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Sample records for activity total protein

  1. Total Cellular RNA Modulates Protein Activity.

    PubMed

    Majumder, Subhabrata; DeMott, Christopher M; Reverdatto, Sergey; Burz, David S; Shekhtman, Alexander

    2016-08-16

    RNA constitutes up to 20% of a cell's dry weight, corresponding to ∼20 mg/mL. This high concentration of RNA facilitates low-affinity protein-RNA quinary interactions, which may play an important role in facilitating and regulating biological processes. In the yeast Pichia pastoris, the level of ubiquitin-RNA colocalization increases when cells are grown in the presence of dextrose and methanol instead of methanol as the sole carbon source. Total RNA isolated from cells grown in methanol increases β-galactosidase activity relative to that seen with RNA isolated from cells grown in the presence of dextrose and methanol. Because the total cellular RNA content changes with growth medium, protein-RNA quinary interactions can alter in-cell protein biochemistry and may play an important role in cell adaptation, critical to many physiological and pathological states. PMID:27456029

  2. Total protein

    MedlinePlus

    The total protein test measures the total amount of two classes of proteins found in the fluid portion of your ... nutritional problems, kidney disease or liver disease . If total protein is abnormal, you will need to have more ...

  3. Total protein

    MedlinePlus

    ... page: //medlineplus.gov/ency/article/003483.htm Total protein To use the sharing features on this page, please enable JavaScript. The total protein test measures the total amount of two classes ...

  4. Effects of gamma irradiation on chickpea seeds vis-a-vis total seed storage proteins, antioxidant activity and protein profiling.

    PubMed

    Bhagyawant, S S; Gupta, N; Shrivastava, N

    2015-01-01

    The present work describes radiation—induced effects on seed composition vis—à—vis total seed proteins, antioxidant levels and protein profiling employing two dimensional gel electrophoresis (2D—GE) in kabuli and desi chickpea varities. Seeds were exposed to the radiation doses of 1,2,3,4 and 5 kGy. The total protein concentrations decreased and antioxidant levels were increased with increasing dose compared to control seed samples. Radiation induced effects were dose dependent to these seed parameters while it showed tolerance to 1 kGy dose. Increase in the dose was complimented with increase in antioxidant levels, like 5 kGy enhanced % scavenging activities in all the seed extracts. Precisely, the investigations reflected that the dose range from 2 to 5 kGy was effective for total seed storage proteins, as depicted quantitatively and qualitative 2D—GE means enhance antioxidant activities in vitro. PMID:26516115

  5. CSF total protein

    MedlinePlus

    CSF total protein is a test to determine the amount of protein in your spinal fluid, also called cerebrospinal fluid (CSF). ... The normal protein range varies from lab to lab, but is typically about 15 to 60 mg/dL. Note: mg/dL = ...

  6. Modular Total Synthesis of Protein Kinase C Activator (-)-Indolactam V.

    PubMed

    Haynes-Smith, Jeremy; Diaz, Italia; Billingsley, Kelvin L

    2016-05-01

    A concise, eight-step total synthesis of (-)-indolactam V, a nanomolar agonist of protein kinase C, is reported. The synthesis relies upon an efficient copper-catalyzed amino acid arylation to establish the indole C4-nitrogen bond. This cross-coupling method is applicable to a range of hydrophobic amino acids, providing a platform for further diversification of indolactam alkaloid scaffolds and studies on their potent biological activity. PMID:27074538

  7. Screening of immunomodulatory activity of total and protein extracts of some Moroccan medicinal plants.

    PubMed

    Daoudi, Abdeljlil; Aarab, Lotfi; Abdel-Sattar, Essam

    2013-04-01

    Herbal and traditional medicines are being widely used in practice in many countries for their benefits of treating different ailments. A large number of plants in Morocco were used in folk medicine to treat immune-related disorders. The objective of this study is to evaluate the immunomodulatory activity of protein extracts (PEs) of 14 Moroccan medicinal plants. This activity was tested on the proliferation of immune cells. The prepared total and PEs of the plant samples were tested using MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) assay on the splenocytes with or without stimulation by concanavalin-A (Con-A), a mitogenic agent used as positive control. The results of this study indicated different activity spectra. Three groups of activities were observed. The first group represented by Citrullus colocynthis, Urtica dioica, Elettaria cardamomum, Capparis spinosa and Piper cubeba showed a significant immunosuppressive activity. The second group that showed a significant immunostimulatory activity was represented by Aristolochia longa, Datura stramonium, Marrubium vulgare, Sinapis nigra, Delphynium staphysagria, Lepidium sativum, Ammi visnaga and Tetraclinis articulata. The rest of the plant extracts did not alter the proliferation induced by Con-A. This result was more important for the PE than for the total extract. In conclusion, this study revealed an interesting immunomodulating action of certain PEs, which could explain their traditional use. The results of this study may also have implications in therapeutic treatment of infections, such as prophylactic and adjuvant with cancer chemotherapy. PMID:22301818

  8. Total protein, animal protein, and physical activity in relation to muscle mass in middle-aged and older Americans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Resistance training is recognized as a good strategy for retarding age-related declines in muscle mass and strength. Recent studies have also highlighted the potential value of protein intakes in excess of current recommendations. The roles that leisure-time physical activity and protein quality mig...

  9. In-vivo assessment of total body protein in rats by prompt-γ neutron activation analysis

    NASA Astrophysics Data System (ADS)

    Stamatelatos, Ion E.; Boozer, Carol N.; Ma, Ruimei; Yasumura, Seiichi

    1997-02-01

    A prompt-(gamma) neutron activation analysis facility for in vivo determination of total body protein (TBP) in rats has been designed. TBP is determined in vivo by assessment of total body nitrogen. The facility is based on a 252Cf radionuclide neutron source within a heavy water moderator assembly and two NaI(Tl) scintillation detectors. The in vivo precision of the technique, as estimated by three repeated measurements of 15 rats is 6 percent, for a radiation dose equivalent of 60 mSv. The radiation dose per measurement is sufficiently low to enable serial measurements on the same animal. MCNP-4A Monte Carlo transport code was utilized to calculate thermal neutron flux correction factors to account for differences in size and shape of the rats and calibration phantoms. Good agrement was observed in comparing body nitrogen assessment by prompt-(gamma) neutron activation and chemical carcass analysis.

  10. Effects of protein kinase C activation on sodium, potassium, chloride, and total CO2 transport in the rabbit cortical collecting tubule.

