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Sample records for centrosome-associated protein required

  1. Cyclin G2 is a centrosome-associated nucleocytoplasmic shuttling protein that influences microtubule stability and induces a p53-dependent cell cycle arrest

    International Nuclear Information System (INIS)

    mature centriole present at microtubule foci, indicates that cyclin G2 resides primarily on the mother centriole. Copurification of cyclin G2 and PP2A subunits with microtubules and centrosomes, together with the effects of ectopic cyclin G2 on cell cycle progression, nuclear morphology and microtubule growth and stability, suggests that cyclin G2 may modulate the cell cycle and cellular division processes through modulation of PP2A and centrosomal associated activities

  2. Activity and regulation of the centrosome-associated proteasome.

    Science.gov (United States)

    Fabunmi, R P; Wigley, W C; Thomas, P J; DeMartino, G N

    2000-01-01

    Regulated proteolysis is important for maintaining appropriate cellular levels of many proteins. The bulk of intracellular protein degradation is catalyzed by the proteasome. Recently, the centrosome was identified as a novel site for concentration of the proteasome and associated regulatory proteins (Wigley, W. C., Fabunmi, R. P., Lee, M. G., Marino, C. R., Muallem, S., DeMartino, G. N., and Thomas, P. J. (1999) J. Cell Biol. 145, 481-490). Here we provide evidence that centrosomes contain the active 26 S proteasome that degrades ubiquitinated-protein and proteasome-specific peptide substrates. Moreover, the centrosomes contain an ubiquitin isopeptidase activity. The proteolytic activity is ATP-dependent and is inhibited by proteasome inhibitors. Notably, treatment of cells with inhibitors of proteasome activity promotes redistribution of the proteasome and associated regulatory proteins to the centrosome independent of an intact microtubule system. These data provide biochemical evidence for active proteasomal complexes at the centrosome, highlighting a novel function for this organizing structure. PMID:10617632

  3. Centrosome-associated regulators of the G2/M checkpoint as targets for cancer therapy

    Directory of Open Access Journals (Sweden)

    Broaddus Russell R

    2009-02-01

    Full Text Available Abstract In eukaryotic cells, control mechanisms have developed that restrain cell-cycle transitions in response to stress. These regulatory pathways are termed cell-cycle checkpoints. The G2/M checkpoint prevents cells from entering mitosis when DNA is damaged in order to afford these cells an opportunity to repair the damaged DNA before propagating genetic defects to the daughter cells. If the damage is irreparable, checkpoint signaling might activate pathways that lead to apoptosis. Since alteration of cell-cycle control is a hallmark of tumorigenesis, cell-cycle regulators represent potential targets for therapy. The centrosome has recently come into focus as a critical cellular organelle that integrates G2/M checkpoint control and repairs signals in response to DNA damage. A growing number of G2/M checkpoint regulators have been found in the centrosome, suggesting that centrosome has an important role in G2/M checkpoint function. In this review, we discuss centrosome-associated regulators of the G2/M checkpoint, the dysregulation of this checkpoint in cancer, and potential candidate targets for cancer therapy.

  4. Protein Requirements during Aging.

    Science.gov (United States)

    Courtney-Martin, Glenda; Ball, Ronald O; Pencharz, Paul B; Elango, Rajavel

    2016-01-01

    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. PMID:27529275

  5. Protein Requirements during Aging

    Directory of Open Access Journals (Sweden)

    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.

  6. 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.

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

    Science.gov (United States)

    Schueler, Markus; Airik, Merlin; Cho, Jang; Ulanowicz, Kelsey A.; Porath, Jonathan D.; Hurd, Toby W.; Bekker-Jensen, Simon; Schrøder, Jacob M.; Andersen, Jens S.; Hildebrandt, Friedhelm

    2016-01-01

    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. PMID:27224062

  8. Protein requirement in critical illness.

    Science.gov (United States)

    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. PMID:26914090

  9. Human Protein and Amino Acid Requirements.

    Science.gov (United States)

    Hoffer, L John

    2016-05-01

    Human protein and amino acid nutrition encompasses a wide, complex, frequently misunderstood, and often contentious area of clinical research and practice. This tutorial explains the basic biochemical and physiologic principles that underlie our current understanding of protein and amino acid nutrition. The following topics are discussed: (1) the identity, measurement, and essentiality of nutritional proteins; (2) the definition and determination of minimum requirements; (3) nutrition adaptation; (4) obligatory nitrogen excretion and the minimum protein requirement; (5) minimum versus optimum protein intakes; (6) metabolic responses to surfeit and deficient protein intakes; (7) body composition and protein requirements; (8) labile protein; (9) N balance; (10) the principles of protein and amino acid turnover, including an analysis of the controversial indicator amino acid oxidation technique; (11) general guidelines for evaluating protein turnover articles; (12) amino acid turnover versus clearance; (13) the protein content of hydrated amino acid solutions; (14) protein requirements in special situations, including protein-catabolic critical illness; (15) amino acid supplements and additives, including monosodium glutamate and glutamine; and (16) a perspective on the future of protein and amino acid nutrition research. In addition to providing practical information, this tutorial aims to demonstrate the importance of rigorous physiologic reasoning, stimulate intellectual curiosity, and encourage fresh ideas in this dynamic area of human nutrition. In general, references are provided only for topics that are not well covered in modern textbooks. PMID:26796095

  10. Protein requirements for long term missions

    Science.gov (United States)

    Stein, T. P.

    1994-01-01

    A key component of the diet for a space mission is protein. This first part of this paper reviews the reasons for emphasizing protein nurtition and then discusses what the requirements are likely to be. The second part discusses potential advantages of modifying these requirements and describes potential potential approaches to effecting these modificatons based on well established ground based models.

  11. Protein and Amino Acid Requirements during Pregnancy.

    Science.gov (United States)

    Elango, Rajavel; Ball, Ronald O

    2016-07-01

    Protein forms an essential component of a healthy diet in humans to support both growth and maintenance. During pregnancy, an exceptional stage of life defined by rapid growth and development, adequate dietary protein is crucial to ensure a healthy outcome. Protein deposition in maternal and fetal tissues increases throughout pregnancy, with most occurring during the third trimester. Dietary protein intake recommendations are based on factorial estimates because the traditional method of determining protein requirements, nitrogen balance, is invasive and undesirable during pregnancy. The current Estimated Average Requirement and RDA recommendations of 0.88 and 1.1 g · kg(-1) · d(-1), respectively, are for all stages of pregnancy. The single recommendation does not take into account the changing needs during different stages of pregnancy. Recently, with the use of the minimally invasive indicator amino acid oxidation method, we defined the requirements to be, on average, 1.2 and 1.52 g · kg(-1) · d(-1) during early (∼16 wk) and late (∼36 wk) stages of pregnancy, respectively. Although the requirements are substantially higher than current recommendations, our values are ∼14-18% of total energy and fit within the Acceptable Macronutrient Distribution Range. Using swine as an animal model we showed that the requirements for several indispensable amino acids increase dramatically during late gestation compared with early gestation. Additional studies should be conducted during pregnancy to confirm the newly determined protein requirements and to determine the indispensable amino acid requirements during pregnancy in humans. PMID:27422521

  12. Protein requirement for Trichogaster lalius, blue variety

    Directory of Open Access Journals (Sweden)

    Jener Alexandre Sampaio Zuanon

    2013-02-01

    Full Text Available The objective of the present study was to evaluate the protein requirement of juvenile Trichogaster lalius, blue variety. The experimental design was of randomised blocks (B1 = initial weight of 1.04±0.05 g and B2 = 1.36±0.02 g, with two replicates within each block and five treatments (230, 270, 310, 350 and 390 g CP/kg diet. The fish were fed to satiation, three times a day for 90 days. The study evaluated: survival rate, weight gain, final length, feed intake, feed conversion ratio, protein efficiency ratio, specific growth rate and condition factor. There was a linear effect of dietary protein levels for protein efficiency ratio, specific growth rate (positive linear effect and feed conversion ratio (negative linear effect. For weight gain, final length, feed intake and condition factor a quadratic effect of dietary protein levels was observed, with estimated values of 409.8, 366.2, 317.4 and 365.0 g CP/kg diet, to improve their performance parameters. Analysis of growth based on the length of the fish shows that 366.2 g of CP/kg diet meets the protein requirement of juvenile Trichogaster lalius, blue variety.

  13. Protein requirements of finishing paca (Cuniculus paca).

    Science.gov (United States)

    Nogueira-Filho, Sérgio Luiz Gama; Bastos, Ivanise da Hora; Mendes, Alcester; Nogueira, Selene Siqueira da Cunha

    2016-06-01

    We conducted a nitrogen balance digestion trial to determine the crude protein requirements of paca (Cuniculus paca) during the last growth phase. In a 4 × 4 Latin square design, four young captive male pacas, aged 5 months, were fed four isoenergetic diets containing four different levels of nitrogen (N) (11.3, 16.6, 21.4, and 26.6 g N/kg of dry matter). After 15 days of adaptation, we collected all feces and urine for five consecutive days. By regression analysis between N intake and N in feces and urine, the metabolic fecal nitrogen (MFN = 4.2 g/kg of dry matter intake) and daily endogenous urinary N (EUN = 91.6 mg/kg(0.75)) were determined. Likewise, by regression analyses between nitrogen intake and nitrogen retention [NR = N intake-(fecal N + urine N)], we estimated the daily requirement of 280.5 mg N/kg(0.75). Therefore, a minimum of 55 g crude protein per kilogram dry matter and 13 MJ/kg of digestible energy are required by finishing paca on unrestricted diets. Such values are similar to those of other wild frugivores and below those of growing rabbits. The data confirm that farmers overfeed protein, and similar growth can be more economically achieved on lower protein diets. PMID:27026234

  14. Protein requirement for Trichogaster lalius, blue variety

    OpenAIRE

    Jener Alexandre Sampaio Zuanon; Antonio Policarpo Souza Carneiro; Lidiane da Silva Nascimento; Donizete Aparecido da Silva; Marcelo Duarte Pontes; Márcio Yoshiyuki Kanashiro; Ana Lúcia Salaro

    2013-01-01

    The objective of the present study was to evaluate the protein requirement of juvenile Trichogaster lalius, blue variety. The experimental design was of randomised blocks (B1 = initial weight of 1.04±0.05 g and B2 = 1.36±0.02 g), with two replicates within each block and five treatments (230, 270, 310, 350 and 390 g CP/kg diet). The fish were fed to satiation, three times a day for 90 days. The study evaluated: survival rate, weight gain, final length, feed intake, feed conversion ratio, prot...

  15. Winter protein requirements of bobwhite quail

    Science.gov (United States)

    Nestler, R.B.; Bailey, W.W.; Llewellyn, L.M.; Rensberger, M.J.

    1944-01-01

    Three experiments involving 714 bobwhite quail were conducted at the Patuxent Research Refuge, Bowie, Maryland, during the winters of 1939-1941 to determine the protein requirement of quail maintained throug'h the winter.....Considering survival, live weights, feed consumption, and subsequent reproduction by the birds, the-9 to 13 per cent levels of crude dietary protein gave as good results as higher levels eggs, which in all cases was over 90 per and in some respects were better.....On the basis of these studies, it is recommended that the winter maintenance diet for bobwhite quail contain . about 11 to 12 per cent of crude protein. The following diet (parts by weight) conforms to these specifications and should be satisfactory:...Ground yellow corn 85.6....Dehvdrated alfalfa leaf meal 5 .O.....Soybean oil meal 7.0.....Special steamed bonemeal 1.2....Salt (or Salt Mixture II,see text) 1.0...Vitamin A and D feeding oil, fortified 0.2.

  16. Protein catabolism and requirements in severe illness.

    Science.gov (United States)

    Genton, L; Pichard, C

    2011-03-01

    Reduced total body protein mass is a marker of protein-energy malnutrition and has been associated with numerous complications. Severe illness is characterized by a loss of total body protein mass, mainly from the skeletal muscle. Studies on protein turnover describe an increased protein breakdown and, to a lesser extent, an increased whole-body protein synthesis, as well as an increased flux of amino acids from the periphery to the liver. Appropriate nutrition could limit protein catabolism. Nutritional support limits but does not stop the loss of total body protein mass occurring in acute severe illness. Its impact on protein kinetics is so far controversial, probably due to the various methodologies and characteristics of nutritional support used in the studies. Maintaining calorie balance alone the days after an insult does not clearly lead to an improved clinical outcome. In contrast, protein intakes between 1.2 and 1.5 g/kg body weight/day with neutral energy balance minimize total body protein mass loss. Glutamine and possibly leucine may improve clinical outcome, but it is unclear whether these benefits occur through an impact on total body protein mass and its turnover, or through other mechanisms. Present recommendations suggest providing 20 - 25 kcal/kg/day over the first 72 - 96 hours and increasing energy intake to target thereafter. Simultaneously, protein intake should be between 1.2 and 1.5 g/kg/day. Enteral immunonutrition enriched with arginine, nucleotides, and omega-3 fatty acids is indicated in patients with trauma, acute respiratory distress syndrome (ARDS), and mild sepsis. Glutamine (0.2 - 0.4 g/kg/day of L-glutamine) should be added to enteral nutrition in burn and trauma patients (ESPEN guidelines 2006) and to parenteral nutrition, in the form of dipeptides, in intensive care unit (ICU) patients in general (ESPEN guidelines 2009). PMID:22139565

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

    OpenAIRE

    Caryl Nowson; Stella O'Connell

    2015-01-01

    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 compensa...

  18. Models of protein and amino acid requirements for cattle

    OpenAIRE

    Luis Orlindo Tedeschi; Danny Gene Fox; Mozart Alves Fonseca; Luigi Francis Lima Cavalcanti

    2015-01-01

    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 comp...

  19. Sperm Motility Requires Wnt/GSK3 Stabilization of Proteins.

    Science.gov (United States)

    De Robertis, Edward M; Ploper, Diego

    2015-11-23

    Inhibition of GSK3 by Wnt signaling stabilizes many cellular proteins, but proof that this effect is independent of β-catenin-mediated transcription is lacking. Koch, Acebron, and colleagues (2015) now demonstrate that transcriptionally silent mammalian sperm require Wnt signaling via exosomes to prevent protein degradation during their lengthy travels through the epididymis. PMID:26609954

  20. Critical assessment of sequence-based protein-protein interaction prediction methods that do not require homologous protein sequences

    OpenAIRE

    Park Yungki

    2009-01-01

    Abstract Background Protein-protein interactions underlie many important biological processes. Computational prediction methods can nicely complement experimental approaches for identifying protein-protein interactions. Recently, a unique category of sequence-based prediction methods has been put forward - unique in the sense that it does not require homologous protein sequences. This enables it to be universally applicable to all protein sequences unlike many of previous sequence-based predi...

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

    Science.gov (United States)

    Dickerson, Roland N.

    2016-01-01

    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. PMID:27096868

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

    Science.gov (United States)

    Dickerson, Roland N

    2016-01-01

    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. PMID:27096868

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    Nowson, Caryl; O'Connell, Stella

    2015-08-01

    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. PMID:26287239

  5. CENP-32 is required to maintain centrosomal dominance in bipolar spindle assembly.

    Science.gov (United States)

    Ohta, Shinya; Wood, Laura; Toramoto, Iyo; Yagyu, Ken-Ichi; Fukagawa, Tatsuo; Earnshaw, William C

    2015-04-01

    Centrosomes nucleate spindle formation, direct spindle pole positioning, and are important for proper chromosome segregation during mitosis in most animal cells. We previously reported that centromere protein 32 (CENP-32) is required for centrosome association with spindle poles during metaphase. In this study, we show that CENP-32 depletion seems to release centrosomes from bipolar spindles whose assembly they had previously initiated. Remarkably, the resulting anastral spindles function normally, aligning the chromosomes to a metaphase plate and entering anaphase without detectable interference from the free centrosomes, which appear to behave as free asters in these cells. The free asters, which contain reduced but significant levels of CDK5RAP2, show weak interactions with spindle microtubules but do not seem to make productive attachments to kinetochores. Thus CENP-32 appears to be required for centrosomes to integrate into a fully functional spindle that not only nucleates astral microtubules, but also is able to nucleate and bind to kinetochore and central spindle microtubules. Additional data suggest that NuMA tethers microtubules at the anastral spindle poles and that augmin is required for centrosome detachment after CENP-32 depletion, possibly due to an imbalance of forces within the spindle. PMID:25657325

  6. Chromosome partition in Escherichia coli requires postreplication protein synthesis.

    OpenAIRE

    Donachie, W. D.; Begg, K J

    1989-01-01

    After inhibition of protein synthesis, the number of nuclear bodies (nucleoids) visible in cells of Escherichia coli B/rA corresponded closely to the number of completely replicated chromosomes. We calculated that nucleoid partition follows almost immediately after replication forks reach the chromosome terminus. We show that such a partition is dependent on protein synthesis and that this may reflect the requirement that cells must achieve a certain minimum length before partition (and subse...

  7. A protein required for prion generation: [URE3] induction requires the Ras-regulated Mks1 protein

    OpenAIRE

    Edskes, Herman K.; Wickner, Reed B.

    2000-01-01

    Infectious proteins (prions) can arise de novo as well as by transmission from another individual. De novo prion generation is believed responsible for most cases of Creutzfeldt–Jakob disease and for initiating the mad cow disease epidemic. However, the cellular components needed for prion generation have not been identified in any system. The [URE3] prion of Saccharomyces cerevisiae is an infectious form of Ure2p, apparently a self-propagating amyloid. We now demonstrate a protein required f...

  8. N protein alone satisfies the requirement for protein synthesis during RNA replication of vesicular stomatitis virus.

    OpenAIRE

    Patton, J T; Davis, N L; Wertz, G W

    1984-01-01

    Genomic replication of the negative-strand RNA viruses is dependent upon protein synthesis. To examine the requirement for protein synthesis in replication, we developed an in vitro system that supports the genome replication of defective interfering particles of the negative-strand rhabdovirus vesicular stomatitis virus (VSV), as a function of protein synthesis (Wertz, J. Virol. 46:513-522, 1983). The system consists of defective interfering nucleocapsid templates and an mRNA-dependent retic...

  9. Energy-protein requirements and adaptation during nutritional recovery

    International Nuclear Information System (INIS)

    Malnutrition is one of the world's most widely prevalent pathological conditions that affects people of all ages. The detrimental effects of malnutrition often become evident in children, in whom both physical and mental development are compromised. The present paper reports preliminary results of a project to asses the protein and energy requirements for nutritional recovery. It is also planned to investigate the adaptive mechanisms resulting from chronic low food intake in severely malnourished children. 12 refs, 2 figs, 7 tabs

  10. Bod1, a novel kinetochore protein required for chromosome biorientation

    OpenAIRE

    Porter, Iain M.; McClelland, Sarah E.; Khoudoli, Guennadi A; Hunter, Christopher J; Andersen, Jens S; McAinsh, Andrew D.; Blow, J Julian; Swedlow, Jason R

    2007-01-01

    We have combined the proteomic analysis of Xenopus laevis in vitro–assembled chromosomes with RNA interference and live cell imaging in HeLa cells to identify novel factors required for proper chromosome segregation. The first of these is Bod1, a protein conserved throughout metazoans that associates with a large macromolecular complex and localizes with kinetochores and spindle poles during mitosis. Small interfering RNA depletion of Bod1 in HeLa cells produces elongated mitotic spindles wit...

  11. Models of protein and amino acid requirements for cattle

    Directory of Open Access Journals (Sweden)

    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

  12. PML tumor suppressor protein is required for HCV production

    International Nuclear Information System (INIS)

    Highlights: ► PML tumor suppressor protein is required for HCV production. ► PML is dispensable for HCV RNA replication. ► HCV could not alter formation of PML-NBs. ► 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.

  13. Insertion of an outer membrane protein in Escherichia coli requires a chaperone-like protein.

    OpenAIRE

    Hardie, K R; Lory, S; Pugsley, A P

    1996-01-01

    Only one of the characterized components of the main terminal branch of the general secretory pathway (GSP) in Gram-negative bacteria, GspD, is an integral outer membrane protein that could conceivably form a channel to permit protein transport across this membrane. PulD, a member of the GspD protein family required for pullulanase secretion by Klebsiella oxytoca, is shown here to form outer membrane-associated complexes which are not readily dissociated by SDS treatment. The outer membrane a...

  14. Memory Impairment in Transgenic Alzheimer Mice Requires Cellular Prion Protein

    OpenAIRE

    Gimbel, David A.; Nygaard, Haakon B.; Coffey, Erin E.; Gunther, Erik C.; Laurén, Juha; Gimbel, Zachary A.; Strittmatter, Stephen M.

    2010-01-01

    Soluble oligomers of the amyloid-β (Aβ) peptide are thought to play a key role in the pathophysiology of Alzheimer’s disease (AD). Recently, we reported that synthetic Aβ oligomers bind to cellular prion protein (PrPC) and that this interaction is required for suppression of synaptic plasticity in hippocampal slices by oligomeric Aβ peptide. We hypothesized that PrPC is essential for the ability of brain-derived Aβ to suppress cognitive function. Here, we crossed familial AD transgenes encodi...

  15. Data requirements for reliable chemical shift assignments in deuterated proteins

    International Nuclear Information System (INIS)

    The information required for chemical shift assignments in large deuterated proteins was investigated using a Monte Carlo approach (Hitchens et al., 2002). In particular, the consequences of missing amide resonances on the reliability of assignments derived from Cα and CO or from Cα and Cβ chemical shifts was investigated. Missing amide resonances reduce both the number of correct assignments as well as the confidence in these assignments. More significantly, a number of undetectable errors can arise when as few as 9% of the amide resonances are missing from the spectra. However, the use of information from residue specific labeling as well as local and long-range distance constraints improves the reliability and extent of assignment. It is also shown that missing residues have only a minor effect on the assignment of protein-ligand complexes using Cα and CO chemical shifts and Cα inter-residue connectivity, provided that the known chemical shifts of the unliganded protein are utilized in the assignment process

  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. Protein methylation is required to maintain optimal HIV-1 infectivity

    OpenAIRE

    Piller Sabine C; Warrilow David; Apolloni Ann; Bodetti Tracey J; Hitchen Eleanor M; Willemsen Nicole M; Harrich David

    2006-01-01

    Abstract Background: Protein methylation is recognized as a major protein modification pathway regulating diverse cellular events such as protein trafficking, transcription, and signal transduction. More recently, protein arginine methyltransferase activity has been shown to regulate HIV-1 transcription via Tat. In this study, adenosine periodate (AdOx) was used to globally inhibit protein methyltransferase activity so that the effect of protein methylation on HIV-1 infectivity could be asses...

  18. Protein "requirements" beyond the RDA: implications for optimizing health.

    Science.gov (United States)

    Phillips, Stuart M; Chevalier, Stéphanie; Leidy, Heather J

    2016-05-01

    Substantial evidence supports the increased consumption of high-quality protein to achieve optimal health outcomes. A growing body of research indicates that protein intakes well above the current Recommended Dietary Allowance help to promote healthy aging, appetite regulation, weight management, and goals aligned with athletic performance. Higher protein intakes may help prevent age-related sarcopenia, the loss of muscle mass, and strength that predisposes older adults to frailty, disability, and loss of autonomy. Higher protein diets also improve satiety and lead to greater reductions in body weight and fat mass compared with standard protein diets, and may therefore serve as a successful strategy to help prevent and/or treat obesity. Athletes can also benefit from higher protein intakes to maximize athletic performance given the critical role protein plays in stimulating muscle protein remodelling after exercise. Protein quality, per meal dose, and timing of ingestion are also important considerations. Despite persistent beliefs to the contrary, we can find no evidence-based link between higher protein diets and renal disease or adverse bone health. This brief synopsis highlights recent learnings based on presentations at the 2015 Canadian Nutrition Society conference, Advances in Protein Nutrition across the Lifespan. Current evidence indicates intakes in the range of at least 1.2 to 1.6 g/(kg·day) of high-quality protein is a more ideal target for achieving optimal health outcomes in adults. PMID:26960445

  19. Protein methylation is required to maintain optimal HIV-1 infectivity

    Directory of Open Access Journals (Sweden)

    Piller Sabine C

    2006-12-01

    Full Text Available Abstract Background: Protein methylation is recognized as a major protein modification pathway regulating diverse cellular events such as protein trafficking, transcription, and signal transduction. More recently, protein arginine methyltransferase activity has been shown to regulate HIV-1 transcription via Tat. In this study, adenosine periodate (AdOx was used to globally inhibit protein methyltransferase activity so that the effect of protein methylation on HIV-1 infectivity could be assessed. Results: Two cell culture models were used: HIV-1-infected CEM T-cells and HEK293T cells transfected with a proviral DNA plasmid. In both models, AdOx treatment of cells increased the levels of virion in culture supernatant. However, these viruses had increased levels of unprocessed or partially processed Gag-Pol, significantly increased diameter, and displayed reduced infectivity in a MAGI X4 assay. AdOx reduced infectivity equally in both dividing and non-dividing cells. However, infectivity was further reduced if Vpr was deleted suggesting virion proteins, other than Vpr, were affected by protein methylation. Endogenous reverse transcription was not inhibited in AdOx-treated HIV-1, and infectivity could be restored by pseudotyping HIV with VSV-G envelope protein. These experiments suggest that AdOx affects an early event between receptor binding and uncoating, but not reverse transcription. Conclusion: Overall, we have shown for the first time that protein methylation contributes towards maximal virus infectivity. Furthermore, our results also indicate that protein methylation regulates HIV-1 infectivity in a complex manner most likely involving the methylation of multiple viral or cellular proteins and/or multiple steps of replication.

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

    Science.gov (United States)

    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. Computing wheat nitrogen requirements from grain yield and protein maps

    Science.gov (United States)

    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 post-harvest information for evaluating water or nitrogen (...

  2. Phase-dependent molecular requirements for memory reconsolidation: differential roles for protein synthesis and protein kinase A activity

    OpenAIRE

    Kemenes, György; Kemenes, Ildikó; Michel, Maximilian; Papp, Andrea; Mueller, Uli

    2006-01-01

    After consolidation, a process that requires gene expression and protein synthesis, memories are stable and highly resistant to disruption by amnestic influences. Recently, consolidated memory has been shown to become labile again after retrieval and to require a phase of reconsolidation to be preserved. New findings, showing that the dependence of reconsolidation on protein synthesis decreases with the age of memory, point to changing molecular requirements for reconsolidation during memory ...

  3. Interactions between acidic proteins and crystals: stereochemical requirements in biomineralization.

    OpenAIRE

    Addadi, L; Weiner, S

    1985-01-01

    Acidic matrix macromolecules are intimately involved in biological crystal growth. In vitro experiments, in which crystals of calcium dicarboxylate salts were grown in the presence of aspartic acid-rich proteins, revealed a stereochemical property common to all the interacting faces. Calcite crystals are nucleated on stereochemically analogous faces when proteins are adsorbed onto a rigid substrate. The importance of this property in biomineralization is discussed.

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

    Science.gov (United States)

    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

  5. Protein composition of malting barley varieties required in brewing industry

    Czech Academy of Sciences Publication Activity Database

    Flodrová, Dana; Benkovská, Dagmar; Bobálová, Janette

    Berlin : Freie Universität Berlin, 2011. s. 354. [Proteomic Forum 2011. 03.04.2011-07.04.2011, Berlin] R&D Projects: GA MŠk 1M0570 Institutional research plan: CEZ:AV0Z40310501 Keywords : barley * varieties * proteins Subject RIV: CB - Analytical Chemistry, Separation

  6. Plastid protein synthesis is required for plant development in tobacco

    OpenAIRE

    Ahlert, Daniela; Ruf, Stephanie; Bock, Ralph

    2003-01-01

    Chloroplasts fulfill important functions in cellular metabolism. The majority of plastid genome-encoded genes is involved in either photosynthesis or chloroplast gene expression. Whether or not plastid genes also can determine extraplastidic functions has remained controversial. We demonstrate here an essential role of plastid protein synthesis in tobacco leaf development. By using chloroplast transformation, we have developed an experimental system that produces recombination-based knockouts...

  7. Mitochondrial uncoupling protein is required for efficient photosynthesis

    OpenAIRE

    Sweetlove, Lee J.; Lytovchenko, Anna; Morgan, Megan; Nunes-Nesi, Adriano; Taylor, Nicolas L.; Baxter, Charles J.; Eickmeier, Ira; Fernie, Alisdair R.

    2006-01-01

    Uncoupling proteins (UCPs) occur in the inner mitochondrial membrane and dissipate the proton gradient across this membrane that is normally used for ATP synthesis. Although the catalytic function and regulation of plant UCPs have been described, the physiological purpose of UCP in plants has not been established. Here, biochemical and physiological analyses of an insertional knockout of one of the Arabidopsis UCP genes (AtUCP1) are presented that resolve this issue. Absence of UCP1 results i...

  8. 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.

  9. 40 CFR 180.1204 - Harpin protein; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... Exemptions From Tolerances § 180.1204 Harpin protein; exemption from the requirement of a tolerance. An... 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...

  10. Identification of protein complexes required for efficient sister chromatid cohesion

    NARCIS (Netherlands)

    Mayer, Melanie L; Pot, Isabelle; Chang, Michael; Xu, Hong; Aneliunas, Victoria; Kwok, Teresa; Newitt, Rick; Aebersold, Ruedi; Boone, Charles; Brown, Grant W; Hieter, Philip

    2004-01-01

    Ctf8p is a component of Ctf18-RFC, an alternative replication factor C-like complex required for efficient sister chromatid cohesion in Saccharomyces cerevisiae. We performed synthetic genetic array (SGA) analysis with a ctf8 deletion strain as a primary screen to identify other nonessential genes r

  11. LAMP proteins are required for fusion of lysosomes with phagosomes.

    Science.gov (United States)

    Huynh, Kassidy K; Eskelinen, Eeva-Liisa; Scott, Cameron C; Malevanets, Anatoly; Saftig, Paul; Grinstein, Sergio

    2007-01-24

    Lysosome-associated membrane proteins 1 and 2 (LAMP-1 and LAMP-2) are delivered to phagosomes during the maturation process. We used cells from LAMP-deficient mice to analyze the role of these proteins in phagosome maturation. Macrophages from LAMP-1- or LAMP-2-deficient mice displayed normal fusion of lysosomes with phagosomes. Because ablation of both the lamp-1 and lamp-2 genes yields an embryonic-lethal phenotype, we were unable to study macrophages from double knockouts. Instead, we reconstituted phagocytosis in murine embryonic fibroblasts (MEFs) by transfection of FcgammaIIA receptors. Phagosomes formed by FcgammaIIA-transfected MEFs obtained from LAMP-1- or LAMP-2- deficient mice acquired lysosomal markers. Remarkably, although FcgammaIIA-transfected MEFs from double-deficient mice ingested particles normally, phagosomal maturation was arrested. LAMP-1 and LAMP-2 double-deficient phagosomes acquired Rab5 and accumulated phosphatidylinositol 3-phosphate, but failed to recruit Rab7 and did not fuse with lysosomes. We attribute the deficiency to impaired organellar motility along microtubules. Time-lapse cinematography revealed that late endosomes/lysosomes as well as phagosomes lacking LAMP-1 and LAMP-2 had reduced ability to move toward the microtubule-organizing center, likely precluding their interaction with each other. PMID:17245426

  12. Relief memory consolidation requires protein synthesis within the nucleus accumbens.

    Science.gov (United States)

    Bruning, Johann E A; Breitfeld, Tino; Kahl, Evelyn; Bergado-Acosta, Jorge R; Fendt, Markus

    2016-06-01

    Relief learning refers to the association of a stimulus with the relief from an aversive event. The thus-learned relief stimulus then can induce, e.g., an attenuation of the startle response or approach behavior, indicating positive valence. Previous studies revealed that the nucleus accumbens is essential for the acquisition and retrieval of relief memory. Here, we ask whether the nucleus accumbens is also the brain site for consolidation of relief memory into a long-term form. In rats, we blocked local protein synthesis within the nucleus accumbens by local infusions of anisomycin at different time points during a relief conditioning experiment. Accumbal anisomycin injections immediately after the relief conditioning session, but not 4 h later, prevented the consolidation into long-term relief memory. The retention of already consolidated relief memory was not affected by anisomycin injections. This identifies a time window and site for relief memory consolidation. These findings should complement our understanding of the full range of effects of adverse experiences, including cases of their distortion in humans such as post-traumatic stress disorder and/or phobias. PMID:26792192

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

    Science.gov (United States)

    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.

  14. 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.

  15. The ATP requiring step in assembly of M13 procoat protein into microsomes is related to preservation of transport competence of the precursor protein

    OpenAIRE

    Wiech, Hans; Sagstetter, Maria; Müller, Günter; Zimmermann, Richard

    1987-01-01

    M13 procoat protein is processed to transmembrane coat protein by dog pancreas microsomes after completion of synthesis and in the absence of the signal recognition particle (SRP)/docking protein system. ATP is required for fast and efficient processing of procoat protein by microsomes in a reticulocyte lysate. Requirement for ATP is also observed in the absence of ribosomes or docking protein. This indicates the existence of a unique assembly pathway for procoat protein into microsomes which...

  16. Distinct Requirement for Two Stages of Protein-Primed Initiation of Reverse Transcription in Hepadnaviruses

    OpenAIRE

    Wang, Xingtai; Hu, Jianming

    2002-01-01

    Reverse transcription in hepadnaviruses is primed by the viral reverse transcriptase (RT) (protein priming) and requires the specific interaction between the RT and a viral RNA signal termed ɛ, which bears the specific template sequence for protein priming. The product of protein priming is a short oligodeoxynucleotide which represents the 5′ end of the viral minus-strand DNA and is covalently attached to the RT. We have now identified truncated RT variants from the duck hepatitis B virus tha...

  17. Requirement for NusG for Transcription Antitermination In Vivo by the λ N Protein

    OpenAIRE

    Zhou, Ying; Filter, Joshua J; Court, Donald L.; Gottesman, Max E.; Friedman, David I

    2002-01-01

    Transcription antitermination by the bacteriophage λ N protein is stimulated in vitro by the Escherichia coli NusG protein. Earlier work suggested that NusG was not required for N activity in vivo. Here we present evidence that NusG also stimulates N-mediated transcription antitermination in intact cells.

  18. General requirements for protein secretion by the F-like conjugation system R1

    OpenAIRE

    Lang, Silvia; Zechner, Ellen L.

    2012-01-01

    Bacterial conjugation disseminates genes among bacteria via a process requiring direct cell contact. The cell envelope spanning secretion apparatus involved belongs to the type IV family of bacterial secretion systems, which transport protein as well as nucleoprotein substrates. This study aims to understand mechanisms leading to the initiation of type IV secretion using conjugative plasmid paradigm R1. We analyze the general requirements for plasmid encoded conjugation proteins and DNA seque...

  19. 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.

  20. Presynaptic protein synthesis required for NT-3-induced long-term synaptic modulation

    Directory of Open Access Journals (Sweden)

    Je H

    2011-01-01

    Full Text Available Abstract Background Neurotrophins elicit both acute and long-term modulation of synaptic transmission and plasticity. Previously, we demonstrated that the long-term synaptic modulation requires the endocytosis of neurotrophin-receptor complex, the activation of PI3K and Akt, and mTOR mediated protein synthesis. However, it is unclear whether the long-term synaptic modulation by neurotrophins depends on protein synthesis in pre- or post-synaptic cells. Results Here we have developed an inducible protein translation blocker, in which the kinase domain of protein kinase R (PKR is fused with bacterial gyrase B domain (GyrB-PKR, which could be dimerized upon treatment with a cell permeable drug, coumermycin. By genetically targeting GyrB-PKR to specific cell types, we show that NT-3 induced long-term synaptic modulation requires presynaptic, but not postsynaptic protein synthesis. Conclusions Our results provide mechanistic insights into the cell-specific requirement for protein synthesis in the long-term synaptic modulation by neurotrophins. The GyrB-PKR system may be useful tool to study protein synthesis in a cell-specific manner.

  1. 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.

  2. 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

  3. 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.

  4. Spike protein assembly into the coronavirion: exploring the limits of its sequence requirements

    International Nuclear Information System (INIS)

    The coronavirus spike (S) protein, required for receptor binding and membrane fusion, is incorporated into the assembling virion by interactions with the viral membrane (M) protein. Earlier we showed that the ectodomain of the S protein is not involved in this process. Here we further defined the requirements of the S protein for virion incorporation. We show that the cytoplasmic domain, not the transmembrane domain, determines the association with the M protein and suffices to effect the incorporation into viral particles of chimeric spikes as well as of foreign viral glycoproteins. The essential sequence was mapped to the membrane-proximal region of the cytoplasmic domain, which is also known to be of critical importance for the fusion function of the S protein. Consistently, only short C-terminal truncations of the S protein were tolerated when introduced into the virus by targeted recombination. The important role of the about 38-residues cytoplasmic domain in the assembly of and membrane fusion by this approximately 1300 amino acids long protein is discussed

  5. Requirement of nucleotide exchange factor for Ypt1 GTPase mediated protein transport

    OpenAIRE

    1995-01-01

    Small GTPases of the rab family are involved in the regulation of vesicular transport. It is believed that cycling between the GTP- and GDP-bound forms, and accessory factors regulating this cycling are crucial for rab function. However, an essential role for rab nucleotide exchange factors has not yet been demonstrated. In this report we show the requirement of nucleotide exchange factor activity for Ypt1 GTPase mediated protein transport. The Ypt1 protein, a member of the rab family, plays ...

  6. 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.

  7. Protein requirements of bobwhite chicks for survival, growth and efficiency of feed utilization

    Science.gov (United States)

    Nestler, R.B.; Bailey, W.W.; McClure, H.E.

    1942-01-01

    During the summer and fall of 1939 four experiments were conducted at the Patuxent Research Refuge, Bowie, Maryland, to determine the protein requirements of bobwhite chicks. A total of 816 chicks were used to compare six levels of protein, namely, 22,24,26, 28, 30, and 32 per cent.....From the three standpoints of survival, rate of growth, and efficiency of feed utilization for the first ten weeks of life, the 28 per cent level of protein gave the best results. During the ninth and tenth weeks, the highest efficiency of feed utilization was obtained on the 22 per cent level. The results indicate that after the birds have reached about twothirds of their mature weight, the difference in efficiency between a diet containing 28 per cent of protein and one containing 22 per cent may be small enough to justify, in the interest of economy, the use of a diet containing the lower percentage of protein.

  8. Protein level affects the relative lysine requirement of growing rainbow trout (Oncorhynchus mykiss) fry

    OpenAIRE

    Bodin, Noelie; Govaerts, Bernadette; Abboudi, Tarik; Detavernier, Christel; De Saeger, Sarah; Larondelle, Yvan; Rollin, Xavier

    2009-01-01

    The effect of two digestible protein levels (310 and 469g/kg DM) on the relative lysine (Lys; g Lys/kg DM or g Lys/100g protein) and the absolute Lys (g Lys intake/kg(0.75) per d) requirements was studied in rainbow trout fry using a dose-response trial. At each protein level, sixteen isoenergetic (22-23 MJ digestible energy/kg DM) diets were tested, involving a full range (2-70 g/kg DM) of sixteen Lys levels. Each diet was given to one group of sixty rainbow trout fry (mean initial body weig...

  9. Maintenance Crude Protein Requirement of Penned Female Korean Spotted Deer (Cervus nippon)

    OpenAIRE

    Yang, S Y; Oh, Y. K.; Ahn, H. S.; Kwak, W. S.

    2014-01-01

    This study was conducted to evaluate the protein requirement for maintenance of 2-year-old female Korean spotted deer. In the course of the experiment, each of three hand-reared female spotted deer was fed three diets that were iso-calorically formulated to contain low (approximately 7%), medium (12%), and high (17%) levels of crude protein (CP). Each of six trials included a 5-day transition, a 10-day preliminary, and a 7-day collection period. Dietary protein levels affected the apparent di...

  10. Protein requirement of Japanese quail (Coturnix coturnix japonica) during rearing and laying periods

    OpenAIRE

    Soares R da TRN; JB Fonseca; Santos AS de O dos; MB Mercandante

    2003-01-01

    Two completely randomized trials were conducted to estimate protein requirements of Japanese quails during the rearing and laying periods. In each trial, 150 quails were distributed in five treatments with five repetitions. Crude protein levels in the rearing period were 18, 20, 22, 24 and 26% (Trial 1) and during the laying period were 16, 18, 20, 22 and 24% (Trial 2). A quadratic effect of protein level was observed on weight gain from seven to 35 days (Trial 1). There were no effects of pr...

  11. Topology of Legionella pneumophila DotA: an inner membrane protein required for replication in macrophages.

    OpenAIRE

    Roy, C R; Isberg, R.R.

    1997-01-01

    The Legionella pneumophila dotA gene is required for intracellular growth of the bacterium in macrophages. In this study, a structure-function analysis of the DotA protein was conducted to elucidate the role of this protein in L. pneumophila pathogenesis. Translational fusions of dotA to the Escherichia coli phoA and lacZ genes indicated that DotA is an integral cytoplasmic membrane protein with eight membrane-spanning domains. DotA contains two large periplasmic domains of approximately 503 ...

  12. 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....

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

    OpenAIRE

    José Jordão Filho; José Humberto Vilar da Silva; Fernando Guilherme Perazzo Costa; Luiz Fernando Teixeira Albino; Thiago de Sousa Melo; Patrícia Barbosa de Lacerda; Gabriela Mafra Dantas; Rejane Pontes Soares

    2012-01-01

    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 pe...

  14. Transition fibre protein FBF1 is required for the ciliary entry of assembled intraflagellar transport complexes

    OpenAIRE

    WEI, QING; Xu, Qingwen; Zhang, Yuxia; Li, Yujie; Zhang, Qing; Hu, Zeng; Harris, Peter C.; Torres, Vicente E.; Ling, Kun; Hu, Jinghua

    2013-01-01

    Sensory organelle cilia play critical roles in mammalian embryonic development and tissue homeostasis. Intraflagellar transport (IFT) machinery is required for the assembly and maintenance of cilia. Yet how this large complex passes through the size-dependent barrier at the ciliary base remains enigmatic. Here we report that FBF1, a highly conserved transition fibre protein, is required for the ciliary import of assembled IFT particles at the cilia base. We cloned dyf-19, the C. elegans homol...

  15. Bacterial protein translocation requires only one copy of the SecY complex in vivo

    OpenAIRE

    Park, Eunyong; Rapoport, Tom A.

    2012-01-01

    The transport of proteins across the plasma membrane in bacteria requires a channel formed from the SecY complex, which cooperates with either a translating ribosome in cotranslational translocation or the SecA ATPase in post-translational translocation. Whether translocation requires oligomers of the SecY complex is an important but controversial issue: it determines channel size, how the permeation of small molecules is prevented, and how the channel interacts with the ribosome and SecA. He...

  16. BH4 domain of bcl-2 protein is required for its proangiogenic function under hypoxic condition.

    Science.gov (United States)

    Gabellini, Chiara; De Luca, Teresa; Trisciuoglio, Daniela; Desideri, Marianna; Di Martile, Marta; Passeri, Daniela; Candiloro, Antonio; Biffoni, Mauro; Rizzo, Maria Giulia; Orlandi, Augusto; Del Bufalo, Donatella

    2013-11-01

    Beyond its classical role as apoptosis inhibitor, bcl-2 protein promotes tumor angiogenesis and the removal of N-terminal bcl-2 homology (BH4) domain abrogates bcl-2-induced hypoxia-inducible factor 1 (HIF-1)-mediated vascular endothelial growth factor (VEGF) expression in hypoxic cancer cells. Using M14 human melanoma cell line and its derivative clones stably overexpressing bcl-2 wild-type or deleted of its BH4 domain, we found that conditioned media (CM) from cells expressing BH4-deleted bcl-2 protein showed a reduced capability to increase in vitro human endothelial cells proliferation and differentiation, and in vivo neovascularization compared with CM from cells overexpressing wild-type bcl-2. Moreover, xenografts derived from cells expressing bcl-2 lacking BH4 domain showed a reduction of metastatic potential compared with tumors derived from wild-type bcl-2 transfectants injection. Stably expressing the Flag-tagged N-terminal sequence of bcl-2 protein, encompassing BH4 domain, we found that this domain is sufficient to enhance the proangiogenic HIF-1/VEGF axis under hypoxic condition. Indeed, lacking of BH4 domain abolishes the interaction between bcl-2 and HIF-1α proteins and the capability of exogenous bcl-2 protein to localize in the nucleus. Moreover, when endoplasmic reticulum-targeted bcl-2 protein is overexpressed in cells, this protein lost the capability to synergize with hypoxia to induce the proangiogenic HIF-1/VEGF axis as shown by wild-type bcl-2 protein. These results demonstrate that BH4 domain of bcl-2 is required for the ability of this protein to increase tumor angiogenesis and progression and indicate that bcl-2 nuclear localization may be required for bcl-2-mediated induction of HIF-1/VEGF axis. PMID:23836782

  17. Protein synthetic requirements for caffeine amelioration of radiation-induced G/sub 2/-arrest

    International Nuclear Information System (INIS)

    Irradiated cells are arrested in G/sub 2/ (transition point [TP] = 32 min before cell selection in mitosis). Irradiated cells do not recover from G/sub 2/ arrest in the presence of cycloheximide (CHM) indicating dependence of recovery on protein synthesis. Irradiated cells in the presence of caffeine progress to mitosis without arrest. The authors investigate whether irradiated cells in the presence of caffeine require protein synthesis to progress to mitosis. Mitotic cell selection was used to monitor the progression of irradiated CHO cells (150 rad) during exposure to 5 mM caffeine and/or 50 μg/ml CHM. Protein synthesis inhibition was confirmed using /sup 3/H-leucine incorporation. Cells exposed to CHM alone are arrested in G/sub 2/ (TP=49 min), thus cells beyond this point have synthesized all proteins necessary for entry into mitosis. In the presence of caffeine, unirradiated cells exposed to CHM are not arrested at all in G/sub 2/, instead arrest occurs near the S/G/sub 2/ boundary (TP=95 min) indicating that caffeine alleviates the dependence of G/sub 2/ cell progression on protein synthesis. However, irradiated cells exposed to both caffeine and CHM are only able to progress to mitosis if beyond the CHM-TP. Irradiated cells in the presence of caffeine thus behave as untreated cells and require protein synthesis for progression to mitosis when prior to the CHM-TP

  18. Both ATP and an energized inner membrane are required to import a purified precursor protein into mitochondria.

    OpenAIRE

    Eilers, M; Oppliger, W.; Schatz, G

    1987-01-01

    We have investigated the energy requirement of mitochondrial protein import with a simplified system containing only isolated yeast mitochondria, energy sources and a purified precursor protein. This precursor was a fusion protein composed of 22 residues of the cytochrome oxidase subunit IV pre-sequence fused to mouse dihydrofolate reductase. Import of this protein required not only an energized inner membrane, but also ATP. ATP could be replaced by GTP, but not by CTP, TTP or non-hydrolyzabl...

  19. 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.

  20. RAN/TC4 mutants identify a common requirement for snRNP and protein import into the nucleus

    OpenAIRE

    1996-01-01

    Kinetic competition experiments have demonstrated that at least some factors required for the nuclear import of proteins and U snRNPs are distinct. Both import processes require energy, and in the case of protein import, the energy requirement is known to be at least partly met by GTP hydrolysis by the Ran GTPase. We have compared the effects of nonhydrolyzable GTP analogues and two mutant Ran proteins on the nuclear import of proteins and U snRNPs in vitro. The mutant Ran proteins have diffe...

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

    OpenAIRE

    Escobar, Martha L.

    2011-01-01

    It has been proposed that long-term memory 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 long-term memo...

  2. KAR5 Encodes a Novel Pheromone-inducible Protein Required for Homotypic Nuclear Fusion

    OpenAIRE

    Beh, Christopher T.; Brizzio, Valeria; Rose, Mark D.

    1997-01-01

    KAR5 is required for membrane fusion during karyogamy, the process of nuclear fusion during yeast mating. To investigate the molecular mechanism of nuclear fusion, we cloned and characterized the KAR5 gene and its product. KAR5 is a nonessential gene, and deletion mutations produce a bilateral defect in the homotypic fusion of yeast nuclei. KAR5 encodes a novel protein that shares similarity with a protein in Schizosaccharomyces pombe that may play a similar role in nuclear fusion. Kar5p is i...

  3. G-Protein α-Subunit Gsα Is Required for Craniofacial Morphogenesis

    OpenAIRE

    Run Lei; Ke Zhang; Yanxia Wei; Min Chen; Weinstein, Lee S.; Yang Hong; Minyan Zhu; Hongchang Li; Huashun Li

    2016-01-01

    The heterotrimeric G protein subunit Gsα couples receptors to activate adenylyl cyclase and is required for the intracellular cAMP response and protein kinase A (PKA) activation. Gsα is ubiquitously expressed in many cell types; however, the role of Gsα in neural crest cells (NCCs) remains unclear. Here we report that NCCs-specific Gsα knockout mice die within hours after birth and exhibit dramatic craniofacial malformations, including hypoplastic maxilla and mandible, cleft palate and cranio...

  4. A conserved OmpA-like protein in Legionella pneumophila required for efficient intracellular replication.

    Science.gov (United States)

    Goodwin, Ian P; Kumova, Ogan K; Ninio, Shira

    2016-08-01

    The OmpA-like protein domain has been associated with peptidoglycan-binding proteins, and is often found in virulence factors of bacterial pathogens. The intracellular pathogen Legionella pneumophila encodes for six proteins that contain the OmpA-like domain, among them the highly conserved uncharacterized protein we named CmpA. Here we set out to characterize the CmpA protein and determine its contribution to intracellular survival of L. pneumophila Secondary structure analysis suggests that CmpA is an inner membrane protein with a peptidoglycan-binding domain at the C-teminus. A cmpA mutant was able to replicate normally in broth, but failed to compete with an isogenic wild-type strain in an intracellular growth competition assay. The cmpA mutant also displayed significant intracellular growth defects in both the protozoan host Acanthamoeba castellanii and in primary bone marrow-derived macrophages, where uptake into the cells was also impaired. The cmpA phenotypes were completely restored upon expression of CmpA in trans The data presented here establish CmpA as a novel virulence factor of L. pneumophila that is required for efficient intracellular replication in both mammalian and protozoan hosts. PMID:27421957

  5. Complementary food: international comparison on protein and energy requirement/intakes.

    Science.gov (United States)

    Agostoni, Carlo; Riva, Enrica; Giovannini, Marcello

    2006-01-01

    The possible role of early dietary habits as the origin of later consequences on health has raised questions on the optimal macronutrient intakes of the growing infant. Infants and toddlers in developed countries usually show a high dietary protein: energy ratio during the complementary feeding period, averaging 2.5-3, because of the protein density of solid weaning foods and the low percentage of mothers still breastfeeding beyond the first 6 months of life. In conditions of very high protein intakes, those in the higher classes of consumption seem to carry a higher risk of becoming obese later on. Over the limit of 14% energy from proteins in the 8- to 24- month period, some mechanisms may begin to operate leading young children towards an early adiposity rebound and overweight development. On the other hand, in many developing countries the only available weaning foods are cereals, with a low protein: energy ratio value. When the protein concentration of weaning foods falls below the limits of human milk (that is, from 1 g/100 kcal to lower levels), the infants' dietary requirements cannot be met. In planning interventions, the coverage of infants' dietary needs through all the various world regions should be considered together with the opportunity not to exceed higher limits. PMID:16902332

  6. Determination of optimal protein quantity required to identify abundant and less abundant soybean seed proteins by 2D-PAGE and MS

    Science.gov (United States)

    Optimizing the amounts of proteins required to separate and characterize both abundant and less abundant proteins by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is critical for conducting proteomic research. In this study, we tested five different levels of soybean seed proteins (7...

  7. Late protein synthesis-dependent phases in CTA long-term memory: BDNF requirement

    Directory of Open Access Journals (Sweden)

    Martha L Escobar

    2011-09-01

    Full Text Available It has been proposed that long-term memory 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 long-term memory 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 hours 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.

