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Sample records for endospore biogenesis factor

  1. ENDOSPORES OF THERMOPHILIC FERMENTATIVE BACTERIA

    DEFF Research Database (Denmark)

    Volpi, Marta

    2016-01-01

    solely based on endospores of sulphate-reducing bacteria (SRB), which presumably constitute only a small fraction of the total thermophilic endospore community reaching cold environments. My PhD project developed an experimental framework for using thermophilic fermentative endospores (TFEs) to trace...

  2. Adhering Pasteuria penetrans endospores affect movements of root-knot nematode juveniles

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    Ioannis VAGELAS

    2013-01-01

    Full Text Available Pasteuria penetrans is a biological control agent of root-knot nematodes (Meloidogyne spp., preventing root invasion by second-stage juveniles (J2s, and eventually causing females sterility and death. greatest control effects for P. penetrans depend on the numbers of endospores attached to nematode cuticles. a method based on digital image analysis was used to record the effects of endospore attachment on the movements of juvenile root-knot nematodes, using a model based on the centroid point. Data showed that the numbers of endospores attached to the cuticle influenced nematode movement. At high endospore attachment levels (20‒30 per J2, nematodes did not show directional movement, whereas nematodes encumbered with five to eight spores showed limited directional movement, compared to those without endospores. nematode cephalic region turns were modelled using a markov chain, showing that P. penetrans endospores affected movements. Less nematodes invaded and established on tomato root systems when encumbered with low (five to eight or high numbers (20‒30 of P. penetrans endospores, compared with unencumbered nematodes.

  3. Insertion of the Biogenesis Factor Rei1 Probes the Ribosomal Tunnel during 60S Maturation.

    Science.gov (United States)

    Greber, Basil Johannes; Gerhardy, Stefan; Leitner, Alexander; Leibundgut, Marc; Salem, Michèle; Boehringer, Daniel; Leulliot, Nicolas; Aebersold, Ruedi; Panse, Vikram Govind; Ban, Nenad

    2016-01-14

    Eukaryotic ribosome biogenesis depends on several hundred assembly factors to produce functional 40S and 60S ribosomal subunits. The final phase of 60S subunit biogenesis is cytoplasmic maturation, which includes the proofreading of functional centers of the 60S subunit and the release of several ribosome biogenesis factors. We report the cryo-electron microscopy (cryo-EM) structure of the yeast 60S subunit in complex with the biogenesis factors Rei1, Arx1, and Alb1 at 3.4 Å resolution. In addition to the network of interactions formed by Alb1, the structure reveals a mechanism for ensuring the integrity of the ribosomal polypeptide exit tunnel. Arx1 probes the entire set of inner-ring proteins surrounding the tunnel exit, and the C terminus of Rei1 is deeply inserted into the ribosomal tunnel, where it forms specific contacts along almost its entire length. We provide genetic and biochemical evidence that failure to insert the C terminus of Rei1 precludes subsequent steps of 60S maturation. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. A Method for Isolation of Pasteuria penetrans Endospores for Bioassay and Genomic Studies.

    Science.gov (United States)

    Waterman, Jenora T; Bird, David McK; Opperman, Charles H

    2006-03-01

    A rapid method for collection of Pasteuria penetrans endospores was developed. Roots containing P. penetrans-infected root-knot nematode females were softened by pectinase digestion, mechanically processed, and filtered to collect large numbers of viable endospores. This method obviates laborious handpicking of Pasteuria-infected females and yields endospores competent to attach to and infect nematodes. Endospores are suitable for morphology studies and DNA preparations.

  5. Exploitation of immunofluorescence for the quantification and characterization of small numbers of Pasteuria endospores.

    Science.gov (United States)

    Costa, Sofia R; Kerry, Brian R; Bardgett, Richard D; Davies, Keith G

    2006-12-01

    The Pasteuria group of endospore-forming bacteria has been studied as a biocontrol agent of plant-parasitic nematodes. Techniques have been developed for its detection and quantification in soil samples, and these mainly focus on observations of endospore attachment to nematodes. Characterization of Pasteuria populations has recently been performed with DNA-based techniques, which usually require the extraction of large numbers of spores. We describe a simple immunological method for the quantification and characterization of Pasteuria populations. Bayesian statistics were used to determine an extraction efficiency of 43% and a threshold of detection of 210 endospores g(-1) sand. This provided a robust means of estimating numbers of endospores in small-volume samples from a natural system. Based on visual assessment of endospore fluorescence, a quantitative method was developed to characterize endospore populations, which were shown to vary according to their host.

  6. Resveratrol induces mitochondrial biogenesis in endothelial cells.

    Science.gov (United States)

    Csiszar, Anna; Labinskyy, Nazar; Pinto, John T; Ballabh, Praveen; Zhang, Hanrui; Losonczy, Gyorgy; Pearson, Kevin; de Cabo, Rafael; Pacher, Pal; Zhang, Cuihua; Ungvari, Zoltan

    2009-07-01

    Pathways that regulate mitochondrial biogenesis are potential therapeutic targets for the amelioration of endothelial dysfunction and vascular disease. Resveratrol was shown to impact mitochondrial function in skeletal muscle and the liver, but its role in mitochondrial biogenesis in endothelial cells remains poorly defined. The present study determined whether resveratrol induces mitochondrial biogenesis in cultured human coronary arterial endothelial cells (CAECs). In CAECs resveratrol increased mitochondrial mass and mitochondrial DNA content, upregulated protein expression of electron transport chain constituents, and induced mitochondrial biogenesis factors (proliferator-activated receptor-coactivator-1alpha, nuclear respiratory factor-1, mitochondrial transcription factor A). Sirtuin 1 (SIRT1) was induced, and endothelial nitric oxide (NO) synthase (eNOS) was upregulated in a SIRT1-dependent manner. Knockdown of SIRT1 (small interfering RNA) or inhibition of NO synthesis prevented resveratrol-induced mitochondrial biogenesis. In aortas of type 2 diabetic (db/db) mice impaired mitochondrial biogenesis was normalized by chronic resveratrol treatment, showing the in vivo relevance of our findings. Resveratrol increases mitochondrial content in endothelial cells via activating SIRT1. We propose that SIRT1, via a pathway that involves the upregulation of eNOS, induces mitochondrial biogenesis. Resveratrol induced mitochondrial biogenesis in the aortas of type 2 diabetic mice, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases.

  7. Biogenesis of the Saccharomyces cerevisiae Pheromone a-Factor, from Yeast Mating to Human Disease

    Science.gov (United States)

    Barrowman, Jemima

    2012-01-01

    Summary: The mating pheromone a-factor secreted by Saccharomyces cerevisiae is a farnesylated and carboxylmethylated peptide and is unusually hydrophobic compared to other extracellular signaling molecules. Mature a-factor is derived from a precursor with a C-terminal CAAX motif that directs a series of posttranslational reactions, including prenylation, endoproteolysis, and carboxylmethylation. Historically, a-factor has served as a valuable model for the discovery and functional analysis of CAAX-processing enzymes. In this review, we discuss the three modules comprising the a-factor biogenesis pathway: (i) the C-terminal CAAX-processing steps carried out by Ram1/Ram2, Ste24 or Rce1, and Ste14; (ii) two sequential N-terminal cleavage steps, mediated by Ste24 and Axl1; and (iii) export by a nonclassical mechanism, mediated by the ATP binding cassette (ABC) transporter Ste6. The small size and hydrophobicity of a-factor present both challenges and advantages for biochemical analysis, as discussed here. The enzymes involved in a-factor biogenesis are conserved from yeasts to mammals. Notably, studies of the zinc metalloprotease Ste24 in S. cerevisiae led to the discovery of its mammalian homolog ZMPSTE24, which cleaves the prenylated C-terminal tail of the nuclear scaffold protein lamin A. Mutations that alter ZMPSTE24 processing of lamin A in humans cause the premature-aging disease progeria and related progeroid disorders. Intriguingly, recent evidence suggests that the entire a-factor pathway, including all three biogenesis modules, may be used to produce a prenylated, secreted signaling molecule involved in germ cell migration in Drosophila. Thus, additional prenylated signaling molecules resembling a-factor, with as-yet-unknown roles in metazoan biology, may await discovery. PMID:22933563

  8. Pasteuria endospore attachment to Pratylenchus species

    Science.gov (United States)

    Pasteuria spp. are endospore-forming bacteria, and most of them are obligate parasites of nematodes. A number of studies have demonstrated that Pasteuria can effectively suppress nematode populations in natural fields and have promising potential as biocontrol agents. However, parasitism of nematode...

  9. The effects of biocides (antiseptics and disinfectants on the endospores of Rhinosporidium seeberi

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    Arseculeratne S

    2006-01-01

    Full Text Available No data exists on the activity of biocides (antiseptics and disinfectants on Rhinosporidium seeberi that causes rhinosporidiosis in humans and animals. On account of the inability to culture R. seeberi, in vitro , dyes were used to assess the morphological integrity and viability of biocide-treated endospores that are considered to be the infective stage of this pathogen. Evan′s Blue (EvB identifies the morphological integrity of the endospores while MTT (3-[4, 5-dimethylthiazol-2yl]-2, 5-diphenyl tetrazolium bromide identifies metabolic activity through its reduction by cellular dehydrogenases to microscopically visible deposits of insoluble formazan. MTT-negativity has earlier been shown to correlate with absence of growth of yeast and mycelial fungi in culture and could thus indicate the loss of viability of MTT-negative rhinosporidial endospores. Hydrogen peroxide, glutaraldehyde, chloroxylenol, chlorhexidine, cetrimide, thimerosal, 70% ethanol, iodine in 70% ethanol, 10% formalin, povidone-iodine, sodium azide and silver nitrate were tested on freshly-harvested endospores and all biocides caused metabolic inactivation with or without altered structural integrity as shown by absence of MTT-staining after 3, 24 or 36 hour after exposure, while EvB stained only the endospores treated with sodium azide, ethanol, thimerosal, chloroxylenol, glutaraldehyde and hydrogen peroxide. With clinically useful biocides - chlorhexidine, cetrimide-chlorhexidine, 70% ethanol, povidone-iodine and silver nitrate, a total period of exposure of endospores to the biocide, for seven minutes, produced metabolic inactivation of the endospores. Anti-rhinosporidial antiseptics that could be used in surgery on rhinosporidial patients include povidone-iodine in nasal packs for nasal and naso-pharyngeal surgery, chlorhexidine and cetrimide-chlorhexidine on the skin, while povidone-iodine and silver nitrate could have application in ocular rhinosporidiosis.

  10. Expression Profiling of Ribosome Biogenesis Factors Reveals Nucleolin as a Novel Potential Marker to Predict Outcome in AML Patients.

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    Virginie Marcel

    Full Text Available Acute myeloid leukemia (AML is a heterogeneous disease. Prognosis is mainly influenced by patient age at diagnosis and cytogenetic alterations, two of the main factors currently used in AML patient risk stratification. However, additional criteria are required to improve the current risk classification and better adapt patient care. In neoplastic cells, ribosome biogenesis is increased to sustain the high proliferation rate and ribosome composition is altered to modulate specific gene expression driving tumorigenesis. Here, we investigated the usage of ribosome biogenesis factors as clinical markers in adult patients with AML. We showed that nucleoli, the nucleus compartments where ribosome production takes place, are modified in AML by analyzing a panel of AML and healthy donor cells using immunofluorescence staining. Using four AML series, including the TCGA dataset, altogether representing a total of about 270 samples, we showed that not all factors involved in ribosome biogenesis have clinical values although ribosome biogenesis is increased in AML. Interestingly, we identified the regulator of ribosome production nucleolin (NCL as over-expressed in AML blasts. Moreover, we found in two series that high NCL mRNA expression level was associated with a poor overall survival, particular in elderly patients. Multivariate analyses taking into account age and cytogenetic risk indicated that NCL expression in blast cells is an independent marker of reduced survival. Our study identifies NCL as a potential novel prognostic factor in AML. Altogether, our results suggest that the ribosome biogenesis pathway may be of interest as clinical markers in AML.

  11. Dispersal of thermophilic Desulfotomaculum endospores into Baltic Sea sediments over thousands of years.

    Science.gov (United States)

    de Rezende, Júlia Rosa; Kjeldsen, Kasper Urup; Hubert, Casey R J; Finster, Kai; Loy, Alexander; Jørgensen, Bo Barker

    2013-01-01

    Patterns of microbial biogeography result from a combination of dispersal, speciation and extinction, yet individual contributions exerted by each of these mechanisms are difficult to isolate and distinguish. The influx of endospores of thermophilic microorganisms to cold marine sediments offers a natural model for investigating passive dispersal in the ocean. We investigated the activity, diversity and abundance of thermophilic endospore-forming sulfate-reducing bacteria (SRB) in Aarhus Bay by incubating pasteurized sediment between 28 and 85 °C, and by subsequent molecular diversity analyses of 16S rRNA and of the dissimilatory (bi)sulfite reductase (dsrAB) genes within the endospore-forming SRB genus Desulfotomaculum. The thermophilic Desulfotomaculum community in Aarhus Bay sediments consisted of at least 23 species-level 16S rRNA sequence phylotypes. In two cases, pairs of identical 16S rRNA and dsrAB sequences in Arctic surface sediment 3000 km away showed that the same phylotypes are present in both locations. Radiotracer-enhanced most probable number analysis revealed that the abundance of endospores of thermophilic SRB in Aarhus Bay sediment was ca. 10(4) per cm(3) at the surface and decreased exponentially to 10(0) per cm(3) at 6.5 m depth, corresponding to 4500 years of sediment age. Thus, a half-life of ca. 300 years was estimated for the thermophilic SRB endospores deposited in Aarhus Bay sediments. These endospores were similarly detected in the overlying water column, indicative of passive dispersal in water masses preceding sedimentation. The sources of these thermophiles remain enigmatic, but at least one source may be common to both Aarhus Bay and Arctic sediments.

  12. Exploiting the aerobic endospore-forming bacterial diversity in saline and hypersaline environments for biosurfactant production.

    Science.gov (United States)

    de Almeida Couto, Camila Rattes; Alvarez, Vanessa Marques; Marques, Joana Montezano; de Azevedo Jurelevicius, Diogo; Seldin, Lucy

    2015-10-28

    Biosurfactants are surface-active biomolecules with great applicability in the food, pharmaceutical and oil industries. Endospore-forming bacteria, which survive for long periods in harsh environments, are described as biosurfactant producers. Although the ubiquity of endospore-forming bacteria in saline and hypersaline environments is well known, studies on the diversity of the endospore-forming and biosurfactant-producing bacterial genera/species in these habitats are underrepresented. In this study, the structure of endospore-forming bacterial communities in sediment/mud samples from Vermelha Lagoon, Massambaba, Dois Rios and Abraão Beaches (saline environments), as well as the Praia Seca salterns (hypersaline environments) was determined via denaturing gradient gel electrophoresis. Bacterial strains were isolated from these environmental samples and further identified using 16S rRNA gene sequencing. Strains presenting emulsification values higher than 30 % were grouped via BOX-PCR, and the culture supernatants of representative strains were subjected to high temperatures and to the presence of up to 20 % NaCl to test their emulsifying activities in these extreme conditions. Mass spectrometry analysis was used to demonstrate the presence of surfactin. A diverse endospore-forming bacterial community was observed in all environments. The 110 bacterial strains isolated from these environmental samples were molecularly identified as belonging to the genera Bacillus, Thalassobacillus, Halobacillus, Paenibacillus, Fictibacillus and Paenisporosarcina. Fifty-two strains showed emulsification values of at least 30%, and they were grouped into 18 BOX groups. The stability of the emulsification values varied when the culture supernatants of representative strains were subjected to high temperatures and to the presence of up to 20% NaCl. The presence of surfactin was demonstrated in one of the most promising strains. The environments studied can harbor endospore

  13. Endospores of B subtilis are pyrogenic and activate Mono Mac 6 cells

    DEFF Research Database (Denmark)

    Moesby, Lise; Hansen, Erik W; Christensen, Jens D

    2003-01-01

    interleukin-6. Lipopolysaccharides (LPS) dose-dependently induce interleukin-6 release, but the curve differs from that of LTA both in shape and offset. The interleukin-6 secretion induced by LPS, LTA and B. subtilis bacteria can be blocked by 73-85% by an antibody directed against CD14, whereas the antibody......The monocytic cell line Mono Mac 6 is sensitive to pyrogens and interleukin-6 secretion is induced after exposure to pyrogens. The aim of this study is to examine the pyrogenic activity and the interleukin-6-inducing capacity of the Gram-positive B. subtilis bacteria, endospores and isolated cell...... in a sandwich immunoassay. B. subtilis bacteria and endospores induce interleukin-6 in a dose-dependent manner. Endospores are less potent than bacteria. Lipoteichoic acid (LTA) isolated from B. subtilis induces interleukin-6 in a dose-dependent manner, whereas muramyl dipeptide (MDP) is unable to induce...

  14. A monoclonal antibody that tracks endospore formation in the microsporidium Nosema bombycis.

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    Yanhong Li

    Full Text Available Nosema bombycis, the first identified microsporidium, is a destructive pathogen of the silkworm Bombyx mori and causes severe worldwide economic losses in sericulture. Major microsporidian structural proteins, such as the spore wall protein (SWP, are known to be involved in host invasion. In this study, the reactivity of the monoclonal antibody 2B10 was tested against an endospore protein of N. bombycis with a molecular weight size at 50-kDa, using Western blotting. The antigen was purified after immunoprecipitation and was further identified as EOB13320 according to MALDI-TOF MS assay. We found that EOB13320 locates to the surface of the different developmental stages of the parasite, mostly the sporoblast stage and the mature spore after immunoelectron microscopy examination. EOB13320 was also widely distributed in the developing endospore, especially at the sporoblast stage. This endospore protein also accumulated in the cytoplasm of both the merogony and sporoblast stages. These results imply that EOB13320 detected by monoclonal antibody 2B10 is expressed throughout the life cycle of the parasite, notably during the stage when the endospore is formed, and that this protein is important for spore-coat formation and parasite maintenance. Our study could be instrumental in the understanding of spore wall formation and will help to gain greater insight into the biology of this parasite.

  15. A monoclonal antibody that tracks endospore formation in the microsporidium Nosema bombycis.

    Science.gov (United States)

    Li, Yanhong; Tao, Meiling; Ma, Fuping; Pan, Guoqing; Zhou, Zeyang; Wu, Zhengli

    2015-01-01

    Nosema bombycis, the first identified microsporidium, is a destructive pathogen of the silkworm Bombyx mori and causes severe worldwide economic losses in sericulture. Major microsporidian structural proteins, such as the spore wall protein (SWP), are known to be involved in host invasion. In this study, the reactivity of the monoclonal antibody 2B10 was tested against an endospore protein of N. bombycis with a molecular weight size at 50-kDa, using Western blotting. The antigen was purified after immunoprecipitation and was further identified as EOB13320 according to MALDI-TOF MS assay. We found that EOB13320 locates to the surface of the different developmental stages of the parasite, mostly the sporoblast stage and the mature spore after immunoelectron microscopy examination. EOB13320 was also widely distributed in the developing endospore, especially at the sporoblast stage. This endospore protein also accumulated in the cytoplasm of both the merogony and sporoblast stages. These results imply that EOB13320 detected by monoclonal antibody 2B10 is expressed throughout the life cycle of the parasite, notably during the stage when the endospore is formed, and that this protein is important for spore-coat formation and parasite maintenance. Our study could be instrumental in the understanding of spore wall formation and will help to gain greater insight into the biology of this parasite.

  16. Environmental quantification of Pasteuria penetrans endospores using in situ antigen extraction and immunodetection with a monoclonal antibody.

    Science.gov (United States)

    Schmidt, L M; Preston, J F; Dickson, D W; Rice, J D; Hewlett, T E

    2003-05-01

    Abstract Pasteuria penetrans is an obligate parasite of root-knot nematodes (Meloidogyne spp.) that has attracted significant attention as a promising biocontrol agent. The inability to culture P. penetrans has invoked the need for a quantitative detection capability to facilitate biocontrol studies. A chemical extraction method using urea, dithiothreitol and CHES buffer (UDC) is shown to release soluble endospore envelope antigen from endospores present in complex matrices, generating an extract that can be used to determine the levels of spores when compared to a standard in an enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody, MAb 2A41D10. Extractions can be performed in less than 1 h. Linear regression analysis routinely produced line fits with r(2)>0.90. Antigen extraction efficiency was not influenced by soil type. Three ELISA formats were analyzed for quantitative detection of P. penetrans endospores. A tertiary ELISA immunodetection system provided the lowest level of detection at approximately 300 spores per gram of soil. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Western blots of soil extracts containing P. penetrans endospore antigen produced signature peptides bearing a common epitope characteristic of endospores of Pasteuria spp. MAb 2A41D10 was specific for Pasteuria spp. and did not react with extracts of Pasteuria-free soil or with spore extracts of native Gram-positive endospore-forming bacteria. Immunofluorescent microscopy revealed that MAb 2A41D10 recognizes an epitope uniformly distributed on the endospore surface. The development of a rapid extraction method and analysis of solubilized antigen by immunodetection has the potential for broad application in food and environmental microbiology.

  17. Assessment of bacterial endospore viability with fluorescent dyes.

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    Laflamme, C; Lavigne, S; Ho, J; Duchaine, C

    2004-01-01

    To validate three fluorescence viability assays designed primarily for vegetative cells on pure Bacillus endospores. Purified fresh and gamma-irradiated Bacillus endospores (Bacillus cereus, B. coagulans and two strains of B. subtilis) were used. The viability assays were: 5-cyano-2,3-diotolyl tetrazolium chloride (CTC) to test respiratory activity and early germination, DiBAC4(3) and Live/Dead BacLight to measure membrane energization and permeabilization, respectively. Gamma irradiation treatment completely eliminated spore culturability and was used as negative control. The untreated spores showed respiratory activity after 1 h of incubation and this was characteristic of almost 100% of spores after 24 h. The membrane potential assessment gave no answer about spore viability. A lower proportion of untreated spores had permeabilized membrane compared with gamma-irradiated spores using Live/Dead BacLight (P plate count. This study shows that fluorescence tests could be applied to assess viability in potentially pathogenic Bacillus spore preparations within 1 h.

  18. Automated thermochemolysis reactor for detection of Bacillus anthracis endospores by gas chromatography–mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Li, Dan [Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602 (United States); Rands, Anthony D.; Losee, Scott C. [Torion Technologies, American Fork, UT 84003 (United States); Holt, Brian C. [Department of Statistics, Brigham Young University, Provo, UT 84602 (United States); Williams, John R. [Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602 (United States); Lammert, Stephen A. [Torion Technologies, American Fork, UT 84003 (United States); Robison, Richard A. [Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602 (United States); Tolley, H. Dennis [Department of Statistics, Brigham Young University, Provo, UT 84602 (United States); Lee, Milton L., E-mail: milton_lee@byu.edu [Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602 (United States)

    2013-05-02

    Graphical abstract: -- Highlights: •An automated sample preparation system for Bacillus anthracis endospores was developed. •A thermochemolysis method was applied to produce and derivatize biomarkers for Bacillus anthracis detection. •The autoreactor controlled the precise delivery of reagents, and TCM reaction times and temperatures. •Solid phase microextraction was used to extract biomarkers, and GC–MS was used for final identification. •This autoreactor was successfully applied to the identification of Bacillus anthracis endospores. -- Abstract: An automated sample preparation system was developed and tested for the rapid detection of Bacillus anthracis endospores by gas chromatography–mass spectrometry (GC–MS) for eventual use in the field. This reactor is capable of automatically processing suspected bio-threat agents to release and derivatize unique chemical biomarkers by thermochemolysis (TCM). The system automatically controls the movement of sample vials from one position to another, crimping of septum caps onto the vials, precise delivery of reagents, and TCM reaction times and temperatures. The specific operations of introduction of sample vials, solid phase microextraction (SPME) sampling, injection into the GC–MS system, and ejection of used vials from the system were performed manually in this study, although they can be integrated into the automated system. Manual SPME sampling is performed by following visual and audible signal prompts for inserting the fiber into and retracting it from the sampling port. A rotating carousel design allows for simultaneous sample collection, reaction, biomarker extraction and analysis of sequential samples. Dipicolinic acid methyl ester (DPAME), 3-methyl-2-butenoic acid methyl ester (a fragment of anthrose) and two methylated sugars were used to compare the performance of the autoreactor with manual TCM. Statistical algorithms were used to construct reliable bacterial endospore signatures, and 24

  19. Automated thermochemolysis reactor for detection of Bacillus anthracis endospores by gas chromatography–mass spectrometry

    International Nuclear Information System (INIS)

    Li, Dan; Rands, Anthony D.; Losee, Scott C.; Holt, Brian C.; Williams, John R.; Lammert, Stephen A.; Robison, Richard A.; Tolley, H. Dennis; Lee, Milton L.

    2013-01-01

    Graphical abstract: -- Highlights: •An automated sample preparation system for Bacillus anthracis endospores was developed. •A thermochemolysis method was applied to produce and derivatize biomarkers for Bacillus anthracis detection. •The autoreactor controlled the precise delivery of reagents, and TCM reaction times and temperatures. •Solid phase microextraction was used to extract biomarkers, and GC–MS was used for final identification. •This autoreactor was successfully applied to the identification of Bacillus anthracis endospores. -- Abstract: An automated sample preparation system was developed and tested for the rapid detection of Bacillus anthracis endospores by gas chromatography–mass spectrometry (GC–MS) for eventual use in the field. This reactor is capable of automatically processing suspected bio-threat agents to release and derivatize unique chemical biomarkers by thermochemolysis (TCM). The system automatically controls the movement of sample vials from one position to another, crimping of septum caps onto the vials, precise delivery of reagents, and TCM reaction times and temperatures. The specific operations of introduction of sample vials, solid phase microextraction (SPME) sampling, injection into the GC–MS system, and ejection of used vials from the system were performed manually in this study, although they can be integrated into the automated system. Manual SPME sampling is performed by following visual and audible signal prompts for inserting the fiber into and retracting it from the sampling port. A rotating carousel design allows for simultaneous sample collection, reaction, biomarker extraction and analysis of sequential samples. Dipicolinic acid methyl ester (DPAME), 3-methyl-2-butenoic acid methyl ester (a fragment of anthrose) and two methylated sugars were used to compare the performance of the autoreactor with manual TCM. Statistical algorithms were used to construct reliable bacterial endospore signatures, and 24

  20. MiRNA Biogenesis and Intersecting Pathways

    DEFF Research Database (Denmark)

    Ben Chaabane, Samir

    MicroRNAs (miRNAs) are small non-coding RNAs that function as guide molecules in RNA silencing. Plant miRNAs are critical for plant growth, development and stress response, and are processed in Arabidopsis from primary miRNA transcripts (pri-miRNAs) by the endonuclease activity of the DICER-LIKE1...... questions need to be addressed to establish a valid link, we provide encouraging evidence of the involvement of chromatin remodeling factors FAS1 and FAS2 in miRNA biogenesis. Together, we have expanded our understanding of the intersections between miRNA biogenesis and other pathways....

  1. Laser induced fluorescence lifetime characterization of Bacillus endospore species using time correlated single photon counting analysis with the multi-exponential fit method

    Science.gov (United States)

    Smith, Clint; Edwards, Jarrod; Fisher, Andmorgan

    2010-04-01

    Rapid detection of biological material is critical for determining presence/absence of bacterial endospores within various investigative programs. Even more critical is that if select material tests positive for bacillus endospores then tests should provide data at the species level. Optical detection of microbial endospore formers such as Bacillus sp. can be heavy, cumbersome, and may only identify at the genus level. Data provided from this study will aid in characterization needed by future detection systems for further rapid breakdown analysis to gain insight into a more positive signature collection of Bacillus sp. Literature has shown that fluorescence spectroscopy of endospores could be statistically separated from other vegetative genera, but could not be separated among one another. Results of this study showed endospore species separation is possible using laser-induce fluorescence with lifetime decay analysis for Bacillus endospores. Lifetime decays of B. subtilis, B. megaterium, B. coagulans, and B. anthracis Sterne strain were investigated. Using the Multi-Exponential fit method data showed three distinct lifetimes for each species within the following ranges, 0.2-1.3 ns; 2.5-7.0 ns; 7.5-15.0 ns, when laser induced at 307 nm. The four endospore species were individually separated using principle component analysis (95% CI).

  2. Geobacillus thermoglucosidasius Endospores Function as Nuclei for the Formation of Single Calcite Crystals

    Science.gov (United States)

    Murai, Rie

    2013-01-01

    Geobacillus thermoglucosidasius colonies were placed on an agar hydrogel containing acetate, calcium ions, and magnesium ions, resulting in the formation of single calcite crystals (calcites) within and peripheral to the plating area or parent colony. Microscopic observation of purified calcites placed on the surface of soybean casein digest (SCD) nutrient medium revealed interior crevices from which bacterial colonies originated. Calcites formed on the gel contained [1-13C]- and [2-13C]acetate, demonstrating that G. thermoglucosidasius utilizes carbon derived from acetate for calcite formation. During calcite formation, vegetative cells swam away from the parent colony in the hydrogel. Hard-agar hydrogel inhibited the formation of calcites peripheral to the parent colony. The calcite dissolved completely in 1 M HCl, with production of bubbles, and the remaining endospore-like particles were easily stained with Brilliant green dye. The presence of DNA and protein in calcites was demonstrated by electrophoresis. We propose that endospores initiate the nucleation of calcites. Endospores of G. thermoglucosidasius remain alive and encapsulated in calcites. PMID:23455343

  3. Temporal Formation and Immunolocalization of an Endospore Surface Epitope During Pasteuria penetrans Sporogenesis.

    Science.gov (United States)

    Brito, J A; Preston, J F; Dickson, D W; Giblin-Davis, R M; Williams, D S; Aldrich, H C; Rice, J D

    2003-09-01

    The synthesis and localization of an endospore surface epitope associated with the development of Pasteuria penetrans was determined using a monoclonal antibody (MAb) as a probe. Nematodes, uninfected or infected with P. penetrans, were harvested at 12, 16, 24, and 38 days after inoculation (DAI) and then examined to determine the developmental stage of the bacterium. Vegetative growth of P. penetrans was observed only in infected nematodes harvested at 12 and 16 DAI, whereas cells at different stages of sporulation and mature endospores were observed at 24 and 38 DAI. ELISA and immunoblot analysis revealed that the adhesin-associated epitope was first detected at 24 DAI, and increased in the later stages of sporogenesis. These results indicate that the synthesis of adhesin-related proteins occurred at a certain developmental stage relative to the sporulation process, and was associated with endospore maturation. Immunofluorescence microscopy indicated that the distribution of the epitope is nearly uniform on the periphery of each spore, as defined by parasporal fibers. Immunocytochemistry at the ultrastructural level indicated a distribution of the epitope over the parasporal fibers. The epitope also was detected over other structures such as sporangium and exosporium during the sporogenesis process, but it was not observed over the cortex, inner-spore coat, outer-spore coat, or protoplasm. The appearance of the adhesin epitope first at stage III of sporogenesis and its presence on the parasporal fibers are consistent with an adhesin-related role in the attachment of the mature endospore to the cuticle of the nematode host.

  4. Placeholder factors in ribosome biogenesis: please, pave my way

    Directory of Open Access Journals (Sweden)

    Francisco J. Espinar-Marchena

    2017-04-01

    Full Text Available The synthesis of cytoplasmic eukaryotic ribosomes is an extraordinarily energy-demanding cellular activity that occurs progressively from the nucleolus to the cytoplasm. In the nucleolus, precursor rRNAs associate with a myriad of trans-acting factors and some ribosomal proteins to form pre-ribosomal particles. These factors include snoRNPs, nucleases, ATPases, GTPases, RNA helicases, and a vast list of proteins with no predicted enzymatic activity. Their coordinate activity orchestrates in a spatiotemporal manner the modification and processing of precursor rRNAs, the rearrangement reactions required for the formation of productive RNA folding intermediates, the ordered assembly of the ribosomal proteins, and the export of pre-ribosomal particles to the cytoplasm; thus, providing speed, directionality and accuracy to the overall process of formation of translation-competent ribosomes. Here, we review a particular class of trans-acting factors known as “placeholders”. Placeholder factors temporarily bind selected ribosomal sites until these have achieved a structural context that is appropriate for exchanging the placeholder with another site-specific binding factor. By this strategy, placeholders sterically prevent premature recruitment of subsequently binding factors, premature formation of structures, avoid possible folding traps, and act as molecular clocks that supervise the correct progression of pre-ribosomal particles into functional ribosomal subunits. We summarize the current understanding of those factors that delay the assembly of distinct ribosomal proteins or subsequently bind key sites in pre-ribosomal particles. We also discuss recurrent examples of RNA-protein and protein-protein mimicry between rRNAs and/or factors, which have clear functional implications for the ribosome biogenesis pathway.

  5. Endospore production allows using spray-drying as a possible formulation system of the biocontrol agent Bacillus subtilis CPA-8.

    Science.gov (United States)

    Yánez-Mendizabal, V; Viñas, I; Usall, J; Cañamás, T; Teixidó, N

    2012-04-01

    The role of endospore production by Bacillus subtilis CPA-8 on survival during spray-drying was investigated by comparison with a non-spore-forming biocontrol agent Pantoea agglomerans CPA-2. Endospore formation promoted heat resistance in CPA-8 depending on growth time (72 h cultures were more resistant than 24 h ones). The survival of CPA-8 and CPA-2 after spray-drying was determined after being grown in optimised media for 24 and 72 h. Spray-dried 72 h CPA-8 had the best survival (32%), while CPA-2 viability was less than 2%. CPA-8 survival directly related with its ability to produce endospores. Spray-dried CPA-8 reduced Monilinia fructicola conidia germination similarly to fresh cells, demonstrating that spray-drying did not adversely affect biocontrol efficacy. Endospore production thus improves CPA-8 resistance to spray-drying. These results can provide a reliable basis for optimising of the spray-drying formulation process for CPA-8 and other microorganisms.

  6. A New Sensitive GC-MS-based Method for Analysis of Dipicolinic Acid and Quantifying Bacterial Endospores in Deep Marine Subsurface Sediment

    Science.gov (United States)

    Fang, J.

    2015-12-01

    Marine sediments cover more than two-thirds of the Earth's surface and represent a major part of the deep biosphere. Microbial cells and microbial activity appear to be widespread in these sediments. Recently, we reported the isolation of gram-positive anaerobic spore-forming piezophilic bacteria and detection of bacterial endospores in marine subsurface sediment from the Shimokita coalbed, Japan. However, the modern molecular microbiological methods (e.g., DNA-based microbial detection techniques) cannot detect bacterial endospore, because endospores are impermeable and are not stained by fluorescence DNA dyes or by ribosomal RNA staining techniques such as catalysed reporter deposition fluorescence in situ hybridization. Thus, the total microbial cell abundance in the deep biosphere may has been globally underestimated. This emphasizes the need for a new cultivation independent approach for the quantification of bacterial endospores in the deep subsurface. Dipicolinic acid (DPA, pyridine-2,6-dicarboxylic acid) is a universal and specific component of bacterial endospores, representing 5-15wt% of the dry spore, and therefore is a useful indicator and quantifier of bacterial endospores and permits to estimate total spore numbers in the subsurface biosphere. We developed a sensitive analytical method to quantify DPA content in environmental samples using gas chromatography-mass spectrometry. The method is sensitive and more convenient in use than other traditional methods. We applied this method to analyzing sediment samples from the South China Sea (obtained from IODP Exp. 349) to determine the abundance of spore-forming bacteria in the deep marine subsurface sediment. Our results suggest that gram-positive, endospore-forming bacteria may be the "unseen majority" in the deep biosphere.

  7. Selenium supplementation induces mitochondrial biogenesis in trophoblasts

    Czech Academy of Sciences Publication Activity Database

    Khera, A.; Dong, L. F.; Holland, O.; Vanderlelie, J.; Pasdar, E.A.; Neužil, Jiří; Perkins, A.V.

    2015-01-01

    Roč. 36, č. 8 (2015), s. 363-369 ISSN 0143-4004 Institutional support: RVO:86652036 Keywords : Selenium * Reactive oxygen species * Mitochondrial biogenesis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.972, year: 2015

  8. Insights into mRNP biogenesis provided by new genetic interactions among export and transcription factors

    Directory of Open Access Journals (Sweden)

    Estruch Francisco

    2012-09-01

    Full Text Available Abstract Background The various steps of mRNP biogenesis (transcription, processing and export are interconnected. It has been shown that the transcription machinery plays a pivotal role in mRNP assembly, since several mRNA export factors are recruited during transcription and physically interact with components of the transcription machinery. Although the shuttling DEAD-box protein Dbp5p is concentrated on the cytoplasmic fibrils of the NPC, previous studies demonstrated that it interacts physically and genetically with factors involved in transcription initiation. Results We investigated the effect of mutations affecting various components of the transcription initiation apparatus on the phenotypes of mRNA export mutant strains. Our results show that growth and mRNA export defects of dbp5 and mex67 mutant strains can be suppressed by mutation of specific transcription initiation components, but suppression was not observed for mutants acting in the very first steps of the pre-initiation complex (PIC formation. Conclusions Our results indicate that mere reduction in the amount of mRNP produced is not sufficient to suppress the defects caused by a defective mRNA export factor. Suppression occurs only with mutants affecting events within a narrow window of the mRNP biogenesis process. We propose that reducing the speed with which transcription converts from initiation and promoter clearance to elongation may have a positive effect on mRNP formation by permitting more effective recruitment of partially-functional mRNP proteins to the nascent mRNP.

  9. Effects of formulation and host nematode density on the ability of in vitro-produced pasteuria endospores to control its host Belonolaimus longicaudatus.

    Science.gov (United States)

    Luc, John E; Pang, Wenjing; Crow, William T; Giblin-Davis, Robin M

    2010-06-01

    The effect of nematode population density at the time of application and formulations of in vitro-produced Pasteuria spp. endospores on the final population density of Belonolaimus longicaudatus was studied in an 84-d-long pot bioassay. The experiment utilized a factorial design consisting of 30 or 300 B. longicaudatus /100 cm(3) of sandy soil and three formulations of in vitro-produced Pasteuria spp. endospores (nontreated, granular, or liquid). No differences were observed in percent endospore attachment between nematode inoculum levels during either trial. Granular and liquid formulations of in vitro-produced endospores suppressed nematode population densities by 22% and 59% in the first trial and 20% and 63% in the second, respectively compared with the nontreated control. The liquid formulation increased percent endospore attachment by 147% and 158%, respectively, compared with the granular formulation. The greatest root retention by the host plant was observed at the lower B. longicaudatus inoculation level following application of the liquid formulation. While both the granular and liquid formulations reduced B. longicaudatus population densities in the soil, the liquid spore suspension was most effective.

  10. Post-transcriptional regulation of ribosome biogenesis in yeast

    Directory of Open Access Journals (Sweden)

    Isabelle C. Kos-Braun

    2017-05-01

    Full Text Available Most microorganisms are exposed to the constantly and often rapidly changing environment. As such they evolved mechanisms to balance their metabolism and energy expenditure with the resources available to them. When resources become scarce or conditions turn out to be unfavourable for growth, cells reduce their metabolism and energy usage to survive. One of the major energy consuming processes in the cell is ribosome biogenesis. Unsurprisingly, cells encountering adverse conditions immediately shut down production of new ribosomes. It is well established that nutrient depletion leads to a rapid repression of transcription of the genes encoding ribosomal proteins, ribosome biogenesis factors as well as ribosomal RNA (rRNA. However, if pre-rRNA processing and ribosome assembly are regulated post-transcriptionally remains largely unclear. We have recently uncovered that the yeast Saccharomyces cerevisiae rapidly switches between two alternative pre-rRNA processing pathways depending on the environmental conditions. Our findings reveal a new level of complexity in the regulation of ribosome biogenesis.

  11. Differential expression of THOC1 and ALY mRNP biogenesis/export factors in human cancers

    International Nuclear Information System (INIS)

    Domínguez-Sánchez, María S; Sáez, Carmen; Japón, Miguel A; Aguilera, Andrés; Luna, Rosa

    2011-01-01

    One key step in gene expression is the biogenesis of mRNA ribonucleoparticle complexes (mRNPs). Formation of the mRNP requires the participation of a number of conserved factors such as the THO complex. THO interacts physically and functionally with the Sub2/UAP56 RNA-dependent ATPase, and the Yra1/REF1/ALY RNA-binding protein linking transcription, mRNA export and genome integrity. Given the link between genome instability and cancer, we have performed a comparative analysis of the expression patterns of THOC1, a THO complex subunit, and ALY in tumor samples. The mRNA levels were measured by quantitative real-time PCR and hybridization of a tumor tissue cDNA array; and the protein levels and distribution by immunostaining of a custom tissue array containing a set of paraffin-embedded samples of different tumor and normal tissues followed by statistical analysis. We show that the expression of two mRNP factors, THOC1 and ALY are altered in several tumor tissues. THOC1 mRNA and protein levels are up-regulated in ovarian and lung tumors and down-regulated in those of testis and skin, whereas ALY is altered in a wide variety of tumors. In contrast to THOC1, ALY protein is highly detected in normal proliferative cells, but poorly in high-grade cancers. These results suggest a differential connection between tumorogenesis and the expression levels of human THO and ALY. This study opens the possibility of defining mRNP biogenesis factors as putative players in cell proliferation that could contribute to tumor development

  12. Dispersal of thermophilic Desulfotomaculum endospores into Baltic Sea sediments over thousands of years

    DEFF Research Database (Denmark)

    Rezende, Julia Rosa de; Kjeldsen, Kasper Urup; Hubert, Casey RJ

    2013-01-01

    S rRNA and of the dissimilatory (bi)sulfite reductase (dsrAB) genes within the endospore-forming SRB genus Desulfotomaculum. The thermophilic Desulfotomaculum community in Aarhus Bay sediments consisted of at least 23 species-level 16S rRNA sequence phylotypes. In two cases, pairs of identical 16S r......RNA and dsrAB sequences in Arctic surface sediment 3000km away showed that the same phylotypes are present in both locations. Radiotracer-enhanced most probable number analysis revealed that the abundance of endospores of thermophilic SRB in Aarhus Bay sediment was ca. 104 per cm3 at the surface and decreased...... in water masses preceding sedimentation. The sources of these thermophiles remain enigmatic, but at least one source may be common to both Aarhus Bay and Arctic sediments....

  13. PMA-Linked Fluorescence for Rapid Detection of Viable Bacterial Endospores

    Science.gov (United States)

    LaDuc, Myron T.; Venkateswaran, Kasthuri; Mohapatra, Bidyut

    2012-01-01

    The most common approach for assessing the abundance of viable bacterial endospores is the culture-based plating method. However, culture-based approaches are heavily biased and oftentimes incompatible with upstream sample processing strategies, which make viable cells/spores uncultivable. This shortcoming highlights the need for rapid molecular diagnostic tools to assess more accurately the abundance of viable spacecraft-associated microbiota, perhaps most importantly bacterial endospores. Propidium monoazide (PMA) has received a great deal of attention due to its ability to differentiate live, viable bacterial cells from dead ones. PMA gains access to the DNA of dead cells through compromised membranes. Once inside the cell, it intercalates and eventually covalently bonds with the double-helix structures upon photoactivation with visible light. The covalently bound DNA is significantly altered, and unavailable to downstream molecular-based manipulations and analyses. Microbiological samples can be treated with appropriate concentrations of PMA and exposed to visible light prior to undergoing total genomic DNA extraction, resulting in an extract comprised solely of DNA arising from viable cells. This ability to extract DNA selectively from living cells is extremely powerful, and bears great relevance to many microbiological arenas.

  14. Insulin-like growth factor 1 signaling is essential for mitochondrial biogenesis and mitophagy in cancer cells.

    Science.gov (United States)

    Lyons, Amy; Coleman, Michael; Riis, Sarah; Favre, Cedric; O'Flanagan, Ciara H; Zhdanov, Alexander V; Papkovsky, Dmitri B; Hursting, Stephen D; O'Connor, Rosemary

    2017-10-13

    Mitochondrial activity and metabolic reprogramming influence the phenotype of cancer cells and resistance to targeted therapy. We previously established that an insulin-like growth factor 1 (IGF-1)-inducible mitochondrial UTP carrier (PNC1/SLC25A33) promotes cell growth. This prompted us to investigate whether IGF signaling is essential for mitochondrial maintenance in cancer cells and whether this contributes to therapy resistance. Here we show that IGF-1 stimulates mitochondrial biogenesis in a range of cell lines. In MCF-7 and ZR75.1 breast cancer cells, IGF-1 induces peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β) and PGC-1α-related coactivator (PRC). Suppression of PGC-1β and PRC with siRNA reverses the effects of IGF-1 and disrupts mitochondrial morphology and membrane potential. IGF-1 also induced expression of the redox regulator nuclear factor-erythroid-derived 2-like 2 (NFE2L2 alias NRF-2). Of note, MCF-7 cells with acquired resistance to an IGF-1 receptor (IGF-1R) tyrosine kinase inhibitor exhibited reduced expression of PGC-1β, PRC, and mitochondrial biogenesis. Interestingly, these cells exhibited mitochondrial dysfunction, indicated by reactive oxygen species expression, reduced expression of the mitophagy mediators BNIP3 and BNIP3L, and impaired mitophagy. In agreement with this, IGF-1 robustly induced BNIP3 accumulation in mitochondria. Other active receptor tyrosine kinases could not compensate for reduced IGF-1R activity in mitochondrial protection, and MCF-7 cells with suppressed IGF-1R activity became highly dependent on glycolysis for survival. We conclude that IGF-1 signaling is essential for sustaining cancer cell viability by stimulating both mitochondrial biogenesis and turnover through BNIP3 induction. This core mitochondrial protective signal is likely to strongly influence responses to therapy and the phenotypic evolution of cancer. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Regulation of chloroplast biogenesis: the immutans mutant of Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Rodermel, Steven

    2015-11-16

    The immutans (im) variegation mutant of Arabidopsis is an ideal model to gain insight into factors that control chloroplast biogenesis. im defines the gene for PTOX, a plastoquinol terminal oxidase that participates in control of thylakoid redox. Here, we report that the im defect can be suppressed during the late stages of plant development by gigantea (gi2), which defines the gene for GIGANTEA (GI), a central component of the circadian clock that plays a poorly-understood role in diverse plant developmental processes. imgi2 mutants are late-flowering and display other well-known phenotypes associated with gi2, such as starch accumulation and resistance to oxidative stress. We show that the restoration of chloroplast biogenesis in imgi2 is caused by a developmental-specific de-repression of cytokinin signaling that involves crosstalk with signaling pathways mediated by gibberellin (GA) and SPINDLY (SPY), a GA response inhibitor. Suppression of the plastid defect in imgi2 is likely caused by a relaxation of excitation pressures in developing plastids by factors contributed by gi2, including enhanced rates of photosynthesis and increased resistance to oxidative stress. Interestingly, the suppression phenotype of imgi can be mimicked by crossing im with the starch accumulation mutant, sex1, perhaps because sex1 utilizes pathways similar to gi. We conclude that our studies provide a direct genetic linkage between GIGANTEA and chloroplast biogenesis, and we construct a model of interactions between signaling pathways mediated by gi, GA, SPY, cytokinins, and sex1 that are required for chloroplast biogenesis.

  16. Erythrocyte nanovesicles: Biogenesis, biolo

    Directory of Open Access Journals (Sweden)

    Gamaleldin I. Harisa

    2017-01-01

    Full Text Available Nanovesicles (NVs represent a novel transporter for cell signals to modify functions of target cells. Therefore, NVs play many roles in both physiological and pathological processes. This report highlights biogenesis, composition and biological roles of erythrocytes derived nanovesicles (EDNVs. Furthermore, we address utilization of EDNVs as novel drug delivery cargo as well as therapeutic target. EDNVs are lipid bilayer vesicles rich in phospholipids, proteins, lipid raft, and hemoglobin. In vivo EDNVs biogenesis is triggered by an increase of intracellular calcium levels, ATP depletion and under effect of oxidative stress conditions. However, in vitro production of EDNVs can be achieved via hypotonic treatment and extrusion of erythrocyte. NVs can be used as biomarkers for diagnosis, monitoring of therapy and drug delivery system. Many therapeutic agents are suggested to decrease NVs biogenesis.

  17. Endospore-forming bacteria as an indicator of pollution in sediments of Lake Geneva

    Directory of Open Access Journals (Sweden)

    Bueche M.

    2013-04-01

    Full Text Available Treated wastewater and runoff-water is released by the outlet of the sewage treatment plant of Vidy (Lausanne directly into the Lake of Geneva via a pipe located 300m from the shore. Even if this water is properly treated with modern technologies, we can observe an accumulation of micro pollutants into the sediments, and particularly heavy-metals. The main objective of this project is to investigate how these elevated concentrations of heavy metals affect both abundance and diversity of prokaryotes in the sediments. A special emphasis was given to endospore-forming bacteria, which could use sporulation as a survival strategy to resist in highly contaminated areas. This study could have implications both for understanding the role of endospore-forming bacteria in the environment as well as in terms of improving the bioremediation processes.

  18. Formulation of stable Bacillus subtilis AH18 against temperature fluctuation with highly heat-resistant endospores and micropore inorganic carriers.

    Science.gov (United States)

    Chung, Soohee; Lim, Hyung Mi; Kim, Sang-Dal

    2007-08-01

    To survive the commercial market and to achieve the desired effect of beneficial organisms, the strains in microbial products must be cost-effectively formulated to remain dormant and hence survive through high and low temperatures of the environment during transportation and storage. Dormancy and stability of Bacillus subtilis AH18 was achieved by producing endospores with enhanced heat resistance and using inorganic carriers. Heat stability assays, at 90 degrees C for 1 h, showed that spores produced under a sublethal temperature of 57 degrees C was 100 times more heat-resistant than the ones produced by food depletion at the growing temperature of 37 degrees C. When these highly heat-resistant endospores were formulated with inorganic carriers of natural and synthetic zeolite or kaolin clay minerals having substantial amount of micropores, the dormancy of the endospores was maintained for 6 months at 15-25 degrees C. Meanwhile, macroporous perlite carriers with average pore diameter larger than 3.7 microm stimulated the germination of the spores and rapid proliferation of the bacteria. These results indicated that a B. subtilis AH18 product that can remain dormant and survive through environmental temperature fluctuation can be formulated by producing heat-stressed endospores and incorporating inorganic carriers with micropores in the formulation step.

  19. A combination of extreme environmental conditions favor the prevalence of Endospore-forming Firmicutes

    Directory of Open Access Journals (Sweden)

    Sevasti Filippidou

    2016-11-01

    Full Text Available Environmental conditions unsuitable for microbial growth are the rule rather than the exception in most habitats. In response to this, microorganisms have developed various strategies to withstand environmental conditions that limit active growth. Endospore-forming Firmicutes (EFF deploy a myriad of survival strategies in order to resist adverse conditions. Like many bacterial groups, they can form biofilms and detect nutrient scarcity through chemotaxis. Moreover, within this paraphyletic group of Firmicutes, ecophysiological optima are diverse. Nonetheless, a response to adversity that delimits this group is the formation of wet-heat resistant spores. These strategies are energetically demanding and therefore might affect the biological success of EFF. Therefore, we hypothesize that abundance and diversity of EFF should be maximized in those environments in which the benefits of these survival strategies offsets the energetic cost. In order to address this hypothesis, geothermal and mineral springs and drillings were selected because in these environments of steep physicochemical gradients, diversified survival strategies may become a successful strategy. We collected 71 samples from geothermal and mineral environments characterized by none (null, single or multiple limiting environmental factors (temperature, pH, UV radiation and specific mineral composition. To measure success, we quantified EFF gene copy numbers (GCN; spo0A gene in relation to total bacterial GCN (16S rRNA gene, as well as the contribution of EFF to community composition. The quantification showed that relative GCN for EFF reached up to 20% at sites characterized by multiple limiting environmental factors, whereas it corresponded to less than 1% at sites with one or no limiting environmental factor. Pyrosequencing of the 16S rRNA gene supports a higher contribution of EFF at sites with multiple limiting factors. Community composition suggested a combination of phylotypes

  20. Reactive oxygen species mediates homocysteine-induced mitochondrial biogenesis in human endothelial cells: Modulation by antioxidants

    International Nuclear Information System (INIS)

    Perez-de-Arce, Karen; Foncea, Rocio; Leighton, Federico

    2005-01-01

    It has been proposed that homocysteine (Hcy)-induces endothelial dysfunction and atherosclerosis by generation of reactive oxygen species (ROS). A previous report has shown that Hcy promotes mitochondrial damage. Considering that oxidative stress can affect mitochondrial biogenesis, we hypothesized that Hcy-induced ROS in endothelial cells may lead to increased mitochondrial biogenesis. We found that Hcy-induced ROS (1.85-fold), leading to a NF-κB activation and increase the formation of 3-nitrotyrosine. Furthermore, expression of the mitochondrial biogenesis factors, nuclear respiratory factor-1 and mitochondrial transcription factor A, was significantly elevated in Hcy-treated cells. These changes were accompanied by increase in mitochondrial mass and higher mRNA and protein expression of the subunit III of cytochrome c oxidase. These effects were significantly prevented by pretreatment with the antioxidants, catechin and trolox. Taken together, our results suggest that ROS is an important mediator of mitochondrial biogenesis induced by Hcy, and that modulation of oxidative stress by antioxidants may protect against the adverse vascular effects of Hcy

  1. Isolation and structure elucidation of avocado seed (Persea americana) lipid derivatives that inhibit Clostridium sporogenes endospore germination.

    Science.gov (United States)

    Rodríguez-Sánchez, Dariana Graciela; Pacheco, Adriana; García-Cruz, María Isabel; Gutiérrez-Uribe, Janet Alejandra; Benavides-Lozano, Jorge Alejandro; Hernández-Brenes, Carmen

    2013-07-31

    Avocado fruit extracts are known to exhibit antimicrobial properties. However, the effects on bacterial endospores and the identity of antimicrobial compounds have not been fully elucidated. In this study, avocado seed extracts were tested against Clostridium sporogenes vegetative cells and active endospores. Bioassay-guided purification of a crude extract based on inhibitory properties linked antimicrobial action to six lipid derivatives from the family of acetogenin compounds. Two new structures and four compounds known to exist in nature were identified as responsible for the activity. Structurally, most potent molecules shared features of an acetyl moiety and a trans-enone group. All extracts produced inhibition zones on vegetative cells and active endospores. Minimum inhibitory concentrations (MIC) of isolated molecules ranged from 7.8 to 15.6 μg/mL, and bactericidal effects were observed for an enriched fraction at 19.5 μg/mL. Identified molecules showed potential as natural alternatives to additives and antibiotics used by the food and pharmaceutical industries to inhibit Gram-positive spore-forming bacteria.

  2. Pasteuria endospores from Heterodera cajani (Nematoda: Heteroderidae) exhibit inverted attachment and altered germination in cross-infection studies with Globodera pallida (Nematoda: Heteroderidae).

    Science.gov (United States)

    Mohan, Sharad; Mauchline, Tim H; Rowe, Janet; Hirsch, Penny R; Davies, Keith G

    2012-03-01

    The Pasteuria group of Gram-positive, endospore-forming bacteria are parasites of invertebrates and exhibit differences in host specificity. We describe a cross-infection study between an isolate of Pasteuria from pigeon pea cyst nematode, Heterodera cajani, which also infects the potato cyst nematode, Globodera pallida, from the United Kingdom. A proportion of the attached endospores, 13% on H. cajani and 22% on G. pallida adhere to the cuticle in an inverted orientation. Inverted and conventionally attached endospores germinated and produced bacillus-like rods that completed their life cycle in Pasteuria population was systematically followed in two different nematode genera. A 1430-base pair fragment of the 16S rRNA gene sequence of the Pasteuria isolate from H. cajani revealed 98.6% similarity to the orthologous gene in Pasteuria nishizawae. Additionally, their respective endospore sizes were not significantly different, in contrast their host ranges are. Potential reasons for this remain unclear and are discussed. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  3. Minotaur is critical for primary piRNA biogenesis.

    Science.gov (United States)

    Vagin, Vasily V; Yu, Yang; Jankowska, Anna; Luo, Yicheng; Wasik, Kaja A; Malone, Colin D; Harrison, Emily; Rosebrock, Adam; Wakimoto, Barbara T; Fagegaltier, Delphine; Muerdter, Felix; Hannon, Gregory J

    2013-08-01

    Piwi proteins and their associated small RNAs are essential for fertility in animals. In part, this is due to their roles in guarding germ cell genomes against the activity of mobile genetic elements. piRNA populations direct Piwi proteins to silence transposon targets and, as such, form a molecular code that discriminates transposons from endogenous genes. Information ultimately carried by piRNAs is encoded within genomic loci, termed piRNA clusters. These give rise to long, single-stranded, primary transcripts that are processed into piRNAs. Despite the biological importance of this pathway, neither the characteristics that define a locus as a source of piRNAs nor the mechanisms that catalyze primary piRNA biogenesis are well understood. We searched an EMS-mutant collection annotated for fertility phenotypes for genes involved in the piRNA pathway. Twenty-seven homozygous sterile strains showed transposon-silencing defects. One of these, which strongly impacted primary piRNA biogenesis, harbored a causal mutation in CG5508, a member of the Drosophila glycerol-3-phosphate O-acetyltransferase (GPAT) family. These enzymes catalyze the first acylation step on the path to the production of phosphatidic acid (PA). Though this pointed strongly to a function for phospholipid signaling in the piRNA pathway, a mutant form of CG5508, which lacks the GPAT active site, still functions in piRNA biogenesis. We have named this new biogenesis factor Minotaur.

  4. Minotaur is critical for primary piRNA biogenesis

    Science.gov (United States)

    Vagin, Vasily V.; Yu, Yang; Jankowska, Anna; Luo, Yicheng; Wasik, Kaja A.; Malone, Colin D.; Harrison, Emily; Rosebrock, Adam; Wakimoto, Barbara T.; Fagegaltier, Delphine; Muerdter, Felix; Hannon, Gregory J.

    2013-01-01

    Piwi proteins and their associated small RNAs are essential for fertility in animals. In part, this is due to their roles in guarding germ cell genomes against the activity of mobile genetic elements. piRNA populations direct Piwi proteins to silence transposon targets and, as such, form a molecular code that discriminates transposons from endogenous genes. Information ultimately carried by piRNAs is encoded within genomic loci, termed piRNA clusters. These give rise to long, single-stranded, primary transcripts that are processed into piRNAs. Despite the biological importance of this pathway, neither the characteristics that define a locus as a source of piRNAs nor the mechanisms that catalyze primary piRNA biogenesis are well understood. We searched an EMS-mutant collection annotated for fertility phenotypes for genes involved in the piRNA pathway. Twenty-seven homozygous sterile strains showed transposon-silencing defects. One of these, which strongly impacted primary piRNA biogenesis, harbored a causal mutation in CG5508, a member of the Drosophila glycerol-3-phosphate O-acetyltransferase (GPAT) family. These enzymes catalyze the first acylation step on the path to the production of phosphatidic acid (PA). Though this pointed strongly to a function for phospholipid signaling in the piRNA pathway, a mutant form of CG5508, which lacks the GPAT active site, still functions in piRNA biogenesis. We have named this new biogenesis factor Minotaur. PMID:23788724

  5. Applicability of UV resistant Bacillus pumilus endospores as a human adenovirus surrogate for evaluating the effectiveness of virus inactivation in low-pressure UV treatment systems

    Science.gov (United States)

    Recent studies have demonstrated the potential to use Bacillus pumilus endospores as a surrogate of human adenovirus (HAdV) in UV disinfection studies. The use of endospores has been limited by observations of batch-to-batch variation in UV sensitivity. This study reports on a pr...

  6. Resistance of Bacillus Endospores to Extreme Terrestrial and Extraterrestrial Environments

    Science.gov (United States)

    Nicholson, Wayne L.; Munakata, Nobuo; Horneck, Gerda; Melosh, Henry J.; Setlow, Peter

    2000-01-01

    Endospores of Bacillus spp., especially Bacillus subtilis, have served as experimental models for exploring the molecular mechanisms underlying the incredible longevity of spores and their resistance to environmental insults. In this review we summarize the molecular laboratory model of spore resistance mechanisms and attempt to use the model as a basis for exploration of the resistance of spores to environmental extremes both on Earth and during postulated interplanetary transfer through space as a result of natural impact processes. PMID:10974126

  7. Gene activation of heavy ion treated bacillus subtilis 168 endospores during germination involved DNA-repair

    International Nuclear Information System (INIS)

    Moeller, R.; Berger, T.; Reitz, G.; Okayasu, Ryuichi

    2006-01-01

    This research project is aimed at correlating radiation effects induced DNA damage in Bacillus subtilis endospores with the linear energy transfer (LET) of the used radiation by investigating survival and gene activation after irradiation with high-LET particles. During the stationary growth phase Bacillus subtilis change their metabolic active state from the vegetative cells to the metabolic inactive but even more resistant endospores. If spores find optimal conditions, they could germinate and switch to the vegetative growth. With these outgrowth spores can and/or must repair the induced formed DNA damage. During germination spores lose their most resistance. In more detail, DNA repair and mutation induction events investigated will include the survivability, behaviour against specific antibiotics and their germination. DNA repair pattern will be detected during germination by using DNA microarrays, which contain the whole genome of Bacillus subtilis 168. (author)

  8. A Simple Method for the Extraction, PCR-amplification, Cloning, and Sequencing of Pasteuria 16S rDNA from Small Numbers of Endospores.

    Science.gov (United States)

    Atibalentja, N; Noel, G R; Ciancio, A

    2004-03-01

    For many years the taxonomy of the genus Pasteuria has been marred with confusion because the bacterium could not be cultured in vitro and, therefore, descriptions were based solely on morphological, developmental, and pathological characteristics. The current study sought to devise a simple method for PCR-amplification, cloning, and sequencing of Pasteuria 16S rDNA from small numbers of endospores, with no need for prior DNA purification. Results show that DNA extracts from plain glass bead-beating of crude suspensions containing 10,000 endospores at 0.2 x 10 endospores ml(-1) were sufficient for PCR-amplification of Pasteuria 16S rDNA, when used in conjunction with specific primers. These results imply that for P. penetrans and P. nishizawae only one parasitized female of Meloidogyne spp. and Heterodera glycines, respectively, should be sufficient, and as few as eight cadavers of Belonolaimus longicaudatus with an average number of 1,250 endospores of "Candidatus Pasteuria usgae" are needed for PCR-amplification of Pasteuria 16S rDNA. The method described in this paper should facilitate the sequencing of the 16S rDNA of the many Pasteuria isolates that have been reported on nematodes and, consequently, expedite the classification of those isolates through comparative sequence analysis.

  9. A method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious bacterium Pasteuria penetrans.

    Science.gov (United States)

    Mauchline, T H; Mohan, S; Davies, K G; Schaff, J E; Opperman, C H; Kerry, B R; Hirsch, P R

    2010-05-01

    To establish a reliable protocol to extract DNA from Pasteuria penetrans endospores for use as template in multiple strand amplification, thus providing sufficient material for genetic analyses. To develop a highly sensitive PCR-based diagnostic tool for P. penetrans. An optimized method to decontaminate endospores, release and purify DNA enabled multiple strand amplification. DNA purity was assessed by cloning and sequencing gyrB and 16S rRNA gene fragments obtained from PCR using generic primers. Samples indicated to be 100%P. penetrans by the gyrB assay were estimated at 46% using the 16S rRNA gene. No bias was detected on cloning and sequencing 12 housekeeping and sporulation gene fragments from amplified DNA. The detection limit by PCR with Pasteuria-specific 16S rRNA gene primers following multiple strand amplification of DNA extracted using the method was a single endospore. Generation of large quantities DNA will facilitate genomic sequencing of P. penetrans. Apparent differences in sample purity are explained by variations in 16S rRNA gene copy number in Eubacteria leading to exaggerated estimations of sample contamination. Detection of single endospores will facilitate investigations of P. penetrans molecular ecology. These methods will advance studies on P. penetrans and facilitate research on other obligate and fastidious micro-organisms where it is currently impractical to obtain DNA in sufficient quantity and quality.

  10. Bioenergetics of lung tumors: Alteration of mitochondrial biogenesis and respiratory kapacity

    Czech Academy of Sciences Publication Activity Database

    Bellance, N.; Benard, G.; Furt, F.; Begueret, H.; Smolková, Katarína; Passerieux, E.; Delage, J.P.; Baste, J.M.; Moreau, P.; Rossignol, R.

    2009-01-01

    Roč. 41, č. 12 (2009), s. 2566-2577 ISSN 1357-2725 Institutional research plan: CEZ:AV0Z50110509 Keywords : tumors * bioenergetics * biogenesis Subject RIV: ED - Physiology Impact factor: 4.887, year: 2009

  11. Maintaining ancient organelles: mitochondrial biogenesis and maturation.

    Science.gov (United States)

    Vega, Rick B; Horton, Julie L; Kelly, Daniel P

    2015-05-22

    The ultrastructure of the cardiac myocyte is remarkable for the high density of mitochondria tightly packed between sarcomeres. This structural organization is designed to provide energy in the form of ATP to fuel normal pump function of the heart. A complex system comprised of regulatory factors and energy metabolic machinery, encoded by both mitochondrial and nuclear genomes, is required for the coordinate control of cardiac mitochondrial biogenesis, maturation, and high-capacity function. This process involves the action of a transcriptional regulatory network that builds and maintains the mitochondrial genome and drives the expression of the energy transduction machinery. This finely tuned system is responsive to developmental and physiological cues, as well as changes in fuel substrate availability. Deficiency of components critical for mitochondrial energy production frequently manifests as a cardiomyopathic phenotype, underscoring the requirement to maintain high respiration rates in the heart. Although a precise causative role is not clear, there is increasing evidence that perturbations in this regulatory system occur in the hypertrophied and failing heart. This review summarizes current knowledge and highlights recent advances in our understanding of the transcriptional regulatory factors and signaling networks that serve to regulate mitochondrial biogenesis and function in the mammalian heart. © 2015 American Heart Association, Inc.

  12. The ribosome biogenesis factor Nol11 is required for optimal rDNA transcription and craniofacial development in Xenopus.

    Directory of Open Access Journals (Sweden)

    John N Griffin

    2015-03-01

    Full Text Available The production of ribosomes is ubiquitous and fundamental to life. As such, it is surprising that defects in ribosome biogenesis underlie a growing number of symptomatically distinct inherited disorders, collectively called ribosomopathies. We previously determined that the nucleolar protein, NOL11, is essential for optimal pre-rRNA transcription and processing in human tissue culture cells. However, the role of NOL11 in the development of a multicellular organism remains unknown. Here, we reveal a critical function for NOL11 in vertebrate ribosome biogenesis and craniofacial development. Nol11 is strongly expressed in the developing cranial neural crest (CNC of both amphibians and mammals, and knockdown of Xenopus nol11 results in impaired pre-rRNA transcription and processing, increased apoptosis, and abnormal development of the craniofacial cartilages. Inhibition of p53 rescues this skeletal phenotype, but not the underlying ribosome biogenesis defect, demonstrating an evolutionarily conserved control mechanism through which ribosome-impaired craniofacial cells are removed. Excessive activation of this mechanism impairs craniofacial development. Together, our findings reveal a novel requirement for Nol11 in craniofacial development, present the first frog model of a ribosomopathy, and provide further insight into the clinically important relationship between specific ribosome biogenesis proteins and craniofacial cell survival.

  13. The Importance of Endospore-Forming Bacteria Originating from Soil for Contamination of Industrial Food Processing

    Directory of Open Access Journals (Sweden)

    Marc Heyndrickx

    2011-01-01

    Full Text Available Specific endospore formers have become important contaminants in industrial food processing. The direct or indirect soil route of contamination or dispersal is the start of events or processes in the agrofood chain that eventually leads to important problems or concerns for food safety and/or quality. Three important food sectors are discussed in this paper. In the dairy sector, Bacillus cereus, the most important pathogen or spoilage organism in this sector, and Clostridium tyrobutyricum, the most important spoiler in certain cheeses, both contaminate pasteurized milk through the faecal and/or (at least for B. cereus the direct soil route. In the fruit juice industry, Alicyclobacillus acidoterrestris, present on raw fruits, has become a major quality-target organism. In the ready-to-eat food sector, B. cereus and other aerobic endospore formers are introduced via vegetables, fruits, or herbs and spices, while anaerobic spore formers like nonproteolytic Clostridium botulinum and Clostridium estertheticum pose safety and spoilage risks in chilled packaged foods, respectively.

  14. The Clostridium sporulation programs: diversity and preservation of endospore differentiation.

    Science.gov (United States)

    Al-Hinai, Mohab A; Jones, Shawn W; Papoutsakis, Eleftherios T

    2015-03-01

    Bacillus and Clostridium organisms initiate the sporulation process when unfavorable conditions are detected. The sporulation process is a carefully orchestrated cascade of events at both the transcriptional and posttranslational levels involving a multitude of sigma factors, transcription factors, proteases, and phosphatases. Like Bacillus genomes, sequenced Clostridium genomes contain genes for all major sporulation-specific transcription and sigma factors (spo0A, sigH, sigF, sigE, sigG, and sigK) that orchestrate the sporulation program. However, recent studies have shown that there are substantial differences in the sporulation programs between the two genera as well as among different Clostridium species. First, in the absence of a Bacillus-like phosphorelay system, activation of Spo0A in Clostridium organisms is carried out by a number of orphan histidine kinases. Second, downstream of Spo0A, the transcriptional and posttranslational regulation of the canonical set of four sporulation-specific sigma factors (σ(F), σ(E), σ(G), and σ(K)) display different patterns, not only compared to Bacillus but also among Clostridium organisms. Finally, recent studies demonstrated that σ(K), the last sigma factor to be activated according to the Bacillus subtilis model, is involved in the very early stages of sporulation in Clostridium acetobutylicum, C. perfringens, and C. botulinum as well as in the very late stages of spore maturation in C. acetobutylicum. Despite profound differences in initiation, propagation, and orchestration of expression of spore morphogenetic components, these findings demonstrate not only the robustness of the endospore sporulation program but also the plasticity of the program to generate different complex phenotypes, some apparently regulated at the epigenetic level. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  15. Coordination of plant mitochondrial biogenesis: keeping pace with cellular requirements

    Science.gov (United States)

    Welchen, Elina; García, Lucila; Mansilla, Natanael; Gonzalez, Daniel H.

    2014-01-01

    Plant mitochondria are complex organelles that carry out numerous metabolic processes related with the generation of energy for cellular functions and the synthesis and degradation of several compounds. Mitochondria are semiautonomous and dynamic organelles changing in shape, number, and composition depending on tissue or developmental stage. The biogenesis of functional mitochondria requires the coordination of genes present both in the nucleus and the organelle. In addition, due to their central role, all processes held inside mitochondria must be finely coordinated with those in other organelles according to cellular demands. Coordination is achieved by transcriptional control of nuclear genes encoding mitochondrial proteins by specific transcription factors that recognize conserved elements in their promoter regions. In turn, the expression of most of these transcription factors is linked to developmental and environmental cues, according to the availability of nutrients, light–dark cycles, and warning signals generated in response to stress conditions. Among the signals impacting in the expression of nuclear genes, retrograde signals that originate inside mitochondria help to adjust mitochondrial biogenesis to organelle demands. Adding more complexity, several nuclear encoded proteins are dual localized to mitochondria and either chloroplasts or the nucleus. Dual targeting might establish a crosstalk between the nucleus and cell organelles to ensure a fine coordination of cellular activities. In this article, we discuss how the different levels of coordination of mitochondrial biogenesis interconnect to optimize the function of the organelle according to both internal and external demands. PMID:24409193

  16. Coordination of plant mitochondrial biogenesis: keeping pace with cellular requirements.

    Directory of Open Access Journals (Sweden)

    Elina eWelchen

    2014-01-01

    Full Text Available Plant mitochondria are complex organelles that carry out numerous metabolic processes related with the generation of energy for cellular functions and the synthesis and degradation of several compounds. Mitochondria are semiautonomous and dynamic organelles changing in shape, number and composition depending on tissue or developmental stage. The biogenesis of functional mitochondria requires the coordination of genes present both in the nucleus and the organelle. In addition, due to their central role, all processes held inside mitochondria must be finely coordinated with those in other organelles according to cellular demands. Coordination is achieved by transcriptional control of nuclear genes encoding mitochondrial proteins by specific transcription factors that recognize conserved elements in their promoter regions. In turn, the expression of most of these transcription factors is linked to developmental and environmental cues, according to the availability of nutrients, light-dark cycles and warning signals generated in response to stress conditions. Among the signals impacting in the expression of nuclear genes, retrograde signals that originate inside mitochondria help to adjust mitochondrial biogenesis to organelle demands. Adding more complexity, several nuclear encoded proteins are dual localized to mitochondria and either chloroplasts or the nucleus. Dual targeting might establish a crosstalk between the nucleus and cell organelles to ensure a fine coordination of cellular activities. In this article, we discuss how the different levels of coordination of mitochondrial biogenesis interconnect to optimize the function of the organelle according to both internal and external demands.

  17. Genome Sequence of Anoxybacillus geothermalis Strain GSsed3, a Novel Thermophilic Endospore-Forming Species

    Science.gov (United States)

    Filippidou, Sevasti; Jaussi, Marion; Junier, Thomas; Wunderlin, Tina; Roussel-Delif, Ludovic; Jeanneret, Nicole; Vieth-Hillebrand, Andrea; Vetter, Alexandra; Regenspurg, Simona; McMurry, Kim; Gleasner, Cheryl D.; Lo, Chien-Chi; Li, Paul; Vuyisich, Momchilo; Chain, Patrick S.

    2015-01-01

    Anoxybacillus geothermalis strain GSsed3 is an endospore-forming thermophilic bacterium isolated from filter deposits in a geothermal site. This novel species has a larger genome size (7.2 Mb) than that of any other Anoxybacillus species, and it possesses genes that support its phenotypic metabolic characterization and suggest an intriguing link to metals. PMID:26067952

  18. Dynamics of bacterial endospores, and microbial activities in soils and sediments

    DEFF Research Database (Denmark)

    Tamez-Hidalgo, Paulina

    Prokaryotes (Bacteria and Archaea) are essential for life on Earth. They catalyze unique and necessary chemical transformations, which shape the biogeochemical cycles, develop stable and labile pools of carbon (C) and nitrogen (N), produce essential molecules that multicellular organisms live upon...... between being considered slow growers, dormant or even immortal. Despite discussions about minimum energy requirements and metabolic categories, there is a paraphyletic group of Bacteria (the Firmicutes or low-GC Gram positive) able to create the toughest dormant forms on Earth, the endospores. Bacterial...

  19. 14,15-EET promotes mitochondrial biogenesis and protects cortical neurons against oxygen/glucose deprivation-induced apoptosis

    International Nuclear Information System (INIS)

    Wang, Lai; Chen, Man; Yuan, Lin; Xiang, Yuting; Zheng, Ruimao; Zhu, Shigong

    2014-01-01

    Highlights: • 14,15-EET inhibits OGD-induced apoptosis in cortical neurons. • Mitochondrial biogenesis of cortical neurons is promoted by 14,15-EET. • 14,15-EET preserves mitochondrial function of cortical neurons under OGD. • CREB mediates effect of 14,15-EET on mitochondrial biogenesis and function. - Abstract: 14,15-Epoxyeicosatrienoic acid (14,15-EET), a metabolite of arachidonic acid, is enriched in the brain cortex and exerts protective effect against neuronal apoptosis induced by ischemia/reperfusion. Although apoptosis has been well recognized to be closely associated with mitochondrial biogenesis and function, it is still unclear whether the neuroprotective effect of 14,15-EET is mediated by promotion of mitochondrial biogenesis and function in cortical neurons under the condition of oxygen–glucose deprivation (OGD). In this study, we found that 14,15-EET improved cell viability and inhibited apoptosis of cortical neurons. 14,15-EET significantly increased the mitochondrial mass and the ratio of mitochondrial DNA to nuclear DNA. Key makers of mitochondrial biogenesis, peroxisome proliferator activator receptor gamma-coactivator 1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM), were elevated at both mRNA and protein levels in the cortical neurons treated with 14,15-EET. Moreover, 14,15-EET markedly attenuated the decline of mitochondrial membrane potential, reduced ROS, while increased ATP synthesis. Knockdown of cAMP-response element binding protein (CREB) by siRNA blunted the up-regulation of PGC-1α and NRF-1 stimulated by 14,15-EET, and consequently abolished the neuroprotective effect of 14,15-EET. Our results indicate that 14,15-EET protects neurons from OGD-induced apoptosis by promoting mitochondrial biogenesis and function through CREB mediated activation of PGC-1α and NRF-1

  20. 14,15-EET promotes mitochondrial biogenesis and protects cortical neurons against oxygen/glucose deprivation-induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Lai; Chen, Man; Yuan, Lin; Xiang, Yuting [Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing (China); Zheng, Ruimao, E-mail: rmzheng@pku.edu.cn [Department of Anatomy, Histology and Embryology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing (China); Zhu, Shigong, E-mail: sgzhu@bjmu.edu.cn [Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing (China)

    2014-07-18

    Highlights: • 14,15-EET inhibits OGD-induced apoptosis in cortical neurons. • Mitochondrial biogenesis of cortical neurons is promoted by 14,15-EET. • 14,15-EET preserves mitochondrial function of cortical neurons under OGD. • CREB mediates effect of 14,15-EET on mitochondrial biogenesis and function. - Abstract: 14,15-Epoxyeicosatrienoic acid (14,15-EET), a metabolite of arachidonic acid, is enriched in the brain cortex and exerts protective effect against neuronal apoptosis induced by ischemia/reperfusion. Although apoptosis has been well recognized to be closely associated with mitochondrial biogenesis and function, it is still unclear whether the neuroprotective effect of 14,15-EET is mediated by promotion of mitochondrial biogenesis and function in cortical neurons under the condition of oxygen–glucose deprivation (OGD). In this study, we found that 14,15-EET improved cell viability and inhibited apoptosis of cortical neurons. 14,15-EET significantly increased the mitochondrial mass and the ratio of mitochondrial DNA to nuclear DNA. Key makers of mitochondrial biogenesis, peroxisome proliferator activator receptor gamma-coactivator 1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM), were elevated at both mRNA and protein levels in the cortical neurons treated with 14,15-EET. Moreover, 14,15-EET markedly attenuated the decline of mitochondrial membrane potential, reduced ROS, while increased ATP synthesis. Knockdown of cAMP-response element binding protein (CREB) by siRNA blunted the up-regulation of PGC-1α and NRF-1 stimulated by 14,15-EET, and consequently abolished the neuroprotective effect of 14,15-EET. Our results indicate that 14,15-EET protects neurons from OGD-induced apoptosis by promoting mitochondrial biogenesis and function through CREB mediated activation of PGC-1α and NRF-1.

  1. Genes Involved in Human Ribosome Biogenesis areTranscriptionally Upregulated in Colorectal Cancer

    DEFF Research Database (Denmark)

    Mansilla, Francisco; Lamy, Philippe; Ørntoft, Torben Falck

    2009-01-01

    Microarray gene expression profiling comprising 168 colorectal adenocarcinomas and 10 normal mucosas showed that over 79% of the genes involved in human ribosome biogenesis are significantly upregulated (log2>0.5, p<10-3) when compared to normal mucosa. Overexpression was independent of microsate......Microarray gene expression profiling comprising 168 colorectal adenocarcinomas and 10 normal mucosas showed that over 79% of the genes involved in human ribosome biogenesis are significantly upregulated (log2>0.5, p... of microsatellite status. The promoters of the genes studied showed a significant enrichment for several transcription factor binding sites. There was a significant correlation between the number of binding site targets for these transcription factors and the observed gene transcript upregulation. The upregulation...

  2. MicroRNAs upregulated during HIV infection target peroxisome biogenesis factors: Implications for virus biology, disease mechanisms and neuropathology.

    Directory of Open Access Journals (Sweden)

    Zaikun Xu

    2017-06-01

    Full Text Available HIV-associated neurocognitive disorders (HAND represent a spectrum neurological syndrome that affects up to 25% of patients with HIV/AIDS. Multiple pathogenic mechanisms contribute to the development of HAND symptoms including chronic neuroinflammation and neurodegeneration. Among the factors linked to development of HAND is altered expression of host cell microRNAs (miRNAs in brain. Here, we examined brain miRNA profiles among HIV/AIDS patients with and without HAND. Our analyses revealed differential expression of 17 miRNAs in brain tissue from HAND patients. A subset of the upregulated miRNAs (miR-500a-5p, miR-34c-3p, miR-93-3p and miR-381-3p, are predicted to target peroxisome biogenesis factors (PEX2, PEX7, PEX11B and PEX13. Expression of these miRNAs in transfected cells significantly decreased levels of peroxisomal proteins and concomitantly decreased peroxisome numbers or affected their morphology. The levels of miR-500a-5p, miR-34c-3p, miR-93-3p and miR-381-3p were not only elevated in the brains of HAND patients, but were also upregulated during HIV infection of primary macrophages. Moreover, concomitant loss of peroxisomal proteins was observed in HIV-infected macrophages as well as in brain tissue from HIV-infected patients. HIV-induced loss of peroxisomes was abrogated by blocking the functions of the upregulated miRNAs. Overall, these findings point to previously unrecognized miRNA expression patterns in the brains of HIV patients. Targeting peroxisomes by up-regulating miRNAs that repress peroxisome biogenesis factors may represent a novel mechanism by which HIV-1 subverts innate immune responses and/or causes neurocognitive dysfunction.

  3. Bioenergetics of lung tumors: alteration of mitochondrial biogenesis and respiratory capacity.

    Science.gov (United States)

    Bellance, N; Benard, G; Furt, F; Begueret, H; Smolková, K; Passerieux, E; Delage, J P; Baste, J M; Moreau, P; Rossignol, R

    2009-12-01

    Little is known on the metabolic profile of lung tumors and the reminiscence of embryonic features. Herein, we determined the bioenergetic profiles of human fibroblasts taken from lung epidermoid carcinoma (HLF-a) and fetal lung (MRC5). We also analysed human lung tumors and their surrounding healthy tissue from four patients with adenocarcinoma. On these different models, we measured functional parameters (cell growth rates in oxidative and glycolytic media, respiration, ATP synthesis and PDH activity) as well as compositional features (expression level of various energy proteins and upstream transcription factors). The results demonstrate that both the lung fetal and cancer cell lines produced their ATP predominantly by glycolysis, while oxidative phosphorylation was only capable of poor ATP delivery. This was explained by a decreased mitochondrial biogenesis caused by a lowered expression of PGC1alpha (as shown by RT-PCR and Western blot) and mtTFA. Consequently, the relative expression of glycolytic versus OXPHOS markers was high in these cells. Moreover, the re-activation of mitochondrial biogenesis with resveratrol induced cell death specifically in cancer cells. A consistent reduction of mitochondrial biogenesis and the subsequent alteration of respiratory capacity was also observed in lung tumors, associated with a lower expression level of bcl2. Our data give a better characterization of lung cancer cells' metabolic alterations which are essential for growth and survival. They designate mitochondrial biogenesis as a possible target for anti-cancer therapy.

  4. crRNA biogenesis

    NARCIS (Netherlands)

    Charpentier, E.; Oost, van der J.; White, M.

    2013-01-01

    Mature crRNAs are key elements in CRISPR-Cas defense against genome invaders. These short RNAs are composed of unique repeat/spacer sequences that guide the Cas protein(s) to the cognate invading nucleic acids for their destruction. The biogenesis of mature crRNAs involves highly precise processing

  5. Reactive Oxygen Species-Mediated Control of Mitochondrial Biogenesis

    Directory of Open Access Journals (Sweden)

    Edgar D. Yoboue

    2012-01-01

    Full Text Available Mitochondrial biogenesis is a complex process. It necessitates the contribution of both the nuclear and the mitochondrial genomes and therefore crosstalk between the nucleus and mitochondria. It is now well established that cellular mitochondrial content can vary according to a number of stimuli and physiological states in eukaryotes. The knowledge of the actors and signals regulating the mitochondrial biogenesis is thus of high importance. The cellular redox state has been considered for a long time as a key element in the regulation of various processes. In this paper, we report the involvement of the oxidative stress in the regulation of some actors of mitochondrial biogenesis.

  6. Nebivolol stimulates mitochondrial biogenesis in 3T3-L1 adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Chenglin; Chen, Dongrui; Xie, Qihai [State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Vascular Biology, Department of Hypertension, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China); Yang, Ying, E-mail: yangying_sh@yahoo.com [Department of Endocrine and Metabolic Diseases, Shanghai Institute of Endocrine and Metabolic Diseases, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China); Shen, Weili, E-mail: weili_shen@hotmail.com [State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Vascular Biology, Department of Hypertension, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China)

    2013-08-16

    Highlights: •Nebivolol may act as a partial agonist of β3-adrenergic receptor (AR). •Nebivolol stimulates mitochondrial DNA replication and protein expression. •Nebivolol promotes mitochondrial synthesis via activation of eNOS by β3-AR. -- Abstract: Nebivolol is a third-generation β-adrenergic receptor (β-AR) blocker with additional beneficial effects, including the improvement of lipid and glucose metabolism in obese individuals. However, the underlying mechanism of nebivolol’s role in regulating the lipid profile remains largely unknown. In this study, we investigated the role of nebivolol in mitochondrial biogenesis in 3T3-L1 adipocytes. Exposure of 3T3-L1 cells to nebivolol for 24 h increased mitochondrial DNA copy number, mitochondrial protein levels and the expression of transcription factors involved in mitochondrial biogenesis, including PPAR-γ coactivator-1α (PGC-1α), Sirtuin 3 (Sirt3), mitochondrial transcription factor A (Tfam) and nuclear related factor 1 (Nrf1). These changes were accompanied by an increase in oxygen consumption and in the expression of genes involved in fatty acid oxidation and antioxidant enzymes in 3T3-L1 adipocytes, including nebivolol-induced endothelial nitric oxide synthase (eNOS), as well as an increase in the formation of cyclic guanosine monophosphate (cGMP). Pretreatment with NG-nitro-L-arginine methyl ester (l-NAME) attenuated nebivolol-induced mitochondrial biogenesis, as did the soluble guanylate cyclase inhibitor, ODQ. Treatment with nebivolol and β3-AR blocker SR59230A markedly attenuated PGC-1α, Sirt3 and manganese superoxide dismutase (MnSOD) protein levels in comparison to treatment with nebivolol alone. These data indicate that the mitochondrial synthesis and metabolism in adipocytes that is promoted by nebivolol is primarily mediated through the eNOS/cGMP-dependent pathway and is initiated by the activation of β3-AR receptors.

