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Sample records for enzymatic mutant proteins

  1. Inactive enzymatic mutant proteins (phosphoglycerate mutase and enolase as sugar binders for ribulose-1,5-bisphosphate regeneration reactors

    Directory of Open Access Journals (Sweden)

    Giri Ashok

    2005-02-01

    Full Text Available Abstract Background Carbon dioxide fixation bioprocess in reactors necessitates recycling of D-ribulose1,5-bisphosphate (RuBP for continuous operation. A radically new close loop of RuBP regenerating reactor design has been proposed that will harbor enzyme-complexes instead of purified enzymes. These reactors will need binders enabling selective capture and release of sugar and intermediate metabolites enabling specific conversions during regeneration. In the current manuscript we describe properties of proteins that will act as potential binders in RuBP regeneration reactors. Results We demonstrate specific binding of 3-phosphoglycerate (3PGA and 3-phosphoglyceraldehyde (3PGAL from sugar mixtures by inactive mutant of yeast enzymes phosphoglycerate mutase and enolase. The reversibility in binding with respect to pH and EDTA has also been shown. No chemical conversion of incubated sugars or sugar intermediate metabolites were found by the inactive enzymatic proteins. The dissociation constants for sugar metabolites are in the micromolar range, both proteins showed lower dissociation constant (Kd for 3-phosphoglycerate (655–796 μM compared to 3-phosphoglyceraldehyde (822–966 μM indicating higher affinity for 3PGA. The proteins did not show binding to glucose, sucrose or fructose within the sensitivity limits of detection. Phosphoglycerate mutase showed slightly lower stability on repeated use than enolase mutants. Conclusions The sugar and their intermediate metabolite binders may have a useful role in RuBP regeneration reactors. The reversibility of binding with respect to changes in physicochemical factors and stability when subjected to repeated changes in these conditions are expected to make the mutant proteins candidates for in-situ removal of sugar intermediate metabolites for forward driving of specific reactions in enzyme-complex reactors.

  2. Non-enzymatic translocation in ribosomes from streptomycin-resistant mutants of Escherichia coli.

    Science.gov (United States)

    Asatryan, L S; Spirin, A S

    1975-07-10

    The capablity of ribosomes of four types of streptomycin-resistant mutants (A1, A2, A40 and A60) for non-enzymatic (EF-G--GTP-independent) translocation was tested. It was found that an A40 type mutation (amino acid replacement in position 87 of the protein S12 polypeptide chain) leads to activation of the capablity of the ribosome to perform spontaneous non-enzymatic translocation, while type A1, A2 and A60 mutations (amino acid replacements in position 42 of protein S12) does not give such an effect. Thus, it is shown that non-enzymatic translocation can be activated not only by the earlier described damage of the protein S12 by para-chloromercuribenzoate or by the complete removal of protein S12, but also by a definate mutational alteration of the protein. Preliminary data are also reported on the possibility of activating non-enzymatic translocation by combinations of mutational alterations of the ribosomal proteins other than protein S12 but interdepending with it (such as S4 and S5).

  3. Analysis of the iso enzymatic variability in rice mutants obtained by means of mutation radioinduction

    International Nuclear Information System (INIS)

    Gonzalez Nunez, L.M.; Inglesias Andreu, L.

    1998-01-01

    The iso enzymatic variability was studied in a group of mutants with tolerance to salinity, which were obtained by mutation induction with Co-60 gamma rays from the J-112 rice variety. The existence of esterase and peroxidase was confirmed, which permitted their utilization in differentiation works with the mutants and the existing variety in the examined material. In addition, the variables that contributed most variability were established

  4. Induced mutants for cereal grain protein improvement

    International Nuclear Information System (INIS)

    1982-01-01

    Out of 17 papers and one summary presented, six dealing with the genetic improvement of seed protein using ionizing radiations fall within the INIS subject scope. Other topics discussed were non-radiation induced mutants used for cereal grain protein improvement

  5. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

    Energy Technology Data Exchange (ETDEWEB)

    Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; Vakeel, Padmanabhan; Span, Elise A.; Kalous, Kelsey S.; Kutty, Raman G.; Jensen, Davin R.; Pokkuluri, Phani Raj; Sem, Daniel S.; Rathore, Rajendra; Ramchandran, Ramani

    2014-12-01

    Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function. We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.

  6. The agronomic characters of a high protein rice mutant

    International Nuclear Information System (INIS)

    Harn, C.; Won, J.L.; Choi, K.T.

    1975-01-01

    Mutant lines (M 5 -M 9 ) of macro-phenotypic traits from several varieties were screened for the protein content. Mutant 398 (M 9 ) is one of the high protein mutants selected from Hokwang. Three years' tests revealed that it has a high protein line under any condition of cultivation. Except for early maturity and short culmness, other agronomic and yield characters were similar to the original variety. There was no difference between the mutant 398 and its mother variety in grain shape and weight, and also the size and protein content of the embryo. The high protein content of the mutant is attributable to the increase of protein in the endosperm. About 150 normal-looking or a few days-earlier-maturing selections were made from Jinheung variety in the M 3 and screened for protein. Promising lines in terms of the plant type, yield and protein were obtained. (author)

  7. A Multiplex Enzymatic Machinery for Cellular Protein S-nitrosylation.

    Science.gov (United States)

    Seth, Divya; Hess, Douglas T; Hausladen, Alfred; Wang, Liwen; Wang, Ya-Juan; Stamler, Jonathan S

    2018-02-01

    S-nitrosylation, the oxidative modification of Cys residues by nitric oxide (NO) to form S-nitrosothiols (SNOs), modifies all main classes of proteins and provides a fundamental redox-based cellular signaling mechanism. However, in contrast to other post-translational protein modifications, S-nitrosylation is generally considered to be non-enzymatic, involving multiple chemical routes. We report here that endogenous protein S-nitrosylation in the model organism E. coli depends principally upon the enzymatic activity of the hybrid cluster protein Hcp, employing NO produced by nitrate reductase. Anaerobiosis on nitrate induces both Hcp and nitrate reductase, thereby resulting in the S-nitrosylation-dependent assembly of a large interactome including enzymes that generate NO (NO synthase), synthesize SNO-proteins (SNO synthase), and propagate SNO-based signaling (trans-nitrosylases) to regulate cell motility and metabolism. Thus, protein S-nitrosylation by NO in E. coli is essentially enzymatic, and the potential generality of the multiplex enzymatic mechanism that we describe may support a re-conceptualization of NO-based cellular signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Seed protein and nitrogen fixation in chickpea mutant variety Hyprosola

    International Nuclear Information System (INIS)

    Schroeder, H.E.; Gibson, A.H.; Oram, R.N.; Shaikh, M.A.Q.

    1989-01-01

    Full text: 'Hyprosola' is a high yielding, high protein mutant cultivar obtained after gamma irradiation from the variety 'Faridpur-1'. The mutant yields 45 % more protein per unit area. The essential amino acid index is unchanged. It is likely that the high nutritional value in 'Hyprosola' seed protein arises from an increase in the albumin:globulin ratio. Nitrogen fixation rates of the mutant during the first 7 weeks of growth were found to be similar to 'Faridpur-1'. Under field conditions, the mutant may be able to nodulate more rapidly and more extensively than the parent variety. (author)

  9. Enzymatic Hydrolysis of Various Proteins of Wheat in Heterogeneous Conditions

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    Hasan Hasanov

    2010-12-01

    Full Text Available Enzymatic hydrolysis of different proteins isolated from wheat flour by neutral proteinase (neutraza "Novozymes" was studied. It was shown, that hydrolysis of alkaline proteins was 10-11 times higher as compared with albumin from wheat, 3-4 times higher than alcohol soluble proteins and 2-2.5 times higher than globulins. It was found that, hydrothermal treatment of wheat flour decreased the rate of protein hydrolysis. The rate of hydrolysis of native alkaline soluble proteins was 4-6 times higher than denaturized proteins. The rate of hydrolysis of denatured water-soluble proteins is 3-5 times higher as compared with native protein (albumin. It was shown that product of thermal degradation of raw materials also influence on the rate of protein hydrolysis.

  10. Genetic Screening Identifies Cyanogenesis-Deficient Mutants of Lotus japonicus and Reveals Enzymatic Specificity in Hydroxynitrile Glucoside Metabolism

    DEFF Research Database (Denmark)

    Takos, A.; Lai, D.; Mikkelsen, L.

    2010-01-01

    Cyanogenesis, the release of hydrogen cyanide from damaged plant tissues, involves the enzymatic degradation of amino acid-derived cyanogenic glucosides (alpha-hydroxynitrile glucosides) by specific beta-glucosidases. Release of cyanide functions as a defense mechanism against generalist herbivores....... We developed a high-throughput screening method and used it to identify cyanogenesis deficient (cyd) mutants in the model legume Lotus japonicus. Mutants in both biosynthesis and catabolism of cyanogenic glucosides were isolated and classified following metabolic profiling of cyanogenic glucoside...... content. L. japonicus produces two cyanogenic glucosides: linamarin (derived from Val) and lotaustralin (derived from Ile). Their biosynthesis may involve the same set of enzymes for both amino acid precursors. However, in one class of mutants, accumulation of lotaustralin and linamarin was uncoupled...

  11. Estimates of selection parameters in protein mutants of spring barley

    International Nuclear Information System (INIS)

    Gaul, H.; Walther, H.; Seibold, K.H.; Brunner, H.; Mikaelsen, K.

    1976-01-01

    Detailed studies have been made with induced protein mutants regarding a possible genetic advance in selection including the estimation of the genetic variation and heritability coefficients. Estimates were obtained for protein content and protein yield. The variation of mutant lines in different environments was found to be many times as large as the variation of the line means. The detection of improved protein mutants seems therefore possible only in trials with more than one environment. The heritability of protein content and protein yield was estimated in different sets of environments and was found to be low. However, higher values were found with an increasing number of environments. At least four environments seem to be necessary to obtain reliable heritability estimates. The geneticall component of the variation between lines was significant for protein content in all environmental combinations. For protein yield some environmental combinations only showed significant differences. The expected genetic advance with one selection step was small for both protein traits. Genetically significant differences between protein micromutants give, however, a first indication that selection among protein mutants with small differences seems also possible. (author)

  12. Streptomycin-induced synthesis of abnormal protein in an Escherichia coli mutant.

    Science.gov (United States)

    Pinkett, M O; Brownstein, B L

    1974-08-01

    To determine directly the effects of streptomycin on translational fidelity in intact cells, we studied the synthesis of beta-galactosidase and of the coat protein of bacteriophage R17 in an Escherichia coli mutant in which the bactericidal effects of streptomycin are delayed. After the addition of streptomycin to exponentially growing mutant cells, protein synthesis continues at an undiminished rate for approximately an hour; however, as measured by enzyme assays, little functional protein is produced. Serological assays designed to detect beta-galactosidase and bacteriophage R17 coat protein show that substantial amounts of the protein synthesized can react with antisera prepared against active beta-galactosidase and phage R17, indicating the aberrance of the protein produced in the presence of the antibiotic. The polypeptides synthesized in the presence of streptomycin are degraded in the cell to a much greater extent than protein synthesized in the absence of the antibiotic. The proteolytic attack on this protein is not affected by inhibitors of serine proteases, suggesting that enzymes other than those involved in "normal turnover" of cellular protein are responsible. In this strain, certain of the multiple effects of streptomycin are separated in time and the production of abnormal protein (enzymatically inactive and susceptible to proteolytic attack) could be studied in the absence of the lethal effect of the drug.

  13. Enzymatic Characterization of a Mutant of Escherichia coli with an Altered DNA Ligase

    Science.gov (United States)

    Modrich, Paul; Lehman, I. R.

    1971-01-01

    A temperature-sensitive, radiation-sensitive mutant of Escherichia coli has been assayed for DNA ligase activity in vitro. The strain contains a markedly reduced amount of DNA-joining activity, which is thermolabile. The formation of the ligase-adenylate intermediate is also temperature-sensitive in vitro. Two temperature-resistant revertants of the mutant contain normal amounts of a thermostable ligase. The mutant is killed by growth at 42°C, a temperature at which it displays aberrant DNA synthesis. These results suggest that the ligase is necessary for normal DNA metabolism and viability in this strain. PMID:4995816

  14. Structural and Antihypertensive Properties of Enzymatic Hemp Seed Protein Hydrolysates.

    Science.gov (United States)

    Malomo, Sunday A; Onuh, John O; Girgih, Abraham T; Aluko, Rotimi E

    2015-09-10

    The aim of this work was to produce antihypertensive protein hydrolysates through different forms of enzymatic hydrolysis (2% pepsin, 4% pepsin, 1% alcalase, 2% alcalase, 2% papain, and 2% pepsin + pancreatin) of hemp seed proteins (HSP). The hemp seed protein hydrolysates (HPHs) were tested for in vitro inhibitions of renin and angiotensin-converting enzyme (ACE), two of the enzymes that regulate human blood pressure. The HPHs were then administered orally (200 mg/kg body weight) to spontaneously hypertensive rats and systolic blood pressure (SBP)-lowering effects measured over a 24 h period. Size exclusion chromatography mainly showed a 300-9560 Da peptide size range for the HPHs, while amino acid composition data had the 2% pepsin HPH with the highest cysteine content. Fluorescence spectroscopy revealed higher fluorescence intensities for the peptides when compared to the unhydrolyzed hemp seed protein. Overall, the 1% alcalase HPH was the most effective (p alcalase) with the longer-lasting HPHs (2% and 4% pepsin) could provide daily effective SBP reductions.

  15. Structural and Antihypertensive Properties of Enzymatic Hemp Seed Protein Hydrolysates

    Directory of Open Access Journals (Sweden)

    Sunday A. Malomo

    2015-09-01

    Full Text Available The aim of this work was to produce antihypertensive protein hydrolysates through different forms of enzymatic hydrolysis (2% pepsin, 4% pepsin, 1% alcalase, 2% alcalase, 2% papain, and 2% pepsin + pancreatin of hemp seed proteins (HSP. The hemp seed protein hydrolysates (HPHs were tested for in vitro inhibitions of renin and angiotensin-converting enzyme (ACE, two of the enzymes that regulate human blood pressure. The HPHs were then administered orally (200 mg/kg body weight to spontaneously hypertensive rats and systolic blood pressure (SBP-lowering effects measured over a 24 h period. Size exclusion chromatography mainly showed a 300–9560 Da peptide size range for the HPHs, while amino acid composition data had the 2% pepsin HPH with the highest cysteine content. Fluorescence spectroscopy revealed higher fluorescence intensities for the peptides when compared to the unhydrolyzed hemp seed protein. Overall, the 1% alcalase HPH was the most effective (p < 0.05 SBP-reducing agent (−32.5 ± 0.7 mmHg after 4 h, while the pepsin HPHs produced longer-lasting effects (−23.0 ± 1.4 mmHg after 24 h. We conclude that an optimized combination of the fast-acting HPH (1% alcalase with the longer-lasting HPHs (2% and 4% pepsin could provide daily effective SBP reductions.

  16. Enzymatic pre-treatment increases the protein bioaccessibility and extractability in dulse (Palmaria palmata)

    OpenAIRE

    Mæhre, Hanne K; Jensen, Ida-Johanne; Eilertsen, Karl-Erik

    2016-01-01

    Several common protein extraction protocols have been applied on seaweeds, but extraction yields have been limited. The aims of this study were to further develop and optimize existing extraction protocols and to examine the effect of enzymatic pre-treatment on bioaccessibility and extractability of seaweed proteins. Enzymatic pre-treatment of seaweed samples resulted in a three-fold increase in amino acids available for extraction. Combining enzymatic pre-treatment with a...

  17. Enzymatic Mercury Detoxification: The Regulatory Protein MerR

    CERN Multimedia

    Ctortecka, B; Walsh, C T; Comess, K M

    2002-01-01

    Mercury ions and organomercurial reagents are extremely toxic due to their affinity for thiol groups. Many bacteria contain an elaborate detoxification system for a metabolic conversion of toxic Hg$^{2+}$ or organomercurials to less toxic elemental Hg$^0$. The main components of the enzymatic mercury detoxification (see Fig. 1) are the regulatory protein MerR (mercury responsive genetic switch), the organomercurial lyase MerB (cleavage of carbon mercury bonds), and the mercuric ion reductase MerA (reduction of mercuric ions). In these proteins Hg$^{2+}$ is usually coordinated by the thiol groups of cysteines. We utilize the nuclear quadrupole interaction (NQI) of ${\\rm^{199m}}$Hg detected by time differential perturbed angular correlation (TDPAC) to identify the Hg metal site geometries in these proteins in order to elucidate the molecular origin of the ultrasensitivity, selectivity and reaction mechanism of this detoxification system. The short lived TDPAC probe ${\\rm^{199m}}$Hg ($\\tau_{1/2} =$ 43 min) is su...

  18. Enzymatic functionalization of a nanobody using protein insertion technology.

    Science.gov (United States)

    Crasson, O; Rhazi, N; Jacquin, O; Freichels, A; Jérôme, C; Ruth, N; Galleni, M; Filée, P; Vandevenne, M

    2015-10-01

    Antibody-based products constitute one of the most attractive biological molecules for diagnostic, medical imagery and therapeutic purposes with very few side effects. Their development has become a major priority of biotech and pharmaceutical industries. Recently, a growing number of modified antibody-based products have emerged including fragments, multi-specific and conjugate antibodies. In this study, using protein engineering, we have functionalized the anti-hen egg-white lysozyme (HEWL) camelid VHH antibody fragment (cAb-Lys3), by insertion into a solvent-exposed loop of the Bacillus licheniformis β-lactamase BlaP. We showed that the generated hybrid protein conserved its enzymatic activity while the displayed nanobody retains its ability to inhibit HEWL with a nanomolar affinity range. Then, we successfully implemented the functionalized cAb-Lys3 in enzyme-linked immunosorbent assay, potentiometric biosensor and drug screening assays. The hybrid protein was also expressed on the surface of phage particles and, in this context, was able to interact specifically with HEWL while the β-lactamase activity was used to monitor phage interactions. Finally, using thrombin-cleavage sites surrounding the permissive insertion site in the β-lactamase, we reported an expression system in which the nanobody can be easily separated from its carrier protein. Altogether, our study shows that insertion into the BlaP β-lactamase constitutes a suitable technology to functionalize nanobodies and allows the creation of versatile tools that can be used in innovative biotechnological assays. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  19. Effects of enzymatic hydrolysis on the allergenicity of whey protein concentrates.

    OpenAIRE

    Cuicui Duan; Lijie Yang; Aili Li; Rui Zhao; Guicheng Huo

    2014-01-01

    Cow's milk whey consists of many protein components and some of them are antigens to human and known to modulate immune responses. Enzymatic hydrolysis is a useful method to modify proteins with allergenicity. The objective of this study was to identify whether the in vitro enzymatic hydrolysis could reduce the allergenicity of whey protein concentrates (WPC). In this study, WPC were hydrolyzed by trypsin and twenty-four BALB/c mice were divided into three groups and fed with WPC formula and ...

  20. Optimum conditions for enzymatic degradation of some oilseed proteins

    Directory of Open Access Journals (Sweden)

    El-Zanaty, E. A.

    2002-09-01

    Full Text Available Soybean, sesame seed, and rice bran meal proteins were hydrolyzed with two enzymes, namely, papain and bromelain. Experiments were carried out to elucidate the optimum condition for each enzyme when acting on each substrate seperately. Results revealed that the highest relative activities for papain were achieved with E/S 0.06 , 0.29, 0.19 and pH 7.2, 7.0, 7.0 for soybean, sesame,and rice bran meal proteins, respectively. Optimum temperature for papain while hydrolysing the three substrates was 50 ºC. When using bromelain optimum E/S resulting in highest relative activities were 0.067, 0.058 and 0.21 for soybean, sesame,and rice bran meal protein, respectively. Optimum pH was 6.0 and optimum temperature was 45 ºC for bromelain when hydrolysing the protein of the three substrates. A numerical correlation of enzymatic behaviour for the different substrates was calculated.Proteínas de haba de soja, semilla de sésamo y harina de germen de arroz se hidrolizaron con dos enzimas, denominadas, papaina y bromelaina. Se han llevado a cabo experimentos para determinar las condiciones óptimas de cada enzima cuando actúan separadamente sobre cada sustrato. Los resultados mostraron que las mayores actividades relativas para la papaina se consiguieron con una E/S 0,06, 0,29, 0,19 y un pH 7.2, 7.0, 7.0 para las proteínas de haba de soja, sésamo y harina de germen de arroz, respectivamente. La temperatura óptima para la papaina durante la hidrólisis de los tres sustratos fue de 50 ºC. Cuando se usa bromelaina las relaciones E/S óptimas que proporcionaron mayor actividad relativa fueron 0.067, 0.058 y 0.21 para las proteínas de habas de soja, sésamo y harina de germen de arroz respectivamente. El pH óptimo fue 6.0 y la temperatura óptima 45 ºC para la bromelaina cuando se hidroliza la proteína de los tres sustratos. Con estos datos se hizo una correlación numérica del comportamiento enzimático para los diferentes sustratos.

  1. Phyto-agglutinin, total proteins and amino assimilating enzymatic ...

    African Journals Online (AJOL)

    Jane

    2011-09-28

    2K cultivars extract showed highly phyto-agglutination of human erythrocytes with reproductive organs and other tissues, which represents the presence of potent lectins (phyto-agglutinin). The amino assimilating enzymatic.

  2. Enzymatic aqueous technology for simultaneous coconut protein and oil extraction

    Directory of Open Access Journals (Sweden)

    Coelho, Maria A. Z.

    2003-03-01

    Full Text Available In this study, the following commercial enzymes were evaluated in the enzymatic extraction of oil and protein from coconut: Celluclast, Termamyl, Viscozyme, Neutrase y Protease. Preliminary experiments were carried out for selection of enzymes, enzyme concentration and incubation time. In order to verify the effects of pH and water/substrate ratio, a response surface methodology was applied based in a 32 full factorial experimental design. The pH is the most meaningful parameter on oil and protein extraction yields, with a significance level higher than 90%. The maximal extraction yields of oil and protein emulsion (83% was reached using Viscozyme L and subsequently Neutrase 1.5 MG at concentrations of 0.6% (w/w and 0.3% (w/w, respectively, total incubation time of 60 minutes, substrate/water ratio 1:6 and pH around 7 (no adjustment.En este trabajo diferentes enzimas hidroliticos fueron usados para la extracción de aceite y proteína de coco: Celluclast, Termamyl, Viscozyme, Neutrase y Protease producidos por Novo Nordisk. Experimientos preliminares fueron encaminados a la selección de los extractos enzimaticos, de las concentraciones de las enzimas y de los tiempos de incubación. La técnica de análisis de varianza, mediante un delineamiento factorial en tres niveles, fue usada para la optimización de los procesos con relación a los parámetros pH y razón sustrato/agua. Comparando con la extracción acuosa todos los enzimas hidroliticos incrementan la extracción de aceite y proteína vegetal de coco. Los coeficientes de correlación muestran que el pH fué el parámetro mas significativo (pViscozyme y Neutrase 0.6 % (w/w y 0.3 % (w/w, respectivamente, tiempo de incubación 30 minutos, razón sustrato/agua 1:6 y pH 7.

  3. Adaptive response in Drosophila melanogaster heat shock proteins mutant strains

    International Nuclear Information System (INIS)

    Shaposhnikov, M.V.; Moskalev, A.A.; Turysheva, E.V.

    2007-01-01

    Complete text of publication follows. The members of the heat shock proteins (Hsp) family function as molecular chaperones and assist intracellular folding of newly synthesized proteins. Also it is possible that molecular chaperones are induced during adaptive response to oxidative stress and radiation. The aim of our research was to exam the role of heat shock proteins in adaptive response to oxidative stress after low dose rate gamma-irradiation in Drosophila melanogaster. Drosophilamelanogaster strains were kindly provided by Bloomington Drosophila Stock Center (University of state of Indiana, Bloomington, USA). We used wild type strain (CS), heat shock protein mutant strains (Hsp22, Hsp70, Hsp83), and heat shock factor mutant strain (Hsf). Strains were chronically exposured to adaptive dose of gamma-irradiation in dose rate of 0.17 cGy/h during all stages of life history (from the embrional stage to the stage of matured imago). The rate of absorbed dose was 60 cGy. For oxidative-stress challenge twodays old flies were starved in empty vials for 6 h and then transferred to vials containing only filter paper soaked with 20 mM paraquat in 5% sucrose solution. Survival data were collected after 26 h of treatment. Dead flies were counted daily. The obtained data were subjected to survival analysis by Kaplan and Meier method and presented as survival curves. Statistical analysis was held by non-parametric methods. To test the significance of the difference between the two age distributions Kolmogorov-Smirnov test was applied. Gehan-Braslow- Wilcoxon and Cox-Mantel tests were used for estimation of median life span differences. In addition the minimal and maximal life span, time of 90% death, and mortality rate doubling time (MRDT) were estimated. The obtained results will be discussed in presentation.

  4. A mutant of phosphomannomutase1 retains full enzymatic activity, but is not activated by IMP: Possible implications for the disease PMM2-CDG.

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    Valentina Citro

    Full Text Available The most frequent disorder of glycosylation, PMM2-CDG, is caused by a deficiency of phosphomannomutase activity. In humans two paralogous enzymes exist, both of them require mannose 1,6-bis-phosphate or glucose 1,6-bis-phosphate as activators, but only phospho-mannomutase1 hydrolyzes bis-phosphate hexoses. Mutations in the gene encoding phosphomannomutase2 are responsible for PMM2-CDG. Although not directly causative of the disease, the role of the paralogous enzyme in the disease should be clarified. Phosphomannomutase1 could have a beneficial effect, contributing to mannose 6-phosphate isomerization, or a detrimental effect, hydrolyzing the bis-phosphate hexose activator. A pivotal role in regulating mannose-1phosphate production and ultimately protein glycosylation might be played by inosine monophosphate that enhances the phosphatase activity of phosphomannomutase1. In this paper we analyzed human phosphomannomutases by conventional enzymatic assays as well as by novel techniques such as 31P-NMR and thermal shift assay. We characterized a triple mutant of phospomannomutase1 that retains mutase and phosphatase activity, but is unable to bind inosine monophosphate.

  5. Enzymatic Pre-Treatment Increases the Protein Bioaccessibility and Extractability in Dulse (Palmaria palmata

    Directory of Open Access Journals (Sweden)

    Hanne K. Mæhre

    2016-10-01

    Full Text Available Several common protein extraction protocols have been applied on seaweeds, but extraction yields have been limited. The aims of this study were to further develop and optimize existing extraction protocols and to examine the effect of enzymatic pre-treatment on bioaccessibility and extractability of seaweed proteins. Enzymatic pre-treatment of seaweed samples resulted in a three-fold increase in amino acids available for extraction. Combining enzymatic pre-treatment with alkaline extraction resulted in a 1.6-fold increase in the protein extraction yield compared to a standard alkaline extraction protocol. A simulated in vitro gastrointestinal digestion model showed that enzymatic pre-treatment of seaweed increased the amount of amino acids available for intestinal absorption 3.2-fold. In conclusion, enzymatic pre-treatment of seaweeds is effective for increasing the amount of amino acids available for utilization and may thus be an effective means for increasing the utilization potential of seaweed proteins. However, both the enzymatic pre-treatment protocol and the protein extraction protocol need further optimization in order to obtain optimal cost-benefit and results from the in vitro gastrointestinal digestion model need to be confirmed in clinical models.

  6. Enzymatic Pre-Treatment Increases the Protein Bioaccessibility and Extractability in Dulse (Palmaria palmata).

    Science.gov (United States)

    Mæhre, Hanne K; Jensen, Ida-Johanne; Eilertsen, Karl-Erik

    2016-10-26

    Several common protein extraction protocols have been applied on seaweeds, but extraction yields have been limited. The aims of this study were to further develop and optimize existing extraction protocols and to examine the effect of enzymatic pre-treatment on bioaccessibility and extractability of seaweed proteins. Enzymatic pre-treatment of seaweed samples resulted in a three-fold increase in amino acids available for extraction. Combining enzymatic pre-treatment with alkaline extraction resulted in a 1.6-fold increase in the protein extraction yield compared to a standard alkaline extraction protocol. A simulated in vitro gastrointestinal digestion model showed that enzymatic pre-treatment of seaweed increased the amount of amino acids available for intestinal absorption 3.2-fold. In conclusion, enzymatic pre-treatment of seaweeds is effective for increasing the amount of amino acids available for utilization and may thus be an effective means for increasing the utilization potential of seaweed proteins. However, both the enzymatic pre-treatment protocol and the protein extraction protocol need further optimization in order to obtain optimal cost-benefit and results from the in vitro gastrointestinal digestion model need to be confirmed in clinical models.

  7. Use of wheat and maize protein mutants in breeding for improved protein quantity and quality

    International Nuclear Information System (INIS)

    Denic, M.; Dumanovic, J.; Misevic, D.; Konstantinov, K.; Fidler, D.; Stojanovic, Z.

    1984-01-01

    Selected offspring progenies (50 mutant lines) originating from mutation experiments with hexaploid wheat (cv. Bezostaya 1) were analysed for induced heritable variation in protein content, lysine content, grain yield and protein and lysine yields. Ten of these mutant lines were crossed with 11 local varieties. The protein and lysine contents were measured in the progenies of these crossings. The data showed better correlations of grain yield with protein and lysine yields than the protein and lysine contents with their corresponding yields. F 1 seeds showed higher lysine and protein contents than local varieties. Data with maize showed that: (1) the total endosperm protein content of modified opaque-2 types increases with an increase in the degree of normalization; (2) the lysine content in dry matter and protein in normalized o 2 kernels usually decreases with the increasing degree of normalization; (3) the lysine content in protein of modified o 2 kernels, is, in general, satisfactory up to the normalization of about 50% of endosperm. A desirable modification of o 2 endosperm within line A632o 2 was selected and crossed with o 2 lines. Most of the tested hybrids had a good protein quality, but endosperm modification was not evident in all hybrids. The o 2 gene was incorporated into high protein backgrounds. Besides a high protein content and quality, some of the hybrids tested had a comparable or higher yield than the o 2 check. (author)

  8. Glycosaminoglycan sulphation affects the seeded misfolding of a mutant prion protein.

    Directory of Open Access Journals (Sweden)

    Victoria A Lawson

    Full Text Available BACKGROUND: The accumulation of protease resistant conformers of the prion protein (PrP(res is a key pathological feature of prion diseases. Polyanions, including RNA and glycosaminoglycans have been identified as factors that contribute to the propagation, transmission and pathogenesis of prion disease. Recent studies have suggested that the contribution of these cofactors to prion propagation may be species specific. METHODOLOGY/PRINCIPAL FINDING: In this study a cell-free assay was used to investigate the molecular basis of polyanion stimulated PrP(res formation using brain tissue or cell line derived murine PrP. Enzymatic depletion of endogenous nucleic acids or heparan sulphate (HS from the PrP(C substrate was found to specifically prevent PrP(res formation seeded by mouse derived PrP(Sc. Modification of the negative charge afforded by the sulphation of glycosaminoglycans increased the ability of a familial PrP mutant to act as a substrate for PrP(res formation, while having no effect on PrP(res formed by wildtype PrP. This difference may be due to the observed differences in the binding of wild type and mutant PrP for glycosaminoglycans. CONCLUSIONS/SIGNIFICANCE: Cofactor requirements for PrP(res formation are host species and prion strain specific and affected by disease associated mutations of the prion protein. This may explain both species and strain dependent propagation characteristics and provide insights into the underlying mechanisms of familial prion disease. It further highlights the challenge of designing effective therapeutics against a disease which effects a range of mammalian species, caused by range of aetiologies and prion strains.

  9. Effects of Ionic Strength on the Enzymatic Hydrolysis of Diluted and Concentrated Whey Protein Isolate

    NARCIS (Netherlands)

    Butré, C.I.; Wierenga, P.A.; Gruppen, H.

    2012-01-01

    To identify the parameters that affect enzymatic hydrolysis at high substrate concentrations, whey protein isolate (1–30% w/v) was hydrolyzed by Alcalase and Neutrase at constant enzyme-to-substrate ratio. No changes were observed in the solubility and the aggregation state of the proteins. With

  10. Analysis of substructural variation in families of enzymatic proteins with applications to protein function prediction

    Directory of Open Access Journals (Sweden)

    Fofanov Viacheslav Y

    2010-05-01

    Full Text Available Abstract Background Structural variations caused by a wide range of physico-chemical and biological sources directly influence the function of a protein. For enzymatic proteins, the structure and chemistry of the catalytic binding site residues can be loosely defined as a substructure of the protein. Comparative analysis of drug-receptor substructures across and within species has been used for lead evaluation. Substructure-level similarity between the binding sites of functionally similar proteins has also been used to identify instances of convergent evolution among proteins. In functionally homologous protein families, shared chemistry and geometry at catalytic sites provide a common, local point of comparison among proteins that may differ significantly at the sequence, fold, or domain topology levels. Results This paper describes two key results that can be used separately or in combination for protein function analysis. The Family-wise Analysis of SubStructural Templates (FASST method uses all-against-all substructure comparison to determine Substructural Clusters (SCs. SCs characterize the binding site substructural variation within a protein family. In this paper we focus on examples of automatically determined SCs that can be linked to phylogenetic distance between family members, segregation by conformation, and organization by homology among convergent protein lineages. The Motif Ensemble Statistical Hypothesis (MESH framework constructs a representative motif for each protein cluster among the SCs determined by FASST to build motif ensembles that are shown through a series of function prediction experiments to improve the function prediction power of existing motifs. Conclusions FASST contributes a critical feedback and assessment step to existing binding site substructure identification methods and can be used for the thorough investigation of structure-function relationships. The application of MESH allows for an automated

  11. A Nearly Non-Functional Mutant Allele of the Storage Protein Locus Hor2 in Barley

    DEFF Research Database (Denmark)

    Doll, Hans

    1980-01-01

    The low content of the storage protein fraction hordein-2 in the high-lysine mutant Risø 56 is due to a mutation at or near the locus Hor2 coding for hordein-2 polypeptides. The mutant gene is recessive in its qualitative effect on the electrophoretic banding pattern of hordein-2, but co-dominant......The low content of the storage protein fraction hordein-2 in the high-lysine mutant Risø 56 is due to a mutation at or near the locus Hor2 coding for hordein-2 polypeptides. The mutant gene is recessive in its qualitative effect on the electrophoretic banding pattern of hordein-2, but co......-dominant with respect to the relative amount of the hordein fractions. Homozygous mutant seeds were about 10 % smaller than normal seeds. Nd linkage was detected between the mutant gene and the translocation between chromosomes 2 and 5 in mutant 56....

  12. Cetyltrimethylammonium bromide discontinuous gel electrophoresis: Mr-based separation of proteins with retention of enzymatic activity.

    Science.gov (United States)

    Akins, R E; Levin, P M; Tuan, R S

    1992-04-01

    A discontinuous polyacrylamide and agarose gel electrophoresis system is presented here which allows the fine separation of proteins based on molecular weight with the concomitant retention of native enzymatic activity. This system, referred to as the CAT gel, uses the cationic detergent cetyltrimethylammonium bromide (CTAB) and includes a stacking gel based on the zwitterion arginine and the buffer N-tris(hydroxymethyl)-methylglycine. The CAT gel system allows specific enzyme histochemical detection and localization of proteins after gel electrophoresis. We present evidence that the CAT system stacked and separated a broad range of proteins into discrete bands which migrate as a linear function of log Mr. We have also assessed the effect of CTAB solubilization on the activity of several proteins and showed that some proteins separated by CAT electrophoresis maintain high levels of native enzymatic activity and may be detected histochemically in situ.

  13. Non-enzymatic action of RRM1 protein upregulates PTEN leading to inhibition of colorectal cancer metastasis.

    Science.gov (United States)

    Qi, Hongyan; Lou, Meng; Chen, Yuexia; Liu, Xiyong; Chen, Naiming; Shan, Jianzhen; Ling, Zhiqiang; Shen, Jing; Zhu, Lijun; Yen, Yun; Zheng, Shu; Shao, Jimin

    2015-06-01

    Ribonucleotide reductase large subunit M1 (RRM1) forms a holoenzyme with small subunits to provide deoxyribonucleotides for DNA synthesis and cell proliferation. Here, we reported a non-RR role of the catalytic subunit protein RRM1 and related pathway in inhibiting colorectal cancer (CRC) metastasis. Ectopic overexpression of the wild-type RRM1, and importantly, its Y738F mutant that lacks RR enzymatic activity, prevented the migration and invasion of CRC cells by promoting phosphatase and tensin homolog on chromosome 10 (PTEN) transactivation. Furthermore, overexpression of the wild-type and RR-inactive mutant RRM1 similarly reduced the phosphorylation of Akt and increased the E-cadherin expression in CRC cells, which were blocked by PTEN knockdown attenuation. Examination of clinical CRC specimens demonstrated that both RRM1 protein expression and RR activity were elevated in most cancer tissues compared to the paired normal tissues. However, while RR activity did not change significantly in different cancer stages, the RRM1 protein level was significantly increased at stages T1-3 but decreased at stage T4, in parallel with the PTEN expression level and negatively correlated with invasion and liver metastasis. Thus, we propose that RRM1 protein can inhibit CRC invasion and metastasis at the advanced stage by regulating PTEN transactivation and its downstream pathways in addition to forming an RR holoenzyme for supporting cancer proliferation. Understanding of the seemingly contrary dual roles of RRM1 protein may further help to explain the complex mechanisms by which this key enzyme and its components are involved in cancer development.

  14. Crystal structure of a mutant of archaeal ribosomal protein L1 from Methanococcus jannaschii

    Science.gov (United States)

    Sarskikh, A. V.; Gabdulkhakov, A. G.; Kostareva, O. S.; Shklyaeva, A. A.; Tishchenko, S. V.

    2014-05-01

    The crystal structure of a mutant of archaeal ribosomal protein L1 from Methanococcus jannaschii with the deletion of a nonconserved positively charged cluster consisting of eight C-terminal amino acid residues is determined by the molecular replacement method at 1.75 Å resolution. This mutant is shown to form more stable and ordered crystals belonging to a space group other than that of the wild-type protein crystals. The positively charged C-terminal region has only a slight effect on the interaction between protein L1 and RNA molecules. Hence, this mutant can be used to prepare protein-RNA complexes and obtain their crystals.

  15. A comparison of protein kinases inhibitor screening methods using both enzymatic activity and binding affinity determination

    DEFF Research Database (Denmark)

    Rudolf, Amalie Frederikke; Skovgaard, Tine; Knapp, Stefan

    2014-01-01

    Binding assays are increasingly used as a screening method for protein kinase inhibitors; however, as yet only a weak correlation with enzymatic activity-based assays has been demonstrated. We show that the correlation between the two types of assays can be improved using more precise screening...

  16. Enzymatic extractability of soybean meal proteins and carbohydrates : heat and humidity effects

    NARCIS (Netherlands)

    Fischer, M.; Kofod, L.V.; Schols, H.A.; Piersma, S.R.; Gruppen, H.; Voragen, A.G.J.

    2001-01-01

    To study the incomplete enzymatic extractability of proteins and carbohydrates of thermally treated soybean meals, one unheated and three heat-treated soybean meals were produced. To obtain truly enzyme-resistant material, the meals were extracted by a repeated hydrolysis procedure using excessive

  17. Apolipoprotein A-I mutant proteins having cysteine substitutions and polynucleotides encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Oda, Michael N [Benicia, CA; Forte, Trudy M [Berkeley, CA

    2007-05-29

    Functional Apolipoprotein A-I mutant proteins, having one or more cysteine substitutions and polynucleotides encoding same, can be used to modulate paraoxonase's arylesterase activity. These ApoA-I mutant proteins can be used as therapeutic agents to combat cardiovascular disease, atherosclerosis, acute phase response and other inflammatory related diseases. The invention also includes modifications and optimizations of the ApoA-I nucleotide sequence for purposes of increasing protein expression and optimization.

  18. Apolipoprotein A-I mutant proteins having cysteine substitutions and polynucleotides encoding same

    Science.gov (United States)

    Oda, Michael N [Benicia, CA; Forte, Trudy M [Berkeley, CA

    2007-05-29

    Functional Apolipoprotein A-I mutant proteins, having one or more cysteine substitutions and polynucleotides encoding same, can be used to modulate paraoxonase's arylesterase activity. These ApoA-I mutant proteins can be used as therapeutic agents to combat cardiovascular disease, atherosclerosis, acute phase response and other inflammatory related diseases. The invention also includes modifications and optimizations of the ApoA-I nucleotide sequence for purposes of increasing protein expression and optimization.

  19. Increased Protein Stability and Decreased Protein Turnover in the Caenorhabditis elegans Ins/IGF-1 daf-2 Mutant.

    Science.gov (United States)

    Depuydt, Geert; Shanmugam, Nilesh; Rasulova, Madina; Dhondt, Ineke; Braeckman, Bart P

    2016-12-01

    In Caenorhabditis elegans, cellular proteostasis is likely essential for longevity. Autophagy has been shown to be essential for lifespan extension of daf-2 insulin/IGF mutants. Therefore, it can be hypothesized that daf-2 mutants achieve this phenotype by increasing protein turnover. However, such a mechanism would exert a substantial energy cost. By using classical 35 S pulse-chase labeling, we observed that protein synthesis and degradation rates are decreased in young adults of the daf-2 insulin/IGF mutants. Although reduction of protein turnover may be energetically favorable, it may lead to accumulation and aggregation of damaged proteins. As this has been shown not to be the case in daf-2 mutants, another mechanism must exist to maintain proteostasis in this strain. We observed that proteins isolated from daf-2 mutants are more soluble in acidic conditions due to increased levels of trehalose. This suggests that trehalose may decrease the potential for protein aggregation and increases proteostasis in the daf-2 mutants. We postulate that daf-2 mutants save energy by decreasing protein turnover rates and instead stabilize their proteome by trehalose. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America.

  20. Production of enzymatic protein hydrolysates from freshwater catfish (Clarias batrachus)

    Science.gov (United States)

    Seniman, Maizatul Sarah Md; Yusop, Salma Mohamad; Babji, Abdul Salam

    2014-09-01

    Fish protein hydrolysate (FPH) was prepared from freshwater catfish (Clarias batrachus) by using Alcalase® 2.4L and Papain. The effect of hydrolysis time (30, 60, 120, 180 min) with enzyme concentration of 1% (v/w substrate); pH = 8.0, 7.0 was studied to determine the degree of hydrolysis (DH), peptide content, proximate composition and amino acid profile. Results showed that the highest DH of Alcalase and Papain FPH were 58.79% and 53.48% after 180 min at 55°C incubation respectively. The peptide content of both FPH increased as hydrolysis time increases. FPH showed higher crude protein content and lower fat, moisture and ash content compared to raw catfish. The major amino acids of both hydrolysates were Glu, Lys and Asp. Content of essential amino acids of Alcalase and Papain hydrolysates were 44.05% and 43.31% respectively.

  1. A Nearly Non-Functional Mutant Allele of the Storage Protein Locus Hor2 in Barley

    DEFF Research Database (Denmark)

    Doll, Hans

    1980-01-01

    The low content of the storage protein fraction hordein-2 in the high-lysine mutant Risø 56 is due to a mutation at or near the locus Hor2 coding for hordein-2 polypeptides. The mutant gene is recessive in its qualitative effect on the electrophoretic banding pattern of hordein-2, but co...

  2. Rapid and efficient protein enzymatic digestion: An experimental comparison

    Czech Academy of Sciences Publication Activity Database

    Dyčka, Filip; Bobáľ, P.; Mazanec, Karel; Bobálová, Janette

    2012-01-01

    Roč. 33, č. 2 (2012), s. 288-295 ISSN 0173-0835 R&D Projects: GA MŠk 1M0570; GA MŠk 1M06030; GA AV ČR IAA600040701 Institutional research plan: CEZ:AV0Z40310501 Keywords : in-gel digestion * MALDI - TOF MS * protein Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.261, year: 2012

  3. Allergenicity of Peanut Proteins is Retained Following Enzymatic Hydrolysis

    Science.gov (United States)

    Rationale: Hydrolysis of peanut proteins by food-grade enzymes may reduce allergenicity and could lead to safer forms of immunotherapy. Methods: Light roasted peanut flour extracts were digested with pepsin (37°C, pH 2), Alcalase (60°C pH 8), or Flavourzyme (50°C, pH 7) up to 1 hr, or sequentially w...

  4. Efficient production of infectious viruses requires enzymatic activity of Epstein-Barr virus protein kinase.

    Science.gov (United States)

    Murata, Takayuki; Isomura, Hiroki; Yamashita, Yoriko; Toyama, Shigenori; Sato, Yoshitaka; Nakayama, Sanae; Kudoh, Ayumi; Iwahori, Satoko; Kanda, Teru; Tsurumi, Tatsuya

    2009-06-20

    The Epstein-Barr virus (EBV) BGLF4 gene product is the only protein kinase encoded by the virus genome. In order to elucidate its physiological roles in viral productive replication, we here established a BGLF4-knockout mutant and a revertant virus. While the levels of viral DNA replication of the deficient mutant were equivalent to those of the wild-type and the revertant, virus production was significantly impaired. Expression of the BGLF4 protein in trans fully complemented the low yield of the mutant virus, while expression of a kinase-dead (K102I) form of the protein failed to restore the virus titer. These results demonstrate that BGLF4 plays a significant role in production of infectious viruses and that the kinase activity is crucial.

  5. Calculation of the redox potential of the protein azurin and some mutants

    NARCIS (Netherlands)

    van den Bosch, M; Swart, M; Snijders, JG; Berendsen, HJC; Mark, AE; Oostenbrink, C; van Gunsteren, WF; Canters, GW

    Azurin from Pseudomonas aeruginosa is a small 128-residue, copper-containing protein. Its redox potential can be modified by mutating the protein. Free-energy calculations based on classical molecular-dynamics simulations of the protein and from mutants in aqueous solution at different pH values

  6. Nutrient value protein of mutant J3 of agaricus blazei by irradiation

    International Nuclear Information System (INIS)

    Jiang Zhihe; Lin Yong; Xiao Shuxia

    2003-01-01

    The studies of nutrient value protein of mutant J 3 of Agaricus blazei were carried out. The results showed that mutant J 3 had 9 kinds of essential amino acids, the compositions of essential amino acids is 46.0% of total amino acids. The amino acid score is 94.5, the essential amino acid index is 92.4, biological value is 89.0, the ratio coefficient of amino acids is 72.3, the nutrient value is 32.5. The chemical score is 74.18. These results proved that the protein of mutant J 3 had high nutrient value

  7. Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants

    International Nuclear Information System (INIS)

    Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M.; Wehnert, Manfred; Huebner, Stefan

    2009-01-01

    Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associated proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.

  8. Protein Conformational Gating of Enzymatic Activity in Xanthine Oxidoreductase

    Energy Technology Data Exchange (ETDEWEB)

    Ishikita, Hiroshi; Eger, Bryan T.; Okamoto, Ken; Nishino, Takeshi; Pai, Emil F. (Toronto); (Kyoto)

    2012-05-24

    In mammals, xanthine oxidoreductase can exist as xanthine dehydrogenase (XDH) and xanthine oxidase (XO). The two enzymes possess common redox active cofactors, which form an electron transfer (ET) pathway terminated by a flavin cofactor. In spite of identical protein primary structures, the redox potential difference between XDH and XO for the flavin semiquinone/hydroquinone pair (E{sub sq/hq}) is {approx}170 mV, a striking difference. The former greatly prefers NAD{sup +} as ultimate substrate for ET from the iron-sulfur cluster FeS-II via flavin while the latter only accepts dioxygen. In XDH (without NAD{sup +}), however, the redox potential of the electron donor FeS-II is 180 mV higher than that for the acceptor flavin, yielding an energetically uphill ET. On the basis of new 1.65, 2.3, 1.9, and 2.2 {angstrom} resolution crystal structures for XDH, XO, the NAD{sup +}- and NADH-complexed XDH, E{sub sq/hq} were calculated to better understand how the enzyme activates an ET from FeS-II to flavin. The majority of the E{sub sq/hq} difference between XDH and XO originates from a conformational change in the loop at positions 423-433 near the flavin binding site, causing the differences in stability of the semiquinone state. There was no large conformational change observed in response to NAD{sup +} binding at XDH. Instead, the positive charge of the NAD{sup +} ring, deprotonation of Asp429, and capping of the bulk surface of the flavin by the NAD{sup +} molecule all contribute to altering E{sub sq/hq} upon NAD{sup +} binding to XDH.

  9. ISOELECTRIC FOCUSING OF MEMBRANE PROTEINS OF PROBIOTIC B. COAGULANS AND ITS BACTERIOPHAGE RESISTANT MUTANTS

    Directory of Open Access Journals (Sweden)

    Kavita Rajesh Pandey

    2016-09-01

    Full Text Available Bacteriophages are the most notorious type of infection in the probiotic and dairy fermentations. Two phage resistant mutants viz. B. co PIII and B. co MIII (B. coagulans mutants PIII and MIII obtained in previous studies (Dubey and Vakil, 2010, were further characterized for their protein profile in comparison with the parental probiotic strain –B. coagulans. The cell lysates were subjected to ultra-centrifugation and the purified membrane fractions were resolved using 2D gel electrophoresis. The Isoelectric focussing showed 187, 202 and 154 protein spots for the parental strain, mutant B. co PIII and mutant B. co MIII, respectively. Ten and 18 protein spots were missing as compared to parent for mutants B.co PIII and B.co MIII whereas there were 21 and 14 new spots noticed for these two mutants. Eight membrane proteins present only in the phage sensitive parental culture could be tentatively identified by comparison with the complete proteome of B. coagulans by use of UniprotKB and then CELLO database It is quite likely that some of these identified membrane proteins may be also functioning as receptors for phage adsorption followed by entry of nucleic acid into the phage sensitive host cell.

  10. Enteral Tube Feeding Nutritional Protein Hydrolysate Production Under Different Factors By Enzymatic Hydrolysis

    Directory of Open Access Journals (Sweden)

    Nguyen ThiQuynhHoa

    2015-01-01

    Full Text Available Abstract Hydrolysis of proteins involves the cleavage of peptide bonds to give peptides of varying sizes and amino acid composition. There are a number of types of hydrolysis enzymatic acid or alkali hydrolysis. Chemical hydrolysis is difficult to control and reduces the nutritional quality of products destroying L-form amino acids and producing toxic substances such as lysino-alanine. Enzymatic hydrolysis works without destructing amino acids and by avoiding the extreme temperatures and pH levels required for chemical hydrolysis the nutritional properties of the protein hydrolysates remain largely unaffected. In this research we investigate the fat removal and protein hydrolysis from pork meat to produce the enteral tube feeding nutritional protein hydrolysate for patient. Our results are as follows meat moisture 75.1 protein 22.6 lipid 1.71 ash 0.5 vitamin B1 1.384mg100g n hexantreatment at 80oCin 45 minutes and drying 30 minutes in 90oC.Viscosity of the hydrolysate is very low 2.240 0.092 cPand high degree of hydrolysis 31.390 0.138 . The final protein powder has balance nutritional components and acid amines low microorganisms which are safety for human consumption.

  11. Germination-Assisted Enzymatic Hydrolysis Can Improve the Quality of Soybean Protein.

    Science.gov (United States)

    Yang, Hui; Li, Xin; Gao, Jinyan; Tong, Ping; Yang, Anshu; Chen, Hongbing

    2017-08-01

    This study aimed to investigate the effects of combined germination and Alcalase hydrolysis on the quality of soybean protein. Protein profiles, water solubility, foaming and emulsifying properties, thixotropic properties, and in vitro protein digestibility (IVPD) were tested, the chemical score (CS), essential amino acid index (EAAI), and protein efficiency ratio (PER) of soybean protein were also defined. The combined treatment of germination and Alcalase hydrolysis remarkably improved the solubility, emulsification activity index, emulsion stability index, and foaming capacity of soybean protein. Notably, a decrease in foaming stability was detected. The electrophoretic profile showed a weak breakdown of soybean protein during germination. However, a strong breakdown of protein was observed after the hydrolysis with Alcalase. The combined treatment also decreased the CS and EAAI of soybean protein, but only by 18%. Meanwhile, the IVPD and PER of soybean protein were significantly improved. Moreover, the protein of the germinated and hydrolyzed soybean flour demonstrated better swallowing properties. These findings indicated that the combined treatment of germination and enzymatic hydrolysis can improve the quality of soybean protein. © 2017 Institute of Food Technologists®.

  12. Protein assembly onto patterned microfabricated devices through enzymatic activation of fusion pro-tag.

    Science.gov (United States)

    Lewandowski, Angela T; Yi, Hyunmin; Luo, Xiaolong; Payne, Gregory F; Ghodssi, Reza; Rubloff, Gary W; Bentley, William E

    2008-02-15

    We report a versatile approach for covalent surface-assembly of proteins onto selected electrode patterns of pre-fabricated devices. Our approach is based on electro-assembly of the aminopolysaccharide chitosan scaffold as a stable thin film onto patterned conductive surfaces of the device, which is followed by covalent assembly of the target protein onto the scaffold surface upon enzymatic activation of the protein's "pro-tag." For our demonstration, the model target protein is green fluorescent protein (GFP) genetically fused with a pentatyrosine pro-tag at its C-terminus, which assembles onto both two-dimensional chips and within fully packaged microfluidic devices in situ and under flow. Our surface-assembly approach enables spatial selectivity and orientational control under mild experimental conditions. We believe that our integrated approach harnessing genetic manipulation, in situ enzymatic activation, and electro-assembly makes it advantageous for a wide variety of bioMEMS and biosensing applications that require facile "biofunctionalization" of microfabricated devices. (c) 2007 Wiley Periodicals, Inc.

  13. The dual effects of Maillard reaction and enzymatic hydrolysis on the antioxidant activity of milk proteins.

    Science.gov (United States)

    Oh, N S; Lee, H A; Lee, J Y; Joung, J Y; Lee, K B; Kim, Y; Lee, K W; Kim, S H

    2013-08-01

    The objective of this study was to determine the enhanced effects on the biological characteristics and antioxidant activity of milk proteins by the combination of the Maillard reaction and enzymatic hydrolysis. Maillard reaction products were obtained from milk protein preparations, such as whey protein concentrates and sodium caseinate with lactose, by heating at 55°C for 7 d in sodium phosphate buffer (pH 7.4). The Maillard reaction products, along with untreated milk proteins as controls, were hydrolyzed for 0 to 3h with commercial proteases Alcalase, Neutrase, Protamex, and Flavorzyme (Novozymes, Bagsværd, Denmark). The antioxidant activity of hydrolyzed Maillard reaction products was determined by reaction with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, their 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, and the ability to reduce ferric ions. Further characteristics were evaluated by the o-phthaldialdehyde method and sodium dodecyl sulfate-PAGE. The degree of hydrolysis gradually increased in a time-dependent manner, with the Alcalase-treated Maillard reaction products being the most highly hydrolyzed. Radical scavenging activities and reducing ability of hydrolyzed Maillard reaction products increased with increasing hydrolysis time. The combined products of enzymatic hydrolysis and Maillard reaction showed significantly greater antioxidant activity than did hydrolysates or Maillard reaction products alone. The hydrolyzed Maillard reaction products generated by Alcalase showed significantly higher antioxidant activity when compared with the other protease products and the antioxidant activity was higher for the whey protein concentrate groups than for the sodium caseinate groups. These findings indicate that Maillard reaction products, coupled with enzymatic hydrolysis, could act as potential antioxidants in the pharmaceutical, food, and dairy industries. Copyright © 2013 American Dairy Science Association

  14. Induction of mutants with higher protein content in soy bean

    International Nuclear Information System (INIS)

    Hiraiwa, S.; Tanaka, S.; Nakamura, S.

    1976-01-01

    Two experiments were carried out to examine the effects of ionizing radiation on protein content of soy beans and to estimate the potential value of ionizing radiation in soy bean breeding. In the first experiment seeds of three cultivars were acutely irradiated by gamma ray doses of 8 krad and 16 krad. Seeds of M 3 plants were analysed for protein content by the Biuret method. Selection within each variety was made to isolate plants having about 2% higher protein content than the control populations. In the second experiment growing plants of a soy bean cultivar were irradiated with 2.9 krad and 6.1 krad, from germination to maturation, in a 60 Co gamma field. Seeds on individual lines in M 5 , M 6 and M 7 plants were determined for their protein contents by the Kjeldahl method and the upper 10% of the treated lines were selected for progeny testing. From the results of the analysis of variance for each trait, significant differences were observed between the control variety and selected lines for all the traits. Six out of 19 lines showed significant increases in both 100 seed weight and protein content, and four lines promising. The mean protein content of selected lines increased 0.68% compared with the control variety. Genetic gain expected from selection for high protein content was therefore considered at about 1%; however, the likelihood of a large increase in protein content would greatly increase if large populations derived from irradiated seeds were observed

  15. Functional analysis of Burkitt's lymphoma mutant c-Myc proteins

    NARCIS (Netherlands)

    Smith-Sørensen, B.; Hijmans, E.M.; Bernards, R.A.

    1996-01-01

    The c-myc gene encodes a sequence-specific DNA binding protein that activates transcription of cellular genes. Transcription activation by Myc proteins is regulated by phosphorylation of serine and threonine residues within the transactivation domain and by complex formation with the

  16. Intracellular localization and movement phenotypes of alfalfa mosaic virus movement protein mutants

    NARCIS (Netherlands)

    Huang, M.; Jongejan, L.; Zheng, H.; Zhang, L.; Bol, J. F.

    2001-01-01

    Thirteen mutations were introduced in the movement protein (MP) gene of Alfalfa mosaic virus (AMV) fused to the green fluorescent protein (GFP) gene and the mutant MP-GFP fusions were expressed transiently in tobacco protoplasts, tobacco suspension cells, and epidermal cells of tobacco leaves. In

  17. Supplementing with non-glycoside hydrolase proteins enhances enzymatic deconstruction of plant biomass.

    Science.gov (United States)

    Su, Xiaoyun; Zhang, Jing; Mackie, Roderick I; Cann, Isaac K O

    2012-01-01

    The glycoside hydrolases (GH) of Caldicellulosiruptor bescii are thermophilic enzymes, and therefore they can hydrolyze plant cell wall polysaccharides at high temperatures. Analyses of two C. bescii glycoside hydrolases, CbCelA-TM1 and CbXyn10A with cellulase and endoxylanase activity, respectively, demonstrated that each enzyme is highly thermostable under static incubation at 70°C. Both enzymes, however, rapidly lost their enzymatic activities when incubated at 70°C with end-over-end shaking. Since crowding conditions, even at low protein concentrations, seem to influence enzymatic properties, three non-glycoside hydrolase proteins were tested for their capacity to stabilize the thermophilic proteins at high temperatures. The three proteins investigated were a small heat shock protein CbHsp18 from C. bescii, a histone MkHistone1 from Methanopyrus kandleri, and bovine RNase A, from a commercial source. Fascinatingly, each of these proteins increased the thermostability of the glycoside hydrolases at 70°C during end-over-end shaking incubation, and this property translated into increases in hydrolysis of several substrates including the bioenergy feedstock Miscanthus. Furthermore, MkHistone1 and RNase A also altered the initial products released from the cello-oligosaccharide cellopentaose during hydrolysis with the cellodextrinase CbCdx1A, which further demonstrated the capacity of the three non-GH proteins to influence hydrolysis of substrates by the thermophilic glycoside hydrolases. The non-GH proteins used in the present report were small proteins derived from each of the three lineages of life, and therefore expand the space from which different polypeptides can be tested for their influence on plant cell wall hydrolysis, a critical step in the emerging biofuel industry.

  18. Supplementing with non-glycoside hydrolase proteins enhances enzymatic deconstruction of plant biomass.

    Directory of Open Access Journals (Sweden)

    Xiaoyun Su

    Full Text Available The glycoside hydrolases (GH of Caldicellulosiruptor bescii are thermophilic enzymes, and therefore they can hydrolyze plant cell wall polysaccharides at high temperatures. Analyses of two C. bescii glycoside hydrolases, CbCelA-TM1 and CbXyn10A with cellulase and endoxylanase activity, respectively, demonstrated that each enzyme is highly thermostable under static incubation at 70°C. Both enzymes, however, rapidly lost their enzymatic activities when incubated at 70°C with end-over-end shaking. Since crowding conditions, even at low protein concentrations, seem to influence enzymatic properties, three non-glycoside hydrolase proteins were tested for their capacity to stabilize the thermophilic proteins at high temperatures. The three proteins investigated were a small heat shock protein CbHsp18 from C. bescii, a histone MkHistone1 from Methanopyrus kandleri, and bovine RNase A, from a commercial source. Fascinatingly, each of these proteins increased the thermostability of the glycoside hydrolases at 70°C during end-over-end shaking incubation, and this property translated into increases in hydrolysis of several substrates including the bioenergy feedstock Miscanthus. Furthermore, MkHistone1 and RNase A also altered the initial products released from the cello-oligosaccharide cellopentaose during hydrolysis with the cellodextrinase CbCdx1A, which further demonstrated the capacity of the three non-GH proteins to influence hydrolysis of substrates by the thermophilic glycoside hydrolases. The non-GH proteins used in the present report were small proteins derived from each of the three lineages of life, and therefore expand the space from which different polypeptides can be tested for their influence on plant cell wall hydrolysis, a critical step in the emerging biofuel industry.

  19. Proteomic profiling of barley spent grains guides enzymatic solubilization of the remaining proteins.

    Science.gov (United States)

    Bi, Xuezhi; Ye, Lijuan; Lau, Ally; Kok, Yee Jiun; Zheng, Lu; Ng, Daniel; Tan, Kelly; Ow, Dave; Ananta, Edwin; Vafiadi, Christina; Muller, Jeroen

    2018-03-17

    Within the brewing industry, malted barley is being increasingly replaced by raw barley supplemented with exogenous enzymes to lessen reliance on the time-consuming, high water and energy cost of malting. Regardless of the initial grain of choice, malted or raw, the resultant bulk spent grains are rich in proteins (up to 25% dry weight). Efficient enzymatic solubilization of these proteins requires knowledge of the protein composition within the spent grains. Therefore, a comprehensive proteomic profiling was performed on spent grains derived from (i) malted barley (spent grain A, SGA) and (ii) enzymatically treated raw barley (spent grain B, SGB); data are available via ProteomeXchange with identifier PXD008090. Results from complementary shotgun proteomics and 2D gel electrophoresis showed that the most abundant proteins in both spent grains were storage proteins (hordeins and embryo globulins); these were present at an average of two fold higher in spent grain B. Quantities of other major proteins were generally consistent in both spent grains A and B. Subsequent in silico protein sequence analysis of the predominant proteins facilitated knowledge-based protease selection to enhance spent grain solubilization. Among tested proteases, Alcalase 2.4 L digestion resulted in the highest remaining protein solubilization with 9.2 and 11.7% net dry weight loss in SGA and SGB respectively within 2 h. Thus, Alcalase alone can significantly reduce spent grain side stream, which makes it a possible solution to increase the value of this low-value side stream from the brewing and malt extract beverage manufacturing industry.

  20. Evaluation of Enzymatically Modified Soy Protein Isolate Film Forming Solution and Film at Different Manufacturing Conditions.

    Science.gov (United States)

    Mohammad Zadeh, Elham; O'Keefe, Sean F; Kim, Young-Teck; Cho, Jin-Hun

    2018-04-01

    The effects of transglutaminase on soy protein isolate (SPI) film forming solution and films were investigated by rheological behavior and physicochemical properties based on different manufacturing conditions (enzyme treatments, enzyme incubation times, and protein denaturation temperatures). Enzymatic crosslinking reaction and changes in molecular weight distribution were confirmed by viscosity measurement and SDS-PAGE, respectively, compared to 2 controls: the nonenzyme treated and the deactivated enzyme treated. Films treated with both the enzyme and the deactivated enzyme showed significant increase in tensile strength (TS), percent elongation (%E), and initial contact angle of films compared to the nonenzyme control film due to the bulk stabilizers in the commercial enzyme. Water absorption property, protein solubility, Fourier transform infrared (FTIR) and X-ray diffraction (XRD) spectroscopy revealed that enzyme treated SPI film matrix in the molecular structure level, resulted in the changes in physicochemical properties. Based on our observation, the enzymatic treatment at appropriate conditions is a practical and feasible way to control the physical properties of protein based biopolymeric film for many different scientific and industrial areas. Enzymes can make bridges selectively among different amino acids in the structure of protein matrix. Therefore, protein network is changed after enzyme treatment. The behavior of biopolymeric materials is dependent on the network structure to be suitable in different applications such as bioplastics applied in food and pharmaceutical products. In the current research, transglutaminase, as an enzyme, applied in soy protein matrix in different types of forms, activated and deactivated, and different preparation conditions to investigate its effects on different properties of the new bioplastic film. © 2018 Institute of Food Technologists®.

  1. Induced protein polymorphisms and nutritional quality of gamma irradiation mutants of sorghum

    International Nuclear Information System (INIS)

    Mehlo, Luke; Mbambo, Zodwa; Bado, Souleymane; Lin, Johnson; Moagi, Sydwell M.; Buthelezi, Sindisiwe; Stoychev, Stoyan; Chikwamba, Rachel

    2013-01-01

    Highlights: • We analyse kafirin protein polymorphisms induced by gamma irradiation in sorghum. • One mutant with suppressed kafirins in the endosperm accumulated them in the germ. • Kafirin polymorphisms were associated with high levels of free amino acids. • Nutritional value of sorghum can be improved significantly by induced mutations. - Abstract: Physical and biochemical analysis of protein polymorphisms in seed storage proteins of a mutant population of sorghum revealed a mutant with redirected accumulation of kafirin proteins in the germ. The change in storage proteins was accompanied by an unusually high level accumulation of free lysine and other essential amino acids in the endosperm. This mutant further displayed a significant suppression in the synthesis and accumulation of the 27 kDa γ-, 24 kDa α-A1 and the 22 kDa α-A2 kafirins in the endosperm. The suppression of kafirins was counteracted by an upsurge in the synthesis and accumulation of albumins, globulins and other proteins. The data collectively suggest that sorghum has huge genetic potential for nutritional biofortification and that induced mutations can be used as an effective tool in achieving premium nutrition in staple cereals

  2. Accumulation of the PX domain mutant Frank-ter Haar syndrome protein Tks4 in aggresomes.

    Science.gov (United States)

    Ádám, Csaba; Fekete, Anna; Bőgel, Gábor; Németh, Zsuzsanna; Tőkési, Natália; Ovádi, Judit; Liliom, Károly; Pesti, Szabolcs; Geiszt, Miklós; Buday, László

    2015-07-17

    Cells deploy quality control mechanisms to remove damaged or misfolded proteins. Recently, we have reported that a mutation (R43W) in the Frank-ter Haar syndrome protein Tks4 resulted in aberrant intracellular localization. Here we demonstrate that the accumulation of Tks4(R43W) depends on the intact microtubule network. Detergent-insoluble Tks4 mutant colocalizes with the centrosome and its aggregate is encaged by the intermediate filament protein vimentin. Both the microtubule inhibitor nocodazole and the histone deacetylase inhibitor Trichostatin A inhibit markedly the aggresome formation in cells expressing Tks4(R43W). Finally, pretreatment of cells with the proteasome inhibitor MG132 markedly increases the level of aggresomes formed by Tks4(R43W). Furthermore, two additional mutant Tks4 proteins (Tks4(1-48) or Tks4(1-341)) have been investigated. Whereas the shorter Tks4 mutant, Tks4(1-48), shows no expression at all, the longer Tks4 truncation mutant accumulates in the nuclei of the cells. Our results suggest that misfolded Frank-ter Haar syndrome protein Tks4(R43W) is transported via the microtubule system to the aggresomes. Lack of expression of Tks4(1-48) or aberrant intracellular expressions of Tks4(R43W) and Tks4(1-341) strongly suggest that these mutations result in dysfunctional proteins which are not capable of operating properly, leading to the development of FTHS.

  3. Effects of enzymatic hydrolysis on conformational and functional properties of chickpea protein isolate.

    Science.gov (United States)

    Mokni Ghribi, Abir; Maklouf Gafsi, Ines; Sila, Assaâd; Blecker, Christophe; Danthine, Sabine; Attia, Hamadi; Bougatef, Ali; Besbes, Souhail

    2015-11-15

    The impact of enzymatic hydrolysis by Alcalase on the conformational and functional properties of chickpea protein isolate (CPI) was investigated. The physicochemical, interfacial tension and surface characteristics of CPI and their hydrolysates (CPH) according to the degree of hydrolysis (DH) were also determined. These parameters were then related to the changes in the emulsification activity (EAI) and stability (ESI). The enzymatic hydrolysis was found to improve protein recovery and solubility, leading to a reduction in the molecular weight bands with a concomitant increase in the intensity and appearance of protein bands having apparent molecular mass below 20 kDa. The interfacial tension decreased from ∼ 66.5 mN m(-1) for CPI to ∼ 59.1 m Nm(-1) for CPH. A similar trend was observed for the surface charge which declined from -27.55 mV to -16.4 mV for the CPI and CPH, respectively. These changes were found to have a detrimental effect on the EAI and ESI values. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Effects of enzymatic hydrolysis on the allergenicity of whey protein concentrates.

    Science.gov (United States)

    Duan, Cuicui; Yang, Lijie; Li, Aili; Zhao, Rui; Huo, Guicheng

    2014-08-01

    Cow's milk whey consists of many protein components and some of them are antigens to human and known to modulate immune responses. Enzymatic hydrolysis is a useful method to modify proteins with allergenicity. The objective of this study was to identify whether the in vitro enzymatic hydrolysis could reduce the allergenicity of whey protein concentrates (WPC). In this study, WPC were hydrolyzed by trypsin and twenty-four BALB/c mice were divided into three groups and fed with WPC formula and WPC hydrolysates formula, while the control mice received milk-free diet. The results revealed that there was no significant difference between the body weights among all groups. WPC-fed mice produced an elevated spleen lymphocyte proliferation level than WPC hydrolysates-fed mice and also produced higher levels of WPC-specific IgE in intestinal tract and serum in comparison to WPC hydrolysates-fed mice and control group. Significant up-regulation of plasma histamine levels were also observed and showed the same trend with IgE. The secretions of IL-4 and IL-5 were significantly enhanced by WPC. WPC significantly suppressed the secretion of IFN-γ while hydrolysates of WPC significantly increased the secretion of IFN-γ compared to control group. These results suggest that hydrolysis may play a role to reduce the allergenicity of WPC.

  5. Production and characterization of cowpea protein hydrolysate with optimum nitrogen solubility by enzymatic hydrolysis using pepsin.

    Science.gov (United States)

    Mune Mune, Martin Alain; Minka, Samuel René

    2017-06-01

    Cowpea is a source of low-cost and good nutritional quality protein for utilization in food formulations in replacement of animal proteins. Therefore it is necessary that cowpea protein exhibits good functionality, particularly protein solubility which affects the other functional properties. The objective of this study was to produce cowpea protein hydrolysate exhibiting optimum solubility by the adequate combination of hydrolysis parameters, namely time, solid/liquid ratio (SLR) and enzyme/substrate ratio (ESR), and to determine its functional properties and molecular characteristics. A Box-Behnken experimental design was used for the experiments, and a second-order polynomial to model the effects of hydrolysis time, SLR and ESR on the degree of hydrolysis and nitrogen solubility index. The optimum hydrolysis conditions of time 208.61 min, SLR 1/15 (w/w) and ESR 2.25% (w/w) yielded a nitrogen solubility of 75.71%. Protein breakdown and the peptide profile following enzymatic hydrolysis were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and size exclusion chromatography. Cowpea protein hydrolysate showed higher oil absorption capacity, emulsifying activity and foaming ability compared with the concentrate. The solubility of cowpea protein hydrolysate was adequately optimized by response surface methodology, and the hydrolysate showed adequate functionality for use in food. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  6. Characterization of Treponema denticola mutants defective in the major antigenic proteins, Msp and TmpC.

    Science.gov (United States)

    Abiko, Yuki; Nagano, Keiji; Yoshida, Yasuo; Yoshimura, Fuminobu

    2014-01-01

    Treponema denticola, a gram-negative and anaerobic spirochete, is associated with advancing severity of chronic periodontitis. In this study, we confirmed that two major antigenic proteins were Msp and TmpC, and examined their physiological and pathological roles using gene-deletion mutants. Msp formed a large complex that localized to the outer membrane, while TmpC existed as a monomer and largely localized to the inner membrane. However, TmpC was also detected in the outer membrane fraction, but its cell-surface exposure was not detected. Msp defects increased cell-surface hydrophobicity and secretion of TNF-α from macrophage-like cells, whereas TmpC defects decreased autoagglutination and chymotrypsin-like protease activities. Both mutants adhered to gingival epithelial cells similarly to the wild-type and showed slightly decreased motility. In addition, in Msp-defective mutants, the TDE1072 protein, which is a major membrane protein, was abolished; therefore, phenotypic changes in the mutant can be, at least in part, attributed to the loss of the TDE1072 protein. Thus, the major antigenic proteins, Msp and TmpC, have significant and diverse impacts on the characteristics of T. denticola, especially cell surface properties.

  7. Effects of sonication and high-pressure carbon dioxide processing on enzymatic hydrolysis of egg white proteins

    Directory of Open Access Journals (Sweden)

    Knežević-Jugović Zorica D.

    2012-01-01

    Full Text Available The objectives of this study were to examine the effect of sonication and high-pressure carbon dioxide processing on proteolytic hydrolysis of egg white proteins and antioxidant activity of the obtained hydrolysates. It appeared that the ultrasound pretreatment resulted in an increase in the degree of hydrolysis of the enzymatic reaction while the high-pressure carbon dioxide processing showed an inhibition effect on the enzymatic hydrolysis of egg white proteins to some extent. The antioxidant activity of the obtained hydrolysates was improved by ultrasound pretreatment of egg white proteins at the pH 8.3. Thus, the combination of ultrasound pretreatment at the pH 8.3 and subsequent enzymatic hydrolysis with alcalase at 50°C and pH 8.0 could offer a new approach to the improvement of the functional properties of egg white proteins and their biological activity. [Projekat Ministarstva nauke Republike Srbije, br. E!6750

  8. Effects of surface proteins and lipids on molecular structure, thermal properties, and enzymatic hydrolysis of rice starch

    Directory of Open Access Journals (Sweden)

    Pan HU

    Full Text Available Abstract Rice starches with different amylose contents were treated with sodium dodecyl sulfate (SDS to deplete surface proteins and lipids, and the changes in molecular structure, thermal properties, and enzymatic hydrolysis were evaluated. SDS treatment did not significantly change the molecular weight distribution, crystalline structure, short-range ordered degree, and gelatinization properties of starch, but significantly altered the pasting properties and increased the swelling power of starch. The removal of surface proteins and lipids increased the enzymatic hydrolysis and in vitro digestion of starch. The influences of removing surface proteins and lipids from starch on swelling power, pasting properties, and enzymatic hydrolysis were different among the various starches because of the differences in molecular structures of different starch styles. The aforementioned results indicated that removing the surface proteins and lipids from starch did not change the molecular structure but had significant effects on some functional properties.

  9. Standardization and quality control in quantifying non-enzymatic oxidative protein modifications in relation to ageing and disease

    DEFF Research Database (Denmark)

    Nedić, Olgica; Rogowska-Wrzesinska, Adelina; Rattan, Suresh I S

    2015-01-01

    Post-translational modifications (PTM) of proteins determine the activity, stability, specificity, transportability and lifespan of a protein. Some PTM are highly specific and regulated involving various enzymatic pathways, but there are other non-enzymatic PTM (nePTM), which occur stochastically...... being fully active to totally inactive molecules. Determination of the type and the amount (abundance) of nePTM is essential for establishing connection between specific protein structure and specific biological role. This article summarizes analytical demands for reliable quantification of ne......PTM, including requirements for the assay performance, standardization and quality control, and points to the difficulties, uncertainties and un-resolved issues....