  8. The BRCA1-Interacting Protein Abraxas Is Required for Genomic Stability and Tumor Suppression

    Directory of Open Access Journals (Sweden)

    Andy Castillo

    2014-08-01

    Full Text Available Germline mutations of BRCA1 confer hereditary susceptibility to breast and ovarian cancer. However, somatic mutation of BRCA1 is infrequent in sporadic breast cancers. The BRCA1 protein C terminus (BRCT domains interact with multiple proteins and are required for BRCA1’s tumor-suppressor function. In this study, we demonstrated that Abraxas, a BRCA1 BRCT domain-interacting protein, plays a role in tumor suppression. Abraxas exerts its function through binding to BRCA1 to regulate DNA repair and maintain genome stability. Both homozygous and heterozygous Abraxas knockout mice exhibited decreased survival and increased tumor incidence. The gene encoding Abraxas suffers from gene copy loss and somatic mutations in multiple human cancers including breast, ovarian, and endometrial cancers, suggesting that mutation and loss of function of Abraxas may contribute to tumor development in human patients.

  9. Nucleolar development and allocation of key nucleolar proteins require de novo transcription in bovine embryos

    DEFF Research Database (Denmark)

    Svarcova, Olga; Laurincik, Jozef; Avery, Birthe;

    2007-01-01

    The goal of the present study was to investigate whether key nucleolar proteins involved in ribosomal RNA (rRNA) transcription and processing are transcribed de novo or from maternally inherited messenger RNAs (mRNA) in bovine embryos, and to which extent de novo transcription of these proteins m......RNA is required for the development of functional nucleoli during the major activation of the embryonic genome. Immunofluorescence for localization of key nucleolar proteins, autoradiography for detection of transcriptional activity, and transmission electron microscopy were applied to in vitro produc ed...... bovine embryos cultured from the 2-cell stage with or without (control groups) a-amanitin, which blocks the RNA plymerases II and III transcription and, thus the synthesis of mRNA. In the control groups, weak autoradiographic labelling was initially observed in the periphery of few nuclei at the 4-cell...

  10. Embryonic poly(A)-binding protein (ePAB) phosphorylation is required for Xenopus oocyte maturation.

    Science.gov (United States)

    Friend, Kyle; Brook, Matthew; Bezirci, F Betül; Sheets, Michael D; Gray, Nicola K; Seli, Emre

    2012-07-01

    Oocyte maturation and early embryonic development require the cytoplasmic polyadenylation and concomitant translational activation of stored maternal mRNAs. ePAB [embryonic poly(A)-binding protein, also known as ePABP and PABPc1-like] is a multifunctional post-transcriptional regulator that binds to poly(A) tails. In the present study we find that ePAB is a dynamically modified phosphoprotein in Xenopus laevis oocytes and show by mutation that phosphorylation at a four residue cluster is required for oocyte maturation. We further demonstrate that these phosphorylations are critical for cytoplasmic polyadenylation, but not for ePAB's inherent ability to promote translation. Our results provide the first insight into the role of post-translational modifications in regulating PABP protein activity in vivo. PMID:22497250

  11. The Saccharomyces cerevisiae centromere protein Slk19p is required for two successive divisions during meiosis.

    OpenAIRE

    Zeng, X.; Saunders, W S

    2000-01-01

    Meiotic cell division includes two separate and distinct types of chromosome segregation. In the first segregational event the sister chromatids remain attached at the centromere; in the second the chromatids are separated. The factors that control the order of chromosome segregation during meiosis have not yet been identified but are thought to be confined to the centromere region. We showed that the centromere protein Slk19p is required for the proper execution of meiosis in Saccharomyces c...

  12. The long-term stability of new hippocampal place fields requires new protein synthesis

    OpenAIRE

    Agnihotri, Naveen T.; Hawkins, Robert D.; Kandel, Eric R.; Kentros, Clifford

    2004-01-01

    The hippocampus is critical for formation of spatial memories. Hippocampal pyramidal neurons in freely behaving animals exhibit spatially selective firing patterns, which taken together form an internal representation of the environment. This representation is thought to contribute to the hippocampal spatial memory system. Behavioral long-term memories differ from short-term memories in requiring the synthesis of new proteins. Does the development of the internal hippocampal representation al...

  13. 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....

  14. Concurrent protein synthesis is required for in vivo chitin synthesis in postmolt blue crabs

    International Nuclear Information System (INIS)

    Chitin synthesis in crustaceans involves the deposition of a protein-polysaccharide complex at the apical surface of epithelial cells which secrete the cuticle or exoskeleton. The present study involves an examination of in vivo incorporation of radiolabeled amino acids and amino sugars into the cuticle of postmolt blue crabs, Callinectes sapidus. Rates of incorporation of both 3H leucine and 3H threonine were linear with respect to time of incubation. Incorporation of 3H threonine into the endocuticle was inhibited greater than 90% in the presence of the protein synthesis inhibitor, puromycin. Linear incorporation of 14C glucosamine into the cuticle was also demonstrated; a significant improvement of radiolabeling was achieved by using 14C-N-acetylglucosamine as the labeled precursor. Incorporation of 3H-N-acetylglucosamine into the cuticle of postmolt blue crabs was inhibited 89% by puromycin, indicating that concurrent protein synthesis is required for the deposition of chitin in the blue crab. Autoradiographic analysis of control vs. puromycin-treated crabs indicates that puromycin totally blocks labeling of the new endocuticle with 3H glucosamine. These results are consistent with the notion that crustacean chitin is synthesized as a protein-polysaccharide complex. Analysis of the postmolt and intermolt blue crab cuticle indicates that the exoskeleton contains about 60% protein and 40% chitin. The predominant amino acids are arginine, glutamic acid, alanine, aspartic acid, and threonine

  15. 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.

  16. 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...

  17. 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...

  18. 40 CFR 174.515 - Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ...; exemption from the requirement of a tolerance. Residues of Coat Protein of Papaya Ringspot Virus are exempt... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance. 174.515 Section 174.515 Protection of...

  19. 40 CFR 174.531 - Coat protein of plum pox virus; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ...; exemption from the requirement of a tolerance. Residues of the coat protein of plum pox virus in or on the... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat protein of plum pox virus; exemption from the requirement of a tolerance. 174.531 Section 174.531 Protection of...

  20. Expression of bactericidal/permeability-increasing protein requires C/EBP epsilon.

    Science.gov (United States)

    Tanaka, Miyuki; Gombart, Adrian F; Koeffler, H Phillip; Shiohara, Masaaki

    2007-05-01

    Bactericidal/permeability-increasing protein (BPI) is a 55-kd cationic protein found mainly in neutrophil primary granules. BPI shows cytotoxicity against Gram-negative bacteria. In this study, we studied the role of a myeloid-specific transcription factor, CCAAT/enhancer binding protein epsilon (C/EBP epsilon), in the regulation of BPI gene expression. A patient with neutrophil-specific granule deficiency with a homozygous inactivating mutation in the CEBP epsilon gene showed severely impaired expression of both BPI messenger RNA (mRNA) and BPI protein. Both U937 and NB4 cells treated with 10-7 M all-trans retinoic acid (ATRA) for 6 days displayed increased levels of BPI protein and accompanying up-regulated C/EBP epsilon expression. Chromatin-immunoprecipitation analysis and electrophoretic mobility shift assays revealed binding of the C/EBP epsilon protein to the C/EBP-binding site in the BPI gene promoter. U937 cells stably transfected with a zinc-inducible C/EBP epsilon expression vector showed a 30-fold increase in BPI mRNA levels compared with cells transfected with control empty vector after culturing for 48 hours with 100 microM ZnSO4. BPI mRNA expression was severely reduced in the bone marrow of C/EBP epsilon-deficient mice compared with wild-type mice. Expression of BPI in human cord blood cells was increased by incubation with 10-7 MATRA for 48 hours. These results demonstrate the requirement for C/EBP epsilon in mediating BPI gene expression in myeloid cells in vitro and in vivo. PMID:17483073

  1. 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.

  2. Removal of damaged proteins during ES cell fate specification requires the proteasome activator PA28

    DEFF Research Database (Denmark)

    Hernebring, Malin; Fredriksson, Asa; Liljevald, Maria; Cvijovic, Marija; Norrman, Karin; Wiseman, John; Semb, Tor Henrik; Nyström, Thomas

    2013-01-01

    In embryonic stem cells, removal of oxidatively damaged proteins is triggered upon the first signs of cell fate specification but the underlying mechanism is not known. Here, we report that this phase of differentiation encompasses an unexpected induction of genes encoding the proteasome activato...... that PA28aß has a hitherto unidentified role required for resetting the levels of protein damage at the transition from self-renewal to cell differentiation.......In embryonic stem cells, removal of oxidatively damaged proteins is triggered upon the first signs of cell fate specification but the underlying mechanism is not known. Here, we report that this phase of differentiation encompasses an unexpected induction of genes encoding the proteasome activator...... PA28aß (11S), subunits of the immunoproteasome (20Si), and the 20Si regulator TNFa. This induction is accompanied by assembly of mature PA28-20S(i) proteasomes and elevated proteasome activity. Inhibiting accumulation of PA28a using miRNA counteracted the removal of damaged proteins demonstrating...

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

    International Nuclear Information System (INIS)

    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

  4. Activation of resting human T cells requires prolonged stimulation of protein kinase C

    Energy Technology Data Exchange (ETDEWEB)

    Berry, N.; Ase, K.; Kishimoto, A.; Nishizuka, Y. (Kobe Univ. School of Medicine (Japan))

    1990-03-01

    Purified resting human T cells can be induced to express the {alpha} subunit of the interleukin 2 receptor and to proliferate by treatment with 12-0-tetradecanoylphorbol-13-acetate plus ionomycin but not with 1,2-dioctanoylglycerol plus ionomycin. Determination of the translocation of protein kinase C showed that 12-0-tetradecanoylphorbol-13-acetate plus ionomycin caused a prolonged membrane association of the enzyme for more than 4 hr, whereas 1,2-dioctanoylglycerol plus ionomycin induced a transient membrane association, which was maximal at 20 min. Delivery of multiple additions of 1,2-dioctanoylglycerol plus ionomycin to the T cells resulted in progressively increased expression of the {alpha} subunit of the interleukin 2 receptor and proliferation commensurate with the number of multiple additions delivered, suggesting that prolonged protein kinase C activity is required for T-cell activation.

  5. 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...

  6. 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.

  7. The Unfolded Protein Response Is Induced by the Cell Wall Integrity Mitogen-activated Protein Kinase Signaling Cascade and Is Required for Cell Wall Integrity in Saccharomyces cerevisiae

    OpenAIRE

    Scrimale, Thomas; DiDone, Louis; de Mesy Bentley, Karen L.; Krysan, Damian J.

    2009-01-01

    The yeast cell wall is an extracellular structure that is dependent on secretory and membrane proteins for its construction. We investigated the role of protein quality control mechanisms in cell wall integrity and found that the unfolded protein response (UPR) and, to a lesser extent, endoplasmic reticulum (ER)-associated degradation (ERAD) pathways are required for proper cell wall construction. Null mutation of IRE1, double mutation of ERAD components (hrd1Δ and ubc7Δ) and ire1Δ, or expres...

  8. AP1S3 is required for hepatitis C virus infection by stabilizing E2 protein.

    Science.gov (United States)

    Li, Xiang; Niu, Yuqiang; Cheng, Min; Chi, Xiaojing; Liu, Xiuying; Yang, Wei

    2016-07-01

    Hepatitis C virus (HCV) infects 130 million people worldwide and is a leading cause of liver cirrhosis, end-stage liver disease and hepatocellular carcinoma. The interactions between viral elements and host factors play critical role on HCV invade, replication and release. Here, we identified adaptor protein complex 1 sigma 3 subunit (AP1S3) as a dependency factor for the efficient HCV infection in hepatoma cells. AP1S3 silencing in cultivated Huh7.5.1 cells significantly reduced the production of HCV progeny particles. Immunoprecipitation analysis revealed that AP1S3 interacted with the HCV E2 protein. With this interaction, AP1S3 could protect HCV E2 from ubiquitin-mediated proteasomal degradation. Using in vivo ubiquitylation assay, we identified that E6-Associated Protein (E6AP) was associated with HCV E2. In addition, treatment with synthetic peptide that contains the AP1S3-recognized motif inhibited HCV infection in Huh7.5.1 cells. Our data reveal AP1 as a novel host network that is required by viruses during infection and provides a potential target for developing broad-spectrum anti-virus strategies. PMID:27079945

  9. Lectin-induced activation of platelets may require only limited phosphorylation of the 47K protein

    International Nuclear Information System (INIS)

    Wheat germ agglutinin (WGA) is an N-acetylglucosamine (Glc-NAc) specific lectin which can activate platelets. Like thrombin, stimulation of platelets by WGA is accompanied by enhanced phosphorylation of two polypeptides of M/sub r/ 47K and 20K. Addition of GlcNAc at different time intervals arrested that aggregation of platelets by WGA and paralleled the modification of phosphorylation of the 47K polypeptide. So, the phosphorylation of the 47K polypeptide may regulate the WGA-receptor mediated stimulation of platelets. However, the ratio of phosphoserine to phosphothreonine in the 47K protein was markedly different in WGA-activated than thrombin-stimulated platelets. Thus, the molecular mechanism of action of thrombin and WGA could be different. To explore this idea, 32P/sub i/-labeled platelets were stimulated with WGA and the activation arrested with N-acetyl-glucosamine at different times. Two-dimensional gel electrophoresis of total protein at 5s showed only two phosphorylated species of 47K protein. At 60s, maximally four phosphorylated species were noted. In contrast, with thrombin using the same technique, seven to nine phosphorylated components have been reported. These results suggest that the different activators of platelets may act by different mechanisms. In addition, activation of platelets may require only limited levels of phosphorylation of the 47K polypeptide

  10. Unfolded protein response is required for Aspergillus oryzae growth under conditions inducing secretory hydrolytic enzyme production.

    Science.gov (United States)

    Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

    2015-12-01

    Unfolded protein response (UPR) is an intracellular signaling pathway for adaptation to endoplasmic reticulum (ER) stress. In yeast UPR, Ire1 cleaves the unconventional intron of HAC1 mRNA, and the functional Hac1 protein translated from the spliced HAC1 mRNA induces the expression of ER chaperone genes and ER-associated degradation genes for the refolding or degradation of unfolded proteins. In this study, we constructed an ireA (IRE1 ortholog) conditionally expressing strain of Aspergillus oryzae, a filamentous fungus producing a large amount of amylolytic enzymes, and examined the contribution of UPR to ER stress adaptation under physiological conditions. Repression of ireA completely blocked A. oryzae growth under conditions inducing the production of hydrolytic enzymes, such as amylases and proteases. This growth defect was restored by the introduction of unconventional intronless hacA (hacA-i). Furthermore, UPR was observed to be induced by amylolytic gene expression, and the disruption of the transcriptional activator for amylolytic genes resulted in partial growth restoration of the ireA-repressing strain. In addition, a homokaryotic ireA disruption mutant was successfully generated using the strain harboring hacA-i as a parental host. These results indicated that UPR is required for A. oryzae growth to alleviate ER stress induced by excessive production of hydrolytic enzymes. PMID:26496881

  11. 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. PMID:25916847

  12. Intracellular transport of secretory proteins in the pancreatic exocrine cell. IV. Metabolic requirements.

    Science.gov (United States)

    Jamieson, J D; Palade, G E

    1968-12-01

    Since in the pancreatic exocrine cell synthesis and intracellular transport of secretory proteins can be uncoupled (1), it is possible to examine separately the metabolic requirements of the latter process. To this intent, guinea pig pancreatic slices were pulse labeled with leucine-(3)H for 3 min and incubated post-pulse for 37 min in chase medium containing 5 x 10(-4)M cycloheximide and inhibitors of glycolysis, respiration, or oxidative phosphorylation. In each case, the effect on transport was assessed by measuring the amount of labeled secretory proteins found in zymogen granule fractions isolated from the corresponding slices. This assay is actually a measure of the efficiency of transport of secretory proteins from the cisternae of the rough endoplasmic reticulum (RER) to the condensing vacuoles of the Golgi complex which are recovered in the zymogen granule fraction (16). The results indicate that transport is insensitive to glycolytic inhibitors (fluoride, iodoacetate) but is blocked by respiratory inhibitors (N(2), cyanide, Antimycin A) and by inhibitors of oxidative phosphorylation (dinitrophenol, oligomycin). Except for Antimycin A, the effect is reversible. Parallel radioautographic studies and cell fractionation procedures applied to microsomal subfractions have indicated that the energy-dependent step is located between the transitional elements of the RER and the small, smooth-surfaced vesicles at the periphery of the Golgi complex. Radiorespirometric data indicate that the substrates oxidized to support transport are endogenous long-chain fatty acids. PMID:5699933

  13. Hadp1, a newly identified pleckstrin homology domain protein, is required for cardiac contractility in zebrafish

    Directory of Open Access Journals (Sweden)

    Joshua D. Wythe

    2011-09-01

    The vertebrate heart is one of the first organs to form, and its early function and morphogenesis are crucial for continued embryonic development. Here we analyze the effects of loss of Heart adaptor protein 1 (Hadp1, which we show is required for normal function and morphogenesis of the embryonic zebrafish heart. Hadp1 is a pleckstrin homology (PH-domain-containing protein whose expression is enriched in embryonic cardiomyocytes. Knockdown of hadp1 in zebrafish embryos reduced cardiac contractility and altered late myocyte differentiation. By using optical mapping and submaximal levels of hadp1 knockdown, we observed profound effects on Ca2+ handling and on action potential duration in the absence of morphological defects, suggesting that Hadp1 plays a major role in the regulation of intracellular Ca2+ handling in the heart. Hadp1 interacts with phosphatidylinositol 4-phosphate [PI4P; also known as PtdIns(4P] derivatives via its PH domain, and its subcellular localization is dependent upon this motif. Pharmacological blockade of the synthesis of PI4P derivatives in vivo phenocopied the loss of hadp1 in zebrafish. Collectively, these results demonstrate that hadp1 is required for normal cardiac function and morphogenesis during embryogenesis, and suggest that hadp1 modulates Ca2+ handling in the heart through its interaction with phosphatidylinositols.

  14. G-Protein α-Subunit Gsα Is Required for Craniofacial Morphogenesis.

    Science.gov (United States)

    Lei, Run; Zhang, Ke; Wei, Yanxia; Chen, Min; Weinstein, Lee S; Hong, Yang; Zhu, Minyan; Li, Hongchang; Li, Huashun

    2016-01-01

    The heterotrimeric G protein subunit Gsα couples receptors to activate adenylyl cyclase and is required for the intracellular cAMP response and protein kinase A (PKA) activation. Gsα is ubiquitously expressed in many cell types; however, the role of Gsα in neural crest cells (NCCs) remains unclear. Here we report that NCCs-specific Gsα knockout mice die within hours after birth and exhibit dramatic craniofacial malformations, including hypoplastic maxilla and mandible, cleft palate and craniofacial skeleton defects. Histological and anatomical analysis reveal that the cleft palate in Gsα knockout mice is a secondary defect resulting from craniofacial skeleton deficiencies. In Gsα knockout mice, the morphologies of NCCs-derived cranial nerves are normal, but the development of dorsal root and sympathetic ganglia are impaired. Furthermore, loss of Gsα in NCCs does not affect cranial NCCs migration or cell proliferation, but significantly accelerate osteochondrogenic differentiation. Taken together, our study suggests that Gsα is required for neural crest cells-derived craniofacial development. PMID:26859889

  15. G-Protein α-Subunit Gsα Is Required for Craniofacial Morphogenesis.

    Directory of Open Access Journals (Sweden)

    Run Lei

    Full Text Available The heterotrimeric G protein subunit Gsα couples receptors to activate adenylyl cyclase and is required for the intracellular cAMP response and protein kinase A (PKA activation. Gsα is ubiquitously expressed in many cell types; however, the role of Gsα in neural crest cells (NCCs remains unclear. Here we report that NCCs-specific Gsα knockout mice die within hours after birth and exhibit dramatic craniofacial malformations, including hypoplastic maxilla and mandible, cleft palate and craniofacial skeleton defects. Histological and anatomical analysis reveal that the cleft palate in Gsα knockout mice is a secondary defect resulting from craniofacial skeleton deficiencies. In Gsα knockout mice, the morphologies of NCCs-derived cranial nerves are normal, but the development of dorsal root and sympathetic ganglia are impaired. Furthermore, loss of Gsα in NCCs does not affect cranial NCCs migration or cell proliferation, but significantly accelerate osteochondrogenic differentiation. Taken together, our study suggests that Gsα is required for neural crest cells-derived craniofacial development.

  16. The AUXIN BINDING PROTEIN 1 is required for differential auxin responses mediating root growth.

    Directory of Open Access Journals (Sweden)

    Alexandre Tromas

    Full Text Available BACKGROUND: In plants, the phytohormone auxin is a crucial regulator sustaining growth and development. At the cellular level, auxin is interpreted differentially in a tissue- and dose-dependent manner. Mechanisms of auxin signalling are partially unknown and the contribution of the AUXIN BINDING PROTEIN 1 (ABP1 as an auxin receptor is still a matter of debate. METHODOLOGY/PRINCIPAL FINDINGS: Here we took advantage of the present knowledge of the root biological system to demonstrate that ABP1 is required for auxin response. The use of conditional ABP1 defective plants reveals that the protein is essential for maintenance of the root meristem and acts at least on the D-type CYCLIN/RETINOBLASTOMA pathway to control entry into the cell cycle. ABP1 affects PLETHORA gradients and confers auxin sensitivity to root cells thus defining the competence of the cells to be maintained within the meristem or to elongate. ABP1 is also implicated in the regulation of gene expression in response to auxin. CONCLUSIONS/SIGNIFICANCE: Our data support that ABP1 is a key regulator for root growth and is required for auxin-mediated responses. Differential effects of ABP1 on various auxin responses support a model in which ABP1 is the major regulator for auxin action on the cell cycle and regulates auxin-mediated gene expression and cell elongation in addition to the already well known TIR1-mediated ubiquitination pathway.

  17. Dynamic binding of replication protein a is required for DNA repair

    Science.gov (United States)

    Chen, Ran; Subramanyam, Shyamal; Elcock, Adrian H.; Spies, Maria; Wold, Marc S.

    2016-01-01

    Replication protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA) binding protein, is essential for replication, repair and recombination. High-affinity ssDNA-binding by RPA depends on two DNA binding domains in the large subunit of RPA. Mutation of the evolutionarily conserved aromatic residues in these two domains results in a separation-of-function phenotype: aromatic residue mutants support DNA replication but are defective in DNA repair. We used biochemical and single-molecule analyses, and Brownian Dynamics simulations to determine the molecular basis of this phenotype. Our studies demonstrated that RPA binds to ssDNA in at least two modes characterized by different dissociation kinetics. We also showed that the aromatic residues contribute to the formation of the longer-lived state, are required for stable binding to short ssDNA regions and are needed for RPA melting of partially duplex DNA structures. We conclude that stable binding and/or the melting of secondary DNA structures by RPA is required for DNA repair, including RAD51 mediated DNA strand exchange, but is dispensable for DNA replication. It is likely that the binding modes are in equilibrium and reflect dynamics in the RPA–DNA complex. This suggests that dynamic binding of RPA to DNA is necessary for different cellular functions. PMID:27131385

  18. The ATM homologue MEC1 is required for phosphorylation of replication protein A in yeast

    International Nuclear Information System (INIS)

    Replication protein A (RPA) is a highly conserved single-stranded DNA-binding protein, required for cellular DNA replication, repair, and recombination. In human cells, RPA is phosphorylated during the S and G2 phases of the cell cycle and also in response to ionizing or ultraviolet radiation. Saccharomyces cerevisiae exhibits a similar pattern of cell cycle-regulated RPA phosphorylation, and our studies indicate that the radiation-induced reactions occur in yeast as well. We have examined yeast RPA phosphorylation during the normal cell cycle and in response to environmental insult, and have demonstrated that the checkpoint gene MEC1 is required for the reaction under all conditions tested. Through examination of several checkpoint mutants, we have placed RPA phosphorylation in a novel pathway of the DNA damage response. MEC1 is similar in sequence to human ATM, the gene mutated in patients with ataxia-telangiectasia (A-T). A-T cells are deficient in multiple checkpoint pathways and are hypersensitive to killing by ionizing radiation. Because A-T cells exhibit a delay in ionizing radiation-induced RPA phosphorylation, our results indicate a functional similarity between MEC1 and ATM, and suggest that RPA phosphorylation is involved in a conserved eukaryotic DNA damage-response pathway defective in A-T

  19. 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.

  20. Nonvesicular sterol movement from plasma membrane to ER requires oxysterol-binding protein–related proteins and phosphoinositides

    OpenAIRE

    Raychaudhuri, Sumana; Im, Young Jun; Hurley, James H.; Prinz, William A.

    2006-01-01

    Sterols are moved between cellular membranes by nonvesicular pathways whose functions are poorly understood. In yeast, one such pathway transfers sterols from the plasma membrane (PM) to the endoplasmic reticulum (ER). We show that this transport requires oxysterol-binding protein (OSBP)–related proteins (ORPs), which are a large family of conserved lipid-binding proteins. We demonstrate that a representative member of this family, Osh4p/Kes1p, specifically facilitates the nonvesicular transf...

  1. Ethanol extraction requirement for purification of protein labeled with [3H]leucine in aquatic bacterial production studies

    International Nuclear Information System (INIS)

    The trichloroacetic acid (TCA)-insoluble fraction of water column bacteria labeled with [3H]leucine contained an ethanol-soluble fraction accounting for up to 44% of the label. A component of the ethanol-soluble fraction is [3H]leucine. Labeled-protein purification requires an ethanol wash step. Cold TCA can replace hot TCA for precipitation of labeled proteins

  2. 77 FR 6471 - Bacillus thuringiensis Cry2Ae Protein in Cotton; Exemption from the Requirement of a Tolerance

    Science.gov (United States)

    2012-02-08

    ... Register. September 10, 2008. (73 FR 52591) (FRL-8380-1). Bacillus thuringiensis Cry2Ae in Cotton... AGENCY 40 CFR Part 174 Bacillus thuringiensis Cry2Ae Protein in Cotton; Exemption from the Requirement of... residues of Bacillus thuringiensis Cry2Ae protein in cotton under the FFDCA. DATES: This regulation...

  3. Prm3p Is a Pheromone-induced Peripheral Nuclear Envelope Protein Required for Yeast Nuclear Fusion

    OpenAIRE

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

    2009-01-01

    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 pheromon...

  4. Transition fibre protein FBF1 is required for the ciliary entry of assembled intraflagellar transport complexes.

    Science.gov (United States)

    Wei, Qing; Xu, Qingwen; Zhang, Yuxia; Li, Yujie; Zhang, Qing; Hu, Zeng; Harris, Peter C; Torres, Vicente E; Ling, Kun; Hu, Jinghua

    2013-01-01

    Sensory organelle cilia have critical roles in mammalian embryonic development and tissue homeostasis. Intraflagellar transport (IFT) machinery is required for the assembly and maintenance of cilia. Yet, how this large complex passes through the size-dependent barrier at the ciliary base remains enigmatic. Here we report that FBF1, a highly conserved transition fibre protein, is required for the ciliary import of assembled IFT particles at the ciliary base. We cloned dyf-19, the Caenorhabditis elegans homologue of human FBF1, in a whole-genome screen for ciliogenesis mutants. DYF-19 localizes specifically to transition fibres and interacts directly with the IFT-B component DYF-11/IFT54. Although not a structural component of transition fibres, DYF-19 is required for the transit of assembled IFT particles through the ciliary base. Furthermore, we found that human FBF1 shares conserved localization and function with its worm counterpart. We conclude that FBF1 is a key functional transition fibre component that actively facilitates the ciliary entry of assembled IFT machinery. PMID:24231678

  5. Maturin is a novel protein required for differentiation during primary neurogenesis

    Science.gov (United States)

    Martinez-De Luna, Reyna I.; Ku, Ray Yueh; Lyou, Yung; Zuber, Michael E.

    2013-01-01

    Proliferation and differentiation are tightly controlled during neural development. In the embryonic neural plate, primary neurogenesis is driven by the proneural pathway. Here we report the characterization of Maturin, a novel, evolutionarily conserved protein that is required for normal primary neurogenesis. Maturin is detected throughout the early nervous system, yet it is most strongly expressed in differentiating neurons of the embryonic fish, frog and mouse nervous systems. Maturin expression can be induced by the proneural transcription factors Neurog2, Neurod1, and Ebf3. Maturin overexpression promotes neurogenesis, while loss-of-function inhibits the differentiation of neuronal progenitors, resulting in neural plate expansion. Maturin knockdown blocks the ability of Neurog2, Neurod1, and Ebf3 to drive ectopic neurogenesis. Maturin and Pak3, are both required for, and can synergize to promote differentiation of the primary neurons in vivo. Together, our results suggest that Maturin functions during primary neurogenesis and is required for the proneural pathway to regulate neural differentiation. PMID:24095902

  6. 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

  7. A Single Amino Acid Change in the Newcastle Disease Virus Fusion Protein Alters the Requirement for HN Protein in Fusion

    OpenAIRE

    Sergel, Theresa A.; McGinnes, Lori W.; Morrison, Trudy G

    2000-01-01

    The role of a leucine heptad repeat motif between amino acids 268 and 289 in the structure and function of the Newcastle disease virus (NDV) F protein was explored by introducing single point mutations into the F gene cDNA. The mutations affected either folding of the protein or the fusion activity of the protein. Two mutations, L275A and L282A, likely interfered with folding of the molecule since these proteins were not proteolytically cleaved, were minimally expressed at the cell surface, a...

  8. Functional requirements of AID's higher order structures and their interaction with RNA-binding proteins.

    Science.gov (United States)

    Mondal, Samiran; Begum, Nasim A; Hu, Wenjun; Honjo, Tasuku

    2016-03-15

    Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. Although both the N and C termini of AID have unique functions in DNA cleavage and recombination, respectively, during SHM and CSR, their molecular mechanisms are poorly understood. Using a bimolecular fluorescence complementation (BiFC) assay combined with glycerol gradient fractionation, we revealed that the AID C terminus is required for a stable dimer formation. Furthermore, AID monomers and dimers form complexes with distinct heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers associate with DNA cleavage cofactor hnRNP K whereas AID dimers associate with recombination cofactors hnRNP L, hnRNP U, and Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein associations are RNA-dependent. We propose that AID's structure-specific cofactor complex formations differentially contribute to its DNA-cleavage and recombination functions. PMID:26929374

  9. Stearoyl CoA Desaturase Is Required to Produce Active, Lipid-Modified Wnt Proteins

    Directory of Open Access Journals (Sweden)

    Jessica Rios-Esteves

    2013-09-01

    Full Text Available Wnt proteins contain palmitoleic acid, an unusual lipid modification. Production of an active Wnt signal requires the acyltransferase Porcupine and depends on the attachment of palmitoleic acid to Wnt. The source of this monounsaturated fatty acid has not been identified, and it is not known how Porcupine recognizes its substrate and whether desaturation occurs before or after fatty acid transfer to Wnt. Here, we show that stearoyl desaturase (SCD generates a monounsaturated fatty acid substrate that is then transferred by Porcupine to Wnt. Treatment of cells with SCD inhibitors blocked incorporation of palmitate analogs into Wnt3a and Wnt5a and reduced Wnt secretion as well as autocrine and paracrine Wnt signaling. The SCD inhibitor effects were rescued by exogenous addition of monounsaturated fatty acids. We propose that SCD is a key molecular player responsible for Wnt biogenesis and processing and that SCD inhibition provides an alternative mechanism for blocking Wnt pathway activation.

  10. 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.

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

    Science.gov (United States)

    Bloksgaard, Maria; Bek, Signe; Marcher, Ann-Britt; Neess, Ditte; Brewer, Jonathan; Hannibal-Bach, Hans Kristian; Helledie, Torben; Fenger, Christina; Due, Marianne; Berzina, Zane; Neubert, Reinhard; Chemnitz, John; Finsen, Bente; Clemmensen, Anders; Wilbertz, Johannes; Saxtorph, Henrik; Knudsen, Jens; Bagatolli, Luis; Mandrup, Susanne

    2012-10-01

    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 with age. Morphology and development of skin and appendages are normal in ACBP(-/-) mice; however, the stratum corneum display altered biophysical properties with reduced proton activity and decreased water content. Mass spectrometry analyses of lipids from epidermis and stratum corneum of ACBP(+/+) and ACBP(-/-) mice showed very similar composition, except for a significant and specific decrease in the very long chain free fatty acids (VLC-FFA) in stratum corneum of ACBP(-/-) mice. This finding indicates that ACBP is critically involved in the processes that lead to production of stratum corneum VLC-FFAs via complex phospholipids in the lamellar bodies. Importantly, we show that ACBP(-/-) mice display a ∼50% increased transepidermal water loss compared with ACBP(+/+) mice. Furthermore, skin and fur sebum monoalkyl diacylglycerol (MADAG) levels are significantly increased, suggesting that ACBP limits MADAG synthesis in sebaceous glands. In summary, our study shows that ACBP is required for production of VLC-FFA for stratum corneum and for maintaining normal epidermal barrier function. PMID:22829653

  12. Temporal Requirements of cMyc Protein for Reprogramming Mouse Fibroblasts

    Directory of Open Access Journals (Sweden)

    Corey Heffernan

    2012-01-01

    Full Text Available Exogenous expression of Oct4, Sox2, Klf4, and cMyc forces mammalian somatic cells to adopt molecular and phenotypic characteristics of embryonic stem cells, commencing with the required suppression of lineage-associated genes (e.g., Thy1 in mouse. Although omitting cMyc from the reprogramming cocktail minimizes risks of uncontrolled proliferation, its exclusion results in fold reductions in reprogramming efficiency. Thus, the feasibility of substituting cMyc transgene with (non-integrative recombinant “pTAT-mcMyc” protein delivery was assessed, without compromising reprogramming efficiency or the pluripotent phenotype. Purification and delivery of semisoluble/particulate pTAT-mcMyc maintained Oct4-GFP+ colony formation (i.e., reprogramming efficiency whilst supporting pluripotency by various criteria. Differential repression of Thy1 by pTAT-mcMyc ± Oct4, Sox2, and Klf4 (OSK suggested differential (and non-additive mechanisms of repression. Extending these findings, attempts to enhance reprogramming efficiency through a staggered approach (prerepression of Thy1 failed to improve reprogramming efficiency. We consider protein delivery a useful tool to decipher temporal/molecular events characterizing somatic cell reprogramming.

  13. 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.

  14. Worniu, a Snail family zinc-finger protein, is required for brain development in Drosophila.

    Science.gov (United States)

    Ashraf, Shovon I; Ganguly, Atish; Roote, John; Ip, Y Tony

    2004-10-01

    The Snail family of zinc-finger transcriptional repressors is essential for morphogenetic cell movements, mesoderm formation, and neurogenesis during embryonic development. These proteins also control cell cycle, cell death, and cancer progression. In Drosophila, three members of this protein family, Snail, Escargot, and Worniu, have essential but redundant functions in asymmetric cell division of neuroblasts. In addition, Snail is critical for early mesoderm formation and Escargot is required for maintaining diploidy in wing imaginal disc cells. In this report, we demonstrate that Worniu plays a role in brain development. We show that alleles of the l(2)35Da complementation group are mutants of worniu. The developing larvae of these mutant alleles fail to shorten their brainstems. The brain phenotype, as well as the lethality, of these mutants can be rescued by worniu transgenes. Moreover, RNAi experiments targeting the worniu transcript show the same nonshortening phenotype in larval brains. worniu is expressed in the neuroblasts of brain hemispheres and ventral ganglions. The results suggest that the loss of Worniu function within the neuroblasts ultimately causes the larval brainstem to fail to go through shortening during development. PMID:15366015

  15. RipA, a cytoplasmic membrane protein conserved among Francisella species, is required for intracellular survival.

    Science.gov (United States)

    Fuller, James R; Craven, Robin R; Hall, Joshua D; Kijek, Todd M; Taft-Benz, Sharon; Kawula, Thomas H

    2008-11-01

    Francisella tularensis is a highly virulent bacterial pathogen that invades and replicates within numerous host cell types, including macrophages and epithelial cells. In an effort to better understand this process, we screened a transposon insertion library of the F. tularensis live vaccine strain (LVS) for mutant strains that invaded but failed to replicate within alveolar epithelial cell lines. One such strain isolated from this screen contained an insertion in the gene FTL_1914, which is conserved among all sequenced Francisella species yet lacks significant homology to any gene with known function. A deletion strain lacking FTL_1914 was constructed. This strain did not replicate in either epithelial or macrophage-like cells, and intracellular replication was restored by the wild-type allele in trans. Based on the deletion mutant phenotype, FTL_1914 was termed ripA (required for intracellular proliferation, factor A). Following uptake by J774.A1 cells, F. tularensis LVS Delta ripA colocalized with LAMP-1 then escaped the phagosome at the same rate and frequency as wild-type LVS-infected cells. Electron micrographs of the F. tularensis LVS Delta ripA mutant demonstrated the reentry of the mutant bacteria into double membrane vacuoles characteristic of autophagosomes in a process that was not dependent on replication. The F. tularensis LVS Delta ripA mutant was significantly impaired in its ability to persist in the lung and in its capacity to disseminate and colonize the liver and spleen in a mouse model of pulmonary tularemia. The RipA protein was expressed during growth in laboratory media and localized to the cytoplasmic membrane. Thus, RipA is a cytoplasmic membrane protein conserved among Francisella species that is required for intracellular replication within the host cell cytoplasm as well as disease progression, dissemination, and virulence. PMID:18765722

  16. The centrosomin protein is required for centrosome assembly and function during cleavage in Drosophila.

    Science.gov (United States)

    Megraw, T L; Li, K; Kao, L R; Kaufman, T C

    1999-07-01

    Centrosomin is a 150 kDa centrosomal protein of Drosophila melanogaster. To study the function of Centrosomin in the centrosome, we have recovered mutations that are viable but male and female sterile (cnnmfs). We have shown that these alleles (1, 2, 3, 7, 8 and hk21) induce a maternal effect on early embryogenesis and result in the accumulation of low or undetectable levels of Centrosomin in the centrosomes of cleavage stage embryos. Hemizygous cnn females produce embryos that show dramatic defects in chromosome segregation and spindle organization during the syncytial cleavage divisions. In these embryos the syncytial divisions proceed as far as the twelfth cycle, and embryos fail to cellularize. Aberrant divisions and nuclear fusions occur in the early cycles of the nuclear divisions, and become more prominent at later stages. Giant nuclei are seen in late stage embryos. The spindles that form in mutant embryos exhibit multiple anomalies. There is a high occurrence of apparently linked spindles that share poles, indicating that Centrosomin is required for the proper spacing and separation of mitotic spindles within the syncytium. Spindle poles in the mutants contain little or no detectable amounts of the centrosomal proteins CP60, CP190 and (gamma)-tubulin and late stage embryos often do not have astral microtubules at their spindle poles. Spindle morphology and centrosomal composition suggest that the primary cause of these division defects in mutant embryos is centrosomal malfunction. These results suggest that Centrosomin is required for the assembly and function of centrosomes during the syncytial cleavage divisions. PMID:10357928

  17. Protein delivery to vacuole requires SAND protein-dependent Rab GTPase conversion for MVB-vacuole fusion

    NARCIS (Netherlands)

    M.K. Singh; F. Krüger; H. Beckmann; S. Brumm; J.E.M. Vermeer; T. Munnik; U. Mayer; Y.D. Stierhof; C. Grefen; K. Schumacher; G. Jürgens

    2014-01-01

    Plasma-membrane proteins such as ligand-binding receptor kinases, ion channels, or nutrient transporters are turned over by targeting to a lytic compartment--lysosome or vacuole--for degradation. After their internalization, these proteins arrive at an early endosome, which then matures into a late

  18. 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 protein; exemption from the requirement of a tolerance. 174.502 Section 174.502 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances...

  19. 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 in cotton; exemption from the requirement of a tolerance. 174.504 Section 174.504 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances...

  20. 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; exemption from the requirement of a tolerance. 174.509 Section 174.509 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and...

  1. 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 in corn; exemption from the requirement of a tolerance. 174.520 Section 174.520 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances...

  2. 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 in cotton; temporary exemption from the requirement of a tolerance. 174.530 Section 174.530 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED...

  3. 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 in corn; exemption from the requirement of a tolerance. 174.517 Section 174.517 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances...

  4. 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.

  5. Differential Requirement of the Extracellular Domain in Activation of Class B G Protein-coupled Receptors.

    Science.gov (United States)

    Zhao, Li-Hua; Yin, Yanting; Yang, Dehua; Liu, Bo; Hou, Li; Wang, Xiaoxi; Pal, Kuntal; Jiang, Yi; Feng, Yang; Cai, Xiaoqing; Dai, Antao; Liu, Mingyao; Wang, Ming-Wei; Melcher, Karsten; Xu, H Eric

    2016-07-15

    G protein-coupled receptors (GPCRs) from the secretin-like (class B) family are key players in hormonal homeostasis and are important drug targets for the treatment of metabolic disorders and neuronal diseases. They consist of a large N-terminal extracellular domain (ECD) and a transmembrane domain (TMD) with the GPCR signature of seven transmembrane helices. Class B GPCRs are activated by peptide hormones with their C termini bound to the receptor ECD and their N termini bound to the TMD. It is thought that the ECD functions as an affinity trap to bind and localize the hormone to the receptor. This in turn would allow the hormone N terminus to insert into the TMD and induce conformational changes of the TMD to activate downstream signaling. In contrast to this prevailing model, we demonstrate that human class B GPCRs vary widely in their requirement of the ECD for activation. In one group, represented by corticotrophin-releasing factor receptor 1 (CRF1R), parathyroid hormone receptor (PTH1R), and pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1R), the ECD requirement for high affinity hormone binding can be bypassed by induced proximity and mass action effects, whereas in the other group, represented by glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), the ECD is required for signaling even when the hormone is covalently linked to the TMD. Furthermore, the activation of GLP-1R by small molecules that interact with the intracellular side of the receptor is dependent on the presence of its ECD, suggesting a direct role of the ECD in GLP-1R activation. PMID:27226600

  6. 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.

  7. Two Outer Membrane Proteins Are Required for Maximal Type I Secretion of the Caulobacter crescentus S-Layer Protein

    OpenAIRE

    Toporowski, Michael C.; Nomellini, John F.; Awram, Peter; Smit, John

    2004-01-01

    Transport of RsaA, the crystalline S-layer subunit protein of Caulobacter crescentus, is mediated by a type I secretion mechanism. Two proteins have been identified that play the role of the outer membrane protein (OMP) component in the RsaA secretion machinery. The genes rsaFa and rsaFb were identified by similarity to the Escherichia coli hemolysin secretion OMP TolC by using the C. crescentus genome sequence. The rsaFa gene is located several kilobases downstream of the other transporter g...

  8. SecA is required for membrane targeting of the cell division protein DivIVA in vivo

    Directory of Open Access Journals (Sweden)

    SvenHalbedel

    2014-02-01

    Full Text Available The conserved protein DivIVA is involved in different morphogenetic processes in Gram-positive bacteria. In Bacillus subtilis, the protein localises to the cell division site and cell poles, and functions as a scaffold for proteins that regulate division site selection, and for proteins that are required for sporulation. To identify other proteins that bind to DivIVA, we performed an in vivo cross-linking experiment. A possible candidate that emerged was the secretion motor ATPase SecA. SecA mutants have been described that inhibit sporulation, and since DivIVA is necessary for sporulation, we examined the localisation of DivIVA in these mutants. Surprisingly, DivIVA was delocalised, suggesting that SecA is required for DivIVA targeting. To further corroborate this, we performed SecA depletion and inhibition experiments, which provided further indications that DivIVA localisation depends on SecA. Cell fractionation experiments showed that SecA is important for binding of DivIVA to the cell membrane. This was unexpected since DivIVA does not contain a signal sequence, and is able to bind to artificial lipid membranes in vitro without support of other proteins. SecA is required for protein secretion and membrane insertion, and therefore its role in DivIVA localisation is likely indirect. Possible alternative roles of SecA in DivIVA folding and/or targeting are discussed.

  9. TTC26/DYF13 is an intraflagellar transport protein required for transport of motility-related proteins into flagella

    Science.gov (United States)

    Ishikawa, Hiroaki; Ide, Takahiro; Yagi, Toshiki; Jiang, Xue; Hirono, Masafumi; Sasaki, Hiroyuki; Yanagisawa, Haruaki; Wemmer, Kimberly A; Stainier, Didier YR; Qin, Hongmin; Kamiya, Ritsu; Marshall, Wallace F

    2014-01-01

    Cilia/flagella are assembled and maintained by the process of intraflagellar transport (IFT), a highly conserved mechanism involving more than 20 IFT proteins. However, the functions of individual IFT proteins are mostly unclear. To help address this issue, we focused on a putative IFT protein TTC26/DYF13. Using live imaging and biochemical approaches we show that TTC26/DYF13 is an IFT complex B protein in mammalian cells and Chlamydomonas reinhardtii. Knockdown of TTC26/DYF13 in zebrafish embryos or mutation of TTC26/DYF13 in C. reinhardtii, produced short cilia with abnormal motility. Surprisingly, IFT particle assembly and speed were normal in dyf13 mutant flagella, unlike in other IFT complex B mutants. Proteomic and biochemical analyses indicated a particular set of proteins involved in motility was specifically depleted in the dyf13 mutant. These results support the concept that different IFT proteins are responsible for different cargo subsets, providing a possible explanation for the complexity of the IFT machinery. DOI: http://dx.doi.org/10.7554/eLife.01566.001 PMID:24596149

  10. ZMS regulation of M2 muscarinic receptor mRNA stability requires protein factor

    International Nuclear Information System (INIS)

    Aim The aim of this work is to study the elevation mechanism of ZMS on muscarinic M2 receptor mRNA expression. Methods Actinomycin D was added to cultured CHOm2 cells to stop the de novo synthesis of M2 receptor mRNA and samples were taken at various times to determine the time course of mRNA of M2 receptor with real-time quantitative RT-PCR. Half-life of M2 receptor mRNA and the effect of ZMS on the half-life was obtained from the slope of the exponential curves. Cycloheximide was added at 4 h prior to and 24 h after the addition of ZMS to examine the effect of de novo protein synthesis on the action of ZMS. Results The half-life of m2 mRNA was prolonged by ZMS treatment without cycloheximide (4.75±0.54 h and 2.13 h±0.23 h for ZMS and vehicle treated groups, respectively, P<0.05). When cycloheximide was added to the culture medium 4h prior to the addition of ZMS, the effect of ZMS in prolonging the half-life of m2 mRNA disappeared (3.06 h±0.23 h and 3.00 h±l.20 h for cells with and without ZMS, respectively). However, when the ZMS was added to the medium 24h prior to the addition of cycloheximide, the action of ZMS was not abolished by cycloheximide (half-life was 5.43 h±1.13 h and 2.46 h±0.09 h for cells with and without ZMS, respectively). Conclusion These data suggest that de novo protein synthesis was required for the increase in M2 mRNA stability induced by ZMS. (authors)

  11. 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

  12. Protein requirements of growing steers limit-fed corn-based diets.

    Science.gov (United States)

    Wessels, R H; Titgemeyer, E C

    1997-12-01

    In Exp. 1, six steers (254 kg) were used in a 6 x 4 incomplete Latin square to determine the effects of solvent-extracted soybean meal alone or in combination with rumen-protected methionine and lysine on N balance in steers limit-fed a high-corn diet to gain 1.1 kg/d. The basal diet contained (DM basis) 80% rolled corn, 15% alfalfa, and .9% urea (13.9% CP), and 2 or 4% soybean meal replaced corn to give CP concentrations of 14.8 and 15.6%, respectively. Each diet was fed with and without 5 g/d of Smartamine-ML (.75 and 2.0 g of rumen-protected methionine and lysine, respectively). Nitrogen retention increased linearly (P = .09) with level of soybean meal. Rumen-protected methionine and lysine had no effect on N balance. In Exp. 2, seven steers (233 kg) were used in a 7 x 4 incomplete Latin square experiment to investigate optimal levels and sources of CP for steers limit-fed to gain 1 kg/d. Treatments included a negative-control diet (urea; 11.7% CP) and six diets containing either 13.5, 15.4, or 17.2% CP with either solvent-extracted or expeller-processed soybean meal. Diets provided 75, 87.5, 100, or 112.5% of estimated CP requirement for a gain of 1 kg/d. The basal diet contained 83% rolled corn, 15% alfalfa, and .2% urea. Nitrogen retention increased linearly (P = .006) with soybean meal addition, and no differences were observed between CP sources. The CP system underpredicted the protein requirements of limit-fed steers under our conditions. PMID:9420002

  13. Requirement of JIP scaffold proteins for NMDA-mediated signal transduction

    OpenAIRE

    Kennedy, Norman J.; Martin, Gilles; Ehrhardt, Anka G.; Cavanagh-Kyros, Julie; Kuan, Chia-Yi; Rakic, Pasko; Richard A Flavell; Treistman, Steven N.; Davis, Roger J

    2007-01-01

    JIP scaffold proteins are implicated in the regulation of protein kinase signal transduction pathways. To test the physiological role of these scaffold proteins, we examined the phenotype of compound mutant mice that lack expression of JIP proteins. These mice were found to exhibit severe defects in N-methyl-D-aspartic acid (NMDA) receptor function, including decreased NMDA-evoked current amplitude, cytoplasmic Ca++, and gene expression. The decreased NMDA receptor activity in JIP-deficient n...

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

    OpenAIRE

    J.R. Poortmans; Carpentier, A; L.O. Pereira-Lancha; A. Lancha Jr.

    2012-01-01

    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 differe...

  15. ATP hydrolysis is required for DEAD-box protein recycling but not for duplex unwinding

    OpenAIRE

    Liu, Fei; Putnam, Andrea; Jankowsky, Eckhard

    2008-01-01

    DEAD-box proteins, the largest helicase family, catalyze ATP-dependent remodeling of RNA–protein complexes and the unwinding of RNA duplexes. Because DEAD-box proteins hydrolyze ATP in an RNA-dependent fashion, the energy provided by ATP hydrolysis is commonly assumed to drive the energetically unfavorable duplex unwinding. Here, we show efficient unwinding of stable duplexes by several DEAD-box proteins in the presence of the nonhydrolyzable ATP analog ADP-beryllium fluoride. Another ATP ana...

  16. Enhanced skeletal muscle protein synthesis rates in pigs treated with somatotropin requires fed amino acids levels

    Science.gov (United States)

    Chronic somatotropin (pST) treatment in pigs increases skeletal muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin alone could not account for the pST-induced increase in protein synthesis. This study...

  17. 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

  18. Alpha-fetoprotein, the major fetal serum protein, is not essential for embryonic development but is required for female fertility

    OpenAIRE

    Gabant, Philippe; Forrester, Lesley; Nichols, Jennifer; Van Reeth, Thierry; De Mees, Christelle; Pajack, Bernard; Watt, Alistair; Smitz, Johan; Alexandre, Henri; Szpirer, Claude; Szpirer, Josiane

    2002-01-01

    The alpha-fetoprotein gene (Afp) is a member of a multigenic family that comprises the related genes encoding albumin, alpha-albumin, and vitamin D binding protein. The biological role of this major embryonic serum protein is unknown although numerous speculations have been made. We have used gene targeting to show that AFP is not required for embryonic development. AFP null embryos develop normally, and individually transplanted homozygous embryos can develop in an AFP-deficient microenviron...

  19. Protein synthesis required for long-term memory is induced by PKC activation on days before associative learning

    OpenAIRE

    Alkon, Daniel L.; Epstein, Herman; Kuzirian, Alan; Bennett, M. Catherine; Nelson, Thomas J.

    2005-01-01

    Protein synthesis has long been known to be required for associative learning to consolidate into long-term memory. Here we demonstrate that PKC isozyme activation on days before training can induce the synthesis of proteins necessary and sufficient for subsequent long-term memory consolidation. Bryostatin (Bryo), a macrolide lactone with efficacy in subnanomolar concentrations and a potential therapeutic for Alzheimer's disease, is a potent activator of PKC, some of whose isozymes undergo pr...