  7. PLK4 trans-Autoactivation Controls Centriole Biogenesis in Space.

    Science.gov (United States)

    Lopes, Carla A M; Jana, Swadhin Chandra; Cunha-Ferreira, Inês; Zitouni, Sihem; Bento, Inês; Duarte, Paulo; Gilberto, Samuel; Freixo, Francisco; Guerrero, Adán; Francia, Maria; Lince-Faria, Mariana; Carneiro, Jorge; Bettencourt-Dias, Mónica

    2015-10-26

    Centrioles are essential for cilia and centrosome assembly. In centriole-containing cells, centrioles always form juxtaposed to pre-existing ones, motivating a century-old debate on centriole biogenesis control. Here, we show that trans-autoactivation of Polo-like kinase 4 (PLK4), the trigger of centriole biogenesis, is a critical event in the spatial control of that process. We demonstrate that centrioles promote PLK4 activation through its recruitment and local accumulation. Though centriole removal reduces the proportion of active PLK4, this is rescued by concentrating PLK4 to the peroxisome lumen. Moreover, while mild overexpression of PLK4 only triggers centriole amplification at the existing centriole, higher PLK4 levels trigger both centriolar and cytoplasmatic (de novo) biogenesis. Hence, centrioles promote their assembly locally and disfavor de novo synthesis. Similar mechanisms enforcing the local concentration and/or activity of other centriole components are likely to contribute to the spatial control of centriole biogenesis under physiological conditions. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Respuesta de frijol al Endospor 33® a dosis 50% de fertilizante nitrogenado/fosfatado en agricultura protegida

    Directory of Open Access Journals (Sweden)

    Juan Manuel Sanchez-Yañez

    2014-01-01

    Full Text Available El frijol requiere fertilización nitrogenada y fosfatada (FNP, cuya indiscriminada aplicación causa pérdida de productividad del suelo, una alternativa de solución para este problema es reducir y optimizar la dosis de FNP con un inoculante mixto con bacterias promotoras de crecimiento vegetal (BPCV y hongos micorricicos vesículo arbusculares (HMA. El objetivo de esta investigación fue analizar la respuesta del frijol al inoculante mixto Endospor 33® a dosis reducida al 50% del FNP. En un suelo ferralítico rojo pobre de Nitrógeno (N y materia orgánica. Con un diseño experimental de bloques al azar con 5 tratamientos y 6 repeticiones. Los resultados indican que el porcentaje de germinación del frijol con Endospor 33® a la dosis 20 mg/semilla fue de 100%. A plántula con 10 mg/planta, tuvo 6,13g de peso fresco total (PFT y de 0,66g de peso seco total (PST comparado con el frijol control relativo (CR con 0,28g de PST con el 100% del FNP. A floración con 30 mg/planta Endospore 33® tuvo 1,5g de PST comparado con 1,07g de PST del frijol CR. A madurez fisiológica con 30mg/planta registro 34,83g/100 semillas, en contraste a su homologo CR con 20,39g/100 semillas. Lo anterior indica que este inoculante mixto es una opción en la producción del frijol a dosis reducida del FNP, sin afectar negativamente su crecimiento y/o rendimiento.

  9. Role of AAA(+)-proteins in peroxisome biogenesis and function.

    Science.gov (United States)

    Grimm, Immanuel; Erdmann, Ralf; Girzalsky, Wolfgang

    2016-05-01

    Mutations in the PEX1 gene, which encodes a protein required for peroxisome biogenesis, are the most common cause of the Zellweger spectrum diseases. The recognition that Pex1p shares a conserved ATP-binding domain with p97 and NSF led to the discovery of the extended family of AAA+-type ATPases. So far, four AAA+-type ATPases are related to peroxisome function. Pex6p functions together with Pex1p in peroxisome biogenesis, ATAD1/Msp1p plays a role in membrane protein targeting and a member of the Lon-family of proteases is associated with peroxisomal quality control. This review summarizes the current knowledge on the AAA+-proteins involved in peroxisome biogenesis and function.

  10. Genome Sequence of Aeribacillus pallidus Strain GS3372, an Endospore-Forming Bacterium Isolated in a Deep Geothermal Reservoir

    OpenAIRE

    Sevasti Filippidou; Marion Jaussi; Thomas Junier; Tina Wunderlin; Nicole Jeanneret; Simona Regenspurg; Po-E Li; Chien-Chi Lo; Shannon Johnson; Kim McMurry; Cheryl D. Gleasner; Momchilo Vuyisich; Patrick S. Chain; Pilar Junier

    2015-01-01

    The genome of strain GS3372 is the first publicly available strain of Aeribacillus pallidus. This endospore-forming thermophilic strain was isolated from a deep geothermal reservoir. The availability of this genome can contribute to the clarification of the taxonomy of the closely related Anoxybacillus, Geobacillus, and Aeribacillus genera.

  11. 14,15-EET promotes mitochondrial biogenesis and protects cortical neurons against oxygen/glucose deprivation-induced apoptosis.

    Science.gov (United States)

    Wang, Lai; Chen, Man; Yuan, Lin; Xiang, Yuting; Zheng, Ruimao; Zhu, Shigong

    2014-07-18

    14,15-Epoxyeicosatrienoic acid (14,15-EET), a metabolite of arachidonic acid, is enriched in the brain cortex and exerts protective effect against neuronal apoptosis induced by ischemia/reperfusion. Although apoptosis has been well recognized to be closely associated with mitochondrial biogenesis and function, it is still unclear whether the neuroprotective effect of 14,15-EET is mediated by promotion of mitochondrial biogenesis and function in cortical neurons under the condition of oxygen-glucose deprivation (OGD). In this study, we found that 14,15-EET improved cell viability and inhibited apoptosis of cortical neurons. 14,15-EET significantly increased the mitochondrial mass and the ratio of mitochondrial DNA to nuclear DNA. Key makers of mitochondrial biogenesis, peroxisome proliferator activator receptor gamma-coactivator 1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM), were elevated at both mRNA and protein levels in the cortical neurons treated with 14,15-EET. Moreover, 14,15-EET markedly attenuated the decline of mitochondrial membrane potential, reduced ROS, while increased ATP synthesis. Knockdown of cAMP-response element binding protein (CREB) by siRNA blunted the up-regulation of PGC-1α and NRF-1 stimulated by 14,15-EET, and consequently abolished the neuroprotective effect of 14,15-EET. Our results indicate that 14,15-EET protects neurons from OGD-induced apoptosis by promoting mitochondrial biogenesis and function through CREB mediated activation of PGC-1α and NRF-1. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Cilostazol promotes mitochondrial biogenesis in human umbilical vein endothelial cells through activating the expression of PGC-1α

    International Nuclear Information System (INIS)

    Zuo, Luning; Li, Qiang; Sun, Bei; Xu, Zhiying; Ge, Zhiming

    2013-01-01

    Highlights: ► First time to show that cilostazol promotes the expressions of PGC-1α. ► First time to show that cilostazol stimulates mitochondrial biogenesis in HUVECs. ► PKA/CREB pathway mediates the effect of cilostazol on PGC-1α expression. ► Suggesting the roles of cilostazol in mitochondrial dysfunction related disease. -- Abstract: Mitochondrial dysfunction is frequently observed in vascular diseases. Cilostazol is a drug approved by the US Food and Drug Administration for the treatment of intermittent claudication. Cilostazol increases intracellular cyclic adenosine monophosphate (cAMP) levels through inhibition of type III phosphodiesterase. The effects of cilostazol in mitochondrial biogenesis in human umbilical vein endothelial cells (HUVECs) were investigated in this study. Cilostazol treated HUVECs displayed increased levels of ATP, mitochondrial DNA/nuclear DNA ratio, expressions of cytochrome B, and mitochondrial mass, suggesting an enhanced mitochondrial biogenesis induced by cilostazol. The promoted mitochondrial biogenesis could be abolished by Protein kinase A (PKA) specific inhibitor H-89, implying that PKA pathway played a critical role in increased mitochondrial biogenesis after cilostazol treatment. Indeed, expression levels of peroxisome proliferator activator receptor gamma-coactivator 1α (PGC-1α), NRF 1 and mitochondrial transcription factor A (TFAM) were significantly increased in HUVECs after incubation with cilostazol at both mRNA levels and protein levels. Importantly, knockdown of PGC-1α could abolish cilostazol-induced mitochondrial biogenesis. Enhanced expression of p-CREB and PGC-1α induced by cilostazol could be inhibited by H-89. Moreover, the increased expression of PGC-1α induced by cilostazol could be inhibited by downregulation of CREB using CREB siRNA at both mRNA and protein levels. All the results indicated that cilostazol promoted mitochondrial biogenesis through activating the expression of PGC-1α in

  13. Detection of Bacterial Endospores in Soil by Terbium Fluorescence

    Directory of Open Access Journals (Sweden)

    Andrea Brandes Ammann

    2011-01-01

    Full Text Available Spore formation is a survival mechanism of microorganisms when facing unfavorable environmental conditions resulting in “dormant” states. We investigated the occurrence of bacterial endospores in soils from various locations including grasslands (pasture, meadow, allotment gardens, and forests, as well as fluvial sediments. Bacterial spores are characterized by their high content of dipicolinic acid (DPA. In the presence of terbium, DPA forms a complex showing a distinctive photoluminescence spectrum. DPA was released from soil by microwaving or autoclaving. The addition of aluminium chloride reduced signal quenching by interfering compounds such as phosphate. The highest spore content (up to 109 spores per gram of dry soil was found in grassland soils. Spore content is related to soil type, to soil depth, and to soil carbon-to-nitrogen ratio. Our study might provide a basis for the detection of “hot spots” of bacterial spores in soil.

  14. Repositioning of antibiotic levofloxacin as a mitochondrial biogenesis inhibitor to target breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Min [Galactophore Department, JingZhou Central Hospital, JingZhou (China); Li, Ruishu, E-mail: liruishu2016@yahoo.com [Forensic Surgery Department, JingZhou Traditional Chinese Medicine Hospital, JingZhou (China); Zhang, Juan [Endocrinology Department, JingZhou Central Hospital, JingZhou (China)

    2016-03-18

    Targeting mitochondrial biogenesis has become a potential therapeutic strategy in cancer due to their unique metabolic dependencies. In this study, we show that levofloxacin, a FDA-approved antibiotic, is an attractive candidate for breast cancer treatment. This is achieved by the inhibition of proliferation and induction of apoptosis in a panel of breast cancer cell lines while sparing normal breast cells. It also acts synergistically with conventional chemo drug in two independent in vivo breast xenograft mouse models. Importantly, levofloxacin inhibits mitochondrial biogenesis as shown by the decreased level of mitochondrial respiration, membrane potential and ATP. In addition, the anti-proliferative and pro-apoptotic effects of levofloxacin are reversed by acetyl-L-Carnitine (ALCAR, a mitochondrial fuel), confirming that levofloxacin's action in breast cancer cells is through inhibition of mitochondrial biogenesis. A consequence of mitochondrial biogenesis inhibition by levofloxacin in breast cancer cells is the deactivation of PI3K/Akt/mTOR and MAPK/ERK pathways. We further demonstrate that breast cancer cells have increased mitochondrial biogenesis than normal breast cells, and this explains their different sensitivity to levofloxacin. Our work suggest that levofloxacin is a useful addition to breast cancer treatment. Our work also establish the essential role of mitochondrial biogenesis on the activation of PI3K/Akt/mTOR and MAPK/ERK pathways in breast cancer cells. - Highlights: • Levofloxacin targets a panel of breast cancer cell lines in vitro and in vivo. • Levofloxacin acts synergistically with 5-Fluorouracil in breast cancer. • Levofloxacin targets breast cancer cells via inhibiting mitochondrial biogenesis. • Breast cancer cells have increased mitochondrial biogenesis than normal cells. • Mitochondrial biogenesis inhibition lead to deactivation of PI3K/Akt/mTOR pathway.

  15. Repositioning of antibiotic levofloxacin as a mitochondrial biogenesis inhibitor to target breast cancer

    International Nuclear Information System (INIS)

    Yu, Min; Li, Ruishu; Zhang, Juan

    2016-01-01

    Targeting mitochondrial biogenesis has become a potential therapeutic strategy in cancer due to their unique metabolic dependencies. In this study, we show that levofloxacin, a FDA-approved antibiotic, is an attractive candidate for breast cancer treatment. This is achieved by the inhibition of proliferation and induction of apoptosis in a panel of breast cancer cell lines while sparing normal breast cells. It also acts synergistically with conventional chemo drug in two independent in vivo breast xenograft mouse models. Importantly, levofloxacin inhibits mitochondrial biogenesis as shown by the decreased level of mitochondrial respiration, membrane potential and ATP. In addition, the anti-proliferative and pro-apoptotic effects of levofloxacin are reversed by acetyl-L-Carnitine (ALCAR, a mitochondrial fuel), confirming that levofloxacin's action in breast cancer cells is through inhibition of mitochondrial biogenesis. A consequence of mitochondrial biogenesis inhibition by levofloxacin in breast cancer cells is the deactivation of PI3K/Akt/mTOR and MAPK/ERK pathways. We further demonstrate that breast cancer cells have increased mitochondrial biogenesis than normal breast cells, and this explains their different sensitivity to levofloxacin. Our work suggest that levofloxacin is a useful addition to breast cancer treatment. Our work also establish the essential role of mitochondrial biogenesis on the activation of PI3K/Akt/mTOR and MAPK/ERK pathways in breast cancer cells. - Highlights: • Levofloxacin targets a panel of breast cancer cell lines in vitro and in vivo. • Levofloxacin acts synergistically with 5-Fluorouracil in breast cancer. • Levofloxacin targets breast cancer cells via inhibiting mitochondrial biogenesis. • Breast cancer cells have increased mitochondrial biogenesis than normal cells. • Mitochondrial biogenesis inhibition lead to deactivation of PI3K/Akt/mTOR pathway.

  16. The Effects of NAD+ on Apoptotic Neuronal Death and Mitochondrial Biogenesis and Function after Glutamate Excitotoxicity

    Science.gov (United States)

    Wang, Xiaowan; Li, Hailong; Ding, Shinghua

    2014-01-01

    NAD+ is an essential co-enzyme for cellular energy metabolism and is also involved as a substrate for many cellular enzymatic reactions. It has been shown that NAD+ has a beneficial effect on neuronal survival and brain injury in in vitro and in vivo ischemic models. However, the effect of NAD+ on mitochondrial biogenesis and function in ischemia has not been well investigated. In the present study, we used an in vitro glutamate excitotoxicity model of primary cultured cortical neurons to study the effect of NAD+ on apoptotic neuronal death and mitochondrial biogenesis and function. Our results show that supplementation of NAD+ could effectively reduce apoptotic neuronal death, and apoptotic inducing factor translocation after neurons were challenged with excitotoxic glutamate stimulation. Using different approaches including confocal imaging, mitochondrial DNA measurement and Western blot analysis of PGC-1 and NRF-1, we also found that NAD+ could significantly attenuate glutamate-induced mitochondrial fragmentation and the impairment of mitochondrial biogenesis. Furthermore, NAD+ treatment effectively inhibited mitochondrial membrane potential depolarization and NADH redistribution after excitotoxic glutamate stimulation. Taken together, our results demonstrated that NAD+ is capable of inhibiting apoptotic neuronal death after glutamate excitotoxicity via preserving mitochondrial biogenesis and integrity. Our findings provide insights into potential neuroprotective strategies in ischemic stroke. PMID:25387075

  17. Regulatory Multidimensionality of Gas Vesicle Biogenesis in Halobacterium salinarum NRC-1

    Directory of Open Access Journals (Sweden)

    Andrew I. Yao

    2011-01-01

    Full Text Available It is becoming clear that the regulation of gas vesicle biogenesis in Halobacterium salinarum NRC-1 is multifaceted and appears to integrate environmental and metabolic cues at both the transcriptional and posttranscriptional levels. The mechanistic details underlying this process, however, remain unclear. In this manuscript, we quantify the contribution of light scattering made by both intracellular and released gas vesicles isolated from Halobacterium salinarum NRC-1, demonstrating that each form can lead to distinct features in growth curves determined by optical density measured at 600 nm (OD600. In the course of the study, we also demonstrate the sensitivity of gas vesicle accumulation in Halobacterium salinarum NRC-1 on small differences in growth conditions and reevaluate published works in the context of our results to present a hypothesis regarding the roles of the general transcription factor tbpD and the TCA cycle enzyme aconitase on the regulation of gas vesicle biogenesis.

  18. Genome Sequence of Aeribacillus pallidus Strain GS3372, an Endospore-Forming Bacterium Isolated in a Deep Geothermal Reservoir.

    Science.gov (United States)

    Filippidou, Sevasti; Jaussi, Marion; Junier, Thomas; Wunderlin, Tina; Jeanneret, Nicole; Regenspurg, Simona; Li, Po-E; Lo, Chien-Chi; Johnson, Shannon; McMurry, Kim; Gleasner, Cheryl D; Vuyisich, Momchilo; Chain, Patrick S; Junier, Pilar

    2015-08-27

    The genome of strain GS3372 is the first publicly available strain of Aeribacillus pallidus. This endospore-forming thermophilic strain was isolated from a deep geothermal reservoir. The availability of this genome can contribute to the clarification of the taxonomy of the closely related Anoxybacillus, Geobacillus, and Aeribacillus genera. Copyright © 2015 Filippidou et al.

  19. The miRNA biogenesis in marine bivalves

    Directory of Open Access Journals (Sweden)

    Umberto Rosani

    2016-03-01

    Full Text Available Small non-coding RNAs include powerful regulators of gene expression, transposon mobility and virus activity. Among the various categories, mature microRNAs (miRNAs guide the translational repression and decay of several targeted mRNAs. The biogenesis of miRNAs depends on few gene products, essentially conserved from basal to higher metazoans, whose protein domains allow specific interactions with dsRNA. Here, we report the identification of key genes responsible of the miRNA biogenesis in 32 bivalves, with particular attention to the aquaculture species Mytilus galloprovincialis and Crassostrea gigas. In detail, we have identified and phylogenetically compared eight evolutionary conserved proteins: DROSHA, DGCR8, EXP5, RAN, DICER TARBP2, AGO and PIWI. In mussels, we recognized several other proteins participating in the miRNA biogenesis or in the subsequent RNA silencing. According to digital expression analysis, these genes display low and not inducible expression levels in adult mussels and oysters whereas they are considerably expressed during development. As miRNAs play an important role also in the antiviral responses, knowledge on their production and regulative effects can shed light on essential molecular processes and provide new hints for disease prevention in bivalves.

  20. Impaired TFEB-mediated Lysosome Biogenesis and Autophagy Promote Chronic Ethanol-induced Liver Injury and Steatosis in Mice.

    Science.gov (United States)

    Chao, Xiaojuan; Wang, Shaogui; Zhao, Katrina; Li, Yuan; Williams, Jessica A; Li, Tiangang; Chavan, Hemantkumar; Krishnamurthy, Partha; He, Xi C; Li, Linheng; Ballabio, Andrea; Ni, Hong-Min; Ding, Wen-Xing

    2018-05-18

    Defects in lysosome function and autophagy contribute to pathogenesis of alcoholic liver disease. We investigated the mechanisms by which alcohol consumption affects these processes, evaluating the functions transcription factor EB (TFEB), which regulates lysosomal biogenesis. We performed studies with GFP-LC3 mice, mice with liver-specific deletion of transcription factor EB (TFEB), mice with disruption of the transcription factor E3 gene (TFE3-knockout mice), mice with disruption of the Tefb and Tfe3 genes (TFEB, TFE3 double-knockout mice), and Tfeb flox/flox albumin cre-negative mice (controls). TFEB was overexpressed from adenoviral vectors or knocked down with small interfering RNAs in mouse livers. Mice were placed on diets of chronic ethanol feeding plus an acute binge to induce liver damage (ethanol diet); some mice were also given injections of torin1, an inhibitor of the kinase activity of the mechanistic target of rapamycin (mTOR). Liver tissues were collected and analyzed by immunohistochemistry, immunoblots, and quantitative real-time PCR to monitor lysosome biogenesis. We analyzed levels of TFEB in liver tissues from patients with alcoholic hepatitis and from healthy donors (controls) by immunohistochemistry. Liver tissues from mice on the ethanol diet had lower levels of total and nuclear TFEB, compared with control mice, and hepatocytes had reduced lysosome biogenesis and autophagy. Hepatocytes from mice on the ethanol diet had increased translocation of mTOR into lysosomes, resulting increased mTOR activation. Administration of torin1 increased liver levels of TFEB and reduced steatosis and liver injury induced by ethanol. Mice that overexpressed TFEB in liver developed less-severe ethanol-induced liver injury and had increased lysosomal biogenesis and mitochondrial bioenergetics compared to mice carrying a control vector. Mice with knockdown of TFEB, as well as TFEB, TFE3 double-knockout mice, developed more severe liver injury in response to the

  1. Evolutionary Conservation of the Ribosomal Biogenesis Factor Rbm19/Mrd1: Implications for Function

    OpenAIRE

    Kallberg, Yvonne; Segerstolpe, Åsa; Lackmann, Fredrik; Persson, Bengt; Wieslander, Lars

    2012-01-01

    Ribosome biogenesis in eukaryotes requires coordinated folding and assembly of a pre-rRNA into sequential pre-rRNA-protein complexes in which chemical modifications and RNA cleavages occur. These processes require many small nucleolar RNAs (snoRNAs) and proteins. Rbm19/Mrd1 is one such protein that is built from multiple RNA-binding domains (RBDs). We find that Rbm19/Mrd1 with five RBDs is present in all branches of the eukaryotic phylogenetic tree, except in animals and Choanoflagellates, th...

  2. Suboptimal T-cell receptor signaling compromises protein translation, ribosome biogenesis, and proliferation of mouse CD8 T cells.

    Science.gov (United States)

    Tan, Thomas C J; Knight, John; Sbarrato, Thomas; Dudek, Kate; Willis, Anne E; Zamoyska, Rose

    2017-07-25

    Global transcriptomic and proteomic analyses of T cells have been rich sources of unbiased data for understanding T-cell activation. Lack of full concordance of these datasets has illustrated that important facets of T-cell activation are controlled at the level of translation. We undertook translatome analysis of CD8 T-cell activation, combining polysome profiling and microarray analysis. We revealed that altering T-cell receptor stimulation influenced recruitment of mRNAs to heavy polysomes and translation of subsets of genes. A major pathway that was compromised, when TCR signaling was suboptimal, was linked to ribosome biogenesis, a rate-limiting factor in both cell growth and proliferation. Defective TCR signaling affected transcription and processing of ribosomal RNA precursors, as well as the translation of specific ribosomal proteins and translation factors. Mechanistically, IL-2 production was compromised in weakly stimulated T cells, affecting the abundance of Myc protein, a known regulator of ribosome biogenesis. Consequently, weakly activated T cells showed impaired production of ribosomes and a failure to maintain proliferative capacity after stimulation. We demonstrate that primary T cells respond to various environmental cues by regulating ribosome biogenesis and mRNA translation at multiple levels to sustain proliferation and differentiation.

  3. Meloidogyne incognita Fatty Acid- and Retinol- Binding Protein (Mi-FAR-1) Affects Nematode Infection of Plant Roots and the Attachment of Pasteuria penetrans Endospores.

    Science.gov (United States)

    Phani, Victor; Shivakumara, Tagginahalli N; Davies, Keith G; Rao, Uma

    2017-01-01

    Root-knot nematode (RKN) Meloidogyne incognita is an economically important pest of crops. Pasteuria penetrans , is a nematode hyperparasitic bacterium capable of suppressing the reproduction of RKN and thereby useful for its management. Secreted fatty acid and retinol-binding proteins are unique in nematodes and are engaged in nutrient acquisition, development and reproduction; they are also a component of the nematode cuticle and thought to be involved in the interface between hosts and parasites. Attachment of endospores to the cuticle of second stage juveniles of RKN is the primary step of infection and several factors have been identified to facilitate attachment. In this study, the full length of Mi-far-1 (573 bp) was cloned from M. incognita and characterized. Analysis revealed that the Mi-far-1 was rich in α-helix structure, contained a predicted consensus casein kinase II phosphorylation site and a glycosylation site. Quantitative PCR showed the highest expression in the fourth stage juveniles and in situ hybridization revealed the presence of Mi-far-1 mRNA in the hypodermis below the cuticle. Single copy insertion pattern of Mi-far-1 in M. incognita genome was detected by Southern blotting. Knockdown of Mi-far-1 showed significantly increased attachment of P. penetrans' endospores on juvenile cuticle surface and also affected host finding, root infection and nematode fecundity.

  4. Meloidogyne incognita Fatty Acid- and Retinol- Binding Protein (Mi-FAR-1 Affects Nematode Infection of Plant Roots and the Attachment of Pasteuria penetrans Endospores

    Directory of Open Access Journals (Sweden)

    Victor Phani

    2017-11-01

    Full Text Available Root-knot nematode (RKN Meloidogyne incognita is an economically important pest of crops. Pasteuria penetrans, is a nematode hyperparasitic bacterium capable of suppressing the reproduction of RKN and thereby useful for its management. Secreted fatty acid and retinol-binding proteins are unique in nematodes and are engaged in nutrient acquisition, development and reproduction; they are also a component of the nematode cuticle and thought to be involved in the interface between hosts and parasites. Attachment of endospores to the cuticle of second stage juveniles of RKN is the primary step of infection and several factors have been identified to facilitate attachment. In this study, the full length of Mi-far-1 (573 bp was cloned from M. incognita and characterized. Analysis revealed that the Mi-far-1 was rich in α-helix structure, contained a predicted consensus casein kinase II phosphorylation site and a glycosylation site. Quantitative PCR showed the highest expression in the fourth stage juveniles and in situ hybridization revealed the presence of Mi-far-1 mRNA in the hypodermis below the cuticle. Single copy insertion pattern of Mi-far-1 in M. incognita genome was detected by Southern blotting. Knockdown of Mi-far-1 showed significantly increased attachment of P. penetrans’ endospores on juvenile cuticle surface and also affected host finding, root infection and nematode fecundity.

  5. Potential roles for ubiquitin and the proteasome during ribosome biogenesis

    Czech Academy of Sciences Publication Activity Database

    Stavreva, D. A.; Kawasaki, M.; Dundr, M.; Koberna, Karel; Müller, W. G.; Tsujimura-Takahashi, T.; Komatsu, W.; Hayano, T.; Isobe, T.; Raška, Ivan; Misteli, T.; Takahashi, N.; McNally, J. G.

    2006-01-01

    Roč. 26, č. 13 (2006), s. 5131-5145 ISSN 0270-7306 R&D Projects: GA MŠk(CZ) LC535; GA ČR(CZ) GA304/05/0374; GA ČR(CZ) GA304/04/0692 Grant - others:NIH(US) Intramural Research Program; Ministry of Education(JP) Pioneer Research grant Institutional research plan: CEZ:AV0Z50110509 Keywords : the role of the ubikvitin * proteasome system in ribosome biogenesis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.773, year: 2006

  6. Genome-wide analysis of effectors of peroxisome biogenesis.

    Directory of Open Access Journals (Sweden)

    Ramsey A Saleem

    2010-08-01

    Full Text Available Peroxisomes are intracellular organelles that house a number of diverse metabolic processes, notably those required for beta-oxidation of fatty acids. Peroxisomes biogenesis can be induced by the presence of peroxisome proliferators, including fatty acids, which activate complex cellular programs that underlie the induction process. Here, we used multi-parameter quantitative phenotype analyses of an arrayed mutant collection of yeast cells induced to proliferate peroxisomes, to establish a comprehensive inventory of genes required for peroxisome induction and function. The assays employed include growth in the presence of fatty acids, and confocal imaging and flow cytometry through the induction process. In addition to the classical phenotypes associated with loss of peroxisomal functions, these studies identified 169 genes required for robust signaling, transcription, normal peroxisomal development and morphologies, and transmission of peroxisomes to daughter cells. These gene products are localized throughout the cell, and many have indirect connections to peroxisome function. By integration with extant data sets, we present a total of 211 genes linked to peroxisome biogenesis and highlight the complex networks through which information flows during peroxisome biogenesis and function.

  7. Proteome distribution between nucleoplasm and nucleolus and its relation to ribosome biogenesis in Arabidopsis thaliana.

    Science.gov (United States)

    Palm, Denise; Simm, Stefan; Darm, Katrin; Weis, Benjamin L; Ruprecht, Maike; Schleiff, Enrico; Scharf, Christian

    2016-01-01

    Ribosome biogenesis is an essential process initiated in the nucleolus. In eukaryotes, multiple ribosome biogenesis factors (RBFs) can be found in the nucleolus, the nucleus and in the cytoplasm. They act in processing, folding and modification of the pre-ribosomal (r)RNAs, incorporation of ribosomal proteins (RPs), export of pre-ribosomal particles to the cytoplasm, and quality control mechanisms. Ribosome biogenesis is best established for Saccharomyces cerevisiae. Plant ortholog assignment to yeast RBFs revealed the absence of about 30% of the yeast RBFs in plants. In turn, few plant specific proteins have been identified by biochemical experiments to act in plant ribosome biogenesis. Nevertheless, a complete inventory of plant RBFs has not been established yet. We analyzed the proteome of the nucleus and nucleolus of Arabidopsis thaliana and the post-translational modifications of these proteins. We identified 1602 proteins in the nucleolar and 2544 proteins in the nuclear fraction with an overlap of 1429 proteins. For a randomly selected set of proteins identified by the proteomic approach we confirmed the localization inferred from the proteomics data by the localization of GFP fusion proteins. We assigned the identified proteins to various complexes and functions and found about 519 plant proteins that have a potential to act as a RBFs, but which have not been experimentally characterized yet. Last, we compared the distribution of RBFs and RPs in the various fractions with the distribution established for yeast.

  8. Role of ATP in the regulation of cholesterol biogenesis

    International Nuclear Information System (INIS)

    Subba Rao, G.; Ramasarma, T.

    1974-01-01

    Intraperitoneal administration of glucose (4oomg/rat) stimulated the biogenesis of sterols in starved rats while citrate or pyruvate (20mg/rat) did not have any effect. ATP (10mg/ rat) administered intraperitoneally stimulated the incorporation of acetate-1- 14 C into sterols but not of mevalonate-2- 14 C into sterols in starved rats. The results indicate that ATP may play a role in regulating cholesterol biogenesis and it is not acting merely as an energy source. (author)

  9. Augmentation of aerobic respiration and mitochondrial biogenesis in skeletal muscle by hypoxia preconditioning with cobalt chloride

    Energy Technology Data Exchange (ETDEWEB)

    Saxena, Saurabh [Experimental Biology Division, Defence Institute of Physiology and Allied Sciences, Lucknow Road, Timarpur, Delhi, 110054 (India); Shukla, Dhananjay [Department of Biotechnology, Gitam University, Gandhi Nagar, Rushikonda, Visakhapatnam-530 045 Andhra Pradesh (India); Bansal, Anju, E-mail: anjubansaldipas@gmail.com [Experimental Biology Division, Defence Institute of Physiology and Allied Sciences, Lucknow Road, Timarpur, Delhi, 110054 (India)

    2012-11-01

    High altitude/hypoxia training is known to improve physical performance in athletes. Hypoxia induces hypoxia inducible factor-1 (HIF-1) and its downstream genes that facilitate hypoxia adaptation in muscle to increase physical performance. Cobalt chloride (CoCl{sub 2}), a hypoxia mimetic, stabilizes HIF-1, which otherwise is degraded in normoxic conditions. We studied the effects of hypoxia preconditioning by CoCl{sub 2} supplementation on physical performance, glucose metabolism, and mitochondrial biogenesis using rodent model. The results showed significant increase in physical performance in cobalt supplemented rats without (two times) or with training (3.3 times) as compared to control animals. CoCl{sub 2} supplementation in rats augmented the biological activities of enzymes of TCA cycle, glycolysis and cytochrome c oxidase (COX); and increased the expression of glucose transporter-1 (Glut-1) in muscle showing increased glucose metabolism by aerobic respiration. There was also an increase in mitochondrial biogenesis in skeletal muscle observed by increased mRNA expressions of mitochondrial biogenesis markers which was further confirmed by electron microscopy. Moreover, nitric oxide production increased in skeletal muscle in cobalt supplemented rats, which seems to be the major reason for peroxisome proliferator activated receptor-gamma coactivator-1α (PGC-1α) induction and mitochondrial biogenesis. Thus, in conclusion, we state that hypoxia preconditioning by CoCl{sub 2} supplementation in rats increases mitochondrial biogenesis, glucose uptake and metabolism by aerobic respiration in skeletal muscle, which leads to increased physical performance. The significance of this study lies in understanding the molecular mechanism of hypoxia adaptation and improvement of work performance in normal as well as extreme conditions like hypoxia via hypoxia preconditioning. -- Highlights: ► We supplemented rats with CoCl{sub 2} for 15 days along with training. ► Co

  10. Augmentation of aerobic respiration and mitochondrial biogenesis in skeletal muscle by hypoxia preconditioning with cobalt chloride

    International Nuclear Information System (INIS)

    Saxena, Saurabh; Shukla, Dhananjay; Bansal, Anju

    2012-01-01

    High altitude/hypoxia training is known to improve physical performance in athletes. Hypoxia induces hypoxia inducible factor-1 (HIF-1) and its downstream genes that facilitate hypoxia adaptation in muscle to increase physical performance. Cobalt chloride (CoCl 2 ), a hypoxia mimetic, stabilizes HIF-1, which otherwise is degraded in normoxic conditions. We studied the effects of hypoxia preconditioning by CoCl 2 supplementation on physical performance, glucose metabolism, and mitochondrial biogenesis using rodent model. The results showed significant increase in physical performance in cobalt supplemented rats without (two times) or with training (3.3 times) as compared to control animals. CoCl 2 supplementation in rats augmented the biological activities of enzymes of TCA cycle, glycolysis and cytochrome c oxidase (COX); and increased the expression of glucose transporter-1 (Glut-1) in muscle showing increased glucose metabolism by aerobic respiration. There was also an increase in mitochondrial biogenesis in skeletal muscle observed by increased mRNA expressions of mitochondrial biogenesis markers which was further confirmed by electron microscopy. Moreover, nitric oxide production increased in skeletal muscle in cobalt supplemented rats, which seems to be the major reason for peroxisome proliferator activated receptor-gamma coactivator-1α (PGC-1α) induction and mitochondrial biogenesis. Thus, in conclusion, we state that hypoxia preconditioning by CoCl 2 supplementation in rats increases mitochondrial biogenesis, glucose uptake and metabolism by aerobic respiration in skeletal muscle, which leads to increased physical performance. The significance of this study lies in understanding the molecular mechanism of hypoxia adaptation and improvement of work performance in normal as well as extreme conditions like hypoxia via hypoxia preconditioning. -- Highlights: ► We supplemented rats with CoCl 2 for 15 days along with training. ► CoCl 2 supplementation

  11. Elucidation of the therapeutic role of mitochondrial biogenesis transducers NRF-1 in the regulation of renal fibrosis

    Energy Technology Data Exchange (ETDEWEB)

    Hsieh, Pei-Fang [Graduate Institute of Biomedical Science, National Sun Yat-Sen University, Kaohsiung, Taiwan (China); Graduate Institute of Medical Laboratory Science and Biotechnology, Chung Hwa University of Medical Technology, Tainan, Taiwan (China); Liu, Shu-Fen [Department of Internal Medicine, Kaohsiung Medical University Chung-Ho Memorial Hospital, Kaohsiung, Taiwan (China); Hung, Tsung-Jen [Graduate Institute of Biomedical Science, Chung Hwa University of Medical Technology, Tainan, Taiwan (China); Hung, Chien-Ya [Department of Food Nutrition, Chung Hwa University of Medical Technology, Tainan, Taiwan (China); Liu, Guo-Zheng [Graduate Institute of Medical Laboratory Science and Biotechnology, Chung Hwa University of Medical Technology, Tainan, Taiwan (China); Chuang, Lea-Yea [Department of Biochemistry, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Chen, Mei-Fen [Department of Acupressure Technology, Chung Hwa University of Medical Technology, Tainan, Taiwan (China); Wang, Jue-Long [Department of Physical Medicine and Rehabilitation, Kaohsiung Veterans General Hospital, Taiwan (China); Shi, Ming-Der [Graduate Institute of Medical Laboratory Science and Biotechnology, Chung Hwa University of Medical Technology, Tainan, Taiwan (China); Department of Medical Technology, Kaohsiung Veterans General Hospital Tainan Branch, Tainan, Taiwan (China); Hsu, Chen Hung [Department of Biological Science and Technology, Chung Hwa University of Medical Technology, Tainan, Taiwan (China); Shiue, Yow-Ling, E-mail: shiue.shirley@gmail.com [Graduate Institute of Biomedical Science, National Sun Yat-Sen University, Kaohsiung, Taiwan (China); Yang, Yu-Lin, E-mail: Call0955443221@gmail.com [Graduate Institute of Medical Laboratory Science and Biotechnology, Chung Hwa University of Medical Technology, Tainan, Taiwan (China); Graduate Institute of Biomedical Science, Chung Hwa University of Medical Technology, Tainan, Taiwan (China)

    2016-11-15

    Background: Mitochondrial dysfunction is a newly established risk factor for the development of renal fibrosis. Cell survival and injury repair is facilitated by mitochondrial biogenesis. Nuclear respiratory factor 1 (NRF-1) is a transcriptional regulation factor that plays a central role in the regulation of mitochondrial biogenesis. However, the transcription factor of this process in renal fibrosis is unknown. Thus, we hereby discussed the correlations of NRF-1 and renal interstitial fibrosis. Materials and methods: In vitro fibrosis model was established by treatment with transforming growth factor-β1 (TGF-β1) in NRK-49F (Normal Rat kidney fibroblast). We investigated the ROS production, mitochondrial biogenesis and fibrogenic marker (e.q. fibronectin) during the progression of renal fibrosis by kit and Western blotting assay. Here, we used that two distinct mechanisms regulate NRF-1 activation and degradation of NRF-1. NRF-1 was transfect by pcDNA-NRF-1 overexpression gene to evaluate the NRF-1 activity of the therapeutic effect in renal fibrosis. In addition, NRF-1 was silenced by shRNA-NRF-1 to evaluate the significance of NRF-1. ELISA was used to evaluate the secreted fibronectin. Immunofluorescence staining was used to assay the in situ expression of proteins (e.g. fibronectin, NRF-1). Results: Under renal fibrosis conditions, TGF-β1 (5 ng/ml) increased ROS. Simultaneously, TGF-β1-induced extracellular fibronectin by ELISA assay. In addition, TGF-β1 decreased expression of mitochondrial biogenesis. This is the first time to demonstrate that expression of NRF-1 is significantly decreased in renal fibrosis. However, NRK49F was a transfection with pcDNA-NRF-1 (2 μg/ml) expression vector dramatically reverse TGF-β1-induced cellular fibrosis concomitantly with the suppression of fibronectin (both intracellular and extracellular fibronectin). More importantly, transfection with shRNA-NRF-1 (2 μg/ml) significantly increased the expression of fibronectin

  12. Mitochondrial biogenesis in the pulmonary vasculature during inhalation lung injury and fibrosis

    Science.gov (United States)

    Cell survival and injury repair is facilitated by mitochondrial biogenesis; however, the role of this process in lung repair is unknown. We evaluated mitochondrial biogenesis in the mouse lung in two injuries that cause acute inflammation and in two that cause chronic inflammatio...

  13. Mitochondrial iron-sulfur cluster biogenesis from molecular understanding to clinical disease

    Science.gov (United States)

    Alfadhel, Majid; Nashabat, Marwan; Ali, Qais Abu; Hundallah, Khalid

    2017-01-01

    Iron–sulfur clusters (ISCs) are known to play a major role in various protein functions. Located in the mitochondria, cytosol, endoplasmic reticulum and nucleus, they contribute to various core cellular functions. Until recently, only a few human diseases related to mitochondrial ISC biogenesis defects have been described. Such diseases include Friedreich ataxia, combined oxidative phosphorylation deficiency 19, infantile complex II/III deficiency defect, hereditary myopathy with lactic acidosis and mitochondrial muscle myopathy, lipoic acid biosynthesis defects, multiple mitochondrial dysfunctions syndromes and non ketotic hyperglycinemia due to glutaredoxin 5 gene defect. Disorders of mitochondrial import, export and translation, including sideroblastic anemia with ataxia, EVEN-PLUS syndrome and mitochondrial complex I deficiency due to nucleotide-binding protein-like protein gene defect, have also been implicated in ISC biogenesis defects. With advances in next generation sequencing technologies, more disorders related to ISC biogenesis defects are expected to be elucidated. In this article, we aim to shed the light on mitochondrial ISC biogenesis, related proteins and their function, pathophysiology, clinical phenotypes of related disorders, diagnostic approach, and future implications. PMID:28064324

  14. Diverse Regulators of Human Ribosome Biogenesis Discovered by Changes in Nucleolar Number

    Directory of Open Access Journals (Sweden)

    Katherine I. Farley-Barnes

    2018-02-01

    Full Text Available Ribosome biogenesis is a highly regulated, essential cellular process. Although studies in yeast have established some of the biological principles of ribosome biogenesis, many of the intricacies of its regulation in higher eukaryotes remain unknown. To understand how ribosome biogenesis is globally integrated in human cells, we conducted a genome-wide siRNA screen for regulators of nucleolar number. We found 139 proteins whose depletion changed the number of nucleoli per nucleus from 2–3 to only 1 in human MCF10A cells. Follow-up analyses on 20 hits found many (90% to be essential for the nucleolar functions of rDNA transcription (7, pre-ribosomal RNA (pre-rRNA processing (16, and/or global protein synthesis (14. This genome-wide analysis exploits the relationship between nucleolar number and function to discover diverse cellular pathways that regulate the making of ribosomes and paves the way for further exploration of the links between ribosome biogenesis and human disease.

  15. Validation of a Rapid Bacteria Endospore Enumeration System for Planetary Protection Application

    Science.gov (United States)

    Chen, Fei; Kern, Roger; Kazarians, Gayane; Venkateswaran, Kasthuri

    NASA monitors spacecraft surfaces to assure that the presence of bacterial endospores meets strict criteria at launch, to minimize the risk of inadvertent contamination of the surface of Mars. Currently, the only approved method for enumerating the spores is a culture based assay that requires three days to produce results. In order to meet the demanding schedules of spacecraft assembly, a more rapid spore detection assay is being considered as an alternate method to the NASA standard culture-based assay. The Millipore Rapid Microbiology Detection System (RMDS) has been used successfully for rapid bioburden enumeration in the pharmaceutical and food industries. The RMDS is rapid and simple, shows high sensitivity (to 1 colony forming unit [CFU]/sample), and correlates well with traditional culture-based methods. It combines membrane filtration, adenosine triphosphate (ATP) bioluminescence chemistry, and image analysis based on photon detection with a Charge Coupled Device (CCD) camera. In this study, we have optimized the assay conditions and evaluated the use of the RMDS as a rapid spore detection tool for NASA applications. In order to select for spores, the samples were subjected to a heat shock step before proceeding with the RMDS incubation protocol. Seven species of Bacillus (nine strains) that have been repeatedly isolated from clean room environments were assayed. All strains were detected by the RMDS in 5 hours and these assay times were repeatedly demonstrated along with low image background noise. Validation experiments to compare the Rapid Sore Assay (RSA) and NASA standard assay (NSA) were also performed. The evaluation criteria were modeled after the FDA Guideline of Process Validation, and Analytical Test Methods. This body of research demonstrates that the Rapid Spore Assay (RSA) is quick, and of equivalent sensitivity to the NASA standard assay, potentially reducing the assay time for bacterial endospores from over 72 hours to less than 8 hours

  16. The Complexity of Mitochondrial Complex IV: An Update of Cytochrome c Oxidase Biogenesis in Plants

    Science.gov (United States)

    Mansilla, Natanael; Racca, Sofia; Gras, Diana E.; Gonzalez, Daniel H.

    2018-01-01

    Mitochondrial respiration is an energy producing process that involves the coordinated action of several protein complexes embedded in the inner membrane to finally produce ATP. Complex IV or Cytochrome c Oxidase (COX) is the last electron acceptor of the respiratory chain, involved in the reduction of O2 to H2O. COX is a multimeric complex formed by multiple structural subunits encoded in two different genomes, prosthetic groups (heme a and heme a3), and metallic centers (CuA and CuB). Tens of accessory proteins are required for mitochondrial RNA processing, synthesis and delivery of prosthetic groups and metallic centers, and for the final assembly of subunits to build a functional complex. In this review, we perform a comparative analysis of COX composition and biogenesis factors in yeast, mammals and plants. We also describe possible external and internal factors controlling the expression of structural proteins and assembly factors at the transcriptional and post-translational levels, and the effect of deficiencies in different steps of COX biogenesis to infer the role of COX in different aspects of plant development. We conclude that COX assembly in plants has conserved and specific features, probably due to the incorporation of a different set of subunits during evolution. PMID:29495437

  17. Cholesterol in myelin biogenesis and hypomyelinating disorders.

    Science.gov (United States)

    Saher, Gesine; Stumpf, Sina Kristin

    2015-08-01

    The largest pool of free cholesterol in mammals resides in myelin membranes. Myelin facilitates rapid saltatory impulse propagation by electrical insulation of axons. This function is achieved by ensheathing axons with a tightly compacted stack of membranes. Cholesterol influences myelination at many steps, from the differentiation of myelinating glial cells, over the process of myelin membrane biogenesis, to the functionality of mature myelin. Cholesterol emerged as the only integral myelin component that is essential and rate-limiting for the development of myelin in the central and peripheral nervous system. Moreover, disorders that interfere with sterol synthesis or intracellular trafficking of cholesterol and other lipids cause hypomyelination and neurodegeneration. This review summarizes recent results on the roles of cholesterol in CNS myelin biogenesis in normal development and under different pathological conditions. This article is part of a Special Issue entitled Brain Lipids. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Cholesterol and myelin biogenesis.

    Science.gov (United States)

    Saher, Gesine; Simons, Mikael

    2010-01-01

    Myelin consists of several layers of tightly compacted membranes wrapped around axons in the nervous system. The main function of myelin is to provide electrical insulation around the axon to ensure the rapid propagation of nerve conduction. As the myelinating glia terminally differentiates, they begin to produce myelin membranes on a remarkable scale. This membrane is unique in its composition being highly enriched in lipids, in particular galactosylceramide and cholesterol. In this review we will summarize the role of cholesterol in myelin biogenesis in the central and peripheral nervous system.

  19. Biodiversity of aerobic endospore-forming bacterial species occurring in Yanyanku and Ikpiru, fermented seeds of Hibiscus sabdariffa used to produce food condiments in Benin

    DEFF Research Database (Denmark)

    Agbobatinkpo, Pélagie B.; Thorsen, Line; Nielsen, Dennis Sandris

    2013-01-01

    Yanyanku and Ikpiru made by the fermentation of Malcavene bean (Hibiscus sabdariffa) are used as functional additives for Parkia biglobosa seed fermentations in Benin. A total of 355 aerobic endospore-forming bacteria (AEFB) isolated from Yanyanku and Ikpiru produced in northern and southern Benin...

  20. Microprocessor Activity Controls Differential miRNA Biogenesis In Vivo

    Directory of Open Access Journals (Sweden)

    Thomas Conrad

    2014-10-01

    Full Text Available In miRNA biogenesis, pri-miRNA transcripts are converted into pre-miRNA hairpins. The in vivo properties of this process remain enigmatic. Here, we determine in vivo transcriptome-wide pri-miRNA processing using next-generation sequencing of chromatin-associated pri-miRNAs. We identify a distinctive Microprocessor signature in the transcriptome profile from which efficiency of the endogenous processing event can be accurately quantified. This analysis reveals differential susceptibility to Microprocessor cleavage as a key regulatory step in miRNA biogenesis. Processing is highly variable among pri-miRNAs and a better predictor of miRNA abundance than primary transcription itself. Processing is also largely stable across three cell lines, suggesting a major contribution of sequence determinants. On the basis of differential processing efficiencies, we define functionality for short sequence features adjacent to the pre-miRNA hairpin. In conclusion, we identify Microprocessor as the main hub for diversified miRNA output and suggest a role for uncoupling miRNA biogenesis from host gene expression.

  1. Mitochondrial cytochrome c biogenesis: no longer an enigma.

    Science.gov (United States)

    Babbitt, Shalon E; Sutherland, Molly C; San Francisco, Brian; Mendez, Deanna L; Kranz, Robert G

    2015-08-01

    Cytochromes c (cyt c) and c1 are heme proteins that are essential for aerobic respiration. Release of cyt c from mitochondria is an important signal in apoptosis initiation. Biogenesis of c-type cytochromes involves covalent attachment of heme to two cysteines (at a conserved CXXCH sequence) in the apocytochrome. Heme attachment is catalyzed in most mitochondria by holocytochrome c synthase (HCCS), which is also necessary for the import of apocytochrome c (apocyt c). Thus, HCCS affects cellular levels of cyt c, impacting mitochondrial physiology and cell death. Here, we review the mechanisms of HCCS function and the roles of heme and residues in the CXXCH motif. Additionally, we consider concepts emerging within the two prokaryotic cytochrome c biogenesis pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. MicroRNA-761 regulates mitochondrial biogenesis in mouse skeletal muscle in response to exercise

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yanli [Affiliated Hospital of Hebei Engineering University, Handan, 056002, Hebei (China); Zhao, Chaoxian; Sun, Xuewen [Medical College of Hebei Engineering University, Handan, 056002, Hebei (China); Liu, Zhijun, E-mail: liuzhij1207@163.com [Affiliated Hospital of Hebei Engineering University, Handan, 056002, Hebei (China); Zhang, Jianzhong, E-mail: zhangjianzhong@icdc.cn [National Institute for Communicable Disease Control and Prevention (ICDC), Chinese Center for Disease Control and Prevention (China CDC), Beijing, 102206 (China)

    2015-11-06

    MicroRNAs (miRNAs) have been suggested to play critical roles in skeletal muscle in response to exercise. Previous study has shown that miR-761 was involved in a novel model regulating the mitochondrial network. However, its role in mitochondrial biogenesis remains poorly understood. Therefore, the current study was aimed to examine the effect of miR-761 on mitochondrial biogenesis in skeletal muscle. Real-time quantitative PCR analysis demonstrated that aberrantly expressed miR-761 is involved in exercise activity and miR-761 is decreased by exercise training compared with the sedentary control mice. miR-761 suppresses mitochondrial biogenesis of C{sub 2}C{sub 12} myocytes by targeting the 3′-UTR of peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 (PGC-1α). Overexpression of miR-761 was capable of inhibiting the protein expression levels of PGC-1α. Moreover, miR-761 overexpression suppressed the p38 MAPK signaling pathway and down-regulated the expression of phosphorylated MAPK-activated protein kinase-2 (P-MK2), a downstream kinase of p38 MAPK. The phosphorylation of activating transcription factors 2 (ATF2) that plays a functional role in linking the activation of the p38 MAPK pathway to enhanced transcription of the PGC-1α was also inhibited by the overexpression of miR-761. These findings revealed a novel regulation mechanism for miR-761 in skeletal myocytes, and contributed to a better understanding of the modulation of skeletal muscle in response to exercise. - Highlights: • Endurance exercise decreases miR-761 expression in skeletal muscle. • MiR-761 suppresses mitochondrial biogenesis in C{sub 2}C{sub 12} myocytes. • MiR-761 directly targeted PGC-1α expression. • MiR-761 suppresses p38 MAPK signaling pathways in C{sub 2}C{sub 12} myocytes. • A novel mechanism for miR-761 in skeletal myocytes is demonstrated.

  3. Anaerobic Copper Toxicity and Iron-Sulfur Cluster Biogenesis in Escherichia coli.

    Science.gov (United States)

    Tan, Guoqiang; Yang, Jing; Li, Tang; Zhao, Jin; Sun, Shujuan; Li, Xiaokang; Lin, Chuxian; Li, Jianghui; Zhou, Huaibin; Lyu, Jianxin; Ding, Huangen

    2017-08-15

    While copper is an essential trace element in biology, pollution of groundwater from copper has become a threat to all living organisms. Cellular mechanisms underlying copper toxicity, however, are still not fully understood. Previous studies have shown that iron-sulfur proteins are among the primary targets of copper toxicity in Escherichia coli under aerobic conditions. Here, we report that, under anaerobic conditions, iron-sulfur proteins in E. coli cells are even more susceptible to copper in medium. Whereas addition of 0.2 mM copper(II) chloride to LB (Luria-Bertani) medium has very little or no effect on iron-sulfur proteins in wild-type E. coli cells under aerobic conditions, the same copper treatment largely inactivates iron-sulfur proteins by blocking iron-sulfur cluster biogenesis in the cells under anaerobic conditions. Importantly, proteins that do not have iron-sulfur clusters (e.g., fumarase C and cysteine desulfurase) in E. coli cells are not significantly affected by copper treatment under aerobic or anaerobic conditions, indicating that copper may specifically target iron-sulfur proteins in cells. Additional studies revealed that E. coli cells accumulate more intracellular copper under anaerobic conditions than under aerobic conditions and that the elevated copper content binds to the iron-sulfur cluster assembly proteins IscU and IscA, which effectively inhibits iron-sulfur cluster biogenesis. The results suggest that the copper-mediated inhibition of iron-sulfur proteins does not require oxygen and that iron-sulfur cluster biogenesis is the primary target of anaerobic copper toxicity in cells. IMPORTANCE Copper contamination in groundwater has become a threat to all living organisms. However, cellular mechanisms underlying copper toxicity have not been fully understood up to now. The work described here reveals that iron-sulfur proteins in Escherichia coli cells are much more susceptible to copper in medium under anaerobic conditions than they

  4. Mild mitochondrial uncoupling and calorie restriction increase fasting eNOS, akt and mitochondrial biogenesis.

    Science.gov (United States)

    Cerqueira, Fernanda M; Laurindo, Francisco R M; Kowaltowski, Alicia J

    2011-03-31

    Enhanced mitochondrial biogenesis promoted by eNOS activation is believed to play a central role in the beneficial effects of calorie restriction (CR). Since treatment of mice with dinitrophenol (DNP) promotes health and lifespan benefits similar to those observed in CR, we hypothesized that it could also impact biogenesis. We found that DNP and CR increase citrate synthase activity, PGC-1α, cytochrome c oxidase and mitofusin-2 expression, as well as fasting plasma levels of NO• products. In addition, eNOS and Akt phosphorylation in skeletal muscle and visceral adipose tissue was activated in fasting CR and DNP animals. Overall, our results indicate that systemic mild uncoupling activates eNOS and Akt-dependent pathways leading to mitochondrial biogenesis.

  5. CDK1 Prevents Unscheduled PLK4-STIL Complex Assembly in Centriole Biogenesis.

    Science.gov (United States)

    Zitouni, Sihem; Francia, Maria E; Leal, Filipe; Montenegro Gouveia, Susana; Nabais, Catarina; Duarte, Paulo; Gilberto, Samuel; Brito, Daniela; Moyer, Tyler; Kandels-Lewis, Steffi; Ohta, Midori; Kitagawa, Daiju; Holland, Andrew J; Karsenti, Eric; Lorca, Thierry; Lince-Faria, Mariana; Bettencourt-Dias, Mónica

    2016-05-09

    Centrioles are essential for the assembly of both centrosomes and cilia. Centriole biogenesis occurs once and only once per cell cycle and is temporally coordinated with cell-cycle progression, ensuring the formation of the right number of centrioles at the right time. The formation of new daughter centrioles is guided by a pre-existing, mother centriole. The proximity between mother and daughter centrioles was proposed to restrict new centriole formation until they separate beyond a critical distance. Paradoxically, mother and daughter centrioles overcome this distance in early mitosis, at a time when triggers for centriole biogenesis Polo-like kinase 4 (PLK4) and its substrate STIL are abundant. Here we show that in mitosis, the mitotic kinase CDK1-CyclinB binds STIL and prevents formation of the PLK4-STIL complex and STIL phosphorylation by PLK4, thus inhibiting untimely onset of centriole biogenesis. After CDK1-CyclinB inactivation upon mitotic exit, PLK4 can bind and phosphorylate STIL in G1, allowing pro-centriole assembly in the subsequent S phase. Our work shows that complementary mechanisms, such as mother-daughter centriole proximity and CDK1-CyclinB interaction with centriolar components, ensure that centriole biogenesis occurs once and only once per cell cycle, raising parallels to the cell-cycle regulation of DNA replication and centromere formation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Structural insights into methyltransferase KsgA function in 30S ribosomal subunit biogenesis.

    Science.gov (United States)

    Boehringer, Daniel; O'Farrell, Heather C; Rife, Jason P; Ban, Nenad

    2012-03-23

    The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3'-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation.

  7. Structural Insights into Methyltransferase KsgA Function in 30S Ribosomal Subunit Biogenesis*

    Science.gov (United States)

    Boehringer, Daniel; O'Farrell, Heather C.; Rife, Jason P.; Ban, Nenad

    2012-01-01

    The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3′-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation. PMID:22308031

  8. Pumping up the volume - vacuole biogenesis in Arabidopsis thaliana.

    Science.gov (United States)

    Krüger, Falco; Schumacher, Karin

    2017-07-08

    Plant architecture follows the need to collect CO 2, solar energy, water and mineral nutrients via large surface areas. It is by the presence of a central vacuole that fills much of the cell volume that plants manage to grow at low metabolic cost. In addition vacuoles buffer the fluctuating supply of essential nutrients and help to detoxify the cytosol when plants are challenged by harmful molecules. Despite their large size and multiple important functions, our knowledge of vacuole biogenesis and the machinery underlying their amazing dynamics is still fragmentary. In this review, we try to reconcile past and present models for vacuole biogenesis with the current knowledge of multiple parallel vacuolar trafficking pathways and the molecular machineries driving membrane fusion and organelle shape. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Perfluorooctanoic acid stimulated mitochondrial biogenesis and gene transcription in rats

    International Nuclear Information System (INIS)

    Walters, M.W.; Bjork, J.A.; Wallace, K.B.

    2009-01-01

    Perfluorooctanoic acid (PFOA), used in the production of non-stick surface compounds, exhibits a worldwide distribution in the serum of humans and wildlife. In rodents PFOA transactivates PPARα and PPARγ nuclear receptors and increases mitochondrial DNA (mtDNA) copy number, which may be critical to the altered metabolic state of affected animals. A key regulator of mitochondrial biogenesis and transcription of mitochondrial genes is the PPARγ coactivator-1α (Pgc-1α) protein. The purpose of this study was to determine if Pgc-1α is implicated in the stimulation of mitochondrial biogenesis that occurs following the treatment of rats with PFOA. Livers from adult male Sprague-Dawley rats that received a 30 mg/kg daily oral dose of PFOA for 28 days were used for all experiments. Analysis of mitochondrial replication and transcription was performed by real time PCR, and proteins were detected using western blotting. PFOA treatment caused a transcriptional activation of the mitochondrial biogenesis pathway leading to a doubling of mtDNA copy number. Further, transcription of OXPHOS genes encoded by mtDNA was 3-4 times greater than that of nuclear encoded genes, suggestive of a preferential induction of mtDNA transcription. Western blot analysis revealed an increase in Pgc-1α, unchanged Tfam and decreased Cox II and Cox IV subunit protein expression. We conclude that PFOA treatment in rats induces mitochondrial biogenesis at the transcriptional level with a preferential stimulation of mtDNA transcription and that this occurs by way of activation of the Pgc-1α pathway. Implication of the Pgc-1α pathway is consistent with PPARγ transactivation by PFOA and reveals new understanding and possibly new critical targets for assessing or averting the associated metabolic disease.

  10. Investigation into spore coat properties for the rapid identification of endospores in marine sediments

    Science.gov (United States)

    Rattray, J. E.; Chakraborty, A.; Bernard, B. B.; Brooks, J.; Hubert, C. R.

    2017-12-01

    Understanding the sediment biogeography of dormant marine thermophilic bacterial endospores (thermospores) has the potential to assist locating and characterising working petroleum systems. The presence of thermospores in cold ocean environments suggests that distribution occurs via hydrocarbon seepage from thermally active reservoirs. Low abundance and endospore coat physiology mean nucleic acid based techniques have limited success for in situ detection of thermospores. Alternative rapid analytical methods are needed so we investigated using the Schaeffer-Fulton (malachite green and safranin) and DAPI (4',6-diamidino-2-phenylindole) staining techniques on thermospores from cultures and marine sediments. Sediment samples from 111 locations in the Eastern Gulf of Mexico (100 to 3300 m water depth; 6 to 600 km apart) were incubated at high temperature, followed by construction of 16S rRNA gene amplicon libraries (V3-V4 region; Illumina MiSeq) revealing enrichment of species-level thermospore OTUs. A sulfate reducing bacterium from site EGM080 was purified and classified based on its rRNA gene sequence as Desulfotomaculum geothermicum. Prior to thermospore staining the culture was kept in the death/ decline phase for 16 weeks to promote sporulation. Samples of D. geothermicum and the source marine sediment were fixed, stained then analysed using brightfield, phase contrast or fluorescence microscopy. Thermospores in pure culture were identified using phase contrast but were difficult to observe in the sediment sample due to particle aggregation. The Schaeffer-Fulton technique aided thermospore identification in a complex sediment sample matrix as thermospores were stained bright green, and also revealed that there were only spores and no (red stained) vegetative cells in the culture. Treatment with DAPI gave dull fluorescing cells but also provided insight into the behaviour of thermospores in sediment suspensions. Spores in the culture medium were free floating but

  11. Mild mitochondrial uncoupling and calorie restriction increase fasting eNOS, akt and mitochondrial biogenesis.

    Directory of Open Access Journals (Sweden)

    Fernanda M Cerqueira

    2011-03-01

    Full Text Available Enhanced mitochondrial biogenesis promoted by eNOS activation is believed to play a central role in the beneficial effects of calorie restriction (CR. Since treatment of mice with dinitrophenol (DNP promotes health and lifespan benefits similar to those observed in CR, we hypothesized that it could also impact biogenesis. We found that DNP and CR increase citrate synthase activity, PGC-1α, cytochrome c oxidase and mitofusin-2 expression, as well as fasting plasma levels of NO• products. In addition, eNOS and Akt phosphorylation in skeletal muscle and visceral adipose tissue was activated in fasting CR and DNP animals. Overall, our results indicate that systemic mild uncoupling activates eNOS and Akt-dependent pathways leading to mitochondrial biogenesis.

  12. A dynamic ribosomal biogenesis response is not required for IGF-1-mediated hypertrophy of human primary myotubes.

    Science.gov (United States)

    Crossland, Hannah; Timmons, James A; Atherton, Philip J

    2017-12-01

    Increased ribosomal DNA transcription has been proposed to limit muscle protein synthesis, making ribosome biogenesis central to skeletal muscle hypertrophy. We examined the relationship between ribosomal RNA (rRNA) production and IGF-1-mediated myotube hypertrophy in vitro Primary skeletal myotubes were treated with IGF-1 (50 ng/ml) with or without 0.5 µM CX-5461 (CX), an inhibitor of RNA polymerase I. Myotube diameter, total protein, and RNA and DNA levels were measured along with markers of RNA polymerase I regulatory factors and regulators of protein synthesis. CX treatment reduced 45S pre-rRNA expression (-64 ± 5% vs. IGF-1; P IGF-1; P IGF-1-treated myotubes. IGF-1-mediated increases in myotube diameter (1.27 ± 0.09-fold, P IGF-1 treatment did not prevent early increases in AKT (+203 ± 39% vs. CX; P IGF-1, myotube diameter and protein accretion were sustained. Thus, while ribosome biogenesis represents a potential site for the regulation of skeletal muscle protein synthesis and muscle mass, it does not appear to be a prerequisite for IGF-1-induced myotube hypertrophy in vitro. -Crossland, H., Timmons, J. A., Atherton, P. J. A dynamic ribosomal biogenesis response is not required for IGF-1-mediated hypertrophy of human primary myotubes. © The Author(s).

  13. Adipose tissue conditioned media support macrophage lipid-droplet biogenesis by interfering with autophagic flux.

    Science.gov (United States)

    Bechor, Sapir; Nachmias, Dikla; Elia, Natalie; Haim, Yulia; Vatarescu, Maayan; Leikin-Frenkel, Alicia; Gericke, Martin; Tarnovscki, Tanya; Las, Guy; Rudich, Assaf

    2017-09-01

    Obesity promotes the biogenesis of adipose tissue (AT) foam cells (FC), which contribute to AT insulin resistance. Autophagy, an evolutionarily-conserved house-keeping process, was implicated in cellular lipid handling by either feeding and/or degrading lipid-droplets (LDs). We hypothesized that beyond phagocytosis of dead adipocytes, AT-FC biogenesis is supported by the AT microenvironment by regulating autophagy. Non-polarized ("M0") RAW264.7 macrophages exposed to AT conditioned media (AT-CM) exhibited a markedly enhanced LDs biogenesis rate compared to control cells (8.3 Vs 0.3 LDs/cells/h, p<0.005). Autophagic flux was decreased by AT-CM, and fluorescently following autophagosomes over time revealed ~20% decline in new autophagic vesicles' formation rate, and 60-70% decrease in autophagosomal growth rate, without marked alternations in the acidic lysosomal compartment. Suppressing autophagy by either targeting autophagosome formation (pharmacologically, with 3-methyladenine or genetically, with Atg12±Atg7-siRNA), decreased the rate of LD formation induced by oleic acid. Conversely, interfering with late autophago-lysosomal function, either pharmacologically with bafilomycin-A1, chloroquine or leupeptin, enhanced LD formation in macrophages without affecting LD degradation rate. Similarly enhanced LD biogenesis rate was induced by siRNA targeting Lamp-1 or the V-ATPase. Collectively, we propose that secreted products from AT interrupt late autophagosome maturation in macrophages, supporting enhanced LDs biogenesis and AT-FC formation, thereby contributing to AT dysfunction in obesity. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

  14. High pressure thermal inactivation of Clostridium botulinum type E endospores – kinetic modeling and mechanistic insights

    Directory of Open Access Journals (Sweden)

    Christian Andreas Lenz

    2015-07-01

    Full Text Available Cold-tolerant, neurotoxigenic, endospore forming Clostridium (C. botulinum type E belongs to the non-proteolytic physiological C. botulinum group II, is primarily associated with aquatic environments, and presents a safety risk for seafood. High pressure thermal (HPT processing exploiting the synergistic effect of pressure and temperature can be used to inactivate bacterial endospores.We investigated the inactivation of C. botulinum type E spores by (near isothermal HPT treatments at 300 – 1200 MPa at 30 – 75 °C for 1 s – 10 min. The occurrence of heat and lysozyme susceptible spore fractions after such treatments was determined. The experimental data were modeled to obtain kinetic parameters and represented graphically by isoeffect lines. In contrast to findings for spores of other species and within the range of treatment parameters applied, zones of spore stabilization (lower inactivation than heat treatments alone, large heat susceptible (HPT-induced germinated or lysozyme-dependently germinable (damaged coat layer spore fractions were not detected. Inactivation followed 1st order kinetics. DPA release kinetics allowed for insights into possible inactivation mechanisms suggesting a (poorly effective physiologic-like (similar to nutrient-induced germination at ≤ 450 MPa/≤ 45 °C and non-physiological germination at >500 MPa/>60 – 70 °C.Results of this study support the existence of some commonalities in the HPT inactivation mechanism of C. botulinum type E spores and Bacillus spores although both organisms have significantly different HPT resistance properties. The information presented here contributes to closing the gap in knowledge regarding the HPT inactivation of spore formers relevant to food safety and may help industrial implementation of HPT processing. The markedly lower HPT resistance of C. botulinum type E spores than spores from other C. botulinum types, could allow for the implementation of milder processes without

  15. Exercise-mediated wall shear stress increases mitochondrial biogenesis in vascular endothelium.

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    Boa Kim

    Full Text Available Enhancing structural and functional integrity of mitochondria is an emerging therapeutic option against endothelial dysfunction. In this study, we sought to investigate the effect of fluid shear stress on mitochondrial biogenesis and mitochondrial respiratory function in endothelial cells (ECs using in vitro and in vivo complementary studies.Human aortic- or umbilical vein-derived ECs were exposed to laminar shear stress (20 dyne/cm2 for various durations using a cone-and-plate shear apparatus. We observed significant increases in the expression of key genes related to mitochondrial biogenesis and mitochondrial quality control as well as mtDNA content and mitochondrial mass under the shear stress conditions. Mitochondrial respiratory function was enhanced when cells were intermittently exposed to laminar shear stress for 72 hrs. Also, shear-exposed cells showed diminished glycolysis and decreased mitochondrial membrane potential (ΔΨm. Likewise, in in vivo experiments, mice that were subjected to a voluntary wheel running exercise for 5 weeks showed significantly higher mitochondrial content determined by en face staining in the conduit (greater and lesser curvature of the aortic arch and thoracic aorta and muscle feed (femoral artery arteries compared to the sedentary control mice. Interestingly, however, the mitochondrial biogenesis was not observed in the mesenteric artery. This region-specific adaptation is likely due to the differential blood flow redistribution during exercise in the different vessel beds.Taken together, our findings suggest that exercise enhances mitochondrial biogenesis in vascular endothelium through a shear stress-dependent mechanism. Our findings may suggest a novel mitochondrial pathway by which a chronic exercise may be beneficial for vascular function.

  16. The Role of Exportin-5 in MicroRNA Biogenesis and Cancer

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    Ke Wu

    2018-04-01

    Full Text Available MicroRNAs (miRNAs are conserved small non-coding RNAs that play an important role in the regulation of gene expression and participate in a variety of biological processes. The biogenesis of miRNAs is tightly controlled at multiple steps, such as transcription of miRNA genes, processing by Drosha and Dicer, and transportation of precursor miRNAs (pre-miRNAs from the nucleus to the cytoplasm by exportin-5 (XPO5. Given the critical role of nuclear export of pre-miRNAs in miRNA biogenesis, any alterations of XPO5, resulting from either genetic mutation, epigenetic change, abnormal expression level or posttranslational modification, could affect miRNA expression and thus have profound effects on tumorigenesis. Importantly, XPO5 phosphorylation by ERK kinase and its cis/trans isomerization by the prolyl isomerase Pin1 impair XPO5′s nucleo-to-cytoplasmic transport ability of pre-miRNAs, leading to downregulation of mature miRNAs in hepatocellular carcinoma. In this review, we focus on how XPO5 transports pre-miRNAs in the cells and summarize the dysregulation of XPO5 in human tumors. Keywords: Exportin-5, MicroRNA, Biogenesis, Dysregulation, Cancer

  17. Two Nucleolar Proteins, GDP1 and OLI2, Function As Ribosome Biogenesis Factors and Are Preferentially Involved in Promotion of Leaf Cell Proliferation without Strongly Affecting Leaf Adaxial–Abaxial Patterning in Arabidopsis thaliana

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    Koji Kojima

    2018-01-01

    Full Text Available Leaf abaxial–adaxial patterning is dependent on the mutual repression of leaf polarity genes expressed either adaxially or abaxially. In Arabidopsis thaliana, this process is strongly affected by mutations in ribosomal protein genes and in ribosome biogenesis genes in a sensitized genetic background, such as asymmetric leaves2 (as2. Most ribosome-related mutants by themselves do not show leaf abaxialization, and one of their typical phenotypes is the formation of pointed rather than rounded leaves. In this study, we characterized two ribosome-related mutants to understand how ribosome biogenesis is linked to several aspects of leaf development. Previously, we isolated oligocellula2 (oli2 which exhibits the pointed-leaf phenotype and has a cell proliferation defect. OLI2 encodes a homolog of Nop2 in Saccharomyces cerevisiae, a ribosome biogenesis factor involved in pre-60S subunit maturation. In this study, we found another pointed-leaf mutant that carries a mutation in a gene encoding an uncharacterized protein with a G-patch domain. Similar to oli2, this mutant, named g-patch domain protein1 (gdp1, has a reduced number of leaf cells. In addition, gdp1 oli2 double mutants showed a strong genetic interaction such that they synergistically impaired cell proliferation in leaves and produced markedly larger cells. On the other hand, they showed additive phenotypes when combined with several known ribosomal protein mutants. Furthermore, these mutants have a defect in pre-rRNA processing. GDP1 and OLI2 are strongly expressed in tissues with high cell proliferation activity, and GDP1-GFP and GFP-OLI2 are localized in the nucleolus. These results suggest that OLI2 and GDP1 are involved in ribosome biogenesis. We then examined the effects of gdp1 and oli2 on adaxial–abaxial patterning by crossing them with as2. Interestingly, neither gdp1 nor oli2 strongly enhanced the leaf polarity defect of as2. Similar results were obtained with as2 gdp1 oli2

  18. Germ Plasm Biogenesis--An Oskar-Centric Perspective.

    Science.gov (United States)

    Lehmann, Ruth

    2016-01-01

    Germ granules are the hallmark of all germ cells. These membrane-less, electron-dense structures were first observed over 100 years ago. Today, their role in regulating and processing transcripts critical for the establishment, maintenance, and protection of germ cells is well established, and pathways outlining the biochemical mechanisms and physical properties associated with their biogenesis are emerging. © 2016 Elsevier Inc. All rights reserved.

  19. Periplasmic quality control in biogenesis of outer membrane proteins.

    Science.gov (United States)

    Lyu, Zhi Xin; Zhao, Xin Sheng

    2015-04-01

    The β-barrel outer membrane proteins (OMPs) are integral membrane proteins that reside in the outer membrane of Gram-negative bacteria and perform a diverse range of biological functions. Synthesized in the cytoplasm, OMPs must be transported across the inner membrane and through the periplasmic space before they are assembled in the outer membrane. In Escherichia coli, Skp, SurA and DegP are the most prominent factors identified to guide OMPs across the periplasm and to play the role of quality control. Although extensive genetic and biochemical analyses have revealed many basic functions of these periplasmic proteins, the mechanism of their collaboration in assisting the folding and insertion of OMPs is much less understood. Recently, biophysical approaches have shed light on the identification of the intricate network. In the present review, we summarize recent advances in the characterization of these key factors, with a special emphasis on the multifunctional protein DegP. In addition, we present our proposed model on the periplasmic quality control in biogenesis of OMPs.

  20. Early stages in the biogenesis of eukaryotic β-barrel proteins.

    Science.gov (United States)

    Jores, Tobias; Rapaport, Doron

    2017-09-01

    The endosymbiotic organelles mitochondria and chloroplasts harbour, similarly to their prokaryotic progenitors, β-barrel proteins in their outer membrane. These proteins are encoded on nuclear DNA, translated on cytosolic ribosomes and imported into their target organelles by a dedicated machinery. Recent studies have provided insights into the import into the organelles and the membrane insertion of these proteins. Although the cytosolic stages of their biogenesis are less well defined, it is speculated that upon their synthesis, chaperones prevent β-barrel proteins from aggregation and keep them in an import-competent conformation. In this Review, we summarize the current knowledge about the biogenesis of β-barrel proteins, focusing on the early stages from the translation on cytosolic ribosomes to the recognition on the surface of the organelle. © 2017 Federation of European Biochemical Societies.

  1. Karrikins delay soybean seed germination by mediating abscisic acid and gibberellin biogenesis under shaded conditions

    Science.gov (United States)

    Meng, Yongjie; Chen, Feng; Shuai, Haiwei; Luo, Xiaofeng; Ding, Jun; Tang, Shengwen; Xu, Shuanshuan; Liu, Jianwei; Liu, Weiguo; Du, Junbo; Liu, Jiang; Yang, Feng; Sun, Xin; Yong, Taiwen; Wang, Xiaochun; Feng, Yuqi; Shu, Kai; Yang, Wenyu

    2016-01-01

    Karrikins (KAR) are a class of signal compounds, discovered in wildfire smoke, which affect seed germination. Currently, numerous studies have focused on the model plant Arabidopsis in the KAR research field, rather than on crops. Thus the regulatory mechanisms underlying KAR regulation of crop seed germination are largely unknown. Here, we report that KAR delayed soybean seed germination through enhancing abscisic acid (ABA) biosynthesis, while impairing gibberellin (GA) biogenesis. Interestingly, KAR only retarded soybean seed germination under shaded conditions, rather than under dark and white light conditions, which differs from in Arabidopsis. Phytohormone quantification showed that KAR enhanced ABA biogenesis while impairing GA biosynthesis during the seed imbibition process, and subsequently, the ratio of active GA4 to ABA was significantly reduced. Further qRT-PCR analysis showed that the transcription pattern of genes involved in ABA and GA metabolic pathways are consistent with the hormonal measurements. Finally, fluridone, an ABA biogenesis inhibitor, remarkably rescued the delayed-germination phenotype of KAR-treatment; and paclobutrazol, a GA biosynthesis inhibitor, inhibited soybean seed germination. Taken together, these evidences suggest that KAR inhibit soybean seed germination by mediating the ratio between GA and ABA biogenesis. PMID:26902640

  2. Karrikins delay soybean seed germination by mediating abscisic acid and gibberellin biogenesis under shaded conditions.

    Science.gov (United States)

    Meng, Yongjie; Chen, Feng; Shuai, Haiwei; Luo, Xiaofeng; Ding, Jun; Tang, Shengwen; Xu, Shuanshuan; Liu, Jianwei; Liu, Weiguo; Du, Junbo; Liu, Jiang; Yang, Feng; Sun, Xin; Yong, Taiwen; Wang, Xiaochun; Feng, Yuqi; Shu, Kai; Yang, Wenyu

    2016-02-23

    Karrikins (KAR) are a class of signal compounds, discovered in wildfire smoke, which affect seed germination. Currently, numerous studies have focused on the model plant Arabidopsis in the KAR research field, rather than on crops. Thus the regulatory mechanisms underlying KAR regulation of crop seed germination are largely unknown. Here, we report that KAR delayed soybean seed germination through enhancing abscisic acid (ABA) biosynthesis, while impairing gibberellin (GA) biogenesis. Interestingly, KAR only retarded soybean seed germination under shaded conditions, rather than under dark and white light conditions, which differs from in Arabidopsis. Phytohormone quantification showed that KAR enhanced ABA biogenesis while impairing GA biosynthesis during the seed imbibition process, and subsequently, the ratio of active GA4 to ABA was significantly reduced. Further qRT-PCR analysis showed that the transcription pattern of genes involved in ABA and GA metabolic pathways are consistent with the hormonal measurements. Finally, fluridone, an ABA biogenesis inhibitor, remarkably rescued the delayed-germination phenotype of KAR-treatment; and paclobutrazol, a GA biosynthesis inhibitor, inhibited soybean seed germination. Taken together, these evidences suggest that KAR inhibit soybean seed germination by mediating the ratio between GA and ABA biogenesis.

  3. Targeting Pin1 by inhibitor API-1 regulates microRNA biogenesis and suppresses hepatocellular carcinoma development.

    Science.gov (United States)

    Pu, Wenchen; Li, Jiao; Zheng, Yuanyuan; Shen, Xianyan; Fan, Xin; Zhou, Jian-Kang; He, Juan; Deng, Yulan; Liu, Xuesha; Wang, Chun; Yang, Shengyong; Chen, Qiang; Liu, Lunxu; Zhang, Guolin; Wei, Yu-Quan; Peng, Yong

    2018-01-30

    Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide, but there are few effective treatments. Aberrant microRNA (miRNA) biogenesis is correlated with HCC development. We previously demonstrated that peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) participates in miRNA biogenesis and is a potential HCC treatment target. However, how Pin1 modulates miRNA biogenesis remains obscure. Here, we present in vivo evidence that Pin1 overexpression is directly linked to the development of HCC. Administration with the Pin1 inhibitor (API-1), a specific small molecule targeting Pin1 peptidyl-prolyl isomerase domain and inhibiting Pin1 cis-trans isomerizing activity, suppresses in vitro cell proliferation and migration of HCC cells. But API-1-induced Pin1 inhibition is insensitive to HCC cells with low Pin1 expression and/or low exportin-5 (XPO5) phosphorylation. Mechanistically, Pin1 recognizes and isomerizes the phosphorylated serine-proline motif of phosphorylated XPO5 and passivates phosphorylated XPO5. Pin1 inhibition by API-1 maintains the active conformation of phosphorylated XPO5 and restores XPO5-driven precursor miRNA nuclear-to-cytoplasm export, activating anticancer miRNA biogenesis and leading to both in vitro HCC suppression and HCC suppression in xenograft mice. Experimental evidence suggests that Pin1 inhibition by API-1 up-regulates miRNA biogenesis by retaining active XPO5 conformation and suppresses HCC development, revealing the mechanism of Pin1-mediated miRNA biogenesis and unequivocally supporting API-1 as a drug candidate for HCC therapy, especially for Pin1-overexpressing, extracellular signal-regulated kinase-activated HCC. (Hepatology 2018). © 2018 by the American Association for the Study of Liver Diseases.

  4. Biogenesis of light harvesting proteins.

    Science.gov (United States)

    Dall'Osto, Luca; Bressan, Mauro; Bassi, Roberto

    2015-09-01

    The LHC family includes nuclear-encoded, integral thylakoid membrane proteins, most of which coordinate chlorophyll and xanthophyll chromophores. By assembling with the core complexes of both photosystems, LHCs form a flexible peripheral moiety for enhancing light-harvesting cross-section, regulating its efficiency and providing protection against photo-oxidative stress. Upon its first appearance, LHC proteins underwent evolutionary diversification into a large protein family with a complex genetic redundancy. Such differentiation appears as a crucial event in the adaptation of photosynthetic organisms to changing environmental conditions and land colonization. The structure of photosystems, including nuclear- and chloroplast-encoded subunits, presented the cell with a number of challenges for the control of the light harvesting function. Indeed, LHC-encoding messages are translated in the cytosol, and pre-proteins imported into the chloroplast, processed to their mature size and targeted to the thylakoids where are assembled with chromophores. Thus, a tight coordination between nuclear and plastid gene expression, in response to environmental stimuli, is required to adjust LHC composition during photoacclimation. In recent years, remarkable progress has been achieved in elucidating structure, function and regulatory pathways involving LHCs; however, a number of molecular details still await elucidation. In this review, we will provide an overview on the current knowledge on LHC biogenesis, ranging from organization of pigment-protein complexes to the modulation of gene expression, import and targeting to the photosynthetic membranes, and regulation of LHC assembly and turnover. Genes controlling these events are potential candidate for biotechnological applications aimed at optimizing light use efficiency of photosynthetic organisms. This article is part of a Special Issue entitled: Chloroplast biogenesis. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation.

    Science.gov (United States)

    Al-Hadid, Qais; White, Jonelle; Clarke, Steven

    2016-02-12

    A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Peroxynitrite induced mitochondrial biogenesis following MnSOD knockdown in normal rat kidney (NRK cells

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    Akira Marine

    2014-01-01

    Full Text Available Superoxide is widely regarded as the primary reactive oxygen species (ROS which initiates downstream oxidative stress. Increased oxidative stress contributes, in part, to many disease conditions such as cancer, atherosclerosis, ischemia/reperfusion, diabetes, aging, and neurodegeneration. Manganese superoxide dismutase (MnSOD catalyzes the dismutation of superoxide into hydrogen peroxide which can then be further detoxified by other antioxidant enzymes. MnSOD is critical in maintaining the normal function of mitochondria, thus its inactivation is thought to lead to compromised mitochondria. Previously, our laboratory observed increased mitochondrial biogenesis in a novel kidney-specific MnSOD knockout mouse. The current study used transient siRNA mediated MnSOD knockdown of normal rat kidney (NRK cells as the in vitro model, and confirmed functional mitochondrial biogenesis evidenced by increased PGC1α expression, mitochondrial DNA copy numbers and integrity, electron transport chain protein CORE II, mitochondrial mass, oxygen consumption rate, and overall ATP production. Further mechanistic studies using mitoquinone (MitoQ, a mitochondria-targeted antioxidant and L-NAME, a nitric oxide synthase (NOS inhibitor demonstrated that peroxynitrite (at low micromolar levels induced mitochondrial biogenesis. These findings provide the first evidence that low levels of peroxynitrite can initiate a protective signaling cascade involving mitochondrial biogenesis which may help to restore mitochondrial function following transient MnSOD inactivation.

  7. Identification of a new complementation group of the peroxisome biogenesis disorders and PEX14 as the mutated gene

    NARCIS (Netherlands)

    Shimozawa, Nobuyuki; Tsukamoto, Toshiro; Nagase, Tomoko; Takemoto, Yasuhiko; Koyama, Naoki; Suzuki, Yasuyuki; Komori, Masayuki; Osumi, Takashi; Jeannette, Gootjes; Wanders, Ronald J. A.; Kondo, Naomi

    2004-01-01

    Peroxisome biogenesis disorders (PBD) are lethal hereditary diseases caused by abnormalities in the biogenesis of peroxisomes. At present, 12 different complementation groups have been identified and to date, all genes responsible for each of these complementation groups have been identified. The

  8. THE REDOX PATHWAY OF Pseudomonas aeruginosa CYTOCHROME C BIOGENESIS

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    Eva Di Silvio

    2012-06-01

    Full Text Available Cytochrome c contains heme covalently bound to the polypeptide chain through two thioether bonds between the heme vinyl groups and the two cysteines of the conserved heme- binding motif of the apoprotein. Surprisingly, the biochemical events leading to the synthesis of the functional holoprotein in the cell are largely unknown. In the human pathogen Pseudomonas aeruginosa, the biogenesis of Cytc is mediated by a group of membrane or membrane-anchored proteins (CcmABCDEFGHI, exposing their active site to the periplasm. The Ccm proteins involved in the necessary reduction of apoCyt disulfide bond are CcmG and CcmH. Here we present the structural and functional characterization of these two redox-active proteins. We determined the crystal structure of CcmG, both in the oxidized and the reduced state. CcmG is a membrane-anchored thioredoxinlike protein acting as a mild reductant in the redox pathway of Cytc biogenesis. The 3D structure of the soluble periplasmic domain of CcmH revealed that it adopts a peculiar three-helix bundle fold that is different from that of canonical thiol-oxidoreductases. Moreover, we present protein-protein interaction experiments aiming at elucidating the molecular mechanism of the reduction of apoCyt disulfide bond for heme attachment in vivo. On the basis of the structural and functional data on CcmG, CcmH and their interactions, we propose an assembly line for Cytc biogenesis in P. aeruginosa in which reduced CcmH specifically recognizes, binds and reduces oxidized apoCyt via the formation of a mixed disulfide complex, which is subsequently resolved by CcmG.

