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Sample records for bi-lobe protein required

  1. A comparative proteomic analysis reveals a new bi-lobe protein required for bi-lobe duplication and cell division in Trypanosoma brucei.

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    Qing Zhou

    Full Text Available A Golgi-associated bi-lobed structure was previously found to be important for Golgi duplication and cell division in Trypanosoma brucei. To further understand its functions, comparative proteomics was performed on extracted flagellar complexes (including the flagellum and flagellum-associated structures such as the basal bodies and the bi-lobe and purified flagella to identify new bi-lobe proteins. A leucine-rich repeats containing protein, TbLRRP1, was characterized as a new bi-lobe component. The anterior part of the TbLRRP1-labeled bi-lobe is adjacent to the single Golgi apparatus, and the posterior side is tightly associated with the flagellar pocket collar marked by TbBILBO1. Inducible depletion of TbLRRP1 by RNA interference inhibited duplication of the bi-lobe as well as the adjacent Golgi apparatus and flagellar pocket collar. Formation of a new flagellum attachment zone and subsequent cell division were also inhibited, suggesting a central role of bi-lobe in Golgi, flagellar pocket collar and flagellum attachment zone biogenesis.

  2. Formation of bi-lobed 67P/C-G-like shapes by sub-catastrophic collisions

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    Jutzi, Martin; Benz, Willy

    2016-10-01

    Small bodies with a bi-lobe shape such as comet 67P/C-G have been argued to form as the result of low velocity (v ≈ vesc) collisional mergers of similar-sized bodies very early on in the history of the solar system [1]. However, the recent analysis of the subsequent collisional survival of the global structure and shape of comet 67P/C-G strongly suggests that such a structure will not survive until today [2]. Hence, the comet must have acquired its present characteristics as a result of a collision occurring at later time when the relative velocities between small bodies are much higher (v >> vesc).One possible scenario would be that 67P/C-G-like bi-lobe structures form as the result of collisional disruptions of larger parent bodies [3]. Whether the internal properties of such larger parent bodies, the timing of such a collision, and the subsequent survival of the shape produced is compatible with observations will remain to be seen.Here, we propose a scenario in which the final bi-lobe shapes result from low-energy, sub-catastrophic impacts. We start with bodies of about the same mass as comet 67P/C-G, which are rotating and are slightly elongated (i.e., with properties which are consistent with the outcome of the disruptions of larger bodies). We use a SPH shock physics code to model the impacts, the subsequent re-accumulation of material and the reconfiguration into a stable final shape. Our modelling results suggest that these kind of collisions result in "splitting" events which frequently lead to formation of bi-lobe 67P/C-G-like shapes.The frequency of such small-scale impact events is consistent with a young (less than 1 Gy) age of the shape of comet 67P/C-G [2]. Equally important, the probability for such a shape-forming event to take place without a subsequent shape-destroying event occurring until today is reasonably high.Although the collisions considered in this work can alter the global shape, their respective energy is small enough not to lead to

  3. Formation of bi-lobed shapes by sub-catastrophic collisions. A late origin of comet 67P's structure

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    Jutzi, M.; Benz, W.

    2017-01-01

    Context. The origin of the particular shape of comet 67P/Churyumov-Gerasimenko (67P) is a topic of active research. How and when it acquired its peculiar characteristics has distinct implications on the origin of the solar system and its dynamics. Aims: We investigate how shapes such as that of comet 67P can result from a new type of low-energy, sub-catastrophic impact involving elongated, rotating bodies. We focus on parameters potentially leading to bi-lobed structures. We also estimate the probability of such structures surviving subsequent impacts. Methods: We used a smooth particle hydrodynamics (SPH) shock physics code to model the impacts, the subsequent re-accumulation of material and the reconfiguration into a stable final shape. The energy increase as well as the degree of compaction of the resulting bodies were tracked in the simulations. Results: Our modelling results suggest that the formation of bi-lobed structures like 67P is a natural outcome of the low-energy, sub-catastrophic collisions considered here. Conclusions: Sub-catastrophic impacts have the potential to alter the shape of a small body significantly, without leading to major heating or compaction. The currently observed shapes of cometary nuclei, such as 67P, may be a result of such a major shape forming impact.

  4. Protein Requirements during Aging

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    Glenda Courtney-Martin

    2016-08-01

    Full Text Available Protein recommendations for elderly, both men and women, are based on nitrogen balance studies. They are set at 0.66 and 0.8 g/kg/day as the estimated average requirement (EAR and recommended dietary allowance (RDA, respectively, similar to young adults. This recommendation is based on single linear regression of available nitrogen balance data obtained at test protein intakes close to or below zero balance. Using the indicator amino acid oxidation (IAAO method, we estimated the protein requirement in young adults and in both elderly men and women to be 0.9 and 1.2 g/kg/day as the EAR and RDA, respectively. This suggests that there is no difference in requirement on a gender basis or on a per kg body weight basis between younger and older adults. The requirement estimates however are ~40% higher than the current protein recommendations on a body weight basis. They are also 40% higher than our estimates in young men when calculated on the basis of fat free mass. Thus, current recommendations may need to be re-assessed. Potential rationale for this difference includes a decreased sensitivity to dietary amino acids and increased insulin resistance in the elderly compared with younger individuals.

  5. Formation of bi-lobed shapes by sub-catastrophic collisions: A late origin of comet 67P/C-G's structure

    CERN Document Server

    Jutzi, M

    2016-01-01

    The origin of the particular shape of a small body like comet 67P/Churyumov-Gerasimenko (67P/C-G) is a topic of active research. How and when it acquired its peculiar characteristics has distinct implications on the origin of the solar system and its dynamics. We investigate how shapes like the one of comet 67P/C-G can result from a new type of low-energy, sub-catastrophic impacts involving elongated, rotating bodies. We focus on parameters potentially leading to bi-lobed structures. We also estimate the probability for such structures to survive subsequent impacts. We use a smooth particle hydrodynamics (SPH) shock physics code to model the impacts, the subsequent reaccumulation of material and the reconfiguration into a stable final shape. The energy increase as well as the degree of compaction of the resulting bodies are tracked in the simulations. Our modelling results suggest that the formation of bi-lobed structures like 67P/C-G is a natural outcome of the low energy, sub-catastrophic collisions conside...

  6. Protein requirement in critical illness.

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    Hoffer, Leonard John

    2016-05-01

    How much protein do critically ill patients require? For the many decades that nutritional support has been used there was a broad consensus that critically ill patients need much more protein than required for normal health. Now, however, some clinical investigators recommend limiting all macronutrient provision during the early phase of critical illness. How did these conflicting recommendations emerge? Which of them is correct? This review explains the longstanding recommendation for generous protein provision in critical illness, analyzes the clinical trials now being claimed to refute it, and concludes with suggestions for clinical investigation and practice.

  7. BIOMARKERS TO DEFINE OPTIMAL PROTEIN REQUIREMENT

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    Di Girolamo, Filippo Giorgio

    2015-01-01

    Dietary proteins are the source of the amino acids required by the body for tissue growth and maintenance. The Population Reference Intake (PRI) for proteins, as defined by the European Food Safety Authority (EFSA) for healthy adults, including the elderly, is 0.83 g/kg body weight/day. This amount is defined on the net balance of body protein (or “nitrogen balance”, given by the difference between dietary nitrogen intake and losses) equivalent to 0.66 g/kg/day plus a safety factor for interp...

  8. Dynamic requirements for a functional protein hinge.

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    Kempf, James G; Jung, Ju-Yeon; Ragain, Christina; Sampson, Nicole S; Loria, J Patrick

    2007-04-20

    The enzyme triosephosphate isomerase (TIM) is a model of catalytic efficiency. The 11 residue loop 6 at the TIM active site plays a major role in this enzymatic prowess. The loop moves between open and closed states, which facilitate substrate access and catalysis, respectively. The N and C-terminal hinges of loop 6 control this motion. Here, we detail flexibility requirements for hinges in a comparative solution NMR study of wild-type (WT) TIM and a quintuple mutant (PGG/GGG). The latter contained glycine substitutions in the N-terminal hinge at Val167 and Trp168, which follow the essential Pro166, and in the C-terminal hinge at Lys174, Thr175, and Ala176. Previous work demonstrated that PGG/GGG has a tenfold higher Km value and 10(3)-fold reduced k(cat) relative to WT with either d-glyceraldehyde 3-phosphate or dihyrdroxyacetone phosphate as substrate. Our NMR results explain this in terms of altered loop-6 dynamics in PGG/GGG. In the mutant, loop 6 exhibits conformational heterogeneity with corresponding motional rates hinge design in proteins: structural rigidity is essential for focused motional freedom of active-site loops.

  9. The specificity of protection against cationic antimicrobial peptides by lactoferrin binding protein B.

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    Morgenthau, Ari; Partha, Sarathy K; Adamiak, Paul; Schryvers, Anthony B

    2014-10-01

    A variety of Gram-negative pathogens possess host-specific lactoferrin (Lf) receptors that mediate the acquisition of iron from host Lf. The integral membrane protein component of the receptor, lactoferrin binding protein A specifically binds host Lf and is required for acquisition of iron from Lf. In contrast, the role of the bi-lobed surface lipoprotein, lactoferrin binding protein B (LbpB), in Lf binding and iron acquisition is uncertain. A common feature of LbpBs from most species is the presence of clusters of negatively charged amino acids in the protein's C-terminal lobe. Recently it has been shown that the negatively charged regions from the Neisseria meningitidis LbpB are responsible for protecting against an 11 amino acid cationic antimicrobial peptide (CAP), lactoferricin (Lfcin), derived from human Lf. In this study we investigated whether the LbpB confers resistance to other CAPs since N. meningitidis is likely to encounter other CAPs from the host. LbpB provided protection against the cathelicidin derived peptide, cathelicidin related antimicrobial peptide (mCRAMP), but did not confer protection against Tritrp 1 or LL37 under our experimental conditions. When tested against a range of rationally designed synthetic peptides, LbpB was shown to protect against IDR-1002 and IDR-0018 but not against HH-2 or HHC10.

  10. Nitrogen Balance and Protein Requirements for Critically Ill Older Patients.

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    Dickerson, Roland N

    2016-04-18

    Critically ill older patients with sarcopenia experience greater morbidity and mortality than younger patients. It is anticipated that unabated protein catabolism would be detrimental for the critically ill older patient. Healthy older subjects experience a diminished response to protein supplementation when compared to their younger counterparts, but this anabolic resistance can be overcome by increasing protein intake. Preliminary evidence suggests that older patients may respond differently to protein intake than younger patients during critical illness as well. If sufficient protein intake is given, older patients can achieve a similar nitrogen accretion response as younger patients even during critical illness. However, there is concern among some clinicians that increasing protein intake in older patients during critical illness may lead to azotemia due to decreased renal functional reserve which may augment the propensity towards worsened renal function and worsened clinical outcomes. Current evidence regarding protein requirements, nitrogen balance, ureagenesis, and clinical outcomes during nutritional therapy for critically ill older patients is reviewed.

  11. Nitrogen Balance and Protein Requirements for Critically Ill Older Patients

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    Roland N. Dickerson

    2016-04-01

    Full Text Available Critically ill older patients with sarcopenia experience greater morbidity and mortality than younger patients. It is anticipated that unabated protein catabolism would be detrimental for the critically ill older patient. Healthy older subjects experience a diminished response to protein supplementation when compared to their younger counterparts, but this anabolic resistance can be overcome by increasing protein intake. Preliminary evidence suggests that older patients may respond differently to protein intake than younger patients during critical illness as well. If sufficient protein intake is given, older patients can achieve a similar nitrogen accretion response as younger patients even during critical illness. However, there is concern among some clinicians that increasing protein intake in older patients during critical illness may lead to azotemia due to decreased renal functional reserve which may augment the propensity towards worsened renal function and worsened clinical outcomes. Current evidence regarding protein requirements, nitrogen balance, ureagenesis, and clinical outcomes during nutritional therapy for critically ill older patients is reviewed.

  12. Protein Requirements and Recommendations for Older People: A Review

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    Caryl Nowson

    2015-08-01

    Full Text Available Declines in skeletal muscle mass and strength are major contributors to increased mortality, morbidity and reduced quality of life in older people. Recommended Dietary Allowances/Intakes have failed to adequately consider the protein requirements of the elderly with respect to function. The aim of this paper was to review definitions of optimal protein status and the evidence base for optimal dietary protein. Current recommended protein intakes for older people do not account for the compensatory loss of muscle mass that occurs on lower protein intakes. Older people have lower rates of protein synthesis and whole-body proteolysis in response to an anabolic stimulus (food or resistance exercise. Recommendations for the level of adequate dietary intake of protein for older people should be informed by evidence derived from functional outcomes. Randomized controlled trials report a clear benefit of increased dietary protein on lean mass gain and leg strength, particularly when combined with resistance exercise. There is good consistent evidence (level III-2 to IV that consumption of 1.0 to 1.3 g/kg/day dietary protein combined with twice-weekly progressive resistance exercise reduces age-related muscle mass loss. Older people appear to require 1.0 to 1.3 g/kg/day dietary protein to optimize physical function, particularly whilst undertaking resistance exercise recommendations.

  13. Receptor component protein (RCP): a member of a multi-protein complex required for G-protein-coupled signal transduction.

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    Prado, M A; Evans-Bain, B; Dickerson, I M

    2002-08-01

    The calcitonin-gene-related peptide (CGRP) receptor component protein (RCP) is a 148-amino-acid intracellular protein that is required for G-protein-coupled signal transduction at receptors for the neuropeptide CGRP. RCP works in conjunction with two other proteins to constitute a functional CGRP receptor: calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying protein 1 (RAMP1). CRLR has the stereotypical seven-transmembrane topology of a G-protein-coupled receptor; it requires RAMP1 for trafficking to the cell surface and for ligand specificity, and requires RCP for coupling to the cellular signal transduction pathway. We have made cell lines that expressed an antisense construct of RCP and determined that CGRP-mediated signal transduction was reduced, while CGRP binding was unaffected. Furthermore, signalling at two other endogenous G-protein-coupled receptors was unaffected, suggesting that RCP was specific for a limited subset of receptors.

  14. The Drosophila HOAP protein is required for telomere capping.

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    Cenci, Giovanni; Siriaco, Giorgia; Raffa, Grazia D; Kellum, Rebecca; Gatti, Maurizio

    2003-01-01

    HOAP (HP1/ORC-associated protein) has recently been isolated from Drosophila melanogaster embryos as part of a cytoplasmic complex that contains heterochromatin protein 1 (HP1) and the origin recognition complex subunit 2 (ORC2). Here, we show that caravaggio, a mutation in the HOAP-encoding gene, causes extensive telomere-telomere fusions in larval brain cells, indicating that HOAP is required for telomere capping. Our analyses indicate that HOAP is specifically enriched at mitotic chromosome telomeres, and strongly suggest that HP1 and HOAP form a telomere-capping complex that does not contain ORC2.

  15. Models of protein and amino acid requirements for cattle

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    Luis Orlindo Tedeschi

    2015-03-01

    Full Text Available Protein supply and requirements by ruminants have been studied for more than a century. These studies led to the accumulation of lots of scientific information about digestion and metabolism of protein by ruminants as well as the characterization of the dietary protein in order to maximize animal performance. During the 1980s and 1990s, when computers became more accessible and powerful, scientists began to conceptualize and develop mathematical nutrition models, and to program them into computers to assist with ration balancing and formulation for domesticated ruminants, specifically dairy and beef cattle. The most commonly known nutrition models developed during this period were the National Research Council (NRC in the United States, Agricultural Research Council (ARC in the United Kingdom, Institut National de la Recherche Agronomique (INRA in France, and the Commonwealth Scientific and Industrial Research Organization (CSIRO in Australia. Others were derivative works from these models with different degrees of modifications in the supply or requirement calculations, and the modeling nature (e.g., static or dynamic, mechanistic, or deterministic. Circa 1990s, most models adopted the metabolizable protein (MP system over the crude protein (CP and digestible CP systems to estimate supply of MP and the factorial system to calculate MP required by the animal. The MP system included two portions of protein (i.e., the rumen-undegraded dietary CP - RUP - and the contributions of microbial CP - MCP as the main sources of MP for the animal. Some models would explicitly account for the impact of dry matter intake (DMI on the MP required for maintenance (MPm; e.g., Cornell Net Carbohydrate and Protein System - CNCPS, the Dutch system - DVE/OEB, while others would simply account for scurf, urinary, metabolic fecal, and endogenous contributions independently of DMI. All models included milk yield and its components in estimating MP required for lactation

  16. PML tumor suppressor protein is required for HCV production

    Energy Technology Data Exchange (ETDEWEB)

    Kuroki, Misao [Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Okayama 700-8558 (Japan); Research Fellow of the Japan Society for the Promotion of Science (Japan); Center for AIDS Research, Kumamoto University, Kumamoto 860-0811 (Japan); Ariumi, Yasuo, E-mail: ariumi@kumamoto-u.ac.jp [Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Okayama 700-8558 (Japan); Center for AIDS Research, Kumamoto University, Kumamoto 860-0811 (Japan); Hijikata, Makoto [Department of Viral Oncology, Institute for Virus Research, Kyoto University, Kyoto 606-8507 (Japan); Ikeda, Masanori; Dansako, Hiromichi [Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Okayama 700-8558 (Japan); Wakita, Takaji [Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640 (Japan); Shimotohno, Kunitada [Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Chiba 272-8516 (Japan); Kato, Nobuyuki [Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Okayama 700-8558 (Japan)

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer PML tumor suppressor protein is required for HCV production. Black-Right-Pointing-Pointer PML is dispensable for HCV RNA replication. Black-Right-Pointing-Pointer HCV could not alter formation of PML-NBs. Black-Right-Pointing-Pointer INI1 and DDX5, PML-related proteins, are involved in HCV life cycle. -- Abstract: PML tumor suppressor protein, which forms discrete nuclear structures termed PML-nuclear bodies, has been associated with several cellular functions, including cell proliferation, apoptosis and antiviral defense. Recently, it was reported that the HCV core protein colocalizes with PML in PML-NBs and abrogates the PML function through interaction with PML. However, role(s) of PML in HCV life cycle is unknown. To test whether or not PML affects HCV life cycle, we examined the level of secreted HCV core and the infectivity of HCV in the culture supernatants as well as the level of HCV RNA in HuH-7-derived RSc cells, in which HCV-JFH1 can infect and efficiently replicate, stably expressing short hairpin RNA targeted to PML. In this context, the level of secreted HCV core and the infectivity in the supernatants from PML knockdown cells was remarkably reduced, whereas the level of HCV RNA in the PML knockdown cells was not significantly affected in spite of very effective knockdown of PML. In fact, we showed that PML is unrelated to HCV RNA replication using the subgenomic HCV-JFH1 replicon RNA, JRN/3-5B. Furthermore, the infectivity of HCV-like particle in the culture supernatants was significantly reduced in PML knockdown JRN/3-5B cells expressing core to NS2 coding region of HCV-JFH1 genome using the trans-packaging system. Finally, we also demonstrated that INI1 and DDX5, the PML-related proteins, are involved in HCV production. Taken together, these findings suggest that PML is required for HCV production.

  17. Bistability: requirements on cell-volume, protein diffusion, and thermodynamics.

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    Robert G Endres

    Full Text Available Bistability is considered wide-spread among bacteria and eukaryotic cells, useful, e.g., for enzyme induction, bet hedging, and epigenetic switching. However, this phenomenon has mostly been described with deterministic dynamic or well-mixed stochastic models. Here, we map known biological bistable systems onto the well-characterized biochemical Schlögl model, using analytical calculations and stochastic spatiotemporal simulations. In addition to network architecture and strong thermodynamic driving away from equilibrium, we show that bistability requires fine-tuning towards small cell volumes (or compartments and fast protein diffusion (well mixing. Bistability is thus fragile and hence may be restricted to small bacteria and eukaryotic nuclei, with switching triggered by volume changes during the cell cycle. For large volumes, single cells generally loose their ability for bistable switching and instead undergo a first-order phase transition.

  18. Haematopoietic stem cells require a highly regulated protein synthesis rate.

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    Signer, Robert A J; Magee, Jeffrey A; Salic, Adrian; Morrison, Sean J

    2014-05-01

    Many aspects of cellular physiology remain unstudied in somatic stem cells, for example, there are almost no data on protein synthesis in any somatic stem cell. Here we set out to compare protein synthesis in haematopoietic stem cells (HSCs) and restricted haematopoietic progenitors. We found that the amount of protein synthesized per hour in HSCs in vivo was lower than in most other haematopoietic cells, even if we controlled for differences in cell cycle status or forced HSCs to undergo self-renewing divisions. Reduced ribosome function in Rpl24(Bst/+) mice further reduced protein synthesis in HSCs and impaired HSC function. Pten deletion increased protein synthesis in HSCs but also reduced HSC function. Rpl24(Bst/+) cell-autonomously rescued the effects of Pten deletion in HSCs; blocking the increase in protein synthesis, restoring HSC function, and delaying leukaemogenesis. Pten deficiency thus depletes HSCs and promotes leukaemia partly by increasing protein synthesis. Either increased or decreased protein synthesis impairs HSC function.

  19. Evaluation of the optimum protein requirements for Vietnamese pigs

    NARCIS (Netherlands)

    Pham, K.T.

    2010-01-01

    The studies reported in this thesis were carried out in Central Vietnam where pig production plays an important role and pig farmers face a general dietary protein shortage for their animals. The objectives of the work presented were (1) to investigate the crude protein (CP) supply to local Mong Cai

  20. Computing wheat nitrogen requirements from grain yield and protein maps

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    Optical protein sensors and mass-flow yield monitors provide the opportunity to continuously measure grain quality and quantity during harvesting. This chapter illustrates how yield monitor and grain protein measurements may provide useful postharvest information for evaluating water or nitrogen (N)...

  1. The bioinformatics of nucleotide sequence coding for proteins requiring metal coenzymes and proteins embedded with metals

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    Tremberger, G.; Dehipawala, Sunil; Cheung, E.; Holden, T.; Sullivan, R.; Nguyen, A.; Lieberman, D.; Cheung, T.

    2015-09-01

    All metallo-proteins need post-translation metal incorporation. In fact, the isotope ratio of Fe, Cu, and Zn in physiology and oncology have emerged as an important tool. The nickel containing F430 is the prosthetic group of the enzyme methyl coenzyme M reductase which catalyzes the release of methane in the final step of methano-genesis, a prime energy metabolism candidate for life exploration space mission in the solar system. The 3.5 Gyr early life sulfite reductase as a life switch energy metabolism had Fe-Mo clusters. The nitrogenase for nitrogen fixation 3 billion years ago had Mo. The early life arsenite oxidase needed for anoxygenic photosynthesis energy metabolism 2.8 billion years ago had Mo and Fe. The selection pressure in metal incorporation inside a protein would be quantifiable in terms of the related nucleotide sequence complexity with fractal dimension and entropy values. Simulation model showed that the studied metal-required energy metabolism sequences had at least ten times more selection pressure relatively in comparison to the horizontal transferred sequences in Mealybug, guided by the outcome histogram of the correlation R-sq values. The metal energy metabolism sequence group was compared to the circadian clock KaiC sequence group using magnesium atomic level bond shifting mechanism in the protein, and the simulation model would suggest a much higher selection pressure for the energy life switch sequence group. The possibility of using Kepler 444 as an example of ancient life in Galaxy with the associated exoplanets has been proposed and is further discussed in this report. Examples of arsenic metal bonding shift probed by Synchrotron-based X-ray spectroscopy data and Zn controlled FOXP2 regulated pathways in human and chimp brain studied tissue samples are studied in relationship to the sequence bioinformatics. The analysis results suggest that relatively large metal bonding shift amount is associated with low probability correlation R

  2. A mechanism regulating G protein-coupled receptor signaling that requires cycles of protein palmitoylation and depalmitoylation.

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    Jia, Lixia; Chisari, Mariangela; Maktabi, Mohammad H; Sobieski, Courtney; Zhou, Hao; Konopko, Aaron M; Martin, Brent R; Mennerick, Steven J; Blumer, Kendall J

    2014-02-28

    Reversible attachment and removal of palmitate or other long-chain fatty acids on proteins has been hypothesized, like phosphorylation, to control diverse biological processes. Indeed, palmitate turnover regulates Ras trafficking and signaling. Beyond this example, however, the functions of palmitate turnover on specific proteins remain poorly understood. Here, we show that a mechanism regulating G protein-coupled receptor signaling in neuronal cells requires palmitate turnover. We used hexadecyl fluorophosphonate or palmostatin B to inhibit enzymes in the serine hydrolase family that depalmitoylate proteins, and we studied R7 regulator of G protein signaling (RGS)-binding protein (R7BP), a palmitoylated allosteric modulator of R7 RGS proteins that accelerate deactivation of Gi/o class G proteins. Depalmitoylation inhibition caused R7BP to redistribute from the plasma membrane to endomembrane compartments, dissociated R7BP-bound R7 RGS complexes from Gi/o-gated G protein-regulated inwardly rectifying K(+) (GIRK) channels and delayed GIRK channel closure. In contrast, targeting R7BP to the plasma membrane with a polybasic domain and an irreversibly attached lipid instead of palmitate rendered GIRK channel closure insensitive to depalmitoylation inhibitors. Palmitate turnover therefore is required for localizing R7BP to the plasma membrane and facilitating Gi/o deactivation by R7 RGS proteins on GIRK channels. Our findings broaden the scope of biological processes regulated by palmitate turnover on specific target proteins. Inhibiting R7BP depalmitoylation may provide a means of enhancing GIRK activity in neurological disorders.

  3. Contemporary Issues in Protein Requirements and Consumption for Resistance Trained Athletes

    Directory of Open Access Journals (Sweden)

    Wilson Jacob

    2006-06-01

    Full Text Available Abstract In recent years an explosion of research papers concerning protein consumption has been published. The need to consolidate this information has become critical from both practical and future research standpoints. For this reason, the following paper presents an in depth analysis of contemporary issues in protein requirements and consumption for resistance trained athletes. Specifically, the paper covers: 1. protein requirements for resistance trained athletes; 2. the effect of the digestion rate of protein on muscular protein balance; 3. the optimal timing of protein intake relative to exercise; 4. the optimal pattern of protein ingestion, relative to how an individual should consume their protein throughout a 24 hour period, and what sources are utilized during this time frame; 5. protein composition and its interaction with measures of protein balance and strength performance; 6. the combination of protein and carbohydrates on plasma insulin levels and protein balance; 7. the efficacy of protein supplements and whole food protein sources. Our goal is to provide the reader with practical information in optimizing protein intake as well as for provision of sound advice to their clients. Finally, special care was taken to provide future research implications.

  4. Osteogenic protein-1 is required for mammalian eye development.

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    Solursh, M; Langille, R M; Wood, J; Sampath, T K

    1996-01-17

    Osteogenic Protein-1 (OP-1/BMP-7) is a bone morphogenetic protein in the transforming growth factor-beta superfamily and has been shown to be expressed temporally and spatially during epithelial-mesenchymal interactions mediating tissue morphogenesis in early embryogenesis. In order to identify the primary role(s) for OP-1 in development, we carried out whole rat embryo cultures, over a 72-h period from primitive streak stages to early limb bud stages, in rat sera containing either OP-1 blocking antibodies (10 micrograms/ml) or nonreactive IgG. Rat embryos cultured with control antibodies developed normally, while those cultured with anti-OP-1 antibodies consistently exhibited over-all reduced size and absence of eyes. Histological sections revealed a greater reduction in neural retina development in the embryos treated with anti-OP-1 blocking antibodies. In situ hybridization and immunolocalization analyses indicate that OP-1 is expressed in the neuroepithelium of the optic vesicle at E11.5, is limited to the presumptive neural retina and developing lens placode, and is subsequently expressed in the neural retina, lens and developing cornea at E12.5-E13.5. Our results indicate that OP-1 mediates the inductive signals involved in mammalian eye development.

  5. Requirement for erythroblast-macrophage protein (Emp) in definitive erythropoiesis.

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    Soni, Shivani; Bala, Shashi; Hanspal, Manjit

    2008-01-01

    Emp, erythroblast-macrophage protein was initially identified as a mediator of erythroblast-macrophage interactions during erythroid differentiation. More recent studies have shown that targeted disruption of Emp leads to abnormal erythropoiesis in the fetal liver, and fetal demise. To further address the activity of Emp in the hematopoietic lineage in adult bone marrow, we conducted fetal liver HSC reconstitution assay. Emp null fetal liver cells were transplanted into lethally irradiated wild-type sibling mice, and assessed the erythropoietic activity. We found that Emp null cells rescued lethally irradiated mice with efficiency comparable to that of wild-type cells. However, the recipients of Emp null cells showed abnormal erythropoiesis as indicated by the presence of persistent anemia, extensive extramedullary erythropoiesis, and increased apoptosis of erythroid precursors. Extramedullary erythropoiesis suggests perturbed interactions between the Emp-deficient hematopoietic cells and the wild-type niche. Furthermore, in spleen colony-forming unit assays, proliferation rates of the Emp null cells were greater than those of the wild-type cells. Similarly, in vitro burst-forming unit-erythroid and colony-forming unit-erythroid assays showed increased erythroid colony numbers from Emp null livers. Morphologic examination showed that Emp null CFU-E-derived erythroblasts were immature compared to those derived from wild-type CFU-Es, suggesting that loss of Emp function in erythroid cells results in impaired proliferation and terminal differentiation. These results demonstrate that Emp plays a cell intrinsic role in the erythroid lineage.

  6. Studies on the protein and sulfur amino acid requirements of young bobwhite quail

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    Serafin, J.A.

    1977-01-01

    Four experiments were conducted with purified diets to examine the influence of protein level and to estimate the sulfur amino acid (S.A.A.) requirement of young Bobwhite quail (Colinus virginianus). These studies demonstrated (I) that 26% protein was sufficient for rapid growth when the diet was supplemented with methionine; (2) that diets containing higher levels of protein (29.3% and 31.3%) failed to support satisfactory growth unless they contained supplemental methionine; and (3) that young Bobwhite quail require no more than 1.0% sulfur-containing amino acids for optimal growth and efficiency of feed utilization. A fifth experiment was conducted to examine the protein and S.A.A. requirements of young Bobwhite quail using practical rations and to compare results with those obtained with purified diets. Diets containing 24%, 26% and 28% protein were supplied with and without supplemental methionine in a five week study. Results showed significant growth responses to protein and supplemental methionine. Responses showed that Bobwhite quail require no more than 26% protein for maximum growth and efficiency of feed utilization when the S.A.A. level of the diet was approximately 1.0%. The results were in close agreement with those obtained with purified diets. These findings define more precisely than had been known the quantitative requirements of young Bobwhite quail for protein and for the S.A.A. necessary for optimal growth.

  7. Effect of energy and protein levels on nutrient utilization and their requirements in growing Murrah buffaloes.

    Science.gov (United States)

    Prusty, Sonali; Kundu, Shivlal Singh; Mondal, Goutam; Sontakke, Umesh; Sharma, Vijay Kumar

    2016-04-01

    To evaluate different levels of energy and protein for optimum growth of Murrah male buffalo calves, a growth trial (150 days) was conducted on 30 calves (body weight 202.5 ± 6.8 kg). Six diets were formulated to provide 90, 100 and 110% protein level and 90 and 110% energy level requirements for buffalo calves, derived from ICAR 2013 recommendations for buffaloes. The crude protein (CP) intake was increased with higher dietary CP, whereas no effect of energy levels or interaction between protein and energy was observed on CP intake. There were significant effects (P interaction between protein and energy (P nutrient intake (protein or energy) per kg body weight (BW)(0.75) at various fortnight intervals was regressed linearly from the average daily gain (ADG) per kg BW(0.75). By setting the average daily gain at zero in the developed regression equation, a maintenance requirement was obtained, i.e. 133.1 kcal ME, 6.45 g CP and 3.95 g metabolizable protein (MP) per kg BW(0.75). Requirement for growth was 6.12 kcal ME, 0.46 g CP and 0.32 g MP per kg BW(0.75) per day. Metabolizable amino acid requirement was estimated from partitioning of MP intake and ADG. The ME requirements were lower, whereas the MP requirement of Murrah buffaloes was higher than ICAR (2013) recommendations.

  8. Replication of murine coronavirus requires multiple cysteines in the endodomain of spike protein

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Jinhua; Lv, Jun; Wang, Yuyan; Gao, Shuang; Yao, Qianqian; Qu, Di; Ye, Rong, E-mail: yerong24@fudan.edu.cn

    2012-06-05

    A conserved cysteine-rich motif located between the transmembrane domain and the endodomain is essential for membrane fusion and assembly of coronavirus spike (S) protein. Here, we proved that three cysteines within the motif, but not dependent on position, are minimally required for the survival of the recombinant mouse hepatitis virus. When the carboxy termini with these mutated motifs of S proteins were respectively introduced into a heterogeneous protein, both incorporation into lipid rafts and S-palmitoylation of these recombinant proteins showed a similar quantity requirement to cysteine residues. Meanwhile, the redistribution of these proteins on cellular surface indicated that the absence of the positively charged rather than cysteine residues in the motif might lead the dramatic reduction in syncytial formation of some mutants with the deleted motifs. These results suggest that multiple cysteine as well as charged residues concurrently improves the membrane-associated functions of S protein in viral replication and cytopathogenesis.

  9. Effect of diet composition on protein requirements of children and adults in northern Mexico.

    Science.gov (United States)

    Ballesteros, M N; Valencia, M E; Brown, D S

    1993-01-01

    The overall vegetable and animal protein combinations of the diet in Northern Mexico were determined through a dietary population survey. Vegetable sources made up 45% and animal protein was 55% (45V/55A). Further combinations of up to 100% vegetable protein dietary mixtures (100V) were studied to test the sensibility of the variations on protein requirements of pre-school, school children and adults. Diets were analyzed for amino acid composition and in vivo protein digestibility in rats to estimate true protein requirements according to FAO/WHO/UNU (1985). The effect on the pre-school group showed the widest variation with 1.46 g/kg/day in the 45V/55A to 2.63 in the 100V. For the school-aged children and adults the variations were 1.15-1.79 and 0.94-0.84 g/kg/day respectively.

  10. The Pseudorabies Virus VP1/2 Tegument Protein Is Required for Intracellular Capsid Transport†

    OpenAIRE

    Luxton, G.W. Gant; Lee, Joy I-Hsuan; Haverlock-Moyns, Sarah; Schober, Joseph Martin; Smith, Gregory Allan

    2006-01-01

    Transport of capsids in cells is critical to alphaherpesvirus infection and pathogenesis; however, viral factors required for transport have yet to be identified. Here we provide a detailed examination of capsid dynamics during the egress phase of infection in Vero cells infected with pseudorabies virus. We demonstrate that the VP1/2 tegument protein is required for processive microtubule-based transport of capsids in the cytoplasm. A second tegument protein that binds to VP1/2, UL37, was nec...

  11. Structural Requirements for Membrane Assembly of Proteins Spanning the Membrane Several Times

    OpenAIRE

    Lipp, Joachim; Flint, Nicholas; Haeuptle, Marie-Theres; Dobberstein, Bernhard

    1989-01-01

    We have investigated the structural requirements for the biogenesis of proteins spanning the membrane several times. Proteins containing various combinations of topological signals (signal anchor and stop transfer sequences) were synthesized in a cell-free translation system and their membrane topology was determined. Proteins spanning the membrane twice were obtained when a signal anchor sequence was followed by either a stop transfer sequence or a second signal anchor sequence. Thus, a sig...

  12. 40 CFR 180.1204 - Harpin protein; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Harpin protein; exemption from the requirement of a tolerance. 180.1204 Section 180.1204 Protection of Environment ENVIRONMENTAL PROTECTION... no more than one cystine residue. (b) The source(s) of genetic material encoding the protein...

  13. Protein Requirements in Healthy Adults:A Meta-analysis of Nitrogen Balance Studies

    Institute of Scientific and Technical Information of China (English)

    LI Min; SUN Feng; PIAO Jian Hua; YANG Xiao Guang

    2014-01-01

    Objective The goal of this study was to analyze protein requirements in healthy adults through a meta-analysis of nitrogen balance studies. Methods A comprehensive search for nitrogen balance studies of healthy adults published up to October 2012 was performed, each study were reviewed, and data were abstracted. The studies were first evaluated for heterogeneity. The average protein requirements were analyzed by using the individual data of each included studies. Study site climate, age, sex, and dietary protein source were compared. Results Data for 348 subjects were gathered from 28 nitrogen balance studies. The natural logarithm of requirement for 348 individuals had a normal distribution with a mean of 4.66. The estimated average requirement was the exponentiation of the mean of the log requirement, 105.64 mg N/kg·d. No significant differences between adult age, source of dietary protein were observed. But there was significant difference between sex and the climate of the study site (P Conclusion The estimated average requirement and recommended nutrient intake of the healthy adult population was 105.64 mg N/kg·d (0.66 g high quality protein/kg·d) and 132.05 mg N/kg·d (0.83 g high quality protein/kg·d), respectively.

  14. Evaluation of the Protein Requirement in Chinese Young Adults Using the Indicator Amino Acid Oxidation Technique

    Institute of Scientific and Technical Information of China (English)

    LI Min; ZHANG Yu Hui; WANG Zhi Ling; GOU Ling Yan; LI Wei Dong; TIAN Yuan; HU Yi Chun; WANG Rui; PIAO Jian Hua; YANG Xiao Guang

    2013-01-01

    Objective To accurately calculate the protein requirements in Chinese young adults using the indicator amino acid oxidation technique. Methods Nine women and ten men received a restricted daily level of protein intake (0.75, 0.82, 0.89, 0.97, and 1.05 g/kg), along with L-[1-13C]-leucine. Subjects’ protein requirement was determined by a biphasic linear regression crossover analysis of F13CO2 data. In doing so, a breakpoint at the minimal rate of appearance of 13CO2 expiration specific to each level of dietary protein was identified. This trial was registered with the Chinese clinical trial registry as ChiCTR-ONC-11001407. Results The Estimated Average Requirement (EAR) and the Recommended Nutrient Intake (RNI) of protein for healthy Chinese young adults were determined to be 0.87 and 0.98 g/(kg·d), respectively, based on the indicator amino acid oxidation technique. Conclusion The EAR and RNI of mixed protein are 5% and 16% that are lower than the current proposed EAR and RNI (0.92 and 1.16 g/(kg·d), respectively), as determined by the nitrogen balance method. The respective EAR and RNI recommendations of 0.87 and 0.98 g/(kg·d) of mixed protein are estimated to be reasonable and suitable for Chinese young adults.

  15. Characterization of EssB, a protein required for secretion of ESAT-6 like proteins in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Chen Yi-Hsing

    2012-09-01

    Full Text Available Abstract Background Staphylococcus aureus secretes EsxA and EsxB, two small polypeptides of the WXG100 family of proteins. Genetic analyses have shown that production and secretion of EsxA and EsxB require an intact ESAT-6 Secretion System (ESS, a cluster of genes that is conserved in many Firmicutes and encompasses esxA and esxB . Here, we characterize EssB, one of the proteins encoded by the ESS cluster. EssB is highly conserved in Gram-positive bacteria and belongs to the Cluster of Orthologous Groups of protein COG4499 with no known function. Results By generating an internal deletion in essB , we demonstrate that EssB is required for secretion of EsxA. We use a polyclonal antibody to identify EssB and show that the protein fractionates with the plasma membrane of S. aureus . Yet, when produced in Escherichia coli, EssB remains mostly soluble and the purified protein assembles into a highly organized oligomer that can be visualized by electron microscopy. Production of truncated EssB variants in wild-type S. aureus confers a dominant negative phenotype on EsxA secretion. Conclusions The data presented here support the notion that EssB may oligomerize and interact with other membrane components to form the WXG100-specific translocon in S. aureus .

  16. Mammalian Clusterin associated protein 1 is an evolutionarily conserved protein required for ciliogenesis

    Directory of Open Access Journals (Sweden)

    Pasek Raymond C

    2012-11-01

    Full Text Available Abstract Background Clusterin associated protein 1 (CLUAP1 was initially characterized as a protein that interacts with clusterin, and whose gene is frequently upregulated in colon cancer. Although the consequences of these observations remain unclear, research of CLUAP1 homologs in C. elegans and zebrafish indicates that it is needed for cilia assembly and maintenance in these models. To begin evaluating whether Cluap1 has an evolutionarily conserved role in cilia in mammalian systems and to explore the association of Cluap1 with disease pathogenesis and developmental abnormalities, we generated Cluap1 mutant mice. Methods Cluap1 mutant embryos were generated and examined for gross morphological and anatomical defects using light microscopy. Reverse transcription PCR, β-galactosidase staining assays, and immunofluorescence analysis were used to determine the expression of the gene and localization of the protein in vivo and in cultured cell lines. We also used immunofluorescence analysis and qRT-PCR to examine defects in the Sonic hedgehog signaling pathway in mutant embryos. Results Cluap1 mutant embryos die in mid-gestation, indicating that it is necessary for proper development. Mutant phenotypes include a failure of embryonic turning, an enlarged pericardial sac, and defects in neural tube development. Consistent with the diverse phenotypes, Cluap1 is widely expressed. Furthermore, the Cluap1 protein localizes to primary cilia, and mutant embryos were found to lack cilia at embryonic day 9.5. The phenotypes observed in Cluap1 mutant mice are indicative of defects in Sonic hedgehog signaling. This was confirmed by analyzing hedgehog signaling activity in Cluap1 mutants, which revealed that the pathway is repressed. Conclusions These data indicate that the function of Cluap1 is evolutionarily conserved with regard to ciliogenesis. Further, the results implicate mammalian Cluap1 as a key regulator of hedgehog signaling and as an

  17. Strategic study on energy-protein requirements for local sheep: 5. Ewes during lactation phase

    Directory of Open Access Journals (Sweden)

    I-W Mathius

    2004-03-01

    Full Text Available Thirty-six Javanese thin-tail ewes in the end of late pregnancy phase were set out to study the energy and crude protein requirements during the first eight-week of lactation phase. The ewes were penned individually in doors and randomly assigned to a 3 x 3 factorial arrangement, consisting of three levels of energy (low, medium and high and three levels of crude protein (low, medium and high diets with four ewes per treatment. The diets were pelleted and offered four times daily in approximately equal amount. Feed intake, nutrient digestibility, body weight and milk production were recorded. Results showed that, total lamb birth weights was not affected, but protein content on the ration treatments significantly altered (P0.05, while crude protein content on the ration highly significantly affected (P<0.01. Based on data recorded, the energy and protein requirements for ewes during lactation phase are highly significantly depended on ewes’ live weight, milk production and the ratio of energy metabolism and crude protein of the ration. It was concluded that in order to fulfil the crude protein and energy needs of the ewes during lactation phase, the ration given should contain crude protein and energy as much as 16% (based on dry matter and 13.4 MJ/kg dry matter respectively.

  18. Discovery of cellular proteins required for the early steps of HCV infection using integrative genomics.

    Directory of Open Access Journals (Sweden)

    Ji Hoon Park

    Full Text Available Successful viral infection requires intimate communication between virus and host cell, a process that absolutely requires various host proteins. However, current efforts to discover novel host proteins as therapeutic targets for viral infection are difficult. Here, we developed an integrative-genomics approach to predict human genes involved in the early steps of hepatitis C virus (HCV infection. By integrating HCV and human protein associations, co-expression data, and tight junction-tetraspanin web specific networks, we identified host proteins required for the early steps in HCV infection. Moreover, we validated the roles of newly identified proteins in HCV infection by knocking down their expression using small interfering RNAs. Specifically, a novel host factor CD63 was shown to directly interact with HCV E2 protein. We further demonstrated that an antibody against CD63 blocked HCV infection, indicating that CD63 may serve as a new therapeutic target for HCV-related diseases. The candidate gene list provides a source for identification of new therapeutic targets.

  19. The fission yeast heterochromatin protein Rik1 is required for telomere clustering during meiosis

    DEFF Research Database (Denmark)

    Tuzon, Creighton T; Borgstrøm, Britta; Weilguny, Dietmar

    2004-01-01

    Telomeres share the ability to silence nearby transcription with heterochromatin, but the requirement of heterochromatin proteins for most telomere functions is unknown. The fission yeast Rik1 protein is required for heterochromatin formation at centromeres and the mating-type locus, as it recrui...... meiosis. However, Rik1 is dispensable for the protective roles of telomeres in preventing chromosome end-fusion. Thus, a Swi6-independent heterochromatin function distinct from that at centromeres and the mating-type locus operates at telomeres during sexual differentiation....

  20. Poly (ADP-ribose polymerase 1 is required for protein localization to Cajal body.

    Directory of Open Access Journals (Sweden)

    Elena Kotova

    2009-02-01

    Full Text Available Recently, the nuclear protein known as Poly (ADP-ribose Polymerase1 (PARP1 was shown to play a key role in regulating transcription of a number of genes and controlling the nuclear sub-organelle nucleolus. PARP1 enzyme is known to catalyze the transfer of ADP-ribose to a variety of nuclear proteins. At present, however, while we do know that the main acceptor for pADPr in vivo is PARP1 protein itself, by PARP1 automodification, the significance of PARP1 automodification for in vivo processes is not clear. Therefore, we investigated the roles of PARP1 auto ADP-ribosylation in dynamic nuclear processes during development. Specifically, we discovered that PARP1 automodification is required for shuttling key proteins into Cajal body (CB by protein non-covalent interaction with pADPr in vivo. We hypothesize that PARP1 protein shuttling follows a chain of events whereby, first, most unmodified PARP1 protein molecules bind to chromatin and accumulate in nucleoli, but then, second, upon automodification with poly(ADP-ribose, PARP1 interacts non-covalently with a number of nuclear proteins such that the resulting protein-pADPr complex dissociates from chromatin into CB.

  1. Requirement for maintenance and gain of crude protein for two genotypes of growing quails

    Directory of Open Access Journals (Sweden)

    José Jordão Filho

    2012-09-01

    Full Text Available The objective of this study was to estimate the requirements for maintenance and gain of crude protein in Japanese and European quails aged 16-36 days. To determine the maintenance requirements, one experiment was conducted in a completely randomized design with four decreasing feeding levels (100, 75, 50 and 25% and four replicates per treatment. The method of comparative slaughter was used, through a feeding assay. A total of 352 quails from each strain were housed by supply level in 16 pens measuring 1.0 × 1.5 m, totaling 22 birds per cage under ambient temperature conditions (26±0.5 ºC. To estimate the requirement gains, one experiment was conducted with five groups of quails fed ad libitum and housed under controlled temperature of 18 ºC. All poultry were slaughtered at 16, 21, 26, 31 and 36 days of testing for determination of body composition in protein throughout growth. The requirement for maintenance of the Japanese quail differs from that obtained with the European quail. The protein was retained at the proportion of 32% for European quails and 25% for the Japanese quails. This difference in retention promoted estimate of 0.65 g/g gain in European quails and 0.84 g/g gain in Japanese quails. Quails should be fed diets formulated considering the requirement for protein of each genotype.

  2. A non-sequence-specific requirement for SMN protein activity: the role of aminoglycosides in inducing elevated SMN protein levels.

    Science.gov (United States)

    Wolstencroft, Elizabeth C; Mattis, Virginia; Bajer, Anna A; Young, Philip J; Lorson, Christian L

    2005-05-01

    Spinal muscular atrophy (SMA) is caused by homozygous loss of the survival motor neuron (SMN1) gene. In virtually all SMA patients, a nearly identical copy gene is present, SMN2. SMN2 cannot fully compensate for the loss of SMN1 because the majority of transcripts derived from SMN2 lack a critical exon (exon 7), resulting in a dysfunctional SMN protein. Therefore, the critical distinction between a functional and a dysfunctional SMN protein is the inclusion or the exclusion of the exon 7 encoded peptide. To determine the role of the 16 amino acids encoded by SMN exon 7, a panel of synthetic mutations were transiently expressed in SMA patient fibroblasts and HeLa cells. Consistent with previous reports, the protein encoded by SMN exons 1-6 was primarily restricted to the nucleus. However, a variety of heterologous sequences fused to the C-terminus of SMN exons 1-6 allowed mutant SMN proteins to properly distribute to the cytoplasm and to the nuclear gems. These data demonstrate that the SMN exon 7 sequence is not specifically required, rather this region functions as a non-specific 'tail' that facilitates proper localization. Therefore, a possible means to restore additional activity to the SMNDelta7 protein could be to induce a longer C-terminus by suppressing recognition of the native stop codon. To address this possibility, aminoglycosides were examined for their ability to restore detectable levels of SMN protein in SMA patient fibroblasts. Aminoglycosides can suppress the accurate identification of translation termination codons in eukaryotic cells. Consistent with this, treatment of SMA patient fibroblasts with tobramycin and amikacin resulted in a quantitative increase in SMN-positive gems and an overall increase in detectable SMN protein. Taken together, this work describes the role of the critical exon 7 region and identifies a possible alternative approach for therapeutic intervention.

  3. An apicoplast localized ubiquitylation system is required for the import of nuclear-encoded plastid proteins.

    Directory of Open Access Journals (Sweden)

    Swati Agrawal

    Full Text Available Apicomplexan parasites are responsible for numerous important human diseases including toxoplasmosis, cryptosporidiosis, and most importantly malaria. There is a constant need for new antimalarials, and one of most keenly pursued drug targets is an ancient algal endosymbiont, the apicoplast. The apicoplast is essential for parasite survival, and several aspects of its metabolism and maintenance have been validated as targets of anti-parasitic drug treatment. Most apicoplast proteins are nuclear encoded and have to be imported into the organelle. Recently, a protein translocon typically required for endoplasmic reticulum associated protein degradation (ERAD has been proposed to act in apicoplast protein import. Here, we show ubiquitylation to be a conserved and essential component of this process. We identify apicoplast localized ubiquitin activating, conjugating and ligating enzymes in Toxoplasma gondii and Plasmodium falciparum and observe biochemical activity by in vitro reconstitution. Using conditional gene ablation and complementation analysis we link this activity to apicoplast protein import and parasite survival. Our studies suggest ubiquitylation to be a mechanistic requirement of apicoplast protein import independent to the proteasomal degradation pathway.

  4. Small heat shock protein Hsp27 is required for proper heart tube formation.

    Science.gov (United States)

    Brown, Daniel D; Christine, Kathleen S; Showell, Christopher; Conlon, Frank L

    2007-11-01

    The small heat shock protein Hsp27 has been shown to be involved in a diverse array of cellular processes, including cellular stress response, protein chaperone activity, regulation of cellular glutathione levels, apoptotic signaling, and regulation of actin polymerization and stability. Furthermore, mutation within Hsp27 has been associated with the human congenital neuropathy Charcot-Marie Tooth (CMT) disease. Hsp27 is known to be expressed in developing embryonic tissues; however, little has been done to determine the endogenous requirement for Hsp27 in developing embryos. In this study, we show that depletion of XHSP27 protein results in a failure of cardiac progenitor fusion resulting in cardia bifida. Furthermore, we demonstrate a concomitant disorganization of actin filament organization and defects in myofibril assembly. Moreover, these defects are not associated with alterations in specification or differentiation. We have thus demonstrated a critical requirement for XHSP27 in developing cardiac and skeletal muscle tissues.

  5. The orphan adapter protein SLY1 as a novel anti-apoptotic protein required for thymocyte development

    Directory of Open Access Journals (Sweden)

    Beer-Hammer Sandra

    2009-07-01

    Full Text Available Abstract Background SH3 containing Lymphocyte Protein (SLY1 is a putative adapter protein exclusively expressed in lymphocytes which is involved in antigen receptor induced activation. We previously have generated SLY1Δ/Δ mice harbouring a partial deletion in the N-terminal region of SLY1 which revealed profound immunological defects in T and B cell functions. Results In this study, T cell development in SLY1-/- and SLY1Δ/Δ mice was analysed ex vivo and upon cultivation with the bone marrow stromal cell line OP9. SLY1-deficient thymocytes were compromised in inducing nutrient receptor expression and ribosomal protein S6 phosphorylation, indicating a defect in mTOR complex activation. Furthermore, SLY1 was identified as a novel anti-apoptotic protein required for developmental progression of T cell precursors to the CD4+CD8+ double-positive stage by protecting from premature programmed cell death initiation in developing CD4-CD8- double-negative thymocytes. In addition, SLY1 phosphorylation was differentially regulated upon Notch ligand-mediated stimulation and expression of the preTCR. Conclusion Thus, our results suggest a non-redundant role for SLY1 in integrating signals from both receptors in early T cell progenitors in the thymus.

  6. Genome-wide RNAi screen identifies novel host proteins required for alphavirus entry.

    Directory of Open Access Journals (Sweden)

    Yaw Shin Ooi

    Full Text Available The enveloped alphaviruses include important and emerging human pathogens such as Chikungunya virus and Eastern equine encephalitis virus. Alphaviruses enter cells by clathrin-mediated endocytosis, and exit by budding from the plasma membrane. While there has been considerable progress in defining the structure and function of the viral proteins, relatively little is known about the host factors involved in alphavirus infection. We used a genome-wide siRNA screen to identify host factors that promote or inhibit alphavirus infection in human cells. Fuzzy homologue (FUZ, a protein with reported roles in planar cell polarity and cilia biogenesis, was required for the clathrin-dependent internalization of both alphaviruses and the classical endocytic ligand transferrin. The tetraspanin membrane protein TSPAN9 was critical for the efficient fusion of low pH-triggered virus with the endosome membrane. FUZ and TSPAN9 were broadly required for infection by the alphaviruses Sindbis virus, Semliki Forest virus, and Chikungunya virus, but were not required by the structurally-related flavivirus Dengue virus. Our results highlight the unanticipated functions of FUZ and TSPAN9 in distinct steps of alphavirus entry and suggest novel host proteins that may serve as targets for antiviral therapy.

  7. Sex effects on net protein and energy requirements for growth of Saanen goats.

    Science.gov (United States)

    Souza, A P; St-Pierre, N R; Fernandes, M H R M; Almeida, A K; Vargas, J A C; Resende, K T; Teixeira, I A M A

    2017-03-22

    Requirements for growth in the different sexes remain poorly quantified in goats. The objective of this study was to develop equations for estimating net protein (NPG) and net energy (NEG) for growth in Saanen goats of different sexes from 5 to 45 kg of body weight (BW). A data set from 7 comparative slaughter studies (238 individual records) of Saanen goats was used. Allometric equations were developed to determine body protein and energy contents in the empty BW (EBW) as dependent variables and EBW as the allometric predictor. Parameter estimates were obtained using a linearized (log-transformation) expression of the allometric equations using the MIXED procedure in SAS software (SAS Institute Inc., Cary, NC). The model included the random effect of the study and the fixed effects of sex (intact male, castrated male, and female; n = 94, 73, and 71, respectively), EBW, and their interactions. Net requirements for growth were estimated as the first partial derivative of the allometric equations with respect to EBW. Additionally, net requirements for growth were evaluated based on the degree of maturity. Monte Carlo techniques were used to estimate the uncertainty of the calculated net requirement values. Sex affected allometric relationships for protein and energy in Saanen goats. The allometric equation for protein content in the EBW of intact and castrated males was log10 protein (g) = 2.221 (±0.0224) + 1.015 (±0.0165) × log10 EBW (kg). For females, the relationship was log10 protein (g) = 2.277 (±0.0288) + 0.958 (±0.0218) × log10 EBW (kg). Therefore, NPG for males was greater than for females. The allometric equation for the energy content in the EBW of intact males was log10 energy (kcal) = 2.988 (±0.0323) + 1.240 (±0.0238) × log10 EBW (kg); of castrated males, log10 energy (kcal) = 2.873 (±0.0377) + 1.359 (±0.0283) × log10 EBW (kg); and of females, log10 energy (kcal) = 2.820 (±0.0377) + 1.442 (±0.0281) × log10 EBW (kg). The NEG of castrated

  8. Determination of energy and protein requirements for crossbred Holstein × Gyr preweaned dairy calves.

    Science.gov (United States)

    Silva, A L; Marcondes, M I; Detmann, E; Campos, M M; Machado, F S; Filho, S C Valadares; Castro, M M D; Dijkstra, J

    2017-02-01

    The objective was to quantify the energy and protein nutritional requirements of Holstein × Gyr crossbred preweaned dairy calves until 64 d of age. Thirty-nine Holstein × Gyr crossbred male calves with an average initial live weight (mean ± SEM; for all next values) of 36 ± 1.0 kg were used. Five calves were slaughtered at 4 d of life to estimate the animals' initial body composition (reference group). The remaining 34 calves were distributed in a completely randomized design in a 3 × 2 factorial arrangement consisting of 3 levels of milk (2, 4, or 8 L/d) and 2 levels of starter feed (presence or absence in diet). At 15 and 45 d of life, 4 animals from each treatment were subjected to digestibility trials with total collection of feces (for 72 h) and urine (for 24 h). At 64 d of age, all animals were slaughtered, their gastro-intestinal tract was washed to determine the empty body weight (EBW; kg), and their body tissues were sampled for subsequent analyses. The net energy requirement for maintenance was estimated using an exponential regression between metabolizable energy intake and heat production (both in Mcal/EBW(0.75) per d) and was 74.3 ± 5.7 kcal/EBW(0.75) per d, and was not affected by inclusion of starter feed in the diet. The metabolizable energy requirement for maintenance was determined at the point of zero energy retention in the body and was 105.2 ± 5.8 kcal/EBW(0.75) per d. The net energy for gain was estimated using the EBW and the empty body gain (EBG; kg/d) as 0.0882 ± 0.0028 × EBW(0.75) × EBG(0.9050±0.0706). The metabolizable energy efficiency for gain (kg) of the milk was 57.4 ± 3.45%, and the kg of the starter feed was 39.3 ± 2.09%. The metabolizable protein requirement for maintenance was 3.52 ± 0.34 g/BW(0.75) per d. The net protein required for each kilogram gained was estimated as 119.1 ± 32.9 × EBW(0.0663±0.059). The metabolizable protein efficiency for gain was 77 ± 8.5% and was not affected by inclusion of starter feed

  9. Late Protein Synthesis-Dependent Phases in CTA Long-Term Memory: BDNF Requirement

    Science.gov (United States)

    Martínez-Moreno, Araceli; Rodríguez-Durán, Luis F.; Escobar, Martha L.

    2011-01-01

    It has been proposed that long-term memory (LTM) persistence requires a late protein synthesis-dependent phase, even many hours after memory acquisition. Brain-derived neurotrophic factor (BDNF) is an essential protein synthesis product that has emerged as one of the most potent molecular mediators for long-term synaptic plasticity. Studies in the rat hippocampus have been shown that BDNF is capable to rescue the late-phase of long-term potentiation as well as the hippocampus-related LTM when protein synthesis was inhibited. Our previous studies on the insular cortex (IC), a region of the temporal cortex implicated in the acquisition and storage of conditioned taste aversion (CTA), have demonstrated that intracortical delivery of BDNF reverses the deficit in CTA memory caused by the inhibition of IC protein synthesis due to anisomycin administration during early acquisition. In this work, we first analyze whether CTA memory storage is protein synthesis-dependent in different time windows. We observed that CTA memory become sensible to protein synthesis inhibition 5 and 7 h after acquisition. Then, we explore the effect of BDNF delivery (2 μg/2 μl per side) in the IC during those late protein synthesis-dependent phases. Our results show that BDNF reverses the CTA memory deficit produced by protein synthesis inhibition in both phases. These findings support the notion that recurrent rounds of consolidation-like events take place in the neocortex for maintenance of CTA memory trace and that BDNF is an essential component of these processes. PMID:21960964

  10. Current issues in determining dietary protein and amino-acid requirements

    DEFF Research Database (Denmark)

    Pencharz, P; Jahoor, F; Kurpad, A

    2014-01-01

    Pregnancy and the first two years of life are periods of rapid growth and yet the knowledge of requirements for protein and dietary indispensable amino acids is very limited. The development of carbon oxidation methods opens the way to studies that should fill these important gaps in knowledge.Eu.......European Journal of Clinical Nutrition advance online publication, 15 January 2014; doi:10.1038/ejcn.2013.297....

  11. Debra, a protein mediating lysosomal degradation, is required for long-term memory in Drosophila.

    Science.gov (United States)

    Kottler, Benjamin; Lampin-Saint-Amaux, Aurélie; Comas, Daniel; Preat, Thomas; Goguel, Valérie

    2011-01-01

    A central goal of neuroscience is to understand how neural circuits encode memory and guide behavior changes. Many of the molecular mechanisms underlying memory are conserved from flies to mammals, and Drosophila has been used extensively to study memory processes. To identify new genes involved in long-term memory, we screened Drosophila enhancer-trap P(Gal4) lines showing Gal4 expression in the mushroom bodies, a specialized brain structure involved in olfactory memory. This screening led to the isolation of a memory mutant that carries a P-element insertion in the debra locus. debra encodes a protein involved in the Hedgehog signaling pathway as a mediator of protein degradation by the lysosome. To study debra's role in memory, we achieved debra overexpression, as well as debra silencing mediated by RNA interference. Experiments conducted with a conditional driver that allowed us to specifically restrict transgene expression in the adult mushroom bodies led to a long-term memory defect. Several conclusions can be drawn from these results: i) debra levels must be precisely regulated to support normal long-term memory, ii) the role of debra in this process is physiological rather than developmental, and iii) debra is specifically required for long-term memory, as it is dispensable for earlier memory phases. Drosophila long-term memory is the only long-lasting memory phase whose formation requires de novo protein synthesis, a process underlying synaptic plasticity. It has been shown in several organisms that regulation of proteins at synapses occurs not only at translation level of but also via protein degradation, acting in remodeling synapses. Our work gives further support to a role of protein degradation in long-term memory, and suggests that the lysosome plays a role in this process.

  12. Flexibility and rigidity, requirements for the function of proteins and protein pigment complexes. Eleventh Keilin memorial lecture.

    Science.gov (United States)

    Huber, R

    1987-12-01

    Proteins may be rigid or flexible to various degrees as required for optimum function. Flexibility at the level of amino acid side-chains occurs universally and is important for binding and catalysis. Flexibility of large parts of a protein which rearrange or move are particularly interesting and will be discussed here. We differentiate between certain categories of large-scale flexibility although the boundaries between them are diffuse: flexibility of peptide segments, domain motions and order-disorder transitions of spatially contigous regions. The domains may be flexibly linked to allow rather unrestricted motion or the motion may be constrained to certain modes. The polypeptide segments linking the domains show characteristic structural features. The various categories of flexibility will be illustrated with the following examples. (a) Small protein proteinase inhibitors which are rather rigid molecules which provide binding surfaces complementary to their cognate proteases, but also show limited segmental flexibility and adaptation. (b) Large plasma inhibitors which exhibit large conformational changes upon interaction with proteases probably for regulatory purposes. (c) Pancreatic serine proteases which employ a disorder-order transition of their activation domain as a means to regulate enzymic activity. (d) Immunoglobulins in which rather unrestricted and also hinged domain motions occur in different parts of the molecule probably to allow binding to antigens in different arrangements. (e) Citrate synthase which adopts open and closed forms by a hinged domain motion to bind substrates and release products and to perform the catalytic condensation reaction, respectively. (f) The bifunctional multienzyme complex riboflavin synthase in which two enzymes (alpha and beta) catalyse two consecutive enzymic reactions. The beta-subunits form a shell, in which the alpha-subunits are enclosed. Diffusional motion of the catalytic intermediates is therefore restricted

  13. DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1-catalyzed Reaction.

    Science.gov (United States)

    Algasaier, Sana I; Exell, Jack C; Bennet, Ian A; Thompson, Mark J; Gotham, Victoria J B; Shaw, Steven J; Craggs, Timothy D; Finger, L David; Grasby, Jane A

    2016-04-08

    Human flap endonuclease-1 (hFEN1) catalyzes the essential removal of single-stranded flaps arising at DNA junctions during replication and repair processes. hFEN1 biological function must be precisely controlled, and consequently, the protein relies on a combination of protein and substrate conformational changes as a prerequisite for reaction. These include substrate bending at the duplex-duplex junction and transfer of unpaired reacting duplex end into the active site. When present, 5'-flaps are thought to thread under the helical cap, limiting reaction to flaps with free 5'-terminiin vivo Here we monitored DNA bending by FRET and DNA unpairing using 2-aminopurine exciton pair CD to determine the DNA and protein requirements for these substrate conformational changes. Binding of DNA to hFEN1 in a bent conformation occurred independently of 5'-flap accommodation and did not require active site metal ions or the presence of conserved active site residues. More stringent requirements exist for transfer of the substrate to the active site. Placement of the scissile phosphate diester in the active site required the presence of divalent metal ions, a free 5'-flap (if present), a Watson-Crick base pair at the terminus of the reacting duplex, and the intact secondary structure of the enzyme helical cap. Optimal positioning of the scissile phosphate additionally required active site conserved residues Tyr(40), Asp(181), and Arg(100)and a reacting duplex 5'-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound and 5'-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage.

  14. Efficient nuclear export of p65-IkappaBalpha complexes requires 14-3-3 proteins.

    Science.gov (United States)

    Aguilera, Cristina; Fernández-Majada, Vanessa; Inglés-Esteve, Julia; Rodilla, Verónica; Bigas, Anna; Espinosa, Lluís

    2006-09-01

    IkappaB are responsible for maintaining p65 in the cytoplasm under non-stimulating conditions and promoting the active export of p65 from the nucleus following NFkappaB activation to terminate the signal. We now show that 14-3-3 proteins regulate the NFkappaB signaling pathway by physically interacting with p65 and IkappaBalpha proteins. We identify two functional 14-3-3 binding domains in the p65 protein involving residues 38-44 and 278-283, and map the interaction region of IkappaBalpha in residues 60-65. Mutation of these 14-3-3 binding domains in p65 or IkappaBalpha results in a predominantly nuclear distribution of both proteins. TNFalpha treatment promotes recruitment of 14-3-3 and IkappaBalpha to NFkappaB-dependent promoters and enhances the binding of 14-3-3 to p65. Disrupting 14-3-3 activity by transfection with a dominant-negative 14-3-3 leads to the accumulation of nuclear p65-IkappaBalpha complexes and the constitutive association of p65 with the chromatin. In this situation, NFkappaB-dependent genes become unresponsive to TNFalpha stimulation. Together our results indicate that 14-3-3 proteins facilitate the nuclear export of IkappaBalpha-p65 complexes and are required for the appropriate regulation of NFkappaB signaling.

  15. Pocket proteins pRb and p107 are required for cortical lamination independent of apoptosis.

    Science.gov (United States)

    Svoboda, D S; Paquin, A; Park, D S; Slack, R S

    2013-12-01

    Pocket proteins (pRb, p107 and p130) are well studied in their role of regulating cell cycle progression. Increasing evidence suggests that these proteins also control early differentiation and even later stages of cell maturation, such as migration. However, pocket proteins also regulate apoptosis, and many of the developmental defects in knock out models have been attributed to increased cell death. Here, we eliminate ectopic apoptosis in the developing brain through the deletion of Bax, and show that pocket proteins are required for radial migration independent of their role in cell death regulation. Following loss of pRb and p107, a population of cortical neurons fails to pass through the intermediate zone into the cortical plate. Importantly, these neurons are born at the appropriate time and this migration defect cannot be rescued by eliminating ectopic cell death. In addition, we show that pRb and p107 regulate radial migration through a cell autonomous mechanism since pRb/p107 deficient neurons fail to migrate to the correct cortical layer within a wild type brain. These results define a novel role of pocket proteins in regulating cortical lamination through a cell autonomous mechanism independent of their role in apoptosis.

  16. Formation of rigid, non-flight forewings (elytra of a beetle requires two major cuticular proteins.

    Directory of Open Access Journals (Sweden)

    Yasuyuki Arakane

    Full Text Available Insect cuticle is composed primarily of chitin and structural proteins. To study the function of structural cuticular proteins, we focused on the proteins present in elytra (modified forewings that become highly sclerotized and pigmented covers for the hindwings of the red flour beetle, Tribolium castaneum. We identified two highly abundant proteins, TcCPR27 (10 kDa and TcCPR18 (20 kDa, which are also present in pronotum and ventral abdominal cuticles. Both are members of the Rebers and Riddiford family of cuticular proteins and contain RR2 motifs. Transcripts for both genes dramatically increase in abundance at the pharate adult stage and then decline quickly thereafter. Injection of specific double-stranded RNAs for each gene into penultimate or last instar larvae had no effect on larval-larval, larval-pupal, or pupal-adult molting. The elytra of the resulting adults, however, were shorter, wrinkled, warped, fenestrated, and less rigid than those from control insects. TcCPR27-deficient insects could not fold their hindwings properly and died prematurely approximately one week after eclosion, probably because of dehydration. TcCPR18-deficient insects exhibited a similar but less dramatic phenotype. Immunolocalization studies confirmed the presence of TcCPR27 in the elytral cuticle. These results demonstrate that TcCPR27 and TcCPR18 are major structural proteins in the rigid elytral, dorsal thoracic, and ventral abdominal cuticles of the red flour beetle, and that both proteins are required for morphogenesis of the beetle's elytra.

  17. Protein levels in enteral feeds: do these meet requirements in children with severe cerebral palsy?

    Science.gov (United States)

    Schoendorfer, Niikee; Tinggi, Ujang; Sharp, Nita; Boyd, Roslyn; Vitetta, Luis; Davies, Peter S W

    2012-05-01

    Children with cerebral palsy (CP) have been documented to have feeding difficulties, which increase in line with condition severity and result in lowered growth potential. Much nutrition literature surrounds energy intake and expenditure in these children, with less information available on other parameters such as protein and micronutrients, which are also important for growth and development. We examined differences in protein intake and a variety of protein metabolism indices in children with CP compared with controls. A total of twenty-four children aged 4-12 years with marked CP fed orally (O, n 15) or enterally (E, n 9) were recruited, including age-matched typically developing children (C, n 24). Fasting blood samples were analysed for levels of albumin, creatinine, urea and urate. Parents collected an exact food replica for three consecutive days of their child's actual intake, which were directly analysed for protein content. Significant differences were found in protein intakes between the groups (mean percentage minimum requirements: E = 178 (sd 47); O = 208 (sd 95); C = 311 (sd 119), P = 0·005). Despite all children consuming over recommended levels, children with CP had significantly reduced levels of the protein metabolic indices compared with controls. These include as z-scores: albumin mean C = 0·71 (sd 1·04) and CP = - 0·17 (sd 1·60), P = 0·03; creatinine C = - 2·06 (sd 0·46) and CP = - 3·11 (sd 0·98), P < 0·001; urate C = 0·18 (sd 0·62) and CP = - 0·58 (sd 0·93), P = 0·002. Post hoc analysis, the present data show potentially greater protein metabolism issues in enterally fed children, compared with the other groups. This may also support recent literature that suggests shortfalls in current recommendations.

  18. Sprinter: a novel transmembrane protein required for Wg secretion and signaling.

    Science.gov (United States)

    Goodman, Robyn M; Thombre, Shreya; Firtina, Zeynep; Gray, Dione; Betts, Daniella; Roebuck, Jamie; Spana, Eric P; Selva, Erica M

    2006-12-01

    Wingless (Wg) is a secreted ligand that differentially activates gene expression in target tissues. It belongs to the Wnt family of secreted signaling molecules that regulate cell-to-cell interactions during development. Activation of Wg targets is dependent on the ligand concentration in the extracellular milieu; cellular mechanisms that govern the synthesis, delivery and receipt of Wg are elaborate and complex. We have identified sprinter (srt), which encodes a novel, evolutionarily conserved transmembrane protein required for the transmission of the Wg signal. Mutations in srt cause the accumulation of Wg in cells that express it, and retention of the ligand prevents activation of its target genes in signal-receiving cells. In the absence of Srt activity, levels of Wg targets (including Engrailed in embryos lacking maternal and zygotic srt, and Senseless and Achaete in wing discs) are reduced. Activation of Wg targets in the receiving cells does not require srt. Hence, the function of Srt is restricted to events occurring within the Wg-producing cells. We show that srt is not required for any aspect of Hedgehog (Hh) signal transduction, suggesting specificity of srt for the Wg pathway. We propose that srt encodes a protein required for Wg secretion that regulates maturation, membrane targeting or delivery of Wg. Loss of srt function in turn diminishes Wg-pathway activation in receiving cells.

  19. Essential genetic interactors of SIR2 required for spatial sequestration and asymmetrical inheritance of protein aggregates.

    Directory of Open Access Journals (Sweden)

    Jia Song

    2014-07-01

    Full Text Available Sir2 is a central regulator of yeast aging and its deficiency increases daughter cell inheritance of stress- and aging-induced misfolded proteins deposited in aggregates and inclusion bodies. Here, by quantifying traits predicted to affect aggregate inheritance in a passive manner, we found that a passive diffusion model cannot explain Sir2-dependent failures in mother-biased segregation of either the small aggregates formed by the misfolded Huntingtin, Htt103Q, disease protein or heat-induced Hsp104-associated aggregates. Instead, we found that the genetic interaction network of SIR2 comprises specific essential genes required for mother-biased segregation including those encoding components of the actin cytoskeleton, the actin-associated myosin V motor protein Myo2, and the actin organization protein calmodulin, Cmd1. Co-staining with Hsp104-GFP demonstrated that misfolded Htt103Q is sequestered into small aggregates, akin to stress foci formed upon heat stress, that fail to coalesce into inclusion bodies. Importantly, these Htt103Q foci, as well as the ATPase-defective Hsp104Y662A-associated structures previously shown to be stable stress foci, co-localized with Cmd1 and Myo2-enriched structures and super-resolution 3-D microscopy demonstrated that they are associated with actin cables. Moreover, we found that Hsp42 is required for formation of heat-induced Hsp104Y662A foci but not Htt103Q foci suggesting that the routes employed for foci formation are not identical. In addition to genes involved in actin-dependent processes, SIR2-interactors required for asymmetrical inheritance of Htt103Q and heat-induced aggregates encode essential sec genes involved in ER-to-Golgi trafficking/ER homeostasis.

  20. 77 FR 6471 - Bacillus thuringiensis Cry2Ae Protein in Cotton; Exemption from the Requirement of a Tolerance

    Science.gov (United States)

    2012-02-08

    ... AGENCY 40 CFR Part 174 Bacillus thuringiensis Cry2Ae Protein in Cotton; Exemption from the Requirement of... regulation establishes an exemption from the requirement of a tolerance for residues of Bacillus... residues of Bacillus thuringiensis Cry2Ae protein in cotton under the FFDCA. DATES: This regulation...

  1. Retention of Ejaculate by Drosophila melanogaster Females Requires the Male-Derived Mating Plug Protein PEBme.

    Science.gov (United States)

    Avila, Frank W; Cohen, Allie B; Ameerudeen, Fatima S; Duneau, David; Suresh, Shruthi; Mattei, Alexandra L; Wolfner, Mariana F

    2015-08-01

    Within the mated reproductive tracts of females of many taxa, seminal fluid proteins (SFPs) coagulate into a structure known as the mating plug (MP). MPs have diverse roles, including preventing female remating, altering female receptivity postmating, and being necessary for mated females to successfully store sperm. The Drosophila melanogaster MP, which is maintained in the mated female for several hours postmating, is comprised of a posterior MP (PMP) that forms quickly after mating begins and an anterior MP (AMP) that forms later. The PMP is composed of seminal proteins from the ejaculatory bulb (EB) of the male reproductive tract. To examine the role of the PMP protein PEBme in D. melanogaster reproduction, we identified an EB GAL4 driver and used it to target PEBme for RNA interference (RNAi) knockdown. PEBme knockdown in males compromised PMP coagulation in their mates and resulted in a significant reduction in female fertility, adversely affecting postmating uterine conformation, sperm storage, mating refractoriness, egg laying, and progeny generation. These defects resulted from the inability of females to retain the ejaculate in their reproductive tracts after mating. The uncoagulated MP impaired uncoupling by the knockdown male, and when he ultimately uncoupled, the ejaculate was often pulled out of the female. Thus, PEBme and MP coagulation are required for optimal fertility in D. melanogaster. Given the importance of the PMP for fertility, we identified additional MP proteins by mass spectrometry and found fertility functions for two of them. Our results highlight the importance of the MP and the proteins that comprise it in reproduction and suggest that in Drosophila the PMP is required to retain the ejaculate within the female reproductive tract, ensuring the storage of sperm by mated females.

  2. 40 CFR 174.509 - Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry3A protein...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.509 Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3A protein are...

  3. 40 CFR 174.515 - Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat Protein of Papaya Ringspot Virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.515 Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance. Residues of Coat Protein of Papaya Ringspot Virus are...

  4. 40 CFR 174.512 - Coat Protein of Potato Virus Y; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat Protein of Potato Virus Y...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.512 Coat Protein of Potato Virus Y; exemption from the requirement of a tolerance. Residues of Coat Protein of Potato Virus Y are exempt...

  5. 40 CFR 174.516 - Coat protein of cucumber mosaic virus; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat protein of cucumber mosaic virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.516 Coat protein of cucumber mosaic virus; exemption from the requirement of a tolerance. Residues of Coat Protein of Cucumber Mosaic Virus are...