    PubMed Central

    Hays, S R; Baum, M; Kokko, J P

    1987-01-01

    Several hormones induce phosphatidylinositol turnover in cell membranes and thus activate protein kinase C. Activation of protein kinase C can, in turn, have effects on epithelial transport. These experiments were designed to investigate the effects of two activators of protein kinase C, phorbol 12-myristate,13-acetate (PMA) and L-alpha-1,2-dioctanoylglycerol (L-alpha-1,2-DOG), and two inactive analogues, 4 alpha-phorbol and 4-O-methyl phorbol 12-myristate,13-acetate, on sodium, potassium, chloride, and total CO2 transport in the rabbit cortical collecting tubule. Utilizing in vitro microperfusion techniques, we found that activation of protein kinase C with either PMA or L-alpha-1,2-DOG significantly inhibited net sodium absorption, net potassium secretion and transepithelial voltage in a dose-dependent manner. There was no effect on net chloride or total CO2 transport. In contrast, the inactive phorbol analogues did not alter either sodium or potassium transport. These studies demonstrate that in the rabbit cortical collecting tubule sodium and potassium transport can be inhibited by compounds known to activate proteins kinase C. Thus, hormones that induce phosphatidylinositol turnover in the rabbit cortical collecting tubule may lead to inhibition of sodium transport by activation of protein kinase C. PMID:3680514

  11. MAFbx, MuRF1, and the stress-activated protein kinases are upregulated in muscle cells during total knee arthroplasty

    PubMed Central

    Bailey, Ashley N.; Hocker, Austin D.; Vermillion, Benjamin R.; Smolkowski, Keith; Shah, Steven N.; Jewett, Brian A.

    2012-01-01

    Total knee arthroplasty (TKA) is the most common and a cost-effective surgical remediation for older adults with long-standing osteoarthritis. In parallel with the expanding population of older adults, the number of TKAs performed annually is projected to be 3.48 million by 2030. During this surgery, a tourniquet is used to stop blood flow to the operative leg. However, the molecular pathways that are affected by tourniquet use during TKA continue to be elucidated. We hypothesized that components of the catabolic FoxO3a (i.e., MuRF1, MAFbx, and Bnip3) pathway, as well as the cellular stress pathways [i.e., stress-activated protein kinase (SAPK)/JNK and MAPKs], are upregulated during TKA. The purpose of this study was to measure changes in transcripts and proteins involved in muscle cell catabolic and stress-activated pathways. We obtained muscle biopsies from subjects, 70 ± 1.3 yr, during TKA, from the vastus lateralis at baseline (before tourniquet inflation), during maximal ischemia (just before tourniquet release), and during reperfusion. Total tourniquet time was 43 ± 2 min and reperfusion time was 16 ± 1. Significant increases in FoxO3a downstream targets, MAFbx and MuRF1, were present for mRNA levels during ischemia (MAFbx, P = 0.04; MuRF1, P = 0.04), and protein expression during ischemia (MAFbx, P = 0.002; MuRF1, P = 0.001) and reperfusion (MuRF1, P = 0.002). Additionally, stress-activated JNK gene expression (P = 0.01) and protein were elevated during ischemia (P = 0.001). The results of this study support our hypothesis that protein degradation pathways are stimulated during TKA. Muscle protein catabolism is likely to play a role in the rapid loss of muscle volume measured within 2 wk of this surgery. PMID:22761181

  12. Effects of soybean trypsin inhibitor on hypopharyngeal gland protein content, total midgut protease activity and survival of the honey bee (Apis mellifera L.).

    PubMed

    Sagili, Ramesh R; Pankiw, Tanya; Zhu-Salzman, Keyan

    2005-09-01

    Insecticidal properties of protease inhibitors have been established in transgenic plants. In the wake of continuous research and rapid development of protease inhibitors it is important to assess possible effects on beneficial insects like the honey bee (Apis mellifera L.). In this study, newly emerged caged bees were fed pollen diets containing three different concentrations (0.1%, 0.5% and 1% w:w) of soybean trypsin inhibitor (SBTI). Hypopharyngeal gland protein content, total midgut proteolytic enzyme activity of these bees, and survival were measured. Bees fed 1% SBTI had significantly reduced hypopharyngeal gland protein content and midgut proteolytic enzyme activity. There were no significant differences between control, 0.1% and 0.5% SBTI treatments. Bees fed a diet containing 1% SBTI had the lowest survival, followed by 0.5% and 0.1%, over a 30-day period. We concluded that nurse bees fed a pollen diet containing at least 1% SBTI would be poor producers of larval food, potentially threatening colony growth and maintenance. PMID:15927200

  13. 21 CFR 862.1635 - Total protein test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Total protein test system. 862.1635 Section 862....1635 Total protein test system. (a) Identification. A total protein test system is a device intended to measure total protein(s) in serum or plasma. Measurements obtained by this device are used in...

  14. 21 CFR 862.1635 - Total protein test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Total protein test system. 862.1635 Section 862....1635 Total protein test system. (a) Identification. A total protein test system is a device intended to measure total protein(s) in serum or plasma. Measurements obtained by this device are used in...

  15. Catalase and superoxide dismutase activities and the total protein content of protocorm-like bodies of Dendrobium sonia-28 subjected to vitrification.

    PubMed

    Poobathy, Ranjetta; Sinniah, Uma Rani; Xavier, Rathinam; Subramaniam, Sreeramanan

    2013-07-01

    Dendrobium sonia-28 is an important ornamental orchid in the Malaysian flower industry. However, the genus faces both low germination rates and the risk of producing heterozygous progenies. Cryopreservation is currently the favoured long-term storage method for orchids with propagation problems. Vitrification, a frequently used cryopreservation technique, involves the application of pretreatments and cryoprotectants to protect and recover explants during and after storage in liquid nitrogen. However, cryopreservation may cause osmotic injuries and toxicity to cryopreserved explants from the use of highly concentrated additives, and cellular injuries from thawing, devitrification and ice formation. Reactive oxygen species (ROS), occurring during dehydration and cryopreservation, may also cause membrane damage. Plants possess efficient antioxidant systems such as the superoxide dismutase (SOD) and catalase (CAT) enzymes to scavenge ROS during low temperature stress. In this study, protocorm-like bodies (PLBs) of Dendrobium sonia-28 were assayed for the total protein content, and both SOD and CAT activities, at each stage of a vitrification exercise to observe for deleterious stages in the protocol. The results indicated that cryopreserved PLBs of Dendrobium sonia-28 underwent excessive post-thawing oxidative stress due to decreased levels of the CAT enzyme at the post-thawing recovery stage, which contributed to the poor survival rates of the cryopreserved PLBs. PMID:23640259

  16. Outdoor Activities for a Total Lunar Eclipse

    ERIC Educational Resources Information Center

    Glenn, William H.

    1977-01-01

    Describes several activities involving a total eclipse of the moon. Included are observations with binoculars, naked eye, and telescope; timing activities and observations of color changes during penumbral and umbral phases; and photography activities. (CS)

  17. Total Protein and Albumin/Globulin Ratio Test

    MedlinePlus

    ... limited. Home Visit Global Sites Search Help? Total Protein and Albumin/Globulin (A/G) Ratio Share this ... Globulin Ratio; A/G Ratio Formal name: Total Protein; Albumin to Globulin Ratio Related tests: Albumin ; Liver ...