  9. A Novel Non-Apoptotic Role of Procaspase-3 in the Regulation of Mitochondrial Biogenesis Activators.

    Science.gov (United States)

    Kim, Ji-Soo; Ha, Ji-Young; Yang, Sol-Ji; Son, Jin H

    2018-01-01

    The executioner caspase-3 has been proposed as a pharmacological intervention target to preserve degenerating dopaminergic (DA) neurons because apoptotic mechanisms involving caspase-3 contribute, at least in part, to the loss of DA neurons in patients and experimental models of Parkinson's disease (PD). Here, we determined that genetic intervention of caspase-3 was sufficient to prevent cell death against oxidative stress (OS), accompanied by unexpected severe mitochondrial dysfunction. Specifically, as we expected, caspase-3-deficient DA neuronal cells were very significantly resistant to OS-induced cell death, while the activation of the initiator caspase-9 by OS was preserved. Moreover, detailed phenotypic characterization of caspase-3-deficient DA cells revealed severe mitochondrial dysfunction, including an accumulation of damaged mitochondria with a characteristic swollen structure and broken cristae, reduced membrane potential, increased levels of reactive oxygen species (ROS), and deficits in mitochondrial oxidative phosphorylation (OXPHOS) enzymes. Of great interest, we found that mitochondrial biogenesis was dramatically decreased in caspase-3-deficient DA cells, whereas their capability of mitophagy was normal. In accordance with this observation, caspase-3 gene knock down (KD) resulted in dramatically decreased expression of the key transcriptional activators of mitochondrial biogenesis, such as Tfam and Nrf-1, implicating a non-apoptotic role of procaspase-3 in mitochondrial biogenesis. Therefore, a prolonged anti-apoptotic intervention targeting caspase-3 should be considered with caution due to the potential adverse effects in mitochondria dynamics resulting from a novel potential functional role of procaspase-3 in mitochondrial biogenesis via regulating the expression of mitochondrial biogenesis activators. J. Cell. Biochem. 119: 347-357, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  10. Biogenesis and function of T cell-derived exosomes

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    Miguel Angel Alonso

    2016-08-01

    Full Text Available Exosomes are a particular type of extracellular vesicle, characterized by their endosomal origin as intraluminal vesicles present in large endosomes with a multivesicular structure. After these endosomes fuse with the plasma membrane, exosomes are secreted into the extracellular space. The ability of exosomes to carry and selectively deliver bioactive molecules (e.g., lipids, proteins and nucleic acids confers on them the capacity to modulate the activity of receptor cells, even if these cells are located in distant tissues or organs. Since exosomal cargo depends on cell type, a detailed understanding of the mechanisms that regulate the biochemical composition of exosomes is fundamental to a comprehensive view of exosome function. Here, we review the latest advances concerning exosome function and biogenesis in T cells, with particular focus on the mechanism of protein sorting at multivesicular endosomes. Exosomes secreted by specific T-cell subsets can modulate the activity of immune cells, including other T-cell subsets. Ceramide, tetraspanins and MAL have been revealed to be important in exosome biogenesis by T cells. These molecules, therefore, constitute potential molecular targets for artificially modulating exosome production and, hence, the immune response for therapeutic purposes.

  11. A novel mechanism involved in the coupling of mitochondrial biogenesis to oxidative phosphorylation

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    Jelena Ostojić

    2014-01-01

    Full Text Available Mitochondria are essential organelles that are central to a multitude of cellular processes, including oxidative phosphorylation (OXPHOS, which produces most of the ATP in animal cells. Thus it is important to understand not only the mechanisms and biogenesis of this energy production machinery but also how it is regulated in both physiological and pathological contexts. A recent study by Ostojić et al. [Cell Metabolism (2013 18, 567-577] has uncovered a regulatory loop by which the biogenesis of a major enzyme of the OXPHOS pathway, the respiratory complex III, is coupled to the energy producing activity of the mitochondria.

  12. Respective Functions of Two Distinct Siwi Complexes Assembled during PIWI-Interacting RNA Biogenesis in Bombyx Germ Cells

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    Kazumichi M. Nishida

    2015-01-01

    Full Text Available PIWI-interacting RNA (piRNA biogenesis consists of two sequential steps: primary piRNA processing and the ping-pong cycle that depends on reciprocal Slicer-mediated RNA cleavage by PIWI proteins. However, the molecular functions of the factors involved remain elusive. Here, we show that RNAs cleaved by a Bombyx mori PIWI, Siwi, remain bound to the protein upon cleavage but are released by a DEAD box protein BmVasa. BmVasa copurifies with Siwi but not another PIWI BmAgo3. A lack of BmVasa does not affect primary piRNA processing but abolishes the ping-pong cycle. Siwi also forms a complex with BmSpn-E and BmQin. This complex is physically separable from the Siwi/BmVasa complex. BmSpn-E, unlike BmVasa, is necessary for primary piRNA production. We propose a model for piRNA biogenesis, where the BmSpn-E/BmQin dimer binds Siwi to function in primary piRNA processing, whereas BmVasa, by associating with Siwi, ensures target RNA release upon cleavage to facilitate the ping-pong cycle.

  13. The Potential of Targeting Ribosome Biogenesis in High-Grade Serous Ovarian Cancer

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    Shunfei Yan

    2017-01-01

    Full Text Available Overall survival for patients with ovarian cancer (OC has shown little improvement for decades meaning new therapeutic options are critical. OC comprises multiple histological subtypes, of which the most common and aggressive subtype is high-grade serous ovarian cancer (HGSOC. HGSOC is characterized by genomic structural variations with relatively few recurrent somatic mutations or dominantly acting oncogenes that can be targeted for the development of novel therapies. However, deregulation of pathways controlling homologous recombination (HR and ribosome biogenesis has been observed in a high proportion of HGSOC, raising the possibility that targeting these basic cellular processes may provide improved patient outcomes. The poly (ADP-ribose polymerase (PARP inhibitor olaparib has been approved to treat women with defects in HR due to germline BRCA mutations. Recent evidence demonstrated the efficacy of targeting ribosome biogenesis with the specific inhibitor of ribosomal RNA synthesis, CX-5461 in v-myc avian myelocytomatosis viral oncogene homolog (MYC-driven haematological and prostate cancers. CX-5461 has now progressed to a phase I clinical trial in patients with haematological malignancies and phase I/II trial in breast cancer. Here we review the currently available targeted therapies for HGSOC and discuss the potential of targeting ribosome biogenesis as a novel therapeutic approach against HGSOC.

  14. Genomic and non-genomic regulation of PGC1 isoforms by estrogen to increase cerebral vascular mitochondrial biogenesis and reactive oxygen species protection

    Science.gov (United States)

    Kemper, Martin F.; Stirone, Chris; Krause, Diana N.; Duckles, Sue P.; Procaccio, Vincent

    2014-01-01

    We previously found that estrogen exerts a novel protective effect on mitochondria in brain vasculature. Here we demonstrate in rat cerebral blood vessels that 17β-estradiol (estrogen), both in vivo and ex vivo, affects key transcriptional coactivators responsible for mitochondrial regulation. Treatment of ovariectomized rats with estrogen in vivo lowered mRNA levels of peroxisome proliferator-activated receptor-γ coactivator-1 alpha (PGC-1α) but increased levels of the other PGC-1 isoforms: PGC-1β and PGC-1 related coactivator (PRC). In vessels ex vivo, estrogen decreased protein levels of PGC-1α via activation of phosphatidylinositol 3-kinase (PI3K). Estrogen treatment also increased phosphorylation of forkhead transcription factor, FoxO1, a known pathway for PGC-1α downregulation. In contrast to the decrease in PGC-1α, estrogen increased protein levels of nuclear respiratory factor 1, a known PGC target and mediator of mitochondrial biogenesis. The latter effect of estrogen was independent of PI3K, suggesting a separate mechanism consistent with increased expression of PGC-1β and PRC. We demonstrated increased mitochondrial biogenesis following estrogen treatment in vivo; cerebrovascular levels of mitochondrial transcription factor A and electron transport chain subunits as well as the mitochondrial/ nuclear DNA ratio were increased. We examined a downstream target of PGC-1β, glutamate-cysteine ligase (GCL), the rate-limiting enzyme for glutathione synthesis. In vivo estrogen increased protein levels of both GCL subunits and total glutathione levels. Together these data show estrogen differentially regulates PGC-1 isoforms in brain vasculature, underscoring the importance of these coactivators in adapting mitochondria in specific tissues. By upregulating PGC-1β and/or PRC, estrogen appears to enhance mitochondrial biogenesis, function and reactive oxygen species protection. PMID:24275351

  15. Dual localized AtHscB involved in iron sulfur protein biogenesis in Arabidopsis.

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    Xiang Ming Xu

    2009-10-01

    Full Text Available Iron-sulfur clusters are ubiquitous structures which act as prosthetic groups for numerous proteins involved in several fundamental biological processes including respiration and photosynthesis. Although simple in structure both the assembly and insertion of clusters into apoproteins requires complex biochemical pathways involving a diverse set of proteins. In yeast, the J-type chaperone Jac1 plays a key role in the biogenesis of iron sulfur clusters in mitochondria.In this study we demonstrate that AtHscB from Arabidopsis can rescue the Jac1 yeast knockout mutant suggesting a role for AtHscB in iron sulfur protein biogenesis in plants. In contrast to mitochondrial Jac1, AtHscB localizes to both mitochondria and the cytosol. AtHscB interacts with AtIscU1, an Isu-like scaffold protein involved in iron-sulfur cluster biogenesis, and through this interaction AtIscU1 is most probably retained in the cytosol. The chaperone AtHscA can functionally complement the yeast Ssq1knockout mutant and its ATPase activity is enhanced by AtHscB and AtIscU1. Interestingly, AtHscA is also localized in both mitochondria and the cytosol. Furthermore, AtHscB is highly expressed in anthers and trichomes and an AtHscB T-DNA insertion mutant shows reduced seed set, a waxless phenotype and inappropriate trichome development as well as dramatically reduced activities of the iron-sulfur enzymes aconitase and succinate dehydrogenase.Our data suggest that AtHscB together with AtHscA and AtIscU1 plays an important role in the biogenesis of iron-sulfur proteins in both mitochondria and the cytosol.

  16. TLR-activated repression of Fe-S cluster biogenesis drives a metabolic shift and alters histone and tubulin acetylation.

    Science.gov (United States)

    Tong, Wing-Hang; Maio, Nunziata; Zhang, De-Liang; Palmieri, Erika M; Ollivierre, Hayden; Ghosh, Manik C; McVicar, Daniel W; Rouault, Tracey A

    2018-05-22

    Given the essential roles of iron-sulfur (Fe-S) cofactors in mediating electron transfer in the mitochondrial respiratory chain and supporting heme biosynthesis, mitochondrial dysfunction is a common feature in a growing list of human Fe-S cluster biogenesis disorders, including Friedreich ataxia and GLRX5-related sideroblastic anemia. Here, our studies showed that restriction of Fe-S cluster biogenesis not only compromised mitochondrial oxidative metabolism but also resulted in decreased overall histone acetylation and increased H3K9me3 levels in the nucleus and increased acetylation of α-tubulin in the cytosol by decreasing the lipoylation of the pyruvate dehydrogenase complex, decreasing levels of succinate dehydrogenase and the histone acetyltransferase ELP3, and increasing levels of the tubulin acetyltransferase MEC17. Previous studies have shown that the metabolic shift in Toll-like receptor (TLR)-activated myeloid cells involves rapid activation of glycolysis and subsequent mitochondrial respiratory failure due to nitric oxide (NO)-mediated damage to Fe-S proteins. Our studies indicated that TLR activation also actively suppresses many components of the Fe-S cluster biogenesis machinery, which exacerbates NO-mediated damage to Fe-S proteins by interfering with cluster recovery. These results reveal new regulatory pathways and novel roles of the Fe-S cluster biogenesis machinery in modifying the epigenome and acetylome and provide new insights into the etiology of Fe-S cluster biogenesis disorders.

  17. The RNA-binding protein HOS5 and serine/arginine-rich proteins RS40 and RS41 participate in miRNA biogenesis in Arabidopsis

    KAUST Repository

    Chen, Tao

    2015-07-30

    MicroRNAs are a class of small regulatory RNAs that are generated from primary miRNA (pri-miRNA) transcripts with a stem-loop structure. Accuracy of the processing of pri-miRNA into mature miRNA in plants can be enhanced by SERRATE (SE) and HYPONASTIC LEAVES 1 (HYL1). HYL1 activity is regulated by the FIERY2 (FRY2)/RNA polymerase II C-terminal domain phosphatase-like 1 (CPL1). Here, we discover that HIGH OSMOTIC STRESS GENE EXPRESSION 5 (HOS5) and two serine/arginine-rich splicing factors RS40 and RS41, previously shown to be involved in pre-mRNA splicing, affect the biogenesis of a subset of miRNA. These proteins are required for correct miRNA strand selection and the maintenance of miRNA levels. FRY2 dephosphorylates HOS5 whose phosphorylation status affects its subnuclear localization. HOS5 and the RS proteins bind both intronless and intron-containing pri-miRNAs. Importantly, all of these splicing-related factors directly interact with both HYL1 and SE in nuclear splicing speckles. Our results indicate that these splicing factors are directly involved in the biogenesis of a group of miRNA.

  18. ER-plasma membrane contact sites contribute to autophagosome biogenesis by regulation of local PI3P synthesis.

    Science.gov (United States)

    Nascimbeni, Anna Chiara; Giordano, Francesca; Dupont, Nicolas; Grasso, Daniel; Vaccaro, Maria I; Codogno, Patrice; Morel, Etienne

    2017-07-14

    The double-membrane-bound autophagosome is formed by the closure of a structure called the phagophore, origin of which is still unclear. The endoplasmic reticulum (ER) is clearly implicated in autophagosome biogenesis due to the presence of the omegasome subdomain positive for DFCP1, a phosphatidyl-inositol-3-phosphate (PI3P) binding protein. Contribution of other membrane sources, like the plasma membrane (PM), is still difficult to integrate in a global picture. Here we show that ER-plasma membrane contact sites are mobilized for autophagosome biogenesis, by direct implication of the tethering extended synaptotagmins (E-Syts) proteins. Imaging data revealed that early autophagic markers are recruited to E-Syt-containing domains during autophagy and that inhibition of E-Syts expression leads to a reduction in autophagosome biogenesis. Furthermore, we demonstrate that E-Syts are essential for autophagy-associated PI3P synthesis at the cortical ER membrane via the recruitment of VMP1, the stabilizing ER partner of the PI3KC3 complex. These results highlight the contribution of ER-plasma membrane tethers to autophagosome biogenesis regulation and support the importance of membrane contact sites in autophagy. © 2017 The Authors.

  19. Knockout of Tmem70 alters biogenesis of ATP synthase and leads to embryonal lethality in mice

    Czech Academy of Sciences Publication Activity Database

    Vrbacký, Marek; Kovalčíková, Jana; Chawengsaksophak, Kallayanee; Beck, Inken; Mráček, Tomáš; Nůsková, Hana; Sedmera, David; Papoušek, František; Kolář, František; Sobol, Margaryta; Hozák, Pavel; Sedláček, Radislav; Houštěk, Josef

    2016-01-01

    Roč. 25, č. 21 (2016), s. 4674-4685 ISSN 0964-6906 R&D Projects: GA ČR(CZ) GB14-36804G; GA MŠk(CZ) LL1204; GA MŠk(CZ) ED1.1.00/02.0109; GA TA ČR(CZ) TE01020118; GA MŠk(CZ) LM2015062; GA MZd(CZ) NV16-33018A; GA MŠk(CZ) LM2015040 Institutional support: RVO:67985823 ; RVO:68378050 Keywords : mouse knockout * mitochondria * ATP synthase * TMEM70 * biogenesis * mitochondrial diseases Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.340, year: 2016

  20. Development of Therapeutics That Induce Mitochondrial Biogenesis for the Treatment of Acute and Chronic Degenerative Diseases.

    Science.gov (United States)

    Cameron, Robert B; Beeson, Craig C; Schnellmann, Rick G

    2016-12-08

    Mitochondria have various roles in cellular metabolism and homeostasis. Because mitochondrial dysfunction is associated with many acute and chronic degenerative diseases, mitochondrial biogenesis (MB) is a therapeutic target for treating such diseases. Here, we review the role of mitochondrial dysfunction in acute and chronic degenerative diseases and the cellular signaling pathways by which MB is induced. We then review existing work describing the development and application of drugs that induce MB in vitro and in vivo. In particular, we discuss natural products and modulators of transcription factors, kinases, cyclic nucleotides, and G protein-coupled receptors.

  1. COPII-Dependent ER Export: A Critical Component of Insulin Biogenesis and β-Cell ER Homeostasis.

    Science.gov (United States)

    Fang, Jingye; Liu, Ming; Zhang, Xuebao; Sakamoto, Takeshi; Taatjes, Douglas J; Jena, Bhanu P; Sun, Fei; Woods, James; Bryson, Tim; Kowluru, Anjaneyulu; Zhang, Kezhong; Chen, Xuequn

    2015-08-01

    Pancreatic β-cells possess a highly active protein synthetic and export machinery in the endoplasmic reticulum (ER) to accommodate the massive production of proinsulin. ER homeostasis is vital for β-cell functions and is maintained by the delicate balance between protein synthesis, folding, export, and degradation. Disruption of ER homeostasis by diabetes-causing factors leads to β-cell death. Among the 4 components to maintain ER homeostasis in β-cells, the role of ER export in insulin biogenesis is the least understood. To address this knowledge gap, the present study investigated the molecular mechanism of proinsulin ER export in MIN6 cells and primary islets. Two inhibitory mutants of the secretion-associated RAS-related protein (Sar)1 small GTPase, known to specifically block coat protein complex II (COPII)-dependent ER export, were overexpressed in β-cells using recombinant adenoviruses. Results from this approach, as well as small interfering RNA-mediated Sar1 knockdown, demonstrated that defective Sar1 function blocked proinsulin ER export and abolished its conversion to mature insulin in MIN6 cells, isolated mouse, and human islets. It is further revealed, using an in vitro vesicle formation assay, that proinsulin was packaged into COPII vesicles in a GTP- and Sar1-dependent manner. Blockage of COPII-dependent ER exit by Sar1 mutants strongly induced ER morphology change, ER stress response, and β-cell apoptosis. These responses were mediated by the PKR (double-stranded RNA-dependent kinase)-like ER kinase (PERK)/eukaryotic translation initiation factor 2α (p-eIF2α) and inositol-requiring protein 1 (IRE1)/x-box binding protein 1 (Xbp1) pathways but not via activating transcription factor 6 (ATF6). Collectively, results from the study demonstrate that COPII-dependent ER export plays a vital role in insulin biogenesis, ER homeostasis, and β-cell survival.

  2. Outer membrane lipoprotein biogenesis: Lol is not the end.

    Science.gov (United States)

    Konovalova, Anna; Silhavy, Thomas J

    2015-10-05

    Bacterial lipoproteins are lipid-anchored proteins that contain acyl groups covalently attached to the N-terminal cysteine residue of the mature protein. Lipoproteins are synthesized in precursor form with an N-terminal signal sequence (SS) that targets translocation across the cytoplasmic or inner membrane (IM). Lipid modification and SS processing take place at the periplasmic face of the IM. Outer membrane (OM) lipoproteins take the localization of lipoproteins (Lol) export pathway, which ends with the insertion of the N-terminal lipid moiety into the inner leaflet of the OM. For many lipoproteins, the biogenesis pathway ends here. We provide examples of lipoproteins that adopt complex topologies in the OM that include transmembrane and surface-exposed domains. Biogenesis of such lipoproteins requires additional steps beyond the Lol pathway. In at least one case, lipoprotein sequences reach the cell surface by being threaded through the lumen of a beta-barrel protein in an assembly reaction that requires the heteropentomeric Bam complex. The inability to predict surface exposure reinforces the importance of experimental verification of lipoprotein topology and we will discuss some of the methods used to study OM protein topology. © 2015 The Author(s).

  3. A Trimeric Lipoprotein Assists in Trimeric Autotransporter Biogenesis in Enterobacteria*

    Science.gov (United States)

    Grin, Iwan; Hartmann, Marcus D.; Sauer, Guido; Hernandez Alvarez, Birte; Schütz, Monika; Wagner, Samuel; Madlung, Johannes; Macek, Boris; Felipe-Lopez, Alfonso; Hensel, Michael; Lupas, Andrei; Linke, Dirk

    2014-01-01

    Trimeric autotransporter adhesins (TAAs) are important virulence factors of many Gram-negative bacterial pathogens. TAAs form fibrous, adhesive structures on the bacterial cell surface. Their N-terminal extracellular domains are exported through a C-terminal membrane pore; the insertion of the pore domain into the bacterial outer membrane follows the rules of β-barrel transmembrane protein biogenesis and is dependent on the essential Bam complex. We have recently described the full fiber structure of SadA, a TAA of unknown function in Salmonella and other enterobacteria. In this work, we describe the structure and function of SadB, a small inner membrane lipoprotein. The sadB gene is located in an operon with sadA; orthologous operons are only found in enterobacteria, whereas other TAAs are not typically associated with lipoproteins. Strikingly, SadB is also a trimer, and its co-expression with SadA has a direct influence on SadA structural integrity. This is the first report of a specific export factor of a TAA, suggesting that at least in some cases TAA autotransport is assisted by additional periplasmic proteins. PMID:24369174

  4. Staphylococcus aureus and Escherichia coli have disparate dependences on KsgA for growth and ribosome biogenesis

    Directory of Open Access Journals (Sweden)

    O’Farrell Heather C

    2012-10-01

    Full Text Available Abstract Background The KsgA methyltransferase has been conserved throughout evolution, methylating two adenosines in the small subunit rRNA in all three domains of life as well as in eukaryotic organelles that contain ribosomes. Understanding of KsgA’s important role in ribosome biogenesis has been recently expanded in Escherichia coli; these studies help explain why KsgA is so highly conserved and also suggest KsgA’s potential as an antimicrobial drug target. Results We have analyzed KsgA’s contribution to ribosome biogenesis and cell growth in Staphylococcus aureus. We found that deletion of ksgA in S. aureus led to a cold-sensitive growth phenotype, although KsgA was not as critical for ribosome biogenesis as it was shown to be in E. coli. Additionally, the ksgA knockout strain showed an increased sensitivity to aminoglycoside antibiotics. Overexpression of a catalytically inactive KsgA mutant was deleterious in the knockout strain but not the wild-type strain; this negative phenotype disappeared at low temperature. Conclusions This work extends the study of KsgA, allowing comparison of this aspect of ribosome biogenesis between a Gram-negative and a Gram-positive organism. Our results in S. aureus are in contrast to results previously described in E. coli, where the catalytically inactive protein showed a negative phenotype in the presence or absence of endogenous KsgA.

  5. Troxerutin attenuates diet-induced oxidative stress, impairment of mitochondrial biogenesis and respiratory chain complexes in mice heart.

    Science.gov (United States)

    Rajagopalan, Geetha; Chandrasekaran, Sathiya Priya; Carani Venkatraman, Anuradha

    2017-01-01

    Mitochondrial abnormality is thought to play a key role in cardiac disease originating from the metabolic syndrome (MS). We evaluated the effect of troxerutin (TX), a semi-synthetic derivative of the natural bioflavanoid rutin, on the respiratory chain complex activity, oxidative stress, mitochondrial biogenesis and dynamics in heart of high fat, high fructose diet (HFFD) -induced mouse model of MS. Adult male Mus musculus mice of body weight 25-30 g were fed either control diet or HFFD for 60 days. Mice from each dietary regimen were divided into two groups on the 16th day and were treated or untreated with TX (150 mg/kg body weight [bw], per oral) for the next 45 days. At the end of experimental period, respiratory chain complex activity, uncoupling proteins (UCP)-2 and -3, mtDNA content, mitochondrial biogenesis and dynamics, oxidative stress markers and reactive oxygen species (ROS) generation were analyzed. Reduced mtDNA abundance with alterations in the expression of genes related to mitochondrial biogenesis and fission and fusion processes were observed in HFFD-fed mice. Disorganized and smaller mitochondria, reduction in complexes I, III and IV activities (by about 55%) and protein levels of UCP-2 (52%) and UCP-3 (46%) were noted in these mice. TX administration suppressed oxidative stress, improved the oxidative capacity and biogenesis and restored fission/fusion imbalance in the cardiac mitochondria of HFFD-fed mice. TX protects the myocardium by modulating the putative molecules of mitochondrial biogenesis and dynamics and by its anti-oxidant function in a mouse model of MS. © 2016 John Wiley & Sons Australia, Ltd.

  6. Therapeutic dosages of aspirin counteract the IL-6 induced pro-tumorigenic effects by slowing down the ribosome biogenesis rate.

    Science.gov (United States)

    Brighenti, Elisa; Giannone, Ferdinando Antonino; Fornari, Francesca; Onofrillo, Carmine; Govoni, Marzia; Montanaro, Lorenzo; Treré, Davide; Derenzini, Massimo

    2016-09-27

    Chronic inflammation is a risk factor for the onset of cancer and the regular use of aspirin reduces the risk of cancer development. Here we showed that therapeutic dosages of aspirin counteract the pro-tumorigenic effects of the inflammatory cytokine interleukin(IL)-6 in cancer and non-cancer cell lines, and in mouse liver in vivo. We found that therapeutic dosages of aspirin prevented IL-6 from inducing the down-regulation of p53 expression and the acquisition of the epithelial mesenchymal transition (EMT) phenotypic changes in the cell lines. This was the result of a reduction in c-Myc mRNA transcription which was responsible for a down-regulation of the ribosomal protein S6 expression which, in turn, slowed down the rRNA maturation process, thus reducing the ribosome biogenesis rate. The perturbation of ribosome biogenesis hindered the Mdm2-mediated proteasomal degradation of p53, throughout the ribosomal protein-Mdm2-p53 pathway. P53 stabilization hindered the IL-6 induction of the EMT changes. The same effects were observed in livers from mice stimulated with IL-6 and treated with aspirin. It is worth noting that aspirin down-regulated ribosome biogenesis, stabilized p53 and up-regulated E-cadherin expression in unstimulated control cells also. In conclusion, these data showed that therapeutic dosages of aspirin increase the p53-mediated tumor-suppressor activity of the cells thus being in this way able to reduce the risk of cancer onset, either or not linked to chronic inflammatory processes.

  7. Therapeutic dosages of aspirin counteract the IL-6 induced pro-tumorigenic effects by slowing down the ribosome biogenesis rate

    Science.gov (United States)

    Brighenti, Elisa; Giannone, Ferdinando Antonino; Fornari, Francesca; Onofrillo, Carmine; Govoni, Marzia; Montanaro, Lorenzo; Treré, Davide; Derenzini, Massimo

    2016-01-01

    Chronic inflammation is a risk factor for the onset of cancer and the regular use of aspirin reduces the risk of cancer development. Here we showed that therapeutic dosages of aspirin counteract the pro-tumorigenic effects of the inflammatory cytokine interleukin(IL)-6 in cancer and non-cancer cell lines, and in mouse liver in vivo. We found that therapeutic dosages of aspirin prevented IL-6 from inducing the down-regulation of p53 expression and the acquisition of the epithelial mesenchymal transition (EMT) phenotypic changes in the cell lines. This was the result of a reduction in c-Myc mRNA transcription which was responsible for a down-regulation of the ribosomal protein S6 expression which, in turn, slowed down the rRNA maturation process, thus reducing the ribosome biogenesis rate. The perturbation of ribosome biogenesis hindered the Mdm2-mediated proteasomal degradation of p53, throughout the ribosomal protein-Mdm2-p53 pathway. P53 stabilization hindered the IL-6 induction of the EMT changes. The same effects were observed in livers from mice stimulated with IL-6 and treated with aspirin. It is worth noting that aspirin down-regulated ribosome biogenesis, stabilized p53 and up-regulated E-cadherin expression in unstimulated control cells also. In conclusion, these data showed that therapeutic dosages of aspirin increase the p53-mediated tumor-suppressor activity of the cells thus being in this way able to reduce the risk of cancer onset, either or not linked to chronic inflammatory processes. PMID:27557515

  8. Molecular mechanism of a temperature-sensitive phenotype in peroxisomal biogenesis disorder

    NARCIS (Netherlands)

    Hashimoto, Kazuyuki; Kato, Zenichiro; Nagase, Tomoko; Shimozawa, Nobuyuki; Kuwata, Kazuo; Omoya, Kentaro; Li, Ailian; Matsukuma, Eiji; Yamamoto, Yutaka; Ohnishi, Hidenori; Tochio, Hidehito; Shirakawa, Masahiro; Suzuki, Yasuyuki; Wanders, Ronald J. A.; Kondo, Naomi

    2005-01-01

    Peroxisomal biogenesis disorders include Zellweger syndrome and milder phenotypes, such as neonatal adrenoleukodystrophy (NALD). Our previous study of a NALD patient with a marked deterioration by a fever revealed a mutation (Ile326Thr) within a SH3 domain of PEX13 protein (Pex13p), showing a

  9. Iron-sulfur cluster biogenesis in mammalian cells: new insights into the molecular mechanisms of cluster delivery

    Science.gov (United States)

    Maio, Nunziata; Rouault, Tracey. A.

    2014-01-01

    Iron-sulfur (Fe-S) clusters are ancient, ubiquitous cofactors composed of iron and inorganic sulfur. The combination of the chemical reactivity of iron and sulfur, together with many variations of cluster composition, oxidation states and protein environments, enables Fe-S clusters to participate in numerous biological processes. Fe-S clusters are essential to redox catalysis in nitrogen fixation, mitochondrial respiration and photosynthesis, to regulatory sensing in key metabolic pathways (i. e. cellular iron homeostasis and oxidative stress response), and to the replication and maintenance of the nuclear genome. Fe-S cluster biogenesis is a multistep process that involves a complex sequence of catalyzed protein- protein interactions and coupled conformational changes between the components of several dedicated multimeric complexes. Intensive studies of the assembly process have clarified key points in the biogenesis of Fe-S proteins. However several critical questions still remain, such as: what is the role of frataxin? Why do some defects of Fe-S cluster biogenesis cause mitochondrial iron overload? How are specific Fe-S recipient proteins recognized in the process of Fe-S transfer? This review focuses on the basic steps of Fe-S cluster biogenesis, drawing attention to recent advances achieved on the identification of molecular features that guide selection of specific subsets of nascent Fe-S recipients by the cochaperone HSC20. Additionally, it outlines the distinctive phenotypes of human diseases due to mutations in the components of the basic pathway. PMID:25245479

  10. Biogenesis of the demarcation membrane system (DMS) in megakaryocytes.

    Science.gov (United States)

    Eckly, Anita; Heijnen, Harry; Pertuy, Fabien; Geerts, Willie; Proamer, Fabienne; Rinckel, Jean-Yves; Léon, Catherine; Lanza, François; Gachet, Christian

    2014-02-06

    The demarcation membrane system (DMS) in megakaryocytes forms the plasma membrane (PM) of future platelets. Using confocal microscopy, electron tomography, and large volume focused ion beam/scanning electron microscopy (FIB/SEM), we determined the sequential steps of DMS formation. We identified a pre-DMS that initiated at the cell periphery and was precisely located between the nuclear lobes. At all developmental stages, the DMS remained continuous with the cell surface. The number of these connections correlated well with the nuclear lobulation, suggesting a relationship with cleavage furrow formation and abortive cytokinesis. On DMS expansion, Golgi complexes assembled around the pre-DMS, and fusion profiles between trans-golgi network-derived vesicles and the DMS were observed. Brefeldin-A reduced DMS expansion, indicating that the exocytic pathway is essential for DMS biogenesis. Close contacts between the endoplasmic reticulum (ER) and the DMS were detected, suggesting physical interaction between the 2 membrane systems. FIB/SEM revealed that the DMS forms an intertwined tubular membrane network resembling the platelet open canalicular system. We thus propose the following steps in DMS biogenesis: (1) focal membrane assembly at the cell periphery; (2) PM invagination and formation of a perinuclear pre-DMS; (3) expansion through membrane delivery from Golgi complexes; and (4) ER-mediated lipid transfer.

  11. Mitochondrial DNA deletion and impairment of mitochondrial biogenesis are mediated by reactive oxygen species in ionizing radiation-induced premature senescence

    Energy Technology Data Exchange (ETDEWEB)

    Eom, Hyeon Soo; Jung, U Hee; Jo, Sung Kee [Radiation Biotechnology Research Division, Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Kim, Young Sang [College of Natural Sciences, Chungnam National University, Daejeon (Korea, Republic of)

    2011-09-15

    Mitochondrial DNA (mtDNA) deletion is a well-known marker for oxidative stress and aging, and contributes to harmful effects in cultured cells and animal tissues. mtDNA biogenesis genes (NRF-1, TFAM) are essential for the maintenance of mtDNA, as well as the transcription and replication of mitochondrial genomes. Considering that oxidative stress is known to affect mitochondrial biogenesis, we hypothesized that ionizing radiation (IR)-induced reactive oxygen species (ROS) causes mtDNA deletion by modulating the mitochondrial biogenesis, thereby leading to cellular senescence. Therefore, we examined the effects of IR on ROS levels, cellular senescence, mitochondrial biogenesis, and mtDNA deletion in IMR-90 human lung fibroblast cells. Young IMR-90 cells at population doubling (PD) 39 were irradiated at 4 or 8 Gy. Old cells at PD55, and H2O2-treated young cells at PD 39, were compared as a positive control. The IR increased the intracellular ROS level, senescence-associated {beta}-galactosidase (SA-{beta}-gal) activity, and mtDNA common deletion (4977 bp), and it decreased the mRNA expression of NRF-1 and TFAM in IMR-90 cells. Similar results were also observed in old cells (PD 55) and H{sub 2}O{sub 2}-treated young cells. To confirm that a increase in ROS level is essential for mtDNA deletion and changes of mitochondrial biogenesis in irradiated cells, the effects of N-acetylcysteine (NAC) were examined. In irradiated and H{sub 2}O{sub 2}-treated cells, 5 mM NAC significantly attenuated the increases of ROS, mtDNA deletion, and SA-{beta}-gal activity, and recovered from decreased expressions of NRF-1 and TFAM mRNA. These results suggest that ROS is a key cause of IR-induced mtDNA deletion, and the suppression of the mitochondrial biogenesis gene may mediate this process.

  12. Mitochondrial DNA deletion and impairment of mitochondrial biogenesis are mediated by reactive oxygen species in ionizing radiation-induced premature senescence

    International Nuclear Information System (INIS)

    Eom, Hyeon Soo; Jung, U Hee; Jo, Sung Kee; Kim, Young Sang

    2011-01-01

    Mitochondrial DNA (mtDNA) deletion is a well-known marker for oxidative stress and aging, and contributes to harmful effects in cultured cells and animal tissues. mtDNA biogenesis genes (NRF-1, TFAM) are essential for the maintenance of mtDNA, as well as the transcription and replication of mitochondrial genomes. Considering that oxidative stress is known to affect mitochondrial biogenesis, we hypothesized that ionizing radiation (IR)-induced reactive oxygen species (ROS) causes mtDNA deletion by modulating the mitochondrial biogenesis, thereby leading to cellular senescence. Therefore, we examined the effects of IR on ROS levels, cellular senescence, mitochondrial biogenesis, and mtDNA deletion in IMR-90 human lung fibroblast cells. Young IMR-90 cells at population doubling (PD) 39 were irradiated at 4 or 8 Gy. Old cells at PD55, and H2O2-treated young cells at PD 39, were compared as a positive control. The IR increased the intracellular ROS level, senescence-associated β-galactosidase (SA-β-gal) activity, and mtDNA common deletion (4977 bp), and it decreased the mRNA expression of NRF-1 and TFAM in IMR-90 cells. Similar results were also observed in old cells (PD 55) and H 2 O 2 -treated young cells. To confirm that a increase in ROS level is essential for mtDNA deletion and changes of mitochondrial biogenesis in irradiated cells, the effects of N-acetylcysteine (NAC) were examined. In irradiated and H 2 O 2 -treated cells, 5 mM NAC significantly attenuated the increases of ROS, mtDNA deletion, and SA-β-gal activity, and recovered from decreased expressions of NRF-1 and TFAM mRNA. These results suggest that ROS is a key cause of IR-induced mtDNA deletion, and the suppression of the mitochondrial biogenesis gene may mediate this process.

  13. Cardiomyocyte-Restricted Deletion of PPARβ/δ in PPARα-Null Mice Causes Impaired Mitochondrial Biogenesis and Defense, but No Further Depression of Myocardial Fatty Acid Oxidation

    Directory of Open Access Journals (Sweden)

    Jian Liu

    2011-01-01

    Full Text Available It is well documented that PPARα and PPARβ/δ share overlapping functions in regulating myocardial lipid metabolism. However, previous studies demonstrated that cardiomyocyte-restricted PPARβ/δ deficiency in mice leads to severe cardiac pathological development, whereas global PPARα knockout shows a benign cardiac phenotype. It is unknown whether a PPARα-null background would alter the pathological development in mice with cardiomyocyte-restricted PPARβ/δ deficiency. In the present study, a mouse model with long-term PPARβ/δ deficiency in PPARα-null background showed a comparably reduced cardiac expression of lipid metabolism to those of single PPAR-deficient mouse models. The PPARα-null background did not rescue or aggravate the cardiac pathological development linked to cardiomyocyte-restricted PPARβ/δ deficiency. Moreover, PPARα-null did not alter the phenotypic development in adult mice with the short-term deletion of PPARβ/δ in their hearts, which showed mitochondrial abnormalities, depressed cardiac performance, and cardiac hypertrophy with attenuated expression of key factors in mitochondrial biogenesis and defense. The present study demonstrates that cardiomyocyte-restricted deletion of PPARβ/δ in PPARα-null mice causes impaired mitochondrial biogenesis and defense, but no further depression of fatty acid oxidation. Therefore, PPARβ/δ is essential for maintaining mitochondrial biogenesis and defense in cardiomyocytes independent of PPARα.

  14. The energetic state of mitochondria modulates complex III biogenesis through the ATP-dependent activity of Bcs1.

    Science.gov (United States)

    Ostojić, Jelena; Panozzo, Cristina; Lasserre, Jean-Paul; Nouet, Cécile; Courtin, Florence; Blancard, Corinne; di Rago, Jean-Paul; Dujardin, Geneviève

    2013-10-01

    Our understanding of the mechanisms involved in mitochondrial biogenesis has continuously expanded during the last decades, yet little is known about how they are modulated to optimize the functioning of mitochondria. Here, we show that mutations in the ATP binding domain of Bcs1, a chaperone involved in the assembly of complex III, can be rescued by mutations that decrease the ATP hydrolytic activity of the ATP synthase. Our results reveal a Bcs1-mediated control loop in which the biogenesis of complex III is modulated by the energy-transducing activity of mitochondria. Although ATP is well known as a regulator of a number of cellular activities, we show here that ATP can be also used to modulate the biogenesis of an enzyme by controlling a specific chaperone involved in its assembly. Our study further highlights the intramitochondrial adenine nucleotide pool as a potential target for the treatment of Bcs1-based disorders. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Dynamics of miRNA biogenesis and nuclear transport

    Directory of Open Access Journals (Sweden)

    Kotipalli Aneesh

    2016-12-01

    Full Text Available MicroRNAs (miRNAs are short noncoding RNA sequences ~22 nucleotides in length that play an important role in gene regulation-transcription and translation. The processing of these miRNAs takes place in both the nucleus and the cytoplasm while the final maturation occurs in the cytoplasm. Some mature miRNAs with nuclear localisation signals (NLS are transported back to the nucleus and some remain in the cytoplasm. The functional roles of these miRNAs are seen in both the nucleus and the cytoplasm. In the nucleus, miRNAs regulate gene expression by binding to the targeted promoter sequences and affect either the transcriptional gene silencing (TGS or transcriptional gene activation (TGA. In the cytoplasm, targeted mRNAs are translationally repressed or cleaved based on the complementarity between the two sequences at the seed region of miRNA and mRNA. The selective transport of mature miRNAs to the nucleus follows the classical nuclear import mechanism. The classical nuclear import mechanism is a highly regulated process, involving exportins and importins. The nuclear pore complex (NPC regulates all these transport events like a gate keeper. The half-life of miRNAs is rather low, so within a short time miRNAs perform their function. Temporal studies of miRNA biogenesis are, therefore, useful. We have carried out simulation studies for important miRNA biogenesis steps and also classical nuclear import mechanism using ordinary differential equation (ODE solver in the Octave software.

  16. The Biogenesis of Lysosomes and Lysosome-Related Organelles

    Science.gov (United States)

    Luzio, J. Paul; Hackmann, Yvonne; Dieckmann, Nele M.G.; Griffiths, Gillian M.

    2014-01-01

    Lysosomes were once considered the end point of endocytosis, simply used for macromolecule degradation. They are now recognized to be dynamic organelles, able to fuse with a variety of targets and to be re-formed after fusion events. They are also now known to be the site of nutrient sensing and signaling to the cell nucleus. In addition, lysosomes are secretory organelles, with specialized machinery for regulated secretion of proteins in some cell types. The biogenesis of lysosomes and lysosome-related organelles is discussed, taking into account their dynamic nature and multiple roles. PMID:25183830

  17. Complete topology inversion can be part of normal membrane protein biogenesis.

    Science.gov (United States)

    Woodall, Nicholas B; Hadley, Sarah; Yin, Ying; Bowie, James U

    2017-04-01

    The topology of helical membrane proteins is generally defined during insertion of the transmembrane helices, yet it is now clear that it is possible for topology to change under unusual circumstances. It remains unclear, however, if topology reorientation is part of normal biogenesis. For dual topology dimer proteins such as the multidrug transporter EmrE, there may be evolutionary pressure to allow topology flipping so that the populations of both orientations can be equalized. We previously demonstrated that when EmrE is forced to insert in a distorted topology, topology flipping of the first transmembrane helix can occur during translation. Here, we show that topological malleability also extends to the C-terminal helix and that even complete topology inversion of the entire EmrE protein can occur after the full protein is translated and inserted. Thus, topology rearrangements are possible during normal biogenesis. Wholesale topology flipping is remarkable given the physical constraints of the membrane and expands the range of possible membrane protein folding pathways, both productive and detrimental. © 2017 The Protein Society.

  18. Anaplastic Thyroid Carcinoma: A ceRNA Analysis Pointed to a Crosstalk between SOX2, TP53, and microRNA Biogenesis

    Directory of Open Access Journals (Sweden)

    Walter Arancio

    2015-01-01

    Full Text Available It has been suggested that cancer stem cells (CSC may play a central role in oncogenesis, especially in undifferentiated tumours. Anaplastic thyroid carcinoma (ATC has characteristics suggestive of a tumour enriched in CSC. Previous studies suggested that the stem cell factor SOX2 has a preeminent hierarchical role in determining the characteristics of stem cells in SW1736 ATC cell line. In detail, silencing SOX2 in SW1736 is able to suppress the expression of the stem markers analysed, strongly sensitizing the line to treatment with chemotherapeutic agents. Therefore, in order to further investigate the role of SOX2 in ATC, a competing endogenous RNA (ceRNA analysis was conducted in order to isolate new functional partners of SOX2. Among the interactors, of particular interest are genes involved in the biogenesis of miRNAs (DICER1, RNASEN, and EIF2C2, in the control cell cycle (TP53, CCND1, and in mitochondrial activity (COX8A. The data suggest that stemness, microRNA biogenesis and functions, p53 regulatory network, cyclin D1, and cell cycle control, together with mitochondrial activity, might be coregulated.

  19. Identification and expression profiles of sRNAs and their biogenesis and action-related genes in male and female cones of Pinus tabuliformis.

    Science.gov (United States)

    Niu, Shi-Hui; Liu, Chang; Yuan, Hu-Wei; Li, Pei; Li, Yue; Li, Wei

    2015-09-15

    Small RNA (sRNA) play pivotal roles in reproductive development, and their biogenesis and action mechanisms are well characterised in angiosperm plants; however, corresponding studies in conifers are very limited. To improve our understanding of the roles of sRNA pathways in the reproductive development of conifers, the genes associated with sRNA biogenesis and action pathways were identified and analysed, and sRNA sequencing and parallel analysis of RNA ends (PARE) were performed in male and female cones of the Chinese pine (Pinus tabuliformis). Based on high-quality reference transcriptomic sequences, 21 high-confidence homologues involved in sRNA biogenesis and action in P. tabuliformis were identified, including two different DCL3 genes and one AGO4 gene. More than 75 % of genes involved in sRNA biogenesis and action have higher expression levels in female than in male cones. Twenty-six microRNA (miRNA) families and 74 targets, including 46 24-nt sRNAs with a 5' A, which are specifically expressed in male cones or female cones and probably bind to AGO4, were identified. The sRNA pathways have higher activity in female than in male cones, and the miRNA pathways are the main sRNA pathways in P. tabuliformis. The low level of 24-nt short-interfering RNAs in conifers is not caused by the absence of biogenesis-related genes or AGO-binding proteins, but most likely caused by the low accumulation of these key components. The identification of sRNAs and their targets, as well as genes associated with sRNA biogenesis and action, will provide a good starting point for investigations into the roles of sRNA pathways in cone development in conifers.

  20. Mitochondrial biogenesis and energy production in differentiating murine stem cells: a functional metabolic study.

    Science.gov (United States)

    Han, Sungwon; Auger, Christopher; Thomas, Sean C; Beites, Crestina L; Appanna, Vasu D

    2014-02-01

    The significance of metabolic networks in guiding the fate of the stem cell differentiation is only beginning to emerge. Oxidative metabolism has been suggested to play a major role during this process. Therefore, it is critical to understand the underlying mechanisms of metabolic alterations occurring in stem cells to manipulate the ultimate outcome of these pluripotent cells. Here, using P19 murine embryonal carcinoma cells as a model system, the role of mitochondrial biogenesis and the modulation of metabolic networks during dimethyl sulfoxide (DMSO)-induced differentiation are revealed. Blue native polyacrylamide gel electrophoresis (BN-PAGE) technology aided in profiling key enzymes, such as hexokinase (HK) [EC 2.7.1.1], glucose-6-phosphate isomerase (GPI) [EC 5.3.1.9], pyruvate kinase (PK) [EC 2.7.1.40], Complex I [EC 1.6.5.3], and Complex IV [EC 1.9.3.1], that are involved in the energy budget of the differentiated cells. Mitochondrial adenosine triphosphate (ATP) production was shown to be increased in DMSO-treated cells upon exposure to the tricarboxylic acid (TCA) cycle substrates, such as succinate and malate. The increased mitochondrial activity and biogenesis were further confirmed by immunofluorescence microscopy. Collectively, the results indicate that oxidative energy metabolism and mitochondrial biogenesis were sharply upregulated in DMSO-differentiated P19 cells. This functional metabolic and proteomic study provides further evidence that modulation of mitochondrial energy metabolism is a pivotal component of the cellular differentiation process and may dictate the final destiny of stem cells.

  1. Hyperglycemia decreases mitochondrial function: The regulatory role of mitochondrial biogenesis

    International Nuclear Information System (INIS)

    Palmeira, Carlos M.; Rolo, Anabela P.; Berthiaume, Jessica; Bjork, James A.; Wallace, Kendall B.

    2007-01-01

    Increased generation of reactive oxygen species (ROS) is implicated in 'glucose toxicity' in diabetes. However, little is known about the action of glucose on the expression of transcription factors in hepatocytes, especially those involved in mitochondrial DNA (mtDNA) replication and transcription. Since mitochondrial functional capacity is dynamically regulated, we hypothesized that stressful conditions of hyperglycemia induce adaptations in the transcriptional control of cellular energy metabolism, including inhibition of mitochondrial biogenesis and oxidative metabolism. Cell viability, mitochondrial respiration, ROS generation and oxidized proteins were determined in HepG2 cells cultured in the presence of either 5.5 mM (control) or 30 mM glucose (high glucose) for 48 h, 96 h and 7 days. Additionally, mtDNA abundance, plasminogen activator inhibitor-1 (PAI-1), mitochondrial transcription factor A (TFAM) and nuclear respiratory factor-1 (NRF-1) transcripts were evaluated by real time PCR. High glucose induced a progressive increase in ROS generation and accumulation of oxidized proteins, with no changes in cell viability. Increased expression of PAI-1 was observed as early as 96 h of exposure to high glucose. After 7 days in hyperglycemia, HepG2 cells exhibited inhibited uncoupled respiration and decreased MitoTracker Red fluorescence associated with a 25% decrease in mtDNA and 16% decrease in TFAM transcripts. These results indicate that glucose may regulate mtDNA copy number by modulating the transcriptional activity of TFAM in response to hyperglycemia-induced ROS production. The decrease of mtDNA content and inhibition of mitochondrial function may be pathogenic hallmarks in the altered metabolic status associated with diabetes

  2. Shear stress-induced mitochondrial biogenesis decreases the release of microparticles from endothelial cells

    OpenAIRE

    Kim, Ji-Seok; Kim, Boa; Lee, Hojun; Thakkar, Sunny; Babbitt, Dianne M.; Eguchi, Satoru; Brown, Michael D.; Park, Joon-Young

    2015-01-01

    This study assesses effects of aerobic exercise training on the release of microparticles from endothelial cells and corroborates these findings using an in vitro experimental exercise stimulant, laminar shear stress. Furthermore, this study demonstrated that shear stress-induced mitochondrial biogenesis mediates these effects against endothelial cell activation and injury.

  3. Shear stress-induced mitochondrial biogenesis decreases the release of microparticles from endothelial cells.

    Science.gov (United States)

    Kim, Ji-Seok; Kim, Boa; Lee, Hojun; Thakkar, Sunny; Babbitt, Dianne M; Eguchi, Satoru; Brown, Michael D; Park, Joon-Young

    2015-08-01

    The concept of enhancing structural integrity of mitochondria has emerged as a novel therapeutic option for cardiovascular disease. Flow-induced increase in laminar shear stress is a potent physiological stimulant associated with exercise, which exerts atheroprotective effects in the vasculature. However, the effect of laminar shear stress on mitochondrial remodeling within the vascular endothelium and its related functional consequences remain largely unknown. Using in vitro and in vivo complementary studies, here, we report that aerobic exercise alleviates the release of endothelial microparticles in prehypertensive individuals and that these salutary effects are, in part, mediated by shear stress-induced mitochondrial biogenesis. Circulating levels of total (CD31(+)/CD42a(-)) and activated (CD62E(+)) microparticles released by endothelial cells were significantly decreased (∼40% for both) after a 6-mo supervised aerobic exercise training program in individuals with prehypertension. In cultured human endothelial cells, laminar shear stress reduced the release of endothelial microparticles, which was accompanied by an increase in mitochondrial biogenesis through a sirtuin 1 (SIRT1)-dependent mechanism. Resveratrol, a SIRT1 activator, treatment showed similar effects. SIRT1 knockdown using small-interfering RNA completely abolished the protective effect of shear stress. Disruption of mitochondrial integrity by either antimycin A or peroxisome proliferator-activated receptor-γ coactivator-1α small-interfering RNA significantly increased the number of total, and activated, released endothelial microparticles, and shear stress restored these back to basal levels. Collectively, these data demonstrate a critical role of endothelial mitochondrial integrity in preserving endothelial homeostasis. Moreover, prolonged laminar shear stress, which is systemically elevated during aerobic exercise in the vessel wall, mitigates endothelial dysfunction by promoting

  4. Dynamic evolution and biogenesis of small RNAs during sex reversal.

    Science.gov (United States)

    Liu, Jie; Luo, Majing; Sheng, Yue; Hong, Qiang; Cheng, Hanhua; Zhou, Rongjia

    2015-05-06

    Understanding origin, evolution and functions of small RNA (sRNA) genes has been a great challenge in the past decade. Molecular mechanisms underlying sexual reversal in vertebrates, particularly sRNAs involved in this process, are largely unknown. By deep-sequencing of small RNA transcriptomes in combination with genomic analysis, we identified a large amount of piRNAs and miRNAs including over 1,000 novel miRNAs, which were differentially expressed during gonad reversal from ovary to testis via ovotesis. Biogenesis and expressions of miRNAs were dynamically changed during the reversal. Notably, phylogenetic analysis revealed dynamic expansions of miRNAs in vertebrates and an evolutionary trajectory of conserved miR-17-92 cluster in the Eukarya. We showed that the miR-17-92 cluster in vertebrates was generated through multiple duplications from ancestor miR-92 in invertebrates Tetranychus urticae and Daphnia pulex from the Chelicerata around 580 Mya. Moreover, we identified the sexual regulator Dmrt1 as a direct target of the members miR-19a and -19b in the cluster. These data suggested dynamic biogenesis and expressions of small RNAs during sex reversal and revealed multiple expansions and evolutionary trajectory of miRNAs from invertebrates to vertebrates, which implicate small RNAs in sexual reversal and provide new insight into evolutionary and molecular mechanisms underlying sexual reversal.

  5. Effect of regional muscle location but not adiposity on mitochondrial biogenesis-regulating proteins

    DEFF Research Database (Denmark)

    Ponce-González, Jesús Gustavo; Ara, Ignacio; Larsen, Steen

    2016-01-01

    PURPOSE: The aim of this study was to determine if the expression of the mitochondrial biogenesis-regulating proteins SIRT1, SIRT3 and PGC-1alpha in human skeletal muscle is influenced by adiposity. METHOD: Twenty-nine male subjects were recruited into three groups: control (n = 10), obese (n = 10...

  6. Pilus Biogenesis in Lactococcus lactis: Molecular Characterization and Role in Aggregation and Biofilm Formation

    Science.gov (United States)

    Oxaran, Virginie; Ledue-Clier, Florence; Dieye, Yakhya; Herry, Jean-Marie; Péchoux, Christine; Meylheuc, Thierry; Briandet, Romain; Juillard, Vincent; Piard, Jean-Christophe

    2012-01-01

    The genome of Lactococcus lactis strain IL1403 harbors a putative pilus biogenesis cluster consisting of a sortase C gene flanked by 3 LPxTG protein encoding genes (yhgD, yhgE, and yhhB), called here pil. However, pili were not detected under standard growth conditions. Over-expression of the pil operon resulted in production and display of pili on the surface of lactococci. Functional analysis of the pilus biogenesis machinery indicated that the pilus shaft is formed by oligomers of the YhgE pilin, that the pilus cap is formed by the YhgD pilin and that YhhB is the basal pilin allowing the tethering of the pilus fibers to the cell wall. Oligomerization of pilin subunits was catalyzed by sortase C while anchoring of pili to the cell wall was mediated by sortase A. Piliated L. lactis cells exhibited an auto-aggregation phenotype in liquid cultures, which was attributed to the polymerization of major pilin, YhgE. The piliated lactococci formed thicker, more aerial biofilms compared to those produced by non-piliated bacteria. This phenotype was attributed to oligomers of YhgE. This study provides the first dissection of the pilus biogenesis machinery in a non-pathogenic Gram-positive bacterium. Analysis of natural lactococci isolates from clinical and vegetal environments showed pili production under standard growth conditions. The identification of functional pili in lactococci suggests that the changes they promote in aggregation and biofilm formation may be important for the natural lifestyle as well as for applications in which these bacteria are used. PMID:23236417

  7. Control of gene expression and mitochondrial biogenesis in the muscular adaption to endurance exercise

    DEFF Research Database (Denmark)

    Joseph, A. M.; Pilegaard, H.; Leick, L.

    2006-01-01

    of these adaptations is an increase in mitochondrial content, which confers a greater resistance to muscle fatigue. This essay reviews current knowledge on the regulation of exercise-induced mitochondrial biogenesis at the molecular level. The major steps involved include, (i) transcriptional regulation of nuclear......-encoded genes encoding mitochondrial proteins by the coactivator peroxisome-proliferatoractivated receptor coactivator-1, (ii) control of mitochondrial DNA gene 1To whom correspondence should be addressed (email dhood@yorku.ca). 13 © 2006 The Biochemical Society Ch-02_essbiochem_hood.indd Page 13 11/13/06 10......:27:15 PM elhi /Volumes/ju108/POIN001/essbiochem_indd%0/Chapter 2 © 2006 The Biochemical Society 14 Essays in Biochemistry volume 42 2006 expression by the transcription factor Tfam, (iii) mitochondrial fi ssion and fusion mechanisms, and (iv) import of nuclear-derived gene products into the mitochondrion...

  8. Yersinia pestis Targets the Host Endosome Recycling Pathway during the Biogenesis of the Yersinia-Containing Vacuole To Avoid Killing by Macrophages

    Science.gov (United States)

    Connor, Michael G.; Pulsifer, Amanda R.; Ceresa, Brian K.

    2018-01-01

    ABSTRACT Yersinia pestis has evolved many strategies to evade the innate immune system. One of these strategies is the ability to survive within macrophages. Upon phagocytosis, Y. pestis prevents phagolysosome maturation and establishes a modified compartment termed the Yersinia-containing vacuole (YCV). Y. pestis actively inhibits the acidification of this compartment, and eventually, the YCV transitions from a tight-fitting vacuole into a spacious replicative vacuole. The mechanisms to generate the YCV have not been defined. However, we hypothesized that YCV biogenesis requires Y. pestis interactions with specific host factors to subvert normal vesicular trafficking. In order to identify these factors, we performed a genome-wide RNA interference (RNAi) screen to identify host factors required for Y. pestis survival in macrophages. This screen revealed that 71 host proteins are required for intracellular survival of Y. pestis. Of particular interest was the enrichment for genes involved in endosome recycling. Moreover, we demonstrated that Y. pestis actively recruits Rab4a and Rab11b to the YCV in a type three secretion system-independent manner, indicating remodeling of the YCV by Y. pestis to resemble a recycling endosome. While recruitment of Rab4a was necessary to inhibit YCV acidification and lysosomal fusion early during infection, Rab11b appeared to contribute to later stages of YCV biogenesis. We also discovered that Y. pestis disrupts global host endocytic recycling in macrophages, possibly through sequestration of Rab11b, and this process is required for bacterial replication. These data provide the first evidence that Y. pestis targets the host endocytic recycling pathway to avoid phagolysosomal maturation and generate the YCV. PMID:29463656

  9. Leucine Modulates Mitochondrial Biogenesis and SIRT1-AMPK Signaling in C2C12 Myotubes

    Directory of Open Access Journals (Sweden)

    Chunzi Liang

    2014-01-01

    Full Text Available Previous studies from this laboratory demonstrate that dietary leucine protects against high fat diet-induced mitochondrial impairments and stimulates mitochondrial biogenesis and energy partitioning from adipocytes to muscle cells through SIRT1-mediated mechanisms. Moreover, β-hydroxy-β-methyl butyrate (HMB, a metabolite of leucine, has been reported to activate AMPK synergistically with resveratrol in C2C12 myotubes. Therefore, we hypothesize that leucine-induced activation of SIRT1 and AMPK is the central event that links the upregulated mitochondrial biogenesis and fatty acid oxidation in skeletal muscle. Thus, C2C12 myotubes were treated with leucine (0.5 mM, alanine (0.5 mM, valine (0.5 mM, EX527 (SIRT1 inhibitor, 25 μM, and Compound C (AMPK inhibitor, 25 μM alone or in combination to determine the roles of AMPK and SIRT1 in leucine-modulation of energy metabolism. Leucine significantly increased mitochondrial content, mitochondrial biogenesis-related genes expression, fatty acid oxidation, SIRT1 activity and gene expression, and AMPK phosphorylation in C2C12 myotubes compared to the controls, while EX527 and Compound C markedly attenuated these effects. Furthermore, leucine treatment for 24 hours resulted in time-dependent increases in cellular NAD+, SIRT1 activity, and p-AMPK level, with SIRT1 activation preceding that of AMPK, indicating that leucine activation of SIRT1, rather than AMPK, is the primary event.

  10. A fluorescent probe distinguishes between inhibition of early and late steps of lipopolysaccharide biogenesis in whole cells

    Science.gov (United States)

    Moison, Eileen; Xie, Ran; Zhang, Ge; Lebar, Matthew D.; Meredith, Timothy C.; Kahne, Daniel

    2017-01-01

    Lipopolysaccharide (LPS) biogenesis in Gram-negative organisms involves its biosynthesis in the cytoplasm and subsequent transport across three cellular compartments to the cell surface. We developed a fluorescent probe that allows us to determine the spatial distribution of LPS in whole cells. We show that polymyxin B nonapeptide (PMBN) containing a dansyl fluorophore specifically binds to LPS in membranes. We show that this probe detects decreases in LPS levels on the cell surface when LPS biosynthesis is inhibited at an early step. We also can detect accumulation of LPS in particular subcellular locations when LPS assembly is blocked during transport, allowing us to differentiate inhibitors targeting early and late stages of LPS biogenesis. PMID:28248483

  11. The synthesis of glutamic acid in the absence of enzymes: Implications for biogenesis

    Science.gov (United States)

    Morowitz, Harold; Peterson, Eta; Chang, Sherwood

    1995-01-01

    This paper reports on the non-enzymatic aqueous phase synthesis of amino acids from keto acids, ammonia and reducing agents. The facile synthesis of key metabolic intermediates, particularly in the glycolytic pathway, the citric acid cycle, and the first step of amino acid synthesis, lead to new ways of looking at the problem of biogenesis.

  12. Bioinformatic analysis of microRNA biogenesis and function related proteins in eleven animal genomes.

    Science.gov (United States)

    Liu, Xiuying; Luo, GuanZheng; Bai, Xiujuan; Wang, Xiu-Jie

    2009-10-01

    MicroRNAs are approximately 22 nt long small non-coding RNAs that play important regulatory roles in eukaryotes. The biogenesis and functional processes of microRNAs require the participation of many proteins, of which, the well studied ones are Dicer, Drosha, Argonaute and Exportin 5. To systematically study these four protein families, we screened 11 animal genomes to search for genes encoding above mentioned proteins, and identified some new members for each family. Domain analysis results revealed that most proteins within the same family share identical or similar domains. Alternative spliced transcript variants were found for some proteins. We also examined the expression patterns of these proteins in different human tissues and identified other proteins that could potentially interact with these proteins. These findings provided systematic information on the four key proteins involved in microRNA biogenesis and functional pathways in animals, and will shed light on further functional studies of these proteins.

  13. A Heme-Sensing Mechanism in the Translational Regulation of Mitochondrial Cytochrome c Oxidase Biogenesis

    Science.gov (United States)

    Soto, Iliana C.; Fontanesi, Flavia; Myers, Richard S.; Hamel, Patrice; Barrientos, Antoni

    2012-01-01

    Heme plays fundamental roles as cofactor and signaling molecule in multiple pathways devoted to oxygen sensing and utilization in aerobic organisms. For cellular respiration, heme serves as a prosthetic group in electron transfer proteins and redox enzymes. Here we report that in the yeast Saccharomyces cerevisiae a heme-sensing mechanism translationally controls the biogenesis of cytochrome c oxidase (COX), the terminal mitochondrial respiratory chain enzyme. We show that Mss51, a COX1 mRNA-specific translational activator and Cox1 chaperone, which coordinates Cox1 synthesis in mitoribosomes with its assembly in COX, is a heme-binding protein. Mss51 contains two heme regulatory motifs or Cys-Pro-X domains located in its N-terminus. Using a combination of in vitro and in vivo approaches, we have demonstrated that these motifs are important for heme binding and efficient performance of Mss51 functions. We conclude that heme sensing by Mss51 regulates COX biogenesis and aerobic energy production. PMID:23217259

  14. Acid sphingomyelinase modulates the autophagic process by controlling lysosomal biogenesis in Alzheimer's disease.

    Science.gov (United States)

    Lee, Jong Kil; Jin, Hee Kyung; Park, Min Hee; Kim, Bo-ra; Lee, Phil Hyu; Nakauchi, Hiromitsu; Carter, Janet E; He, Xingxuan; Schuchman, Edward H; Bae, Jae-sung

    2014-07-28

    In Alzheimer's disease (AD), abnormal sphingolipid metabolism has been reported, although the pathogenic consequences of these changes have not been fully characterized. We show that acid sphingomyelinase (ASM) is increased in fibroblasts, brain, and/or plasma from patients with AD and in AD mice, leading to defective autophagic degradation due to lysosomal depletion. Partial genetic inhibition of ASM (ASM(+/-)) in a mouse model of familial AD (FAD; amyloid precursor protein [APP]/presenilin 1 [PS1]) ameliorated the autophagocytic defect by restoring lysosomal biogenesis, resulting in improved AD clinical and pathological findings, including reduction of amyloid-β (Aβ) deposition and improvement of memory impairment. Similar effects were noted after pharmacologic restoration of ASM to the normal range in APP/PS1 mice. Autophagic dysfunction in neurons derived from FAD patient induced pluripotent stem cells (iPSCs) was restored by partial ASM inhibition. Overall, these results reveal a novel mechanism of ASM pathogenesis in AD that leads to defective autophagy due to impaired lysosomal biogenesis and suggests that partial ASM inhibition is a potential new therapeutic intervention for the disease. © 2014 Lee et al.

  15. PGC-1α is dispensable for exercise-induced mitochondrial biogenesis in skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Glenn C Rowe

    Full Text Available Exercise confers numerous health benefits, many of which are thought to stem from exercise-induced mitochondrial biogenesis (EIMB in skeletal muscle. The transcriptional coactivator PGC-1α, a potent regulator of metabolism in numerous tissues, is widely believed to be required for EIMB. We show here that this is not the case. Mice engineered to lack PGC-1α specifically in skeletal muscle (Myo-PGC-1αKO mice retained intact EIMB. The exercise capacity of these mice was comparable to littermate controls. Induction of metabolic genes after 2 weeks of in-cage voluntary wheel running was intact. Electron microscopy revealed no gross abnormalities in mitochondria, and the mitochondrial biogenic response to endurance exercise was as robust in Myo-PGC-1αKO mice as in wildtype mice. The induction of enzymatic activity of the electron transport chain by exercise was likewise unperturbed in Myo-PGC-1αKO mice. These data demonstrate that PGC-1α is dispensable for exercise-induced mitochondrial biogenesis in skeletal muscle, in sharp contrast to the prevalent assumption in the field.

  16. High-throughput identification of chemical inhibitors of E. coli Group 2 capsule biogenesis as anti-virulence agents.

    Directory of Open Access Journals (Sweden)

    Carlos C Goller

    Full Text Available Rising antibiotic resistance among Escherichia coli, the leading cause of urinary tract infections (UTIs, has placed a new focus on molecular pathogenesis studies, aiming to identify new therapeutic targets. Anti-virulence agents are attractive as chemotherapeutics to attenuate an organism during disease but not necessarily during benign commensalism, thus decreasing the stress on beneficial microbial communities and lessening the emergence of resistance. We and others have demonstrated that the K antigen capsule of E. coli is a preeminent virulence determinant during UTI and more invasive diseases. Components of assembly and export are highly conserved among the major K antigen capsular types associated with UTI-causing E. coli and are distinct from the capsule biogenesis machinery of many commensal E. coli, making these attractive therapeutic targets. We conducted a screen for anti-capsular small molecules and identified an agent designated "C7" that blocks the production of K1 and K5 capsules, unrelated polysaccharide types among the Group 2-3 capsules. Herein lies proof-of-concept that this screen may be implemented with larger chemical libraries to identify second-generation small-molecule inhibitors of capsule biogenesis. These inhibitors will lead to a better understanding of capsule biogenesis and may represent a new class of therapeutics.

  17. Gender-specific hierarchy in nuage localization of PIWI-interacting RNA factors in Drosophila

    Directory of Open Access Journals (Sweden)

    Mikiko C Siomi

    2011-08-01

    Full Text Available PIWI-interacting RNAs (piRNAs are germline-specific small non-coding RNAs that form piRNA-induced silencing complexes (piRISCs by associating with PIWI proteins, a subclade of the Argonaute proteins predominantly expressed in the germline. piRISCs protect the integrity of the germline genome from invasive transposable DNA elements by silencing them. Multiple piRNA biogenesis factors have been identified in Drosophila. The majority of piRNA factors are localized in the nuage, electron-dense non-membranous cytoplasmic structures located in the perinuclear regions of germ cells. Thus, piRNA biogenesis is thought to occur in the nuage in germ cells. Immunofluorescence analyses of ovaries from piRNA factor mutants have revealed a localization hierarchy of piRNA factors in female nuage. However, whether this hierarchy is female-specific or can also be applied in male gonads remains undetermined. Here, we show by immunostaining of both ovaries and testes from piRNA factor mutants that the molecular hierarchy of piRNA factors shows gender-specificity, especially for Krimper (Krimp, a Tudor-domain containing protein of unknown function(s: Krimp is dispensable for PIWI protein Aubergine (Aub nuage localization in ovaries but Krimp and Aub require each other for their proper nuage localization in testes. This suggests that the functional requirement of Krimp in piRNA biogenesis may be different in male and female gonads.

  18. Studies on the role of NonA in mRNA biogenesis

    International Nuclear Information System (INIS)

    Kozlova, Natalia; Braga, Jose; Lundgren, Josefin; Rino, Jose; Young, Patrick; Carmo-Fonseca, Maria; Visa, Neus

    2006-01-01

    The NonA protein of Drosophila melanogaster is an abundant nuclear protein that belongs to the DBHS (Drosophila behavior, human splicing) protein family. The DBHS proteins bind both DNA and RNA in vitro and have been involved in different aspects of gene expression, including pre-mRNA splicing, transcription regulation and nuclear retention of mRNA. We have used double-stranded RNA interference in Drosophila S2 cells to silence the expression of NonA and to investigate its role in mRNA biogenesis. We show that knockdown of NonA does not affect transcription nor splicing. We demonstrate that NonA forms a complex with the essential nuclear export factor NXF1 in an RNA-dependent manner. We have constructed stable S2 cell lines that express full-length and truncated NXF1 fused to GFP in order to perform fluorescence recovery after photobleaching experiments. We show that knockdown of NonA reduces the intranuclear mobility of NXF1-GFP associated with poly(A) + RNA in vivo, while the mobility of the truncated NXF1-GFP that does not bind RNA is not affected. Our data suggest that NonA facilitates the intranuclear mobility of mRNP particles

  19. A dsRNA-binding protein MdDRB1 associated with miRNA biogenesis modifies adventitious rooting and tree architecture in apple.

    Science.gov (United States)

    You, Chun-Xiang; Zhao, Qiang; Wang, Xiao-Fei; Xie, Xing-Bin; Feng, Xiao-Ming; Zhao, Ling-Ling; Shu, Huai-Rui; Hao, Yu-Jin

    2014-02-01

    Although numerous miRNAs have been already isolated from fruit trees, knowledge about miRNA biogenesis is largely unknown in fruit trees. Double-strand RNA-binding (DRB) protein plays an important role in miRNA processing and maturation; however, its role in the regulation of economically important traits is not clear yet in fruit trees. EST blast and RACE amplification were performed to isolate apple MdDRB1 gene. Following expression analysis, RNA binding and protein interaction assays, MdDRB1 was transformed into apple callus and in vitro tissue cultures to characterize the functions of MdDRB1 in miRNA biogenesis, adventitious rooting, leaf development and tree growth habit. MdDRB1 contained two highly conserved DRB domains. Its transcripts existed in all tissues tested and are induced by hormones. It bound to double-strand RNAs and interacted with AtDCL1 (Dicer-Like 1) and MdDCL1. Chip assay indicated its role in miRNA biogenesis. Transgenic analysis showed that MdDRB1 controls adventitious rooting, leaf curvature and tree architecture by modulating the accumulation of miRNAs and the transcript levels of miRNA target genes. Our results demonstrated that MdDRB1 functions in the miRNA biogenesis in a conserved way and that it is a master regulator in the formation of economically important traits in fruit trees. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  20. Alternative Sigma Factors SigF, SigE, and SigG Are Essential for Sporulation in Clostridium botulinum ATCC 3502

    OpenAIRE

    Kirk, David G.; Zhang, Zhen; Korkeala, Hannu; Lindström, Miia

    2014-01-01

    Clostridium botulinum produces heat-resistant endospores that may germinate and outgrow into neurotoxic cultures in foods. Sporulation is regulated by the transcription factor Spo0A and the alternative sigma factors SigF, SigE, SigG, and SigK in most spore formers studied to date. We constructed mutants of sigF, sigE, and sigG in C. botulinum ATCC 3502 and used quantitative reverse transcriptase PCR and electron microscopy to assess their expression of the sporulation pathway on transcription...

  1. Cox1 mutation abrogates need for Cox23 in cytochrome c oxidase biogenesis

    Directory of Open Access Journals (Sweden)

    Richard Dela Cruz

    2016-06-01

    Full Text Available Cox23 is a known conserved assembly factor for cytochrome c oxidase, although its role in cytochrome c oxidase (CcO biogenesis remains unresolved. To gain additional insights into its role, we isolated spontaneous suppressors of the respiratory growth defect in cox23∆ yeast cells. We recovered independent colonies that propagated on glycerol/lactate medium for cox23∆ cells at 37°C. We mapped these mutations to the mitochondrial genome and specifically to COX1 yielding an I101F substitution. The I101F Cox1 allele is a gain-of-function mutation enabling yeast to respire in the absence of Cox23. CcO subunit steady-state levels were restored with the I101F Cox1 suppressor mutation and oxygen consumption and CcO activity were likewise restored. Cells harboring the mitochondrial genome encoding I101F Cox1 were used to delete genes for other CcO assembly factors to test the specificity of the Cox1 mutation as a suppressor of cox23∆ cells. The Cox1 mutant allele fails to support respiratory growth in yeast lacking Cox17, Cox19, Coa1, Coa2, Cox14 or Shy1, demonstrating its specific suppressor activity for cox23∆ cells.

  2. Interleukin 6 downregulates p53 expression and activity by stimulating ribosome biogenesis: a new pathway connecting inflammation to cancer

    Science.gov (United States)

    Brighenti, E; Calabrese, C; Liguori, G; Giannone, F A; Trerè, D; Montanaro, L; Derenzini, M

    2014-01-01

    Chronic inflammation is an established risk factor for the onset of cancer, and the inflammatory cytokine IL-6 has a role in tumorigenesis by enhancing proliferation and hindering apoptosis. As factors stimulating proliferation also downregulate p53 expression by enhancing ribosome biogenesis, we hypothesized that IL-6 may cause similar changes in inflamed tissues, thus activating a mechanism that favors neoplastic transformation. Here, we showed that IL-6 downregulated the expression and activity of p53 in transformed and untransformed human cell lines. This was the consequence of IL-6-dependent stimulation of c-MYC mRNA translation, which was responsible for the upregulation of rRNA transcription. The enhanced rRNA transcription stimulated the MDM2-mediated proteasomal degradation of p53, by reducing the availability of ribosome proteins for MDM2 binding. The p53 downregulation induced the acquisition of cellular phenotypic changes characteristic of epithelial–mesenchymal transition, such as a reduced level of E-cadherin expression, increased cell invasiveness and a decreased response to cytotoxic stresses. We found that these changes also occurred in colon epithelial cells of patients with ulcerative colitis, a very representative example of chronic inflammation at high risk for tumor development. Histochemical and immunohistochemical analysis of colon biopsy samples showed an upregulation of ribosome biogenesis, a reduced expression of p53, together with a focal reduction or absence of E-cadherin expression in chronic colitis in comparison with normal mucosa samples. These changes disappeared after treatment with anti-inflammatory drugs. Taken together, the present results highlight a new mechanism that may link chronic inflammation to cancer, based on p53 downregulation, which is activated by the enhancement of rRNA transcription upon IL-6 exposure. PMID:24531714

  3. Common genetic polymorphisms of microRNA biogenesis pathway genes and breast cancer survival

    International Nuclear Information System (INIS)

    Sung, Hyuna; Ahn, Sei-Hyun; Kang, Daehee; Jeon, Sujee; Lee, Kyoung-Mu; Han, Sohee; Song, Minkyo; Choi, Ji-Yeob; Park, Sue K; Yoo, Keun-Young; Noh, Dong-Young

    2012-01-01

    Although the role of microRNA’s (miRNA’s) biogenesis pathway genes in cancer development and progression has been well established, the association between genetic variants of this pathway genes and breast cancer survival is still unknown. We used genotype data available from a previously conducted case–control study to investigate association between common genetic variations in miRNA biogenesis pathway genes and breast cancer survival. We investigated the possible associations between 41 germ-line single-nucleotide polymorphisms (SNPs) and both disease free survival (DFS) and overall survival (OS) among 488 breast cancer patients. During the median follow-up of 6.24 years, 90 cases developed disease progression and 48 cases died. Seven SNPs were significantly associated with breast cancer survival. Two SNPs in AGO2 (rs11786030 and rs2292779) and DICER1 rs1057035 were associated with both DFS and OS. Two SNPs in HIWI (rs4759659 and rs11060845) and DGCR8 rs9606250 were associated with DFS, while DROSHA rs874332 and GEMIN4 rs4968104 were associated with only OS. The most significant association was observed in variant allele of AGO2 rs11786030 with 2.62-fold increased risk of disease progression (95% confidence interval (CI), 1.41-4.88) and in minor allele homozygote of AGO2 rs2292779 with 2.94-fold increased risk of death (95% CI, 1.52-5.69). We also found cumulative effects of SNPs on DFS and OS. Compared to the subjects carrying 0 to 2 high-risk genotypes, those carrying 3 or 4–6 high-risk genotypes had an increased risk of disease progression with a hazard ratio of 2.16 (95% CI, 1.18- 3.93) and 4.47 (95% CI, 2.45- 8.14), respectively (P for trend, 6.11E-07). Our results suggest that genetic variants in miRNA biogenesis pathway genes may be associated with breast cancer survival. Further studies in larger sample size and functional characterizations are warranted to validate these results

  4. Effect of dietary resveratrol supplementation on meat quality, muscle antioxidative capacity and mitochondrial biogenesis of broilers.

    Science.gov (United States)

    Zhang, Cheng; Yang, Lei; Zhao, Xiaohui; Chen, Xingyong; Wang, Li; Geng, Zhaoyu

    2018-02-01

    The naturally occurring polyphenol resveratrol has been acknowledged with many beneficial biological effects. The aim of this study was to evaluate the influence of dietary resveratrol supplementation on meat quality, muscle antioxidative capacity and mitochondrial biogenesis of broilers. One hundred and eighty 21-day-old male Cobb broilers were randomly assigned to two groups and fed on a 0 mg kg -1 or 400 mg kg -1 resveratrol-supplemented diet for 21 days. Then, chickens were slaughtered and pectoralis major muscle (PM) samples were collected for analysis. The results showed that resveratrol not only tended to increase (P resveratrol, while malondialdehyde content was decreased (P resveratrol significantly increased (P Resveratrol can be used as a feed additive to improve meat quality of broilers, which may be associated with improved muscle antioxidative status and mitochondrial biogenesis. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  5. ABI4 regulates primary seed dormancy by regulating the biogenesis of abscisic acid and gibberellins in arabidopsis.

    Directory of Open Access Journals (Sweden)

    Kai Shu

    2013-06-01

    Full Text Available Seed dormancy is an important economic trait for agricultural production. Abscisic acid (ABA and Gibberellins (GA are the primary factors that regulate the transition from dormancy to germination, and they regulate this process antagonistically. The detailed regulatory mechanism involving crosstalk between ABA and GA, which underlies seed dormancy, requires further elucidation. Here, we report that ABI4 positively regulates primary seed dormancy, while negatively regulating cotyledon greening, by mediating the biogenesis of ABA and GA. Seeds of the Arabidopsis abi4 mutant that were subjected to short-term storage (one or two weeks germinated significantly more quickly than Wild-Type (WT, and abi4 cotyledons greened markedly more quickly than WT, while the rates of germination and greening were comparable when the seeds were subjected to longer-term storage (six months. The ABA content of dry abi4 seeds was remarkably lower than that of WT, but the amounts were comparable after stratification. Consistently, the GA level of abi4 seeds was increased compared to WT. Further analysis showed that abi4 was resistant to treatment with paclobutrazol (PAC, a GA biosynthesis inhibitor, during germination, while OE-ABI4 was sensitive to PAC, and exogenous GA rescued the delayed germination phenotype of OE-ABI4. Analysis by qRT-PCR showed that the expression of genes involved in ABA and GA metabolism in dry and germinating seeds corresponded to hormonal measurements. Moreover, chromatin immunoprecipitation qPCR (ChIP-qPCR and transient expression analysis showed that ABI4 repressed CYP707A1 and CYP707A2 expression by directly binding to those promoters, and the ABI4 binding elements are essential for this repression. Accordingly, further genetic analysis showed that abi4 recovered the delayed germination phenotype of cyp707a1 and cyp707a2 and further, rescued the non-germinating phenotype of ga1-t. Taken together, this study suggests that ABI4 is a key

  6. Induction of mitochondrial biogenesis and respiration is associated with mTOR regulation in hepatocytes of rats treated with the pan-PPAR activator tetradecylthioacetic acid (TTA)

    Energy Technology Data Exchange (ETDEWEB)

    Hagland, Hanne R.; Nilsson, Linn I.H. [Department of Biomedicine, University of Bergen (Norway); Burri, Lena [Institute of Medicine, University of Bergen, Haukeland University Hospital (Norway); Nikolaisen, Julie [Department of Biomedicine, University of Bergen (Norway); Berge, Rolf K. [Institute of Medicine, University of Bergen, Haukeland University Hospital (Norway); Department of Heart Disease, Haukeland University Hospital (Norway); Tronstad, Karl J., E-mail: karl.tronstad@biomed.uib.no [Department of Biomedicine, University of Bergen (Norway)

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer We investigated mechanisms of mitochondrial regulation in rat hepatocytes. Black-Right-Pointing-Pointer Tetradecylthioacetic acid (TTA) was employed to activate mitochondrial oxidation. Black-Right-Pointing-Pointer Mitochondrial biogenesis and respiration were induced. Black-Right-Pointing-Pointer It was confirmed that PPAR target genes were induced. Black-Right-Pointing-Pointer The mechanism involved activation mTOR. -- Abstract: The hypolipidemic effect of peroxisome proliferator-activated receptor (PPAR) activators has been explained by increasing mitochondrial fatty acid oxidation, as observed in livers of rats treated with the pan-PPAR activator tetradecylthioacetic acid (TTA). PPAR-activation does, however, not fully explain the metabolic adaptations observed in hepatocytes after treatment with TTA. We therefore characterized the mitochondrial effects, and linked this to signalling by the metabolic sensor, the mammalian target of rapamycin (mTOR). In hepatocytes isolated from TTA-treated rats, the changes in cellular content and morphology were consistent with hypertrophy. This was associated with induction of multiple mitochondrial biomarkers, including mitochondrial DNA, citrate synthase and mRNAs of mitochondrial proteins. Transcription analysis further confirmed activation of PPAR{alpha}-associated genes, in addition to genes related to mitochondrial biogenesis and function. Analysis of mitochondrial respiration revealed that the capacity of both electron transport and oxidative phosphorylation were increased. These effects coincided with activation of the stress related factor, ERK1/2, and mTOR. The protein level and phosphorylation of the downstream mTOR actors eIF4G and 4E-BP1 were induced. In summary, TTA increases mitochondrial respiration by inducing hypertrophy and mitochondrial biogenesis in rat hepatocytes, via adaptive regulation of PPARs as well as mTOR.

  7. Isolation of Penicillium chrysogenum PEX1 and PEX6 encoding AAA proteins involved in peroxisome biogenesis

    NARCIS (Netherlands)

    Kiel, JAKW; Hilbrands, RE; Bovenberg, RAL; Veenhuis, M

    In Penicillium chrysogenum, key enzymes involved in the production of penicillin reside in peroxisomes. As a first step to understand the role of these organelles in penicillin biosynthesis, we set out to isolate the genes involved in peroxisome biogenesis. Here we report the cloning and

  8. AKT3 controls mitochondrial biogenesis and autophagy via regulation of the major nuclear export protein CRM-1.

    Science.gov (United States)

    Corum, Daniel G; Tsichlis, Philip N; Muise-Helmericks, Robin C

    2014-01-01

    Our previous work has shown that Akt3 is required for mitochondrial biogenesis in primary human endothelial cells (ECs) and in Akt3-null mice; Akt3 affects subcellular localization of peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1α), the master regulator of mitochondrial biogenesis. The purpose of this study is to determine the mechanism by which Akt3 controls the subcellular distribution of PGC-1α and to explore the effect on mitochondrial biogenesis and turnover during angiogenesis. Here we use standard biochemical analyses and Akt3-knockdown strategies to show that Akt3 controls the stabilization of chromosome maintenance region-1 (CRM-1), the major nuclear export receptor. Site-directed mutagenesis and association analyses show that PGC-1α nuclear export is CRM-1 dependent. Akt3 knockdown and CRM-1 overexpression cause 3-fold reductions in PGC-1α target gene expression, compared to control levels. Akt3 inhibition causes autophagy, as measured by autophagosome formation, in a CRM-1-dependent, Akt1/mTOR-independent pathway. In vivo, Akt3-null and heterozygous mice show dose-dependent decreases in angiogenesis compared to wild-type littermates (~5- and 2.5-fold decreases, respectively), as assessed by Matrigel plug assays. This correlates with an ~1.5-fold decrease in mitochondrial Cox IV expression. Our studies suggest that Akt3 is a regulator of mitochondrial dynamics in the vasculature via regulation of CRM-1-dependent nuclear export.

  9. The Extracellular Vesicles of the Helminth Pathogen, Fasciola hepatica: Biogenesis Pathways and Cargo Molecules Involved in Parasite Pathogenesis*

    Science.gov (United States)

    Cwiklinski, Krystyna; de la Torre-Escudero, Eduardo; Trelis, Maria; Bernal, Dolores; Dufresne, Philippe J.; Brennan, Gerard P.; O'Neill, Sandra; Tort, Jose; Paterson, Steve; Marcilla, Antonio; Dalton, John P.; Robinson, Mark W.