  20. The Drosophila fork head domain protein crocodile is required for the establishment of head structures.

    Science.gov (United States)

    Häcker, U; Kaufmann, E; Hartmann, C; Jürgens, G; Knöchel, W; Jäckle, H

    1995-01-01

    The fork head (fkh) domain defines the DNA-binding region of a family of transcription factors which has been implicated in regulating cell fate decisions across species lines. We have cloned and molecularly characterized the crocodile (croc) gene which encodes a new family member from Drosophila. croc is expressed in the head anlagen of the blastoderm embryo under the control of the anterior, the dorsoventral and the terminal maternal organizer systems. The croc mutant phenotype indicates that the croc wild-type gene is required to function as an early patterning gene in the anterior-most blastoderm head segment anlage and for the establishment of a specific head skeletal structure that derives from the non-adjacent intercalary segment at a later stage of embryogenesis. As an early patterning gene, croc exerts unusual properties which do not allow it to be grouped among the established segmentation genes. A single-site mutation within the croc fkh domain, which causes a replacement of the first out of four conserved amino acid residues thought to be involved in the coordinate binding of Mg2+, abolishes the DNA binding of the protein in vitro. In view of the resulting lack-of-function mutant phenotype, it appears likely that metal binding by the affected region of the fkh domain is crucial for proper folding of the DNA-binding structure. Images PMID:7489720

  1. A nuclear export signal within the structural Gag protein is required for prototype foamy virus replication

    Directory of Open Access Journals (Sweden)

    Coiffic Audrey

    2011-01-01

    Full Text Available Abstract Background The Gag polyproteins play distinct roles during the replication cycle of retroviruses, hijacking many cellular machineries to fulfill them. In the case of the prototype foamy virus (PFV, Gag structural proteins undergo transient nuclear trafficking after their synthesis, returning back to the cytoplasm for capsid assembly and virus egress. The functional role of this nuclear stage as well as the molecular mechanism(s responsible for Gag nuclear export are not understood. Results We have identified a leptomycin B (LMB-sensitive nuclear export sequence (NES within the N-terminus of PFV Gag that is absolutely required for the completion of late stages of virus replication. Point mutations of conserved residues within this motif lead to nuclear redistribution of Gag, preventing subsequent virus egress. We have shown that a NES-defective PFV Gag acts as a dominant negative mutant by sequestrating its wild-type counterpart in the nucleus. Trans-complementation experiments with the heterologous NES of HIV-1 Rev allow the cytoplasmic redistribution of FV Gag, but fail to restore infectivity. Conclusions PFV Gag-Gag interactions are finely tuned in the cytoplasm to regulate their functions, capsid assembly, and virus release. In the nucleus, we have shown Gag-Gag interactions which could be involved in the nuclear export of Gag and viral RNA. We propose that nuclear export of unspliced and partially spliced PFV RNAs relies on two complementary mechanisms, which take place successively during the replication cycle.

  2. FGF21-Mediated Improvements in Glucose Clearance Require Uncoupling Protein 1

    Directory of Open Access Journals (Sweden)

    Michelle M. Kwon

    2015-11-01

    Full Text Available Fibroblast growth factor 21 (FGF21-mediated weight loss and improvements in glucose metabolism correlate with increased uncoupling protein 1 (Ucp1 levels in adipose tissues, suggesting that UCP1-dependent thermogenesis may drive FGF21 action. It was reported that FGF21 is equally effective at reducing body weight and improving glucose homeostasis without UCP1. We find while FGF21 can lower body weight in both wild-type and Ucp1 knockout mice, rapid clearance of glucose by FGF21 is defective in the absence of UCP1. Furthermore, in obese wild-type mice there is a fall in brown adipose tissue (BAT temperature during glucose excursion, and FGF21 improves glucose clearance while preventing the fall in BAT temperature. In Ucp1 knockout mice, the fall in BAT temperature during glucose excursion and FGF21-mediated changes in BAT temperature are lost. We conclude FGF21-mediated improvements in clearance of a glucose challenge require UCP1 and evoke UCP1-dependent thermogenesis as a method to increase glucose disposal.

  3. 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

  4. Protein level affects the relative lysine requirement of growing rainbow trout (O ncorhynchus mykiss) fry. Br. J. Nutr.

    OpenAIRE

    Bodin, Noelie

    2009-01-01

    The effect of two digestible protein levels (310 and 469 g/kg DM) on the relative lysine (Lys; g Lys/kg DM or g Lys/100 g protein) and the absolute Lys (g Lys intake/kg0.75 per d) requirements was studied in rainbow trout fry using a dose-response trial. At each protein level, sixteen isoenergetic (22-23 MJ digestible energy/kg DM) diets were tested, involving a full range (2-70 g/kg DM) of sixteen Lys levels. Each diet was given to one group of sixty rainbow trout fry (mean initial body weig...

  5. Protein level affects the relative lysine requirement of growing rainbow trout (Oncorhynchus mykiss) fry

    OpenAIRE

    Bodin, N.; Govaerts, B; Abboudi, T.; Detavernier, C.; De Saeger, S.; Larondelle, Y; Rollin, X.

    2009-01-01

    The effect of two digestible protein levels (310 and 469 g/kg DM) on the relative lysine (Lys; g Lys/kg DM or g Lys/100 g protein) and the absolute Lys (g Lys intake/kg0·75 per d) requirements was studied in rainbow trout fry using a dose–response trial. At each protein level, sixteen isoenergetic (22–23 MJ digestible energy/kg DM) diets were tested, involving a full range (2–70 g/kg DM) of sixteen Lys levels. Each diet was given to one group of sixty rainbow trout fry (mean initial body weig...

  6. 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. PMID:27478691

  7. 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.

  8. 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

    /day (CV=9.6%), respectively. The SID protein and lysine requirements were lowest at week 1, intermediate at week 2 and 4 and the highest at week 3 of lactation. The model is a valuable tool to develop new feeding strategies by taking into account the variable requirement between groups of sows and changes...... 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...

  9. 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.

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

    Science.gov (United States)

    Poortmans, J R; Carpentier, A; Pereira-Lancha, L O; Lancha Jr, A

    2012-10-01

    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 ((13)C-lysine, (15)N-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. PMID:22666780

  11. 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.

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

    International Nuclear Information System (INIS)

    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

  13. Genes required for formation of the apoMoFe protein of Klebsiella pneumoniae nitrogenase in Escherichia coli.

    Science.gov (United States)

    Harris, G S; White, T C; Flory, J E; Orme-Johnson, W H

    1990-09-15

    A binary plasmid system was used to produce nitrogenase components in Escherichia coli and subsequently to define a minimum set of nitrogen fixation (nif) genes required for the production of the iron-molybdenum cofactor (FeMoco) reactivatable apomolybdenum-iron (apoMoFe) protein of nitrogenase. The active MoFe protein is an alpha 2 beta 2 tetramer containing two FeMoco clusters and 4 Fe4S4 P centers (for review see, Orme-Johnson, W.H. (1985) Annu. Rev. Biophys. Biophys. Chem. 14, 419-459). The plasmid pVL15, carrying a tac-promoted nifA activator gene, was coharbored in E. coli with the plasmid pGH1 which contained nifHDKTYENXUSVWZMF' derived from the chromosome of the nitrogen fixing bacterium Klebsiella pneumoniae. The apoMoFe protein produced in E. coli by pGH1 + VL15 was identical to the apoprotein in derepressed cells of the nifB- mutant of K. pneumoniae (UN106) in its electrophoretic properties on nondenaturing polyacrylamide gels as well as in its ability to be activated by FeMoco. The constituent peptides migrated identically to those from purified MoFe protein during electrophoresis on denaturing gels. The concentrations of apoMoFe protein produced in nif-transformed strains of E. coli were greater than 50% of the levels of MoFe protein observed in derepressed wild-type K. pneumoniae. Systematic deletion of individual nif genes carried by pGH1 has established the requirements for the maximal production of the FeMoco-reactivatable apoMoFe protein to be the following gene products, NifHDKTYUSWZM+A. It appears that several of the genes (nifT, Y, U, W, and Z) are only required for maximal production of the apoMoFe protein, while others (nifH, D, K, and S) are absolutely required for synthesis of this protein in E. coli. One curious result is that the nifH gene product, the peptide of the Fe protein, but not active Fe protein itself, is required for formation of the apoMoFe protein. This suggests the possibility of a ternary complex of the NifH, D, and K

  14. 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

  15. Protein and amino acid requirements of warm-water fishes: a tool to efficient and low-cost fish feed production in Nigeria

    OpenAIRE

    Balogun, A.M.; Bello-Olusoji, O.; Fasakin, E.A.

    1993-01-01

    Numerous investigations have utilized various semi-purified and purified diets to estimate the protein and amino acid requirements of several temperate fishes. The vast literature on the protein and amino acid requirements of fishes has continued to omit that of the tropical warm water species. The net effect is that fish feed formulation in Nigeria have relied on the requirement for temperate species. This paper attempts to review the state of knowledge on the protein amino acid requirements...

  16. The Persistence of Hippocampal-Based Memory Requires Protein Synthesis Mediated by the Prion-like Protein CPEB3.

    Science.gov (United States)

    Fioriti, Luana; Myers, Cory; Huang, Yan-You; Li, Xiang; Stephan, Joseph S; Trifilieff, Pierre; Colnaghi, Luca; Kosmidis, Stylianos; Drisaldi, Bettina; Pavlopoulos, Elias; Kandel, Eric R

    2015-06-17

    Consolidation of long-term memories depends on de novo protein synthesis. Several translational regulators have been identified, and their contribution to the formation of memory has been assessed in the mouse hippocampus. None of them, however, has been implicated in the persistence of memory. Although persistence is a key feature of long-term memory, how this occurs, despite the rapid turnover of its molecular substrates, is poorly understood. Here we find that both memory storage and its underlying synaptic plasticity are mediated by the increase in level and in the aggregation of the prion-like translational regulator CPEB3 (cytoplasmic polyadenylation element-binding protein). Genetic ablation of CPEB3 impairs the maintenance of both hippocampal long-term potentiation and hippocampus-dependent spatial memory. We propose a model whereby persistence of long-term memory results from the assembly of CPEB3 into aggregates. These aggregates serve as functional prions and regulate local protein synthesis necessary for the maintenance of long-term memory. PMID:26074003

  17. The budding yeast protein Chl1p is required to preserve genome integrity upon DNA damage in S-phase

    OpenAIRE

    Laha, Suparna; Das, Shankar Prasad; Hajra, Sujata; Sau, Soumitra; Sinha, Pratima

    2006-01-01

    The budding yeast protein, Chl1p, is required for sister-chromatid cohesion, transcriptional silencing, rDNA recombination and aging. In this work, we show that Chl1p is also required for viability when DNA replication is stressed, either due to mutations or if cells are treated with genotoxic agents like methylmethane sulfonate (MMS) and ultraviolet (UV) rays. The chl1 mutation caused synthetic growth defects with mutations in DNA replication genes. At semi-permissive temperatures, the doubl...

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

    OpenAIRE

    Wilson Massamitu Furuya; Mariana Michelato; Ana Lúcia Salaro; Thais Pereira da Cruz; Valéria Rossetto Barriviera-Furuya

    2015-01-01

    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 a...

  19. The Development of Digestion and Absorption and the Energy Protein Lipid and Carbohydrate Requirements of the Premature Infants

    OpenAIRE

    Aydın, Ahmet; Çam, Halit; Fıçıcıoğlu, Can; Mıkla, Şerare

    1994-01-01

    Premature infants have high energy and nutrient requirements for their rapid growth The knowledge of the development of salivary gastric biliary and intestinal enzymes and factors involved in digestion and absorption plays important roles in the choice of the feeding constituents In this review we shall discuss the protein lipid and carbohydrate requirements of the premature infants and the importance of breast feeding Key words: Premature Infant Nutriton Gastrointestinal Functions

  20. The tomato xylem sap protein XSP10 is required for full susceptibility to Fusarium wilt disease

    OpenAIRE

    Krasikov, Vladimir; Dekker, Henk L.; Rep, Martijn; Takken, Frank L. W.

    2010-01-01

    XSP10 is an abundant 10 kDa protein found in the xylem sap of tomato. The protein displays structural similarity to plant lipid transfer proteins (LTPs). LTPs are involved in various physiological processes, including disease resistance, and some are able to bind and transfer diverse lipid molecules. XSP10 abundance in xylem sap declines upon infection with Fusarium oxysporum f. sp. lycopersici (Fol), implying involvement of XSP10 in the plant–pathogen interaction. Here, the biochemical chara...

  1. Proteolytic activation of human pancreatitis associated protein is required for peptidoglycan binding and bacterial aggregation

    OpenAIRE

    Medveczky, Péter; Szmola, Richárd; Sahin-Tóth, Miklós

    2009-01-01

    Pancreatitis associated protein (PAP) is a 16 kDa lectin-like protein, which becomes robustly upregulated in the pancreatic juice during acute pancreatitis. Trypsin cleaves the N terminus of PAP, which in turn forms insoluble fibrils. PAP and its paralog the pancreatic stone protein induce bacterial aggregation and, more recently, PAP was shown to bind to the peptidoglycan of Gram positive bacteria and exert a direct bactericidal effect. However, the role of N-terminal processing in the antib...

  2. A hydrophobic domain within the small capsid protein of Kaposi's sarcoma-associated herpesvirus is required for assembly.

    Science.gov (United States)

    Capuano, Christopher M; Grzesik, Peter; Kreitler, Dale; Pryce, Erin N; Desai, Keshal V; Coombs, Gavin; McCaffery, J Michael; Desai, Prashant J

    2014-08-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) capsids can be produced in insect cells using recombinant baculoviruses for protein expression. All six capsid proteins are required for this process to occur and, unlike for alphaherpesviruses, the small capsid protein (SCP) ORF65 is essential for this process. This protein decorates the capsid shell by virtue of its interaction with the capsomeres. In this study, we have explored the SCP interaction with the major capsid protein (MCP) using GFP fusions. The assembly site within the nucleus of infected cells was visualized by light microscopy using fluorescence produced by the SCP-GFP polypeptide, and the relocalization of the SCP to these sites was evident only when the MCP and the scaffold protein were also present - indicative of an interaction between these proteins that ensures delivery of the SCP to assembly sites. Biochemical assays demonstrated a physical interaction between the SCP and MCP, and also between this complex and the scaffold protein. Self-assembly of capsids with the SCP-GFP polypeptide was evident. Potentially, this result can be used to engineer fluorescent KSHV particles. A similar SCP-His6 polypeptide was used to purify capsids from infected cell lysates using immobilized affinity chromatography and to directly label this protein in capsids using chemically derivatized gold particles. Additional studies with SCP-GFP polypeptide truncation mutants identified a domain residing between aa 50 and 60 of ORF65 that was required for the relocalization of SCP-GFP to nuclear assembly sites. Substitution of residues in this region and specifically at residue 54 with a polar amino acid (lysine) disrupted or abolished this localization as well as capsid assembly, whereas substitution with non-polar residues did not affect the interaction. Thus, this study identified a small conserved hydrophobic domain that is important for the SCP-MCP interaction. PMID:24824860

  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. 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.

  5. TTC26/DYF13 is an intraflagellar transport protein required for transport of motility-related proteins into flagella

    OpenAIRE

    Ishikawa, Hiroaki; Ide, Takahiro; Yagi, Toshiki; Jiang, Xue; Hirono, Masafumi; Sasaki, Hiroyuki; Yanagisawa, Haruaki; Wemmer, Kimberly A; Stainier, Didier YR; Qin, Hongmin; Kamiya, Ritsu; Marshall, Wallace F.

    2014-01-01

    eLife digest Sperm cells have tails called flagella that propel them towards an egg. Other cells have similar, but shorter, structures called cilia that sway back and forth on their surface. In addition to sweeping dust and debris out of our lungs and airways, cilia have a number of other crucial roles during development. This means that faulty cilia can lead to serious birth defects, as well as diseases of the kidneys and respiratory system. Cilia and flagella are made from proteins that are...

  6. Ethylene-induced senescence-related gene expression requires protein synthesis

    International Nuclear Information System (INIS)

    We have investigated the effects of inhibiting protein synthesis on the ethylene-induced expression of 3 carnation senescence-related genes, pSR5, pSR8, and pSR12. Treatment of preclimacteric carnation petal discs with 1μg/ml of cycloheximide, a cytoplasmic protein synthesis inhibitor, for 3h inhibited protein synthesis by >80% as quantitated by the incorporation of [35S]methionine into protein. Pre-treatment of petal discs with cycloheximide prevented ethylene-induced SR transcript accumulation. Cycloheximide treatment of petal discs held in air did not result in increased levels of SR mRNA. These results indicate that ethylene does not interact with pre-formed factors but rather that the activation of SR gene expression by ethylene is mediated by labile protein factor(s) synthesized on cytoplasmic ribosomes. Experiments are currently underway to determine if cycloheximide exerts its effect at the transcriptional or post-transcriptional level

  7. 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

    activity was detected in the deeper layers of a P. aeruginosa biofilm using a PPA3309-gfp (green fluorescent protein gene) fusion and confocal laser-scanning microscopy. This is the first description of an Anr-dependent, anaerobically induced, and functional Usp-like protein in bacteria....... 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...... proteins (Usp). Long-term survival of a PA3309 knockout mutant by pyruvate fermentation was found drastically reduced. The oxygen-sensing regulator Anr controls expression of the PPA3309-lacZ reporter gene fusion after a shift to anaerobic conditions and further pyruvate fermentation. PA3309 expression...

  8. Structural features of the GroEL-GroES nano-cage required for rapid folding of encapsulated protein

    DEFF Research Database (Denmark)

    Tang, Sheila Tuyet; Chang, HC; Roeben, A; Wischnewski, D; Wischnewski, N; Kerner, MJ; Hartl, FU; Hayer-Hartl, M

    2006-01-01

    . Additionally, interactions with the C-terminal, mildly hydrophobic Gly-Gly-Met repeat sequences of GroEL protruding into the cavity, and repulsion effects from the negatively charged cavity wall were required for rapid folding of some proteins. We suggest that by combining these features, the chaperonin cage......GroEL and GroES form a chaperonin nano-cage for proteins up to similar to 60 kDa to fold in isolation. Here we explored the structural features of the chaperonin cage critical for rapid folding of encapsulated substrates. Modulating the volume of the GroEL central cavity affected folding speed in...... accordance with confinement theory. Small proteins (similar to 30 kDa) folded more rapidly as the size of the cage was gradually reduced to a point where restriction in space slowed folding dramatically. For larger proteins (similar to 40-50 kDa), either expanding or reducing cage volume decelerated folding...

  9. Requirements for non-food applications of pea proteins. A review.

    Science.gov (United States)

    De Graaf, L A; Harmsen, P F; Vereijken, J M; Mönikes, M

    2001-10-01

    So far, limited research is performed on technical applications of pea proteins and no applications have yet been reported. At ATO, three technical applications were investigated: surfactants, films, and microspheres as encapsulation matrices. Pea protein hydrolysates are surfactants with good emulsifying and foaming properties. By variation of enzyme type and degree of hydrolysis, the surfactant properties can be tailored toward specific applications. Pea protein films could be prepared by casting from dispersions at pH 7 and 10, and by compression moulding at 140 degrees C. Opposite to many other proteins, pea protein films combine strength (5-7.5 MPa) with high elongation at break (150%). A protein isolate derived from peas was applied as matrix material for the microencapsulation of beta-carotene, intended for cosmetic applications. Supercritical CO2 technology appeared to be a promising encapsulation technique for beta-carotene in porous pea protein microspheres. Advantages of this method are that no organic solvents are used, and that encapsulation is achieved under mild conditions, thereby preventing the sensitive beta-carotene from degradation. PMID:11712244

  10. The V Protein of Canine Distemper Virus Is Required for Virus Replication in Human Epithelial Cells

    OpenAIRE

    Noriyuki Otsuki; Yuichiro Nakatsu; Toru Kubota; Tsuyoshi Sekizuka; Fumio Seki; Kouji Sakai; Makoto Kuroda; Ryoji Yamaguchi; Makoto Takeda

    2013-01-01

    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 posit...

  11. An Accessory Protein Required for Anchoring and Assembly of Amyloid Fibers in B. subtilis Biofilms

    OpenAIRE

    Romero, Diego; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

    2011-01-01

    Cells within Bacillus subtilis biofilms are held in place by an extracellular matrix that contains cell-anchored amyloid fibers, composed of the amyloidogenic protein TasA. As biofilms age they disassemble because the cells release the amyloid fibers. This release appears to be the consequence of incorporation of D-tyrosine, D-leucine, D-tryptophan and D-methionine into the cell wall. Here, we characterize the in vivo roles of an accessory protein TapA (TasA anchoring/assembly protein; previo...

  12. Effect of genotype on the protein and energy requirements of growing cattle

    International Nuclear Information System (INIS)

    The effect of energy and protein intake on energy retention and lean production in cattle is discussed. The differences between genotypes in body gain, protein and fat retention are summarized. Interactions between some breeds and diets are described in order to assess the energy retention and daily gain efficiency. For a reliable comparison between breed, sex and feeding regimes it is necessary to refer to age at puberty. The possibility of extending the main energy and protein systems to other breeds and genotypes is also investigated. (author)

  13. Poly (ADP-Ribose) Polymerase 1 Is Required for Protein Localization to Cajal Body

    OpenAIRE

    Kotova, Elena; Jarnik, Michael; Tulin, Alexei V.

    2009-01-01

    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 processe...

  14. Activation of AMP-Activated Protein Kinase Is Required for Berberine-Induced Reduction of Atherosclerosis in Mice: The Role of Uncoupling Protein 2

    OpenAIRE

    Qilong Wang; Miao Zhang; Bin Liang; Najeeb Shirwany; Yi Zhu; Ming-Hui Zou

    2011-01-01

    AIMS: Berberine, a botanical alkaloid purified from Coptidis rhizoma, is reported to activate the AMP-activated protein kinase (AMPK). Whether AMPK is required for the protective effects of berberine in cardiovascular diseases remains unknown. This study was designed to determine whether AMPK is required for berberine-induced reduction of oxidative stress and atherosclerosis in vivo. METHODS: ApoE (ApoE⁻/⁻) mice and ApoE⁻/⁻/AMPK alpha 2⁻/⁻ mice that were fed Western diets were treated with be...

  15. Mechanisms that uncouple growth and differentiation in myeloid leukemia cells: restoration of requirement for normal growth-inducing protein without restoring induction of differentiation-inducing protein.

    OpenAIRE

    Lotem, J; Sachs, L

    1982-01-01

    There are different macrophage- and granulocyte-inducing (MGI) proteins. Normal myeloid precursors are induced to multiply by one form (MGI-1) and to differentiate by another form (MGI-2). There are clones of myeloid leukemia cells that no longer require MGI-1 for growth but can still be induced to differentiate by MGI-2. After induction of differentiation in these leukemia cells by adding MCI-2 or inducing endogenous production of MGI-2 by lipopolysaccharide, the differentiating leukemia cel...

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

    Directory of Open Access Journals (Sweden)

    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.

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

    DEFF Research Database (Denmark)

    Schreiber, K; Boes, N; Escbach, M; Jaensch, L; Wehland, J; Bjarnsholt, Thomas; Givskov, Michael Christian; Hentzer, M; Schobert, M

    2006-01-01

    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...... proteins (Usp). Long-term survival of a PA3309 knockout mutant by pyruvate fermentation was found drastically reduced. The oxygen-sensing regulator Anr controls expression of the PPA3309-lacZ reporter gene fusion after a shift to anaerobic conditions and further pyruvate fermentation. PA3309 expression was...... also found induced during the anaerobic and aerobic stationary phases. This aerobic stationary-phase induction is independent of the regulatory proteins Anr, RpoS, RelA, GacA, RhlR, and LasR, indicating a currently unknown mechanism of stationary-phase-dependent gene activation. PA3309 promoter...

  18. Protein stabilization explains the gag requirement for transformation of lymphoid cells by Abelson murine leukemia virus

    OpenAIRE

    Prywes, R; Hoag, J; Rosenberg, N; Baltimore, D

    1985-01-01

    The single protein encoded by Abelson murine leukemia virus is a fusion of sequence from the retroviral gag genes with the v-abl sequence. Deletion of most of the gag region from the transforming protein results in a virus capable of transforming fibroblasts but no longer capable of transforming lymphoid cells. Smaller deletions in gag reveal that p15 gag sequences are responsible for this effect, whereas deletion of p12 sequences had no effect on lymphoid transformation. In transformed fibro...

  19. Myocyte remodelling in response to hypertrophic stimuli requires nucleocytoplasmic shuttling of muscle LIM protein

    OpenAIRE

    Boateng, Samuel Y; Senyo, Samuel E.; Qi, Lixin; Goldspink, Paul H.; Russell, Brenda

    2009-01-01

    CSRP3 or Muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein and a mechanosensor in cardiac myocytes. MLP regulation and function was studied in cultured neonatal rat myocytes treated with pharmacological or mechanical stimuli. Either verapamil or BDM decreased nuclear MLP while phenylephrine and cyclic strain increased it. These results suggest that myocyte contractility regulates MLP subcellular localization. When RNA polymerase II was inhibited with α-amanitin, nuclear MLP wa...

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

    International Nuclear Information System (INIS)

    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

  1. 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.

  2. The J Domain of Simian Virus 40 Large T Antigen Is Required To Functionally Inactivate RB Family Proteins

    OpenAIRE

    Zalvide, Juan; Stubdal, Hilde; DeCaprio, James A.

    1998-01-01

    Transformation by simian virus 40 large T antigen (TAg) is dependent on the inactivation of cellular tumor suppressors. Transformation minimally requires the following three domains: (i) a C-terminal domain that mediates binding to p53; (ii) the LXCXE domain (residues 103 to 107), necessary for binding to the retinoblastoma tumor suppressor protein, pRB, and the related p107 and p130; and (iii) an N-terminal domain that is homologous to the J domain of DnaJ molecular chaperone proteins. We ha...

  3. Fusarium verticillioides SGE1 is required for full virulence and regulates expression of protein effector and secondary metabolite biosynthetic genes.

    Science.gov (United States)

    Brown, Daren W; Busman, Mark; Proctor, Robert H

    2014-08-01

    The transition from one lifestyle to another in some fungi is initiated by a single orthologous gene, SGE1, that regulates markedly different genes in different fungi. Despite these differences, many of the regulated genes encode effector proteins or proteins involved in the synthesis of secondary metabolites (SM), both of which can contribute to pathogenicity. Fusarium verticillioides is both an endophyte and a pathogen of maize and can grow as a saprophyte on dead plant material. During growth on live maize plants, the fungus can synthesize a number of toxic SM, including fumonisins, fusarins, and fusaric acid, that can contaminate kernels and kernel-based food and feed. In this study, the role of F. verticillioides SGE1 in pathogenicity and secondary metabolism was examined by gene deletion analysis and transcriptomics. SGE1 is not required for vegetative growth or conidiation but is required for wild-type pathogenicity and affects synthesis of multiple SM, including fumonisins and fusarins. Induced expression of SGE1 enhanced or reduced expression of hundreds of genes, including numerous putative effector genes that could contribute to growth in planta; genes encoding cell surface proteins; gene clusters required for synthesis of fusarins, bikaverin, and an unknown metabolite; as well as the gene encoding the fumonisin cluster transcriptional activator. Together, our results indicate that SGE1 has a role in global regulation of transcription in F. verticillioides that impacts but is not absolutely required for secondary metabolism and pathogenicity on maize. PMID:24742071

  4. 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.

  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. Utilization of alimentary protein and amino acids in satisfying the nitrogen requirements of monogastric mammals

    International Nuclear Information System (INIS)

    The nitrogenous matter in the food of monogastric animals consists mainly of proteins, which are rapidly hydrolized in the intestinal tract when they have left the gastric reservoir. The digestive tube has several roles: it provides for hydrolysis of the food proteins and for a supply of endogenous nitrogen; it enables a certain digestive function to be performed by the intestinal flora and permits the transport of amino acids into the blood, selecting those which are needed for protein synthesis. The digestion products appear mainly in the form of free amino acids in the portal blood. A large proportion of these amino acids is taken up by the liver, so that intense protein synthesis takes place in the latter, coupled with a decrease in catabolism leading to a rhythmic increase in the liver content of proteins and RNA. The labile proteins retained are mainly enzymes, which catabolize the amino acids, and the liver is the site of the catabolism of most of the excess amino acids except those with chain branching. Alimentary deficiencies do not markedly reduce protein synthesis in this organ, since the rate of re-utilization of the amino acids is increased and the liver thus plays a regulatory role. The utilization of amino acids in muscle also follows a certain rhythm, partly connected with feeding, and under hormonal control. The muscle is the seat of catabolism of a large part of the branched chain amino acids, and like the liver it contributes to the energy utilization of amino acids. The rate of utilization of certain essential amino acids can be measured by metabolic criteria, including determination of blood and muscle concentrations and excretion of 14CO2 labels in the exhaled air or of 35S labels in urine. (author)

  7. 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.; Nielsen, H.B.; Harris, C.A.; Beale, M.H.; Andersen, M.; Mant, A.; Scheller, H.; Camara, B.; Mattsson, O.; Mundy, J.

    2004-01-01

    The stable, recessive Arabidopsis variegated 3 (var3) mutant exhibits a variegated phenotype due to somatic areas lacking or containing developmentally retarded chloroplasts and greatly reduced numbers of palisade cells. The VAR3 gene, isolated by transposon tagging, encodes the 85.9 kDa VAR3 pro...... pigment profiles are qualitatively similar in wild type and var3, although var3 accumulates lower levels of chlorophylls and carotenoids. These results indicate that VAR3 is a part of a protein complex required for normal chloroplast and palisade cell development....... 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 that...

  8. 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

    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...... 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....... 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....

  9. 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.

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

    International Nuclear Information System (INIS)

    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 R49 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 Kd 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 R49 that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding

  11. The adenovirus tripartite leader may eliminate the requirement for cap-binding protein complex during translation initiation.

    OpenAIRE

    Dolph, P J; Racaniello, V; Villamarin, A; Palladino, F.; Schneider, R J

    1988-01-01

    The adenovirus tripartite leader is a 200-nucleotide 5' noncoding region that is found on all late viral mRNAs. This segment is required for preferential translation of viral mRNAs at late times during infection. Most tripartite leader-containing mRNAs appear to exhibit little if any requirement for intact cap-binding protein complex, a property previously established only for uncapped poliovirus mRNAs and capped mRNAs with minimal secondary structure. The tripartite leader also permits the t...

  12. Smad2/3 Proteins Are Required for Immobilization-induced Skeletal Muscle Atrophy.

    Science.gov (United States)

    Tando, Toshimi; Hirayama, Akiyoshi; Furukawa, Mitsuru; Sato, Yuiko; Kobayashi, Tami; Funayama, Atsushi; Kanaji, Arihiko; Hao, Wu; Watanabe, Ryuichi; Morita, Mayu; Oike, Takatsugu; Miyamoto, Kana; Soga, Tomoyoshi; Nomura, Masatoshi; Yoshimura, Akihiko; Tomita, Masaru; Matsumoto, Morio; Nakamura, Masaya; Toyama, Yoshiaki; Miyamoto, Takeshi

    2016-06-01

    Skeletal muscle atrophy promotes muscle weakness, limiting activities of daily living. However, mechanisms underlying atrophy remain unclear. Here, we show that skeletal muscle immobilization elevates Smad2/3 protein but not mRNA levels in muscle, promoting atrophy. Furthermore, we demonstrate that myostatin, which negatively regulates muscle hypertrophy, is dispensable for denervation-induced muscle atrophy and Smad2/3 protein accumulation. Moreover, muscle-specific Smad2/3-deficient mice exhibited significant resistance to denervation-induced muscle atrophy. In addition, expression of the atrogenes Atrogin-1 and MuRF1, which underlie muscle atrophy, did not increase in muscles of Smad2/3-deficient mice following denervation. We also demonstrate that serum starvation promotes Smad2/3 protein accumulation in C2C12 myogenic cells, an in vitro muscle atrophy model, an effect inhibited by IGF1 treatment. In vivo, we observed IGF1 receptor deactivation in immobilized muscle, even in the presence of normal levels of circulating IGF1. Denervation-induced muscle atrophy was accompanied by reduced glucose intake and elevated levels of branched-chain amino acids, effects that were Smad2/3-dependent. Thus, muscle immobilization attenuates IGF1 signals at the receptor rather than the ligand level, leading to Smad2/3 protein accumulation, muscle atrophy, and accompanying metabolic changes. PMID:27129272

  13. Comments on the minimum number of observations required in the study of protein turnover

    International Nuclear Information System (INIS)

    This paper describes a simple two-compartment model for the analysis of clinical data on protein turnover. The first compartment of the model consists of measuring the catabolic rate by either the clearance method or by using a whole body counter. The second compartment of the model uses the extravascular pool and synthesis rate. A discussion of the model follows the explanation

  14. An accessory protein required for anchoring and assembly of amyloid fibres in B. subtilis biofilms.

    Science.gov (United States)

    Romero, Diego; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

    2011-06-01

    Cells within Bacillus subtilis biofilms are held in place by an extracellular matrix that contains cell-anchored amyloid fibres, composed of the amyloidogenic protein TasA. As biofilms age they disassemble because the cells release the amyloid fibres. This release appears to be the consequence of incorporation of D-tyrosine, D-leucine, D-tryptophan and D-methionine into the cell wall. Here, we characterize the in vivo roles of an accessory protein TapA (TasA anchoring/assembly protein; previously YqxM) that serves both to anchor the fibres to the cell wall and to assemble TasA into fibres. TapA is found in discrete foci in the cell envelope and these foci disappear when cells are treated with a mixture of D-amino acids. Purified cell wall sacculi retain a functional form of this anchoring protein such that purified fibres can be anchored to the sacculi in vitro. In addition, we show that TapA is essential for the proper assembly of the fibres. Its absence results in a dramatic reduction in TasA levels and what little TasA is left produces only thin fibres that are not anchored to the cell. PMID:21477127

  15. 75 FR 29431 - Coat Protein of Plum Pox Virus; Exemption from the Requirement of a Tolerance

    Science.gov (United States)

    2010-05-26

    ... Findings In the Federal Register of November 14, 2008 (73 FR 67512) (FRL- 8388-3), EPA issued a notice... fact that plum pox virus coat protein has been in the human diet without adverse effects, and the... the human diet and exist in the human intestine without negative effects was reviewed by the...

  16. The dyf-3 gene encodes a novel protein required for sensory cilium formation in Caenorhabditis elegans.

    Science.gov (United States)

    Murayama, Takashi; Toh, Yoshihiro; Ohshima, Yasumi; Koga, Makoto

    2005-02-25

    Ciliated neurons in animals are important for the reception of environmental stimuli. To understand the mechanism of cilium morphogenesis in Caenorhabditis elegans, we analyzed dyf-3 mutants that are defective in uptake of a fluorescent dye and abnormal in sensory cilium structure. Expression of green fluorescent protein in sensory neurons of a dyf-3 mutant revealed that the mutant has stunted cilia and abnormal posterior projections in some sensory neurons. The dyf-3 gene encodes three proteins with different N-terminals. The largest DYF-3 protein has 404 amino acid residues that are 38% identical with those of a predicted human protein of unknown function. Expression of a functional dyf-3Colon, two colonsgfp fusion gene is detected in 26 chemosensory neurons, including six IL2 neurons, eight pairs of amphid neurons (ASE, ADF, ASG, ASH, ASI, ASJ, ASK and ADL) and two pairs of phasmid neurons (PHA and PHB). Expression of a dyf-3 cDNA in specific neurons of dyf-3 animals indicated that dyf-3 acts cell-autonomously for fluorescent dye uptake. Reduction of dyf-3Colon, two colonsgfp expression in a daf-19 mutant suggests that dyf-3 expression is regulated by DAF-19 transcription factor, and DYF-3 may be involved in the intraflagellar transport system. PMID:15713455

  17. 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...

  18. THE TSC COMPLEX IS REQUIRED FOR THE BENEFITS OF DIETARY PROTEIN RESTRICTION ON STRESS RESISTANCE IN VIVO

    OpenAIRE

    Eylul Harputlugil; Christopher Hine; Dorathy Vargas; Lauren Robertson; Brendan D. Manning; James R. Mitchell

    2014-01-01

    Protein restriction (PR) is important for the benefits of dietary restriction on longevity and stress resistance, but relevant nutrient sensors and downstream effectors in mammals remain poorly defined. We used PR-mediated protection from hepatic ischemia reperfusion injury to probe genetic requirements for the evolutionarily conserved nutrient sensors GCN2 and mTORC1 in stress resistance. One week of PR reduced free amino acids and circulating growth factors, activating GCN2 and mTORC1 repre...

  19. Characterization of three protein components required for functional reconstitution of the epoxide carboxylase multienzyme complex from Xanthobacter strain Py2.

    OpenAIRE

    Allen, J. R.; Ensign, S A

    1997-01-01

    Epoxide carboxylase from Xanthobacter strain Py2 catalyzes the reductant- and NAD+-dependent carboxylation of aliphatic epoxides to beta-keto acids. Epoxide carboxylase from Xanthobacter strain Py2 has been resolved from cell extracts by anion-exchange chromatography into three protein components, designated I, II, and III, that are obligately required for functional reconstitution of epoxide carboxylase activity. Component II has been purified to homogeneity on the basis of its ability to co...

  20. Molecular and functional analyses of a maize autoactive NB-LRR protein identify precise structural requirements for activity.

    Directory of Open Access Journals (Sweden)

    Guan-Feng Wang

    2015-02-01

    Full Text Available Plant disease resistance is often mediated by nucleotide binding-leucine rich repeat (NLR proteins which remain auto-inhibited until recognition of specific pathogen-derived molecules causes their activation, triggering a rapid, localized cell death called a hypersensitive response (HR. Three domains are recognized in one of the major classes of NLR proteins: a coiled-coil (CC, a nucleotide binding (NB-ARC and a leucine rich repeat (LRR domains. The maize NLR gene Rp1-D21 derives from an intergenic recombination event between two NLR genes, Rp1-D and Rp1-dp2 and confers an autoactive HR. We report systematic structural and functional analyses of Rp1 proteins in maize and N. benthamiana to characterize the molecular mechanism of NLR activation/auto-inhibition. We derive a model comprising the following three main features: Rp1 proteins appear to self-associate to become competent for activity. The CC domain is signaling-competent and is sufficient to induce HR. This can be suppressed by the NB-ARC domain through direct interaction. In autoactive proteins, the interaction of the LRR domain with the NB-ARC domain causes de-repression and thus disrupts the inhibition of HR. Further, we identify specific amino acids and combinations thereof that are important for the auto-inhibition/activity of Rp1 proteins. We also provide evidence for the function of MHD2, a previously uncharacterized, though widely conserved NLR motif. This work reports several novel insights into the precise structural requirement for NLR function and informs efforts towards utilizing these proteins for engineering disease resistance.

  1. 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...

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

    Science.gov (United States)

    Leite, Sérgio Carvalho; Sampaio, Paula; Sousa, Vera Filipe; Nogueira-Rodrigues, Joana; Pinto-Costa, Rita; Peters, Luanne Laurel; Brites, Pedro; Sousa, Mónica Mendes

    2016-04-19

    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. PMID:27068466

  3. Poly(A)-binding proteins are required for diverse biological processes in metazoans.

    Science.gov (United States)

    Smith, Richard W P; Blee, Tajekesa K P; Gray, Nicola K

    2014-08-01

    PABPs [poly(A)-binding proteins] bind to the poly(A) tail of eukaryotic mRNAs and are conserved in species ranging from yeast to human. The prototypical cytoplasmic member, PABP1, is a multifunctional RNA-binding protein with roles in global and mRNA-specific translation and stability, consistent with a function as a central regulator of mRNA fate in the cytoplasm. More limited insight into the molecular functions of other family members is available. However, the consequences of disrupting PABP function in whole organisms is less clear, particularly in vertebrates, and even more so in mammals. In the present review, we discuss current and emerging knowledge with respect to the functions of PABP family members in whole animal studies which, although incomplete, already underlines their biological importance and highlights the need for further intensive research in this area. PMID:25110030

  4. 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...... with age. Morphology and development of skin and appendages are normal in ACBP(-/-) mice; however, the stratum corneum display altered biophysical properties with reduced proton activity and decreased water content. Mass spectrometry analyses of lipids from epidermis and stratum corneum of ACBP...... VLC-FFAs via complex phospholipids in the lamellar bodies. Importantly, we show that ACBP(-/-) mice display a ∼50% increased transepidermal water loss compared with ACBP(+/+) mice. Furthermore, skin and fur sebum monoalkyl diacylglycerol (MADAG) levels are significantly increased, suggesting that ACBP...

  5. Mitogen-activated protein kinase phosphatase is required for genotoxic stress relief in Arabidopsis

    OpenAIRE

    Ulm, Roman; Revenkova, Ekaterina; Di Sansebastiano, Gian-Pietro; Bechtold, Nicole; Paszkowski, Jerzy

    2001-01-01

    Genotoxic stress activates complex cellular responses allowing for the repair of DNA damage and proper cell recovery. Although plants are exposed constantly to increasing solar UV irradiation, the signaling cascades activated by genotoxic environments are largely unknown. We have identified an Arabidopsis mutant (mkp1) hypersensitive to genotoxic stress treatments (UV-C and methyl methanesulphonate) due to disruption of a gene that encodes an Arabidopsis homolog of mitogen-activated protein k...

  6. NOA1 is an essential GTPase required for mitochondrial protein synthesis

    OpenAIRE

    Kolanczyk, M; Pech, M.; Zemojtel, T.; Yamamoto, H.; Mikula, I.; Calvaruso, M.A.; Brand, M.A.M. van den; R. Richter; Fischer, B.; Ritz, A.; Kossler, N.; Thurisch, B; Spoerle, R.; Smeitink, J A; Kornak, U.

    2011-01-01

    Nitric oxide associated-1 (NOA1) is an evolutionarily conserved guanosine triphosphate (GTP) binding protein that localizes predominantly to mitochondria in mammalian cells. On the basis of bioinformatic analysis, we predicted its possible involvement in ribosomal biogenesis, although this had not been supported by any experimental evidence. Here we determine NOA1 function through generation of knockout mice and in vitro assays. NOA1-deficient mice exhibit midgestation lethality associated wi...

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

    OpenAIRE

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

    2009-01-01

    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 (TERT), the telomerase RNA (TERC), and dyskerin. Here, we identify a holoenzyme subunit, TCAB1 (telomerase Cajal body protein1), uniquely enriched in Cajal bodies, nuclear sites of RNP processing important for telomerase function. TCAB1 associates with...

  8. A chromodomain protein, Chp1, is required for the establishment of heterochromatin in fission yeast

    OpenAIRE

    Sadaie, Mahito; Iida, Tetsushi; URANO, TAKESHI; Nakayama, Jun-ichi

    2004-01-01

    The chromodomain is a conserved motif that functions in the epigenetic control of gene expression. Here, we report the functional characterization of a chromodomain protein, Chp1, in the heterochromatin assembly in fission yeast. We show that Chp1 is a structural component of three heterochromatic regions—centromeres, the mating-type region, and telomeres—and that its localization in these regions is dependent on the histone methyltransferase Clr4. Although deletion of the chp1+ gene causes c...

  9. 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.

  10. Activation of resting human T cells requires prolonged stimulation of protein kinase C.

    OpenAIRE

    Berry, N; Ase, K; Kishimoto, A.; Nishizuka, Y

    1990-01-01

    Purified resting human T cells can be induced to express the alpha subunit of the interleukin 2 receptor and to proliferate by treatment with 12-O-tetradecanoylphorbol-13-acetate plus ionomycin but not with 1,2-dioctanoylglycerol plus ionomycin. Determination of the translocation of protein kinase C showed that 12-O-tetradecanoylphorbol-13-acetate plus ionomycin caused a prolonged membrane association of the enzyme for more than 4 hr, whereas 1,2-dioctanoylglycerol plus ionomycin induced a tr...

  11. DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes

    OpenAIRE

    Bailey, Susan M.; Meyne, Julianne; Chen, David J.; Kurimasa, Akihiro; Li, Gloria C.; Lehnert, Bruce E.; Goodwin, Edwin H.

    1999-01-01

    Recent findings intriguingly place DNA double-strand break repair proteins at chromosome ends in yeast, where they help maintain normal telomere length and structure. In the present study, an essential telomere function, the ability to cap and thereby protect chromosomes from end-to-end fusions, was assessed in repair-deficient mouse cell lines. By using fluorescence in situ hybridization with a probe to telomeric DNA, spontaneously occurring chromosome aberrations were examined for telomere ...

  12. ATM protein purified from vaccinia virus expression system: DNA binding requirements for kinase activation

    International Nuclear Information System (INIS)

    The ataxia-telangiectasia mutated (ATM) gene product plays a role in responding to double stand DNA breaks. Some biochemical studies of ATM function have been hampered by lack of an efficient expression system and abundant purified ATM protein. We report the construction of a vaccinia virus expressing ATM, vWR-ATM, which was used to produce large amounts of functional FLAG-tagged ATM protein (FLAG-ATM) in HeLa cells. Kinase activity of the purified FLAG-ATM was dependent on manganese and inhibited with wortmannin. Using the FLAG-ATM recombinant protein, GST-p53 serine 15 phosphorylation increased in the presence of damaged DNA. PHAS-1 phosphorylation was found to be DNA independent. Purified FLAG-ATM was recovered in the autophosphorylated form, as demonstrated by phosphorylation of ATM serine 1981. As shown by atomic force microscopy, FLAG-ATM bound to linear DNA both at broken ends and in mid-strands. Vaccinia virus is the most efficient ATM expression system described to date

  13. 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.

  14. Somitogenesis clock-wave initiation requires differential decay and multiple binding sites for clock protein.

    Directory of Open Access Journals (Sweden)

    Mark Campanelli

    2010-04-01

    Full Text Available Somitogenesis is a process common to all vertebrate embryos in which repeated blocks of cells arise from the presomitic mesoderm (PSM to lay a foundational pattern for trunk and tail development. Somites form in the wake of passing waves of periodic gene expression that originate in the tailbud and sweep posteriorly across the PSM. Previous work has suggested that the waves result from a spatiotemporally graded control protein that affects the oscillation rate of clock-gene expression. With a minimally constructed mathematical model, we study the contribution of two control mechanisms to the initial formation of this gene-expression wave. We test four biologically motivated model scenarios with either one or two clock protein transcription binding sites, and with or without differential decay rates for clock protein monomers and dimers. We examine the sensitivity of wave formation with respect to multiple model parameters and robustness to heterogeneity in cell population. We find that only a model with both multiple binding sites and differential decay rates is able to reproduce experimentally observed waveforms. Our results show that the experimentally observed characteristics of somitogenesis wave initiation constrain the underlying genetic control mechanisms.

  15. Dietary requirements of synthesizable amino acids by animals: a paradigm shift in protein nutrition.

    Science.gov (United States)

    Wu, Guoyao

    2014-01-01

    Amino acids are building blocks for proteins in all animals. Based on growth or nitrogen balance, amino acids were traditionally classified as nutritionally essential or nonessential for mammals, birds and fish. It was assumed that all the "nutritionally nonessential amino acids (NEAA)" were synthesized sufficiently in the body to meet the needs for maximal growth and optimal health. However, careful analysis of the scientific literature reveals that over the past century there has not been compelling experimental evidence to support this assumption. NEAA (e.g., glutamine, glutamate, proline, glycine and arginine) play important roles in regulating gene expression, cell signaling, antioxidative responses, fertility, neurotransmission, and immunity. Additionally, glutamate, glutamine and aspartate are major metabolic fuels for the small intestine to maintain its digestive function and to protect the integrity of the intestinal mucosa. Thus, diets for animals must contain all NEAA to optimize their survival, growth, development, reproduction, and health. Furthermore, NEAA should be taken into consideration in revising the "ideal protein" concept that is currently used to formulate swine and poultry diets. Adequate provision of all amino acids (including NEAA) in diets enhances the efficiency of animal production. In this regard, amino acids should not be classified as nutritionally essential or nonessential in animal or human nutrition. The new Texas A&M University's optimal ratios of dietary amino acids for swine and chickens are expected to beneficially reduce dietary protein content and improve the efficiency of their nutrient utilization, growth, and production performance. PMID:24999386

  16. 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.

  17. 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…

  18. Protein import into mitochondria: the requirement for external ATP is precursor-specific whereas intramitochondrial ATP is universally needed for translocation into the matrix.

    OpenAIRE

    Wachter, C.; Schatz, G.; Glick, B S

    1994-01-01

    ATP is needed for the import of precursor proteins into mitochondria. However, the role of ATP and its site of action have been unclear. We have now investigated the ATP requirements for protein import into the mitochondrial matrix. These experiments employed an in vitro system that allowed ATP levels to be manipulated both inside and outside the mitochondrial inner membrane. Our results indicate that there are two distinct ATP requirements for mitochondrial protein import. ATP in the matrix ...

  19. Grsf1-induced translation of the SNARE protein Use1 is required for expansion of the erythroid compartment.

    Directory of Open Access Journals (Sweden)

    Andrzej Nieradka

    Full Text Available Induction of cell proliferation requires a concomitant increase in the synthesis of glycosylated lipids and membrane proteins, which is dependent on ER-Golgi protein transport by CopII-coated vesicles. In this process, retrograde transport of ER resident proteins from the Golgi is crucial to maintain ER integrity, and allows for anterograde transport to continue. We previously showed that expression of the CopI specific SNARE protein Use1 (Unusual SNARE in the ER 1 is tightly regulated by eIF4E-dependent translation initiation of Use1 mRNA. Here we investigate the mechanism that controls Use1 mRNA translation. The 5'UTR of mouse Use1 contains a 156 nt alternatively spliced intron. The non-spliced form is the predominantly translated mRNA. The alternatively spliced sequence contains G-repeats that bind the RNA-binding protein G-rich sequence binding factor 1 (Grsf1 in RNA band shift assays. The presence of these G-repeats rendered translation of reporter constructs dependent on the Grsf1 concentration. Down regulation of either Grsf1 or Use1 abrogated expansion of erythroblasts. The 5'UTR of human Use1 lacks the splice donor site, but contains an additional upstream open reading frame in close proximity of the translation start site. Similar to mouse Use1, also the human 5'UTR contains G-repeats in front of the start codon. In conclusion, Grsf1 controls translation of the SNARE protein Use1, possibly by positioning the 40S ribosomal subunit and associated translation factors in front of the translation start site.

  20. 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...... 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...

  1. Efficient excision of phage lambda from the Escherichia coli chromosome requires the Fis protein.

    OpenAIRE

    Ball, C A; Johnson, R C

    1991-01-01

    The Escherichia coli protein Fis has been shown to bind a single site in the recombination region of phage lambda and to stimulate excisive recombination in vitro (J. F. Thompson, L. Moitoso de Vargas, C. Koch, R. Kahmann, and A. Landy, Cell 50:901-908, 1987). We demonstrate that mutant strains deficient in fis expression show dramatically reduced rates of lambda excision in vivo. Phage yields after induction of a stable lysogen are reduced more than 200-fold in fis cells. The defect observed...

  2. Different Training Procedures Recruit Either One or Two Critical Periods for Contextual Memory Consolidation, Each of Which Requires Protein Synthesis and PKA

    OpenAIRE

    Bourtchouladze, Roussoudan; Abel, Ted; Berman, Nathaniel; Gordon, Rachael; Lapidus, Kyle; Kandel, Eric R.

    1998-01-01

    We have used a combined genetic and pharmacological approach to define the time course of the requirement for protein kinase A (PKA) and protein synthesis in long-term memory for contextual fear conditioning in mice. The time course of amnesia in transgenic mice that express R(AB) and have genetically reduced PKA activity in the hippocampus parallels that observed both in mice treated with inhibitors of PKA and mice treated with inhibitors of protein synthesis. This PKA- and protein synthesis...

  3. 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.

  4. RepA and DnaA proteins are required for initiation of R1 plasmid replication in vitro and interact with the oriR sequence.

    OpenAIRE

    Masai, H.; Arai, K.