  18. Protein extraction from activated sludge.

    PubMed

    Denecke, M

    2006-01-01

    Two methods for the separation of protein originating from activated sludge were compared. In one method, the total protein was isolated out of the activated sludge (crude extract). These samples included all dissolved proteins originating from the bacterial cells and biofilm made up of extracellular polymeric substances (EPS). Every time polyacrylamide gel electrophoresis (PAGE) was done, the protein bands from samples of crude extract were covered by polymeric substances including carbohydrates, uronic acids or humic compounds. Using the immunoblot technique it was possible to demonstrate the presence of the heat shock protein HSP70 in crude extracts of activated sludge. The comparison of protein fingerprints required that clear and distinct bands appear on the PAGE analysis. To this end, a procedure to separates bacterial cells from the EPS was developed. Bacterial cells were separated by incubation with EDTA and subsequent filtration. The isolated cells were directly incubated in a sample buffer. PMID:16898150

  19. An amplified electrochemical proximity immunoassay for the total protein of Nosema bombycis based on the catalytic activity of Fe3O4NPs towards methylene blue.

    PubMed

    Wang, Qin; Gan, Xianxue; Zang, Ruhua; Chai, Yaqin; Yuan, Yali; Yuan, Ruo

    2016-07-15

    A simple electrochemical proximity immunoassay (ECPA) system for the total protein of Nosema bombycis (TP N.b) detection has been developed on the basis of a new amplification strategy combined with target-induced proximity hybridization. The desirable ECPA system was achieved through following process: firstly, the methylene blue (MB) labeled hairpin DNA (MB-DNA) were immobilized on electrode through Au-S bonding. Then, the antibody labeled complementary single-stranded DNA probe (Ab1-S1) hybridized with MB-DNA to open its hairpin structure, which led to the labeled MB far away from electrode surface. After that, the presence of target biomarker (TP N.b) and antibody labeled single-stranded DNA (Ab2-S2) triggered the typical sandwich reaction and proximity hybridization, which resulted in the dissociation of Ab1-S1 from electrode and the transformation of the MB-DNA into a hairpin structure with MB approaching to electrode surface. In consequence, the hairpin-closed MB was electrocatalyzed by the modified magnetic nanoparticles (Fe3O4NPs), leading to an increased and amplified electrochemical signal for the quantitative detection of TP N.b. In the present work, Fe3O4NPs were acted as catalyst to electrocatalyze the reduction of electron mediator MB for signal amplification, which could not only overcome the drawbacks of protein enzyme in electrocatalytic signal amplification but also shorten the interaction distance between catalyst and substance. Under optimal condition, the proposed ECPA system exhibited a wide linear range from 0.001ngmL(-1) to 100ngmL(-)(1) with a detection limit (LOD) of 0.54pgmL(-1). Considering the desirable sensitivity and specificity, as well as the novel and simple features, this signal amplified ECPA system opened an opportunity for quantitative analysis of many other kinds of protein biomarker. PMID:26994365

  20. Physical Activity After Total Joint Arthroplasty

    PubMed Central

    Vogel, Laura A.; Carotenuto, Giuseppe; Basti, John J.; Levine, William N.

    2011-01-01

    Context: Total joint arthroplasty (TJA) is a common surgical option to treat painful degenerative joint disease. However, there is currently no consensus on the appropriate intensity of physical activity after TJA or how physical activity level affects the rate of revision surgery. Materials and Methods: A systematic review of the literature regarding physical or athletic activity after TJA was performed to determine current clinical opinion and recommendations regarding appropriate activity levels after TJA, as well as variables affecting successful surgery and improved outcomes. Results: Many studies in the literature regarding athletic activity after TJA focus on total hip arthroplasty and total knee arthroplasty. The literature reports contradictory results regarding rates of physical activity after TJA as well as the relationship between physical activity and rates of revision surgery. The current trend in expert opinion shows more liberal recommendations for patients to engage in athletic activity after TJA. Conclusions: Individual characteristics, lifestyle, and patient preferences must be taken into account when one considers appropriate recommendations for athletic activity after TJA. Current trends in clinical opinion favor a higher level of athletic activity after TJA, but clinicians should caution patients not to participate in contact sports or sports that create high joint loads in the replaced joint. PMID:23016041

  1. 21 CFR 862.1635 - Total protein test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Total protein test system. 862.1635 Section 862.1635 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1635 Total protein test system....

  2. 21 CFR 862.1635 - Total protein test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Total protein test system. 862.1635 Section 862.1635 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1635 Total protein test system....

  3. 21 CFR 862.1635 - Total protein test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Total protein test system. 862.1635 Section 862.1635 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1635 Total protein test system....

  4. Total body nitrogen analysis. [neutron activation analysis

    NASA Technical Reports Server (NTRS)

    Palmer, H. E.

    1975-01-01

    Studies of two potential in vivo neutron activation methods for determining total and partial body nitrogen in animals and humans are described. A method using the CO-11 in the expired air as a measure of nitrogen content was found to be adequate for small animals such as rats, but inadequate for human measurements due to a slow excretion rate. Studies on the method of measuring the induced N-13 in the body show that with further development, this method should be adequate for measuring muscle mass changes occurring in animals or humans during space flight.

  5. Quantification Assays for Total and Polyglutamine-Expanded Huntingtin Proteins

    PubMed Central

    Boogaard, Ivette; Smith, Melanie; Pulli, Kristiina; Szynol, Agnieszka; Albertus, Faywell; Lamers, Marieke B. A. C.; Dijkstra, Sipke; Kordt, Daniel; Reindl, Wolfgang; Herrmann, Frank; McAllister, George; Fischer, David F.; Munoz-Sanjuan, Ignacio

    2014-01-01

    The expansion of a CAG trinucleotide repeat in the huntingtin gene, which produces huntingtin protein with an expanded polyglutamine tract, is the cause of Huntington's disease (HD). Recent studies have reported that RNAi suppression of polyglutamine-expanded huntingtin (mutant HTT) in HD animal models can ameliorate disease phenotypes. A key requirement for such preclinical studies, as well as eventual clinical trials, aimed to reduce mutant HTT exposure is a robust method to measure HTT protein levels in select tissues. We have developed several sensitive and selective assays that measure either total human HTT or polyglutamine-expanded human HTT proteins on the electrochemiluminescence Meso Scale Discovery detection platform with an increased dynamic range over other methods. In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions. We demonstrate the application of these assays to measure HTT protein in several HD in vitro cellular and in vivo animal model systems as well as in HD patient biosamples. Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples. PMID:24816435

  6. Determination of IgG and IgM levels in sera of newborn calves until the 10th day of life by ELISA and description of their correlation to total plasma protein concentration and GGT activity.