    2015-01-01

    Extracellular vesicles (EVs) released by parasites have important roles in establishing and maintaining infection. Analysis of the soluble and vesicular secretions of adult Fasciola hepatica has established a definitive characterization of the total secretome of this zoonotic parasite. Fasciola secretes at least two subpopulations of EVs that differ according to size, cargo molecules and site of release from the parasite. The larger EVs are released from the specialized cells that line the parasite gastrodermus and contain the zymogen of the 37 kDa cathepsin L peptidase that performs a digestive function. The smaller exosome-like vesicle population originate from multivesicular bodies within the tegumental syncytium and carry many previously described immunomodulatory molecules that could be delivered into host cells. By integrating our proteomics data with recently available transcriptomic data sets we have detailed the pathways involved with EV biogenesis in F. hepatica and propose that the small exosome biogenesis occurs via ESCRT-dependent MVB formation in the tegumental syncytium before being shed from the apical plasma membrane. Furthermore, we found that the molecular “machinery” required for EV biogenesis is constitutively expressed across the intramammalian development stages of the parasite. By contrast, the cargo molecules packaged within the EVs are developmentally regulated, most likely to facilitate the parasites migration through host tissue and to counteract host immune attack. PMID:26486420

  10. Increased biogenesis of glucagon-containing secretory granules and glucagon secretion in BIG3-knockout mice

    Directory of Open Access Journals (Sweden)

    Hongyu Li

    2015-03-01

    Conclusions: Together with our previous studies, the current data reveal a conserved role for BIG3 in regulating alpha- and beta-cell functions. We propose that BIG3 negatively regulates hormone production at the secretory granule biogenesis stage and that such regulatory mechanism may be used in secretory pathways of other endocrine cells.

  11. Progranulin regulates lysosomal function and biogenesis through acidification of lysosomes.

    Science.gov (United States)

    Tanaka, Yoshinori; Suzuki, Genjiro; Matsuwaki, Takashi; Hosokawa, Masato; Serrano, Geidy; Beach, Thomas G; Yamanouchi, Keitaro; Hasegawa, Masato; Nishihara, Masugi

    2017-03-01

    Progranulin (PGRN) haploinsufficiency resulting from loss-of-function mutations in the PGRN gene causes frontotemporal lobar degeneration accompanied by TDP-43 accumulation, and patients with homozygous mutations in the PGRN gene present with neuronal ceroid lipofuscinosis. Although it remains unknown why PGRN deficiency causes neurodegenerative diseases, there is increasing evidence that PGRN is implicated in lysosomal functions. Here, we show PGRN is a secretory lysosomal protein that regulates lysosomal function and biogenesis by controlling the acidification of lysosomes. PGRN gene expression and protein levels increased concomitantly with the increase of lysosomal biogenesis induced by lysosome alkalizers or serum starvation. Down-regulation or insufficiency of PGRN led to the increased lysosomal gene expression and protein levels, while PGRN overexpression led to the decreased lysosomal gene expression and protein levels. In particular, the level of mature cathepsin D (CTSDmat) dramatically changed depending upon PGRN levels. The acidification of lysosomes was facilitated in cells transfected with PGRN. Then, this caused degradation of CTSDmat by cathepsin B. Secreted PGRN is incorporated into cells via sortilin or cation-independent mannose 6-phosphate receptor, and facilitated the acidification of lysosomes and degradation of CTSDmat. Moreover, the change of PGRN levels led to a cell-type-specific increase of insoluble TDP-43. In the brain tissue of FTLD-TDP patients with PGRN deficiency, CTSD and phosphorylated TDP-43 accumulated in neurons. Our study provides new insights into the physiological function of PGRN and the role of PGRN insufficiency in the pathogenesis of neurodegenerative diseases. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  12. A comparison of the endotoxin biosynthesis and protein oxidation pathways in the biogenesis of the outer membrane of Escherichia coli and Neisseria meningitidis

    Directory of Open Access Journals (Sweden)

    Susannah ePiek

    2012-12-01

    Full Text Available The Gram-negative bacterial cell envelope consists of an inner membrane (IM that surrounds the cytoplasm, and an asymmetrical outer-membrane (OM that forms a protective barrier to the external environment. The OM consists of lipopolysaccahride (LPS, phospholipids, outer membrane proteins (OMPs and lipoproteins. Oxidative protein folding mediated by periplasmic oxidoreductases is required for the correct biogenesis of the protein components, mainly constituents of virulence determinants such as pili, flagella and toxins, of the Gram-negative OM. Recently, periplasmic oxidoreductases have been implicated in LPS biogenesis of Escherichia coli and Neisseria meningitidis. Differences in OM biogenesis, in particular the transport pathways for endotoxin to the OM, the composition and role of the protein oxidation and isomerisation pathways and the regulatory networks that control them have been found in these two Gram-negative species suggesting that although form and function of the OM is conserved, these conserved pathways have been modified to suit the lifestyle of each organism.

  13. The Arabidopsis gene DIG6 encodes a large 60S subunit nuclear export GTPase 1 that is involved in ribosome biogenesis and affects multiple auxin-regulated development processes

    KAUST Repository

    Zhao, Huayan

    2015-08-13

    The circularly permuted GTPase large subunit GTPase 1 (LSG1) is involved in the maturation step of the 60S ribosome and is essential for cell viability in yeast. Here, an Arabidopsis mutant dig6 (drought inhibited growth of lateral roots) was isolated. The mutant exhibited multiple auxin-related phenotypes, which included reduced lateral root number, altered leaf veins, and shorter roots. Genetic mapping combined with next-generation DNA sequencing identified that the mutation occurred in AtLSG1-2. This gene was highly expressed in regions of auxin accumulation. Ribosome profiling revealed that a loss of function of AtLSG1-2 led to decreased levels of monosomes, further demonstrating its role in ribosome biogenesis. Quantitative proteomics showed that the expression of certain proteins involved in ribosome biogenesis was differentially regulated, indicating that ribosome biogenesis processes were impaired in the mutant. Further investigations showed that an AtLSG1-2 deficiency caused the alteration of auxin distribution, response, and transport in plants. It is concluded that AtLSG1-2 is integral to ribosome biogenesis, consequently affecting auxin homeostasis and plant development.

  14. The Arabidopsis gene DIG6 encodes a large 60S subunit nuclear export GTPase 1 that is involved in ribosome biogenesis and affects multiple auxin-regulated development processes

    KAUST Repository

    Zhao, Huayan; Lü , Shiyou; Li, Ruixi; Chen, Tao; Zhang, Huoming; Cui, Peng; Ding, Feng; Liu, Pei; Wang, Guangchao; Xia, Yiji; Running, Mark P.; Xiong, Liming

    2015-01-01

    The circularly permuted GTPase large subunit GTPase 1 (LSG1) is involved in the maturation step of the 60S ribosome and is essential for cell viability in yeast. Here, an Arabidopsis mutant dig6 (drought inhibited growth of lateral roots) was isolated. The mutant exhibited multiple auxin-related phenotypes, which included reduced lateral root number, altered leaf veins, and shorter roots. Genetic mapping combined with next-generation DNA sequencing identified that the mutation occurred in AtLSG1-2. This gene was highly expressed in regions of auxin accumulation. Ribosome profiling revealed that a loss of function of AtLSG1-2 led to decreased levels of monosomes, further demonstrating its role in ribosome biogenesis. Quantitative proteomics showed that the expression of certain proteins involved in ribosome biogenesis was differentially regulated, indicating that ribosome biogenesis processes were impaired in the mutant. Further investigations showed that an AtLSG1-2 deficiency caused the alteration of auxin distribution, response, and transport in plants. It is concluded that AtLSG1-2 is integral to ribosome biogenesis, consequently affecting auxin homeostasis and plant development.

  15. Emerging roles and regulation of MiT/TFE transcriptional factors.

    Science.gov (United States)

    Yang, Min; Liu, En; Tang, Li; Lei, Yuanyuan; Sun, Xuemei; Hu, Jiaxi; Dong, Hui; Yang, Shi-Ming; Gao, Mingfa; Tang, Bo

    2018-06-15

    The MiT/TFE transcription factors play a pivotal role in the regulation of autophagy and lysosomal biogenesis. The subcellular localization and activity of MiT/TFE proteins are primarily regulated through phosphorylation. And the phosphorylated protein is retained in the cytoplasm and subsequently translocates to the nucleus upon dephosphorylation, where it stimulates the expression of hundreds of genes, leading to lysosomal biogenesis and autophagy induction. The transcription factor-mediated lysosome-to-nucleus signaling can be directly controlled by several signaling molecules involved in the mTORC1, PKC, and AKT pathways. MiT/TFE family members have attracted much attention owing to their intracellular clearance of pathogenic factors in numerous diseases. Recently, multiple studies have also revealed the MiT/TFE proteins as master regulators of cellular metabolic reprogramming, converging on autophagic and lysosomal function and playing a critical role in cancer, suggesting that novel therapeutic strategies could be based on the modulation of MiT/TFE family member activity. Here, we present an overview of the latest research on MiT/TFE transcriptional factors and their potential mechanisms in cancer.

  16. Adaptation of motor unit contractile properties in rat medial gastrocnemius to treadmill endurance training: Relationship to muscle mitochondrial biogenesis.

    Science.gov (United States)

    Kryściak, Katarzyna; Majerczak, Joanna; Kryściak, Jakub; Łochyński, Dawid; Kaczmarek, Dominik; Drzymała-Celichowska, Hanna; Krutki, Piotr; Gawedzka, Anna; Guzik, Magdalena; Korostynski, Michał; Szkutnik, Zbigniew; Pyza, Elżbieta; Jarmuszkiewicz, Wiesława; Zoladz, Jerzy A; Celichowski, Jan

    2018-01-01

    This study aimed at investigating the effects of 2, 4 and 8 weeks of endurance training on the contractile properties of slow (S), fast fatigue resistant (FR) and fast fatigable (FF) motor units (MUs) in rat medial gastrocnemius (MG) in relation to the changes in muscle mitochondrial biogenesis. The properties of functionally isolated MUs were examined in vivo. Mitochondrial biogenesis was judged based on the changes in mitochondrial DNA copy number (mtDNA), the content of the electron transport chain (ETC) proteins and PGC-1α in the MG. Moreover, the markers of mitochondria remodeling mitofusins (Mfn1, Mfn2) and dynamin-like protein (Opa1) were studied using qPCR. A proportion of FR MUs increased from 37.9% to 50.8% and a proportion of FF units decreased from 44.7% to 26.6% after 8 weeks of training. The increased fatigue resistance, shortened twitch duration, and increased ability to potentiate force were found as early as after 2 weeks of endurance training, predominantly in FR MUs. Moreover, just after 2 weeks of the training an enhancement of the mitochondrial network remodeling was present as judged by an increase in expression of Mfn1, Opa1 and an increase in PGC-1α in the slow part of MG. Interestingly, no signs of intensification of mitochondrial biogenesis assessed by ETC proteins content and mtDNA in slow and fast parts of gastrocnemius were found at this stage of the training. Nevertheless, after 8 weeks of training an increase in the ETC protein content was observed, but mainly in the slow part of gastrocnemius. Concluding, the functional changes in MUs' contractile properties leading to the enhancement of muscle performance accompanied by an activation of signalling that controls the muscle mitochondrial network reorganisation and mitochondrial biogenesis belong to an early muscle adaptive responses that precede an increase in mitochondrial ETC protein content.

  17. Subcellular localization and logistics of integral membrane protein biogenesis in Escherichia coli.

    Science.gov (United States)

    Bogdanov, Mikhail; Aboulwafa, Mohammad; Saier, Milton H

    2013-01-01

    Transporters catalyze entry and exit of molecules into and out of cells and organelles, and protein-lipid interactions influence their activities. The bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS) catalyzes transport-coupled sugar phosphorylation as well as nonvectorial sugar phosphorylation in the cytoplasm. The vectorial process is much more sensitive to the lipid environment than the nonvectorial process. Moreover, cytoplasmic micellar forms of these enzyme-porters have been identified, and non-PTS permeases have similarly been shown to exist in 'soluble' forms. The latter porters exhibit lipid-dependent activities and can adopt altered topologies by simply changing the lipid composition. Finally, intracellular membranes and vesicles exist in Escherichia coli leading to the following unanswered questions: (1) what determines whether a PTS permease catalyzes vectorial or nonvectorial sugar phosphorylation? (2) How do phospholipids influence relative amounts of the plasma membrane, intracellular membrane, inner membrane-derived vesicles and cytoplasmic micelles? (3) What regulates the route(s) of permease insertion and transfer into and between the different subcellular sites? (4) Do these various membranous forms have distinct physiological functions? (5) What methods should be utilized to study the biogenesis and interconversion of these membranous structures? While research concerning these questions is still in its infancy, answers will greatly enhance our understanding of protein-lipid interactions and how they control the activities, conformations, cellular locations and biogenesis of integral membrane proteins. Copyright © 2013 S. Karger AG, Basel.

  18. Guarana (Paullinia cupana Stimulates Mitochondrial Biogenesis in Mice Fed High-Fat Diet

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    Natália da Silva Lima

    2018-01-01

    Full Text Available The aim of this study was to evaluate the effects of guarana on mitochondrial biogenesis in a high-fat diet (HFD-fed mice. C57BL6J mice were divided in two groups: high-fat diet HFD and high-fat diet + guarana (HFD-GUA. Both groups received HFD and water ad libitum and the HFD-GUA group also received a daily gavage of guarana (1 g/kg weight. Body weight and food intake was measured weekly. Glycemic, triglyceride, and cholesterol levels were determined. VO2 and energy expenditure (EE were determined by indirect calorimetry. Gene expression was evaluated by real-time PCR and protein content by western blotting. The HFD-GUA group presented lower body weight, subcutaneous, retroperitoneal, visceral, and epididyimal adipose tissue depots, and glycemic and triglyceride levels, with no change in food intake and cholesterol levels. Furthermore, the HFD-GUA group presented an increase in VO2 and basal energy expenditure (EE, as well as Pgc1α, Creb1, Ampka1, Nrf1, Nrf2, and Sirt1 expression in the muscle and brown adipose tissue. In addition, the HFD-GUA group presented an increase in mtDNA (mitochondrial deoxyribonucleic acid content in the muscle when compared to the HFD group. Thus, our data showed that guarana leads to an increase in energetic metabolism and stimulates mitochondrial biogenesis, contributing to control of weight gain, even when associated with high-fat diet.

  19. Genome-Wide Identification, Characterization, and Expression Analysis of Small RNA Biogenesis Purveyors Reveal Their Role in Regulation of Biotic Stress Responses in Three Legume Crops

    Directory of Open Access Journals (Sweden)

    Rajeev K. Varshney

    2017-04-01

    Full Text Available Biotic stress in legume crops is one of the major threats to crop yield and productivity. Being sessile organisms, plants have evolved a myriad of mechanisms to combat different stresses imposed on them. One such mechanism, deciphered in the last decade, is small RNA (sRNA mediated defense in plants. Small RNAs (sRNAs have emerged as one of the major players in gene expression regulation in plants during developmental stages and under stress conditions. They are known to act both at transcriptional and post-transcriptional levels. Dicer-like (DCL, Argonaute (AGO, and RNA dependent RNA polymerase (RDR constitute the major components of sRNA biogenesis machinery and are known to play a significant role in combating biotic and abiotic stresses. This study is, therefore, focused on identification and characterization of sRNA biogenesis proteins in three important legume crops, namely chickpea, pigeonpea, and groundnut. Phylogenetic analysis of these proteins between legume species classified them into distinct clades and suggests the evolutionary conservation of these genes across the members of Papillionidoids subfamily. Variable expression of sRNA biogenesis genes in response to the biotic stresses among the three legumes indicate the possible existence of specialized regulatory mechanisms in different legumes. This is the first ever study to understand the role of sRNA biogenesis genes in response to pathogen attacks in the studied legumes.

  20. Mitochondrial Cytochrome c Oxidase Biogenesis Is Regulated by the Redox State of a Heme-Binding Translational Activator.

    Science.gov (United States)

    Soto, Iliana C; Barrientos, Antoni

    2016-02-20

    Mitochondrial cytochrome c oxidase (COX), the last enzyme of the respiratory chain, catalyzes the reduction of oxygen to water and therefore is essential for cell function and viability. COX is a multimeric complex, whose biogenesis is extensively regulated. One type of control targets cytochrome c oxidase subunit 1 (Cox1), a key COX enzymatic core subunit translated on mitochondrial ribosomes. In Saccharomyces cerevisiae, Cox1 synthesis and COX assembly are coordinated through a negative feedback regulatory loop. This coordination is mediated by Mss51, a heme-sensing COX1 mRNA-specific processing factor and translational activator that is also a Cox1 chaperone. In this study, we investigated whether Mss51 hemylation and Mss51-mediated Cox1 synthesis are both modulated by the reduction-oxidation (redox) environment. We report that Cox1 synthesis is attenuated under oxidative stress conditions and have identified one of the underlying mechanisms. We show that in vitro and in vivo exposure to hydrogen peroxide induces the formation of a disulfide bond in Mss51 involving CPX motif heme-coordinating cysteines. Mss51 oxidation results in a heme ligand switch, thereby lowering heme-binding affinity and promoting its release. We demonstrate that in addition to affecting Mss51-dependent heme sensing, oxidative stress compromises Mss51 roles in COX1 mRNA processing and translation. H2O2-induced downregulation of mitochondrial translation has so far not been reported. We show that high H2O2 concentrations induce a global attenuation effect, but milder concentrations specifically affect COX1 mRNA processing and translation in an Mss51-dependent manner. The redox environment modulates Mss51 functions, which are essential for regulation of COX biogenesis and aerobic energy production.

  1. Bacterial lipoproteins; biogenesis, sorting and quality control.

    Science.gov (United States)

    Narita, Shin-Ichiro; Tokuda, Hajime

    2017-11-01

    Bacterial lipoproteins are a subset of membrane proteins localized on either leaflet of the lipid bilayer. These proteins are anchored to membranes through their N-terminal lipid moiety attached to a conserved Cys. Since the protein moiety of most lipoproteins is hydrophilic, they are expected to play various roles in a hydrophilic environment outside the cytoplasmic membrane. Gram-negative bacteria such as Escherichia coli possess an outer membrane, to which most lipoproteins are sorted. The Lol pathway plays a central role in the sorting of lipoproteins to the outer membrane after lipoprotein precursors are processed to mature forms in the cytoplasmic membrane. Most lipoproteins are anchored to the inner leaflet of the outer membrane with their protein moiety in the periplasm. However, recent studies indicated that some lipoproteins further undergo topology change in the outer membrane, and play critical roles in the biogenesis and quality control of the outer membrane. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. From centriole biogenesis to cellular function: centrioles are essential for cell division at critical developmental stages.

    Science.gov (United States)

    Rodrigues-Martins, Ana; Riparbelli, Maria; Callaini, Giuliano; Glover, David M; Bettencourt-Dias, Monica

    2008-01-01

    Centrioles are essential for the formation of cilia, flagella and centrosome organization. Abnormalities in centrosome structure and number in many cancers can be associated with aberrant cell division and genomic instability.(1,2) Canonical centriole duplication occurs in coordination with the cell division cycle, such that a single new "daughter" centriole arises next to each "mother" centriole. If destroyed, or eliminated during development, centrioles can form de novo.(3-5) Here we discuss our recent data demonstrating a molecular pathway that operates in both de novo and canonical centriole biogenesis involving SAK/PLK4, SAS-6 and SAS-4.(6) We showed that centriole biogenesis is a self-assembly process locally triggered by high SAK/PLK4 activity that may or not be associated with an existing centriole. SAS-6 acts downstream of SAK/PLK4 to organize nine precentriolar units, which we call here enatosomes, fitting together laterally and longitudinally, specifying a tube-like centriole precursor.(7,8) The identification of mutants impaired in centriole biogenesis has permitted the study of the physiological consequences of their absence in the whole organism. In Drosophila, centrioles are not necessary for somatic cell divisions.(9,10) However, we show here that mitotic abnormalities arise in syncytial SAK/PLK4-derived mutant embryos resulting in lethality. Moreover male meiosis fails in both SAK/PLK4 and DSAS-4 mutant spermatids that have no centrioles. These results show diversity in the need for centrioles in cell division. This suggests that tissue specific constraints selected for different contributions of centrosome-independent and dependent mechanisms in spindle function. This heterogeneity should be taken into account both in reaching an understanding of spindle function and when designing drugs that target cell division.

  3. Drosophila Vps16A is required for trafficking to lysosomes and biogenesis of pigment granules.

    Science.gov (United States)

    Pulipparacharuvil, Suprabha; Akbar, Mohammed Ali; Ray, Sanchali; Sevrioukov, Evgueny A; Haberman, Adam S; Rohrer, Jack; Krämer, Helmut

    2005-08-15

    Mutations that disrupt trafficking to lysosomes and lysosome-related organelles cause multiple diseases, including Hermansky-Pudlak syndrome. The Drosophila eye is a model system for analyzing such mutations. The eye-color genes carnation and deep orange encode two subunits of the Vps-C protein complex required for endosomal trafficking and pigment-granule biogenesis. Here we demonstrate that dVps16A (CG8454) encodes another Vps-C subunit. Biochemical experiments revealed a specific interaction between the dVps16A C-terminus and the Sec1/Munc18 homolog Carnation but not its closest homolog, dVps33B. Instead, dVps33B interacted with a related protein, dVps16B (CG18112). Deep orange bound both Vps16 homologs. Like a deep orange null mutation, eye-specific RNAi-induced knockdown of dVps16A inhibited lysosomal delivery of internalized ligands and interfered with biogenesis of pigment granules. Ubiquitous knockdown of dVps16A was lethal. Together, these findings demonstrate that Drosophila Vps16A is essential for lysosomal trafficking. Furthermore, metazoans have two types of Vps-C complexes with non-redundant functions.

  4. Transpeptidase activity of penicillin-binding protein SpoVD in peptidoglycan synthesis conditionally depends on the disulfide reductase StoA.

    Science.gov (United States)

    Bukowska-Faniband, Ewa; Hederstedt, Lars

    2017-07-01

    Endospore cortex peptidoglycan synthesis is not required for bacterial growth but essential for endospore heat resistance. It therefore constitutes an amenable system for research on peptidoglycan biogenesis. The Bacillus subtilis sporulation-specific class B penicillin-binding protein (PBP) SpoVD and many homologous PBPs contain two conserved cysteine residues of unknown function in the transpeptidase domain - one as residue x in the SxN catalytic site motif and the other in a flexible loop near the catalytic site. A disulfide bond between these residues blocks the function of SpoVD in cortex synthesis. With a combination of experiments with purified proteins and B. subtilis mutant cells, it was shown that in active SpoVD the two cysteine residues most probably interact by hydrogen bonding and that this is important for peptidoglycan synthesis in vivo. It was furthermore demonstrated that the sporulation-specific thiol-disulfide oxidoreductase StoA reduces SpoVD and that requirement of StoA for cortex synthesis can be suppressed by two completely different types of structural alterations in SpoVD. It is concluded that StoA plays a critical role mainly during maturation of SpoVD in the forespore outer membrane. The findings advance our understanding of essential PBPs and redox control of extra-cytoplasmic protein disulfides in bacterial cells. © 2017 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.

  5. Lifting the mask: identification of new small molecule inhibitors of uropathogenic Escherichia coli group 2 capsule biogenesis.

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    Carlos C Goller

    Full Text Available Uropathogenic Escherichia coli (UPEC is the leading cause of community-acquired urinary tract infections (UTIs, with over 100 million UTIs occurring annually throughout the world. Increasing antimicrobial resistance among UPEC limits ambulatory care options, delays effective treatment, and may increase overall morbidity and mortality from complications such as urosepsis. The polysaccharide capsules of UPEC are an attractive target a therapeutic, based on their importance in defense against the host immune responses; however, the large number of antigenic types has limited their incorporation into vaccine development. The objective of this study was to identify small-molecule inhibitors of UPEC capsule biogenesis. A large-scale screening effort entailing 338,740 compounds was conducted in a cell-based, phenotypic screen for inhibition of capsule biogenesis in UPEC. The primary and concentration-response assays yielded 29 putative inhibitors of capsule biogenesis, of which 6 were selected for further studies. Secondary confirmatory assays identified two highly active agents, named DU003 and DU011, with 50% inhibitory concentrations of 1.0 µM and 0.69 µM, respectively. Confirmatory assays for capsular antigen and biochemical measurement of capsular sugars verified the inhibitory action of both compounds and demonstrated minimal toxicity and off-target effects. Serum sensitivity assays demonstrated that both compounds produced significant bacterial death upon exposure to active human serum. DU011 administration in mice provided near complete protection against a lethal systemic infection with the prototypic UPEC K1 isolate UTI89. This work has provided a conceptually new class of molecules to combat UPEC infection, and future studies will establish the molecular basis for their action along with efficacy in UTI and other UPEC infections.

  6. Arabidopsis EMB1990 Encoding a Plastid-Targeted YlmG Protein Is Required for Chloroplast Biogenesis and Embryo Development

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    Hongyu Chen

    2018-02-01

    Full Text Available In higher plants, embryo development originated from fertilized egg cell is the first step of the life cycle. The chloroplast participates in many essential metabolic pathways, and its function is highly associated with embryo development. However, the mechanisms and relevant genetic components by which the chloroplast functions in embryogenesis are largely uncharacterized. In this paper, we describe the Arabidopsis EMB1990 gene, encoding a plastid-targeted YlmG protein which is required for chloroplast biogenesis and embryo development. Loss of the EMB1990/YLMG1-1 resulted in albino seeds containing abortive embryos, and the morphological development of homozygous emb1990 embryos was disrupted after the globular stage. Our results showed that EMB1990/YLMG1-1 was expressed in the primordia and adaxial region of cotyledon during embryogenesis, and the encoded protein was targeted to the chloroplast. TEM observation of cellular ultrastructure showed that chloroplast biogenesis was impaired in emb1990 embryo cells. Expression of certain plastid genes was also affected in the loss-of-function mutants, including genes encoding core protein complex subunits located in the thylakoid membrane. Moreover, the tissue-specific genes of embryo development were misexpressed in emb1990 mutant, including genes known to delineate cell fate decisions in the SAM (shoot apical meristem, cotyledon and hypophysis. Taken together, we propose that the nuclear-encoded YLMG1-1 is targeted to the chloroplast and required for normal plastid gene expression. Hence, YLMG1-1 plays a critical role in Arabidopsis embryogenesis through participating in chloroplast biogenesis.

  7. 20-Hydroxyecdysone stimulates nuclear accumulation of BmNep1, a nuclear ribosome biogenesis-related protein in the silkworm, Bombyx mori.

    Science.gov (United States)

    Ji, M-M; Liu, A-Q; Sima, Y-H; Xu, S-Q

    2016-10-01

    The pathway of communication between endocrine hormones and ribosome biogenesis critical for physiological adaptation is largely unknown. Nucleolar essential protein 1 (Nep1) is an essential gene for ribosome biogenesis and is functionally conserved in many in vertebrate and invertebrate species. In this study, we cloned Bombyx mori Nep1 (BmNep1) due to its high expression in silk glands of silkworms on day 3 of the fifth instar. We found that BmNep1 mRNA and protein levels were upregulated in silk glands during fourth-instar ecdysis and larval-pupal metamorphosis. By immunoprecipitation with the anti-BmNep1 antibody and liquid chromatography-tandem mass spectrometry analyses, it was shown that BmNep1 probably interacts with proteins related to ribosome structure formation. Immunohistochemistry, biochemical fractionation and immunocytochemistry revealed that BmNep1 is localized to the nuclei in Bombyx cells. Using BmN cells originally derived from ovaries, we demonstrated that 20-hydroxyecdysone (20E) induced BmNep1 expression and stimulated nuclear accumulation of BmNep1. Under physiological conditions, BmNep1 was also upregulated in ovaries during larval-pupal metamorphosis. Overall, our results indicate that the endocrine hormone 20E facilitates nuclear accumulation of BmNep1, which is involved in nuclear ribosome biogenesis in Bombyx. © 2016 The Royal Entomological Society.

  8. BLOC-3 mutated in Hermansky-Pudlak syndrome is a Rab32/38 guanine nucleotide exchange factor.

    Science.gov (United States)

    Gerondopoulos, Andreas; Langemeyer, Lars; Liang, Jin-Rui; Linford, Andrea; Barr, Francis A

    2012-11-20

    Hermansky-Pudlak syndrome (HPS) is a human disease characterized by partial loss of pigmentation and impaired blood clotting. These symptoms are caused by defects in the biogenesis of melanosomes and platelet dense granules, often referred to as lysosome-related organelles. Genes mutated in HPS encode subunits of the biogenesis of lysosome-related organelles complexes (BLOCs). BLOC-1 and BLOC-2, together with the AP-3 clathrin adaptor complex, act at early endosomes to sort components required for melanin formation and melanosome biogenesis away from the degradative lysosomal pathway toward early stage melanosomes. However the molecular functions of the Hps1-Hps4 complex BLOC-3 remain mysterious. Like other trafficking pathways, melanosome biogenesis and transport of enzymes involved in pigmentation involves specific Rab GTPases, in this instance Rab32 and Rab38. We now demonstrate that BLOC-3 is a Rab32 and Rab38 guanine nucleotide exchange factor (GEF). Silencing of the BLOC-3 subunits Hps1 and Hps4 results in the mislocalization of Rab32 and Rab38 and reduction in pigmentation. In addition, we show that BLOC-3 can promote specific membrane recruitment of Rab32/38. BLOC-3 therefore defines a novel Rab GEF family with a specific function in the biogenesis of lysosome-related organelles. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Sam37 is crucial for formation of the mitochondrial TOM-SAM supercomplex, thereby promoting β-barrel biogenesis.

    Science.gov (United States)

    Wenz, Lena-Sophie; Ellenrieder, Lars; Qiu, Jian; Bohnert, Maria; Zufall, Nicole; van der Laan, Martin; Pfanner, Nikolaus; Wiedemann, Nils; Becker, Thomas

    2015-09-28

    Biogenesis of mitochondrial β-barrel proteins requires two preprotein translocases, the general translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). TOM and SAM form a supercomplex that promotes transfer of β-barrel precursors. The SAM core complex contains the channel protein Sam50, which cooperates with Sam35 in precursor recognition, and the peripheral membrane protein Sam37. The molecular function of Sam37 has been unknown. We report that Sam37 is crucial for formation of the TOM-SAM supercomplex. Sam37 interacts with the receptor domain of Tom22 on the cytosolic side of the mitochondrial outer membrane and links TOM and SAM complexes. Sam37 thus promotes efficient transfer of β-barrel precursors to the SAM complex. We conclude that Sam37 functions as a coupling factor of the translocase supercomplex of the mitochondrial outer membrane. © 2015 Wenz et al.

  10. The helicase and RNaseIIIa domains of Arabidopsis Dicer-Like1 modulate catalytic parameters during MicroRNA biogenesis

    KAUST Repository

    Liu, Chenggang; Axtell, Michael J.; Fedoroff, Nina V.

    2012-01-01

    Dicer-Like1 (DCL1), an RNaseIII endonuclease, and Hyponastic Leaves1 (HYL1), a double-stranded RNA-binding protein, are core components of the plant microRNA (miRNA) biogenesis machinery. hyl1 mutants accumulate low levels of miRNAs and display

  11. Impact of Resistance Training on Skeletal Muscle Mitochondrial Biogenesis, Content, and Function

    Directory of Open Access Journals (Sweden)

    Thomas Groennebaek

    2017-09-01

    Full Text Available Skeletal muscle metabolic and contractile properties are reliant on muscle mitochondrial and myofibrillar protein turnover. The turnover of these specific protein pools is compromised during disease, aging, and inactivity. Oppositely, exercise can accentuate muscle protein turnover, thereby counteracting decay in muscle function. According to a traditional consensus, endurance exercise is required to drive mitochondrial adaptations, while resistance exercise is required to drive myofibrillar adaptations. However, concurrent practice of traditional endurance exercise and resistance exercise regimens to achieve both types of muscle adaptations is time-consuming, motivationally demanding, and contended to entail practice at intensity levels, that may not comply with clinical settings. It is therefore of principle interest to identify effective, yet feasible, exercise strategies that may positively affect both mitochondrial and myofibrillar protein turnover. Recently, reports indicate that traditional high-load resistance exercise can stimulate muscle mitochondrial biogenesis and mitochondrial respiratory function. Moreover, fatiguing low-load resistance exercise has been shown capable of promoting muscle hypertrophy and expectedly entails greater metabolic stress to potentially enhance mitochondrial adaptations. Consequently, fatiguing low-load resistance exercise regimens may possess the ability to stimulate muscle mitochondrial adaptations without compromising muscle myofibrillar accretion. However, the exact ability of resistance exercise to drive mitochondrial adaptations is debatable, not least due to some methodological challenges. The current review therefore aims to address the evidence on the effects of resistance exercise on skeletal muscle mitochondrial biogenesis, content and function. In prolongation, a perspective is taken on the specific potential of low-load resistance exercise on promoting mitochondrial adaptations.

  12. Coccidioides Endospores and Spherules Draw Strong Chemotactic, Adhesive, and Phagocytic Responses by Individual Human Neutrophils.

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    Cheng-Yuk Lee

    Full Text Available Coccidioides spp. are dimorphic pathogenic fungi whose parasitic forms cause coccidioidomycosis (Valley fever in mammalian hosts. We use an innovative interdisciplinary approach to analyze one-on-one encounters between human neutrophils and two forms of Coccidioides posadasii. To examine the mechanisms by which the innate immune system coordinates different stages of the host response to fungal pathogens, we dissect the immune-cell response into chemotaxis, adhesion, and phagocytosis. Our single-cell technique reveals a surprisingly strong response by initially quiescent neutrophils to close encounters with C. posadasii, both from a distance (by complement-mediated chemotaxis as well as upon contact (by serum-dependent adhesion and phagocytosis. This response closely resembles neutrophil interactions with Candida albicans and zymosan particles, and is significantly stronger than the neutrophil responses to Cryptococcus neoformans, Aspergillus fumigatus, and Rhizopus oryzae under identical conditions. The vigorous in vitro neutrophil response suggests that C. posadasii evades in vivo recognition by neutrophils through suppression of long-range mobilization and recruitment of the immune cells. This observation elucidates an important paradigm of the recognition of microbes, i.e., that intact immunotaxis comprises an intricate spatiotemporal hierarchy of distinct chemotactic processes. Moreover, in contrast to earlier reports, human neutrophils exhibit vigorous chemotaxis toward, and frustrated phagocytosis of, the large spherules of C. posadasii under physiological-like conditions. Finally, neutrophils from healthy donors and patients with chronic coccidioidomycosis display subtle differences in their responses to antibody-coated beads, even though the patient cells appear to interact normally with C. posadasii endospores.

  13. Global SUMO proteome responses guide gene regulation, mRNA biogenesis, and plant stress responses

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    Magdalena eMazur

    2012-09-01

    Full Text Available Small-ubiquitin-like MOdifier (SUMO is a key regulator of abiotic stress, disease resistance and development in plants. The identification of >350 plant SUMO targets has revealed many processes modulated by SUMO and potential consequences of SUMO on its targets. Importantly, highly related proteins are SUMO-modified in plants, yeast, and metazoans. Overlapping SUMO targets include heat-shock proteins, transcription regulators, histones, histone-modifying enzymes, proteins involved in DNA damage repair, but also proteins involved in mRNA biogenesis and nucleo-cytoplasmic transport. Proteomics studies indicate key roles for SUMO in gene repression by controlling histone (deacetylation activity at genomic loci. The responsible heavily sumoylated transcriptional repressor complexes are recruited by EAR (Ethylene-responsive element binding factor [ERF]-associated Amphiphilic Repression-motif containing transcription factors in plants. These transcription factors are not necessarily themselves a SUMO target. Conversely, SUMO acetylation prevents binding of downstream partners by preventing binding of SIMs (SUMO-interaction peptide motifs presents in these partners, while SUMO acetylation has emerged as mechanism to recruit specifically bromodomains; bromodomain are generally linked with gene activation. These findings strengthen the idea of a bidirectional sumo-/acetylation switch in gene regulation. Quantitative proteomics has highlighted that global sumoylation provides a dynamic response to protein damage involving SUMO chain-mediated protein degradation, but also SUMO E3 ligase-dependent transcription of HSP (Heat-shock protein genes. With these insights in SUMO function and novel technical advancements, we can now study SUMO dynamics in responses to (abiotic stress in plants.

  14. The Absence of the Transcription Factor Yrr1p, Identified from Comparative Genome Profiling, Increased Vanillin Tolerance Due to Enhancements of ABC Transporters Expressing, rRNA Processing and Ribosome Biogenesis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Wang, Xinning; Liang, Zhenzhen; Hou, Jin; Shen, Yu; Bao, Xiaoming

    2017-01-01

    Enhancing the tolerance of Saccharomyces cerevisiae to inhibitors derived from lignocellulose is conducive to producing biofuel and chemicals using abundant lignocellulosic materials. Vanillin is a major type of phenolic inhibitor in lignocellulose hydrolysates for S. cerevisiae . In the present work, the factors beneficial to vanillin resistance in yeast were identified from the vanillin-resistant strain EMV-8, which was derived from strain NAN-27 by adaptive evolution. We found 450 SNPs and 44 genes with InDels in the vanillin-tolerant strain EMV-8 by comparing the genome sequences of EMV-8 and NAN-27. To investigate the effects of InDels, InDels were deleted in BY4741, respectively. We demonstrated that the deletion of YRR1 improved vanillin tolerance of strain. In the presence of 6 mM vanillin, deleting YRR1 increase the maximum specific growth rate and the vanillin consumption rate by 142 and 51%, respectively. The subsequent transcriptome analysis revealed that deleting YRR1 resulted in changed expression of over 200 genes in the presence of 5 mM vanillin. The most marked changes were the significant up-regulation of the dehydrogenase ADH7 , several ATP-binding cassette (ABC) transporters, and dozens of genes involved in ribosome biogenesis and rRNA processing. Coincidently, the crude enzyme solution of BY4741( yrr1 Δ) exhibited higher NADPH-dependent vanillin reduction activity than control. In addition, overexpressing the ABC transporter genes PDR5, YOR1 , and SNQ2 , as well as the RNA helicase gene DBP2 , increased the vanillin tolerance of strain. Interestingly, unlike the marked changes we mentioned above, under vanillin-free conditions, there are only limited transcriptional differences between wildtype and yrr1 Δ. This indicated that vanillin might act as an effector in Yrr1p-related regulatory processes. The new findings of the relationship between YRR1 and vanillin tolerance, as well as the contribution of rRNA processing and ribosome biogenesis to

  15. Karrikins delay soybean seed germination by mediating abscisic acid and gibberellin biogenesis under shaded conditions

    OpenAIRE

    Meng, Yongjie; Chen, Feng; Shuai, Haiwei; Luo, Xiaofeng; Ding, Jun; Tang, Shengwen; Xu, Shuanshuan; Liu, Jianwei; Liu, Weiguo; Du, Junbo; Liu, Jiang; Yang, Feng; Sun, Xin; Yong, Taiwen; Wang, Xiaochun

    2016-01-01

    Karrikins (KAR) are a class of signal compounds, discovered in wildfire smoke, which affect seed germination. Currently, numerous studies have focused on the model plant Arabidopsis in the KAR research field, rather than on crops. Thus the regulatory mechanisms underlying KAR regulation of crop seed germination are largely unknown. Here, we report that KAR delayed soybean seed germination through enhancing abscisic acid (ABA) biosynthesis, while impairing gibberellin (GA) biogenesis. Interest...

  16. Triiodothyronine induces lipid oxidation and mitochondrial biogenesis in rat Harderian gland.

    Science.gov (United States)

    Santillo, A; Burrone, L; Falvo, S; Senese, R; Lanni, A; Chieffi Baccari, G

    2013-10-01

    The rat Harderian gland (HG) is an orbital gland producing a copious lipid secretion. Recent studies indicate that its secretory activity is regulated by thyroid hormones. In this study, we found that both isoforms of the thyroid hormone receptor (Trα (Thra) and Trβ (Thrb)) are expressed in rat HGs. Although Thra is expressed at a higher level, only Thrb is regulated by triiodothyronine (T3). Because T3 induces an increase in lipid metabolism in rat HGs, we investigated the effects of an animal's thyroid state on the expression levels of carnitine palmitoyltransferase-1A (Cpt1a) and carnitine palmitoyltransferase-1B (Cpt1b) and acyl-CoA oxidase (Acox1) (rate-limiting enzymes in mitochondrial and peroxisomal fatty acid oxidation respectively), as well as on the mitochondrial compartment, thereby correlating mitochondrial activity and biogenesis with morphological analysis. We found that hypothyroidism decreased the expression of Cpt1b and Acox1 mRNA, whereas the administration of T3 to hypothyroid rats increased transcript levels. Respiratory parameters and catalase protein levels provided further evidence that T3 modulates mitochondrial and peroxisomal activities. Furthermore, in hypothyroid rat HGs, the mitochondrial number and their total area decreased with respect to the controls, whereas the average area of the individual mitochondrion did not change. However, the average area of the individual mitochondrion was reduced by ∼50% in hypothyroid T3-treated HGs, and the mitochondrial number and the total area of the mitochondrial compartment increased. The mitochondrial morphometric data correlated well with the molecular results. Indeed, hypothyroid status did not modify the expression of mitochondrial biogenesis genes such as Ppargc1a, Nrf1 and Tfam, whereas T3 treatment increased the expression level of these genes.

  17. Neisserial surface lipoproteins: structure, function and biogenesis.

    Science.gov (United States)

    Hooda, Yogesh; Shin, Hyejin E; Bateman, Thomas J; Moraes, Trevor F

    2017-03-01

    The surface of many Gram-negative bacteria contains lipidated protein molecules referred to as surface lipoproteins or SLPs. SLPs play critical roles in host immune evasion, nutrient acquisition and regulation of the bacterial stress response. The focus of this review is on the SLPs present in Neisseria, a genus of bacteria that colonise the mucosal surfaces of animals. Neisseria contains two pathogens of medical interest, namely Neisseria meningitidis and N. gonorrhoeae. Several SLPs have been identified in Neisseria and their study has elucidated key strategies used by these pathogens to survive inside the human body. Herein, we focus on the identification, structure and function of SLPs that have been identified in Neisseria. We also survey the translocation pathways used by these SLPs to reach the cell surface. Specifically, we elaborate on the strategies used by neisserial SLPs to translocate across the outer membrane with an emphasis on Slam, a novel outer membrane protein that has been implicated in SLP biogenesis. Taken together, the study of SLPs in Neisseria illustrates the widespread roles played by this family of proteins in Gram-negative bacteria. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Roles of Oxidative Stress, Apoptosis, PGC-1α and Mitochondrial Biogenesis in Cerebral Ischemia

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    Ding-I Yang

    2011-10-01

    Full Text Available The primary physiological function of mitochondria is to generate adenosine triphosphate through oxidative phosphorylation via the electron transport chain. Overproduction of reactive oxygen species (ROS as byproducts generated from mitochondria have been implicated in acute brain injuries such as stroke from cerebral ischemia. It was well-documented that mitochondria-dependent apoptotic pathway involves pro- and anti-apoptotic protein binding, release of cytochrome c, leading ultimately to neuronal death. On the other hand, mitochondria also play a role to counteract the detrimental effects elicited by excessive oxidative stress. Recent studies have revealed that oxidative stress and the redox state of ischemic neurons are also implicated in the signaling pathway that involves peroxisome proliferative activated receptor-γ (PPARγ co-activator 1α (PGC1-α. PGC1-α is a master regulator of ROS scavenging enzymes including manganese superoxide dismutase 2 and the uncoupling protein 2, both are mitochondrial proteins, and may contribute to neuronal survival. PGC1-α is also involved in mitochondrial biogenesis that is vital for cell survival. Experimental evidence supports the roles of mitochondrial dysfunction and oxidative stress as determinants of neuronal death as well as endogenous protective mechanisms after stroke. This review aims to summarize the current knowledge focusing on the molecular mechanisms underlying cerebral ischemia involving ROS, mitochondrial dysfunction, apoptosis, mitochondrial proteins capable of ROS scavenging, and mitochondrial biogenesis.

  19. Mitophagy is required for mitochondrial biogenesis and myogenic differentiation of C2C12 myoblasts.

    Science.gov (United States)

    Sin, Jon; Andres, Allen M; Taylor, David J R; Weston, Thomas; Hiraumi, Yoshimi; Stotland, Aleksandr; Kim, Brandon J; Huang, Chengqun; Doran, Kelly S; Gottlieb, Roberta A

    2016-01-01

    Myogenesis is a crucial process governing skeletal muscle development and homeostasis. Differentiation of primitive myoblasts into mature myotubes requires a metabolic switch to support the increased energetic demand of contractile muscle. Skeletal myoblasts specifically shift from a highly glycolytic state to relying predominantly on oxidative phosphorylation (OXPHOS) upon differentiation. We have found that this phenomenon requires dramatic remodeling of the mitochondrial network involving both mitochondrial clearance and biogenesis. During early myogenic differentiation, autophagy is robustly upregulated and this coincides with DNM1L/DRP1 (dynamin 1-like)-mediated fragmentation and subsequent removal of mitochondria via SQSTM1 (sequestosome 1)-mediated mitophagy. Mitochondria are then repopulated via PPARGC1A/PGC-1α (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha)-mediated biogenesis. Mitochondrial fusion protein OPA1 (optic atrophy 1 [autosomal dominant]) is then briskly upregulated, resulting in the reformation of mitochondrial networks. The final product is a myotube replete with new mitochondria. Respirometry reveals that the constituents of these newly established mitochondrial networks are better primed for OXPHOS and are more tightly coupled than those in myoblasts. Additionally, we have found that suppressing autophagy with various inhibitors during differentiation interferes with myogenic differentiation. Together these data highlight the integral role of autophagy and mitophagy in myogenic differentiation.

  20. Outer membrane biogenesis in Helicobacter pylori: A deviation from the paradigm

    Directory of Open Access Journals (Sweden)

    George W. Liechti

    2012-04-01

    Full Text Available The bacterial pathogen Helicobacter pylori is capable of colonizing the gastric mucosa of the human stomach using a variety of factors associated with or secreted from its outer membrane (OM. Lipopolysaccharide (LPS and numerous outer membrane proteins have been shown to be involved in adhesion and immune stimulation/evasion. Many of these factors are essential for colonization and/or pathogenesis in a variety of animal models. Despite this wide array of potential targets present on the bacterial surface, the ability of H. pylori to vary its outer membrane profile limits the effectiveness of vaccines that use any single one of these components. However, it has become evident that the proteins comprising the complexes that transport the majority of these molecules to the OM are highly conserved and often essential. The field of membrane biogenesis has progressed remarkably in the last few years, and the possibility now exists for targeting the mechanisms by which β-barrel proteins, lipoproteins, and LPS are transported to the OM, resulting in loss of bacterial fitness and significant altering of membrane permeability. In this review, the OM transport machinery for LPS, lipoproteins, and outer membrane proteins are discussed. While the principal investigations of these transport mechanisms have been conducted in Escherichia coli and Neisseria meningitidis, here these systems will be presented in the genetic context of ε- proteobacteria. Bioinformatic analysis reveals that minimalist genomes, such as that of Helicobacter pylori, offer insight into the smallest number of components required for these essential pathways to function. Interestingly, in the majority of ε-proteobacteria, while the inner and outer membrane associated apparatus of LPS, lipoprotein, and OM protein transport pathways appear to all be intact, most of the components associated with the periplasmic compartment are either missing or are almost unrecognizable when compared to

  1. The Mkk2 MAPKK Regulates Cell Wall Biogenesis in Cooperation with the Cek1-Pathway in Candida albicans

    NARCIS (Netherlands)

    Román, Elvira; Alonso-Monge, Rebeca; Miranda Bedate, A.; Pla, Jesús

    2015-01-01

    The cell wall integrity pathway (CWI) plays an important role in the biogenesis of the cell wall in Candida albicans and other fungi. In the present work, the C. albicans MKK2 gene that encodes the putative MAPKK of this pathway was deleted in different backgrounds and the phenotypes of the

  2. The 5S RNP couples p53 homeostasis to ribosome biogenesis and nucleolar stress.

    Science.gov (United States)

    Sloan, Katherine E; Bohnsack, Markus T; Watkins, Nicholas J

    2013-10-17

    Several proto-oncogenes and tumor suppressors regulate the production of ribosomes. Ribosome biogenesis is a major consumer of cellular energy, and defects result in p53 activation via repression of mouse double minute 2 (MDM2) homolog by the ribosomal proteins RPL5 and RPL11. Here, we report that RPL5 and RPL11 regulate p53 from the context of a ribosomal subcomplex, the 5S ribonucleoprotein particle (RNP). We provide evidence that the third component of this complex, the 5S rRNA, is critical for p53 regulation. In addition, we show that the 5S RNP is essential for the activation of p53 by p14(ARF), a protein that is activated by oncogene overexpression. Our data show that the abundance of the 5S RNP, and therefore p53 levels, is determined by factors regulating 5S complex formation and ribosome integration, including the tumor suppressor PICT1. The 5S RNP therefore emerges as the critical coordinator of signaling pathways that couple cell proliferation with ribosome production. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Helicase-like transcription factor (Hltf regulates G2/M transition, Wt1/Gata4/Hif-1a cardiac transcription networks, and collagen biogenesis.

    Directory of Open Access Journals (Sweden)

    Rebecca A Helmer

    Full Text Available HLTF/Hltf regulates transcription, remodels chromatin, and coordinates DNA damage repair. Hltf is expressed in mouse brain and heart during embryonic and postnatal development. Silencing Hltf is semilethal. Seventy-four percent of congenic C57BL/6J Hltf knockout mice died, 75% within 12-24 hours of birth. Previous studies in neonatal (6-8 hour postpartum brain revealed silencing Hltf disrupted cell cycle progression, and attenuated DNA damage repair. An RNA-Seq snapshot of neonatal heart transcriptome showed 1,536 of 20,000 total transcripts were altered (p < 0.05 - 10 up- and 1,526 downregulated. Pathway enrichment analysis with MetaCore™ showed Hltf's regulation of the G2/M transition (p=9.726E(-15 of the cell cycle in heart is nearly identical to its role in brain. In addition, Brca1 and 12 members of the Brca1 associated genome surveillance complex are also downregulated. Activation of caspase 3 coincides with transcriptional repression of Bcl-2. Hltf loss caused downregulation of Wt1/Gata4/Hif-1a signaling cascades as well as Myh7b/miR499 transcription. Hltf-specific binding to promoters and/or regulatory regions of these genes was authenticated by ChIP-PCR. Hif-1a targets for prolyl (P4ha1, P4ha2 and lysyl (Plod2 collagen hydroxylation, PPIase enzymes (Ppid, Ppif, Ppil3 for collagen trimerization, and lysyl oxidase (Loxl2 for collagen-elastin crosslinking were downregulated. However, transcription of genes for collagens, fibronectin, Mmps and their inhibitors (Timps was unaffected. The collective downregulation of genes whose protein products control collagen biogenesis caused disorganization of the interstitial and perivascular myocardial collagen fibrillar network as viewed with picrosirius red-staining, and authenticated with spectral imaging. Wavy collagen bundles in control hearts contrasted with collagen fibers that were thin, short and disorganized in Hltf null hearts. Collagen bundles in Hltf null hearts were tangled and

  4. In vivo roles of BamA, BamB and BamD in the biogenesis of BamA, a core protein of the β-barrel assembly machine of Escherichia coli

    Science.gov (United States)

    Misra, Rajeev; Stikeleather, Ryan; Gabriele, Rebecca

    2014-01-01

    Assembly of the β-barrel outer membrane proteins (OMPs) is an essential cellular process in Gram negative bacteria and in the mitochondria and chloroplasts of eukaryotes—two organelles of bacterial origin. Central to this process is the conserved β-barrel OMP that belongs to the Omp85 superfamily. In Escherichia coli, BamA is the core β-barrel OMP, and together with four outer membrane lipoproteins, BamBCDE, constitute the β-barrel assembly machine (BAM). In this paper, we investigated the roles of BamD, an essential lipoprotein, and BamB in BamA biogenesis. Depletion of BamD caused impairment in BamA biogenesis and cessation of cell growth. These defects of BamD depletion were partly reversed by single amino acid substitutions mapping within the β-barrel domain of BamA. However, in the absence of BamB, the positive effects of the β-barrel substitutions on BamA biogenesis under BamD depletion conditions were nullified. By employing a BamA protein bearing one such substitution, F494L, it was demonstrated that the mutant BamA protein could not only assemble without BamD, but it could also facilitate the assembly of wild-type BamA expressed in trans. Based on these data, we propose a model in which the Bam lipoproteins, which are localized to the outer membrane by the BAM-independent Lol pathway, aid in the creation of new BAM complexes by serving as outer membrane receptors and folding factors for nascent BamA molecules. The newly assembled BAM holocomplex then catalyzes the assembly of substrate OMPs and BamA. These in vivo findings are corroborated by recently published in vitro data. PMID:24792419

  5. In vivo roles of BamA, BamB and BamD in the biogenesis of BamA, a core protein of the β-barrel assembly machine of Escherichia coli.

    Science.gov (United States)

    Misra, Rajeev; Stikeleather, Ryan; Gabriele, Rebecca

    2015-03-13

    Assembly of the β-barrel outer membrane proteins (OMPs) is an essential cellular process in Gram-negative bacteria and in the mitochondria and chloroplasts of eukaryotes--two organelles of bacterial origin. Central to this process is the conserved β-barrel OMP that belongs to the Omp85 superfamily. In Escherichia coli, BamA is the core β-barrel OMP and, together with four outer membrane lipoproteins, BamBCDE, constitutes the β-barrel assembly machine (BAM). In this paper, we investigated the roles of BamD, an essential lipoprotein, and BamB in BamA biogenesis. Depletion of BamD caused impairment in BamA biogenesis and cessation of cell growth. These defects of BamD depletion were partly reversed by single-amino-acid substitutions mapping within the β-barrel domain of BamA. However, in the absence of BamB, the positive effects of the β-barrel substitutions on BamA biogenesis under BamD depletion conditions were nullified. By employing a BamA protein bearing one such substitution, F474L, it was demonstrated that the mutant BamA protein could not only assemble without BamD but also facilitate the assembly of wild-type BamA expressed in trans. Based on these data, we propose a model in which the Bam lipoproteins, which are localized to the outer membrane by the BAM-independent Lol pathway, aid in the creation of new BAM complexes by serving as outer membrane receptors and folding factors for nascent BamA molecules. The newly assembled BAM holocomplex then catalyzes the assembly of substrate OMPs and BamA. These in vivo findings are corroborated by recently published in vitro data. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Twinkle overexpression prevents cardiac rupture after myocardial infarction by alleviating impaired mitochondrial biogenesis.

    Science.gov (United States)

    Inoue, Takahiro; Ikeda, Masataka; Ide, Tomomi; Fujino, Takeo; Matsuo, Yuka; Arai, Shinobu; Saku, Keita; Sunagawa, Kenji

    2016-09-01

    Cardiac rupture is a fatal complication after myocardial infarction (MI). However, the detailed mechanism underlying cardiac rupture after MI remains to be fully elucidated. In this study, we investigated the role of mitochondrial DNA (mtDNA) and mitochondria in the pathophysiology of cardiac rupture by analyzing Twinkle helicase overexpression mice (TW mice). Twinkle overexpression increased mtDNA copy number approximately twofold and ameliorated ischemic cardiomyopathy at day 28 after MI. Notably, Twinkle overexpression markedly prevented cardiac rupture and improved post-MI survival, accompanied by the suppression of MMP-2 and MMP-9 in the MI border area at day 5 after MI when cardiac rupture frequently occurs. Additionally, these cardioprotective effects of Twinkle overexpression were abolished in transgenic mice overexpressing mutant Twinkle with an in-frame duplication of amino acids 353-365, which resulted in no increases in mtDNA copy number. Furthermore, although apoptosis and oxidative stress were induced and mitochondria were damaged in the border area, these injuries were improved in TW mice. Further analysis revealed that mitochondrial biogenesis, including mtDNA copy number, transcription, and translation, was severely impaired in the border area at day 5 In contrast, Twinkle overexpression maintained mtDNA copy number and restored the impaired transcription and translation of mtDNA in the border area. These results demonstrated that Twinkle overexpression alleviated impaired mitochondrial biogenesis in the border area through maintained mtDNA copy number and thereby prevented cardiac rupture accompanied by the reduction of apoptosis and oxidative stress, and suppression of MMP activity. Copyright © 2016 the American Physiological Society.

  7. Absence of biochemical evidence at an early age delays diagnosis in a patient with a clinically severe peroxisomal biogenesis disorder

    NARCIS (Netherlands)

    Lüsebrink, Natalia; Porto, Luciana; Waterham, Hans R.; Ferdinandusse, Sacha; Rosewich, Hendrik; Kurlemann, Gerd; Kieslich, Matthias

    2016-01-01

    Analysis of the plasma levels of very long chain fatty acids (VLCFA) is a primary screening method for peroxisomal disorders and usually identifies severe peroxisomal biogenesis defects reliably. We report a patient presenting with typical facial stigmata, a treatment resistant seizure disorder and

  8. The dynein regulatory complex is required for ciliary motility and otolith biogenesis in the inner ear.

    Science.gov (United States)

    Colantonio, Jessica R; Vermot, Julien; Wu, David; Langenbacher, Adam D; Fraser, Scott; Chen, Jau-Nian; Hill, Kent L

    2009-01-08

    In teleosts, proper balance and hearing depend on mechanical sensors in the inner ear. These sensors include actin-based microvilli and microtubule-based cilia that extend from the surface of sensory hair cells and attach to biomineralized 'ear stones' (or otoliths). Otolith number, size and placement are under strict developmental control, but the mechanisms that ensure otolith assembly atop specific cells of the sensory epithelium are unclear. Here we demonstrate that cilia motility is required for normal otolith assembly and localization. Using in vivo video microscopy, we show that motile tether cilia at opposite poles of the otic vesicle create fluid vortices that attract otolith precursor particles, thereby biasing an otherwise random distribution to direct localized otolith seeding on tether cilia. Independent knockdown of subunits for the dynein regulatory complex and outer-arm dynein disrupt cilia motility, leading to defective otolith biogenesis. These results demonstrate a requirement for the dynein regulatory complex in vertebrates and show that cilia-driven flow is a key epigenetic factor in controlling otolith biomineralization.

  9. Proximity Interactions among Basal Body Components in Trypanosoma brucei Identify Novel Regulators of Basal Body Biogenesis and Inheritance

    Directory of Open Access Journals (Sweden)

    Hung Quang Dang

    2017-01-01

    Full Text Available The basal body shares similar architecture with centrioles in animals and is involved in nucleating flagellar axonemal microtubules in flagellated eukaryotes. The early-branching Trypanosoma brucei possesses a motile flagellum nucleated from the basal body that consists of a mature basal body and an adjacent pro-basal body. Little is known about the basal body proteome and its roles in basal body biogenesis and flagellar axoneme assembly in T. brucei. Here, we report the identification of 14 conserved centriole/basal body protein homologs and 25 trypanosome-specific basal body proteins. These proteins localize to distinct subdomains of the basal body, and several of them form a ring-like structure surrounding the basal body barrel. Functional characterization of representative basal body proteins revealed distinct roles in basal body duplication/separation and flagellar axoneme assembly. Overall, this work identified novel proteins required for basal body duplication and separation and uncovered new functions of conserved basal body proteins in basal body duplication and separation, highlighting an unusual mechanism of basal body biogenesis and inheritance in this early divergent eukaryote.

  10. Nitrite reductase activity and inhibition of H₂S biogenesis by human cystathionine ß-synthase.

    Directory of Open Access Journals (Sweden)

    Carmen Gherasim

    Full Text Available Nitrite was recognized as a potent vasodilator >130 years and has more recently emerged as an endogenous signaling molecule and modulator of gene expression. Understanding the molecular mechanisms that regulate nitrite metabolism is essential for its use as a potential diagnostic marker as well as therapeutic agent for cardiovascular diseases. In this study, we have identified human cystathionine ß-synthase (CBS as a new player in nitrite reduction with implications for the nitrite-dependent control of H₂S production. This novel activity of CBS exploits the catalytic property of its unusual heme cofactor to reduce nitrite and generate NO. Evidence for the possible physiological relevance of this reaction is provided by the formation of ferrous-nitrosyl (Fe(II-NO CBS in the presence of NADPH, the human diflavin methionine synthase reductase (MSR and nitrite. Formation of Fe(II-NO CBS via its nitrite reductase activity inhibits CBS, providing an avenue for regulating biogenesis of H₂S and cysteine, the limiting reagent for synthesis of glutathione, a major antioxidant. Our results also suggest a possible role for CBS in intracellular NO biogenesis particularly under hypoxic conditions. The participation of a regulatory heme cofactor in CBS in nitrite reduction is unexpected and expands the repertoire of proteins that can liberate NO from the intracellular nitrite pool. Our results reveal a potential molecular mechanism for cross-talk between nitrite, NO and H₂S biology.

  11. Role of loop L5-6 connecting transmembrane segments M5 and M6 in biogenesis and functioning of yeast Pma1 H+-ATPase.

    Science.gov (United States)

    Petrov, V V

    2015-01-01

    The L5-6 loop is a short extracytoplasmic stretch (714-DNSLDID) connecting transmembrane segments M5 and M6 and forming along with segments M4 and M8 the core through which cations are transported by H+-, Ca2+-, K+,Na+-, H+,K+-, and other P2-ATPases. To study structure-function relationships within this loop of the yeast plasma membrane Pma1 H+-ATPase, alanine- and cysteine-scanning mutagenesis has been employed. Ala and Cys substitutions for the most conserved residue (Leu717) led to complete block in biogenesis preventing the enzyme from reaching secretory vesicles. The Ala replacement at Asp714 led to five-fold decrease in the mutant expression and loss of its activity, while the Cys substitution blocked biogenesis completely. Replacements of other residues did not lead to loss of enzymatic activity. Additional replacements were made for Asp714 and Asp720 (Asp®Asn/Glu). Of the substitutions made at Asp714, only D714N partially restored the mutant enzyme biogenesis and functioning. However, all mutant enzymes with substituted Asp720 were active. The expressed mutants (34-95% of the wild-type level) showed activity high enough (35-108%) to be analyzed in detail. One of the mutants (I719A) had three-fold reduced coupling ratio between ATP hydrolysis and H+ transport; however, the I719C mutation was rather indistinguishable from the wild-type enzyme. Thus, substitutions at two of the seven positions seriously affected biogenesis and/or functioning of the enzyme. Taken together, these results suggest that the M5-M6 loop residues play an important role in protein stability and function, and they are probably responsible for proper arrangement of transmembrane segments M5 and M6 and other domains of the enzyme. This might also be important for the regulation of the enzyme.

  12. Coordinated Upregulation of Mitochondrial Biogenesis and Autophagy in Breast Cancer Cells: The Role of Dynamin Related Protein-1 and Implication for Breast Cancer Treatment

    Directory of Open Access Journals (Sweden)

    Peng Zou

    2016-01-01

    Full Text Available Overactive mitochondrial fission was shown to promote cell transformation and tumor growth. It remains elusive how mitochondrial quality is regulated in such conditions. Here, we show that upregulation of mitochondrial fission protein, dynamin related protein-1 (Drp1, was accompanied with increased mitochondrial biogenesis markers (PGC1α, NRF1, and Tfam in breast cancer cells. However, mitochondrial number was reduced, which was associated with lower mitochondrial oxidative capacity in breast cancer cells. This contrast might be owing to enhanced mitochondrial turnover through autophagy, because an increased population of autophagic vacuoles engulfing mitochondria was observed in the cancer cells. Consistently, BNIP3 (a mitochondrial autophagy marker and autophagic flux were significantly upregulated, indicative of augmented mitochondrial autophagy (mitophagy. The upregulation of Drp1 and BNIP3 was also observed in vivo (human breast carcinomas. Importantly, inhibition of Drp1 significantly suppressed mitochondrial autophagy, metabolic reprogramming, and cancer cell viability. Together, this study reveals coordinated increase of mitochondrial biogenesis and mitophagy in which Drp1 plays a central role regulating breast cancer cell metabolism and survival. Given the emerging evidence of PGC1α contributing to tumor growth, it will be of critical importance to target both mitochondrial biogenesis and mitophagy for effective cancer therapeutics.

  13. Peroxisome biogenesis disorders: identification of a new complementation group distinct from peroxisome-deficient CHO mutants and not complemented by human PEX 13

    NARCIS (Netherlands)

    Shimozawa, N.; Suzuki, Y.; Zhang, Z.; Imamura, A.; Tsukamoto, T.; Osumi, T.; Tateishi, K.; Okumoto, K.; Fujiki, Y.; Orii, T.; Barth, P. G.; Wanders, R. J.; Kondo, N.

    1998-01-01

    Ten complementation groups of generalized peroxisome biogenesis disorders (PBD), (excluding rhizomelic chondrodysplasia punctata) have been identified using complementation analysis. Four of the genes involved have been identified using two different methods of (1) genetic functional complementation

  14. Sirtuin 3, a new target of PGC-1alpha, plays an important role in the suppression of ROS and mitochondrial biogenesis.

    Directory of Open Access Journals (Sweden)

    Xingxing Kong

    2010-07-01

    Full Text Available Sirtuin 3 (SIRT3 is one of the seven mammalian sirtuins, which are homologs of the yeast Sir2 gene. SIRT3 is the only sirtuin with a reported association with the human life span. Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha plays important roles in adaptive thermogenesis, gluconeogenesis, mitochondrial biogenesis and respiration. PGC-1alpha induces several key reactive oxygen species (ROS-detoxifying enzymes, but the molecular mechanism underlying this is not well understood.Here we show that PGC-1alpha strongly stimulated mouse Sirt3 gene expression in muscle cells and hepatocytes. Knockdown of PGC-1alpha led to decreased Sirt3 gene expression. PGC-1alpha activated the mouse SIRT3 promoter, which was mediated by an estrogen-related receptor (ERR binding element (ERRE (-407/-399 mapped to the promoter region. Chromatin immunoprecipitation and electrophoretic mobility shift assays confirmed that ERRalpha bound to the identified ERRE and PGC-1alpha co-localized with ERRalpha in the mSirt3 promoter. Knockdown of ERRalpha reduced the induction of Sirt3 by PGC-1alpha in C(2C(12 myotubes. Furthermore, Sirt3 was essential for PGC-1alpha-dependent induction of ROS-detoxifying enzymes and several components of the respiratory chain, including glutathione peroxidase-1, superoxide dismutase 2, ATP synthase 5c, and cytochrome c. Overexpression of SIRT3 or PGC-1alpha in C(2C(12 myotubes decreased basal ROS level. In contrast, knockdown of mSIRT3 increased basal ROS level and blocked the inhibitory effect of PGC-1alpha on cellular ROS production. Finally, SIRT3 stimulated mitochondrial biogenesis, and SIRT3 knockdown decreased the stimulatory effect of PGC-1alpha on mitochondrial biogenesis in C(2C(12 myotubes.Our results indicate that Sirt3 functions as a downstream target gene of PGC-1alpha and mediates the PGC-1alpha effects on cellular ROS production and mitochondrial biogenesis. Thus, SIRT3 integrates cellular energy

  15. Role of G3BP1 in glucocorticoid receptor-mediated microRNA-15b and microRNA-23a biogenesis in endothelial cells

    KAUST Repository

    Kwok, Hoi-Hin

    2017-05-18

    MicroRNAs (miRNAs) are a family of non-coding RNAs that play crucial roles in regulating various normal cellular responses. Recent studies revealed that the canonical miRNA biogenesis pathway is subject to sophisticated regulation. Hormonal control of miRNA biogenesis by androgen and estrogen has been demonstrated, but the direct effects of the glucocorticoid receptor (GR) on miRNA biogenesis are unknown. This study revealed the role of GR in miRNA maturation. We showed that two GR agonists, dexamethasone and ginsenoside-Rg1 rapidly suppressed the expression of mature miR-15b, miR-23a, and miR-214 in human endothelial cells. RNA pulldown coupled with proteomic analysis identified GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) as one of the RNA-binding proteins mediating GR-regulated miRNA maturation. Activated GR induced phosphorylation of v-AKT Murine Thymoma Viral Oncogene Homologue (AKT) kinase, which in turn phosphorylated and promoted nuclear translocation of G3BP1. The nuclear G3BP1 bound to the G3BP1 consensus sequence located on primary miR-15b~16-2 and miR-23a~27a~24-2 to inhibit their maturation. The findings from this study have advanced our understanding of the non-genomic effects of GR in the vascular system.

  16. Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury.

    Science.gov (United States)

    Maejima, Ikuko; Takahashi, Atsushi; Omori, Hiroko; Kimura, Tomonori; Takabatake, Yoshitsugu; Saitoh, Tatsuya; Yamamoto, Akitsugu; Hamasaki, Maho; Noda, Takeshi; Isaka, Yoshitaka; Yoshimori, Tamotsu

    2013-08-28

    Diverse causes, including pathogenic invasion or the uptake of mineral crystals such as silica and monosodium urate (MSU), threaten cells with lysosomal rupture, which can lead to oxidative stress, inflammation, and apoptosis or necrosis. Here, we demonstrate that lysosomes are selectively sequestered by autophagy, when damaged by MSU, silica, or the lysosomotropic reagent L-Leucyl-L-leucine methyl ester (LLOMe). Autophagic machinery is recruited only on damaged lysosomes, which are then engulfed by autophagosomes. In an autophagy-dependent manner, low pH and degradation capacity of damaged lysosomes are recovered. Under conditions of lysosomal damage, loss of autophagy causes inhibition of lysosomal biogenesis in vitro and deterioration of acute kidney injury in vivo. Thus, we propose that sequestration of damaged lysosomes by autophagy is indispensable for cellular and tissue homeostasis.

  17. Tim50a, a nuclear isoform of the mitochondrial Tim50, interacts with proteins involved in snRNP biogenesis

    Directory of Open Access Journals (Sweden)

    Robinson Melvin L

    2005-07-01

    Full Text Available Abstract Background The Cajal body (CB is a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs, which are vital for pre-mRNA splicing. Newly imported Sm-class snRNPs traffic through CBs, where the snRNA component of the snRNP is modified, and then target to other nuclear domains such as speckles and perichromatin fibrils. It is not known how nascent snRNPs localize to the CB and are released from this structure after modification. The marker protein for CBs, coilin, may play a role in snRNP biogenesis given that it can interact with snRNPs and SMN, the protein mutated in Spinal Muscular Atrophy. Loss of coilin function in mice leads to significant viability and fertility problems and altered CB formation. Results In this report, we identify a minor isoform of the mitochondrial Tim50, Tim50a, as a coilin interacting protein. The Tim50a transcript can be detected in some cancer cell lines and normal brain tissue. The Tim50a protein differs only from Tim50 in that it contains an additional 103 aa N-terminal to the translation start of Tim50. Importantly, a putative nuclear localization signal is found within these 103 residues. In contrast to Tim50, which localizes to the cytoplasm and mitochondria, Tim50a is strictly nuclear and is enriched in speckles with snRNPs. In addition to coilin, Tim50a interacts with snRNPs and SMN. Competition binding experiments demonstrate that coilin competes with Sm proteins of snRNPs and SMN for binding sites on Tim50a. Conclusion Tim50a may play a role in snRNP biogenesis given its cellular localization and protein interaction characteristics. We hypothesize that Tim50a takes part in the release of snRNPs and SMN from the CB.

  18. Signaling Pathways in Exosomes Biogenesis, Secretion and Fate

    Directory of Open Access Journals (Sweden)

    Carla Emiliani

    2013-03-01

    Full Text Available Exosomes are small extracellular vesicles (30–100 nm derived from the endosomal system, which have raised considerable interest in the last decade. Several studies have shown that they mediate cell-to-cell communication in a variety of biological processes. Thus, in addition to cell-to-cell direct interaction or secretion of active molecules, they are now considered another class of signal mediators. Exosomes can be secreted by several cell types and retrieved in many body fluids, such as blood, urine, saliva and cerebrospinal fluid. In addition to proteins and lipids, they also contain nucleic acids, namely mRNA and miRNA. These features have prompted extensive research to exploit them as a source of biomarkers for several pathologies, such as cancer and neurodegenerative disorders. In this context, exosomes also appear attractive as gene delivery vehicles. Furthermore, exosome immunomodulatory and regenerative properties are also encouraging their application for further therapeutic purposes. Nevertheless, several issues remain to be addressed: exosome biogenesis and secretion mechanisms have not been clearly understood, and physiological functions, as well as pathological roles, are far from being satisfactorily elucidated.

  19. The pre-existing population of 5S rRNA effects p53 stabilization during ribosome biogenesis inhibition.

    Science.gov (United States)

    Onofrillo, Carmine; Galbiati, Alice; Montanaro, Lorenzo; Derenzini, Massimo

    2017-01-17

    Pre-ribosomal complex RPL5/RPL11/5S rRNA (5S RNP) is considered the central MDM2 inhibitory complex that control p53 stabilization during ribosome biogenesis inhibition. Despite its role is well defined, the dynamic of 5S RNP assembly still requires further characterization. In the present work, we report that MDM2 inhibition is dependent by a pre-existing population of 5S rRNA.

  20. Regulation of cardiac microRNAs by serum response factor

    Directory of Open Access Journals (Sweden)

    Wei Jeanne Y

    2011-02-01

    Full Text Available Abstract Serum response factor (SRF regulates certain microRNAs that play a role in cardiac and skeletal muscle development. However, the role of SRF in the regulation of microRNA expression and microRNA biogenesis in cardiac hypertrophy has not been well established. In this report, we employed two distinct transgenic mouse models to study the impact of SRF on cardiac microRNA expression and microRNA biogenesis. Cardiac-specific overexpression of SRF (SRF-Tg led to altered expression of a number of microRNAs. Interestingly, downregulation of miR-1, miR-133a and upregulation of miR-21 occurred by 7 days of age in these mice, long before the onset of cardiac hypertrophy, suggesting that SRF overexpression impacted the expression of microRNAs which contribute to cardiac hypertrophy. Reducing cardiac SRF level using the antisense-SRF transgenic approach (Anti-SRF-Tg resulted in the expression of miR-1, miR-133a and miR-21 in the opposite direction. Furthermore, we observed that SRF regulates microRNA biogenesis, specifically the transcription of pri-microRNA, thereby affecting the mature microRNA level. The mir-21 promoter sequence is conserved among mouse, rat and human; one SRF binding site was found to be in the mir-21 proximal promoter region of all three species. The mir-21 gene is regulated by SRF and its cofactors, including myocardin and p49/Strap. Our study demonstrates that the downregulation of miR-1, miR-133a, and upregulation of miR-21 can be reversed by one single upstream regulator, SRF. These results may help to develop novel therapeutic interventions targeting microRNA biogenesis.

  1. Co-and post-translational events in the biogenesis of pig small intestinal aminopeptidase N

    DEFF Research Database (Denmark)

    Danielsen, Erik Michael; Norén, O; Sjöström, H

    1982-01-01

    The biogenesis of pig small intestinal aminopeptidase N (EC 3. 4. 11. 2) was studied by cell-free translation of intestinal mRNA and by labelling of organ cultured intestinal explants. In cell-free translation, the primary mRNA translation product of aminopeptidase N was a polypeptide of Mr 115......,000. When translation was performed in the presence of dog pancreatic microsomes, a Mr 140,000 polypeptide was also observed. A polypeptide of Mr 115,000 was seen for the enzyme, purified from tunicamycin exposed explants. This result suggests that aminopeptidase N is co-translationally inserted...

  2. Role of G3BP1 in glucocorticoid receptor-mediated microRNA-15b and microRNA-23a biogenesis in endothelial cells

    KAUST Repository

    Kwok, Hoi-Hin; Poon, Po-Ying; Mak, Kylie Hin-Man; Zhang, Lin-Yao; Liu, Pei; Zhang, Huoming; Mak, Nai-Ki; Yue, Patrick Ying-Kit; Wong, Ricky Ngok-Shun

    2017-01-01

    MicroRNAs (miRNAs) are a family of non-coding RNAs that play crucial roles in regulating various normal cellular responses. Recent studies revealed that the canonical miRNA biogenesis pathway is subject to sophisticated regulation. Hormonal control

  3. Mirna biogenesis pathway is differentially regulated during adipose derived stromal/stem cell differentiation.

    Science.gov (United States)

    Martin, E C; Qureshi, A T; Llamas, C B; Burow, M E; King, A G; Lee, O C; Dasa, V; Freitas, M A; Forsberg, J A; Elster, E A; Davis, T A; Gimble, J M

    2018-02-07

    Stromal/stem cell differentiation is controlled by a vast array of regulatory mechanisms. Included within these are methods of mRNA gene regulation that occur at the level of epigenetic, transcriptional, and/or posttranscriptional modifications. Current studies that evaluate the posttranscriptional regulation of mRNA demonstrate microRNAs (miRNAs) as key mediators of stem cell differentiation through the inhibition of mRNA translation. miRNA expression is enhanced during both adipogenic and osteogenic differentiation; however, the mechanism by which miRNA expression is altered during stem cell differentiation is less understood. Here we demonstrate for the first time that adipose-derived stromal/stem cells (ASCs) induced to an adipogenic or osteogenic lineage have differences in strand preference (-3p and -5p) for miRNAs originating from the same primary transcript. Furthermore, evaluation of miRNA expression in ASCs demonstrates alterations in both miRNA strand preference and 5'seed site heterogeneity. Additionally, we show that during stem cell differentiation there are alterations in expression of genes associated with the miRNA biogenesis pathway. Quantitative RT-PCR demonstrated changes in the Argonautes (AGO1-4), Drosha, and Dicer at intervals of ASC adipogenic and osteogenic differentiation compared to untreated ASCs. Specifically, we demonstrated altered expression of the AGOs occurring during both adipogenesis and osteogenesis, with osteogenesis increasing AGO1-4 expression and adipogenesis decreasing AGO1 gene and protein expression. These data demonstrate changes to components of the miRNA biogenesis pathway during stromal/stem cell differentiation. Identifying regulatory mechanisms for miRNA processing during ASC differentiation may lead to novel mechanisms for the manipulation of lineage differentiation of the ASC through the global regulation of miRNA as opposed to singular regulatory mechanisms.

  4. New insights in the bacterial spore resistance to extreme terrestrial and extraterrestrial factors

    Science.gov (United States)

    Moeller, Ralf; Horneck, Gerda; Reitz, Guenther

    Based on their unique resistance to various space parameters, Bacillus endospores are one of the model systems used for astrobiological studies. The extremely high resistance of bacterial endospores to environmental stress factors has intrigued researchers since long time and many characteristic spore features, especially those involved in the protection of spore DNA, have already been uncovered. The disclosure of the complete genomic sequence of Bacillus subtilis 168, one of the often used astrobiological model system, and the rapid development of tran-scriptional microarray techniques have opened new opportunities of gaining further insights in the enigma of spore resistance. Spores of B. subtilis were exposed to various extreme ter-restrial and extraterrestrial stressors to reach a better understanding of the DNA protection and repair strategies, which them to cope with the induced DNA damage. Following physical stress factors of environmental importance -either on Earth or in space -were selected for this thesis: (i) mono-and polychromatic UV radiation, (ii) ionizing radiation, (iii) exposure to ultrahigh vacuum; and (iv) high shock pressures simulating meteorite impacts. To reach a most comprehensive understanding of spore resistance to those harsh terrestrial or simulated extraterrestrial conditions, a standardized experimental protocol of the preparation and ana-lyzing methods was established including the determination of the following spore responses: (i) survival, (ii) induced mutations, (iii) DNA damage, (iv) role of different repair pathways by use of a set of repair deficient mutants, and (v) transcriptional responses during spore germi-nation by use of genome-wide transcriptome analyses and confirmation by RT-PCR. From this comprehensive set of data on spore resistance to a variety of environmental stress parameters a model of a "built-in" transcriptional program of bacterial spores in response to DNA damaging treatments to ensure DNA restoration

  5. Evidence for a Role of Chloroplastic m-Type Thioredoxins in the Biogenesis of Photosystem II in Arabidopsis1[C][W][OPEN

    Science.gov (United States)

    Wang, Peng; Liu, Jun; Liu, Bing; Feng, Dongru; Da, Qingen; Wang, Peng; Shu, Shengying; Su, Jianbin; Zhang, Yang; Wang, Jinfa; Wang, Hong-Bin

    2013-01-01

    Chloroplastic m-type thioredoxins (TRX m) are essential redox regulators in the light regulation of photosynthetic metabolism. However, recent genetic studies have revealed novel functions for TRX m in meristem development, chloroplast morphology, cyclic electron flow, and tetrapyrrole synthesis. The focus of this study is on the putative role of TRX m1, TRX m2, and TRX m4 in the biogenesis of the photosynthetic apparatus in Arabidopsis (Arabidopsis thaliana). To that end, we investigated the impact of single, double, and triple TRX m deficiency on chloroplast development and the accumulation of thylakoid protein complexes. Intriguingly, only inactivation of three TRX m genes led to pale-green leaves and specifically reduced stability of the photosystem II (PSII) complex, implying functional redundancy between three TRX m isoforms. In addition, plants silenced for three TRX m genes displayed elevated levels of reactive oxygen species, which in turn interrupted the transcription of photosynthesis-related nuclear genes but not the expression of chloroplast-encoded PSII core proteins. To dissect the function of TRX m in PSII biogenesis, we showed that TRX m1, TRX m2, and TRX m4 interact physically with minor PSII assembly intermediates as well as with PSII core subunits D1, D2, and CP47. Furthermore, silencing three TRX m genes disrupted the redox status of intermolecular disulfide bonds in PSII core proteins, most notably resulting in elevated accumulation of oxidized CP47 oligomers. Taken together, our results suggest an important role for TRX m1, TRX m2, and TRX m4 proteins in the biogenesis of PSII, and they appear to assist the assembly of CP47 into PSII. PMID:24151299

  6. The Endospore-Forming Pathogen Bacillus cereus Exploits a Small Colony Variant-Based Diversification Strategy in Response to Aminoglycoside Exposure.

    Science.gov (United States)

    Frenzel, Elrike; Kranzler, Markus; Stark, Timo D; Hofmann, Thomas; Ehling-Schulz, Monika

    2015-12-08

    Bacillus cereus is among the microorganisms most often isolated from cases of food spoilage and causes gastrointestinal diseases as well as nongastrointestinal infections elicited by the emetic toxin cereulide, enterotoxins, and a panel of tissue-destructive virulence factors. This opportunistic pathogen is increasingly associated with rapidly fatal clinical infections especially linked to neonates and immunocompromised individuals. Fatality results from either the misdiagnosis of B. cereus as a contaminant of the clinical specimen or from failure of antibiotic therapy. Here we report for the first time that exposure to aminoglycoside antibiotics induces a phenotype switching of emetic B. cereus subpopulations to a slow-growing small colony variant (SCV) state. Along with altered antibiotic resistance, SCVs showed distinct phenotypic and metabolic properties, bearing the risk of antibiotic treatment failure and of clinical misdiagnosis by standard identification tests used in routine diagnostic. The SCV subpopulation is characterized by enhanced production of the toxin cereulide, but it does not secrete tissue-destructive and immune system-affecting enzymes such as sphingomyelinase and phospholipase. SCVs showed significantly prolonged persistence and decreased virulence in the Galleria mellonella model for bacterial infections, indicating diversification concerning their ecological lifestyle. Importantly, diversification into coexisting wild-type and SCV subpopulations also emerged during amikacin pressure during in vivo infection experiments. This study shows for the first time that pathogenic spore-forming B. cereus strains are able to switch to a so far unreported slow-growing lifestyle, which differs substantially in terms of developmental, phenotypic, metabolic, and virulence traits from the wild-type populations. This underpins the necessity of molecular-based differential diagnostics and a well-chosen therapeutic treatment strategy in clinical

  7. A PEX6-defective peroxisomal biogenesis disorder with severe phenotype in an infant, versus mild phenotype resembling Usher syndrome in the affected parents

    NARCIS (Netherlands)

    Raas-Rothschild, Annick; Wanders, Ronald J. A.; Mooijer, Petra A. W.; Gootjes, Jeannette; Waterham, Hans R.; Gutman, Alisa; Suzuki, Yasuyuki; Shimozawa, Nobuyuki; Kondo, Naomi; Eshel, Gideon; Espeel, Marc; Roels, Frank; Korman, Stanley H.

    2002-01-01

    Sensorineural deafness and retinitis pigmentosa (RP) are the hallmarks of Usher syndrome (USH) but are also prominent features in peroxisomal biogenesis defects (PBDs); both are autosomal recessively inherited. The firstborn son of unrelated parents, who both had sensorineural deafness and RP

  8. Equatorin is not essential for acrosome biogenesis but is required for the acrosome reaction

    Energy Technology Data Exchange (ETDEWEB)

    Hao, Jianxiu [State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Graduate School of the Chinese Academy of Sciences, Beijing 100049 (China); Chen, Min [State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Ji, Shaoyang; Wang, Xiaona; Wang, Yanbo [State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Graduate School of the Chinese Academy of Sciences, Beijing 100049 (China); Huang, Xingxu [MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, Nanjing Biomedical Research Institute, National Resource Center for Mutant Mice, Nanjing 210061 (China); Yang, Lin; Wang, Yaqing [State Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101 (China); Cui, Xiuhong; Lv, Limin [State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Liu, Yixun, E-mail: liuyx@ioz.ac.cn [State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Gao, Fei, E-mail: gaof@ioz.ac.cn [State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China)

    2014-02-21

    Highlights: • Eqtn knockout mice were used for these experiments. • In vivo and in vitro fertilization analyses were performed. • Eqtn-deficient sperm were evaluated by transmission electron microscopy (TEM) and an A23187-induced acrosome reaction (AR) assay. • Co-immunoprecipitation (Co-IP) was performed to assess the interaction between Eqtn and the SNARE complex. - Abstract: The acrosome is a specialized organelle that covers the anterior part of the sperm nucleus and plays an essential role in mammalian fertilization. However, the regulatory mechanisms controlling acrosome biogenesis and acrosome exocytosis during fertilization are largely unknown. Equatorin (Eqtn) is a membrane protein that is specifically localized to the acrosomal membrane. In the present study, the physiological functions of Eqtn were investigated using a gene knockout mouse model. We found that Eqtn{sup −/−} males were subfertile. Only approximately 50% of plugged females were pregnant after mating with Eqtn{sup −/−} males, whereas more than 90% of plugged females were pregnant after mating with control males. Sperm and acrosomes from Eqtn{sup −/−} mice presented normal motility and morphology. However, the fertilization and induced acrosome exocytosis rates of Eqtn-deficient sperm were dramatically reduced. Further studies revealed that the Eqtn protein might interact with Syntaxin1a and SNAP25, but loss of Eqtn did not affect the protein levels of these genes. Therefore, our study demonstrates that Eqtn is not essential for acrosome biogenesis but is required for the acrosome reaction. Eqtn is involved in the fusion of the outer acrosomal membrane and the sperm plasma membrane during the acrosome reaction, most likely via an interaction with the SNARE complex.