    1987-01-01

    RepA, an initiation protein of R1 plasmid replication, was purified from an Escherichia coli strain overproducing the protein. The purified RepA protein specifically initiated replication in vitro of plasmid DNA bearing the replication origin of R1 plasmid (oriR). The replication, strictly dependent on added RepA protein, was independent of host RNA polymerase but required other host replication functions (DnaB and DnaC proteins, the single-stranded-DNA-binding protein SSB, and DNA gyrase). T...

  5. The extracellular matrix protein artichoke is required for integrity of ciliated mechanosensory and chemosensory organs in Drosophila embryos.

    Science.gov (United States)

    Andrés, Marta; Turiégano, Enrique; Göpfert, Martin C; Canal, Inmaculada; Torroja, Laura

    2014-04-01

    Sensory cilia are often encapsulated by an extracellular matrix (ECM). In Caenorhabditis elegans, Drosophila melanogaster, and vertebrates, this ECM is thought to be directly involved in ciliary mechanosensing by coupling external forces to the ciliary membrane. Drosophila mechano- and chemosensory cilia are both associated with an ECM, indicating that the ECM may have additional roles that go beyond mechanosensory cilium function. Here, we identify Artichoke (ATK), an evolutionarily conserved leucine-rich repeat ECM protein that is required for normal morphogenesis and function of ciliated sensilla in Drosophila. atk is transiently expressed in accessory cells in all ciliated sensory organs during their late embryonic development. Antibody stainings show ATK protein in the ECM that surrounds sensory cilia. Loss of ATK protein in atk null mutants leads to cilium deformation and disorientation in chordotonal organs, apparently without uncoupling the cilia from the ECM, and consequently to locomotion defects. Moreover, impaired chemotaxis in atk mutant larvae suggests that, based on ATK protein localization, the ECM is also crucial for the correct assembly of chemosensory receptors. In addition to defining a novel ECM component, our findings show the importance of ECM integrity for the proper morphogenesis of ciliated organs in different sensory modalities. PMID:24496014

  6. DEADSouth protein localizes to germ plasm and is required for the development of primordial germ cells in Xenopus laevis

    Directory of Open Access Journals (Sweden)

    Takeshi Yamaguchi

    2012-11-01

    DEADSouth mRNA is a component of germ plasm in Xenopus laevis and encodes a DDX25 DEAD-box RNA helicase. To determine the intracellular localization of DEADSouth protein, we injected mRNA encoding DEADSouth tagged with mCherry fluorescent protein into fertilized eggs from transgenic Xenopus expressing EGFP fused with a mitochondrial targeting signal. The DEADSouth-mCherry fusion protein was localized to the germ plasm, a mitochondria-rich region in primordial germ cells (PGCs. DEADSouth overexpression resulted in a reduction of PGC numbers after stage 20. Conversely, DEADSouth knockdown using an antisense locked nucleic acid gapmer inhibited movement of the germ plasm from the cortex to the perinuclear region, resulting in inhibition of PGC division at stage 12 and a decrease in PGC numbers at later stages. The knockdown phenotype was rescued by intact DEADSouth mRNA, but not mutant mRNA encoding inactive DEADSouth helicase. Surprisingly, it was also rescued by mouse vasa homolog and Xenopus vasa-like gene 1 mRNAs that encode DDX4 RNA helicases. The rescue was dependent on the 3′ untranslated region (3′UTR of DEADSouth mRNA, which was used for PGC-specific expression. The 3′UTR contributed to localization of the injected mRNA to the germ plasm, resulting in effective localization of DEADSouth protein. These results demonstrate that localization of DEADSouth helicase to the germ plasm is required for proper PGC development in Xenopus laevis.

  7. 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.

  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. The Rho-GTPase binding protein IQGAP2 is required for the glomerular filtration barrier.

    Science.gov (United States)

    Sugano, Yuya; Lindenmeyer, Maja T; Auberger, Ines; Ziegler, Urs; Segerer, Stephan; Cohen, Clemens D; Neuhauss, Stephan C F; Loffing, Johannes

    2015-11-01

    Podocyte dysfunction impairs the size selectivity of the glomerular filter, leading to proteinuria, hypoalbuminuria, and edema, clinically defined as nephrotic syndrome. Hereditary forms of nephrotic syndrome are linked to mutations in podocyte-specific genes. To identify genes contributing to podocyte dysfunction in acquired nephrotic syndrome, we studied human glomerular gene expression data sets for glomerular-enriched gene transcripts differentially regulated between pretransplant biopsy samples and biopsies from patients with nephrotic syndrome. Candidate genes were screened by in situ hybridization for expression in the zebrafish pronephros, an easy-to-use in vivo assay system to assess podocyte function. One glomerulus-enriched product was the Rho-GTPase binding protein, IQGAP2. Immunohistochemistry found a strong presence of IQGAP2 in normal human and zebrafish podocytes. In zebrafish larvae, morpholino-based knockdown of iqgap2 caused a mild foot process effacement of zebrafish podocytes and a cystic dilation of the urinary space of Bowman's capsule upon onset of urinary filtration. Moreover, the glomerulus of zebrafish morphants showed a glomerular permeability for injected high-molecular-weight dextrans, indicating an impaired size selectivity of the glomerular filter. Thus, IQGAP2 is a Rho-GTPase binding protein, highly abundant in human and zebrafish podocytes, which controls normal podocyte structure and function as evidenced in the zebrafish pronephros. PMID:26154927

  10. 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.

  11. 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.

  12. A Salmonella protein that is required for resistance to antimicrobial peptides and transport of potassium.

    OpenAIRE

    Parra-Lopez, C; Lin, R; Aspedon, A; Groisman, E A

    1994-01-01

    The ability of invading pathogens to proliferate within host tissues requires the capacity to resist the killing effects of a wide variety of host defense molecules. sap mutants of the facultative intracellular parasite Salmonella typhimurium exhibit hypersensitivity to antimicrobial peptides, cannot survive within macrophages in vitro and are attenuated for mouse virulence in vivo. We conducted a molecular genetic analysis of the sapG locus and showed that it encodes a product that is 99% id...

  13. Co-administration of non-carrier nanoparticles boosts antigen immune response without requiring protein conjugation.

    Science.gov (United States)

    Wibowo, Nani; Chuan, Yap P; Seth, Arjun; Cordoba, Yoann; Lua, Linda H L; Middelberg, Anton P J

    2014-06-17

    Nanotechnology promises a revolution in medicine including through new vaccine approaches. The use of nanoparticles in vaccination has, to date, focused on attaching antigen directly to or within nanoparticle structures to enhance antigen uptake by immune cells. Here we question whether antigen incorporation with the nanoparticle is actually necessary to boost vaccine effectiveness. We show that the immunogenicity of a sub-unit protein antigen was significantly boosted by formulation with silica nanoparticles even without specific conjugation of antigen to the nanoparticle. We further show that this effect was observed only for virus-sized nanoparticles (50 nm) but not for larger (1,000 nm) particles, demonstrating a pronounced effect of nanoparticle size. This non-attachment approach has potential to radically simplify the development and application of nanoparticle-based formulations, leading to safer and simpler nanoparticle applications in vaccine development. PMID:24793947

  14. Protein SUMOylation Is Required for Regulatory T Cell Expansion and Function.

    Science.gov (United States)

    Ding, Xiao; Wang, Aibo; Ma, Xiaopeng; Demarque, Maud; Jin, Wei; Xin, Huawei; Dejean, Anne; Dong, Chen

    2016-07-26

    Foxp3-expressing regulatory T (Treg) cells are essential for immune tolerance; however, the molecular mechanisms underlying Treg cell expansion and function are still not well understood. SUMOylation is a protein post-translational modification characterized by covalent attachment of SUMO moieties to lysines. UBC9 is the only E2 conjugating enzyme involved in this process, and loss of UBC9 completely abolishes the SUMOylation pathway. Here, we report that selective deletion of Ubc9 within the Treg lineage results in fatal early-onset autoimmunity similar to Foxp3 mutant mice. Ubc9-deficient Treg cells exhibit severe defects in TCR-driven homeostatic proliferation, accompanied by impaired activation and compromised suppressor function. Importantly, TCR ligation enhanced SUMOylation of IRF4, a critical regulator of Treg cell function downstream of TCR signals, which regulates its stability in Treg cells. Our data thus have demonstrated an essential role of SUMOylation in the expansion and function of Treg cells. PMID:27425617

  15. 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. PMID:25100858

  16. Outer membrane protein OmpQ of Bordetella bronchiseptica is required for mature biofilm formation.

    Science.gov (United States)

    Cattelan, Natalia; Villalba, María Inés; Parisi, Gustavo; Arnal, Laura; Serra, Diego Omar; Aguilar, Mario; Yantorno, Osvaldo

    2016-02-01

    Bordetella bronchiseptica, an aerobic Gram-negative bacterium, is capable of colonizing the respiratory tract of diverse animals and chronically persists inside the hosts by forming biofilm. Most known virulence factors in Bordetella species are regulated by the BvgAS two-component transduction system. The Bvg-activated proteins play a critical role during host infection. OmpQ is an outer membrane porin protein which is expressed under BvgAS control. Here, we studied the contribution of OmpQ to the biofilm formation process by B. bronchiseptica. We found that the lack of expression of OmpQ did not affect the growth kinetics and final biomass of B. bronchiseptica under planktonic growth conditions. The ΔompQ mutant strain displayed no differences in attachment level and in early steps of biofilm formation. However, deletion of the ompQ gene attenuated the ability of B. bronchiseptica to form a mature biofilm. Analysis of ompQ gene expression during the biofilm formation process by B. bronchiseptica showed a dynamic expression pattern, with an increase of biofilm culture at 48 h. Moreover, we demonstrated that the addition of serum anti-OmpQ had the potential to reduce the biofilm biomass formation in a dose-dependent manner. In conclusion, we showed for the first time, to the best of our knowledge, evidence of the contribution of OmpQ to a process of importance for B. bronchiseptica pathobiology. Our results indicate that OmpQ plays a role during the biofilm development process, particularly at later stages of development, and that this porin could be a potential target for strategies of biofilm formation inhibition. PMID:26673448

  17. 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.

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

    Science.gov (United States)

    González, Natalia; Moresco, James J.; Bustos, Francisco; Yates, John R.; Olguín, Hugo C.

    2016-01-01

    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 (re)acquiring 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. PMID:27144531

  19. Evaluating lysine requirements of nursery pigs fed low protein diets with different sources of nonessential amino acids.

    Science.gov (United States)

    Jones, C K; Tokach, M D; Usry, J L; Neill, C R; Patience, J F

    2014-08-01

    The Lys requirement of nursery pigs may be dependent on the source of nonessential AA (NEAA) nitrogen or the source of Lys itself. However, little peer-reviewed data examines these phenomena. The objectives of these experiments were to determine if the Lys requirement of pigs is altered when 1) low protein diets are supplemented with different sources of NEAA nitrogen or 2) Lys is supplied as a crystalline source instead of intact protein such as soybean meal (SBM). Two 14-d experiments were conducted using 450 (Exp. 1) and 540 (Exp. 2) pigs (PIC C22/C29 × 337). There were 10 treatments in each experiment, each aligned as a 2 × 5 factorial. In Exp. 1, there were 2 sources of NEAA (l-Gln + l-Gly or l-Gly + l-Ala + l-Pro + l-His) and 5 levels of Lys (1.2, 1.3, 1.4, 1.5, and 1.6%). In Exp. 2, there were 2 sources of proteins providing additional Lys (l-Lys HCl or SBM) and the same 5 levels of Lys. Following weaning at 18 to 22 d of age, pigs were fed a common starter diet for 5 d postweaning followed by a 14-d treatment period. Pigs were weighed and feed disappearance determined on d 0, 7, and 14 of the experiment. Data were analyzed using the MIXED and NLIN procedures of SAS (SAS Inst., Cary, NC). In Exp. 1, increasing CP and Lys resulted in a quadratic increase (P improvement (P 0.10) growth performance during the treatment period. In Exp. 2, both ADG and G:F increased linearly (P 0.10) ADG, but pigs fed additional Lys from crystalline sources had improved (P < 0.05) G:F than those fed additional Lys from intact protein at 1.50% Lys; however, the analyzed Lys values at this level differ. Overall, these data show that the standardized ileal digestibility Lys requirement of pigs is not altered when low protein diets are supplemented with different sources of NEAA nitrogen. Feed efficiency appears to be maximized when additional Lys is supplied by l-Lys HCl instead of SBM, but more research is needed to confirm this phenomenon. PMID:25074452

  20. Evaluating lysine requirements of nursery pigs fed low protein diets with different sources of non-essential amino acids.

    Science.gov (United States)

    Jones, C K; Tokach, M D; Usry, J L; Neill, C R; Patience, J F

    2014-07-01

    The Lys requirement of nursery pigs may be dependent upon the source of non-essential AA nitrogen (NEAA) or the source of Lys itself. However, little peer-reviewed data examines these phenomena. The objectives of these experiments were to determine if the Lys requirement of pigs is altered when 1) low protein diets are supplemented with different sources NEAA nitrogen or 2) Lys is supplied as a crystalline source instead of intact protein such as soybean meal (SBM). Two 14-d experiments were conducted using 450 (Exp. 1) and 540 (Exp. 2) pigs (PIC C22/C29 × 337). There were 10 treatments in each experiment, each aligned as a 2 × 5 factorial. In Exp. 1, there were 2 sources of NEAA (L-Gln + L-Gly or L-Gly + L-Ala + L-Pro + L-His) and 5 levels of Lys (1.2, 1.3, 1.4, 1.5, 1.6). In Exp. 2, there were 2 sources of proteins providing additional Lys, (L-Lys•HCl or SBM) and the same 5 levels of Lys. Following weaning at 18 to 22 days of age, pigs were fed a common starter diet for 5 d post-weaning, followed by a 14-d treatment period. Pigs were weighed and feed disappearance determined on d 0, 7, and 14 of the experiment. Data were analyzed using the MIXED and NLIN procedures of SAS (SAS Inst., Cary, NC). In Exp. 1, increasing CP and Lys resulted in a quadratic increase (P improvement (P 0.10) growth performance during the treatment period. In Exp. 2, both ADG and G:F increased linearly (P 0.10) ADG, but pigs fed additional Lys from crystalline sources had improved (P < 0.05) G:F than those fed additional Lys from intact protein at 1.50% Lys; however the analyzed Lys values at this level differ. Overall, these data show that the standardized ileal digestibility (SID) Lys requirement of pigs is not altered when low protein diets are supplemented with different sources of NEAA nitrogen. Feed efficiency appears to be maximized when additional Lys is supplied by L-Lys·HCl instead of SBM, but more research is needed to confirm this phenomenon. PMID:24987075

  1. Exigências nutricionais de zebuínos: proteína Nutritional requirements of Zebu cattle: protein

    Directory of Open Access Journals (Sweden)

    Pedro Veiga Rodrigues Paulino

    2004-06-01

    objective of determining their protein requirements. Four steers were slaughtered at the beginning of the trial, compounding the reference group, three were fed at maintenance level and the remaining were uniformly allotted to a complete randomized design, of three treatments, with different levels of concentrate in the diets (5, 35, 65 %, in the total dry matter basis. As roughage was used Brachiaria brizantha and Cynodon sp. haylage. The diets were isonitrogenous and the animals were fed ad libitum. The protein content retained in the body was estimated by a regression equation obtained between the logarithm of the body content of protein and the logarithm of the empty body weight (EBW. The net requirements of protein for 1 kg of empty body gain (EBG were determined as the derivative of the equation of prediction of protein body content. The protein requirements for gain decreased as the live weight increased. The protein requirement found for an animal of 400 kg of live weight was 106.84 g/kg EBW, while for an animal of 200 kg, was 129.01 g/kg EBW. Considering a steer of 400 kg of live weight, the estimated metabolizable requirements of protein for maintenance (MPm and gain (MPg were, respectively, 339.88 and 217.14 g/kg LW. For this type of animal, the daily protein requirements in terms of degradable and undegradable protein were, respectively, 830.86 and 4.46 g. The requirement of crude protein was 835.32 g/d.

  2. Autophosphorylation of the DNA-dependent protein kinase catalytic subunit is required for rejoining of DNA double-strand breaks

    OpenAIRE

    Chan, Doug W.; Chen, Benjamin Ping-Chi; Prithivirajsingh, Sheela; Kurimasa, Akihiro; Story, Michael D.; Qin, Jun; Chen, David J.

    2002-01-01

    Nonhomologous end-joining (NHEJ) is the predominant pathway that repairs DNA double-strand breaks (DSBs) in mammalian cells. The DNA-dependent protein kinase (DNA-PK), consisting of Ku and DNA-PK catalytic subunit (DNA-PKcs), is activated by DNA in vitro and is required for NHEJ. We report that DNA-PKcs is autophosphorylated at Thr2609 in vivo in a Ku-dependent manner in response to ionizing radiation. Phosphorylated DNA-PKcs colocalizes with both γ-H2AX and 53BP1 after DNA damage. Mutation o...

  3. A Type II Protein Secretory Pathway Required for Levansucrase Secretion by Gluconacetobacter diazotrophicus

    OpenAIRE

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

    2004-01-01

    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 ac...

  4. Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

    Science.gov (United States)

    Lee, Rebecca; Xu, Bin; Rame, J. Eduardo; Felkin, Leanne E.; Barton, Paul; Dries, Daniel L.

    2014-01-01

    Background The compensatory actions of the endogenous natriuretic peptide system require adequate processing of natriuretic peptide pro‐hormones into biologically active, carboxyl‐terminal fragments. Natriuretic peptide pro‐peptide processing is accomplished by corin, a transmembrane serine protease expressed by cardiomyocytes. Brain natriuretic peptide (BNP) processing is inadequate in advanced heart failure and is independently associated with adverse outcomes; however, the molecular mechanisms causing impaired BNP processing are not understood. We hypothesized that the development of endoplasmic reticulum stress in cardiomyocytes in advanced heart failure triggers inositol‐requiring protein 1 (IRE1)‐dependent corin mRNA decay, which would favor a molecular substrate favoring impaired natriuretic peptide pro‐peptide processing. Methods and Results Two independent samples of hearts obtained from patients with advanced heart failure at transplant demonstrated that corin RNA was reduced as Atrial natriuretic peptide (ANP)/BNP RNA increased. Increases in spliced X‐box protein 1, a marker for IRE1‐endoribonuclease activity, were associated with decreased corin RNA. Moreover, ≈50% of the hearts demonstrated significant reductions in corin RNA and protein as compared to the nonfailing control sample. In vitro experiments demonstrated that induction of endoplasmic reticulum stress in cultured cardiomyocytes with thapsigargin activated IRE1's endoribonuclease activity and time‐dependent reductions in corin mRNA. In HL‐1 cells, overexpression of IRE1 activated IRE1 endoribonuclease activity and caused corin mRNA decay, whereas IRE1‐RNA interference with shRNA attenuated corin mRNA decay after induction of endoplasmic reticulum stress with thapsigargin. Pre‐treatment of cells with Actinomycin D to inhibit transcription did not alter the magnitude or time course of thapsigargin‐induced corin mRNA decline, supporting the hypothesis that this was

  5. The Dysferlin Domain-Only Protein, Spo73, Is Required for Prospore Membrane Extension in Saccharomyces cerevisiae.

    Science.gov (United States)

    Okumura, Yuuya; Nakamura, Tsuyoshi S; Tanaka, Takayuki; Inoue, Ichiro; Suda, Yasuyuki; Takahashi, Tetsuo; Nakanishi, Hideki; Nakamura, Shugo; Gao, Xiao-Dong; Tachikawa, Hiroyuki

    2016-01-01

    Sporulation of Saccharomyces cerevisiae is a developmental process in which an ascus containing four haploid spores forms from a diploid cell. During this process, newly formed membrane structures called prospore membranes extend along the nuclear envelope and engulf and package daughter nuclei along with cytosol and organelles to form precursors of spores. Proteins involved in prospore membrane extension, Vps13 and Spo71, have recently been reported; however, the overall mechanism of membrane extension remains unclear. Here, we identified Spo73 as an additional factor involved in prospore membrane extension. Analysis of a spo73∆ mutant revealed that it shows defects similar to those of a spo71∆ mutant during prospore membrane formation. Spo73 localizes to the prospore membrane, and this localization is independent of Spo71 and Vps13. In contrast, a Spo73 protein carrying mutations in a surface basic patch mislocalizes to the cytoplasm and overexpression of Spo71 can partially rescue localization to the prospore membrane. Similar to spo71∆ mutants, spo73∆ mutants display genetic interactions with the mutations in the SMA2 and SPO1 genes involved in prospore membrane bending. Further, our bioinformatic analysis revealed that Spo73 is a dysferlin domain-only protein. Thus, these results suggest that a dysferlin domain-only protein, Spo73, functions with a dual pleckstrin homology domain protein, Spo71, in prospore membrane extension. Analysis of Spo73 will provide insights into the conserved function of dysferlin domains, which is related to dysferlinopathy. IMPORTANCE Prospore membrane formation consists of de novo double-membrane formation, which occurs during the developmental process of sporulation in Saccharomyces cerevisiae. Membranes are formed into their proper size and shape, and thus, prospore membrane formation has been studied as a general model of membrane formation. We identified SPO73, previously shown to be required for spore wall formation

  6. The insulator protein BEAF-32 is required for Hippo pathway activity in the terminal differentiation of neuronal subtypes.

    Science.gov (United States)

    Jukam, David; Viets, Kayla; Anderson, Caitlin; Zhou, Cyrus; DeFord, Peter; Yan, Jenny; Cao, Jinshuai; Johnston, Robert J

    2016-07-01

    The Hippo pathway is crucial for not only normal growth and apoptosis but also cell fate specification during development. What controls Hippo pathway activity during cell fate specification is incompletely understood. In this article, we identify the insulator protein BEAF-32 as a regulator of Hippo pathway activity in Drosophila photoreceptor differentiation. Though morphologically uniform, the fly eye is composed of two subtypes of R8 photoreceptor neurons defined by expression of light-detecting Rhodopsin proteins. In one R8 subtype, active Hippo signaling induces Rhodopsin 6 (Rh6) and represses Rhodopsin 5 (Rh5), whereas in the other subtype, inactive Hippo signaling induces Rh5 and represses Rh6. The activity state of the Hippo pathway in R8 cells is determined by the expression of warts, a core pathway kinase, which interacts with the growth regulator melted in a double-negative feedback loop. We show that BEAF-32 is required for expression of warts and repression of melted Furthermore, BEAF-32 plays a second role downstream of Warts to induce Rh6 and prevent Rh5 fate. BEAF-32 is dispensable for Warts feedback, indicating that BEAF-32 differentially regulates warts and Rhodopsins. Loss of BEAF-32 does not noticeably impair the functions of the Hippo pathway in eye growth regulation. Our study identifies a context-specific regulator of Hippo pathway activity in post-mitotic neuronal fate, and reveals a developmentally specific role for a broadly expressed insulator protein. PMID:27226322

  7. The Evolutionarily Conserved Protein PHOTOSYNTHESIS AFFECTED MUTANT71 Is Required for Efficient Manganese Uptake at the Thylakoid Membrane in Arabidopsis.

    Science.gov (United States)

    Schneider, Anja; Steinberger, Iris; Herdean, Andrei; Gandini, Chiara; Eisenhut, Marion; Kurz, Samantha; Morper, Anna; Hoecker, Natalie; Rühle, Thilo; Labs, Mathias; Flügge, Ulf-Ingo; Geimer, Stefan; Schmidt, Sidsel Birkelund; Husted, Søren; Weber, Andreas P M; Spetea, Cornelia; Leister, Dario

    2016-04-01

    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 lumen remains poorly understood. Here, we show that Arabidopsis thaliana PHOTOSYNTHESIS AFFECTED MUTANT71 (PAM71) is an integral thylakoid membrane protein involved in Mn(2+) and Ca(2+) homeostasis in chloroplasts. This protein is required for normal operation of the oxygen-evolving complex (as evidenced by oxygen evolution rates) and for manganese incorporation. Manganese binding to PSII was severely reduced in pam71 thylakoids, particularly in PSII supercomplexes. In cation partitioning assays with intact chloroplasts, Mn(2+) and Ca(2+) ions were differently sequestered in pam71, with Ca(2+) enriched in pam71 thylakoids relative to the wild type. The changes in Ca(2+) 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 Mn(2+), but not Ca(2+) Furthermore, PAM71 suppressed the Mn(2+)-sensitive phenotype of the yeast mutant Δpmr1 Therefore, PAM71 presumably functions in Mn(2+) uptake into thylakoids to ensure optimal PSII performance. PMID:27020959

  8. Heat shock protein 90 is required for sexual and asexual development, virulence, and heat shock response in Fusarium graminearum

    Science.gov (United States)

    Bui, Duc-Cuong; Lee, Yoonji; Lim, Jae Yun; Fu, Minmin; Kim, Jin-Cheol; Choi, Gyung Ja; Son, Hokyoung; Lee, Yin-Won

    2016-01-01

    Eukaryotic cells repress global translation and selectively upregulate stress response proteins by altering multiple steps in gene expression. In this study, genome-wide transcriptome analysis of cellular adaptation to thermal stress was performed on the plant pathogenic fungus Fusarium graminearum. The results revealed that profound alterations in gene expression were required for heat shock responses in F. graminearum. Among these proteins, heat shock protein 90 (FgHsp90) was revealed to play a central role in heat shock stress responses in this fungus. FgHsp90 was highly expressed and exclusively localised to nuclei in response to heat stress. Moreover, our comprehensive functional characterisation of FgHsp90 provides clear genetic evidence supporting its crucial roles in the vegetative growth, reproduction, and virulence of F. graminearum. In particular, FgHsp90 performs multiple functions as a transcriptional regulator of conidiation. Our findings provide new insight into the mechanisms underlying adaptation to heat shock and the roles of Hsp90 in fungal development.

  9. 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.

  10. Heat shock protein 90 is required for sexual and asexual development, virulence, and heat shock response in Fusarium graminearum.

    Science.gov (United States)

    Bui, Duc-Cuong; Lee, Yoonji; Lim, Jae Yun; Fu, Minmin; Kim, Jin-Cheol; Choi, Gyung Ja; Son, Hokyoung; Lee, Yin-Won

    2016-01-01

    Eukaryotic cells repress global translation and selectively upregulate stress response proteins by altering multiple steps in gene expression. In this study, genome-wide transcriptome analysis of cellular adaptation to thermal stress was performed on the plant pathogenic fungus Fusarium graminearum. The results revealed that profound alterations in gene expression were required for heat shock responses in F. graminearum. Among these proteins, heat shock protein 90 (FgHsp90) was revealed to play a central role in heat shock stress responses in this fungus. FgHsp90 was highly expressed and exclusively localised to nuclei in response to heat stress. Moreover, our comprehensive functional characterisation of FgHsp90 provides clear genetic evidence supporting its crucial roles in the vegetative growth, reproduction, and virulence of F. graminearum. In particular, FgHsp90 performs multiple functions as a transcriptional regulator of conidiation. Our findings provide new insight into the mechanisms underlying adaptation to heat shock and the roles of Hsp90 in fungal development. PMID:27306495

  11. Sip1, an AP-1 accessory protein in fission yeast, is required for localization of Rho3 GTPase.

    Directory of Open Access Journals (Sweden)

    Yang Yu

    Full Text Available Rho family GTPases act as molecular switches to regulate a range of physiological functions, including the regulation of the actin-based cytoskeleton, membrane trafficking, cell morphology, nuclear gene expression, and cell growth. Rho function is regulated by its ability to bind GTP and by its localization. We previously demonstrated functional and physical interactions between Rho3 and the clathrin-associated adaptor protein-1 (AP-1 complex, which revealed a role of Rho3 in regulating Golgi/endosomal trafficking in fission yeast. Sip1, a conserved AP-1 accessory protein, recruits the AP-1 complex to the Golgi/endosomes through physical interaction. In this study, we showed that Sip1 is required for Rho3 localization. First, overexpression of rho3⁺ suppressed defective membrane trafficking associated with sip1-i4 mutant cells, including defects in vacuolar fusion, Golgi/endosomal trafficking and secretion. Notably, Sip1 interacted with Rho3, and GFP-Rho3, similar to Apm1-GFP, did not properly localize to the Golgi/endosomes in sip1-i4 mutant cells at 27°C. Interestingly, the C-terminal region of Sip1 is required for its localization to the Golgi/endosomes, because Sip1-i4-GFP protein failed to properly localize to Golgi/endosomes, whereas the fluorescence of Sip1ΔN mutant protein co-localized with that of FM4-64. Consistently, in the sip1-i4 mutant cells, which lack the C-terminal region of Sip1, binding between Apm1 and Rho3 was greatly impaired, presumably due to mislocalization of these proteins in the sip1-i4 mutant cells. Furthermore, the interaction between Apm1 and Rho3 as well as Rho3 localization to the Golgi/endosomes were significantly rescued in sip1-i4 mutant cells by the expression of Sip1ΔN. Taken together, these results suggest that Sip1 recruits Rho3 to the Golgi/endosomes through physical interaction and enhances the formation of the Golgi/endosome AP-1/Rho3 complex, thereby promoting crosstalk between AP-1 and Rho3 in

  12. Sip1, an AP-1 accessory protein in fission yeast, is required for localization of Rho3 GTPase.

    Science.gov (United States)

    Yu, Yang; Li, Cuifang; Kita, Ayako; Katayama, Yuta; Kubouchi, Koji; Udo, Masako; Imanaka, Yukako; Ueda, Shiho; Masuko, Takashi; Sugiura, Reiko

    2013-01-01

    Rho family GTPases act as molecular switches to regulate a range of physiological functions, including the regulation of the actin-based cytoskeleton, membrane trafficking, cell morphology, nuclear gene expression, and cell growth. Rho function is regulated by its ability to bind GTP and by its localization. We previously demonstrated functional and physical interactions between Rho3 and the clathrin-associated adaptor protein-1 (AP-1) complex, which revealed a role of Rho3 in regulating Golgi/endosomal trafficking in fission yeast. Sip1, a conserved AP-1 accessory protein, recruits the AP-1 complex to the Golgi/endosomes through physical interaction. In this study, we showed that Sip1 is required for Rho3 localization. First, overexpression of rho3⁺ suppressed defective membrane trafficking associated with sip1-i4 mutant cells, including defects in vacuolar fusion, Golgi/endosomal trafficking and secretion. Notably, Sip1 interacted with Rho3, and GFP-Rho3, similar to Apm1-GFP, did not properly localize to the Golgi/endosomes in sip1-i4 mutant cells at 27°C. Interestingly, the C-terminal region of Sip1 is required for its localization to the Golgi/endosomes, because Sip1-i4-GFP protein failed to properly localize to Golgi/endosomes, whereas the fluorescence of Sip1ΔN mutant protein co-localized with that of FM4-64. Consistently, in the sip1-i4 mutant cells, which lack the C-terminal region of Sip1, binding between Apm1 and Rho3 was greatly impaired, presumably due to mislocalization of these proteins in the sip1-i4 mutant cells. Furthermore, the interaction between Apm1 and Rho3 as well as Rho3 localization to the Golgi/endosomes were significantly rescued in sip1-i4 mutant cells by the expression of Sip1ΔN. Taken together, these results suggest that Sip1 recruits Rho3 to the Golgi/endosomes through physical interaction and enhances the formation of the Golgi/endosome AP-1/Rho3 complex, thereby promoting crosstalk between AP-1 and Rho3 in the regulation of

  13. 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.

  14. 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.

  15. 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.

  16. Microcephalin and pericentrin regulate mitotic entry via centrosome-associated Chk1

    DEFF Research Database (Denmark)

    Tibelius, Alexandra; Marhold, Joachim; Zentgraf, Hanswalter;

    2009-01-01

    Primary microcephaly, Seckel syndrome, and microcephalic osteodysplastic primordial dwarfism type II (MOPD II) are disorders exhibiting marked microcephaly, with small brain sizes reflecting reduced neuron production during fetal life. Although primary microcephaly can be caused by mutations...

  17. A G-Protein β Subunit Required for Sexual and Vegetative Development and Maintenance of Normal Gα Protein Levels in Neurospora crassa

    OpenAIRE

    Yang, Qi; Poole, Sheven I.; Borkovich, Katherine A.

    2002-01-01

    The genome of the filamentous fungus Neurospora crassa contains a single gene encoding a heterotrimeric G-protein β subunit, gnb-1. The predicted GNB-1 protein sequence is most identical to Gβ proteins from the filamentous fungi Cryphonectria parasitica and Aspergillus nidulans. N. crassa GNB-1 is also 65% identical to the human GNB-1 protein but only 38 and 45% identical to Gβ proteins from budding and fission yeasts. Previous studies in animal and fungal systems have elucidated phenotypes o...

  18. Three wall-associated kinases required for rice basal immunity form protein complexes in the plasma membrane.

    Science.gov (United States)

    Cayrol, Bastien; Delteil, Amandine; Gobbato, Enrico; Kroj, Thomas; Morel, Jean-Benoit

    2016-04-01

    Receptor-like kinases (RLKs) play key roles in disease resistance, in particular basal immunity. They recognize patterns produced by the pathogen invasion and often work as complexes in the plasma membrane. Among these RLKs, there is increasing evidence in several plant species of the key role of Wall-associated kinases (WAKs) in disease resistance. We recently showed using rice (Oryza sativa) loss-of-function mutants of three transcriptionaly co-regulated OsWAK genes that individual OsWAKs are positively required for quantitative resistance to the rice blast fungus, Magnaporthe oryzae. This finding was unexpected since WAK genes belong to large gene families where functional redundancy is expected. Here we provide evidence that this may be due to complex physical interaction between OsWAK proteins. PMID:26853099

  19. Dimerization of the DYT6 dystonia protein, THAP1, requires residues within the coiled-coil domain.

    Science.gov (United States)

    Sengel, Cem; Gavarini, Sophie; Sharma, Nutan; Ozelius, Laurie J; Bragg, D Cristopher

    2011-09-01

    Thanatos-associated [THAP] domain-containing apoptosis-associated protein 1 (THAP1) is a DNA-binding protein that has been recently associated with DYT6 dystonia, a hereditary movement disorder involving sustained, involuntary muscle contractions. A large number of dystonia-related mutations have been identified in THAP1 in diverse patient populations worldwide. Previous reports have suggested that THAP1 oligomerizes with itself via a C-terminal coiled-coil domain, raising the possibility that DYT6 mutations in this region might affect this interaction. In this study, we examined the ability of wild-type THAP1 to bind itself and the effects on this interaction of the following disease mutations: C54Y, F81L, ΔF132, T142A, I149T, Q154fs180X, and A166T. The results confirmed that wild-type THAP1 associated with itself and most of the DYT6 mutants tested, except for the Q154fs180X variant, which loses most of the coiled-coil domain because of a frameshift at position 154. However, deletion of C-terminal residues after position 166 produced a truncated variant of THAP1 that was able to bind the wild-type protein. The interaction of THAP1 with itself therefore required residues within a 13-amino acid region (aa 154-166) of the coiled-coil domain. Further inspection of this sequence revealed elements highly consistent with previous descriptions of leucine zippers, which serve as dimerization domains in other transcription factor families. Based on this similarity, a structural model was generated to predict how hydrophobic residues in this region may mediate dimerization. These observations offer additional insight into the role of the coiled-coil domain in THAP1, which may facilitate future analyses of DYT6 mutations in this region. PMID:21752024

  20. Proteins and carbohydrates in nipple aspirate fluid predict the presence of atypia and cancer in women requiring diagnostic breast biopsy

    Directory of Open Access Journals (Sweden)

    Qin Wenyi

    2012-02-01

    Full Text Available Abstract Background Herein we present the results of two related investigations. The first study determined if concentrations in breast nipple discharge (ND of two proteins (urinary plasminogen activator, uPA and its inhibitor, PAI-1 predicted the presence of breast atypia and cancer in pre- and/or postmenopausal women requiring surgery because of a suspicious breast lesion. The second study assessed if these proteins increased the predictive ability of a carbohydrate (Thomsen Friedenreich, TF which we previously demonstrated predicted the presence of disease in postmenopausal women requiring surgery. Methods In the first study we prospectively enrolled 79 participants from whom we collected ND, measured uPA and PAI-1 and correlated expression with pathologic findings. In the second study we analyzed 35 (uPA and PAI-1 in 24, uPA in an additional 11 ND samples collected from different participants requiring breast surgery, all of whom also had TF results. Results uPA expression was higher in pre- and PAI-1 in postmenopausal women with 1 cancer (DCIS or invasive vs. either no cancer (atypia or benign pathology, p = .018 and .025, respectively, or benign pathology (p = .017 and .033, respectively; and 2 abnormal (atypia or cancer versus benign pathology (p = .018 and .052, respectively. High uPA and PAI-1 concentrations and age were independent predictors of disease in premenopausal women, with an area under the curve (AUC of 83-87% when comparing diseased vs. benign pathology. uPA, TF, and age correctly classified 35 pre- and postmenopausal women as having disease or not 84-91% of the time, whereas combining uPA+PAI-1+TF correctly classified 24 women 97-100% of the time. Conclusions uPA and PAI-1 concentrations in ND were higher in women with atypia and cancer compared to women with benign disease. Combining uPA, PAI-1 and TF in the assessment of women requiring diagnostic breast surgery maximized disease prediction. The assessment of these

  1. Slow cluster formation of purified human or rhesus T cells requires protein kinase C and LFA-1.

    Science.gov (United States)

    Eylar, E H; Molina, C; Báez, I; Kessler, M

    1996-03-01

    Homotropic T cell adhesion, as generally studied, consists of a rapid, transient binding process that is measured over a 15-120 min. period. Here we report a slow type of adhesion process occurring with human or rhesus T cells, purified from peripheral blood, that manifests itself by the formation of rounded, multi-layer clusters which may contain hundreds of cells. The maximal number and size of the clusters peak 1-2 days after the addition of phorbol ester, an absolute requirement. The number of clusters formed is proportional to phorbol ester concentration up to 1.25 ng/mL. Phorbol esters such as phorbol myristate acetate (PMA), phorbol dibutyrate (PDB), and 7-octylindolactam (OIL) induced optimal cluster formation at 1-13 ng/mL, levels slightly higher than that required to induce mitogenesis of purified T cells. Phorbol itself and the alpha-form of the ester were inactive. Both cluster formation and mitogenesis (stimulated by Con A or anti-CD3) are completely inhibited by staurosporin at 12.5 ng/mL. Even at 2.5 ng/mL, 74% of cluster formation was inhibited, which strongly implies a crucial role for protein kinase C. In the presence of accessory cells, T cell clusters were suppressed. Monoclonal Ab such as anti-CD3, mouse anti-CD3 followed by anti-mouse IgG, anti-CD4, anti-CD4A, anti-CD2, anti-CD8, and anti-CD45 did not induce cluster formation. None were inhibitory or stimulatory in the presence of PMA, except for anti-CD3 which enhanced cluster formation by 26%. However, anti-LFA-1 beta-chain (mouse monoclonal) completely blocked cluster formation over the range studied (63-1000 ng/mL) for both human and rhesus cells; rat anti-LFA-1 only blocked human cell adhesion. Anti LFA-1 only partially inhibited T cell mitogenesis. These results show that slow cluster formation shares the LFA-1 and phorbol ester requirements of the rapid adhesion of T cells requiring LFA-1 and ICAM-1. However, cluster occurs at a very low phorbol ester concentration, appears more

  2. The cleverSuite approach for protein characterization: predictions of structural properties, solubility, chaperone requirements and RNA-binding abilities

    Science.gov (United States)

    Klus, Petr; Bolognesi, Benedetta; Agostini, Federico; Marchese, Domenica; Zanzoni, Andreas; Tartaglia, Gian Gaetano

    2014-01-01

    Motivation: The recent shift towards high-throughput screening is posing new challenges for the interpretation of experimental results. Here we propose the cleverSuite approach for large-scale characterization of protein groups. Description: The central part of the cleverSuite is the cleverMachine (CM), an algorithm that performs statistics on protein sequences by comparing their physico-chemical propensities. The second element is called cleverClassifier and builds on top of the models generated by the CM to allow classification of new datasets. Results: We applied the cleverSuite to predict secondary structure properties, solubility, chaperone requirements and RNA-binding abilities. Using cross-validation and independent datasets, the cleverSuite reproduces experimental findings with great accuracy and provides models that can be used for future investigations. Availability: The intuitive interface for dataset exploration, analysis and prediction is available at http://s.tartaglialab.com/clever_suite. Contact: gian.tartaglia@crg.es Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24493033

  3. The DotA protein from Legionella pneumophila is secreted by a novel process that requires the Dot/Icm transporter

    OpenAIRE

    Nagai, Hiroki; Roy, Craig R.

    2001-01-01

    Legionella pneumophila requires the dot/icm genes to create an organelle inside eukaryotic host cells that will support bacterial replication. The dot/icm genes are predicted to encode a type IV-related secretion apparatus. However, no proteins have been identified that require the dot/icm genes for secretion. In this study we show that the DotA protein, which was previously found to be a polytopic membrane protein, is secreted by the Dot/Icm transporter into culture supernatants. Secreted Do...

  4. The interactions of GW182 proteins with PABP and deadenylases are required for both translational repression and degradation of miRNA targets.

    Science.gov (United States)

    Huntzinger, Eric; Kuzuoglu-Öztürk, Duygu; Braun, Joerg E; Eulalio, Ana; Wohlbold, Lara; Izaurralde, Elisa

    2013-01-01

    Animal miRNAs silence the expression of mRNA targets through translational repression, deadenylation and subsequent mRNA degradation. Silencing requires association of miRNAs with an Argonaute protein and a GW182 family protein. In turn, GW182 proteins interact with poly(A)-binding protein (PABP) and the PAN2-PAN3 and CCR4-NOT deadenylase complexes. These interactions are required for the deadenylation and decay of miRNA targets. Recent studies have indicated that miRNAs repress translation before inducing target deadenylation and decay; however, whether translational repression and deadenylation are coupled or represent independent repressive mechanisms is unclear. Another remaining question is whether translational repression also requires GW182 proteins to interact with both PABP and deadenylases. To address these questions, we characterized the interaction of Drosophila melanogaster GW182 with deadenylases and defined the minimal requirements for a functional GW182 protein. Functional assays in D. melanogaster and human cells indicate that miRNA-mediated translational repression and degradation are mechanistically linked and are triggered through the interactions of GW182 proteins with PABP and deadenylases. PMID:23172285

  5. The lissencephaly protein Lis1 is present in motile mammalian cilia and requires outer arm dynein for targeting to Chlamydomonas flagella

    DEFF Research Database (Denmark)

    Pedersen, Lotte B; Rompolas, Panteleimon; Christensen, Søren T;

    2007-01-01

    . Several proteins required for nuclear migration in Aspergillus bind directly to Lis1, including NudC. Mammalian NudC is highly expressed in ciliated epithelia, and localizes to motile cilia in various tissues. Moreover, a NudC ortholog is upregulated upon deflagellation in Chlamydomonas. We found that...... mammalian Lis1 localizes to motile cilia in trachea and oviduct, but is absent from non-motile primary cilia. Furthermore, we cloned a gene encoding a Lis1-like protein (CrLis1) from Chlamydomonas. CrLis1 is a approximately 37 kDa protein that contains seven WD-repeat domains, similar to Lis1 proteins from...

  6. Possibilities and limiting conditions for the genetic improvement of protein yield in cereals with particular reference to nitrogen balance and requirements

    International Nuclear Information System (INIS)

    It has proved much more difficult than originally expected to convert gains in grain protein content into improved protein yields per unit area, and decisive success in breeding for high protein yield in cereals has not yet been achieved. The reasons are rather complex. It is the opinion of the author that a more realistic situation is realised if the nitrogen balance and requirements of the crop are considered. All the protein produced derives from nitrogen taken up from the soil. More grain protein per unit area can only be produced if more nitrogen is available. Some rough numerical calculations illustrate that this additional nitrogen requirement may cause serious limitations in breeding for improved protein yield. By providing unchanged manure conditions, protein yield can nevertheless be increased (even if only slightly) in two ways: by improved nitrogen uptake from the soil, and/or by improved nitrogen mobilization and translocation from vegetative organs to the grain. On the other hand, in breeding for improved protein quality, i.e. a changed amino acid composition, no major problems should arise from the changed nitrogen requirements, as shown in supplementary calculations. (author)

  7. Male Sterile2 Encodes a Plastid-Localized Fatty Acyl Carrier Protein Reductase Required for Pollen Exine Development in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Chen, W.; Shanklin, J.; Yu, X.-H.; Zhang, K.; Shi, J.; De Oliveira, S.; Schreiber, L.; Zhang, D.

    2011-10-01

    Male Sterile2 (MS2) is predicted to encode a fatty acid reductase required for pollen wall development in Arabidopsis (Arabidopsis thaliana). Transient expression of MS2 in tobacco (Nicotiana benthamiana) leaves resulted in the accumulation of significant levels of C16 and C18 fatty alcohols. Expression of MS2 fused with green fluorescent protein revealed that an amino-terminal transit peptide targets the MS2 to plastids. The plastidial localization of MS2 is biologically important because genetic complementation of MS2 in ms2 homozygous plants was dependent on the presence of its amino-terminal transit peptide or that of the Rubisco small subunit protein amino-terminal transit peptide. In addition, two domains, NAD(P)H-binding domain and sterile domain, conserved in MS2 and its homologs were also shown to be essential for MS2 function in pollen exine development by genetic complementation testing. Direct biochemical analysis revealed that purified recombinant MS2 enzyme is able to convert palmitoyl-Acyl Carrier Protein to the corresponding C16:0 alcohol with NAD(P)H as the preferred electron donor. Using optimized reaction conditions (i.e. at pH 6.0 and 30 C), MS2 exhibits a K{sub m} for 16:0-Acyl Carrier Protein of 23.3 {+-} 4.0 {mu}m, a V{sub max} of 38.3 {+-} 4.5 nmol mg{sup -1} min{sup -1}, and a catalytic efficiency/K{sub m} of 1,873 m{sup -1} s{sup -1}. Based on the high homology of MS2 to other characterized fatty acid reductases, it was surprising that MS2 showed no activity against palmitoyl- or other acyl-coenzyme A; however, this is consistent with its plastidial localization. In summary, genetic and biochemical evidence demonstrate an MS2-mediated conserved plastidial pathway for the production of fatty alcohols that are essential for pollen wall biosynthesis in Arabidopsis.

  8. 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 protein in corn; temporary exemption from the requirement of a tolerance. 174.532 Section 174.532 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED...

  9. 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 in corn and cotton; exemption from the requirement of a tolerance. 174.501 Section 174.501 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED...

  10. 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 in corn; exemption from the requirement of a tolerance. 174.518 Section 174.518 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances...

  11. 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 in all plants; exemption from the requirement of a tolerance. 174.511 Section 174.511 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS...

  12. 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 protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED...

  13. 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 in all plants; exemption from the requirement of a tolerance. 174.510 Section 174.510 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS...

  14. 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 in corn and cotton; exemption from the requirement of a tolerance. 174.519 Section 174.519 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED...

  15. 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 Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. 174.506 Section 174.506 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED...

  16. Dynamin-related protein Drp1 is required for Bax translocation to mitochondria in response to irradiation-induced apoptosis.

    Science.gov (United States)

    Wang, Ping; Wang, Peiguo; Liu, Becky; Zhao, Jing; Pang, Qingsong; Agrawal, Samir G; Jia, Li; Liu, Feng-Ting

    2015-09-01

    Translocation of the pro-apoptotic protein Bax from the cytosol to the mitochondria is a crucial step in DNA damage-mediated apoptosis, and is also found to be involved in mitochondrial fragmentation. Irradiation-induced cytochrome c release and apoptosis was associated with Bax activation, but not mitochondrial fragmentation. Both Bax and Drp1 translocated from the cytosol to the mitochondria in response to irradiation. However, Drp1 mitochondrial translocation and oligomerization did not require Bax, and failed to induce apoptosis in Bax deficient diffuse large B-cell lymphoma (DLBCL) cells. Using fluorescent microscopy and the intensity correlation analysis, we demonstrated that Bax and Drp1 were colocalized and the levels of colocalization were increased by UV irradiation. Using co-immuno-precipitation, we confirmed that Bax and Drp1 were binding partners. Irradiation induced a time-associated increase in the interaction between active Bax and Drp1. Knocking down Drp1 using siRNA blocked UV irradiation-mediated Bax mitochondrial translocation. In conclusion, our findings demonstrate for the first time, that Drp1 is required for Bax mitochondrial translocation, but Drp1-induced mitochondrial fragmentation alone is not sufficient to induce apoptosis in DLBCL cells. PMID:26093086

  17. The conserved proteins CHE-12 and DYF-11 are required for sensory cilium function in Caenorhabditis elegans.

    Science.gov (United States)

    Bacaj, Taulant; Lu, Yun; Shaham, Shai

    2008-02-01

    Sensory neuron cilia are evolutionarily conserved dendritic appendages that convert environmental stimuli into neuronal activity. Although several cilia components are known, the functions of many remain uncharacterized. Furthermore, the basis of morphological and functional differences between cilia remains largely unexplored. To understand the molecular basis of cilia morphogenesis and function, we studied the Caenorhabditis elegans mutants che-12 and dyf-11. These mutants fail to concentrate lipophilic dyes from their surroundings in sensory neurons and are chemotaxis defective. In che-12 mutants, sensory neuron cilia lack distal segments, while in dyf-11 animals, medial and distal segments are absent. CHE-12 and DYF-11 are conserved ciliary proteins that function cell-autonomously and are continuously required for maintenance of cilium morphology and function. CHE-12, composed primarily of HEAT repeats, may not be part of the intraflagellar transport (IFT) complex and is not required for the localization of some IFT components. DYF-11 undergoes IFT-like movement and may function at an early stage of IFT-B particle assembly. Intriguingly, while DYF-11 is expressed in all C. elegans ciliated neurons, CHE-12 expression is restricted to some amphid sensory neurons, suggesting a specific role in these neurons. Our results provide insight into general and neuron-specific aspects of cilium development and function. PMID:18245347

  18. Efficient translation of alfamovirus RNAs requires the binding of coat protein dimers to the 3' termini of the viral RNAs.

    Science.gov (United States)

    Neeleman, Lyda; Linthorst, Huub J M; Bol, John F

    2004-01-01

    The coat protein (CP) of Alfalfa mosaic virus (AMV) is required to initiate infection by the viral tripartite RNA genome whereas infection by the tripartite Brome mosaic virus (BMV) genome is independent of CP. AMV CP stimulates translation of AMV RNA in vivo 50- to 100-fold. The 3' untranslated region (UTR) of the AMV subgenomic CP messenger RNA 4 contains at least two CP binding sites. A CP binding site in the 3'-terminal 112 nucleotides of RNA 4 was found to be required for efficient translation of the RNA whereas an upstream binding site was not. Binding of CP to the AMV 3' UTR induces a conformational change of the RNA but this change alone was not sufficient to stimulate translation. CP mutant R17A is unable to bind to the 3' UTR and translation in vivo of RNA 4 encoding this mutant occurs at undetectable levels. Replacement of the 3' UTR of this mutant RNA 4 by the 3' UTR of BMV RNA 4 restored translation of R17A-CP to wild-type levels. Apparently, the BMV 3' UTR stimulates translation independently of CP. AMV CP mutant N199 is defective in the formation of CP dimers and did not stimulate translation of RNA 4 in vivo although the mutant CP did bind to the 3' UTR. The finding that N199-CP does not promote AMV infection corroborates the notion that the requirement of CP in the inoculum reflects its role in translation of the viral RNAs. PMID:14718638

  19. The MAP kinase Pmk1 and protein kinase A are required for rotenone resistance in the fission yeast, Schizosaccharomyces pombe

    International Nuclear Information System (INIS)

    Research highlights: → Rotenone induces generation of ROS and mitochondrial fragmentation in fission yeast. → The MAPK Pmk1 and PKA are required for rotenone resistance in fission yeast. → Pmk1 and PKA are required for ROS clearance in rotenone treated fission yeast cells. → PKA plays a role in ROS clearance under normal growth conditions in fission yeast. -- Abstract: Rotenone is a widely used pesticide that induces Parkinson's disease-like symptoms in rats and death of dopaminergic neurons in culture. Although rotenone is a potent inhibitor of complex I of the mitochondrial electron transport chain, it can induce death of dopaminergic neurons independently of complex I inhibition. Here we describe effects of rotenone in the fission yeast, Schizosaccharomyces pombe, which lacks complex I and carries out rotenone-insensitive cellular respiration. We show that rotenone induces generation of reactive oxygen species (ROS) as well as fragmentation of mitochondrial networks in treated S. pombe cells. While rotenone is only modestly inhibitory to growth of wild type S. pombe cells, it is strongly inhibitory to growth of mutants lacking the ERK-type MAP kinase, Pmk1, or protein kinase A (PKA). In contrast, cells lacking the p38 MAP kinase, Spc1, exhibit modest resistance to rotenone. Consistent with these findings, we provide evidence that Pmk1 and PKA, but not Spc1, are required for clearance of ROS in rotenone treated S. pombe cells. Our results demonstrate the usefulness of S. pombe for elucidating complex I-independent molecular targets of rotenone as well as mechanisms conferring resistance to the toxin.

