-terminal tail (CTT), which enters the nucleus. To determine whether PC-1 CTT cleavage is influenced by PC-2, a quantitative cleavage assay was utilized, in which the DNA binding and activation domains of Gal4 and VP16, respectively, were appended to PC-1 downstream of its CTT domain (PKDgalvp). Cells cotransfected with the resultant PKDgalvp fusion protein and PC-2 showed an increase in luciferase activity and in CTT expression, indicating that the C-terminal tail of PC-1 is cleaved and enters the nucleus. To assess whether CTT cleavage depends upon Ca2+ signaling, cells transfected with PKDgalvp alone or together with PC-2 were incubated with several agents that alter intracellular Ca2+ concentrations. PC-2 enhancement of luciferase activity was not altered by any of these treatments. Using a series of PC-2 C-terminal truncated mutations, we identified a portion of the PC-2 protein that is required to stimulate PC-1 CTT accumulation. These data demonstrate that release of the CTT from PC-1 is influenced and stabilized by PC-2. This effect is independent of Ca2+ but is regulated by sequences contained within the PC-2 C-terminal tail, suggesting a mechanism through which PC-1 and PC-2 may modulate a novel signaling pathway. PMID:19491093

Phthalocyanine tetrasulfonates affect the amyloid formation and cytotoxicity of alpha-synuclein.

PubMed

Lee, Eui-Nam; Cho, Hyun-Ju; Lee, Choong-Hwan; Lee, Daekyun; Chung, Kwang Chul; Paik, Seung R

2004-03-30

Alpha-synuclein is a pathological component of Parkinson's disease by constituting the filamentous component of Lewy bodies. Phthalocyanine (Pc) effects on the amyloidosis of alpha-synuclein have been examined. The copper complex of phthalocyanine tetrasulfonate (PcTS-Cu(2+)) caused the self-oligomerization of alpha-synuclein while Pc-Cu(2+) did not affect the protein, indicating that introduction of the sulfonate groups was critical for the selective protein interaction. The PcTS-Cu(2+) interaction with alpha-synuclein has occurred predominantly at the N-terminal region of the protein with a K(d) of 0.83 microM apart from the hydrophobic NAC (non-Abeta component of Alzheimer's disease amyloid) segment. Phthalocyanine tetrasulfonate (PcTS) lacking the intercalated copper ion also showed a considerable affinity toward alpha-synuclein with a K(d) of 3.12 microM, and its binding site, on the other hand, was located at the acidic C-terminus. These mutually exclusive interactions between PcTS and PcTS-Cu(2+) toward alpha-synuclein resulted in distinctive features on the kinetics of protein aggregation, morphologies of the final aggregates, and their in vitro cytotoxicities. The PcTS actually suppressed the fibrous amyloid formation of alpha-synuclein, but it produced the chopped-wood-looking protein aggregates. The aggregates showed rather low toxicity (9.5%) on human neuroblastoma cells (SH-SY5Y). In fact, the PcTS was shown to effectively rescue the cell death of alpha-synuclein overexpressing cells caused by the lactacystin treatment as a proteasome inhibitor. The anti-aggregative and anti-amyloidogenic properties of PcTS were also demonstrated with alcohol dehydrogenase, glutathione S-transferase, and amyloid beta/A4 protein under their aggregative conditions. The PcTS-Cu(2+), on the other hand, promoted the protein aggregation of alpha-synuclein, which gave rise to the fibrillar protein aggregates whose cytotoxicity became significant to 35.8%. Taken together, the data provided in this study indicate that PcTS/PcTS-Cu(2+) could be considered as possible candidates for the development of therapeutic or prophylactic strategies against the alpha-synuclein-related neurodegenerative disorders.

A clinical and molecular review of ubiquitous glucose-6-phosphatase deficiency caused by G6PC3 mutations

PubMed Central

2013-01-01

The G6PC3 gene encodes the ubiquitously expressed glucose-6-phosphatase enzyme (G-6-Pase β or G-6-Pase 3 or G6PC3). Bi-allelic G6PC3 mutations cause a multi-system autosomal recessive disorder of G6PC3 deficiency (also called severe congenital neutropenia type 4, MIM 612541). To date, at least 57 patients with G6PC3 deficiency have been described in the literature. G6PC3 deficiency is characterized by severe congenital neutropenia, recurrent bacterial infections, intermittent thrombocytopenia in many patients, a prominent superficial venous pattern and a high incidence of congenital cardiac defects and uro-genital anomalies. The phenotypic spectrum of the condition is wide and includes rare manifestations such as maturation arrest of the myeloid lineage, a normocellular bone marrow, myelokathexis, lymphopaenia, thymic hypoplasia, inflammatory bowel disease, primary pulmonary hypertension, endocrine abnormalities, growth retardation, minor facial dysmorphism, skeletal and integument anomalies amongst others. Dursun syndrome is part of this extended spectrum. G6PC3 deficiency can also result in isolated non-syndromic severe neutropenia. G6PC3 mutations in result in reduced enzyme activity, endoplasmic reticulum stress response, increased rates of apoptosis of affected cells and dysfunction of neutrophil activity. In this review we demonstrate that loss of function in missense G6PC3 mutations likely results from decreased enzyme stability. The condition can be diagnosed by sequencing the G6PC3 gene. A number of G6PC3 founder mutations are known in various populations and a possible genotype-phenotype relationship also exists. G6PC3 deficiency should be considered as part of the differential diagnoses in any patient with unexplained congenital neutropenia. Treatment with G-CSF leads to improvement in neutrophil numbers, prevents infections and improves quality of life. Mildly affected patients can be managed with prophylactic antibiotics. Untreated G6PC3 deficiency can be fatal. Echocardiogram, renal and pelvic ultrasound scans should be performed in all cases of suspected or confirmed G6PC3 deficiency. Routine assessment should include biochemical profile, growth profile and monitoring for development of varicose veins or venous ulcers. PMID:23758768

Mutant PFN1 causes ALS phenotypes and progressive motor neuron degeneration in mice by a gain of toxicity

PubMed Central

Yang, Chunxing; Danielson, Eric W.; Qiao, Tao; Metterville, Jake; Brown, Robert H.; Landers, John E.; Xu, Zuoshang

2016-01-01

Mutations in the profilin 1 (PFN1) gene cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease caused by the loss of motor neurons leading to paralysis and eventually death. PFN1 is a small actin-binding protein that promotes formin-based actin polymerization and regulates numerous cellular functions, but how the mutations in PFN1 cause ALS is unclear. To investigate this problem, we have generated transgenic mice expressing either the ALS-associated mutant (C71G) or wild-type protein. Here, we report that mice expressing the mutant, but not the wild-type, protein had relentless progression of motor neuron loss with concomitant progressive muscle weakness ending in paralysis and death. Furthermore, mutant, but not wild-type, PFN1 forms insoluble aggregates, disrupts cytoskeletal structure, and elevates ubiquitin and p62/SQSTM levels in motor neurons. Unexpectedly, the acceleration of motor neuron degeneration precedes the accumulation of mutant PFN1 aggregates. These results suggest that although mutant PFN1 aggregation may contribute to neurodegeneration, it does not trigger its onset. Importantly, these experiments establish a progressive disease model that can contribute toward identifying the mechanisms of ALS pathogenesis and the development of therapeutic treatments. PMID:27681617

Identification and characterization of Escherichia coli RS218-derived islands in the pathogenesis of E. coli meningitis.

PubMed

Xie, Yi; Kolisnychenko, Vitaliy; Paul-Satyaseela, Maneesh; Elliott, Simon; Parthasarathy, Geetha; Yao, Yufeng; Plunkett, Guy; Blattner, Frederick R; Kim, Kwang Sik

2006-08-01

Escherichia coli K1 is the most common gram-negative bacterium causing neonatal meningitis, but the mechanisms by which E. coli K1 causes meningitis are not clear. We identified 22 E. coli RS218-derived genomic islands (RDIs), using a comparative genome analysis of meningitis-causing E. coli K1 strain RS218 (O18:K1:H7) and laboratory K-12 strain MG1655. Series of RDI deletion mutants were constructed and examined for phenotypes relevant to E. coli K1 meningitis. We identified 9 RDI deletion mutants (RDI 1, 4, 7, 12, 13, 16, 20, 21, and 22) that exhibited defects in meningitis development. RDI 16 and 21 mutants had profound defects in the induction of a high level of bacteremia in neonatal rats, and RDI 4 mutants exhibited a moderate defect in the induction of bacteremia. RDI 1 and 22 mutants showed defects in the ability to invade human brain microvascular endothelial cells (HBMECs), and RDI 12 mutants were defective in the ability to bind to HBMECs. RDI 13 and 20 mutants were defective in the ability to both bind to and invade HBMECs. RDI 7 mutants were defective in the induction of bacteremia and in the ability to both bind to and invade HBMECs. These results provide a framework for the future discovery and analysis of bacteremia and meningitis caused by E. coli K1 strain RS218.

Ninjin'yoeito and ginseng extract prevent oxaliplatin-induced neurodegeneration in PC12 cells.

PubMed

Suzuki, Toshiaki; Yamamoto, Ayano; Ohsawa, Masahiro; Motoo, Yoshiharu; Mizukami, Hajime; Makino, Toshiaki

2015-10-01

Ninjin'yoeito (NYT) is a formula of Japanese traditional kampo medicine composed of 12 crude drugs, and is designed to improve the decline in constitution after recovery from disease, fatigue, anemia, anorexia, perspiration during sleep, cold limbs, slight fever, chills, persistent cough, malaise, mental disequilibrium, insomnia, and constipation. Oxaliplatin (L-OHP) is a platinum-based anticancer drug used to treat colorectal, pancreatic, and stomach cancers. However, it often causes acute and chronic peripheral neuropathies including cold allodynia and mechanical hyperalgesia. In this study, we investigated the preventive effects of NYT on neuronal degeneration caused by L-OHP using PC12 cells, which are derived from the rat adrenal medulla and differentiate into nerve-like cells after exposure to nerve growth factor. L-OHP treatment decreased the elongation of neurite-like projection outgrowths in differentiated PC12 cells. When PC12 cells were treated with NYT hot water extract, neurodegeneration caused by L-OHP was significantly prevented in a concentration-dependent manner. Among the 12 crude drugs composing NYT, the extract of Ginseng (the root of Panax ginseng) exhibited the strongest preventive effects on neurodegeneration in differentiated PC12 cells. By activity-guided fractionation, we found that the fraction containing ginsenosides displayed preventive activity and, among several ginsenosides, ginsenoside F2 exhibited significant preventive effects on L-OHP-induced decreases in neurite-like outgrowths in differentiated PC12 cells. These results suggest that NYT and ginseng are promising agents for preventing L-OHP-induced neuropathies and present ginsenoside F2 as one of the active ingredients in ginseng.

Tetra-methyl substituted copper (II) phthalocyanine as a hole injection enhancer in organic light-emitting diodes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yu-Long; Xu, Jia-Ju; Lin, Yi-Wei

2015-10-15

We have enhanced hole injection and lifetime in organic light-emitting diodes (OLEDs) by incorporating the isomeric metal phthalocyanine, CuMePc, as a hole injection enhancer. The OLED devices containing CuMePc as a hole injection layer (HIL) exhibited higher luminous efficiency and operational lifetime than those using a CuPc layer and without a HIL. The effect of CuMePc thickness on device performance was investigated. Atomic force microscope (AFM) studies revealed that the thin films were smooth and uniform because the mixture of CuMePc isomers depressed crystallization within the layer. This may have caused the observed enhanced hole injection, indicating that CuMePc ismore » a promising HIL material for highly efficient OLEDs.« less

Awareness of Prostate Cancer and Screening Modalities Among Long Island Men.

PubMed

Leonard, James A; Wells, Jenna B; Brandler, Ethan S

2017-03-01

Despite advances in prostate cancer (PC) treatment and outcomes, it remains one of the most commonly diagnosed cancers and the second leading cause of cancer mortality in men. Furthermore, there exist little data about patient awareness of PC and the frequency of screening. Adult men (470) presenting to the emergency department at a tertiary care center (Stony Brook University) between 2014 and 2015 were surveyed orally for their perceived risk of PC, awareness of PC and screening modalities, and screening history. In a population that mirrors the racial demographics of the United States, it was observed that significant disparities in awareness of PC exist among several populations including those at greatest risk. This study revealed an important opportunity for education on PC and screening modalities among the population of men at risk.

Evaluation Of prestressed concrete beams in shear : [part 1 and 2].

DOT National Transportation Integrated Search

2014-09-01

This research investigates possible design-related causes of apparent shear cracks on MDOT prestressed : concrete (PC) bridge girders; assesses the adequacy of PC shear design and rating methods; and : recommends changes to these procedures. It invol...

Erwinia amylovora pyrC mutant causes fire blight despite pyrimidine auxotrophy.

PubMed

Ramos, L S; Sinn, J P; Lehman, B L; Pfeufer, E E; Peter, K A; McNellis, T W

2015-06-01

Erwinia amylovora bacteria cause fire blight disease, which affects apple and pear production worldwide. The Erw. amylovora pyrC gene encodes a predicted dihydroorotase enzyme involved in pyrimidine biosynthesis. Here, we discovered that the Erw. amylovora pyrC244::Tn5 mutant was a uracil auxotroph. Unexpectedly, the Erw. amylovora pyrC244::Tn5 mutant grew as well as the wild-type in detached immature apple and pear fruits. Fire blight symptoms caused by the pyrC244::Tn5 mutant in immature apple and pear fruits were attenuated compared to those caused by the wild-type. The pyrC244::Tn5 mutant also caused severe fire blight symptoms in apple tree shoots. A plasmid-borne copy of the wild-type pyrC gene restored prototrophy and symptom induction in apple and pear fruit to the pyrC244::Tn5 mutant. These results suggest that Erw. amylovora can obtain sufficient pyrimidine from the host to support bacterial growth and fire blight disease development, although de novo pyrimidine synthesis by Erw. amylovora is required for full symptom development in fruits. Significance and impact of the study: This study provides information about the fire blight host-pathogen interaction. Although the Erwinia amylovora pyrC mutant was strictly auxotrophic for pyrimidine, it grew as well as the wild-type in immature pear and apple fruits and caused severe fire blight disease in apple trees. This suggests that Erw. amylovora can obtain sufficient pyrimidines from host tissue to support growth and fire blight disease development. This situation contrasts with findings in some human bacterial pathogens, which require de novo pyrimidine synthesis for growth in host blood, for example. © 2015 The Society for Applied Microbiology.

Low testosterone at first prostate-specific antigen failure and assessment of risk of death in men with unfavorable-risk prostate cancer treated on prospective clinical trials.

PubMed

Atkins, Katelyn M; Chen, Ming-Hui; Wu, Jing; Renshaw, Andrew A; Loffredo, Marian; Kantoff, Philip W; Small, Eric J; D'Amico, Anthony V

2018-04-01

Low testosterone at the time of diagnosis of prostate cancer has been associated with a worse prognosis. Whether this is true and how to define the best treatment approach at the time of first prostate-specific antigen (PSA) failure to the authors' knowledge has not been elucidated to date and was studied herein. Between 1995 and 2001, a total of 58 men with unfavorable-risk PC who were treated on clinical trials with radiotherapy and androgen deprivation therapy (ADT) had available testosterone levels at the time of PSA failure. Cox and Fine and Gray regressions were performed to ascertain whether low versus normal testosterone was associated with the risk of PC-specific mortality, other-cause mortality, and all-cause mortality adjusting for age, salvage ADT, and known PC prognostic factors. After a median follow-up of 6.68 years after PSA failure, 31 men (53.4%) had died; 10 of PC (32.3%), of which 8 of 11 (72.7%) versus 2 of 47 (4.3%) deaths occurred in men with low versus normal testosterone at the time of PSA failure, respectively. A significant increase in the risk of all-cause mortality (adjusted hazard ratio [AHR], 2.54; 95% confidence interval [95% CI], 1.04-6.21 [P = .04]) and PC-specific mortality (AHR, 13.71; 95% CI, 2.4-78.16 [P = .003]), with a reciprocal trend toward a decreased risk of other-cause mortality (AHR, 0.18; 95% CI, 0.02-1.55 [P = .12]) was observed in men with low versus normal testosterone. Low, but not necessarily castrate, testosterone levels at the time of PSA failure confer a very poor prognosis. These observations provide evidence to support testosterone testing at the time of PSA failure. Given prolonged survival when abiraterone or docetaxel is added to ADT in men with castrate-sensitive metastatic PC and possibly localized high-risk PC provides a rationale supporting their use with ADT in men with low testosterone in the setting of a phase 2 trial. Cancer 2018;124:1383-90. © 2017 American Cancer Society. © 2017 American Cancer Society.

The polycystins are modulated by cellular oxygen-sensing pathways and regulate mitochondrial function

PubMed Central

Padovano, Valeria; Kuo, Ivana Y.; Stavola, Lindsey K.; Aerni, Hans R.; Flaherty, Benjamin J.; Chapin, Hannah C.; Ma, Ming; Somlo, Stefan; Boletta, Alessandra; Ehrlich, Barbara E.; Rinehart, Jesse; Caplan, Michael J.

2017-01-01

Autosomal dominant polycystic kidney disease is caused by mutations in the genes encoding polycystin-1 (PC1) and polycystin-2 (PC2), which form an ion channel complex that may mediate ciliary sensory processes and regulate endoplasmic reticulum (ER) Ca2+ release. Loss of PC1 expression profoundly alters cellular energy metabolism. The mechanisms that control the trafficking of PC1 and PC2, as well as their broader physiological roles, are poorly understood. We found that O2 levels regulate the subcellular localization and channel activity of the polycystin complex through its interaction with the O2-sensing prolyl hydroxylase domain containing protein EGLN3 (or PHD3), which hydroxylates PC1. Moreover, cells lacking PC1 expression use less O2 and show less mitochondrial Ca2+ uptake in response to bradykinin-induced ER Ca2+ release, indicating that PC1 can modulate mitochondrial function. These data suggest a novel role for the polycystins in sensing and responding to cellular O2 levels. PMID:27881662

Analysis of the V2 Vasopressin Receptor (V2R) Mutations Causing Partial Nephrogenic Diabetes Insipidus Highlights a Sustainable Signaling by a Non-peptide V2R Agonist.

PubMed

Makita, Noriko; Sato, Tomohiko; Yajima-Shoji, Yuki; Sato, Junichiro; Manaka, Katsunori; Eda-Hashimoto, Makiko; Ootaki, Masanori; Matsumoto, Naoki; Nangaku, Masaomi; Iiri, Taroh

2016-10-21

Disease-causing mutations in G protein-coupled receptor (GPCR) genes, including the V2 vasopressin receptor (V2R) gene, often cause misfolded receptors, leading to a defect in plasma membrane trafficking. A novel V2R mutation, T273M, identified in a boy with partial nephrogenic diabetes insipidus (NDI), shows intracellular localization and partial defects similar to the two mutants we described previously (10). Although non-peptide V2R antagonists have been shown to rescue the membrane localization of V2R mutants, their level of functional rescue is weak. Interestingly, it has been reported that a non-peptide agonist, OPC51803, activates misfolded V2R mutants intracellularly without degradation, thus potentially serving as a therapeutic agent against NDI (14). In our current experiments, however, a peptide antagonist blocked arginine vasopressin (AVP)- or OPC51803-stimulated cAMP accumulation both in COS-7 and MDCK cells, suggesting that OPC51803 mainly stimulates cell surface V2R mutants. In addition, our analyses revealed that OPC51803 works not only as a non-peptide agonist that causes activation/β-arrestin-dependent desensitization of V2R mutants expressed at the plasma membrane but also as a pharmacochaperone that promotes the endoplasmic reticulum-retained mutant maturation and trafficking to the plasma membrane. The ratio of the pharmacochaperone effect to the desensitization effect likely correlates negatively with the residual function of the tested mutants, suggesting that OPC5 has a more favorable effect on the V2R mutants with a less residual function. We speculated that the canceling of the desensitization effect of OPC51803 by the pharmacochaperone effect after long-term treatment may produce sustainable signaling, and thus pharmacochaperone agonists such as OPC51803 may serve as promising therapeutics for NDI caused by misfolded V2R mutants. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Analysis of the V2 Vasopressin Receptor (V2R) Mutations Causing Partial Nephrogenic Diabetes Insipidus Highlights a Sustainable Signaling by a Non-peptide V2R Agonist*

PubMed Central

Makita, Noriko; Sato, Tomohiko; Yajima-Shoji, Yuki; Sato, Junichiro; Manaka, Katsunori; Eda-Hashimoto, Makiko; Ootaki, Masanori; Matsumoto, Naoki; Nangaku, Masaomi; Iiri, Taroh

2016-01-01

Disease-causing mutations in G protein-coupled receptor (GPCR) genes, including the V2 vasopressin receptor (V2R) gene, often cause misfolded receptors, leading to a defect in plasma membrane trafficking. A novel V2R mutation, T273M, identified in a boy with partial nephrogenic diabetes insipidus (NDI), shows intracellular localization and partial defects similar to the two mutants we described previously (10). Although non-peptide V2R antagonists have been shown to rescue the membrane localization of V2R mutants, their level of functional rescue is weak. Interestingly, it has been reported that a non-peptide agonist, OPC51803, activates misfolded V2R mutants intracellularly without degradation, thus potentially serving as a therapeutic agent against NDI (14). In our current experiments, however, a peptide antagonist blocked arginine vasopressin (AVP)- or OPC51803-stimulated cAMP accumulation both in COS-7 and MDCK cells, suggesting that OPC51803 mainly stimulates cell surface V2R mutants. In addition, our analyses revealed that OPC51803 works not only as a non-peptide agonist that causes activation/β-arrestin-dependent desensitization of V2R mutants expressed at the plasma membrane but also as a pharmacochaperone that promotes the endoplasmic reticulum-retained mutant maturation and trafficking to the plasma membrane. The ratio of the pharmacochaperone effect to the desensitization effect likely correlates negatively with the residual function of the tested mutants, suggesting that OPC5 has a more favorable effect on the V2R mutants with a less residual function. We speculated that the canceling of the desensitization effect of OPC51803 by the pharmacochaperone effect after long-term treatment may produce sustainable signaling, and thus pharmacochaperone agonists such as OPC51803 may serve as promising therapeutics for NDI caused by misfolded V2R mutants. PMID:27601473

Elucidating the Mechanism of Gain of Toxic Function from Mutant C1 Inhibitor Proteins in Hereditary Angioedema

DTIC Science & Technology

2016-10-01

this is due, at least in part, to an additional acquired GOTF defect caused by the mutant protein that interferes with the secretion of WT C1INH. Our...overall hypothesis is that mutant C1INH proteins exert a variable GOTF phenotype that inhibit secretion of WT C1INH protein and worsen disease...will assess the mechanisms of the GOTF with a hypothesis that misfolding of mutant C1INH protein in the ER causes impairment of WT C1INH secretion

The CMB Topography Beneath Cook Inlet And Alaskan Kenai Peninsula

NASA Astrophysics Data System (ADS)

Wu, W.; Ni, S.

2009-05-01

It has long been known that the PcP-to-P amplitude ratios demonstrate strong scatter in some regions. Rost and Revenaugh studied PcP amplitudes which sample the core-mantle boundary (CMB) beneath the Alaskan Kenai peninsula and the Cook inlet and found a ˜1° region on the CMB with very large PcP/P amplitude ratios. For some events, the ratios are several tens times larger than the theoretical ratios. After analyzing different possible mechanisms, they concluded the CMB topography is the major cause of the high amplitude ratios, but they did not give an quantitative topography model because of the lack of short period synthetic waveforms tools. We generate short period PcP synthetics using representation theorems and study the PcP phases theoretically for a core-mantle boundary (CMB) with single sinusoidal topography. After testing different combinations of the sinusoid wavelength L and amplitude H, we conclude that a dent in CMB with diameter of L=300km and height H=1˜2km best fits the observed data and can partly explain the high amplitude ratios. The PcP reflected from the CMB dip with L=300km and H=2km will be amplified by 2˜3 times which is smaller than the value the observed data needed. We primarily have three causes to determine L=300km and H=1˜2km. First, a dip with L=300km and H=1˜2km will focus the PcP significantly in a ˜1° region on the CMB which is consistent with the observed data. Certainly, the northern limit of the region is not clear, therefore more data are needed to constraint the northern limit and give a more reliable model. Second, there are not obvious travel time anomalies coupled with the high ratios in the observed PcP and our synthetic travel time anomalies are just about 0.4s too. Lastly, the strong similarities of P and PcP for some simple events excludes such large value of H. A dip with larger L and corresponding H surly produces stronger focusing effect, for example, a dip with L= 300km and H=3km will amplifies the PcP by 4˜5 times, but the waveform will be distorted seriously, contradictory to the similarity of P and PcP. So we speculate that the topography and other causes are combined to produce the high amplitude ratios together.

Deletion of Ku80 causes early aging independent of chronic inflammation and Rag-1-induced DSBs.

PubMed

Holcomb, Valerie B; Vogel, Hannes; Hasty, Paul

2007-01-01

Animal models of premature aging are often defective for DNA repair. Ku80-mutant mice are disabled for nonhomologous end joining; a pathway that repairs both spontaneous DNA double-strand breaks (DSBs) and induced DNA DSBs generated by the action of a complex composed of Rag-1 and Rag-2 (Rag). Rag is essential for inducing DSBs important for assembling V(D)J segments of antigen receptor genes that are required for lymphocyte development. Thus, deletion of either Rag-1 or Ku80 causes severe combined immunodeficiency (scid) leading to chronic inflammation. In addition, Rag-1 induces breaks at non-B DNA structures. Previously we reported Ku80-mutant mice undergo premature aging, yet we do not know the root cause of this phenotype. Early aging may be caused by either defective repair of spontaneous DNA damage, defective repair of Rag-1-induced breaks or chronic inflammation caused by scid. To address this issue, we analyzed aging in control and Ku80-mutant mice deleted for Rag-1 such that both cohorts are scid and suffer from chronic inflammation. We make two observations: (1) chronic inflammation does not cause premature aging in these mice and (2) Ku80-mutant mice exhibit early aging independent of Rag-1. Therefore, this study supports defective repair of spontaneous DNA damage as the root cause of early aging in Ku80-mutant mice.

Genetic inactivation of prohormone convertase (PC1) causes a reduction in cholecystokinin (CCK) levels in the hippocampus, amygdala, pons and medulla in mouse brain that correlates with the degree of colocalization of PC1 and CCK mRNA in these structures in rat brain.

PubMed

Cain, B M; Connolly, K; Blum, A C; Vishnuvardhan, D; Marchand, J E; Zhu, X; Steiner, D F; Beinfeld, M C

2004-04-01

Prohormone convertase (PC1) is found in endocrine cell lines that express cholecystokinin (CCK) mRNA and process pro CCK to biologically active products. Other studies have demonstrated that PC1 may be a one of the enzymes responsible for the endoproteolytic cleavages that occur in pro CCK during its biosynthesis and processing. Prohormone convertase 1 (PC1) has a distribution that is similar to cholecystokinin (CCK) in rat brain. A moderate to high percentage of CCK mRNA-positive neurons express PC1 mRNA. CCK levels were measured in PC1 knockout and control mice to assess the degree to which loss of PC1 changed CCK content. CCK levels were decreased 62% in hippocampus, 53% in amygdala and 57% in pons-medulla in PC1 knockout mice as compared to controls. These results are highly correlated with the colocalization of CCK and PC1. The majority of CCK mRNA-positive neurons in the pyramidal cell layer of the hippocampus express PC1 mRNA and greater than 50% of CCK mRNA-positive neurons in several nuclei of the amygdala also express PC1. These results demonstrate that PC1 is important for CCK processing. PC2 and PC5 are also widely colocalized with CCK. It may be that PC2, PC5 or another non-PC enzyme are able to substitute for PC1 and sustain production of some amidated CCK. Together these enzymes may represent a redundant system to insure the production of CCK.

Investigation of the Photocurrent in Hot-Wall-Epitaxy-Grown BaIn2S4 Layers

NASA Astrophysics Data System (ADS)

You, S. H.; Hong, K. J.; Jeong, T. S.; Youn, C. J.

2015-12-01

The photocurrent (PC) of hot-wall-epitaxy-grown BaIn2S4 layers was studied at different temperatures and for different photoresponse intensities. With increasing temperature, the position of the PC spectra tended to shift toward longer wavelength. These PC peaks corresponded to band-to-band transitions caused by intrinsic transitions from the valence band states to the conduction band states. Also, the bandgap variations were well matched by the equation E g( T) = E g(0) - 3.79 × 10-3 T 2/( T + 499), where E g(0) was estimated to be 3.0597 eV, 3.2301 eV, and 3.2606 eV for transitions corresponding to the valence band states Γ 4(z), Γ 5(x), and Γ 5(y), respectively. By use of the selection rule and results from the PC spectroscopy, the crystal field and the spin-orbit splitting were found to be 0.1703 and 0.0306 eV, respectively. Thus, the PC intensity gradually decreased with decreasing temperature. The decrease of PC intensity was caused by the presence of trapping centers associated with native defects in the BaIn2S4 layers. The trap level was found to be a shallow donor-level type of 20.4 meV, 1.6 meV below the conduction band. Consequently, these trap levels, which are related to native defects in BaIn2S4 layers, are believed to limit PC intensity with decreasing temperature.

Distinct cerebellar engrams in short-term and long-term motor learning.

PubMed

Wang, Wen; Nakadate, Kazuhiko; Masugi-Tokita, Miwako; Shutoh, Fumihiro; Aziz, Wajeeha; Tarusawa, Etsuko; Lorincz, Andrea; Molnár, Elek; Kesaf, Sebnem; Li, Yun-Qing; Fukazawa, Yugo; Nagao, Soichi; Shigemoto, Ryuichi

2014-01-07

Cerebellar motor learning is suggested to be caused by long-term plasticity of excitatory parallel fiber-Purkinje cell (PF-PC) synapses associated with changes in the number of synaptic AMPA-type glutamate receptors (AMPARs). However, whether the AMPARs decrease or increase in individual PF-PC synapses occurs in physiological motor learning and accounts for memory that lasts over days remains elusive. We combined quantitative SDS-digested freeze-fracture replica labeling for AMPAR and physical dissector electron microscopy with a simple model of cerebellar motor learning, adaptation of horizontal optokinetic response (HOKR) in mouse. After 1-h training of HOKR, short-term adaptation (STA) was accompanied with transient decrease in AMPARs by 28% in target PF-PC synapses. STA was well correlated with AMPAR decrease in individual animals and both STA and AMPAR decrease recovered to basal levels within 24 h. Surprisingly, long-term adaptation (LTA) after five consecutive daily trainings of 1-h HOKR did not alter the number of AMPARs in PF-PC synapses but caused gradual and persistent synapse elimination by 45%, with corresponding PC spine loss by the fifth training day. Furthermore, recovery of LTA after 2 wk was well correlated with increase of PF-PC synapses to the control level. Our findings indicate that the AMPARs decrease in PF-PC synapses and the elimination of these synapses are in vivo engrams in short- and long-term motor learning, respectively, showing a unique type of synaptic plasticity that may contribute to memory consolidation.

Dianionic Titanyl and Vanadyl (Cation+ )2 [MIV O(Pc4- )]2- Phthalocyanine Salts Containing Pc4- Macrocycles.

PubMed

Konarev, Dmitri V; Kuzmin, Alexey V; Khasanov, Salavat S; Litvinov, Alexey L; Otsuka, Akihiro; Yamochi, Hideki; Kitagawa, Hiroshi; Lyubovskaya, Rimma N

2018-06-18

In this study, the titanyl and vanadyl phthalocyanine (Pc) salts (Bu 4 N + ) 2 [M IV O(Pc 4- )] 2- (M=Ti, V) and (Bu 3 MeP + ) 2 [M IV O(Pc 4- )] 2- (M=Ti, V) with [M IV O(Pc 4- )] 2- dianions were synthesized and characterized. Reduction of M IV O(Pc 2- ) carried out with an excess of sodium fluorenone ketyl in the presence of Bu 4 N + or Bu 3 MeP + is exclusive to the phthalocyanine centers, forming Pc 4- species. During reduction, the metal +4 charge did not change, implying that Pc is an non-innocent ligand. The Pc negative charge increase caused the C-N(pyr) bonds to elongate and the C-N(imine) bonds to alternate, thus increasing the distortion of Pc. Jahn-Teller effects are significant in the [eg(π*)] 2 dianion ground state and can additionally distort the Pc macrocycles. Blueshifts of the Soret and Q-bands were observed in the UV/Vis/NIR when M IV O(Pc 2- ) was reduced to [M IV O(Pc . 3- )] . - and [M IV O(Pc 4- )] 2- . From magnetic measurements, [Ti IV O(Pc 4- )] 2- was found to be diamagnetic and (Bu 4 N + ) 2 [V IV O(Pc 4- )] 2- and (Bu 3 MeP + ) 2 [V IV O(Pc 4- )] 2- were found to have magnetic moments of 1.72-1.78 μ B corresponding to an S=1/2 spin state owing to V IV electron spin. As a result, two latter salts show EPR signals with V IV hyperfine coupling. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhanced photothermal lens using a photonic crystal surface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Yunfei; Liu, Longju; Zhao, Xiangwei

2016-08-15

A photonic crystal (PC)-enhanced photothermal lens (PTL) is demonstrated for the detection of optically thin light absorption materials. The PC-enhanced PTL system is based on a pump-probe scheme consisting of a PC surface, pump laser beam, and probe laser beam. Heated by the pump beam, light absorption materials on the PC surface generate the PTL and cause a substantial change to the guided-mode resonance supported by the PC structure. The change of the PC resonance is detected using the probe laser beam by measuring its reflectivity from the PC surface. When applied to analyze dye molecules deposited on the PCmore » substrate, the developed system is capable of enhancing the PTL signal by 10-fold and reducing the lowest distinguishable concentration by 8-fold, in comparison to measuring without utilizing the PC resonance. The PC-enhanced PTL was also used to detect gold nanoparticles on the PC surface and exhibited a 20-fold improvement of the lowest distinguishable concentration. The PC-enhanced PTL technology offers a potential tool to obtain the absorption signatures of thin films in a broad spectral range with high sensitivity and inexpensive instrumentation. As a result, this technology will enable a broad range of applications of photothermal spectroscopy in chemical analysis and biomolecule sensing.« less

The intrinsic cause of color fading in sliced cooked cured beef during chilled storage.

PubMed

Ma, Jing; Yu, Chensha; Guo, Juanjuan; Wu, Mengting; Xu, Yunqiang; Yi, Hongchao; Sun, Weiqing

2017-10-01

The relationship between color change and other physical and chemical characteristics of sliced cooked cured beef (SCCB) during chilled storage were investigated using principal components analysis (PCA) to determine the color fading causes. Samples were prepared and stored at 8°C for up to 35 days in a vacuum package to simulate a supermarket storage environment; related indicators were measured periodically every week. The results showed that the first PC explained 59.82% of the total variation, and the second explained 22.28%. PC1 was a concentrated reflection of color changes of SCCB during storage and PC2 was an environment factor causing the change of color. The change in apparent redness is mainly caused by redox reaction of the nitroso hemochromogen (NH) (eigenvectors of a*, C and NH in PC1 were all the maximum value of 0.28); a* was correlated with NH (0.96), free sulfhydryls (0.98), carbonyl derivatives (-0.95) formed during protein oxidation, and malondialdehyde (-0.98) and dienes (-0.92) formed by lipid oxidation. Color fading was significantly correlated with oxidizing and reducing power, existing forms of nitrogen and with the pH of the meat matrix. Changes in the internal environment of the sample initially influenced L* and b* values, and subsequently a*. © 2017 Japanese Society of Animal Science.

Misfolded opsin mutants display elevated β -sheet structure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller, Lisa M.; Gragg, Megan; Kim, Tae Gyun

Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinitis pigmentosa, a progressive retinal degenerative disease. The structure adopted by misfolded opsin mutants and the associated cell toxicity is poorly understood. Förster resonance energy transfer (FRET) and Fourier transform infrared (FTIR) microspectroscopy were utilized to probe within cells the structures formed by G188R and P23H opsins, which are misfolding mutants that cause autosomal dominant retinitis pigmentosa. Also, both mutants formed aggregates in the endoplasmic reticulum and exhibited altered secondary structure with elevated β-sheet and reduced α-helical content. The newly formed β-sheet structure may facilitate themore » aggregation of misfolded opsin mutants. In conclusion, the effects observed for the mutants were unrelated to retention of opsin molecules in the endoplasmic reticulum itself.« less

Misfolded opsin mutants display elevated β -sheet structure

DOE PAGES

Miller, Lisa M.; Gragg, Megan; Kim, Tae Gyun; ...

2015-09-07

Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinitis pigmentosa, a progressive retinal degenerative disease. The structure adopted by misfolded opsin mutants and the associated cell toxicity is poorly understood. Förster resonance energy transfer (FRET) and Fourier transform infrared (FTIR) microspectroscopy were utilized to probe within cells the structures formed by G188R and P23H opsins, which are misfolding mutants that cause autosomal dominant retinitis pigmentosa. Also, both mutants formed aggregates in the endoplasmic reticulum and exhibited altered secondary structure with elevated β-sheet and reduced α-helical content. The newly formed β-sheet structure may facilitate themore » aggregation of misfolded opsin mutants. In conclusion, the effects observed for the mutants were unrelated to retention of opsin molecules in the endoplasmic reticulum itself.« less

A cataract-causing connexin 50 mutant is mislocalized to the ER due to loss of the fourth transmembrane domain and cytoplasmic domain.

PubMed

Somaraju Chalasani, Madhavi Latha; Muppirala, Madhavi; G Ponnam, Surya Prakash; Kannabiran, Chitra; Swarup, Ghanshyam

2013-01-01

Mutations in the eye lens gap junction protein connexin 50 cause cataract. Earlier we identified a frameshift mutant of connexin 50 (c.670insA; p.Thr203AsnfsX47) in a family with autosomal recessive cataract. The mutant protein is smaller and contains 46 aberrant amino acids at the C-terminus after amino acid 202. Here, we have analysed this frameshift mutant and observed that it localized to the endoplasmic reticulum (ER) but not in the plasma membrane. Moreover, overexpression of the mutant resulted in disintegration of the ER-Golgi intermediate compartment (ERGIC), reduction in the level of ERGIC-53 protein and breakdown of the Golgi in many cells. Overexpression of the frameshift mutant partially inhibited the transport of wild type connexin 50 to the plasma membrane. A deletion mutant lacking the aberrant sequence showed predominant localization in the ER and inhibited anterograde protein transport suggesting, therefore, that the aberrant sequence is not responsible for improper localization of the frameshift mutant. Further deletion analysis showed that the fourth transmembrane domain and a membrane proximal region (231-294 amino acids) of the cytoplasmic domain are needed for transport from the ER and localization to the plasma membrane. Our results show that a frameshift mutant of connexin 50 mislocalizes to the ER and causes disintegration of the ERGIC and Golgi. We have also identified a sequence of connexin 50 crucial for transport from the ER and localization to the plasma membrane.

Crystallographic Analysis Reveals a Novel Second Binding Site for Trimethoprim in Active Site Double Mutants of Human Dihydrofolate Reductase†,‡

PubMed Central

Cody, Vivian; Pace, Jim; Piraino, Jennifer; Queener, Sherry F.

2011-01-01

In order to produce a more potent replacement for trimethoprim (TMP) used as a therapy for Pneumocystis pneumonia and targets dihydrofolate reductase from Pneumocystis jirovecii (pjDHFR), it is necessary to understand the determinants of potency and selectivity against DHFR from the mammalian host and fungal pathogen cells. To this end, active site residues in human (h)DHFR were replaced with those from pjDHFR. Structural data are reported for two complexes of TMP with the double mutants Gln35Ser/Asn64Phe (Q35S/N64F) and Gln35Lys/Asn64Phe (Q35K/N64F) of hDHFR that unexpectedly show evidence for the binding of two molecules of TMP: one molecule that binds in the normal folate binding site and the second molecule that binds in a novel subpocket site such that the mutated residue Phe64 is involved in van der Waals contacts to the trimethoxyphenyl ring of the second TMP molecule. Kinetic data for the binding of TMP to hDHFR and pjDHFR reveal an 84-fold selectivity of TMP against pjDHFR (Ki 49 nM) compared to hDHFR (Ki 4093 nM). Two mutants that contain one substitution from pj- and one from the closely related Pneumocystis carinii DHFR (pcDHFR) (Q35K/N64F and Q35S/N64F) show Ki values of 593 and 617 nM, respectively; these Ki values are well above both the Ki for pjDHFR and are similar to pcDHFR (Q35K/N64F) and Q35S/N64F) (305 nM). These results suggest that active site residues 35 and 64 play key roles in determining selectivity for pneumocystis DHFR, but that other residues contribute to the unique binding of inhibitors to these enzymes. PMID:21684339

The selective and inducible activation of endogenous PI 3-kinase in PC12 cells results in efficient NGF-mediated survival but defective neurite outgrowth.

PubMed

Ashcroft, M; Stephens, R M; Hallberg, B; Downward, J; Kaplan, D R

1999-08-12

The Trk/Nerve Growth Factor receptor mediates the rapid activation of a number of intracellular signaling proteins, including phosphatidylinositol 3-kinase (PI 3-kinase). Here, we describe a novel, NGF-inducible system that we used to specifically address the signaling potential of endogenous PI 3-kinase in NGF-mediated neuronal survival and differentiation processes. This system utilizes a Trk receptor mutant (Trk(def)) lacking sequences Y490, Y785 and KFG important for the activation of the major Trk targets; SHC, PLC-gammal, Ras, PI 3-kinase and SNT. Trk(def) was kinase active but defective for NGF-induced responses when stably expressed in PC12nnr5 cells (which lack detectable levels of TrkA and are non-responsive to NGF). The PI 3-kinase consensus binding site, YxxM (YVPM), was introduced into the insert region within the kinase domain of Trk(def). NGF-stimulated tyrosine phosphorylation of the Trk(def)+PI 3-kinase addback receptor, resulted in the direct association and selective activation of PI 3-kinase in vitro and the production of PI(3,4)P2 and PI(3,4,5)P3 in vivo (comparable to wild-type). PC12nnr5 cells stably expressing Trk(def) + PI 3-kinase, initiated neurite outgrowth but failed to stably extend and maintain these neurites in response to NGF as compared to PC12 parental cells, or PC12nnr5 cells overexpressing wild-type Trk. However, Trk(def) + PI 3-kinase was fully competent in mediating NGF-induced survival processes. We propose that while endogenous PI 3-kinase can contribute in part to neurite initiation processes, its selective activation and subsequent signaling to downstream effectors such as Akt, functions mainly to promote cell survival in the PC12 system.

Prion Protein Regulates Iron Transport by Functioning as a Ferrireductase

PubMed Central

Singh, Ajay; Haldar, Swati; Horback, Katharine; Tom, Cynthia; Zhou, Lan; Meyerson, Howard; Singh, Neena

2017-01-01

Prion protein (PrPC) is implicated in the pathogenesis of prion disorders, but its normal function is unclear. We demonstrate that PrPC is a ferrireductase (FR), and its absence causes systemic iron deficiency in PrP knock-out mice (PrP−/−). When exposed to non-transferrin-bound (NTB) radioactive-iron (59FeCl3) by gastric-gavage, PrP−/− mice absorb significantly more 59Fe from the intestinal lumen relative to controls, indicating appropriate systemic response to the iron deficiency. Chronic exposure to excess dietary iron corrects this deficiency, but unlike wild-type (PrP+/+) controls that remain iron over-loaded, PrP−/− mice revert back to the iron deficient phenotype after 5 months of chase on normal diet. Bone marrow (BM) preparations of PrP−/− mice on normal diet show relatively less stainable iron, and this phenotype is only partially corrected by intraperitoneal administration of excess iron-dextran. Cultured PrP−/− BM-macrophages incorporate significantly less NTB-59Fe in the absence or presence of excess extracellular iron, indicating reduced uptake and/or storage of available iron in the absence of PrPC. When expressed in neuroblastoma cells, PrPC exhibits NAD(P)H-dependent cell-surface and intracellular FR activity that requires the copper-binding octa-peptide-repeat region and linkage to the plasma membrane for optimal function. Incorporation of NTB-59Fe by neuroblastoma cells correlates with FR activity of PrPC, implicating PrPC in cellular iron uptake and metabolism. These observations explain the correlation between PrPC expression and cellular iron levels, and the cause of iron imbalance in sporadic-Creutzfeldt-Jakob-disease brains where PrPC accumulates as insoluble aggregates. PMID:23478311

Disparate effects of oxidation on plasma acyltransferase activities: inhibition of cholesterol esterification but stimulation of transesterification of oxidized phospholipids.

PubMed

Subbaiah, P V; Liu, M

1996-05-31

Oxidation of lipoproteins results in the formation of several polar phospholipids with pro-inflammatory and pro-atherogenic properties. To examine the possible role of lecithin/cholesterol acyltransferase (LCAT) in the metabolism of these oxidized phospholipids, we oxidized whole plasma with either Cu(2+) or a free-radical generator, and determined the various activities of LCAT. Oxidation caused a reduction in plasma phosphatidylcholine (PC), an increase in a short-chain polar PC (SCP-PC), and an inhibition of the transfer of long-chain acyl groups to cholesterol (LCAT activity) or to lyso PC (lysolecithin acyltransferase (LAT) I activity). However, the transfer of short-chain acyl groups from SCP-PC to lyso PCLAT II activity) was stimulated several fold, in direct correlation with the degree of oxidation. LAT II activity was not stimulated by oxidation in LCAT-deficient plasma, showing that it is carried out by LCAT. Oxidized normal plasma exhibited low LCAT activity even in the presence of exogenous proteoliposome substrate, indicating that the depletion of substrate PC was not responsible for the loss of activity. Oxidation of isolated LDL or HDL abolished their ability to support LCAT and LAT I activities of exogenous enzyme, but promoted the LAT II activity. Purified LCAT lost its LCAT and LAT I functions, but not its LAT II function, when oxidized in vitro. These results show that while oxidation of plasma causes a loss of LCAT's ability to transfer long-chain acyl groups, its ability to transfer short-chain acyl groups, from SCP-PC is retained, and even stimulated, suggesting that LCAT may have a physiological role in the metabolism of oxidized PC in plasma.

Preclinical Characterization of PC786, an Inhaled Small-Molecule Respiratory Syncytial Virus L Protein Polymerase Inhibitor

PubMed Central

Coates, Matthew; Brookes, Daniel; Kim, Young-In; Allen, Heather; Fordyce, Euan A. F.; Meals, Elizabeth A.; Colley, Thomas; Ciana, Claire-Lise; Parra, Guillaume F.; Sherbukhin, Vladimir; Stockwell, Jennifer A.; Thomas, Jennifer C.; Hunt, S. Fraser; Anderson-Dring, Lauren; Onions, Stuart T.; Cass, Lindsey; Murray, Peter J.; Strong, Pete; DeVincenzo, John P.; Rapeport, Garth

2017-01-01

ABSTRACT Although respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in infants and young children, attempts to develop an effective therapy have so far proved unsuccessful. Here we report the preclinical profiles of PC786, a potent nonnucleoside RSV L protein polymerase inhibitor, designed for inhalation treatment of RSV infection. PC786 demonstrated a potent and selective antiviral activity against laboratory-adapted or clinical isolates of RSV-A (50% inhibitory concentration [IC50], <0.09 to 0.71 nM) and RSV-B (IC50, 1.3 to 50.6 nM), which were determined by inhibition of cytopathic effects in HEp-2 cells without causing detectable cytotoxicity. The underlying inhibition of virus replication was confirmed by PCR analysis. The effects of PC786 were largely unaffected by the multiplicity of infection (MOI) and were retained in the face of established RSV replication in a time-of-addition study. Persistent anti-RSV effects of PC786 were also demonstrated in human bronchial epithelial cells. In vivo intranasal once daily dosing with PC786 was able to reduce the virus load to undetectable levels in lung homogenates from RSV-infected mice and cotton rats. Treatment with escalating concentrations identified a dominant mutation in the L protein (Y1631H) in vitro. In addition, PC786 potently inhibited RSV RNA-dependent RNA polymerase (RdRp) activity in a cell-free enzyme assay and minigenome assay in HEp-2 cells (IC50, 2.1 and 0.5 nM, respectively). Thus, PC786 was shown to be a potent anti-RSV agent via inhibition of RdRp activity, making topical treatment with this compound a novel potential therapy for the treatment of human RSV infections. PMID:28652242

Protective effects of protopine on hydrogen peroxide-induced oxidative injury of PC12 cells via Ca(2+) antagonism and antioxidant mechanisms.

PubMed

Xiao, Xianghua; Liu, Juntian; Hu, Jingwen; Zhu, Xiuping; Yang, Hua; Wang, Chaoyun; Zhang, Yuanhui

2008-09-04

Calcium and lipid peroxidation play important roles in oxidative stress-induced cellular injury and apoptosis, which ultimately cause cell death. In this study we examined whether protopine had a neuroprotection against H(2)O(2)-induced injury in PC12 cells. Pretreatment of PC12 cells with protopine improved the cell viability, enhanced activities of superoxide dismutase, glutathione peroxidase and catalase, and decreased malondialdehyde level in the H(2)O(2) injured cells. Protopine also reversed the increased intracellular Ca(2+) concentration and the reduced mitochondrial membrane potential caused by H(2)O(2) in the cells. Furthermore, protopine was able to inhibit caspase-3 expression and cell apoptosis induced by H(2)O(2). In summary, this study demonstrates that protopine is able to relieve H(2)O(2)-induced oxidative stress and apoptosis in PC12 cells, at least in part, by Ca(2+) antagonism and antioxidant mechanisms.

Targeting NK-1 Receptors to Prevent and Treat Pancreatic Cancer: A New Therapeutic Approach

PubMed Central

Muñoz, Miguel; Coveñas, Rafael

2015-01-01

Pancreatic cancer (PC) is the fourth leading cause of cancer related-deaths in both men and women, and the 1- and 5-year relative survival rates are 25% and 6%, respectively. It is known that smoking, alcoholism and psychological stress are risk factors that can promote PC and increase PC progression. To date, the prevention of PC is crucial because there is no curative treatment. After binding to the neurokinin-1 (NK-1) receptor (a receptor coupled to the stimulatory G-protein Gαs that activates adenylate cyclase), the peptide substance P (SP)—at high concentrations—is involved in many pathophysiological functions, such as depression, smoking, alcoholism, chronic inflammation and cancer. It is known that PC cells and samples express NK-1 receptors; that the NK-1 receptor is overexpressed in PC cells in comparison with non-tumor cells, and that nanomolar concentrations of SP induce PC cell proliferation. By contrast, NK-1 receptor antagonists exert antidepressive, anxiolytic and anti-inflammatory effects and anti-alcohol addiction. These antagonists also exert an antitumor action since in vitro they inhibit PC cell proliferation (PC cells death by apoptosis), and in a xenograft PC mouse model they exert both antitumor and anti-angiogenic actions. NK-1 receptor antagonists could be used for the treatment of PC and hence the NK-1 receptor could be a new promising therapeutic target in PC. PMID:26154566

Ammonia modification for flotation separation of polycarbonate and polystyrene waste plastics.

PubMed

Wang, Chong-Qing; Wang, Hui; Gu, Guo-Hua; Lin, Qing-Quan; Zhang, Ling-Ling; Huang, Luo-Luo; Zhao, Jun-Yao

2016-05-01

A promising method, ammonia modification, was developed for flotation separation of polycarbonate (PC) and polystyrene (PS) waste plastics. Ammonia modification has little effect on flotation behavior of PS, while it changes significantly that of PC. The PC recovery in the floated product drops from 100% to 3.17% when modification time is 13min and then rises to 100% after longer modification. The mechanism of ammonia modification was studied by contact angle, and Fourier transform infrared (FT-IR) and X-ray photoelectron spectroscopy (XPS) measurements. Contact angle of PC indicates the decline of PC recovery in the floated product is ascribed to an increase in surface wettability. FT-IR and XPS spectra suggest that ammonia modification causes chemical reactions occurred on PC surface. Flotation behavior of ammonia-modified PC and PS was investigated with respect to flotation time, frother concentration and particle sizes. Flotation separation of PC and PS waste plastics was conducted based on the flotation behavior of single plastic. PC and PS mixtures with different particle sizes are separated efficiently, implying that the technology possesses superior applicability to particle sizes of plastics. The purity of PS and PC is up to 99.53% and 98.21%, respectively, and the recovery of PS and PC is larger than 92.06%. A reliable, cheap and effective process is proposed for separation of PC and PS waste plastics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Neurite outgrowth on a fibronectin isoform expressed during peripheral nerve regeneration is mediated by the interaction of paxillin with α4β1 integrins

PubMed Central

Vogelezang, Mariette; Forster, Ulrike B; Han, Jaewon; Ginsberg, Mark H; ffrench-Constant, Charles

2007-01-01

Background The regeneration of peripheral nerve is associated with a change in the alternative splicing of the fibronectin primary gene transcript to re-express embryonic isoforms containing a binding site for α4β1 integrins that promote neurite outgrowth. Here we use PC12 cells to examine the role of the interaction between paxillin and the α4 integrin cytoplasmic domain in neurite outgrowth. Results Expression of α4 with mutations in the paxillin-binding domain reduced neurite outgrowth on recombinant embryonic fibronectin fragments relative to wild type α4. Over-expression of paxillin promoted neurite outgrowth while a mutant isoform lacking the LD4 domain implicated in the regulation of ARF and Rac GTPases was less effective. Optimal α4-mediated migration in leucocytes requires spatial regulation of α4 phosphorylation at Ser988, a post-translational modification that blocks paxillin binding to the integrin cytoplasmic domain. In keeping with this α4(S988D), which mimics phosphorylated α4, did not promote neurite outgrowth. However, α4 was not phosphorylated in the PC12 cells, and a non-phosphorylatable α4(S988A) mutant promoted neurite outgrowth indistinguishably from the wild type integrin. Conclusion We establish the importance of the α4 integrin-paxillin interaction in a model of axonal regeneration and highlight differing dependence on phosphorylation of α4 for extension of neuronal growth cones and migration of non-neural cells. PMID:17603879

Control of cell division and the spatial localization of assembled gene products in Caulobacter crescentus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nathan, P.D.

Experiments are described that examine the role of penicillin-binding proteins (PBPs) in the regulation of cell division in Caulobacter crescentus; and the spatial localization of methyl-accepting chemotaxis proteins (MCPs) in C. crescentus swarmer and predivisional cells. In the analysis of PBP function, in vivo and in vitro assays are used to directly label C. crescentus PBPs with (/sup 3/H) penicillin G in wild type strain CB15, in a series of conditional cell division mutants and in new temperature sensitive cephalosporin C resistant mutants PC8002 and PC8003. 14 PBPs are characterized and a high molecular weight PBP (PBP 1B) that ismore » required for cell division is identified. PBP 1B competes for ..beta..-lactams that induce filament formation and may be a high affinity binding protein. A second high molecular weight PBP (PBP 1C) is also associated with defective cell division. The examination of PBP patterns in synchronous swarmer cells reveals that the in vivo activity of PBP 1B and PBP 1C increases at the time that the cell division pathway is initiated. None of the PBPs, however, appear to be differentially localized in the C. crescentus cell. In the analysis of MCP localization, in vivo and in vitro assays are used to directly label C. crescentus MCPs with methyl-/sup 3/H. MCPs are examined in flagellated and non-flagellated vesicles prepared from cells by immunoaffinity chromatography.« less

Amyloid precursor protein-induced axonopathies are independent of amyloid-beta peptides.

PubMed

Stokin, Gorazd B; Almenar-Queralt, Angels; Gunawardena, Shermali; Rodrigues, Elizabeth M; Falzone, Tomás; Kim, Jungsu; Lillo, Concepción; Mount, Stephanie L; Roberts, Elizabeth A; McGowan, Eileen; Williams, David S; Goldstein, Lawrence S B

2008-11-15

Overexpression of amyloid precursor protein (APP), as well as mutations in the APP and presenilin genes, causes rare forms of Alzheimer's disease (AD). These genetic changes have been proposed to cause AD by elevating levels of amyloid-beta peptides (Abeta), which are thought to be neurotoxic. Since overexpression of APP also causes defects in axonal transport, we tested whether defects in axonal transport were the result of Abeta poisoning of the axonal transport machinery. Because directly varying APP levels also alters APP domains in addition to Abeta, we perturbed Abeta generation selectively by combining APP transgenes in Drosophila and mice with presenilin-1 (PS1) transgenes harboring mutations that cause familial AD (FAD). We found that combining FAD mutant PS1 with FAD mutant APP increased Abeta42/Abeta40 ratios and enhanced amyloid deposition as previously reported. Surprisingly, however, this combination suppressed rather than increased APP-induced axonal transport defects in both Drosophila and mice. In addition, neuronal apoptosis induced by expression of FAD mutant human APP in Drosophila was suppressed by co-expressing FAD mutant PS1. We also observed that directly elevating Abeta with fusions to the Familial British and Danish Dementia-related BRI protein did not enhance axonal transport phenotypes in APP transgenic mice. Finally, we observed that perturbing Abeta ratios in the mouse by combining FAD mutant PS1 with FAD mutant APP did not enhance APP-induced behavioral defects. A potential mechanism to explain these findings was suggested by direct analysis of axonal transport in the mouse, which revealed that axonal transport or entry of APP into axons is reduced by FAD mutant PS1. Thus, we suggest that APP-induced axonal defects are not caused by Abeta.

Unwrapping eddy current compensation: improved compensation of eddy current induced baseline shifts in high-resolution phase-contrast MRI at 9.4 Tesla.

PubMed

Espe, Emil K S; Zhang, Lili; Sjaastad, Ivar

2014-10-01

Phase-contrast MRI (PC-MRI) is a versatile tool allowing evaluation of in vivo motion, but is sensitive to eddy current induced phase offsets, causing errors in the measured velocities. In high-resolution PC-MRI, these offsets can be sufficiently large to cause wrapping in the baseline phase, rendering conventional eddy current compensation (ECC) inadequate. The purpose of this study was to develop an improved ECC technique (unwrapping ECC) able to handle baseline phase discontinuities. Baseline phase discontinuities are unwrapped by minimizing the spatiotemporal standard deviation of the static-tissue phase. Computer simulations were used for demonstrating the theoretical foundation of the proposed technique. The presence of baseline wrapping was confirmed in high-resolution myocardial PC-MRI of a normal rat heart at 9.4 Tesla (T), and the performance of unwrapping ECC was compared with conventional ECC. Areas of phase wrapping in static regions were clearly evident in high-resolution PC-MRI. The proposed technique successfully eliminated discontinuities in the baseline, and resulted in significantly better ECC than the conventional approach. We report the occurrence of baseline phase wrapping in PC-MRI, and provide an improved ECC technique capable of handling its presence. Unwrapping ECC offers improved correction of eddy current induced baseline shifts in high-resolution PC-MRI. Copyright © 2013 Wiley Periodicals, Inc.

Hereditary pancreatic cancer: molecular bases and their application in diagnosis and clinical management: a guideline of the TTD group.

PubMed

Segura, P Pérez; Ponce, C Guillén; Ramón Y Cajal, T; Blanch, R Serrano; Aranda, E

2012-08-01

Pancreatic carcinoma (PC) represents the fourth leading cause of cancer death in Spain with a death rate of 2,400 males and 2,000 females per year. Poor outcome related to its silent nature and the lack of reliable secondary prevention measures translate into advanced-stage diagnosis, 75 % of deaths within the first year of diagnosis and 5-year survival rate of <5 %. Family history was first recognized as a risk factor for PC. Further population-based and case-control studies subsequently found that 7.8 % of patients with PC have a family history of the same tumor and individuals with a first-degree relative with PC have a 3.2-fold increased risk of developing PC. Overall, it is estimated that up to 10 % of PC have a familial component. However, known genetic syndromes account for <20 % of the observed familial aggregation of PC. We review the most important aspects in epidemiology, molecular biology and clinical management of familial PC.

Effect of Fermentation and Cooking on Soluble and Bound Phenolic Profiles of Finger Millet Sour Porridge.

PubMed

Gabaza, Molly; Shumoy, Habtu; Muchuweti, Maud; Vandamme, Peter; Raes, Katleen

2016-10-12

The aim of this study was to evaluate the soluble and bound phenolic content of finger millet and the impact of process induced changes on phenolic profiles of their sour porridge. Finger millet porridge and intermediate products were collected from four groups of households in the Hwedza communal area, Zimbabwe, after which soluble and bound phenolic compounds (PC) including condensed tannins (CT) were quantified. Bound PC and CT contributed 95% of the total PC and CT. The CT were only detected in the red varieties. Major individual PC identified were catechin occurring in the soluble fraction only, while ferulic, sinapic, and salicylic acid were mainly present in the bound fraction. Fermentation and cooking caused a more than 2-fold increase in soluble PC, CT, and individual PC. Improved traditional processing techniques optimized for improved bioavailability and health benefits of phenolics are highly relevant for the low income populations.