  10. Destabilizing protein polymorphisms in the genetic background direct phenotypic expression of mutant SOD1 toxicity.

    Directory of Open Access Journals (Sweden)

    Tali Gidalevitz

    2009-03-01

    Full Text Available Genetic background exerts a strong modulatory effect on the toxicity of aggregation-prone proteins in conformational diseases. In addition to influencing the misfolding and aggregation behavior of the mutant proteins, polymorphisms in putative modifier genes may affect the molecular processes leading to the disease phenotype. Mutations in SOD1 in a subset of familial amyotrophic lateral sclerosis (ALS cases confer dominant but clinically variable toxicity, thought to be mediated by misfolding and aggregation of mutant SOD1 protein. While the mechanism of toxicity remains unknown, both the nature of the SOD1 mutation and the genetic background in which it is expressed appear important. To address this, we established a Caenorhabditis elegans model to systematically examine the aggregation behavior and genetic interactions of mutant forms of SOD1. Expression of three structurally distinct SOD1 mutants in C. elegans muscle cells resulted in the appearance of heterogeneous populations of aggregates and was associated with only mild cellular dysfunction. However, introduction of destabilizing temperature-sensitive mutations into the genetic background strongly enhanced the toxicity of SOD1 mutants, resulting in exposure of several deleterious phenotypes at permissive conditions in a manner dependent on the specific SOD1 mutation. The nature of the observed phenotype was dependent on the temperature-sensitive mutation present, while its penetrance reflected the specific combination of temperature-sensitive and SOD1 mutations. Thus, the specific toxic phenotypes of conformational disease may not be simply due to misfolding/aggregation toxicity of the causative mutant proteins, but may be defined by their genetic interactions with cellular pathways harboring mildly destabilizing missense alleles.

  11. Induction of high yielding and high protein containing chickpea mutant variety through gamma radiation

    International Nuclear Information System (INIS)

    Hassan, S.; Javed, M.A.; Khan, A.J.; Tariq, M.

    1997-01-01

    Pure seeds of a blight susceptible but high yielding chickpea variety 6153 were irradiated at 20 Kr(0.2 kGy) dose of gamma radiation and the mutant line CMN-446-4 was selected in M3 generation on the basis of high yield and disease resistance. After confirmation of its resistance to blight in M4 and M5, the mutant line CMN-446-4 along with other promising chickpea mutants were evaluated in various yield trials at different locations. The mutant line CMN-446-4 was got evaluated in chickpea national uniform yield trial conducted over two locations in the country during 1993-94. The mutant line, on average, ranked 3rd by producing significantly higher yield of 1528 kg/ha as compared to the two checked varieties Punjab-91 and Paidar-91 which yielded 1316 and 1391 kg/ha respectively. The mutant CMN-446-4 has significantly greater percentage of protein content (25.22%) compared to its parental variety having (20.12%). (author)

  12. Soybean bio-refinery platform: enzymatic process for production of soy protein concentrate, soy protein isolate and fermentable sugar syrup.

    Science.gov (United States)

    Loman, Abdullah Al; Islam, S M Mahfuzul; Li, Qian; Ju, Lu-Kwang

    2016-10-01

    Soybean carbohydrate is often found to limit the use of protein in soy flour as food and animal feed due to its indigestibility to monogastric animal. In the current study, an enzymatic process was developed to produce not only soy protein concentrate and soy protein isolate without indigestible carbohydrate but also soluble reducing sugar as potential fermentation feedstock. For increasing protein content in the product and maximizing protein recovery, the process was optimized to include the following steps: hydrolysis of soy flour using an Aspergillus niger enzyme system; separation of the solid and liquid by centrifugation (10 min at 7500×g); an optional step of washing to remove entrapped hydrolysate from the protein-rich wet solid stream by ethanol (at an ethanol-to-wet-solid ratio (v/w) of 10, resulting in a liquid phase of approximately 60 % ethanol); and a final precipitation of residual protein from the sugar-rich liquid stream by heat treatment (30 min at 95 °C). Starting from 100 g soy flour, this process would produce approximately 54 g soy protein concentrate with 70 % protein (or, including the optional solid wash, 43 g with 80 % protein), 9 g soy protein isolate with 89 % protein, and 280 ml syrup of 60 g/l reducing sugar. The amino acid composition of the soy protein concentrate produced was comparable to that of the starting soy flour. Enzymes produced by three fungal species, A. niger, Trichoderma reesei, and Aspergillus aculeatus, were also evaluated for effectiveness to use in this process.

  13. Characterization and enzymatic properties of protein kinase ACR4 from Arabidopsis thaliana.

    Science.gov (United States)

    Zhao, Yu; Liu, Xuehe; Xu, Ziyan; Yang, Hui; Li, Jixi

    2017-07-22

    Serine/threonine-protein kinase-like protein ARABIDOPSIS CRINKLY4 (ACR4), a transmembrane protein of Arabidopsis thaliana, plays important roles in cell division and differentiation. Although accumulating studies shed light on the function of ACR4, the structure and catalytic mechanism of ACR4 remain to be elucidated. Here, we report the purification and enzymatic properties of the intracellular kinase domain (residues 464-799) of ACR4 (ACR4 IKD ). Through Ni-affinity chromatography and gel filter chromatography methods, we successfully obtain high-purity ACR4 IKD protein from Escherichia coli. Dynamic light scattering and gel-filtration methods reveal that ACR4 IKD distributes with high homogeneity and exists as a monomer in solution. In addition, the ACR4 IKD protein has typical kinase activity with myelin basic protein (MBP) as the substrate. Our study may lay the foundation for structure determination of ACR4 IKD and further functional research, for example, screening significant substrates of ACR4 in Arabidopsis thaliana. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Selective processing and metabolism of disease-causing mutant prion proteins.

    Directory of Open Access Journals (Sweden)

    Aarthi Ashok

    2009-06-01

    Full Text Available Prion diseases are fatal neurodegenerative disorders caused by aberrant metabolism of the cellular prion protein (PrP(C. In genetic forms of these diseases, mutations in the globular C-terminal domain are hypothesized to favor the spontaneous generation of misfolded PrP conformers (including the transmissible PrP(Sc form that trigger downstream pathways leading to neuronal death. A mechanistic understanding of these diseases therefore requires knowledge of the quality control pathways that recognize and degrade aberrant PrPs. Here, we present comparative analyses of the biosynthesis, trafficking, and metabolism of a panel of genetic disease-causing prion protein mutants in the C-terminal domain. Using quantitative imaging and biochemistry, we identify a misfolded subpopulation of each mutant PrP characterized by relative detergent insolubility, inaccessibility to the cell surface, and incomplete glycan modifications. The misfolded populations of mutant PrPs were neither recognized by ER quality control pathways nor routed to ER-associated degradation despite demonstrable misfolding in the ER. Instead, mutant PrPs trafficked to the Golgi, from where the misfolded subpopulation was selectively trafficked for degradation in acidic compartments. Surprisingly, selective re-routing was dependent not only on a mutant globular domain, but on an additional lysine-based motif in the highly conserved unstructured N-terminus. These results define a specific trafficking and degradation pathway shared by many disease-causing PrP mutants. As the acidic lysosomal environment has been implicated in facilitating the conversion of PrP(C to PrP(Sc, our identification of a mutant-selective trafficking pathway to this compartment may provide a cell biological basis for spontaneous generation of PrP(Sc in familial prion disease.

  15. Comparative analysis of the protein compositions between wild type and body color mutant of helicoverpa armigera adult

    International Nuclear Information System (INIS)

    He Lihua; Chen Jin'e; Liu Yan; Wang Yongqiang; Liu Peigang; Meng Zhiqi

    2012-01-01

    To gain an in-depth understanding of the fineness and regulation mechanism of body color mutant of Helicoverpa armigera Hbner, the protein composition differences between adult of dominant mutant, recessive mutant and wild type were studied using the SDS-PAGE combined with MALDI-TOF-TOF/MS and bioinformatics analysis. The results indicated that the protein composition of the dominant mutant and wild type had little difference. However, there were obvious differences between the recessive mutant and wild-type. Three specific stripe were chosen for mass spectrometry and bioinformatics analysis, and two types of proteins related to energy metabolism and cytoskeleton were identified. These findings suggested that the two types of proteins may be associated with occurrence and regulation of body color mutant traits of H. armigera. (authors)

  16. Towards plant protein refinery: Review on protein extraction using alkalo and potential enzymatic assistance

    NARCIS (Netherlands)

    Sari, Y.W.; Mulder, W.J.; Sanders, J.P.M.; Bruins, M.E.

    2015-01-01

    The globally increasing protein demands require additional resources to those currently available. Furthermore, the optimal usage of protein fractions from both traditional and new protein resources, such as algae and leaves, is essential. Here, we present an overview on alkaline plant protein

  17. The cellular Mre11 protein interferes with adenovirus E4 mutant DNA replication

    International Nuclear Information System (INIS)

    Mathew, Shomita S.; Bridge, Eileen

    2007-01-01

    Adenovirus type 5 (Ad5) relocalizes and degrades the host DNA repair protein Mre11, and efficiently initiates viral DNA replication. Mre11 associates with Ad E4 mutant DNA replication centers and is important for concatenating viral genomes. We have investigated the role of Mre11 in the E4 mutant DNA replication defect. RNAi-mediated knockdown of Mre11 dramatically rescues E4 mutant DNA replication in cells that do or do not concatenate viral genomes, suggesting that Mre11 inhibits DNA replication independent of genome concatenation. The mediator of DNA damage checkpoint 1 (Mdc1) protein is involved in recruiting and sustaining Mre11 at sites of DNA damage following ionizing radiation. We observe foci formation by Mdc1 in response to viral infection, indicating that this damage response protein is activated. However, knockdown of Mdc1 does not prevent Mre11 from localizing at viral DNA replication foci or rescue E4 mutant DNA replication. Our results are consistent with a model in which Mre11 interferes with DNA replication when it is localized at viral DNA replication foci

  18. New non detrimental DNA binding mutants of the Escherichia coli initiator protein DnaA

    DEFF Research Database (Denmark)

    Asklund, Marlene; Atlung, Tove

    2004-01-01

    The initiator protein DnaA has several unique DNA-binding features. It binds with high affinity as a monomer to the nonamer DnaA box. In the ATP form, DnaA binds cooperatively to the low-affinity ATP-DnaA boxes, and to single-stranded DNA in the 13mer region of the origin. We have carried out...... an extensive mutational analysis of the DNA-binding domain of the Escherichia coli DnaA protein using mutagenic PCR. We analyzed mutants exhibiting more or less partial activity by selecting for complementation of a dnaA(Ts) mutant strain at different expression levels of the new mutant proteins. The selection...... gave rise to 30 single amino acid substitutions and, including double substitutions, more than 100 mutants functional in initiation of chromosome replication were characterized. The analysis indicated that all regions of the DNA-binding domain are involved in DNA binding, but the most important amino...

  19. Invitro Synthesis of Barley Endosperm Proteins on Wild Type and Mutant Templates

    DEFF Research Database (Denmark)

    Brandt, A.; Ingversen, J.

    1976-01-01

    Membrane bound and free polyribosomes were isolated from 20 day old barley endosperms. Sucrose gradient analysis revealed distinct polysomal peaks up to heptamers. The isolated polysomes were active in a cell-free protein synthesizing system employing wheat germ extract. SDS-polyacrylamide gel...... electrophoresis showed that proteins with molecular weights ranging from 200,000 to 10,000 daltons were synthesized. A substantial part of the polypeptides coded for by the template associated with the membrane bound polysomes was identified as hordeins by their solubility in 55% isopropanol and by their co......-migration with native hordein on SDS-polyacrylamide gels. Membrane bound endosperm polysomes from a barley mutant defective in hordein synthesis produced in the cell-free protein synthesizing system only a small amount of hordein. Conversely membrane bound polysomes from the endosperm of a mutant giving rise...

  20. Stromal protein degradation is incomplete in Arabidopsis thaliana autophagy mutants undergoing natural senescence

    Directory of Open Access Journals (Sweden)

    Lee Travis A

    2013-01-01

    Full Text Available Abstract Background Degradation of highly abundant stromal proteins plays an important role in the nitrogen economy of the plant during senescence. Lines of evidence supporting proteolysis within the chloroplast and outside the chloroplast have been reported. Two extra-plastidic degradation pathways, chlorophagy and Rubisco Containing Bodies, rely on cytoplasmic autophagy. Results In this work, levels of three stromal proteins (Rubisco large subunit, chloroplast glutamine synthetase and Rubisco activase and one thylakoid protein (the major light harvesting complex protein of photosystem II were measured during natural senescence in WT and in two autophagy T-DNA insertion mutants (atg5 and atg7. Thylakoid-localized protein decreased similarly in all genotypes, but stromal protein degradation was incomplete in the two atg mutants. In addition, degradation of two stromal proteins was observed in chloroplasts isolated from mid-senescence leaves. Conclusions These data suggest that autophagy does contribute to the complete proteolysis of stromal proteins, but does not play a major degenerative role. In addition, support for in organello degradation is provided.

  1. Enzymatic characterisation of the immobilised Alcalase to hydrolyse egg white protein for potential allergenicity reduction.

    Science.gov (United States)

    Yang, Anshu; Long, Caiyun; Xia, Jiaheng; Tong, Ping; Cheng, Youfei; Wang, Yan; Chen, Hongbing

    2017-01-01

    This study examined technique characteristics of the immobilised Alcalase to hydrolyse egg white protein for potential allergenicity reduction. Alcalase was immobilised covalently on carboxyl-functionalised magnetic beads by carbodiimide activation. The technique characteristics of the immobilised Alcalase were investigated, followed by determining the degrees of hydrolysis (DH), immunoglobulin G (IgG) binding, and IgE binding of the digested egg white protein by immobilised Alcalase. Enzymatic activity, enzyme loading, and immobilisation yield of the prepared immobilised Alcalase were 20.55 U mg -1 , 925 mg g -1 , and 45%, respectively. Immobilised Alcalase showed maximum activity at pH 8.0 and 60 °C. Compared with free Alcalase, immobilised Alcalase exhibited better thermal and storage stability. Moreover, immobilised Alcalase can be reused 10 times and still maintained 55% of its initial activity. Partial hydrolysis of egg white protein by immobilised Alcalase can effectively reduce IgG and IgE binding of the hydrolysates. This study indicates that the immobilised Alcalase can be used to hydrolyse continuously egg white protein for potential allergenicity reduction. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  2. 125I-labeled cortisol radioimmunoassay in which serum binding protein are enzymatically denatured

    International Nuclear Information System (INIS)

    Hasler, M.J.; Painter, K.; Niswender, G.D.

    1976-01-01

    We report an iodine-125 radioimmunoassay for cortisol in biological fluids, in which interfering binding proteins are enzymatically denatured. An antiserum to cortisol-3-carboxymethyloxime-bovine serum albumin, extremely low cross-reacting with other corticosteroids, was raised in rabbits. A cortisol-3-carboxymethyloxime tyrosine methyl ester derivative was synthesized and labeled with iodine-125 by standard radioiodination techniques. To eliminate the need for extraction and recovery procedures, we digested interfering binding with a proteolytic enzyme, which then was heat-inactivated before adding the labeled derivative and the premixed, preincubated antiserum complex. There was quantitative analytical recovery of esogenous cortisol added to sera from a normal man, a normal woman, and a pregnant woman. Values for the same samples agreed after extraction and chromatographic purification and agreed well with values obtained by other techniques by independent reference laboratories. The five-step assay can be done in 6 h or less

  3. Enzymatic hydrolysis of heated whey: iron-binding ability of peptides and antigenic protein fractions.

    Science.gov (United States)

    Kim, S B; Seo, I S; Khan, M A; Ki, K S; Lee, W S; Lee, H J; Shin, H S; Kim, H S

    2007-09-01

    This study evaluated the influence of various enzymes on the hydrolysis of whey protein concentrate (WPC) to reduce its antigenic fractions and to quantify the peptides having iron-binding ability in its hydrolysates. Heated (for 10 min at 100 degrees C) WPC (2% protein solution) was incubated with 2% each of Alcalase, Flavourzyme, papain, and trypsin for 30, 60, 90, 120, 150, 180, and 240 min at 50 degrees C. The highest hydrolysis of WPC was observed after 240 min of incubation with Alcalase (12.4%), followed by Flavourzyme (12.0%), trypsin (10.4%), and papain (8.53%). The nonprotein nitrogen contents of WPC hydrolysate followed the hydrolytic pattern of whey. The major antigenic fractions (beta-lactoglobulin) in WPC were degraded within 60 min of its incubation with Alcalase, Flavourzyme, or papain. Chromatograms of enzymatic hydrolysates of heated WPC also indicated complete degradation of beta-lactoglobulin, alpha-lactalbumin, and BSA. The highest iron solubility was noticed in hydrolysates derived with Alcalase (95%), followed by those produced with trypsin (90%), papain (87%), and Flavourzyme (81%). Eluted fraction 1 (F-1) and fraction 2 (F-2) were the respective peaks for the 0.25 and 0.5 M NaCl chromatographic step gradient for analysis of hydrolysates. Iron-binding ability was noticeably higher in F-1 than in F-2 of all hydrolysates of WPC. The highest iron contents in F-1 were observed in WPC hydrolysates derived with Alcalase (0.2 mg/kg), followed by hydrolysates derived with Flavourzyme (0.14 mg/kg), trypsin (0.14 mg/kg), and papain (0.08 mg/kg). Iron concentrations in the F-2 fraction of all enzymatic hydrolysates of WPC were low and ranged from 0.03 to 0.05 mg/kg. Fraction 1 may describe a new class of iron chelates based on the reaction of FeSO4 x 7 H2O with a mixture of peptides obtained by the enzymatic hydrolysis of WPC. The chromatogram of Alcalase F-1 indicated numerous small peaks of shorter wavelengths, which probably indicated a variety of

  4. Preparation, aroma characteristics and volatile compounds of flavorings from enzymatic hydrolyzed rice bran protein concentrate.

    Science.gov (United States)

    Arsa, Supeeraya; Theerakulkait, Chockchai

    2018-02-19

    Rice bran is a by-product obtained from the rice milling industry. The aims of this research were to add value to rice bran by preparation of enzymatic hydrolyzed rice bran protein concentrate (HRPC) as a flavoring agent and the flavoring which was produced by HRPC has not been investigated. Different drying methods (freeze-drying and spray-drying) and fructose additions were studied for improvement of rice bran protein sensorial aroma characteristics. The most abundant amino acids in liquid HRPC (LH) were glutamic acid, arginine, aspartic acid and leucine. The intensity of desirable aromas, such as cereal-like, nut-like, milk-powder-like, sweet, and cocoa-like aroma, were higher in spray-dried HRPC powder (SHP) than in LH and freeze-dried HRPC. Volatile compounds, such as aldehydes, pyrazines and ketones, were significantly increased in HRPC powders in which fructose was added before spray-drying (SHP-F). Higher amounts of 2-methylbutanal, 3-methylbutanal, phenylacetaldehyde, 2,5-dimethylpyrazine, vanillin, 2-acetylpyrrole and maltol were detected in SHP-F. Moreover, these compounds had high odor active values, which accounted for the cocoa-like, sweet, nut-like, and milk-powder-like characteristics of SHP-F. These findings could lead to the creation of desirable aroma characteristics of rice bran protein concentrate by different preparation methods. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

  5. Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity.

    Science.gov (United States)

    Petzold, Christine; Marceau, Aimee H; Miller, Katherine H; Marqusee, Susan; Keck, James L

    2015-06-05

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity*

    Science.gov (United States)

    Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L.

    2015-01-01

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. PMID:25903123

  7. Biochemical characterization of protein composition of high-lysine mutant ML-109 of winter barley (Hordeum vulgare L.)

    International Nuclear Information System (INIS)

    Kapala, A.; Yankoulov, M.

    1994-01-01

    The objective of the present studies was to determine the biochemical basis of a high lysine content in the mutant ML-109. The protein composition of kernels of high-lysine mutant of winter barley and its initial breeding line were analysed. The portion of individual fractions differing by their solubility in total proteins as well as amino acid composition of kernel protein and separated main protein fractions were also determined. The performed studies showed that a high lysine content in the kernels of the analysed mutant results from a decrease in the hordein level and from an increase in the portion of salt soluble proteins - albumins and globulins. Besides that, the obtained results indicate that the amino acid composition of the main protein fractions of the analysed mutant was advantageously changed; these fractions contained more lysine and other exogenic amino acids. (author). 15 refs, 6 tabs

  8. Performance of coupled enzymatic hydrolysis and membrane separation bioreactor for antihypertensive peptides production from Porphyra yezoensis protein

    Science.gov (United States)

    To explore more efficient production methods of antihypertensive peptides from Porphyra yezoensis protein, three methods of coupling of enzymatic hydrolysis and membrane separation (CEH-MS) were studied and compared with the traditional EH and offline MS method. The results showed that the conversio...

  9. Molecular pathogenesis of Spondylocheirodysplastic Ehlers-Danlos syndrome caused by mutant ZIP13 proteins

    Science.gov (United States)

    Bin, Bum-Ho; Hojyo, Shintaro; Hosaka, Toshiaki; Bhin, Jinhyuk; Kano, Hiroki; Miyai, Tomohiro; Ikeda, Mariko; Kimura-Someya, Tomomi; Shirouzu, Mikako; Cho, Eun-Gyung; Fukue, Kazuhisa; Kambe, Taiho; Ohashi, Wakana; Kim, Kyu-Han; Seo, Juyeon; Choi, Dong-Hwa; Nam, Yeon-Ju; Hwang, Daehee; Fukunaka, Ayako; Fujitani, Yoshio; Yokoyama, Shigeyuki; Superti-Furga, Andrea; Ikegawa, Shiro; Lee, Tae Ryong; Fukada, Toshiyuki

    2014-01-01

    The zinc transporter protein ZIP13 plays critical roles in bone, tooth, and connective tissue development, and its dysfunction is responsible for the spondylocheirodysplastic form of Ehlers-Danlos syndrome (SCD-EDS, OMIM 612350). Here, we report the molecular pathogenic mechanism of SCD-EDS caused by two different mutant ZIP13 proteins found in human patients: ZIP13G64D, in which Gly at amino acid position 64 is replaced by Asp, and ZIP13ΔFLA, which contains a deletion of Phe-Leu-Ala. We demonstrated that both the ZIP13G64D and ZIP13ΔFLA protein levels are decreased by degradation via the valosin-containing protein (VCP)-linked ubiquitin proteasome pathway. The inhibition of degradation pathways rescued the protein expression levels, resulting in improved intracellular Zn homeostasis. Our findings uncover the pathogenic mechanisms elicited by mutant ZIP13 proteins. Further elucidation of these degradation processes may lead to novel therapeutic targets for SCD-EDS. PMID:25007800

  10. Theoretical Analysis of Allosteric and Operator Binding for Cyclic-AMP Receptor Protein Mutants

    Science.gov (United States)

    Einav, Tal; Duque, Julia; Phillips, Rob

    2018-02-01

    Allosteric transcription factors undergo binding events both at their inducer binding sites as well as at distinct DNA binding domains, and it is often difficult to disentangle the structural and functional consequences of these two classes of interactions. In this work, we compare the ability of two statistical mechanical models - the Monod-Wyman-Changeux (MWC) and the Koshland-N\\'emethy-Filmer (KNF) models of protein conformational change - to characterize the multi-step activation mechanism of the broadly acting cyclic-AMP receptor protein (CRP). We first consider the allosteric transition resulting from cyclic-AMP binding to CRP, then analyze how CRP binds to its operator, and finally investigate the ability of CRP to activate gene expression. In light of these models, we examine data from a beautiful recent experiment that created a single-chain version of the CRP homodimer, thereby enabling each subunit to be mutated separately. Using this construct, six mutants were created using all possible combinations of the wild type subunit, a D53H mutant subunit, and an S62F mutant subunit. We demonstrate that both the MWC and KNF models can explain the behavior of all six mutants using a small, self-consistent set of parameters. In comparing the results, we find that the MWC model slightly outperforms the KNF model in the quality of its fits, but more importantly the parameters inferred by the MWC model are more in line with structural knowledge of CRP. In addition, we discuss how the conceptual framework developed here for CRP enables us to not merely analyze data retrospectively, but has the predictive power to determine how combinations of mutations will interact, how double mutants will behave, and how each construct would regulate gene expression.

  11. SUMO-2 and PIAS1 Modulate Insoluble Mutant Huntingtin Protein Accumulation

    Directory of Open Access Journals (Sweden)

    Jacqueline Gire O’Rourke

    2013-07-01

    Full Text Available A key feature in Huntington disease (HD is the accumulation of mutant Huntingtin (HTT protein, which may be regulated by posttranslational modifications. Here, we define the primary sites of SUMO modification in the amino-terminal domain of HTT, show modification downstream of this domain, and demonstrate that HTT is modified by the stress-inducible SUMO-2. A systematic study of E3 SUMO ligases demonstrates that PIAS1 is an E3 SUMO ligase for both HTT SUMO-1 and SUMO-2 modification and that reduction of dPIAS in a mutant HTT Drosophila model is protective. SUMO-2 modification regulates accumulation of insoluble HTT in HeLa cells in a manner that mimics proteasome inhibition and can be modulated by overexpression and acute knockdown of PIAS1. Finally, the accumulation of SUMO-2-modified proteins in the insoluble fraction of HD postmortem striata implicates SUMO-2 modification in the age-related pathogenic accumulation of mutant HTT and other cellular proteins that occurs during HD progression.

  12. FTIR Spectroscopy of Protein Isolates of Salt-Tolerant Soybean Mutants

    Science.gov (United States)

    Akyuz, S.; Akyuz, T.; Celik, O.; Atak, C.

    2018-01-01

    The effect of salinity on the conformation of proteins of four salt-tolerant M2 generation mutants of soybean plants (S04-05/150-2, S04-05/150-8, S04-05/150-106, and S04-05/150-114) was investigated using Fourier transform infrared (FTIR) spectroscopy. Salinity is one of the important abiotic stress factors that limits growth and productivity of plants. The mutants belonging to the M2 generation were determined as tolerant to 90 mM NaCl. The relative contents of α-helix, β-sheet, turn, and irregular conformations for the soybean protein isolates were determined depending on the analysis of the amide I region. The comparison of the secondary structures of soybean proteins of the mutants with those of the control group indicated that the α-helix structure percentage was diminished while β-turn and disordered structures were increased as a result of the salt stress.

  13. ProCMD: a database and 3D web resource for protein C mutants

    Directory of Open Access Journals (Sweden)

    D'Ursi Pasqualina

    2007-03-01

    Full Text Available Abstract Background Activated Protein C (ProC is an anticoagulant plasma serine protease which also plays an important role in controlling inflammation and cell proliferation. Several mutations of the gene are associated with phenotypic functional deficiency of protein C, and with the risk of developing venous thrombosis. Structure prediction and computational analysis of the mutants have proven to be a valuable aid in understanding the molecular aspects of clinical thrombophilia. Results We have built a specialized relational database and a search tool for natural mutants of protein C. It contains 195 entries that include 182 missense and 13 stop mutations. A menu driven search engine allows the user to retrieve stored information for each variant, that include genetic as well as structural data and a multiple alignment highlighting the substituted position. Molecular models of variants can be visualized with interactive tools; PDB coordinates of the models are also available for further analysis. Furthermore, an automatic modelling interface allows the user to generate multiple alignments and 3D models of new variants. Conclusion ProCMD is an up-to-date interactive mutant database that integrates phenotypical descriptions with functional and structural data obtained by computational approaches. It will be useful in the research and clinical fields to help elucidate the chain of events leading from a molecular defect to the related disease. It is available for academics at the URL http://www.itb.cnr.it/procmd/.

  14. Protein extraction and enzymatic hydrolysis of ammonia-treated cassava leaves (Manihot esculenta Crantz).

    Science.gov (United States)

    Urribarrí, Lauris; Chacón, David; González, Orlaidy; Ferrer, Alexis

    2009-05-01

    In the present work, cassava leaves were treated with 0.5 kg ammonia/kg dry matter at 78 degrees C and 30% moisture content in a 2-kg reactor. Protein extraction was carried out with a calcium hydroxide solution (pH 10) for 30 min at several temperatures (30 degrees C, 45 degrees C, 60 degrees C, 75 degrees C, and 90 degrees C) and solid/liquid ratios (1:10 and 1:15) in a thermostatized bath. Soluble protein content of the extracts was determined by Lowry's method. Dry substrate concentrations of 5%, 7.5%, and 10% and enzyme doses of 2 and 5 IU/g dry matter were used for the enzymatic hydrolysis in an orbital incubator at 50 degrees C and 100 rpm. Both cellulase and xylanase were used. Reducing sugars produced were determined with the dinitrosalicylic acid method. The highest protein extraction yield for the ammonia-treated leaves was 29.10%, which was 50% higher than with the untreated leaves (20%), and was obtained at 90 degrees C with a 1:10 solid/liquid ratio. The concentrate had a protein content of 36.35% and the amino acid profile was suitable for swine and poultry. The highest sugar yield was 54.72% with respect to theoretical and was obtained with 5% solids and an enzyme dose of 5 IU/g dry matter. This yield was 3.4 times higher than the yield of the untreated leaves (16.13%). These results indicate that cassava leaves have a great potential for animal feeding and ethanol production. Both protein extraction and sugar yields may be enhanced by optimizing the ammonia treatment.

  15. The G46S-hPAH mutant protein: a model to study the rescue of aggregation-prone PKU mutations by chaperones.

    Science.gov (United States)

    Leandro, João; Saraste, Jaakko; Leandro, Paula; Flatmark, Torgeir

    2011-01-01

    Phenylketonuria (PKU), the most common inborn error of metabolism, is caused by dysfunction of the liver enzyme phenylalanine hydroxylase (PAH), with more than 550 PAH gene mutations identified to date. A large number of these mutations result in mutant forms of the enzyme displaying reduced stability, increased propensity to aggregate, and accelerated in cellulo degradation. Loss or reduction of human PAH activity results in hyperphenylalaninemia (HPA) which, if untreated, results in severe mental retardation and impaired cognitive development. Until now, strict low phenylalanine diet has been the most effective therapy, but as a protein misfolding disease PKU is a good candidate for treatment by natural/chemical/pharmacological chaperones. The natural cofactor of human PAH, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)), has already been approved for oral treatment of HPA, giving a positive response in mild forms of the disease showing considerable residual enzymatic activity. In the case of the most severe forms of PKU, ongoing studies with chemical and pharmacological chaperones to rescue misfolded mutant proteins from aggregation and degradation are providing promising results. The PKU mutation G46S is associated with a severe form of the disease, resulting in an aggregation-prone protein. The human PAH mutant G46S is rapidly degraded in the cellular environment and, in vitro (upon removal of its stabilizing fusion partner maltose binding protein (MBP)) self-associates to form higher-order oligomers/fibrils. Here, we present an in vitro experimental model system to study the modulation of G46S aggregation by chemical/pharmacological chaperones, which may represent a useful approach to study the rescue of other severe PKU mutations by chemical/pharmacological chaperones. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. Resveratrol Modulation of Protein Expression in parkin-Mutant Human Skin Fibroblasts: A Proteomic Approach

    Science.gov (United States)

    Gaballo, Antonio; Signorile, Anna; Tanzarella, Paola; Pacelli, Consiglia; Di Paola, Marco

    2017-01-01

    In this study, we investigated by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) analysis the effects of resveratrol treatment on skin primary fibroblasts from a healthy subject and from a parkin-mutant early onset Parkinson's disease patient. Parkin, an E3 ubiquitin ligase, is the most frequently mutated gene in hereditary Parkinson's disease. Functional alteration of parkin leads to impairment of the ubiquitin-proteasome system, resulting in the accumulation of misfolded or aggregated proteins accountable for the neurodegenerative process. The identification of proteins differentially expressed revealed that resveratrol treatment can act on deregulated specific biological process and molecular function such as cellular redox balance and protein homeostasis. In particular, resveratrol was highly effective at restoring the heat-shock protein network and the protein degradation systems. Moreover, resveratrol treatment led to a significant increase in GSH level, reduction of GSSG/GSH ratio, and decrease of reduced free thiol content in patient cells compared to normal fibroblasts. Thus, our findings provide an experimental evidence of the beneficial effects by which resveratrol could contribute to preserve the cellular homeostasis in parkin-mutant fibroblasts. PMID:29138676

  17. Resveratrol Modulation of Protein Expression in parkin-Mutant Human Skin Fibroblasts: A Proteomic Approach

    Directory of Open Access Journals (Sweden)

    Daniele Vergara

    2017-01-01

    Full Text Available In this study, we investigated by two-dimensional gel electrophoresis (2-DE and mass spectrometry (MS analysis the effects of resveratrol treatment on skin primary fibroblasts from a healthy subject and from a parkin-mutant early onset Parkinson’s disease patient. Parkin, an E3 ubiquitin ligase, is the most frequently mutated gene in hereditary Parkinson’s disease. Functional alteration of parkin leads to impairment of the ubiquitin-proteasome system, resulting in the accumulation of misfolded or aggregated proteins accountable for the neurodegenerative process. The identification of proteins differentially expressed revealed that resveratrol treatment can act on deregulated specific biological process and molecular function such as cellular redox balance and protein homeostasis. In particular, resveratrol was highly effective at restoring the heat-shock protein network and the protein degradation systems. Moreover, resveratrol treatment led to a significant increase in GSH level, reduction of GSSG/GSH ratio, and decrease of reduced free thiol content in patient cells compared to normal fibroblasts. Thus, our findings provide an experimental evidence of the beneficial effects by which resveratrol could contribute to preserve the cellular homeostasis in parkin-mutant fibroblasts.

  18. A New Bacillus licheniformis Mutant Strain Producing Serine Protease Efficient for Hvdrolvqis of Sov Meal Proteins.

    Science.gov (United States)

    Kostyleva, E V; Sereda, A S; Velikoretskaya, I A; Nefedova, L I; Sharikov, A Yu; Tsurikova, N V; Lobanov, N S; Semenova, M V; Sinitsyn, A P

    2016-07-01

    Induced mutagenesis with y-irradiation of the industrial strain Bacillus licheniformis-60 VKM B-2366,D was used to obtain a new highly active producer of an extracellular serine protease, Bacillus licheni- formis7 145. Samples of dry.concentrated preparations of serine protease produced by the original and mutant strains were obtained, and identity of their protein composition was'established. Alkaline serine protease sub- tilisin DY was the main component of the preparations. The biochemical and physicochemical properties of the Protolkheterm-145 enzyme preparation obtained from the mutant strain were studied. It exhibited pro- teolytic activity (1.5 times higher than the preparation from the initial strain) within broad ranges of pH (5- 11) and temperature (30-70'C).-Efficient hydrolysis of extruded soy meal protein at high concentrations (2 to 50%) in-the reaction mixture was.the main advantage of the Protolikheterm 145 preparation. Compared to,. the preparation obtained using the initial strain, the new preparation with increased proteolytic-activity pro- vided for more complete hydrolysis of the main non-nutritious soy,proteins.(glycinin and 0-conglycinin) with the yield of soluble protein increased by 19-28%, which decreased the cost of bioconversion of the protein- aceous material and indicated promise of the new preparation in resource-saving technologies for processing soy meals and cakes.

  19. Genotypic variability and mutant identification in cicer arietinum L. by seed storage protein profiling

    International Nuclear Information System (INIS)

    Hameed, A.; Iqbal, N.; Shah, T.M.

    2012-01-01

    A collection of thirty-four chickpea genotypes, including five kabuli and twenty-nine desi, were analyzed by SDS-PAGE for seed storage protein profiling. Total soluble seed proteins were resolved on 12% gels. A low level of variability was observed in desi as compared to kabuli genotypes. Dendrogram based on electrophoretic data clustered the thirty-four genotypes in four major groups. As large number of desi genotypes illustrated identical profiles, therefore could not be differentiated on the basis of seed storage protein profiles. One kabuli genotype ILC-195 found to be the most divergent showing 86% similarity with all other genotypes. ILC-195 can be distinguished from its mutant i.e., CM-2000 and other kabuli genotypes on the basis of three peptides i.e. SSP-66, SSP-43 and SSP-39. Some proteins peptides were found to be genotype specific like SSP-26 for ICCV-92311. Uniprot and NCBI protein databases were searched for already reported and characterized seed storage proteins in chickpea. Among 33 observed peptides, only six seed storages proteins from chickpea source were available in databases. On the basis of molecular weight similarity, identified peptides were SSP-64 as Serine/Threonine dehydratase, SSP-56 as Alpha-amylase inhibitor, SSP-50 as Provicillin, SSP-39 as seed imbibition protein, SSP-35 as Isoflavane reductase and SSP-19 as lipid transport protein. Highest variability was observed in vicillin subunits and beta subunits of legumins and its polymorphic forms. In conclusion, seed storage profiling can be economically used to asses the genetic variation, phylogenetic relationship and as markers to differentiate mutants from their parents. (author)

  20. Enzymatic Properties of Populus α- and β-NAD-ME Recombinant Proteins

    Directory of Open Access Journals (Sweden)

    Nabil I. Elsheery

    2013-06-01

    Full Text Available Plant mitochondrial NAD-malic enzyme (NAD-ME, which is composed of α- and β-subunits in many species, participates in many plant biosynthetic pathways and in plant respiratory metabolism. However, little is known about the properties of woody plant NAD-MEs. In this study, we analyzed four NAD-ME genes (PtNAD-ME1 through PtNAD-ME4 in the genome of Populus trichocarpa. PtNAD-ME1 and -2 encode putative α-subunits, while PtNAD-ME3 and -4 encode putative β-subunits. The Populus NAD-MEs were expressed in Escherichia coli cells as GST-tagged fusion proteins. Each recombinant GST-PtNAD-ME protein was purified to near homogeneity by glutathione-Sepharose 4B affinity chromatography. Milligram quantities of each native protein were obtained from 1 L bacterial cultures after cleavage of the GST tag. Analysis of the enzymatic properties of these proteins in vitro indicated that α-NAD-MEs are more active than β-NAD-MEs and that α- and β-NAD-MEs presented different kinetic properties (Vmax, kcat and kcat/Km. The effect of different amounts of metabolites on the activities of Populus α- and β-NAD-MEs was assessed in vitro. While none of the metabolites evaluated in our assays activated Populus NAD-ME, oxalacetate and citrate inhibited all α- and β-NAD-MEs and glucose-6-P and fructose inhibited only the α-NAD-MEs.

  1. Na(v)1.8 channelopathy in mutant mice deficient for myelin protein zero is detrimental to motor axons

    DEFF Research Database (Denmark)

    Moldovan, Mihai; Alvarez Herrero, Susana; Pinchenko, Volodymyr

    2011-01-01

    deficient homozygous mutants to an extent that precipitates conduction failure in severely affected axons. Our data suggest that a Na(V)1.8 channelopathy contributed to the poor motor function of protein zero deficient homozygous mutants, and that the conduction failure was associated with partially...

  2. Comparison between medium-chain acyl-CoA dehydrogenase mutant proteins overexpressed in bacterial and mammalian cells

    DEFF Research Database (Denmark)

    Jensen, T G; Bross, P; Andresen, B S

    1995-01-01

    ." Upon expression in E. coli, these mutant proteins produce activity levels in the range of the wild-type enzyme only if the chaperonins GroESL are co-overproduced. When overexpressed in COS cells, the pure folding mutants display enzyme activities comparable to the wild-type enzyme. The results suggest...

  3. Addition of Surfactants and Non-Hydrolytic Proteins and Their Influence on Enzymatic Hydrolysis of Pretreated Sugarcane Bagasse.

    Science.gov (United States)

    Méndez Arias, Johanna; de Oliveira Moraes, Anelize; Modesto, Luiz Felipe Amarante; de Castro, Aline Machado; Pereira, Nei

    2017-02-01

    Poly(ethylene glycol) (PEG 4000) and bovine serum albumin (BSA) were investigated with the purpose of evaluating their influence on enzymatic hydrolysis of sugarcane bagasse. Effects of these supplements were assayed for different enzymatic cocktails (Trichoderma harzianum and Penicillium funiculosum) that acted on lignocellulosic material submitted to different pretreatment methods with varying solid (25 and 100 g/L) and protein (7.5 and 20 mg/g cellulose) loadings. The highest levels of glucose release were achieved using partially delignified cellulignin as substrate, along with the T. harzianum cocktail: increases of 14 and 18 % for 25 g/L solid loadings and of 33 and 43 % for 100 g/L solid loadings were reached for BSA and PEG supplementation, respectively. Addition of these supplements could maintain hydrolysis yield even for higher solid loadings, but for higher enzymatic cocktail protein loadings, increases in glucose release were not observed. Results indicate that synergism might occur among these additives and cellulase and xylanases. The use of these supplements, besides depending on factors such as pretreatment method of sugarcane bagasse, enzymatic cocktails composition, and solid and protein loadings, may not always lead to positive effects on the hydrolysis of lignocellulosic material, making it necessary further statistical studies, according to process conditions.

  4. Characterization of enzymatically extracted sunflower seed oil as well as the protein residues

    Directory of Open Access Journals (Sweden)

    Sitohy, M. Z.

    1993-12-01

    Full Text Available Sunflower seed oil was enzymatically extracted with six different enzymes: cellulase, hemicellulase, animal proteinase, acid proteinase, pectinase and pectinex under the following conditions: substrate concentration in phosphate buffer (0.5M, pH 5 30%, enzyme concentration 2% (E/S, temperature 50°C and time 3 hours. The obtained oils were analyzed for physicochemical properties and fatty acid profiles. The protein residues were analyzed for amino acid compositions. The results showed that the enzymatic extraction with cellulase or hemicellulase could maintain good oil quality of the extracted oils as their levels of linoleic and oleic acids recorded similar values to those of the control oil extracted with organic solvents. Also the level of iodine value was in the same level of control. On the other hand, the use of proteases in the enzymatic extraction of sunflower seed oil caused some reductions in the levels of the unsaturated fatty acids as well as the iodine value. The pectinases showed a similar trend to that of the proteinase with the least recovery of linoleic acid among the different oils under study. Similarly, the use of cellulases did not change the amino acid composition of the protein residue as compared to the control, in the contrary to the extraction with the proteinases which caused reduction of some amino acids from the protein residues especially lysine, leucine, iso-leucine, alanine, arginine and aspartic. In that respect the use of pectinases behaved similar to cellulases.

    Aceite de semilla de girasol fue extraído enzimáticamente con seis enzimas diferentes: celulasa, hemicelulasa, proteinasa animal, proteinase acida, pectinasa y pectinex bajo las condiciones siguientes: concentración de sustrato en tampón fosfato (0,5M, pH 5 30%, concentración enzimática 2% (E/S, temperatura 50°C y tiempo 3 horas. Los aceites obtenidos fueron analizados por sus propiedades fisicoquímicas y perfiles de ácidos grasos

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

    Directory of Open Access Journals (Sweden)

    Brian P Grone

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

  6. Solubilization of industrial grade plant protein by enzymatic hydrolysis monitored by vibrational and nuclear magnetic resonance spectroscopy

    DEFF Research Database (Denmark)

    Bevilacqua, Marta; Pratico, Giulia; Plesner, Johanne

    2017-01-01

    by enzymatic hydrolysis, but during this process several collateral properties of the protein hydrolysates changes. It is therefore important to determine the end-point of the process and to monitor its development. In this feasibility study, we demonstrated the potential of different spectroscopic techniques......Protein hydrolysates are of great interest in the food industry due to their nutritional and functional properties, but their use often implies solubilization in water and therefore hamper the use of plant proteins with inherent low water solubility. Protein solubility in water can be modified...

  7. Food safety assessment of Cry8Ka5 mutant protein using Cry1Ac as a control Bt protein.

    Science.gov (United States)

    Farias, Davi Felipe; Viana, Martônio Ponte; Oliveira, Gustavo Ramos; Santos, Vanessa Olinto; Pinto, Clidia Eduarda Moreira; Viana, Daniel Araújo; Vasconcelos, Ilka Maria; Grossi-de-Sa, Maria Fátima; Carvalho, Ana Fontenele Urano

    2015-07-01

    Cry8Ka5 is a mutant protein from Bacillus thuringiensis (Bt) that has been proposed for developing transgenic plants due to promising activity against coleopterans, like Anthonomus grandis (the major pest of Brazilian cotton culture). Thus, an early food safety assessment of Cry8Ka5 protein could provide valuable information to support its use as a harmless biotechnological tool. This study aimed to evaluate the food safety of Cry8Ka5 protein following the two-tiered approach, based on weights of evidence, proposed by ILSI. Cry1Ac protein was used as a control Bt protein. The history of safe use revealed no convincing hazard reports for Bt pesticides and three-domain Cry proteins. The bioinformatics analysis with the primary amino acids sequence of Cry8Ka5 showed no similarity to any known toxic, antinutritional or allergenic proteins. The mode of action of Cry proteins is well understood and their fine specificity is restricted to insects. Cry8Ka5 and Cry1Ac proteins were rapidly degraded in simulated gastric fluid, but were resistant to simulated intestinal fluid and heat treatment. The LD50 for Cry8Ka5 and Cry1Ac was >5000 mg/kg body weight when administered by gavage in mice. Thus, no expected relevant risks are associated with the consumption of Cry8Ka5 protein. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Crystallization and preliminary X-ray analysis of Ebola VP35 interferon inhibitory domain mutant proteins

    International Nuclear Information System (INIS)

    Leung, Daisy W.; Borek, Dominika; Farahbakhsh, Mina; Ramanan, Parameshwaran; Nix, Jay C.; Wang, Tianjiao; Prins, Kathleen C.; Otwinowski, Zbyszek; Honzatko, Richard B.; Helgeson, Luke A.; Basler, Christopher F.; Amarasinghe, Gaya K.

    2010-01-01

    Three mutant forms of Ebola VP35 interferon inhibitory domain were crystallized in three different space groups. VP35 is one of seven structural proteins encoded by the Ebola viral genome and mediates viral replication, nucleocapsid formation and host immune suppression. The C-terminal interferon inhibitory domain (IID) of VP35 is critical for dsRNA binding and interferon inhibition. The wild-type VP35 IID structure revealed several conserved residues that are important for dsRNA binding and interferon antagonism. Here, the expression, purification and crystallization of recombinant Zaire Ebola VP35 IID mutants R312A, K319A/R322A and K339A in space groups P6 1 22, P2 1 2 1 2 1 and P2 1 , respectively, are described. Diffraction data were collected using synchrotron sources at the Advanced Light Source and the Advanced Photon Source

  9. Crystal Structure of Human Senescence Marker Protein 30: Insights Linking Structural, Enzymatic, and Physiological Functions

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborti, Subhendu; Bahnson, Brian J. (Delaware)

    2010-05-25

    Human senescence marker protein 30 (SMP30), which functions enzymatically as a lactonase, hydrolyzes various carbohydrate lactones. The penultimate step in vitamin-C biosynthesis is catalyzed by this enzyme in nonprimate mammals. It has also been implicated as an organophosphate hydrolase, with the ability to hydrolyze diisopropyl phosphofluoridate and other nerve agents. SMP30 was originally identified as an aging marker protein, whose expression decreased androgen independently in aging cells. SMP30 is also referred to as regucalcin and has been suggested to have functions in calcium homeostasis. The crystal structure of the human enzyme has been solved from X-ray diffraction data collected to a resolution of 1.4 {angstrom}. The protein has a 6-bladed {beta}-propeller fold, and it contains a single metal ion. Crystal structures have been solved with the metal site bound with either a Ca{sup 2+} or a Zn{sup 2+} atom. The catalytic role of the metal ion has been confirmed by mutagenesis of the metal coordinating residues. Kinetic studies using the substrate gluconolactone showed a k{sub cat} preference of divalent cations in the order Zn{sup 2+} > Mn{sup 2+} > Ca{sup 2+} > Mg{sup 2+}. Notably, the Ca{sup 2+} had a significantly higher value of K{sub d} compared to those of the other metal ions tested (566, 82, 7, and 0.6 {micro}m for Ca{sup 2+}, Mg{sup 2+}, Zn{sup 2+}, and Mn{sup 2+}, respectively), suggesting that the Ca{sup 2+}-bound form may be physiologically relevant for stressed cells with an elevated free calcium level.

  10. Quantification of mutant SPOP proteins in prostate cancer using mass spectrometry-based targeted proteomics

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hui; Barbieri, Christopher E.; He, Jintang; Gao, Yuqian; Shi, Tujin; Wu, Chaochao; Schepmoes, Athena A.; Fillmore, Thomas L.; Chae, Sung-Suk; Huang, Dennis; Mosquera, Juan Miguel; Qian, Wei-Jun; Smith, Richard D.; Srivastava, Sudhir; Kagan, Jacob; Camp, David G.; Rodland, Karin D.; Rubin, Mark A.; Liu, Tao

    2017-08-15

    Speckle-type POZ protein (SPOP) is an E3 ubiquitin ligase adaptor protein that functions as a potential tumor suppressor, and SPOP mutations have been identified in ~10% of human prostate cancers. However, it remains unclear if mutant SPOP proteins can be utilized as biomarkers for early detection, diagnosis, prognosis or targeted therapy of prostate cancer. Moreover, the SPOP mutation sites are distributed in a relatively short region where multiple lysine residues, posing significant challenges for bottom-up proteomics analysis of the SPOP mutations. To address this issue, PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing)-SRM (selected reaction monitoring) mass spectrometry assays have been developed for quantifying wild-type SPOP protein and 11 prostate cancer-derived SPOP mutations. Despite inherent limitations due to amino acid sequence constraints, all the PRISM-SRM assays developed using Arg-C digestion showed a linear dynamic range of at least two orders of magnitude, with limits of quantification range from 0.1 to 1 fmol/μg of total protein in the cell lysate. Applying these SRM assays to analyze HEK293T cells with and without expression of the three most frequent SPOP mutations in prostate cancer (Y87N, F102C or F133V) led to confident detection of all three SPOP mutations in corresponding positive cell lines but not in the negative cell lines. Expression of the F133V mutation and wild-type SPOP was at much lower levels compared to that of F102C and Y87N mutations; however, at present it is unknown if this also affects the activity of the SPOP protein. In summary, PRISM-SRM enables multiplexed, isoform-specific detection of mutant SPOP proteins in cell lysates, which holds great potential in biomarker development for prostate cancer.

  11. Induced variation in protein mutants after multiple EMS and X-ray treatments

    International Nuclear Information System (INIS)

    Walther, H.; Seibold, K.H.

    1978-01-01

    Results from two experiments with spring barley in the M 3 and M 8 generations gave information on the efficiency of selected mutants, improved in protein yield by different mutagenic treatments. A selection rate of 1-2% was found to be realistic according to a 5% significance level. However, differences between mutagenic treatments with EMS and X-rays and between varieties were found to be not only incidental. To evaluate the results within a protein mutation breeding programme a bivariate selection model was applied and was found to allow clear decisions on mutagenic improvements in protein production, measured in g protein/m 2 . When substituting protein yield in g/seed for protein yield in g/m 2 in the early generations, all relations to protein and grain yield in g/m 2 were found to be low and negative. We conclude that this substituted selection character can be of only limited aid. But very high positive correlations exist between protein yield/m 2 , lysine yield/m 2 and grain yield/m 2 , which means that these selection characters would render a more reliable basis for selection in early generations. (author)

  12. Enzymatic crosslinking and degradation of gelatin as a switch for bone morphogenetic protein-2 activity.

    Science.gov (United States)

    Kuwahara, Kenrick; Fang, Josephine Y; Yang, Zhi; Han, Bo

    2011-12-01

    Current therapies for tissue regeneration rely on the presence or direct delivery of growth factors to sites of repair. Bone morphogenetic protein-2 (BMP-2), combined with a carrier (usually collagen), is clinically proven to induce new bone formation during spinal fusion and nonunion repair. However, due to BMP-2's short half-life and its diffusive properties, orders of magnitude above physiological levels are required to ensure effectiveness. In addition, a high dose of this multifunctional growth factor is known to induce adverse effects in patients. To circumvent these challenges, we proposed and tested a new approach for BMP-2 delivery, by controlling BMP activity via carrier binding and localized proteolysis. BMP-2 was covalently bound to gelatin through site-specific enzymatic crosslinking using a microbial transglutaminase. Binding of BMP-2 to gelatin can completely switch off BMP-2 activity, as evidenced by loss of its transdifferentiating ability toward C2C12 promyoblasts. When gelatin sequestered BMP-2 is incubated with either microbial collagenase or tissue-derived matrix metalloproteinases, BMP-2 activity is fully restored. The activity of released BMP-2 correlates with the protease activity in a dose- and time-dependent manner. This observation suggests a novel way of delivering BMP-2 and controlling its activity. This improved delivery method, which relies on a physiological feedback, should enhance the known potential of this and other growth factors for tissue repair and regeneration.

  13. Using an enzymatic galactose assay to detect lactose glycation extents of two proteins caseinate and soybean protein isolate via the Maillard reaction.

    Science.gov (United States)

    Wang, Xiao-Peng; Zhao, Xin-Huai

    2017-06-01

    Glycation of food proteins via the Maillard reaction has been widely studied in the recent years; however, the amount of saccharide connected to proteins is usually not determined. An enzymatic galactose assay was proposed firstly in this study to detect lactose glycation extents of caseinate and soybean protein isolate (SPI) during the Maillard reaction at two temperatures and different times. The separated glycated proteins were hydrolysed to release galactose necessary for the enzymatic assay and glycation calculation. Caseinate and SPI both obtained the highest lactose glycation extents at 100 °C or 121 °C by a reaction time of 180 or 20 min. Short- and long-time reaction resulted in lower glycation extents. During the reaction, three chemical indices (absorbences at 294/490 nm and fluorescence intensities) of reaction mixtures increased continually, but another index reactable NH 2 of glycated proteins showed the opposite trend. In general, changing profiles of the four indices were inconsistent with those profiles of lactose glycation extents of glycated proteins, implying practical limitation of the four indices in studies. This proposed enzymatic assay could directly detect lactose glycation of the two proteins, and thus was more useful than the four chemical indices to monitor glycation of the two proteins. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  14. Protein nutrient value of Agaricus bazei murrill mutant J3 induced by 60Co γ-irradiation in different generations

    International Nuclear Information System (INIS)

    Jiang Zhihe; Lin Yong; Xiao Shuxia

    2004-01-01

    Protein nutritional values of Agaricus bazei Murrill mutant J 3 and original strain J 1 were compared by non-biological evaluation methods. The results showed that five protein indexes in the fruitbodies of M 1 and M 6 generations of Agaricus bazei Murrill mutant J 3 were higher than that of original strain J 1 ; four protein indexes in M 4 and M 5 generations were higher than that of original strain J 1 ; and six protein indexes in M 2 and M 3 generations were higher than that of original strain J 1 . It was concluded that the protein nutritional values of Agaricus bazei Murrill mutant J 3 was better than that of original strain J 1 . Except the ratio scores of amino acids had little change in the different generations, the other indexes in strain J 3 Agaricus bazei Murrill showed that the heredity efficiencies of the proteins was rather stable. (authors)

  15. Induced Levels of Heat Shock Proteins in dnaK mutants of Lactococcus lactis

    DEFF Research Database (Denmark)

    Koch, Birgit; Hammer, Karin; Vogensen, Finn K.

    1998-01-01

    of the inferred substrate binding site of the DnaK protein, exhibits a pronounced temperature sensitive phenotype and shows altered regulation of the heat shock response. The expression of the heat shock proteins are increased at the normal growth temperature measured both as protein synthesis rates and m......The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes and proteases including the DnaK-GrpE-DnaJ and the GroELS chaperone complexes. In order to investigate the importance of the DnaK chaperone complex for the growth and the heat shock...... regulation in Lactococcus lactis we have constructed two dnaK mutants with C-terminal deletions in dnaK. The minor deletion of 65 amino acids in the dnaK2 mutant, results in a slightly temperature sensitive phenotype. BK6 containing the larger deletion of 174 amino acids (dnaK1) removing the major part...

  16. Influence of enzymatic hydrolysis on the allergenicity of roasted peanut protein extract.

    Science.gov (United States)

    Cabanillas, Beatriz; Pedrosa, Mercedes M; Rodríguez, Julia; Muzquiz, Mercedes; Maleki, Soheila J; Cuadrado, Carmen; Burbano, Carmen; Crespo, Jesús F

    2012-01-01

    Peanut allergy is recognized as one of the most severe food allergies. Some studies have investigated the effects of enzymatic treatments on the in vitro immunological reactivity of members of the Leguminosae family, such as the soybean, chickpea and lentil. Nevertheless, there are only a few studies carried out with sera from patients with a well-documented allergy. Roasted peanut protein extract was hydrolyzed by the sequential and individual action of 2 food-grade enzymes, an endoprotease (Alcalase) and an exoprotease (Flavourzyme). Immunoreactivity to roasted peanut extract and hydrolyzed samples was evaluated by means of IgE immunoblot, ELISA and 2-dimensional electrophoresis using sera from 5 patients with a clinical allergy to peanuts and anti-Ara h 1, anti-Ara h 2 and anti-Ara h 3 immunoblots. Immunoblot and ELISA assays showed an important decrease of IgE reactivity and Ara h 1, Ara h 2 and Ara h 3 levels in the first 30 min of hydrolyzation with Alcalase. In contrast, individual treatment with Flavourzyme caused an increase in IgE reactivity detected by ELISA at 30 min and led to a 65% inhibition of IgE reactivity at the end of the assay (300 min). Ara h 1 and the basic subunit of Ara h 3 were still recognized after treatment with Flavourzyme for 300 min. Hydrolysis with the endoprotease Alcalase decreases IgE reactivity in the soluble protein fraction of roasted peanut better than hydrolysis with the exoprotease Flavourzyme. Copyright © 2011 S. Karger AG, Basel.

  17. Protein kinase CK2 mutants defective in substrate recognition. Purification and kinetic analysis

    DEFF Research Database (Denmark)

    Sarno, S; Vaglio, P; Meggio, F

    1996-01-01

    Five mutants of protein kinase CK2 alpha subunit in which altogether 14 basic residues were singly to quadruply replaced by alanines (K74A,K75A,K76A,K77A; K79A, R80A,K83A; R191A,R195A,K198A; R228A; and R278A, K279A,R280A) have been purified to near homogeneity either as such or after addition...... downstream from serine, the other basic residues seem to play a more elusive and/or indirect role in catalysis....

  18. Structural analyses of a constitutively active mutant of exchange protein directly activated by cAMP.

    Science.gov (United States)

    White, Mark A; Li, Sheng; Tsalkova, Tamara; Mei, Fang C; Liu, Tong; Woods, Virgil L; Cheng, Xiaodong

    2012-01-01

    Exchange proteins directly activated by cAMP (EPACs) are important allosteric regulators of cAMP-mediated signal transduction pathways. To understand the molecular mechanism of EPAC activation, we have combined site-directed mutagenesis, X-ray crystallography, and peptide amide hydrogen/deuterium exchange mass spectrometry (DXMS) to probe the structural and conformational dynamics of EPAC2-F435G, a constitutively active EPAC2 mutant. Our study demonstrates that conformational dynamics plays a critical role in cAMP-induced EPAC activation. A glycine mutation at 435 position shifts the equilibrium of conformational dynamics towards the extended active conformation.

  19. Structural analyses of a constitutively active mutant of exchange protein directly activated by cAMP.

    Directory of Open Access Journals (Sweden)

    Mark A White

    Full Text Available Exchange proteins directly activated by cAMP (EPACs are important allosteric regulators of cAMP-mediated signal transduction pathways. To understand the molecular mechanism of EPAC activation, we have combined site-directed mutagenesis, X-ray crystallography, and peptide amide hydrogen/deuterium exchange mass spectrometry (DXMS to probe the structural and conformational dynamics of EPAC2-F435G, a constitutively active EPAC2 mutant. Our study demonstrates that conformational dynamics plays a critical role in cAMP-induced EPAC activation. A glycine mutation at 435 position shifts the equilibrium of conformational dynamics towards the extended active conformation.

  20. Effect of methanol/acetone/water extraction and enzymatic hydrolysis on the nutritional value of unheated rapeseed proteins.

    Science.gov (United States)

    Lacroix, M; Amiot, J; Cheour, F; De La Noüe, J; Goulet, G; Brisson, G J

    1988-01-01

    The objective of this work was to study the effect of the extraction of phenols by methanol/acetone/water and proteolysis (pepsin 1 hour; trypsin 2 hours) on the nutritional characteristics of unheated rapeseed protein as measured by weight gain, protein intake, net protein ratio, apparent digestibility and absorbed protein. The effect of proteolysis of the methanol/acetone/water extracted rapeseed protein, and the effect of mixing the methanol/acetone/water extract back with the extracted rapeseed protein was also studied. Extraction of phenolic compounds from rapeseed flour significantly improved weight gain, protein intake, net protein ratio and absorbed protein value. However, the mixing of phenolic extract with the extracted rapeseed protein did not appear to have a significant effect. Enzymatic hydrolysis (1 hour with pepsin and 2 hours with trypsin) of the raw material significantly improved the weight gain and protein intake. The combined methanol/acetone/water extraction and protein hydrolysis treatments were beneficial on all nutritional quality parameters of rapeseed protein. These results suggest that the protein-bound phenolic compounds, rather than the free phenolic compounds contribute mainly to the decrease in the nutritional values of proteins associated with phenolic compounds in raw flour before extraction.

  1. ATM mutants

    Indian Academy of Sciences (India)

    First page Back Continue Last page Graphics. ATM mutants. ATM (Ataxia Telangiectasia Mutated). AT2BE and AT5B1 cells – fibroblast cell lines from Ataxia telangiectasia patients. Deletion mutants expressing truncated ATM protein which is inactive. Have been used in studies looking at the role of ATM in DNA damage ...

  2. Ultrafast electronic and vibrational dynamics of stabilized A state mutants of the green fluorescent protein (GFP): Snipping the proton wire

    Science.gov (United States)

    Stoner-Ma, Deborah; Jaye, Andrew A.; Ronayne, Kate L.; Nappa, Jérôme; Tonge, Peter J.; Meech, Stephen R.

    2008-06-01

    Two blue absorbing and emitting mutants (S65G/T203V/E222Q and S65T at pH 5.5) of the green fluorescent protein (GFP) have been investigated through ultrafast time resolved infra-red (TRIR) and fluorescence spectroscopy. In these mutants, in which the excited state proton transfer reaction observed in wild-type GFP has been blocked, the photophysics are dominated by the neutral A state. It was found that the A∗ excited state lifetime is short, indicating that it is relatively less stabilised in the protein matrix than the anionic form. However, the lifetime of the A state can be increased through modifications to the protein structure. The TRIR spectra show that a large shifts in protein vibrational modes on excitation of the A state occurs in both these GFP mutants. This is ascribed to a change in H-bonding interactions between the protein matrix and the excited state.

  3. Changes in the secondary structure of proteins labeled with 125I. CD spectroscopy and enzymatic activity studies

    International Nuclear Information System (INIS)

    Efimova, Y.M.; Wierczinski, B.; Haemers, S.; Well, A.A. van

    2005-01-01

    Bovine serum albumin (BSA) and lysozyme (LSZ) were radiolabeled with 125 I. Three different methods for protein iodination with 125 I were optimized. Parameters like incubation time and ratio of oxidizing agent and amount of protein were established. During protein iodination with 125 I, structural damages caused by the introduction of iodine into the protein may occur. These damages depend on the oxidizing agent used and may lead to considerable changes in the protein structure and, hence, their biological activity. Changes in secondary structure of LSZ and BSA were examined by circular dichroism (CD). Enzymatic activity tests were performed with lysozyme to check its biological activity. The Iodo Bead was found the best oxidizing agent for protein iodination. (author)

  4. Expression and purification of single cysteine-containing mutant variants of the mouse prion protein by oxidative refolding.

    Science.gov (United States)

    Sengupta, Ishita; Udgaonkar, Jayant B

    2017-12-01

    The folding and aggregation of proteins has been studied extensively, using multiple probes. To facilitate such experiments, introduction of spectroscopically-active moieties in to the protein of interest is often necessary. This is commonly achieved by specifically labelling cysteine residues in the protein, which are either present naturally or introduced artificially by site-directed mutagenesis. In the case of the recombinant prion protein, which is normally expressed in inclusion bodies, the presence of the native disulfide bond complicates the correct refolding of single cysteine-containing mutant variants of the protein. To overcome this major bottleneck, a simple purification strategy for single tryptophan, single cysteine-containing mutant variants of the mouse prion protein is presented, with yields comparable to that of the wild type protein. The protein(s) obtained by this method are correctly folded, with a single reduced cysteine, and the native disulfide bond between residues C178 and C213 intact. The β-sheet rich oligomers formed from these mutant variant protein(s) are identical to the wild type protein oligomer. The current strategy facilitates sample preparation for a number of high resolution spectroscopic measurements for the prion protein, which specifically require thiol labelling. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. 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.

  6. Accurate prediction of stability changes in protein mutants by combining machine learning with structure based computational mutagenesis.

    Science.gov (United States)

    Masso, Majid; Vaisman, Iosif I

    2008-09-15

    Accurate predictive models for the impact of single amino acid substitutions on protein stability provide insight into protein structure and function. Such models are also valuable for the design and engineering of new proteins. Previously described methods have utilized properties of protein sequence or structure to predict the free energy change of mutants due to thermal (DeltaDeltaG) and denaturant (DeltaDeltaG(H2O)) denaturations, as well as mutant thermal stability (DeltaT(m)), through the application of either computational energy-based approaches or machine learning techniques. However, accuracy associated with applying these methods separately is frequently far from optimal. We detail a computational mutagenesis technique based on a four-body, knowledge-based, statistical contact potential. For any mutation due to a single amino acid replacement in a protein, the method provides an empirical normalized measure of the ensuing environmental perturbation occurring at every residue position. A feature vector is generated for the mutant by considering perturbations at the mutated position and it's ordered six nearest neighbors in the 3-dimensional (3D) protein structure. These predictors of stability change are evaluated by applying machine learning tools to large training sets of mutants derived from diverse proteins that have been experimentally studied and described. Predictive models based on our combined approach are either comparable to, or in many cases significantly outperform, previously published results. A web server with supporting documentation is available at http://proteins.gmu.edu/automute.

  7. The evolutionarily conserved protein PHOTOSYNTHESIS AFFECTED MUTANT71 is required for efficient manganese uptake at the thylakoid membrane in Arabidopsis

    DEFF Research Database (Denmark)

    Schneider, Anja; Steinberger, Iris; Herdean, Andrei

    2016-01-01

    lumen remains poorly understood. Here, we show that Arabidopsis thaliana PHOTOSYNTHESIS AFFECTED MUTANT71 (PAM71) is an integral thylakoid membrane protein involved in Mn2+ and Ca2+ homeostasis in chloroplasts. This protein is required for normal operation of the oxygen-evolving complex (as evidenced...

  8. Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion

    Directory of Open Access Journals (Sweden)

    Lijuan Han

    2016-05-01

    Full Text Available Abstract Background Somatic calreticulin (CALR, Janus kinase 2 (JAK2, and thrombopoietin receptor (MPL mutations essentially show mutual exclusion in myeloproliferative neoplasms (MPN, suggesting that they activate common oncogenic pathways. Recent data have shown that MPL function is essential for CALR mutant-driven MPN. However, the exact role and the mechanisms of action of CALR mutants have not been fully elucidated. Methods The murine myeloid cell line 32D and human HL60 cells overexpressing the most frequent CALR type 1 and type 2 frameshift mutants were generated to analyze the first steps of cellular transformation, in the presence and absence of MPL expression. Furthermore, mutant CALR protein stability and secretion were examined using brefeldin A, MG132, spautin-1, and tunicamycin treatment. Results The present study demonstrates that the expression of endogenous Mpl, CD41, and the key megakaryocytic transcription factor NF-E2 is stimulated by type 1 and type 2 CALR mutants, even in the absence of exogenous MPL. Mutant CALR expressing 32D cells spontaneously acquired cytokine independence, and this was associated with increased Mpl mRNA expression, CD41, and NF-E2 protein as well as constitutive activation of downstream signaling and response to JAK inhibitor treatment. Exogenous expression of MPL led to constitutive activation of STAT3 and 5, ERK1/2, and AKT, cytokine-independent growth, and reduction of apoptosis similar to the effects seen in the spontaneously outgrown cells. We observed low CALR-mutant protein amounts in cellular lysates of stably transduced cells, and this was due to accelerated protein degradation that occurred independently from the ubiquitin-proteasome system as well as autophagy. CALR-mutant degradation was attenuated by MPL expression. Interestingly, we found high levels of mutated CALR and loss of downstream signaling after blockage of the secretory pathway and protein glycosylation. Conclusions These

  9. Mutant Brucella abortus membrane fusogenic protein induces protection against challenge infection in mice.

    Science.gov (United States)

    de Souza Filho, Job Alves; de Paulo Martins, Vicente; Campos, Priscila Carneiro; Alves-Silva, Juliana; Santos, Nathalia V; de Oliveira, Fernanda Souza; Menezes, Gustavo B; Azevedo, Vasco; Cravero, Silvio Lorenzo; Oliveira, Sergio Costa

    2015-04-01

    Brucella species can cause brucellosis, a zoonotic disease that causes serious livestock economic losses and represents a public health threat. The mechanism of virulence of Brucella spp. is not yet fully understood. Therefore, it is crucial to identify new molecules that serve as virulence factors to better understand this host-pathogen interplay. Here, we evaluated the role of the Brucella membrane fusogenic protein (Mfp) and outer membrane protein 19 (Omp19) in bacterial pathogenesis. In this study, we showed that B. abortus Δmfp::kan and Δomp19::kan deletion mutant strains have reduced persistence in vivo in C57BL/6 and interferon regulatory factor 1 (IRF-1) knockout (KO) mice. Additionally, 24 h after macrophage infection with a Δmfp::kan or Δomp19::kan strain expressing green fluorescent protein (GFP) approximately 80% or 65% of Brucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP-1, respectively, whereas around 60% of BCVs containing wild-type S2308 were found in LAMP-1-negative compartments. B. abortus Δomp19::kan was attenuated in vivo but had a residual virulence in C57BL/6 and IRF-1 KO mice, whereas the Δmfp::kan strain had a lower virulence in these same mouse models. Furthermore, Δmfp::kan and Δomp19::kan strains were used as live vaccines. Challenge experiments revealed that in C57BL/6 and IRF-1 KO mice, the Δmfp::kan strain induced greater protection than the vaccine RB51 and protection similar that of vaccine S19. However, a Δomp19::kan strain induced protection similar to that of RB51. Thus, these results demonstrate that Brucella Mfp and Omp19 are critical for full bacterial virulence and that the Δmfp::kan mutant may serve as a potential vaccine candidate in future studies. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  10. Plasma mutant α-galactosidase A protein and globotriaosylsphingosine level in Fabry disease

    Directory of Open Access Journals (Sweden)

    Takahiro Tsukimura

    2014-01-01

    Full Text Available Fabry disease is an X-linked genetic disorder characterized by deficient activity of α-galactosidase A (GLA and accumulation of glycolipids, and various GLA gene mutations lead to a wide range of clinical phenotypes from the classic form to the later-onset one. To investigate the biochemical heterogeneity and elucidate the basis of the disease using available clinical samples, we measured GLA activity, GLA protein and accumulated globotriaosylsphingosine (Lyso-Gb3, a biomarker of this disease, in plasma samples from Fabry patients. The analysis revealed that both the enzyme activity and the protein level were apparently decreased, and the enzyme activity was well correlated with the protein level in many Fabry patients. In these cases, a defect of biosynthesis or excessive degradation of mutant GLAs should be involved in the pathogenesis, and the residual protein level would determine the accumulation of Lyso-Gb3 and the severity of the disease. However, there are some exceptional cases, i.e., ones harboring p.C142Y, p.R112H and p.M296I, who exhibit a considerable amount of GLA protein. Especially, a subset of Fabry patients with p.R112H or p.M296I has been attracted interest because the patients exhibit almost normal plasma Lyso-Gb3 concentration. Structural analysis revealed that C142Y causes a structural change at the entrance of the active site. It will lead to a complete enzyme activity deficiency, resulting in a high level of plasma Lyso-Gb3 and the classic Fabry disease. On the other hand, it is thought that R112H causes a relatively large structural change on the molecular surface, and M296I a small one in a restricted region from the core to the surface, both the structural changes being far from the active site. These changes will cause not only partial degradation but also degeneration of the mutant GLA proteins, and the degenerated enzymes exhibiting small and residual activity remain and probably facilitate degradation of Lyso-Gb3

  11. Structure of a Highly Active Cephalopod S-crystallin Mutant: New Molecular Evidence for Evolution from an Active Enzyme into Lens-Refractive Protein.

    Science.gov (United States)

    Tan, Wei-Hung; Cheng, Shu-Chun; Liu, Yu-Tung; Wu, Cheng-Guo; Lin, Min-Han; Chen, Chiao-Che; Lin, Chao-Hsiung; Chou, Chi-Yuan

    2016-08-08

    Crystallins are found widely in animal lenses and have important functions due to their refractive properties. In the coleoid cephalopods, a lens with a graded refractive index provides good vision and is required for survival. Cephalopod S-crystallin is thought to have evolved from glutathione S-transferase (GST) with various homologs differentially expressed in the lens. However, there is no direct structural information that helps to delineate the mechanisms by which S-crystallin could have evolved. Here we report the structural and biochemical characterization of novel S-crystallin-glutathione complex. The 2.35-Å crystal structure of a S-crystallin mutant from Octopus vulgaris reveals an active-site architecture that is different from that of GST. S-crystallin has a preference for glutathione binding, although almost lost its GST enzymatic activity. We've also identified four historical mutations that are able to produce a "GST-like" S-crystallin that has regained activity. This protein recapitulates the evolution of S-crystallin from GST. Protein stability studies suggest that S-crystallin is stabilized by glutathione binding to prevent its aggregation; this contrasts with GST-σ, which do not possess this protection. We suggest that a tradeoff between enzyme activity and the stability of the lens protein might have been one of the major driving force behind lens evolution.

  12. Lipid composition of cAMP-dependent protein kinase mutants of Aspergillus niger.

    Science.gov (United States)

    Jernejc, Katarina; Bencina, Mojca

    2003-08-29

    Lipid composition of cAMP-dependent protein kinase (PKA) Aspergillus niger mutants with overexpressed or deleted genes for either regulatory and/or the catalytic subunit of PKA was analyzed. Disruption of the gene encoding the PKA regulatory subunit resulted in 20% less total lipids, 30% less neutral lipids, four times more glycolipids and two-fold higher triacylglycerol lipase activity compared to the control strain. Concomitantly a five-fold decrease in phosphatidylcholine, accompanied with 1.5-, 1.8- and 2.8-fold increases in phosphatidylethanolamine, lysophosphatidylethanolamine and phosphatidylinositol, was determined, respectively. The lack of PKA activity, due to the disruption of a gene encoding the PKA catalytic subunit, resulted in a 1.6-times increase in total lipids with two times more neutral lipids associated with lower triacylglycerol lipase activity and a decrease in phospholipids. The mutants with unrestricted PKA activity synthesized twice as much citric acid as the control strain and three times more than strains lacking PKA activity. The results indicate the involvement of cAMP-mediated PKA activity in regulation of lipid biosynthesis as well as citric acid synthesis.