    PubMed

    Bender, Petra; Bostedt, Hartwig

    2009-02-01

    The aim of this study was the determination of IgG and IgM concentrations in sera of 15 vital and healthy calves from the day of birth to the 10th day of life using two ELISAs exclusively developed for this purpose. We investigated if and to which extent the sera profiles were correlated with antibody levels in the colostral milk administered, with GGT activity and with total plasma protein content. Due to the assays' high sensitivity, traces of IgG and IgM in calf sera could be determined prior to the first uptake of the foremilk. Throughout the colostrum administration period until the 12th living hour, IgG and IgM levels remarkably increased (P < 0.0001).The correlation between IgG concentrations in sera determined 24 h post natum and the IgG content of the colostrum administered was highly significant (P < 0.001; r = 0.851), while the correlation of seral IgM levels 24 h post natum and the IgM content of the foremilk was significant (P = 0.009; r = 0.651). The sum of the IgG and IgM concentrations in calf serum 24 h post natum was significantly correlated with the neonatal plasma protein level (P = 0.01; r = 0.642). With P = 0.012; r = 0.629 and P = 0.029; r = 0.561 respectively, there was also a significant correlation between the subjects' IgG and IgM concentrations at 24 h post natum and the GGT activity in calf serum. By looking at individual cases, it became evident that the administration of colostrum containing maximum or minimum immunoglobulin concentrations does not necessarily result in the respective sera immunoglobulin concentrations. From these findings, as well as from the fact that numerous subjects displayed their highest IgG and IgM sera concentrations well after the gut closure, we conclude that individually diverse resorption patterns are in place which cannot be characterized by immunoglobulin measurements only. The determination of the total plasma protein content or GGT activity in calf serum at 24 h post natum only give a rough idea about

  7. The association between oxidative stress, activator protein-1, inflammatory, total antioxidant status and artery stiffness and the efficacy of olmesartan in elderly patients with mild-to-moderate essential hypertension.

    PubMed

    Liu, Qunwei; Han, Limin; Du, Qiufan; Zhang, Ming; Zhou, Shenghua; Shen, Xiangqian

    2016-01-01

    This study investigated the change of oxidative stress, activator protein-1 (AP-1), inflammatory, total antioxidant status (TAS) and artery stiffness, and explored the relationship between these characteristics and the efficacy of olmesartan intervention in elderly patients with mild-to-moderate essential hypertension (EH). In total, 386 elderly patients with EH and 353 normotensive controls were recruited. All study subjects had oxidative stress markers, AP-1, inflammatory factors, TAS and brancial-ankle artery pulse wave velocity (ba-PWV) measured. In total, 193 elderly patients with EH were randomized to olmesartan and were matched with 193 normotensive controls to observe the change of index above mentioned before and after the treatment. Compared with the controls, superoxide dismutase (SOD) and TAS were significantly reduced in patients with EH, and malondialdehyde (MDA), AP-1, high-sensitivity C-reactive protein (Hs-CRP), Monocyte Chemoattractant Protein-1 (MCP-1), heart rate, endothelin-1 (ET-1), TAS and ba-PWV were significantly increased (P < 0.01 for all). Pearson's correlation analysis showed that SOD and TAS were negatively related to AP-1 (P < 0.05 for all), and that blood pressure (BP), age, MDA, Hs-CRP, MCP-1, ET-1 were positively related to AP-1 (P < 0.01 for all). Multivariate linear regression analysis showed that BP, SOD, MDA, AP-1, Hs-CRP, MCP-1, ET-1, TAS, heart rate and age were independent risk factors for ba-PWV. After treatment with olmesartan, SOD and TAS were increased, while BP, heart rate, AP-1 and inflammatory factors were reduced with significant improvement in ba-PWV (P < 0.05 for all). More increase of arterial stiffness was reported in elderly hypertensive patients with greater oxidative stress, inflammatory, AP-1, heart rate, and lower TAS. Higher oxidative stress, AP-1 and inflammatory may predict higher arterial stiffness. Olmesartan may increase TAS, yet inhibit oxidative stress, AP-1, inflammatory, and heart rate with

  8. New quantitative total protein S-assay system for diagnosing protein S type II deficiency: clinical application of the screening system for protein S type II deficiency.

    PubMed

    Tsuda, Tomohide; Jin, Xiuri; Tsuda, Hiroko; Ieko, Masahiro; Morishita, Eriko; Adachi, Tomoko; Hamasaki, Naotaka

    2012-01-01

    Venous thromboembolism (VTE) incidence is rising rapidly in Japan with lifestyle westernization and aging. Deficiency of protein S, an important blood coagulation regulator, is a risk factor for VTE. Protein S deficiency prevalence in Asians is approximately 10 times that in Caucasians and that of protein S type II deficiency, associated with the protein S Tokushima mutation (K155E), is quite high in Japan. However, currently available methods for measuring protein S are not precise enough for detection of this deficiency. We developed a novel assay system for precise simultaneous determinations of total protein S activity and total protein S antigen level, using a general-purpose automated analyzer, allowing protein S-specific activity (ratio of total protein S activity to total protein S antigen level) to be calculated. Mean specific activity was 0.99 for samples from healthy individuals but 0.69 or less (mean-3SD) in protein S type II-deficient and warfarin-treated samples, but was 1.0 in an estrogen-treated sample with significantly decreased protein S antigen. Protein S gene analyses in healthy individuals with specific activity 0.69 or less revealed the K155E mutation in all three. These results show our new assay system to be an effective screening tool for protein S type II deficiency. This system can also be used in an automated analyzer, facilitating numerous sample measurements, and is, thus, applicable to regular medical checkups and diagnosing VTE. Such applications would potentially contribute to early detection of protein S type II deficiency, and, thereby, to thrombosis prevention. PMID:22157257

  9. Active Robotics for Total Hip Arthroplasty.

    PubMed

    Dungy, Danton S; Netravali, Nathan A

    2016-01-01

    Robotics and computer-assisted navigation have been developed to increase the accuracy of hip implant placement and improve long-term outcomes of total hip arthroplasty (THA). These technologies have shown significant improvements in implant positioning when compared to conventional techniques. Currently, 3 robotic systems are cleared for use for THA in the US. The lead author (DSD) describes his preferred technique for using one of these systems, the TSolution One® (Think Surgical, Inc.). PMID:27327918

  10. Total protein or high-abundance protein: Which offers the best loading control for Western blotting?

    PubMed

    Thacker, Jonathan S; Yeung, Derrick H; Staines, W Richard; Mielke, John G

    2016-03-01

    Western blotting routinely involves a control for variability in the amount of protein across immunoblot lanes. Normalizing a target signal to one found for an abundantly expressed protein is widely regarded as a reliable loading control; however, this approach is being increasingly questioned. As a result, we compared blotting for two high-abundance proteins (actin and glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) and two total protein membrane staining methods (Ponceau and Coomassie Brilliant Blue) to determine the best control for loading variability. We found that Ponceau staining optimally balanced accuracy and precision, and we suggest that this approach be considered as an alternative to normalizing with a high-abundance protein. PMID:26706797

  11. Purification of total DNA extracted from activated sludge.

    PubMed

    Shan, Guobin; Jin, Wenbiao; Lam, Edward K H; Xing, Xinhui

    2008-01-01

    Purification of the total DNA extracted from activated sludge samples was studied. The effects of extraction buffers and lysis treatments (lysozyme, sodium dodecyl sulfate (SDS), sonication, mechanical mill and thermal shock) on yield and purity of the total DNA extracted from activated sludge were investigated. It was found that SDS and mechanical mill were the most effective ways for cell lysis, and both gave the highest DNA yields, while by SDS and thermal shock, the purest DNA extract could be obtained. The combination of SDS with other lysis treatment, such as sonication and thermal shock, could apparently increase the DNA yields but also result in severe shearing. For the purification of the crude DNA extract, polyvinyl polypyrrolidone was used for the removal of humic contaminants. Cetyltrimethyl ammonium bromide, potassium acetate and phenol/chloroform were used to remove proteins and polysaccharides from crude DNA. Crude DNA was further purified by isopropanol precipitation. Thus, a suitable protocol was proposed for DNA extraction, yielding about 49.9 mg (total DNA)/g volatile suspended solids, and the DNA extracts were successfully used in PCR amplifications for 16S rDNA and 16S rDNA V3 region. The PCR products of 16S rDNA V3 region allowed the DGGE analysis (denatured gradient gel electrophoresis) to be possible. PMID:18572527