Evaluation of aluminum phthalocyanine chloride and DNA interactions for the design of an advanced drug delivery system in photodynamic therapy.

PubMed

Jayme, Cristiano Ceron; Calori, Italo Rodrigo; Cunha, Elise Marques Freire; Tedesco, Antonio Claudio

2018-08-05

The aim of this study was to evaluate the interaction of aluminum phthalocyanine chloride (AlClPc) with double-stranded DNA. Absorption and fluorescence spectra, resonance light scattering, and circular dichroism were evaluated in water and water/ethanol mixtures with different concentrations of DNA or AlClPc. AlClPc showed a high ability to bind to DNA in both water and 4/6 water/ethanol mixture (v/v), with a majority of monomeric and aggregated initial forms of AlClPc, respectively. In this interaction, AlClPc bound preferentially to the grooves of DNA. The monomeric/aggregate state of AlClPc in DNA was dependent on the AlClPc/DNA ratio. At low concentrations of AlClPc, the interaction of AlClPc with few DNA sites caused a curvature in the DNA structure that provided a favorable environment for the intercalation of AlClPc aggregates. Increase in AlClPc concentration induced interactions with a high number of binding sites on DNA, which prevented bending and therefore aggregation of AlClPc molecules throughout the double-stranded DNA. These results are relevant to the understanding of the behavior and interaction of AlClPc with double-stranded DNA in the design of novel drug delivery systems for clinical application in photodynamic therapy as a new approach to treat skin or oral cancer, scars, or wound healing. Copyright © 2018 Elsevier B.V. All rights reserved.

Unique and shared functions of nuclear lamina LEM domain proteins in Drosophila.

PubMed

Barton, Lacy J; Wilmington, Shameika R; Martin, Melinda J; Skopec, Hannah M; Lovander, Kaylee E; Pinto, Belinda S; Geyer, Pamela K

2014-06-01

The nuclear lamina is an extensive protein network that contributes to nuclear structure and function. LEM domain (LAP2, emerin, MAN1 domain, LEM-D) proteins are components of the nuclear lamina, identified by a shared ∼45-amino-acid motif that binds Barrier-to-autointegration factor (BAF), a chromatin-interacting protein. Drosophila melanogaster has three nuclear lamina LEM-D proteins, named Otefin (Ote), Bocksbeutel (Bocks), and dMAN1. Although these LEM-D proteins are globally expressed, loss of either Ote or dMAN1 causes tissue-specific defects in adult flies that differ from each other. The reason for such distinct tissue-restricted defects is unknown. Here, we generated null alleles of bocks, finding that loss of Bocks causes no overt adult phenotypes. Next, we defined phenotypes associated with lem-d double mutants. Although the absence of individual LEM-D proteins does not affect viability, loss of any two proteins causes lethality. Mutant phenotypes displayed by lem-d double mutants differ from baf mutants, suggesting that BAF function is retained in animals with a single nuclear lamina LEM-D protein. Interestingly, lem-d double mutants displayed distinct developmental and cellular mutant phenotypes, suggesting that Drosophila LEM-D proteins have developmental functions that are differentially shared with other LEM-D family members. This conclusion is supported by studies showing that ectopically produced LEM-D proteins have distinct capacities to rescue the tissue-specific phenotypes found in single lem-d mutants. Our findings predict that cell-specific mutant phenotypes caused by loss of LEM-D proteins reflect both the constellation of LEM-D proteins within the nuclear lamina and the capacity of functional compensation of the remaining LEM-D proteins. Copyright © 2014 by the Genetics Society of America.

Unique and Shared Functions of Nuclear Lamina LEM Domain Proteins in Drosophila

PubMed Central

Barton, Lacy J.; Wilmington, Shameika R.; Martin, Melinda J.; Skopec, Hannah M.; Lovander, Kaylee E.; Pinto, Belinda S.; Geyer, Pamela K.

2014-01-01

The nuclear lamina is an extensive protein network that contributes to nuclear structure and function. LEM domain (LAP2, emerin, MAN1 domain, LEM-D) proteins are components of the nuclear lamina, identified by a shared ∼45-amino-acid motif that binds Barrier-to-autointegration factor (BAF), a chromatin-interacting protein. Drosophila melanogaster has three nuclear lamina LEM-D proteins, named Otefin (Ote), Bocksbeutel (Bocks), and dMAN1. Although these LEM-D proteins are globally expressed, loss of either Ote or dMAN1 causes tissue-specific defects in adult flies that differ from each other. The reason for such distinct tissue-restricted defects is unknown. Here, we generated null alleles of bocks, finding that loss of Bocks causes no overt adult phenotypes. Next, we defined phenotypes associated with lem-d double mutants. Although the absence of individual LEM-D proteins does not affect viability, loss of any two proteins causes lethality. Mutant phenotypes displayed by lem-d double mutants differ from baf mutants, suggesting that BAF function is retained in animals with a single nuclear lamina LEM-D protein. Interestingly, lem-d double mutants displayed distinct developmental and cellular mutant phenotypes, suggesting that Drosophila LEM-D proteins have developmental functions that are differentially shared with other LEM-D family members. This conclusion is supported by studies showing that ectopically produced LEM-D proteins have distinct capacities to rescue the tissue-specific phenotypes found in single lem-d mutants. Our findings predict that cell-specific mutant phenotypes caused by loss of LEM-D proteins reflect both the constellation of LEM-D proteins within the nuclear lamina and the capacity of functional compensation of the remaining LEM-D proteins. PMID:24700158

Different metabolic profiles of K1 serotype and non-serotype K1 and K2 Klebsiella pneumoniae isolates in oral infection mice model.

PubMed

Chen, Nan; Wang, Lin-Lin; Xue, Juan; Ma, Xiang-Bo; Zhao, Sheng; Rong, Rui-Xue; Li, Hong-Quan; Ding, Liang; Zheng, Ming-Zhi; Chen, Ying-Ying; Duan, Fei; Shen, Yue-Liang

2014-10-01

K1 or K2 serotype Klebsiella pneumoniae isolate caused clinical pyogenic liver abscess (KLA) infection is prevalent in many areas. It has been identified that K1 or K2 serotype K. pneumoniae isolates caused KLA infection in mice by oral inoculation. In our study, K1 serotype K. pneumoniae isolate Kp1002 with hypermucoviscosity (HV)-positive phenotype caused KLA infection in C57BL/6 mice by oral inoculation. Simultaneously, non-serotype K1 and K2 isolate Kp1014 with HV-negative phenotype failed to cause KLA infection in the same manner. It seems that gastrointestinal tract translocation is the pathway by which K1 or K2 serotype K. pneumoniae caused KLA infection. Liquid chromatography-tandem mass spectrometry was used to further analyze metabolic profile changes in mice with KLA infection. Data showed that after Kp1002 or Kp1014 oral inoculation, serum Phosphatidylcholine (PC) and Lysophosphatidylcholine (LPC) levels significantly changed in mice. Some PC and LPC molecules showed changes both in the Kp1002 KLA group and the Kp1014 no-KLA group compared with the control group. The level of 18:1/18:2-PC significantly changed in the Kp1002 KLA group compared with the control group, but showed no change between the Kp1014 no-KLA group and the control group. The level of 18:1/18:2-PC might have been particularly affected by KLA infection caused by K1 serotype K. pneumoniae Kp1002. It may be a potential biomarker for KLA infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparative clinicopathological and cytomorphological analyses of peritoneal carcinomatosis associated with metastatic breast carcinoma and primary peritoneal/ovarian carcinoma in patients with a history of breast carcinoma.

PubMed

Na, Kiyong; Lee, Jung-Yun; Sung, Ji-Youn; Kim, Gun Min; Koo, Ja Seung; Kim, Hyun-Soo

2018-06-20

Causes of peritoneal carcinomatosis (PC) in patients with a history of breast carcinoma include both metastatic breast carcinoma (MBC) and primary peritoneal/ovarian carcinoma (PPOC). The origin of PC is important to determine the appropriate treatment strategy. Cytological examination of the peritoneal fluid (PF), which may be the first diagnostic approach to PC, is of distinct value in confirming the presence of malignant cells and determining the origin of PC. We analyzed the clinicopathological and cytomorphological characteristics of 33 patients with a history of breast carcinoma whose PF cytology contained malignant cells. Cases showing positive immunoreactivity for PAX8 and a lack of GATA3 expression were considered as PPOC. Sixteen patients developed PC caused by PPOC. PPOC patients were characterized by early-stage primary breast carcinoma, absence of non-peritoneal MBC before PC, and normal serum levels of CEA and CA15-3. Fourteen PPOC patients had pathogenic germline BRCA mutations. Cytological examination revealed that most of the PPOC cases had a dominant papillary arrangement of the tumor cells with severe nuclear pleomorphism, occasional bizarre nuclei, and atypical mitotic figures. Patients with PPOC who underwent cytoreductive surgery had a significantly longer survival time compared to those who did not, or MBC patients. In patients with a history of breast carcinoma presenting with PC, the presence of early-stage primary breast carcinoma, no prior non-peritoneal MBC, and a dominant papillary cellular arrangement pattern in the PF cytology were independent predictors of PPOC. Cytoreductive surgery significantly improved survival for patients with PPOC.

Prognostic significance of platelet count changes during hospitalization for community-acquired pneumonia.

PubMed

Gorelik, Oleg; Izhakian, Shimon; Barchel, Dana; Almoznino-Sarafian, Dorit; Tzur, Irma; Swarka, Muhareb; Beberashvili, Ilia; Feldman, Leonid; Cohen, Natan; Shteinshnaider, Miriam

2017-06-01

The prognostic significance of platelet count (PC) changes during hospitalization for community-acquired pneumonia (CAP) has not been investigated. For 976 adults, clinical data during hospitalization for CAP and all-cause mortality following discharge were compared according to ΔPC (PC on discharge minus PC on admission): groups A (declining PC, ΔPC < -50 × 10 9 /l), B (stable PC, ΔPC ± 50 × 10 9 /l), and C (rising PC, ΔPC >50 × 10 9 /l), and according to the presence of thrombocytopenia, normal PC, and thrombocytosis on admission/discharge. Groups A, B, and C comprised 7.9%, 46.5%, and 45.6% of patients, respectively. On hospital admission/discharge, thrombocytopenia, normal PC, and thrombocytosis were observed in 12.8%/6.4%, 84.1%/84.4%, and 3.1%/9.2% of patients, respectively. The respective 90-day, 3-year, and total (median follow-up of 54 months) mortality rates were significantly higher: in group A (40.3%, 63.6%, and 72.7%), compared to groups B (12.3%, 31.5%, and 39.0%) and C (4.9%, 17.3%, and 25.4%), p < 0.001; and in patients with thrombocytopenia at discharge (27.4%, 48.4%, and 51.6%), compared to those with normal PC (10.2%, 26.9%, and 35.4%) and thrombocytosis (8.9%, 17.8%, and 24.4%) at discharge (p < 0.001). Mortality rates were comparable among groups with thrombocytopenia, normal PC, and thrombocytosis at admission (p = 0.6). In the entire sample, each 100 × 10 9 /l increment of ΔPC strongly predicted lower mortality (p < 0.001, relative risk 0.73, 95% confidence interval 0.64-0.83). In conclusion, PC changes are common among CAP inpatients. Rising PC throughout hospitalization is a powerful predictor of better survival, while declining PC predicts poor outcome. Evaluation of PC changes during hospitalization for CAP may provide useful prognostic information.

Novel histone deacetylase inhibitor AR-42 exhibits antitumor activity in pancreatic cancer cells by affecting multiple biochemical pathways.

PubMed

Chen, Yi-Jin; Wang, Wen-Hung; Wu, Wan-Yu; Hsu, Chia-Chi; Wei, Ling-Rung; Wang, Sheng-Fan; Hsu, Ya-Wen; Liaw, Chih-Chuang; Tsai, Wan-Chi

2017-01-01

Pancreatic cancer is one of the most lethal types of cancer with a 5-year survival rate of ~5%. Histone deacetylases (HDACs) participate in many cellular processes, including carcinogenesis, and pharmacological inhibition of HDACs has emerged as a potential therapeutic strategy. In this study, we explored antitumor activity of the novel HDAC inhibitor AR-42 in pancreatic cancer. Human pancreatic cancer cell lines BxPC-3 and PANC-1 were used in this study. Real-time PCR, RT-PCR, and western blotting were employed to investigate expression of specific genes and proteins, respectively. Translocation of apoptosis-inducing factor was investigated by immunofluorescence and subcellular fractionation. The number of apoptotic cells, cell cycle stages, and reactive oxygen species (ROS) generation levels were determined by flow cytometry. Cell invasiveness was examined by the Matrigel invasion assay. Efficacy of AR-42 in vivo was evaluated by utilizing BxPC-3 xenograft mouse model. AR-42 inhibited pancreatic cancer cell proliferation by causing G2/M cell cycle arrest via regulating expression levels of genes and proteins involved in cell cycle. AR-42 also induced ROS generation and DNA damage, triggering apoptosis of pancreatic cancer cells via both caspase-3-dependent and caspase-3-independent pathways. In addition, AR-42 increased expression levels of negative regulators of p53 (miR-125b, miR-30d, and miR33), which could contribute to lower expression level of mutant p53 in pancreatic cancer cells. Cell invasion assay showed that AR-42 reduced cancer cell aggressiveness and significantly diminished BxPC-3 xenograft tumor growth in vivo. AR-42, a novel HDAC inhibitor, inhibited pancreatic cancer cells by regulating p53 expression, inducing cell cycle arrest, particularly at the G2/M stage, and activating multiple apoptosis pathways. Additionally, AR-42 inhibited cell invasiveness and potently suppressed pancreatic cancer tumors in vivo. We conclude that by virtue of its multiple mechanisms of action, AR-42 possesses a considerable potential as an antitumor agent in pancreatic cancer.

Improvement in motor performance of Weaver mutant mice following lesions of the cerebellum.

PubMed

Grüsser, C; Grüsser-Cornehls, U

1998-12-01

In Weaver mutants (B6CBA wv/wv) cerebellar granule cells degenerate almost completely postnatally. A partial loss of Purkinje cells (PC) and a degeneration of dopaminergic cells in the substantia nigra have also been found. Weaver mice suffer from striking motor symptoms, including difficulty in walking without toppling over. In an attempt to influence the poor motor performance, the cerebellum in young animals was removed, thus eliminating the faulty output of surviving PCs, demonstrated electrophysiologically. Unoperated Weaver, lesioned wildtypes and one sham mouse were used as controls. Before and after operation, a battery of behavioural tests was performed. In Weaver mice, tumbling to the side (t) and the relation of t to the motor activity (k) while traversing an open-field matrix, (t/k), improved considerably, as did manoeuvring on a slanted wire mesh, but keeping balance on a wooden bench did not to the same degree. Locomotor activity alone improved in some animals. In wildtypes no significant changes occurred after operation, with the exception of a strong reduction in locomotor activity. The experiments demonstrate that the motor performance in Weaver mutant mice benefits from removal of their cerebellum.

Glycyl-alanyl-histidine protects PC12 cells against hydrogen peroxide toxicity.

PubMed

Shimura, Hideki; Tanaka, Ryota; Shimada, Yoshiaki; Yamashiro, Kazuo; Hattori, Nobutaka; Urabe, Takao

2017-11-22

Peptides with cytoprotective functions, including antioxidants and anti-infectives, could be useful therapeutics. Carnosine, β-alanine-histidine, is a dipeptide with anti-oxidant properties. Tripeptides of Ala-His-Lys, Pro-His-His, or Tyr-His-Tyr are also of interest in this respect. We synthesized several histidine-containing peptides including glycine or alanine, and tested their cytoprotective effects on hydrogen peroxide toxicity for PC12 cells. Of all these peptides (Gly-His-His, Ala-His-His, Ala-His-Ala, Ala-Ala-His, Ala-Gly-His, Gly-Ala-His (GAH), Ala-His-Gly, His-Ala-Gly, His-His-His, Gly-His-Ala, and Gly-Gly-His), GAH was found to have the strongest cytoprotective activity. GAH decreased lactate dehydrogenase (LDH) leakage, apoptosis, morphological changes, and nuclear membrane permeability changes against hydrogen peroxide toxicity in PC12 cells. The cytoprotective activity of GAH was superior to that of carnosine against hydrogen peroxide toxicity in PC12 cells. GAH also protected PC12 cells against damage caused by actinomycin D and staurosporine. Additionally, it was found that GAH also protected SH-SY5Y and Jurkat cells from damage caused by hydrogen peroxide, as assessed by LDH leakage. Thus, a novel tripeptide, GAH, has been identified as having broad cytoprotective effects against hydrogen peroxide-induced cell damage.

Mitochondrial targets of photodynamic therapy and their contribution to cell death

NASA Astrophysics Data System (ADS)

Oleinick, Nancy L.; Usuda, Jitsuo; Xue, Liang-yan; Azizuddin, Kashif; Chiu, Song-mao; Lam, Minh C.; Morris, Rachel L.; Nieminen, Anna-Liisa

2002-06-01

In response to photodynamic therapy (PDT), many cells in culture or within experimental tumors are eliminated by apoptosis. PDT with photosensitizers that localize in or target mitochondria, such as the phthalocyanine Pc 4, causes prompt release of cytochrome c into the cytoplasm and activation of caspases-9 and -3, among other caspases, that are responsible for initiating cell degradation. Some cells appear resistant to apoptosis after PDT; however, if they have sustained sufficient damage, they will die by a necrotic process or through a different apoptotic pathway. In the case of PDT, the distinction between apoptosis and necrosis may be less important than the mechanism that triggers both processes, since critical lethal damage appears to occur during treatment and does not require the major steps in apoptosis to be expressed. We earlier showed, for example, that human breast cancer MCF-7 cells that lack caspase-3 are resistant to the induction of apoptosis by PDT, but are just as sensitive to the loss of clonogenicity as MCF-7 cells stably expressing transfected procaspase-3. Many photosensitizers that target mitochondria specifically attack the anti-apoptotic protein Bcl-2, generating a variety of crosslinked and cleaved photoproducts. Recent evidence suggests that the closely related protein Bcl-xL is also a target of Pc 4-PDT. Transient transfection of an expression vector encoding deletion mutants of Bcl-2 have identified the critical sensitive site in the protein that is required for photodamage. This region contains two alpha helices that form a secondary membrane anchorage site and are thought to be responsible for pore formation by Bcl-2. As specific protein targets are identified, we are becoming better able to model the critical events in PDT-induced cell death.

Diet, Lifestyles, Family History, and Prostate Cancer Incidence in an East Algerian Patient Group

PubMed Central

Lassed, Somia; Deus, Cláudia M.; Lourenço, Nuno; Dahdouh, Abderrezak

2016-01-01

Prostate cancer (PC) is the fourth most common cancer in men and the sixth leading cause of death in Algeria. To examine the relationship between lifestyle factors, including diet, and family history and PC risk, a case-control study was performed in an eastern Algerian population, comprising 90 patients with histologically confirmed PC and 190 controls. Data collection was carried out through a structured questionnaire and statistical analysis was performed to evaluate the different variables. The data showed that consumption of lamb and beef meat and high intake of animal fat and dairy products increased PC risk. Seven to thirteen vegetables servings per week and fourteen or more servings decreased PC risk by 62% and 96%, respectively. Seven to fourteen fruit servings per week decrease PC risk by 98%. Green tea consumption reduced the risk of PC but the results were statistically borderline. Increased risk was observed for individuals with family history of PC in first and in second degree. A positive strong association was also found for alcohol and smoking intake and a dose-response relationship existed for quantity and history of smoking. This study suggests that dietary habits, lifestyle factors, and family history have influence on the development of PC in Algerian population. PMID:27975054

Diet, Lifestyles, Family History, and Prostate Cancer Incidence in an East Algerian Patient Group.

PubMed

Lassed, Somia; Deus, Cláudia M; Lourenço, Nuno; Dahdouh, Abderrezak; Rizvanov, Albert A; Oliveira, Paulo J; Zama, Djamila

2016-01-01

Prostate cancer (PC) is the fourth most common cancer in men and the sixth leading cause of death in Algeria. To examine the relationship between lifestyle factors, including diet, and family history and PC risk, a case-control study was performed in an eastern Algerian population, comprising 90 patients with histologically confirmed PC and 190 controls. Data collection was carried out through a structured questionnaire and statistical analysis was performed to evaluate the different variables. The data showed that consumption of lamb and beef meat and high intake of animal fat and dairy products increased PC risk. Seven to thirteen vegetables servings per week and fourteen or more servings decreased PC risk by 62% and 96%, respectively. Seven to fourteen fruit servings per week decrease PC risk by 98%. Green tea consumption reduced the risk of PC but the results were statistically borderline. Increased risk was observed for individuals with family history of PC in first and in second degree. A positive strong association was also found for alcohol and smoking intake and a dose-response relationship existed for quantity and history of smoking. This study suggests that dietary habits, lifestyle factors, and family history have influence on the development of PC in Algerian population.

Characterization of the PcCdc42 small G protein from Pneumocystis carinii, which interacts with the PcSte20 life cycle regulatory kinase

PubMed Central

Krajicek, Bryan J.; Kottom, Theodore J.; Villegas, Leah

2010-01-01

Pneumocystis carinii (Pc) causes severe pneumonia in immunocompromised hosts. The binding of Pc trophic forms to alveolar epithelial cells is a central feature of infection, inducing the expression and activation of PcSte20, a gene participating in mating, proliferation, and pseudohyphal growth. In related fungi, Ste20 proteins are generally activated by immediate upstream small G proteins of the Cdc42-like family. PcCdc42 has not been previously described in Pneumocystis. To address the potential role of such a G protein in Pneumocystis, PcCdc42 was cloned from a Pc cDNA library. Using the full-length 576-bp PcCdc42 cDNA sequence, a CHEF blot of genomic DNA yielded a single band, providing evidence that this gene is present as a single copy within the genome. The total length of PcCdc42 cDNA was 576 bp with an estimated molecular mass of ∼38 kDa. BLASTP analysis demonstrated greater than 80% homology with other fungal Cdc42p proteins. Northern analysis indicated equal mRNA expression in both cystic and trophic life forms. Heterologous expression of PcCdc42 in Saccharomyces cerevisiae (Sc) demonstrated that PcCdc42p was able to restore growth in an ScCdc42Δ yeast strain. Additional assays with purified PcCdc42 protein demonstrated GTP binding and intrinsic GTPase activity, which was partially but significantly suppressed by Clostridium difficile toxin B, characteristic of Cdc42 GTPases. Furthermore, PcCdc42 protein was also shown to bind to the downstream PCSte20 kinase partner in the presence (but not the absence) of GTP. These data indicate that Pc possesses a Cdc42 gene expressing an active G protein, which binds the downstream regulatory kinase PcSte20, important in Pc life cycle regulation. PMID:19915161

Delayed Triplet-State Formation through Hybrid Charge Transfer Exciton at Copper Phthalocyanine/GaAs Heterojunction.

PubMed

Lim, Heeseon; Kwon, Hyuksang; Kim, Sang Kyu; Kim, Jeong Won

2017-10-05

Light absorption in organic molecules on an inorganic substrate and subsequent electron transfer to the substrate create so-called hybrid charge transfer exciton (HCTE). The relaxation process of the HCTE states largely determines charge separation efficiency or optoelectronic device performance. Here, the study on energy and time-dispersive behavior of photoelectrons at the hybrid interface of copper phthalocyanine (CuPc)/p-GaAs(001) upon light excitation of GaAs reveals a clear pathway for HCTE relaxation and delayed triplet-state formation. According to the ground-state energy level alignment at the interface, CuPc/p-GaAs(001) shows initially fast hole injection from GaAs to CuPc. Thus, the electrons in GaAs and holes in CuPc form an unusual HCTE state manifold. Subsequent electron transfer from GaAs to CuPc generates the formation of the triplet state in CuPc with a few picoseconds delay. Such two-step charge transfer causes delayed triplet-state formation without singlet excitation and subsequent intersystem crossing within the CuPc molecules.

Curcumin AntiCancer Studies in Pancreatic Cancer.

PubMed

Bimonte, Sabrina; Barbieri, Antonio; Leongito, Maddalena; Piccirillo, Mauro; Giudice, Aldo; Pivonello, Claudia; de Angelis, Cristina; Granata, Vincenza; Palaia, Raffaele; Izzo, Francesco

2016-07-16

Pancreatic cancer (PC) is one of the deadliest cancers worldwide. Surgical resection remains the only curative therapeutic treatment for this disease, although only the minority of patients can be resected due to late diagnosis. Systemic gemcitabine-based chemotherapy plus nab-paclitaxel are used as the gold-standard therapy for patients with advanced PC; although this treatment is associated with a better overall survival compared to the old treatment, many side effects and poor results are still present. Therefore, new alternative therapies have been considered for treatment of advanced PC. Several preclinical studies have demonstrated that curcumin, a naturally occurring polyphenolic compound, has anticancer effects against different types of cancer, including PC, by modulating many molecular targets. Regarding PC, in vitro studies have shown potent cytotoxic effects of curcumin on different PC cell lines including MiaPaCa-2, Panc-1, AsPC-1, and BxPC-3. In addition, in vivo studies on PC models have shown that the anti-proliferative effects of curcumin are caused by the inhibition of oxidative stress and angiogenesis and are due to the induction of apoptosis. On the basis of these results, several researchers tested the anticancer effects of curcumin in clinical trials, trying to overcome the poor bioavailability of this agent by developing new bioavailable forms of curcumin. In this article, we review the results of pre-clinical and clinical studies on the effects of curcumin in the treatment of PC.

Curcumin AntiCancer Studies in Pancreatic Cancer

PubMed Central

Bimonte, Sabrina; Barbieri, Antonio; Leongito, Maddalena; Piccirillo, Mauro; Giudice, Aldo; Pivonello, Claudia; de Angelis, Cristina; Granata, Vincenza; Palaia, Raffaele; Izzo, Francesco

2016-01-01

Pancreatic cancer (PC) is one of the deadliest cancers worldwide. Surgical resection remains the only curative therapeutic treatment for this disease, although only the minority of patients can be resected due to late diagnosis. Systemic gemcitabine-based chemotherapy plus nab-paclitaxel are used as the gold-standard therapy for patients with advanced PC; although this treatment is associated with a better overall survival compared to the old treatment, many side effects and poor results are still present. Therefore, new alternative therapies have been considered for treatment of advanced PC. Several preclinical studies have demonstrated that curcumin, a naturally occurring polyphenolic compound, has anticancer effects against different types of cancer, including PC, by modulating many molecular targets. Regarding PC, in vitro studies have shown potent cytotoxic effects of curcumin on different PC cell lines including MiaPaCa-2, Panc-1, AsPC-1, and BxPC-3. In addition, in vivo studies on PC models have shown that the anti-proliferative effects of curcumin are caused by the inhibition of oxidative stress and angiogenesis and are due to the induction of apoptosis. On the basis of these results, several researchers tested the anticancer effects of curcumin in clinical trials, trying to overcome the poor bioavailability of this agent by developing new bioavailable forms of curcumin. In this article, we review the results of pre-clinical and clinical studies on the effects of curcumin in the treatment of PC. PMID:27438851

Quantifying the relative amounts of PrP polymorphisms present in prions isolated from heterozygous prion-infected animals

USDA-ARS?s Scientific Manuscript database

Prions cause protein misfolding diseases, such as transmissible spongiform encephalopathy. They propagate infections by converting a normal cellular prion protein into a prion (PrPSc). PrPC and PrPSc are isosequential and differ only in their respective conformations. PrPC is monomeric and sensit...

Two novel de novo mutations of KRT6A and KRT16 genes in two Chinese pachyonychia congenita pedigrees with fissured tongue or diffuse plantar keratoderma.

PubMed

Du, Zhen-Fang; Xu, Chen-Ming; Zhao, Yan; Liu, Wen-Ting; Chen, Xiao-Ling; Chen, Chun-Yue; Fang, Hong; Ke, Hai-Ping; Zhang, Xian-Ning

2012-01-01

Mutations in the KRT6A or KRT16 gene cause pachyonychia congenita type 1 (PC-1), while mutations in KRT16 or KRT6C underlie focal palmoplantar keratoderma (FPPK). A new classification system of PC has been adopted based on the mutated gene. PC rarely presents the symptoms of diffuse plantar keratoderma. Mutation in the tail domain of keratins is rarely reported. PC combined with fissured tongue has never been described. To investigate the genotype-phenotype correlations between clinical features and gene mutational sites in two unrelated southern Chinese PC pedigrees (one family presented with specific fissured tongue, the other with diffuse plantar keratoderma). The whole coding regions of the KRT6A/KRT16/KRT17/KRT6B genes were amplified and directly sequenced to detect the mutation. To confirm the effect of the IVS8-2A>C mutation in KRT6A at the mRNA level, total RNA from the plantar lesion of a patient was extracted and reverse-transcribed to cDNA for sequence analysis. Two novel de novo mutations, a splice acceptor site variant IVS8-2A>C (p.S487FfsX72) in KRT6A and a heterozygous substitution c.AA373_374GG (p.N125G) within exon 1 of KRT16, were found separately in the two PC families. Genotype-phenotype correlations among PC patients with codon-125 mutation in KRT16 were established, while the phenotypes caused by the IVS8-2A>C mutation in KRT6A need further studies to confirm the rare feature of fissured tongue.

A cytoplasmically transmissible hypovirulence phenotype associated with mitochondrial DNA mutations in the chestnut blight fungus Cryphonectria parasitica.

PubMed Central

Monteiro-Vitorello, C B; Bell, J A; Fulbright, D W; Bertrand, H

1995-01-01

Mutations causing mitochondrial defects were induced in a virulent strain of the chestnut blight fungus Cryphonectria parasitica (Murr.) Barr. Virulence on apples and chestnut trees was reduced in four of six extensively characterized mutants. Relative to the virulent progenitor, the attenuated mutants had reduced growth rates, abnormal colony morphologies, and few asexual spores, and they resembled virus-infected strains. The respiratory defects and attenuated virulence phenotypes (hypovirulence) were transmitted from two mutants to a virulent strain by hyphal contact. The infectious transmission of hypovirulence occurred independently of the transfer of nuclei, did not involve a virus, and dynamically reflects fungal diseases caused by mitochondrial mutations. In these mutants, mitochondrial mutations are further implicated in generation of the attenuated state by (i) uniparental (maternal) inheritance of the trait, (ii) presence of high levels of cyanide-insensitive mitochondrial alternative oxidase activity, (iii) cytochrome deficiencies, and (iv) structural abnormalities in the mtDNA. Hence, cytoplasmically transmissible hypovirulence phenotypes found in virus-free strains of C. parasitica from recovering trees may be caused by mutant forms of mtDNA. Images Fig. 2 Fig. 4 PMID:11607549

Structural Factors and Mechanisms Underlying the Improved Photodynamic Cell Killing with Silicon Phthalocyanine Photosensitizers Directed to Lysosomes Versus Mitochondria

PubMed Central

Rodriguez, Myriam E.; Zhang, Ping; Azizuddin, Kashif; Delos Santos, Grace B.; Chiu, Song-mao; Xue, Liang-yan; Berlin, Jeffery C.; Peng, Xinzhan; Wu, Hongqiao; Lam, Minh; Nieminen, Anna-Liisa; Kenney, Malcolm E.; Oleinick, Nancy L.

2012-01-01

The phthalocyanine photosensitizer Pc 4 has been shown to bind preferentially to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components, especially Bcl-2, are photodamaged and apoptosis, as indicated by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, is triggered. A series of analogs of Pc 4 were synthesized, and the results demonstrate that Pcs with the aminopropylsiloxy ligand of Pc 4 or a similar one on one side of the Pc ring and a second large axial ligand on the other side of the ring have unexpected properties, including enhanced cell uptake, greater monomerization resulting in greater intracellular fluorescence and three-fold higher affinity constants for liposomes. The hydroxyl-bearing axial ligands tend to reduce aggregation of the Pc and direct it to lysosomes, resulting in four to six times more killing of cells, as defined by loss of clonogenicity, than with Pc 4. Whereas Pc 4-PDT photodamages Bcl-2 and Bcl-xL, Pc 181-PDT causes much less photodamage to Bcl-2 over the same dose–response range relative to cell killing, with earlier cleavage of Bid and slower caspase-3-dependent apoptosis. Therefore, within this series of photosensitizers, these hydroxyl-bearing axial ligands are less aggregated than is Pc 4, tend to localize to lysosomes and are more effective in overall cell killing than is Pc 4, but induce apoptosis more slowly and by a modified pathway. PMID:19508642

Mutant TDP-43 within motor neurons drives disease onset but not progression in amyotrophic lateral sclerosis.

PubMed

Ditsworth, Dara; Maldonado, Marcus; McAlonis-Downes, Melissa; Sun, Shuying; Seelman, Amanda; Drenner, Kevin; Arnold, Eveline; Ling, Shuo-Chien; Pizzo, Donald; Ravits, John; Cleveland, Don W; Da Cruz, Sandrine

2017-06-01

Mutations in TDP-43 cause amyotrophic lateral sclerosis (ALS), a fatal paralytic disease characterized by degeneration and premature death of motor neurons. The contribution of mutant TDP-43-mediated damage within motor neurons was evaluated using mice expressing a conditional allele of an ALS-causing TDP-43 mutant (Q331K) whose broad expression throughout the central nervous system mimics endogenous TDP-43. TDP-43 Q331K mice develop age- and mutant-dependent motor deficits from degeneration and death of motor neurons. Cre-recombinase-mediated excision of the TDP-43 Q331K gene from motor neurons is shown to delay onset of motor symptoms and appearance of TDP-43-mediated aberrant nuclear morphology, and abrogate subsequent death of motor neurons. However, reduction of mutant TDP-43 selectively in motor neurons did not prevent age-dependent degeneration of axons and neuromuscular junction loss, nor did it attenuate astrogliosis or microgliosis. Thus, disease mechanism is non-cell autonomous with mutant TDP-43 expressed in motor neurons determining disease onset but progression defined by mutant acting within other cell types.

Distinct roles of two anaplerotic pathways in glutamate production induced by biotin limitation in Corynebacterium glutamicum.

PubMed

Sato, Hiroki; Orishimo, Keita; Shirai, Tomokazu; Hirasawa, Takashi; Nagahisa, Keisuke; Shimizu, Hiroshi; Wachi, Masaaki

2008-07-01

Corynebacterium glutamicum is a biotin auxotrophic bacterium in which glutamate production is induced under biotin-limited conditions. During glutamate production, anaplerotic reactions catalyzed by phosphoenolpyruvate carboxylase (PEPC) and a biotin-containing enzyme pyruvate carboxylase (PC) are believed to play an important role in supplying oxaloacetate in the tricarboxylic acid cycle. To understand the distinct roles of PEPC and PC on glutamate production by C. glutamicum, we observed glutamate production induced under biotin-limited conditions in the disruptants of the genes encoding PEPC (ppc) and PC (pyc), respectively. The pyc disruptant retained the ability to produce high amounts of glutamate, and lactate was simultaneously produced probably due to the increased intracellular pyruvate levels. On the other hand, the ppc knockout mutant could not produce glutamate. Additionally, glutamate production in the pyc disruptant was enhanced by overexpression of ppc rather than disruption of the lactate dehydrogenase gene (ldh), which is involved in lactate production. Metabolic flux analysis based on the 13C-labeling experiment and measurement of 13C-enrichment in glutamate using nuclear magnetic resonance spectroscopy revealed that the flux for anaplerotic reactions in the pyc disruptant was lower than that in the wild type, concomitantly increasing the flux for lactate formation. Moreover, overexpression of ppc increased this flux in both the pyc disruptant and the wild type. Our results suggest that the PEPC-catalyzed anaplerotic reaction is necessary for glutamate production induced under biotin-limited conditions, because PC is not active during glutamate production, and overexpression of ppc effectively enhances glutamate production under biotin-limited conditions.

Mutant FGF-23 responsible for autosomal dominant hypophosphatemic rickets is resistant to proteolytic cleavage and causes hypophosphatemia in vivo.

PubMed

Shimada, Takashi; Muto, Takanori; Urakawa, Itaru; Yoneya, Takashi; Yamazaki, Yuji; Okawa, Katsuya; Takeuchi, Yasuhiro; Fujita, Toshiro; Fukumoto, Seiji; Yamashita, Takeyoshi

2002-08-01

FGF-23 is involved in the pathogenesis of two similar hypophosphatemic diseases, autosomal dominant hypophosphatemic rickets/osteomalacia (ADHR) and tumor-induced osteomalacia (TIO). We have shown that the overproduction of FGF-23 by tumors causes TIO. In contrast, ADHR derives from missense mutations in FGF-23 gene. However, it has been unclear how those mutations affect phosphate metabolism. Therefore, we produced mutant as well as wild-type FGF-23 proteins and examined their biological activity. Western blot analysis using site-specific antibodies showed that wild-type FGF-23 secreted into conditioned media was partially cleaved between Arg(179) and Ser(180). In addition, further processing of the cleaved N-terminal portion was observed. In constrast, mutant FGF-23 proteins found in ADHR were resistant to the cleavage. In order to clarify which molecule has the biological activity to induce hypophosphatemia, we separated full-length protein, the N-terminal and C-terminal fragments of wild-type FGF-23. When the activity of each fraction was examined in vivo, only the full-length FGF-23 decreased serum phosphate. Mutant FGF-23 protein that was resistant to the cleavage also retained the activity to induce hypophosphatemia. The extent of hypophosphatemia induced by the single administration of either wild-type or the mutant full-length FGF-23 protein was similar. In addition, implantation of CHO cells expressing the mutant FGF-23 protein caused hypophosphatemia and the decrease of bone mineral content. We conclude that ADHR is caused by hypophosphatemic action of mutant full-length FGF-23 proteins that are resistant to the cleavage between Arg(179) and Ser(180).

Mitochondrial deficits and abnormal mitochondrial retrograde axonal transport play a role in the pathogenesis of mutant Hsp27-induced Charcot Marie Tooth Disease.

PubMed

Kalmar, Bernadett; Innes, Amy; Wanisch, Klaus; Kolaszynska, Alicia Koyen; Pandraud, Amelie; Kelly, Gavin; Abramov, Andrey Y; Reilly, Mary M; Schiavo, Giampietro; Greensmith, Linda

2017-09-01

Mutations in the small heat shock protein Hsp27, encoded by the HSPB1 gene, have been shown to cause Charcot Marie Tooth Disease type 2 (CMT-2) or distal hereditary motor neuropathy (dHMN). Protein aggregation and axonal transport deficits have been implicated in the disease. In this study, we conducted analysis of bidirectional movements of mitochondria in primary motor neuron axons expressing wild type and mutant Hsp27. We found significantly slower retrograde transport of mitochondria in Ser135Phe, Pro39Leu and Arg140Gly mutant Hsp27 expressing motor neurons than in wild type Hsp27 neurons, although anterograde movement velocities remained normal. Retrograde transport of other important cargoes, such as the p75 neurotrophic factor receptor was minimally altered in mutant Hsp27 neurons, implicating that axonal transport deficits primarily affect mitochondria and the axonal transport machinery itself is less affected. Investigation of mitochondrial function revealed a decrease in mitochondrial membrane potential in mutant Hsp27 expressing motor axons, as well as a reduction in mitochondrial complex 1 activity, increased vulnerability of mitochondria to mitochondrial stressors, leading to elevated superoxide release and reduced mitochondrial glutathione (GSH) levels, although cytosolic GSH remained normal. This mitochondrial redox imbalance in mutant Hsp27 motor neurons is likely to cause low level of oxidative stress, which in turn will contribute to, and indeed may be the underlying cause of the deficits in mitochondrial axonal transport. Together, these findings suggest that the mitochondrial abnormalities in mutant Hsp27-induced neuropathies may be a primary cause of pathology, leading to further deficits in the mitochondrial axonal transport and onset of disease. © The Author 2017. Published by Oxford University Press.

Mitochondrial deficits and abnormal mitochondrial retrograde axonal transport play a role in the pathogenesis of mutant Hsp27-induced Charcot Marie Tooth Disease

PubMed Central

Innes, Amy; Wanisch, Klaus; Kolaszynska, Alicia Koyen; Pandraud, Amelie; Kelly, Gavin; Abramov, Andrey Y.; Reilly, Mary M.; Schiavo, Giampietro; Greensmith, Linda

2017-01-01

Abstract Mutations in the small heat shock protein Hsp27, encoded by the HSPB1 gene, have been shown to cause Charcot Marie Tooth Disease type 2 (CMT-2) or distal hereditary motor neuropathy (dHMN). Protein aggregation and axonal transport deficits have been implicated in the disease. In this study, we conducted analysis of bidirectional movements of mitochondria in primary motor neuron axons expressing wild type and mutant Hsp27. We found significantly slower retrograde transport of mitochondria in Ser135Phe, Pro39Leu and Arg140Gly mutant Hsp27 expressing motor neurons than in wild type Hsp27 neurons, although anterograde movement velocities remained normal. Retrograde transport of other important cargoes, such as the p75 neurotrophic factor receptor was minimally altered in mutant Hsp27 neurons, implicating that axonal transport deficits primarily affect mitochondria and the axonal transport machinery itself is less affected. Investigation of mitochondrial function revealed a decrease in mitochondrial membrane potential in mutant Hsp27 expressing motor axons, as well as a reduction in mitochondrial complex 1 activity, increased vulnerability of mitochondria to mitochondrial stressors, leading to elevated superoxide release and reduced mitochondrial glutathione (GSH) levels, although cytosolic GSH remained normal. This mitochondrial redox imbalance in mutant Hsp27 motor neurons is likely to cause low level of oxidative stress, which in turn will contribute to, and indeed may be the underlying cause of the deficits in mitochondrial axonal transport. Together, these findings suggest that the mitochondrial abnormalities in mutant Hsp27-induced neuropathies may be a primary cause of pathology, leading to further deficits in the mitochondrial axonal transport and onset of disease. PMID:28595321

Negative differential resistance in nickel octabutoxy phthalocyanine and nickel octabutoxy phthalocyanine/graphene oxide ultrathin films

NASA Astrophysics Data System (ADS)

Sarkar, Arup; Suresh, K. A.

2018-04-01

We find negative differential resistance (NDR) at room temperature in ultrathin films of nickel (II) 1,4,8,11,15,18,22,25-octabutoxy-29H,31H-phthalocyanine [NiPc(OBu)8] deposited on highly ordered pyrolytic graphite (HOPG) substrate [NiPc(OBu)8/HOPG] and NiPc(OBu)8 on graphene oxide (GO) deposited on HOPG [NiPc(OBu)8/GO/HOPG]. For the NiPc(OBu)8/HOPG system, NiPc(OBu)8 was transferred four times onto HOPG by the Langmuir-Blodgett (LB) technique. We have prepared a stable Langmuir monolayer of amphiphilic GO at the air-water interface and transferred it onto HOPG by the LB technique. Further, the monolayer of NiPc(OBu)8 was transferred four times for good coverage on GO to obtain the NiPc(OBu)8/GO/HOPG system. The current-voltage characteristics were carried out using a current sensing atomic force microscope (CSAFM) with a platinum (Pt) tip that forms Pt/NiPc(OBu)8/HOPG and Pt/NiPc(OBu)8/GO/HOPG junctions. The CSAFM, UV-visible spectroscopy, and cyclic voltammetry studies show that the NDR effect occurs due to molecular resonant tunneling. In the Pt/NiPc(OBu)8/GO/HOPG junction, we find that due to the presence of GO, the features of NDR become more prominent. Also, GO causes a shift in NDR voltage towards a lower value in the negative bias direction. We attribute this behavior to the role of GO in injecting holes into the NiPc(OBu)8 film.

Mutant SOD1 in cell types other than motor neurons and oligodendrocytes accelerates onset of disease in ALS mice

PubMed Central

Yamanaka, Koji; Boillee, Severine; Roberts, Elizabeth A.; Garcia, Michael L.; McAlonis-Downes, Melissa; Mikse, Oliver R.; Cleveland, Don W.; Goldstein, Lawrence S. B.

2008-01-01

Dominant mutations in ubiquitously expressed superoxide dismutase (SOD1) cause familial ALS by provoking premature death of adult motor neurons. To test whether mutant damage to cell types beyond motor neurons is required for the onset of motor neuron disease, we generated chimeric mice in which all motor neurons and oligodendrocytes expressed mutant SOD1 at a level sufficient to cause fatal, early-onset motor neuron disease when expressed ubiquitously, but did so in a cellular environment containing variable numbers of non-mutant, non-motor neurons. Despite high-level mutant expression within 100% of motor neurons and oligodendrocytes, in most of these chimeras, the presence of WT non-motor neurons substantially delayed onset of motor neuron degeneration, increasing disease-free life by 50%. Disease onset is therefore non-cell autonomous, and mutant SOD1 damage within cell types other than motor neurons and oligodendrocytes is a central contributor to initiation of motor neuron degeneration. PMID:18492803

Cardiovascular Comorbidity and Mortality in Men With Prostate Cancer Treated With Brachytherapy-Based Radiation With or Without Hormonal Therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nanda, Akash, E-mail: akash.nanda@orlandohealth.com; Chen, Ming-Hui; Moran, Brian J.

Purpose: To assess the impact of coronary artery disease (CAD) risk factors and sequelae on the risk of all-cause mortality (ACM) in men treated for prostate cancer (PC). Methods and Materials: The study cohort comprised 5077 men with PC consecutively treated with curative intent between 1997 and 2006 at the Chicago Prostate Cancer Center. Cox and Fine and Gray's competing risks regression multivariable analyses were performed, assessing whether cardiovascular comorbidity impacted the risk of ACM and PC-specific mortality, respectively, adjusting for CAD risk factors (diabetes mellitus, hypercholesterolemia, or hypertension) and sequelae (congestive heart failure or myocardial infarction), age, year andmore » type of treatment, and known PC prognostic factors. Results: When compared with men with no comorbidity there was a significantly increased risk of ACM in men with congestive heart failure or myocardial infarction (adjusted hazard ratio [AHR] 1.96, P<.001) and in men with diabetes mellitus (AHR 1.60, P=.03) and hypertension (AHR 1.25, P=.04). In contrast, men with hypercholesterolemia had a similar risk of ACM (AHR 0.68, P=.17) when compared with men with no comorbidity. Other factors associated with a significantly increased risk of ACM included age (AHR 1.09, P<.001), prostate-specific antigen level (AHR 1.25, P=.008), and Gleason score 8-10 disease (AHR 1.71, P=.003). Cardiovascular comorbidity did not impact the risk of PC-specific mortality. Conclusions: In addition to age and unfavorable PC prognostic factors, select CAD risk factors and sequelae are associated with an increased risk of ACM in men treated for PC. These comorbidity prognostic factors predict time courses of mortality from competing causes, which may be factored into the decision-making process when considering management options for PC in a given individual.« less

Role of RpoS in virulence and stress tolerance of the plant pathogen Erwinia carotovora subsp. carotovora.

PubMed

Andersson, R A; Kõiv, V; Norman-Setterblad, C; Pirhonen, M

1999-12-01

The plant-pathogenic bacterium Erwinia carotovora subsp. carotovora causes plant disease mainly through a number of extracellular plant-cell-wall-degrading enzymes. In this study, the ability of an rpoS mutant of the Er. carotovora subsp. carotovora strain SCC3193 to infect plants and withstand environmental stress was characterized. This mutant was found to be sensitive to osmotic and oxidative stresses in vitro and to be deficient in glycogen accumulation. The production of extracellular enzymes in vitro was similar in the mutant and in the wild-type strains. However, the rpoS mutant caused more severe symptoms than the wild-type strain on tobacco plants and also produced more extracellular enzymes in planta, but did not grow to higher cell density in planta compared to the wild-type strain. When tested on plants with reduced catalase activities, which show higher levels of reactive oxygen species, the rpoS mutant was found to cause lower symptom levels and to have impaired growth. In addition, the mutant was unable to compete with the wild-type strain in planta and in vitro. These results suggest that a functional rpoS gene is needed mainly for survival in a competitive environment and during stress conditions, and not for effective infection of plants.

Variability of methacholine bronchoprovocation and the effect of inhaled corticosteroids in mild asthma

PubMed Central

Sumino, Kaharu; Sugar, Elizabeth A.; Irvin, Charles G.; Kaminsky, David A.; Shade, Dave; Wei, Christine Y.; Holbrook, Janet T.; Wise, Robert A.; Castro, Mario

2014-01-01

Background The methacholine challenge test quantifies airway hyper-responsiveness, which is measured by the provocative concentration of methacholine causing a 20% decrease in forced expiration volume in 1 second (PC20). The dose–response effect of inhaled corticosteroids (ICS) on PC20 has been inconsistent and within-patient variability of PC20 is not well established. Objectives To determine the effect of high- vs low-dose ICS on PC20 and within-patient variability in those with repeated measurements of PC20. Methods A randomized, double-masked, crossover trial was conducted in patients with asthma on controller medications with PC20 of 8 mg/mL or lower (n = 64) to evaluate the effect of high-dose (1,000 μg/d) vs low-dose (250 μg/d) fluticasone for 4 weeks on PC20. In addition, the variability of PC20 was assessed in participants who underwent 2 or 3 PC20 measurements on the same dose of ICS (n = 27) over a 4-week interval. Results Because there was a significant period effect, dose comparison of the change in PC20 was assessed in the first treatment period. There was no significant difference in the change in PC20 for high- vs low-dose ICS (39% vs 30% increase, respectively; P = .87). The within- and between-participant variances for log PC20 were 0.84 and 0.96, respectively, with an intra-class correlation of 0.53, and 37% of participants had more than 2 doubling dose changes in PC20 in those with repeated measurements. Conclusion The effect of ICS on PC20 is not dose dependent at fluticasone levels of 250 and 1,000 μg/d. Interpersonal variability for PC20 is large. A lack of precise measurements should be taken into account when interpreting any change in PC20. PMID:24507830

Amino Alcohol- (NPS-2143) and Quinazolinone-Derived Calcilytics (ATF936 and AXT914) Differentially Mitigate Excessive Signalling of Calcium-Sensing Receptor Mutants Causing Bartter Syndrome Type 5 and Autosomal Dominant Hypocalcemia

PubMed Central

Letz, Saskia; Haag, Christine; Schulze, Egbert; Frank-Raue, Karin; Raue, Friedhelm; Hofner, Benjamin; Mayr, Bernhard; Schöfl, Christof

2014-01-01

Introduction Activating calcium sensing receptor (CaSR) mutations cause autosomal dominant hypocalcemia (ADH) characterized by low serum calcium, inappropriately low PTH and relative hypercalciuria. Four activating CaSR mutations cause additional renal wasting of sodium, chloride and other salts, a condition called Bartter syndrome (BS) type 5. Until today there is no specific medical treatment for BS type 5 and ADH. We investigated the effects of different allosteric CaSR antagonists (calcilytics) on activating CaSR mutants. Methods All 4 known mutations causing BS type 5 and five ADH mutations were expressed in HEK 293T cells and receptor signalling was studied by measurement of intracellular free calcium in response to extracellular calcium ([Ca2+]o). To investigate the effect of calcilytics, cells were stimulated with 3 mM [Ca2+]o in the presence or absence of NPS-2143, ATF936 or AXT914. Results All BS type 5 and ADH mutants showed enhanced signalling activity to [Ca2+]o with left shifted dose response curves. In contrast to the amino alcohol NPS-2143, which was only partially effective, the quinazolinone calcilytics ATF936 and AXT914 significantly mitigated excessive cytosolic calcium signalling of all BS type 5 and ADH mutants studied. When these mutants were co-expressed with wild-type CaSR to approximate heterozygosity in patients, ATF936 and AXT914 were also effective on all mutants. Conclusion The calcilytics ATF936 and AXT914 are capable of attenuating enhanced cytosolic calcium signalling activity of CaSR mutations causing BS type 5 and ADH. Quinazolinone calcilytics might therefore offer a novel treatment option for patients with activating CaSR mutations. PMID:25506941

Identification of Biomarkers for PKD1 Using Urinary Exosomes

PubMed Central

Hogan, Marie C.; Bakeberg, Jason L.; Gainullin, Vladimir G.; Irazabal, Maria V.; Harmon, Amber J.; Lieske, John C.; Charlesworth, M. Cristine; Johnson, Kenneth L.; Madden, Benjamin J.; Zenka, Roman M.; McCormick, Daniel J.; Sundsbak, Jamie L.; Heyer, Christina M.; Torres, Vicente E.; Harris, Peter C.

2015-01-01

Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of ESRD. Affected individuals inherit a defective copy of either PKD1 or PKD2, which encode polycystin-1 (PC1) or polycystin-2 (PC2), respectively. PC1 and PC2 are secreted on urinary exosome-like vesicles (ELVs) (100-nm diameter vesicles), in which PC1 is present in a cleaved form and may be complexed with PC2. Here, label-free quantitative proteomic studies of urine ELVs in an initial discovery cohort (13 individuals with PKD1 mutations and 18 normal controls) revealed that of 2008 ELV proteins, 9 (0.32%) were expressed at significantly different levels in samples from individuals with PKD1 mutations compared to controls (P<0.03). In samples from individuals with PKD1 mutations, levels of PC1 and PC2 were reduced to 54% (P<0.02) and 53% (P<0.001), respectively. Transmembrane protein 2 (TMEM2), a protein with homology to fibrocystin, was 2.1-fold higher in individuals with PKD1 mutations (P<0.03). The PC1/TMEM2 ratio correlated inversely with height-adjusted total kidney volume in the discovery cohort, and the ratio of PC1/TMEM2 or PC2/TMEM2 could be used to distinguish individuals with PKD1 mutations from controls in a confirmation cohort. In summary, results of this study suggest that a test measuring the urine exosomal PC1/TMEM2 or PC2/TMEM2 ratio may have utility in diagnosis and monitoring of polycystic kidney disease. Future studies will focus on increasing sample size and confirming these studies. The data were deposited in the ProteomeXchange (identifier PXD001075). PMID:25475747

Palliative Care is Associated with Reduced Aggressive End-of-Life Care in Patients with Gastrointestinal Cancer.

PubMed

Merchant, Shaila J; Brogly, Susan B; Goldie, Craig; Booth, Christopher M; Nanji, Sulaiman; Patel, Sunil V; Lajkosz, Katherine; Baxter, Nancy N

2018-06-01

We examined the delivery of physician palliative care (PC) services and its association with aggressive end-of-life care (EOLC) in patients with gastrointestinal (GI) cancer in Ontario, Canada. All patients with primary cause of death from esophageal, gastric, colon, and anorectal cancer from January 2003 to December 2013 were identified. PC services within 2 years of death were classified: (1) any PC; (2) timing of first PC (≤ 7, 8-90, 91-180, and 181-730 days before death); and (3) intensity of PC measured by number of days used (1st-25th, 26th-50th, 51st-75th, and 76th-100th percentiles). Aggressive EOLC was defined as any of the following: chemotherapy, emergency department visits, hospital or intensive care unit (ICU) admissions (all ≤ 30 days of death), and death in hospital and in the ICU; these were combined as a composite outcome (any aggressive EOLC). The cohort included 34,630 patients, of whom 74% had at least one PC service. Timing of the first PC service varied: ≤ 7 (12%), 8-90 (42%), 91-180 (16%), and 181-730 (30%) days before death. Compared with patients not receiving PC, any PC was associated with a reduction in any aggressive EOLC (risk ratio [RR] 0.75, 95% confidence interval [CI] 0.74-0.76); this association was similar regardless of timing of the first PC service. The most dramatic reduction in aggressive EOLC occurred in patients who received the greatest number of days of PC (RR 0.65, 95% CI 0.63-0.67). The majority of patients received PC within 2 years of death. A larger number of days of PC was associated with a greater reduction in aggressive EOLC.

Reversed polarized delivery of an aquaporin-2 mutant causes dominant nephrogenic diabetes insipidus

PubMed Central

Kamsteeg, Erik-Jan; Bichet, Daniel G.; Konings, Irene B.M.; Nivet, Hubert; Lonergan, Michelle; Arthus, Marie-Françoise; van Os, Carel H.; Deen, Peter M.T.

2003-01-01

Vasopressin regulates body water conservation by redistributing aquaporin-2 (AQP2) water channels from intracellular vesicles to the apical surface of renal collecting ducts, resulting in water reabsorption from urine. Mutations in AQP2 cause autosomal nephrogenic diabetes insipidus (NDI), a disease characterized by the inability to concentrate urine. Here, we report a frame-shift mutation in AQP2 causing dominant NDI. This AQP2 mutant is a functional water channel when expressed in Xenopus oocytes. However, expressed in polarized renal cells, it is misrouted to the basolateral instead of apical plasma membrane. Additionally, this mutant forms heterotetramers with wild-type AQP2 and redirects this complex to the basolateral surface. The frame shift induces a change in the COOH terminus of AQP2, creating both a leucine- and a tyrosine-based motif, which cause the reversed sorting of AQP2. Our data reveal a novel cellular phenotype in dominant NDI and show that dominance of basolateral sorting motifs in a mutant subunit can be the molecular basis for disease. PMID:14662748

Melt crystallization of bisphenol A polycarbonate in PC/zinc sulfonated polystyrene ionomer blend

NASA Astrophysics Data System (ADS)

Xu, Liang

The effects of zinc sulfonated polystyrene ionomer (ZnSPS) on the melt crystallization of bisphenol A polycarbonate (PC) were investigated. Melt crystallization of pure PC is extremely slow due to its rigid chain. In the blend of PC and ZnSPS (PC-ZnSPS), the melt crystallization rate of PC can be enhanced. DSC was used to study the crystallization kinetics of PC in PC-ZnSPS blend. The crystallization of PC at 190°C increased in both partially miscible and miscible blends with ZnSPS. For PC-ZnSPS blend with same PC composition as 80%, the crystallization rate was affected by the sulfonation level of ZnSPS. The induction time of crystallization for a partially miscible blend PC-ZnSPS9.98 (80/20) was 40 minutes, and the crystallization reaches 27% crystallinity within 14 hrs. The induction time for pure PC with the same thermal history was more than 24 hrs. The crystal structure of PC crystal formed in PC-ZnSPS blend was studied by WAXD, which showed no difference from the reported WAXD pattern for pure PC. Molecular weight change of PC was found during the thermal annealing of PC-ZnSPS blend at 190°C, but molecular weight alone cannot explain the change of crystallization rate of PC in PC-ZnSPS blend. Discussion was made to address the mechanisms that are responsible for the crystallization rate enhancement of PC in PC-ZnSPS blend. In order to understand and elucidate the reason for the molecular weight change of PC in PC-ZnSPS blend and its effect on the crystallization of PC, TG, GPC and GC-MS were used to investigate the stability of PC-ZnSPS blend and mixtures of PC with sodium tosylate (NaTS), zinc tosylate (ZnTS) and sodium benzoate (NaBZ). ZnSPS, NaTS and ZnTS undergo desulfonation of the sulfonate group at temperatures above 350°C. The desulfonation process can destabilize PC and lower the maximum mass loss rate temperature of PC for more than 70°C. NaTS, ZnTS and NaBZ have quite different effect on the thermal stability of PC at temperatures below 250°C. NaBZ can significantly degrade PC both at 190°C and 250°C. PC does not show any molecular weight (M w) change in the presence of NaTS at 250°C and 190°C for up to 1hr and 16 hrs respectively. ZnTS can also cause Mw change of PC at 250°C and 190°C, but the changing of Mw of PC in the presence of ZnTS is less than that in the presence of NaBZ. The reason for the molecular weight change of PC in PC-ZnSPS blend can be explained based on Davis's ionic ester exchange reaction mechanism.