  9. Equatorin is not essential for acrosome biogenesis but is required for the acrosome reaction

    International Nuclear Information System (INIS)

    Hao, Jianxiu; Chen, Min; Ji, Shaoyang; Wang, Xiaona; Wang, Yanbo; Huang, Xingxu; Yang, Lin; Wang, Yaqing; Cui, Xiuhong; Lv, Limin; Liu, Yixun; Gao, Fei

    2014-01-01

    Highlights: • Eqtn knockout mice were used for these experiments. • In vivo and in vitro fertilization analyses were performed. • Eqtn-deficient sperm were evaluated by transmission electron microscopy (TEM) and an A23187-induced acrosome reaction (AR) assay. • Co-immunoprecipitation (Co-IP) was performed to assess the interaction between Eqtn and the SNARE complex. - Abstract: The acrosome is a specialized organelle that covers the anterior part of the sperm nucleus and plays an essential role in mammalian fertilization. However, the regulatory mechanisms controlling acrosome biogenesis and acrosome exocytosis during fertilization are largely unknown. Equatorin (Eqtn) is a membrane protein that is specifically localized to the acrosomal membrane. In the present study, the physiological functions of Eqtn were investigated using a gene knockout mouse model. We found that Eqtn −/− males were subfertile. Only approximately 50% of plugged females were pregnant after mating with Eqtn −/− males, whereas more than 90% of plugged females were pregnant after mating with control males. Sperm and acrosomes from Eqtn −/− mice presented normal motility and morphology. However, the fertilization and induced acrosome exocytosis rates of Eqtn-deficient sperm were dramatically reduced. Further studies revealed that the Eqtn protein might interact with Syntaxin1a and SNAP25, but loss of Eqtn did not affect the protein levels of these genes. Therefore, our study demonstrates that Eqtn is not essential for acrosome biogenesis but is required for the acrosome reaction. Eqtn is involved in the fusion of the outer acrosomal membrane and the sperm plasma membrane during the acrosome reaction, most likely via an interaction with the SNARE complex

  10. Molecular basis of usher pore gating in Escherichia coli pilus biogenesis.

    Science.gov (United States)

    Volkan, Ender; Kalas, Vasilios; Pinkner, Jerome S; Dodson, Karen W; Henderson, Nadine S; Pham, Thieng; Waksman, Gabriel; Delcour, Anne H; Thanassi, David G; Hultgren, Scott J

    2013-12-17

    Extracellular fibers called chaperone-usher pathway pili are critical virulence factors in a wide range of Gram-negative pathogenic bacteria that facilitate binding and invasion into host tissues and mediate biofilm formation. Chaperone-usher pathway ushers, which catalyze pilus assembly, contain five functional domains: a 24-stranded transmembrane β-barrel translocation domain (TD), a β-sandwich plug domain (PLUG), an N-terminal periplasmic domain, and two C-terminal periplasmic domains (CTD1 and 2). Pore gating occurs by a mechanism whereby the PLUG resides stably within the TD pore when the usher is inactive and then upon activation is translocated into the periplasmic space, where it functions in pilus assembly. Using antibiotic sensitivity and electrophysiology experiments, a single salt bridge was shown to function in maintaining the PLUG in the TD channel of the P pilus usher PapC, and a loop between the 12th and 13th beta strands of the TD (β12-13 loop) was found to facilitate pore opening. Mutation of the β12-13 loop resulted in a closed PapC pore, which was unable to efficiently mediate pilus assembly. Deletion of the PapH terminator/anchor resulted in increased OM permeability, suggesting a role for the proper anchoring of pili in retaining OM integrity. Further, we introduced cysteine residues in the PLUG and N-terminal periplasmic domains that resulted in a FimD usher with a greater propensity to exist in an open conformation, resulting in increased OM permeability but no loss in type 1 pilus assembly. These studies provide insights into the molecular basis of usher pore gating and its roles in pilus biogenesis and OM permeability.

  11. Distribution and Downward Movement of Pasteuria penetrans in Field Soil.

    Science.gov (United States)

    Cetintas, R; Dickson, D W

    2005-06-01

    Endospores of Pasteuria penetrans were evaluated for their vertical distribution in field soil and their downward movement through soil in the laboratory. In the field trial, the number of endospores attached to second-stage juveniles (J2) of Meloidogyne arenaria race 1 varied greatly in different soil depths. There were higher percentages of J2 with endospores attached in former weed fallow plots during the first 3 years of growing peanut than in former bahiagrass and rhizomal peanut plots (P 0.05). In laboratory trials, P. penetrans endospores were observed to move throughout the soil through the percolation of water. After one application of water, some endospores were detected 25 to 37.5 cm deep. Endospores were present at the greatest depth, 37.5 to 50 cm, after the third application of water. These results indicate that rain or water applications by irrigation are likely to move endospores to deeper levels of the soil, but the majority of endospores remain in the upper 0-to-30-cm depth.

  12. Dual Role for Hsc70 in the Biogenesis and Regulation of the Heme-Regulated Kinase of the α Subunit of Eukaryotic Translation Initiation Factor 2

    Science.gov (United States)

    Uma, Sheri; Thulasiraman, Vanitha; Matts, Robert L.

    1999-01-01

    The heme-regulated kinase of the α subunit of eukaryotic initiation factor 2 (HRI) is activated in rabbit reticulocyte lysate (RRL) in response to a number of environmental conditions, including heme deficiency, heat shock, and oxidative stress. Activation of HRI causes an arrest of initiation of protein synthesis. Recently, we have demonstrated that the heat shock cognate protein Hsc70 negatively modulates the activation of HRI in RRL in response to these environmental conditions. Hsc70 is also known to be a critical component of the Hsp90 chaperone machinery in RRL, which plays an obligatory role for HRI to acquire and maintain a conformation that is competent to activate. Using de novo-synthesized HRI in synchronized pulse-chase translations, we have examined the role of Hsc70 in the regulation of HRI biogenesis and activation. Like Hsp90, Hsc70 interacted with nascent HRI and HRI that was matured to a state which was competent to undergo stimulus-induced activation (mature-competent HRI). Interaction of HRI with Hsc70 was required for the transformation of HRI, as the Hsc70 antagonist clofibric acid inhibited the folding of HRI into a mature-competent conformation. Unlike Hsp90, Hsc70 also interacted with transformed HRI. Clofibric acid disrupted the interaction of Hsc70 with transformed HRI that had been matured and transformed in the absence of the drug. Disruption of Hsc70 interaction with transformed HRI in heme-deficient RRL resulted in its hyperactivation. Furthermore, activation of HRI in response to heat shock or denatured proteins also resulted in a similar blockage of Hsc70 interaction with transformed HRI. These results indicate that Hsc70 is required for the folding and transformation of HRI into an active kinase but is subsequently required to negatively attenuate the activation of transformed HRI. PMID:10454533

  13. Autophagy in the test tube: In vitro reconstitution of aspects of autophagosome biogenesis.

    Science.gov (United States)

    Rao, Yijian; Matscheko, Nena; Wollert, Thomas

    2016-06-01

    Autophagy is a versatile recycling pathway that delivers cytoplasmic contents to lysosomal compartments for degradation. It involves the formation of a cup-shaped membrane that expands to capture cargo. After the cargo has been entirely enclosed, the membrane is sealed to generate a double-membrane-enclosed compartment, termed the autophagosome. Depending on the physiological state of the cell, the cargo is selected either specifically or non-specifically. The process involves a highly conserved set of autophagy-related proteins. Reconstitution of their action on model membranes in vitro has contributed tremendously to our understanding of autophagosome biogenesis. This review will focus on various in vitro techniques that have been employed to decipher the function of the autophagic core machinery. © 2016 Federation of European Biochemical Societies.

  14. Epigenetic up-regulation of ribosome biogenesis and more aggressive phenotype triggered by the lack of the histone demethylase JHDM1B in mammary epithelial cells.

    Science.gov (United States)

    Galbiati, Alice; Penzo, Marianna; Bacalini, Maria Giulia; Onofrillo, Carmine; Guerrieri, Ania Naila; Garagnani, Paolo; Franceschi, Claudio; Treré, Davide; Montanaro, Lorenzo

    2017-06-06

    The alterations of ribosome biogenesis and protein synthesis play a direct role in the development of tumors. The accessibility and transcription of ribosomal genes is controlled at several levels, with their epigenetic regulation being one of the most important. Here we explored the JmjC domain-containing histone demethylase 1B (JHDM1B) function in the epigenetic control of rDNA transcription. Since JHDM1B is a negative regulator of gene transcription, we focused on the effects induced by JHDM1B knock-down (KD). We studied the consequences of stable inducible JHDM1B silencing in cell lines derived from transformed and untransformed mammary epithelial cells. In these cellular models, prolonged JHDM1B downregulation triggered a surge of 45S pre-rRNA transcription and processing, associated with a re-modulation of the H3K36me2 levels at rDNA loci and with changes in DNA methylation of specific CpG sites in rDNA genes. We also found that after JHDM1B KD, cells showed a higher ribosome content: which were engaged in mRNA translation. JHDM1B KD and the consequent stimulation of ribosomes biogenesis conferred more aggressive features to the tested cellular models, which acquired a greater clonogenic, staminal and invasive potential. Taken together, these data indicate that the reduction of JHDM1B leads to a more aggressive cellular phenotype in mammary gland cells, by virtue of its negative regulatory activity on ribosome biogenesis.

  15. Aluminium induced oxidative stress results in decreased mitochondrial biogenesis via modulation of PGC-1α expression.

    Science.gov (United States)

    Sharma, Deep Raj; Sunkaria, Aditya; Wani, Willayat Yousuf; Sharma, Reeta Kumari; Kandimalla, Ramesh J L; Bal, Amanjit; Gill, Kiran Dip

    2013-12-01

    The present investigation was carried out to elucidate a possible molecular mechanism related to the effects of aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of Peroxisome proliferator activated receptor gamma co-activator 1α (PGC-1α) and its downstream targets i.e. Nuclear respiratory factor-1(NRF-1), Nuclear respiratory factor-2(NRF-2) and Mitochondrial transcription factor A (Tfam) in mitochondrial biogenesis. Aluminium lactate (10mg/kgb.wt./day) was administered intragastrically to rats for 12 weeks. After 12 weeks of exposure, we found an increase in ROS levels, mitochondrial DNA oxidation and decrease in citrate synthase activity in the Hippocampus (HC) and Corpus striatum (CS) regions of rat brain. On the other hand, there was a decrease in the mRNA levels of the mitochondrial encoded subunits-NADH dehydrogenase (ND) subunits i.e. ND1, ND2, ND3, Cytochrome b (Cytb), Cytochrome oxidase (COX) subunits i.e. COX1, COX3, ATP synthase (ATPase) subunit 6 along with reduced expression of nuclear encoded subunits COX4, COX5A, COX5B of Electron transport chain (ETC). Besides, a decrease in mitochondrial DNA copy number and mitochondrial content in both regions of rat brain was observed. The PGC-1α was down-regulated in aluminium treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α in aluminium treated rats. Electron microscopy results revealed a significant increase in the mitochondrial swelling, loss of cristae, chromatin condensation and decreases in mitochondrial number in case of aluminium treated rats as compared to control. So, PGC-1α seems to be a potent target for aluminium neurotoxicity, which makes it an almost ideal target to control or limit the damage that has been associated with the defective mitochondrial function seen in neurodegenerative diseases. © 2013.

  16. The Prohormone VGF Regulates β Cell Function via Insulin Secretory Granule Biogenesis

    Directory of Open Access Journals (Sweden)

    Samuel B. Stephens

    2017-09-01

    Full Text Available The prohormone VGF is expressed in neuroendocrine and endocrine tissues and regulates nutrient and energy status both centrally and peripherally. We and others have shown that VGF-derived peptides have direct action on the islet β cell as secretagogues and cytoprotective agents; however, the endogenous function of VGF in the β cell has not been described. Here, we demonstrate that VGF regulates secretory granule formation. VGF loss-of-function studies in both isolated islets and conditional knockout mice reveal a profound decrease in stimulus-coupled insulin secretion. Moreover, VGF is necessary to facilitate efficient exit of granule cargo from the trans-Golgi network and proinsulin processing. It also functions to replenish insulin granule stores following nutrient stimulation. Our data support a model in which VGF operates at a critical node of granule biogenesis in the islet β cell to coordinate insulin biosynthesis with β cell secretory capacity.

  17. SNP Variation in MicroRNA Biogenesis Pathway Genes as a New Innovation Strategy for Alzheimer Disease Diagnostics: A Study of 10 Candidate Genes in an Understudied Population From the Eastern Mediterranean.

    Science.gov (United States)

    Görücü Yilmaz, Şenay; Erdal, Mehmet E; Avci Özge, Aynur; Sungur, Mehmet A

    2016-01-01

    Alzheimer disease (AD) is a common complex neurodegenerative disorder accounting for nearly 50% to 70% of dementias worldwide. Yet the current diagnostic options for AD are limited. New diagnostic innovation strategies focusing on novel molecules and pathways are sorely needed. In this connection, microRNAs (miRNAs) are conserved small noncoding RNAs that regulate posttranscriptional gene expression and are vital for neuronal development and its functional sustainability. Conceivably, biological pathways responsible for the biogenesis of miRNAs represent a veritable set of upstream candidate genes that can be potentially associated with the AD pathophysiology. Notably, whereas functional single-nucleotide polymorphisms (SNPs) in miRNA biogenesis pathway genes have been studied in other complex diseases, surprisingly, virtually no such study has been conducted on their relevance in AD. Moreover, novel diagnostics identified in easily accessible peripheral tissues such as the whole blood samples represent the initial entry or gateway points on the biomarker discovery critical path for AD. To the best of our knowledge, we report here the first association study of functional SNPs, as measured by real-time PCR in 10 "upstream" candidate genes critically situated on the miRNA biogenesis pathway, in a large sample of AD patients (N=172) and healthy controls (N=109) in a hitherto understudied world population from the Mersin region of the Eastern Mediterranean. We observed a significant association between 2 candidate genes and AD, TARBP2 rs784567 genotype and AD (χ=6.292, P=0.043), and a trend for RNASEN rs10719 genotype (χ=4.528, P=0.104) and allele (P=0.035). Functional SNP variations in the other 8 candidate genes (DGCR8, XPO5, RAN, DICER1, AGO1, AGO2, GEMIN3, and GEMIN4) did not associate with AD in our sample. Given the putative biological importance of miRNA biogenesis pathways, these emerging data can provide a new foundation to stimulate future debate and

  18. Gemfibrozil pretreatment resulted in a sexually dimorphic outcome in the rat models of global cerebral ischemia-reperfusion via modulation of mitochondrial pro-survival and apoptotic cell death factors as well as MAPKs.

    Science.gov (United States)

    Mohagheghi, Fatemeh; Ahmadiani, Abolhassan; Rahmani, Behrouz; Moradi, Fatemeh; Romond, Nathalie; Khalaj, Leila

    2013-07-01

    Inducers of mitochondrial biogenesis are widely under investigation for use in a novel therapeutic approach in neurodegenerative disorders. The ability of Gemfibrozil, a fibrate, is investigated for the first time to modulate mitochondrial pro-survival factors involved in the mitochondrial biogenesis signaling pathway, including peroxisome proliferator-activated receptor coactivator-1α (PGC-1α), nuclear respiratory factor (NRF-1), and mitochondrial transcription factor A (TFAM) in the brain. Gemfibozil is clinically administered to control hyperlipidemia. It secondarily prevents cardiovascular events such as cardiac arrest in susceptible patients. In this study, pretreatment of animals with gemfibrozil prior to ischemia-reperfusion (I/R) resulted in a sexually dimorphic outcome. While the expression of NRF-1 and TFAM were induced in gemfibrozil-pretreated met-estrous females, they were suppressed in males. Gemfibrozil also proved to be neuroprotective in met-estrous females, as it inhibited caspase-dependent apoptosis while in males it led to hippocampal neurodegeneration via activation of both the caspase-dependent and caspase-independent apoptosis. In the mitogen-activated protein kinase (MAPKs) pathway, gemfibrozil pretreatment induced the expression of extracellular signal-regulated kinases (ERK1/2) in met-estrous females and reduced it in males. These findings correlatively point to the sexual-dimorphic effects of gemfibrozil in global cerebral I/R context by affecting important factors involved in the mitochondrial biogenesis, MAPKs, and apoptotic cell death pathways.

  19. Pasteuria spp.: Systematics and Phylogeny of These Bacterial Parasites of Phytopathogenic Nematodes.

    Science.gov (United States)

    Preston, J F; Dickson, D W; Maruniak, J E; Nong, G; Brito, J A; Schmidt, L M; Giblin-Davis, R M

    2003-06-01

    Pasteuria spp. include endospore-forming bacterial pathogens of cladoceran crustaceans and plant-parasitic nematodes. Propagation of these nematode pathogens requires attachment of soilborne endospores to nematode hosts, infection, growth, sporulation, and release of endospores to repeat the cycle of infection and propagation. The ability of these bacteria to suppress the levels of plant-parasitic nematodes in the field has made them particularly promising candidates for biocontrol of nematode diseases of plants. Genes encoding 16S ribosomal RNA have been sequenced for the cladoceran (water flea) parasite and type species, Pasteuria ramosa, and for Pasteuria spp. isolated from root-knot (Meloidogyne arenaria race 1 and Meloidogyne sp.), soybean cyst (Heterodera glycines), and sting (Belonolaimus longicaudatus) nematodes. These have provided a phylogenetic basis for their designation to a distinct clade within the family Alicyclobacillaceae of the gram-positive endospore-forming bacteria. Two apparent biotypes of P. penetrans demonstrating a host preference for different Meloidogyne spp. showed identical 16S rDNA sequences, suggesting host-recognition evolves within a given species. The sequences of genes encoding sporulation transcription factors, sigE and sigF, from P. penetrans biotype P-20 show different phylogenetic relationships to other endospore-forming bacteria, supporting their application to further discriminate Pasteuria spp. and biotypes. Distribution of an adhesin-associated epitope on polypeptides from different Pasteuria isolates provides an immunochemical approach to differentiate species and biotypes with specific host preferences. Application of bioinformatics to genomic data, as well as further characterization of the biochemical basis for host recognition, will facilitate development of Pasteuria spp. as benign alternatives to chemical nematicides.

  20. Calorie restriction increases muscle mitochondrial biogenesis in healthy humans.

    Directory of Open Access Journals (Sweden)

    Anthony E Civitarese

    2007-03-01

    Full Text Available Caloric restriction without malnutrition extends life span in a range of organisms including insects and mammals and lowers free radical production by the mitochondria. However, the mechanism responsible for this adaptation are poorly understood.The current study was undertaken to examine muscle mitochondrial bioenergetics in response to caloric restriction alone or in combination with exercise in 36 young (36.8 +/- 1.0 y, overweight (body mass index, 27.8 +/- 0.7 kg/m(2 individuals randomized into one of three groups for a 6-mo intervention: Control, 100% of energy requirements; CR, 25% caloric restriction; and CREX, caloric restriction with exercise (CREX, 12.5% CR + 12.5% increased energy expenditure (EE. In the controls, 24-h EE was unchanged, but in CR and CREX it was significantly reduced from baseline even after adjustment for the loss of metabolic mass (CR, -135 +/- 42 kcal/d, p = 0.002 and CREX, -117 +/- 52 kcal/d, p = 0.008. Participants in the CR and CREX groups had increased expression of genes encoding proteins involved in mitochondrial function such as PPARGC1A, TFAM, eNOS, SIRT1, and PARL (all, p < 0.05. In parallel, mitochondrial DNA content increased by 35% +/- 5% in the CR group (p = 0.005 and 21% +/- 4% in the CREX group (p < 0.004, with no change in the control group (2% +/- 2%. However, the activity of key mitochondrial enzymes of the TCA (tricarboxylic acid cycle (citrate synthase, beta-oxidation (beta-hydroxyacyl-CoA dehydrogenase, and electron transport chain (cytochrome C oxidase II was unchanged. DNA damage was reduced from baseline in the CR (-0.56 +/- 0.11 arbitrary units, p = 0.003 and CREX (-0.45 +/- 0.12 arbitrary units, p = 0.011, but not in the controls. In primary cultures of human myotubes, a nitric oxide donor (mimicking eNOS signaling induced mitochondrial biogenesis but failed to induce SIRT1 protein expression, suggesting that additional factors may regulate SIRT1 content during CR.The observed increase in

  1. The atlA operon of Streptococcus mutans: role in autolysin maturation and cell surface biogenesis.

    Science.gov (United States)

    Ahn, Sang-Joon; Burne, Robert A

    2006-10-01

    The Smu0630 protein (AtlA) was recently shown to be involved in cell separation, biofilm formation, and autolysis. Here, transcriptional studies revealed that atlA is part of a multigene operon under the control of at least three promoters. The morphology and biofilm-forming capacity of a nonpolar altA mutant could be restored to that of the wild-type strain by adding purified AtlA protein to the medium. A series of truncated derivatives of AtlA revealed that full activity required the C terminus and repeat regions. AtlA was cell associated and readily extractable from with sodium dodecyl sulfate. Of particular interest, the surface protein profile of AtlA-deficient strains was dramatically altered compared to the wild-type strain, as was the nature of the association of the multifunctional adhesin P1 with the cell wall. In addition, AtlA-deficient strains failed to develop competence as effectively as the parental strain. Mutation of thmA, which can be cotranscribed with atlA and encodes a putative pore-forming protein, resulted in a phenotype very similar to that of the AtlA-deficient strain. ThmA was also shown to be required for efficient processing of AtlA to its mature form, and treatment of the thmA mutant strain with full-length AtlA protein did not restore normal cell separation and biofilm formation. The effects of mutating other genes in the operon on cell division, biofilm formation, or AtlA biogenesis were not as profound. This study reveals that AtlA is a surface-associated protein that plays a critical role in the network connecting cell surface biogenesis, biofilm formation, genetic competence, and autolysis.

  2. Training-induced acceleration of O(2) uptake on-kinetics precedes muscle mitochondrial biogenesis in humans.

    Science.gov (United States)

    Zoladz, Jerzy A; Grassi, Bruno; Majerczak, Joanna; Szkutnik, Zbigniew; Korostyński, Michał; Karasiński, Janusz; Kilarski, Wincenty; Korzeniewski, Bernard

    2013-04-01

    The effects of 5 weeks of moderate-intensity endurance training on pulmonary oxygen uptake kinetics (V(O(2)) on-kinetics) were studied in 15 healthy men (mean ± SD: age 22.7 ± 1.8 years, body weight 76.4 ± 8.9 kg and maximal V(O(2)) 46.0 ± 3.7 ml kg(-1) min(-1)). Training caused a significant acceleration (P = 0.003) of V(O(2)) on-kinetics during moderate-intensity cycling (time constant of the 'primary' component 30.0 ± 6.6 versus 22.8 ± 5.6 s before and after training, respectively) and a significant decrease (P = 0.04) in the amplitude of the primary component (837 ± 351 versus 801 ± 330 ml min(-1)). No changes in myosin heavy chain distribution, muscle fibre capillarization, level of peroxisome proliferator-activated receptor γ coactivator 1α and other markers of mitochondrial biogenesis (mitochondrial DNA copy number, cytochrome c and cytochrome oxidase subunit I contents) in the vastus lateralis were found after training. A significant downregulation in the content of the sarcoplasmic reticulum ATPase 2 (SERCA2; P = 0.03) and a tendency towards a decrease in SERCA1 (P = 0.055) was found after training. The decrease in SERCA1 was positively correlated (P = 0.05) with the training-induced decrease in the gain of the V(O(2)) on-kinetics (ΔV(O(2)) at steady state/Δpower output). In the early stage of training, the acceleration in V(O(2)) on-kinetics during moderate-intensity cycling can occur without enhanced mitochondrial biogenesis or changes in muscle myosin heavy chain distribution and in muscle fibre capillarization. The training-induced decrease of the O(2) cost of cycling could be caused by the downregulation of SERCA pumps.

  3. Mono-uridylation of pre-microRNA as a key step in the biogenesis of group II let-7 microRNAs.

    Science.gov (United States)

    Heo, Inha; Ha, Minju; Lim, Jaechul; Yoon, Mi-Jeong; Park, Jong-Eun; Kwon, S Chul; Chang, Hyeshik; Kim, V Narry

    2012-10-26

    RNase III Drosha initiates microRNA (miRNA) maturation by cleaving a primary miRNA transcript and releasing a pre-miRNA with a 2 nt 3' overhang. Dicer recognizes the 2 nt 3' overhang structure to selectively process pre-miRNAs. Here, we find that, unlike prototypic pre-miRNAs (group I), group II pre-miRNAs acquire a shorter (1 nt) 3' overhang from Drosha processing and therefore require a 3'-end mono-uridylation for Dicer processing. The majority of let-7 and miR-105 belong to group II. We identify TUT7/ZCCHC6, TUT4/ZCCHC11, and TUT2/PAPD4/GLD2 as the terminal uridylyl transferases responsible for pre-miRNA mono-uridylation. The TUTs act specifically on dsRNAs with a 1 nt 3' overhang, thereby creating a 2 nt 3' overhang. Depletion of TUTs reduces let-7 levels and disrupts let-7 function. Although the let-7 suppressor, Lin28, induces inhibitory oligo-uridylation in embryonic stem cells, mono-uridylation occurs in somatic cells lacking Lin28 to promote let-7 biogenesis. Our study reveals functional duality of uridylation and introduces TUT7/4/2 as components of the miRNA biogenesis pathway. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Biogenesis of the yeast cytochrome bc1 complex.

    Science.gov (United States)

    Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L

    2009-01-01

    The mitochondrial respiratory chain is composed of four different protein complexes that cooperate in electron transfer and proton pumping across the inner mitochondrial membrane. The cytochrome bc1 complex, or complex III, is a component of the mitochondrial respiratory chain. This review will focus on the biogenesis of the bc1 complex in the mitochondria of the yeast Saccharomyces cerevisiae. In wild type yeast mitochondrial membranes the major part of the cytochrome bc1 complex was found in association with one or two copies of the cytochrome c oxidase complex. The analysis of several yeast mutant strains in which single genes or pairs of genes encoding bc1 subunits had been deleted revealed the presence of a common set of bc1 sub-complexes. These sub-complexes are represented by the central core of the bc1 complex, consisting of cytochrome b bound to subunit 7 and subunit 8, by the two core proteins associated with each other, by the Rieske protein associated with subunit 9, and by those deriving from the unexpected interaction of each of the two core proteins with cytochrome c1. Furthermore, a higher molecular mass sub-complex is that composed of cytochrome b, cytochrome c1, core protein 1 and 2, subunit 6, subunit 7 and subunit 8. The identification and characterization of all these sub-complexes may help in defining the steps and the molecular events leading to bc1 assembly in yeast mitochondria.

  5. Increase of mitochondrial DNA content and transcripts in early bovine embryogenesis associated with upregulation of mtTFA and NRF1 transcription factors

    Directory of Open Access Journals (Sweden)

    Heyman Yvan

    2005-11-01

    Full Text Available Abstract Background Recent work has shown that mitochondrial biogenesis and mitochondrial functions are critical determinants of embryonic development. However, the expression of the factors controlling mitochondrial biogenesis in early embryogenesis has received little attention so far. Methods We used real-time quantitative PCR to quantify mitochondrial DNA (mtDNA in bovine oocytes and in various stages of in vitro produced embryos. To investigate the molecular mechanisms responsible for the replication and the transcriptional activation of mtDNA, we quantified the mRNA corresponding to the mtDNA-encoded cytochrome oxidase 1 (COX1, and two nuclear-encoded factors, i.e. the Nuclear Respiratory Factor 1 (NRF1, and the nuclear-encoded Mitochondrial Transcription Factor A (mtTFA. Results Unlike findings reported in mouse embryos, the mtDNA content was not constant during early bovine embryogenesis. We found a sharp, 60% decrease in mtDNA content between the 2-cell and the 4/8-cell stages. COX1 mRNA was constant until the morula stage after which it increased dramatically. mtTFA mRNA was undetectable in oocytes and remained so until the 8/16-cell stage; it began to appear only at the morula stage, suggesting de novo synthesis. In contrast, NRF1 mRNA was detectable in oocytes and the quantity remained constant until the morula stage. Conclusion Our results revealed a reduction of mtDNA content in early bovine embryos suggesting an active process of mitochondrial DNA degradation. In addition, de novo mtTFA expression associated with mitochondrial biogenesis activation and high levels of NRF1 mRNA from the oocyte stage onwards argue for the essential function of these factors during the first steps of bovine embryogenesis.

  6. Nitazoxanide Inhibits Pilus Biogenesis by Interfering with Folding of the Usher Protein in the Outer Membrane.

    Science.gov (United States)

    Chahales, Peter; Hoffman, Paul S; Thanassi, David G

    2016-04-01

    Many bacterial pathogens assemble surface fibers termed pili or fimbriae that facilitate attachment to host cells and colonization of host tissues. The chaperone/usher (CU) pathway is a conserved secretion system that is responsible for the assembly of virulence-associated pili by many different Gram-negative bacteria. Pilus biogenesis by the CU pathway requires a dedicated periplasmic chaperone and an integral outer membrane (OM) assembly and secretion platform termed the usher. Nitazoxanide (NTZ), an antiparasitic drug, was previously shown to inhibit the function of aggregative adherence fimbriae and type 1 pili assembled by the CU pathway in enteroaggregativeEscherichia coli, an important causative agent of diarrhea. We show here that NTZ also inhibits the function of type 1 and P pili from uropathogenicE. coli(UPEC). UPEC is the primary causative agent of urinary tract infections, and type 1 and P pili mediate colonization of the bladder and kidneys, respectively. By analysis of the different stages of the CU pilus biogenesis pathway, we show that treatment of bacteria with NTZ causes a reduction in the number of usher molecules in the OM, resulting in a loss of pilus assembly on the bacterial surface. In addition, we determine that NTZ specifically prevents proper folding of the usher β-barrel domain in the OM. Our findings demonstrate that NTZ is a pilicide with a novel mechanism of action and activity against diverse CU pathways. This suggests that further development of the NTZ scaffold may lead to new antivirulence agents that target the usher to prevent pilus assembly. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  7. Alterations in antioxidant system, mitochondrial biogenesis and autophagy in preeclamptic myometrium

    Directory of Open Access Journals (Sweden)

    Polina A. Vishnyakova

    2017-12-01

    Full Text Available Preeclampsia is a pregnancy complication which causes significant maternal and fetal morbidity and mortality worldwide. Although intensive research has been performed in the last 40 years, the pathology of preeclampsia is still poorly understood. The present work is a comparative study of the myometrium of women with normal pregnancy, and those with late- and early-onset preeclampsia (n = 10 for each group. We observed significant changes in the levels of antioxidant enzymes, markers of mitochondrial biogenesis and autophagy proteins in preeclamptic myometrium. Levels of superoxide dismutase 1 and catalase were lower in both preeclamptic groups than the control group. In late-onset preeclampsia, expression levels of essential mitochondria-related proteins VDAC1, TFAM, hexokinase 1, PGC-1α and PGC-1β, and autophagy marker LC3A, were significantly elevated. In the myometrium of the early-onset preeclampsia group OPA1 and Bcl-2 were up-regulated compared to those of the control (p < 0.05. These findings suggest that crucial molecular changes in the maternal myometrium occur with the development of preeclampsia.

  8. Lipopolysaccharide biogenesis and transport at the outer membrane of Gram-negative bacteria.

    Science.gov (United States)

    Sperandeo, Paola; Martorana, Alessandra M; Polissi, Alessandra

    2017-11-01

    The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer containing a unique glycolipid, lipopolysaccharide (LPS) in its outer leaflet. LPS molecules confer to the OM peculiar permeability barrier properties enabling Gram-negative bacteria to exclude many toxic compounds, including clinically useful antibiotics, and to survive harsh environments. Transport of LPS poses several problems to the cells due to the amphipatic nature of this molecule. In this review we summarize the current knowledge on the LPS transport machinery, discuss the challenges associated with this process and present the solutions that bacterial cells have evolved to address the problem of LPS transport and assembly at the cell surface. Finally, we discuss how knowledge on LPS biogenesis can be translated for the development of novel antimicrobial therapies. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop. Copyright © 2016. Published by Elsevier B.V.

  9. Quality control mechanisms of protein biogenesis: proteostasis dies hard

    Directory of Open Access Journals (Sweden)

    Timothy Jan Bergmann

    2016-10-01

    Full Text Available The biosynthesis of proteins entails a complex series of chemical reactions that transform the information stored in the nucleic acid sequence into a polypeptide chain that needs to properly fold and reach its functional location in or outside the cell. It is of no surprise that errors might occur that alter the polypeptide sequence leading to a non-functional proteins or that impede delivery of proteins at the appropriate site of activity. In order to minimize such mistakes and guarantee the synthesis of the correct amount and quality of the proteome, cells have developed folding, quality control, degradation and transport mechanisms that ensure and tightly regulate protein biogenesis. Genetic mutations, harsh environmental conditions or attack by pathogens can subvert the cellular quality control machineries and perturb cellular proteostasis leading to pathological conditions. This review summarizes basic concepts of the flow of information from DNA to folded and active proteins and to the variable fidelity (from incredibly high to quite sloppy characterizing these processes. We will give particular emphasis on events that maintain or recover the homeostasis of the endoplasmic reticulum (ER, a major site of proteins synthesis and folding in eukaryotic cells. Finally, we will report on how cells can adapt to stressful conditions, how perturbation of ER homeostasis may result in diseases and how these can be treated.

  10. Rrp12 and the Exportin Crm1 participate in late assembly events in the nucleolus during 40S ribosomal subunit biogenesis.

    Science.gov (United States)

    Moriggi, Giulia; Nieto, Blanca; Dosil, Mercedes

    2014-12-01

    During the biogenesis of small ribosomal subunits in eukaryotes, the pre-40S particles formed in the nucleolus are rapidly transported to the cytoplasm. The mechanisms underlying the nuclear export of these particles and its coordination with other biogenesis steps are mostly unknown. Here we show that yeast Rrp12 is required for the exit of pre-40S particles to the cytoplasm and for proper maturation dynamics of upstream 90S pre-ribosomes. Due to this, in vivo elimination of Rrp12 leads to an accumulation of nucleoplasmic 90S to pre-40S transitional particles, abnormal 35S pre-rRNA processing, delayed elimination of processing byproducts, and no export of intermediate pre-40S complexes. The exportin Crm1 is also required for the same pre-ribosome maturation events that involve Rrp12. Thus, in addition to their implication in nuclear export, Rrp12 and Crm1 participate in earlier biosynthetic steps that take place in the nucleolus. Our results indicate that, in the 40S subunit synthesis pathway, the completion of early pre-40S particle assembly, the initiation of byproduct degradation and the priming for nuclear export occur in an integrated manner in late 90S pre-ribosomes.

  11. Assembling Fe/S-clusters and modifying tRNAs: ancient co-factors meet ancient adaptors

    Czech Academy of Sciences Publication Activity Database

    Alfonzo, J. D.; Lukeš, Julius

    2011-01-01

    Roč. 27, č. 6 (2011), 234-237 ISSN 1471-4922 R&D Projects: GA MŠk LC07032; GA MŠk 2B06129; GA ČR GA204/09/1667 Institutional research plan: CEZ:AV0Z60220518 Keywords : IRON-SULFUR CLUSTERS * TRYPANOSOMA-BRUCEI * MITOCHONDRIAL * PROTEIN * FRATAXIN * BIOSYNTHESIS * SYNTHETASES * BIOGENESIS * THIOLATION * ANTICODON Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.144, year: 2011

  12. New localization and function of calpain-2 in nucleoli of colorectal cancer cells in ribosomal biogenesis: effect of KRAS status.

    Science.gov (United States)

    Telechea-Fernández, Marcelino; Rodríguez-Fernández, Lucia; García, Concha; Zaragozá, Rosa; Viña, Juan; Cervantes, Andrés; García-Trevijano, Elena R

    2018-02-06

    Calpain-2 belongs to a family of pleiotropic Cys-proteases with modulatory rather than degradative functions. Calpain (CAPN) overexpression has been controversially correlated with poor prognosis in several cancer types, including colorectal carcinoma (CRC). However, the mechanisms of substrate-recognition, calpain-2 regulation/deregulation and specific functions in CRC remain elusive. Herein, calpain subcellular distribution was studied as a key event for substrate-recognition and consequently, for calpain-mediated function. We describe a new localization for calpain-2 in the nucleoli of CRC cells. Calpain-2 nucleolar distribution resulted dependent on its enzymatic activity and on the mutational status of KRAS. In KRASWT/- cells serum-starvation induced CAPN2 expression, nucleolar accumulation and increased binding to the rDNA-core promoter and intergenic spacer (IGS), concomitant with a reduction in pre-rRNA levels. Depletion of calpain-2 by specific siRNA prevented pre-rRNA down-regulation after serum removal. Conversely, ribosomal biogenesis proceeded in the absence of serum in unresponsive KRASG13D/- cells whose CAPN2 expression, nucleolar localization and rDNA-occupancy remained unchanged during the time-course of serum starvation. We propose here that nucleolar calpain-2 might be a KRAS-dependent sensor to repress ribosomal biogenesis in growth limiting conditions. Under constitutive activation of the pathway commonly found in CRC, calpain-2 is deregulated and tumor cells become insensitive to the extracellular microenvironment.

  13. Active participation of Hsp90 in the biogenesis of the trimeric reovirus cell attachment protein sigma1.

    Science.gov (United States)

    Gilmore, R; Coffey, M C; Lee, P W

    1998-06-12

    The reovirus cell attachment protein, sigma1, is a lollipop-shaped homotrimer with an N-terminal fibrous tail and a C-terminal globular head. Biogenesis of this protein involves two trimerization events: N-terminal trimerization, which occurs cotranslationally and is Hsp70/ATP-independent, and C-terminal trimerization, which occurs posttranslationally and is Hsp70/ATP-dependent. To determine if Hsp90 also plays a role in sigma1 biogenesis, we analyzed sigma1 synthesized in rabbit reticulocyte lysate. Coprecipitation experiments using anti-Hsp90 antibodies revealed that Hsp90 was associated with immature sigma1 trimers (hydra-like intermediates with assembled N termini and unassembled C termini) but not with mature trimers. The use of truncated sigma1 further demonstrated that only the C-terminal half of sigma1 associated with Hsp90. In the presence of the Hsp90 binding drug geldanamycin, N-terminal trimerization proceeded normally, but C-terminal trimerization was blocked. Geldanamycin did not inhibit the association of Hsp90 with sigma 1 but prevented the subsequent release of Hsp90 from the immature sigma1 complex. We also examined the status of p23, an Hsp90-associated cochaperone. Like Hsp90, p23 only associated with immature sigma1 trimers, and this association was mapped to the C-terminal half of sigma1. However, unlike Hsp90, p23 was released from the sigma1 complex upon the addition of geldanamycin. These results highlight an all-or-none concept of chaperone involvement in different oligomerization domains within a single protein and suggest a possible common usage of chaperones in the regulation of general protein folding and of steroid receptor activation.

  14. The role of mitochondria in cellular iron-sulfur protein biogenesis and iron metabolism.

    Science.gov (United States)

    Lill, Roland; Hoffmann, Bastian; Molik, Sabine; Pierik, Antonio J; Rietzschel, Nicole; Stehling, Oliver; Uzarska, Marta A; Webert, Holger; Wilbrecht, Claudia; Mühlenhoff, Ulrich

    2012-09-01

    Mitochondria play a key role in iron metabolism in that they synthesize heme, assemble iron-sulfur (Fe/S) proteins, and participate in cellular iron regulation. Here, we review the latter two topics and their intimate connection. The mitochondrial Fe/S cluster (ISC) assembly machinery consists of 17 proteins that operate in three major steps of the maturation process. First, the cysteine desulfurase complex Nfs1-Isd11 as the sulfur donor cooperates with ferredoxin-ferredoxin reductase acting as an electron transfer chain, and frataxin to synthesize an [2Fe-2S] cluster on the scaffold protein Isu1. Second, the cluster is released from Isu1 and transferred toward apoproteins with the help of a dedicated Hsp70 chaperone system and the glutaredoxin Grx5. Finally, various specialized ISC components assist in the generation of [4Fe-4S] clusters and cluster insertion into specific target apoproteins. Functional defects of the core ISC assembly machinery are signaled to cytosolic or nuclear iron regulatory systems resulting in increased cellular iron acquisition and mitochondrial iron accumulation. In fungi, regulation is achieved by iron-responsive transcription factors controlling the expression of genes involved in iron uptake and intracellular distribution. They are assisted by cytosolic multidomain glutaredoxins which use a bound Fe/S cluster as iron sensor and additionally perform an essential role in intracellular iron delivery to target metalloproteins. In mammalian cells, the iron regulatory proteins IRP1, an Fe/S protein, and IRP2 act in a post-transcriptional fashion to adjust the cellular needs for iron. Thus, Fe/S protein biogenesis and cellular iron metabolism are tightly linked to coordinate iron supply and utilization. This article is part of a Special Issue entitled: Cell Biology of Metals. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Disrupted Skeletal Muscle Mitochondrial Dynamics, Mitophagy, and Biogenesis during Cancer Cachexia: A Role for Inflammation

    Science.gov (United States)

    VanderVeen, Brandon N.; Fix, Dennis K.

    2017-01-01

    Chronic inflammation is a hallmark of cancer cachexia in both patients and preclinical models. Cachexia is prevalent in roughly 80% of cancer patients and accounts for up to 20% of all cancer-related deaths. Proinflammatory cytokines IL-6, TNF-α, and TGF-β have been widely examined for their regulation of cancer cachexia. An established characteristic of cachectic skeletal muscle is a disrupted capacity for oxidative metabolism, which is thought to contribute to cancer patient fatigue, diminished metabolic function, and muscle mass loss. This review's primary objective is to highlight emerging evidence linking cancer-induced inflammation to the dysfunctional regulation of mitochondrial dynamics, mitophagy, and biogenesis in cachectic muscle. The potential for either muscle inactivity or exercise to alter mitochondrial dysfunction during cancer cachexia will also be discussed. PMID:28785374

  16. Fe-S Cluster Biogenesis in Isolated Mammalian Mitochondria

    Science.gov (United States)

    Pandey, Alok; Pain, Jayashree; Ghosh, Arnab K.; Dancis, Andrew; Pain, Debkumar

    2015-01-01

    Iron-sulfur (Fe-S) clusters are essential cofactors, and mitochondria contain several Fe-S proteins, including the [4Fe-4S] protein aconitase and the [2Fe-2S] protein ferredoxin. Fe-S cluster assembly of these proteins occurs within mitochondria. Although considerable data exist for yeast mitochondria, this biosynthetic process has never been directly demonstrated in mammalian mitochondria. Using [35S]cysteine as the source of sulfur, here we show that mitochondria isolated from Cath.A-derived cells, a murine neuronal cell line, can synthesize and insert new Fe-35S clusters into aconitase and ferredoxins. The process requires GTP, NADH, ATP, and iron, and hydrolysis of both GTP and ATP is necessary. Importantly, we have identified the 35S-labeled persulfide on the NFS1 cysteine desulfurase as a genuine intermediate en route to Fe-S cluster synthesis. In physiological settings, the persulfide sulfur is released from NFS1 and transferred to a scaffold protein, where it combines with iron to form an Fe-S cluster intermediate. We found that the release of persulfide sulfur from NFS1 requires iron, showing that the use of iron and sulfur for the synthesis of Fe-S cluster intermediates is a highly coordinated process. The release of persulfide sulfur also requires GTP and NADH, probably mediated by a GTPase and a reductase, respectively. ATP, a cofactor for a multifunctional Hsp70 chaperone, is not required at this step. The experimental system described here may help to define the biochemical basis of diseases that are associated with impaired Fe-S cluster biogenesis in mitochondria, such as Friedreich ataxia. PMID:25398879

  17. A model for tetrapyrrole synthesis as the primary mechanism for plastid-to-nucleus signaling during chloroplast biogenesis

    Directory of Open Access Journals (Sweden)

    Matthew J. Terry

    2013-02-01

    Full Text Available Chloroplast biogenesis involves the co-ordinated expression of the chloroplast and nuclear genomes, requiring information to be sent from the developing chloroplasts to the nucleus. This is achieved through retrograde signaling pathways and can be demonstrated experimentally using the photobleaching herbicide, Norflurazon, which results in chloroplast damage and the reduced expression of many photosynthesis-related, nuclear genes in seedlings. Genetic analysis of this pathway points to a major role for tetrapyrrole synthesis in retrograde signaling, as well as a strong interaction with light-signaling pathways. Currently, the best model to explain the genetic data is that a specific heme pool generated by flux through ferrochelatase-1 functions as a positive signal to promote the expression of genes required for chloroplast development. We propose that this heme-related signal is the primary positive signal during chloroplast biogenesis, and that treatments and mutations affecting chloroplast transcription, RNA editing, translation, or protein import all impact on the synthesis and/or processing of this signal. A positive signal is consistent with the need to provide information on chloroplast status at all times. We further propose that GUN1 normally serves to restrict the production of the heme signal. In addition to a positive signal re-enforcing chloroplast development under normal conditions, aberrant chloroplast development may produce a negative signal due to accumulation of unbound chlorophyll biosynthesis intermediates, such as Mg-porphyrins. Under these conditions a rapid shut-down of tetrapyrrole synthesis is required. We propose that accumulation of these intermediates results in a rapid light-dependent inhibition of nuclear gene expression that is most likely mediated via singlet oxygen generated by photo-excitation of Mg-porphyrins. Thus, the tetrapyrrole pathway may provide both positive and inhibitory signals to control

  18. The Role of Chaperone-subunit Usher Domain Interactions in the Mechanism of Bacterial Pilus Biogenesis Revealed by ESI-MS*

    Science.gov (United States)

    Morrissey, Bethny; Leney, Aneika C.; Toste Rêgo, Ana; Phan, Gilles; Allen, William J.; Verger, Denis; Waksman, Gabriel; Ashcroft, Alison E.; Radford, Sheena E.

    2012-01-01

    The PapC usher is a β-barrel outer membrane protein essential for assembly and secretion of P pili that are required for adhesion of pathogenic E. coli, which cause the development of pyelonephritis. Multiple protein subunits form the P pilus, the highly specific assembly of which is coordinated by the usher. Despite a wealth of structural knowledge, how the usher catalyzes subunit polymerization and orchestrates a correct and functional order of subunit assembly remain unclear. Here, the ability of the soluble N-terminal (UsherN), C-terminal (UsherC2), and Plug (UsherP) domains of the usher to bind different chaperone-subunit (PapDPapX) complexes is investigated using noncovalent electrospray ionization mass spectrometry. The results reveal that each usher domain is able to bind all six PapDPapX complexes, consistent with an active role of all three usher domains in pilus biogenesis. Using collision induced dissociation, combined with competition binding experiments and dissection of the adhesin subunit, PapG, into separate pilin and adhesin domains, the results reveal why PapG has a uniquely high affinity for the usher, which is consistent with this subunit always being displayed at the pilus tip. In addition, we show how the different soluble usher domains cooperate to coordinate and control efficient pilus assembly at the usher platform. As well as providing new information about the protein-protein interactions that determine pilus biogenesis, the results highlight the power of noncovalent MS to interrogate biological mechanisms, especially in complex mixtures of species. PMID:22371487

  19. The role of chaperone-subunit usher domain interactions in the mechanism of bacterial pilus biogenesis revealed by ESI-MS.

    Science.gov (United States)

    Morrissey, Bethny; Leney, Aneika C; Toste Rêgo, Ana; Phan, Gilles; Allen, William J; Verger, Denis; Waksman, Gabriel; Ashcroft, Alison E; Radford, Sheena E

    2012-07-01

    The PapC usher is a β-barrel outer membrane protein essential for assembly and secretion of P pili that are required for adhesion of pathogenic E. coli, which cause the development of pyelonephritis. Multiple protein subunits form the P pilus, the highly specific assembly of which is coordinated by the usher. Despite a wealth of structural knowledge, how the usher catalyzes subunit polymerization and orchestrates a correct and functional order of subunit assembly remain unclear. Here, the ability of the soluble N-terminal (UsherN), C-terminal (UsherC2), and Plug (UsherP) domains of the usher to bind different chaperone-subunit (PapDPapX) complexes is investigated using noncovalent electrospray ionization mass spectrometry. The results reveal that each usher domain is able to bind all six PapDPapX complexes, consistent with an active role of all three usher domains in pilus biogenesis. Using collision induced dissociation, combined with competition binding experiments and dissection of the adhesin subunit, PapG, into separate pilin and adhesin domains, the results reveal why PapG has a uniquely high affinity for the usher, which is consistent with this subunit always being displayed at the pilus tip. In addition, we show how the different soluble usher domains cooperate to coordinate and control efficient pilus assembly at the usher platform. As well as providing new information about the protein-protein interactions that determine pilus biogenesis, the results highlight the power of noncovalent MS to interrogate biological mechanisms, especially in complex mixtures of species.

  20. A quorum-sensing molecule acts as a morphogen controlling gas vesicle organelle biogenesis and adaptive flotation in an enterobacterium

    Science.gov (United States)

    Ramsay, Joshua P.; Williamson, Neil R.; Spring, David R.; Salmond, George P. C.

    2011-01-01

    Gas vesicles are hollow intracellular proteinaceous organelles produced by aquatic Eubacteria and Archaea, including cyanobacteria and halobacteria. Gas vesicles increase buoyancy and allow taxis toward air–liquid interfaces, enabling subsequent niche colonization. Here we report a unique example of gas vesicle-mediated flotation in an enterobacterium; Serratia sp. strain ATCC39006. This strain is a member of the Enterobacteriaceae previously studied for its production of prodigiosin and carbapenem antibiotics. Genes required for gas vesicle synthesis mapped to a 16.6-kb gene cluster encoding three distinct homologs of the main structural protein, GvpA. Heterologous expression of this locus in Escherichia coli induced copious vesicle production and efficient cell buoyancy. Gas vesicle morphogenesis in Serratia enabled formation of a pellicle-like layer of highly vacuolated cells, which was dependent on oxygen limitation and the expression of ntrB/C and cheY-like regulatory genes within the gas-vesicle gene cluster. Gas vesicle biogenesis was strictly controlled by intercellular chemical signaling, through an N-acyl homoserine lactone, indicating that in this system the quorum-sensing molecule acts as a morphogen initiating organelle development. Flagella-based motility and gas vesicle morphogenesis were also oppositely regulated by the small RNA-binding protein, RsmA, suggesting environmental adaptation through physiological control of the choice between motility and flotation as alternative taxis modes. We propose that gas vesicle biogenesis in this strain represents a distinct mechanism of mobility, regulated by oxygen availability, nutritional status, the RsmA global regulatory system, and the quorum-sensing morphogen. PMID:21873216

  1. A quorum-sensing molecule acts as a morphogen controlling gas vesicle organelle biogenesis and adaptive flotation in an enterobacterium.

    Science.gov (United States)

    Ramsay, Joshua P; Williamson, Neil R; Spring, David R; Salmond, George P C

    2011-09-06

    Gas vesicles are hollow intracellular proteinaceous organelles produced by aquatic Eubacteria and Archaea, including cyanobacteria and halobacteria. Gas vesicles increase buoyancy and allow taxis toward air-liquid interfaces, enabling subsequent niche colonization. Here we report a unique example of gas vesicle-mediated flotation in an enterobacterium; Serratia sp. strain ATCC39006. This strain is a member of the Enterobacteriaceae previously studied for its production of prodigiosin and carbapenem antibiotics. Genes required for gas vesicle synthesis mapped to a 16.6-kb gene cluster encoding three distinct homologs of the main structural protein, GvpA. Heterologous expression of this locus in Escherichia coli induced copious vesicle production and efficient cell buoyancy. Gas vesicle morphogenesis in Serratia enabled formation of a pellicle-like layer of highly vacuolated cells, which was dependent on oxygen limitation and the expression of ntrB/C and cheY-like regulatory genes within the gas-vesicle gene cluster. Gas vesicle biogenesis was strictly controlled by intercellular chemical signaling, through an N-acyl homoserine lactone, indicating that in this system the quorum-sensing molecule acts as a morphogen initiating organelle development. Flagella-based motility and gas vesicle morphogenesis were also oppositely regulated by the small RNA-binding protein, RsmA, suggesting environmental adaptation through physiological control of the choice between motility and flotation as alternative taxis modes. We propose that gas vesicle biogenesis in this strain represents a distinct mechanism of mobility, regulated by oxygen availability, nutritional status, the RsmA global regulatory system, and the quorum-sensing morphogen.

  2. Triglyceride Blisters in Lipid Bilayers: Implications for Lipid Droplet Biogenesis and the Mobile Lipid Signal in Cancer Cell Membranes

    DEFF Research Database (Denmark)

    Khandelia, Himanshu; Duelund, Lars; Pakkanen, Kirsi Inkeri

    2010-01-01

    triolein molecules to the bilayer center in the form of a disordered, isotropic, mobile neutral lipid aggregate, at least 17 nm in diameter, which forms spontaneously, and remains stable on at least the microsecond time scale. The results give credence to the hotly debated existence of mobile neutral lipid...... aggregates of unknown function present in malignant cells, and to the early biogenesis of lipid droplets accommodated between the two leaflets of the endoplasmic reticulum membrane. The TO aggregates give the bilayer a blister-like appearance, and will hinder the formation of multi-lamellar phases in model...

  3. Vertical Distribution of Pasteuria penetrans Parasitizing Meloidogyne incognita on Pittosporum tobira in Florida.

    Science.gov (United States)

    Baidoo, Richard; Mengistu, Tesfamariam Mekete; Brito, Janete A; McSorley, Robert; Stamps, Robert H; Crow, William T

    2017-09-01

    Pasteuria penetrans is considered as the primary agent responsible for soil suppressiveness to root-knot nematodes widely distributed in many agricultural fields. A preliminary survey on a Pittosporum tobira field where the grower had experienced a continuous decline in productivity caused by Meloidogyne incognita showed that the nematode was infected with Pasteuria penetrans . For effective control of the nematode, the bacterium and the host must coexist in the same root zone. The vertical distribution of Pasteuria penetrans and its relationship with the nematode host in the soil was investigated to identify (i) the vertical distribution of P. penetrans endospores in an irrigated P. tobira field and (ii) the relationship among P. penetrans endospore density, M. incognita J2 population density, and host plant root distribution over time. Soil bioassays revealed that endospore density was greater in the upper 18 cm of the top soil compared with the underlying depths. A correlation analysis showed that the endospore density was positively related to the J2 population density and host plant root distribution. Thus, the vertical distribution of P. penetrans was largely dependent on its nematode host which in turn was determined by the distribution of the host plant roots. The Pasteuria was predominant mostly in the upper layers of the soil where their nematode host and the plant host roots are abundant, a factor which may be a critical consideration when using P. penetrans as a nematode biological control agent.

  4. Glucose-Modulated Mitochondria Adaptation in Tumor Cells: A Focus on ATP Synthase and Inhibitor Factor 1

    Directory of Open Access Journals (Sweden)

    Irene Mavelli

    2012-02-01

    Full Text Available Warburg’s hypothesis has been challenged by a number of studies showing that oxidative phosphorylation is repressed in some tumors, rather than being inactive per se. Thus, treatments able to shift energy metabolism by activating mitochondrial pathways have been suggested as an intriguing basis for the optimization of antitumor strategies. In this study, HepG2 hepatocarcinoma cells were cultivated with different metabolic substrates under conditions mimicking “positive” (activation/biogenesis or “negative” (silencing mitochondrial adaptation. In addition to the expected up-regulation of mitochondrial biogenesis, glucose deprivation caused an increase in phosphorylating respiration and a rise in the expression levels of the ATP synthase β subunit and Inhibitor Factor 1 (IF1. Hyperglycemia, on the other hand, led to a markedly decreased level of the transcriptional coactivator PGC-α suggesting down-regulation of mitochondrial biogenesis, although no change in mitochondrial mass and no impairment of phosphorylating respiration were observed. Moreover, a reduction in mitochondrial networking and in ATP synthase dimer stability was produced. No effect on β-ATP synthase expression was elicited. Notably, hyperglycemia caused an increase in IF1 expression levels, but it did not alter the amount of IF1 associated with ATP synthase. These results point to a new role of IF1 in relation to high glucose utilization by tumor cells, in addition to its well known effect upon mitochondrial ATP synthase regulation.

  5. Biogenesis and the growth of DNA-like polymer chains: a computer simulation

    International Nuclear Information System (INIS)

    Herrmann, H.J.; Tsallis, C.

    1987-01-01

    We study, through computer simulation, a crucial step of Biogenesis, namely the growth of self-replicating codified DNA-like polymers starting from a mixture of oligomers. We have adopted the growth scheme that has been recently proposed by Ferreira and Tsallis which incorporates usual ideas of autocatalysis through complementary pairs and within which a central role is played by the hydrogen-like links (characterized by the probabilities p AT and p CG of chemical bonding of the A-T and C-G pairs respectively) between the two chains of the growing polymer. We find that the average equilibrium polymeric length ξ diverges, for any fixed ratio (1-p AT )/(1-p sub (CG)), as ξ ∝ 1/r1-p AT . Selection of patterns may happen at all stages and in particular at chemical equilibrium. Selection occurs via two different mechanisms: (i) away from the critical point p AT = p CG = 1 if p AT ≠ p CG ; (ii) both on and away from the critical point if the initial concentrations of nucleotides (A, T, C and G or their precursors) are different. (author) [pt

  6. Characterization of yeast extracellular vesicles: evidence for the participation of different pathways of cellular traffic in vesicle biogenesis.

    Directory of Open Access Journals (Sweden)

    Débora L Oliveira

    2010-06-01

    Full Text Available Extracellular vesicles in yeast cells are involved in the molecular traffic across the cell wall. In yeast pathogens, these vesicles have been implicated in the transport of proteins, lipids, polysaccharide and pigments to the extracellular space. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown.We characterized extracellular vesicle production in wild type (WT and mutant strains of the model yeast Saccharomyces cerevisiae using transmission electron microscopy in combination with light scattering analysis, lipid extraction and proteomics. WT cells and mutants with defective expression of Sec4p, a secretory vesicle-associated Rab GTPase essential for Golgi-derived exocytosis, or Snf7p, which is involved in multivesicular body (MVB formation, were analyzed in parallel. Bilayered vesicles with diameters at the 100-300 nm range were found in extracellular fractions from yeast cultures. Proteomic analysis of vesicular fractions from the cells aforementioned and additional mutants with defects in conventional secretion pathways (sec1-1, fusion of Golgi-derived exocytic vesicles with the plasma membrane; bos1-1, vesicle targeting to the Golgi complex or MVB functionality (vps23, late endosomal trafficking revealed a complex and interrelated protein collection. Semi-quantitative analysis of protein abundance revealed that mutations in both MVB- and Golgi-derived pathways affected the composition of yeast extracellular vesicles, but none abrogated vesicle production. Lipid analysis revealed that mutants with defects in Golgi-related components of the secretory pathway had slower vesicle release kinetics, as inferred from intracellular accumulation of sterols and reduced detection of these lipids in vesicle fractions in comparison with WT cells.Our results suggest that both conventional and unconventional pathways of secretion are required for biogenesis of extracellular vesicles, which demonstrate the

  7. Mitochondrial biogenesis in brown adipose tissue is associated with differential expression of transcription regulatory factors

    Czech Academy of Sciences Publication Activity Database

    Villena, J. A.; Carmona, M. C.; Rodriguez de la Concepción, M.; Rossmeisl, Martin; Vinas, O.; Mampel, T.; Iglesias, R.; Giralt, M.; Villarroya, F.

    2002-01-01

    Roč. 59, č. 11 (2002), s. 1934-1944 ISSN 1420-682X Grant - others:Ministerio de Ciencia y Tecnología (ES) PM98.0188 Institutional research plan: CEZ:AV0Z5011922 Keywords : brown adipose tissue * mitochondria * transcription factors Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.259, year: 2002

  8. Outer membrane biogenesis in Escherichia coli, Neisseria meningitidis, and Helicobacter pylori: paradigm deviations in H. pylori.

    Science.gov (United States)

    Liechti, George; Goldberg, Joanna B

    2012-01-01

    The bacterial pathogen Helicobacter pylori is capable of colonizing the gastric mucosa of the human stomach using a variety of factors associated with or secreted from its outer membrane (OM). Lipopolysaccharide (LPS) and numerous OM proteins have been shown to be involved in adhesion and immune stimulation/evasion. Many of these factors are essential for colonization and/or pathogenesis in a variety of animal models. Despite this wide array of potential targets present on the bacterial surface, the ability of H. pylori to vary its OM profile limits the effectiveness of vaccines or therapeutics that target any single one of these components. However, it has become evident that the proteins comprising the complexes that transport the majority of these molecules to the OM are highly conserved and often essential. The field of membrane biogenesis has progressed remarkably in the last few years, and the possibility now exists for targeting the mechanisms by which β-barrel proteins, lipoproteins, and LPS are transported to the OM, resulting in loss of bacterial fitness and significant altering of membrane permeability. In this review, the OM transport machinery for LPS, lipoproteins, and outer membrane proteins (OMPs) are discussed. While the principal investigations of these transport mechanisms have been conducted in Escherichia coli and Neisseria meningitidis, here these systems will be presented in the genetic context of ε proteobacteria. Bioinformatic analysis reveals that minimalist genomes, such as that of Helicobacter pylori, offer insight into the smallest number of components required for these essential pathways to function. Interestingly, in the majority of ε proteobacteria, while the inner and OM associated apparatus of LPS, lipoprotein, and OMP transport pathways appear to all be intact, most of the components associated with the periplasmic compartment are either missing or are almost unrecognizable when compared to their E. coli counterparts. Eventual

  9. Literature-Related Discovery: Common Factors for Parkinson’s Disease and Crohn’s Disease

    Science.gov (United States)

    2010-01-01

    cytoplasm of human cells. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. MicroRNAs: genomics, biogenesis, mechanism, and...tetrahydrocannabinol 0.7%, marijuana 0.6%, delta.thc 0.6%, extinct 0.6%, cannabinoid.receptors 0.6%, acid 0.5%, 940 0.5%, cisaprid 0.5%, antagonist 0.4...0.6%, receptor 0.6%, patient 0.6%, endogen 0.5%, tetrahydrocannabinol 0.5%, delta 0.5%, genom 0.4%, kinas 0.4%, factor 0.4%, marijuana 0.4% Single

  10. Correlation between Ribosome Biogenesis and the Magnitude of Hypertrophy in Overloaded Skeletal Muscle.

    Directory of Open Access Journals (Sweden)

    Satoshi Nakada

    Full Text Available External loads applied to skeletal muscle cause increases in the protein translation rate, which leads to muscle hypertrophy. Although some studies have demonstrated that increases in the capacity and efficiency of translation are involved in this process, it remains unclear how these two factors are related to the magnitude of muscle hypertrophy. The present study aimed to clarify the roles played by the capacity and efficiency of translation in muscle hypertrophy. We used an improved synergist ablation in which the magnitude of compensatory hypertrophy could be controlled by partial removal of synergist muscles. Male rats were assigned to four groups in which the plantaris muscle was unilaterally subjected to weak (WK, moderate (MO, middle (MI, and strong (ST overloading by four types of synergist ablation. Fourteen days after surgery, the weight of the plantaris muscle per body weight increased by 8%, 22%, 32% and 45%, in the WK, MO, MI and ST groups, respectively. Five days after surgery, 18+28S rRNA content (an indicator of translational capacity increased with increasing overload, with increases of 1.8-fold (MO, 2.2-fold (MI, and 2.5-fold (ST, respectively, relative to non-overloaded muscle (NL in the WK group. rRNA content showed a strong correlation with relative muscle weight measured 14 days after surgery (r = 0.98. The phosphorylated form of p70S6K (a positive regulator of translational efficiency showed a marked increase in the MO group, but no further increase was observed with further increase in overload (increases of 22.6-fold (MO, 17.4-fold (MI, and 18.2-fold (ST, respectively, relative to NL in the WK group. These results indicate that increases in ribosome biogenesis at the early phase of overloading are strongly dependent on the amount of overloading, and may play an important role in increasing the translational capacity for further gain of muscular size.

  11. Correlation between Ribosome Biogenesis and the Magnitude of Hypertrophy in Overloaded Skeletal Muscle.

    Science.gov (United States)

    Nakada, Satoshi; Ogasawara, Riki; Kawada, Shigeo; Maekawa, Takahiro; Ishii, Naokata

    2016-01-01

    External loads applied to skeletal muscle cause increases in the protein translation rate, which leads to muscle hypertrophy. Although some studies have demonstrated that increases in the capacity and efficiency of translation are involved in this process, it remains unclear how these two factors are related to the magnitude of muscle hypertrophy. The present study aimed to clarify the roles played by the capacity and efficiency of translation in muscle hypertrophy. We used an improved synergist ablation in which the magnitude of compensatory hypertrophy could be controlled by partial removal of synergist muscles. Male rats were assigned to four groups in which the plantaris muscle was unilaterally subjected to weak (WK), moderate (MO), middle (MI), and strong (ST) overloading by four types of synergist ablation. Fourteen days after surgery, the weight of the plantaris muscle per body weight increased by 8%, 22%, 32% and 45%, in the WK, MO, MI and ST groups, respectively. Five days after surgery, 18+28S rRNA content (an indicator of translational capacity) increased with increasing overload, with increases of 1.8-fold (MO), 2.2-fold (MI), and 2.5-fold (ST), respectively, relative to non-overloaded muscle (NL) in the WK group. rRNA content showed a strong correlation with relative muscle weight measured 14 days after surgery (r = 0.98). The phosphorylated form of p70S6K (a positive regulator of translational efficiency) showed a marked increase in the MO group, but no further increase was observed with further increase in overload (increases of 22.6-fold (MO), 17.4-fold (MI), and 18.2-fold (ST), respectively, relative to NL in the WK group). These results indicate that increases in ribosome biogenesis at the early phase of overloading are strongly dependent on the amount of overloading, and may play an important role in increasing the translational capacity for further gain of muscular size.

  12. ADAR1 attenuates allogeneic graft rejection by suppressing miR-21 biogenesis in macrophages and promoting M2 polarization.

    Science.gov (United States)

    Li, Junjie; Xie, Jiangang; Liu, Shanshou; Li, Xiao; Zhang, Dongliang; Wang, Xianqi; Jiang, Jinquan; Hu, Wei; Zhang, Yuan; Jin, Boquan; Zhuang, Ran; Yin, Wen

    2018-04-25

    ADAR1 (adenosine deaminase acting on double-stranded RNA 1) is an RNA-editing enzyme that mediates adenosine-to-inosine RNA editing events, an important post-transcriptional modification mechanism that can alter the coding properties of mRNA or regulate microRNA biogenesis. ADAR1 also regulates the innate immune response. Here, we have demonstrated that ADAR1 expression increased in LPS-stimulated macrophages. Silencing ADAR1 by using small interfering RNA in macrophages resulted in the pronounced polarization of macrophages to M1, whereas ADAR1 overexpression promoted M2 polarization, which indicated that ADAR1 can inhibit macrophage hyperpolarization and prevent immune hyperactivity. The RNA-RNP immunoprecipitation binding assay demonstrated a direct interaction between ADAR1 and miR-21 precursor. Significant up-regulation in IL-10 and down-regulation in miR-21 were observed in ADAR1-overexpressing macrophages. We evaluated miR-21 target mRNAs and macrophage polarization signaling pathways and found that forkhead box protein O1 (Foxo1) was up-regulated in cells that overexpressed ADAR1. In a mouse allogeneic skin transplantation model, grafts in the ADAR1-overexpressed group survived longer and suffered less immune cell infiltration. In ADAR1-overexpressed recipients, splenic macrophages were significantly polarized to M2, and levels of sera IL-10 were markedly higher than those in the control group. In summary, ADAR1 modulates macrophage M2 polarization via the ADAR1-miR-21-Foxo1-IL-10 axis, thereby suppressing allogeneic graft rejection.-Li, J., Xie, J., Liu, S., Li, X., Zhang, D., Wang, X., Jiang, J., Hu, W., Zhang, Y., Jin, B., Zhuang, R., Yin, W. ADAR1 attenuates allogeneic graft rejection by suppressing miR-21 biogenesis in macrophages and promoting M2 polarization.

  13. Nucleolus Precursor Bodies and Ribosome Biogenesis in Early Mammalian Embryos: Old Theories and New Discoveries.

    Science.gov (United States)

    Fulka, Helena; Aoki, Fugaku

    2016-06-01

    In mammals, mature oocytes and early preimplantation embryos contain transcriptionally inactive structures termed nucleolus precursor bodies instead of the typical fibrillo-granular nucleoli. These nuclear organelles are essential and strictly of maternal origin. If they are removed from oocytes, the resulting embryos are unable to replace them and consequently fail to develop. Historically, nucleolus precursor bodies have been perceived as a passive repository site of nucleolar proteins that are required for embryos to form fully functional nucleoli. Recent results, however, contradict this long-standing dogma and show that these organelles are dispensable for nucleologenesis and ribosome biogenesis. In this article, we discuss the possible roles of nucleolus precursor bodies and propose how they might be involved in embryogenesis. Furthermore, we argue that these organelles are essential only shortly after fertilization and suggest that they might actively participate in centromeric chromatin establishment. © 2016 by the Society for the Study of Reproduction, Inc.

  14. Vacuolar biogenesis and aquaporin expression at early germination of broad bean seeds.

    Science.gov (United States)

    Novikova, Galina V; Tournaire-Roux, Colette; Sinkevich, Irina A; Lityagina, Snejana V; Maurel, Christophe; Obroucheva, Natalie

    2014-09-01

    A key event in seed germination is water uptake-mediated growth initiation in embryonic axes. Vicia faba var. minor (broad bean) seeds were used for studying cell growth, vacuolar biogenesis, expression and function of tonoplast water channel proteins (aquaporins) in embryonic axes during seed imbibition, radicle emergence and growth. Hypocotyl and radicle basal cells showed vacuole restoration from protein storage vacuoles, whereas de novo vacuole formation from provacuoles was observed in cells newly produced by root meristem. cDNA fragments of seven novel aquaporin isoforms including five Tonoplast Intrinsic Proteins (TIP) from three sub-types were amplified by PCR. The expression was probed using q-RT-PCR and when possible with isoform-specific antibodies. Decreased expression of TIP3s was associated to the transformation of protein storage vacuoles to vacuoles, whereas enhanced expression of a TIP2 homologue was closely linked to the fast cell elongation. Water channel functioning checked by inhibitory test with mercuric chloride showed closed water channels prior to growth initiation and active water transport into elongating cells. The data point to a crucial role of tonoplast aquaporins during germination, especially during growth of embryonic axes, due to accelerated water uptake and vacuole enlargement resulting in rapid cell elongation. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  15. A family of membrane-shaping proteins at ER subdomains regulates pre-peroxisomal vesicle biogenesis.

    Science.gov (United States)

    Joshi, Amit S; Huang, Xiaofang; Choudhary, Vineet; Levine, Tim P; Hu, Junjie; Prinz, William A

    2016-11-21

    Saccharomyces cerevisiae contains three conserved reticulon and reticulon-like proteins that help maintain ER structure by stabilizing high membrane curvature in ER tubules and the edges of ER sheets. A mutant lacking all three proteins has dramatically altered ER morphology. We found that ER shape is restored in this mutant when Pex30p or its homologue Pex31p is overexpressed. Pex30p can tubulate membranes both in cells and when reconstituted into proteoliposomes, indicating that Pex30p is a novel ER-shaping protein. In contrast to the reticulons, Pex30p is low abundance, and we found that it localizes to subdomains in the ER. We show that these ER subdomains are the sites where most preperoxisomal vesicles (PPVs) are generated. In addition, overproduction or deletion of Pex30p or Pex31p alters the size, shape, and number of PPVs. Our findings suggest that Pex30p and Pex31p help shape and generate regions of the ER where PPV biogenesis occurs.

  16. Alternative function for the mitochondrial SAM complex in biogenesis of alpha-helical TOM proteins.

    Science.gov (United States)

    Stojanovski, Diana; Guiard, Bernard; Kozjak-Pavlovic, Vera; Pfanner, Nikolaus; Meisinger, Chris

    2007-12-03

    The mitochondrial outer membrane contains two preprotein translocases: the general translocase of outer membrane (TOM) and the beta-barrel-specific sorting and assembly machinery (SAM). TOM functions as the central entry gate for nuclear-encoded proteins. The channel-forming Tom40 is a beta-barrel protein, whereas all Tom receptors and small Tom proteins are membrane anchored by a transmembrane alpha-helical segment in their N- or C-terminal portion. Synthesis of Tom precursors takes place in the cytosol, and their import occurs via preexisting TOM complexes. The precursor of Tom40 is then transferred to SAM for membrane insertion and assembly. Unexpectedly, we find that the biogenesis of alpha-helical Tom proteins with a membrane anchor in the C-terminal portion is SAM dependent. Each SAM protein is necessary for efficient membrane integration of the receptor Tom22, whereas assembly of the small Tom proteins depends on Sam37. Thus, the substrate specificity of SAM is not restricted to beta-barrel proteins but also includes the majority of alpha-helical Tom proteins.

  17. The peroxisomal AAA ATPase complex prevents pexophagy and development of peroxisome biogenesis disorders.

    Science.gov (United States)

    Law, Kelsey B; Bronte-Tinkew, Dana; Di Pietro, Erminia; Snowden, Ann; Jones, Richard O; Moser, Ann; Brumell, John H; Braverman, Nancy; Kim, Peter K

    2017-05-04

    Peroxisome biogenesis disorders (PBDs) are metabolic disorders caused by the loss of peroxisomes. The majority of PBDs result from mutation in one of 3 genes that encode for the peroxisomal AAA ATPase complex (AAA-complex) required for cycling PEX5 for peroxisomal matrix protein import. Mutations in these genes are thought to result in a defect in peroxisome assembly by preventing the import of matrix proteins. However, we show here that loss of the AAA-complex does not prevent matrix protein import, but instead causes an upregulation of peroxisome degradation by macroautophagy, or pexophagy. The loss of AAA-complex function in cells results in the accumulation of ubiquitinated PEX5 on the peroxisomal membrane that signals pexophagy. Inhibiting autophagy by genetic or pharmacological approaches rescues peroxisome number, protein import and function. Our findings suggest that the peroxisomal AAA-complex is required for peroxisome quality control, whereas its absence results in the selective degradation of the peroxisome. Thus the loss of peroxisomes in PBD patients with mutations in their peroxisomal AAA-complex is a result of increased pexophagy. Our study also provides a framework for the development of novel therapeutic treatments for PBDs.

  18. MicroRNA-directed siRNA biogenesis in Caenorhabditis elegans.

    Science.gov (United States)

    Corrêa, Régis L; Steiner, Florian A; Berezikov, Eugene; Ketting, René F

    2010-04-08

    RNA interference (RNAi) is a post-transcriptional silencing process, triggered by double-stranded RNA (dsRNA), leading to the destabilization of homologous mRNAs. A distinction has been made between endogenous RNAi-related pathways and the exogenous RNAi pathway, the latter being essential for the experimental use of RNAi. Previous studies have shown that, in Caenorhabditis elegans, a complex containing the enzymes Dicer and the Argonaute RDE-1 process dsRNA. Dicer is responsible for cleaving dsRNA into short interfering RNAs (siRNAs) while RDE-1 acts as the siRNA acceptor. RDE-1 then guides a multi-protein complex to homologous targets to trigger mRNA destabilization. However, endogenous role(s) for RDE-1, if any, have remained unexplored. We here show that RDE-1 functions as a scavenger protein, taking up small RNA molecules from many different sources, including the microRNA (miRNA) pathway. This is in striking contrast to Argonaute proteins functioning directly in the miRNA pathway, ALG-1 and ALG-2: these proteins exclusively bind miRNAs. While playing no significant role in the biogenesis of the main pool of miRNAs, RDE-1 binds endogenous miRNAs and triggers RdRP activity on at least one perfectly matching, endogenous miRNA target. The resulting secondary siRNAs are taken up by a set of Argonaute proteins known to act as siRNA acceptors in exogenous RNAi, resulting in strong mRNA destabilization. Our results show that RDE-1 in an endogenous setting is actively screening the transcriptome using many different small RNAs, including miRNAs, as a guide, with implications for the evolution of transcripts with a potential to be recognized by Dicer.

  19. MicroRNA–Directed siRNA Biogenesis in Caenorhabditis elegans

    Science.gov (United States)

    Corrêa, Régis L.; Steiner, Florian A.; Berezikov, Eugene; Ketting, René F.

    2010-01-01

    RNA interference (RNAi) is a post-transcriptional silencing process, triggered by double-stranded RNA (dsRNA), leading to the destabilization of homologous mRNAs. A distinction has been made between endogenous RNAi–related pathways and the exogenous RNAi pathway, the latter being essential for the experimental use of RNAi. Previous studies have shown that, in Caenorhabditis elegans, a complex containing the enzymes Dicer and the Argonaute RDE-1 process dsRNA. Dicer is responsible for cleaving dsRNA into short interfering RNAs (siRNAs) while RDE-1 acts as the siRNA acceptor. RDE-1 then guides a multi-protein complex to homologous targets to trigger mRNA destabilization. However, endogenous role(s) for RDE-1, if any, have remained unexplored. We here show that RDE-1 functions as a scavenger protein, taking up small RNA molecules from many different sources, including the microRNA (miRNA) pathway. This is in striking contrast to Argonaute proteins functioning directly in the miRNA pathway, ALG-1 and ALG-2: these proteins exclusively bind miRNAs. While playing no significant role in the biogenesis of the main pool of miRNAs, RDE-1 binds endogenous miRNAs and triggers RdRP activity on at least one perfectly matching, endogenous miRNA target. The resulting secondary siRNAs are taken up by a set of Argonaute proteins known to act as siRNA acceptors in exogenous RNAi, resulting in strong mRNA destabilization. Our results show that RDE-1 in an endogenous setting is actively screening the transcriptome using many different small RNAs, including miRNAs, as a guide, with implications for the evolution of transcripts with a potential to be recognized by Dicer. PMID:20386745

  20. The spore differentiation pathway in the enteric pathogen Clostridium difficile.

    Directory of Open Access Journals (Sweden)

    Fátima C Pereira

    Full Text Available Endosporulation is an ancient bacterial developmental program that culminates with the differentiation of a highly resistant endospore. In the model organism Bacillus subtilis, gene expression in the forespore and in the mother cell, the two cells that participate in endospore development, is governed by cell type-specific RNA polymerase sigma subunits. σ(F in the forespore, and σ(E in the mother cell control early stages of development and are replaced, at later stages, by σ(G and σ(K, respectively. Starting with σ(F, the activation of the sigma factors is sequential, requires the preceding factor, and involves cell-cell signaling pathways that operate at key morphological stages. Here, we have studied the function and regulation of the sporulation sigma factors in the intestinal pathogen Clostridium difficile, an obligate anaerobe in which the endospores are central to the infectious cycle. The morphological characterization of mutants for the sporulation sigma factors, in parallel with use of a fluorescence reporter for single cell analysis of gene expression, unraveled important deviations from the B. subtilis paradigm. While the main periods of activity of the sigma factors are conserved, we show that the activity of σ(E is partially independent of σ(F, that σ(G activity is not dependent on σ(E, and that the activity of σ(K does not require σ(G. We also show that σ(K is not strictly required for heat resistant spore formation. In all, our results indicate reduced temporal segregation between the activities of the early and late sigma factors, and reduced requirement for the σ(F-to-σ(E, σ(E-to-σ(G, and σ(G-to-σ(K cell-cell signaling pathways. Nevertheless, our results support the view that the top level of the endosporulation network is conserved in evolution, with the sigma factors acting as the key regulators of the pathway, established some 2.5 billion years ago upon its emergence at the base of the Firmicutes Phylum.

  1. Aluminium induced oxidative stress results in decreased mitochondrial biogenesis via modulation of PGC-1α expression

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Deep Raj; Sunkaria, Aditya; Wani, Willayat Yousuf; Sharma, Reeta Kumari; Kandimalla, Ramesh J.L. [Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh 160012 (India); Bal, Amanjit [Department of Histopathology, Postgraduate Institute of Medical Education and Research, Chandigarh (India); Gill, Kiran Dip, E-mail: kdgill2002@yahoo.co.in [Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh 160012 (India)

    2013-12-01

    The present investigation was carried out to elucidate a possible molecular mechanism related to the effects of aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of Peroxisome proliferator activated receptor gamma co-activator 1α (PGC-1α) and its downstream targets i.e. Nuclear respiratory factor-1(NRF-1), Nuclear respiratory factor-2(NRF-2) and Mitochondrial transcription factor A (Tfam) in mitochondrial biogenesis. Aluminium lactate (10 mg/kg b.wt./day) was administered intragastrically to rats for 12 weeks. After 12 weeks of exposure, we found an increase in ROS levels, mitochondrial DNA oxidation and decrease in citrate synthase activity in the Hippocampus (HC) and Corpus striatum (CS) regions of rat brain. On the other hand, there was a decrease in the mRNA levels of the mitochondrial encoded subunits–NADH dehydrogenase (ND) subunits i.e. ND1, ND2, ND3, Cytochrome b (Cytb), Cytochrome oxidase (COX) subunits i.e. COX1, COX3, ATP synthase (ATPase) subunit 6 along with reduced expression of nuclear encoded subunits COX4, COX5A, COX5B of Electron transport chain (ETC). Besides, a decrease in mitochondrial DNA copy number and mitochondrial content in both regions of rat brain was observed. The PGC-1α was down-regulated in aluminium treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α in aluminium treated rats. Electron microscopy results revealed a significant increase in the mitochondrial swelling, loss of cristae, chromatin condensation and decreases in mitochondrial number in case of aluminium treated rats as compared to control. So, PGC-1α seems to be a potent target for aluminium neurotoxicity, which makes it an almost ideal target to control or limit the damage that has been associated with the defective mitochondrial function seen in neurodegenerative diseases. - Highlights: • Aluminium decreases the mRNA levels of mitochondrial and nuclear encoded

  2. Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat.

    Science.gov (United States)

    Martins, Ligia O; Soares, Claudio M; Pereira, Manuela M; Teixeira, Miguel; Costa, Teresa; Jones, George H; Henriques, Adriano O

    2002-05-24

    The Bacillus subtilis endospore coat protein CotA shows laccase activity. By using comparative modeling techniques, we were able to derive a model for CotA based on the known x-ray structures of zucchini ascorbate oxidase and Cuprinus cereneus laccase. This model of CotA contains all the structural features of a laccase, including the reactive surface-exposed copper center (T1) and two buried copper centers (T2 and T3). Single amino acid substitutions in the CotA T1 copper center (H497A, or M502L) did not prevent assembly of the mutant proteins into the coat and did not alter the pattern of extractable coat polypeptides. However, in contrast to a wild type strain, both mutants produced unpigmented colonies and spores unable to oxidize syringaldazine (SGZ) and 2'2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The CotA protein was purified to homogeneity from an overproducing Escherichia coli strain. The purified CotA shows an absorbance and a EPR spectra typical of blue multicopper oxidases. Optimal enzymatic activity was found at < or =pH 3.0 and at pH 7.0 for ABTS or SGZ oxidation, respectively. The apparent K(m) values for ABTS and SGZ at 37 degrees C were of 106 +/- 11 and 26 +/- 2 microm, respectively, with corresponding k(cat) values of 16.8 +/- 0.8 and 3.7 +/- 0.1 s(-1). Maximal enzyme activity was observed at 75 degrees C with ABTS as substrate. Remarkably, the coat-associated or the purified enzyme showed a half-life of inactivation at 80 degrees C of about 4 and 2 h, respectively, indicating that CotA is intrinsically highly thermostable.

  3. Quercetin protects against aluminium induced oxidative stress and promotes mitochondrial biogenesis via activation of the PGC-1α signaling pathway.