  13. Enzymatic decontamination

    Directory of Open Access Journals (Sweden)

    Edyta Prusińska-Kurstak

    2014-12-01

    Full Text Available [b]Abstract[/b]. This paper is devoted to the methods of decontamination of weapons of mass destruction (biological and chemical, based on the use of protein catalysts of chemical reactions — enzymes. This paper presents the possibility of using enzymes to neutralize the harmful and destructive to the environment and human chemicals used in weapons of mass destruction. The mechanism of the enzymatic reaction is showed. These are the possibilities of using lysozyme as destructor dangerous bacteria (E. coli, anthrax Bacillus anthracis and their spores. The advantages and disadvantages of chemical and enzymatic methods of decontamination have been compared. It was found that under certain conditions the enzymes can be an alternative to chemical methods of decontamination of weapons of mass destruction.[b]Keywords[/b]: decontamination, weapons of mass destruction, enzymes

  14. PROXiMATE: a database of mutant protein-protein complex thermodynamics and kinetics.

    Science.gov (United States)

    Jemimah, Sherlyn; Yugandhar, K; Michael Gromiha, M

    2017-09-01

    We have developed PROXiMATE, a database of thermodynamic data for more than 6000 missense mutations in 174 heterodimeric protein-protein complexes, supplemented with interaction network data from STRING database, solvent accessibility, sequence, structural and functional information, experimental conditions and literature information. Additional features include complex structure visualization, search and display options, download options and a provision for users to upload their data. The database is freely available at http://www.iitm.ac.in/bioinfo/PROXiMATE/ . The website is implemented in Python, and supports recent versions of major browsers such as IE10, Firefox, Chrome and Opera. gromiha@iitm.ac.in. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  15. Recyclability of PET/WPI/PE Multilayer Films by Removal of Whey Protein Isolate-Based Coatings with Enzymatic Detergents

    Directory of Open Access Journals (Sweden)

    Patrizia Cinelli

    2016-06-01

    Full Text Available Multilayer plastic films provide a range of properties, which cannot be obtained from monolayer films but, at present, their recyclability is an open issue and should be improved. Research to date has shown the possibility of using whey protein as a layer material with the property of acting as an excellent barrier against oxygen and moisture, replacing petrochemical non-recyclable materials. The innovative approach of the present research was to achieve the recyclability of the substrate films by separating them, with a simple process compatible with industrial procedures, in order to promote recycling processes leading to obtain high value products that will beneficially impact the packaging and food industries. Hence, polyethyleneterephthalate (PET/polyethylene (PE multi-layer film was prepared based on PET coated with a whey protein layer, and then the previous structure was laminated with PE. Whey proteins, constituting the coating, can be degraded by enzymes so that the coating films can be washed off from the plastic substrate layer. Enzyme types, dosage, time, and temperature optima, which are compatible with procedures adopted in industrial waste recycling, were determined for a highly-efficient process. The washing of samples based on PET/whey and PET/whey/PE were efficient when performed with enzymatic detergent containing protease enzymes, as an alternative to conventional detergents used in recycling facilities. Different types of enzymatic detergents tested presented positive results in removing the protein layer from the PET substrate and from the PET/whey/PE multilayer films at room temperature. These results attested to the possibility of organizing the pre-treatment of the whey-based multilayer film by washing with different available commercial enzymatic detergents in order to separate PET and PE, thus allowing a better recycling of the two different polymers. Mechanical properties of the plastic substrate, such as stress at

  16. Mechanism of the discrepancy in the enzymatic hydrolysis efficiency between defatted peanut flour and peanut protein isolate by Flavorzyme.

    Science.gov (United States)

    Zheng, Lin; Zhao, Yijun; Xiao, Chuqiao; Sun-Waterhouse, Dongxiao; Zhao, Mouming; Su, Guowan

    2015-02-01

    Both defatted peanut flour (DPF) and peanut protein isolate (PPI) are widely used to prepare peanut protein hydrolysates. To compare their enzymatic hydrolysis efficiencies, DPF and PPI were hydrolysed by Alcalase, Neutrase, Papain, Protamex and Flavorzyme. Alcalase and Flavorzyme were found to be the most efficient proteases to hydrolyse both DPF and PPI. The efficiency was comparable to each other when using Alcalase, while PPI was hydrolysed less efficiently than DPF when using Flavorzyme. Analysis of changes in the protein solubility, subunit and conformation, and amino acid composition of DPF, PPI and their Flavorzyme hydrolysis residues indicated that the PPI preparation process had minimal effect on it, but peptide aggregation via non-covalent bonding (including hydrophobic interactions and hydrogen bonds) during hydrolysis and/or thermal treatment after hydrolysis were likely responsible for the reduced hydrolysis efficiency of PPI by Flavorzyme. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Biochemical activities of T-antigen proteins encoded by simian virus 40 A gene deletion mutants.

    OpenAIRE

    Clark, R; Peden, K; Pipas, J M; Nathans, D; Tjian, R

    1983-01-01

    We have analyzed T antigens produced by a set of simian virus 40 (SV40) A gene deletion mutants for ATPase activity and for binding to the SV40 origin of DNA replication. Virus stocks of nonviable SV40 A gene deletion mutants were established in SV40-transformed monkey COS cells. Mutant T antigens were produced in mutant virus-infected CV1 cells. The structures of the mutant T antigens were characterized by immunoprecipitation with monoclonal antibodies directed against distinct regions of th...

  18. Thermodynamics of protein denaturation at temperatures over 100 °C: CutA1 mutant proteins substituted with hydrophobic and charged residues.

    Science.gov (United States)

    Matsuura, Yoshinori; Takehira, Michiyo; Joti, Yasumasa; Ogasahara, Kyoko; Tanaka, Tomoyuki; Ono, Naoko; Kunishima, Naoki; Yutani, Katsuhide

    2015-10-26

    Although the thermodynamics of protein denaturation at temperatures over 100 °C is essential for the rational design of highly stable proteins, it is not understood well because of the associated technical difficulties. We designed certain hydrophobic mutant proteins of CutA1 from Escherichia coli, which have denaturation temperatures (Td) ranging from 101 to 113 °C and show a reversible heat denaturation. Using a hydrophobic mutant as a template, we successfully designed a hyperthermostable mutant protein (Td = 137 °C) by substituting six residues with charged ones. Thermodynamic analyses of these mutant proteins indicated that the hydrophobic mutants were stabilized by the accumulation of denaturation enthalpy (ΔH) with no entropic gain from hydrophobic solvation around 100 °C, and that the stabilization due to salt bridges resulted from both the increase in ΔH from ion-ion interactions and the entropic effect of the electrostatic solvation over 113 °C. This is the first experimental evidence that has successfully overcome the typical technical difficulties.

  19. Quantifying protein adsorption and function at nanostructured materials: enzymatic activity of glucose oxidase at GLAD structured electrodes.

    Science.gov (United States)

    Jensen, Uffe B; Ferapontova, Elena E; Sutherland, Duncan S

    2012-07-31

    Nanostructured materials strongly modulate the behavior of adsorbed proteins; however, the characterization of such interactions is challenging. Here we present a novel method combining protein adsorption studies at nanostructured quartz crystal microbalance sensor surfaces (QCM-D) with optical (surface plasmon resonance SPR) and electrochemical methods (cyclic voltammetry CV) allowing quantification of both bound protein amount and activity. The redox enzyme glucose oxidase is studied as a model system to explore alterations in protein functional behavior caused by adsorption onto flat and nanostructured surfaces. This enzyme and such materials interactions are relevant for biosensor applications. Novel nanostructured gold electrode surfaces with controlled curvature were fabricated using colloidal lithography and glancing angle deposition (GLAD). The adsorption of enzyme to nanostructured interfaces was found to be significantly larger compared to flat interfaces even after normalization for the increased surface area, and no substantial desorption was observed within 24 h. A decreased enzymatic activity was observed over the same period of time, which indicates a slow conformational change of the adsorbed enzyme induced by the materials interface. Additionally, we make use of inherent localized surface plasmon resonances in these nanostructured materials to directly quantify the protein binding. We hereby demonstrate a QCM-D-based methodology to quantify protein binding at complex nanostructured materials. Our approach allows label free quantification of protein binding at nanostructured interfaces.

  20. Modulation of proteolytic polyprotein processing by coxsackievirus mutants resistant to inhibitors targeting phosphatidylinositol-4-kinase IIIβ or oxysterol binding protein

    NARCIS (Netherlands)

    Lyoo, Heyrhyoung; Dorobantu, Cristina M; van der Schaar, Hilde M; van Kuppeveld, Frank J M

    2017-01-01

    Enteroviruses (e.g. poliovirus, coxsackievirus, and rhinovirus) require several host factors for genome replication. Among these host factors are phosphatidylinositol-4-kinase IIIβ (PI4KB) and oxysterol binding protein (OSBP). Enterovirus mutants resistant to inhibitors of PI4KB and OSBP were

  1. Properties of adenyl cyclase and cyclic adenosine 3',5'-monophosphate receptor protein-deficient mutants of Escherichia coli

    International Nuclear Information System (INIS)

    Kumar, S.

    1976-01-01

    Several spontaneous cya and crp mutants of Escherichia coli have been selected as clones simultaneously resistant to phage lambda and nalidixic acid and characterized. Both cya and crp mutants have been found to grow as cocci with increased doubling times. They have increased resistance to some mutagens (methylmethanesulfonate, ultraviolet light, gamma rays), antibiotics (nalidixic acid, ampicillin), phages (lambda, T6), sublethal heat and hypotonic shock, and decreased resistance to neutral detergents (sodium dodecyl sulfate, sodium deoxycholate), a protein synthesis inhibitor (streptomycin), and a respiratory inhibitor (sodium azide). The nature of changes in cell parameters indicate fundamental alterations in the envelope structure of the cya and crp mutant cells. The new cya and crp mutants have been found to be multiply carbohydrate negative and nonmotile in conformity with similar previously isolated mutants. Studies of revertants and phi 80 cya + and phi 80 cya transductants indicated that the pleiotropic phenotype is related to a single mutational event at the cya or the crp locus in the mutants

  2. Functional display of proteins, mutant proteins, fragments of proteins and peptides on the surface of filamentous (bacterio) phages: A review

    NARCIS (Netherlands)

    Pannekoek, H.; van Meijer, M.; Gaardsvoll, H.; van Zonneveld, A. J.

    1995-01-01

    Cytoplasmic expression of complex eukaryotic proteins inEscherichia coli usually yields inactive protein preparations. In some cases, (part) of the biological activity can be recovered by rather inefficient denaturation-renaturation procedures. Recently, novel concepts have been developed for the

  3. Mutant huntingtin protein expression and blood-spinal cord barrier dysfunction in huntington disease.

    Science.gov (United States)

    Sciacca, Giacomo; Cicchetti, Francesca

    2017-12-01

    The aim of the study was to assess the distribution, frequency, and specific location of mutant huntingtin protein (mHTT) aggregates-the pathological hallmark of Huntington disease (HD)-within the various compartments of the spinal cord and their potential impact on the local vasculature and blood-spinal cord barrier (BSCB). We performed a series of postmortem immunohistochemical and immunofluorescent stainings, as well as Western blot analyses, on cervical and lumbar sections of the spinal cord in patients diagnosed with HD (n = 11 of all grades of disease severity) along with sex- and age-matched healthy controls (n = 9). We observed that mHTT was preferably expressed within the anterior horn of the gray matter, in both cervical and lumbar sections. At the cellular level, mHTT aggregates were more often encountered in the extracellular matrix but could also be observed within cell bodies and neurites as well as within the endothelium of blood vessels with an increase in the density of small blood vessels in cervical sections of HD cases. These vasculature changes were accompanied with features of BSCB leakage, as assessed by the presence of increased levels of fibrinogen in the surrounding parenchyma and enhanced leukocyte infiltration. This alteration in BSCB integrity may be explained, in part, by the dysregulation we found in some of the main proteins associated with it such as junctional adhesion molecule-1 and vascular endothelial cadherin. These observations have important implications for our understanding of HD pathology and may also have significant therapeutic implications. Ann Neurol 2017;82:981-994. © 2017 American Neurological Association.

  4. Manifestation in F3 of pleiotropic gene in the cross between mother variety and its high protein mutant pt. 2

    International Nuclear Information System (INIS)

    Harn, C.; Won, J.L.

    1977-01-01

    Rice variety Hokwang was X-rayed in 1965 and a high protein line No. 398 was selected. This mutant, besides high protein, accompanied two visible mutated characters, short culm and early heading. In 1973 crossing was made between mutant 398 and its mother variety and in 1976 F 3 generations were grown to examine the manifestation of the mutated gene. It was confirmed that the above-mentioned mutated characters were controlled by a single recessive major gene of pleiotropic nature. Recessive deficit was observed in the segregation ratio of mutated characters. Besides the parental types, there were deviants which were assumed to be caused by the changed genetic background. It was considered possible to make a selection, from among the deviants, of mother-like type with high protein content. (author)

  5. Concerted asynchronous hula-twist photoisomerization in the S65T/H148D mutant of green fluorescent protein.

    Science.gov (United States)

    Zhang, Qiangqiang; Chen, Xuebo; Cui, Ganglong; Fang, Wei-Hai; Thiel, Walter

    2014-08-11

    Fluorescence emission of wild-type green fluorescent protein (GFP) is lost in the S65T mutant, but partly recovered in the S65T/H148D double mutant. These experimental findings are rationalized by a combined quantum mechanics/molecular mechanics (QM/MM) study at the QM(CASPT2//CASSCF)/AMBER level. A barrierless excited-state proton transfer, which is exclusively driven by the Asp148 residue introduced in the double mutant, is responsible for the ultrafast formation of the anionic fluorescent state, which can be deactivated through a concerted asynchronous hula-twist photoisomerization. This causes the lower fluorescence quantum yield in S65T/H148D compared to wild-type GFP. Hydrogen out-of-plane motion plays an important role in the deactivation of the S65T/H148D fluorescent state. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Grp78 is involved in retention of mutant low density lipoprotein receptor protein in the endoplasmic reticulum

    DEFF Research Database (Denmark)

    Jørgensen, M M; Jensen, Ole Nørregaard; Holst, H U

    2000-01-01

    The low density lipoprotein (LDL) receptor is responsible for removing the majority of the LDL cholesterol from the plasma. Mutations in the LDL receptor gene cause the disease familial hypercholesterolemia (FH). Approximately 50% of the mutations in the LDL receptor gene in patients with FH lead...... to receptor proteins that are retained in the endoplasmic reticulum (ER). Misfolding of mutant LDL receptors is a probable cause of this ER retention, resulting in no functional LDL receptors at the cell surface. However, the specific factors and mechanisms responsible for retention of mutant LDL receptors...... is involved in retention of mutant LDL receptors in the ER. Overexpression of Grp78 causes no major alterations on the steady state level of active LDL receptors at the cell surface. However, overexpression of Grp78 decreases the processing rate of newly synthesized wild type LDL receptors. This indicates...

  7. Generation of Helper Plasmids Encoding Mutant Adeno-associated Virus Type 2 Capsid Proteins with Increased Resistance against Proteasomal Degradation

    Directory of Open Access Journals (Sweden)

    Naghmeh Ahmadiankia

    2013-07-01

    Full Text Available   Objective(s: Adeno-associated virus type 2 (AAV2 vectors are widely used for both experimental and clinical gene therapy. A recent research has shown that the performance of these vectors can be greatly improved by substitution of specific surface-exposed tyrosine residues with phenylalanines. In this study, a fast and simple method is presented to generate AAV2 vector helper plasmids encoding capsid proteins with single, double or triple Y→F mutations.   Materials and Methods: A one-step, high-fidelity polymerase chain reaction (PCR cloning procedure involving the use of two partially overlapping primers to amplify a circular DNA template was applied to produce AAV2 cap genes encoding VP1 mutants with Y→F substitutions in residues 444, 500 or 730. The resulting constructs were used to make the different double and triple mutant by another round of PCR (Y444500F mutant, subcloning (Y444730F and Y500730F mutants or a combination of both techniques (Y444500730F mutant. Results: Nucleotide sequence analysis revealed successful introduction of the desired mutations in the AAV2 cap gene and showed the absence of any unintended mutations in the DNA fragments used to assemble the final set of AAV2 vector helper plasmids. The correctness of these plasmids was further confirmed by restriction mapping. Conclusion: PCR-based, single-step site-directed mutagenesis of circular DNA templates is a highly efficient and cost-effective method to generate AAV2 vector helper plasmids encoding mutant Cap proteins for the production of vector particles with increased gene transfer efficiency.

  8. Recovery of Whey Proteins and Enzymatic Hydrolysis of Lactose Derived from Casein Whey Using a Tangential Flow Ultrafiltration Module

    Science.gov (United States)

    Das, Bipasha; Bhattacharjee, Sangita; Bhattacharjee, Chiranjib

    2013-09-01

    In this study, ultrafiltration (UF) of pretreated casein whey was carried out in a cross-flow module fitted with 5 kDa molecular weight cut-off polyethersulfone membrane to recover whey proteins in the retentate and lactose in the permeate. Effects of processing conditions, like transmembrane pressure and pH on permeate flux and rejection were investigated and reported. The polarised layer resistance was found to increase with time during UF even in this high shear device. The lactose concentration in the permeate was measured using dinitro salicylic acid method. Enzymatic kinetic study for lactose hydrolysis was carried out at three different temperatures ranging from 30 to 50 °C using β-galactosidase enzyme. The glucose formed during lactose hydrolysis was analyzed using glucose oxidase-peroxidase method. Kinetics of enzymatic hydrolysis of lactose solution was found to follow Michaelis-Menten model and the model parameters were estimated by Lineweaver-Burk plot. The hydrolysis rate was found to be maximum (with Vmax = 5.5091 mmol/L/min) at 30 °C.

  9. Study on Optimal Conditions of Alcalase Enzymatic Hydrolysis of Soybean Protein Isolate

    OpenAIRE

    Yongsheng Ma; Xianhui Sun; Lintong Wang

    2015-01-01

    Soybean protein isolate was hydrolyzed to obtain soybean polypeptide solution using Alcalase as hydrolase. Degree of hydrolysis and the recovery rate of protein were used to characterize the soybean protein hydrolysis reaction result. Influence factors of soybean protein hydrolysis reaction including the substrate concentration, temperature, pH, enzyme concentration characterized by E/S (ratio of Enzyme and Substrate) and hydrolysis time were systematically studied with single factor and mult...

  10. Sensitizing potential of enzymatically cross-linked peanut proteins in a mouse model of peanut allergy.

    NARCIS (Netherlands)

    Smit, Joost

    2014-01-01

    SCOPE: The cross-linking of proteins by enzymes to form high-molecular-weight protein, aggregates can be used to tailor the technological or physiological functionality of food products. Aggregation of dietary proteins by food processing may promote allergic sensitization, but the effects of

  11. Leaf phenolics and seaweed tannins : analysis, enzymatic oxidation and non-covalent protein binding

    NARCIS (Netherlands)

    Vissers, Anne M.

    2017-01-01

    Upon extraction of proteins from sugar beet leaves (Beta vulgaris L.) and oarweed (Laminaria digitata) for animal food and feed purposes, endogenous phenolics and proteins can interact with each other, which might affect the protein’s applicability. Sugar beet leaf proteins might become

  12. A protein phosphatase methylesterase (PME-1) is one of several novel proteins stably associating with two inactive mutants of protein phosphatase 2A.

    Science.gov (United States)

    Ogris, E; Du, X; Nelson, K C; Mak, E K; Yu, X X; Lane, W S; Pallas, D C

    1999-05-14

    Carboxymethylation of proteins is a highly conserved means of regulation in eukaryotic cells. The protein phosphatase 2A (PP2A) catalytic (C) subunit is reversibly methylated at its carboxyl terminus by specific methyltransferase and methylesterase enzymes which have been purified, but not cloned. Carboxymethylation affects PP2A activity and varies during the cell cycle. Here, we report that substitution of glutamine for either of two putative active site histidines in the PP2A C subunit results in inactivation of PP2A and formation of stable complexes between PP2A and several cellular proteins. One of these cellular proteins, herein named protein phosphatase methylesterase-1 (PME-1), was purified and microsequenced, and its cDNA was cloned. PME-1 is conserved from yeast to human and contains a motif found in lipases having a catalytic triad-activated serine as their active site nucleophile. Bacterially expressed PME-1 demethylated PP2A C subunit in vitro, and okadaic acid, a known inhibitor of the PP2A methylesterase, inhibited this reaction. To our knowledge, PME-1 represents the first mammalian protein methylesterase to be cloned. Several lines of evidence indicate that, although there appears to be a role for C subunit carboxyl-terminal amino acids in PME-1 binding, amino acids other than those at the extreme carboxyl terminus of the C subunit also play an important role in PME-1 binding to a catalytically inactive mutant.

  13. Integrating alkaline extraction of proteins with enzymatic hydrolysis of cellulose from wet distiller's grains and solubles.

    Science.gov (United States)

    Bals, Bryan; Balan, Venkatesh; Dale, Bruce

    2009-12-01

    Fractionation of distiller's grains into value added products may serve to improve the economic viability of dry grind corn ethanol facilities in the wake of variable corn and ethanol prices. This research is aimed at creating a high protein, high lysine product from the grain using alkaline protein extractions in conjunction with hydrolysis of the remaining fiber to sugars which are then fermented to ethanol. Alkaline extractions improved the lysine content in protein products, although protein solubility did not exceed 45% of the total protein. In addition, oligomeric carbohydrates, starch, and other water solubles were also extracted, leading to a low purity protein product. Resulting sugar yields following ammonia fiber expansion (AFEX) pretreatment were also lower for extracted distiller's grains. From these experiments, it does not appear likely that alkaline extraction is a useful tool for fractionation of distiller's grains. However, pretreatment and hydrolysis can be an effective tool for further fractionation of protein.

  14. Enzymatic treatment of duck hepatitis B virus: Topology of the surface proteins for virions and noninfectious subviral particles

    International Nuclear Information System (INIS)

    Franke, Claudia; Matschl, Urte; Bruns, Michael

    2007-01-01

    The large surface antigen L of duck hepatitis B virus exhibits a mixed topology with the preS domains of the protein alternatively exposed to the particles' interior or exterior. After separating virions from subviral particles (SVPs), we compared their L topologies and showed that both particle types exhibit the same amount of L with the following differences: 1-preS of intact virions was enzymatically digested with chymotrypsin, whereas in SVPs only half of preS was accessible, 2-phosphorylation of L at S118 was completely removed by phosphatase treatment only in virions, 3-iodine-125 labeling disclosed a higher ratio of exposed preS to S domains in virions compared to SVPs. These data point towards different surface architectures of virions and SVPs. Because the preS domain acts in binding to a cellular receptor of hepatocytes, our findings implicate the exclusion of SVPs as competitors for the receptor binding and entry of virions

  15. Exploring the effect of different plant lignin content and composition on ionic liquid pretreatment efficiency and enzymatic saccharification of Eucalyptus globulus L. mutants.

    Science.gov (United States)

    Papa, G; Varanasi, P; Sun, L; Cheng, G; Stavila, V; Holmes, B; Simmons, B A; Adani, F; Singh, S

    2012-08-01

    There are several approaches being investigated to improve the efficiency of biomass conversion into fermentable sugars, including those that engineer the feedstocks to enhance digestibility. In this study it was evaluated the impact of genotype modifications of three mutants of Eucalyptus globulus L., and of the corresponding wild type on cellulose hydrolyzability before and after ionic liquid (IL) pretreatment. Both untreated and IL-treated samples were chemically characterized and tested for cellulose hydrolizability. Results obtained indicate that genetic modifications altered wood lignin-S/G ratio. This alteration resulted in a different hydrolyzability of cellulose for untreated samples, i.e. high lignin-S/G ratio produced low glucose yield (r=-0.97; Pglobulus. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Identification of Streptococcus equi ssp. zooepidemicus surface associated proteins by enzymatic shaving.

    Science.gov (United States)

    Wei, Zigong; Fu, Qiang; Liu, Xiaohong; Xiao, Pingping; Lu, Zhaohui; Chen, Yaosheng

    2012-10-12

    Streptococcus equi ssp. zooepidemicus (Streptococcus zooepidemicus, SEZ) is responsible for a wide variety of infections in many species. Attempts to control the infection caused by this agent are hampered by a lack of effective vaccines and useful diagnostic kits. Surface proteins of bacterial species are usually involved in interaction with host and hopefully act as biomarkers for serodiagnosis and subunit vaccine components. In this study, the surface proteins of SEZ C55138 strain were systematically identified by surface shaving with trypsin and a total of 20 surface associated proteins were found. Further analysis of five selected novel proteins (SzM, FBP, SAP, CSP and 5'-Nu) revealed that they all expressed in vivo and their recombinant derived proteins could be reactive with convalescent sera. These identified immunogenic surface proteins have potential as SEZ vaccine candidates and diagnostic markers. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Crystal structure of the left-handed archaeal RadA helical filament: identification of a functional motif for controlling quaternary structures and enzymatic functions of RecA family proteins

    Science.gov (United States)

    Chen, Li-Tzu; Ko, Tzu-Ping; Chang, Yuan-Chih; Lin, Kuei-An; Chang, Chia-Seng; Wang, Andrew H.-J.; Wang, Ting-Fang

    2007-01-01

    The RecA family of proteins mediates homologous recombination, an evolutionarily conserved pathway that maintains genomic stability by protecting against DNA double strand breaks. RecA proteins are thought to facilitate DNA strand exchange reactions as closed-rings or as right-handed helical filaments. Here, we report the crystal structure of a left-handed Sulfolobus solfataricus RadA helical filament. Each protomer in this left-handed filament is linked to its neighbour via interactions of a β-strand polymerization motif with the neighbouring ATPase domain. Immediately following the polymerization motif, we identified an evolutionarily conserved hinge region (a subunit rotation motif) in which a 360° clockwise axial rotation accompanies stepwise structural transitions from a closed ring to the AMP–PNP right-handed filament, then to an overwound right-handed filament and finally to the left-handed filament. Additional structural and functional analyses of wild-type and mutant proteins confirmed that the subunit rotation motif is crucial for enzymatic functions of RecA family proteins. These observations support the hypothesis that RecA family protein filaments may function as rotary motors. PMID:17329376

  18. Antihypertensive properties of tilapia (Oreochromis spp.) frame and skin enzymatic protein hydrolysates

    OpenAIRE

    Lin, Hsin-Chieh; Alashi, Adeola M.; Aluko, Rotimi E.; Sun Pan, Bonnie; Chang, Yu-Wei

    2017-01-01

    ABSTRACT Proteins from tilapia frame and skin can potentially be precursors of antihypertensive peptides according to the result of BIOPEP analyses. The aim was to generate peptides with inhibitory effects against angiotensin-converting enzyme (ACE) and renin from tilapia frame and skin protein isolates (FPI and SPI). The most active hydrolysate was then tested for blood pressure-lowering ability in spontaneously hypertensive rats (SHRs). Tilapia frame and skin protein hydrolysates (FPHs and ...

  19. PTH1R Mutants Found in Patients with Primary Failure of Tooth Eruption Disrupt G-Protein Signaling.

    Science.gov (United States)

    Subramanian, Hariharan; Döring, Frank; Kollert, Sina; Rukoyatkina, Natalia; Sturm, Julia; Gambaryan, Stepan; Stellzig-Eisenhauer, Angelika; Meyer-Marcotty, Philipp; Eigenthaler, Martin; Wischmeyer, Erhard

    2016-01-01

    Primary failure of tooth eruption (PFE) is causally linked to heterozygous mutations of the parathyroid hormone receptor (PTH1R) gene. The mutants described so far lead to exchange of amino acids or truncation of the protein that may result in structural changes of the expressed PTH1R. However, functional effects of these mutations have not been investigated yet. In HEK293 cells, PTH1R wild type was co-transfected with selected PTH1R mutants identified in patients with PFE. The effects on activation of PTH-regulated intracellular signaling pathways were analyzed by ELISA and Western immunoblotting. Differential effects of wild type and mutated PTH1R on TRESK ion channel regulation were analyzed by electrophysiological recordings in Xenopus laevis oocytes. In HEK293 cells, activation of PTH1R wild type increases cAMP and in response activates cAMP-stimulated protein kinase as detected by phosphorylation of the vasodilator stimulated phosphoprotein (VASP). In contrast, the PTH1R mutants are functionally inactive and mutant PTH1R/Gly452Glu has a dominant negative effect on the signaling of PTH1R wild type. Confocal imaging revealed that wild type PTH1R is expressed on the cell surface, whereas PTH1R/Gly452Glu mutant is mostly retained inside the cell. Furthermore, in contrast to wild type PTH1R which substantially augmented K+ currents of TRESK channels, coupling of mutated PTH1R to TRESK channels was completely abolished. PTH1R mutations affect intracellular PTH-regulated signaling in vitro. In patients with primary failure of tooth eruption defective signaling of PTH1R mutations is suggested to occur in dento-alveolar cells and thus may lead to impaired tooth movement.

  20. ALS mutant FUS proteins are recruited into stress granules in induced pluripotent stem cell-derived motoneurons

    Directory of Open Access Journals (Sweden)

    Jessica Lenzi

    2015-07-01

    Full Text Available Patient-derived induced pluripotent stem cells (iPSCs provide an opportunity to study human diseases mainly in those cases for which no suitable model systems are available. Here, we have taken advantage of in vitro iPSCs derived from patients affected by amyotrophic lateral sclerosis (ALS and carrying mutations in the RNA-binding protein FUS to study the cellular behavior of the mutant proteins in the appropriate genetic background. Moreover, the ability to differentiate iPSCs into spinal cord neural cells provides an in vitro model mimicking the physiological conditions. iPSCs were derived from FUSR514S and FUSR521C patient fibroblasts, whereas in the case of the severe FUSP525L mutation, in which fibroblasts were not available, a heterozygous and a homozygous iPSC line were raised by TALEN-directed mutagenesis. We show that aberrant localization and recruitment of FUS into stress granules (SGs is a prerogative of the FUS mutant proteins and occurs only upon induction of stress in both undifferentiated iPSCs and spinal cord neural cells. Moreover, we show that the incorporation into SGs is proportional to the amount of cytoplasmic FUS, strongly correlating with the cytoplasmic delocalization phenotype of the different mutants. Therefore, the available iPSCs represent a very powerful system for understanding the correlation between FUS mutations, the molecular mechanisms of SG formation and ALS ethiopathogenesis.

  1. Crystal Structures of the uL3 Mutant Ribosome: Illustration of the Importance of Ribosomal Proteins for Translation Efficiency

    Science.gov (United States)

    Mailliot, Justine; de Loubresse, Nicolas Garreau; Yusupova, Gulnara; Meskauskas, Arturas; Dinman, Jonathan D.; Yusupov, Marat

    2017-01-01

    The ribosome has been described as a ribozyme in which ribosomal RNA is responsible for peptidyl-transferase reaction catalysis. The W255C mutation of the universally conserved ribosomal protein uL3 has diverse effects on ribosome function (e.g., increased affinities for transfer RNAs, decreased rates of peptidyl-transfer), and cells harboring this mutation are resistant to peptidyl-transferase inhibitors (e.g., anisomycin). These observations beg the question of how a single amino acid mutation may have such wide ranging consequences. Here, we report the structure of the vacant yeast uL3 W255C mutant ribosome by X-ray crystallography, showing a disruption of the A-site side of the peptidyl-transferase center (PTC). An additional X-ray crystallographic structure of the anisomycin-containing mutant ribosome shows that high concentrations of this inhibitor restore a “WT-like” configuration to this region of the PTC, providing insight into the resistance mechanism of the mutant. Globally, our data demonstrate that ribosomal protein uL3 is structurally essential to ensure an optimal and catalytically efficient organization of the PTC, highlighting the importance of proteins in the RNA-centered ribosome. PMID:26906928

  2. Orthogonal enzymatic reactions for the assembly of proteins at electrode addresses.

    Science.gov (United States)

    Yang, Xiaohua; Shi, Xiao-Wen; Liu, Yi; Bentley, William E; Payne, Gregory F

    2009-01-06

    The ability to interface proteins to device surfaces is important for a range of applications. Here, we enlist the unique capabilities of enzymes and biologically derived polymers to assemble target proteins to electrode addresses. First, the stimuli-responsive aminopolysaccharide chitosan is directed to assemble at the electrode address in response to electrode-imposed signals. The electrodeposited chitosan film serves as the biodevice interface for subsequent protein assembly. Next, tyrosinase is used to catalyze grafting of a protein or peptide tether to the chitosan film. Finally, microbial transglutaminase (mTG) catalyzes the assembly of target proteins to the tether. mTG covalently links proteins through their glutamine (Gln) and lysine (Lys) residues. Since Gln and Lys residues of globular proteins are often inaccessible to mTG, we engineered our target proteins to have fusion tags with added Gln or Lys residues. This assembly method employs the electrical signal to confer spatial selectivity (during chitosan electrodeposition) and employs the enzymes to confer chemical selectivity (i.e., amino acid residue selectivity). Further, this method is mild, since no reactive reagents or protection steps are required, and all steps are performed in aqueous solution. These results demonstrate the potential for employing biological materials and mechanisms to biofabricate the biodevice interface.

  3. Antihypertensive properties of tilapia (Oreochromis spp.) frame and skin enzymatic protein hydrolysates.

    Science.gov (United States)

    Lin, Hsin-Chieh; Alashi, Adeola M; Aluko, Rotimi E; Sun Pan, Bonnie; Chang, Yu-Wei

    2017-01-01

    Proteins from tilapia frame and skin can potentially be precursors of antihypertensive peptides according to the result of BIOPEP analyses. The aim was to generate peptides with inhibitory effects against angiotensin-converting enzyme (ACE) and renin from tilapia frame and skin protein isolates (FPI and SPI). The most active hydrolysate was then tested for blood pressure-lowering ability in spontaneously hypertensive rats (SHRs). Tilapia frame and skin protein hydrolysates (FPHs and SPHs) were respectively produced from FPI and SPI hydrolysis using pepsin, papain, or bromelain. The ACE-inhibitory activities of tilapia protein hydrolysates with varying degree of hydrolysis (DH) were evaluated. In order to enhance the activity, the hydrolysate was fractionated into four fractions (tilapia processing coproducts may be valuable protein raw materials for producing antihypertensive peptides.

  4. Identification of a 34 kDa protein altered in the LF-1 mutant as the herbicide-binding D1 protein of photosystem II

    International Nuclear Information System (INIS)

    Metz, J.; Pakrasi, H.; Seibert, M.; Arntzen, C.

    1986-01-01

    The LF-1 mutant of Scenedesmus has a complete block on the oxidizing side of its PSII reaction center. However, the reaction center as well as the reducing side of PSII is fully functional in this mutant. Compared to the wildtype (WT) the only detected protein difference in the PSII complex of LF-1 is the change in mobility of a 34 kDa protein to 36 kDa. This protein has been implicated to have a major role in Mn-binding and water-oxidation. The authors have recently shown that photoaffinity labeling of thylakoids with azido-[ 14 C]-atrazine tags the 34 kDa protein in WT and the 36 kDa protein in LF-1. It has been shown that the azido-atrazine labeled protein, called D1, functions in herbicide binding and Q/sub A/ to Q/sub B/ electron transfer on the reducing side of PSII. Polyclonal antibodies directed against the D1 protein of Amaranthus hybridus (Ohad, et al., EMBOJ 1985) were found to recognize the Scenedesmus 34 kDa (WT) and 36 kDa (LF-1) proteins. The implied dual function for the D1 protein on the reducing as well as the oxidizing side of PSII reaction center will be discussed

  5. Fish protein hydrolysate production from sardine solid waste by crude pepsin enzymatic hydrolysis in a bioreactor coupled to an ultrafiltration unit

    Energy Technology Data Exchange (ETDEWEB)

    Benhabiles, M.S.; Abdi, N. [National Polytechnic school of Algiers, B.P. 182-16200, El Harrach, Algiers (Algeria); Drouiche, N., E-mail: nadjibdrouiche@yahoo.fr [National Polytechnic school of Algiers, B.P. 182-16200, El Harrach, Algiers (Algeria); Silicon Technology Development Unit (UDTS) 2, Bd Frantz Fanon BP140, Alger-7 Merveilles, 16000 (Algeria); Lounici, H. [National Polytechnic school of Algiers, B.P. 182-16200, El Harrach, Algiers (Algeria); Pauss, A. [University of Technology of Compiegne, Departement Genie chimique,B.P. 20.509, 60205 Compiegne cedex (France); Goosen, M.F.A. [Alfaisal University, Riyadh (Saudi Arabia); Mameri, N. [University of Technology of Compiegne, Departement Genie chimique,B.P. 20.509, 60205 Compiegne cedex (France)

    2012-05-01

    The aims of the study were to optimize the production a fish protein hydrolysate (FPH) by enzymatic hydrolysis of sardine solid waste using crude pepsin, and to scale up the process in a bioreactor coupled to an ultrafiltration unit for product recovery. Results showed that the crude pepsin prepared by autolysis of the mucous membranes of a sheep stomach at optimal conditions (i. e. pH = 1.5-2 and incubation time of 6 h) could be satisfactory used for the enzymatic hydrolysis of fish solid waste. The optimal conditions for enzymatic reaction were: temperature 48 Degree-Sign C, and pH 1.5. The scale up of the enzymatic hydrolysis and the coupling of the reactor an ultrafiltration unit to concentrate the hydrolysate gave good results with a rejection coefficient for the protein hydrolysate product in the range of 90%. The volumetric concentration factor was 2.5, with a permeate flux of 200 L m{sup -2} bar{sup -1}. However, the results also suggest that the ultrafiltration product concentration process may be operating beyond the critical flux at which point irreversible membrane fouling occurs. - Highlights: Black-Right-Pointing-Pointer Evaluating to produce a (FPH) by enzymatic hydrolysis of sardine solid wastes was achieved. Black-Right-Pointing-Pointer Investigation of key parameters for optimal conditions for enzymatic hydrolysis have been studied. Black-Right-Pointing-Pointer Valorization of sardine waste was realized by enzymatic hydrolysis process. Black-Right-Pointing-Pointer Performances of this enzyme gave comparable results to those obtained with commercial pepsin. Black-Right-Pointing-Pointer The nutritional quality of the FPH produced appears to be satisfactory.

  6. Fish protein hydrolysate production from sardine solid waste by crude pepsin enzymatic hydrolysis in a bioreactor coupled to an ultrafiltration unit

    International Nuclear Information System (INIS)

    Benhabiles, M.S.; Abdi, N.; Drouiche, N.; Lounici, H.; Pauss, A.; Goosen, M.F.A.; Mameri, N.

    2012-01-01

    The aims of the study were to optimize the production a fish protein hydrolysate (FPH) by enzymatic hydrolysis of sardine solid waste using crude pepsin, and to scale up the process in a bioreactor coupled to an ultrafiltration unit for product recovery. Results showed that the crude pepsin prepared by autolysis of the mucous membranes of a sheep stomach at optimal conditions (i. e. pH = 1.5–2 and incubation time of 6 h) could be satisfactory used for the enzymatic hydrolysis of fish solid waste. The optimal conditions for enzymatic reaction were: temperature 48 °C, and pH 1.5. The scale up of the enzymatic hydrolysis and the coupling of the reactor an ultrafiltration unit to concentrate the hydrolysate gave good results with a rejection coefficient for the protein hydrolysate product in the range of 90%. The volumetric concentration factor was 2.5, with a permeate flux of 200 L m −2 bar −1 . However, the results also suggest that the ultrafiltration product concentration process may be operating beyond the critical flux at which point irreversible membrane fouling occurs. - Highlights: ► Evaluating to produce a (FPH) by enzymatic hydrolysis of sardine solid wastes was achieved. ► Investigation of key parameters for optimal conditions for enzymatic hydrolysis have been studied. ► Valorization of sardine waste was realized by enzymatic hydrolysis process. ► Performances of this enzyme gave comparable results to those obtained with commercial pepsin. ► The nutritional quality of the FPH produced appears to be satisfactory.

  7. Soya bean Gα proteins with distinct biochemical properties exhibit differential ability to complement Saccharomyces cerevisiae gpa1 mutant.

    Science.gov (United States)

    Roy Choudhury, Swarup; Wang, Yuqi; Pandey, Sona

    2014-07-01

    Signalling pathways mediated by heterotrimeric G-proteins are common to all eukaryotes. Plants have a limited number of each of the G-protein subunits, with the most elaborate G-protein network discovered so far in soya bean (Glycine max, also known as soybean) which has four Gα, four Gβ and ten Gγ proteins. Biochemical characterization of Gα proteins from plants suggests significant variation in their properties compared with the well-characterized non-plant proteins. Furthermore, the four soya bean Gα (GmGα) proteins exhibit distinct biochemical activities among themselves, but the extent to which such biochemical differences contribute to their in vivo function is also not known. We used the yeast gpa1 mutant which displays constitutive signalling and growth arrest in the pheromone-response pathway as an in vivo model to evaluate the effect of distinct biochemical activities of GmGα proteins. We showed that specific GmGα proteins can be activated during pheromone-dependent receptor-mediated signalling in yeast and they display different strengths towards complementation of yeast gpa1 phenotypes. We also identified amino acids that are responsible for differential complementation abilities of specific Gα proteins. These data establish that specific plant Gα proteins are functional in the receptor-mediated pheromone-response pathway in yeast and that the subtle biochemical differences in their activity are physiologically relevant.

  8. Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity

    Energy Technology Data Exchange (ETDEWEB)

    Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L. (UW-MED); (UCB)

    2015-04-22

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome.

  9. Comparison the effect of three commercial enzymes for enzymatic hydrolysis of two substrates (rice bran protein concentrate and soy-been protein) with SDS-PAGE

    OpenAIRE

    Ahmadifard, Nasrollah; Murueta, Julio Humberto Cordova; Abedian-Kenari, Abdolmohammad; Motamedzadegan, Ali; Jamali, Hadi

    2015-01-01

    In this research enzymatic hydrolysis of rice bran protein concentrate (RBPC) and soybean Protein (SBP) as control were studied with 3 commercial enzymes (Alcalase®, Papain and acommercial 3-enzyme cocktail containing of 1.6 mg ml−1 Trypsin, 3.1 mg ml−1 Chymotrypsin, 1.3 mg ml−1Aminopeptidase (SIGMA P7500) and 7.95 mg ml−1pronase type XIV (SIGMA P5147) by the pH stat method. The hydrolysis was carried out at temperature of 28 C, 60 min and pH 8.00. Results were showed that RBPC, and SBP had h...

  10. Characterization of Leber Congenital Amaurosis-associated NMNAT1 Mutants.

    Science.gov (United States)

    Sasaki, Yo; Margolin, Zachary; Borgo, Benjamin; Havranek, James J; Milbrandt, Jeffrey

    2015-07-10

    Leber congenital amaurosis 9 (LCA9) is an autosomal recessive retinal degeneration condition caused by mutations in the NAD(+) biosynthetic enzyme NMNAT1. This condition leads to early blindness but no other consistent deficits have been reported in patients with NMNAT1 mutations despite its central role in metabolism and ubiquitous expression. To study how these mutations affect NMNAT1 function and ultimately lead to the retinal degeneration phenotype, we performed detailed analysis of LCA-associated NMNAT1 mutants, including the expression, nuclear localization, enzymatic activity, secondary structure, oligomerization, and promotion of axonal and cellular integrity in response to injury. In many assays, most mutants produced results similar to wild type NMNAT1. Indeed, NAD(+) synthetic activity is unlikely to be a primary mechanism underlying retinal degeneration as most LCA-associated NMNAT1 mutants had normal enzymatic activity. In contrast, the secondary structure of many NMNAT1 mutants was relatively less stable as they lost enzymatic activity after heat shock, whereas wild type NMNAT1 retains significant activity after this stress. These results suggest that LCA-associated NMNAT1 mutants are more vulnerable to stressful conditions that lead to protein unfolding, a potential contributor to the retinal degeneration observed in this syndrome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Characterization of Leber Congenital Amaurosis-associated NMNAT1 Mutants*

    Science.gov (United States)

    Sasaki, Yo; Margolin, Zachary; Borgo, Benjamin; Havranek, James J.; Milbrandt, Jeffrey

    2015-01-01

    Leber congenital amaurosis 9 (LCA9) is an autosomal recessive retinal degeneration condition caused by mutations in the NAD+ biosynthetic enzyme NMNAT1. This condition leads to early blindness but no other consistent deficits have been reported in patients with NMNAT1 mutations despite its central role in metabolism and ubiquitous expression. To study how these mutations affect NMNAT1 function and ultimately lead to the retinal degeneration phenotype, we performed detailed analysis of LCA-associated NMNAT1 mutants, including the expression, nuclear localization, enzymatic activity, secondary structure, oligomerization, and promotion of axonal and cellular integrity in response to injury. In many assays, most mutants produced results similar to wild type NMNAT1. Indeed, NAD+ synthetic activity is unlikely to be a primary mechanism underlying retinal degeneration as most LCA-associated NMNAT1 mutants had normal enzymatic activity. In contrast, the secondary structure of many NMNAT1 mutants was relatively less stable as they lost enzymatic activity after heat shock, whereas wild type NMNAT1 retains significant activity after this stress. These results suggest that LCA-associated NMNAT1 mutants are more vulnerable to stressful conditions that lead to protein unfolding, a potential contributor to the retinal degeneration observed in this syndrome. PMID:26018082

  12. Modulation of proteolytic polyprotein processing by coxsackievirus mutants resistant to inhibitors targeting phosphatidylinositol-4-kinase IIIβ or oxysterol binding protein

    OpenAIRE

    Lyoo, Heyrhyoung; Dorobantu, Cristina M; van der Schaar, Hilde M; van Kuppeveld, Frank J M

    2017-01-01

    Enteroviruses (e.g. poliovirus, coxsackievirus, and rhinovirus) require several host factors for genome replication. Among these host factors are phosphatidylinositol-4-kinase IIIβ (PI4KB) and oxysterol binding protein (OSBP). Enterovirus mutants resistant to inhibitors of PI4KB and OSBP were previously isolated, which demonstrated a role of single substitutions in the non-structural 3A protein in conferring resistance. Besides the 3A substitutions (i.e., 3A-I54F and 3A-H57Y) in coxsackieviru...

  13. Production of protein hydrolysates from fish byproduct prepared by enzymatic hydrolysis

    OpenAIRE

    Murna Muzaifa; Novi Safriani; Fahrizal Zakaria

    2012-01-01

    The objective of this research was to study the production of fish protein hydrolysate (FPH) from fish by-product prepared by enzymatichydrolysis. Fish by-product were prepared using Alcalase and Flavourzyme enzyme and properties of FPH were analyzed. The resultsshowed that FPH prepared using Alcalase enzyme had greater amount of protein (82.66%) than FPH prepared using Flavourzyme enzyme(73.51%). Solubility, emulsifying and foaming properties of FPH prepared using Alcalase were also better t...

  14. Analysis of Protein Expression inBrucella abortusMutants with Different Growth Rates by Two Dimensional Gel Electrophoresis and LC-MS/MS Peptide Analysis.

    Science.gov (United States)

    Park, Woo Bin; Im, Young Bin; Soojin, Shim; Yoo, Han Sang

    2017-10-13

    Brucella abortus is a bacterium that causes brucellosis and is the causative agent of worldwide zoonosis. Pathogenesis of the Brucella abortus infection is so complicates. Thus, several researches have made to solve mechanism in infection of Brucella abortus . While several proteins are revealed as the pathogenic factors by previous researches, underlying mechanism of the Brucella abortus infection is still left to be resolved. We identify the proteins showing the different expression level in Brucella abortus mutants with different biological characteristics that generated by random insertion of transposon. Five mutants were selected based on the biological characteristics, especially the growth features. Total proteins of mutants and wild type were purified and subjected to two dimensional gel electrophoresis. 30 spots of proteins were selected each with an increase and decrease, a change of more than 2-fold was confirmed as compared with the wild type. Selected spots were analyzed using LC-MS/MS peptide analysis. DnaK and ClpB involved in protein aggregation were simultaneously increased. Proteins associated with energy metabolism, SecA and GAPDH were reduced in the expression in some mutants that slower growth rates than wild type. Mutants with slower growth showed decrease with energy metabolism related proteins, while mutants with faster growth showed increase in the pathogenicity related proteins.

  15. Pretreatment of flaxseed protein isolate by high hydrostatic pressure: Impacts on protein structure, enzymatic hydrolysis and final hydrolysate antioxidant capacities.

    Science.gov (United States)

    Perreault, Véronique; Hénaux, Loïc; Bazinet, Laurent; Doyen, Alain

    2017-04-15

    The effect of high hydrostatic pressure (HHP) on flaxseed protein structure and peptide profiles, obtained after protein hydrolysis, was investigated. Isolated flaxseed protein (1%, m/v) was subjected to HHP (600MPa, 5min or 20min at 20°C) prior to hydrolysis with trypsin only and trypsin-pronase. The results demonstrated that HHP treatment induced dissociation of flaxseed proteins and generated higher molecular weight aggregates as a function of processing duration. Fluorescence spectroscopy showed that HHP treatment, as well as processing duration, had an impact on flaxseed protein structure since exposition of hydrophobic amino acid tyrosine was modified. Except for some specific peptides, the concentrations of which were modified, similar peptide profiles were obtained after hydrolysis of pressure-treated proteins using trypsin. Finally, hydrolysates obtained using trypsin-pronase had a greater antioxidant capacity (ORAC) than control samples; these results confirmed that HHP enhanced the generation of antioxidant peptides. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Identification and functional clustering of genes regulating muscle protein degradation from amongst the known C. elegans muscle mutants.

    Directory of Open Access Journals (Sweden)

    Freya Shephard

    Full Text Available Loss of muscle mass via protein degradation is an important clinical problem but we know little of how muscle protein degradation is regulated genetically. To gain insight our labs developed C. elegans into a model for understanding the regulation of muscle protein degradation. Past studies uncovered novel functional roles for genes affecting muscle and/or involved in signalling in other cells or tissues. Here we examine most of the genes previously identified as the sites of mutations affecting muscle for novel roles in regulating degradation. We evaluate genomic (RNAi knockdown approaches and combine them with our established genetic (mutant and pharmacologic (drugs approaches to examine these 159 genes. We find that RNAi usually recapitulates both organismal and sub-cellular mutant phenotypes but RNAi, unlike mutants, can frequently be used acutely to study gene function solely in differentiated muscle. In the majority of cases where RNAi does not produce organismal level phenotypes, sub-cellular defects can be detected; disrupted proteostasis is most commonly observed. We identify 48 genes in which mutation or RNAi knockdown causes excessive protein degradation; myofibrillar and/or mitochondrial morphologies are also disrupted in 19 of these 48 cases. These 48 genes appear to act via at least three sub-networks to control bulk degradation of protein in muscle cytosol. Attachment to the extracellular matrix regulates degradation via unidentified proteases and affects myofibrillar and mitochondrial morphology. Growth factor imbalance and calcium overload promote lysosome based degradation whereas calcium deficit promotes proteasome based degradation, in both cases myofibrillar and mitochondrial morphologies are largely unaffected. Our results provide a framework for effectively using RNAi to identify and functionally cluster novel regulators of degradation. This clustering allows prioritization of candidate genes/pathways for future

  17. Cancer-associated PTEN mutants act in a dominant negative manner to suppress PTEN protein function

    OpenAIRE

    Papa, Antonella; Wan, Lixin; Bonora, Massimo; Salmena, Leonardo; Song, Min Sup; Hobbs, Robin M.; Lunardi, Andrea; Webster, Kaitlyn; Ng, Christopher; Newton, Ryan H.; Knoblauch, Nicholas; Guarnerio, Jlenia; Ito, Keisuke; Turka, Laurence A.; Beck, Andy H.

    2014-01-01

    PTEN dysfunction plays a crucial role in the pathogenesis of hereditary and sporadic cancers. Here we show that PTEN homo-dimerizes, and in this active conformation exerts lipid phosphatase activity on PtdIns(3,4,5)P3. We demonstrate that catalytically inactive cancer-associated PTEN mutants hetero-dimerize with wild-type PTEN and constrain its phosphatase activity in a dominant-negative manner. To study the consequences of homo- and hetero-dimerization of wild-type and mutant PTEN in vivo, w...

  18. [Construction and characterization of a recombinant mutant homolog of the CheW protein from Thermotoga petrophila RKU-1].

    Science.gov (United States)

    Grishin, D V; Zhdanov, D D; Gladilina, Ju A; Pokrovsky, V S; Podobed, O V; Pokrovskaya, M V; Aleksandrova, S S; Milyushkina, A L; Vigovskiy, M A; Sokolov, N N

    2018-01-01

    In the work a recombinant chemotaxis protein CheW from Thermotoga petrophila RKU-1 (TpeCheW) and its mutant homolog (TpeCheW-mut) were created. It was shown that, despite the low homology with CheW prototypes from intestinal bacteria, these proteins didn't cause metabolic overload and were well expressed by cells of E. coli laboratory strains. We have discovered a broad spectrum of industrial valuable properties of the TpeCheW-mut protein such as stability in a wide range of temperatures and pH, high expression level, solubility and possibility of the application of a simple low-stage purification methodology with the use of preliminary heat treatment. Possible directions of the scientific and industrial application of this protein were claimed.

  19. Change in the enzymatic dual function of the peroxiredoxin protein by gamma irradiation

    International Nuclear Information System (INIS)

    An, Byung Chull; Lee, Seung Sik; Lee, Jae Taek; Park, Chul-Hong; Lee, Sang Yeol; Chung, Byung Yeoup

    2012-01-01

    PP1084 protein was exposed to gamma irradiation ranging from 5 to 500 kGy. Native PAGE showed minor structural changes in PP1084 at 5 kGy, and major structural changes at >15 kGy. Size-exclusion chromatography (SEC) showed the formation of a new shoulder peak when the protein was irradiated with 15 and 30 kGy, and a double peak appeared at 100 kGy. The results of PAGE and SEC imply that PP1084 protein is degraded by gamma irradiation, with simultaneous oligomerization. PP1084 chaperone activity reached the highest level at 30 kGy of gamma irradiation, and then, decreased in a dose-dependent manner with increasing gamma irradiation. However, the peroxidase activity significantly decreased following exposure to all intensities of gamma irradiation. The improvement of chaperone activity using gamma irradiation might be promoted by the oligomeric structures containing covalently cross-linked amino acids. Consequently, PP1084 modification using gamma irradiation could elevate chaperone activity by about 3–4 folds compared to the non-irradiated protein. - Highlights: ► The structure of PP1084 protein was drastically changed above 15 kGy gamma irradiation. ► PP1084 chaperone activity reached the highest level at 30 kGy of gamma irradiation. ► PP1084 modification using gamma irradiation could elevate chaperone activity by about 3–4 folds.

  20. Overexpression of the autophagic beclin-1 protein clears mutant ataxin-3 and alleviates Machado-Joseph disease.

    Science.gov (United States)

    Nascimento-Ferreira, Isabel; Santos-Ferreira, Tiago; Sousa-Ferreira, Lígia; Auregan, Gwennaëlle; Onofre, Isabel; Alves, Sandro; Dufour, Noëlle; Colomer Gould, Veronica F; Koeppen, Arnulf; Déglon, Nicole; Pereira de Almeida, Luís

    2011-05-01

    Machado-Joseph disease, also known as spinocerebellar ataxia type 3, is the most common of the dominantly inherited ataxias worldwide and is characterized by mutant ataxin-3 misfolding, intracellular accumulation of aggregates and neuronal degeneration. Here we investigated the implication of autophagy, the major pathway for organelle and protein turnover, in the accumulation of mutant ataxin-3 aggregates and neurodegeneration found in Machado-Joseph disease and we assessed whether specific stimulation of this pathway could mitigate the disease. Using tissue from patients with Machado-Joseph disease, transgenic mice and a lentiviral-based rat model, we found an abnormal expression of endogenous autophagic markers, accumulation of autophagosomes and decreased levels of beclin-1, a crucial protein in the early nucleation step of autophagy. Lentiviral vector-mediated overexpression of beclin-1 led to stimulation of autophagic flux, mutant ataxin-3 clearance and overall neuroprotective effects in neuronal cultures and in a lentiviral-based rat model of Machado-Joseph disease. These data demonstrate that autophagy is a key degradation pathway, with beclin-1 playing a significant role in alleviating Machado-Joseph disease pathogenesis.

  1. A mutant of SWAP-70, a phosphatidylinositoltrisphosphate binding protein, transforms mouse embryo fibroblasts, which is inhibited by sanguinarine.

    Directory of Open Access Journals (Sweden)

    Yasuhisa Fukui

    2010-12-01

    Full Text Available SWAP-70, a phosphatidylinositol trisphosphate (PtdIns(3,4,5P(3 binding protein, has been suggested to be involved in transformation of mouse embryo fibroblasts (MEFs as well as membrane ruffling after growth factor stimulation of the cells. A mutant, SWAP-70-374, was found to be able to bind to F-actin in vitro, whereas wild-type SWAP-70 failed to do so. This mutant was present at the plasma membrane without any stimulation while the wild-type protein was present only in the cytosol unless cells were stimulated with EGF. Expression of this mutant in MEFs resulted in morphologic transformation, fast growth, and loss of contact inhibition, suggesting that SWAP-70 with this mutation can transform the cells. ERK1/2 was activated in SWAP-70-374-transformed cells. Use of MEK inhibitors revealed that the ERK1/2 pathway does not affect the cell growth of MEFs but is responsible for loss of contact inhibition. To investigate the function of SWAP-70 further, drugs that can inhibit SWAP-70-dependent cell responses were screened. Among various drugs, sanguinarine was found to inhibit transformation of MEFs by SWAP-70-374. This drug was able to inhibit SWAP-70-mediated membrane ruffling as well, suggesting that its effect was closely related to the SWAP-70 signaling pathway. These results suggest that SWAP-70-374 can activate some signaling pathways, including the ERK1/2 pathway, to transform MEFs.

  2. Molecular Investigation of the Mechanism of Non-Enzymatic Hydrolysis of Proteins and the Predictive Algorithm for Susceptibility.

    Science.gov (United States)

    Lauer, Timothy M; Wood, Geoffrey P F; Farkas, David; Sathish, Hasige A; Samra, Hardeep S; Trout, Bernhardt L

    2016-06-14

    A number of potential degradation routes can limit the shelf life of a biotherapeutic. While these are experimentally measurable, the tests to do so require a substantial investment in both time and material, resources rarely available early in the drug development process. To address the potential degradation route of non-enzymatic hydrolysis, we performed a molecular modeling analysis, together with an experimental study, to gain detailed insight into the reaction. On the basis of the mechanism, an algorithm for predicting the likely cleavage sites of a protein has been created. This algorithm measures four key properties during a molecular dynamics simulation, which relate to the key steps of the hydrolysis mechanism, in particular the rate-determining step (which can vary depending on the local environment). The first two properties include the secondary structure and the surface exposure of the amide bond, both of which help detect if the addition of the proton to the amide bond is possible. The second two properties relate to whether the side chain can cyclize and form a furane ring. These two properties are the orientation of the side chain relative to the amide bond and the number of hydrogen bonds between the side chain and the surrounding protein. Overall, the algorithm performs well at identifying reactive versus nonreactive bonds. The algorithm correctly classifies nearly 90% of all amide bonds following an aspartic or glutamic acid residue as reactive or nonreactive.

  3. Seafood-like flavour obtained from the enzymatic hydrolysis of the protein by-products of seaweed (Gracilaria sp.).

    Science.gov (United States)

    Laohakunjit, Natta; Selamassakul, Orrapun; Kerdchoechuen, Orapin

    2014-09-01

    An enzymatic bromelain seaweed protein hydrolysate (eb-SWPH) was characterised as the precursor for thermally processed seafood flavour. Seaweed (Gracilaria fisheri) protein after agar extraction was hydrolysed using bromelain (enzyme activity=119,325 U/g) at 0-20% (w/w) for 0.5-24 h. Optimal hydrolysis conditions were determined using response surface methodology. The proposed model took into account the interaction effect of the enzyme concentration and hydrolysis time on the physicochemical properties and volatile components of eb-SWPH. The optimal hydrolysis conditions for the production of eb-SWPH were 10% bromelain for 3h, which resulted in a 38.15% yield and a 62.91% degree of hydrolysis value. Three free amino acids, arginine, lysine, and leucine, were abundant in the best hydrolysate. Ten volatile flavours of the best eb-SWPH were identified using gas chromatography/mass spectrometry. The predominant odourants were hexanal, hexanoic acid, nonanoic acid, and dihydroactinidiolide. The thermally processed seafood flavour produced from eb-SWPH exhibited a roasted seafood-like flavouring. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Comparison of mitochondrial and nucleolar RNase MRP reveals identical RNA components with distinct enzymatic activities and protein components.

    Science.gov (United States)

    Lu, Qiaosheng; Wierzbicki, Sara; Krasilnikov, Andrey S; Schmitt, Mark E

    2010-03-01

    RNase MRP is a ribonucleoprotein endoribonuclease found in three cellular locations where distinct substrates are processed: the mitochondria, the nucleolus, and the cytoplasm. Cytoplasmic RNase MRP is the nucleolar enzyme that is transiently relocalized during mitosis. Nucleolar RNase MRP (NuMRP) was purified to homogeneity, and we extensively purified the mitochondrial RNase MRP (MtMRP) to a single RNA component identical to the NuMRP RNA. Although the protein components of the NuMRP were identified by mass spectrometry successfully, none of the known NuMRP proteins were found in the MtMRP preparation. Only trace amounts of the core NuMRP protein, Pop4, were detected in MtMRP by Western blot. In vitro activity of the two enzymes was compared. MtMRP cleaved only mitochondrial ORI5 substrate, while NuMRP cleaved all three substrates. However, the NuMRP enzyme cleaved the ORI5 substrate at sites different than the MtMRP enzyme. In addition, enzymatic differences in preferred ionic strength confirm these enzymes as distinct entities. Magnesium was found to be essential to both enzymes. We tested a number of reported inhibitors including puromycin, pentamidine, lithium, and pAp. Puromycin inhibition suggested that it binds directly to the MRP RNA, reaffirming the role of the RNA component in catalysis. In conclusion, our study confirms that the NuMRP and MtMRP enzymes are distinct entities with differing activities and protein components but a common RNA subunit, suggesting that the RNA must be playing a crucial role in catalytic activity.

  5. Enzymatic and protein differences between infective larvae of Trichostrongylus colubriformis conditioned or not conditioned to hypobiosis

    Czech Academy of Sciences Publication Activity Database

    Vadlejch, J.; Langrová, I.; Borovský, M.; Sedmíková, M.; Jankovská, I.; Fechtner, J.; Lytvynets, Andrej

    2006-01-01

    Roč. 43, č. 2 (2006), s. 64-68 ISSN 0440-6605 Institutional research plan: CEZ:AV0Z50110509 Keywords : Trichostrongylus colubriformis * hypobiosis * protein * enzymes * SDS - PAGE * API-ZYM Subject RIV: GJ - Animal Vermins ; Diseases, Veterinary Medicine Impact factor: 0.500, year: 2006

  6. Rumen escape protein in concentrate ingredients determined with the nylon bag and enzymatic techniques

    NARCIS (Netherlands)

    Cone, J.W.; Kamman, A.A.; Gelder, A.H. van; Hindle, V.A.

    2002-01-01

    Rumen escape protein (REP) was determined for 26 concentrate feed ingredients using the nylon bag technique and in vitro using a proteolytic enzyme preparation of Streptomyces griseus. In vitro, the samples were incubated for 0, 1, 6 and 24 h. The highest correlation was observed between percentage

  7. Characterization of PUD-1 and PUD-2, two proteins up-regulated in a long-lived daf-2 mutant.

    Science.gov (United States)

    Ding, Yue-He; Du, Yun-Guang; Luo, Shukun; Li, Yu-Xin; Li, Tie-Mei; Yoshina, Sawako; Wang, Xing; Klage, Karsten; Mitani, Shohei; Ye, Keqiong; Dong, Meng-Qiu

    2013-01-01

    C. elegans PUD-1 and PUD-2, two proteins up-regulated in daf-2(loss-of-function) (PUD), are homologous 17-kD proteins with a large abundance increase in long-lived daf-2 mutant animals of reduced insulin signaling. In this study, we show that both PUD-1 and PUD-2 are abundantly expressed in the intestine and hypodermis, and form a heterodimer. We have solved their crystal structure to 1.9-Å resolution and found that both proteins adopt similar β-sandwich folds in the V-shaped dimer. In contrast, their homologs PUD-3, PUD-4, PUDL-1 and PUDL-2 are all monomeric proteins with distinct expression patterns in C. elegans. Thus, the PUD-1/PUD-2 heterodimer probably has a function distinct from their family members. Neither overexpression nor deletion of pud-1 and pud-2 affected the lifespan of WT or daf-2 mutant animals, suggesting that their induction in daf-2 worms does not contribute to longevity. Curiously, deletion of pud-1 and pud-2 was associated with a protective effect against paralysis induced by the amyloid β-peptide (1-42), which further enhanced the protection conferred by daf-2(RNAi) against Aβ.

  8. Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants.

    Science.gov (United States)

    Lee, Hyelee; Liu, Shih-Yuan

    2017-03-25

    We describe a general synthesis of 1,2-azaborines using standard air-free techniques and protein complex preparation with T4 lysozyme mutants by vapor diffusion. Oxygen- and moisture-sensitive compounds are prepared and isolated under an inert atmosphere (N2) using either a vacuum gas manifold or a glove box. As an example of azaborine synthesis, we demonstrate the synthesis and purification of the volatile N-H-B-ethyl-1,2-azaborine by a five-step sequence involving distillation and column chromatography for the isolation of products. T4 lysozyme mutants L99A and L99A/M102Q are expressed with Escherichia coli RR1 strain. Standard protocols for chemical cell lysis followed by purification using carboxymethyl ion exchange column affords protein of sufficiently high purity for crystallization. Protein crystallization is performed in various concentrations of precipitant at different pH ranges using the hanging drop vapor diffusion method. Complex preparation with the small molecules is carried out by vapor diffusion method under an inert atmosphere. X-ray diffraction analysis of the crystal complex provides unambiguous structural evidence of binding interactions between the protein binding site and 1,2-azaborines.

  9. The Protein Cost of Metabolic Fluxes: Prediction from Enzymatic Rate Laws and Cost Minimization.

    Directory of Open Access Journals (Sweden)

    Elad Noor

    2016-11-01

    Full Text Available Bacterial growth depends crucially on metabolic fluxes, which are limited by the cell's capacity to maintain metabolic enzymes. The necessary enzyme amount per unit flux is a major determinant of metabolic strategies both in evolution and bioengineering. It depends on enzyme parameters (such as kcat and KM constants, but also on metabolite concentrations. Moreover, similar amounts of different enzymes might incur different costs for the cell, depending on enzyme-specific properties such as protein size and half-life. Here, we developed enzyme cost minimization (ECM, a scalable method for computing enzyme amounts that support a given metabolic flux at a minimal protein cost. The complex interplay of enzyme and metabolite concentrations, e.g. through thermodynamic driving forces and enzyme saturation, would make it hard to solve this optimization problem directly. By treating enzyme cost as a function of metabolite levels, we formulated ECM as a numerically tractable, convex optimization problem. Its tiered approach allows for building models at different levels of detail, depending on the amount of available data. Validating our method with measured metabolite and protein levels in E. coli central metabolism, we found typical prediction fold errors of 4.1 and 2.6, respectively, for the two kinds of data. This result from the cost-optimized metabolic state is significantly better than randomly sampled metabolite profiles, supporting the hypothesis that enzyme cost is important for the fitness of E. coli. ECM can be used to predict enzyme levels and protein cost in natural and engineered pathways, and could be a valuable computational tool to assist metabolic engineering projects. Furthermore, it establishes a direct connection between protein cost and thermodynamics, and provides a physically plausible and computationally tractable way to include enzyme kinetics into constraint-based metabolic models, where kinetics have usually been ignored or

  10. A two-step, one-pot enzymatic method for preparation of duck egg white protein hydrolysates with high antioxidant activity.

    Science.gov (United States)

    Ren, Yao; Wu, Hui; Li, Xiaofeng; Lai, Furao; Zhao, Guanglei; Xiao, Xinglong

    2014-02-01

    Biocatalytic hydrolysis reactions were designed for preparation of bioactive hydrolysate of duck egg white protein (DEWP) employing two enzymes in one pot. Firstly, the fresh DEWP was thermal treated at 95 °C, for 40 min at pH 10, to effectively deactivate enzyme inhibitors thus facilitating the following two-step enzymatic hydrolysis. Compared with single-enzyme processes, the two-step enzymatic procedures showed much higher reaction efficiency. The first enzymatic step (in the presence of Alcalase or hydrolase SEEP) allowed to hydrolyze DEWP with degree of hydrolysis (DH) of 8.8-10% and soluble peptide yield (SEP) of 60.5-70.2% in a short period (4 h). The second enzymatic step (in the presence of Trypsin or Alcalase) gave a further degradation of DEWP with DH and SEP being more than 26.2% and 90.4%, respectively. The final hydrolysates exhibited high antioxidant activity in an evident DH dependent manner. The hydrolysates achieved by sequential addition of the proteinase SEEP and Alcalase at DH value 21% gave the highest antioxidant activity, which was mainly ascribed to the changes in the amino acid compositions that the contents of some key amino acids and total hydrophobic amino acids were significantly improved by the enzymatic hydrolysis.

  11. Enzymatic protein hydrolysates from high pressure-pretreated isolated pea proteins have better antioxidant properties than similar hydrolysates produced from heat pretreatment.

    Science.gov (United States)

    Girgih, Abraham T; Chao, Dongfang; Lin, Lin; He, Rong; Jung, Stephanie; Aluko, Rotimi E

    2015-12-01

    Isolated pea protein (IPP) dispersions (1%, w/v) were pretreated with high pressure (HP) of 200, 400, or 600 MPa for 5 min at 24 °C or high temperature (HT) for 30 min at 100 °C prior to hydrolysis with 1% (w/w) Alcalase. HP pretreatment of IPP at 400 and 600 MPa levels led to significantly (P40%) oxygen radical absorption capacity (ORAC) of hydrolysates. 2,2-Diphenyl-1-picrylhydrazyl, superoxide radical and hydroxyl radical scavenging activities of pea protein hydrolysates were also significantly (P<0.05) improved (25%, 20%, and 40%, respectively) by HP pretreatment of IPP. Protein hydrolysates from HT IPP showed no ORAC, superoxide or hydroxyl scavenging activity but had significantly (P<0.05) improved (80%) ferric reducing antioxidant power. The protein hydrolysates had weaker antioxidant properties than glutathione but overall, the HP pretreatment was superior to HT pretreatment in facilitating enzymatic release of antioxidant peptides from IPP. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Enzymatic Release and Characterization of Novel Bioactive Peptides from Milk Proteins

    DEFF Research Database (Denmark)

    De Gobba, Cristian

    -inhibitory, antioxidant and antimicrobial peptides) released from milk proteins by mean of enzyme-catalysed hydrolysis. Goat milk fractions (produced using microfiltration membranes) and bovine casein were used as substrates. The goat milk fractions (retentate, permeate and skimmed milk) were hydrolysed with two...... protein hydrolysates made in other studies. Regarding radical scavenging activity, the bovine casein hydrolysates also showed a positive correlation between extent of hydrolysis and activity, although the difference between the unhydrolysed sample and the hydrolysates was less marked. The goat milk....... The bovine casein did not show an increase in iron chelation capacity after hydrolysis, while the goat milk hydrolysates showed again a difference in the substrates and not in degree of hydrolysis, with the permeate as the most active substrate. Partial hydrolysis of the bovine casein increased...

  13. A yeast mutant lacking mitochondrial porin is respiratory-deficient, but can recover respiration with simultaneous accumulation of an 86-kd extramitochondrial protein.

    Science.gov (United States)

    Dihanich, M; Suda, K; Schatz, G

    1987-03-01

    A yeast mutant lacking the only known pore-forming protein of the mitochondrial outer membrane was constructed by gene disruption. The mutant retained all other major proteins of the mitochondrial outer membrane, but was severely deficient in mitochondrial cytochromes and initially did not grow on the non-fermentable carbon source, glycerol. However, it could slowly adapt to glycerol; adaptation was accompanied by the partial restoration of cytochrome levels and massive accumulation of an 86-kd polypeptide in extramitochondrial cell fractions.

  14. Chemical composition and immunomodulatory effects of enzymatic protein hydrolysates from common carp (Cyprinus carpio) egg.

    Science.gov (United States)

    Chalamaiah, M; Hemalatha, R; Jyothirmayi, T; Diwan, Prakash V; Bhaskarachary, K; Vajreswari, A; Ramesh Kumar, R; Dinesh Kumar, B

    2015-02-01

    The aim of this study was to prepare protein hydrolysates from underutilized common carp (Cyprinus carpio) egg and to investigate their immunomodulatory effects in vivo. Common carp (Cyprinus carpio) egg (roe) was hydrolysed by pepsin, trypsin, and Alcalase. Chemical composition (proximate, amino acid, mineral and fatty acid compositions) and molecular mass distribution of the three hydrolysates were determined. The carp egg protein hydrolysates (CEPHs) were evaluated for their immunomodulatory effects in BALB/c mice. CEPHs (0.25, 0.5 and 1 g/kg body weight) were orally administered daily to female BALB/c mice (4-6 wk, 18-20 g) for a period of 45 d. After 45 d, mice were sacrificed and different tissues were collected for the immunologic investigations. The three hydrolysates contained high protein content (64%-73%) with all essential amino acids, and good proportion of ω-3 fatty acids, especially docosahexaenoic acid. Molecular mass analysis of hydrolysates confirmed the conversion of large-molecular-weight roe proteins into peptides of different sizes (5-90 kDa). The three hydrolysates significantly enhanced the proliferation of spleen lymphocytes. Pepsin hydrolysate (0.5 g/kg body weight) significantly increased the splenic natural killer cell cytotoxicity, mucosal immunity (secretory immunoglobulin A) in the gut and level of serum immunoglobulin A. Whereas Alcalase hydrolysate induced significant increases in the percentages of CD4+ and CD8+ cells in spleen. The results demonstrate that CEPHs are able to improve the immune system and further reveal that different CEPHs may exert differential influences on the immune function. These results indicate that CEPHs could be useful for several applications in the health food, pharmaceutical, and nutraceutical industries. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Fractionation and identification of antioxidant peptides from an enzymatically hydrolysed Palmaria palmata protein isolate.

    Science.gov (United States)

    Harnedy, Pádraigín A; O'Keeffe, Martina B; FitzGerald, Richard J

    2017-10-01

    Proteins derived from the macroalgal species Palmaria palmata have emerged as potential substrates for the generation of bioactive peptides. The aim of this study was to fractionate, identify and characterize antioxidant peptides from a P. palmata protein hydrolysate. The P. palmata protein hydrolysate generated with the food-grade proteolytic enzyme Corolase PP was sequentially fractionated using solid phase extraction and semi-preparative (SP) RP-HPLC. The most active SP-RP-HPLC peptide fraction (SP-RP-HPLC-30-F26) was analysed by ESI-MS/MS. Seventeen novel peptide sequences were identified in this fraction. Of the peptides selected for synthesis, Ser-Asp-Ile-Thr-Arg-Pro-Gly-Gly-Asn-Met, showed the highest oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) activity with values of 152.43±2.73 and 21.23±0.90nmolTE/μmol peptide, respectively. The results presented herein indicate that P. palmata derived peptides may have potential applications as health enhancing ingredients and as food preservatives due to their antioxidant activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Cellular Trafficking of the Pathogenic Prion Protein PrPSc and Phenotypic Characterisation of Deletion Mutants in the Hydrophobic Domain of the Normal Prion Protein PrPC

    OpenAIRE

    Veith, Nathalie Monika

    2008-01-01

    The localisation of the pathogenic prion protein PrPSc was investigated with light and electron microscopy. The PrPSc specific antibody 15B3 was tested for its efficiency in immuncytochemistry. Subsequently, an appropriate method was found to stain PrPSc selectively. PrPSc was detected in clathrin coated pits, early endosomes, late endosomes/lysosomes nad exosomes. PrPSc could not be observed in lipid droplets.In the next part of the thesis different mutants of the prion protein carrying micr...

  17. Latent membrane protein 1 deletion mutants accumulate in reed-sternberg cells of human immunodeficiency virus-related Hodgkin's lymphoma.