Production of cattle lacking prion protein

PubMed Central

Richt, Jürgen A; Kasinathan, Poothappillai; Hamir, Amir N; Castilla, Joaquin; Sathiyaseelan, Thillai; Vargas, Francisco; Sathiyaseelan, Janaki; Wu, Hua; Matsushita, Hiroaki; Koster, Julie; Kato, Shinichiro; Ishida, Isao; Soto, Claudio; Robl, James M; Kuroiwa, Yoshimi

2010-01-01

Prion diseases are caused by propagation of misfolded forms of the normal cellular prion protein PrPC, such as PrPBSE in bovine spongiform encephalopathy (BSE) in cattle and PrPCJD in Creutzfeldt-Jakob disease (CJD) in humans1. Disruption of PrPC expression in mice, a species that does not naturally contract prion diseases, results in no apparent developmental abnormalities2–5. However, the impact of ablating PrPC function in natural host species of prion diseases is unknown. Here we report the generation and characterization of PrPC-deficient cattle produced by a sequential gene-targeting system6. At over 20 months of age, the cattle are clinically, physiologically, histopathologically, immunologically and reproductively normal. Brain tissue homogenates are resistant to prion propagation in vitro as assessed by protein misfolding cyclic amplification7. PrPC-deficient cattle may be a useful model for prion research and could provide industrial bovine products free of prion proteins. PMID:17195841

A critical note on the IAGA-endorsed Polar Cap (PC) indices: excessive excursions in the real-time index values

NASA Astrophysics Data System (ADS)

Stauning, Peter

2018-04-01

The Polar Cap (PC) indices were approved by the International Association for Geomagnetism and Aeronomy (IAGA) in 2013 and made available at the web portal http://pcindex.org holding prompt (real-time) as well as archival index values. The present note provides the first reported examination of the validity of the IAGA-endorsed method to generate real-time PC index values. It is demonstrated that features of the derivation procedure defined by Janzhura and Troshichev (2011) may cause considerable excursions in the real-time PC index values compared to the final index values. In examples based on occasional downloads of index values, the differences between real-time and final values of PC indices were found to exceed 3 mV m-1, which is a magnitude level that may indicate (or hide) strong magnetic storm activity.

Acorus tatarinowii Schott extract protects PC12 cells from amyloid-beta induced neurotoxicity.

PubMed

An, Hong-Mei; Li, Guo-Wen; Lin, Chen; Gu, Chao; Jin, Miao; Sun, Wen-Xian; Qiu, Ming-Feng; Hu, Bing

2014-05-01

Amyloid-beta induced neurotoxicity has been identified as a major cause of Alzheimer's disease. Acorus tatarinowii Schott is one of the most frequently used Chinese herbs for Alzheimer's disease treatment. However, the effects of Acorus tatarinowii Schott on amyloid-beta mediated nerve cell damage remains unknown. In the present study, neuronal differentiated PC12 cells were used as a model to evaluate the effects of A. tatarinowii Schott extract (ATSE) against Abeta25-35 induced neurotoxicity. The results showed pretreatment with ATSE significantly protected PC12 cells from Abeta25-35 induced cell death, lactate dehydrogenase release, DNA damage, mitochondrial dysfunction and cytochrome c release from mitochondria. In addition, pretreatment with ATSE also significantly inhibited Abeta25-35 induced caspase-3 activation and reactive oxygen species generation in PC12 cells. These observations suggested that ATSE protects PC12 cells from amyloid-beta induced neurotoxicity.

Differential effects of divalent cations on elk prion protein fibril formation and stability

USDA-ARS?s Scientific Manuscript database

Misfolding of the normally folded prion protein of mammals (PrPC) into infectious fibrils causes a variety of different diseases, from scrapie in sheep to bovine spongiform encephalopathy in cattle to chronic wasting disease (CWD) in deer and elk. The misfolded form of PrPC, termed PrPSc, or in this...

Clinical and Molecular Features of Renal and Pheochromocytoma/Paraganglioma Tumor Association Syndrome (RAPTAS): Case Series and Literature Review.

PubMed

Casey, Ruth T; Warren, Anne Y; Martin, Jose Ezequiel; Challis, Benjamin G; Rattenberry, Eleanor; Whitworth, James; Andrews, Katrina A; Roberts, Thomas; Clark, Graeme R; West, Hannah; Smith, Philip S; Docquier, France M; Rodger, Fay; Murray, Vicki; Simpson, Helen L; Wallis, Yvonne; Giger, Olivier; Tran, Maxine; Tomkins, Susan; Stewart, Grant D; Park, Soo-Mi; Woodward, Emma R; Maher, Eamonn R

2017-11-01

The co-occurrence of pheochromocytoma (PC) and renal tumors was linked to the inherited familial cancer syndrome von Hippel-Lindau (VHL) disease more than six decades ago. Subsequently, other shared genetic causes of predisposition to renal tumors and to PC, paraganglioma (PGL), or head and neck paraganglioma (HNPGL) have been described, but case series of non-VHL-related cases of renal tumor and pheochromocytoma/paraganglioma tumor association syndrome (RAPTAS) are rare. To determine the clinical and molecular features of non-VHL RAPTAS by literature review and characterization of a case series. A review of the literature was performed and a retrospective study of referrals for investigation of genetic causes of RAPTAS. Literature review revealed evidence of an association, in addition to VHL disease, between germline mutations in SDHB, SDHC, SDHD, TMEM127, and MAX genes and RAPTAS [defined here as the co-occurrence of tumors from both classes (PC/PGL/HNPGL and renal tumors) in the same individual or in first-degree relatives]. In both the literature review and our case series of 22 probands with non-VHL RAPTAS, SDHB mutations were the most frequent cause of non-VHL RAPTAS. A genetic cause was identified in 36.3% (8/22) of kindreds. Renal tumors and PC/PGL/HNPGL tumors share common molecular features and their co-occurrence in an individual or family should prompt genetic investigations. We report a case of MAX-associated renal cell carcinoma and confirm the role of TMEM127 mutations with renal cell carcinoma predisposition. Copyright © 2017 Endocrine Society

Ordering of Zn-centered porphyrin and phthalocyanine on TiO2(011): STM studies

PubMed Central

Godlewski, Szymon; Such, Bartosz; Pawlak, Rémy; Hinaut, Antoine; Jöhr, Res; Glatzel, Thilo; Meyer, Ernst; Szymonski, Marek

2017-01-01

Zn(II)phthalocyanine molecules (ZnPc) were thermally deposited on a rutile TiO2(011) surface and on Zn(II)meso-tetraphenylporphyrin (ZnTPP) wetting layers at room temperature and after elevated temperature thermal processing. The molecular homo- and heterostructures were characterized by high-resolution scanning tunneling microscopy (STM) at room temperature and their geometrical arrangement and degree of ordering are compared with the previously studied copper phthalocyanine (CuPc) and ZnTPP heterostructures. It was found that the central metal atom may play some role in ordering and growth of phthalocyanine/ZnTPP heterostructures, causing differences in stability of upright standing ZnPc versus CuPc molecular chains at given thermal annealing conditions. PMID:28144569

Liquid chromatography mass spectrometry-based profiling of phosphatidylcholine and phosphatidylethanolamine in the plasma and liver of acetaminophen-induced liver injured mice.

PubMed

Ming, Ya-Nan; Zhang, Jing-Yi; Wang, Xiao-Lin; Li, Chun-Min; Ma, Si-Cong; Wang, Zheng-Yang; Liu, Xiao-Lin; Li, Xiao-Bo; Mao, Yi-Min

2017-08-14

Acetaminophen (APAP) overdose is one of the most common causes of acute liver failure in many countries. The aim of the study was to describe the profiling of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in the plasma and liver of Acetaminophen -induced liver injured mice. A time course study was carried out using C57BL/6 mice after intraperitoneal administration of 300 mg/kg Acetaminophen 1 h, 3 h, 6 h, 12 h and 24 h. A high-throughput liquid chromatography mass spectrometry (LC-MS) lipidomic method was utilized to detect phosphatidylcholine and phosphatidylethanolamine species in the plasma and liver. The expressions of phosphatidylcholine and phosphatidylethanolamine metabolism related genes in liver were detected by quantitative Reverse transcription polymerase chain reaction (qRT-PCR) and Western-blot. Following Acetaminophen treatment, the content of many PC and PE species in plasma increased from 1 h time point, peaked at 3 h or 6 h, and tended to return to baseline at 24 h time point. The relative contents of almost all PC species in liver decreased from 1 h, appeared to be lowest at 6 h, and then return to normality at 24 h, which might be partly explained by the suppression of phospholipases mRNA expressions and the induction of choline kinase (Chka) expression. Inconsistent with PC profile, the relative contents of many PE species in liver increased upon Acetaminophen treatment, which might be caused by the down-regulation of phosphatidylethanolamine N-methyltransferase (Pemt). Acetaminophen overdose induced dramatic change of many PC and PE species in plasma and liver, which might be caused by damaging hepatocytes and interfering the phospholipid metabolism in Acetaminophen -injured liver.

Immunization-induced perturbation of human blood plasma cell pool: progressive maturation, IL-6 responsiveness, and high PRDI-BF1/BLIMP1 expression are critical distinctions between antigen-specific and nonspecific plasma cells.

PubMed

González-García, Inés; Ocaña, Esther; Jiménez-Gómez, Gema; Campos-Caro, Antonio; Brieva, José A

2006-04-01

The present study shows that reimmunization with tetanus toxoid (tet) caused a transient increase of the human blood plasma cell (PC) pool, detectable from 6th to 15th day postboost, as well as the temporal alteration of several PC features. Labeling of specific PC with FITC-tet C fragment (tetC) allowed kinetics analysis of the tetC(+) and tetC(-) PC, and revealed remarkable differences between them: 1) the kinetics of tetC(+) PC occurrence was exponential, and most of them appeared in a narrow time frame (5th to 8th day postboost), whereas the tetC(-) PC increase was lower (three to five times) and more prolonged (4th to 15th day postboost). 2) The tetC(+) PC subset contained a fraction of cycling cells, expressed high levels of DR, CD138, and CD126, and responded to IL-6 by improving their survival and Ig secretion; in contrast, the tetC(-) PC showed higher CXCR4 and lower DR and CD138, did not respond to IL-6, and contained a fraction of apoptotic cells. 3) Sequential phenotypic analysis revealed maturational changes within the tetC(+), but not tetC(-), PC subset; sequencing of tetC(+) PC IgVH genes showed clear features of Ag selection. 4) The tetC(+) PC expressed several times more positive regulatory domain I- binding factor 1/B lymphocyte-induced maturation protein 1 transcription factor than the tetC(-) PC. 5) The tetC(-) PC and bone marrow resident PC similarly expressed low DR and high CXCR4, but differed in that the latter exhibited higher levels of CD31, CD138, and positive regulatory domain I- binding factor 1/B lymphocyte-induced maturation protein 1. These findings support the view that tetC(+) PC contain bone marrow PC precursors, and tetC(-) PC probably belong to a removable compartment of aged PC.

Partial wave spectroscopic microscopy can predict prostate cancer progression and mitigate over-treatment (Conference Presentation)

NASA Astrophysics Data System (ADS)

Zhang, Di; Graff, Taylor; Crawford, Susan; Subramanian, Hariharan; Thompson, Sebastian; Derbas, Justin R.; Lyengar, Radha; Roy, Hemant K.; Brendler, Charles B.; Backman, Vadim

2016-02-01

Prostate Cancer (PC) is the second leading cause of cancer deaths in American men. While prostate specific antigen (PSA) test has been widely used for screening PC, >60% of the PSA detected cancers are indolent, leading to unnecessary clinical interventions. An alternative approach, active surveillance (AS), also suffer from high expense, discomfort and complications associated with repeat biopsies (every 1-3 years), limiting its acceptance. Hence, a technique that can differentiate indolent from aggressive PC would attenuate the harms from over-treatment. Combining microscopy with spectroscopy, our group has developed partial wave spectroscopic (PWS) microscopy, which can quantify intracellular nanoscale organizations (e.g. chromatin structures) that are not accessible by conventional microscopy. PWS microscopy has previously been shown to predict the risk of cancer in seven different organs (N ~ 800 patients). Herein we use PWS measurement of label-free histologically-normal prostatic epithelium to distinguish indolent from aggressive PC and predict PC risk. Our results from 38 men with low-grade PC indicated that there is a significant increase in progressors compared to non-progressors (p=0.002, effect size=110%, AUC=0.80, sensitivity=88% and specificity=72%), while the baseline clinical characteristics were not significantly different. We further improved the diagnostic power by performing nuclei-specific measurements using an automated system that separates in real-time the cell nuclei from the remaining prostate epithelium. In the long term, we envision that the PWS based prognostication can be coupled with AS without any change to the current procedure to mitigate the harms caused by over-treatment.

Functional recovery in rat spinal cord injury induced by hyperbaric oxygen preconditioning.

PubMed

Lu, Pei-Gang; Hu, Sheng-Li; Hu, Rong; Wu, Nan; Chen, Zhi; Meng, Hui; Lin, Jiang-Kai; Feng, Hua

2012-12-01

It is a common belief that neurosurgical interventions can cause inevitable damage resulting from the procedure itself in surgery especially for intramedullary spinal cord tumors. The present study was designed to examine if hyperbaric oxygen preconditioning (HBO-PC) was neuroprotective against surgical injuries using a rat model of spinal cord injury (SCI). Sprague-Dawley rats were randomly divided into three groups: HBO-PC group, hypobaric hypoxic preconditioning (HH-PC) control group, and normobaric control group. All groups were subjected to SCI by weight drop device. Rats from each group were examined for neurological behavior and electrophysiological function. Tissue sections were analyzed by using immunohistochemistry, TdT-mediated dUTP-biotin nick end labeling, and axonal tract tracing. Significant neurological deficits were observed after SCI and HBO-PC and HH-PC improved neurological deficits 1 week post-injury. The latencies of motor-evoked potential and somatosensory-evoked potential were significantly delayed after SCI, which was attenuated by HBO-PC and HH-PC. Compared with normobaric control group, pretreatment with HBO and hypobaric hypoxia significantly reduced the number of TdT-mediated dUTP-biotin nick end labeling-positive cells, and increased nestin-positive cells. HBO-PC and HH-PC enhanced axonal growth after SCI. In conclusion, preconditioning with HBO and hypobaric hypoxia can facilitate functional recovery and suppress cell apoptosis after SCI and may prove to be a useful preventive strategy to neurosurgical SCI.

Cellular prion protein controls stem cell-like properties of human glioblastoma tumor-initiating cells.

PubMed

Corsaro, Alessandro; Bajetto, Adriana; Thellung, Stefano; Begani, Giulia; Villa, Valentina; Nizzari, Mario; Pattarozzi, Alessandra; Solari, Agnese; Gatti, Monica; Pagano, Aldo; Würth, Roberto; Daga, Antonio; Barbieri, Federica; Florio, Tullio

2016-06-21

Prion protein (PrPC) is a cell surface glycoprotein whose misfolding is responsible for prion diseases. Although its physiological role is not completely defined, several lines of evidence propose that PrPC is involved in self-renewal, pluripotency gene expression, proliferation and differentiation of neural stem cells. Moreover, PrPC regulates different biological functions in human tumors, including glioblastoma (GBM). We analyzed the role of PrPC in GBM cell pathogenicity focusing on tumor-initiating cells (TICs, or cancer stem cells, CSCs), the subpopulation responsible for development, progression and recurrence of most malignancies. Analyzing four GBM CSC-enriched cultures, we show that PrPC expression is directly correlated with the proliferation rate of the cells. To better define its role in CSC biology, we knocked-down PrPC expression in two of these GBM-derived CSC cultures by specific lentiviral-delivered shRNAs. We provide evidence that CSC proliferation rate, spherogenesis and in vivo tumorigenicity are significantly inhibited in PrPC down-regulated cells. Moreover, PrPC down-regulation caused loss of expression of the stemness and self-renewal markers (NANOG, Sox2) and the activation of differentiation pathways (i.e. increased GFAP expression). Our results suggest that PrPC controls the stemness properties of human GBM CSCs and that its down-regulation induces the acquisition of a more differentiated and less oncogenic phenotype.

Cellular prion protein controls stem cell-like properties of human glioblastoma tumor-initiating cells

PubMed Central

Corsaro, Alessandro; Bajetto, Adriana; Thellung, Stefano; Begani, Giulia; Villa, Valentina; Nizzari, Mario; Pattarozzi, Alessandra; Solari, Agnese; Gatti, Monica; Pagano, Aldo; Würth, Roberto; Daga, Antonio; Barbieri, Federica; Florio, Tullio

2016-01-01

Prion protein (PrPC) is a cell surface glycoprotein whose misfolding is responsible for prion diseases. Although its physiological role is not completely defined, several lines of evidence propose that PrPC is involved in self-renewal, pluripotency gene expression, proliferation and differentiation of neural stem cells. Moreover, PrPC regulates different biological functions in human tumors, including glioblastoma (GBM). We analyzed the role of PrPC in GBM cell pathogenicity focusing on tumor-initiating cells (TICs, or cancer stem cells, CSCs), the subpopulation responsible for development, progression and recurrence of most malignancies. Analyzing four GBM CSC-enriched cultures, we show that PrPC expression is directly correlated with the proliferation rate of the cells. To better define its role in CSC biology, we knocked-down PrPC expression in two of these GBM-derived CSC cultures by specific lentiviral-delivered shRNAs. We provide evidence that CSC proliferation rate, spherogenesis and in vivo tumorigenicity are significantly inhibited in PrPC down-regulated cells. Moreover, PrPC down-regulation caused loss of expression of the stemness and self-renewal markers (NANOG, Sox2) and the activation of differentiation pathways (i.e. increased GFAP expression). Our results suggest that PrPC controls the stemness properties of human GBM CSCs and that its down-regulation induces the acquisition of a more differentiated and less oncogenic phenotype. PMID:27229535

Normative and descriptive accounts of the influence of power and contingency on causal judgement.

PubMed

Perales, José C; Shanks, David R

2003-08-01

The power PC theory (Cheng, 1997) is a normative account of causal inference, which predicts that causal judgements are based on the power p of a potential cause, where p is the cause-effect contingency normalized by the base rate of the effect. In three experiments we demonstrate that both cause-effect contingency and effect base-rate independently affect estimates in causal learning tasks. In Experiment 1, causal strength judgements were directly related to power p in a task in which the effect base-rate was manipulated across two positive and two negative contingency conditions. In Experiments 2 and 3 contingency manipulations affected causal estimates in several situations in which power p was held constant, contrary to the power PC theory's predictions. This latter effect cannot be explained by participants' conflation of reliability and causal strength, as Experiment 3 demonstrated independence of causal judgements and confidence. From a descriptive point of view, the data are compatible with Pearce's (1987) model, as well as with several other judgement rules, but not with the Rescorla-Wagner (Rescorla & Wagner, 1972) or power PC models.

Identical substitutions in magnesium chelatase paralogs result in chlorophyll deficient soybean mutants

USDA-ARS?s Scientific Manuscript database

The soybean (Glycine max (L.) Merr.) chlorophyll deficient line MinnGold is a spontaneous mutant characterized by yellow foliage. Map-based cloning and transgenic complementation revealed that the mutant phenotype is caused by a non-synonymous nucleotide substitution in the third exon of a Mg-chelat...

The prion-ZIP connection: From cousins to partners in iron uptake

PubMed Central

Singh, Neena; Asthana, Abhishek; Baksi, Shounak; Desai, Vilok; Haldar, Swati; Hari, Sahi; Tripathi, Ajai K

2015-01-01

ABSTRACT Converging observations from disparate lines of inquiry are beginning to clarify the cause of brain iron dyshomeostasis in sporadic Creutzfeldt-Jakob disease (sCJD), a neurodegenerative condition associated with the conversion of prion protein (PrPC), a plasma membrane glycoprotein, from α-helical to a β-sheet rich PrP-scrapie (PrPSc) isoform. Biochemical evidence indicates that PrPC facilitates cellular iron uptake by functioning as a membrane-bound ferrireductase (FR), an activity necessary for the transport of iron across biological membranes through metal transporters. An entirely different experimental approach reveals an evolutionary link between PrPC and the Zrt, Irt-like protein (ZIP) family, a group of proteins involved in the transport of zinc, iron, and manganese across the plasma membrane. Close physical proximity of PrPC with certain members of the ZIP family on the plasma membrane and increased uptake of extracellular iron by cells that co-express PrPC and ZIP14 suggest that PrPC functions as a FR partner for certain members of this family. The connection between PrPC and ZIP proteins therefore extends beyond common ancestry to that of functional cooperation. Here, we summarize evidence supporting the facilitative role of PrPC in cellular iron uptake, and implications of this activity on iron metabolism in sCJD brains. PMID:26689487

APC-PC Combined Scheme in Gilbert Two State Model: Proposal and Study

NASA Astrophysics Data System (ADS)

Bulo, Yaka; Saring, Yang; Bhunia, Chandan Tilak

2017-04-01

In an automatic repeat request (ARQ) scheme, a packet is retransmitted if it gets corrupted due to transmission errors caused by the channel. However, an erroneous packet may contain both erroneous bits and correct bits and hence it may still contain useful information. The receiver may be able to combine this information from multiple erroneous copies to recover the correct packet. Packet combining (PC) is a simple and elegant scheme of error correction in transmitted packet, in which two received copies are XORed to obtain the bit location of erroneous bits. Thereafter, the packet is corrected by bit inversion of bit located as erroneous. Aggressive packet combining (APC) is a logic extension of PC primarily designed for wireless communication with objective of correcting error with low latency. PC offers higher throughput than APC, but PC does not correct double bit errors if occur in same bit location of erroneous copies of the packet. A hybrid technique is proposed to utilize the advantages of both APC and PC while attempting to remove the limitation of both. In the proposed technique, applications of APC-PC on Gilbert two state model has been studied. The simulation results show that the proposed technique offers better throughput than the conventional APC and lesser packet error rate than PC scheme.

Coronary revascularization and mortality in men with congestive heart failure or prior myocardial infarction who receive androgen deprivation.

PubMed

Nguyen, Paul L; Chen, Ming H; Goldhaber, Samuel Z; Martin, Neil E; Beard, Clair J; Dosoretz, Daniel E; Katin, Michael J; Ross, Rudi; Salenius, Sharon A; D'Amico, Anthony V

2011-01-15

A study was undertaken to determine the impact of prior coronary revascularization (angioplasty, stent, or coronary artery bypass graft) on the risk of all-cause mortality after neoadjuvant hormonal therapy (HT) for prostate cancer (PC) in men with a history of coronary artery disease (CAD)-induced congestive heart failure (CHF) or myocardial infarction (MI). Among 7839 men who received radiation with or without a median of 4 months of HT for PC from 1991 to 2006, 495 (6.3%) had CAD-induced CHF or MI and formed the study cohort. Of these men, 250 (50.5%) had been revascularized before treatment for PC. Cox regression was used to determine whether HT increased the risk of all-cause mortality, and whether revascularization altered this risk, after adjusting for known PC prognostic factors and a propensity score for revascularization. Median follow-up was 4.1 years. Neoadjuvant HT was associated with an increased risk of all-cause mortality (28.9% vs 15.7% at 5 years; adjusted hazard ratio [HR], 1.73; 95% confidence interval [CI], 1.13-2.64; P = .01). Men who received HT without revascularization had the highest risk of all-cause mortality (33.3%; adjusted HR, 1.48; 95% CI, 1.01-2.18; P = .047), whereas men who were revascularized and did not receive HT had the lowest risk of all-cause mortality (9.4%; adjusted HR, 0.51; 95% CI, 0.28-0.93; P = .028). The reference group had an intermediate risk of all-cause mortality (23.4%) and was comprised of men in whom HT use and revascularization were either both given or both withheld. In men with a history of CAD-induced CHF or MI, neoadjuvant HT is associated with an excess risk of mortality, which appears to be reduced but not eliminated by prior revascularization. Copyright © 2010 American Cancer Society.

PC4/Tis7/IFRD1 Stimulates Skeletal Muscle Regeneration and Is Involved in Myoblast Differentiation as a Regulator of MyoD and NF-κB*

PubMed Central

Micheli, Laura; Leonardi, Luca; Conti, Filippo; Maresca, Giovanna; Colazingari, Sandra; Mattei, Elisabetta; Lira, Sergio A.; Farioli-Vecchioli, Stefano; Caruso, Maurizia; Tirone, Felice

2011-01-01

In skeletal muscle cells, the PC4 (Tis7/Ifrd1) protein is known to function as a coactivator of MyoD by promoting the transcriptional activity of myocyte enhancer factor 2C (MEF2C). In this study, we show that up-regulation of PC4 in vivo in adult muscle significantly potentiates injury-induced regeneration by enhancing myogenesis. Conversely, we observe that PC4 silencing in myoblasts causes delayed exit from the cell cycle, accompanied by delayed differentiation, and we show that such an effect is MyoD-dependent. We provide evidence revealing a novel mechanism underlying the promyogenic actions of PC4, by which PC4 functions as a negative regulator of NF-κB, known to inhibit MyoD expression post-transcriptionally. In fact, up-regulation of PC4 in primary myoblasts induces the deacetylation, and hence the inactivation and nuclear export of NF-κB p65, in concomitance with induction of MyoD expression. On the contrary, PC4 silencing in myoblasts induces the acetylation and nuclear import of p65, in parallel with a decrease of MyoD levels. We also observe that PC4 potentiates the inhibition of NF-κB transcriptional activity mediated by histone deacetylases and that PC4 is able to form trimolecular complexes with p65 and HDAC3. This suggests that PC4 stimulates deacetylation of p65 by favoring the recruitment of HDAC3 to p65. As a whole, these results indicate that PC4 plays a role in muscle differentiation by controlling the MyoD pathway through multiple mechanisms, and as such, it positively regulates regenerative myogenesis. PMID:21127072

Theoretical and experimental analysis of analyte transport in a fiber-optic, protein C immuno-biosensor.

PubMed

Tang, Liang; Kwon, Hyun J; Kang, Kyung A

2004-12-30

Protein C (PC) is an important anticoagulant in human blood plasma, and early diagnosis of PC deficiency is critical for preventing dangerous thromboembolic complications. A fiber-optic PC immuno-biosensor has been under development in our research group for real-time PC-deficiency diagnosis. The sensor has demonstrated a good sensitivity and specificity for quantifying PC in buffered solutions. However, for plasma samples, with a limited sample reaction time, the sensor produced only 30% of the signal intensity of PC in buffer. The high plasma viscosity (1.9 cP) was speculated as the major reason for signal intensity reduction. In this investigation, the sensing performance of the fiber-optic PC biosensor is systematically characterized in terms of physical and chemical properties of the sample media. Theoretical and experimental analyses indicate that the reduced diffusion rate of PC molecules in viscous samples caused the sensing system to be more mass-transfer-limited. Convective flow of sample/reagent solutions during immunoreactions can increase the rate of the analyte mass transport from the bulk solution to the sensor surface, with reaction kinetics changing from mass-transfer-limited to reaction-limited as flow velocity increases. It was shown that PC sensor performance was significantly improved for plasma samples with convection. The effect of the flow velocity and incubation times for samples and reagents on the sensor performance was also systematically analyzed to optimize the assay protocol for PC sensing. Currently, a 6-cm-long immuno-biosensor is capable of quantifying PC in plasma (1 mL) in the heterozygous PC deficiency range (0.5 to 2.5 microg/mL) within 5 minutes, at an average signal-to-noise ratio of 50. 2004 Wiley Periodicals, Inc.

Clinical response of the novel activating ALK-I1171T mutation in neuroblastoma to the ALK inhibitor ceritinib.

PubMed

Guan, Jikui; Fransson, Susanne; Siaw, Joachim Tetteh T; Treis, Diana; Van den Eynden, Jimmy; Chand, Damini; Umapathy, Ganesh; Svenberg, Petter; Ruuth, Kristina; Wessman, Sandra; Shamikh, Alia; Jacobsson, Hans; Gordon, Lena; Stenman, Jakob; Larsson, Erik; Svensson, Par-Johan; Hansson, Magnus; Martinsson, Tommy; Kogner, Per; Palmer, Ruth H; Hallberg, Bengt

2018-06-15

Tumors with Anaplastic Lymphoma Kinase (ALK) fusion rearrangements, including non-small cell lung cancer and anaplastic large cell lymphoma, are highly sensitive to ALK tyrosine kinase inhibitors (TKIs), underscoring the notion that such cancers are addicted to ALK activity. While mutations in ALK are heavily implicated in childhood neuroblastoma, response to the ALK TKI crizotinib has been disappointing. Embryonal tumors in patients with DNA repair defects such as Fanconi anemia (FA) often have a poor prognosis, due to lack of therapeutic options. Here we report a child with underlying FA and ALK mutant high-risk neuroblastoma responding strongly to precision therapy with the ALK TKI ceritinib. Conventional chemotherapy treatment caused severe, life-threatening toxicity. Genomic analysis of the initial biopsy identified germ-line FANCA mutations as well as a novel ALK-I1171T variant. ALK-I1171T generates a potent gain-of-function mutant, as measured in PC12 cell neurite outgrowth and NIH3T3 transformation. Pharmacological inhibition profiling of ALK-I1171T in response to various ALK TKIs identified an 11-fold improved inhibition of ALK-I1171T with ceritinib when compared with crizotinib. Immunoaffinity-coupled LC-MS/MS phosphoproteomics analysis indicated a decrease in ALK signaling in response to ceritinib. Ceritinib was therefore selected for treatment in this child. Mono-therapy with ceritinib was well tolerated and resulted in normalized catecholamine markers and tumor shrinkage. After 7.5 months treatment, residual primary tumor was surgically removed and exhibited hallmarks of differentiation together with reduced Ki67 levels. Clinical follow-up after 21 months treatment revealed complete clinical remission including all metastatic sites. Therefore, ceritinib presents a viable therapeutic option for ALK-positive neuroblastoma. Cold Spring Harbor Laboratory Press.

Methods of producing protoporphyrin IX and bacterial mutants therefor

DOEpatents

Zhou, Jizhong; Qiu, Dongru; He, Zhili; Xie, Ming

2016-03-01

The presently disclosed inventive concepts are directed in certain embodiments to a method of producing protoporphyrin IX by (1) cultivating a strain of Shewanella bacteria in a culture medium under conditions suitable for growth thereof, and (2) recovering the protoporphyrin IX from the culture medium. The strain of Shewanella bacteria comprises at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX. In certain embodiments of the method, the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or of shew_1140. In other embodiments, the presently disclosed inventive concepts are directed to mutant strains of Shewanella bacteria having at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX during cultivation of the bacteria. In certain embodiments the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or shew_1140.

Starvation induced cell death in autophagy-defective yeast mutants is caused by mitochondria dysfunction.

PubMed

Suzuki, Sho W; Onodera, Jun; Ohsumi, Yoshinori

2011-02-25

Autophagy is a highly-conserved cellular degradation and recycling system that is essential for cell survival during nutrient starvation. The loss of viability had been used as an initial screen to identify autophagy-defective (atg) mutants of the yeast Saccharomyces cerevisiae, but the mechanism of cell death in these mutants has remained unclear. When cells grown in a rich medium were transferred to a synthetic nitrogen starvation media, secreted metabolites lowered the extracellular pH below 3.0 and autophagy-defective mutants mostly died. We found that buffering of the starvation medium dramatically restored the viability of atg mutants. In response to starvation, wild-type (WT) cells were able to upregulate components of the respiratory pathway and ROS (reactive oxygen species) scavenging enzymes, but atg mutants lacked this synthetic capacity. Consequently, autophagy-defective mutants accumulated the high level of ROS, leading to deficient respiratory function, resulting in the loss of mitochondria DNA (mtDNA). We also showed that mtDNA deficient cells are subject to cell death under low pH starvation conditions. Taken together, under starvation conditions non-selective autophagy, rather than mitophagy, plays an essential role in preventing ROS accumulation, and thus in maintaining mitochondria function. The failure of response to starvation is the major cause of cell death in atg mutants.

A novel familial mutation in the PCSK1 gene that alters the oxyanion hole residue of proprotein convertase 1/3 and impairs its enzymatic activity.

PubMed

Wilschanski, Michael; Abbasi, Montaser; Blanco, Elias; Lindberg, Iris; Yourshaw, Michael; Zangen, David; Berger, Itai; Shteyer, Eyal; Pappo, Orit; Bar-Oz, Benjamin; Martín, Martin G; Elpeleg, Orly

2014-01-01

Four siblings presented with congenital diarrhea and various endocrinopathies. Exome sequencing and homozygosity mapping identified five regions, comprising 337 protein-coding genes that were shared by three affected siblings. Exome sequencing identified a novel homozygous N309K mutation in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene, encoding the neuroendocrine convertase 1 precursor (PC1/3) which was recently reported as a cause of Congenital Diarrhea Disorder (CDD). The PCSK1 mutation affected the oxyanion hole transition state-stabilizing amino acid within the active site, which is critical for appropriate proprotein maturation and enzyme activity. Unexpectedly, the N309K mutant protein exhibited normal, though slowed, prodomain removal and was secreted from both HEK293 and Neuro2A cells. However, the secreted enzyme showed no catalytic activity, and was not processed into the 66 kDa form. We conclude that the N309K enzyme is able to cleave its own propeptide but is catalytically inert against in trans substrates, and that this variant accounts for the enteric and systemic endocrinopathies seen in this large consanguineous kindred.

A Novel Familial Mutation in the PCSK1 Gene That Alters the Oxyanion Hole Residue of Proprotein Convertase 1/3 and Impairs Its Enzymatic Activity

PubMed Central

Wilschanski, Michael; Abbasi, Montaser; Blanco, Elias; Lindberg, Iris; Yourshaw, Michael; Zangen, David; Berger, Itai; Shteyer, Eyal; Pappo, Orit; Bar-Oz, Benjamin; Martín, Martin G.; Elpeleg, Orly

2014-01-01

Four siblings presented with congenital diarrhea and various endocrinopathies. Exome sequencing and homozygosity mapping identified five regions, comprising 337 protein-coding genes that were shared by three affected siblings. Exome sequencing identified a novel homozygous N309K mutation in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene, encoding the neuroendocrine convertase 1 precursor (PC1/3) which was recently reported as a cause of Congenital Diarrhea Disorder (CDD). The PCSK1 mutation affected the oxyanion hole transition state-stabilizing amino acid within the active site, which is critical for appropriate proprotein maturation and enzyme activity. Unexpectedly, the N309K mutant protein exhibited normal, though slowed, prodomain removal and was secreted from both HEK293 and Neuro2A cells. However, the secreted enzyme showed no catalytic activity, and was not processed into the 66 kDa form. We conclude that the N309K enzyme is able to cleave its own propeptide but is catalytically inert against in trans substrates, and that this variant accounts for the enteric and systemic endocrinopathies seen in this large consanguineous kindred. PMID:25272002

Adam10 Mediates the Choice between Principal Cells and Intercalated Cells in the Kidney

PubMed Central

Guo, Qiusha; Wang, Yinqiu; Tripathi, Piyush; Manda, Kalyan R.; Mukherjee, Malini; Chaklader, Malay; Austin, Paul F.

2015-01-01

A disintegrin and metalloproteinase domain 10 (Adam10), a member of the ADAM family of cell membrane–anchored proteins, has been linked to the regulation of the Notch, EGF, E-cadherin, and other signaling pathways. However, it is unclear what role Adam10 has in the kidney in vivo. In this study, we showed that Adam10 deficiency in ureteric bud (UB) derivatives leads to a decrease in urinary concentrating ability, polyuria, and hydronephrosis in mice. Furthermore, Adam10 deficiency led to a reduction in the percentage of aquaporin 2 (Aqp2)+ principal cells (PCs) in the collecting ducts that was accompanied by a proportional increase in the percentage of intercalated cells (ICs). This increase was more prominent in type A ICs than in type B ICs. Foxi1, a transcription factor important for the differentiation of ICs, was upregulated in the Adam10 mutants. The observed reduction of Notch activity in Adam10 mutant collecting duct epithelium and the similar reduction of PC/IC ratios in the collecting ducts in mice deficient for mindbomb E3 ubiquitin protein ligase 1, a key regulator of the Notch and Wnt/receptor-like tyrosine kinase signaling pathways, suggest that Adam10 regulates cell fate determination through the activation of Notch signaling, probably through the regulation of Foxi1 expression. However, phenotypic differences between the Adam10 mutants, the Mib1 mutants, and the Foxi1 mutants suggest that the functions of Adam10 in determining the fate of collecting duct cells are more complex than those of a simple upstream factor in a linear pathway involving Notch and Foxi1. PMID:24904084

CCDC103 mutations cause primary ciliary dyskinesia by disrupting assembly of ciliary dynein arms

PubMed Central

Panizzi, Jennifer R.; Becker-Heck, Anita; Castleman, Victoria H.; Al-Mutairi, Dalal; Liu, Yan; Loges, Niki T.; Pathak, Narendra; Austin-Tse, Christina; Sheridan, Eamonn; Schmidts, Miriam; Olbrich, Heike; Werner, Claudius; Häffner, Karsten; Hellman, Nathan; Chodhari, Rahul; Gupta, Amar; Kramer-Zucker, Albrecht; Olale, Felix; Burdine, Rebecca D.; Schier, Alexander F.; O’Callaghan, Christopher; Chung, Eddie MK; Reinhardt, Richard; Mitchison, Hannah M.; King, Stephen M.; Omran, Heymut; Drummond, Iain A.

2012-01-01

Cilia are essential for fertilization, respiratory clearance, cerebrospinal fluid circulation, and to establish laterality1. Cilia motility defects cause Primary Ciliary Dyskinesia (PCD, MIM 242650), a disorder affecting 1:15-30,000 births. Cilia motility requires the assembly of multisubunit dynein arms that drive cilia bending2. Despite progress in understanding the genetic basis of PCD, mutations remain to be identified for several PCD linked loci3. Here we show that the zebrafish cilia paralysis mutant schmalhanstn222 (smh) mutant encodes the coiled-coil domain containing 103 protein (Ccdc103), a foxj1a regulated gene. Screening 146 unrelated PCD families identified patients in six families with reduced outer dynein arms, carrying mutations in CCDC103. Dynein arm assembly in smh mutant zebrafish was rescued by wild-type but not mutant human CCDC103. Chlamydomonas Ccdc103 functions as a tightly bound, axoneme-associated protein. The results identify Ccdc103 as a novel dynein arm attachment factor that when mutated causes Primary Ciliary Dyskinesia. PMID:22581229

Novel epitopes identified by Anti-PrP monoclonal antibodies produced following immunization of Prnp0/0 Balb/cJ mice with purified scrapie prions

USDA-ARS?s Scientific Manuscript database

Prions, or infectious proteins, cause a class of uniformly fatal neurodegenerative diseases. Prions are composed solely of an aberrantly folded isoform(PrPSc)of a normal cellular protein (PrPC). Shared sequence identity of PrPSc with PrPC has limited the detection sensitivity of immunochemical assay...

The Arabidopsis arc5 and arc6 mutations differentially affect plastid morphology in pavement and guard cells in the leaf epidermis.

PubMed

Fujiwara, Makoto T; Yasuzawa, Mana; Kojo, Kei H; Niwa, Yasuo; Abe, Tomoko; Yoshida, Shigeo; Nakano, Takeshi; Itoh, Ryuuichi D

2018-01-01

Chloroplasts, or photosynthetic plastids, multiply by binary fission, forming a homogeneous population in plant cells. In Arabidopsis thaliana, the division apparatus (or division ring) of mesophyll chloroplasts includes an inner envelope transmembrane protein ARC6, a cytoplasmic dynamin-related protein ARC5 (DRP5B), and members of the FtsZ1 and FtsZ2 families of proteins, which co-assemble in the stromal mid-plastid division ring (FtsZ ring). FtsZ ring placement is controlled by several proteins, including a stromal factor MinE (AtMinE1). During leaf mesophyll development, ARC6 and AtMinE1 are necessary for FtsZ ring formation and thus plastid division initiation, while ARC5 is essential for a later stage of plastid division. Here, we examined plastid morphology in leaf epidermal pavement cells (PCs) and stomatal guard cells (GCs) in the arc5 and arc6 mutants using stroma-targeted fluorescent proteins. The arc5 PC plastids were generally a bit larger than those of the wild type, but most had normal shapes and were division-competent, unlike mutant mesophyll chloroplasts. The arc6 PC plastids were heterogeneous in size and shape, including the formation of giant and mini-plastids, plastids with highly developed stromules, and grape-like plastid clusters, which varied on a cell-by-cell basis. Moreover, unique plastid phenotypes for stomatal GCs were observed in both mutants. The arc5 GCs rarely lacked chlorophyll-bearing plastids (chloroplasts), while they accumulated minute chlorophyll-less plastids, whereas most GCs developed wild type-like chloroplasts. The arc6 GCs produced large chloroplasts and/or chlorophyll-less plastids, as previously observed, but unexpectedly, their chloroplasts/plastids exhibited marked morphological variations. We quantitatively analyzed plastid morphology and partitioning in paired GCs from wild-type, arc5, arc6, and atminE1 plants. Collectively, our results support the notion that ARC5 is dispensable in the process of equal division of epidermal plastids, and indicate that dysfunctions in ARC5 and ARC6 differentially affect plastid replication among mesophyll cells, PCs, and GCs within a single leaf.

The Aqueous Extract of Rhizome of Gastrodia elata Protected Drosophila and PC12 Cells against Beta-Amyloid-Induced Neurotoxicity

PubMed Central

Ng, Chun-Fai; Ko, Chun-Hay; Koon, Chi-Man; Xian, Jia-Wen; Leung, Ping-Chung; Fung, Kwok-Pui; Chan, Ho Yin Edwin; Lau, Clara Bik-San

2013-01-01

This study aims to investigate the neuroprotective effect of the rhizome of Gastrodia elata (GE) aqueous extract on beta-amyloid(Aβ)-induced toxicity in vivo and in vitro. Transgenic Drosophila mutants with Aβ-induced neurodegeneration in pan-neuron and ommatidia were used to determine the efficacy of GE. The antiapoptotic and antioxidative mechanisms of GE were also studied in Aβ-treated pheochromocytoma (PC12) cells. In vivo studies demonstrated that GE (5 mg/g Drosophila media)-treated Drosophila possessed a longer lifespan, better locomotor function, and less-degenerated ommatidia when compared with the Aβ-expressing control (all P < 0.05). In vitro studies illustrated that GE increased the cell viability of Aβ-treated PC12 cells in dose-dependent manner, probably through attenuation of Aβ-induced oxidative and apoptotic stress. GE also significantly upregulated the enzymatic activities of catalase, superoxide dismutase, and glutathione peroxidase, leading to the decrease of reactive oxidation species production and apoptotic marker caspase-3 activity. In conclusion, our current data presented the first evidence that the aqueous extract of GE was capable of reducing the Aβ-induced neurodegeneration in Drosophila, possibly through inhibition of apoptosis and reduction of oxidative stress. GE aqueous extract could be developed as a promising herbal agent for neuroprotection and novel adjuvant therapies for Alzheimer's disease. PMID:24174977

The molecular genetic analysis of the expanding pachyonychia congenita case collection

PubMed Central

Wilson, NJ; O'Toole, EA; Milstone, LM; Hansen, CD; Shepherd, AA; Al-Asadi, E; Schwartz, ME; McLean, WHI; Sprecher, E; Smith, FJD

2014-01-01

Background Pachyonychia congenita (PC) is a rare autosomal dominant keratinizing disorder characterized by severe, painful, palmoplantar keratoderma and nail dystrophy, often accompanied by oral leucokeratosis, cysts and follicular keratosis. It is caused by mutations in one of five keratin genes: KRT6A, KRT6B, KRT6C, KRT16 or KRT17. Objectives To identify mutations in 84 new families with a clinical diagnosis of PC, recruited by the International Pachyonychia Congenita Research Registry during the last few years. Methods Genomic DNA isolated from saliva or peripheral blood leucocytes was amplified using primers specific for the PC-associated keratin genes and polymerase chain reaction products were directly sequenced. Results Mutations were identified in 84 families in the PC-associated keratin genes, comprising 46 distinct keratin mutations. Fourteen were previously unreported mutations, bringing the total number of different keratin mutations associated with PC to 105. Conclusions By identifying mutations in KRT6A, KRT6B, KRT6C, KRT16 or KRT17, this study has confirmed, at the molecular level, the clinical diagnosis of PC in these families. PMID:24611874

The Neuroprotective Effects of Decursin Isolated from Angelica gigas Nakai Against Amyloid β-Protein-Induced Apoptosis in PC 12 Cells via a Mitochondria-Related Caspase Pathway.

PubMed

Li, Li; Du, Jikun; Zou, Liyi; Xia, Haishan; Wu, Tie; Kim, Yongho; Lee, Yongwoo

2015-08-01

Decursin, purified from Angelica gigas Nakai, has been proven to exert neuroprotective property. Previous study revealed decursin protected the PC12 cells from Aβ25-35-induced oxidative cytotoxicity. The present study aimed to investigate whether decursin could protect PC12 cells from apoptosis caused by Aβ. Our results indicated that pretreatment of PC12 cells with decursin significantly inhibited Aβ25-35-induced cytotoxicity and apoptosis. The mechanism of action is likely to reverse Aβ25-35-induced mitochondrial dysfunction, including the reduction of mitochondrial membrane potential, the inhibition of reactive oxygen species production, and the decrease of mitochondrial release of cytochrome c in PC12 cells. In addition, decursin significantly suppressed the activity of caspase-3 and moderated the ratio of Bcl-2/Bax induced by Aβ25-35. These findings indicate that decursin exerts a neuroprotective effect against Aβ25-35-induced neurotoxicity in PC12 cells, at least in part, via suppressing the mitochondrial pathway of cellular apoptosis.

HTLV-1 Tax Effects on Cellular Mitotic Regulation

DTIC Science & Technology

2007-04-12

39 2 MutagenesisTax mutagenesis and selection for tax mutants...43 Isolation of tax mutants that do not cause growth arrest in S. cerevisiae. .......... 43 Tax mutants selected in W303a are functional in...114). One currently-approved therapeutic agent , Indinavir, is an effective inhibitor of both proteases. New protease inhibitors are currently being

Structure and Expression of Hybrid Dysgenesis-Induced Alleles of the Ovarian Tumor (Otu) Gene in Drosophila Melanogaster

PubMed Central

Sass, G. L.; Mohler, J. D.; Walsh, R. C.; Kalfayan, L. J.; Searles, L. L.

1993-01-01

Mutations at the ovarian tumor (otu) gene of Drosophila melanogaster cause female sterility and generate a range of ovarian phenotypes. Quiescent (QUI) mutants exhibit reduced germ cell proliferation; in oncogenic (ONC) mutants germ cells undergo uncontrolled proliferation generating excessive numbers of undifferentiated cells; the egg chambers of differentiated (DIF) mutants differentiate to variable degrees but fail to complete oogenesis. We have examined mutations caused by insertion and deletion of P elements at the otu gene. The P element insertion sites are upstream of the major otu transcription start sites. In deletion derivatives, the P element, regulatory regions and/or protein coding sequences have been removed. In both insertion and deletion mutants, the level of otu expression correlates directly with the severity of the phenotype: the absence of otu function produces the most severe QUI phenotype while the ONC mutants express lower levels of otu than those which are DIF. The results of this study demonstrate that the diverse mutant phenotypes of otu are the consequence of different levels of otu function. PMID:8436274

Mutations in Prickle Orthologs Cause Seizures in Flies, Mice, and Humans

PubMed Central

Tao, Hirotaka; Manak, J. Robert; Sowers, Levi; Mei, Xue; Kiyonari, Hiroshi; Abe, Takaya; Dahdaleh, Nader S.; Yang, Tian; Wu, Shu; Chen, Shan; Fox, Mark H.; Gurnett, Christina; Montine, Thomas; Bird, Thomas; Shaffer, Lisa G.; Rosenfeld, Jill A.; McConnell, Juliann; Madan-Khetarpal, Suneeta; Berry-Kravis, Elizabeth; Griesbach, Hilary; Saneto, Russell P.; Scott, Matthew P.; Antic, Dragana; Reed, Jordan; Boland, Riley; Ehaideb, Salleh N.; El-Shanti, Hatem; Mahajan, Vinit B.; Ferguson, Polly J.; Axelrod, Jeffrey D.; Lehesjoki, Anna-Elina; Fritzsch, Bernd; Slusarski, Diane C.; Wemmie, John; Ueno, Naoto; Bassuk, Alexander G.

2011-01-01

Epilepsy is heritable, yet few causative gene mutations have been identified, and thus far no human epilepsy gene mutations have been found to produce seizures in invertebrates. Here we show that mutations in prickle genes are associated with seizures in humans, mice, and flies. We identified human epilepsy patients with heterozygous mutations in either PRICKLE1 or PRICKLE2. In overexpression assays in zebrafish, prickle mutations resulted in aberrant prickle function. A seizure phenotype was present in the Prickle1-null mutant mouse, two Prickle1 point mutant (missense and nonsense) mice, and a Prickle2-null mutant mouse. Drosophila with prickle mutations displayed seizures that were responsive to anti-epileptic medication, and homozygous mutant embryos showed neuronal defects. These results suggest that prickle mutations have caused seizures throughout evolution. PMID:21276947

ALS-causing profilin-1-mutant forms a non-native helical structure in membrane environments.

PubMed

Lim, Liangzhong; Kang, Jian; Song, Jianxing

2017-11-01

Despite having physiological functions completely different from superoxide dismutase 1 (SOD1), profilin 1 (PFN1) also carries mutations causing amyotrophic lateral sclerosis (ALS) with a striking similarity to that triggered by SOD1 mutants. Very recently, the C71G-PFN1 has been demonstrated to cause ALS by a gain of toxicity and the acceleration of motor neuron degeneration preceded the accumulation of its aggregates. Here by atomic-resolution NMR determination of conformations and dynamics of WT-PFN1 and C71G-PFN1 in aqueous buffers and in membrane mimetics DMPC/DHPC bicelle and DPC micelle, we deciphered that: 1) the thermodynamic destabilization by C71G transforms PFN1 into coexistence with the unfolded state, which is lacking of any stable tertiary/secondary structures as well as restricted ps-ns backbone motions, thus fundamentally indistinguishable from ALS-causing SOD1 mutants. 2) Most strikingly, while WT-PFN1 only weakly interacts with DMPC/DHPC bicelle without altering the native structure, C71G-PFN1 acquires abnormal capacity in strongly interacting with DMPC/DHPC bicelle and DPC micelle, energetically driven by transforming the highly disordered unfolded state into a non-native helical structure, similar to what has been previously observed on ALS-causing SOD1 mutants. Our results imply that one potential mechanism for C71G-PFN1 to initiate ALS might be the abnormal interaction with membranes as recently established for SOD1 mutants. Copyright © 2017 Elsevier B.V. All rights reserved.

Regulation of stem cell maintenance by the Polycomb protein FIE has been conserved during land plant evolution.

PubMed

Mosquna, Assaf; Katz, Aviva; Decker, Eva L; Rensing, Stefan A; Reski, Ralf; Ohad, Nir

2009-07-01

The Polycomb group (PcG) complex is involved in the epigenetic control of gene expression profiles. In flowering plants, PcG proteins regulate vegetative and reproductive programs. Epigenetically inherited states established in the gametophyte generation are maintained after fertilization in the sporophyte generation, having a profound influence on seed development. The gametophyte size and phase dominance were dramatically reduced during angiosperm evolution, and have specialized in flowering plants to support the reproductive process. The moss Physcomitrella patens is an ideal organism in which to study epigenetic processes during the gametophyte stage, as it possesses a dominant photosynthetic gametophytic haploid phase and efficient homologous recombination, allowing targeted gene replacement. We show that P. patens PcG protein FIE (PpFIE) accumulates in haploid meristematic cells and in cells that undergo fate transition during dedifferentiation programs in the gametophyte. In the absence of PpFIE, meristems overproliferate and are unable to develop leafy gametophytes or reach the reproductive phase. This aberrant phenotype might result from failure of the PcG complex to repress proliferation and differentiation of three-faced apical stem cells, which are designated to become lateral shoots. The PpFIE phenotype can be partially rescued by FIE of Arabidopsis thaliana, a flowering plant that diverged >450 million years ago from bryophytes. PpFIE can partially complement the A. thaliana fie mutant, illustrating functional conservation of the protein during evolution in regulating the differentiation of meristematic cells in gametophyte development, both in bryophytes and angiosperms. This mechanism was harnessed at the onset of the evolution of alternating generations, facilitating the establishment of sporophytic developmental programs.

Induction of neurite extension and survival in pheochromocytoma cells by the Rit GTPase.

PubMed

Spencer, Michael L; Shao, Haipeng; Andres, Douglas A

2002-06-07

The Rit, Rin, and Ric proteins comprise a distinct and evolutionarily conserved subfamily of the Ras-like small G-proteins. Although these proteins share the majority of core effector domain residues with Ras, recent studies suggest that Rit uses novel effector pathways to regulate NIH3T3 cell proliferation and transformation, while the functions of Rin and Ric remain largely unknown. Since we demonstrate that Rit is expressed in neurons, we investigated the role of Rit signaling in promoting the differentiation and survival of pheochromocytoma cells. In this study, we show that expression of constitutively active Rit (RitL79) in PC6 cells results in neuronal differentiation, characterized by the elaboration of an extensive network of neurite-like processes that are morphologically distinct from those mediated by the expression of oncogenic Ras. Although activated Rit fails to stimulate mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) signaling pathways in COS cells, RitL79 induced the phosphorylation of ERK1/2 in PC6 cells. We also find that Rit-mediated effects on neurite outgrowth can be blocked by co-expression of dominant-negative mutants of C-Raf1 or mitogen-activated protein kinase kinase 1 (MEK1). Moreover, expression of dominant-negative Rit is sufficient to inhibit NGF-induced neurite outgrowth. Expression of active Rit inhibits growth factor-withdrawal mediated apoptosis of PC6 cells, but does not induce phosphorylation of Akt/protein kinase B, suggesting that survival does not utilize the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Instead, pharmacological inhibitors of MEK block Rit-stimulated cell survival. Taken together, these studies suggest that Rit represents a distinct regulatory protein, capable of mediating differentiation and cell survival in PC6 cells using a MEK-dependent signaling pathway to achieve its effects.

Constitutive Overexpression of the Basic Helix-Loop-Helix Nex1/MATH-2 Transcription Factor Promotes Neuronal Differentiation of PC12 Cells and Neurite Regeneration

PubMed Central

Uittenbogaard, Martine; Chiaramello, Anne

2009-01-01

Elucidation of the intricate transcriptional pathways leading to neural differentiation and the establishment of neuronal identity is critical to the understanding and design of therapeutic approaches. Among the important players, the basic helix-loop-helix (bHLH) transcription factors have been found to be pivotal regulators of neurogenesis. In this study, we investigate the role of the bHLH differentiation factor Nex1/MATH-2 in conjunction with the nerve growth factor (NGF) signaling pathway using the rat phenochromocytoma PC12 cell line. We report that the expression of Nex1 protein is induced after 5 hr of NGF treatment and reaches maximal levels at 24 hr, when very few PC12 cells have begun extending neurites and ceased cell division. Furthermore, our study demonstrates that Nex1 has the ability to trigger neuronal differentiation of PC12 cells in the absence of neurotrophic factor. We show that Nex1 plays an important role in neurite outgrowth and has the capacity to regenerate neurite outgrowth in the absence of NGF. These results are corroborated by the fact that Nex1 targets a repertoire of distinct types of genes associated with neuronal differentiation, such as GAP-43, βIII-tubulin, and NeuroD. In addition, our findings show that Nex1 up-regulates the expression of the mitotic inhibitor p21WAF1, thus linking neuronal differentiation to cell cycle withdrawal. Finally, our studies show that overexpression of a Nex1 mutant has the ability to block the execution of NGF-induced differentiation program, suggesting that Nex1 may be an important effector of the NGF signaling pathway. PMID:11782967

Resolving Phenylalanine Metabolism Sheds Light on Natural Synthesis of Penicillin G in Penicillium chrysogenum

PubMed Central

Veiga, Tânia; Solis-Escalante, Daniel; Romagnoli, Gabriele; ten Pierick, Angela; Hanemaaijer, Mark; Deshmuhk, Amit; Wahl, Aljoscha; Pronk, Jack T.

2012-01-01

The industrial production of penicillin G by Penicillium chrysogenum requires the supplementation of the growth medium with the side chain precursor phenylacetate. The growth of P. chrysogenum with phenylalanine as the sole nitrogen source resulted in the extracellular production of phenylacetate and penicillin G. To analyze this natural pathway for penicillin G production, chemostat cultures were switched to [U-13C]phenylalanine as the nitrogen source. The quantification and modeling of the dynamics of labeled metabolites indicated that phenylalanine was (i) incorporated in nascent protein, (ii) transaminated to phenylpyruvate and further converted by oxidation or by decarboxylation, and (iii) hydroxylated to tyrosine and subsequently metabolized via the homogentisate pathway. The involvement of the homogentisate pathway was supported by the comparative transcriptome analysis of P. chrysogenum cultures grown with phenylalanine and with (NH4)2SO4 as the nitrogen source. This transcriptome analysis also enabled the identification of two putative 2-oxo acid decarboxylase genes (Pc13g9300 and Pc18g01490). cDNAs of both genes were cloned and expressed in the 2-oxo-acid-decarboxylase-free Saccharomyces cerevisiae strain CEN.PK711-7C (pdc1 pdc5 pdc6Δ aro10Δ thi3Δ). The introduction of Pc13g09300 restored the growth of this S. cerevisiae mutant on glucose and phenylalanine, thereby demonstrating that Pc13g09300 encodes a dual-substrate pyruvate and phenylpyruvate decarboxylase, which plays a key role in an Ehrlich-type pathway for the production of phenylacetate in P. chrysogenum. These results provide a basis for the metabolic engineering of P. chrysogenum for the production of the penicillin G side chain precursor phenylacetate. PMID:22158714

Chromosome painting reveals asynaptic full alignment of homologs and HIM-8-dependent remodeling of X chromosome territories during Caenorhabditis elegans meiosis.

PubMed

Nabeshima, Kentaro; Mlynarczyk-Evans, Susanna; Villeneuve, Anne M

2011-08-01

During early meiotic prophase, a nucleus-wide reorganization leads to sorting of chromosomes into homologous pairs and to establishing associations between homologous chromosomes along their entire lengths. Here, we investigate global features of chromosome organization during this process, using a chromosome painting method in whole-mount Caenorhabditis elegans gonads that enables visualization of whole chromosomes along their entire lengths in the context of preserved 3D nuclear architecture. First, we show that neither spatial proximity of premeiotic chromosome territories nor chromosome-specific timing is a major factor driving homolog pairing. Second, we show that synaptonemal complex-independent associations can support full lengthwise juxtaposition of homologous chromosomes. Third, we reveal a prominent elongation of chromosome territories during meiotic prophase that initiates prior to homolog association and alignment. Mutant analysis indicates that chromosome movement mediated by association of chromosome pairing centers (PCs) with mobile patches of the nuclear envelope (NE)-spanning SUN-1/ZYG-12 protein complexes is not the primary driver of territory elongation. Moreover, we identify new roles for the X chromosome PC (X-PC) and X-PC binding protein HIM-8 in promoting elongation of X chromosome territories, separable from their role(s) in mediating local stabilization of pairing and association of X chromosomes with mobile SUN-1/ZYG-12 patches. Further, we present evidence that HIM-8 functions both at and outside of PCs to mediate chromosome territory elongation. These and other data support a model in which synapsis-independent elongation of chromosome territories, driven by PC binding proteins, enables lengthwise juxtaposition of chromosomes, thereby facilitating assessment of their suitability as potential pairing partners.

Chromosome Painting Reveals Asynaptic Full Alignment of Homologs and HIM-8–Dependent Remodeling of X Chromosome Territories during Caenorhabditis elegans Meiosis

PubMed Central

Nabeshima, Kentaro; Mlynarczyk-Evans, Susanna; Villeneuve, Anne M.

2011-01-01

During early meiotic prophase, a nucleus-wide reorganization leads to sorting of chromosomes into homologous pairs and to establishing associations between homologous chromosomes along their entire lengths. Here, we investigate global features of chromosome organization during this process, using a chromosome painting method in whole-mount Caenorhabditis elegans gonads that enables visualization of whole chromosomes along their entire lengths in the context of preserved 3D nuclear architecture. First, we show that neither spatial proximity of premeiotic chromosome territories nor chromosome-specific timing is a major factor driving homolog pairing. Second, we show that synaptonemal complex-independent associations can support full lengthwise juxtaposition of homologous chromosomes. Third, we reveal a prominent elongation of chromosome territories during meiotic prophase that initiates prior to homolog association and alignment. Mutant analysis indicates that chromosome movement mediated by association of chromosome pairing centers (PCs) with mobile patches of the nuclear envelope (NE)–spanning SUN-1/ZYG-12 protein complexes is not the primary driver of territory elongation. Moreover, we identify new roles for the X chromosome PC (X-PC) and X-PC binding protein HIM-8 in promoting elongation of X chromosome territories, separable from their role(s) in mediating local stabilization of pairing and association of X chromosomes with mobile SUN-1/ZYG-12 patches. Further, we present evidence that HIM-8 functions both at and outside of PCs to mediate chromosome territory elongation. These and other data support a model in which synapsis-independent elongation of chromosome territories, driven by PC binding proteins, enables lengthwise juxtaposition of chromosomes, thereby facilitating assessment of their suitability as potential pairing partners. PMID:21876678

Clinical usefulness of novel tag-less Agile patency capsule prior to capsule endoscopy for patients with suspected small bowel stenosis.