  6. 40 CFR 174.531 - Coat protein of plum pox virus; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat protein of plum pox virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.531 Coat protein of plum pox virus; exemption from the requirement of a tolerance. Residues of the coat protein of plum pox virus in or on...

  7. Host ESCRT proteins are required for bromovirus RNA replication compartment assembly and function.

    Directory of Open Access Journals (Sweden)

    Arturo Diaz

    2015-03-01

    Full Text Available Positive-strand RNA viruses genome replication invariably is associated with vesicles or other rearranged cellular membranes. Brome mosaic virus (BMV RNA replication occurs on perinuclear endoplasmic reticulum (ER membranes in ~70 nm vesicular invaginations (spherules. BMV RNA replication vesicles show multiple parallels with membrane-enveloped, budding retrovirus virions, whose envelopment and release depend on the host ESCRT (endosomal sorting complexes required for transport membrane-remodeling machinery. We now find that deleting components of the ESCRT pathway results in at least two distinct BMV phenotypes. One group of genes regulate RNA replication and the frequency of viral replication complex formation, but had no effect on spherule size, while a second group of genes regulate RNA replication in a way or ways independent of spherule formation. In particular, deleting SNF7 inhibits BMV RNA replication > 25-fold and abolishes detectable BMV spherule formation, even though the BMV RNA replication proteins accumulate and localize normally on perinuclear ER membranes. Moreover, BMV ESCRT recruitment and spherule assembly depend on different sets of protein-protein interactions from those used by multivesicular body vesicles, HIV-1 virion budding, or tomato bushy stunt virus (TBSV spherule formation. These and other data demonstrate that BMV requires cellular ESCRT components for proper formation and function of its vesicular RNA replication compartments. The results highlight growing but diverse interactions of ESCRT factors with many viruses and viral processes, and potential value of the ESCRT pathway as a target for broad-spectrum antiviral resistance.

  8. Host ESCRT proteins are required for bromovirus RNA replication compartment assembly and function.

    Science.gov (United States)

    Diaz, Arturo; Zhang, Jiantao; Ollwerther, Abigail; Wang, Xiaofeng; Ahlquist, Paul

    2015-03-01

    Positive-strand RNA viruses genome replication invariably is associated with vesicles or other rearranged cellular membranes. Brome mosaic virus (BMV) RNA replication occurs on perinuclear endoplasmic reticulum (ER) membranes in ~70 nm vesicular invaginations (spherules). BMV RNA replication vesicles show multiple parallels with membrane-enveloped, budding retrovirus virions, whose envelopment and release depend on the host ESCRT (endosomal sorting complexes required for transport) membrane-remodeling machinery. We now find that deleting components of the ESCRT pathway results in at least two distinct BMV phenotypes. One group of genes regulate RNA replication and the frequency of viral replication complex formation, but had no effect on spherule size, while a second group of genes regulate RNA replication in a way or ways independent of spherule formation. In particular, deleting SNF7 inhibits BMV RNA replication > 25-fold and abolishes detectable BMV spherule formation, even though the BMV RNA replication proteins accumulate and localize normally on perinuclear ER membranes. Moreover, BMV ESCRT recruitment and spherule assembly depend on different sets of protein-protein interactions from those used by multivesicular body vesicles, HIV-1 virion budding, or tomato bushy stunt virus (TBSV) spherule formation. These and other data demonstrate that BMV requires cellular ESCRT components for proper formation and function of its vesicular RNA replication compartments. The results highlight growing but diverse interactions of ESCRT factors with many viruses and viral processes, and potential value of the ESCRT pathway as a target for broad-spectrum antiviral resistance.

  9. Zebrafish Thsd7a is a neural protein required for angiogenic patterning during development.

    Science.gov (United States)

    Wang, Chieh-Huei; Chen, I-Hui; Kuo, Meng-Wei; Su, Pei-Tsu; Lai, Zih-Yin; Wang, Chian-Huei; Huang, Wei-Chang; Hoffman, Jana; Kuo, Calvin J; You, May-Su; Chuang, Yung-Jen

    2011-06-01

    Angiogenesis is a highly organized process under the control of guidance cues that direct endothelial cell (EC) migration. Recently, many molecules that were initially described as regulators of neural guidance were subsequently shown to also direct EC migration. Here, we report a novel protein, thrombospondin type I domain containing 7A (Thsd7a), that is a neural molecule required for directed EC migration during embryonic angiogenesis in zebrafish. Thsd7a is a vertebrate conserved protein. Zebrafish thsd7a transcript was detected along the ventral edge of the neural tube in the developing zebrafish embryos, correlating with the growth path of angiogenic intersegmental vessels (ISVs). Morpholino-knockdown of Thsd7a caused a lateral deviation of angiogenic ECs below the thsd7a-expressing sites, resulting in aberrant ISV patterning. Collectively, our study shows that zebrafish Thsd7a is a neural protein required for ISV angiogenesis, and suggests an important role of Thsd7a in the neurovascular interaction during zebrafish development.

  10. The ribosomal biogenesis protein Utp21 interacts with Hsp90 and has differing requirements for Hsp90-associated proteins.

    Directory of Open Access Journals (Sweden)

    Victoria R Tenge

    Full Text Available The molecular chaperone Hsp90 buffers the effects of genetic variation by assisting the stabilization and folding of multiple clients critical for cell signaling and growth. We identified an interaction of Hsp90 and associated proteins with the essential nucleolar protein, Utp21, part of a large complex required for biogenesis of the small ribosomal subunit. The utp21-S602F mutation, which causes minor defects in otherwise wild-type yeast, exhibited severe or lethal growth defects when combined with mutations in Hsp90 or co-chaperones. WT Utp21 and Utp21-S602F exhibited similar interactions with Hsp90, and steady-state levels of WT Utp21 were reduced upon Hsp90 mutation or inhibition. Mutations in the human homolog of UTP21, WDR36, have been associated with adult-onset primary open-angle glaucoma, a leading cause of blindness worldwide. Three different mutant forms of Utp21 analogous to glaucoma-associated WDR36 mutations exhibit reduced levels in yeast cells expressing mutations in Hsp90 or associated chaperones, suggesting that Hsp90 and co-chaperones buffer the effects of those mutations.

  11. TYLCV-Is movement in planta does not require V2 protein

    Energy Technology Data Exchange (ETDEWEB)

    Hak, Hagit [Institute of Plant Sciences, Agricultural Research Organization, The Volcani Center, Bet Dagan (Israel); Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem (Israel); Levy, Yael; Chandran, Sam A.; Belausov, Eduard [Institute of Plant Sciences, Agricultural Research Organization, The Volcani Center, Bet Dagan (Israel); Loyter, Abraham [Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem (Israel); Lapidot, Moshe [Institute of Plant Sciences, Agricultural Research Organization, The Volcani Center, Bet Dagan (Israel); Gafni, Yedidya, E-mail: ygafni@volcani.agri.gov.il [Institute of Plant Sciences, Agricultural Research Organization, The Volcani Center, Bet Dagan (Israel)

    2015-03-15

    Tomato yellow leaf curl virus (TYLCV), a major tomato pathogen causing extensive crop losses, is a whitefly-transmitted geminivirus. V2 mutants of TYLCV-Is and related viruses tend to induce symptomless infection with attenuated viral DNA levels, while accumulating close to wild-type DNA levels in protoplasts, suggesting V2 as a movement protein. The discovery of plant-silencing mechanisms and viral silencing suppressors, V2 included, led us to reconsider V2's involvement in viral movement. We studied two mutant versions of the virus, one impaired in V2 silencing-suppression activity, and another carrying a non-translatable V2. While both mutant viruses spread in the infected plant to newly emerged leaves at the same rate as the wild-type virus, their DNA-accumulation levels were tenfold lower than in the wild-type virus. Thus, we suggest that the setback in virus proliferation, previously ascribed to a movement impediment, is due to lack of silencing-suppression activity. - Highlights: • TYLCV-Is V2 protein is localized in distinct microbodies throughout the cell cytoplasm, around the nucleus and in association with cytoplasmic strands but is not associated with the plasmodesmata. • Disruption of RNA-silencing suppression activity of TYLCV-Is V2 protein causes low titer of the virus in the infected plants. • The movement of TYLCV-Is in planta does not require a functional V2 protein.

  12. Provision of protein and energy in relation to measured requirements in intensive care patients

    DEFF Research Database (Denmark)

    Allingstrup, Matilde Jo; Esmailzadeh, Negar; Knudsen, Anne Wilkens

    2012-01-01

    BACKGROUND & AIMS: Adequacy of nutritional support in intensive care patients is still a matter of investigation. This study aimed to relate mortality to provision, measured requirements and balances for energy and protein in ICU patients. DESIGN: Prospective observational cohort study of 113 ICU......, also when adjusted for baseline prognostic variables (APACHE II, SOFA scores and age). Provision of energy, measured resting energy expenditure or energy and nitrogen balance was not related to mortality. The possible cause-effect relationship is discussed after a more detailed analysis of the initial...... part of the admission. CONCLUSION: In these severely ill ICU patients, a higher provision of protein and amino acids was associated with a lower mortality. This was not the case for provision of energy or measured resting energy expenditure or energy or nitrogen balances. The hypothesis that higher...

  13. Determination of the amino acid requirements for a protein hinge in triosephosphate isomerase.

    OpenAIRE

    Sun, J.; Sampson, N. S.

    1998-01-01

    We have determined the sequence requirements for a protein hinge in triosephosphate isomerase. The codons encoding the hinge at the C-terminus of the active-site lid of triosephosphate isomerase were replaced with a genetic library of all possible 8,000 amino acid combinations. The most active of these 8,000 mutants were selected using in vivo complementation of a triosephosphate isomerase deficient strain of E. coli, DF502. Approximately 3% of the mutants complement DF502 with an activity th...

  14. Obif, a Transmembrane Protein, Is Required for Bone Mineralization and Spermatogenesis in Mice.

    Directory of Open Access Journals (Sweden)

    Koji Mizuhashi

    Full Text Available Various kinds of transmembrane and secreted proteins play pivotal roles in development through cell-cell communication. We previously reported that Obif (Osteoblast induction factor, Tmem119, encoding a single transmembrane protein, is expressed in differentiating osteoblasts, and that Obif-/- mice exhibit significantly reduced bone volume in the femur. In the current study, we characterized the Obif protein and further investigated the biological phenotypes of a variety of tissues in Obif-/- mice.First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain. Next, we observed that Obif-/- mice exhibit bone dysplasia in association with significantly increased osteoid volume per osteoid surface (OV/OS and osteoid maturation time (Omt, and significantly decreased mineral apposition rate (MAR and bone formation rate per bone surface (BFR/BS. In addition, we observed that Obif-/- mice show a significant decrease in testis weight as well as in sperm number. By histological analysis, we found that Obif is expressed in spermatocytes and spermatids in the developing testis and that spermatogenesis is halted at the round spermatid stage in the Obif-/- testis that lacks sperm. However, the number of litters fathered by male mice was slightly reduced in Obif-/- mice compared with wild-type mice, although this was not statistically significant.Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated. In addition, we found that Obif is required for normal bone mineralization and late testicular differentiation in vivo. These findings suggest that Obif plays essential roles in the development of multiple tissues.

  15. Protein, calcium and phosphorus requirements in the maintenance of captive-bred capybaras

    Directory of Open Access Journals (Sweden)

    Sérgio Luiz Gama Nogueira-Filho

    2013-05-01

    Full Text Available The objective was to determine the nitrogen-protein, calcium and phosphorus requirements of adult capybaras (Hydrochoerus hydrochaeris bred in captivity through a nutritional balance study under conditions that favored the occurrence of cecotrophy. In a completely randomized design with four replicates per treatment, 16 adult female capybaras were fed isocaloric diets containing four levels of nitrogen (11.4, 12.3, 15.4 and 18.4 g N/kg dry matter-DM; calcium (3.0, 3.5, 4.0 and 4.5 g Ca/kg-DM; and phosphorus (2.0, 2.3, 2.7 and 3.0 g P/kg-DM. After 10 days of adaptation, the collection of feces was carried out for five consecutive days. Immediately following fecal collection, the animals were housed in metabolic cages for two days for urine collection. During the fecal collection, capybaras were observed to perform on average 0.32±0.27 cecotrophy acts per hour. Regression analyses allowed for estimating daily requirements of 0.25 g digestible nitrogen/kg LW0.75, which equals 1.56 g of digestible protein/kg LW0.75. Likewise, the available calcium and phosphorus requirements in 0.30 g Ca/kg LW0.75 and 0.15 g P/kg LW0.75 were estimated. The requirements of digestible nitrogen, as well as those of available calcium and phosphorus for the maintenance of capybaras are lower than those of domestic and other wild animal species with zootechnical potential.

  16. Iron-regulated biofilm formation in Staphylococcus aureus Newman requires ica and the secreted protein Emp.

    Science.gov (United States)

    Johnson, Miranda; Cockayne, Alan; Morrissey, Julie A

    2008-04-01

    Staphylococcus aureus biofilm formation is induced in iron-restricted growth conditions in vitro. In this study, we showed that Emp and Eap play important roles in low-iron-induced biofilm formation of S. aureus Newman. Eap and Emp are secreted proteins which are non-covalently attached to the S. aureus cell surface and have previously been implicated in a number of aspects of S. aureus pathogenesis. We showed here that the transcription of these important virulence factors is induced by growth in low-iron medium, reflective of the in vivo environment. Our results show that iron regulation of Eap and Emp is Fur independent. However, Fur is required for full induction of eap and emp expression in low-iron conditions. In this study, we demonstrated that in addition to Fur, low-iron-induced biofilm formation requires Sae, Agr, and SarA. In iron-restricted growth conditions, Sae and Agr are essential for Emp and Eap expression and hence for biofilm formation, whereas SarA appears to have a less-significant role. We also showed that expression of the ica operon is required for biofilm formation in iron-restricted growth conditions. We demonstrated that in fact, ica is required for the expression of the important multifunctional virulence determinants eap and emp.

  17. Vif proteins of human and simian immunodeficiency viruses require cellular CBFβ to degrade APOBEC3 restriction factors.

    Science.gov (United States)

    Hultquist, Judd F; Binka, Mawuena; LaRue, Rebecca S; Simon, Viviana; Harris, Reuben S

    2012-03-01

    HIV-1 requires the cellular transcription factor CBFβ to stabilize its accessory protein Vif and promote APOBEC3G degradation. Here, we demonstrate that both isoforms of CBFβ allow for increased steady-state levels of Vif, enhanced APOBEC3G degradation, and increased viral infectivity. This conserved functional interaction enhances the steady-state levels of Vif proteins from multiple HIV-1 subtypes and is required for the degradation of all human and rhesus Vif-sensitive APOBEC3 proteins by their respective lentiviral Vif proteins.

  18. A Protein Complex Required for Polymerase V Transcripts and RNA- Directed DNA Methylation in Arabidopsis

    KAUST Repository

    Law, Julie A.

    2010-05-01

    DNA methylation is an epigenetic modification associated with gene silencing. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which is targeted by small interfering RNAs through a pathway termed RNA-directed DNA methylation (RdDM) [1, 2]. Recently, RdDM was shown to require intergenic noncoding (IGN) transcripts that are dependent on the Pol V polymerase. These transcripts are proposed to function as scaffolds for the recruitment of downstream RdDM proteins, including DRM2, to loci that produce both siRNAs and IGN transcripts [3]. However, the mechanism(s) through which Pol V is targeted to specific genomic loci remains largely unknown. Through affinity purification of two known RdDM components, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1) [4] and DEFECTIVE IN MERISTEM SILENCING 3 (DMS3) [5, 6], we found that they copurify with each other and with a novel protein, RNA-DIRECTED DNA METHYLATION 1 (RDM1), forming a complex we term DDR. We also found that DRD1 copurified with Pol V subunits and that RDM1, like DRD1 [3] and DMS3 [7], is required for the production of Pol V-dependent transcripts. These results suggest that the DDR complex acts in RdDM at a step upstream of the recruitment or activation of Pol V. © 2010 Elsevier Ltd. All rights reserved.

  19. Inca: a novel p21-activated kinase-associated protein required for cranial neural crest development.

    Science.gov (United States)

    Luo, Ting; Xu, Yanhua; Hoffman, Trevor L; Zhang, Tailin; Schilling, Thomas; Sargent, Thomas D

    2007-04-01

    Inca (induced in neural crest by AP2) is a novel protein discovered in a microarray screen for genes that are upregulated in Xenopus embryos by the transcriptional activator protein Tfap2a. It has no significant similarity to any known protein, but is conserved among vertebrates. In Xenopus, zebrafish and mouse embryos, Inca is expressed predominantly in the premigratory and migrating neural crest (NC). Knockdown experiments in frog and fish using antisense morpholinos reveal essential functions for Inca in a subset of NC cells that form craniofacial cartilage. Cells lacking Inca migrate successfully but fail to condense into skeletal primordia. Overexpression of Inca disrupts cortical actin and prevents formation of actin "purse strings", which are required for wound healing in Xenopus embryos. We show that Inca physically interacts with p21-activated kinase 5 (PAK5), a known regulator of the actin cytoskeleton that is co-expressed with Inca in embryonic ectoderm, including in the NC. These results suggest that Inca and PAK5 cooperate in restructuring cytoskeletal organization and in the regulation of cell adhesion in the early embryo and in NC cells during craniofacial development.

  20. The nuclear protein Waharan is required for endosomal-lysosomal trafficking in Drosophila.

    Science.gov (United States)

    Lone, Mohiddin; Kungl, Theresa; Koper, Andre; Bottenberg, Wolfgang; Kammerer, Richard; Klein, Melanie; Sweeney, Sean T; Auburn, Richard P; O'Kane, Cahir J; Prokop, Andreas

    2010-07-15

    Here we report Drosophila Waharan (Wah), a 170-kD predominantly nuclear protein with two potential human homologues, as a newly identified regulator of endosomal trafficking. Wah is required for neuromuscular-junction development and muscle integrity. In muscles, knockdown of Wah caused novel accumulations of tightly packed electron-dense tubules, which we termed 'sausage bodies'. Our data suggest that sausage bodies coincide with sites at which ubiquitylated proteins and a number of endosomal and lysosomal markers co-accumulate. Furthermore, loss of Wah function generated loss of the acidic LysoTracker compartment. Together with data demonstrating that Wah acts earlier in the trafficking pathway than the Escrt-III component Drosophila Shrb (snf7 in Schizosaccharomyces pombe), our results indicate that Wah is essential for endocytic trafficking at the late endosome. Highly unexpected phenotypes result from Wah knockdown, in that the distribution of ubiquitylated cargos and endolysosomal morphologies are affected despite Wah being a predominant nuclear protein. This finding suggests the existence of a relationship between nuclear functions and endolysosomal trafficking. Future studies of Wah function will give us insights into this interesting phenomenon.

  1. A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria.

    Science.gov (United States)

    Donovan, Catriona; Heyer, Antonia; Pfeifer, Eugen; Polen, Tino; Wittmann, Anja; Krämer, Reinhard; Frunzke, Julia; Bramkamp, Marc

    2015-05-26

    In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin.

  2. The interaction between the yeast telomerase RNA and the Est1 protein requires three structural elements.

    Science.gov (United States)

    Lubin, Johnathan W; Tucey, Timothy M; Lundblad, Victoria

    2012-09-01

    In the budding yeast Saccharomyces cerevisiae, the telomerase enzyme is composed of a 1.3-kb TLC1 RNA that forms a complex with Est2 (the catalytic subunit) and two regulatory proteins, Est1 and Est3. Previous work has identified a conserved 5-nt bulge, present in a long helical arm of TLC1, which mediates binding of Est1 to TLC1. However, increased expression of Est1 can bypass the consequences of removal of this RNA bulge, indicating that there are additional binding site(s) for Est1 on TLC1. We report here that a conserved single-stranded internal loop immediately adjacent to the bulge is also required for the Est1-RNA interaction; furthermore, a TLC1 variant that lacks this internal loop but retains the bulge cannot be suppressed by Est1 overexpression, arguing that the internal loop may be a more critical element for Est1 binding. An additional structural feature consisting of a single-stranded region at the base of the helix containing the bulge and internal loop also contributes to recognition of TLC1 by Est1, potentially by providing flexibility to this helical arm. Association of Est1 with each of these TLC1 motifs was assessed using a highly sensitive biochemical assay that simultaneously monitors the relative levels of the Est1 and Est2 proteins in the telomerase complex. The identification of three elements of TLC1 that are required for Est1 association provides a detailed view of this particular protein-RNA interaction.

  3. Niche-specific requirement for hyphal wall protein 1 in virulence of Candida albicans.

    Directory of Open Access Journals (Sweden)

    Janet F Staab

    Full Text Available Specialized Candida albicans cell surface proteins called adhesins mediate binding of the fungus to host cells. The mammalian transglutaminase (TG substrate and adhesin, Hyphal wall protein 1 (Hwp1, is expressed on the hyphal form of C. albicans where it mediates fungal adhesion to epithelial cells. Hwp1 is also required for biofilm formation and mating thus the protein functions in both fungal-host and self-interactions. Hwp1 is required for full virulence of C. albicans in murine models of disseminated candidiasis and of esophageal candidiasis. Previous studies correlated TG activity on the surface of oral epithelial cells, produced by epithelial TG (TG1, with tight binding of C. albicans via Hwp1 to the host cell surfaces. However, the contribution of other Tgs, specifically tissue TG (TG2, to disseminated candidiasis mediated by Hwp1 was not known. A newly created hwp1 null strain in the wild type SC5314 background was as virulent as the parental strain in C57BL/6 mice, and virulence was retained in C57BL/6 mice deleted for Tgm2 (TG2. Further, the hwp1 null strains displayed modestly reduced virulence in BALB/c mice as did strain DD27-U1, an independently created hwp1Δ/Δ in CAI4 corrected for its ura3Δ defect at the URA3 locus. Hwp1 was still needed to produce wild type biofilms, and persist on murine tongues in an oral model of oropharyngeal candidiasis consistent with previous studies by us and others. Finally, lack of Hwp1 affected the translocation of C. albicans from the mouse intestine into the bloodstream of mice. Together, Hwp1 appears to have a minor role in disseminated candidiasis, independent of tissue TG, but a key function in host- and self-association to the surface of oral mucosa.

  4. The Arabidopsis thaliana vernalization response requires a polycomb-like protein complex that also includes VERNALIZATION INSENSITIVE 3.

    Science.gov (United States)

    Wood, Craig C; Robertson, Masumi; Tanner, Greg; Peacock, W James; Dennis, Elizabeth S; Helliwell, Chris A

    2006-09-26

    In Arabidopsis thaliana, the promotion of flowering by cold temperatures, vernalization, is regulated via a floral-repressive MADS box transcription factor, FLOWERING LOCUS C (FLC). Vernalization leads to the epigenetic repression of FLC expression, a process that requires the polycomb group (PcG) protein VERNALIZATION 2 (VRN2) and the plant homeodomain protein VERNALIZATION INSENSITIVE 3 (VIN3). We demonstrate that the repression of FLC by vernalization requires homologues of other Polycomb Repressive Complex 2 proteins and VRN2. We show in planta that VRN2 and VIN3 are part of a large protein complex that can include the PcG proteins FERTILIZATION INDEPENDENT ENDOSPERM, CURLY LEAF, and SWINGER. These findings suggest a single protein complex is responsible for histone deacetylation at FLC and histone methylation at FLC in vernalized plants. The abundance of the complex increases during vernalization and declines after plants are returned to higher temperatures, consistent with the complex having a role in establishing FLC repression.

  5. Requirement for the eIF4E binding proteins for the synergistic down-regulation of protein synthesis by hypertonic conditions and mTOR inhibition.

    Science.gov (United States)

    Clemens, Michael J; Elia, Androulla; Morley, Simon J

    2013-01-01

    The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely.

  6. 40 CFR 174.517 - Bacillus thuringiensis Cry9C protein in corn; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry9C protein... PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.517 Bacillus thuringiensis... Bacillus thuringiensis Cry9C protein in corn is exempted from the requirement of a tolerance for...

  7. 40 CFR 174.502 - Bacillus thuringiensis Cry1A.105 protein; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry1A.105...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.502 Bacillus thuringiensis Cry1A.105 protein; exemption from the requirement of a tolerance. (a) Residues of Bacillus thuringiensis Cry1A.105 protein...

  8. 40 CFR 174.530 - Bacillus thuringiensis Cry2Ae protein in cotton; temporary exemption from the requirement of a...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry2Ae protein... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.530 Bacillus... Bacillus thuringiensis Cry2Ae protein in or on the food commodities of cotton, cotton; cotton,...

  9. 40 CFR 174.504 - Bacillus thuringiensis Cry1F protein in cotton; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry1F protein... PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.504 Bacillus thuringiensis Cry1F protein in cotton; exemption from the requirement of a tolerance. Residues of...

  10. 40 CFR 174.520 - Bacillus thuringiensis Cry1F protein in corn; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry1F protein... PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.520 Bacillus thuringiensis Cry1F protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus...

  11. The eco-nutrition requirements for dietary protein and its rhomb characteristics in juvenile turbot (Scophthalmus maximus L.)

    Institute of Scientific and Technical Information of China (English)

    LI Yong; JIANG Keyong; SUN Guoxiang; GAO Tingting; ZHOU Bangwei

    2011-01-01

    We evaluated the dietary protein requirements of juvenile turbot (Scophthalmus maximus L.)and their effects on aquatic quality.Five experimental diets were formulated containing 450,480,500,520,and 540 g/kg.Each diet was randomly assigned to triplicate groups of juvenile turbot (mean initial body weight 34.5±5.5 g) for 88 d.Both the weight gain ratio and feed efficiency increased with increasing dietary protein up to 500 g/kg,but no further improvement was detected when dietary protein levels were >500 g/kg.Protein intake and digestion increased with protein levels,while fecal nitrogen and nitrogen content in seawater increased only when dietary protein exceeded 500 g/kg.Protein digestibility was highest at intermediate dietary protein levels.Chemical oxygen demand,nitrite-nitrogen (NO2- -N) and phosphatic-phosphor (PO43 -P) levels increased in the rearing water as dietary protein levels increased.The optimum eco-nutrition level of dietary protein for juvenile turbot was 500 g/kg under the current experimental conditions.The diets containing 540 and 500 g/kg protein had similar growth rates and feed conversion ratios,but levels of ammonia (NH4+) and nitrogen were considerably higher in the water and feces,respectively,at the higher level of dietary protein.The difference in the pattem of change between body weight gain and ammonia concentration in water with increasing dietary protein is described by rhomb characteristics.

  12. Protein crystals in Adenovirus type 5-infected cells: requirements for intranuclear crystallogenesis, structural and functional analysis.

    Directory of Open Access Journals (Sweden)

    Laure Franqueville

    Full Text Available Intranuclear crystalline inclusions have been observed in the nucleus of epithelial cells infected with Adenovirus serotype 5 (Ad5 at late steps of the virus life cycle. Using immuno-electron microscopy and confocal microscopy of cells infected with various Ad5 recombinants modified in their penton base or fiber domains, we found that these inclusions represented crystals of penton capsomers, the heteromeric capsid protein formed of penton base and fiber subunits. The occurrence of protein crystals within the nucleus of infected cells required the integrity of the fiber knob and part of the shaft domain. In the knob domain, the region overlapping residues 489-492 in the FG loop was found to be essential for crystal formation. In the shaft, a large deletion of repeats 4 to 16 had no detrimental effect on crystal inclusions, whereas deletion of repeats 8 to 21 abolished crystal formation without altering the level of fiber protein expression. This suggested a crucial role of the five penultimate repeats in the crystallisation process. Chimeric pentons made of Ad5 penton base and fiber domains from different serotypes were analyzed with respect to crystal formation. No crystal was found when fiber consisted of shaft (S from Ad5 and knob (K from Ad3 (heterotypic S5-K3 fiber, but occurred with homotypic S3K3 fiber. However, less regular crystals were observed with homotypic S35-K35 fiber. TB5, a monoclonal antibody directed against the Ad5 fiber knob was found by immunofluorescence microscopy to react with high efficiency with the intranuclear protein crystals in situ. Data obtained with Ad fiber mutants indicated that the absence of crystalline inclusions correlated with a lower infectivity and/or lower yields of virus progeny, suggesting that the protein crystals might be involved in virion assembly. Thus, we propose that TB5 staining of Ad-infected 293 cells can be used as a prognostic assay for the viability and productivity of fiber-modified Ad5

  13. Effect of protein-energy malnutrition on erythropoietin requirement in maintenance hemodialysis patients.

    Science.gov (United States)

    Akgul, Arzu; Bilgic, Ayse; Sezer, Siren; Ozdemir, Fatma Nurhan; Olcay, Irem; Arat, Zubeyde; Haberal, Mehmet

    2007-04-01

    Possible interactions between inflammatory and nutritional markers and their impact on recombinant human erythropoietin (rHuEPO) hyporesponsiveness are not well understood. We investigated the role of nutritional status in rHuEPO requirement in maintenance hemodialysis (MHD) patients without evidence of inflammation. This cross-sectional study included 88 MHD patients. The associations between required rHuEPO dose and malnutrition-inflammation score (MIS) and several laboratory values known to be related to nutrition and/or inflammation were analyzed. Anthropometric measures including body mass index, triceps skinfold thickness, and midarm circumferences were also measured. Twenty-three patients with serum C-reactive protein levels >10 mg/L were excluded from the analysis. The remaining 65 patients (male/female, 41/24; age 49.1+/-11.4 years; dialysis duration 99.7+/-63.0 months) were studied. These patients had moderate malnutrition and the average MIS was 7.4 (range 3-17). The average weekly dose of administered rHuEPO was 69.1+/-63.1 U/kg. Malnutrition-inflammation score had a positive correlation with the serum concentration of tumor necrosis factor-alpha, whereas it had a negative correlation with anthropometric measures, total iron-binding capacity, prealbumin, phosphorus, creatinine, and triglyceride. According to Pearson's correlation analysis, significant relationships of increased MIS with increased required rHuEPO dose and rHuEPO responsiveness index (EPO divided by hematocrit) were observed (p=0.008, r=-0.326; p=0.017, r=-0.306, respectively). Recombinant human erythropoietin dose requirement is correlated with MIS and adverse nutritional status in MHD patients without evidence of inflammation. Further research should focus on reversing the undergoing microinflammation for a better outcome in dialysis patients.

  14. A human telomerase holoenzyme protein required for Cajal body localization and telomere synthesis.

    Science.gov (United States)

    Venteicher, Andrew S; Abreu, Eladio B; Meng, Zhaojing; McCann, Kelly E; Terns, Rebecca M; Veenstra, Timothy D; Terns, Michael P; Artandi, Steven E

    2009-01-30

    Telomerase is a ribonucleoprotein (RNP) complex that synthesizes telomere repeats in tissue progenitor cells and cancer cells. Active human telomerase consists of at least three principal subunits, including the telomerase reverse transcriptase, the telomerase RNA (TERC), and dyskerin. Here, we identify a holoenzyme subunit, TCAB1 (telomerase Cajal body protein 1), that is notably enriched in Cajal bodies, nuclear sites of RNP processing that are important for telomerase function. TCAB1 associates with active telomerase enzyme, established telomerase components, and small Cajal body RNAs that are involved in modifying splicing RNAs. Depletion of TCAB1 by using RNA interference prevents TERC from associating with Cajal bodies, disrupts telomerase-telomere association, and abrogates telomere synthesis by telomerase. Thus, TCAB1 controls telomerase trafficking and is required for telomere synthesis in human cancer cells.

  15. Plant science. Morphinan biosynthesis in opium poppy requires a P450-oxidoreductase fusion protein.

    Science.gov (United States)

    Winzer, Thilo; Kern, Marcelo; King, Andrew J; Larson, Tony R; Teodor, Roxana I; Donninger, Samantha L; Li, Yi; Dowle, Adam A; Cartwright, Jared; Bates, Rachel; Ashford, David; Thomas, Jerry; Walker, Carol; Bowser, Tim A; Graham, Ian A

    2015-07-17

    Morphinan alkaloids from the opium poppy are used for pain relief. The direction of metabolites to morphinan biosynthesis requires isomerization of (S)- to (R)-reticuline. Characterization of high-reticuline poppy mutants revealed a genetic locus, designated STORR [(S)- to (R)-reticuline] that encodes both cytochrome P450 and oxidoreductase modules, the latter belonging to the aldo-keto reductase family. Metabolite analysis of mutant alleles and heterologous expression demonstrate that the P450 module is responsible for the conversion of (S)-reticuline to 1,2-dehydroreticuline, whereas the oxidoreductase module converts 1,2-dehydroreticuline to (R)-reticuline rather than functioning as a P450 redox partner. Proteomic analysis confirmed that these two modules are contained on a single polypeptide in vivo. This modular assembly implies a selection pressure favoring substrate channeling. The fusion protein STORR may enable microbial-based morphinan production.

  16. BRCA1 interaction of centrosomal protein Nlp is required for successful mitotic progression.

    Science.gov (United States)

    Jin, Shunqian; Gao, Hua; Mazzacurati, Lucia; Wang, Yang; Fan, Wenhong; Chen, Qiang; Yu, Wei; Wang, Mingrong; Zhu, Xueliang; Zhang, Chuanmao; Zhan, Qimin

    2009-08-21

    Breast cancer susceptibility gene BRCA1 is implicated in the control of mitotic progression, although the underlying mechanism(s) remains to be further defined. Deficiency of BRCA1 function leads to disrupted mitotic machinery and genomic instability. Here, we show that BRCA1 physically interacts and colocalizes with Nlp, an important molecule involved in centrosome maturation and spindle formation. Interestingly, Nlp centrosomal localization and its protein stability are regulated by normal cellular BRCA1 function because cells containing BRCA1 mutations or silenced for endogenous BRCA1 exhibit disrupted Nlp colocalization to centrosomes and enhanced Nlp degradation. Its is likely that the BRCA1 regulation of Nlp stability involves Plk1 suppression. Inhibition of endogenous Nlp via the small interfering RNA approach results in aberrant spindle formation, aborted chromosomal segregation, and aneuploidy, which mimic the phenotypes of disrupted BRCA1. Thus, BRCA1 interaction of Nlp might be required for the successful mitotic progression, and abnormalities of Nlp lead to genomic instability.

  17. Truncation of LEAFY COTYLEDON1 protein is required for asexual reproduction in Kalanchoë daigremontiana.

    Science.gov (United States)

    Garcês, Helena M P; Koenig, Daniel; Townsley, Brad T; Kim, Minsung; Sinha, Neelima R

    2014-05-01

    Kalanchoë daigremontiana reproduces asexually by generating numerous plantlets on its leaf margins. The formation of plantlets requires the somatic initiation of organogenic and embryogenic developmental programs in the leaves. However, unlike normal embryogenesis in seeds, leaf somatic embryogenesis bypasses seed dormancy to form viable plantlets. In Arabidopsis (Arabidopsis thaliana), seed dormancy and embryogenesis are initiated by the transcription factor LEAFY COTYLEDON1 (LEC1). The K. daigremontiana ortholog of LEC1 is expressed during leaf somatic embryo development. However, KdLEC1 encodes for a LEC1-type protein that has a unique B domain, with 11 unique amino acids and a premature stop codon. Moreover, the truncated KdLEC1 protein is not functional in Arabidopsis. Here, we show that K. daigremontiana transgenic plants expressing a functional, chimeric KdLEC1 gene under the control of Arabidopsis LEC1 promoter caused several developmental defects to leaf somatic embryos, including seed dormancy characteristics. The dormant plantlets also behaved as typical dormant seeds. Transgenic plantlets accumulated oil bodies and responded to the abscisic acid biosynthesis inhibitor fluridone, which broke somatic-embryo dormancy and promoted their normal development. Our results indicate that having a mutated form of LEC1 gene in K. daigremontiana is essential to bypass dormancy in the leaf embryos and generate viable plantlets, suggesting that the loss of a functional LEC1 promotes viviparous leaf somatic embryos and thus enhances vegetative propagation in K. daigremontiana. Mutations resulting in truncated LEC1 proteins may have been of a selective advantage in creating somatic propagules, because such mutations occurred independently in several Kalanchoë species, which form plantlets constitutively.

  18. Rybp, a polycomb complex-associated protein, is required for mouse eye development

    Directory of Open Access Journals (Sweden)

    Schreiber-Agus Nicole

    2007-04-01

    Full Text Available Abstract Background Rybp (Ring1 and YY1 binding protein is a zinc finger protein which interacts with the members of the mammalian polycomb complexes. Previously we have shown that Rybp is critical for early embryogenesis and that haploinsufficiency of Rybp in a subset of embryos causes failure of neural tube closure. Here we investigated the requirement for Rybp in ocular development using four in vivo mouse models which resulted in either the ablation or overexpression of Rybp. Results Our results demonstrate that loss of a single Rybp allele in conventional knockout mice often resulted in retinal coloboma, an incomplete closure of the optic fissure, characterized by perturbed localization of Pax6 but not of Pax2. In addition, about one half of Rybp-/- Rybp+/+ chimeric embryos also developed retinal colobomas and malformed lenses. Tissue-specific transgenic overexpression of Rybp in the lens resulted in abnormal fiber cell differentiation and severe lens opacification with increased levels of AP-2α and Sox2, and reduced levels of βA4-crystallin gene expression. Ubiquitous transgenic overexpression of Rybp in the entire eye caused abnormal retinal folds, corneal neovascularization, and lens opacification. Additional changes included defects in anterior eye development. Conclusion These studies establish Rybp as a novel gene that has been associated with coloboma. Other genes linked to coloboma encode various classes of transcription factors such as BCOR, CBP, Chx10, Pax2, Pax6, Six3, Ski, Vax1 and Vax2. We propose that the multiple functions for Rybp in regulating mouse retinal and lens development are mediated by genetic, epigenetic and physical interactions between these genes and proteins.

  19. The planar cell polarity protein Vangl2 is required for retinal axon guidance.

    Science.gov (United States)

    Leung, Vicki; Iliescu, Alexandra; Jolicoeur, Christine; Gravel, Michel; Apuzzo, Sergio; Torban, Elena; Cayouette, Michel; Gros, Philippe

    2016-02-01

    Vangl2 plays a critical role in the establishment of planar cell polarity (PCP). Previously, we detected expression of Vangl2 in the developing retina during late embryogenesis, which led us to investigate the possible role of Vangl2-mediated PCP signaling in eye development. We have generated a Vangl2(BGeo) knock-in mouse allowing us to evaluate Vangl2 mRNA expression during retinal development, and used an isoform-specific antibody to examine Vangl2 protein expression in cryosections. To investigate the role of Vangl2 in retinal development, we examined eyes taken from embryos homozygous for independent alleles of Looptail (Lp, Lp(m1jus) ) mutant mice. We found that Vangl2 mRNA and protein are dynamically expressed in the developing embryonic and postnatal retina, with Vangl2 expression becoming progressively restricted to the ganglion cell layer and optic nerve as the retina matures. The expression pattern of Vangl2 transcript and protein is most prominent in retinal ganglion cells (RGC), and their axons. Additionally, we show that Vangl2 is required for retinal and optic nerve development as Vangl2 (Lp/Lp) mutant embryos display a significantly reduced eye size, marked thickening of the retina, and striking abnormalities in the morphology of the optic nerve (significant hypoplasia, and aberrant exit trajectory). Notably, we identified a salient intraretinal axon guidance defect in Vangl2 (Lp/Lp) mutant embryos through which axon bundles traverse the entire thickness of the retina and become trapped within the subretinal space. Our observations identify a new and essential role for Vangl2-dependent PCP signaling in the intraretinal path-finding of RGC axons.

  20. The CXXC finger 5 protein is required for DNA damage-induced p53 activation

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    The tumor suppressor p53 is a critical component of the DNA damage response pathway that induces a set of genes responsible for cell cycle arrest,senescence,apoptosis,and DNA repair.The ataxia te-langiectasia mutated protein kinase(ATM) responds to DNA-damage stimuli and signals p53 stabiliza-tion and activation,thereby facilitating transactivation of p53 inducible genes and maintainence of genome integrity.In this study,we identified a CXXC zinc finger domain containing protein termed CF5 as a critical component in the DNA damage signaling pathway.CF5 induces p53 transcriptional activity and apoptosis in cells expressing wild type p53 but not in p53-deficient cells.Knockdown of CF5 in-hibits DNA damage-induced p53 activation as well as cell cycle arrest.Furthermore,CF5 physically interacts with ATM and is required for DNA damage-induced ATM phosphorylation but not its recruitment to chromatin.These findings suggest that CF5 plays a crucial role in ATM-p53 signaling in response to DNA damage.

  1. CSL protein regulates transcription of genes required to prevent catastrophic mitosis in fission yeast.

    Science.gov (United States)

    Převorovský, Martin; Oravcová, Martina; Zach, Róbert; Jordáková, Anna; Bähler, Jürg; Půta, František; Folk, Petr

    2016-11-16

    For every eukaryotic cell to grow and divide, intricately coordinated action of numerous proteins is required to ensure proper cell-cycle progression. The fission yeast Schizosaccharomyces pombe has been instrumental in elucidating the fundamental principles of cell-cycle control. Mutations in S. pombe 'cut' (cell untimely torn) genes cause failed coordination between cell and nuclear division, resulting in catastrophic mitosis. Deletion of cbf11, a fission yeast CSL transcription factor gene, triggers a 'cut' phenotype, but the precise role of Cbf11 in promoting mitotic fidelity is not known. We report that Cbf11 directly activates the transcription of the acetyl-coenzyme A carboxylase gene cut6, and the biotin uptake/biosynthesis genes vht1 and bio2, with the former 2 implicated in mitotic fidelity. Cbf11 binds to a canonical, metazoan-like CSL response element (GTGGGAA) in the cut6 promoter. Expression of Cbf11 target genes shows apparent oscillations during the cell cycle using temperature-sensitive cdc25-22 and cdc10-M17 block-release experiments, but not with other synchronization methods. The penetrance of catastrophic mitosis in cbf11 and cut6 mutants is nutrient-dependent. We also show that drastic decrease in biotin availability arrests cell proliferation but does not cause mitotic defects. Taken together, our results raise the possibility that CSL proteins play conserved roles in regulating cell-cycle progression, and they could guide experiments into mitotic CSL functions in mammals.

  2. Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein.

    Directory of Open Access Journals (Sweden)

    Berlin Londono-Renteria

    2015-10-01

    Full Text Available Dengue virus (DENV is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were ≥5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379, whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.

  3. Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein.

    Science.gov (United States)

    Londono-Renteria, Berlin; Troupin, Andrea; Conway, Michael J; Vesely, Diana; Ledizet, Michael; Roundy, Christopher M; Cloherty, Erin; Jameson, Samuel; Vanlandingham, Dana; Higgs, Stephen; Fikrig, Erol; Colpitts, Tonya M

    2015-10-01

    Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were ≥5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379), whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.

  4. CGRP-RCP, a novel protein required for signal transduction at calcitonin gene-related peptide and adrenomedullin receptors.

    Science.gov (United States)

    Evans, B N; Rosenblatt, M I; Mnayer, L O; Oliver, K R; Dickerson, I M

    2000-10-06

    It is becoming clear that receptors that initiate signal transduction by interacting with G-proteins do not function as monomers, but often require accessory proteins for function. Some of these accessory proteins are chaperones, required for correct transport of the receptor to the cell surface, but the function of many accessory proteins remains unknown. We determined the role of an accessory protein for the receptor for calcitonin gene-related peptide (CGRP), a potent vasodilator neuropeptide. We have previously shown that this accessory protein, the CGRP-receptor component protein (RCP), is expressed in CGRP responsive tissues and that RCP protein expression correlates with the biological efficacy of CGRP in vivo. However, the function of RCP has remained elusive. In this study stable cell lines were made that express antisense RCP RNA, and CGRP- and adrenomedullin-mediated signal transduction were greatly reduced. However, the loss of RCP did not effect CGRP binding or receptor density, indicating that RCP did not behave as a chaperone but was instead coupling the CGRP receptor to downstream effectors. A candidate CGRP receptor named calcitonin receptor-like receptor (CRLR) has been identified, and in this study RCP co-immunoprecipitated with CRLR indicating that these two proteins interact directly. Since CGRP and adrenomedullin can both signal through CRLR, which has been previously shown to require a chaperone protein for function, we now propose that a functional CGRP or adrenomedullin receptor consists of at least three proteins: the receptor (CRLR), the chaperone protein (RAMP), and RCP that couples the receptor to the cellular signal transduction pathway.

  5. SHANK-associated RH domain interacting protein (SHARPIN) is required for optimal NLRP3 inflammasome activation

    Science.gov (United States)

    Gurung, Prajwal; Lamkanfi, Mohamed; Kanneganti, Thirumala-Devi

    2015-01-01

    The NLRP3 inflammasome is a multimeric protein complex assembled in response to a wide array of pathogens and danger-associated molecular patterns. Despite the ability of NLRP3 to respond to diverse cues, the mechanisms controlling assembly of this complex are contested. Recently published studies show that HOIL-1, a member of the linear ubiquitin chain assembly complex (LUBAC), contributes to activation of the NLRP3 inflammasome. SHARPIN, along with HOIP and HOIL-1 assembles the LUBAC complex. Herein, we examined whether SHARPIN is required for the activation of NLRP3 inflammasome. Utilizing Sharpincpdm macrophages (deficient in SHARPIN expression), we demonstrate that SHARPIN is required for optimal activation of the NLRP3 inflammasome by both canonical and non-canonical stimuli. Furthermore, Sharpincpdm macrophages had dramatic defects in both NFκB and MAP kinase pathways, suggesting a role in transcriptional priming of the NLRP3 inflammasome. In conclusion, our study identified SHARPIN as a novel regulator of the NLRP3 inflammasome. PMID:25637014

  6. A cellulose synthase-like protein is required for osmotic stress tolerance in Arabidopsis

    KAUST Repository

    Zhu, Jianhua

    2010-04-16

    Osmotic stress imposed by soil salinity and drought stress significantly affects plant growth and development, but osmotic stress sensing and tolerance mechanisms are not well understood. Forward genetic screens using a root-bending assay have previously identified salt overly sensitive (sos) mutants of Arabidopsis that fall into five loci, SOS1 to SOS5. These loci are required for the regulation of ion homeostasis or cell expansion under salt stress, but do not play a major role in plant tolerance to the osmotic stress component of soil salinity or drought. Here we report an additional sos mutant, sos6-1, which defines a locus essential for osmotic stress tolerance. sos6-1 plants are hypersensitive to salt stress and osmotic stress imposed by mannitol or polyethylene glycol in culture media or by water deficit in the soil. SOS6 encodes a cellulose synthase-like protein, AtCSLD5. Only modest differences in cell wall chemical composition could be detected, but we found that sos6-1 mutant plants accumulate high levels of reactive oxygen species (ROS) under osmotic stress and are hypersensitive to the oxidative stress reagent methyl viologen. The results suggest that SOS6/AtCSLD5 is not required for normal plant growth and development but has a critical role in osmotic stress tolerance and this function likely involves its regulation of ROS under stress. © 2010 Blackwell Publishing Ltd.

  7. The Cladosporium fulvum virulence protein Avr2 inhibits host proteases required for basal defense.

    Science.gov (United States)

    van Esse, H Peter; Van't Klooster, John W; Bolton, Melvin D; Yadeta, Koste A; van Baarlen, Peter; Boeren, Sjef; Vervoort, Jacques; de Wit, Pierre J G M; Thomma, Bart P H J

    2008-07-01

    Cladosporium fulvum (syn. Passalora fulva) is a biotrophic fungal pathogen that causes leaf mold of tomato (Solanum lycopersicum). During growth in the apoplast, the fungus establishes disease by secreting effector proteins, 10 of which have been characterized. We have previously shown that the Avr2 effector interacts with the apoplastic tomato Cys protease Rcr3, which is required for Cf-2-mediated immunity. We now show that Avr2 is a genuine virulence factor of C. fulvum. Heterologous expression of Avr2 in Arabidopsis thaliana causes enhanced susceptibility toward extracellular fungal pathogens, including Botrytis cinerea and Verticillium dahliae, and microarray analysis showed that Avr2 expression triggers a global transcriptome reflecting pathogen challenge. Cys protease activity profiling showed that Avr2 inhibits multiple extracellular Arabidopsis Cys proteases. In tomato, Avr2 expression caused enhanced susceptibility toward Avr2-defective C. fulvum strains and also toward B. cinerea and V. dahliae. Cys protease activity profiling in tomato revealed that, in this plant also, Avr2 inhibits multiple extracellular Cys proteases, including Rcr3 and its close relative Pip1. Finally, silencing of Avr2 significantly compromised C. fulvum virulence on tomato. We conclude that Avr2 is a genuine virulence factor of C. fulvum that inhibits several Cys proteases required for plant basal defense.

  8. Lysine requirement of starting barrows from two genetic groups fed on low crude protein diets

    Directory of Open Access Journals (Sweden)

    Alessandro Luís Fraga

    2008-02-01

    Full Text Available A trial was carried out to determine the lysine requirement for starting barrows fed on ideal protein concept-based diets. Thirty-two pigs from a commercial crossbred genetic group (CCGG, BW=15.9 ± 1.4 kg and 32 pigs from a dam line one (DLGG, BW= 14.8 ± 1.0 kg were used. Pigs were allotted to 4 treatments with diets containing increasing levels of total lysine (0.80, 1.00, 1.20 and 1.40%. Methionine+cystine, threonine and tryptophan were adjusted according to ideal protein profile. Data from performance, plasma urea nitrogen (PUN and carcass composition were analyzed. CCGG showed higher daily feed intake, daily weight gain, PUN and protein:fat ratio in carcass, while DLGG showed higher fat carcass content and nitrogen retention. Fat content and protein:fat ratio in carcass for CCCGG and PUN and crude protein carcass content for DLGG showed quadratic response to increasing total lysine levels. Derivations of the quadratic equations indicated the total lysine requirement for CCGG starting barrows is 1.15% and for DLGG starting barrows is 1.09%.Foi realizado um trabalho com o objetivo de determinar a exigência em lisina para suínos castrados em fase inicial, alimentados com dietas formuladas de acordo com o conceito de proteína ideal. Trinta e dois suínos provenientes de cruzamento comercial (CC, PV = 15,9 kg e 32 suínos provenientes de linhagem materna (LM, PV= 14,8 kg foram alimentados com quatro dietas contendo níveis crescentes de lisina total (0,80; 1,00; 1,20 e 1,40%. Metionina + cistina, triptofano e treonina foram adicionados às dietas para manter constante o padrão de proteína ideal. Foram analisados dados de desempenho, nitrogênio da uréia plasmática (NUP e carcaça. Suínos do grupo CC apresentaram maior consumo diário de ração, ganho diário de peso, NUP e relação proteína: gordura na carcaça, enquanto que os animais do grupo LM apresentaram maiores teores de gordura na carcaça e retenção de nitrogênio. Teor de

  9. Food and nutritional security requires adequate protein as well as energy, delivered from whole-year crop production

    DEFF Research Database (Denmark)

    Coles, Graeme D; Wratten, Stephen D; Porter, John Roy

    2016-01-01

    Human food security requires the production of sufficient quantities of both high-quality protein and dietary energy. In a series of case-studies from New Zealand, we show that while production of food ingredients from crops on arable land can meet human dietary energy requirements effectively......, requirements for high-quality protein are met more efficiently by animal production from such land. We present a model that can be used to assess dietary energy and quality-corrected protein production from various crop and crop/animal production systems, and demonstrate its utility. We extend our analysis...... with an accompanying economic analysis of commercially-available, pre-prepared or simply-cooked foods that can be produced from our case-study crop and animal products. We calculate the per-person, per-day cost of both quality-corrected protein and dietary energy as provided in the processed foods. We conclude...

  10. Increasing dietary crude protein does not increase the methionine requirement in kittens.

    Science.gov (United States)

    Strieker, M J; Morris, J G; Kass, P H; Rogers, Q R

    2007-12-01

    The objective of this study was to determine if the methionine (met) requirement of kittens is correlated with the concentration of dietary crude protein (CP). The study used 48 male kittens in two replications of six 4 x 4 Latin squares, each representing one concentration of met (1.5, 2.5, 3.5, 4.5, 6.0 or 9.0 g/kg diet) with four CP concentrations (150, 200, 300 and 500 g/kg diet) in 2-week periods. Cystine was present in the lowest CP diet at 5.3 g/kg diet and increased as dietary CP increased. Body weight gain, food intake, nitrogen balance and plasma amino acids, glucose, insulin, cortisol, somatomedin C, T(3) and T(4) concentrations on day 12 were measured. From breakpoint analysis of the nitrogen retention curves, the met requirement of kittens was found to be 3.1, 3.8, 3.1 and 2.4 g met/kg for the 150, 200, 300 and 500 g CP/kg diets, respectively. When met was limiting (1.5 or 2.5 g/kg diet), increasing dietary CP did not decrease, but rather increased food intake, body weight gain and nitrogen retention. Plasma met concentrations increased as dietary met increased and at 2.5-3.5 g met/kg diet were not different among kittens fed the various CP diets. Total plasma T(3) and T(4) increased significantly as dietary CP increased in kittens given the 2.5 and 4.5 g met/kg diets. Results indicate that food intake and possibly altered hormonal secretion play a role in this growth response. In conclusion, the met requirement of growing kittens, unlike omnivores and herbivores studied, was not positively correlated with the concentration of dietary CP.

  11. Temporal requirements of the fragile X mental retardation protein in modulating circadian clock circuit synaptic architecture

    Directory of Open Access Journals (Sweden)

    Cheryl L Gatto

    2009-08-01

    Full Text Available Loss of fragile X mental retardation 1 (FMR1 gene function is the most common cause of inherited mental retardation and autism spectrum disorders, characterized by attention disorder, hyperactivity and disruption of circadian activity cycles. Pursuit of effective intervention strategies requires determining when the FMR1 product (FMRP is required in the regulation of neuronal circuitry controlling these behaviors. In the well-characterized Drosophila disease model, loss of the highly conserved dFMRP causes circadian arrhythmicity and conspicuous abnormalities in the circadian clock circuitry. Here, a novel Sholl Analysis was used to quantify over-elaborated synaptic architecture in dfmr1-null small ventrolateral neurons (sLNvs, a key subset of clock neurons. The transgenic Gene-Switch system was employed to drive conditional neuronal dFMRP expression in the dfmr1-null mutant background in order to dissect temporal requirements within the clock circuit. Introduction of dFMRP during early brain development, including the stages of neurogenesis, neuronal fate specification and early pathfinding, provided no rescue of dfmr1 mutant phenotypes. Similarly, restoring normal dFMRP expression in the adult failed to restore circadian circuit architecture. In sharp contrast, supplying dFMRP during a transient window of very late brain development, wherein synaptogenesis and substantial subsequent synaptic reorganization (e.g. use-dependent pruning occur, provided strong morphological rescue to reestablish normal sLNvs synaptic arbors. We conclude that dFMRP plays a developmentally restricted role in sculpting synaptic architecture in these neurons that cannot be compensated for by later reintroduction of the protein at maturity.

  12. Protein histochemistry using coronaviral spike proteins: studying binding profiles and sialic acid requirements for attachment to tissues

    NARCIS (Netherlands)

    Ambepitiya Wickramasinghe, I.N.; Verheije, M.H.

    2015-01-01

    Protein histochemistry is a tissue-based technique that enables the analysis of viral attachment patterns as well as the identification of specific viral and host determinants involved in the first step in the infection of a host cell by a virus. Applying recombinantly expressed spike proteins of in

  13. Cholesterol and F-actin are required for clustering of recycling synaptic vesicle proteins in the presynaptic plasma membrane.

    Science.gov (United States)

    Dason, Jeffrey S; Smith, Alex J; Marin, Leo; Charlton, Milton P

    2014-02-15

    Synaptic vesicles (SVs) and their proteins must be recycled for sustained synaptic transmission. We tested the hypothesis that SV cholesterol is required for proper sorting of SV proteins during recycling in live presynaptic terminals. We used the reversible block of endocytosis in the Drosophila temperature-sensitive dynamin mutant shibire-ts1 to trap exocytosed SV proteins, and then examined the effect of experimental treatments on the distribution of these proteins within the presynaptic plasma membrane by confocal microscopy. SV proteins synaptotagmin, vglut and csp were clustered following SV trapping in control experiments but dispersed in samples treated with the cholesterol chelator methyl-β-cyclodextrin to extract SV cholesterol. There was accumulation of phosphatidylinositol (4,5)-bisphosphate (PIP2) in presynaptic terminals following SV trapping and this was reduced following SV cholesterol extraction. Reduced PIP2 accumulation was associated with disrupted accumulation of actin in presynaptic terminals. Similar to vesicular cholesterol extraction, disruption of actin by latrunculin A after SV proteins had been trapped on the plasma membrane resulted in the dispersal of SV proteins and prevented recovery of synaptic transmission due to impaired endocytosis following relief of the endocytic block. Our results demonstrate that vesicular cholesterol is required for aggregation of exocytosed SV proteins in the presynaptic plasma membrane and are consistent with a mechanism involving regulation of PIP2 accumulation and local actin polymerization by cholesterol. Thus, alteration of membrane or SV lipids may affect the ability of synapses to undergo sustained synaptic transmission by compromising the recycling of SV proteins.

  14. A delicate balance between functionally required flexibility and aggregation risk in a β-rich protein

    OpenAIRE

    Ferrolino, Mylene C.; Zhuravleva, Anastasia; Budyak, Ivan L.; Krishnan, Beena; Gierasch, Lila M.

    2013-01-01

    Susceptibility to aggregation is general to proteins because of the potential for intermolecular interactions between hydrophobic stretches in their amino acid sequences. Protein aggregation has been implicated in several catastrophic diseases, yet we still lack in-depth understanding about how proteins are channeled to this state. Using a predominantly β-sheet protein whose folding has been explored in detail: cellular retinoic acid-binding protein 1 (CRABP1), as a model, we have tackled the...

  15. Adhesion protein VSIG1 is required for the proper differentiation of glandular gastric epithelia.

    Directory of Open Access Journals (Sweden)

    Odgerel Oidovsambuu

    Full Text Available VSIG1, a cell adhesion protein of the immunoglobulin superfamily, is preferentially expressed in stomach, testis, and certain gastric, esophageal and ovarian cancers. Here, we describe the expression patterns of three alternatively spliced isoforms of mouse Vsig1 during pre- and postnatal development of stomach and potential function of Vsig1 in differentiation of gastric epithelia. We show that isoforms Vsig1A and Vsig1B, which differ in the 3'untranslated region, are expressed in the early stages of stomach development. Immunohistochemical analysis revealed that VSIG1 is restricted to the adherens junction of the glandular epithelium. The shorter transcript Vsig1C is restricted to the testis, encodes an N-terminal truncated protein and is presumably regulated by an internal promoter, which is located upstream of exon 1b. To determine whether the 5' flanking region of exon 1a specifically targets the expression of Vsig1 to stomach epithelia, we generated and analyzed transgenic mice. The 4.8-kb fragment located upstream of exon 1a was sufficient to direct the expression of the reporter gene to the glandular epithelia of transgenic stomach. To determine the role of VSIG1 during the development of stomach epithelia, an X-linked Vsig1 was inactivated in embryonic stem cells (ESCs. Although Vsig1(-/Y ESCs were only able to generate low coat color chimeric mice, no male chimeras transmitted the targeted allele to their progeny suggesting that the high contribution of Vsig1(-/Y cells leads to the lethality of chimeric embryos. Analysis of chimeric stomachs revealed the differentiation of VSIG1-null cells into squamous epithelia inside the glandular region. These results suggest that VSIG1 is required for the establishment of glandular versus squamous epithelia in the stomach.

  16. Adhesion protein VSIG1 is required for the proper differentiation of glandular gastric epithelia.

    Science.gov (United States)

    Oidovsambuu, Odgerel; Nyamsuren, Gunsmaa; Liu, Shuai; Göring, Wolfgang; Engel, Wolfgang; Adham, Ibrahim M

    2011-01-01

    VSIG1, a cell adhesion protein of the immunoglobulin superfamily, is preferentially expressed in stomach, testis, and certain gastric, esophageal and ovarian cancers. Here, we describe the expression patterns of three alternatively spliced isoforms of mouse Vsig1 during pre- and postnatal development of stomach and potential function of Vsig1 in differentiation of gastric epithelia. We show that isoforms Vsig1A and Vsig1B, which differ in the 3'untranslated region, are expressed in the early stages of stomach development. Immunohistochemical analysis revealed that VSIG1 is restricted to the adherens junction of the glandular epithelium. The shorter transcript Vsig1C is restricted to the testis, encodes an N-terminal truncated protein and is presumably regulated by an internal promoter, which is located upstream of exon 1b. To determine whether the 5' flanking region of exon 1a specifically targets the expression of Vsig1 to stomach epithelia, we generated and analyzed transgenic mice. The 4.8-kb fragment located upstream of exon 1a was sufficient to direct the expression of the reporter gene to the glandular epithelia of transgenic stomach. To determine the role of VSIG1 during the development of stomach epithelia, an X-linked Vsig1 was inactivated in embryonic stem cells (ESCs). Although Vsig1(-/Y) ESCs were only able to generate low coat color chimeric mice, no male chimeras transmitted the targeted allele to their progeny suggesting that the high contribution of Vsig1(-/Y) cells leads to the lethality of chimeric embryos. Analysis of chimeric stomachs revealed the differentiation of VSIG1-null cells into squamous epithelia inside the glandular region. These results suggest that VSIG1 is required for the establishment of glandular versus squamous epithelia in the stomach.

  17. Dengue virus NS1 protein interacts with the ribosomal protein RPL18: this interaction is required for viral translation and replication in Huh-7 cells.

    Science.gov (United States)

    Cervantes-Salazar, Margot; Angel-Ambrocio, Antonio H; Soto-Acosta, Ruben; Bautista-Carbajal, Patricia; Hurtado-Monzon, Arianna M; Alcaraz-Estrada, Sofia L; Ludert, Juan E; Del Angel, Rosa M

    2015-10-01

    Given dengue virus (DENV) genome austerity, it uses cellular molecules and structures for virion entry, translation and replication of the genome. NS1 is a multifunctional protein key to viral replication and pathogenesis. Identification of cellular proteins that interact with NS1 may help in further understanding the functions of NS1. In this paper we isolated a total of 64 proteins from DENV infected human hepatic cells (Huh-7) that interact with NS1 by affinity chromatography and immunoprecipitation assays. The subcellular location and expression levels during infection of the ribosomal proteins RPS3a, RPL7, RPL18, RPL18a plus GAPDH were determined. None of these proteins changed their expression levels during infection; however, RPL-18 was redistributed to the perinuclear region after 48hpi. Silencing of the RPL-18 does not affect cell translation efficiency or viability, but it reduces significantly viral translation, replication and viral yield, suggesting that the RPL-18 is required during DENV replicative cycle.

  18. Energy and Protein Requirements for - to -Week-Age Gushi Chicks

    Institute of Scientific and Technical Information of China (English)

    KANG Xiang-tao; TIAN Ya-dong; SONG Su-fang

    2002-01-01

    In this experiment, 540 male and female chicks of 1-day-age were selected respectively and reared separately. 3 × 3 factorial experiment was designed. Gushi chicks were fed with nine diets under different metabolizable energy (ME) and crude protein (CP). Effects of different diets on the performances and carcass ingredients of 0-to 4-week-age Gushi chicks were studied by rearing, comparative slaughter and metabolism experiments. Based on the results of three experiments, ME intake per kilogram metabolic weight was regarded as dependent variable (Y), and its corresponding net energy (NE) deposition per kilogram metabolic weight was regarded as independent variable (X), respectively. Lineal regressive analysis was made according to the mathematic model: Y=a+bX. Maintenance ME(MEm) and the converted coefficient from net energy for gain(NEg) to ME for gain(MEg) were found out, then ME requirement was divided into MErm+ MEg,which is ME = 359.14 W0.75 + 10.47/△W; CP intake and corresponding metabolic weight were regarded as dependent variable and independent variable respectively, and then regressive analysis was made. The result was CP = 0. 57 + 8.21 W0.75. Consequently, average daily gain (ADG), average daily feed intake and metabolic weight were put into the regressive equations, and then ME and CP requirements for 0- to 4-week-age Gushi chicks were obtained, 12.38 MJ kg-1 and 20.13%, respectively, the ratio of CP to ME was 16.26 g MJ-1.

  19. Identification of regions required for apical membrane localization of human multidrug resistance protein 2.

    Science.gov (United States)

    Bandler, Paul E; Westlake, Christopher J; Grant, Caroline E; Cole, Susan P C; Deeley, Roger G

    2008-07-01

    Multidrug resistance proteins MRP1 and MRP2 transport a wide range of endo- and xenobiotics. However, with the exception of certain parts of the brain, MRP1 traffics to basolateral membranes of polarized cells, whereas MRP2 is apical in location and thus it is particularly important for systemic elimination of such compounds. Different regions of MRP1 and MRP2 seem to target them to their respective membrane locations. In addition to two "core" membrane spanning domains (MSDs) characteristic of ATP-binding cassette transporters, MRP1 and MRP2 have a third NH2-terminal MSD (MSD0), which is not required for basolateral targeting of MRP1, or for transport of at least some substrates. Here, we demonstrate that all elements necessary for apical targeting of MRP2 reside in MSD0 and the adjacent cytoplasmic loop (CL) 3. Furthermore, we show that this region of MRP2 can target the core of MRP1 to an exclusively apical location. Within MRP2 CL3, we identified a lysine-rich element that is essential for apical targeting. When introduced into MRP1, this element alone is sufficient to result in partial apical localization. However, exclusive targeting to the apical membrane seems to require the integrity of the entire region encompassing MSD0 and CL3 of MRP2. Because CL3 of MRP1 is critical for binding, transport, or both of several compounds, we also examined the function of hybrids containing all, or portions of MRP2 MSD0 and CL3. Our results indicate that CL3 is important for interaction with both the glutathione and glucuronide conjugates tested, but that different regions may be involved.

  20. Analysis of the thermal energy requirements for the extraction of leaf protein concentrate from some green plants

    Energy Technology Data Exchange (ETDEWEB)

    Tangka, Julius K. [Dschang Univ., Dept. of Agricultural Engineering, Dschang (Cameroon)

    2003-12-01

    Extraction of protein from the leaves of green plants is very important because of the high cost of conventional forms of protein such as meat, milk and fish. In order to design machinery for this extraction, and also to embark on leaf protein concentrate extraction, it is necessary to measure and analyse the energy requirements to carry out each process involved in the extraction, using different plant species. Experiments were carried out to determine the amount of crude protein, and the thermal energy required to extract leaf protein concentrate, from juices obtained from the leaves of some plant species. Leaves from the following plants were selected: cassava (Manihot esculanta), Siam weed (Chromolaena odorata), bitter leaf (Vernonia amygdalina), gliricidia (Gliricidia maculata) and thorny tree (Hura crepetans). The leaves from the plant species were macerated in a laboratory pulper. Juice was obtained from the samples using perforated cylinders and a hydraulic press. The specific heat capacity of the juices was determined using the cooling curve method. The values of the heat capacities were used to calculate the amount of thermal energy required to raise the temperature of each juice from its normal temperature of about 25 deg C to a total protein coagulation temperature of about 80 deg C. The crude protein content of the extract was determined using the Kjeldal method. Results indicate that the green coagulum extracted from all the juices all have a protein content of at least 37%. The thermal energy required to coagulate protein from the juices ranges from 1.59 kJ kg{sup -1} for Hura crepetans to 2.7 kJ kg{sup -1} for Vernonia amygdalina. The energy requirement to obtain crude protein (CP) ranges from 8 kJ kg{sup -1} [CP] with Bura crepetans to 182 kJ kg{sup -1} [CP] with Vernonia amygdalina. Both results are statistically significant at the 0.01 confidence interval. It is concluded that the choice of plant species can significantly lower the thermal energy

  1. A Rice Ca2+ Binding Protein Is Required for Tapetum Function and Pollen Formation.

    Science.gov (United States)

    Yu, Jing; Meng, Zhaolu; Liang, Wanqi; Behera, Smrutisanjita; Kudla, Jörg; Tucker, Matthew R; Luo, Zhijing; Chen, Mingjiao; Xu, Dawei; Zhao, Guochao; Wang, Jie; Zhang, Siyi; Kim, Yu-Jin; Zhang, Dabing

    2016-11-01

    In flowering plants, successful male reproduction requires the sophisticated interaction between somatic anther wall layers and reproductive cells. Timely degradation of the innermost tissue of the anther wall layer, the tapetal layer, is critical for pollen development. Ca(2+) is a well-known stimulus for plant development, but whether it plays a role in affecting male reproduction remains elusive. Here we report a role of Defective in Exine Formation 1 (OsDEX1) in rice (Oryza sativa), a Ca(2+) binding protein, in regulating rice tapetal cell degradation and pollen formation. In osdex1 anthers, tapetal cell degeneration is delayed and degradation of the callose wall surrounding the microspores is compromised, leading to aborted pollen formation and complete male sterility. OsDEX1 is expressed in tapetal cells and microspores during early anther development. Recombinant OsDEX1 is able to bind Ca(2+) and regulate Ca(2+) homeostasis in vitro, and osdex1 exhibited disturbed Ca(2+) homeostasis in tapetal cells. Phylogenetic analysis suggested that OsDEX1 may have a conserved function in binding Ca(2+) in flowering plants, and genetic complementation of pollen wall defects of an Arabidopsis (Arabidopsis thaliana) dex1 mutant confirmed its evolutionary conservation in pollen development. Collectively, these findings suggest that OsDEX1 plays a fundamental role in the development of tapetal cells and pollen formation, possibly via modulating the Ca(2+) homeostasis during pollen development.

  2. Plasmodium berghei calcium dependent protein kinase 1 is not required for host cell invasion.

    Science.gov (United States)

    Jebiwott, Sylvia; Govindaswamy, Kavitha; Mbugua, Amos; Bhanot, Purnima

    2013-01-01

    Plasmodium Calcium Dependent Protein Kinase (CDPK1) is required for the development of sexual stages in the mosquito. In addition, it is proposed to play an essential role in the parasite's invasive stages possibly through the regulation of the actinomyosin motor and micronemal secretion. We demonstrate that Plasmodium berghei CDPK1 is dispensable in the parasite's erythrocytic and pre-erythrocytic stages. We successfully disrupted P. berghei CDPK1 (PbCDPK1) by homologous recombination. The recovery of erythrocytic stage parasites lacking PbCDPK1 (PbCDPK1-) demonstrated that PbCDPK1 is not essential for erythrocytic invasion or intra-erythrocytic development. To study PbCDPK1's role in sporozoites and liver stage parasites, we generated a conditional mutant (CDPK1 cKO). Phenotypic characterization of CDPK1 cKO sporozoites demonstrated that CDPK1 is redundant or dispensable for the invasion of mammalian hepatocytes, the egress of parasites from infected hepatocytes and through the subsequent erythrocytic cycle. We conclude that P. berghei CDPK1 plays an essential role only in the mosquito sexual stages.

  3. Plasmodium berghei calcium dependent protein kinase 1 is not required for host cell invasion.

    Directory of Open Access Journals (Sweden)

    Sylvia Jebiwott

    Full Text Available Plasmodium Calcium Dependent Protein Kinase (CDPK1 is required for the development of sexual stages in the mosquito. In addition, it is proposed to play an essential role in the parasite's invasive stages possibly through the regulation of the actinomyosin motor and micronemal secretion. We demonstrate that Plasmodium berghei CDPK1 is dispensable in the parasite's erythrocytic and pre-erythrocytic stages. We successfully disrupted P. berghei CDPK1 (PbCDPK1 by homologous recombination. The recovery of erythrocytic stage parasites lacking PbCDPK1 (PbCDPK1- demonstrated that PbCDPK1 is not essential for erythrocytic invasion or intra-erythrocytic development. To study PbCDPK1's role in sporozoites and liver stage parasites, we generated a conditional mutant (CDPK1 cKO. Phenotypic characterization of CDPK1 cKO sporozoites demonstrated that CDPK1 is redundant or dispensable for the invasion of mammalian hepatocytes, the egress of parasites from infected hepatocytes and through the subsequent erythrocytic cycle. We conclude that P. berghei CDPK1 plays an essential role only in the mosquito sexual stages.

  4. The Alpha Catalytic Subunit of Protein Kinase CK2 Is Required for Mouse Embryonic Development▿

    Science.gov (United States)

    Lou, David Y.; Dominguez, Isabel; Toselli, Paul; Landesman-Bollag, Esther; O'Brien, Conor; Seldin, David C.

    2008-01-01

    Protein kinase CK2 (formerly casein kinase II) is a highly conserved and ubiquitous serine/threonine kinase that is composed of two catalytic subunits (CK2α and/or CK2α′) and two CK2β regulatory subunits. CK2 has many substrates in cells, and key roles in yeast cell physiology have been uncovered by introducing subunit mutations. Gene-targeting experiments have demonstrated that in mice, the CK2β gene is required for early embryonic development, while the CK2α′ subunit appears to be essential only for normal spermatogenesis. We have used homologous recombination to disrupt the CK2α gene in the mouse germ line. Embryos lacking CK2α have a marked reduction in CK2 activity in spite of the presence of the CK2α′ subunit. CK2α−/− embryos die in mid-gestation, with abnormalities including open neural tubes and reductions in the branchial arches. Defects in the formation of the heart lead to hydrops fetalis and are likely the cause of embryonic lethality. Thus, CK2α appears to play an essential and uncompensated role in mammalian development. PMID:17954558

  5. Transmembrane protein 108 is required for glutamatergic transmission in dentate gyrus.

    Science.gov (United States)

    Jiao, Hui-Feng; Sun, Xiang-Dong; Bates, Ryan; Xiong, Lei; Zhang, Lei; Liu, Fang; Li, Lei; Zhang, Hong-Sheng; Wang, Shun-Qi; Xiong, Ming-Tao; Patel, Mihir; Stranahan, Alexis M; Xiong, Wen-Cheng; Li, Bao-Ming; Mei, Lin

    2017-01-31

    Neurotransmission in dentate gyrus (DG) is critical for spatial coding, learning memory, and emotion processing. Although DG dysfunction is implicated in psychiatric disorders, including schizophrenia, underlying pathological mechanisms remain unclear. Here we report that transmembrane protein 108 (Tmem108), a novel schizophrenia susceptibility gene, is highly enriched in DG granule neurons and its expression increased at the postnatal period critical for DG development. Tmem108 is specifically expressed in the nervous system and enriched in the postsynaptic density fraction. Tmem108-deficient neurons form fewer and smaller spines, suggesting that Tmem108 is required for spine formation and maturation. In agreement, excitatory postsynaptic currents of DG granule neurons were decreased in Tmem108 mutant mice, indicating a hypofunction of glutamatergic activity. Further cell biological studies indicate that Tmem108 is necessary for surface expression of AMPA receptors. Tmem108-deficient mice display compromised sensorimotor gating and cognitive function. Together, these observations indicate that Tmem108 plays a critical role in regulating spine development and excitatory transmission in DG granule neurons. When Tmem108 is mutated, mice displayed excitatory/inhibitory imbalance and behavioral deficits relevant to schizophrenia, revealing potential pathophysiological mechanisms of schizophrenia.

  6. Lactobacillus gasseri requires peptides, not proteins or free amino acids, for growth in milk.

    Science.gov (United States)

    Arakawa, K; Matsunaga, K; Takihiro, S; Moritoki, A; Ryuto, S; Kawai, Y; Masuda, T; Miyamoto, T

    2015-03-01

    Lactobacillus gasseri is a widespread commensal lactic acid bacterium inhabiting human mucosal niches and has many beneficial effects as a probiotic. However, L. gasseri is difficult to grow in milk, which hurts usability for the food industry. It had been previously reported that supplementation with yeast extract or proteose peptone, including peptides, enables L. gasseri to grow well in milk. In this study, our objective was to confirm peptide requirement of L. gasseri and evaluate efficacy of peptide release by enzymatic proteolysis on growth of L. gassei in milk. Three strains of L. gasseri did not grow well in modified DeMan, Rogosa, Sharpe broth without any nitrogen sources (MRS-N), but addition of a casein-derived peptide mixture, tryptone, promoted growth. In contrast, little effect was observed after adding casein or a casein-derived amino acid mixture, casamino acids. These results indicate that L. gasseri requires peptides, not proteins or free amino acids, among milk-derived nitrogen sources for growth. Lactobacillus gasseri JCM 1131T hardly had growth capacity in 6 kinds of milk-based media: bovine milk, human milk, skim milk, cheese whey, modified MRS-N (MRSL-N) supplemented with acid whey, and MRSL-N supplemented with casein. Moreover, treatment with digestive proteases, particularly pepsin, to release peptides made it grow well in each milk-based medium. The pepsin treatment was the most effective for growth of strain JCM 1131T in skim milk among the tested food-grade proteases such as trypsin, α-chymotrypsin, calf rennet, ficin, bromelain, and papain. As well as strain JCM 1131T, pepsinolysis of milk improved growth of other L. gasseri strains and some strains of enteric lactobacilli such as Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus johnsonii, and Lactobacillus reuteri. These results suggest that some relatives of L. gasseri also use peptides as desirable nitrogen sources, and that milk may be a good supplier of nutritious

  7. Predictors for achieving protein and energy requirements in undernourished hospital patients

    NARCIS (Netherlands)

    Leistra, Eva; Willeboordse, Floor; Bokhorst - de van der Schueren, A.E. van Marian; Visser, Marjolein; Weijs, J.M. Peter; Haans - van den Oord, Annelie; Oostenbrink, Jan; Evers , M. Anja; Kruizenga, M. Hinke

    2011-01-01

    Background & aims Providing sufficient protein an energy is considered crucial in the treatment of undernutrition. Still, the majority of undernourished hospital patients have a suboptimal protein and energy intake. The aim of this study was to investigate predictors for achieving protein and energy

  8. Protein Requirements Are Elevated in Endurance Athletes after Exercise as Determined by the Indicator Amino Acid Oxidation Method.