    Science.gov (United States)

    Sharma, Deep Raj; Sunkaria, Aditya; Wani, Willayat Yousuf; Sharma, Reeta Kumari; Verma, Deepika; Priyanka, Kumari; Bal, Amanjit; Gill, Kiran Dip

    2015-12-01

    The present investigation was carried out to elucidate a possible molecular mechanism related to the protective effect of quercetin administration against aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of PGC-1α and its downstream targets, i.e. NRF-1, NRF-2 and Tfam in mitochondrial biogenesis. Aluminium lactate (10mg/kg b.wt./day) was administered intragastrically to rats, which were pre-treated with quercetin 6h before aluminium (10mg/kg b.wt./day, intragastrically) for 12 weeks. We found a decrease in ROS levels, mitochondrial DNA oxidation and citrate synthase activity in the hippocampus (HC) and corpus striatum (CS) regions of rat brain treated with quercetin. Besides this an increase in the mRNA levels of the mitochondrial encoded subunits - ND1, ND2, ND3, Cyt b, COX1, COX3 and ATPase6 along with increased expression of nuclear encoded subunits COX4, COX5A and COX5B of electron transport chain (ETC). In quercetin treated group an increase in the mitochondrial DNA copy number and mitochondrial content in both the regions of rat brain was observed. The PGC-1α was up regulated in quercetin treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α. Electron microscopy results revealed a significant decrease in the mitochondrial cross-section area, mitochondrial perimeter length and increase in mitochondrial number in case of quercetin treated rats as compared to aluminium treated ones. Therefore it seems quercetin increases mitochondrial biogenesis and makes it an almost ideal flavanoid to control or limit the damage that has been associated with the defective mitochondrial function seen in many neurodegenerative diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Mitochondrial Biogenesis in Diverse Cauliflower Cultivars under Mild and Severe Drought. Impaired Coordination of Selected Transcript and Proteomic Responses, and Regulation of Various Multifunctional Proteins

    Directory of Open Access Journals (Sweden)

    Michał Rurek

    2018-04-01

    Full Text Available Mitochondrial responses under drought within Brassica genus are poorly understood. The main goal of this study was to investigate mitochondrial biogenesis of three cauliflower (Brassica oleracea var. botrytis cultivars with varying drought tolerance. Diverse quantitative changes (decreases in abundance mostly in the mitochondrial proteome were assessed by two-dimensional gel electrophoresis (2D PAGE coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS. Respiratory (e.g., complex II, IV (CII, CIV and ATP synthase subunits, transporter (including diverse porin isoforms and matrix multifunctional proteins (e.g., components of RNA editing machinery were diversely affected in their abundance under two drought levels. Western immunoassays showed additional cultivar-specific responses of selected mitochondrial proteins. Dehydrin-related tryptic peptides (found in several 2D spots immunopositive with dehydrin-specific antisera highlighted the relevance of mitochondrial dehydrin-like proteins for the drought response. The abundance of selected mRNAs participating in drought response was also determined. We conclude that mitochondrial biogenesis was strongly, but diversely affected in various cauliflower cultivars, and associated with drought tolerance at the proteomic and functional levels. However, discussed alternative oxidase (AOX regulation at the RNA and protein level were largely uncoordinated due to the altered availability of transcripts for translation, mRNA/ribosome interactions, and/or miRNA impact on transcript abundance and translation.

  5. Mitochondrial Biogenesis in Diverse Cauliflower Cultivars under Mild and Severe Drought. Impaired Coordination of Selected Transcript and Proteomic Responses, and Regulation of Various Multifunctional Proteins

    Science.gov (United States)

    Rurek, Michał; Czołpińska, Magdalena; Staszak, Aleksandra Maria; Nowak, Witold; Krzesiński, Włodzimierz; Spiżewski, Tomasz

    2018-01-01

    Mitochondrial responses under drought within Brassica genus are poorly understood. The main goal of this study was to investigate mitochondrial biogenesis of three cauliflower (Brassica oleracea var. botrytis) cultivars with varying drought tolerance. Diverse quantitative changes (decreases in abundance mostly) in the mitochondrial proteome were assessed by two-dimensional gel electrophoresis (2D PAGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Respiratory (e.g., complex II, IV (CII, CIV) and ATP synthase subunits), transporter (including diverse porin isoforms) and matrix multifunctional proteins (e.g., components of RNA editing machinery) were diversely affected in their abundance under two drought levels. Western immunoassays showed additional cultivar-specific responses of selected mitochondrial proteins. Dehydrin-related tryptic peptides (found in several 2D spots) immunopositive with dehydrin-specific antisera highlighted the relevance of mitochondrial dehydrin-like proteins for the drought response. The abundance of selected mRNAs participating in drought response was also determined. We conclude that mitochondrial biogenesis was strongly, but diversely affected in various cauliflower cultivars, and associated with drought tolerance at the proteomic and functional levels. However, discussed alternative oxidase (AOX) regulation at the RNA and protein level were largely uncoordinated due to the altered availability of transcripts for translation, mRNA/ribosome interactions, and/or miRNA impact on transcript abundance and translation. PMID:29642585

  6. A Novel Association between Two Trypanosome-Specific Factors and the Conserved L5-5S rRNA Complex

    Science.gov (United States)

    Ciganda, Martin; Prohaska, Kimberly; Hellman, Kristina; Williams, Noreen

    2012-01-01

    P34 and P37 are two previously identified RNA binding proteins in the flagellate protozoan Trypanosoma brucei. RNA interference studies have determined that the proteins are involved in and essential for ribosome biogenesis. The proteins interact with the 5S rRNA with nearly identical binding characteristics. We have shown that this interaction is achieved mainly through the LoopA region of the RNA, but P34 and P37 also protect the L5 binding site located on LoopC. We now provide evidence to show that these factors form a novel pre-ribosomal particle through interactions with both 5S rRNA and the L5 ribosomal protein. Further in silico and in vitro analysis of T. brucei L5 indicates a lower affinity for 5S rRNA than expected, based on other eukaryotic L5 proteins. We hypothesize that P34 and P37 complement L5 and bridge the interaction with 5S rRNA, stabilizing it and aiding in the early steps of ribosome biogenesis. PMID:22859981

  7. Prevalence, isolation and characterization of Bacillus cereus strains from rice of local cultivators of Sabah, Sarawak, and Peninsular Malaysia

    Science.gov (United States)

    Sawei, Jelin; Sani, Norrakiah Abdullah

    2016-11-01

    Bacillus cereus is a spore-forming, facultative anaerobic, motile microorganism that has been identified as a causative agent of two types of gastrointestinal diseases such as emetic and diarrhea. This foodborne pathogen is found in both vegetative cells and endospores form in foods such as rice either raw or cooked. The aim of this study is to investigate and determine the prevalence, characterize and identify the isolation of vegetative cells and endospores of B. cereus in thirty varieties (n=3) of raw rice from Sabah, Sarawak and Peninsular Malaysia. A total of 90 (n=90) raw rice were examined and 84 (93.33%) samples were positive to vegetative cells of B. cereus. However, only 32 (35.56%) samples were positive for endospore cells that able to germinate after samples were heated at 75°C for 15 mins. The mean log cfu/g for vegetative cells were higher range (0.00 - 4.1533) than visible endospores (0.00 - 3.7533 mean log cfu/g). Sample of raw red rice (UKMRC9) had significantly higher contamination by both vegetative cells and endospores at p<0.05, than the other raw rice samples.

  8. The Signal Transducer and Activator of Transcription 1 (STAT1 Inhibits Mitochondrial Biogenesis in Liver and Fatty Acid Oxidation in Adipocytes.

    Directory of Open Access Journals (Sweden)

    Jennifer D Sisler

    Full Text Available The transcription factor STAT1 plays a central role in orchestrating responses to various pathogens by activating the transcription of nuclear-encoded genes that mediate the antiviral, the antigrowth, and immune surveillance effects of interferons and other cytokines. In addition to regulating gene expression, we report that STAT1-/- mice display increased energy expenditure and paradoxically decreased release of triglycerides from white adipose tissue (WAT. Liver mitochondria from STAT1-/- mice show both defects in coupling of the electron transport chain (ETC and increased numbers of mitochondria. Consistent with elevated numbers of mitochondria, STAT1-/- mice expressed increased amounts of PGC1α, a master regulator of mitochondrial biogenesis. STAT1 binds to the PGC1α promoter in fed mice but not in fasted animals, suggesting that STAT1 inhibited transcription of PGC1α. Since STAT1-/- mice utilized more lipids we examined white adipose tissue (WAT stores. Contrary to expectations, fasted STAT1-/- mice did not lose lipid from WAT. β-adrenergic stimulation of glycerol release from isolated STAT1-/- WAT was decreased, while activation of hormone sensitive lipase was not changed. These findings suggest that STAT1-/- adipose tissue does not release glycerol and that free fatty acids (FFA re-esterify back to triglycerides, thus maintaining fat mass in fasted STAT1-/- mice.

  9. Proteomic Analysis of Lipid Droplets from Arabidopsis Aging Leaves Brings New Insight into Their Biogenesis and Functions

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    Lysiane Brocard

    2017-05-01

    Full Text Available Lipid droplets (LDs are cell compartments specialized for oil storage. Although their role and biogenesis are relatively well documented in seeds, little is known about their composition, structure and function in senescing leaves where they also accumulate. Here, we used a label free quantitative mass spectrometry approach to define the LD proteome of aging Arabidopsis leaves. We found that its composition is highly different from that of seed/cotyledon and identified 28 proteins including 9 enzymes of the secondary metabolism pathways involved in plant defense response. With the exception of the TRIGALACTOSYLDIACYLGLYCEROL2 protein, we did not identify enzymes implicated in lipid metabolism, suggesting that growth of leaf LDs does not occur by local lipid synthesis but rather through contact sites with the endoplasmic reticulum (ER or other membranes. The two most abundant proteins of the leaf LDs are the CALEOSIN3 and the SMALL RUBBER PARTICLE1 (AtSRP1; both proteins have structural functions and participate in plant response to stress. CALEOSIN3 and AtSRP1 are part of larger protein families, yet no other members were enriched in the LD proteome suggesting a specific role of both proteins in aging leaves. We thus examined the function of AtSRP1 at this developmental stage and found that AtSRP1 modulates the expression of CALEOSIN3 in aging leaves. Furthermore, AtSRP1 overexpression induces the accumulation of triacylglycerol with an unusual composition compared to wild-type. We demonstrate that, although AtSRP1 expression is naturally increased in wild type senescing leaves, its overexpression in senescent transgenic lines induces an over-accumulation of LDs organized in clusters at restricted sites of the ER. Conversely, atsrp1 knock-down mutants displayed fewer but larger LDs. Together our results reveal that the abundancy of AtSRP1 regulates the neo-formation of LDs during senescence. Using electron tomography, we further provide evidence

  10. Tob38, a novel essential component in the biogenesis of β-barrel proteins of mitochondria

    Science.gov (United States)

    Waizenegger, Thomas; Habib, Shukry J; Lech, Maciej; Mokranjac, Dejana; Paschen, Stefan A; Hell, Kai; Neupert, Walter; Rapaport, Doron

    2004-01-01

    Insertion of β-barrel proteins into the outer membrane of mitochondria is mediated by the TOB complex. Known constituents of this complex are Tob55 and Mas37. We identified a novel component, Tob38. It is essential for viability of yeast and the function of the TOB complex. Tob38 is exposed on the surface of the mitochondrial outer membrane. It interacts with Mas37 and Tob55 and is associated with Tob55 even in the absence of Mas37. The Tob38–Tob55 core complex binds precursors of β-barrel proteins and facilitates their insertion into the outer membrane. Depletion of Tob38 results in strongly reduced levels of Tob55 and Mas37 and the residual proteins no longer form a complex. Tob38-depleted mitochondria are deficient in the import of β-barrel precursor proteins, but not of other outer membrane proteins or proteins of other mitochondrial subcompartments. We conclude that Tob38 has a crucial function in the biogenesis of β-barrel proteins of mitochondria. PMID:15205677

  11. Emended description of Pasteuria nishizawae.

    Science.gov (United States)

    Noel, Gregory R; Atibalentja, N; Domier, Leslie L

    2005-07-01

    The description of the Gram-positive, obligately parasitic, mycelial and endospore-forming bacterium, Pasteuria nishizawae, is emended to include additional observations on the life cycle, host specificity and endospore morphology. The nucleotide sequence of the 16S rRNA gene is also provided.

  12. Functional analysis of a tyrosinase gene involved in early larval shell biogenesis in Crassostrea angulata and its response to ocean acidification.

    Science.gov (United States)

    Yang, Bingye; Pu, Fei; Li, Lingling; You, Weiwei; Ke, Caihuan; Feng, Danqing

    2017-04-01

    The formation of the primary shell is a vital process in marine bivalves. Ocean acidification largely influences shell formation. It has been reported that enzymes involved in phenol oxidation, such as tyrosinase and phenoloxidases, participate in the formation of the periostracum. In the present study, we cloned a tyrosinase gene from Crassostrea angulata named Ca-tyrA1, and its potential function in early larval shell biogenesis was investigated. The Ca-tyrA1 gene has a full-length cDNA of 2430bp in size, with an open reading frame of 1896bp in size, which encodes a 631-amino acid protein that includes a 24-amino acid putative signal peptide. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed that Ca-tyrA1 transcription mainly occurs at the trochophore stage, and the Ca-tyrA1 mRNA levels in the 3000ppm treatment group were significantly upregulated in the early D-veliger larvae. WMISH and electron scanning microscopy analyses showed that the expression of Ca-tyrA1 occurs at the gastrula stage, thereby sustaining the early D-veliger larvae, and the shape of its signal is saddle-like, similar to that observed under an electron scanning microscope. Furthermore, the RNA interference has shown that the treatment group has a higher deformity rate than that of the control, thereby indicating that Ca-tyrA1 participates in the biogenesis of the primary shell. In conclusion, and our results indicate that Ca-tyrA1 plays a vital role in the formation of the larval shell and participates in the response to larval shell damages in Crassostrea angulata that were induced by ocean acidification. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Impaired Telomere Maintenance and Decreased Canonical WNT Signaling but Normal Ribosome Biogenesis in Induced Pluripotent Stem Cells from X-Linked Dyskeratosis Congenita Patients.

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    Bai-Wei Gu

    Full Text Available Dyskeratosis congenita (DC is an inherited bone marrow failure syndrome characterized by the presence of short telomeres at presentation. Mutations in ten different genes, whose products are involved in the telomere maintenance pathway, have been shown to cause DC. The X-linked form is the most common form of the disease and is caused by mutations in the gene DKC1, encoding the protein dyskerin. Dyskerin is required for the assembly and stability of telomerase and is also involved in ribosomal RNA (rRNA processing where it converts specific uridines to pseudouridine. DC is thought to result from failure to maintain tissues, like blood, that are renewed by stem cell activity, but research into pathogenic mechanisms has been hampered by the difficulty of obtaining stem cells from patients. We reasoned that induced pluripotent stem (iPS cells from X-linked DC patients may provide information about the mechanisms involved. Here we describe the production of iPS cells from DC patients with DKC1 mutations Q31E, A353V and ΔL37. In addition we constructed "corrected" lines with a copy of the wild type dyskerin cDNA expressed from the AAVS1 safe harbor locus. We show that in iPS cells with DKC1 mutations telomere maintenance is compromised with short telomere lengths and decreased telomerase activity. The degree to which telomere lengths are affected by expression of telomerase during reprograming, or with ectopic expression of wild type dyskerin, is variable. The recurrent mutation A353V shows the most severe effect on telomere maintenance. A353V cells but not Q31E or ΔL37 cells, are refractory to correction by expression of wild type DKC1 cDNA. Because dyskerin is involved in both telomere maintenance and ribosome biogenesis it has been postulated that defective ribosome biogenesis and translation may contribute to the disease phenotype. Evidence from mouse and zebra fish models has supported the involvement of ribosome biogenesis but primary cells

  14. Impaired Telomere Maintenance and Decreased Canonical WNT Signaling but Normal Ribosome Biogenesis in Induced Pluripotent Stem Cells from X-Linked Dyskeratosis Congenita Patients.

    Science.gov (United States)

    Gu, Bai-Wei; Apicella, Marisa; Mills, Jason; Fan, Jian-Meng; Reeves, Dara A; French, Deborah; Podsakoff, Gregory M; Bessler, Monica; Mason, Philip J

    2015-01-01

    Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by the presence of short telomeres at presentation. Mutations in ten different genes, whose products are involved in the telomere maintenance pathway, have been shown to cause DC. The X-linked form is the most common form of the disease and is caused by mutations in the gene DKC1, encoding the protein dyskerin. Dyskerin is required for the assembly and stability of telomerase and is also involved in ribosomal RNA (rRNA) processing where it converts specific uridines to pseudouridine. DC is thought to result from failure to maintain tissues, like blood, that are renewed by stem cell activity, but research into pathogenic mechanisms has been hampered by the difficulty of obtaining stem cells from patients. We reasoned that induced pluripotent stem (iPS) cells from X-linked DC patients may provide information about the mechanisms involved. Here we describe the production of iPS cells from DC patients with DKC1 mutations Q31E, A353V and ΔL37. In addition we constructed "corrected" lines with a copy of the wild type dyskerin cDNA expressed from the AAVS1 safe harbor locus. We show that in iPS cells with DKC1 mutations telomere maintenance is compromised with short telomere lengths and decreased telomerase activity. The degree to which telomere lengths are affected by expression of telomerase during reprograming, or with ectopic expression of wild type dyskerin, is variable. The recurrent mutation A353V shows the most severe effect on telomere maintenance. A353V cells but not Q31E or ΔL37 cells, are refractory to correction by expression of wild type DKC1 cDNA. Because dyskerin is involved in both telomere maintenance and ribosome biogenesis it has been postulated that defective ribosome biogenesis and translation may contribute to the disease phenotype. Evidence from mouse and zebra fish models has supported the involvement of ribosome biogenesis but primary cells from human

  15. Impact of Anthropogenic Factor on Urboecological Space Development

    Directory of Open Access Journals (Sweden)

    Kuprina Tamara

    2016-01-01

    Full Text Available The article discusses the issues of the impact of the anthropogenic factor on urboecological space development. The issues are considered taking into account retrospective theoretical data to show the process of Anthropoecology development as a new branch of sociological science. At present the noosphere acquires features of anthropoecosystems having a number of parameters from the endogenous and exogenous point of view. Anthropoecology has special socio-cultural significance as considers the interaction of all actors of international space. There introduced the new branch Ecopsycology as the outer world is the reflection of the inner human world. There is a definition of the sustainability of ecological system. In the practical part of the article there is an example of academic mobility as the basis of the human potential with possible transfer into the human capital supporting by survey data. In conclusion there are recommendations on management and adaptation of the anthropogenic factor (a kind of biogenesis in modern urboecological space.

  16. GAIP interacting protein C-terminus regulates autophagy and exosome biogenesis of pancreatic cancer through metabolic pathways.

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    Santanu Bhattacharya

    Full Text Available GAIP interacting protein C terminus (GIPC is known to play an important role in a variety of physiological and disease states. In the present study, we have identified a novel role for GIPC as a master regulator of autophagy and the exocytotic pathways in cancer. We show that depletion of GIPC-induced autophagy in pancreatic cancer cells, as evident from the upregulation of the autophagy marker LC3II. We further report that GIPC regulates cellular trafficking pathways by modulating the secretion, biogenesis, and molecular composition of exosomes. We also identified the involvement of GIPC on metabolic stress pathways regulating autophagy and microvesicular shedding, and observed that GIPC status determines the loading of cellular cargo in the exosome. Furthermore, we have shown the overexpression of the drug resistance gene ABCG2 in exosomes from GIPC-depleted pancreatic cancer cells. We also demonstrated that depletion of GIPC from cancer cells sensitized them to gemcitabine treatment, an avenue that can be explored as a potential therapeutic strategy to overcome drug resistance in cancer.

  17. Maternal stress reduces the susceptibility of Meloidogyne arenaria progeny to Pasteuria penetrans

    Science.gov (United States)

    Pasteuria penetrans is an obligate parasite of Meloidogyne spp. Endospores of P. penetrans attach to the cuticle of the second-stage juvenile (J2) and the bacterium completes its life cycle in the mature female nematode; infected females are filled with millions of endospores and produce few to no ...

  18. Review of Pasteuria penetrans: Biology, Ecology, and Biological Control Potential.

    Science.gov (United States)

    Chen, Z X; Dickson, D W

    1998-09-01

    Pasteuria penetrans is a mycelial, endospore-forming, bacterial parasite that has shown great potential as a biological control agent of root-knot nematodes. Considerable progress has been made during the last 10 years in understanding its biology and importance as an agent capable of effectively suppressing root-knot nematodes in field soil. The objective of this review is to summarize the current knowledge of the biology, ecology, and biological control potential of P. penetrans and other Pasteuria members. Pasteuria spp. are distributed worldwide and have been reported from 323 nematode species belonging to 116 genera of free-living, predatory, plant-parasitic, and entomopathogenic nematodes. Artificial cultivation of P. penetrans has met with limited success; large-scale production of endospores depends on in vivo cultivation. Temperature affects endospore attachment, germination, pathogenesis, and completion of the life cycle in the nematode pseudocoelom. The biological control potential of Pasteuria spp. have been demonstrated on 20 crops; host nematodes include Belonolaimus longicaudatus, Heterodera spp., Meloidogyne spp., and Xiphinema diversicaudatum. Pasteuria penetrans plays an important role in some suppressive soils. The efficacy of the bacterium as a biological control agent has been examined. Approximately 100,000 endospores/g of soil provided immediate control of the peanut root-knot nematode, whereas 1,000 and 5,000 endospores/g of soil each amplified in the host nematode and became suppressive after 3 years.

  19. WHAMM Directs the Arp2/3 Complex to the ER for Autophagosome Biogenesis through an Actin Comet Tail Mechanism.

    Science.gov (United States)

    Kast, David J; Zajac, Allison L; Holzbaur, Erika L F; Ostap, E Michael; Dominguez, Roberto

    2015-06-29

    Nucleation-promoting factors (NPFs) control the spatio-temporal activity of Arp2/3 complex in cells]. Thus, WASP and the WAVE complex direct the formation of branched actin networks at the leading edge during cell motility and endo/exocytosis, whereas the WASH complex is involved in endosomal transport. Less understood are WHAMM and JMY, two NPFs with similar domain architecture. JMY is found in the nucleus and the cytosol and is involved in transcriptional regulation, cell motility, and trans-Golgi transport. WHAMM was reported to bind microtubules and to be involved in ER to cis-Golgi transport. Here, we show that WHAMM directs the activity of Arp2/3 complex for autophagosome biogenesis through an actin-comet tail motility mechanism. Macroautophagy--the process by which cytosolic material is engulfed into autophagosomes for degradation and/or recycling--was recently shown to involve actin, but the mechanism is unknown. We found that WHAMM forms puncta that colocalize and comigrate with the autophagy markers LC3, DFCP1, and p62 through a WHAMM-dependent actin-comet tail mechanism. Under starvation, WHAMM and actin are observed at the interface between neighboring autophagosomes, whose number and size increase with WHAMM expression. Interfering with actin polymerization, inhibiting Arp2/3 complex, knocking down WHAMM, or blocking its interaction with Arp2/3 complex through mutagenesis all inhibit comet tail formation and reduce the size and number of autophagosomes. Finally, JMY shows similar localization to WHAMM and could be involved in similar processes. These results reveal a link between Arp2/3-complex-dependent actin assembly and autophagy. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Cre-mediated stress affects sirtuin expression levels, peroxisome biogenesis and metabolism, antioxidant and proinflammatory signaling pathways.

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    Yu Xiao

    Full Text Available Cre-mediated excision of loxP sites is widely used in mice to manipulate gene function in a tissue-specific manner. To analyze phenotypic alterations related to Cre-expression, we have used AMH-Cre-transgenic mice as a model system. Different Cre expression levels were obtained by investigation of C57BL/6J wild type as well as heterozygous and homozygous AMH-Cre-mice. Our results indicate that Cre-expression itself in Sertoli cells already has led to oxidative stress and lipid peroxidation (4-HNE lysine adducts, inducing PPARα/γ, peroxisome proliferation and alterations of peroxisome biogenesis (PEX5, PEX13 and PEX14 as well as metabolic proteins (ABCD1, ABCD3, MFP1, thiolase B, catalase. In addition to the strong catalase increase, a NRF2- and FOXO3-mediated antioxidative response (HMOX1 of the endoplasmic reticulum and mitochondrial SOD2 and a NF-κB activation were noted. TGFβ1 and proinflammatory cytokines like IL1, IL6 and TNFα were upregulated and stress-related signaling pathways were induced. Sertoli cell mRNA-microarray analysis revealed an increase of TNFR2-signaling components. 53BP1 recruitment and expression levels for DNA repair genes as well as for p53 were elevated and the ones for related sirtuin deacetylases affected (SIRT 1, 3-7 in Sertoli cells. Under chronic Cre-mediated DNA damage conditions a strong downregulation of Sirt1 was observed, suggesting that the decrease of this important coordinator between DNA repair and metabolic signaling might induce the repression release of major transcription factors regulating metabolic and cytokine-mediated stress pathways. Indeed, caspase-3 was activated and increased germ cell apoptosis was observed, suggesting paracrine effects. In conclusion, the observed wide stress-induced effects and metabolic alterations suggest that it is essential to use the correct control animals (Cre/Wt with matched Cre expression levels to differentiate between Cre-mediated and specific gene-knock out

  1. Cre-Mediated Stress Affects Sirtuin Expression Levels, Peroxisome Biogenesis and Metabolism, Antioxidant and Proinflammatory Signaling Pathways

    Science.gov (United States)

    Xiao, Yu; Karnati, Srikanth; Qian, Guofeng; Nenicu, Anca; Fan, Wei; Tchatalbachev, Svetlin; Höland, Anita; Hossain, Hamid; Guillou, Florian; Lüers, Georg H.; Baumgart-Vogt, Eveline

    2012-01-01

    Cre-mediated excision of loxP sites is widely used in mice to manipulate gene function in a tissue-specific manner. To analyze phenotypic alterations related to Cre-expression, we have used AMH-Cre-transgenic mice as a model system. Different Cre expression levels were obtained by investigation of C57BL/6J wild type as well as heterozygous and homozygous AMH-Cre-mice. Our results indicate that Cre-expression itself in Sertoli cells already has led to oxidative stress and lipid peroxidation (4-HNE lysine adducts), inducing PPARα/γ, peroxisome proliferation and alterations of peroxisome biogenesis (PEX5, PEX13 and PEX14) as well as metabolic proteins (ABCD1, ABCD3, MFP1, thiolase B, catalase). In addition to the strong catalase increase, a NRF2- and FOXO3-mediated antioxidative response (HMOX1 of the endoplasmic reticulum and mitochondrial SOD2) and a NF-κB activation were noted. TGFβ1 and proinflammatory cytokines like IL1, IL6 and TNFα were upregulated and stress-related signaling pathways were induced. Sertoli cell mRNA-microarray analysis revealed an increase of TNFR2-signaling components. 53BP1 recruitment and expression levels for DNA repair genes as well as for p53 were elevated and the ones for related sirtuin deacetylases affected (SIRT 1, 3-7) in Sertoli cells. Under chronic Cre-mediated DNA damage conditions a strong downregulation of Sirt1 was observed, suggesting that the decrease of this important coordinator between DNA repair and metabolic signaling might induce the repression release of major transcription factors regulating metabolic and cytokine-mediated stress pathways. Indeed, caspase-3 was activated and increased germ cell apoptosis was observed, suggesting paracrine effects. In conclusion, the observed wide stress-induced effects and metabolic alterations suggest that it is essential to use the correct control animals (Cre/Wt) with matched Cre expression levels to differentiate between Cre-mediated and specific gene-knock out

  2. Vitamin D Impacts the Expression of Runx2 Target Genes and Modulates Inflammation, Oxidative Stress and Membrane Vesicle Biogenesis Gene Networks in 143B Osteosarcoma Cells

    Science.gov (United States)

    Garimella, Rama; Tadikonda, Priyanka; Tawfik, Ossama; Gunewardena, Sumedha; Rowe, Peter; Van Veldhuizen, Peter

    2017-01-01

    Osteosarcoma (OS) is an aggressive malignancy of bone affecting children, adolescents and young adults. Understanding vitamin D metabolism and vitamin D regulated genes in OS is an important aspect of vitamin D/cancer paradigm, and in evaluating vitamin D as adjuvant therapy for human OS. Vitamin D treatment of 143B OS cells induced significant and novel changes in the expression of genes that regulate: (a) inflammation and immunity; (b) formation of reactive oxygen species, metabolism of cyclic nucleotides, sterols, vitamins and mineral (calcium), quantity of gap junctions and skeletogenesis; (c) bone mineral density; and (d) cell viability of skeletal cells, aggregation of bone cancer cells and exocytosis of secretory vesicles. Ingenuity pathway analysis revealed significant reduction in Runx2 target genes such as fibroblast growth factor -1, -12 (FGF1 and FGF12), bone morphogenetic factor-1 (BMP1), SWI/SNF related, matrix associated actin dependent regulator of chromatin subfamily a, member 4 (SMARCA4), Matrix extracellular phosphoglycoprotein (MEPE), Integrin, β4 (ITGBP4), Matrix Metalloproteinase -1, -28 (MMP1 and MMP28), and signal transducer and activator of transcription-4 (STAT4) in vitamin D treated 143B OS cells. These genes interact with the inflammation, oxidative stress and membrane vesicle biogenesis gene networks. Vitamin D not only inhibited the expression of Runx2 target genes MMP1, MMP28 and kallikrein related peptidase-7 (KLK7), but also migration and invasion of 143B OS cells. Vitamin D regulated Runx2 target genes or their products represent potential therapeutic targets and laboratory biomarkers for applications in translational oncology. PMID:28300755

  3. Biogenesis and dynamics of mitochondria during the cell cycle: significance of 3'UTRs.

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    Marta Martínez-Diez

    Full Text Available Nowadays, we are facing a renaissance of mitochondria in cancer biology. However, our knowledge of the basic cell biology and on the timing and mechanisms that control the biosynthesis of mitochondrial constituents during progression through the cell cycle of mammalian cells remain largely unknown. Herein, we document the in vivo changes on mitochondrial morphology and dynamics that accompany cellular mitosis, and illustrate the following key points of the biogenesis of mitochondria during progression of liver cells through the cycle: (i the replication of nuclear and mitochondrial genomes is synchronized during cellular proliferation, (ii the accretion of OXPHOS proteins is asynchronously regulated during proliferation being the synthesis of beta-F1-ATPase and Hsp60 carried out also at G2/M and, (iii the biosynthesis of cardiolipin is achieved during the S phase, although full development of the mitochondrial membrane potential (DeltaPsim is attained at G2/M. Furthermore, we demonstrate using reporter constructs that the mechanism regulating the accretion of beta-F1-ATPase during cellular proliferation is controlled at the level of mRNA translation by the 3'UTR of the transcript. The 3'UTR-driven synthesis of the protein at G2/M is essential for conferring to the daughter cells the original phenotype of the parental cell. Our findings suggest that alterations on this process may promote deregulated beta-F1-ATPase expression in human cancer.

  4. An in silico approach combined with in vivo experiments enables the identification of a new protein whose overexpression can compensate for specific respiratory defects in Saccharomyces cerevisiae

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    Meunier Brigitte

    2011-10-01

    Full Text Available Abstract Background The mitochondrial inner membrane contains five large complexes that are essential for oxidative phosphorylation. Although the structure and the catalytic mechanisms of the respiratory complexes have been progressively established, their biogenesis is far from being fully understood. Very few complex III assembly factors have been identified so far. It is probable that more factors are needed for the assembly of a functional complex, but that the genetic approaches used to date have not been able to identify them. We have developed a systems biology approach to identify new factors controlling complex III biogenesis. Results We collected all the physical protein-protein interactions (PPI involving the core subunits, the supernumerary subunits and the assembly factors of complex III and used Cytoscape 2.6.3 and its plugins to construct a network. It was then divided into overlapping and highly interconnected sub-graphs with clusterONE. One sub-graph contained the core and the supernumerary subunits of complex III, it also contained some subunits of complex IV and proteins participating in the assembly of complex IV. This sub-graph was then split with another algorithm into two sub-graphs. The subtraction of these two sub-graphs from the previous sub-graph allowed us to identify a protein of unknown function Usb1p/Ylr132p that interacts with the complex III subunits Qcr2p and Cor1p. We then used genetic and cell biology approaches to investigate the function of Usb1p. Preliminary results indicated that Usb1p is an essential protein with a dual localization in the nucleus and in the mitochondria, and that the over-expression of this protein can compensate for defects in the biogenesis of the respiratory complexes. Conclusions Our systems biology approach has highlighted the multiple associations between subunits and assembly factors of complexes III and IV during their biogenesis. In addition, this approach has allowed the

  5. Development of a functionalized coating for inhibition of marine corrosion and biofouling

    Science.gov (United States)

    Gittens, Jeanette Elizabeth

    The financial loss incurred by corrosion of metals in the marine environment has led to a need to develop effective, economic and environmentally friendly methods of protection. Traditional methods of counteracting the development of surface biofilms and biofouling within aqueous environments have involved implementing chemical biocides, often with a deleterious effect on non-target organisms. Sol gel coating technology offers a convenient route for immobilizing functional additives, such as inhibitors or, in the case of this study, biologically active microorganisms. Paenibacillus polymyxa biofilms inhibit the corrosion of metal substrates and this strain has the advantage of forming endospores can withstand the solvent and acid concentrations required in sol-gel formulation. Encapsulation of viable P. polymyxa endospores within the sol-gel matrix allowed germination on exposure to nutrients, when germinating endospores and vegetative cells were seen after fluorescence microscopy to be distributed throughout the coating. Laboratory electrochemical impedance tests were used to characterize the corrosion behaviour of the endospore-containing (biotic) sol-gel coating in comparison to an abiotic (no endospores) sol-gel only coating and one containing non-viable (killed) endospores. The technology enabled manipulation of the sol-gel formulation and the method of application to produce biotic sol-gel with enhanced corrosion inhibition properties on aluminium alloy. Field trials in a marine environment confirmed the corrosion protecting properties of the biotic coating and that the biotic coatings inhibited macroscopic biofouling for at least 29 weeks relative to the controls without encapsulated live endospores. Production of polymyxin by the encapsulated bacteria, which was proposed as a mechanism by which they inhibit MIC, was less than 1 mug per ml and below the threshold of detection by liquid chromatography mass spectrometry and antimicrobial bioassay. Microcosm

  6. A Comprehensive Analysis of Chromoplast Differentiation Reveals Complex Protein Changes Associated with Plastoglobule Biogenesis and Remodeling of Protein Systems in Sweet Orange Flesh1[OPEN

    Science.gov (United States)

    Wang, Lun; Deng, Xiuxin

    2015-01-01

    Globular and crystalloid chromoplasts were observed to be region specifically formed in sweet orange (Citrus sinensis) flesh and converted from amyloplasts during fruit maturation, which was associated with the composition of specific carotenoids and the expression of carotenogenic genes. Subsequent isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analyses of purified plastids from the flesh during chromoplast differentiation and senescence identified 1,386 putative plastid-localized proteins, 1,016 of which were quantified by spectral counting. The iTRAQ values reflecting the expression abundance of three identified proteins were validated by immunoblotting. Based on iTRAQ data, chromoplastogenesis appeared to be associated with three major protein expression patterns: (1) marked decrease in abundance of the proteins participating in the translation machinery through ribosome assembly; (2) increase in abundance of the proteins involved in terpenoid biosynthesis (including carotenoids), stress responses (redox, ascorbate, and glutathione), and development; and (3) maintenance of the proteins for signaling and DNA and RNA. Interestingly, a strong increase in abundance of several plastoglobule-localized proteins coincided with the formation of plastoglobules in the chromoplast. The proteomic data also showed that stable functioning of protein import, suppression of ribosome assembly, and accumulation of chromoplast proteases are correlated with the amyloplast-to-chromoplast transition; thus, these processes may play a collective role in chromoplast biogenesis and differentiation. By contrast, the chromoplast senescence process was inferred to be associated with significant increases in stress response and energy supply. In conclusion, this comprehensive proteomic study identified many potentially new plastid-localized proteins and provides insights into the potential developmental and molecular mechanisms underlying chromoplast

  7. Genetic and Immunological Comparison of the Cladoceran Parasite Pasteuria ramosa with the Nematode Parasite Pasteuria penetrans▿

    Science.gov (United States)

    Schmidt, Liesbeth M.; Mouton, Laurence; Nong, Guang; Ebert, Dieter; Preston, James F.

    2008-01-01

    Pasteuria penetrans, an obligate endospore-forming parasite of Meloidogyne spp. (root knot nematodes), has been identified as a promising agent for biocontrol of these destructive agricultural crop pests. Pasteuria ramosa, an obligate parasite of water fleas (Daphnia spp.), has been shown to modulate cladoceran populations in natural ecosystems. Selected sporulation genes and an epitope associated with the spore envelope of these related species were compared. The sigE and spoIIAA/spoIIAB genes differentiate the two species to a greater extent than 16S rRNA and may serve as probes to differentiate the species. Single-nucleotide variations were observed in several conserved genes of five distinct populations of P. ramosa, and while most of these variations are silent single-nucleotide polymorphisms, a few result in conservative amino acid substitutions. A monoclonal antibody directed against an adhesin epitope present on P. penetrans P20 endospores, previously determined to be specific for Pasteuria spp. associated with several phytopathogenic nematodes, also detects an epitope associated with P. ramosa endospores. Immunoblotting provided patterns that differentiate P. ramosa from other Pasteuria spp. This monoclonal antibody thus provides a probe with which to detect and discriminate endospores of different Pasteuria spp. The presence of a shared adhesin epitope in two species with such ecologically distant hosts suggests that there is an ancient and ecologically significant recognition process in these endospore-forming bacilli that contributes to the virulence of both species in their respective hosts. PMID:17933927

  8. Genetic and immunological comparison of the cladoceran parasite Pasteuria ramosa with the nematode parasite Pasteuria penetrans.

    Science.gov (United States)

    Schmidt, Liesbeth M; Mouton, Laurence; Nong, Guang; Ebert, Dieter; Preston, James F

    2008-01-01

    Pasteuria penetrans, an obligate endospore-forming parasite of Meloidogyne spp. (root knot nematodes), has been identified as a promising agent for biocontrol of these destructive agricultural crop pests. Pasteuria ramosa, an obligate parasite of water fleas (Daphnia spp.), has been shown to modulate cladoceran populations in natural ecosystems. Selected sporulation genes and an epitope associated with the spore envelope of these related species were compared. The sigE and spoIIAA/spoIIAB genes differentiate the two species to a greater extent than 16S rRNA and may serve as probes to differentiate the species. Single-nucleotide variations were observed in several conserved genes of five distinct populations of P. ramosa, and while most of these variations are silent single-nucleotide polymorphisms, a few result in conservative amino acid substitutions. A monoclonal antibody directed against an adhesin epitope present on P. penetrans P20 endospores, previously determined to be specific for Pasteuria spp. associated with several phytopathogenic nematodes, also detects an epitope associated with P. ramosa endospores. Immunoblotting provided patterns that differentiate P. ramosa from other Pasteuria spp. This monoclonal antibody thus provides a probe with which to detect and discriminate endospores of different Pasteuria spp. The presence of a shared adhesin epitope in two species with such ecologically distant hosts suggests that there is an ancient and ecologically significant recognition process in these endospore-forming bacilli that contributes to the virulence of both species in their respective hosts.

  9. Radiation microbiology relevant to the food industry

    International Nuclear Information System (INIS)

    Holzapfel, W.H.

    1985-01-01

    Destruction or inactivation of most microbial cells takes place at relatively low doses of gamma irradiation, making 'pasteurising' treatment of several food commodities in the dose range of 1 to 10 kGy a feasible decontamination method. Several factors may influence the effectiveness of an irradiation process, and should be taken into account when radurisation of foods is practised. Damage to microbes is enhanced in the presence of oxygen and at low pH levels, whereas substances such as sulfhydryl compounds tend to act as protectors. Living organisms may be arranged in the order of increasing resistance to ionising radiation, as follows: higher animals, insects, vegetative bacteria, yeasts and fungi, bacterial endospores and viruses. Most food spoilage organisms as well as food-borne pathogens (D 10 -values for the majority ranging between 0,08 and 0,5 kGy) are sensitive to relatively small irradiation doses. Although several bacterial endospores may survive in radurised foods (D 10 -values ranging between 1,5 and 5,0 kGy), additional safety factors (e.g. low pH, refrigeration, reduced Eh, reduced a w , preservatives) may contribute to the shelf stability of a given food

  10. Prospects for improving CO2 fixation in C3-crops through understanding C4-Rubisco biogenesis and catalytic diversity.

    Science.gov (United States)

    Sharwood, Robert E; Ghannoum, Oula; Whitney, Spencer M

    2016-06-01

    By operating a CO2 concentrating mechanism, C4-photosynthesis offers highly successful solutions to remedy the inefficiency of the CO2-fixing enzyme Rubisco. C4-plant Rubisco has characteristically evolved faster carboxylation rates with low CO2 affinity. Owing to high CO2 concentrations in bundle sheath chloroplasts, faster Rubisco enhances resource use efficiency in C4 plants by reducing the energy and carbon costs associated with photorespiration and lowering the nitrogen investment in Rubisco. Here, we show that C4-Rubisco from some NADP-ME species, such as maize, are also of potential benefit to C3-photosynthesis under current and future atmospheric CO2 pressures. Realizing this bioengineering endeavour necessitates improved understanding of the biogenesis requirements and catalytic variability of C4-Rubisco, as well as the development of transformation capabilities to engineer Rubisco in a wider variety of food and fibre crops. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Triglyceride blisters in lipid bilayers: implications for lipid droplet biogenesis and the mobile lipid signal in cancer cell membranes.

    Directory of Open Access Journals (Sweden)

    Himanshu Khandelia

    Full Text Available Triglycerides have a limited solubility, around 3%, in phosphatidylcholine lipid bilayers. Using millisecond-scale course grained molecular dynamics simulations, we show that the model lipid bilayer can accommodate a higher concentration of triolein (TO than earlier anticipated, by sequestering triolein molecules to the bilayer center in the form of a disordered, isotropic, mobile neutral lipid aggregate, at least 17 nm in diameter, which forms spontaneously, and remains stable on at least the microsecond time scale. The results give credence to the hotly debated existence of mobile neutral lipid aggregates of unknown function present in malignant cells, and to the early biogenesis of lipid droplets accommodated between the two leaflets of the endoplasmic reticulum membrane. The TO aggregates give the bilayer a blister-like appearance, and will hinder the formation of multi-lamellar phases in model, and possibly living membranes. The blisters will result in anomalous membrane probe partitioning, which should be accounted for in the interpretation of probe-related measurements.

  12. Silencing of RpATG6 impaired the yolk accumulation and the biogenesis of the yolk organelles in the insect vector R. prolixus.

    Directory of Open Access Journals (Sweden)

    Priscila H Vieira

    2018-05-01

    Full Text Available In oviparous animals, the egg yolk is synthesized by the mother in a major metabolic challenge, where the different yolk components are secreted to the hemolymph and delivered to the oocytes mostly by endocytosis. The yolk macromolecules are then stored in a wide range of endocytic-originated vesicles which are collectively referred to as yolk organelles and occupy most of the mature oocytes cytoplasm. After fertilization, the contents of these organelles are degraded in a regulated manner to supply the embryo cells with fundamental molecules for de novo synthesis. Yolk accumulation and its regulated degradation are therefore crucial for successful development, however, most of the molecular mechanisms involved in the biogenesis, sorting and degradation of targeted yolk organelles are still poorly understood. ATG6 is part of two PI3P-kinase complexes that can regulate the recruitment of the endocytic or the autophagy machineries. Here, we investigate the role of RpATG6 in the endocytosis of the yolk macromolecules and in the biogenesis of the yolk organelles in the insect vector Rhodnius prolixus. We found that vitellogenic females express high levels of RpATG6 in the ovaries, when compared to the levels detected in the midgut and fat body. RNAi silencing of RpATG6 resulted in yolk proteins accumulated in the vitellogenic hemolymph, as a consequence of poor uptake by the oocytes. Accordingly, the silenced oocytes are unviable, white (contrasting to the control pink oocytes, smaller (62% of the control oocyte volume and accumulate only 40% of the yolk proteins, 80% of the TAG and 50% of the polymer polyphosphate quantified in control oocytes. The cortex of silenced oocytes present atypical smaller vesicles indicating that the yolk organelles were not properly formed and/or sorted, which was supported by the lack of endocytic vesicles near the plasma membrane of silenced oocytes as seen by TEM. Altogether, we found that RpATG6 is central for the

  13. Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells.

    Science.gov (United States)

    Kwon, Ilmin; Xiang, Siheng; Kato, Masato; Wu, Leeju; Theodoropoulos, Pano; Wang, Tao; Kim, Jiwoong; Yun, Jonghyun; Xie, Yang; McKnight, Steven L

    2014-09-05

    Many RNA regulatory proteins controlling pre-messenger RNA splicing contain serine:arginine (SR) repeats. Here, we found that these SR domains bound hydrogel droplets composed of fibrous polymers of the low-complexity domain of heterogeneous ribonucleoprotein A2 (hnRNPA2). Hydrogel binding was reversed upon phosphorylation of the SR domain by CDC2-like kinases 1 and 2 (CLK1/2). Mutated variants of the SR domains changing serine to glycine (SR-to-GR variants) also bound to hnRNPA2 hydrogels but were not affected by CLK1/2. When expressed in mammalian cells, these variants bound nucleoli. The translation products of the sense and antisense transcripts of the expansion repeats associated with the C9orf72 gene altered in neurodegenerative disease encode GRn and PRn repeat polypeptides. Both peptides bound to hnRNPA2 hydrogels independent of CLK1/2 activity. When applied to cultured cells, both peptides entered cells, migrated to the nucleus, bound nucleoli, and poisoned RNA biogenesis, which caused cell death. Copyright © 2014, American Association for the Advancement of Science.

  14. TTC19 Plays a Husbandry Role on UQCRFS1 Turnover in the Biogenesis of Mitochondrial Respiratory Complex III.

    Science.gov (United States)

    Bottani, Emanuela; Cerutti, Raffaele; Harbour, Michael E; Ravaglia, Sabrina; Dogan, Sukru Anil; Giordano, Carla; Fearnley, Ian M; D'Amati, Giulia; Viscomi, Carlo; Fernandez-Vizarra, Erika; Zeviani, Massimo

    2017-07-06

    Loss-of-function mutations in TTC19 (tetra-tricopeptide repeat domain 19) have been associated with severe neurological phenotypes and mitochondrial respiratory chain complex III deficiency. We previously demonstrated the mitochondrial localization of TTC19 and its link with complex III biogenesis. Here we provide detailed insight into the mechanistic role of TTC19, by investigating a Ttc19 ?/? mouse model that shows progressive neurological and metabolic decline, decreased complex III activity, and increased production of reactive oxygen species. By using both the Ttc19 ?/? mouse model and a range of human cell lines, we demonstrate that TTC19 binds to the fully assembled complex III dimer, i.e., after the incorporation of the iron-sulfur Rieske protein (UQCRFS1). The in situ maturation of UQCRFS1 produces N-terminal polypeptides, which remain bound to holocomplex III. We show that, in normal conditions, these UQCRFS1 fragments are rapidly removed, but when TTC19 is absent they accumulate within complex III, causing its structural and functional impairment. Copyright © 2017. Published by Elsevier Inc.

  15. Vitamin D Impacts the Expression of Runx2 Target Genes and Modulates Inflammation, Oxidative Stress and Membrane Vesicle Biogenesis Gene Networks in 143B Osteosarcoma Cells

    Directory of Open Access Journals (Sweden)

    Rama Garimella

    2017-03-01

    Full Text Available Osteosarcoma (OS is an aggressive malignancy of bone affecting children, adolescents and young adults. Understanding vitamin D metabolism and vitamin D regulated genes in OS is an important aspect of vitamin D/cancer paradigm, and in evaluating vitamin D as adjuvant therapy for human OS. Vitamin D treatment of 143B OS cells induced significant and novel changes in the expression of genes that regulate: (a inflammation and immunity; (b formation of reactive oxygen species, metabolism of cyclic nucleotides, sterols, vitamins and mineral (calcium, quantity of gap junctions and skeletogenesis; (c bone mineral density; and (d cell viability of skeletal cells, aggregation of bone cancer cells and exocytosis of secretory vesicles. Ingenuity pathway analysis revealed significant reduction in Runx2 target genes such as fibroblast growth factor -1, -12 (FGF1 and FGF12, bone morphogenetic factor-1 (BMP1, SWI/SNF related, matrix associated actin dependent regulator of chromatin subfamily a, member 4 (SMARCA4, Matrix extracellular phosphoglycoprotein (MEPE, Integrin, β4 (ITGBP4, Matrix Metalloproteinase -1, -28 (MMP1 and MMP28, and signal transducer and activator of transcription-4 (STAT4 in vitamin D treated 143B OS cells. These genes interact with the inflammation, oxidative stress and membrane vesicle biogenesis gene networks. Vitamin D not only inhibited the expression of Runx2 target genes MMP1, MMP28 and kallikrein related peptidase-7 (KLK7, but also migration and invasion of 143B OS cells. Vitamin D regulated Runx2 target genes or their products represent potential therapeutic targets and laboratory biomarkers for applications in translational oncology.

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

    Science.gov (United States)

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

    2016-03-01

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

  17. The role of protein crystallography in defining the mechanisms of biogenesis and catalysis in copper amine oxidase.

    Science.gov (United States)

    Klema, Valerie J; Wilmot, Carrie M

    2012-01-01

    Copper amine oxidases (CAOs) are a ubiquitous group of enzymes that catalyze the conversion of primary amines to aldehydes coupled to the reduction of O(2) to H(2)O(2). These enzymes utilize a wide range of substrates from methylamine to polypeptides. Changes in CAO activity are correlated with a variety of human diseases, including diabetes mellitus, Alzheimer's disease, and inflammatory disorders. CAOs contain a cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), that is required for catalytic activity and synthesized through the post-translational modification of a tyrosine residue within the CAO polypeptide. TPQ generation is a self-processing event only requiring the addition of oxygen and Cu(II) to the apoCAO. Thus, the CAO active site supports two very different reactions: TPQ synthesis, and the two electron oxidation of primary amines. Crystal structures are available from bacterial through to human sources, and have given insight into substrate preference, stereospecificity, and structural changes during biogenesis and catalysis. In particular both these processes have been studied in crystallo through the addition of native substrates. These latter studies enable intermediates during physiological turnover to be directly visualized, and demonstrate the power of this relatively recent development in protein crystallography.

  18. Functional identification of conserved residues involved in Lactobacillus rhamnosus strain GG sortase specificity and pilus biogenesis.

    Science.gov (United States)

    Douillard, François P; Rasinkangas, Pia; von Ossowski, Ingemar; Reunanen, Justus; Palva, Airi; de Vos, Willem M

    2014-05-30

    In Gram-positive bacteria, sortase-dependent pili mediate the adhesion of bacteria to host epithelial cells and play a pivotal role in colonization, host signaling, and biofilm formation. Lactobacillus rhamnosus strain GG, a well known probiotic bacterium, also displays on its cell surface mucus-binding pilus structures, along with other LPXTG surface proteins, which are processed by sortases upon specific recognition of a highly conserved LPXTG motif. Bioinformatic analysis of all predicted LPXTG proteins encoded by the L. rhamnosus GG genome revealed a remarkable conservation of glycine residues juxtaposed to the canonical LPXTG motif. Here, we investigated and defined the role of this so-called triple glycine (TG) motif in determining sortase specificity during the pilus assembly and anchoring. Mutagenesis of the TG motif resulted in a lack or an alteration of the L. rhamnosus GG pilus structures, indicating that the TG motif is critical in pilus assembly and that they govern the pilin-specific and housekeeping sortase specificity. This allowed us to propose a regulatory model of the L. rhamnosus GG pilus biogenesis. Remarkably, the TG motif was identified in multiple pilus gene clusters of other Gram-positive bacteria, suggesting that similar signaling mechanisms occur in other, mainly pathogenic, species. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Biogenesis of «fusty» defect in virgin olive oils

    Directory of Open Access Journals (Sweden)

    Angerosa, F.

    1996-06-01

    Full Text Available The biogenesis of «fusty» defect was studied by chemical and microbial analyses on olives stored in piles for different times and their resulting oils. The fusty defect was perceived by tasters after four days of storage. The quali-quantitative composition of oil volatile fraction was a very suitable way to emphasize metabolites produced by microorganisms involved during the fruit storage. Some volatile compounds, such as 2- and 3- methyl butan-1-al, their corresponding alcohols and propionic acid, 2- methyl propionic acid and 3-methyl butanoic acid, were produced. At the same time, it was observed a dramatic development of Clostridium sp. and, in a lower proportion, of Pseudomonas sp. Furthermore, during the storage the evident softening of fruits has to be attributed to microorganisms in rapid growth belonging to Enterobacter sp. and moulds, that have in its enzymatic store pectinolytic enzymes.

    La biogénesis del atrojado ha sido estudiada química y microbiológicamente en aceitunas amontonadas durante diferentes períodos de tiempo; además ha sido evaluado el aceite procedente de la elaboración de dichos frutos. La percepción de atrojado era percibida por el panel de catadores sólo después de cuatro días de almacenamiento de las aceitunas. La determinación cuanti-cualitativa de los compuestos aromáticos del aceite resultante se ha relacionado con los microorganismos implicados en la degradación de los frutos. Se han encontrado algunos compuestos volátiles como el 2- y 3- metil butan-1-al y sus correspondientes alcoholes, el ácido propiónico, el ácido 2-metil propiónico y el 3-metil butanoico. Al mismo tiempo, ha sido observado el fuerte desarrollo de especies del género Clostridium y, en menor proporción, de Pseudomonas. Asimismo, durante el almacenamiento se produjo un ablandamiento de los frutos a causa de la acción de las enzimas pectolíticas de microorganismos de r

  20. Transcription Factor EB Expression in Early Breast Cancer Relates to Lysosomal/Autophagosomal Markers and Prognosis.

    Science.gov (United States)

    Giatromanolaki, Alexandra; Sivridis, Efthimios; Kalamida, Dimitra; Koukourakis, Michael I

    2017-06-01

    Disrupting the autophagic balance to trigger autophagic death may open new strategies for cancer therapy. Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and may play a role in cancer biology and clinical behavior. The expression of TFEB and the lysosomal cancer cell content (expression of lysosomal associated membrane protein 2a [LAMP2a] and cathepsin D) was studied in a series of 100 T1-stage breast carcinomas. Expression patterns were correlated with autophagy/hypoxia-related proteins, angiogenesis, and clinical outcome. The effect of hypoxic/acidic conditions on TFEB kinetics was studied in the MCF-7 cancer cell line. Overexpression of TFEB in cancer cell cytoplasm and the perinuclear/nuclear area was noted in 23 (23%) of 100 cases. High LAMP2a and cathepsin D expression was noted in 30 (30%) of 100 and 28 (28%) of 100 cases, respectively. TFEB expression was directly linked with LAMP2a (P factor 2-alpha (HIF-2α) (P = .01, r = 0.25) expression and inversely with progesterone receptor (P = .01, r = 0.22). High vascular density was directly linked with LAMP2a (P = .05, r = 0.18) and cathepsin D (P = .005, r = 0.28). In Kaplan-Meier survival analysis, TFEB and cathepsin D expression were related to an ominous prognosis (P = .001 and P = .03, respectively). In multivariate analysis, TFEB expression sustained its independent prognostic significance (P = .05, hazard ratio 2.1). In in vitro experiments, acidity triggered overexpression of TFEB and nuclear translocation. Intense TFEB expression and lysosomal biogenesis, evident in one fourth of early breast carcinomas, define poor prognosis. Tumor acidity is among the microenvironmental conditions that trigger TFEB overactivity. TFEB is a sound target for the development of lysosomal targeting therapies. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Sensor kinase KinB and its pathway-associated key factors sense the signal of nutrition starvation in sporulation of Bacillus subtilis.

    Science.gov (United States)

    Liu, Weipeng; He, Zeying; Gao, Feng; Yan, Jinyuan; Huang, Xiaowei

    2018-01-03

    Bacillus subtilis responds to environmental stress cues and develops endospores for survival. In the process of endospore formation, sporulation initiation is a vital stage and this stage is governed by autophosphorylation of the sensor histidine kinases. The second major sensor kinase KinB perceives the intracellular changes of GTP and ATP during sporulation. However, determination of the environmental signals as well as its related signaling pathway of KinB requires further elucidation. Our current study found that, contrary to the sporulation failure induced by ΔkinA in the nutrient-rich 2× SG medium, the sensor kinase KinB sensed the environmental cues in the nutrient-poor MM medium. Two other membrane proteins, KapB and KbaA, also responded similarly to the same external signal as KinB. Both KapB and KbaA acted upstream of KinB, but they exerted their regulation upon KinB independently. Furthermore, we demonstrated that both the SH3 domain and the α-helix structure in KapB are required for sensing or transducing the signal of sporulation initiation. Collectively, our work here supplied the direct evidences that KinB and its pathway sense the external signal of nutrient starvation in MM medium, and further analyzes the interrelationship among KinB, KbaA, and KapB. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  2. Enzymes in biogenesis of plant cell wall polysaccharides. Enzyme characterization using tracer techniques

    International Nuclear Information System (INIS)

    Dickinson, D.B.

    1975-01-01

    Enzymes and metabolic pathways, by which starch and cell wall polysaccharides are formed, were investigated in order to learn how these processes are regulated and to identify the enzymatic regulatory mechanisms involved. Germinating lily pollen was used for studies of cell wall formation, and pollen and maize endosperm for studies of starch biosynthesis. Hexokinase being the first step in conversion of hexoses to starch, wall polysaccharides and respiratory substrates, maize endosperm enzyme was assayed by its conversion of 14 C-hexose to 14 C-hexose-6-P, and rapid separation of the two labelled compounds on anion-exchange paper. This enzyme did not appear to be under tight regulation by feed-back inhibition or activation, nor to be severely inhibited by glucose-6-P or activated by citrate. ADP-glucose pyrophosphorylase and other pyrophosphorylases were assayed radiochemically with 14 C-glucose-1-P (forward direction) or 32-PPsub(i) (reverse direction). They showed that the maize endosperm enzyme was activated by the glycolytic intermediates fructose-6-P and 3-phosphoglycerate, and that low levels of the enzyme were present in the high sucrose-low starch mutant named shrunken-2. Under optimal in-vitro assay conditions, the pollen enzyme reacted four times faster than the observed in-vivo rate of starch accumulation. Biogenesis of plant cell wall polysaccharides requires the conversion of hexose phosphates to various sugar nucleotides and utilization of the latter by the appropriate polysaccharide synthetases. Lily pollen possesses a β-1,3-glucan synthetase which is activated up to six-fold by β-linked oligosaccharides. Hence, the in-vivo activity of this enzyme may be modulated by such effector molecules

  3. miR-125b affects mitochondrial biogenesis and impairs brite adipocyte formation and function

    Directory of Open Access Journals (Sweden)

    Maude Giroud

    2016-08-01

    Full Text Available Objective: In rodents and humans, besides brown adipose tissue (BAT, islands of thermogenic adipocytes, termed “brite” (brown-in-white or beige adipocytes, emerge within white adipose tissue (WAT after cold exposure or β3-adrenoceptor stimulation, which may protect from obesity and associated diseases. microRNAs are novel modulators of adipose tissue development and function. The purpose of this work was to characterize the role of microRNAs in the control of brite adipocyte formation. Methods/Results: Using human multipotent adipose derived stem cells, we identified miR-125b-5p as downregulated upon brite adipocyte formation. In humans and rodents, miR-125b-5p expression was lower in BAT than in WAT. In vitro, overexpression and knockdown of miR-125b-5p decreased and increased mitochondrial biogenesis, respectively. In vivo, miR-125b-5p levels were downregulated in subcutaneous WAT and interscapular BAT upon β3-adrenergic receptor stimulation. Injections of an miR-125b-5p mimic and LNA inhibitor directly into WAT inhibited and increased β3-adrenoceptor-mediated induction of UCP1, respectively, and mitochondrial brite adipocyte marker expression and mitochondriogenesis. Conclusion: Collectively, our results demonstrate that miR-125b-5p plays an important role in the repression of brite adipocyte function by modulating oxygen consumption and mitochondrial gene expression. Author Video: Author Video Watch what authors say about their articles Keywords: miR-125b-5p, White adipocyte, Brite adipocyte, Mitochondriogenesis

  4. Symportin 1 chaperones 5S RNP assembly during ribosome biogenesis by occupying an essential rRNA-binding site.

    Science.gov (United States)

    Calviño, Fabiola R; Kharde, Satyavati; Ori, Alessandro; Hendricks, Astrid; Wild, Klemens; Kressler, Dieter; Bange, Gert; Hurt, Ed; Beck, Martin; Sinning, Irmgard

    2015-04-07

    During 60S biogenesis, mature 5S RNP consisting of 5S RNA, RpL5 and RpL11, assembles into a pre-60S particle, where docking relies on RpL11 interacting with helix 84 (H84) of the 25S RNA. How 5S RNP is assembled for recruitment into the pre-60S is not known. Here we report the crystal structure of a ternary symportin Syo1-RpL5-N-RpL11 complex and provide biochemical and structural insights into 5S RNP assembly. Syo1 guards the 25S RNA-binding surface on RpL11 and competes with H84 for binding. Pull-down experiments show that H84 releases RpL11 from the ternary complex, but not in the presence of 5S RNA. Crosslinking mass spectrometry visualizes structural rearrangements on incorporation of 5S RNA into the Syo1-RpL5-RpL11 complex supporting the formation of a pre-5S RNP. Our data underline the dual role of Syo1 in ribosomal protein transport and as an assembly platform for 5S RNP.

  5. The helicase and RNaseIIIa domains of Arabidopsis Dicer-Like1 modulate catalytic parameters during MicroRNA biogenesis

    KAUST Repository

    Liu, Chenggang

    2012-04-03

    Dicer-Like1 (DCL1), an RNaseIII endonuclease, and Hyponastic Leaves1 (HYL1), a double-stranded RNA-binding protein, are core components of the plant microRNA (miRNA) biogenesis machinery. hyl1 mutants accumulate low levels of miRNAs and display pleiotropic developmental phenotypes. We report the identification of five new hyl1 suppressor mutants, all of which are alleles of DCL1. These new alleles affect either the helicase or the RNaseIIIa domains of DCL1, highlighting the critical functions of these domains. Biochemical analysis of the DCL1 suppressor variants reveals that they process the primary transcript (pri-miRNA) more efficiently than wild-type DCL1, with both higher Kcat and lower Km values. The DCL1 variants largely rescue wild-type miRNA accumulation levels in vivo, but do not rescue the MIRNA processing precision defects of the hyl1 mutant. In vitro, the helicase domain confers ATP dependence on DCL1-catalyzed MIRNA processing, attenuates DCL1 cleavage activity, and is required for precise MIRNA processing of some substrates. © 2012 American Society of Plant Biologists.

  6. Lipoprotein Transport: Greasing the Machines of Outer Membrane Biogenesis: Re-Examining Lipoprotein Transport Mechanisms Among Diverse Gram-Negative Bacteria While Exploring New Discoveries and Questions.

    Science.gov (United States)

    Grabowicz, Marcin

    2018-04-01

    The Gram-negative outer membrane (OM) is a potent permeability barrier against antibiotics, limiting clinical options amid mounting rates of resistance. The Lol transport pathway delivers lipoproteins to the OM. All the OM assembly machines require one or more OM lipoprotein to function, making the Lol pathway central for all aspects of OM biogenesis. The Lol pathways of many medically important species clearly deviate from the Escherichia coli paradigm, perhaps with implications for efforts to develop novel antibiotics. Moreover, recent work reveals the existence of an undiscovered alternate route for bringing lipoproteins to the OM. Here, lipoprotein transport mechanisms, and the quality control systems that underpin them, is re-examined in context of their diversity. © 2018 WILEY Periodicals, Inc.

  7. Biogenesis of mitochondria in cauliflower (Brassica oleracea var. botrytis) curds subjected to temperature stress and recovery involves regulation of the complexome, respiratory chain activity, organellar translation and ultrastructure.

    Science.gov (United States)

    Rurek, Michal; Woyda-Ploszczyca, Andrzej M; Jarmuszkiewicz, Wieslawa

    2015-01-01

    The biogenesis of the cauliflower curd mitochondrial proteome was investigated under cold, heat and the recovery. For the first time, two dimensional fluorescence difference gel electrophoresis was used to study the plant mitochondrial complexome in heat and heat recovery. Particularly, changes in the complex I and complex III subunits and import proteins, and the partial disintegration of matrix complexes were observed. The presence of unassembled subunits of ATP synthase was accompanied by impairment in mitochondrial translation of its subunit. In cold and heat, the transcription profiles of mitochondrial genes were uncorrelated. The in-gel activities of respiratory complexes were particularly affected after stress recovery. Despite a general stability of respiratory chain complexes in heat, functional studies showed that their activity and the ATP synthesis yield were affected. Contrary to cold stress, heat stress resulted in a reduced efficiency of oxidative phosphorylation likely due to changes in alternative oxidase (AOX) activity. Stress and stress recovery differently modulated the protein level and activity of AOX. Heat stress induced an increase in AOX activity and protein level, and AOX1a and AOX1d transcript level, while heat recovery reversed the AOX protein and activity changes. Conversely, cold stress led to a decrease in AOX activity (and protein level), which was reversed after cold recovery. Thus, cauliflower AOX is only induced by heat stress. In heat, contrary to the AOX activity, the activity of rotenone-insensitive internal NADH dehydrogenase was diminished. The relevance of various steps of plant mitochondrial biogenesis to temperature stress response and recovery is discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. [Characteristics of Bacillus cereus dissociants].

    Science.gov (United States)

    Doroshenko, E V; Loĭko, N G; Il'inskaia, O N; Kolpakov, A I; Gornova, I B; Klimanova, E V; El'-Registan, G I

    2001-01-01

    The autoregulation of the phenotypic (populational) variability of the Bacillus cereus strain 504 was studied. The isolated colonial morphotypes of this bacterium were found to differ in their growth characteristics and the synthesis of extracellular proteases. The phenotypic variabilities of vegetative proliferating cells and those germinated from endospores and cystlike refractory cells were different. Bacterial variants also differed in the production of the d1 and d2 factors (the autoinducers of dormancy and autolysis, respectively) and sensitivity to them. The possible role of these factors in the dissociation of microorganisms is discussed.

  9. The Role of Protein Crystallography in Defining the Mechanisms of Biogenesis and Catalysis in Copper Amine Oxidase

    Directory of Open Access Journals (Sweden)

    Carrie M. Wilmot

    2012-05-01

    Full Text Available Copper amine oxidases (CAOs are a ubiquitous group of enzymes that catalyze the conversion of primary amines to aldehydes coupled to the reduction of O2 to H2O2. These enzymes utilize a wide range of substrates from methylamine to polypeptides. Changes in CAO activity are correlated with a variety of human diseases, including diabetes mellitus, Alzheimer’s disease, and inflammatory disorders. CAOs contain a cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ, that is required for catalytic activity and synthesized through the post-translational modification of a tyrosine residue within the CAO polypeptide. TPQ generation is a self-processing event only requiring the addition of oxygen and Cu(II to the apoCAO. Thus, the CAO active site supports two very different reactions: TPQ synthesis, and the two electron oxidation of primary amines. Crystal structures are available from bacterial through to human sources, and have given insight into substrate preference, stereospecificity, and structural changes during biogenesis and catalysis. In particular both these processes have been studied in crystallo through the addition of native substrates. These latter studies enable intermediates during physiological turnover to be directly visualized, and demonstrate the power of this relatively recent development in protein crystallography.

  10. Plant Nucleolar Stress Response, a New Face in the NAC-Dependent Cellular Stress Responses

    OpenAIRE

    Iwai Ohbayashi; Munetaka Sugiyama

    2018-01-01

    The nucleolus is the most prominent nuclear domain, where the core processes of ribosome biogenesis occur vigorously. All these processes are finely orchestrated by many nucleolar factors to build precisely ribosome particles. In animal cells, perturbations of ribosome biogenesis, mostly accompanied by structural disorders of the nucleolus, cause a kind of cellular stress to induce cell cycle arrest, senescence, or apoptosis, which is called nucleolar stress response. The best-characterized p...

  11. Conformational Flexibility of Proteins Involved in Ribosome Biogenesis: Investigations via Small Angle X-ray Scattering (SAXS

    Directory of Open Access Journals (Sweden)

    Dritan Siliqi

    2018-02-01

    Full Text Available The dynamism of proteins is central to their function, and several proteins have been described as flexible, as consisting of multiple domains joined by flexible linkers, and even as intrinsically disordered. Several techniques exist to study protein structures, but small angle X-ray scattering (SAXS has proven to be particularly powerful for the quantitative analysis of such flexible systems. In the present report, we have used SAXS in combination with X-ray crystallography to highlight their usefulness at characterizing flexible proteins, using as examples two proteins involved in different steps of ribosome biogenesis. The yeast BRCA2 and CDKN1A-interactig protein, Bcp1, is a chaperone for Rpl23 of unknown structure. We showed that it consists of a rigid, slightly elongated protein, with a secondary structure comprising a mixture of alpha helices and beta sheets. As an example of a flexible molecule, we studied the SBDS (Shwachman-Bodian-Diamond Syndrome protein that is involved in the cytoplasmic maturation of the 60S subunit and constitutes the mutated target in the Shwachman-Diamond Syndrome. In solution, this protein coexists in an ensemble of three main conformations, with the N- and C-terminal ends adopting different orientations with respect to the central domain. The structure observed in the protein crystal corresponds to an average of those predicted by the SAXS flexibility analysis.

  12. Two novel genes, fanA and fanB, involved in the biogenesis of K99 fimbriae.

    Science.gov (United States)

    Roosendaal, E; Boots, M; de Graaf, F K

    1987-08-11

    The nucleotide sequence of the region located transcriptionally upstream of the K99 fimbrial subunit gene (fanC) was determined. Several putative transcription signals and two open reading frames, designated fanA and fanB, became apparent. Frameshift mutations in fanA and fanB reduced K99 fimbriae expression 8-fold and 16-fold, respectively. Complementation of the mutants in trans restored the K99 expression to about 75% of the wild type level, indicating that fanA and fanB code for transacting polypeptides involved in the biogenesis of K99 fimbriae. The fanA and fanB gene products FanA and FanB were not detectable in minicell preparations, indicating that both polypeptides are synthesized in very small amounts. However, in an in vitro DNA directed translation system FanA and FanB could be identified. The deduced amino acid sequences of FanA and FanB showed that both polypeptides contain no signal peptides, indicating a cytoplasmic location. Furthermore, the polypeptides are very hydrophilic, mainly basic, and exhibit remarkable homology to each other and to a regulatory protein (papB) encoded by the pap-operon (1). Some of these features are characteristics of nucleic acid binding proteins, which suggests that FanA and FanB have a regulatory function in the synthesis of FanC and the auxiliary polypeptides FanD-H.

  13. Population dynamics of Meloidogyne arenaria and Pasteuria penetrans in a long-term crop rotation study.

    Science.gov (United States)

    Timper, Patricia

    2009-12-01

    The endospore-forming bacterium Pasteuria penetrans is an obligate parasite of root-knot nematodes (Meloidogyne spp.). The primary objective of this study was to determine the effect of crop sequence on abundance of P. penetrans. The experiment was conducted from 2000 to 2008 at a field site naturally infested with both the bacterium and its host Meloidogyne arenaria and included the following crop sequences: continuous peanut (Arachis hypogaea) (P-P-P) and peanut rotated with either 2 years of corn (Zea mays) (C-C-P), 1 year each of cotton (Gossypium hirsutum) and corn (Ct-C-P), or 1 year each of corn and a vegetable (V-C-P). The vegetable was a double crop of sweet corn and eggplant (Solanum melongena). A bioassay with second-stage juveniles (J2) of M. arenaria from a greenhouse (GH) population was used to estimate endospore abundance under the different crop sequences. A greater numerical increase in endospore densities was expected in the P-P-P and V-C-P sequences than in the other sequences because both peanut and eggplant are good hosts for M. arenaria. However, endospore densities, as determined by bioassay, did not substantially increase in any of the sequences during the 9-year experiment. To determine whether the nematode population had developed resistance to the resident P. penetrans, five single egg-mass (SEM) lines from the field population of M. arenaria were tested alongside the GH population for acquisition of endospores from the field soil. Four of the five SEM lines acquired 9 to 14 spores/J2 whereas the GH population and one of the SEM lines acquired 3.5 and 1.8 spores/J2, respectively. Endospore densities estimated with the four receptive SEM lines were highest in the P-P-P plots (14-20 spores/J2), intermediate in the V-C-P plots (6-7 spores/J2), and lowest in the Ct-C-P plots (< 1 spore/J2). These results indicate that the field population of M. arenaria is heterogeneous for attachment of P. penetrans endospores. Moreover, spore densities

  14. Population Dynamics of Meloidogyne arenaria and Pasteuria penetrans in a Long-Term Crop Rotation Study

    Science.gov (United States)

    2009-01-01

    The endospore-forming bacterium Pasteuria penetrans is an obligate parasite of root-knot nematodes (Meloidogyne spp.). The primary objective of this study was to determine the effect of crop sequence on abundance of P. penetrans. The experiment was conducted from 2000 to 2008 at a field site naturally infested with both the bacterium and its host Meloidogyne arenaria and included the following crop sequences: continuous peanut (Arachis hypogaea) (P-P-P) and peanut rotated with either 2 years of corn (Zea mays) (C-C-P), 1 year each of cotton (Gossypium hirsutum) and corn (Ct-C-P), or 1 year each of corn and a vegetable (V-C-P). The vegetable was a double crop of sweet corn and eggplant (Solanum melongena). A bioassay with second-stage juveniles (J2) of M. arenaria from a greenhouse (GH) population was used to estimate endospore abundance under the different crop sequences. A greater numerical increase in endospore densities was expected in the P-P-P and V-C-P sequences than in the other sequences because both peanut and eggplant are good hosts for M. arenaria. However, endospore densities, as determined by bioassay, did not substantially increase in any of the sequences during the 9-year experiment. To determine whether the nematode population had developed resistance to the resident P. penetrans, five single egg-mass (SEM) lines from the field population of M. arenaria were tested alongside the GH population for acquisition of endospores from the field soil. Four of the five SEM lines acquired 9 to 14 spores/J2 whereas the GH population and one of the SEM lines acquired 3.5 and 1.8 spores/J2, respectively. Endospore densities estimated with the four receptive SEM lines were highest in the P-P-P plots (14-20 spores/J2), intermediate in the V-C-P plots (6-7 spores/J2), and lowest in the Ct-C-P plots (< 1 spore/J2). These results indicate that the field population of M. arenaria is heterogeneous for attachment of P. penetrans endospores. Moreover, spore densities

  15. Defects of mtDNA Replication Impaired Mitochondrial Biogenesis During Trypanosoma cruzi Infection in Human Cardiomyocytes and Chagasic Patients: The Role of Nrf1/2 and Antioxidant Response

    Science.gov (United States)

    Wan, Xianxiu; Gupta, Shivali; Zago, Maria P.; Davidson, Mercy M.; Dousset, Pierre; Amoroso, Alejandro; Garg, Nisha Jain

    2012-01-01

    Background Mitochondrial dysfunction is a key determinant in chagasic cardiomyopathy development in mice; however, its relevance in human Chagas disease is not known. We determined if defects in mitochondrial biogenesis and dysregulation of peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 (PGC-1)–regulated transcriptional pathways constitute a mechanism or mechanisms underlying mitochondrial oxidative-phosphorylation (OXPHOS) deficiency in human Chagas disease. Methods and Results We utilized human cardiomyocytes and left-ventricular tissue from chagasic and other cardiomyopathy patients and healthy donors (n>6/group). We noted no change in citrate synthase activity, yet mRNA and/or protein levels of subunits of the respiratory complexes were significantly decreased in Trypanosoma cruzi–infected cardiomyocytes (0 to 24 hours) and chagasic hearts. We observed increased mRNA and decreased nuclear localization of PGC-1-coactivated transcription factors, yet the expression of genes for PPARγ-regulated fatty acid oxidation and nuclear respiratory factor (NRF1/2)–regulated mtDNA replication and transcription machinery was enhanced in infected cardiomyocytes and chagasic hearts. The D-loop formation was normal or higher, but mtDNA replication and mtDNA content were decreased by 83% and 40% to 65%, respectively. Subsequently, we noted that reactive oxygen species (ROS), oxidative stress, and mtDNA oxidation were significantly increased, yet NRF1/2-regulated antioxidant gene expression remained compromised in infected cardiomyocytes and chagasic hearts. Conclusions The replication of mtDNA was severely compromised, resulting in a significant loss of mtDNA and expression of OXPHOS genes in T cruzi–infected cardiomyocytes and chagasic hearts. Our data suggest increased ROS generation and selective functional incapacity of NRF2-mediated antioxidant gene expression played a role in the defects in mtDNA replication and unfitness of mtDNA for

  16. Role of IscX in Iron-Sulfur Cluster Biogenesis in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jin Hae; Bothe, Jameson R.; Frederick, Ronnie O.; Holder, Johneisa C.; Markley, John L. [UW

    2014-08-20

    The Escherichia coli isc operon encodes key proteins involved in the biosynthesis of iron–sulfur (Fe–S) clusters. Whereas extensive studies of most ISC proteins have revealed their functional properties, the role of IscX (also dubbed YfhJ), a small acidic protein encoded by the last gene in the operon, has remained in question. Previous studies showed that IscX binds iron ions and interacts with the cysteine desulfurase (IscS) and the scaffold protein for cluster assembly (IscU), and it has been proposed that IscX functions either as an iron supplier or a regulator of Fe–S cluster biogenesis. We have used a combination of NMR spectroscopy, small-angle X-ray scattering (SAXS), chemical cross-linking, and enzymatic assays to enlarge our understanding of the interactions of IscX with iron ions, IscU, and IscS. We used chemical shift perturbation to identify the binding interfaces of IscX and IscU in their complex. NMR studies showed that Fe2+ from added ferrous ammonium sulfate binds IscX much more avidly than does Fe3+ from added ferric ammonium citrate and that Fe2+ strengthens the interaction between IscX and IscU. We found that the addition of IscX to the IscU–IscS binary complex led to the formation of a ternary complex with reduced cysteine desulfurase activity, and we determined a low-resolution model for that complex from a combination of NMR and SAXS data. We postulate that the inhibition of cysteine desulfurase activity by IscX serves to reduce unproductive conversion of cysteine to alanine. By incorporating these new findings with results from prior studies, we propose a detailed mechanism for Fe–S cluster assembly in which IscX serves both as a donor of Fe2+ and as a regulator of cysteine desulfurase activity.

  17. Interaction between bacterial outer membrane proteins and periplasmic quality control factors: a kinetic partitioning mechanism.

    Science.gov (United States)

    Wu, Si; Ge, Xi; Lv, Zhixin; Zhi, Zeyong; Chang, Zengyi; Zhao, Xin Sheng

    2011-09-15

    The OMPs (outer membrane proteins) of Gram-negative bacteria have to be translocated through the periplasmic space before reaching their final destination. The aqueous environment of the periplasmic space and high permeability of the outer membrane engender such a translocation process inevitably challenging. In Escherichia coli, although SurA, Skp and DegP have been identified to function in translocating OMPs across the periplasm, their precise roles and their relationship remain to be elucidated. In the present paper, by using fluorescence resonance energy transfer and single-molecule detection, we have studied the interaction between the OMP OmpC and these periplasmic quality control factors. The results of the present study reveal that the binding rate of OmpC to SurA or Skp is much faster than that to DegP, which may lead to sequential interaction between OMPs and different quality control factors. Such a kinetic partitioning mechanism for the chaperone-substrate interaction may be essential for the quality control of the biogenesis of OMPs.

  18. A phosphatidylinositol transfer protein integrates phosphoinositide signaling with lipid droplet metabolism to regulate a developmental program of nutrient stress-induced membrane biogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Jihui; Lin, Coney Pei-Chen; Pathak, Manish C.; Temple, Brenda R.S.; Nile, Aaron H.; Mousley, Carl J.; Duncan, Mara C.; Eckert, Debra M.; Leiker, Thomas J.; Ivanova, Pavlina T.; Myers, David S.; Murphy, Robert C.; Brown, H. Alex; Verdaasdonk, Jolien; Bloom, Kerry S.; Ortlund, Eric A.; Neiman, Aaron M.; Bankaitis, Vytas A. [Emory-MED; (SBU); (TAM); (UNC); (Vanderbilt-MED); (Utah); (UCHSC)

    2014-07-11

    Lipid droplet (LD) utilization is an important cellular activity that regulates energy balance and release of lipid second messengers. Because fatty acids exhibit both beneficial and toxic properties, their release from LDs must be controlled. Here we demonstrate that yeast Sfh3, an unusual Sec14-like phosphatidylinositol transfer protein, is an LD-associated protein that inhibits lipid mobilization from these particles. We further document a complex biochemical diversification of LDs during sporulation in which Sfh3 and select other LD proteins redistribute into discrete LD subpopulations. The data show that Sfh3 modulates the efficiency with which a neutral lipid hydrolase-rich LD subclass is consumed during biogenesis of specialized membrane envelopes that package replicated haploid meiotic genomes. These results present novel insights into the interface between phosphoinositide signaling and developmental regulation of LD metabolism and unveil meiosis-specific aspects of Sfh3 (and phosphoinositide) biology that are invisible to contemporary haploid-centric cell biological, proteomic, and functional genomics approaches.

  19. A phosphatidylinositol transfer protein integrates phosphoinositide signaling with lipid droplet metabolism to regulate a developmental program of nutrient stress-induced membrane biogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Jihui; Lin, Coney Pei-Chen; Pathak, Manish C.; Temple, Brenda R.S.; Nile, Aaron H.; Mousley, Carl J.; Duncan, Mara C.; Eckert, Debra M.; Leiker, Thomas J.; Ivanova, Pavlina T.; Myers, David S.; Murphy, Robert C.; Brown, H. Alex; Verdaasdonk, Jolien; Bloom, Kerry S.; Ortlund, Eric A.; Neiman, Aaron M.; Bankaitis, Vytas A. (Emory-MED); (UNCSM); (UNC); (UCHSC); (TAM); (Vanderbilt-MED); (SBU); (Utah)

    2016-07-06

    Lipid droplet (LD) utilization is an important cellular activity that regulates energy balance and release of lipid second messengers. Because fatty acids exhibit both beneficial and toxic properties, their release from LDs must be controlled. Here we demonstrate that yeast Sfh3, an unusual Sec14-like phosphatidylinositol transfer protein, is an LD-associated protein that inhibits lipid mobilization from these particles. We further document a complex biochemical diversification of LDs during sporulation in which Sfh3 and select other LD proteins redistribute into discrete LD subpopulations. The data show that Sfh3 modulates the efficiency with which a neutral lipid hydrolase-rich LD subclass is consumed during biogenesis of specialized membrane envelopes that package replicated haploid meiotic genomes. These results present novel insights into the interface between phosphoinositide signaling and developmental regulation of LD metabolism and unveil meiosis-specific aspects of Sfh3 (and phosphoinositide) biology that are invisible to contemporary haploid-centric cell biological, proteomic, and functional genomics approaches.

  20. Phylogenetic Analysis of Pasteuria penetrans by 16S rRNA Gene Cloning and Sequencing.

    Science.gov (United States)

    Anderson, J M; Preston, J F; Dickson, D W; Hewlett, T E; Williams, N H; Maruniak, J E

    1999-09-01

    Pasteuria penetrans is an endospore-forming bacterial parasite of Meloidogyne spp. This organism is among the most promising agents for the biological control of root-knot nematodes. In order to establish the phylogenetic position of this species relative to other endospore-forming bacteria, the 16S ribosomal genes from two isolates of P. penetrans, P-20, which preferentially infects M. arenaria race 1, and P-100, which preferentially infects M. incognita and M. javanica, were PCR-amplified from a purified endospore extraction. Universal primers for the 16S rRNA gene were used to amplify DNA which was cloned, and a nucleotide sequence was obtained for 92% of the gene (1,390 base pairs) encoding the 16S rDNA from each isolate. Comparison of both isolates showed identical sequences that were compared to 16S rDNA sequences of 30 other endospore-forming bacteria obtained from GenBank. Parsimony analyses indicated that P. penetrans is a species within a clade that includes Alicyclobacillus acidocaldarius, A. cycloheptanicus, Sulfobacillus sp., Bacillus tusciae, B. schlegelii, and P. ramosa. Its closest neighbor is P. ramosa, a parasite of Daphnia spp. (water fleas). This study provided a genomic basis for the relationship of species assigned to the genus Pasteuria, and for comparison of species that are parasites of different phytopathogenic nematodes.

  1. The Mkk2 MAPKK Regulates Cell Wall Biogenesis in Cooperation with the Cek1-Pathway in Candida albicans.

    Directory of Open Access Journals (Sweden)

    Elvira Román

    Full Text Available The cell wall integrity pathway (CWI plays an important role in the biogenesis of the cell wall in Candida albicans and other fungi. In the present work, the C. albicans MKK2 gene that encodes the putative MAPKK of this pathway was deleted in different backgrounds and the phenotypes of the resultant mutants were characterised. We show here that Mkk2 mediates the phosphorylation of the Mkc1 MAPK in response to cell wall assembly interfering agents such as zymolyase or tunicamycin and also to oxidative stress. Remarkably, mkk2 and mkc1 mutants display related but distinguishable- cell wall associated phenotypes and differ in the pattern of MAPK phosphorylation under different stress conditions. mkk2 and mkc1 mutants display an altered expression of GSC1, CEK1 and CRH11 genes at different temperatures. Combined deletion of MKK2 with HST7 supports a cooperative role for the Cek1-mediated and CWI pathways in regulating cell wall architecture under vegetative growth. However, and in contrast to Mkc1, Mkk2 does not seem to play a role in the virulence of C. albicans in the mouse systemic model or the Galleria mellonella model of infection.

  2. Inactivation of Geobacillus stearothermophilus in canned food and coconut milk samples by addition of enterocin AS-48.

    Science.gov (United States)

    Viedma, Pilar Martínez; Abriouel, Hikmate; Ben Omar, Nabil; López, Rosario Lucas; Valdivia, Eva; Gálvez, Antonio

    2009-05-01

    The cyclic bacteriocin enterocin AS-48 was tested on a cocktail of two Geobacillus stearothermophilus strains in canned food samples (corn and peas), and in coconut milk. AS-48 (7 microg/g) reduced viable cell counts below detection levels in samples from canned corn and peas stored at 45 degrees C for 30 days. In coconut milk, bacterial inactivation by AS-48 (1.75 microg/ml) was even faster. In all canned food and drink samples inoculated with intact G. stearothermophilus endospores, bacteriocin addition (1.75 microg per g or ml of food sample) rapidly reduced viable cell counts below detection levels and avoided regrowth during storage. After a short-time bacteriocin treatment of endospores, trypsin addition markedly increased G. stearothermophilus survival, supporting the effect of residual bacteriocin on the observed loss of viability for endospores. Results from this study support the potential of enterocin AS-48 as a biopreservative against G. stearothermophilus.

  3. Arabidopsis cotyledon chloroplast biogenesis factor CYO1 uses glutathione as an electron donor and interacts with PSI (A1 and A2) and PSII (CP43 and CP47) subunits.