PubMed

Nakamura, Masanao; Hirooka, Yoshiki; Yamamura, Takeshi; Miyahara, Ryoji; Watanabe, Osamu; Ando, Takafumi; Ohmiya, Naoki; Goto, Hidemi

2015-01-01

The Agile patency capsule (PC; Given Imaging Ltd, Yoqneam, Israel) is used as a dummy prior to capsule endoscopy (CE) to avoid retention of the CE capsule. However, impaction of the PC's inner radio frequency identification (RFID) tag in a stricture could cause small-bowel ileus. Recently, the RFID tag-less PC was introduced into clinical practice. Herein, we aimed to retrospectively evaluate the usefulness of the tag-less PC. Of 154 patients who were scheduled to undergo CE, 100 consecutive patients (65%) who underwent PC evaluation were enrolled in the present study. Primary study end point was the retention rate of the CE capsule after successful passage of the PC. Secondary end point was analysis of the significant factors affecting the passage of the PC. In total, 87 patients (87%) had bowel patency confirmed by PC evaluation. There was no capsule retention in any of these 87 patients during CE. Abnormal findings were obtained from 60 CE, and 41 patients received new or modified treatment. Multivariate analysis of factors related to the confirmation of patency demonstrated that stenosis on imaging was the most influential factor (P = 0.002, odds ratio 16.387). The results confirmed that passage of the PC depends on stenosis on imaging. Use of the tag-less PC confirmed gastrointestinal tract patency for most of the patients who did not have stenosis on imaging and allowed estimation of the patency for patients who did have stenosis on imaging. © 2014 The Authors. Digestive Endoscopy © 2014 Japan Gastroenterological Endoscopy Society.

The Methylation of the PcMYB10 Promoter Is Associated with Green-Skinned Sport in Max Red Bartlett Pear1[C][W

PubMed Central

Wang, Zhigang; Meng, Dong; Wang, Aide; Li, Tianlai; Jiang, Shuling; Cong, Peihua; Li, Tianzhong

2013-01-01

Varieties of the European pear (Pyrus communis) can produce trees with both red- and green-skinned fruits, such as the Max Red Bartlett (MRB) variety, although little is known about the mechanism behind this differential pigmentation. In this study, we investigated the pigmentation of MRB and its green-skinned sport (MRB-G). The results suggest that a reduction in anthocyanin concentration causes the MRB-G sport. Transcript levels of PcUFGT (for UDP-glucose:flavonoid 3-O-glucosyltransferase), the key structural gene in anthocyanin biosynthesis, paralleled the change of anthocyanin concentration in both MRB and MRB-G fruit. We cloned the PcMYB10 gene, a transcription factor associated with the promoter of PcUFGT. An investigation of the 2-kb region upstream of the ATG translation start site of PcMYB10 showed the regions −604 to −911 bp and −1,218 to −1,649 bp to be highly methylated. A comparison of the PcMYB10 promoter methylation level between the MRB and MRB-G forms indicated a correlation between hypermethylation and the green-skin phenotype. An Agrobacterium tumefaciens infiltration assay was conducted on young MRB fruits by using a plasmid constructed to silence endogenous PcMYB10 via DNA methylation. The infiltrated fruits showed blocked anthocyanin biosynthesis, higher methylation of the PcMYB10 promoter, and lower expression of PcMYB10 and PcUFGT. We suggest that the methylation level of PcMYB10 is associated with the formation of the green-skinned sport in the MRB pear. The potential mechanism behind the regulation of anthocyanin biosynthesis is discussed. PMID:23629835

Developmental Hypothyroxinemia and Hypothyroidism Reduce Parallel Fiber-Purkinje Cell Synapses in Rat Offspring by Downregulation of Neurexin1/Cbln1/GluD2 Tripartite Complex.

PubMed

Wang, Yuan; Dong, Jing; Wang, Yi; Wei, Wei; Song, Binbin; Shan, Zhongyan; Teng, Weiping; Chen, Jie

2016-10-01

Iodine is a significant micronutrient. Iodine deficiency (ID)-induced hypothyroxinemia and hypothyroidism during developmental period can cause cerebellar dysfunction. However, mechanisms are still unclear. Therefore, the present research aims to study effects of developmental hypothyroxinemia caused by mild ID and hypothyroidism caused by severe ID or methimazole (MMZ) on parallel fiber-Purkinje cell (PF-PC) synapses in filial cerebellum. Maternal hypothyroxinemia and hypothyroidism models were established in Wistar rats using ID diet and deionized water supplemented with different concentrations of potassium iodide or MMZ water. Birth weight and cerebellum weight were measured. We also examined PF-PC synapses using immunofluorescence, and western blot analysis was conducted to investigate the activity of Neurexin1/cerebellin1 (Cbln1)/glutamate receptor d2 (GluD2) tripartite complex. Our results showed that hypothyroxinemia and hypothyroidism decreased birth weight and cerebellum weight and reduced the PF-PC synapses on postnatal day (PN) 14 and PN21. Accordingly, the mean intensity of vesicular glutamate transporter (VGluT1) and Calbindin immunofluorescence was reduced in mild ID, severe ID, and MMZ groups. Moreover, maternal hypothyroxinemia and hypothyroidism reduced expression of Neurexin1/Cbln1/GluD2 tripartite complex. Our study supports the hypothesis that developmental hypothyroxinemia and hypothyroidism reduce PF-PC synapses, which may be attributed to the downregulation of Neurexin1/Cbln1/GluD2 tripartite complex.

Homozygosity Mapping and Whole Exome Sequencing to Detect SLC45A2 and G6PC3 Mutations in a Single Patient with Oculocutaneous Albinism and Neutropenia

PubMed Central

Cullinane, Andrew R.; Vilboux, Thierry; O’Brien, Kevin; Curry, James A.; Maynard, Dawn M.; Carlson-Donohoe, Hannah; Ciccone, Carla; Markello, Thomas C.; Gunay-Aygun, Meral; Huizing, Marjan; Gahl, William A.

2011-01-01

We evaluated a 32 year-old woman whose oculocutaneous albinism, bleeding diathesis, neutropenia, and history of recurrent infections prompted consideration of the diagnosis of Hermansky-Pudlak syndrome type 2 (HPS-2). This was ruled out due to the presence of platelet delta granules and absence of AP3B1 mutations. Since parental consanguinity suggested an autosomal recessive mode of inheritance, we employed homozygosity mapping, followed by whole exome sequencing, to identify two candidate disease-causing genes, SLC45A2 and G6PC3. Conventional di-deoxy sequencing confirmed pathogenic mutations in SLC45A2, associated with oculocutaneous albinism type 4 (OCA-4), and G6PC3, associated with neutropenia. The substantial reduction of SLC45A2 protein in the patient’s melanocytes caused the mis-localization of tyrosinase from melanosomes to the plasma membrane and also led to the incorporation of tyrosinase into exosomes and secretion into the culture medium, explaining the hypopigmentation in OCA-4. Our patient’s G6PC3 mRNA expression level was also reduced, leading to increased apoptosis of her fibroblasts under ER stress. This report describes the first North American patient with OCA-4, the first culture of human OCA-4 melanocytes, and the use of homozygosity mapping followed by whole exome sequencing to identify disease-causing mutations in multiple genes in a single affected individual. PMID:21677667

Loss of PTEN causes SHP2 activation, making lung cancer cells unresponsive to IFN-γ

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Chia-Ling; Chiang, Tzu-Hui; Tseng, Po-Chun

Src homology-2 domain-containing phosphatase (SHP) 2, an oncogenic phosphatase, inhibits type II immune interferon (IFN)-γ signaling by subverting signal transducers and activators of transcription 1 tyrosine phosphorylation and activation. For cancer immunoediting, this study aimed to investigate the decrease of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor protein, leading to cellular impairment of IFN-γ signaling. In comparison with human lung adenocarcinoma A549 cells, the natural PTEN loss in another human lung adenocarcinoma line, PC14PE6/AS2 cells, presents reduced responsiveness in IFN-γ-induced IFN regulatory factor 1 activation and CD54 expression. Artificially silencing PTEN expression in A549 cellsmore » also caused cells to be unresponsive to IFN-γ without affecting IFN-γ receptor expression. IFN-γ-induced inhibition of cell proliferation and cytotoxicity were demonstrated in A549 cells but were defective in PC14PE6/AS2 cells and in PTEN-deficient A549 cells. Aberrant activation of SHP2 by ROS was specifically shown in PC14PE6/AS2 cells and PTEN-deficient A549 cells. Inhibiting ROS and SHP2 rescued cellular responses to IFN-γ-induced cytotoxicity and inhibition of cell proliferation in PC14PE6/AS2 cells. These results demonstrate that a decrease in PTEN facilitates ROS/SHP2 signaling, causing lung cancer cells to become unresponsive to IFN-γ. - Highlights: • This study demonstrates that PTEN decrease causes cellular unresponsive to IFN-γ. • Lung cancer cells with PTEN deficiency show unresponsive to IFN-γ signaling. • PTEN decrease inhibits IFN-γ-induced CD54, cell proliferation inhibition, and cytotoxicity. • ROS-mediated SHP2 activation makes PTEN-deficient cells unresponsive to IFN-γ.« less

Southern Brazilian native fruit shows neurochemical, metabolic and behavioral benefits in an animal model of metabolic syndrome.

PubMed

Oliveira, Pathise Souto; Chaves, Vitor Clasen; Soares, Mayara Sandrielly Pereira; Bona, Natália Pontes; Mendonça, Lorenço Torres; Carvalho, Fabiano Barbosa; Gutierres, Jessié Martins; Vasconcellos, Flávia Aleixo; Vizzotto, Marcia; Vieira, Andriele; Spanevello, Roselia Maria; Reginatto, Flávio Henrique; Lencina, Claiton Leoneti; Stefanello, Francieli Moro

2018-06-07

In this work, we evaluated the effects of Psidium cattleianum (Red Type) (PcRT) fruit extract on metabolic, behavioral, and neurochemical parameters in rats fed with a highly palatable diet (HPD) consisted of sucrose (65% carbohydrates being 34% from condensed milk, 8% from sucrose and 23% from starch, 25% protein and 10% fat). Animals were divided into 4 groups: standard chow, standard chow + PcRT extract (200 mg/Kg/day by gavage), HPD, HPD + extract. The animals were treated for 150 days. Concerning chemical profiling, LC/PDA/MS/MS analysis revealed cyanidin-3-O-glucoside as the only anthocyanin in the PcRT extract. Our results showed that the animals exposed to HPD presented glucose intolerance, increased weight gain and visceral fat, as well as higher serum levels of glucose, triacylglycerol, total cholesterol, LDL-cholesterol and interleukin-6. These alterations were prevented by PcRT. In addition, HPD caused an increase in immobility time in a forced swimming test and the fruit extract prevented this alteration, indicating an antidepressant-like effect. PcRT treatment also prevented increased acetylcholinesterase activity in the prefrontal cortex caused by HPD consumption. Moreover, PcRT extract was able to restore Ca 2+ -ATPase activity in the prefrontal cortex, hippocampus, and striatum, as well as Na + ,K + -ATPase activity in the prefrontal cortex and hippocampus. PcRT treatment decreased thiobarbituric acid-reactive substances, nitrite, and reactive oxygen species levels and prevented the reduction of superoxide dismutase activity in all cerebral structures of the HPD group. Additionally, HPD decreased catalase in the hippocampus and striatum. However, the extract prevented this change in the hippocampus. Our results showed that this berry extract has antihyperglycemic and antihyperlipidemic effects, and neuroprotective properties, proving to be a potential therapeutic agent for individuals with metabolic syndrome.

Pneumocystis jirovecii Rtt109, a Novel Drug Target for Pneumocystis Pneumonia in Immunosuppressed Humans

PubMed Central

Dahlin, Jayme L.; Kottom, Theodore; Han, Junhong; Zhou, Hui; Walters, Michael A.; Zhang, Zhiguo

2014-01-01

Pneumocystis pneumonia (PcP) is a significant cause of morbidity and mortality in immunocompromised patients. In humans, PcP is caused by the opportunistic fungal species Pneumocystis jirovecii. Progress in Pneumocystis research has been hampered by a lack of viable in vitro culture methods, which limits laboratory access to human-derived organisms for drug testing. Consequently, most basic drug discovery research for P. jirovecii is performed using related surrogate organisms such as Pneumocystis carinii, which is derived from immunosuppressed rodents. While these studies provide useful insights, important questions arise about interspecies variations and the relative utility of identified anti-Pneumocystis agents against human P. jirovecii. Our recent work has identified the histone acetyltransferase (HAT) Rtt109 in P. carinii (i.e., PcRtt109) as a potential therapeutic target for PcP, since Rtt109 HATs are widely conserved in fungi but are absent in humans. To further address the potential utility of this target in human disease, we now demonstrate the presence of a functional Rtt109 orthologue in the clinically relevant fungal pathogen P. jirovecii (i.e., PjRtt109). In a fashion similar to that of Pcrtt109, Pjrtt109 restores H3K56 acetylation and genotoxic resistance in rtt109-null yeast. Recombinant PjRtt109 is an active HAT in vitro, with activity comparable to that of PcRtt109 and yeast Rtt109. PjRtt109 HAT activity is also enhanced by the histone chaperone Asf1 in vitro. PjRtt109 and PcRtt109 showed similar low micromolar sensitivities to two reported small-molecule HAT inhibitors in vitro. Together, these results demonstrate that PjRtt109 is a functional Rtt109 HAT, and they support the development of anti-Pneumocystis agents directed at Rtt109-catalyzed histone acetylation as a novel therapeutic target for human PcP. PMID:24733475

Pneumocystis jirovecii Rtt109, a novel drug target for Pneumocystis pneumonia in immunosuppressed humans.

PubMed

Dahlin, Jayme L; Kottom, Theodore; Han, Junhong; Zhou, Hui; Walters, Michael A; Zhang, Zhiguo; Limper, Andrew H

2014-07-01

Pneumocystis pneumonia (PcP) is a significant cause of morbidity and mortality in immunocompromised patients. In humans, PcP is caused by the opportunistic fungal species Pneumocystis jirovecii. Progress in Pneumocystis research has been hampered by a lack of viable in vitro culture methods, which limits laboratory access to human-derived organisms for drug testing. Consequently, most basic drug discovery research for P. jirovecii is performed using related surrogate organisms such as Pneumocystis carinii, which is derived from immunosuppressed rodents. While these studies provide useful insights, important questions arise about interspecies variations and the relative utility of identified anti-Pneumocystis agents against human P. jirovecii. Our recent work has identified the histone acetyltransferase (HAT) Rtt109 in P. carinii (i.e., PcRtt109) as a potential therapeutic target for PcP, since Rtt109 HATs are widely conserved in fungi but are absent in humans. To further address the potential utility of this target in human disease, we now demonstrate the presence of a functional Rtt109 orthologue in the clinically relevant fungal pathogen P. jirovecii (i.e., PjRtt109). In a fashion similar to that of Pcrtt109, Pjrtt109 restores H3K56 acetylation and genotoxic resistance in rtt109-null yeast. Recombinant PjRtt109 is an active HAT in vitro, with activity comparable to that of PcRtt109 and yeast Rtt109. PjRtt109 HAT activity is also enhanced by the histone chaperone Asf1 in vitro. PjRtt109 and PcRtt109 showed similar low micromolar sensitivities to two reported small-molecule HAT inhibitors in vitro. Together, these results demonstrate that PjRtt109 is a functional Rtt109 HAT, and they support the development of anti-Pneumocystis agents directed at Rtt109-catalyzed histone acetylation as a novel therapeutic target for human PcP. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Differential inhibitory effects of 2-azafluorenones on PI-PLC activation but not on PC-PLC- or PC-PLD-activation induced by histamine, PAF, PMA or A23187 in C6 glioma cells.

PubMed

Wang, Hai-Long; Wang, Li-Chuan; Wei, Jiann-Wu

2013-02-28

In this study, C6 glioma cells were used to test the effects of 2-azafluorenone and its related compounds on membrane phosphatidylinositol (PI) and phosphatidylcholine (PC) turnover. An increase of [³H]-labeled inositol phosphate (IP1) formation by histamine (100 μM) or A23187 (100 nM) via the activation of phosphatidylinositol-specific phospholipase C (PI-PLC) to breakdown labeled substrate was observed, and this effect could be partially blocked by about half at 100 μM of 2-azafluorenones. Histamine induced the increase of IP1 formation, but failed to cause an increase in extracellularly releasing of [3H]choline metabolites, or intracellular accumulation of [³H]phosphscholine. However, platelet activation factor (PAF) from 0.2 to 1 μM, and phorbol 12-myristate-13-acetate (PMA) at 1 μM caused an increase in extracellularly releasing of [³H]choline metabolites, and intracellular accumulation of [³H]phosphocholine via the activation on phosphatidylcholine (PC)-PLC. These responses of PAF and PMA were not affected by 2-azafluorenone or 4-methyl-2-azafluorenone even at high concentration (10⁻⁴ M). A23187 induced an increase of intracellular [³H]choline release via the activation of PCphospholipase D (PLD). This increasing effect of 100 nM A23187 was not affected by 2-azafluorenone or 4-methyl-2-azafluorenone even at a high concentration of 10⁻⁴ M. In summary, the inhibitory effect of 2-azafluorenone and its related compound 4-methyl-2-azafluorenone was observed selectively on PIPLC, but not on PC-PLC or PC-PLD based on changes of products after the activation of these enzymes.

Puerarin protects differentiated PC12 cells from H₂O₂-induced apoptosis through the PI3K/Akt signalling pathway.

PubMed

Zhang, Qin; Huang, Wei-Dong; Lv, Xue-Ying; Yang, Yun-Mei

2012-05-01

Oxidative stress has been implicated as a major mechanism underlying the pathogenesis of neurodegenerative disorders. ROS (reactive oxygen species) can cause cell death via apoptosis. NGF (nerve growth factor) differentiated rat PC12 cells have been extensively used to study the differentiation and apoptosis of neurons. This study has investigated the protective effects of puerarin in H2O2-induced apoptosis of differentiated PC12 cells, and the possible molecular mechanisms involved. Differentiated PC12 cells were incubated with 700 μM H2O2 in the absence or presence of different doses of puerarin (4, 8 and 16 μM). Apoptosis was assessed by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) analysis and Annexin V-PI (propidium iodide) double staining flow cytometry. Protein levels of phospho-Akt and phospho-BAD (Bcl-2/Bcl-XL-antagonist, causing cell death) were assayed by Western blotting. After stimulation with H2O2 for 18 h, the viability of differentiated PC12 cells decreased significantly and a large number of cells underwent apoptosis. Differentiated PC12 cells were rescued from H2O2-induced apoptosis at different concentrations of puerarin in a dose-dependent manner. This was through increased production of phospho-Akt and phospho-BAD, an effect that could be reversed by wortmannin, an inhibitor of PI3K (phosphoinositide 3-kinase). The results suggest that puerarin may have neuroprotective effect through activation of the PI3K/Akt signalling pathway.

Acute hepatitis B caused by a vaccine-escape HBV strain in vaccinated subject: sequence analysis and therapeutic strategy.

PubMed

Luongo, Monica; Critelli, Rosina; Grottola, Antonella; Gitto, Stefano; Bernabucci, Veronica; Bevini, Mirco; Vecchi, Chiara; Montagnani, Giuliano; Villa, Erica

2015-01-01

HBV vaccine contains the 'a' determinant region, the major immune-target of antibodies (anti-HBs). Failure of immunization may be caused by vaccine-induced or spontaneous 'a' determinant surface gene mutants. Here, we evaluate the possible lack of protection by HBV vaccine, describing the case of an acute hepatitis B diagnosed in a 55-year-old Caucasian male unpaid blood donor, vaccinated against HBV. Sequencing data for preS-S region revealed multiple point mutations. Of all the substitutions found, Q129H, located in the "a" determinant region of HBsAg, can alter antigenicity, leading to mutants. This mutant may cause vaccine failure especially when associated with high viremia of infecting source. Copyright © 2014 Elsevier B.V. All rights reserved.

Increased Steady-State Mutant Huntingtin mRNA in Huntington's Disease Brain.

PubMed

Liu, Wanzhao; Chaurette, Joanna; Pfister, Edith L; Kennington, Lori A; Chase, Kathryn O; Bullock, Jocelyn; Vonsattel, Jean Paul G; Faull, Richard L M; Macdonald, Douglas; DiFiglia, Marian; Zamore, Phillip D; Aronin, Neil

2013-01-01

Huntington's disease is caused by expansion of CAG trinucleotide repeats in the first exon of the huntingtin gene, which is essential for both development and neurogenesis. Huntington's disease is autosomal dominant. The normal allele contains 6 to 35 CAG triplets (average, 18) and the mutant, disease-causing allele contains >36 CAG triplets (average, 42). We examined 279 postmortem brain samples, including 148 HD and 131 non-HD controls. A total of 108 samples from 87 HD patients that are heterozygous at SNP rs362307, with a normal allele (18 to 27 CAG repeats) and a mutant allele (39 to 73 CAG repeats) were used to measure relative abundance of mutant and wild-type huntingtin mRNA. We used allele-specific, quantitative RT-PCR based on SNP heterozygosity to estimate the relative amount of mutant versus normal huntingtin mRNA in postmortem brain samples from patients with Huntington's disease. In the cortex and striatum, the amount of mRNA from the mutant allele exceeds that from the normal allele in 75% of patients. In the cerebellum, no significant difference between the two alleles was evident. Brain tissues from non-HD controls show no significant difference between two alleles of huntingtin mRNAs. Allelic differences were more pronounced at early neuropathological grades (grades 1 and 2) than at late grades (grades 3 and 4). More mutant HTT than normal could arise from increased transcription of mutant HTT allele, or decreased clearance of mutant HTT mRNA, or both. An implication is that equimolar silencing of both alleles would increase the mutant HTT to normal HTT ratio.

Osteopontin regulates the cross-talk between phosphatidylcholine and cholesterol metabolism in mouse liver.

PubMed

Nuñez-Garcia, Maitane; Gomez-Santos, Beatriz; Buqué, Xabier; García-Rodriguez, Juan L; Romero, Marta R; Marin, Jose J G; Arteta, Beatriz; García-Monzón, Carmelo; Castaño, Luis; Syn, Wing-Kin; Fresnedo, Olatz; Aspichueta, Patricia

2017-09-01

Osteopontin (OPN) is involved in different liver pathologies in which metabolic dysregulation is a hallmark. Here, we investigated whether OPN could alter liver, and more specifically hepatocyte, lipid metabolism and the mechanism involved. In mice, lack of OPN enhanced cholesterol 7α-hydroxylase (CYP7A1) levels and promoted loss of phosphatidylcholine (PC) content in liver; in vivo treatment with recombinant (r)OPN caused opposite effects. rOPN directly decreased CYP7A1 levels through activation of focal adhesion kinase-AKT signaling in hepatocytes. PC content was also decreased in OPN-deficient (OPN-KO) hepatocytes in which de novo FA and PC synthesis was lower, whereas cholesterol (CHOL) synthesis was higher, than in WT hepatocytes. In vivo inhibition of cholesterogenesis normalized liver PC content in OPN-KO mice, demonstrating that OPN regulates the cross-talk between liver CHOL and PC metabolism. Matched liver and serum samples showed a positive correlation between serum OPN levels and liver PC and CHOL concentration in nonobese patients with nonalcoholic fatty liver. In conclusion, OPN regulates CYP7A1 levels and the metabolic fate of liver acetyl-CoA as a result of CHOL and PC metabolism interplay. The results suggest that CYP7A1 is a main axis and that serum OPN could disrupt liver PC and CHOL metabolism, contributing to nonalcoholic fatty liver disease progression in nonobese patients.

Gain-of-function profilin 1 mutations linked to familial amyotrophic lateral sclerosis cause seed-dependent intracellular TDP-43 aggregation.

PubMed

Tanaka, Yoshinori; Nonaka, Takashi; Suzuki, Genjiro; Kametani, Fuyuki; Hasegawa, Masato

2016-04-01

Profilin 1 (PFN1) is an actin monomer-binding protein essential for regulating cytoskeletal dynamics in all cell types. Recently, mutations in the PFN1 gene have been identified as a cause of familial amyotrophic lateral sclerosis (ALS). The co-aggregation of PFN1 bearing mutations that cause ALS with TDP-43 (a key molecule in both sporadic and some familial forms of ALS), together with the classical TDP-43 pathology detected in post-mortem tissues of patients with autosomal dominant PFN1 mutation, imply that gain-of-toxic-function of PFN1 mutants is associated with the onset of ALS. However, it remains unknown how PFN1 mutants cause ALS. We found mutant PFN1 that causes ALS formed cytoplasmic aggregates positive for ubiquitin and p62, and these aggregates sequestered endogenous TDP-43. In cells harboring PFN1 aggregates, formation of aggresome-like structures was inhibited in the presence of proteasome inhibitor, and conversion of LC3-I to LC3-II was suppressed in the presence of lysosome inhibitor. Further, insoluble TDP-43 was increased in both cases. Co-expression of ALS-linked mutant PFN1 and TDP-43 increased insoluble and phosphorylated TDP-43 levels. The C-terminal region of TDP-43, essential for aggregation of TDP-43, was also indispensable for the interaction with PFN1. Interestingly, insoluble fractions prepared from cells expressing ALS-linked mutant PFN1 functioned as a seed to induce accumulation and phosphorylation of TDP-43, indicating that TDP-43 accumulated in the presence of the PFN1 mutants is converted to prion-like species. These findings provide new insight into the mechanisms of neurodegeneration in ALS, suggesting that gain-of-toxic-function PFN1 gene mutation leads to conformational change of TDP-43. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Survival and differentiation defects contribute to neutropenia in glucose-6-phosphatase-β (G6PC3) deficiency in a model of mouse neutrophil granulocyte differentiation.

PubMed

Gautam, S; Kirschnek, S; Gentle, I E; Kopiniok, C; Henneke, P; Häcker, H; Malleret, L; Belaaouaj, A; Häcker, G

2013-08-01

Differentiation of neutrophil granulocytes (neutrophils) occurs through several steps in the bone marrow and requires a coordinate regulation of factors determining survival and lineage-specific development. A number of genes are known whose deficiency disrupts neutrophil generation in humans and in mice. One of the proteins encoded by these genes, glucose-6-phosphatase-β (G6PC3), is involved in glucose metabolism. G6PC3 deficiency causes neutropenia in humans and in mice, linked to enhanced apoptosis and ER stress. We used a model of conditional Hoxb8 expression to test molecular and functional differentiation as well as survival defects in neutrophils from G6PC3(-/-) mice. Progenitor lines were established and differentiated into neutrophils when Hoxb8 was turned off. G6PC3(-/-) progenitor cells underwent substantial apoptosis when differentiation was started. Transgenic expression of Bcl-XL rescued survival; however, Bcl-XL-protected differentiated cells showed reduced proliferation, immaturity and functional deficiency such as altered MAP kinase signaling and reduced cytokine secretion. Impaired glucose utilization was found and was associated with ER stress and apoptosis, associated with the upregulation of Bim and Bax; downregulation of Bim protected against apoptosis during differentiation. ER-stress further caused a profound loss of expression and secretion of the main neutrophil product neutrophil elastase during differentiation. Transplantation of wild-type Hoxb8-progenitor cells into irradiated mice allowed differentiation into neutrophils in the bone marrow in vivo. Transplantation of G6PC3(-/-) cells yielded few mature neutrophils in bone marrow and peripheral blood. Transgenic Bcl-XL permitted differentiation of G6PC3(-/-) cells in vivo. However, functional deficiencies and differentiation abnormalities remained. Differentiation of macrophages from Hoxb8-dependent progenitors was only slightly disturbed. A combination of defects in differentiation and survival thus underlies neutropenia in G6PC3(-/-) deficiency, both originating from a reduced ability to utilize glucose. Hoxb8-dependent cells are a model to study differentiation and survival of the neutrophil lineage.

Data supporting mitochondrial morphological changes by SPG13-associated HSPD1 mutants.

PubMed

Miyamoto, Yuki; Megumi, Funakoshi-Tago; Hasegawa, Nanami; Eguchi, Takahiro; Tanoue, Akito; Tamura, Hiroomi; Yamauchi, Junji

2016-03-01

The data is related to the research article entitled "Hypomyelinating leukodystrophy-associated missense mutation in HSPD1 blunts mitochondrial dynamics" [1]. In addition to hypomyelinating leukodystrophy (HLD) 4 (OMIM no. 612233), it is known that spastic paraplegia (SPG) 13 (OMIM no. 605280) is caused by HSPD1's amino acid mutation. Two amino acid mutations Val-98-to-Ile (V98I) and Gln-461-to-Glu (Q461E) are associated with SPG13 [2]. In order to investigate the effects of HSPD1's V98I or Q461E mutant on mitochondrial morphological changes, we transfected each of the respective mutant-encoding genes into Cos-7 cells. Either of V98I or Q461E mutant exhibited increased number of mitochondria and short length mitochondrial morphologies. Using MitoTracker dye-incorporating assay, decreased mitochondrial membrane potential was also observed in both cases. The data described here supports that SPG13-associated HSPD1 mutant participates in causing aberrant mitochondrial morphological changes with decreased activities.

Analysis of Mycobacterium avium subsp. paratuberculosis mutant libraries reveals loci-dependent transcription biases and strategies to novel mutant discovery

USDA-ARS?s Scientific Manuscript database

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease in ruminants and it has been implicated as a cause of Crohn’s disease in humans. The generation of comprehensive random mutant banks by transposon mutagenesis is a fundamental wide genomic technology utilized...

Plastocyanin Controls the Stabilization of the Thylakoid Cu-transporting P-type ATPase PAA2/HMA8 in Response to Low Copper in Arabidopsis*

PubMed Central

Tapken, Wiebke; Ravet, Karl; Pilon, Marinus

2012-01-01

PAA2/HMA8 (P-type ATPase of Arabidopsis/Heavy-metal-associated 8) is a thylakoid located copper (Cu)-transporter in Arabidopsis thaliana. In tandem with PAA1/HMA6, which is located in the inner chloroplast envelope, it supplies Cu to plastocyanin (PC), an essential cuproenzyme of the photosynthetic machinery. We investigated whether the chloroplast Cu transporters are affected by Cu addition to the growth media. Immunoblots showed that PAA2 protein abundance decreased significantly and specifically when Cu in the media was increased, while PAA1 remained unaffected. The function of SPL7, the transcriptional regulator of Cu homeostasis, was not required for this regulation of PAA2 protein abundance and Cu addition did not affect PAA2 transcript levels, as determined by qRT-PCR. We used the translational inhibitor cycloheximide to analyze turnover and observed that the stability of the PAA2 protein was decreased in plants grown with elevated Cu. Interestingly, PAA2 protein abundance was significantly increased in paa1 mutants, in which the Cu content in the chloroplast is half of that of the wild-type, due to impaired Cu import into the organelle. In contrast in a pc2 insertion mutant, which has strongly reduced plastocyanin expression, the PAA2 protein levels were low regardless of Cu addition to the growth media. Together, these data indicate that plastid Cu levels control PAA2 stability and that plastocyanin, which is the target of PAA2 mediated Cu delivery in thylakoids, is a major determinant of this regulatory mechanism. PMID:22493454

Abnormal cerebellar development and Purkinje cell defects in Lgl1-Pax2 conditional knockout mice.

PubMed

Hou, Congzhe; Ding, Lingcui; Zhang, Jian; Jin, Yecheng; Sun, Chen; Li, Zhenzu; Sun, Xiaoyang; Zhang, Tingting; Zhang, Aizhen; Li, Huashun; Gao, Jiangang

2014-11-01

Lgl1 was initially identified as a tumour suppressor in flies and is characterised as a key regulator of epithelial polarity and asymmetric cell division. A previous study indicated that More-Cre-mediated Lgl1 knockout mice exhibited significant brain dysplasia and died within 24h after birth. To overcome early neonatal lethality, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in almost all cells in the cerebellum, and we examined the functions of Lgl1 in the cerebellum. Impaired motor coordination was detected in the mutant mice. Consistent with this abnormal behaviour, homozygous mice possessed a smaller cerebellum with fewer lobes, reduced granule precursor cell (GPC) proliferation, decreased Purkinje cell (PC) quantity and dendritic dysplasia. Loss of Lgl1 in the cerebellum led to hyperproliferation and impaired differentiation of neural progenitors in ventricular zone. Based on the TUNEL assay, we observed increased apoptosis in the cerebellum of mutant mice. We proposed that impaired differentiation and increased apoptosis may contribute to decreased PC quantity. To clarify the effect of Lgl1 on cerebellar granule cells, we used Math1-Cre to specifically delete Lgl1 in granule cells. Interestingly, the Lgl1-Math1 conditional knockout mice exhibited normal proliferation of GPCs and cerebellar development. Thus, we speculated that the reduction in the proliferation of GPCs in Lgl1-Pax2 conditional knockout mice may be secondary to the decreased number of PCs, which secrete the mitogenic factor Sonic hedgehog to regulate GPC proliferation. Taken together, these findings suggest that Lgl1 plays a key role in cerebellar development and folia formation by regulating the development of PCs. Copyright © 2014. Published by Elsevier Inc.

Tracking synthesis and turnover of triacylglycerol in leaves

PubMed Central

Tjellström, Henrik; Strawsine, Merissa; Ohlrogge, John B.

2015-01-01

Triacylglycerol (TAG), typically represents <1% of leaf glycerolipids but can accumulate under stress and other conditions or if leaves are supplied with fatty acids, or in plants transformed with regulators or enzymes of lipid metabolism. To better understand the metabolism of TAG in leaves, pulse–chase radiolabelling experiments were designed to probe its synthesis and turnover. When Arabidopsis leaves were incubated with [14C]lauric acid (12:0), a major initial product was [14C]TAG. Thus, despite low steady-state levels, leaves possess substantial TAG biosynthetic capacity. The contributions of diacylglycerol acyltransferase1 and phospholipid:diacylglycerol acyltransferase1 to leaf TAG synthesis were examined by labelling of dgat1 and pdat1 mutants. The dgat1 mutant displayed a major (76%) reduction in [14C]TAG accumulation whereas pdat1 TAG labelling was only slightly reduced. Thus, DGAT1 has a principal role in TAG biosynthesis in young leaves. During a 4h chase period, radioactivity in TAG declined 70%, whereas the turnover of [14C]acyl chains of phosphatidylcholine (PC) and other polar lipids was much lower. Sixty percent of [14C]12:0 was directly incorporated into glycerolipids without modification, whereas 40% was elongated and desaturated to 16:0 and 18:1 by plastids. The unmodified [14C]12:0 and the plastid products of [14C]12:0 metabolism entered different pathways. Although plastid-modified 14C-labelled products accumulated in monogalactosyldiacylglycerol, PC, phosphatidylethanolamine, and diacylglcerol (DAG), there was almost no accumulation of [14C]16:0 and [14C]18:1 in TAG. Because DAG and acyl-CoA are direct precursors of TAG, the differential labelling of polar glycerolipids and TAG by [14C]12:0 and its plastid-modified products provides evidence for multiple subcellular pools of both acyl-CoA and DAG. PMID:25609824

Experimental study on the neurotoxic effect of β-amyloid on the cytoskeleton of PC12 cells

PubMed Central

Shi, Zhenyu; Fan, Wenjuan; Liu, Hongliang; Deng, Jinbo; Deng, Jiexin

2018-01-01

The aim of the present study was to establish a cell model of Alzheimer's disease (AD) and investigate the neurotoxic effects of β-amyloid (Aβ) on the cytoskeleton. PC12 cells were cultured and treated with Aβ25-35, and cell survival was analyzed with the MTT assay. Cell apoptosis was visualized using 4′,6-diamidino-2-phenylindole staining and the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Immunocytochemistry and phalloidin staining were used to label the cytoskeleton of PC12 cells. Aβ25-35 was found to induce PC12 cell apoptosis in a dose-dependent manner (P<0.05). Moreover, Aβ25-35 also caused dose-dependent disintegration of the cytoskeleton (P<0.05). Therefore, the PC12 cell cytoskeleton was found to be sensitive to Aβ25-35 neurotoxicity. The disintegration of the cytoskeleton is likely an important pathological alteration in AD, and Aβ is a key molecule involved in AD pathogenesis. PMID:29436599

Structure of the polycystic kidney disease TRP channel Polycystin-2 (PC2).

PubMed

Grieben, Mariana; Pike, Ashley C W; Shintre, Chitra A; Venturi, Elisa; El-Ajouz, Sam; Tessitore, Annamaria; Shrestha, Leela; Mukhopadhyay, Shubhashish; Mahajan, Pravin; Chalk, Rod; Burgess-Brown, Nicola A; Sitsapesan, Rebecca; Huiskonen, Juha T; Carpenter, Elisabeth P

2017-02-01

Mutations in either polycystin-1 (PC1 or PKD1) or polycystin-2 (PC2, PKD2 or TRPP1) cause autosomal-dominant polycystic kidney disease (ADPKD) through unknown mechanisms. Here we present the structure of human PC2 in a closed conformation, solved by electron cryomicroscopy at 4.2-Å resolution. The structure reveals a novel polycystin-specific 'tetragonal opening for polycystins' (TOP) domain tightly bound to the top of a classic transient receptor potential (TRP) channel structure. The TOP domain is formed from two extensions to the voltage-sensor-like domain (VSLD); it covers the channel's endoplasmic reticulum lumen or extracellular surface and encloses an upper vestibule, above the pore filter, without blocking the ion-conduction pathway. The TOP-domain fold is conserved among the polycystins, including the homologous channel-like region of PC1, and is the site of a cluster of ADPKD-associated missense variants. Extensive contacts among the TOP-domain subunits, the pore and the VSLD provide ample scope for regulation through physical and chemical stimuli.

Manipulating the dipole layer of polar organic molecules on metal surfaces via different charge-transfer channels

NASA Astrophysics Data System (ADS)

Lin, Meng-Kai; Nakayama, Yasuo; Zhuang, Ying-Jie; Wang, Chin-Yung; Pi, Tun-Wen; Ishii, Hisao; Tang, S.-J.

The key properties of organic films such as energy level alignment (ELA), work functions, and injection barriers are closely linked to this dipole layer. Using angle resolved photoemission spectroscopy (ARPES), we systemically investigate the coverage-dependent work functions and spectra line shapes of occupied molecular orbital states of a polar molecule, chloroaluminium phthalocyanine (ClAlPc), grown on Ag(111) to show that the orientations of the first ClAlPc layer can be manipulated via the molecule deposition rate and post annealing, causing ELA at organic-metal interface to differ for about 0.3 eV between Cl-up and Cl-down configuration. Moreover, by comparing the experimental results with the calculations based on both gas-phase model and realistic model of ClAlPc on Ag(111) , we evidence that the different orientations of ClAlPc dipole layers lead to different charge-transfer channels between ClAlPc and Ag, a key factor that controls the ELA at organic-metal interface.

MtDNA depleted PC3 cells exhibit Warburg effect and cancer stem cell features

PubMed Central

Li, Xiaoran; Zhong, Yali; Lu, Jie; Axcrona, Karol; Eide, Lars; Syljuåsen, Randi G.; Peng, Qian; Wang, Junbai; Zhang, Hongquan; Goscinski, Mariusz Adam; Kvalheim, Gunnar; Nesland, Jahn M.; Suo, Zhenhe

2016-01-01

Reducing mtDNA content was considered as a critical step in the metabolism restructuring for cell stemness restoration and further neoplastic development. However, the connections between mtDNA depletion and metabolism reprograming-based cancer cell stemness in prostate cancers are still lack of studies. Here, we demonstrated that human CRPC cell line PC3 tolerated high concentration of the mtDNA replication inhibitor ethidium bromide (EtBr) and the mtDNA depletion triggered a universal metabolic remodeling process. Failure in completing that process caused lethal consequences. The mtDNA depleted (MtDP) PC3 cells could be steadily maintained in the special medium in slow cycling status. The MtDP PC3 cells contained immature mitochondria and exhibited Warburg effect. Furthermore, the MtDP PC3 cells were resistant to therapeutic treatments and contained greater cancer stem cell-like subpopulations: CD44+, ABCG2+, side-population and ALDHbright. In conclusion, these results highlight the association of mtDNA content, mitochondrial function and cancer cell stemness features. PMID:27248169

Generation of Pc 1 waves by the ion temperature anisotropy associated with fast shocks caused by sudden impulses

NASA Technical Reports Server (NTRS)

Mandt, M. E.; Lee, L. C.

1991-01-01

The high correlation of Pc 1 events with magnetospheric compressions is known. A mechanism is proposed which leads to the generation of Pc 1 waves. The interaction of a dynamic pressure pulse with the earth's bow shock leads to the formation of a weak fast-mode shock propagating into the magnetoshealth. The shock wave can pass right through a tangential discontinuity (magnetopause) and into the magnetosphere, without disturbing either of the structures. In a quasiperpendicular geometry, the shock wave exhibits anisotropic heating. This anisotropy drives unstable ion-cyclotron waves which can contribute to the generation of the Pc 1 waves which are detected. The viability of the mechanism is demonstrated with simulations. This mechanism could explain the peak in the occurrence of observed Pc 1 waves in the postnoon sector where a field-aligned discontinuity in the solar wind would most often be parallel to the magnetopause surface due to the average Parker-spiral magnetic-field configuration.

Tl(I) and Tl(III) activate both mitochondrial and extrinsic pathways of apoptosis in rat pheochromocytoma (PC12) cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hanzel, Cecilia Eliana; Verstraeten, Sandra Viviana

2009-04-01

Thallium (Tl) is a highly toxic metal though yet its mechanisms are poorly understood. Previously, we demonstrated that rat pheochromocytoma (PC12) cells exposure to thallous (Tl(I)) or thallic (Tl(III)) cations leads to mitochondrial damage and reduced cell viability. In the present work we comparatively characterized the possible pathways involved in Tl(I)- and Tl(III)- (10-100 {mu}M) mediated decrease in PC12 cells viability. We observed that these cations do not cause cell necrosis but significantly increased the number of cells with apoptotic features. Both cations lead to Bax oligomerization and caused apoptosis inducing factor (AIF), endonuclease G (Endo G), and cytochrome cmore » release from mitochondria, but they did not activate caspase dependent DNAse (CAD). Tl(I)- and Tl(III)-dependent caspases 9 and 3 activation followed similar kinetics, with maximal effects at 18 h of incubation. In addition, Tl(I) promoted phosphatidylserine (PS) exposure. Tl(III) induced 2- and 18-fold increase in Fas content and caspase 8 activity, respectively. Together, experimental results show that Tl(I) and Tl(III) induce PC12 cells apoptosis, although differential pathways are involved. While Tl(I)-mediated cell apoptosis was mainly associated with mitochondrial damage, Tl(III) showed a mixed effect triggering both the intrinsic and extrinsic pathways of apoptosis. These findings contribute to a better understanding of the mechanisms underlying Tl-induced loss of cell viability in PC12 cells.« less

Selection of lys2 Mutants of the Yeast SACCHAROMYCES CEREVISIAE by the Utilization of α-AMINOADIPATE

PubMed Central

Chattoo, Bharat B.; Sherman, Fred; Azubalis, Dalia A.; Fjellstedt, Thorsten A.; Mehnert, David; Ogur, Maurice

1979-01-01

Normal strains of Saccharomyces cerevisiae do not use α-aminoadipate as a principal nitrogen source. However, α-aminoadipate is utilized as a nitrogen source by lys2 and lys5 strains having complete or partial deficiencies of α-aminoadipate reductase and, to a limited extent, by heterozygous lys2/+ strains. Lys2 mutants were conveniently selected on media containing α-aminoadipate as a nitrogen source, lysine, and other supplements to furnish other possible auxotrophic requirements. The lys2 mutations were obtained in a variety of laboratory strains containing other markers, including other lysine mutations. In addition to the predominant class of lys2 mutants, low frequencies of lys5 mutants and mutants not having any obvious lysine requirement were recovered on α-aminoadipate medium. The mutants not requiring lysine appeared to have mutations at the lys2 locus that caused partial deficiencies of α-aminoadipate reductase. Such partial deficiencies are believed to be sufficiently permissive to allow lysine biosynthesis, but sufficiently restrictive to allow for the utilization of α-aminoadipate. Although it is unknown why partial or complete deficiencies of α-aminoadipate reductase cause utilization of α-aminoadipate as a principal nitrogen source, the use of α-aminoadipate medium has considerable utility as a selective medium for lys2 and lys5 mutants. PMID:17248969

Munc18-1 is a molecular chaperone for α-synuclein, controlling its self-replicating aggregation.

PubMed

Chai, Ye Jin; Sierecki, Emma; Tomatis, Vanesa M; Gormal, Rachel S; Giles, Nichole; Morrow, Isabel C; Xia, Di; Götz, Jürgen; Parton, Robert G; Collins, Brett M; Gambin, Yann; Meunier, Frédéric A

2016-09-12

Munc18-1 is a key component of the exocytic machinery that controls neurotransmitter release. Munc18-1 heterozygous mutations cause developmental defects and epileptic phenotypes, including infantile epileptic encephalopathy (EIEE), suggestive of a gain of pathological function. Here, we used single-molecule analysis, gene-edited cells, and neurons to demonstrate that Munc18-1 EIEE-causing mutants form large polymers that coaggregate wild-type Munc18-1 in vitro and in cells. Surprisingly, Munc18-1 EIEE mutants also form Lewy body-like structures that contain α-synuclein (α-Syn). We reveal that Munc18-1 binds α-Syn, and its EIEE mutants coaggregate α-Syn. Likewise, removal of endogenous Munc18-1 increases the aggregative propensity of α-Syn(WT) and that of the Parkinson's disease-causing α-Syn(A30P) mutant, an effect rescued by Munc18-1(WT) expression, indicative of chaperone activity. Coexpression of the α-Syn(A30P) mutant with Munc18-1 reduced the number of α-Syn(A30P) aggregates. Munc18-1 mutations and haploinsufficiency may therefore trigger a pathogenic gain of function through both the corruption of native Munc18-1 and a perturbed chaperone activity for α-Syn leading to aggregation-induced neurodegeneration. © 2016 Chai et al.

Genes Required for Vacuolar Acidity in Saccharomyces Cerevisiae

PubMed Central

Preston, R. A.; Reinagel, P. S.; Jones, E. W.

1992-01-01

Mutations that cause loss of acidity in the vacuole (lysosome) of Saccharomyces cerevisiae were identified by screening colonies labeled with the fluorescent, pH-sensitive, vacuolar labeling agent, 6-carboxyfluorescein. Thirty nine vacuolar pH (Vph(-)) mutants were identified. Four of these contained mutant alleles of the previously described PEP3, PEP5, PEP6 and PEP7 genes. The remaining mutants defined eight complementation groups of vph mutations. No alleles of the VAT2 or TFP1 genes (known to encode subunits of the vacuolar H(+)-ATPase) were identified in the Vph(-) screen. Strains bearing mutations in any of six of the VPH genes failed to grow on medium buffered at neutral pH; otherwise, none of the vph mutations caused notable growth inhibition on standard yeast media. Expression of the vacuolar protease, carboxypeptidase Y, was defective in strains bearing vph4 mutations but was apparently normal in strains bearing any of the other vph mutations. Defects in vacuolar morphology at the light microscope level were evident in all Vph(-) mutants. Strains that contained representative mutant alleles of the 17 previously described PEP genes were assayed for vacuolar pH; mutations in seven of the PEP genes (including PEP3, PEP5, PEP6 and PEP7) caused loss of vacuolar acidity. PMID:1628805

Immunization by intrabronchial administration to 1-week-old foals of an unmarked double gene disruption strain of Rhodococcus equi strain 103+.

PubMed

Pei, Yanlong; Nicholson, Vivian; Woods, Katharine; Prescott, John F

2007-11-15

Rhodococcus equi causes fatal granulomatous pneumonia in foals and immunocompromised animals and humans. However, there is no effective vaccine against this infection. In this study, the chromosomal genes isocitrate lyase (icl) and cholesterol oxidase (choE) were chosen as targets for mutation and assessment of the double mutant as an intrabronchial vaccine in 1-week-old foals. Using a modification of a suicide plasmid previously developed in this laboratory, we developed a choE-icl unmarked deletion mutant of R. equi strain 103+. Five 1-week-old foals were infected intrabronchially with the mutant and challenged intrabronchially with the parent, virulent, strain 2 weeks later. Three of the foals were protected against pneumonia caused by the virulent strain, but the other two foals developed pneumonia caused by the mutant strain during the post-challenge period. Since infection of 3-week-old foals by an icl mutant in an earlier study had shown complete attenuation of the strain, we conclude that a proportion of foals in the 1st week or so of life are predisposed to developing R. equi pneumonia because of an inability to mount an effective immune response. This has been suspected previously but this is the first time that this has been demonstrated experimentally.

Munc18-1 is a molecular chaperone for α-synuclein, controlling its self-replicating aggregation

PubMed Central

Giles, Nichole; Morrow, Isabel C.; Collins, Brett M.

2016-01-01

Munc18-1 is a key component of the exocytic machinery that controls neurotransmitter release. Munc18-1 heterozygous mutations cause developmental defects and epileptic phenotypes, including infantile epileptic encephalopathy (EIEE), suggestive of a gain of pathological function. Here, we used single-molecule analysis, gene-edited cells, and neurons to demonstrate that Munc18-1 EIEE-causing mutants form large polymers that coaggregate wild-type Munc18-1 in vitro and in cells. Surprisingly, Munc18-1 EIEE mutants also form Lewy body–like structures that contain α-synuclein (α-Syn). We reveal that Munc18-1 binds α-Syn, and its EIEE mutants coaggregate α-Syn. Likewise, removal of endogenous Munc18-1 increases the aggregative propensity of α-SynWT and that of the Parkinson’s disease–causing α-SynA30P mutant, an effect rescued by Munc18-1WT expression, indicative of chaperone activity. Coexpression of the α-SynA30P mutant with Munc18-1 reduced the number of α-SynA30P aggregates. Munc18-1 mutations and haploinsufficiency may therefore trigger a pathogenic gain of function through both the corruption of native Munc18-1 and a perturbed chaperone activity for α-Syn leading to aggregation-induced neurodegeneration. PMID:27597756

5-Fluoroindole Resistance Identifies Tryptophan Synthase Beta Subunit Mutants in Arabidopsis Thaliana

PubMed Central

Barczak, A. J.; Zhao, J.; Pruitt, K. D.; Last, R. L.

1995-01-01

A study of the biochemical genetics of the Arabidopsis thaliana tryptophan synthase beta subunit was initiated by characterization of mutants resistant to the inhibitor 5-fluoroindole. Thirteen recessive mutations were recovered that are allelic to trp2-1, a mutation in the more highly expressed of duplicate tryptophan synthase beta subunit genes (TSB1). Ten of these mutations (trp2-2 through trp2-11) cause a tryptophan requirement (auxotrophs), whereas three (trp2-100 through trp2-102) remain tryptophan prototrophs. The mutations cause a variety of changes in tryptophan synthase beta expression. For example, two mutations (trp2-5 and trp2-8) cause dramatically reduced accumulation of TSB mRNA and immunologically detectable protein, whereas trp2-10 is associated with increased mRNA and protein. A correlation exists between the quantity of mutant beta and wild-type alpha subunit levels in the trp2 mutant plants, suggesting that the synthesis of these proteins is coordinated or that the quantity or structure of the beta subunit influences the stability of the alpha protein. The level of immunologically detectable anthranilate synthase alpha subunit protein is increased in the trp2 mutants, suggesting the possibility of regulation of anthranilate synthase levels in response to tryptophan limitation. PMID:7635295

Gain and loss of function of ALS-related mutations of TARDBP (TDP-43) cause motor deficits in vivo.

PubMed

Kabashi, Edor; Lin, Li; Tradewell, Miranda L; Dion, Patrick A; Bercier, Valérie; Bourgouin, Patrick; Rochefort, Daniel; Bel Hadj, Samar; Durham, Heather D; Vande Velde, Christine; Rouleau, Guy A; Drapeau, Pierre

2010-02-15

TDP-43 has been found in inclusion bodies of multiple neurological disorders, including amyotrophic lateral sclerosis, frontotemporal dementia, Parkinson's disease and Alzheimer's disease. Mutations in the TDP-43 encoding gene, TARDBP, have been subsequently reported in sporadic and familial ALS patients. In order to investigate the pathogenic nature of these mutants, the effects of three consistently reported TARDBP mutations (A315T, G348C and A382T) were tested in cell lines, primary cultured motor neurons and living zebrafish embryos. Each of the three mutants and wild-type (WT) human TDP-43 localized to nuclei when expressed in COS1 and Neuro2A cells by transient transfection. However, when expressed in motor neurons from dissociated spinal cord cultures these mutant TARDBP alleles, but less so for WT TARDBP, were neurotoxic, concomitant with perinuclear localization and aggregation of TDP-43. Finally, overexpression of mutant, but less so of WT, human TARDBP caused a motor phenotype in zebrafish (Danio rerio) embryos consisting of shorter motor neuronal axons, premature and excessive branching as well as swimming deficits. Interestingly, knock-down of zebrafisfh tardbp led to a similar phenotype, which was rescued by co-expressing WT but not mutant human TARDBP. Together these approaches showed that TARDBP mutations cause motor neuron defects and toxicity, suggesting that both a toxic gain of function as well as a novel loss of function may be involved in the molecular mechanism by which mutant TDP-43 contributes to disease pathogenesis.

Modulation of the antioxidant/pro-oxidant balance, cytotoxicity and antiviral actions of grape seed extracts.

PubMed

Ignea, Codruţa; Dorobanţu, Cristina Mihaela; Mintoff, Christopher Paul; Branza-Nichita, Norica; Ladomery, Michael R; Kefalas, Panagiotis; Chedea, Veronica Sanda

2013-12-15

Grape seed extracts (GSEs) were investigated in yeast cells harbouring defects in their antioxidant system (regarding the cellular growth and growth recovery from H2O2 insult). GSEs antioxidant activity was detected in wild-type and mutant strains Δcta1, Δgsh1 and Δoye2glr1, while pro-oxidant activity in Δsod1 cells was seen. Assessment of proliferation of prostate cancer PC3 and HBV-replicating HepG2 2.2.15 cells treated with GSEs has shown higher cytotoxicity of red grape seed extract (RW) than white grape seed extract (WW) subjective to dose and period of administration. No antiviral effect was detected by measuring the secreted virion particles in HepG2 2.2.15 cells treated with GSEs. The GSEs play a dual antioxidant/pro-oxidant role in vivo according with the cellular antioxidant system deficiencies and exhibit cytotoxic properties in PC3 and HepG2 2.2.15 cell lines, but no antiviral action against HBV. Copyright © 2013 Elsevier Ltd. All rights reserved.

Poliovirus Mutants Resistant to Neutralization with Soluble Cell Receptors

NASA Astrophysics Data System (ADS)

Kaplan, Gerardo; Peters, David; Racaniello, Vincent R.

1990-12-01

Poliovirus mutants resistant to neutralization with soluble cellular receptor were isolated. Replication of soluble receptor-resistant (srr) mutants was blocked by a monoclonal antibody directed against the HeLa cell receptor for poliovirus, indicating that the mutants use this receptor to enter cells. The srr mutants showed reduced binding to HeLa cells and cell membranes. However, the reduced binding phenotype did not have a major impact on viral replication, as judged by plaque size and one-step growth curves. These results suggest that the use of soluble receptors as antiviral agents could lead to the selection of neutralization-resistant mutants that are able to bind cell surface receptors, replicate, and cause disease.

Role of Iron Uptake Systems in Pseudomonas aeruginosa Virulence and Airway Infection

PubMed Central

Minandri, Fabrizia; Imperi, Francesco; Frangipani, Emanuela; Bonchi, Carlo; Visaggio, Daniela; Facchini, Marcella; Pasquali, Paolo; Bragonzi, Alessandra

2016-01-01

Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia and chronic lung infections in cystic fibrosis patients. Iron is essential for bacterial growth, and P. aeruginosa expresses multiple iron uptake systems, whose role in lung infection deserves further investigation. P. aeruginosa Fe3+ uptake systems include the pyoverdine and pyochelin siderophores and two systems for heme uptake, all of which are dependent on the TonB energy transducer. P. aeruginosa also has the FeoB transporter for Fe2+ acquisition. To assess the roles of individual iron uptake systems in P. aeruginosa lung infection, single and double deletion mutants were generated in P. aeruginosa PAO1 and characterized in vitro, using iron-poor media and human serum, and in vivo, using a mouse model of lung infection. The iron uptake-null mutant (tonB1 feoB) and the Fe3+ transport mutant (tonB1) did not grow aerobically under low-iron conditions and were avirulent in the mouse model. Conversely, the wild type and the feoB, hasR phuR (heme uptake), and pchD (pyochelin) mutants grew in vitro and caused 60 to 90% mortality in mice. The pyoverdine mutant (pvdA) and the siderophore-null mutant (pvdA pchD) grew aerobically in iron-poor media but not in human serum, and they caused low mortality in mice (10 to 20%). To differentiate the roles of pyoverdine in iron uptake and virulence regulation, a pvdA fpvR double mutant defective in pyoverdine production but expressing wild-type levels of pyoverdine-regulated virulence factors was generated. Deletion of fpvR in the pvdA background partially restored the lethal phenotype, indicating that pyoverdine contributes to the pathogenesis of P. aeruginosa lung infection by combining iron transport and virulence-inducing capabilities. PMID:27271740

The effect of novel rhenium compounds on lymphosarcoma, PC-3 prostate and myeloid leukemia cancer cell lines and an investigation on the DNA binding properties of one of these compounds through electronic spectroscopy

PubMed Central

Parson, Carl; Smith, Valerie; Krauss, Christopher; Banerjee, Hirendra N.; Reilly, Christopher; Krause, Jeanette A.; Wachira, James M.; Giri, Dipak; Winstead, Angela; Mandal, Santosh K.

2014-01-01

Despite the tremendous success of cisplatin and other platinum-based anticancer drugs, severe toxicity and resistance to tumors limit their applications. It is believed that the coordination (formation of covalent bond) of the metal (platinum) to the nitrogen bases of DNA cause the ruptures of the cancer as well as normal cells. A search for anticancer drugs with different modes of action resulted in the synthesis of variety of novel compounds. Many of them are in clinical trials now. Recently we synthesized a series of novel rhenium pentylcarbonato compounds (PC1–PC6). The rhenium atom in each compound is coordinated (bonded) to a planar polypyridyl aromatic ligand, thereby forcing each compound to intercalate between the DNA bases. We have investigated the DNA binding properties of one of the PC-series of compounds (PC6) using electronic spectroscopy. The UV absorption titration of PC6 with DNA shows hypochromic effect with concomitant bathochromic shift of the charge transfer band at 290 nm. These results suggest that the compound PC6 binds to DNA through intercalation. It is therefore likely that the other PC-series of compounds will behave in a similar manner. Thus it is expected that these compounds will exhibit negligible or no side effect. We have observed that the PC-series of compounds are strong cytotoxic agents against lymphosarcoma (average GI50 ≈ 2±2.6 µM), PC-3 prostate (average GI50 ≈ 3±2.8 µM) and myeloid leukemia (average GI50 ≈ 3±2.8 µM) cancer cell lines. The average GI50 values of the PC-series of compounds are 2–3 less than the corresponding GI50 values of cisplatin. Also each of the PC-series of compounds exhibits less toxicity than cisplatin in the glomerular mesangial cells. PMID:25221731

A Deafness- and Diabetes-associated tRNA Mutation Causes Deficient Pseudouridinylation at Position 55 in tRNAGlu and Mitochondrial Dysfunction*

PubMed Central

Wang, Meng; Liu, Hao; Zheng, Jing; Chen, Bobei; Zhou, Mi; Fan, Wenlu; Wang, Hen; Liang, Xiaoyang; Zhou, Xiaolong; Eriani, Gilbert; Jiang, Pingping; Guan, Min-Xin

2016-01-01

Several mitochondrial tRNA mutations have been associated with maternally inherited diabetes and deafness. However, the pathophysiology of these tRNA mutations remains poorly understood. In this report, we identified the novel homoplasmic 14692A→G mutation in the mitochondrial tRNAGlu gene among three Han Chinese families with maternally inherited diabetes and deafness. The m.14692A→G mutation affected a highly conserved uridine at position 55 of the TΨC loop of tRNAGlu. The uridine is modified to pseudouridine (Ψ55), which plays an important role in the structure and function of this tRNA. Using lymphoblastoid cell lines derived from a Chinese family, we demonstrated that the m.14692A→G mutation caused loss of Ψ55 modification and increased angiogenin-mediated endonucleolytic cleavage in mutant tRNAGlu. The destabilization of base-pairing (18A-Ψ55) caused by the m.14692A→G mutation perturbed the conformation and stability of tRNAGlu. An approximately 65% decrease in the steady-state level of tRNAGlu was observed in mutant cells compared with control cells. A failure in tRNAGlu metabolism impaired mitochondrial translation, especially for polypeptides with a high proportion of glutamic acid codons such as ND1, ND6, and CO2 in mutant cells. An impairment of mitochondrial translation caused defective respiratory capacity, especially reducing the activities of complexes I and IV. Furthermore, marked decreases in the levels of mitochondrial ATP and membrane potential were observed in mutant cells. These mitochondrial dysfunctions caused an increasing production of reactive oxygen species in the mutant cells. Our findings may provide new insights into the pathophysiology of maternally inherited diabetes and deafness, which is primarily manifested by the deficient nucleotide modification of mitochondrial tRNAGlu. PMID:27519417

Isolation of Novel Synthetic Prion Strains by Amplification in Transgenic Mice Coexpressing Wild-Type and Anchorless Prion Proteins

PubMed Central

Raymond, Gregory J.; Race, Brent; Hollister, Jason R.; Offerdahl, Danielle K.; Moore, Roger A.; Kodali, Ravindra; Raymond, Lynne D.; Hughson, Andrew G.; Rosenke, Rebecca; Long, Dan; Dorward, David W.