    Science.gov (United States)

    Guidoboni, Massimo; Ponzoni, Maurilio; Caggiari, Laura; Lettini, Antonia A; Vago, Luca; De Re, Valli; Gloghini, Annunziata; Zancai, Paola; Carbone, Antonino; Boiocchi, Mauro; Dolcetti, Riccardo

    2005-02-01

    The origin and biological significance of deletions at the 3' end of the Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP-1) gene are still controversial. We herein demonstrate that LMP-1 deletion mutants are highly associated with human immunodeficiency virus-related Hodgkin's lymphoma (HIV-HL) of Italian patients (29 of 31 cases; 93.5%), a phenomenon that is not due to a peculiar distribution of EBV strains in this area. In fact, although HIV-HL patients are infected by multiple EBV variants, we demonstrate that LMP-1 deletion mutants preferentially accumulate within neoplastic tissues. Subcloning and sequencing of the 3' LMP-1 ends of two HIV-HL genes in which both variants were present showed the presence of molecular signatures suggestive of a likely derivation of the LMP-1 deletion mutant from a nondeletion ancestor. This phenomenon likely occurs within tumor cells in vivo, as shown by the detection of both LMP-1 variants in single microdissected Reed-Sternberg cells, and may at least in part explain the high prevalence of LMP-1 deletions associated with HIV-HL.

  18. Latent Membrane Protein 1 Deletion Mutants Accumulate in Reed-Sternberg Cells of Human Immunodeficiency Virus-Related Hodgkin's Lymphoma

    OpenAIRE

    Guidoboni, Massimo; Ponzoni, Maurilio; Caggiari, Laura; Lettini, Antonia A.; Vago, Luca; De Re, Valli; Gloghini, Annunziata; Zancai, Paola; Carbone, Antonino; Boiocchi, Mauro; Dolcetti, Riccardo

    2005-01-01

    The origin and biological significance of deletions at the 3′ end of the Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP-1) gene are still controversial. We herein demonstrate that LMP-1 deletion mutants are highly associated with human immunodeficiency virus-related Hodgkin's lymphoma (HIV-HL) of Italian patients (29 of 31 cases; 93.5%), a phenomenon that is not due to a peculiar distribution of EBV strains in this area. In fact, although HIV-HL patients are infected by multi...

  19. Co-activation of STAT3 and YES-Associated Protein 1 (YAP1) Pathway in EGFR-Mutant NSCLC.

    Science.gov (United States)

    Chaib, Imane; Karachaliou, Niki; Pilotto, Sara; Codony Servat, Jordi; Cai, Xueting; Li, Xuefei; Drozdowskyj, Ana; Servat, Carles Codony; Yang, Jie; Hu, Chunping; Cardona, Andres Felipe; Vivanco, Guillermo Lopez; Vergnenegre, Alain; Sanchez, Jose Miguel; Provencio, Mariano; de Marinis, Filipo; Passaro, Antonio; Carcereny, Enric; Reguart, Noemi; Campelo, Charo Garcia; Teixido, Christina; Sperduti, Isabella; Rodriguez, Sonia; Lazzari, Chiara; Verlicchi, Alberto; de Aguirre, Itziar; Queralt, Cristina; Wei, Jia; Estrada, Roger; Puig de la Bellacasa, Raimon; Ramirez, Jose Luis; Jacobson, Kirstine; Ditzel, Henrik J; Santarpia, Mariacarmela; Viteri, Santiago; Molina, Migual Angel; Zhou, Caicun; Cao, Peng; Ma, Patrick C; Bivona, Trever G; Rosell, Rafael

    2017-09-01

    The efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC) is limited by adaptive activation of cell survival signals. We hypothesized that both signal transducer and activator of transcription 3 (STAT3) and Src-YES-associated protein 1 (YAP1) signaling are dually activated during EGFR TKI treatment to limit therapeutic response. We used MTT and clonogenic assays, immunoblotting, and quantitative polymerase chain reaction to evaluate the efficacy of EGFR TKI alone and in combination with STAT3 and Src inhibition in three EGFR-mutant NSCLC cell lines. The Chou-Talalay method was used for the quantitative determination of drug interaction. We examined tumor growth inhibition in one EGFR-mutant NSCLC xenograft model (n = 4 mice per group). STAT3 and YAP1 expression was evaluated in tumors from 119 EGFR-mutant NSCLC patients (64 in an initial cohort and 55 in a validation cohort) by quantitative polymerase chain reaction. Kaplan-Meier and Cox regression analyses were used to assess the correlation between survival and gene expression. All statistical tests were two-sided. We discovered that lung cancer cells survive initial EGFR inhibitor treatment through activation of not only STAT3 but also Src-YAP1 signaling. Cotargeting EGFR, STAT3, and Src was synergistic in two EGFR-mutant NSCLC cell lines with a combination index of 0.59 (95% confidence interval [CI] = 0.54 to 0.63) for the PC-9 and 0.59 (95% CI = 0.54 to 0.63) for the H1975 cell line. High expression of STAT3 or YAP1 predicted worse progression-free survival (hazard ratio [HR] = 3.02, 95% CI = 1.54 to 5.93, P = .001, and HR = 2.57, 95% CI = 1.30 to 5.09, P = .007, respectively) in an initial cohort of 64 EGFR-mutant NSCLC patients treated with firstline EGFR TKIs. Similar results were observed in a validation cohort. Our study uncovers a coordinated signaling network centered on both STAT3 and Src-YAP signaling

  20. Autotaxin (ATX): a multi-functional and multi-modular protein possessing enzymatic lysoPLD activity and matricellular properties.

    Science.gov (United States)

    Yuelling, Larra M; Fuss, Babette

    2008-09-01

    Recent studies have established that autotaxin (ATX), also known as phosphodiesterase Ialpha/autotaxin (PD-Ialpha/ATX) or (ecto)nucleotide pyrophosphatase/phosphodiesterase 2 [(E)NPP2], represents a multi-functional and multi-modular protein. ATX was initially thought to function exclusively as a phosphodiesterase/pyrophosphatase. However, it has become apparent that this enzymatically active site, which is ultimately responsible for ATX's originally discovered property of tumor cell motility stimulation, mediates the conversion of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA). In addition, a separate functionally active domain, here referred to as the Modulator of Oligodendrocyte Remodeling and Focal adhesion Organization (MORFO) domain, was discovered in studies analyzing the role of ATX during the differentiation of myelinating cells of the central nervous system (CNS), namely oligodendrocytes. This novel domain was found to mediate anti-adhesive, i.e. matricellular, properties and to promote morphological maturation of oligodendrocytes. In this review, we summarize our current understanding of ATX's structure-function domains and discuss their contribution to the presently known main functional roles of ATX.

  1. pH-stat vs. free-fall pH techniques in the enzymatic hydrolysis of whey proteins.

    Science.gov (United States)

    Fernández, Ayoa; Kelly, Phil

    2016-05-15

    Enzymatic hydrolysis of a commercial whey protein isolate (WPI) using either trypsin or Protamex® was compared using controlled (pH-stat) and uncontrolled (free-fall) pH conditions. pH-stat control at the enzyme's optimum value led to a more rapid rate of WPI hydrolysis by trypsin, while the opposite was the case when Protamex® was used. Furthermore, the choice of alkaline solution used to maintain constant pH during pH-stat experiments appeared to affect the reaction rate, being higher when KOH is added to the reaction mixture instead of NaOH. It would appear that potassium may play a role as co-factor or activator for the activity of this particular protease preparation. Although pH-stat techniques are usually considered to yield better hydrolysis kinetics, these findings suggest that the response of proteolytic enzyme preparations to static or free-fall pH control should be checked in advance, particularly when undertaking large scale production of WPI hydrolysates. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. An autoactive mutant of the M flax rust resistance protein has a preference for binding ATP, whereas wild-type M protein binds ADP.

    Science.gov (United States)

    Williams, Simon J; Sornaraj, Pradeep; deCourcy-Ireland, Emma; Menz, R Ian; Kobe, Bostjan; Ellis, Jeffrey G; Dodds, Peter N; Anderson, Peter A

    2011-08-01

    Resistance (R) proteins are key regulators of the plant innate immune system and are capable of pathogen detection and activation of the hypersensitive cell death immune response. To understand the molecular mechanism of R protein activation, we undertook a phenotypic and biochemical study of the flax nucleotide binding (NB)-ARC leucine-rich repeat protein, M. Using Agrobacterium-mediated transient expression in flax cotyledons, site-directed mutations of key residues within the P-loop, kinase 2, and MHD motifs within the NB-ARC domain of M were shown to affect R protein function. When purified using a yeast expression system and assayed for ATP and ADP, these mutated proteins exhibited marked differences in the quantity and identity of the bound nucleotide. ADP was bound to recombinant wild-type M protein, while the nonfunctional P-loop mutant did not have any nucleotides bound. In contrast, ATP was bound to an autoactive M protein mutated in the highly conserved MHD motif. These data provide direct evidence supporting a model of R protein function in which the "off" R protein binds ADP and activation of R protein defense signaling involves the exchange of ADP for ATP.

  3. Protein-Rich Fraction of Cnidoscolus urens (L. Arthur Leaves: Enzymatic Characterization and Procoagulant and Fibrinogenolytic Activities

    Directory of Open Access Journals (Sweden)

    Yamara A. S. de Menezes

    2014-03-01

    Full Text Available Proteolytic enzymes are important macromolecules in the regulation of biochemical processes in living organisms. Additionally, these versatile biomolecules have numerous applications in the industrial segment. In this study we have characterized a protein-rich fraction of Cnidoscolus urens (L. Arthur leaves, rich in proteolytic enzymes, and evaluated its effects on the coagulation cascade. Three protein-rich fractions were obtained from the crude extract of C. urens leaves by precipitation with acetone. Fraction F1.0 showed higher proteolytic activity upon azocasein, and thus, was chosen for subsequent tests. The proteolytic activity of F1.0 on fibrinogen was dose-dependent and time-dependent. The extract demonstrated procoagulant activity on citrated plasma and reduced the APTT, not exerting effects on PT. Despite the fibrin(ogenolytic activity, F1.0 showed no defibrinogenating activity in vivo. The fraction F1.0 did not express hemorrhagic nor hemolytic activities. The proteolytic activity was inhibited by E-64, EDTA and in the presence of metal ions, and increased when pretreated with reducing agents, suggesting that the observed activity was mostly due to cysteine proteases. Several bands with proteolytic activity were detected by zymography with gelatin, albumin and fibrinogen. The optimal enzymatic activity was observed in temperature of 60 °C and pH 5.0, demonstrating the presence of acidic proteases. In conclusion, these results could provide basis for the pharmacological application of C. urens proteases as a new source of bioactive molecules to treat bleeding and thrombotic disorders.

  4. GALT protein database, a bioinformatics resource for the management and analysis of structural features of a galactosemia-related protein and its mutants.

    Science.gov (United States)

    d'Acierno, Antonio; Facchiano, Angelo; Marabotti, Anna

    2009-06-01

    We describe the GALT-Prot database and its related web-based application that have been developed to collect information about the structural and functional effects of mutations on the human enzyme galactose-1-phosphate uridyltransferase (GALT) involved in the genetic disease named galactosemia type I. Besides a list of missense mutations at gene and protein sequence levels, GALT-Prot reports the analysis results of mutant GALT structures. In addition to the structural information about the wild-type enzyme, the database also includes structures of over 100 single point mutants simulated by means of a computational procedure, and the analysis to each mutant was made with several bioinformatics programs in order to investigate the effect of the mutations. The web-based interface allows querying of the database, and several links are also provided in order to guarantee a high integration with other resources already present on the web. Moreover, the architecture of the database and the web application is flexible and can be easily adapted to store data related to other proteins with point mutations. GALT-Prot is freely available at http://bioinformatica.isa.cnr.it/GALT/.

  5. Effect of high pressures on the enzymatic activity of commercial milk protein coagulants

    Science.gov (United States)

    Wiśniewska, Krystyna; Reps, Arnold; Jankowska, Agnieszka

    2014-04-01

    This study was aimed at determining the effect of high pressures in the range of 100-1000 MPa/15 min, applied in 100 MPa increments, on the coagulating and proteolytic activity of commercial coagulants produced with genetic engineering methods: Maxiren, Chymogen, Chymax and of a natural rennin preparation, Hala. The coagulating activity of Hala preparation differed compared with the other preparations, due to greater resistance to high pressures, especially in the range of 500-600 MPa. The preparations produced with genetic engineering methods lost their capability for milk protein coagulation by 500 MPa. Pressurization at 200 MPa contributed to their reduced capability for casein macroproteolysis. In contrast, an increase in Chymax, Chymogen, Maxiren and Hala preparations' hydrolytic capability for the macroproteolysis of isoelectric casein was observed upon pressure treatment at 100 and 400 MPa and for microproteolysis after pressure treatment at 200 MPa. Storage (48 h/5°C) of the pressurized preparations had an insignificant effect on their coagulating and proteolytic activities.

  6. Improvement of antioxidant properties of egg white protein enzymatic hydrolysates by membrane ultrafiltration

    Directory of Open Access Journals (Sweden)

    Jovanović Jelena R.

    2016-01-01

    Full Text Available The production of bioactive peptides from egg white proteins (EWPs and their separation are emerging areas with many new applications. The objective of this study was to compare antioxidant activity of three distinct EWP hydrolysates and their peptide fractions prepared by membrane ultrafiltration using membranes with 30, 10 and 1 kDa molecular weight cut-off. The hydrolysates were obtained by thermal and ultrasound pretreated EWPs hydrolyzed with a bacterial protease, Alcalase. It appeared that the pretreatment significantly affected peptide profiles and antioxidant activity of the hydrolysates measured by ABTS, DPPH and FRAP methods. The hydrolysate prepared using Alcalase and ultrasound pretreatment at 40 kHz - 15 min has shown to be the most effective in scavenging both DPPH and ABTS radicals (28.10±1.38 and 79.44±2.31%, respectively. It has been noticed that this hydrolysate had a nutritionally more adequate peptide profile than the other hydrolysates with a much lower amount of peptides <1 kDa (11.19±0.53 % and the greatest content of the peptide fraction in the molecular weight (MW range of 1-10 kDa (28.80±0.07 %. This peptide fraction has been exactly showed the highest DPPH and ABTS antioxidant activity compared to all other fractions having a potential to be used as a functional food ingredient. [Projekat Ministarstva nauke Republike Srbije, br. III46010

  7. Schistosoma mansoni: Heterologous complementation of a yeast null mutant by SmRbx, a protein similar to a RING box protein involved in ubiquitination.

    Science.gov (United States)

    Santos, Débora N; Aguiar, Pedro H N; Lobo, Francisco P; Mourão, Marina M; Tambor, José H M; Valadão, Analina F; Vilas-Boas, Adlane; Nobrega, Francisco G; LoVerde, Philip T; Macedo, Andréa M; Pena, Sérgio D J; Machado, Carlos R; Franco, Glória R

    2007-08-01

    The SCF (Skp1-Cul1-F-box) complex is one of the several E3 ligase enzymes and it catalyzes protein ubiquitination and degradation by the 26S proteasome. Rbx1 is a member of the SCF complex in humans and HRT1 is its yeast orthologue. A cDNA encoding a Schistosoma mansoni Rbx1 homolog was cloned and functionally characterized. Heterologous functional complementation in yeast showed that the worm SmRbx gene was able to complement the HRT1yeast null mutation. Gene deletion constructs for N- and C-termini truncated proteins were used to transform hrt1(-) yeast mutant strains, allowing us to observe that regions reported to be involved in the interaction with cullin1 (Cul1) were essential for SmRbx function. Yeast two-hybrid assays using SmRbx and yeast Cul1 confirmed that SmRbx, but not the mutant SmRbxDelta24N, lacking the N-terminus of the protein, was capable of interacting with Cul1. These results suggest that SmRbx protein is involved in the SCF complex formation.

  8. Protein nutrient value evaluation of mutant strain J5 fruitbody Agaricus bazei murrill by 60Co γ-irradiation

    International Nuclear Information System (INIS)

    Weng Boqi; Huang Tingjun

    2004-01-01

    Protein nutrient value evaluation of mutant strain J 5 fruitbody Agaricus bazei Murrill by 60 Co γ-irradiation was studied. The results showed its total content of amino acids Agaricus bazei murrill was 48.20%; chemical score 58.10; amino acids score 88.60; necessary amino acids index 89.94; biologic value 86.29; nutrient index 29.15. All the index above were higher than that in original strain J 1 , but the ratio score of amino acids of J 5 fruitbody (39.48) was lower than strain J 1 . The results indicated that nutrient value of protein in J 5 was higher than in J 1 . (authors)

  9. Mutant HbpR transcription activator isolation for 2-chlorobiphenyl via green fluorescent protein-based flow cytometry and cell sorting.

    Science.gov (United States)

    Beggah, Siham; Vogne, Christelle; Zenaro, Elena; Van Der Meer, Jan Roelof

    2008-01-01

    Mutants were produced in the A-domain of HbpR, a protein belonging to the XylR family of σ(54)-dependent transcription activators, with the purpose of changing its effector recognition specificity from 2-hydroxybiphenyl (2-HBP, the cognate effector) to 2-chlorobiphenyl (2-CBP). Mutations were introduced in the hbpR gene part for the A-domain via error-prone polymerase chain reaction, and assembled on a gene circuitry plasmid in Escherichia coli, permitting HbpR-dependent induction of the enhanced green fluorescent protein (egfp). Cells with mutant HbpR proteins responsive to 2-CBP were enriched and separated in a flow cytometry-assisted cell-sorting procedure. Some 70 mutants were isolated and the A-domain mutations mapped. One of these had acquired true 2-CBP recognition but reacted hypersensitively to 2-HBP (20-fold more than the wild type), whereas others had reduced sensitivity to 2-HBP but a gain of 2-CBP recognition. Sequencing showed that most mutants carried double or triple mutations in the A-domain gene part, and were not located in previously recognized conserved residues within the XylR family members. Further selection from a new mutant pool prepared of the hypersensitive mutant did not result in increased 2-CBP or reduced 2-HBP recognition. Our data thus demonstrate that a one-step in vitro 'evolutionary' adaptation of the HbpR protein can result in both enhancement and reduction of the native effector recognition.

  10. Defective folding and rapid degradation of mutant proteins is a common disease mechanism in genetic disorders

    DEFF Research Database (Denmark)

    Gregersen, N; Bross, P; Jørgensen, M M

    2000-01-01

    Many disease-causing point mutations do not seriously compromise synthesis of the affected polypeptide but rather exert their effects by impairing subsequent protein folding or stability of the folded protein. This often results in rapid degradation of the affected protein. The concepts of such '......Many disease-causing point mutations do not seriously compromise synthesis of the affected polypeptide but rather exert their effects by impairing subsequent protein folding or stability of the folded protein. This often results in rapid degradation of the affected protein. The concepts...... of such 'conformational disease' are illustrated by reference to cystic fibrosis, phenylketonuria and short-chain acyl-CoA dehydrogenase deficiency. Other cellular components such as chaperones and proteases, as well as environmental factors, may combine to modulate the phenotype of such disorders and this may open up...

  11. Nine mutants of Chlorobium tepidum each unable to synthesize a different chlorosome protein still assemble functional chlorosomes

    DEFF Research Database (Denmark)

    Frigaard, Niels-Ulrik; Li, Hui; Milks, Kirstin J

    2004-01-01

    H, CsmI, CsmJ, and CsmX) but contain relatively little total protein compared to other photosynthetic antenna complexes. Except for CsmA, which has been suggested to bind BChl a, the functions of the chlorosome proteins are not known. Nine mutants in which a single csm gene was inactivated were created......Chlorosomes of the green sulfur bacterium Chlorobium tepidum comprise mostly bacteriochlorophyll c (BChl c), small amounts of BChl a, carotenoids, and quinones surrounded by a lipid-protein envelope. These structures contain 10 different protein species (CsmA, CsmB, CsmC, CsmD, CsmE, CsmF, Csm......, and the BChl c absorbance maximum was blue-shifted about 8 nm, indicating that the structure of the BChl c aggregates in these chlorosomes is altered. The results of the present study establish that, except with CsmA, when the known chlorosome proteins are eliminated individually, none of them are essential...

  12. Modulation of proteolytic polyprotein processing by coxsackievirus mutants resistant to inhibitors targeting phosphatidylinositol-4-kinase IIIβ or oxysterol binding protein.

    Science.gov (United States)

    Lyoo, Heyrhyoung; Dorobantu, Cristina M; van der Schaar, Hilde M; van Kuppeveld, Frank J M

    2017-11-01

    Enteroviruses (e.g. poliovirus, coxsackievirus, and rhinovirus) require several host factors for genome replication. Among these host factors are phosphatidylinositol-4-kinase IIIβ (PI4KB) and oxysterol binding protein (OSBP). Enterovirus mutants resistant to inhibitors of PI4KB and OSBP were previously isolated, which demonstrated a role of single substitutions in the non-structural 3A protein in conferring resistance. Besides the 3A substitutions (i.e., 3A-I54F and 3A-H57Y) in coxsackievirus B3 (CVB3), substitution N2D in 2C was identified in each of the PI4KB-inhibitor resistant CVB3 pools, but its possible benefit has not been investigated yet. In this study, we set out to investigate the possible role of 2C-N2D in the resistance to PI4KB and OSBP inhibition. We show that 2C-N2D by itself did not confer any resistance to inhibitors of PI4KB and OSBP. However, the double mutant (i.e., 2C-N2D/3A-H57Y) showed better replication than the 3A-H57Y single mutant in the presence of inhibitors. Growing evidence suggests that alterations in lipid homeostasis affect the proteolytic processing of the poliovirus polyprotein. Therefore, we studied the effect of PI4KB or OSBP inhibition on proteolytic processing of the CVB3 polyprotein during infection as well as in a replication-independent system. We show that both PI4KB and OSBP inhibitors specifically affected the cleavage at the 3A-3B junction, and that mutation 3A-H57Y recovered impaired proteolytic processing at this junction. Although 2C-N2D enhanced replication of the 3A-H57Y single mutant, we did not detect additional effects of this substitution on polyprotein processing, which leaves the mechanism of how 2C-N2D contributes to the resistance to be revealed. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  13. Induction of expression and co-localization of heat shock polypeptides with the polyalanine expansion mutant of poly(A)-binding protein N1 after chemical stress

    International Nuclear Information System (INIS)

    Wang Qishan; Bag, Jnanankur

    2008-01-01

    Formation of nuclear inclusions consisting of aggregates of a polyalanine expansion mutant of nuclear poly(A)-binding protein (PABPN1) is the hallmark of oculopharyngeal muscular dystrophy (OPMD). OPMD is a late onset autosomal dominant disease. Patients with this disorder exhibit progressive swallowing difficulty and drooping of their eye lids, which starts around the age of 50. Previously we have shown that treatment of cells expressing the mutant PABPN1 with a number of chemicals such as ibuprofen, indomethacin, ZnSO 4 , and 8-hydroxy-quinoline induces HSP70 expression and reduces PABPN1 aggregation. In these studies we have shown that expression of additional HSPs including HSP27, HSP40, and HSP105 were induced in mutant PABPN1 expressing cells following exposure to the chemicals mentioned above. Furthermore, all three additional HSPs were translocated to the nucleus and probably helped to properly fold the mutant PABPN1 by co-localizing with this protein

  14. Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3)

    DEFF Research Database (Denmark)

    Baumgarten, Thomas; Schlegel, Susan; Wagner, Samuel

    2017-01-01

    Membrane protein production is usually toxic to E. coli. However, using genetic screens strains can be isolated in which the toxicity of membrane protein production is reduced, thereby improving production yields. Best known examples are the C41(DE3) and C43(DE3) strains, which are both derived...

  15. Protein from pea mutants as a co-product in starch separation : isolates from wet and dry separation : yield, composition and solubility

    NARCIS (Netherlands)

    Bergthaller, W.; Dijkink, B.H.; Langelaan, H.C.; Vereijken, J.M.

    2001-01-01

    The dry separation behaviour of cotyledon material of certain pea genotypes (wild type and 5 r mutants) with different metabolic background in starch biosynthesis allowed satisfying protein recovery only with smooth seeded genotypes. Structural effects and protein distribution (starch granule size

  16. Data for high-throughput estimation of specific activities of enzyme/mutants in cell lysates through immunoturbidimetric assay of proteins

    Directory of Open Access Journals (Sweden)

    Yiran Feng

    2017-10-01

    Full Text Available Data in this article are associated with the research article “Highthroughput estimation of specific activities of enzyme/mutants in cell lysates through immunoturbidimetric assay of proteins” (Yang et al., 2017 [1]. This article provided data on how to develop an immunoturbidimetric assay (ITA of enzyme/mutants as proteins in cell lysates in high-throughput (HTP mode together with HTP assay of their activities to derive their specific activities in cell lysates for comparison, with Pseudomonas aeruginosa arylsulfatase (PAAS and Bacillus fastidious uricase (BFU plus their mutants as models. Data were made publicly available for further analyses.

  17. Endoglucanase enzyme protein engineering by site-directed mutagenesis to improve the enzymatic properties and its expression in yeast

    Directory of Open Access Journals (Sweden)

    Farnaz Nikzad Jamnani

    2013-11-01

    Full Text Available Introduction: Fossil fuel is an expensive and finite energy source. Therefore, the use of renewable energy and biofuels production has been taken into consideration. One of the most suitable raw materials for biofuels is cellulosic compounds. Only microorganisms that contain cellulose enzymes can decompose cellulose and fungus of Trichodermareesei is the most important producer of this enzyme. Methods: In this study the nucleotide sequence of endoglucanase II, which is the starter of attack to cellulose chains, synthesized from amino acid sequence of this enzyme in fungus T.reesei and based on codon usage in the host; yeast Pichiapastoris. To produce optimized enzyme and to decrease the production time and enzyme price, protein engineering will be used. There are some methods to improve the enzymatic properties like site-directed mutagenesis in which amino-acid replacement occur. In this study two mutations were induced in endoglucanase enzyme gene by PCR in which free syctein positions 169 and 393 were switched to valine and histidine respectively. Then this gene was inserted into the pPinka expression vector and cloned in Escherichia coli. The recombinant plasmids were transferred into P.pastoris competent cells with electroporation, recombinant yeasts were cultured in BMMY medium and induced with methanol. Results: The sequencing of gene proved the induction of the two mutations and the presence of recombinant enzyme was confirmed by dinitrosalicilic acid method and SDS-PAGE. Conclusion: Examination of biochemical properties revealed that the two mutations simultaneously decreased catalytic power, thermal stability and increased the affinity of enzyme and substrate.

  18. Identification of the Major ACE-Inhibitory Peptides Produced by Enzymatic Hydrolysis of a Protein Concentrate from Cuttlefish Wastewater

    Directory of Open Access Journals (Sweden)

    Isabel Rodríguez Amado

    2014-03-01

    Full Text Available The aim of this work was the purification and identification of the major angiotensin converting enzyme (ACE inhibitory peptides produced by enzymatic hydrolysis of a protein concentrate recovered from a cuttlefish industrial manufacturing effluent. This process consisted on the ultrafiltration of cuttlefish softening wastewater, with a 10 kDa cut-off membrane, followed by the hydrolysis with alcalase of the retained fraction. Alcalase produced ACE inhibitors reaching the highest activity (IC50 = 76.8 ± 15.2 μg mL−1 after 8 h of proteolysis. Sequential ultrafiltration of the 8 h hydrolysate with molecular weight cut-off (MWCO membranes of 10 and 1 kDa resulted in the increased activity of each permeate, with a final IC50 value of 58.4 ± 4.6 μg mL−1. Permeate containing peptides lower than 1 kDa was separated by reversed-phase high performance liquid chromatography (RP-HPLC. Four fractions (A–D with potent ACE inhibitory activity were isolated and their main peptides identified using high performance liquid chromatography coupled to an electrospray ion trap Fourier transform ion cyclotron resonance-mass spectrometer (HPLC-ESI-IT-FTICR followed by comparison with databases and de novo sequencing. The amino acid sequences of the identified peptides contained at least one hydrophobic and/or a proline together with positively charged residues in at least one of the three C-terminal positions. The IC50 values of the fractions ranged from 1.92 to 8.83 μg mL−1, however this study fails to identify which of these peptides are ultimately responsible for the potent antihypertensive activity of these fractions.

  19. Photosystem II repair and plant immunity: Lessons learned from Arabidopsis mutant lacking the THYLAKOID LUMEN PROTEIN 18.3

    Directory of Open Access Journals (Sweden)

    Sari eJärvi

    2016-03-01

    Full Text Available Chloroplasts play an important role in the cellular sensing of abiotic and biotic stress. Signals originating from photosynthetic light reactions, in the form of redox and pH changes, accumulation of reactive oxygen and electrophile species or stromal metabolites are of key importance in chloroplast retrograde signaling. These signals initiate plant acclimation responses to both abiotic and biotic stresses. To reveal the molecular responses activated by rapid fluctuations in growth light intensity, gene expression analysis was performed with Arabidopsis thaliana wild type and the tlp18.3 mutant plants, the latter showing a stunted growth phenotype under fluctuating light conditions (Biochem. J, 406, 415-425. Expression pattern of genes encoding components of the photosynthetic electron transfer chain did not differ between fluctuating and constant light conditions, neither in wild type nor in tlp18.3 plants, and the composition of the thylakoid membrane protein complexes likewise remained unchanged. Nevertheless, the fluctuating light conditions repressed in wild-type plants a broad spectrum of genes involved in immune responses, which likely resulted from shade-avoidance responses and their intermixing with hormonal signaling. On the contrary, in the tlp18.3 mutant plants there was an imperfect repression of defense-related transcripts upon growth under fluctuating light, possibly by signals originating from minor malfunction of the photosystem II (PSII repair cycle, which directly or indirectly modulated the transcript abundances of genes related to light perception via phytochromes. Consequently, a strong allocation of resources to defense reactions in the tlp18.3 mutant plants presumably results in the stunted growth phenotype under fluctuating light.

  20. Photosystem II Repair and Plant Immunity: Lessons Learned from Arabidopsis Mutant Lacking the THYLAKOID LUMEN PROTEIN 18.3.

    Science.gov (United States)

    Järvi, Sari; Isojärvi, Janne; Kangasjärvi, Saijaliisa; Salojärvi, Jarkko; Mamedov, Fikret; Suorsa, Marjaana; Aro, Eva-Mari

    2016-01-01

    Chloroplasts play an important role in the cellular sensing of abiotic and biotic stress. Signals originating from photosynthetic light reactions, in the form of redox and pH changes, accumulation of reactive oxygen and electrophile species or stromal metabolites are of key importance in chloroplast retrograde signaling. These signals initiate plant acclimation responses to both abiotic and biotic stresses. To reveal the molecular responses activated by rapid fluctuations in growth light intensity, gene expression analysis was performed with Arabidopsis thaliana wild type and the tlp18.3 mutant plants, the latter showing a stunted growth phenotype under fluctuating light conditions (Biochem. J, 406, 415-425). Expression pattern of genes encoding components of the photosynthetic electron transfer chain did not differ between fluctuating and constant light conditions, neither in wild type nor in tlp18.3 plants, and the composition of the thylakoid membrane protein complexes likewise remained unchanged. Nevertheless, the fluctuating light conditions repressed in wild-type plants a broad spectrum of genes involved in immune responses, which likely resulted from shade-avoidance responses and their intermixing with hormonal signaling. On the contrary, in the tlp18.3 mutant plants there was an imperfect repression of defense-related transcripts upon growth under fluctuating light, possibly by signals originating from minor malfunction of the photosystem II (PSII) repair cycle, which directly or indirectly modulated the transcript abundances of genes related to light perception via phytochromes. Consequently, a strong allocation of resources to defense reactions in the tlp18.3 mutant plants presumably results in the stunted growth phenotype under fluctuating light.

  1. Crystallization and preliminary crystallographic analysis of a calcineurin B-like protein 1 (CBL1) mutant from Ammopiptanthus mongolicus

    International Nuclear Information System (INIS)

    Shang, Guijun; Cang, Huaixing; Liu, Zhijie; Gao, Wei; Bi, Ruchang

    2010-01-01

    Recombinant calcineurin B-like protein 1 from Ammopiptanthus mongolicus (AmCBL1) was overexpressed, purified and crystallized. Calcineurin B-like protein 1 (CBL1) is a calcium sensor in plants. It transmits the calcium signal through the downstream protein CBL-interacting protein kinase (CIPK). CBL1 and CIPK play crucial roles in the response to environmental stresses such as low K + , osmotic shock, high salt, cold and drought. Recombinant CBL1 from Ammopiptanthus mongolicus (AmCBL1) was overexpressed, purified and crystallized. However, the crystal did not diffract well. A mutant prepared using the surface-entropy method and crystallized using the hanging-drop method at 298 K with PEG 2000 MME as a precipitant diffracted to 2.90 Å resolution. The crystal belonged to space group P2 1 2 1 2, with unit-cell parameters a = 99.87, b = 114.42, c = 63.80 Å, α = β = γ = 90.00° and three molecules per asymmetric unit

  2. Pharmacologic Approach to Defective Protein Trafficking in the E637K-hERG Mutant with PD-118057 and Thapsigargin.

    Directory of Open Access Journals (Sweden)

    Haiyan Mao

    Full Text Available Treatment of LQT2 is inadequate. Many drugs which can pharmacologically rescue defective protein trafficking in LQT2 also result in potent blockade of HERG current, negating their therapeutic benefit. It is reported that PD-118057 and thapsigargin can rescue LQT2 without hERG channel blockade, but the precise mechanism of action is unknown. Furthermore, the effect of PD-118057 and thapsigargin on the dominant negative E637K-hERG mutant has not been previously investigated.IN THIS STUDY, WE INVESTIGATED: (a the effect of PD-118057 and thapsigargin on the current amplitudes of WT-hERG and WT/E637K-hERG channels; (b the effect of PD-118057 and thapsigargin on the biophysical properties of WT-hERG and WT/E637K-hERG channels; (c whether drug treatment can rescue channel processing and trafficking defects of the WT/E637K-hERG mutant.The whole-cell Patch-clamp technique was used to assess the effect of PD-118057 and thapsigargin on the electrophysiological characteristics of the rapidly activating delayed rectifier K(+ current (Ikr of the hERG protein channel. Western blot was done to investigate pharmacological rescue on hERG protein channel function.In our study, PD-118057 was shown to significantly enhance both the maximum current amplitude and tail current amplitude, but did not alter the gating and kinetic properties of the WT-hERG channel, with the exception of accelerating steady-state inactivation. Additionally, thapsigargin shows a similar result as PD-118057 for the WT-hERG channel, but with the exception of attenuating steady-state inactivation. However, for the WT/E637K-hERG channel, PD-118057 had no effect on either the current or on the gating and kinetic properties. Furthermore, thapsigargin treatment did not alter the current or the gating and kinetic properties of the WT/E637K-hERG channel, with the exception of opening at more positive voltages.Our findings illustrate that neither PD-118057 nor thapsigargin play a role in correcting

  3. Induced protein polymorphisms and nutritional quality of gamma irradiation mutants of sorghum

    CSIR Research Space (South Africa)

    Mehlo, L

    2013-09-01

    Full Text Available in the endosperm. The suppression of kafirins was counteracted by an upsurge in the synthesis and accumulation of albumins, globulins and other proteins. The data collectively suggest that sorghum has huge genetic potential for nutritional biofortification...

  4. Laminopathy-inducing lamin A mutants can induce redistribution of lamin binding proteins into nuclear aggregates.

    Science.gov (United States)

    Hübner, S; Eam, J E; Hübner, A; Jans, D A

    2006-01-15

    Lamins, members of the family of intermediate filaments, form a supportive nucleoskeletal structure underlying the nuclear envelope and can also form intranuclear structures. Mutations within the A-type lamin gene cause a variety of degenerative diseases which are collectively referred to as laminopathies. At the molecular level, laminopathies have been shown to be linked to a discontinuous localization pattern of A-type lamins, with some laminopathies containing nuclear lamin A aggregates. Since nuclear aggregate formation could lead to the mislocalization of proteins interacting with A-type lamins, we set out to examine the effects of FLAG-lamin A N195K and R386K protein aggregate formation on the subnuclear distribution of the retinoblastoma protein (pRb) and the sterol responsive element binding protein 1a (SREBP1a) after coexpression as GFP-fusion proteins in HeLa cells. We observed strong recruitment of both proteins into nuclear aggregates. Nuclear aggregate recruitment of the NPC component nucleoporin NUP153 was also observed and found to be dependent on the N-terminus. That these effects were specific was implied by the fact that a number of other coexpressed karyophilic GFP-fusion proteins, such as the nucleoporin NUP98 and kanadaptin, did not coaggregate with FLAG-lamin A N195K or R386K. Immunofluorescence analysis further indicated that the precursor form of lamin A, pre-lamin A, could be found in intranuclear aggregates. Our results imply that redistribution into lamin A-/pre-lamin A-containing aggregates of proteins such as pRb and SREBP1a could represent a key aspect underlying the molecular pathogenesis of certain laminopathies.

  5. An aberrant protein synthesis activity is linked with antibiotic overproduction in rpsL mutants of Streptomyces coelicolor A3(2).

    Science.gov (United States)

    Okamoto-Hosoya, Yoshiko; Hosaka, Takeshi; Ochi, Kozo

    2003-11-01

    Certain mutations in the rpsL gene (encoding the ribosomal protein S12) activate or enhance antibiotic production in various bacteria. K88E and P91S rpsL mutants of Streptomyces coelicolor A3(2), with an enhanced actinorhodin production, were found to exhibit an aberrant protein synthesis activity. While a high level of this activity (as determined by the incorporation of labelled leucine) was detected at the late stationary phase in the mutants, it decreased with age of the cells in the wild-type strain. In addition, the aberrant protein synthesis was particularly pronounced when cells were subjected to amino acid shift-down, and was independent of their ability to accumulate ppGpp. Ribosomes of K88E and P91S mutants displayed an increased accuracy in protein synthesis as demonstrated by the poly(U)-directed cell-free translation system, but so did K43N, K43T, K43R and K88R mutants, which were streptomycin resistant but showed no effect on actinorhodin production. This eliminates the possibility that the increased accuracy level is a cause of the antibiotic overproduction in the K88E and P91S mutants. The K88E and P91S mutant ribosomes exhibited an increased stability of the 70S complex under low concentrations of magnesium. The authors propose that the aberrant activation of protein synthesis caused by the increased stability of the ribosome is responsible for the remarkable enhancement of antibiotic production in the K88E and P91S mutants.

  6. Inhibition of Mutant αB Crystallin-Induced Protein Aggregation by a Molecular Tweezer.

    Science.gov (United States)

    Xu, Na; Bitan, Gal; Schrader, Thomas; Klärner, Frank-Gerrit; Osinska, Hanna; Robbins, Jeffrey

    2017-08-08

    Compromised protein quality control causes the accumulation of misfolded proteins and intracellular aggregates, contributing to cardiac disease and heart failure. The development of therapeutics directed at proteotoxicity-based pathology in heart disease is just beginning. The molecular tweezer CLR01 is a broad-spectrum inhibitor of abnormal self-assembly of amyloidogenic proteins, including amyloid β-protein, tau, and α-synuclein. This small molecule interferes with aggregation by binding selectively to lysine side chains, changing the charge distribution of aggregation-prone proteins and thereby disrupting aggregate formation. However, the effects of CLR01 in cardiomyocytes undergoing proteotoxic stress have not been explored. Here we assess whether CLR01 can decrease cardiac protein aggregation catalyzed by cardiomyocyte-specific expression of mutated αB-crystallin (CryAB R 120G ). A proteotoxic model of desmin-related cardiomyopathy caused by cardiomyocyte-specific expression of CryAB R 120G was used to test the efficacy of CLR01 therapy in the heart. Neonatal rat cardiomyocytes were infected with adenovirus expressing either wild-type CryAB or CryAB R 120G . Subsequently, the cells were treated with different doses of CLR01 or a closely related but inactive derivative, CLR03. CLR01 decreased aggregate accumulation and attenuated cytotoxicity caused by CryAB R 120G expression in a dose-dependent manner, whereas CLR03 had no effect. Ubiquitin-proteasome system function was analyzed using a ubiquitin-proteasome system reporter protein consisting of a short degron, CL1, fused to the COOH-terminus of green fluorescent protein. CLR01 improved proteasomal function in CryAB R 120G cardiomyocytes but did not alter autophagic flux. In vivo, CLR01 administration also resulted in reduced protein aggregates in CryAB R 120G transgenic mice. CLR01 can inhibit CryAB R 120G aggregate formation and decrease cytotoxicity in cardiomyocytes undergoing proteotoxic stress

  7. Cotton leaf curl Burewala virus with intact or mutant transcriptional activator proteins: complexity of cotton leaf curl disease.

    Science.gov (United States)

    Kumar, Jitendra; Gunapati, Samatha; Alok, Anshu; Lalit, Adarsh; Gadre, Rekha; Sharma, Naresh C; Roy, Joy K; Singh, Sudhir P

    2015-05-01

    Cotton leaf curl disease (CLCuD) is a serious disease of cotton on the Indian subcontinent. In the present study, three cotton leaf curl viruses, cotton leaf curl Burewala virus (CLCuBuV), cotton leaf curl Kokhran virus (CLCuKoV) and cotton leaf curl Multan virus (CLCuMV), and their associated satellites, cotton leaf curl Multan betasatellite (CLCuMB) and cotton leaf curl Multan alphasatellite (CLCuMA), were detected. CLCuBuV with either intact (CLCuBuV-1) or mutant (CLCuBuV-2) transcriptional activator protein (TrAP) were detected in different plants. Agroinoculation with CLCuBuV-1 or CLCuBuV-2 together with CLCuMB and CLCuMA, resulted in typical leaf curling and stunting of tobacco plants. Inoculation with CLCuKoV or an isolate of CLCuMV (CLCuMV-2), together with CLCuMB and CLCuMA, induced severe leaf curling, while the other isolate of CLCuMV (CLCuMV-1), which was recombinant in origin, showed mild leaf curling in tobacco. To investigate the effect of intact or mutant TrAP and also the recombination events, CLCuBuV-1, CLCuBuV-2, CLCuMV-1 or CLCuMV-2 together with the satellites (CLCuMA and CLCuMB) were transferred to cotton via whitefly-mediated transmission. Cotton plants containing CLCuBuV-1, CLCuBuV-2 or CLCuMV-2 together with satellites showed curling and stunting, whereas the plants having CLCuMV-1 and the satellites showed only mild and indistinguishable symptoms. CLCuBuV-1 (intact TrAP) showed severe symptoms in comparison to CLCuBuV-2 (mutant TrAP). The present study reveals that two types of CLCuBuV, one with an intact TrAP and the other with a mutant TrAP, exist in natural infection of cotton in India. Additionally, CLCuMuV-1, which has a recombinant origin, induces mild symptoms in comparison to the other CLCuMV isolates.

  8. Transformation frequency of γ irradiated plasmid DNA and the enzymatic double strand break formation by incubation in a protein extract of Escherichia coli

    International Nuclear Information System (INIS)

    Schulte-Frohlinde, D.; Mark, F.; Ventur, Y.

    1994-01-01

    It was found that incubation of γ-irradiated or DNaseI-treated plasmid DNA in a protein extract of Escherichia coli leads to enzyme-induced formation of double strand breaks (dsb) in competition with repair of precursors of these dsb. A survival curve of the plasmid DNA (as determined by transformation of E. coli) was calculated on the basis of enzyme-induced dsb as well as those produced by irradiation assuming that they are lethal. The calculated D O value was the same as that measured directly by transformation of irradiated plasmid DNA. Two models are presented that fit the experimental survival data as a function of dose. One is based on damage formation in the plasmid DNA including enzymatic conversion of single strand damage into dsb (U-model), the other is an enzymatic repair saturation model based on Michaelis-Menten kinetics. (Author)

  9. Transformation frequency of [gamma] irradiated plasmid DNA and the enzymatic double strand break formation by incubation in a protein extract of Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Schulte-Frohlinde, D.; Mark, F.; Ventur, Y. (Max-Planck-Institut fuer Strahlenchemie, Muelheim an der Ruhr (Germany))

    1994-01-01

    It was found that incubation of [gamma]-irradiated or DNaseI-treated plasmid DNA in a protein extract of Escherichia coli leads to enzyme-induced formation of double strand breaks (dsb) in competition with repair of precursors of these dsb. A survival curve of the plasmid DNA (as determined by transformation of E. coli) was calculated on the basis of enzyme-induced dsb as well as those produced by irradiation assuming that they are lethal. The calculated D[sub O] value was the same as that measured directly by transformation of irradiated plasmid DNA. Two models are presented that fit the experimental survival data as a function of dose. One is based on damage formation in the plasmid DNA including enzymatic conversion of single strand damage into dsb (U-model), the other is an enzymatic repair saturation model based on Michaelis-Menten kinetics. (Author).

  10. Functional testing of keratin 14 mutant proteins associated with the three major subtypes of epidermolysis bullosa simplex

    DEFF Research Database (Denmark)

    Sørensen, Charlotte B; Andresen, Brage S; Jensen, Uffe B

    2003-01-01

    in either the keratin 5 (KRT5) or keratin 14 (KRT14) gene. Previously, we identified three novel KRT14 missense mutations in Danish EBS patients associated with the three different forms of EBS (1). The identified KRT14 mutations represent the full spectrum of the classical EBS subtypes. In the present...... study we investigated these mutations in a cellular expression system in order to analyse their effects on the keratin cytoskeleton. KRT14 expression vectors were constructed by fusing the nucleotide sequence encoding the FLAG reporter peptide to the 3' end of the KRT14 cDNA sequences. The expression...... vectors were transiently transfected into normal human primary keratinocytes (NHK), HaCaT or HeLa cells in order to analyze the ability of the mutant K14 proteins to integrate into the existing endogenous keratin filament network (KFN). No effect on the keratin cytoskeleton was observed upon transfection...

  11. Shutdown of HIV-1 Transcription in T Cells by Nullbasic, a Mutant Tat Protein

    OpenAIRE

    Jin, Hongping; Li, Dongsheng; Sivakumaran, Haran; Lor, Mary; Rustanti, Lina; Cloonan, Nicole; Wani, Shivangi; Harrich, David

    2016-01-01

    ABSTRACT Nullbasic is a derivative of the HIV-1 transactivator of transcription (Tat) protein that strongly inhibits HIV-1 replication in lymphocytes. Here we show that lentiviral vectors that constitutively express a Nullbasic-ZsGreen1 (NB-ZSG1) fusion protein by the eEF1? promoter led to robust long-term inhibition of HIV-1 replication in Jurkat cells. Although Jurkat-NB-ZSG1 cells were infected by HIV-1, no virus production could be detected and addition of phorbol ester 12-myristate 13-ac...

  12. Hybrid model for an enzymatic reactor: hydrolysis of cheese whey proteins by alcalase immobilized in agarose gel particles.

    Science.gov (United States)

    Sousa, Ruy; Resende, Mariam M; Giordano, Raquel L C; Giordano, Roberto C

    2003-01-01

    Cheese whey proteolysis, carried out by immobilized enzymes, can either change or evidence functional properties of the produced peptides, increasing the potential applications of this byproduct of the dairy industry. Optimization and scale-up of the enzymatic reactor relies on its mathematical model-a set of mass balance equations, with reaction rates usually given by Michaelis-Menten-like kinetics; no information about the distribution of peptides' molecular sizes is supplied. In this article, a hybrid model of a batch enzymatic reactor is presented, consisting of differential mass balances coupled to a "neural-kinetic model," which provides the molecular weight distributions of the resulting peptides.

  13. Low-Density Lipoprotein Receptor-Related Protein-1 (LRP1) C4408R Mutant Promotes Amyloid Precursor Protein (APP) α-Cleavage in Vitro.

    Science.gov (United States)

    Hou, Huayan; Habib, Ahsan; Zi, Dan; Tian, Kathy; Tian, Jun; Giunta, Brian; Sawmiller, Darrell; Tan, Jun

    2017-09-01

    Previous studies have demonstrated that the low-density lipoprotein receptor-related protein-1 (LRP1) plays conflicting roles in Alzheimer's disease (AD) pathogenesis, clearing β-amyloid (Aβ) from the brain while also enhancing APP endocytosis and resultant amyloidogenic processing. We have recently discovered that co-expression of mutant LRP1 C-terminal domain (LRP1-CT C4408R) with Swedish mutant amyloid precursor protein (APPswe) in Chinese hamster ovary (CHO) cells decreases Aβ production, while also increasing sAPPα and APP α-C-terminal fragment (α-CTF), compared with CHO cells expressing APPswe alone. Surprisingly, the location of this mutation on LRP1 corresponded with the α-secretase cleavage site of APP. Further experimentation confirmed that in CHO cells expressing APPswe or wild-type APP (APPwt), co-expression of LRP1-CT C4408R decreases Aβ and increases sAPPα and α-CTF compared with co-expression of wild-type LRP1-CT. In addition, LRP1-CT C4408R enhanced the unglycosylated form of LRP1-CT and reduced APP endocytosis as determined by flow cytometry. This finding identifies a point mutation in LRP1 which slows LRP1-CT-mediated APP endocytosis and amyloidogenic processing, while enhancing APP α-secretase cleavage, thus demonstrating a potential novel target for slowing AD pathogenesis.

  14. Non-spa-typeable clinical Staphylococcus aureus strains are naturally occurring protein A mutants

    DEFF Research Database (Denmark)

    Baum, Cathrin; Haslinger-Löffler, Bettina; Westh, Henrik

    2009-01-01

    Staphylococcus aureus is a major human pathogen responsible for increasing the prevalence of community- and hospital-acquired infections. Protein A (SpA) is a key virulence factor of S. aureus and is highly conserved. Sequencing of the variable-number tandem-repeat region of SpA (spa typing) prov...

  15. Characterization of mutagen-activated cellular oncogenes that confer anchorage independence to human fibroblasts and tumorigenicity to NIH 3T3 cells: Sequence analysis of an enzymatically amplified mutant HRAS allele

    International Nuclear Information System (INIS)

    Stevens, C.W.; Manoharan, T.H.; Fahl, W.E.

    1988-01-01

    Treatment of diploid human fibroblasts with an alkylating mutagen has been shown to induce stable, anchorage-independent cell populations at frequencies consistent with an activating mutation. After treatment of human foreskin fibroblasts with the mutagen benzo[α]pyrene (±)anti-7,8-dihydrodiol 9,10-epoxide and selection in soft agar, 17 anchorage-independent clones were isolated and expanded, and their cellular DNA was used to cotransfect NIH 3T3 cells along with pSV2neo. DNA from 11 of the 17 clones induced multiple NIH 3T3 cell tumors in recipient nude mice. Southern blot analyses showed the presence of human Alu repetitive sequences in all of the NIH 3T3 tumor cell DNAs. Intact, human HRAS sequences were observed in 2 of the 11 tumor groups, whereas no hybridization was detected when human KRAS or NRAS probes were used. Slow-migrating ras p21 proteins, consistent with codon 12 mutations, were observed in the same two NIH 3T3 tumor cell groups that contained the human HRAS bands. Genomic DNA from one of these two human anchorage-independent cell populations (clone 21A) was used to enzymatically amplify a portion of exon 1 of the HRAS gene. The results demonstrate that exposure of normal human cells to a common environmental mutagen yields HRAS GC → TA codon 12 transversions that have been commonly observed in human tumors

  16. β-Hydroxyacyl-acyl Carrier Protein Dehydratase (FabZ) from Francisella tularensis and Yersinia pestis : Structure Determination, Enzymatic Characterization, and Cross-Inhibition Studies

    Energy Technology Data Exchange (ETDEWEB)

    McGillick, Brian E.; Kumaran, Desigan; Vieni, Casey; Swaminathan, Subramanyam

    2016-02-23

    The bacterial system for fatty acid biosynthesis (FAS) contains several enzymes whose sequence and structure are highly conserved across a vast array of pathogens. This, coupled with their low homology and difference in organization compared to the equivalent system in humans, makes the FAS pathway an excellent target for antimicrobial drug development. To this end, we have cloned, expressed, and purified the β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from both Francisella tularensis (FtFabZ) and Yersinia pestis (YpFabZ). We also solved the crystal structures and performed an enzymatic characterization of both enzymes and several mutant forms of YpFabZ. Additionally, we have discovered two novel inhibitors of FabZ, mangostin and stictic acid, which show similar potencies against both YpFabZ and FtFabZ. Lastly, we selected several compounds from the literature that have been shown to be active against single homologues of FabZ and tested them against both YpFabZ and FtFabZ. These results have revealed clues as to which scaffolds are likely to lead to broad-spectrum antimicrobials targeted against FabZ as well as modifications to existing FabZ inhibitors that may improve potency.

  17. Analysis of mouse polyomavirus mutants with lesions in the minor capsid proteins

    Czech Academy of Sciences Publication Activity Database

    Mannová, P.; Liebl, D.; Krauzewitz, N.; Fejtová, A.; Štokrová, Jitka; Palková, Z.; Griffin, B. E.; Forstová, J.

    2002-01-01

    Roč. 83, - (2002), s. 2309-2319 ISSN 0022-1317 R&D Projects: GA ČR GA204/00/0271 Grant - others:HHMI USA(US) 75195-540501 Institutional research plan: CEZ:AV0Z5052915; CEZ:MSM 113100003 Keywords : polyomavirus * minor capsid proteins * mutation Subject RIV: EE - Microbiology, Virology Impact factor: 3.300, year: 2002

  18. Prediction on the risk population of idiosyncratic adverse reactions based on molecular docking with mutant proteins.

    Science.gov (United States)

    Xie, Hongbo; Zeng, Diheng; Chen, Xiujie; Huo, Diwei; Liu, Lei; Zhang, Denan; Jin, Qing; Ke, Kehui; Hu, Ming

    2017-11-10

    Idiosyncratic adverse drug reactions are drug reactions that occur rarely and unpredictably among the population. These reactions often occur after a drug is marketed, which means that they are strongly related to the genotype of the population. The prediction of such adverse reactions is a major challenge because of the lack of appropriate test models during the drug development process. In this study, we chose withdrawn drugs because the reasons why they were withdrawn and from which countries or regions is easily obtained. We selected Dilevalol and its chiral drug (Labetalol) as the investigatory drugs, as they have been withdrawn from a European market (Britain) because of serious hepatotoxicity. First, we searched for and obtained the Dilevalol-induced- liver-injury related protein, multidrug resistance protein 1 (MDR1), from the Comparative Toxicogenomics Database (CTD). Then, we searched and extracted 477 non-synonymous single nucleotide polymorphisms (nsSNP) on MDR1 in the dbSNP database. Second, we used the VarMod tool to predict the functional changes of MDR1 induced by these nsSNPs, from which we extracted the nsSNPs that significantly change the functions of this protein. Third, we built the three-dimensional structures of those variant proteins and used AutoDock to perform a docking study, choosing the best model to determine the sites of nsSNPs. Finally, we used the data from the 1000 Genomes Project to verify the dominant population distribution of the risk SNP. We applied the same strategy to the post-marketing drug-induced liver injury drugs to further test the feasibility of our method.

  19. The splicing mutant of the human tumor suppressor protein DFNA5 induces programmed cell death when expressed in the yeast Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Sofie eVan Rossom

    2012-07-01

    Full Text Available DFNA5 was first identified as a gene responsible for autosomal dominant deafness. Different mutations were found, but they all resulted in exon 8 skipping during splicing and premature termination of the protein. Later, it became clear that the protein also has a tumor suppression function and that it can induce apoptosis. Epigenetic silencing of the DFNA5 gene is associated with different types of cancers, including gastric and colorectal cancers as well as breast tumors. We introduced the wild-type and mutant DFNA5 allele in the yeast Saccharomyces cerevisiae. The expression of the wild-type protein was well tolerated by the yeast cells, although the protein was subject of degradation and often deposited in distinct foci when cells entered the diauxic shift. In contrast, cells had problems to cope with mutant DFNA5 and despite an apparent compensatory reduction in expression levels, the mutant protein still triggered a marked growth defect, which in part can be ascribed to its interaction with mitochondria. Consistently, cells with mutant DFNA5 displayed significantly increased levels of ROS and signs of programmed cell death. The latter occurred independently of the yeast caspase, Mca1, but involved the mitochondrial fission protein, Fis1, the voltage-dependent anion channel protein, Por1 and the mitochondrial adenine-nucleotide translocators, Aac1 and Aac3. Recent data proposed DFNA5 toxicity to be associated to a globular domain encoded by exon 2 to 6. We confirmed these data by showing that expression of solely this domain confers a strong growth phenotype. In addition, we identified a point mutant in this domain that completely abrogated its cytotoxicity in yeast as well as human HEK293T cells. Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.

  20. Overexpression of the herpes simplex virus type 1 immediate-early regulatory protein, ICP27, is responsible for the aberrant localization of ICP0 and mutant forms of ICP4 in ICP4 mutant virus-infected cells.

    OpenAIRE

    Zhu, Z; DeLuca, N A; Schaffer, P A

    1996-01-01

    ICP0 and ICP4 are immediate-early regulatory proteins of herpes simplex virus type 1. Previous studies by Knipe and Smith demonstrated that these two proteins are characteristically observed in the nuclei of wild-type virus-infected cells but predominantly in the cytoplasms of cells infected with several ICP4 temperature-sensitive (ts) mutant viruses at the nonpermissive temperature (NPT) (D. M. Knipe and J. L. Smith, Mol. Cell. Biol. 6:2371-2381, 1986). Consistent with this observation, it h...

  1. The role of mutant protein level in autosomal recessive catecholamine dependent polymorphic ventricular tachycardia (CPVT2)

    OpenAIRE

    Katz, Guy; Shainberg, Asher; Hochhauser, Edith; Kurtzwald-Josefson, Efrat; Issac, Ahuva; El-Ani, Dalia; Aravot, Dan; Afek, Arnon; Seidman, Jonathan G.; Seidman, Christine E.; Eldar, Michael; Arad, Michael

    2013-01-01

    Humans and genetically engineered mice with recessively inherited CPVT develop arrhythmia which may arise due to malfunction or degradation of calsequestrin (CASQ2). We investigated the relation between protein level and arrhythmia severity in CASQ2D307H/D307H (D307H), compared to CASQ2Δ/Δ (KO) and wild type (WT) mice. CASQ2 expression and Ca2+ transients were recorded in cardiomyocytes from neonatal or adult mice. Arrhythmia was studied in vivo using heart rhythm telemetry at rest, exercise ...

  2. A comparison of the enzymatic properties of three recombinant isoforms of thrombolytic and antibacterial protein--Destabilase-Lysozyme from medicinal leech.

    Science.gov (United States)

    Kurdyumov, Alexey S; Manuvera, Valentin A; Baskova, Isolda P; Lazarev, Vassili N

    2015-11-21

    Destabilase-Lysozyme (mlDL) is a multifunctional i-type enzyme that has been found in the secretions from the salivary glands of medicinal leeches. mlDL has been shown to exhibit isopeptidase, muramidase and antibacterial activity. This enzyme attracts interest because it expresses thrombolytic activity through isopeptidolysis of the ε-(γ-Glu)-Lys bonds that cross-link polypeptide chains in stabilised fibrin. To date, three isoforms of mlDL have been identified. The enzymatic properties of pure mlDL isoforms have not yet been described because only destabilase complexes containing other proteins could be isolated from the salivary gland secretion and because low product yield from the generation of recombinant proteins has made comprehensive testing difficult. In the present study, we optimised the procedures related to the expression, isolation and purification of active mlDL isoforms (mlDL-Ds1, mlDL-Ds2, mlDL-Ds3) using an Escherichia coli expression system, and we detected and compared their muramidase, lytic, isopeptidase and antimicrobial activities. After optimisation, the product yield was 30 mg per litre of culture. The data obtained in our study led to the suggestion that the recombinant mlDL isoforms isolated from inclusion bodies form stable oligomeric complexes. Analyses of the tested activities revealed that all isoforms exhibited almost identical patterns of pH and ionic strength effects on the activities. We determined that mlDL-Ds1, 2, 3 possessed non-enzymatic antibacterial activity independent of their muramidase activity. For the first time, we demonstrated the fibrinolytic activity of the recombinant mlDL and showed that only intact proteins possessed this activity, suggesting their enzymatic nature. The recombinant Destabilase-Lysozyme isoforms obtained in our study may be considered potential thrombolytic agents that act through a mechanism different from that of common thrombolytics.

  3. Probing Early Misfolding Events in Prion Protein Mutants by NMR Spectroscopy

    Directory of Open Access Journals (Sweden)

    Gregor Ilc

    2013-08-01

    Full Text Available The post-translational conversion of the ubiquitously expressed cellular form of the prion protein, PrPC, into its misfolded and pathogenic isoform, known as prion or PrPSc, plays a key role in prion diseases. These maladies are denoted transmissible spongiform encephalopathies (TSEs and affect both humans and animals. A prerequisite for understanding TSEs is unraveling the molecular mechanism leading to the conversion process whereby most α-helical motifs are replaced by β-sheet secondary structures. Importantly, most point mutations linked to inherited prion diseases are clustered in the C-terminal domain region of PrPC and cause spontaneous conversion to PrPSc. Structural studies with PrP variants promise new clues regarding the proposed conversion mechanism and may help identify “hot spots” in PrPC involved in the pathogenic conversion. These investigations may also shed light on the early structural rearrangements occurring in some PrPC epitopes thought to be involved in modulating prion susceptibility. Here we present a detailed overview of our solution-state NMR studies on human prion protein carrying different pathological point mutations and the implications that such findings may have for the future of prion research.

  4. Opposing effects of cancer-type-specific SPOP mutants on BET protein degradation and sensitivity to BET inhibitors.

    Science.gov (United States)

    Janouskova, Hana; El Tekle, Geniver; Bellini, Elisa; Udeshi, Namrata D; Rinaldi, Anna; Ulbricht, Anna; Bernasocchi, Tiziano; Civenni, Gianluca; Losa, Marco; Svinkina, Tanya; Bielski, Craig M; Kryukov, Gregory V; Cascione, Luciano; Napoli, Sara; Enchev, Radoslav I; Mutch, David G; Carney, Michael E; Berchuck, Andrew; Winterhoff, Boris J N; Broaddus, Russell R; Schraml, Peter; Moch, Holger; Bertoni, Francesco; Catapano, Carlo V; Peter, Matthias; Carr, Steven A; Garraway, Levi A; Wild, Peter J; Theurillat, Jean-Philippe P

    2017-09-01

    It is generally assumed that recurrent mutations within a given cancer driver gene elicit similar drug responses. Cancer genome studies have identified recurrent but divergent missense mutations affecting the substrate-recognition domain of the ubiquitin ligase adaptor SPOP in endometrial and prostate cancers. The therapeutic implications of these mutations remain incompletely understood. Here we analyzed changes in the ubiquitin landscape induced by endometrial cancer-associated SPOP mutations and identified BRD2, BRD3 and BRD4 proteins (BETs) as SPOP-CUL3 substrates that are preferentially degraded by endometrial cancer-associated SPOP mutants. The resulting reduction of BET protein levels sensitized cancer cells to BET inhibitors. Conversely, prostate cancer-specific SPOP mutations resulted in impaired degradation of BETs, promoting their resistance to pharmacologic inhibition. These results uncover an oncogenomics paradox, whereby mutations mapping to the same domain evoke opposing drug susceptibilities. Specifically, we provide a molecular rationale for the use of BET inhibitors to treat patients with endometrial but not prostate cancer who harbor SPOP mutations.

  5. Modelling in Escherichia coli of mutations in mitoribosomal protein S12: novel mutant phenotypes of rpsL.

    Science.gov (United States)

    Toivonen, J M; Boocock, M R; Jacobs, H T

    1999-03-01

    The rpsL gene of Escherichia coli encodes the highly conserved rps12 protein of the ribosomal accuracy centre. We have used the E. coli gene to model the phenotypic effects of specific substitutions found in the mitochondrial gene for rps12. Variants created by in vitro mutagenesis were tested in two different plasmid vector systems, in both streptomycin-sensitive and streptomycin-resistant hosts. A substitution with respect to eubacterial rps12 (K87-->Q), found in all metazoan and fungal mitochondrial orthologues thus far studied, is associated with low-level resistance to streptomycin and a modest (15%) drop in translational elongation rate, but without significant effects on translational accuracy. An amino-acid replacement at a highly conserved leucine residue (L56-->H), associated with the phenotype of sensitivity to mechanical vibration and hemizygous female lethality in Drosophila, creates a functionally inactive but structurally stable protein that is not assembled into ribosomes. The presence in the cell of the mutant, but not wild-type, rpsL greatly downregulates the level of a prominent polypeptide of approximately 50 kDa. These results indicate novel structure-function relationships in rps12 genes affecting translational function, ribosome assembly and drug sensitivity, and indicate a novel regulatory pathway that may influence ribosome biogenesis.

  6. The enzymatic hydrolysis of soy protein isolate by Corolase PP under high hydrostatic pressure and its effect on bioactivity and characteristics of hydrolysates.

    Science.gov (United States)

    Guan, Haining; Diao, Xiaoqin; Jiang, Fan; Han, Jianchun; Kong, Baohua

    2018-04-15

    Enzymatic hydrolysis of soy protein isolate by Corolase PP under high hydrostatic pressure conditions was studied and the effects of hydrolysis on antioxidant and antihypertensive activities were investigated. As observed, high hydrostatic pressure (80-300MPa) enhanced the hydrolytic efficiency of Corolase PP and decreased the surface hydrophobicity of the hydrolysates. Hydrolysates obtained at 200MPa for 4h had higher bioactivities (reducing power, ABTS radical-scavenging and ACE inhibitory activities). The molecular weight (MW) determination indicated that hydrolysis at high hydrostatic pressure could increase the production of small peptides (isolate. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Shutdown of HIV-1 Transcription in T Cells by Nullbasic, a Mutant Tat Protein

    Directory of Open Access Journals (Sweden)

    Hongping Jin

    2016-07-01

    Full Text Available Nullbasic is a derivative of the HIV-1 transactivator of transcription (Tat protein that strongly inhibits HIV-1 replication in lymphocytes. Here we show that lentiviral vectors that constitutively express a Nullbasic-ZsGreen1 (NB-ZSG1 fusion protein by the eEF1α promoter led to robust long-term inhibition of HIV-1 replication in Jurkat cells. Although Jurkat-NB-ZSG1 cells were infected by HIV-1, no virus production could be detected and addition of phorbol ester 12-myristate 13-acetate (PMA and JQ1 had no effect, while suberanilohydroxamic acid (SAHA modestly stimulated virus production but at levels 300-fold lower than those seen in HIV-1-infected Jurkat-ZSG1 cells. Virus replication was not recovered by coculture of HIV-1-infected Jurkat-NB-ZSG1 cells with uninfected Jurkat cells. Latently infected Jurkat latent 6.3 and ACH2 cells treated with latency-reversing agents produced measurable viral capsid (CA, but little or none was made when they expressed NB-ZSG1. When Jurkat cells chronically infected with HIV-1 were transduced with lentiviral virus-like particles conveying NB-ZSG1, a >3-log reduction in CA production was observed. Addition of PMA increased virus CA production but at levels 500-fold lower than those seen in nontransduced Jurkat cells. Transcriptome sequencing analysis confirmed that HIV-1 mRNA was strongly inhibited by NB-ZSG1 but indicated that full-length viral mRNA was made. Analysis of HIV-1-infected Jurkat cells expressing NB-ZSG1 by chromatin immunoprecipitation assays indicated that recruitment of RNA polymerase II (RNAPII and histone 3 lysine 9 acetylation were inhibited. The reduction of HIV-1 promoter-associated RNAPII and epigenetic changes in viral nucleosomes indicate that Nullbasic can inhibit HIV-1 replication by enforcing viral silencing in cells.

  8. Nitrate levels and stages of growth in hypernodulating mutants of Lupinus albus. II. Enzymatic activity and transport of N in the xylem sap Diferentes níveis de nitrato e estágio de crescimento em mutantes hipernodulantes de Lupinus albus II. Atividade enzimática e transporte de N na seiva do xilema

    Directory of Open Access Journals (Sweden)

    Hélio Almeida Burity

    1999-04-01

    Full Text Available The enzymatic study and transport of N in the xylem sap was carried out with a view to observing the influence of different nitrate levels and growth stages of the plant in chemically treated mutants of Lupinus albus. Several stresses induce a reduction in plant growth, resulting in the accumulation of free amino acids, amides or ureides, not only in the shoot, but also in the roots and nodules. Although enzyme activity is decisive in avoiding products that inhibit nitrogenase by ammonium, little is known about the mechanism by which the xylem carries these products. However, this process may be the key to the function of avoiding the accumulation of amino acids in the cells of infected nodules. The behaviour of the enzymes nitrate reductase (NR, phosphoenolpyruvate carboxylase (PEPC, glutamine synthetase (GS and nitrogen compounds derived from fixation, such as N-a-amino, N-ureides and N-amide in mutant genotypes were observed. The NR enzyme activity was highly influenced by the application of nitrate showing much higher values than those in the non-application of nitrate, independently of genotype, being that the NR, the best evaluation period was in the tenth week. The L-62 genotype characterized with nitrate- resistance, clearly showed that the enzyme PEPC is inhibited by presence of nitrate. The L-135 genotype (nod- fix- showed GS activity extremely low, thus demonstrating that GS is an enzyme highly correlated with fixation. With regard to the best growth stage for GS, Lupinus albus should be evaluated in the seventh week.O estudo enzimático e o transporte de N na seiva do xilema foi realizado visando observar a influência de diferentes níveis de nitrato e estágios de crescimento da planta em mutantes tratadas quimicamente. Vários estresses induzem a redução no crescimento da planta da qual resulta na acumulação de aminoácidos livres, amidas ou ureídos, tanto na parte aérea como nas raízes e nódulos. A atividade de enzimas

  9. Reduced sweetness of a monellin (MNEI) mutant results from increased protein flexibility and disruption of a distant poly-(L-proline) II helix.

    Science.gov (United States)

    Templeton, Catherine M; Ostovar pour, Saeideh; Hobbs, Jeanette R; Blanch, Ewan W; Munger, Steven D; Conn, Graeme L

    2011-06-01

    Monellin is a highly potent sweet-tasting protein but relatively little is known about how it interacts with the sweet taste receptor. We determined X-ray crystal structures of 3 single-chain monellin (MNEI) proteins with alterations at 2 core residues (G16A, V37A, and G16A/V37A) that induce 2- to 10-fold reductions in sweetness relative to the wild-type protein. Surprisingly, no changes were observed in the global protein fold or the positions of surface amino acids important for MNEI sweetness that could explain these differences in protein activity. Differential scanning calorimetry showed that while the thermal stability of each mutant MNEI was reduced, the least sweet mutant, G16A-MNEI, was not the least stable protein. In contrast, solution spectroscopic measurements revealed that changes in protein flexibility and the C-terminal structure correlate directly with protein activity. G16A mutation-induced disorder in the protein core is propagated via changes to hydrophobic interactions that disrupt the formation and/or position of a critical C-terminal poly-(L-proline) II helix. These findings suggest that MNEI interaction with the sweet taste receptor is highly sensitive to the relative positions of key residues across its protein surface and that loss of sweetness in G16A-MNEI may result from an increased entropic cost of binding.

  10. Transcriptomic and proteomic approach to identify differentially expressed genes and proteins in Arabidopsis thaliana mutants lacking chloroplastic 1 and cytosolic FBPases reveals several levels of metabolic regulation.

    Science.gov (United States)

    Soto-Suárez, Mauricio; Serrato, Antonio J; Rojas-González, José A; Bautista, Rocío; Sahrawy, Mariam

    2016-12-01

    During the photosynthesis, two isoforms of the fructose-1,6-bisphosphatase (FBPase), the chloroplastidial (cFBP1) and the cytosolic (cyFBP), catalyse the first irreversible step during the conversion of triose phosphates (TP) to starch or sucrose, respectively. Deficiency in cyFBP and cFBP1 isoforms provokes an imbalance of the starch/sucrose ratio, causing a dramatic effect on plant development when the plastidial enzyme is lacking. We study the correlation between the transcriptome and proteome profile in rosettes and roots when cFBP1 or cyFBP genes are disrupted in Arabidopsis thaliana knock-out mutants. By using a 70-mer oligonucleotide microarray representing the genome of Arabidopsis we were able to identify 1067 and 1243 genes whose expressions are altered in the rosettes and roots of the cfbp1 mutant respectively; whilst in rosettes and roots of cyfbp mutant 1068 and 1079 genes are being up- or down-regulated respectively. Quantitative real-time PCR validated 100% of a set of 14 selected genes differentially expressed according to our microarray analysis. Two-dimensional (2-D) gel electrophoresis-based proteomic analysis revealed quantitative differences in 36 and 26 proteins regulated in rosettes and roots of cfbp1, respectively, whereas the 18 and 48 others were regulated in rosettes and roots of cyfbp mutant, respectively. The genes differentially expressed and the proteins more or less abundant revealed changes in protein metabolism, RNA regulation, cell signalling and organization, carbon metabolism, redox regulation, and transport together with biotic and abiotic stress. Notably, a significant set (25%) of the proteins identified were also found to be regulated at a transcriptional level. This transcriptomic and proteomic analysis is the first comprehensive and comparative study of the gene/protein re-adjustment that occurs in photosynthetic and non-photosynthetic organs of Arabidopsis mutants lacking FBPase isoforms.

  11. Differential expression of SOS genes in an E. coli mutant producing unstable lexA protein enhances excision repair but inhibits mutagenesis

    International Nuclear Information System (INIS)

    Peterson, K.R.; Ganesan, A.K.; Mount, D.W.; Stanford Univ., CA)

    1986-01-01

    The SOS response is displayed following treatments which damage DNA or inhibit DNA replication. Two associated activities include enhanced capacity for DNA repair resulting from derepression of the recA, uvrA, uvrB and uvrD genes and increased mutagenesis due to derepression of recA, umuC and umuD. These changes are the consequence of the derepression of at least seventeen unlinked operons negatively regulated by LexA repressor. Following treatments that induce the SOS response, a signal molecule interacts with RecA protein, converting it to an activated form. Activated RecA protein facilitates the proteolytic cleavage of LexA repressor, which results in derepression of the regulon. The cell then enters a new physiological state during which time DNA repair processes are augmented. The lexA41 mutant of E. coli is a uv-resistant derivative of another mutant, lexA3, which produces a repressor that is not cleaved following inducing treatments. The resultant protein is unstable. Lac operon fusions to most of the genes in the SOS regulon were used to show that the various damage-inducible genes were derepressed to different extents. uvrA, B, and D were almost fully derepressed. Consistent with this finding, the rate of removal of T4 endonuclease V-sensitive sites was more rapid in the uv-irradiated lexA41 mutant than in normal cells, suggesting a more active excision repair system. We propose that the instability of the LexA41 protein reduces the intracellular concentration of repressor to a level that allows a high level of excision repair. The additional observation that SOS mutagenesis was only weakly induced in a lexA41 uvrA - mutant implies that the mutant protein partially represses one or more genes whose products promote SOS mutagenesis. 17 refs., 4 figs., 1 tab

  12. An attempt towards simultaneous biobased solvent based extraction of proteins and enzymatic saccharification of cellulosic materials from distiller's grains and solubles.

    Energy Technology Data Exchange (ETDEWEB)

    Datta, S.; Bals, B. D.; Lin, Y. J.; Negri, M. C.; Datta, R.; Pasieta, L.; Ahmad, S. F.; Moradia, A. A.; Dale, B. E.; Snyder, S. W.; Energy Systems; Michigan State Univ.; Vertec BioSolvents Inc.; Illinois Mathematics and Science Academy

    2010-07-01

    Distiller's grains and solubles (DGS) is the major co-product of corn dry mill ethanol production, and is composed of 30% protein and 30-40% polysaccharides. We report a strategy for simultaneous extraction of protein with food-grade biobased solvents (ethyl lactate, d-limonene, and distilled methyl esters) and enzymatic saccharification of glucan in DGS. This approach would produce a high-value animal feed while simultaneously producing additional sugars for ethanol production. Preliminary experiments on protein extraction resulted in recovery of 15-45% of the protein, with hydrophobic biobased solvents obtaining the best results. The integrated hydrolysis and extraction experiments showed that biobased solvent addition did not inhibit hydrolysis of the cellulose. However, only 25-33% of the total protein was extracted from DGS, and the extracted protein largely resided in the aqueous phase, not the solvent phase. We hypothesize that the hydrophobic solvent could not access the proteins surrounded by the aqueous phase inside the fibrous structure of DGS due to poor mass transfer. Further process improvements are needed to overcome this obstacle.