  20. 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

  1. Identification of AML-1 and the (8;21) translocation protein (AML-1/ETO) as sequence-specific DNA-binding proteins: the runt homology domain is required for DNA binding and protein-protein interactions.

    OpenAIRE

    Meyers, S; Downing, J R; Hiebert, S W

    1993-01-01

    The AML1 gene on chromosome 21 is disrupted in the (8;21)(q22;q22) translocation associated with acute myelogenous leukemia and encodes a protein with a central 118-amino-acid domain with 69% homology to the Drosophila pair-rule gene, runt. We demonstrate that AML-1 is a DNA-binding protein which specifically interacts with a sequence belonging to the group of enhancer core motifs, TGT/cGGT. Electrophoretic mobility shift analysis of cell extracts identified two AML-1-containing protein-DNA c...

  2. Plasmodium falciparum erythrocytic stage parasites require the putative autophagy protein PfAtg7 for normal growth.

    Directory of Open Access Journals (Sweden)

    Dawn M Walker

    Full Text Available Analysis of the Plasmodium falciparum genome reveals a limited number of putative autophagy genes, specifically the four genes involved in ATG8 lipidation, an essential step in formation of autophagosomes. In yeast, Atg8 lipidation requires the E1-type ligase Atg7, an E2-type ligase Atg3, and a cysteine protease Atg4. These four putative P. falciparum ATG (PfATG genes are transcribed during the parasite's erythrocytic stages. PfAtg7 has relatively low identity and similarity to yeast Atg7 (14.7% and 32.2%, respectively, due primarily to long insertions typical of P. falciparum. Excluding the insertions the identity and similarity are higher (38.0% and 70.8%, respectively. This and the fact that key residues are conserved, including the catalytic cysteine and ATP binding domain, we hypothesize that PfAtg7 is the activating enzyme of PfAtg8. To assess the role of PfAtg7 we have generated two transgenic parasite lines. In one, the PfATG7 locus was modified to introduce a C-terminal hemagglutinin tag. Western blotting reveals two distinct protein species, one migrating near the predicted 150 kDa and one at approximately 65 kDa. The second transgenic line introduces an inducible degradation domain into the PfATG7 locus, allowing us to rapidly attenuate PfAtg7 protein levels. Corresponding species are also observed in this parasite line at approximately 200 kDa and 100 kDa. Upon PfATG7 attenuation parasites exhibit a slow growth phenotype indicating the essentiality of this putative enzyme for normal growth.

  3. The Unstructured N-terminal Region of Arabidopsis Group 4 Late Embryogenesis Abundant (LEA) Proteins Is Required for Folding and for Chaperone-like Activity under Water Deficit.

    Science.gov (United States)

    Cuevas-Velazquez, Cesar L; Saab-Rincón, Gloria; Reyes, José Luis; Covarrubias, Alejandra A

    2016-05-13

    Late embryogenesis abundant (LEA) proteins are a conserved group of proteins widely distributed in the plant kingdom that participate in the tolerance to water deficit of different plant species. In silico analyses indicate that most LEA proteins are structurally disordered. The structural plasticity of these proteins opens the question of whether water deficit modulates their conformation and whether these possible changes are related to their function. In this work, we characterized the secondary structure of Arabidopsis group 4 LEA proteins. We found that they are disordered in aqueous solution, with high intrinsic potential to fold into α-helix. We demonstrate that complete dehydration is not required for these proteins to sample ordered structures because milder water deficit and macromolecular crowding induce high α-helix levels in vitro, suggesting that prevalent conditions under water deficit modulate their conformation. We also show that the N-terminal region, conserved across all group 4 LEA proteins, is necessary and sufficient for conformational transitions and that their protective function is confined to this region, suggesting that folding into α-helix is required for chaperone-like activity under water limitation. We propose that these proteins can exist as different conformers, favoring functional diversity, a moonlighting property arising from their structural dynamics. PMID:27006402

  4. Identification of domains in apolipoprotein B100 that confer a high requirement for the microsomal triglyceride transfer protein.

    Science.gov (United States)

    Nicodeme, E; Benoist, F; McLeod, R; Yao, Z; Scott, J; Shoulders, C C; Grand-Perret, T

    1999-01-22

    The microsomal triglyceride transfer protein (MTP) is required for the assembly and secretion of apoB-containing lipoproteins. To investigate the role of MTP in lipoprotein assembly, we determined the ability of carboxyl-terminally truncated forms of apoB to be secreted from cells treated with the MTP inhibitor 4'-bromo-3'-methylmetaqualone (Benoist, F., Nicodeme, E., and Grand-Perret, T. (1996) Eur. J. Biochem. 240, 713-720). In Caco-2 and mhAT3F cells that produce apoB100 and apoB48, the inhibitor preferentially blocked apoB100 secretion. When the inhibitor was tested on McA-RH7777 cells stably transfected with cDNAs encoding human apoB100, apoB72, apoB53, apoB29, and apoB18, the secretion of apoB100, apoB72, and apoB53 was preferentially impaired relative to apoB48 and shorter forms. To delineate the region between apoB48 and apoB53 that has a high requirement for MTP, we used puromycin to generate a range of truncated forms of apoB in HepG2 cells. The secretion of apoB53 and longer forms of apoB was markedly affected by low concentrations of the MTP inhibitor (approximately 1 microM), whereas apoB51 and smaller forms of apoB were only affected at higher concentrations (> 10 microM). The size-related sensitivity to MTP inhibitor was not due to late processing or retention, since the same result was observed when nascent lipoproteins were isolated from the endoplasmic reticulum. The MTP inhibitor did not alter the density of the secreted lipoproteins, indicating that each apoB polypeptide requires a minimally defined amount of lipid to attain a secretable conformation. Our results suggest that the folding of the domain between apoB51 and apoB53 has a high requirement for lipid. This domain is predicted to form amphipathic alpha-helices and to bind lipid reversibly. It proceeds and is followed by rigid amphipathic beta-sheets that are predicted to associate with lipid irreversibly. We speculate that these domains enable apoB to switch from a stable lipid

  5. Exigências proteicas de bovinos de corte suplementados a pasto Protein requirements of pasture supplemented beef cattle

    Directory of Open Access Journals (Sweden)

    Maykel Franklin Lima Sales

    2010-09-01

    Full Text Available Objetivou-se determinar as exigências proteicas de bovinos anelorados, em pastagem com Brachiaria decumbens suplementada com concentrado. Foram utilizados 28 novilhos zebuínos não-castrados com média de 7 meses de idade e 180 kg. Foram abatidos oitos animais-referência para determinação da composição corporal inicial. Os 20 animais restantes foram divididos em cinco lotes de quatro animais, em delineamento inteiramente ao acaso, de modo que cada lote recebeu um dos cinco planos nutricionais: mantença; mistura mineral; e suplementação nos níveis de 0,75; 1,50 e 2,25 kg/animal/dia. A meia-carcaça direita de todos os animais foi dissecada em músculo, gordura e ossos e todos os constituintes do corpo foram pesados para determinação do peso de corpo vazio e da composição corporal. De posse desses dados, procedeu-se ao cálculo das exigências líquidas para ganho de peso em diversas faixas de peso corporal dos animais. Os conteúdos de proteína retidos no corpo dos animais foram regredidos em função do ganho de peso de corpo vazio e da energia retida. As exigências de proteína metabolizável total de um animal de 350 kg e ganho diário de 1,0 kg, foram de 593,59 g/dia. Esse mesmo animal, consumindo 7,40 kg de MS/dia, necessita de dietas com 12,97% de PB na MS total. A exigência líquida de proteína para ganho de peso de animais anelorados não-castrados em pastejo, com até 350 kg de peso corporal, reduzem conforme o peso corporal ou a energia retida aumentam e pode ser estimada a partir da equação: PR (g/dia = -26,2946 + 247,4853 x GPCVZ - 24,8436 x ER.The objective of this trial was to determine the protein requirements of Zebu bovines on Brachiaria decumbens Stapf. pasture supplemented with concentrate. It was used 28 non-castrated Zebu steers at an average age of 7 months and 180 kg average weight. Eight baseline animals were slaughtered to determine the initial body composition. The 20 remaining animals were divided

  6. G-protein stimulatory subunit alpha and Gq/11α G-proteins are both required to maintain quiescent stem-like chondrocytes

    OpenAIRE

    Chagin, Andrei S; Vuppalapati, Karuna K; Kobayashi, Tatsuya; Guo, Jun; Hirai, Takao; Chen, Min; Offermanns, Stefan; Lee S Weinstein; Kronenberg, Henry M.

    2014-01-01

    Round chondrocytes in the resting zone of the growth plate provide precursors for columnar chondrocytes and have stem-like properties. Here we demonstrate that these stem-like chondrocytes undergo apoptosis in the absence of the receptor (PPR) for parathyroid hormone-related protein. We examine the possible roles of heterotrimeric G-proteins activated by the PPR. Inactivation of the G-protein stimulatory α-subunit (Gsα) leads to accelerated differentiation of columnar chondrocytes, as seen in...

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

    OpenAIRE

    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 ...

  8. Memory formation for trace fear conditioning requires ubiquitin-proteasome mediated protein degradation in the prefrontal cortex.

    OpenAIRE

    Helmstetter, Fred J.

    2013-01-01

    The cellular mechanisms supporting plasticity during memory consolidation have been a subject of considerable interest. De novo protein and mRNA synthesis in several brain areas are critical, and more recently protein degradation, mediated by the ubiquitin-proteasome system (UPS), has been shown to be important. Previous work clearly establishes a relationship between protein synthesis and protein degradation in the amygdala, but it is unclear whether cortical mechanisms of memory consolidati...

  9. Dimerization of the Papillomavirus E2 Protein Is Required for Efficient Mitotic Chromosome Association and Brd4 Binding▿

    OpenAIRE

    Cardenas-Mora, Juan; Spindler, Jonathan E.; Jang, Moon Kyoo; Alison A McBride

    2008-01-01

    The E2 proteins of several papillomaviruses link the viral genome to mitotic chromosomes to ensure retention and the efficient partitioning of genomes into daughter cells following cell division. Bovine papillomavirus type 1 E2 binds to chromosomes in a complex with Brd4, a cellular bromodomain protein. Interaction with Brd4 is also important for E2-mediated transcriptional regulation. The transactivation domain of E2 is crucial for interaction with the Brd4 protein; proteins lacking or mutat...

  10. Energy-requiring translocation of the OmpA protein and alkaline phosphatase of Escherichia coli into inner membrane vesicles.

    OpenAIRE

    Rhoads, D B; Tai, P C; Davis, B D

    1984-01-01

    In developing a reliable in vitro system for translocating bacterial proteins, we found that the least dense subfraction of the membrane of Escherichia coli was superior to the total inner membrane, both for a secreted protein (alkaline phosphatase) and for an outer membrane protein (OmpA). Compounds that eliminated the proton motive force inhibited translocation, as already observed in cells; since protein synthesis continued, the energy for translocation appears to be derived from the energ...

  11. A DNA Damage Response Screen Identifies RHINO: a 9-1-1 and TopBP1 interacting protein required for ATR signaling

    OpenAIRE

    Cotta-Ramusino, Cecilia; 3, E. Robert McDonald; Hurov, Kristen; Sowa, Mathew E.; Harper, J. Wade; Elledge, Stephen J.

    2011-01-01

    The DNA damage response (DDR) is a protein kinase cascade that orchestrates DNA repair processes via transcriptional and post-translational mechanisms. Cell cycle arrest is a hallmark of the DDR. We performed a damage-induced cell cycle arrest screen and uncovered a critical role for Fanconi anemia (FA) and homologous recombination (HR) proteins in ATR signaling. HR was required to maintain prolonged cell cycle arrest and to prevent massive genomic instability. Over 100 high scoring DDR candi...

  12. Membrane topology and mutational analysis of the TolQ protein of Escherichia coli required for the uptake of macromolecules and cell envelope integrity.

    OpenAIRE

    Vianney, A; Lewin, T M; Beyer, W F; Lazzaroni, J C; Portalier, R; Webster, R E

    1994-01-01

    TolQ is a 230-amino-acid protein required to maintain the integrity of the bacterial envelope and to facilitate the import of both filamentous bacteriophage and group A colicins. Cellular fractionation experiments showed TolQ to be localized to the cytoplasmic membrane. Bacteria expressing a series of TolQ-beta-galactosidase and TolQ-alkaline phosphatase fusion proteins were analyzed for the appropriate enzyme activity, membrane location, and sensitivity to exogenously added protease. The res...

  13. Export of malaria proteins requires co-translational processing of the PEXEL motif independent of phosphatidylinositol-3-phosphate binding.

    Science.gov (United States)

    Boddey, Justin A; O'Neill, Matthew T; Lopaticki, Sash; Carvalho, Teresa G; Hodder, Anthony N; Nebl, Thomas; Wawra, Stephan; van West, Pieter; Ebrahimzadeh, Zeinab; Richard, Dave; Flemming, Sven; Spielmann, Tobias; Przyborski, Jude; Babon, Jeff J; Cowman, Alan F

    2016-01-01

    Plasmodium falciparum exports proteins into erythrocytes using the Plasmodium export element (PEXEL) motif, which is cleaved in the endoplasmic reticulum (ER) by plasmepsin V (PMV). A recent study reported that phosphatidylinositol-3-phosphate (PI(3)P) concentrated in the ER binds to PEXEL motifs and is required for export independent of PMV, and that PEXEL motifs are functionally interchangeable with RxLR motifs of oomycete effectors. Here we show that the PEXEL does not bind PI(3)P, and that this lipid is not concentrated in the ER. We find that RxLR motifs cannot mediate export in P. falciparum. Parasites expressing a mutated version of KAHRP, with the PEXEL motif repositioned near the signal sequence, prevented PMV cleavage. This mutant possessed the putative PI(3)P-binding residues but is not exported. Reinstatement of PEXEL to its original location restores processing by PMV and export. These results challenge the PI(3)P hypothesis and provide evidence that PEXEL position is conserved for co-translational processing and export. PMID:26832821

  14. 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

    2016-04-15

    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 planarianSchmidtea mediterraneaand 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

  15. Actin-associated protein palladin is required for migration behavior and differentiation potential of C2C12 myoblast cells

    International Nuclear Information System (INIS)

    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

  16. 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.

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

    Science.gov (United States)

    Natsume, Toyoaki; Tsutsui, Yasuhiro; Sutani, Takashi; Dunleavy, Elaine M; Pidoux, Alison L; Iwasaki, Hiroshi; Shirahige, Katsuhiko; Allshire, Robin C; Yamao, Fumiaki

    2008-01-01

    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. PMID:18493607

  18. 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.

  19. Adaptor protein complexes AP-1 and AP-3 are required by the HHV-7 Immunoevasin U21 for rerouting of class I MHC molecules to the lysosomal compartment.

    Directory of Open Access Journals (Sweden)

    Lisa A Kimpler

    Full Text Available The human herpesvirus-7 (HHV-7 U21 gene product binds to class I major histocompatibility complex (MHC molecules and reroutes them to a lysosomal compartment. Trafficking of integral membrane proteins to lysosomes is mediated through cytoplasmic sorting signals that recruit heterotetrameric clathrin adaptor protein (AP complexes, which in turn mediate protein sorting in post-Golgi vesicular transport. Since U21 can mediate rerouting of class I molecules to lysosomes even when lacking its cytoplasmic tail, we hypothesize the existence of a cellular protein that contains the lysosomal sorting information required to escort class I molecules to the lysosomal compartment. If such a protein exists, we expect that it might recruit clathrin adaptor protein complexes as a means of lysosomal sorting. Here we describe experiments demonstrating that the μ adaptins from AP-1 and AP-3 are involved in U21-mediated trafficking of class I molecules to lysosomes. These experiments support the idea that a cellular protein(s is necessary for U21-mediated lysosomal sorting of class I molecules. We also examine the impact of transient versus chronic knockdown of these adaptor protein complexes, and show that the few remaining μ subunits in the cells are eventually able to reroute class I molecules to lysosomes.

  20. Repair of DNA double-strand breaks in Escherichia coli cells requires synthesis of proteins that can be induced by UV light

    International Nuclear Information System (INIS)

    The repair of DNA double-strand breaks in Escherichia coli cells irradiated with γ rays occurs only after new proteins are synthesized in response to damage introduced in the genome DNA. One protein whose synthesis is thus induced is the recA protein, and previous work has shown that recA- cells do not repair double-strand breaks. However, inducing recA protein by treating cells with nalidixic acid does not induce repair of double-strand breaks, so this repair requires more than the presence of the recA protein. When repair of double-strand breaks is blocked, the genome DNA is degraded by an endonuclease-like action. Evidence is presented to show that the inducible inhibition of DNA degradation after x-irradiation [Pollard, E.C. and Randall, E.P. (1973) Radiat. Res. 55, 265] is probably caused by the inducible repair of DNA double-strand breaks

  1. Truncation Analysis of TatA and TatB Defines the Minimal Functional Units Required for Protein Translocation

    OpenAIRE

    Lee, Philip A.; Buchanan, Grant; Stanley, Nicola R.; Berks, Ben C.; Palmer, Tracy

    2002-01-01

    The TatA and TatB proteins are essential components of the twin arginine protein translocation pathway in Escherichia coli. C-terminal truncation analysis of the TatA protein revealed that a plasmid-expressed TatA protein shortened by 40 amino acids is still fully competent to support protein translocation. Similar truncation analysis of TatB indicated that the final 30 residues of TatB are dispensable for function. Further deletion experiments with TatB indicated that removal of even 70 resi...

  2. 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.

  3. Requirement of energy and protein of beef cattle on tropical pasture - doi: 10.4025/actascianimsci.v35i4.21143

    OpenAIRE

    Eriton Egidio Lisboa Valente; Mário Fonseca Paulino; Edenio Detmann; Sebastião de Campos Valadares Filho; Javier Enrique Garces Cardenas; Isabela Fernanda Teixeira Dias

    2013-01-01

    Protein and energy requirements of beef cattle, aged between 4 and 18 months old, on tropical pastures, were estimated. Forty-six Nellore calves (138.3 ± 3.4 kg BW and 90-150 days old), kept on pasture, were distributed in a maintenance group (restricted feeding) or in nutritional plans, receiving a supplements with different amounts of protein and carbohydrate or non-supplemented. The net energy requirement for weight gain (NEg) was obtained by linear regression of logarithm of retained ener...

  4. 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

  5. Requirement of energy and protein of beef cattle on tropical pasture - doi: 10.4025/actascianimsci.v35i4.21143

    Directory of Open Access Journals (Sweden)

    Eriton Egidio Lisboa Valente

    2013-10-01

    Full Text Available Protein and energy requirements of beef cattle, aged between 4 and 18 months old, on tropical pastures, were estimated. Forty-six Nellore calves (138.3 ± 3.4 kg BW and 90-150 days old, kept on pasture, were distributed in a maintenance group (restricted feeding or in nutritional plans, receiving a supplements with different amounts of protein and carbohydrate or non-supplemented. The net energy requirement for weight gain (NEg was obtained by linear regression of logarithm of retained energy as a function of logarithm of empty body weight gain. The net energy requirement for maintenance (NEm was estimated by the exponential relation between heat production and metabolizable energy intake. Net protein requirement for weight gain (RP was estimated by multiple linear regression of retained protein in the weight gain of empty body (EBWG and retained energy. The efficiency of metabolizable energy (ME used for maintenance and for weight gain was 0.55 and 0.26, respectively. The ME requirement for maintenance was 124 kcal EBW-0.75. RP decreased in proportion to body weight increase. NEg and RP may be obtained by equations: RE (Mcal kg-1 = 0.044 x EBW0.75 x EBWG1.1302 and RP (g day-1 = -31.45 + 229.69 x EBWG – 8.75 x RE, respectively.  

  6. Direct interaction of TFIIB and the IE protein of equine herpesvirus 1 is required for maximal trans-activation function

    International Nuclear Information System (INIS)

    Recently, we reported that the immediate-early (IE) protein of equine herpesvirus 1 (EHV-1) associates with transcription factor TFIIB [J. Virol. 75 (2001), 10219]. In the current study, the IE protein purified as a glutathione-S-transferase (GST) fusion protein was shown to interact directly with purified TFIIB in GST-pulldown assays. A panel of TFIIB mutants employed in protein-binding assays revealed that residues 125 to 174 within the first direct repeat of TFIIB mediate its interaction with the IE protein. This interaction is physiologically relevant as transient transfection assays demonstrated that (1) exogenous native TFIIB did not perturb IE protein function, and (2) ectopic expression of a TFIIB mutant that lacked the IE protein interactive domain significantly diminished the ability of the IE protein to trans-activate EHV-1 promoters. These results suggest that an interaction of the IE protein with TFIIB is an important aspect of the regulatory role of the IE protein in the trans-activation of EHV-1 promoters

  7. 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

  8. Interaction of the TGGCA-binding protein with upstream sequences is required for efficient transcription of mouse mammary tumor virus.

    OpenAIRE

    Miksicek, R; Borgmeyer, U; Nowock, J

    1987-01-01

    A high-affinity binding site for the TGGCA-binding protein, also known as nuclear factor I, has previously been shown to reside within the mouse mammary tumor virus (MMTV) long terminal repeat. We have introduced mutations into this binding site to test the importance of this ubiquitous nuclear protein in MMTV transcription. Mutations which abolish the binding of the TGGCA protein in vitro are shown to impair strongly glucocorticoid-induced transcription from this promoter in vivo. These data...

  9. Three distinct domains of SSI-1/SOCS-1/JAB protein are required for its suppression of interleukin 6 signaling

    OpenAIRE

    Narazaki, Masashi; Fujimoto, Minoru; Matsumoto, Tomoshige; Morita, Yoshiaki; SAITO, HIROSHI; Kajita, Tadahiro; Yoshizaki, Kazuyuki; Naka, Tetsuji; Kishimoto, Tadamitsu

    1998-01-01

    Cytokine-inducible protein SSI-1 [signal transducers and activators of transcription (STAT)-induced STAT inhibitor 1, also referred to as SOCS-1 (suppressor of cytokine signaling 1) or JAB (Janus kinase-binding protein)] negatively regulates cytokine receptor signaling by inhibition of JAK kinases. The SSI family of proteins includes eight members that are structurally characterized by an SH2 domain and a C-terminal conserved region that we have called the SC-motif. In this study, we investig...

  10. 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

  11. Activation of Hormone-sensitive Lipase Requires Two Steps, Protein Phosphorylation and Binding to the PAT-1 Domain of Lipid Droplet Coat Proteins*

    OpenAIRE

    Wang, Hong; Hu, Liping; Dalen, Knut; Dorward, Heidi; Marcinkiewicz, Amy; Russell, Deanna; Gong, Dawei; Londos, Constantine; Yamaguchi, Tomohiro; Holm, Cecilia; Rizzo, Mark A.; Brasaemle, Dawn; Sztalryd, Carole

    2009-01-01

    Lipolysis is an important metabolic pathway controlling energy homeostasis through degradation of triglycerides stored in lipid droplets and release of fatty acids. Lipid droplets of mammalian cells are coated with one or more members of the PAT protein family, which serve important functions in regulating lipolysis. In this study, we investigate the mechanisms by which PAT family members, perilipin A, adipose differentiation-related protein (ADFP), and LSDP5, control lipolysis catalyzed by h...

  12. Viral protein requirements for assembly and release of human parainfluenza virus type 3 virus-like particles.

    Science.gov (United States)

    Bracken, Megan K; Hayes, Brandon C; Kandel, Suresh R; Scott-Shemon, Deja; Ackerson, Larissa; Hoffman, Michael A

    2016-06-01

    To understand the roles of human parainfluenza virus 3 (HPIV3) proteins in assembly and release, viral proteins were expressed individually and in combination in 293T cells. Expression of the matrix (M) protein triggered release of enveloped, matrix-containing virus-like particles (VLPs) from cells. When M was co-expressed with the nucleocapsid (N), fusion (F) or haemagglutinin-neuraminidase (HN) proteins, VLPs that contained M+N, M+F and M+HN, respectively, were generated, suggesting that M can independently interact with each protein to facilitate assembly and release. Additionally, expression of N protein enabled incorporation of the phosphoprotein (P) into VLPs, likely due to known N-P interactions. Finally, the HPIV3 C protein did not enhance VLP release, in contrast to observations with the related Sendai virus. These findings reinforce the central importance of the M protein in virus assembly and release, but also illustrate the variable roles of other paramyxovirus proteins during these processes. PMID:26960133

  13. Role of RecA protein in untargeted UV mutagenesis of bacteriophage lambda: evidence for the requirement for the dinB gene

    International Nuclear Information System (INIS)

    Untargeted UV mutagenesis of bacteriophage lambda--i.e., the increased recovery of lambda mutants when unirradiated lambda infects UV-irradiated Escherichia coli--is thought to be mediated by a transient decrease in DNA replication fidelity, generating mutations in the newly synthesized strands. Using the bacteriophage lambda cI857----lambda c mutation system, we provide evidence that the RecA protein, shown previously to be required for this mutagenic pathway, is no longer needed when the LexA protein is inactivated by mutation. We suggest that the error-prone DNA replication responsible for UV-induced untargeted mutagenesis is turned on by the presence of replication-blocking lesions in the host cell DNA and that the RecA protein is required only to derepress the relevant din gene(s). This is in contrast to mutagenesis of irradiated bacteria or irradiated phage lambda, in which activated RecA protein has a second role in mutagenesis in addition to the cleavage of the LexA protein. Among the tested din genes, the dinB gene product (in addition to the uvrA and uvrB gene products) was found to be required for untargeted mutagenesis of bacteriophage lambda. To our knowledge, a phenotype associated with the dinB gene has not been reported previously

  14. FBXO22 Protein Is Required for Optimal Synthesis of the N-Methyl-d-Aspartate (NMDA) Receptor Coagonist d-Serine

    DEFF Research Database (Denmark)

    Dikopoltsev, Elena; Foltyn, Veronika N; Zehl, Martin; Jensen, Ole Nørregaard; Mori, Hisashi; Radzishevsky, Inna; Wolosker, Herman

    2014-01-01

    is known about the regulation of d-serine synthesis. We now demonstrate that the F-box only protein 22 (FBXO22) interacts with SR and is required for optimal d-serine synthesis in cells. Although FBXO22 is classically associated with the ubiquitin system and is recruited to the Skip1-Cul1-F-box E3...

  15. Association between receptor protein-tyrosine phosphatase RPTPalpha and the Grb2 adaptor. Dual Src homology (SH) 2/SH3 domain requirement and functional consequences

    DEFF Research Database (Denmark)

    Su, J; Yang, L T; Sap, J

    1996-01-01

    binding site in RPTPalpha was studied further by expression of wild type or mutant RPTPalpha proteins in PC12 cells. In these cells, wild type RPTPalpha interferes with acidic fibroblast growth factor-induced neurite outgrowth; this effect requires both the catalytic activity and the Grb2 binding Tyr798...

  16. 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 c...

  17. The impact of vernalization requirement, photoperiod sensitivity, and earliness per se on grain protein content of bread wheat (Triticum aestivum L.)

    Science.gov (United States)

    In wheat, a shorter pre-anthesis phase is often associated with increased grain protein content (GPC) but decreased grain yield. Cultivar differences in pre-anthesis development are mainly determined by vernalization requirement, photoperiod sensitivity and earliness per se. This research examines w...

  18. 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.

  19. Application of 15N-glycine to study the protein requirements of young Filipino children consuming local diets

    International Nuclear Information System (INIS)

    This report presents the results obtained from nitrogen balance studies and protein turnover rate measurements on a total of 7 children who served as subjects in this study which aims to evaluate the adequacy of the current Recommended Dietary Allowance for dietary protein for 2-4 y old children at or around the safe level of energy intake. (author). 1 fig., 5 tabs

  20. Drainin required for membrane fusion of the contractile vacuole in Dictyostelium is the prototype of a protein family also represented in man.

    Science.gov (United States)

    Becker, M; Matzner, M; Gerisch, G

    1999-01-01

    The contractile vacuole expels water by forming a channel with the plasma membrane and thus enables cells to survive in a hypo-osmotic environment. Here we characterize drainin, a Dictyostelium protein involved in this process, as the first member of a protein family represented in fission yeast, Caenorhabditis elegans and man. Gene replacement in Dictyostelium shows that drainin acts at a checkpoint of channel formation between the contractile vacuole and the plasma membrane. A green fluorescent protein fusion of drainin localizes specifically to the contractile vacuole and rescues its periodic discharge in drainin-null cells. Drainin is a peripheral membrane protein, requiring a short hydrophobic stretch in its C-terminal region for localization and function. We suggest that drainin acts in a signaling cascade that couples a volume-sensing device in the vacuolar membrane to the membrane fusion machinery. PMID:10369671

  1. Classic nuclear localization signals and a novel nuclear localization motif are required for nuclear transport of porcine parvovirus capsid proteins.

    Science.gov (United States)

    Boisvert, Maude; Bouchard-Lévesque, Véronique; Fernandes, Sandra; Tijssen, Peter

    2014-10-01

    Nuclear targeting of capsid proteins (VPs) is important for genome delivery and precedes assembly in the replication cycle of porcine parvovirus (PPV). Clusters of basic amino acids, corresponding to potential nuclear localization signals (NLS), were found only in the unique region of VP1 (VP1up, for VP1 unique part). Of the five identified basic regions (BR), three were important for nuclear localization of VP1up: BR1 was a classic Pat7 NLS, and the combination of BR4 and BR5 was a classic bipartite NLS. These NLS were essential for viral replication. VP2, the major capsid protein, lacked these NLS and contained no region with more than two basic amino acids in proximity. However, three regions of basic clusters were identified in the folded protein, assembled into a trimeric structure. Mutagenesis experiments showed that only one of these three regions was involved in VP2 transport to the nucleus. This structural NLS, termed the nuclear localization motif (NLM), is located inside the assembled capsid and thus can be used to transport trimers to the nucleus in late steps of infection but not for virions in initial infection steps. The two NLS of VP1up are located in the N-terminal part of the protein, externalized from the capsid during endosomal transit, exposing them for nuclear targeting during early steps of infection. Globally, the determinants of nuclear transport of structural proteins of PPV were different from those of closely related parvoviruses. Importance: Most DNA viruses use the nucleus for their replication cycle. Thus, structural proteins need to be targeted to this cellular compartment at two distinct steps of the infection: in early steps to deliver viral genomes to the nucleus and in late steps to assemble new viruses. Nuclear targeting of proteins depends on the recognition of a stretch of basic amino acids by cellular transport proteins. This study reports the identification of two classic nuclear localization signals in the minor capsid

  2. Protein Factor Requirements of the Apaf-1 Internal Ribosome Entry Segment: Roles of Polypyrimidine Tract Binding Protein and upstream of N-ras

    OpenAIRE

    Mitchell, Sally A.; Brown, Emma C; Coldwell, Mark J; Jackson, Richard J.; Willis, Anne E.

    2001-01-01

    It has been reported previously that the 5′ untranslated region of the mRNA encoding Apaf-1 (apoptotic protease-activating factor 1) has an internal ribosome entry site (IRES), whose activity varies widely among different cell types. Here it is shown that the Apaf-1 IRES is active in rabbit reticulocyte lysates, provided that the system is supplemented with polypyrimidine tract binding protein (PTB) and upstream of N-ras (unr), two cellular RNA binding proteins previously identified to be req...

  3. Polyglutamylated Tubulin Binding Protein C1orf96/CSAP Is Involved in Microtubule Stabilization in Mitotic Spindles

    Science.gov (United States)

    Ohta, Shinya; Hamada, Mayako; Sato, Nobuko; Toramoto, Iyo

    2015-01-01

    The centrosome-associated C1orf96/Centriole, Cilia and Spindle-Associated Protein (CSAP) targets polyglutamylated tubulin in mitotic microtubules (MTs). Loss of CSAP causes critical defects in brain development; however, it is unclear how CSAP association with MTs affects mitosis progression. In this study, we explored the molecular mechanisms of the interaction of CSAP with mitotic spindles. Loss of CSAP caused MT instability in mitotic spindles and resulted in mislocalization of Nuclear protein that associates with the Mitotic Apparatus (NuMA), with defective MT dynamics. Thus, CSAP overload in the spindles caused extensive MT stabilization and recruitment of NuMA. Moreover, MT stabilization by CSAP led to high levels of polyglutamylation on MTs. MT depolymerization by cold or nocodazole treatment was inhibited by CSAP binding. Live-cell imaging analysis suggested that CSAP-dependent MT-stabilization led to centrosome-free MT aster formation immediately upon nuclear envelope breakdown without γ-tubulin. We therefore propose that CSAP associates with MTs around centrosomes to stabilize MTs during mitosis, ensuring proper bipolar spindle formation and maintenance. PMID:26562023

  4. 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. PMID:17346982

  5. Multimerization of the cytoplasmic domain of syndecan-4 is required for its ability to activate protein kinase C

    DEFF Research Database (Denmark)

    Oh, E S; Woods, A; Couchman, J R

    1997-01-01

    corresponding to the central region of syndecan-4 cytoplasmic domain (4V) also oligomerizes. The degree of oligomerization correlates with the previously reported ability to bind protein kinase C (PKC) and regulate its activity. Only multimeric recombinant syndecan-4 core protein, but not the monomeric protein......, potentiated the activity of PKCalpha, and only oligomeric syndecan-4 cytoplasmic peptides were active. Changes in peptide sequence caused parallel loss of stable oligomeric status and ability to regulate a mixture of PKCalphabetagamma activity. A synthetic peptide encompassing the whole cytoplasmic domain of...

  6. The gene encoding cAMP receptor protein is required for competence development in Haemophilus influenzae Rd.

    OpenAIRE

    Chandler, M S

    1992-01-01

    The Haemophilus influenzae Rd strain JG87 contains a single mini-Tn10kan insertion that causes a deficiency in the development of competence for genetic transformation. The DNA fragment containing this insertion mutation, as well as the wild-type locus, was cloned, mapped, and sequenced. The sequence contained an open reading frame for a protein of 224 amino acids with a predicted Mr of 25,152. The deduced protein sequence showed strong similarity to the Escherichia coli cAMP receptor protein...

  7. 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.

  8. Vernalization requirement duration in winter wheat is controlled by TaVRN-A1 at the protein level

    OpenAIRE

    Li, Genqiao; Yu, Ming; Fang, Tilin; Cao, Shuanghe; Carver, Brett F; Yan, Liuling

    2013-01-01

    Winter wheat requires a period of low temperatures to accelerate flowering (vernalization). This requirement could make winter wheat more vulnerable to elevated global temperature via insufficient vernalization. All known vernalization genes are cloned according to qualitative variation in vernalization requirement between spring and winter wheat, but the genes controlling quantitative variation for more or less vernalization requirement among winter wheat cultivars remain unknown. We report ...

  9. 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.

  10. DNA cleavage by CgII and NgoAVII requires interaction between N- and R-proteins and extensive nucleotide hydrolysis.

    Science.gov (United States)

    Zaremba, Mindaugas; Toliusis, Paulius; Grigaitis, Rokas; Manakova, Elena; Silanskas, Arunas; Tamulaitiene, Giedre; Szczelkun, Mark D; Siksnys, Virginijus

    2014-12-16

    The stress-sensitive restriction-modification (RM) system CglI from Corynebacterium glutamicum and the homologous NgoAVII RM system from Neisseria gonorrhoeae FA1090 are composed of three genes: a DNA methyltransferase (M.CglI and M.NgoAVII), a putative restriction endonuclease (R.CglI and R.NgoAVII, or R-proteins) and a predicted DEAD-family helicase/ATPase (N.CglI and N.NgoAVII or N-proteins). Here we report a biochemical characterization of the R- and N-proteins. Size-exclusion chromatography and SAXS experiments reveal that the isolated R.CglI, R.NgoAVII and N.CglI proteins form homodimers, while N.NgoAVII is a monomer in solution. Moreover, the R.CglI and N.CglI proteins assemble in a complex with R2N2 stoichiometry. Next, we show that N-proteins have ATPase activity that is dependent on double-stranded DNA and is stimulated by the R-proteins. Functional ATPase activity and extensive ATP hydrolysis (∼170 ATP/s/monomer) are required for site-specific DNA cleavage by R-proteins. We show that ATP-dependent DNA cleavage by R-proteins occurs at fixed positions (6-7 nucleotides) downstream of the asymmetric recognition sequence 5'-GCCGC-3'. Despite similarities to both Type I and II restriction endonucleases, the CglI and NgoAVII enzymes may employ a unique catalytic mechanism for DNA cleavage. PMID:25429977

  11. Gonococcal transferrin-binding protein 1 is required for transferrin utilization and is homologous to TonB-dependent outer membrane receptors.

    Science.gov (United States)

    Cornelissen, C N; Biswas, G D; Tsai, J; Paruchuri, D K; Thompson, S A; Sparling, P F

    1992-09-01

    The pathogenic Neisseria species are capable of utilizing transferrin as their sole source of iron. A neisserial transferrin receptor has been identified and its characteristics defined; however, the biochemical identities of proteins which are required for transferrin receptor function have not yet been determined. We identified two iron-repressible transferrin-binding proteins in Neisseria gonorrhoeae, TBP1 and TBP2. Two approaches were taken to clone genes required for gonococcal transferrin receptor function. First, polyclonal antiserum raised against TBP1 was used to identify clones expressing TBP1 epitopes. Second, a wild-type gene copy was cloned that repaired the defect in a transferrin receptor function (trf) mutant. The clones obtained by these two approaches were shown to overlap by DNA sequencing. Transposon mutagenesis of both clones and recombination of mutagenized fragments into the gonococcal chromosome generated mutants that showed reduced binding of transferrin to whole cells and that were incapable of growth on transferrin. No TBP1 was produced in these mutants, but TBP2 expression was normal. The DNA sequence of the gene encoding gonococcal TBP1 (tbpA) predicted a protein sequence homologous to the Escherichia coli and Pseudomonas putida TonB-dependent outer membrane receptors. Thus, both the function and the predicted protein sequence of TBP1 were consistent with this protein serving as a transferrin receptor. PMID:1325963

  12. Fragile X Mental Retardation Protein is Required for Synapse Elimination by the Activity-Dependent Transcription Factor MEF2

    OpenAIRE

    Pfeiffer, Brad E.; Zang, Tong; Wilkerson, Julia R.; Taniguchi, Makoto; Maksimova, Marina A.; Smith, Laura N.; Cowan, Christopher W.; Huber, Kimberly M.

    2010-01-01

    Fragile X Syndrome (FXS), the most common genetic form of mental retardation and autism, is caused by loss of function mutations in an RNA binding protein, Fragile X Mental Retardation Protein (FMRP). Patients’ neurons, as well as those of the mouse model, Fmr1 knockout (KO), are characterized by an excess of dendritic spines, suggesting a deficit in excitatory synapse elimination. In response to neuronal activity, myocyte enhancing factor 2 (MEF2) transcription factors induce robust synapse ...

  13. Daple is a novel non-receptor GEF required for trimeric G protein activation in Wnt signaling.

    Science.gov (United States)

    Aznar, Nicolas; Midde, Krishna K; Dunkel, Ying; Lopez-Sanchez, Inmaculada; Pavlova, Yelena; Marivin, Arthur; Barbazán, Jorge; Murray, Fiona; Nitsche, Ulrich; Janssen, Klaus-Peter; Willert, Karl; Goel, Ajay; Abal, Miguel; Garcia-Marcos, Mikel; Ghosh, Pradipta

    2015-01-01

    Wnt signaling is essential for tissue homeostasis and its dysregulation causes cancer. Wnt ligands trigger signaling by activating Frizzled receptors (FZDRs), which belong to the G-protein coupled receptor superfamily. However, the mechanisms of G protein activation in Wnt signaling remain controversial. In this study, we demonstrate that FZDRs activate G proteins and trigger non-canonical Wnt signaling via the Dishevelled-binding protein, Daple. Daple contains a Gα-binding and activating (GBA) motif, which activates Gαi proteins and an adjacent domain that directly binds FZDRs, thereby linking Wnt stimulation to G protein activation. This triggers non-canonical Wnt responses, that is, suppresses the β-catenin/TCF/LEF pathway and tumorigenesis, but enhances PI3K-Akt and Rac1 signals and tumor cell invasiveness. In colorectal cancers, Daple is suppressed during adenoma-to-carcinoma transformation and expressed later in metastasized tumor cells. Thus, Daple activates Gαi and enhances non-canonical Wnt signaling by FZDRs, and its dysregulation can impact both tumor initiation and progression to metastasis. PMID:26126266

  14. Several Days of CPEB-Dependent Local Protein Synthesis Are Required to Stabilize Synaptic Growth for Persistence of Long-Term Facilitation in Aplysia

    OpenAIRE

    Miniaci, Maria Concetta; Kim, Joung-Hun; Puthenveettil, Sathyanarayanan; Si, Kausik; Zhu, Huixiang; Kandel, Eric R.; Bailey, Craig H.

    2008-01-01

    The time course of the requirement for local protein synthesis in the stabilization of learning-related synaptic growth and the persistence of long-term memory was examined using Aplysia bifurcated sensory neuron-motor neuron cultures. We find that following repeated pulses of serotonin (5-HT) the local perfusion of emetine, an inhibitor of protein synthesis, or a TAT-AS oligonucleotide directed against ApCPEB blocks long-term facilitation (LTF) at either 24 hr or 48 hr and leads to a selecti...

  15. HHR23A, a human homolog of Saccharomyces cerevisiae Rad23, regulates xeroderma pigmentosum C protein and is required for nucleotide excision repair

    International Nuclear Information System (INIS)

    HHR23A and hHR23B are the human homologs of Saccharomyces cerevisiae Rad23. hHR23B is associated with the nucleotide excision repair (NER) factor xeroderma pigmentosum C (XPC) protein and is required for global genome repair. The function of hHR23A is not yet clear. In this study, the potential function of the hHR23A protein was investigated using RNA interference techniques. The hHR23A knock-down (KD) construct diminished the RNA level of hHR23A protein by approximately 60%, and it did not interfere with expression of the hHR23B gene. Based on Southwestern immunoblot and host-cell reactivation assays, hHR23AKD cells were found to be deficient in DNA repair activity against the DNA damage caused by UVC irradiation. In these hHR23AKD cells, the XPC gene was not normally induced by UVC irradiation, indicating that the hHR23A protein is involved in NER through regulation of the DNA damage recognition protein XPC. Co-immunoprecipitation experiments revealed that hHR23A was associated with a small portion of hHR23B and the majority of p53 protein, indicating that hHR23A regulates the function of XPC by its association with the NER activator p53

  16. Memory formation for trace fear conditioning requires ubiquitin-proteasome mediated protein degradation in the prefrontal cortex.

    Directory of Open Access Journals (Sweden)

    David S Reis

    2013-10-01

    Full Text Available The cellular mechanisms supporting plasticity during memory consolidation have been a subject of considerable interest. De novo protein and mRNA synthesis in several brain areas are critical, and more recently protein degradation, mediated by the ubiquitin-proteasome system (UPS, has been shown to be important. Previous work clearly establishes a relationship between protein synthesis and protein degradation in the amygdala, but it is unclear whether cortical mechanisms of memory consolidation are similar to those in the amygdala. Recent work demonstrating a critical role for prefrontal cortex (PFC in the acquisition and consolidation of fear memory allows us to address this question. Here we use a PFC-dependent fear conditioning protocol to determine whether UPS mediated protein degradation is necessary for memory consolidation in PFC. Groups of rats were trained with auditory delay or trace fear conditioning and sacrificed 60 min after training. PFC tissue was then analyzed to quantify the amount of polyubiquinated protein. Other animals were trained with similar procedures but were infused with either a proteasome inhibitor (clasto-lactacystin β-lactone or a translation inhibitor (anisomycin in the PFC immediately after training. Our results show increased UPS-mediated protein degradation in the PFC following trace but not delay fear conditioning. Additionally, post-training proteasome or translation inhibition significantly impaired trace but not delay fear memory when tested the next day. Our results further support the idea that the PFC is critical for trace but not delay fear conditioning highlight the role of UPS-mediated degradation as critical for synaptic plasticity.

  17. RNA:protein ratio of the unicellular organism as a characteristic of phosphorous and nitrogen stoichiometry and of the cellular requirement of ribosomes for protein synthesis

    OpenAIRE

    Sams Carl E; Greenwood Duncan J; Karpinets Tatiana V; Ammons John T

    2006-01-01

    Abstract Background Mean phosphorous:nitrogen (P:N) ratios and relationships of P:N ratios with the growth rate of organisms indicate a surprising similarity among and within microbial species, plants, and insect herbivores. To reveal the cellular mechanisms underling this similarity, the macromolecular composition of seven microorganisms and the effect of specific growth rate (SGR) on RNA:protein ratio, the number of ribosomes, and peptide elongation rate (PER) were analyzed under different ...

  18. Adjuvant requirement for successful immunization with recombinant derivatives of Plasmodium vivax merozoite surface protein-1 delivered via the intranasal route

    Directory of Open Access Journals (Sweden)

    Daniel Y Bargieri

    2007-06-01

    Full Text Available Recently, we generated two bacterial recombinant proteins expressing 89 amino acids of the C-terminal domain of the Plasmodium vivax merozoite surface protein-1 and the hexa-histidine tag (His6MSP1(19. One of these recombinant proteins contained also the amino acid sequence of the universal pan allelic T-cell epitope (His6MSP1(19-PADRE. In the present study, we evaluated the immunogenic properties of these antigens when administered via the intra-nasal route in the presence of distinct adjuvant formulations. We found that C57BL/6 mice immunized with either recombinant proteins in the presence of the adjuvants cholera toxin (CT or the Escherichia coli heat labile toxin (LT developed high and long lasting titers of specific serum antibodies. The induced immune responses reached maximum levels after three immunizing doses with a prevailing IgG1 subclass response. In contrast, mice immunized by intranasal route with His6MSP1(19-PADRE in the presence of the synthetic oligonucleotides adjuvant CpG ODN 1826 developed lower antibody titers but when combined to CT, CpG addition resulted in enhanced IgG responses characterized by lower IgG1 levels. Considering the limitations of antigens formulations that can be used in humans, mucosal adjuvants can be a reliable alternative for the development of new strategies of immunization using recombinant proteins of P. vivax.

  19. Promoter opening (melting) and transcription initiation by RNA polymerase I requires neither nucleotide beta,gamma hydrolysis nor protein phosphorylation.

    OpenAIRE

    Lofquist, A K; Li, H; Imboden, M A; Paule, M. R.

    1993-01-01

    With some bacterial RNA polymerases and in eukaryotic RNA polymerase II, DNA melting during initiation requires the coupling of energy derived from beta,gamma hydrolysis of ATP. A detailed analysis of this possible requirement for eukaryotic RNA polymerase I reveals no such requirement. However, in some cases, beta,gamma non-hydrolyzable derivatives (beta,gamma imido or methylene) of nucleotide substrates have been found to significantly inhibit transcription initiation because of their ineff...

  20. The DNA replication priming protein, PriA, is required for homologous recombination and double-strand break repair.

    OpenAIRE

    Kogoma, T; Cadwell, G W; Barnard, K G; Asai, T

    1996-01-01

    The PriA protein, a component of the phiX174-type primosome, was previously shown to be essential for damage-inducible DNA replication in Escherichia coli, termed inducible stable DNA replication. Here, we show that priA::kan null mutants are defective in transductional and conjugational homologous recombination and are hypersensitive to mitomycin C and gamma rays, which cause double-strand breaks. The introduction of a plasmid carrying the priA300 allele, which encodes a mutant PriA protein ...

  1. The Legionella pneumophila major secretory protein, a protease, is not required for intracellular growth or cell killing.

    OpenAIRE

    Szeto, L.; Shuman, H A

    1990-01-01

    The Legionella pneumophila major secretory protein (Msp) is a Zn2+ metalloprotease whose function in pathogenesis is unknown. The structural gene for the Msp protease, mspA, was isolated from an L. pneumophila genomic library. In Escherichia coli which contain plasmids with the mspA gene, Msp protein and activity are found in the periplasmic space and the cytoplasm. Transposon mutagenesis with Tn9 of an mspA-containing plasmid in E. coli yielded mutants which no longer expressed protease acti...

  2. Casein kinase I-like protein kinases encoded by YCK1 and YCK2 are required for yeast morphogenesis.

    OpenAIRE

    Robinson, L. C.; Menold, M. M.; Garrett, S.; Culbertson, M R

    1993-01-01

    Casein kinase I is an acidotropic protein kinase class that is widely distributed among eukaryotic cell types. In the yeast Saccharomyces cerevisiae, the casein kinase I isoform encoded by the gene pair YCK1 and YCK2 is a 60- to 62-kDa membrane-associated form. The Yck proteins perform functions essential for growth and division; either alone supports growth, but loss of function of both is lethal. We report here that casein kinase I-like activity is associated with a soluble Yck2-beta-galact...

  3. 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; Nielsen, Henrik Bjørn; Harris, Cassandra A.; Beale, Michael H.; Andersen, Mathias; Mant, Alexandra; Scheller, Henrik Vibe; Camara, Bilal; Mattsson, Ole; Mundy, John

    2004-01-01

    The stable, recessive Arabidopsis variegated 3 (var3) mutant exhibits a variegated phenotype due to somatic areas lacking or containing developmentally retarded chloroplasts and greatly reduced numbers of palisade cells. The VAR3 gene, isolated by transposon tagging, encodes the 85.9 kDa VAR3 pro...... pigment profiles are qualitatively similar in wild type and var3, although var3 accumulates lower levels of chlorophylls and carotenoids. These results indicate that VAR3 is a part of a protein complex required for normal chloroplast and palisade cell development....... 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 that...

  4. A methodological approach to estimate the lactation curve and net energy and protein requirements of beef cows using nonlinear mixed-effects modeling.

    Science.gov (United States)

    Albertini, T Z; Medeiros, S R; Torres, R A A; Zocchi, S S; Oltjen, J W; Strathe, A B; Lanna, D P D

    2012-11-01

    The objective of this study was to evaluate methods to predict the secretion of milk and net energy and protein requirements of beef cows (Bos indicus and B. taurus) after approximately 1 mo postpartum using nonlinear mixed-effect modeling (NLME). Twenty Caracu × Nellore (CN) and 10 Nellore (NL) cows were inseminated to Red Angus bulls, and 10 Angus × Nellore (AN) were bred to Canchim bulls. Cows were evaluated from just after calving (25 ± 11 d) to weaning (220 d). Milk yield was estimated by weighing calves before and after suckling (WSW) and by machine milking (MM) methods at 25, 52, 80, 109, 136, 164, 193, and 220 ± 11 d of lactation. Brody and simple linear equations were consecutively fitted to the data and compared using information criteria. For the Brody equation, a NLME model was used to estimate all lactation profiles incorporating different sources of variation (calf sex and breed of cow, cow as a nested random effect, and within-cow auto-correlation). The CV for the MM method (29%) was less than WSW (45%). Consequently, the WSW method was responsible for reducing the variance about 1.5 times among individuals, which minimized the ability to detect differences among cows. As a result, only milk yield MM data were used in the NLME models. The Brody equation provided the best fit to this dataset, and inclusion of a continuous autoregressive process improved fit (P energy and protein yield at the beginning of lactation were affected by cow genotype and calf sex (P energy and net protein requirements for milk yield followed a similar ranking. Male calves stimulated their dams to produce 11.7, 11.4, and 11.9% more milk, energy and protein, respectively (P energy and protein requirements. The NLME is a powerful tool to describe differences in the secretion of milk due to heterosis and cell mammary external stimulus in beef cows. PMID:22665632

  5. Phospholipid translocation and miltefosine potency require both L. donovani miltefosine transporter and the new protein LdRos3 in Leishmania parasites.