2012-01-01

Mammalian prions are thought to consist of misfolded aggregates (protease-resistant isoform of the prion protein [PrPres]) of the cellular prion protein (PrPC). Transmissible spongiform encephalopathy (TSE) can be induced in animals inoculated with recombinant PrP (rPrP) amyloid fibrils lacking mammalian posttranslational modifications, but this induction is inefficient in hamsters or transgenic mice overexpressing glycosylphosphatidylinositol (GPI)-anchored PrPC. Here we show that TSE can be initiated by inoculation of misfolded rPrP into mice that express wild-type (wt) levels of PrPC and that synthetic prion strain propagation and selection can be affected by GPI anchoring of the host's PrPC. To create prions de novo, we fibrillized mouse rPrP in the absence of molecular cofactors, generating fibrils with a PrPres-like protease-resistant banding profile. These fibrils induced the formation of PrPres deposits in transgenic mice coexpressing wt and GPI-anchorless PrPC (wt/GPI−) at a combined level comparable to that of PrPC expression in wt mice. Secondary passage into mice expressing wt, GPI−, or wt plus GPI− PrPC induced TSE disease with novel clinical, histopathological, and biochemical phenotypes. Contrary to laboratory-adapted mouse scrapie strains, the synthetic prion agents exhibited a preference for conversion of GPI− PrPC and, in one case, caused disease only in GPI− mice. Our data show that novel TSE agents can be generated de novo solely from purified mouse rPrP after amplification in mice coexpressing normal levels of wt and anchorless PrPC. These observations provide insight into the minimal elements required to create prions in vitro and suggest that the PrPC GPI anchor can modulate the propagation of synthetic TSE strains. PMID:22915801

Identification of novel putative-binding proteins for cellular prion protein and a specific interaction with the STIP1 homology and U-Box-containing protein 1

PubMed Central

Gimenez, Ana Paula Lappas; Richter, Larissa Morato Luciani; Atherino, Mariana Campos; Beirão, Breno Castello Branco; Fávaro, Celso; Costa, Michele Dietrich Moura; Zanata, Silvio Marques; Malnic, Bettina; Mercadante, Adriana Frohlich

2015-01-01

ABSTRACT Prion diseases involve the conversion of the endogenous cellular prion protein, PrPC, into a misfolded infectious isoform, PrPSc. Several functions have been attributed to PrPC, and its role has also been investigated in the olfactory system. PrPC is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp−/− mice showed impaired behavior in olfactory tests. Given the high PrPC expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrPC-binding partners. Ten different putative PrPC ligands were identified, which were involved in functions such as cellular proliferation and apoptosis, cytoskeleton and vesicle transport, ubiquitination of proteins, stress response, and other physiological processes. In vitro binding assays confirmed the interaction of PrPC with STIP1 homology and U-Box containing protein 1 (Stub1) and are reported here for the first time. Stub1 is a co-chaperone with ubiquitin E3-ligase activity, which is associated with neurodegenerative diseases characterized by protein misfolding and aggregation. Physiological and pathological implications of PrPC-Stub1 interaction are under investigation. The PrPC-binding proteins identified here are not exclusive to the OE, suggesting that these interactions may occur in other tissues and play general biological roles. These data corroborate the proposal that PrPC is part of a multiprotein complex that modulates several cellular functions and provide a platform for further studies on the physiological and pathological roles of prion protein. PMID:26237451

Evaluation of Silicon Phthalocyanine 4 Photodynamic Therapy Against Human Cervical Cancer Cells In Vitro and in Mice

PubMed Central

Gadzinski, Jill A.; Guo, Jianxia; Philips, Brian J.; Basse, Per; Craig, Ethan K.; Bailey, Lisa; Comerci, John T.; Eiseman, Julie L.

2017-01-01

Background Cervical cancer is the second most common cancer in women worldwide [1]. Photodynamic therapy has been used for cervical intraepithelial neoplasia with good responses, but few studies have used newer phototherapeutics. We evaluated the effectiveness of photodynamic therapy using Pc 4 in vitro and in vivo against human cervical cancer cells. Methods CaSki and ME-180 cancer cells were grown as monolayers and spheroids. Cell growth and cytotoxicity were measured using a methylthiazol tetrazolium assay. Pc 4 cellular uptake and intracellular distrubtion were determined. For in vitro Pc 4 photodynamic therapy cells were irradiated at 667nm at a fluence of 2.5 J/cm2 at 48 h. SCID mice were implanted with CaSki and ME-180 cells both subcutaneously and intracervically. Forty-eight h after Pc 4 photodynamic therapy was administered at 75 and 150 J/cm2. Results The IC50s for Pc 4 and Pc 4 photodynamic therapy for CaSki and ME-180 cells as monolayers were, 7.6μM and 0.016μM and >10μM and 0.026μM; as spheroids, IC50s of Pc 4 photodynamic therapy were, 0.26μM and 0.01μM. Pc 4 was taken up within cells and widely distributed in tumors and tissues. Intracervical photodynamic therapy resulted in tumor death, however mice died due to gastrointestinal toxicity. Photodynamic therapy resulted in subcutaneous tumor death and growth delay. Conclusions Pc 4 photodynamic therapy caused death within cervical cancer cells and xenografts, supporting development of Pc 4 photodynamic therapy for treatment of cervical cancer. Support: P30-CA47904, CTSI BaCCoR Pilot Program. PMID:28890844

An epidemiological survey on the prevalence of equine peripheral dental caries in the United Kingdom and possible risk factors for its development.

PubMed

Borkent, D; Reardon, R J M; McLachlan, G; Smith, S; Dixon, P M

2017-07-01

Equine peripheral caries (PC) is an increasingly recognised disorder that causes premature wear of teeth and dental fractures and thus has major welfare implications. Little information is available on its prevalence or severity in UK horses and there are no proven associations with any risk factors. To document the prevalence of PC over a wide area of the UK, assess its intraoral distribution and severity in affected horses and examine for potential risk factors for its development. Cross sectional study. Experienced personnel were recruited for a UK wide dental survey on their patients during dental examinations. Established guidelines were used for grading PC. Frequency of PC occurrence was compared between teeth and dental arcades using McNemar's tests. Potential risk factors for PC were screened using univariable logistic regression prior to building a multivariable model. A total of 706 horses were examined by 25 participants, showing a 51.7% prevalence of PC (365/706). Some regional differences in prevalence were found. The PC primarily affected the cheek teeth with the 12 caudal being significantly more commonly affected than the 12 rostral cheek teeth. Most of the hypothesised risk factors including age, breed, sex, time at pasture and feeding of silage (haylage) were unproven. A limited association with moderate levels of concentrate feeding was observed. The presence of concurrent dental abnormalities were significantly associated with the likelihood of having PC. Not all regions in UK were included and there may be inconsistencies between examiners. Peripheral caries is common in British horses, primarily affecting the caudal cheek teeth. There was limited evidence of an association between feeding and PC. The association between PC and concurrent dental disorders indicates that these should be addressed in affected horses. © 2016 EVJ Ltd.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M.

Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associatedmore » proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.« less

Phototoxic effect of aluminium-chlorine and aluminium-hydroxide phthalocyanines on Leishmania (l.) amazonensis.

PubMed

Nesi-Reis, V; Navasconi, T R; Lera-Nosone, D S S L; Oliveira, E L; Barbosa, P M; Caetano, W; Silveira, T G V; Aristides, S M A; Hioka, N; Lonardoni, M V C

2018-03-01

This study investigated the activity of photosensitive phthalocyanines on promastigotes and amastigotes of Leishmania (L.) amazonensis. Aluminum phthalocyanine chloride (AlPcCl), Aluminum phthalocyanine hydroxide (AlPcOH) and zinc phthalocyanine (PcZn) were tested in the presence (matte red LED, potency of 2.5-2.3 μW for 30 min) and absence of light against L. amazonensis promastigotes and the parasite viability was evaluated after 24, 48 and 72 h. The amastigote forms were treated with AlPcCl and AlPcOH, following the same lighting protocols described for the promastigote forms, being evaluated after 24 h. Cytotoxicity to human erythrocytes and peritoneal macrophages was also evaluated. The results showed that AlPcCl and AlPcOH in the presence of light have antileishmania activity, with leishmanistatic effects on promastigotes and amastigotes of L. amazonensis, without causing cytotoxicity to peritoneal macrophages and erythrocytes. The concentrations that inhibited 50% of the promastigote forms after 24 h of light exposure were 0.21 ± 0.08 μM for AlPcCl and 0.23 ± 0.06 μM for AlPcOH. In 48 h and 72 h after the treatment, the IC 50 of AlPcCl was 0.13 ± 0.02 and 0.12 ± 0.03 μM and for AlPcOH was 0.14 ± 0.01 μM and 0.11 ± 0.01 μM, respectively. PcZn showed no activity on promastigotes of L. amazonensis. This study showed a substantial photodynamic activity of the phthalocyanines AlPcCl and AlPcOH against intracellular amastigotes forms of L. amazonensis after irradiation, presenting IC 50 values of 0.62 ± 0.06 μM and 0.92 ± 0.12 μM, respectively. These results support the possibility of using photodynamic therapy for the treatment of cutaneous leishmaniasis. Copyright © 2017 Elsevier B.V. All rights reserved.

(2S)-2-(3-(1-Carboxy-5-(4-211At-Astatobenzamido)Pentyl)Ureido)-Pentanedioic Acid for PSMA-Targeted α-Particle Radiopharmaceutical Therapy

PubMed Central

Kiess, Ana P.; Minn, Il; Vaidyanathan, Ganesan; Hobbs, Robert F.; Josefsson, Anders; Shen, Colette; Brummet, Mary; Chen, Ying; Choi, Jaeyeon; Koumarianou, Eftychia; Baidoo, Kwamena; Brechbiel, Martin W.; Mease, Ronnie C.; Sgouros, George; Zalutsky, Michael R.

2016-01-01

Alpha-particle emitters have a high linear energy transfer and short range, offering the potential for treating micrometastases while sparing normal tissues. We developed a urea-based, 211At-labeled small molecule targeting prostate-specific membrane antigen (PSMA) for the treatment of micrometastases due to prostate cancer (PC). Methods: PSMA-targeted (2S)-2-(3-(1-carboxy-5-(4-211At-astatobenzamido)pentyl)ureido)-pentanedioic acid (211At-6) was synthesized. Cellular uptake and clonogenic survival were tested in PSMA-positive (PSMA+) PC3 PIP and PSMA-negative (PSMA−) PC3 flu human PC cells after 211At-6 treatment. The antitumor efficacy of 211At-6 was evaluated in mice bearing PSMA+ PC3 PIP and PSMA– PC3 flu flank xenografts at a 740-kBq dose and in mice bearing PSMA+, luciferase-expressing PC3-ML micrometastases. Biodistribution was determined in mice bearing PSMA+ PC3 PIP and PSMA– PC3 flu flank xenografts. Suborgan distribution was evaluated using α-camera images, and microscale dosimetry was modeled. Long-term toxicity was assessed in mice for 12 mo. Results: 211At-6 treatment resulted in PSMA-specific cellular uptake and decreased clonogenic survival in PSMA+ PC3 PIP cells and caused significant tumor growth delay in PSMA+ PC3 PIP flank tumors. Significantly improved survival was achieved in the newly developed PSMA+ micrometastatic PC model. Biodistribution showed uptake of 211At-6 in PSMA+ PC3 PIP tumors and in kidneys. Microscale kidney dosimetry based on α-camera images and a nephron model revealed hot spots in the proximal renal tubules. Long-term toxicity studies confirmed that the dose-limiting toxicity was late radiation nephropathy. Conclusion: PSMA-targeted 211At-6 α-particle radiotherapy yielded significantly improved survival in mice bearing PC micrometastases after systemic administration. 211At-6 also showed uptake in renal proximal tubules resulting in late nephrotoxicity, highlighting the importance of long-term toxicity studies and microscale dosimetry. PMID:27230930

Synergistic Toxicity of Polyglutamine-Expanded TATA-Binding Protein in Glia and Neuronal Cells: Therapeutic Implications for Spinocerebellar Ataxia 17

PubMed Central

Yang, Yang; Cui, Yiting; Tang, Beisha

2017-01-01

Spinocerebellar ataxia 17 (SCA17) is caused by polyglutamine (polyQ) repeat expansion in the TATA-binding protein (TBP) and is among a family of neurodegenerative diseases in which polyQ expansion leads to preferential neuronal loss in the brain. Although previous studies have demonstrated that expression of polyQ-expanded proteins in glial cells can cause neuronal injury via noncell-autonomous mechanisms, these studies investigated animal models that overexpress transgenic mutant proteins. Since glial cells are particularly reactive to overexpressed mutant proteins, it is important to investigate the in vivo role of glial dysfunction in neurodegeneration when mutant polyQ proteins are endogenously expressed. In the current study, we generated two conditional TBP-105Q knock-in mouse models that specifically express mutant TBP at the endogenous level in neurons or in astrocytes. We found that mutant TBP expression in neuronal cells or astrocytes alone only caused mild neurodegeneration, whereas severe neuronal toxicity requires the expression of mutant TBP in both neuronal and glial cells. Coculture of neurons and astrocytes further validated that mutant TBP in astrocytes promoted neuronal injury. We identified activated inflammatory signaling pathways in mutant TBP-expressing astrocytes, and blocking nuclear factor κB (NF-κB) signaling in astrocytes ameliorated neurodegeneration. Our results indicate that the synergistic toxicity of mutant TBP in neuronal and glial cells plays a critical role in SCA17 pathogenesis and that targeting glial inflammation could be a potential therapeutic approach for SCA17 treatment. SIGNIFICANCE STATEMENT Mutant TBP with polyglutamine expansion preferentially affects neuronal viability in SCA17 patients. Whether glia, the cells that support and protect neurons, contribute to neurodegeneration in SCA17 remains mostly unexplored. In this study, we provide both in vivo and in vitro evidence arguing that endogenous expression of mutant TBP in neurons and glia synergistically impacts neuronal survival. Hyperactivated inflammatory signaling pathways, particularly the NF-κB pathway, underlie glia-mediated neurotoxicity. Moreover, blocking NF-κB activity with small chemical inhibitors alleviated such neurotoxicity. Our study establishes glial dysfunction as an important component of SCA17 pathogenesis and suggests targeting glial inflammation as a potential therapeutic approach for SCA17 treatment. PMID:28821675

Attenuation of Cisplatin-Induced Neurotoxicity by Cyanidin, a Natural Inhibitor of ROS-Mediated Apoptosis in PC12 Cells.

PubMed

Li, Da-wei; Sun, Jing-yi; Wang, Kun; Zhang, Shuai; Hou, Ya-jun; Yang, Ming-feng; Fu, Xiao-yan; Zhang, Zong-yong; Mao, Lei-lei; Yuan, Hui; Fang, Jie; Fan, Cun-dong; Zhu, Mei-jia; Sun, Bao-liang

2015-10-01

Cisplatin-based chemotherapy in clinic is severely limited by its adverse effect, including neurotoxicity. Oxidative damage contributes to cisplatin-induced neurotoxicity, but the mechanism remains unclearly. Cyanidin, a natural flavonoid compound, exhibits powerful antioxidant activity. Hence, we investigated the protective effects of cyanidin on PC12 cells against cisplatin-induced neurotoxicity and explored the underlying mechanisms. The results showed that cisplatin-induced cytotoxicity was completely reversed by cyanidin through inhibition of PC12 cell apoptosis, as proved by the attenuation of Sub-G1 peak, PARP cleavage, and caspases-3 activation. Mechanistically, cyanidin significantly inhibited reactive oxygen species (ROS)-induced DNA damage in cisplatin-treated PC12 cells. Our findings revealed that cyanidin as an apoptotic inhibitor effectively blocked cisplatin-induced neurotoxicity through inhibition of ROS-mediated DNA damage and apoptosis, predicating its therapeutic potential in prevention of chemotherapy-induced neurotoxicity. Cisplatin caused DNA damage, activated p53, and subsequently induced PC12 cells apoptosis by triggering ROS overproduction. However, cyanidin administration effectively inhibited DNA damage, attenuated p53 phosphorylation, and eventually reversed cisplatin-induced PC12 cell apoptosis through inhibition ROS accumulation.

Modeling and optimization of phospholipase A₁-catalyzed hydrolysis of phosphatidylcholine using response surface methodology for lysophosphatidylcholine production.

PubMed

Lim, Chang Wan; Kim, Byung Hee; Kim, In-Hwan; Lee, Moon-Won

2015-01-01

Modeling the phospholipase A1 (PLA1 )-catalyzed partial hydrolysis of soy phosphatidylcholine (PC) in hexane for the production of lysophosphatidylcholine (LPC) and optimizing the reaction conditions using response surface methodology were described. The reaction was performed with 4 g of PC in a stirred batch reactor using a commercial PLA1 (Lecitase Ultra) as the biocatalyst. The effects of temperature, reaction time, water content, and enzyme loading on LPC and glycerylphosphorylcholine (GPC) content in the reaction products were elucidated using the models established. Optimal reaction conditions for maximizing the LPC content while suppressing acyl migration, which causes GPC formation, were as follows: temperature, 60°C; reaction time, 3 h; water content, 10% of PC; and enzyme loading, 1% of PC. When the reaction was conducted with 40 g of PC under these conditions, the reaction products contained 83.7 mol % LPC and were free of GPC. LPC had a higher total unsaturated fatty acid content than original PC had and was mainly composed of linoleic acid (78.0 mol % of the total fatty acids). © 2014 American Institute of Chemical Engineers.

Global Prostate Cancer Incidence and Mortality Rates According to the Human Development Index.

PubMed

Khazaei, Salman; Rezaeian, Shahab; Ayubi, Erfan; Gholamaliee, Behzad; Pishkuhi, Mahin Ahmadi; Khazaei, Somayeh; Mansori, Kamyar; Nematollahi, Shahrzad; Sani, Mohadeseh; Hanis, Shiva Mansouri

2016-01-01

Prostate cancer (PC) is one of the leading causes of death, especially in developed countries. The human development index (HDI) and its dimensions seem correlated with incidence and mortality rates of PC. This study aimed to assess the association of the specific components of HDI (life expectancy at birth, education, gross national income per 1000 capita, health, and living standards) with burden indicators of PC worldwide. Information of the incidence and mortality rates of PC was obtained from the GLOBOCAN cancer project in year 2012 and data about the HDI 2013 were obtained from the World Bank database. The correlation between incidence, mortality rates, and the HDI parameters were assessed using STATA software. A significant inequality of PC incidence rates was observed according to concentration indexes=0.25 with 95% CI (0.22, 0.34) and a negative mortality concentration index of -0.04 with 95% CI (-0.09, 0.01) was observed. A positive significant correlation was detected between the incidence rates of PC and the HDI and its dimensions including life expectancy at birth, education, income, urbanization level and obesity. However, there was a negative significant correlation between the standardized mortality rates and the life expectancy, income and HDI.

Effect of polymer blend types and gamma radiation on the physico-chemical properties of polycarbonate

NASA Astrophysics Data System (ADS)

Nouh, S. A.; Magida, M. M.; Al-Shekify, L. S.; Bashter, I. I.

2016-12-01

Samples from polycarbonate (PC) were classified into two main groups. The first group was blended with Abietic acid (Rosin) with different compositions, while the second group was blended with ethylene-vinyl acetate (EVA) copolymer with almost the same composition. A comparative study of the effect of both Rosin and EVA concentration on the thermal stability of PC has been investigated using thermogravimetric analysis. The results show that blending with Rosin causes the dominance of degradation. Samples from PC with 20% Rosin blend were irradiated with gamma at the dose of range 20-300 kGy. The resultant effect of gamma radiation on the structural properties of the 20% Rosin/ 80%PC blend has been investigated using Fourier Transform Infrared FTIR spectroscopy. The results indicate that degradation of the polymer blend dominates, thus increases the creation of hot free radicals that leads to the formation of color centers in PC. In addition, the transmission of the 20% Rosin/80%PC samples as well as color changes was studied. The color intensity ΔE was greatly increased with increasing the gamma dose up to 300 kGy, accompanied by a significant increase in the blue color component.

Prostate-Specific Membrane Antigen Targeted Gold Nanoparticles for Theranostics of Prostate Cancer.

PubMed

Mangadlao, Joey Dacula; Wang, Xinning; McCleese, Christopher; Escamilla, Maria; Ramamurthy, Gopalakrishnan; Wang, Ziying; Govande, Mukul; Basilion, James P; Burda, Clemens

2018-04-24

Prostate cancer is one of the most common cancers and among the leading causes of cancer deaths in the United States. Men diagnosed with the disease typically undergo radical prostatectomy, which often results in incontinence and impotence. Recurrence of the disease is often experienced by most patients with incomplete prostatectomy during surgery. Hence, the development of a technique that will enable surgeons to achieve a more precise prostatectomy remains an open challenge. In this contribution, we report a theranostic agent (AuNP-5kPEG-PSMA-1-Pc4) based on prostate-specific membrane antigen (PSMA-1)-targeted gold nanoparticles (AuNPs) loaded with a fluorescent photodynamic therapy (PDT) drug, Pc4. The fabricated nanoparticles are well-characterized by spectroscopic and imaging techniques and are found to be stable over a wide range of solvents, buffers, and media. In vitro cellular uptake experiments demonstrated significantly higher nanoparticle uptake in PSMA-positive PC3pip cells than in PSMA-negative PC3flu cells. Further, more complete cell killing was observed in Pc3pip than in PC3flu cells upon exposure to light at different doses, demonstrating active targeting followed by Pc4 delivery. Likewise, in vivo studies showed remission on PSMA-expressing tumors 14 days post-PDT. Atomic absorption spectroscopy revealed that targeted AuNPs accumulate 4-fold higher in PC3pip than in PC3flu tumors. The nanoparticle system described herein is envisioned to provide surgical guidance for prostate tumor resection and therapeutic intervention when surgery is insufficient.

Design of anti- and pro-aggregation variants to assess the effects of methionine oxidation in human prion protein

PubMed Central

Wolschner, Christina; Giese, Armin; Kretzschmar, Hans A.; Huber, Robert; Moroder, Luis; Budisa, Nediljko

2009-01-01

Prion disease is characterized by the α→β structural conversion of the cellular prion protein (PrPC) into the misfolded and aggregated “scrapie” (PrPSc) isoform. It has been speculated that methionine (Met) oxidation in PrPC may have a special role in this process, but has not been detailed and assigned individually to the 9 Met residues of full-length, recombinant human PrPC [rhPrPC(23-231)]. To better understand this oxidative event in PrP aggregation, the extent of periodate-induced Met oxidation was monitored by electrospray ionization-MS and correlated with aggregation propensity. Also, the Met residues were replaced with isosteric and chemically stable, nonoxidizable analogs, i.e., with the more hydrophobic norleucine (Nle) and the highly hydrophilic methoxinine (Mox). The Nle-rhPrPC variant is an α-helix rich protein (like Met-rhPrPC) resistant to oxidation that lacks the in vitro aggregation properties of the parent protein. Conversely, the Mox-rhPrPC variant is a β-sheet rich protein that features strong proaggregation behavior. In contrast to the parent Met-rhPrPC, the Nle/Mox-containing variants are not sensitive to periodate-induced in vitro aggregation. The experimental results fully support a direct correlation of the α→β secondary structure conversion in rhPrPC with the conformational preferences of Met/Nle/Mox residues. Accordingly, sporadic prion and other neurodegenerative diseases, as well as various aging processes, might also be caused by oxidative stress leading to Met oxidation. PMID:19416900

Proprotein convertase 1 mediated proneuropeptide proteolytic processing in ischemic neuron injury.

PubMed

Tang, S S; Liang, Z Y; Guo, L R; Zhang, J H; Zhou, D

2017-01-01

Pro-protein processing mechanism plays an important role in neuron injury. To study the protein convertase 1 (PC1) mediated processing mechanism, the ischemic cellular or tissue proPC1/PC1 or proCgA/CgA (pro-chromogranin A) was analyzed. NS20Y differentiated cells were stressed by 0-6 h of oxygen and glucose deprivation (OGD) in glucose-free DMEM and an anaerobic jar environment. Ischemic C57BL/J mouse model was established by performing 60-min of middle cerebral artery occlusion (MCAO) operation and subsequent 4 or 24-h reperfusion. The TUNEL, immunochemistry, and Western blot methods were used to detect protein expression in ischemic cells or tissues. The OGD or MCAO stress caused substantial cell death in a dose-dependent manner (p < 0.05 or 0.01). With the increasing OGD dose, proPC1 and PC1 proteins gradually increased (p < 0.05 or 0.01) whereas proCgA and CgA proteins decreased (p < 0.05). In vivo the proPC1 and PC1 expressions presented with a peak at 4-h and then decreased at 24-h reperfusion (p < 0.05 or 0.01). The tissue proCgA and CgA proteins decreased with the increasing reperfusion time (p < 0.05). The results suggest that the increasing PC1 expression promoted the transformation of proCgA into CgA or smaller peptides, i.e. Pancreastatin or Secretoneurin, and the PC1 mediated processing plays a critical role (Fig. 4, Ref. 15).

Effects of Phos-Chek® G75-F and Silv-Ex® on red-winged blackbird (Agelaius phoeniceus) embryos

USGS Publications Warehouse

Buscemi, D.M.; Hoffman, D.J.; Vyas, N.B.; Spann, J.W.; Kuenzel, W.J.

2007-01-01

Effects of field application levels of wildfire control chemicals, Phos-Chek® G75-F (PC) and Silv-Ex® (SE), were examined on red-winged blackbird (Agelaius phoeniceus) embryos. Embryos were more sensitive to PC and SE when eggs were immersed for 10 s at an early developmental stage (days 3–5 of incubation) than at a later stage (days 6–9 of incubation). The LC50 (concentration causing 50% mortality) for early stage embryos exposed to PC was 213.3 g/L (slope = 1.6; 95% confidence interval [CI] = 129.1–326.1). The no observed effect concentration (NOEC) was below 135 g PC/L, which caused a significant increase in embryonic mortality and represents the lowest field coverage level of 1 gal/100 feet2. The LC50 for early stage embryos exposed to SE was 19.8 g/L (slope = 1.5; 95% CI = 11.7–52.2). Significant mortality was observed at 10 g SE/L and marginal at 7.5 g SE/L with an apparent NOEC around 5 g SE/L. Neither chemical resulted in apparent developmental malformations.

Inactivation and deficiency of core proteins of photosystems I and II caused by genetical phylloquinone and plastoquinone deficiency but retained lamellar structure in a T-DNA mutant of Arabidopsis.

PubMed

Shimada, Hiroshi; Ohno, Ryoichi; Shibata, Masaru; Ikegami, Isamu; Onai, Kiyoshi; Ohto, Masa-aki; Takamiya, Ken-ichiro

2005-02-01

Phylloquinone, a substituted 1,4-naphthoquinone with an 18-carbon-saturated phytyl tail, functions as a bound one-electron carrier cofactor at the A1 site of photosystem I (PSI). A Feldmann tag line mutant, no. 2755 (designated as abc4 hereafter), showed pale-green young leaves and white old leaves. The mutated nuclear gene encoded 1,4-dihydroxy-2-naphtoic acid phytyltransferase, an enzyme of phylloquinone biosynthesis, and high-performance liquid chromatography analysis revealed that the abc4 mutant contained no phylloquinone, and only about 3% plastoquinone. Photooxidation of P700 of PSI in the abc4 mutant was not observed, and reduced-versus-oxidized difference spectroscopy indicated that the abc4 mutant had no P700. The maximum quantum yield of photosystem II (PSII) in the abc4 mutant was much decreased, and the electron transfer from PSII to PSI in the abc4 mutant did not occur. For the pale-green leaves of the abc4 mutant plant, the ultrastructure of the chloroplasts was almost the same as that of the wild-type plant. However, the chloroplasts in the albino leaves of the mutant were smaller and had a lot of grana thylakoids and few stroma thylakoids. The amounts of PSI and PSII core subunits in the abc4 mutant were significantly decreased compared with those in the wild type. These results suggested that a deficiency of phylloquinone in PSI caused the abolishment of PSI and a partial defect of PSII due to a significant decrease of plastoquinone, but did not influence the ultrastructure of the chloroplasts in young leaves.

Mutant Huntingtin Causes a Selective Decrease in the Expression of Synaptic Vesicle Protein 2C.

PubMed

Peng, Chaohua; Zhu, Gaochun; Liu, Xiangqian; Li, He

2018-04-30

Huntington's disease (HD) is a neurodegenerative disease caused by a polyglutamine expansion in the huntingtin (Htt) protein. Mutant Htt causes synaptic transmission dysfunctions by interfering in the expression of synaptic proteins, leading to early HD symptoms. Synaptic vesicle proteins 2 (SV2s), a family of synaptic vesicle proteins including 3 members, SV2A, SV2B, and SV2C, plays important roles in synaptic physiology. Here, we investigated whether the expression of SV2s is affected by mutant Htt in the brains of HD transgenic (TG) mice and Neuro2a mouse neuroblastoma cells (N2a cells) expressing mutant Htt. Western blot analysis showed that the protein levels of SV2A and SV2B were not significantly changed in the brains of HD TG mice expressing mutant Htt with 82 glutamine repeats. However, in the TG mouse brain there was a dramatic decrease in the protein level of SV2C, which has a restricted distribution pattern in regions particularly vulnerable in HD. Immunostaining revealed that the immunoreactivity of SV2C was progressively weakened in the basal ganglia and hippocampus of TG mice. RT-PCR demonstrated that the mRNA level of SV2C progressively declined in the TG mouse brain without detectable changes in the mRNA levels of SV2A and SV2B, indicating that mutant Htt selectively inhibits the transcriptional expression of SV2C. Furthermore, we found that only SV2C expression was progressively inhibited in N2a cells expressing a mutant Htt containing 120 glutamine repeats. These findings suggest that the synaptic dysfunction in HD results from the mutant Htt-mediated inhibition of SV2C transcriptional expression. These data also imply that the restricted distribution and decreased expression of SV2C contribute to the brain region-selective pathology of HD.

Evaluation of vaccine candidate potential of deltaaroA, deltahtrA and deltaaroAdeltahtrA mutants of Salmonella enterica subspecies enterica serovar Abortusequi in guinea pigs.

PubMed

Singh, Bhoj Raj; Chandra, Mudit; Hansda, Dhananjoy; Alam, Javed; Babu, Narayanan; Siddiqui, Mehtab Z; Agrawal, Ravi K; Sharma, Gautam

2013-04-01

Salmonella enterica subspecies enterica serovar Abortusequi (S. Abortusequi), a host adapted Salmonella causes abortions, still births and foal mortality in equids. Though known since more than 100 years, it is still a problem in many of the developing countries including India. There is dearth of really good vaccine affording immunity lasting at least for one full gestation. In search of a potential vaccine candidate, three defined deletion mutants (deltaaroA, deltahtrA and deltaaroAdeltahtrA) of S. Abortusequi were tested in guinea pig model for attenuation, safety, immunogenicity, humoral immune response, protective efficacy and persistence in host. The deltahtrA and deltaaroAdeltahtrA mutants were found to be safe on oral inoculation in doses as high as 4.2 x 10(9) cfu/animal. Also through subcutaneous inoculation deltaaroAdeltahtrA mutant did not induce any abortion in pregnant guinea pigs. All the three mutants did not induce any illness or death in 1-2 week-old baby guinea pigs except deltahtrA mutant which caused mortality on intraperitoneal inoculation. Inoculation with mutants protected against challenge and increased breeding efficiency of guinea pigs. After >4.5 months of mutant inoculation, guinea pigs were protected against abortifacient dose of wild type S. Abortusequi and mother guinea pigs also conferred resistance to their babies to the similar challenge. Early humoral immune response of S. Abortusequi mutants was characteristic. Faecal excretion of deltaaroA and htrA mutants was detected up to 45 days of inoculation in guinea pigs while deltaaroAdeltahtrA mutant could not be detected after 21 days of inoculation. The results indicated that the double deletion mutant (deltaaroAdeltahtrA) was the most effective and safe candidate for vaccination against S. Abortusequi through mucosal route of inoculation.

Heterozygous Mutations Causing Partial Prohormone Convertase 1 Deficiency Contribute to Human Obesity

PubMed Central

Creemers, John W.M.; Choquet, Hélène; Stijnen, Pieter; Vatin, Vincent; Pigeyre, Marie; Beckers, Sigri; Meulemans, Sandra; Than, Manuel E.; Yengo, Loïc; Tauber, Maithé; Balkau, Beverley; Elliott, Paul; Jarvelin, Marjo-Riitta; Van Hul, Wim; Van Gaal, Luc; Horber, Fritz; Pattou, François; Froguel, Philippe; Meyre, David

2012-01-01

Null mutations in the PCSK1 gene, encoding the proprotein convertase 1/3 (PC1/3), cause recessive monogenic early onset obesity. Frequent coding variants that modestly impair PC1/3 function mildly increase the risk for common obesity. The aim of this study was to determine the contribution of rare functional PCSK1 mutations to obesity. PCSK1 exons were sequenced in 845 nonconsanguineous extremely obese Europeans. Eight novel nonsynonymous PCSK1 mutations were identified, all heterozygous. Seven mutations had a deleterious effect on either the maturation or the enzymatic activity of PC1/3 in cell lines. Of interest, five of these novel mutations, one of the previously described frequent variants (N221D), and the mutation found in an obese mouse model (N222D), affect residues at or near the structural calcium binding site Ca-1. The prevalence of the newly identified mutations was assessed in 6,233 obese and 6,274 lean European adults and children, which showed that carriers of any of these mutations causing partial PCSK1 deficiency had an 8.7-fold higher risk to be obese than wild-type carriers. These results provide the first evidence of an increased risk of obesity in heterozygous carriers of mutations in the PCSK1 gene. Furthermore, mutations causing partial PCSK1 deficiency are present in 0.83% of extreme obesity phenotypes. PMID:22210313

Heterozygous mutations causing partial prohormone convertase 1 deficiency contribute to human obesity.

PubMed

Creemers, John W M; Choquet, Hélène; Stijnen, Pieter; Vatin, Vincent; Pigeyre, Marie; Beckers, Sigri; Meulemans, Sandra; Than, Manuel E; Yengo, Loïc; Tauber, Maithé; Balkau, Beverley; Elliott, Paul; Jarvelin, Marjo-Riitta; Van Hul, Wim; Van Gaal, Luc; Horber, Fritz; Pattou, François; Froguel, Philippe; Meyre, David

2012-02-01

Null mutations in the PCSK1 gene, encoding the proprotein convertase 1/3 (PC1/3), cause recessive monogenic early onset obesity. Frequent coding variants that modestly impair PC1/3 function mildly increase the risk for common obesity. The aim of this study was to determine the contribution of rare functional PCSK1 mutations to obesity. PCSK1 exons were sequenced in 845 nonconsanguineous extremely obese Europeans. Eight novel nonsynonymous PCSK1 mutations were identified, all heterozygous. Seven mutations had a deleterious effect on either the maturation or the enzymatic activity of PC1/3 in cell lines. Of interest, five of these novel mutations, one of the previously described frequent variants (N221D), and the mutation found in an obese mouse model (N222D), affect residues at or near the structural calcium binding site Ca-1. The prevalence of the newly identified mutations was assessed in 6,233 obese and 6,274 lean European adults and children, which showed that carriers of any of these mutations causing partial PCSK1 deficiency had an 8.7-fold higher risk to be obese than wild-type carriers. These results provide the first evidence of an increased risk of obesity in heterozygous carriers of mutations in the PCSK1 gene. Furthermore, mutations causing partial PCSK1 deficiency are present in 0.83% of extreme obesity phenotypes.

Molecular genetics of syndromic and non-syndromic forms of parathyroid carcinoma.

PubMed

Cardoso, Luís; Stevenson, Mark; Thakker, Rajesh V

2017-12-01

Parathyroid carcinoma (PC) may occur as part of a complex hereditary syndrome or an isolated (i.e., non-syndromic) non-hereditary (i.e., sporadic) endocrinopathy. Studies of hereditary and syndromic forms of PC, which include the hyperparathyroidism-jaw tumor syndrome (HPT-JT), multiple endocrine neoplasia types 1 and 2 (MEN1 and MEN2), and familial isolated primary hyperparathyroidism (FIHP), have revealed some genetic mechanisms underlying PC. Thus, cell division cycle 73 (CDC73) germline mutations cause HPT-JT, and CDC73 mutations occur in 70% of sporadic PC, but in only ∼2% of parathyroid adenomas. Moreover, CDC73 germline mutations occur in 20%-40% of patients with sporadic PC and may reveal unrecognized HPT-JT. This indicates that CDC73 mutations are major driver mutations in the etiology of PCs. However, there is no genotype-phenotype correlation and some CDC73 mutations (e.g., c.679_680insAG) have been reported in patients with sporadic PC, HPT-JT, or FIHP. Other genes involved in sporadic PC include germline MEN1 and rearranged during transfection (RET) mutations and somatic alterations of the retinoblastoma 1 (RB1) and tumor protein P53 (TP53) genes, as well as epigenetic modifications including DNA methylation and histone modifications, and microRNA misregulation. This review summarizes the genetics and epigenetics of the familial syndromic and non-syndromic (sporadic) forms of PC. © 2017 The Authors. Human Mutation published by Wiley Periodicals, Inc.

Effect of biotin on activity and gene expression of biotin-dependent carboxylases in the liver of dairy cows.

PubMed

Ferreira, G; Weiss, W P

2007-03-01

Biotin is a cofactor of the gluconeogenic enzymes pyruvate carboxylase (PC) and propionyl-coenzyme A carboxylase (PCC). We hypothesized that biotin supplementation increases the activity and gene expression of PC and PCC and the gene expression of phosphoenol-pyruvate carboxykinase (PEPCK) in the liver of lactating dairy cows. Eight multiparous Holstein cows (40 +/- 2 kg/d of milk yield and 162 +/- 35 d in milk) were randomly assigned to 1 of 2 diet sequences in a crossover design with two 22-d periods. Treatments consisted of a basal diet (60% concentrate) containing 0 or 0.96 mg/kg of supplemental biotin. On d 21 of each period, liver tissue was collected by percutaneous liver biopsy. Activities of PC and PCC were determined by measuring the fixation of [14C]O2 in liver homogenates. Abundance of mRNA for PCC, PC, and PEPCK was determined by quantitative reverse-transcription PCR. Biotin supplementation did not affect milk production or composition. Biotin supplementation increased the activity of PC but had no effect on PCC activity. Biotin supplementation did not affect the gene expression of PC, PCC, and PEPCK. The increased activity of PC without changes in mRNA abundance may have been caused by increased activation of the apoenzymes by holocarboxylase synthetase. In conclusion, biotin supplementation affected the activity of PC in the liver of lactating dairy cows, but whether biotin supplementation increases glucose production in the liver remains to be determined.

A Founder Mutation in VPS11 Causes an Autosomal Recessive Leukoencephalopathy Linked to Autophagic Defects.

PubMed

Zhang, Jinglan; Lachance, Véronik; Schaffner, Adam; Li, Xianting; Fedick, Anastasia; Kaye, Lauren E; Liao, Jun; Rosenfeld, Jill; Yachelevich, Naomi; Chu, Mary-Lynn; Mitchell, Wendy G; Boles, Richard G; Moran, Ellen; Tokita, Mari; Gorman, Elizabeth; Bagley, Kaytee; Zhang, Wei; Xia, Fan; Leduc, Magalie; Yang, Yaping; Eng, Christine; Wong, Lee-Jun; Schiffmann, Raphael; Diaz, George A; Kornreich, Ruth; Thummel, Ryan; Wasserstein, Melissa; Yue, Zhenyu; Edelmann, Lisa

2016-04-01

Genetic leukoencephalopathies (gLEs) are a group of heterogeneous disorders with white matter abnormalities affecting the central nervous system (CNS). The causative mutation in ~50% of gLEs is unknown. Using whole exome sequencing (WES), we identified homozygosity for a missense variant, VPS11: c.2536T>G (p.C846G), as the genetic cause of a leukoencephalopathy syndrome in five individuals from three unrelated Ashkenazi Jewish (AJ) families. All five patients exhibited highly concordant disease progression characterized by infantile onset leukoencephalopathy with brain white matter abnormalities, severe motor impairment, cortical blindness, intellectual disability, and seizures. The carrier frequency of the VPS11: c.2536T>G variant is 1:250 in the AJ population (n = 2,026). VPS11 protein is a core component of HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering) protein complexes involved in membrane trafficking and fusion of the lysosomes and endosomes. The cysteine 846 resides in an evolutionarily conserved cysteine-rich RING-H2 domain in carboxyl terminal regions of VPS11 proteins. Our data shows that the C846G mutation causes aberrant ubiquitination and accelerated turnover of VPS11 protein as well as compromised VPS11-VPS18 complex assembly, suggesting a loss of function in the mutant protein. Reduced VPS11 expression leads to an impaired autophagic activity in human cells. Importantly, zebrafish harboring a vps11 mutation with truncated RING-H2 domain demonstrated a significant reduction in CNS myelination following extensive neuronal death in the hindbrain and midbrain. Thus, our study reveals a defect in VPS11 as the underlying etiology for an autosomal recessive leukoencephalopathy disorder associated with a dysfunctional autophagy-lysosome trafficking pathway.

RNA interference-mediated silencing of mutant superoxide dismutase rescues cyclosporin A-induced death in cultured neuroblastoma cells

PubMed Central

Maxwell, Michele M.; Pasinelli, Piera; Kazantsev, Aleksey G.; Brown, Robert H.

2004-01-01

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disorder resulting from selective death of motor neurons in the brain and spinal cord. In ≈25% of familial ALS cases, the disease is caused by dominantly acting point mutations in the gene encoding cytosolic Cu,Zn superoxide dismutase (SOD1). In cell culture and in rodent models of ALS, mutant SOD1 proteins exhibit dose-dependent toxicity; thus, agents that reduce mutant protein expression would be powerful therapeutic tools. A wealth of recent evidence has demonstrated that the mechanism of RNA-mediated interference (RNAi) can be exploited to achieve potent and specific gene silencing in vitro and in vivo. We have evaluated the utility of RNAi for selective silencing of mutant SOD1 expression in cultured cells and have identified small interfering RNAs capable of specifically inhibiting expression of ALS-linked mutant, but not wild-type, SOD1. We have investigated the functional effects of RNAi-mediated silencing of mutant SOD1 in cultured murine neuroblastoma cells. In this model, stable expression of mutant, but not wild-type, human SOD1 sensitizes cells to cytotoxic stimuli. We find that silencing of mutant SOD1 protects these cells against cyclosporin A-induced cell death. These results demonstrate a positive physiological effect caused by RNAi-mediated silencing of a dominant disease allele. The present study further supports the therapeutic potential of RNAi-based methods for the treatment of inherited human diseases, including ALS. PMID:14981234

Drosophila Fatty Acid Transport Protein Regulates Rhodopsin-1 Metabolism and Is Required for Photoreceptor Neuron Survival

PubMed Central

Dourlen, Pierre; Bertin, Benjamin; Chatelain, Gilles; Robin, Marion; Napoletano, Francesco; Roux, Michel J.; Mollereau, Bertrand

2012-01-01

Tight regulation of the visual response is essential for photoreceptor function and survival. Visual response dysregulation often leads to photoreceptor cell degeneration, but the causes of such cell death are not well understood. In this study, we investigated a fatty acid transport protein (fatp) null mutation that caused adult-onset and progressive photoreceptor cell death. Consistent with fatp having a role in the retina, we showed that fatp is expressed in adult photoreceptors and accessory cells and that its re-expression in photoreceptors rescued photoreceptor viability in fatp mutants. The visual response in young fatp-mutant flies was abnormal with elevated electroretinogram amplitudes associated with high levels of Rhodopsin-1 (Rh1). Reducing Rh1 levels in rh1 mutants or depriving flies of vitamin A rescued photoreceptor cell death in fatp mutant flies. Our results indicate that fatp promotes photoreceptor survival by regulating Rh1 abundance. PMID:22844251

One amino acid in mouse activated factor VII defines its endothelial protein C receptor (EPCR) binding and modulates its EPCR-dependent hemostatic activity in vivo.

PubMed

Pavani, G; Zintner, S M; Ivanciu, L; Small, J C; Stafford, K A; Szeto, J H; Margaritis, P

2017-03-01

Essentials The lack of factor (F) VIIa-endothelial protein C receptor (EPCR) binding in mice is unresolved. A single substitution of Leu4 to Phe in mouse FVIIa (mFVIIa) enables its interaction with EPCR. mFVIIa with a Phe4 shows EPCR binding-dependent enhanced hemostatic function in vivo vs. mFVIIa. Defining the FVIIa-EPCR interaction in mice allows for further investigating its biology in vivo. Background Human activated factor VII (hFVIIa), which is used in hemophilia treatment, binds to the endothelial protein C (PC) receptor (EPCR) with unclear hemostatic consequences. Interestingly, mice lack the activated FVII (FVIIa)-EPCR interaction. Therefore, to investigate the hemostatic consequences of this interaction in hemophilia, we previously engineered a mouse FVIIa (mFVIIa) molecule that bound mouse EPCR (mEPCR) by using three substitutions from mouse PC (mPC), i.e. Leu4→Phe, Leu8→Met, and Trp9→Arg. The resulting molecule, mFVIIa-FMR, modeled the EPCR-binding properties of hFVIIa and showed enhanced hemostatic capacity in hemophilic mice versus mFVIIa. These data implied a role of EPCR in the action of hFVIIa in hemophilia treatment. However, the substitutions in mFVIIa-FMR only broadly defined the sequence determinants for its mEPCR interaction and enhanced function in vivo. Objectives To determine the individual contributions of mPC Phe4, Met8 and Arg9 to the in vitro/in vivo properties of mFVIIa-FMR. Methods The mEPCR-binding properties of single amino acid variants of mFVIIa or mPC at position 4, 8 or 9 were investigated. Results and conclusions Phe4 in mFVIIa or mPC was solely critical for interaction with mEPCR. In hemophilic mice, administration of mFVIIa harboring a Phe4 resulted in a 1.9-2.5-fold increased hemostatic capacity versus mFVIIa that was EPCR binding-dependent. This recapitulated previous observations made with triple-mutant mFVIIa-FMR. As Leu8 is crucial for hFVIIa-EPCR binding, we describe the sequence divergence of this interaction in mice, now allowing its further characterization in vivo. We also illustrate that modulation of the EPCR-FVIIa interaction may lead to improved FVIIa therapeutics. © 2016 International Society on Thrombosis and Haemostasis.

CDDO-Me inhibits tumor growth and prevents recurrence of pancreatic ductal adenocarcinoma.

PubMed

Gao, Xiaohua; Deeb, Dorrah; Liu, Yongbo; Liu, Patricia; Zhang, Yiguan; Shaw, Jiajiu; Gautam, Subhash C

2015-12-01

Methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me) has shown potent antitumorigenic activity against a wide range of cancer cell lines in vitro and inhibited the growth of liver, lung and prostate cancer in vivo. In the present study, we examined the antitumor activity of CDDO-Me for pancreatic ductal adenocarcinoma (PDAC) cells with and without activating K-ras mutations. Treatment of K-ras mutant MiaPaCa-2 and K-ras normal BxPC-3 cells with CDDO-Me elicited strong antiproliferative and proapoptopic responses in both cell lines in culture. The inhibition of cell proliferation and induction of apoptosis was accompanied by the inhibition of antiapoptotic/prosurvival p-Akt, NF-кB and p-mTOR signaling proteins. For testing efficacy of CDDO-Me in vivo heterotopic and orthotopic xenografts were generated by implanting BxPC-3 and MiaPaCa-2 cells subcutaneously and in the pancreatic tail, respectively. Treatment with CDDO-Me significantly inhibited the growth of BxPC-3 xenografts and reduced the levels of p-Akt and p-mTOR in tumor tissue. In mice with orthotopic MiaPaCa-2 xenografts, treatment with CDDO-Me prolonged the survival of mice when administered following the surgical resection of tumors. The latter was attributed to the eradication of residual PDAC remaining after resection of tumors. These preclinical data demonstrate the potential of CDDO-Me for treating primary PDAC tumors and for preventing relapse/recurrence through the destruction of residual disease.

The fire blight pathogen Erwinia amylovora requires the rpoN gene for pathogenicity in apple.

PubMed

Ramos, Laura S; Lehman, Brian L; Sinn, Judith P; Pfeufer, Emily E; Halbrendt, Noemi O; McNellis, Timothy W

2013-10-01

RpoN is a σ(54) factor regulating essential virulence gene expression in several plant pathogenic bacteria, including Pseudomonas syringae and Pectobacterium carotovorum. In this study, we found that mutation of rpoN in the fire blight pathogen Erwinia amylovora caused a nonpathogenic phenotype. The E. amylovora rpoN Tn5 transposon mutant rpoN1250::Tn5 did not cause fire blight disease symptoms on shoots of mature apple trees. In detached immature apple fruits, the rpoN1250::Tn5 mutant failed to cause fire blight disease symptoms and grew to population levels 12 orders of magnitude lower than the wild-type. In addition, the rpoN1250::Tn5 mutant failed to elicit a hypersensitive response when infiltrated into nonhost tobacco plant leaves, and rpoN1250::Tn5 cells failed to express HrpN protein when grown in hrp (hypersensitive response and pathogenicity)-inducing liquid medium. A plasmid-borne copy of the wild-type rpoN gene complemented all the rpoN1250::Tn5 mutant phenotypes tested. The rpoN1250::Tn5 mutant was prototrophic on minimal solid and liquid media, indicating that the rpoN1250::Tn5 nonpathogenic phenotype was not caused by a defect in basic metabolism or growth. This study provides clear genetic evidence that rpoN is an essential virulence gene of E. amylovora, suggesting that rpoN has the same function in E. amylovora as in P. syringae and Pe. carotovorum. 2013 BSPP and JOHN WILEY & SONS LTD

Effects of different fatty acids composition of phosphatidylcholine on brain function of dementia mice induced by scopolamine.

PubMed

Zhou, Miao-Miao; Xue, Yong; Sun, Shu-Hong; Wen, Min; Li, Zhao-Jie; Xu, Jie; Wang, Jing-Feng; Yanagita, Teruyoshi; Wang, Yu-Ming; Xue, Chang-Hu

2016-08-24

Phosphatidylcholine (PC), the major source of dietary choline, has been demonstrated to improve the capability of learning and memory in rodent and the amelioration of long-chain n-3 polyunsaturated fatty acids (PUFA) on anti-aging and anti-oxidation is widely known as well. In this study, three kinds of PC were chose to demonstrate the role of different fatty acids composition on glycerol backbone in improving the brain function of mice induced by scopolamine which was used to impair cholinergic system and cause oxidative stress. Male BALB/c mice were randomly divided into 5 groups: model (M) group, control (Con) group, egg yolk lecithin (EL) group, squid PC (SQ-PC) group and sea cucumber PC (SC-PC) group. The intraperitoneal injection of scopolamine hydrobromide (5 mg/kg) was carried out on the 8(th) of group feeding and sustained daily until the end of test. Morris water maze test was used to evaluate the improvement of cognitive decline and the activity of acetylcholinesterase (AchE), superoxide dismutase (SOD) and monoamine oxidase (MAO) and malondialdehyde (MDA) content in brain were measured to assess the physiological changes. In behavior test, the latency of PC groups was significantly reduced, while number of crossing the platform and time in target quadrant were increased in comparison with M group and the improvements of SQ-PC and SC-PC were better than that of EL (P < 0.05). Similar trend was observed in physiological changes. The AchE activity was effectively decreased and the SOD activity increased in hippocampus, cortex and white matter when comparing PC groups with M group. SQ-PC, SC-PC and EL respectively showed 22.82, 28.80 and 11.81 % decrease in MDA level in brain compared with M group. The MAO activity in white matter of SQ-PC, SC-PC and EL group separately depressed 33.05, 33.64 and 19.73 % in comparison with M group. No significance between SQ-PC and SC-PC was found in these indicators except the SOD activity in hippocampus and white matter. SQ-PC group had a higher SOD activity in hippocampus (103.68U/mg · prot.) and lower in white matter (120.57 U/mg · prot.) than SC-PC group (95.53 U/mg · prot. in hippocampus, 134.49 U/mg · prot. in white matter). PC rich in n-3 PUFA acted more ameliorative effects than that barely contained on the indicators above. Different fatty acids composition of PC all could diminish the cognitive decline and biological damage and protect the brain. EPA and DHA partly enhaced to the advantageous effects.

The Influence of channel length to the characteristics of CuPc based OFET thin films

NASA Astrophysics Data System (ADS)

Sujarwata; Handayani, L.; Mosik; Fianti

2018-03-01

The main focus of this research is to characterize organic field effect transistor (OFET) thin films based on CuPc with a bottom-contact structure and varied channel length. OFET was prepared by Si substrate cleaning in the ultrasonic cleaner first, then deposition of the source and drain electrodes on the substrate with vacuum evaporation at room temperature, and finally CuPc thin film deposition among the source, drain, and gate electrodes. The distance between source anddrain electrodes is the channel length of the CuPc thin film. In this research, the channel length was varied; 100 μm, 200 μm and 300 μm, with the same active areas of 2.9-3.42 V and different current, IDS. The result showed that the shorter channel length causes, the bigger IDS flowing on the OFET

Genetic and functional characterization of PCSK1.

PubMed

Choquet, Hélène; Stijnen, Pieter; Creemers, John W M

2011-01-01

PC1/3 is a neuroendocrine-specific member of the mammalian subtilisin-like proprotein convertase family. This seven-member family is involved in the endoproteolytic cleavage of a large number of precursor proteins including prohormones, proneuropeptides, zymogens, and proreceptors. PC1/3 is synthesized as a zymogen, proPC1/3, and its propeptide is rapidly and autocatalytically cleaved in the endoplasmic reticulum. The mature protein is sorted and stored in dense-core secretory vesicles, together with its substrates. Compound-inactivating mutations in the PCSK1 gene, which encodes PC1/3, cause monogenic obesity. Furthermore, the contribution of two common nonsynonymous variants in PCSK1 to polygenic obesity risk has recently been established. Additional rare variants have been identified in non-consanguineous extremely obese Europeans but functional characterization has not yet been described. Sequencing efforts of larger cohorts of obese patients might reveal more variants conferring risk of obesity.

Alteration in levels of unsaturated fatty acids in mutants of Escherichia coli defective in DNA replication.

PubMed

Suzuki, E; Kondo, T; Makise, M; Mima, S; Sakamoto, K; Tsuchiya, T; Mizushima, T

1998-07-01

We previously reported that mutations in the dnaA gene which encodes the initiator of chromosomal DNA replication in Escherichia coli caused an alteration in the levels of unsaturated fatty acids of phospholipids in membranes. In this study, we examined fatty acid compositions in other mutants which are defective in DNA replication. As in the case of temperature-sensitive dnaA mutants, temperature-sensitive dnaC and dnaE mutants, which have defects in initiation and elongation, respectively, of DNA replication showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) compared with the wild-type strain, especially at high temperatures. On the other hand, temperature-sensitive mutants defective in cellular processes other than DNA replication, such as RNA synthesis and cell division, did not show a lower level of unsaturation of fatty acids compared with the wild-type strain. These results suggest that the inhibition of DNA replication causes a lower level of unsaturation of fatty acids in Escherichia coli cells.

acn-1, a C. elegans homologue of ACE, genetically interacts with the let-7 microRNA and other heterochronic genes.

PubMed

Metheetrairut, Chanatip; Ahuja, Yuri; Slack, Frank J

2017-10-02

The heterochronic pathway in C. elegans controls the relative timing of cell fate decisions during post-embryonic development. It includes a network of microRNAs (miRNAs), such as let-7, and protein-coding genes, such as the stemness factors, LIN-28 and LIN-41. Here we identified the acn-1 gene, a homologue of mammalian angiotensin-converting enzyme (ACE), as a new suppressor of the stem cell developmental defects of let-7 mutants. Since acn-1 null mutants die during early larval development, we used RNAi to characterize the role of acn-1 in C. elegans seam cell development, and determined its interaction with heterochronic factors, including let-7 and its downstream interactors - lin-41, hbl-1, and apl-1. We demonstrate that although RNAi knockdown of acn-1 is insufficient to cause heterochronic defects on its own, loss of acn-1 suppresses the retarded phenotypes of let-7 mutants and enhances the precocious phenotypes of hbl-1, though not lin-41, mutants. Conversely, the pattern of acn-1 expression, which oscillates during larval development, is disrupted by lin-41 mutants but not by hbl-1 mutants. Finally, we show that acn-1(RNAi) enhances the let-7-suppressing phenotypes caused by loss of apl-1, a homologue of the Alzheimer's disease-causing amyloid precursor protein (APP), while significantly disrupting the expression of apl-1 during the L4 larval stage. In conclusion, acn-1 interacts with heterochronic genes and appears to function downstream of let-7 and its target genes, including lin-41 and apl-1.

Antitumor activity of curcumin is involved in down-regulation of YAP/TAZ expression in pancreatic cancer cells.

PubMed

Zhou, Xiuxia; Su, Jingna; Feng, Shaoyan; Wang, Lixia; Yin, Xuyuan; Yan, Jingzhe; Wang, Zhiwei

2016-11-29

Pancreatic cancer (PC) is one of the most aggressive human malignancies worldwide and is the fourth leading cause of cancer-related deaths. Curcumin (diferuloylmethane) is a polyphenol derived from the Curcuma longa plant. Certain studies have demonstrated that curcumin exerts its anti-tumor function in a variety of human cancers including PC, via targeting multiple therapeutically important cancer signaling pathways. However, the detailed molecular mechanisms are not fully understood. Two transcriptional co-activators, YAP (Yes-associated protein) and its close paralog TAZ (transcriptional coactivator with PDZ-binding motif) exert oncogenic activities in various cancers. Therefore, in this study we aimed to determine the molecular basis of curcumin-induced cell proliferation inhibition in PC cells. First, we detected the anti-tumor effects of curcumin on PC cell lines using CTG assay, Flow cytometry, clonogenic assay, wound healing assay and Transwell invasion assay. We found that curcumin significantly suppressed cell growth, weakened clonogenic potential, inhibited migration and invasion, and induced apoptosis and cell cycle arrest in PC cells. We further measured that overexpression of YAP enhanced cell proliferation and abrogated the cytotoxic effects of curcumin on PC cells. Moreover, we found that curcumin markedly down-regulated YAP and TAZ expression and subsequently suppressed Notch-1 expression. Collectively, these findings suggest that pharmacological inhibition of YAP and TAZ activity may be a promising anticancer strategy for the treatment of PC patients.

Targeted deletion of kidney glucose-6 phosphatase leads to nephropathy.