  12. Separating proteins with activated carbon.

    PubMed

    Stone, Matthew T; Kozlov, Mikhail

    2014-07-15

    Activated carbon is applied to separate proteins based on differences in their size and effective charge. Three guidelines are suggested for the efficient separation of proteins with activated carbon. (1) Activated carbon can be used to efficiently remove smaller proteinaceous impurities from larger proteins. (2) Smaller proteinaceous impurities are most efficiently removed at a solution pH close to the impurity's isoelectric point, where they have a minimal effective charge. (3) The most efficient recovery of a small protein from activated carbon occurs at a solution pH further away from the protein's isoelectric point, where it is strongly charged. Studies measuring the binding capacities of individual polymers and proteins were used to develop these three guidelines, and they were then applied to the separation of several different protein mixtures. The ability of activated carbon to separate proteins was demonstrated to be broadly applicable with three different types of activated carbon by both static treatment and by flowing through a packed column of activated carbon. PMID:24898563

  13. A method for measuring total thiaminase activity in fish tissues

    USGS Publications Warehouse

    Zajicek, J.L.; Tillitt, D.E.; Honeyfield, D.C.; Brown, S.B.; Fitzsimons, J.D.

    2005-01-01

    An accurate, quantitative, and rapid method for the measurement of thiaminase activity in fish samples is required to provide sufficient information to characterize the role of dietary thiaminase in the onset of thiamine deficiency in Great Lakes salmonines. A radiometric method that uses 14C-thiamine was optimized for substrate and co-substrate (nicotinic acid) concentrations, incubation time, and sample dilution. Total thiaminase activity was successfully determined in extracts of selected Great Lakes fishes and invertebrates. Samples included whole-body and selected tissues of forage fishes. Positive control material prepared from frozen alewives Alosa pseudoharengus collected in Lake Michigan enhanced the development and application of the method. The method allowed improved discrimination of thiaminolytic activity among forage fish species and their tissues. The temperature dependence of the thiaminase activity observed in crude extracts of Lake Michigan alewives followed a Q10 = 2 relationship for the 1-37??C temperature range, which is consistent with the bacterial-derived thiaminase I protein. ?? Copyright by the American Fisheries Society 2005.

  14. Free radical scavenging activity, total phenolic content, total antioxidant status, and total oxidant status of endemic Thermopsis turcica

    PubMed Central

    Aksoy, Laçine; Kolay, Erdi; Ağılönü, Yasin; Aslan, Zeyneb; Kargıoğlu, Mustafa

    2013-01-01

    Thermopsis turcica, endemic to Turkey, is in danger of extinction. Studies on this species are very few due to the fact that it was only discovered in 1983 and grows in a small circumscribed area in Turkey. In this study, free radical scavenging activity, total phenolic content, total oxidant status (TOS), and total antioxidant status (TAS) of methanol (TTM) and acetone (TTA) extracts of T. turcica were measured spectroscopically. Free radical scavenging activity was determined according to the elimination of DPPH radicals and total phenol content was determined by the Folin–Ciocalteu reaction. Total oxidant status (TOS) and total antioxidant status (TAS) were measured with commercially available kits. Methanol and acetone extracts of T. turcica were found to have a specific radical scavenging effect. This effect was found to be related to the total phenolic content of the extracts. Since the TTA had a higher phenolic content than the methanol extract, it had a stronger radical scavenging effect. In addition, the total antioxidant capacity of the methanol extract was observed to be higher than that of its acetone counterpart. As a result, due to its antioxidative properties, T. turcica is thought to be a natural source of antioxidants. PMID:23961240

  15. Comparison of Urinary Total Proteins by Four Different Methods.

    PubMed

    Yalamati, Padma; Karra, Madhu Latha; Bhongir, Aparna V

    2016-10-01

    The total proteins in human urine have been compared by sulfosalicylic acid, sulfosalicylic acid with sodium sulphate and trichloroacetic acid methods with pyrogallol red molybdate method as there are no studies found quantifying imprecision and bias components. Fresh urine of 36 patients was analyzed by four methods. Imprecision and inaccuracy were determined by repeated analysis and method comparison studies using correlation plots, Bland and Altman, and Passing and Bablok regression analyses respectively. The coefficient of variation was 5.07 % for pyrogallol red molybdate; 6.84 % for sulfosalicylic acid; 3.97 % for sulfosalicylic acid with sodium sulphate and 5.93 % for trichloroacetic acid methods. Bland and Altman analysis showed a bias of 5.8, 1.7 and -5.4 for pyrogallol red molybdate versus sulfosalicylic acid, sulfosalicylic acid with sodium sulphate and trichloroacetic acid methods respectively. Passing and Bablok regression revealed a constant bias for pyrogallol red molybdate versus all turbidimetric methods but a proportional bias only with trichloroacetic acid method. Sulfosalicylic acid with sodium sulphate method is preferred to sulfosalicylic acid and trichloroacetic acid methods. PMID:27605745

  16. Bacterial Heat Shock Protein Activity

    PubMed Central

    Maleki, Farajollah; Khosravi, Afra; Nasser, Ahmad; Taghinejad, Hamid

    2016-01-01

    Bacteria are exposed to different types of stress in their growth conditions. They have developed appropriate responses, modulated by the re-modeling of protein complexes and by phosphorylation dependent signal transduction systems, to adapt and to survive in a variety range of nature. Proteins are essential components for biologic activity in the eukaryotic and prokaryotic cell. Heat Shock Proteins (HSP) have been identified from various organisms and have critical role in cell hemostasis. Chaperone can sense environment and have different potential role in the organism evolution. PMID:27134861

  17. Total Phenolic, Total Flavonoids, Antioxidant and Antimicrobial Activities of Scrophularia Striata Boiss Extracts

    PubMed Central

    Mahboubi, Mohaddese; Kazempour, Nastaran; Boland Nazar, Ali Reza

    2013-01-01

    Background Scrophularia striata (Scrophulariaceae family) is an herbaceous plant that is traditionally used for treatment of microbial infections. Objectives Antimicrobial and antioxidant activity of different extracts (methanolic, ethanolic, aqueous and ethyl acetate) from S. striata aerial parts was evaluated. Materials and Methods The antimicrobial activity of different extracts from S. striata was evaluated against a large number of bacteria and fungi by micro broth dilution. Total phenolic and flavonoid contents were measured and their antioxidant activities evaluated by DPPH assay and beta carotene linoleic acid test. Results Antimicrobial screening exhibited the positive relation between the total phenolic content and its antimicrobial activity but their antioxidant activity had a negative relation. Conclusions Further studies are recommended against clinical isolate of sensitive bacteria and deep investigation on flavonoid and phenolic compounds of S. striata and detecting the antioxidant portion in aqueous extract. PMID:24624181

  18. Truly Absorbed Microbial Protein Synthesis, Rumen Bypass Protein, Endogenous Protein, and Total Metabolizable Protein from Starchy and Protein-Rich Raw Materials: Model Comparison and Predictions.