    Directory of Open Access Journals (Sweden)

    Hiroyuki Kato

    Full Text Available A higher protein intake has been recommended for endurance athletes compared with healthy non-exercising individuals based primarily on nitrogen balance methodology. The aim of this study was to determine the estimated average protein requirement and recommended protein intake in endurance athletes during an acute 3-d controlled training period using the indicator amino acid oxidation method. After 2-d of controlled diet (1.4 g protein/kg/d and training (10 and 5km/d, respectively, six male endurance-trained adults (28±4 y of age; Body weight, 64.5±10.0 kg; VO2peak, 60.3±6.7 ml·kg-1·min-1; means±SD performed an acute bout of endurance exercise (20 km treadmill run prior to consuming test diets providing variable amounts of protein (0.2-2.8 g·kg-1·d-1 and sufficient energy. Protein was provided as a crystalline amino acid mixture based on the composition of egg protein with [1-13C]phenylalanine provided to determine whole body phenylalanine flux, 13CO2 excretion, and phenylalanine oxidation. The estimated average protein requirement was determined as the breakpoint after biphasic linear regression analysis with a recommended protein intake defined as the upper 95% confidence interval. Phenylalanine flux (68.8±8.5 μmol·kg-1·h-1 was not affected by protein intake. 13CO2 excretion displayed a robust bi-phase linear relationship (R2 = 0.86 that resulted in an estimated average requirement and a recommended protein intake of 1.65 and 1.83 g protein·kg-1·d-1, respectively, which was similar to values based on phenylalanine oxidation (1.53 and 1.70 g·kg-1·d-1, respectively. We report a recommended protein intake that is greater than the RDA (0.8 g·kg-1·d-1 and current recommendations for endurance athletes (1.2-1.4 g·kg-1·d-1. Our results suggest that the metabolic demand for protein in endurance-trained adults on a higher volume training day is greater than their sedentary peers and current recommendations for athletes based

  9. Cancer-Related NEET Proteins Transfer 2Fe-2S Clusters to Anamorsin, a Protein Required for Cytosolic Iron-Sulfur Cluster Biogenesis

    Science.gov (United States)

    Lipper, Colin H.; Paddock, Mark L.; Onuchic, José N.; Mittler, Ron; Nechushtai, Rachel; Jennings, Patricia A.

    2015-01-01

    Iron-sulfur cluster biogenesis is executed by distinct protein assembly systems. Mammals have two systems, the mitochondrial Fe-S cluster assembly system (ISC) and the cytosolic assembly system (CIA), that are connected by an unknown mechanism. The human members of the NEET family of 2Fe-2S proteins, nutrient-deprivation autophagy factor-1 (NAF-1) and mitoNEET (mNT), are located at the interface between the mitochondria and the cytosol. These proteins have been implicated in cancer cell proliferation, and they can transfer their 2Fe-2S clusters to a standard apo-acceptor protein. Here we report the first physiological 2Fe-2S cluster acceptor for both NEET proteins as human Anamorsin (also known as cytokine induced apoptosis inhibitor-1; CIAPIN-1). Anamorsin is an electron transfer protein containing two iron-sulfur cluster-binding sites that is required for cytosolic Fe-S cluster assembly. We show, using UV-Vis spectroscopy, that both NAF-1 and mNT can transfer their 2Fe-2S clusters to apo-Anamorsin with second order rate constants similar to those of other known human 2Fe-2S transfer proteins. A direct protein-protein interaction of the NEET proteins with apo-Anamorsin was detected using biolayer interferometry. Furthermore, electrospray mass spectrometry of holo-Anamorsin prepared by cluster transfer shows that it receives both of its 2Fe-2S clusters from the NEETs. We propose that mNT and NAF-1 can provide parallel routes connecting the mitochondrial ISC system and the CIA. 2Fe-2S clusters assembled in the mitochondria are received by NEET proteins and when needed transferred to Anamorsin, activating the CIA. PMID:26448442

  10. Food and nutritional security requires adequate protein as well as energy, delivered from whole-year crop production.

    Science.gov (United States)

    Coles, Graeme D; Wratten, Stephen D; Porter, John R

    2016-01-01

    Human food security requires the production of sufficient quantities of both high-quality protein and dietary energy. In a series of case-studies from New Zealand, we show that while production of food ingredients from crops on arable land can meet human dietary energy requirements effectively, requirements for high-quality protein are met more efficiently by animal production from such land. We present a model that can be used to assess dietary energy and quality-corrected protein production from various crop and crop/animal production systems, and demonstrate its utility. We extend our analysis with an accompanying economic analysis of commercially-available, pre-prepared or simply-cooked foods that can be produced from our case-study crop and animal products. We calculate the per-person, per-day cost of both quality-corrected protein and dietary energy as provided in the processed foods. We conclude that mixed dairy/cropping systems provide the greatest quantity of high-quality protein per unit price to the consumer, have the highest food energy production and can support the dietary requirements of the highest number of people, when assessed as all-year-round production systems. Global food and nutritional security will largely be an outcome of national or regional agroeconomies addressing their own food needs. We hope that our model will be used for similar analyses of food production systems in other countries, agroecological zones and economies.

  11. Vascular endothelin ET(B) receptor-mediated contraction requires phosphorylation of ERK1/2 proteins

    DEFF Research Database (Denmark)

    Luo, Guogang; Jamali, Roya; Cao, Yong-Xiao;

    2006-01-01

    RNA and protein expressions. The endothelin ET(B) receptor-mediated contraction was associated with increase in phosphorylation of extracellular regulation kinase 1 and 2 (ERK1/2) proteins and elevated levels of intracellular calcium. The elevation curve of intracellular calcium consisted of two phases: one rapid...... and one sustained. Inhibition of ERK1/2 phosphorylation by SB386023 or blockage of calcium channels by nifedipine significantly reduced the endothelin ET(B) receptor-mediated contraction (P..., phosphorylation of ERK1/2 proteins and elevation of intracellular calcium level are required for endothelin ET(B) receptor-mediated contraction in rat mesenteric artery....

  12. Human-Chromatin-Related Protein Interactions Identify a Demethylase Complex Required for Chromosome Segregation

    Directory of Open Access Journals (Sweden)

    Edyta Marcon

    2014-07-01

    Full Text Available Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division.

  13. Human-chromatin-related protein interactions identify a demethylase complex required for chromosome segregation.

    Science.gov (United States)

    Marcon, Edyta; Ni, Zuyao; Pu, Shuye; Turinsky, Andrei L; Trimble, Sandra Smiley; Olsen, Jonathan B; Silverman-Gavrila, Rosalind; Silverman-Gavrila, Lorelei; Phanse, Sadhna; Guo, Hongbo; Zhong, Guoqing; Guo, Xinghua; Young, Peter; Bailey, Swneke; Roudeva, Denitza; Zhao, Dorothy; Hewel, Johannes; Li, Joyce; Gräslund, Susanne; Paduch, Marcin; Kossiakoff, Anthony A; Lupien, Mathieu; Emili, Andrew; Wodak, Shoshana J; Greenblatt, Jack

    2014-07-10

    Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision) network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division.

  14. A type II protein secretory pathway required for levansucrase secretion by Gluconacetobacter diazotrophicus.

    Science.gov (United States)

    Arrieta, Juan G; Sotolongo, Mailin; Menéndez, Carmen; Alfonso, Dubiel; Trujillo, Luis E; Soto, Melvis; Ramírez, Ricardo; Hernández, Lázaro

    2004-08-01

    The endophytic diazotroph Gluconacetobacter diazotrophicus secretes a constitutively expressed levansucrase (LsdA, EC 2.4.1.10) to utilize plant sucrose. LsdA, unlike other extracellular levansucrases from gram-negative bacteria, is transported to the periplasm by a signal-peptide-dependent pathway. We identified an unusually organized gene cluster encoding at least the components LsdG, -O, -E, -F, -H, -I, -J, -L, -M, -N, and -D of a type II secretory system required for LsdA translocation across the outer membrane. Another open reading frame, designated lsdX, is located between the operon promoter and lsdG, but it was not identified in BLASTX searches of the DDBJ/EMBL/GenBank databases. The lsdX, -G, and -O genes were isolated from a cosmid library of strain SRT4 by complementation of an ethyl methanesulfonate mutant unable to transport LsdA across the outer membrane. The downstream genes lsdE, -F, -H, -I, -J, -L, -M, -N, and -D were isolated through chromosomal walking. The high G+C content (64 to 74%) and the codon usage of the genes identified are consistent with the G+C content and codon usage of the standard G. diazotrophicus structural gene. Sequence analysis of the gene cluster indicated that a polycistronic transcript is synthesized. Targeted disruption of lsdG, lsdO, or lsdF blocked LsdA secretion, and the bacterium failed to grow on sucrose. Replacement of Cys(162) by Gly at the C terminus of the pseudopilin LsdG abolished the protein functionality, suggesting that there is a relationship with type IV pilins. Restriction fragment length polymorphism analysis revealed conservation of the type II secretion operon downstream of the levansucrase-levanase (lsdA-lsdB) locus in 14 G. diazotrophicus strains representing 11 genotypes recovered from four different host plants in diverse geographical regions. To our knowledge, this is the first report of a type II pathway for protein secretion in the Acetobacteraceae.

  15. Prm3p is a pheromone-induced peripheral nuclear envelope protein required for yeast nuclear fusion.

    Science.gov (United States)

    Shen, Shu; Tobery, Cynthia E; Rose, Mark D

    2009-05-01

    Nuclear membrane fusion is the last step in the mating pathway of the yeast Saccharomyces cerevisiae. We adapted a bioinformatics approach to identify putative pheromone-induced membrane proteins potentially required for nuclear membrane fusion. One protein, Prm3p, was found to be required for nuclear membrane fusion; disruption of PRM3 caused a strong bilateral defect, in which nuclear congression was completed but fusion did not occur. Prm3p was localized to the nuclear envelope in pheromone-responding cells, with significant colocalization with the spindle pole body in zygotes. A previous report, using a truncated protein, claimed that Prm3p is localized to the inner nuclear envelope. Based on biochemistry, immunoelectron microscopy and live cell microscopy, we find that functional Prm3p is a peripheral membrane protein exposed on the cytoplasmic face of the outer nuclear envelope. In support of this, mutations in a putative nuclear localization sequence had no effect on full-length protein function or localization. In contrast, point mutations and deletions in the highly conserved hydrophobic carboxy-terminal domain disrupted both protein function and localization. Genetic analysis, colocalization, and biochemical experiments indicate that Prm3p interacts directly with Kar5p, suggesting that nuclear membrane fusion is mediated by a protein complex.

  16. Protein turnover, amino acid requirements and recommendations for athletes and active populations

    Energy Technology Data Exchange (ETDEWEB)

    Poortmans, J.R.; Carpentier, A. [Laboratory for Biometry and Sport Nutrition, Faculty of Motor Sciences, Free University of Brussels, Brussels (Belgium); Pereira-Lancha, L.O. [Departamento de Nutrição, Instituto Vita, São Paulo, SP (Brazil); Lancha, A. Jr. [Laboratório de Nutrição Aplicada à Atividade Motora, Escola de Educação Física e Esporte, Universidade de São Paulo, São Paulo, SP (Brazil)

    2012-06-08

    Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers ({sup 13}C-lysine, {sup 15}N-glycine, {sup 2}H{sub 5}-phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g·kg{sup −1}·day{sup −1} compared to 0.8 g·kg{sup −1}·day{sup −1} in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h.

  17. Defining meal requirements for protein to optimize metabolic roles of amino acids.

    Science.gov (United States)

    Layman, Donald K; Anthony, Tracy G; Rasmussen, Blake B; Adams, Sean H; Lynch, Christopher J; Brinkworth, Grant D; Davis, Teresa A

    2015-04-29

    Dietary protein provides essential amino acids (EAAs) for the synthesis of new proteins plus an array of other metabolic functions; many of these functions are sensitive to postprandial plasma and intracellular amino acid concentrations. Recent research has focused on amino acids as metabolic signals that influence the rate of protein synthesis, inflammation responses, mitochondrial activity, and satiety, exerting their influence through signaling systems including mammalian/mechanistic target of rapamycin complex 1 (mTORC1), general control nonrepressed 2 (GCN2), glucagon-like peptide 1 (GLP-1), peptide YY (PYY), serotonin, and insulin. These signals represent meal-based responses to dietary protein. The best characterized of these signals is the leucine-induced activation of mTORC1, which leads to the stimulation of skeletal muscle protein synthesis after ingestion of a meal that contains protein. The response of this metabolic pathway to dietary protein (i.e., meal threshold) declines with advancing age or reduced physical activity. Current dietary recommendations for protein are focused on total daily intake of 0.8 g/kg body weight, but new research suggests daily needs for older adults of ≥1.0 g/kg and identifies anabolic and metabolic benefits to consuming at least 20-30 g protein at a given meal. Resistance exercise appears to increase the efficiency of EAA use for muscle anabolism and to lower the meal threshold for stimulation of protein synthesis. Applying this information to a typical 3-meal-a-day dietary plan results in protein intakes that are well within the guidelines of the Dietary Reference Intakes for acceptable macronutrient intakes. The meal threshold concept for dietary protein emphasizes a need for redistribution of dietary protein for optimum metabolic health.

  18. Protein turnover, amino acid requirements and recommendations for athletes and active populations

    Directory of Open Access Journals (Sweden)

    J.R. Poortmans

    2012-10-01

    Full Text Available Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers (13C-lysine, 15N-glycine, ²H5-phenylalanine and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes compared to resistance exercise, which induces fiber hypertrophy (myofibrils. Nitrogen balance (difference between protein intake and protein degradation for athletes is usually balanced when the intake of protein reaches 1.2 g·kg-1·day-1 compared to 0.8 g·kg-1·day-1 in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins and carbohydrate (± 30 g maltodextrine drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h.

  19. Basic residues within the ebolavirus VP35 protein are required for its viral polymerase cofactor function.

    Science.gov (United States)

    Prins, Kathleen C; Binning, Jennifer M; Shabman, Reed S; Leung, Daisy W; Amarasinghe, Gaya K; Basler, Christopher F

    2010-10-01

    The ebolavirus (EBOV) VP35 protein binds to double-stranded RNA (dsRNA), inhibits host alpha/beta interferon (IFN-α/β) production, and is an essential component of the viral polymerase complex. Structural studies of the VP35 C-terminal IFN inhibitory domain (IID) identified specific structural features, including a central basic patch and a hydrophobic pocket, that are important for dsRNA binding and IFN inhibition. Several other conserved basic residues bordering the central basic patch and a separate cluster of basic residues, called the first basic patch, were also identified. Functional analysis of alanine substitution mutants indicates that basic residues outside the central basic patch are not required for dsRNA binding or for IFN inhibition. However, minigenome assays, which assess viral RNA polymerase complex function, identified these other basic residues to be critical for viral RNA synthesis. Of these, a subset located within the first basic patch is important for VP35-nucleoprotein (NP) interaction, as evidenced by the inability of alanine substitution mutants to coimmunoprecipitate with NP. Therefore, first basic patch residues are likely critical for replication complex formation through interactions with NP. Coimmunoprecipitation studies further demonstrate that the VP35 IID is sufficient to interact with NP and that dsRNA can modulate VP35 IID interactions with NP. Other basic residue mutations that disrupt the VP35 polymerase cofactor function do not affect interaction with NP or with the amino terminus of the viral polymerase. Collectively, these results highlight the importance of conserved basic residues from the EBOV VP35 C-terminal IID and validate the VP35 IID as a potential therapeutic target.

  20. Protein phosphatases decrease their activity during capacitation: a new requirement for this event.

    Directory of Open Access Journals (Sweden)

    Janetti R Signorelli

    Full Text Available There are few reports on the role of protein phosphatases during capacitation. Here, we report on the role of PP2B, PP1, and PP2A during human sperm capacitation. Motile sperm were resuspended in non-capacitating medium (NCM, Tyrode's medium, albumin- and bicarbonate-free or in reconstituted medium (RCM, NCM plus 2.6% albumin/25 mM bicarbonate. The presence of the phosphatases was evaluated by western blotting and the subcellular localization by indirect immunofluorescence. The function of these phosphatases was analyzed by incubating the sperm with specific inhibitors: okadaic acid, I2, endothall, and deltamethrin. Different aliquots were incubated in the following media: 1 NCM; 2 NCM plus inhibitors; 3 RCM; and 4 RCM plus inhibitors. The percent capacitated sperm and phosphatase activities were evaluated using the chlortetracycline assay and a phosphatase assay kit, respectively. The results confirm the presence of PP2B and PP1 in human sperm. We also report the presence of PP2A, specifically, the catalytic subunit and the regulatory subunits PR65 and B. PP2B and PP2A were present in the tail, neck, and postacrosomal region, and PP1 was present in the postacrosomal region, neck, middle, and principal piece of human sperm. Treatment with phosphatase inhibitors rapidly (≤1 min increased the percent of sperm depicting the pattern B, reaching a maximum of ∼40% that was maintained throughout incubation; after 3 h, the percent of capacitated sperm was similar to that of the control. The enzymatic activity of the phosphatases decreased during capacitation without changes in their expression. The pattern of phosphorylation on threonine residues showed a sharp increase upon treatment with the inhibitors. In conclusion, human sperm express PP1, PP2B, and PP2A, and the activity of these phosphatases decreases during capacitation. This decline in phosphatase activities and the subsequent increase in threonine phosphorylation may be an important

  1. Plastid chaperonin proteins Cpn60α and Cpn60β are required for plastid division in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Osteryoung Katherine W

    2009-04-01

    Full Text Available Abstract Background Plastids arose from a free-living cyanobacterial endosymbiont and multiply by binary division as do cyanobacteria. Plastid division involves nucleus-encoded homologs of cyanobacterial division proteins such as FtsZ, MinD, MinE, and ARC6. However, homologs of many other cyanobacterial division genes are missing in plant genomes and proteins of host eukaryotic origin, such as a dynamin-related protein, PDV1 and PDV2 are involved in the division process. Recent identification of plastid division proteins has started to elucidate the similarities and differences between plastid division and cyanobacterial cell division. To further identify new proteins that are required for plastid division, we characterized previously and newly isolated plastid division mutants of Arabidopsis thaliana. Results Leaf cells of two mutants, br04 and arc2, contain fewer, larger chloroplasts than those of wild type. We found that ARC2 and BR04 are identical to nuclear genes encoding the plastid chaperonin 60α (ptCpn60α and chaperonin 60β (ptCpn60β proteins, respectively. In both mutants, plastid division FtsZ ring formation was partially perturbed though the level of FtsZ2-1 protein in plastids of ptcpn60β mutants was similar to that in wild type. Phylogenetic analyses showed that both ptCpn60 proteins are derived from ancestral cyanobacterial proteins. The A. thaliana genome encodes two members of ptCpn60α family and four members of ptCpn60β family respectively. We found that a null mutation in ptCpn60α abolished greening of plastids and resulted in an albino phenotype while a weaker mutation impairs plastid division and reduced chlorophyll levels. The functions of at least two ptCpn60β proteins are redundant and the appearance of chloroplast division defects is dependent on the number of mutant alleles. Conclusion Our results suggest that both ptCpn60α and ptCpn60β are required for the formation of a normal plastid division apparatus, as

  2. Fertilization in C. elegans requires an intact C-terminal RING finger in sperm protein SPE-42

    Directory of Open Access Journals (Sweden)

    Rumbley Jon N

    2011-02-01

    Full Text Available Abstract Background The C. elegans sperm protein SPE-42, a membrane protein of unknown structure and molecular function, is required for fertilization. Sperm from worms with spe-42 mutations appear normal but are unable to fertilize eggs. Sequence analysis revealed the presence of 8 conserved cysteine residues in the C-terminal cytoplasmic domain of this protein suggesting these residues form a zinc-coordinating RING finger structure. Results We made an in silico structural model of the SPE-42 RING finger domain based on primary sequence analysis and previously reported RING structures. To test the model, we created spe-42 transgenes coding for mutations in each of the 8 cysteine residues predicted to coordinate Zn++ ions in the RING finger motif. Transgenes were crossed into a spe-42 null background and protein function was measured by counting progeny. We found that all 8 cysteines are required for protein function. We also showed that sequence differences between the C-terminal 29 and 30 amino acids in C. elegans and C. briggsae SPE-42 following the RING finger domain are not responsible for the failure of the C. briggsae SPE-42 homolog to rescue C. elegans spe-42 mutants. Conclusions The results suggest that a bona fide RING domain is present at the C-terminus of the SPE-42 protein and that this motif is required for sperm-egg interactions during C. elegans fertilization. Our structural model of the RING domain provides a starting point for further structure-function analysis of this critical region of the protein. The C-terminal domain swap experiment suggests that the incompatibility between the C. elegans and C. briggsae SPE-42 proteins is caused by small amino acid differences outside the C-terminal domain.

  3. Distinct determinants in HIV-1 Vif and human APOBEC3 proteins are required for the suppression of diverse host anti-viral proteins.

    Directory of Open Access Journals (Sweden)

    Wenyan Zhang

    Full Text Available BACKGROUND: APOBEC3G (A3G and related cytidine deaminases of the APOBEC3 family of proteins are potent inhibitors of many retroviruses, including HIV-1. Formation of infectious HIV-1 requires the suppression of multiple cytidine deaminases by Vif. HIV-1 Vif suppresses various APOBEC3 proteins through the common mechanism of recruiting the Cullin5-ElonginB-ElonginC E3 ubiquitin ligase to induce target protein polyubiquitination and proteasome-mediated degradation. The domains in Vif and various APOBEC3 proteins required for APOBEC3 recognition and degradation have not been fully characterized. METHODS AND FINDINGS: In the present study, we have demonstrated that the regions of APOBEC3F (A3F that are required for its HIV-1-mediated binding and degradation are distinct from those reported for A3G. We found that the C-terminal cytidine deaminase domain (C-CDD of A3F alone is sufficient for its interaction with HIV-1 Vif and its Vif-mediated degradation. We also observed that the domains of HIV-1 Vif that are uniquely required for its functional interaction with full-length A3F are also required for the degradation of the C-CDD of A3F; in contrast, those Vif domains that are uniquely required for functional interaction with A3G are not required for the degradation of the C-CDD of A3F. Interestingly, the HIV-1 Vif domains required for the degradation of A3F are also required for the degradation of A3C and A3DE. On the other hand, the Vif domains uniquely required for the degradation of A3G are dispensable for the degradation of cytidine deaminases A3C and A3DE. CONCLUSIONS: Our data suggest that distinct regions of A3F and A3G are targeted by HIV-1 Vif molecules. However, HIV-1 Vif suppresses A3F, A3C, and A3DE through similar recognition determinants, which are conserved among Vif molecules from diverse HIV-1 strains. Mapping these determinants may be useful for the design of novel anti-HIV inhibitors.

  4. Collagenase-3 binds to a specific receptor and requires the low density lipoprotein receptor-related protein for internalization

    Science.gov (United States)

    Barmina, O. Y.; Walling, H. W.; Fiacco, G. J.; Freije, J. M.; Lopez-Otin, C.; Jeffrey, J. J.; Partridge, N. C.

    1999-01-01

    We have previously identified a specific receptor for collagenase-3 that mediates the binding, internalization, and degradation of this ligand in UMR 106-01 rat osteoblastic osteosarcoma cells. In the present study, we show that collagenase-3 binding is calcium-dependent and occurs in a variety of cell types, including osteoblastic and fibroblastic cells. We also present evidence supporting a two-step mechanism of collagenase-3 binding and internalization involving both a specific collagenase-3 receptor and the low density lipoprotein receptor-related protein. Ligand blot analysis shows that (125)I-collagenase-3 binds specifically to two proteins ( approximately 170 kDa and approximately 600 kDa) present in UMR 106-01 cells. Western blotting identified the 600-kDa protein as the low density lipoprotein receptor-related protein. Our data suggest that the 170-kDa protein is a specific collagenase-3 receptor. Low density lipoprotein receptor-related protein-null mouse embryo fibroblasts bind but fail to internalize collagenase-3, whereas UMR 106-01 and wild-type mouse embryo fibroblasts bind and internalize collagenase-3. Internalization, but not binding, is inhibited by the 39-kDa receptor-associated protein. We conclude that the internalization of collagenase-3 requires the participation of the low density lipoprotein receptor-related protein and propose a model in which the cell surface interaction of this ligand requires a sequential contribution from two receptors, with the collagenase-3 receptor acting as a high affinity primary binding site and the low density lipoprotein receptor-related protein mediating internalization.

  5. SDCCAG8 Interacts with RAB Effector Proteins RABEP2 and ERC1 and Is Required for Hedgehog Signaling

    DEFF Research Database (Denmark)

    Airik, Rannar; Schueler, Markus; Airik, Merlin

    2016-01-01

    Hedgehog (Hh) signaling. Indeed, cell culture studies demonstrate the requirement of SDCCAG8 for ciliogenesis and Hh signaling. Using an affinity proteomics approach, we demonstrate that SDCCAG8 interacts with proteins of the centriolar satellites (OFD1, AZI1), of the endosomal sorting complex (RABEP2, ERC...

  6. Characterization of EssB, a protein required for secretion of ESAT-6 like proteins in Staphylococcus aureus

    OpenAIRE

    Chen Yi-Hsing; Anderson Mark; Hendrickx Antoni PA; Missiakas Dominique

    2012-01-01

    Abstract Background Staphylococcus aureus secretes EsxA and EsxB, two small polypeptides of the WXG100 family of proteins. Genetic analyses have shown that production and secretion of EsxA and EsxB require an intact ESAT-6 Secretion System (ESS), a cluster of genes that is conserved in many Firmicutes and encompasses esxA and esxB . Here, we characterize EssB, one of the proteins encoded by the ESS cluster. EssB is highly conserved in Gram-positive bacteria and belongs to the Cluster of ...

  7. A transmembrane ubiquitin ligase required to sort membrane proteins into multivesicular bodies

    NARCIS (Netherlands)

    Reggiori, Fulvio; Pelham, Hugh R B; Reggiori, Fulvio

    2002-01-01

    Membrane proteins with transmembrane domains (TMDs) that contain polar residues exposed to the lipid bilayer are selectively sorted into multivesicular bodies (MVBs) and delivered to the yeast vacuole. Sorting of some, although not all, proteins into these structures is mediated by ubiquitination. W

  8. The acyl-CoA binding protein is required for normal epidermal barrier function in mice

    DEFF Research Database (Denmark)

    Bloksgaard, Maria; Bek, Signe; Marcher, Ann-Britt;

    2012-01-01

    The acyl-CoA binding protein (ACBP) is a 10 kDa intracellular protein expressed in all eukaryotic species. Mice with targeted disruption of Acbp (ACBP(-/-) mice) are viable and fertile but present a visible skin and fur phenotype characterized by greasy fur and development of alopecia and scaling...

  9. Drosophila Vps13 Is Required for Protein Homeostasis in the Brain

    Science.gov (United States)

    Vonk, Jan J.; Lahaye, Liza L.; Kanon, Bart; van der Zwaag, Marianne; Velayos-Baeza, Antonio; Freire, Raimundo; van IJzendoorn, Sven C.; Grzeschik, Nicola A.; Sibon, Ody C. M.

    2017-01-01

    Chorea-Acanthocytosis is a rare, neurodegenerative disorder characterized by progressive loss of locomotor and cognitive function. It is caused by loss of function mutations in the Vacuolar Protein Sorting 13A (VPS13A) gene, which is conserved from yeast to human. The consequences of VPS13A dysfunction in the nervous system are still largely unspecified. In order to study the consequences of VPS13A protein dysfunction in the ageing central nervous system we characterized a Drosophila melanogaster Vps13 mutant line. The Drosophila Vps13 gene encoded a protein of similar size as human VPS13A. Our data suggest that Vps13 is a peripheral membrane protein located to endosomal membranes and enriched in the fly head. Vps13 mutant flies showed a shortened life span and age associated neurodegeneration. Vps13 mutant flies were sensitive to proteotoxic stress and accumulated ubiquitylated proteins. Levels of Ref(2)P, the Drosophila orthologue of p62, were increased and protein aggregates accumulated in the central nervous system. Overexpression of the human Vps13A protein in the mutant flies partly rescued apparent phenotypes. This suggests a functional conservation of human VPS13A and Drosophila Vps13. Our results demonstrate that Vps13 is essential to maintain protein homeostasis in the larval and adult Drosophila brain. Drosophila Vps13 mutants are suitable to investigate the function of Vps13 in the brain, to identify genetic enhancers and suppressors and to screen for potential therapeutic targets for Chorea-Acanthocytosis. PMID:28107480

  10. Social Recognition Memory Requires Two Stages of Protein Synthesis in Mice

    Science.gov (United States)

    Wolf, Gerald; Engelmann, Mario; Richter, Karin

    2005-01-01

    Olfactory recognition memory was tested in adult male mice using a social discrimination task. The testing was conducted to begin to characterize the role of protein synthesis and the specific brain regions associated with activity in this task. Long-term olfactory recognition memory was blocked when the protein synthesis inhibitor anisomycin was…

  11. Defining meal requirements for protein to optimize metabolic roles of amino acids

    Science.gov (United States)

    Dietary protein provides essential amino acids (EAAs) for the synthesis of new proteins plus an array of other metabolic functions; many of these functions are sensitive to postprandial plasma and intracellular amino acid concentrations. Recent research has focused on amino acids as metabolic signal...

  12. The V protein of canine distemper virus is required for virus replication in human epithelial cells.

    Directory of Open Access Journals (Sweden)

    Noriyuki Otsuki

    Full Text Available Canine distemper virus (CDV becomes able to use human receptors through a single amino acid substitution in the H protein. In addition, CDV strains possessing an intact C protein replicate well in human epithelial H358 cells. The present study showed that CDV strain 007Lm, which was isolated from lymph node tissue of a dog with distemper, failed to replicate in H358 cells, although it possessed an intact C protein. Sequence analyses suggested that a cysteine-to-tyrosine substitution at position 267 of the V protein caused this growth defect. Analyses using H358 cells constitutively expressing the CDV V protein showed that the V protein with a cysteine, but not that with a tyrosine, at this position effectively blocked the interferon-stimulated signal transduction pathway, and supported virus replication of 007Lm in H358 cells. Thus, the V protein as well as the C protein appears to be functional and essential for CDV replication in human epithelial cells.

  13. Vaccinia virus virion membrane biogenesis protein A11 associates with viral membranes in a manner that requires the expression of another membrane biogenesis protein, A6.

    Science.gov (United States)

    Wu, Xiang; Meng, Xiangzhi; Yan, Bo; Rose, Lloyd; Deng, Junpeng; Xiang, Yan

    2012-10-01

    A group of vaccinia virus (VACV) proteins, including A11, L2, and A6, are required for biogenesis of the primary envelope of VACV, specifically, for the acquisition of viral membrane precursors. However, the interconnection among these proteins is unknown and, with the exception of L2, the connection of these proteins with membranes is also unknown. In this study, prompted by the findings that A6 coprecipitated A11 and that the cellular distribution of A11 was dramatically altered by repression of A6 expression, we studied the localization of A11 in cells by using immunofluorescence and cell fractionation analysis. A11 was found to associate with membranes and colocalize with virion membrane proteins in viral replication factories during normal VACV replication. A11 partitioned almost equally between the detergent and aqueous phases upon Triton X-114 phase separation, demonstrating an intrinsic affinity with lipids. However, in the absence of infection or VACV late protein synthesis, A11 did not associate with cellular membranes. Furthermore, when A6 expression was repressed, A11 did not colocalize with any viral membrane proteins or associate with membranes. In contrast, when virion envelope formation was blocked at a later step by repression of A14 expression or by rifampin treatment, A11 colocalized with virion membrane proteins in the factories. Altogether, our data showed that A11 associates with viral membranes during VACV replication, and this association requires A6 expression. This study provides a physical connection between A11 and viral membranes and suggests that A6 regulates A11 membrane association.

  14. An NB-LRR protein required for HR signalling mediated by both extra- and intracellular resistance proteins.

    NARCIS (Netherlands)

    S.H.E.J. Gabriels; J.H. Vossen; S.K. Ekengren; G. van Ooijen; A.M. Abd-El-Haliem; G.C.M. van den Berg; D.Y. Rainey; G.B. Martin; F.L.W. Takken; P.J.G.M. de Wit; M.H.A.J. Joosten

    2007-01-01

    Tomato (Solanum lycopersicum) Cf resistance genes confer hypersensitive response (HR)-associated resistance to strains of the pathogenic fungus Cladosporium fulvum that express the matching avirulence (Avr) gene. Previously, we identified an Avr4-responsive tomato (ART) gene that is required for Cf-

  15. An NB-LRR protein required for HR signalling mediated by both extra- and intracellular resistance proteins

    NARCIS (Netherlands)

    Gabriëls, S.H.E.J.; Vossen, J.H.; Ekengren, S.K.; Ooijen, van G.; Abd-El-Haliem, A.M.; Berg, van den G.C.M.; Rainey, D.Y.; Martin, G.B.; Takken, F.L.W.; Wit, de P.J.G.M.; Joosten, M.H.A.J.

    2007-01-01

    Tomato (Solanum lycopersicum) Cf resistance genes confer hypersensitive response (HR)-associated resistance to strains of the pathogenic fungus Cladosporium fulvum that express the matching avirulence (Avr) gene. Previously, we identified an Avr4-responsive tomato (ART) gene that is required for Cf-

  16. Protein secretion is required for pregnancy-associated plasma protein-A to promote lung cancer growth in vivo.

    Directory of Open Access Journals (Sweden)

    Hong Pan

    Full Text Available Pregnancy-associated plasma protein-A (PAPPA has been reported to regulate the activity of insulin-like growth factor (IGF signal pathway through proteolytic degradation of IGF binding proteins (IGFBPs thereby increasing the local concentration of free IGFs available to receptors. In this study we found that PAPPA is secreted from two out of seven lung cancer cell lines examined. None of immortalized normal bronchial epithelial cells (HBE tested secrets PAPPA. There is no correlation between expression level and secretion of PAPPA in these cells. A cell line over-expressing PAPPA accompanied with secretion shows no notable changes in proliferation under cell culture conditions in vitro, but displays significantly augmentation of tumor growth in vivo in a xenograft model. In contrast, a cell line over-expressing PAPPA without secretion exhibits reduction of tumor growth both in vitro and in vivo. Down-regulation of PAPPA expression and secretion by RNAi knockdown decreases tumor growth after implanted in vivo. The tumor promoting activity of PAPPA appears to be mediated mainly through augmentation of the IGF signaling pathway as indicated by notable increases in downstream Akt kinase phosphorylation in tumor samples. Our results indicate that PAPPA secretion may play an important role in lung cancer growth and progression.

  17. Sporophyte Formation and Life Cycle Completion in Moss Requires Heterotrimeric G-Proteins1[OPEN

    Science.gov (United States)

    Hackenberg, Dieter; Quatrano, Ralph

    2016-01-01

    In this study, we report the functional characterization of heterotrimeric G-proteins from a nonvascular plant, the moss Physcomitrella patens. In plants, G-proteins have been characterized from only a few angiosperms to date, where their involvement has been shown during regulation of multiple signaling and developmental pathways affecting overall plant fitness. In addition to its unparalleled evolutionary position in the plant lineages, the P. patens genome also codes for a unique assortment of G-protein components, which includes two copies of Gβ and Gγ genes, but no canonical Gα. Instead, a single gene encoding an extra-large Gα (XLG) protein exists in the P. patens genome. Here, we demonstrate that in P. patens the canonical Gα is biochemically and functionally replaced by an XLG protein, which works in the same genetic pathway as one of the Gβ proteins to control its development. Furthermore, the specific G-protein subunits in P. patens are essential for its life cycle completion. Deletion of the genomic locus of PpXLG or PpGβ2 results in smaller, slower growing gametophores. Normal reproductive structures develop on these gametophores, but they are unable to form any sporophyte, the only diploid stage in the moss life cycle. Finally, the mutant phenotypes of ΔPpXLG and ΔPpGβ2 can be complemented by the homologous genes from Arabidopsis, AtXLG2 and AtAGB1, respectively, suggesting an overall conservation of their function throughout the plant evolution. PMID:27550997

  18. HSC90 is required for nascent hepatitis C virus core protein stability in yeast cells.

    Science.gov (United States)

    Kubota, Naoko; Inayoshi, Yasutaka; Satoh, Naoko; Fukuda, Takashi; Iwai, Kenta; Tomoda, Hiroshi; Kohara, Michinori; Kataoka, Kazuhiro; Shimamoto, Akira; Furuichi, Yasuhiro; Nomoto, Akio; Naganuma, Akira; Kuge, Shusuke

    2012-07-30

    Hepatitis C virus core protein (Core) contributes to HCV pathogenicity. Here, we demonstrate that Core impairs growth in budding yeast. We identify HSP90 inhibitors as compounds that reduce intracellular Core protein level and restore yeast growth. Our results suggest that HSC90 (Hsc82) may function in the protection of the nascent Core polypeptide against degradation in yeast and the C-terminal region of Core corresponding to the organelle-interaction domain was responsible for Hsc82-dependent stability. The yeast system may be utilized to select compounds that can direct the C-terminal region to reduce the stability of Core protein.

  19. Requirement for sex comb on midleg protein interactions in Drosophila polycomb group repression.

    OpenAIRE

    Aidan J Peterson; Mallin, Daniel R.; Francis, Nicole J.; Ketel, Carrie S.; Stamm, Joyce; Voeller, Rochus K.; Kingston, Robert E.; Jeffrey A Simon

    2004-01-01

    The Drosophila Sex Comb on Midleg (SCM) protein is a transcriptional repressor of the Polycomb group (PcG). Although genetic studies establish SCM as a crucial PcG member, its molecular role is not known. To investigate how SCM might link to PcG complexes, we analyzed the in vivo role of a conserved protein interaction module, the SPM domain. This domain is found in SCM and in another PcG protein, Polyhomeotic (PH), which is a core component of Polycomb repressive complex 1 (PRC1). SCM-PH int...

  20. Cytoplasmic Dynein Is Required for the Spatial Organization of Protein Aggregates in Filamentous Fungi

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    Martin J. Egan

    2015-04-01

    Full Text Available Eukaryotes have evolved multiple strategies for maintaining cellular protein homeostasis. One such mechanism involves neutralization of deleterious protein aggregates via their defined spatial segregation. Here, using the molecular disaggregase Hsp104 as a marker for protein aggregation, we describe the spatial and temporal dynamics of protein aggregates in the filamentous fungus Aspergillus nidulans. Filamentous fungi, such as A. nidulans, are a diverse group of species of major health and economic importance and also serve as model systems for studying highly polarized eukaryotic cells. We find that microtubules promote the formation of Hsp104-positive aggregates, which coalesce into discrete subcellular structures in a process dependent on the microtubule-based motor cytoplasmic dynein. Finally, we find that impaired clearance of these inclusions negatively impacts retrograde trafficking of endosomes, a conventional dynein cargo, indicating that microtubule-based transport can be overwhelmed by chronic cellular stress.

  1. Lauric acid dependent enhancement in hepatic SCPx protein requires an insulin deficient environment.

    Science.gov (United States)

    Lopez, Dayami; Niesen, Melissa; Bedi, Mohini; McLean, Mark P

    2008-02-01

    Sterol carrier protein X (SCPx) is a peroxisomal protein with both lipid transfer and thiolase activity. Treating with the fatty acid, lauric acid, induced SCPx mRNA levels in rat liver and in rat hepatoma H4IIE cells but enhanced protein levels of SCPx and the thiolase produced as a post-translational modification of SCPx were only seen in H4IIE cells. Further investigation revealed that the presence of insulin can mask lauric acid effects on the SCPx gene especially at the protein level. These data are in agreement with the findings that diabetes, a medical condition characterized by high levels of fatty acids in an insulin deficient environment, enhances the hepatic expression of SCPx.

  2. Double-Stranded RNA-Binding Protein 4 Is Required for Resistance Signaling against Viral and Bacterial Pathogens

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    Shifeng Zhu

    2013-09-01

    Full Text Available Plant viruses often encode suppressors of host RNA silencing machinery, which occasionally function as avirulence factors that are recognized by host resistance (R proteins. For example, the Arabidopsis R protein, hypersensitive response to TCV (HRT, recognizes the turnip crinkle virus (TCV coat protein (CP. HRT-mediated resistance requires the RNA-silencing component double-stranded RNA-binding protein 4 (DRB4 even though it neither is associated with the accumulation of TCV-specific small RNA nor requires the RNA silencing suppressor function of CP. HRT interacts with the cytosolic fraction of DRB4. Interestingly, TCV infection both increases the cytosolic DRB4 pool and inhibits the HRT-DRB4 interaction. The virulent R8A CP derivative, which induces a subset of HRT-derived responses, also disrupts this interaction. The differential localization of DRB4 in the presence of wild-type and R8A CP implies the importance of subcellular compartmentalization of DRB4. The requirement of DRB4 in resistance to bacterial infection suggests a universal role in R-mediated defense signaling.

  3. Critical lysine residues of Klf4 required for protein stabilization and degradation

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Key-Hwan; Kim, So-Ra; Ramakrishna, Suresh; Baek, Kwang-Hyun, E-mail: baek@cha.ac.kr

    2014-01-24

    Highlights: • Klf4 undergoes the 26S proteasomal degradation by ubiquitination on its multiple lysine residues. • Essential Klf4 ubiquitination sites are accumulated between 190–263 amino acids. • A mutation of lysine at 232 on Klf4 elongates protein turnover. • Klf4 mutants dramatically suppress p53 expression both under normal and UV irradiated conditions. - Abstract: The transcription factor, Krüppel-like factor 4 (Klf4) plays a crucial role in generating induced pluripotent stem cells (iPSCs). As the ubiquitination and degradation of the Klf4 protein have been suggested to play an important role in its function, the identification of specific lysine sites that are responsible for protein degradation is of prime interest to improve protein stability and function. However, the molecular mechanism regulating proteasomal degradation of the Klf4 is poorly understood. In this study, both the analysis of Klf4 ubiquitination sites using several Klf4 deletion fragments and bioinformatics predictions showed that the lysine sites which are signaling for Klf4 protein degradation lie in its N-terminal domain (aa 1–296). The results also showed that Lys32, 52, 232, and 252 of Klf4 are responsible for the proteolysis of the Klf4 protein. These results suggest that Klf4 undergoes proteasomal degradation and that these lysine residues are critical for Klf4 ubiquitination.

  4. A tetanus toxin sensitive protein other than VAMP 2 is required for exocytosis in the pancreatic acinar cell.

    Science.gov (United States)

    Padfield, P J

    2000-11-01

    The neurotoxin sensitivity of regulated exocytosis in the pancreatic acinar cell was investigated using streptolysin-O permeabilized pancreatic acini. Treatment of permeabilized acini with botulinum toxin B (BoNT/B) or botulinum toxin D (BoNT/D) had no detectable effect on Ca(2+)-dependent amylase secretion but did result in the complete cleavage of VAMP 2. In comparison, tetanus toxin (TeTx) treatment both significantly inhibited Ca(2+)-dependent amylase secretion and cleaved VAMP 2. These results indicate that regulated exocytosis in the pancreatic acinar cell requires a tetanus toxin sensitive protein(s) other than VAMP 2.

  5. The N-Terminal of Aquareovirus NS80 Is Required for Interacting with Viral Proteins and Viral Replication.

    Directory of Open Access Journals (Sweden)

    Jie Zhang

    Full Text Available Reovirus replication and assembly occurs within viral inclusion bodies that formed in specific intracellular compartments of cytoplasm in infected cells. Previous study indicated that aquareovirus NS80 is able to form inclusion bodies, and also can retain viral proteins within its inclusions. To better understand how NS80 performed in viral replication and assembly, the functional regions of NS80 associated with other viral proteins in aquareovirus replication were investigated in this study. Deletion mutational analysis and rotavirus NSP5-based protein association platform were used to detect association regions. Immunofluorescence images indicated that different N-terminal regions of NS80 could associate with viral proteins VP1, VP4, VP6 and NS38. Further co-immunoprecipitation analysis confirmed the interaction between VP1, VP4, VP6 or NS38 with different regions covering the N-terminal amino acid (aa, 1-471 of NS80, respectively. Moreover, removal of NS80 N-terminal sequences required for interaction with proteins VP1, VP4, VP6 or NS38 not only prevented the capacity of NS80 to support viral replication in NS80 shRNA-based replication complementation assays, but also inhibited the expression of aquareovirus proteins, suggesting that N-terminal regions of NS80 are necessary for viral replication. These results provided a foundational basis for further understanding the role of NS80 in viral replication and assembly during aquareovirus infection.

  6. Gli2 and Gli3 Localize to Cilia and Require the Intraflagellar Transport Protein Polaris for Processing and Function

    Energy Technology Data Exchange (ETDEWEB)

    Michaud III, Edward J [ORNL; Haycraft, Courtney J [University of Alabama, Birmingham; Aydin Son, Yesim [University of Tennessee, Knoxville (UTK); Zhang, Qihong [University of Alabama, Birmingham; Yoder, Bradley [University of Alabama, Birmingham

    2005-01-01

    Intraflagellar transport (IFT) proteins are essential for cilia assembly and have recently been associated with a number of developmental processes, such as left-right axis specification and limb and neural tube patterning. Genetic studies indicate that IFT proteins are required for Sonic hedgehog (Shh)signaling downstream of the Smoothened and Patched membrane proteins but upstream of the Glioma (Gli) transcription factors. However, the role that IFT proteins play in transduction of Shh signaling and the importance of cilia in this process remain unknown. Here we provide insights into the mechanism by which defects in an IFT protein, Tg737/Polaris, affect Shh signaling in the murine limb bud. Our data show that loss of Tg737 results in altered Gli3 processing that abrogates Gli3-mediated repression of Gli1 transcriptional activity. In contrast to the conclusions drawn from genetic analysis, the activity of Gli1 and truncated forms of Gli3 (Gli3R) are unaffected in Tg737 mutants at the molecular level, indicating that Tg737/Polaris is differentially involved in specific activities of the Gli proteins. Most important, a negative regulator of Shh signaling, Suppressor of fused, and the three full-length Gli transcription factors localize to the distal tip of cilia in addition to the nucleus. Thus, our data support a model where cilia have a direct role in Gli processing and Shh signal transduction.

  7. Stimulation of TLR4 by recombinant HSP70 requires structural integrity of the HSP70 protein itself

    Directory of Open Access Journals (Sweden)

    Luong Michael

    2012-03-01

    Full Text Available Abstract Background Toll-like receptor 4 (TLR4 is activated by bacterial endotoxin, a prototypical pathogen-associated molecular pattern (PAMP. It has been suggested that TLR4 can also be activated by damage-associated molecular pattern (DAMP proteins such as HSP70. It remains a challenge to provide unequivocal evidence that DAMP proteins themselves play a role in TLR4 activation, as the DAMP proteins used are often contaminated with endotoxin and other TLR ligands introduced during protein expression and/or purification. Results Here we report that the activation of TLR4 on primary human macrophage cultures by recombinant HSP70 is not solely due to contaminating endotoxin. Polymyxin B pretreatment of HSP70 preparations to neutralize contaminating endotoxin caused significant reductions in the amount of TNF-α induced by the recombinant protein as determined by ELISA. However, digestion of HSP70 with Proteinase K-agarose beads also dramatically reduced the TNF-α response of macrophages to HSP70, while leaving levels of contaminating endotoxin largely unchanged relative to controls. Conclusions These results indicate that the stimulatory effect of recombinant HSP70 requires both the presence of endotoxin and structural integrity of the heat shock protein itself.

  8. Rab18 is required for viral assembly of hepatitis C virus through trafficking of the core protein to lipid droplets.

    Science.gov (United States)

    Dansako, Hiromichi; Hiramoto, Hiroki; Ikeda, Masanori; Wakita, Takaji; Kato, Nobuyuki

    2014-08-01

    During persistent infection of HCV, the HCV core protein (HCV-JFH-1 strain of genotype 2a) is recruited to lipid droplets (LDs) for viral assembly, but the mechanism of recruitment of the HCV core protein is uncertain. Here, we demonstrated that one of the Ras-related small GTPases, Rab18, was required for trafficking of the core protein around LDs. The knockdown of Rab18 reduced intracellular and extracellular viral infectivity, but not intracellular viral replication in HCV-JFH-1-infected RSc cells (an HuH-7-derived cell line). Exogenous expression of Rab18 increased extracellular viral infectivity almost two-fold. Furthermore, Rab18 was co-localized with the core protein in HCV-JFH-1-infected RSc cells, and the knockdown of Rab18 blocked recruitment of the HCV-JFH-1 core protein to LDs. These results suggest that Rab18 has an important role in viral assembly through the trafficking of the core protein to LDs.

  9. Gli2 and gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function.

    Directory of Open Access Journals (Sweden)

    2005-10-01

    Full Text Available Intraflagellar transport (IFT proteins are essential for cilia assembly and have recently been associated with a number of developmental processes, such as left-right axis specification and limb and neural tube patterning. Genetic studies indicate that IFT proteins are required for Sonic hedgehog (Shh signaling downstream of the Smoothened and Patched membrane proteins but upstream of the Glioma (Gli transcription factors. However, the role that IFT proteins play in transduction of Shh signaling and the importance of cilia in this process remain unknown. Here we provide insights into the mechanism by which defects in an IFT protein, Tg737/Polaris, affect Shh signaling in the murine limb bud. Our data show that loss of Tg737 results in altered Gli3 processing that abrogates Gli3-mediated repression of Gli1 transcriptional activity. In contrast to the conclusions drawn from genetic analysis, the activity of Gli1 and truncated forms of Gli3 (Gli3R are unaffected in Tg737 mutants at the molecular level, indicating that Tg737/Polaris is differentially involved in specific activities of the Gli proteins. Most important, a negative regulator of Shh signaling, Suppressor of fused, and the three full-length Gli transcription factors localize to the distal tip of cilia in addition to the nucleus. Thus, our data support a model where cilia have a direct role in Gli processing and Shh signal transduction.

  10. A protein kinase from Colletotrichum trifolii is induced by plant cutin and is required for appressorium formation.

    Science.gov (United States)

    Dickman, M B; Ha, Y S; Yang, Z; Adams, B; Huang, C

    2003-05-01

    When certain phytopathogenic fungi contact plant surfaces, specialized infection structures (appressoria) are produced that facilitate penetration of the plant external barrier; the cuticle. Recognition of this hydrophobic host surface must be sensed by the fungus, initiating the appropriate signaling pathway or pathways for pathogenic development. Using polymerase chain reaction and primers designed from mammalian protein kinase C sequences (PKC), we have isolated, cloned, and characterized a protein kinase from Colletotrichum trifolii, causal agent of alfalfa anthracnose. Though sequence analysis indicated conserved sequences in mammalian PKC genes, we were unable to induce activity of the fungal protein using known activators of PKC. Instead, we show that the C. trifolii gene, designated LIPK (lipid-induced protein kinase) is induced specifically by purified plant cutin or long-chain fatty acids which are monomeric constituents of cutin. PKC inhibitors prevented appressorium formation and, to a lesser extent, spore germination. Overexpression of LIPK resulted in multiple, abnormally shaped appressoria. Gene replacement of lipk yielded strains which were unable to develop appressoria and were unable to infect intact host plant tissue. However, these mutants were able to colonize host tissue following artificial wounding, resulting in typical anthracnose lesions. Taken together, these data indicate a central role in triggering infection structure formation for this protein kinase, which is induced specifically by components of the plant cuticle. Thus, the fungus is able to sense and use host surface chemistry to induce a protein kinase-mediated pathway that is required for pathogenic development.

  11. Transmembrane Protein 184A Is a Receptor Required for Vascular Smooth Muscle Cell Responses to Heparin.

    Science.gov (United States)

    Pugh, Raymond J; Slee, Joshua B; Farwell, Sara Lynn N; Li, Yaqiu; Barthol, Trista; Patton, Walter A; Lowe-Krentz, Linda J

    2016-03-01

    Vascular cell responses to exogenous heparin have been documented to include decreased vascular smooth muscle cell proliferation following decreased ERK pathway signaling. However, the molecular mechanism(s) by which heparin interacts with cells to induce those responses has remained unclear. Previously characterized monoclonal antibodies that block heparin binding to vascular cells have been found to mimic heparin effects. In this study, those antibodies were employed to isolate a heparin binding protein. MALDI mass spectrometry data provide evidence that the protein isolated is transmembrane protein 184A (TMEM184A). Commercial antibodies against three separate regions of the TMEM184A human protein were used to identify the TMEM184A protein in vascular smooth muscle cells and endothelial cells. A GFP-TMEM184A construct was employed to determine colocalization with heparin after endocytosis. Knockdown of TMEM184A eliminated the physiological responses to heparin, including effects on ERK pathway activity and BrdU incorporation. Isolated GFP-TMEM184A binds heparin, and overexpression results in additional heparin uptake. Together, these data support the identification of TMEM184A as a heparin receptor in vascular cells.

  12. DIGESTIBLE VALINE REQUIREMENTS IN LOW-PROTEIN DIETS FOR BROILERS CHICKS

    Directory of Open Access Journals (Sweden)

    GR Nascimento

    Full Text Available ABSTRACT Three experiments were carried out to evaluate the levels of digestible valine in diets with reduced crude protein on the performance, carcass yield and muscle fiber diameter of male broilers during the pre-starter (1 to 8 d of age, starter (9 to 21 d of age and grower phases (21 to 42 d of age. A total of 1,080 chickens in the pre-starter phase, 900 in the starter phase and 864 in the grower phase were distributed in a completely randomized design with six treatments, consisting of a control positive diet (C+ and diets with 2 percentage points (p.p. reduction of crude protein level and five graded levels of digestible valine (Val, with six replicates of 30, 25 and 24 birds per experimental unit, respectively. The recommended level of TID Val in the low-protein diets for broilers in the pre-starter, starter and grower stages were 1.028; 0.905 and 0.789%, respectively. The reduction of 2 percentage points of the crude protein level in diets based on corn and soybean meal impaired (p ≤0.05 the feed conversion ratio in the starter and grower stages. Likewise, the reduced-protein diets decreased (p ≤0.05 muscle fiber diameter, but did not affect (p> 0.05 carcass and parts yields, or abdominal fat percentage at 42 days.

  13. Resolution of a low molecular weight G protein in neutrophil cytosol required for NADPH oxidase activation and reconstitution by recombinant Krev-1 protein.

    Science.gov (United States)

    Eklund, E A; Marshall, M; Gibbs, J B; Crean, C D; Gabig, T G

    1991-07-25

    Activation of the membrane-associated NADPH oxidase in intact human neutrophils requires a receptor-associated heterotrimeric GTP-binding protein that is sensitive to pertussis toxin. Activation of this NADPH oxidase by arachidonate in a cell-free system requires an additional downstream pertussis toxin-insensitive G protein (Gabig, T. G., English, D., Akard, L. P., and Schell, M. J. (1987) (J. Biol. Chem. 262, 1685-1690) that is located in the cytosolic fraction of unstimulated cells (Gabig, T. G., Eklund, E. A., Potter, G. B., and Dykes, J. R. (1990) J. Immunol. 145, 945-951). In the present study, immunodepletion of G proteins from the cytosolic fraction of unstimulated neutrophils resulted in a loss of the ability to activate NADPH oxidase in the membrane fraction. The activity in immunodepleted cytosol was fully reconstituted by a partially purified fraction from neutrophil cytosol that contained a 21-kDa GTP-binding protein. Purified human recombinant Krev-1 p21 also completely reconstituted immunodepleted cytosol whereas recombinant human H-ras p21 or yeast RAS GTP-binding proteins had no reconstitutive activity. Rabbit antisera raised against a synthetic peptide corresponding to the effector region of Krev-1 (amino acids 31-43) completely inhibited cell-free NADPH oxidase activation, and this inhibition was blocked by the synthetic 31-43 peptide. An inhibitory monoclonal antibody specific for ras p21 amino acids 60-77 (Y13-259) had no effect on cell-free NADPH oxidase activation. Activation of the NADPH oxidase in intact neutrophils by stimulation with phorbol myristate acetate caused a marked increase in the amount of membrane-associated antigen recognized by 151 antiserum on Western blot. Thus a G protein in the cytosol of unstimulated neutrophils antigenically and functionally related to Krev-1 may be the downstream effector G protein for NADPH oxidase activation. This system represents a unique model to study molecular interactions of a ras-like G

  14. Food and nutritional security requires adequate protein as well as energy, delivered from whole-year crop production

    DEFF Research Database (Denmark)

    Coles, Graeme D; Wratten, Stephen D; Porter, John Roy

    2016-01-01

    Human food security requires the production of sufficient quantities of both high-quality protein and dietary energy. In a series of case-studies from New Zealand, we show that while production of food ingredients from crops on arable land can meet human dietary energy requirements effectively...... with an accompanying economic analysis of commercially-available, pre-prepared or simply-cooked foods that can be produced from our case-study crop and animal products. We calculate the per-person, per-day cost of both quality-corrected protein and dietary energy as provided in the processed foods. We conclude...... and nutritional security will largely be an outcome of national or regional agroeconomies addressing their own food needs. We hope that our model will be used for similar analyses of food production systems in other countries, agroecological zones and economies....

  15. Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Leder, Verena [Molecular Cell Physiology, Faculty of Biology, Bielefeld University (Germany); Biomolecular Photonics, Faculty of Physics, Bielefeld University (Germany); Lummer, Martina [Molecular Cell Physiology, Faculty of Biology, Bielefeld University (Germany); Tegeler, Kathrin [Molecular Cell Physiology, Faculty of Biology, Bielefeld University (Germany); Biomolecular Photonics, Faculty of Physics, Bielefeld University (Germany); Humpert, Fabian [Biomolecular Photonics, Faculty of Physics, Bielefeld University (Germany); Lewinski, Martin [Molecular Cell Physiology, Faculty of Biology, Bielefeld University (Germany); Schüttpelz, Mark [Biomolecular Photonics, Faculty of Physics, Bielefeld University (Germany); Staiger, Dorothee, E-mail: dorothee.staiger@uni-bielefeld.de [Molecular Cell Physiology, Faculty of Biology, Bielefeld University (Germany)

    2014-10-10

    Highlights: • We use FCS to investigate binding site requirements for the hnRNP-like protein AtGRP7. • We identify three nucleotides critical for AtGRP7 binding to its own intron. • Mutation of the conserved R{sup 49} abolishes binding altogether. • The paralogue AtGRP8 binds to an overlapping motif with different sequence requirement. • The glycine-rich stretch of a plant hnRNP-like protein contributes to binding. - Abstract: Arabidopsis thaliana glycine-rich RNA binding protein 7 (AtGRP7) is part of a negative feedback loop through which it regulates alternative splicing and steady-state abundance of its pre-mRNA. Here we use fluorescence correlation spectroscopy to investigate the requirements for AtGRP7 binding to its intron using fluorescently-labelled synthetic oligonucleotides. By systematically introducing point mutations we identify three nucleotides that lead to an increased K{sub d} value when mutated and thus are critical for AtGRP7 binding. Simultaneous mutation of all three residues abrogates binding. The paralogue AtGRP8 binds to an overlapping motif but with a different sequence preference, in line with overlapping but not identical functions of this protein pair. Truncation of the glycine-rich domain reduces the binding affinity of AtGRP7, showing for the first time that the glycine-rich stretch of a plant hnRNP-like protein contributes to binding. Mutation of the conserved R{sup 49} that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding.

  16. CAS-1, a C. elegans cyclase-associated protein, is required for sarcomeric actin assembly in striated muscle

    OpenAIRE

    Nomura, Kazumi; Ono, Kanako; Ono, Shoichiro

    2012-01-01

    Assembly of contractile apparatuses in striated muscle requires precisely regulated reorganization of the actin cytoskeletal proteins into sarcomeric organization. Regulation of actin filament dynamics is one of the essential processes of myofibril assembly, but the mechanism of actin regulation in striated muscle is not clearly understood. Actin depolymerizing factor (ADF)/cofilin is a key enhancer of actin filament dynamics in striated muscle in both vertebrates and nematodes. Here, we repo...

  17. Nuclear actin-related protein is required for chromosome segregation in Toxoplasma gondii.

    Science.gov (United States)

    Suvorova, Elena S; Lehmann, Margaret M; Kratzer, Stella; White, Michael W

    2012-01-01

    Apicomplexa parasites use complex cell cycles to replicate that are not well understood mechanistically. We have established a robust forward genetic strategy to identify the essential components of parasite cell division. Here we describe a novel temperature sensitive Toxoplasma strain, mutant 13-20C2, which growth arrests due to a defect in mitosis. The primary phenotype is the mis-segregation of duplicated chromosomes with chromosome loss during nuclear division. This defect is conditional-lethal with respect to temperature, although relatively mild in regard to the preservation of the major microtubule organizing centers. Despite severe DNA loss many of the physical structures associated with daughter budding and the assembly of invasion structures formed and operated normally at the non-permissive temperature before completely arresting. These results suggest there are coordinating mechanisms that govern the timing of these events in the parasite cell cycle. The defect in mutant 13-20C2 was mapped by genetic complementation to Toxoplasma chromosome III and to a specific mutation in the gene encoding an ortholog of nuclear actin-related protein 4. A change in a conserved isoleucine to threonine in the helical structure of this nuclear actin related protein leads to protein instability and cellular mis-localization at the higher temperature. Given the age of this protist family, the results indicate a key role for nuclear actin-related proteins in chromosome segregation was established very early in the evolution of eukaryotes.

  18. Canavanine-induced longevity in mice may require diets with greater than 15.7% protein

    Directory of Open Access Journals (Sweden)

    Brown Dan L

    2005-02-01

    Full Text Available Abstract Background Dietary administration of 1% canavanine had been shown to improve survival in female BALB/c mice consuming diets containing 23.4% protein (dry matter basis. Methods In order to determine if this effect also obtains at more moderate dietary protein concentrations, 30 female BALB/c mice were fed a basal diet with 14% protein (15.7% dry matter basis and another 30 were fed the same diet plus 1% canavanine. Results Neither mean (Control 873.2 d, Canavanine 870.0 d; SEM = 34.2 d; P = 0.949 from ANOVA nor median (Control 902 d, Canavanine 884.5 d; P = 0.9058 from Mann-Whitney lifespans differed between groups. Although mean antinuclear antibody (ANA titers did not differ between control and canavanine-treated mice at 833 days of age (19.84 vs 20.39 respectively; SEM = 2.64; P = 0.889 from ANOVA, one canavanine-treated mouse displayed an outlying ANA value of 50 (next lower value = 30 denoting possible early sign of incipient autoimmune disease in that individual. Conclusion There may be an interaction between dietary protein level and canavanine with respect to lifespan in mice.

  19. Mitogen requirement for cell cycle progression in the absence of pocket protein activity

    NARCIS (Netherlands)

    Foijer, Floris; Wolthuis, Rob M F; Doodeman, Valerie; Medema, René H; te Riele, Hein

    2005-01-01

    Primary mouse embryonic fibroblasts lacking expression of all three retinoblastoma protein family members (TKO MEFs) have lost the G1 restriction point. However, in the absence of mitogens these cells become highly sensitive to apoptosis. Here, we show that TKO MEFs that survive serum depletion pass

  20. The neuronal PAS domain protein 4 (Npas4 is required for new and reactivated fear memories.

    Directory of Open Access Journals (Sweden)

    Jonathan E Ploski

    Full Text Available The Neuronal PAS domain protein 4 (Npas4 is a neuronal activity-dependent immediate early gene that has recently been identified as a transcription factor which regulates the transcription of genes that control inhibitory synapse development and synaptic plasticity. The role Npas4 in learning and memory, however, is currently unknown. Here, we systematically examine the role of Npas4 in auditory Pavlovian fear conditioning, an amygdala-dependent form of emotional learning. In our first series of experiments, we show that Npas4 mRNA and protein are regulated in the rat lateral nucleus of the amygdala (LA in a learning-dependent manner. Further, knockdown of Npas4 protein in the LA via adeno-associated viral (AAV mediated gene delivery of RNAi was observed to impair fear memory formation, while innate fear and the expression of fear memory were not affected. In our second series of experiments, we show that Npas4 protein is regulated in the LA by retrieval of an auditory fear memory and that knockdown of Npas4 in the LA impairs retention of a reactivated, but not a non-reactivated, fear memory. Collectively, our findings provide the first comprehensive look at the functional role of Npas4 in learning and memory.

  1. Adult neurogenesis requires Smad4-mediated bone morphogenic protein signaling in stem cells.

    NARCIS (Netherlands)

    Colak, D.; Mori, T.; Brill, M.S; Pfeifer, A.; Falk, S.; Deng, C.; Monteiro, R.; Mummery, C.L.; Sommer, L.; Gotz, M.

    2008-01-01

    In the mammalian brain, neurogenesis continues only in few regions of the forebrain. The molecular signals governing neurogenesis in these unique neurogenic niches, however, are still ill defined. Here, we show that bone morphogenic protein (BMP)-mediated signaling is active in adult neural stem cel

  2. CREB binding protein is required for both short-term and long-term memory formation

    NARCIS (Netherlands)

    Chen, G.; Zou, X.; Watanabe, H.; Deursen, J.M.A. van; Shen, J.

    2010-01-01

    CREB binding protein (CBP) is a transcriptional coactivator with histone acetyltransferase activity. Our prior study suggested that CBP might be a key target of presenilins in the regulation of memory formation and neuronal survival. To elucidate the role of CBP in the adult brain, we generated cond

  3. Virulence of the fungal pathogen Candida albicans requires the five isoforms of protein mannosyltransferases.

    Science.gov (United States)

    Rouabhia, Mahmoud; Schaller, Martin; Corbucci, Cristina; Vecchiarelli, Anna; Prill, Stephan K-H; Giasson, Luc; Ernst, Joachim F

    2005-08-01

    The PMT gene family in Candida albicans encodes five isoforms of protein mannosyltransferases (Pmt proteins Pmt1p, Pmt2p, Pmt4p, Pmt5p, and Pmt6p) that initiate O mannosylation of secretory proteins. We compared virulence characteristics of pmt mutants in two complex, three-dimensional models of localized candidiasis, using reconstituted human epithelium (RHE) and engineered human oral mucosa (EHOM); in addition, mutants were tested in a mouse model of hematogenously disseminated candidiasis (HDC). All pmt mutants showed attenuated virulence in the HDC model and at least one model of localized candidiasis. The pmt5 mutant, which lacks in vitro growth phenotypes, was less virulent in the EHOM and HDC assays but had no consistent phenotype in the RHE assay. In contrast, the pmt4 and pmt6 mutants were less virulent in the RHE and HDC assays but not in the EHOM assay. The results stress the contribution of all Pmt isoforms to the virulence of C. albicans and suggest that the importance of individual Pmt isoforms may differ in specific host niches. We propose that Pmt proteins may be suitable targets for future novel classes of antifungal agents.

  4. Reconsolidation of a Context Long-Term Memory in the Terrestrial Snail Requires Protein Synthesis

    Science.gov (United States)

    Gainutdinova, Tatiana H.; Tagirova, Rosa R.; Ismailova, Asja I.; Muranova, Lyudmila N.; Samarova, Elena I.; Gainutdinov, Khalil L.; Balaban, Pavel M.

    2005-01-01

    We investigated the influence of the protein synthesis blocker anisomycin on contextual memory in the terrestrial snail "Helix." Prior to the training session, the behavioral responses in two contexts were similar. Two days after a session of electric shocks (5 d) in one context only, the context conditioning was observed as the significant…

  5. Capping protein beta is required for actin cytoskeleton organisation and cell migration during Drosophila oogenesis.

    Science.gov (United States)

    Ogienko, Anna A; Karagodin, Dmitry A; Lashina, Valentina V; Baiborodin, Sergey I; Omelina, Eugeniya S; Baricheva, Elina M

    2013-02-01

    Capping protein (CP) is a well-characterised actin-binding protein important for regulation of actin filament (AF) assembly. CP caps the barbed end of AFs, inhibiting the addition and loss of actin monomers. In Drosophila melanogaster, the gene encoding CP β-subunit is named capping protein beta (cpb; see Hopmann et al. [1996] J Cell Biol 133: 1293-305). The cpb level is reduced in the Drosophila bristle actin cytoskeleton and becomes disorganised with abnormal morphology. A reduced level of the CP protein in ovary results in disruption of oocyte determination, and disturbance of nurse cell (NC) cortical integrity and dumping. We describe novel defects appearing in cpb mutants during oogenesis, in which cpb plays an important role in border and centripetal follicle cell migration, ring canal development and cytoplasmic AF formation. The number of long cytoplasmic AFs was dramatically reduced in cpb hypomorphs and abnormal actin aggregates was seen on the inner side of NC membranes. A hypothesis to explain the formation of abnormal short-cut cytoplasmic AFs and actin aggregates in the cpb mutant NCs was proffered, along with a discussion of the reasons for 'dumpless' phenotype formation in the mutants.

  6. The Cladosporium fulvum virulence protein Avr2 inhibits host proteases require for basal defense

    Science.gov (United States)

    Cladosporium fulvum (syn. Passalora fulva) is a biotrophic fungal pathogen that causes leaf mold of tomato. During growth in the apoplast, the fungus secretes effector proteins enabling it to establish disease. For most of these effectors, cognate C. fulvum resistance (Cf) loci have been identified...

  7. CAS-1, a C. elegans cyclase-associated protein, is required for sarcomeric actin assembly in striated muscle.

    Science.gov (United States)

    Nomura, Kazumi; Ono, Kanako; Ono, Shoichiro

    2012-09-01

    Assembly of contractile apparatuses in striated muscle requires precisely regulated reorganization of the actin cytoskeletal proteins into sarcomeric organization. Regulation of actin filament dynamics is one of the essential processes of myofibril assembly, but the mechanism of actin regulation in striated muscle is not clearly understood. Actin depolymerizing factor (ADF)/cofilin is a key enhancer of actin filament dynamics in striated muscle in both vertebrates and nematodes. Here, we report that CAS-1, a cyclase-associated protein in Caenorhabditis elegans, promotes ADF/cofilin-dependent actin filament turnover in vitro and is required for sarcomeric actin organization in striated muscle. CAS-1 is predominantly expressed in striated muscle from embryos to adults. In vitro, CAS-1 binds to actin monomers and enhances exchange of actin-bound ATP/ADP even in the presence of UNC-60B, a muscle-specific ADF/cofilin that inhibits the nucleotide exchange. As a result, CAS-1 and UNC-60B cooperatively enhance actin filament turnover. The two proteins also cooperate to shorten actin filaments. A cas-1 mutation is homozygous lethal with defects in sarcomeric actin organization. cas-1-mutant embryos and worms have aggregates of actin in muscle cells, and UNC-60B is mislocalized to the aggregates. These results provide genetic and biochemical evidence that cyclase-associated protein is a critical regulator of sarcomeric actin organization in striated muscle.

  8. Slam is an outer membrane protein that is required for the surface display of lipidated virulence factors in Neisseria.

    Science.gov (United States)

    Hooda, Yogesh; Lai, Christine Chieh-Lin; Judd, Andrew; Buckwalter, Carolyn M; Shin, Hyejin Esther; Gray-Owen, Scott D; Moraes, Trevor F

    2016-02-29

    Lipoproteins decorate the surface of many Gram-negative bacterial pathogens, playing essential roles in immune evasion and nutrient acquisition. In Neisseria spp., the causative agents of gonorrhoea and meningococcal meningitis, surface lipoproteins (SLPs) are required for virulence and have been extensively studied as prime candidates for vaccine development. However, the machinery and mechanism that allow for the surface display of SLPs are not known. Here, we describe a transposon (Tn5)-based search for the proteins required to deliver SLPs to the surface of Neisseria meningitidis, revealing a family of proteins that we have named the surface lipoprotein assembly modulator (Slam). N. meningitidis contains two Slam proteins, each exhibiting distinct substrate preferences. The Slam proteins are sufficient to reconstitute SLP transport in laboratory strains of Escherichia coli, which are otherwise unable to efficiently display these lipoproteins on their cell surface. Immunoprecipitation and domain probing experiments suggest that the SLP, TbpB, interacts with Slam during the transit process; furthermore, the membrane domain of Slam is sufficient for selectivity and proper surface display of SLPs. Rather than being a Neisseria-specific factor, our bioinformatic analysis shows that Slam can be found throughout proteobacterial genomes, indicating a conserved but until now unrecognized virulence mechanism.

  9. PPE Surface Proteins Are Required for Heme Utilization by Mycobacterium tuberculosis

    Science.gov (United States)

    Mitra, Avishek; Speer, Alexander; Lin, Kan; Ehrt, Sabine

    2017-01-01

    ABSTRACT Iron is essential for replication of Mycobacterium tuberculosis, but iron is efficiently sequestered in the human host during infection. Heme constitutes the largest iron reservoir in the human body and is utilized by many bacterial pathogens as an iron source. While heme acquisition is well studied in other bacterial pathogens, little is known in M. tuberculosis. To identify proteins involved in heme utilization by M. tuberculosis, a transposon mutant library was screened for resistance to the toxic heme analog gallium(III)-porphyrin (Ga-PIX). Inactivation of the ppe36, ppe62, and rv0265c genes resulted in resistance to Ga-PIX. Growth experiments using isogenic M. tuberculosis deletion mutants showed that PPE36 is essential for heme utilization by M. tuberculosis, while the functions of PPE62 and Rv0265c are partially redundant. None of the genes restored growth of the heterologous M. tuberculosis mutants, indicating that the proteins encoded by the genes have separate functions. PPE36, PPE62, and Rv0265c bind heme as shown by surface plasmon resonance spectroscopy and are associated with membranes. Both PPE36 and PPE62 proteins are cell surface accessible, while the Rv0265c protein is probably located in the periplasm. PPE36 and PPE62 are, to our knowledge, the first proline-proline-glutamate (PPE) proteins of M. tuberculosis that bind small molecules and are involved in nutrient acquisition. The absence of a virulence defect of the ppe36 deletion mutant indicates that the different iron acquisition pathways of M. tuberculosis may substitute for each other during growth and persistence in mice. The emerging model of heme utilization by M. tuberculosis as derived from this study is substantially different from those of other bacteria. PMID:28119467

  10. Temporal Requirements of the Fragile X Mental Retardation Protein in Modulating Circadian Clock Circuit Synaptic Architecture

    OpenAIRE

    Gatto, Cheryl L.; Broadie, Kendal

    2009-01-01

    Loss of fragile X mental retardation 1 (FMR1) gene function is the most common cause of inherited mental retardation and autism spectrum disorders, characterized by attention disorder, hyperactivity and disruption of circadian activity cycles. Pursuit of effective intervention strategies requires determining when the FMR1 product (FMRP) is required in the regulation of neuronal circuitry controlling these behaviors. In the well-characterized Drosophila disease model, loss of the highly conser...

  11. Distinct domains of the spinal muscular atrophy protein SMN are required for targeting to Cajal bodies in mammalian cells.

    Science.gov (United States)

    Renvoisé, Benoît; Khoobarry, Kevinee; Gendron, Marie-Claude; Cibert, Christian; Viollet, Louis; Lefebvre, Suzie

    2006-02-15

    Mutations of the survival motor neuron gene SMN1 cause the inherited disease spinal muscular atrophy (SMA). The ubiquitous SMN protein facilitates the biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs). The protein is detected in the cytoplasm, nucleoplasm and enriched with snRNPs in nuclear Cajal bodies. It is structurally divided into at least an amino-terminal region rich in basic amino acid residues, a central Tudor domain, a self-association tyrosine-glycine-box and an exon7-encoded C-terminus. To examine the domains required for the intranuclear localization of SMN, we have used fluorescently tagged protein mutants transiently overexpressed in mammalian cells. The basic amino acid residues direct nucleolar localization of SMN mutants. The Tudor domain promotes localization of proteins in the nucleus and it cooperates with the basic amino acid residues and the tyrosine-glycine-box for protein localization in Cajal bodies. Moreover, the most frequent disease-linked mutant SMNDeltaex7 reduces accumulation of snRNPs in Cajal bodies, suggesting that the C-terminus of SMN participates in targeting to Cajal bodies. A reduced number of Cajal bodies in patient fibroblasts associates with the absence of snRNPs in Cajal bodies, revealing that intranuclear snRNA organization is modified in disease. These results indicate that direct and indirect mechanisms regulate localization of SMN in Cajal bodies.