PubMed

Clar, Julie; Gri, Blandine; Calderaro, Julien; Birling, Marie-Christine; Hérault, Yann; Smit, G Peter A; Mithieux, Gilles; Rajas, Fabienne

2014-10-01

Renal failure is a major complication that arises with aging in glycogen storage disease type 1a and type 1b patients. In the kidneys, glucose-6 phosphatase catalytic subunit (encoded by G6pc) deficiency leads to the accumulation of glycogen, an effect resulting in marked nephromegaly and progressive glomerular hyperperfusion and hyperfiltration preceding the development of microalbuminuria and proteinuria. To better understand the end-stage nephropathy in glycogen storage disease type 1a, we generated a novel kidney-specific G6pc knockout (K-G6pc(-/-)) mouse, which exhibited normal life expectancy. After 6 months, K-G6pc(-/-) mice showed glycogen overload leading to nephromegaly and tubular dilation. Moreover, renal accumulation of lipids due to activation of de novo lipogenesis was observed. This led to the activation of the renin-angiotensin system and the development of epithelial-mesenchymal transition process and podocyte injury by transforming growth factor β1 signaling. The K-G6pc(-/-) mice developed microalbuminuria caused by the impairment of the glomerular filtration barrier. Thus, renal G6pc deficiency alone is sufficient to induce the development of the early-onset nephropathy observed in glycogen storage disease type 1a, independent of the liver disease. The K-G6pc(-/-) mouse model is a unique tool to decipher the molecular mechanisms underlying renal failure and to evaluate potential therapeutic strategies.

Antibodies associated with heparin-induced thrombocytopenia (HIT) inhibit activated protein C generation: new insights into the prothrombotic nature of HIT.

PubMed

Kowalska, M Anna; Krishnaswamy, Sriram; Rauova, Lubica; Zhai, Li; Hayes, Vincent; Amirikian, Karine; Esko, Jeffrey D; Bougie, Daniel W; Aster, Richard H; Cines, Douglas B; Poncz, Mortimer

2011-09-08

Heparin-induced thrombocytopenia (HIT) is caused by antibodies that recognize complexes between platelet factor 4 (PF4) and heparin or glycosaminoglycan side chains. These antibodies can lead to a limb- and life-threatening prothrombotic state. We now show that HIT antibodies are able to inhibit generation of activated protein C (aPC) by thrombin/thrombomodulin (IIa/TM) in the presence of PF4. Tetrameric PF4 potentiates aPC generation by formation of complexes with chondroitin sulfate (CS) on TM. Formation of these complexes occurs at a specific molar ratio of PF4 to glycosaminoglycan. This observation and the finding that the effect of heparin on aPC generation depends on the concentration of PF4 suggest similarity between PF4/CS complexes and those that bind HIT antibodies. HIT antibodies reduced the ability of PF4 to augment aPC formation. Cationic protamine sulfate, which forms similar complexes with heparin, also enhanced aPC generation, but its activity was not blocked by HIT antibodies. Our studies provide evidence that complexes formed between PF4 and TM's CS may play a physiologic role in potentiating aPC generation. Recognition of these complexes by HIT antibodies reverses the PF4-dependent enhancement in aPC generation and may contribute to the prothrombotic nature of HIT.

Antitumor activity of curcumin is involved in down-regulation of YAP/TAZ expression in pancreatic cancer cells

PubMed Central

Wang, Lixia; Yin, Xuyuan; Yan, Jingzhe; Wang, Zhiwei

2016-01-01

Pancreatic cancer (PC) is one of the most aggressive human malignancies worldwide and is the fourth leading cause of cancer-related deaths. Curcumin (diferuloylmethane) is a polyphenol derived from the Curcuma longa plant. Certain studies have demonstrated that curcumin exerts its anti-tumor function in a variety of human cancers including PC, via targeting multiple therapeutically important cancer signaling pathways. However, the detailed molecular mechanisms are not fully understood. Two transcriptional co-activators, YAP (Yes-associated protein) and its close paralog TAZ (transcriptional coactivator with PDZ-binding motif) exert oncogenic activities in various cancers. Therefore, in this study we aimed to determine the molecular basis of curcumin-induced cell proliferation inhibition in PC cells. First, we detected the anti-tumor effects of curcumin on PC cell lines using CTG assay, Flow cytometry, clonogenic assay, wound healing assay and Transwell invasion assay. We found that curcumin significantly suppressed cell growth, weakened clonogenic potential, inhibited migration and invasion, and induced apoptosis and cell cycle arrest in PC cells. We further measured that overexpression of YAP enhanced cell proliferation and abrogated the cytotoxic effects of curcumin on PC cells. Moreover, we found that curcumin markedly down-regulated YAP and TAZ expression and subsequently suppressed Notch-1 expression. Collectively, these findings suggest that pharmacological inhibition of YAP and TAZ activity may be a promising anticancer strategy for the treatment of PC patients. PMID:27738325

Locus of Control, Self-Reported Depression, and Perceived Causes of Depression

ERIC Educational Resources Information Center

Calhoun, Lawrence G.; And Others

1974-01-01

Examines the relation of depression to locus of control and to the perceived causes of depression in a nonpsychiatric population. Findings suggest that adolescent females tend to hold themselves more responsible than males for unsatisfactory personal situations, and this extends to the attribution of causes for unhappy moods. (Author/PC)

Application of signature-tagged mutagenesis to the study of virulence of Erwinia amylovora.

PubMed

Wang, Limei; Beer, Steven V

2006-12-01

To identify genes that contribute to the virulence of Erwinia amylovora in plants, 1892 mutants were created and screened in pools of < or =96 mutants using signature-tagged mutagenesis. Nineteen mutants were not recovered from apple shoots following inoculation, which suggested that the insertions in these mutants affected genes important for bacterial survival in planta. DNA flanking the Tn5 insertions in the 19 mutants was sequenced and analysed by blast. One mutant had a Tn5 insertion in amsE, a gene involved in the biosynthesis of exopolysaccaride (EPS). Fourteen mutants had insertions in loci that were implicated in biosynthesis or transport of particular amino acids or nucleotides, a site-specific recombinase active during cell division and several putative proteins of unknown function; the flanking DNA of the remaining four mutants lacked significant homology with any DNA in the database. When inoculated individually to hosts, 10 of the 19 mutants caused significantly less disease and multiplied less, as compared with the wild-type strain.

OGO 5 observations of Pc 5 waves - Particle flux modulations

NASA Technical Reports Server (NTRS)

Kokubun, S.; Kivelson, M. G.; Mcpherron, R. L.; Russell, C. T.; West, H. I., Jr.

1977-01-01

An investigation is conducted concerning the modulations of particle fluxes associated with Pc 5 waves in the region beyond the plasmapause. A study of thermal flux modulations indicates that some of the density enhancements observed are not spatial structures but are spurious features caused by temporal flux variations associated with hydromagnetic waves. A resonance model of the energetic particle flux modulations is discussed. Energetic particle modulations are also considered. The reported observations reveal that modulations are dominant at energies of about 100 keV for electrons and at 100 keV to 1 MeV for protons. This may indicate that the bounce resonance interaction is not important for Pc 5 waves.

Contribution of Bordetella bronchiseptica Filamentous Hemagglutinin and Pertactin to Respiratory Disease in Swine▿ †

PubMed Central

Nicholson, Tracy L.; Brockmeier, Susan L.; Loving, Crystal L.

2009-01-01

Bordetella bronchiseptica is pervasive in swine populations and plays multiple roles in respiratory disease. Most studies addressing virulence factors of B. bronchiseptica are based on isolates derived from hosts other than pigs. Two well-studied virulence factors implicated in the adhesion process are filamentous hemagglutinin (FHA) and pertactin (PRN). We hypothesized that both FHA and PRN would serve critical roles in the adhesion process and be necessary for colonization of the swine respiratory tract. To investigate the role of FHA and PRN in Bordetella pathogenesis in swine, we constructed mutants containing an in-frame deletion of the FHA or the PRN structural gene in a virulent B. bronchiseptica swine isolate. Both mutants were compared to the wild-type swine isolate for their ability to colonize and cause disease in swine. Colonization of the FHA mutant was lower than that of the wild type at all respiratory tract sites and time points examined and caused limited to no disease. In contrast, the PRN mutant caused similar disease severity relative to the wild type; however, colonization of the PRN mutant was reduced relative to the wild type during early and late infection and induced higher anti-Bordetella antibody titers. Together, our results indicate that despite inducing different pathologies and antibody responses, both FHA and PRN are necessary for optimal colonization of the swine respiratory tract. PMID:19237531

Pseudomonas protegens Pf-5 causes discoloration and pitting of mushroom caps due to the production of antifungal metabolites.

PubMed

Henkels, Marcella D; Kidarsa, Teresa A; Shaffer, Brenda T; Goebel, Neal C; Burlinson, Peter; Mavrodi, Dmitri V; Bentley, Michael A; Rangel, Lorena I; Davis, Edward W; Thomashow, Linda S; Zabriskie, T Mark; Preston, Gail M; Loper, Joyce E

2014-07-01

Bacteria in the diverse Pseudomonas fluorescens group include rhizosphere inhabitants known for their antifungal metabolite production and biological control of plant disease, such as Pseudomonas protegens Pf-5, and mushroom pathogens, such as Pseudomonas tolaasii. Here, we report that strain Pf-5 causes brown, sunken lesions on peeled caps of the button mushroom (Agaricus bisporus) that resemble brown blotch symptoms caused by P. tolaasii. Strain Pf-5 produces six known antifungal metabolites under the control of the GacS/GacA signal transduction system. A gacA mutant produces none of these metabolites and did not cause lesions on mushroom caps. Mutants deficient in the biosynthesis of the antifungal metabolites 2,4-diacetylphloroglucinol and pyoluteorin caused less-severe symptoms than wild-type Pf-5 on peeled mushroom caps, whereas mutants deficient in the production of lipopeptide orfamide A caused similar symptoms to wild-type Pf-5. Purified pyoluteorin and 2,4-diacetylphloroglucinol mimicked the symptoms caused by Pf-5. Both compounds were isolated from mushroom tissue inoculated with Pf-5, providing direct evidence for their in situ production by the bacterium. Although the lipopeptide tolaasin is responsible for brown blotch of mushroom caused by P. tolaasii, P. protegens Pf-5 caused brown blotch-like symptoms on peeled mushroom caps through a lipopeptide-independent mechanism involving the production of 2,4-diacetylphloroglucinol and pyoluteorin.

Diagnostic Challenges in Prostate Cancer and 68Ga-PSMA PET Imaging: A Game Changer?

PubMed

Zaman, Maseeh uz; Fatima, Nosheen; Zaman, Areeba; Sajid, Mahwsih; Zaman, Unaiza; Zaman, Sidra

2017-10-26

Prostate cancer (PC) is the most frequent solid tumor in men and the third most common cause of cancer mortality among men in developed countries. Current imaging modalities like ultrasound (US), computerized tomography (CT), magnetic resonance imaging (MRI) and choline based positron emission (PET) tracing have disappointing sensitivity for detection of nodal metastasis and small tumor recurrence. This poses a diagnostic challenge in staging of intermediate to high risk PC and restaging of patients with biochemical recurrence (PSA >0.2 ng/ml). Gallium-68 labeled prostate specific membrane antigen (68Ga-PSMA) PET imaging has now emerged with a higher diagnostic yield. 68Ga-PSMA PET/CT or PET/MRI can be expected to offer a one-stop-shop for staging and restaging of PC. PSMA ligands labeled with alpha and beta emitters have also shown promising therapeutic efficacy for nodal, bone and visceral metastasis. Therefore a PSMA based theranostics approach for detection, staging, treatment, and follow-up of PC would appear to be highly valuable to achieve personalized PC treatment. Creative Commons Attribution License

Adjuvant effects of recombinant giant panda (Ailuropoda melanoleuca) IL-18 on the canine distemper disease vaccine in mice.

PubMed

Yan, Yue; Niu, Lili; Deng, Jiabo; Wang, Qiang; Yu, Jianqiu; Zhang, Yizheng; Wang, Jianxi; Chen, Jiao; Wei, Changhe; Tan, Xuemei

2015-02-01

Canine distemper virus (CDV) is a morbillivirus known to cause morbidity and mortality in a broad range of animals. Giant pandas (Ailuropoda melanoleuca), especially captive ones, are susceptible to natural infection with CDV. Interleukin-18 (IL-18) is a powerful adjuvant molecule that can enhance the development of antigen-specific immunity and vaccine efficacy. In this study, a giant panda IL-18 gene eukaryotic expression plasmid (pcAmIL-18) was constructed. Female BALB/c mice were muscularly inoculated with the plasmids pcAmIL-18, pcDNA3.1 and PBS, respectively. They were subsequently injected with an attenuated CDV vaccine for dogs, and the induced humoral and cellular responses were evaluated. The results showed that pcAmIL-18 remarkably improved the level of specific antibody, IFN-γ and IL-2 in mice sera, the T lymphocyte proliferation index and the percentage of CD4(+) and CD8(+) cells. These data indicated that pcAmIL-18 is a potential adjuvant that promotes specific immunity.

Adjuvant effects of recombinant giant panda (Ailuropoda melanoleuca) IL-18 on the canine distemper disease vaccine in mice

PubMed Central

YAN, Yue; NIU, Lili; DENG, Jiabo; WANG, Qiang; YU, Jianqiu; ZHANG, Yizheng; WANG, Jianxi; CHEN, Jiao; WEI, Changhe; TAN, Xuemei

2014-01-01

Canine distemper virus (CDV) is a morbillivirus known to cause morbidity and mortality in a broad range of animals. Giant pandas (Ailuropoda melanoleuca), especially captive ones, are susceptible to natural infection with CDV. Interleukin-18 (IL-18) is a powerful adjuvant molecule that can enhance the development of antigen-specific immunity and vaccine efficacy. In this study, a giant panda IL-18 gene eukaryotic expression plasmid (pcAmIL-18) was constructed. Female BALB/c mice were muscularly inoculated with the plasmids pcAmIL-18, pcDNA3.1 and PBS, respectively. They were subsequently injected with an attenuated CDV vaccine for dogs, and the induced humoral and cellular responses were evaluated. The results showed that pcAmIL-18 remarkably improved the level of specific antibody, IFN-γ and IL-2 in mice sera, the T lymphocyte proliferation index and the percentage of CD4+ and CD8+ cells. These data indicated that pcAmIL-18 is a potential adjuvant that promotes specific immunity. PMID:25399820

Azaphenalene phthalocyanines: phthalocyanine analogues with six-membered-ring units instead of five-membered-ring units.

PubMed

Shimizu, Soji; Zhu, Hua; Kobayashi, Nagao

2010-09-24

Mixed-condensation reaction of 1,8-naphthalenedicarbonitrile and a 4,5-disubstituted phthalonitrile provided a series of phthalocyanine (Pc) analogues with azaphenalene (AP) moieties in place of the isoindole moieties. Monosubstituted species, APPc, and the two structural isomers of disubstituted species, adj-AP(2)Pc and opp-AP(2)Pc, were successfully isolated by gel-permeation chromatography on HPLC apparatus. Their structures were elucidated by (1)H NMR spectroscopy and X-ray crystallographic analysis. Replacement of the isoindole moieties with azaphenalene moieties created six-membered-ring units in the core and caused distortion of the molecular structures. The Q-band absorption shifted to the red upon an increase in the number of azaphenalene units; the shape of the absorption spectra depended on the molecular symmetries. APPc and opp-AP(2)Pc showed a large splitting of the Q band, whereas adj-AP(2)Pc exhibited a single broad Q band. These changes in the absorption spectra, as well as the unique electronic structures, are discussed in detail, based on magnetic circular dichroism spectra, electrochemical measurements, and density functional theory calculations.

CGP 55398, a liposomal Ge(IV) phthalocyanine bearing two axially ligated cholesterol moieties: a new potential agent for photodynamic therapy of tumours.

PubMed Central

Segalla, A.; Milanesi, C.; Jori, G.; Capraro, H. G.; Isele, U.; Schieweck, K.

1994-01-01

Ge(IV) phthalocyanine (GePc) with two axially ligated cholesterol moieties was prepared by chemical synthesis and incorporated in a monomeric state into small unilamellar liposomes (CGP 55398). Upon photoexcitation with light wavelengths around its intense absorption peak at 680 nm, GePc shows an efficient photosensitising activity towards biological substrates through a mechanism which largely involves the intermediacy of singlet oxygen. GePc injected systemically into mice bearing an intramuscularly implanted MS-2 fibrosarcoma is quantitatively transferred to serum lipoproteins and localises in the tumour tissue with good efficiency: at 24 h post injection the GePc content in the tumour is 0.74 and 1.87 micrograms per g of tissue with a tumour/peritumoral ratio of 4.35 and 5.67 for injected doses of 0.76 and 1.52 mg kg-1 respectively. At this time the red-light irradiation of the GePc-loaded fibrosarcoma causes a fast and massive tumour necrosis involving both malignant cells and blood vessels. Images Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 PMID:8180009

Preclinical evaluation of zinc phthalocyanine tetrasulfonate-based PDT

NASA Astrophysics Data System (ADS)

Borgatti-Jeffreys, Antonella; Hooser, Stephen B.; Miller, Margaret A.; Thomas, Rose M.; deGortari, Amalia; Lucroy, Michael D.

2005-04-01

Photodynamic therapy (PDT) involves the light activation of a drug within a tumor causing selective tumor cell death. Unfortunately, some photosensitizing drugs have been associated with adverse reactions in veterinary patients. Zinc phthalocyanine tetrasulfonate (ZnPcS4) is a promising second-generation photosensitizer for use in veterinary medicine, however, it cannot be applied clinically until safety and efficacy data are available. ZnPcS4 was given to Swiss Webster mice to assess acute toxicity. Doses >100 mg/kg were associated with acute toxicity and mortality, and doses >100 mg/kg resulted in renal tubular nephrosis, suggesting that the minimum toxic dose is approximately 100 mg/kg. Based on these data, a Phase I clinical trial of ZnPcS4-based PDT in tumor-bearing dogs is underway, with ZnPcS4 doses up to 2 mg/kg producing no apparent toxicity. Tumor response has been observed after ZnPcS4-based PDT using doses as low as 0.25 mg/kg, suggesting that conventional phase I clinical trials may not be appropriate for PDT protocols.

A role for SIRT1 in cell growth and chemoresistance in prostate cancer PC3 and DU145 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kojima, Keitaro; Department of Longevity and Aging Research, Gifu International Institute of Biotechnology, 1-1 Naka-fudogaoka, Kakamigahara, Gifu 504-0838; Ohhashi, Riyako

2008-08-29

SIRT1, which belongs to the family of type III histone deacetylase, is implicated in diverse cellular processes. We have determined the expression levels of SIRT1 in human prostate cancer cell lines and have examined the roles of SIRT1 in cell growth and chemoresistance. SIRT1 expression was markedly up-regulated in androgen-refractory PC3 and DU145 cells compared with androgen-sensitive LNCaP cells and its expression level was correlated with cell growth in PC3 cells. Treatment with a SIRT1 inhibitor, sirtinol, inhibited cell growth and increased sensitivity to camptothecin and cisplatin. Silencing of SIRT1 expression by siRNA also suppressed cell proliferation and reduced camptothecinmore » resistance in PC3 cells, mimicking the chemosensitizing effect caused by sirtinol. Also in DU145 cells, sirtinol treatment enhanced sensitivity to camptothecin and cisplatin. These results suggest that up-regulation of SIRT1 expression may play an important role in promoting cell growth and chemoresistance in androgen-refractory PC3 and DU145 cells.« less

Autophagosome-mediated EGFR down-regulation induced by the CK2 inhibitor enhances the efficacy of EGFR-TKI on EGFR-mutant lung cancer cells with resistance by T790M.

PubMed

So, Kwang Sup; Kim, Cheol Hyeon; Rho, Jin Kyung; Kim, Sun Ye; Choi, Yun Jung; Song, Joon Seon; Kim, Woo Sung; Choi, Chang Min; Chun, Young Jin; Lee, Jae Cheol

2014-01-01

Protein kinase CK2 has diverse functions promoting and maintaining cancer phenotypes. We investigated the effect of CK2 inhibition in lung cancer cells with T790M-mediated resistance to the EGFR-TK inhibitor. Resistant sublines of PC-9 to gefitinib (PC-9/GR) and erlotinib (PC-9/ER) were established by previous study, and T790M secondary mutation was found in both resistant sublines. A decrease of EGFR by siRNA treatment effectively controlled the growth of resistant cells, thus suggesting that they still have EGFR-dependency. CX-4945, a potent and selective CK2 inhibitor, induced autophagy in PC-9/GR and PC-9/ER, and which was supported by the induction of autophagic vacuoles and microtubule-associated protein 1 light chain 3 (LC3) expression, and the increase of punctate fluorescent signals in resistant cells pre-transfected with green fluorescent protein (GFP)-tagged LC3. However, the withdrawal of CX-4945 led to the recovery of cancer cells with autophagy. We found that the induction of autophagy by CX-4945 in both resistant cells was CK2 dependent by using small interfering RNA against CK2. The treatment with CX-4945 alone induced a minimal growth inhibition in resistant cells. However, combined treatment of CX-4945 and EGFR-TKI effectively inhibited cancer-cell proliferation and induced apoptosis. CX-4945 increased the translocation of EGFR from the cell surface into the autophagosome, subsequently leading to the decrease of EGFR while inhibition of autophagy by 3MA or Atg7-targeted siRNA pretreatment reduced the decrease of EGFR by CX-4945. Accordingly, apoptosis by a combination of CX-4945 and EGFR-TKI was suppressed by 3MA or Atg7-targeted siRNA pretreatment, thus suggesting that autophagosome-mediated EGFR down-regulation would have an important role regarding apoptotic cell death by EGFR-TKI. Combined treatment of the CK2 inhibitor and EGFR-TKI may be a promising strategy for overcoming T790M-mediated resistance.

Effect of interleukin 13 on bronchial hyperresponsiveness and the bronchoprotective effect of beta-adrenergic bronchodilators and corticosteroids.

PubMed

Townley, Robert G; Gendapodi, Pradeep R; Qutna, Nidal; Evans, Joseph; Romero, Francisco A; Abel, Peter

2009-03-01

Fluticasone affects airway bronchial hyperresponsiveness (BHR) and enhances bronchodilation and bronchoprotection induced by beta-adrenergic agonists. Interleukin 13 (IL-13), however, induces BHR. To test the hypotheses that fluticasone inhibits BHR after either allergen sensitization or IL-13 administration and that fluticasone restores the bronchodilation and bronchoprotective effects of beta-agonists. The BHR to methacholine induced by IL-13 or ovalbumin was determined in BALB/c mice, and the provocation concentration of methacholine that caused an increase in enhanced pause in expiration of 200% (PC200) was calculated. We compared this response to methacholine in control mice with the response after treatment with IL-13 receptor alpha 2-IgGFc fusion protein (IL-13R alpha 2) (an IL-13 blocker), fluticasone, albuterol, salmeterol, fluticasone-albuterol, and fluticasone-salmeterol. IL-13R alpha 2 (PC200, 17.59) completely blocks the BHR-induced effects of IL-13 (PC200, 7.28; P < .005). After IL-13 therapy (PC200, 5.90; P < .005), 1 mg/mL of albuterol (PC200, 3.38; P = .33), fluticasone (PC200, 4.59; P = .40), or fluticasone plus 50 microg/mL of salmeterol (PC200, 5.59; P = .11) showed no significant bronchoprotection. In nonsensitized mice, fluticasone plus 0.25 microg/mL of salmeterol (PC200, 25.90; P < .005) showed significantly greater bronchoprotection than did salmeterol alone (PC200, 11.08; P = .26). Fluticasone plus 0.3 mg/mL of albuterol and fluticasone plus 1 mg/mL of albuterol were significantly more protective than was fluticasone or albuterol alone in ovalbumin-sensitized mice. The protective effects of fluticasone, beta-agonists, and fluticasone plus beta-agonists are significantly less in IL-13-treated mice than in nonsensitized or ovalbumin-sensitized mice.

Germline mutation prevalence in individuals with pancreatic cancer and a history of previous malignancy.

PubMed

Dudley, Beth; Karloski, Eve; Monzon, Federico A; Singhi, Aatur D; Lincoln, Stephen E; Bahary, Nathan; Brand, Randall E

2018-04-15

Approximately 10% of pancreatic adenocarcinoma (PC) cases are attributed to hereditary causes. Individuals with PC and a personal history of another cancer associated with hereditary breast and ovarian cancer (HBOC) or Lynch syndrome (LS) may be more likely to carry germline mutations. Participants with PC and a history of cancer were selected from a pancreatic disease registry. Of 1296 individuals with PC, 149 had a relevant history of cancer. If banked DNA was available, a multigene panel was performed for individuals who had not 1) previously had a mutation identified through clinical testing or 2) undergone clinical multigene panel testing with no mutations detected. Twenty-two of 124 individuals with PC and another HBOC- or LS-related cancer who underwent genetic testing had a mutation identified in a PC susceptibility gene (18%). If prostate cancer is excluded, the mutation prevalence increased to 23% (21/93). Mutation carriers were more likely to have more than 1 previous cancer diagnosis (P = .001), to have had clinical genetic testing (P = .001), and to meet National Comprehensive Cancer Network (NCCN) genetic testing criteria (P < .001). Approximately 23% of mutation carriers did not meet NCCN HBOC or LS testing guidelines based on their personal cancer history and reported cancer history in first-degree relatives. At least 18% of individuals with PC and a personal history of other HBOC- or LS-related cancers carry mutations in a PC susceptibility gene based on our data, suggesting that criteria for genetic testing in individuals with PC should include consideration of previous cancer history. Cancer 2018;124:1691-700. © 2018 American Cancer Society. © 2018 American Cancer Society.

Intensity-Modulated Radiotherapy of Head and Neck Cancer Aiming to Reduce Dysphagia: Early Dose-Effect Relationships for the Swallowing Structures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feng, Felix Y.; Kim, Hyungjin M.; Lyden, Teresa H.

2007-08-01

Purpose: To present initial results of a clinical trial of intensity-modulated radiotherapy (IMRT) aiming to spare the swallowing structures whose dysfunction after chemoradiation is a likely cause of dysphagia and aspiration, without compromising target doses. Methods and Materials: This was a prospective, longitudinal study of 36 patients with Stage III-IV oropharyngeal (31) or nasopharyngeal (5) cancer. Definitive chemo-IMRT spared salivary glands and swallowing structures: pharyngeal constrictors (PC), glottic and supraglottic larynx (GSL), and esophagus. Lateral but not medial retropharyngeal nodes were considered at risk. Dysphagia endpoints included objective swallowing dysfunction (videofluoroscopy), and both patient-reported and observer-rated scores. Correlations between dosesmore » and changes in these endpoints from pre-therapy to 3 months after therapy were assessed. Results: Significant correlations were observed between videofluoroscopy-based aspirations and the mean doses to the PC and GSL, as well as the partial volumes of these structures receiving 50-65 Gy; the highest correlations were associated with doses to the superior PC (p = 0.005). All patients with aspirations received mean PC doses >60 Gy or PC V{sub 65} >50%, and GSL V{sub 50} >50%. Reduced laryngeal elevation and epiglottic inversion were correlated with mean PC and GSL doses (p < 0.01). All 3 patients with strictures had PC V{sub 70} >50%. Worsening patient-reported liquid swallowing was correlated with mean PC (p = 0.05) and esophageal (p 0.02) doses. Only mean PC doses were correlated with worsening patient-reported solid swallowing (p = 0.04) and observer-rated swallowing scores (p = 0.04). Conclusions: These dose-volume-effect relationships provide initial IMRT optimization goals and motivate further efforts to reduce swallowing structures doses to reduce dysphagia and aspiration.« less

Development of an adenoviral vector with robust expression driven by p53

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bajgelman, Marcio C.; Biotechnology Program, Biomedical Sciences Institute, University of Sao Paulo; Millennium Institute-Gene Therapy Network, Ministry of Science and Technology

2008-02-05

Here we introduce a new adenoviral vector where transgene expression is driven by p53. We first developed a synthetic promoter, referred to as PGTx{beta}, containing a p53-responsive element, a minimal promoter and the first intron of the rabbit {beta}-globin gene. Initial assays using plasmid-based vectors indicated that expression was tightly controlled by p53 and was 5-fold stronger than the constitutive CMV immediate early promoter/enhancer. The adenoviral vector, AdPG, was also shown to offer p53-responsive expression in prostate carcinoma cells LNCaP (wt p53), DU-145 (temperature sensitive mutant of p53) and PC3 (p53-null, but engineered to express temperature-sensitive p53 mutants). AdPG servedmore » as a sensor of p53 activity in LNCaP cells treated with chemotherapeutic agents. Since p53 can be induced by radiotherapy and chemotherapy, this new vector could be further developed for use in combination with conventional therapies to bring about cooperation between the genetic and pharmacologic treatment modalities.« less

A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation.

PubMed

Dikic, I; Tokiwa, G; Lev, S; Courtneidge, S A; Schlessinger, J

1996-10-10

The mechanisms by which mitogenic G-protein-coupled receptors activate the MAP kinase signalling pathway are poorly understood. Candidate protein tyrosine kinases that link G-protein-coupled receptors with MAP kinase include Src family kinases, the epidermal growth factor receptor, Lyn and Syk. Here we show that lysophosphatidic acid (LPA) and bradykinin induce tyrosine phosphorylation of Pyk2 and complex formation between Pyk2 and activated Src. Moreover, tyrosine phosphorylation of Pyk2 leads to binding of the SH2 domain of Src to tyrosine 402 of Pyk2 and activation of Src. Transient overexpression of a dominant interfering mutant of Pyk2 or the protein tyrosine kinase Csk reduces LPA- or bradykinin-induced activation of MAP kinase. LPA- or bradykinin-induced MAP kinase activation was also inhibited by overexpression of dominant interfering mutants of Grb2 and Sos. We propose that Pyk2 acts with Src to link Gi- and Gq-coupled receptors with Grb2 and Sos to activate the MAP kinase signalling pathway in PC12 cells.

Isolation of a novel mutant gene for soil-surface rooting in rice (Oryza sativa L.)

PubMed Central

2013-01-01

Background Root system architecture is an important trait affecting the uptake of nutrients and water by crops. Shallower root systems preferentially take up nutrients from the topsoil and help avoid unfavorable environments in deeper soil layers. We have found a soil-surface rooting mutant from an M2 population that was regenerated from seed calli of a japonica rice cultivar, Nipponbare. In this study, we examined the genetic and physiological characteristics of this mutant. Results The primary roots of the mutant showed no gravitropic response from the seedling stage on, whereas the gravitropic response of the shoots was normal. Segregation analyses by using an F2 population derived from a cross between the soil-surface rooting mutant and wild-type Nipponbare indicated that the trait was controlled by a single recessive gene, designated as sor1. Fine mapping by using an F2 population derived from a cross between the mutant and an indica rice cultivar, Kasalath, revealed that sor1 was located within a 136-kb region between the simple sequence repeat markers RM16254 and 2935-6 on the terminal region of the short arm of chromosome 4, where 13 putative open reading frames (ORFs) were found. We sequenced these ORFs and detected a 33-bp deletion in one of them, Os04g0101800. Transgenic plants of the mutant transformed with the genomic fragment carrying the Os04g0101800 sequence from Nipponbare showed normal gravitropic responses and no soil-surface rooting. Conclusion These results suggest that sor1, a rice mutant causing soil-surface rooting and altered root gravitropic response, is allelic to Os04g0101800, and that a 33-bp deletion in the coding region of this gene causes the mutant phenotypes. PMID:24280269

Isolation of a novel mutant gene for soil-surface rooting in rice (Oryza sativa L.).

PubMed

Hanzawa, Eiko; Sasaki, Kazuhiro; Nagai, Shinsei; Obara, Mitsuhiro; Fukuta, Yoshimichi; Uga, Yusaku; Miyao, Akio; Hirochika, Hirohiko; Higashitani, Atsushi; Maekawa, Masahiko; Sato, Tadashi

2013-11-20

Root system architecture is an important trait affecting the uptake of nutrients and water by crops. Shallower root systems preferentially take up nutrients from the topsoil and help avoid unfavorable environments in deeper soil layers. We have found a soil-surface rooting mutant from an M2 population that was regenerated from seed calli of a japonica rice cultivar, Nipponbare. In this study, we examined the genetic and physiological characteristics of this mutant. The primary roots of the mutant showed no gravitropic response from the seedling stage on, whereas the gravitropic response of the shoots was normal. Segregation analyses by using an F2 population derived from a cross between the soil-surface rooting mutant and wild-type Nipponbare indicated that the trait was controlled by a single recessive gene, designated as sor1. Fine mapping by using an F2 population derived from a cross between the mutant and an indica rice cultivar, Kasalath, revealed that sor1 was located within a 136-kb region between the simple sequence repeat markers RM16254 and 2935-6 on the terminal region of the short arm of chromosome 4, where 13 putative open reading frames (ORFs) were found. We sequenced these ORFs and detected a 33-bp deletion in one of them, Os04g0101800. Transgenic plants of the mutant transformed with the genomic fragment carrying the Os04g0101800 sequence from Nipponbare showed normal gravitropic responses and no soil-surface rooting. These results suggest that sor1, a rice mutant causing soil-surface rooting and altered root gravitropic response, is allelic to Os04g0101800, and that a 33-bp deletion in the coding region of this gene causes the mutant phenotypes.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dunach, M.; Marti, T.; Khorana, H.G.

The light-dark adaptation reactions of a set of bacteriorhodopsin (bR) mutants that affect function and color of the chromophore were examined by using visible absorption spectroscopy. The absorbance spectra of the mutants Arg-82 in equilibrium Ala (Gln), Asp-85 in equilibrium Ala (Asn, Glu), Tyr-185 in equilibrium Phe, and Asp-212 in equilibrium Ala (Asn, Glu) were measured at different pH values during and after illumination. None of these mutants exhibited a normal dark-light adaptation, which in wild-type bR causes a red shift of the visible absorption maximum from 558 nm (dark-adapted bR) to 568 nm (light-adapted bR). Instead a reversible lightmore » reaction occurs in the Asp-85 and Asp-212 mutants from a blue form with lambda max near 600 nm to a pink form with lambda max near 480 nm. This light-induced shift explains the appearance of a reversed light adaptation previously observed for the Asp-212 mutants. In the case of the Tyr-185 and Arg-82 mutants, light causes a purple-to-blue transformation similar to the effect of lowering the pH. However, the blue forms observed in these mutants are not identical to those formed by acid titration or deionization of wild-type bR. It is suggested that in all of these mutants, the chromophore has lost the ability to undergo the normal 13-cis, 15-syn to all-trans, 15-anti light-driven isomerization, which occurs in native bR. Instead these mutants may have as stable forms all-trans,syn and 13-cis,anti chromophores, which are not allowed in native bR, except transiently.« less

Pseudomonas aeruginosa gshA Mutant Is Defective in Biofilm Formation, Swarming, and Pyocyanin Production

PubMed Central

Van Laar, Tricia A.; Esani, Saika; Birges, Tyler J.; Hazen, Bethany; Thomas, Jason M.

2018-01-01

ABSTRACT Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium that can cause severe opportunistic infections. The principal redox buffer employed by this organism is glutathione (GSH). To assess the role of GSH in the virulence of P. aeruginosa, a number of analyses were performed using a mutant strain deficient in gshA, which does not produce GSH. The mutant strain exhibited a growth delay in minimal medium compared to the wild-type strain. Furthermore, the gshA mutant was defective in biofilm and persister cell formation and in swimming and swarming motility and produced reduced levels of pyocyanin, a key virulence factor. Finally, the gshA mutant strain demonstrated increased sensitivity to methyl viologen (a redox cycling agent) as well as the thiol-reactive antibiotics fosfomycin and rifampin. Taken together, these data suggest a key role for GSH in the virulence of P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is a ubiquitous bacterium that can cause severe opportunistic infections, including many hospital-acquired infections. It is also a major cause of infections in patients with cystic fibrosis. P. aeruginosa is intrinsically resistant to a number of drugs and is capable of forming biofilms that are difficult to eradicate with antibiotics. The number of drug-resistant strains is also increasing, making treatment of P. aeruginosa infections very difficult. Thus, there is an urgent need to understand how P. aeruginosa causes disease in order to find novel ways to treat infections. We show that the principal redox buffer, glutathione (GSH), is involved in intrinsic resistance to the fosfomycin and rifampin antibiotics. We further demonstrate that GSH plays a role in P. aeruginosa disease and infection, since a mutant lacking GSH has less biofilm formation, is less able to swarm, and produces less pyocyanin, a pigment associated with infection. PMID:29669887

Phenotypic and genetic analysis of Lymantria dispar nucleopolyhedrovirus few polyhedra mutants: Mutations in the 25K FP gene may be caused by DNA replication errors

Treesearch

David S. Bischoff; James M. Slavicek

1997-01-01

We previously demonstrated that polyhedron formation (PF) mutants arise at a high frequency during serial passage of the Lymantria dispar nucleopolyhedrovirus (LdMNPV) in the L. dispar 652Y cell line (J.M. Slavicek, N. Hayes-Plazolles, and M.E. Kelly, Biol. Control 5:251-261, 1995). Most of these PF mutants...

Inactivation of the Catalytic Subunit of cAMP-Dependent Protein Kinase A Causes Delayed Appressorium Formation and Reduced Pathogenicity of Colletotrichum gloeosporioides

PubMed Central

Priyatno, Tri Puji; Abu Bakar, Farah Diba; Kamaruddin, Nurhaida; Mahadi, Nor Muhammad; Abdul Murad, Abdul Munir

2012-01-01

The cyclic AMP- (cAMP-) dependent protein kinase A signaling pathway is one of the major signaling pathways responsible for regulation of the morphogenesis and pathogenesis of several pathogenic fungi. To evaluate the role of this pathway in the plant pathogenic fungus, Colletotrichum gloeosporioides, the gene encoding the catalytic subunit of cAMP-dependent protein kinase A, CgPKAC, was cloned, inactivated, and the mutant was analyzed. Analysis of the Cgpkac mutant generated via gene replacement showed that the mutants were able to form appressoria; however, their formation was delayed compared to the wild type. In addition, the mutant conidia underwent bipolar germination after appressoria formation, but no appressoria were generated from the second germ tube. The mutants also showed reduced ability to adhere to a hydrophobic surface and to degrade lipids localized in the appressoria. Based on the number of lesions produced during a pathogenicity test, the mutant's ability to cause disease in healthy mango fruits was reduced, which may be due to failure to penetrate into the fruit. These findings indicate that cAMP-dependent protein kinase A has an important role in regulating morphogenesis and is required for pathogenicity of C. gloeosporioides. PMID:22666136

A nuclear mutation defective in mitochondrial recombination in yeast.

PubMed

Ling, F; Makishima, F; Morishima, N; Shibata, T

1995-08-15

Homologous recombination (crossing over and gene conversion) is generally essential for heritage and DNA repair, and occasionally causes DNA aberrations, in nuclei of eukaryotes. However, little is known about the roles of homologous recombination in the inheritance and stability of mitochondrial DNA which is continuously damaged by reactive oxygen species, by-products of respiration. Here, we report the first example of a nuclear recessive mutation which suggests an essential role for homologous recombination in the stable inheritance of mitochondrial DNA. For the detection of this class of mutants, we devised a novel procedure, 'mitochondrial crossing in haploid', which has enabled us to examine many mutant clones. Using this procedure, we examined mutants of Saccharomyces cerevisiae that showed an elevated UV induction of respiration-deficient mutations. We obtained a mutant that was defective in both the omega-intron homing and Endo.SceI-induced homologous gene conversion. We found that the mutant cells are temperature sensitive in the maintenance of mitochondrial DNA. A tetrad analysis indicated that elevated UV induction of respiration-deficient mutations, recombination deficiency and temperature sensitivity are all caused by a single nuclear mutation (mhr1) on chromosome XII. The pleiotropic characteristics of the mutant suggest an essential role for the MHR1 gene in DNA repair, recombination and the maintenance of DNA in mitochondria.

Neuroprotective effects of overexpressed cyclophilin B against Aβ-induced neurotoxicity in PC12 cells.

PubMed

Oh, Yoojung; Kim, Eun Young; Kim, Yeonghwan; Jin, Jizi; Jin, Byung Kwan; Jahng, Geon-Ho; Jung, Min Hyung; Park, Chan; Kang, Insug; Ha, Joohun; Choe, Wonchae

2011-08-15

Accumulated amyloid-β (Aβ) is a well-known cause of neuronal apoptosis in Alzheimer disease and functions in part by generating oxidative stress. Our previous work suggested that cyclophilin B (CypB) protects against endoplasmic reticulum (ER) stress. Therefore, in this study we examined the ability of CypB to protect against Aβ toxicity. CypB is present in the neurons of rat and mouse brains, and treating neural cells with Aβ(25-35) mediates apoptotic cell death. Aβ(25-35)-induced neuronal toxicity was inhibited by the overexpression of CypB as measured by cell viability, apoptotic morphology, sub-G1 cell population, intracellular reactive oxygen species accumulation, activated caspase-3, PARP cleavage, Bcl-2 proteins, mitogen-activated protein kinase (MAPK) activation, and phosphoinositide 3-kinase (PI-3-K) activation. CypB/R95A PPIase mutants did not reduce Aβ(25-35) toxicity. We showed that Aβ(25-35)-induced apoptosis is more severe in a CypB knockdown model, confirming that CypB protects against Aβ(25-35)-induced toxicity. Consequently, these findings suggest that CypB may protect against Aβ toxicity by its antioxidant properties, by regulating MAPK and PI-3-K signaling, and through the ER stress pathway. Copyright © 2011 Elsevier Inc. All rights reserved.

Deficiency of RgpG Causes Major Defects in Cell Division and Biofilm Formation, and Deficiency of LytR-CpsA-Psr Family Proteins Leads to Accumulation of Cell Wall Antigens in Culture Medium by Streptococcus mutans.

PubMed

De, Arpan; Liao, Sumei; Bitoun, Jacob P; Roth, Randy; Beatty, Wandy L; Wu, Hui; Wen, Zezhang T

2017-09-01

Streptococcus mutans is known to possess rhamnose-glucose polysaccharide (RGP), a major cell wall antigen. S. mutans strains deficient in rgpG , encoding the first enzyme of the RGP biosynthesis pathway, were constructed by allelic exchange. The rgpG deficiency had no effect on growth rate but caused major defects in cell division and altered cell morphology. Unlike the coccoid wild type, the rgpG mutant existed primarily in chains of swollen, "squarish" dividing cells. Deficiency of rgpG also causes significant reduction in biofilm formation ( P < 0.01). Double and triple mutants with deficiency in brpA and/or psr , genes coding for the LytR-CpsA-Psr family proteins BrpA and Psr, which were previously shown to play important roles in cell envelope biogenesis, were constructed using the rgpG mutant. There were no major differences in growth rates between the wild-type strain and the rgpG brpA and rgpG psr double mutants, but the growth rate of the rgpG brpA psr triple mutant was reduced drastically ( P < 0.001). Under transmission electron microscopy, both double mutants resembled the rgpG mutant, while the triple mutant existed as giant cells with multiple asymmetric septa. When analyzed by immunoblotting, the rgpG mutant displayed major reductions in cell wall antigens compared to the wild type, while little or no signal was detected with the double and triple mutants and the brpA and psr single mutants. These results suggest that RgpG in S. mutans plays a critical role in cell division and biofilm formation and that BrpA and Psr may be responsible for attachment of cell wall antigens to the cell envelope. IMPORTANCE Streptococcus mutans , a major etiological agent of human dental caries, produces rhamnose-glucose polysaccharide (RGP) as the major cell wall antigen. This study provides direct evidence that deficiency of RgpG, the first enzyme of the RGP biosynthesis pathway, caused major defects in cell division and morphology and reduced biofilm formation by S. mutans , indicative of a significant role of RGP in cell division and biofilm formation in S. mutans These results are novel not only in S. mutans , but also other streptococci that produce RGP. This study also shows that the LytR-CpsA-Psr family proteins BrpA and Psr in S. mutans are involved in attachment of RGP and probably other cell wall glycopolymers to the peptidoglycan. In addition, the results also suggest that BrpA and Psr may play a direct role in cell division and biofilm formation in S. mutans This study reveals new potential targets to develop anticaries therapeutics. Copyright © 2017 American Society for Microbiology.

Marrow transplantation in the treatment of a murine heritable hemolytic anemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barker, J.E.; McFarland-Starr, E.C.

1989-05-15

Mice with hemolytic anemia, sphha/sphha, have extremely fragile RBCs with a lifespan of approximately one day. Neither splenectomy nor simple transplantation of normal marrow after lethal irradiation cures the anemia but instead causes rapid deterioration and death of the mutant unless additional prophylactic procedures are used. In this report, we show that normal marrow transplantation preceded by sublethal irradiation increases but does not normalize RBC count. The mutant RBCs but not all the WBCs are replaced by donor cells. Splenectomy of the improved recipient causes a dramatic decrease in RBC count, indicating that the mutant spleen is a site ofmore » donor-origin erythropoiesis as well as of RBC destruction. Injections of iron dextran did not improve RBC counts. Transplantation of primary recipient marrow cells into a secondary host with a heritable stem cell deficiency (W/Wv) corrects the defect caused by residence of the normal cells in the sphha/sphha host. The original +/+ donor cells replace the RBCs of the secondary host, and the RBC count is normalized. Results indicate that the environment in the sphha/sphha host is detrimental to normal (as well as mutant) erythroid cells but the restriction is not transmitted.« less

Controlling aggregation propensity in A53T mutant of alpha-synuclein causing Parkinson's disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Sonu; Sarkar, Anita; Sundar, Durai, E-mail: sundar@dbeb.iitd.ac.in

2009-09-18

Understanding {alpha}-synuclein in terms of fibrillization, aggregation, solubility and stability is fundamental in Parkinson's disease (PD). The three familial mutations, namely, A30P, E46K and A53T cause PD because the hydrophobic regions in {alpha}-synuclein acquire {beta}-sheet configuration, and have a propensity to fibrillize and form amyloids that cause cytotoxicity and neurodegeneration. On simulating the native form and mutants (A30P, E46K and A53T) of {alpha}-synuclein in water solvent, clear deviations are observed in comparison to the all-helical 1XQ8 PDB structure. We have identified two crucial residues, {sup 40}Val and {sup 74}Val, which play key roles in {beta}-sheet aggregation in the hydrophobic regionsmore » 36-41 and 68-78, respectively, leading to fibrillization and amyloidosis in familial (A53T) PD. We have also identified V40D{sub V}74D, a double mutant of A53T (the most amyloidogenic mutant). The simultaneous introduction of these two mutations in A53T nearly ends its aggregation propensity, increases its solubility and positively enhances its thermodynamic stability.« less

Analysis of trafficking, stability and function of human connexin 26 gap junction channels with deafness-causing mutations in the fourth transmembrane helix.

PubMed

Ambrosi, Cinzia; Walker, Amy E; Depriest, Adam D; Cone, Angela C; Lu, Connie; Badger, John; Skerrett, I Martha; Sosinsky, Gina E

2013-01-01

Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased tendency to aggregate. Thus, mutations in TM4 cause a range of phenotypes of dysfunctional gap junction channels that are discussed within the context of the X-ray crystallographic structure.

Surface antigens contribute differently to the pathophysiological features in serotype K1 and K2 Klebsiella pneumoniae strains isolated from liver abscesses.

PubMed

Yeh, Kuo-Ming; Chiu, Sheng-Kung; Lin, Chii-Lan; Huang, Li-Yueh; Tsai, Yu-Kuo; Chang, Jen-Chang; Lin, Jung-Chung; Chang, Feng-Yee; Siu, Leung-Kei

2016-01-01

The virulence role of surface antigens in a single serotype of Klebsiella pneumoniae strain have been studied, but little is known about whether their contribution will vary with serotype. To investigate the role of K and O antigen in hyper-virulent strains, we constructed O and K antigen deficient mutants from serotype K1 STL43 and K2 TSGH strains from patients with liver abscess, and characterized their virulence in according to the abscess formation and resistance to neutrophil phagocytosis, serum, and bacterial clearance in liver. Both of K1 and K2-antigen mutants lost their wildtype resistance to neutrophil phagocytosis and hepatic clearance, and failed to cause abscess formation. K2-antigen mutant became serum susceptible while K1-antigen mutant maintained its resistance to serum killing. The amount of glucuronic acid, indicating the amount of capsular polysaccharide (CPS, K antigen), was inversed proportional to the rate of phagocytosis. O-antigen mutant of serotype K1 strains had significantly more amount of CPS, and more resistant to neutrophil phagocytosis than its wildtype counterpart. O-antigen mutants of serotype K1 and K2 strains lost their wildtype serum resistance, and kept resistant to neutrophil phagocytosis. While both mutants lacked the same O1 antigen, O-antigen mutant of serotype K1 became susceptible to liver clearance and cause mild abscess formation, but its serotype K2 counterpart maintained these wildtype virulence. We conclude that the contribution of surface antigens to virulence of K. pneumoniae strains varies with serotypes.

New Late Gene, dar, Involved in DNA Replication of Bacteriophage T4 I. Isolation, Characterization, and Genetic Location.

PubMed

Wu, J R; Yeh, Y C

1975-05-01

Suppressors of gene 59-defective mutants were isolated by screening spontaneous, temperature-sensitive (ts) revertants of the amber mutant, amC5, in gene 59. Six ts revertants were isolated. No gene 59-defective ts recombinant was obtained by crossing each ts revertant with the wild type, T4D. However, suppressors of gene 59-defective mutants were obtained from two of these ts revertants. These suppressor mutants are referred to as dar (DNA arrested restoration). dar mutants specifically restored the abnormalities, both in DNA synthesis and burst size, caused by gene 59-defective mutants to normal levels. It is unlikely that dar mutants are nonsense suppressors since theý failed to suppress amber mutations in 11 other genes investigated. The genetic expression of dar is controlled by gene 55; therefore, dar is a late gene. The genetic location of dar has been mapped between genes 24 and 25, a region contiguous to late genes. dar appears to be another nonessential gene of T4 since burst sizes of dar were almost identical to those of the wild type. Mutations in dar did not affect genetic recombination and repair of UV-damaged DNA, but caused a sensitivity to hydroxyurea in progeny formation. The effect of the dar mutation on host DNA degradation cannot account for its hydroxyurea sensitivity. dar mutant alleles were recessive to the wild-type allele as judged by restoration of arrested DNA synthesis. The possible mechanisms for the suppression of defects in gene 59 are discussed.

Calpain-mediated proteolysis of polycystin-1 C-terminus induces JAK2 and ERK signal alterations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Hyunho; Department of Medicine, University of Maryland, Baltimore, MD; Kang, Ah-Young

2014-01-01

Autosomal dominant polycystic kidney disease (ADPKD), a hereditary renal disease caused by mutations in PKD1 (85%) or PKD2 (15%), is characterized by the development of gradually enlarging multiple renal cysts and progressive renal failure. Polycystin-1 (PC1), PKD1 gene product, is an integral membrane glycoprotein which regulates a number of different biological processes including cell proliferation, apoptosis, cell polarity, and tubulogenesis. PC1 is a target of various proteolytic cleavages and proteosomal degradations, but its role in intracellular signaling pathways remains poorly understood. Herein, we demonstrated that PC1 is a novel substrate for μ- and m-calpains, which are calcium-dependent cysteine proteases. Overexpressionmore » of PC1 altered both Janus-activated kinase 2 (JAK2) and extracellular signal-regulated kinase (ERK) signals, which were independently regulated by calpain-mediated PC1 degradation. They suggest that the PC1 function on JAK2 and ERK signaling pathways might be regulated by calpains in response to the changes in intracellular calcium concentration. - Highlights: • Polycystin-1 is a target of ubiquitin-independent degradation by calpains. • The PEST domain is required for calpain-mediated degradation of polycystin-1. • Polycystin-1 may independently regulate JAK2 and ERK signaling pathways.« less

Protein C and protein S deficiencies: similarities and differences between two brothers playing in the same game.

PubMed

Bereczky, Zsuzsanna; Kovács, Kitti B; Muszbek, László

2010-12-01

Protein C (PC) and protein S (PS) are vitamin K-dependent glycoproteins that play an important role in the regulation of blood coagulation as natural anticoagulants. PC is activated by thrombin and the resulting activated PC (APC) inactivates membrane-bound activated factor VIII and factor V. The free form of PS is an important cofactor of APC. Deficiencies in these proteins lead to an increased risk of venous thromboembolism; a few reports have also associated these deficiencies with arterial diseases. The degree of risk and the prevalence of PC and PS deficiency among patients with thrombosis and in those in the general population have been examined by several population studies with conflicting results, primarily due to methodological variability. The molecular genetic background of PC and PS deficiencies is heterogeneous. Most of the mutations cause type I deficiency (quantitative disorder). Type II deficiency (dysfunctional molecule) is diagnosed in approximately 5%-15% of cases. The diagnosis of PC and PS deficiencies is challenging; functional tests are influenced by several pre-analytical and analytical factors, and the diagnosis using molecular genetics also has special difficulties. Large gene segment deletions often remain undetected by DNA sequencing methods. The presence of the PS pseudogene makes genetic diagnosis even more complicated.

Reassessing the Efficiency Penalty from Carbon Capture in Coal-Fired Power Plants.

PubMed

Supekar, Sarang D; Skerlos, Steven J

2015-10-20

This paper examines thermal efficiency penalties and greenhouse gas as well as other pollutant emissions associated with pulverized coal (PC) power plants equipped with postcombustion CO2 capture for carbon sequestration. We find that, depending on the source of heat used to meet the steam requirements in the capture unit, retrofitting a PC power plant that maintains its gross power output (compared to a PC power plant without a capture unit) can cause a drop in plant thermal efficiency of 11.3-22.9%-points. This estimate for efficiency penalty is significantly higher than literature values and corresponds to an increase of about 5.3-7.7 US¢/kWh in the levelized cost of electricity (COE) over the 8.4 US¢/kWh COE value for PC plants without CO2 capture. The results follow from the inclusion of mass and energy feedbacks in PC power plants with CO2 capture into previous analyses, as well as including potential quality considerations for safe and reliable transportation and sequestration of CO2. We conclude that PC power plants with CO2 capture are likely to remain less competitive than natural gas combined cycle (without CO2 capture) and on-shore wind power plants, both from a levelized and marginal COE point of view.

Impact-induced fracture mechanisms of immiscible PC/ABS (50/50) blends

NASA Astrophysics Data System (ADS)

Machmud, M. N.; Omiya, M.; Inoue, H.; Kishimoto, K.

2018-03-01

This paper presents a study on fracture mechanisms of polycarbonate (PC)/acrylonitrile-butadiene-styrene (ABS) (50/50) blends with different ABS types under a drop weight impact test (DWIT) using a circular sheet specimen. Formation of secondary crack indicated by a stress-whitening layer on the mid-plane of scattered specimens and secondary surface of fracture perpendicular to primary fracture surface were captured under scanning electron microscope (SEM). Although the both blends finally failed in brittle modes, SEM observation showed that their secondary fracture mechanisms were completely different. Observation through the thickness of the etched PC/ABS specimen samples using SEM also clearly showed that PC and ABS were immiscible. The immiscibility between PC and ABS was indicated by presence of their layer structures through the thickness of the blends. It was revealed that layer of ABS structure was influenced by size of rubber particle and this latter parameter then affected microstructure and fracture mechanisms of the blends. Impact-induced fracture mechanisms of the blends due to such microstructures are discussed in this paper. It was also pointed out that the secondary cracking was likely caused by interface delamination between PC and ABS layers in the core due to transverse shear stress generated during the impact test.

Homophilic and heterophilic polycystin 1 interactions regulate E-cadherin recruitment and junction assembly in MDCK cells

PubMed Central

Streets, Andrew J.; Wagner, Bart E.; Harris, Peter C.; Ward, Christopher J.; Ong, Albert C. M.

2009-01-01

Summary Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited human renal disease and is caused by mutations in two genes, PKD1 (85%) and PKD2 (15%). Cyst epithelial cells are characterised by a complex cellular phenotype including changes in proliferation, apoptosis, basement membrane composition and apicobasal polarity. Since polycystin 1 (PC1), the PKD1 protein, has been located in the basolateral membrane of kidney epithelial cells, we hypothesised that it might have a key role in mediating or stabilising cell-cell interactions. In non-ciliated L929 cells, stable or transient surface expression of the PC1 extracellular domain was sufficient to confer an adhesive phenotype and stimulate junction formation. In MDCK cells, we found that PC1 was recruited to the lateral membranes coincident with E-cadherin within 30 minutes after a `calcium switch'. Recruitment of both proteins was significantly delayed when cells were treated with a PC1 blocking antibody raised to the PKD domains. Finally, PC1 and E-cadherin could be coimmunoprecipitated together from MDCK cells. We conclude that PC1 has a key role in initiating junction formation via initial homophilic interactions and facilitates junction assembly and the establishment of apicobasal polarity by E-cadherin recruitment. PMID:19351715

In-Silico Integration Approach to Identify a Key miRNA Regulating a Gene Network in Aggressive Prostate Cancer

PubMed Central

Colaprico, Antonio; Bontempi, Gianluca; Castiglioni, Isabella

2018-01-01

Like other cancer diseases, prostate cancer (PC) is caused by the accumulation of genetic alterations in the cells that drives malignant growth. These alterations are revealed by gene profiling and copy number alteration (CNA) analysis. Moreover, recent evidence suggests that also microRNAs have an important role in PC development. Despite efforts to profile PC, the alterations (gene, CNA, and miRNA) and biological processes that correlate with disease development and progression remain partially elusive. Many gene signatures proposed as diagnostic or prognostic tools in cancer poorly overlap. The identification of co-expressed genes, that are functionally related, can identify a core network of genes associated with PC with a better reproducibility. By combining different approaches, including the integration of mRNA expression profiles, CNAs, and miRNA expression levels, we identified a gene signature of four genes overlapping with other published gene signatures and able to distinguish, in silico, high Gleason-scored PC from normal human tissue, which was further enriched to 19 genes by gene co-expression analysis. From the analysis of miRNAs possibly regulating this network, we found that hsa-miR-153 was highly connected to the genes in the network. Our results identify a four-gene signature with diagnostic and prognostic value in PC and suggest an interesting gene network that could play a key regulatory role in PC development and progression. Furthermore, hsa-miR-153, controlling this network, could be a potential biomarker for theranostics in high Gleason-scored PC. PMID:29562723

Repair of Ultraviolet Radiation Damage in Sensitive Mutants of Micrococcus radiodurans

PubMed Central

Moseley, B. E. B.

1969-01-01

Various aspects of the repair of ultraviolet (UV) radiation-induced damage were compared in wild-type Micrococcus radiodurans and two UV-sensitive mutants. Unlike the wild type, the mutants are more sensitive to radiation at 265 nm than at 280 nm. The delay in deoxyribonucleic acid (DNA) synthesis following exposure to UV is about seven times as long in the mutants as in the wild type. All three strains excise UV-induced pyrimidine dimers from their DNA, although the rate at which cytosine-thymine dimers are excised is slower in the mutants. The three strains also mend the single-strand breaks that appear in the irradiated DNA as a result of dimer excision, although the process is less efficient in the mutants. It is suggested that the increased sensitivity of the mutants to UV radiation may be caused by a partial defect in the second step of dimer excision. PMID:5773016

Use of NAP gene to manipulate leaf senescence in plants

DOEpatents

Gan, Susheng; Guo, Yongfeng

2013-04-16

The present invention discloses transgenic plants having an altered level of NAP protein compared to that of a non-transgenic plant, where the transgenic plants display an altered leaf senescence phenotype relative to a non-transgenic plant, as well as mutant plants comprising an inactivated NAP gene, where mutant plants display a delayed leaf senescence phenotype compared to that of a non-mutant plant. The present invention also discloses methods for delaying leaf senescence in a plant, as well as methods of making a mutant plant having a decreased level of NAP protein compared to that of a non-mutant plant, where the mutant plant displays a delayed leaf senescence phenotype relative to a non-mutant plant. Methods for causing precocious leaf senescence or promoting leaf senescence in a plant are also disclosed. Also disclosed are methods of identifying a candidate plant suitable for breeding that displays a delayed leaf senescence and/or enhanced yield phenotype.

Induction of a massive endoplasmic reticulum and perinuclear space expansion by expression of lamin B receptor mutants and the related sterol reductases TM7SF2 and DHCR7.