  13. Abnormal behaviors and developmental disorder of hippocampus in zinc finger protein 521 (ZFP521 mutant mice.

    Directory of Open Access Journals (Sweden)

    Nobutaka Ohkubo

    Full Text Available Zinc finger protein 521 (ZFP521 regulates a number of cellular processes in a wide range of tissues, such as osteoblast formation and adipose commitment and differentiation. In the field of neurobiology, it is reported to be an essential factor for transition of epiblast stem cells into neural progenitors in vitro. However, the role of ZFP521 in the brain in vivo still remains elusive. To elucidate the role of ZFP521 in the mouse brain, we generated mice lacking exon 4 of the ZFP521 gene. The birth ratio of our ZFP521Δ/Δ mice was consistent with Mendel's laws. Although ZFP521Δ/Δ pups had no apparent defect in the body and were indistinguishable from ZFP521+/+ and ZFP521+/Δ littermates at the time of birth, ZFP521Δ/Δ mice displayed significant weight reduction as they grew, and most of them died before 10 weeks of age. They displayed abnormal behavior, such as hyper-locomotion, lower anxiety and impaired learning, which correspond to the symptoms of schizophrenia. The border of the granular cell layer of the dentate gyrus in the hippocampus of the mice was indistinct and granular neurons were reduced in number. Furthermore, Sox1-positive neural progenitor cells in the dentate gyrus and cerebellum were significantly reduced in number. Taken together, these findings indicate that ZFP521 directly or indirectly affects the formation of the neuronal cell layers of the dentate gyrus in the hippocampus, and thus ZFP521Δ/Δ mice displayed schizophrenia-relevant symptoms. ZFP521Δ/Δ mice may be a useful research tool as an animal model of schizophrenia.

  14. Immunogenicity in African Green Monkeys of M Protein Mutant Vesicular Stomatitis Virus Vectors and Contribution of Vector-Encoded Flagellin

    Directory of Open Access Journals (Sweden)

    Marlena M. Westcott

    2018-03-01

    Full Text Available Recombinant vesicular stomatitis virus (VSV is a promising platform for vaccine development. M51R VSV, an attenuated, M protein mutant strain, is an effective inducer of Type I interferon and dendritic cell (DC maturation, which are desirable properties to exploit for vaccine design. We have previously evaluated M51R VSV (M51R and M51R VSV that produces flagellin (M51R-F as vaccine vectors using murine models, and found that flagellin enhanced DC activation and VSV-specific antibody production after low-dose vaccination. In this report, the immunogenicity of M51R vectors and the adjuvant effect of virus-produced flagellin were evaluated in nonhuman primates following high-dose (108 pfu and low-dose (105 pfu vaccination. A single intramuscular vaccination of African green monkeys with M51R or M51R-F induced VSV-specific, dose-dependent humoral immune responses. Flagellin induced a significant increase in antibody production (IgM, IgG and neutralizing antibody at the low vaccination dose. A VSV-specific cellular response was detected at 6 weeks post-vaccination, but was neither dose-dependent nor enhanced by flagellin; similar numbers of VSV-specific, IFNγ-producing cells were detected in lymph node and spleen of all animals. These results indicate that virus-directed, intracellular flagellin production may improve VSV-based vaccines encoding heterologous antigens by lowering the dose required to achieve humoral immunity.

  15. Opposing Roles of Double-Stranded RNA Effector Pathways and Viral Defense Proteins Revealed with CRISPR-Cas9 Knockout Cell Lines and Vaccinia Virus Mutants.

    Science.gov (United States)

    Liu, Ruikang; Moss, Bernard

    2016-09-01

    Vaccinia virus (VACV) decapping enzymes and cellular exoribonuclease Xrn1 catalyze successive steps in mRNA degradation and prevent double-stranded RNA (dsRNA) accumulation, whereas the viral E3 protein can bind dsRNA. We showed that dsRNA and E3 colocalized within cytoplasmic viral factories in cells infected with a decapping enzyme mutant as well as with wild-type VACV and that they coprecipitated with antibody. An E3 deletion mutant induced protein kinase R (PKR) and eukaryotic translation initiation factor alpha (eIF2α) phosphorylation earlier and more strongly than a decapping enzyme mutant even though less dsRNA was made, leading to more profound effects on viral gene expression. Human HAP1 and A549 cells were genetically modified by clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) to determine whether the same pathways restrict E3 and decapping mutants. The E3 mutant replicated in PKR knockout (KO) HAP1 cells in which RNase L is intrinsically inactive but only with a double knockout (DKO) of PKR and RNase L in A549 cells, indicating that both pathways decreased replication equivalently and that no additional dsRNA pathway was crucial. In contrast, replication of the decapping enzyme mutant increased significantly (though less than that of wild-type virus) in DKO A549 cells but not in DKO HAP1 cells where a smaller increase in viral protein synthesis occurred. Xrn1 KO A549 cells were viable but nonpermissive for VACV; however, wild-type and mutant viruses replicated in triple-KO cells in which RNase L and PKR were also inactivated. Since KO of PKR and RNase L was sufficient to enable VACV replication in the absence of E3 or Xrn1, the poor replication of the decapping mutant, particularly in HAP1 DKO, cells indicated additional translational defects. Viruses have evolved ways of preventing or counteracting the cascade of antiviral responses that double-stranded RNA (dsRNA) triggers in host cells. We showed that the dsRNA produced in

  16. A method of radiocompetitive assay of total thyroxine in the serum by means of enzymatic release of thyroxine from the transporting proteins

    International Nuclear Information System (INIS)

    Snarski, A.; Wyrwinski, J.

    1978-01-01

    Pepsin causes denaturation of the transporting proteins and liberates thyroxine which can be assayed by the radiocompetitive method. Change of the pH of the medium from acid to alkaline inactivates irreveribly pepsin. The enzymatic release of thyroxine is much simpler that the method of ethanol extraction and thermal denaturation of the transporting proteins applied up to now. The new technique of thyroxine release has been introduced for radiocompetitive determination of thyroxine using dextran coated charcoal for adsorption of the free hormone. A new method has been elaborated for preparation of working standards of thyroxine in a mixture of pepsin solution with hormone-free serum. The method is efficient and rapid. The normal range is from 50 to 130 nanomol/l. Over 7 000 determinations were done as yet in patients with suspected thyroid function disturbances. (author)

  17. Identification of Atg2 and ArfGAP1 as Candidate Genetic Modifiers of the Eye Pigmentation Phenotype of Adaptor Protein-3 (AP-3) Mutants in Drosophila melanogaster.

    Science.gov (United States)

    Rodriguez-Fernandez, Imilce A; Dell'Angelica, Esteban C

    2015-01-01

    The Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions--which together covered most of the autosomal chromosomes-to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in

  18. Structures of the N47A and E109Q mutant proteins of pyruvoyl-dependent arginine decarboxylase from Methanococcus jannaschii.

    Science.gov (United States)

    Soriano, Erika V; McCloskey, Diane E; Kinsland, Cynthia; Pegg, Anthony E; Ealick, Steven E

    2008-04-01

    Pyruvoyl-dependent arginine decarboxylase (PvlArgDC) catalyzes the first step of the polyamine-biosynthetic pathway in plants and some archaebacteria. The pyruvoyl group of PvlArgDC is generated by an internal autoserinolysis reaction at an absolutely conserved serine residue in the proenzyme, resulting in two polypeptide chains. Based on the native structure of PvlArgDC from Methanococcus jannaschii, the conserved residues Asn47 and Glu109 were proposed to be involved in the decarboxylation and autoprocessing reactions. N47A and E109Q mutant proteins were prepared and the three-dimensional structure of each protein was determined at 2.0 A resolution. The N47A and E109Q mutant proteins showed reduced decarboxylation activity compared with the wild-type PvlArgDC. These residues may also be important for the autoprocessing reaction, which utilizes a mechanism similar to that of the decarboxylation reaction.

  19. Further enhanced production of heterologous proteins by double-gene disruption (ΔAosedD ΔAovps10) in a hyper-producing mutant of Aspergillus oryzae.

    Science.gov (United States)

    Zhu, Lin; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

    2013-07-01

    The filamentous fungus Aspergillus oryzae is used as one of the most favored hosts for heterologous protein production due to its ability to secrete large amounts of proteins into the culture medium. We previously generated a hyper-producing mutant strain of A. oryzae, AUT1, which produced 3.2- and 2.6-fold higher levels of bovine chymosin (CHY) and human lysozyme (HLY), respectively, compared with the wild-type strain. However, further enhancement of heterologous protein production by multiple gene disruption is difficult because of the low gene-targeting efficiency in strain AUT1. Here, we disrupted the ligD gene, which is involved in nonhomologous recombination, and the pyrG gene to create uridine/uracil auxotrophy in strain AUT1, to generate a hyper-producing mutant applicable to pyrG marker recycling with highly efficient gene targeting. We generated single and double disruptants of the tripeptidyl peptidase gene AosedD and vacuolar sorting receptor gene Aovps10 in the hyper-producing mutant background, and found that all disruptants showed significant increases in heterologous protein production. Particularly, double disruption of the Aovps10 and AosedD genes increased the production levels of CHY and HLY by 1.6- and 2.1-fold, respectively, compared with the parental strain. Thus, we successfully generated a fungal host for further enhancing the heterologous protein production ability by combining mutational and molecular breeding techniques.

  20. Crystal structure of a C-terminal deletion mutant of human protein kinase CK2 catalytic subunit

    DEFF Research Database (Denmark)

    Ermakova, Inessa; Boldyreff, Brigitte; Issinger, Olaf-Georg

    2003-01-01

    structure of a C-terminal deletion mutant of human CK2alpha was solved and refined to 2.5A resolution. In the crystal the CK2alpha mutant exists as a monomer in agreement with the organization of the subunits in the CK2 holoenzyme. The refined structure shows the helix alphaC and the activation segment, two...

  1. Role of Flexibility in Protein-DNA-Drug Recognition: The Case of Asp677Gly-Val703Ile Topoisomerase Mutant Hypersensitive to Camptothecin

    Science.gov (United States)

    D'Annessa, Ilda; Tesauro, Cinzia; Fiorani, Paola; Chillemi, Giovanni; Castelli, Silvia; Vassallo, Oscar; Capranico, Giovanni; Desideri, Alessandro

    2012-01-01

    Topoisomerases I are ubiquitous enzymes that control DNA topology within the cell. They are the unique target of the antitumor drug camptothecin that selectively recognizes the DNA-topoisomerase covalent complex and reversibly stabilizes it. The biochemical and structural-dynamical properties of the Asp677Gly-Val703Ile double mutant with enhanced CPT sensitivity have been investigated. The mutant displays a lower religation rate of the DNA substrate when compared to the wild-type protein. Analyses of the structural dynamical properties by molecular dynamics simulation show that the mutant has reduced flexibility and an active site partially destructured at the level of the Lys532 residue. These results demonstrate long-range communication mechanism where reduction of the linker flexibility alters the active site geometry with the consequent lowering of the religation rate and increase in drug sensitivity. PMID:22315664

  2. Role of Flexibility in Protein-DNA-Drug Recognition: The Case of Asp677Gly-Val703Ile Topoisomerase Mutant Hypersensitive to Camptothecin

    Directory of Open Access Journals (Sweden)

    Ilda D'Annessa

    2012-01-01

    Full Text Available Topoisomerases I are ubiquitous enzymes that control DNA topology within the cell. They are the unique target of the antitumor drug camptothecin that selectively recognizes the DNA-topoisomerase covalent complex and reversibly stabilizes it. The biochemical and structural-dynamical properties of the Asp677Gly-Val703Ile double mutant with enhanced CPT sensitivity have been investigated. The mutant displays a lower religation rate of the DNA substrate when compared to the wild-type protein. Analyses of the structural dynamical properties by molecular dynamics simulation show that the mutant has reduced flexibility and an active site partially destructured at the level of the Lys532 residue. These results demonstrate long-range communication mechanism where reduction of the linker flexibility alters the active site geometry with the consequent lowering of the religation rate and increase in drug sensitivity.

  3. A new approach to protein enzymatic digestion for fast protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

    Czech Academy of Sciences Publication Activity Database

    Dyčka, Filip; Laštovičková, Markéta; Bobálová, Janette

    2008-01-01

    Roč. 102, č. 15 (2008), s967-s968 ISSN 1803-2389. [Meeting on Chemistry and Life /4./. Brno, 09.09.2008-11.09.2008] R&D Projects: GA AV ČR IAA600040701 Institutional research plan: CEZ:AV0Z40310501 Keywords : enzymatic digest ion * fast approach Subject RIV: CB - Analytical Chemistry, Separation

  4. Analyses of the in vitro non-enzymatic glycation of peptides/proteins by matrix-assisted laser desorption/ionization mass spectrometry

    Science.gov (United States)

    Lee, Bao-Shiang; Krishnanchettiar, Sangeeth; Lateef, Syed Salman; Gupta, Shalini

    2007-01-01

    Non-enzymatic glycation of proteins with the reducing agent glucose is implicated to be responsible for diabetes-derived complications, food browning, and aging. However, the non-enzymatic glycation process of peptides/proteins is not well understood and further research is needed to gain an understanding of the underlying principles involved in diabetes-related complications. In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is used to analyze the in vitro glycation of peptides/proteins. In addition to the physiological conditions, harsh conditions (higher concentration of glucose, higher or lower pH, and higher temperature) are also used in this study. Peptides/proteins are reacted with glucose for up to 120 h at 4 [degree sign]C, 37 [degree sign]C, or 65 [degree sign]C. Single and/or multiple glycations are observed using broad pH conditions (from 10% TFA with pKa of 0.5 to pH 10) at various glucose concentrations (from 0.01 M to 1 M). Data suggest that glucose reacts readily with both peptides and proteins, and the efficiency of the glycation increases with higher temperature, higher pH, higher glucose concentration, or longer incubation time. However, influence of the buffer pH on the efficiency of the glycation of peptides is less pronounced compared to that of proteins. This effect could result from denaturation of proteins at higher pH and the resultant exposure of potential glycation sites. This data could lead to the inference that the glycation process of peptides/proteins would occur but proceed very slowly under the diabetes conditions in vivo (37 [degree sign]C, ~neutral pH, ~0.007 M glucose). Postsource decay and MS/MS results of singly glycated angiotensin I, P14R (PPPPPPPPPPPPPPR), and human adrenocorticotropic hormone (ATCH) fragments 1-13 indicate that glucose reacts with the amino group of the N-terminal of ATCH 1-13 and the guanidino group of the arginine residue of both angiotensin I and P14R.

  5. Mechanistic and Structural Studies of Protein-Only RNase P Compared to Ribonucleoproteins Reveal the Two Faces of the Same Enzymatic Activity

    Directory of Open Access Journals (Sweden)

    Cédric Schelcher

    2016-06-01

    Full Text Available RNase P, the essential activity that performs the 5′ maturation of tRNA precursors, can be achieved either by ribonucleoproteins containing a ribozyme present in the three domains of life or by protein-only enzymes called protein-only RNase P (PRORP that occur in eukaryote nuclei and organelles. A fast growing list of studies has investigated three-dimensional structures and mode of action of PRORP proteins. Results suggest that similar to ribozymes, PRORP proteins have two main domains. A clear functional analogy can be drawn between the specificity domain of the RNase P ribozyme and PRORP pentatricopeptide repeat domain, and between the ribozyme catalytic domain and PRORP N4BP1, YacP-like Nuclease domain. Moreover, both types of enzymes appear to dock with the acceptor arm of tRNA precursors and make specific contacts with the corner of pre-tRNAs. While some clear differences can still be delineated between PRORP and ribonucleoprotein (RNP RNase P, the two types of enzymes seem to use, fundamentally, the same catalytic mechanism involving two metal ions. The occurrence of PRORP and RNP RNase P represents a remarkable example of convergent evolution. It might be the unique witness of an ongoing replacement of catalytic RNAs by proteins for enzymatic activities.

  6. Oligomeric structure and chaperone-like activity of Drosophila melanogaster mitochondrial small heat shock protein Hsp22 and arginine mutants in the alpha-crystallin domain.

    Science.gov (United States)

    Dabbaghizadeh, Afrooz; Finet, Stéphanie; Morrow, Genevieve; Moutaoufik, Mohamed Taha; Tanguay, Robert M

    2017-07-01

    The structure and chaperone function of DmHsp22WT, a small Hsp of Drosophila melanogaster localized within mitochondria were examined. Mutations of conserved arginine mutants within the alpha-crystallin domain (ACD) domain (R105G, R109G, and R110G) were introduced, and their effects on oligomerization and chaperone function were assessed. Arginine to glycine mutations do not induce significant changes in tryptophan fluorescence, and the mutated proteins form oligomers that are of equal or smaller size than the wild-type protein. They all form oligomer with one single peak as determined by size exclusion chromatography. While all mutants demonstrate the same efficiency as the DmHsp22WT in a DTT-induced insulin aggregation assay, all are more efficient chaperones to prevent aggregation of malate dehydrogenase. Arginine mutants of DmHsp22 are efficient chaperones to retard aggregation of CS and Luc. In summary, this study shows that mutations of arginine to glycine in DmHsp22 ACD induce a number of structural changes, some of which differ from those described in mammalian sHsps. Interestingly, only the R110G-DmHsp22 mutant, and not the expected R109G equivalent to human R140-HspB1, R116-HspB4, and R120-HspB5, showed different structural properties compared with the DmHsp22WT.

  7. Structures of the N47A and E109Q mutant proteins of pyruvoyl-dependent arginine decarboxylase from Methanococcus jannaschii

    Energy Technology Data Exchange (ETDEWEB)

    Soriano, Erika V. [Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14850-1301 (United States); McCloskey, Diane E. [Department of Cellular and Molecular Physiology, Pennsylvania State University, College of Medicine, Hershey, PA 17033 (United States); Kinsland, Cynthia [Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14850-1301 (United States); Pegg, Anthony E. [Department of Cellular and Molecular Physiology, Pennsylvania State University, College of Medicine, Hershey, PA 17033 (United States); Ealick, Steven E., E-mail: see3@cornell.edu [Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14850-1301 (United States)

    2008-04-01

    The crystal structures of two arginine decarboxylase mutant proteins provide insights into the mechanisms of pyruvoyl-group formation and the decarboxylation reaction. Pyruvoyl-dependent arginine decarboxylase (PvlArgDC) catalyzes the first step of the polyamine-biosynthetic pathway in plants and some archaebacteria. The pyruvoyl group of PvlArgDC is generated by an internal autoserinolysis reaction at an absolutely conserved serine residue in the proenzyme, resulting in two polypeptide chains. Based on the native structure of PvlArgDC from Methanococcus jannaschii, the conserved residues Asn47 and Glu109 were proposed to be involved in the decarboxylation and autoprocessing reactions. N47A and E109Q mutant proteins were prepared and the three-dimensional structure of each protein was determined at 2.0 Å resolution. The N47A and E109Q mutant proteins showed reduced decarboxylation activity compared with the wild-type PvlArgDC. These residues may also be important for the autoprocessing reaction, which utilizes a mechanism similar to that of the decarboxylation reaction.

  8. A δ38 deletion variant of human transketolase as a model of transketolase-like protein 1 exhibits no enzymatic activity.

    Directory of Open Access Journals (Sweden)

    Stefan Schneider

    Full Text Available Besides transketolase (TKT, a thiamin-dependent enzyme of the pentose phosphate pathway, the human genome encodes for two closely related transketolase-like proteins, which share a high sequence identity with TKT. Transketolase-like protein 1 (TKTL1 has been implicated in cancerogenesis as its cellular expression levels were reported to directly correlate with invasion efficiency of cancer cells and patient mortality. It has been proposed that TKTL1 exerts its function by catalyzing an unusual enzymatic reaction, a hypothesis that has been the subject of recent controversy. The most striking difference between TKTL1 and TKT is a deletion of 38 consecutive amino acids in the N-terminal domain of the former, which constitute part of the active site in authentic TKT. Our structural and sequence analysis suggested that TKTL1 might not possess transketolase activity. In order to test this hypothesis in the absence of a recombinant expression system for TKTL1 and resilient data on its biochemical properties, we have engineered and biochemically characterized a "pseudo-TKTL1" Δ38 deletion variant of human TKT (TKTΔ38 as a viable model of TKTL1. Although the isolated protein is properly folded under in vitro conditions, both thermal stability as well as stability of the TKT-specific homodimeric assembly are markedly reduced. Circular dichroism and NMR spectroscopic analysis further indicates that TKTΔ38 is unable to bind the thiamin cofactor in a specific manner, even at superphysiological concentrations. No transketolase activity of TKTΔ38 can be detected for conversion of physiological sugar substrates thus arguing against an intrinsically encoded enzymatic function of TKTL1 in tumor cell metabolism.

  9. Reduced Activity of Mutant Calcium-Dependent Protein Kinase 1 Is Compensated in Plasmodium falciparum through the Action of Protein Kinase G

    Directory of Open Access Journals (Sweden)

    Abhisheka Bansal

    2016-12-01

    Full Text Available We used a sensitization approach that involves replacement of the gatekeeper residue in a protein kinase with one with a different side chain. The activity of the enzyme with a bulky gatekeeper residue, such as methionine, cannot be inhibited using bumped kinase inhibitors (BKIs. Here, we have used this approach to study Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1. The methionine gatekeeper substitution, T145M, although it led to a 47% reduction in transphosphorylation, was successfully introduced into the CDPK1 locus using clustered regularly interspaced short palindromic repeat (CRISPR/Cas9. As methionine is a bulky residue, BKI 1294 had a 10-fold-greater effect in vitro on the wild-type enzyme than on the methionine mutant. However, in contrast to in vitro data with recombinant enzymes, BKI 1294 had a slightly greater inhibition of the growth of CDPK1 T145M parasites than the wild type. Moreover, the CDPK1 T145M parasites were more sensitive to the action of compound 2 (C2, a specific inhibitor of protein kinase G (PKG. These results suggest that a reduction in the activity of CDPK1 due to methionine substitution at the gatekeeper position is compensated through the direct action of PKG or of another kinase under the regulation of PKG. The transcript levels of CDPK5 and CDPK6 were significantly upregulated in the CDPK1 T145M parasites. The increase in CDPK6 or some other kinase may compensate for decrease in CDPK1 activity during invasion. This study suggests that targeting two kinases may be more effective in chemotherapy to treat malaria so as not to select for mutations in one of the enzymes.

  10. The Zea mays mutants opaque-2 and opaque-7 disclose extensive changes in endosperm metabolism as revealed by protein, amino acid, and transcriptome-wide analyses

    Directory of Open Access Journals (Sweden)

    Pirona Raul

    2011-01-01

    Full Text Available Abstract Background The changes in storage reserve accumulation during maize (Zea mays L. grain maturation are well established. However, the key molecular determinants controlling carbon flux to the grain and the partitioning of carbon to starch and protein are more elusive. The Opaque-2 (O2 gene, one of the best-characterized plant transcription factors, is a good example of the integration of carbohydrate, amino acid and storage protein metabolisms in maize endosperm development. Evidence also indicates that the Opaque-7 (O7 gene plays a role in affecting endosperm metabolism. The focus of this study was to assess the changes induced by the o2 and o7 mutations on maize endosperm metabolism by evaluating protein and amino acid composition and by transcriptome profiling, in order to investigate the functional interplay between these two genes in single and double mutants. Results We show that the overall amino acid composition of the mutants analyzed appeared similar. Each mutant had a high Lys and reduced Glx and Leu content with respect to wild type. Gene expression profiling, based on a unigene set composed of 7,250 ESTs, allowed us to identify a series of mutant-related down (17.1% and up-regulated (3.2% transcripts. Several differentially expressed ESTs homologous to genes encoding enzymes involved in amino acid synthesis, carbon metabolism (TCA cycle and glycolysis, in storage protein and starch metabolism, in gene transcription and translation processes, in signal transduction, and in protein, fatty acid, and lipid synthesis were identified. Our analyses demonstrate that the mutants investigated are pleiotropic and play a critical role in several endosperm-related metabolic processes. Pleiotropic effects were less evident in the o7 mutant, but severe in the o2 and o2o7 backgrounds, with large changes in gene expression patterns, affecting a broad range of kernel-expressed genes. Conclusion Although, by necessity, this paper is

  11. The Zea mays mutants opaque-2 and opaque-7 disclose extensive changes in endosperm metabolism as revealed by protein, amino acid, and transcriptome-wide analyses.

    Science.gov (United States)

    Hartings, Hans; Lauria, Massimiliano; Lazzaroni, Nadia; Pirona, Raul; Motto, Mario

    2011-01-18

    The changes in storage reserve accumulation during maize (Zea mays L.) grain maturation are well established. However, the key molecular determinants controlling carbon flux to the grain and the partitioning of carbon to starch and protein are more elusive. The Opaque-2 (O2) gene, one of the best-characterized plant transcription factors, is a good example of the integration of carbohydrate, amino acid and storage protein metabolisms in maize endosperm development. Evidence also indicates that the Opaque-7 (O7) gene plays a role in affecting endosperm metabolism. The focus of this study was to assess the changes induced by the o2 and o7 mutations on maize endosperm metabolism by evaluating protein and amino acid composition and by transcriptome profiling, in order to investigate the functional interplay between these two genes in single and double mutants. We show that the overall amino acid composition of the mutants analyzed appeared similar. Each mutant had a high Lys and reduced Glx and Leu content with respect to wild type. Gene expression profiling, based on a unigene set composed of 7,250 ESTs, allowed us to identify a series of mutant-related down (17.1%) and up-regulated (3.2%) transcripts. Several differentially expressed ESTs homologous to genes encoding enzymes involved in amino acid synthesis, carbon metabolism (TCA cycle and glycolysis), in storage protein and starch metabolism, in gene transcription and translation processes, in signal transduction, and in protein, fatty acid, and lipid synthesis were identified. Our analyses demonstrate that the mutants investigated are pleiotropic and play a critical role in several endosperm-related metabolic processes. Pleiotropic effects were less evident in the o7 mutant, but severe in the o2 and o2o7 backgrounds, with large changes in gene expression patterns, affecting a broad range of kernel-expressed genes. Although, by necessity, this paper is descriptive and more work is required to define gene functions

  12. Role of protein synthesis in the repair of sublethal x-ray damage in a mutant Chinese hamster ovary cell line

    Energy Technology Data Exchange (ETDEWEB)

    Yezzi, M.J.

    1985-04-01

    A temperature-sensitive mutant for protein synthesis, CHO-TSH1, has been compared to the wild-type cell, CHO-sC1, in single- and split-radiation-dose schemes. When the exponentially growing TS mutant and the wild-type cells were treated at 40/sub 0/C for up to 2 hrs prior to graded doses of x rays, the survival curves were identical and were the same as those obtained without heat treatment. If the cultures were incubated at 40/sup 0/C for 2 hrs before a first dose and maintained at 40/sup 0/C during a 2 hr dose fractionation interval, repair of radiation damage was reduced in the mutant compared to the wild type. These observations implied that a pool of proteins was involved in the repair of sublethal x-ray damage. However, if repair was measured by the alkaline-unwinding technique under the same time and temperature schemes, no difference in the kientics of DNA strand rejoining was observed. Misrepair processes may permit restoration of DNA strand integrity but not allow functional repair. The effect of diminished repair under conditions of inhibition of protein synthesis was found to be cell-cycle dependent in survival studies with synchronized mutant cell populations. Repair was found to be almost completely eliminated if the temperature sequence described above was applied in the middle of the DNA synthetic phase. Treatment of cell populations in the middle of G/sub 1/-phase yielded repair inhibition comparable to that observed with the asynchronous cells. Splitdose experiments were done using pre-incubation with cycloheximide to chemically inhibit protein synthesis. WT cells and TS cells were treated with cycloheximide at 35/sup 0/C for 2 hrs before a first dose and during a 2 hr dose fractionation interval. 23 figs., 7 tabs.

  13. Role of protein synthesis in the repair of sublethal x-ray damage in a mutant Chinese hamster ovary cell line

    International Nuclear Information System (INIS)

    Yezzi, M.J.

    1985-04-01

    A temperature-sensitive mutant for protein synthesis, CHO-TSH1, has been compared to the wild-type cell, CHO-sC1, in single- and split-radiation-dose schemes. When the exponentially growing TS mutant and the wild-type cells were treated at 40 0 C for up to 2 hrs prior to graded doses of x rays, the survival curves were identical and were the same as those obtained without heat treatment. If the cultures were incubated at 40 0 C for 2 hrs before a first dose and maintained at 40 0 C during a 2 hr dose fractionation interval, repair of radiation damage was reduced in the mutant compared to the wild type. These observations implied that a pool of proteins was involved in the repair of sublethal x-ray damage. However, if repair was measured by the alkaline-unwinding technique under the same time and temperature schemes, no difference in the kientics of DNA strand rejoining was observed. Misrepair processes may permit restoration of DNA strand integrity but not allow functional repair. The effect of diminished repair under conditions of inhibition of protein synthesis was found to be cell-cycle dependent in survival studies with synchronized mutant cell populations. Repair was found to be almost completely eliminated if the temperature sequence described above was applied in the middle of the DNA synthetic phase. Treatment of cell populations in the middle of G 1 -phase yielded repair inhibition comparable to that observed with the asynchronous cells. Splitdose experiments were done using pre-incubation with cycloheximide to chemically inhibit protein synthesis. WT cells and TS cells were treated with cycloheximide at 35 0 C for 2 hrs before a first dose and during a 2 hr dose fractionation interval. 23 figs., 7 tabs

  14. QM/MM Geometry Optimization on Extensive Free-Energy Surfaces for Examination of Enzymatic Reactions and Design of Novel Functional Properties of Proteins.

    Science.gov (United States)

    Hayashi, Shigehiko; Uchida, Yoshihiro; Hasegawa, Taisuke; Higashi, Masahiro; Kosugi, Takahiro; Kamiya, Motoshi

    2017-05-05

    Many remarkable molecular functions of proteins use their characteristic global and slow conformational dynamics through coupling of local chemical states in reaction centers with global conformational changes of proteins. To theoretically examine the functional processes of proteins in atomic detail, a methodology of quantum mechanical/molecular mechanical (QM/MM) free-energy geometry optimization is introduced. In the methodology, a geometry optimization of a local reaction center is performed with a quantum mechanical calculation on a free-energy surface constructed with conformational samples of the surrounding protein environment obtained by a molecular dynamics simulation with a molecular mechanics force field. Geometry optimizations on extensive free-energy surfaces by a QM/MM reweighting free-energy self-consistent field method designed to be variationally consistent and computationally efficient have enabled examinations of the multiscale molecular coupling of local chemical states with global protein conformational changes in functional processes and analysis and design of protein mutants with novel functional properties.

  15. Novel induced mlo mutant alleles in combination with site-directed mutagenesis reveal functionally important domains in the heptahelical barley Mlo protein

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    Piffanelli Pietro

    2010-02-01

    Full Text Available Abstract Background Recessively inherited natural and induced mutations in the barley Mlo gene confer durable broad-spectrum resistance against the powdery mildew pathogen, Blumeria graminis f.sp. hordei. Mlo codes for a member of a plant-specific family of polytopic integral membrane proteins with unknown biochemical activity. Resistant barley mlo mutant alleles identify amino acid residues that are critical for Mlo function in the context of powdery mildew susceptibility. Results We molecularly analyzed a novel set of induced barley mlo mutants and used site-directed mutagenesis in combination with transient gene expression to unravel novel amino acid residues of functional significance. We integrate these results with previous findings to map functionally important regions of the heptahelical Mlo protein. Our data reveal the second and third cytoplasmic loop as being particularly sensitive to functional impediment by mutational perturbation, suggesting that these regions are critical for the susceptibility-conferring activity of the Mlo protein. In contrast, only mutations in the second but not the third cytoplasmic loop appear to trigger the Endoplasmic Reticulum-localized quality control machinery that ensures the biogenesis of properly folded membrane proteins. Conclusion Our findings identify functionally important regions of the polytopic barley Mlo protein and reveal the differential sensitivity of individual protein domains to cellular quality control.

  16. Expression, purification and characterization of a novel double-sites mutant of the single-chain sweet-tasting protein monellin (MNEI) with both improved sweetness and stability.

    Science.gov (United States)

    Zheng, Weiwei; Yang, Liu; Cai, Chenggu; Ni, Jinfeng; Liu, Bo

    2018-03-01

    The sweet protein monellin has high sweet potency with limited stability. In this study, 3 double-sites mutants (E2N/E23A, E2N/Y65R and E23A/Y65R) of the single-chain monellin (MNEI) were constructed. The proteins were expressed in E. coli BL21 and purified to homogeneity by nickel affinity chromatography with yields above 10 mg/L cell culture. Introduction of a sweeter mutant E2N into E23A or Y65R (E2N/E23A and E2N/Y65R) led to about 3-fold increase of sweetness, while addition of a more stable mutant E23A into E2N or Y65R (E2N/E23A and E23A/Y65R) resulted in improved thermal stability (about 10 °C). The results indicate that residues E2 and E23 mediate the sweetness and thermal stability of the protein, respectively. Multiple mutations of different residues (E2N/E23A) led to an additive performance with both improved sweetness and stability, suggesting that the sweetness and stability could be modulated by the independent molecular mechanism. The sweeter and thermal stable variant has a potential in further industrial applications. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. An attenuated Shigella mutant lacking the RNA-binding protein Hfq provides cross-protection against Shigella strains of broad serotype.

    Science.gov (United States)

    Mitobe, Jiro; Sinha, Ritam; Mitra, Soma; Nag, Dhrubajyoti; Saito, Noriko; Shimuta, Ken; Koizumi, Nobuo; Koley, Hemanta

    2017-07-01

    Few live attenuated vaccines protect against multiple serotypes of bacterial pathogen because host serotype-specific immune responses are limited to the serotype present in the vaccine strain. Here, immunization with a mutant of Shigella flexneri 2a protected guinea pigs against subsequent infection by S. dysenteriae type 1 and S. sonnei strains. This deletion mutant lacked the RNA-binding protein Hfq leading to increased expression of the type III secretion system via loss of regulation, resulting in attenuation of cell viability through repression of stress response sigma factors. Such increased antigen production and simultaneous attenuation were expected to elicit protective immunity against Shigella strains of heterologous serotypes. Thus, the vaccine potential of this mutant was tested in two guinea pig models of shigellosis. Animals vaccinated in the left eye showed fewer symptoms upon subsequent challenge via the right eye, and even survived subsequent intestinal challenge. In addition, oral vaccination effectively induced production of immunoglobulins without severe side effects, again protecting all animals against subsequent intestinal challenge with S. dysenteriae type 1 or S. sonnei strains. Antibodies against common virulence proteins and the O-antigen of S. flexneri 2a were detected by immunofluorescence microscopy. Reaction of antibodies with various strains, including enteroinvasive Escherichia coli, suggested that common virulence proteins induced protective immunity against a range of serotypes. Therefore, vaccination is expected to cover not only the most prevalent serotypes of S. sonnei and S. flexneri 2a, but also various Shigella strains, including S. dysenteriae type 1, which produces Shiga toxin.

  18. Structure–Biological Function Relationship Extended to Mitotic Arrest-Deficient 2-Like Protein Mad2 Native and Mutants-New Opportunity for Genetic Disorder Control

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    Speranta Avram

    2014-11-01

    Full Text Available Overexpression of mitotic arrest-deficient proteins Mad1 and Mad2, two components of spindle assembly checkpoint, is a risk factor for chromosomal instability (CIN and a trigger of many genetic disorders. Mad2 transition from inactive open (O-Mad2 to active closed (C-Mad2 conformations or Mad2 binding to specific partners (cell-division cycle protein 20 (Cdc20 or Mad1 were targets of previous pharmacogenomics studies. Here, Mad2 binding to Cdc20 and the interconversion rate from open to closed Mad2 were predicted and the molecular features with a critical contribution to these processes were determined by extending the quantitative structure-activity relationship (QSAR method to large-size proteins such as Mad2. QSAR models were built based on available published data on 23 Mad2 mutants inducing CIN-related functional changes. The most relevant descriptors identified for predicting Mad2 native and mutants action mechanism and their involvement in genetic disorders are the steric (van der Waals area and solvent accessible area and their subdivided and energetic van der Waals energy descriptors. The reliability of our QSAR models is indicated by significant values of statistical coefficients: Cross-validated correlation q2 (0.53–0.65 and fitted correlation r2 (0.82–0.90. Moreover, based on established QSAR equations, we rationally design and analyze nine de novo Mad2 mutants as possible promoters of CIN.

  19. 4-Hydroxy-2-nonenal Alkylated and Peroxynitrite Nitrated Proteins Localize to the Fused Mitochondria in Malpighian Epithelial Cells of Type IV Collagen Drosophila Mutants

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    András A. Kiss

    2018-01-01

    Full Text Available Background. Human type IV collagenopathy is associated with mutations within the COL4A1 and to a less extent the COL4A2 genes. The proteins encoded by these genes form heterotrimers and are the highest molar ratio components of the ubiquitous basement membrane. The clinical manifestations of the COL4A1/A2 mutations are systemic affecting many tissues and organs among these kidneys. In order to uncover the cellular and biochemical alterations associated with aberrant type IV collagen, we have explored the phenotype of the Malpighian tubules, the secretory organ and insect kidney model, in col4a1 collagen gene mutants of the fruit fly Drosophila melanogaster. In Malpighian epithelial cells of col4a1 mutants, robust mitochondrial fusion indicated mutation-induced stress. Immunohistochemistry detected proteins nitrated by peroxynitrite that localized to the enlarged mitochondria and increased level of membrane peroxidation, assessed by the amount of proteins alkylated by 4-hydroxy-2-nonenal that similarly localized to the fused mitochondria. Nuclei within the Malpighian epithelium showed TUNEL-positivity suggesting cell degradation. The results demonstrated that col4a1 mutations affect the epithelia and, consequently, secretory function of the Malpighian tubules and provide mechanistic insight into col4a1 mutation-associated functional impairments not yet reported in human patients and in mouse models with mutant COL4A1.

  20. Xyloketal-derived small molecules show protective effect by decreasing mutant Huntingtin protein aggregates in Caenorhabditis elegans model of Huntington's disease.

    Science.gov (United States)

    Zeng, Yixuan; Guo, Wenyuan; Xu, Guangqing; Wang, Qinmei; Feng, Luyang; Long, Simei; Liang, Fengyin; Huang, Yi; Lu, Xilin; Li, Shichang; Zhou, Jiebin; Burgunder, Jean-Marc; Pang, Jiyan; Pei, Zhong

    2016-01-01

    Huntington's disease is an autosomal-dominant neurodegenerative disorder, with chorea as the most prominent manifestation. The disease is caused by abnormal expansion of CAG codon repeats in the IT15 gene, which leads to the expression of a glutamine-rich protein named mutant Huntingtin (Htt). Because of its devastating disease burden and lack of valid treatment, development of more effective therapeutics for Huntington's disease is urgently required. Xyloketal B, a natural product from mangrove fungus, has shown protective effects against toxicity in other neurodegenerative disease models such as Parkinson's and Alzheimer's diseases. To identify potential neuroprotective molecules for Huntington's disease, six derivatives of xyloketal B were screened in a Caenorhabditis elegans Huntington's disease model; all six compounds showed a protective effect. Molecular docking studies indicated that compound 1 could bind to residues GLN369 and GLN393 of the mutant Htt protein, forming a stable trimeric complex that can prevent the formation of mutant Htt aggregates. Taken together, we conclude that xyloketal derivatives could be novel drug candidates for treating Huntington's disease. Molecular target analysis is a good method to simulate the interaction between proteins and drug compounds. Further, protective candidate drugs could be designed in future using the guidance of molecular docking results.

  1. Xyloketal-derived small molecules show protective effect by decreasing mutant Huntingtin protein aggregates in Caenorhabditis elegans model of Huntington’s disease

    Science.gov (United States)

    Zeng, Yixuan; Guo, Wenyuan; Xu, Guangqing; Wang, Qinmei; Feng, Luyang; Long, Simei; Liang, Fengyin; Huang, Yi; Lu, Xilin; Li, Shichang; Zhou, Jiebin; Burgunder, Jean-Marc; Pang, Jiyan; Pei, Zhong

    2016-01-01

    Huntington’s disease is an autosomal-dominant neurodegenerative disorder, with chorea as the most prominent manifestation. The disease is caused by abnormal expansion of CAG codon repeats in the IT15 gene, which leads to the expression of a glutamine-rich protein named mutant Huntingtin (Htt). Because of its devastating disease burden and lack of valid treatment, development of more effective therapeutics for Huntington’s disease is urgently required. Xyloketal B, a natural product from mangrove fungus, has shown protective effects against toxicity in other neurodegenerative disease models such as Parkinson’s and Alzheimer’s diseases. To identify potential neuroprotective molecules for Huntington’s disease, six derivatives of xyloketal B were screened in a Caenorhabditis elegans Huntington’s disease model; all six compounds showed a protective effect. Molecular docking studies indicated that compound 1 could bind to residues GLN369 and GLN393 of the mutant Htt protein, forming a stable trimeric complex that can prevent the formation of mutant Htt aggregates. Taken together, we conclude that xyloketal derivatives could be novel drug candidates for treating Huntington’s disease. Molecular target analysis is a good method to simulate the interaction between proteins and drug compounds. Further, protective candidate drugs could be designed in future using the guidance of molecular docking results. PMID:27110099

  2. Oculocutaneous albinism type 1: link between mutations, tyrosinase conformational stability, and enzymatic activity.

    Science.gov (United States)

    Dolinska, Monika B; Kus, Nicole J; Farney, S Katie; Wingfield, Paul T; Brooks, Brian P; Sergeev, Yuri V

    2017-01-01

    Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disorder caused by mutations in the tyrosinase gene. Two subtypes of OCA1 have been described: severe OCA1A with complete absence of tyrosinase activity and less severe OCA1B with residual tyrosinase activity. Here, we characterize the recombinant human tyrosinase intramelanosomal domain and mutant variants, which mimic genetic changes in both subtypes of OCA1 patients. Proteins were prepared using site-directed mutagenesis, expressed in insect larvae, purified by chromatography, and characterized by enzymatic activities, tryptophan fluorescence, and Gibbs free energy changes. The OCA1A mutants showed very low protein expression and protein yield and are enzymatically inactive. Mutants mimicking OCA1B were biochemically similar to the wild type, but exhibited lower specific activities and protein stabilities. The results are consistent with clinical data, which indicates that OCA1A mutations inactivate tyrosinase and result in severe phenotype, while OCA1B mutations partially inactivate tyrosinase and result in OCA1B albinism. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  3. The effect of the lamin A and its mutants on nuclear structure, cell proliferation, protein stability, and mobility in embryonic cells.

    Science.gov (United States)

    Piekarowicz, Katarzyna; Machowska, Magdalena; Dratkiewicz, Ewelina; Lorek, Daria; Madej-Pilarczyk, Agnieszka; Rzepecki, Ryszard

    2017-08-01

    LMNA gene encodes for nuclear intermediate filament proteins lamin A/C. Mutations in this gene lead to a spectrum of genetic disorders, collectively referred to as laminopathies. Lamin A/C are widely expressed in most differentiated somatic cells but not in early embryos and some undifferentiated cells. To investigate the role of lamin A/C in cell phenotype maintenance and differentiation, which could be a determinant of the pathogenesis of laminopathies, we examined the role played by exogenous lamin A and its mutants in differentiated cell lines (HeLa, NHDF) and less-differentiated HEK 293 cells. We introduced exogenous wild-type and mutated (H222P, L263P, E358K D446V, and ∆50) lamin A into different cell types and analyzed proteins' impact on proliferation, protein mobility, and endogenous nuclear envelope protein distribution. The mutants give rise to a broad spectrum of nuclear phenotypes and relocate lamin C. The mutations ∆50 and D446V enhance proliferation in comparison to wild-type lamin A and control cells, but no changes in exogenous protein mobility measured by FRAP were observed. Interestingly, although transcripts for lamins A and C are at similar level in HEK 293 cells, only lamin C protein is detected in western blots. Also, exogenous lamin A and its mutants, when expressed in HEK 293 cells underwent posttranscriptional processing. Overall, our results provide new insight into the maintenance of lamin A in less-differentiated cells. Embryonic cells are very sensitive to lamin A imbalance, and its upregulation disturbs lamin C, which may influence gene expression and many regulatory pathways.

  4. Modelling Tethered Enzymatic Reactions

    Science.gov (United States)

    Solis Salas, Citlali; Goyette, Jesse; Coker-Gordon, Nicola; Bridge, Marcus; Isaacson, Samuel; Allard, Jun; Maini, Philip; Dushek, Omer

    Enzymatic reactions are key to cell functioning, and whilst much work has been done in protein interaction in cases where diffusion is possible, interactions of tethered proteins are poorly understood. Yet, because of the large role cell membranes play in enzymatic reactions, several reactions may take place where one of the proteins is bound to a fixed point in space. We develop a model to characterize tethered signalling between the phosphatase SHP-1 interacting with a tethered, phosphorylated protein. We compare our model to experimental data obtained using surface plasmon resonance (SPR). We show that a single SPR experiment recovers 5 independent biophysical/biochemical constants. We also compare the results between a three dimensional model and a two dimensional model. The work gives the opportunity to use known techniques to learn more about signalling processes, and new insights into how enzyme tethering alters cellular signalling. With support from the Mexican Council for Science and Technology (CONACyT), the Public Education Secretariat (SEP), and the Mexican National Autonomous University's Foundation (Fundacion UNAM).

  5. An Ash1-Like Protein MoKMT2H Null Mutant Is Delayed for Conidium Germination and Pathogenesis in Magnaporthe oryzae

    Directory of Open Access Journals (Sweden)

    Zhaojun Cao

    2016-01-01

    Full Text Available Ash1 is a known H3K36-specific histone demethylase that is required for normal Hox gene expression and fertility in Drosophila and mammals. However, little is known about the expression and function of the fungal ortholog of Ash1 in phytopathogenic fungus Magnaporthe oryzae. Here we report that MoKMT2H, an Ash1-like protein, is required for conidium germination and virulence in rice. We obtained MoKMT2H null mutant (ΔMoKMT2H using a target gene replacement strategy. In the ΔMoKMT2H null mutants, global histone methyltransferase modifications (H3K4me3, H3K9me3, H3K27me3, and H3K36me2/3 of the genome were unaffected. The ΔMoKMT2H mutants showed no defect in vegetative hyphal growth, conidium morphology, conidiation, or disease lesion formation on rice leaves. However, the MoKMT2H deletion mutants were delayed for conidium germination and consequently had decreased virulence. Taken together, our results indicated that MoKMT2H plays an important role in conidium germination during appressorium formation in the rice blast fungus and perhaps other pathogenic plant fungi.

  6. The rye mutants identify a role for Ssn/Srb proteins of the RNA polymerase II holoenzyme during stationary phase entry in Saccharomyces cerevisiae.

    Science.gov (United States)

    Chang, Y W; Howard, S C; Budovskaya, Y V; Rine, J; Herman, P K

    2001-01-01

    Saccharomyces cerevisiae cells enter into a distinct resting state, known as stationary phase, in response to specific types of nutrient deprivation. We have identified a collection of mutants that exhibited a defective transcriptional response to nutrient limitation and failed to enter into a normal stationary phase. These rye mutants were isolated on the basis of defects in the regulation of YGP1 expression. In wild-type cells, YGP1 levels increased during the growth arrest caused by nutrient deprivation or inactivation of the Ras signaling pathway. In contrast, the levels of YGP1 and related genes were significantly elevated in the rye mutants during log phase growth. The rye defects were not specific to this YGP1 response as these mutants also exhibited multiple defects in stationary phase properties, including an inability to survive periods of prolonged starvation. These data indicated that the RYE genes might encode important regulators of yeast cell growth. Interestingly, three of the RYE genes encoded the Ssn/Srb proteins, Srb9p, Srb10p, and Srb11p, which are associated with the RNA polymerase II holoenzyme. Thus, the RNA polymerase II holoenzyme may be a target of the signaling pathways responsible for coordinating yeast cell growth with nutrient availability.

  7. The Mutant KRAS Gene Up-regulates BCL-XL Protein via STAT3 to Confer Apoptosis Resistance That Is Reversed by BIM Protein Induction and BCL-XL Antagonism.

    Science.gov (United States)

    Zaanan, Aziz; Okamoto, Koichi; Kawakami, Hisato; Khazaie, Khashayarsha; Huang, Shengbing; Sinicrope, Frank A

    2015-09-25

    In colorectal cancers with oncogenic GTPase Kras (KRAS) mutations, inhibition of downstream MEK/ERK signaling has shown limited efficacy, in part because of failure to induce a robust apoptotic response. We studied the mechanism of apoptosis resistance in mutant KRAS cells and sought to enhance the efficacy of a KRAS-specific MEK/ERK inhibitor, GDC-0623. GDC-0623 was shown to potently up-regulate BIM expression to a greater extent versus other MEK inhibitors in isogenic KRAS HCT116 and mutant KRAS SW620 colon cancer cells. ERK silencing enhanced BIM up-regulation by GDC-0623 that was due to its loss of phosphorylation at Ser(69), confirmed by a BIM-EL phosphorylation-defective mutant (S69G) that increased protein stability and blocked BIM induction. Despite BIM and BIK induction, the isogenic KRAS mutant versus wild-type cells remained resistant to GDC-0623-induced apoptosis, in part because of up-regulation of BCL-XL. KRAS knockdown by a doxycycline-inducible shRNA attenuated BCL-XL expression. BCL-XL knockdown sensitized KRAS mutant cells to GDC-0623-mediated apoptosis, as did the BH3 mimetic ABT-263. GDC-0623 plus ABT-263 induced a synergistic apoptosis by a mechanism that includes release of BIM from its sequestration by BCL-XL. Furthermore, mutant KRAS activated p-STAT3 (Tyr(705)) in the absence of IL-6 secretion, and STAT3 knockdown reduced BCL-XL mRNA and protein expression. These data suggest that BCL-XL up-regulation by STAT3 contributes to mutant KRAS-mediated apoptosis resistance. Such resistance can be overcome by potent BIM induction and concurrent BCL-XL antagonism to enable a synergistic apoptotic response. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. The novel influenza A virus protein PA-X and its naturally deleted variant show different enzymatic properties in comparison to the viral endonuclease PA.

    Science.gov (United States)

    Bavagnoli, Laura; Cucuzza, Stefano; Campanini, Giulia; Rovida, Francesca; Paolucci, Stefania; Baldanti, Fausto; Maga, Giovanni

    2015-10-30

    The PA protein of Influenza A virus (IAV) encoded by segment 3 acts as a specialized RNA endonuclease in the transcription of the viral genome. The same genomic segment encodes for a second shorter protein, termed PA-X, with the first 191 N-terminal aminoacids (aa) identical to PA, but with a completely different C-ter domain of 61 aa, due to a ribosomal frameshifting. In addition, it has been shown that several IAV isolates encode for a naturally truncated PA-X variant, PAXΔC20, missing the last 20 aa. The biochemical properties of PA-X and PAXΔC20 have been poorly investigated so far. Here, we have carried out an enzymatic characterization of PA-X and its naturally deleted form, in comparison with PA from the human IAV strain A/WSN/33 (H1N1). Our results showed, to the best of our knowledge for the first time, that PA-X possesses an endonucleolytic activity. Both PA and PA-X preferentially cut single stranded RNA regions, but with some differences. In addition, we showed that PAXΔC20 has severely reduced nuclease activity. These results point to a previously undetected role of the last C-ter 20 aa for the catalytic activity of PA-X and support distinct roles for these proteins in the viral life cycle. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Bimolecular interaction of argpyrimidine (a Maillard reaction product) in in vitro non-enzymatic protein glycation model and its potential role as an antiglycating agent.

    Science.gov (United States)

    Bhattacherjee, Abhishek; Dhara, Kaliprasanna; Chakraborti, Abhay Sankar

    2017-09-01

    Non- enzymatic glycation, also known as Maillard reaction, is one of the most important and investigated reactions in biochemistry. Maillard reaction products (MRPs) like protein-derived advanced glycation end products (AGEs) are often referred to cause pathophysiological complications in human systems. On contrary, several MRPs are exogenously used as antioxidant, antimicrobial and flavouring agents. In the preset study, we have shown that argpyrimidine, a well-established AGE, interacts with bovine serum albumin (BSA) and glucose individually in standard BSA-glucose model system and successfully inhibits glycation of the protein. Bimolecular interaction of argpyrimidine with glucose or BSA has been studied independently. Chromatographic purification, different spectroscopic studies and molecular modeling have been used to evaluate the nature and pattern of interactions. Binding of argpyrimidine with BSA prevents incorporation of glucose inside the native protein. Argpyrimidine can also directly entrap glucose. Both these interactions may be associated with the antiglycation potential of argpyrimidine, indicating a beneficial function of an AGE. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Brucella ovis PA mutants for outer membrane proteins Omp10, Omp19, SP41, and BepC are not altered in their virulence and outer membrane properties.

    Science.gov (United States)

    Sidhu-Muñoz, Rebeca S; Sancho, Pilar; Vizcaíno, Nieves

    2016-04-15

    Mutants in several genes have been obtained on the genetic background of virulent rough (lacking O-polysaccharide) Brucella ovis PA. The target genes encode outer membrane proteins previously associated with the virulence of smooth (bearing O-polysaccharide chains in the lipopolysaccharide) Brucella strains. Multiple attempts to delete omp16, coding for a homologue to peptidoglycan-associated lipoproteins, were unsuccessful, which suggests that Omp16 is probably essential for in vitro survival of B. ovis PA. Single deletion of omp10 or omp19-that encode two other outer membrane lipoproteins--was achieved, but the simultaneous removal of both genes failed, suggesting an essential complementary function between both proteins. Two other deletion mutants, defective in the Tol-C-homologue BepC or in the SP41 adhesin, were also obtained. Surprisingly when compared to previous results obtained with smooth Brucella, none of the B. ovis mutants showed attenuation in the virulence, either in the mouse model or in cellular models of professional and non-professional phagocytes. Additionally, and in contrast to the observations reported with smooth Brucella strains, several properties related to the outer membrane remained almost unaltered. These results evidence new distinctive traits between naturally rough B. ovis and smooth brucellae. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Recruitment of DNA replication and damage response proteins to viral replication centers during infection with NS2 mutants of Minute Virus of Mice (MVM).

    Science.gov (United States)

    Ruiz, Zandra; Mihaylov, Ivailo S; Cotmore, Susan F; Tattersall, Peter

    2011-02-20

    MVM NS2 is essential for viral DNA amplification, but its mechanism of action is unknown. A classification scheme for autonomous parvovirus-associated replication (APAR) center development, based on NS1 distribution, was used to characterize abnormal APAR body maturation in NS2null mutant infections, and their organization examined for defects in host protein recruitment. Since acquisition of known replication factors appeared normal, we looked for differences in invoked DNA damage responses. We observed widespread association of H2AX/MDC1 damage response foci with viral replication centers, and sequestration and complex hyperphosphorylation of RPA(32), which occurred in wildtype and mutant infections. Quantifying these responses by western transfer indicated that both wildtype and NS2 mutant MVM elicited ATM activation, while phosphorylation of ATR, already basally activated in asynchronous A9 cells, was downregulated. We conclude that MVM infection invokes multiple damage responses that influence the APAR environment, but that NS2 does not modify the recruitment of cellular proteins. Copyright © 2010 Elsevier Inc. All rights reserved.

  12. Identification of Atg2 and ArfGAP1 as Candidate Genetic Modifiers of the Eye Pigmentation Phenotype of Adaptor Protein-3 (AP-3 Mutants in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Imilce A Rodriguez-Fernandez

    Full Text Available The Adaptor Protein (AP-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions--which together covered most of the autosomal chromosomes-to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with

  13. Enzymatic reaction paths as determined by transition path sampling

    Science.gov (United States)

    Masterson, Jean Emily

    Enzymes are biological catalysts capable of enhancing the rates of chemical reactions by many orders of magnitude as compared to solution chemistry. Since the catalytic power of enzymes routinely exceeds that of the best artificial catalysts available, there is much interest in understanding the complete nature of chemical barrier crossing in enzymatic reactions. Two specific questions pertaining to the source of enzymatic rate enhancements are investigated in this work. The first is the issue of how fast protein motions of an enzyme contribute to chemical barrier crossing. Our group has previously identified sub-picosecond protein motions, termed promoting vibrations (PVs), that dynamically modulate chemical transformation in several enzymes. In the case of human heart lactate dehydrogenase (hhLDH), prior studies have shown that a specific axis of residues undergoes a compressional fluctuation towards the active site, decreasing a hydride and a proton donor--acceptor distance on a sub-picosecond timescale to promote particle transfer. To more thoroughly understand the contribution of this dynamic motion to the enzymatic reaction coordinate of hhLDH, we conducted transition path sampling (TPS) using four versions of the enzymatic system: a wild type enzyme with natural isotopic abundance; a heavy enzyme where all the carbons, nitrogens, and non-exchangeable hydrogens were replaced with heavy isotopes; and two versions of the enzyme with mutations in the axis of PV residues. We generated four separate ensembles of reaction paths and analyzed each in terms of the reaction mechanism, time of barrier crossing, dynamics of the PV, and residues involved in the enzymatic reaction coordinate. We found that heavy isotopic substitution of hhLDH altered the sub-picosecond dynamics of the PV, changed the favored reaction mechanism, dramatically increased the time of barrier crossing, but did not have an effect on the specific residues involved in the PV. In the mutant systems

  14. Gene expression profile of zeitlupe/lov kelch protein1 T-DNA insertion mutants in Arabidopsis thaliana: Downregulation of auxin-inducible genes in hypocotyls.

    Science.gov (United States)

    Saitoh, Aya; Takase, Tomoyuki; Kitaki, Hiroyuki; Miyazaki, Yuji; Kiyosue, Tomohiro

    2015-01-01

    Elongation of hypocotyl cells has been studied as a model for elucidating the contribution of cellular expansion to plant organ growth. ZEITLUPE (ZTL) or LOV KELCH PROTEIN1 (LKP1) is a positive regulator of warmth-induced hypocotyl elongation under white light in Arabidopsis, although the molecular mechanisms by which it promotes hypocotyl cell elongation remain unknown. Microarray analysis showed that 134 genes were upregulated and 204 genes including 15 auxin-inducible genes were downregulated in the seedlings of 2 ztl T-DNA insertion mutants grown under warm conditions with continuous white light. Application of a polar auxin transport inhibitor, an auxin antagonist or an auxin biosynthesis inhibitor inhibited hypocotyl elongation of control seedlings to the level observed with the ztl mutant. Our data suggest the involvement of auxin and auxin-inducible genes in ZTL-mediated hypocotyl elongation.

  15. Spontaneous nisin-resistant Listeria monocytogenes mutants with increased expression of a putative penicillin-binding protein and their sensitivity to various antibiotics

    DEFF Research Database (Denmark)

    Gravesen, Anne; Sorensen, K.; Aarestrup, Frank Møller

    2001-01-01

    A concern regarding the use of bacteriocins, as for example the lantibiotic nisin, for biopreservation of certain food products is the possibility of resistance development and potential cross-resistance to antibiotics in the target organism. The genetic basis for nisin resistance development...... is as yet unknown. We analyzed changes in gene expression following nisin resistance development in Listeria monocytogenes 412 by restriction fragment differential display. The mutant had increased expression of a protein with strong homology to the glycosyltransferase domain of high...... a prevalent nisin resistance mechanism under the employed isolation conditions. Increased expression of the putative PBP may affect the cell wall composition and thereby alter the sensitivity to cell wall-targeting compounds, The mutants had an isolate-specific increase in sensitivity to different beta...

  16. Co-activation of STAT3 and YES-Associated Protein 1 (YAP1) Pathway in EGFR-Mutant NSCLC

    DEFF Research Database (Denmark)

    Chaib, Imane; Karachaliou, Niki; Pilotto, Sara

    2017-01-01

    Background: The efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC) is limited by adaptive activation of cell survival signals. We hypothesized that both signal transducer and activator of transcription 3 (STAT3) ...

  17. Uncoupling in Secondary Transport Proteins. A Mechanistic Explanation for Mutants of lac Permease with an Uncoupled Phenotype

    NARCIS (Netherlands)

    Lolkema, J.S.; Poolman, B.

    1995-01-01

    The kinetic behavior of a H+-substrate symporter has been studied in which in addition to the unloaded (E) and fully loaded states (E.S.H) of the carrier also one of the binary complexes (E.S or E.H) may reorient its binding sites. This results in two types of uncoupled mutants, the ES leak and the

  18. Cooperative assembly of proteins in the ribosomal GTPase centre demonstrated by their interactions with mutant 23S rRNAs

    DEFF Research Database (Denmark)

    Rosendahl, G; Douthwaite, S

    1995-01-01

    ) and numerous multiple mutations were generated. Expression of mutant 23S rRNAs in vivo shows that all the mutations detectably alter the phenotype, with effects ranging from a slight growth rate reduction to lack of viability. Temperature sensitivity is conferred by 1071G-->A and 1092C-->U substitutions...

  19. Induced High Lysine Mutants in Barley

    DEFF Research Database (Denmark)

    Doll, Hans; Køie, B.; Eggum, B. O.

    1974-01-01

    variety. Comparisons of six high lysine mutants with the parent variety showed that grain yield and seed size of the mutants are reduced between 10 and 30 per cent. However, the most promising mutant had the lowest reduction in grain yield, and the absolute lysine yield of this mutant was some 30 per cent...... above that of the parent variety. Feeding tests with rats revealed substantial increases in the biological value of the high lysine mutant protein. Also the net protein utilization was improved but less so because of a somewhat reduced digestibility of the mutant protein....

  20. Amyotrophic lateral sclerosis mutant vesicle-associated membrane protein-associated protein-B transgenic mice develop TAR-DNA-binding protein-43 pathology.

    LENUS (Irish Health Repository)

    Tudor, E L

    2010-05-19

    Cytoplasmic ubiquitin-positive inclusions containing TAR-DNA-binding protein-43 (TDP-43) within motor neurons are the hallmark pathology of sporadic amyotrophic lateral sclerosis (ALS). TDP-43 is a nuclear protein and the mechanisms by which it becomes mislocalized and aggregated in ALS are not properly understood. A mutation in the vesicle-associated membrane protein-associated protein-B (VAPB) involving a proline to serine substitution at position 56 (VAPBP56S) is the cause of familial ALS type-8. To gain insight into the molecular mechanisms by which VAPBP56S induces disease, we created transgenic mice that express either wild-type VAPB (VAPBwt) or VAPBP56S in the nervous system. Analyses of both sets of mice revealed no overt motor phenotype nor alterations in survival. However, VAPBP56S but not VAPBwt transgenic mice develop cytoplasmic TDP-43 accumulations within spinal cord motor neurons that were first detected at 18 months of age. Our results suggest a link between abnormal VAPBP56S function and TDP-43 mislocalization.

  1. Functional analysis of a frame-shift mutant of the dihydropyridine receptor pore subunit (α1S expressing two complementary protein fragments

    Directory of Open Access Journals (Sweden)

    Mortenson Lindsay

    2001-12-01

    Full Text Available Abstract Background The L-type Ca2+ channel formed by the dihydropyridine receptor (DHPR of skeletal muscle senses the membrane voltage and opens the ryanodine receptor (RyR1. This channel-to-channel coupling is essential for Ca2+ signaling but poorly understood. We characterized a single-base frame-shift mutant of α1S, the pore subunit of the DHPR, that has the unusual ability to function voltage sensor for excitation-contraction (EC coupling by virtue of expressing two complementary hemi-Ca2+ channel fragments. Results Functional analysis of cDNA transfected dysgenic myotubes lacking α1S were carried out using voltage-clamp, confocal Ca2+ indicator fluoresence, epitope immunofluorescence and immunoblots of expressed proteins. The frame-shift mutant (fs-α1S expressed the N-terminal half of α1S (M1 to L670 and the C-terminal half starting at M701 separately. The C-terminal fragment was generated by an unexpected restart of translation of the fs-α1S message at M701 and was eliminated by a M701I mutation. Protein-protein complementation between the two fragments produced recovery of skeletal-type EC coupling but not L-type Ca2+ current. Discussion A premature stop codon in the II-III loop may not necessarily cause a loss of DHPR function due to a restart of translation within the II-III loop, presumably by a mechanism involving leaky ribosomal scanning. In these cases, function is recovered by expression of complementary protein fragments from the same cDNA. DHPR-RyR1 interactions can be achieved via protein-protein complementation between hemi-Ca2+ channel proteins, hence an intact II-III loop is not essential for coupling the DHPR voltage sensor to the opening of RyR1 channel.

  2. Flavonoid Interaction with a Chitinase from Grape Berry Skin: Protein Identification and Modulation of the Enzymatic Activity

    Directory of Open Access Journals (Sweden)

    Antonio Filippi

    2016-09-01

    Full Text Available In the present study, an antibody raised against a peptide sequence of rat bilitranslocase (anti-peptide Ab was tested on microsomal proteins obtained from red grape berry skin. Previously, this antibody had demonstrated to recognize plant membrane proteins associated with flavonoid binding and transport. Immuno-proteomic assays identified a number of proteins reacting with this particular antibody, suggesting that the flavonoid binding and interaction may be extended not only to carriers of these molecules, but also to enzymes with very different functions. One of these proteins is a pathogenesis-related (PR class IV chitinase, whose in vitro chitinolytic activity was modulated by two of the most representative flavonoids of grape, quercetin and catechin, as assessed by both spectrophotometric and fluorimetric assays in grape microsomes and commercial enzyme preparations. The effect of these flavonoids on the catalysis and its kinetic parameters was also evaluated, evidencing that they determine a hormetic dose-dependent response. These results highlight the importance of flavonoids not only as antioxidants or antimicrobial effectors, but also as modulators of plant growth and stress response. Implications of the present suggestion are here discussed in the light of environment and pesticide-reduction concerns.

  3. Enzymatic-fluorometric analyses for glutamine, glutamate and free amino groups in protein-free plasma and milk

    DEFF Research Database (Denmark)

    Larsen, Torben; Fernández, Carlos J.

    2017-01-01

    This Technical Research Communication describes new analytical methods for free, unbound glutamic acid and glutamine in protein-free blood plasma and milk and introduces the use of quantitation of free amino groups in the same matrices for descriptive and analytical purposes. The present enzymati...

  4. Can electromagnetic fields influence the structure and enzymatic digest of proteins? A critical evaluation of microwave-assisted proteomics protocols

    Science.gov (United States)

    Damm, Markus; Nusshold, Christoph; Cantillo, David; Rechberger, Gerald N.; Gruber, Karl; Sattler, Wolfgang; Kappe, C. Oliver

    2012-01-01

    This study reevaluates the putative advantages of microwave-assisted tryptic digests compared to conventionally heated protocols performed at the same temperature. An initial investigation of enzyme stability in a temperature range of 37–80 °C demonstrated that trypsin activity declines sharply at temperatures above 60 °C, regardless if microwave dielectric heating or conventional heating is employed. Tryptic digests of three proteins of different size (bovine serum albumin, cytochrome c and β-casein) were thus performed at 37 °C and 50 °C using both microwave and conventional heating applying accurate internal fiber-optic probe reaction temperature measurements. The impact of the heating method on protein degradation and peptide fragment generation was analyzed by SDS-PAGE and MALDI-TOF-MS. Time-dependent tryptic digestion of the three proteins and subsequent analysis of the corresponding cleavage products by MALDI-TOF provided virtually identical results for both microwave and conventional heating. In addition, the impact of electromagnetic field strength on the tertiary structure of trypsin and BSA was evaluated by molecular mechanics calculations. These simulations revealed that the applied field in a typical laboratory microwave reactor is 3–4 orders of magnitude too low to induce conformational changes in proteins or enzymes. PMID:22889711

  5. Involvement of the Eukaryote-Like Kinase-Phosphatase System and a Protein That Interacts with Penicillin-Binding Protein 5 in Emergence of Cephalosporin Resistance in Cephalosporin-Sensitive Class A Penicillin-Binding Protein Mutants in Enterococcus faecium

    Directory of Open Access Journals (Sweden)

    Charlene Desbonnet

    2016-04-01

    Full Text Available The intrinsic resistance of Enterococcus faecium to ceftriaxone and cefepime (here referred to as “cephalosporins” is reliant on the presence of class A penicillin-binding proteins (Pbps PbpF and PonA. Mutants lacking these Pbps exhibit cephalosporin susceptibility that is reversible by exposure to penicillin and by selection on cephalosporin-containing medium. We selected two cephalosporin-resistant mutants (Cro1 and Cro2 of class A Pbp-deficient E. faecium CV598. Genome analysis revealed changes in the serine-threonine kinase Stk in Cro1 and a truncation in the associated phosphatase StpA in Cro2 whose respective involvements in resistance were confirmed in separate complementation experiments. In an additional effort to identify proteins linked to cephalosporin resistance, we performed tandem affinity purification using Pbp5 as bait in penicillin-exposed E. faecium; these experiments yielded a protein designated Pbp5-associated protein (P5AP. Transcription of the P5AP gene was increased after exposure to penicillin in wild-type strains and in Cro2 and suppressed in Cro2 complemented with the wild-type stpA. Transformation of class A Pbp-deficient strains with the plasmid-carried P5AP gene conferred cephalosporin resistance. These data suggest that Pbp5-associated cephalosporin resistance in E. faecium devoid of typical class A Pbps is related to the presence of P5AP, whose expression is influenced by the activity of the serine-threonine phosphatase/kinase system.

  6. A dominant-negative mutant of Max that inhibits sequence-specific DNA binding by Myc proteins

    NARCIS (Netherlands)

    Billaud, Marc; Isselbacher, Kurt J.; Bernards, R.A.

    1993-01-01

    Myc proteins are basic helix-loop-helix/ leucine-zipper proteins that bind to specific DNA sequences. In vivo, Myc proteins have been found associated with Max, another basic helix4oop-helix/leucine-zipper protein. However, it is not known to what extent the dimerization of Myc with Max is

  7. An attenuated Shigella mutant lacking the RNA-binding protein Hfq provides cross-protection against Shigella strains of broad serotype.

    Directory of Open Access Journals (Sweden)

    Jiro Mitobe

    2017-07-01

    Full Text Available Few live attenuated vaccines protect against multiple serotypes of bacterial pathogen because host serotype-specific immune responses are limited to the serotype present in the vaccine strain. Here, immunization with a mutant of Shigella flexneri 2a protected guinea pigs against subsequent infection by S. dysenteriae type 1 and S. sonnei strains. This deletion mutant lacked the RNA-binding protein Hfq leading to increased expression of the type III secretion system via loss of regulation, resulting in attenuation of cell viability through repression of stress response sigma factors. Such increased antigen production and simultaneous attenuation were expected to elicit protective immunity against Shigella strains of heterologous serotypes. Thus, the vaccine potential of this mutant was tested in two guinea pig models of shigellosis. Animals vaccinated in the left eye showed fewer symptoms upon subsequent challenge via the right eye, and even survived subsequent intestinal challenge. In addition, oral vaccination effectively induced production of immunoglobulins without severe side effects, again protecting all animals against subsequent intestinal challenge with S. dysenteriae type 1 or S. sonnei strains. Antibodies against common virulence proteins and the O-antigen of S. flexneri 2a were detected by immunofluorescence microscopy. Reaction of antibodies with various strains, including enteroinvasive Escherichia coli, suggested that common virulence proteins induced protective immunity against a range of serotypes. Therefore, vaccination is expected to cover not only the most prevalent serotypes of S. sonnei and S. flexneri 2a, but also various Shigella strains, including S. dysenteriae type 1, which produces Shiga toxin.

  8. Molecular Mechanisms Leading to Null-protein Product from Retinoschisin (RS1) Signal-sequence Mutants in X-linked Retinoschisis (XLRS) Disease

    Science.gov (United States)

    Vijayasarathy, Camasamudram; Sui, Ruifang; Zeng, Yong; Yang, Guoxing; Xu, Fei; Caruso, Rafael C.; Lewis, Richard A.; Ziccardi, Lucia; Sieving, Paul A.

    2010-01-01

    Retinoschisin (RS1) is a cell-surface adhesion molecule expressed by photoreceptor and bipolar cells of the retina. The 24-kDa protein encodes two conserved sequence motifs: the initial signal sequence targets the protein for secretion while the larger discoidin domain is implicated in cell adhesion. RS1 helps to maintain the structural organization of the retinal cell layers and promotes visual signal transduction. RS1 gene mutations cause X-linked retinoschisis disease (XLRS) in males, characterized by early-onset central vision loss. We analyzed the biochemical consequences of several RS1 signal-sequence mutants (c.1A>T, c.35T>A, c.38T>C, and c.52G>A) found in our subjects. Expression analysis in COS-7 cells demonstrates that they affect RS1 biosynthesis and result in an RS1 null phenotype by several different mechanisms. By comparison, discoidin-domain mutations generally lead to nonfunctional conformational variants that remain trapped inside the cell. XLRS disease has a broad heterogeneity in general, but subjects with the RS1 null-protein signal-sequence mutations are on the more severe end of the clinical phenotype. Results from the signal-sequence mutants are discussed in the context of the discoidin-domain mutations, clinical phenotypes, genotype–phenotype correlations, and implications for RS1 gene replacement therapy. PMID:20809529

  9. The protein kinase MBK-1 contributes to lifespan extension in daf-2 mutant and germline-deficient Caenorhabditis elegans.

    Science.gov (United States)

    Mack, Hildegard I D; Zhang, Peichuan; Fonslow, Bryan R; Yates, John R

    2017-05-25

    In Caenorhabditis elegans , reduction of insulin/IGF-1 like signaling and loss of germline stem cells both increase lifespan by activating the conserved transcription factor DAF-16 (FOXO). While the mechanisms that regulate DAF-16 nuclear localization in response to insulin/IGF-1 like signaling are well characterized, the molecular pathways that act in parallel to regulate DAF-16 transcriptional activity, and the pathways that couple DAF-16 activity to germline status, are not fully understood at present. Here, we report that inactivation of MBK-1, the C. elegans ortholog of the human FOXO1-kinase DYRK1A substantially shortens the prolonged lifespan of daf-2 and glp-1 mutant animals while decreasing wild-type lifespan to a lesser extent. On the other hand, lifespan-reduction by mutation of the MBK-1-related kinase HPK-1 was not preferential for long-lived mutants. Interestingly, mbk-1 loss still allowed for DAF-16 nuclear accumulation but reduced expression of certain DAF-16 target genes in germline-less, but not in daf-2 mutant animals. These findings indicate that mbk-1 and daf-16 functionally interact in the germline- but not in the daf-2 pathway. Together, our data suggest mbk-1 as a novel regulator of C. elegans longevity upon both, germline ablation and DAF-2 inhibition, and provide evidence for mbk-1 regulating DAF-16 activity in germline-deficient animals.

  10. Involvement of the Eukaryote-Like Kinase-Phosphatase System and a Protein That Interacts with Penicillin-Binding Protein 5 in Emergence of Cephalosporin Resistance in Cephalosporin-Sensitive Class A Penicillin-Binding Protein Mutants in Enterococcus faecium.