    Science.gov (United States)

    Muranaka, Atsuko; Watanabe, Shunsuke; Sakamoto, Atsushi; Shimada, Hiroshi

    2012-08-15

    CYO1 is required for thylakoid biogenesis in cotyledons of Arabidopsis thaliana. To elucidate the enzymatic characteristics of CYO1, we analyzed the protein disulfide isomerase (PDI) activity of CYO1 using dieosin glutathione disulfide (Di-E-GSSG) as a substrate. The reductase activity of CYO1 increased as a function of Di-E-GSSG, with an apparent K(m) of 824nM and K(cat) of 0.53min(-1). PDI catalyzes dithiol/disulfide interchange reactions, and the cysteine residues in PDI proteins are very important. To analyze the significance of the cysteine residues for the PDI activity of CYO1, we estimated the kinetic parameters of point-mutated CYO1 proteins. C117S, C124S, C135S, and C156S had higher values for K(m) than did wild-type CYO1. C158S had a similar K(m) but a higher K(cat), and C138S and C161S had similar K(m) values but lower K(cat) values than did wild-type CYO1. These results suggested that the cysteine residues at positions 138 and 161 were important for PDI activity. Low PDI activity of CYO1 was observed when NADPH or NADH was used as an electron donor. However, PDI activity was observed with CYO1 and glutathione, suggesting that glutathione may serve as a reducing agent for CYO1 in vivo. Based on analysis with the split-ubiquitin system, CYO1 interacted with the A1 and A2 subunits of PSI and the CP43 and CP47 subunits of PSII. Thus, CYO1 may accelerate the folding of cysteine residue--containing PSI and PSII subunits by repeatedly breaking and creating disulfide bonds. Copyright © 2012 Elsevier GmbH. All rights reserved.

  4. Eating habits modulate short term memory and epigenetical regulation of brain derived neurotrophic factor in hippocampus of low- and high running capacity rats.

    Science.gov (United States)

    Torma, Ferenc; Bori, Zoltan; Koltai, Erika; Felszeghy, Klara; Vacz, Gabriella; Koch, Lauren; Britton, Steven; Boldogh, Istvan; Radak, Zsolt

    2014-08-01

    Exercise capacity and dietary restriction (DR) are linked to improved quality of life, including enhanced brain function and neuro-protection. Brain derived neurotrophic factor (BDNF) is one of the key proteins involved in the beneficial effects of exercise on brain. Low capacity runner (LCR) and high capacity runner (HCR) rats were subjected to DR in order to investigate the regulation of BDNF. HCR-DR rats out-performed other groups in a passive avoidance test. BDNF content increased significantly in the hippocampus of HCR-DR groups compared to control groups (p<0.05). The acetylation of H3 increased significantly only in the LCR-DR group. However, chip-assay revealed that the specific binding between acetylated histone H3 and BNDF promoter was increased in both LCR-DR and HCR-DR groups. In spite of these increases in binding, at the transcriptional level only, the LCR-DR group showed an increase in BDNF mRNA content. Additionally, DR also induced the activity of cAMP response element-binding protein (CREB), while the content of SIRT1 was not altered. Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) was elevated in HCR-DR groups. But, based on the levels of nuclear respiratory factor-1 and cytocrome c oxidase, it appears that DR did not cause mitochondrial biogenesis. The data suggest that DR-mediated induction of BDNF levels includes chromatin remodeling. Moreover, DR does not induce mitochondrial biogenesis in the hippocampus of LCR/HCR rats. DR results in different responses to a passive avoidance test, and BDNF regulation in LCR and HCR rats. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Essential Assembly Factor Rpf2 Forms Novel Interactions within the 5S RNP in Trypanosoma brucei.

    Science.gov (United States)

    Kamina, Anyango D; Jaremko, Daniel; Christen, Linda; Williams, Noreen

    2017-01-01

    Ribosome biogenesis is a highly complex and conserved cellular process that is responsible for making ribosomes. During this process, there are several assembly steps that function as regulators to ensure proper ribosome formation. One of these steps is the assembly of the 5S ribonucleoprotein particle (5S RNP) in the central protuberance of the 60S ribosomal subunit. In eukaryotes, the 5S RNP is composed of 5S rRNA, ribosomal proteins L5 and L11, and assembly factors Rpf2 and Rrs1. Our laboratory previously showed that in Trypanosoma brucei , the 5S RNP is composed of 5S rRNA, L5, and trypanosome-specific RNA binding proteins P34 and P37. In this study, we characterize an additional component of the 5S RNP, the T. brucei homolog of Rpf2. This is the first study to functionally characterize interactions mediated by Rpf2 in an organism other than fungi. T . brucei Rpf2 (TbRpf2) was identified from tandem affinity purification using extracts prepared from protein A-tobacco etch virus (TEV)-protein C (PTP)-tagged L5, P34, and P37 cell lines, followed by mass spectrometry analysis. We characterized the binding interactions between TbRpf2 and the previously characterized members of the T. brucei 5S RNP. Our studies show that TbRpf2 mediates conserved binding interactions with 5S rRNA and L5 and that TbRpf2 also interacts with trypanosome-specific proteins P34 and P37. We performed RNA interference (RNAi) knockdown of TbRpf2 and showed that this protein is essential for the survival of the parasites and is critical for proper ribosome formation. These studies provide new insights into a critical checkpoint in the ribosome biogenesis pathway in T. brucei . IMPORTANCE Trypanosoma brucei is the parasitic protozoan that causes African sleeping sickness. Ribosome assembly is essential for the survival of this parasite through the different host environments it encounters during its life cycle. The assembly of the 5S ribonucleoprotein particle (5S RNP) functions as one of

  6. Δ(1-pyrroline-5-carboxylate/glutamate biogenesis is required for fungal virulence and sporulation.

    Directory of Open Access Journals (Sweden)

    Ziting Yao

    Full Text Available Proline dehydrogenase (Prodh and Δ(1-pyrroline-5-carboxylate dehydrogenase (P5Cdh are two key enzymes in the cellular biogenesis of glutamate. Recombinant Prodh and P5Cdh proteins of the chestnut blight fungus Cryphonectria parasitica were investigated and showed activity in in vitro assays. Additionally, the C. parasitica Prodh and P5Cdh genes were able to complement the Saccharomyces cerevisiae put1 and put2 null mutants, respectively, to allow these proline auxotrophic yeast mutants to grow on media with proline as the sole source of nitrogen. Deletion of the Prodh gene in C. parasitica resulted in hypovirulence and a lower level of sporulation, whereas deletion of P5Cdh resulted in hypovirulence though no effect on sporulation; both Δprodh and Δp5cdh mutants were unable to grow on minimal medium with proline as the sole nitrogen source. In a wild-type strain, the intracellular level of proline and the activity of Prodh and P5Cdh increased after supplementation of exogenous proline, though the intracellular Δ(1-pyrroline-5-carboxylate (P5C content remained unchanged. Prodh and P5Cdh were both transcriptionally down-regulated in cells infected with hypovirus. The disruption of other genes with products involved in the conversion of arginine to ornithine, ornithine and glutamate to P5C, and P5C to proline in the cytosol did not appear to affect virulence; however, asexual sporulation was reduced in the Δpro1 and Δpro2 mutants. Taken together, our results showed that Prodh, P5Cdh and related mitochondrial functions are essential for virulence and that proline/glutamate pathway components may represent down-stream targets of hypovirus regulation in C. parasitica.

  7. A cyclic nucleotide-gated channel mutation associated with canine daylight blindness provides insight into a role for the S2 segment tri-Asp motif in channel biogenesis.

    Directory of Open Access Journals (Sweden)

    Naoto Tanaka

    Full Text Available Cone cyclic nucleotide-gated channels are tetramers formed by CNGA3 and CNGB3 subunits; CNGA3 subunits function as homotetrameric channels but CNGB3 exhibits channel function only when co-expressed with CNGA3. An aspartatic acid (Asp to asparagine (Asn missense mutation at position 262 in the canine CNGB3 (D262N subunit results in loss of cone function (daylight blindness, suggesting an important role for this aspartic acid residue in channel biogenesis and/or function. Asp 262 is located in a conserved region of the second transmembrane segment containing three Asp residues designated the Tri-Asp motif. This motif is conserved in all CNG channels. Here we examine mutations in canine CNGA3 homomeric channels using a combination of experimental and computational approaches. Mutations of these conserved Asp residues result in the absence of nucleotide-activated currents in heterologous expression. A fluorescent tag on CNGA3 shows mislocalization of mutant channels. Co-expressing CNGB3 Tri-Asp mutants with wild type CNGA3 results in some functional channels, however, their electrophysiological characterization matches the properties of homomeric CNGA3 channels. This failure to record heteromeric currents suggests that Asp/Asn mutations affect heteromeric subunit assembly. A homology model of S1-S6 of the CNGA3 channel was generated and relaxed in a membrane using molecular dynamics simulations. The model predicts that the Tri-Asp motif is involved in non-specific salt bridge pairings with positive residues of S3/S4. We propose that the D262N mutation in dogs with CNGB3-day blindness results in the loss of these inter-helical interactions altering the electrostatic equilibrium within in the S1-S4 bundle. Because residues analogous to Tri-Asp in the voltage-gated Shaker potassium channel family were implicated in monomer folding, we hypothesize that destabilizing these electrostatic interactions impairs the monomer folding state in D262N mutant CNG

  8. MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5.

    Science.gov (United States)

    Nezich, Catherine L; Wang, Chunxin; Fogel, Adam I; Youle, Richard J

    2015-08-03

    The kinase PINK1 and ubiquitin ligase Parkin can regulate the selective elimination of damaged mitochondria through autophagy (mitophagy). Because of the demand on lysosomal function by mitophagy, we investigated a role for the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, in this process. We show that during mitophagy TFEB translocates to the nucleus and displays transcriptional activity in a PINK1- and Parkin-dependent manner. MITF and TFE3, homologues of TFEB belonging to the same microphthalmia/transcription factor E (MiT/TFE) family, are similarly regulated during mitophagy. Unlike TFEB translocation after starvation-induced mammalian target of rapamycin complex 1 inhibition, Parkin-mediated TFEB relocalization required Atg9A and Atg5 activity. However, constitutively active Rag guanosine triphosphatases prevented TFEB translocation during mitophagy, suggesting cross talk between these two MiT/TFE activation pathways. Analysis of clustered regularly interspaced short palindromic repeats-generated TFEB/MITF/TFE3/TFEC single, double, and triple knockout cell lines revealed that these proteins partly facilitate Parkin-mediated mitochondrial clearance. These results illuminate a pathway leading to MiT/TFE transcription factor activation, distinct from starvation-induced autophagy, which occurs during mitophagy.

  9. Enhanced oxidative stress and aberrant mitochondrial biogenesis in human neuroblastoma SH-SY5Y cells during methamphetamine induced apoptosis

    International Nuclear Information System (INIS)

    Wu, C.-W.; Ping, Y.-H.; Yen, J.-C.; Chang, C.-Y.; Wang, S.-F.; Yeh, C.-L.; Chi, C.-W.; Lee, H.-C.

    2007-01-01

    Methamphetamine (METH) is an abused drug that may cause psychiatric and neurotoxic damage, including degeneration of monoaminergic terminals and apoptosis of non-monoaminergic cells in Brain. The cellular and molecular mechanisms underlying these METH-induced neurotoxic effects remain to be clarified. In this study, we performed a time course assessment to investigate the effects of METH on intracellular oxidative stress and mitochondrial alterations in a human dopaminergic neuroblastoma SH-SY5Y cell line. We characterized that METH induces a temporal sequence of several cellular events including, firstly, a decrease in mitochondrial membrane potential within 1 h of the METH treatment, secondly, an extensive decline in mitochondrial membrane potential and increase in the level of reactive oxygen species (ROS) after 8 h of the treatment, thirdly, an increase in mitochondrial mass after the drug treatment for 24 h, and finally, a decrease in mtDNA copy number and mitochondrial proteins per mitochondrion as well as the occurrence of apoptosis after 48 h of the treatment. Importantly, vitamin E attenuated the METH-induced increases in intracellular ROS level and mitochondrial mass, and prevented METH-induced cell death. Our observations suggest that enhanced oxidative stress and aberrant mitochondrial biogenesis may play critical roles in METH-induced neurotoxic effects

  10. Rab GTPases and the Autophagy Pathway: Bacterial Targets for a Suitable Biogenesis and Trafficking of Their Own Vacuoles

    Directory of Open Access Journals (Sweden)

    María Milagros López de Armentia

    2016-03-01

    Full Text Available Autophagy is an intracellular process that comprises degradation of damaged organelles, protein aggregates and intracellular pathogens, having an important role in controlling the fate of invading microorganisms. Intracellular pathogens are internalized by professional and non-professional phagocytes, localizing in compartments called phagosomes. To degrade the internalized microorganism, the microbial phagosome matures by fusion events with early and late endosomal compartments and lysosomes, a process that is regulated by Rab GTPases. Interestingly, in order to survive and replicate in the phagosome, some pathogens employ different strategies to manipulate vesicular traffic, inhibiting phagolysosomal biogenesis (e.g., Staphylococcus aureus and Mycobacterium tuberculosis or surviving in acidic compartments and forming replicative vacuoles (e.g., Coxiella burnetti and Legionella pneumophila. The bacteria described in this review often use secretion systems to control the host’s response and thus disseminate. To date, eight types of secretion systems (Type I to Type VIII are known. Some of these systems are used by bacteria to translocate pathogenic proteins into the host cell and regulate replicative vacuole formation, apoptosis, cytokine responses, and autophagy. Herein, we have focused on how bacteria manipulate small Rab GTPases to control many of these processes. The growing knowledge in this field may facilitate the development of new treatments or contribute to the prevention of these types of bacterial infections.

  11. High-Level Accumulation of Exogenous Small RNAs Not Affecting Endogenous Small RNA Biogenesis and Function in Plants

    Institute of Scientific and Technical Information of China (English)

    SHEN Wan-xia; Neil A Smith; ZHOU Chang-yong; WANG Ming-bo

    2014-01-01

    RNA silencing is a fundamental plant defence and gene control mechanism in plants that are directed by 20-24 nucleotide (nt) small interfering RNA (siRNA) and microRNA (miRNA). Infection of plants with viral pathogens or transformation of plants with RNA interference (RNAi) constructs is usually associated with high levels of exogenous siRNAs, but it is unclear if these siRNAs interfere with endogenous small RNA pathways and hence affect plant development. Here we provide evidence that viral satellite RNA (satRNA) infection does not affect siRNA and miRNA biogenesis or plant growth despite the extremely high level of satRNA-derived siRNAs. We generated transgenic Nicotiana benthamiana plants that no longer develop the speciifc yellowing symptoms generally associated with infection by Cucumber mosaic virus (CMV) Y-satellite RNA (Y-Sat). We then used these plants to show that CMV Y-Sat infection did not cause any visible phenotypic changes in comparison to uninfected plants, despite the presence of high-level Y-Sat siRNAs. Furthermore, we showed that the accumulation of hairpin RNA (hpRNA)-derived siRNAs or miRNAs, and the level of siRNA-directed transgene silencing, are not signiifcantly affected by CMV Y-Sat infection. Taken together, our results suggest that the high levels of exogenous siRNAs associated with viral infection or RNAi-inducing transgenes do not saturate the endogenous RNA silencing machineries and have no signiifcant impact on normal plant development.

  12. Heme Oxygenase-1/Carbon Monoxide System and Embryonic Stem Cell Differentiation and Maturation into Cardiomyocytes

    Science.gov (United States)

    Suliman, Hagir B.; Zobi, Fabio

    2016-01-01

    Abstract Aims: The differentiation of embryonic stem (ES) cells into energetically efficient cardiomyocytes contributes to functional cardiac repair and is envisioned to ameliorate progressive degenerative cardiac diseases. Advanced cell maturation strategies are therefore needed to create abundant mature cardiomyocytes. In this study, we tested whether the redox-sensitive heme oxygenase-1/carbon monoxide (HO-1/CO) system, operating through mitochondrial biogenesis, acts as a mechanism for ES cell differentiation and cardiomyocyte maturation. Results: Manipulation of HO-1/CO to enhance mitochondrial biogenesis demonstrates a direct pathway to ES cell differentiation and maturation into beating cardiomyocytes that express adult structural markers. Targeted HO-1/CO interventions up- and downregulate specific cardiogenic transcription factors, transcription factor Gata4, homeobox protein Nkx-2.5, heart- and neural crest derivatives-expressed protein 1, and MEF2C. HO-1/CO overexpression increases cardiac gene expression for myosin regulatory light chain 2, atrial isoform, MLC2v, ANP, MHC-β, and sarcomere α-actinin and the major mitochondrial fusion regulators, mitofusin 2 and MICOS complex subunit Mic60. This promotes structural mitochondrial network expansion and maturation, thereby supporting energy provision for beating embryoid bodies. These effects are prevented by silencing HO-1 and by mitochondrial reactive oxygen species scavenging, while disruption of mitochondrial biogenesis and mitochondrial DNA depletion by loss of mitochondrial transcription factor A compromise infrastructure. This leads to failure of cardiomyocyte differentiation and maturation and contractile dysfunction. Innovation: The capacity to augment cardiomyogenesis via a defined mitochondrial pathway has unique therapeutic potential for targeting ES cell maturation in cardiac disease. Conclusion: Our findings establish the HO-1/CO system and redox regulation of mitochondrial biogenesis as

  13. Storage of factor VIII variants with impaired von Willebrand factor binding in Weibel-Palade bodies in endothelial cells.

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    Maartje van den Biggelaar

    Full Text Available BACKGROUND: Point mutations resulting in reduced factor VIII (FVIII binding to von Willebrand factor (VWF are an important cause of mild/moderate hemophilia A. Treatment includes desmopressin infusion, which concomitantly increases VWF and FVIII plasma levels, apparently from storage pools containing both proteins. The source of these VWF/FVIII co-storage pools and the mechanism of granule biogenesis are not fully understood. METHODOLOGY/PRINCIPAL FINDINGS: We studied intracellular trafficking of FVIII variants implicated in mild/moderate hemophilia A together with VWF in HEK293 cells and primary endothelial cells. The role of VWF binding was addressed using FVIII variants displaying reduced VWF interaction. Binding studies using purified FVIII proteins revealed moderate (Arg2150His, Del2201, Pro2300Ser to severe (Tyr1680Phe, Ser2119Tyr VWF binding defects. Expression studies in HEK293 cells and primary endothelial cells revealed that all FVIII variants were present within VWF-containing organelles. Quantitative studies showed that the relative amount of FVIII storage was independent of various mutations. Substantial amounts of FVIII variants are co-stored in VWF-containing storage organelles, presumably by virtue of their ability to interact with VWF at low pH. CONCLUSIONS: Our data suggest that the potential of FVIII co-storage with VWF is not affected in mild/moderate hemophilia A caused by reduced FVIII/VWF interaction in the circulation. These data support the hypothesis that Weibel-Palade bodies comprise the desmopressin-releasable FVIII storage pool in vivo.

  14. Low-Intensity Ultrasound-Induced Anti-inflammatory Effects Are Mediated by Several New Mechanisms Including Gene Induction, Immunosuppressor Cell Promotion, and Enhancement of Exosome Biogenesis and Docking

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    Qian Yang

    2017-10-01

    Full Text Available Background: Low-intensity ultrasound (LIUS was shown to be beneficial in mitigating inflammation and facilitating tissue repair in various pathologies. Determination of the molecular mechanisms underlying the anti-inflammatory effects of LIUS allows to optimize this technique as a therapy for the treatment of malignancies and aseptic inflammatory disorders.Methods: We conducted cutting-edge database mining approaches to determine the anti-inflammatory mechanisms exerted by LIUS.Results: Our data revealed following interesting findings: (1 LIUS anti-inflammatory effects are mediated by upregulating anti-inflammatory gene expression; (2 LIUS induces the upregulation of the markers and master regulators of immunosuppressor cells including MDSCs (myeloid-derived suppressor cells, MSCs (mesenchymal stem cells, B1-B cells and Treg (regulatory T cells; (3 LIUS not only can be used as a therapeutic approach to deliver drugs packed in various structures such as nanobeads, nanospheres, polymer microspheres, and lipidosomes, but also can make use of natural membrane vesicles as small as exosomes derived from immunosuppressor cells as a novel mechanism to fulfill its anti-inflammatory effects; (4 LIUS upregulates the expression of extracellular vesicle/exosome biogenesis mediators and docking mediators; (5 Exosome-carried anti-inflammatory cytokines and anti-inflammatory microRNAs inhibit inflammation of target cells via multiple shared and specific pathways, suggesting exosome-mediated anti-inflammatory effect of LIUS feasible; and (6 LIUS-mediated physical effects on tissues may activate specific cellular sensors that activate downstream transcription factors and signaling pathways.Conclusions: Our results have provided novel insights into the mechanisms underlying anti-inflammatory effects of LIUS, and have provided guidance for the development of future novel therapeutic LIUS for cancers, inflammatory disorders, tissue regeneration and tissue repair.

  15. Importance of Campylobacter jejuni FliS and FliW in Flagella Biogenesis and Flagellin Secretion

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    Katarzyna A. Radomska

    2017-06-01

    Full Text Available Flagella-driven motility enables bacteria to reach their favorable niche within the host. The human foodborne pathogen Campylobacter jejuni produces two heavily glycosylated structural flagellins (FlaA and FlaB that form the flagellar filament. It also encodes the non-structural FlaC flagellin which is secreted through the flagellum and has been implicated in host cell invasion. The mechanisms that regulate C. jejuni flagellin biogenesis and guide the proteins to the export apparatus are different from those in most other enteropathogens and are not fully understood. This work demonstrates the importance of the putative flagellar protein FliS in C. jejuni flagella assembly. A constructed fliS knockout strain was non-motile, displayed reduced levels of FlaA/B and FlaC flagellin, and carried severely truncated flagella. Pull-down and Far Western blot assays showed direct interaction of FliS with all three C. jejuni flagellins (FlaA, FlaB, and FlaC. This is in contrast to, the sensor and regulator of intracellular flagellin levels, FliW, which bound to FlaA and FlaB but not to FlaC. The FliS protein but not FliW preferred binding to glycosylated C. jejuni flagellins rather than to their non-glycosylated recombinant counterparts. Mapping of the binding region of FliS and FliW using a set of flagellin fragments showed that the C-terminal subdomain of the flagellin was required for FliS binding, whereas the N-terminal subdomain was essential for FliW binding. The separate binding subdomains required for FliS and FliW, the different substrate specificity, and the differential preference for binding of glycosylated flagellins ensure optimal processing and assembly of the C. jejuni flagellins.

  16. Structural Dynamics Control Allosteric Activation of Cytohesin Family Arf GTPase Exchange Factors

    Energy Technology Data Exchange (ETDEWEB)

    Malaby, Andrew W.; Das, Sanchaita; Chakravarthy, Srinivas; Irving, Thomas C.; Bilsel, Osman; Lambright, David G.

    2018-01-01

    Membrane dynamic processes including vesicle biogenesis depend on Arf guanosine triphosphatase (GTPase) activation by guanine nucleotide exchange factors (GEFs) containing a catalytic Sec7 domain and a membrane-targeting module such as a pleckstrin homology (PH) domain. The catalytic output of cytohesin family Arf GEFs is controlled by autoinhibitory interactions that impede accessibility of the exchange site in the Sec7 domain. These restraints can be relieved through activator Arf-GTP binding to an allosteric site comprising the PH domain and proximal autoinhibitory elements (Sec7-PH linker and C-terminal helix). Small-angle X-ray scattering and negative-stain electron microscopy were used to investigate the structural organization and conformational dynamics of cytohesin-3 (Grp1) in autoinhibited and active states. The results support a model in which hinge dynamics in the autoinhibited state expose the activator site for Arf-GTP binding, while subsequent C-terminal helix unlatching and repositioning unleash conformational entropy in the Sec7-PH linker to drive exposure of the exchange site.

  17. Temporal increase of platelet mitochondrial respiration is negatively associated with clinical outcome in patients with sepsis

    DEFF Research Database (Denmark)

    Sjövall, Fredrik; Morota, Saori; Hansson, Magnus J

    2010-01-01

    Mitochondrial dysfunction has been suggested as a contributing factor to the pathogenesis of sepsis-induced multiple organ failure. Also, restoration of mitochondrial function, known as mitochondrial biogenesis, has been implicated as a key factor for the recovery of organ function in patients wi...

  18. Amla Enhances Mitochondrial Spare Respiratory Capacity by Increasing Mitochondrial Biogenesis and Antioxidant Systems in a Murine Skeletal Muscle Cell Line

    Directory of Open Access Journals (Sweden)

    Hirotaka Yamamoto

    2016-01-01

    Full Text Available Amla is one of the most important plants in Indian traditional medicine and has been shown to improve various age-related disorders while decreasing oxidative stress. Mitochondrial dysfunction is a proposed cause of aging through elevated oxidative stress. In this study, we investigated the effects of Amla on mitochondrial function in C2C12 myotubes, a murine skeletal muscle cell model with abundant mitochondria. Based on cell flux analysis, treatment with an extract of Amla fruit enhanced mitochondrial spare respiratory capacity, which enables cells to overcome various stresses. To further explore the mechanisms underlying these effects on mitochondrial function, we analyzed mitochondrial biogenesis and antioxidant systems, both proposed regulators of mitochondrial spare respiratory capacity. We found that Amla treatment stimulated both systems accompanied by AMPK and Nrf2 activation. Furthermore, we found that Amla treatment exhibited cytoprotective effects and lowered reactive oxygen species (ROS levels in cells subjected to t-BHP-induced oxidative stress. These effects were accompanied by increased oxygen consumption, suggesting that Amla protected cells against oxidative stress by using enhanced spare respiratory capacity to produce more energy. Thus we identified protective effects of Amla, involving activation of mitochondrial function, which potentially explain its various effects on age-related disorders.

  19. Simulated microgravity, Mars gravity, and 2g hypergravity affect cell cycle regulation, ribosome biogenesis, and epigenetics in Arabidopsis cell cultures.

    Science.gov (United States)

    Kamal, Khaled Y; Herranz, Raúl; van Loon, Jack J W A; Medina, F Javier

    2018-04-23

    Gravity is the only component of Earth environment that remained constant throughout the entire process of biological evolution. However, it is still unclear how gravity affects plant growth and development. In this study, an in vitro cell culture of Arabidopsis thaliana was exposed to different altered gravity conditions, namely simulated reduced gravity (simulated microgravity, simulated Mars gravity) and hypergravity (2g), to study changes in cell proliferation, cell growth, and epigenetics. The effects after 3, 14, and 24-hours of exposure were evaluated. The most relevant alterations were found in the 24-hour treatment, being more significant for simulated reduced gravity than hypergravity. Cell proliferation and growth were uncoupled under simulated reduced gravity, similarly, as found in meristematic cells from seedlings grown in real or simulated microgravity. The distribution of cell cycle phases was changed, as well as the levels and gene transcription of the tested cell cycle regulators. Ribosome biogenesis was decreased, according to levels and gene transcription of nucleolar proteins and the number of inactive nucleoli. Furthermore, we found alterations in the epigenetic modifications of chromatin. These results show that altered gravity effects include a serious disturbance of cell proliferation and growth, which are cellular functions essential for normal plant development.

  20. Molecular phylogenetics and comparative modeling of HEN1, a methyltransferase involved in plant microRNA biogenesis

    Directory of Open Access Journals (Sweden)

    Obarska Agnieszka

    2006-01-01

    Full Text Available Abstract Background Recently, HEN1 protein from Arabidopsis thaliana was discovered as an essential enzyme in plant microRNA (miRNA biogenesis. HEN1 transfers a methyl group from S-adenosylmethionine to the 2'-OH or 3'-OH group of the last nucleotide of miRNA/miRNA* duplexes produced by the nuclease Dicer. Previously it was found that HEN1 possesses a Rossmann-fold methyltransferase (RFM domain and a long N-terminal extension including a putative double-stranded RNA-binding motif (DSRM. However, little is known about the details of the structure and the mechanism of action of this enzyme, and about its phylogenetic origin. Results Extensive database searches were carried out to identify orthologs and close paralogs of HEN1. Based on the multiple sequence alignment a phylogenetic tree of the HEN1 family was constructed. The fold-recognition approach was used to identify related methyltransferases with experimentally solved structures and to guide the homology modeling of the HEN1 catalytic domain. Additionally, we identified a La-like predicted RNA binding domain located C-terminally to the DSRM domain and a domain with a peptide prolyl cis/trans isomerase (PPIase fold, but without the conserved PPIase active site, located N-terminally to the catalytic domain. Conclusion The bioinformatics analysis revealed that the catalytic domain of HEN1 is not closely related to any known RNA:2'-OH methyltransferases (e.g. to the RrmJ/fibrillarin superfamily, but rather to small-molecule methyltransferases. The structural model was used as a platform to identify the putative active site and substrate-binding residues of HEN and to propose its mechanism of action.

  1. Biogenesis of the mitochondrial TOM complex: Mim1 promotes insertion and assembly of signal-anchored receptors.

    Science.gov (United States)

    Becker, Thomas; Pfannschmidt, Sylvia; Guiard, Bernard; Stojanovski, Diana; Milenkovic, Dusanka; Kutik, Stephan; Pfanner, Nikolaus; Meisinger, Chris; Wiedemann, Nils

    2008-01-04

    The translocase of the outer membrane (TOM complex) is the central entry gate for nuclear-encoded mitochondrial precursor proteins. All Tom proteins are also encoded by nuclear genes and synthesized as precursors in the cytosol. The channel-forming beta-barrel protein Tom40 is targeted to mitochondria via Tom receptors and inserted into the outer membrane by the sorting and assembly machinery (SAM complex). A further outer membrane protein, Mim1, plays a less defined role in assembly of Tom40 into the TOM complex. The three receptors Tom20, Tom22, and Tom70 are anchored in the outer membrane by a single transmembrane alpha-helix, located at the N terminus in the case of Tom20 and Tom70 (signal-anchored) or in the C-terminal portion in the case of Tom22 (tail-anchored). Insertion of the precursor of Tom22 into the outer membrane requires pre-existing Tom receptors while the import pathway of the precursors of Tom20 and Tom70 is only poorly understood. We report that Mim1 is required for efficient membrane insertion and assembly of Tom20 and Tom70, but not Tom22. We show that Mim1 associates with SAM(core) components to a large SAM complex, explaining its role in late steps of the assembly pathway of Tom40. We conclude that Mim1 is not only required for biogenesis of the beta-barrel protein Tom40 but also for membrane insertion and assembly of signal-anchored Tom receptors. Thus, Mim1 plays an important role in the efficient assembly of the mitochondrial TOM complex.

  2. The mammalian phosphate carrier SLC25A3 is a mitochondrial copper transporter required for cytochrome c oxidase biogenesis.

    Science.gov (United States)

    Boulet, Aren; Vest, Katherine E; Maynard, Margaret K; Gammon, Micah G; Russell, Antoinette C; Mathews, Alexander T; Cole, Shelbie E; Zhu, Xinyu; Phillips, Casey B; Kwong, Jennifer Q; Dodani, Sheel C; Leary, Scot C; Cobine, Paul A

    2018-02-09

    Copper is required for the activity of cytochrome c oxidase (COX), the terminal electron-accepting complex of the mitochondrial respiratory chain. The likely source of copper used for COX biogenesis is a labile pool found in the mitochondrial matrix. In mammals, the proteins that transport copper across the inner mitochondrial membrane remain unknown. We previously reported that the mitochondrial carrier family protein Pic2 in budding yeast is a copper importer. The closest Pic2 ortholog in mammalian cells is the mitochondrial phosphate carrier SLC25A3. Here, to investigate whether SLC25A3 also transports copper, we manipulated its expression in several murine and human cell lines. SLC25A3 knockdown or deletion consistently resulted in an isolated COX deficiency in these cells, and copper addition to the culture medium suppressed these biochemical defects. Consistent with a conserved role for SLC25A3 in copper transport, its heterologous expression in yeast complemented copper-specific defects observed upon deletion of PIC2 Additionally, assays in Lactococcus lactis and in reconstituted liposomes directly demonstrated that SLC25A3 functions as a copper transporter. Taken together, these data indicate that SLC25A3 can transport copper both in vitro and in vivo . © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Effect of limb demand ischemia on autophagy and morphology in mice.

    Science.gov (United States)

    Albadawi, Hassan; Oklu, Rahmi; Milner, John D; Uong, Thuy P; Yoo, Hyung-Jin; Austen, William G; Watkins, Michael T

    2015-10-01

    Obesity is a major risk factor for diabetes and peripheral arterial disease, which frequently leads to lower limb demand ischemia. Skeletal muscle autophagy and mitochondrial biogenesis are important processes for proper oxidative capacity and energy metabolism, which are compromised in diabetes. This study compares autophagy, mitochondrial biogenesis, energy metabolism, and morphology in the hind limbs of obese diabetic mice subjected to demand or sedentary ischemia. Unilateral hind limb demand ischemia was created in a group of diet-induced obese mice after femoral artery ligation and 4 wk of daily exercise. A parallel group of mice underwent femoral artery ligation but remained sedentary for 4 wk. Hind limb muscles were analyzed for markers of autophagy, mitochondrial biogenesis, adenosine triphosphate, and muscle tissue morphology. At the end of the 4-wk exercise period, demand ischemia increased the autophagy mediator Beclin-1, but it did not alter the autophagy indicator, LC3B-II/I ratio, or markers of mitochondrial biogenesis, optic atrophy/dynamin-related protein. In contrast, exercise significantly increased the level of mitochondrial protein-succinate dehydrogenase subunit-A and reduced adipocyte accumulation and the percentage of centrally nucleated myofibers in the demand ischemia limb. In addition, demand ischemia resulted in decreased uncoupling protein-3 levels without altering muscle adenosine triphosphate or pS473-Akt levels. Limb demand ischemia markedly decreased adipocyte accumulation and enhanced muscle regeneration in obese mice, but it did not appear to enhance autophagy, mitochondrial biogenesis, energy metabolism, or insulin sensitivity. Future studies aimed at evaluating novel therapies that enhance autophagy and mitochondrial biogenesis in diabetes with peripheral arterial disease are warranted. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Pivotal Role of O-GlcNAc Modification in Cold-Induced Thermogenesis by Brown Adipose Tissue Through Mitochondrial Biogenesis.

    Science.gov (United States)

    Ohashi, Natsuko; Morino, Katsutaro; Ida, Shogo; Sekine, Osamu; Lemecha, Mengistu; Kume, Shinji; Park, Shi-Young; Choi, Cheol Soo; Ugi, Satoshi; Maegawa, Hiroshi

    2017-09-01

    Adipose tissues considerably influence metabolic homeostasis, and both white (WAT) and brown (BAT) adipose tissue play significant roles in lipid and glucose metabolism. O -linked N -acetylglucosamine ( O -GlcNAc) modification is characterized by the addition of N -acetylglucosamine to various proteins by O -GlcNAc transferase (Ogt), subsequently modulating various cellular processes. However, little is known about the role of O -GlcNAc modification in adipose tissues. Here, we report the critical role of O -GlcNAc modification in cold-induced thermogenesis. Deletion of Ogt in WAT and BAT using adiponectin promoter-driven Cre recombinase resulted in severe cold intolerance with decreased uncoupling protein 1 (Ucp1) expression. Furthermore, Ogt deletion led to decreased mitochondrial protein expression in conjunction with decreased peroxisome proliferator-activated receptor γ coactivator 1-α protein expression. This phenotype was further confirmed by deletion of Ogt in BAT using Ucp1 promoter-driven Cre recombinase, suggesting that O -GlcNAc modification in BAT is responsible for cold-induced thermogenesis. Hypothermia was significant under fasting conditions. This effect was mitigated after normal diet consumption but not after consumption of a fatty acid-rich ketogenic diet lacking carbohydrates, suggesting impaired diet-induced thermogenesis, particularly by fat. In conclusion, O -GlcNAc modification is essential for cold-induced thermogenesis and mitochondrial biogenesis in BAT. Glucose flux into BAT may be a signal to maintain BAT physiological responses. © 2017 by the American Diabetes Association.

  5. Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription.

    Science.gov (United States)

    Xie, Qiu; Li, Caihua; Song, Xiaozhen; Wu, Lihua; Jiang, Qian; Qiu, Zhiyong; Cao, Haiyan; Yu, Kaihui; Wan, Chunlei; Li, Jianting; Yang, Feng; Huang, Zebing; Niu, Bo; Jiang, Zhengwen; Zhang, Ting

    2017-03-17

    The biogenesis of ribosomes in vivo is an essential process for cellular functions. Transcription of ribosomal RNA (rRNA) genes is the rate-limiting step in ribosome biogenesis controlled by environmental conditions. Here, we investigated the role of folate antagonist on changes of DNA double-strand breaks (DSBs) landscape in mouse embryonic stem cells. A significant DSB enhancement was detected in the genome of these cells and a large majority of these DSBs were found in rRNA genes. Furthermore, spontaneous DSBs in cells under folate deficiency conditions were located exclusively within the rRNA gene units, representing a H3K4me1 hallmark. Enrichment H3K4me1 at the hot spots of DSB regions enhanced the recruitment of upstream binding factor (UBF) to rRNA genes, resulting in the increment of rRNA genes transcription. Supplement of folate resulted in a restored UBF binding across DNA breakage sites of rRNA genes, and normal rRNA gene transcription. In samples from neural tube defects (NTDs) with low folate level, up-regulation of rRNA gene transcription was observed, along with aberrant UBF level. Our results present a new view by which alterations in folate levels affects DNA breakage through epigenetic control leading to the regulation of rRNA gene transcription during the early stage of development. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. An Ancient Transcription Factor Initiates the Burst of piRNA Production During Early Meiosis in Mouse Testes

    Science.gov (United States)

    Li, Xin Zhiguo; Roy, Christian K.; Dong, Xianjun; Bolcun-Filas, Ewelina; Wang, Jie; Han, Bo W.; Xu, Jia; Moore, Melissa J.; Schimenti, John C.; Weng, Zhiping; Zamore, Phillip D.

    2013-01-01

    SUMMARY Animal germ cells produce PIWI-interacting RNAs (piRNAs), small silencing RNAs that suppress transposons and enable gamete maturation. Mammalian transposon-silencing piRNAs accumulate early in spermatogenesis, whereas pachytene piRNAs are produced later during post-natal spermatogenesis and account for >95% of all piRNAs in the adult mouse testis. Mutants defective for pachytene piRNA pathway proteins fail to produce mature sperm, but neither the piRNA precursor transcripts nor the trigger for pachytene piRNA production is known. Here, we show that the transcription factor A-MYB initiates pachytene piRNA production. A-MYB drives transcription of both pachytene piRNA precursor RNAs and the mRNAs for core piRNA biogenesis factors, including MIWI, the protein through which pachytene piRNAs function. A-MYB regulation of piRNA pathway proteins and piRNA genes creates a coherent feed-forward loop that ensures the robust accumulation of pachytene piRNAs. This regulatory circuit, which can be detected in rooster testes, likely predates the divergence of birds and mammals. PMID:23523368

  7. The pre-rRNA processing factor DEF is rate limiting for the pathogenesis of MYCN-driven neuroblastoma.

    Science.gov (United States)

    Tao, T; Sondalle, S B; Shi, H; Zhu, S; Perez-Atayde, A R; Peng, J; Baserga, S J; Look, A T

    2017-07-06

    The nucleolar factor, digestive organ expansion factor (DEF), has a key role in ribosome biogenesis, functioning in pre-ribosomal RNA (pre-rRNA) processing as a component of the small ribosomal subunit (SSU) processome. Here we show that the peripheral sympathetic nervous system (PSNS) is very underdeveloped in def-deficient zebrafish, and that def haploinsufficiency significantly decreases disease penetrance and tumor growth rate in a MYCN-driven transgenic zebrafish model of neuroblastoma that arises in the PSNS. Consistent with these findings, DEF is highly expressed in human neuroblastoma, and its depletion in human neuroblastoma cell lines induces apoptosis. Interestingly, overexpression of MYCN in zebrafish and in human neuroblastoma cells results in the appearance of intermediate pre-rRNAs species that reflect the processing of pre-rRNAs through Pathway 2, a pathway that processes pre-rRNAs in a different temporal order than the more often used Pathway 1. Our results indicate that DEF and possibly other components of the SSU processome provide a novel site of vulnerability in neuroblastoma cells that could be exploited for targeted therapy.

  8. CONTROLE DE MELOIDOGYNE JAVANICA COM PASTEURIA PENETRANS CONTROL OF MELOIDOGYNE JAVANICA BY PASTEURIA PENETRANS

    Directory of Open Access Journals (Sweden)

    RAVI DATT SHARMA

    1999-11-01

    Full Text Available Objetivou-se, com esse trabalho, avaliar a eficiência de Pasteuria penetrans no controle de Meloidogyne javanica em condições de casa de vegetação. Os tratamentos eram compostos de quatro níveis de inóculo de P. penetrans, 0, 10x10(5, 50x10(5 e 100x10(5 endósporos/kg de solo autoclavado. Imediatamente após a inoculação da bactéria P. penetrans no solo autoclavado, 1.000 juvenis de segundo estádio de M. javanica foram inoculados em cada vaso. Quarenta e oito horas após a inoculação do nematóide, uma plântula de soja cv. FT-Cristalina, com três dias de idade, foi transplantada para cada vaso. O experimento foi avaliado em duas etapas: a primeira, 89 dias após o transplantio da soja, e a segunda, 90 dias após um segundo(plantio de soja, em seqüência a um pousio de 30 dias. Na primeira avaliação, o maior peso da matéria fresca da planta foi obtido no tratamento com 100x10(5 endósporos/kg de solo, o que diferiu significativamente (PIn a greenhouse experiment, the efficiency of Pasteuria penetrans against Meloidogyne javanica was evaluated on soybean cv. FT-Cristalina using four inoculum levels of P. penetrans viz: 0, 10x10(5, 50x10(5 and 100x10(5 endospores/kg of soil. Immediately after inoculating the autoclaved soil with P. penetrans, 1,000 second-stage juveniles were inoculated in each pot. After 48 hours of nematode inoculation, a 3-day old soybean seedling was transplanted in each pot. The experiment was evaluated in two steps of which the first evaluation was made after 89 days of transplanting the seedlings; and the second after 90 days of soybean sowing in sequence with a following period of 30 days. In the first evaluation, the highest fresh plant weight was observed in treatment with 100x10(5 endospores/kg of soil which differed significantly (P<0.05 from other treatments except the untreated control. The maximum increase in fresh pod weight of treatment with 100x10(5 endospores/kg of soil differed significantly

  9. Life Cycle, Ultrastructure, and Host Specificity of the North American Isolate of Pasteuria that Parasitizes the Soybean Cyst Nematode, Heterodera glycines.

    Science.gov (United States)

    Atibalentja, N; Jakstys, B P; Noel, G R

    2004-06-01

    Light and transmission electron microscopy were used to investigate the life cycle and ultrastructure of an undescribed isolate of Pasteuria that parasitizes the soybean cyst nematode, Heterodera glycines. Studies also were conducted to determine the host specificity of Pasteuria. The endospores that attached to the cuticle of second-stage juveniles (J2) of H. glycines in soil did not germinate until the encumbered nematodes invaded soybean roots. Thereafter, the bacterium developed and completed its life cycle only in females. The stages of endosporogenesis were typical of Pasteuria spp. The mature endospore, like that of P. nishizawae, the only other Pasteuria known to infect H. glycines, produces an epicortical layer that completely surrounds the cortex, an outer spore coat that tapers progressively from the top to the base of the central body, and a double basal adhesion layer. However, subtle differences exist between the Pasteuria from North America and P. nishizawae with regard to size of the central body, nature and function of the mesosomes observed in the earlier stages of endosporogenesis, and appearance of the fibers lining the basal adhesion layer and the exosporium of the mature endospore. Endospores of the North American Pasteuria attached to J2 of H. schachtii, H. trifolii, and H. lespedezae but not to Meloidogyne arenaria race 1, Tylenchorhynchus nudus, and Labronema sp. Results from this study indicate that the North American Pasteuria is more similar to P. nishizawae than to any other known member of the genus. Additional evidence from comparative analysis of 16S rDNA sequences is needed to clarify whether these two Pasteuria belong to the same species.

  10. Occurrence, hosts, morphology, and molecular characterisation of Pasteuria bacteria parasitic in nematodes of the family Plectidae.

    Science.gov (United States)

    Sturhan, Dieter; Shutova, Tatyana S; Akimov, Vladimir N; Subbotin, Sergei A

    2005-01-01

    Parasitic bacteria of the genus Pasteuria are reported for three Anaplectus and four identified and several unidentified Plectus species found in eight countries in various habitats. The pasteurias from plectids agree in essential morphological characters of sporangia and endospores as well as in developmental cycle with those of the Pasteuria species and strains described from tylenchid nematodes, but appear to be mainly distinguished from these by absence of a distinct perisporium in the spores and the endospores obviously not being cup- or saucer-shaped. The wide range of measurements and morphological peculiarities of sporangia and endospores suggest that probably several Pasteuria species have to be distinguished as parasites in Plectidae. From an infected juvenile of an unidentified plectid species the 16S rRNA gene sequence of Pasteuria sp. was obtained. Substantial sequence divergence from described Pasteuria species and its phylogenetic position on molecular trees indicate that this Pasteuria sp. could be considered as a new species. Preliminary results of the analysis of DNA phylogeny of Pasteuria spp. and their nematode hosts provide evidence for incongruence of their phylogenetic history and of host switching events during evolution of the bacterial parasites.

  11. Detection of Pasteuria penetrans infection in Meloidogyne arenaria race 1 in planta by polymerase chain reaction.

    Science.gov (United States)

    Schmidt, L M; Preston, J F; Nong, G; Dickson, D W; Aldrich, H C

    2004-06-01

    We report on the development of a PCR-based assay to detect Pasteuria penetrans infection of Meloidogyne arenaria in planta using specific primers for recently sequenced sigE, spoIIAB and atpF genes of P. penetrans biotype P20. Amplification of these genes in crude DNA extracts of ground tomato root galls using real-time kinetic PCR distinguished infected from uninfected M. arenaria race 1 by analysis of consensus thresholds for single copy genes. Fluorescent in situ hybridization (FISH) using the sigE primer sequence as a probe shows hybridization to P. penetrans cells in various stages of vegetative (pre-endospore) development. Ratios of gene copies for sigE and 16S rDNA were obtained for P. penetrans and compared to Bacillus subtilis as a genomic paradigm of endospore-forming bacteria. Phylogenetic analysis of the sigE gene from Gram-positive, endospore-forming bacteria finds P. penetrans most closely related Paenbacillus polymyxa. The sporulation genes (spo genes), particularly sigE, have sequence diversity that recommends them for species and biotype differentiation of the numerous Pasteuria isolates that infect a large number of plant-parasitic nematodes.

  12. Blocking miRNA Biogenesis in Adult Forebrain Neurons Enhances Seizure Susceptibility, Fear Memory, and Food Intake by Increasing Neuronal Responsiveness.

    Science.gov (United States)

    Fiorenza, Anna; Lopez-Atalaya, Jose P; Rovira, Victor; Scandaglia, Marilyn; Geijo-Barrientos, Emilio; Barco, Angel

    2016-04-01

    The RNase Dicer is essential for the maturation of most microRNAs, a molecular system that plays an essential role in fine-tuning gene expression. To gain molecular insight into the role of Dicer and the microRNA system in brain function, we conducted 2 complementary RNA-seq screens in the hippocampus of inducible forebrain-restricted Dicer1 mutants aimed at identifying the microRNAs primarily affected by Dicer loss and their targets, respectively. Functional genomics analyses predicted the main biological processes and phenotypes associated with impaired microRNA maturation, including categories related to microRNA biology, signal transduction, seizures, and synaptic transmission and plasticity. Consistent with these predictions, we found that, soon after recombination, Dicer-deficient mice exhibited an exaggerated seizure response, enhanced induction of immediate early genes in response to different stimuli, stronger and more stable fear memory, hyperphagia, and increased excitability of CA1 pyramidal neurons. In the long term, we also observed slow and progressive excitotoxic neurodegeneration. Overall, our results indicate that interfering with microRNA biogenesis causes an increase in neuronal responsiveness and disrupts homeostatic mechanisms that protect the neuron against overactivation, which may explain both the initial and late phenotypes associated with the loss of Dicer in excitatory neurons. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  13. eNOS se correlaciona com a biogênese mitocondrial em corações com cardiopatia congênita e cianose eNOS correlates with mitochondrial biogenesis in hearts of congenital heart disease with cyanosis

    Directory of Open Access Journals (Sweden)

    Juan Xiao

    2012-09-01

    cardiopatia congênita com cianose, que poderia ser a resposta adaptativa à hipóxia crônica e possivelmente envolve suprarregulação da eNOS. (Arq Bras Cardiol. 2012; [online].ahead print, PP.0-0BACKGROUND: Mitochondrial biogenesis program in heart appears to exhibit adaptive remodeling following biomechanical and oxidative stress. The adaptive mechanisms that protect myocardium metabolism during hypoxia are coordinated in part by nitric oxide (NO. OBJECTIVE: To observe mitochondrial biogenesis and nitric oxide synthase (NOS expression in hearts of congenital heart disease with cyanosis, discuss mitochondrial response to chronic hypoxia in myocardium. METHODS: 20 patients with cyanotic (n=10 or acyanotic cardiac defects (n=10 were investigated. Samples from the right ventricular outflow tract myocardium taken during operation were studied. Morphometric analysis of mitochondria was performed with transmission electron microscope. Relative mtDNA/nDNA ratio was determined with real-time PCR. Cytochrome c oxidase subunit I (COXI, peroxisome-proliferator-activated receptor γ coactivator-1α (PGC-1α, nuclear respiratory factor 1 (NRF1, and mitochondrial transcription factor A (Tfam transcript levels were detected by real-time fluorescent RT-PCR. COXI and nNOS, iNOS and eNOS protein levels were measured with western blot. RESULTS: Mitochondrial volume density (Vv and numerical density (Nv were significantly elevated in patients with cyanotic compared to acyanotic congenital heart disease. Elevated mtDNA and up-regulated COXI, PGC-1α, NRF1 and Tfam mRNA levels were observed in cyanotic patients. Protein levels of COXI and eNOS were significantly higher in the myocardium of cyanotic than of acyanotic patients. PGC-1α transcript levels correlated with the levels of eNOS. Conclusion: Mitochondrial biogenesis is activated in right ventricular outflow tract myocardium in congenital heart disease with cyanosis, which could be the adaptive response to chronic hypoxia and possibly

  14. The importance of ribosome production, and the 5S RNP-MDM2 pathway, in health and disease.

    Science.gov (United States)

    Pelava, Andria; Schneider, Claudia; Watkins, Nicholas J

    2016-08-15

    Ribosomes are abundant, large RNA-protein complexes that are the source of all protein synthesis in the cell. The production of ribosomes is an extremely energetically expensive cellular process that has long been linked to human health and disease. More recently, it has been shown that ribosome biogenesis is intimately linked to multiple cellular signalling pathways and that defects in ribosome production can lead to a wide variety of human diseases. Furthermore, changes in ribosome production in response to nutrient levels in the diet lead to metabolic re-programming of the liver. Reduced or abnormal ribosome production in response to cellular stress or mutations in genes encoding factors critical for ribosome biogenesis causes the activation of the tumour suppressor p53, which leads to re-programming of cellular transcription. The ribosomal assembly intermediate 5S RNP (ribonucleoprotein particle), containing RPL5, RPL11 and the 5S rRNA, accumulates when ribosome biogenesis is blocked. The excess 5S RNP binds to murine double minute 2 (MDM2), the main p53-suppressor in the cell, inhibiting its function and leading to p53 activation. Here, we discuss the involvement of ribosome biogenesis in the homoeostasis of p53 in the cell and in human health and disease. © 2016 The Author(s).

  15. Genes Important for Catalase Activity in Enterococcus faecalis

    Science.gov (United States)

    Baureder, Michael; Hederstedt, Lars

    2012-01-01

    Little in general is known about how heme proteins are assembled from their constituents in cells. The Gram-positive bacterium Enterococcus faecalis cannot synthesize heme and does not depend on it for growth. However, when supplied with heme in the growth medium the cells can synthesize two heme proteins; catalase (KatA) and cytochrome bd (CydAB). To identify novel factors important for catalase biogenesis libraries of E. faecalis gene insertion mutants were generated using two different types of transposons. The libraries of mutants were screened for clones deficient in catalase activity using a colony zymogram staining procedure. Analysis of obtained clones identified, in addition to katA (encoding the catalase enzyme protein), nine genes distributed over five different chromosomal loci. No factors with a dedicated essential role in catalase biogenesis or heme trafficking were revealed, but the results indicate the RNA degradosome (srmB, rnjA), an ABC-type oligopeptide transporter (oppBC), a two-component signal transducer (etaR), and NADH peroxidase (npr) as being important for expression of catalase activity in E. faecalis. It is demonstrated that catalase biogenesis in E. faecalis is independent of the CydABCD proteins and that a conserved proline residue in the N-terminal region of KatA is important for catalase assembly. PMID:22590595

  16. Nucleolar Proteome Analysis and Proteasomal Activity Assays Reveal a Link between Nucleolus and 26S Proteasome in A. thaliana

    Science.gov (United States)

    Montacié, Charlotte; Durut, Nathalie; Opsomer, Alison; Palm, Denise; Comella, Pascale; Picart, Claire; Carpentier, Marie-Christine; Pontvianne, Frederic; Carapito, Christine; Schleiff, Enrico; Sáez-Vásquez, Julio

    2017-01-01

    In all eukaryotic cells, the nucleolus is functionally and structurally linked to rRNA synthesis and ribosome biogenesis. This compartment contains as well factors involved in other cellular activities, but the functional interconnection between non-ribosomal activities and the nucleolus (structure and function) still remains an open question. Here, we report a novel mass spectrometry analysis of isolated nucleoli from Arabidopsis thaliana plants using the FANoS (Fluorescence Assisted Nucleolus Sorting) strategy. We identified many ribosome biogenesis factors (RBF) and proteins non-related with ribosome biogenesis, in agreement with the recognized multi-functionality of the nucleolus. Interestingly, we found that 26S proteasome subunits localize in the nucleolus and demonstrated that proteasome activity and nucleolus organization are intimately linked to each other. Proteasome subunits form discrete foci in the disorganized nucleolus of nuc1.2 plants. Nuc1.2 protein extracts display reduced proteasome activity in vitro compared to WT protein extracts. Remarkably, proteasome activity in nuc1.2 is similar to proteasome activity in WT plants treated with proteasome inhibitors (MG132 or ALLN). Finally, we show that MG132 treatment induces disruption of nucleolar structures in WT but not in nuc1.2 plants. Altogether, our data suggest a functional interconnection between nucleolus structure and proteasome activity. PMID:29104584

  17. Fibrillarin from Archaea to human

    Czech Academy of Sciences Publication Activity Database

    Rodriguez-Corona, U.; Sobol, Margaryta; Rodriguez-Zapata, L.C.; Hozák, Pavel; Castano, E.

    2015-01-01

    Roč. 107, č. 6 (2015), s. 159-174 ISSN 0248-4900 Institutional support: RVO:68378050 Keywords : Cancer * Methylation * p53 * Ribosomal biogenesis * RNA processing Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.552, year: 2015

  18. A new General Purpose Decontamination System for Chemical and Biological Warfare and Terrorism Agents

    National Research Council Canada - National Science Library

    Khetan, Sushil; Banerjee, xdDeboshri; Chanda, Arani; Collins, Terry

    2003-01-01

    Partial contents: Fe-TAML Activator of Peroxide,Activators of Hydrogen peroxide,Biological Warfare Agents,Bacterial Endospore,Bacterial Spore Deactivation,Modeling Studies,Deactivation Studies with Bacillus spores...

  19. mtDNA as a Mediator for Expression of Hypoxia-Inducible Factor 1α and ROS in Hypoxic Neuroblastoma Cells.

    Science.gov (United States)

    Kuo, Chung-Wen; Tsai, Meng-Han; Lin, Tsu-Kung; Tiao, Mao-Meng; Wang, Pei-Wen; Chuang, Jiin-Haur; Chen, Shang-Der; Liou, Chia-Wei

    2017-06-07

    Mitochondria consume O₂ to produce ATP and are critical for adaption of hypoxia, but the role of mitochondria in HIF-1α pathway is as yet unclear. In this study, mitochondrial DNA (mtDNA) enriched (SK-N-AS) and depleted (ρ⁰) cells of neuroblastoma were cultured in a hypoxic chamber to simulate a hypoxic condition and then the major components involved in mitochondrial related pathways, hypoxia-inducible factor 1α (HIF-1α) and reactive oxygen species (ROS) were measured. The results showed that hypoxia-stimulated exposure elevated expression of HIF-1α, which was additionally influenced by level of generated ROS within the cytosol. Moreover, elevation of HIF-1α also resulted in increases of lactate dehydrogenase A (LDH-A) and pyruvate dehydrogenase kinase 1 (PDK1) in both hypoxic cells. The expression of mitochondrial biogenesis related proteins and metabolic components were noted to increase significantly in hypoxic SK-N-AS cells, indicating that mtDNA was involved in mitochondrial retrograde signaling and metabolic pathways. An analysis of dynamic proteins found elevated levels of HIF-1α causing an increased expression of dynamin-related protein 1 (DRP1) during hypoxia; further, the existence of mtDNA also resulted in higher expression of DRP1 during hypoxia. By using siRNA of HIF-1α or DRP1, expression of DRP1 decreased after suppression of HIF-1α; moreover, the expression of HIF-1α was also affected by the suppression of DRP1. In this study, we demonstrated that mtDNA is a mediator of HIF-1α in eliciting metabolic reprogramming, and mitochondrial biogenesis. Identification of a mutual relationship between HIF-1α and DRP1 may be a critical tool in the future development of clinical applications.

  20. Mutational analysis of the glycosylphosphatidylinositol (GPI) anchor pathway demonstrates that GPI-anchored proteins are required for cell wall biogenesis and normal hyphal growth in Neurospora crassa.

    Science.gov (United States)

    Bowman, Shaun M; Piwowar, Amy; Al Dabbous, Mash'el; Vierula, John; Free, Stephen J

    2006-03-01

    Using mutational and proteomic approaches, we have demonstrated the importance of the glycosylphosphatidylinositol (GPI) anchor pathway for cell wall synthesis and integrity and for the overall morphology of the filamentous fungus Neurospora crassa. Mutants affected in the gpig-1, gpip-1, gpip-2, gpip-3, and gpit-1 genes, which encode components of the N. crassa GPI anchor biosynthetic pathway, have been characterized. GPI anchor mutants exhibit colonial morphologies, significantly reduced rates of growth, altered hyphal growth patterns, considerable cellular lysis, and an abnormal "cell-within-a-cell" phenotype. The mutants are deficient in the production of GPI-anchored proteins, verifying the requirement of each altered gene for the process of GPI-anchoring. The mutant cell walls are abnormally weak, contain reduced amounts of protein, and have an altered carbohydrate composition. The mutant cell walls lack a number of GPI-anchored proteins, putatively involved in cell wall biogenesis and remodeling. From these studies, we conclude that the GPI anchor pathway is critical for proper cell wall structure and function in N. crassa.

  1. Understanding the interaction between an obligate hyperparasitic bacterium, Pasteuria penetrans and its obligate plant-parasitic nematode host, Meloidogyne spp.

    Science.gov (United States)

    Davies, Keith G

    2009-01-01

    Pasteuria penetrans is an endospore-forming bacterium, which is a hyperparasite of root-knot nematodes Meloidogyne spp. that are economically important pests of a wide range of crops. The life cycle of the bacterium and nematode are described with emphasis on the bacterium's potential as a biocontrol agent. Two aspects that currently prohibit the commercial development of the bacterium as a biocontrol agent are the inability to culture it outside its host and its host specificity. Vegetative growth of the bacterium is possible in vitro; however, getting the vegetative stages of the bacterium to enter sporogenesis has been problematic. Insights from genomic survey sequences regarding the role of cation concentration and the phosphorylation of Spo0F have proved useful in inducing vegetative bacteria to sporulate. Similarly, genomic data have also proved useful in understanding the attachment of endospores to the cuticle of infective nematode juveniles, and a Velcro-like model of spore attachment is proposed that involves collagen-like fibres on the surface of the endospore interacting with mucins on the nematode cuticle. Ecological studies of the interactions between Daphnia and Pasteuria ramosa are examined and similarities are drawn between the co-evolution of virulence in the Daphnia system and that of plant-parasitic nematodes.

  2. Isolation and characterization of differentially expressed genes in ...

    African Journals Online (AJOL)

    Among them, six proteins (putative fatty acid oxygenase, heat shock sks2, PriA homologue, Ap-1 like transcription factor YAP7, mung bean seed albumin, and C2H2 Zinc finger domain protein) and one protein (peroxisomal biogenesis factor 6) showed increased expression levels at the fruiting process and the mycelial ...

  3. PICK1 deficiency impairs secretory vesicle biogenesis and leads to growth retardation and decreased glucose tolerance.

    Directory of Open Access Journals (Sweden)

    Birgitte Holst

    Full Text Available Secretory vesicles in endocrine cells store hormones such as growth hormone (GH and insulin before their release into the bloodstream. The molecular mechanisms governing budding of immature secretory vesicles from the trans-Golgi network (TGN and their subsequent maturation remain unclear. Here, we identify the lipid binding BAR (Bin/amphiphysin/Rvs domain protein PICK1 (protein interacting with C kinase 1 as a key component early in the biogenesis of secretory vesicles in GH-producing cells. Both PICK1-deficient Drosophila and mice displayed somatic growth retardation. Growth retardation was rescued in flies by reintroducing PICK1 in neurosecretory cells producing somatotropic peptides. PICK1-deficient mice were characterized by decreased body weight and length, increased fat accumulation, impaired GH secretion, and decreased storage of GH in the pituitary. Decreased GH storage was supported by electron microscopy showing prominent reduction in secretory vesicle number. Evidence was also obtained for impaired insulin secretion associated with decreased glucose tolerance. PICK1 localized in cells to immature secretory vesicles, and the PICK1 BAR domain was shown by live imaging to associate with vesicles budding from the TGN and to possess membrane-sculpting properties in vitro. In mouse pituitary, PICK1 co-localized with the BAR domain protein ICA69, and PICK1 deficiency abolished ICA69 protein expression. In the Drosophila brain, PICK1 and ICA69 co-immunoprecipitated and showed mutually dependent expression. Finally, both in a Drosophila model of type 2 diabetes and in high-fat-diet-induced obese mice, we observed up-regulation of PICK1 mRNA expression. Our findings suggest that PICK1, together with ICA69, is critical during budding of immature secretory vesicles from the TGN and thus for vesicular storage of GH and possibly other hormones. The data link two BAR domain proteins to membrane remodeling processes in the secretory pathway of

  4. Apoptosis inducing factor (AIF) mediates lethal redox stress induced by menadione.

    Science.gov (United States)

    Wiraswati, Hesti Lina; Hangen, Emilie; Sanz, Ana Belén; Lam, Ngoc-Vy; Reinhardt, Camille; Sauvat, Allan; Mogha, Ariane; Ortiz, Alberto; Kroemer, Guido; Modjtahedi, Nazanine

    2016-11-22

    Mitochondrial apoptosis inducing factor (AIF) is a redox-active enzyme that participates to the biogenesis/maintenance of complex I of the respiratory chain, yet also contributes to catabolic reactions in the context of regulated cell death when AIF translocates to the cytosol and to the nucleus. Here we explore the contribution of AIF to cell death induced by menadione (2-methyl-1,4-naphtoquinone; also called vitamin K3) in conditions in which this pro-oxidant does not cause the mitochondrial release of AIF, yet causes caspase-independent cell killing. Depletion of AIF from human cancer cells reduced the cytotoxicity of menadione. This cytoprotective effect was accompanied by the maintenance of high levels of reduced glutathione (GSH), which are normally depleted by menadione. In addition, AIF depletion reduced the arylation of cellular proteins induced by menadione. This menadione-triggered arylation, which can be measured by a fluorescence assay, is completely suppressed by addition of exogenous glutathione or N-acetyl cysteine. Complex I inhibition by Rotenone did not mimic the cytoprotective action of AIF depletion. Altogether, these results are compatible with the hypothesis that mitochondrion-sessile AIF facilitates lethal redox cycling of menadione, thereby precipitating protein arylation and glutathione depletion.

  5. p53- and ERK7-dependent ribosome surveillance response regulates Drosophila insulin-like peptide secretion.

    Directory of Open Access Journals (Sweden)

    Kiran Hasygar

    2014-11-01

    Full Text Available Insulin-like signalling is a conserved mechanism that coordinates animal growth and metabolism with nutrient status. In Drosophila, insulin-producing median neurosecretory cells (IPCs regulate larval growth by secreting insulin-like peptides (dILPs in a diet-dependent manner. Previous studies have shown that nutrition affects dILP secretion through humoral signals derived from the fat body. Here we uncover a novel mechanism that operates cell autonomously in the IPCs to regulate dILP secretion. We observed that impairment of ribosome biogenesis specifically in the IPCs strongly inhibits dILP secretion, which consequently leads to reduced body size and a delay in larval development. This response is dependent on p53, a known surveillance factor for ribosome biogenesis. A downstream effector of this growth inhibitory response is an atypical MAP kinase ERK7 (ERK8/MAPK15, which is upregulated in the IPCs following impaired ribosome biogenesis as well as starvation. We show that ERK7 is sufficient and essential to inhibit dILP secretion upon impaired ribosome biogenesis, and it acts epistatically to p53. Moreover, we provide evidence that p53 and ERK7 contribute to the inhibition of dILP secretion upon starvation. Thus, we conclude that a cell autonomous ribosome surveillance response, which leads to upregulation of ERK7, inhibits dILP secretion to impede tissue growth under limiting dietary conditions.

  6. Sensitivity of hiPSC-derived neural stem cells (NSC) to Pyrroloquinoline quinone depends on their developmental stage.

    Science.gov (United States)

    Augustyniak, J; Lenart, J; Zychowicz, M; Lipka, G; Gaj, P; Kolanowska, M; Stepien, P P; Buzanska, L

    2017-12-01

    Pyrroloquinoline quinone (PQQ) is a factor influencing on the mitochondrial biogenesis. In this study the PQQ effect on viability, total cell number, antioxidant capacity, mitochondrial biogenesis and differentiation potential was investigated in human induced Pluripotent Stem Cells (iPSC) - derived: neural stem cells (NSC), early neural progenitors (eNP) and neural progenitors (NP). Here we demonstrated that sensitivity to PQQ is dependent upon its dose and neural stage of development. Induction of the mitochondrial biogenesis by PQQ at three stages of neural differentiation was evaluated at mtDNA, mRNA and protein level. Changes in NRF1, TFAM and PPARGC1A gene expression were observed at all developmental stages, but only at eNP were correlated with the statistically significant increase in the mtDNA copy numbers and enhancement of SDHA, COX-1 protein level. Thus, the "developmental window" of eNP for PQQ-evoked mitochondrial biogenesis is proposed. This effect was independent of high antioxidant capacity of PQQ, which was confirmed in all tested cell populations, regardless of the stage of hiPSC neural differentiation. Furthermore, a strong induction of GFAP, with down regulation of MAP2 gene expression upon PQQ treatment was observed. This indicates a possibility of shifting the balance of cell differentiation in the favor of astroglia, but more research is needed at this point. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Comparative metabolism of branched-chain amino acids to precursors of juvenile hormone biogenesis in corpora allata of lepidopterous versus nonlepidopterous insects

    Energy Technology Data Exchange (ETDEWEB)

    Brindle, P.A.; Schooley, D.A.; Tsai, L.W.; Baker, F.C.

    1988-08-05

    Comparative studies were performed on the role of branched-chain amino acids (BCAA) in juvenile hormone (JH) biosynthesis using several lepidopterous and nonlepidopterous insects. Corpora cardiaca-corpora allata complexes (CC-CA, the corpora allata being the organ of JH biogenesis) were maintained in culture medium containing a uniformly /sup 14/C-labeled BCAA, together with (methyl-/sup 3/H)methionine as mass marker for JH quantification. BCAA catabolism was quantified by directly analyzing the medium for the presence of /sup 14/C-labeled propionate and/or acetate, while JHs were extracted, purified by liquid chromatography, and subjected to double-label liquid scintillation counting. Our results indicate that active BCAA catabolism occurs within the CC-CA of lepidopterans, and this efficiently provides propionyl-CoA (from isoleucine or valine) for the biosynthesis of the ethyl branches of JH I and II. Acetyl-CoA, formed from isoleucine or leucine catabolism, is also utilized by lepidopteran CC-CA for biosynthesizing JH III and the acetate-derived portions of the ethyl-branched JHs. In contrast, CC-CA of nonlepidopterans fail to catabolize BCAA. Consequently, exogenous isoleucine or leucine does not serve as a carbon source for the biosynthesis of JH III by these glands, and no propionyl-CoA is produced for genesis of ethyl-branched JHs. This is the first observation of a tissue-specific metabolic difference which in part explains why these novel homosesquiterpenoids exist in lepidopterans, but not in nonlepidopterans.

  8. Comparative metabolism of branched-chain amino acids to precursors of juvenile hormone biogenesis in corpora allata of lepidopterous versus nonlepidopterous insects

    International Nuclear Information System (INIS)

    Brindle, P.A.; Schooley, D.A.; Tsai, L.W.; Baker, F.C.

    1988-01-01

    Comparative studies were performed on the role of branched-chain amino acids (BCAA) in juvenile hormone (JH) biosynthesis using several lepidopterous and nonlepidopterous insects. Corpora cardiaca-corpora allata complexes (CC-CA, the corpora allata being the organ of JH biogenesis) were maintained in culture medium containing a uniformly 14 C-labeled BCAA, together with [methyl- 3 H]methionine as mass marker for JH quantification. BCAA catabolism was quantified by directly analyzing the medium for the presence of 14 C-labeled propionate and/or acetate, while JHs were extracted, purified by liquid chromatography, and subjected to double-label liquid scintillation counting. Our results indicate that active BCAA catabolism occurs within the CC-CA of lepidopterans, and this efficiently provides propionyl-CoA (from isoleucine or valine) for the biosynthesis of the ethyl branches of JH I and II. Acetyl-CoA, formed from isoleucine or leucine catabolism, is also utilized by lepidopteran CC-CA for biosynthesizing JH III and the acetate-derived portions of the ethyl-branched JHs. In contrast, CC-CA of nonlepidopterans fail to catabolize BCAA. Consequently, exogenous isoleucine or leucine does not serve as a carbon source for the biosynthesis of JH III by these glands, and no propionyl-CoA is produced for genesis of ethyl-branched JHs. This is the first observation of a tissue-specific metabolic difference which in part explains why these novel homosesquiterpenoids exist in lepidopterans, but not in nonlepidopterans

  9. Sex-specific differences in mitochondria biogenesis, morphology, respiratory function, and ROS homeostasis in young mouse heart and brain.

    Science.gov (United States)

    Khalifa, Abdel Rahman M; Abdel-Rahman, Engy A; Mahmoud, Ali M; Ali, Mohamed H; Noureldin, Maha; Saber, Saber H; Mohsen, Mahmoud; Ali, Sameh S

    2017-03-01

    Sex-specific differences in mitochondrial function and free radical homeostasis are reported in the context of aging but not well-established in pathogeneses occurring early in life. Here, we examine if sex disparity in mitochondria function, morphology, and redox status starts early and hence can be implicated in sexual dimorphism in cardiac as well as neurological disorders prevalent at young age. Although mitochondrial activity in the heart did not significantly vary between sexes, female brain exhibited enhanced respiration and higher reserve capacity. This was associated with lower H 2 O 2 production in female cardiac and brain tissues. Using transmission electron microscopy, we found that the number of female cardiac mitochondria is moderately greater (117 ± 3%, P  = 0.049, N  = 4) than male's, which increased significantly for cortical mitochondria (134 ± 4%, P  = 0.001, N  = 4). However, male's cardiac mitochondria exhibited fragmented, circular, and smaller mitochondria relative to female's mitochondria, while no morphologic sex-dependent differences were observed in cortical mitochondria. No sex differences were detected in Nox2 and Nox4 proteins or O 2 -consuming/H 2 O 2 -producing activities in brain homogenate or synaptosomes. However, a strong trend of increased EPR-detected NOX superoxide in male synaptosomes hinted at higher superoxide dismutase activity in female brains, which was confirmed by two independent protocols. We also provide direct evidence that respiring mitochondria generally produce an order-of-magnitude lower reactive oxygen species (ROS) proportions than currently estimated. Our results indicate that sex differences in mitochondrial biogenesis, bioenergetics, and morphology may start at young age and that sex-dependent SOD capacity may be responsible for differences in ROS homeostasis in heart and brain. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological

  10. Identification of Oxa1 Homologs Operating in the Eukaryotic Endoplasmic Reticulum

    Directory of Open Access Journals (Sweden)

    S. Andrei Anghel

    2017-12-01

    Full Text Available Members of the evolutionarily conserved Oxa1/Alb3/YidC family mediate membrane protein biogenesis at the mitochondrial inner membrane, chloroplast thylakoid membrane, and bacterial plasma membrane, respectively. Despite their broad phylogenetic distribution, no Oxa1/Alb3/YidC homologs are known to operate in eukaryotic cells outside the endosymbiotic organelles. Here, we present bioinformatic evidence that the tail-anchored protein insertion factor WRB/Get1, the “endoplasmic reticulum (ER membrane complex” subunit EMC3, and TMCO1 are ER-resident homologs of the Oxa1/Alb3/YidC family. Topology mapping and co-evolution-based modeling demonstrate that Get1, EMC3, and TMCO1 share a conserved Oxa1-like architecture. Biochemical analysis of human TMCO1, the only homolog not previously linked to membrane protein biogenesis, shows that it associates with the Sec translocon and ribosomes. These findings suggest a specific biochemical function for TMCO1 and define a superfamily of proteins—the “Oxa1 superfamily”—whose shared function is to facilitate membrane protein biogenesis.

  11. Neurotoxin synthesis is positively regulated by the sporulation transcription factor Spo0A in Clostridium botulinum type E.

    Science.gov (United States)

    Mascher, Gerald; Mertaoja, Anna; Korkeala, Hannu; Lindström, Miia

    2017-10-01

    Clostridium botulinum produces the most potent natural toxin, the botulinum neurotoxin (BoNT), probably to create anaerobiosis and nutrients by killing the host, and forms endospores that facilitate survival in harsh conditions and transmission. Peak BoNT production coincides with initiation of sporulation in C. botulinum cultures, which suggests common regulation. Here, we show that Spo0A, the master regulator of sporulation, positively regulates BoNT production. Insertional inactivation of spo0A in C. botulinum type E strain Beluga resulted in significantly reduced BoNT production and in abolished or highly reduced sporulation in relation to wild-type controls. Complementation with spo0A restored BoNT production and sporulation. Recombinant DNA-binding domain of Spo0A directly bound to a putative Spo0A-binding box (CTTCGAA) within the BoNT/E operon promoter, demonstrating direct regulation. Spo0A is the first neurotoxin regulator reported in C. botulinum type E. Unlike other C. botulinum strains that are terrestrial and employ the alternative sigma factor BotR in directing BoNT expression, C. botulinum type E strains are adapted to aquatic ecosystems, possess distinct epidemiology and lack BotR. Our results provide fundamental new knowledge on the genetic control of BoNT production and demonstrate common regulation of BoNT production and sporulation, providing a key intervention point for control. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  12. Absorption edge imaging of sporocide-treated and non-treated bacterial spores

    International Nuclear Information System (INIS)

    Panessa-Warren, B.J.; Tortora, G.T.; Warren, J.B.

    1987-01-01

    When deprived of nutrients, spore forming bacilli produce endospores which are remarkably resistant to chemical sterilization. Little is known about the morphology and response fo these spores following exposure to sporocidal agents. Light microscopy does not provide sufficient resolution for studying the rupture of the spore coat and fate of intracellular material. Transmission and scanning electron microscopy offer superior resolution but require specimen preparation methods that induce physiologic as well as morphologic changes in the spores, thereby making accurate interpretation of micrographs difficult. To eliminate the possible artifacts induced by chemical fixation, dehydration, embeddment, staining and sectioning, treated and non-sporocide-treated endospores of B. thuringiensis and B. subtilis were imaged by x-ray contact microscopy using monochromatic x-rays. 6 refs., 2 figs

  13. Plant Nucleolar Stress Response, a New Face in the NAC-Dependent Cellular Stress Responses

    Directory of Open Access Journals (Sweden)

    Iwai Ohbayashi

    2018-01-01

    Full Text Available The nucleolus is the most prominent nuclear domain, where the core processes of ribosome biogenesis occur vigorously. All these processes are finely orchestrated by many nucleolar factors to build precisely ribosome particles. In animal cells, perturbations of ribosome biogenesis, mostly accompanied by structural disorders of the nucleolus, cause a kind of cellular stress to induce cell cycle arrest, senescence, or apoptosis, which is called nucleolar stress response. The best-characterized pathway of this stress response involves p53 and MDM2 as key players. p53 is a crucial transcription factor that functions in response to not only nucleolar stress but also other cellular stresses such as DNA damage stress. These cellular stresses release p53 from the inhibition by MDM2, an E3 ubiquitin ligase targeting p53, in various ways, which leads to p53-dependent activation of a set of genes. In plants, genetic impairments of ribosome biogenesis factors or ribosome components have been shown to cause characteristic phenotypes, including a narrow and pointed leaf shape, implying a common signaling pathway connecting ribosomal perturbations and certain aspects of growth and development. Unlike animals, however, plants have neither p53 nor MDM2 family proteins. Then the question arises whether plant cells have a nucleolar stress response pathway. In recent years, it has been reported that several members of the plant-specific transcription factor family NAC play critical roles in the pathways responsive to various cellular stresses. In this mini review, we outline the plant cellular stress response pathways involving NAC transcription factors with reference to the p53-MDM2-dependent pathways of animal cells, and discuss the possible involvement of a plant-unique, NAC-mediated pathway in the nucleolar stress response in plants.

  14. Plant Nucleolar Stress Response, a New Face in the NAC-Dependent Cellular Stress Responses.

    Science.gov (United States)

    Ohbayashi, Iwai; Sugiyama, Munetaka

    2017-01-01

    The nucleolus is the most prominent nuclear domain, where the core processes of ribosome biogenesis occur vigorously. All these processes are finely orchestrated by many nucleolar factors to build precisely ribosome particles. In animal cells, perturbations of ribosome biogenesis, mostly accompanied by structural disorders of the nucleolus, cause a kind of cellular stress to induce cell cycle arrest, senescence, or apoptosis, which is called nucleolar stress response. The best-characterized pathway of this stress response involves p53 and MDM2 as key players. p53 is a crucial transcription factor that functions in response to not only nucleolar stress but also other cellular stresses such as DNA damage stress. These cellular stresses release p53 from the inhibition by MDM2, an E3 ubiquitin ligase targeting p53, in various ways, which leads to p53-dependent activation of a set of genes. In plants, genetic impairments of ribosome biogenesis factors or ribosome components have been shown to cause characteristic phenotypes, including a narrow and pointed leaf shape, implying a common signaling pathway connecting ribosomal perturbations and certain aspects of growth and development. Unlike animals, however, plants have neither p53 nor MDM2 family proteins. Then the question arises whether plant cells have a nucleolar stress response pathway. In recent years, it has been reported that several members of the plant-specific transcription factor family NAC play critical roles in the pathways responsive to various cellular stresses. In this mini review, we outline the plant cellular stress response pathways involving NAC transcription factors with reference to the p53-MDM2-dependent pathways of animal cells, and discuss the possible involvement of a plant-unique, NAC-mediated pathway in the nucleolar stress response in plants.

  15. Respiratory chain complex I, a main regulatory target of the cAMP/PKA pathway is defective in different human diseases

    DEFF Research Database (Denmark)

    Papa, S.; De Rasmo, D.; Technikova-Dobrova, Z.

    2012-01-01

    In mammals, complex I (NADH-ubiquinone oxidoreductase) of the mitochondrial respiratory chain has 31 supernumerary subunits in addition to the 14 conserved from prokaryotes to humans. Multiplicity of structural protein components, as well as of biogenesis factors, makes complex I a sensible pace-...

  16. Biogenesis and function of Porphyromonas gingivalis outer membrane vesicles

    Science.gov (United States)

    Xie, H

    2015-01-01

    Porphyromonas gingivalis is one of the keystone pathogens associated with chronic periodontitis. All P. gingivalis strains examined thus far produce outer membrane vesicles. Recent studies have found that vesicles possess some well-known virulence factors of P. gingivalis such as adhesins, toxins and proteolytic enzymes. Carrying most of the characteristic features of their parent P. gingivalis cells, vesicles communicate with host cells and other members of microbial biofilms, resulting in the transmission of virulence factors into these host cells and the formation of pathogenic bacteria-dominated microbial communities. An in-depth understanding of both the nature and role of vesicles in the pathogenicity of P. gingivalis is both important and timely, particularly when speaking of periodontitis and its related systemic effects. PMID:26343879

  17. SUN Family Proteins Sun4p, Uth1p and Sim1p Are Secreted from Saccharomyces cerevisiae and Produced Dependently on Oxygen Level

    Czech Academy of Sciences Publication Activity Database

    Kuznetsov, E.; Kučerová, Helena; Váchová, Libuše; Palková, Z.

    2013-01-01

    Roč. 8, č. 9 (2013) E-ISSN 1932-6203 R&D Projects: GA ČR GA13-08605S Institutional support: RVO:61388971 Keywords : YEAST COLONY DEVELOPMENT * CELL-WALL BIOGENESIS * CANDIDA-ALBICANS Subject RIV: EE - Microbiology, Virology Impact factor: 3.534, year: 2013

  18. Directed proteomic analysis of the human nucleolus

    DEFF Research Database (Denmark)

    Andersen, Jens S; Lyon, Carol E; Fox, Archa H

    2002-01-01

    BACKGROUND: The nucleolus is a subnuclear organelle containing the ribosomal RNA gene clusters and ribosome biogenesis factors. Recent studies suggest it may also have roles in RNA transport, RNA modification, and cell cycle regulation. Despite over 150 years of research into nucleoli, many aspec...

  19. A network of paralogous stress response transcription factors in the human pathogen Candida glabrata.

    Directory of Open Access Journals (Sweden)

    Jawad eMerhej

    2016-05-01

    Full Text Available The yeast Candida glabrata has become the second cause of systemic candidemia in humans. However, relatively few genome-wide studies have been conducted in this organism and our knowledge of its transcriptional regulatory network is quite limited. In the present work, we combined genome-wide chromatin immunoprecipitation (ChIP-seq, transcriptome analyses and DNA binding motif predictions to describe the regulatory interactions of the seven Yap (Yeast AP1 transcription factors of C. glabrata. We described a transcriptional network containing 255 regulatory interactions and 309 potential target genes. We predicted with high confidence the preferred DNA binding sites for 5 of the 7 CgYaps and showed a strong conservation of the Yap DNA binding properties between S. cerevisiae and C. glabrata. We provided reliable functional annotation for 3 of the 7 Yaps and identified for Yap1 and Yap5 a core regulon which is conserved in S. cerevisiae, C. glabrata and C. albicans. We uncovered new roles for CgYap7 in the regulation of iron-sulfur cluster biogenesis, for CgYap1 in the regulation of heme biosynthesis and for CgYap5 in the repression of GRX4 in response to iron starvation. These transcription factors define an interconnected transcriptional network at the cross-roads between redox homeostasis, oxygen consumption and iron metabolism.

  20. Sporulation in Bacteria: Beyond the Standard Model.

    Science.gov (United States)

    Hutchison, Elizabeth A; Miller, David A; Angert, Esther R

    2014-10-01

    Endospore formation follows a complex, highly regulated developmental pathway that occurs in a broad range of Firmicutes. Although Bacillus subtilis has served as a powerful model system to study the morphological, biochemical, and genetic determinants of sporulation, fundamental aspects of the program remain mysterious for other genera. For example, it is entirely unknown how most lineages within the Firmicutes regulate entry into sporulation. Additionally, little is known about how the sporulation pathway has evolved novel spore forms and reproductive schemes. Here, we describe endospore and internal offspring development in diverse Firmicutes and outline progress in characterizing these programs. Moreover, comparative genomics studies are identifying highly conserved sporulation genes, and predictions of sporulation potential in new isolates and uncultured bacteria can be made from these data. One surprising outcome of these comparative studies is that core regulatory and some structural aspects of the program appear to be universally conserved. This suggests that a robust and sophisticated developmental framework was already in place in the last common ancestor of all extant Firmicutes that produce internal offspring or endospores. The study of sporulation in model systems beyond B. subtilis will continue to provide key information on the flexibility of the program and provide insights into how changes in this developmental course may confer advantages to cells in diverse environments.

  1. Inter- and intra-specific cuticle variation between amphimictic and parthenogenetic species of root-knot nematode (Meloidogyne spp.) as revealed by a bacterial parasite (Pasteuria penetrans).

    Science.gov (United States)

    Davies, K G; Rowe, J A; Williamson, V M

    2008-06-01

    Specific host-parasite interactions exist between species and strains of plant parasitic root-knot nematodes and the Gram-positive bacterial hyperparasite Pasteuria penetrans. This bacterium produces endospores that adhere to the cuticle of migrating juveniles, germinate and colonise the developing female within roots. Endospore attachment of P. penetrans populations to second-stage juveniles of the root-knot nematode species Meloidogyne incognita and Meloidogyne hapla showed there were interactive differences between bacterial populations and nematode species. Infected females of M. incognita produced a few progeny which were used to establish two nematode lines from single infective juveniles encumbered with either three or 26 endospores. Single juvenile descent lines of each nematode species were produced to test whether cuticle variation was greater within M. hapla lines that reproduce by facultative meiotic parthenogenesis than within lines of M. incognita, which reproduces by obligate parthenogenesis. Assays revealed variability between broods of individual females derived from single second-stage juvenile descent lines of both M. incognita and M. hapla suggesting that progeny derived from a single individual can differ in spore adhesion in both sexual and asexual nematode species. These results suggest that special mechanisms that produced these functional differences in the cuticle surface may have evolved in both sexually and asexually reproducing nematodes as a strategy to circumvent infection by this specialised hyperparasite.