PubMed

Zwerger, Monika; Kolb, Thorsten; Richter, Karsten; Karakesisoglou, Iakowos; Herrmann, Harald

2010-01-15

Lamin B receptor (LBR) is an inner nuclear membrane protein involved in tethering the nuclear lamina and the underlying chromatin to the nuclear envelope. In addition, LBR exhibits sterol reductase activity. Mutations in the LBR gene cause two different human diseases: Pelger-Huët anomaly and Greenberg skeletal dysplasia, a severe chrondrodystrophy causing embryonic death. Our study aimed at investigating the effect of five LBR disease mutants on human cultured cells. Three of the tested LBR mutants caused a massive compaction of chromatin coincidental with the formation of a large nucleus-associated vacuole (NAV) in several human cultured cell lines. Live cell imaging and electron microscopy revealed that this structure was generated by the separation of the inner and outer nuclear membrane. During NAV formation, nuclear pore complexes and components of the linker of nucleoskeleton and cytoskeleton complex were lost in areas of membrane separation. Concomitantly, a large number of smaller vacuoles formed throughout the cytoplasm. Notably, forced expression of the two structurally related sterol reductases transmembrane 7 superfamily member 2 and 7-dehydrocholesterol reductase caused, even in their wild-type form, a comparable phenotype in susceptible cell lines. Hence, LBR mutant variants and sterol reductases can severely interfere with the regular organization of the nuclear envelope and the endoplasmic reticulum.

DNA Misfolding Found to Cause Cancer in IDH-mutant Gliomas

Cancer.gov

Researchers studying IDH-mutant brain tumors have identified a previously unknown genetic mechanism that may contribute to cancer. A change in how DNA is arranged, or packaged, in the cell nucleus may inappropriately activate a gene associated with brain cancer.

Noonan syndrome-causing SHP2 mutants impair ERK-dependent chondrocyte differentiation during endochondral bone growth.

PubMed

Tajan, Mylène; Pernin-Grandjean, Julie; Beton, Nicolas; Gennero, Isabelle; Capilla, Florence; Neel, Benjamin G; Araki, Toshiyuki; Valet, Philippe; Tauber, Maithé; Salles, Jean-Pierre; Yart, Armelle; Edouard, Thomas

2018-04-12

Growth retardation is a constant feature of Noonan syndrome (NS) but its physiopathology remains poorly understood. We previously reported that hyperactive NS-causing SHP2 mutants impair the systemic production of insulin-like growth factor 1 (IGF1) through hyperactivation of the RAS/extracellular signal-regulated kinases (ERK) signalling pathway. Besides endocrine defects, a direct effect of these mutants on growth plate has not been explored, although recent studies have revealed an important physiological role for SHP2 in endochondral bone growth. We demonstrated that growth plate length was reduced in NS mice, mostly due to a shortening of the hypertrophic zone and to a lesser extent of the proliferating zone. These histological features were correlated with decreased expression of early chondrocyte differentiation markers, and with reduced alkaline phosphatase staining and activity, in NS murine primary chondrocytes. Although IGF1 treatment improved growth of NS mice, it did not fully reverse growth plate abnormalities, notably the decreased hypertrophic zone. In contrast, we documented a role of RAS/ERK hyperactivation at the growth plate level since 1) NS-causing SHP2 mutants enhance RAS/ERK activation in chondrocytes in vivo (NS mice) and in vitro (ATDC5 cells) and 2) inhibition of RAS/ERK hyperactivation by U0126 treatment alleviated growth plate abnormalities and enhanced chondrocyte differentiation. Similar effects were obtained by chronic treatment of NS mice with statins.In conclusion, we demonstrated that hyperactive NS-causing SHP2 mutants impair chondrocyte differentiation during endochondral bone growth through a local hyperactivation of the RAS/ERK signalling pathway, and that statin treatment may be a possible therapeutic approach in NS.

Separating genetic and hemodynamic defects in neuropilin 1 knockout embryos.

PubMed

Jones, Elizabeth A V; Yuan, Li; Breant, Christine; Watts, Ryan J; Eichmann, Anne

2008-08-01

Targeted inactivation of genes involved in murine cardiovascular development frequently leads to abnormalities in blood flow. As blood fluid dynamics play a crucial role in shaping vessel morphology, the presence of flow defects generally prohibits the precise assignment of the role of the mutated gene product in the vasculature. In this study, we show how to distinguish between genetic defects caused by targeted inactivation of the neuropilin 1 (Nrp1) receptor and hemodynamic defects occurring in homozygous knockout embryos. Our analysis of a Nrp1 null allele bred onto a C57BL/6 background shows that vessel remodeling defects occur concomitantly with the onset of blood flow and cause death of homozygous mutants at E10.5. Using mouse embryo culture, we establish that hemodynamic defects are already present at E8.5 and continuous circulation is never established in homozygous mutants. The geometry of yolk sac blood vessels is altered and remodeling into yolk sac arteries and veins does not occur. To separate flow-induced deficiencies from those caused by the Nrp1 mutation, we arrested blood flow in cultured wild-type and mutant embryos and followed their vascular development. We find that loss of Nrp1 function rather than flow induces the altered geometry of the capillary plexus. Endothelial cell migration, but not replication, is altered in Nrp1 mutants. Gene expression analysis of endothelial cells isolated from freshly dissected wild-type and mutants and after culture in no-flow conditions showed down-regulation of the arterial marker genes connexin 40 and ephrin B2 related to the loss of Nrp1 function. This method allows genetic defects caused by loss-of-function of a gene important for cardiovascular development to be isolated even in the presence of hemodynamic defects.

Novel toxic shock syndrome toxin-1 amino acids required for biological activity.

PubMed

Brosnahan, Amanda J; Schaefers, Matthew M; Amundson, William H; Mantz, Mary J; Squier, Christopher A; Peterson, Marnie L; Schlievert, Patrick M

2008-12-09

Superantigens interact with T lymphocytes and macrophages to cause T lymphocyte proliferation and overwhelming cytokine production, which lead to toxic shock syndrome. Staphylococcus aureus superantigen toxic shock syndrome toxin-1 is a major cause of menstrual toxic shock syndrome. In general, superantigen-secreting S. aureus remains localized at the vaginal surface, and the superantigen must therefore penetrate the vaginal mucosa to interact with underlying immune cells to cause toxic shock syndrome. A dodecapeptide region (toxic shock syndrome toxin-1 amino acids F119-D130), relatively conserved among superantigens, has been implicated in superantigen penetration of the epithelium. The purpose of this study was to determine amino acids within this dodecapeptide region that are required for interaction with vaginal epithelium. Alanine mutations were constructed in S. aureus toxic shock syndrome toxin-1 amino acids D120 to D130. All mutants maintained superantigenicity, and selected mutants were lethal when given intravenously to rabbits. Toxic shock syndrome toxin-1 induces interleukin-8 from immortalized human vaginal epithelial cells; however, three toxin mutants (S127A, T128A, and D130A) induced low levels of interleukin-8 compared to wild type toxin. When carboxy-terminal mutants (S127A to D130A) were administered vaginally to rabbits, D130A was nonlethal, while S127A and T128A demonstrated delayed lethality compared to wild type toxin. In a porcine ex vivo permeability model, mutant D130A penetrated the vaginal mucosa more quickly than wild type toxin. Toxic shock syndrome toxin-1 residue D130 may contribute to binding an epithelial receptor, which allows it to penetrate the vaginal mucosa, induce interleukin-8, and cause toxic shock syndrome.

Repulsion between Lys258 and upstream arginines explains the missorting of the AQP2 mutant p.Glu258Lys in nephrogenic diabetes insipidus.

PubMed

Kamsteeg, Erik-Jan; Stoffels, Monique; Tamma, Grazia; Konings, Irene B M; Deen, Peter M T

2009-10-01

Regulation of body water homeostasis occurs by the vasopressin-dependent sorting of aquaporin-2 (AQP2) water channels to and from the apical membrane of renal principal cells. Mutations in AQP2 cause autosomal nephrogenic diabetes insipidus (NDI), a disease that renders the kidney unresponsive to vasopressin, resulting in polyuria and polydipsia. The AQP2 mutant c.772G>A; p.Glu258Lys (AQP2-E258K) causes dominant NDI by oligomerizing with wild-type AQP2 and missorting of this AQP2 complex to multivesicular bodies instead of the apical membrane. The motif causing this missorting of AQP2-E258K was identified here. Functional analyses and plasma membrane expression studies of truncation mutants in oocytes revealed that AQP2-E258K shortened to Leu259 is still intracellular retained. Alanine scanning and glutamic acid to arginine exchanges revealed increased function and plasma membrane expression for AQP2-E258K mutants with the following additional changes: Leu259Ala, Arg252Glu, Arg253Glu, or Arg252Ala-Arg254Ala, or for the AQP2 mutant p.Glu258Ala, indicating that the motif RRRxxxK(258)L confers AQP2-E258K retention. Fusion of this motif to aquaporin-1 also resulted in missorting of that water channel, indicating that this retention motif is transferable. In conclusion, our data reveal that the RRRxxxKL motif and repulsion between K258 and the arginine-triplet within this motif are the primary cause of missorting of AQP2-E258K in NDI.

The Arthroderma benhamiae hydrophobin HypA mediates hydrophobicity and influences recognition by human immune effector cells.

PubMed

Heddergott, Christoph; Bruns, Sandra; Nietzsche, Sandor; Leonhardt, Ines; Kurzai, Oliver; Kniemeyer, Olaf; Brakhage, Axel A

2012-05-01

Dermatophytes are the most common cause of superficial mycoses in humans and animals. They can coexist with their hosts for many years without causing significant symptoms but also cause highly inflammatory diseases. To identify mechanisms involved in the modulation of the host response during infection caused by the zoophilic dermatophyte Arthroderma benhamiae, cell wall-associated surface proteins were studied. By two-dimensional gel electrophoresis, we found that a hydrophobin protein designated HypA was the dominant cell surface protein. HypA was also detected in the supernatant during the growth and conidiation of the fungus. The A. benhamiae genome harbors only a single hydrophobin gene, designated hypA. A hypA deletion mutant was generated, as was a complemented hypA mutant strain (hypA(C)). In contrast to the wild type and the complemented strain, the hypA deletion mutant exhibited "easily wettable" mycelia and conidia, indicating the loss of surface hydrophobicity of both morphotypes. Compared with the wild type, the hypA deletion mutant triggered an increased activation of human neutrophil granulocytes and dendritic cells, characterized by an increased release of the immune mediators interleukin-6 (IL-6), IL-8, IL-10, and tumor necrosis factor alpha (TNF-α). For the first time, we observed the formation of neutrophil extracellular traps against dermatophytes, whose level of formation was increased by the ΔhypA mutant compared with the wild type. Furthermore, conidia of the ΔhypA strain were killed more effectively by neutrophils. Our data suggest that the recognition of A. benhamiae by the cellular immune defense system is notably influenced by the presence of the surface rodlet layer formed by the hydrophobin HypA.

The Arthroderma benhamiae Hydrophobin HypA Mediates Hydrophobicity and Influences Recognition by Human Immune Effector Cells

PubMed Central

Heddergott, Christoph; Bruns, Sandra; Nietzsche, Sandor; Leonhardt, Ines; Kurzai, Oliver; Kniemeyer, Olaf

2012-01-01

Dermatophytes are the most common cause of superficial mycoses in humans and animals. They can coexist with their hosts for many years without causing significant symptoms but also cause highly inflammatory diseases. To identify mechanisms involved in the modulation of the host response during infection caused by the zoophilic dermatophyte Arthroderma benhamiae, cell wall-associated surface proteins were studied. By two-dimensional gel electrophoresis, we found that a hydrophobin protein designated HypA was the dominant cell surface protein. HypA was also detected in the supernatant during the growth and conidiation of the fungus. The A. benhamiae genome harbors only a single hydrophobin gene, designated hypA. A hypA deletion mutant was generated, as was a complemented hypA mutant strain (hypAC). In contrast to the wild type and the complemented strain, the hypA deletion mutant exhibited “easily wettable” mycelia and conidia, indicating the loss of surface hydrophobicity of both morphotypes. Compared with the wild type, the hypA deletion mutant triggered an increased activation of human neutrophil granulocytes and dendritic cells, characterized by an increased release of the immune mediators interleukin-6 (IL-6), IL-8, IL-10, and tumor necrosis factor alpha (TNF-α). For the first time, we observed the formation of neutrophil extracellular traps against dermatophytes, whose level of formation was increased by the ΔhypA mutant compared with the wild type. Furthermore, conidia of the ΔhypA strain were killed more effectively by neutrophils. Our data suggest that the recognition of A. benhamiae by the cellular immune defense system is notably influenced by the presence of the surface rodlet layer formed by the hydrophobin HypA. PMID:22408226

Expression of recombinant CD59 with an N-terminal peptide epitope facilitates analysis of residues contributing to its complement-inhibitory function.

PubMed

Zhou, Q; Zhao, J; Hüsler, T; Sims, P J

1996-10-01

CD59 is a plasma membrane-anchored glycoprotein that serves to protect human cells from lysis by the C5b-9 complex of complement. The immunodominant epitopes of CD59 are known to be sensitive to disruption of native tertiary structure, complicating immunological measurement of expressed mutant constructs for structure function analysis. In order to quantify cell-surface expression of wild-type and mutant forms of this complement inhibitor, independent of CD59 antigen, an 11-residue peptide (TAG) recognized by monoclonal antibody (mAb) 9E10 was inserted before the N-terminal codon (L1) of mature CD59, in a pcDNA3 expression plasmid. SV-T2 cells were transfected with this plasmid, yielding cell lines expressing 0 to > 10(5) CD59/cell. The TAG-CD59 fusion protein was confirmed to be GPI-anchored, N-glycosylated and showed identical complement-inhibitory function to wild-type CD59, lacking the TAG peptide sequence. Using this construct, the contribution of each of four surface-localized aromatic residues (4Y, 47F, 61Y, and 62Y) to CD59's complement-inhibitory function was examined. These assays revealed normal surface expression with complete loss of complement-inhibitory function in the 4Y --> S, 47F --> G and 61Y --> S mutants. By contrast, 62Y --> S mutants retained approximately 40% of function of wild-type CD59. These studies confirmed the utility of the TAG-CD59 construct for quantifying CD59 surface expression and activity, and implicate surface aromatic residues 4Y, 47F, 61Y and 62Y as essential to maintenance of CD59's normal complement-regulatory function.

The Bmi-1 helix–turn and ring finger domains are required for Bmi-1 antagonism of (–) epigallocatechin-3-gallate suppression of skin cancer cell survival

PubMed Central

Balasubramanian, Sivaprakasam; Scharadin, Tiffany M.; Han, Bingshe; Xu, Wen; Eckert, Richard L.

2016-01-01

The Bmi-1 Polycomb group (PcG) protein is an important epigenetic regulator of chromatin status. Elevated Bmi-1 expression is observed in skin cancer and contributes to cancer cell survival. (–) Epigallocatechin-3-gallate (EGCG), an important green tea-derived cancer prevention agent, reduces Bmi-1 level resulting in reduced skin cancer cell survival. This is associated with increased p21Cip1 and p27Kip1 expression, reduced cyclin, and cyclin dependent kinase expression, and increased cleavage of apoptotic markers. These EGCG-dependent changes are attenuated by vector-mediated maintenance of Bmi-1 expression. In the present study, we identify Bmi-1 functional domains that are required for this response. Bmi-1 expression reverses the EGCG-dependent reduction in SCC-13 cell survival, but Bmi-1 mutants lacking the helix–turn–helix–turn–helix–turn (Bmi-1ΔHT) or ring finger (Bmi-1ΔRF) domains do not reverse the EGCG impact. The reduction in Ring1B ubiquitin ligase activity, observed in the presence of mutant Bmi-1, is associated with reduced ability of these mutants to interact with and activate Ring1B ubiquitin ligase, the major ligase responsible for the ubiquitination of histone H2A during chromatin condensation. This results in less chromatin condensation leading to increased tumor suppressor gene expression and reduced cell survival; thereby making the cells more susceptible to the anti-survival action of EGCG. We further show that these mutants act in a dominant-negative manner to inhibit the action of endogenous Bmi-1. Our results suggest that the HT and RF domains are required for Bmi-1 ability to maintain skin cancer cell survival in response to cancer preventive agents. PMID:25843776

The antiandrogenic effect of finasteride against a mutant androgen receptor

PubMed Central

Chhipa, Rishi Raj; Zhang, Haitao; Ip, Clement

2011-01-01

Finasteride is known to inhibit Type 2 5α-reductase and thus block the conversion of testosterone to dihydrotestosterone (DHT). The structural similarity of finasteride to DHT raises the possibility that finasteride may also interfere with the function of the androgen receptor (AR). Experiments were carried out to evaluate the antiandrogenic effect of finasteride in LNCaP, C4-2 and VCaP human prostate cancer cells. Finasteride decreased DHT binding to AR, and DHT-stimulated AR activity and cell growth in LNCaP and C4-2 cells, but not in VCaP cells. LNCaP and C4-2 (derived from castration-resistant LNCaP) cells express the T877A mutant AR, while VCaP cells express the wild-type AR. When PC-3 cells, which are AR-null, were transfected with either the wild-type or the T877A mutant AR, only the mutant AR-expressing cells were sensitive to finasteride inhibition of DHT binding. Peroxiredoxin-1 (Prx1) is a novel endogenous facilitator of AR binding to DHT. In Prx1-rich LNCaP cells, the combination of Prx1 knockdown and finasteride was found to produce a greater inhibitory effect on AR activity and cell growth than either treatment alone. The observation suggests that cells with a low expression of Prx1 are likely to be more responsive to the antiandrogenic effect of finasteride. Additional studies showed that the efficacy of finasteride was comparable to that of bicalutamide (a widely used non-steroidal antiandrogen). The implication of the above findings is discussed in the context of developing strategies to improve the outcome of androgen deprivation therapy. PMID:21386657

α-Mangostin: a dietary antioxidant derived from the pericarp of Garcinia mangostana L. inhibits pancreatic tumor growth in xenograft mouse model.

PubMed

Hafeez, Bilal Bin; Mustafa, Ala; Fischer, Joseph W; Singh, Ashok; Zhong, Weixiong; Shekhani, Mohammed Ozair; Meske, Louise; Havighurst, Thomas; Kim, KyungMann; Verma, Ajit Kumar

2014-08-10

Pancreatic cancer (PC) is the most aggressive malignant disease, ranking as the fourth most leading cause of cancer-related death among men and women in the United States. In this study, we provide evidence of chemotherapeutic effects of α-mangostin, a dietary antioxidant isolated from the pericarp of Garcinia mangostana L. against human PC. The chemotherapeutic effect of α-mangostin was determined using four human PC cells (PL-45, PANC1, BxPC3, and ASPC1). α-Mangostin resulted in a significant inhibition of PC cells viability without having any effects on normal human pancreatic duct epithelial cells. α-Mangostin showed a dose-dependent increase of apoptosis in PC cells. Also, α-mangostin inhibited the expression levels of pNF-κB/p65Ser552, pStat3Ser727, and pStat3Tyr705. α-Mangostin inhibited DNA binding activity of nuclear factor kappa B (NF-κB) and signal transducer and activator 3 (Stat3). α-Mangostin inhibited the expression levels of matrix metallopeptidase 9 (MMP9), cyclin D1, and gp130; however, increased expression of tissue inhibitor of metalloproteinase 1 (TIMP1) was observed in PC cells. In addition, i.p. administration of α-mangostin (6 mg/kg body weight, 5 days a week) resulted in a significant inhibition of both primary (PL-45) and secondary (ASPC1) human PC cell-derived orthotopic and ectopic xenograft tumors in athymic nude mice. No sign of toxicity was observed in any of the mice administered with α-mangostin. α-Mangostin treatment inhibited the biomarkers of cell proliferation (Ki-67 and proliferating cell nuclear antigen [PCNA]) in the xenograft tumor tissues. We present, for the first time, that dietary antioxidant α-mangostin inhibits the growth of PC cells in vitro and in vivo. These results suggest the potential therapeutic efficacy of α-mangostin against human PC.

(1-3)-beta-D-glucan in association with lactate dehydrogenase as biomarkers of Pneumocystis pneumonia (PcP) in HIV-infected patients.

PubMed

Esteves, F; Lee, C-H; de Sousa, B; Badura, R; Seringa, M; Fernandes, C; Gaspar, J F; Antunes, F; Matos, O

2014-07-01

Pneumocystis pneumonia (PcP) is a major HIV-related illness caused by Pneumocystis jirovecii. Definitive diagnosis of PcP requires microscopic detection of P. jirovecii in pulmonary specimens. The objective of this study was to evaluate the usefulness of two serum markers in the diagnosis of PcP. Serum levels of (1-3)-beta-d-glucan (BG) and lactate dehydrogenase (LDH) were investigated in 100 HIV-positive adult patients and 50 healthy blood donors. PcP cases were confirmed using indirect immunofluorescence with monoclonal anti-Pneumocystis antibodies and nested-PCR to amplify the large subunit mitochondrial rRNA gene of P. jirovecii in pulmonary specimens. BG and LDH levels in serum were measured using quantitative microplate-based assays. BG and LDH positive sera were statistically associated with PcP cases (P ≤ 0.001). Sensitivity, specificity, positive/negative predictive values (PPV/NPV), and positive/negative likelihood ratios (PLR/NLR) were 91.3 %, 61.3 %, 85.1 %, 79.2 %, 2.359, and 0.142, respectively, for the BG kit assay, and 91.3 %, 35.5 %, 75.9 %, 64.7 %, 1.415 and 0.245, respectively, for the LDH test. Serologic markers levels combined with the clinical diagnostic criteria for PcP were evaluated for their usefulness in diagnosis of PcP. The most promising cutoff levels for diagnosis of PcP were determined to be 400 pg/ml of BG and 350 U/l of LDH, which combined with clinical data presented 92.8 % sensitivity, 83.9 % specificity, 92.8 % PPV, 83.9 % NPV, 5.764 PLR and 0.086 NLR (P < 0.001). This study confirmed that BG is a reliable indicator for detecting P. jirovecii infection. The combination between BG/LDH levels and clinical data is a promising alternative approach for PcP diagnosis.

Metal-based phthalocyanines as a potential photosensitizing agent in photodynamic therapy for the treatment of melanoma skin cancer

NASA Astrophysics Data System (ADS)

Maduray, Kaminee; Odhav, B.

2014-03-01

Photodynamic therapy (PDT) is an emerging medical treatment that uses photosensitizers (drug) which are activated by laser light for the generation of cytotoxic free radicals and singlet oxygen molecules that cause tumor cell death. In the recent years, there has been a focus on using and improving an industrial colorant termed phthalocyanines as a prospective photosensitizer because of its unique properties. This in vitro study investigated the photodynamic effect of indium (InPcCl) and iron (FePcCl) phthalocyanine chlorides on human skin cancer cells (melanoma). Experimentally, 2 x 104 cells/ml were seeded in 24-well tissue culture plates and allowed to attach overnight, after which cells were treated with different concentrations (2 μg/ml - 100 μg/ml) of InPcCl and FePcCl. After 2 h, cells were irradiated with constant light doses of 2.5 J/cm2, 4.5 J/cm2 and 8.5 J/cm2 delivered from a diode laser. Post-irradiated cells were incubated for 24 h before cell viability was measured using the MTT Assay. At 24 h after PDT, irradiation with a light dose of 2.5 J/cm2 for each photosensitizing concentration of InPcCl and FePcCl produced a significant decrease in cell viability, but when the treatment light dose was further increased to 4.5 J/cm2 and 8.5 J/cm2 the cell survival was less than 55% for photosensitizing concentrations of InPcCl and FePcCl from 4 μg/ml to 100 μg/ml. This PDT study concludes that low concentrations on InPcCl and FePcCl activated with low level light doses can be used for the effective in vitro killing of melanoma cancer cells.

Optimization of a Nanomedicine-based Pc 4-PDT Strategy for Targeted Treatment of EGFR-Overexpressing Cancers

PubMed Central

Master, Alyssa M.; Livingston, Megan; Oleinick, Nancy L.; Gupta, Anirban Sen

2012-01-01

The current clinical mainstays for cancer treatment, namely, surgical resection, chemotherapy and radiotherapy, can cause significant trauma, systemic toxicity, and functional/cosmetic debilitation of tissue, especially if repetitive treatment becomes necessary due to tumor recurrence. Hence there is significant clinical interest in alternate treatment strategies like photodynamic therapy (PDT) which can effectively and selectively eradicate tumors and can be safely repeated if needed. We have previously demonstrated that the second-generation photosensitizer Pc 4 can be formulated within polymeric micelles, and these micelles can be specifically targeted to EGFR-overexpressing cancer cells using GE11 peptide ligands, to enhance cell-specific Pc 4 delivery and internalization. In the current study, we report on the in vitro optimization of the EGFR-targeting, Pc 4 loading of the micellar nanoformulation, along with optimization of the corresponding photoirradiation conditions to maximize Pc 4 delivery, internalization and subsequent PDT-induced cytotoxicity in EGFR-overexpressing cells in vitro. In our studies, absorption and fluorescence spectroscopy were used to monitor the cell-specific uptake of the GE11-decorated Pc 4-loaded micelles and the cytotoxic singlet oxygen production from the micelle-encapsulated Pc 4, to determine the optimum ligand density and Pc 4 loading. It was found that the micelle formulations bearing 10 mole% of GE11-modified polymer component resulted in the highest cellular uptake in EGFR-overexpressing A431 cells within the shortest incubation periods. Also, the loading of ~50 μg Pc 4 per mg of polymer in these micellar formulations resulted in the highest levels of singlet oxygen production. When formulations bearing these optimized parameters were tested in vitro on A431 cells for PDT effect, a formulation dose containing 400 nM Pc 4 and photoirradiation duration of 400 seconds at a fluence of 200 mJ/cm2 yielded close to 100% cell death. PMID:22775587

Ozone-Sensitive Arabidopsis Mutants with Deficiencies in Photorespiratory Enzymes.

PubMed

Saji, Shoko; Bathula, Srinivas; Kubo, Akihiro; Tamaoki, Masanori; Aono, Mitsuko; Sano, Tomoharu; Tobe, Kazuo; Timm, Stefan; Bauwe, Hermann; Nakajima, Nobuyoshi; Saji, Hikaru

2017-05-01

An ozone-sensitive mutant was isolated from T-DNA-tagged lines of Arabidopsis thaliana. The T-DNA was inserted at a locus on chromosome 3, where two genes encoding glycolate oxidases, GOX1 and GOX2, peroxisomal enzymes involved in photorespiration, reside contiguously. The amounts of the mutant's foliar transcripts for these genes were reduced, and glycolate oxidase activity was approximately 60% of that of the wild-type plants. No difference in growth and appearance was observed between the mutant and the wild-type plants under normal conditions with ambient air under a light intensity of 100 µmol photons m-2 s-1. However, signs of severe damage, such as chlorosis and ion leakage from the tissue, rapidly appeared in mutant leaves in response to ozone treatment at a concentration of 0.2 µl l-1 under a higher light intensity of 350 µmol photons m-2 s-1 that caused no such symptoms in the wild-type plant. The mutant also exhibited sensitivity to sulfur dioxide and long-term high-intensity light. Arabidopsis mutants with deficiencies in other photorespiratory enzymes such as glutamate:glyoxylate aminotransferase and hydroxypyruvate reductase also exhibited ozone sensitivities. Therefore, photorespiration appears to be involved in protection against photooxidative stress caused by ozone and other abiotic factors under high-intensity light. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Structure-Based Systematic Isolation of Conditional-Lethal Mutations in the Single Yeast Calmodulin Gene

PubMed Central

Ohya, Y.; Botstein, D.

1994-01-01

Conditional-lethal mutations of the single calmodulin gene in Saccharomyces cerevisiae have been very difficult to isolate by random and systematic methods, despite the fact that deletions cause recessive lethality. We report here the isolation of numerous conditional-lethal mutants that were recovered by systematically altering phenylalanine residues. The phenylalanine residues of calmodulin were implicated in function both by structural studies of calmodulin bound to target peptides and by their extraordinary conservation in evolution. Seven single and 26 multiple Phe -> Ala mutations were constructed. Mutant phenotypes were examined in a haploid cmd1 disrupted strain under three conditions: single copy, low copy, and overexpressed. Whereas all but one of the single mutations caused no obvious phenotype, most of the multiple mutations caused obvious growth phenotypes. Five were lethal, 6 were lethal only in synthetic medium, 13 were temperature-sensitive lethal and 2 had no discernible phenotypic consequences. Overexpression of some of the mutant genes restored the phenotype to nearly wild type. Several temperature-sensitive calmodulin mutations were suppressed by elevated concentration of CaCl(2) in the medium. Mutant calmodulin protein was detected at normal levels in extracts of most of the lethal mutant cells, suggesting that the deleterious phenotypes were due to loss of the calmodulin function and not protein instability. Analysis of diploid strains heterozygous for all combinations of cmd1-ts alleles revealed four intragenic complementation groups. The contributions of individual phe->ala changes to mutant phenotypes support the idea of internal functional redundancy in the symmetrical calmodulin protein molecule. These results suggest that the several phenylalanine residues in calmodulin are required to different extents in different combinations in order to carry out each of the several essential tasks. PMID:7896089

Pharmacochaperoning in a Drosophila model system rescues human dopamine transporter variants associated with infantile/juvenile parkinsonism

PubMed Central

Asjad, H. M. Mazhar; Kasture, Ameya; El-Kasaby, Ali; Sackel, Michael; Hummel, Thomas; Freissmuth, Michael; Sucic, Sonja

2017-01-01

Point mutations in the gene encoding the human dopamine transporter (hDAT, SLC6A3) cause a syndrome of infantile/juvenile dystonia and parkinsonism. To unravel the molecular mechanism underlying these disorders and investigate possible pharmacological therapies, here we examined 13 disease-causing DAT mutants that were retained in the endoplasmic reticulum when heterologously expressed in HEK293 cells. In three of these mutants, i.e. hDAT-V158F, hDAT-G327R, and hDAT-L368Q, the folding deficit was remedied with the pharmacochaperone noribogaine or the heat shock protein 70 (HSP70) inhibitor pifithrin-μ such that endoplasmic reticulum export of and radioligand binding and substrate uptake by these DAT mutants were restored. In Drosophila melanogaster, DAT deficiency results in reduced sleep. We therefore exploited the power of targeted transgene expression of mutant hDAT in Drosophila to explore whether these hDAT mutants could also be pharmacologically rescued in an intact organism. Noribogaine or pifithrin-μ treatment supported hDAT delivery to the presynaptic terminals of dopaminergic neurons and restored sleep to normal length in DAT-deficient (fumin) Drosophila lines expressing hDAT-V158F or hDAT-G327R. In contrast, expression of hDAT-L368Q in the Drosophila DAT mutant background caused developmental lethality, indicating a toxic action not remedied by pharmacochaperoning. Our observations identified those mutations most likely amenable to pharmacological rescue in the affected children. In addition, our findings also highlight the challenges of translating insights from pharmacochaperoning in cell culture to the clinical situation. Because of the evolutionary conservation in dopaminergic neurotransmission between Drosophila and people, pharmacochaperoning of DAT in D. melanogaster may allow us to bridge that gap. PMID:28972153

Heterozygous inactivation of tsc2 enhances tumorigenesis in p53 mutant zebrafish

PubMed Central

Kim, Seok-Hyung; Kowalski, Marie L.; Carson, Robert P.; Bridges, L. Richard; Ess, Kevin C.

2013-01-01

SUMMARY Tuberous sclerosis complex (TSC) is a multi-organ disorder caused by mutations of the TSC1 or TSC2 genes. A key function of these genes is to inhibit mTORC1 (mechanistic target of rapamycin complex 1) kinase signaling. Cells deficient for TSC1 or TSC2 have increased mTORC1 signaling and give rise to benign tumors, although, as a rule, true malignancies are rarely seen. In contrast, other disorders with increased mTOR signaling typically have overt malignancies. A better understanding of genetic mechanisms that govern the transformation of benign cells to malignant ones is crucial to understand cancer pathogenesis. We generated a zebrafish model of TSC and cancer progression by placing a heterozygous mutation of the tsc2 gene in a p53 mutant background. Unlike tsc2 heterozygous mutant zebrafish, which never exhibited cancers, compound tsc2;p53 mutants had malignant tumors in multiple organs. Tumorigenesis was enhanced compared with p53 mutant zebrafish. p53 mutants also had increased mTORC1 signaling that was further enhanced in tsc2;p53 compound mutants. We found increased expression of Hif1-α, Hif2-α and Vegf-c in tsc2;p53 compound mutant zebrafish compared with p53 mutant zebrafish. Expression of these proteins probably underlies the increased angiogenesis seen in compound mutant zebrafish compared with p53 mutants and might further drive cancer progression. Treatment of p53 and compound mutant zebrafish with the mTORC1 inhibitor rapamycin caused rapid shrinkage of tumor size and decreased caliber of tumor-associated blood vessels. This is the first report using an animal model to show interactions between tsc2, mTORC1 and p53 during tumorigenesis. These results might explain why individuals with TSC rarely have malignant tumors, but also suggest that cancer arising in individuals without TSC might be influenced by the status of TSC1 and/or TSC2 mutations and be potentially treatable with mTORC1 inhibitors. PMID:23580196

A Disease-associated Mutant of NLRC4 Shows Enhanced Interaction with SUG1 Leading to Constitutive FADD-dependent Caspase-8 Activation and Cell Death.

PubMed

Raghawan, Akhouri Kishore; Sripada, Anand; Gopinath, Gayathri; Pushpanjali, Pendyala; Kumar, Yatender; Radha, Vegesna; Swarup, Ghanshyam

2017-01-27

Nod-like receptor family card containing 4 (NLRC4)/Ipaf is involved in recognition of pathogen-associated molecular patterns leading to caspase-1 activation and cytokine release, which mediate protective innate immune response. Point mutations in NLRC4 cause autoinflammatory syndromes. Although all the mutations result in constitutive caspase-1 activation, their phenotypic presentations are different, implying that these mutations cause different alterations in properties of NLRC4. NLRC4 interacts with SUG1 and induces caspase-8-mediated cell death. Here, we show that one of the autoinflammatory syndrome-causing mutants of NLRC4, H443P, but not T337A and V341A, constitutively activates caspase-8 and induces apoptotic cell death in human lung epithelial cells. Compared with wild type NLRC4, the H443P mutant shows stronger interaction with SUG1 and with ubiquitinated cellular proteins. Phosphorylation of NLRC4 at Ser 533 plays a crucial role in caspase-8 activation and cell death. However, H443P mutant does not require Ser 533 phosphorylation for caspase-8 activation and cell death. Caspase-8 activation by NLRC4 and its H443P mutant are dependent on the adaptor protein FADD. A phosphomimicking mutant of NLRC4, S533D does not require SUG1 activity for inducing cell death. Ubiquitin-tagged NLRC4 could induce cell death and activate caspase-8 independent of Ser 533 phosphorylation. Our work suggests that SUG1-mediated signaling results in enhanced ubiquitination and regulates FADD-dependent caspase-8 activation by NLRC4. We show that the autoinflammation-associated H443P mutant is altered in interaction with SUG1 and ubiquitinated proteins, triggering constitutive caspase-8-mediated cell death dependent on FADD but independent of Ser 533 phosphorylation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

EPHA2 MUTATIONS CONTRIBUTE TO CONGENITAL CATARACT THROUGH DIVERSE MECHANISMS.

PubMed

Dave, Alpana; Martin, Sarah; Kumar, Raman; Craig, Jamie E; Burdon, Kathryn P; Sharma, Shiwani

2016-01-01

Congenital cataract is a leading cause of childhood blindness. Mutations in the EPHA2 gene are one of the causes of inherited congenital cataract. The EPHA2 gene encodes a membrane-bound tyrosine kinase receptor and is highly expressed in epithelial cells, including in the ocular lens. Signaling through the EPHA2 receptor plays a pivotal role in epithelial cell homeostasis. The aim of this study was to determine the effect of congenital cataract causing mutations in the EPHA2 gene on the encoded protein in epithelial cells. The effect of five disease-causing mutations, p.P584L (c.1751C>T), p.T940I (c.2819C>T), p.D942fsXC71 (c.2826-9G>A), p.A959T (c.2875G>A), and p.V972GfsX39 (c.2915_2916delTG), on localization of the protein was examined in two in vitro epithelial cell culture systems: Madin-Darby Canine Kidney (MDCK) and human colorectal adenocarcinoma (Caco-2) epithelial cells. Myc-tagged mutant constructs were generated by polymerase chain reaction (PCR)-based mutagenesis. The Myc-tagged wild-type construct was used as a control. The Myc-tagged wild-type and mutant proteins were ectopically expressed and detected by immunofluorescence labeling. Two of the mutations, p.T940I and p.D942fsXC71, located within the cytoplasmic sterile-α-motif (SAM) domain of EPHA2, led to mis-localization of the protein to the perinuclear space and co-localization with the cis-golgi apparatus, indicating sub-organellar/cellular retention of the mutant proteins. The mutant proteins carrying the remaining three mutations, similar to the wild-type EPHA2, localized to the cell membrane. Mis-localization of two of the mutant proteins in epithelial cells suggests that some disease-causing mutations in EPHA2 likely affect lens epithelial cell homeostasis and contribute to cataract. This study suggests that mutations in EPHA2 contribute to congenital cataract through diverse mechanisms.

An FGF3-BMP Signaling Axis Regulates Caudal Neural Tube Closure, Neural Crest Specification and Anterior-Posterior Axis Extension

PubMed Central

Anderson, Matthew J.; Schimmang, Thomas; Lewandoski, Mark

2016-01-01

During vertebrate axis extension, adjacent tissue layers undergo profound morphological changes: within the neuroepithelium, neural tube closure and neural crest formation are occurring, while within the paraxial mesoderm somites are segmenting from the presomitic mesoderm (PSM). Little is known about the signals between these tissues that regulate their coordinated morphogenesis. Here, we analyze the posterior axis truncation of mouse Fgf3 null homozygotes and demonstrate that the earliest role of PSM-derived FGF3 is to regulate BMP signals in the adjacent neuroepithelium. FGF3 loss causes elevated BMP signals leading to increased neuroepithelium proliferation, delay in neural tube closure and premature neural crest specification. We demonstrate that elevated BMP4 depletes PSM progenitors in vitro, phenocopying the Fgf3 mutant, suggesting that excessive BMP signals cause the Fgf3 axis defect. To test this in vivo we increased BMP signaling in Fgf3 mutants by removing one copy of Noggin, which encodes a BMP antagonist. In such mutants, all parameters of the Fgf3 phenotype were exacerbated: neural tube closure delay, premature neural crest specification, and premature axis termination. Conversely, genetically decreasing BMP signaling in Fgf3 mutants, via loss of BMP receptor activity, alleviates morphological defects. Aberrant apoptosis is observed in the Fgf3 mutant tailbud. However, we demonstrate that cell death does not cause the Fgf3 phenotype: blocking apoptosis via deletion of pro-apoptotic genes surprisingly increases all Fgf3 defects including causing spina bifida. We demonstrate that this counterintuitive consequence of blocking apoptosis is caused by the increased survival of BMP-producing cells in the neuroepithelium. Thus, we show that FGF3 in the caudal vertebrate embryo regulates BMP signaling in the neuroepithelium, which in turn regulates neural tube closure, neural crest specification and axis termination. Uncovering this FGF3-BMP signaling axis is a major advance toward understanding how these tissue layers interact during axis extension with important implications in human disease. PMID:27144312

Early Clinical Diagnosis of PC1/3 Deficiency in a Patient With a Novel Homozygous PCSK1 Splice-Site Mutation.

PubMed

Härter, Bettina; Fuchs, Irene; Müller, Thomas; Akbulut, Ulas Emre; Cakir, Murat; Janecke, Andreas R

2016-04-01

Autosomal recessive proprotein convertase 1/3 (PC1/3) deficiency, caused by mutations in the PCSK1 gene, is characterized by severe congenital malabsorptive diarrhea, early-onset obesity, and certain endocrine abnormalities. We suspected PC1/3 deficiency in a 4-month-old girl based on the presence of congenital diarrhea and polyuria. Sequencing the whole coding region and splice sites detected a novel homozygous PCSK1 splice-site mutation, c.544-2A>G, in the patient. The mutation resulted in the skipping of exon 5, the generation of a premature termination codon, and nonsense-mediated PCSK1 messenger ribonucleic acid decay, which was demonstrated in complementary DNA derived from fibroblasts.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Dongbo; Zhao, Jinfeng, E-mail: jinfeng.zhao@tongji.edu.cn; Li, Libing

In this work, we applied a robust and fully air-coupled method to investigate the propagation of the lowest-order antisymmetric Lamb (A{sub 0}) mode in both a stubbed and an air-drilled phononic-crystal (PC) plate. By measuring simply the radiative acoustic waves of A{sub 0} mode close to the plate surface, we observed the band gaps for the stubbed PC plate caused by either the local resonance or the Bragg scattering, in frequency ranges in good agreement with theoretical predictions. We measured then the complete band gap of A{sub 0} mode for the air-drilled PC plate, in good agreement with the bandmore » structures. Finally, we compared the measurements made using the air-coupled method with those obtained by the laser ultrasonic technique.« less

Coherent response of the Indo-African boreal summer monsoon to Pacific SST captured in Ethiopian rain δ18O

NASA Astrophysics Data System (ADS)

Madhavan, M.; Palliyil, L. R.; Ramesh, R.

2017-12-01

Pacific Sea Surface Temperature (SST) plays an important role in the inter-annual to inter-decadal variability of boreal monsoons. We identified a common mode of inter annual variability in the Indian and African boreal summer monsoon (June to September) rainfalls, which is linked to Pacific SSTs, using Empirical Orthogonal Function (EOF) analysis. Temporal coefficients (Principle component: PC1) of the leading mode of variability (EOF-1) is well correlated with the Indian summer monsoon rainfall and Sahel rainfall. About forty year long monthly observations of δ18O (and δD) at Addis Ababa, Ethiopia show a strong association with PC1 (r=0.69 for δ18O and r=0.75 for δD). Analysis of SST, sea level pressure and lower tropospheric winds suggest that 18O depletion in Ethiopian rainfall (and wet phases of PC1) is associated with cooler eastern tropical Pacific and warmer western Pacific and strengthening of Pacific subtropical high in both the hemispheres. Associated changes in the trade winds cause enhanced westerly moisture transport into the Indian subcontinent and northern Africa and cause enhanced rainfall. The intrusion of Atlantic westerly component of moisture transport at Addis Ababa during wet phases of PC1 is clearly recorded in δ18O of rain. We also observe the same common mode of variability (EOF1) of Indo-African boreal summer monsoon rain on decadal time scales. A 100 year long δ18O record of actively growing speleothem from the Mechara cave, Ethiopia, matches very well with the PC1 on the decadal time scale. This highlights the potential of speleothem δ18O and leaf wax δD from Ethiopia to investigate the natural variability and teleconnections of Indo-African boreal monsoon.

Mutations that abolish the ability of Ha-Ras to associate with Raf-1.

PubMed

Shirouzu, M; Koide, H; Fujita-Yoshigaki, J; Oshio, H; Toyama, Y; Yamasaki, K; Fuhrman, S A; Villafranca, E; Kaziro, Y; Yokoyama, S

1994-08-01

Recent studies have revealed that Ras can associate physically with Raf. In the present study, we tested 34 mutants of Ha-Ras carrying substitution(s) in the region of residues 23-71 for their ability to associate with Raf-1. Mouse Ba/F3 cell lysates were incubated with each mutant Ras protein, in either the guanosine 5'-[gamma-thio]triphosphate (GTP gamma S)- or the guanosine 5'-[beta-thio]diphosphate (GDP beta S)-bound form, and the anti-Ras antibody Y13-238. The immunoprecipitates were analysed for the presence of Raf-1 by Western blotting with an anti-Raf-1 antibody. Six mutants of Ras, E31K, P34G, T35S, D38N, D57A and A59T, failed to bind Raf-1. Mutations N26G, V29A, S39A, Y40W, R41A, V44A, V45E, L56A and T58A partially reduced the ability to bind Raf-1. All the other mutants could associate with Raf-1 with nearly the same efficiency as that of wild-type Ras. Thus, the Raf-I-binding ability of Ras appears to be affected by mutations in the N-terminal region, and in particular, by those in and neighboring the effector region (residues 32-40) and in the region (residues 56-59) flanking the N-terminal of Switch II. The abilities to bind Raf-1 and to induce neurite outgrowth of pheochromocytoma (PC) 12 cells correlate to each other for 22 Ras mutants. However, mutation A59T, which does not reduce the neurite-inducing or transforming activities, abolishes the ability to bind Raf-1. In contrast, mutations Y32F, K42A and L53A, which impair the neurite-inducing activity of Ras, have no effect on the Ras.Raf-1 association. Partially reduced Raf-1-binding ability was observed for mutants V29A, S39A, Y40W, R41A, V44A, L56A and T58A, which exhibit full neurite-inducing activity, and also for mutant V45E, which has no activity of neurite induction.

Positional cloning of the sex-linked giant egg (Ge) locus in the silkworm, Bombyx mori.

PubMed

Fujii, T; Abe, H; Kawamoto, M; Banno, Y; Shimada, T

2015-04-01

The giant egg (Ge) locus is a Z-linked mutation that leads to the production of large eggs. Cytological observations suggest that an unusual translocation of a large fragment of the W chromosome bearing a putative egg size-determining gene, Esd, gave rise to giant egg mutants. However, there is currently no molecular evidence confirming either a W-Z translocation or the presence of Esd on the W chromosome. To elucidate the origin of giant egg mutants, we performed positional cloning. We observed that the Bombyx mori. orthologue of the human Phytanoyl-CoA dioxygenase domain containing 1 gene (PHYHD1) is disrupted in giant egg mutants. PHYHD1 is highly conserved in eukaryotes and is predicted to be a Fe(II) and 2-oxoglutarate-dependent oxygenase. Exon skipping in one of the two available Ge mutants is probably caused by the insertion of a non-long terminal repeat transposon into intron 4 in the vicinity of the 5' splice site. Segmental duplication in Ge(2) , an independent allele, was caused by unequal recombination between short interspersed elements inserted into introns 3 and 5. Our results indicate that (1) Bombyx PHYHD1 is responsible for the Ge mutants and that (2) the Ge locus is unrelated to the W-linked putative Esd. To our knowledge, this is the first report describing the phenotypic defects caused by mutations in PHYHD1 orthologues. © 2014 The Royal Entomological Society.

CcpN Controls Central Carbon Fluxes in Bacillus subtilis▿ ‡

PubMed Central

Tännler, Simon; Fischer, Eliane; Le Coq, Dominique; Doan, Thierry; Jamet, Emmanuel; Sauer, Uwe; Aymerich, Stéphane

2008-01-01

The transcriptional regulator CcpN of Bacillus subtilis has been recently characterized as a repressor of two gluconeogenic genes, gapB and pckA, and of a small noncoding regulatory RNA, sr1, involved in arginine catabolism. Deletion of ccpN impairs growth on glucose and strongly alters the distribution of intracellular fluxes, rerouting the main glucose catabolism from glycolysis to the pentose phosphate (PP) pathway. Using transcriptome analysis, we show that during growth on glucose, gapB and pckA are the only protein-coding genes directly repressed by CcpN. By quantifying intracellular fluxes in deletion mutants, we demonstrate that derepression of pckA under glycolytic condition causes the growth defect observed in the ccpN mutant due to extensive futile cycling through the pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and pyruvate kinase. Beyond ATP dissipation via this cycle, PckA activity causes a drain on tricarboxylic acid cycle intermediates, which we show to be the main reason for the reduced growth of a ccpN mutant. The high flux through the PP pathway in the ccpN mutant is modulated by the flux through the alternative glyceraldehyde-3-phosphate dehydrogenases, GapA and GapB. Strongly increased concentrations of intermediates in upper glycolysis indicate that GapB overexpression causes a metabolic jamming of this pathway and, consequently, increases the relative flux through the PP pathway. In contrast, derepression of sr1, the third known target of CcpN, plays only a marginal role in ccpN mutant phenotypes. PMID:18586936

Exploration of Structural and Functional Variations Owing to Point Mutations in α-NAGA.

PubMed

Meshach Paul, D; Rajasekaran, R

2018-03-01

Schindler disease is a lysosomal storage disorder caused due to deficiency or defective activity of alpha-N-acetylgalactosaminidase (α-NAGA). Mutations in gene encoding α-NAGA cause wide range of diseases, characterized with mild to severe clinical features. Molecular effects of these mutations are yet to be explored in detail. Therefore, this study was focused on four missense mutations of α-NAGA namely, S160C, E325K, R329Q and R329W. Native and mutant structures of α-NAGA were analysed to determine geometrical deviations such as the contours of root mean square deviation, root mean square fluctuation, percentage of residues in allowed regions of Ramachandran plot and solvent accessible surface area, using conformational sampling technique. Additionally, global energy-minimized structures of native and mutants were further analysed to compute their intra-molecular interactions, hydrogen bond dilution and distribution of secondary structure. In addition, docking studies were also performed to determine variations in binding energies between native and mutants. The deleterious effects of mutants were evident due to variations in their active site residues pertaining to spatial conformation and flexibility, comparatively. Hence, variations exhibited by mutants, namely S160C, E325K, R329Q and R329W to that of native, consequently, lead to the detrimental effects causing Schindler disease. This study computationally explains the underlying reasons for the pathogenesis of the disease, thereby aiding future researchers in drug development and disease management.

In Vitro Expansion of CAG, CAA, and Mixed CAG/CAA Repeats.

PubMed

Figura, Grzegorz; Koscianska, Edyta; Krzyzosiak, Wlodzimierz J

2015-08-11

Polyglutamine diseases, including Huntington's disease and a number of spinocerebellar ataxias, are caused by expanded CAG repeats that are located in translated sequences of individual, functionally-unrelated genes. Only mutant proteins containing polyglutamine expansions have long been thought to be pathogenic, but recent evidence has implicated mutant transcripts containing long CAG repeats in pathogenic processes. The presence of two pathogenic factors prompted us to attempt to distinguish the effects triggered by mutant protein from those caused by mutant RNA in cellular models of polyglutamine diseases. We used the SLIP (Synthesis of Long Iterative Polynucleotide) method to generate plasmids expressing long CAG repeats (forming a hairpin structure), CAA-interrupted CAG repeats (forming multiple unstable hairpins) or pure CAA repeats (not forming any secondary structure). We successfully modified the original SLIP protocol to generate repeats of desired length starting from constructs containing short repeat tracts. We demonstrated that the SLIP method is a time- and cost-effective approach to manipulate the lengths of expanded repeat sequences.

Intron retention and nuclear loss of SFPQ are molecular hallmarks of ALS.

PubMed

Luisier, Raphaelle; Tyzack, Giulia E; Hall, Claire E; Mitchell, Jamie S; Devine, Helen; Taha, Doaa M; Malik, Bilal; Meyer, Ione; Greensmith, Linda; Newcombe, Jia; Ule, Jernej; Luscombe, Nicholas M; Patani, Rickie

2018-05-22

Mutations causing amyotrophic lateral sclerosis (ALS) strongly implicate ubiquitously expressed regulators of RNA processing. To understand the molecular impact of ALS-causing mutations on neuronal development and disease, we analysed transcriptomes during in vitro differentiation of motor neurons (MNs) from human control and patient-specific VCP mutant induced-pluripotent stem cells (iPSCs). We identify increased intron retention (IR) as a dominant feature of the splicing programme during early neural differentiation. Importantly, IR occurs prematurely in VCP mutant cultures compared with control counterparts. These aberrant IR events are also seen in independent RNAseq data sets from SOD1- and FUS-mutant MNs. The most significant IR is seen in the SFPQ transcript. The SFPQ protein binds extensively to its retained intron, exhibits lower nuclear abundance in VCP mutant cultures and is lost from nuclei of MNs in mouse models and human sporadic ALS. Collectively, we demonstrate SFPQ IR and nuclear loss as molecular hallmarks of familial and sporadic ALS.

The Shift of the Intestinal Microbiome in the Innate Immunity-Deficient Mutant rde-1 Strain of C. elegans upon Orsay Virus Infection.

PubMed

Guo, Yuanyuan; Xun, Zhe; Coffman, Stephanie R; Chen, Feng

2017-01-01

The status of intestinal microbiota is a determinant of host health. However, the alteration of the gut microbiota caused by the innate immune response to virus infection is unclear. Caenorhabditis elegans and its natural virus Orsay provide an excellent model of host-virus interactions. We evaluated the intestinal microbial community complexity of the wild-type N2 and the innate immunity-deficient mutant rde-1 ( ne219 ) strains of C. elegans upon Orsay virus infection. The gut microbiota diversity was decreased in rde-1 ( ne219 ) mutant animals, and a large number of genes were associated with the difference between infected and uninfected rde-1 ( ne219 ) mutant animals. Therefore, this study provides the first evaluation of the alterations caused by Orsay virus on intestinal microbiota in wildtype and innate immunity-deficient animals using C. elegans as the model species. Our findings indicate that virus infection may alters the microbiome in animals with defective immune response.

The Shift of the Intestinal Microbiome in the Innate Immunity-Deficient Mutant rde-1 Strain of C. elegans upon Orsay Virus Infection

PubMed Central

Guo, Yuanyuan; Xun, Zhe; Coffman, Stephanie R.; Chen, Feng

2017-01-01

The status of intestinal microbiota is a determinant of host health. However, the alteration of the gut microbiota caused by the innate immune response to virus infection is unclear. Caenorhabditis elegans and its natural virus Orsay provide an excellent model of host–virus interactions. We evaluated the intestinal microbial community complexity of the wild-type N2 and the innate immunity-deficient mutant rde-1 (ne219) strains of C. elegans upon Orsay virus infection. The gut microbiota diversity was decreased in rde-1 (ne219) mutant animals, and a large number of genes were associated with the difference between infected and uninfected rde-1 (ne219) mutant animals. Therefore, this study provides the first evaluation of the alterations caused by Orsay virus on intestinal microbiota in wildtype and innate immunity-deficient animals using C. elegans as the model species. Our findings indicate that virus infection may alters the microbiome in animals with defective immune response. PMID:28611740

Complementation between polymerase- and exonuclease-deficient mitochondrial DNA polymerase mutants in genomically engineered flies

PubMed Central

Bratic, Ana; Kauppila, Timo E. S.; Macao, Bertil; Grönke, Sebastian; Siibak, Triinu; Stewart, James B.; Baggio, Francesca; Dols, Jacqueline; Partridge, Linda; Falkenberg, Maria; Wredenberg, Anna; Larsson, Nils-Göran

2015-01-01

Replication errors are the main cause of mitochondrial DNA (mtDNA) mutations and a compelling approach to decrease mutation levels would therefore be to increase the fidelity of the catalytic subunit (POLγA) of the mtDNA polymerase. Here we genomically engineer the tamas locus, encoding fly POLγA, and introduce alleles expressing exonuclease- (exo−) and polymerase-deficient (pol−) POLγA versions. The exo− mutant leads to accumulation of point mutations and linear deletions of mtDNA, whereas pol− mutants cause mtDNA depletion. The mutant tamas alleles are developmentally lethal but can complement each other in trans resulting in viable flies with clonally expanded mtDNA mutations. Reconstitution of human mtDNA replication in vitro confirms that replication is a highly dynamic process where POLγA goes on and off the template to allow complementation during proofreading and elongation. The created fly models are valuable tools to study germ line transmission of mtDNA and the pathophysiology of POLγA mutation disease. PMID:26554610

La Crosse virus (LACV) Gc fusion peptide mutants have impaired growth and fusion phenotypes, but remain neurotoxic

DOE Office of Scientific and Technical Information (OSTI.GOV)

Soldan, Samantha S., E-mail: sssoldan@mail.med.upenn.ed; Hollidge, Bradley S.; Department of Neuroscience Graduate Group, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4283

La Crosse virus is a leading cause of pediatric encephalitis in the Midwestern United States and an emerging pathogen in the American South. The LACV glycoprotein Gc plays a critical role in entry as the virus attachment protein. A 22 amino acid hydrophobic region within Gc (1066-1087) was recently identified as the LACV fusion peptide. To further define the role of Gc (1066-1087) in virus entry, fusion, and neuropathogenesis, a panel of recombinant LACV (rLACV) fusion peptide mutant viruses was generated. Replication of mutant rLACVs was significantly reduced. In addition, the fusion peptide mutants demonstrated decreased fusion phenotypes relative tomore » LACV-WT. Interestingly, these viruses maintained their ability to cause neuronal loss in culture, suggesting that the fusion peptide of LACV Gc is a determinant of properties associated with neuroinvasion (growth to high titer in muscle cells and a robust fusion phenotype), but not necessarily of neurovirulence.« less

Multiple organ gigantism caused by mutation in VmPPD gene in blackgram (Vigna mungo).

PubMed

Naito, Ken; Takahashi, Yu; Chaitieng, Bubpa; Hirano, Kumi; Kaga, Akito; Takagi, Kyoko; Ogiso-Tanaka, Eri; Thavarasook, Charaspon; Ishimoto, Masao; Tomooka, Norihiko

2017-03-01

Seed size is one of the most important traits in leguminous crops. We obtained a recessive mutant of blackgram that had greatly enlarged leaves, stems and seeds. The mutant produced 100% bigger leaves, 50% more biomass and 70% larger seeds though it produced 40% less number of seeds. We designated the mutant as multiple-organ-gigantism ( mog ) and found the mog phenotype was due to increase in cell numbers but not in cell size. We also found the mog mutant showed a rippled leaf ( rl ) phenotype, which was probably caused by a pleiotropic effect of the mutation. We performed a map-based cloning and successfully identified an 8 bp deletion in the coding sequence of VmPPD gene, an orthologue of Arabidopsis PEAPOD ( PPD ) that regulates arrest of cell divisions in meristematic cells . We found no other mutations in the neighboring genes between the mutant and the wild type. We also knocked down GmPPD genes and reproduced both the mog and rl phenotypes in soybean. Controlling PPD genes to produce the mog phenotype is highly valuable for breeding since larger seed size could directly increase the commercial values of grain legumes.

Multiple organ gigantism caused by mutation in VmPPD gene in blackgram (Vigna mungo)

PubMed Central

Naito, Ken; Takahashi, Yu; Chaitieng, Bubpa; Hirano, Kumi; Kaga, Akito; Takagi, Kyoko; Ogiso-Tanaka, Eri; Thavarasook, Charaspon; Ishimoto, Masao; Tomooka, Norihiko

2017-01-01

Seed size is one of the most important traits in leguminous crops. We obtained a recessive mutant of blackgram that had greatly enlarged leaves, stems and seeds. The mutant produced 100% bigger leaves, 50% more biomass and 70% larger seeds though it produced 40% less number of seeds. We designated the mutant as multiple-organ-gigantism (mog) and found the mog phenotype was due to increase in cell numbers but not in cell size. We also found the mog mutant showed a rippled leaf (rl) phenotype, which was probably caused by a pleiotropic effect of the mutation. We performed a map-based cloning and successfully identified an 8 bp deletion in the coding sequence of VmPPD gene, an orthologue of Arabidopsis PEAPOD (PPD) that regulates arrest of cell divisions in meristematic cells. We found no other mutations in the neighboring genes between the mutant and the wild type. We also knocked down GmPPD genes and reproduced both the mog and rl phenotypes in soybean. Controlling PPD genes to produce the mog phenotype is highly valuable for breeding since larger seed size could directly increase the commercial values of grain legumes. PMID:28588392

PC-based high-speed video-oculography for measuring rapid eye movements in mice.

PubMed

Sakatani, Tomoya; Isa, Tadashi

2004-05-01

We newly developed an infrared video-oculographic system for on-line tracking of the eye position in awake and head-fixed mice, with high temporal resolution (240 Hz). The system consists of a commercially available high-speed CCD camera and an image processing software written in LabVIEW run on IBM-PC with a plug-in video grabber board. This software calculates the center and area of the pupil by fitting circular function to the pupil boundary, and allows robust and stable tracking of the eye position in small animals like mice. On-line calculation is performed to obtain reasonable circular fitting of the pupil boundary even if a part of the pupil is covered with shadows or occluded by eyelids or corneal reflections. The pupil position in the 2-D video plane is converted to the rotation angle of the eyeball by estimating its rotation center based on the anatomical eyeball model. By this recording system, it is possible to perform quantitative analysis of rapid eye movements such as saccades in mice. This will provide a powerful tool for analyzing molecular basis of oculomotor and cognitive functions by using various lines of mutant mice.

Degradation of misfolded proteins by autophagy: is it a strategy for Huntington's disease treatment?

PubMed

Lin, Fang; Qin, Zheng-Hong

2013-01-01

Autophagy is a degradation pathway for long-lived cytoplasmic proteins, protein complexes, or damaged organelles. The accumulation and aggregation of misfolded proteins are hallmarks of several neurodegenerative diseases. Many researchers have reported that autophagy degrades disease-causing misfolded and aggregated proteins, including mutant huntingtin (Htt) in Huntington's disease, mutant synuclein in familial Parkingson's disease, mutant Cu, Zn-Superoxide dismutase (SOD1) in familial amyotrophic lateral sclerosis. In this review, we will bring up new evidence to elucidate the involvement of autophagy in degradation of mutant Htt, discuss the mechanisms regulating the degradation of mutant Htt by autophagy and the therapeutic effects of drugs that enhance autophagy to improve clearance of mutant Htt. We propose that enhancement of autophagy by drugs may be a strategy to treat or retard progression of Huntington's disease.