  12. The Gos28 SNARE protein mediates intra-Golgi transport of rhodopsin and is required for photoreceptor survival.

    Science.gov (United States)

    Rosenbaum, Erica E; Vasiljevic, Eva; Cleland, Spencer C; Flores, Carlos; Colley, Nansi Jo

    2014-11-21

    SNARE proteins play indispensable roles in membrane fusion events in many cellular processes, including synaptic transmission and protein trafficking. Here, we characterize the Golgi SNARE protein, Gos28, and its role in rhodopsin (Rh1) transport through Drosophila photoreceptors. Mutations in gos28 lead to defective Rh1 trafficking and retinal degeneration. We have pinpointed a role for Gos28 in the intra-Golgi transport of Rh1, downstream from α-mannosidase-II in the medial- Golgi. We have confirmed the necessity of key residues in Gos28's SNARE motif and demonstrate that its transmembrane domain is not required for vesicle fusion, consistent with Gos28 functioning as a t-SNARE for Rh1 transport. Finally, we show that human Gos28 rescues both the Rh1 trafficking defects and retinal degeneration in Drosophila gos28 mutants, demonstrating the functional conservation of these proteins. Our results identify Gos28 as an essential SNARE protein in Drosophila photoreceptors and provide mechanistic insights into the role of SNAREs in neurodegenerative disease.

  13. The Arabidopsis Thylakoid Protein PAM68 Is Required for Efficient D1 Biogenesis and Photosystem II Assembly[W

    Science.gov (United States)

    Armbruster, Ute; Zühlke, Jessica; Rengstl, Birgit; Kreller, Renate; Makarenko, Elina; Rühle, Thilo; Schünemann, Danja; Jahns, Peter; Weisshaar, Bernd; Nickelsen, Jörg; Leister, Dario

    2010-01-01

    Photosystem II (PSII) is a multiprotein complex that functions as a light-driven water:plastoquinone oxidoreductase in photosynthesis. Assembly of PSII proceeds through a number of distinct intermediate states and requires auxiliary proteins. The photosynthesis affected mutant 68 (pam68) of Arabidopsis thaliana displays drastically altered chlorophyll fluorescence and abnormally low levels of the PSII core subunits D1, D2, CP43, and CP47. We show that these phenotypes result from a specific decrease in the stability and maturation of D1. This is associated with a marked increase in the synthesis of RC (the PSII reaction center-like assembly complex) at the expense of PSII dimers and supercomplexes. PAM68 is a conserved integral membrane protein found in cyanobacterial and eukaryotic thylakoids and interacts in split-ubiquitin assays with several PSII core proteins and known PSII assembly factors. Biochemical analyses of thylakoids from Arabidopsis and Synechocystis sp PCC 6803 suggest that, during PSII assembly, PAM68 proteins associate with an early intermediate complex that might contain D1 and the assembly factor LPA1. Inactivation of cyanobacterial PAM68 destabilizes RC but does not affect larger PSII assembly complexes. Our data imply that PAM68 proteins promote early steps in PSII biogenesis in cyanobacteria and plants, but their inactivation is differently compensated for in the two classes of organisms. PMID:20923938

  14. The Arabidopsis thylakoid protein PAM68 is required for efficient D1 biogenesis and photosystem II assembly.

    Science.gov (United States)

    Armbruster, Ute; Zühlke, Jessica; Rengstl, Birgit; Kreller, Renate; Makarenko, Elina; Rühle, Thilo; Schünemann, Danja; Jahns, Peter; Weisshaar, Bernd; Nickelsen, Jörg; Leister, Dario

    2010-10-01

    Photosystem II (PSII) is a multiprotein complex that functions as a light-driven water:plastoquinone oxidoreductase in photosynthesis. Assembly of PSII proceeds through a number of distinct intermediate states and requires auxiliary proteins. The photosynthesis affected mutant 68 (pam68) of Arabidopsis thaliana displays drastically altered chlorophyll fluorescence and abnormally low levels of the PSII core subunits D1, D2, CP43, and CP47. We show that these phenotypes result from a specific decrease in the stability and maturation of D1. This is associated with a marked increase in the synthesis of RC (the PSII reaction center-like assembly complex) at the expense of PSII dimers and supercomplexes. PAM68 is a conserved integral membrane protein found in cyanobacterial and eukaryotic thylakoids and interacts in split-ubiquitin assays with several PSII core proteins and known PSII assembly factors. Biochemical analyses of thylakoids from Arabidopsis and Synechocystis sp PCC 6803 suggest that, during PSII assembly, PAM68 proteins associate with an early intermediate complex that might contain D1 and the assembly factor LPA1. Inactivation of cyanobacterial PAM68 destabilizes RC but does not affect larger PSII assembly complexes. Our data imply that PAM68 proteins promote early steps in PSII biogenesis in cyanobacteria and plants, but their inactivation is differently compensated for in the two classes of organisms.

  15. BDNF stimulation of protein synthesis in cortical neurons requires the MAP kinase-interacting kinase MNK1.

    Science.gov (United States)

    Genheden, Maja; Kenney, Justin W; Johnston, Harvey E; Manousopoulou, Antigoni; Garbis, Spiros D; Proud, Christopher G

    2015-01-21

    Although the MAP kinase-interacting kinases (MNKs) have been known for >15 years, their roles in the regulation of protein synthesis have remained obscure. Here, we explore the involvement of the MNKs in brain-derived neurotrophic factor (BDNF)-stimulated protein synthesis in cortical neurons from mice. Using a combination of pharmacological and genetic approaches, we show that BDNF-induced upregulation of protein synthesis requires MEK/ERK signaling and the downstream kinase, MNK1, which phosphorylates eukaryotic initiation factor (eIF) 4E. Translation initiation is mediated by the interaction of eIF4E with the m(7)GTP cap of mRNA and with eIF4G. The latter interaction is inhibited by the interactions of eIF4E with partner proteins, such as CYFIP1, which acts as a translational repressor. We find that BDNF induces the release of CYFIP1 from eIF4E, and that this depends on MNK1. Finally, using a novel combination of BONCAT and SILAC, we identify a subset of proteins whose synthesis is upregulated by BDNF signaling via MNK1 in neurons. Interestingly, this subset of MNK1-sensitive proteins is enriched for functions involved in neurotransmission and synaptic plasticity. Additionally, we find significant overlap between our subset of proteins whose synthesis is regulated by MNK1 and those encoded by known FMRP-binding mRNAs. Together, our data implicate MNK1 as a key component of BDNF-mediated translational regulation in neurons.

  16. An Organelle RNA Recognition Motif Protein Is Required for Photosystem II Subunit psbF Transcript Editing1[OPEN

    Science.gov (United States)

    Lucas, Meriah K.

    2017-01-01

    Loss-of-function mutations in ORGANELLE RNA RECOGNITION MOTIF PROTEIN6 (ORRM6) result in the near absence of RNA editing of psbF-C77 and the reduction in accD-C794 editing in Arabidopsis (Arabidopsis thaliana). The orrm6 mutants have decreased levels of photosystem II (PSII) proteins, especially PsbF, lower PSII activity, pale green pigmentation, smaller leaf and plant sizes, and retarded growth. Stable expression of ORRM6 rescues the orrm6 editing defects and mutant phenotype. Unlike ORRM1, the other known ORRM plastid editing factor, ORRM6, does not contain RNA editing interacting protein/multiple organellar RNA editing factor (RIP/MORF) boxes, which are required for ORRM1 to interact with site-specific pentatricopeptide repeat protein editing factors. ORRM6 interacts with RIP1/MORF8, RIP2/MORF2, and RIP9/MORF9, known components of RNA editosomes. While some plastid RRM proteins are involved in other forms of RNA processing and translation, the primary function of ORRM6 is evidently to mediate psbF-C77 editing, like the essential site-specific pentatricopeptide repeat protein LOW PSII ACCUMULATION66. Stable expression in the orrm6 mutants of a nucleus-encoded, plastid-targeted PsbF protein from a psbF gene carrying a T at nucleotide 77 significantly increases leaf and plant sizes, chlorophyll content, and PSII activity. These transformants demonstrate that plastid RNA editing can be bypassed through the expression of nucleus-encoded, edited forms of plastid genes. PMID:28213559

  17. An Organelle RNA Recognition Motif Protein Is Required for Photosystem II Subunit psbF Transcript Editing.

    Science.gov (United States)

    Hackett, Justin B; Shi, Xiaowen; Kobylarz, Amy T; Lucas, Meriah K; Wessendorf, Ryan L; Hines, Kevin M; Bentolila, Stephane; Hanson, Maureen R; Lu, Yan

    2017-04-01

    Loss-of-function mutations in ORGANELLE RNA RECOGNITION MOTIF PROTEIN6 (ORRM6) result in the near absence of RNA editing of psbF-C77 and the reduction in accD-C794 editing in Arabidopsis (Arabidopsis thaliana). The orrm6 mutants have decreased levels of photosystem II (PSII) proteins, especially PsbF, lower PSII activity, pale green pigmentation, smaller leaf and plant sizes, and retarded growth. Stable expression of ORRM6 rescues the orrm6 editing defects and mutant phenotype. Unlike ORRM1, the other known ORRM plastid editing factor, ORRM6, does not contain RNA editing interacting protein/multiple organellar RNA editing factor (RIP/MORF) boxes, which are required for ORRM1 to interact with site-specific pentatricopeptide repeat protein editing factors. ORRM6 interacts with RIP1/MORF8, RIP2/MORF2, and RIP9/MORF9, known components of RNA editosomes. While some plastid RRM proteins are involved in other forms of RNA processing and translation, the primary function of ORRM6 is evidently to mediate psbF-C77 editing, like the essential site-specific pentatricopeptide repeat protein LOW PSII ACCUMULATION66. Stable expression in the orrm6 mutants of a nucleus-encoded, plastid-targeted PsbF protein from a psbF gene carrying a T at nucleotide 77 significantly increases leaf and plant sizes, chlorophyll content, and PSII activity. These transformants demonstrate that plastid RNA editing can be bypassed through the expression of nucleus-encoded, edited forms of plastid genes.

  18. Anaerobic survival of Pseudomonas aeruginosa by pyruvate fermentation requires an Usp-type stress protein

    DEFF Research Database (Denmark)

    Schreiber, K; Boes, N; Escbach, M;

    2006-01-01

    Recently, we identified a pyruvate fermentation pathway in Pseudomonas aeruginosa sustaining anaerobic survival in the absence of alternative anaerobic respiratory and fermentative energy generation systems (M. Eschbach, K. Schreiber, K. Trunk, J. Buer, D. Jahn, and M. Schobert, J. Bacteriol. 186......:4596-4604, 2004). Anaerobic long-term survival of P. aeruginosa might be essential for survival in deeper layers of a biofilm and the persistent infection of anaerobic mucus plaques in the cystic fibrosis lung. Proteome analysis of P. aeruginosa cells during a 7-day period of pyruvate fermentation revealed...... the induced synthesis of three enzymes involved in arginine fermentation, ArcA, ArcB, and ArcC, and the outer membrane protein OprL. Moreover, formation of two proteins of unknown function, PA3309 and PA4352, increased by factors of 72- and 22-fold, respectively. Both belong to the group of universal stress...

  19. Protein turnover and plant RNA and phosphorus requirements in relation to nitrogen fixation.

    Science.gov (United States)

    Raven, John A

    2012-06-01

    Phosphorus (P) is the proximate (immediate) limiting element for primary productivity in some habitats, and is generally the ultimate limiting element for primary productivity. Although RNA can account for over half of the non-storage P in photosynthetic organisms, some primary producers have more ribosomes than the minimum needed for the observed rate of net protein synthesis; some of this RNA may be needed for protein turnover. Two cases of protein turnover which can occur at a much faster rate than the bulk protein turnover are those of photodamaged photosystem II and O(2)-damaged nitrogenase. While RNA involved in photosystem II repair accounts for less than 1% of the non-storage P in photosynthetic organisms, a maximum, of 12% of non-storage P could occur in RNA associated with replacement of damaged nitrogenase and/or O(2) damage avoidance mechanism in diazotrophic (N(2) fixing) organisms. There is a general trend in published data towards lower P use efficiency (g dry matter gain per day per mol P in the organism) for photosynthetic diazotrophic organisms growing under P limitation with N(2) as their nitrogen source, rather than with NH(4)(+), urea or NO(3)(-). Additional work is needed to examine the generality of a statistically verified decrease in P use efficiency for diazotrophic growth relative to growth on other nitrogen sources and, if this is confirmed, further investigation of the mechanism is needed. The outcome of such work would be important for relating the global distribution of diazotrophy to P availability. There are no known P acquisition mechanisms specific to diazotrophs. Phosphorus (P) is the proximate (immediate) limiting element for primary productivity in some habitats, and is generally the ultimate limiting element for primary productivity. Although RNA can account for over half of the non-storage P in photosynthetic organisms, some primary producers have more ribosomes than the minimum needed for the observed rate of net protein

  20. Insulin Is Required to Maintain Albumin Expression by Inhibiting Forkhead Box O1 Protein.

    Science.gov (United States)

    Chen, Qing; Lu, Mingjian; Monks, Bobby R; Birnbaum, Morris J

    2016-01-29

    Diabetes is accompanied by dysregulation of glucose, lipid, and protein metabolism. In recent years, much effort has been spent on understanding how insulin regulates glucose and lipid metabolism, whereas the effect of insulin on protein metabolism has received less attention. In diabetes, hepatic production of serum albumin decreases, and it has been long established that insulin positively controls albumin gene expression. In this study, we used a genetic approach in mice to identify the mechanism by which insulin regulates albumin gene transcription. Albumin expression was decreased significantly in livers with insulin signaling disrupted by ablation of the insulin receptor or Akt. Concomitant deletion of Forkhead Box O1 (Foxo1) in these livers rescued the decreased albumin secretion. Furthermore, activation of Foxo1 in the liver is sufficient to suppress albumin expression. These results suggest that Foxo1 acts as a repressor of albumin expression.

  1. Protein Tpr is required for establishing nuclear pore-associated zones of heterochromatin exclusion.

    Science.gov (United States)

    Krull, Sandra; Dörries, Julia; Boysen, Björn; Reidenbach, Sonja; Magnius, Lars; Norder, Helene; Thyberg, Johan; Cordes, Volker C

    2010-05-19

    Amassments of heterochromatin in somatic cells occur in close contact with the nuclear envelope (NE) but are gapped by channel- and cone-like zones that appear largely free of heterochromatin and associated with the nuclear pore complexes (NPCs). To identify proteins involved in forming such heterochromatin exclusion zones (HEZs), we used a cell culture model in which chromatin condensation induced by poliovirus (PV) infection revealed HEZs resembling those in normal tissue cells. HEZ occurrence depended on the NPC-associated protein Tpr and its large coiled coil-forming domain. RNAi-mediated loss of Tpr allowed condensing chromatin to occur all along the NE's nuclear surface, resulting in HEZs no longer being established and NPCs covered by heterochromatin. These results assign a central function to Tpr as a determinant of perinuclear organization, with a direct role in forming a morphologically distinct nuclear sub-compartment and delimiting heterochromatin distribution.

  2. Rsp5 ubiquitin ligase is required for protein trafficking in Saccharomyces cerevisiae COPI mutants.

    Directory of Open Access Journals (Sweden)

    Katarzyna Jarmoszewicz

    Full Text Available Retrograde trafficking from the Golgi to the endoplasmic reticulum (ER depends on the formation of vesicles coated with the multiprotein complex COPI. In Saccharomyces cerevisiae ubiquitinated derivatives of several COPI subunits have been identified. The importance of this modification of COPI proteins is unknown. With the exception of the Sec27 protein (β'COP neither the ubiquitin ligase responsible for ubiquitination of COPI subunits nor the importance of this modification are known. Here we find that the ubiquitin ligase mutation, rsp5-1, has a negative effect that is additive with ret1-1 and sec28Δ mutations, in genes encoding α- and ε-COP, respectively. The double ret1-1 rsp5-1 mutant is also more severely defective in the Golgi-to-ER trafficking compared to the single ret1-1, secreting more of the ER chaperone Kar2p, localizing Rer1p mostly to the vacuole, and increasing sensitivity to neomycin. Overexpression of ubiquitin in ret1-1 rsp5-1 mutant suppresses vacuolar accumulation of Rer1p. We found that the effect of rsp5 mutation on the Golgi-to-ER trafficking is similar to that of sla1Δ mutation in a gene encoding actin cytoskeleton proteins, an Rsp5p substrate. Additionally, Rsp5 and Sla1 proteins were found by co-immunoprecipitation in a complex containing COPI subunits. Together, our results show that Rsp5 ligase plays a role in regulating retrograde Golgi-to-ER trafficking.

  3. Sec24D-dependent transport of extracellular matrix proteins is required for zebrafish skeletal morphogenesis.

    Directory of Open Access Journals (Sweden)

    Swapnalee Sarmah

    Full Text Available Protein transport from endoplasmic reticulum (ER to Golgi is primarily conducted by coated vesicular carriers such as COPII. Here, we describe zebrafish bulldog mutations that disrupt the function of the cargo adaptor Sec24D, an integral component of the COPII complex. We show that Sec24D is essential for secretion of cartilage matrix proteins, whereas the preceding development of craniofacial primordia and pre-chondrogenic condensations does not depend on this isoform. Bulldog chondrocytes fail to secrete type II collagen and matrilin to extracellular matrix (ECM, but membrane bound receptor beta1-Integrin and Cadherins appear to leave ER in Sec24D-independent fashion. Consequently, Sec24D-deficient cells accumulate proteins in the distended ER, although a subset of ER compartments and Golgi complexes as visualized by electron microscopy and NBD C(6-ceramide staining appear functional. Consistent with the backlog of proteins in the ER, chondrocytes activate the ER stress response machinery and significantly upregulate BiP transcription. Failure of ECM secretion hinders chondroblast intercalations thus resulting in small and malformed cartilages and severe craniofacial dysmorphology. This defect is specific to Sec24D mutants since knockdown of Sec24C, a close paralog of Sec24D, does not result in craniofacial cartilage dysmorphology. However, craniofacial development in double Sec24C/Sec24D-deficient animals is arrested earlier than in bulldog/sec24d, suggesting that Sec24C can compensate for loss of Sec24D at initial stages of chondrogenesis, but Sec24D is indispensable for chondrocyte maturation. Our study presents the first developmental perspective on Sec24D function and establishes Sec24D as a strong candidate for cartilage maintenance diseases and craniofacial birth defects.

  4. Arabidopsis R-SNARE proteins VAMP721 and VAMP722 are required for cell plate formation.

    Directory of Open Access Journals (Sweden)

    Liang Zhang

    Full Text Available BACKGROUND: Cell plate formation during plant cytokinesis is facilitated by SNARE complex-mediated vesicle fusion at the cell-division plane. However, our knowledge regarding R-SNARE components of membrane fusion machinery for cell plate formation remains quite limited. METHODOLOGY/PRINCIPAL FINDINGS: We report the in vivo function of Arabidopsis VAMP721 and VAMP722, two closely sequence-related R-SNAREs, in cell plate formation. Double homozygous vamp721vamp722 mutant seedlings showed lethal dwarf phenotypes and were characterized by rudimentary roots, cotyledons and hypocotyls. Furthermore, cell wall stubs and incomplete cytokinesis were frequently observed in vamp721vamp722 seedlings. Confocal images revealed that green fluorescent protein-tagged VAMP721 and VAMP722 were preferentially localized to the expanding cell plates in dividing cells. Drug treatments and co-localization analyses demonstrated that punctuate organelles labeled with VAMP721 and VAMP722 represented early endosomes overlapped with VHA-a1-labeled TGN, which were distinct from Golgi stacks and prevacuolar compartments. In addition, protein traffic to the plasma membrane, but not to the vacuole, was severely disrupted in vamp721vamp722 seedlings by subcellular localization of marker proteins. CONCLUSION/SIGNIFICANCE: These observations suggest that VAMP721 and VAMP722 are involved in secretory trafficking to the plasma membrane via TGN/early endosomal compartment, which contributes substantially to cell plate formation during plant cytokinesis.

  5. Transport of the outer dynein arm complex to cilia requires a cytoplasmic protein Lrrc6.

    Science.gov (United States)

    Inaba, Yasuko; Shinohara, Kyosuke; Botilde, Yanick; Nabeshima, Ryo; Takaoka, Katsuyoshi; Ajima, Rieko; Lamri, Lynda; Takeda, Hiroyuki; Saga, Yumiko; Nakamura, Tetsuya; Hamada, Hiroshi

    2016-07-01

    Lrrc6 encodes a cytoplasmic protein that is expressed specifically in cells with motile cilia including the node, trachea and testes of the mice. A mutation of Lrrc6 has been identified in human patients with primary ciliary dyskinesia (PCD). Mutant mice lacking Lrrc6 show typical PCD defects such as hydrocephalus and laterality defects. We found that in the absence of Lrrc6, the morphology of motile cilia remained normal, but their motility was completely lost. The 9 + 2 arrangement of microtubules remained normal in Lrrc6(-/-) mice, but the outer dynein arms (ODAs), the structures essential for the ciliary beating, were absent from the cilia. In the absence of Lrrc6, ODA proteins such as DNAH5, DNAH9 and IC2, which are assembled in the cytoplasm and transported to the ciliary axoneme, remained in the cytoplasm and were not transported to the ciliary axoneme. The IC2-IC1 interaction, which is the first step of ODA assembly, was normal in Lrrc6(-/-) mice testes. Our results suggest that ODA proteins may be transported from the cytoplasm to the cilia by an Lrrc6-dependent mechanism.

  6. Requirements on paramagnetic relaxation enhancement data for membrane protein structure determination by NMR.

    Science.gov (United States)

    Gottstein, Daniel; Reckel, Sina; Dötsch, Volker; Güntert, Peter

    2012-06-06

    Nuclear magnetic resonance (NMR) structure calculations of the α-helical integral membrane proteins DsbB, GlpG, and halorhodopsin show that distance restraints from paramagnetic relaxation enhancement (PRE) can provide sufficient structural information to determine their structure with an accuracy of about 1.5 Å in the absence of other long-range conformational restraints. Our systematic study with simulated NMR data shows that about one spin label per transmembrane helix is necessary for obtaining enough PRE distance restraints to exclude wrong topologies, such as pseudo mirror images, if only limited other NMR restraints are available. Consequently, an experimentally realistic amount of PRE data enables α-helical membrane protein structure determinations that would not be feasible with the very limited amount of conventional NOESY data normally available for these systems. These findings are in line with our recent first de novo NMR structure determination of a heptahelical integral membrane protein, proteorhodopsin, that relied extensively on PRE data.

  7. Sorbitol dehydrogenase is a cytosolic protein required for sorbitol metabolism in Arabidopsis thaliana.

    Science.gov (United States)

    Aguayo, María Francisca; Ampuero, Diego; Mandujano, Patricio; Parada, Roberto; Muñoz, Rodrigo; Gallart, Marta; Altabella, Teresa; Cabrera, Ricardo; Stange, Claudia; Handford, Michael

    2013-05-01

    Sorbitol is converted to fructose in Rosaceae species by SORBITOL DEHYDROGENASE (SDH, EC 1.1.1.14), especially in sink organs. SDH has also been found in non-Rosaceae species and here we show that the protein encoded by At5g51970 in Arabidopsis thaliana (L.) Heynh. possesses the molecular characteristics of an SDH. Using a green fluorescent protein-tagged version and anti-SDH antisera, we determined that SDH is cytosolically localized, consistent with bioinformatic predictions. We also show that SDH is widely expressed, and that SDH protein accumulates in both source and sink organs. In the presence of NAD+, recombinant SDH exhibited greatest oxidative activity with sorbitol, ribitol and xylitol as substrates; other sugar alcohols were oxidized to a lesser extent. Under standard growth conditions, three independent sdh- mutants developed as wild-type. Nevertheless, all three exhibited reduced dry weight and primary root length compared to wild-type when grown in the presence of sorbitol. Additionally, under short-day conditions, the mutants were more resistant to dehydration stress, as shown by a reduced loss of leaf water content when watering was withheld, and a greater survival rate on re-watering. This evidence suggests that limitations in the metabolism of sugar alcohols alter the growth of Arabidopsis and its response to drought.

  8. Requirement for sex comb on midleg protein interactions in Drosophila polycomb group repression.

    Science.gov (United States)

    Peterson, Aidan J; Mallin, Daniel R; Francis, Nicole J; Ketel, Carrie S; Stamm, Joyce; Voeller, Rochus K; Kingston, Robert E; Simon, Jeffrey A

    2004-07-01

    The Drosophila Sex Comb on Midleg (SCM) protein is a transcriptional repressor of the Polycomb group (PcG). Although genetic studies establish SCM as a crucial PcG member, its molecular role is not known. To investigate how SCM might link to PcG complexes, we analyzed the in vivo role of a conserved protein interaction module, the SPM domain. This domain is found in SCM and in another PcG protein, Polyhomeotic (PH), which is a core component of Polycomb repressive complex 1 (PRC1). SCM-PH interactions in vitro are mediated by their respective SPM domains. Yeast two-hybrid and in vitro binding assays were used to isolate and characterize >30 missense mutations in the SPM domain of SCM. Genetic rescue assays showed that SCM repressor function in vivo is disrupted by mutations that impair SPM domain interactions in vitro. Furthermore, overexpression of an isolated, wild-type SPM domain produced PcG loss-of-function phenotypes in flies. Coassembly of SCM with a reconstituted PRC1 core complex shows that SCM can partner with PRC1. However, gel filtration chromatography showed that the bulk of SCM is biochemically separable from PH in embryo nuclear extracts. These results suggest that SCM, although not a core component of PRC1, interacts and functions with PRC1 in gene silencing.

  9. Mitogen-activated protein kinase pathways are required for melatonin-mediated defense responses in plants.

    Science.gov (United States)

    Lee, Hyoung Yool; Back, Kyoungwhan

    2016-04-01

    Melatonin enhances pathogen resistance by inducing the expression of a number of plant defense-related genes. To examine whether the melatonin-mediated pathogen resistance is associated with mitogen-activated protein kinase (MAPK) cascades, Arabidopsis and tobacco leaves were treated with melatonin and investigated for MAPK activation using an antiphospho-p44/42 MAPK (Erk1/2) monoclonal antibody. Two MAPKs, MPK3 and MPK6, were activated rapidly and transiently by 1 μm melatonin treatment in Arabidopsis. Its tobacco ortholog MAPKs were also activated. The activation of MPK3 and MPK6 by 2-hydroxymelatonin and N-acetylserotonin was also observed, albeit to a lesser degree than that by melatonin. Furthermore, MAPK activation by melatonin was uncoupled from G-protein signaling, because melatonin efficiently activated two MAPKs in a G-protein β knockout mutant (agb1). Suppression of both MPK3 and MPK6 in transgenic Arabidopsis exhibited significant decreases in the induction of defense-related gene expression and pathogen resistance relative to wild-type plants. Using an array of MAP kinase kinase (MKK) knockout mutants, we found that four MKKs, namely MKK4, MKK5, MKK7, and MKK9, are responsible for the activation of MPK3 and MPK6 by melatonin, indicating that melatonin-mediated innate immunity is triggered by MAPK signaling through MKK4/5/7/9-MPK3/6 cascades.

  10. 40 CFR 174.519 - Bacillus thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry2Ab2 protein... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.519 Bacillus... Bacillus thuringiensis Cry2Ab2 protein in or on corn or cotton are exempt from the requirement of...

  11. 40 CFR 174.501 - Bacillus thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Vip3Aa protein... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.501 Bacillus... Bacillus thuringiensis Vip3Aa proteins in or on corn or cotton are exempt from the requirement of...

  12. The Timing of Multiple Retrieval Events Can Alter GluR1 Phosphorylation and the Requirement for Protein Synthesis in Fear Memory Reconsolidation

    Science.gov (United States)

    Jarome, Timothy J.; Kwapis, Janine L.; Werner, Craig T.; Parsons, Ryan G.; Gafford, Georgette M.; Helmstetter, Fred J.

    2012-01-01

    Numerous studies have indicated that maintaining a fear memory after retrieval requires de novo protein synthesis. However, no study to date has examined how the temporal dynamics of repeated retrieval events affect this protein synthesis requirement. The present study varied the timing of a second retrieval of an established auditory fear memory…

  13. Phosphatidylinositol and phosphatidic acid transport between the ER and plasma membrane during PLC activation requires the Nir2 protein.

    Science.gov (United States)

    Kim, Yeun Ju; Guzman-Hernandez, Maria Luisa; Wisniewski, Eva; Echeverria, Nicolas; Balla, Tamas

    2016-02-01

    Phospholipase C (PLC)-mediated hydrolysis of the limited pool of plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] requires replenishment from a larger pool of phosphatidylinositol (PtdIns) via sequential phosphorylation by PtdIns 4-kinases and phosphatidylinositol 4-phosphate (PtdIns4P) 5-kinases. Since PtdIns is synthesized in the endoplasmic reticulum (ER) and PtdIns(4,5)P2 is generated in the PM, it has been postulated that PtdIns transfer proteins (PITPs) provide the means for this lipid transfer function. Recent studies identified the large PITP protein, Nir2 as important for PtdIns transfer from the ER to the PM. It was also found that Nir2 was required for the transfer of phosphatidic acid (PtdOH) from the PM to the ER. In Nir2-depleted cells, activation of PLC leads to PtdOH accumulation in the PM and PtdIns synthesis becomes severely impaired. In quiescent cells, Nir2 is localized to the ER via interaction of its FFAT domain with ER-bound VAMP-associated proteins VAP-A and-B. After PLC activation, Nir2 also binds to the PM via interaction of its C-terminal domains with diacylglycerol (DAG) and PtdOH. Through these interactions, Nir2 functions in ER-PM contact zones. Mutations in VAP-B that have been identified in familial forms of amyotrophic lateral sclerosis (ALS or Lou-Gehrig's disease) cause aggregation of the VAP-B protein, which then impairs its binding to several proteins, including Nir2. These findings have shed new lights on the importance of non-vesicular lipid transfer of PtdIns and PtdOH in ER-PM contact zones with a possible link to a devastating human disease.

  14. 77 FR 47287 - Bacillus thuringiensis eCry3.1Ab Protein in Corn; Exemption From the Requirement of a Tolerance

    Science.gov (United States)

    2012-08-08

    ... AGENCY 40 CFR Part 174 Bacillus thuringiensis eCry3.1Ab Protein in Corn; Exemption From the Requirement...-incorporated protectant (PIP), Bacillus thuringiensis eCry3.1Ab protein in corn, in or on the food and feed... permissible level for residues of Bacillus thuringiensis eCry3.1Ab protein in corn. DATES: This regulation...

  15. Solution structure of the Equine Infectious Anemia Virus p9 protein: a rationalization of its different ALIX binding requirements compared to the analogous HIV-p6 protein

    Directory of Open Access Journals (Sweden)

    Henklein Peter

    2009-12-01

    Full Text Available Abstract Background The equine infection anemia virus (EIAV p9 Gag protein contains the late (L- domain required for efficient virus release of nascent virions from the cell membrane of infected cell. Results In the present study the p9 protein and N- and C-terminal fragments (residues 1-21 and 22-51, respectively were chemically synthesized and used for structural analyses. Circular dichroism and 1H-NMR spectroscopy provide the first molecular insight into the secondary structure and folding of this 51-amino acid protein under different solution conditions. Qualitative 1H-chemical shift and NOE data indicate that in a pure aqueous environment p9 favors an unstructured state. In its most structured state under hydrophobic conditions, p9 adopts a stable helical structure within the C-terminus. Quantitative NOE data further revealed that this α-helix extends from Ser-27 to Ser-48, while the N-terminal residues remain unstructured. The structural elements identified for p9 differ substantially from that of the functional homologous HIV-1 p6 protein. Conclusions These structural differences are discussed in the context of the different types of L-domains regulating distinct cellular pathways in virus budding. EIAV p9 mediates virus release by recruiting the ALG2-interacting protein X (ALIX via the YPDL-motif to the site of virus budding, the counterpart of the YPXnL-motif found in p6. However, p6 contains an additional PTAP L-domain that promotes HIV-1 release by binding to the tumor susceptibility gene 101 (Tsg101. The notion that structures found in p9 differ form that of p6 further support the idea that different mechanisms regulate binding of ALIX to primary versus secondary L-domains types.

  16. Analysis of Phosphorylation-dependent Protein Interactions of Adhesion and Degranulation Promoting Adaptor Protein (ADAP) Reveals Novel Interaction Partners Required for Chemokine-directed T cell Migration.

    Science.gov (United States)

    Kuropka, Benno; Witte, Amelie; Sticht, Jana; Waldt, Natalie; Majkut, Paul; Hackenberger, Christian P R; Schraven, Burkhart; Krause, Eberhard; Kliche, Stefanie; Freund, Christian

    2015-11-01

    Stimulation of T cells leads to distinct changes of their adhesive and migratory properties. Signal propagation from activated receptors to integrins depends on scaffolding proteins such as the adhesion and degranulation promoting adaptor protein (ADAP)(1). Here we have comprehensively investigated the phosphotyrosine interactome of ADAP in T cells and define known and novel interaction partners of functional relevance. While most phosphosites reside in unstructured regions of the protein, thereby defining classical SH2 domain interaction sites for master regulators of T cell signaling such as SLP76, Fyn-kinase, and NCK, other binding events depend on structural context. Interaction proteomics using different ADAP constructs comprising most of the known phosphotyrosine motifs as well as the structured domains confirm that a distinct set of proteins is attracted by pY571 of ADAP, including the ζ-chain-associated protein kinase of 70 kDa (ZAP70). The interaction of ADAP and ZAP70 is inducible upon stimulation either of the T cell receptor (TCR) or by chemokine. NMR spectroscopy reveals that the N-terminal SH2 domains within a ZAP70-tandem-SH2 construct is the major site of interaction with phosphorylated ADAP-hSH3(N) and microscale thermophoresis (MST) indicates an intermediate binding affinity (Kd = 2.3 μm). Interestingly, although T cell receptor dependent events such as T cell/antigen presenting cell (APC) conjugate formation and adhesion are not affected by mutation of Y571, migration of T cells along a chemokine gradient is compromised. Thus, although most phospho-sites in ADAP are linked to T cell receptor related functions we have identified a unique phosphotyrosine that is solely required for chemokine induced T cell behavior.

  17. SGT1 interacts with the Prf resistance protein and is required for Prf accumulation and Prf-mediated defense signaling.

    Science.gov (United States)

    Kud, Joanna; Zhao, Zhulu; Du, Xinran; Liu, Yule; Zhao, Yun; Xiao, Fangming

    2013-02-15

    The highly conserved eukaryotic co-chaperone SGT1 (suppressor of the G2 allele of skp1) is an important signaling component of plant defense responses and positively regulates disease resistance conferred by many resistance (R) proteins. In this study, we investigated the contribution of SGT1 in the Prf-mediated defense responses in both Nicotiana benthamiana and tomato (Solanum lycopersicum). SGT1 was demonstrated to interact with Prf in plant cells by co-immunoprecipitation. The requirement of SGT1 in the accumulation of Prf or autoactive Prf(D1416V) was determined by the degradation of these proteins in N. benthamiana, in which SGT1 was repressed by virus-induced gene silencing (VIGS). Pseudomonas pathogen assay on the SGT1-silenced tomato plants implicates SGT1 is required for the Prf-mediated full resistance to Pseudomonas syringae pv. tomato (Pst). These results suggest that, in both N. benthamiana and tomato, SGT1 contributes to the Prf-mediated defense responses by stabilizing Prf protein via its co-chaperone activity.

  18. Maintenance of muscle myosin levels in adult C. elegans requires both the double bromodomain protein BET-1 and sumoylation

    Directory of Open Access Journals (Sweden)

    Kate Fisher

    2013-10-01

    Attenuation of RAS-mediated signalling is a conserved process essential to control cell proliferation, differentiation, and apoptosis. Cooperative interactions between histone modifications such as acetylation, methylation and sumoylation are crucial for proper attenuation in C. elegans, implying that the proteins recognising these histone modifications could also play an important role in attenuation of RAS-mediated signalling. We sought to systematically identify these proteins and found BET-1. BET-1 is a conserved double bromodomain protein that recognises acetyl-lysines on histone tails and maintains the stable fate of various lineages. Unexpectedly, adults lacking both BET-1 and SUMO-1 are depleted of muscle myosin, an essential component of myofibrils. We also show that this muscle myosin depletion does not occur in all animals at a specific time, but rather that the penetrance of the phenotype increases with age. To gain mechanistic insights into this process, we sought to delay the occurrence of the muscle myosin depletion phenotype and found that it requires caspase activity and MEK-dependent signalling. We also performed transcription profiling on these mutants and found an up-regulation of the FGF receptor, egl-15, a tyrosine kinase receptor acting upstream of MEK. Consistent with a MEK requirement, we could delay the muscle phenotype by systemic or hypodermal knock down of egl-15. Thus, this work uncovered a caspase- and MEK-dependent mechanism that acts specifically on ageing adults to maintain the appropriate net level of muscle myosin.

  19. BARREN STALK FASTIGIATE1 is an AT-hook protein required for the formation of maize ears.

    Science.gov (United States)

    Gallavotti, Andrea; Malcomber, Simon; Gaines, Craig; Stanfield, Sharon; Whipple, Clinton; Kellogg, Elizabeth; Schmidt, Robert J

    2011-05-01

    Ears are the seed-bearing inflorescences of maize (Zea mays) plants and represent a crucial component of maize yield. The first step in the formation of ears is the initiation of axillary meristems in the axils of developing leaves. In the classic maize mutant barren stalk fastigiate1 (baf1), first discovered in the 1950s, ears either do not form or, if they do, are partially fused to the main stalk. We positionally cloned Baf1 and found that it encodes a transcriptional regulator containing an AT-hook DNA binding motif. Single coorthologs of Baf1 are found in syntenic regions of brachypodium (Brachypodium distachyon), rice (Oryza sativa), and sorghum (Sorghum bicolor), suggesting that the gene is likely present in all cereal species. Protein-protein interaction assays suggest that BAF1 is capable of forming homodimers and heterodimers with other members of the AT-hook family. Another transcriptional regulator required for ear initiation is the basic helix-loop-helix protein BARREN STALK1 (BA1). Genetic and expression analyses suggest that Baf1 is required to reach a threshold level of Ba1 expression for the initiation of maize ears. We propose that Baf1 functions in the demarcation of a boundary region essential for the specification of a stem cell niche.

  20. Protein

    Science.gov (United States)

    ... Food Service Resources Additional Resources About FAQ Contact Protein Protein is found throughout the body—in muscle, ... the heart and respiratory system, and death. All Protein Isn’t Alike Protein is built from building ...

  1. Production of recombinant snakehead rhabdovirus: the NV protein is not required for viral replication.

    Science.gov (United States)

    Johnson, M C; Simon, B E; Kim, C H; Leong, J A

    2000-03-01

    Snakehead rhabdovirus (SHRV) affects warm water fish in Southeast Asia and belongs to the genus Novirhabdovirus by virtue of its nonvirion gene (NV). Because SHRV grows best at temperatures between 28 and 31 degrees C, we were able to use the T7 expression system to produce viable recombinant SHRV from a cloned cDNA copy of the viral genome. Expression of a positive-strand RNA copy of the 11, 550-nucleotide SHRV genome along with the viral nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins resulted in the generation of infectious SHRV in cells preinfected with a vaccinia virus vector for T7 polymerase expression. Recombinant virus production was verified by detection of a unique restriction site engineered into the SHRV genome between the NV and L genes. Since we were now able to begin examining the function of the NV gene, we constructed a recombinant virus containing a nonsense mutation located 22 codons into the coding sequence of the NV protein. The NV knockout virus was produced at a concentration as high as that of wild-type virus in cultured fish cells, and the resulting virions appeared to be identical to the wild-type virions in electron micrographs. These initial studies suggest that NV has no critical function in SHRV replication in cultured fish cells.

  2. Survival Motor Neuron (SMN) protein is required for normal mouse liver development

    Science.gov (United States)

    Szunyogova, Eva; Zhou, Haiyan; Maxwell, Gillian K.; Powis, Rachael A.; Francesco, Muntoni; Gillingwater, Thomas H.; Parson, Simon H.

    2016-01-01

    Spinal Muscular Atrophy (SMA) is caused by mutation or deletion of the survival motor neuron 1 (SMN1) gene. Decreased levels of, cell-ubiquitous, SMN protein is associated with a range of systemic pathologies reported in severe patients. Despite high levels of SMN protein in normal liver, there is no comprehensive study of liver pathology in SMA. We describe failed liver development in response to reduced SMN levels, in a mouse model of severe SMA. The SMA liver is dark red, small and has: iron deposition; immature sinusoids congested with blood; persistent erythropoietic elements and increased immature red blood cells; increased and persistent megakaryocytes which release high levels of platelets found as clot-like accumulations in the heart. Myelopoiesis in contrast, was unaffected. Further analysis revealed significant molecular changes in SMA liver, consistent with the morphological findings. Antisense treatment from birth with PMO25, increased lifespan and ameliorated all morphological defects in liver by postnatal day 21. Defects in the liver are evident at birth, prior to motor system pathology, and impair essential liver function in SMA. Liver is a key recipient of SMA therapies, and systemically delivered antisense treatment, completely rescued liver pathology. Liver therefore, represents an important therapeutic target in SMA. PMID:27698380

  3. The NG2 Protein Is Not Required for Glutamatergic Neuron-NG2 Cell Synaptic Signaling.

    Science.gov (United States)

    Passlick, Stefan; Trotter, Jacqueline; Seifert, Gerald; Steinhäuser, Christian; Jabs, Ronald

    2016-01-01

    NG2 glial cells (as from now NG2 cells) are unique in receiving synaptic input from neurons. However, the components regulating formation and maintenance of these neuron-glia synapses remain elusive. The transmembrane protein NG2 has been considered a potential mediator of synapse formation and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) clustering, because it contains 2 extracellular Laminin G/Neurexin/Sex Hormone-Binding Globulin domains, which in neurons are crucial for formation of transsynaptic neuroligin-neurexin complexes. NG2 is connected via Glutamate Receptor-Interacting Protein with GluA2/3-containing AMPARs, thereby possibly mediating receptor clustering in glial postsynaptic density. To elucidate the role of NG2 in neuron-glia communication, we investigated glutamatergic synaptic transmission in juvenile and aged hippocampal NG2 cells of heterozygous and homozygous NG2 knockout mice. Neuron-NG2 cell synapses readily formed in the absence of NG2. Short-term plasticity, synaptic connectivity, postsynaptic AMPAR current kinetics, and density were not affected by NG2 deletion. During development, an NG2-independent acceleration of AMPAR current kinetics and decreased synaptic connectivity were observed. Our results indicate that the lack of NG2 does not interfere with genesis and basic properties of neuron-glia synapses. In addition, we demonstrate frequent expression of neuroligins 1-3 in juvenile and aged NG2 cells, suggesting a role of these molecules in synapse formation between NG2 glia and neurons.

  4. The adapter protein ADAP is required for selected dendritic cell functions

    Directory of Open Access Journals (Sweden)

    Togni Mauro

    2012-06-01

    Full Text Available Abstract Background The cytosolic adaptor protein ADAP (adhesion and degranulation promoting adapter protein is expressed by T cells, natural killer cells, myeloid cells and platelets. ADAP is involved in T-cell-receptor-mediated inside-out signaling, which leads to integrin activation, adhesion and reorganization of the actin cytoskeleton. However, little is known about the role of ADAP in myeloid cells. In the present study, we analyzed the function of ADAP in bone-marrow-derived dendritic cells (BMDCs from ADAP-deficient mice. Results ADAP-deficient BMDCs showed almost normal levels of antigen uptake, adhesion, maturation, migration from the periphery to the draining lymph nodes, antigen-specific T-cell activation, and production of the proinflammatory cytokines IL-6 and TNF-∝. Furthermore, we provide evidence that the activation of signaling pathways after lipopolysaccharide (LPS stimulation are not affected by the loss of ADAP. In contrast, ADAP-deficient BMDCs showed defects in CD11c-mediated cellular responses, with significantly diminished production of IL-6, TNF-∝ and IL-10. Actin polymerization was enhanced after CD11c integrin stimulation. Conclusions In summary, we propose that the adapter molecule ADAP is critical for selected CD11c integrin-mediated functions of dendritic cells.

  5. Histone acetylation and CREB binding protein are required for neuronal resistance against ischemic injury.

    Directory of Open Access Journals (Sweden)

    Ferah Yildirim

    Full Text Available Epigenetic transcriptional regulation by histone acetylation depends on the balance between histone acetyltransferase (HAT and deacetylase activities (HDAC. Inhibition of HDAC activity provides neuroprotection, indicating that the outcome of cerebral ischemia depends crucially on the acetylation status of histones. In the present study, we characterized the changes in histone acetylation levels in ischemia models of focal cerebral ischemia and identified cAMP-response element binding protein (CREB-binding protein (CBP as a crucial factor in the susceptibility of neurons to ischemic stress. Both neuron-specific RNA interference and neurons derived from CBP heterozygous knockout mice showed increased damage after oxygen-glucose deprivation (OGD in vitro. Furthermore, we demonstrated that ischemic preconditioning by a short (5 min subthreshold occlusion of the middle cerebral artery (MCA, followed 24 h afterwards by a 30 min occlusion of the MCA, increased histone acetylation levels in vivo. Ischemic preconditioning enhanced CBP recruitment and histone acetylation at the promoter of the neuroprotective gene gelsolin leading to increased gelsolin expression in neurons. Inhibition of CBP's HAT activity attenuated neuronal ischemic preconditioning. Taken together, our findings suggest that the levels of CBP and histone acetylation determine stroke outcome and are crucially associated with the induction of an ischemia-resistant state in neurons.

  6. Sperm postacrosomal WW domain-binding protein is not required for mouse egg activation.

    Science.gov (United States)

    Satouh, Yuhkoh; Nozawa, Kaori; Ikawa, Masahito

    2015-10-01

    To begin embryonic development, the zygote must resume the cell cycle correctly after stimulation by sperm-borne oocyte-activating factors (SOAFs). The postacrosomal WW domain-binding protein (PAWP) is one of the strongest SOAF candidates and is widely conserved among eutherian mammals. It has been reported that the microinjection of recombinant PAWP protein can trigger not only Ca(2+) oscillations in mammalian eggs but also intracellular Ca(2+) release in amphibian eggs. It was also suggested that PAWP is involved in the formation of high-quality spermatozoa. On the other hand, negligible SOAF activity for PAWP cRNA has also been reported. In this study, we generated PAWP null mice and examined the fertilizing ability of male mice. Electron microscopy showed no aberrant morphology in spermatogenesis. Intracytoplasmic injection of a single spermatozoon from the null mouse line showed that depletion of PAWP elicited no quantitative differences in Ca(2+) oscillations or in subsequent development of the embryos. We conclude that PAWP does not play an essential role in mouse fertilization.

  7. 15-zinc finger protein Bloody Fingers is required for zebrafish morphogenetic movements during neurulation.

    Science.gov (United States)

    Sumanas, Saulius; Zhang, Bo; Dai, Rujuan; Lin, Shuo

    2005-07-01

    A novel zebrafish gene bloody fingers (blf) encoding a 478 amino acid protein containing fifteen C(2)H(2) type zinc fingers was identified by expression screening. As determined by in situ hybridization, blf RNA displays strong ubiquitous early zygotic expression, while during late gastrulation and early somitogenesis, blf expression becomes transiently restricted to the posterior dorsal and lateral mesoderm. During later somitogenesis, blf expression appears only in hematopoietic cells. It is completely eliminated in cloche, moonshine but not in vlad tepes (gata1) mutant embryos. Morpholino (MO) knockdown of the Blf protein results in the defects of morphogenetic movements. Blf-MO-injected embryos (morphants) display shortened and widened axial tissues due to defective convergent extension. Unlike other convergent extension mutants, blf morphants display a split neural tube, resulting in a phenotype similar to the human open neural tube defect spina bifida. In addition, dorsal ectodermal cells delaminate in blf morphants during late somitogenesis. We propose a model explaining the role of blf in convergent extension and neurulation. We conclude that blf plays an important role in regulating morphogenetic movements during gastrulation and neurulation while its role in hematopoiesis may be redundant.

  8. A novel chloroplast-localized protein EMB1303 is required for chloroplast development in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Xiaozhen Huang; Xiaoyan Zhang; Shuhua Yang

    2009-01-01

    To understand the molecular mechanisms underlying chloroplast development, we isolated and characterized the albino mutant emb1303-1 in Arabidopsis. The mutant displayed a severe dwarf phenotype with small albino rosette leaves and short roots on a synthetic medium containing sucrose. It is pigment-deficient and seedling lethal when grown in soil. Embryo development was delayed in the mutant, although seed germination was not significantly im-paired. The plastids of emb1303-1 were arrested in early developmental stages without the classical stack of thylakoid membrane. Genetic and molecular analyses uncovered that the EMB1303 gene encodes a novel chloroplast-localized protein. Mieroarray and RT-PCR analyses revealed that a number of nuclear-and plastid-encoded genes involved in photosynthesis and chloroplast biogenesis were substantially downregulated in the mutant. Moreover, the accu-mulation of several major chloroplast proteins was severely compromised in emb1303-1. These results suggest that EMBI303 is essential for chloroplast development.

  9. tassel-less1 encodes a boron channel protein required for inflorescence development in maize.

    Science.gov (United States)

    Leonard, April; Holloway, Beth; Guo, Mei; Rupe, Mary; Yu, GongXin; Beatty, Mary; Zastrow-Hayes, Gina; Meeley, Robert; Llaca, Victor; Butler, Karlene; Stefani, Tony; Jaqueth, Jennifer; Li, Bailin

    2014-06-01

    tassel-less1 (tls1) is a classical maize (Zea mays) inflorescence mutant. Homozygous mutant plants have no tassels or very small tassels, and ear development is also impaired. Using a positional cloning approach, ZmNIP3;1 (a NOD26-like intrinsic protein) was identified as the candidate gene for tls1. The ZmNIP3;1 gene is completely deleted in the tls1 mutant genome. Two Mutator-insertional TUSC alleles of ZmNIP3;1 exhibited tls1-like phenotypes, and allelism tests confirmed that the tls1 gene encodes ZmNIP3;1. Transgenic plants with an RNA interference (RNAi) construct to down-regulate ZmNIP3;1 also showed tls1-like phenotypes, further demonstrating that TLS1 is ZmNIP3;1. Sequence analysis suggests that ZmNIP3;1 is a boron channel protein. Foliar application of boron could rescue the tls1 phenotypes and restore the normal tassel and ear development. Gene expression analysis indicated that in comparison with that of the wild type or tls1 plants treated with boron, the transition from the vegetative to reproductive phase or the development of the floral meristem is impaired in the shoot apical meristem of the tls1 mutant plants. It is concluded that the tls1 mutant phenotypes are caused by impaired boron transport, and boron is essential for inflorescence development in maize.

  10. The Actin-Binding Protein α-Adducin Is Required for Maintaining Axon Diameter

    Directory of Open Access Journals (Sweden)

    Sérgio Carvalho Leite

    2016-04-01

    Full Text Available The actin-binding protein adducin was recently identified as a component of the neuronal subcortical cytoskeleton. Here, we analyzed mice lacking adducin to uncover the function of this protein in actin rings. α-adducin knockout mice presented progressive axon enlargement in the spinal cord and optic and sciatic nerves, followed by axon degeneration and loss. Using stimulated emission depletion super-resolution microscopy, we show that a periodic subcortical actin cytoskeleton is assembled in every neuron type inspected including retinal ganglion cells and dorsal root ganglia neurons. In neurons devoid of adducin, the actin ring diameter increased, although the inter-ring periodicity was maintained. In vitro, the actin ring diameter adjusted as axons grew, suggesting the lattice is dynamic. Our data support a model in which adducin activity is not essential for actin ring assembly and periodicity but is necessary to control the diameter of both actin rings and axons and actin filament growth within rings.

  11. The polycomb group protein Suz12 is required for embryonic stem cell differentiation

    DEFF Research Database (Denmark)

    Pasini, Diego; Bracken, Adrian P; Hansen, Jacob Bo Højberg;

    2007-01-01

    results in early lethality of mouse embryos. Here, we demonstrate that Suz12(-/-) mouse embryonic stem (ES) cells can be established and expanded in tissue culture. The Suz12(-/-) ES cells are characterized by global loss of H3K27 trimethylation (H3K27me3) and higher expression levels of differentiation......-specific genes. Moreover, Suz12(-/-) ES cells are impaired in proper differentiation, resulting in a lack of repression of ES cell markers as well as activation of differentiation-specific genes. Finally, we demonstrate that the PcGs are actively recruited to several genes during ES cell differentiation, which...... despite an increase in H3K27me3 levels is not always sufficient to prevent transcriptional activation. In summary, we demonstrate that Suz12 is required for the establishment of specific expression programs required for ES cell differentiation. Furthermore, we provide evidence that PcGs have different...

  12. Protein N-glycosylation and N-glycan trimming are required for postembryonic development of the pest beetle Tribolium castaneum

    Science.gov (United States)

    Walski, Tomasz; Van Damme, Els J. M.; Smargiasso, Nicolas; Christiaens, Olivier; De Pauw, Edwin; Smagghe, Guy

    2016-01-01

    In holometabolous insects the transition from larva to adult requires a complete body reorganization and relies on N-glycosylated proteins. N-glycosylation is an important posttranslational modification that influences protein activity but its impact on the metamorphosis has not been studied yet. Here we used the red flour beetle, Tribolium castaneum, to perform a first comprehensive study on the involvement of the protein N-glycosylation pathway in metamorphosis. The transcript levels for genes encoding N-glycan processing enzymes increased during later developmental stages and, in turn, transition from larva to adult coincided with an enrichment of more extensively modified paucimannose glycans, including fucosylated ones. Blockage of N-glycan attachment resulted in larval mortality, while RNAi of α-glucosidases involved in early N-glycan trimming and quality control disrupted the larva to pupa transition. Additionally, simultaneous knockdown of multiple genes responsible for N-glycan processing towards paucimannose structures revealed their novel roles in pupal appendage formation and adult eclosion. Our findings revealed that, next to hormonal control, insect post-embryonic development and metamorphosis depend on protein N-glycan attachment and efficient N-glycan processing. Consequently, disruption of these processes could be an effective new approach for insect control. PMID:27731363

  13. Phospholipase C not protein kinase C is required for the activation of TRPC5 channels by cholecystokinin.

    Science.gov (United States)

    Grisanti, Laurel A; Kurada, Lalitha; Cilz, Nicholas I; Porter, James E; Lei, Saobo

    2012-08-15

    Cholecystokinin (CCK) is one of the most abundant neuropeptides in the brain where it interacts with two G protein-coupled receptors (CCK1 and CCK2). Both types of CCK receptors are coupled to G(q/11) proteins resulting in increased function of phospholipase C (PLC) pathway. Whereas CCK has been suggested to increase neuronal excitability in the brain via activation of cationic channels, the types of cationic channels have not yet been identified. Here, we co-expressed CCK2 receptors and TRPC5 channels in human embryonic kidney (HEK) 293 cells and studied the effects of CCK on TRPC5 channels using patch-clamp techniques. Our results demonstrate that activation of CCK2 receptors robustly potentiates the function of TRPC5 channels. CCK-induced activation of TRPC5 channels requires the functions of G-proteins and PLC and depends on extracellular Ca(2+). The activation of TRPC5 channels mediated by CCK2 receptors is independent of IP(3) receptors and protein kinase C. CCK-induced opening of TRPC5 channels is not store-operated because application of thapsigargin to deplete intracellular Ca(2+) stores failed to alter CCK-induced TRPC5 channel currents significantly. Bath application of CCK also significantly increased the open probability of TRPC5 single channel currents in cell-attached patches. Because CCK exerts extensive effects in the brain, our results may provide a novel mechanism to explain its roles in modulating neuronal excitability.

  14. Structure of Yeast OSBP-Related Protein Osh1 Reveals Key Determinants for Lipid Transport and Protein Targeting at the Nucleus-Vacuole Junction.

    Science.gov (United States)

    Manik, Mohammad Kawsar; Yang, Huiseon; Tong, Junsen; Im, Young Jun

    2017-03-10

    Yeast Osh1 belongs to the oxysterol-binding protein (OSBP) family of proteins and contains multiple targeting modules optimized for lipid transport at the nucleus-vacuole junction (NVJ). The key determinants for NVJ targeting and the role of Osh1 at NVJs have remained elusive because of unknown lipid specificities. In this study, we determined the structures of the ankyrin repeat domain (ANK), and OSBP-related domain (ORD) of Osh1, in complex with Nvj1 and ergosterol, respectively. The Osh1 ANK forms a unique bi-lobed structure that recognizes a cytosolic helical segment of Nvj1. We discovered that Osh1 ORD binds ergosterol and phosphatidylinositol 4-phosphate PI(4)P in a competitive manner, suggesting counter-transport function of the two lipids. Ergosterol is bound to the hydrophobic pocket in a head-down orientation, and the structure of the PI(4)P-binding site in Osh1 is well conserved. Our results suggest that Osh1 performs non-vesicular transport of ergosterol and PI(4)P at the NVJ.

  15. Estimation of the dietary essential amino acid requirements of colliroja Astyanax fasciatus by using the ideal protein concept

    Directory of Open Access Journals (Sweden)

    Wilson Massamitu Furuya

    2015-11-01

    Full Text Available Colliroja, Astyanax fasciatus, is a new aquaculture species, and information on its dietary essential amino acid requirements is lacking. The whole body composition of 120 farmed fish (16.2 ± 8.8 g was determined to estimate the dietary essential amino acid requirement based on the ideal protein concept ((each essential amino acid/lysine x100, and the findings were correlated to the whole body essential amino acid content of Nile tilapia Oreochromis niloticus. The dietary essential amino acids, including cysteine and tyrosine, accounted for 5.46, 4.62, 1.16, 3.28, 5.63, 2.01, 2.59, 2.84, 4.66, 3.39, 0.65, and 3.51% of the total protein for lysine, arginine, histidine, isoleucine, leucine, methionine, methionine+tyrosine, phenylalanine, phenylalanine+tyrosine, threonine, tryptophan, and valine, respectively. There were positive linear and high correlations (r = 0.971 between the whole body amino acid profiles of colliroja and Nile tilapia. Thus, the whole body amino acid profile of colliroja might be used to estimate accurately the essential amino acid requirement.

  16. Stem-loop binding protein is required for retinal cell proliferation, neurogenesis, and intraretinal axon pathfinding in zebrafish.

    Science.gov (United States)

    Imai, Fumiyasu; Yoshizawa, Asuka; Matsuzaki, Ayako; Oguri, Eri; Araragi, Masato; Nishiwaki, Yuko; Masai, Ichiro

    2014-10-01

    In the developing retina, neurogenesis and cell differentiation are coupled with cell proliferation. However, molecular mechanisms that coordinate cell proliferation and differentiation are not fully understood. In this study, we found that retinal neurogenesis is severely delayed in the zebrafish stem-loop binding protein (slbp) mutant. SLBP binds to a stem-loop structure at the 3'-end of histone mRNAs, and regulates a replication-dependent synthesis and degradation of histone proteins. Retinal cell proliferation becomes slower in the slbp1 mutant, resulting in cessation of retinal stem cell proliferation. Although retinal stem cells cease proliferation by 2 days postfertilization (dpf) in the slbp mutant, retinal progenitor cells in the central retina continue to proliferate and generate neurons until at least 5dpf. We found that this progenitor proliferation depends on Notch signaling, suggesting that Notch signaling maintains retinal progenitor proliferation when faced with reduced SLBP activity. Thus, SLBP is required for retinal stem cell maintenance. SLBP and Notch signaling are required for retinal progenitor cell proliferation and subsequent neurogenesis. We also show that SLBP1 is required for intraretinal axon pathfinding, probably through morphogenesis of the optic stalk, which expresses attractant cues. Taken together, these data indicate important roles of SLBP in retinal development.

  17. TATA-binding protein (TBP)-like protein is required for p53-dependent transcriptional activation of upstream promoter of p21Waf1/Cip1 gene.

    Science.gov (United States)

    Suzuki, Hidefumi; Ito, Ryo; Ikeda, Kaori; Tamura, Taka-Aki

    2012-06-01

    TATA-binding protein-like protein (TLP) is involved in development, checkpoint, and apoptosis through potentiation of gene expression. TLP-overexpressing human cells, especially p53-containing cells, exhibited a decreased growth rate and increased proportion of G(1) phase cells. TLP stimulated expression of several growth-related genes including p21 (p21(Waf1/Cip1)). TLP-mediated activation of the p21 upstream promoter in cells was shown by a promoter-luciferase reporter assay. The p53-binding sequence located in the p21 upstream promoter and p53 itself are required for TLP-mediated transcriptional activation. TLP and p53 bound to each other and synergistically enhanced activity of the upstream promoter. TLP specifically activated transcription from the endogenous upstream promoter, and p53 was required for this activation. Etoposide treatment also resulted in activation of the upstream promoter as well as nuclear accumulation of TLP and p53. Moreover, the upstream promoter was associated with endogenous p53 and TLP, and the p53 recruitment was enhanced by TLP. The results of the present study suggest that TLP mediates p53-governed transcriptional activation of the p21 upstream promoter.

  18. A Clostridium difficile-Specific, Gel-Forming Protein Required for Optimal Spore Germination

    Science.gov (United States)

    Donnelly, M. Lauren; Li, William; Li, Yong-qing; Hinkel, Lauren; Setlow, Peter

    2017-01-01

    ABSTRACT Clostridium difficile is a Gram-positive spore-forming obligate anaerobe that is a leading cause of antibiotic-associated diarrhea worldwide. In order for C. difficile to initiate infection, its aerotolerant spore form must germinate in the gut of mammalian hosts. While almost all spore-forming organisms use transmembrane germinant receptors to trigger germination, C. difficile uses the pseudoprotease CspC to sense bile salt germinants. CspC activates the related subtilisin-like protease CspB, which then proteolytically activates the cortex hydrolase SleC. Activated SleC degrades the protective spore cortex layer, a step that is essential for germination to proceed. Since CspC incorporation into spores also depends on CspA, a related pseudoprotease domain, Csp family proteins play a critical role in germination. However, how Csps are incorporated into spores remains unknown. In this study, we demonstrate that incorporation of the CspC, CspB, and CspA germination regulators into spores depends on CD0311 (renamed GerG), a previously uncharacterized hypothetical protein. The reduced levels of Csps in gerG spores correlate with reduced responsiveness to bile salt germinants and increased germination heterogeneity in single-spore germination assays. Interestingly, asparagine-rich repeat sequences in GerG’s central region facilitate spontaneous gel formation in vitro even though they are dispensable for GerG-mediated control of germination. Since GerG is found exclusively in C. difficile, our results suggest that exploiting GerG function could represent a promising avenue for developing C. difficile-specific anti-infective therapies. PMID:28096487

  19. Klebsiella pneumoniae outer membrane protein A is required to prevent the activation of airway epithelial cells.

    Science.gov (United States)

    March, Catalina; Moranta, David; Regueiro, Verónica; Llobet, Enrique; Tomás, Anna; Garmendia, Junkal; Bengoechea, José A

    2011-03-25

    Outer membrane protein A (OmpA) is a class of proteins highly conserved among the Enterobacteriaceae family and throughout evolution. Klebsiella pneumoniae is a capsulated gram-negative pathogen. It is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that K. pneumoniae infections are characterized by a lack of an early inflammatory response. Data from our laboratory indicate that K. pneumoniae CPS helps to suppress the host inflammatory response. However, it is unknown whether K. pneumoniae employs additional factors to modulate host inflammatory responses. Here, we report that K. pneumoniae OmpA is important for immune evasion in vitro and in vivo. Infection of A549 and normal human bronchial cells with 52OmpA2, an ompA mutant, increased the levels of IL-8. 52145-Δwca(K2)ompA, which does not express CPS and ompA, induced the highest levels of IL-8. Both mutants could be complemented. In vivo, 52OmpA2 induced higher levels of tnfα, kc, and il6 than the wild type. ompA mutants activated NF-κB, and the phosphorylation of p38, p44/42, and JNK MAPKs and IL-8 induction was via NF-κB-dependent and p38- and p44/42-dependent pathways. 52OmpA2 engaged TLR2 and -4 to activate NF-κB, whereas 52145-Δwca(K2)ompA activated not only TLR2 and TLR4 but also NOD1. Finally, we demonstrate that the ompA mutant is attenuated in the pneumonia mouse model. The results of this study indicate that K. pneumoniae OmpA contributes to attenuate airway cell responses. This may facilitate pathogen survival in the hostile environment of the lung.

  20. Huntingtin interacting proteins 14 and 14-like are required for chorioallantoic fusion during early placental development.

    Science.gov (United States)

    Sanders, Shaun S; Hou, Juan; Sutton, Liza M; Garside, Victoria C; Mui, Katherine K N; Singaraja, Roshni R; Hayden, Michael R; Hoodless, Pamela A

    2015-01-15

    Huntington disease (HD) is an adult-onset neurodegenerative disease characterized by motor, cognitive, and psychiatric symptoms that is caused by a CAG expansion in the HTT gene. Palmitoylation is the addition of saturated fatty acids to proteins by DHHC palmitoylacyl transferases. HTT is palmitoylated by huntingtin interacting proteins 14 and 14-like (HIP14 and HIP14L or ZDHHC17 and 13 respectively). Mutant HTT is less palmitoylated and this reduction of palmitoylation accelerates its aggregation and increases cellular toxicity. Mouse models deficient in either Hip14 (Hip14(-/-)) or Hip14l (Hip14l(-/-)) develop HD-like phenotypes. The biological function of HTT palmitoylation and the role that loss of HTT palmitoylation plays in the pathogenesis of HD are unknown. To address these questions mice deficient for both genes were created. Loss of Hip14 and Hip14l leads to early embryonic lethality at day embryonic day 10-11 due to failed chorioallantoic fusion. The chorion is thickened and disorganized and the allantois does not fuse correctly with the chorion and forms a balloon-like shape compared to Hip14l(-/-); Hip14(+/+) littermate control embryos. Interestingly, the Hip14(-/-) ; Hip14(-/-) embryos share many features with the Htt(-/-) embryos, including folding of the yolk sac, a bulb shaped allantois, and a thickened and disorganized chorion. This may be due to a decrease in HTT palmitoylation. In Hip14(-/-); Hip14l(-/-) mouse embryonic fibroblasts show a 25% decrease in HTT palmitoylation compared to wild type cells. This is the first description of a double PAT deficient mouse model where loss of a PAT or multiple PATs results in embryonic lethality in mammals. These results reinforce the physiological importance of palmitoylation during embryogenesis.

  1. Ck2-Dependent Phosphorylation Is Required to Maintain Pax7 Protein Levels in Proliferating Muscle Progenitors.

    Directory of Open Access Journals (Sweden)

    Natalia González

    Full Text Available Skeletal muscle regeneration and long term maintenance is directly link to the balance between self-renewal and differentiation of resident adult stem cells known as satellite cells. In turn, satellite cell fate is influenced by a functional interaction between the transcription factor Pax7 and members of the MyoD family of muscle regulatory factors. Thus, changes in the Pax7-to-MyoD protein ratio may act as a molecular rheostat fine-tuning acquisition of lineage identity while preventing precocious terminal differentiation. Pax7 is expressed in quiescent and proliferating satellite cells, while its levels decrease sharply in differentiating progenitors Pax7 is maintained in cells (reacquiring quiescence. While the mechanisms regulating Pax7 levels based on differentiation status are not well understood, we have recently described that Pax7 levels are directly regulated by the ubiquitin-ligase Nedd4, thus promoting proteasome-dependent Pax7 degradation in differentiating satellite cells. Here we show that Pax7 levels are maintained in proliferating muscle progenitors by a mechanism involving casein kinase 2-dependent Pax7 phosphorylation at S201. Point mutations preventing S201 phosphorylation or casein kinase 2 inhibition result in decreased Pax7 protein in proliferating muscle progenitors. Accordingly, this correlates directly with increased Pax7 ubiquitination. Finally, Pax7 down regulation induced by casein kinase 2 inhibition results in precocious myogenic induction, indicating early commitment to terminal differentiation. These observations highlight the critical role of post translational regulation of Pax7 as a molecular switch controlling muscle progenitor fate.

  2. Chromosome movements promoted by the mitochondrial protein SPD-3 are required for homology search during Caenorhabditis elegans meiosis.

    Directory of Open Access Journals (Sweden)

    Leticia Labrador

    2013-05-01

    Full Text Available Pairing of homologous chromosomes during early meiosis is essential to prevent the formation of aneuploid gametes. Chromosome pairing includes a step of homology search followed by the stabilization of homolog interactions by the synaptonemal complex (SC. These events coincide with dramatic changes in nuclear organization and rapid chromosome movements that depend on cytoskeletal motors and are mediated by SUN-domain proteins on the nuclear envelope, but how chromosome mobility contributes to the pairing process remains poorly understood. We show that defects in the mitochondria-localizing protein SPD-3 cause a defect in homolog pairing without impairing nuclear reorganization or SC assembly, which results in promiscuous installation of the SC between non-homologous chromosomes. Preventing SC assembly in spd-3 mutants does not improve homolog pairing, demonstrating that SPD-3 is required for homology search at the start of meiosis. Pairing center regions localize to SUN-1 aggregates at meiosis onset in spd-3 mutants; and pairing-promoting proteins, including cytoskeletal motors and polo-like kinase 2, are normally recruited to the nuclear envelope. However, quantitative analysis of SUN-1 aggregate movement in spd-3 mutants demonstrates a clear reduction in mobility, although this defect is not as severe as that seen in sun-1(jf18 mutants, which also show a stronger pairing defect, suggesting a correlation between chromosome-end mobility and the efficiency of pairing. SUN-1 aggregate movement is also impaired following inhibition of mitochondrial respiration or dynein knockdown, suggesting that mitochondrial function is required for motor-driven SUN-1 movement. The reduced chromosome-end mobility of spd-3 mutants impairs coupling of SC assembly to homology recognition and causes a delay in meiotic progression mediated by HORMA-domain protein HTP-1. Our work reveals how chromosome mobility impacts the different early meiotic events that promote

  3. Estimation of the leucine and histidine requirements for piglets fed a low-protein diet.

    Science.gov (United States)

    Wessels, A G; Kluge, H; Mielenz, N; Corrent, E; Bartelt, J; Stangl, G I

    2016-11-01

    Reduction of the CP content in the diets of piglets requires supplementation with crystalline essential amino acids (AA). Data on the leucine (Leu) and histidine (His) requirements of young pigs fed low-CP diets are limited and have primarily been obtained from nonlinear models. However, these models do not consider the possible decline in appetite and growth that can occur when pigs are fed excessive amounts of AA such as Leu. Therefore, two dose-response studies were conducted to estimate the standardised ileal digestible (SID) Leu : lysine (Lys) and His : Lys required to optimise the growth performance of young pigs. In both studies, the average daily gain (ADG), average daily feed intake (ADFI) and gain-to-feed ratio (G : F) were determined during a 6-week period. To ensure that the diets had sub-limiting Lys levels, a preliminary Lys dose-response study was conducted. In the Leu study, 60 35-day-old piglets of both sexes were randomly assigned to one of five treatments and fed a low-CP diet (15%) with SID Leu : Lys levels of 83%, 94%, 104%, 115% or 125%. The His study used 120 31-day-old piglets of both sexes, which were allotted to one of five treatments and fed a low-CP diet (14%) with SID His : Lys levels of 22%, 26%, 30%, 34% or 38%. Linear broken-line, curvilinear-plateau and quadratic-function models were used for estimations of SID Leu : Lys and SID His : Lys. The minimum SID Leu : Lys level needed to maximise ADG, ADFI and G : F was, on average, 101% based on the linear broken-line and curvilinear-plateau models. Using the quadratic-function model, the minimum SID Leu : Lys level needed to maximise ADG, ADFI and G : F was 108%. Data obtained from the quadratic-function analysis further showed that a ±10% deviation from the identified Leu requirement was accompanied by a small decline in the ADG (-3%). The minimum SID His : Lys level needed to maximise ADG, ADFI and G : F was 27% and 28% using the linear broken-line and curvilinear-plateau models

  4. Trichoplusia ni Kinesin-1 Associates with Autographa californica Multiple Nucleopolyhedrovirus Nucleocapsid Proteins and Is Required for Production of Budded Virus

    Science.gov (United States)

    Biswas, Siddhartha; Blissard, Gary W.

    2016-01-01

    ABSTRACT The mechanism by which nucleocapsids of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) egress from the nucleus to the plasma membrane, leading to the formation of budded virus (BV), is not known. AC141 is a nucleocapsid-associated protein required for BV egress and has previously been shown to be associated with β-tubulin. In addition, AC141 and VP39 were previously shown by fluorescence resonance energy transfer by fluorescence lifetime imaging to interact directly with the Drosophila melanogaster kinesin-1 light chain (KLC) tetratricopeptide repeat (TPR) domain. These results suggested that microtubule transport systems may be involved in baculovirus nucleocapsid egress and BV formation. In this study, we investigated the role of lepidopteran microtubule transport using coimmunoprecipitation, colocalization, yeast two-hybrid, and small interfering RNA (siRNA) analyses. We show that nucleocapsid AC141 associates with the lepidopteran Trichoplusia ni KLC and kinesin-1 heavy chain (KHC) by coimmunoprecipitation and colocalization. Kinesin-1, AC141, and microtubules colocalized predominantly at the plasma membrane. In addition, the nucleocapsid proteins VP39, FP25, and BV/ODV-C42 were also coimmunoprecipitated with T. ni KLC. Direct analysis of the role of T. ni kinesin-1 by downregulation of KLC by siRNA resulted in a significant decrease in BV production. Nucleocapsids labeled with VP39 fused with three copies of the mCherry fluorescent protein also colocalized with microtubules. Yeast two-hybrid analysis showed no evidence of a direct interaction between kinesin-1 and AC141 or VP39, suggesting that either other nucleocapsid proteins or adaptor proteins may be required. These results further support the conclusion that microtubule transport is required for AcMNPV BV formation. IMPORTANCE In two key processes of the replication cycle of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), nucleocapsids are

  5. Exigências de proteína de bovinos anelorados em pastejo Protein requirements of grazing Zebu steers

    Directory of Open Access Journals (Sweden)

    Eduardo Henrique Bevitori Kling de Moraes

    2010-03-01

    Full Text Available Objetivou-se determinar as exigências proteicas de bovinos anelorados sob pastejo em cinco piquetes de Brachiaria decumbens. Utilizaram-se 27 animais não-castrados, com peso corporal (PC médio inicial de 311,0 kg e idade média de 14 meses. Três animais foram abatidos após o período de adaptação para servirem como referência para as estimativas do peso de corpo vazio (PCVZ e da composição corporal iniciais dos animais mantidos no experimento. Dos 24 animais restantes, quatro foram designados ao grupo mantença, que teve tempo de pastejo restrito. Os 20 demais foram distribuídos em quatro tratamentos: mistura mineral, autocontrole e duas frequências de alimentação: três vezes/semana (às segunda, quartas e sextas-feiras ou diariamente. As exigências líquidas de proteína para ganho diminuíram com o aumento do peso vivo (PV dos animais. A exigência líquida de proteína encontrada para um animal com peso corporal de 250 kg foi de 153,71 g/kg GPCVZ, enquanto para um animal de 400 kg foi de 141,86 g/kg GPCVZ. A seguinte equação foi obtida para estimativa da proteína retida em relação ao ganho de peso vivo em jejum (GPVJ e da energia retida (ER: PR (g/dia = -34,6109 + 257,956*GPVJ – 17,01*ER. As exigências de proteína metabolizável estimadas para mantença e ganho de peso foram de 357,77 e 288,33 g/kg PC, respectivamente, para um bovino não-castrado de 400 kg de PC sob pastejo. Animais que consomem suplementos proteicos apresentam maiores exigências de proteína degradável no rúmen (PDR em comparação a animais sem suplementação, em virtude do maior consumo de nutrientes digestíveis totais. Para um animal de 400 kg de PV sob suplementação, as exigências de proteína degradável no rúmen e proteína não-degradável no rúmen são de 764,22 e 73,89, respectivamente, que correspondem à exigência de proteína bruta de 838,10 g/dia.The objective of this study was to determine the protein requirements of Zebu

  6. Mitotic destruction of the cell cycle regulated NIMA protein kinase of Aspergillus nidulans is required for mitotic exit.