    Science.gov (United States)

    Desbonnet, Charlene; Tait-Kamradt, Amelia; Garcia-Solache, Monica; Dunman, Paul; Coleman, Jeffrey; Arthur, Michel; Rice, Louis B

    2016-04-05

    The intrinsic resistance of Enterococcus faecium to ceftriaxone and cefepime (here referred to as "cephalosporins") is reliant on the presence of class A penicillin-binding proteins (Pbps) PbpF and PonA. Mutants lacking these Pbps exhibit cephalosporin susceptibility that is reversible by exposure to penicillin and by selection on cephalosporin-containing medium. We selected two cephalosporin-resistant mutants (Cro1 and Cro2) of class A Pbp-deficient E. faecium CV598. Genome analysis revealed changes in the serine-threonine kinase Stk in Cro1 and a truncation in the associated phosphatase StpA in Cro2 whose respective involvements in resistance were confirmed in separate complementation experiments. In an additional effort to identify proteins linked to cephalosporin resistance, we performed tandem affinity purification using Pbp5 as bait in penicillin-exposed E. faecium; these experiments yielded a protein designated Pbp5-associated protein (P5AP). Transcription of the P5AP gene was increased after exposure to penicillin in wild-type strains and in Cro2 and suppressed in Cro2 complemented with the wild-type stpA Transformation of class A Pbp-deficient strains with the plasmid-carried P5AP gene conferred cephalosporin resistance. These data suggest that Pbp5-associated cephalosporin resistance in E. faecium devoid of typical class A Pbps is related to the presence of P5AP, whose expression is influenced by the activity of the serine-threonine phosphatase/kinase system. β-Lactam antibiotics remain our most effective therapies against susceptible Gram-positive bacteria. The intrinsic resistance of Enterococcus faecium to β-lactams, particularly to cephalosporins, therefore represents a major limitation of therapy. Although the primary mechanism of resistance to β-lactams in E. faecium is the presence of low-affinity monofunctional transpeptidase (class B) penicillin-binding protein Pbp5, the interaction of Pbp5 with other proteins is fundamental to maintain a

  11. Tetrahymena telomerase protein p65 induces conformational changes throughout telomerase RNA (TER) and rescues telomerase reverse transcriptase and TER assembly mutants.

    Science.gov (United States)

    Berman, Andrea J; Gooding, Anne R; Cech, Thomas R

    2010-10-01

    The biogenesis of the Tetrahymena telomerase ribonucleoprotein particle (RNP) is enhanced by p65, a La family protein. Single-molecule and biochemical studies have uncovered a hierarchical assembly of the RNP, wherein the binding of p65 to stems I and IV of telomerase RNA (TER) causes a conformational change that facilitates the subsequent binding of telomerase reverse transcriptase (TERT) to TER. We used purified p65 and variants of TERT and TER to investigate the conformational rearrangements that occur during RNP assembly. Nuclease protection assays and mutational analysis revealed that p65 interacts with and stimulates conformational changes in regions of TER beyond stem IV. Several TER mutants exhibited telomerase activity only in the presence of p65, revealing the importance of p65 in promoting the correct RNP assembly pathway. In addition, p65 rescued TERT assembly mutants but not TERT activity mutants. Taken together, these results suggest that p65 stimulates telomerase assembly and activity in two ways. First, by sequestering stems I and IV, p65 limits the ensemble of structural conformations of TER, thereby presenting TERT with the active conformation of TER. Second, p65 acts as a molecular buttress within the assembled RNP, mutually stabilizing TER and TERT in catalytically active conformations.

  12. Isolation of Escherichia coli rpoB mutants resistant to killing by lambda cII protein and altered in pyrE gene attenuation

    DEFF Research Database (Denmark)

    Hammer, Karin; Jensen, Kaj Frank; Poulsen, Peter

    1987-01-01

    Escherichia coli mutants simultaneously resistant to rifampin and to the lethal effects of bacteriophage lambda cII protein were isolated. The sck mutant strains carry alterations in rpoB that allow them to survive cII killing (thus the name sck), but that do not impair either the expression of c....... In contrast to their effect on lambda growth, the three mutations reduce transcription termination in bacterial operons. The E. coli pyrE gene, which is normally regulated by attenuation, is expressed constitutively in the mutant strains. The sck mutations appear to prevent pyrE attenuation by slowing......II or the activation by cII of the lambda promoters pE and pI. The sck-1, sck-2, and sck-3 mutations modify transcription termination. The growth of lambda, but not of the N-independent lambda variant, lambda nin-5, is hindered by these mutations, which act either alone or in concert with the bacterial nusA1 mutation...

  13. Mutant HbpR transcription activator isolation for 2-chlorobiphenyl via green fluorescent protein-based flow cytometry and cell sorting.

    OpenAIRE

    Beggah, S.; Vogne, C.; Zenaro, E.; Van Der Meer, J.R.

    2008-01-01

    Summary Mutants were produced in the A‐domain of HbpR, a protein belonging to the XylR family of σ54‐dependent transcription activators, with the purpose of changing its effector recognition specificity from 2‐hydroxybiphenyl (2‐HBP, the cognate effector) to 2‐chlorobiphenyl (2‐CBP). Mutations were introduced in the hbpR gene part for the A‐domain via error‐prone polymerase chain reaction, and assembled on a gene circuitry plasmid in Escherichia coli, permitting HbpR‐dependent induction of th...

  14. Characterization of the Pseudomonas aeruginosa recA analog and its protein product: rec-102 is a mutant allele of the P. aeruginosa PAO recA gene

    Energy Technology Data Exchange (ETDEWEB)

    Kokjohn, T.A.; Miller, R.V.

    1987-04-01

    We cloned a 2.3-kilobase-pair fragment of the Pseudomonas aeruginosa PAO chromosome which is capable of complementing recA mutations of Escherichia coli. The recA-complementing activity was further localized to a 1.5-kilobase-pair PvuII-HindIII fragment. Southern blot analysis under conditions of high stringency indicated that DNA sequence homology is shared by the E. coli recA gene and the P. aeruginosa recA analog. The cloned recA analog was shown to restore resistance to methyl methanesulfonate, nitrofurantoin, and UV irradiation to E. coli recA mutants. Upon introduction of the cloned P. aeruginosa gene, these mutants regained recombination proficiency in HfrH-mediated conjugation and the ability to induce lambda prophages and SOS functions (din gene transcription) after exposure to DNA-damaging agents. Lambda prophage carrying a cI ind mutation was not inducible, suggesting that the mechanism of induction of these SOS functions by the P. aeruginosa RecA analog is similar to that by the activated E. coli RecA protein. The product of the recA analog was identified in minicells as a protein of approximately 47,000 daltons. Western blot analysis using anti-E. coli RecA antibody demonstrated that this protein is antigenically cross-reactive with the E. coli recA protein. The recA-containing fragment was cloned into the broad-host-range vector pCP13 and introduced into Rec- strains of P. aeruginosa containing the rec-102 allele. The plasmid was shown to restore recombination proficiency in FP5-mediated conjugations and to restore resistance to UV irradiation and methyl methanesulfonate to these Rec- mutants. It was shown that a wild-type allele of rec-102 is necessary for UV-mediated induction of D3 and F116 prophages. The cloned recA analog restored the UV inducibility of these prophages in rec-102 mutants.

  15. Xyloketal-derived small molecules show protective effect by decreasing mutant Huntingtin protein aggregates in Caenorhabditis elegans model of Huntington’s disease

    Directory of Open Access Journals (Sweden)

    Zeng YX

    2016-04-01

    Full Text Available Yixuan Zeng,1,2,* Wenyuan Guo,1,* Guangqing Xu,3 Qinmei Wang,4 Luyang Feng,1,2 Simei Long,1 Fengyin Liang,1 Yi Huang,1 Xilin Lu,1 Shichang Li,5 Jiebin Zhou,5 Jean-Marc Burgunder,6 Jiyan Pang,5 Zhong Pei1,2 1Department of Neurology, National Key Clinical Department and Key Discipline of Neurology, Guangdong Key Laboratory for Diagnosis and Treatment of Major Neurological Disease, The First Affiliated Hospital, Sun Yat-sen University, 2Guangzhou Center, Chinese Huntington’s Disease Network, 3Department of Rehabilitation, The First Affiliated Hospital, 4Key laboratory on Assisted Circulation, Ministry of Health, Department of Cardiovascular Medicine of the First Affiliated Hospital, 5School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou, Guangdong, People’s Republic of China; 6Swiss Huntington’s Disease Center, Department of Neurology, University of Bern, Bern, Switzerland *These authors contributed equally to this work Abstract: Huntington’s disease is an autosomal-dominant neurodegenerative disorder, with chorea as the most prominent manifestation. The disease is caused by abnormal expansion of CAG codon repeats in the IT15 gene, which leads to the expression of a glutamine-rich protein named mutant Huntingtin (Htt. Because of its devastating disease burden and lack of valid treatment, development of more effective therapeutics for Huntington’s disease is urgently required. Xyloketal B, a natural product from mangrove fungus, has shown protective effects against toxicity in other neurodegenerative disease models such as Parkinson’s and Alzheimer’s diseases. To identify potential neuroprotective molecules for Huntington’s disease, six derivatives of xyloketal B were screened in a Caenorhabditis elegans Huntington’s disease model; all six compounds showed a protective effect. Molecular docking studies indicated that compound 1 could bind to residues GLN369 and GLN393 of the mutant Htt protein, forming a

  16. Hepatitis B virus X protein mutant HBxΔ127 promotes proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Fabao [Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); You, Xiaona [Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Chi, Xiumei [Department of Hepatology, The First Hospital, Jilin University, Changchun 130021 (China); Wang, Tao [Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ye, Lihong [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); Niu, Junqi, E-mail: junqiniu@yahoo.com.cn [Department of Hepatology, The First Hospital, Jilin University, Changchun 130021 (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China)

    2014-02-07

    Highlights: • Relative to wild type HBx, HBX mutant HBxΔ127 strongly enhances cell proliferation. • Relative to wild type HBx, HBxΔ127 remarkably up-regulates miR-215 in hepatoma cells. • HBxΔ127-elevated miR-215 promotes cell proliferation via targeting PTPRT mRNA. - Abstract: The mutant of virus is a frequent event. Hepatitis B virus X protein (HBx) plays a vital role in the development of hepatocellular carcinoma (HCC). Therefore, the identification of potent mutant of HBx in hepatocarcinogenesis is significant. Previously, we identified a natural mutant of the HBx gene (termed HBxΔ127). Relative to wild type HBx, HBxΔ127 strongly enhanced cell proliferation and migration in HCC. In this study, we aim to explore the mechanism of HBxΔ127 in promotion of proliferation of hepatoma cells. Our data showed that both wild type HBx and HBxΔ127 could increase the expression of miR-215 in hepatoma HepG2 and H7402 cells. However, HBxΔ127 was able to significantly increase miR-215 expression relative to wild type HBx in the cells. We identified that protein tyrosine phosphatase, receptor type T (PTPRT) was one of the target genes of miR-215 through targeting 3′UTR of PTPRT mRNA. In function, miR-215 was able to promote the proliferation of hepatoma cells. Meanwhile anti-miR-215 could partially abolish the enhancement of cell proliferation mediated by HBxΔ127 in vitro. Knockdown of PTPRT by siRNA could distinctly suppress the decrease of cell proliferation mediated by anti-miR-215 in HepG2-XΔ127/H7402-XΔ127 cells. Moreover, we found that anti-miR-215 remarkably inhibited the tumor growth of hepatoma cells in nude mice. Collectively, relative to wild type HBx, HBxΔ127 strongly enhances proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT. Our finding provides new insights into the mechanism of HBx mutant HBxΔ127 in promotion of proliferation of hepatoma cells.

  17. Molecular dissection of placental malaria protein VAR2CSA interaction with a chemo-enzymatically synthesized chondroitin sulfate library

    DEFF Research Database (Denmark)

    Sugiura, Nobuo; Clausen, Thomas Mandel; Shioiri, Tatsuasa

    2016-01-01

    Placental malaria, a serious infection caused by the parasite Plasmodium falciparum, is characterized by the selective accumulation of infected erythrocytes (IEs) in the placentas of the pregnant women. Placental adherence is mediated by the malarial VAR2CSA protein, which interacts......-sulfated CSA dodecasaccharide, and found that a highly sulfated CSA eicosasaccharide is a more potent inhibitor of rVAR2 binding than the dodecasaccharides. These results suggest that CSA derivatives may potentially serve as targets in therapeutic strategies against placental malaria....

  18. Mutant copper-zinc superoxide dismutase (SOD1) induces protein secretion pathway alterations and exosome release in astrocytes: implications for disease spreading and motor neuron pathology in amyotrophic lateral sclerosis.

    Science.gov (United States)

    Basso, Manuela; Pozzi, Silvia; Tortarolo, Massimo; Fiordaliso, Fabio; Bisighini, Cinzia; Pasetto, Laura; Spaltro, Gabriella; Lidonnici, Dario; Gensano, Francesco; Battaglia, Elisa; Bendotti, Caterina; Bonetto, Valentina

    2013-05-31

    Amyotrophic lateral sclerosis is the most common motor neuron disease and is still incurable. The mechanisms leading to the selective motor neuron vulnerability are still not known. The interplay between motor neurons and astrocytes is crucial in the outcome of the disease. We show that mutant copper-zinc superoxide dismutase (SOD1) overexpression in primary astrocyte cultures is associated with decreased levels of proteins involved in secretory pathways. This is linked to a general reduction of total secreted proteins, except for specific enrichment in a number of proteins in the media, such as mutant SOD1 and valosin-containing protein (VCP)/p97. Because there was also an increase in exosome release, we can deduce that astrocytes expressing mutant SOD1 activate unconventional secretory pathways, possibly as a protective mechanism. This may help limit the formation of intracellular aggregates and overcome mutant SOD1 toxicity. We also found that astrocyte-derived exosomes efficiently transfer mutant SOD1 to spinal neurons and induce selective motor neuron death. We conclude that the expression of mutant SOD1 has a substantial impact on astrocyte protein secretion pathways, contributing to motor neuron pathology and disease spread.

  19. Novel causative mutations in patients with Nance-Horan syndrome and altered localization of the mutant NHS-A protein isoform.

    Science.gov (United States)

    Sharma, Shiwani; Burdon, Kathryn P; Dave, Alpana; Jamieson, Robyn V; Yaron, Yuval; Billson, Frank; Van Maldergem, Lionel; Lorenz, Birgit; Gécz, Jozef; Craig, Jamie E

    2008-01-01

    Nance-Horan syndrome is typically characterized by severe bilateral congenital cataracts and dental abnormalities. Truncating mutations in the Nance-Horan syndrome (NHS) gene cause this X-linked genetic disorder. NHS encodes two isoforms, NHS-A and NHS-1A. The ocular lens expresses NHS-A, the epithelial and neuronal cell specific isoform. The NHS-A protein localizes in the lens epithelium at the cellular periphery. The data to date suggest a role for this isoform at cell-cell junctions in epithelial cells. This study aimed to identify the causative mutations in new patients diagnosed with Nance-Horan syndrome and to investigate the effect of mutations on subcellular localization of the NHS-A protein. All coding exons of NHS were screened for mutations by polymerase chain reaction (PCR) and sequencing. PCR-based mutagenesis was performed to introduce three independent mutations in the NHS-A cDNA. Expression and localization of the mutant proteins was determined in mammalian epithelial cells. Truncating mutations were found in 6 out of 10 unrelated patients from four countries. Each of four patients carried a novel mutation (R248X, P264fs, K1198fs, and I1302fs), and each of the two other patients carried two previously reported mutations (R373X and R879X). No mutation was found in the gene in four patients. Two disease-causing mutations (R134fs and R901X) and an artificial mutation (T1357fs) resulted in premature truncation of the NHS-A protein. All three mutant proteins failed to localize to the cellular periphery in epithelial cells and instead were found in the cytoplasm. This study brings the total number of mutations identified in NHS to 18. The mislocalization of the mutant NHS-A protein, revealed by mutation analysis, is expected to adversely affect cell-cell junctions in epithelial cells such as the lens epithelium, which may explain cataractogenesis in Nance-Horan syndrome patients. Mutation analysis also shed light on the significance of NHS-A regions for

  20. LC-ESI-QTOF-MS identification of novel antioxidant peptides obtained by enzymatic and microbial hydrolysis of vegetable proteins.

    Science.gov (United States)

    Babini, Elena; Tagliazucchi, Davide; Martini, Serena; Dei Più, Lucilla; Gianotti, Andrea

    2017-08-01

    Bioactive antioxidant peptides are more and more attracting the attention of food manufacturers for their potential to transform food in functional food, able to prevent a variety of chronic diseases associated with oxidative stress. In the present study proteins extracted from different vegetable sources (KAMUT® khorasan wheat, emmer, lupine and pea) were hydrolyzed with commercial enzymes and Lactobacillus spp. strains. Hydrolysates were separated by size exclusion chromatography and purified fractions were analyzed for their antioxidant activity. Peptides from the fractions with the highest activity were identified by nanoLC-ESI-QTOF-MS and thirteen peptides were selected for synthesis on the basis of their sequence. Four peptides (VLPPQQQY, TVTSLDLPVLRW, VTSLDLPVLRW, FVPY) were found able to scavenge superoxide anion and hydroxyl radicals, organic nitro-radicals (ABTS, DPPH) and to inhibit lipid peroxidation. The impact of this work is targeted to add hydrolysed vegetable proteins to reformulated functional food or to produce health-promoting ingredients and nutraceuticals. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Enzymatic turnover of macromolecules generates long-lasting protein-water-coupled motions beyond reaction steady state.

    Science.gov (United States)

    Dielmann-Gessner, Jessica; Grossman, Moran; Conti Nibali, Valeria; Born, Benjamin; Solomonov, Inna; Fields, Gregg B; Havenith, Martina; Sagi, Irit

    2014-12-16

    The main focus of enzymology is on the enzyme rates, substrate structures, and reactivity, whereas the role of solvent dynamics in mediating the biological reaction is often left aside owing to its complex molecular behavior. We used integrated X-ray- and terahertz- based time-resolved spectroscopic tools to study protein-water dynamics during proteolysis of collagen-like substrates by a matrix metalloproteinase. We show equilibration of structural kinetic transitions in the millisecond timescale during degradation of the two model substrates collagen and gelatin, which have different supersecondary structure and flexibility. Unexpectedly, the detected changes in collective enzyme-substrate-water-coupled motions persisted well beyond steady state for both substrates while displaying substrate-specific behaviors. Molecular dynamics simulations further showed that a hydration funnel (i.e., a gradient in retardation of hydrogen bond (HB) dynamics toward the active site) is substrate-dependent, exhibiting a steeper gradient for the more complex enzyme-collagen system. The long-lasting changes in protein-water dynamics reflect a collection of local energetic equilibrium states specifically formed during substrate conversion. Thus, the observed long-lasting water dynamics contribute to the net enzyme reactivity, impacting substrate binding, positional catalysis, and product release.

  2. Congo red, an amyloid-inhibiting compound, alleviates various types of cellular dysfunction triggered by mutant protein kinase cγ that causes spinocerebellar ataxia type 14 (SCA14) by inhibiting oligomerization and aggregation.

    Science.gov (United States)

    Seki, Takahiro; Takahashi, Hideyuki; Yamamoto, Kazuhiro; Ogawa, Kota; Onji, Tomoya; Adachi, Naoko; Tanaka, Shigeru; Hide, Izumi; Saito, Naoaki; Sakai, Norio

    2010-01-01

    Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that the mutant γPKC found in SCA14 is susceptible to aggregation that induces apoptotic cell death. Congo red is widely used as a histological dye for amyloid detection. Recent evidence has revealed that Congo red has the property to inhibit amyloid oligomers and fibril formation of misfolded proteins. In the present study, we examine whether Congo red inhibits aggregate formation and cytotoxicity of mutant γPKC. Congo red likely inhibits aggregate formation of mutant γPKC – green fluorescent protein (GFP) without affecting its expression level in SH-SY5Y cells. Congo red counteracts the insolubilization of recombinant mutant γPKC, suggesting that the dye inhibits aggregation of mutant γPKC by a direct mechanism. Congo red also inhibits aggregation and oligomerization of mutant γPKC-GFP in primary cultured cerebellar Purkinje cells. Moreover, the dye reverses the improper development of dendrites and inhibits apoptotic cell death in Purkinje cells that express mutant γPKC-GFP. These results indicate that amyloid-inhibiting compounds like Congo red may be novel therapeutics for SCA14.

  3. Use of a Cellulase-Derepressed Mutant of Cellulomonas in the Production of a Single-Cell Protein Product from Cellulose †

    Science.gov (United States)

    Hitchner, E. V.; Leatherwood, J. M.

    1980-01-01

    A cellulase-derepressed mutant of a Cellulomonas species was used to produce single-cell protein from crystalline cellulose. In preliminary tests, maximum yield of single-cell protein was obtained at 30°C (pH 7.0) with urea as the nitrogen source. A continuous-flow foam flotation procedure was developed for rapid and efficient separation of bacteria from the culture liquid and cellulose residue. A pH of 4.5 was optimum for foam flotation of this organism. In preliminary trials, recovery was 85% of the cells with the flotation procedure. Cellulomonas was 68% true protein and had an essential amino acid profile featuring a high lysine content (6.5% of protein). The Cellulomonas product was evaluated nutritionally with weanling rats. The net protein utilization value for the protein supplemented with methionine was 50.4% Weight gain of rats on the Cellulomonas diet was similar to that of rats fed a casein diet. PMID:16345511

  4. A cost-effective protocol for the parallel production of libraries of 13CH3-specifically labeled mutants for NMR studies of high molecular weight proteins.

    Science.gov (United States)

    Crublet, Elodie; Kerfah, Rime; Mas, Guillaume; Noirclerc-Savoye, Marjolaine; Lantez, Violaine; Vernet, Thierry; Boisbouvier, Jerome

    2014-01-01

    There is increasing interest in applying NMR spectroscopy to the study of large protein assemblies. Development of methyl-specific labeling protocols combined with improved NMR spectroscopy enable nowadays studies of proteins complexes up to 1 MDa. For such large complexes, the major interest lies in obtaining structural, dynamic and interaction information in solution, which requires sequence-specific resonance assignment of NMR signals. While such analysis is quite standard for small proteins, it remains one of the major bottlenecks when the size of the protein increases. Here, we describe implementation and latest improvements of SeSAM, a fast and user-friendly approach for assignment of methyl resonances in large proteins using mutagenesis. We have improved culture medium to boost the production of methyl-specifically labeled proteins, allowing us to perform small-scale parallel production and purification of a library of (13)CH3-specifically labeled mutants. This optimized protocol is illustrated by assignment of Alanine, Isoleucine, and Valine methyl groups of the homododecameric aminopeptidase PhTET2. We estimated that this improved method allows assignment of ca. 100 methyl cross-peaks in 2 weeks, including 4 days of NMR time and less than 2 k€ of isotopic materials.

  5. Quantitative analysis of wild-type and V600E mutant BRAF proteins in colorectal carcinoma using immunoenrichment and targeted mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Hang [Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan (China); Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang 110122 (China); Hsiao, Yung-Chin [Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan (China); Department of Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan (China); Chiang, Sum-Fu [Division of Colon and Rectal Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan (China); Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Wu, Chia-Chun; Lin, Yu-Tsun [Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Liu, Hsuan [Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan (China); Division of Colon and Rectal Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan (China); Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Department of Cell and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Zhao, Hong [Experimental Center of Functional Subjects, College of Basic Medicine, China Medical University, Shenyang, Liaoning 110122 (China); Chen, Jinn-Shiun [Division of Colon and Rectal Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan (China); Chang, Yu-Sun [Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan (China); Department of Otolaryngology, Chang Gung Memorial Hospital, Linkou, Taiwan (China); and others

    2016-08-24

    The BRAF V600E mutation is one of the most common mutations implicated in the development of several types of cancer including colorectal cancer (CRC), where it is associated with aggressive disease phenotypes and poor outcomes. The status of the BRAF V600E mutation is frequently determined by direct DNA sequencing. However, no previous study has sought to quantify the BRAF V600E protein in cancer specimens. Here, we evaluated immunoenrichment coupled with two MS-based quantitative techniques, namely multiple reaction monitoring (MRM) and single ion monitoring conjugated accurate inclusion mass screening (SIM-AIMS), to detect and precisely quantify wild-type (WT) and V600E mutant BRAF proteins in DNA sequence-confirmed CRC tissue specimens. WT and V600E BRAF proteins were immunoprecipitated from a CRC cell line (HT-29), and their representative peptides ({sup 592}IGDFGLATVK{sup 601} and {sup 592}IGDFGLATEK{sup 601}, respectively) were confirmed by LC-MS/MS analysis and then quantified by MRM or SIM-AIMS with spiked stable isotope-labeled peptide standards. Both assays worked well for measuring WT BRAF from different amounts of HT-29 cell lysates, but the MRM assay was more sensitive than SIM-AIMS assay for quantifying lower levels of V600E BRAF. In protein extracts (2 mg) from 11 CRC tissue specimens, the MRM assay could measure WT BRAF in all 11 cases (0.32–1.66 ng) and the V600E BRAF in two cases (0.1–0.13 ng; mutant-to-WT ratio, 0.16–0.17). The SIM-AIMS assay could also detect WT and V600E BRAF in CRC specimens, but the measured levels of both targets were lower than those determined by MRM assay. Collectively, this study provides an effective method to precisely quantify WT and V600E BRAF proteins in complex biological samples using immunoenrichment-coupled targeted MS. Since the V600E BRAF protein has emerged as an important therapeutic target for cancer, the developed assay should facilitate future BRAF-related basic and clinical studies

  6. Weak Intermolecular Hydrogen Bonds with Fluorine: Detection and Implications for Enzymatic/Chemical Reactions, Chemical Properties, and Ligand/Protein Fluorine NMR Screening.

    Science.gov (United States)

    Dalvit, Claudio; Vulpetti, Anna

    2016-05-23

    It is known that strong hydrogen-bonding interactions play an important role in many chemical and biological systems. However, weak or very weak hydrogen bonds, which are often difficult to detect and characterize, may also be relevant in many recognition and reaction processes. Fluorine serving as a hydrogen-bond acceptor has been the subject of many controversial discussions and there are different opinions about it. It now appears that there is compelling experimental evidence for the involvement of fluorine in weak intramolecular or intermolecular hydrogen bonds. Using established NMR methods, we have previously characterized and measured the strengths of intermolecular hydrogen-bond complexes involving the fluorine moieties CH2 F, CHF2 , and CF3 , and have compared them with the well-known hydrogen-bond complex formed between acetophenone and the strong hydrogen-bond donor p-fluorophenol. We now report evidence for the formation of hydrogen bonds involving fluorine with significantly weaker donors, namely 5-fluoroindole and water. A simple NMR method is proposed for the simultaneous measurement of the strengths of hydrogen bonds between an acceptor and a donor or water. Important implications of these results for enzymatic/chemical reactions involving fluorine, for chemical and physical properties, and for ligand/protein (19) F NMR screening are analyzed through experiments and theoretical simulations. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Complementation analysis of mutants of 1-aminocyclopropane- 1-carboxylate synthase reveals the enzyme is a dimer with shared active sites.

    Science.gov (United States)

    Tarun, A S; Theologis, A

    1998-05-15

    The pyridoxal phosphate-dependent enzyme 1-aminocyclopropane-1-carboxylate synthase (ACS, EC 4.4.1.14) catalyzes the rate-limiting step in the ethylene biosynthetic pathway. ACS shares the conservation of 11 invariant residues with a family of aminotransferases that includes aspartate aminotransferase. Site-directed mutagenesis on two of these residues, Tyr-92 and Lys-278, in the tomato isoenzyme Le-ACS2 greatly reduces enzymatic activity, indicating their importance in catalysis. These mutants have been used in complementation experiments either in vivo in Escherichia coli or in an in vitro transcription/translation assay to study whether the enzyme functions as a dimer. When the Y92L mutant is coexpressed with the K278A mutant protein, there is partial restoration of enzyme activity, suggesting that the mutant proteins can dimerize and form active heterodimers. Coexpressing a double mutant with the wild-type protein reduces wild-type activity, indicating that inactive heterodimers are formed between the wild-type and the double mutant protein subunits. Furthermore, hybrid complementation shows that another tomato isoenzyme, Le-ACS4, can dimerize and that Le-ACS2 and Le-ACS4 have limited capacity for heterodimerization. The data suggest that ACS functions as a dimer with shared active sites.

  8. The structure of mAG, a monomeric mutant of the green fluorescent protein Azami-Green, reveals the structural basis of its stable green emission

    International Nuclear Information System (INIS)

    Ebisawa, Tatsuki; Yamamura, Akihiro; Kameda, Yasuhiro; Hayakawa, Kou; Nagata, Koji; Tanokura, Masaru

    2010-01-01

    The crystal structure of a monomeric mutant of Azami-Green (mAG) from G. fascicularis was determined at 2.2 Å resolution. Monomeric Azami-Green (mAG) from the stony coral Galaxea fascicularis is the first known monomeric green-emitting fluorescent protein that is not a variant of Aequorea victoria green fluorescent protein (avGFP). These two green fluorescent proteins are only 27% identical in their amino-acid sequences. mAG is more similar in its amino-acid sequence to four fluorescent proteins: Dendra2 (a green-to-red irreversibly photoconverting fluorescent protein), Dronpa (a bright-and-dark reversibly photoswitchable fluorescent protein), KikG (a tetrameric green-emitting fluorescent protein) and Kaede (another green-to-red irreversibly photoconverting fluorescent protein). To reveal the structural basis of stable green emission by mAG, the 2.2 Å crystal structure of mAG has been determined and compared with the crystal structures of avGFP, Dronpa, Dendra2, Kaede and KikG. The structural comparison revealed that the chromophore formed by Gln62-Tyr63-Gly64 (QYG) and the fixing of the conformation of the imidazole ring of His193 by hydrogen bonds and van der Waals contacts involving His193, Arg66 and Thr69 are likely to be required for the stable green emission of mAG. The crystal structure of mAG will contribute to the design and development of new monomeric fluorescent proteins with faster maturation, brighter fluorescence, improved photostability, new colours and other preferable properties as alternatives to avGFP and its variants

  9. Large scale comparative proteomics of a chloroplast Clp protease mutant reveals folding stress, altered protein homeostasis, and feedback regulation of metabolism.

    Science.gov (United States)

    Zybailov, Boris; Friso, Giulia; Kim, Jitae; Rudella, Andrea; Rodríguez, Verenice Ramírez; Asakura, Yukari; Sun, Qi; van Wijk, Klaas J

    2009-08-01

    The clpr2-1 mutant is delayed in development due to reduction of the chloroplast ClpPR protease complex. To understand the role of Clp proteases in plastid biogenesis and homeostasis, leaf proteomes of young seedlings of clpr2-1 and wild type were compared using large scale mass spectrometry-based quantification using an LTQ-Orbitrap and spectral counting with significance determined by G-tests. Virtually only chloroplast-localized proteins were significantly affected, indicating that the molecular phenotype was confined to the chloroplast. A comparative chloroplast stromal proteome analysis of fully developed plants was used to complement the data set. Chloroplast unfoldase ClpB3 was strongly up-regulated in both young and mature leaves, suggesting widespread and persistent protein folding stress. The importance of ClpB3 in the clp2-1 mutant was demonstrated by the observation that a CLPR2 and CLPB3 double mutant was seedling-lethal. The observed up-regulation of chloroplast chaperones and protein sorting components further illustrated destabilization of protein homeostasis. Delayed rRNA processing and up-regulation of a chloroplast DEAD box RNA helicase and polynucleotide phosphorylase, but no significant change in accumulation of ribosomal subunits, suggested a bottleneck in ribosome assembly or RNA metabolism. Strong up-regulation of a chloroplast translational regulator TypA/BipA GTPase suggested a specific response in plastid gene expression to the distorted homeostasis. The stromal proteases PreP1,2 were up-regulated, likely constituting compensation for reduced Clp protease activity and possibly shared substrates between the ClpP and PreP protease systems. The thylakoid photosynthetic apparatus was decreased in the seedlings, whereas several structural thylakoid-associated plastoglobular proteins were strongly up-regulated. Two thylakoid-associated reductases involved in isoprenoid and chlorophyll synthesis were up-regulated reflecting feedback from rate

  10. Structural characterization of respiratory syncytial virus fusion inhibitor escape mutants: homology model of the F protein and a syncytium formation assay

    International Nuclear Information System (INIS)

    Morton, Craig J.; Cameron, Rachel; Lawrence, Lynne J.; Lin Bo; Lowe, Melinda; Luttick, Angela; Mason, Anthony; McKimm-Breschkin, Jenny; Parker, Michael W.; Ryan, Jane; Smout, Michael; Sullivan, Jayne; Tucker, Simon P.; Young, Paul R.

    2003-01-01

    Respiratory syncytial virus (RSV) is a ubiquitous human pathogen and the leading cause of lower respiratory tract infections in infants. Infection of cells and subsequent formation of syncytia occur through membrane fusion mediated by the RSV fusion protein (RSV-F). A novel in vitro assay of recombinant RSV-F function has been devised and used to characterize a number of escape mutants for three known inhibitors of RSV-F that have been isolated. Homology modeling of the RSV-F structure has been carried out on the basis of a chimera derived from the crystal structures of the RSV-F core and a fragment from the orthologous fusion protein from Newcastle disease virus (NDV). The structure correlates well with the appearance of RSV-F in electron micrographs, and the residues identified as contributing to specific binding sites for several monoclonal antibodies are arranged in appropriate solvent-accessible clusters. The positions of the characterized resistance mutants in the model structure identify two promising regions for the design of fusion inhibitors

  11. Oligomerization and chaperone-like activity of Drosophila melanogaster small heat shock protein DmHsp27 and three arginine mutants in the alpha-crystallin domain.

    Science.gov (United States)

    Moutaoufik, Mohamed Taha; Morrow, Geneviève; Maaroufi, Halim; Férard, Céline; Finet, Stéphanie; Tanguay, Robert M

    2017-07-01

    The small Hsp DmHsp27 from Drosophila melanogaster is one of the few small heat shock proteins (sHsps) found within the nucleus. We report that its dimerization is independent of disulfide bond formation and seems to rely on salt bridges. Unlike metazoan sHsps, DmHsp27 forms two populations of oligomers not in equilibrium. Mutations at highly conserved arginine residues in mammalian sHsps have been reported to be associated with protein conformational defects and intracellular aggregation. Independent mutation of three highly conserved arginines (R122, R131, and R135) to glycine in DmHsp27 results in only one population of higher molecular weight form. In vitro, the chaperone-like activity of wild-type DmHsp27 was comparable with that of its two isolated populations and to the single population of the R122G, R131G, and R135G using luciferase as substrate. However, using insulin, the chaperone-like activity of wild-type DmHsp27 was lower than that of R122G and R131G mutants. Altogether, the results characterize wild-type DmHsp27 and its alpha-crystallin domain (ACD) arginine mutants and may give insight into protection mechanism of sHsps.

  12. Effect of permethrin, anthracene and mixture exposure on shell components, enzymatic activities and proteins status in the Mediterranean clam Venerupis decussata

    Energy Technology Data Exchange (ETDEWEB)

    Sellami, Badreddine, E-mail: sellamibadreddine@gmail.com [Laboratory of Environment Biomonitoring, Coastal Ecology Unit, Faculty of Sciences of Bizerta, University of Carthage, 7021 Zarzouna (Tunisia); Khazri, Abdelhafidh [Laboratory of Environment Biomonitoring, Coastal Ecology Unit, Faculty of Sciences of Bizerta, University of Carthage, 7021 Zarzouna (Tunisia); Mezni, Amine [Unit of Research 99/UR12-30, Department of Chemistry, Faculty of Sciences of Bizerte, 7021 Jarzouna (Tunisia); Louati, Héla; Dellali, Mohamed; Aissa, Patricia; Mahmoudi, Ezzeddine; Beyrem, Hamouda [Laboratory of Environment Biomonitoring, Coastal Ecology Unit, Faculty of Sciences of Bizerta, University of Carthage, 7021 Zarzouna (Tunisia); Sheehan, David, E-mail: d.sheehan@ucc.ie [Environmental Research Institute and Department of Biochemistry, University College Cork, Western Gateway Building, Western Road, Cork (Ireland)

    2015-01-15

    Highlights: • We assessed toxicity of anthracene, permethrin and their mixture on clams. • Tissue and stressor-dependent changes were observed in biochemical responses. • Permethrin induces phase transition from aragonite to calcite in shell structure. • Interactive effects were observed on digestive gland and gill biomarkers. • Both approaches give new vision to risk assessment of organic pollution. - Abstract: Anthracene (ANT) and permethrin (PER) are two of the more toxic compounds reaching the marine environment. This study aimed to determine the impact of these molecules on Venerupis decussata, an economically important species cultured on the Tunisian coast. Shell structure and its possible transformation upon exposure to the two contaminants were studied by X-ray diffraction and gravimetric analyses. Results revealed a phase transition in shell composition from aragonite to calcite after PER exposure, to a mixture of PER and ANT (Mix) but not for ANT alone. Catalase (CAT), superoxide dismutase (SOD) and glutathione transferase (GST) activities were determined in digestive gland and gills after exposure to ANT, PER and Mix to assess the impact of the contamination on the oxidative status of V. decussata. Enzyme activities increased in the digestive gland after PER treatment and in the gills after ANT treatment. PER exposure significantly reduced the levels of free thiols and increased levels of carbonylated proteins in the digestive gland, as compared to controls. In contrast, ANT exposure significantly reduced free thiols and increased the number of carbonylated proteins in the gills. Mix induced additive effects as measured by both enzymatic and proteomic approaches. The present study suggests that PER has a strong effect on shell structure; that PER and ANT exposure generate compound-dependent oxidative stress in the tissues of V. decussata and that a mixture of the two compounds has synergistic effects on biochemical response.

  13. Effect of permethrin, anthracene and mixture exposure on shell components, enzymatic activities and proteins status in the Mediterranean clam Venerupis decussata

    International Nuclear Information System (INIS)

    Sellami, Badreddine; Khazri, Abdelhafidh; Mezni, Amine; Louati, Héla; Dellali, Mohamed; Aissa, Patricia; Mahmoudi, Ezzeddine; Beyrem, Hamouda; Sheehan, David

    2015-01-01

    Highlights: • We assessed toxicity of anthracene, permethrin and their mixture on clams. • Tissue and stressor-dependent changes were observed in biochemical responses. • Permethrin induces phase transition from aragonite to calcite in shell structure. • Interactive effects were observed on digestive gland and gill biomarkers. • Both approaches give new vision to risk assessment of organic pollution. - Abstract: Anthracene (ANT) and permethrin (PER) are two of the more toxic compounds reaching the marine environment. This study aimed to determine the impact of these molecules on Venerupis decussata, an economically important species cultured on the Tunisian coast. Shell structure and its possible transformation upon exposure to the two contaminants were studied by X-ray diffraction and gravimetric analyses. Results revealed a phase transition in shell composition from aragonite to calcite after PER exposure, to a mixture of PER and ANT (Mix) but not for ANT alone. Catalase (CAT), superoxide dismutase (SOD) and glutathione transferase (GST) activities were determined in digestive gland and gills after exposure to ANT, PER and Mix to assess the impact of the contamination on the oxidative status of V. decussata. Enzyme activities increased in the digestive gland after PER treatment and in the gills after ANT treatment. PER exposure significantly reduced the levels of free thiols and increased levels of carbonylated proteins in the digestive gland, as compared to controls. In contrast, ANT exposure significantly reduced free thiols and increased the number of carbonylated proteins in the gills. Mix induced additive effects as measured by both enzymatic and proteomic approaches. The present study suggests that PER has a strong effect on shell structure; that PER and ANT exposure generate compound-dependent oxidative stress in the tissues of V. decussata and that a mixture of the two compounds has synergistic effects on biochemical response

  14. Rapid de novo generation of defective interfering RNA by cucumber necrosis virus mutants that do not express the 20-kDa nonstructural protein.

    Science.gov (United States)

    Rochon, D M

    1991-01-01

    It is generally believed that serial passage at high multiplicity of infection (moi) is required for the generation of defective interfering (DI) particles. High levels of DI RNAs are found associated with persistent infections initiated with laboratory cultures of cucumber necrosis virus (CNV). Two synthetic CNV transcripts that were derived through site-directed mutagenesis of a highly infectious CNV cDNA clone and that do not express the CNV 20-kDa nonstructural protein were found to generate high levels of symptom-attenuating DI RNAs de novo without serial high-moi passage in transcript-inoculated plants. Such de novo generation of DI RNAs did not occur in infections initiated with wild-type transcript until at least eight serial high-moi passages. The observation that a CNV nonstructural protein mutant rapidly generates DI RNA de novo may provide insight into mechanisms that underly DI particle formation in RNA viruses in general. Images PMID:1722320

  15. ALS-linked mutant SOD1 proteins promote Aβ aggregates in ALS through direct interaction with Aβ.

    Science.gov (United States)

    Jang, Ja-Young; Cho, Hyungmin; Park, Hye-Yoon; Rhim, Hyangshuk; Kang, Seongman

    2017-11-04

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of motor neurons. Aggregation of ALS-linked mutant Cu/Zn superoxide dismutase (SOD1) is a hallmark of a subset of familial ALS (fALS). Recently, intracellular amyloid-β (Aβ) is detected in motor neurons of both sporadic and familial ALS. We have previously shown that intracellular Aβ specifically interacts with G93A, an ALS-linked SOD1 mutant. However, little is known about the pathological and biological effect of this interaction in neurons. In this study, we have demonstrated that the Aβ-binding region is exposed on the SOD1 surface through the conformational changes due to misfolding of SOD1. Interestingly, we found that the intracellular aggregation of Aβ is enhanced through the direct interaction of Aβ with the Aβ-binding region exposed to misfolded SOD1. Ultimately, increased Aβ aggregation by this interaction promotes neuronal cell death. Consistent with this result, Aβ aggregates was three-fold higher in the brains of G93A transgenic mice than those of non Tg. Our study provides the first direct evidence that Aβ, an AD-linked factor, is associated to the pathogenesis of ALS and provides molecular clues to understand common aggregation mechanisms in the pathogenesis of neurodegenerative diseases. Furthermore, it will provide new insights into the development of therapeutic approaches for ALS. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Decreased interactions of mutant muscle LIM protein (MLP) with N-RAP and alpha-actinin and their implication for hypertrophic cardiomyopathy.

    Science.gov (United States)

    Gehmlich, Katja; Geier, Christian; Osterziel, Karl Josef; Van der Ven, Peter F M; Fürst, Dieter O

    2004-08-01

    Previous work has shown that mutations in muscle LIM protein (MLP) can cause hypertrophic cardiomyopathy (HCM). In order to gain an insight into the molecular basis of the disease phenotype, we analysed the binding characteristics of wild-type MLP and of the (C58G) mutant MLP that causes hypertrophic cardiomyopathy. We show that MLP can form a ternary complex with two of its previously documented myofibrillar ligand proteins, N-RAP and alpha-actinin, which indicates the presence of distinct, non-overlapping binding sites. Our data also show that, in comparison to wild-type MLP, the capacity of the mutated MLP protein to bind both N-RAP and alpha-actinin is significantly decreased. In addition, this single point mutation prevents zinc coordination and proper folding of the second zinc-finger in the first LIM domain, which consequently renders the protein less stable and more susceptible to proteolysis. The molecular basis for HCM-causing mutations in the MLP gene might therefore be an alteration in the equilibrium of interactions of the ternary complex MLP-N-RAP-alpha-actinin. This assumption is supported by the previous observation that in the pathological situation accompanied by MLP down regulation, cardiomyocytes try to compensate for the decreased stability of MLP protein by increasing the expression of its ligand N-RAP, which might finally result in the development of myocyte disarray that is characteristic of this disease. Copyright 2004 Springer-Verlag

  17. Cerebral ischemia is exacerbated by extracellular nicotinamide phosphoribosyltransferase via a non-enzymatic mechanism.

    Directory of Open Access Journals (Sweden)

    Bing Zhao

    Full Text Available Intracellular nicotinamide phosphoribosyltransferase (iNAMPT in neuron has been known as a protective factor against cerebral ischemia through its enzymatic activity, but the role of central extracellular NAMPT (eNAMPT is not clear. Here we show that eNAMPT protein level was elevated in the ischemic rat brain after middle-cerebral-artery occlusion (MCAO and reperfusion, which can be traced to at least in part from blood circulation. Administration of recombinant NAMPT protein exacerbated MCAO-induced neuronal injury in rat brain, while exacerbated oxygen-glucose-deprivation (OGD induced neuronal injury only in neuron-glial mixed culture, but not in neuron culture. In the mixed culture, NAMPT protein promoted TNF-α release in a time- and concentration-dependent fashion, while TNF-α neutralizing antibody protected OGD-induced, NAMPT-enhanced neuronal injury. Importantly, H247A mutant of NAMPT with essentially no enzymatic activity exerted similar effects on ischemic neuronal injury and TNF-α release as the wild type protein. Thus, eNAMPT is an injurious and inflammatory factor in cerebral ischemia and aggravates ischemic neuronal injury by triggering TNF-α release from glia cells, via a mechanism not related to NAMPT enzymatic activity.

  18. Alzheimer’s disease against peptides products of enzymatic cleavage APP protein: Biological, pathobiological and physico-chemical properties of fibrillating peptides

    Directory of Open Access Journals (Sweden)

    Małgorzata Marszałek

    2017-05-01

    Full Text Available Various peptides products of enzymatic cleavage of key for Alzheimer’s disease Amyloid Precursor Protein (APP are well known, but still are matter of scientific debate. The Aβ type products are especially challenging for experimental and medical research. This paper outlines several, still poorly known, biological and medical processes such as peptides biology, i.e., formation, biodistribution, translocation, transport and finally removal from brain compartments and body fluids like Intracellular Fluid (ICF, Cerebrospinal Fluid (CSF, Interstitial Fluid (ISF, blood serum or urine. In addition, the following studies concerning AD patients might prove challenging and simultaneously promising: peptides translocation through Blood-Brain – Barrier (BBB and Blood–Cerebrospinal Fluid Barrier (BCSFB and their removal from the brain according to a new concept of glymphatic system; – diagnostic difficulties that stem from physico-chemical properties and the nature of proteins or fibrillating peptides itself like low concentration, short half-live and from experimental-technical problems as well like high adsorption or low solubility of Aβ, tau or amylin. The study of diagnostic parameters is very important, as it may better reflect early changes before the disease develops; one such parameter is the Aβ42/Aβ40 ratio, or the ratio with the total tau concentration combination and other new biomarkers like Aβ1-38; other factors include oxidative stress and inflammation process proteins, complement factor H, alpha-2-macroglobulin, or clusterin. The study of various forms of pathological amyloid deposits that emerge in different but specific brain regions AD patients seems to be crucial as well. The composition of the first initial pathological, pre-fibrillating monomers of fibrillating peptides and their role in AD development and disease progression have been described as well. They are even more challenging for science and simultaneously might be

  19. Food safety knowledge on the Bt mutant protein Cry8Ka5 employed in the development of coleopteran-resistant transgenic cotton plants.

    Science.gov (United States)

    Farias, Davi F; Peijnenburg, Ad A C M; Grossi-de-Sá, Maria F; Carvalho, Ana F U

    2015-01-01

    Insecticidal Cry proteins from Bacillus thuringiensis (Bt) have been exploited in the development of genetically modified (GM) crops for pest control. However, several pests are still difficult to control such as the coleopteran boll weevil Anthonomus grandis. By applying in vitro molecular evolution to the cry8Ka1 gene sequence, variants were generated with improved activity against A. grandis. Among them, Cry8Ka5 mutant protein showed coleoptericidal activity 3-fold higher (LC50 2.83 μg/mL) than that of the original protein (Cry8Ka1). Cry8Ka5 has been used in breeding programs in order to obtain coleopteran-resistant cotton plants. Nevertheless, there is some concern in relation to the food safety of transgenic crops, especially to the heterologously expressed proteins. In this context, our research group has performed risk assessment studies on Cry8Ka5, using the tests recommended by Codex as well as tests that we proposed as alternative and/or complementary approaches. Our results on the risk analysis of Cry8Ka5 taken together with those of other Cry proteins, point out that there is a high degree of certainty on their food safety. It is reasonable to emphasize that most safety studies on Cry proteins have essentially used the Codex approach. However, other methodologies would potentially provide additional information such as studies on the effects of Cry proteins and derived peptides on the indigenous gastrointestinal microbiota and on intestinal epithelial cells of humans. Additionally, emerging technologies such as toxicogenomics potentially will offer sensitive alternatives for some current approaches or methods.

  20. Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies

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    Edelheit Oded

    2009-06-01

    Full Text Available Abstract Background In protein engineering, site-directed mutagenesis methods are used to generate DNA sequences with mutated codons, insertions or deletions. In a widely used method, mutations are generated by PCR using a pair of oligonucleotide primers designed with mismatching nucleotides at the center of the primers. In this method, primer-primer annealing may prevent cloning of mutant cDNAs. To circumvent this problem we developed an alternative procedure that does not use forward-reverse primer pair in the same reaction. Results In initial studies we used a double-primer PCR mutagenesis protocol, but sequencing of products showed tandem repeats of primer in cloned DNA. We developed an alternative method that starts with two Single-Primer Reactions IN Parallel using high-fidelity Pwo DNA polymerase. Thus, we call the method with the acronym SPRINP. The SPRINP reactions are then combined, denatured at 95°C, and slowly cooled, promoting random annealing of the parental DNA and the newly synthesized strands. The products are digested with DpnI that digests methylated parental strands, and then transformed into E. coli. Using this method we generated >40 mutants in cDNAs coding for human Epithelial Na+ Channel (ENaC subunits. The method has been tested for 1–3 bp codon mutation and insertion of a 27 bp epitope tag into cDNAs. Conclusion The SPRINP mutagenesis protocol yields mutants reliably and with high fidelity. The use of a single primer in each amplification reaction increases the probability of success of primers relative to previous methods employing a forward and reverse primer pair in the same reaction.

  1. Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies.

    Science.gov (United States)

    Edelheit, Oded; Hanukoglu, Aaron; Hanukoglu, Israel

    2009-06-30

    In protein engineering, site-directed mutagenesis methods are used to generate DNA sequences with mutated codons, insertions or deletions. In a widely used method, mutations are generated by PCR using a pair of oligonucleotide primers designed with mismatching nucleotides at the center of the primers. In this method, primer-primer annealing may prevent cloning of mutant cDNAs. To circumvent this problem we developed an alternative procedure that does not use forward-reverse primer pair in the same reaction. In initial studies we used a double-primer PCR mutagenesis protocol, but sequencing of products showed tandem repeats of primer in cloned DNA. We developed an alternative method that starts with two Single-Primer Reactions IN Parallel using high-fidelity Pwo DNA polymerase. Thus, we call the method with the acronym SPRINP. The SPRINP reactions are then combined, denatured at 95 degrees C, and slowly cooled, promoting random annealing of the parental DNA and the newly synthesized strands. The products are digested with DpnI that digests methylated parental strands, and then transformed into E. coli. Using this method we generated >40 mutants in cDNAs coding for human Epithelial Na+ Channel (ENaC) subunits. The method has been tested for 1-3 bp codon mutation and insertion of a 27 bp epitope tag into cDNAs. The SPRINP mutagenesis protocol yields mutants reliably and with high fidelity. The use of a single primer in each amplification reaction increases the probability of success of primers relative to previous methods employing a forward and reverse primer pair in the same reaction.

  2. Tunable Control of an Escherichia coli Expression System for the Overproduction of Membrane Proteins by Titrated Expression of a Mutant lac Repressor.

    Science.gov (United States)

    Kim, Seong Keun; Lee, Dae-Hee; Kim, Oh Cheol; Kim, Jihyun F; Yoon, Sung Ho

    2017-09-15

    Most inducible expression systems suffer from growth defects, leaky basal induction, and inhomogeneous expression levels within a host cell population. These difficulties are most prominent with the overproduction of membrane proteins that are toxic to host cells. Here, we developed an Escherichia coli inducible expression system for membrane protein production based on titrated expression of a mutant lac repressor (mLacI). Performance of the mLacI inducible system was evaluated in conjunction with commonly used lac operator-based expression vectors using a T7 or tac promoter. Remarkably, expression of a target gene can be titrated by the dose-dependent addition of l-rhamnose, and the expression levels were homogeneous in the cell population. The developed system was successfully applied to overexpress three membrane proteins that were otherwise difficult to produce in E. coli. This gene expression control system can be easily applied to a broad range of existing protein expression systems and should be useful in constructing genetic circuits that require precise output signals.

  3. Complementation of a yeast cell cycle mutant by an alfalfa cDNA encoding a protein kinase homologous to p34cdc2.

    Science.gov (United States)

    Hirt, H; Páy, A; Györgyey, J; Bakó, L; Németh, K; Bögre, L; Schweyen, R J; Heberle-Bors, E; Dudits, D

    1991-03-01

    The cdc2 protein kinase plays a central role in control of the eukaryotic cell cycle of animals and yeasts. We have isolated a cDNA clone (cdc2Ms) from alfalfa (Medicago sativa L.) that is homologous to the yeast cdc2/CDC28 genes. The encoded protein is 64% identical to the yeast and mammalian counterparts and shows all the prominent structural features known from these organisms. Antibody raised against a 16-amino acid synthetic peptide with crossreactivity against p34 proteins recognized a 34-kilodalton protein in extracts of alfalfa cells. When transferred into a fission yeast, the plant cdc2 homolog can complement a temperature-sensitive cdc2 mutant. Northern analysis revealed higher transcript levels in shoots and suspension cultures than in roots. In addition to the dominant transcript of 1.4 kilobases detected in the poly(A)+fraction, 2.5- and 1.2-kilobase transcripts were detected in total RNA preparations from shoots or somatic embryos. Suspension cultures that were induced to form somatic embryos by an auxin (2,4-dichlorophenoxyacetic acid) showed fluctuations in transcription pattern during the induction period and embryogenesis.

  4. Immune escape mutants of Highly Pathogenic Avian Influenza H5N1 selected using polyclonal sera: identification of key amino acids in the HA protein.

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    Ioannis Sitaras

    Full Text Available Evolution of Avian Influenza (AI viruses--especially of the Highly Pathogenic Avian Influenza (HPAI H5N1 subtype--is a major issue for the poultry industry. HPAI H5N1 epidemics are associated with huge economic losses and are sometimes connected to human morbidity and mortality. Vaccination (either as a preventive measure or as a means to control outbreaks is an approach that splits the scientific community, due to the risk of it being a potential driving force in HPAI evolution through the selection of mutants able to escape vaccination-induced immunity. It is therefore essential to study how mutations are selected due to immune pressure. To this effect, we performed an in vitro selection of mutants from HPAI A/turkey/Turkey/1/05 (H5N1, using immune pressure from homologous polyclonal sera. After 42 rounds of selection, we identified 5 amino acid substitutions in the Haemagglutinin (HA protein, most of which were located in areas of antigenic importance and suspected to be prone to selection pressure. We report that most of the mutations took place early in the selection process. Finally, our antigenic cartography studies showed that the antigenic distance between the selected isolates and their parent strain increased with passage number.

  5. Site-directed enzymatic PEGylation of the human granulocyte colony-stimulating factor.

    Science.gov (United States)

    Maullu, Carlo; Raimondo, Domenico; Caboi, Francesca; Giorgetti, Alejandro; Sergi, Mauro; Valentini, Maria; Tonon, Giancarlo; Tramontano, Anna

    2009-11-01

    Poly(ethylene glycol) (PEG) is a widely used polymer employed to increase the circulating half-life of proteins in blood and to decrease their immunogenicity and antigenicity. PEG attaches to free amines, typically at lysine residues or at the N-terminal amino acid. This lack of selectivity can present problems when a PEGylated protein therapeutic is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval. Enzymatic modification of proteins is one route to overcome this limitation. Bacterial transglutaminases are enzyme candidates for site-specific modification, but they also have rather broad specificity. The need arises to be able to predict a priori potential PEGylation sites on the protein of interest and, especially, to be able to design mutants where unique PEGylation sites can be introduced when needed. We investigated the feasibility of a computational approach to the problem, using human granulocyte colony-stimulating factor as a test case. The selected protein is therapeutically relevant and represents a challenging problem, as it contains 17 potential PEGylation sites. Our results show that a combination of computational methods allows the identification of the specific glutamines that are substrates for enzymatic PEGylation by a microbial transglutaminase, and that it is possible to rationally modify the protein and introduce PEG moieties at desired sites, thus allowing the selection of regions that are unlikely to interfere with the biological activity of a therapeutic protein.

  6. Motor neuron disease-associated mutant vesicle-associated membrane protein-associated protein (VAP) B recruits wild-type VAPs into endoplasmic reticulum-derived tubular aggregates

    NARCIS (Netherlands)

    Teuling, Eva; Ahmed, Suaad; Haasdijk, Elize; Demmers, Jeroen; Steinmetz, Michel O; Akhmanova, Anna; Jaarsma, Dick; Hoogenraad, Casper C

    2007-01-01

    The vesicle-associated membrane protein-associated proteins (VAPs) VAPA and VAPB interact with lipid-binding proteins carrying a short motif containing two phenylalanines in an acidic tract (FFAT motif) and targets them to the cytosolic surface of the endoplasmic reticulum (ER). A genetic mutation

  7. Problem-Solving Test: Tryptophan Operon Mutants

    Science.gov (United States)

    Szeberenyi, Jozsef

    2010-01-01

    This paper presents a problem-solving test that deals with the regulation of the "trp" operon of "Escherichia coli." Two mutants of this operon are described: in mutant A, the operator region of the operon carries a point mutation so that it is unable to carry out its function; mutant B expresses a "trp" repressor protein unable to bind…

  8. Protein preparation and preliminary X-ray crystallographic analysis of a putative glucosamine 6-phosphate deaminase from Streptococcus mutants

    International Nuclear Information System (INIS)

    Hu, Guan-Jing; Li, Lan-Fen; Li, Dan; Liu, Cong; Wei, Shi-Cheng; Liang, Yu-He; Su, Xiao-Dong

    2007-01-01

    A glucosamine 6-phosphate deaminase homologue from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.4 Å resolution. The SMU.636 protein from Streptococcus mutans is a putative glucosamine 6-phosphate deaminase with 233 residues. The smu.636 gene was PCR-amplified from S. mutans genomic DNA and cloned into the expression vector pET-28a(+). The resultant His-tagged fusion protein was expressed in Escherichia coli and purified to homogeneity in two steps. Crystals of the fusion protein were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 Å resolution and belong to space group P2 1 2 1 2 1 , with unit-cell parameters a = 53.83, b = 82.13, c = 134.70 Å

  9. Protein preparation and preliminary X-ray crystallographic analysis of a putative glucosamine 6-phosphate deaminase from Streptococcus mutants

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Guan-Jing; Li, Lan-Fen; Li, Dan; Liu, Cong [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Wei, Shi-Cheng, E-mail: kqsc-wei@bjmu.edu.cn [Peking University School of Stomatology, Beijing 100081 (China); Liang, Yu-He, E-mail: kqsc-wei@bjmu.edu.cn; Su, Xiao-Dong [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China)

    2007-09-01

    A glucosamine 6-phosphate deaminase homologue from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.4 Å resolution. The SMU.636 protein from Streptococcus mutans is a putative glucosamine 6-phosphate deaminase with 233 residues. The smu.636 gene was PCR-amplified from S. mutans genomic DNA and cloned into the expression vector pET-28a(+). The resultant His-tagged fusion protein was expressed in Escherichia coli and purified to homogeneity in two steps. Crystals of the fusion protein were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 Å resolution and belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.83, b = 82.13, c = 134.70 Å.

  10. Protein profiles construction and differential expressed proteins of the Arabidopsis thaliana quadruple mutant phyA phyB cry1 cry2.

    OpenAIRE

    Fox, Ana Romina; Muschietti, Jorge P; Mazzella, Agustina; XXVIII Argentinean Reunion of Vegetal Physiology

    2010-01-01

    En Arabidopsis phyA phyB cry1 y cry2, son los cuatro fotorreceptores más importantes que controlan el crecimiento y desarrollo por la luz. La técnica de geles bidimensionales provee información sobre la abundancia de una proteína y sus modificaciones postraduccionales. Con el objetivo de identificar nuevos componentes en la fototrasnducción de señales estudiamos el perfil proteómico del cuádruple mutante phyA phyB cry1 cry2 (tet). Para esto obtuvimos y comparamos los proteomas específicos del...

  11. Proteomic analysis reveals differential accumulation of small heat shock proteins and late embryogenesis abundant proteins between ABA-deficient mutant vp5 seeds and wild-type Vp5 seeds in maize

    Directory of Open Access Journals (Sweden)

    Xiaolin eWu

    2015-01-01

    Full Text Available ABA is a major plant hormone that plays important roles during many phases of plant life cycle, including seed development, maturity and dormancy, and especially the acquisition of desiccation tolerance. Understanding of the molecular basis of ABA-mediated plant response to stress is of interest not only in basic research on plant adaptation but also in applied research on plant productivity. Maize mutant viviparous-5 (vp5, deficient in ABA biosynthesis in seeds, is a useful material for studying ABA-mediated response in maize. Due to carotenoid deficiency, vp5 endosperm is white, compared to yellow Vp5 endosperm. However, the background difference at proteome level between vp5 and Vp5 seeds is unclear. This study aimed to characterize proteome alterations of maize vp5 seeds and to identify ABA-dependent proteins during seed maturation. We compared the embryo and endosperm proteomes of vp5 and Vp5 seeds by gel-based proteomics. Up to 46 protein spots, most in embryos, were found to be differentially accumulated between vp5 and Vp5. The identified proteins included small heat shock proteins (sHSPs, late embryogenesis abundant (LEA proteins, stress proteins, storage proteins and enzymes among others. However, EMB564, the most abundant LEA protein in maize embryo, accumulated in comparable levels between vp5 and Vp5 embryos, which contrasted to previously characterized, greatly lowered expression of emb564 mRNA in vp5 embryos. Moreover, LEA proteins and sHSPs displayed differential accumulations in vp5 embryos: six out of eight identified LEA proteins decreased while nine sHSPs increased in abundance. Finally, we discussed the possible causes of global proteome alterations, especially the observed differential accumulation of identified LEA proteins and sHSPs in vp5 embryos. The data derived from this study provides new insight into ABA-dependent proteins and ABA-mediated response during maize seed maturation.

  12. Differential interaction of Apolipoprotein-E isoforms with insulin receptors modulates brain insulin signaling in mutant human amyloid precursor protein transgenic mice.

    Science.gov (United States)

    Chan, Elizabeth S; Chen, Christopher; Cole, Gregory M; Wong, Boon-Seng

    2015-09-08

    It is unclear how human apolipoprotein E4 (ApoE4) increases the risk for Alzheimer's disease (AD). Although Aβ levels can lead to insulin signaling impairment, these experiments were done in the absence of human ApoE. To examine ApoE role, we crossed the human ApoE-targeted replacement mice with mutant human amyloid precursor protein (APP) mice. In 26 week old mice with lower Aβ levels, the expression and phosphorylation of insulin signaling proteins remained comparable among APP, ApoE3xAPP and ApoE4xAPP mouse brains. When the mice aged to 78 weeks, these proteins were markedly reduced in APP and ApoE4xAPP mouse brains. While Aβ can bind to insulin receptor, how ApoE isoforms modulate this interaction remains unknown. Here, we showed that ApoE3 had greater association with insulin receptor as compared to ApoE4, regardless of Aβ42 concentration. In contrast, ApoE4 bound more Aβ42 with increasing peptide levels. Using primary hippocampal neurons, we showed that ApoE3 and ApoE4 neurons are equally sensitive to physiological levels of insulin. However, in the presence of Aβ42, insulin failed to elicit a downstream response only in ApoE4 hippocampal neurons. Taken together, our data show that ApoE genotypes can modulate this Aβ-mediated insulin signaling impairment.

  13. Mutation of a Cuticle Protein Gene, BmCPG10, Is Responsible for Silkworm Non-Moulting in the 2nd Instar Mutant.

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    Fan Wu

    Full Text Available In the silkworm, metamorphosis and moulting are regulated by ecdysone hormone and juvenile hormone. The subject in the present study is a silkworm mutant that does not moult in the 2nd instar (nm2. Genetic analysis indicated that the nm2 mutation is controlled by a recessive gene and is homozygous lethal. Based on positional cloning, nm2 was located in a region approximately 275 kb on the 5th linkage group by eleven SSR polymorphism markers. In this specific range, according to the transcriptional expression of thirteen genes and cloning, the relative expression level of the BmCPG10 gene that encodes a cuticle protein was lower than the expression level of the wild-type gene. Moreover, this gene's structure differs from that of the wild-type gene: there is a deletion of 217 bp in its open reading frame, which resulted in a change in the protein it encoded. The BmCPG10 mRNA was detectable throughout silkworm development from the egg to the moth. This mRNA was low in the pre-moulting and moulting stages of each instar but was high in the gluttonous stage and in newly exuviated larvae. The BmCPG10 mRNA showed high expression levels in the epidermis, head and trachea, while the expression levels were low in the midgut, Malpighian tubule, prothoracic gland, haemolymph and ventral nerve cord. The ecdysone titre was determined by ELISA, and the results demonstrated that the ecdysone titre of nm2 larvae was lower than that of the wild-type larvae. The nm2 mutant could be rescued by feeding 20-hydroxyecdysone, cholesterol and 7-dehydrocholesterol (7dC, but the rescued nm2 only developed to the 4th instar and subsequently died. The moulting time of silkworms could be delayed by BmCPG10 RNAi. Thus, we speculated that the mutation of BmCPG10 was responsible for the silkworm mutant that did not moult in the 2nd instar.

  14. Differential Expression of Claudin Family Proteins in Mouse Ovarian Serous Papillary Epithelial Adenoma in Aging FSH Receptor-Deficient Mutants

    Directory of Open Access Journals (Sweden)

    Jayaprakash Aravindakshan

    2006-12-01

    Full Text Available Ovarian cancer is a deadly disease with long latency. To understand the consequences of loss of folliclestimulating hormone receptor (FSH-R signaling and to explore why the atrophic and anovulatory ovaries of follitropin receptor knockout (FORKO mice develop different types of ovarian tumors, including serous papillary epithelial adenoma later in life, we used mRNA expression profiling to gain a comprehensive view of misregulated genes. Using real-time quantitative reverse transcription-polymerase chain reaction, protein analysis, and cellular localization, we show, for the first time, in vivo evidence that, in the absence of FSH-R signaling, claudin-3, claudin-4, and claudin-11 are selectively upregulated, whereas claudin-1 decreases in ovarian surface epithelium and tumors in comparison to wild type. In vitro experiments using a mouse ovarian surface epithelial cell line derived from wild-type females reveal direct hormonal influence on claudin proteins. Although recent studies suggest that cell junction proteins are differentially expressed in ovarian tumors in women, the etiology of such changes remains unclear. Our results suggest an altered hormonal environment resulting from FSH-R loss as a cause of early changes in tight junction proteins that predispose the ovary to late-onset tumors that occur with aging. More importantly, this study identifies claudin-11 overexpression in mouse ovarian serous cystadenoma.

  15. Coat protein deletion mutants elicit more severe symptoms than wild-type virus in multiple cereal hosts

    Science.gov (United States)

    The coat protein (CP) of Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) tolerates deletion of amino acids 36 to 84 for efficient systemic infection of wheat. This study demonstrates that deletion of CP amino acids 58 to 84, but not 36 to 57, from WSMV genome induced severe ...

  16. Protein Expression Modifications in Phage-Resistant Mutants of Aeromonas salmonicida after AS-A Phage Treatment

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    Catarina Moreirinha

    2018-03-01

    Full Text Available The occurrence of infections by pathogenic bacteria is one of the main sources of financial loss for the aquaculture industry. This problem often cannot be solved with antibiotic treatment or vaccination. Phage therapy seems to be an alternative environmentally-friendly strategy to control infections. Recognizing the cellular modifications that bacteriophage therapy may cause to the host is essential in order to confirm microbial inactivation, while understanding the mechanisms that drive the development of phage-resistant strains. The aim of this work was to detect cellular modifications that occur after phage AS-A treatment in A. salmonicida, an important fish pathogen. Phage-resistant and susceptible cells were subjected to five successive streak-plating steps and analysed with infrared spectroscopy, a fast and powerful tool for cell study. The spectral differences of both populations were investigated and compared with a phage sensitivity profile, obtained through the spot test and efficiency of plating. Changes in protein associated peaks were found, and these results were corroborated by 1-D electrophoresis of intracellular proteins analysis and by phage sensitivity profiles. Phage AS-A treatment before the first streaking-plate step clearly affected the intracellular proteins expression levels of phage-resistant clones, altering the expression of distinct proteins during the subsequent five successive streak-plating steps, making these clones recover and be phenotypically more similar to the sensitive cells.

  17. Extração enzimática das proteínas da farinha de arroz Enzymatic extraction of proteins from rice flour

    Directory of Open Access Journals (Sweden)

    Claudia Regina Vieira

    2008-09-01

    Full Text Available O presente trabalho teve como objetivo extrair enzimaticamente as proteínas de uma farinha comercial de arroz. Visando aumentar o Rendimento de Extração Protéica (REP, os seguintes parâmetros foram avaliados: tipo de enzima (protease alcalina e neutra; temperatura (40, 50 e 60 °C; pH (9,5, 10,5 e 11,0; tratamento físico da amostra (sem tratamento, ultra-turrax a 16.000 rpm e ultra-som a 120 W, ambos por 5, 10 e 15 minutos; relação Enzima:Substrato (E:S de 5:100 e 10:100; e concentração inicial de matéria-prima (1:3, 1:5 e 1:10 p/v. Os teores de proteínas da farinha de arroz e dos resíduos foram determinados para o cálculo do REP. Os resultados mostraram que a melhor condição de extração protéica, que levou ao maior REP, foi a que empregou a concentração inicial de matéria-prima a 1:10 (p/v, sem tratamento físico, com pH 10,5, com a protease alcalina na relação E:S de 10:100, a 50 °C, tendo atingido um REP de 63,4%.The enzymatic extraction of proteins from a commercial rice flour was studied in this work. In order to increase the Protein Extraction Yield (PEY, the following parameters were evaluated: enzyme type (alkaline and neutral protease; temperature (40, 50 and 60 °C; pH (9.5, 10.5 and 11.0; physical treatment of the sample (no treatment; Ultra-Turrax at 16.000 rpm and ultrasound at 120 W, both for 5, 10 and 15 minutes; enzyme:substrate ratio (E:S of 5:100 and 10:100 and initial concentration of raw material (1:3, 1:5 and 1:10 w/v. The PEY was calculated using the protein contents of rice flour and the extraction residues. The results showed that the best condition for protein extraction, which gave the highest PEY (63.4%, was that using an initial concentration of raw material of 1:10 (w/v, no physical treatment, pH 10.5, the alkaline protease, an E:S of 10:100, at 50 °C.

  18. Bifunctional anti-huntingtin proteasome-directed intrabodies mediate efficient degradation of mutant huntingtin exon 1 protein fragments.

    Directory of Open Access Journals (Sweden)

    David C Butler

    Full Text Available Huntington's disease (HD is a fatal autosomal dominant neurodegenerative disorder caused by a trinucleotide (CAG(n repeat expansion in the coding sequence of the huntingtin gene, and an expanded polyglutamine (>37Q tract in the protein. This results in misfolding and accumulation of huntingtin protein (htt, formation of neuronal intranuclear and cytoplasmic inclusions, and neuronal dysfunction/degeneration. Single-chain Fv antibodies (scFvs, expressed as intrabodies that bind htt and prevent aggregation, show promise as immunotherapeutics for HD. Intrastriatal delivery of anti-N-terminal htt scFv-C4 using an adeno-associated virus vector (AAV2/1 significantly reduces the size and number of aggregates in HDR6/1 transgenic mice; however, this protective effect diminishes with age and time after injection. We therefore explored enhancing intrabody efficacy via fusions to heterologous functional domains. Proteins containing a PEST motif are often targeted for proteasomal degradation and generally have a short half life. In ST14A cells, fusion of the C-terminal PEST region of mouse ornithine decarboxylase (mODC to scFv-C4 reduces htt exon 1 protein fragments with 72 glutamine repeats (httex1-72Q by ~80-90% when compared to scFv-C4 alone. Proteasomal targeting was verified by either scrambling the mODC-PEST motif, or via proteasomal inhibition with epoxomicin. For these constructs, the proteasomal degradation of the scFv intrabody proteins themselves was reduced<25% by the addition of the mODC-PEST motif, with or without antigens. The remaining intrabody levels were amply sufficient to target N-terminal httex1-72Q protein fragment turnover. Critically, scFv-C4-PEST prevents aggregation and toxicity of httex1-72Q fragments at significantly lower doses than scFv-C4. Fusion of the mODC-PEST motif to intrabodies is a valuable general approach to specifically target toxic antigens to the proteasome for degradation.

  19. Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype.

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    Stephanie Plog

    Full Text Available The human CLCA4 (chloride channel regulator, calcium-activated modulates the intestinal phenotype of cystic fibrosis (CF patients via an as yet unknown pathway. With the generation of new porcine CF models, species-specific differences between human modifiers of CF and their porcine orthologs are considered critical for the translation of experimental data. Specifically, the porcine ortholog to the human CF modulator gene CLCA4 has recently been shown to be duplicated into two separate genes, CLCA4a and CLCA4b. Here, we characterize the duplication product, CLCA4b, in terms of its genomic structure, tissue and cellular expression patterns as well as its in vitro electrophysiological properties. The CLCA4b gene is a pig-specific duplication product of the CLCA4 ancestor and its protein is exclusively expressed in small and large intestinal crypt epithelial cells, a niche specifically occupied by no other porcine CLCA family member. Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals. Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins. The apparently pig-specific duplication of the CLCA4 gene with unique expression of the CLCA4b protein variant in intestinal crypt epithelial cells where the porcine CFTR is also present raises the question of whether it may modulate the porcine CF phenotype. Moreover, the naturally occurring null variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.

  20. Characterization of pneumococcal Ser/Thr protein phosphatase phpP mutant and identification of a novel PhpP substrate, putative RNA binding protein Jag

    Czech Academy of Sciences Publication Activity Database

    Ulrych, Aleš; Holečková, Nela; Goldová, Jana; Doubravová, Linda; Benada, Oldřich; Kofroňová, Olga; Halada, Petr; Branny, Pavel

    2016-01-01

    Roč. 16, OCT 24 (2016), s. 247 ISSN 1471-2180 R&D Projects: GA ČR GAP302/12/0256; GA ČR GAP207/12/1568; GA MŠk LH12055 Institutional support: RVO:61388971 Keywords : Signal transduction * Protein phosphatase * Protein kinase Subject RIV: EE - Microbiology, Virology Impact factor: 2.644, year: 2016

  1. ENZYMATIC PREPARATION OF 1,6-ANHYDRO-MUROPEPTIDES BY IMMOBILIZED MUREIN HYDROLASES FROM ESCHERICHIA-COLI FUSED TO STAPHYLOCOCCAL PROTEIN-A

    NARCIS (Netherlands)

    Engel, Hendrik; van Leeuwen, A; Dijkstra, A; Keck, W

    In order to produce biologically active 1,6-anhydro-muropeptides in large amounts by enzymatic degradation of isolated bacterial murein polymer highly specific periplasmic murein-metabolizing enzymes from Escherichia coli are made available. The genes slt, dacB, and mepA, encoding the soluble lytic

  2. Deregulation of the actin cytoskeleton and macropinocytosis in response to phorbol ester by the mutant protein kinase C gamma that causes spinocerebellar ataxia type 14.