    Science.gov (United States)

    Pérez-Victoria, F Javier; Sánchez-Cañete, María P; Castanys, Santiago; Gamarro, Francisco

    2006-08-18

    The antitumor drug miltefosine has been recently approved as the first oral drug active against visceral leishmaniasis. We have previously identified the L. donovani miltefosine transporter (LdMT) as a P-type ATPase involved in phospholipid translocation at the plasma membrane of Leishmania parasites. Here we show that this protein is essential but not sufficient for the phospholipid translocation activity and, thus, for the potency of the drug. Based on recent findings in yeast, we have identified the putative beta subunit of LdMT, named LdRos3, as another protein factor required for the translocation activity. LdRos3 belongs to the CDC50/Lem3 family, proposed as likely beta subunits for P4-ATPases. The phenotype of LdRos3-defective parasites was identical to that of the LdMT-/-, including a defect in the uptake of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)-phosphatidylserine, generally considered as not affected in Lem3p-deficient yeast. Both LdMT and LdRos3 normally localized to the plasma membrane but were retained inside the endoplasmic reticulum in the absence of the other protein or when inactivating point mutations were introduced in LdMT. Modulating the expression levels of either protein independently, we show that any one of them could behave as the protein limiting the level of flippase activity. Thus, LdMT and LdRos3 seem to form part of the same translocation machinery that determines flippase activity and miltefosine sensitivity in Leishmania, further supporting the consideration of CDC50/Lem3 proteins as beta subunits required for the normal functioning of P4-ATPases. PMID:16785229

  6. Regulation of protein biosynthesis by non-lymphoid cells requires the participation of receptors, which recognize the same protein through a center analogous to the antibody active center

    International Nuclear Information System (INIS)

    This paper studies the mechanism for regulating the biosynthesis of one of the complement components (anti-idiotypic antibodies CI /SUB q/ ) by macrophages. The experiments were conducted on mouse resident peritoneal macrophages cultivated in medium containing C 14-glycine. The synthesis of CI /SUB q/ was evaluated according to the content of protein which was bound by rabbit antibodies against mouse CI /SUB q/ immobilized on bromocyan-Sepharose 4B. The study of the kinetics of the biosynthesis of CI /SUB q/ by propagated macrophages shows that the biosynthesis was initially recorded and in the subsequent period the culture contained no other cells apart from macrophages

  7. Induction of p53-Independent Apoptosis by the Adenovirus E4orf4 Protein Requires Binding to the Bα Subunit of Protein Phosphatase 2A

    OpenAIRE

    Marcellus, Richard C.; Chan, Helen; Paquette, Denis; Thirlwell, Sarah; Boivin, Dominique; Branton, Philip E.

    2000-01-01

    Previous studies have indicated that the E4orf4 protein of human adenovirus type 2 (Ad2) induces p53-independent apoptosis. We believe that this process may play a role in cell death and viral spread at the final stages of productive infection. E4orf4 may also be of therapeutic value in treating some diseases, including cancer, through its ability to induce apoptosis when expressed individually. The only previously identified biochemical function of E4orf4 is its ability to associate with the...

  8. Studies on water turnover and water requirement in buffalo calves fed on different levels of energy and protein in the diet

    International Nuclear Information System (INIS)

    Experiments were conducted for measuring water turnover and water requirement in buffalo calves fed on different levels of energy and protein in the diet. There was significant difference (P<0.01) in the biological half life of tritiated water between various groups of animals. Water turnover rates were statistically (P<0.05) more in groups I and II (106.52 and 99.74 ml/kg/24 hr) than groups III and IV (93.03 and 77.09 ml/kg/24 hr). There was no significant difference in water requirement due to various treatments. (author)

  9. 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.

  10. 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.

  11. CD1d-mediated presentation of endogenous lipid antigens by adipocytes requires microsomal triglyceride transfer protein (MTP)

    DEFF Research Database (Denmark)

    Rakhshandehroo, Maryam; Gijzel, Sanne M W; Siersbæk, Rasmus;

    2014-01-01

    microsomal triglyceride transfer protein (MTP), 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...

  12. MavN is a Legionella pneumophila vacuole-associated protein required for efficient iron acquisition during intracellular growth

    Science.gov (United States)

    Isaac, Dervla T.; Laguna, Rita K.; Valtz, Nicole; Isberg, Ralph R.

    2015-01-01

    Iron is essential for the growth and virulence of most intravacuolar pathogens. The mechanisms by which microbes bypass host iron restriction to gain access to this metal across the host vacuolar membrane are poorly characterized. In this work, we identify a unique intracellular iron acquisition strategy used by Legionella pneumophila. The bacterial Icm/Dot (intracellular multiplication/defect in organelle trafficking) type IV secretion system targets the bacterial-derived MavN (more regions allowing vacuolar colocalization N) protein to the surface of the Legionella-containing vacuole where this putative transmembrane protein facilitates intravacuolar iron acquisition. The ΔmavN mutant exhibits a transcriptional iron-starvation signature before its growth is arrested during the very early stages of macrophage infection. This intracellular growth defect is rescued only by the addition of excess exogenous iron to the culture medium and not a variety of other metals. Consistent with MavN being a translocated substrate that plays an exclusive role during intracellular growth, the mutant shows no defect for growth in broth culture, even under severe iron-limiting conditions. Putative iron-binding residues within the MavN protein were identified, and point mutations in these residues resulted in defects specific for intracellular growth that are indistinguishable from the ΔmavN mutant. This model of a bacterial protein inserting into host membranes to mediate iron transport provides a paradigm for how intravacuolar pathogens can use virulence-associated secretion systems to manipulate and acquire host iron. PMID:26330609

  13. 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.

  14. Expression of Uncoupling Protein 1 in Mouse Brown Adipose Tissue Is Thyroid Hormone Receptor-β Isoform Specific and Required for Adaptive Thermogenesis

    OpenAIRE

    Ribeiro, Miriam O.; Bianco, Suzy D. C.; Kaneshige, Masahiro; Schultz, James J.; Cheng, Sheue-yann; Bianco, Antonio C.; Brent, Gregory A.

    2009-01-01

    Cold-induced adaptive (or nonshivering) thermogenesis in small mammals is produced primarily in brown adipose tissue (BAT). BAT has been identified in humans and becomes more active after cold exposure. Heat production from BAT requires sympathetic nervous system stimulation, T3, and uncoupling protein 1 (UCP1) expression. Our previous studies with a thyroid hormone receptor-β (TRβ) isoform-selective agonist demonstrated that after TRβ stimulation alone, adaptive thermogenesis was markedly im...

  15. A structural comparison of Listeria monocytogenes protein chaperones PrsA1 and PrsA2 reveals molecular features required for virulence.

    Science.gov (United States)

    Cahoon, Laty A; Freitag, Nancy E; Prehna, Gerd

    2016-07-01

    Listeria monocytogenes is a Gram-positive environmental bacterium that lives within soil but transitions into a pathogen upon contact with a mammalian host. The transition of L. monocytogenes from soil dweller to cytosolic pathogen is dependent upon secreted virulence factors that mediate cell invasion and intracellular growth. PrsA1 and PrsA2 are secreted bacterial lipoprotein chaperones that contribute to the folding of proteins translocated across the bacterial membrane; PrsA2 is required for L. monocytogenes virulence, whereas the function of PrsA1 remains to be determined. We have solved an X-ray crystal structure of PrsA1 and have used this model to guide comparison structure-based mutagenesis studies with PrsA2. Targeted mutagenesis of PrsA2 demonstrates that oligomerization of PrsA2 as well as molecular features of the foldase domain are required for protein secretion and virulence, whereas a functional role was uncovered for PrsA1 in bacterial resistance to alcohol. Interestingly, PrsA2 membrane localization is not required for all PrsA2-dependent activities, suggesting that the lipoprotein retains function when released from the bacterial cell. PrsA chaperones are thus multifaceted proteins with distinct domains adapted to accommodate the functional needs of a diverse array of secreted substrates. PMID:27007641

  16. Seed germination of GA-insensitive sleepy1 mutants does not require RGL2 protein disappearance in Arabidopsis

    Science.gov (United States)

    Seed germination is a complex developmental process regulated by phytohormones. The phytohormone abscisic acid (ABA) inhibits seed germination, whereas gibberellin (GA) stimulates seed germination. In tomato and Arabidopsis, GA is clearly required for seed germination. Recent evidence suggests tha...

  17. 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.

  18. The stress granule protein Vgl1 and poly(A)-binding protein Pab1 are required for doxorubicin resistance in the fission yeast Schizosaccharomyces pombe

    International Nuclear Information System (INIS)

    Highlights: ► Stress granules (SGs) as a mechanism of doxorubicin tolerance. ► We characterize the role of stress granules in doxorubicin tolerance. ► Deletion of components of SGs enhances doxorubicin sensitivity in fission yeast. ► Doxorubicin promotes SG formation when combined with heat shock. ► Doxorubicin regulates stress granule assembly independent of eIF2α 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+, which encodes a multi-KH type RNA-binding protein, and pab1+, which encodes a poly(A)-binding protein, resulted in greater sensitivity to doxorubicin than seen in wild-type cells. Disruption of the vgl1+ and pab1+ 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 eIF2α, suggesting that doxorubicin is involved in stress granule assembly independent of eIF2α 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.

  19. De Novo Protein Synthesis Is Required for Lytic Cycle Reactivation of Epstein-Barr Virus, but Not Kaposi's Sarcoma-Associated Herpesvirus, in Response to Histone Deacetylase Inhibitors and Protein Kinase C Agonists▿

    OpenAIRE

    Ye, Jianjiang; Gradoville, Lyndle; Daigle, Derek; Miller, George

    2007-01-01

    The oncogenic human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), are latent in cultured lymphoma cells. We asked whether reactivation from latency of either virus requires de novo protein synthesis. Using Northern blotting and quantitative reverse transcriptase PCR, we measured the kinetics of expression of the lytic cycle activator genes and determined whether abundance of mRNAs encoding these genes from either virus was reduced by treatmen...

  20. 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

    In cardiovascular diseases, endothelin type B (ET(B)) receptors in arterial smooth muscle cells are upregulated. The present study revealed that organ culture of rat mesenteric artery segments enhanced endothelin ET(B) receptor-mediated contraction paralleled with increase in the receptor mRNA 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<0.05) and decreased the sustained phase of intracellular calcium level, but not the rapid phase. Thus...

  1. TOM1, an Arabidopsis gene required for efficient multiplication of a tobamovirus, encodes a putative transmembrane protein.

    Science.gov (United States)

    Yamanaka, T; Ohta, T; Takahashi, M; Meshi, T; Schmidt, R; Dean, C; Naito, S; Ishikawa, M

    2000-08-29

    Host-encoded factors play an important role in virus multiplication, acting in concert with virus-encoded factors. However, information regarding the host factors involved in this process is limited. Here we report the map-based cloning of an Arabidopsis thaliana gene, TOM1, which is necessary for the efficient multiplication of tobamoviruses, positive-strand RNA viruses infecting a wide variety of plants. The TOM1 mRNA is suggested to encode a 291-aa polypeptide that is predicted to be a multipass transmembrane protein. The Sos recruitment assay supported the hypothesis that TOM1 is associated with membranes, and in addition, that TOM1 interacts with the helicase domain of tobamovirus-encoded replication proteins. Taken into account that the tobamovirus replication complex is associated with membranes, we propose that TOM1 participates in the in vivo formation of the replication complex by serving as a membrane anchor. PMID:10944200

  2. Requirements for Recruitment of a G Protein-coupled Receptor to Clathrin-coated Pits in Budding Yeast

    OpenAIRE

    Toshima, Junko Y.; Nakanishi, Jun-ichi; Mizuno, Kensaku; Toshima, Jiro; Drubin, David G.

    2009-01-01

    Endocytic internalization of G protein-coupled receptors (GPCRs) plays a critical role in down-regulation of GPCR signaling. The yeast mating pheromone receptor Ste2p has been used as a model to investigate mechanisms of signal transduction, modification, and endocytic internalization of GPCRs. We previously used a fluorescently labeled mating pheromone derivative to reveal unappreciated molecular and spatiotemporal features of GPCR endocytosis in budding yeast. Here, we identify recruitment ...

  3. Trypanosoma brucei: Differential requirement of membrane potential for import of proteins into mitochondria in two developmental stages

    OpenAIRE

    Williams, Shuntae; Saha, Lipi; Singha, Ujjal K; Chaudhuri, Minu

    2007-01-01

    Trypanosome alternative oxidase (TAO) and the cytochrome oxidase (COX) are two developmentally regulated terminal oxidases of the mitochondrial electron transport chain in Trypanosoma brucei. Here, we have compared the import of TAO and cytochrome oxidase subunit IV (COIV), two stage specific nuclear encoded mitochondrial proteins, into the bloodstream and procyclic form mitochondria of T. brucei to understand the import processes in two different developmental stages. Under in vitro conditio...

  4. Activation of Mitogen-Activated Protein Kinase Is Required for Migration and Invasion of Placental Site Trophoblastic Tumor

    OpenAIRE

    Köbel, Martin; Pohl, Gudrun; Schmitt, Wolfgang D.; Hauptmann, Steffen; Wang, Tian-Li; Shih, Ie-Ming

    2005-01-01

    Placental site trophoblastic tumor (PSTT) is a gestational neoplasm derived from the extravillous (intermediate) trophoblast of the implantation site. PSTT is characterized by a highly invasive phenotype, but the molecular mechanisms are poorly understood. In this report, we demonstrate that PSTTs expressed the activated (phosphorylated) form of mitogen-activated protein kinase (MAPK) in 84% of cases, whereas the normal extravillous trophoblastic cells did not. To characterize the role of MAP...

  5. Sip1, an AP-1 Accessory Protein in Fission Yeast, Is Required for Localization of Rho3 GTPase

    OpenAIRE

    Yang Yu; Cuifang Li; Ayako Kita; Yuta Katayama; Koji Kubouchi; Masako Udo; Yukako Imanaka; Shiho Ueda; Takashi Masuko; Reiko Sugiura

    2013-01-01

    Rho family GTPases act as molecular switches to regulate a range of physiological functions, including the regulation of the actin-based cytoskeleton, membrane trafficking, cell morphology, nuclear gene expression, and cell growth. Rho function is regulated by its ability to bind GTP and by its localization. We previously demonstrated functional and physical interactions between Rho3 and the clathrin-associated adaptor protein-1 (AP-1) complex, which revealed a role of Rho3 in regulating Golg...

  6. Induction of alternative lengthening of telomeres-associated PML bodies by p53/p21 requires HP1 proteins

    OpenAIRE

    Jiang, Wei-Qin; Zhong, Ze-Huai; Nguyen, Akira; Henson, Jeremy D.; Toouli, Christian D.; Braithwaite, Antony W.; Reddel, Roger R

    2009-01-01

    Alternative lengthening of telomeres (ALT) is a recombination-mediated process that maintains telomeres in telomerase-negative cancer cells. In asynchronously dividing ALT-positive cell populations, a small fraction of the cells have ALT-associated promyelocytic leukemia nuclear bodies (APBs), which contain (TTAGGG)n DNA and telomere-binding proteins. We found that restoring p53 function in ALT cells caused p21 up-regulation, growth arrest/senescence, and a large increase in cells containing ...

  7. 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...

  8. Mitogen-Activated Protein Kinase Cascade Required for Regulation of Development and Secondary Metabolism in Neurospora crassa▿

    OpenAIRE

    Park, Gyungsoon; Pan, Songqin; Borkovich, Katherine A.

    2008-01-01

    Mitogen-activated protein kinase (MAPK) signaling cascades are composed of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In this study, we characterize components of a MAPK cascade in Neurospora crassa (mik-1, MAPKKK; mek-1, MAPKK; and mak-1, MAPK) homologous to that controlling cell wall integrity in Saccharomyces cerevisiae. Growth of basal hyphae is significantly reduced in mik-1, mek-1, and mak-1 deletion mutants on solid medium. All three mutants formed short aerial hy...

  9. Identification and characterization of LFD-2, a predicted fringe protein required for membrane integrity during cell fusion in neurospora crassa.

    Science.gov (United States)

    Palma-Guerrero, Javier; Zhao, Jiuhai; Gonçalves, A Pedro; Starr, Trevor L; Glass, N Louise

    2015-03-01

    The molecular mechanisms of membrane merger during somatic cell fusion in eukaryotic species are poorly understood. In the filamentous fungus Neurospora crassa, somatic cell fusion occurs between genetically identical germinated asexual spores (germlings) and between hyphae to form the interconnected network characteristic of a filamentous fungal colony. In N. crassa, two proteins have been identified to function at the step of membrane fusion during somatic cell fusion: PRM1 and LFD-1. The absence of either one of these two proteins results in an increase of germling pairs arrested during cell fusion with tightly appressed plasma membranes and an increase in the frequency of cell lysis of adhered germlings. The level of cell lysis in ΔPrm1 or Δlfd-1 germlings is dependent on the extracellular calcium concentration. An available transcriptional profile data set was used to identify genes encoding predicted transmembrane proteins that showed reduced expression levels in germlings cultured in the absence of extracellular calcium. From these analyses, we identified a mutant (lfd-2, for late fusion defect-2) that showed a calcium-dependent cell lysis phenotype. lfd-2 encodes a protein with a Fringe domain and showed endoplasmic reticulum and Golgi membrane localization. The deletion of an additional gene predicted to encode a low-affinity calcium transporter, fig1, also resulted in a strain that showed a calcium-dependent cell lysis phenotype. Genetic analyses showed that LFD-2 and FIG1 likely function in separate pathways to regulate aspects of membrane merger and repair during cell fusion. PMID:25595444

  10. A Coherent Organization of Differentiation Proteins Is Required to Maintain an Appropriate Thyroid Function in the Pendred Thyroid

    OpenAIRE

    Senou, Maximin; Khalifa, Céline; Thimmesch, Matthieu; Jouret, François; Devuyst, Olivier; Col, Vincent; Audinot, Jean-Nicolas; Lipnik, Pascale; Moreno, Jose C.; Van Sande, Jacqueline; Dumont, Jacques E.; Many, Marie-Christine; Colin, Ides M.; Gérard, Anne-Catherine

    2010-01-01

    CONTEXT: Pendred syndrome is caused by mutations in the gene coding for pendrin, an apical Cl-/I- exchanger. OBJECTIVE: To analyze intrathyroidal compensatory mechanisms when pendrin is lacking, we investigated the thyroid of a patient with Pendred syndrome. The expression of proteins involved in thyroid hormone synthesis, markers of oxidative stress (OS), cell proliferation, apoptosis, and antioxidant enzymes were analyzed. RESULTS: Three morphological zones were identified: nearly normal fo...

  11. Rhodococcus equi Virulence-Associated Protein A Is Required for Diversion of Phagosome Biogenesis but Not for Cytotoxicity▿

    OpenAIRE

    von Bargen, Kristine; Polidori, Marco; Becken, Ulrike; Huth, Gitta; Prescott, John F.; Haas, Albert

    2009-01-01

    Rhodococcus equi is a gram-positive facultative intracellular pathogen that can cause severe bronchopneumonia in foals and AIDS patients. Virulence is plasmid regulated and is accompanied by phagosome maturation arrest and host cell necrosis. A replacement mutant in the gene for VapA (virulence-associated protein A), a major virulence factor of R. equi, was tested for its activities during macrophage infection. Early in infection, phagosomes containing the vapA mutant did not fuse with lysoso...

  12. The bone morphogenetic protein receptor-1A pathway is required for lactogenic differentiation of mammary epithelial cells in vitro

    OpenAIRE

    Perotti, C.; Karayazi, Ö.; Moffat, S.; Shemanko, C. S.

    2012-01-01

    Bone morphogenetic proteins (BMPs) have been implicated in the control of proliferation, tissue formation, and differentiation. BMPs regulate the biology of stem and progenitor cells and can promote cellular differentiation, depending on the cell type and context. Although the BMP pathway is known to be involved in early embryonic development of the mammary gland via mesenchymal cells, its role in later epithelial cellular differentiation has not been examined. The majority of the mammary gla...

  13. 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

    We tested the hypothesis that repeated activation of AMPK induces mitochondrial and glucose membrane transporter gene/protein expression via a peroxisome proliferator activated receptor Upsilon co-activator (PGC)-1alpha dependent mechanism. Whole body PGC-1alpha knockout (KO) and littermate wild ...... in both WT and PGC-1alpha KO mice. In conclusion, we here provide genetic evidence for a major role of PGC-1alpha in AMPK mediated regulation of mitochondrial and glucose membrane transport protein expression in skeletal muscle....... type (WT) mice were given either a single or repeated subcutaneous injection of the AMPK activator AICAR or saline. Skeletal muscles were dissected either 1h or 4h after the single AICAR treatment or 24h after the last injection following the repeated AICAR treatment. Repeated AICAR treatment increased...... 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...

  14. Fatal Hepatitis Mediated by TNFα Requires Caspase-8 and Involves the BH3-only Proteins Bid and Bim

    Science.gov (United States)

    Kaufmann, Thomas; Jost, Philipp J; Pellegrini, Marc; Puthalakath, Hamsa; Gugasyan, Raffi; Gerondakis, Steve; Cretney, Erika; Smyth, Mark J; Silke, John; Hakem, Razq; Bouillet, Philippe; Mak, Tak W; Dixit, Vishva M; Strasser, Andreas

    2009-01-01

    SUMMARY Apoptotic death of hepatocytes, a feature and contributing factor of many chronic and acute liver diseases, can be a consequence of over-activation of the immune system. Injection with lipopolysaccharide (LPS) plus the transcriptional inhibitor D(+)-galactosamine (GalN) or mitogenic T cell activation cause fatal hepatocyte apoptosis in mice. In both settings hepatocyte killing is mediated by TNFα/TNF-R signaling but the effector mechanisms remain unclear. Our analysis of gene-targeted mice showed that caspase-8 is essential for hepatocyte killing in both settings. Loss of Bid, the pro-apoptotic BH3-only protein activated by caspase-8, and essential for Fas ligand-induced hepatocyte killing, resulted only in a minor reduction of liver damage. However, combined loss of Bid and another BH3-only protein, Bim, activated by JNK, protected mice from LPS+GalN-induced hepatitis. These observations identify caspase-8 and the BH3-only proteins Bid and Bim as potential therapeutic targets for treatment of inflammatory liver diseases. PMID:19119023

  15. Requirements for stress granule recruitment of fused in sarcoma (FUS) and TAR DNA-binding protein of 43 kDa (TDP-43).

    Science.gov (United States)

    Bentmann, Eva; Neumann, Manuela; Tahirovic, Sabina; Rodde, Ramona; Dormann, Dorothee; Haass, Christian

    2012-06-29

    Cytoplasmic inclusions containing TAR DNA-binding protein of 43 kDa (TDP-43) or Fused in sarcoma (FUS) are a hallmark of amyotrophic lateral sclerosis (ALS) and several subtypes of frontotemporal lobar degeneration (FTLD). FUS-positive inclusions in FTLD and ALS patients are consistently co-labeled with stress granule (SG) marker proteins. Whether TDP-43 inclusions contain SG markers is currently still debated. We determined the requirements for SG recruitment of FUS and TDP-43 and found that cytoplasmic mislocalization is a common prerequisite for SG recruitment of FUS and TDP-43. For FUS, the arginine-glycine-glycine zinc finger domain, which is the protein's main RNA binding domain, is most important for SG recruitment, whereas the glycine-rich domain and RNA recognition motif (RRM) domain have a minor contribution and the glutamine-rich domain is dispensable. For TDP-43, both the RRM1 and the C-terminal glycine-rich domain are required for SG localization. ALS-associated point mutations located in the glycine-rich domain of TDP-43 do not affect SG recruitment. Interestingly, a 25-kDa C-terminal fragment of TDP-43, which is enriched in FTLD/ALS cortical inclusions but not spinal cord inclusions, fails to be recruited into SG. Consistently, inclusions in the cortex of FTLD patients, which are enriched for C-terminal fragments, are not co-labeled with the SG marker poly(A)-binding protein 1 (PABP-1), whereas inclusions in spinal cord, which contain full-length TDP-43, are frequently positive for this marker protein. PMID:22563080

  16. 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.

  17. Cytidine deaminase motifs within the DYW domain of two pentatricopeptide repeat-containing proteins are required for site-specific chloroplast RNA editing.

    Science.gov (United States)

    Wagoner, Jessica A; Sun, Tao; Lin, Lin; Hanson, Maureen R

    2015-01-30

    In angiosperm organelles, cytidines are converted to uridines by a deamination reaction in the process termed RNA editing. The C targets of editing are recognized by members of the pentatricopeptide repeat (PPR) protein family. Although other members of the editosome have begun to be identified, the enzyme that catalyzes the C-U conversion is still unknown. The DYW motif at the C terminus of many PPR editing factors contains residues conserved with known cytidine deaminase active sites; however, some PPR editing factors lack a DYW motif. Furthermore, in many PPR-DYW editing factors, the truncation of the DYW motif does not affect editing efficiency, so the role of the DYW motif in RNA editing is unclear. Here, a chloroplast PPR-DYW editing factor, quintuple editing factor 1 (QED1), was shown to affect five different plastid editing sites, the greatest number of chloroplast C targets known to be affected by a single PPR protein. Loss of editing at the five sites resulted in stunted growth and accumulation of apparent photodamage. Adding a C-terminal protein tag to QED1 was found to severely inhibit editing function. QED1 and RARE1, another plastid PPR-DYW editing factor, were discovered to require their DYW motifs for efficient editing. To identify specific residues critical for editing, conserved deaminase residues in each PPR protein were mutagenized. The mutant PPR proteins, when expressed in qed1 or rare1 mutant protoplasts, could not complement the editing defect. Therefore, the DYW motif, and specifically, the deaminase residues, of QED1 and RARE1 are required for editing efficiency. PMID:25512379

  18. Innate Stat3-mediated induction of the antimicrobial protein Reg3γ is required for host defense against MRSA pneumonia

    OpenAIRE

    Choi, Sun-Mi; McAleer, Jeremy P.; Zheng, Mingquan; Pociask, Derek A.; Kaplan, Mark H.; Qin, Shulin; Reinhart, Todd A.; Kolls, Jay K.

    2013-01-01

    Pulmonary Staphylococcus aureus (SA) infections are a public health concern and a major complication of hyper-IgE syndrome, caused by mutations in STAT3. In contrast to previous findings of skin infection, we observed that clearance of SA from the lung did not require T, B, or NK cells but did require Stat3 activation. Immunohistochemistry showed robust Stat3 phosphorylation in the lung epithelium. We identified that a critical Stat3 target gene in lung epithelium is Reg3g (regenerating islet...

  19. Requirement for the POZ/BTB protein NAC1 in acute but not chronic psychomotor stimulant response

    OpenAIRE

    Mackler, Scott; Pacchioni, Alejandra; Degnan, Ryan; Homan, Ying; Conti, Alana C.; Kalivas, Peter; Blendy, Julie A.

    2007-01-01

    NAC1 is a novel member of the POZ/BTB (Pox virus and Zinc finger/Bric-a-brac Tramtrack 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 v...

  20. Classic nuclear localization signals and a novel nuclear localization motif are required for nuclear transport of porcine parvovirus capsid proteins.

    OpenAIRE

    Boisvert, Maude; Bouchard-Lévesque, Véronique; Fernandes, Sandra; Tijssen, Peter

    2014-01-01

    Nuclear targeting of capsid proteins (VPs) is important for genome delivery and precedes assembly in the replication cycle of porcine parvovirus (PPV). Clusters of basic amino acids, corresponding to potential nuclear localization signals (NLS), were found only in the unique region of VP1 (VP1up, for VP1 unique part). Of the five identified basic regions (BR), three were important for nuclear localization of VP1up: BR1 was a classic Pat7 NLS, and the combination of BR4 and BR5 was a classic b...

  1. Non-structural protein NS3/NS3a is required for propagation of bluetongue virus in Culicoides sonorensis

    OpenAIRE

    Feenstra, Femke; Drolet, B.S.; Boonstra, Jan; Rijn, van, C.J.M.

    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. However, deletion of NS3/NS3a leads to delayed virus release from mammalian cells and largely reduces virus release from insect cells. NS3/NS3a knockout BTV in sheep causes no viremia, but induces sterile...

  2. 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

  3. AMP-activated protein kinase is required for the anti-adipogenic effects of alpha-linolenic acid

    OpenAIRE

    Zhou, Xihong; Wu, Weiche; Chen, Jingqing; Wang, Xinxia; Wang, Yizhen

    2015-01-01

    Background n-3 long chain polyunsaturated fatty acid (n-3 LC PUFA) increases β-oxidation and limits lipid accumulation in adipocytes. The current study was conducted to determine whether their precursor alpha-linolenic acid (ALA) could also exert the above effects and how AMP-activated protein kinase (AMPK) was involved. Methods AMPKα1−/−, AMPKα2−/− mice and wild-type (WT) mice were fed a high-fat diet (HFD) or HFD with ALA. Body weight was recorded weekly and serum was collected. Adipocytes ...

  4. Primary-structure requirements for inhibition by the heat-stable inhibitor of the cAMP-dependent protein kinase.

    OpenAIRE

    Scott, J D; Glaccum, M B; Fischer, E H; Krebs, E G

    1986-01-01

    The amino acid sequence of the heat-stable inhibitor of the cAMP-dependent protein kinase (PKI) was determined recently [Scott, J. D., Fischer, E. H., Takio, K., Demaille, J. G. & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. USA 82, 5732-5736]. An earlier report [Scott, J. D., Fischer, E.H., Demaille, J. G. & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. USA 82, 4379-4383] showed that at least part of the inhibitory domain of PKI is located in a 20-residue segment extending from residue 11 to resi...

  5. The yeast actin-related protein Arp2p is required for the internalization step of endocytosis.

    OpenAIRE

    Moreau, V; Galan, J M; Devilliers, G; Haguenauer-Tsapis, R; Winsor, B

    1997-01-01

    The Saccharomyces cerevisiae actin-related protein Arp2p is an essential component of the actin cytoskeleton. We have tested its potential role in the endocytic and exocytic pathways by using a temperature-sensitive allele, arp2-1. The fate of the plasma membrane transporter uracil permease was followed to determine whether Arp2p plays a role in the endocytic pathway. Inhibition of normal endocytosis as revealed by maintenance of active uracil permease at the plasma membrane and strong protec...

  6. Whole-body protein turnover and energy expenditure in post-viral hepatocirrhotic patients. A study using multiple stable isotope tracers to estimate protein and energy requirements and the efficacy of a new diet therapy based on Chinese food

    International Nuclear Information System (INIS)

    L-[1-13C]-leucine and 15N-glycine doubly-labelled tracer experiments revealed accelerated kinetics of leucine, glycine and whole-body protein in post-viral hepatocirrhotic patients. Together with the results of nitrogen balance measurement, the daily protein requirement of these patients was estimated to be higher than 1.2 g/kg/d. Doubly labelled water experiments and NaH13CO3 experiments revealed that the freely living and basal energy expenditure of post-viral hepatocirrhotic patients was not different from that in normal subjects with comparable physical and mental activities. For those freely living in hospital, the energy requirements is estimated to be 150-160 kJ/kg/d. According to the above results, a therapeutic diet formulation based on Chinese food was designed for the patients which contained 1.5 g/kg/d of protein and 150-160 kJ/kg/d. 60-70% of the dietary protein was of vegetable origin, with a branched chain amino acid/aromatic amino acid ratio slightly but significantly higher than the common hospital diet. Patients with compensated post-viral hepatocirrhosis adapted to the diet rapidly. After two months' therapy, the negative nitrogen balance turned positive along with an increase of body weight and urinary creatinine, indicating and improvement of general nutritional status, probably with accumulation of muscle protein. The diet is relatively cheap, can be easily handled by the patients themselves, and hence is also applicable to outpatients. 54 refs, 8 tabs

  7. The N-terminal extension domain of the C. elegans half-molecule ABC transporter, HMT-1, is required for protein-protein interactions and function.

    Directory of Open Access Journals (Sweden)

    Sungjin Kim

    Full Text Available BACKGROUND: Members of the HMT-1 (heavy metal tolerance factor 1 subfamily of the ATP-binding cassette (ABC transporter superfamily detoxify heavy metals and have unique topology: they are half-molecule ABC transporters that, in addition to a single transmembrane domain (TMD1 and a single nucleotide-binding domain (NBD1, possess a hydrophobic NH2-terminal extension (NTE. These structural features distinguish HMTs from other ABC transporters in different species including Drosophila and humans. Functional ABC transporters, however, are comprised of at least four-domains (two TMDs and two NDBs formed from either a single polypeptide or by the association of two or four separate subunits. Whether HMTs act as oligomers and what role the NTE domain plays in their function have not been determined. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we examined the oligomeric status of Caenorhabditis elegans HMT-1 and the functional significance of its NTE using gel-filtration chromatography in combination with the mating-based split-ubiquitin yeast two-hybrid system (mbSUS and functional in vivo assays. We found that HMT-1 exists in a protein complex in C. elegans. Studies in S. cerevisiae showed that HMT-1 at a minimum homodimerizes and that oligomerization is essential for HMT-1 to confer cadmium tolerance. We also established that the NTE domain plays an important structural and functional role: it is essential for HMT-1 oligomerization and Cd-detoxification function. However, the NTE itself was not sufficient for oligomerization suggesting that multiple structural features of HMT-1 must associate to form a functional transporter. CONCLUSIONS: The prominence of heavy metals as environmental toxins and the remarkable conservation of HMT-1 structural architecture and function in different species reinforce the value of continued studies of HMT-1 in model systems for identifying functional domains in HMT1 of humans.

  8. 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.

  9. Expression of cytoskeletal and matrix genes following exposure to ionizing radiation: Dose-rate effects and protein synthesis requirements

    International Nuclear Information System (INIS)

    Experiments were designed to examine the effects of radiation dose-rate and of the protein synthesis inhibitor cycloheximide on expression of cytoskeletal elements (γ- and β-actin and α-tubulin) and matrix elements (fibronectin) in Syrian hamster embryo cells. Past work from our laboratory had already demonstrated optimum time points and doses for examination of radiation effects on accumulation of specific transcripts. Our results here demonstrated little effect of dose-rate for JANUS fission spectrum neutrons when comparing expression of either α-tubulin or fibronectin genes. Past work had already documented similar results for expression of actin transcripts. Effects of cycloheximide, however, revealed several interesting and novel findings: (1) Cycloheximide repressed accumulation of α-tubulin following exposure to high dose-rate neutrons or γ rays; this did not occur following similar low dose-rate exposure (2) Cycloheximide did not affect accumulation of mRNA for actin genes. Cycloheximide abrogated the moderate induction of fibronectin-mRNA which occurred following exposure to γ rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of α-tubulin and fibronectin mRNA accumulation following exposure to ionizing radiation. In addition, they suggest that the cellular/molecular response to low dose-rate neutrons may be different from the response to high dose-rate neutrons

  10. The requirement of the DEAD-box protein DDX24 for the packaging of human immunodeficiency virus type 1 RNA

    International Nuclear Information System (INIS)

    RNA helicases play important roles in RNA metabolism. Human immunodeficiency virus type 1 (HIV-1) does not carry its own RNA helicase, the virus thus needs to exploit cellular RNA helicases to promote the replication of its RNA at various steps such as transcription, folding and transport. In this study, we report that knockdown of a DEAD-box protein named DDX24 inhibits the packaging of HIV-1 RNA and thus diminishes viral infectivity. The decreased viral RNA packaging as a result of DDX24-knockdown is observed only in the context of the Rev/RRE (Rev response element)-dependent but not the CTE (constitutive transport element)-mediated nuclear export of viral RNA, which is explained by the specific interaction of DDX24 with the Rev protein. We propose that DDX24 acts at the early phase of HIV-1 RNA metabolism prior to nuclear export and the consequence of this action extends to the viral RNA packaging stage during virus assembly

  11. The MYST-containing protein Chameau is required for proper sensory organ specification during Drosophila thorax morphogenesis.

    Directory of Open Access Journals (Sweden)

    Matthieu Hainaut

    Full Text Available The adult thorax of Drosophila melanogaster is covered by a stereotyped pattern of mechanosensory bristles called macrochaetes. Here, we report that the MYST containing protein Chameau (Chm contributes to the establishment of this pattern in the most dorsal part of the thorax. Chm mutant pupae present extra-dorsocentral (DC and scutellar (SC macrochaetes, but a normal number of the other macrochaetes. We provide evidences that chm restricts the singling out of sensory organ precursors from proneural clusters and genetically interacts with transcriptional regulators involved in the regulation of achaete and scute in the DC and SC proneural cluster. This function of chm likely relies on chromatin structure regulation since a protein with a mutation in the conserved catalytic site fails to rescue the formation of supernumerary DC and SC bristles in chm mutant flies. This is further supported by the finding that mutations in genes encoding chromatin modifiers and remodeling factors, including Polycomb group (PcG and Trithorax group (TrxG members, dominantly modulate the penetrance of chm extra bristle phenotype. These data support a critical role for chromatin structure modulation in the establishment of the stereotyped sensory bristle pattern in the fly thorax.

  12. The novel protein DELAYED PALE-GREENING1 is required for early chloroplast biogenesis in Arabidopsis thaliana.

    Science.gov (United States)

    Liu, Dong; Li, Weichun; Cheng, Jianfeng

    2016-01-01

    Chloroplast biogenesis is one of the most important subjects in plant biology. In this study, an Arabidopsis early chloroplast biogenesis mutant with a delayed pale-greening phenotype (dpg1) was isolated from a T-DNA insertion mutant collection. Both cotyledons and true leaves of dpg1 mutants were initially albino but gradually became pale green as the plant matured. Transmission electron microscopic observations revealed that the mutant displayed a delayed proplastid-to-chloroplast transition. Sequence and transcription analyses showed that AtDPG1 encodes a putatively chloroplast-localized protein containing three predicted transmembrane helices and that its expression depends on both light and developmental status. GUS staining for AtDPG1::GUS transgenic lines showed that this gene was widely expressed throughout the plant and that higher expression levels were predominantly found in green tissues during the early stages of Arabidopsis seedling development. Furthermore, quantitative real-time RT-PCR analyses revealed that a number of chloroplast- and nuclear-encoded genes involved in chlorophyll biosynthesis, photosynthesis and chloroplast development were substantially down-regulated in the dpg1 mutant. These data indicate that AtDPG1 plays an essential role in early chloroplast biogenesis, and its absence triggers chloroplast-to-nucleus retrograde signalling, which ultimately down-regulates the expression of nuclear genes encoding chloroplast-localized proteins. PMID:27160321

  13. Mitogen-activated protein kinase cascade required for regulation of development and secondary metabolism in Neurospora crassa.

    Science.gov (United States)

    Park, Gyungsoon; Pan, Songqin; Borkovich, Katherine A

    2008-12-01

    Mitogen-activated protein kinase (MAPK) signaling cascades are composed of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In this study, we characterize components of a MAPK cascade in Neurospora crassa (mik-1, MAPKKK; mek-1, MAPKK; and mak-1, MAPK) homologous to that controlling cell wall integrity in Saccharomyces cerevisiae. Growth of basal hyphae is significantly reduced in mik-1, mek-1, and mak-1 deletion mutants on solid medium. All three mutants formed short aerial hyphae and the formation of asexual macroconidia was reduced in Deltamik-1 mutants and almost abolished in Deltamek-1 and Deltamak-1 strains. In contrast, the normally rare asexual spores, arthroconidia, were abundant in cultures of the three mutants. Deltamik-1, Deltamek-1, and Deltamak-1 mutants were unable to form protoperithecia or perithecia when used as females in a sexual cross. The MAK-1 MAPK was not phosphorylated in Deltamik-1 and Deltamek-1 mutants, consistent with the involvement of MIK-1, MEK-1, and MAK-1 in the same signaling cascade. Interestingly, we observed increased levels of mRNA and protein for tyrosinase in the mutants under nitrogen starvation, a condition favoring sexual differentiation. Tyrosinase is an enzyme that catalyzes production of the secondary metabolite l-DOPA melanin. These results implicate the MAK-1 pathway in regulation of development and secondary metabolism in filamentous fungi. PMID:18849472

  14. Expression of cytoskeletal and matrix genes following exposure to ionizing radiation: Dose-rate effects and protein synthesis requirements

    International Nuclear Information System (INIS)

    Experiments were designed to examine the effects Of radiation dose-rate and of the protein synthesis inhibitor cycloheximide on expression of cytoskeletal elements (γ- and β-actin and α-tubulin) and matrix elements (fibronectin) in Syrian hamster embryo cells. Past work from our laboratory had already demonstrated optimum time points and doses for examination of radiation effects on accumulation of specific transcripts. Our results here demonstrated little effect of dose-rate for JANUS fission spectrum neutrons when comparing expression of either α-tubulin or fibronectin genes. Past work had already documented similar results for expression of actin transcripts. Effects of cycloheximide revealed that cycloheximide repressed accumulation of α-tubulin following exposure to high dose-rate neutrons or γ rays; this did not occur following similar low dose-rate exposure. (2) Cycloheximide did not affect accumulation of MRNA for actin genes; and that cycloheximide abrogated the moderate induction of fibronectin-mRNA which occurred following exposure to γ rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of α-tubulin and fibronectin MRNA accumulation following exposure to ionizing radiation. in addition, they suggest that the cellular/molecular response to low dose-rate neutrons may be different from the response to high dose-rate neutrons

  15. Idas, a novel phylogenetically conserved geminin-related protein, binds to geminin and is required for cell cycle progression.

    Science.gov (United States)

    Pefani, Dafni-Eleutheria; Dimaki, Maria; Spella, Magda; Karantzelis, Nickolas; Mitsiki, Eirini; Kyrousi, Christina; Symeonidou, Ioanna-Eleni; Perrakis, Anastassis; Taraviras, Stavros; Lygerou, Zoi

    2011-07-01

    Development and homeostasis of multicellular organisms relies on an intricate balance between cell proliferation and differentiation. Geminin regulates the cell cycle by directly binding and inhibiting the DNA replication licensing factor Cdt1. Geminin also interacts with transcriptional regulators of differentiation and chromatin remodelling factors, and its balanced interactions are implicated in proliferation-differentiation decisions during development. Here, we describe Idas (Idas being a cousin of the Gemini in Ancient Greek Mythology), a previously uncharacterised coiled-coil protein related to Geminin. We show that human Idas localizes to the nucleus, forms a complex with Geminin both in cells and in vitro through coiled-coil mediated interactions, and can change Geminin subcellular localization. Idas does not associate with Cdt1 and prevents Geminin from binding to Cdt1 in vitro. Idas depletion from cells affects cell cycle progression; cells accumulate in S phase and are unable to efficiently progress to mitosis. Idas protein levels decrease in anaphase, whereas its overexpression causes mitotic defects. During development, we show that Idas exhibits high level expression in the choroid plexus and the cortical hem of the mouse telencephalon. Our data highlight Idas as a novel Geminin binding partner, implicated in cell cycle progression, and a putative regulator of proliferation-differentiation decisions during development. PMID:21543332

  16. Negative selection of semimature CD4+8-HSA+ thymocytes requires the BH3-only protein Bim but is independent of death receptor signaling

    Science.gov (United States)

    Villunger, Andreas; Marsden, Vanessa S.; Zhan, Yifan; Erlacher, Miriam; Lew, Andrew M.; Bouillet, Philippe; Berzins, Stuart; Godfrey, Dale I.; Heath, William R.; Strasser, Andreas

    2004-01-01

    T cell receptor/CD3 ligation induces apoptosis in semimature CD4+8-HSA+ thymocytes, and this helps establish immunological tolerance and constitutes one of the safeguards against autoimmune disease. We analyzed several knockout and transgenic mouse lines and found that T cell receptor/CD3-ligation-induced killing of semimature thymocytes occurred independently of Fas and “death receptor” signaling in general but required the proapoptotic BH3-only protein Bim and could be inhibited by Bcl-2. Loss of Apaf-1 or caspase-9, which act downstream of the Bcl-2 family protein family, provided only minor protection, indicating that the “apoptosome” functions as an amplifier rather than as an essential initiator of this death program. These results reveal the mechanisms of apoptosis in negative selection of semimature thymocytes and have implications for immunological tolerance and autoimmunity. PMID:15118096

  17. Multiple activities of the plant pathogen type III effector proteins WtsE and AvrE require WxxxE motifs.

    Science.gov (United States)

    Ham, Jong Hyun; Majerczak, Doris R; Nomura, Kinya; Mecey, Christy; Uribe, Francisco; He, Sheng-Yang; Mackey, David; Coplin, David L

    2009-06-01

    The broadly conserved AvrE-family of type III effectors from gram-negative plant-pathogenic bacteria includes important virulence factors, yet little is known about the mechanisms by which these effectors function inside plant cells to promote disease. We have identified two conserved motifs in AvrE-family effectors: a WxxxE motif and a putative C-terminal endoplasmic reticulum membrane retention/retrieval signal (ERMRS). The WxxxE and ERMRS motifs are both required for the virulence activities of WtsE and AvrE, which are major virulence factors of the corn pathogen Pantoea stewartii subsp. stewartii and the tomato or Arabidopsis pathogen Pseudomonas syringae pv. tomato, respectively. The WxxxE and the predicted ERMRS motifs are also required for other biological activities of WtsE, including elicitation of the hypersensitive response in nonhost plants and suppression of defense responses in Arabidopsis. A family of type III effectors from mammalian bacterial pathogens requires WxxxE and subcellular targeting motifs for virulence functions that involve their ability to mimic activated G-proteins. The conservation of related motifs and their necessity for the function of type III effectors from plant pathogens indicates that disturbing host pathways by mimicking activated host G-proteins may be a virulence mechanism employed by plant pathogens as well. PMID:19445595

  18. Characterization of 47 Cys2 -His2 zinc finger proteins required for the development and pathogenicity of the rice blast fungus Magnaporthe oryzae.

    Science.gov (United States)

    Cao, Huijuan; Huang, Pengyun; Zhang, Lilin; Shi, Yongkai; Sun, Dandan; Yan, Yuxin; Liu, Xiaohong; Dong, Bo; Chen, Guoqing; Snyder, John Hugh; Lin, Fucheng; Lu, Jianping

    2016-08-01

    The Cys2 -His2 (C2H2) zinc finger protein family is the second-largest family of transcription factors (TFs) in Magnaporthe oryzae, the causal fungus responsible for the destructive rice blast disease. However, little is known about the roles of most C2H2 TFs in the development and pathogenicity of M. oryzae. The roles of 47 C2H2 genes in development and pathogenicity were investigated by gene deletion in M. oryzae. The TF-dependent genes in mycelia or appressoria were analyzed with RNA sequencing and quantitative PCR (qPCR). Forty-four C2H2 genes are involved in growth (20 genes), conidiation (28 genes), appressorium formation (four genes) and pathogenicity (22 genes) in M. oryzae. Of these, MGG_14931, named as VRF1, is required for pathogenicity, specifically controlling appressorium maturation by affecting the expression of genes related to appressorial structure and function, including melanin biosynthesis, chitin catabolism, lipid metabolism, proteolysis, transmembrane transport, and response to oxidative stress; MGG_01776, named as VRF2, is required for plant penetration and invasive growth; conidiation-related gene CON7 is required for conidial differentiation; and MoCREA, encoding a carbon catabolite repression protein, is a novel repressor of lipid catabolism when glucose obtainable in M. oryzae. This study provides many insights into the regulation of growth, asexual development, appressorium formation, and pathogenicity by C2H2 TFs in M. oryzae. PMID:27041000

  19. 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. PMID:26431709

  20. Wdpcp, a PCP protein required for ciliogenesis, regulates directional cell migration and cell polarity by direct modulation of the actin cytoskeleton.

    Directory of Open Access Journals (Sweden)

    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

  1. Identification of the promoter region required for human adiponectin gene transcription: Association with CCAAT/enhancer binding protein-β and tumor necrosis factor-α

    International Nuclear Information System (INIS)

    Adiponectin, an adipose tissue-specific plasma protein, is involved in insulin sensitizing and has anti-atherosclerotic properties. Plasma levels of adiponectin are decreased in obese individuals and patients with type 2 diabetes with insulin resistance. Tumor necrosis factor-α (TNF-α) decreases the expression of adiponectin in adipocytes. The aims of the present study were: (1) to identify the promoter region responsible for basal transcription of the human adiponectin gene, and (2) to investigate the mechanism by which adiponectin was regulated by TNF-α. The human adiponectin promoter (2.1 kb) was isolated and used for luciferase reporter analysis by transient transfection into 3T3-L1 adipocytes. Deletion analysis demonstrated that the promoter region from -676 to +41 was sufficient for basal transcriptional activity. Mutation analysis of putative response elements for sterol regulatory element binding protein (SREBP) (-431 to -423) and CCAAT/enhancer binding protein (C/EBP) (-230 to -224) showed that both elements were required for basal promoter activity. Adiponectin transcription was increased 3-fold in cells that over-expressed constitutively active C/EBP-β. Electrophoretic mobility shift assay, using nuclear extract from 3T3-L1 cells and the -258 to -199 region as a probe, demonstrated specific DNA-protein binding, which was abolished by TNF-α treatment. The present data indicate that the putative response elements for SREBP and C/EBP are required for human adiponectin promoter activity, and that suppression by TNF-α may, at least in part, be associated with inactivation of C/EBP-β

  2. Non-bilayer lipids are required for efficient protein transport across the plasma membrane of Escherichia coli.

    OpenAIRE

    Rietveld, A G; Koorengevel, M C; de Kruijff, B.

    1995-01-01

    The construction of a mutant Escherichia coli strain which cannot synthesize phosphatidylethanolamine provides a tool to study the involvement of non-bilayer lipids in membrane function. This strain produces phosphatidylglycerol and cardiolipin (CL) as major membrane constituents and requires millimolar concentrations of divalent cations for growth. In this strain, the lipid phase behaviour is tightly regulated by adjustment of the level of CL which favours a nonbilayer organization in the pr...

  3. Yeast spore germination: a requirement for Ras protein activity during re-entry into the cell cycle.

    OpenAIRE

    Herman, P K; Rine, J.

    1997-01-01

    Saccharomyces cerevisiae spore germination is a process in which quiescent, non-dividing spores become competent for mitotic cell division. Using a novel assay for spore uncoating, we found that spore germination was a multi-step process whose nutritional requirements differed from those for mitotic division. Although both processes were controlled by nutrient availability, efficient spore germination occurred in conditions that did not support cell division. In addition, germination did not ...

  4. The three rows gene of Drosophila melanogaster encodes a novel protein that is required for chromosome disjunction during mitosis.

    OpenAIRE

    D'Andrea, R J; Stratmann, R.; Lehner, C F; John, U P; Saint, R

    1993-01-01

    Zygotic expression of the three rows (thr) gene of Drosophila melanogaster is required for normal cell proliferation during embryogenesis. Mitotic defects in thr mutant embryos begin during mitosis 15, and all subsequent divisions are disrupted. Chromosome disjunction and consequently cytokinesis fail during these defective mitoses, although the initial mitotic processes (chromosome condensation, spindle assembly, metaphase plate formation, and cyclin degradation) are not affected. Despite th...

  5. Environmental pH and the Requirement for the Extrinsic Proteins of Photosystem II in the Function of Cyanobacterial Photosynthesis.

    Science.gov (United States)

    Morris, Jaz N; Eaton-Rye, Julian J; Summerfield, Tina C

    2016-01-01

    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 as well as 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 impairment 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. PMID:27555848

  6. Environmental pH and the Requirement for the Extrinsic Proteins of Photosystem II in the Function of Cyanobacterial Photosynthesis

    Science.gov (United States)

    Morris, Jaz N.; Eaton-Rye, Julian J.; Summerfield, Tina C.

    2016-01-01

    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 as well as 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 impairment 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.

  7. Functional diversification of hsp40: distinct j-protein functional requirements for two prions allow for chaperone-dependent prion selection.