    OpenAIRE

    Pu, R T; Osmani, S A

    1995-01-01

    NIMA is a cell cycle regulated protein kinase required, in addition to p34cdc2/cyclin B, for initiation of mitosis in Aspergillus nidulans. Like cyclin B, NIMA accumulates when cells are arrested in G2 and is degraded as cells traverse mitosis. However, it is stable in cells arrested in mitosis. NIMA, and related kinases, have an N-terminal kinase domain and a C-terminal extension. Deletion of the C-terminus does not completely inactivate NIMA kinase activity but does prevent functional compl...

  7. mTORC1-dependent protein synthesis underlying rapid antidepressant effect requires GABABR signaling.

    Science.gov (United States)

    Workman, E R; Niere, Farr; Raab-Graham, Kimberly F

    2013-10-01

    Administration of N-methyl-D-aspartate receptors (NMDAR) antagonists initiates a rapid anti-depressant response requiring mammalian Target of Rapamycin Complex 1 (mTORC1) kinase; however the molecular mechanism is unknown. We have determined that upon NMDAR blockade, dendritic γ-amino-butyric acid B receptors (GABABR) facilitate dendritic calcium entry. The GABABR-mediated increase in calcium signal requires the availability of dendritic L-type calcium channels. Moreover, GABABR can activate mTOR and increase mTOR dependent expression of BDNF under the same NMDAR blocked conditions. In vivo, blocking GABABR prevents the fast-acting, anti-depressant effect of the NR2B antagonist, Ro-25-6891, decreases active mTORC1 kinase, and reduces expression of BDNF and the AMPA receptor subunit GluA1. These findings propose a novel role for GABABRs in the antidepressant action of NR2B antagonists and as an initiator/regulator of mTORC1-mediated translation.

  8. The protein domains of the Dictyostelium microprocessor that are required for correct subcellular localization and for microRNA maturation.

    Science.gov (United States)

    Kruse, Janis; Meier, Doreen; Zenk, Fides; Rehders, Maren; Nellen, Wolfgang; Hammann, Christian

    2016-10-02

    The maturation pathways of microRNAs (miRNAs) have been delineated for plants and several animals, belonging to the evolutionary supergroups of Archaeplastida and Opisthokonta, respectively. Recently, we reported the discovery of the microprocessor complex in Dictyostelium discoideum of the Amoebozoa supergroup. The complex is composed of the Dicer DrnB and the dsRBD (double-stranded RNA binding domain) containing protein RbdB. Both proteins localize at nucleoli, where they physically interact, and both are required for miRNA maturation. Here we show that the miRNA phenotype of a ΔdrnB gene deletion strain can be rescued by ectopic expression of a series of DrnB GFP fusion proteins, which consistently showed punctate perinucleolar localization in fluorescence microscopy. These punctate foci appear surprisingly stable, as they persist both disintegration of nucleoli and degradation of cellular nucleic acids. We observed that DrnB expression levels influence the number of microprocessor foci and alter RbdB accumulation. An investigation of DrnB variants revealed that its newly identified nuclear localization signal is necessary, but not sufficient for the perinucleolar localization. Biogenesis of miRNAs, which are RNA Pol II transcripts, is correlated with that localization. Besides its bidentate RNase III domains, DrnB contains only a dsRBD, which surprisingly is dispensable for miRNA maturation. This dsRBD can, however, functionally replace the homologous domain in RbdB. Based on the unique setup of the Dictyostelium microprocessor with a subcellular localization similar to plants, but a protein domain composition similar to animals, we propose a model for the evolutionary origin of RNase III proteins acting in miRNA maturation.

  9. Transcriptional activation requires protection of the TATA-binding protein Tbp1 by the ubiquitin-specific protease Ubp3.

    Science.gov (United States)

    Chew, Boon Shang; Siew, Wee Leng; Xiao, Benjamin; Lehming, Norbert

    2010-11-01

    Tbp1, the TATA-binding protein, is essential for transcriptional activation, and Gal4 and Gcn4 are unable to fully activate transcription in a Saccharomyces cerevisiae TBP1E86D mutant strain. In the present study we have shown that the Tbp1E186D mutant protein is proteolytically instable, and we have isolated intragenic and extragenic suppressors of the transcription defects of the TBP1E186D mutant strain. The TBP1R6S mutation stabilizes the Tbp1E186D mutant protein and suppresses the defects of the TBP1E186D mutant strain. Furthermore, we found that the overexpression of the de-ubiquitinating enzyme Ubp3 (ubiquitin-specific protease 3) also stabilizes the Tbp1E186D mutant protein and suppresses of the defects of the TBP1E186D mutant strain. Importantly, the deletion of UBP3 and its cofactor BRE5 lead to increased degradation of wild-type Tbp1 protein and to defects in transcriptional activation by Gal4 and Gcn4. Purified GST (glutathione transferase)-Ubp3 reversed Tbp1 ubiquitination, and the deletion of UBP3 lead to the accumulation of poly-ubiquitinated species of Tbp1 in a proteaseome-deficient genetic background, demonstrating that Ubp3 reverses ubiquitination of Tbp1 in vitro and in vivo. Chromatin immunoprecipitation showed that Ubp3 was recruited to the GAL1 and HIS3 promoters upon the induction of the respective gene, indicating that protection of promoter-bound Tbp1 by Ubp3 is required for transcriptional activation.

  10. p38 Mitogen-Activated Protein Kinase Is Required for Central Nervous System Myelination

    Institute of Scientific and Technical Information of China (English)

    GABRIELA FRAGOSO; JEFFERY D. HAINES; JANICE ROBERSTON; LILIANA PEDRAZA; WALTER E. MUSHYNSKI; GUILLERMINA ALMAZAN

    2008-01-01

    p38MAPKs是一个激酶家族,负责调节包括细胞迁移、增生和分化在内的多种细胞功能.本文主要介绍p38对少突胶质细胞分化的调节作用.采用PD169316和SB203580抑制p38后,不同分化阶段少突胶质细胞特异性标志物的蛋白和mRNA的聚集减少,包括髓鞘碱性蛋白、髓鞘相关糖蛋白、鞘糖脂、半乳糖酰基鞘氨醇和硫脂.同时,细胞周期调节因子p27kip1和转录因子Sox10的表达也有显著的下降.最为重要的是,p38抑制剂能够通过少突胶质细胞完全和不可逆地阻断背根神经节神经元的髓鞘形成,并阻止轴-胶粘附分子Caspr的轴膜组装.本实验结果提示p38MAPKs在OLGs成熟和启动髓鞘形成的关键调控步骤中扮演了重要角色.%The p38 MAPKs are a family of kinases that regulate a number of cellular functions including cell migration, proliferation, and differentiation. Here, we report that p38 regulates oligodendrocyte differentiation. Inhibition of p38 with PD169316 and SB203580 prevented accumulation of protein and mRNA of cell-stage specific markers characteristic of differentiated oligodendrocytes, including myelin basic protein, myelin-associated glycoprotein, and the glycosphingolipids, galactosylceramide and sulfatide. In addition, the cell cycle regulator p27kip1 and the transcription factor Sox10 were also significantly reduced. Most significantly, p38 inhibitors completely and irreversibly blocked myelination of dorsal root ganglion neurons by oligodendrocytes and prevented the axolemmal organization of the axo-glial adhesion molecule Caspr. Our results suggest a role(s) for this kinase in key regulatory steps in the maturation of OLGs and initiation of myelination.

  11. Surface protein IsdC and Sortase B are required for heme-iron scavenging of Bacillus anthracis.

    Science.gov (United States)

    Maresso, Anthony W; Chapa, Travis J; Schneewind, Olaf

    2006-12-01

    Bacillus anthracis, the spore-forming agent of anthrax, requires iron for growth and is capable of scavenging heme-iron during infection. We show here that the B. anthracis iron-regulated surface determinants (isd) locus encompasses isdC, specifying a heme-iron binding surface protein. Anchoring of IsdC to the cell wall envelopes of vegetative bacilli requires srtB, which encodes sortase B. Purified sortase B cleaves IsdC between the threonine and the glycine of its NPKTG motif sorting signal. B. anthracis variants lacking either isdC or srtB display defects in heme-iron scavenging, suggesting that IsdC binding to heme-iron in the cell wall envelope contributes to bacterial uptake of heme.

  12. The polarity protein Pard3 is required for centrosome positioning during neurulation.

    Science.gov (United States)

    Hong, Elim; Jayachandran, Pradeepa; Brewster, Rachel

    2010-05-15

    Microtubules are essential regulators of cell polarity, architecture and motility. The organization of the microtubule network is context-specific. In non-polarized cells, microtubules are anchored to the centrosome and form radial arrays. In most epithelial cells, microtubules are noncentrosomal, align along the apico-basal axis and the centrosome templates a cilium. It follows that cells undergoing mesenchyme-to-epithelium transitions must reorganize their microtubule network extensively, yet little is understood about how this process is orchestrated. In particular, the pathways regulating the apical positioning of the centrosome are unknown, a central question given the role of cilia in fluid propulsion, sensation and signaling. In zebrafish, neural progenitors undergo progressive epithelialization during neurulation, and thus provide a convenient in vivo cellular context in which to address this question. We demonstrate here that the microtubule cytoskeleton gradually transitions from a radial to linear organization during neurulation and that microtubules function in conjunction with the polarity protein Pard3 to mediate centrosome positioning. Pard3 depletion results in hydrocephalus, a defect often associated with abnormal cerebrospinal fluid flow that has been linked to cilia defects. These findings thus bring to focus cellular events occurring during neurulation and reveal novel molecular mechanisms implicated in centrosome positioning.

  13. MreC and MreD Proteins Are Not Required for Growth of Staphylococcus aureus.

    Science.gov (United States)

    Tavares, Andreia C; Fernandes, Pedro B; Carballido-López, Rut; Pinho, Mariana G

    2015-01-01

    The transmembrane proteins MreC and MreD are present in a wide variety of bacteria and are thought to be involved in cell shape determination. Together with the actin homologue MreB and other morphological elements, they play an essential role in the synthesis of the lateral cell wall in rod-shaped bacteria. In ovococcus, which lack MreB homologues, mreCD are also essential and have been implicated in peripheral cell wall synthesis. In this work we addressed the possible roles of MreC and MreD in the spherical pathogen Staphylococcus aureus. We show that MreC and MreD are not essential for cell viability and do not seem to affect cell morphology, cell volume or cell cycle control. MreC and MreD localize preferentially to the division septa, but do not appear to influence peptidoglycan composition, nor the susceptibility to different antibiotics and to oxidative and osmotic stress agents. Our results suggest that the function of MreCD in S. aureus is not critical for cell division and cell shape determination.

  14. The MCM-binding protein ETG1 aids sister chromatid cohesion required for postreplicative homologous recombination repair.

    Directory of Open Access Journals (Sweden)

    Naoki Takahashi

    2010-01-01

    Full Text Available The DNA replication process represents a source of DNA stress that causes potentially spontaneous genome damage. This effect might be strengthened by mutations in crucial replication factors, requiring the activation of DNA damage checkpoints to enable DNA repair before anaphase onset. Here, we demonstrate that depletion of the evolutionarily conserved minichromosome maintenance helicase-binding protein ETG1 of Arabidopsis thaliana resulted in a stringent late G2 cell cycle arrest. This arrest correlated with a partial loss of sister chromatid cohesion. The lack-of-cohesion phenotype was intensified in plants without functional CTF18, a replication fork factor needed for cohesion establishment. The synergistic effect of the etg1 and ctf18 mutants on sister chromatid cohesion strengthened the impact on plant growth of the replication stress caused by ETG1 deficiency because of inefficient DNA repair. We conclude that the ETG1 replication factor is required for efficient cohesion and that cohesion establishment is essential for proper development of plants suffering from endogenous DNA stress. Cohesion defects observed upon knockdown of its human counterpart suggest an equally important developmental role for the orthologous mammalian ETG1 protein.

  15. The requirement for the Dam1 complex is dependent upon the number of kinetochore proteins and microtubules.

    Science.gov (United States)

    Burrack, Laura S; Applen, Shelly E; Berman, Judith

    2011-05-24

    The Dam1 complex attaches the kinetochore to spindle microtubules and is a processivity factor in vitro. In Saccharomyces cerevisiae, which has point centromeres that attach to a single microtubule, deletion of any Dam1 complex member results in chromosome segregation failures and cell death. In Schizosaccharomyces pombe, which has epigenetically defined regional centromeres that each attach to 3-5 kinetochore microtubules, Dam1 complex homologs are not essential. To determine why the complex is essential in some organisms and not in others, we used Candida albicans, a multimorphic yeast with regional centromeres that attach to a single microtubule. Interestingly, the Dam1 complex was essential in C. albicans, suggesting that the number of microtubules per centromere is critical for its requirement. Importantly, by increasing CENP-A expression levels, more kinetochore proteins and microtubules were recruited to the centromeres, which remained fully functional. Furthermore, Dam1 complex members became less crucial for growth in cells with extra kinetochore proteins and microtubules. Thus, the requirement for the Dam1 complex is not due to the DNA-specific nature of point centromeres. Rather, the Dam1 complex is less critical when chromosomes have multiple kinetochore complexes and microtubules per centromere, implying that it functions as a processivity factor in vivo as well as in vitro.

  16. Herpes simplex virus virion host shutoff protein requires a mammalian factor for efficient in vitro endoribonuclease activity.

    Science.gov (United States)

    Lu, P; Jones, F E; Saffran, H A; Smiley, J R

    2001-02-01

    The virion host shutoff protein (vhs) of herpes simplex virus (HSV) triggers global shutoff of host protein synthesis and accelerated mRNA turnover during virus infection and induces endoribonucleolytic cleavage of exogenous RNA substrates when it is produced in a rabbit reticulocyte (RRL) in vitro translation system. Although vhs induces RNA turnover in the absence of other HSV gene products, it is not yet known whether cellular factors are required for its activity. As one approach to addressing this question, we expressed vhs in the budding yeast Saccharomyces cerevisiae. Expression of vhs inhibited colony formation, and the severity of this effect varied with the carbon source. The biological relevance of this effect was assessed by examining the activity of five mutant forms of vhs bearing previously characterized in-frame linker insertions. The results indicated a complete concordance between the growth inhibition phenotype in yeast and mammalian host cell shutoff. Despite these results, expression of vhs did not trigger global mRNA turnover in vivo, and cell extracts of yeast expressing vhs displayed little if any vhs-dependent endoribonuclease activity. However, activity was readily detected when such extracts were mixed with RRL. These data suggest that the vhs-dependent endoribonuclease requires one or more mammalian macromolecular factors for efficient activity.

  17. The Microcephaly-Associated Protein Wdr62/CG7337 Is Required to Maintain Centrosome Asymmetry in Drosophila Neuroblasts

    Directory of Open Access Journals (Sweden)

    Anjana Ramdas Nair

    2016-02-01

    Full Text Available Centrosome asymmetry has been implicated in stem cell fate maintenance in both flies and vertebrates, but the underlying molecular mechanisms are incompletely understood. Here, we report that loss of CG7337, the fly ortholog of WDR62, compromises interphase centrosome asymmetry in fly neural stem cells (neuroblasts. Wdr62 maintains an active interphase microtubule-organizing center (MTOC by stabilizing microtubules (MTs, which are necessary for sustained recruitment of Polo/Plk1 to the pericentriolar matrix (PCM and downregulation of Pericentrin-like protein (Plp. The loss of an active MTOC in wdr62 mutants compromises centrosome positioning, spindle orientation, and biased centrosome segregation. wdr62 mutant flies also have an ∼40% reduction in brain size as a result of cell-cycle delays. We propose that CG7337/Wdr62, a microtubule-associated protein, is required for the maintenance of interphase microtubules, thereby regulating centrosomal Polo and Plp levels. Independent of this function, Wdr62 is also required for the timely mitotic entry of neural stem cells.

  18. BARREN STALK FASTIGIATE1 Is an AT-Hook Protein Required for the Formation of Maize Ears[W][OA

    Science.gov (United States)

    Gallavotti, Andrea; Malcomber, Simon; Gaines, Craig; Stanfield, Sharon; Whipple, Clinton; Kellogg, Elizabeth; Schmidt, Robert J.

    2011-01-01

    Ears are the seed-bearing inflorescences of maize (Zea mays) plants and represent a crucial component of maize yield. The first step in the formation of ears is the initiation of axillary meristems in the axils of developing leaves. In the classic maize mutant barren stalk fastigiate1 (baf1), first discovered in the 1950s, ears either do not form or, if they do, are partially fused to the main stalk. We positionally cloned Baf1 and found that it encodes a transcriptional regulator containing an AT-hook DNA binding motif. Single coorthologs of Baf1 are found in syntenic regions of brachypodium (Brachypodium distachyon), rice (Oryza sativa), and sorghum (Sorghum bicolor), suggesting that the gene is likely present in all cereal species. Protein–protein interaction assays suggest that BAF1 is capable of forming homodimers and heterodimers with other members of the AT-hook family. Another transcriptional regulator required for ear initiation is the basic helix-loop-helix protein BARREN STALK1 (BA1). Genetic and expression analyses suggest that Baf1 is required to reach a threshold level of Ba1 expression for the initiation of maize ears. We propose that Baf1 functions in the demarcation of a boundary region essential for the specification of a stem cell niche. PMID:21540434

  19. Requirements of Slm proteins for proper eisosome organization, endocytic trafficking and recycling in the yeast Saccharomyces cerevisiae

    Indian Academy of Sciences (India)

    Chitra Kamble; Sandhya Jain; Erin Murphy; Kyoungtae Kim

    2011-03-01

    Eisosomes are large immobile assemblies at the cortex of a cell under the membrane compartment of Can1 (MCC) in yeast. Slm1 has recently been identified as an MCC component that acts downstream of Mss4 in a pathway that regulates actin cytoskeleton organization in response to stress. In this study, we showed that inactivation of Slm proteins disrupts proper localization of the primary eisosome marker Pil1, providing evidence that Slm proteins play a role in eisosome organization. Furthermore, we found that slmts mutant cells exhibit actin defects in both the ability to polarize cortical F-actin and the formation of cytoplasmic actin cables even at the permissive temperature (30°C). We further demonstrated that the actin defect accounts for the slow traffic of FM4-64-labelled endosome in the cytoplasm, supporting the notion that intact actin is essential for endosome trafficking. However, our real-time microscopic analysis of Abp1-RFP revealed that the actin defect in slmts cells was not accompanied by a noticeable defect in actin patch internalization during receptor-mediated endocytosis. In addition, we found that slmts cells displayed impaired membrane recycling and that recycling occurred in an actin-independent manner. Our data provide evidence for the requirement of Slm proteins in eisosome organization and endosome trafficking and recycling.

  20. SDCCAG8 Interacts with RAB Effector Proteins RABEP2 and ERC1 and Is Required for Hedgehog Signaling.

    Directory of Open Access Journals (Sweden)

    Rannar Airik

    Full Text Available Recessive mutations in the SDCCAG8 gene cause a nephronophthisis-related ciliopathy with Bardet-Biedl syndrome-like features in humans. Our previous characterization of the orthologous Sdccag8gt/gt mouse model recapitulated the retinal-renal disease phenotypes and identified impaired DNA damage response signaling as an underlying disease mechanism in the kidney. However, several other phenotypic and mechanistic features of Sdccag8gt/gt mice remained unexplored. Here we show that Sdccag8gt/gt mice exhibit developmental and structural abnormalities of the skeleton and limbs, suggesting impaired Hedgehog (Hh signaling. Indeed, cell culture studies demonstrate the requirement of SDCCAG8 for ciliogenesis and Hh signaling. Using an affinity proteomics approach, we demonstrate that SDCCAG8 interacts with proteins of the centriolar satellites (OFD1, AZI1, of the endosomal sorting complex (RABEP2, ERC1, and with non-muscle myosin motor proteins (MYH9, MYH10, MYH14 at the centrosome. Furthermore, we show that RABEP2 localization at the centrosome is regulated by SDCCAG8. siRNA mediated RABEP2 knockdown in hTERT-RPE1 cells leads to defective ciliogenesis, indicating a critical role for RABEP2 in this process. Together, this study identifies several centrosome-associated proteins as novel SDCCAG8 interaction partners, and provides new insights into the function of SDCCAG8 at this structure.

  1. Requirement of cellular DDX3 for hepatitis C virus replication is unrelated to its interaction with the viral core protein.

    Science.gov (United States)

    Angus, Allan G N; Dalrymple, David; Boulant, Steeve; McGivern, David R; Clayton, Reginald F; Scott, Martin J; Adair, Richard; Graham, Susan; Owsianka, Ania M; Targett-Adams, Paul; Li, Kui; Wakita, Takaji; McLauchlan, John; Lemon, Stanley M; Patel, Arvind H

    2010-01-01

    The cellular DEAD-box protein DDX3 was recently shown to be essential for hepatitis C virus (HCV) replication. Prior to that, we had reported that HCV core binds to DDX3 in yeast-two hybrid and transient transfection assays. Here, we confirm by co-immunoprecipitation that this interaction occurs in cells replicating the JFH1 virus. Consistent with this result, immunofluorescence staining of infected cells revealed a dramatic redistribution of cytoplasmic DDX3 by core protein to the virus assembly sites around lipid droplets. Given this close association of DDX3 with core and lipid droplets, and its involvement in virus replication, we investigated the importance of this host factor in the virus life cycle. Mutagenesis studies located a single amino acid in the N-terminal domain of JFH1 core that when changed to alanine significantly abrogated this interaction. Surprisingly, this mutation did not alter infectious virus production and RNA replication, indicating that the core-DDX3 interaction is dispensable in the HCV life cycle. Consistent with previous studies, siRNA-led knockdown of DDX3 lowered virus production and RNA replication levels of both WT JFH1 and the mutant virus unable to bind DDX3. Thus, our study shows for the first time that the requirement of DDX3 for HCV replication is unrelated to its interaction with the viral core protein.

  2. A Novel C2-Domain Phospholipid-Binding Protein,OsPBP1.Is Required for Pollen Fertility in Rice

    Institute of Scientific and Technical Information of China (English)

    Wen-Qiang Yang; Ying Lai; Mei-Na Li; Wen-Ying Xu; Yong-Biao Xue

    2008-01-01

    Pollen fertility is a crucial factor for successful pollination and essential for seed formation.Recent studies have suggested that a diverse range of internal and external factors,signaling components and their related pathways are likely involved in pollen fertility.Here,we reporta single C2-domain containing protein.OsPBPl.initially identified through cDNA microarray analysis.OsP8P1 is a single copy gene and preferentially expressed in pistil and pollen but downregulated by pollination.OsPBP1 had a calcium concentration-dependent phospholipid-binding activity and was localized mainly in cytoplasm and nucleus,but translocated onto the plasma membrane in response to an intracellular Ca2+increase.Pollen grains of antisense OsPBP1 transgenic Iines were largely nonviable.germinated poorly in vitro and of low fertility,OsPBP1 protein was localized in a region peripheral to pollen wall and vesicles of elongating pollen tube.and its repressed expression reduced substantially this association and led to alteration of microfilament polymerization during pollen germination.Taken together,these results indicate that OsPBP1 is a novel functional C2-domain phosphoIipids-binding protein that is required for pollen fertility likely by regulating Ca2+ and phospholipid signaling pathways.

  3. The apical complex protein Pals1 is required to maintain cerebellar progenitor cells in a proliferative state.

    Science.gov (United States)

    Park, Jun Young; Hughes, Lucinda J; Moon, Uk Yeol; Park, Raehee; Kim, Sang-Bae; Tran, Khoi; Lee, Ju-Seog; Cho, Seo-Hee; Kim, Seonhee

    2016-01-01

    Through their biased localization and function within the cell, polarity complex proteins are necessary to establish the cellular asymmetry required for tissue organization. Well-characterized germinal zones, mitogenic signals and cell types make the cerebellum an excellent model for addressing the crucial function of polarity complex proteins in the generation and organization of neural tissues. Deletion of the apical polarity complex protein Pals1 in the developing cerebellum results in a remarkably undersized cerebellum with disrupted layers in poorly formed folia and strikingly reduced granule cell production. We demonstrate that Pals1 is not only essential for cerebellum organogenesis, but also for preventing premature differentiation and thus maintaining progenitor pools in cerebellar germinal zones, including cerebellar granule neuron precursors in the external granule layer. In the Pals1 mouse mutants, the expression of genes that regulate the cell cycle was diminished, correlating with the loss of the proliferating cell population of germinal zones. Furthermore, enhanced Shh signaling through activated Smo cannot overcome impaired cerebellar cell generation, arguing for an epistatic role of Pals1 in proliferation capacity. Our study identifies Pals1 as a novel intrinsic factor that regulates the generation of cerebellar cells and Pals1 deficiency as a potential inhibitor of overactive mitogenic signaling.

  4. The SLO1 PPR protein is required for RNA editing at multiple sites with similar upstream sequences in Arabidopsis mitochondria.

    Science.gov (United States)

    Sung, Tzu-Ying; Tseng, Ching-Chih; Hsieh, Ming-Hsiun

    2010-08-01

    In Arabidopsis, RNA editing changes more than 500 cytidines to uridines in mitochondrial transcripts. The editing enzyme and co-factors involved in these processes are largely unknown. We have identified a nuclear gene SLOW GROWTH1 (SLO1) encoding an E motif-containing pentatricopeptide repeat protein that is required for RNA editing of nad4 and nad9 in Arabidopsis mitochondria. The SLO1 protein is localized to the mitochondrion, and its absence gives rise to small plants with slow growth and delayed development. A survey of approximately 500 mitochondrial RNA editing sites in Arabidopsis reveals that the editing of two sites, nad4-449 and nad9-328, is abolished in the slo1 mutants. Sequence comparison in the upstream (from -1 to -15 bp) of nad4-449 and nad9-328 editing sites shows that nine of the 15 nucleotides are identical. In addition to RNA editing, we used RNA gel blot analysis to compare the abundance and banding patterns of mitochondrial transcripts between the wild type and slo1 mutants. Of the 79 genes and open reading frames examined, steady-state levels of 56 mitochondrial transcripts are increased in the slo1 mutants. These results suggest that the SLO1 protein may indirectly regulate plant growth and development via affecting mitochondrial RNA editing and gene expression.

  5. Talpid3-binding centrosomal protein Cep120 is required for centriole duplication and proliferation of cerebellar granule neuron progenitors.

    Directory of Open Access Journals (Sweden)

    Chuanqing Wu

    Full Text Available Granule neuron progenitors (GNPs are the most abundant neuronal type in the cerebellum. GNP proliferation and thus cerebellar development require Sonic hedgehog (Shh secreted from Purkinje cells. Shh signaling occurs in primary cilia originating from the mother centriole. Centrioles replicate only once during a typical cell cycle and are responsible for mitotic spindle assembly and organization. Recent studies have linked cilia function to cerebellar morphogenesis, but the role of centriole duplication in cerebellar development is not known. Here we show that centrosomal protein Cep120 is asymmetrically localized to the daughter centriole through its interaction with Talpid3 (Ta3, another centrosomal protein. Cep120 null mutant mice die in early gestation with abnormal heart looping. Inactivation of Cep120 in the central nervous system leads to both hydrocephalus, due to the loss of cilia on ependymal cells, and severe cerebellar hypoplasia, due to the failed proliferation of GNPs. The mutant GNPs lack Hedgehog pathway activity. Cell biological studies show that the loss of Cep120 results in failed centriole duplication and consequently ciliogenesis, which together underlie Cep120 mutant cerebellar hypoplasia. Thus, our study for the first time links a centrosomal protein necessary for centriole duplication to cerebellar morphogenesis.

  6. The intermediate filament network protein, vimentin, is required for parvoviral infection

    Energy Technology Data Exchange (ETDEWEB)

    Fay, Nikta; Panté, Nelly, E-mail: pante@zoology.ubc.ca

    2013-09-15

    Intermediate filaments (IFs) have recently been shown to serve novel roles during infection by many viruses. Here we have begun to study the role of IFs during the early steps of infection by the parvovirus minute virus of mice (MVM). We found that during early infection with MVM, after endosomal escape, the vimentin IF network was considerably altered, yielding collapsed immunofluorescence staining near the nuclear periphery. Furthermore, we found that vimentin plays an important role in the life cycle of MVM. The number of cells, which successfully replicated MVM, was reduced in infected cells in which the vimentin network was genetically or pharmacologically modified; viral endocytosis, however, remained unaltered. Perinuclear accumulation of MVM-containing vesicles was reduced in cells lacking vimentin. Our data suggests that vimentin is required for the MVM life cycle, presenting possibly a dual role: (1) following MVM escape from endosomes and (2) during endosomal trafficking of MVM. - Highlights: • MVM infection changes the distribution of the vimentin network to perinuclear regions. • Disrupting the vimentin network with acrylamide decreases MVM replication. • MVM replication is significantly reduced in vimentin-null cells. • Distribution of MVM-containing vesicles is affected in MVM infected vimentin-null cells.

  7. Chronic Myelogenous Leukemia- Initiating Cells Require Polycomb Group Protein EZH2.

    Science.gov (United States)

    Xie, Huafeng; Peng, Cong; Huang, Jialiang; Li, Bin E; Kim, Woojin; Smith, Elenoe C; Fujiwara, Yuko; Qi, Jun; Cheloni, Giulia; Das, Partha P; Nguyen, Minh; Li, Shaoguang; Bradner, James E; Orkin, Stuart H

    2016-11-01

    Tyrosine kinase inhibitors (TKI) have revolutionized chronic myelogenous leukemia (CML) management. Disease eradication, however, is hampered by innate resistance of leukemia-initiating cells (LIC) to TKI-induced killing, which also provides the basis for subsequent emergence of TKI-resistant mutants. We report that EZH2, the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), is overexpressed in CML LICs and required for colony formation and survival and cell-cycle progression of CML cell lines. A critical role for EZH2 is supported by genetic studies in a mouse CML model. Inactivation of Ezh2 in conventional conditional mice and through CRISPR/Cas9-mediated gene editing prevents initiation and maintenance of disease and survival of LICs, irrespective of BCR-ABL1 mutational status, and extends survival. Expression of the EZH2 homolog EZH1 is reduced in EZH2-deficient CML LICs, creating a scenario resembling complete loss of PRC2. EZH2 dependence of CML LICs raises prospects for improved therapy of TKI-resistant CML and/or eradication of disease by addition of EZH2 inhibitors.

  8. Survival of motor neurone protein is required for normal postnatal development of the spleen.

    Science.gov (United States)

    Thomson, Alison K; Somers, Eilidh; Powis, Rachael A; Shorrock, Hannah K; Murphy, Kelley; Swoboda, Kathryn J; Gillingwater, Thomas H; Parson, Simon H

    2017-02-01

    Spinal muscular atrophy (SMA), traditionally described as a predominantly childhood form of motor neurone disease, is the leading genetic cause of infant mortality. Although motor neurones are undoubtedly the primary affected cell type, the severe infantile form of SMA (Type I SMA) is now widely recognised to represent a multisystem disorder where a variety of organs and systems in the body are also affected. Here, we report that the spleen is disproportionately small in the 'Taiwanese' murine model of severe SMA (Smn(-/-) ;SMN2(tg/0) ), correlated to low levels of cell proliferation and increased cell death. Spleen lacks its distinctive red appearance and presents with a degenerated capsule and a disorganised fibrotic architecture. Histologically distinct white pulp failed to form and this was reflected in an almost complete absence of B lymphocytes necessary for normal immune function. In addition, megakaryoctyes persisted in the red pulp. However, the vascular density remained unchanged in SMA spleen. Assessment of the spleen in SMA patients with the infantile form of the disease indicated a range of pathologies. We conclude that development of the spleen fails to occur normally in SMA mouse models and human patients. Thus, further analysis of immune function is likely to be required to fully understand the full extent of systemic disease pathology in SMA.

  9. A component of the Sec61 ER protein transporting pore is required for plant susceptibility to powdery mildew

    Directory of Open Access Journals (Sweden)

    Wen-Jing eZhang

    2013-05-01

    Full Text Available Biotrophic pathogens, like the powdery mildew fungi, require living plant cells for their growth and reproduction. During infection, a specialized structure called the haustorium is formed by the fungus. The haustorium is surrounded by a plant cell-derived extrahaustorial membrane (EHM. Over the EHM, the fungus obtains nutrients from and secretes effectors into the plant cell. In the plant cell these effectors interfere with cellular processes such as pathogen defense and membrane trafficking. However, the mechanisms behind effector delivery are largely unknown. This paper provides a model for and new insights into a putative transfer mechanism of effectors into the plant cell. We show that silencing of the barley Sec61βa protein results in decreased susceptibility to the powdery mildew fungus. HvSec61βa is a component of both the endoplasmic reticulum (ER translocon and retrotranslocon pores, the latter being part of the ER-associated protein degradation machinery (ERAD. We provide support for a model suggesting that the retrotranslocon function of HvSec61βa is required for successful powdery mildew fungal infection. HvSec61βa-GFP and a luminal ER marker were co-localized to the ER, which was found to be in close proximity to the EHM around the haustorial body, but not the haustorial fingers. This differential EHM proximity suggests that the ER, including HvSec61βa, may be actively recruited by the haustorium, potentially to provide efficient effector transfer to the cytosol. Effector transport across this EHM-ER interface may occur by a vesicle-mediated process, while the Sec61 retrotranslocon pore potentially provides an escape route for these proteins to reach the cytosol.

  10. A component of the Sec61 ER protein transporting pore is required for plant susceptibility to powdery mildew.

    Science.gov (United States)

    Zhang, Wen-Jing; Hanisch, Susanne; Kwaaitaal, Mark; Pedersen, Carsten; Thordal-Christensen, Hans

    2013-01-01

    Biotrophic pathogens, like the powdery mildew fungi, require living plant cells for their growth and reproduction. During infection, a specialized structure called the haustorium is formed by the fungus. The haustorium is surrounded by a plant cell-derived extrahaustorial membrane (EHM). Over the EHM, the fungus obtains nutrients from and secretes effector proteins into the plant cell. In the plant cell these effectors interfere with cellular processes such as pathogen defense and membrane trafficking. However, the mechanisms behind effector delivery are largely unknown. This paper provides a model for and new insights into a putative transfer mechanism of effectors into the plant cell. We show that silencing of the barley Sec61βa transcript results in decreased susceptibility to the powdery mildew fungus. HvSec61βa is a component of both the endoplasmic reticulum (ER) translocon and retrotranslocon pores, the latter being part of the ER-associated protein degradation machinery. We provide support for a model suggesting that the retrotranslocon function of HvSec61βa is required for successful powdery mildew fungal infection. HvSec61βa-GFP and a luminal ER marker were co-localized to the ER, which was found to be in close proximity to the EHM around the haustorial body, but not the haustorial fingers. This differential EHM proximity suggests that the ER, including HvSec61βa, may be actively recruited by the haustorium, potentially to provide efficient effector transfer to the cytosol. Effector transport across this EHM-ER interface may occur by a vesicle-mediated process, while the Sec61 retrotranslocon pore potentially provides an escape route for these proteins to reach the cytosol.

  11. Protein kinase A regulation of P2X(4) receptors: requirement for a specific motif in the C-terminus.

    Science.gov (United States)

    Brown, David A; Yule, David I

    2010-02-01

    The P2X purinergic receptor sub-family of ligand-gated ion channels are subject to protein kinase modulation. We have previously demonstrated that P2X(4)R signaling can be positively regulated by increasing intracellular cAMP levels. The molecular mechanism underlying this effect was, however, unknown. The present study initially addressed whether protein kinase A (PKA) activation was required. Subsequently a mutational approach was utilized to determine which region of the receptor was required for this potentiation. In both DT-40 3KO and HEK-293 cells transiently expressing P2X(4)R, forskolin treatment enhanced ATP-mediated signaling. Specific PKA inhibitors prevented the forskolin-induced enhancement of ATP-mediated inward currents in P2X(4)R expressing HEK-293 cells. To define which region of the P2X(4)R was required for the potentiation, mutations were generated in the cytoplasmic C-terminal tail. It was determined that a limited region of the C-terminus, consisting of a non-canonical tyrosine based sorting motif, was required for the effects of PKA. Of note, this region does not harbor any recognizable PKA phosphorylation motifs, and no direct phosphorylation of P2X(4)R was detected, suggesting that PKA phosphorylation of an accessory protein interacts with the endocytosis motif in the C-terminus of the P2X(4)R. In support of this notion, using Total Internal Reflection Fluorescence Microscopy (TIRF)\\ P2X(4)-EGFP was shown to accumulate at/near the plasma membrane following forskolin treatment. In addition, disrupting the endocytosis machinery using a dominant-negative dynamin construct also prevented the PKA-mediated enhancement of ATP-stimulated Ca(2+) signals. Our results are consistent with a novel mechanism of P2XR regulation, whereby PKA activity, without directly phosphorylating P2X(4)R, markedly enhances ATP-stimulated P2X(4)R currents and hence cytosolic Ca(2+) signals. This may occur at least in part, by altering the trafficking of a population of

  12. Autonomous requirements of the Menkes disease protein in the nervous system.

    Science.gov (United States)

    Hodgkinson, Victoria L; Zhu, Sha; Wang, Yanfang; Ladomersky, Erik; Nickelson, Karen; Weisman, Gary A; Lee, Jaekwon; Gitlin, Jonathan D; Petris, Michael J

    2015-11-15

    Menkes disease is a fatal neurodegenerative disorder arising from a systemic copper deficiency caused by loss-of-function mutations in a ubiquitously expressed copper transporter, ATP7A. Although this disorder reveals an essential role for copper in the developing human nervous system, the role of ATP7A in the pathogenesis of signs and symptoms in affected patients, including severe mental retardation, ataxia, and excitotoxic seizures, remains unknown. To directly examine the role of ATP7A within the central nervous system, we generated Atp7a(Nes) mice, in which the Atp7a gene was specifically deleted within neural and glial cell precursors without impairing systemic copper homeostasis, and compared these mice with the mottled brindle (mo-br) mutant, a murine model of Menkes disease in which Atp7a is defective in all cells. Whereas mo-br mice displayed neurodegeneration, demyelination, and 100% mortality prior to weaning, the Atp7a(Nes) mice showed none of these phenotypes, exhibiting only mild sensorimotor deficits, increased anxiety, and susceptibility to NMDA-induced seizure. Our results indicate that the pathophysiology of severe neurological signs and symptoms in Menkes disease is the result of copper deficiency within the central nervous system secondary to impaired systemic copper homeostasis and does not arise from an intrinsic lack of ATP7A within the developing brain. Furthermore, the sensorimotor deficits, hypophagia, anxiety, and sensitivity to NMDA-induced seizure in the Atp7a(Nes) mice reveal unique autonomous requirements for ATP7A in the nervous system. Taken together, these data reveal essential roles for copper acquisition in the central nervous system in early development and suggest novel therapeutic approaches in affected patients.

  13. The Microtubule Regulatory Protein Stathmin Is Required to Maintain the Integrity of Axonal Microtubules in Drosophila.

    Directory of Open Access Journals (Sweden)

    Jason E Duncan

    Full Text Available Axonal transport, a form of long-distance, bi-directional intracellular transport that occurs between the cell body and synaptic terminal, is critical in maintaining the function and viability of neurons. We have identified a requirement for the stathmin (stai gene in the maintenance of axonal microtubules and regulation of axonal transport in Drosophila. The stai gene encodes a cytosolic phosphoprotein that regulates microtubule dynamics by partitioning tubulin dimers between pools of soluble tubulin and polymerized microtubules, and by directly binding to microtubules and promoting depolymerization. Analysis of stai function in Drosophila, which has a single stai gene, circumvents potential complications with studies performed in vertebrate systems in which mutant phenotypes may be compensated by genetic redundancy of other members of the stai gene family. This has allowed us to identify an essential function for stai in the maintenance of the integrity of axonal microtubules. In addition to the severe disruption in the abundance and architecture of microtubules in the axons of stai mutant Drosophila, we also observe additional neurological phenotypes associated with loss of stai function including a posterior paralysis and tail-flip phenotype in third instar larvae, aberrant accumulation of transported membranous organelles in stai deficient axons, a progressive bang-sensitive response to mechanical stimulation reminiscent of the class of Drosophila mutants used to model human epileptic seizures, and a reduced adult lifespan. Reductions in the levels of Kinesin-1, the primary anterograde motor in axonal transport, enhance these phenotypes. Collectively, our results indicate that stai has an important role in neuronal function, likely through the maintenance of microtubule integrity in the axons of nerves of the peripheral nervous system necessary to support and sustain long-distance axonal transport.

  14. Autonomous requirements of the Menkes disease protein in the nervous system

    Science.gov (United States)

    Hodgkinson, Victoria L.; Zhu, Sha; Wang, Yanfang; Ladomersky, Erik; Nickelson, Karen; Weisman, Gary A.; Lee, Jaekwon; Gitlin, Jonathan D.

    2015-01-01

    Menkes disease is a fatal neurodegenerative disorder arising from a systemic copper deficiency caused by loss-of-function mutations in a ubiquitously expressed copper transporter, ATP7A. Although this disorder reveals an essential role for copper in the developing human nervous system, the role of ATP7A in the pathogenesis of signs and symptoms in affected patients, including severe mental retardation, ataxia, and excitotoxic seizures, remains unknown. To directly examine the role of ATP7A within the central nervous system, we generated Atp7aNes mice, in which the Atp7a gene was specifically deleted within neural and glial cell precursors without impairing systemic copper homeostasis, and compared these mice with the mottled brindle (mo-br) mutant, a murine model of Menkes disease in which Atp7a is defective in all cells. Whereas mo-br mice displayed neurodegeneration, demyelination, and 100% mortality prior to weaning, the Atp7aNes mice showed none of these phenotypes, exhibiting only mild sensorimotor deficits, increased anxiety, and susceptibility to NMDA-induced seizure. Our results indicate that the pathophysiology of severe neurological signs and symptoms in Menkes disease is the result of copper deficiency within the central nervous system secondary to impaired systemic copper homeostasis and does not arise from an intrinsic lack of ATP7A within the developing brain. Furthermore, the sensorimotor deficits, hypophagia, anxiety, and sensitivity to NMDA-induced seizure in the Atp7aNes mice reveal unique autonomous requirements for ATP7A in the nervous system. Taken together, these data reveal essential roles for copper acquisition in the central nervous system in early development and suggest novel therapeutic approaches in affected patients. PMID:26269458

  15. RNA synthesis by the brome mosaic virus RNA-dependent RNA polymerase in human cells reveals requirements for de novo initiation and protein-protein interaction.

    Science.gov (United States)

    Subba-Reddy, Chennareddy V; Tragesser, Brady; Xu, Zhili; Stein, Barry; Ranjith-Kumar, C T; Kao, C Cheng

    2012-04-01

    Brome mosaic virus (BMV) is a model positive-strand RNA virus whose replication has been studied in a number of surrogate hosts. In transiently transfected human cells, the BMV polymerase 2a activated signaling by the innate immune receptor RIG-I, which recognizes de novo-initiated non-self-RNAs. Active-site mutations in 2a abolished RIG-I activation, and coexpression of the BMV 1a protein stimulated 2a activity. Mutations previously shown to abolish 1a and 2a interaction prevented the 1a-dependent enhancement of 2a activity. New insights into 1a-2a interaction include the findings that helicase active site of 1a is required to enhance 2a polymerase activity and that negatively charged amino acid residues between positions 110 and 120 of 2a contribute to interaction with the 1a helicase-like domain but not to the intrinsic polymerase activity. Confocal fluorescence microscopy revealed that the BMV 1a and 2a colocalized to perinuclear region in human cells. However, no perinuclear spherule-like structures were detected in human cells by immunoelectron microscopy. Sequencing of the RNAs coimmunoprecipitated with RIG-I revealed that the 2a-synthesized short RNAs are derived from the message used to translate 2a. That is, 2a exhibits a strong cis preference for BMV RNA2. Strikingly, the 2a RNA products had initiation sequences (5'-GUAAA-3') identical to those from the 5' sequence of the BMV genomic RNA2 and RNA3. These results show that the BMV 2a polymerase does not require other BMV proteins to initiate RNA synthesis but that the 1a helicase domain, and likely helicase activity, can affect RNA synthesis by 2a.

  16. Fragile X protein FMRP is required for homeostatic plasticity and regulation of synaptic strength by retinoic acid.

    Science.gov (United States)

    Soden, Marta E; Chen, Lu

    2010-12-15

    Homeostatic synaptic plasticity adjusts the strength of synapses during global changes in neural activity, thereby stabilizing the overall activity of neural networks. Suppression of synaptic activity increases synaptic strength by inducing synthesis of retinoic acid (RA), which activates postsynaptic synthesis of AMPA-type glutamate receptors (AMPARs) in dendrites and promotes synaptic insertion of newly synthesized AMPARs. Here, we show that fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates dendritic protein synthesis, is essential for increases in synaptic strength induced by RA or by blockade of neural activity in the mouse hippocampus. Although activity-dependent RA synthesis is maintained in Fmr1 knock-out neurons, RA-dependent dendritic translation of GluR1-type AMPA receptors is impaired. Intriguingly, FMRP is only required for the form of homeostatic plasticity that is dependent on both RA signaling and local protein synthesis. Postsynaptic expression of wild-type or mutant FMRP(I304N) in knock-out neurons reduced the total, surface, and synaptic levels of AMPARs, implying a role for FMRP in regulating AMPAR abundance. Expression of FMRP lacking the RGG box RNA-binding domain had no effect on AMPAR levels. Importantly, postsynaptic expression of wild-type FMRP, but not FMRP(I304N) or FMRPΔRGG, restored synaptic scaling when expressed in knock-out neurons. Together, these findings identify an unanticipated role for FMRP in regulating homeostatic synaptic plasticity downstream of RA. Our results raise the possibility that at least some of the symptoms of fragile X syndrome reflect impaired homeostatic plasticity and impaired RA signaling.

  17. Dissecting CNBP, a zinc-finger protein required for neural crest development, in its structural and functional domains.

    Science.gov (United States)

    Armas, Pablo; Agüero, Tristán H; Borgognone, Mariana; Aybar, Manuel J; Calcaterra, Nora B

    2008-10-17

    Cellular nucleic-acid-binding protein (CNBP) plays an essential role in forebrain and craniofacial development by controlling cell proliferation and survival to mediate neural crest expansion. CNBP binds to single-stranded nucleic acids and displays nucleic acid chaperone activity in vitro. The CNBP family shows a conserved modular organization of seven Zn knuckles and an arginine-glycine-glycine (RGG) box between the first and second Zn knuckles. The participation of these structural motifs in CNBP biochemical activities has still not been addressed. Here, we describe the generation of CNBP mutants that dissect the protein into regions with structurally and functionally distinct properties. Mutagenesis approaches were followed to generate: (i) an amino acid replacement that disrupted the fifth Zn knuckle; (ii) N-terminal deletions that removed the first Zn knuckle and the RGG box, or the RGG box alone; and (iii) a C-terminal deletion that eliminated the three last Zn knuckles. Mutant proteins were overexpressed in Escherichia coli, purified, and used to analyze their biochemical features in vitro, or overexpressed in Xenopus laevis embryos to study their function in vivo during neural crest cell development. We found that the Zn knuckles are required, but not individually essential, for CNBP biochemical activities, whereas the RGG box is essential for RNA-protein binding and nucleic acid chaperone activity. Removal of the RGG box allowed CNBP to preserve a weak single-stranded-DNA-binding capability. A mutant mimicking the natural N-terminal proteolytic CNBP form behaved as the RGG-deleted mutant. By gain-of-function and loss-of-function experiments in Xenopus embryos, we confirmed the participation of CNBP in neural crest development, and we demonstrated that the CNBP mutants lacking the N-terminal region or the RGG box alone may act as dominant negatives in vivo. Based on these data, we speculate about the existence of a specific proteolytic mechanism for the

  18. Tribolium castaneum RR-1 cuticular protein TcCPR4 is required for formation of pore canals in rigid cuticle.

    Directory of Open Access Journals (Sweden)

    Mi Young Noh

    2015-02-01

    Full Text Available Insect cuticle is composed mainly of structural proteins and the polysaccharide chitin. The CPR family is the largest family of cuticle proteins (CPs, which can be further divided into three subgroups based on the presence of one of the three presumptive chitin-binding sequence motifs denoted as Rebers-Riddiford (R&R consensus sequence motifs RR-1, RR-2 and RR-3. The TcCPR27 protein containing the RR-2 motif is one of the most abundant CPs present both in the horizontal laminae and in vertical pore canals in the procuticle of rigid cuticle found in the elytron of the red flour beetle, Tribolium castaneum. Depletion of TcCPR27 by RNA interference (RNAi causes both unorganized laminae and pore canals, resulting in malformation and weakening of the elytron. In this study, we investigated the function(s of another CP, TcCPR4, which contains the RR-1 motif and is easily extractable from elytra after RNAi to deplete the level of TcCPR27. Transcript levels of the TcCPR4 gene are dramatically increased in 3 d-old pupae when adult cuticle synthesis begins. Immunohistochemical studies revealed that TcCPR4 protein is present in the rigid cuticles of the dorsal elytron, ventral abdomen and leg but not in the flexible cuticles of the hindwing and dorsal abdomen of adult T. castaneum. Immunogold labeling and transmission electron microscopic analyses revealed that TcCPR4 is predominantly localized in pore canals and regions around the apical plasma membrane protrusions into the procuticle of rigid adult cuticles. RNAi for TcCPR4 resulted in an abnormal shape of the pore canals with amorphous pore canal fibers (PCFs in their lumen. These results support the hypothesis that TcCPR4 is required for achieving proper morphology of the vertical pore canals and PCFs that contribute to the assembly of a cuticle that is both lightweight and rigid.

  19. Inactivation of retinoblastoma protein does not overcome the requirement for human cytomegalovirus UL97 in lamina disruption and nuclear egress.

    Science.gov (United States)

    Reim, Natalia I; Kamil, Jeremy P; Wang, Depeng; Lin, Alison; Sharma, Mayuri; Ericsson, Maria; Pesola, Jean M; Golan, David E; Coen, Donald M

    2013-05-01

    Human cytomegalovirus (HCMV) encodes one conventional protein kinase, UL97. During infection, UL97 phosphorylates the retinoblastoma tumor suppressor protein (pRb) on sites ordinarily phosphorylated by cyclin-dependent kinases (CDK), inactivating the ability of pRb to repress host genes required for cell cycle progression to S phase. UL97 is important for viral DNA synthesis in quiescent cells, but this function can be replaced by human papillomavirus type 16 E7, which targets pRb for degradation. However, viruses in which E7 replaces UL97 are still defective for virus production. UL97 is also required for efficient nuclear egress of viral nucleocapsids, which is associated with disruption of the nuclear lamina during infection, and phosphorylation of lamin A/C on serine 22, which antagonizes lamin polymerization. We investigated whether inactivation of pRb might overcome the requirement of UL97 for these roles, as pRb inactivation induces CDK1, and CDK1 phosphorylates lamin A/C on serine 22. We found that lamin A/C serine 22 phosphorylation during HCMV infection correlated with expression of UL97 and was considerably delayed in UL97-null mutants, even when E7 was expressed. E7 failed to restore gaps in the nuclear lamina seen in wild-type but not UL97-null virus infections. In electron microscopy analyses, a UL97-null virus expressing E7 was as impaired as a UL97-null mutant in cytoplasmic accumulation of viral nucleocapsids. Our results demonstrate that pRb inactivation is insufficient to restore efficient viral nuclear egress of HCMV in the absence of UL97 and instead argue further for a direct role of UL97 in this stage of the infectious cycle.

  20. Differential requirement of the epidermal growth factor receptor for G protein-mediated activation of transcription factors by lysophosphatidic acid

    Directory of Open Access Journals (Sweden)

    Dent Paul

    2010-01-01

    Full Text Available Abstract Background The role of the epidermal growth factor receptor (EGFR and other receptor tyrosine kinases (RTKs in provoking biological actions of G protein-coupled receptors (GPCRs has been one of the most disputed subjects in the field of GPCR signal transduction. The purpose of the current study is to identify EGFR-mediated mechanisms involved in activation of G protein cascades and the downstream transcription factors by lysophosphatidic acid (LPA. Results In ovarian cancer cells highly responsive to LPA, activation of AP-1 by LPA was suppressed by inhibition of EGFR, an effect that could be reversed by co-stimulation of another receptor tyrosine kinase c-Met with hepatocyte growth factor, indicating that LPA-mediated activation of AP-1 requires activity of a RTK, not necessarily EGFR. Induction of AP-1 components by LPA lied downstream of Gi, G12/13, and Gq. Activation of the effectors of Gi, but not Gq or G12/13 was sensitive to inhibition of EGFR. In contrast, LPA stimulated another prominent transcription factor NF-κB via the Gq-PKC pathway in an EGFR-independent manner. Consistent with the importance of Gi-elicited signals in a plethora of biological processes, LPA-induced cytokine production, cell proliferation, migration and invasion require intact EGFR. Conclusions An RTK activity is required for activation of the AP-1 transcription factor and other Gi-dependent cellular responses to LPA. In contrast, activation of G12/13, Gq and Gq-elicited NF-κB by LPA is independent of such an input. These results provide a novel insight into the role of RTK in GPCR signal transduction and biological functions.

  1. THE REQUIREMENTS OF CRUDE PROTEIN BY LARGE WHITE BREEDING SOWS AND PIGLETS IN GHANA

    Directory of Open Access Journals (Sweden)

    S.W.A. RHULE

    2012-07-01

    Full Text Available Eighteen Large White gilts at an average initial live weight of 100.39kg were distributed by completely randomized block design over three treatments. There were three cereals-based diets with three different calculated levels of crude protein (CP namely control breeder diet, Diet 1 (13.16% CP, Diet 2 (12.56% CP and Diet 3 (12.01% CP with corresponding lactation diets namely control lactation Diet 1 (LD1 (16.00% CP, Diet 2 (LD2 (14.50%CP and Diet 3 (LD3 (13.20%CP. Additionally there were three creep diets comprising control Diet 1 (CD1 with 23.01%CP, Diet 2 (CD2 with 21.46% CP and Diet 3 (CD3 with 20.00% CP. The average live litter size of sows were 8.50, 8.40 and 6.67 on Diets 1, 2, and 3 respectively (P>0.05. The number of piglets weaned decreased with decreasing CP in the diet being 7.83. 7.80 and 5.33 on Diets 1, 2 and 3 respectively but were not found to be significantly (P>0.05 different. The average birth weights of the piglets were similar at 1.45, 1.34 and 1.40 kg on Diets 1, 2 and 3 respectively (P>0.05. The milk yield by the sows at 28 days of lactation were found to be 3.06, 3.03 and 5.44kg/day on LD1, 2 and 3 and significantly (P<0.05 different being higher on the lowest CP diet. There was inconsistency with the milk yield at 42 days of lactation with significantly (P<0.05 different values of 4.87, 8.33 and 3.60kg/day on lactation with lactation diet 1, 2, and 3 respectively. It was observed that live weight gains by the sows during gestation significantly (P<0.05 increased with decreasing CP levels in the diets and were 30.3, 36.3 and 34.0 kg on Diets 1, 2 and 3 respectively. The ADGs of the piglets on the creep diets were 0.22, 0.17 and 0.19 kg/day on CD1, CD2 and CD3 respectively, and found to be significantly (P<0.05 different. The study indicated that decreasing levels of CP in the diet at gestation could affect the reproductive performance of the Sow. Decreasing CP in the lactation diet significantly affected sow performance

  2. Major basic protein from eosinophils and myeloperoxidase from neutrophils are required for protective immunity to Strongyloides stercoralis in mice.

    Science.gov (United States)

    O'Connell, Amy E; Hess, Jessica A; Santiago, Gilberto A; Nolan, Thomas J; Lok, James B; Lee, James J; Abraham, David

    2011-07-01

    Eosinophils and neutrophils contribute to larval killing during the primary immune response, and neutrophils are effector cells in the secondary response to Strongyloides stercoralis in mice. The objective of this study was to determine the molecular mechanisms used by eosinophils and neutrophils to control infections with S. stercoralis. Using mice deficient in the eosinophil granule products major basic protein (MBP) and eosinophil peroxidase (EPO), it was determined that eosinophils kill the larvae through an MBP-dependent mechanism in the primary immune response if other effector cells are absent. Infecting PHIL mice, which are eosinophil deficient, with S. stercoralis resulted in development of primary and secondary immune responses that were similar to those of wild-type mice, suggesting that eosinophils are not an absolute requirement for larval killing or development of secondary immunity. Treating PHIL mice with a neutrophil-depleting antibody resulted in a significant impairment in larval killing. Naïve and immunized mice with neutrophils deficient in myeloperoxidase (MPO) infected with S. stercoralis had significantly decreased larval killing. It was concluded that there is redundancy in the primary immune response, with eosinophils killing the larvae through an MBP-dependent mechanism and neutrophils killing the worms through an MPO-dependent mechanism. Eosinophils are not required for the development or function of secondary immunity, but MPO from neutrophils is required for protective secondary immunity.

  3. Analysis of the spacial requirements for RNA-protein interactions within the N antitermination complex of bacteriophage lambda.

    Science.gov (United States)

    Horiya, Satoru; Inaba, Mitsuru; Koh, Chang-Song; Uehara, Hiroaki; Masui, Naomi; Ishibashi, Masaya; Matsufuji, Senya; Harada, Kazuo

    2009-01-01

    In bacteriophage lambda, formation of a transcriptional antitermination complex consisting of the lambda N protein, nut RNA transcript (boxA-boxB), host factors, and RNA polymerase is mediated by the interaction of the boxB RNA with the RNA-binding domain of N. In order to understand the spacial requirements of this boxB/N interaction within the complex, the effects of changes in the length of the nut site linker, the boxB stem, and the peptide spacer connecting the RNA-binding domain and activation domain of N were examined using a bacterial reporter system. As a result, we found that the requirements for the boxB stem length and N peptide linker length were optimized and strict. In contrast, when the boxB/N interaction was replaced by heterologous RNA/peptide interactions, the strict requirement for the length of the peptide linker and the RNA stem was relaxed, presumably due to the absence of the interaction between boxB/N and the host factor NusA in the wild-type complex. It was also shown that the decrease in activity upon stem lengthening could be partially suppressed by simultaneous lengthening of the RNA spacer, suggesting that a further understanding of the organization of the antitermination complex may provide insights into the engineering of functional ribonucleoprotein complexes.

  4. 40 CFR 174.518 - Bacillus thuringiensis Cry3Bb1 protein in corn; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry3Bb1 protein... PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.518 Bacillus thuringiensis Cry3Bb1 protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus...

  5. 40 CFR 174.511 - Bacillus thuringiensis Cry1Ab protein in all plants; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry1Ab protein... PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.511 Bacillus thuringiensis Cry1Ab protein in all plants; exemption from the requirement of a tolerance. Residues of...

  6. 40 CFR 174.510 - Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry1Ac protein... PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.510 Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance. Residues of...

  7. A component of the Sec61 ER protein transporting pore is required for plant susceptibility to powdery mildew

    DEFF Research Database (Denmark)

    Zhang, Wen-Jing; Hanisch, Susanne; Kwaaitaal, Mark Adrianus Cornelis J;

    2013-01-01

    the EHM, the fungus obtains nutrients from and secretes effector proteins into the plant cell. In the plant cell these effectors interfere with cellular processes such as pathogen defense and membrane trafficking. However, the mechanisms behind effector delivery are largely unknown. This paper provides......Biotrophic pathogens, like the powdery mildew fungi, require living plant cells for their growth and reproduction. During infection, a specialized structure called the haustorium is formed by the fungus. The haustorium is surrounded by a plant cell-derived extrahaustorial membrane (EHM). Over...... a model for and new insights into a putative transfer mechanism of effectors into the plant cell. We show that silencing of the barley Sec61βa transcript results in decreased susceptibility to the powdery mildew fungus. HvSec61βa is a component of both the endoplasmic reticulum (ER) translocon...

  8. Normocyte-binding protein required for human erythrocyte invasion by the zoonotic malaria parasitePlasmodium knowlesi

    KAUST Repository

    Moon, Robert W.

    2016-06-15

    The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen.

  9. DEAD-box protein Ddx46 is required for the development of the digestive organs and brain in zebrafish.

    Directory of Open Access Journals (Sweden)

    Shunya Hozumi

    Full Text Available Spatially and temporally controlled gene expression, including transcription, several mRNA processing steps, and the export of mature mRNA to the cytoplasm, is essential for developmental processes. It is well known that RNA helicases of the DExD/H-box protein family are involved in these gene expression processes, including transcription, pre-mRNA splicing, and rRNA biogenesis. Although one DExD/H-box protein, Prp5, a homologue of vertebrate Ddx46, has been shown to play important roles in pre-mRNA splicing in yeast, the in vivo function of Ddx46 remains to be fully elucidated in metazoans. In this study, we isolated zebrafish morendo (mor, a mutant that shows developmental defects in the digestive organs and brain, and found that it encodes Ddx46. The Ddx46 transcript is maternally supplied, and as development proceeds in zebrafish larvae, its ubiquitous expression gradually becomes restricted to those organs. The results of whole-mount in situ hybridization showed that the expression of various molecular markers in these organs is considerably reduced in the Ddx46 mutant. Furthermore, splicing status analysis with RT-PCR revealed unspliced forms of mRNAs in the digestive organ and brain tissues of the Ddx46 mutant, suggesting that Ddx46 may be required for pre-mRNA splicing during zebrafish development. Therefore, our results suggest a model in which zebrafish Ddx46 is required for the development of the digestive organs and brain, possibly through the control of pre-mRNA splicing.

  10. SH-2-containing protein tyrosine phosphatase 1 is required for IL-4-induced IL-4R expression in spleen cells

    Institute of Scientific and Technical Information of China (English)

    CAO Xin; HUANG Zan; FAN Jingyi

    2005-01-01

    To investigate the role of SH-2-containing protein tyrosine phosphatase 1, SHP-1, in IL-4-induced IL-4 receptor (IL-4R) expression, we examined IL-4 receptor α-chain (IL-4Rα) mRNA expression in Na3VO4-treated wild type (WT) spleen cells and measured IL-4R mRNA in IL-4-stimulated spleen cells of viable motheaten mice (mev/mev). It is found that IL-4-induced IL-4R mRNA expression was impaired in Na3VO4-treated WT spleen cells and IL-4-stimulated mev/mev spleen cells. Here we show that the impaired IL-4-induced IL-4RαmRNA expression was due to reduced expression of IL-4R that led to impaired STAT6 signaling. We further demonstrate that reduction of IL-4Rαprotein expression in mev/mev spleen cells was due to alteration in cell compositions. In mev/mev spleen, the percentages of CD4+, CD8+, and CD19+ cells expressing relatively high levels of IL-4R were reduced dramatically while the percentages of Mac-1+ and Gr-1+ cells with relative low levels of IL-4R increased greatly. Despite the profound effect of reduced expression of IL-4R protein, the IL-4RαmRNA expression was comparable in spleen cells of littermate control mice (+/() and mev/mev mice and no differences were found in B cells, T cells, and macrophages, suggesting cell type-specific downregulation of IL-4R expression in macrophages through a posttranscriptional mechanism. Our study suggests that SHP-1 is required for IL-4-meidated function and indirectly regulates IL-4-meidated function in spleen cells by affecting hematopoiesis.

  11. The N-terminus of murine leukaemia virus p12 protein is required for mature core stability.

    Directory of Open Access Journals (Sweden)

    Darren J Wight

    2014-10-01

    Full Text Available The murine leukaemia virus (MLV gag gene encodes a small protein called p12 that is essential for the early steps of viral replication. The N- and C-terminal regions of p12 are sequentially acting domains, both required for p12 function. Defects in the C-terminal domain can be overcome by introducing a chromatin binding motif into the protein. However, the function of the N-terminal domain remains unknown. Here, we undertook a detailed analysis of the effects of p12 mutation on incoming viral cores. We found that both reverse transcription complexes and isolated mature cores from N-terminal p12 mutants have altered capsid complexes compared to wild type virions. Electron microscopy revealed that mature N-terminal p12 mutant cores have different morphologies, although immature cores appear normal. Moreover, in immunofluorescent studies, both p12 and capsid proteins were lost rapidly from N-terminal p12 mutant viral cores after entry into target cells. Importantly, we determined that p12 binds directly to the MLV capsid lattice. However, we could not detect binding of an N-terminally altered p12 to capsid. Altogether, our data imply that p12 stabilises the mature MLV core, preventing premature loss of capsid, and that this is mediated by direct binding of p12 to the capsid shell. In this manner, p12 is also retained in the pre-integration complex where it facilitates tethering to mitotic chromosomes. These data also explain our previous observations that modifications to the N-terminus of p12 alter the ability of particles to abrogate restriction by TRIM5alpha and Fv1, factors that recognise viral capsid lattices.

  12. The N-Terminus of Murine Leukaemia Virus p12 Protein Is Required for Mature Core Stability

    Science.gov (United States)

    Wight, Darren J.; Boucherit, Virginie C.; Wanaguru, Madushi; Elis, Efrat; Hirst, Elizabeth M. A.; Li, Wilson; Ehrlich, Marcelo; Bacharach, Eran; Bishop, Kate N.

    2014-01-01

    The murine leukaemia virus (MLV) gag gene encodes a small protein called p12 that is essential for the early steps of viral replication. The N- and C-terminal regions of p12 are sequentially acting domains, both required for p12 function. Defects in the C-terminal domain can be overcome by introducing a chromatin binding motif into the protein. However, the function of the N-terminal domain remains unknown. Here, we undertook a detailed analysis of the effects of p12 mutation on incoming viral cores. We found that both reverse transcription complexes and isolated mature cores from N-terminal p12 mutants have altered capsid complexes compared to wild type virions. Electron microscopy revealed that mature N-terminal p12 mutant cores have different morphologies, although immature cores appear normal. Moreover, in immunofluorescent studies, both p12 and capsid proteins were lost rapidly from N-terminal p12 mutant viral cores after entry into target cells. Importantly, we determined that p12 binds directly to the MLV capsid lattice. However, we could not detect binding of an N-terminally altered p12 to capsid. Altogether, our data imply that p12 stabilises the mature MLV core, preventing premature loss of capsid, and that this is mediated by direct binding of p12 to the capsid shell. In this manner, p12 is also retained in the pre-integration complex where it facilitates tethering to mitotic chromosomes. These data also explain our previous observations that modifications to the N-terminus of p12 alter the ability of particles to abrogate restriction by TRIM5alpha and Fv1, factors that recognise viral capsid lattices. PMID:25356837

  13. The Loricrin-Like Protein (LLP) of Phytophthora infestans Is Required for Oospore Formation and Plant Infection

    Science.gov (United States)

    Guo, Ting; Wang, Xiao-Wen; Shan, Kun; Sun, Wenxian; Guo, Li-Yun

    2017-01-01

    Loricrin-like protein (LLP) is characterized by a high content of glycine residues and is a major component of plant cell wall. Here, we identified a Phytophthora infestans ortholog of plant LLP, named PiLLP. In P. infestans, PiLLP is strongly expressed in asexual and sexual developmental stages, including in sporangia, zoospores and germinating cysts, and during oospore formation, as well as in the early stages of infection and during hydrogen peroxide stress. Compared with the wild type, the PiLLP-silenced transformants were defective in oospore formation, had slower colony expansion rates, produced less sporangia with lower germination and zoospore-release rates, and were more sensitive to hydrogen peroxide. Moreover, Nile red staining, and PiLLP-red fluorescent protein fusions indicated that PiLLP is involved in oogonia formation. The silenced transformants also had severely diminished virulence levels that could be partially restored with diphenyleneiodium treatments. The analysis of catalase activity showed a decrease of catalase activity in silenced transformants. Thus, PiLLP is important for sexual and asexual reproduction, and is required for oxidative stress tolerance and plant infection. PMID:28232841

  14. Sterol Regulatory Element Binding Protein (Srb1) Is Required for Hypoxic Adaptation and Virulence in the Dimorphic Fungus Histoplasma capsulatum

    Science.gov (United States)

    DuBois, Juwen C.; Smulian, A. George

    2016-01-01

    The Histoplasma capsulatum sterol regulatory element binding protein (SREBP), Srb1 is a member of the basic helix-loop-helix (bHLH), leucine zipper DNA binding protein family of transcription factors that possess a unique tyrosine (Y) residue instead of an arginine (R) residue in the bHLH region. We have determined that Srb1 message levels increase in a time dependent manner during growth under oxygen deprivation (hypoxia). To further understand the role of Srb1 during infection and hypoxia, we silenced the gene encoding Srb1 using RNA interference (RNAi); characterized the resulting phenotype, determined its response to hypoxia, and its ability to cause disease within an infected host. Silencing of Srb1 resulted in a strain of H. capsulatum that is incapable of surviving in vitro hypoxia. We found that without complete Srb1 expression, H. capsulatum is killed by murine macrophages and avirulent in mice given a lethal dose of yeasts. Additionally, silencing Srb1 inhibited the hypoxic upregulation of other known H. capsulatum hypoxia-responsive genes (HRG), and genes that encode ergosterol biosynthetic enzymes. Consistent with these regulatory functions, Srb1 silenced H. capsulatum cells were hypersensitive to the antifungal azole drug itraconazole. These data support the theory that the H. capsulatum SREBP is critical for hypoxic adaptation and is required for H. capsulatum virulence. PMID:27711233

  15. Identification of DSB-1, a protein required for initiation of meiotic recombination in Caenorhabditis elegans, illuminates a crossover assurance checkpoint.

    Directory of Open Access Journals (Sweden)

    Ericca L Stamper

    Full Text Available Meiotic recombination, an essential aspect of sexual reproduction, is initiated by programmed DNA double-strand breaks (DSBs. DSBs are catalyzed by the widely-conserved Spo11 enzyme; however, the activity of Spo11 is regulated by additional factors that are poorly conserved through evolution. To expand our understanding of meiotic regulation, we have characterized a novel gene, dsb-1, that is specifically required for meiotic DSB formation in the nematode Caenorhabditis elegans. DSB-1 localizes to chromosomes during early meiotic prophase, coincident with the timing of DSB formation. DSB-1 also promotes normal protein levels and chromosome localization of DSB-2, a paralogous protein that plays a related role in initiating recombination. Mutations that disrupt crossover formation result in prolonged DSB-1 association with chromosomes, suggesting that nuclei may remain in a DSB-permissive state. Extended DSB-1 localization is seen even in mutants with defects in early recombination steps, including spo-11, suggesting that the absence of crossover precursors triggers the extension. Strikingly, failure to form a crossover precursor on a single chromosome pair is sufficient to extend the localization of DSB-1 on all chromosomes in the same nucleus. Based on these observations we propose a model for crossover assurance that acts through DSB-1 to maintain a DSB-permissive state until all chromosome pairs acquire crossover precursors. This work identifies a novel component of the DSB machinery in C. elegans, and sheds light on an important pathway that regulates DSB formation for crossover assurance.

  16. Identification of DSB-1, a protein required for initiation of meiotic recombination in Caenorhabditis elegans, illuminates a crossover assurance checkpoint.

    Science.gov (United States)

    Stamper, Ericca L; Rodenbusch, Stacia E; Rosu, Simona; Ahringer, Julie; Villeneuve, Anne M; Dernburg, Abby F

    2013-01-01

    Meiotic recombination, an essential aspect of sexual reproduction, is initiated by programmed DNA double-strand breaks (DSBs). DSBs are catalyzed by the widely-conserved Spo11 enzyme; however, the activity of Spo11 is regulated by additional factors that are poorly conserved through evolution. To expand our understanding of meiotic regulation, we have characterized a novel gene, dsb-1, that is specifically required for meiotic DSB formation in the nematode Caenorhabditis elegans. DSB-1 localizes to chromosomes during early meiotic prophase, coincident with the timing of DSB formation. DSB-1 also promotes normal protein levels and chromosome localization of DSB-2, a paralogous protein that plays a related role in initiating recombination. Mutations that disrupt crossover formation result in prolonged DSB-1 association with chromosomes, suggesting that nuclei may remain in a DSB-permissive state. Extended DSB-1 localization is seen even in mutants with defects in early recombination steps, including spo-11, suggesting that the absence of crossover precursors triggers the extension. Strikingly, failure to form a crossover precursor on a single chromosome pair is sufficient to extend the localization of DSB-1 on all chromosomes in the same nucleus. Based on these observations we propose a model for crossover assurance that acts through DSB-1 to maintain a DSB-permissive state until all chromosome pairs acquire crossover precursors. This work identifies a novel component of the DSB machinery in C. elegans, and sheds light on an important pathway that regulates DSB formation for crossover assurance.

  17. Neto1 is a novel CUB-domain NMDA receptor-interacting protein required for synaptic plasticity and learning.

    Directory of Open Access Journals (Sweden)

    David Ng

    2009-02-01

    Full Text Available The N-methyl-D-aspartate receptor (NMDAR, a major excitatory ligand-gated ion channel in the central nervous system (CNS, is a principal mediator of synaptic plasticity. Here we report that neuropilin tolloid-like 1 (Neto1, a complement C1r/C1s, Uegf, Bmp1 (CUB domain-containing transmembrane protein, is a novel component of the NMDAR complex critical for maintaining the abundance of NR2A-containing NMDARs in the postsynaptic density. Neto1-null mice have depressed long-term potentiation (LTP at Schaffer collateral-CA1 synapses, with the subunit dependency of LTP induction switching from the normal predominance of NR2A- to NR2B-NMDARs. NMDAR-dependent spatial learning and memory is depressed in Neto1-null mice, indicating that Neto1 regulates NMDA receptor-dependent synaptic plasticity and cognition. Remarkably, we also found that the deficits in LTP, learning, and memory in Neto1-null mice were rescued by the ampakine CX546 at doses without effect in wild-type. Together, our results establish the principle that auxiliary proteins are required for the normal abundance of NMDAR subunits at synapses, and demonstrate that an inherited learning defect can be rescued pharmacologically, a finding with therapeutic implications for humans.

  18. An essential requirement for β1 integrin in the assembly of extracellular matrix proteins within the vascular wall.

    Science.gov (United States)

    Turlo, Kirsten A; Noel, Onika D V; Vora, Roshni; LaRussa, Marie; Fassler, Reinhard; Hall-Glenn, Faith; Iruela-Arispe, M Luisa

    2012-05-01

    β1 integrin has been shown to contribute to vascular smooth muscle cell differentiation, adhesion and mechanosensation in vitro. Here we showed that deletion of β1 integrin at the onset of smooth muscle differentiation resulted in interrupted aortic arch, aneurysms and failure to assemble extracellular matrix proteins. These defects result in lethality prior to birth. Our data indicates that β1 integrin is not required for the acquisition, but it is essential for the maintenance of the smooth muscle cell phenotype, as levels of critical smooth muscle proteins are gradually reduced in mutant mice. Furthermore, while deposition of extracellular matrix was not affected, its structure was disrupted. Interestingly, defects in extracellular matrix and vascular wall assembly, were restricted to the aortic arch and its branches, compromising the brachiocephalic and carotid arteries and to the exclusion of the descending aorta. Additional analysis of β1 integrin in the pharyngeal arch smooth muscle progenitors was performed using wnt1Cre. Neural crest cells deleted for β1 integrin were able to migrate to the pharyngeal arches and associate with endothelial lined arteries; but exhibited vascular remodeling defects and early lethality. This work demonstrates that β1 integrin is dispensable for migration and initiation of the smooth muscle differentiation program, however, it is essential for remodeling of the pharyngeal arch arteries and for the assembly of the vessel wall of their derivatives. It further establishes a critical role of β1 integrin in the protection against aneurysms that is particularly confined to the ascending aorta and its branches.

  19. Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain.

    Science.gov (United States)

    Nezis, Ioannis P; Simonsen, Anne; Sagona, Antonia P; Finley, Kim; Gaumer, Sébastien; Contamine, Didier; Rusten, Tor Erik; Stenmark, Harald; Brech, Andreas

    2008-03-24

    P62 has been proposed to mark ubiquitinated protein bodies for autophagic degradation. We report that the Drosophila melanogaster p62 orthologue, Ref(2)P, is a regulator of protein aggregation in the adult brain. We demonstrate that Ref(2)P localizes to age-induced protein aggregates as well as to aggregates caused by reduced autophagic or proteasomal activity. A similar localization to protein aggregates is also observed in D. melanogaster models of human neurodegenerative diseases. Although atg8a autophagy mutant flies show accumulation of ubiquitin- and Ref(2)P-positive protein aggregates, this is abrogated in atg8a/ref(2)P double mutants. Both the multimerization and ubiquitin binding domains of Ref(2)P are required for aggregate formation in vivo. Our findings reveal a major role for Ref(2)P in the formation of ubiquitin-positive protein aggregates both under physiological conditions and when normal protein turnover is inhibited.