    Science.gov (United States)

    Yamamoto, Kazuhiro; Seki, Takahiro; Yamamoto, Hikaru; Adachi, Naoko; Tanaka, Shigeru; Hide, Izumi; Saito, Naoaki; Sakai, Norio

    2014-01-01

    Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. γPKC is a neuron-specific member of the classical PKCs and is activated and translocated to subcellular regions as a result of various stimuli, including diacylglycerol synthesis, increased intracellular Ca(2+) and phorbol esters. We investigated whether SCA14 mutations affect the γPKC-related functions by stimulating HeLa cells with TPA (12-O-tetradecanoylpholbol 13-acetate), a type of phorbol ester. Wild-type (WT) γPKC-GFP was translocated to the plasma membrane within 10 min of TPA stimulation, followed by its perinuclear translocation and cell shrinkage, in a PKC kinase activity- and microtubule-dependent manner. On the other hand, although SCA14 mutant γPKC-GFP exhibited a similar translocation to the plasma membrane, the subsequent perinuclear translocation and cell shrinkage were significantly impaired in response to TPA. Translocated WT γPKC colocalized with F-actin and formed large vesicular structures in the perinuclear region. The uptake of FITC-dextran, a marker of macropinocytosis, was promoted by TPA stimulation in cells expressing WT γPKC, and FITC-dextran was surrounded by γPKC-positive vesicles. Moreover, TPA induced the phosphorylation of MARCKS, which is a membrane-substrate of PKC, resulting in the translocation of phosphorylated MARCKS to the perinuclear region, suggesting that TPA induces macropinocytosis via γPKC activation. However, TPA failed to activate macropinocytosis and trigger the translocation of phosphorylated MARCKS in cells expressing the SCA14 mutant γPKC. These findings suggest that γPKC is involved in the regulation of the actin cytoskeleton and macropinocytosis in HeLa cells, while SCA14 mutant γPKC fails to regulate these processes due to its reduced kinase activity at the plasma membrane. This property might be involved in pathogenesis

  3. Deregulation of the actin cytoskeleton and macropinocytosis in response to phorbol ester by the mutant protein kinase C gamma that causes spinocerebellar ataxia type 14

    Directory of Open Access Journals (Sweden)

    Kazuhiro eYamamoto

    2014-04-01

    Full Text Available Several missense mutations in the protein kinase Cγ (γPKC gene have been found to cause spinocerebellar ataxia type 14 (SCA14, an autosomal dominant neurodegenerative disease. γPKC is a neuron-specific member of the classical PKCs and is activated and translocated to subcellular regions as a result of various stimuli, including diacylglycerol synthesis, increased intracellular Ca2+ and phorbol esters. We investigated whether SCA14 mutations affect the γPKC-related functions by stimulating HeLa cells with TPA (12-O-tetradecanoylpholbol 13-acetate, a type of phorbol ester. Wild-type (WT γPKC-GFP was translocated to the plasma membrane within 10 min of TPA stimulation, followed by its perinuclear translocation and cell shrinkage, in a PKC kinase activity- and microtubule-dependent manner. On the other hand, although SCA14 mutant γPKC-GFP exhibited a similar translocation to the plasma membrane, the subsequent perinuclear translocation and cell shrinkage were significantly impaired in response to TPA. Translocated WT γPKC colocalized with F-actin and formed large vesicular structures in the perinuclear region. The uptake of FITC-dextran, a marker of macropinocytosis, was promoted by TPA stimulation in cells expressing WT γPKC, and FITC-dextran was surrounded by γPKC-positive vesicles. Moreover, TPA induced the phosphorylation of MARCKS, which is a membrane-substrate of PKC, resulting in the translocation of phosphorylated MARCKS to the perinuclear region, suggesting that TPA induces macropinocytosis via γPKC activation. However, TPA failed to activate macropinocytosis and trigger the translocation of phosphorylated MARCKS in cells expressing the SCA14 mutant γPKC. These findings suggest that γPKC is involved in the regulation of the actin cytoskeleton and macropinocytosis in HeLa cells, while SCA14 mutant γPKC fails to regulate these processes due to its reduced kinase activity at the plasma membrane. This property might be involved in

  4. Role of IGF-binding protein 3 in the resistance of EGFR mutant lung cancer cells to EGFR-tyrosine kinase inhibitors.

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    Yun Jung Choi

    Full Text Available Most patients treated with EGFR-tyrosine kinase inhibitors (EGFR-TKIs eventually develop acquired resistance. Loss of expression of insulin-like growth factor (IGF-binding protein-3 (IGFBP-3 has been suggested as a possible mechanism of resistance to EGFR-TKIs in the A431 and HN11 cell lines. Here, we investigated IGFBP-3 expression in two EGFR mutant lung cancer cell lines with resistance to EGFR-TKIs and examined the value of serum IGFBP-3 level as a marker of resistance. The effect of the induction or suppression of IGFBP-3 expression on resistance was also evaluated. HCC827 sublines with resistance to gefitinib (HCC827/GR and erlotinib (HCC827/ER were established. Loss of IGFBP-3 expression was detected by Western blotting in both cell lines without changes in transcriptional activity, and ELISA showed significantly lower amounts of secreted IGFBP-3 in the culture media of the mutant cell lines than in that of the parental line. Despite the loss of IGFBP-3 expression, IGFR signalling activity remained unchanged. Forced expression of IGFBP-3 by adenovirus-mediated transfection or recombinant IGFBP-3 slightly increased the growth-inhibitory and apoptotic effects of EGFR-TKIs, whereas suppression of IGFBP-3 did not affect sensitivity to EGFR-TKI. Serum IGFBP-3 levels measured by ELISA before and after the development of EGFR-TKI resistance in 20 patients showed no significant changes (1815.3±94.6 ng/mL before treatment vs. 1778.9±87.8 ng/mL after EGFR-TKI resistance. In summary, although IGFBP-3 downregulation is associated with the acquisition of resistance to EGFR-TKIs regardless of the mechanism, its effect on resistance was not significant, indicating that IGFBP-3 may not play an important role in resistance to EGFR-TKIs and serum IGFBP-3 level is not a reliable indicator of resistance.

  5. Global Systems-Level Analysis of Hfq and SmpB Deletion Mutants in Salmonella: Implications for Virulence and Global Protein Translation

    Energy Technology Data Exchange (ETDEWEB)

    Ansong, Charles; Yoon, Hyunjin; Porwollik, Steffen; Mottaz-Brewer, Heather; Petritis, Brianne O.; Jaitly, Navdeep; Adkins, Joshua N.; Mcclelland, Michael; Heffron, Fred; Smith, Richard D.

    2009-03-11

    In recent years the profound importance of sRNA-mediated translational/post-transcriptional regulation has been increasingly appreciated. However, the global role played by translational regulation in control of gene expression has never been elucidated in any organism for the simple reason that global proteomics methods required to accurately characterize post-transcriptional processes and the knowledge of translational control mechanisms have only become available within the last few years. The proteins Hfq and SmpB are essential for the biological activity of a range of regulatory sRNAs and thus provide a means to identify potential targets of sRNA regulation. We performed a sample-matched global proteomics and transcriptional analysis to examine the role of Hfq and SmpB in global protein translation and virulence using the Salmonella typhimurium model system. Samples were analyzed from bacteria grown under four different conditions; two laboratory conditions and two that are thought to mimic the intracellular environment. We show that mutants of hfq and smpB directly or indirectly modulate at least 20% and 4% of all Salmonella proteins, respectively, with limited correlation between transcription and protein expression. This is the first report suggesting that SmpB could be a general translational regulator. The broad spectrum of proteins modulated by Hfq was also surprising including central metabolism, LPS biosynthesis, two-component regulatory systems, quorum sensing, SP1-TTSS, oxidative stress, fatty acid metabolism, nucleoside and nucleotide metabolism, envelope stress, aminoacyl-tRNA synthetases, amino acid biosynthesis, peptide transport, and motility.. The extent of global regulation of translation by Hfq is unexpected, with profound effects in all stages of Salmonella’s life cycle. Our results represent the first global systems-level analysis of translational regulation; the elucidated potential targets of sRNA regulation from our analysis will

  6. Identification of distinct specificity determinants in resistance protein Cf-4 allows construction of a Cf-9 mutant that confers recognition of avirulence protein AVR4

    NARCIS (Netherlands)

    Hoorn, Van der R.A.L.; Roth, R.; Wit, De P.J.G.M.

    2001-01-01

    The tomato resistance genes Cf-4 and Cf-9 confer specific, hypersensitive response-associated recognition of Cladosporium carrying the avirulence genes Avr4 and Avr9, respectively. Cf-4 and Cf-9 encode type I transmembrane proteins with extracellular leucine-rich repeats (LRRs). Compared with Cf-9,

  7. Peroxisomal matrix protein import - Suppression of protein import defects in Hansenula polymorpha pex mutants by overproduction of the PTS1 receptor pex5p

    NARCIS (Netherlands)

    Kiel, JAKW; Veenhuis, M

    2000-01-01

    In the past decade, much progress has been made in understanding the mechanisms that govern sorting of proteins to the peroxisomal lumen. This article summarizes the principal features of how peroxisomal matrix enzymes are thought to reach the peroxisome. In addition, it describes recent data that

  8. Exploring the Oxidation of Lignin-Derived Phenols by a Library of Laccase Mutants

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    Isabel Pardo

    2015-09-01

    Full Text Available Saturation mutagenesis was performed over six residues delimiting the substrate binding pocket of a fungal laccase previously engineered in the lab. Mutant libraries were screened using sinapic acid as a model substrate, and those mutants presenting increased activity were selected for exploring the oxidation of lignin-derived phenols. The latter comprised a battery of phenolic compounds of interest due to their use as redox mediators or precursors of added-value products and their biological activity. The new laccase variants were investigated in a multi-screening assay and the structural determinants, at both the substrate and the protein level, for the oxidation of the different phenols are discussed. Laccase activity greatly varied only by changing one or two residues of the enzyme pocket. Our results suggest that once the redox potential threshold is surpassed, the contribution of the residues of the enzymatic pocket for substrate recognition and binding strongly influence the overall rate of the catalytic reaction.

  9. Client Proteins and Small Molecule Inhibitors Display Distinct Binding Preferences for Constitutive and Stress-Induced HSP90 Isoforms and Their Conformationally Restricted Mutants.

    Directory of Open Access Journals (Sweden)

    Thomas L Prince

    Full Text Available The two cytosolic/nuclear isoforms of the molecular chaperone HSP90, stress-inducible HSP90α and constitutively expressed HSP90β, fold, assemble and maintain the three-dimensional structure of numerous client proteins. Because many HSP90 clients are important in cancer, several HSP90 inhibitors have been evaluated in the clinic. However, little is known concerning possible unique isoform or conformational preferences of either individual HSP90 clients or inhibitors. In this report, we compare the relative interaction strength of both HSP90α and HSP90β with the transcription factors HSF1 and HIF1α, the kinases ERBB2 and MET, the E3-ubiquitin ligases KEAP1 and RHOBTB2, and the HSP90 inhibitors geldanamycin and ganetespib. We observed unexpected differences in relative client and drug preferences for the two HSP90 isoforms, with HSP90α binding each client protein with greater apparent affinity compared to HSP90β, while HSP90β bound each inhibitor with greater relative interaction strength compared to HSP90α. Stable HSP90 interaction was associated with reduced client activity. Using a defined set of HSP90 conformational mutants, we found that some clients interact strongly with a single, ATP-stabilized HSP90 conformation, only transiently populated during the dynamic HSP90 chaperone cycle, while other clients interact equally with multiple HSP90 conformations. These data suggest different functional requirements among HSP90 clientele that, for some clients, are likely to be ATP-independent. Lastly, the two inhibitors examined, although sharing the same binding site, were differentially able to access distinct HSP90 conformational states.

  10. BCR and its mutants, the reciprocal t(9;22-associated ABL/BCR fusion proteins, differentially regulate the cytoskeleton and cell motility

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    Puccetti Elena

    2006-11-01

    Full Text Available Abstract Background The reciprocal (9;22 translocation fuses the bcr (breakpoint cluster region gene on chromosome 22 to the abl (Abelson-leukemia-virus gene on chromosome 9. Depending on the breakpoint on chromosome 22 (the Philadelphia chromosome – Ph+ the derivative 9+ encodes either the p40(ABL/BCR fusion transcript, detectable in about 65% patients suffering from chronic myeloid leukemia, or the p96(ABL/BCR fusion transcript, detectable in 100% of Ph+ acute lymphatic leukemia patients. The ABL/BCRs are N-terminally truncated BCR mutants. The fact that BCR contains Rho-GEF and Rac-GAP functions strongly suggest an important role in cytoskeleton modeling by regulating the activity of Rho-like GTPases, such as Rho, Rac and cdc42. We, therefore, compared the function of the ABL/BCR proteins with that of wild-type BCR. Methods We investigated the effects of BCR and ABL/BCRs i. on the activation status of Rho, Rac and cdc42 in GTPase-activation assays; ii. on the actin cytoskeleton by direct immunofluorescence; and iii on cell motility by studying migration into a three-dimensional stroma spheroid model, adhesion on an endothelial cell layer under shear stress in a flow chamber model, and chemotaxis and endothelial transmigration in a transwell model with an SDF-1α gradient. Results Here we show that both ABL/BCRs lost fundamental functional features of BCR regarding the regulation of small Rho-like GTPases with negative consequences on cell motility, in particular on the capacity to adhere to endothelial cells. Conclusion Our data presented here describe for the first time an analysis of the biological function of the reciprocal t(9;22 ABL/BCR fusion proteins in comparison to their physiological counterpart BCR.

  11. BCR and its mutants, the reciprocal t(9;22)-associated ABL/BCR fusion proteins, differentially regulate the cytoskeleton and cell motility

    International Nuclear Information System (INIS)

    Zheng, Xiaomin; Güller, Saskia; Beissert, Tim; Puccetti, Elena; Ruthardt, Martin

    2006-01-01

    The reciprocal (9;22) translocation fuses the bcr (breakpoint cluster region) gene on chromosome 22 to the abl (Abelson-leukemia-virus) gene on chromosome 9. Depending on the breakpoint on chromosome 22 (the Philadelphia chromosome – Ph+) the derivative 9+ encodes either the p40 (ABL/BCR) fusion transcript, detectable in about 65% patients suffering from chronic myeloid leukemia, or the p96 (ABL/BCR) fusion transcript, detectable in 100% of Ph+ acute lymphatic leukemia patients. The ABL/BCRs are N-terminally truncated BCR mutants. The fact that BCR contains Rho-GEF and Rac-GAP functions strongly suggest an important role in cytoskeleton modeling by regulating the activity of Rho-like GTPases, such as Rho, Rac and cdc42. We, therefore, compared the function of the ABL/BCR proteins with that of wild-type BCR. We investigated the effects of BCR and ABL/BCRs i.) on the activation status of Rho, Rac and cdc42 in GTPase-activation assays; ii.) on the actin cytoskeleton by direct immunofluorescence; and iii) on cell motility by studying migration into a three-dimensional stroma spheroid model, adhesion on an endothelial cell layer under shear stress in a flow chamber model, and chemotaxis and endothelial transmigration in a transwell model with an SDF-1α gradient. Here we show that both ABL/BCRs lost fundamental functional features of BCR regarding the regulation of small Rho-like GTPases with negative consequences on cell motility, in particular on the capacity to adhere to endothelial cells. Our data presented here describe for the first time an analysis of the biological function of the reciprocal t(9;22) ABL/BCR fusion proteins in comparison to their physiological counterpart BCR

  12. Otimização dos parâmetros de produção de hidrolisados protéicos enzimáticos utilizando pescado de baixo valor comercial Optimization of the production parameters for enzymatic protein hydrolyzed using low commercial value fish

    Directory of Open Access Journals (Sweden)

    Sarita D´Avila dos Santos

    2009-01-01

    Full Text Available The enzymatic modification of proteins has been widely studied with the aim of add value to low commercial value fish. The objective of this work was to evaluate and optimize the parameters involved in the production process of an enzymatic protein hydrolyzed with high protein content. The results showed that for Alcalase the most significant parameters were temperature, pH and substrate concentration and for Flavourzyme were pH, substrate concentration and enzyme concentration. It was obtained for Alcalase a predictive model for the recovered nitrogen and for Flavourzyme a predictive model for the hydrolysis degree.

  13. Cryptococcus gattii urease as a virulence factor and the relevance of enzymatic activity in cryptococcosis pathogenesis.

    Science.gov (United States)

    Feder, Vanessa; Kmetzsch, Lívia; Staats, Charley Christian; Vidal-Figueiredo, Natalia; Ligabue-Braun, Rodrigo; Carlini, Célia Regina; Vainstein, Marilene Henning

    2015-04-01

    Ureases (EC 3.5.1.5) are Ni(2+) -dependent metalloenzymes produced by plants, fungi and bacteria that hydrolyze urea to produce ammonia and CO2 . The insertion of nickel atoms into the apo-urease is better characterized in bacteria, and requires at least three accessory proteins: UreD, UreF, and UreG. Our group has demonstrated that ureases possess ureolytic activity-independent biological properties that could contribute to the pathogenicity of urease-producing microorganisms. The presence of urease in pathogenic bacteria strongly correlates with pathogenesis in some human diseases. Some medically important fungi also produce urease, including Cryptococcus neoformans and Cryptococcus gattii. C. gattii is an etiological agent of cryptococcosis, most often affecting immunocompetent individuals. The cryptococcal urease might play an important role in pathogenesis. It has been proposed that ammonia produced via urease action might damage the host endothelium, which would enable yeast transmigration towards the central nervous system. To analyze the role of urease as a virulence factor in C. gattii, we constructed knockout mutants for the structural urease-coding gene URE1 and for genes that code the accessory proteins Ure4 and Ure6. All knockout mutants showed reduced multiplication within macrophages. In intranasally infected mice, the ure1Δ (lacking urease protein) and ure4Δ (enzymatically inactive apo-urease) mutants caused reduced blood burdens and a delayed time of death, whereas the ure6Δ (enzymatically inactive apo-urease) mutant showed time and dose dependency with regard to fungal burden. Our results suggest that C. gattii urease plays an important role in virulence, in part possibly through enzyme activity-independent mechanism(s). © 2015 FEBS.

  14. The single N-glycan deletion mutant of soluble ErbB3 protein attenuates heregulin β1-induced tumor progression by blocking of the HIF-1 and Nrf2 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Takamiya, Rina, E-mail: rinataka0429@gmail.com; Takahashi, Motoko; Uehara, Yasuaki; Ariki, Shigeru; Hashimoto, Jiro; Hasegawa, Yoshihiro; Kuroki, Yoshio

    2014-11-21

    Highlights: • The sErbB3 N418Q mutant blocks heregulin β1 induced nuclear accumulation of HIF-1α. • The sErbB3 N418Q mutant attenuates cancer cell migration induced by heregulin β1. • The sErbB3 N418Q mutant blocks heregulin β1 induced nuclear accumulation of Nrf2. • The sErbB3 N418Q mutant may be a potential therapeutic application for tumor. - Abstract: It has been well documented that activation of the ErbB3–PI3K–Akt pathway is implicated in tumor survival and progression. We previously demonstrated that the single N-glycan deletion mutant of soluble ErbB3 protein (sErbB3 N418Q) attenuates heregulin β1-induced ErbB3 signaling. The active PI3K–Akt pathway augments the nuclear accumulation of hypoxia inducible factor (HIF)-1α, which activates the transcription of many target genes and drives cancer progression. In this study, we focused on the effects of sErbB3 N418Q mutant on nuclear accumulation of HIF-1α. Pretreatment with the sErbB3 N418Q mutant suppressed heregulin β1-induced HIF-1α activation in MCF7 cells. Similar results were also obtained in other breast cancer cell lines, T47D and BT474. Interestingly, these suppressive effects were not observed with the sErbB3 wild type. In addition, pretreatment with the sErbB3 N418Q mutant suppressed the cell migration of MCF7 cells induced by heregulin β1. Furthermore, incubation with heregulin β1 also induced the nuclear accumulation of Nrf2, and this effect was also reduced by the sErbB3 N418Q mutant, but not the sErbB3 wild type. These findings indicated that the sErbB3 N418Q mutant suppressed malignant formation of cancer cells by blocking of the HIF-1α and Nrf2 pathways.

  15. A tomato mutant that shows stunting, wilting, progressive necrosis and constitutive expression of defence genes contains a recombinant Hcr9 gene encoding an autoactive protein.

    Science.gov (United States)

    Barker, Claire L; Talbot, Stephen J; Jones, Jonathan D G; Jones, David A

    2006-05-01

    The tomato Cf-9 gene confers resistance to races of the leaf mould fungus Cladosporium fulvum that carry the Avr9 avirulence gene. Cf-9 resides at a locus containing five paralogous genes and was isolated by transposon tagging using a modified maize Dissociation (Ds) element. The tagging experiment generated an allelic series of Ds-induced mutations of Cf-9, most of which were wild type in appearance. However, one mutant, designated M205, showed stunted growth, wilting, progressive leaf chlorosis and necrosis and constitutive expression of defence genes. The phenotype of M205 was caused by a semidominant, Avr9-independent mutation that co-segregated with a Ds element insertion at the Cf-9 locus. Molecular genetic analysis indicated that the Cf-9 locus of M205 had undergone recombination, generating a chimeric gene, designated Hcr9-M205, that comprised an in-frame fusion between the 5' coding region of the Cf-9 paralogue, Hcr9-9A, and the 3' coding region of Cf-9. The presence of a possible excision footprint adjacent to the junction between Hcr9-9A and Cf-9, and a Ds insertion at the homologous position in the downstream paralogue Hcr9-9D, is consistent with recombination between Hcr9-9A and Cf-9 promoted by transposition of Ds from Cf-9 into Hcr9-9D. Agrobacterium tumefaciens-mediated transient expression of Hcr9-M205 in Nicotiana tabacum caused chlorosis and the accumulation of defence gene transcripts, indicating that the protein encoded by this novel Hcr9 gene is autoactive.

  16. Mutants of alfalfa mosaic virus

    International Nuclear Information System (INIS)

    Roosien, J.

    1983-01-01

    In this thesis the isolation and characterization of a number of mutants of alfalfa mosaic virus, a plant virus with a coat protein dependent genome, is described. Thermo-sensitive (ts) mutants were selected since, at least theoretically, ts mutations can be present in all virus coded functions. It was found that a high percentage of spontaneous mutants, isolated because of their aberrant symptoms, were ts. The majority of these isolates could grow at the non-permissive temperature in the presence of a single wild type (wt) component. To increase the mutation rate virus preparations were treated with several mutagens. After nitrous acid treatment or irradiation with ultraviolet light, an increase in the level of mutations was observed. UV irradiation was preferred since it did not require large amounts of purified viral components. During the preliminary characterization of potential ts mutants the author also obtained one structural and several symptom mutants which were analysed further (chapter 7, 8 and 9). The properties of the ts mutants are described in chapter 3-7. (Auth.)

  17. Use of the rep technique for allele replacement to construct mutants with deletions of the pstSCAB-phoU operon: evidence of a new role for the PhoU protein in the phosphate regulon.

    Science.gov (United States)

    Steed, P M; Wanner, B L

    1993-01-01

    The phosphate regulon is negatively regulated by the PstSCAB transporter and PhoU protein by a mechanism that may involve protein-protein interaction(s) between them and the Pi sensor protein, PhoR. In order to study such presumed interaction(s), mutants with defined deletions of the pstSCAB-phoU operon were made. This was done by construction of M13 recombinant phage carrying these mutations and by recombination of them onto the chromosome by using a rep host (which cannot replicate M13) for allele replacement. These mutants were used to show that delta (pstSCAB-phoU) and delta (pstB-phoU) mutations abolished Pi uptake by the PstSCAB transporter, as expected, and that delta phoU mutations had no effect on uptake. Unexpectedly, delta phoU mutations had a severe growth defect, and this growth defect was (largely) alleviated by a compensatory mutation in the pstSCAB genes or in the phoBR operon, whose gene products positively regulate expression of the pstSCAB-phoU operon. Because delta phoU mutants that synthesize a functional PstSCAB transporter constitutively grew extremely poorly, the PhoU protein must have a new role, in addition to its role as a negative regulator. A role for the PhoU protein in intracellular Pi metabolism is proposed. Further, our results contradict those of M. Muda, N. N. Rao, and A. Torriani (J. Bacteriol. 174:8057-8064, 1992), who reported that the PhoU protein was required for Pi uptake. Images PMID:8226621

  18. Characteristics of the repair - deficient mutants 1435 plague microbe strain

    International Nuclear Information System (INIS)

    Temiralieva, G.A.

    1977-01-01

    Repair-deficient mutants 1435 A uvr - hcr - , 1435-17 uvr - hcr + and 1435-35 lon have been obtained from 1435 plague microbe strain, isolated from a large gerbil living in the Central Asian desert region. The mutants have the same cultural-morphological and enzymatic characteristics, the same need in growth factors and similar virulence determinants as the original strain, but they do not cause death of the experimental animals

  19. Photoelectrochemical enzymatic biosensors.

    Science.gov (United States)

    Zhao, Wei-Wei; Xu, Jing-Juan; Chen, Hong-Yuan

    2017-06-15

    Enzymatic biosensors have been valuable bioanalytical devices for analysis of diverse targets in disease diagnosis, biological and biomedical research, etc. Photoelectrochemical (PEC) bioanalysis is a recently emerged method that promptly becoming a subject of new research interests due to its attractive potential for future bioanalysis with high sensitivity and specificity. PEC enzymatic biosensors integrate the inherent sensitivities of PEC bioanalysis and the selectivity of enzymes and thus share their both advantages. Currently, PEC enzymatic biosensors have become a hot topic of significant research and the recent impetus has grown rapidly as demonstrated by increased research papers. Given the pace of advances in this area, this review will make a thorough discussion and survey on the fundamentals, sensing strategies, applications and the state of the art in PEC enzymatic biosensors, followed by future prospects based on our own opinions. We hope this work could provide an accessible introduction to PEC enzymatic biosensors for any scientist. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Comprehensive analysis to explain reduced or increased SOD1 enzymatic activity in ALS patients and their relatives.

    Science.gov (United States)

    Keskin, Isil; Birve, Anna; Berdynski, Mariusz; Hjertkvist, Karin; Rofougaran, Reza; Nilsson, Torbjörn K; Glass, Jonathan D; Marklund, Stefan L; Andersen, Peter M

    2017-08-01

    To characterise stabilities in erythrocytes of mutant SOD1 proteins, compare SOD1 enzymatic activities between patients with different genetic causes of ALS and search for underlying causes of deviant SOD1 activities in individuals lacking SOD1 mutations. Blood samples from 4072 individuals, ALS patients with or without a SOD1 mutation, family members and controls were studied. Erythrocyte SOD1 enzymatic activities normalised to haemoglobin content were determined, and effects of haemoglobin disorders on dismutation assessed. Coding SOD1 sequences were analysed by Sanger sequencing, exon copy number variations by fragment length analysis and by TaqMan Assay. Of the 44 SOD1 mutations found, 75% caused severe destabilisation of the mutant protein but in 25% it was physically stable. Mutations producing structural changes caused halved erythrocyte SOD1 activities. There were no differences in SOD1 activities between patients without a SOD1 mutation and control individuals or carriers of TBK1 mutations and C9orf72 HRE . In the low and high SOD1 activity groups no deviations were found in exon copy numbers and intron gross structures. Thalassemias and iron deficiency were associated with increased SOD1 activity/haemoglobin ratios. Adjunct erythrocyte SOD1 activity analysis reliably signals destabilising SOD1 mutations including intronic mutations that are missed by exon sequencing.

  1. Anti-inflammatory activities of enzymatic (alcalase) hydrolysate of a ...

    African Journals Online (AJOL)

    Anti-inflammatory activities of enzymatic (alcalase) hydrolysate of a whey protein concentrate. LB de Carvalho-Silva, MTB Pacheco, R Bertoldo, C de Carvalho Veloso, LC Teodoro, A Giusti-Paiva, PCB Lollo, R Soncini ...

  2. Crystals of DhaA mutants from Rhodococcus rhodochrous NCIMB 13064 diffracted to ultrahigh resolution: crystallization and preliminary diffraction analysis

    International Nuclear Information System (INIS)

    Stsiapanava, Alena; Koudelakova, Tana; Lapkouski, Mikalai; Pavlova, Martina; Damborsky, Jiri; Kuta Smatanova, Ivana

    2008-01-01

    Three mutants of the haloalkane dehalogenase DhaA derived from R. rhodochrous NCIMB 13064 were crystallized and diffracted to ultrahigh resolution. The enzyme DhaA from Rhodococcus rhodochrous NCIMB 13064 belongs to the haloalkane dehalogenases, which catalyze the hydrolysis of haloalkanes to the corresponding alcohols. The haloalkane dehalogenase DhaA and its variants can be used to detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Three mutants named DhaA04, DhaA14 and DhaA15 were constructed in order to study the importance of tunnels connecting the buried active site with the surrounding solvent to the enzymatic activity. All protein mutants were crystallized using the sitting-drop vapour-diffusion method. The crystals of DhaA04 belonged to the orthorhombic space group P2 1 2 1 2 1 , while the crystals of the other two mutants DhaA14 and DhaA15 belonged to the triclinic space group P1. Native data sets were collected for the DhaA04, DhaA14 and DhaA15 mutants at beamline X11 of EMBL, DESY, Hamburg to the high resolutions of 1.30, 0.95 and 1.15 Å, respectively

  3. ¹H, ¹³C, and ¹⁵N backbone resonance assignments of the L124D mutant of StAR-related lipid transfer domain protein 4 (StARD4).

    Science.gov (United States)

    Dikiy, Igor; Ramlall, Trudy F; Eliezer, David

    2013-10-01

    Protein-mediated cholesterol trafficking is central to maintaining cholesterol homeostasis in cells. START (Steroidogenic acute regulatory protein-related lipid transfer) domains constitute a sterol and lipid binding motif and the START domain protein StARD4 typifies a small family of mammalian sterol transport proteins. StARD4 consists of a single START domain and has been reported to act as a general cholesterol transporter in cells. However, the structural basis of cholesterol uptake and transport is not well understood and no cholesterol-bound START domain structures have been reported. We have undertaken the study of cholesterol binding and transport by StARD4 using solution state NMR spectroscopy. To this end, we report nearly complete (1)H, (15)N, and (13)C backbone resonance assignments of an inactive but well behaved mutant (L124D) of StARD4.

  4. 1H, 13C, and 15N backbone resonance assignments of the L124D mutant of StAR-related lipid transfer domain protein 4 (StARD4)

    Science.gov (United States)

    Dikiy, Igor; Ramlall, Trudy F.; Eliezer, David

    2012-01-01

    Protein-mediated cholesterol trafficking is central to maintaining cholesterol homeostasis in cells. START (Steroidogenic acute regulatory protein-related lipid transfer) domains constitute a sterol and lipid binding motif and the START domain protein StARD4 typifies a small family of mammalian sterol transport proteins. StARD4 consists of a single START domain and has been reported to act as a general cholesterol transporter in cells. However, the structural basis of cholesterol uptake and transport is not well understood and no cholesterol-bound START domain structures have been reported. We have undertaken the study of cholesterol binding and transport by StARD4 using solution state NMR spectroscopy. To this end, we report nearly complete 1H, 15N, and 13C backbone resonance assignments of an inactive but well behaved mutant (L124D) of StARD4. PMID:22918595

  5. Enzymatic synthesis of vanillin

    NARCIS (Netherlands)

    van den Heuvel, RHH; Fraaije, MW; Laane, C; van Berkel, WJH; Heuvel, Robert H.H. van den; Berkel, Willem J.H. van

    Due to increasing interest in natural vanillin, two enzymatic routes for the synthesis of vanillin were developed. The flavoprotein vanillyl alcohol oxidase (VAO) acts on a wide range of phenolic compounds and converts both creosol and vanillylamine to vanillin with high yield. The VAO-mediated

  6. Enzymatic synthesis of vanillin

    NARCIS (Netherlands)

    Heuvel, van den R.H.H.; Fraaije, M.W.; Laane, C.; Berkel, van W.J.H.

    2001-01-01

    Due to increasing interest in natural vanillin, two enzymatic routes for the synthesis of vanillin were developed. The flavoprotein vanillyl alcohol oxidase (VAO) acts on a wide range of phenolic compounds and converts both creosol and vanillylamine to vanillin with high yield. The VAO-mediated

  7. Neurotoxicity and other pharmacological activities of the snake venom phospholipase A2 OS2: The N-terminal region is more important than enzymatic activity

    Science.gov (United States)

    Rouault, Morgane; Rash, Lachlan D.; Escoubas, Pierre; Boilard, Eric; Bollinger, James; Lomonte, Bruno; Maurin, Thomas; Guillaume, Carole; Canaan, Stéphane; Deregnaucourt, Christiane; Schrével, Joseph; Doglio, Alain; Gutiérrez, José María; Lazdunski, Michel; Gelb, Michael H.; Lambeau, Gérard

    2009-01-01

    Several snake venom secreted phospholipases A2 (sPLA2s) including OS2 exert a variety of pharmacological effects ranging from central neurotoxicity to anti-HIV activity by mechanisms that are not yet fully understood. To conclusively address the role of enzymatic activity and map the key structural elements of OS2 responsible for its pharmacological properties, we have prepared single point OS2 mutants at the catalytic site and large chimeras between OS2 and OS1, an homologous but non toxic sPLA2. Most importantly, we found that the enzymatic activity of the active site mutant H48Q is 500-fold lower than that of the wild-type protein, while central neurotoxicity is only 16-fold lower, providing convincing evidence that catalytic activity is at most a minor factor that determines central neurotoxicity. The chimera approach has identified the N-terminal region (residues 1–22) of OS2, but not the central one (residues 58–89), as crucial for both enzymatic activity and pharmacological effects. The C-terminal region of OS2 (residues 102–119) was found to be critical for enzymatic activity, but not for central neurotoxicity and anti-HIV activity, allowing us to further dissociate enzymatic activity and pharmacological effects. Finally, direct binding studies with the C-terminal chimera which poorly binds to phospholipids while it is still neurotoxic, led to the identification of a subset of brain N-type receptors which may be directly involved in central neurotoxicity. PMID:16669624

  8. Brucella abortus mutants lacking ATP-binding cassette transporter proteins are highly attenuated in virulence and confer protective immunity against virulent B. abortus challenge in BALB/c mice.

    Science.gov (United States)

    Truong, Quang Lam; Cho, Youngjae; Park, Soyeon; Park, Bo-Kyoung; Hahn, Tae-Wook

    2016-06-01

    Brucella abortus RB51 is an attenuated vaccine strain that has been most frequently used for bovine brucellosis. Although it is known to provide good protection in cattle, it still has some drawbacks including resistance to rifampicin, residual virulence and pathogenicity in humans. Thus, there has been a continuous interest on new safe and effective bovine vaccine candidates. In the present study, we have constructed unmarked mutants by deleting singly cydD and cydC genes, which encode ATP-binding cassette transporter proteins, from the chromosome of the virulent Brucella abortus isolate from Korean cow (referred to as IVK15). Both IVK15ΔcydD and ΔcydC mutants showed increased sensitivity to metal ions, hydrogen peroxide and acidic pH, which are mimic to intracellular environment during host infection. Additionally, the mutants exhibited a significant growth defect in RAW264.7 cells and greatly attenuated in mice. Vaccination of mice with either IVK15ΔcydC or IVK15ΔcydD mutant could elicit an anti-Brucella specific immunoglobulin G (IgG) and IgG subclass responses as well as enhance the secretion of interferon-gamma, and provided better protection against challenge with B. abortus strain 2308 than with the commercial B. abortus strain RB51 vaccine. Collectively, these results suggest that both IVK15ΔcydC and IVK15ΔcydD mutants could be an attenuated vaccine candidate against B. abortus. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Biochemical and structural analysis of 14 mutant adsl enzyme complexes and correlation to phenotypic heterogeneity of adenylosuccinate lyase deficiency.

    Science.gov (United States)

    Zikanova, Marie; Skopova, Vaclava; Hnizda, Ales; Krijt, Jakub; Kmoch, Stanislav

    2010-04-01

    Adenylosuccinate lyase (ADSL) deficiency is neurometabolic disease characterized by accumulation of dephosphorylated enzyme substrates SAICA-riboside (SAICAr) and succinyladenosine (S-Ado) in body fluids of affected individuals. The phenotypic severity differs considerably among patients: neonatal fatal, severe childhood, and moderate phenotypic forms correlating with different values for the ratio between S-Ado and SAICAr concentrations in cerebrospinal fluid have been distinguished. To reveal the biochemical and structural basis for this phenotypic heterogeneity, we expressed and characterized 19 ADSL mutant proteins identified in 16 patients representing clinically distinct subgroups. Respecting compound heterozygosity and considering the homotetrameric structure of ADSL, we used intersubunit complementation and prepared and characterized genotype-specific heteromeric mutant ADSL complexes. We correlated clinical phenotypes with biochemical properties of the mutant proteins and predicted structural impacts of the mutations. We found that phenotypic severity in ADSL deficiency is correlated with residual enzymatic activity and structural stability of the corresponding mutant ADSL complexes and does not seem to result from genotype-specific disproportional catalytic activities toward one of the enzyme substrates. This suggests that the S-Ado/SAICAr ratio is probably not predictive of phenotype severity; rather, it may be secondary to the degree of the patient's development (i.e., to the age of the patient at the time of sample collection). (c) 2010 Wiley-Liss, Inc.

  10. Bypassing Iron Storage in Endodermal Vacuoles Rescues the Iron Mobilization Defect in the natural resistance associated-macrophage protein3natural resistance associated-macrophage protein4 Double Mutant.

    Science.gov (United States)

    Mary, Viviane; Schnell Ramos, Magali; Gillet, Cynthia; Socha, Amanda L; Giraudat, Jérôme; Agorio, Astrid; Merlot, Sylvain; Clairet, Colin; Kim, Sun A; Punshon, Tracy; Guerinot, Mary Lou; Thomine, Sébastien

    2015-09-01

    To improve seed iron (Fe) content and bioavailability, it is crucial to decipher the mechanisms that control Fe storage during seed development. In Arabidopsis (Arabidopsis thaliana) seeds, most Fe is concentrated in insoluble precipitates, with phytate in the vacuoles of cells surrounding the vasculature of the embryo. NATURAL RESISTANCE ASSOCIATED-MACROPHAGE PROTEIN3 (AtNRAMP3) and AtNRAMP4 function redundantly in Fe retrieval from vacuoles during germination. When germinated under Fe-deficient conditions, development of the nramp3nramp4 double mutant is arrested as a consequence of impaired Fe mobilization. To identify novel genes involved in seed Fe homeostasis, we screened an ethyl methanesulfonate-mutagenized population of nramp3nramp4 seedlings for mutations suppressing their phenotypes on low Fe. Here, we report that, among the suppressors, two independent mutations in the VACUOLAR IRON TRANSPORTER1 (AtVIT1) gene caused the suppressor phenotype. The AtVIT1 transporter is involved in Fe influx into vacuoles of endodermal and bundle sheath cells. This result establishes a functional link between Fe loading in vacuoles by AtVIT1 and its remobilization by AtNRAMP3 and AtNRAMP4. Moreover, analysis of subcellular Fe localization indicates that simultaneous disruption of AtVIT1, AtNRAMP3, and AtNRAMP4 limits Fe accumulation in vacuolar globoids. © 2015 American Society of Plant Biologists. All Rights Reserved.

  11. A novel screening method for cell wall mutants in Aspergillus niger identifies UDP-galactopyranose mutase as an important protein in fungal cell wall biosynthesis

    NARCIS (Netherlands)

    Damveld, R.A.; Franken, A.; Arentshorst, M.; Punt, P.J.; Klis, F.M.; Hondel, C.A.M.J.J. van den; Ram, A.F.J.

    2008-01-01

    To identify cell wall biosynthetic genes in filamentous fungi and thus potential targets for the discovery of new antifungals, we developed a novel screening method for cell wall mutants. It is based on our earlier observation that the Aspergillus niger agsA gene, which encodes a putative a-glucan

  12. Chaperone protein HYPK interacts with the first 17 amino acid region of Huntingtin and modulates mutant HTT-mediated aggregation and cytotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Choudhury, Kamalika Roy [Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064 (India); Centre for Neuroscience, Indian Institute of Science, Bangalore 560012 (India); Bhattacharyya, Nitai P., E-mail: nitai_sinp@yahoo.com [Biomedical Genomics Centre, PG Polyclinic Building, 5, Suburbun Hospital Road, Kolkata 700020 (India)

    2015-01-02

    Highlights: • HYPK reduces mutant HTT-mediated aggregate formation and cytotoxicity. • Interaction of HYPK with HTT requires N-terminal 17 amino acid of HTT (HTT-N17). • Deletion of HTT-N17 leads to SDS-soluble, smaller, nuclear aggregates. • These smaller aggregates do not associate with HYPK and are more cytotoxic. • Maybe, interaction of HYPK with amphipathic HTT-N17 block HTT aggregate formation. - Abstract: Huntington’s disease is a polyglutamine expansion disorder, characterized by mutant HTT-mediated aggregate formation and cytotoxicity. Many reports suggests roles of N-terminal 17 amino acid domain of HTT (HTT-N17) towards subcellular localization, aggregate formation and subsequent pathogenicity induced by N-terminal HTT harboring polyQ stretch in pathogenic range. HYPK is a HTT-interacting chaperone which can reduce N-terminal mutant HTT-mediated aggregate formation and cytotoxicity in neuronal cell lines. However, how HYPK interacts with N-terminal fragment of HTT remained unknown. Here we report that specific interaction of HYPK with HTT-N17 is crucial for the chaperone activity of HYPK. Deletion of HTT-N17 leads to formation of tinier, SDS-soluble nuclear aggregates formed by N-terminal mutant HTT. The increased cytotoxicity imparted by these tiny aggregates might be contributed due to loss of interaction with HYPK.

  13. The Hansenula polymorpha per6 mutant is affected in two adjacent genes which encode dihydroxyacetone kinase and a novel protein, Pak1p, involved in peroxisome integrity

    NARCIS (Netherlands)

    Klei, Ida J. van der; Heide, Meis van der; Baerends, Richard J.S.; Rechinger, Karl-Björn; Nicolay, Klaas; Kiel, Jan A.K.W.; Veenhuis, Marten

    The Hansenula polymorpha per6-210 mutant is impaired in respect of growth on methanol (Mut–) and is characterized by aberrant peroxisome formation. The functionally complementing DNA fragment contains two open reading frames. The first encodes dihydroxyacetone kinase (DAK), a cytosolic enzyme

  14. Analyzing the 3D Structure of Human Carbonic Anhydrase II and Its Mutants Using Deep View and the Protein Data Bank

    Science.gov (United States)

    Ship, Noam J.; Zamble, Deborah B.

    2005-01-01

    The self directed study of a 3D image of a biomolecule stresses the complex nature of the intra- and intermolecular interactions that come together to define its structure. This is made up of a series of in vitro experiments with a wild-type and mutants forms of human carbonic anhydrase II (hCAII) that examine the structure function relationship…

  15. Evaluation of the Expression of Amyloid Precursor Protein and the Ratio of Secreted Amyloid Beta 42 to Amyloid Beta 40 in SH-SY5Y Cells Stably Transfected with Wild-Type, Single-Mutant and Double-Mutant Forms of the APP Gene for the Study of Alzheimer's Disease Pathology.

    Science.gov (United States)

    Pahrudin Arrozi, Aslina; Shukri, Siti Nur Syazwani; Wan Ngah, Wan Zurinah; Mohd Yusof, Yasmin Anum; Ahmad Damanhuri, Mohd Hanafi; Makpol, Suzana

    2017-11-01

    Neuroblastoma cell lines such as SH-SY5Y are the most frequently utilized models in neurodegenerative research, and their use has advanced the understanding of the pathology of neurodegeneration over the past few decades. In Alzheimer's disease (AD), several pathogenic mutations have been described, all of which cause elevated levels of pathological hallmarks such as amyloid-beta (Aβ). Although the genetics of Alzheimer's disease is well known, familial AD only accounts for a small number of cases in the population, with the rest being sporadic AD, which contains no known mutations. Currently, most of the in vitro models used to study AD pathogenesis only examine the level of Aβ42 as a confirmation of successful model generation and only perform comparisons between wild-type APP and single mutants of the APP gene. Recent findings have shown that the Aβ42/40 ratio in cerebrospinal fluid (CSF) is a better diagnostic indicator for AD patients than is Aβ42 alone and that more extensive Aβ formation, such as accumulation of intraneuronal Aβ, Aβ plaques, soluble oligomeric Aβ (oAβ), and insoluble fibrillar Aβ (fAβ) occurs in TgCRND8 mice expressing a double-mutant form (Swedish and Indiana) of APP, later leading to greater progressive impairment of the brain. In this study, we generated SH-SY5Y cells stably transfected separately with wild-type APP, the Swedish mutation of APP, and the Swedish and Indiana mutations of APP and evaluated the APP expression as well as the Aβ42/40 ratio in those cells. The double-mutant form of APP (Swedish/Indiana) expressed markedly high levels of APP protein and showed a high Aβ2/40 ratio compared to wild-type and single-mutant cells.

  16. Novel aggregate formation of a frame-shift mutant protein of tissue-nonspecific alkaline phosphatase is ascribed to three cysteine residues in the C-terminal extension. Retarded secretion and proteasomal degradation.

    Science.gov (United States)

    Komaru, Keiichi; Ishida, Yoko; Amaya, Yoshihiro; Goseki-Sone, Masae; Orimo, Hideo; Oda, Kimimitsu

    2005-04-01

    In the majority of hypophosphatasia patients, reductions in the serum levels of alkaline phosphatase activity are caused by various missense mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. A unique frame-shift mutation due to a deletion of T at cDNA number 1559 [TNSALP (1559delT)] has been reported only in Japanese patients with high allele frequency. In this study, we examined the molecular phenotype of TNSALP (1559delT) using in vitro translation/translocation system and COS-1 cells transiently expressing this mutant protein. We showed that the mutant protein not only has a larger molecular size than the wild type enzyme by approximately 12 kDa, reflecting an 80 amino acid-long extension at its C-terminus, but that it also lacks a glycosylphosphatidylinositol anchor. In support of this, alkaline phosphatase activity of the cells expressing TNSALP (1559delT) was localized at the juxtanucleus position, but not on the cell surface. However, only a limited amount of the newly synthesized protein was released into the medium and the rest was polyubiquitinated, followed by degradation in the proteasome. SDS/PAGE and analysis by sucrose-density-gradient analysis indicated that TNSALP (1559delT) forms a disulfide-bonded high-molecular-mass aggregate. Interestingly, the aggregate form of TNSALP (1559delT) exhibited a significant enzyme activity. When all three cysteines at positions of 506, 521 and 577 of TNSALP (1559delT) were replaced with serines, the aggregation disappeared and instead this modified mutant protein formed a noncovalently associated dimer, strongly indicating that these cysteine residues in the C-terminal region are solely responsible for aggregate formation by cross-linking the catalytically active dimers. Thus, complete absence of TNSALP on cell surfaces provides a plausible explanation for a severe lethal phenotype of a homozygote hypophosphatasia patient carrying TNSALP (1559delT).

  17. Enzymatic desulfurization of coal

    Energy Technology Data Exchange (ETDEWEB)

    Boyer, Y.N.; Crooker, S.C.; Kitchell, J.P.; Nochur, S.V.

    1991-05-16

    The overall objective of this program was to investigate the feasibility of an enzymatic desulfurization process specifically intended for organic sulfur removal from coal. Toward that end, a series of specific objectives were defined: (1) establish the feasibility of (bio)oxidative pretreatment followed by biochemical sulfate cleavage for representative sulfur-containing model compounds and coals using commercially-available enzymes; (2) investigate the potential for the isolation and selective use of enzyme preparations from coal-utilizing microbial systems for desulfurization of sulfur-containing model compounds and coals; and (3) develop a conceptual design and economic analysis of a process for enzymatic removal of organic sulfur from coal. Within the scope of this program, it was proposed to carry out a portion of each of these efforts concurrently. (VC)

  18. Enzymatic glycosylation of lincomycin

    Czech Academy of Sciences Publication Activity Database

    Weignerová, Lenka; Spížek, Jaroslav; Najmanová, Lucie; Křen, Vladimír

    2001-01-01

    Roč. 65, č. 8 (2001), s. 1897-1899 ISSN 0916-8451 R&D Projects: GA ČR GA303/99/1382; GA ČR GA203/01/1018; GA MPO PZ-Z2/23 Institutional research plan: CEZ:AV0Z5020903 Keywords : lincomycin * enzymatic glycosylation * monnosylation Subject RIV: EE - Microbiology, Virology Impact factor: 0.968, year: 2001

  19. PENICILLIN-BINDING PROTEIN 2X OF STREPTOCOCCUS-PNEUMONIAE - EXPRESSION IN ESCHERICHIA-COLI AND PURIFICATION OF A SOLUBLE ENZYMATICALLY ACTIVE DERIVATIVE

    NARCIS (Netherlands)

    LAIBLE, G; KECK, W; LURZ, R; MOTTL, H; FRERE, JM; JAMIN, M; HAKENBECK, R

    1992-01-01

    A 2.5-kb DNA fragment including the structural gene coding for the penicillin-binding protein 2x (PBP 2x) of Streptococcus pneumoniae has been cloned into the vector pJDC9 and expressed in Escherichia coli. Mapping of RNA polymerase binding sites by electron microscopy indicated that the pbpX

  20. Examination of the ligand-binding and enzymatic properties of a bilin-binding protein from the poisonous caterpillar Lonomia obliqua.

    Directory of Open Access Journals (Sweden)

    Ana B G Veiga

    Full Text Available The bilin-binding proteins (BBP from lepidopteran insects are members of the lipocalin family of proteins and play a special role in pigmentation through the binding of biliverdin IXγ. Lopap, a BBP-like protein from the venom of the toxic caterpillar Lonomia obliqua has been reported to act as a serine protease that activates the coagulation proenzyme prothrombin. Here we show that BBPLo, a variant of lopap from the same organism binds biliverdin IXγ, forming a complex that is spectrally identical with previously described BBP proteins. Although BBPLo is nearly identical in sequence to lopap, no prothrombinase activity was detected in our recombinant preparations using reconstituted systems containing coagulation factors Xa and Va, as well as anionic phospholipids. In addition to biliverdin, BBPLo was found to form a 1:1 complex with heme prompting us to examine whether the unusual biliverdin IXγ ligand of BBPs forms as a result of oxidation of bound heme in situ rather than by a conventional heme oxygenase. Using ascorbate or a NADPH(+-ferredoxin reductase-ferredoxin system as a source of reducing equivalents, spectral changes are seen that suggest an initial reduction of heme to the Fe(II state and formation of an oxyferrous complex. The complex then disappears and a product identified as a 5-coordinate carbonyl complex of verdoheme, an intermediate in the biosynthesis of biliverdin, is formed. However, further reaction to form biliverdin was not observed, making it unlikely that biliverdin IXγ is formed by this pathway.

  1. Sequence-Independent Cloning and Post-Translational Modification of Repetitive Protein Polymers through Sortase and Sfp-Mediated Enzymatic Ligation.

    Science.gov (United States)

    Ott, Wolfgang; Nicolaus, Thomas; Gaub, Hermann E; Nash, Michael A

    2016-04-11

    Repetitive protein-based polymers are important for many applications in biotechnology and biomaterials development. Here we describe the sequential additive ligation of highly repetitive DNA sequences, their assembly into genes encoding protein-polymers with precisely tunable lengths and compositions, and their end-specific post-translational modification with organic dyes and fluorescent protein domains. Our new Golden Gate-based cloning approach relies on incorporation of only type IIS BsaI restriction enzyme recognition sites using PCR, which allowed us to install ybbR-peptide tags, Sortase c-tags, and cysteine residues onto either end of the repetitive gene polymers without leaving residual cloning scars. The assembled genes were expressed in Escherichia coli and purified using inverse transition cycling (ITC). Characterization by cloud point spectrophotometry, and denaturing polyacrylamide gel electrophoresis with fluorescence detection confirmed successful phosphopantetheinyl transferase (Sfp)-mediated post-translational N-terminal labeling of the protein-polymers with a coenzyme A-647 dye (CoA-647) and simultaneous sortase-mediated C-terminal labeling with a GFP domain containing an N-terminal GG-motif in a one-pot reaction. In a further demonstration, we installed an N-terminal cysteine residue into an elastin-like polypeptide (ELP) that was subsequently conjugated to a single chain poly(ethylene glycol)-maleimide (PEG-maleimide) synthetic polymer, noticeably shifting the ELP cloud point. The ability to straightforwardly assemble repetitive DNA sequences encoding ELPs of precisely tunable length and to post-translationally modify them specifically at the N- and C- termini provides a versatile platform for the design and production of multifunctional smart protein-polymeric materials.

  2. [Anti-HBV effects of genetically engineered replication-defective HBV with combined expression of antisense RNA and dominant negative mutants of core protein and construction of first-generation packaging cell line for HBV vector].

    Science.gov (United States)

    Sun, Dian Xing; Hu, Da Rong; Wu, Guang Hui; Hu, Xue Ling; Li, Juan; Fan, Gong Ren

    2002-08-01

    To explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular combined expression of antisense RNA and dominant negative mutants of core protein. Full length of mutant HBV genome, which expresses core-partial P fusion protein and/or antisense RNA, was transfected into HepG2.2.15 cell lines. Positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA method. Intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR. Free of packaging signal, HBV genome, which express the HBV structural proteins including core, pol and preS/S proteins, was inserted into pCI-neo vector. HepG2 cell lines were employed to transfect with the construct. G418 selection was done at the concentration of 400mug/ml in the culture medium. The G418-resistant clones with the best expression of HBsAg and HBcAg were theoretically considered as packaging cell lines and propagated under the same conditions. It was transfected with plasmid pMEP-CPAS and then selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR. The mean inhibitory rates of HBsAg were 2.74% 3.83%, 40.08 2.05% (t=35.5, PDNA were 0, 82.0%, 59.9%, and 96.6%, respectively. Recombinant HB virion was detectable in the culture medium of all the three treatment groups. G418-resistant HBV packaging cell line, which harbored an HBV mutant whose packaging signal had been deleted, was generated. Expression of HBsAg and HBcAg was detectable. Transfected with plasmid pMEP-CPAS, it was found to secrete recombinant HB virion and no wild-type HBV was detectable in the culture medium. It has stronger anti-HBV effects by combined expression of antisense RNA and dominant negative mutants than by individual

  3. Expression of a Mutant SEMA3A Protein with Diminished Signalling Capacity Does Not Alter ALS-Related Motor Decline, or Confer Changes in NMJ Plasticity after BotoxA-Induced Paralysis of Male Gastrocnemic Muscle.

    Directory of Open Access Journals (Sweden)

    Elizabeth B Moloney

    Full Text Available Terminal Schwann cells (TSCs are specialized cells that envelop the motor nerve terminal, and play a role in the maintenance and regeneration of neuromuscular junctions (NMJs. The chemorepulsive protein semaphorin 3A (SEMA3A is selectively up-regulated in TSCs on fast-fatigable muscle fibers following experimental denervation of the muscle (BotoxA-induced paralysis or crush injury to the sciatic nerve or in the motor neuron disease amyotrophic lateral sclerosis (ALS. Re-expression of SEMA3A in this subset of TSCs is thought to play a role in the selective plasticity of nerve terminals as observed in ALS and following BotoxA-induced paralysis. Using a mouse model expressing a mutant SEMA3A with diminished signaling capacity, we studied the influence of SEMA3A signaling at the NMJ with two denervation paradigms; a motor neuron disease model (the G93A-hSOD1 ALS mouse line and an injury model (BotoxA-induced paralysis. ALS mice that either expressed 1 or 2 mutant SEMA3A alleles demonstrated no difference in ALS-induced decline in motor behavior. We also investigated the effects of BotoxA-induced paralysis on the sprouting capacity of NMJs in the K108N-SEMA3A mutant mouse, and observed no change in the differential neuronal plasticity found at NMJs on fast-fatigable or slow muscle fibers due to the presence of the SEMA3A mutant protein. Our data may be explained by the residual repulsive activity of the mutant SEMA3A, or it may imply that SEMA3A alone is not a key component of the molecular signature affecting NMJ plasticity in ALS or BotoxA-induced paralysis. Interestingly, we did observe a sex difference in motor neuron sprouting behavior after BotoxA-induced paralysis in WT mice which we speculate may be an important factor in the sex dimorphic differences seen in ALS.

  4. Utilization of baker's yeast (Saccharomyces cerevisiae for the production of yeast extract: effects of different enzymatic treatments on solid, protein and carbohydrate recovery

    Directory of Open Access Journals (Sweden)

    TATJANA VUKASINOVIC MILIC

    2007-05-01

    Full Text Available Yeast extract (YE was produced from commercial pressed baker's yeast (active and inactivated using two enzymes: papain and lyticase. The effects of enzyme concentration and hydrolysis time on the recovery of solid, protein and carbohydrate were investigated. Autolysis, as a basic method for cell lysis was also used and the results compared. The optimal extraction conditions were investigated. The optimal concentrations of papain and lyticase were found to be 2.5 % and 0.025 %, respectively.

  5. Evanescent wave-initiated photopolymerisation as a new way to create monolithic open-tubular capillary columns: use as enzymatic microreactor for on-line protein digestion

    Czech Academy of Sciences Publication Activity Database

    Abele, S.; Smejkal, Petr; Yavorska, O.; Foret, František; Macka, M.

    2010-01-01

    Roč. 135, č. 3 (2010), s. 477-481 ISSN 0003-2654 R&D Projects: GA MŠk LC06023; GA AV ČR KAN400310651 Institutional research plan: CEZ:AV0Z40310501 Keywords : photopolymerisation * light-emitting -diode * on-line protein digestion Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.913, year: 2010

  6. The properties and kinetics of enzymatic reaction in the process of the enzymatic extraction of fish oil.

    Science.gov (United States)

    Qian, Junqing; Zhang, Hongyong; Liao, Qiyuan

    2011-06-01

    The technology of enzymatic extraction of fish oil has many advantages, such as moderate operating conditions, lower energy consumption and high efficiency. Moreover, it could reduce the breakage for the functional component of fish oil. In enzymatic extraction of oil, the investigation of the property of enzymatic reaction is propitious to improve the enzymolysis efficiency. In this study, the 1398 neutrase was used for hydrolyzing fish protein, then analyzed the impacts to the enzymolysis efficiency which were induced by the different initial substrate concentration and different initial enzyme concentration, the result showed that the higher substrate concentration generate inhibition to the enzyme. And then, the properties of enzymatic hydrolysis were studied by the Michaelis-Menten equation of substrate inhibition and the enzymatic hydrolysis kinetics equation which is derived by theory. By means of the verification to the enzymatic hydrolysis kinetics model, it could see that the model in line with the actual situation better at a lower degree of hydrolysis. Lastly, the critical enzyme concentration and critical substrate concentration of enzymatic reaction could be obtained by deducing the enzymatic hydrolysis kinetics model.

  7. Mutant IDH inhibits HNF-4α to block hepatocyte differentiation and promote biliary cancer.

    Science.gov (United States)

    Saha, Supriya K; Parachoniak, Christine A; Ghanta, Krishna S; Fitamant, Julien; Ross, Kenneth N; Najem, Mortada S; Gurumurthy, Sushma; Akbay, Esra A; Sia, Daniela; Cornella, Helena; Miltiadous, Oriana; Walesky, Chad; Deshpande, Vikram; Zhu, Andrew X; Hezel, Aram F; Yen, Katharine E; Straley, Kimberly S; Travins, Jeremy; Popovici-Muller, Janeta; Gliser, Camelia; Ferrone, Cristina R; Apte, Udayan; Llovet, Josep M; Wong, Kwok-Kin; Ramaswamy, Sridhar; Bardeesy, Nabeel

    2014-09-04

    Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly liver cancer. Mutant IDH proteins in IHCC and other malignancies acquire an abnormal enzymatic activity allowing them to convert α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), which inhibits the activity of multiple αKG-dependent dioxygenases, and results in alterations in cell differentiation, survival, and extracellular matrix maturation. However, the molecular pathways by which IDH mutations lead to tumour formation remain unclear. Here we show that mutant IDH blocks liver progenitor cells from undergoing hepatocyte differentiation through the production of 2HG and suppression of HNF-4α, a master regulator of hepatocyte identity and quiescence. Correspondingly, genetically engineered mouse models expressing mutant IDH in the adult liver show an aberrant response to hepatic injury, characterized by HNF-4α silencing, impaired hepatocyte differentiation, and markedly elevated levels of cell proliferation. Moreover, IDH and Kras mutations, genetic alterations that co-exist in a subset of human IHCCs, cooperate to drive the expansion of liver progenitor cells, development of premalignant biliary lesions, and progression to metastatic IHCC. These studies provide a functional link between IDH mutations, hepatic cell fate, and IHCC pathogenesis, and present a novel genetically engineered mouse model of IDH-driven malignancy.

  8. Expression of a high sweetness and heat-resistant mutant of sweet-tasting protein, monellin, in Pichia pastoris with a constitutive GAPDH promoter and modified N-terminus.

    Science.gov (United States)

    Cai, Chenggu; Li, Lei; Lu, Nan; Zheng, Weiwei; Yang, Liu; Liu, Bo

    2016-11-01

    To improve the stability and sweetness of the sweet-tasting protein, monellin, by using site-directed mutagenesis and a Pichia pastoris expression system with a GAPDH constitutive promoter. Both wild-type and E2 N mutant of single-chain monellin gene were cloned into the PGAPZαA vector and expressed in Pichia pastoris. The majority of the secreted recombinant protein, at 0.15 g/l supernatant, was monellin. This was purified by Sephadex G50 chromatography. The sweetness threshold of wild-type and E2 N were 30 μg/ml and 20 μg/ml, respectively. Compared with the proteins expressed in Escherichia coli, the thermostability of both proteins was improved. The N-terminal sequence is determinative for the sweetness of the proteins expressed in yeast strains. Site-directed mutagenesis, modification of the N-terminus of monellin, and without the need of methanol induction in P. pastoris expression system, indicate the possibility for large-scale production of this sweet-tasting protein.

  9. Crystals of DhaA mutants from Rhodococcus rhodochrous NCIMB 13064 diffracted to ultrahigh resolution: crystallization and preliminary diffraction analysis.

    Science.gov (United States)

    Stsiapanava, Alena; Koudelakova, Tana; Lapkouski, Mikalai; Pavlova, Martina; Damborsky, Jiri; Smatanova, Ivana Kuta

    2008-02-01

    The enzyme DhaA from Rhodococcus rhodochrous NCIMB 13064 belongs to the haloalkane dehalogenases, which catalyze the hydrolysis of haloalkanes to the corresponding alcohols. The haloalkane dehalogenase DhaA and its variants can be used to detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Three mutants named DhaA04, DhaA14 and DhaA15 were constructed in order to study the importance of tunnels connecting the buried active site with the surrounding solvent to the enzymatic activity. All protein mutants were crystallized using the sitting-drop vapour-diffusion method. The crystals of DhaA04 belonged to the orthorhombic space group P2(1)2(1)2(1), while the crystals of the other two mutants DhaA14 and DhaA15 belonged to the triclinic space group P1. Native data sets were collected for the DhaA04, DhaA14 and DhaA15 mutants at beamline X11 of EMBL, DESY, Hamburg to the high resolutions of 1.30, 0.95 and 1.15 A, respectively.

  10. Enzymatic cascade bioreactor

    Science.gov (United States)

    Simmons, Blake A.; Volponi, Joanne V.; Ingersoll, David; Walker, Andrew

    2007-09-04

    Disclosed is an apparatus and method for continuously converting sucrose to .beta.-D-glucose. The method comprises a three stage enzymatic reactor in which an aqueous solution of sucrose is first converted into a solution of fructose and .alpha.-D-glucose by passing it through a porous, packed column containing an inert media on which invertase is immobilized. This solution is then sent through a second packed column containing glucose isomerase and finally a third packed column containing mutarotase. Solution temperature and pH are adjusted to maximize glucose output.

  11. Separation of foot-and-mouth disease virus leader protein activities; identification of mutants that retain efficient self-processing activity but poorly induce eIF4G cleavage

    DEFF Research Database (Denmark)

    Belsham, Graham; Hua Guan, Su

    2017-01-01

    amino acid residues in the functions of this protein. These selected residues are either highly conserved or are charged and exposed on the protein surface. Using transient expression assays within BHK cells, it was found that residues around the active site (W52, L53 and A149) of Lpro and others...... located elsewhere (K38, K39, R44, H138 and W159) are involved in the induction of eIF4G cleavage but not in the processing of the L/P1 junction. Modified viruses, encoding such amino acid substitutions within Lpro can replicate in BHK cells but did not grow well in primary bovine thyroid cells. This study...... characterizes mutant viruses that are deficient in blocking host cell responses to infection (e.g. interferon induction) and can assist in the rational design of antiviral agents targeting this process and in the production of attenuated viruses....

  12. An integrated mechanical-enzymatic reverse osmosis treatment of dairy industry wastewater and milk protein recovery as a fat replacer: a closed loop approach

    Directory of Open Access Journals (Sweden)

    F. Sarghini

    2013-09-01

    Full Text Available The dairy industry can be classified among the most polluting of the food industries in volume in regard to its large water consumption, generating from 0.2 to 10 L of effluent per liter of processed milk. Dairy industry effluents usually include highly dissolved organic matter with varying characteristics, and a correct waste management project is required to handle. In a framework of natural water resource availability and cost increase, wastewater treatment for water reuse can lower the overall water consumption and the global effluent volume of industrial plants. Moreover, correct dismissal of dairy industry wastewater is sometimes neglected by the operators , increasing the environmental impact due to the chemical and biological characteristics of such effluents. On the other hand, in the case of whey effluents, several by-products are still present inside, such as lactose and milk proteins. Membrane technology has some advantages including a high degree of reliability in removing dissolved, colloidal and particulate matter, like the selectivity in size of pollutants to be removed and the possibility of very compact treatment plants. For example, Reverse Osmosis (RO technology has been successfully applied for the treatment of dairy wastes (1, and as a technology for concentration and fractionation of whey. In this work a membrane treatment approach using reverse osmosis technology is investigated and implemented: the permeate obtained can be reused as clean warm water for cleaning and sanitation of production plants, while concentrated milk proteins are modified by using transglutaminase enzyme obtaining a high temperature resistant fat replacer to be used in different low-fat products like for example mozzarella cheese.

  13. Promising rice mutants

    International Nuclear Information System (INIS)

    Hakim, L.; Azam, M.A.; Miah, A.J.; Mansur, M.A.; Akanda, H.R.

    1988-01-01

    Two induced mutants namely, Mut NS 1 (tall) and Mut NS 5 (semi-dwarf) derived from rice variety Nizersail were evaluated for various agronomic characters at four locations in Bangladesh. Both the mutants matured about three weeks earlier and yielded significantly higher than the parent variety Nizersail. (author). 3 tabs., 9 refs

  14. Two and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins

    Energy Technology Data Exchange (ETDEWEB)

    Suhadolnik, R.J.; Kariko, K.; Sobol, R.W. Jr.; Li, S.W.; Reichenbach, N.L.; Haley, B.E.

    1988-11-29

    The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymatically synthesized from (..gamma..-/sup 32/P)2-azidoATP and (..cap alpha..-/sup 32/P)8-azidoAPT by 2-5A synthetase from rabbit reticulocyte lysates. Identification and structural determination of the 2- and 8-azido adenylate trimer 5'-triphosphates were accomplished by enzymatic hydrolyses with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase. Hydrolysis products were identified by HPLC and PEI-cellulose TLC analyses. The 8-azido photoprobe of 2-5A displaces p/sub 3/A/sub 4/(/sup 32/P)pCp from RNase L with affinity equivalent to p/sub 3/A/sub 3/. The 8-azido photoprobe also activates RNase L to hydrolyze poly(U)(/sup 32/P)pCp 50% at 7 /times/ 10/sup /minus/9/ M in core-cellulose assays. The 2- and 8-azido photoprobes and authentic p/sub 3/A/sub 3/ activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10/sup /minus/9/ M in rRNA cleavage assays. The nucleotide binding site(s) of RNase L and/or other 2-5A binding proteins in extracts of interferon-treated L929 cells were investigated by photoaffinity labeling. Dramatically different photolabeling patterns were observed with the 2- and 8-azido photoprobes. The (..gamma..-/sup 32/P)2-azido adenylate trimer 5'-triphosphate photolabels only one polypeptide with a molecular weight of 185,000 as determined by SDS gel electrophoresis, whereas the (..cap alpha..-/sup 32/P)8-azido adenylate trimer 5'-triphosphate covalently photolabels six polypeptides with molecular weights of 46,000, 63,000, 80,000, 89,000, 109,000, and 158,000. Evidence that the photolabeling by 2- and 8-azido 2-5A photoprobes was highly specific for the p/sub 3/A/sub 3/ allosteric binding site was obtained.

  15. Microarray analysis of rice d1 (RGA1 mutant reveals the potential role of G-protein alpha subunit in regulating multiple abiotic stresses such as drought, salinity, heat and cold

    Directory of Open Access Journals (Sweden)

    Annie Prasanna Jangam

    2016-01-01

    Full Text Available The genome-wide role of heterotrimeric G-proteins in abiotic stress response in rice has not been examined from a functional genomics perspective, despite the availability of mutants and evidences involving individual genes/processes/stresses. Our rice whole transcriptome microarray analysis (GSE 20925 at NCBI GEO using the G-alpha subunit (RGA1 null mutant (Daikoku 1 or d1 and its corresponding wild type (O. sativa Japonica Nipponbare identified 2270 unique differentially expressed genes (DEGs. Out of them, we mined for all the potentially abiotic stress-responsive genes using Gene Ontology terms, STIFDB2.0 and Rice DB. The first two approaches produced smaller subsets of the 1886 genes found at Rice DB. The GO approach revealed similar regulation of several families of stress-responsive genes in RGA1 mutant. The Genevestigator analysis of the stress-responsive subset of the RGA1-regulated genes from STIFDB revealed cold and drought-responsive clusters. Meta data analysis at Rice DB revealed large stress-response categories such as cold (878 up /810 down, drought (882 up /837 down, heat (913 up /777 down, and salt stress (889 up /841 down. 1498 of them are common to all the four abiotic stresses, followed by fewer genes common to smaller groups of stresses. The RGA1-regulated genes that uniquely respond to individual stresses include 111 in heat stress, 8 each in cold only and drought only stresses and 2 genes in salt stress only. The common DEGs (1498 belong to pathways such as the synthesis of polyamine, glycine-betaine, proline and trehalose. Some of the common DEGs belong to abiotic stress signaling pathways such as calcium-dependent pathway, ABA independent and dependent pathway and MAP kinase pathway in the RGA1 mutant. Gene ontology of the common stress responsive DEGs revealed 62 unique molecular functions such as transporters, enzyme regulators, transferases, hydrolases, carbon and protein metabolism, binding to nucleotides

  16. Use of chitin and chitosan to produce new chitooligosaccharides by chitinase Chit42: enzymatic activity and structural basis of protein specificity.

    Science.gov (United States)

    Kidibule, Peter Elias; Santos-Moriano, Paloma; Jiménez-Ortega, Elena; Ramírez-Escudero, Mercedes; Limón, M Carmen; Remacha, Miguel; Plou, Francisco José; Sanz-Aparicio, Julia; Fernández-Lobato, María

    2018-03-22

    Chitinases are ubiquitous enzymes that have gained a recent biotechnological attention due to their ability to transform biological waste from chitin into valued chito-oligomers with wide agricultural, industrial or medical applications. The biological activity of these molecules is related to their size and acetylation degree. Chitinase Chit42 from Trichoderma harzianum hydrolyses chitin oligomers with a minimal of three N-acetyl-D-glucosamine (GlcNAc) units. Gene chit42 was previously characterized, and according to its sequence, the encoded protein included in the structural Glycoside Hydrolase family GH18. Chit42 was expressed in Pichia pastoris using fed-batch fermentation to about 3 g/L. Protein heterologously expressed showed similar biochemical properties to those expressed by the natural producer (42 kDa, optima pH 5.5-6.5 and 30-40 °C). In addition to hydrolyse colloidal chitin, this enzyme released reducing sugars from commercial chitosan of different sizes and acetylation degrees. Chit42 hydrolysed colloidal chitin at least 10-times more efficiently (defined by the k cat /K m ratio) than any of the assayed chitosan. Production of partially acetylated chitooligosaccharides was confirmed in reaction mixtures using HPAEC-PAD chromatography and mass spectrometry. Masses corresponding to (D-glucosamine) 1-8 -GlcNAc were identified from the hydrolysis of different substrates. Crystals from Chit42 were grown and the 3D structure determined at 1.8 Å resolution, showing the expected folding described for other GH18 chitinases, and a characteristic groove shaped substrate-binding site, able to accommodate at least six sugar units. Detailed structural analysis allows depicting the features of the Chit42 specificity, and explains the chemical nature of the partially acetylated molecules obtained from analysed substrates. Chitinase Chit42 was expressed in a heterologous system to levels never before achieved. The enzyme produced small partially acetylated

  17. Mutant heterosis in rice

    International Nuclear Information System (INIS)

    1987-01-01

    In the variety TKM6 a high yielding semidwarf mutant has been induced. This TKM6 mutant was used in test crosses with a number of other varieties and mutants to examine the extent of heterosis of dwarfs in rice and to select superior crosses. An excerpt of the published data is given. It appears from the backcross of the mutant with its original variety, that an increase in number of productive tillers occurs in the hybrid, leading to a striking grain yield increase, while the semi-dwarf culm length (the main mutant character) reverts to the normal phenotype. In the cross with IR8 on the other hand, there is only a minimal increase in tiller number but a substantial increase in TGW leading to more than 30% yield increase over the better parent

  18. Production of Fish Protein Hydrolysates from Scyliorhinus canicula Discards with Antihypertensive and Antioxidant Activities by Enzymatic Hydrolysis and Mathematical Optimization Using Response Surface Methodology

    Directory of Open Access Journals (Sweden)

    José A. Vázquez

    2017-10-01

    Full Text Available Fish discards are of major concern in new EU policies. Alternatives for the management of the new biomass that has to be landed is compulsory. The production of bioactive compounds from fish protein hydrolysates (FPH has been explored in recent years. However, the viability of Scyliorhinus canicula discards, which might account for up to 90–100% of captures in mixed trawler, gillnet, and longline industrial fisheries, to produce FPH from the muscle with bioactivities has still not been studied in terms of the optimization of the experimental conditions to enhance its production. The effect of pH and temperature on the hydrolysis of the S. canicula muscle was mediated by three commercial proteases using response surface methodology. Temperatures of 64.6 °C and 60.8 °C and pHs of 9.40 and 8.90 were established as the best hydrolysis conditions for Alcalase and Esperase, respectively. Optimization of the best conditions for the maximization of antihypertensive and antioxidant activities was performed. Higher Angiotensin-converting enzyme (ACE activity was found with Esperase. The pH optimum and temperature optimum for antioxidants were 55 °C/pH8.0 for ABTS/DPPH-Esperase, 63.1 °C/pH9.0 for DPPH-Alcalase, and 55 °C/pH9.0 for ABTS-Alcalase. No hydrolysis was detected when using Protamex.

  19. Human GLTP and mutant forms of ACD11 suppress cell death in the Arabidopsis acd11 mutant

    DEFF Research Database (Denmark)

    Petersen, Nikolaj H T; McKinney, Lea V; Pike, Helen

    2008-01-01

    The Arabidopsis acd11 mutant exhibits runaway, programmed cell death due to the loss of a putative sphingosine transfer protein (ACD11) with homology to mammalian GLTP. We demonstrate that transgenic expression in Arabidopsis thaliana of human GLTP partially suppressed the phenotype of the acd11...... null mutant, resulting in delayed programmed cell death development and plant survival. Surprisingly, a GLTP mutant form impaired in glycolipid transfer activity also complemented the acd11 mutants. To understand the relationship between functional complementarity and transfer activity, we generated...... site-specific mutants in ACD11 based on homologous GLTP residues required for glycolipid transfer. We show that these ACD11 mutant forms are impaired in their in vitro transfer activity of sphingolipids. However, transgenic expression of these mutant forms fully complemented acd11 mutant cell death...