  2. A PEX6-defective peroxisomal biogenesis disorder with severe phenotype in an infant, versus mild phenotype resembling Usher syndrome in the affected parents.

    Science.gov (United States)

    Raas-Rothschild, Annick; Wanders, Ronald J A; Mooijer, Petra A W; Gootjes, Jeannette; Waterham, Hans R; Gutman, Alisa; Suzuki, Yasuyuki; Shimozawa, Nobuyuki; Kondo, Naomi; Eshel, Gideon; Espeel, Marc; Roels, Frank; Korman, Stanley H

    2002-04-01

    Sensorineural deafness and retinitis pigmentosa (RP) are the hallmarks of Usher syndrome (USH) but are also prominent features in peroxisomal biogenesis defects (PBDs); both are autosomal recessively inherited. The firstborn son of unrelated parents, who both had sensorineural deafness and RP diagnosed as USH, presented with sensorineural deafness, RP, dysmorphism, developmental delay, hepatomegaly, and hypsarrhythmia and died at age 17 mo. The infant was shown to have a PBD, on the basis of elevated plasma levels of very-long- and branched-chain fatty acids (VLCFAs and BCFAs), deficiency of multiple peroxisomal functions in fibroblasts, and complete absence of peroxisomes in fibroblasts and liver. Surprisingly, both parents had elevated plasma levels of VLCFAs and BCFAs. Fibroblast studies confirmed that both parents had a PBD. The parents' milder phenotypes correlated with relatively mild peroxisomal biochemical dysfunction and with catalase immunofluorescence microscopy demonstrating mosaicism and temperature sensitivity in fibroblasts. The infant and both of his parents belonged to complementation group C. PEX6 gene sequencing revealed mutations on both alleles, in the infant and in his parents. This unique family is the first report of a PBD with which the parents are themselves affected individuals rather than asymptomatic carriers. Because of considerable overlap between USH and milder PBD phenotypes, individuals suspected to have USH should be screened for peroxisomal dysfunction.

  3. Common miR-590 Variant rs6971711 Present Only in African Americans Reduces miR-590 Biogenesis.

    Directory of Open Access Journals (Sweden)

    Xiaoping Lin

    Full Text Available MicroRNAs (miRNAs are recognized as important regulators of cardiac development, hypertrophy and fibrosis. Recent studies have demonstrated that genetic variations which cause alterations in miRNA:target interactions can lead to disease. We hypothesized that genetic variations in miRNAs that regulate cardiac hypertrophy/fibrosis might be involved in generation of the cardiac phenotype in patients diagnosed with hypertrophic cardiomyopathy (HCM. To investigate this question, we Sanger sequenced 18 miRNA genes previously implicated in myocyte hypertrophy/fibrosis and apoptosis, using genomic DNA isolated from the leukocytes of 199 HCM patients. We identified a single nucleotide polymorphism (rs6971711, C57T SNP at the 17th position of mature miR-590-3p (= 57th position of pre-miR-590 that is common in individuals of African ancestry. SNP frequency was higher in African American HCM patients (n = 55 than ethnically-matched controls (n = 100, but the difference was not statistically significant (8.2% vs. 6.5%; p = 0.5. Using a cell culture system, we discovered that presence of this SNP resulted in markedly lower levels of mature miR-590-5p (39 ± 16%, p<0.003 and miR-590-3p (20 ± 2%, p<0.003, when compared with wild-type (WT miR-590, without affecting levels of pri-miR-590 and pre-miR-590. Consistent with this finding, the SNP resulted in reduced target suppression when compared to WT miR-590 (71% suppression by WT vs 60% suppression by SNP, p<0.03. Since miR-590 can regulate TGF-β, Activin A and Akt signaling, SNP-induced reduction in miR-590 biogenesis could influence cardiac phenotype by de-repression of these signaling pathways. Since the SNP is only present in African Americans, population studies in this patient population would be valuable to investigate effects of this SNP on myocyte function and cardiac physiology.

  4. The pulsed light inactivation of veterinary relevant microbial biofilms ...

    African Journals Online (AJOL)

    tulyasys

    2016-01-27

    Jan 27, 2016 ... functioning innate and adaptive immune responses, biofilm-based infections ... of parasite species and bacterial endospores has shown this system to be highly ..... chlorine commonly observed with these complex structures ...

  5. Amino acid-dependent signaling via S6K1 and MYC is essential for regulation of rDNA transcription

    Science.gov (United States)

    Kang, Jian; Kusnadi, Eric P.; Ogden, Allison J.; Hicks, Rodney J.; Bammert, Lukas; Kutay, Ulrike; Hung, Sandy; Sanij, Elaine; Hannan, Ross D.; Hannan, Katherine M.; Pearson, Richard B.

    2016-01-01

    Dysregulation of RNA polymerase I (Pol I)-dependent ribosomal DNA (rDNA) transcription is a consistent feature of malignant transformation that can be targeted to treat cancer. Understanding how rDNA transcription is coupled to the availability of growth factors and nutrients will provide insight into how ribosome biogenesis is maintained in a tumour environment characterised by limiting nutrients. We demonstrate that modulation of rDNA transcription initiation, elongation and rRNA processing is an immediate, co-regulated response to altered amino acid abundance, dependent on both mTORC1 activation of S6K1 and MYC activity. Growth factors regulate rDNA transcription initiation while amino acids modulate growth factor-dependent rDNA transcription by primarily regulating S6K1-dependent rDNA transcription elongation and processing. Thus, we show for the first time amino acids regulate rRNA synthesis by a distinct, post-initiation mechanism, providing a novel model for integrated control of ribosome biogenesis that has implications for understanding how this process is dysregulated in cancer. PMID:27385002

  6. Transcriptomic and Hormonal Analyses Reveal that YUC-Mediated Auxin Biogenesis Is Involved in Shoot Regeneration from Rhizome in Cymbidium.

    Science.gov (United States)

    Liu, Yang; Zhang, Hai-Liang; Guo, He-Rong; Xie, Li; Zeng, Rui-Zhen; Zhang, Xiang-Qian; Zhang, Zhi-Sheng

    2017-01-01

    Cymbidium , one of the most important orchid genera in horticulture, can be classified into epiphytic and terrestrial species. Generally, epiphytic Cymbidium seedlings can be easily propagated by tissue culture, but terrestrial seedlings are difficult to propagate. To date, the molecular mechanisms underlying the differences in the ease with which terrestrial and epiphytic cymbidiums can be propagated are largely unknown. Using RNA-sequencing, quantitative reverse transcription PCR and enzyme-linked immunosorbent assay, Cymbidium 'Xiaofeng' (CXF), which can be efficiently micropropagated, and terrestrial Cymbidium sinense 'Qijianbaimo' (CSQ), which has a low regeneration ability, were used to explore the molecular mechanisms underlying the micropropagation ability of Cymbidium species. To this end, 447 million clean short reads were generated, and 31,264 annotated unigenes were obtained from 10 cDNA libraries. A total of 1,290 differentially expressed genes (DEGs) were identified between CXF and CSQ during shoot induction. Gene ontology (GO) enrichment analysis indicated that the DEGs were significantly enriched in auxin pathway-related GO terms. Further analysis demonstrated that YUC and GH3 family genes, which play crucial roles in the regulation of auxin/IAA (indole-3-acetic acid) metabolism, acted quickly in response to shoot induction culture in vitro and were closely correlated with variation in shoot regeneration between CXF and CSQ. In addition, the study showed that IAA accumulated rapidly and significantly during shoot induction in CXF compared to that in CSQ; in contrast, no significant changes in other hormones were observed between CXF and CSQ. Furthermore, shoot regeneration in CXF was inhibited by a yucasin-auxin biosynthesis inhibitor, indicating that increased IAA level is required for high-frequency shoot regeneration in CXF. In conclusion, our study revealed that YUC-mediated auxin biogenesis is involved in shoot regeneration from rhizome in

  7. Transcriptomic and Hormonal Analyses Reveal that YUC-Mediated Auxin Biogenesis Is Involved in Shoot Regeneration from Rhizome in Cymbidium

    Directory of Open Access Journals (Sweden)

    Yang Liu

    2017-10-01

    Full Text Available Cymbidium, one of the most important orchid genera in horticulture, can be classified into epiphytic and terrestrial species. Generally, epiphytic Cymbidium seedlings can be easily propagated by tissue culture, but terrestrial seedlings are difficult to propagate. To date, the molecular mechanisms underlying the differences in the ease with which terrestrial and epiphytic cymbidiums can be propagated are largely unknown. Using RNA-sequencing, quantitative reverse transcription PCR and enzyme-linked immunosorbent assay, Cymbidium ‘Xiaofeng’ (CXF, which can be efficiently micropropagated, and terrestrial Cymbidium sinense ‘Qijianbaimo’ (CSQ, which has a low regeneration ability, were used to explore the molecular mechanisms underlying the micropropagation ability of Cymbidium species. To this end, 447 million clean short reads were generated, and 31,264 annotated unigenes were obtained from 10 cDNA libraries. A total of 1,290 differentially expressed genes (DEGs were identified between CXF and CSQ during shoot induction. Gene ontology (GO enrichment analysis indicated that the DEGs were significantly enriched in auxin pathway-related GO terms. Further analysis demonstrated that YUC and GH3 family genes, which play crucial roles in the regulation of auxin/IAA (indole-3-acetic acid metabolism, acted quickly in response to shoot induction culture in vitro and were closely correlated with variation in shoot regeneration between CXF and CSQ. In addition, the study showed that IAA accumulated rapidly and significantly during shoot induction in CXF compared to that in CSQ; in contrast, no significant changes in other hormones were observed between CXF and CSQ. Furthermore, shoot regeneration in CXF was inhibited by a yucasin-auxin biosynthesis inhibitor, indicating that increased IAA level is required for high-frequency shoot regeneration in CXF. In conclusion, our study revealed that YUC-mediated auxin biogenesis is involved in shoot

  8. Fiber type conversion by PGC-1α activates lysosomal and autophagosomal biogenesis in both unaffected and Pompe skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Shoichi Takikita

    2010-12-01

    Full Text Available PGC-1α is a transcriptional co-activator that plays a central role in the regulation of energy metabolism. Our interest in this protein was driven by its ability to promote muscle remodeling. Conversion from fast glycolytic to slow oxidative fibers seemed a promising therapeutic approach in Pompe disease, a severe myopathy caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA which is responsible for the degradation of glycogen. The recently approved enzyme replacement therapy (ERT has only a partial effect in skeletal muscle. In our Pompe mouse model (KO, the poor muscle response is seen in fast but not in slow muscle and is associated with massive accumulation of autophagic debris and ineffective autophagy. In an attempt to turn the therapy-resistant fibers into fibers amenable to therapy, we made transgenic KO mice expressing PGC-1α in muscle (tgKO. The successful switch from fast to slow fibers prevented the formation of autophagic buildup in the converted fibers, but PGC-1α failed to improve the clearance of glycogen by ERT. This outcome is likely explained by an unexpected dramatic increase in muscle glycogen load to levels much closer to those observed in patients, in particular infants, with the disease. We have also found a remarkable rise in the number of lysosomes and autophagosomes in the tgKO compared to the KO. These data point to the role of PGC-1α in muscle glucose metabolism and its possible role as a master regulator for organelle biogenesis - not only for mitochondria but also for lysosomes and autophagosomes. These findings may have implications for therapy of lysosomal diseases and other disorders with altered autophagy.

  9. MR-1S Interacts with PET100 and PET117 in Module-Based Assembly of Human Cytochrome c Oxidase

    Directory of Open Access Journals (Sweden)

    Sara Vidoni

    2017-02-01

    Full Text Available The biogenesis of human cytochrome c oxidase (COX is an intricate process in which three mitochondrial DNA (mtDNA-encoded core subunits are assembled in a coordinated way with at least 11 nucleus-encoded subunits. Many chaperones shared between yeast and humans are involved in COX assembly. Here, we have used a MT-CO3 mutant cybrid cell line to define the composition of assembly intermediates and identify new human COX assembly factors. Quantitative mass spectrometry analysis led us to modify the assembly model from a sequential pathway to a module-based process. Each module contains one of the three core subunits, together with different ancillary components, including HIGD1A. By the same analysis, we identified the short isoform of the myofibrillogenesis regulator 1 (MR-1S as a new COX assembly factor, which works with the highly conserved PET100 and PET117 chaperones to assist COX biogenesis in higher eukaryotes.

  10. MR-1S Interacts with PET100 and PET117 in Module-Based Assembly of Human Cytochrome c Oxidase.

    Science.gov (United States)

    Vidoni, Sara; Harbour, Michael E; Guerrero-Castillo, Sergio; Signes, Alba; Ding, Shujing; Fearnley, Ian M; Taylor, Robert W; Tiranti, Valeria; Arnold, Susanne; Fernandez-Vizarra, Erika; Zeviani, Massimo

    2017-02-14

    The biogenesis of human cytochrome c oxidase (COX) is an intricate process in which three mitochondrial DNA (mtDNA)-encoded core subunits are assembled in a coordinated way with at least 11 nucleus-encoded subunits. Many chaperones shared between yeast and humans are involved in COX assembly. Here, we have used a MT-CO3 mutant cybrid cell line to define the composition of assembly intermediates and identify new human COX assembly factors. Quantitative mass spectrometry analysis led us to modify the assembly model from a sequential pathway to a module-based process. Each module contains one of the three core subunits, together with different ancillary components, including HIGD1A. By the same analysis, we identified the short isoform of the myofibrillogenesis regulator 1 (MR-1S) as a new COX assembly factor, which works with the highly conserved PET100 and PET117 chaperones to assist COX biogenesis in higher eukaryotes. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. RNA helicase HEL-1 promotes longevity by specifically activating DAF-16/FOXO transcription factor signaling in Caenorhabditis elegans

    Science.gov (United States)

    Seo, Mihwa; Seo, Keunhee; Hwang, Wooseon; Koo, Hee Jung; Hahm, Jeong-Hoon; Yang, Jae-Seong; Han, Seong Kyu; Hwang, Daehee; Kim, Sanguk; Jang, Sung Key; Lee, Yoontae; Nam, Hong Gil; Lee, Seung-Jae V.

    2015-01-01

    The homeostatic maintenance of the genomic DNA is crucial for regulating aging processes. However, the role of RNA homeostasis in aging processes remains unknown. RNA helicases are a large family of enzymes that regulate the biogenesis and homeostasis of RNA. However, the functional significance of RNA helicases in aging has not been explored. Here, we report that a large fraction of RNA helicases regulate the lifespan of Caenorhabditis elegans. In particular, we show that a DEAD-box RNA helicase, helicase 1 (HEL-1), promotes longevity by specifically activating the DAF-16/forkhead box O (FOXO) transcription factor signaling pathway. We find that HEL-1 is required for the longevity conferred by reduced insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS) and is sufficient for extending lifespan. We further show that the expression of HEL-1 in the intestine and neurons contributes to longevity. HEL-1 enhances the induction of a large fraction of DAF-16 target genes. Thus, the RNA helicase HEL-1 appears to promote longevity in response to decreased IIS as a transcription coregulator of DAF-16. Because HEL-1 and IIS are evolutionarily well conserved, a similar mechanism for longevity regulation via an RNA helicase-dependent regulation of FOXO signaling may operate in mammals, including humans. PMID:26195740

  12. Giardia mitosomes and trichomonad hydrogenosomes share a common mode of protein targeting

    Czech Academy of Sciences Publication Activity Database

    Doležal, P.; Šmíd, O.; Rada, P.; Zubáčová, Z.; Bursać, D.; Šuťák, R.; Nebesářová, Jana; Lithgow, T.; Tachezy, J.

    2005-01-01

    Roč. 102, č. 31 (2005), s. 10924-10929 ISSN 0027-8424 R&D Projects: GA ČR GA204/04/0435 Institutional research plan: CEZ:AV0Z60220518 Keywords : biogenesis * FeS cluster assembly * Pam 18 Subject RIV: EE - Microbiology, Virology Impact factor: 10.231, year: 2005

  13. MiRNA-Related SNPs and Risk of Esophageal Adenocarcinoma and Barrett's Esophagus: Post Genome-Wide Association Analysis in the BEACON Consortium.

    Directory of Open Access Journals (Sweden)

    Matthew F Buas

    Full Text Available Incidence of esophageal adenocarcinoma (EA has increased substantially in recent decades. Multiple risk factors have been identified for EA and its precursor, Barrett's esophagus (BE, such as reflux, European ancestry, male sex, obesity, and tobacco smoking, and several germline genetic variants were recently associated with disease risk. Using data from the Barrett's and Esophageal Adenocarcinoma Consortium (BEACON genome-wide association study (GWAS of 2,515 EA cases, 3,295 BE cases, and 3,207 controls, we examined single nucleotide polymorphisms (SNPs that potentially affect the biogenesis or biological activity of microRNAs (miRNAs, small non-coding RNAs implicated in post-transcriptional gene regulation, and deregulated in many cancers, including EA. Polymorphisms in three classes of genes were examined for association with risk of EA or BE: miRNA biogenesis genes (157 SNPs, 21 genes; miRNA gene loci (234 SNPs, 210 genes; and miRNA-targeted mRNAs (177 SNPs, 158 genes. Nominal associations (P0.50, and we did not find evidence for interactions between variants analyzed and two risk factors for EA/BE (smoking and obesity. This analysis provides the most extensive assessment to date of miRNA-related SNPs in relation to risk of EA and BE. While common genetic variants within components of the miRNA biogenesis core pathway appear unlikely to modulate susceptibility to EA or BE, further studies may be warranted to examine potential associations between unassessed variants in miRNA genes and targets with disease risk.

  14. Synergistic interaction of fatty acids and oxysterols impairs mitochondrial function and limits liver adaptation during nafld progression

    Directory of Open Access Journals (Sweden)

    Francesco Bellanti

    2018-05-01

    Full Text Available The complete mechanism accounting for the progression from simple steatosis to steatohepatitis in nonalcoholic fatty liver disease (NAFLD has not been elucidated. Lipotoxicity refers to cellular injury caused by hepatic free fatty acids (FFAs and cholesterol accumulation. Excess cholesterol autoxidizes to oxysterols during oxidative stress conditions. We hypothesize that interaction of FAs and cholesterol derivatives may primarily impair mitochondrial function and affect biogenesis adaptation during NAFLD progression. We demonstrated that the accumulation of specific non-enzymatic oxysterols in the liver of animals fed high-fat+high-cholesterol diet induces mitochondrial damage and depletion of proteins of the respiratory chain complexes. When tested in vitro, 5α-cholestane-3β,5,6β-triol (triol combined to FFAs was able to reduce respiration in isolated liver mitochondria, induced apoptosis in primary hepatocytes, and down-regulated transcription factors involved in mitochondrial biogenesis. Finally, a lower protein content in the mitochondrial respiratory chain complexes was observed in human non-alcoholic steatohepatitis. In conclusion, hepatic accumulation of FFAs and non-enzymatic oxysterols synergistically facilitates development and progression of NAFLD by impairing mitochondrial function, energy balance and biogenesis adaptation to chronic injury.

  15. Comparison of preribosomal RNA processing pathways in yeast, plant and human cells - focus on coordinated action of endo- and exoribonucleases.

    Science.gov (United States)

    Tomecki, Rafal; Sikorski, Pawel J; Zakrzewska-Placzek, Monika

    2017-07-01

    Proper regulation of ribosome biosynthesis is mandatory for cellular adaptation, growth and proliferation. Ribosome biogenesis is the most energetically demanding cellular process, which requires tight control. Abnormalities in ribosome production have severe consequences, including developmental defects in plants and genetic diseases (ribosomopathies) in humans. One of the processes occurring during eukaryotic ribosome biogenesis is processing of the ribosomal RNA precursor molecule (pre-rRNA), synthesized by RNA polymerase I, into mature rRNAs. It must not only be accurate but must also be precisely coordinated with other phenomena leading to the synthesis of functional ribosomes: RNA modification, RNA folding, assembly with ribosomal proteins and nucleocytoplasmic RNP export. A multitude of ribosome biogenesis factors ensure that these events take place in a correct temporal order. Among them are endo- and exoribonucleases involved in pre-rRNA processing. Here, we thoroughly present a wide spectrum of ribonucleases participating in rRNA maturation, focusing on their biochemical properties, regulatory mechanisms and substrate specificity. We also discuss cooperation between various ribonucleolytic activities in particular stages of pre-rRNA processing, delineating major similarities and differences between three representative groups of eukaryotes: yeast, plants and humans. © 2017 Federation of European Biochemical Societies.

  16. A Regulatory Circuit Composed of a Transcription Factor, IscR, and a Regulatory RNA, RyhB, Controls Fe-S Cluster Delivery.

    Science.gov (United States)

    Mandin, Pierre; Chareyre, Sylvia; Barras, Frédéric

    2016-09-20

    Fe-S clusters are cofactors conserved through all domains of life. Once assembled by dedicated ISC and/or SUF scaffolds, Fe-S clusters are conveyed to their apo-targets via A-type carrier proteins (ATCs). Escherichia coli possesses four such ATCs. ErpA is the only ATC essential under aerobiosis. Recent studies reported a possible regulation of the erpA mRNA by the small RNA (sRNA) RyhB, which controls the expression of many genes under iron starvation. Surprisingly, erpA has not been identified in recent transcriptomic analysis of the iron starvation response, thus bringing into question the actual physiological significance of the putative regulation of erpA by RyhB. Using an sRNA library, we show that among 26 sRNAs, only RyhB represses the expression of an erpA-lacZ translational fusion. We further demonstrate that this repression occurs during iron starvation. Using mutational analysis, we show that RyhB base pairs to the erpA mRNA, inducing its disappearance. In addition, IscR, the master regulator of Fe-S homeostasis, represses expression of erpA at the transcriptional level when iron is abundant, but depleting iron from the medium alleviates this repression. The conjunction of transcriptional derepression by IscR and posttranscriptional repression by RyhB under Fe-limiting conditions is best described as an incoherent regulatory circuit. This double regulation allows full expression of erpA at iron concentrations for which Fe-S biogenesis switches from the ISC to the SUF system. We further provide evidence that this regulatory circuit coordinates ATC usage to iron availability. Regulatory small RNAs (sRNAs) have emerged as major actors in the control of gene expression in the last few decades. Relatively little is known about how these regulators interact with classical transcription factors to coordinate genetic responses. We show here how an sRNA, RyhB, and a transcription factor, IscR, regulate expression of an essential gene, erpA, in the bacterium E

  17. Hierarchical recruitment of ribosomal proteins and assembly factors remodels nucleolar pre-60S ribosomes.

    Science.gov (United States)

    Biedka, Stephanie; Micic, Jelena; Wilson, Daniel; Brown, Hailey; Diorio-Toth, Luke; Woolford, John L

    2018-04-24

    Ribosome biogenesis involves numerous preribosomal RNA (pre-rRNA) processing events to remove internal and external transcribed spacer sequences, ultimately yielding three mature rRNAs. Removal of the internal transcribed spacer 2 spacer RNA is the final step in large subunit pre-rRNA processing and begins with endonucleolytic cleavage at the C 2 site of 27SB pre-rRNA. C 2 cleavage requires the hierarchical recruitment of 11 ribosomal proteins and 14 ribosome assembly factors. However, the function of these proteins in C 2 cleavage remained unclear. In this study, we have performed a detailed analysis of the effects of depleting proteins required for C 2 cleavage and interpreted these results using cryo-electron microscopy structures of assembling 60S subunits. This work revealed that these proteins are required for remodeling of several neighborhoods, including two major functional centers of the 60S subunit, suggesting that these remodeling events form a checkpoint leading to C 2 cleavage. Interestingly, when C 2 cleavage is directly blocked by depleting or inactivating the C 2 endonuclease, assembly progresses through all other subsequent steps. © 2018 Biedka et al.

  18. Catalytic properties of the Gas family β-(1,3)-glucanosyltransferases active in fungal cell-wall biogenesis as determined by a novel fluorescent assay.

    Science.gov (United States)

    Mazáň, Marián; Ragni, Enrico; Popolo, Laura; Farkaš, Vladimír

    2011-09-01

    BGTs [β-(1,3)-glucanosyltransglycosylases; EC 2.4.1.-] of the GH72 (family 72 of glycosylhydrolases) are GPI (glycosylphosphatidylinositol)-anchored proteins that play an important role in the biogenesis of fungal cell walls. They randomly cleave glycosidic linkages in β-(1,3)-glucan chains and ligate the polysaccharide portions containing newly formed reducing ends to C(3)(OH) at non-reducing ends of other β-(1,3)-glucan molecules. We have developed a sensitive fluorescence-based method for the assay of transglycosylating activity of GH72 enzymes. In the new assay, laminarin [β-(1,3)-glucan] is used as the glucanosyl donor and LamOS (laminarioligosaccharides) fluorescently labelled with SR (sulforhodamine) serve as the acceptors. The new fluorescent assay was employed for partial biochemical characterization of the heterologously expressed Gas family proteins from the yeast Saccharomyces cerevisiae. All the Gas enzymes specifically used laminarin as the glucanosyl donor and a SR-LamOS of DP (degree of polymerization) ≥5 as the acceptors. Gas proteins expressed in distinct stages of the yeast life cycle showed differences in their pH optima. Gas1p and Gas5p, which are expressed during vegetative growth, had the highest activity at pH 4.5 and 3.5 respectively, whereas the sporulation-specific Gas2p and Gas4p were most active between pH 5 and 6. The novel fluorescent assay provides a suitable tool for the screening of potential glucanosyltransferases or their inhibitors.

  19. Stimulation of Pol III-dependent 5S rRNA and U6 snRNA gene expression by AP-1 transcription factors.

    Science.gov (United States)

    Ahuja, Richa; Kumar, Vijay

    2017-07-01

    RNA polymerase III transcribes structurally diverse group of essential noncoding RNAs including 5S ribosomal RNA (5SrRNA) and U6 snRNA. These noncoding RNAs are involved in RNA processing and ribosome biogenesis, thus, coupling Pol III activity to the rate of protein synthesis, cell growth, and proliferation. Even though a few Pol II-associated transcription factors have been reported to participate in Pol III-dependent transcription, its activation by activator protein 1 (AP-1) factors, c-Fos and c-Jun, has remained unexplored. Here, we show that c-Fos and c-Jun bind to specific sites in the regulatory regions of 5S rRNA (type I) and U6 snRNA (type III) gene promoters and stimulate their transcription. Our chromatin immunoprecipitation studies suggested that endogenous AP-1 factors bind to their cognate promoter elements during the G1/S transition of cell cycle apparently synchronous with Pol III transcriptional activity. Furthermore, the interaction of c-Jun with histone acetyltransferase p300 promoted the recruitment of p300/CBP complex on the promoters and facilitated the occupancy of Pol III transcriptional machinery via histone acetylation and chromatin remodeling. The findings of our study, together, suggest that AP-1 factors are novel regulators of Pol III-driven 5S rRNA and U6 snRNA expression with a potential role in cell proliferation. © 2017 Federation of European Biochemical Societies.

  20. Ecology and metagenomics of soil microorganisms

    Czech Academy of Sciences Publication Activity Database

    Baldrian, Petr; Head, I. M.; Prosser, J. I.; Schloter, M.; Smalla, K.; Tebbe, C. C.

    2011-01-01

    Roč. 78, č. 1 (2011), s. 1-2 ISSN 0168-6496 R&D Projects: GA MŠk LC06066; GA MŠk(CZ) LA10001 Institutional research plan: CEZ:AV0Z50200510 Keywords : microorganism * bioremediation * biogenesis of soil Subject RIV: EE - Microbiology, Virology Impact factor: 3.408, year: 2011

  1. The Role of Prokaryotes in Sediment Carbon Cycling

    DEFF Research Database (Denmark)

    Piil, Kristoffer

    in the sediment. In particular, the work has focused on estimating how rapidly amino acids derived from plankton are degraded and replaced by amino acids from prokaryotes and how extensive this reworking of amino acids is in surface sediments. Another part of my work has focused on establishing reliable estimates...... of cell specific amino acid and muramic acid concentrations in sediment bacteria. Such estimates are important tools when studying the reworking of amino acids by bacteria and the preservation of bacterial cell walls. In addition, it has been an aim of the work to investigate how abundant endospores...... are in marine sediment and how dynamic the endospore population is, as very little is known about this compartment of the prokaryotic community. Prokaryotic reworking of amino acids was investigated by two independent methods. The first approach involved estimating the amount of amino acids produced...

  2. Evaluation of a new device for sterilizing dental high-speed handpieces

    DEFF Research Database (Denmark)

    Larsen, T; Andersen, H K; Fiehn, N E

    1997-01-01

    Dental high-speed turbines and handpieces can take up and expel microorganisms during operation and thus need regular sterilization. This study established a method for validating devices used to sterilize high-speed turbines and handpieces. The air and water channels and turbine chambers were...... contaminated with suspensions of Streptococcus salivarius or endospores of Bacillus stearothermophilus. The effect of flushing and/or autoclaving performed by a new device combining both procedures was evaluated by counting the number of viable bacteria recovered from these devices. Further, the effect...... on clinically used handpieces was evaluated. In an initial experiment, the device partially reduced S. salivarius, and the endospores survived. In a second experiment, a 5 to 6 log reduction of S. salivarius in air and water channels was obtained. No growth was observed in clinically used high-speed handpieces...

  3. Isolation of alkaline protease from Bacillus subtilis AKRS3

    African Journals Online (AJOL)

    ashok

    2012-08-28

    Aug 28, 2012 ... production proved high protease production than the other tested ... Crude alkaline protease was most active at 55°C, pH 9 with casein as ... 13416 Afr. J. Biotechnol. ... The Gram-positive, aerobic, rod-shaped endospore-.

  4. Isolation of thermophilic Desulfotomaculum strains with methanol and sulfite from solfataric mud pools, and characterization of Desulfotomaculum solfataficum sp nov

    NARCIS (Netherlands)

    Goorissen, H.P.; Boschker, H.T.S.; Stams, A.J.M.; Hansen, T.A.

    2003-01-01

    Four strains of thermophilic, endospore-forming, sulfate-reducing bacteria were enriched and isolated from hot solfataric fields in the Krafla area of north-east Iceland, using methanol and sulfite as substrates. Morphologically, these strains resembled thermophilic Desulfotomaculum species. The

  5. Isolation of thermophilic Desulfotomaculum strains with methanol and sulfite from solfataric mud pools, and characterization of Desulfotomaculum solfataficum sp nov

    NARCIS (Netherlands)

    Goorissen, HP; Boschker, HTS; Stams, AJM; Hansen, TA

    Four strains of thermophilic, endospore-forming, sulfate-reducing bacteria were enriched and isolated from hot solfataric fields in the Krafla area of north-east Iceland, using methanol and sulfite as substrates. Morphologically, these strains resembled thermophilic Desulfotomaculum species. The

  6. Isolation of thermophilic Desulfotomaculum strains with methanol and sulphite from solfataric mud pools, and characterization of Desulfotomaculum solfataricum

    NARCIS (Netherlands)

    Goorissen, H.P.; Stams, A.J.M.; Hansen, T.A.

    2003-01-01

    Four strains of thermophilic, endospore-forming, sulfate-reducing bacteria were enriched and isolated from hot solfataric fields in the Krafla area of north-east Iceland, using methanol and sulfite as substrates. Morphologically, these strains resembled thermophilic Desulfotomaculum species. The

  7. Fe/S protein biogenesis in trypanosomes — A review

    Czech Academy of Sciences Publication Activity Database

    Lukeš, Julius; Basu, Somsuvro

    2015-01-01

    Roč. 1853, č. 6 (2015), s. 1481-1492 ISSN 0167-4889 R&D Projects: GA ČR GAP305/12/2261; GA MŠk(CZ) EE2.3.30.0032; GA ČR(CZ) GA14-23986S EU Projects: European Commission(XE) 316304 Institutional support: RVO:60077344 Keywords : Fe/S cluster * Trypanosoma brucei * protists * Kinetoplastida Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.128, year: 2015

  8. Detecting genotypic variation among the single spore isolates of Pasteuria penetrans population occuring in Florida using SNP-based markers

    Science.gov (United States)

    Pasteuria penetrans is a naturally occurring soil-borne endospore-forming bacterium, which functions as a castrating parasite of plant-parasitic nematodes belonging to the genus Meloidogyne. Pasteuria penetrans is established as an effective biological control agent for control and management o...

  9. Photosystem II Assembly Steps Take Place in the Thylakoid Membrane of the Cyanobacterium Synechocystis sp PCC6803

    Czech Academy of Sciences Publication Activity Database

    Sealo, T.T.; Zhang, L.; Knoppová, Jana; Komenda, Josef; Norling, B.

    2016-01-01

    Roč. 57, č. 1 (2016), s. 95-104 ISSN 0032-0781 R&D Projects: GA ČR GBP501/12/G055; GA MŠk LO1416 Institutional support: RVO:61388971 Keywords : Aqueous two-phase partitioning * Cyanobacteria * Photosystem II biogenesis Subject RIV: EE - Microbiology, Virology Impact factor: 4.760, year: 2016

  10. A model for the biosynthesis and transport of plasma membrane-associated signaling receptors to the cell surface

    Directory of Open Access Journals (Sweden)

    Sorina Claudia Popescu

    2012-04-01

    Full Text Available Intracellular protein transport is emerging as critical in determining the outcome of receptor-activated signal transduction pathways. In plants, relatively little is known about the nature of the molecular components and mechanisms involved in coordinating receptor synthesis and transport to the cell surface. Recent advances in this field indicate that signaling pathways and intracellular transport machinery converge and coordinate to render receptors competent for signaling at their plasma membrane activity sites. The biogenesis and transport to the cell surface of signaling receptors appears to require both general trafficking and receptor-specific factors. Several molecular determinants, residing or associated with compartments of the secretory pathway and known to influence aspects in receptor biogenesis, are discussed and integrated into a predictive cooperative model for the functional expression of signaling receptors at the plasma membrane.

  11. Separation of the gluconeogenic and mitochondrial functions of pgc-1α through s6 kinase

    DEFF Research Database (Denmark)

    Lustig, Y.; Ruas, J.L.; Estall, J.L.

    2011-01-01

    PGC-1α is a transcriptional coactivator that powerfully regulates many pathways linked to energy homeostasis. Specifically, PGC-1α controls mitochondrial biogenesis in most tissues but also initiates important tissue-specific functions, including fiber type switching in skeletal muscle and glucon......PGC-1α is a transcriptional coactivator that powerfully regulates many pathways linked to energy homeostasis. Specifically, PGC-1α controls mitochondrial biogenesis in most tissues but also initiates important tissue-specific functions, including fiber type switching in skeletal muscle...... of gluconeogenesis in cultured hepatocytes and in vivo, while leaving the functions of PGC-1α as an activator of mitochondrial and fatty acid oxidation genes completely intact. These phosphorylations interfere with the ability of PGC-1α to bind to HNF4α, a transcription factor required for gluconeogenesis, while...

  12. Proteomic analyses reveal the key roles of BrlA and AbaA in biogenesis of gliotoxin in Aspergillus fumigatus

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Kwang-Soo, E-mail: shinks@dju.kr [Division of Life Science, Daejeon University, Daejeon, 300-716 (Korea, Republic of); Kim, Young Hwan [Biomedical Omics Team, Korea Basic Science Institute (KBSI), Ohcang, 368-883 (Korea, Republic of); Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, 305-764 (Korea, Republic of); Department of Bio-Analytical Science, University of Science and Technology, Daejeon, 305-333 (Korea, Republic of); Yu, Jae-Hyuk, E-mail: jyu1@wisc.edu [Departments of Bacteriology and Genetics, The University of Wisconsin–Madison, Madison, WI, 53706 (United States)

    2015-07-31

    The opportunistic human pathogenic fungus Aspergillus fumigatus primarily reproduces by forming a large number of asexual spores (conidia). Sequential activation of the central regulators BrlA, AbaA and WetA is necessary for the fungus to undergo asexual development. In this study, to address the presumed roles of these key developmental regulators during proliferation of the fungus, we analyzed and compared the proteomes of vegetative cells of wild type (WT) and individual mutant strains. Approximately 1300 protein spots were detectable from 2-D electrophoresis gels. Among these, 13 proteins exhibiting significantly altered accumulation levels were further identified by ESI-MS/MS. Markedly, we found that the GliM and GliT proteins associated with gliotoxin (GT) biosynthesis and self-protection of the fungus from GT were significantly down-regulated in the ΔabaA and ΔbrlA mutants. Moreover, mRNA levels of other GT biosynthetic genes including gliM, gliP, gliT, and gliZ were significantly reduced in both mutant strains, and no and low levels of GT were detectable in the ΔbrlA and ΔabaA mutant strains, respectively. As GliT is required for the protection of the fungus from GT, growth of the ΔbrlA mutant with reduced levels of GliT was severely impaired by exogenous GT. Our studies demonstrate that AbaA and BrlA positively regulate expression of the GT biosynthetic gene cluster in actively growing vegetative cells, and likely bridge morphological and chemical development during the life-cycle of A. fumigatus. - Highlights: • Proteome analyses of WT and mutants reveal 13 differentially expressed proteins. • The GliT and GliM proteins are significantly down-regulated by ΔabaA and ΔbrlA. • Expression of other gliotoxin biosynthetic genes is lowered by ΔabaA and ΔbrlA. • Growth of ΔbrlA strain lacking GliT is completely inhibited by exogenous gliotoxin. • BrlA and AbaA play key roles in biogenesis of gliotoxin in Aspergillus fumigatus.

  13. Proteomic analyses reveal the key roles of BrlA and AbaA in biogenesis of gliotoxin in Aspergillus fumigatus

    International Nuclear Information System (INIS)

    Shin, Kwang-Soo; Kim, Young Hwan; Yu, Jae-Hyuk

    2015-01-01

    The opportunistic human pathogenic fungus Aspergillus fumigatus primarily reproduces by forming a large number of asexual spores (conidia). Sequential activation of the central regulators BrlA, AbaA and WetA is necessary for the fungus to undergo asexual development. In this study, to address the presumed roles of these key developmental regulators during proliferation of the fungus, we analyzed and compared the proteomes of vegetative cells of wild type (WT) and individual mutant strains. Approximately 1300 protein spots were detectable from 2-D electrophoresis gels. Among these, 13 proteins exhibiting significantly altered accumulation levels were further identified by ESI-MS/MS. Markedly, we found that the GliM and GliT proteins associated with gliotoxin (GT) biosynthesis and self-protection of the fungus from GT were significantly down-regulated in the ΔabaA and ΔbrlA mutants. Moreover, mRNA levels of other GT biosynthetic genes including gliM, gliP, gliT, and gliZ were significantly reduced in both mutant strains, and no and low levels of GT were detectable in the ΔbrlA and ΔabaA mutant strains, respectively. As GliT is required for the protection of the fungus from GT, growth of the ΔbrlA mutant with reduced levels of GliT was severely impaired by exogenous GT. Our studies demonstrate that AbaA and BrlA positively regulate expression of the GT biosynthetic gene cluster in actively growing vegetative cells, and likely bridge morphological and chemical development during the life-cycle of A. fumigatus. - Highlights: • Proteome analyses of WT and mutants reveal 13 differentially expressed proteins. • The GliT and GliM proteins are significantly down-regulated by ΔabaA and ΔbrlA. • Expression of other gliotoxin biosynthetic genes is lowered by ΔabaA and ΔbrlA. • Growth of ΔbrlA strain lacking GliT is completely inhibited by exogenous gliotoxin. • BrlA and AbaA play key roles in biogenesis of gliotoxin in Aspergillus fumigatus

  14. Influence of crop production practices on Pasteuria penetrans and suppression of Meloidogyne incognita

    Science.gov (United States)

    Pasteuria penetrans is a parasite of root-knot nematodes (Meloidogyne spp.). Infected nematodes are not killed by the bacterium, but instead of producing eggs, females produce millions of infectious endospores. In addition to sterilizing females, P. penetrans can reduce nematode infection of roots...

  15. AAA-ATPase NVL2 acts on MTR4-exosome complex to dissociate the nucleolar protein WDR74

    Energy Technology Data Exchange (ETDEWEB)

    Hiraishi, Nobuhiro; Ishida, Yo-ichi; Nagahama, Masami, E-mail: nagahama@my-pharm.ac.jp

    2015-11-20

    Nuclear VCP-like 2 (NVL2) is a chaperone-like nucleolar ATPase of the AAA (ATPase associated with diverse cellular activities) family, which exhibits a high level of amino acid sequence similarity with the cytosolic AAA-ATPase VCP/p97. These proteins generally act on macromolecular complexes to stimulate energy-dependent release of their constituents. We previously showed that NVL2 interacts with RNA processing/degradation machinery containing an RNA helicase MTR4/DOB1 and an exonuclease complex, nuclear exosome, and involved in the biogenesis of 60S ribosomal subunits. These observations implicate NVL2 as a remodeling factor for the MTR4-exosome complex during the maturation of pre-ribosomal particles. Here, we used a proteomic screen and identified a WD repeat-containing protein 74 (WDR74) as a factor that specifically dissociates from this complex depending on the ATPase activity of NVL2. WDR74 shows weak amino acid sequence similarity with the yeast ribosome biogenesis protein Nsa1 and is co-localized with NVL2 in the nucleolus. Knockdown of WDR74 decreases 60S ribosome levels. Taken together, our results suggest that WDR74 is a novel regulatory protein of the MTR4-exsosome complex whose interaction is regulated by NVL2 and is involved in ribosome biogenesis. - Highlights: • WDR74 accumulates in MTR4-exosome complex upon expression of dominant-negative NVL2. • WDR74 is co-localized with NVL2 in the nucleolus. • WDR74, along with NVL2, is involved in the synthesis of 60S ribosomal subunits.

  16. AAA-ATPase NVL2 acts on MTR4-exosome complex to dissociate the nucleolar protein WDR74

    International Nuclear Information System (INIS)

    Hiraishi, Nobuhiro; Ishida, Yo-ichi; Nagahama, Masami

    2015-01-01

    Nuclear VCP-like 2 (NVL2) is a chaperone-like nucleolar ATPase of the AAA (ATPase associated with diverse cellular activities) family, which exhibits a high level of amino acid sequence similarity with the cytosolic AAA-ATPase VCP/p97. These proteins generally act on macromolecular complexes to stimulate energy-dependent release of their constituents. We previously showed that NVL2 interacts with RNA processing/degradation machinery containing an RNA helicase MTR4/DOB1 and an exonuclease complex, nuclear exosome, and involved in the biogenesis of 60S ribosomal subunits. These observations implicate NVL2 as a remodeling factor for the MTR4-exosome complex during the maturation of pre-ribosomal particles. Here, we used a proteomic screen and identified a WD repeat-containing protein 74 (WDR74) as a factor that specifically dissociates from this complex depending on the ATPase activity of NVL2. WDR74 shows weak amino acid sequence similarity with the yeast ribosome biogenesis protein Nsa1 and is co-localized with NVL2 in the nucleolus. Knockdown of WDR74 decreases 60S ribosome levels. Taken together, our results suggest that WDR74 is a novel regulatory protein of the MTR4-exsosome complex whose interaction is regulated by NVL2 and is involved in ribosome biogenesis. - Highlights: • WDR74 accumulates in MTR4-exosome complex upon expression of dominant-negative NVL2. • WDR74 is co-localized with NVL2 in the nucleolus. • WDR74, along with NVL2, is involved in the synthesis of 60S ribosomal subunits.

  17. Initiation of sporulation in Clostridium difficile: a twist on the classic model.

    Science.gov (United States)

    Edwards, Adrianne N; McBride, Shonna M

    2014-09-01

    The formation of dormant endospores is a complex morphological process that permits long-term survival in inhospitable environments for many Gram-positive bacteria. Sporulation for the anaerobic gastrointestinal pathogen Clostridium difficile is necessary for survival outside of the gastrointestinal tract of its host. While the developmental stages of spore formation are largely conserved among endospore-forming bacteria, the genus Clostridium appears to be missing a number of conserved regulators required for efficient sporulation in other spore-forming bacteria. Several recent studies have discovered novel mechanisms and distinct regulatory pathways that control the initiation of sporulation and early-sporulation-specific gene expression. These differences in regulating the decision to undergo sporulation reflects the unique ecological niche and environmental conditions that C. difficile inhabits and encounters within the mammalian host. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  18. 'Candidatus Pasteuria aldrichii', an obligate endoparasite of the bacterivorous nematode Bursilla.

    Science.gov (United States)

    Giblin-Davis, R M; Nong, G; Preston, J F; Williams, D S; Center, B J; Brito, J A; Dickson, D W

    2011-09-01

    A novel bacterium of the genus Pasteuria was discovered parasitizing bacterivorous nematodes of the genus Bursilla, in selected bermudagrass (Cynodon) field plots in Davie, FL, USA. Soil containing this bacterium was sampled and supplied with bi-weekly inoculations of cultured species of the genus Bursilla in order to build and maintain a source of endospores for continuous in vivo conservation of the bacteria for further study and characterization. 16S rRNA gene sequence similarities supported its congeneric ranking with other members of the genus Pasteuria that have been identified from nematodes and cladocerans. There were, however, no clear sister candidates for this organism, which supported the evidence of endospore ultrastructure and host-range studies, suggesting it belonged to a novel taxon. Because members of the genus Pasteuria cannot yet be isolated, definitive type strains could not be maintained; therefore, the name 'Candidatus Pasteuria aldrichii' is proposed for this organism.

  19. Serum-free culture alters the quantity and protein composition of neuroblastoma-derived extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Jinghuan Li

    2015-05-01

    Full Text Available Extracellular vesicles (EVs play a significant role in cell–cell communication in numerous physiological processes and pathological conditions, and offer promise as novel biomarkers and therapeutic agents for genetic diseases. Many recent studies have described different molecular mechanisms that contribute to EV biogenesis and release from cells. However, little is known about how external stimuli such as cell culture conditions can affect the quantity and content of EVs. While N2a neuroblastoma cells cultured in serum-free (OptiMEM conditions did not result in EVs with significant biophysical or size differences compared with cells cultured in serum-containing (pre-spun conditions, the quantity of isolated EVs was greatly increased. Moreover, the expression levels of certain vesicular proteins (e.g. small GTPases, G-protein complexes, mRNA processing proteins and splicing factors, some of which were previously reported to be involved in EV biogenesis, were found to be differentially expressed in EVs under different culture conditions. These data, therefore, contribute to the understanding of how extracellular factors and intracellular molecular pathways affect the composition and release of EVs.

  20. Purple sweet potato color attenuates domoic acid-induced cognitive deficits by promoting estrogen receptor-α-mediated mitochondrial biogenesis signaling in mice.

    Science.gov (United States)

    Lu, Jun; Wu, Dong-Mei; Zheng, Yuan-Lin; Hu, Bin; Cheng, Wei; Zhang, Zi-Feng

    2012-02-01

    Recent findings suggest that endoplasmic reticulum stress may be involved in the pathogenesis of domoic acid-induced neurodegeneration. Purple sweet potato color, a class of naturally occurring anthocyanins, has beneficial health and biological effects. Recent studies have also shown that anthocyanins have estrogenic activity and can enhance estrogen receptor-α expression. In this study, we evaluated the effect of purple sweet potato color on cognitive deficits induced by hippocampal mitochondrial dysfunction in domoic acid-treated mice and explored the potential mechanisms underlying this effect. Our results showed that the oral administration of purple sweet potato color to domoic acid-treated mice significantly improved their behavioral performance in a step-through passive avoidance task and a Morris water maze task. These improvements were mediated, at least in part, by a stimulation of estrogen receptor-α-mediated mitochondrial biogenesis signaling and by decreases in the expression of p47phox and gp91phox. Decreases in reactive oxygen species and protein carbonylation were also observed, along with a blockade of the endoplasmic reticulum stress pathway. Furthermore, purple sweet potato color significantly suppressed endoplasmic reticulum stress-induced apoptosis, which prevented neuron loss and restored the expression of memory-related proteins. However, knockdown of estrogen receptor-α using short hairpin RNA only partially blocked the neuroprotective effects of purple sweet potato color in the hippocampus of mice cotreated with purple sweet potato color and domoic acid, indicating that purple sweet potato color acts through multiple pathways. These results suggest that purple sweet potato color could be a possible candidate for the prevention and treatment of cognitive deficits in excitotoxic and other brain disorders. Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.

  1. A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement*

    Science.gov (United States)

    Chuang, Tzu-Wei; Lee, Kuo-Ming; Lou, Yuan-Chao; Lu, Chia-Chen; Tarn, Woan-Yuh

    2016-01-01

    Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis. PMID:26887951

  2. A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement.

    Science.gov (United States)

    Chuang, Tzu-Wei; Lee, Kuo-Ming; Lou, Yuan-Chao; Lu, Chia-Chen; Tarn, Woan-Yuh

    2016-04-15

    Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. The Pseudo signal peptide of the corticotropin-releasing factor receptor type 2A prevents receptor oligomerization.

    Science.gov (United States)

    Teichmann, Anke; Rutz, Claudia; Kreuchwig, Annika; Krause, Gerd; Wiesner, Burkhard; Schülein, Ralf

    2012-08-03

    N-terminal signal peptides mediate the interaction of native proteins with the translocon complex of the endoplasmic reticulum membrane and are cleaved off during early protein biogenesis. The corticotropin-releasing factor receptor type 2a (CRF(2(a))R) possesses an N-terminal pseudo signal peptide, which represents a so far unique domain within the large protein family of G protein-coupled receptors (GPCRs). In contrast to a conventional signal peptide, the pseudo signal peptide remains uncleaved and consequently forms a hydrophobic extension at the N terminus of the receptor. The functional consequence of the presence of the pseudo signal peptide is not understood. Here, we have analyzed the significance of this domain for receptor dimerization/oligomerization in detail. To this end, we took the CRF(2(a))R and the homologous corticotropin-releasing factor receptor type 1 (CRF(1)R) possessing a conventional cleaved signal peptide and conducted signal peptide exchange experiments. Using single cell and single molecule imaging methods (fluorescence resonance energy transfer and fluorescence cross-correlation spectroscopy, respectively) as well as biochemical experiments, we obtained two novel findings; we could show that (i) the CRF(2(a))R is expressed exclusively as a monomer, and (ii) the presence of the pseudo signal peptide prevents its oligomerization. Thus, we have identified a novel functional domain within the GPCR protein family, which plays a role in receptor oligomerization and which may be useful to study the functional significance of this process in general.

  4. C. elegans nucleostemin is required for larval growth and germline stem cell division.

    Directory of Open Access Journals (Sweden)

    Michelle M Kudron

    2008-08-01

    Full Text Available The nucleolus has shown to be integral for many processes related to cell growth and proliferation. Stem cells in particular are likely to depend upon nucleolus-based processes to remain in a proliferative state. A highly conserved nucleolar factor named nucleostemin is proposed to be a critical link between nucleolar function and stem-cell-specific processes. Currently, it is unclear whether nucleostemin modulates proliferation by affecting ribosome biogenesis or by another nucleolus-based activity that is specific to stem cells and/or highly proliferating cells. Here, we investigate nucleostemin (nst-1 in the nematode C. elegans, which enables us to examine nst-1 function during both proliferation and differentiation in vivo. Like mammalian nucleostemin, the NST-1 protein is localized to the nucleolus and the nucleoplasm; however, its expression is found in both differentiated and proliferating cells. Global loss of C. elegans nucleostemin (nst-1 leads to a larval arrest phenotype due to a growth defect in the soma, while loss of nst-1 specifically in the germ line causes germline stem cells to undergo a cell cycle arrest. nst-1 mutants exhibit reduced levels of rRNAs, suggesting defects in ribosome biogenesis. However, NST-1 is generally not present in regions of the nucleolus where rRNA transcription and processing occurs, so this reduction is likely secondary to a different defect in ribosome biogenesis. Transgenic studies indicate that NST-1 requires its N-terminal domain for stable expression and both its G1 GTPase and intermediate domains for proper germ line function. Our data support a role for C. elegans nucleostemin in cell growth and proliferation by promoting ribosome biogenesis.

  5. Gel-free proteomic identification of the Bacillus subtilis insoluble spore coat protein fraction.

    NARCIS (Netherlands)

    Abhyankar, W; Beek, A.T.; Dekker, H.; Kort, R.; Brul, S.; de Koster, C. G.

    2011-01-01

    Species from the genus Bacillus have the ability to form endospores, dormant cellular forms that are able to survive heat and acid preservation techniques commonly used in the food industry. Resistance characteristics of spores towards various environmental stresses are in part attributed to their

  6. Spo0A regulates chromosome copy number during sporulation by directly binding to the origin of replication in Bacillus subtilis

    NARCIS (Netherlands)

    Boonstra, Mirjam; de Jong, Imke G.; Scholefield, Graham; Murray, Heath; Kuipers, Oscar P.; Veening, Jan-Willem

    When starved, Bacillus subtilis cells can enter the developmental programme of endospore formation by activation of the master transcriptional regulator Spo0A. Correct chromosome copy number is crucial for the production of mature and fully resistant spores. The production and maintenance of one

  7. Natural suppression of Meloidogyne incognita by Pasteuria penetrans in cotton

    Science.gov (United States)

    The endospore-forming bacterium Pasteuria penetrans is an obligate parasite of root-knot nematodes (Meloidogyne spp.). This bacterium is commonly found in agricultural soils and has been associated with suppression of Meloidogyne spp. In a field site naturally infested with both P. penetrans and M...

  8. A real-time PCR assay for the detection of Pasteuria nishizawae in soil

    Science.gov (United States)

    Pasteuria nishizawae is a mycelial, endospore-forming, and obligately parasitic bacterium that has shown a great potential for the biological control of the soybean cyst nematode, Heterodera glycines, in both microplot and field studies. Other than China, Japan, and South Korea, P. nishizawae has be...

  9. Gel-free proteomic identification of the Bacillus subtilis insoluble spore coat protein fraction

    NARCIS (Netherlands)

    Abhyankar, W.; Beek, A.T.; Dekker, H.; Kort, R.; Brul, S.; Koster, C.G. de

    2011-01-01

    Species from the genus Bacillus have the ability to form endospores, dormant cellular forms that are able to survive heat and acid preservation techniques commonly used in the food industry. Resistance characteristics of spores towards various environmental stresses are in part attributed to their

  10. Disinfection of Airborne Organisms by Ultraviolet-C Radiation and Sunlight

    Science.gov (United States)

    2012-07-01

    organisms used in this test were E. coli cells, B. subtilis spores, Candida famata var. flareri cells, and Penicillium citrinum spores. A few studies...in Protecting Bacillus sp . Endospores Against Environmental UV Radiation. FEMS Microbiol. Ecol. 2005, 51, 231–236. Munakata, N. Comparative

  11. Antibiosis plays a role in the context of direct interaction during antagonism of Paenibacillus polymyxa towards Fusarium oxysporum

    NARCIS (Netherlands)

    Dijksterhuis, J; Sanders, M; Gorris, L G; Smid, E J

    Interaction of Fusarium oxysporum and Paenibacillus polymyxa starts with polar attachment of bacteria to the fungal hyphae followed by the formation of a large cluster of non-motile cells embedded in an extracellular matrix in which the bacteria develop endospores. Enumeration of fungal viable

  12. σK of Clostridium acetobutylicum is the first known sporulation-specific sigma factor with two developmentally separated roles, one early and one late in sporulation.

    Science.gov (United States)

    Al-Hinai, Mohab A; Jones, Shawn W; Papoutsakis, Eleftherios T

    2014-01-01

    Sporulation in the model endospore-forming organism Bacillus subtilis proceeds via the sequential and stage-specific activation of the sporulation-specific sigma factors, σ(H) (early), σ(F), σ(E), σ(G), and σ(K) (late). Here we show that the Clostridium acetobutylicum σ(K) acts both early, prior to Spo0A expression, and late, past σ(G) activation, thus departing from the B. subtilis model. The C. acetobutylicum sigK deletion (ΔsigK) mutant was unable to sporulate, and solventogenesis, the characteristic stationary-phase phenomenon for this organism, was severely diminished. Transmission electron microscopy demonstrated that the ΔsigK mutant does not develop an asymmetric septum and produces no granulose. Complementation of sigK restored sporulation and solventogenesis to wild-type levels. Spo0A and σ(G) proteins were not detectable by Western analysis, while σ(F) protein levels were significantly reduced in the ΔsigK mutant. spo0A, sigF, sigE, sigG, spoIIE, and adhE1 transcript levels were all downregulated in the ΔsigK mutant, while those of the sigH transcript were unaffected during the exponential and transitional phases of culture. These data show that σ(K) is necessary for sporulation prior to spo0A expression. Plasmid-based expression of spo0A in the ΔsigK mutant from a nonnative promoter restored solventogenesis and the production of Spo0A, σ(F), σ(E), and σ(G), but not sporulation, which was blocked past the σ(G) stage of development, thus demonstrating that σ(K) is also necessary in late sporulation. sigK is expressed very early at low levels in exponential phase but is strongly upregulated during the middle to late stationary phase. This is the first sporulation-specific sigma factor shown to have two developmentally separated roles.

  13. Exercise induces transient transcriptional activation of the PGC-1a gene in human skeletal muscle

    DEFF Research Database (Denmark)

    Pilegaard, Henriette; Saltin, Bengt; Neufer, P. Darrell

    2003-01-01

    Endurance exercise training induces mitochondrial biogenesis in skeletal muscle. The peroxisome proliferator activated receptor co-activator 1a (PGC-1a) has recently been identified as a nuclear factor critical for coordinating the activation of genes required for mitochondrial biogenesis in cell...... culture and rodent skeletal muscle. To determine whether PGC-1a transcription is regulated by acute exercise and exercise training in human skeletal muscle, seven male subjects performed 4 weeks of one-legged knee extensor exercise training. At the end of training, subjects completed 3 h of two......-legged knee extensor exercise. Biopsies were obtained from the vastus lateralis muscle of both the untrained and trained legs before exercise and after 0, 2, 6 and 24 h of recovery. Time to exhaustion (2 min maximum resistance), as well as hexokinase II (HKII), citrate synthase and 3-hydroxyacyl...

  14. Subsurface Endospore-Forming Bacteria Possess Bio-Sealant Properties.

    Science.gov (United States)

    Basha, Sreenivasulu; Lingamgunta, Lakshman Kumar; Kannali, Jayakumar; Gajula, Swarna Kumari; Bandikari, Ramesh; Dasari, Sreenivasulu; Dalavai, Veena; Chinthala, Paramageetham; Gundala, Prasada Babu; Kutagolla, Peera; Balaji, Vinodh Kumar

    2018-04-24

    Concrete is a strong and fairly inexpensive building substance, but has several disadvantages like cracking that allows corrosion, thus reducing its lifespan. To mitigate these complications, long-lasting microbial self-healing cement is an alternative that is eco-friendly and also actively repairs cracks. The present paper describes the detailed experimental investigation on compressive strength of cement mortars, mixed with six alkaliphilic bacteria, isolated from subsurface mica mines of high alkalinity. The experiments showed that the addition of alkaliphilic isolates at different cell concentrations (10 4 and 10 6 cells/ml) enhanced the compressive strength of cement mortar, because the rapid growth of bacteria at high alkalinity precipitates calcite crystals that lead to filling of pores and densifying the concrete mix. Thus, Bacillus subtilis (SVUNM4) showed the highest compressive strength (28.61%) of cement mortar at 10 4 cells/ml compared to those of other five alkaliphilic isolates (Brevibacillus sp., SVUNM15-22.1%; P. dendritiformis, SVUNM11-19.9%; B. methylotrophicus, SVUNM9-16%; B. licheniformis, SVUNM14-12.7% and S. maltophilia, SVUNM13-9.6%) and controlled cement mortar as well. This method resulted in the filling of cracks in concrete with calcite (CaCO 3 ), which was observed by scanning electron microscopy (SEM). Our results showed that the alkaliphilic bacterial isolates used in the study are effective in self-healing and repair of concrete cracks.

  15. Characterization of germination receptors of Bacillus cereus ATCC 14579

    NARCIS (Netherlands)

    Hornstra, L.M.; Vries, de Y.P.; Wells-Bennik, M.H.J.; Vos, de W.M.; Abee, T.

    2006-01-01

    Specific amino acids, purine ribonucleosides, or a combination of the two is required for efficient germination of endospores of Bacillus cereus ATCC 14579. A survey including 20 different amino acids showed that L-alanine, L-cysteine, L-threonine, and L-glutamine are capable of initiating the

  16. Evidence for Reciprocal Selection between Populations of Meloidogyne arenaria and Pasteuria penetrans in a Field Study

    Science.gov (United States)

    Beginning in 1998, a bioassay using second-stage juveniles (J2) from a greenhouse (GH) population of Meloidogyne arenaria (Ma) was used to monitor endospore densities of the bacterium Pasteuria penetrans, which was parasitizing Ma in a crop rotation study. Spore densities of the bacterium were very...

  17. [Anthrax due to deliberate infection

    NARCIS (Netherlands)

    Dissel, J.T. van; Kullberg, B.J.; Berg, P.C. van den; Steenbergen, J.E. van

    2001-01-01

    Anthrax is a zoonosis which is particularly prevalent in cattle, goats and sheep and is caused by Bacillus anthracis, a Gram-positive spore forming aerobic microorganism. The endospores can survive outside of the body for many decades. The natural form of anthrax has a cutaneous, pulmonary and

  18. Expression and characterization of a novel spore wall protein from ...

    African Journals Online (AJOL)

    Microsporidia are obligate intracellular, eukaryotic, spore-forming parasites. The environmentally resistant spores, which harbor a rigid cell wall, are critical for their survival outside their host cells and host-to-host transmission. The spore wall comprises two major layers: the exospore and the endospore. In Nosema ...

  19. Diesel exhaust particulate extracts inhibit transcription of nuclear respiratory factor-1 and cell viability in human umbilical vein endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Mattingly, Kathleen A.; Klinge, Carolyn M. [University of Louisville School of Medicine, Department of Biochemistry and Molecular Biology, Center for Genetics and Molecular Medicine, Louisville, KY (United States)

    2012-04-15

    Endothelial dysfunction precedes cardiovascular disease and is accompanied by mitochondrial dysfunction. Here we tested the hypothesis that diesel exhaust particulate extracts (DEPEs), prepared from a truck run at different speeds and engine loads, would inhibit genomic estrogen receptor activation of nuclear respiratory factor-1 (NRF-1) transcription in human umbilical vein endothelial cells (HUVECs). Additionally, we examined how DEPEs affect NRF-1-regulated TFAM expression and, in turn, Tfam-regulated mtDNA-encoded cytochrome c oxidase subunit I (COI, MTCO1) and NADH dehydrogenase subunit I (NDI) expression as well as cell proliferation and viability. We report that 17{beta}-estradiol (E{sub 2}), 4-hydroxytamoxifen (4-OHT), and raloxifene increased NRF-1 transcription in HUVECs in an ER-dependent manner. DEPEs inhibited NRF-1 transcription, and this suppression was not ablated by concomitant treatment with E{sub 2}, 4-OHT, or raloxifene, indicating that the effect was not due to inhibition of ER activity. While E{sub 2} increased HUVEC proliferation and viability, DEPEs inhibited viability but not proliferation. Resveratrol increased NRF-1 transcription in an ER-dependent manner in HUVECs, and ablated DEPE inhibition of basal NRF-1 expression. Given that NRF-1 is a key nuclear transcription factor regulating genes involved in mitochondrial activity and biogenesis, these data suggest that DEPEs may adversely affect mitochondrial function leading to endothelial dysfunction and resveratrol may block these effects. (orig.)

  20. Inhibition of toxicogenic Bacillus cereus in rice-based foods by enterocin AS-48.

    Science.gov (United States)

    Grande, Maria J; Lucas, Rosario; Abriouel, Hikmate; Valdivia, Eva; Omar, Nabil Ben; Maqueda, Mercedes; Martínez-Bueno, Manuel; Martínez-Cañamero, Magdalena; Gálvez, Antonio

    2006-02-01

    The antimicrobial effect of the broad-spectrum bacteriocin enterocin AS-48 against the toxicogenic psychrotrophic strain Bacillus cereus LWL1 has been investigated in a model food system consisting of boiled rice and in a commercial infant rice-based gruel dissolved in whole milk stored at temperatures of 37 degrees C, 15 degrees C and 6 degrees C. In food samples supplemented with enterocin AS-48 (in a concentration range of 20-35 mug/ml), viable cell counts decreased rapidly over incubation time, depending on the bacteriocin concentration, the temperature of incubation and the food sample. Enterotoxin production at 37 degrees C was also inhibited. Heat sensitivity of endospores increased markedly in food samples supplemented with enterocin AS-48: inactivation of endospores was achieved by heating for 1 min at 90 degrees C in boiled rice or at 95 degrees C in rice-based gruel. Activity of enterocin AS-48 in rice gruel was potentiated by sodium lactate in a concentration-dependent way.

  1. A mother cell-to-forespore channel: current understanding and future challenges.

    Science.gov (United States)

    Crawshaw, Adam D; Serrano, Mónica; Stanley, Will A; Henriques, Adriano O; Salgado, Paula S

    2014-09-01

    Formation of endospores allows some bacteria to survive extreme nutrient limitation. The resulting dormant cell, the spore, persists in the environment and is highly resistant to physical and chemical stresses. During spore formation, cells divide asymmetrically and the mother cell engulfs the developing spore, encasing it within a double membrane and isolating it from the medium. Communication between mother cell and isolated forespore involves a specialised connection system that allows nurturing of the forespore and continued macromolecular synthesis, required to finalise spore maturation. Here, we review current understanding of this feeding channel formed by a forespore protein, SpoIIQ, and a mother cell protein, SpoIIIAH, in the model organism Bacillus subtilis and the important human pathogen Clostridium difficile. We also analyse the presence of this channel across endospore-forming bacteria and highlight the main questions still remaining. © 2014 The Authors FEMS Microbiology Letters published by John Wiley & Sons Ltd on behalf of Federation of European Microbiological Societies.

  2. Biogenesis of antibiotics-viewing its history and glimpses of the future

    Czech Academy of Sciences Publication Activity Database

    Spížek, Jaroslav; Sigler, Karel; Řezanka, Tomáš; Demain, A.

    2016-01-01

    Roč. 61, č. 4 (2016), s. 347-358 ISSN 0015-5632 Institutional support: RVO:61388971 Keywords : STREPTOMYCES-GLAUCESCENS * POLYKETIDE SYNTHASE * SYNTHETIC BIOLOGY Subject RIV: EI - Biotechnology ; Bionics Impact factor: 1.521, year: 2016

  3. Minimal cytosolic iron-sulfur cluster assembly machinery of Giardia intestinalis is partially associated with mitosomes

    Czech Academy of Sciences Publication Activity Database

    Pyrih, J.; Pyrihová, E.; Kolisko, M.; Stojanovova, D.; Basu, Somsuvro; Harant, K.; Haindrich, Alexander C.; Doležal, P.; Lukeš, Julius; Roger, A.; Tachezy, J.

    2016-01-01

    Roč. 102, č. 4 (2016), s. 701-714 ISSN 0950-382X R&D Projects: GA ČR GA15-21974S Institutional support: RVO:60077344 Keywords : Trypanosoma brucei * protein maturation * abc transporter * Trichomonas vaginalis * cfd1-nbp35 complex * DNA metabolism * Fe/S proteins * mitochondrion * biogenesis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.898, year: 2016

  4. HIF and reactive oxygen species regulate oxidative phosphorylation in cancer

    Czech Academy of Sciences Publication Activity Database

    Hervouet, E.; Čížková, Alena; Demont, J.; Vojtíšková, Alena; Pecina, Petr; Franssen-van Hal, N.; Keijer, J.; Simonnet, H.; Ivánek, Robert; Kmoch, S.; Godinot, C.; Houštěk, Josef

    2008-01-01

    Roč. 29, č. 8 (2008), s. 1528-1537 ISSN 0143-3334 R&D Projects: GA MŠk(CZ) 1M0520; GA ČR GA303/07/0781 Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z50520514 Keywords : carcinoma * mitochondrial biogenesis * reactive oxygen species Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.930, year: 2008

  5. Mitochondrial membrane assembly of TMEM70 protein

    Czech Academy of Sciences Publication Activity Database

    Kratochvílová, H.; Hejzlarová, Kateřina; Vrbacký, Marek; Mráček, Tomáš; Karbanová, Vendula; Tesařová, M.; Gombitová, A.; Cmarko, D.; Wittig, I.; Zeman, J.; Houštěk, Josef

    2014-01-01

    Roč. 15, Mar 2014 (2014), s. 1-9 ISSN 1567-7249 R&D Projects: GA ČR(CZ) GAP303/11/0970; GA MŠk(CZ) LL1204; GA ČR(CZ) GB14-36804G Institutional support: RVO:67985823 Keywords : mitochondria * ATP synthase * TMEM70 * biogenesis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.249, year: 2014

  6. AN 11-YEAR FIELD STUDY WITH PASTEURIA PENETRANS: LESSONS LEARNED THE HARD WAY.

    Science.gov (United States)

    Beginning in 1998, a bioassay using second-stage juveniles (J2) from a greenhouse (GH) population of the root-knot nematode Meloidogyne arenaria (Ma) was used to monitor endospore densities of the bacterium Pasteuria penetrans, which was parasitizing Ma in a long-term rotation study (begun in 1991)....

  7. High-Level Heat Resistance of Spores of Bacillus amyloliquefaciens and Bacillus licheniformis Results from the Presence of a spoVA Operon in a Tn1546 Transposon

    NARCIS (Netherlands)

    Berendsen, Erwin M; Koning, Rosella A; Boekhorst, Jos; de Jong, Anne; Kuipers, Oscar P; Wells-Bennik, Marjon H J

    2016-01-01

    Bacterial endospore formers can produce spores that are resistant to many food processing conditions, including heat. Some spores may survive heating processes aimed at production of commercially sterile foods. Recently, it was shown that a spoVA operon, designated spoVA(2mob), present on a Tn1546

  8. Influence of the irradiation by the γ rays on various factors of immunity

    International Nuclear Information System (INIS)

    Gaude, G.; Coursaget, J.

    1955-01-01

    Exposed animals often present, in the days or weeks following the irradiation, heavy infectious lesions. The goal of this survey was to search the influence of the total irradiation by the gamma radiation from cobalt-60, on the organism, on the phagocytic power of the leukocytes and on the biogenesis of antibodies. The experiences were done on hundred animals, rabbits and guinea pigs. All irradiations have been achieved with eight sources of Cobalt 60 of 10 curies each, disposed on a sphere of 1 m, the animal living the region neighboring of the center. The intensity of the radiation, in these conditions, is about 100 roentgens per hour. (authors) [fr

  9. Potassium ferrate [Fe(VI] does not mediate self-sterilization of a surrogate mars soil

    Directory of Open Access Journals (Sweden)

    Paszczynski Andrzej

    2003-03-01

    Full Text Available Abstract Background Martian soil is thought to be enriched with strong oxidants such as peroxides and/or iron in high oxidation states that might destroy biological materials. There is also a high flux of ultraviolet radiation at the surface of Mars. Thus, Mars may be inhospitable to life as we know it on Earth. We examined the hypothesis that if the soil of Mars contains ferrates [Fe(VI], the strongest of the proposed oxidizing species, and also is exposed to high fluxes of UV radiation, it will be self-sterilizing. Results Under ambient conditions (25°C, oxygen and water present K2FeO4 mixed into sand mineralized some reactive organic molecules to CO2, while less reactive compounds were not degraded. Dried endospores of Bacillus subtilis incubated in a Mars surrogate soil comprised of dry silica sand containing 20% by weight K2FeO4 and under conditions similar to those now on Mars (extreme desiccation, cold, and a CO2-dominated atmosphere were resistant to killing by the ferrate-enriched sand. Similar results were observed with permanganate. Spores in oxidant-enriched sand exposed to high fluxes of UV light were protected from the sporocidal activity of the radiation below about 5 mm depths. Conclusion Based on our data and previously published descriptions of ancient but dormant life forms on Earth, we suggest that if entities resembling bacterial endospores were produced at some point by life forms on Mars, they might still be present and viable, given appropriate germination conditions. Endospores delivered to Mars on spacecraft would possibly survive and potentially compromise life detection experiments.

  10. Pasteuria penetrans for Control of Meloidogyne incognita on Tomato and Cucumber, and M. arenaria on Snapdragon.

    Science.gov (United States)

    Kokalis-Burelle, Nancy

    2015-09-01

    Meloidogyne incognita and Meloidogyne arenaria are important parasitic nematodes of vegetable and ornamental crops. Microplot and greenhouse experiments were conducted to test commercial formulations of the biocontrol agent Pasteuria penetrans for control of M. incognita on tomato and cucumber and M. arenaria on snapdragon. Three methods of application for P. penetrans were assessed including seed, transplant, and post-plant treatments. Efficacy in controlling galling and reproduction of the two root-knot nematode species was evaluated. Seed treatment application was assessed only for M. incognita on cucumber. Pasteuria treatment rates of a granular transplant formulation ranged from 1.5 × 10(5) endospores/cm(3) to 3 × 10(5) endospores/cm(3) of transplant mix applied at seeding. Additional applications of 1.5 × 10(5) endospores/cm(3) of soil were applied as a liquid formulation to soil post-transplant for both greenhouse and microplot trials. In greenhouse cucumber trials, all Pasteuria treatments were equivalent to steamed soil for reducing M. incognita populations in roots and soil, and reducing nematode reproduction and galling. In cucumber microplot trials there were no differences among treatments for M. incognita populations in roots or soil, eggs/g root, or root condition ratings. Nematode reproduction on cucumber was low with Telone II and with the seed treatment plus post-plant application of Pasteuria, which had the lowest nematode reproduction. However, galling for all Pasteuria treatments was higher than galling with Telone II. Root-knot nematode control with Pasteuria in greenhouse and microplot trials varied on tomato and snapdragon. Positive results were achieved for control of M. incognita with the seed treatment application on cucumber.

  11. Proteomic Analysis of Chloroplast-to-Chromoplast Transition in Tomato Reveals Metabolic Shifts Coupled with Disrupted Thylakoid Biogenesis Machinery and Elevated Energy-Production Components1[W

    Science.gov (United States)

    Barsan, Cristina; Zouine, Mohamed; Maza, Elie; Bian, Wanping; Egea, Isabel; Rossignol, Michel; Bouyssie, David; Pichereaux, Carole; Purgatto, Eduardo; Bouzayen, Mondher; Latché, Alain; Pech, Jean-Claude

    2012-01-01

    A comparative proteomic approach was performed to identify differentially expressed proteins in plastids at three stages of tomato (Solanum lycopersicum) fruit ripening (mature-green, breaker, red). Stringent curation and processing of the data from three independent replicates identified 1,932 proteins among which 1,529 were quantified by spectral counting. The quantification procedures have been subsequently validated by immunoblot analysis of six proteins representative of distinct metabolic or regulatory pathways. Among the main features of the chloroplast-to-chromoplast transition revealed by the study, chromoplastogenesis appears to be associated with major metabolic shifts: (1) strong decrease in abundance of proteins of light reactions (photosynthesis, Calvin cycle, photorespiration) and carbohydrate metabolism (starch synthesis/degradation), mostly between breaker and red stages and (2) increase in terpenoid biosynthesis (including carotenoids) and stress-response proteins (ascorbate-glutathione cycle, abiotic stress, redox, heat shock). These metabolic shifts are preceded by the accumulation of plastid-encoded acetyl Coenzyme A carboxylase D proteins accounting for the generation of a storage matrix that will accumulate carotenoids. Of particular note is the high abundance of proteins involved in providing energy and in metabolites import. Structural differentiation of the chromoplast is characterized by a sharp and continuous decrease of thylakoid proteins whereas envelope and stroma proteins remain remarkably stable. This is coincident with the disruption of the machinery for thylakoids and photosystem biogenesis (vesicular trafficking, provision of material for thylakoid biosynthesis, photosystems assembly) and the loss of the plastid division machinery. Altogether, the data provide new insights on the chromoplast differentiation process while enriching our knowledge of the plant plastid proteome. PMID:22908117

  12. A Novel Iron Chelator-Radical Scavenger Ameliorates Motor Dysfunction and Improves Life Span and Mitochondrial Biogenesis in SOD1G93A ALS Mice.

    Science.gov (United States)

    Golko-Perez, Sagit; Amit, Tamar; Bar-Am, Orit; Youdim, Moussa B H; Weinreb, Orly

    2017-02-01

    The aim of the present study was to evaluate the therapeutic effect of the novel neuroprotective multitarget brain permeable monoamine oxidase inhibitor/iron chelating-radical scavenging drug, VAR10303 (VAR), co-administered with high-calorie/energy-supplemented diet (ced) in SOD1 G93A transgenic amyotrophic lateral sclerosis (ALS) mice. Administration of VAR-ced was initiated after the appearance of disease symptoms (at day 88), as this regimen is comparable with the earliest time at which drug therapy could start in ALS patients. Using this rescue protocol, we demonstrated in the current study that VAR-ced treatment provided several beneficial effects in SOD1 G93A mice, including improvement in motor performance, elevation of survival time, and attenuation of iron accumulation and motoneuron loss in the spinal cord. Moreover, VAR-ced treatment attenuated neuromuscular junction denervation and exerted a significant preservation of myofibril regular morphology, associated with a reduction in the expression levels of genes related to denervation and atrophy in the gastrocnemius (GNS) muscle in SOD1 G93A mice. These effects were accompanied by upregulation of mitochondrial DNA and elevated activities of complexes I and II in the GNS muscle. We have also demonstrated that VAR-ced treatment upregulated the mitochondrial biogenesis master regulator, peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α) and increased PGC-1α-targeted metabolic genes and proteins, such as, PPARγ, UCP1/3, NRF1/2, Tfam, and ERRα in GNS muscle. These results provide evidence of therapeutic potential of VAR-ced in SOD1 G93A mice with underlying molecular mechanisms, further supporting the importance role of multitarget iron chelators in ALS treatment.

  13. Effect of tillage and fumigation on Pasteuria penetrans

    Science.gov (United States)

    The endospore-forming bacterium Pasteuria penetrans (Pp) is a parasite of Meloidogyne spp. In this study, the effect of tillage and the fumigant 1,3-dichloropropene (1,3-D) on numbers of Pp and suppression of M. incognita (Mi) was evaluated from 2011-2013. A split-plot experiment was established i...

  14. C. difficile 630Δerm Spo0A regulates sporulation, but does not contribute to toxin production, by direct high-affinity binding to target DNA.

    Directory of Open Access Journals (Sweden)

    Katharina E Rosenbusch

    Full Text Available Clostridium difficile is a Gram positive, anaerobic bacterium that can form highly resistant endospores. The bacterium is the causative agent of C. difficile infection (CDI, for which the symptoms can range from a mild diarrhea to potentially fatal pseudomembranous colitis and toxic megacolon. Endospore formation in Firmicutes, including C. difficile, is governed by the key regulator for sporulation, Spo0A. In Bacillus subtilis, this transcription factor is also directly or indirectly involved in various other cellular processes. Here, we report that C. difficile Spo0A shows a high degree of similarity to the well characterized B. subtilis protein and recognizes a similar binding sequence. We find that the laboratory strain C. difficile 630Δerm contains an 18bp-duplication near the DNA-binding domain compared to its ancestral strain 630. In vitro binding assays using purified C-terminal DNA binding domain of the C. difficile Spo0A protein demonstrate direct binding to DNA upstream of spo0A and sigH, early sporulation genes and several other putative targets. In vitro binding assays suggest that the gene encoding the major clostridial toxin TcdB may be a direct target of Spo0A, but supernatant derived from a spo0A negative strain was no less toxic towards Vero cells than that obtained from a wild type strain, in contrast to previous reports. These results identify for the first time direct (putative targets of the Spo0A protein in C. difficile and make a positive effect of Spo0A on production of the large clostridial toxins unlikely.

  15. The regulation of mitochondrial transcription factor A (Tfam) expression during skeletal muscle cell differentiation.

    Science.gov (United States)

    Collu-Marchese, Melania; Shuen, Michael; Pauly, Marion; Saleem, Ayesha; Hood, David A

    2015-05-19

    The ATP demand required for muscle development is accommodated by elevations in mitochondrial biogenesis, through the co-ordinated activities of the nuclear and mitochondrial genomes. The most important transcriptional activator of the mitochondrial genome is mitochondrial transcription factor A (Tfam); however, the regulation of Tfam expression during muscle differentiation is not known. Thus, we measured Tfam mRNA levels, mRNA stability, protein expression and localization and Tfam transcription during the progression of muscle differentiation. Parallel 2-fold increases in Tfam protein and mRNA were observed, corresponding with 2-3-fold increases in mitochondrial content. Transcriptional activity of a 2051 bp promoter increased during this differentiation period and this was accompanied by a 3-fold greater Tfam mRNA stabilization. Interestingly, truncations of the promoter at 1706 bp, 978 bp and 393 bp promoter all exhibited 2-3-fold higher transcriptional activity than the 2051 bp construct, indicating the presence of negative regulatory elements within the distal 350 bp of the promoter. Activation of AMP kinase augmented Tfam transcription within the proximal promoter, suggesting the presence of binding sites for transcription factors that are responsive to cellular energy state. During differentiation, the accumulating Tfam protein was progressively distributed to the mitochondrial matrix where it augmented the expression of mtDNA and COX (cytochrome c oxidase) subunit I, an mtDNA gene product. Our data suggest that, during muscle differentiation, Tfam protein levels are regulated by the availability of Tfam mRNA, which is controlled by both transcription and mRNA stability. Changes in energy state and Tfam localization also affect Tfam expression and action in differentiating myotubes. © 2015 Authors.

  16. Biophysical analysis of bacterial and viral systems. A shock tube study of bio-aerosols and a correlated AFM/nanosims investigation of vaccinia virus

    Energy Technology Data Exchange (ETDEWEB)

    Gates, Sean Damien [Stanford Univ., CA (United States)

    2013-05-01

    The work presented herein is concerned with the development of biophysical methodology designed to address pertinent questions regarding the behavior and structure of select pathogenic agents. Two distinct studies are documented: a shock tube analysis of endospore-laden bio-aerosols and a correlated AFM/NanoSIMS study of the structure of vaccinia virus.

  17. The Anti-Warburg Effect Elicited by the cAMP-PGC1α Pathway Drives Differentiation of Glioblastoma Cells into Astrocytes

    Directory of Open Access Journals (Sweden)

    Fan Xing

    2017-01-01

    Full Text Available Summary: Glioblastoma multiforme (GBM is among the most aggressive of human cancers. Although differentiation therapy has been proposed as a potential approach to treat GBM, the mechanisms of induced differentiation remain poorly defined. Here, we established an induced differentiation model of GBM using cAMP activators that specifically directed GBM differentiation into astroglia. Transcriptomic and proteomic analyses revealed that oxidative phosphorylation and mitochondrial biogenesis are involved in induced differentiation of GBM. Dibutyryl cyclic AMP (dbcAMP reverses the Warburg effect, as evidenced by increased oxygen consumption and reduced lactate production. Mitochondrial biogenesis induced by activation of the CREB-PGC1α pathway triggers metabolic shift and differentiation. Blocking mitochondrial biogenesis using mdivi1 or by silencing PGC1α abrogates differentiation; conversely, overexpression of PGC1α elicits differentiation. In GBM xenograft models and patient-derived GBM samples, cAMP activators also induce tumor growth inhibition and differentiation. Our data show that mitochondrial biogenesis and metabolic switch to oxidative phosphorylation drive the differentiation of tumor cells. : Xing et al. show that the metabolic shift from glycolysis to oxidative phosphorylation drives differentiation of GBM cells into astrocytes by cAMP activation. Mechanistically, the cAMP-CREB-PGC1α signal mediates mitochondrial biogenesis, which leads to metabolic reprogramming, induced differentiation, and tumor growth inhibition. Keywords: glioblastoma, induced differentiation, Warburg effect, metabolic reprogramming, oxidative phosphorylation, glycolysis, mitochondrial biogenesis, cyclic adenosine monophosphate, cAMP, PPARγ coactivator-1α, PGC1α

  18. NCBI nr-aa BLAST: CBRC-AGAM-01-0038 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-01-0038 ref|NP_938793.1| Putative cytochrome C biogenesis protein [Corynebacterium diphtheria...e NCTC 13129] emb|CAE48916.1| Putative cytochrome C biogenesis protein [Corynebacterium diphtheriae] NP_938793.1 1.6 24% ...

  19. Evolution of Fe/S cluster biogenesis in the anaerobic parasite Blastocystis

    Czech Academy of Sciences Publication Activity Database

    Tsaousis, A.D.; de Choudens, S. O.; Gentekaki, E.; Long, Shaojun; Gaston, D.; Stechmann, A.; Vinella, D.; Py, B.; Fontecave, M.; Barras, F.; Lukeš, Julius; Roger, A. J.

    2012-01-01

    Roč. 109, č. 26 (2012), s. 10426-10431 ISSN 0027-8424 R&D Projects: GA ČR GA204/09/1667; GA ČR(CZ) GAP305/11/2179 Institutional support: RVO:60077344 Keywords : iron/sulfur cluster biosynthesis * lateral gene transfer * parasite evolution * sulfur-mobilization machinery * oxygen stress adaptation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 9.737, year: 2012

  20. The NFL-TBS.40-63 anti-glioblastoma peptide disrupts microtubule and mitochondrial networks in the T98G glioma cell line.

    Directory of Open Access Journals (Sweden)

    Romain Rivalin

    Full Text Available Despite aggressive therapies, including combinations of surgery, radiotherapy and chemotherapy, glioblastoma remains a highly aggressive brain cancer with the worst prognosis of any central nervous system disease. We have previously identified a neurofilament-derived cell-penetrating peptide, NFL-TBS.40-63, that specifically enters by endocytosis in glioblastoma cells, where it induces microtubule destruction and inhibits cell proliferation. Here, we explore the impact of NFL-TBS.40-63 peptide on the mitochondrial network and its functions by using global cell respiration, quantitative PCR analysis of the main actors directing mitochondrial biogenesis, western blot analysis of the oxidative phosphorylation (OXPHOS subunits and confocal microscopy. We show that the internalized peptide disturbs mitochondrial and microtubule networks, interferes with mitochondrial dynamics and induces a rapid depletion of global cell respiration. This effect may be related to reduced expression of the NRF-1 transcription factor and of specific miRNAs, which may impact mitochondrial biogenesis, in regard to default mitochondrial mobility.