    Directory of Open Access Journals (Sweden)

    Julia M Harris

    2014-07-01

    Full Text Available 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

  8. Identification of functional domains of the IR2 protein of equine herpesvirus 1 required for inhibition of viral gene expression and replication

    International Nuclear Information System (INIS)

    The equine herpesvirus 1 (EHV-1) negative regulatory IR2 protein (IR2P), an early 1,165-amino acid (aa) truncated form of the 1487-aa immediate-early protein (IEP), lacks the trans-activation domain essential for IEP activation functions but retains domains for binding DNA, TFIIB, and TBP and the nuclear localization signal. IR2P mutants of the N-terminal region which lack either DNA-binding activity or TFIIB-binding activity were unable to down-regulate EHV-1 promoters. In EHV-1-infected cells expressing full-length IR2P, transcription and protein expression of viral regulatory IE, early EICP0, IR4, and UL5, and late ETIF genes were dramatically inhibited. Viral DNA levels were reduced to 2.1% of control infected cells, but were vey weakly affected in cells that express the N-terminal 706 residues of IR2P. These results suggest that IR2P function requires the two N-terminal domains for binding DNA and TFIIB as well as the C-terminal residues 707 to 1116 containing the TBP-binding domain. - Highlights: → We examine the functional domains of IR2P that mediates negative regulation. → IR2P inhibits at the transcriptional level. → DNA-binding mutant or TFIIB-binding mutant fails to inhibit. → C-terminal aa 707 to 1116 are required for full inhibition. → Inhibition requires the DNA-binding domain, TFIIB-binding domain, and C-terminus.

  9. Amyloid fibril formation requires a chemically discriminating nucleation event: studies of an amyloidogenic sequence from the bacterial protein OsmB.

    Science.gov (United States)

    Jarrett, J T; Lansbury, P T

    1992-12-15

    The sequence of the Escherichia coli OsmB protein was found to resemble that of the C-terminal region of the beta amyloid protein of Alzheimer's disease, which seems to be the major determinant of its unusual structural and solubility properties. A peptide corresponding to residues 28-44 of the OsmB protein was synthesized, and its conformational properties and aggregation behavior were analyzed. The peptide OsmB(28-44) was shown to form amyloid fibrils, as did two sequence analogs designed to test the sequence specificity of fibril formation. These fibrils bound Congo red, and two of the peptides showed birefringence. The peptide fibrils were analyzed by electron microscopy and Fourier transform infrared spectroscopy. Subtle differences were observed which were not interpretable at the molecular level. The rate of fibril formation by each peptide was followed by monitoring the turbidity of supersaturated aqueous solutions. The kinetics of aggregation were characterized by a delay period during which the solution remained clear, followed by a nucleation event which led to a growth phase, during which the solution became viscous and turbid due to the presence of insoluble fibrils. The observation of a kinetic barrier to aggregation is typical of a crystallization event. The delay period could be eliminated by seeding the supersaturated solution with previously formed fibrils. Each peptide could be nucleated by fibrils formed from that same peptide, but not by fibrils from closely related sequences, suggesting that fibril growth requires specific hydrophobic interactions. It appears likely that this repeated sequence motif, which comprises most of the OsmB protein sequence, dictates the structure and possibly the function of that protein.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1463722

  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. PMID:22100554

  11. WtsE, an AvrE-family effector protein from Pantoea stewartii subsp. stewartii, causes disease-associated cell death in corn and requires a chaperone protein for stability.

    Science.gov (United States)

    Ham, Jong Hyun; Majerczak, Doris R; Arroyo-Rodriguez, Angel S; Mackey, David M; Coplin, David L

    2006-10-01

    The pathogenicity of Pantoea stewartii subsp. stewartii to sweet corn and maize requires a Hrp type III secretion system. In this study, we genetically and functionally characterized a disease-specific (Dsp) effector locus, composed of wtsE and wtsF, that is adjacent to the hrp gene cluster. WtsE, a member of the AvrE family of effector proteins, was essential for pathogenesis on corn and was complemented by DspA/E from Erwinia amylovora. An intact C-terminus of WtsE, which contained a putative endoplasmic reticulum membrane retention signal, was important for function of WtsE. Delivery of WtsE into sweet corn leaves by an Escherichia coli strain carrying the hrp cluster of Erwinia chrysanthemi caused water-soaking and necrosis. WtsE-induced cell death was not inhibited by cycloheximide treatment, unlike the hypersensitive response caused by a known Avr protein, AvrRxol. WtsF, the putative chaperone of WtsE, was not required for secretion of WtsE from P. stewartii, and the virulence of wtsF mutants was reduced only at low inoculum concentrations. However, WtsF was required for full accumulation of WtsE within the bacteria at low temperatures. In contrast, WtsF was needed for efficient delivery of WtsE from E. coli via the Erwinia chrysanthemi Hrp system. PMID:17022173

  12. Vitamin D receptor interaction with specific DNA requires a nuclear protein and 1,25-dihydroxyvitamin D3.

    OpenAIRE

    Liao, J.; Ozono, K; Sone, T.; McDonnell, D P; Pike, J W

    1990-01-01

    The regulation of osteocalcin gene expression by 1,25-dihydroxyvitamin D3 is mediated by the vitamin D receptor and a cis-acting DNA response element that has been identified within the 5' region of the osteocalcin promoter. In this report, we show that vitamin D receptors derived from nuclear extracts of mammalian cells bind directly to this cis-acting element in vitro and do so in a manner requiring hormone. Vitamin D receptors derived from reticulocyte lysate translations in vitro or from ...

  13. Biosynthesis of intestinal microvillar proteins. Processing of N-linked carbohydrate is not required for surface expression

    DEFF Research Database (Denmark)

    Danielsen, Erik Michael; Cowell, G M

    1986-01-01

    Castanospermine, an inhibitor of glucosidase I, the initial enzyme in the trimming of N-linked carbohydrate, was used to study the importance of carbohydrate processing in the biosynthesis of microvillar enzymes in organ-cultured pig intestinal explants. For aminopeptidase N (EC 3...... microvillar enzymes, it does not receive post-translational O-linked carbohydrate. Castanospermine suppressed the synthesis of the four enzymes, but did not block their transport to the microvillar membrane, showing that processing of N-linked carbohydrate is not required for microvillar expression. The...

  14. Heterodimerization of the transcription factors E2F-1 and DP-1 is required for binding to the adenovirus E4 (ORF6/7) protein

    DEFF Research Database (Denmark)

    Helin, K; Harlow, E

    1994-01-01

    , and we demonstrate here that heterodimerization of two of these proteins, E2F-1 and DP-1, is required for stable binding to E4. This complex is formed independently of DNA binding and requires the C-terminal 20 amino acids of E4. Furthermore, the binding is dependent on a region of E2F-1 between amino...... acids 284 and 358. This region of E2F-1 is conserved in E2F-2 and E2F-3, and deletion of this region drastically reduces the transcriptional activity of the molecule without affecting DP-1 binding, suggesting that this region of the E2F transcription factors is involved in regulating their activity. Our...... experiments also demonstrate that pRB binding to the E2F-1/DP-1 heterodimer prevents the formation of an E2F-1/DP-1/E4 complex....

  15. Differentiating a Ligand's Chemical Requirements for Allosteric Interactions from Those for Protein Binding. Phenylalanine Inhibition of Pyruvate Kinase

    International Nuclear Information System (INIS)

    The isoform of pyruvate kinase from brain and muscle of mammals (M1-PYK) is allosterically inhibited by phenylalanine. Initial observations in this model allosteric system indicate that Ala binds competitively with Phe, but elicits a minimal allosteric response. Thus, the allosteric ligand of this system must have requirements for eliciting an allosteric response in addition to the requirements for binding. Phe analogues have been used to dissect what chemical properties of Phe are responsible for eliciting the allosteric response. We first demonstrate that the L-2-aminopropanaldehyde substructure of the amino acid ligand is primarily responsible for binding to M1-PYK. Since the allosteric response to Ala is minimal and linear addition of methyl groups beyond the -carbon increase the magnitude of the allosteric response, we conclude that moieties beyond the -carbon are primarily responsible for allostery. Instead of an all-or-none mechanism of allostery, these findings support the idea that the bulk of the hydrophobic side chain, but not the aromatic nature, is the primary determinant of the magnitude of the observed allosteric inhibition. The use of these results to direct structural studies has resulted in a 1.65 Angstroms structure of M1-PYK with Ala bound. The coordination of Ala in the allosteric amino acid binding site confirms the binding role of the L-2-aminopropanaldehyde substructure of the ligand. Collectively, this study confirms that a ligand can have chemical regions specific for eliciting the allosteric signal in addition to the chemical regions necessary for binding

  16. Riboflavin-Induced Disease Resistance Requires the Mitogen-Activated Protein Kinases 3 and 6 in Arabidopsis thaliana

    Science.gov (United States)

    Nie, Shengjun; Xu, Huilian

    2016-01-01

    As a resistance elicitor, riboflavin (vitamin B2) protects plants against a wide range of pathogens. At molecular biological levels, it is important to elucidate the signaling pathways underlying the disease resistance induced by riboflavin. Here, riboflavin was tested to induce resistance against virulent Pseudomonas syringae pv. Tomato DC3000 (Pst DC3000) in Arabidopsis. Results showed that riboflavin induced disease resistance based on MAPK-dependent priming for the expression of PR1 gene. Riboflavin induced transient expression of PR1 gene. However, following Pst DC3000 inoculation, riboflavin potentiated stronger PR1 gene transcription. Further was suggested that the transcript levels of mitogen-activated protein kinases, MPK3 and MPK6, were primed under riboflavin. Upon infection by Pst DC3000, these two enzymes were more strongly activated. The elevated activation of both MPK3 and MPK6 was responsible for enhanced defense gene expression and resistance after riboflavin treatment. Moreover, riboflavin significantly reduced the transcript levels of MPK3 and MPK6 by application of AsA and BAPTA, an H2O2 scavenger and a calcium (Ca2+) scavenger, respectively. In conclusion, MPK3 and MPK6 were responsible for riboflavin-induced resistance, and played an important role in H2O2- and Ca2+-related signaling pathways, and this study could provide a new insight into the mechanistic study of riboflavin-induced defense responses. PMID:27054585

  17. CRL6, a member of the CHD protein family, is required for crown root development in rice.

    Science.gov (United States)

    Wang, Yihua; Wang, Di; Gan, Ting; Liu, Linglong; Long, Wuhua; Wang, Yunlong; Niu, Mei; Li, Xiaohui; Zheng, Ming; Jiang, Ling; Wan, Jianmin

    2016-08-01

    The root system in monocotyledonous plants is largely composed of postembryonic shoot-borne roots named crown roots, which are important for nutrients and water uptake. The molecular mechanism underlying regulation of crown root development is not fully explored. In this study, we characterized a rice (Oryza sativa) mutant defective in crown root formation, designated as crown rootless6 (crl6). Histological analysis showed that CRL6 influences crown root formation by regulating primordial initiation and development. Map-based cloning and subsequent complementation tests verified that the CRL6 gene encodes a member of the large chromodomain, helicase/ATPase, and DNA-binding domain (CHD) family protein. Realtime RT-PCR analysis showed that CRL6 was most highly expressed in the stem base region where crown roots initiated. In addition, auxin-action inhibited phenotype was observed during crl6 development. The expressions of OsIAA genes were down-regulated in crl6. Our results provide evidence that CRL6 plays an important role in crown root development in rice via auxin-related signaling pathway. PMID:27108205

  18. Slack sodium-activated potassium channel membrane expression requires p38 mitogen-activated protein kinase phosphorylation.

    Science.gov (United States)

    Gururaj, Sushmitha; Fleites, John; Bhattacharjee, Arin

    2016-04-01

    p38 MAPK has long been understood as an inducible kinase under conditions of cellular stress, but there is now increasing evidence to support its role in the regulation of neuronal function. Several phosphorylation targets have been identified, an appreciable number of which are ion channels, implicating the possible involvement of p38 MAPK in neuronal excitability. The KNa channel Slack is an important protein to be studied as it is highly and ubiquitously expressed in DRG neurons and is important in the maintenance of their firing accommodation. We sought to examine if the Slack channel could be a substrate of p38 MAPK activity. First, we found that the Slack C-terminus contains two putative p38 MAPK phosphorylation sites that are highly conserved across species. Second, we show via electrophysiology experiments that KNa currents and further, Slack currents, are subject to tonic modulation by p38 MAPK. Third, biochemical approaches revealed that Slack channel regulation by p38 MAPK occurs through direct phosphorylation at the two putative sites of interaction, and mutating both sites prevented surface expression of Slack channels. Based on these results, we conclude that p38 MAPK is an obligate regulator of Slack channel function via the trafficking of channels into the membrane. The present study identifies Slack KNa channels as p38 MAPK substrates. PMID:26721627

  19. Persistent directional cell migration requires ion transport proteins as direction sensors and membrane potential differences in order to maintain directedness

    Directory of Open Access Journals (Sweden)

    Sharma Priyanka

    2011-01-01

    Full Text Available Abstract Background Ion transport proteins generate small electric fields that can induce directional cell motility; however, little is known about their mechanisms that lead to directedness. We investigated Na, K-ATPase (NaKA and Na+/H+ exchanger isoforms (NHE1 and 3 in SaOS-2 and Calvarial osteoblasts, which present anode- and cathode- directed motility, during electrotaxis. Results Significant colocalizations of NaKA with vinculin and pNHE3 with ß-actin were observed to occur at the leading edges of cells. The directedness were attenuated when NaKA or NHE3 was inhibited, confirming their implication in directional sensing. Depending on the perceived direction, a divergent regulation in PIP2 levels as a function of NHE3 and NaKA levels was observed, suggesting that PIP2 may act as a spatiotemporal regulator of the cell membrane during electrotaxis. Moreover, at the same places where pNHE3 accumulates, bubble-shaped H+ clouds were observed, suggesting a physio-mechanical role for NHE3. The cell membrane becomes hyperpolarized at the front and depolarized at the back, which confirms NaKA activity at the leading edge. Conclusion We suggest a novel role for both NaKA and NHE3 that extends beyond ion translocation and conclude that they can act as directional sensors and Vmem as a regulatory cue which maintain the persistent direction in electrotaxis.

  20. 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.

  1. Energy and protein requirements of 3/4 Zebu × 1/4 Holstein crossbreds fed different calcium and phosphorus levels in the diet

    Directory of Open Access Journals (Sweden)

    L.F. Prados

    2015-04-01

    Full Text Available The aim of this study was to determine the nutritional requirements of energy and protein for maintenance and weight gain of crossbred cattle, as well as their efficiencies. Fifty 3/4 Zebu × 1/4 Holstein crossbred bulls with initial weights of 214±4kg and aged 11±0.2 months on average were used in this experiment. Four animals were used in the reference group; ten bulls were fed at the maintenance level; and the remaining 36 bulls were fed ad libitum and distributed in a completely randomized design in a 3×3 factorial arrangement, which had three feedlot periods (56, 112 or 168 days and three calcium and phosphorus levels (low, medium and normal in the diet. Four of the maintenance animals had their heat production measured by respirometry at the Laboratory of Metabolism and Calorimetry of UFMG. After slaughter, composite samples, referred to as carcass and noncarcass samples were obtained from each animal. The net energy requirements for maintenance (NEm and metabolizable energy for maintenance (MEm were 68.9 and 90.1 kcal/EBW0.75/day, respectively. The efficiency (km was 76.41%. The NEm requirement determined in the respirometry chamber was 85.5 kcal/kg0.75. The following equations were obtained for net energy for gain (NEg and net protein for gain (NPg: NEg (Mcal/day = 0.0505±0.000986 × EBW0.75 × EBWG1.095 and NPg (g/day = 162.79±18.2546 × EBWG - 1.30±5.3010 × RE. The efficiencies of fat and protein deposition were 70.04 and 15.12%, respectively. In conclusion, the requirements of NEm for growing and finishing non-castrated 3/4 Zebu × 1/4 Holstein crossbred cattle are 68.9 kcal/EBW0.75/day. Requirements of NEg and NPg can be obtained by the following equations: NEg (Mcal/day = 0.05050.000986 × EBW0.75 × EBWG1.095 and NPg (g/day = 162.79±18.2546 × EBWG - 1.30±5.3010 × RE.

  2. The yeast HRS1 gene encodes a polyglutamine-rich nuclear protein required for spontaneous and hpr1-induced deletions between direct repeats

    Energy Technology Data Exchange (ETDEWEB)

    Santos-Rosa, H.; Aguilera, A. [Universidad de Sevilla (Spain); Clever, B. [Univ. of Bern (Switzerland)] [and others

    1996-03-01

    The hrs1-1 mutation was isolated as an extragenic suppressor of the hyperrecombination phenotype of hpr1{Delta} cells. We have cloned, sequenced and deleted from the genome the HRS1 gene. The DNA sequence of the HRS1 gene reveals that it is identical to PGD1, a gene with no reported function, and that the Hrs1p protein contains polyglutamine stretches typically found in transcription factors. We have purified a His(6) tagged version of Hrs1p protein from E. coli and have obtained specific anti-Hrs1p polyclonal antibodies. We show that Hrs1p is a 49-kD nuclear protein, as determined bv indirect immunofluorescence microscopy and Western blot analysis. The hrs1{Delta} null mutation reduces the frequency of deletions in wild-type and hpr1{Delta} backgrounds sevenfold below wild-type and rad52 levels. Furthermore, hrs1{Delta} cells show reduced induction of the GAL1,10 promoter relative to wild-type cells. Our results suggest that Hrs1p is required for the formation of deletions between direct repeats and that it may function in gene expression. This suggests a connection between gene expression and direct repeat recombination. In this context, we discuss the possible roles of Hrs1p and Hpr1p in initiation of direct-repeat recombination. 62 refs., 5 figs., 5 tabs.

  3. Stimulation of translation by human Unr requires cold shock domains 2 and 4, and correlates with poly(A) binding protein interaction

    Science.gov (United States)

    Ray, Swagat; Anderson, Emma C.

    2016-01-01

    The RNA binding protein Unr, which contains five cold shock domains, has several specific roles in post-transcriptional control of gene expression. It can act as an activator or inhibitor of translation initiation, promote mRNA turnover, or stabilise mRNA. Its role depends on the mRNA and other proteins to which it binds, which includes cytoplasmic poly(A) binding protein 1 (PABP1). Since PABP1 binds to all polyadenylated mRNAs, and is involved in translation initiation by interaction with eukaryotic translation initiation factor 4G (eIF4G), we investigated whether Unr has a general role in translational control. We found that Unr strongly stimulates translation in vitro, and mutation of cold shock domains 2 or 4 inhibited its translation activity. The ability of Unr and its mutants to stimulate translation correlated with its ability to bind RNA, and to interact with PABP1. We found that Unr stimulated the binding of PABP1 to mRNA, and that Unr was required for the stable interaction of PABP1 and eIF4G in cells. siRNA-mediated knockdown of Unr reduced the overall level of cellular translation in cells, as well as that of cap-dependent and IRES-dependent reporters. These data describe a novel role for Unr in regulating cellular gene expression. PMID:26936655

  4. Stimulation of translation by human Unr requires cold shock domains 2 and 4, and correlates with poly(A) binding protein interaction.

    Science.gov (United States)

    Ray, Swagat; Anderson, Emma C

    2016-01-01

    The RNA binding protein Unr, which contains five cold shock domains, has several specific roles in post-transcriptional control of gene expression. It can act as an activator or inhibitor of translation initiation, promote mRNA turnover, or stabilise mRNA. Its role depends on the mRNA and other proteins to which it binds, which includes cytoplasmic poly(A) binding protein 1 (PABP1). Since PABP1 binds to all polyadenylated mRNAs, and is involved in translation initiation by interaction with eukaryotic translation initiation factor 4G (eIF4G), we investigated whether Unr has a general role in translational control. We found that Unr strongly stimulates translation in vitro, and mutation of cold shock domains 2 or 4 inhibited its translation activity. The ability of Unr and its mutants to stimulate translation correlated with its ability to bind RNA, and to interact with PABP1. We found that Unr stimulated the binding of PABP1 to mRNA, and that Unr was required for the stable interaction of PABP1 and eIF4G in cells. siRNA-mediated knockdown of Unr reduced the overall level of cellular translation in cells, as well as that of cap-dependent and IRES-dependent reporters. These data describe a novel role for Unr in regulating cellular gene expression. PMID:26936655

  5. Lysine residues K66, K109, and K110 in the bovine foamy virus transactivator protein are required for transactivation and viral replication.

    Science.gov (United States)

    Zhang, Suzhen; Cui, Xiaoxu; Li, Jing; Liang, Zhibin; Qiao, Wentao; Tan, Juan

    2016-04-01

    Bovine foamy virus (BFV) is a complex retrovirus that infects cattle. Like all retroviruses, BFV encodes a transactivator Tas protein (BTas) that increases gene transcription from viral promoters. BFV encodes two promoters that can interact with BTas, a conserved promoter in the 5' long terminal repeat (LTR) and a unique internal promoter (IP). Our previous study showed that BTas is acetylated by p300 at residues K66, K109, and K110, which markedly enhanced the ability of BTas to bind to DNA. However, whether these residues are important for BFV replication was not determined. Therefore, in this study we provide direct evidence that BTas is required for BFV replication and demonstrate that residues K66, K109, and K110 are critical for BTas function and BFV replication. Full-length infectious clones were generated, which were BTas deficient or contained lysine to arginine (K→R) mutations at position 66, 109, and/or 110. In vivo data indicated that K→R mutations at positions 66, 109, and 110 in BTas impaired transactivation of both the LTR and IP promoters. In addition, the K→R mutations in full-length infectious clones reduced expression of viral proteins, and the triple mutant and BTas deletion completely abrogated viral replication. Taken together, these results indicate that lysine residues at positions 66, 109, and 110 in the BTas protein are crucial for BFV replication and suggest a potential role for BTas acetylation in regulating the viral life cycle. PMID:26980333

  6. Inhibitor of apoptosis-stimulating protein of p53 (iASPP is required for neuronal survival after axonal injury.

    Directory of Open Access Journals (Sweden)

    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.

  7. cAMP response element binding protein is required for differentiation of respiratory epithelium during murine development.

    Directory of Open Access Journals (Sweden)

    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.

  8. A study of the protein and amino acid requirements of the growing New Zealand White rabbit with emphasis on lysine and the sulphur-containing amino acids.

    Science.gov (United States)

    Spreadbury, D

    1978-05-01

    1. New Zealand White (NZW) rabbits were given, between 4 and 8 weeks of age, a range of diets, based on oats and fish meal, containing from 104 to 255 g crude protein (nitrogen x 6.25; CP)/kg to establish the level of CP below which growth was retarded. 2. In three experiments each diet was fed to four animals and food intake, growth and N balance were measured over 4 weeks. Body analysis was also carried out after two of the experiments. 3. The rates of food intake and growth of animals increased with dietary CP concentration until a CP concentration of approximately 150 g/kg diet had been reached. Beyond this there was little further improvement. N balance studies showed that once this dietary concentration of CP had been reached, there was a reduced rate of N retention. 4. Good agreement was found between N retention measured by balance methods and by body analysis: body composition showed a tendency towards an increase 5. Microbial protein produced in the caecum and eaten during coprophagy, was found to supplement the dietary protein by approximately 2 g CP/d, or by only 0.1 of a normal dietary intake of CP. 6. In the second part of the study NZW rabbits were offered, between 5 and 8 weeks of age, diets based on oats containing 150 g CP/kg. The protein supplied by oats was supplemented with maize gluten, gelatin, groundnut meal, casein, soya-bean meal or fish meal. 7. Rabbits offered diets containing casein, soya-bean meal and fish meal gained 40-50 g/d similar, to animals given a well-balanced control diet, while those given diets containing maize gluten, gelatin or groundnut meal gained approximately 30 g/d. This indicated that amino acid balance in dietary protein was important to the growing rabbit. 8. In later experiments, diets based on cereals and groundnut meal supplemented with varying amounts of lysine and methionine were offered during a 3-week-post-weaning period in order to assess requirements for those limiting amino acids. 9. The addition of both

  9. The relationship between dietary crude protein and dietary lysine requirement by broiler chicks on diets with and without the "ideal" amino acid balance.

    Science.gov (United States)

    Surisdiarto; Farrell, D J

    1991-04-01

    Experiments were designed with groups of broiler chickens to test whether the requirement for dietary lysine was related to the CP content of diets that had either a surplus or the recommended requirement of critical amino acids. Diets of equal energy were formulated using a summit and basal diet to obtain a range of CP contents from 14 to 26% (Experiment 1) with surplus critical amino acids or from 12 to 25% (Experiment 2) with the "ideal" amino acid balance. In both experiments at each CP level, there was an optimum lysine content for weight gain and feed conversion ratio (FCR). Diets with ideal amino acid balances gave less of a response as measured by growth rate and FCR than those with surpluses. Combining all data gave a significant improvement in both weight gain and FCR with increasing intake of dietary lysine. It would appear that because performance was highest on diets with the highest crude protein, there may be a requirement for CP per se. Alternatively, because all diets contained substantial amounts of synthetic amino acids, broilers may have a requirement for amino acid peptides that is not met by free amino acids. PMID:1908577

  10. Exploring the structural requirements for inhibition of the ubiquitin E3 ligase breast cancer associated protein 2 (BCA2) as a treatment for breast cancer.

    Science.gov (United States)

    Brahemi, Ghali; Kona, Fathima R; Fiasella, Annalisa; Buac, Daniela; Soukupová, Jitka; Brancale, Andrea; Burger, Angelika M; Westwell, Andrew D

    2010-04-01

    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 against recombinant BCA2 protein. Notably, the DSF analogs (3a and 3c) and dithio(peroxo)thioate compounds (5d and 5f) were found to have potent activity (submicromolar IC(50)) in BCA2 positive MCF-7 and T47D cells but were inactive (IC(50) > 10 microM) in BCA2 negative MDA-MB-231 breast cancer cells and the normal breast epithelial cell line MCF10A. Testing in the isogenic BCA2 +ve MDA-MB-231/ER cell line restored antitumor activity for compounds that were inactive in the BCA2 -ve MDA-MB-231 cell line. In contrast, structurally related dithiocarbamates and benzisothiazolones (lacking the disulfide bond) were all inactive. Compounds 5d and 5f were additionally found to lack ALDH-inhibitory activity, suggestive of selective E3 ligase-inhibitory activity and worthy of further development. PMID:20222671

  11. Transcriptional activation of peroxisome proliferator-activated receptor-γ requires activation of both protein kinase A and Akt during adipocyte differentiation

    International Nuclear Information System (INIS)

    Research highlights: → Elevated cAMP activates both PKA and Epac. → PKA activates CREB transcriptional factor and Epac activates PI3K/Akt pathway via Rap1. → Akt modulates PPAR-γ transcriptional activity in concert with CREB. -- Abstract: Peroxisome proliferator-activated receptor-γ (PPAR-γ) is required for the conversion of pre-adipocytes. However, the mechanism underlying activation of PPAR-γ is unclear. Here we showed that cAMP-induced activation of protein kinase A (PKA) and Akt is essential for the transcriptional activation of PPAR-γ. Hormonal induction of adipogenesis was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), by a protein kinase A (PKA) inhibitor (H89), and by a Rap1 inhibitor (GGTI-298). Transcriptional activity of PPAR-γ was markedly enhanced by 3-isobutyl-1-methylxanthine (IBMX), but not insulin and dexamethasone. In addition, IBMX-induced PPAR-γ transcriptional activity was blocked by PI3K/Akt, PKA, or Rap1 inhibitors. 8-(4-Chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-Me-cAMP) which is a specific agonist for exchanger protein directly activated by cAMP (Epac) significantly induced the activation of Akt. Furthermore, knock-down of Akt1 markedly attenuated PPAR-γ transcriptional activity. These results indicate that both PKA and Akt signaling pathways are required for transcriptional activation of PPAR-γ, suggesting post-translational activation of PPAR-γ might be critical step for adipogenic gene expression.

  12. Transcriptional activation of peroxisome proliferator-activated receptor-{gamma} requires activation of both protein kinase A and Akt during adipocyte differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sang-pil [Department of Thoracic and Cardiovascular Surgery, Pusan National University School of Medicine (Korea, Republic of); Ha, Jung Min; Yun, Sung Ji; Kim, Eun Kyoung [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of); Chung, Sung Woon [Department of Thoracic and Cardiovascular Surgery, Pusan National University School of Medicine (Korea, Republic of); Hong, Ki Whan; Kim, Chi Dae [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of); Bae, Sun Sik, E-mail: sunsik@pusan.ac.kr [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of)

    2010-08-13

    Research highlights: {yields} Elevated cAMP activates both PKA and Epac. {yields} PKA activates CREB transcriptional factor and Epac activates PI3K/Akt pathway via Rap1. {yields} Akt modulates PPAR-{gamma} transcriptional activity in concert with CREB. -- Abstract: Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is required for the conversion of pre-adipocytes. However, the mechanism underlying activation of PPAR-{gamma} is unclear. Here we showed that cAMP-induced activation of protein kinase A (PKA) and Akt is essential for the transcriptional activation of PPAR-{gamma}. Hormonal induction of adipogenesis was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), by a protein kinase A (PKA) inhibitor (H89), and by a Rap1 inhibitor (GGTI-298). Transcriptional activity of PPAR-{gamma} was markedly enhanced by 3-isobutyl-1-methylxanthine (IBMX), but not insulin and dexamethasone. In addition, IBMX-induced PPAR-{gamma} transcriptional activity was blocked by PI3K/Akt, PKA, or Rap1 inhibitors. 8-(4-Chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-Me-cAMP) which is a specific agonist for exchanger protein directly activated by cAMP (Epac) significantly induced the activation of Akt. Furthermore, knock-down of Akt1 markedly attenuated PPAR-{gamma} transcriptional activity. These results indicate that both PKA and Akt signaling pathways are required for transcriptional activation of PPAR-{gamma}, suggesting post-translational activation of PPAR-{gamma} might be critical step for adipogenic gene expression.

  13. Double-stranded RNA-dependent protein kinase is required for bone calcification in MC3T3-E1 cells in vitro.

    Science.gov (United States)

    Yoshida, Kaya; Okamura, Hirohiko; Amorim, Bruna Rabelo; Ozaki, Akiko; Tanaka, Hiroaki; Morimoto, Hiroyuki; Haneji, Tatsuji

    2005-11-15

    In this study, we demonstrated that double-stranded RNA-dependent protein kinase (PKR) is required for the calcification of osteoblasts via the signal transducers and activators of transcription 1alpha (STAT1alpha) signaling in vitro. A dominant-negative mutant PKR cDNA, in which the amino acid lysine at 296 was replaced with arginine and which does not have catalytic activity, was transfected into mouse osteoblastic MC3T3-E1 cells; thereby, we established cells that stably expressed the PKR mutant gene (PKR-K/R). Phosphorylation of PKR was not stimulated by polyinosic-polycytidylic acid in the mutant cells. The PKR-K/R mutant cells exhibited up-regulated cell growth and had low alkaline phosphatase (ALP) activity. The PKR-K/R mutant cells were not able to form bone nodules in vitro. In the PKR-K/R mutant cells, runt-related gene 2 (Runx2)-mediated transcription decreased compared with the levels in the control cells. The expression of STAT1alpha protein increased and the protein was translocated to the nucleus in the PKR-K/R mutant cells. When the expression of STAT1alpha protein in PKR mutant cells was suppressed using RNAi, the activity of Runx2-mediated transcription recovered to the control level. Our results indicate that PKR is a stimulator of Runx2 transcription and is a negative modulator of STAT1alpha expression. Our findings also suggest that PKR plays important roles in the differentiation and calcification of osteoblasts by modulating STAT1alpha and/or Runx2 expression. PMID:16216244

  14. The MTL1 Pentatricopeptide Repeat Protein Is Required for Both Translation and Splicing of the Mitochondrial NADH DEHYDROGENASE SUBUNIT7 mRNA in Arabidopsis.

    Science.gov (United States)

    Haïli, Nawel; Planchard, Noelya; Arnal, Nadège; Quadrado, Martine; Vrielynck, Nathalie; Dahan, Jennifer; des Francs-Small, Catherine Colas; Mireau, Hakim

    2016-01-01

    Mitochondrial translation involves a complex interplay of ancient bacteria-like features and host-derived functionalities. Although the basic components of the mitochondrial translation apparatus have been recognized, very few protein factors aiding in recruiting ribosomes on mitochondria-encoded messenger RNA (mRNAs) have been identified in higher plants. In this study, we describe the identification of the Arabidopsis (Arabidopsis thaliana) MITOCHONDRIAL TRANSLATION FACTOR1 (MTL1) protein, a new member of the Pentatricopeptide Repeat family, and show that it is essential for the translation of the mitochondrial NADH dehydrogenase subunit7 (nad7) mRNA. We demonstrate that mtl1 mutant plants fail to accumulate the Nad7 protein, even though the nad7 mature mRNA is produced and bears the same 5' and 3' extremities as in wild-type plants. We next observed that polysome association of nad7 mature mRNA is specifically disrupted in mtl1 mutants, indicating that the absence of Nad7 results from a lack of translation of nad7 mRNA. These findings illustrate that mitochondrial translation requires the intervention of gene-specific nucleus-encoded PPR trans-factors and that their action does not necessarily involve the 5' processing of their target mRNA, as observed previously. Interestingly, a partial decrease in nad7 intron 2 splicing was also detected in mtl1 mutants, suggesting that MTL1 is also involved in group II intron splicing. However, this second function appears to be less essential for nad7 expression than its role in translation. MTL1 will be instrumental to understand the multifunctionality of PPR proteins and the mechanisms governing mRNA translation and intron splicing in plant mitochondria. PMID:26537562

  15. Loss of mitogen-activated protein kinase kinase kinase 4 (MAP3K4 reveals a requirement for MAPK signalling in mouse sex determination.

    Directory of Open Access Journals (Sweden)

    Debora Bogani

    2009-09-01

    Full Text Available Sex determination in mammals is controlled by the presence or absence of the Y-linked gene SRY. In the developing male (XY gonad, sex-determining region of the Y (SRY protein acts to up-regulate expression of the related gene, SOX9, a transcriptional regulator that in turn initiates a downstream pathway of testis development, whilst also suppressing ovary development. Despite the requirement for a number of transcription factors and secreted signalling molecules in sex determination, intracellular signalling components functioning in this process have not been defined. Here we report a role for the phylogenetically ancient mitogen-activated protein kinase (MAPK signalling pathway in mouse sex determination. Using a forward genetic screen, we identified the recessive boygirl (byg mutation. On the C57BL/6J background, embryos homozygous for byg exhibit consistent XY gonadal sex reversal. The byg mutation is an A to T transversion causing a premature stop codon in the gene encoding MAP3K4 (also known as MEKK4, a mitogen-activated protein kinase kinase kinase. Analysis of XY byg/byg gonads at 11.5 d post coitum reveals a growth deficit and a failure to support mesonephric cell migration, both early cellular processes normally associated with testis development. Expression analysis of mutant XY gonads at the same stage also reveals a dramatic reduction in Sox9 and, crucially, Sry at the transcript and protein levels. Moreover, we describe experiments showing the presence of activated MKK4, a direct target of MAP3K4, and activated p38 in the coelomic region of the XY gonad at 11.5 d post coitum, establishing a link between MAPK signalling in proliferating gonadal somatic cells and regulation of Sry expression. Finally, we provide evidence that haploinsufficiency for Map3k4 accounts for T-associated sex reversal (Tas. These data demonstrate that MAP3K4-dependent signalling events are required for normal expression of Sry during testis development, and

  16. Feasibility and efficacy of an isocaloric high-protein vs. standard diet on insulin requirement, body weight and metabolic parameters in patients with type 2 diabetes on insulin therapy

    DEFF Research Database (Denmark)

    Luger, M; Holstein, B; Schindler, K;

    2013-01-01

    To determine the feasibility and efficacy of a high-protein diet compared with a standard diet aiming for weight maintenance in insulin treated type-2 diabetic patients on insulin requirement, body weight and metabolic parameters over 12 weeks.......To determine the feasibility and efficacy of a high-protein diet compared with a standard diet aiming for weight maintenance in insulin treated type-2 diabetic patients on insulin requirement, body weight and metabolic parameters over 12 weeks....

  17. The conserved glycine residues in the transmembrane domain of the Semliki Forest virus fusion protein are not required for assembly and fusion

    International Nuclear Information System (INIS)

    The alphavirus Semliki Forest virus (SFV) infects cells via a low pH-triggered fusion reaction mediated by the viral E1 protein. Both the E1 fusion peptide and transmembrane (TM) domain are essential for membrane fusion, but the functional requirements for the TM domain are poorly understood. Here we explored the role of the five TM domain glycine residues, including the highly conserved glycine pair at E1 residues 415/416. SFV mutants with alanine substitutions for individual or all five glycine residues (5G/A) showed growth kinetics and fusion pH dependence similar to those of wild-type SFV. Mutants with increasing substitution of glycine residues showed an increasingly more stringent requirement for cholesterol during fusion. The 5G/A mutant showed decreased fusion kinetics and extent in fluorescent lipid mixing assays. TM domain glycine residues thus are not required for efficient SFV fusion or assembly but can cause subtle effects on the properties of membrane fusion

  18. Identification and Characterization of a Novel Issatchenkia orientalis GPI-Anchored Protein, IoGas1, Required for Resistance to Low pH and Salt Stress.

    Science.gov (United States)

    Matsushika, Akinori; Negi, Kanako; Suzuki, Toshihiro; Goshima, Tetsuya; Hoshino, Tamotsu

    2016-01-01

    The use of yeasts tolerant to acid (low pH) and salt stress is of industrial importance for several bioproduction processes. To identify new candidate genes having potential roles in low-pH tolerance, we screened an expression genomic DNA library of a multiple-stress-tolerant yeast, Issatchenkia orientalis (Pichia kudriavzevii), for clones that allowed Saccharomyces cerevisiae cells to grow under highly acidic conditions (pH 2.0). A genomic DNA clone containing two putative open reading frames was obtained, of which the putative protein-coding gene comprising 1629 bp was retransformed into the host. This transformant grew significantly at pH 2.0, and at pH 2.5 in the presence of 7.5% Na2SO4. The predicted amino acid sequence of this new gene, named I. orientalis GAS1 (IoGAS1), was 60% identical to the S. cerevisiae Gas1 protein, a glycosylphosphatidylinositol-anchored protein essential for maintaining cell wall integrity, and 58-59% identical to Candida albicans Phr1 and Phr2, pH-responsive proteins implicated in cell wall assembly and virulence. Northern hybridization analyses indicated that, as for the C. albicans homologs, IoGAS1 expression was pH-dependent, with expression increasing with decreasing pH (from 4.0 to 2.0) of the medium. These results suggest that IoGAS1 represents a novel pH-regulated system required for the adaptation of I. orientalis to environments of diverse pH. Heterologous expression of IoGAS1 complemented the growth and morphological defects of a S. cerevisiae gas1Δ mutant, demonstrating that IoGAS1 and the corresponding S. cerevisiae gene play similar roles in cell wall biosynthesis. Site-directed mutagenesis experiments revealed that two conserved glutamate residues (E161 and E262) in the IoGas1 protein play a crucial role in yeast morphogenesis and tolerance to low pH and salt stress. Furthermore, overexpression of IoGAS1 in S. cerevisiae remarkably improved the ethanol fermentation ability at pH 2.5, and at pH 2.0 in the presence of

  19. 陕北白绒山羊羯羊能量和蛋白质需要量%Energy and Protein Requirements of Shanbei White Cashmere Wether Goats

    Institute of Scientific and Technical Information of China (English)

    高晔; 李碧波; 黄帅; 王荣斌; 王冬; 李琴芳; 屈雷

    2016-01-01

    This experiment was conducted to investigate the requirements of energy and protein of Shanbei white cashmere wether goats so as to provide data for establishing them.Thirty-six healthy one-year-old Shan-bei white cashmere wether goats with similar body weight were randomly divided into 9 groups according to a 3×3 ( energy×protein) completely random experiment design, and each group had 4 replicates with 1 goat per replicate.digestible energy (DE) level in diet was formulated at 100%, 110%and 120%of NRC (1981) rec-ommendation, respectively; and crude protein ( CP) level was formulated at 90%, 110%and 130%of NRC (1981) recommendation, respectively.The pretest lasted for 7 days, and the test lasted for 46 days.The re-sults showed as follows:1) energy level in diet had extremely significant effects on dry matter intake ( DMI) and average daily gain ( ADG) ( P<0.01) , and protein level significantly affected DMI ( P<0.05);there was significant interaction on DMI between energy and protein levels in diet ( P<0.05).2) Digestible nitrogen ( N) in low energy group was significantly lower than that in medium and high energy groups ( P<0.05); N intake, digestible N, retained N, N apparent digestibility in low protein group were extremely lower than those of medium and high protein groups ( P<0.01) .3) Gross energy ( GE) digestibility and metabolic rate were extremely significantly increased with energy level improving ( P<0.01 ); metabolic rates of GE and DE in high protein group were significantly lower than these in low protein group ( P<0.05); There was significant interactions of energy×protein on GE digestibility, GE metabolic rate and DE metabolic rate ( P<0.05 or P<0.01) .We establish regression equations of energy and protein requirement of one-year-old Shanbei white cashmere wether goats, the proper range of DE and ME levels in diet are 8.80 to 10.61 MJ/kg and 7.34 to 8.76 MJ/kg, respectively, and the appropriate CP and digestible CP levels are 10.00%and 7

  20. De novo analysis of the transcriptome of Pratylenchus zeae to identify transcripts for proteins required for structural integrity, sensation, locomotion and parasitism.

    Science.gov (United States)

    Fosu-Nyarko, John; Tan, Jo-Anne C H; Gill, Reetinder; Agrez, Vaughan G; Rao, Uma; Jones, Michael G K

    2016-05-01

    The root lesion nematode Pratylenchus zeae, a migratory endoparasite, is an economically important pest of major crop plants (e.g. cereals, sugarcane). It enters host roots, migrates through root tissues and feeds from cortical cells, and defends itself against biotic and abiotic stresses in the soil and in host tissues. We report de novo sequencing of the P. zeae transcriptome using 454 FLX, and the identification of putative transcripts encoding proteins required for movement, response to stimuli, feeding and parasitism. Sequencing generated 347 443 good quality reads which were assembled into 10 163 contigs and 139 104 singletons: 65% of contigs and 28% of singletons matched sequences of free-living and parasitic nematodes. Three-quarters of the annotated transcripts were common to reference nematodes, mainly representing genes encoding proteins for structural integrity and fundamental biochemical processes. Over 15 000 transcripts were similar to Caenorhabditis elegans genes encoding proteins with roles in mechanical and neural control of movement, responses to chemicals, mechanical and thermal stresses. Notably, 766 transcripts matched parasitism genes employed by both migratory and sedentary endoparasites in host interactions, three of which hybridized to the gland cell region, suggesting that they might be secreted. Conversely, transcripts for effectors reported to be involved in feeding site formation by sedentary endoparasites were conspicuously absent. Transcripts similar to those encoding some secretory-excretory products at the host interface of Brugia malayi, the secretome of Meloidogyne incognita and products of gland cells of Heterodera glycines were also identified. This P. zeae transcriptome provides new information for genome annotation and functional analysis of possible targets for control of pratylenchid nematodes. PMID:26292651

  1. Intravital Imaging of Vascular Transmigration by the Lyme Spirochete: Requirement for the Integrin Binding Residues of the B. burgdorferi P66 Protein.

    Directory of Open Access Journals (Sweden)

    Devender Kumar

    2015-12-01

    Full Text Available Vascular extravasation, a key step in systemic infection by hematogenous microbial pathogens, is poorly understood, but has been postulated to encompass features similar to vascular transmigration by leukocytes. The Lyme disease spirochete can cause a variety of clinical manifestations, including arthritis, upon hematogenous dissemination. This pathogen encodes numerous surface adhesive proteins (adhesins that may promote extravasation, but none have yet been implicated in this process. In this work we report the novel use of intravital microscopy of the peripheral knee vasculature to study transmigration of the Lyme spirochete in living Cd1d-/-mice. In the absence of iNKT cells, major immune modulators in the mouse joint, spirochetes that have extravasated into joint-proximal tissue remain in the local milieu and can be enumerated accurately. We show that BBK32, a fibronectin and glycosaminoglycan adhesin of B. burgdorferi involved in early steps of endothelial adhesion, is not required for extravasation from the peripheral knee vasculature. In contrast, almost no transmigration occurs in the absence of P66, an outer membrane protein that has porin and integrin adhesin functions. Importantly, P66 mutants specifically defective in integrin binding were incapable of promoting extravasation. P66 itself does not promote detectable microvascular interactions, suggesting that vascular adhesion of B. burgdorferi mediated by other adhesins, sets the stage for P66-integrin interactions leading to transmigration. Although integrin-binding proteins with diverse functions are encoded by a variety of bacterial pathogens, P66 is the first to have a documented and direct role in vascular transmigration. The emerging picture of vascular escape by the Lyme spirochete shows similarities, but distinct differences from leukocyte transmigration.

  2. Autophagy-Associated Protein SmATG12 Is Required for Fruiting-Body Formation in the Filamentous Ascomycete Sordaria macrospora.

    Science.gov (United States)

    Werner, Antonia; Herzog, Britta; Frey, Stefan; Pöggeler, Stefanie

    2016-01-01

    In filamentous fungi, autophagy functions as a catabolic mechanism to overcome starvation and to control diverse developmental processes under normal nutritional conditions. Autophagy involves the formation of double-membrane vesicles, termed autophagosomes that engulf cellular components and bring about their degradation via fusion with vacuoles. Two ubiquitin-like (UBL) conjugation systems are essential for the expansion of the autophagosomal membrane: the UBL protein ATG8 is conjugated to the lipid phosphatidylethanolamine and the UBL protein ATG12 is coupled to ATG5. We recently showed that in the homothallic ascomycete Sordaria macrospora autophagy-related genes encoding components of the conjugation systems are required for fruiting-body development and/or are essential for viability. In the present work, we cloned and characterized the S. macrospora (Sm)atg12 gene. Two-hybrid analysis revealed that SmATG12 can interact with SmATG7 and SmATG3. To examine its role in S. macrospora, we replaced the open reading frame of Smatg12 with a hygromycin resistance cassette and generated a homokaryotic ΔSmatg12 knockout strain, which displayed slower vegetative growth under nutrient starvation conditions and was unable to form fruiting bodies. In the hyphae of S. macrospora EGFP-labeled SmATG12 was detected in the cytoplasm and as punctate structures presumed to be phagophores or phagophore assembly sites. Delivery of EGFP-labelled SmATG8 to the vacuole was entirely dependent on SmATG12. PMID:27309377

  3. Mutational analysis of the RNA-binding domain of the Prunus necrotic ringspot virus (PNRSV) movement protein reveals its requirement for cell-to-cell movement

    International Nuclear Information System (INIS)

    The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for cell-to-cell movement. MP subcellular localization studies using a GFP fusion protein revealed highly punctate structures between neighboring cells, believed to represent plasmodesmata. Deletion of the RNA-binding domain (RBD) of PNRSV MP abolishes the cell-to-cell movement. A mutational analysis on this RBD was performed in order to identify in vivo the features that govern viral transport. Loss of positive charges prevented the cell-to-cell movement even though all mutants showed a similar accumulation level in protoplasts to those observed with the wild-type (wt) MP. Synthetic peptides representing the mutants and wild-type RBDs were used to study RNA-binding affinities by EMSA assays being approximately 20-fold lower in the mutants. Circular dichroism analyses revealed that the secondary structure of the peptides was not significantly affected by mutations. The involvement of the affinity changes between the viral RNA and the MP in the viral cell-to-cell movement is discussed

  4. Cleavage of the HPV16 Minor Capsid Protein L2 during Virion Morphogenesis Ablates the Requirement for Cellular Furin during De Novo Infection

    Directory of Open Access Journals (Sweden)

    Linda Cruz

    2015-11-01

    Full Text Available Infections by high-risk human papillomaviruses (HPV are the causative agents for the development of cervical cancer. As with other non-enveloped viruses, HPVs are taken up by the cell through endocytosis following primary attachment to the host cell. Through studies using recombinant pseudovirus particles (PsV, many host cellular proteins have been implicated in the process. The proprotein convertase furin has been demonstrated to cleave the minor capsid protein, L2, post-attachment to host cells and is required for infectious entry by HPV16 PsV. In contrast, using biochemical inhibition by a furin inhibitor and furin-negative cells, we show that tissue-derived HPV16 native virus (NV initiates infection independent of cellular furin. We show that HPV16 L2 is cleaved during virion morphogenesis in differentiated tissue. In addition, HPV45 is also not dependent on cellular furin, but two other alpha papillomaviruses, HPV18 and HPV31, are dependent on the activity of cellular furin for infection.

  5. Ciliogenesis in Caenorhabditis elegans requires genetic interactions between ciliary middle segment localized NPHP-2 (inversin) and transition zone-associated proteins

    Science.gov (United States)

    Warburton-Pitt, Simon R. F.; Jauregui, Andrew R.; Li, Chunmei; Wang, Juan; Leroux, Michel R.; Barr, Maureen M.

    2012-01-01

    The cystic kidney diseases nephronophthisis (NPHP), Meckel–Gruber syndrome (MKS) and Joubert syndrome (JBTS) share an underlying etiology of dysfunctional cilia. Patients diagnosed with NPHP type II have mutations in the gene INVS (also known as NPHP2), which encodes inversin, a cilia localizing protein. Here, we show that the C. elegans inversin ortholog, NPHP-2, localizes to the middle segment of sensory cilia and that nphp-2 is partially redundant with nphp-1 and nphp-4 (orthologs of human NPHP1 and NPHP4, respectively) for cilia placement within the head and tail sensilla. nphp-2 also genetically interacts with MKS ciliopathy gene orthologs, including mks-1, mks-3, mks-6, mksr-1 and mksr-2, in a sensilla-dependent manner to control cilia formation and placement. However, nphp-2 is not required for correct localization of the NPHP- and MKS-encoded ciliary transition zone proteins or for intraflagellar transport (IFT). We conclude that INVS/NPHP2 is conserved in C. elegans and that nphp-2 plays an important role in C. elegans cilia by acting as a modifier of the NPHP and MKS pathways to control cilia formation and development. PMID:22393243

  6. Detection of the Endosomal Sorting Complex Required for Transport in Entamoeba histolytica and Characterization of the EhVps4 Protein

    Directory of Open Access Journals (Sweden)

    Israel López-Reyes

    2010-01-01

    Full Text Available Eukaryotic endocytosis involves multivesicular bodies formation, which is driven by endosomal sorting complexes required for transport (ESCRT. Here, we showed the presence and expression of homologous ESCRT genes in Entamoeba histolytica. We cloned and expressed the Ehvps4 gene, an ESCRT member, to obtain the recombinant EhVps4 and generate specific antibodies, which immunodetected EhVps4 in cytoplasm of trophozoites. Bioinformatics and biochemical studies evidenced that rEhVps4 is an ATPase, whose activity depends on the conserved E211 residue. Next, we generated trophozoites overexpressing EhVps4 and mutant EhVps4-E211Q FLAG-tagged proteins. The EhVps4-FLAG was located in cytosol and at plasma membrane, whereas the EhVps4-E211Q-FLAG was detected as abundant cytoplasmic dots in trophozoites. Erythrophagocytosis, cytopathic activity, and hepatic damage in hamsters were not improved in trophozoites overexpressing EhVps4-FLAG. In contrast, EhVps4-E211Q-FLAG protein overexpression impaired these properties. The localization of EhVps4-FLAG around ingested erythrocytes, together with our previous results, strengthens the role for EhVps4 in E. histolytica phagocytosis and virulence.

  7. Autophagy-Associated Protein SmATG12 Is Required for Fruiting-Body Formation in the Filamentous Ascomycete Sordaria macrospora.