    PubMed

    Parand, Ehsan; Vakili, Alireza; Mesgaran, Mohsen Danesh; van Duinkerken, Gert; Yu, Peiqiang

    2015-07-29

    This study was carried out to measure truly absorbed microbial protein synthesis, rumen bypass protein, and endogenous protein loss, as well as total metabolizable protein, from starchy and protein-rich raw feed materials with model comparisons. Predictions by the DVE2010 system as a more mechanistic model were compared with those of two other models, DVE1994 and NRC-2001, that are frequently used in common international feeding practice. DVE1994 predictions for intestinally digestible rumen undegradable protein (ARUP) for starchy concentrates were higher (27 vs 18 g/kg DM, p < 0.05, SEM = 1.2) than predictions by the NRC-2001, whereas there was no difference in predictions for ARUP from protein concentrates among the three models. DVE2010 and NRC-2001 had highest estimations of intestinally digestible microbial protein for starchy (92 g/kg DM in DVE2010 vs 46 g/kg DM in NRC-2001 and 67 g/kg DM in DVE1994, p < 0.05 SEM = 4) and protein concentrates (69 g/kg DM in NRC-2001 vs 31 g/kg DM in DVE1994 and 49 g/kg DM in DVE2010, p < 0.05 SEM = 4), respectively. Potential protein supplies predicted by tested models from starchy and protein concentrates are widely different, and comparable direct measurements are needed to evaluate the actual ability of different models to predict the potential protein supply to dairy cows from different feedstuffs. PMID:26118653

  19. RubisCO is not a major fraction of total protein in marine phytoplankton

    NASA Astrophysics Data System (ADS)

    Losh, J.; Young, J. N.; Morel, F. M.

    2012-12-01

    Ribulose 1,5 bisphosphate carboxylase oxygnenase (Rubisco) concentrations were quantified as a proportion of total protein in diatoms to determine whether Rubisco is as abundant in phytoplankton as previously thought. This enzyme has been assumed to be a major fraction of total protein in phytoplankton, as has been demonstrated in plants, potentially constituting a large sink for cellular nitrogen (N). Marine diatoms were grown in batch cultures, and in N-limited continuous cultures at various carbon dioxide (CO2) levels. Quantitative western blots were performed using commercially available global antibodies and protein standards. Field incubations with natural populations of organisms from the coast of California were conducted under both N-limited and N-replete incubations with varying CO2. In all experiments, Rubisco represented less than 5% of total protein. Within exponentially growing batch cultures, concentrations ranged from 2-4%, while in N-limited laboratory and field cultures, concentrations were less than 1%. In some experiments under N-limiting conditions, Rubisco concentrations decreased with decreasing growth rates or with increasing CO2. These results were used as a basis of a theoretical calculation of maximum Rubisco activity and suggest that phytoplankton contain the minimum amount of Rubisco necessary to operate. Unlike plants, Rubisco is not a major sink of cellular N in phytoplankton. This has implications for phytoplankton's response to increasing CO2 under N-limitation.

  20. Rubisco is a small fraction of total protein in marine phytoplankton.

    PubMed

    Losh, Jenna L; Young, Jodi N; Morel, François M M

    2013-04-01

    Ribulose 1,5 bisphosphate carboxylase oxygenase (Rubisco) concentrations were quantified as a proportion of total protein in eight species of microalgae. This enzyme has been assumed to be a major fraction of total protein in phytoplankton, as has been demonstrated in plants, potentially constituting a large sink for cellular nitrogen. Representative microalgae were grown in batch and continuous cultures under nutrient-replete, nitrogen (N)-limited, or phosphorus (P)-limited conditions with varying CO(2). Quantitative Western blots were performed using commercially available global antibodies and protein standards. Field incubations with natural populations of organisms from the coast of California were conducted under both nutrient-replete and N-limited conditions with varying CO(2). In all experiments, Rubisco represented < 6% of total protein. In nutrient-replete exponentially growing batch cultures, concentrations ranged from 2% to 6%, while in nutrient-limited laboratory and field cultures, concentrations were < 2.5%. Rubisco generally decreased with increasing CO(2) and with decreasing growth rates. Based on a calculation of maximum Rubisco activity, these results suggest that phytoplankton contain the minimum concentration of enzyme necessary to support observed growth rates. Unlike in plants, Rubisco does not account for a major fraction of cellular N in phytoplankton. PMID:23343368

  1. Antiviral activities of whey proteins.

    PubMed

    Ng, Tzi Bun; Cheung, Randy Chi Fai; Wong, Jack Ho; Wang, Yan; Ip, Denis Tsz Ming; Wan, David Chi Cheong; Xia, Jiang

    2015-09-01

    Milk contains an array of proteins with useful bioactivities. Many milk proteins encompassing native or chemically modified casein, lactoferrin, alpha-lactalbumin, and beta-lactoglobulin demonstrated antiviral activities. Casein and alpha-lactalbumin gained anti-HIV activity after modification with 3-hydroxyphthalic anhydride. Many milk proteins inhibited HIV reverse transcriptase. Bovine glycolactin, angiogenin-1, lactogenin, casein, alpha-lactalbumin, beta-lactoglobulin, bovine lactoferrampin, and human lactoferrampin inhibited HIV-1 protease and integrase. Several mammalian lactoferrins prevented hepatitis C infection. Lactoferrin, methylated alpha-lactalbumin and methylated beta-lactoglobulin inhibited human cytomegalovirus. Chemically modified alpha-lactalbumin, beta-lactoglobulin and lysozyme, lactoferrin and lactoferricin, methylated alpha-lactalbumin, methylated and ethylated beta-lactoglobulins inhibited HSV. Chemically modified bovine beta-lactoglobulin had antihuman papillomavirus activity. Beta-lactoglobulin, lactoferrin, esterified beta-lactoglobulin, and esterified lactoferrindisplayed anti-avian influenza A (H5N1) activity. Lactoferrin inhibited respiratory syncytial virus, hepatitis B virus, adenovirus, poliovirus, hantavirus, sindbis virus, semliki forest virus, echovirus, and enterovirus. Milk mucin, apolactoferrin, Fe(3+)-lactoferrin, beta-lactoglobulin, human lactadherin, bovine IgG, and bovine kappa-casein demonstrated antihuman rotavirus activity. PMID:26198883

  2. Active and total prokaryotic communities in dryland soils.

    PubMed

    Angel, Roey; Pasternak, Zohar; Soares, M Ines M; Conrad, Ralf; Gillor, Osnat

    2013-10-01

    The relationship between total and metabolically active soil microbial communities can change drastically with environment. In dry lands, water availability is a key factor limiting cells' activity. We surveyed the diversity of total and active Archaea and Bacteria in soils ranging from arid desert to Mediterranean forests. Thirty composited soil samples were retrieved from five sites along a precipitation gradient, collected from patches located between and under the dominant perennial plant at each site. Molecular fingerprinting was used to site-sort the communities according of their 16S rRNA genes (total community) and their rRNA (active community) amplified by PCR or RT-PCR from directly extracted soil nucleic acids. The differences between soil samples were much higher in total rather than active microbial communities: differences in DNA fingerprints between sites were 1.2 and 2.5 times higher than RNA differences (for Archaea and Bacteria, respectively). Patch-type discrepancies between DNA fingerprints were on average 2.7-19.7 times greater than RNA differences. Moreover, RNA-based community patterns were highly correlated with soil moisture but did not necessarily follow spatial distribution pattern. Our results suggest that in water-limited environments, the spatial patterns obtained by the analysis of active communities are not as robust as those drawn from total communities. PMID:23730745

  3. [Comparative analysis of total cell protein electrophoregram of pathogenic Burkholderia].