Multi-Scale Simulation of Interfacial Phenomena and Nano-Particle Placement in Polymer Matrix Composites

DTIC Science & Technology

2012-08-01

Molecular Dynamics Simulations Coarse-Grain Particle Dynamics Simulations Local structure; Force field parameterization Extended structure...K) C8H18 C12H26 C16H34 Adhesive forces can cause local density gradients and defects " Pronounced layering of polymer near interfaces...reactive end groups (CnH2n+1S) on Cu Gap SubPc on C60 Pentacene on a-SiO2 Cyclopentene on Au Crystalline CuPc on Al Polyimide on Si

Molecular Targets and Synaptic Effects of Carbamates, Organophosphates, Oximes, and Pyridiniums: Implications for Prophylaxis and Therapy of Organophosphate Toxicity

DTIC Science & Technology

1994-03-16

excitability in the latter studies , it does seem that block of gK by PC, is an important factor in the pathopharmacological eltfect- of PC? in causing...muscle following acute and subacute exposure to pyridostigmine. Studies on muscle contractility and cellular mechanisms. Proc. Fourth Annual Chemical...receptor-ionic chauinel complex II. Patch dlamp studies . Mol. Pharmacol. 0 25:102-112, 1984. 7. Albuquerque, E.X., Aguayo, L., Swanson, K.L., Idriss, M

Novel CLCNKB mutations causing Bartter syndrome affect channel surface expression.

PubMed

Keck, Mathilde; Andrini, Olga; Lahuna, Olivier; Burgos, Johanna; Cid, L Pablo; Sepúlveda, Francisco V; L'hoste, Sébastien; Blanchard, Anne; Vargas-Poussou, Rosa; Lourdel, Stéphane; Teulon, Jacques

2013-09-01

Mutations in the CLCNKB gene encoding the ClC-Kb Cl(-) channel cause Bartter syndrome, which is a salt-losing renal tubulopathy. Here, we investigate the functional consequences of seven mutations. When expressed in Xenopus laevis oocytes, four mutants carried no current (c.736G>C, p.Gly246Arg; c.1271G>A, p.Gly424Glu; c.1313G>A, p.Arg438His; c.1316T>C, p.Leu439Pro), whereas others displayed a 30%-60% reduction in conductance as compared with wild-type ClC-Kb (c.242T>C, p.Leu81Pro; c.274C>T, p.Arg92Trp; c.1052G>C, p.Arg351Pro). Anion selectivity and sensitivity to external Ca(2+) and H(+), typical of the ClC-Kb channel, were not modified in the partially active mutants. In oocytes, we found that all the mutations reduced surface expression with a profile similar to that observed for currents. In HEK293 cells, the currents in the mutants had similar profiles to those obtained in oocytes, except for p.Leu81Pro, which produced no current. Furthermore, p.Arg92Trp and p.Arg351Pro mutations did not modify the unit-conductance of closely related ClC-K1. Western blot analysis in HEK293 cells showed that ClC-Kb protein abundance was lower for the nonconducting mutants but similar to wild-type for other mutants. Overall, two classes of mutants can be distinguished: nonconducting mutants associated with low total protein expression, and partially conducting mutants with unaltered channel properties and ClC-Kb protein abundance. © 2013 WILEY PERIODICALS, INC.

Isolation and characterization of low-sulphur-tolerant mutants of Arabidopsis

PubMed Central

Wu, Yu; Zhao, Qing; Gao, Lei; Yu, Xiao-Min; Fang, Ping; Oliver, David J.; Xiang, Cheng-Bin

2010-01-01

Sulphur is an essential element for plant growth and development as well as for defence against biotic and abiotic stresses. Increasing sulphate utilization efficiency (SUE) is an important issue for crop improvement. Little is known about the genetic determinants of sulphate utilization efficiency. No gain-of-function mutants with improved SUE have been reported to date. Here the isolation and characterization of two low-sulphur-tolerant mutants, sue3 and sue4 are reported using a high-throughput genetic screen where a ‘sulphur-free’ solid medium was devised to give the selection pressure necessary to suppress the growth of the wild-type seedlings. Both mutants showed improved tolerance to low sulphur conditions and well-developed root systems. The mutant phenotype of both sue3 and sue4 was specific to sulphate deficiency and the mutants displayed enhanced tolerance to heavy metal and oxidative stress. Genetic analysis revealed that sue3 was caused by a single recessive nuclear mutation while sue4 was caused by a single dominant nuclear mutation. The recessive locus in sue3 is the previously identified VirE2-interacting Protein 1. The dominant locus in sue4 is a function-unknown locus activated by the four enhancers on the T-DNA. The function of SUE3 and SUE4 in low sulphur tolerance was confirmed either by multiple mutant alleles or by recapitulation analysis. Taken together, our results demonstrate that this genetic screen is a reasonable approach to isolate Arabidopsis mutants with improved low sulphur tolerance and potentially with enhanced sulphate utilization efficiency. The two loci identified in sue3 and sue4 should assist in understanding the molecular mechanisms of low sulphur tolerance. PMID:20547563

FUdR extends the lifespan of the short-lived AP endonuclease mutant in Caenorhabditis elegans in a fertility-dependent manner.

PubMed

Kato, Yuichi; Miyaji, Masahiro; Zhang-Akiyama, Qiu-Mei

2017-03-17

The anticancer drug 5-fluorouracil (5-FU) and its metabolite 5-fluoro-2'-deoxyuridine (FUdR) inhibit thymidylate synthase and induce uracil bases in DNA. FUdR is commonly used for inhibiting fertility when measuring the lifespan of the nematode Caenorhabditis elegans. However, it is not known whether DNA damage induced by FUdR affects lifespan. EXO-3 is an apurinic/apyrimidinic endonuclease in C. elegans, and we reported previously that deletion of the exo-3 gene causes reproductive abnormalities and decreased lifespan. In this study, we found that FUdR extended the lifespan of exo-3 mutants. We measured the lifespan of multiple germline mutants to examine whether this lifespan extension effect was dependent on fertility. In the presence of a fem-1 mutation, which causes a deficiency in sperm production, FUdR did not extend the lifespan of the exo-3 mutant. In glp-1 mutants, which do not develop gonads, the exo-3 mutant was not short-lived, and FUdR did not extend its lifespan. These results suggest that the lifespan extension effect of FUdR depends on fertility and the presence of gonads. fem-3 mutants, which do not produce oocytes, had increased lifespan in the presence of FUdR, independent of the exo-3 mutation. It is possible that the fem-3 mutant was susceptible to the lifespan extension effect of FUdR. From these results, we suggest that FUdR affects the lifespan of C. elegans in two ways: by interfering with fertility, which extends lifespan, and by inducing DNA base damage, which reduces lifespan.

A Point Mutation in the Gene for Asparagine-Linked Glycosylation 10B (Alg10b) Causes Nonsyndromic Hearing Impairment in Mice (Mus musculus)

PubMed Central

Probst, Frank J.; Corrigan, Rebecca R.; del Gaudio, Daniela; Salinger, Andrew P.; Lorenzo, Isabel; Gao, Simon S.; Chiu, Ilene; Xia, Anping

2013-01-01

The study of mouse hearing impairment mutants has led to the identification of a number of human hearing impairment genes and has greatly furthered our understanding of the physiology of hearing. The novel mouse mutant neurological/sensory 5 (nse5) demonstrates a significantly reduced or absent startle response to sound and is therefore a potential murine model of human hearing impairment. Genetic analysis of 500 intercross progeny localized the mutant locus to a 524 kilobase (kb) interval on mouse chromosome 15. A missense mutation in a highly-conserved amino acid was found in the asparagine-linked glycosylation 10B gene (Alg10b), which is within the critical interval for the nse5 mutation. A 20.4 kb transgene containing a wildtype copy of the Alg10b gene rescued the mutant phenotype in nse5/nse5 homozygous animals, confirming that the mutation in Alg10b is responsible for the nse5/nse5 mutant phenotype. Homozygous nse5/nse5 mutants had abnormal auditory brainstem responses (ABRs), distortion product otoacoustic emissions (DPOAEs), and cochlear microphonics (CMs). Endocochlear potentials (EPs), on the other hand, were normal. ABRs and DPOAEs also confirmed the rescue of the mutant nse5/nse5 phenotype by the wildtype Alg10b transgene. These results suggested a defect in the outer hair cells of mutant animals, which was confirmed by histologic analysis. This is the first report of mutation in a gene involved in the asparagine (N)-linked glycosylation pathway causing nonsyndromic hearing impairment, and it suggests that the hearing apparatus, and the outer hair cells in particular, are exquisitely sensitive to perturbations of the N-linked glycosylation pathway. PMID:24303013

Structural Effects of Fibulin 5 Missense Mutations Associated with Age-Related Macular Degeneration and Cutis Laxa

PubMed Central

Jones, Richard P. O.; Ridley, Caroline; Jowitt, Thomas A.; Wang, Ming-Chuan; Howard, Marjorie; Bobola, Nicoletta; Wang, Tao; Bishop, Paul N.; Kielty, Cay M.; Baldock, Clair; Lotery, Andrew J.; Trump, Dorothy

2010-01-01

Purpose. AMD has a complex etiology with environmental and genetic risk factors. Ten fibulin 5 sequence variants have been associated with AMD and two other fibulin 5 mutations cause autosomal-recessive cutis laxa. Fibulin 5 is a 52-kDa calcium-binding epidermal growth factor (cbEGF)–rich extracellular matrix protein that is essential for the formation of elastic tissues. Biophysical techniques were used to detect structural changes in the fibulin 5 mutants and to determine whether changes are predictive of pathogenicity. Methods. Native PAGE, nonreduced SDS-PAGE, size-exclusion column multiangle laser light scattering, sedimentation velocity, and circular dichroism (CD) were used to investigate the mobility, hydrodynamic radii, folding, and oligomeric states of the fibulin 5 mutants in the absence and presence of Ca2+. Results. CD showed that all mutants are folded, although perturbations to secondary structure contents were detected. Both cutis laxa mutants increased dimerization. Most other mutants slightly increased self-association in the absence of Ca2+ but this was also demonstrated by G202R, a polymorphism detected in a control individual. The AMD-associated mutant G412E showed lower-than-expected mobility during native-PAGE, the largest hydrodynamic radius for the monomer form and the highest levels of aggregation in both the absence and presence of Ca2+. Conclusions. The results identified structural differences for the disease-causing cutis laxa mutants and for one AMD variant (G412E), suggesting that this may also be pathogenic. Although the other AMD-associated mutants showed no gross structural differences, they cannot be excluded as pathogenic by differences outside the scope of this study—for example, disruption of heterointeractions. PMID:20007835

Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

PubMed Central

Mihic, Anton; Chauhan, Vijay S.; Gao, Xiaodong; Oudit, Gavin Y.; Tsushima, Robert G.

2011-01-01

The Kv11.1 (hERG) K+ channel plays a fundamental role in cardiac repolarization. Missense mutations in KCNH2, the gene encoding Kv11.1, cause long QT syndrome (LQTS) and frequently cause channel trafficking-deficiencies. This study characterized the properties of a novel KCNH2 mutation discovered in a LQT2 patient resuscitated from a ventricular fibrillation arrest. Proband genotyping was performed by SSCP and DNA sequencing. The electrophysiological and biochemical properties of the mutant channel were investigated after expression in HEK293 cells. The proband manifested a QTc of 554 ms prior to electrolyte normalization. Mutation analysis revealed an autosomal dominant frameshift mutation at proline 1086 (P1086fs+32X; 3256InsG). Co-immunoprecipitation demonstrated that wild-type Kv11.1 and mutant channels coassemble. Western blot showed that the mutation did not produce mature complex-glycosylated Kv11.1 channels and coexpression resulted in reduced channel maturation. Electrophysiological recordings revealed mutant channel peak currents to be similar to untransfected cells. Co-expression of channels in a 1∶1 ratio demonstrated dominant negative suppression of peak Kv11.1 currents. Immunocytochemistry confirmed that mutant channels were not present at the plasma membrane. Mutant channel trafficking rescue was attempted by incubation at reduced temperature or with the pharmacological agents E-4031. These treatments did not significantly increase peak mutant currents or induce the formation of mature complex-glycosylated channels. The proteasomal inhibitor lactacystin increased the protein levels of the mutant channels demonstrating proteasomal degradation, but failed to induce mutant Kv11.1 protein trafficking. Our study demonstrates a novel dominant-negative Kv11.1 mutation, which results in degraded non-functional channels leading to a LQT2 phenotype. PMID:21483829

Isolation and characterization of low-sulphur-tolerant mutants of Arabidopsis.

PubMed

Wu, Yu; Zhao, Qing; Gao, Lei; Yu, Xiao-Min; Fang, Ping; Oliver, David J; Xiang, Cheng-Bin

2010-07-01

Sulphur is an essential element for plant growth and development as well as for defence against biotic and abiotic stresses. Increasing sulphate utilization efficiency (SUE) is an important issue for crop improvement. Little is known about the genetic determinants of sulphate utilization efficiency. No gain-of-function mutants with improved SUE have been reported to date. Here the isolation and characterization of two low-sulphur-tolerant mutants, sue3 and sue4 are reported using a high-throughput genetic screen where a 'sulphur-free' solid medium was devised to give the selection pressure necessary to suppress the growth of the wild-type seedlings. Both mutants showed improved tolerance to low sulphur conditions and well-developed root systems. The mutant phenotype of both sue3 and sue4 was specific to sulphate deficiency and the mutants displayed enhanced tolerance to heavy metal and oxidative stress. Genetic analysis revealed that sue3 was caused by a single recessive nuclear mutation while sue4 was caused by a single dominant nuclear mutation. The recessive locus in sue3 is the previously identified VirE2-interacting Protein 1. The dominant locus in sue4 is a function-unknown locus activated by the four enhancers on the T-DNA. The function of SUE3 and SUE4 in low sulphur tolerance was confirmed either by multiple mutant alleles or by recapitulation analysis. Taken together, our results demonstrate that this genetic screen is a reasonable approach to isolate Arabidopsis mutants with improved low sulphur tolerance and potentially with enhanced sulphate utilization efficiency. The two loci identified in sue3 and sue4 should assist in understanding the molecular mechanisms of low sulphur tolerance.

A conserved SREBP-1/phosphatidylcholine feedback circuit regulates lipogenesis in metazoans.

PubMed

Walker, Amy K; Jacobs, René L; Watts, Jennifer L; Rottiers, Veerle; Jiang, Karen; Finnegan, Deirdre M; Shioda, Toshi; Hansen, Malene; Yang, Fajun; Niebergall, Lorissa J; Vance, Dennis E; Tzoneva, Monika; Hart, Anne C; Näär, Anders M

2011-11-11

Sterol regulatory element-binding proteins (SREBPs) activate genes involved in the synthesis and trafficking of cholesterol and other lipids and are critical for maintaining lipid homeostasis. Aberrant SREBP activity, however, can contribute to obesity, fatty liver disease, and insulin resistance, hallmarks of metabolic syndrome. Our studies identify a conserved regulatory circuit in which SREBP-1 controls genes in the one-carbon cycle, which produces the methyl donor S-adenosylmethionine (SAMe). Methylation is critical for the synthesis of phosphatidylcholine (PC), a major membrane component, and we find that blocking SAMe or PC synthesis in C. elegans, mouse liver, and human cells causes elevated SREBP-1-dependent transcription and lipid droplet accumulation. Distinct from negative regulation of SREBP-2 by cholesterol, our data suggest a feedback mechanism whereby maturation of nuclear, transcriptionally active SREBP-1 is controlled by levels of PC. Thus, nutritional or genetic conditions limiting SAMe or PC production may activate SREBP-1, contributing to human metabolic disorders. Copyright © 2011 Elsevier Inc. All rights reserved.

In vivo digestion of bovine milk fat globules: effect of processing and interfacial structural changes. I. Gastric digestion.

PubMed

Gallier, Sophie; Cui, Jack; Olson, Trent D; Rutherfurd, Shane M; Ye, Aiqian; Moughan, Paul J; Singh, Harjinder

2013-12-01

The aim was to study the in vivo gastric digestion of fat globules in bovine cream from raw, pasteurised or pasteurised and homogenised milk. Fasted rats were gavaged once and chyme samples were collected after 30, 120 and 180 min post-gavage. Proteins from raw (RC) and pasteurised (PC) creams appeared to be digested faster and to a greater extent. Free fatty acids (FAs) increased throughout the 3h postprandial period. Short and medium chain FAs were released more rapidly than long chain FAs which were hydrolysed to a greater degree from PC. The size of the fat globules of all creams increased in the stomach. Protein aggregates were observed in pasteurised and homogenised cream chyme. Protrusions, probably caused by the accumulation of insoluble lipolytic products, appeared at the surface of the globules in RC and PC chyme. Overall, PC proteins and lipids appeared to be digested to a greater extent. Copyright © 2013 Elsevier Ltd. All rights reserved.

Controllable fabrication of copper phthalocyanine nanostructure crystals.

PubMed

Liu, Fangmei; Sun, Jia; Xiao, Si; Huang, Wenglong; Tao, Shaohua; Zhang, Yi; Gao, Yongli; Yang, Junliang

2015-06-05

Copper phthalocyanine (CuPc) nanostructure crystals, including nanoflower, nanoribbon, and nanowire, were controllably fabricated by temperature gradient physical vapor deposition (TG-PVD) through controlling the growth parameters. In a controllable growth system with carrier gas N2, nanoflower, nanoribbon, and nanowire crystals were formed in a high-temperature zone, medium-temperature zone, and low-temperature zone, respectively. They were proved to be β-phase, coexist of α-phase and β-phase, and α-phase respectively based on x-ray diffraction results. Furthermore, ultralong CuPc nanowires up to several millimeters could be fabricated by TG-PVD without carrier gas, and they were well-aligned to form large-area CuPc nanowire crystal arrays by the Langmuir-Blodgett method. The nanostructure crystals showed unusual optical absorption spectra from the ultraviolet-visible to near-infrared range, which was explained by the diffraction and scattering caused by the wavelength-sized nanostructures. These CuPc nanostructure crystals show potential applications in organic electronic and optoelectronic devices.

LIQUID EFFLUENT RETENTION FACILITY (LERF) BASIN 42 STUDIES

DOE Office of Scientific and Technical Information (OSTI.GOV)

DUNCAN JB

2004-10-29

This report documents laboratory results obtained under test plan RPP-21533 for samples submitted by the Effluent Treatment Facility (ETF) from the Liquid Effluent Retention Facility (LERF) Basin 42 (Reference 1). The LERF Basin 42 contains process condensate (PC) from the 242-A Evaporator and landfill leachate. The ETF processes one PC campaign approximately every 12 to 18 months. A typical PC campaign volume can range from 1.5 to 2.5 million gallons. During the September 2003 ETF Basin 42 processing campaign, a recurring problem with 'gelatinous buildup' on the outlet filters from 60A-TK-I (surge tank) was observed (Figure 1). This buildup appearedmore » on the filters after the contents of the surge tank were adjusted to a pH of between 5 and 6 using sulfuric acid. Biological activity in the PC feed was suspected to be the cause of the gelatinous material. Due to this buildup, the filters (10 {micro}m CUNO) required daily change out to maintain process throughput.« less

Human FDC express PrPc in vivo and in vitro

PubMed Central

Thielen, Caroline; Antoine, Nadine; Mélot, France; Cesbron, Jean-Yves; Heinen, Ernst

2001-01-01

Prion diseases are fatal neurodegenerative disorders caused by accumulation of abnormal prion protein (protease-resistant prion, PrPres). PrPres accumulation is also detected in lymphoid organs after peripheral infection. Several studies suggest that follicular dendritic cells (FDC) could be the site of PrPres retention and amplification. Here we show that human follicular dendritic cells can express normal cellular prion protein (PrPc) both in situ and in vitro. When tonsillar cryosections were treated with anti-PrP antibody, the label was found on some very delicate cell extensions inside the lymphoid follicles, especially in the germinal centres. These extensions react with DRC1 antibody, used frequently to label FDC. Other structures labelled with anti-PrP antibody were the keratinocytes. To confirm the ability of FDC to synthesise PrPc, we isolated FDC by a non-enzymatic procedure and cultured them. By cytochemistry and flow cytometry it was clearly shown that FDC do produce PrPc. PMID:11785675

The general utilization of scrapped PC board.

PubMed

Liu, Robert; Shieh, R S; Yeh, Ruth Y L; Lin, C H

2009-11-01

The traditional burning process is used to recover copper from scrapped PC board (printed circuit board) but it causes serious environmental problems. In this research a new process was developed which not only prevents pollution problems, but also maximizes the utility of all the materials on the waste board. First, the scrapped PC board was crushed and grounded, then placed in the NH3/NH5CO3 solution with aeration in order to dissolve copper. After distilling the copper NH3/NH5CO3 solution and then heating the distilled residue of copper carbonate, pure copper oxide was obtained with particle size of about 0.2 microm and the shape elliptical. The remaining solid residue after copper removal was then leached with 6N hydrochloric acid to remove tin and lead. The last residue was used as a filler in PVC plastics. The PVC plastics with PC board powder as filling material was found to have the same tensile strength as unfilled plastics, but had higher elastic modulus, higher abrasion resistance, and was cheaper.

Clinical implications of miRNAs in the pathogenesis, diagnosis and therapy of pancreatic cancer

PubMed Central

Rachagani, Satyanarayana; Macha, Muzafar A.; Heimann, Nicholas; Seshacharyulu, Parthasarathy; Haridas, Dhanya; Chugh, Seema; Batra, Surinder K.

2014-01-01

Despite considerable progress being made in understanding pancreatic cancer (PC) pathogenesis, it still remains the 10th most often diagnosed malignancy in the world and 4th leading cause of cancer related deaths in the United States with a five year survival rate of only 6%. The aggressive nature, lack of early diagnostic and prognostic markers, late clinical presentation, and limited efficacy of existing treatment regimens makes PC a lethal cancer with high mortality and poor prognosis. Therefore, novel reliable biomarkers and molecular targets are urgently needed to combat this deadly disease. MicroRNAs (miRNAs) are short (19–24 nucleotides) non-coding RNA molecules implicated in the regulation of gene expression at post-transcriptional level and play significant roles in various physiological and pathological conditions. Aberrant expression of miRNAs has been reported in several cancers including PC and is implicated in PC pathogenesis and progression, suggesting their utility in diagnosis, prognosis and therapy. In this review, we summarize the role of several miRNAs that regulate various oncogenes (KRAS) and tumor suppressor genes (p53, p16, SMAD4 etc) involved in PC development, their prospective roles as diagnostic and prognostic markers and their therapeutic targets. PMID:25453266

Adsorption heights and bonding strength of organic molecules on a Pb-Ag surface alloy

NASA Astrophysics Data System (ADS)

Stadtmüller, Benjamin; Haag, Norman; Seidel, Johannes; van Straaten, Gerben; Franke, Markus; Kumpf, Christian; Cinchetti, Mirko; Aeschlimann, Martin

2016-12-01

The understanding of the fundamental geometric and electronic properties of metal-organic hybrid interfaces is a key issue on the way to improving the performance of organic electronic and spintronic devices. Here, we studied the adsorption heights of copper-II-phthalocyanine (CuPc) and 3,4,9,10-perylene-tetracarboxylic-dianhydride (PTCDA) on a Pb1Ag2 surface alloy on Ag(111) using the normal-incidence x-ray standing waves technique. We find a significantly larger adsorption height of both molecules on the Pb-Ag surface alloy compared to the bare Ag(111) surface which is caused by the larger size of Pb. This increased adsorption height suppresses the partial chemical interaction of both molecules with Ag surface atoms. Instead, CuPc and PTCDA molecules bond only to the Pb atoms with different interaction strength ranging from a van der Waals-like interaction for CuPc to a weak chemical interaction with additional local bonds for PTCDA. The different adsorption heights for CuPc and PTCDA on Pb1Ag2 are the result of local site-specific molecule-surface bonds mediated by functional molecular groups and the different charge donating and accepting character of CuPc and PTCDA.

Molecular Drivers of Pancreatic Cancer Pathogenesis: Looking Inward to Move Forward

PubMed Central

Khan, Mohammad Aslam; Azim, Shafquat; Zubair, Haseeb; Bhardwaj, Arun; Patel, Girijesh Kumar; Khushman, Moh’d; Singh, Seema; Singh, Ajay Pratap

2017-01-01

Pancreatic cancer (PC) continues to rank among the most lethal cancers. The consistent increase in incidence and mortality has made it the seventh leading cause of cancer-associated deaths globally and the third in the United States. The biggest challenge in combating PC is our insufficient understanding of the molecular mechanism(s) underlying its complex biology. Studies during the last several years have helped identify several putative factors and events, both genetic and epigenetic, as well as some deregulated signaling pathways, with implications in PC onset and progression. In this review article, we make an effort to summarize our current understanding of molecular and cellular events involved in the pathogenesis of pancreatic malignancy. Specifically, we provide up-to-date information on the genetic and epigenetic changes that occur during the initiation and progression of PC and their functional involvement in the pathogenic processes. We also discuss the impact of the tumor microenvironment on the molecular landscape of PC and its role in aggressive disease progression. It is envisioned that a better understanding of these molecular factors and the mechanisms of their actions can help unravel novel diagnostic and prognostic biomarkers and can also be exploited for future targeted therapies. PMID:28383487

Diagnosis and management of Pneumocystis jirovecii infection.

PubMed

White, P Lewis; Backx, Matthijs; Barnes, Rosemary A

2017-05-01

Pneumocystis jirovecii is a ubiquitous fungus, which causes pneumonia in humans. Diagnosis was hampered by the inability to culture the organism, and based on microscopic examination of respiratory samples or clinical presentation. New assays can assist in the diagnosis and even aid with the emergence of resistant infections. Areas covered: This manuscript will provide background information on Pneumocystis pneumonia (PcP). Diagnosis, from radiological to non-microbiological (e.g. Lactate dehydrogenase) and microbiological investigations (Microscopy, PCR, β-D-Glucan) will be discussed. Recommendations on prophylactic and therapeutic management will be covered. Expert commentary: PcP diagnosis using microscopy is far from optimal and false negatives will occur. With an incidence of 1% or less, the pre-test probability of not having PcP is 99% and testing is suited to excluding disease. Microscopy provides a high degree of diagnostic confidence but it is not infallible, and its lower sensitivity limits its application. Newer diagnostics (PCR, β-D-Glucan) can aid management and improve performance when testing less invasive specimens, such as upper respiratory samples or blood, alleviating clinical pressure. Combination testing may allow PcP to be both diagnosed and excluded, and molecular testing can assist in the detection of emerging resistant PcP.

Bim directly antagonizes Bcl-xl in doxorubicin-induced prostate cancer cell apoptosis independently of p53.

PubMed

Yang, Min-Chi; Lin, Ru-Wei; Huang, Shih-Bo; Huang, Shin-Yuan; Chen, Wen-Jie; Wang, Shiaw; Hong, Yi-Ren; Wang, Chihuei

2016-01-01

Doxorubicin and other anthracycline compounds exert their anti-cancer effects by causing DNA damage and initiating cell cycle arrest in cancer cells, followed by apoptosis. DNA damage generally activates a p53-mediated pathway to initiate apoptosis by increasing the level of the BH3-only protein, Puma. However, p53-mediated apoptosis in response to DNA damage has not yet been validated in prostate cancers. In the current study, we used LNCaP and PC3 prostate cancer cells, representing wild type p53 and a p53-null model, to determine if DNA damage activates p53-mediated apoptosis in prostate cancers. Our results revealed that PC3 cells were 4 to 8-fold less sensitive than LNCaP cells to doxorubicin-inuced apoptosis. We proved that the differential response of LNCaP and PC3 to doxorubicin was p53-independent by introducing wild-type or dominant negative p53 into PC3 or LNCaP cells, respectively. By comparing several apoptosis-related proteins in both cell lines, we found that Bcl-xl proteins were much more abundant in PC3 cells than in LNCaP cells. We further demonstrated that Bcl-xl protects LNCaP and PC3 cells from doxorubicin-induced apoptosis by using ABT-263, an inhibitor of Bcl-xl, as a single agent or in combination with doxorubicin to treat LNCaP or PC3 cells. Bcl-xl rather than p53, likely contributes to the differential response of LNCaP and PC3 to doxorubicin in apoptosis. Finally, co-immunoprecipitation and siRNA analysis revealed that a BH3-only protein, Bim, is involved in doxorubicin-induced apoptosis by directly counteracting Bcl-xl.

Buffer-induced swelling and vesicle budding in binary lipid mixtures of dioleoylphosphatidylcholine:dioleoylphosphatidylethanolamine and dioleoylphosphatidylcholine:lysophosphatidylcholine using small-angle X-ray scattering and ³¹P static NMR.

PubMed

Barriga, Hanna M G; Bazin, Richard; Templer, Richard H; Law, Robert V; Ces, Oscar

2015-03-17

A large variety of data exists on lipid phase behavior; however, it is mostly in nonbuffered systems over nonbiological temperature ranges. We present biophysical data on lipid mixtures of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), and lysophosphatidylcholine (LysoPC) examining their behaviors in excess water and buffer systems over the temperature range 4-34 °C. These mixtures are commonly used to investigate the effects of spontaneous curvature on integral membrane proteins. Using small-angle X-ray scattering (SAXS) and (31)P NMR, we observed lamellar and vesicle phases, with the buffer causing an increase in the layer spacing. Increasing amounts of DOPE in a DOPC bilayer decreased the layer spacing of the mesophase, while the opposite trend was observed for increasing amounts of LysoPC. (31)P static NMR was used to analyze the DOPC:LysoPC samples to investigate the vesicle sizes present, with evidence of vesicle budding observed at LysoPC concentrations above 30 mol %. NMR line shapes were fitted using an adapted program accounting for the distortion of the lipids within the magnetic field. The distortion of the vesicle, because of magnetic susceptibility, varied with LysoPC content, and a discontinuity was found in both the water and buffer samples. Generally, the distortion increased with LysoPC content; however, at a ratio of DOPC:LysoPC 60:40, the sample showed a level of distortion of the vesicle similar to that of pure DOPC. This implies an increased flexibility in the membrane at this point. Commonly, the assumption is that for increasing LysoPC concentration there is a reduction in membrane tension, implying that estimations of membrane tension based on spontaneous curvature assumptions may not be accurate.

An Investigation of Siloxane Cross-linked Hydroxyapatite-Gelatin/Copolymer Composites for Potential Orthopedic Applications†

PubMed Central

Dyke, Jason Christopher; Knight, Kelly Jane; Zhou, Huaxing; Chiu, Chi-Kai; Ko, Ching-Chang; You, Wei

2012-01-01

Causes of bone deficiency are numerous, but biomimetic alloplastic grafts provide an alternative to repair tissue naturally. Previously, a hydroxyapatite-gelatin modified siloxane (HAp-Gemosil) composite was prepared by cross-linking (N, N′-bis[(3-trimethoxysilyl)propyl]ethylene diamine (enTMOS) around the HAp-Gel nanocomposite particles, to mimic the natural composition and properties of bone. However, the tensile strength remained too low for many orthopedic applications. It was hypothesized that incorporating a polymer chain into the composite could help improve long range interaction. Furthermore, designing this polymer to interact with the enTMOS siloxane cross-linked matrix would provide improved adhesion between the polymer and the ceramic composite, and improve mechanical properties. To this end, copolymers of L-Lactide (LLA), and a novel alkyne derivatized trimethylene carbonate, propargyl carbonate (PC), were synthesized. Incorporation of PC during copolymerization affects properties of copolymers such as molecular weight, Tg, and % PC incorporation. More importantly, PC monomers bear a synthetic handle, allowing copolymers to undergo post-polymerization functionalization with graft monomers to specifically tailor the properties of the final composite. For our investigation, P(LLA-co-PC) copolymers were functionalized by an azido-silane (AS) via copper catalyzed azide-alkyne cycloaddition (CuAAC) through terminal alkyne on PC monomers. The new functionalized polymer, P(LLA-co-PC)(AS) was blended with HAp-Gemosil, with the azido-silane linking the copolymer to the silsesquioxane matrix within the final composite. These HAp-Gemosil/P(LLA-co-PC)(AS) composites were subjected to mechanical and biological testing, and the results were compared with those from the HAp-Gemosil composites. This study revealed that incorporating a cross-linkable polymer served to increase the flexural strength of the composite by 50%, while maintaining the biocompatibility of HAp-Gemosil ceramics. PMID:23139457

Spirulina maxima extract prevents cell death through BDNF activation against amyloid beta 1-42 (Aβ1-42) induced neurotoxicity in PC12 cells.

PubMed

Koh, Eun-Jeong; Kim, Kui-Jin; Choi, Jia; Kang, Do-Hyung; Lee, Boo-Yong

2018-04-23

Spirulina maxima is a blue-green micro alga that contains abundant amounts of proteins (60-70%), vitamins, chlorophyll a, and C-phycocyanin (C-PC). It has been shown to reduce oxidative stress, and prevent diabetes and non-alcoholic fatty liver disease. However, it is unclear whether Spirulina maxima 70% ethanol extract (SM70EE), chlorophyll a, and C-PC prevent Aβ 1-42 -induced neurotoxicity in PC12 cells. The aim of this study was to investigate whether SM70EE, chlorophyll a, and C-PC prevent Aβ 1-42 -induced cell death. SM70EE, chlorophyll a, and C-PC suppressed the Aβ 1-42 -induced increase in poly-ADP ribose polymerase-1 (PARP-1) cleavage and reduced Aβ 1-42 -induced decreases in glutathione and its associated factors. The level of brain-derived neurotrophic factor (BDNF), which plays a critical role in neuronal survival and neuroprotection, was increased by SM70EE, chlorophyll a, and C-PC in Aβ 1-42 -treated cells. SM70EE treatment decreased oxidative stress and cell death in response to Aβ 1-42 treatment, while simultaneously suppressing PARP cleavage and increasing the levels of glutathione (GSH) and its associated factors. Moreover, SM70EE lowered the levels of APP and BACE1, two major factors involved in APP processing, and increased BDNF expression during Aβ 1-42 -induced neurotoxicity in PC12 cells. We suggest that SM70EE prevents cell death caused by Aβ 1-42 -induced neurotoxicity via the activation of BDNF signaling. Copyright © 2018 Elsevier B.V. All rights reserved.

Complexation of C6-Ceramide with Cholesteryl Phosphocholine – A Potent Solvent-Free Ceramide Delivery Formulation for Cells in Culture

PubMed Central

Långvik, Otto; Pulli, Ilari; Törnquist, Kid; Slotte, J. Peter

2013-01-01

Ceramides are potent bioactive molecules in cells. However, they are very hydrophobic molecules, and difficult to deliver efficiently to cells. We have made fluid bilayers from a short-chain D-erythro-ceramide (C6-Cer) and cholesteryl phosphocholine (CholPC), and have used this as a formulation to deliver ceramide to cells. C6-Cer complexed with CholPC led to much larger biological effects in cultured cells (rat thyroid FRTL-5 and human HeLa cells in culture) compared to C6-Cer dissolved in dimethyl sulfoxide (DMSO). Inhibition of cell proliferation and induction of apoptosis was significantly more efficient by C6-Cer/CholPC compared to C6-Cer dissolved in DMSO. C6-Cer/CholPC also permeated cell membranes and caused mitochondrial Ca2+ influx more efficiently than C6-Cer in DMSO. Even though CholPC was taken up by cells to some extent (from C6-Cer/CholPC bilayers), and was partially hydrolyzed to free cholesterol (about 9%), none of the antiproliferative effects were due to CholPC or excess cholesterol. The ceramide effect was not limited to D-erythro-C6-Cer, since L-erythro-C6-Cer and D-erythro-C6-dihydroCer also inhibited cell priolifereation and affected Ca2+ homeostasis. We conclude that C6-Cer complexed to CholPC increased the bioavailability of the short-chain ceramide for cells, and potentiated its effects in comparison to solvent-dissolved C6-Cer. This new ceramide formulation appears to be superior to previous solvent delivery approaches, and may even be useful with longer-chain ceramides. PMID:23620740

Tryptophan 32 potentiates aggregation and cytotoxicity of a copper/zinc superoxide dismutase mutant associated with familial amyotrophic lateral sclerosis.

PubMed

Taylor, David M; Gibbs, Bernard F; Kabashi, Edor; Minotti, Sandra; Durham, Heather D; Agar, Jeffrey N

2007-06-01

One familial form of the neurodegenerative disease, amyotrophic lateral sclerosis, is caused by gain-of-function mutations in the gene encoding copper/zinc superoxide dismutase (SOD-1). This study provides in vivo evidence that normally occurring oxidative modification to SOD-1 promotes aggregation and toxicity of mutant proteins. The oxidation of Trp-32 was identified as a normal modification being present in both wild-type enzyme and SOD-1 with the disease-causing mutation, G93A, isolated from erythrocytes. Mutating Trp-32 to a residue with a slower rate of oxidative modification, phenylalanine, decreased both the cytotoxicity of mutant SOD-1 and its propensity to form cytoplasmic inclusions in motor neurons of dissociated mouse spinal cord cultures.

Role of Tir and Intimin in the Virulence of Rabbit Enteropathogenic Escherichia coli Serotype O103:H2

PubMed Central

Marchès, Olivier; Nougayrède, Jean-Philippe; Boullier, Séverine; Mainil, Jacques; Charlier, Gérard; Raymond, Isabelle; Pohl, Pierre; Boury, Michèle; De Rycke, Jean; Milon, Alain; Oswald, Eric

2000-01-01

Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity. PMID:10722617

Role of tir and intimin in the virulence of rabbit enteropathogenic Escherichia coli serotype O103:H2.

PubMed

Marchès, O; Nougayrède, J P; Boullier, S; Mainil, J; Charlier, G; Raymond, I; Pohl, P; Boury, M; De Rycke, J; Milon, A; Oswald, E

2000-04-01

Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity.

New genes of Xanthomonas citri subsp. citri involved in pathogenesis and adaptation revealed by a transposon-based mutant library

PubMed Central

2009-01-01

Background Citrus canker is a disease caused by the phytopathogens Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolli and Xanthomonas alfalfae subsp. citrumelonis. The first of the three species, which causes citrus bacterial canker type A, is the most widely spread and severe, attacking all citrus species. In Brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. Like most phytobacterioses, there is no efficient way to control citrus canker. Considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis. Results Through transposon insertion mutagenesis, 10,000 mutants of Xanthomonas citri subsp. citri strain 306 (Xcc) were obtained, and 3,300 were inoculated in Rangpur lime (Citrus limonia) leaves. Their ability to cause citrus canker was analyzed every 3 days until 21 days after inoculation; a set of 44 mutants showed altered virulence, with 8 presenting a complete loss of causing citrus canker symptoms. Sequencing of the insertion site in all 44 mutants revealed that 35 different ORFs were hit, since some ORFs were hit in more than one mutant, with mutants for the same ORF presenting the same phenotype. An analysis of these ORFs showed that some encoded genes were previously known as related to pathogenicity in phytobacteria and, more interestingly, revealed new genes never implicated with Xanthomonas pathogenicity before, including hypothetical ORFs. Among the 8 mutants with no canker symptoms are the hrpB4 and hrpX genes, two genes that belong to type III secretion system (TTSS), two hypothetical ORFS and, surprisingly, the htrA gene, a gene reported as involved with the virulence process in animal-pathogenic bacteria but not described as involved in phytobacteria virulence. Nucleic acid hybridization using labeled cDNA probes showed that some of the mutated genes are differentially expressed when the bacterium is grown in citrus leaves. Finally, comparative genomic analysis revealed that 5 mutated ORFs are in new putative pathogenicity islands. Conclusion The identification of these new genes related with Xcc infection and virulence is a great step towards the understanding of plant-pathogen interactions and could allow the development of strategies to control citrus canker. PMID:19149882

Phase-dependent ultrafast third-order optical nonlinearities in metallophthalocyanine thin films

NASA Astrophysics Data System (ADS)

Kumar, Samir; Anil Kumar, K. V.; Dharmaprakash, S. M.; Das, Ritwick

2016-09-01

We present a comprehensive study on the impact of phase transformations of metallophthalocyanine thin films on their third-order nonlinear optical (NLO) properties. The metallophthalocyanine thin films are prepared by thermally evaporating the commercially available Copper(II)2,9,16,23-Tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) and Zinc(II) 2,9,16,23-Tetra-tert-butyl-29H,31H-phthalocyanine (ZnPc) powder on glass substrate. Thermal annealing causes a phase transformation which has a distinct signature in powder X-ray diffraction and UV-Vis-NIR spectroscopy. The NLO characteristics which include nonlinear refractive index n2, as well as nonlinear absorption coefficient (βeff), were measured by using a single beam Z-scan technique. An ultrashort pulsed fiber laser emitting femtosecond pulses (Δτ ≈ 250 fs) at 1064 nm central wavelength is used as a source for the Z-scan experiment. The βeff values in as prepared thin films were ascertained to be smaller as compared to the annealed one due to the smaller value of saturation intensity (Is) which, in turn, is a consequence of ground-state bleaching in the thermally unstable amorphous state of the molecule. Interestingly, the nonlinear refractive indices bear opposite sign for CuPc and ZnPc. The variations in the third-order nonlinearity in CuPc and ZnPc are discussed in terms of molecular packing and geometries of metallophthalocyanine molecules.

Metabolic syndrome in patients with prostate cancer undergoing androgen suppression.

PubMed

Morote, J; Ropero, J; Planas, J; Celma, A; Placer, J; Ferrer, R; de Torres, I

2014-06-01

Cardiovascular mortality is the leading cause of death in patients with prostate cancer (PC), metabolic syndrome (MS) being related to it. The main objective of this study was to determine the prevalence of MS in patients with CP undergoing androgen suppression (AS). We performed a retrospective study of cases and controls that included 159 patients. The study group was made up of 53 patients with PC undergoing SA for a period exceeding 12 months. The control group was formed by 53 patients with PC at the time of diagnosis and 53 patients with negative prostate biopsy. All patients were evaluated for presence of MS according to NCEP-ATPIII criteria. Prevalence of MS in patients without PC was 32.1% and in those with non-treated PC 35.8%, P = .324. In patients with PC undergoing AS, prevalence of MS was 50.9%, P < .001. When AS duration was less than 36 months, prevalence of MS was 44.0% and when greater than 36 months 57.1%, P < .001. Waist circumference and hyperglycemia were the two MS components that significantly increased. AS and its duration were independent predictors factors for the development of MS. Continuous AS therapy increases the prevalence of MS and especially waist circumference and hyperglycemia. Development of MS increases according to AS duration. Copyright © 2013 AEU. Published by Elsevier Espana. All rights reserved.

Structural Characterization of Unsaturated Phosphatidylcholines Using Traveling Wave Ion Mobility Spectrometry

PubMed Central

Kim, Hugh I.; Kim, Hyungjun; Pang, Eric S.; Ryu, Ernest K.; Beegle, Luther W.; Loo, Joseph A.; Goddard, William A.; Kanik, Isik

2009-01-01

A number of phosphatidylcholine (PC) cations spanning a mass range of 400 to 1000 Da are investigated using electrospray ionization mass spectrometry coupled with traveling wave ion mobility spectrometry (TWIMS). A high correlation between mass and mobility is demonstrated with saturated phosphatidylcholine cations in N2. A significant deviation from this mass-mobility correlation line is observed for the unsaturated PC cation. We found that the double bond in the acyl chain causes a 5% reduction in drift time. The drift time is reduced at a rate of ~1% for each additional double bond. Theoretical collision cross sections of PC cations exhibit good agreement with experimentally evaluated values. Collision cross sections are determined using the recently derived relationship between mobility and drift time in TWIMS stacked ring ion guide (SRIG) and compared to estimate collision cross-sections using empiric calibration method. Computational analysis was performed using the modified trajectory (TJ) method with nonspherical N2 molecules as the drift gas. The difference between estimated collision cross-sections and theoretical collision cross-sections of PC cations is related to the sensitivity of the PC cation collision cross-sections to the details of the ion-neutral interactions. The origin of the observed correlation and deviation between mass and mobility of PC cations is discussed in terms of the structural rigidity of these molecules using molecular dynamic simulations. PMID:19764704

Structuring a palliative care service in Brazil: experience report.

PubMed

Garcia, João Batista Santos; Rodrigues, Rayssa Fiterman; Lima, Sara Fiterman

2014-01-01

in Brazil, palliative care (PC) is not properly structured and that reality transforms this theme in a public health problem; therefore, initiatives become relevant in this context. This paper aims to share the experience that occurred in an oncology referral hospital in the State of Maranhão and present initiatives that helped in the development of PC Service. the hospital had an outpatient Pain and PC Service, but without specialized beds. The terminally ill patients stayed in common wards, which caused much unrest. A sensitization process was initiated in the hospital through initiatives, such as a photo contest called Flashes of Life and a ward called Room of Dreams, designed in partnership with the architecture course at the Universidade Estadual do Maranhão. The process culminated in the granting of wards to the PC and in the commitment of the Foundation, sponsor of the hospital, to run the project. this experience was a reproducible local initiative for the establishment of PC in a cancer hospital. Local initiatives are valuable in Brazil because they favor a significant number of patients and show its effectiveness in practice to governments and society. To structure a PC service, it is essential to establish priorities that include the assignment of drugs for management of symptoms, humanization, multidisciplinarity, sensitization and education of professionals. Copyright © 2013 Sociedade Brasileira de Anestesiologia. Published by Elsevier Editora Ltda. All rights reserved.

[Structuring a palliative care service in Brazil: experience report].

PubMed

Garcia, João Batista Santos; Rodrigues, Rayssa Fiterman; Lima, Sara Fiterman

2014-01-01

In Brazil, palliative care (PC) is not properly structured and that reality transforms this theme in a public health problem; therefore, initiatives become relevant in this context. This paper aims to share the experience that occurred in an oncology referral hospital in the State of Maranhão and present initiatives that helped in the development of PC Service. The hospital had an outpatient Pain and PC Service, but without specialized beds. The terminally ill patients stayed in common wards, which caused much unrest. A sensitization process was initiated in the hospital through initiatives, such as a photo contest called Flashes of Life and a ward called Room of Dreams, designed in partnership with the architecture course at the Universidade Estadual do Maranhão. The process culminated in the granting of wards to the PC and in the commitment of the Foundation, sponsor of the hospital, to run the project. This experience was a reproducible local initiative for the establishment of PC in a cancer hospital. Local initiatives are valuable in Brazil because they favor a significant number of patients and show its effectiveness in practice to governments and society. To structure a PC service, it is essential to establish priorities that include the assignment of drugs for management of symptoms, humanization, multidisciplinarity, sensitization and education of professionals. Copyright © 2013 Sociedade Brasileira de Anestesiologia. Publicado por Elsevier Editora Ltda. All rights reserved.

Identification of Deleterious Mutations in Myostatin Gene of Rohu Carp (Labeo rohita) Using Modeling and Molecular Dynamic Simulation Approaches

PubMed Central

Rasal, Kiran Dashrath; Chakrapani, Vemulawada; Patra, Swagat Kumar; Mohapatra, Shibani D.; Nayak, Swapnarani; Jena, Sasmita; Sundaray, Jitendra Kumar; Jayasankar, Pallipuram; Barman, Hirak Kumar

2016-01-01

The myostatin (MSTN) is a known negative growth regulator of skeletal muscle. The mutated myostatin showed a double-muscular phenotype having a positive significance for the farmed animals. Consequently, adequate information is not available in the teleosts, including farmed rohu carp, Labeo rohita. In the absence of experimental evidence, computational algorithms were utilized in predicting the impact of point mutation of rohu myostatin, especially its structural and functional relationships. The four mutations were generated at different positions (p.D76A, p.Q204P, p.C312Y, and p.D313A) of MSTN protein of rohu. The impacts of each mutant were analyzed using SIFT, I-Mutant 2.0, PANTHER, and PROVEAN, wherein two substitutions (p.D76A and p.Q204P) were predicted as deleterious. The comparative structural analysis of each mutant protein with the native was explored using 3D modeling as well as molecular-dynamic simulation techniques. The simulation showed altered dynamic behaviors concerning RMSD and RMSF, for either p.D76A or p.Q204P substitution, when compared with the native counterpart. Interestingly, incorporated two mutations imposed a significant negative impact on protein structure and stability. The present study provided the first-hand information in identifying possible amino acids, where mutations could be incorporated into MSTN gene of rohu carp including other carps for undertaking further in vivo studies. PMID:27019850

Identification of Deleterious Mutations in Myostatin Gene of Rohu Carp (Labeo rohita) Using Modeling and Molecular Dynamic Simulation Approaches.

PubMed

Rasal, Kiran Dashrath; Chakrapani, Vemulawada; Patra, Swagat Kumar; Mohapatra, Shibani D; Nayak, Swapnarani; Jena, Sasmita; Sundaray, Jitendra Kumar; Jayasankar, Pallipuram; Barman, Hirak Kumar

2016-01-01

The myostatin (MSTN) is a known negative growth regulator of skeletal muscle. The mutated myostatin showed a double-muscular phenotype having a positive significance for the farmed animals. Consequently, adequate information is not available in the teleosts, including farmed rohu carp, Labeo rohita. In the absence of experimental evidence, computational algorithms were utilized in predicting the impact of point mutation of rohu myostatin, especially its structural and functional relationships. The four mutations were generated at different positions (p.D76A, p.Q204P, p.C312Y, and p.D313A) of MSTN protein of rohu. The impacts of each mutant were analyzed using SIFT, I-Mutant 2.0, PANTHER, and PROVEAN, wherein two substitutions (p.D76A and p.Q204P) were predicted as deleterious. The comparative structural analysis of each mutant protein with the native was explored using 3D modeling as well as molecular-dynamic simulation techniques. The simulation showed altered dynamic behaviors concerning RMSD and RMSF, for either p.D76A or p.Q204P substitution, when compared with the native counterpart. Interestingly, incorporated two mutations imposed a significant negative impact on protein structure and stability. The present study provided the first-hand information in identifying possible amino acids, where mutations could be incorporated into MSTN gene of rohu carp including other carps for undertaking further in vivo studies.

The Amino-Terminal PrP Domain Is Crucial to Modulate Prion Misfolding and Aggregation

PubMed Central

Cordeiro, Yraima; Kraineva, Julia; Gomes, Mariana P. B.; Lopes, Marilene H.; Martins, Vilma R.; Lima, Luís M. T. R.; Foguel, Débora; Winter, Roland; Silva, Jerson L.

2005-01-01

The main hypothesis for prion diseases is that the cellular protein (PrPC) can be altered into a misfolded, β-sheet-rich isoform (PrPSc), which undergoes aggregation and triggers the onset of transmissible spongiform encephalopathies. Here, we investigate the effects of amino-terminal deletion mutations, rPrPΔ51–90 and rPrPΔ32–121, on the stability and the packing properties of recombinant murine PrP. The region lacking in rPrPΔ51–90 is involved physiologically in copper binding and the other construct lacks more amino-terminal residues (from 32 to 121). The pressure stability is dramatically reduced with decreasing N-domain length and the process is not reversible for rPrPΔ51–90 and rPrPΔ32–121, whereas it is completely reversible for the wild-type form. Decompression to atmospheric pressure triggers immediate aggregation for the mutants in contrast to a slow aggregation process for the wild-type, as observed by Fourier-transform infrared spectroscopy. The temperature-induced transition leads to aggregation of all rPrPs, but the unfolding temperature is lower for the rPrP amino-terminal deletion mutants. The higher susceptibility to pressure of the amino-terminal deletion mutants can be explained by a change in hydration and cavity distribution. Taken together, our results show that the amino-terminal region has a pivotal role on the development of prion misfolding and aggregation. PMID:16040743

Abnormal N-Glycosylation of a Novel Missense Creatine Transporter Mutant, G561R, Associated with Cerebral Creatine Deficiency Syndromes Alters Transporter Activity and Localization.

PubMed

Uemura, Tatsuki; Ito, Shingo; Ohta, Yusuke; Tachikawa, Masanori; Wada, Takahito; Terasaki, Tetsuya; Ohtsuki, Sumio

2017-01-01

Cerebral creatine deficiency syndromes (CCDSs) are caused by loss-of-function mutations in creatine transporter (CRT, SLC6A8), which transports creatine at the blood-brain barrier and into neurons of the central nervous system (CNS). This results in low cerebral creatine levels, and patients exhibit mental retardation, poor language skills and epilepsy. We identified a novel human CRT gene missense mutation (c.1681 G>C, G561R) in Japanese CCDSs patients. The purpose of the present study was to evaluate the reduction of creatine transport in G561R-mutant CRT-expressing 293 cells, and to clarify the mechanism of its functional attenuation. G561R-mutant CRT exhibited greatly reduced creatine transport activity compared to wild-type CRT (WT-CRT) when expressed in 293 cells. Also, the mutant protein is localized mainly in intracellular membrane fraction, while WT-CRT is localized in plasma membrane. Western blot analysis revealed a 68 kDa band of WT-CRT protein in plasma membrane fraction, while G561R-mutant CRT protein predominantly showed bands at 55, 110 and 165 kDa in crude membrane fraction. The bands of both WT-CRT and G561R-mutant CRT were shifted to 50 kDa by N-glycosidase treatment. Our results suggest that the functional impairment of G561R-mutant CRT was probably caused by incomplete N-linked glycosylation due to misfolding during protein maturation, leading to oligomer formation and changes of cellular localization.

Smad3 mutant mice develop colon cancer with overexpression of COX-2

PubMed Central

Zhu, Yu-Ping; Liu, Zhuo; Fu, Zhi-Xuan; Li, De-Chuan

2017-01-01

Colon cancer is the second most common cause of cancer-associated mortality in human populations. The aim of the present study was to identify the role of cyclooxygenase-2 (COX-2) in Smad3 mutant mice, which are known to develop colon cancer. Homozygous Smad3 (−/−) mutant mice were generated from inbred and hybrid Smad3 mouse strains by intercrossing the appropriate heterozygotes. Immunohistochemistry with COX-2 antibody was performed throughout this experiment and the data was validated and cross-checked with reverse transcription-polymerase chain reaction (RT-PCR). Homozygous mutant Smad3 mice were generated and the overexpression pattern of COX-2 was identified by immunohistochemistry and validated with RT-PCR. The results of the present study demonstrated a link between the Smad3 mutant mice, colon cancer and COX-2. In addition, the overexpression pattern of COX-2 in Smad3 mutant mice that develop colon cancer was identified. PMID:28454287

Clozapine protects PC-12 cells from death due to oxidative stress induced by hydrogen peroxide via a cell-type specific mechanism involving inhibition of extracellular signal-regulated kinase phosphorylation.

PubMed

Magliaro, Brian C; Saldanha, Colin J

2009-08-04

Recent evidence suggests that some atypical antipsychotic drugs may protect against oxidative stress and consequent neurodegeneration by mechanisms that remain unclear. Using the neuron-like rat pheochromocytoma (PC-12) cell line, Clozapine and N-desmethylclozapine were tested for their ability to protect against cell death due to oxidative stress induced by hydrogen peroxide (H(2)O(2)). These drugs demonstrated significant protection of PC-12 cells, as measured by both the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) and Alamar Blue cell viability assays. However, neither viability assay detected a protective effect of Clozapine on human embryonic kidney (HEK293), rat primary cortical neurons, or human neuroblastoma (SH-SY5Y) exposed to H(2)O(2) treatment. The mechanism of protection involves a PC-12 cell-specific differential response to H(2)O(2) treatment vs. the other cell lines. Pre-treatment with 250 microM or 125 microM diethyldithiocarbamate (DETC), a superoxide dismutase (SOD) inhibitor, unexpectedly showed protection of the PC-12 cells from H(2)O(2) treatment. Western blots revealed that Clozapine, N-desmethylclozapine, and DETC reduce the phosphorylation of extracellular signal-regulated kinase (ERK) that is caused by H(2)O(2) exposure in PC-12 cells. In both HEK293 and SH-SY5Y cells, H(2)O(2) exposure did not increase ERK phosphorylation over control, demonstrating a different response to H(2)O(2) vs. PC-12 cells, and explaining why Clozapine could not protect these cells. Also, U0126, a specific MEK inhibitor, was able to protect PC-12 cells from H(2)O(2) exposure, showing that inhibiting ERK phosphorylation is sufficient to provide protection. Cumulatively, these results indicate that Clozapine, N-desmethylclozapine, DETC, and U0126 protect PC-12 cells by blocking the cell-type specific H(2)O(2) induced increase in ERK phosphorylation.

The mechanisms of detoxification of As(III), dimethylarsinic acid (DMA) and As(V) in the microalga Chlorella vulgaris.

PubMed

Pantoja Munoz, L; Purchase, D; Jones, H; Raab, A; Urgast, D; Feldmann, J; Garelick, H

2016-06-01

The response of Chlorella vulgaris when challenged by As(III), As(V) and dimethylarsinic acid (DMA) was assessed through experiments on adsorption, efflux and speciation of arsenic (reduction, oxidation, methylation and chelation with glutathione/phytochelatin [GSH/PC]). Our study indicates that at high concentrations of phosphate (1.62mM of HPO4(2-)), upon exposure to As(V), cells are able to shift towards methylation of As(V) rather than PC formation. Treatment with As(V) caused a moderate decrease in intracellular pH and a strong increase in the concentration of free thiols (GSH). Passive surface adsorption was found to be negligible for living cells exposed to DMA and As(V). However, adsorption of As(III) was observed to be an active process in C. vulgaris, because it did not show saturation at any of the exposure periods. Chelation of As(III) with GS/PC and to a lesser extent hGS/hPC is a major detoxification mechanism employed by C. vulgaris cells when exposed to As(III). The increase of bound As-GS/PC complexes was found to be strongly related to an increase in concentration of As(III) in media. C. vulgaris cells did not produce any As-GS/PC complex when exposed to As(V). This may indicate that a reduction step is needed for As(V) complexation with GSH/PC. C. vulgaris cells formed DMAS(V)-GS upon exposure to DMA independent of the exposure period. As(III) triggers the formation of arsenic complexes with PC and homophytochelatins (hPC) and their compartmentalisation to vacuoles. A conceptual model was devised to explain the mechanisms involving ABCC1/2 transport. The potential of C. vulgaris to bio-remediate arsenic from water appeared to be highly selective and effective without the potential hazard of reducing As(V) to As(III), which is more toxic to humans. Copyright © 2016 Elsevier B.V. All rights reserved.

The complexities of obesity, diabetes, and the development and progression of pancreatic cancer

PubMed Central

Bao, Bin; Wang, Zhiwei; Li, Yiwei; Kong, Dejuan; Ali, Shadan; Banerjee, Sanjeev; Ahmad, Aamir; Sarkar, Fazlul H.

2011-01-01

Pancreatic cancer (PC) is one of the most lethal malignant diseases with the worst prognosis. It is ranked as the fourth leading cause of cancer-related deaths in the United States. Many risk factors have been associated with PC. Interestingly, large numbers of epidemiological studies suggest that obesity and diabetes, especially type-2 diabetes, are positively associated with increased risk of PC. Similarly, these chronic diseases (obesity, diabetes and cancer) are also a major public health concern. In the U.S. population, 50 percent are overweight, 30 percent are medically obese and 10 percent have diabetes mellitus (DM). Therefore, obesity and DM have been considered as potential risk factors for cancers; however, the focus of this article is restricted to PC. Although the mechanisms responsible for the development of these chronic diseases leading to the development of PC are not fully understood, the biological importance of the activation of insulin, insulin like growth factor-1 (IGF-1) and its receptor (IGF-1R) signaling pathways in insulin resistance mechanism and subsequent induction of compensatory hyperinsulinemia has been proposed. Therefore, targeting insulin/IGF-1 signaling with anti-diabetic drugs for lowering blood insulin levels and reversal of insulin-resistance could be useful strategy for the prevention and/or treatment of PC. A large number of studies have demonstrated that the administration of anti-diabetic drugs such as metformin and thiazolidinediones (TZD) class of PPAR-γ agonists decreases the risk of cancers, suggesting that these agents might be useful anti-tumor agents for the treatment of PC. In this review article, we will discuss the potential roles of metformin and TZD anti-diabetic drugs as anti-tumor agents in the context of PC, and will further discuss the complexities and the possible roles of microRNAs (miRNAs) in the pathogenesis of obesity, diabetes and PC. PMID:21129444