    Directory of Open Access Journals (Sweden)

    Antonia Werner

    Full Text Available In filamentous fungi, autophagy functions as a catabolic mechanism to overcome starvation and to control diverse developmental processes under normal nutritional conditions. Autophagy involves the formation of double-membrane vesicles, termed autophagosomes that engulf cellular components and bring about their degradation via fusion with vacuoles. Two ubiquitin-like (UBL conjugation systems are essential for the expansion of the autophagosomal membrane: the UBL protein ATG8 is conjugated to the lipid phosphatidylethanolamine and the UBL protein ATG12 is coupled to ATG5. We recently showed that in the homothallic ascomycete Sordaria macrospora autophagy-related genes encoding components of the conjugation systems are required for fruiting-body development and/or are essential for viability. In the present work, we cloned and characterized the S. macrospora (Smatg12 gene. Two-hybrid analysis revealed that SmATG12 can interact with SmATG7 and SmATG3. To examine its role in S. macrospora, we replaced the open reading frame of Smatg12 with a hygromycin resistance cassette and generated a homokaryotic ΔSmatg12 knockout strain, which displayed slower vegetative growth under nutrient starvation conditions and was unable to form fruiting bodies. In the hyphae of S. macrospora EGFP-labeled SmATG12 was detected in the cytoplasm and as punctate structures presumed to be phagophores or phagophore assembly sites. Delivery of EGFP-labelled SmATG8 to the vacuole was entirely dependent on SmATG12.

  8. VPS29 is not an active metallo-phosphatase but is a rigid scaffold required for retromer interaction with accessory proteins.

    Directory of Open Access Journals (Sweden)

    James D Swarbrick

    Full Text Available VPS29 is a key component of the cargo-binding core complex of retromer, a protein assembly with diverse roles in transport of receptors within the endosomal system. VPS29 has a fold related to metal-binding phosphatases and mediates interactions between retromer and other regulatory proteins. In this study we examine the functional interactions of mammalian VPS29, using X-ray crystallography and NMR spectroscopy. We find that although VPS29 can coordinate metal ions Mn(2+ and Zn(2+ in both the putative active site and at other locations, the affinity for metals is low, and lack of activity in phosphatase assays using a putative peptide substrate support the conclusion that VPS29 is not a functional metalloenzyme. There is evidence that structural elements of VPS29 critical for binding the retromer subunit VPS35 may undergo both metal-dependent and independent conformational changes regulating complex formation, however studies using ITC and NMR residual dipolar coupling (RDC measurements show that this is not the case. Finally, NMR chemical shift mapping indicates that VPS29 is able to associate with SNX1 via a conserved hydrophobic surface, but with a low affinity that suggests additional interactions will be required to stabilise the complex in vivo. Our conclusion is that VPS29 is a metal ion-independent, rigid scaffolding domain, which is essential but not sufficient for incorporation of retromer into functional endosomal transport assemblies.

  9. F-box and Leucine-rich Repeat Protein 5 (FBXL5) Is Required for Maintenance of Cellular and Systemic Iron Homeostasis*

    Science.gov (United States)

    Ruiz, Julio C.; Walker, Scott D.; Anderson, Sheila A.; Eisenstein, Richard S.; Bruick, Richard K.

    2013-01-01

    Maintenance of cellular iron homeostasis requires post-transcriptional regulation of iron metabolism genes by iron regulatory protein 2 (IRP2). The hemerythrin-like domain of F-box and leucine-rich repeat protein 5 (FBXL5), an E3 ubiquitin ligase subunit, senses iron and oxygen availability and facilitates IRP2 degradation in iron replete cells. Disruption of the ubiquitously expressed murine Fbxl5 gene results in a failure to sense increased cellular iron availability, accompanied by constitutive IRP2 accumulation and misexpression of IRP2 target genes. FBXL5-null mice die during embryogenesis, although viability is restored by simultaneous deletion of the IRP2, but not IRP1, gene. Mice containing a single functional Fbxl5 allele behave like their wild type littermates when fed an iron-sufficient diet. However, unlike wild type mice that manifest decreased hematocrit and hemoglobin levels when fed a low-iron diet, Fbxl5 heterozygotes maintain normal hematologic values due to increased iron absorption. The responsiveness of IRP2 to low iron is specifically enhanced in the duodena of the heterozygotes and is accompanied by increased expression of the divalent metal transporter-1. These results confirm the role of FBXL5 in the in vivo maintenance of cellular and systemic iron homeostasis and reveal a privileged role for the intestine in their regulation by virtue of its unique FBXL5 iron sensitivity. PMID:23135277

  10. F-box and leucine-rich repeat protein 5 (FBXL5) is required for maintenance of cellular and systemic iron homeostasis.

    Science.gov (United States)

    Ruiz, Julio C; Walker, Scott D; Anderson, Sheila A; Eisenstein, Richard S; Bruick, Richard K

    2013-01-01

    Maintenance of cellular iron homeostasis requires post-transcriptional regulation of iron metabolism genes by iron regulatory protein 2 (IRP2). The hemerythrin-like domain of F-box and leucine-rich repeat protein 5 (FBXL5), an E3 ubiquitin ligase subunit, senses iron and oxygen availability and facilitates IRP2 degradation in iron replete cells. Disruption of the ubiquitously expressed murine Fbxl5 gene results in a failure to sense increased cellular iron availability, accompanied by constitutive IRP2 accumulation and misexpression of IRP2 target genes. FBXL5-null mice die during embryogenesis, although viability is restored by simultaneous deletion of the IRP2, but not IRP1, gene. Mice containing a single functional Fbxl5 allele behave like their wild type littermates when fed an iron-sufficient diet. However, unlike wild type mice that manifest decreased hematocrit and hemoglobin levels when fed a low-iron diet, Fbxl5 heterozygotes maintain normal hematologic values due to increased iron absorption. The responsiveness of IRP2 to low iron is specifically enhanced in the duodena of the heterozygotes and is accompanied by increased expression of the divalent metal transporter-1. These results confirm the role of FBXL5 in the in vivo maintenance of cellular and systemic iron homeostasis and reveal a privileged role for the intestine in their regulation by virtue of its unique FBXL5 iron sensitivity. PMID:23135277

  11. Sperm surface protein PH-20 is bifunctional: one activity is a hyaluronidase and a second, distinct activity is required in secondary sperm-zona binding.

    Science.gov (United States)

    Hunnicutt, G R; Primakoff, P; Myles, D G

    1996-07-01

    In previous studies, we have found that the sperm membrane protein PH-20 acts during two different stages of fertilization. On acrosome-intact sperm, PH-20 has a hyaluronidase activity that is required for sperm penetration through the cumulus cell layer that surrounds the oocyte. On acrosome-reacted sperm, PH-20 has a required function in sperm-zona binding (secondary binding). Because hyaluronic acid (HA) has been detected in the zona pellucida, secondary sperm-zona adhesion could depend on repetitive binding and hydrolysis of HA by PH-20 acting as a hyaluronidase. Alternatively, PH-20 may be bifunctional and have a second, different activity required for secondary binding. To distinguish between these two possibilities, in this study we used reagents that inhibit either PH-20's function in sperm-zona binding or its hyaluronidase activity. We found that an anti-PH-20 monoclonal antibody that inhibited sperm-zona binding (approximately 90%) had no effect on hyaluronidase activity. Conversely, apigenin, a hyaluronidase inhibitor, blocked PH-20 hyaluronidase activity 93% without inhibiting sperm-zona binding. Similarly, another anti-PH-20 monoclonal antibody that inhibited hyaluronidase activity 95% only partially inhibited sperm-zona binding (approximately 45%). We also extensively pretreated oocytes with hyaluronidase to remove all accessible HA on or in the zona pellucida and found little or no effect on secondary sperm-zona binding. Our results suggest that PH-20 is bifunctional and has two activities: a hyaluronidase activity and a second, separate activity required for secondary sperm-zona binding. PMID:8793062

  12. Factors involved in the initiation of phage ø29 DNA replication in vitro: requirement of the gene 2 product for the formation of the protein p3-dAMP complex

    OpenAIRE

    1983-01-01

    To study the requirements for the in vitro formation of the protein p3-dAMP complex, the first step in φ29 DNA replication, extracts from B. subtilis infected with ø29 mutants in genes 2, 3, 5, 6 and 17, involved in DNA synthesis, have been used. The formation of the initiation complex is completely dependent on the presence of a functional gene 2 product, in addition to protein p3 and ø29 DNA-protein p3 as template. ATP is also required, although it can be replaced by other nucleotides. The ...

  13. 海南黑山羊生长期的能量与蛋白质需要量%Energy and Protein Requirements of Growing Hainan Black Goats

    Institute of Scientific and Technical Information of China (English)

    周汉林; 李茂; 字学娟; 徐铁山; 蒋昌顺

    2009-01-01

    Twelve growing waning Hainan Black goats at the age of 4 months were selected for experiments and fed by using the feeding and digestion methods to observe their requirements for energy and protein,and their metabolism. The goats were divided into 4 groups and a 4×4 Latin design was arranged. Diets with 4 ratios of energy to nitrogen,i.e. high energy high N,low energy high N,high energy low N,low energy low N,were prepared according to Keral I.C. (1982) who recommended the nutrient requirements of goats in developing countries. The prediction equations of nutritional requirements for dry matter(DM),Gross energy(GE),digestible energy(DE),crude protein(CP) and digestible crude protein (DCP) by stall-feeding growing Hainan Black goats were: ZM//(g/d)=27.181W~(0.75)+0.83AJF+210.76(r=0.891 6),GEI(MJ/d)=0.82W~(0.75)+0.028△W+3.76(r=0.780 4),DEI(MJ/d)=0.59W~(0.75)+0.021 △W-0.24(r=0.665 1),CPI(g/d) =2.45W~(0.75)+0.26△W+22.14(r=0.5712),DCP(g/d)=2.69W~(0.75)+0.03 △W-0.38(r=0.660 5). For the stall-feeding growing Hainan Black goats,metabolic rate of gross energy of the diets was 58.88%,digestibility of crude protein 57.08%,and the total availability of protein 35.25%. The protein requirement of per unit live weight gain of meat goats is 0.37 g PD/g gain(PD,protein deposited).%试验选用12只海南黑山羊断奶羔羊(约4月龄),采用饲养试验和消化代谢试验,研究生长期海南黑山羊的能量和蛋白质需要量及其代谢规律.试验羊分成4组,采用4x4拉丁方设计.按照Keral(1982)适宜于发展中国家的山羊饲养标准为参考设计了高能高氮、低能高氮、高能低氮、低能低氮4种能氮比的日粮.结果表明:舍饲海南黑山羊生长期干物质采食量与代谢体重和日增重的关系为DMI(g/d)=27.18W0.75+0.83△W+210.76(r=0.8916);总能、消化能、粗蛋白质和可消化蛋白质需要量的估测模型分别为GEI(MJId)=0.82W~(0.75)+0.028△W+3.76(r=0.7804).DEI(MJ/d)=0.59W~(0.75)+0.021 △W-0.24(r=0

  14. Gonadotropin-dependent oocyte maturational competence requires activation of the protein kinase A pathway and synthesis of RNA and protein in ovarian follicles of Nibe, Nibea mitsukurii (Teleostei, Sciaenidae)

    Science.gov (United States)

    Yoshizaki, G.; Shusa, M.; Takeuchi, T.; Patino, R.

    2002-01-01

    Luteinizing hormone- (LH)-dependent ovarian follicle maturation has been recently described in two stages for teleost fishes. The oocyte's ability to respond to the steroidal maturation-inducing hormone (MIH), also known as oocyte maturational competence (OMC), is acquired during the first stage; whereas the MIH-dependent resumption of meiosis occurs during the second stage. However, studies directly addressing OMC have been performed with a limited number of species and therefore the general relevance of the two-stage model and its mechanisms remain uncertain. In this study, we examined the hormonal regulation of OMC and its basic transduction mechanisms in ovarian follicles of the sciaenid teleost, Nibe (Nibea mitsukurii). Exposure to MIH [17,20??-dihydroxy-4-pregnen-3-one or 17,20??,21-trihydroxy-4-pregnen-3-one] stimulated germinal vesicle breakdown (index of meiotic resumption) in full-grown follicles primed with human chorionic gonadotropin (HCG, an LH-like gonadotropin) but not in those pre-cultured in plain incubation medium. The induction of OMC by HCG was mimicked by protein kinase A (PKA) activators (forskolin and dibutyryl cyclic AMP), and blocked by specific inhibitors of PKA (H89 and H8) as well as inhibitors of RNA (actinomycin D) and protein (cycloheximide) synthesis. Forskolin-induced OMC was also inhibited by actinomycin D and cycloheximide. A strong activator of protein kinase C, PMA, inhibited HCG-dependent OMC. In conclusion, OMC in Nibe ovarian follicles is gonadotropin-dependent and requires activation of the PKA pathway followed by gene transcription and translation events. These observations are consistent with the two-stage model of ovarian follicle maturation proposed for other teleosts, and suggest that Nibe can be used as new model species for mechanistic studies of ovarian follicle differentiation and maturation in fishes.

  15. The Protein Arginine Methylase 5 (PRMT5/SKB1) Gene Is Required for the Maintenance of Root Stem Cells in Response to DNA Damage.

    Science.gov (United States)

    Li, Qiuling; Zhao, Yan; Yue, Minghui; Xue, Yongbiao; Bao, Shilai

    2016-04-20

    Plant root stem cells and their surrounding microenvironment, namely the stem cell niche, are hypersensitive to DNA damage. However, the molecular mechanisms that help maintain the genome stability of root stem cells remain elusive. Here we show that the root stem cells in the skb1 (Shk1 kinase binding protein 1) mutant undergoes DNA damage-induced cell death, which is enhanced when combined with a lesion of the Ataxia-telangiectasia mutated (ATM) or the ATM/RAD3-related (ATR) genes, suggesting that the SKB1 plays a synergistically effect with ATM and ATR in DNA damage pathway. We also provide evidence that SKB1 is required for the maintenance of quiescent center (QC), a root stem cell niche, under DNA damage treatments. Furthermore, we report decreased and ectopic expression of SHORTROOT (SHR) in response to DNA damage in the skb1 root tips, while the expression of SCARECROW (SCR) remains unaffected. Our results uncover a new mechanism of plant root stem cell maintenance under DNA damage conditions that requires SKB1. PMID:27090604

  16. The molecular, temporal and region-specific requirements of the beta isoform of Calcium/Calmodulin-dependent protein kinase type 2 (CAMK2B) in mouse locomotion.

    Science.gov (United States)

    Kool, Martijn J; van de Bree, Jolet E; Bodde, Hanna E; Elgersma, Ype; van Woerden, Geeske M

    2016-01-01

    Genetic approaches using temporal and brain region-specific restricted gene deletions have provided a wealth of insight in the brain regions and temporal aspects underlying spatial and associative learning. However, for locomotion such extensive studies are still scarce. Previous studies demonstrated that Camk2b(-/-) mice, which lack the β isoform of Calcium/Calmodulin-dependent protein kinase 2 (CAMK2B), show very severe locomotion deficits. However, where these locomotion deficits originate is unknown. Here we made use of novel Camk2b mutants (Camk2b(f/f) and Camk2b(T287A)), to explore the molecular, temporal and brain region-specific requirements of CAMK2B for locomotion. At the molecular level we found that normal locomotion requires Calcium/Calmodulin mediated activation of CAMK2B, but CAMK2B autonomous activity is largely dispensable. At a systems level, we found that global deletion of Camk2b in the adult mouse causes only mild locomotion deficits, suggesting that the severe locomotion deficits of Camk2b(-/-) mice are largely of developmental origin. However, early onset deletion of Camk2b in cerebellum, striatum or forebrain did not recapitulate the locomotion deficits, suggesting that these deficits cannot be attributed to a single brain area. Taken together, these results provide the first insights into the molecular, temporal and region-specific role of CAMK2B in locomotion. PMID:27244486

  17. Sla1p Is a Functionally Modular Component of the Yeast Cortical Actin Cytoskeleton Required for Correct Localization of Both Rho1p-GTPase and Sla2p, a Protein with Talin Homology

    OpenAIRE

    Ayscough, Kathryn R.; Eby, Jennifer J.; Lila, Thomas; Dewar, Hilary; Kozminski, Keith G.; Drubin, David G.

    1999-01-01

    SLA1 was identified previously in budding yeast in a genetic screen for mutations that caused a requirement for the actin-binding protein Abp1p and was shown to be required for normal cortical actin patch structure and organization. Here, we show that Sla1p, like Abp1p, localizes to cortical actin patches. Furthermore, Sla1p is required for the correct localization of Sla2p, an actin-binding protein with homology to talin implicated in endocytosis, and the Rho1p-GTPase, which is associated wi...

  18. Neuron class-specific requirements for Fragile X Mental Retardation Protein in critical period development of calcium signaling in learning and memory circuitry.

    Science.gov (United States)

    Doll, Caleb A; Broadie, Kendal

    2016-05-01

    Neural circuit optimization occurs through sensory activity-dependent mechanisms that refine synaptic connectivity and information processing during early-use developmental critical periods. Fragile X Mental Retardation Protein (FMRP), the gene product lost in Fragile X syndrome (FXS), acts as an activity sensor during critical period development, both as an RNA-binding translation regulator and channel-binding excitability regulator. Here, we employ a Drosophila FXS disease model to assay calcium signaling dynamics with a targeted transgenic GCaMP reporter during critical period development of the mushroom body (MB) learning/memory circuit. We find FMRP regulates depolarization-induced calcium signaling in a neuron-specific manner within this circuit, suppressing activity-dependent calcium transients in excitatory cholinergic MB input projection neurons and enhancing calcium signals in inhibitory GABAergic MB output neurons. Both changes are restricted to the developmental critical period and rectified at maturity. Importantly, conditional genetic (dfmr1) rescue of null mutants during the critical period corrects calcium signaling defects in both neuron classes, indicating a temporally restricted FMRP requirement. Likewise, conditional dfmr1 knockdown (RNAi) during the critical period replicates constitutive null mutant defects in both neuron classes, confirming cell-autonomous requirements for FMRP in developmental regulation of calcium signaling dynamics. Optogenetic stimulation during the critical period enhances depolarization-induced calcium signaling in both neuron classes, but this developmental change is eliminated in dfmr1 null mutants, indicating the activity-dependent regulation requires FMRP. These results show FMRP shapes neuron class-specific calcium signaling in excitatory vs. inhibitory neurons in developing learning/memory circuitry, and that FMRP mediates activity-dependent regulation of calcium signaling specifically during the early

  19. Dietary protein and energy requirements of juvenile freshwater angelfish Exigências nutricionais de proteína e energia em juvenis de acará-bandeira

    Directory of Open Access Journals (Sweden)

    Jener Alexandre Sampaio Zuanon

    2009-06-01

    Full Text Available Dietary protein and energy requirements of juvenile freshwater angelfish (Pterophyllum scalare were evaluated. A 3 × 2 factorial design was used, with three dietary crude protein levels being tested (26, 30, and 34% of CP combined with two digestible energy levels (3,100 and 3,300 kcal DE/kg of diet in three replicates. Juveniles averaging 2.33 ± 0.26 g were reared in a 25L-aquarium with controlled temperature (26 ± 1ºC, biological filter and stocking density of six fish/aquarium. Fish were fed ad libitum at 09:00 a.m., 2:00 p.m. and 4:30 p.m. The following performance parameters were evaluated: final weight, final length, weight gain, feed intake, feed conversion ratio, specific growth rate, protein efficiency ratio and condition factor. Fish fed diets with 26% CP showed greater protein efficiency values when compared to those fed diets with 34% CP. Diets with 26% of CP and 3100 kcal DE/kg could meet the nutritional requirements of juvenile freshwater angelfish.Avaliaram-se as exigências nutricionais de proteína e energia em juvenis de acará-bandeira (Pterophyllum scalare. Utilizou-se delineamento inteiramente casualizado, em esquema fatorial 3 × 2, com três níveis de proteína bruta (26, 30 e 34%, dois de energia digestível (3.100 e 3.300 kcal/kg de ração e três repetições. Juvenis com peso médio de 2,33 ± 0,26 g foram distribuídos em aquários contendo 25 litros de água, temperatura controlada (26 ± 1ºC e filtro biológico, na densidade de estocagem de seis animais por aquário. Os peixes foram alimentados à vontade às 9, 14 e 16h30. Na análise do desempenho produtivo, foram avaliados o peso final, o comprimento final, o ganho de peso, o consumo de ração, a conversão alimentar, a taxa de crescimento específico, a taxa de eficiência protéica e o fator de condição. As dietas contendo 26% PB proporcionaram maiores valores para taxa de eficiência protéica apenas em relação às dietas contendo 34% PB. As exig

  20. Identification of an amino acid residue required for differential recognition of a viral movement protein by the Tomato mosaic virus resistance gene Tm-2(2).

    Science.gov (United States)

    Kobayashi, Michie; Yamamoto-Katou, Ayako; Katou, Shinpei; Hirai, Katsuyuki; Meshi, Tetsuo; Ohashi, Yuko; Mitsuhara, Ichiro

    2011-07-01

    The Tm-2 gene of tomato and its allelic gene, Tm-2(2), confer resistance to Tomato mosaic virus (ToMV) and encode a member of the coiled-coil/nucleotide binding-ARC/leucine-rich repeat (LRR) protein class of plant resistance (R) genes. Despite exhibiting only four amino acid differences between the products of Tm-2 and Tm-2(2), Tm-2(2) confers resistance to ToMV mutant B7, whereas Tm-2 is broken by ToMV-B7. An Agrobacterium-mediated transient expression system was used to study the mechanism of differential recognition of the movement proteins (MPs), an avirulence factor for ToMV resistance, of ToMV-B7 by Tm-2 and Tm-2(2). Although resistance induced by Tm-2 and Tm-2(2) is not usually accompanied by hypersensitive response (HR), Tm-2 and Tm-2(2) induced HR-like cell death by co-expression with MP of a wild-type ToMV, a strain that causes resistance for these R genes, and Tm-2(2) but not Tm-2 induced cell death with B7-MP in this system. Site-directed amino acid mutagenesis revealed that Tyr-767 in the LRR of Tm-2(2) is required for the specific recognition of the B7-MP. These results suggest that the Tyr residue in LRR contributes to the recognition of B7-MP, and that Tm-2 and Tm-2(2) are involved in HR cell death. PMID:21310506

  1. Diffusible signal factor (DSF) synthase RpfF of Xylella fastidiosa is a multifunction protein also required for response to DSF.

    Science.gov (United States)

    Ionescu, Michael; Baccari, Clelia; Da Silva, Aline Maria; Garcia, Angelica; Yokota, Kenji; Lindow, Steven E

    2013-12-01

    Xylella fastidiosa, like related Xanthomonas species, employs an Rpf cell-cell communication system consisting of a diffusible signal factor (DSF) synthase, RpfF, and a DSF sensor, RpfC, to coordinate expression of virulence genes. While phenotypes of a ΔrpfF strain in Xanthomonas campestris could be complemented by its own DSF, the DSF produced by X. fastidiosa (XfDSF) did not restore expression of the XfDSF-dependent genes hxfA and hxfB to a ΔrpfF strain of X. fastidiosa, suggesting that RpfF is involved in XfDSF sensing or XfDSF-dependent signaling. To test this conjecture, rpfC and rpfF of X. campestris were replaced by those of X. fastidiosa, and the contribution of each gene to the induction of a X. campestris DSF-dependent gene was assessed. As in X. fastidiosa, XfDSF-dependent signaling required both X. fastidiosa proteins RpfF and RpfC. RpfF repressed RpfC signaling activity, which in turn was derepressed by XfDSF. A mutated X. fastidiosa RpfF protein with two substitutions of glutamate to alanine in its active site was incapable of XfDSF production yet enabled a response to XfDSF, indicating that XfDSF production and the response to XfDSF are two separate functions in which RpfF is involved. This mutant was also hypervirulent to grape, demonstrating the antivirulence effects of XfDSF itself in X. fastidiosa. The Rpf system of X. fastidiosa is thus a novel example of a quorum-sensing signal synthase that is also involved in the response to the signal molecule that it synthesizes. PMID:24056101

  2. OsARID3, an AT-rich Interaction Domain-containing protein, is required for shoot meristem development in rice.

    Science.gov (United States)

    Xu, Yan; Zong, Wei; Hou, Xin; Yao, Jialing; Liu, Hongbo; Li, Xianghua; Zhao, Yunde; Xiong, Lizhong

    2015-09-01

    The shoot apical meristem (SAM) produces all of the plant's aerial organs. The SAM is established either during embryogenesis or experimentally in in vitro tissue culture. Although several factors including the Class I KNOTTED1-LIKE HOMEOBOX (KNOXI) proteins, auxin, and cytokinin are known to play essential roles in SAM development, the underlying mechanisms of SAM formation and maintenance are still largely not understood. Herein we demonstrate that OsARID3, a member of the rice (Oryza sativa) AT-rich Interaction Domain (ARID) family, is required for SAM development. Disruption of OsARID3 leads to a defective SAM, early seedling lethality, and impaired capacity of in vitro shoot regeneration. We show that the expression levels of several KNOXI genes and the biosynthetic genes for auxin and cytokinin are significantly altered in the Osarid3 mutant calli. Moreover, we determine that auxin concentrations are increased, whereas cytokinin levels are decreased, in Osarid3 calli. Furthermore, chromatin immunoprecipitation results demonstrate that OsARID3 binds directly to the KNOXI gene OSH71, the auxin biosynthetic genes OsYUC1 and OsYUC6, and the cytokinin biosynthetic genes OsIPT2 and OsIPT7. We also show through electrophoretic mobility shift assays that OsARID3 specifically binds to the AT-rich DNA sequences of the identified target genes. We conclude that OsARID3 is an AT-rich specific DNA-binding protein and that it plays a major role in SAM development in rice. PMID:26121094

  3. Activation of HIV transcription by the viral Tat protein requires a demethylation step mediated by lysine-specific demethylase 1 (LSD1/KDM1.

    Directory of Open Access Journals (Sweden)

    Naoki Sakane

    2011-08-01

    Full Text Available The essential transactivator function of the HIV Tat protein is regulated by multiple posttranslational modifications. Although individual modifications are well characterized, their crosstalk and dynamics of occurrence during the HIV transcription cycle remain unclear.We examine interactions between two critical modifications within the RNA-binding domain of Tat: monomethylation of lysine 51 (K51 mediated by Set7/9/KMT7, an early event in the Tat transactivation cycle that strengthens the interaction of Tat with TAR RNA, and acetylation of lysine 50 (K50 mediated by p300/KAT3B, a later process that dissociates the complex formed by Tat, TAR RNA and the cyclin T1 subunit of the positive transcription elongation factor b (P-TEFb. We find K51 monomethylation inhibited in synthetic Tat peptides carrying an acetyl group at K50 while acetylation can occur in methylated peptides, albeit at a reduced rate. To examine whether Tat is subject to sequential monomethylation and acetylation in cells, we performed mass spectrometry on immunoprecipitated Tat proteins and generated new modification-specific Tat antibodies against monomethylated/acetylated Tat. No bimodified Tat protein was detected in cells pointing to a demethylation step during the Tat transactivation cycle. We identify lysine-specific demethylase 1 (LSD1/KDM1 as a Tat K51-specific demethylase, which is required for the activation of HIV transcription in latently infected T cells. LSD1/KDM1 and its cofactor CoREST associates with the HIV promoter in vivo and activate Tat transcriptional activity in a K51-dependent manner. In addition, small hairpin RNAs directed against LSD1/KDM1 or inhibition of its activity with the monoamine oxidase inhibitor phenelzine suppresses the activation of HIV transcription in latently infected T cells.Our data support the model that a LSD1/KDM1/CoREST complex, normally known as a transcriptional suppressor, acts as a novel activator of HIV transcription through

  4. TmpL, a transmembrane protein required for intracellular redox homeostasis and virulence in a plant and an animal fungal pathogen.

    Directory of Open Access Journals (Sweden)

    Kwang-Hyung Kim

    2009-11-01

    Full Text Available The regulation of intracellular levels of reactive oxygen species (ROS is critical for developmental differentiation and virulence of many pathogenic fungi. In this report we demonstrate that a novel transmembrane protein, TmpL, is necessary for regulation of intracellular ROS levels and tolerance to external ROS, and is required for infection of plants by the necrotroph Alternaria brassicicola and for infection of mammals by the human pathogen Aspergillus fumigatus. In both fungi, tmpL encodes a predicted hybrid membrane protein containing an AMP-binding domain, six putative transmembrane domains, and an experimentally-validated FAD/NAD(P-binding domain. Localization and gene expression analyses in A. brassicicola indicated that TmpL is associated with the Woronin body, a specialized peroxisome, and strongly expressed during conidiation and initial invasive growth in planta. A. brassicicola and A. fumigatus DeltatmpL strains exhibited abnormal conidiogenesis, accelerated aging, enhanced oxidative burst during conidiation, and hypersensitivity to oxidative stress when compared to wild-type or reconstituted strains. Moreover, A. brassicicola DeltatmpL strains, although capable of initial penetration, exhibited dramatically reduced invasive growth on Brassicas and Arabidopsis. Similarly, an A. fumigatus DeltatmpL mutant was dramatically less virulent than the wild-type and reconstituted strains in a murine model of invasive aspergillosis. Constitutive expression of the A. brassicicola yap1 ortholog in an A. brassicicola DeltatmpL strain resulted in high expression levels of genes associated with oxidative stress tolerance. Overexpression of yap1 in the DeltatmpL background complemented the majority of observed developmental phenotypic changes and partially restored virulence on plants. Yap1-GFP fusion strains utilizing the native yap1 promoter exhibited constitutive nuclear localization in the A. brassicicola DeltatmpL background. Collectively, we

  5. Double-muscled and conventional cattle have the same net energy requirements if these are related to mature and current body protein mass, and to gain composition.

    Science.gov (United States)

    Schiavon, S; Bittante, G

    2012-11-01

    The hypothesis tested in this paper is that double-muscled (DBM) and conventional cattle, considerably differing in body composition, have similar NE requirements when: a) NE(m) is scaled as a function of current (P(i)) and adult (P(m)) protein mass; and b) ME for gain (ME(g)) is estimated from protein (Pr) and lipid (Lr) retention and their partial ME use efficiencies, the k(p) and k(l) values, respectively. First, 2 databases were examined: 1 was developed combining well known literature information from comparative slaughter trials conducted on British beef steers; the other was based on a trial conducted using extremely lean DBM Piemontese bulls. From the first database, NE(m) was calculated to be 1.625 × P(i) ÷ P(m) × P(m)(0.73) (MJ/kg(0.73)). From the second database, the daily ME(g) was determined as 22.8 MJ × Pr ÷ k(p) + 38.74 MJ × Lr ÷ k(l), assuming (from prior reports) that k(p) = 0.20 and k(l) = 0.75. Thereafter, ME(m) was defined as ME intake minus ME(g), and, hence, NE(m) was predicted as 1.625 × P(i) ÷ P(m) × P(m)(0.73) (where 1.625 was the value obtained from the first dataset). The resulting k(m) (NE(m)/ME(m)) averaged 0.67. This k(m) value did not differ from that (0.65; P = 0.12) predicted by Garrett's equation, which uses dietary ME content as the only predictive variable. Second, the procedure was tested for the ability to detect effects on k(m) caused by increasing BW and dietary factors not estimable from the dietary ME content only. Data were gathered from a trial involving 48 DBM Piemontese bulls divided into 4 groups fed 1 of 4 diets differing in CP content (145 or 108 g/kg DM), with or without addition of 80 g/d of rumen-protected CLA (rpCLA). Bulls were examined at 3 consecutive periods of growth, corresponding to 365, 512 and 631 kg of average BW. All energy balance items were influenced by increasing BW, except k(m) (P = 0.61), in agreement with the expectation that NE(m) requirement depends on the degree of maturity (P

  6. Recovery from heat, salt and osmotic stress in Physcomitrella patens requires a functional small heat shock protein PpHsp16.4

    OpenAIRE

    Ruibal, Cecilia; Castro, Alexandra; Carballo, Valentina; Szabados, László; Vidal, Sabina

    2013-01-01

    Background Plant small heat shock proteins (sHsps) accumulate in response to various environmental stresses, including heat, drought, salt and oxidative stress. Numerous studies suggest a role for these proteins in stress tolerance by preventing stress-induced protein aggregation as well as by facilitating protein refolding by other chaperones. However, in vivo evidence for the involvement of sHsps in tolerance to different stress factors is still missing, mainly due to the lack of appropriat...

  7. Zygotic Expression of the Double-Stranded RNA Binding Motif Protein Drb2p Is Required for DNA Elimination in the Ciliate Tetrahymena thermophila ▿

    OpenAIRE

    Motl, Jason A.; Chalker, Douglas L.

    2011-01-01

    Double-stranded RNA binding motif (DSRM)-containing proteins play many roles in the regulation of gene transcription and translation, including some with tandem DSRMs that act in small RNA biogenesis. We report the characterization of the genes for double-stranded RNA binding proteins 1 and 2 (DRB1 and DRB2), two genes encoding nuclear proteins with tandem DSRMs in the ciliate Tetrahymena thermophila. Both proteins are expressed throughout growth and development but exhibit distinct peaks of ...

  8. The Seckel syndrome and centrosomal protein Ninein localizes asymmetrically to stem cell centrosomes but is not required for normal development, behavior, or DNA damage response in Drosophila.

    Science.gov (United States)

    Zheng, Yiming; Mennella, Vito; Marks, Steven; Wildonger, Jill; Elnagdi, Esraa; Agard, David; Megraw, Timothy L

    2016-06-01

    Ninein (Nin) is a centrosomal protein whose gene is mutated in Seckel syndrome (SCKL, MIM 210600), an inherited recessive disease that results in primordial dwarfism, cognitive deficiencies, and increased sensitivity to genotoxic stress. Nin regulates neural stem cell self-renewal, interkinetic nuclear migration, and microtubule assembly in mammals. Nin is evolutionarily conserved, yet its role in cell division and development has not been investigated in a model organism. Here we characterize the single Nin orthologue in Drosophila Drosophila Nin localizes to the periphery of the centrosome but not at centriolar structures as in mammals. However, Nin shares the property of its mammalian orthologue of promoting microtubule assembly. In neural and germline stem cells, Nin localizes asymmetrically to the younger (daughter) centrosome, yet it is not required for the asymmetric division of stem cells. In wing epithelia and muscle, Nin localizes to noncentrosomal microtubule-organizing centers. Surprisingly, loss of nin expression from a nin mutant does not significantly affect embryonic and brain development, fertility, or locomotor performance of mutant flies or their survival upon exposure to DNA-damaging agents. Although it is not essential, our data suggest that Nin plays a supportive role in centrosomal and extracentrosomal microtubule organization and asymmetric stem cell division. PMID:27053665

  9. Sequence characteristics required for cooperative binding and efficient in vivo titration of the replication initiator protein DnaA in E. coli

    DEFF Research Database (Denmark)

    Hansen, Flemming G.; Christensen, Bjarke Bak; Atlung, Tove

    2007-01-01

    was eliminated by insertion of half a helical turn between any of the DnaA boxes. Titration strongly depends on the presence and orientation of the promoter distal R6 DnaA box located 104 bp upstream of the R5 box as well as neighbouring sequences downstream of R6. Titration depends on the integrity of a 43 bp......Plasmids carrying the mioC promoter region, which contains two DnaA boxes, R5 and R6 with one misfit to the consensus TT(A)/(T)TNCACA, are as efficient in in vivo titration of the DnaA protein as plasmids carrying a replication-inactivated oriC region with its eight DnaA boxes. Three additional Dna...... was located less than 20 bp upstream of the -35 sequence. Thus, the architectural requirements for titration and for repression of transcription are different. A new set of rules for identifying efficiently titrating DnaA box regions was formulated and used to analyse sequences for which good titration data...

  10. A NASP (N1/N2)-related protein, Sim3, binds CENP-A and is required for its deposition at fission yeast centromeres.

    Science.gov (United States)

    Dunleavy, Elaine M; Pidoux, Alison L; Monet, Marie; Bonilla, Carolina; Richardson, William; Hamilton, Georgina L; Ekwall, Karl; McLaughlin, Paul J; Allshire, Robin C

    2007-12-28

    A defining feature of centromeres is the presence of the histone H3 variant CENP-A(Cnp1). It is not known how CENP-A(Cnp1) is specifically delivered to, and assembled into, centromeric chromatin. Through a screen for factors involved in kinetochore integrity in fission yeast, we identified Sim3. Sim3 is homologous to known histone binding proteins NASP(Human) and N1/N2(Xenopus) and aligns with Hif1(S. cerevisiae), defining the SHNi-TPR family. Sim3 is distributed throughout the nucleoplasm, yet it associates with CENP-A(Cnp1) and also binds H3. Cells defective in Sim3 function have reduced levels of CENP-A(Cnp1) at centromeres (and increased H3) and display chromosome segregation defects. Sim3 is required to allow newly synthesized CENP-A(Cnp1) to accumulate at centromeres in S and G2 phase-arrested cells in a replication-independent mechanism. We propose that one function of Sim3 is to act as an escort that hands off CENP-A(Cnp1) to chromatin assembly factors, allowing its incorporation into centromeric chromatin. PMID:18158900

  11. Exigência Protéica e Relação Energia/Proteína para Alevinos de Piracanjuba (Brycon orbignyanus Dietary Protein Requirement and Energy to Protein Ratio for Piracanjuba (Brycon orbignyanus Fingerlings

    Directory of Open Access Journals (Sweden)

    Marcelo Vinícius do Carmo e Sá

    2002-02-01

    Full Text Available O objetivo do presente estudo foi determinar a exigência protéica e correspondente relação energia/proteína em dietas para alevinos de piracanjuba, Brycon orbignyanus. Seis dietas semi-purificadas isocalóricas foram formuladas para conter 3.000 kcal de energia metabolizável (EM/kg e concentrações de proteína bruta (PB de 24, 26, 29, 32, 36 e 42%. Para essas concentrações, as relações E/P das dietas foram de 12,3; 11,6; 10,4; 9,2; 8,5 e 7,1 kcal EM/g PB, respectivamente. As fontes de proteína, lipídios e carboidratos digestíveis foram, respectivamente, caseína/gelatina, óleo de fígado de bacalhau/óleo de soja e dextrina. Após condicionamento de cinco dias, as dietas foram fornecidas, até a saciedade, em duas alimentações diárias, a 162 alevinos (27 peixes/dieta, que apresentaram 8,38 ± 0,09 g de peso médio inicial, distribuídos em 18 tanques de fibra-de-vidro de 100 L, conectados a um sistema de recirculação de água. A temperatura média da água foi de 26,3°C, com extremos de 23,7 e 30,2°C. Após 90 dias, a concentração de proteína na dieta que proporcionou ganho em peso máximo aos peixes foi 29% PB, com relação E/P igual a 10,4 kcal EM/g PB. As dietas com concentrações de PB iguais a 32, 36 e 42% não se mostraram superiores para conversão alimentar, taxa de eficiência protéica, valor produtivo da proteína e retenção de energia bruta. A deposição corporal de proteína e gordura não sofreu influência da concentração de PB da dietaThe aim of this study was to determine the dietary protein requirement and associated energy to protein (E/P ratio for "Piracanjuba", Brycon orbignyanus, fingerlings. Casein-gelatin semipurified diets were formulated to contain six crude protein (CP concentrations: 24, 26, 29, 32, 36 e 42% at one energy level, 3,000 kcal metabolizable energy (ME/kg. These diets resulted in E/P ratios of 12.3, 11.6, 10.4, 9.2, 8.5 and 7.1 kcal ME/g CP, respectively. The protein, lipid

  12. Luv1p/Rki1p/Tcs3p/Vps54p, a Yeast Protein That Localizes to the Late Golgi and Early Endosome, Is Required for Normal Vacuolar Morphology.

    OpenAIRE

    Conboy, Michael J; Martha S Cyert

    2000-01-01

    We have characterized LUV1/RKI1/TCS3/VPS54, a novel yeast gene required to maintain normal vacuolar morphology. The luv1 mutant was identified in a genetic screen for mutants requiring the phosphatase calcineurin for vegetative growth. luv1 mutants lack a morphologically intact vacuole and instead accumulate small vesicles that are acidified and contain the vacuolar proteins alkaline phosphatase and carboxypeptidase Y and the vacuolar membrane H+-ATPase. Endocytosis appears qualitatively norm...

  13. Recombinant Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin Proteins Are Required To Interact To Inhibit Human Cell Cycle Progression and To Stimulate Human Leukocyte Cytokine Synthesis

    OpenAIRE

    Akifusa, Sumio; Poole, Stephen; Lewthwaite, Jo; Henderson, Brian; Nair, Sean P

    2001-01-01

    It has recently been discovered that Actinobacillus actinomycetemcomitans, an oral bacterium causing periodontitis, produces cytolethal distending toxin (CDT), a cell cycle-modulating toxin that has three protein subunits: CdtA, CdtB, and CdtC. In this study, we have cloned and expressed each toxin gene from A. actinomycetemcomitans in Escherichia coli and purified the recombinant Cdt proteins to homogeneity. Individual Cdt proteins failed to induce cell cycle arrest of the human epithelial c...

  14. 76 FR 57653 - Bacillus thuringiensis eCry3.1Ab Protein in Corn; Temporary Exemption From the Requirement of a...

    Science.gov (United States)

    2011-09-16

    ... AGENCY 40 CFR Part 174 Bacillus thuringiensis eCry3.1Ab Protein in Corn; Temporary Exemption From the... residues of Bacillus thuringiensis eCry3.1Ab protein in corn, in or on the food or feed commodities of corn... temporary tolerance exemption for Bacillus thuringiensis eCry3.1Ab protein in corn that was set to expire...

  15. Potentiation of growth factor signaling by insulin-like growth factor-binding protein-3 in breast epithelial cells requires sphingosine kinase activity.

    Science.gov (United States)

    Martin, Janet L; Lin, Mike Z; McGowan, Eileen M; Baxter, Robert C

    2009-09-18

    We have investigated the mechanism underlying potentiation of epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (IGFR1) signaling by IGF-binding protein-3 (IGFBP-3) in MCF-10A breast epithelial cells, focusing on a possible involvement of the sphingosine kinase (SphK) system. IGFBP-3 potentiated EGF-stimulated EGF receptor activation and DNA synthesis, and this was blocked by inhibitors of SphK activity or small interference RNA-mediated silencing of SphK1, but not SphK2, expression. Similarly, IGFR1 phosphorylation and DNA synthesis stimulated by LR3-IGF-I (an IGF-I analog not bound by IGFBP-3), were enhanced by IGFBP-3, and this was blocked by SphK1 silencing. SphK1 expression and activity were stimulated by IGFBP-3 approximately 2-fold over 24 h. Silencing of sphingosine 1-phosphate receptor 1 (S1P1) or S1P3, but not S1P2, abolished the effect of IGFBP-3 on EGF-stimulated EGFR activation. The effects of IGFBP-3 could be reproduced with exogenous S1P or medium conditioned by cells treated with IGFBP-3, and this was also blocked by inhibition of S1P1 and S1P3. These data indicate that potentiation of growth factor signaling by IGFBP-3 in MCF-10A cells requires SphK1 activity and S1P1/S1P3, suggesting that S1P, the product of SphK activity and ligand for S1P1 and S1P3, is the "missing link" mediating IGF and EGFR transactivation and cell growth stimulation by IGFBP-3. PMID:19633297

  16. Distinct accessory cell requirements define two types of rat T cell hybridomas specific for unique determinants in the encephalitogenic 68-86 region of myelin basic protein

    International Nuclear Information System (INIS)

    Six clonotypically unique T cell hybridomas from Lewis rats were used to study accessory cell activities required for class II MHC restricted T cell responses to the 68-86 encephalitogenic sequence of myelin basic protein (MBP). T cell hybrids which were cultured with GP68-86 68-86 sequence of guinea pig MBP (GPMBP) and naive splenocytes (SPL) were induced to produce IL-2 as measured by the CTLL indicator cell line. The hybrids were categorized into two subsets (designated THYB-1 and THYB-2), because two distinct subset-specific pathways of communication between accessory cells and T cells were involved in GPMBP-induced IL-2 production. These pathways were distinguished by the following six observations. First, when the duration of a pulse of SPL with GPMBP was lengthened from 1 to 4 h, these SPL lost their ability to induce IL-2 production by THYB-2 hybrids yet nevertheless retained full stimulatory activity for THYB-1 hybrids. Second, paraformaldehyde fixation of GPMBP-pulsed SPL abrogated an activity necessary for Ag-induced IL-2 production by THYB-2 hybrids. These fixed SPL were nevertheless able to stimulate THYB-1 hybrids, albeit to a lesser extent than viable unfixed SPL. Third, the addition of either cycloheximide, cytochalasin B, or 2-deoxyglucose to an Ag pulse of SPL with GPMBP dramatically inhibited the subsequent responses of THYB-2 hybrids yet had little or no effect upon the reactivity of THYB-1 hybrids. Fourth, thymocytes lacked necessary activities for GPMBP evoked IL-2 production by THYB-2 hybrids yet strongly promoted THYB-1 hybrid responses. Fifth, exposure of SPL to as little as 500 rad of gamma-irradiation markedly attenuated THYB-2 hybrid response to GPMBP but did not affect THYB-1 responses. Sixth, anti-GPMBP responses by THYB-2 hybrids were observed only in the presence of both radioresistant adherent SPL and a distinct population of radiosensitive nonadherent SPL

  17. TaCPK2-A, a calcium-dependent protein kinase gene that is required for wheat powdery mildew resistance enhances bacterial blight resistance in transgenic rice.

    Science.gov (United States)

    Geng, Shuaifeng; Li, Aili; Tang, Lichuan; Yin, Lingjie; Wu, Liang; Lei, Cailin; Guo, Xiuping; Zhang, Xin; Jiang, Guanghuai; Zhai, Wenxue; Wei, Yuming; Zheng, Youliang; Lan, Xiujin; Mao, Long

    2013-08-01

    Calcium-dependent protein kinases (CPKs) are important Ca2+ signalling components involved in complex immune and stress signalling networks; but the knowledge of CPK gene functions in the hexaploid wheat is limited. Previously, TaCPK2 was shown to be inducible by powdery mildew (Blumeria graminis tritici, Bgt) infection in wheat. Here, its functions in disease resistance are characterized further. This study shows the presence of defence-response and cold-response cis-elements on the promoters of the A subgenome homoeologue (TaCPK2-A) and D subgenome homoeologue (TaCPK2-D), respectively. Their expression patterns were then confirmed by quantitative real-time PCR (qRT-PCR) using genome-specific primers, where TaCPK2-A was induced by Bgt treatment while TaCPK2-D mainly responded to cold treatment. Downregulation of TaCPK2-A by virus-induced gene silencing (VIGS) causes loss of resistance to Bgt in resistant wheat lines, indicating that TaCPK2-A is required for powdery mildew resistance. Furthermore, overexpression of TaCPK2-A in rice enhanced bacterial blight (Xanthomonas oryzae pv. oryzae, Xoo) resistance. qRT-PCR analysis showed that overexpression of TaCPK2-A in rice promoted the expression of OsWRKY45-1, a transcription factor involved in both fungal and bacterial resistance by regulating jasmonic acid and salicylic acid signalling genes. The opposite effect was found in wheat TaCPK2-A VIGS plants, where the homologue of OsWRKY45-1 was significantly repressed. These data suggest that modulation of WRKY45-1 and associated defence-response genes by CPK2 genes may be the common mechanism for multiple disease resistance in grass species, which may have undergone subfunctionalization in promoters before the formation of hexaploid wheat. PMID:23918959

  18. BCL10GFP fusion protein as a substrate for analysis of determinants required for Mucosa-Associated Lymphoid Tissue 1 (MALT1-mediated cleavage

    Directory of Open Access Journals (Sweden)

    Jou Shin-Yi

    2012-10-01

    Full Text Available Abstract Background MALT1 belongs to a family of paracaspase and modulates NF-κB signaling pathways through its scaffolding function and proteolytic activity. MALT1 cleaves protein substrates after a positively charged Arginine residue. BCL10, a 233 amino acids polypeptide, is identified as one of the MALT1 proteolytic substrates. MALT1 cleaves BCL10 at the C-terminal end of Arg228. A mere 5 amino acids difference between the substrate and the proteolytic product made it difficult to tell whether the cleavage event took place by using a simple western blot analysis. Here, BCL10GFP was constructed and utilized to examine the specificity and domain determinants for MALT1 cleavage in cells. Methods Various BCL10GFP constructs were transfected into HEK293T cell with MALT1 construct by using calcium phosphate-DNA precipitation method. Lysates of transfectants were resolved by SDS/PAGE and analyzed by western blot analysis. Results BCL10GFP was proteolytically processed by MALT1 as BCL10. The integrity of caspase recruitment domain (CARD and MALT1-interacting domain on BCL10 were required for MALT1 proteolytic activity. Besides the invariant P1 cleavage site Arg228, P4 Leu225 played a role in defining BCL10 as a good substrate for MALT1. Conclusions We offered a way of monitoring the catalytic activity of MALT1 in HEK293T cells using BCL10GFP as a substrate. BCL10GFP can be utilized as a convenient tool for studying the determinants for efficient MALT1 cleavage in HEK293T cells

  19. Arabidopsis Raf-Like Mitogen-Activated Protein Kinase Kinase Kinase Gene Raf43 Is Required for Tolerance to Multiple Abiotic Stresses.

    Directory of Open Access Journals (Sweden)

    Nasar Virk

    Full Text Available Mitogen-activated protein kinase (MAPK cascades are critical signaling modules that mediate the transduction of extracellular stimuli into intracellular response. A relatively large number of MAPKKKs have been identified in a variety of plant genomes but only a few of them have been studied for their biological function. In the present study, we identified an Arabidopsis Raf-like MAPKKK gene Raf43 and studied its function in biotic and abiotic stress response using a T-DNA insertion mutant raf43-1 and two Raf43-overexpressing lines Raf43-OE#1 and Raf43-OE#13. Expression of Raf43 was induced by multiple abiotic and biotic stresses including treatments with drought, mannitol and oxidative stress or defense signaling molecule salicylic acid and infection with necrotrophic fungal pathogen Botrytis cinerea. Seed germination and seedling root growth of raf43-1 were significantly inhibited on MS medium containing mannitol, NaCl, H2O2 or methyl viologen (MV while seed germination and seedling root growth of the Raf43-OE#1 and Raf43-OE#13 lines was similar to wild type Col-0 under the above stress conditions. Soil-grown raf43-1 plants exhibited reduced tolerance to MV, drought and salt stress. Abscisic acid inhibited significantly seed germination and seedling root growth of the raf43-1 line but had no effect on the two Raf43-overexpressing lines. Expression of stress-responsive RD17 and DREB2A genes was significantly down-regulated in raf43-1 plants. However, the raf43-1 and Raf43-overexpressing plants showed similar disease phenotype to the wild type plants after infection with B. cinerea or Pseudomonas syringae pv. tomato DC3000. Our results demonstrate that Raf43, encoding for a Raf-like MAPKKK, is required for tolerance to multiple abiotic stresses in Arabidopsis.

  20. G1 cell cycle arrest due to the inhibition of erbB family receptor tyrosine kinases does not require the retinoblastoma protein

    International Nuclear Information System (INIS)

    The erbB receptor family (EGFr, erbB-2, erbB-3, and erbB-4) consists of transmembrane glycoproteins that transduce extracellular signals to the nucleus when activated. erbB family members are widely expressed in epithelial, mesenchymal, and neuronal cells and contribute to the proliferation, differentiation, migration, and survival of these cell types. The present study evaluates the effects of erbB family signaling on cell cycle progression and the role that pRB plays in regulating this process. ErbB family RTK activity was inhibited by PD 158780 in the breast epithelial cell line MCF10A. PD 158780 (0.5 μM) inhibited EGF-stimulated and heregulin-stimulated autophosphorylation and caused a G1 cell cycle arrest within 24 h, which correlated with hypophosporylation of pRB. MCF10A cells lacking functional pRB retained the ability to arrest in G1 when treated with PD 158780. Both cell lines showed induction of p27KIP1 protein when treated with PD 158780 and increased association of p27KIP1 with cyclin E-CDK2. Furthermore, CDK2 kinase activity was dramatically inhibited with drug treatment. Changes in other pRB family members were noted with drug treatment, namely a decrease in p107 and an increase in p130. These findings show that the G1 arrest induced through inhibition of erbB family RTK activity does not require functional pRB