  20. Amyloid precursor protein is required for normal function of the rod and cone pathways in the mouse retina.

    Directory of Open Access Journals (Sweden)

    Tracy Ho

    Full Text Available Amyloid precursor protein (APP is a transmembrane glycoprotein frequently studied for its role in Alzheimer's disease. Our recent study in APP knockout (KO mice identified an important role for APP in modulating normal neuronal development in the retina. However the role APP plays in the adult retina and whether it is required for vision is unknown. In this study we evaluated the role of APP in retinal function and morphology comparing adult wildtype (WT and APP-KO mice. APP was expressed on neuronal cells of the inner retina, including horizontal, cone bipolar, amacrine and ganglion cells in WT mice. The function of the retina was assessed using the electroretinogram and although the rod photoreceptor responses were similar in APP-KO and WT mice, the post-photoreceptor, inner retinal responses of both the rod and cone pathways were reduced in APP-KO mice. These changes in inner retinal function did not translate to a substantial change in visual acuity as assessed using the optokinetic response or to changes in the gross cellular structure of the retina. These findings indicate that APP is not required for basic visual function, but that it is involved in modulating inner retinal circuitry.

  1. MiR-155 induction by microbes/microbial ligands requires NF-kB-dependent de novo protein synthesis

    Directory of Open Access Journals (Sweden)

    Susheela eTridandapani

    2012-06-01

    Full Text Available MiR-155 regulates numerous aspects of innate and adaptive immune function. This miR is induced in response to toll-like receptor ligands, cytokines, and microbial infection. We have previously shown that miR-155 is induced in monocytes/macrophages infected with Francisella tularensis and suppresses expression of the inositol phosphatase SHIP to enhance activation of the PI3K/Akt pathway, which in turn promotes favorable responses for the host. Here we examined how miR-155 expression is regulated during infection. First, our data demonstrate that miR-155 can be induced through soluble factors of bacterial origin and not the host. Second, miR-155 induction is not a direct effect of infection and it requires NF-κB signaling to up-regulate fos/jun transcription factors. Finally, we demonstrate that the requirement for NF-κB-dependent de novo protein synthesis is globally shared by microbial ligands and live bacteria. This study provides new insight into the complex regulation of miR-155 during microbial infection.

  2. 40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis modified Cry3A... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.505 Bacillus... of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement...

  3. 40 CFR 174.506 - Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry34Ab1 and... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.506 Bacillus... Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn are exempted from the requirement of...

  4. The expression and crystallization of Cry65Aa require two C-termini, revealing a novel evolutionary strategy of Bacillus thuringiensis Cry proteins

    Science.gov (United States)

    Peng, Dong-hai; Pang, Cui-yun; Wu, Han; Huang, Qiong; Zheng, Jin-shui; Sun, Ming

    2015-01-01

    The insecticidal crystal protein (Cry) genes of Bacillus thuringiensis are a key gene resource for generating transgenic crops with pest resistance. However, many cry genes cannot be expressed or form crystals in mother cells. Here, we report a novel Cry protein gene, cry65Aa1, which exists in an operon that contains a downstream gene encoding a hypothetical protein ORF2. We demonstrated that ORF2 is required for Cry65Aa1 expression and crystallization by function as a C-terminal crystallization domain. The orf2 sequence is also required for Cry65Aa expression, because orf2 transcripts have a stabilizing effect on cry65Aa1 transcripts. Furthermore, we found that the crystallization of Cry65Aa1 required the Cry65Aa1 C-terminus in addition to ORF2 or a typical Cry protein C-terminal region. Finally, we showed that Cry65Aa1 has a selective cytotoxic effect on MDA-MB231 cancer cells. This report is the first description of a 130-kDa mass range Cry protein requiring two C-termini for crystallization. Our findings reveal a novel evolutionary strategy of Cry proteins and provide an explanation for the existence of Cry protein genes that cannot form crystals in B. thuringiensis. This study also provides a potential framework for isolating novel cry genes from “no crystal” B. thuringiensis strains. PMID:25656389

  5. Rice calcium-dependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose phosphate synthase and is required for a proper cold stress response

    KAUST Repository

    Almadanim, M. Cecília

    2017-01-19

    Calcium-dependent protein kinases (CDPKs) are involved in plant tolerance mechanisms to abiotic stresses. Although CDPKs are recognized as key messengers in signal transduction, the specific role of most members of this family remains unknown. Here we test the hypothesis that OsCPK17 plays a role in rice cold stress response by analyzing OsCPK17 knockout, silencing, and overexpressing rice lines under low temperature. Altered OsCPK17 gene expression compromises cold tolerance performance, without affecting the expression of key cold stress-inducible genes. A comparative phosphoproteomic approach led to the identification of six potential in vivo OsCPK17 targets, which are associated with sugar and nitrogen metabolism, and with osmotic regulation. To test direct interaction, in vitro kinase assays were performed, showing that the sucrose phosphate synthase OsSPS4, and the aquaporin OsPIP2;1/OsPIP2;6 are phosphorylated by OsCPK17 in a calcium-dependent manner. Altogether, our data indicates that OsCPK17 is required for a proper cold stress response in rice, likely affecting the activity of membrane channels and sugar metabolism.

  6. windbeutel, a gene required for dorsoventral patterning in Drosophila, encodes a protein that has homologies to vertebrate proteins of the endoplasmic reticulum.

    Science.gov (United States)

    Konsolaki, M; Schüpbach, T

    1998-01-01

    The formation of the dorsoventral axis of the Drosophila embryo depends on cell-cell interactions that take place in the female ovary and involve the activation of transmembrane receptors by secreted ligands. The gene windbeutel functions in the somatic follicle cells of the ovary and is required for the generation of a signal that will determine the ventral side of the embryo. This signal originates in the follicle cells during oogenesis, but its actions are only manifested after fertilization, when the egg has already been laid. We have performed a molecular analysis of windbeutel. We have found that windbeutel encodes a putative resident protein of the endoplasmic reticulum, and has homologs in rats and humans. The gene is expressed for a brief period of time in the follicle cells of the ovary, at around the time when the dorsoventral axis of the egg chamber is first established. We propose that Windbeutel is responsible for the folding and/or modification of a specific factor that is secreted from the follicle cells and participates in the activation of the ventralizing signal.

  7. Maintenance energy requirements of beef cows and relationship with cow and calf performance, metabolic hormones, and functional proteins.

    Science.gov (United States)

    Cooper-Prado, M J; Long, N M; Davis, M P; Wright, E C; Madden, R D; Dilwith, J W; Bailey, C L; Spicer, L J; Wettemann, R P

    2014-08-01

    Gestating Angus, nonlactating, spring-calving cows were used to determine variation in maintenance energy requirements (MR); to evaluate the relationship among MR and cow and calf performance, plasma concentrations of IGF-I, T4, glucose, insulin, and ruminal temperature; and to describe the LM proteome and evaluate protein abundance in cows with different MR. Cows (4 to 7 yr of age) with a BCS of 5.0 ± 0.2 and BW of 582 ± 37 kg in the second to third trimester of gestation were studied in 3 trials (trial 1, n = 23; trial 2, n = 32; trial 3, n = 38). Cows were individually fed a complete diet in amounts to meet predicted MR (Level 1 Model of NRC), and feed intake was adjusted weekly until constant BW was achieved for at least 21 d (maintenance). Cows were classified on the basis of MR as low (>0.5 SD less than mean, LMR), moderate (±0.5 SD of mean, MMR), or high (>0.5 SD more than mean, HMR) MR. Blood samples were taken at maintenance and at 2 mo postpartum in trial 2. Muscle biopsies were taken from LMR and HMR after cows consumed actual MR for 28 d (trial 2) or 21 d (trial 3). Proteins from LM were separated by 2-dimensional difference gel electrophoresis and were identified, and abundance was quantified and compared. The greatest differences in MR between cows were 29%, 24%, and 25% in trials 1, 2, and 3, respectively. Daily MR (NEm, kcal·BW(-0.75)·d(-1)) averaged 89.2 ± 6.3, 93.0 ± 4.9, and 90.4 ± 4.6 in trials 1, 2, and 3, respectively. Postpartum BW and BCS, calf birth and weaning weights, postpartum luteal activity, and ruminal temperature were not influenced by MR of the cows. Concentrations of IGF-I were greater (P = 0.001) in plasma of MMR compared with LMR cows consuming predicted MR diets, and MR was negatively correlated with concentrations of IGF-I in plasma (r = -0.38; P = 0.05) at 2 mo postpartum. A total of 103 proteins were isolated from LM; 52 gene products were identified. Abundance of specific proteins in the LM was not influenced (P > 0

  8. Guanine nucleotide-binding protein (Gα) endocytosis by a cascade of ubiquitin binding domain proteins is required for sustained morphogenesis and proper mating in yeast.

    Science.gov (United States)

    Dixit, Gauri; Baker, Rachael; Sacks, Carly M; Torres, Matthew P; Dohlman, Henrik G

    2014-05-23

    Heterotrimeric G proteins are well known to transmit signals from cell surface receptors to intracellular effector proteins. There is growing appreciation that G proteins are also present at endomembrane compartments, where they can potentially interact with a distinct set of signaling proteins. Here, we examine the cellular trafficking function of the G protein α subunit in yeast, Gpa1. Gpa1 contains a unique 109-amino acid insert within the α-helical domain that undergoes a variety of posttranslational modifications. Among these is monoubiquitination, catalyzed by the NEDD4 family ubiquitin ligase Rsp5. Using a newly optimized method for G protein purification together with biophysical measures of structure and function, we show that the ubiquitination domain does not influence enzyme activity. By screening a panel of 39 gene deletion mutants, each lacking a different ubiquitin binding domain protein, we identify seven that are necessary to deliver Gpa1 to the vacuole compartment including four proteins (Ede1, Bul1, Ddi1, and Rup1) previously not known to be involved in this process. Finally, we show that proper endocytosis of the G protein is needed for sustained cellular morphogenesis and mating in response to pheromone stimulation. We conclude that a cascade of ubiquitin-binding proteins serves to deliver the G protein to its final destination within the cell. In this instance and in contrast to the previously characterized visual system, endocytosis from the plasma membrane is needed for proper signal transduction rather than for signal desensitization.

  9. Transport of the GlcNAc-1-phosphotransferase α/β-subunit precursor protein to the Golgi apparatus requires a combinatorial sorting motif.

    Science.gov (United States)

    Franke, Mine; Braulke, Thomas; Storch, Stephan

    2013-01-11

    The Golgi-resident N-acetylglucosamine-1-phosphotransferase (PT) complex is composed of two α-, β-, and γ-subunits and represents the key enzyme for the biosynthesis of mannose 6-phosphate recognition marker on soluble lysosomal proteins. Mutations in the PT complex cause the lysosomal storage diseases mucolipidosis II and III. A prerequisite for the enzymatic activity is the site-1 protease-mediated cleavage of the PT α/β-subunit precursor protein in the Golgi apparatus. Here, we have investigated structural requirements of the PT α/β-subunit precursor protein for its efficient export from the endoplasmic reticulum (ER). Both wild-type and a cleavage-resistant type III membrane PT α/β-subunit precursor protein are exported whereas coexpressed separate α- and β-subunits failed to reach the cis-Golgi compartment. Mutational analyses revealed combinatorial, non-exchangeable dileucine and dibasic motifs located in a defined sequence context in the cytosolic N- and C-terminal domains that are required for efficient ER exit and subsequent proteolytic activation of the α/β-subunit precursor protein in the Golgi. In the presence of a dominant negative Sar1 mutant the ER exit of the PT α/β-subunit precursor protein is inhibited indicating its transport in coat protein complex II-coated vesicles. Expression studies of missense mutations identified in mucolipidosis III patients that alter amino acids in the N- and C-terminal domains demonstrated that the substitution of a lysine residue in close proximity to the dileucine sorting motif impaired ER-Golgi transport and subsequent activation of the PT α/β-subunit precursor protein. The data suggest that the oligomeric type III membrane protein PT complex requires a combinatorial sorting motif that forms a tertiary epitope to be recognized by distinct sites within the coat protein complex II machinery.

  10. The scaffold protein MEK Partner 1 is required for the survival of estrogen receptor positive breast cancer cells.

    Science.gov (United States)

    Marina, Mihaela; Wang, Limin; Conrad, Susan E

    2012-07-09

    MEK Partner 1 (MP1 or MAPKSP1) is a scaffold protein that has been reported to function in multiple signaling pathways, including the ERK, PAK and mTORC pathways. Several of these pathways influence the biology of breast cancer, but MP1's functional significance in breast cancer cells has not been investigated. In this report, we demonstrate a requirement for MP1 expression in estrogen receptor (ER) positive breast cancer cells. MP1 is widely expressed in both ER-positive and negative breast cancer cell lines, and in non-tumorigenic mammary epithelial cell lines. However, inhibition of its expression using siRNA duplexes resulted in detachment and apoptosis of several ER-positive breast cancer cell lines, but not ER-negative breast cancer cells or non-tumorigenic mammary epithelial cells. Inhibition of MP1 expression in ER-positive MCF-7 cells did not affect ERK activity, but resulted in reduced Akt1 activity and reduced ER expression and activity. Inhibition of ER expression did not result in cell death, suggesting that decreased ER expression is not the cause of cell death. In contrast, pharmacological inhibition of PI3K signaling did induce cell death in MCF-7 cells, and expression of a constitutively active form of Akt1 partially rescued the cell death observed when the MP1 gene was silenced in these cells. Together, these results suggest that MP1 is required for pro-survival signaling from the PI3K/Akt pathway in ER-positive breast cancer cells.

  11. Protection of mice against Trypanosoma cruzi by immunization with paraflagellar rod proteins requires T cell, but not B cell, function.

    Science.gov (United States)

    Miller, M J; Wrightsman, R A; Stryker, G A; Manning, J E

    1997-06-01

    Previous studies have shown that immunization of mice with the paraflagellar rod proteins (PAR) of Trypanosoma cruzi induces an immune response capable of protecting mice against an otherwise lethal challenge with this parasite. Herein, we define immunologic responses that do or do not play a critical role in PAR-mediated protection. Firstly, PAR-immunized Ab-deficient (muMT) strain mice survived an otherwise lethal T. cruzi challenge, indicating that a B cell response is not required for PAR-induced immunity. However, beta2m -/- mice, which are severely deficient in MHC class I and TCR alphabeta+ CD8+ CD4- T cells, did not survive challenge infection following PAR immunization, indicating that MHC class I/CD8+ T cell function is necessary for protection induced by PAR immunization. Surprisingly, PAR-immunized mice depleted of CD4+ T cells survived a T. cruzi challenge for >84 days postinfection while maintaining a parasitemia that is generally thought to be lethal (i.e., >10(6) trypomastigotes/ml), thus associating CD4+ T cell function with the process of parasite clearance. Consistent with this association, CD4+ T cells from PAR-immunized mice released INF-gamma and stimulated T. cruzi-infected macrophages to release nitric oxide. The importance of IFN-gamma in PAR-induced protective immunity is further indicated by the observation that PAR-immunized INF-gamma knockout mice developed an extremely high parasitemia and did not survive a challenge infection. Thus, while Ab-mediated immune mechanisms are not required for protection induced by PAR immunization, T cell responses are necessary for both elimination of bloodstream parasites and survival.

  12. The Myelin and Lymphocyte Protein MAL Is Required for Binding and Activity of Clostridium perfringens ε-Toxin.

    Directory of Open Access Journals (Sweden)

    Kareem Rashid Rumah

    2015-05-01

    Full Text Available Clostridium perfringens ε-toxin (ETX is a potent pore-forming toxin responsible for a central nervous system (CNS disease in ruminant animals with characteristics of blood-brain barrier (BBB dysfunction and white matter injury. ETX has been proposed as a potential causative agent for Multiple Sclerosis (MS, a human disease that begins with BBB breakdown and injury to myelin forming cells of the CNS. The receptor for ETX is unknown. Here we show that both binding of ETX to mammalian cells and cytotoxicity requires the tetraspan proteolipid Myelin and Lymphocyte protein (MAL. While native Chinese Hamster Ovary (CHO cells are resistant to ETX, exogenous expression of MAL in CHO cells confers both ETX binding and susceptibility to ETX-mediated cell death. Cells expressing rat MAL are ~100 times more sensitive to ETX than cells expressing similar levels of human MAL. Insertion of the FLAG sequence into the second extracellular loop of MAL abolishes ETX binding and cytotoxicity. ETX is known to bind specifically and with high affinity to intestinal epithelium, renal tubules, brain endothelial cells and myelin. We identify specific binding of ETX to these structures and additionally show binding to retinal microvasculature and the squamous epithelial cells of the sclera in wild-type mice. In contrast, there is a complete absence of ETX binding to tissues from MAL knockout (MAL-/- mice. Furthermore, MAL-/- mice exhibit complete resistance to ETX at doses in excess of 1000 times the symptomatic dose for wild-type mice. We conclude that MAL is required for both ETX binding and cytotoxicity.

  13. Residues of the UL25 protein of herpes simplex virus that are required for its stable interaction with capsids.

    Science.gov (United States)

    Cockrell, Shelley K; Huffman, Jamie B; Toropova, Katerina; Conway, James F; Homa, Fred L

    2011-05-01

    The herpes simplex virus 1 (HSV-1) UL25 gene product is a minor capsid component that is required for encapsidation, but not cleavage, of replicated viral DNA. UL25 is located on the capsid surface in a proposed heterodimer with UL17, where five copies of the heterodimer are found at each of the capsid vertices. Previously, we demonstrated that amino acids 1 to 50 of UL25 are essential for its stable interaction with capsids. To further define the UL25 capsid binding domain, we generated recombinant viruses with either small truncations or amino acid substitutions in the UL25 N terminus. Studies of these mutants demonstrated that there are two important regions within the capsid binding domain. The first 27 amino acids are essential for capsid binding of UL25, while residues 26 to 39, which are highly conserved in the UL25 homologues of other alphaherpesviruses, were found to be critical for stable capsid binding. Cryo-electron microscopy reconstructions of capsids containing either a small tag on the N terminus of UL25 or the green fluorescent protein (GFP) fused between amino acids 50 and 51 of UL25 demonstrate that residues 1 to 27 of UL25 contact the hexon adjacent to the penton. A second region, most likely centered on amino acids 26 to 39, contacts the triplex that is one removed from the penton. Importantly, both of these UL25 capsid binding regions are essential for the stable packaging of full-length viral genomes.

  14. The Microtubule Plus-End Tracking Protein CLASP2 Is Required for Hematopoiesis and Hematopoietic Stem Cell Maintenance

    Directory of Open Access Journals (Sweden)

    Ksenija Drabek

    2012-10-01

    Full Text Available Mammalian CLASPs are microtubule plus-end tracking proteins whose essential function as regulators of microtubule behavior has been studied mainly in cultured cells. We show here that absence of murine CLASP2 in vivo results in thrombocytopenia, progressive anemia, and pancytopenia, due to defects in megakaryopoiesis, in erythropoiesis, and in the maintenance of hematopoietic stem cell activity. Furthermore, microtubule stability and organization are affected upon attachment of Clasp2 knockout hematopoietic stem-cell-enriched populations, and these cells do not home efficiently toward their bone marrow niche. Strikingly, CLASP2-deficient hematopoietic stem cells contain severely reduced mRNA levels of c-Mpl, which encodes the thrombopoietin receptor, an essential factor for megakaryopoiesis and hematopoietic stem cell maintenance. Our data suggest that thrombopoietin signaling is impaired in Clasp2 knockout mice. We propose that the CLASP2-mediated stabilization of microtubules is required for proper attachment, homing, and maintenance of hematopoietic stem cells and that this is necessary to sustain c-Mpl transcription.

  15. A DNA polymerase alpha accessory protein, Mcl1, is required for propagation of centromere structures in fission yeast.

    Directory of Open Access Journals (Sweden)

    Toyoaki Natsume

    Full Text Available Specialized chromatin exists at centromeres and must be precisely transmitted during DNA replication. The mechanisms involved in the propagation of these structures remain elusive. Fission yeast centromeres are composed of two chromatin domains: the central CENP-A(Cnp1 kinetochore domain and flanking heterochromatin domains. Here we show that fission yeast Mcl1, a DNA polymerase alpha (Pol alpha accessory protein, is critical for maintenance of centromeric chromatin. In a screen for mutants that alleviate both central domain and outer repeat silencing, we isolated several cos mutants, of which cos1 is allelic to mcl1. The mcl1-101 mutation causes reduced CENP-A(Cnp1 in the central domain and an aberrant increase in histone acetylation in both domains. These phenotypes are also observed in a mutant of swi7(+, which encodes a catalytic subunit of Pol alpha. Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Pol alpha. These results suggest that Mcl1 and Pol alpha are required for propagation of centromere chromatin structures during DNA replication.

  16. Multiple ribosomal proteins are expressed at high levels in developing zebrafish endoderm and are required for normal exocrine pancreas development.

    Science.gov (United States)

    Provost, Elayne; Weier, Christopher A; Leach, Steven D

    2013-06-01

    Ribosomal protein L (rpl) genes are essential for assembly of the 60S subunit of the eukaryotic ribosome and may also carry out additional extra-ribosomal functions. We have identified a common expression pattern for rpl genes in developing zebrafish larvae. After initially widespread expression in early embryos, the expression of multiple rpl genes becomes increasingly restricted to the endoderm. With respect to the pancreas, rpl genes are highly expressed in ptf1a-expressing pancreatic progenitors at 48 hpf, suggesting possible functional roles in pancreatic morphogenesis and/or differentiation. Utilizing two available mutant lines, rpl23a(hi2582) and rpl6(hi3655b), we found that ptf1a-expressing pancreatic progenitors fail to properly expand in embryos homozygous for either of these genes. In addition to these durable homozygous phenotypes, we also demonstrated recoverable delays in ptf1a-expressing pancreatic progenitor expansion in rpl23a(hi2582) and rpl6(hi3655b) heterozygotes. Disruptions in ribosome assembly are generally understood to initiate a p53-dependent cellular stress response. However, concomitant p53 knockdown was unable to rescue normal pancreatic progenitor expansion in either rpl23a(hi2582) or rpl6(hi3655b) mutant embryos, suggesting required and p53-independent roles for rpl23a and rpl6 in pancreas development.

  17. Actin-associated protein palladin is required for migration behavior and differentiation potential of C2C12 myoblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Ngoc Uyen Nhi; Liang, Vincent Roderick; Wang, Hao-Ven, E-mail: hvwang@mail.ncku.edu.tw

    2014-09-26

    Highlights: • Palladin is involved in myogenesis in vitro. • Palladin knockdown by siRNA increases myoblast proliferation, viability and differentiation. • Palladin knockdown decreases C2C12 myoblast migration ability. - Abstract: The actin-associated protein palladin has been shown to be involved in differentiation processes in non-muscle tissues. However, but its function in skeletal muscle has rarely been studied. Palladin plays important roles in the regulation of diverse actin-related signaling in a number of cell types. Since intact actin-cytoskeletal remodeling is necessary for myogenesis, in the present study, we pursue to investigate the role of actin-associated palladin in skeletal muscle differentiation. Palladin in C2C12 myoblasts is knocked-down using specific small interfering RNA (siRNA). The results show that down-regulation of palladin decreased migratory activity of mouse skeletal muscle C2C12 myoblasts. Furthermore, the depletion of palladin enhances C2C12 vitality and proliferation. Of note, the loss of palladin promotes C2C12 to express the myosin heavy chain, suggesting that palladin has a role in the modulation of C2C12 differentiation. It is thus proposed that palladin is required for normal C2C12 myogenesis in vitro.

  18. Cryptococcus neoformans requires the ESCRT protein Vps23 for iron acquisition from heme, for capsule formation, and for virulence.

    Science.gov (United States)

    Hu, Guanggan; Caza, Mélissa; Cadieux, Brigitte; Chan, Vivienne; Liu, Victor; Kronstad, James

    2013-01-01

    Iron availability is a key regulator of virulence factor elaboration in Cryptococcus neoformans, the causative agent of fungal meningoencephalitis in HIV/AIDS patients. In addition, iron is an essential nutrient for pathogen proliferation in mammalian hosts but little is known about the mechanisms of iron sensing and uptake in fungal pathogens that attack humans. In this study, we mutagenized C. neoformans by Agrobacterium-mediated T-DNA insertion and screened for mutants with reduced growth on heme as the sole iron source. Among 34 mutants, we identified a subset with insertions in the gene for the ESCRT-I (endosomal sorting complex required for transport) protein Vps23 that resulted in a growth defect on heme, presumably due to a defect in uptake via endocytosis or misregulation of iron acquisition from heme. Remarkably, vps23 mutants were also defective in the elaboration of the cell-associated capsular polysaccharide that is a major virulence factor, while overexpression of Vps23 resulted in cells with a slightly enlarged capsule. These phenotypes were mirrored by a virulence defect in the vps23 mutant in a mouse model of cryptococcosis and by hypervirulence of the overexpression strain. Overall, these results reveal an important role for trafficking via ESCRT functions in both heme uptake and capsule formation, and they further reinforce the connection between iron and virulence factor deployment in C. neoformans.

  19. A prefoldin-associated WD-repeat protein (WDR92) is required for the correct architectural assembly of motile cilia

    Science.gov (United States)

    Patel-King, Ramila S.; King, Stephen M.

    2016-01-01

    WDR92 is a highly conserved WD-repeat protein that has been proposed to be involved in apoptosis and also to be part of a prefoldin-like cochaperone complex. We found that WDR92 has a phylogenetic signature that is generally compatible with it playing a role in the assembly or function of specifically motile cilia. To test this hypothesis, we performed an RNAi-based knockdown of WDR92 gene expression in the planarian Schmidtea mediterranea and were able to achieve a robust reduction in mRNA expression to levels undetectable under our standard RT-PCR conditions. We found that this treatment resulted in a dramatic reduction in the rate of organismal movement that was caused by a switch in the mode of locomotion from smooth, cilia-driven gliding to muscle-based, peristaltic contractions. Although the knockdown animals still assembled cilia of normal length and in similar numbers to controls, these structures had reduced beat frequency and did not maintain hydrodynamic coupling. By transmission electron microscopy we observed that many cilia had pleiomorphic defects in their architecture, including partial loss of dynein arms, incomplete closure of the B-tubule, and occlusion or replacement of the central pair complex by accumulated electron-dense material. These observations suggest that WDR92 is part of a previously unrecognized cytoplasmic chaperone system that is specifically required to fold key components necessary to build motile ciliary axonemes. PMID:26912790

  20. PIAS-like protein Zimp7 is required for the restriction of the zebrafish organizer and mesoderm development.

    Science.gov (United States)

    Moreno-Ayala, Roberto; Schnabel, Denhí; Salas-Vidal, Enrique; Lomelí, Hilda

    2015-07-01

    The Zmiz2 (Zimp7) protein and its homolog Zmiz1 (Zimp10) were initially identified in humans as androgen receptor co-activators. Sequence analysis revealed the presence of an SP-RING/Miz domain, which is highly conserved in members of the PIAS family and confers SUMO-conjugating activity. Zimp7 has been shown to interact with components of the Wnt/β-Catenin signaling pathway and with Brg1 and BAF57, components of the ATP-dependent mammalian SWI/SNF-like BAF chromatin-remodeling complexes. In this work, we analyze the role of zygotic Zimp7 in zebrafish development. We describe evidence indicating that Zimp7 is required for mesoderm development and dorsoventral patterning. Morpholino-mediated reduction of zygotic Zimp7 produced axial mesodermal defects that were preceded by up-regulation of organizer genes such as bozozok, goosecoid and floating head at the onset of gastrulation and by down-regulation of the ventral markers vox, vent and eve1 indicating loss of the ventrolateral mesoderm. Consistently, embryos overexpressing zimp7 RNA exhibited midline defects such as loss of forebrain and cyclopia accompanied by transcriptional changes directly opposite of those found in the morphants. In addition, the patterning of ventralized embryos produced by the overexpression of vox and vent was restored by a reduction of Zimp7 activity. Altogether, our findings indicate that Zimp7 is involved in transcriptional regulation of factors that are essential for patterning in the dorsoventral axis.

  1. The Centriole Cartwheel Protein SAS-6 in Trypanosoma brucei Is Required for Probasal Body Biogenesis and Flagellum Assembly.

    Science.gov (United States)

    Hu, Huiqing; Liu, Yi; Zhou, Qing; Siegel, Sara; Li, Ziyin

    2015-09-01

    The centriole in eukaryotes functions as the cell's microtubule-organizing center (MTOC) to nucleate spindle assembly, and its biogenesis requires an evolutionarily conserved protein, SAS-6, which assembles the centriole cartwheel. Trypanosoma brucei, an early branching protozoan, possesses the basal body as its MTOC to nucleate flagellum biogenesis. However, little is known about the components of the basal body and their roles in basal body biogenesis and flagellum assembly. Here, we report that the T. brucei SAS-6 homolog, TbSAS-6, is localized to the mature basal body and the probasal body throughout the cell cycle. RNA interference (RNAi) of TbSAS-6 inhibited probasal body biogenesis, compromised flagellum assembly, and caused cytokinesis arrest. Surprisingly, overexpression of TbSAS-6 in T. brucei also impaired probasal body duplication and flagellum assembly, contrary to SAS-6 overexpression in humans, which produces supernumerary centrioles. Furthermore, we showed that depletion of T. brucei Polo-like kinase, TbPLK, or inhibition of TbPLK activity did not abolish TbSAS-6 localization to the basal body, in contrast to the essential role of Polo-like kinase in recruiting SAS-6 to centrioles in animals. Altogether, these results identified the essential role of TbSAS-6 in probasal body biogenesis and flagellum assembly and suggest the presence of a TbPLK-independent pathway governing basal body duplication in T. brucei.

  2. Cyclic AMP (cAMP)-mediated stimulation of adipocyte differentiation requires the synergistic action of Epac- and cAMP-dependent protein kinase-dependent processes

    DEFF Research Database (Denmark)

    Petersen, Rasmus Koefoed; Madsen, Lise; Pedersen, Lone Møller;

    2008-01-01

    Cyclic AMP (cAMP)-dependent processes are pivotal during the early stages of adipocyte differentiation. We show that exchange protein directly activated by cAMP (Epac), which functions as a guanine nucleotide exchange factor for the Ras-like GTPases Rap1 and Rap2, was required for cAMP-dependent ......Cyclic AMP (cAMP)-dependent processes are pivotal during the early stages of adipocyte differentiation. We show that exchange protein directly activated by cAMP (Epac), which functions as a guanine nucleotide exchange factor for the Ras-like GTPases Rap1 and Rap2, was required for c......AMP-dependent stimulation of adipocyte differentiation. Epac, working via Rap, acted synergistically with cAMP-dependent protein kinase (protein kinase A [PKA]) to promote adipogenesis. The major role of PKA was to down-regulate Rho and Rho-kinase activity, rather than to enhance CREB phosphorylation. Suppression of Rho...

  3. Metabolizable energy requirement for starting barrow pigs (15 to 30 kg fed on the ideal protein concept based diets

    Directory of Open Access Journals (Sweden)

    Gisele Cristina de Oliveira

    2005-09-01

    Full Text Available The objective of this study was to determine the metabolizable energy (ME requirement for starting barrow pigs. Forty-three animals, selected for their high lean gain, were allotted in a completely randomized block design, divided in four treatments with five blocks and two animals in each experimental unit. The diet in Treatment 1 consisted of 3,264 kcal of ME/kg containing 0.96% of digestible lysine, 0.55% of digestible methionine+cystine, 0.60% of digestible threonine, and 0.188% of digestible tryptophan reaching the ideal protein pattern. The diets in Treatments 2, 3, and 4 were similar to the diet in Treatment 1; nevertheless, the levels of ME in Treatments 2, 3, and 4 were 2, 4, and 6% higher than those in Treatment 1. The lysine:ME ratio, was mantained the same (2.82 g in all treatments. The daily feed intake (DFI and the feed:gain ratio (F:G were not affected by the levels of ME. There was a linear increase of daily weight gain (DWG and of daily energy intake (DEI. Later, a linear reduction in carcass protein percentage (CPP and a linear increase of fat content and daily fat accretion (DFA occurred. Results suggested that the required ME was of 3,264 kcal/kg or less for improved barrows (15 to 30 kg, of the dam line, fed with diets containing 0.96% of digestible lysine, formulated according to the ideal protein concept.A exigência de energia metabolizável (EM para suínos machos castrados foi determinada no presente experimento. Foram utilizados 43 suínos geneticamente melhorados, distribuídos em delineamento experimental de blocos inteiramente casualizados, com quatro tratamentos, cinco blocos e dois animais por unidade experimental. Tratamento 1 constituiu-se de uma dieta contendo 3.264 kcal de EM/kg contendo 0,96% de lisina digestível, 0,55% de metionina + cistina digestíveis, 0,60% de treonina digestível e 0,188% de triptofano digestível, atendendo ao conceito de proteína ideal. Tratamentos 2, 3 e 4 foram semelhantes à do

  4. Infectious bronchitis coronavirus limits interferon production by inducing a host shutoff that requires accessory protein 5b

    NARCIS (Netherlands)

    Kint, Joeri; Langereis, Martijn A.; Maier, Helena J.; Britton, Paul; Kuppeveld, van Frank J.; Koumans, Joseph; Wiegertjes, Geert F.; Forlenza, Maria

    2016-01-01

    During infection of their host cells, viruses often inhibit the production of host proteins, a process that is referred to as host shutoff. By doing this, viruses limit the production of antiviral proteins and increase production capacity for viral proteins. Coronaviruses from the genera Alphacor

  5. Reactive oxygen species decrease cAMP response element binding protein expression in cardiomyocytes via a protein kinase D1-dependent mechanism that does not require Ser133 phosphorylation.

    Science.gov (United States)

    Ozgen, Nazira; Guo, Jianfen; Gertsberg, Zoya; Danilo, Peter; Rosen, Michael R; Steinberg, Susan F

    2009-10-01

    Reactive oxygen species (ROS) exert pleiotropic effects on a wide array of signaling proteins that regulate cellular growth and apoptosis. This study shows that long-term treatment with a low concentration of H2O2 leads to the activation of signaling pathways involving extracellular signal-regulated kinase, ribosomal protein S6 kinase, and protein kinase D (PKD) that increase cAMP binding response element protein (CREB) phosphorylation at Ser(133) in cardiomyocytes. Although CREB-Ser(133) phosphorylation typically mediates cAMP-dependent increases in CREB target gene expression, the H2O2-dependent increase in CREB-Ser(133) phosphorylation is accompanied by a decrease in CREB protein abundance and no change in Cre-luciferase reporter activity. Mutagenesis studies indicate that H2O2 decreases CREB protein abundance via a mechanism that does not require CREB-Ser(133) phosphorylation. Rather, the H2O2-dependent decrease in CREB protein is prevented by the proteasome inhibitor lactacystin, by inhibitors of mitogen-activated protein kinase kinase or protein kinase C activity, or by adenoviral-mediated delivery of a small interfering RNA that decreases PKD1 expression. A PKD1-dependent mechanism that links oxidative stress to decreased CREB protein abundance is predicted to contribute to the pathogenesis of heart failure by influencing cardiac growth and apoptosis responses.

  6. Newly identified phosphorylation site in the vesicular stomatitis virus P protein is required for viral RNA synthesis.

    Science.gov (United States)

    Mondal, Arindam; Victor, Ken G; Pudupakam, R S; Lyons, Charles E; Wertz, Gail W

    2014-02-01

    The vesicular stomatitis virus (VSV) RNA-dependent RNA polymerase consists of two viral proteins; the large (L) protein is the main catalytic subunit, and the phosphoprotein (P) is an essential cofactor for polymerase function. The P protein interacts with the L protein and the N-RNA template, thus connecting the polymerase to the template. P protein also binds to free N protein to maintain it in a soluble, encapsidation-competent form. Previously, five sites of phosphorylation were identified on the P protein and these sites were reported to be differentially important for mRNA synthesis or genomic replication. The previous studies were carried out by biochemical analysis of portions of the authentic viral P protein or by analysis of bacterium-expressed, exogenously phosphorylated P protein by mutagenesis. However, there has been no systematic biochemical search for phosphorylation sites on authentic, virus-expressed P protein. In this study, we analyzed the P protein isolated from VSV-infected cells for sites of phosphorylation by mass spectrometry. We report the identification of Tyr14 as a previously unidentified phosphorylation site of VSV P and show that it is essential for viral transcription and replication. However, our mass spectral analysis failed to observe the phosphorylation of previously reported C-terminal residues Ser226 and Ser227 and mutagenic analyses did not demonstrate a role for these sites in RNA synthesis.

  7. Rotavirus structural proteins and dsRNA are required for the human primary plasmacytoid dendritic cell IFNalpha response.

    Directory of Open Access Journals (Sweden)

    Emily M Deal

    Full Text Available Rotaviruses are the leading cause of severe dehydrating diarrhea in children worldwide. Rotavirus-induced immune responses, especially the T and B cell responses, have been extensively characterized; however, little is known about innate immune mechanisms involved in the control of rotavirus infection. Although increased levels of systemic type I interferon (IFNalpha and beta correlate with accelerated resolution of rotavirus disease, multiple rotavirus strains, including rhesus rotavirus (RRV, have been demonstrated to antagonize type I IFN production in a variety of epithelial and fibroblast cell types through several mechanisms, including degradation of multiple interferon regulatory factors by a viral nonstructural protein. This report demonstrates that stimulation of highly purified primary human peripheral plasmacytoid dendritic cells (pDCs with either live or inactivated RRV induces substantial IFNalpha production by a subset of pDCs in which RRV does not replicate. Characterization of pDC responses to viral stimulus by flow cytometry and Luminex revealed that RRV replicates in a small subset of human primary pDCs and, in this RRV-permissive small subset, IFNalpha production is diminished. pDC activation and maturation were observed independently of viral replication and were enhanced in cells in which virus replicates. Production of IFNalpha by pDCs following RRV exposure required viral dsRNA and surface proteins, but neither viral replication nor activation by trypsin cleavage of VP4. These results demonstrate that a minor subset of purified primary human peripheral pDCs are permissive to RRV infection, and that pDCs retain functionality following RRV stimulus. Additionally, this study demonstrates trypsin-independent infection of primary peripheral cells by rotavirus, which may allow for the establishment of extraintestinal viremia and antigenemia. Importantly, these data provide the first evidence of IFNalpha induction in primary

  8. Vaccinia protein F12 has structural similarity to kinesin light chain and contains a motor binding motif required for virion export.

    Directory of Open Access Journals (Sweden)

    Gareth W Morgan

    2010-02-01

    Full Text Available Vaccinia virus (VACV uses microtubules for export of virions to the cell surface and this process requires the viral protein F12. Here we show that F12 has structural similarity to kinesin light chain (KLC, a subunit of the kinesin-1 motor that binds cargo. F12 and KLC share similar size, pI, hydropathy and cargo-binding tetratricopeptide repeats (TPRs. Moreover, molecular modeling of F12 TPRs upon the crystal structure of KLC2 TPRs showed a striking conservation of structure. We also identified multiple TPRs in VACV proteins E2 and A36. Data presented demonstrate that F12 is critical for recruitment of kinesin-1 to virions and that a conserved tryptophan and aspartic acid (WD motif, which is conserved in the kinesin-1-binding sequence (KBS of the neuronal protein calsyntenin/alcadein and several other cellular kinesin-1 binding proteins, is essential for kinesin-1 recruitment and virion transport. In contrast, mutation of WD motifs in protein A36 revealed they were not required for kinesin-1 recruitment or IEV transport. This report of a viral KLC-like protein containing a KBS that is conserved in several cellular proteins advances our understanding of how VACV recruits the kinesin motor to virions, and exemplifies how viruses use molecular mimicry of cellular components to their advantage.

  9. Procalcitonin and C-reactive protein cannot differentiate bacterial or viral infection in COPD exacerbation requiring emergency department visits

    Directory of Open Access Journals (Sweden)

    Chang CH

    2015-04-01

    Full Text Available Chih-Hao Chang,1 Kuo-Chien Tsao,2,3 Han-Chung Hu,1,4 Chung-Chi Huang,1,4 Kuo-Chin Kao,1,4 Ning-Hung Chen,1,4 Cheng-Ta Yang,1,4 Ying-Huang Tsai,4,5 Meng-Jer Hsieh4,51Department of Pulmonary and Critical Care Medicine, Linkou Chang-Gung Memorial Hospital, Chang-Gung Medical Foundation, Chang-Gung University College of Medicine, Taoyuan, Taiwan; 2Department of Laboratory Medicine, Linkou Chang-Gung Memorial Hospital, Chang-Gung Medical Foundation; 3Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Taoyuan, Taiwan; 4Department of Respiratory Therapy, Chang-Gung University, Taoyuan, Taiwan; 5Department of Pulmonary and Critical Care Medicine, Chiayi Chang-Gung Memorial Hospital, Chang-Gung Medical Foundation, Puzi City, TaiwanBackground: Viral and bacterial infections are the most common causes of chronic obstructive pulmonary disease (COPD exacerbations. Whether serum inflammatory markers can differentiate bacterial from virus infection in patients with COPD exacerbation requiring emergency department (ED visits remains controversial.Methods: Viral culture and polymerase chain reaction (PCR were used to identify the viruses in the oropharynx of patients with COPD exacerbations. The bacteria were identified by the semiquantitative culture of the expectorated sputum. The peripheral blood white blood cell (WBC counts, serum C-reactive protein (CRP, procalcitonin (PCT, and clinical symptoms were compared among patients with different types of infections.Results: Viruses were isolated from 16 (22.2% of the 72 patients enrolled. The most commonly identified viruses were parainfluenza type 3, influenza A, and rhinovirus. A total of 30 (41.7% patients had positive bacterial cultures, with the most commonly found bacteria being Haemophilus influenzae and Haemophilus parainfluenzae. Five patients (6.9% had both positive sputum cultures and virus identification. The WBC, CRP, and PCT levels of the bacteria-positive and bacteria

  10. Knickkopf and retroactive proteins are required for formation of laminar serosal procuticle during embryonic development of Tribolium castaneum.

    Science.gov (United States)

    Chaudhari, Sujata S; Noh, Mi Young; Moussian, Bernard; Specht, Charles A; Kramer, Karl J; Beeman, Richard W; Arakane, Yasuyuki; Muthukrishnan, Subbaratnam

    2015-05-01

    Chitin, a homopolymer of β-1-4-linked N-acetylglucosamine synthesized by chitin synthase A (Chs-A), is organized in the procuticle of the postembryonic cuticle or exoskeleton, which is composed of laminae stacked parallel to the cell surface to give stability and integrity to the underlying insect epidermal and other tissues. Our previous work has revealed an important role for two proteins from Tribolium castaneum named Knickkopf (TcKnk) and Retroactive (TcRtv) in postembryonic cuticular chitin maintenance. TcKnk and TcRtv were shown to be required for protection and organization of newly synthesized procuticular chitin. To study the functions of TcKnk and TcRtv in serosal and larval cuticles produced during embryogenesis in T. castaneum, dsRNAs specific for these two genes were injected into two week-old adult females. The effects of dsRNA treatment on ovarial integrity, oviposition, egg hatching and adult survival were determined. Insects treated with dsRNA for chitin synthase-A (TcChs-A) and tryptophan oxygenase (TcVer) were used as positive and negative controls for these experiments, respectively. Like TcChs-A RNAi, injection of dsRNA for TcKnk or TcRtv into adult females exhibited no adult lethality and oviposition was normal. However, a vast majority of the embryos did not hatch. The remaining (∼10%) of the embryos hatched into first instar larvae that died without molting to the second instar. Chitin content analysis following TcKnk and TcRtv parental RNAi revealed approximately 50% reduction in chitin content of eggs in comparison with control TcVer RNAi, whereas TcChs-A dsRNA-treatment led to >90% loss of chitin. Furthermore, transmission electron microscopic (TEM) analysis of serosal cuticle from TcChs-A, TcKnk and TcRtv dsRNA-treated insects revealed a complete absence of laminar organization of serosal (and larval) procuticle in comparison with TcVer dsRNA-treated controls, which exhibited normal laminar organization of procuticular chitin. The

  11. PROTEIN TARGETING TO STARCH is required for localising GRANULE-BOUND STARCH SYNTHASE to starch granules and for normal amylose synthesis in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    David Seung

    2015-02-01

    Full Text Available The domestication of starch crops underpinned the development of human civilisation, yet we still do not fully understand how plants make starch. Starch is composed of glucose polymers that are branched (amylopectin or linear (amylose. The amount of amylose strongly influences the physico-chemical behaviour of starchy foods during cooking and of starch mixtures in non-food manufacturing processes. The GRANULE-BOUND STARCH SYNTHASE (GBSS is the glucosyltransferase specifically responsible for elongating amylose polymers and was the only protein known to be required for its biosynthesis. Here, we demonstrate that PROTEIN TARGETING TO STARCH (PTST is also specifically required for amylose synthesis in Arabidopsis. PTST is a plastidial protein possessing an N-terminal coiled coil domain and a C-terminal carbohydrate binding module (CBM. We discovered that Arabidopsis ptst mutants synthesise amylose-free starch and are phenotypically similar to mutants lacking GBSS. Analysis of granule-bound proteins showed a dramatic reduction of GBSS protein in ptst mutant starch granules. Pull-down assays with recombinant proteins in vitro, as well as immunoprecipitation assays in planta, revealed that GBSS physically interacts with PTST via a coiled coil. Furthermore, we show that the CBM domain of PTST, which mediates its interaction with starch granules, is also required for correct GBSS localisation. Fluorescently tagged Arabidopsis GBSS, expressed either in tobacco or Arabidopsis leaves, required the presence of Arabidopsis PTST to localise to starch granules. Mutation of the CBM of PTST caused GBSS to remain in the plastid stroma. PTST fulfils a previously unknown function in targeting GBSS to starch. This sheds new light on the importance of targeting biosynthetic enzymes to sub-cellular sites where their action is required. Importantly, PTST represents a promising new gene target for the biotechnological modification of starch composition, as it is

  12. Mycobacterium tuberculosis universal stress protein Rv2623 regulates bacillary growth by ATP-Binding: requirement for establishing chronic persistent infection.

    Directory of Open Access Journals (Sweden)

    Joshua E Drumm

    2009-05-01

    Full Text Available Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii Rv2623 binds ATP; and iv the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.

  13. Fragile X mental retardation protein is required for programmed cell death and clearance of developmentally-transient peptidergic neurons.

    Science.gov (United States)

    Gatto, Cheryl L; Broadie, Kendal

    2011-08-15

    Fragile X syndrome (FXS), caused by loss of fragile X mental retardation 1 (FMR1) gene function, is the most common heritable cause of intellectual disability and autism spectrum disorders. The FMR1 product (FMRP) is an RNA-binding protein best established to function in activity-dependent modulation of synaptic connections. In the Drosophila FXS disease model, loss of functionally-conserved dFMRP causes synaptic overgrowth and overelaboration in pigment dispersing factor (PDF) peptidergic neurons in the adult brain. Here, we identify a very different component of PDF neuron misregulation in dfmr1 mutants: the aberrant retention of normally developmentally-transient PDF tritocerebral (PDF-TRI) neurons. In wild-type animals, PDF-TRI neurons in the central brain undergo programmed cell death and complete, processive clearance within days of eclosion. In the absence of dFMRP, a defective apoptotic program leads to constitutive maintenance of these peptidergic neurons. We tested whether this apoptotic defect is circuit-specific by examining crustacean cardioactive peptide (CCAP) and bursicon circuits, which are similarly developmentally-transient and normally eliminated immediately post-eclosion. In dfmr1 null mutants, CCAP/bursicon neurons also exhibit significantly delayed clearance dynamics, but are subsequently eliminated from the nervous system, in contrast to the fully persistent PDF-TRI neurons. Thus, the requirement of dFMRP for the retention of transitory peptidergic neurons shows evident circuit specificity. The novel defect of impaired apoptosis and aberrant neuron persistence in the Drosophila FXS model suggests an entirely new level of "pruning" dysfunction may contribute to the FXS disease state.

  14. Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP Binding: Requirement for Establishing Chronic Persistent Infection

    Energy Technology Data Exchange (ETDEWEB)

    Drumm, J.; Mi, K; Bilder, P; Sun, M; Lim, J; Bielefeldt-Ohmann, H; Basaraba, R; So, M; Zhu, G; et. al.

    2009-01-01

    Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii Rv2623 binds ATP; and iv the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.

  15. Ethanol metabolism and oxidative stress are required for unfolded protein response activation and steatosis in zebrafish with alcoholic liver disease

    Science.gov (United States)

    Tsedensodnom, Orkhontuya; Vacaru, Ana M.; Howarth, Deanna L.; Yin, Chunyue; Sadler, Kirsten C.

    2013-01-01

    SUMMARY Secretory pathway dysfunction and lipid accumulation (steatosis) are the two most common responses of hepatocytes to ethanol exposure and are major factors in the pathophysiology of alcoholic liver disease (ALD). However, the mechanisms by which ethanol elicits these cellular responses are not fully understood. Recent data indicates that activation of the unfolded protein response (UPR) in response to secretory pathway dysfunction can cause steatosis. Here, we examined the relationship between alcohol metabolism, oxidative stress, secretory pathway stress and steatosis using zebrafish larvae. We found that ethanol was immediately internalized and metabolized by larvae, such that the internal ethanol concentration in 4-day-old larvae equilibrated to 160 mM after 1 hour of exposure to 350 mM ethanol, with an average ethanol metabolism rate of 56 μmol/larva/hour over 32 hours. Blocking alcohol dehydrogenase 1 (Adh1) and cytochrome P450 2E1 (Cyp2e1), the major enzymes that metabolize ethanol, prevented alcohol-induced steatosis and reduced induction of the UPR in the liver. Thus, we conclude that ethanol metabolism causes ALD in zebrafish. Oxidative stress generated by Cyp2e1-mediated ethanol metabolism is proposed to be a major culprit in ALD pathology. We found that production of reactive oxygen species (ROS) increased in larvae exposed to ethanol, whereas inhibition of the zebrafish CYP2E1 homolog or administration of antioxidants reduced ROS levels. Importantly, these treatments also blocked ethanol-induced steatosis and reduced UPR activation, whereas hydrogen peroxide (H2O2) acted as a pro-oxidant that synergized with low doses of ethanol to induce the UPR. Collectively, these data demonstrate that ethanol metabolism and oxidative stress are conserved mechanisms required for the development of steatosis and hepatic dysfunction in ALD, and that these processes contribute to ethanol-induced UPR activation and secretory pathway stress in hepatocytes. PMID

  16. The Rcs stress response and accessory envelope proteins are required for de novo generation of cell shape in Escherichia coli.

    Science.gov (United States)

    Ranjit, Dev K; Young, Kevin D

    2013-06-01

    Interactions with immune responses or exposure to certain antibiotics can remove the peptidoglycan wall of many Gram-negative bacteria. Though the spheroplasts thus created usually lyse, some may survive by resynthesizing their walls and shapes. Normally, bacterial morphology is generated by synthetic complexes directed by FtsZ and MreBCD or their homologues, but whether these classic systems can recreate morphology in the absence of a preexisting template is unknown. To address this question, we treated Escherichia coli with lysozyme to remove the peptidoglycan wall while leaving intact the inner and outer membranes and periplasm. The resulting lysozyme-induced (LI) spheroplasts recovered a rod shape after four to six generations. Recovery proceeded via a series of cell divisions that produced misshapen and branched intermediates before later progeny assumed a normal rod shape. Importantly, mutants defective in mounting the Rcs stress response and those lacking penicillin binding protein 1B (PBP1B) or LpoB could not divide or recover their cell shape but instead enlarged until they lysed. LI spheroplasts from mutants lacking the Lpp lipoprotein or PBP6 produced spherical daughter cells that did not recover a normal rod shape or that did so only after a significant delay. Thus, to regenerate normal morphology de novo, E. coli must supplement the classic FtsZ- and MreBCD-directed cell wall systems with activities that are otherwise dispensable for growth under normal laboratory conditions. The existence of these auxiliary mechanisms implies that they may be required for survival in natural environments, where bacterial walls can be damaged extensively or removed altogether.

  17. The Multimerization of Human Immunodeficiency Virus Type I Vif Protein: A REQUIREMENT FOR Vif FUNCTION IN THE VIRAL LIFE CYCLE*

    OpenAIRE

    Yang, Shicheng; Sun, Yong; Zhang, Hui

    2000-01-01

    The Vif (virion infectivity factor protein of human immunodeficiency virus type I (HIV-1) is essential for viral replication in vivo and productive infection of peripheral blood mononuclear cells, macrophages, and H9 T-cells. However, the molecular mechanism(s) of Vif remains unknown and needs to be further determined. In this report, we show that, like many other proteins encoded by HIV-1, Vif proteins possess a strong tendency toward self-association. In relatively native conditions, Vif pr...

  18. 40 CFR 174.532 - Bacillus thuringiensis eCry3.1Ab protein in corn; temporary exemption from the requirement of a...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis eCry3.1Ab... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.532 Bacillus... Bacillus thuringiensis eCry3.1Ab protein in corn, in or on the food and feed commodities of corn;...

  19. Co-factor insertion and disulfide bond requirements for twin-arginine translocase-dependent export of the Bacillus subtilis Rieske protein QcrA

    NARCIS (Netherlands)

    Goosens, Vivianne J; Monteferrante, Carmine G; van Dijl, Jan Maarten

    2014-01-01

    Background: The Rieske protein QcrA was recently shown to be exported by twin-arginine translocation (Tat) in Bacillus subtilis. Results: QcrA has disulfide bond and co-factor requirements for effective Tat-dependent translocation. Conclusion: A hierarchy exists between disulfide bonding and co-fact

  20. Aspergillus parasiticus CrzA, Which Encodes a Calcineurin Response Zinc-Finger Protein, is Required for Aflatoxin Production Under Calcium Stress

    Science.gov (United States)

    Calcium has been reported to be required for aflatoxin production. Calcium, like cAMP, is a second messenger. Cacineurin, a calmodulin-dependent serine/threonine protein phosphatase, is an important component of the calcium signaling pathway. The control of calcineurin-dependent gene expression is v...

  1. A novel RNA-recognition-motif protein is required for premeiotic G1/S-phase transition in rice (Oryza sativa L..

    Directory of Open Access Journals (Sweden)

    Ken-Ichi Nonomura

    Full Text Available The molecular mechanism for meiotic entry remains largely elusive in flowering plants. Only Arabidopsis SWI1/DYAD and maize AM1, both of which are the coiled-coil protein, are known to be required for the initiation of plant meiosis. The mechanism underlying the synchrony of male meiosis, characteristic to flowering plants, has also been unclear in the plant kingdom. In other eukaryotes, RNA-recognition-motif (RRM proteins are known to play essential roles in germ-cell development and meiosis progression. Rice MEL2 protein discovered in this study shows partial similarity with human proline-rich RRM protein, deleted in Azoospermia-Associated Protein1 (DAZAP1, though MEL2 also possesses ankyrin repeats and a RING finger motif. Expression analyses of several cell-cycle markers revealed that, in mel2 mutant anthers, most germ cells failed to enter premeiotic S-phase and meiosis, and a part escaped from the defect and underwent meiosis with a significant delay or continued mitotic cycles. Immunofluorescent detection revealed that T7 peptide-tagged MEL2 localized at cytoplasmic perinuclear region of germ cells during premeiotic interphase in transgenic rice plants. This study is the first report of the plant RRM protein, which is required for regulating the premeiotic G1/S-phase transition of male and female germ cells and also establishing synchrony of male meiosis. This study will contribute to elucidation of similarities and diversities in reproduction system between plants and other species.

  2. Arabidopsis VARIEGATED 3 encodes a chloroplast-targeted, zinc-finger protein required for chloroplast and palisade cell development

    DEFF Research Database (Denmark)

    Næsted, Henrik; Holm, Agnethe; Jenkins, Tom

    2004-01-01

    protein containing novel repeats and zinc fingers described as protein interaction domains. VAR3 interacts specifically in yeast and in vitro with NCED4, a putative polyene chain or carotenoid dioxygenase, and both VAR3 and NCED4 accumulate in the chloroplast stroma. Metabolic profiling demonstrates...

  3. Arabidopsis VARIEGATED 3 encodes a chloroplasttargeted, zinc-finger protein required for chloroplast and palisade cell development

    DEFF Research Database (Denmark)

    Næsted, Henrik; Holm, A.; Jenkins, T.

    2004-01-01

    protein containing novel repeats and zinc fingers described as protein interaction domains. VAR3 interacts specifically in yeast and in vitro with NCED4, a putative polyene chain or carotenoid dioxygenase, and both VAR3 and NCED4 accumulate in the chloroplast stroma. Metabolic profiling demonstrates...

  4. Protein adsorption is required for stealth effect of poly(ethylene glycol)- and poly(phosphoester)-coated nanocarriers

    Science.gov (United States)

    Schöttler, Susanne; Becker, Greta; Winzen, Svenja; Steinbach, Tobias; Mohr, Kristin; Landfester, Katharina; Mailänder, Volker; Wurm, Frederik R.

    2016-04-01

    The current gold standard to reduce non-specific cellular uptake of drug delivery vehicles is by covalent attachment of poly(ethylene glycol) (PEG). It is thought that PEG can reduce protein adsorption and thereby confer a stealth effect. Here, we show that polystyrene nanocarriers that have been modified with PEG or poly(ethyl ethylene phosphate) (PEEP) and exposed to plasma proteins exhibit a low cellular uptake, whereas those not exposed to plasma proteins show high non-specific uptake. Mass spectrometric analysis revealed that exposed nanocarriers formed a protein corona that contains an abundance of clusterin proteins (also known as apolipoprotein J). When the polymer-modified nanocarriers were incubated with clusterin, non-specific cellular uptake could be reduced. Our results show that in addition to reducing protein adsorption, PEG, and now PEEPs, can affect the composition of the protein corona that forms around nanocarriers, and the presence of distinct proteins is necessary to prevent non-specific cellular uptake.

  5. Requirement of the N-terminal residues of human cytomegalovirus UL112-113 proteins for viral growth and oriLyt-dependent DNA replication.

    Science.gov (United States)

    Kim, Young-Eui; Park, Mi Young; Kang, Kyeong Jin; Han, Tae Hee; Lee, Chan Hee; Ahn, Jin-Hyun

    2015-08-01

    The UL112-113 region of the human cytomegalovirus (HCMV) genome encodes four phosphoproteins of 34, 43, 50, and 84 kDa that promote viral DNA replication. Co-transfection assays have demonstrated that self-interaction of these proteins via the shared N-termini is necessary for their intranuclear distribution as foci and for the efficient relocation of a viral DNA polymerase processivity factor (UL44) to the viral replication sites. However, the requirement of UL112-113 N-terminal residues for viral growth and DNA replication has not been fully elucidated. Here, we investigated the effect of deletion of the N-terminal regions of UL112-113 proteins on viral growth and oriLyt-dependent DNA replication. A deletion of the entire UL112 region or the region encoding the 25 N-terminal amino-acid residues from the HCMV (Towne strain) bacmid impaired viral growth in bacmid-transfected human fibroblast cells, indicating their requirement for viral growth. In co-immunoprecipitation assays using the genomic gene expressing the four UL112-113 proteins together, the 25 N-terminal amino-acid residues were found to be necessary for stable expression of UL112-113 proteins and their self-interaction. These residues were also required for efficient binding to and relocation of UL44, but not for interaction with IE2, an origin-binding transcription factor. In co-transfection/replication assays, replication of the oriLyt-containing plasmid was promoted by expression of intact UL112-113 proteins, but not by the expression of 25-amino-acid residue-deleted proteins. Our results demonstrate that the 25 N-terminal amino-acid residues of UL112-113 proteins that mediate self-interaction contribute to viral growth by promoting their binding to UL44 and the initiation of oriLyt-dependent DNA replication.

  6. Muscle A-Kinase Anchoring Protein-α is an Injury-Specific Signaling Scaffold Required for Neurotrophic- and Cyclic Adenosine Monophosphate-Mediated Survival

    Directory of Open Access Journals (Sweden)

    Yan Wang

    2015-12-01

    Full Text Available Neurotrophic factor and cAMP-dependent signaling promote the survival and neurite outgrowth of retinal ganglion cells (RGCs after injury. However, the mechanisms conferring neuroprotection and neuroregeneration downstream to these signals are unclear. We now reveal that the scaffold protein muscle A-kinase anchoring protein-α (mAKAPα is required for the survival and axon growth of cultured primary RGCs. Although genetic deletion of mAKAPα early in prenatal RGC development did not affect RGC survival into adulthood, nor promoted the death of RGCs in the uninjured adult retina, loss of mAKAPα in the adult increased RGC death after optic nerve crush. Importantly, mAKAPα was required for the neuroprotective effects of brain-derived neurotrophic factor and cyclic adenosine-monophosphate (cAMP after injury. These results identify mAKAPα as a scaffold for signaling in the stressed neuron that is required for RGC neuroprotection after optic nerve injury.

  7. Active cAMP-dependent protein kinase incorporated within highly purified HIV-1 particles is required for viral infectivity and interacts with viral capsid protein.

    Science.gov (United States)

    Cartier, Christine; Hemonnot, Bénédicte; Gay, Bernard; Bardy, Martine; Sanchiz, Céline; Devaux, Christian; Briant, Laurence

    2003-09-12

    Host cell components, including protein kinases such as ERK-2/mitogen-activated protein kinase, incorporated within human immunodeficiency virus type 1 (HIV-1) virions play a pivotal role in the ability of HIV to infect and replicate in permissive cells. The present work provides evidence that the catalytic subunit of cAMP-dependent protein kinase (C-PKA) is packaged within HIV-1 virions as demonstrated using purified subtilisin-digested viral particles. Virus-associated C-PKA was shown to be enzymatically active and able to phosphorylate synthetic substrate in vitro. Suppression of virion-associated C-PKA activity by specific synthetic inhibitor had no apparent effect on viral precursor maturation and virus assembly. However, virus-associated C-PKA activity was demonstrated to regulate HIV-1 infectivity as assessed by single round infection assays performed by using viruses produced from cells expressing an inactive form of C-PKA. In addition, virus-associated C-PKA was found to co-precipitate with and to phosphorylate the CAp24gag protein. Altogether our results indicate that virus-associated C-PKA regulates HIV-1 infectivity, possibly by catalyzing phosphorylation of the viral CAp24gag protein.

  8. Trafficking of the myrosinase-associated protein GLL23 requires NUC/MVP1/GOLD36/ERMO3 and the p24 protein CYB.

    Science.gov (United States)

    Jancowski, Sylwia; Catching, Amanda; Pighin, Jamie; Kudo, Takamasa; Foissner, Ilse; Wasteneys, Geoffrey O

    2014-02-01

    Proteins detrimental to endoplasmic reticulum (ER) morphology need to be efficiently exported. Here, we identify two mechanisms that control trafficking of Arabidopsis thalianaGLL23, a 43 kDa GDSL-like lipase implicated in glucosinolate metabolism through its association with the β-glucosidase myrosinase. Using immunofluorescence, we identified two mutants that showed aberrant accumulation of GLL23: large perinuclear ER aggregates in the nuclear cage (nuc) mutant; and small compartments contiguous with the peripheral ER in the cytoplasmic bodies (cyb) mutant. Live imaging of fluorescently tagged GLL23 confirmed its presence in the nuc and cyb compartments, but lack of fluorescent signals in the wild-type plants suggested that GLL23 is normally post-translationally modified for ER export. NUC encodes the MVP1/GOLD36/ERMO3 myrosinase-associated protein, previously shown to have vacuolar distribution. CYB is an ER and Golgi-localized p24 type I membrane protein component of coat protein complex (COP) vesicles, animal and yeast homologues of which are known to be involved in selective cargo sorting for ER-Golgi export. Without NUC, GLL23 accumulates in the ER this situation suggests that NUC is in fact active in the ER. Without CYB, both GLL23 and NUC were found to accumulate in cyb compartments, consistent with a role for NUC in GLL23 processing and indicated that GLL23 is the likely sorting target of the CYB p24 protein.

  9. Requirement and Functional Redundancy of Ib Subgroup bHLH Proteins for Iron Deficiency Responses and Uptake in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Ning Wang; Yan Cui; Yi Liu; Huajie Fan; Juan Du; Zongan Huang; Youxi Yuan

    2013-01-01

    The Ib subgroup of the bHLH gene family in Arabidopsis contains four members (AtbHLH38,AtbHLH39,AtbHLHIO0,and AtbHLHI01).AtbHLH38 and AtbHLH39 were previously confirmed to interact with FER-like iron deficiency induced transcription factor (FIT),directly functioning in activation of the expression of ferric-chelate reductase FRO2 and high-affinity ferrous iron transporter IRT1.In this work,we characterized the functions of AtbHLH100 and AtbHLH101 in the regulation of the iron-deficiency responses and uptake.Yeast two-hybrid analysis and bimolecular fluorescence complementation assay demonstrated that both AtbHLH100 and AtbHLH101 could interact with FIT.Dual expression of either AtbHLHIO0 or AtbHLHI01 with FIT in yeast cells activated the GUS expression driven by promoters of FRO2 and IRT1.The plants overexpressing FIT together with AtbHLH101 showed constitutive expression of FRO2 and IRT1 in roots,and accumulated more iron in shoots.Further,the single,double,and triple knockout mutants of AtbHLH38,AtbHLH39,AtbHLHIO0,and AtbHLHI01 were generated and characterized.The FRO2 and IRT1 expression in roots and the iron content in shoots were more drastically decreased in the triple knockout mutant of AtbHLH39,AtbHLHI00,and AtbHLHI01 than that of the other available double and triple mutants of the four genes,Comparison of the physiological responses as well as the expression of FRO2 and IRT1 in the multiple knockout mutants under iron deficiency revealed that AtbHLH100,AtbHLH38,AtbHLH101,and AtbHLH39 played the gradually increased important role in the iron-deficiency responses and uptake.Taken all together,we conclude that the four Ib subgroup bHLH proteins are required and possess redundant functions with differential significance for activation of iron-deficiency responses and uptake in Arabidopsis.

  10. DNA damage-processing in E. coli: on-going protein synthesis is required for fixation of UV-induced lethality and mutation.

    Science.gov (United States)

    Burger, Amanda; Raymer, Jenny; Bockrath, R

    2002-10-01

    UV irradiation of E. coli produces photoproducts in the DNA genome. In consequence, some bacteria lose viability (colony-forming ability) or remain viable as mutant cells. However, the end-points of viability inactivation (lethality) or mutation are determined by cellular processes that act on the UV-damaged DNA. We have investigated the in vivo time course for processes that deal with cyclobutane pyrimidine dimers (CPD) which can be specifically removed by photoreactivation (PR). At different times during post-UV incubation, samples were challenged with PR and assayed for viability or mutation. We used excision-defective E. coli B/r cells and worked under yellow light to avoid background PR. During post-UV incubation (0-100min) in fully supplemented defined medium, inactivation and mutation were initially significantly reversed by PR but the extent of this reversal decreased during continued incubation defining "fixation" of lethality or mutation, respectively. In contrast, if protein synthesis was restricted during the post-UV incubation, no fixation developed. When chloramphenicol was added to inhibit protein synthesis after 30min of supplemented post-UV incubation, at a time sufficient for expression of UV-induced protein(s), fixation of lethality or mutation was still annulled (no change in the effectiveness of PR developed). Lethality fixation did progress when protein synthesis was restricted and the cells were incubated in the presence of puromycin or were either clpP or clpX defective. We discuss these and related results to suggest (1) on-going protein synthesis is required in the fixation process for lethality and mutation to sustain an effective level of a hypothetical protein sensitive to ClpXP proteolysis and (2) this protein plays a critical role in the process leading to exchange between Pol III activity and alternative polymerase activities required as each cell deals with damage in template DNA.

  11. Requirements for F-BAR proteins TOCA-1 and TOCA-2 in actin dynamics and membrane trafficking during Caenorhabditis elegans oocyte growth and embryonic epidermal morphogenesis.

    Directory of Open Access Journals (Sweden)

    Chiara Giuliani

    2009-10-01

    Full Text Available The TOCA family of F-BAR-containing proteins bind to and remodel lipid bilayers via their conserved F-BAR domains, and regulate actin dynamics via their N-Wasp binding SH3 domains. Thus, these proteins are predicted to play a pivotal role in coordinating membrane traffic with actin dynamics during cell migration and tissue morphogenesis. By combining genetic analysis in Caenorhabditis elegans with cellular biochemical experiments in mammalian cells, we showed that: i loss of CeTOCA proteins reduced the efficiency of Clathrin-mediated endocytosis (CME in oocytes. Genetic interference with CeTOCAs interacting proteins WSP-1 and WVE-1, and other components of the WVE-1 complex, produced a similar effect. Oocyte endocytosis defects correlated well with reduced egg production in these mutants. ii CeTOCA proteins localize to cell-cell junctions and are required for proper embryonic morphogenesis, to position hypodermal cells and to organize junctional actin and the junction-associated protein AJM-1. iii Double mutant analysis indicated that the toca genes act in the same pathway as the nematode homologue of N-WASP/WASP, wsp-1. Furthermore, mammalian TOCA-1 and C. elegans CeTOCAs physically associated with N-WASP and WSP-1 directly, or WAVE2 indirectly via ABI-1. Thus, we propose that TOCA proteins control tissues morphogenesis by coordinating Clathrin-dependent membrane trafficking with WAVE and N-WASP-dependent actin-dynamics.

  12. Vipp1 is required for basic thylakoid membrane formation but not for the assembly of thylakoid protein complexes.

    Science.gov (United States)

    Aseeva, Elena; Ossenbühl, Friederich; Sippel, Claudia; Cho, Won K; Stein, Bernhard; Eichacker, Lutz A; Meurer, Jörg; Wanner, Gerhard; Westhoff, Peter; Soll, Jürgen; Vothknecht, Ute C

    2007-02-01

    Vipp1 (vesicle inducing protein in plastids 1) is found in cyanobacteria and chloroplasts where it is essential for thylakoid formation. Arabidopsis thaliana mutant plants with a reduction of Vipp1 to about 20% of wild type content become albinotic at an early stage. We propose that this drastic phenotype results from an inability of the remaining Vipp1 protein to assemble into a homo-oligomeric complex, indicating that oligomerization is a prerequisite for Vipp1 function. A Vipp1-ProteinA fusion protein, expressed in the Deltavipp1 mutant background, is able to reinstate oligomerization and restore photoautotrophic growth. Plants containing Vipp1-ProteinA in amounts comparable to Vipp1 in the wild type exhibit a wild type phenotype. However, plants with a reduced amount of Vipp1-ProteinA protein are growth-retarded and significantly paler than the wild type. This phenotype is caused by a decrease in thylakoid membrane content and a concomitant reduction in photosynthetic activity. To the extent that thylakoid membranes are made in these plants they are properly assembled with protein-pigment complexes and are photosynthetically active. This strongly supports a function of Vipp1 in basic thylakoid membrane formation and not in the functional assembly of thylakoid protein complexes. Intriguingly, electron microscopic analysis shows that chloroplasts in the mutant plants are not equally affected by the Vipp1 shortage. Indeed, a wide range of different stages of thylakoid development ranging from wild-type-like chloroplasts to plastids nearly devoid of thylakoids can be observed in organelles of one and the same cell.

  13. Complexes of Usher proteins preassemble at the endoplasmic reticulum and are required for trafficking and ER homeostasis

    Directory of Open Access Journals (Sweden)

    Bernardo Blanco-Sánchez

    2014-05-01

    Full Text Available Usher syndrome (USH, the leading cause of hereditary combined hearing and vision loss, is characterized by sensorineural deafness and progressive retinal degeneration. Mutations in several different genes produce USH, but the proximal cause of sensory cell death remains mysterious. We adapted a proximity ligation assay to analyze associations among three of the USH proteins, Cdh23, Harmonin and Myo7aa, and the microtubule-based transporter Ift88 in zebrafish inner ear mechanosensory hair cells. We found that the proteins are in close enough proximity to form complexes and that these complexes preassemble at the endoplasmic reticulum (ER. Defects in any one of the three USH proteins disrupt formation and trafficking of the complex and result in diminished levels of the other proteins, generalized trafficking defects and ER stress that triggers apoptosis. ER stress, thus, contributes to sensory hair cell loss and provides a new target to explore for protective therapies for USH.

  14. Distinct domains of the spinal muscular atrophy protein SMN are required for targeting to Cajal bodies in mammalian cells.

    OpenAIRE

    Renvoisé, Benoît; Khoobarry, Kevinee; Gendron, Marie-Claude; Cibert, Christian; Viollet, Louis; Lefebvre, Suzie

    2006-01-01

    Mutations of the survival motor neuron gene SMN1 cause the inherited disease spinal muscular atrophy (SMA). The ubiquitous SMN protein facilitates the biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs). The protein is detected in the cytoplasm, nucleoplasm and enriched with snRNPs in nuclear Cajal bodies. It is structurally divided into at least an amino-terminal region rich in basic amino acid residues, a central Tudor domain, a self-association tyrosine-glycine-box and an ...

  15. The SUFBC2 D complex is required for the biogenesis of all major classes of plastid Fe-S proteins.

    Science.gov (United States)

    Hu, Xueyun; Kato, Yukako; Sumida, Akihiro; Tanaka, Ayumi; Tanaka, Ryouichi

    2017-04-01

    Iron-sulfur (Fe-S) proteins play crucial roles in plastids, participating in photosynthesis and other metabolic pathways. Fe-S clusters are thought to be assembled on a scaffold complex composed of SUFB, SUFC and SUFD proteins. However, several additional proteins provide putative scaffold functions in plastids, and, therefore, the contribution of SUFB, C and D proteins to overall Fe-S assembly still remains unclear. In order to gain insights regarding Fe-S cluster biosynthesis in plastids, we analyzed the complex composed of SUFB, C and D in Arabidopsis by blue native-polyacrylamide gel electrophoresis. Using this approach, a major complex of 170 kDa containing all subunits was detected, indicating that these proteins constitute a SUFBC2 D complex similar to their well characterized bacterial counterparts. The functional effects of SUFB, SUFC or SUFD depletion were analyzed using an inducible RNAi silencing system to specifically target the aforementioned components; resulting in a decrease of various plastidic Fe-S proteins including the PsaA/B and PsaC subunits of photosystem I, ferredoxin and glutamine oxoglutarate aminotransferase. In contrast, the knockout of potential Fe-S scaffold proteins, NFU2 and HCF101, resulted in a specific decrease in the PsaA/B and PsaC levels. These results indicate that the functions of SUFB, SUFC and SUFD for Fe-S cluster biosynthesis cannot be replaced by other scaffold proteins and that SUFBC2 D, NFU2 and HCF101 are involved in the same pathway for the biogenesis of PSI. Taken together, our results provide in vivo evidence supporting the hypothesis that SUFBC2 D is the major, and possibly sole scaffold in plastids.

  16. Inhibition of cellular protein secretion by norwalk virus nonstructural protein p22 requires a mimic of an endoplasmic reticulum export signal.

    Directory of Open Access Journals (Sweden)

    Tyler M Sharp

    Full Text Available Protein trafficking between the endoplasmic reticulum (ER and Golgi apparatus is central to cellular homeostasis. ER export signals are utilized by a subset of proteins to rapidly exit the ER by direct uptake into COPII vesicles for transport to the Golgi. Norwalk virus nonstructural protein p22 contains a YXΦESDG motif that mimics a di-acidic ER export signal in both sequence and function. However, unlike normal ER export signals, the ER export signal mimic of p22 is necessary for apparent inhibition of normal COPII vesicle trafficking, which leads to Golgi disassembly and antagonism of Golgi-dependent cellular protein secretion. This is the first reported function for p22. Disassembly of the Golgi apparatus was also observed in cells replicating Norwalk virus, which may contribute to pathogenesis by interfering with cellular processes that are dependent on an intact secretory pathway. These results indicate that the ER export signal mimic is critical to the antagonistic function of p22, shown herein to be a novel antagonist of ER/Golgi trafficking. This unique and well-conserved human norovirus motif is therefore an appealing target for antiviral drug development.

  17. Pluripotency transcription factor Sox2 is strongly adsorbed by heparin but requires a protein transduction domain for cell internalization

    Energy Technology Data Exchange (ETDEWEB)

    Albayrak, Cem [Department of Chemical Engineering, Stanford University, 381 North-South Mall, Stanford, CA 94305 (United States); Yang, William C. [Department of Bioengineering, Stanford University, 318 Campus Drive, Stanford, CA 94305 (United States); Swartz, James R., E-mail: jswartz@stanford.edu [Department of Chemical Engineering, Stanford University, 381 North-South Mall, Stanford, CA 94305 (United States); Department of Bioengineering, Stanford University, 318 Campus Drive, Stanford, CA 94305 (United States)

    2013-02-15

    Highlights: ► Both R9Sox2 and Sox2 bind heparin with comparable affinity. ► Both R9Sox2 and Sox2 bind to fibroblasts, but only R9Sox2 is internalized. ► Internalization efficiency of R9Sox2 is 0.3% of the administered protein. ► Heparan sulfate adsorption may be part of a mechanism for managing cell death. -- Abstract: The binding of protein transduction domain (PTD)-conjugated proteins to heparan sulfate is an important step in cellular internalization of macromolecules. Here, we studied the pluripotency transcription factor Sox2, with or without the nonaarginine (R9) PTD. Unexpectedly, we observed that Sox2 is strongly adsorbed by heparin and by the fibroblasts without the R9 PTD. However, only the R9Sox2 fusion protein is internalized by the cells. These results collectively show that binding to heparan sulfate is not sufficient for cellular uptake, thereby supporting a recent hypothesis that other proteins play a role in cell internalization of PTD-conjugated proteins.