    PubMed

    Budchenko, A A; Iliukhin, V I; Viktorov, D V

    2005-01-01

    Whole-cell proteins of 22 strain of Burkhoderia pseudomallei, including 13 B. mallei, 5 B. cepacia strains and 14 strains of opportunistically pathogenic Pseudomonas defined by 1D SDC-PAAG electrophoresis. Electrophoregrams contained 35 to 45 protein fractions sized 19 to 130 kDa, which were highly reproductive. On the basis of computer-aided comparative analysis of protein patterns the interspecies and intraspecies grouping of studied microorganisms was made. The cluster analysis of the similarity matrix of protein spectra made it possible to allocate two groups of strains at the level of similarity of 78%. Group I was formed by Burkholderia species that previously belonged to the II RNA-DNA homology group of Pseudomonas: B. pseudomallei, B. mallei, B. cepacia. All Pseudomonas species were added to the 2nd Group: P. aeruginosa, P. stutzeri, P. testosterone, P. fluorescens, P. putida, P. mendocina. Four phenons were isolated among the strains of B. pseudomallei and 2 phenons--among the strains of B. mallei at the threshold similarity level (89%). The authors conclude that the comparative analysis of electrophoregrams of whole-cell proteins can be useful in the identification and typing of pathogenic Burkholderia. PMID:15954473

  4. Total Chemical Synthesis of Biologically Active Vascular Endothelial Growth Factor

    SciTech Connect

    Mandal, Kalyaneswar; Kent, Stephen B.H.

    2011-09-15

    The 204-residue covalent-dimer vascular endothelial growth factor (VEGF, see picture) with full mitogenic activity was prepared from three unprotected peptide segments by one-pot native chemical ligations. The covalent structure of the synthetic VEGF was confirmed by precise mass measurement, and the three-dimensional structure of the synthetic protein was determined by high-resolution X-ray crystallography.

  5. The influence of the ratio of protein energy to total energy in the feed on the activity of protein synthesis in vitro, the level of ribosomal RNA and the RNA-DNA ratio in white trunk muscle of Atlantic cod (Gadus morhua).

    PubMed

    Lied, E; Rosenlund, G

    1984-01-01

    Cod (Gadus morhua) were fed diets containing protein energy to total energy levels (PE/TE) of 10.0, 20.6, 29.6, 38.4, 56.2 and 74.1% for 21 days. Ribosomes were isolated from the white trunk muscle tissue, the capacity for protein synthesis in vitro determined and related to muscle tissue wet weight rRNA and DNA. Protein concentrations of less than 47.4% PE/TE in the diets reduce the ribosomal capacity for protein synthesis per g wet weight and per mg DNA, and the tissue contents of rRNA and ratio of rRNA/DNA. The capacity for muscle protein synthesis in vitro is a significant and sensitive parameter of protein inadequacy in fish diets. PMID:6200270

  6. Abrogation of fibroblast activation protein enzymatic activity attenuates tumor growth.

    PubMed

    Cheng, Jonathan D; Valianou, Matthildi; Canutescu, Adrian A; Jaffe, Eileen K; Lee, Hyung-Ok; Wang, Hao; Lai, Jack H; Bachovchin, William W; Weiner, Louis M

    2005-03-01

    Tumor-associated fibroblasts are functionally and phenotypically distinct from normal fibroblasts that are not in the tumor microenvironment. Fibroblast activation protein is a 95 kDa cell surface glycoprotein expressed by tumor stromal fibroblasts, and has been shown to have dipeptidyl peptidase and collagenase activity. Site-directed mutagenesis at the catalytic site of fibroblast activation protein, Ser624 --> Ala624, resulted in an approximately 100,000-fold loss of fibroblast activation protein dipeptidyl peptidase (DPP) activity. HEK293 cells transfected with wild-type fibroblast activation protein, enzymatic mutant (S624A) fibroblast activation protein, or vector alone, were inoculated subcutaneously into immunodeficient mouse to assess the contribution of fibroblast activation protein enzymatic activity to tumor growth. Overexpression of wild-type fibroblast activation protein showed growth potentiation and enhanced tumorigenicity compared with both fibroblast activation protein S624A and vector-transfected HEK293 xenografts. HEK293 cells transfected with fibroblast activation protein S624A showed tumor growth rates and tumorigenicity potential similar only to vector-transfected HEK293. In vivo assessment of fibroblast activation protein DPP activity of these tumors showed enhanced enzymatic activity of wild-type fibroblast activation protein, with only baseline levels of fibroblast activation protein DPP activity in either fibroblast activation protein S624A or vector-only xenografts. These results indicate that the enzymatic activity of fibroblast activation protein is necessary for fibroblast activation protein-driven tumor growth in the HEK293 xenograft model system. This establishes the proof-of-principle that the enzymatic activity of fibroblast activation protein plays an important role in the promotion of tumor growth, and provides an attractive target for therapeutics designed to alter fibroblast activation protein-induced tumor growth by targeting

  7. Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L

    PubMed Central

    2012-01-01

    Background The aim of this study was to screen various solvent extracts of whole plant of Torilis leptophylla to display potent antioxidant activity in vitro and in vivo, total phenolic and flavonoid contents in order to find possible sources for future novel antioxidants in food and pharmaceutical formulations. Material and methods A detailed study was performed on the antioxidant activity of the methanol extract of whole plant of Torilis leptophylla (TLM) and its derived fractions {n-hexane (TLH), chloroform (TLC) ethyl acetate (TLE) n-butanol (TLB) and residual aqueous fraction (TLA)} by in vitro chemical analyses and carbon tetrachloride (CCl4) induced hepatic injuries (lipid peroxidation and glutathione contents) in male Sprague-Dawley rat. The total yield, total phenolic (TPC) and total flavonoid contents (TFC) of all the fractions were also determined. TLM was also subjected to preliminary phytochemical screening test for various constituents. Results The total phenolic contents (TPC) (121.9±3.1 mg GAE/g extract) of TLM while total flavonoid contents (TFC) of TLE (60.9 ±2.2 mg RTE/g extract) were found significantly higher as compared to other solvent fractions. Phytochemical screening of TLM revealed the presence of alkaloids, anthraquinones, cardiac glycosides, coumarins, flavonoids, saponins, phlobatannins, tannins and terpenoids. The EC50 values based on the DPPH (41.0±1 μg/ml), ABTS (10.0±0.9 μg/ml) and phosphomolybdate (10.7±2 μg/ml) for TLB, hydroxyl radicals (8.0±1 μg/ml) for TLC, superoxide radicals (57.0±0.3 μg/ml) for TLM and hydrogen peroxide radicals (68.0±2 μg/ml) for TLE were generally lower showing potential antioxidant properties. A significant but marginal positive correlation was found between TPC and EC50 values for DPPH, hydroxyl, phosphomolybdate and ABTS, whereas another weak and positive correlation was determined between TFC and EC50 values for superoxide anion and hydroxyl radicals. Results of in vivo experiment