  20. Liver tumor formation by a mutant retinoblastoma protein in the transgenic mice is caused by an upregulation of c-Myc target genes

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Bo; Hikosaka, Keisuke; Sultana, Nishat; Sharkar, Mohammad Tofael Kabir [Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Noritake, Hidenao [Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Department of Internal Medicine, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Kimura, Wataru; Wu, Yi-Xin [Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Kobayashi, Yoshimasa [Department of Internal Medicine, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Uezato, Tadayoshi [Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan); Miura, Naoyuki, E-mail: nmiura@hama-med.ac.jp [Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192 (Japan)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Fifty percent of the mutant Rb transgenic mice produced liver tumors. Black-Right-Pointing-Pointer In the tumor, Foxm1, Skp2, Bmi1 and AP-1 mRNAs were up-regulated. Black-Right-Pointing-Pointer No increase in expression of the Myc-target genes was observed in the non-tumorous liver. Black-Right-Pointing-Pointer Tumor formation depends on up-regulation of the Myc-target genes. -- Abstract: The retinoblastoma (Rb) tumor suppressor encodes a nuclear phosphoprotein that regulates cellular proliferation, apoptosis and differentiation. In order to adapt itself to these biological functions, Rb is subjected to modification cycle, phosphorylation and dephosphorylation. To directly determine the effect of phosphorylation-resistant Rb on liver development and function, we generated transgenic mice expressing phosphorylation-resistant human mutant Rb (mt-Rb) under the control of the rat hepatocyte nuclear factor-1 gene promoter/enhancer. Expression of mt-Rb in the liver resulted in macroscopic neoplastic nodules (adenomas) with {approx}50% incidence within 15 months old. Interestingly, quantitative reverse transcriptase-PCR analysis showed that c-Myc was up-regulated in the liver of mt-Rb transgenic mice irrespective of having tumor tissues or no tumor. In tumor tissues, several c-Myc target genes, Foxm1, c-Jun, c-Fos, Bmi1 and Skp2, were also up-regulated dramatically. We determined whether mt-Rb activated the Myc promoter in the HTP9 cells and demonstrated that mt-Rb acted as an inhibitor of wild-type Rb-induced repression on the Myc promoter. Our results suggest that continued upregulation of c-Myc target genes promotes the liver tumor formation after about 1 year of age.

  1. Liver tumor formation by a mutant retinoblastoma protein in the transgenic mice is caused by an upregulation of c-Myc target genes

    International Nuclear Information System (INIS)

    Wang, Bo; Hikosaka, Keisuke; Sultana, Nishat; Sharkar, Mohammad Tofael Kabir; Noritake, Hidenao; Kimura, Wataru; Wu, Yi-Xin; Kobayashi, Yoshimasa; Uezato, Tadayoshi; Miura, Naoyuki

    2012-01-01

    Highlights: ► Fifty percent of the mutant Rb transgenic mice produced liver tumors. ► In the tumor, Foxm1, Skp2, Bmi1 and AP-1 mRNAs were up-regulated. ► No increase in expression of the Myc-target genes was observed in the non-tumorous liver. ► Tumor formation depends on up-regulation of the Myc-target genes. -- Abstract: The retinoblastoma (Rb) tumor suppressor encodes a nuclear phosphoprotein that regulates cellular proliferation, apoptosis and differentiation. In order to adapt itself to these biological functions, Rb is subjected to modification cycle, phosphorylation and dephosphorylation. To directly determine the effect of phosphorylation-resistant Rb on liver development and function, we generated transgenic mice expressing phosphorylation-resistant human mutant Rb (mt-Rb) under the control of the rat hepatocyte nuclear factor-1 gene promoter/enhancer. Expression of mt-Rb in the liver resulted in macroscopic neoplastic nodules (adenomas) with ∼50% incidence within 15 months old. Interestingly, quantitative reverse transcriptase-PCR analysis showed that c-Myc was up-regulated in the liver of mt-Rb transgenic mice irrespective of having tumor tissues or no tumor. In tumor tissues, several c-Myc target genes, Foxm1, c-Jun, c-Fos, Bmi1 and Skp2, were also up-regulated dramatically. We determined whether mt-Rb activated the Myc promoter in the HTP9 cells and demonstrated that mt-Rb acted as an inhibitor of wild-type Rb-induced repression on the Myc promoter. Our results suggest that continued upregulation of c-Myc target genes promotes the liver tumor formation after about 1 year of age.

  2. PLD2 has both enzymatic and cell proliferation-inducing capabilities, that are differentially regulated by phosphorylation and dephosphorylation

    International Nuclear Information System (INIS)

    Henkels, Karen M.; Short, Stephen; Peng, Hong-Juan; Fulvio, Mauricio Di; Gomez-Cambronero, Julian

    2009-01-01

    Phospholipase D2 (PLD2) overexpression in mammalian cells results in cell transformation. We have hypothesized that this is due to an increase of de novo DNA synthesis. We show here that overexpression of PLD2-WT leads to an increased DNA synthesis, as measured by the expression levels of the proliferation markers PCNA, p27 KIP1 and phospho-histone-3. The enhancing effect was even higher with phosphorylation-deficient PLD2-Y179F and PLD2-Y511F mutants. The mechanism for this did not involve the enzymatic activity of the lipase, but, rather, the presence of the protein tyrosine phosphatase CD45, as silencing with siRNA for CD45 abrogated the effect. The two Y→F mutants had in common a YxN consensus site that, in the phosphorylated counterparts, could be recognized by SH2-bearing proteins, such as Grb2. Even though Y179F and Y511F cannot bind Grb2, they could still find other protein partners, one of which, we have reasoned, could be CD45 itself. Affinity purified PLD2 is indeed activated by Grb2 and deactivated by CD45 in vitro. We concluded that phosphorylated PLD2, aided by Grb2, mediates lipase activity, whereas dephosphorylated PLD2 mediates an induction of cell proliferation, and the specific residues involved in this newly discovered regulation of PLD2 are Y 179 and Y 511 .

  3. Mutant Forms of the Azotobacter vinelandii Transcriptional Activator NifA Resistant to Inhibition by the NifL Regulatory Protein

    OpenAIRE

    Reyes-Ramirez, Francisca; Little, Richard; Dixon, Ray

    2002-01-01

    The Azotobacter vinelandii σ54-dependent transcriptional activator protein NifA is regulated by the NifL protein in response to redox, carbon, and nitrogen status. Under conditions inappropriate for nitrogen fixation, NifL inhibits transcription activation by NifA through the formation of the NifL-NifA protein complex. NifL inhibits the ATPase activity of the central AAA+ domain of NifA required to drive open complex formation by σ54-RNA polymerase and may also inhibit the activator-polymeras...

  4. Comparison of a coq7 deletion mutant with other respiration-defective mutants in fission yeast.

    Science.gov (United States)

    Miki, Risa; Saiki, Ryoichi; Ozoe, Yoshihisa; Kawamukai, Makoto

    2008-11-01

    Among the steps in ubiquinone biosynthesis, that catalyzed by the product of the clk-1/coq7 gene has received considerable attention because of its relevance to life span in Caenorhabditis elegans. We analyzed the coq7 ortholog (denoted coq7) in Schizosaccharomyces pombe, to determine whether coq7 has specific roles that differ from those of other coq genes. We first confirmed that coq7 is necessary for the penultimate step in ubiquinone biosynthesis, from the observation that the deletion mutant accumulated the ubiquinone precursor demethoxyubiquinone-10 instead of ubiquinone-10. The coq7 mutant displayed phenotypes characteristic of other ubiquinone-deficient Sc. pombe mutants, namely, hypersensitivity to hydrogen peroxide, a requirement for antioxidants for growth on minimal medium, and an elevated production of sulfide. To compare these phenotypes with those of other respiration-deficient mutants, we constructed cytochrome c (cyc1) and coq3 deletion mutants. We also assessed accumulation of oxidative stress in various ubiquinone-deficient strains and in the cyc1 mutant by measuring mRNA levels of stress-inducible genes and the phosphorylation level of the Spc1 MAP kinase. Induction of ctt1, encoding catalase, and apt1, encoding a 25 kDa protein, but not that of gpx1, encoding glutathione peroxidase, was indistinguishable in four ubiquinone-deficient mutants, indicating that the oxidative stress response operates at similar levels in the tested strains. One new phenotype was observed, namely, loss of viability in stationary phase (chronological life span) in both the ubiquinone-deficient mutant and in the cyc1 mutant. Finally, Coq7 was found to localize in mitochondria, consistent with the possibility that ubiquinone biosynthesis occurs in mitochondria in yeasts. In summary, our results indicate that coq7 is required for ubiquinone biosynthesis and the coq7 mutant is not distinguishable from other ubiquinone-deficient mutants, except that its phenotypes are more

  5. Enzymatic modification of starch

    DEFF Research Database (Denmark)

    Jensen, Susanne Langgård

    In the food industry approaches for using bioengineering are investigated as alternatives to conventional chemical and physical starch modification techniques in development of starches with specific properties. Enzyme-assisted post-harvest modification is an interesting approach to this, since...... it is considered a clean and energy saving technology. This thesis aimed to investigate the effect of using reaction conditions, simulating an industrial process, for enzymatic treatment of starch with branching enzyme (BE) from Rhodothermus obamensis. Thus treatements were conducted at 70°C using very high...... substrate concentration (30-40% dry matter (DM)) and high enzyme activity (750-2250 BE units (BEU)/g sample). Starches from various botanical sources, representing a broad range of properties, were used as substrates. The effects of the used conditions on the BE-reaction were evaluated by characterization...

  6. Enzymatic modification of starch

    DEFF Research Database (Denmark)

    Jensen, Susanne Langgård

    In the food industry approaches for using bioengineering are investigated as alternatives to conventional chemical and physical starch modification techniques in development of starches with specific properties. Enzyme-assisted post-harvest modification is an interesting approach to this, since...... it is considered a clean and energy saving technology. This thesis aimed to investigate the effect of using reaction conditions, simulating an industrial process, for enzymatic treatment of starch with branching enzyme (BE) from Rhodothermus obamensis. Thus treatements were conducted at 70°C using very high...... of the obtained products and were correlated to properties of the starch substrates. It was found that the obtained products differed depending on both the conditions used and the properties of the starch. Products of starch from certain origins completely lost their granular structure during the enzyme treatment...

  7. Enzymatic Modification of Sphingomyelin

    DEFF Research Database (Denmark)

    Zhang, Long; Hellgren, Lars; Xu, Xuebing

    2005-01-01

    . Due to its major role in the water-retaining properties of the epidermis, ceramide is of great commercial potential in cosmetic and pharmaceuticals such as hair and skin care products. In current, ceramide is not easy to synthesis for industrial application and synthetic ceramide is still expensive......Sphingomyelin (SM) is a ubiquitous component of animal cell membranes, and it is the most abundant sphingolipid. Ceramide, a hydrolysis product from SM, has an important role in cellular signaling, and especially in the regulation of apoptosis, cell differentiation, transformation and proliferation....... Therefore, it is desirable to develop alternative cost-efficient, high yield production methods. This study optimized the enzymatic production of ceramide from SM. Phospholipase C from Clostridium perfringens was chosen to catalyze the reaction. Several important factors were considered in optimization....

  8. Enzymatic Modification of Sphingomyelin

    DEFF Research Database (Denmark)

    Due to its major role in maintaining the water-retaining properties of the epidermis, ceramide is of great commercial potential in cosmetic and pharmaceuticals such as hair and skin care products. Currently, chemical synthesis of ceramide is a costly process, and developments of alternative cost......-efficient, high yield production methods are of great interest. In the present study, the potential of producing ceramide through the enzymatic hydrolysis of sphingomyelin have been studied. sphingomyelin is a ubiquitous membrane-lipid and rich in dairy products or by-products. It has been verified...... that sphingomyelin modification gives a feasible approach to the potential production of ceramide. The reaction system has been improved through system evaluation and the optimization of several important factors, and phospholipase C from Clostridium perfringens shows higher activity towards the hydrolysis reaction...

  9. Developing a genetically encoded green fluorescent protein mutant for sensitive light-up fluorescent sensing and cellular imaging of Hg(II).

    Science.gov (United States)

    Jiang, Tao; Guo, Daiping; Wang, Qian; Wu, Xin; Li, Zhao; Zheng, Zhenhua; Yin, Boyuan; Xia, Lin; Tang, Jixian; Luo, Wenxin; Xia, Ningshao; Jiang, Yunbao

    2015-05-30

    Hg(II) is well-known for quenching fluorescence in a distance dependent manner. Nevertheless, when we exposed the fluorophore of a green fluorescent protein (GFP) toward Hg(II), through H148C mutation, the GFP fluorescence could be "lighted up" by Hg(II) down to sub-nM level. The detection linear range is 0.5-3.0 nM for protein solutions at 8.0 nM. The GFPH148C protein displayed a promising selectivity toward Hg(II) and also the cellular imaging capacity. Spectra measurements suggested that the ground-state redistribution of protein contributed to the fluorescence enhancement, which was found not limited to Hg(II), and thus presented an opening for building a pool of GFP-based chemosensors toward other heavy metal ions. Copyright © 2015. Published by Elsevier B.V.

  10. Distribution of soluble amino acids in maize endosperm mutants

    Directory of Open Access Journals (Sweden)

    Toro Alejandro Alberto

    2003-01-01

    Full Text Available For human nutrition the main source of vegetable proteins are cereal and legume seeds. The content of total soluble amino acids in mature endosperm of wild-type, opaque and floury maize (Zea mays L. mutants were determined by HPLC. The total absolute concentration of soluble amino acids among the mutants varied depending on the mutant. The o11 and o13 mutants exhibited the highest average content, whereas o10, fl3 and fl1 exhibited the lowest average content. In general, the mutants exhibited similar concentrations of total soluble amino acids when compared to the wild-type lines, with the clear exception of mutants o11 and fl1, with the o11 mutant exhibiting a higher concentration of total soluble amino acids when compared to its wild-type counterpart W22 and the fl1 mutant a lower concentration when compared to its wild-type counterpart Oh43. For methionine, the mutants o2 and o11 and wild-type Oh43 exhibited the highest concentrations of this amino acid. Significant differences were not observed between mutants for other amino acids such as lysine and threonine. The high lysine concentrations obtained originally for these mutants may be due to the amino acids incorporated into storage proteins, but not those present in the soluble form.

  11. Coproporphyrinogenase in a respiration-deficient mutant of yeast lacking all cytochromes and accumulating coproporphyrin.

    Science.gov (United States)

    Miyake, S; Sugimura, T

    1968-12-01

    In an earlier report, a respiration-deficient mutant of yeast which lacks all cytochromes and hemoproteins and accumulates coproporphyrin was described. This respiration-deficient mutant was temperature-sensitive and resulted from the single chromosomal gene(cyt). In this study, the activity of coproporphyrinogenase, catalyzing the conversion of coproporphyrinogen to protoporphyrinogen, was assayed in the cyt mutant and wild strains. Coproporphyrinogenase activity was 10 times higher in the cyt mutant than in the wild strains. Cells of the cyt mutant grown at 20 C had less activity than those grown at 35 C. The Michaelis constants, pH optima, and temperature activations of the enzymes of the cyt mutant and the wild strains were similar. The significance of the higher activity of this enzyme in the cyt mutant, in which this enzymatic step is apparently blocked in vivo, is discussed.

  12. Hydroimidazolone modification of the conserved Arg12 in small heat shock proteins: studies on the structure and chaperone function using mutant mimics.

    Directory of Open Access Journals (Sweden)

    Ram H Nagaraj

    Full Text Available Methylglyoxal (MGO is an α-dicarbonyl compound present ubiquitously in the human body. MGO reacts with arginine residues in proteins and forms adducts such as hydroimidazolone and argpyrimidine in vivo. Previously, we showed that MGO-mediated modification of αA-crystallin increased its chaperone function. We identified MGO-modified arginine residues in αA-crystallin and found that replacing such arginine residues with alanine residues mimicked the effects of MGO on the chaperone function. Arginine 12 (R12 is a conserved amino acid residue in Hsp27 as well as αA- and αB-crystallin. When treated with MGO at or near physiological concentrations (2-10 µM, R12 was modified to hydroimidazolone in all three small heat shock proteins. In this study, we determined the effect of arginine substitution with alanine at position 12 (R12A to mimic MGO modification on the structure and chaperone function of these proteins. Among the three proteins, the R12A mutation improved the chaperone function of only αA-crystallin. This enhancement in the chaperone function was accompanied by subtle changes in the tertiary structure, which increased the thermodynamic stability of αA-crystallin. This mutation induced the exposure of additional client protein binding sites on αA-crystallin. Altogether, our data suggest that MGO-modification of the conserved R12 in αA-crystallin to hydroimidazolone may play an important role in reducing protein aggregation in the lens during aging and cataract formation.

  13. Resonance assignments of the myristoylated Y28F/Y67F mutant of the Mason-Pfizer monkey virus matrix protein

    Czech Academy of Sciences Publication Activity Database

    Doležal, Michal; Hrabal, R.; Ruml, T.; Rumlová, Michaela

    2015-01-01

    Roč. 9, č. 2 (2015), s. 229-233 ISSN 1874-2718 Institutional support: RVO:61388963 Keywords : isotopic labeling * matrix protein * M-PMV * myristoylation * resonance assignment * reverse labeling Subject RIV: CE - Biochemistry Impact factor: 0.687, year: 2015

  14. Correlation between survival, ability to rejoin DNA and stability of DNA after preirradiation inhibition of protein synthesis in a rec- mutant of Escherichia coli K12

    International Nuclear Information System (INIS)

    Pirsel, M.; Slezarikova, V.

    1977-01-01

    A 90 min inhibition of protein synthesis induced by starvation for amino acids (AA - ) or by chloramphenicol (CAP) treatment prior to UV irradiation (2.5 J m -2 ) increased more than tenfold the resistance of the strain Escherichia coli K12 SR19 to UV radiation. Under these conditions, cultures in which protein synthesis was inhibited before the UV irradiation rejoin short regions of DNA synthesized after the irradiation to a normal-size molecule, whereas an exponentially growing culture does not rejoin DNA synthesized after UV irradiation to a molecule of a normal size. In the exponentially growing culture both the parental and the newly synthesized DNA are unstable after the irradiation. In cultures with inhibited protein synthesis only the parental DNA is somewhat unstable. In Escherichia coli K12 SR19 where protein synthesis was inhibited before the irradiation, a correlation between the survival of cells, the ability to rejoin short regions of DNA synthesized after UV irradiation, and a higher stability of both parental and newly synthesized DNAs could be demonstrated. (author)

  15. Food safety knowledge on the Bt mutant protein Cry8Ka5 employed in the development of coleopteran-resistant transgenic cotton plants

    NARCIS (Netherlands)

    Felipe Farias, Davi; Peijnenburg, A.A.C.M.; Grossi-de-Sá, Maria F.; Carvalho, A.F.U.

    2015-01-01

    Insecticidal Cry proteins from Bacillus thuringiensis (Bt) have been exploited in the development of genetically modified (GM) crops for pest control. However, several pests are still difficult to control such as the coleopteran boll weevil Anthonomus grandis. By applying in vitro molecular

  16. Determination of the catalytic activity of LEOPARD syndrome-associated SHP2 mutants toward parafibromin, a bona fide SHP2 substrate involved in Wnt signaling

    International Nuclear Information System (INIS)

    Noda, Saori; Takahashi, Atsushi; Hayashi, Takeru; Tanuma, Sei-ichi; Hatakeyama, Masanori

    2016-01-01

    SHP2, encoded by the PTPN11 gene, is a protein tyrosine phosphatase that plays a key role in the proliferation of cells via RAS-ERK activation. SHP2 also promotes Wnt signaling by dephosphorylating parafibromin. Germline missense mutations of PTPN11 are found in more than half of patients with Noonan syndrome (NS) and LEOPARD syndrome (LS), both of which are congenital developmental disorders with multiple common symptoms. However, whereas NS-associated PTPN11 mutations give rise to gain-of-function SHP2 mutants, LS-associated SHP2 mutants are reportedly loss-of-function mutants. To determine the phosphatase activity of LS-associated SHP2 more appropriately, we performed an in vitro phosphatase assay using tyrosine-phosphorylated parafibromin, a biologically relevant substrate of SHP2 and the positive regulator of Wnt signaling that is activated through SHP2-mediated dephosphorylation. We found that LS-associated SHP2 mutants (Y279C, T468M, Q506P, and Q510E) exhibited a substantially reduced phosphatase activity toward parafibromin when compared with wild-type SHP2. Furthermore, each of the LS-associated mutants displayed a differential degree of decrease in phosphatase activity. Deviation of the SHP2 catalytic activity from a certain range, either too strong or too weak, may therefore lead to similar clinical outcomes in NS and LS, possibly through an imbalanced Wnt signal caused by inadequate dephosphorylation of parafibromin. - Highlights: • LS-associated SHP2 mutants dephosphorylate parafibromin on Y290, Y293, and Y315. • LS-associated SHP2 mutants display a reduced tyrosine phosphatase activity. • LS-specific SHP2-Y279C is catalytically less active than LS-specific SHP2-T468M. • NS/LS-associated SHP2-Q506P has both hyper- and hypomorphic enzymatic properties.

  17. Determination of the catalytic activity of LEOPARD syndrome-associated SHP2 mutants toward parafibromin, a bona fide SHP2 substrate involved in Wnt signaling

    Energy Technology Data Exchange (ETDEWEB)

    Noda, Saori [Division of Microbiology, Graduate School of Medicine, The University of Tokyo, Tokyo (Japan); Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba (Japan); Takahashi, Atsushi; Hayashi, Takeru [Division of Microbiology, Graduate School of Medicine, The University of Tokyo, Tokyo (Japan); Tanuma, Sei-ichi [Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba (Japan); Hatakeyama, Masanori, E-mail: mhata@m.u-tokyo.ac.jp [Division of Microbiology, Graduate School of Medicine, The University of Tokyo, Tokyo (Japan)

    2016-01-22

    SHP2, encoded by the PTPN11 gene, is a protein tyrosine phosphatase that plays a key role in the proliferation of cells via RAS-ERK activation. SHP2 also promotes Wnt signaling by dephosphorylating parafibromin. Germline missense mutations of PTPN11 are found in more than half of patients with Noonan syndrome (NS) and LEOPARD syndrome (LS), both of which are congenital developmental disorders with multiple common symptoms. However, whereas NS-associated PTPN11 mutations give rise to gain-of-function SHP2 mutants, LS-associated SHP2 mutants are reportedly loss-of-function mutants. To determine the phosphatase activity of LS-associated SHP2 more appropriately, we performed an in vitro phosphatase assay using tyrosine-phosphorylated parafibromin, a biologically relevant substrate of SHP2 and the positive regulator of Wnt signaling that is activated through SHP2-mediated dephosphorylation. We found that LS-associated SHP2 mutants (Y279C, T468M, Q506P, and Q510E) exhibited a substantially reduced phosphatase activity toward parafibromin when compared with wild-type SHP2. Furthermore, each of the LS-associated mutants displayed a differential degree of decrease in phosphatase activity. Deviation of the SHP2 catalytic activity from a certain range, either too strong or too weak, may therefore lead to similar clinical outcomes in NS and LS, possibly through an imbalanced Wnt signal caused by inadequate dephosphorylation of parafibromin. - Highlights: • LS-associated SHP2 mutants dephosphorylate parafibromin on Y290, Y293, and Y315. • LS-associated SHP2 mutants display a reduced tyrosine phosphatase activity. • LS-specific SHP2-Y279C is catalytically less active than LS-specific SHP2-T468M. • NS/LS-associated SHP2-Q506P has both hyper- and hypomorphic enzymatic properties.

  18. Expression of enzymatically inactive wasp venom phospholipase A1 in Pichia pastoris.

    Directory of Open Access Journals (Sweden)

    Irina Borodina

    Full Text Available Wasp venom allergy is the most common insect venom allergy in Europe. It is manifested by large local reaction or anaphylactic shock occurring after a wasp sting. The allergy can be treated by specific immunotherapy with whole venom extracts. Wasp venom is difficult and costly to obtain and is a subject to composition variation, therefore it can be advantageous to substitute it with a cocktail of recombinant allergens. One of the major venom allergens is phospholipase A1, which so far has been expressed in Escherichia coli and in insect cells. Our aim was to produce the protein in secreted form in yeast Pichia pastoris, which can give high yields of correctly folded protein on defined minimal medium and secretes relatively few native proteins simplifying purification.Residual amounts of enzymatically active phospholipase A1 could be expressed, but the venom protein had a deleterious effect on growth of the yeast cells. To overcome the problem we introduced three different point mutations at the critical points of the active site, where serine137, aspartate165 or histidine229 were replaced by alanine (S137A, D165A and H229A. All the three mutated forms could be expressed in P. pastoris. The H229A mutant did not have any detectable phospholipase A1 activity and was secreted at the level of several mg/L in shake flask culture. The protein was purified by nickel-affinity chromatography and its identity was confirmed by MALDI-TOF mass spectrometry. The protein could bind IgE antibodies from wasp venom allergic patients and could inhibit the binding of wasp venom to IgE antibodies specific for phospholipase A1 as shown by Enzyme Allergo-Sorbent Test (EAST. Moreover, the recombinant protein was allergenic in a biological assay as demonstrated by its capability to induce histamine release of wasp venom-sensitive basophils.The recombinant phospholipase A1 presents a good candidate for wasp venom immunotherapy.

  19. Translational control of ribosomal protein S15.

    Science.gov (United States)

    Portier, C; Philippe, C; Dondon, L; Grunberg-Manago, M; Ebel, J P; Ehresmann, B; Ehresmann, C

    1990-08-27

    The expression of ribosomal protein S15 is shown to be translationally and negatively autocontrolled using a fusion within a reporter gene. Isolation and characterization of several deregulated mutants indicate that the regulatory site (the translational operator site) overlaps the ribosome loading site of the S15 messenger. In this region, three domains, each exhibiting a stem-loop structure, were determined using chemical and enzymatic probes. The most downstream hairpin carries the Shine-Dalgarno sequence and the initiation codon. Genetic and structural data derived from mutants constructed by site-directed mutagenesis show that the operator is a dynamic structure, two domains of which can form a pseudoknot. Binding of S15 to these two domains suggests that the pseudoknot could be stabilized by S15. A model is presented in which two alternative structures would explain the molecular basis of the S15 autocontrol.

  20. Enzymatic Ligation of Large Biomolecules to DNA

    DEFF Research Database (Denmark)

    Sørensen, Rasmus Schøler; Okholm, Anders Hauge; Schaffert, David Henning

    2013-01-01

    application. However, conjugation of DNA to large molecular components using classical chemistries often suffers from suboptimal yields. Here, we report the use of terminal deoxynucleotidyl transferase (TdT) for direct enzymatic ligation of native DNA to nucleotide triphosphates coupled to proteins and other...... large macromolecules. We demonstrate facile synthesis routes for a range of NTP-activated macromolecules and subsequent ligation to the 3′ hydroxyl group of oligodeoxynucleotides using TdT. The reaction is highly specific and proceeds rapidly and essentially to completion at micromolar concentrations...

  1. Structure of the enzymatically synthesized fructan inulin

    Energy Technology Data Exchange (ETDEWEB)

    Heyer, A.G.; Schroeer, B. [Max-Planck-Institut fuer Molekulare Pflanzenphysiologie, Karl-Liebknecht-Str. 25, 14476 Golm (Germany); Radosta, S. [Fraunhofer-Institut fuer Angewandte Polymerforschung, Postfach 126, 14504 Teltow (Germany); Wolff, D.; Czapla, S.; Springer, J. [Technische Universitaet Berlin, FG Makromolekulare Chemie, Str. des 17. Juni 135, 10623 Berlin (Germany)

    1998-12-15

    Construction, purification and characterization of a fusion protein of maltose-binding protein of Escherichia coli and the fructosyltransferase of Streptococcus mutans is described. With the purified protein, in vitro synthesis of inulin was performed. The obtained polysaccharide was characterized by high-performance size-exclusion chromatography (HPSEC) and static light scattering (SLS) in dilute aqueous and dimethyl sulfoxide solution. For all samples very high molecular weights between 60x10{sup 6} and 90x10{sup 6} g/mol and a remarkable small polydispersity index of 1.1 have been determined. Small root-mean-square radii of gyration point to a compact conformation in dilute solution. No difference between native and enzymatically synthesized inulin was observed by X-ray powder diffraction and thermoanalysis of solid samples. (Copyright (c) 1998 Elsevier Science B.V., Amsterdam. All rights reserved.)

  2. Structure of the enzymatically synthesized fructan inulin

    International Nuclear Information System (INIS)

    Heyer, A.G.; Schroeer, B.; Radosta, S.; Wolff, D.; Czapla, S.; Springer, J.

    1998-01-01

    Construction, purification and characterization of a fusion protein of maltose-binding protein of Escherichia coli and the fructosyltransferase of Streptococcus mutans is described. With the purified protein, in vitro synthesis of inulin was performed. The obtained polysaccharide was characterized by high-performance size-exclusion chromatography (HPSEC) and static light scattering (SLS) in dilute aqueous and dimethyl sulfoxide solution. For all samples very high molecular weights between 60x10 6 and 90x10 6 g/mol and a remarkable small polydispersity index of 1.1 have been determined. Small root-mean-square radii of gyration point to a compact conformation in dilute solution. No difference between native and enzymatically synthesized inulin was observed by X-ray powder diffraction and thermoanalysis of solid samples. (Copyright (c) 1998 Elsevier Science B.V., Amsterdam. All rights reserved.)

  3. Homogeneous, Heterogeneous, and Enzymatic Catalysis.

    Science.gov (United States)

    Oyama, S. Ted; Somorjai, Gabor A.

    1988-01-01

    Discusses three areas of catalysis: homegeneous, heterogeneous, and enzymatic. Explains fundamentals and economic impact of catalysis. Lists and discusses common industrial catalysts. Provides a list of 107 references. (MVL)

  4. Enzymatic synthesis of organophosphorus compounds

    International Nuclear Information System (INIS)

    Kolodiazhnyi, Oleg I

    2011-01-01

    Data on biocatalytic methods for the preparation of chiral organophosphorus compounds are generalized and described systematically. Various examples of enzymatic and microbiological synthesis of hydroxyphosphonates, aminophosphonates, phosphinites, phosphine oxides and tertiary phosphines are discussed. The bibliography includes 154 references.

  5. Enzymatic synthesis of organophosphorus compounds

    Science.gov (United States)

    Kolodiazhnyi, Oleg I.

    2011-09-01

    Data on biocatalytic methods for the preparation of chiral organophosphorus compounds are generalized and described systematically. Various examples of enzymatic and microbiological synthesis of hydroxyphosphonates, aminophosphonates, phosphinites, phosphine oxides and tertiary phosphines are discussed. The bibliography includes 154 references.

  6. Measles Virus Mutants Possessing the Fusion Protein with Enhanced Fusion Activity Spread Effectively in Neuronal Cells, but Not in Other Cells, without Causing Strong Cytopathology

    Science.gov (United States)

    Ohno, Shinji; Shirogane, Yuta; Suzuki, Satoshi O.; Koga, Ritsuko

    2014-01-01

    ABSTRACT Subacute sclerosing panencephalitis (SSPE) is caused by persistent measles virus (MV) infection in the central nervous system (CNS). Since human neurons, its main target cells, do not express known MV receptors (signaling lymphocyte activation molecule [SLAM] and nectin 4), it remains to be understood how MV infects and spreads in them. We have recently reported that fusion-enhancing substitutions in the extracellular domain of the MV fusion (F) protein (T461I and S103I/N462S/N465S), which are found in multiple SSPE virus isolates, promote MV spread in human neuroblastoma cell lines and brains of suckling hamsters. In this study, we show that hyperfusogenic viruses with these substitutions also spread efficiently in human primary neuron cultures without inducing syncytia. These substitutions were found to destabilize the prefusion conformation of the F protein trimer, thereby enhancing fusion activity. However, these hyperfusogenic viruses exhibited stronger cytopathology and produced lower titers at later time points in SLAM- or nectin 4-expressing cells compared to the wild-type MV. Although these viruses spread efficiently in the brains of SLAM knock-in mice, they did not in the spleens. Taken together, the results suggest that enhanced fusion activity is beneficial for MV to spread in neuronal cells where no cytopathology occurs, but detrimental to other types of cells due to strong cytopathology. Acquisition of enhanced fusion activity through substitutions in the extracellular domain of the F protein may be crucial for MV's extensive spread in the CNS and development of SSPE. IMPORTANCE Subacute sclerosing panencephalitis (SSPE) is a fatal disease caused by persistent measles virus (MV) infection in the central nervous system (CNS). Its cause is not well understood, and no effective therapy is currently available. Recently, we have reported that enhanced fusion activity of MV through the mutations in its fusion protein is a major determinant of

  7. Mutant PTEN in Cancer : Worse Than Nothing

    NARCIS (Netherlands)

    Leslie, Nick R; den Hertog, Jeroen

    2014-01-01

    Tumor suppressors block the development of cancer and are often lost during tumor development. Papa et al. show that partial loss of normal PTEN tumor suppressor function can be compounded by additional disruption caused by the expression of inactive mutant PTEN protein. This has significant

  8. Novel causative mutations in patients with Nance-Horan syndrome and altered localization of the mutant NHS-A protein isoform

    OpenAIRE

    Shama, S.; Burdon, K. P.; Dave, A.; Jamieson, R. V.; Yaron, Y.; Billson, F.; Van Maldergem, Lionel; Lorenz, B.; Gécz, J.; Craig, J. E.

    2008-01-01

    Purpose Nance-Horan syndrome is typically characterized by severe bilateral congenital cataracts and dental abnormalities. Truncating mutations in the Nance-Horan syndrome (NHS) gene cause this X-linked genetic disorder. NHS encodes two isoforms, NHS-A and NHS-1A. The ocular lens expresses NHS-A, the epithelial and neuronal cell specific isoform. The NHS-A protein localizes in the lens epithelium at the cellular periphery. The data to date suggest a role for this isoform at cell-cell junction...

  9. Mitochondrial dysfunction in NnaD mutant flies and Purkinje cell degeneration (pcd) mice reveals a role for Nna proteins in neuronal bioenergetics

    Science.gov (United States)

    Chakrabarti, Lisa; Zahra, Rabaab; Jackson, Stephen M.; Kazemi-Esfarjani, Parsa; Sopher, Bryce L.; Mason, Amanda G.; Toneff, Thomas; Ryu, Soyoung; Shaffer, Scott; Kansy, Janice W.; Eng, Jeremiah; Merrihew, Gennifer; MacCoss, Michael J.; Murphy, Anne; Goodlett, David R.; Hook, Vivian; Bennett, Craig L.; Pallanck, Leo; La Spada, Albert R.

    2011-01-01

    The Purkinje cell degeneration (pcd) mouse is a recessive model of neurodegeneration, involving cerebellum and retina. Purkinje cell death in pcd is dramatic, as >99% of Purkinje neurons are lost in three weeks. Loss-of-function of Nna1 causes pcd, and Nna1 is a highly conserved zinc carboxypeptidase. To determine the basis of pcd, we implemented a two-pronged approach, combining characterization of loss-of-function phenotypes of the Drosophila Nna1 orthologue (NnaD) with proteomics analysis of pcd mice. Reduced NnaD function yielded larval lethality, with survivors displaying phenotypes that mirror disease in pcd. Quantitative proteomics revealed expression alterations for glycolytic and oxidative phosphorylation enzymes. Nna proteins localize to mitochondria, loss of NnaD / Nna1 produces mitochondrial abnormalities, and pcd mice display altered proteolytic processing of Nna1 interacting proteins. Our studies indicate that Nna1 loss-of-function results in altered bioenergetics and mitochondrial dysfunction, and suggest that pcd shares pathogenic features with neurodegenerative disorders such as Parkinson's disease. PMID:20620870

  10. Understanding the relationship between biotherapeutic protein stability and solid-liquid interfacial shear in constant region mutants of IgG1 and IgG4.

    Science.gov (United States)

    Tavakoli-Keshe, Roumteen; Phillips, Jonathan J; Turner, Richard; Bracewell, Daniel G

    2014-02-01

    Relative stability of therapeutic antibody candidates is currently evaluated primarily through their response to thermal degradation, yet this technique is not always predictive of stability in manufacture, shipping, and storage. A rotating disk shear device is proposed that produces defined shear conditions at a known solid-liquid interface to measure stability in this environment. Five variants of IgG1 and IgG4 antibodies were created using combinations of two discrete triple amino acid sequence mutations denoted TM and YTE. Antibodies were ranked for stability based on shear device output (protein decay coefficient, PDC), and compared with accelerated thermal stability data and the melting temperature of the CH2 domain (Tm 1) from differential scanning calorimetry to investigate technique complimentarity. Results suggest that the techniques are orthogonal, with thermal methods based on intramolecular interaction and shear device stability based on localized unfolding revealing less stable regions that drive aggregation. Molecular modeling shows the modifications' effects on the antibody structures and indicates a possible role for Fc conformation and Fab-Fc docking in determining suspended protein stability. The data introduce the PDC value as an orthogonal stability indicator, complementary to traditional thermal methods, allowing lead antibody selection based on a more full understanding of process stability. © 2013 The Authors. Journal of Pharmaceutical Sciences published by Wiley Periodicals, Inc. and the American Pharmacists Association.

  11. Productive mutants of niger

    International Nuclear Information System (INIS)

    Misra, R.C.

    2001-01-01

    Seeds of six niger (Guizotia abyssinica Cass.) varieties ('GA-10', 'ONS-8', 'IGP-72', 'N-71', 'NB-9' and 'UN-4') were treated with 0.5, 0.75 and 1% ethyl methanesulphonate. After four generations of selection, 29 mutant lines were developed and those were evaluated from 1990-92 during Kharif (July to October) and Rabi (December to March) seasons. Average plant characteristics and yield data of four high yielding mutants along with 'IGP-76' (National Check), GA-10 (Zonal Check) and 'Semiliguda Local' (Local Check) are presented

  12. Emery-Dreifuss Muscular Dystrophy-Associated Mutant Forms of Lamin A Recruit the Stress Responsive Protein Ankrd2 into the Nucleus, Affecting the Cellular Response to Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Silvia Angori

    2017-05-01

    Full Text Available Background: Ankrd2 is a stress responsive protein mainly expressed in muscle cells. Upon the application of oxidative stress, Ankrd2 translocates into the nucleus where it regulates the activity of genes involved in cellular response to stress. Emery-Dreifuss Muscular Dystrophy 2 (EDMD2 is a muscular disorder caused by mutations of the gene encoding lamin A, LMNA. As well as many phenotypic abnormalities, EDMD2 muscle cells also feature a permanent basal stress state, the underlying molecular mechanisms of which are currently unclear. Methods: Experiments were performed in EDMD2-lamin A overexpressing cell lines and EDMD2-affected human myotubes. Oxidative stress was produced by H2O2 treatment. Co-immunoprecipitation, cellular subfractionation and immunofluorescence analysis were used to validate the relation between Ankrd2 and forms of lamin A; cellular sensibility to stress was monitored by the analysis of Reactive Oxygen Species (ROS release and cell viability. Results: Our data demonstrate that oxidative stress induces the formation of a complex between Ankrd2 and lamin A. However, EDMD2-lamin A mutants were able to bind and mislocalize Ankrd2 in the nucleus even under basal conditions. Nonetheless, cells co-expressing Ankrd2 and EDMD2-lamin A mutants were more sensitive to oxidative stress than the Ankrd2-wild type lamin A counterpart. Conclusions: For the first time, we present evidence that in muscle fibers from patients affected by EDMD2, Ankrd2 has an unusual nuclear localization. By introducing a plausible mechanism ruling this accumulation, our data hint at a novel function of Ankrd2 in the pathogenesis of EDMD2-affected cells.

  13. Mutant Enpp1asj mice as a model for generalized arterial calcification of infancy

    Directory of Open Access Journals (Sweden)

    Qiaoli Li

    2013-09-01

    Generalized arterial calcification of infancy (GACI, an autosomal recessive disorder, is characterized by early mineralization of blood vessels, often diagnosed by prenatal ultrasound and usually resulting in demise during the first year of life. It is caused in most cases by mutations in the ENPP1 gene, encoding an enzyme that hydrolyzes ATP to AMP and inorganic pyrophosphate, the latter being a powerful anti-mineralization factor. Recently, a novel mouse phenotype was recognized as a result of ENU mutagenesis – those mice developed stiffening of the joints, hence the mutant mouse was named ‘ages with stiffened joints’ (asj. These mice harbor a missense mutation, p.V246D, in the Enpp1 gene. Here we demonstrate that the mutant ENPP1 protein is largely absent in the liver of asj mice, and the lack of enzymatic activity results in reduced inorganic pyrophosphate (PPi levels in the plasma, accompanied by extensive mineralization of a number of tissues, including arterial blood vessels. The progress of mineralization is highly dependent on the mineral composition of the diet, with significant shortening of the lifespan on a diet enriched in phosphorus and low in magnesium. These results suggest that the asj mouse can serve as an animal model for GACI.

  14. Characterization of human glucocerebrosidase from different mutant alleles

    NARCIS (Netherlands)

    Ohashi, T.; Hong, C. M.; Weiler, S.; Tomich, J. M.; Aerts, J. M.; Tager, J. M.; Barranger, J. A.

    1991-01-01

    Human cDNA was mutagenized to duplicate six naturally occurring mutations in the gene for glucocere-brosidase. The mutant genes were expressed in NIH 3T3 cells. The abnormal human enzymes were purified by immunoaffinity chromatography and characterized. The Asn370----Ser mutant protein differed from

  15. Frataxin directly stimulates mitochondrial cysteine desulfurase by exposing substrate-binding sites, and a mutant Fe-S cluster scaffold protein with frataxin-bypassing ability acts similarly.

    Science.gov (United States)

    Pandey, Alok; Gordon, Donna M; Pain, Jayashree; Stemmler, Timothy L; Dancis, Andrew; Pain, Debkumar

    2013-12-27

    For iron-sulfur (Fe-S) cluster synthesis in mitochondria, the sulfur is derived from the amino acid cysteine by the cysteine desulfurase activity of Nfs1. The enzyme binds the substrate cysteine in the pyridoxal phosphate-containing site, and a persulfide is formed on the active site cysteine in a manner depending on the accessory protein Isd11. The persulfide is then transferred to the scaffold Isu, where it combines with iron to form the Fe-S cluster intermediate. Frataxin is implicated in the process, although it is unclear where and how, and deficiency causes Friedreich ataxia. Using purified proteins and isolated mitochondria, we show here that the yeast frataxin homolog (Yfh1) directly and specifically stimulates cysteine binding to Nfs1 by exposing substrate-binding sites. This novel function of frataxin does not require iron, Isu1, or Isd11. Once bound to Nfs1, the substrate cysteine is the source of the Nfs1 persulfide, but this step is independent of frataxin and strictly dependent on Isd11. Recently, a point mutation in Isu1 was found to bypass many frataxin functions. The data presented here show that the Isu1 suppressor mimics the frataxin effects on Nfs1, explaining the bypassing activity. We propose a regulatory mechanism for the Nfs1 persulfide-forming activity. Specifically, at least two separate conformational changes must occur in the enzyme for optimum activity as follows: one is mediated by frataxin interaction that exposes the "buried" substrate-binding sites, and the other is mediated by Isd11 interaction that brings the bound substrate cysteine and the active site cysteine in proximity for persulfide formation.

  16. Enzymatic activities of Dermatophilus congolensis measured by API ZYM.

    Science.gov (United States)

    Hermoso de Mendoza, J; Arenas, A; Alonso, J M; Rey, J M; Gil, M C; Anton, J M; Hermoso de Mendoza, M

    1993-10-01

    API ZYM kit was used to test enzymatic activities on eighteen strains of Dermatophilus congolensis. All strains produced lipase and acid phosphatase, which act on lipids, and leucine arylamidase which act on proteins. Another 10 exoenzymes were present in at least one of the strains.

  17. Proteomic analysis of tylosin-resistant Mycoplasma gallisepticum reveals enzymatic activities associated with resistance.

    Science.gov (United States)

    Xia, Xi; Wu, Congming; Cui, Yaowen; Kang, Mengjiao; Li, Xiaowei; Ding, Shuangyang; Shen, Jianzhong

    2015-11-20

    Mycoplasma gallisepticum is a significant pathogenic bacterium that infects poultry, causing chronic respiratory disease and sinusitis in chickens and turkeys, respectively. M. gallisepticum infection poses a substantial economic threat to the poultry industry, and this threat is made worse by the emergence of antibiotic-resistant strains. The mechanisms of resistance are often difficult to determine; for example, little is known about antibiotic resistance of M. gallisepticum at the proteome level. In this study, we performed comparative proteomic analyses of an antibiotic (tylosin)-resistant M. gallisepticum mutant and a susceptible parent strain using a combination of two-dimensional differential gel electrophoresis and nano-liquid chromatography-quadrupole-time of flight mass spectrometry. Thirteen proteins were identified as differentially expressed in the resistant strain compared to the susceptible strain. Most of these proteins were related to catalytic activity, including catalysis that promotes the formylation of initiator tRNA and energy production. Elongation factors Tu and G were over-expressed in the resistant strains, and this could promote the binding of tRNA to ribosomes and catalyze ribosomal translocation, the coordinated movement of tRNA, and conformational changes in the ribosome. Taken together, our results indicate that M. gallisepticum develops resistance to tylosin by regulating associated enzymatic activities.

  18. Forkhead Box Protein 1 (FoxO1) Inhibits Accelerated β Cell Aging in Pancreas-specific SMAD7 Mutant Mice.

    Science.gov (United States)

    Xiao, Xiangwei; Chen, Congde; Guo, Ping; Zhang, Ting; Fischbach, Shane; Fusco, Joseph; Shiota, Chiyo; Prasadan, Krishna; Dong, Henry; Gittes, George K

    2017-02-24

    The mechanisms underlying the effects of exocrine dysfunction on the development of diabetes remain largely unknown. Here we show that pancreatic depletion of SMAD7 resulted in age-dependent increases in β cell dysfunction with accelerated glucose intolerance, followed by overt diabetes. The accelerated β cell dysfunction and loss of proliferation capacity, two features of β cell aging, appeared to be non-cell-autonomous, secondary to the adjacent exocrine failure as a "bystander effect." Increased Forkhead box protein 1 (FoxO1) acetylation and nuclear retention was followed by progressive FoxO1 loss in β cells that marked the onset of diabetes. Moreover, forced FoxO1 expression in β cells prevented β cell dysfunction and loss in this model. Thus, we present a model of accelerated β cell aging that may be useful for studying the mechanisms underlying β cell failure in diabetes. Moreover, we provide evidence highlighting a critical role of FoxO1 in maintaining β cell identity in the context of SMAD7 failure. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Forkhead Box Protein 1 (FoxO1) Inhibits Accelerated β Cell Aging in Pancreas-specific SMAD7 Mutant Mice*

    Science.gov (United States)

    Xiao, Xiangwei; Chen, Congde; Guo, Ping; Zhang, Ting; Fischbach, Shane; Fusco, Joseph; Shiota, Chiyo; Prasadan, Krishna; Dong, Henry; Gittes, George K.

    2017-01-01

    The mechanisms underlying the effects of exocrine dysfunction on the development of diabetes remain largely unknown. Here we show that pancreatic depletion of SMAD7 resulted in age-dependent increases in β cell dysfunction with accelerated glucose intolerance, followed by overt diabetes. The accelerated β cell dysfunction and loss of proliferation capacity, two features of β cell aging, appeared to be non-cell-autonomous, secondary to the adjacent exocrine failure as a “bystander effect.” Increased Forkhead box protein 1 (FoxO1) acetylation and nuclear retention was followed by progressive FoxO1 loss in β cells that marked the onset of diabetes. Moreover, forced FoxO1 expression in β cells prevented β cell dysfunction and loss in this model. Thus, we present a model of accelerated β cell aging that may be useful for studying the mechanisms underlying β cell failure in diabetes. Moreover, we provide evidence highlighting a critical role of FoxO1 in maintaining β cell identity in the context of SMAD7 failure. PMID:28057752

  20. Common missense mutation G1528C in long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. Characterization and expression of the mutant protein, mutation analysis on genomic DNA and chromosomal localization of the mitochondrial trifunctional protein alpha subunit gene

    NARCIS (Netherlands)

    IJlst, L.; Ruiter, J. P.; Hoovers, J. M.; Jakobs, M. E.; Wanders, R. J.

    1996-01-01

    Mitochondrial trifunctional protein (MTP) is a recently identified enzyme involved in mitochondrial beta-oxidation, harboring long-chain enoyl-CoA hydratase, long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) and long-chain 3-ketothiolase activity. A deficiency of this protein is associated with

  1. Serrated leaf mutant in mungbean (Vigna radiata (L) Wilczek)

    International Nuclear Information System (INIS)

    Malik, I.A.; Ghulam, Sarwar; Yousaf, Ali; Saleem, M.

    1988-01-01

    Dry dormant seeds of mungbean (Vigna radiata (L) Wilczek) were treated with gamma rays (15, 30 and 60 kR). The serrated leaf mutation was noticed in M 2 of cultivar Pak 32 treated with 60 kR. Cf 14 plants, 3 showed the altered leaf structure and the others were normal. The feature of this mutant was the deep serration of leaflet margins. The mutant had large thick leaflets with prominent venation. The mutant bred true in the M 3 and successive generation. Details of the morphological characteristics of the mutant are presented. The mutant exhibited slower growth particularly during the early stages of development, flowered later and attained shorter height. There was an increase in the number of pods, in seed weight and in seed protein content, but number of seed per pod was considerably reduced. The seed coat colour showed a change from green to yellowish green. In the mutant's flowers the stamina were placed much below the stigma level and the stigma sometimes protruded the corolla. Outcrossing of 4% recorded in some of the mutant lines revealed a reduced cleistogamy. The low number of seeds per pod in the mutant could be due to reduced pollen fertility. The mutant behaved as monogenic recessive. The symbols SL/sl are proposed for this allelic pair. The mutant may have use as a green manure crop because of its large foliage and for the breeders as a genetic marker

  2. Regulatory Mechanisms of a Highly Pectinolytic Mutant of Penicillium occitanis and Functional Analysis of a Candidate Gene in the Plant Pathogen Fusarium oxysporum

    Directory of Open Access Journals (Sweden)

    Gustavo Bravo-Ruiz

    2017-09-01

    Full Text Available Penicillium occitanis is a model system for enzymatic regulation. A mutant strain exhibiting constitutive overproduction of different pectinolytic enzymes both under inducing (pectin or repressing conditions (glucose was previously isolated after chemical mutagenesis. In order to identify the molecular basis of this regulatory mechanism, the genomes of the wild type and the derived mutant strain were sequenced and compared, providing the first reference genome for this species. We used a phylogenomic approach to compare P. occitanis with other pectinolytic fungi and to trace expansions of gene families involved in carbohydrate degradation. Genome comparison between wild type and mutant identified seven mutations associated with predicted proteins. The most likely candidate was a mutation in a highly conserved serine residue of a conserved fungal protein containing a GAL4-like Zn2Cys6 binuclear cluster DNA-binding domain and a fungus-specific transcription factor regulatory middle homology region. To functionally characterize the role of this candidate gene, the mutation was recapitulated in the predicted orthologue Fusarium oxysporum, a vascular wilt pathogen which secretes a wide array of plant cell wall degrading enzymes, including polygalacturonases, pectate lyases, xylanases and proteases, all of which contribute to infection. However, neither the null mutant nor a mutant carrying the analogous point mutation exhibited a deregulation of pectinolytic enzymes. The availability, annotation and phylogenomic analysis of the P. occitanis genome sequence represents an important resource for understanding the evolution and biology of this species, and sets the basis for the discovery of new genes of biotechnological interest for the degradation of complex polysaccharides.

  3. Regulatory Mechanisms of a Highly Pectinolytic Mutant ofPenicillium occitanisand Functional Analysis of a Candidate Gene in the Plant PathogenFusarium oxysporum.

    Science.gov (United States)

    Bravo-Ruiz, Gustavo; Sassi, Azza Hadj; Marcet-Houben, Marina; Di Pietro, Antonio; Gargouri, Ali; Gabaldon, Toni; Roncero, M Isabel G

    2017-01-01

    Penicillium occitanis is a model system for enzymatic regulation. A mutant strain exhibiting constitutive overproduction of different pectinolytic enzymes both under inducing (pectin) or repressing conditions (glucose) was previously isolated after chemical mutagenesis. In order to identify the molecular basis of this regulatory mechanism, the genomes of the wild type and the derived mutant strain were sequenced and compared, providing the first reference genome for this species. We used a phylogenomic approach to compare P. occitanis with other pectinolytic fungi and to trace expansions of gene families involved in carbohydrate degradation. Genome comparison between wild type and mutant identified seven mutations associated with predicted proteins. The most likely candidate was a mutation in a highly conserved serine residue of a conserved fungal protein containing a GAL4-like Zn 2 Cys 6 binuclear cluster DNA-binding domain and a fungus-specific transcription factor regulatory middle homology region. To functionally characterize the role of this candidate gene, the mutation was recapitulated in the predicted orthologue Fusarium oxysporum , a vascular wilt pathogen which secretes a wide array of plant cell wall degrading enzymes, including polygalacturonases, pectate lyases, xylanases and proteases, all of which contribute to infection. However, neither the null mutant nor a mutant carrying the analogous point mutation exhibited a deregulation of pectinolytic enzymes. The availability, annotation and phylogenomic analysis of the P. occitanis genome sequence represents an important resource for understanding the evolution and biology of this species, and sets the basis for the discovery of new genes of biotechnological interest for the degradation of complex polysaccharides.

  4. Enzymatic Browning: a practical class

    Directory of Open Access Journals (Sweden)

    Maria Teresa Pedrosa Silva Clerici

    2014-10-01

    Full Text Available This paper presents a practical class about the enzymes polyphenol oxidases, which have been shown to be responsible for the enzymatic browning of fruits and vegetables. Vegetables samples were submitted to enzymatic inactivation process with chemical reagents, as well as by bleaching methods of applying heat by conventional oven and microwave oven. Process efficiency was assessed qualitatively by both observing the guaiacol peroxidase activity and after the storage period under refrigeration or freezing. The practical results obtained in this class allow exploring multidisciplinary knowledge in food science, with practical applications in everyday life.

  5. Angelman syndrome-associated ubiquitin ligase UBE3A/E6AP mutants interfere with the proteolytic activity of the proteasome.

    Science.gov (United States)

    Tomaić, V; Banks, L

    2015-01-29

    Angelman syndrome, a severe neurodevelopmental disease, occurs primarily due to genetic defects, which cause lack of expression or mutations in the wild-type E6AP/UBE3A protein. A proportion of the Angelman syndrome patients bear UBE3A point mutations, which do not interfere with the expression of the full-length protein, however, these individuals still develop physiological conditions of the disease. Interestingly, most of these mutations are catalytically defective, thereby indicating the importance of UBE3A enzymatic activity role in the Angelman syndrome pathology. In this study, we show that Angelman syndrome-associated mutants interact strongly with the proteasome via the S5a proteasomal subunit, resulting in an overall inhibitory effect on the proteolytic activity of the proteasome. Our results suggest that mutated catalytically inactive forms of UBE3A may cause defects in overall proteasome function, which could have an important role in the Angelman syndrome pathology.

  6. Influência do tamanho das partículas na solubilização enzimática da proteína do farelo de soja = The influence of particle size in the enzymatic extraction of soybean meal protein

    Directory of Open Access Journals (Sweden)

    Maura Stenzel

    2009-04-01

    Full Text Available O objetivo deste trabalho foi verificar a influência do tamanho das partículas na quantidade de proteína solubilizada a partir do farelo de soja. O farelo foi peneirado e cada fração foi submetida à hidrólise enzimática com as proteases Alcalase e Flavourzyme. Ashidrólises foram realizadas a 60ºC, agitação de 100 rpm, concentração de Alcalase 0,5% (proteína/proteína por 3h e concentração de Flavourzyme 1% (proteína/proteína por 5h. A quantidade de proteína solubilizada foi determinada pelo método de Lowry. A análisegranulométrica do farelo mostrou que quase metade da amostra corresponde a partículas maiores que 1,68 mm. O modelo que resultou em melhor ajuste dos dados foi o GGS, e o diâmetro médio de Sauter da amostra foi 1,150 mm. Com relação à hidrólise enzimática,para as duas enzimas foi verificada uma tendência de aumento na quantidade solubilizada para as frações mais finas e maior efetividade da Alcalase na solubilização proteica. The objective of this work was to verify the influence of particlesize in the results of enzymatic extraction of soybean meal protein. The meal was classified in homogeneous fractions, and each was subjected to enzymatic hydrolysis with Alcalase and Flavourzyme proteases. The hydrolyses were conducted at 60ºC, with agitation of 100 rpm, 0.5% concentration of Alcalase (protein/protein for three hours, and 1% concentration of Flavourzyme (protein/protein for five hours. The results were analyzed based on the amount of solubilized protein, determined through the Lowry method. The granulometric analysis of the meal showed that almost half of the sample is composed of particles larger that 1.68 mm. The model that resulted in the best adjustment of experimental data was GGS, and the Sauter mean diameter of the sample was 1.150 mm. Regarding enzymatic hydrolysis, a trend of increase in the amount of solubilized proteinwas verified for both enzymes for the finer fractions and greater

  7. Phenotype analysis of male transgenic mice overexpressing mutant IGFBP-2 lacking the Cardin-Weintraub sequence motif: Reduced expression of synaptic markers and myelin basic protein in the brain and a lower degree of anxiety-like behaviour.

    Science.gov (United States)

    Schindler, N; Mayer, J; Saenger, S; Gimsa, U; Walz, C; Brenmoehl, J; Ohde, D; Wirthgen, E; Tuchscherer, A; Russo, V C; Frank, M; Kirschstein, T; Metzger, F; Hoeflich, A

    2017-04-01

    Brain growth and function are regulated by insulin-like growth factors I and II (IGF-I and IGF-II) but also by IGF-binding proteins (IGFBPs), including IGFBP-2. In addition to modulating IGF activities, IGFBP-2 interacts with a number of components of the extracellular matrix and cell membrane via a Cardin-Weintraub sequence or heparin binding domain (HBD1). The nature and the signalling elicited by these interactions are not fully understood. Here, we examined transgenic mice (H1d-hBP2) overexpressing a mutant human IGFBP-2 that lacks a specific heparin binding domain (HBD1) known as the Cardin-Weintraub sequence. H1d-hBP2 transgenic mice have the genetic background of FVB mice and are characterized by severe deficits in brain growth throughout their lifetime (pmice, protein levels of the GTPase dynamin-I were significantly reduced (pmice in the cerebellum but not in the hippocampus. At 80weeks of age, weight reductions were similarly present in the cerebellum (-28%; pmice were challenged in the elevated plus maze, aged but not younger H1d-hBP2 mice displayed significantly less anxiety-like behaviour, which was also observed in a second transgenic mouse model overexpressing mouse IGFBP-2 lacking HBD1 (H1d-mBP2). These in vivo studies provide, for the first time, evidence for a specific role of IGFBP-2 in brain functions associated with anxiety and risk behaviour. These activities of IGFBP-2 could be mediated by the Cardin-Weintraub/HBD1 sequence and are altered in mice expressing IGFBP-2 lacking the HBD1. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  8. Enzymatically crosslinked silk-hyaluronic acid hydrogels.

    Science.gov (United States)

    Raia, Nicole R; Partlow, Benjamin P; McGill, Meghan; Kimmerling, Erica Palma; Ghezzi, Chiara E; Kaplan, David L

    2017-07-01

    In this study, silk fibroin and hyaluronic acid (HA) were enzymatically crosslinked to form biocompatible composite hydrogels with tunable mechanical properties similar to that of native tissues. The formation of di-tyrosine crosslinks between silk fibroin proteins via horseradish peroxidase has resulted in a highly elastic hydrogel but exhibits time-dependent stiffening related to silk self-assembly and crystallization. Utilizing the same method of crosslinking, tyramine-substituted HA forms hydrophilic and bioactive hydrogels that tend to have limited mechanics and degrade rapidly. To address the limitations of these singular component scaffolds, HA was covalently crosslinked with silk, forming a composite hydrogel that exhibited both mechanical integrity and hydrophilicity. The composite hydrogels were assessed using unconfined compression and infrared spectroscopy to reveal of the physical properties over time in relation to polymer concentration. In addition, the hydrogels were characterized by enzymatic degradation and for cytotoxicity. Results showed that increasing HA concentration, decreased gelation time, increased degradation rate, and reduced changes that were observed over time in mechanics, water retention, and crystallization. These hydrogel composites provide a biologically relevant system with controllable temporal stiffening and elasticity, thus offering enhanced tunable scaffolds for short or long term applications in tissue engineering. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Neutralisation of specific surface carboxylates speeds up translocation of botulinum neurotoxin type B enzymatic domain.

    Science.gov (United States)

    Pirazzini, Marco; Henke, Tina; Rossetto, Ornella; Mahrhold, Stefan; Krez, Nadja; Rummel, Andreas; Montecucco, Cesare; Binz, Thomas

    2013-11-29

    Botulinum neurotoxins translocate their enzymatic domain across vesicular membranes. The molecular triggers of this process are unknown. Here, we tested the possibility that this is elicited by protonation of conserved surface carboxylates. Glutamate-48, glutamate-653 and aspartate-877 were identified as possible candidates and changed into amide. This triple mutant showed increased neurotoxicity due to faster cytosolic delivery of the enzymatic domain; membrane translocation could take place at less acidic pH. Thus, neutralisation of specific negative surface charges facilitates membrane contact permitting a faster initiation of the toxin membrane insertion. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  10. Kinetic modelling of enzymatic starch hydrolysis

    NARCIS (Netherlands)

    Bednarska, K.A.

    2015-01-01

    Kinetic modelling of enzymatic starch hydrolysis – a summary

    K.A. Bednarska

    The dissertation entitled ‘Kinetic modelling of enzymatic starch hydrolysis’ describes the enzymatic hydrolysis and kinetic modelling of liquefaction and saccharification of wheat starch.

  11. Production of cellulases and xylanases under catabolic repression conditions from mutant PR-22 of Cellulomonas flavigena.

    Science.gov (United States)

    Rojas-Rejón, Oscar A; Poggi-Varaldo, Héctor M; Ramos-Valdivia, Ana C; Martínez-Jiménez, Alfredo; Cristiani-Urbina, Eliseo; de la Torre Martínez, Mayra; Ponce-Noyola, Teresa

    2011-01-01

    Derepressed mutant PR-22 was obtained by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenic treatment of Cellulomonas flavigena PN-120. This mutant improved its xylanolytic activity from 26.9 to 40 U mg(-1) and cellulolytic activity from 1.9 to 4 U mg(-1); this represented rates almost 2 and 1.5 times higher, respectively, compared to its parent strain growing in sugarcane bagasse. Either glucose or cellobiose was added to cultures of C. flavigena PN-120 and mutant PR-22 induced with sugarcane bagasse in batch culture. The inhibitory effect of glucose on xylanase activity was more noticeable for parent strain PN-120 than for mutant PR-22. When 20 mM glucose was added, the xylanolytic activity decreased 41% compared to the culture grown without glucose in mutant PR-22, whereas in the PN-120 strain the xylanolytic activity decreased by 49% at the same conditions compared to its own control. Addition of 10 and 15 mM of glucose did not adversely affect CMCase activity in PR-22, but glucose at 20 mM inhibited the enzymatic activity by 28%. The CMCase activity of the PN-120 strain was more sensitive to glucose than PR-22, with a reduction of CMCase activity in the range of 20-32%. Cellobiose had a more significant effect on xylanase and CMCase activities than glucose did in the mutant PR-22 and parent strain. Nevertheless, the activities under both conditions were always higher in the mutant PR-22 than in the PN-120 strain. Enzymatic saccharification experiments showed that it is possible to accumulate up to 10 g l(-1) of total soluble sugars from pretreated sugarcane bagasse with the concentrated enzymatic crude extract from mutant PR-22.

  12. Characterization of a Legionella micdadei mip mutant

    DEFF Research Database (Denmark)

    O'Connell, W A; Bangsborg, Jette Marie; Cianciotto, N P

    1995-01-01

    The pathogenesis of Legionella micdadei is dependent upon its ability to infect alveolar phagocytes. To better understand the basis of intracellular infection by this organism, we examined the importance of its Mip surface protein. In Legionella pneumophila, Mip promotes infection of both human m...... into the phagocyte. Similarly, the mutant was less able to parasitize Hartmannella amoebae. Taken together, these data argue that Mip specifically potentiates intracellular growth by L. micdadei....

  13. Photorepair mutants of Arabidopsis

    International Nuclear Information System (INIS)

    Jiang, C.Z.; Yee, J.; Mitchell, D.L.; Britt, A.B.

    1997-01-01

    UV radiation induces two major DNA damage products, the cyclobutane pyrimidine dimer (CPD) and, at a lower frequency, the pyrimidine (6-4) pyrimidinone dimer (6-4 product). Although Escherichia coli and Saccharomyces cerevisiae produce a CPD-specific photolyase that eliminates only this class of dimer, Arabidopsis thaliana, Drosophila melanogaster, Crotalus atrox, and Xenopus laevis have recently been shown to photoreactivate both CPDs and 6-4 products. We describe the isolation and characterization of two new classes of mutants of Arabidopsis, termed uvr2 and uvr3, that are defective in the photoreactivation of CPDs and 6-4 products, respectively. We demonstrate that the CPD photolyase mutation is genetically linked to a DNA sequence encoding a type II (metazoan) CPD photolyase. In addition, we are able to generate plants in which only CPDs or 6-4 products are photoreactivated in the nuclear genome by exposing these mutants to UV light and then allowing them to repair one or the other class of dimers. This provides us with a unique opportunity to study the biological consequences of each of these two major UV-induced photoproducts in an intact living system

  14. Enzymatic and non-enzymatic detoxification of 4-hydroxynonenal: Methodological aspects and biological consequences.

    Science.gov (United States)

    Mol, Marco; Regazzoni, Luca; Altomare, Alessandra; Degani, Genny; Carini, Marina; Vistoli, Giulio; Aldini, Giancarlo

    2017-10-01

    4-Hydroxynonenal (HNE), an electrophilic end-product deriving from lipid peroxidation, undergoes a heterogeneous set of biotransformations including enzymatic and non-enzymatic reactions. The former mostly involve red-ox reactions on the HNE oxygenated functions (phase I metabolism) and GSH conjugations (phase II) while the latter are due to the HNE capacity to spontaneously condense with nucleophilic sites within endogenous molecules such as proteins, nucleic acids and phospholipids. The overall metabolic fate of HNE has recently attracted great interest not only because it clearly determines the HNE disposal, but especially because the generated metabolites and adducts are not inactive molecules (as initially believed) but show biological activities even more pronounced than those of the parent compound as exemplified by potent pro-inflammatory stimulus induced by GSH conjugates. Similarly, several studies revealed that the non-enzymatic reactions, initially considered as damaging processes randomly involving all endogenous nucleophilic reactants, are in fact quite selective in terms of both reactivity of the nucleophilic sites and stability of the generated adducts. Even though many formed adducts retain the expected toxic consequences, some adducts exhibit well-defined beneficial roles as documented by the protective effects of sublethal concentrations of HNE against toxic concentrations of HNE. Clearly, future investigations are required to gain a more detailed understanding of the metabolic fate of HNE as well as to identify novel targets involved in the biological activity of the HNE metabolites. These studies are and will be permitted by the continuous progress in the analytical methods for the identification and quantitation of novel HNE metabolites as well as for proteomic analyses able to offer a comprehensive picture of the HNE-induced adducted targets. On these grounds, the present review will focus on the major enzymatic and non-enzymatic HNE

  15. Shotgun protein sequencing.

    Energy Technology Data Exchange (ETDEWEB)

    Faulon, Jean-Loup Michel; Heffelfinger, Grant S.

    2009-06-01

    A novel experimental and computational technique based on multiple enzymatic digestion of a protein or protein mixture that reconstructs protein sequences from sequences of overlapping peptides is described in this SAND report. This approach, analogous to shotgun sequencing of DNA, is to be used to sequence alternative spliced proteins, to identify post-translational modifications, and to sequence genetically engineered proteins.

  16. Characteristics of mutant lines of sweet potato flour

    International Nuclear Information System (INIS)

    Aryanti

    2012-01-01

    Research on mutation induction of sweet potato Sari variety has been conducted. Flour mutant lines were obtained from selection of M1V5 tubers irradiated by gamma rays at the dose of 10 Gy. Flour was made by peeling of tubers, then dried, blended and sieved. The quality test of flour have been done by measuring degree of whiteness, proximate, amylose contents, water content, soluble water, swelling power, and flour characteristics. The result of this work showed that flour of C6.26.13 mutant line had higher protein content than the parent plant with concentration of 3.62 % and its amylose content was also higher than the other mutant lines. The soluble water value of mutant lines were significant different compared to the parent plant from 1.82 to 2.25 % and swelling power from 4.28 to 5.55 %. The flour granule of the mutant line was different compared to the parent plant. (author)

  17. Quantifying the limits of transition state theory in enzymatic catalysis.

    Science.gov (United States)

    Zinovjev, Kirill; Tuñón, Iñaki

    2017-11-21

    While being one of the most popular reaction rate theories, the applicability of transition state theory to the study of enzymatic reactions has been often challenged. The complex dynamic nature of the protein environment raised the question about the validity of the nonrecrossing hypothesis, a cornerstone in this theory. We present a computational strategy to quantify the error associated to transition state theory from the number of recrossings observed at the equicommittor, which is the best possible dividing surface. Application of a direct multidimensional transition state optimization to the hydride transfer step in human dihydrofolate reductase shows that both the participation of the protein degrees of freedom in the reaction coordinate and the error associated to the nonrecrossing hypothesis are small. Thus, the use of transition state theory, even with simplified reaction coordinates, provides a good theoretical framework for the study of enzymatic catalysis. Copyright © 2017 the Author(s). Published by PNAS.

  18. Isozyme differences in barley mutants

    International Nuclear Information System (INIS)

    AI-Jibouri, A.A.M.; Dham, K.M.

    1990-01-01

    Full text: Thirty mutants (M 11 ) of barley (Hordeum vulgare L.) induced by physical and chemical mutagens were analysed for isozyme composition using polyacrylamide gel electrophoresis. Results show that these mutants were different in the isozymes leucine aminopeptidase, esterase and peroxidase. The differences included the number of forms of each enzyme, relative mobility value and their intensity on the gel. Glutamate oxaloacetate transaminase isozyme was found in six molecular forms and these forms were similar in all mutants. (author)

  19. The functions of grainy head-like proteins in animals and fungi and the evolution of apical extracellular barriers.

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    Adam Paré

    Full Text Available The Grainy head (GRH family of transcription factors are crucial for the development and repair of epidermal barriers in all animals in which they have been studied. This is a high-level functional conservation, as the known structural and enzymatic genes regulated by GRH proteins differ between species depending on the type of epidermal barrier being formed. Interestingly, members of the CP2 superfamily of transcription factors, which encompasses the GRH and LSF families in animals, are also found in fungi--organisms that lack epidermal tissues. To shed light on CP2 protein function in fungi, we characterized a Neurospora crassa mutant lacking the CP2 member we refer to as grainy head-like (grhl. We show that Neurospora GRHL has a DNA-binding specificity similar to that of animal GRH proteins and dissimilar to that of animal LSF proteins. Neurospora grhl mutants are defective in conidial-spore dispersal due to an inability to remodel the cell wall, and we show that grhl mutants and the long-known conidial separation-2 (csp-2 mutants are allelic. We then characterized the transcriptomes of both Neurospora grhl mutants and Drosophila grh mutant embryos to look for similarities in the affected genes. Neurospora grhl appears to play a role in the development and remodeling of the cell wall, as well as in the activation of genes involved in defense and virulence. Drosophila GRH is required to activate the expression of many genes involved in cuticular/epidermal-barrier formation. We also present evidence that GRH plays a role in adult antimicrobial defense. These results, along with previous studies of animal GRH proteins, suggest the fascinating possibility that the apical extracellular barriers of some animals and fungi might share an evolutionary connection, and that the formation of physical barriers in the last common ancestor was under the control of a transcriptional code that included GRH-like proteins.

  20. Enzymatic vegetable organic extracts as soil biochemical biostimulants and atrazine extenders.

    Science.gov (United States)

    García-Martínez, Ana María; Tejada, Manuel; Díaz, Ana Isabel; Rodríguez-Morgado, Bruno; Bautista, Juan; Parrado, Juan

    2010-09-08

    The purpose of this study was to gather information on the potential effects of organic biostimulants on soil activity and atrazine biodegradation. Carob germ enzymatic extract (CGEE) and wheat condensed distiller solubles enzymatic extract (WCDS-EE) have been obtained using an enzymatic process; their main organic components are soluble carbohydrates and proteins in the form of peptides and free amino acids. Their application to soil results in high biostimulation, rapidly increased dehydrogenase, phosphatase and glucosidase activities, and an observed atrazine extender capacity due to inhibition of its mineralization. The extender capacity of both extracts is proportional to the protein/carbohydrate ratio content. As a result, these enzymatic extracts are highly microbially available, leading to two independent phenomena, fertility and an atrazine persistence that is linked to increased soil activity.

  1. Analysis of AtCry1 and Mutants

    Science.gov (United States)

    Burdick, Derek; Purvis, Adam; Ahmad, Margaret; Link, Justin J.; Engle, Dorothy

    Cryptochrome is an incredibly versatile protein that influences numerous biological processes such as plant growth, bird migration, and sleep cycles. Due to the versatility of this protein, understanding the mechanism would allow for advances in numerous fields such as crop growth, animal behavior, and sleep disorders. It is known that cryptochrome requires blue light to function, but the exact processes in the regulation of biological activity are still not fully understood. It is believed that the c-terminal domain of the protein undergoes a conformational change when exposed to blue light which allows for biological function. Three different non-functioning mutants were tested during this study to gain insight on the mechanism of cryptochrome. Absorbance spectra showed a difference between two of the mutants and the wild type with one mutant showing little difference. Immunoprecipitation experiments were also conducted to identify the different c-terminal responses of the mutants. By studying non functioning mutants of this protein, the mechanism of the protein can be further characterized. This two-month research experience in Paris allowed us to experience international and interdisciplinary collaborations in science and immerse in a different culture. The Borcer Fund for Student Research, Xavier University, Cincinnati, OH, and John Hauck Foundation.

  2. Discovery of a proteolytic flagellin family in diverse bacterial phyla that assembles enzymatically active flagella

    OpenAIRE

    Eckhard, Ulrich; Bandukwala, Hina; Mansfield, Michael J.; Marino, Giada; Cheng, Jiujun; Wallace, Iain; Holyoak, Todd; Charles, Trevor C.; Austin, John; Overall, Christopher M.; Doxey, Andrew C.

    2017-01-01

    Bacterial flagella are cell locomotion and occasional adhesion organelles composed primarily of the polymeric protein flagellin, but to date have not been associated with any enzymatic function. Here, we report the bioinformatics-driven discovery of a class of enzymatic flagellins that assemble to form proteolytically active flagella. Originating by a metallopeptidase insertion into the central flagellin hypervariable region, this flagellin family has expanded to at least 74 bacterial species...

  3. Evaluation of tall rice mutant

    International Nuclear Information System (INIS)

    Hakim, L.; Azam, M.A.; Miah, A.J.; Mansur, M.A.; Akanda, H.R.

    1989-01-01

    One tall mutant (Mut NS1) of rice variety Nizersail was put to multilocation on-farm trial. It showed improvement over the parent in respect of by earlier maturity and higher grain yield at all locations and thus it appears as an improved mutant of Nizersail. (author). 6 refs

  4. Dimerization and enzymatic activity of fungal 17β-hydroxysteroid dehydrogenase from the short-chain dehydrogenase/reductase superfamily

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