  18. Hormonal activation of a kinase cascade localized at the mitochondria is required for StAR protein activity.

    Science.gov (United States)

    Poderoso, Cecilia; Maloberti, Paula; Duarte, Alejandra; Neuman, Isabel; Paz, Cristina; Cornejo Maciel, Fabiana; Podesta, Ernesto J

    2009-03-01

    It is known that ERK1/2 and MEK1/2 participate in the regulation of Star gene transcription. However, their role in StAR protein post-transcriptional regulation is not described yet. In this study we analyzed the relationship between the MAPK cascade and StAR protein phosphorylation and function. We have demonstrated that (a) steroidogenesis in MA-10 Leydig cells depends on the specific of ERK1/2 activation at the mitochondria; (b) ERK1/2 phosphorylation is driven by mitochondrial PKA and constitutive MEK1/2 in this organelle; (c) active ERK1/2 interacts with StAR protein, leads to StAR protein phosphorylation at Ser(232) only in the presence of cholesterol; (d) directed mutagenesis of Ser(232) (S232A) inhibited in vitro StAR protein phosphorylation by ERK1; (e) transient transfection of MA-10 cells with StAR S232A cDNA markedly reduced the yield of progesterone production. We show that StAR protein is a substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric complex that regulates cholesterol transport.

  19. Nicotinic stimulation modulates tyrosine hydroxylase mRNA half-life and protein binding to the 3'UTR in a manner that requires transcription.

    Science.gov (United States)

    Roe, David F; Craviso, Gale L; Waymire, Jack C

    2004-01-05

    Tyrosine hydroxylase (TH) expression increases in adrenal chromaffin cells treated with the nicotinic agonist, dimethylphenylpiperazinium (DMPP; 1 microM). We are using this response as a model of the changes in TH level that occur during increased cholinergic neural activity. Here we report a 4-fold increase in TH mRNA half-life in DMPP-treated cells chromaffin cells that is apparent when using a pulse-chase analysis to measure TH mRNA half-life. No increase is apparent using actinomycin D to measure half-life, indicating a requirement for ongoing transcription. Characterization of protein binding to the TH 3'UTR responsible for stabilization using labeled TH 3'UTR probes and electro-mobility shift assays shows the presence of two complexes both of which are increased by DMPP-treatment. The faster migrating complex (FMC) increases 2.5-fold and the slower migrating complex (SMC) increases 1.5-fold. Both changes are prevented by actinomycin D. Characterization of the protein binding to the TH UTR probes indicates SMC is disrupted by polyribonucleotides, poly (A) and poly (U), while binding to FMC is reduced by poly (CU). Separation of UV crosslinked RNA-protein complexes on SDS polyacrylamide gels shows FMC to contain a single protein whereas SMC contains three proteins. Northwesterns yielded similar results. Comparison of DMPP-induced protein binding with the poly C binding protein (PCBP) involved in hypoxia induced rat PC12 TH mRNA stability indicates none of the bovine UTR binding proteins are the PCBP. Thus, nicotinic stimulation produces a transcription-dependent increase in TH mRNA half-life that is mediated by previously unrecognized TH mRNA binding proteins.

  20. Inhibition of tumorigenesis driven by different Wnt proteins requires blockade of distinct ligand-binding regions by LRP6 antibodies

    Science.gov (United States)

    Ettenberg, Seth A.; Charlat, Olga; Daley, Michael P.; Liu, Shanming; Vincent, Karen J.; Stuart, Darrin D.; Schuller, Alwin G.; Yuan, Jing; Ospina, Beatriz; Green, John; Yu, Qunyan; Walsh, Renee; Schmitz, Rita; Heine, Holger; Bilic, Sanela; Ostrom, Lance; Mosher, Rebecca; Hartlepp, K. Felix; Zhu, Zhenping; Fawell, Stephen; Yao, Yung-Mae; Stover, David; Finan, Peter M.; Porter, Jeffery A.; Sellers, William R.; Klagge, Ingo M.; Cong, Feng

    2010-01-01

    Disregulated Wnt/β-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1- and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1- and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wnt-driven cancer. PMID:20713706

  1. Dietary Protein Intake in Young Children in Selected Low-Income Countries Is Generally Adequate in Relation to Estimated Requirements for Healthy Children, Except When Complementary Food Intake Is Low.

    Science.gov (United States)

    Arsenault, Joanne E; Brown, Kenneth H

    2017-02-15

    Background: Previous research indicates that young children in low-income countries (LICs) generally consume greater amounts of protein than published estimates of protein requirements, but this research did not account for protein quality based on the mix of amino acids and the digestibility of ingested protein.Objective: Our objective was to estimate the prevalence of inadequate protein and amino acid intake by young children in LICs, accounting for protein quality.Methods: Seven data sets with information on dietary intake for children (6-35 mo of age) from 6 LICs (Peru, Guatemala, Ecuador, Bangladesh, Uganda, and Zambia) were reanalyzed to estimate protein and amino acid intake and assess adequacy. The protein digestibility-corrected amino acid score of each child's diet was calculated and multiplied by the original (crude) protein intake to obtain an estimate of available protein intake. Distributions of usual intake were obtained to estimate the prevalence of inadequate protein and amino acid intake for each cohort according to Estimated Average Requirements.Results: The prevalence of inadequate protein intake was highest in breastfeeding children aged 6-8 mo: 24% of Bangladeshi and 16% of Peruvian children. With the exception of Bangladesh, the prevalence of inadequate available protein intake decreased by age 9-12 mo and was very low in all sites (0-2%) after 12 mo of age. Inadequate protein intake in children low energy intake from complementary foods, not inadequate protein density.Conclusions: Overall, most children consumed protein amounts greater than requirements, except for the younger breastfeeding children, who were consuming low amounts of complementary foods. These findings reinforce previous evidence that dietary protein is not generally limiting for children in LICs compared with estimated requirements for healthy children, even after accounting for protein quality. However, unmeasured effects of infection and intestinal dysfunction on the

  2. The Fe-type nitrile hydratase from Comamonas testosteroni Ni1 does not require an activator accessory protein for expression in Escherichia coli.

    Science.gov (United States)

    Kuhn, Misty L; Martinez, Salette; Gumataotao, Natalie; Bornscheuer, Uwe; Liu, Dali; Holz, Richard C

    2012-08-03

    We report herein the functional expression of an Fe-type nitrile hydratase (NHase) without the co-expression of an activator protein or the Escherichia coli chaperone proteins GroES/EL. Soluble protein was obtained when the α- and β-subunit genes of the Fe-type NHase Comamonas testosteroni Ni1 (CtNHase) were synthesized with optimized E. coli codon usage and co-expressed. As a control, the Fe-type NHase from Rhodococcus equi TG328-2 (ReNHase) was expressed with (ReNHase(+Act)) and without (ReNHase(-Act)) its activator protein, establishing that expression of a fully functional, metallated ReNHase enzyme requires the co-expression of its activator protein, similar to all other Fe-type NHase enzymes reported to date, whereas the CtNHase does not. The X-ray crystal structure of CtNHase was determined to 2.4Å resolution revealing an αβ heterodimer, similar to other Fe-type NHase enzymes, except for two important differences. First, two His residues reside in the CtNHase active site that are not observed in other Fe-type NHase enzymes and second, the active site Fe(III) ion resides at the bottom of a wide solvent exposed channel. The solvent exposed active site, along with the two active site histidine residues, are hypothesized to play a role in iron incorporation in the absence of an activator protein.

  3. The Fe-type nitrile hydratase from Comamonas testosteroni Ni1 does not require an activator accessory protein for expression in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Kuhn, Misty L.; Martinez, Salette; Gumataotao, Natalie; Bornscheuer, Uwe; Liu, Dali; Holz, Richard C. (Loyola); (Greifswald)

    2012-10-10

    We report herein the functional expression of an Fe-type nitrile hydratase (NHase) without the co-expression of an activator protein or the Escherichia coli chaperone proteins GroES/EL. Soluble protein was obtained when the {alpha}- and {beta}-subunit genes of the Fe-type NHase Comamonas testosteroni Ni1 (CtNHase) were synthesized with optimized E. coli codon usage and co-expressed. As a control, the Fe-type NHase from Rhodococcus equi TG328-2 (ReNHase) was expressed with (ReNHase{sup +Act}) and without (ReNHase{sup -Act}) its activator protein, establishing that expression of a fully functional, metallated ReNHase enzyme requires the co-expression of its activator protein, similar to all other Fe-type NHase enzymes reported to date, whereas the CtNHase does not. The X-ray crystal structure of CtNHase was determined to 2.4 {angstrom} resolution revealing an {alpha}{beta} heterodimer, similar to other Fe-type NHase enzymes, except for two important differences. First, two His residues reside in the CtNHase active site that are not observed in other Fe-type NHase enzymes and second, the active site Fe(III) ion resides at the bottom of a wide solvent exposed channel. The solvent exposed active site, along with the two active site histidine residues, are hypothesized to play a role in iron incorporation in the absence of an activator protein.

  4. Vaccinia virus protein A3 is required for the production of normal immature virions and for the encapsidation of the nucleocapsid protein L4.

    Science.gov (United States)

    Jesus, Desyree Murta; Moussatche, Nissin; McFadden, Baron B D; Nielsen, Casey Paulasue; D'Costa, Susan M; Condit, Richard C

    2015-07-01

    Maturation of the vaccinia virion is an intricate process that results in the organization of the viroplasm contained in immature virions into the lateral bodies, core wall and nucleocapsid observed in the mature particles. It is unclear how this organization takes place and studies with mutants are indispensable in understanding this process. By characterizing an inducible mutant in the A3L gene, we revealed that A3, an inner core wall protein, is important for formation of normal immature viruses and also for the correct localization of L4, a nucleocapsid protein. L4 did not accumulate in the viral factories in the absence of A3 and was not encapsidated in the particles that do not contain A3. These data strengthen our previously suggested hypothesis that A3 and L4 interact and that this interaction is critical for proper formation of the core wall and nucleocapsid.

  5. Hipk2 and PP1c Cooperate to Maintain Dvl Protein Levels Required for Wnt Signal Transduction

    Directory of Open Access Journals (Sweden)

    Nobuyuki Shimizu

    2014-09-01

    Full Text Available The phosphoprotein Dishevelled (Dvl is a common essential component of Wnt/β-catenin and Wnt/planar cell polarity (PCP signaling pathways. However, the regulation and significance of Dvl phosphorylation are not fully understood. Here, we show that homeodomain-interacting protein kinase 2 (Hipk2 facilitates protein phosphatase 1 catalytic subunit (PP1c-mediated dephosphorylation of Dvl via its C-terminal domain and that this dephosphorylation blocks ubiquitination and consequent degradation mediated by the E3 ubiquitin ligase Itch, which targets the phosphorylated form of Dvl proteins. Inhibition of Hipk2 or PP1c function reduces Dvl protein levels and suppresses Wnt/β-catenin and Wnt/PCP pathway-dependent events in mammalian cells and zebrafish embryos, suggesting that Hipk2 and PP1c are essential for maintaining Dvl protein levels that are sufficient to activate Wnt signaling. We also show that Wnt-3a, a Wnt/β-catenin ligand, induces dissociation of the Dvl-Hipk2-PP1c complex and Dvl degradation under high-cell-density conditions. This regulation may be a negative feedback mechanism that fine-tunes Wnt/β-catenin signaling.

  6. Nutritional requirements of energy, protein and macrominerals for maintenance and weight gain of young crossbred Nellore × Holstein bulls on pasture

    Directory of Open Access Journals (Sweden)

    Marlos Oliveira Porto

    2012-03-01

    Full Text Available The objective of this study was to estimate requirements of energy, protein and macrominerals of young Nellore/Holstein crossbreds bulls supplemented on pastures of Brachiaria decumbens Stapf. Thirty-five young bulls, at 8.53±0.18 months of age and with initial body weight of 230.6±6.1 kg were used. Ten animals were slaughtered as reference, in different weight range, and the other animals were slaughtered at the end of the experimental period. For estimate of net energy requirements for weight, a regression equation between log of retained energy (RE and log of empty body weight gain (EBWG was constructed. Net requirements of Ca, P, Mg, Na and K were determined by the equation Y' = a.b.Xb-1, in which a and b represent the intercept and the coefficient of equation of prediction of macrominerals in body content, respectively. Requirements of metabolizable energy for maintenance (MEm were obtained from retained energy in function of metabolizable energy intake (MEI. The requirements of MEm of Nellore/Holstein crossbreds young bulls on pasture was 125 kcal/EBW0.75/day. The efficiency of ME utilization for maintenance (k of grazing Nellore/Holstein crossbred young mbulls was 0.58 and 0.24 for gain. The total metabolizable protein requirements for an animal with 400 kg and with average daily gain of 1.0 kg, were 638.36 g/day. The dietetic requirements of Ca and P for an animal with 400 kg BW were 0.49 and 0.21% of DM, respectively. Daily metabolizable energy requirement for maintenance of grazing Nellore/Holstein crossbred young bulls was 11.6% greater than the values found for cattle in feedlot in Brazil (112 kcal/kg EBW0.75.

  7. Exploring the Structural Requirements for Inhibition of the Ubiquitin E3 Ligase Breast Cancer Associated Protein 2 (BCA2)a as a Treatment for Breast Cancer

    OpenAIRE

    Brahemi, Ghali; Kona, Fathima R; Fiasella, Annalisa; Buac, Daniela; Soukupová, Jitka; Brancale, Andrea; Burger, Angelika M; Westwell, Andrew D.

    2010-01-01

    The zinc-ejecting aldehyde dehydrogenase (ALDH) inhibitory drug disulfiram (DSF) was found to be a breast cancer-associated protein 2 (BCA2) inhibitor with potent antitumor activity. We herein describe our work in the synthesis and evaluation of new series of zinc-affinic molecules to explore the structural requirements for selective BCA2-inhibitory antitumor activity. An N(C=S)S-S motif was found to be required, based on selective activity in BCA2-expressing breast cancer cell lines and agai...

  8. Functional diversification of hsp40: distinct j-protein functional requirements for two prions allow for chaperone-dependent prion selection.

    Science.gov (United States)

    Harris, Julia M; Nguyen, Phil P; Patel, Milan J; Sporn, Zachary A; Hines, Justin K

    2014-07-01

    Yeast prions are heritable amyloid aggregates of functional yeast proteins; their propagation to subsequent cell generations is dependent upon fragmentation of prion protein aggregates by molecular chaperone proteins. Mounting evidence indicates the J-protein Sis1 may act as an amyloid specificity factor, recognizing prion and other amyloid aggregates and enabling Ssa and Hsp104 to act in prion fragmentation. Chaperone interactions with prions, however, can be affected by variations in amyloid-core structure resulting in distinct prion variants or 'strains'. Our genetic analysis revealed that Sis1 domain requirements by distinct variants of [PSI+] are strongly dependent upon overall variant stability. Notably, multiple strong [PSI+] variants can be maintained by a minimal construct of Sis1 consisting of only the J-domain and glycine/phenylalanine-rich (G/F) region that was previously shown to be sufficient for cell viability and [RNQ+] prion propagation. In contrast, weak [PSI+] variants are lost under the same conditions but maintained by the expression of an Sis1 construct that lacks only the G/F region and cannot support [RNQ+] propagation, revealing mutually exclusive requirements for Sis1 function between these two prions. Prion loss is not due to [PSI+]-dependent toxicity or dependent upon a particular yeast genetic background. These observations necessitate that Sis1 must have at least two distinct functional roles that individual prions differentially require for propagation and which are localized to the glycine-rich domains of the Sis1. Based on these distinctions, Sis1 plasmid-shuffling in a [PSI+]/[RNQ+] strain permitted J-protein-dependent prion selection for either prion. We also found that, despite an initial report to the contrary, the human homolog of Sis1, Hdj1, is capable of [PSI+] prion propagation in place of Sis1. This conservation of function is also prion-variant dependent, indicating that only one of the two Sis1-prion functions may have

  9. Stathmin/oncoprotein 18, a microtubule regulatory protein, is required for survival of both normal and cancer cell lines lacking the tumor suppressor, p53.

    Science.gov (United States)

    Carney, Bruce K; Cassimeris, Lynne

    2010-05-01

    Stathmin, a microtubule regulatory protein, is overexpressed in many cancers and required for survival of several cancer lines. In a study of breast cancer cell lines(1) proposed that stathmin is required for survival of cells lacking p53, but this hypothesis was not tested directly. Here we tested their hypothesis by examining cell survival in cells depleted of stathmin, p53 or both proteins. Comparing HCT116 colon cancer cell lines differing in TP53 genotype, stathmin depletion resulted in significant death only in cells lacking p53. As a second experimental system, we compared the effects of stathmin depletion from HeLa cells, which normally lack detectable levels of p53 due to expression of the HPV E6 protein. Stathmin depletion caused a large percentage of HeLa cells to die. Restoring p53, by depletion of HPV E6, rescued HeLa cells from stathmin-depletion induced death. Cleaved PARP was detected in HCT116(p53-/-) cells depleted of stathmin and cell death in stathmin-depleted HeLa cells was blocked by the caspase inhibitor Z-VAD-FMK, consistent with apoptotic death. The stathmin-dependent survival of cells lacking p53 was not confined to cancerous cells because both proteins were required for survival of normal human fibroblasts. In HCT116 and HeLa cells, depletion of both stathmin and p53 leads to a cell cycle delay through G(2). Our results demonstrate that stathmin is required for cell survival in cells lacking p53, suggesting that stathmin depletion could be used therapeutically to induce apoptosis in tumors without functional p53.

  10. Protein characterization by LC-MS/MS may be required for the DNA identification of a fusion hemoglobin: the example of Hb P-Nilotic.

    Science.gov (United States)

    Zanella-Cleon, Isabelle; Delolme, Frédéric; Lacan, Philippe; Garcia, Caroline; Vinatier, Isabelle; Francina, Alain; Joly, Philippe

    2012-02-01

    DNA analysis is currently the easiest way to identify a hemoglobin variant in most cases. Nevertheless, in case of complex gene rearrangements, mass spectrometry studies may be required to orientate the DNA diagnosis. The present report shows the use of mass spectrometry techniques prior to DNA analysis for the identification of the rare P-Nilotic fusion hemoglobin. Complete protein analysis is performed by liquid chromatography-tandem mass spectrometry on the abnormal globin chain isolated by reversed-phase liquid chromatography.

  11. The APP-Interacting Protein FE65 is Required for Hippocampus-Dependent Learning and Long-Term Potentiation

    Science.gov (United States)

    Wang, Yan; Zhang, Ming; Moon, Changjong; Hu, Qubai; Wang, Baiping; Martin, George; Sun, Zhongsheng; Wang, Hongbing

    2009-01-01

    FE65 is expressed predominantly in the brain and interacts with the C-terminal domain of [beta]-amyloid precursor protein (APP). We examined hippocampus-dependent memory and in vivo long-term potentiation (LTP) at the CA1 synapses with isoform-specific FE65 knockout (p97FE65[superscript -/-]) mice. When examined using the Morris water maze,…

  12. Membrane targeting of cGMP-dependent protein kinase is required for cystic fibrosis transmembrane conductance regulator Cl- channel activation

    NARCIS (Netherlands)

    A.B. Vaandrager (Arie); A. Smolenski; B.C. Tilly (Bernard); A.B. Houtsmuller (Adriaan); E.M.E. Ehlert (Ehrich); A.G. Bot (Alice); M.J. Edixhoven (Marcel); W.E. Boomaars (Wendy); S.M. Lohmann (Suzanne); H.R. de Jonge (Hugo)

    1998-01-01

    textabstractA recently cloned isoform of cGMP-dependent protein kinase (cGK), designated type II, was implicated as the mediator of cGMP-provoked intestinal Cl- secretion based on its localization in the apical membrane of enterocytes and on its capacity to activate cys

  13. The Adhesion of Lactobacillus salivarius REN to a Human Intestinal Epithelial Cell Line Requires S-layer Proteins

    Science.gov (United States)

    Wang, Ran; Jiang, Lun; Zhang, Ming; Zhao, Liang; Hao, Yanling; Guo, Huiyuan; Sang, Yue; Zhang, Hao; Ren, Fazheng

    2017-01-01

    Lactobacillus salivarius REN, a novel probiotic isolated from Chinese centenarians, can adhere to intestinal epithelial cells and subsequently colonize the host. We show here that the surface-layer protein choline-binding protein A (CbpA) of L. salivarius REN was involved in adherence to the human colorectal adenocarcinoma cell line HT-29. Adhesion of a cbpA deletion mutant was significantly reduced compared with that of wild-type, suggesting that CbpA acts as an adhesin that mediates the interaction between the bacterium and its host. To identify the molecular mechanism of adhesion, we determined the crystal structure of a truncated form of CbpA that is likely involved in binding to its cell-surface receptor. The crystal structure identified CbpA as a peptidase of the M23 family whose members harbor a zinc-dependent catalytic site. Therefore, we propose that CbpA acts as a multifunctional surface protein that cleaves the host extracellular matrix and participates in adherence. Moreover, we identified enolase as the CbpA receptor on the surface of HT-29 cells. The present study reveals a new class of surface-layer proteins as well as the molecular mechanism that may contribute to the ability of L. salivarius REN to colonize the human gut. PMID:28281568

  14. In Entamoeba histolytica, a BspA family protein is required for chemotaxis toward tumour necrosis factor

    Directory of Open Access Journals (Sweden)

    Anne Silvestre

    2015-07-01

    Full Text Available Background: Entamoeba histolytica cell migration is essential for the development of human amoebiasis (an infectious disease characterized by tissue invasion and destruction. The tissue inflammation associated with tumour necrosis factor (TNF secretion by host cells is a well-documented feature of amoebiasis. Tumour necrosis factor is a chemoattractant for E. histolytica, and the parasite may have a TNF receptor at its cell surface. Methods: confocal microscopy, RNA Sequencing, bioinformatics, RNA antisense techniques and histological analysis of human colon explants were used to characterize the interplay between TNF and E. histolytica. Results: an antibody against human TNF receptor 1 (TNFR1 stained the E. histolytica trophozoite surface and (on immunoblots binds to a 150-kDa protein. Proteome screening with the TNFR1 sequence revealed a BspA family protein in E. histolytica that carries a TNFR signature domain and six leucine-rich repeats (named here as "cell surface protein", CSP, in view of its cellular location. Cell surface protein shares structural homologies with Toll-Like receptors, colocalizes with TNF and is internalized in TNF-containing vesicles. Reduction of cellular CSP levels abolished chemotaxis toward TNF and blocked parasite invasion of human colon. Conclusions: there is a clear link between TNF chemotaxis, CSP and pathogenesis.

  15. Non-structural protein NS3/NS3a is required for propagation of bluetongue virus in Culicoides sonorensis

    NARCIS (Netherlands)

    Feenstra, Femke; Drolet, B.S.; Boonstra, Jan; Rijn, Van P.A.

    2015-01-01

    Background: Bluetongue virus (BTV) causes non-contagious haemorrhagic disease in ruminants and is transmitted by Culicoides spp. biting midges. BTV encodes four non-structural proteins of which NS3/NS3a is functional in virus release. NS3/NS3a is not essential for in vitro virus replication. Howe

  16. BacS: an abundant bacteroid protein in Rhizobium etli whose expression ex planta requires nifA.

    Science.gov (United States)

    Jahn, Olivia J; Davila, Guillermo; Romero, David; Noel, K Dale

    2003-01-01

    Rhizobium etli CFN42 bacteroids from bean nodules possessed an abundant 16-kDa protein (BacS) that was found in the membrane pellet after cell disruption. This protein was not detected in bacteria cultured in tryptone-yeast extract. In minimal media, it was produced at low oxygen concentration but not in a mutant whose nifA was disrupted. N-terminal sequencing of the protein led to isolation of a bacS DNA fragment. DNA hybridization and nucleotide sequencing revealed three copies of the bacS gene, all residing on the main symbiotic plasmid of strain CFN42. A stretch of 304 nucleotides, exactly conserved upstream of all three bacS open reading frames, had very close matches with the NifA and sigma 54 consensus binding sequences. The only bacS homology in the genetic sequence databases was to three hypothetical proteins of unknown function, all from rhizobial species. Mutation and genetic complementation indicated that each of the bacS genes gives rise to a BacS polypeptide. Mutants disrupted or deleted in all three genes did not produce the BacS polypeptide but were Nod+ and Fix+ on Phaseolus vulgaris.

  17. Characterization of a mitogen-activated protein kinase gene from cucumber required for trichoderma-conferred plant resistance.

    Science.gov (United States)

    Shoresh, Michal; Gal-On, Amit; Leibman, Diana; Chet, Ilan

    2006-11-01

    The fungal biocontrol agent Trichoderma asperellum has been recently shown to induce systemic resistance in plants through a mechanism that employs jasmonic acid and ethylene signal transduction pathways. Mitogen-activated protein kinase (MAPK) proteins have been implicated in the signal transduction of a wide variety of plant stress responses. Here we report the identification and characterization of a Trichoderma-induced MAPK (TIPK) gene function in cucumber (Cucumis sativus). Similar to its homologs, wound-induced protein kinase, MPK3, and MPK3a, TIPK is also induced by wounding. Normally, preinoculation of roots with Trichoderma activates plant defense mechanisms, which result in resistance to the leaf pathogen Pseudomonas syringae pv lachrymans. We used a unique attenuated virus vector, Zucchini yellow mosaic virus (ZYMV-AGII), to overexpress TIPK protein and antisense (AS) RNA. Plants overexpressing TIPK were more resistant to pathogenic bacterial attack than control plants, even in the absence of Trichoderma preinoculation. On the other hand, plants expressing TIPK-AS revealed increased sensitivity to pathogen attack. Moreover, Trichoderma preinoculation could not protect these AS plants against subsequent pathogen attack. We therefore demonstrate that Trichoderma exerts its protective effect on plants through activation of the TIPK gene, a MAPK that is involved in signal transduction pathways of defense responses.

  18. The nucleolar GTP-binding proteins Gnl2 and nucleostemin are required for retinal neurogenesis in developing zebrafish

    NARCIS (Netherlands)

    Paridaen, J.T.M.; Janson, E.; Utami, K.H.; Pereboom, T.C.; Essers, P.; van Rooijen, C.R.; Zivkovic, D.; MacInnes, A.W.

    2011-01-01

    Nucleostemin (NS), a member of a family of nucleolar GTP-binding proteins, is highly expressed in proliferating cells such as stem and cancer cells and is involved in the control of cell cycle progression. Both depletion and overexpression of NS result in stabilization of the tumor suppressor p53 pr

  19. Type I J-domain NbMIP1 proteins are required for both Tobacco mosaic virus infection and plant innate immunity.

    Directory of Open Access Journals (Sweden)

    Yumei Du

    Full Text Available Tm-2² is a coiled coil-nucleotide binding-leucine rich repeat resistance protein that confers durable extreme resistance against Tomato mosaic virus (ToMV and Tobacco mosaic virus (TMV by recognizing the viral movement protein (MP. Here we report that the Nicotiana benthamiana J-domain MIP1 proteins (NbMIP1s associate with tobamovirus MP, Tm-2² and SGT1. Silencing of NbMIP1s reduced TMV movement and compromised Tm-2²-mediated resistance against TMV and ToMV. Furthermore, silencing of NbMIP1s reduced the steady-state protein levels of ToMV MP and Tm-2². Moreover, NbMIP1s are required for plant resistance induced by other R genes and the nonhost pathogen Pseudomonas syringae pv. tomato (Pst DC3000. In addition, we found that SGT1 associates with Tm-2² and is required for Tm-2²-mediated resistance against TMV. These results suggest that NbMIP1s function as co-chaperones during virus infection and plant immunity.

  20. The inhibition of the apoptosis pathway by the Coxiella burnetii effector protein CaeA requires the EK repetition motif, but is independent of survivin.

    Science.gov (United States)

    Bisle, Stephanie; Klingenbeck, Leonie; Borges, Vítor; Sobotta, Katharina; Schulze-Luehrmann, Jan; Menge, Christian; Heydel, Carsten; Gomes, João Paulo; Lührmann, Anja

    2016-05-18

    ABSRTACT Coxiella burnetii is an obligate intracellular bacterium that causes Query (Q) fever, a zoonotic disease. It requires a functional type IV secretion system (T4SS) which translocate bacterial effector proteins into the host cell cytoplasm and thereby facilitates bacterial replication. To date, more than 130 effector proteins have been identified, but their functions remain largely unknown. Recently, we demonstrated that one of these proteins, CaeA (CBU1524) localized to the host cell nucleus and inhibited intrinsic apoptosis of HEK293 or CHO cells. In the present study we addressed the question whether CaeA also affects the extrinsic apoptosis pathway. Ectopic expression of CaeA reduced extrinsic apoptosis and prevented the cleavage of the executioner caspase 7, but did not impair the activation of initiator caspase 9. CaeA expression resulted in an up-regulation of survivin (an inhibitor of activated caspases), which, however, was not causal for the anti-apoptotic effect of CaeA. Comparing the sequence of CaeA from 25 different C. burnetii isolates we identified an EK (glutamic acid/ lysine) repetition motif as a site of high genetic variability. The EK motif of CaeA was essential for the anti-apoptotic activity of CaeA. From these data, we conclude that the C. burnetii effector protein CaeA interferes with the intrinsic and extrinsic apoptosis pathway. The process requires the EK repetition motif of CaeA, but is independent of the upregulated expression of survivin.

  1. The Rho1 GTPase-activating Protein CgBem2 Is Required for Survival of Azole Stress in Candida glabrata*

    Science.gov (United States)

    Borah, Sapan; Shivarathri, Raju; Kaur, Rupinder

    2011-01-01

    Invasive fungal infections are common clinical complications of neonates, critically ill, and immunocompromised patients worldwide. Candida species are the leading cause of disseminated fungal infections, with Candida albicans being the most prevalent species. Candida glabrata, the second/third most common cause of candidemia, shows reduced susceptibility to a widely used antifungal drug fluconazole. Here, we present findings from a screen of 9134 C. glabrata Tn7 insertion mutants for altered survival profiles in the presence of fluconazole. We have identified two components of RNA polymerase II mediator complex, three players of Rho GTPase-mediated signaling cascade, and two proteins implicated in actin cytoskeleton biogenesis and ergosterol biosynthesis that are required to sustain viability during fluconazole stress. We show that exposure to fluconazole leads to activation of the protein kinase C (PKC)-mediated cell wall integrity pathway in C. glabrata. Our data demonstrate that disruption of a RhoGAP (GTPase activating protein) domain-containing protein, CgBem2, results in bud-emergence defects, azole susceptibility, and constitutive activation of CgRho1-regulated CgPkc1 signaling cascade and cell wall-related phenotypes. The viability loss of Cgbem2Δ mutant upon fluconazole treatment could be partially rescued by the PKC inhibitor staurosporine. Additionally, we present evidence that CgBEM2 is required for the transcriptional activation of genes encoding multidrug efflux pumps in response to fluconazole exposure. Last, we report that Hsp90 inhibitor geldanamycin renders fluconazole a fungicidal drug in C. glabrata. PMID:21832071

  2. The Scaffold Protein Muscle A-Kinase Anchoring Protein β Orchestrates Cardiac Myocyte Hypertrophic Signaling Required for the Development of Heart Failure

    Science.gov (United States)

    Kritzer, Michael D.; Li, Jinliang; Passariello, Catherine L.; Gayanilo, Marjorie; Thakur, Hrishikesh; Dayan, Joseph; Dodge-Kafka, Kimberly; Kapiloff, Michael S.

    2014-01-01

    Background Cardiac myocyte hypertrophy is regulated by an extensive intracellular signal transduction network. In vitro evidence suggests that the scaffold protein muscle A-kinase anchoring protein β (mAKAPβ) serves as a nodal organizer of hypertrophic signaling. However, the relevance of mAKAPβ signalosomes to pathological remodeling and heart failure in vivo remains unknown. Methods and Results Using conditional, cardiac myocyte–specific gene deletion, we now demonstrate that mAKAPβ expression in mice is important for the cardiac hypertrophy induced by pressure overload and catecholamine toxicity. mAKAPβ targeting prevented the development of heart failure associated with long-term transverse aortic constriction, conferring a survival benefit. In contrast to 29% of control mice (n=24), only 6% of mAKAPβ knockout mice (n=31) died in the 16 weeks of pressure overload (P=0.02). Accordingly, mAKAPβ knockout inhibited myocardial apoptosis and the development of interstitial fibrosis, left atrial hypertrophy, and pulmonary edema. This improvement in cardiac status correlated with the attenuated activation of signaling pathways coordinated by the mAKAPβ scaffold, including the decreased phosphorylation of protein kinase D1 and histone deacetylase 4 that we reveal to participate in a new mAKAP signaling module. Furthermore, mAKAPβ knockout inhibited pathological gene expression directed by myocyte-enhancer factor-2 and nuclear factor of activated T-cell transcription factors that associate with the scaffold. Conclusions mAKAPβ orchestrates signaling that regulates pathological cardiac remodeling in mice. Targeting of the underlying physical architecture of signaling networks, including mAKAPβ signalosome formation, may constitute an effective therapeutic strategy for the prevention and treatment of pathological remodeling and heart failure. PMID:24812305

  3. Determination of protein and amino acid requirements of lactating sows using a population-based factorial approach

    DEFF Research Database (Denmark)

    Strathe, Anja Varmløse; Strathe, Anders Bjerring; Theil, Peter Kappel

    2015-01-01

    for maintenance and milk. The energy balance of the sows was either negative or zero depending on feed intake being a limiting factor. Some parameters in the model were sow-specific and others were population-specific, depending on state of knowledge. Each simulation was for 1000 sows repeated 100 times using......Determination of appropriate nutritional requirements is essential to optimize the productivity and longevity of lactating sows. The current recommendations for requirements do not consider the large variation between animals. Therefore, the aim of this study was to determine the amino acid...... recommendations for lactating sows using a stochastic modeling approach that integrates population variation and uncertainty of key parameters into establishing nutritional recommendations for lactating sows. The requirement for individual sows was calculated using a factorial approach by adding the requirement...

  4. JC virus small T antigen binds phosphatase PP2A and Rb family proteins and is required for efficient viral DNA replication activity.

    Directory of Open Access Journals (Sweden)

    Brigitte Bollag

    Full Text Available BACKGROUND: The human polyomavirus, JC virus (JCV produces five tumor proteins encoded by transcripts alternatively spliced from one precursor messenger RNA. Significant attention has been given to replication and transforming activities of JCV's large tumor antigen (TAg and three T' proteins, but little is known about small tumor antigen (tAg functions. Amino-terminal sequences of tAg overlap with those of the other tumor proteins, but the carboxy half of tAg is unique. These latter sequences are the least conserved among the early coding regions of primate polyomaviruses. METHODOLOGY AND FINDINGS: We investigated the ability of wild type and mutant forms of JCV tAg to interact with cellular proteins involved in regulating cell proliferation and survival. The JCV P99A tAg is mutated at a conserved proline, which in the SV40 tAg is required for efficient interaction with protein phosphatase 2A (PP2A, and the C157A mutant tAg is altered at one of two newly recognized LxCxE motifs. Relative to wild type and C157A tAgs, P99A tAg interacts inefficiently with PP2A in vivo. Unlike SV40 tAg, JCV tAg binds to the Rb family of tumor suppressor proteins. Viral DNAs expressing mutant t proteins replicated less efficiently than did the intact JCV genome. A JCV construct incapable of expressing tAg was replication-incompetent, a defect not complemented in trans using a tAg-expressing vector. CONCLUSIONS: JCV tAg possesses unique properties among the polyomavirus small t proteins. It contributes significantly to viral DNA replication in vivo; a tAg null mutant failed to display detectable DNA replication activity, and a tAg substitution mutant, reduced in PP2A binding, was replication-defective. Our observation that JCV tAg binds Rb proteins, indicates all five JCV tumor proteins have the potential to influence cell cycle progression in infected and transformed cells. It remains unclear how these proteins coordinate their unique and overlapping functions.

  5. windbeutel, a gene required for dorsoventral patterning in Drosophila, encodes a protein that has homologies to vertebrate proteins of the endoplasmic reticulum

    OpenAIRE

    Konsolaki, Mary; Schüpbach, Trudi

    1998-01-01

    The formation of the dorsoventral axis of the Drosophila embryo depends on cell–cell interactions that take place in the female ovary and involve the activation of transmembrane receptors by secreted ligands. The gene windbeutel functions in the somatic follicle cells of the ovary and is required for the generation of a signal that will determine the ventral side of the embryo. This signal originates in the follicle cells during oogenesis, but its actions are only manifested after fertilizati...

  6. Metabolic Responses to Dietary Protein Restriction Require an Increase in FGF21 that Is Delayed by the Absence of GCN2.

    Science.gov (United States)

    Laeger, Thomas; Albarado, Diana C; Burke, Susan J; Trosclair, Lexus; Hedgepeth, John W; Berthoud, Hans-Rudolf; Gettys, Thomas W; Collier, J Jason; Münzberg, Heike; Morrison, Christopher D

    2016-07-19

    FGF21 contributes to the metabolic response to dietary protein restriction, and prior data implicate GCN2 as the amino acid sensor linking protein restriction to FGF21 induction. Here, we demonstrate the persistent and essential role of FGF21 in the metabolic response to protein restriction. We show that Fgf21 KO mice are fully resistant to low protein (LP)-induced changes in food intake, energy expenditure (EE), body weight gain, and metabolic gene expression for 6 months. Gcn2 KO mice recapitulate this phenotype, but LP-induced effects on food intake, EE, and body weight subsequently begin to appear after 14 days on diet. We show that this delayed emergence of LP-induced metabolic effects in Gcn2 KO mice coincides with a delayed but progressive increase of hepatic Fgf21 expression and blood FGF21 concentrations over time. These data indicate that FGF21 is essential for the metabolic response to protein restriction but that GCN2 is only transiently required for LP-induced FGF21.

  7. Metabolic Responses to Dietary Protein Restriction Require an Increase in FGF21 that Is Delayed by the Absence of GCN2

    Directory of Open Access Journals (Sweden)

    Thomas Laeger

    2016-07-01

    Full Text Available FGF21 contributes to the metabolic response to dietary protein restriction, and prior data implicate GCN2 as the amino acid sensor linking protein restriction to FGF21 induction. Here, we demonstrate the persistent and essential role of FGF21 in the metabolic response to protein restriction. We show that Fgf21 KO mice are fully resistant to low protein (LP-induced changes in food intake, energy expenditure (EE, body weight gain, and metabolic gene expression for 6 months. Gcn2 KO mice recapitulate this phenotype, but LP-induced effects on food intake, EE, and body weight subsequently begin to appear after 14 days on diet. We show that this delayed emergence of LP-induced metabolic effects in Gcn2 KO mice coincides with a delayed but progressive increase of hepatic Fgf21 expression and blood FGF21 concentrations over time. These data indicate that FGF21 is essential for the metabolic response to protein restriction but that GCN2 is only transiently required for LP-induced FGF21.

  8. A decay-accelerating factor-binding strain of coxsackievirus B3 requires the coxsackievirus-adenovirus receptor protein to mediate lytic infection of rhabdomyosarcoma cells.

    Science.gov (United States)

    Shafren, D R; Williams, D T; Barry, R D

    1997-12-01

    The composition of the cellular receptor complex for coxsackievirus B3 (CVB3) has been an area of much contention for the last 30 years. Recently, two individual components of a putative CVB3 cellular receptor complex have been identified as (i) decay-accelerating factor (DAF) and (ii) the coxsackievirus-adenovirus receptor protein (CAR). The present study elucidates the individual roles of DAF and CAR in cell entry of CVB3 Nancy. First, we confirm that the DAF-binding phenotype of CVB3 correlates to the presence of key amino acids located in the viral capsid protein, VP2. Second, using antibody blockade, we show that complete protection of permissive cells from infection by high input multiplicities of CVB3 requires a combination of both anti-DAF and anti-CAR antibodies. Finally, it is shown that expression of the CAR protein on the surface of nonpermissive DAF-expressing RD cells renders them highly susceptible to CVB3-mediated lytic infection. Therefore, although the majority of CVB3 Nancy attaches to the cell via DAF, only virus directly interacting with the CAR protein mediates lytic infection. The role of DAF in CVB3 cell infection may be analogous to that recently described for coxsackievirus A21 (D. R. Shafren, D. J. Dorahy, R. A. Ingham, G. F. Burns, and R. D. Barry, J. Virol. 71:4736-4743, 1997), in that DAF may act as a CVB3 sequestration site, enhancing viral presentation to the functional CAR protein.

  9. Transport of proteins to the plant vacuole is not by bulk flow through the secretory system, and requires positive sorting information.

    Science.gov (United States)

    Dorel, C; Voelker, T A; Herman, E M; Chrispeels, M J

    1989-02-01

    Plant cells, like other eukaryotic cells, use the secretory pathway to target proteins to the vacuolar/lysosomal compartment and to the extracellular space. We wished to determine whether the presence of a hydrophobic signal peptide would result in the transport of a reporter protein to vacuoles by bulk flow; to investigate this question, we expressed a chimeric gene in transgenic tobacco. The chimeric gene, Phalb, used for this study consists of the 1,188-bp 5' upstream sequence and the hydrophobic signal sequence of a vacuolar seed protein phytohemagglutinin, and the coding sequence of a cytosolic seed albumin (PA2). The chimeric protein PHALB cross-reacted with antibodies to PA2 and was found in the seeds of the transgenic plants (approximately 0.7% of total protein), but not in the leaves, roots, or flowers. Immunoblot analyses of seed extracts revealed four glycosylated polypeptides ranging in molecular weight from 29,000 to 32,000. The four polypeptides are glycoforms of a single polypeptide of Mr 27,000, and the heterogeneity is due to the presence of high mannose and endoglycosidase H-resistant glycans. The PHALB products reacted with an antiserum specific for complex plant glycans indicating that the glycans had been modified in the Golgi apparatus. Subcellular fractionation of glycerol extracts of mature seeds showed that only small amounts of PHALB accumulated in the protein storage vacuoles of the tobacco seeds. In homogenates made in an isotonic medium, very little PHALB was associated with the organelle fraction containing the endoplasmic reticulum and Golgi apparatus; most of it was in the soluble fraction. We conclude that PHALB passed through the Golgi apparatus, but did not arrive in the vacuoles. Transport to vacuoles is not by a bulk-flow mechanism, once proteins have entered the secretory system, and requires information beyond that provided by a hydrophobic signal peptide.

  10. PriC-mediated DNA replication restart requires PriC complex formation with the single-stranded DNA-binding protein.

    Science.gov (United States)

    Wessel, Sarah R; Marceau, Aimee H; Massoni, Shawn C; Zhou, Ruobo; Ha, Taekjip; Sandler, Steven J; Keck, James L

    2013-06-14

    Frequent collisions between cellular DNA replication complexes (replisomes) and obstacles such as damaged DNA or frozen protein complexes make DNA replication fork progression surprisingly sporadic. These collisions can lead to the ejection of replisomes prior to completion of replication, which, if left unrepaired, results in bacterial cell death. As such, bacteria have evolved DNA replication restart mechanisms that function to reload replisomes onto abandoned DNA replication forks. Here, we define a direct interaction between PriC, a key Escherichia coli DNA replication restart protein, and the single-stranded DNA-binding protein (SSB), a protein that is ubiquitously associated with DNA replication forks. PriC/SSB complex formation requires evolutionarily conserved residues from both proteins, including a pair of Arg residues from PriC and the C terminus of SSB. In vitro, disruption of the PriC/SSB interface by sequence changes in either protein blocks the first step of DNA replication restart, reloading of the replicative DnaB helicase onto an abandoned replication fork. Consistent with the critical role of PriC/SSB complex formation in DNA replication restart, PriC variants that cannot bind SSB are non-functional in vivo. Single-molecule experiments demonstrate that PriC binding to SSB alters SSB/DNA complexes, exposing single-stranded DNA and creating a platform for other proteins to bind. These data lead to a model in which PriC interaction with SSB remodels SSB/DNA structures at abandoned DNA replication forks to create a DNA structure that is competent for DnaB loading.

  11. Different blue-light requirement for the accumulation of transcripts from nuclear genes for thylakoid proteins in Nicotiana tabacum and Lycopersicon esculentum.

    Science.gov (United States)

    Palomares, R; Herrmann, R G; Oelmüller, R

    1991-11-01

    We have isolated recombinant lambda gt11 phages which carry cDNA clones for the major light-harvesting chlorophyll a/b-binding proteins of photosystem I (LHCPI) and II (LHCPII), subunit II of photosystem I, a chlorophyll a/b-binding protein of photosystem II (CP24), the Rieske iron-sulphur protein of the cytochrome b6/f complex, and the 33, 23 and 16 kDa proteins of the water-oxidizing complex of photosystem II from Nicotiana tabacum. The nucleotide sequences of cDNA clones encoding the precursors for LHCPI and the FeS protein are presented. If tobacco or tomato seedlings, or seedlings of a phytochrome-deficient aurea mutant of tomato which lacks more than 95% of the phytochrome of the isogenic wild type, are kept in blue light, the transcript level of each of these genes is higher than in seedlings grown in red light suggesting the involvement of a blue-UVA-light photoreceptor. In the case of LHCPI, a 1 min blue-light pulse applied to red-light-grown seedlings is sufficient to increase the transcript levels to those present in blue-light-grown seedlings, whereas almost no increase is observed for transcripts encoding the FeS and 33 kDa proteins. If dark-grown tomato seedlings receive a single far-red-light pulse, significant stimulation is detected for LHCPI transcripts, whereas transcripts encoding the FeS and 33 kDa proteins are not stimulated. It is concluded that the lower light requirement for the increase in the LHCPI transcript level is not specific for one of the light-dependent signal transduction chains.

  12. Protein kinase C δ signaling is required for dietary prebiotic-induced strengthening of intestinal epithelial barrier function

    Science.gov (United States)

    Wu, Richard Y.; Abdullah, Majd; Määttänen, Pekka; Pilar, Ana Victoria C.; Scruten, Erin; Johnson-Henry, Kathene C.; Napper, Scott; O’Brien, Catherine; Jones, Nicola L.; Sherman, Philip M.

    2017-01-01

    Prebiotics are non-digestible oligosaccharides that promote the growth of beneficial gut microbes, but it is unclear whether they also have direct effects on the intestinal mucosal barrier. Here we demonstrate two commercial prebiotics, inulin and short-chain fructo-oligosaccharide (scFOS), when applied onto intestinal epithelia in the absence of microbes, directly promote barrier integrity to prevent pathogen-induced barrier disruptions. We further show that these effects involve the induction of select tight junction (TJ) proteins through a protein kinase C (PKC) δ-dependent mechanism. These results suggest that in the absence of microbiota, prebiotics can directly exert barrier protective effects by activating host cell signaling in the intestinal epithelium, which represents a novel alternative mechanism of action of prebiotics. PMID:28098206

  13. A specific sorting signal is not required for the polarized secretion of newly synthesized proteins from cultured intestinal epithelial cells.

    Science.gov (United States)

    Rindler, M J; Traber, M G

    1988-08-01

    Caco-2 cells, derived from human colon, have the morphological, functional, and biochemical properties of small intestinal epithelial cells. After infection with enveloped viruses, influenza virions assembled at the apical plasma membrane while vesicular stomatitis virus (VSV) particles appeared exclusively at the basolateral membrane, similar to the pattern observed in virus-infected Madin-Darby canine kidney (MDCK). When grown in Millicell filter chamber devices and labeled with [35S]methionine, Caco-2 monolayers released all of their radiolabeled secretory products preferentially into the basal chamber. Among the proteins identified were apolipoproteins AI and E, transferrin, and alpha-fetoprotein. No proteins were observed to be secreted preferentially from the apical cell surface. The lysosomal enzyme beta-hexosaminidase was also secreted primarily from the basolateral surface of the cells in the presence or absence of lysosomotropic drugs or tunicamycin, which inhibit the targetting of lysosomal enzymes to lysosomes. Neither of these drug treatments significantly affected the polarized secretion of other nonlysosomal proteins. In addition, growth hormone (GH), which is released in a nonpolar fashion from MDCK cells, was secreted exclusively from the basolateral membrane after transfection of Caco-2 cells with GH cDNA in a pSV2-based expression vector. Similar results were obtained in transient expression experiments and after selection of permanently transformed Caco-2 cells expressing GH. Since both beta-hexosaminidase and GH would be expected to lack sorting signals for polarized exocytosis in epithelial cells, these results indicate that in intestinal cells, proteins transported via the basolateral secretory pathway need not have specific sorting signals.

  14. The ciliary proteins Meckelin and Jouberin are required for retinoic acid-dependent neural differentiation of mouse embryonic stem cells.

    Science.gov (United States)

    Romani, Sveva; Illi, Barbara; De Mori, Roberta; Savino, Mauro; Gleeson, Joseph G; Valente, Enza Maria

    2014-01-01

    The dysfunction of the primary cilium, a complex, evolutionarily conserved, organelle playing an important role in sensing and transducing cell signals, is the unifying pathogenetic mechanism of a growing number of diseases collectively termed "ciliopathies", typically characterized by multiorgan involvement. Developmental defects of the central nervous system (CNS) characterize a subset of ciliopathies showing clinical and genetic overlap, such as Joubert syndrome (JS) and Meckel syndrome (MS). Although several knock-out mice lacking a variety of ciliary proteins have shown the importance of primary cilia in the development of the brain and CNS-derived structures, developmental in vitro studies, extremely useful to unravel the role of primary cilia along the course of neural differentiation, are still missing. Mouse embryonic stem cells (mESCs) have been recently proven to mimic brain development, giving the unique opportunity to dissect the CNS differentiation process along its sequential steps. In the present study we show that mESCs express the ciliary proteins Meckelin and Jouberin in a developmentally-regulated manner, and that these proteins co-localize with acetylated tubulin labeled cilia located at the outer embryonic layer. Further, mESCs differentiating along the neuronal lineage activate the cilia-dependent sonic hedgehog signaling machinery, which is impaired in Meckelin knock-out cells but results unaffected in Jouberin-deficient mESCs. However, both lose the ability to acquire a neuronal phenotype. Altogether, these results demonstrate a pivotal role of Meckelin and Jouberin during embryonic neural specification and indicate mESCs as a suitable tool to investigate the developmental impact of ciliary proteins dysfunction.

  15. The insulator protein Suppressor of Hairy wing is required for proper ring canal development during oogenesis in Drosophila.

    Science.gov (United States)

    Hsu, Shih-Jui; Plata, Maria P; Ernest, Ben; Asgarifar, Saghi; Labrador, Mariano

    2015-07-01

    Chromatin insulators orchestrate gene transcription during embryo development and cell differentiation by stabilizing interactions between distant genomic sites. Mutations in genes encoding insulator proteins are generally lethal, making in vivo functional analyses of insulator proteins difficult. In Drosophila, however, mutations in the gene encoding the Suppressor of Hairy wing insulator protein [Su(Hw)] are viable and female sterile, providing an opportunity to study insulator function during oocyte development. Whereas previous reports suggest that the function of Su(Hw) in oogenesis is independent of its insulator activity, many aspects of the role of Su(Hw) in Drosophila oogenesis remain unexplored. Here we show that mutations in su(Hw) result in smaller ring canal lumens and smaller outer ring diameters, which likely obstruct molecular and vesicle passage from nurse cells to the oocyte. Fluorescence microscopy reveals that lack of Su(Hw) leads to excess accumulation of Kelch (Kel) and Filament-actin (F-actin) proteins in the ring canal structures of developing egg chambers. Furthermore, we found that misexpression of the Src oncogene at 64B (Src64B) may cause ring canal development defects as microarray analysis and real-time RT-PCR revealed there is a three fold decrease in Src64B expression in su(Hw) mutant ovaries. Restoration of Src64B expression in su(Hw) mutant female germ cells rescued the ring phenotype but did not restore fertility. We conclude that loss of su(Hw) affects expression of many oogenesis related genes and down-regulates Src64B, resulting in ring canal defects potentially contributing to obstruction of molecular flow and an eventual failure of egg chamber organization.

  16. Efficient extraction of proteins from formalin-fixed paraffin-embedded tissues requires higher concentration of tris(hydroxymethyl)aminomethane

    OpenAIRE

    KAWASHIMA, Yusuke; Kodera, Yoshio; Singh, Anil; Matsumoto, Masaomi; Matsumoto, Hiroyuki

    2014-01-01

    Background Numerous formaldehyde-fixed and paraffin-embedded clinical tissues have been created in the past decades and stored in pathological depositories at hospitals as well as in clinical laboratories worldwide. In addition to the archived tissues, formaldehyde-fixation is also mandatory for preparing proteomics samples from diseased patients or animal models in order to inactivate contagious agents. Protein extraction from formaldehyde-fixed tissues is hampered by the Schiff base formati...

  17. InvA protein is a Nudix hydrolase required for infection by pathogenic Leptospira in cell lines and animals.

    Science.gov (United States)

    Luo, Yihui; Liu, Yan; Sun, Dexter; Ojcius, David M; Zhao, Jinfang; Lin, Xuai; Wu, Dong; Zhang, Rongguang; Chen, Ming; Li, Lanjuan; Yan, Jie

    2011-10-21

    Leptospirosis caused by pathogenic species of the genus Leptospira is a re-emerging zoonotic disease, which affects a wide variety of host species and is transmitted by contaminated water. The genomes of several pathogenic Leptospira species contain a gene named invA, which contains a Nudix domain. However, the function of this gene has never been characterized. Here, we demonstrated that the invA gene was highly conserved in protein sequence and present in all tested pathogenic Leptospira species. The recombinant InvA protein of pathogenic L. interrogans strain Lai hydrolyzed several specific dinucleoside oligophosphate substrates, reflecting the enzymatic activity of Nudix in Leptospira species. Pathogenic leptospires did not express this protein in media but temporarily expressed it at early stages (within 60 min) of infection of macrophages and nephric epithelial cells. Comparing with the wild type, the invA-deficient mutant displayed much lower infectivity and a significantly reduced survival rate in macrophages and nephric epithelial cells. Moreover, the invA-deficient leptospires presented an attenuated virulence in hamsters, caused mild histopathological damage, and were transmitted in lower numbers in the urine, compared with the wild-type strain. The invA revertant, made by complementing the invA-deficient mutant with the invA gene, reacquired virulence similar to the wild type in vitro and in vivo. The LD(50) in hamsters was 1000-fold higher for the invA-deficient mutant than for the invA revertant and wild type. These results demonstrate that the InvA protein is a Nudix hydrolase, and the invA gene is essential for virulence in pathogenic Leptospira species.

  18. PGC-1{alpha} is required for AICAR induced expression of GLUT4 and mitochondrial proteins in mouse skeletal muscle

    DEFF Research Database (Denmark)

    Leick, Lotte; Fentz, Joachim; Biensø, Rasmus S

    2010-01-01

    GLUT4, cytochrome c oxidase (COX)I and cytochrome (cyt) c protein expression ~10-40% relative to saline in white muscles of the WT mice, but not of the PGC-1alpha KO mice. In line, GLUT4 and cyt c mRNA content increased 30-60% 4h after a single AICAR injection relative to saline only in WT mice. One...

  19. Distinct functional domains within the acidic cluster of tegument protein pp28 required for trafficking and cytoplasmic envelopment of human cytomegalovirus.

    Science.gov (United States)

    Seo, Jun-Young; Jeon, Hyejin; Hong, Sookyung; Britt, William J

    2016-10-01

    Human cytomegalovirus UL99-encoded tegument protein pp28 contains a 16 aa acidic cluster that is required for pp28 trafficking to the assembly compartment (AC) and the virus assembly. However, functional signals within the acidic cluster of pp28 remain undefined. Here, we demonstrated that an acidic cluster rather than specific sorting signals was required for trafficking to the AC. Recombinant viruses with chimeric pp28 proteins expressing non-native acidic clusters exhibited delayed viral growth kinetics and decreased production of infectious virus, indicating that the native acidic cluster of pp28 was essential for wild-type virus assembly. These results suggested that the acidic cluster of pp28 has distinct functional domains required for trafficking and for efficient virus assembly. The first half (aa 44-50) of the acidic cluster was sufficient for pp28 trafficking, whereas the native acidic cluster consisting of aa 51-59 was required for the assembly of wild-type levels of infectious virus.

  20. An ABC transporter B family protein, ABCB19, is required for cytoplasmic streaming and gravitropism of the inflorescence stems.

    Science.gov (United States)

    Okamoto, Keishi; Ueda, Haruko; Shimada, Tomoo; Tamura, Kentaro; Koumoto, Yasuko; Tasaka, Masao; Morita, Miyo Terao; Hara-Nishimura, Ikuko

    2016-01-01

    A significant feature of plant cells is the extensive motility of organelles and the cytosol, which was originally defined as cytoplasmic streaming. We suggested previously that a three-way interaction between plant-specific motor proteins myosin XIs, actin filaments, and the endoplasmic reticulum (ER) was responsible for cytoplasmic streaming. (1) Currently, however, there are no reports of molecular components for cytoplasmic streaming other than the actin-myosin-cytoskeleton and ER-related proteins. In the present study, we found that elongated cells of inflorescence stems of Arabidopsis thaliana exhibit vigorous cytoplasmic streaming. Statistical analysis showed that the maximal velocity of plastid movements is 7.26 µm/s, which is much faster than the previously reported velocities of organelles. Surprisingly, the maximal velocity of streaming in the inflorescence stem cells was significantly reduced to 1.11 µm/s in an Arabidopsis mutant, abcb19-101, which lacks ATP BINDING CASSETTE SUBFAMILY B19 (ABCB19) that mediates the polar transport of the phytohormone auxin together with PIN-FORMED (PIN) proteins. Polar auxin transport establishes the auxin concentration gradient essential for plant development and tropisms. Deficiency of ABCB19 activity eventually caused enhanced gravitropic responses of the inflorescence stems and abnormally flexed inflorescence stems. These results suggest that ABCB19-mediated auxin transport plays a role not only in tropism regulation, but also in cytoplasmic streaming.

  1. The chloroplast NifS-like protein of Arabidopsis thaliana is required for iron-sulfur cluster formation in ferredoxin.

    Science.gov (United States)

    Ye, Hong; Garifullina, Gulnara F; Abdel-Ghany, Salah E; Zhang, Lihong; Pilon-Smits, Elizabeth A H; Pilon, Marinus

    2005-02-01

    Plastids are known to be able to synthesize their own iron-sulfur clusters, but the biochemical machinery responsible for this process is not known. In this study it is investigated whether CpNifS, the chloroplastic NifS-like cysteine desulfurase of Arabidopsis thaliana (L.) Heynh. is responsible for the release of sulfur from cysteine for the biogenesis of iron-sulfur (Fe-S) clusters in chloroplasts. Using an in vitro reconstitution assay it was found that purified CpNifS was sufficient for Fe-S cluster formation in ferredoxin in the presence of cysteine and a ferrous iron salt. Antibody-depletion experiments using stromal extract showed that CpNifS is also essential for the Fe-S cluster formation activity of chloroplast stroma. The activity of CpNifS in the stroma was 50- to 80-fold higher than that of purified CpNifS on a per-protein basis, indicating that other stromal factors cooperate in Fe-S cluster formation. When stromal extract was separated on a gel-filtration column, most of the CpNifS eluted as a dimer of 86 kDa, but a minor fraction of the stromal CpNifS eluted at a molecular weight of approx. 600 kDa, suggesting the presence of a multi-protein complex. The possible nature of the interacting proteins is discussed.

  2. A Novel and Conserved Plasmodium Sporozoite Membrane Protein SPELD is Required for Maturation of Exo-erythrocytic Forms

    Science.gov (United States)

    Al-Nihmi, Faisal Mohammed Abdul; Kolli, Surendra Kumar; Reddy, Segireddy Rameswara; Mastan, Babu S.; Togiri, Jyothi; Maruthi, Mulaka; Gupta, Roshni; Sijwali, Puran Singh; Mishra, Satish; Kumar, Kota Arun

    2017-01-01

    Plasmodium sporozoites are the infective forms of malaria parasite to vertebrate host and undergo dramatic changes in their transcriptional repertoire during maturation in mosquito salivary glands. We report here the role of a novel and conserved Plasmodium berghei protein encoded by PBANKA_091090 in maturation of Exo-erythrocytic Forms (EEFs) and designate it as Sporozoite surface Protein Essential for Liver stage Development (PbSPELD). PBANKA_091090 was previously annotated as PB402615.00.0 and its transcript was recovered at maximal frequency in the Serial Analysis of the Gene Expression (SAGE) of Plasmodium berghei salivary gland sporozoites. An orthologue of this transcript was independently identified in Plasmodium vivax sporozoite microarrays and was designated as Sporozoite Conserved Orthologous Transcript-2 (scot-2). Functional characterization through reverse genetics revealed that PbSPELD is essential for Plasmodium liver stage maturation. mCherry transgenic of PbSPELD localized the protein to plasma membrane of sporozoites and early EEFs. Global microarray analysis of pbspeld ko revealed EEF attenuation being associated with down regulation of genes central to general transcription, cell cycle, proteosome and cadherin signaling. pbspeld mutant EEFs induced pre-erythrocytic immunity with 50% protective efficacy. Our studies have implications for attenuating the human Plasmodium liver stages by targeting SPELD locus. PMID:28067322

  3. A DEAD box protein is required for formation of a hidden break in Arabidopsis chloroplast 23S rRNA.

    Science.gov (United States)

    Nishimura, Kenji; Ashida, Hiroki; Ogawa, Taro; Yokota, Akiho

    2010-09-01

    In plant chloroplasts, the ribosomal RNA (rRNA) of the large subunit of the ribosome undergoes post-maturation fragmentation processing. This processing consists of site-specific cleavage that generates gapped, discontinuous rRNA molecules. However, the molecular mechanism underlying introduction of the gap structure (the 'hidden break') is poorly understood. Here, we found that the DEAD box protein RH39 plays a key role in introduction of the hidden break into the 23S rRNA in Arabidopsis chloroplasts. Genetic screening for an Arabidopsis plant with a drastically reduced level of ribulose-1,5-bisphosphate carboxylase/oxygenase identified an RH39 mutant. The levels of other chloroplast-encoded photosynthetic proteins were also severely reduced. The reductions were not due to a failure of transcription, but rather inefficiency in translation. RNA gel blotting revealed incomplete fragmentation of 23S rRNA in chloroplasts during maturation. In vitro analysis with recombinant RH39 suggested that the protein binds to the adjacent sequence upstream of the hidden break site to exert its function. We propose a molecular mechanism for the RH39-mediated fragmentation processing of 23S rRNA in chloroplasts.

  4. The stress granule protein Vgl1 and poly(A)-binding protein Pab1 are required for doxorubicin resistance in the fission yeast Schizosaccharomyces pombe

    Energy Technology Data Exchange (ETDEWEB)

    Morita, Takahiro [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka 577-8502 (Japan); Satoh, Ryosuke [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka 577-8502 (Japan); Japan Society for the Promotion of Science, 1-8 Chiyoda-ku, Tokyo 102-8472 (Japan); Umeda, Nanae; Kita, Ayako [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka 577-8502 (Japan); Sugiura, Reiko, E-mail: sugiurar@phar.kindai.ac.jp [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka 577-8502 (Japan)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Stress granules (SGs) as a mechanism of doxorubicin tolerance. Black-Right-Pointing-Pointer We characterize the role of stress granules in doxorubicin tolerance. Black-Right-Pointing-Pointer Deletion of components of SGs enhances doxorubicin sensitivity in fission yeast. Black-Right-Pointing-Pointer Doxorubicin promotes SG formation when combined with heat shock. Black-Right-Pointing-Pointer Doxorubicin regulates stress granule assembly independent of eIF2{alpha} phosphorylation. -- Abstract: Doxorubicin is an anthracycline antibiotic widely used for chemotherapy. Although doxorubicin is effective in the treatment of several cancers, including solid tumors and leukemias, the basis of its mechanism of action is not completely understood. Here, we describe the effects of doxorubicin and its relationship with stress granules formation in the fission yeast, Schizosaccharomyces pombe. We show that disruption of genes encoding the components of stress granules, including vgl1{sup +}, which encodes a multi-KH type RNA-binding protein, and pab1{sup +}, which encodes a poly(A)-binding protein, resulted in greater sensitivity to doxorubicin than seen in wild-type cells. Disruption of the vgl1{sup +} and pab1{sup +} genes did not confer sensitivity to other anti-cancer drugs such as cisplatin, 5-fluorouracil, and paclitaxel. We also showed that doxorubicin treatment promoted stress granule formation when combined with heat shock. Notably, doxorubicin treatment did not induce hyperphosphorylation of eIF2{alpha}, suggesting that doxorubicin is involved in stress granule assembly independent of eIF2{alpha} phosphorylation. Our results demonstrate the usefulness of fission yeast for elucidating the molecular targets of doxorubicin toxicity and suggest a novel drug-resistance mechanism involving stress granule assembly.

  5. Human Cytomegalovirus UL97 Kinase Activity Is Required for the Hyperphosphorylation of Retinoblastoma Protein and Inhibits the Formation of Nuclear Aggresomes

    Energy Technology Data Exchange (ETDEWEB)

    Prichard, Mark N.; Sztul, Elizabeth; Daily, Shannon L.; Perry, Amie L.; Frederick, Samuel L.; Gill, Rachel B.; Hartline, Caroll B.; Streblow, Daniel N.; Varnum, Susan M.; Smith, Richard D.; Kern, Earl R.

    2008-05-01

    Cells infected with human cytomegalovirus in the absence of UL97 kinase activity produce large nuclear aggregates that sequester considerable quantities of viral proteins. A transient expression assay suggested that pp71 and IE1 were also involved in this process, and this suggestion was significant, since both proteins have been reported to interact with components of promyelocytic leukemia (PML) bodies (ND10) and also interact functionally with retinoblastoma pocket proteins (RB). PML bodies have been linked to the formation of nuclear aggresomes, and colocalization studies suggested that viral proteins were recruited to these structures and that UL97 kinase activity inhibited their formation. Proteins associated with PML bodies were examined by Western blot analysis, and pUL97 appeared to specifically affect the phosphorylation of RB in a kinasedependent manner. Three consensus RB binding motifs were identified in the UL97 kinase, and recombinant viruses were constructed in which each was mutated to assess a potential role in the phosphorylation of RB and the inhibition of nuclear aggresome formation. The mutation of either the conserved LxCxE RB binding moti for the lysine required for kinase activity impaired the ability of the virus to stabilize and phosphorylate RB. We concluded from these studies that both UL97 kinase activity and the LxCxE RB binding motif are required for the phosphorylation and stabilization of RB in infected cells and that this effect can be antagonized by the antiviral drug maribavir. These data also suggest a potential link between RB function and the formation of aggresomes.

  6. echinus, required for interommatidial cell sorting and cell death in the Drosophila pupal retina, encodes a protein with homology to ubiquitin-specific proteases

    Directory of Open Access Journals (Sweden)

    Gorski Sharon M

    2007-07-01

    Full Text Available Abstract Background Programmed cell death is used to remove excess cells between ommatidia in the Drosophila pupal retina. This death is required to establish the crystalline, hexagonal packing of ommatidia that characterizes the adult fly eye. In previously described echinus mutants, interommatidial cell sorting, which precedes cell death, occurred relatively normally. Interommatidial cell death was partially suppressed, resulting in adult eyes that contained excess pigment cells, and in which ommatidia were mildly disordered. These results have suggested that echinus functions in the pupal retina primarily to promote interommatidial cell death. Results We generated a number of new echinus alleles, some likely null mutants. Analysis of these alleles provides evidence that echinus has roles in cell sorting as well as cell death. echinus encodes a protein with homology to ubiquitin-specific proteases. These proteins cleave ubiquitin-conjugated proteins at the ubiquitin C-terminus. The echinus locus encodes multiple splice forms, including two proteins that lack residues thought to be critical for deubiquitination activity. Surprisingly, ubiquitous expression in the eye of versions of Echinus that lack residues critical for ubiquitin specific protease activity, as well as a version predicted to be functional, rescue the echinus loss-of-function phenotype. Finally, genetic interactions were not detected between echinus loss and gain-of-function and a number of known apoptotic regulators. These include Notch, EGFR, the caspases Dronc, Drice, Dcp-1, Dream, the caspase activators, Rpr, Hid, and Grim, the caspase inhibitor DIAP1, and Lozenge or Klumpfuss. Conclusion The echinus locus encodes multiple splice forms of a protein with homology to ubiquitin-specific proteases, but protease activity is unlikely to be required for echinus function, at least when echinus is overexpressed. Characterization of likely echinus null alleles and genetic interactions

  7. DNA cross-link-dependent RAD50/MRE11/NBS1 subnuclear assembly requires the Fanconi anemia C protein.

    Science.gov (United States)

    Pichierri, Pietro; Averbeck, Dietrich; Rosselli, Filippo

    2002-10-01

    Fanconi anemia (FA) is a cancer-predisposition syndrome characterized by hypersensitivity to interstrand-cross-link (ICL) inducers. FA hypersensitivity to ICL has been correlated with alterations in homologous recombination, non-homologous end-joining, telomere maintenance, DNA-damage assessment and checkpoint regulation, processes in which the components of the RAD50/MRE11/NBS1 (RMN) complex are involved. To better characterize the mechanisms by which ICL are processed in human cells and to gain insight into their toxicity in FA, we examined (i). the RMN complex assembling in response to the ICL inducers mitomycin C (MMC) and photoactivated 8-methoxypsoralen and (ii). the proficiency of FA cells to perform RMN activation in response to ICL inducers. We show here that ICL activates the assembly of the RMN proteins into subnuclear foci, and that their formation proceeds independently of ICL incision, a step mainly dependent on XP-F/ERCC1 heterodimer activity. Interestingly, FA cells were unable to form RMN foci in response to either ICL inducer. Analysis by pulsed-field gel electrophoresis and single-cell gel electrophoresis of MMC-treated cells showed that FA cells from complementation group C (FA-C cells, defective in the FANCC gene) form double-strand breaks and unhook MMC-induced ICL similarly to FANCC wild-type cells. These observations imply that the absence of RMN assembly in FA-C cells is not simply due to the absence of DNA ends produced as intermediates of ICL processing, and indicates a direct role for FANCC in RMN focus assembly in response to ICL inducers. Moreover, we show that the formation of foci, including BRCA1 and/or RAD51 proteins, is significantly delayed in FA cells. These alterations in the assembly of DNA-repair proteins in FA provide an interpretation for the DNA-damage processing anomalies observed in FA cells and for the genetic instability and the cancer predisposition of the syndrome.

  8. Activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo.

    Directory of Open Access Journals (Sweden)

    Larry J Bischof

    2008-10-01

    Full Text Available Pore-forming toxins (PFTs constitute the single largest class of proteinaceous bacterial virulence factors and are made by many of the most important bacterial pathogens. Host responses to these toxins are complex and poorly understood. We find that the endoplasmic reticulum unfolded protein response (UPR is activated upon exposure to PFTs both in Caenorhabditis elegans and in mammalian cells. Activation of the UPR is protective in vivo against PFTs since animals that lack either the ire-1-xbp-1 or the atf-6 arms of the UPR are more sensitive to PFT than wild-type animals. The UPR acts directly in the cells targeted by the PFT. Loss of the UPR leads to a normal response against unrelated toxins or a pathogenic bacterium, indicating its PFT-protective role is specific. The p38 mitogen-activated protein (MAPK kinase pathway has been previously shown to be important for cellular defenses against PFTs. We find here that the UPR is one of the key downstream targets of the p38 MAPK pathway in response to PFT since loss of a functional p38 MAPK pathway leads to a failure of PFT to properly activate the ire-1-xbp-1 arm of the UPR. The UPR-mediated activation and response to PFTs is distinct from the canonical UPR-mediated response to unfolded proteins both in terms of its activation and functional sensitivities. These data demonstrate that the UPR, a fundamental intracellular pathway, can operate in intrinsic cellular defenses against bacterial attack.

  9. Basic Residues within the Ebolavirus VP35 Protein Are Required for Its Viral Polymerase Cofactor Function ▿

    OpenAIRE

    Prins, Kathleen C.; Binning, Jennifer M.; Shabman, Reed S.; Leung, Daisy W.; Amarasinghe, Gaya K.; Christopher F Basler

    2010-01-01

    The ebolavirus (EBOV) VP35 protein binds to double-stranded RNA (dsRNA), inhibits host alpha/beta interferon (IFN-α/β) production, and is an essential component of the viral polymerase complex. Structural studies of the VP35 C-terminal IFN inhibitory domain (IID) identified specific structural features, including a central basic patch and a hydrophobic pocket, that are important for dsRNA binding and IFN inhibition. Several other conserved basic residues bordering the central basic patch and ...

  10. Cdc42 Effector Protein 2 (XCEP2 is required for normal gastrulation and contributes to cellular adhesion in Xenopus laevis

    Directory of Open Access Journals (Sweden)

    Nelson Richard W

    2004-10-01

    Full Text Available Abstract Background Rho GTPases and their downstream effector proteins regulate a diverse array of cellular processes during embryonic development, including reorganization of cytoskeletal architecture, cell adhesion, and transcription. Changes in the activation state of Rho GTPases are converted into changes in cellular behavior by a diversity of effector proteins, which are activated in response to changes in the GTP binding state of Rho GTPases. In this study we characterize the expression and function of one such effector, XCEP2, that is present during gastrulation stages in Xenopus laevis. Results In a search for genes whose expression is regulated during early stages of embryonic development in Xenopus laevis, a gene encoding a Rho GTPase effector protein (Xenopus Cdc42 effector protein 2, or XCEP2 was isolated, and found to be highly homologous, but not identical, to a Xenopus sequence previously submitted to the Genbank database. These two gene sequences are likely pseudoalleles. XCEP2 mRNA is expressed at constant levels until mid- to late- gastrula stages, and then strongly down-regulated at late gastrula/early neurula stages. Injection of antisense morpholino oligonucleotides directed at one or both pseudoalleles resulted in a significant delay in blastopore closure and interfered with normal embryonic elongation, suggesting a role for XCEP2 in regulating gastrulation movements. The morpholino antisense effect could be rescued by co-injection with a morpholino-insensitive version of the XCEP2 mRNA. Antisense morpholino oligonucleotides were found to have no effect on mesodermal induction, suggesting that the observed effects were due to changes in the behavior of involuting cells, rather than alterations in their identity. XCEP2 antisense morpholino oligonucleotides were also observed to cause complete disaggregation of cells composing animal cap explants, suggesting a specific role of XCEP2 in maintenance or regulation of cell

  11. Nitrogenase switch-off by ammonium ions in Azospirillum brasilense requires the GlnB nitrogen signal-transducing protein.

    Science.gov (United States)

    Klassen, Giseli; Souza, Emanuel M; Yates, M Geoffrey; Rigo, Liu Un; Costa, Roberta M; Inaba, Juliana; Pedrosa, Fábio O

    2005-09-01

    Nitrogenase activity in several diazotrophs is switched off by ammonium and reactivated after consumption. The signaling pathway to this system in Azospirillum brasilense is not understood. We show that ammonium-dependent switch-off through ADP-ribosylation of Fe protein was partial in a glnB mutant of A. brasilense but absent in a glnB glnZ double mutant. Triggering of inactivation by anaerobic conditions was not affected in either mutant. The results suggest that glnB is necessary for full ammonium-dependent nitrogenase switch-off in A. brasilense.

  12. The crystal structure of human IRE1 luminal domain reveals a conserved dimerization interface required for activation of the unfolded protein response

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Jiahai; Liu, Chuan Yin; Back, Sung Hoon; Clark, Robert L.; Peisach, Daniel; Xu, Zhaohui; Kaufman, Randal J. (Michigan)

    2010-03-08

    The unfolded protein response (UPR) is an evolutionarily conserved mechanism by which all eukaryotic cells adapt to the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Inositol-requiring kinase 1 (IRE1) and PKR-related ER kinase (PERK) are two type I transmembrane ER-localized protein kinase receptors that signal the UPR through a process that involves homodimerization and autophosphorylation. To elucidate the molecular basis of the ER transmembrane signaling event, we determined the x-ray crystal structure of the luminal domain of human IRE1{alpha}. The monomer of the luminal domain comprises a unique fold of a triangular assembly of {beta}-sheet clusters. Structural analysis identified an extensive dimerization interface stabilized by hydrogen bonds and hydrophobic interactions. Dimerization creates an MHC-like groove at the interface. However, because this groove is too narrow for peptide binding and the purified luminal domain forms high-affinity dimers in vitro, peptide binding to this groove is not required for dimerization. Consistent with our structural observations, mutations that disrupt the dimerization interface produced IRE1{alpha} molecules that failed to either dimerize or activate the UPR upon ER stress. In addition, mutations in a structurally homologous region within PERK also prevented dimerization. Our structural, biochemical, and functional studies in vivo altogether demonstrate that IRE1 and PERK have conserved a common molecular interface necessary and sufficient for dimerization and UPR signaling.

  13. Vesicle-associated membrane protein 3 (VAMP-3) and VAMP-8 are present in human platelets and are required for granule secretion.

    Science.gov (United States)

    Polgár, János; Chung, Sul-Hee; Reed, Guy L

    2002-08-01

    Secretion of platelet granules is necessary for normal hemostasis. Platelet secretion requires soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) complex formation between different members of the syntaxin, SNAP-25, and vesicle-associated membrane protein (VAMP) gene families. Using microcapillary reverse-phase high-performance liquid chromatography-nano-electrospray tandem mass spectrometry, we identified VAMP-3 and VAMP-8 as VAMP isoforms coimmunoprecipitated from platelets with syntaxin 4. Immunoblotting experiments confirmed the presence of VAMP-3 and VAMP-8 but not VAMP-1 or VAMP-2 in platelets. To examine the effect of VAMP proteins on platelet secretion, soluble recombinant (r) VAMP-2, rVAMP-3, and rVAMP-8 were incubated with streptolysin O-permeabilized platelets. Secretion of alpha granules (monitored by flow cytometric measurement of P-selectin) was blocked, and dense-granule secretion (assessed by release of carbon 14-serotonin) was almost completely inhibited by rVAMP-3, whereas rVAMP-8 inhibited secretion of dense granules but not alpha granules. In contrast, rVAMP-2, which formed SNARE complexes in vitro, had no effect on platelet exocytosis. We conclude that VAMP-3 and VAMP-8 form SNARE complexes with platelet syntaxin 4 and are required for platelet granule secretion.

  14. The two-domain LysX protein of Mycobacterium tuberculosis is required for production of lysinylated phosphatidylglycerol and resistance to cationic antimicrobial peptides.

    Directory of Open Access Journals (Sweden)

    Erin Maloney

    2009-07-01

    Full Text Available The well-recognized phospholipids (PLs of Mycobacterium tuberculosis (Mtb include several acidic species such as phosphatidylglycerol (PG, cardiolipin, phosphatidylinositol and its mannoside derivatives, in addition to a single basic species, phosphatidylethanolamine. Here we demonstrate that an additional basic PL, lysinylated PG (L-PG, is a component of the PLs of Mtb H37Rv and that the lysX gene encoding the two-domain lysyl-transferase (mprF-lysyl-tRNA synthetase (lysU protein is responsible for L-PG production. The Mtb lysX mutant is sensitive to cationic antibiotics and peptides, shows increased association with lysosome-associated membrane protein-positive vesicles, and it exhibits altered membrane potential compared to wild type. A lysX complementing strain expressing the intact lysX gene, but not one expressing mprF alone, restored the production of L-PG and rescued the lysX mutant phenotypes, indicating that the expression of both proteins is required for LysX function. The lysX mutant also showed defective growth in mouse and guinea pig lungs and showed reduced pathology relative to wild type, indicating that LysX activity is required for full virulence. Together, our results suggest that LysX-mediated production of L-PG is necessary for the maintenance of optimal membrane integrity and for survival of the pathogen upon infection.

  15. Conserved Tryptophan Motifs in the Large Tegument Protein pUL36 Are Required for Efficient Secondary Envelopment of Herpes Simplex Virus Capsids

    Science.gov (United States)

    Ivanova, Lyudmila; Buch, Anna; Döhner, Katinka; Pohlmann, Anja; Binz, Anne; Prank, Ute; Sandbaumhüter, Malte

    2016-01-01

    ABSTRACT Herpes simplex virus (HSV) replicates in the skin and mucous membranes, and initiates lytic or latent infections in sensory neurons. Assembly of progeny virions depends on the essential large tegument protein pUL36 of 3,164 amino acid residues that links the capsids to the tegument proteins pUL37 and VP16. Of the 32 tryptophans of HSV-1-pUL36, the tryptophan-acidic motifs 1766WD1767 and 1862WE1863 are conserved in all HSV-1 and HSV-2 isolates. Here, we characterized the role of these motifs in the HSV life cycle since the rare tryptophans often have unique roles in protein function due to their large hydrophobic surface. The infectivity of the mutants HSV-1(17+)Lox-pUL36-WD/AA-WE/AA and HSV-1(17+)Lox-CheVP26-pUL36-WD/AA-WE/AA, in which the capsid has been tagged with the fluorescent protein Cherry, was significantly reduced. Quantitative electron microscopy shows that there were a larger number of cytosolic capsids and fewer enveloped virions compared to their respective parental strains, indicating a severe impairment in secondary capsid envelopment. The capsids of the mutant viruses accumulated in the perinuclear region around the microtubule-organizing center and were not dispersed to the cell periphery but still acquired the inner tegument proteins pUL36 and pUL37. Furthermore, cytoplasmic capsids colocalized with tegument protein VP16 and, to some extent, with tegument protein VP22 but not with the envelope glycoprotein gD. These results indicate that the unique conserved tryptophan-acidic motifs in the central region of pUL36 are required for efficient targeting of progeny capsids to the membranes of secondary capsid envelopment and for efficient virion assembly. IMPORTANCE Herpesvirus infections give rise to severe animal and human diseases, especially in young, immunocompromised, and elderly individuals. The structural hallmark of herpesvirus virions is the tegument, which contains evolutionarily conserved proteins that are essential for several

  16. Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT Machinery via Ubiquitination To Facilitate Viral Envelopment

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    Rina Barouch-Bentov

    2016-11-01

    Full Text Available Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate, an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses.

  17. Sequence and structure requirements for specific recognition of HIV-1 TAR and DIS RNA by the HIV-1 Vif protein.

    Science.gov (United States)

    Freisz, Séverine; Mezher, Joelle; Hafirassou, Lamine; Wolff, Philippe; Nominé, Yves; Romier, Christophe; Dumas, Philippe; Ennifar, Eric

    2012-07-01

    The HIV-1 Vif protein plays an essential role in the regulation of the infectivity of HIV-1 virion and in vivo pathogenesis. Vif neutralizes the human DNA-editing enzyme APOBEC3 protein, an antiretroviral cellular factor from the innate immune system, allowing the virus to escape the host defence system. It was shown that Vif is packaged into viral particles through specific interactions with the viral genomic RNA. Conserved and structured sequences from the 5'-noncoding region, such as the Tat-responsive element (TAR) or the genomic RNA dimerization initiation site (DIS), are primary binding sites for Vif. In the present study we used isothermal titration calorimetry to investigate sequence and structure determinants important for Vif binding to short viral RNA corresponding to TAR and DIS stem-loops. We showed that Vif specifically binds TAR and DIS in the low nanomolar range. In addition, Vif primarily binds the TAR UCU bulge, but not the apical loop. Determinants for Vif binding to the DIS loop-loop complex are likely more complex and involve the self-complementary loop together with the upper part of the stem. These results suggest that Tat-TAR inhibitors or DIS small molecule binders might be also effective to disturb Vif-TAR and Vif-DIS binding in order to reduce Vif packaging into virions.

  18. The actin-related protein Sac1 is required for morphogenesis and cell wall integrity in Candida albicans.

    Science.gov (United States)

    Zhang, Bing; Yu, Qilin; Jia, Chang; Wang, Yuzhou; Xiao, Chenpeng; Dong, Yijie; Xu, Ning; Wang, Lei; Li, Mingchun

    2015-08-01

    Candida albicans is a common pathogenic fungus and has aroused widespread attention recently. Actin cytoskeleton, an important player in polarized growth, protein secretion and organization of cell shape, displays irreplaceable role in hyphal development and cell integrity. In this study, we demonstrated a homologue of Saccharomyces cerevisiae Sac1, in C. albicans. It is a potential PIP phosphatase with Sac domain which is related to actin organization, hyphal development, biofilm formation and cell wall integrity. Deletion of SAC1 did not lead to insitiol-auxotroph phenotype in C. albicans, but this gene rescued the growth defect of S. cerevisiae sac1Δ in the insitiol-free medium. Hyphal induction further revealed the deficiency of sac1Δ/Δ in hyphal development and biofilm formation. Fluorescence observation and real time PCR (RT-PCR) analysis suggested both actin and the hyphal cell wall protein Hwp1 were overexpressed and mislocated in this mutant. Furthermore, cell wall integrity (CWI) was largely affected by deletion of SAC1, due to the hypersensitivity to cell wall stress, changed content and distribution of chitin in the mutant. As a result, the virulence of sac1Δ/Δ was seriously attenuated. Taken together, this study provides evidence that Sac1, as a potential PIP phosphatase, is essential for actin organization, hyphal development, CWI and pathogenicity in C. albicans.

  19. The meiosis-specific nuclear passenger protein is required for proper assembly of forespore membrane in fission yeast.

    Science.gov (United States)

    Takaine, Masak; Imada, Kazuki; Numata, Osamu; Nakamura, Taro; Nakano, Kentaro

    2014-10-15

    Sporulation, gametogenesis in yeast, consists of meiotic nuclear division and spore morphogenesis. In the fission yeast Schizosaccharomyces pombe, the four haploid nuclei produced after meiosis II are encapsulated by the forespore membrane (FSM), which is newly synthesized from spindle pole bodies (SPBs) in the cytoplasm of the mother cell as spore precursors. Although the coordination between meiosis and FSM assembly is vital for proper sporulation, the underlying mechanism remains unclear. In the present study, we identified a new meiosis-specific protein Npg1, and found that it was involved in the efficient formation of spores and spore viability. The accumulation and organization of the FSM was compromised in npg1-null cells, leading to the error-prone envelopment of nuclei. Npg1 was first seen as internuclear dots and translocated to the SPBs before the FSM assembled. Genetic analysis revealed that Npg1 worked in conjunction with the FSM proteins Spo3 and Meu14. These results suggest a possible signaling link from the nucleus to the meiotic SPBs in order to associate the onset of FSM assembly with meiosis II, which ensures the successful partitioning of gametic nuclei.

  20. Pep1, a secreted effector protein of Ustilago maydis, is required for successful invasion of plant cells.

    Directory of Open Access Journals (Sweden)

    Gunther Doehlemann

    2009-02-01

    Full Text Available The basidiomycete Ustilago maydis causes smut disease in maize. Colonization of the host plant is initiated by direct penetration of cuticle and cell wall of maize epidermis cells. The invading hyphae are surrounded by the plant plasma membrane and proliferate within the plant tissue. We identified a novel secreted protein, termed Pep1, that is essential for penetration. Disruption mutants of pep1 are not affected in saprophytic growth and develop normal infection structures. However, Deltapep1 mutants arrest during penetration of the epidermal cell and elicit a strong plant defense response. Using Affymetrix maize arrays, we identified 116 plant genes which are differentially regulated in Deltapep1 compared to wild type infections. Most of these genes are related to plant defense. By in vivo immunolocalization, live-cell imaging and plasmolysis approaches, we detected Pep1 in the apoplastic space as well as its accumulation at sites of cell-to-cell passages. Site-directed mutagenesis identified two of the four cysteine residues in Pep1 as essential for function, suggesting that the formation of disulfide bridges is crucial for proper protein folding. The barley covered smut fungus Ustilago hordei contains an ortholog of pep1 which is needed for penetration of barley and which is able to complement the U. maydis Deltapep1 mutant. Based on these results, we conclude that Pep1 has a conserved function essential for establishing compatibility that is not restricted to the U. maydis / maize interaction.

  1. Medicago truncatula natural resistance-associated macrophage Protein1 is required for iron uptake by rhizobia-infected nodule cells.

    Science.gov (United States)

    Tejada-Jiménez, Manuel; Castro-Rodríguez, Rosario; Kryvoruchko, Igor; Lucas, M Mercedes; Udvardi, Michael; Imperial, Juan; González-Guerrero, Manuel

    2015-05-01

    Iron is critical for symbiotic nitrogen fixation (SNF) as a key component of multiple ferroproteins involved in this biological process. In the model legume Medicago truncatula, iron is delivered by the vasculature to the infection/maturation zone (zone II) of the nodule, where it is released to the apoplast. From there, plasma membrane iron transporters move it into rhizobia-containing cells, where iron is used as the cofactor of multiple plant and rhizobial proteins (e.g. plant leghemoglobin and bacterial nitrogenase). MtNramp1 (Medtr3g088460) is the M. truncatula Natural Resistance-Associated Macrophage Protein family member, with the highest expression levels in roots and nodules. Immunolocalization studies indicate that MtNramp1 is mainly targeted to the plasma membrane. A loss-of-function nramp1 mutant exhibited reduced growth compared with the wild type under symbiotic conditions, but not when fertilized with mineral nitrogen. Nitrogenase activity was low in the mutant, whereas exogenous iron and expression of wild-type MtNramp1 in mutant nodules increased nitrogen fixation to normal levels. These data are consistent with a model in which MtNramp1 is the main transporter responsible for apoplastic iron uptake by rhizobia-infected cells in zone II.

  2. Protein synthesis is not required for acquisition, consolidation, and extinction of high foot-shock active avoidance training.

    Science.gov (United States)

    González-Salinas, Sofía; Medina, Andrea C; Marín-Vignando, Vera; Ruiz-López, Clyo X; Quirarte, Gina L; Prado-Alcalá, Roberto A

    2015-01-01

    Long-term memory of active avoidance in mice is not disturbed by administration of protein synthesis inhibitors (PSIs) when relatively high levels of training are used, whereas a detrimental effect is produced with lower levels of training. PSIs also disrupt extinction of avoidance behaviors in rodents, but it is not clear whether PSIs also affect this form of learning when the behavior to be extinguished was produced by a high level of training. Experiment 1 demonstrated that rats treated with the PSI cycloheximide (CXM) 30 min before training developed normal acquisition after training with either high or low foot-shock stimulation, but that memory consolidation was hindered only after low foot-shock training. Experiment 2 demonstrated that CXM disrupted extinction when administered before the first of a series of extinction sessions when low foot-shock intensity was used during training; in contrast, after training with a higher foot-shock, the PSI treatment only interfered transiently with extinction. These results indicate that acquisition, consolidation, and extinction of active avoidance learning produced by high aversive stimulation are not dependent on protein synthesis and that these processes are governed by mechanisms different from those underlying moderate forms of learning.

  3. Vesicle associated membrane protein (VAMP)-7 and VAMP-8, but not VAMP-2 or VAMP-3, are required for activation-induced degranulation of mature human mast cells.

    Science.gov (United States)

    Sander, Leif E; Frank, Simon P C; Bolat, Seza; Blank, Ulrich; Galli, Thierry; Bigalke, Hans; Bischoff, Stephan C; Lorentz, Axel

    2008-03-01

    Mediator release from mast cells (MC) is a crucial step in allergic and non-allergic inflammatory disorders. However, the final events in response to activation leading to membrane fusion and thereby facilitating degranulation have hitherto not been analyzed in human MC. Soluble N-ethyl-maleimide-sensitive factor attachment protein receptors (SNARE) represent a highly conserved family of proteins that have been shown to mediate intracellular membrane fusion events. Here, we show that mature MC isolated from human intestinal tissue express soluble N-ethylmaleide sensitive factor attachment protein (SNAP)-23, Syntaxin (STX)-1B, STX-2, STX-3, STX-4, and STX-6 but not SNAP-25. Furthermore, we found that primary human MC express substantial amounts of vesicle associated membrane protein (VAMP)-3, VAMP-7 and VAMP-8 and, in contrast to previous reports about rodent MC, only low levels of VAMP-2. Furthermore, VAMP-7 and VAMP-8 were found to translocate to the plasma membrane and interact with SNAP-23 and STX-4 upon activation. Inhibition of SNAP-23, STX-4, VAMP-7 or VAMP-8, but not VAMP-2 or VAMP-3, resulted in a markedly reduced high-affinity IgE receptor-mediated histamine release. In summary, our data show that mature human MC express a specific pattern of SNARE and that VAMP-7 and VAMP-8, but not VAMP-2, are required for rapid degranulation.

  4. IQGAP and mitotic exit network (MEN) proteins are required for cytokinesis and re-polarization of the actin cytoskeleton in the budding yeast, Saccharomyces cerevisiae.

    Science.gov (United States)

    Corbett, Mark; Xiong, Yulan; Boyne, James R; Wright, Daniel J; Munro, Ewen; Price, Clive

    2006-11-01

    In budding yeast the final stages of the cell division cycle, cytokinesis and cell separation, are distinct events that require to be coupled, both together and with mitotic exit. Here we demonstrate that mutations in genes of the mitotic exit network (MEN) prevent cell separation and are synthetically lethal in combination with both cytokinesis and septation defective mutations. Analysis of the synthetic lethal phenotypes reveals that Iqg1p functions in combination with the MEN components, Tem1p, Cdc15p Dbf20p and Dbf2p to govern the re-polarization of the actin cytoskeleton to either side of the bud neck. In addition phosphorylation of the conserved PCH protein, Hof1p, is dependent upon these activities and requires actin ring assembly. Recruitment of Dbf2p to the bud neck is dependent upon actin ring assembly and correlates with Hof1p phosphorylation. Failure to phosphorylate Hof1p results in the increased stability of the protein and its persistence at the bud neck. These data establish a mechanistic dependency of cell separation upon an intermediate step requiring actomyosin ring assembly.

  5. Protein requirements in larvae and adults of Scaptotrigona postica (Hymenoptera: Apidae [correction of Apidia], Meliponinae): midgut proteolytic activity and pollen digestion.

    Science.gov (United States)

    do Carmo Zerbo, A; Silva de Moraes, R L; Brochetto-Braga, M R

    2001-05-01

    The number and degree of digestion of pollen grains in the midgut and rectum, the midgut proteolytic activity and the time of pollen grain passage through the digestive tract in the stingless bee Scaptotrigona postica (Latreille) have been analyzed. The results show similar protein requirements among larvae, nurse bees and queens, as well as between forager bees and old males, but these requirements are higher in individuals from the former groups than in those from the latter. Although protein requirements have been demonstrated to vary according to a bee's activity in the colony, they are similar among bees from different castes or sexes. These changes in feeding behavior are related to the bee's function and to less competition for nourishment among individuals of the colony. It is also noted that pollen grains took between 6 and 28 h to pass through the digestive tract. Pollen grains are irregularly accumulated in the various regions of the midgut, which may reflect functional differentiation throughout the midgut.

  6. Energy and protein requirements of non-descript breed hair lambs of different sex classes in the semiarid region of Brazil.

    Science.gov (United States)

    Rodrigues, Rafael Torres de Souza; Chizzotti, Mario Luiz; Martins, Samara Rodrigues; da Silva, Ivonete Ferreira; Queiroz, Mário Adriano Ávila; Silva, Tiago Santos; Busato, Karina Costa; Silva, Aderbal Marcos de Azevêdo

    2016-01-01

    The aim of this study was to determine the energy and protein requirements of non-descript breed hair lambs (NDB) reared under Brazilian semiarid conditions. Sixty animals from three sex classes (20 intact males, 20 castrated males, and 20 females) with an average initial body weight of 18.1 ± 0.4 kg and an average age of 5 months were used. The nutritional requirements were estimated using the comparative slaughter. The animals in the final slaughter group were distributed in a completely randomized design with a 3 × 3 factorial scheme (three sex classes and three feeding levels: ad libitum feeding (positive energy balance), 70% feed restriction (maintenance level), and 80% feed restriction (negative energy balance)). The net energy requirement for maintenance (NEm) did not differ between sex classes (P > 0.05) and it was 68 kcal/kg of metabolic empty body weight (EBW(0.75))/day (P  0.05). The net energy requirement for weight gain (NEg) was estimated by NEg (Mcal/day) = 0.29 × EBW(0.75) × EBWG(0.86) for all sex classes (P < 0.05). The net protein requirement for weight gain (NPg) was estimated by NPg (g/day) = 224.45 × EBWG - 0.025 × RE for all sex classes (P < 0.05). The NEg increased and the NPg decreased with the increase in body weight of NDB lambs.

  7. Wdpcp, a PCP protein required for ciliogenesis, regulates directional cell migration and cell polarity by direct modulation of the actin cytoskeleton.

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    Cheng Cui

    2013-11-01

    Full Text Available Planar cell polarity (PCP regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet-Biedl/Meckel-Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin

  8. The evolutionarily conserved protein PHOTOSYNTHESIS AFFECTED MUTANT71 is required for efficient manganese uptake at the thylakoid membrane in Arabidopsis

    DEFF Research Database (Denmark)

    Schneider, Anja; Steinberger, Iris; Herdean, Andrei;

    2016-01-01

    In plants, algae, and cyanobacteria, photosystem II (PSII) catalyzes the light-driven oxidation of water. The oxygen-evolving complex of PSII is a Mn4CaO5 cluster embedded in a well-defined protein environment in the thylakoid membrane. However, transport of manganese and calcium into the thylakoid...... thylakoids relative to the wild type. The changes in Ca2+ homeostasis were accompanied by an increased contribution of the transmembrane electrical potential to the proton motive force across the thylakoid membrane. PSII activity in pam71 plants and the corresponding Chlamydomonas reinhardtii mutant cgld1...... was restored by supplementation with Mn2+, but not Ca2+. Furthermore, PAM71 suppressed the Mn2+-sensitive phenotype of the yeast mutant Δpmr1. Therefore, PAM71 presumably functions in Mn2+ uptake into thylakoids to ensure optimal PSII performance....

  9. Plasma membrane proteins Yro2 and Mrh1 are required for acetic acid tolerance in Saccharomyces cerevisiae.

    Science.gov (United States)

    Takabatake, Akiko; Kawazoe, Nozomi; Izawa, Shingo

    2015-03-01

    Yro2 and its paralogous protein Mrh1 of Saccharomyces cerevisiae have seven predicted transmembrane domains and predominantly localize to the plasma membrane. Their physiological functions and regulation of gene expression have not yet been elucidated in detail. We herein demonstrated that MRH1 was constitutively expressed, whereas the expression of YRO2 was induced by acetic acid stress and entering the stationary phase. Fluorescence microscopic analysis revealed that Mrh1 and Yro2 were distributed as small foci in the plasma membrane under acetic acid stress conditions. The null mutants of these genes (mrh1∆, yro2∆, and mrh1∆yro2∆) showed delayed growth and a decrease in the productivity of ethanol in the presence of acetic acid, indicating that Yro2 and Mrh1 are involved in tolerance to acetic acid stress.

  10. Requirement of Fra proteins for communication channels between cells in the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120.

    Science.gov (United States)

    Omairi-Nasser, Amin; Mariscal, Vicente; Austin, Jotham R; Haselkorn, Robert

    2015-08-11

    The filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 differentiates specialized cells, heterocysts, that fix atmospheric nitrogen and transfer the fixed nitrogen to adjacent vegetative cells. Reciprocally, vegetative cells transfer fixed carbon to heterocysts. Several routes have been described for metabolite exchange within the filament, one of which involves communicating channels that penetrate the septum between adjacent cells. Several fra gene mutants were isolated 25 y ago on the basis of their phenotypes: inability to fix nitrogen and fragmentation of filaments upon transfer from N+ to N- media. Cryopreservation combined with electron tomography were used to investigate the role of three fra gene products in channel formation. FraC and FraG are clearly involved in channel formation, whereas FraD has a minor part. Additionally, FraG was located close to the cytoplasmic membrane and in the heterocyst neck, using immunogold labeling with antibody raised to the N-terminal domain of the FraG protein.

  11. Environmental pH and the requirement for the extrinsic proteins of Photosystem II in the function of cyanobacterial photosynthesis

    Directory of Open Access Journals (Sweden)

    Jaz N Morris

    2016-08-01

    Full Text Available In one of the final stages of cyanobacterial Photosystem II (PS II assembly, binding of up to four extrinsic proteins to PS II stabilizes the oxygen-evolving complex (OEC. Growth of cyanobacterial mutants deficient in certain combinations of these thylakoid-lumen-associated polypeptides is sensitive to changes in environmental pH, despite the physical separation of the membrane-embedded PS II complex from the external environment. In this perspective, we discuss the effect of environmental pH on OEC function and photoautotrophic growth in cyanobacteria, with reference to pH-sensitive PS II mutants lacking extrinsic proteins. We consider the possibilities that, compared to pH 10.0, pH 7.5 increases susceptibility to PS II-generated reactive oxygen species (ROS, causing photoinhibition and reducing PS II assembly in some mutants and that perturbations to channels in the lumenal regions of PS II might alter the accessibility of water to the active site, and egress of oxygen and protons to the thylakoid lumen. Reduced levels of PS II in these mutants and reduced OEC activity arising from the disruption of substrate/product channels could reduce the trans-thylakoid pH gradient (ΔpH, leading to the impairement of photosynthesis. Growth of some PS II mutants at pH 7.5 can be rescued by elevating CO2 levels, suggesting that the pH-sensitive phenotype might primarily be an indirect result of back-pressure in the electron transport chain that results in heightened production of ROS by the impaired photosystem.

  12. CD1d-mediated Presentation of Endogenous Lipid Antigens by Adipocytes Requires Microsomal Triglyceride Transfer Protein*

    Science.gov (United States)

    Rakhshandehroo, Maryam; Gijzel, Sanne M. W.; Siersbæk, Rasmus; Broekema, Marjoleine F.; de Haar, Colin; Schipper, Henk S.; Boes, Marianne; Mandrup, Susanne; Kalkhoven, Eric

    2014-01-01

    Obesity-induced adipose tissue (AT) dysfunction results in a chronic low-grade inflammation that predisposes to the development of insulin resistance and type 2 diabetes. During the development of obesity, the AT-resident immune cell profile alters to create a pro-inflammatory state. Very recently, CD1d-restricted invariant (i) natural killer T (NKT) cells, a unique subset of lymphocytes that are reactive to so called lipid antigens, were implicated in AT homeostasis. Interestingly, recent data also suggest that human and mouse adipocytes can present such lipid antigens to iNKT cells in a CD1d-dependent fashion, but little is known about the lipid antigen presentation machinery in adipocytes. Here we show that CD1d, as well as the lipid antigen loading machinery genes pro-saposin (Psap), Niemann Pick type C2 (Npc2), α-galactosidase (Gla), are up-regulated in early adipogenesis, and are transcriptionally controlled by CCAAT/enhancer-binding protein (C/EBP)-β and -δ. Moreover, adipocyte-induced Th1 and Th2 cytokine release by iNKT cells also occurred in the absence of exogenous ligands, suggesting the display of endogenous lipid antigen-D1d complexes by 3T3-L1 adipocytes. Furthermore, we identified microsomal triglyceride transfer protein, which we show is also under the transcriptional regulation of C/EBPβ and –δ, as a novel player in the presentation of endogenous lipid antigens by adipocytes. Overall, our findings indicate that adipocytes can function as non-professional lipid antigen presenting cells, which may present an important aspect of adipocyte-immune cell communication in the regulation of whole body energy metabolism and immune homeostasis. PMID:24966328

  13. CD1d-mediated presentation of endogenous lipid antigens by adipocytes requires microsomal triglyceride transfer protein.

    Science.gov (United States)

    Rakhshandehroo, Maryam; Gijzel, Sanne M W; Siersbæk, Rasmus; Broekema, Marjoleine F; de Haar, Colin; Schipper, Henk S; Boes, Marianne; Mandrup, Susanne; Kalkhoven, Eric

    2014-08-08

    Obesity-induced adipose tissue (AT) dysfunction results in a chronic low-grade inflammation that predisposes to the development of insulin resistance and type 2 diabetes. During the development of obesity, the AT-resident immune cell profile alters to create a pro-inflammatory state. Very recently, CD1d-restricted invariant (i) natural killer T (NKT) cells, a unique subset of lymphocytes that are reactive to so called lipid antigens, were implicated in AT homeostasis. Interestingly, recent data also suggest that human and mouse adipocytes can present such lipid antigens to iNKT cells in a CD1d-dependent fashion, but little is known about the lipid antigen presentation machinery in adipocytes. Here we show that CD1d, as well as the lipid antigen loading machinery genes pro-saposin (Psap), Niemann Pick type C2 (Npc2), α-galactosidase (Gla), are up-regulated in early adipogenesis, and are transcriptionally controlled by CCAAT/enhancer-binding protein (C/EBP)-β and -δ. Moreover, adipocyte-induced Th1 and Th2 cytokine release by iNKT cells also occurred in the absence of exogenous ligands, suggesting the display of endogenous lipid antigen-D1d complexes by 3T3-L1 adipocytes. Furthermore, we identified microsomal triglyceride transfer protein, which we show is also under the transcriptional regulation of C/EBPβ and -δ, as a novel player in the presentation of endogenous lipid antigens by adipocytes. Overall, our findings indicate that adipocytes can function as non-professional lipid antigen presenting cells, which may present an important aspect of adipocyte-immune cell communication in the regulation of whole body energy metabolism and immune homeostasis.

  14. Inhibition of phagocytosis by Haemophilus ducreyi requires expression of the LspA1 and LspA2 proteins.

    Science.gov (United States)

    Vakevainen, Merja; Greenberg, Steven; Hansen, Eric J

    2003-10-01

    Haemophilus ducreyi previously has been shown to inhibit the phagocytosis of both secondary targets and itself by certain cells in vitro. Wild-type H. ducreyi strain 35000HP contains two genes, lspA1 and lspA2, whose encoded protein products are predicted to be 456 and 543 kDa, respectively. An isogenic mutant of H. ducreyi 35000HP with inactivated lspA1 and lspA2 genes has been shown to exhibit substantially decreased virulence in the temperature-dependent rabbit model for chancroid. This lspA1 lspA2 mutant was tested for its ability to inhibit phagocytosis of immunoglobulin G-opsonized particles by differentiated HL-60 and U-937 cells and by J774A.1 cells. The wild-type strain H. ducreyi 35000HP readily inhibited phagocytosis, whereas the lspA1 lspA2 mutant was unable to inhibit phagocytosis. Similarly, the wild-type strain was resistant to phagocytosis, whereas the lspA1 lspA2 mutant was readily engulfed by phagocytes. This inhibitory effect of wild-type H. ducreyi on phagocytic activity was primarily associated with live bacterial cells but could also be found, under certain conditions, in concentrated H. ducreyi culture supernatant fluids that lacked detectable outer membrane fragments. Both the wild-type strain and the lspA1 lspA2 mutant attached to phagocytes at similar levels. These results indicate that the LspA1 and LspA2 proteins of H. ducreyi are involved, directly or indirectly, in the antiphagocytic activity of this pathogen, and they provide a possible explanation for the greatly reduced virulence of the lspA1 lspA2 mutant.

  15. A low-affinity penicillin-binding protein 2x variant is required for heteroresistance in Streptococcus pneumoniae.

    Science.gov (United States)

    Engel, Hansjürg; Mika, Moana; Denapaite, Dalia; Hakenbeck, Regine; Mühlemann, Kathrin; Heller, Manfred; Hathaway, Lucy J; Hilty, Markus

    2014-07-01

    Heteroresistance to penicillin in Streptococcus pneumoniae is the ability of subpopulations to grow at a higher antibiotic concentration than expected from the MIC. This may render conventional resistance testing unreliable and lead to therapeutic failure. We investigated the role of the primary β-lactam resistance determinants, penicillin-binding protein 2b (PBP2b) and PBP2x, and the secondary resistance determinant PBP1a in heteroresistance to penicillin. Transformants containing PBP genes from the heteroresistant strain Spain(23F) 2349 in the nonheteroresistant strain R6 background were tested for heteroresistance by population analysis profiling (PAP). We found that pbp2x, but not pbp2b or pbp1a alone, conferred heteroresistance to R6. However, a change of pbp2x expression was not observed, and therefore, expression does not correlate with an increased proportion of resistant subpopulations. In addition, the influence of the CiaRH system, mediating PBP-independent β-lactam resistance, was assessed by PAP on ciaR disruption mutants but revealed no heteroresistant phenotype. We also showed that the highly resistant subpopulations (HOM*) of transformants containing low-affinity pbp2x undergo an increase in resistance upon selection on penicillin plates that partially reverts after passaging on selection-free medium. Shotgun proteomic analysis showed an upregulation of phosphate ABC transporter subunit proteins encoded by pstS, phoU, pstB, and pstC in these highly resistant subpopulations. In conclusion, the presence of low-affinity pbp2x enables certain pneumococcal colonies to survive in the presence of β-lactams. Upregulation of phosphate ABC transporter genes may represent a reversible adaptation to antibiotic stress.

  16. Mitogen- and stress-activated protein kinases 1 and 2 are required for maximal trefoil factor 1 induction.

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    Protiti Khan

    Full Text Available Mitogen- and stress-activated protein kinases 1 and 2 (MSK1 and MSK2, activated downstream of the ERK- and p38-mitogen-activated protein kinase pathways are involved in cell survival, proliferation and differentiation. Following mitogenic or stress stimuli, they mediate the nucleosomal response, which includes phosphorylation of histone H3 at serine 10 (H3S10ph coupled with transcriptional activation of immediate-early genes. While MSK1 and MSK2 are closely related, their relative roles may vary with cellular context and/or stimuli. However, our knowledge of MSK2 recruitment to immediate-early genes is limited, as research has primarily focused on MSK1. Here, we demonstrate that both MSK1 and MSK2, regulate the phorbol ester 12-O-tetradecanoylphorbol-13-acetate induced expression of the breast cancer marker gene, trefoil factor 1 (TFF1, by phosphorylating H3S10 at its 5' regulatory regions. The MSK-mediated phosphorylation of H3S10 promotes the recruitment of 14-3-3 isoforms and BRG1, the ATPase subunit of the BAF/PBAF remodeling complex, to the enhancer and upstream promoter elements of TFF1. The recruited chromatin remodeling activity leads to the RNA polymerase II carboxy-terminal domain phosphorylation at the TFF1 promoter, initiating TFF1 expression in MCF-7 breast cancer cells. Moreover, we show that MSK1 or MSK2 is recruited to TFF1 regulatory regions, but as components of different multiprotein complexes.

  17. Human RECQ1 is a DNA damage responsive protein required for genotoxic stress resistance and suppression of sister chromatid exchanges.

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    Sudha Sharma

    Full Text Available BACKGROUND: DNA helicases are ubiquitous enzymes that unwind DNA in an ATP-dependent and directionally specific manner. Unwinding of double-stranded DNA is essential for the processes of DNA repair, recombination, transcription, and DNA replication. Five human DNA helicases sharing sequence similarity with the E. coli RecQ helicase have been identified. Three of the human RecQ helicases are implicated in hereditary diseases (Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome which display clinical symptoms of premature aging and cancer. RECQ1 helicase is the most highly expressed of the human RecQ helicases; however, a genetic disease has yet not been linked to mutations in the RECQ1 gene, and the biological functions of human RECQ1 in cellular DNA metabolism are not known. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we report that RECQ1 becomes phosphorylated upon DNA damage and forms irradiation-induced nuclear foci that associate with chromatin in human cells. Depletion of RECQ1 renders human cells sensitive to DNA damage induced by ionizing radiation or the topoisomerase inhibitor camptothecin, and results in spontaneous gamma-H2AX foci and elevated sister chromatid exchanges, indicating aberrant repair of DNA breaks. Consistent with a role in homologous recombinational repair, endogenous RECQ1 is associated with the strand exchange protein Rad51 and the two proteins directly interact with high affinity. CONCLUSION/SIGNIFICANCE: Collectively, these results provide the first evidence for a role of human RECQ1 in the response to DNA damage and chromosomal stability maintenance and point to the vital importance of RECQ1 in genome homeostasis.

  18. Drosophila Zpr1 (Zinc finger protein 1 is required downstream of both EGFR and FGFR signaling in tracheal subcellular lumen formation.

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    Oscar E Ruiz

    Full Text Available The cellular and molecular cues involved in creating branched tubular networks that transport liquids or gases throughout an organism are not well understood. To identify factors required in branching and lumen formation of Drosophila tracheal terminal cells, a model for branched tubular networks, we performed a forward genetic-mosaic screen to isolate mutations affecting these processes. From this screen, we have identified the first Drosophila mutation in the gene Zpr1 (Zinc finger protein 1 by the inability of Zpr1-mutant terminal cells to form functional, gas-filled lumens. We show that Zpr1 defective cells initiate lumen formation, but are blocked from completing the maturation required for gas filling. Zpr1 is an evolutionarily conserved protein first identified in mammalian cells as a factor that binds the intracellular domain of the unactivated epidermal growth factor receptor (EGFR. We show that down-regulation of EGFR in terminal cells phenocopies Zpr1 mutations and that Zpr1 is epistatic to ectopic lumen formation driven by EGFR overexpression. However, while Zpr1 mutants are fully penetrant, defects observed when reducing EGFR activity are only partially penetrant. These results suggest that a distinct pathway operating in parallel to the EGFR pathway contributes to lumen formation, and this pathway is also dependent on Zpr1. We provide evidence that this alternative pathway may involve fibroblast growth factor receptor (FGFR signaling. We suggest a model in which Zpr1 mediates both EGFR and FGFR signal transduction cascades required for lumen formation in terminal cells. To our knowledge, this is the first genetic evidence placing Zpr1 downstream of EGFR signaling, and the first time Zpr1 has been implicated in FGFR signaling. Finally, we show that down-regulation of Smn, a protein known to interact with Zpr1 in mammalian cells, shows defects similar to Zpr1 mutants.

  19. Residues of the UL25 Protein of Herpes Simplex Virus That Are Required for Its Stable Interaction with Capsids ▿

    OpenAIRE

    Cockrell, Shelley K.; Huffman, Jamie B.; Toropova, Katerina; James F Conway; Homa, Fred L.

    2011-01-01

    The herpes simplex virus 1 (HSV-1) UL25 gene product is a minor capsid component that is required for encapsidation, but not cleavage, of replicated viral DNA. UL25 is located on the capsid surface in a proposed heterodimer with UL17, where five copies of the heterodimer are found at each of the capsid vertices. Previously, we demonstrated that amino acids 1 to 50 of UL25 are essential for its stable interaction with capsids. To further define the UL25 capsid binding domain, we generated reco...

  20. G Protein-Dependent CCR5 Signaling Is Not Required for Efficient Infection of Primary T Lymphocytes and Macrophages by R5 Human Immunodeficiency Virus Type 1 Isolates

    OpenAIRE

    2003-01-01

    The requirement of human immunodeficiency virus (HIV)-induced CCR5 activation for infection by R5 HIV type 1 (HIV-1) strains remains controversial. Ectopic CCR5 expression in CD4+-transformed cells or pharmacological inhibition of Gαi proteins coupled to CCR5 left unsolved whether CCR5-dependent cell activation is necessary for the HIV life cycle. In this study, we investigated the role played by HIV-induced CCR5-dependent cell signaling during infection of primary CD4-expressing leukocytes. ...

  1. Mitogen-activated protein kinase kinase activity is required for the G2/M transition of the cell cycle in mammalian fibroblasts

    OpenAIRE

    Wright, Jocelyn H.; Munar, Erlynda; Jameson, Damon R; Andreassen, Paul R.; Margolis, Robert L.; Seger, Rony; Krebs, Edwin G.

    1999-01-01

    The mitogen-activated protein kinase (MAPK) cascade is required for mitogenesis in somatic mammalian cells and is activated by a wide variety of oncogenic stimuli. Specific roles for this signaling module in growth were dissected by inhibiting MAPK kinase 1 (MAPKK1) activity in highly synchronized NIH 3T3 cells. In addition to the known role of this kinase in cell-cycle entry from G0, the level of MAPKK activity was observed to affect the kinetics of progression through both the G1 and G2 pha...

  2. Activation of p38 and JNK MAPK pathways abrogates requirement for new protein synthesis for phorbol ester mediated induction of select MMP and TIMP genes.

    Science.gov (United States)

    Sampieri, Clara L; Nuttall, Robert K; Young, David A; Goldspink, Deborah; Clark, Ian M; Edwards, Dylan R

    2008-03-01

    The human matrix metalloproteinase (MMP) gene family includes 24 genes whose regulated expression, together with that of four tissue inhibitors of metalloproteinases (TIMPs), is essential in tissue remodelling and cell signalling. Quantitative real-time-PCR (qPCR) analysis was used to evaluate the shared and unique patterns of control of these two gene families in human MRC-5 and WI-38 fibroblasts in response to the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA). The requirement for ongoing translation was analysed using three protein synthesis inhibitors, anisomycin, cycloheximide and emetine. PMA induced MMP1, 3, 8, 9, 10, 12, 13, 14 and TIMP1 and TIMP3 RNAs after 4-8 h, and induction of all except MMP9 and TIMP3 was blocked by all protein synthesis inhibitors. However, even though all inhibitors effectively blocked translation, PMA-induction of MMP9 and TIMP3 was blocked by emetine but was insensitive to cycloheximide and anisomycin. Anisomycin alone induced MMP9 and TIMP3, along with MMP25 and MMP19. The extracellular signal-regulated kinases (ERKs)-1/2 were strongly activated by PMA, while anisomycin activated the c-Jun N-terminal kinase (JNK) and p38 pathways, and cycloheximide activated p38, but emetine had no effect on the stress-activated mitogen-activated protein kinase (MAPK) pathways. The involvement of the p38 and JNK pathways in the selective effects of anisomycin and cycloheximide on MMP/TIMP expression was supported by use of pharmacological inhibitors. These data confirm that most inducible MMPs and TIMP1 behave as "late" activated, protein synthesis-dependent genes in fibroblasts. However, the requirement of protein synthesis for PMA-induction of MMPs and TIMPs is not universal, since it is abrogated for MMP9 and TIMP3 by stimulation of the stress-activated MAPK pathways. The definition of clusters of co-regulated genes among the two gene families will aid in bioinformatic dissection of control mechanisms.

  3. The HhH2/NDD domain of the Drosophila Nod chromokinesin-like protein is required for binding to chromosomes in the oocyte nucleus.

    Science.gov (United States)

    Cui, Wei; Hawley, R Scott

    2005-12-01

    Nod is a chromokinesin-like protein that plays a critical role in segregating achiasmate chromosomes during female meiosis. The C-terminal half of the Nod protein contains two putative DNA-binding domains. The first of these domains, known as the HMGN domain, consists of three tandemly repeated high-mobility group N motifs. This domain was previously shown to be both necessary and sufficient for binding of the C-terminal half of Nod to mitotic chromosomes in embryos. The second putative DNA-binding domain, denoted HhH(2)/NDD, is a helix-hairpin-helix(2)/Nod-like DNA-binding domain. Although the HhH(2)/NDD domain is not required or sufficient for chromosome binding in embryos, several well-characterized nod mutations have been mapped in this domain. To characterize the role of the HhH(2)/NDD domain in mediating Nod function, we created a series of UAS-driven transgene constructs capable of expressing either a wild-type Nod-GFP fusion protein or proteins in which the HhH(2)/NDD domain had been altered by site-directed mutagenesis. Although wild-type Nod-GFP localizes to the oocyte chromosomes and rescues the segregation defect in nod mutant oocytes, two of three proteins carrying mutants in the HhH(2)/NDD domain fail to either rescue the nod mutant phenotype or bind to oocyte chromosomes. However, these mutant proteins do bind to the polytene chromosomes in nurse-cell nuclei and enter the oocyte nucleus. Thus, even though the HhH(2)/NDD domain is not essential for chromosome binding in other cell types, it is required for chromosome binding in the oocyte. These HhH(2)/NDD mutants also block the localization of Nod to the posterior pole of stage 9-10A oocytes, a process that is thought to facilitate the interaction of Nod with the plus ends of microtubules (Cui et al. 2005). This observation suggests that the Nod HhH2/NDD domain may play other roles in addition to binding Nod to meiotic chromosomes.

  4. The milkweed pod1 gene encodes a KANADI protein that is required for abaxial/adaxial patterning in maize leaves.

    Science.gov (United States)

    Candela, Héctor; Johnston, Robyn; Gerhold, Abigail; Foster, Toshi; Hake, Sarah

    2008-08-01

    Leaf primordia initiate from the shoot apical meristem with inherent polarity; the adaxial side faces the meristem, while the abaxial side faces away from the meristem. Adaxial/abaxial polarity is thought to be necessary for laminar growth of leaves, as mutants lacking either adaxial or abaxial cell types often develop radially symmetric lateral organs. The milkweed pod1 (mwp1) mutant of maize (Zea mays) has adaxialized sectors in the sheath, the proximal part of the leaf. Ectopic leaf flaps develop where adaxial and abaxial cell types juxtapose. Ectopic expression of the HD-ZIPIII gene rolled leaf1 (rld1) correlates with the adaxialized regions. Cloning of mwp1 showed that it encodes a KANADI transcription factor. Double mutants of mwp1-R with a microRNA-resistant allele of rld1, Rld1-N1990, show a synergistic phenotype with polarity defects in sheath and blade and a failure to differentiate vascular and photosynthetic cell types in the adaxialized sectors. The sectored phenotype and timing of the defect suggest that mwp1 is required late in leaf development to maintain abaxial cell fate. The phenotype of mwp1; Rld1 double mutants shows that both genes are also required early in leaf development to delineate leaf margins as well as to initiate vascular and photosynthetic tissues.

  5. Functional characterization of ExFadLO, an outer membrane protein required for exporting oxygenated long-chain fatty acids in Pseudomonas aeruginosa.

    Science.gov (United States)

    Martínez, Eriel; Estupiñán, Mónica; Pastor, F I Javier; Busquets, Montserrat; Díaz, Pilar; Manresa, Angeles

    2013-02-01

    Bacterial proteins of the FadL family have frequently been associated to the uptake of exogenous hydrophobic substrates. However, their outer membrane location and involvement in substrate uptake have been inferred mainly from sequence similarity to Escherichia coli FadL, the first well-characterized outer membrane transporters of Long-Chain Fatty Acids (LCFAs) in bacteria. Here we report the functional characterization of a Pseudomonas aeruginosa outer membrane protein (ORF PA1288) showing similarities to the members of the FadL family, for which we propose the name ExFadLO. We demonstrate herein that this protein is required to export LCFAs 10-HOME and 7,10-DiHOME, derived from a diol synthase oxygenation activity on oleic acid, from the periplasm to the extracellular medium. Accumulation of 10-HOME and 7,10-DiHOME in the extracellular medium of P. aeruginosa was abolished by a transposon insertion mutation in exFadLO (ExFadLO¯ mutant). However, intact periplasm diol synthase activity was found in this mutant, indicating that ExFadLO participates in the export of these oxygenated LCFAs across the outer membrane. The capacity of ExFadLO¯ mutant to export 10-HOME and 7,10-DiHOME was recovered after complementation with a wild-type, plasmid-expressed ExFadLO protein. A western blot assay with a variant of ExFadLO tagged with a V5 epitope confirmed the location of ExFadLO in the bacterial outer membrane under the experimental conditions tested. Our results provide the first evidence that FadL family proteins, known to be involved in the uptake of hydrophobic substrates from the extracellular environment, also function as secretion elements for metabolites of biological relevance.

  6. Structure of the cytoplasmic domain of TcpE, the inner membrane core protein required for assembly of the Vibrio cholerae toxin-coregulated pilus.

    Science.gov (United States)

    Kolappan, Subramaniapillai; Craig, Lisa

    2013-04-01

    Type IV pili are long thin surface-displayed polymers of the pilin subunit that are present in a diverse group of bacteria. These multifunctional filaments are critical to virulence for pathogens such as Vibrio cholerae, which use them to form microcolonies and to secrete the colonization factor TcpF. The type IV pili are assembled from pilin subunits by a complex inner membrane machinery. The core component of the type IV pilus-assembly platform is an integral inner membrane protein belonging to the GspF superfamily of secretion proteins. These proteins somehow convert chemical energy from ATP hydrolysis by an assembly ATPase on the cytoplasmic side of the inner membrane to mechanical energy for extrusion of the growing pilus filament out of the inner membrane. Most GspF-family inner membrane core proteins are predicted to have N-terminal and central cytoplasmic domains, cyto1 and cyto2, and three transmembrane segments, TM1, TM2 and TM3. Cyto2 and TM3 represent an internal repeat of cyto1 and TM1. Here, the 1.88 Å resolution crystal structure of the cyto1 domain of V. cholerae TcpE, which is required for assembly of the toxin-coregulated pilus, is reported. This domain folds as a monomeric six-helix bundle with a positively charged membrane-interaction face at one end and a hydrophobic groove at the other end that may serve as a binding site for partner proteins in the pilus-assembly complex.

  7. Fulvestrant-induced cell death and proteasomal degradation of estrogen receptor α protein in MCF-7 cells require the CSK c-Src tyrosine kinase.

    Directory of Open Access Journals (Sweden)

    Wei-Lan Yeh

    Full Text Available Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD. In contrast to the Selective Estrogen Receptor Modulators (SERMs such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK, a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant.

  8. NfuA, a new factor required for maturing Fe/S proteins in Escherichia coli under oxidative stress and iron starvation conditions.

    Science.gov (United States)

    Angelini, Sandra; Gerez, Catherine; Ollagnier-de Choudens, Sandrine; Sanakis, Yiannis; Fontecave, Marc; Barras, Frédéric; Py, Béatrice

    2008-05-16

    Iron/sulfur (Fe/S) proteins are central to the functioning of cells in both prokaryotes and eukaryotes. Here, we show that the yhgI gene, which we renamed nfuA, encodes a two-domain protein that is required for Fe/S biogenesis in Escherichia coli. The N-terminal domain resembles the so-called Fe/S A-type scaffold but, curiously, has lost the functionally important Cys residues. The C-terminal domain shares sequence identity with Nfu proteins. Mössbauer and UV-visible spectroscopic analyses revealed that, upon reconstitution, NfuA binds a [4Fe-4S] cluster. Moreover, NfuA can transfer this cluster to apo-aconitase. Mutagenesis studies indicated that the N- and C-terminal domains are important for NfuA function in vivo. Similarly, the functional importance of Cys residues present in the Nfu-like domain was demonstrated in vivo by introducing Cys-->Ser mutations. In vivo investigations revealed that the nfuA gene is important for E. coli to sustain oxidative stress and iron starvation. Also, combining nfuA with either isc or suf mutations led to additive phenotypic deficiencies, indicating that NfuA is a bona fide new player in Isc- and Suf-dependent Fe/S biogenesis pathways. Taken together, these data demonstrate that NfuA intervenes in the maturation of apoproteins in E. coli, allowing them to acquire Fe/S clusters. By taking into account results from numerous previous transcriptomic studies that had suggested a link between NfuA and protein misfolding, we discuss the possibility that NfuA could act as a scaffold/chaperone for damaged Fe/S proteins.

  9. Protein tyrosine phosphatase PTP1 negatively regulates Dictyostelium STATa and is required for proper cell-type proportioning.

    Science.gov (United States)

    Early, A; Gamper, M; Moniakis, J; Kim, E; Hunter, T; Williams, J G; Firtel, R A

    2001-04-01

    The protein tyrosine phosphatase PTP1, which mediates reversible phosphorylation on tyrosine, has been shown to play an important regulatory role during Dictyostelium development. Mutants lacking PTP1 develop more rapidly than normal, while strains that overexpress PTP1 display aberrant morphology. However, the signalling pathways involved have not been characterised. In reexamining these strains, we have found that there is an inverse correlation between levels of PTP1 activity, the extent of tyrosine phosphorylation on Dictyostelium STATa after treatment with cAMP, and the proportion of the slug population exhibiting STATa nuclear enrichment in vivo. This suggests that PTP1 acts to attenuate the tyrosine phosphorylation of STATa and downstream STATa-mediated pathways. Consistent with this, we show that when PTP1 is overexpressed, there is increased expression of a prestalk cell marker at the slug posterior, a phenocopy of STATa null slugs. In ptp1 null strains, STATa tyrosine phosphorylation and nuclear enrichment in the slug anterior is increased. There is also a change in the prestalk to prespore cell ratio. Synergy experiments suggest that this is due to a cell-autonomous defect in forming the subset of prespore cells that are located in the anterior prespore region.

  10. High Mobility Group Box-1 Protein and Outcomes in Critically Ill Surgical Patients Requiring Open Abdominal Management

    Directory of Open Access Journals (Sweden)

    Michelle S. Malig

    2017-01-01

    Full Text Available Background. Previous studies assessing various cytokines in the critically ill/injured have been uninformative in terms of translating to clinical care management. Animal abdominal sepsis work suggests that enhanced intraperitoneal (IP clearance of Damage-Associated Molecular Patterns (DAMPs improves outcome. Thus measuring the responses of DAMPs offers alternate potential insights and a representative DAMP, High Mobility Group Box-1 protein (HMGB-1, was considered. While IP biomediators are being recognized in critical illness/trauma, HMGB-1 behaviour has not been examined in open abdomen (OA management. Methods. A modified protocol for HMGB-1 detection was used to examine plasma/IP fluid samples from 44 critically ill/injured OA patients enrolled in a randomized controlled trial comparing two negative pressure peritoneal therapies (NPPT: Active NPPT (ANPPT and Barker’s Vacuum Pack NPPT (BVP. Samples were collected and analyzed at the time of laparotomy and at 24 and 48 hours after. Results. There were no statistically significant differences in survivor versus nonsurvivor HMGB-1 plasma or IP concentrations at baseline, 24 hours, or 48 hours. However, plasma HMGB-1 levels tended to increase continuously in the BVP cohort. Conclusions. HMGB-1 appeared to behave differently between NPPT cohorts. Further studies are needed to elucidate the relationship of HMGB-1 and outcomes in septic/injured patients.

  11. Requirement for the POZ/BTB protein NAC1 in acute but not chronic psychomotor stimulant response.

    Science.gov (United States)

    Mackler, Scott; Pacchioni, Alejandra; Degnan, Ryan; Homan, Ying; Conti, Alana C; Kalivas, Peter; Blendy, Julie A

    2008-02-11

    NAC1 is a novel member of the POZ/BTB (Pox virus and Zinc finger/Bric-a-bracTramtrack Broad complex) but varies from other proteins of this class in that it lacks the characteristic DNA-binding motif, suggesting a novel role. We have employed constitutive gene deletion to elucidate the role of NAC1 in vivo. Nac1 mutant mice are viable with no obvious developmental or physiological impairments. Previous studies suggest a role for NAC1 in cocaine-mediated behaviors. Therefore, we evaluated a variety of behaviors associated with psychomotor stimulant effects in Nac1 mutant mice. Acute locomotor activating effects of cocaine or amphetamine are absent in Nac1 mutant mice, however longer exposure to these psychomotor stimulants result in the development of behavioral sensitization. Acute rewarding properties of cocaine and amphetamine are also blunted in mutant mice, yet repeated exposure resulted in conditioned place preference similar to that observed in wild-type mice. Lastly, increases in extracellular dopamine in the nucleus accumbens, which accompany acute cocaine administration, are blunted in mutant mice, but following chronic cocaine extracellular dopamine levels are increased to the same extent as in wild-type mice. Together these data indicate involvement of NAC1 in the acute behavioral and neurochemical responses to psychomotor stimulants.

  12. MAPK-activated protein kinase 2 is required for mouse meiotic spindle assembly and kinetochore-microtubule attachment.

    Directory of Open Access Journals (Sweden)

    Ju Yuan

    Full Text Available MAPK-activated protein kinase 2 (MK2, a direct substrate of p38 MAPK, plays key roles in multiple physiological functions in mitosis. Here, we show for the first time the unique distribution pattern of MK2 in meiosis. Phospho-MK2 was localized on bipolar spindle minus ends and along the interstitial axes of homologous chromosomes extending over centromere regions and arm regions at metaphase of first meiosis (MI stage in mouse oocytes. At metaphase of second meiosis (MII stage, p-MK2 was localized on the bipolar spindle minus ends and at the inner centromere region of sister chromatids as dots. Knockdown or inhibition of MK2 resulted in spindle defects. Spindles were surrounded by irregular nondisjunction chromosomes, which were arranged in an amphitelic or syntelic/monotelic manner, or chromosomes detached from the spindles. Kinetochore-microtubule attachments were impaired in MK2-deficient oocytes because spindle microtubules became unstable in response to cold treatment. In addition, homologous chromosome segregation and meiosis progression were inhibited in these oocytes. Our data suggest that MK2 may be essential for functional meiotic bipolar spindle formation, chromosome segregation and proper kinetochore-microtubule attachments.

  13. Carboxyl terminus of Hsp70-interacting protein (CHIP) is required to modulate cardiac hypertrophy and attenuate autophagy during exercise.

    Science.gov (United States)

    Willis, Monte S; Min, Jin-Na; Wang, Shaobin; McDonough, Holly; Lockyer, Pamela; Wadosky, Kristine M; Patterson, Cam

    2013-12-01

    The carboxyl terminus of Hsp70-interacting protein (CHIP) is a ubiquitin ligase/cochaperone critical for the maintenance of cardiac function. Mice lacking CHIP (CHIP-/-) suffer decreased survival, enhanced myocardial injury and increased arrhythmias compared with wild-type controls following challenge with cardiac ischaemia reperfusion injury. Recent evidence implicates a role for CHIP in chaperone-assisted selective autophagy, a process that is associated with exercise-induced cardioprotection. To determine whether CHIP is involved in cardiac autophagy, we challenged CHIP-/- mice with voluntary exercise. CHIP-/- mice respond to exercise with an enhanced autophagic response that is associated with an exaggerated cardiac hypertrophy phenotype. No impairment of function was identified in the CHIP-/- mice by serial echocardiography over the 5 weeks of running, indicating that the cardiac hypertrophy was physiologic not pathologic in nature. It was further determined that CHIP plays a role in inhibiting Akt signalling and autophagy determined by autophagic flux in cardiomyocytes and in the intact heart. Taken together, cardiac CHIP appears to play a role in regulating autophagy during the development of cardiac hypertrophy, possibly by its role in supporting Akt signalling, induced by voluntary running in vivo.

  14. Bst1 is required for Candida albicans infecting host via facilitating cell wall anchorage of Glycosylphosphatidyl inositol anchored proteins

    Science.gov (United States)

    Liu, Wei; Zou, Zui; Huang, Xin; Shen, Hui; He, Li Juan; Chen, Si Min; Li, Li Ping; Yan, Lan; Zhang, Shi Qun; Zhang, Jun Dong; Xu, Zheng; Xu, Guo Tong; An, Mao Mao; Jiang, Yuan Ying

    2016-01-01

    Glycosylphosphatidyl inositol anchored proteins (GPI-APs) on fungal cell wall are essential for invasive infections. While the function of inositol deacylation of GPI-APs in mammalian cells has been previously characterized the impact of inositol deacylation in fungi and implications to host infection remains largely unexplored. Herein we describe our identification of BST1, an inositol deacylase of GPI-Aps in Candida albicans, was critical for GPI-APs cell wall attachment and host infection. BST1-deficient C. albicans (bst1Δ/Δ) was associated with severely impaired cell wall anchorage of GPI-APs and subsequen unmasked β-(1,3)-glucan. Consistent with the aberrant cell wall structures, bst1Δ/Δ strain did not display an invasive ability and could be recognized more efficiently by host immune systems. Moreover, BST1 null mutants or those expressing Bst1 variants did not display inositol deacylation activity and exhibited severely attenuated virulence and reduced organic colonization in a murine systemic candidiasis model. Thus, Bst1 can facilitate cell wall anchorage of GPI-APs in C. albicans by inositol deacylation, and is critical for host invasion and immune escape. PMID:27708385

  15. Defective Pollen Wall is Required for Anther and Microspore Development in Rice and Encodes a Fatty Acyl Carrier Protein Reductase

    Energy Technology Data Exchange (ETDEWEB)

    Shi, J.; Shanklin, J.; Tan, H.; Yu, X.-H.; Liu, Y.; Liang, W.; Ranathunge, K.; Franke, R. B.; Schreiber, L.; Wang, Y.; Kai, G.; Ma, H.; Zhang, D.

    2011-06-01

    Aliphatic alcohols naturally exist in many organisms as important cellular components; however, their roles in extracellular polymer biosynthesis are poorly defined. We report here the isolation and characterization of a rice (Oryza sativa) male-sterile mutant, defective pollen wall (dpw), which displays defective anther development and degenerated pollen grains with an irregular exine. Chemical analysis revealed that dpw anthers had a dramatic reduction in cutin monomers and an altered composition of cuticular wax, as well as soluble fatty acids and alcohols. Using map-based cloning, we identified the DPW gene, which is expressed in both tapetal cells and microspores during anther development. Biochemical analysis of the recombinant DPW enzyme shows that it is a novel fatty acid reductase that produces 1-hexadecanol and exhibits >270-fold higher specificity for palmiltoyl-acyl carrier protein than for C16:0 CoA substrates. DPW was predominantly targeted to plastids mediated by its N-terminal transit peptide. Moreover, we demonstrate that the monocot DPW from rice complements the dicot Arabidopsis thaliana male sterile2 (ms2) mutant and is the probable ortholog of MS2. These data suggest that DPWs participate in a conserved step in primary fatty alcohol synthesis for anther cuticle and pollen sporopollenin biosynthesis in monocots and dicots.

  16. Midgut-enriched receptor protein tyrosine phosphatase PTP52F is required for Drosophila development during larva-pupa transition.

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    Santhanam, Abirami; Liang, Suh-Yuen; Chen, Dong-Yuan; Chen, Guang-Chao; Meng, Tzu-Ching

    2013-01-01

    To date our understanding of Drosophila receptor protein tyrosine phosphatases (R-PTPs) in the regulation of signal transduction is limited. Of the seven R-PTPs identified in flies, six are involved in the axon guidance that occurs during embryogenesis. However, whether and how R-PTPs may control key steps of Drosophila development is not clear. In this study we investigated the potential role of Drosophila R-PTPs in developmental processes outside the neuronal system and beyond the embryogenesis stage. Through systematic data mining of available microarray databases, we found the mRNA level of PTP52F to be highly enriched in the midgut of flies at the larva-pupa transition. This finding was confirmed by gut tissue staining with a specific antibody. The unique spatiotemporal expression of PTP52F suggests that it is possibly involved in regulating metamorphosis during the transformation from larva to pupa. To test this hypothesis, we employed RNA interference to examine the defects of transgenic flies. We found that ablation of endogenous PTP52F led to high lethality characterized by the pharate adult phenotype, occurring due to post pupal eclosion failure. These results show that PTP52F plays an indispensable role during the larva-pupa transition. We also found that PTP52F could be reclassified as a member of the subtype R3 PTPs instead of as an unclassified R-PTP without a human ortholog, as suggested previously. Together, these findings suggest that Drosophila R-PTPs may control metamorphosis and other biological processes beyond our current knowledge.

  17. Rhn1, a nuclear protein, is required for suppression of meiotic mRNAs in mitotically dividing fission yeast.

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    Sugiyama, Tomoyasu; Sugioka-Sugiyama, Rie; Hada, Kazumasa; Niwa, Ryusuke

    2012-01-01

    In the fission yeast Schizosaccharomyces pombe, many meiotic mRNAs are transcribed during mitosis and meiosis and selectively eliminated in mitotic cells. However, this pathway for mRNA decay, called the determinant of selective removal (DSR)-Mmi1 system, targets only some of the numerous meiotic mRNAs that are transcribed in mitotic cells. Here we describe Rhn1, a nuclear protein involved in meiotic mRNA suppression in vegetative fission yeast. Rhn1 is homologous to budding yeast Rtt103 and localizes to one or a few discrete nuclear dots in growing vegetative cells. Rhn1 colocalizes with a pre-mRNA 3'-end processing factor, Pcf11, and with the 5'-3' exoribonuclease, Dhp1; moreover, Rhn1 coimmunoprecipitates with Pcf11. Loss of rhn1 results in elevated sensitivity to high temperature, to thiabendazole (TBZ), and to UV. Interestingly, meiotic mRNAs--including moa1(+), mcp5(+), and mug96(+)--accumulate in mitotic rhn1Δ cells. Accumulation of meiotic mRNAs also occurs in strains lacking Lsk1, a kinase that phosphorylates serine 2 (Ser-2) in the C-terminal domain (CTD) of RNA polymerase II (Pol II), and in strains lacking Sen1, an ATP-dependent 5'-3' RNA/DNA helicase: notably, both Lsk1 and Sen1 have been implicated in termination of Pol II-dependent transcription. Furthermore, RNAi knockdown of cids-2, a Caenorhabditis elegans ortholog of rhn1(+), leads to elevated expression of a germline-specific gene, pgl-1, in somatic cells. These results indicate that Rhn1 contributes to the suppression of meiotic mRNAs in vegetative fission yeast and that the mechanism by which Rhn1 downregulates germline-specific transcripts may be conserved in unicellular and multicellular organisms.

  18. Rhn1, a nuclear protein, is required for suppression of meiotic mRNAs in mitotically dividing fission yeast.

    Directory of Open Access Journals (Sweden)

    Tomoyasu Sugiyama

    Full Text Available In the fission yeast Schizosaccharomyces pombe, many meiotic mRNAs are transcribed during mitosis and meiosis and selectively eliminated in mitotic cells. However, this pathway for mRNA decay, called the determinant of selective removal (DSR-Mmi1 system, targets only some of the numerous meiotic mRNAs that are transcribed in mitotic cells. Here we describe Rhn1, a nuclear protein involved in meiotic mRNA suppression in vegetative fission yeast. Rhn1 is homologous to budding yeast Rtt103 and localizes to one or a few discrete nuclear dots in growing vegetative cells. Rhn1 colocalizes with a pre-mRNA 3'-end processing factor, Pcf11, and with the 5'-3' exoribonuclease, Dhp1; moreover, Rhn1 coimmunoprecipitates with Pcf11. Loss of rhn1 results in elevated sensitivity to high temperature, to thiabendazole (TBZ, and to UV. Interestingly, meiotic mRNAs--including moa1(+, mcp5(+, and mug96(+--accumulate in mitotic rhn1Δ cells. Accumulation of meiotic mRNAs also occurs in strains lacking Lsk1, a kinase that phosphorylates serine 2 (Ser-2 in the C-terminal domain (CTD of RNA polymerase II (Pol II, and in strains lacking Sen1, an ATP-dependent 5'-3' RNA/DNA helicase: notably, both Lsk1 and Sen1 have been implicated in termination of Pol II-dependent transcription. Furthermore, RNAi knockdown of cids-2, a Caenorhabditis elegans ortholog of rhn1(+, leads to elevated expression of a germline-specific gene, pgl-1, in somatic cells. These results indicate that Rhn1 contributes to the suppression of meiotic mRNAs in vegetative fission yeast and that the mechanism by which Rhn1 downregulates germline-specific transcripts may be conserved in unicellular and multicellular organisms.

  19. Killing of p53-deficient hepatoma cells by parvovirus H-1 and chemotherapeutics requires promyelocytic leukemia protein

    Institute of Scientific and Technical Information of China (English)

    Maike Sieben; Markus Moehler; Kerstin Herzer; Maja Zeidler; Vera Heinrichs; Barbara Leuchs; Martin Schuler; Jan J Cornelis; Peter R Galle; Jean Rommelaere

    2008-01-01

    AIM: To evaluate the synergistic targeting and killing of human hepatocellular carcinoma (HCC) cells lacking p53 by the oncolytic autonomous parvovirus (PV) H-1 and chemotherapeutic agents and its dependence on functional promyelocytic leukemia protein (PML).METHODS: The role of p53 and PML in regulating cytotoxicity and gene transfer mediated by wild-type (wt)PV H-1 were explored in two pairs of isogenic human hepatoma cell lines with different p53 status.Furthermore,H-1 PV infection was combined with cytostatic drug treatment.RESULTS: While the HCC cells with different p53 status studied were all susceptible to H-1 PV-induced apoptosis,the cytotoxicity of H-1 PV was more pronounced in p53-negative than in p53-positive cells.Apoptosis rates in p53-negative cell lines treated by genotoxic drugs were further enhanced by a treatment with H-1 PV.In flow cytometric analyses,H-1 PV infection resulted in a reduction of the mitochondrial transmembrane potential.In addition,H-1 PV cells showed a significant increase in PML expression.Knocking down PML expression resulted in a striking reduction of the level of H-1 PV infected tumor cell death.CONCLUSION: H-1 PV is a suitable agent to circumvent the resistance of p53-negative HCC cells to genotoxic agents,and it enhances the apoptotic process which is dependent on functional PML.Thus,H-1 PV and its oncolytic vector derivatives may be considered as therapeutic options for HCC,particularly for p53-negative tumors.

  20. cAMP response element binding protein is required for differentiation of respiratory epithelium during murine development.

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    A Daniel Bird

    Full Text Available The cAMP response element binding protein 1 (Creb1 transcription factor regulates cellular gene expression in response to elevated levels of intracellular cAMP. Creb1(-/- fetal mice are phenotypically smaller than wildtype littermates, predominantly die in utero and do not survive after birth due to respiratory failure. We have further investigated the respiratory defect of Creb1(-/- fetal mice during development. Lungs of Creb1(-/- fetal mice were pale in colour and smaller than wildtype controls in proportion to their reduced body size. Creb1(-/- lungs also did not mature morphologically beyond E16.5 with little or no expansion of airway luminal spaces, a phenotype also observed with the Creb1(-/- lung on a Crem(-/- genetic background. Creb1 was highly expressed throughout the lung at all stages examined, however activation of Creb1 was detected primarily in distal lung epithelium. Cell differentiation of E17.5 Creb1(-/- lung distal epithelium was analysed by electron microscopy and showed markedly reduced numbers of type-I and type-II alveolar epithelial cells. Furthermore, immunomarkers for specific lineages of proximal epithelium including ciliated, non-ciliated (Clara, and neuroendocrine cells showed delayed onset of expression in the Creb1(-/- lung. Finally, gene expression analyses of the E17.5 Creb1(-/- lung using whole genome microarray and qPCR collectively identified respiratory marker gene profiles and provide potential novel Creb1-regulated genes. Together, these results demonstrate a crucial role for Creb1 activity for the development and differentiation of the conducting and distal lung epithelium.

  1. Inhibitor of apoptosis-stimulating protein of p53 (iASPP is required for neuronal survival after axonal injury.

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    Ariel M Wilson

    Full Text Available The transcription factor p53 mediates the apoptosis of post-mitotic neurons exposed to a wide range of stress stimuli. The apoptotic activity of p53 is tightly regulated by the apoptosis-stimulating proteins of p53 (ASPP family members: ASPP1, ASPP2 and iASPP. We previously showed that the pro-apoptotic members ASPP1 and ASPP2 contribute to p53-dependent death of retinal ganglion cells (RGCs. However, the role of the p53 inhibitor iASPP in the central nervous system (CNS remains to be elucidated. To address this, we asked whether iASPP contributes to the survival of RGCs in an in vivo model of acute optic nerve damage. We demonstrate that iASPP is expressed by injured RGCs and that iASPP phosphorylation at serine residues, which increase iASPP affinity towards p53, is significantly reduced following axotomy. We show that short interference RNA (siRNA-induced iASPP knockdown exacerbates RGC death, whereas adeno-associated virus (AAV-mediated iASPP expression promotes RGC survival. Importantly, our data also demonstrate that increasing iASPP expression in RGCs downregulates p53 activity and blocks the expression of pro-apoptotic targets PUMA and Fas/CD95. This study demonstrates a novel role for iASPP in the survival of RGCs, and provides further evidence of the importance of the ASPP family in the regulation of neuronal loss after axonal injury.

  2. The zinc finger protein Zfr1p is localized specifically to conjugation junction and required for sexual development in Tetrahymena thermophila.

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    Jing Xu

    Full Text Available Conjugation in Tetrahymena thermophila involves a developmental program consisting of three prezygotic nuclear divisions, pronuclear exchange and fusion, and postzygotic and exconjugant stages. The conjugation junction structure appears during the initiation of conjugation development, and disappears during the exconjugant stage. Many structural and functional proteins are involved in the establishment and maintenance of the junction structure in T. thermophila. In the present study, a zinc finger protein-encoding gene ZFR1 was found to be expressed specifically during conjugation and to localize specifically to the conjugation junction region. Truncated Zfr1p localized at the plasma membrane in ordered arrays and decorated Golgi apparatus located adjacent to basal body. The N-terminal zinc finger and C-terminal hydrophobic domains of Zfr1p were found to be required for its specific conjugation junction localization. Conjugation development of ZFR1 somatic knockout cells was aborted at the pronuclear exchange and fusion conjugation stages. Furthermore, Zfr1p was found to be important for conjugation junction stability during the prezygotic nuclear division stage. Taken together, our data reveal that Zfr1p is required for the stability and integrity of the conjugation junction structure and essential for the sexual life cycle of the Tetrahymena cell.

  3. Differential requirement for protein tyrosine kinase Fyn in the functional activation of antigen-specific T lymphocyte clones through the TCR or Thy-1.

    Science.gov (United States)

    Lancki, D W; Qian, D; Fields, P; Gajewski, T; Fitch, F W

    1995-05-01

    The protein tyrosine kinase Fyn has been shown to be involved in signal transduction through the TCR and the glycosyl-phosphatidylinositol-linked surface molecule Thy-1 expressed on T cells. In this study, we examine the requirement for Fyn expression in signaling through the TCR or Thy-1 using a panel of Ag-specific T cell clones derived from fyn-/- mutant mice. These clones do not express normal Fyn protein, as measured by immune-complex kinase reaction using anti-Fyn Ab. Stimulation through the TCR, either by APC bearing relevant Ag or by immobilized anti-CD3 mAb, resulted in comparable levels of proliferation, lymphokine production, and cytolysis by clones from both wild-type and fyn-/- mice. In contrast, stimulation through Thy-1, using soluble (or cross-linked) anti-Thy-1 mAb, was deficient, as measured by these responses. Thus, Fyn expression is selectively required for functional activation through Thy-1 in these T cell clones.

  4. Syntaxin 7 and VAMP-7 are soluble N-ethylmaleimide-sensitive factor attachment protein receptors required for late endosome-lysosome and homotypic lysosome fusion in alveolar macrophages.

    Science.gov (United States)

    Ward, D M; Pevsner, J; Scullion, M A; Vaughn, M; Kaplan, J

    2000-07-01

    Endocytosis in alveolar macrophages can be reversibly inhibited, permitting the isolation of endocytic vesicles at defined stages of maturation. Using an in vitro fusion assay, we determined that each isolated endosome population was capable of homotypic fusion. All vesicle populations were also capable of heterotypic fusion in a temporally specific manner; early endosomes, isolated 4 min after internalization, could fuse with endosomes isolated 8 min after internalization but not with 12-min endosomes or lysosomes. Lysosomes fuse with 12-min endosomes but not with earlier endosomes. Using homogenous populations of endosomes, we have identified Syntaxin 7 as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) required for late endosome-lysosome and homotypic lysosome fusion in vitro. A bacterially expressed human Syntaxin 7 lacking the transmembrane domain inhibited homotypic late endosome and lysosome fusion as well as heterotypic late endosome-lysosome fusion. Affinity-purified antibodies directed against Syntaxin 7 also inhibited lysosome fusion in vitro but had no affect on homotypic early endosome fusion. Previous work suggested that human VAMP-7 (vesicle-associated membrane protein-7) was a SNARE required for late endosome-lysosome fusion. A bacterially expressed human VAMP-7 lacking the transmembrane domain inhibited both late endosome-lysosome fusion and homotypic lysosome fusion in vitro. These studies indicate that: 1) fusion along the endocytic pathway is a highly regulated process, and 2) two SNARE molecules, Syntaxin 7 and human VAMP-7, are involved in fusion of vesicles in the late endocytic pathway in alveolar macrophages.

  5. The leucine-rich repeats of LINGO-1 are not required for self-interaction or interaction with the amyloid precursor protein.

    Science.gov (United States)

    Stein, Thomas; Walmsley, Adrian Robert

    2012-02-10

    LINGO-1 (leucine rich repeat and Ig domain containing Nogo receptor interacting protein-1) is a central nervous system transmembrane protein which simultaneously interacts with the Nogo-66 receptor and p75(NTR) or TROY on neurons to form a receptor complex responsible for myelin-mediated neurite outgrowth inhibition. On oligodendroglial cells, LINGO-1 interacts with p75(NTR) to constitutively inhibit multiple aspects of oligodendrocyte differentiation. Recently, LINGO-1 was identified as an in vivo interacting partner of the amyloid precursor protein (APP) and, correspondingly, cellular LINGO-1 expression was found to augment the release of the Abeta peptide, the potential causative agent of Alzheimer's disease. In addition, the recombinant LINGO-1 ectodomain has been shown to self-interact in solution and after crystallisation. Here, we have used deletional mutagenesis to identify the regions on LINGO-1 that are involved in homo- and heterotypic interactions. We have found that the N-terminal region containing the leucine-rich repeats along with the transmembrane and cytoplasmic domains of LINGO-1 are not required for self-interaction or interaction with APP.

  6. The heat-shock DnaK protein is required for plasmid R1 replication and it is dispensable for plasmid ColE1 replication.

    Science.gov (United States)

    Giraldo-Suárez, R; Fernández-Tresguerres, E; Díaz-Orejas, R; Malki, A; Kohiyama, M

    1993-01-01

    Plasmid R1 replication in vitro is inactive in extracts prepared from a dnaK756 strain but is restored to normal levels upon addition of purified DnaK protein. Replication of R1 in extracts of a dnaKwt strain can be specifically inhibited with polyclonal antibodies