  8. Multiexcitation Fluorogenic Labeling of Surface, Intracellular, and Total Protein Pools in Living Cells.

    PubMed

    Naganbabu, Matharishwan; Perkins, Lydia A; Wang, Yi; Kurish, Jeffery; Schmidt, Brigitte F; Bruchez, Marcel P

    2016-06-15

    Malachite green (MG) is a fluorogenic dye that shows fluorescence enhancement upon binding to its engineered cognate protein, a fluorogen activating protein (FAP). Energy transfer donors such as cyanine and rhodamine dyes have been conjugated with MG to modify the spectral properties of the fluorescent complexes, where the donor dyes transfer energy through Förster resonance energy transfer to the MG complex resulting in binding-conditional fluorescence emission in the far-red region. In this article, we use a violet-excitable dye as a donor to sensitize the far-red emission of the MG-FAP complex. Two blue emitting fluorescent coumarin dyes were coupled to MG and evaluated for energy transfer to the MG-FAP complex via its secondary excitation band. 6,8-Difluoro-7-hydroxycoumarin-3-carboxylic acid (Pacific blue, PB) showed the most efficient energy transfer and maximum brightness in the far-red region upon violet (405 nm) excitation. These blue-red (BluR) tandem dyes are spectrally varied from other tandem dyes and are able to produce fluorescence images of the MG-FAP complex with a large Stokes shift (>250 nm). These dyes are cell-permeable and are used to label intracellular proteins. Used together with a cell-impermeable hexa-Cy3-MG (HCM) dye that labels extracellular proteins, we are able to visualize extracellular, intracellular, and total pools of cellular protein using one fluorogenic tag that combines with distinct dyes to effect different spectral characteristics. PMID:27159569

  9. Making Sense of Total VET Activity: An Initial Market Analysis

    ERIC Educational Resources Information Center

    National Centre for Vocational Education Research (NCVER), 2016

    2016-01-01

    Following the successful first national publication of total vocational education and training (VET) activity and presentation of various informative data products, NCVER has continued to undertake further analysis of the submitted data. This paper is the first in a suite of the National Centre for Vocational Education Research (NCVER) authored…

  10. Customized cooking method improves total antioxidant activity in selected vegetables.

    PubMed

    Ng, Zhi-Xiang; Chai, Jen-Wai; Kuppusamy, Umah Rani

    2011-03-01

    The present study compares water-soluble phenolic content (WPC) and antioxidant activities in Chinese long bean (Vigna unguiculata), bitter gourd (Momordica charantia), water convolvulus (Ipomoea aquatica) and broccoli (Brassica olearacea) prior to and after subjecting to boiling, microwaving and pressure cooking. The total antioxidant activity was increased in cooked water convolvulus, broccoli and bitter gourd, estimated based on the ferric reducing antioxidant power, the Trolox equivalent antioxidant capacity and 2,2-diphenyl-1-picryl-hydrazyl radical scavenging activity. Pressure cooking did not cause any significant decline in the antioxidant property. Boiling generally improved the overall antioxidant activity in all the vegetables. Correlation analysis suggests that WPC contributed to significant antioxidant activities in these vegetables. Thus, prudence in selecting an appropriate cooking method for different vegetables may improve or preserve their nutritional value. PMID:21250903

  11. [The behavior of serum total protein and protein fractions during the use of various hormonal contraceptives].

    PubMed

    Klinger, G; Scheler, R; Tarnick, M; Krause, G; Hesse, A; Carol, W

    1977-01-01

    Total protein fractions in the serum were measured in 213 women who used various contraceptive preparations (53 used Gravistat, 56 Non-Ovlon, 37 Ovosiston, 67 Deposiston). Blood samples were taken before the 1st, 3rd, 6th, 12th, and 24th cycles of use and 4 weeks after discontinuing preparation use. Serum protein concentrations decreased through the first 18 cycles of contraceptive use, after which a continual increase was observed. Serum albumin concentrations deceased during the first 3 cycles of use, after which increases were observed, which in the case of Non-Ovlon and Gravistat exceeded original values. Serum alpha 1 and alpha 2 globulin concentrations showed relative increases during the first 3 cycles, after which a decrease was observed. Serum beta globulin increased in the first 3 cycles of use; a decrease was observed between the 12th and 18 cycles, followed by another increase. Serum gamma globulin levels increased during the first 6 cycles, followed by a marked decrease, which extended partially into the post-treatment period. The changes are comparable to those observed during an undisturbed pregnancy. Deposiston caused the most marked changes in the parameters; this is attributed to the estrogenic nature of the preparation. PMID:73442

  12. Physical activity after total knee arthroplasty: A critical review

    PubMed Central

    Paxton, Roger J; Melanson, Edward L; Stevens-Lapsley, Jennifer E; Christiansen, Cory L

    2015-01-01

    Total knee arthroplasty (TKA) is the most commonly performed elective surgery in the United States. TKA typically improves functional performance and reduces pain associated with knee osteoarthritis. Little is known about the influence of TKA on overall physical activity levels. Physical activity, defined as “any bodily movement produced by skeletal muscles that results in energy expenditure”, confers many health benefits but typically decreases with endstage osteoarthritis. The purpose of this review is to describe the potential benefits (metabolic, functional, and orthopedic) of physical activity to patients undergoing TKA, present results from recent studies aimed to determine the effect of TKA on physical activity, and discuss potential sources of variability and conflicting results for physical activity outcomes. Several studies utilizing self-reported outcomes indicate that patients perceive themselves to be more physically active after TKA than they were before surgery. Accelerometry-based outcomes indicate that physical activity for patients after TKA remains at or below pre-surgical levels. Several different factors likely contributed to these variable results, including the use of different instruments, duration of follow-up, and characteristics of the subjects studied. Comparison to norms, however, suggests that daily physical activity for patients following TKA may fall short of healthy age-matched controls. We propose that further study of the relationship between TKA and physical activity needs to be performed using accelerometry-based outcome measures at multiple post-surgical time points. PMID:26396937

  13. First total synthesis of prasinic acid and its anticancer activity.

    PubMed

    Chakor, Narayan; Patil, Ganesh; Writer, Diana; Periyasamy, Giridharan; Sharma, Rajiv; Roychowdhury, Abhijit; Mishra, Prabhu Dutt

    2012-11-01

    The first total synthesis of prasinic acid is being reported along with its biological evaluation. The ten step synthesis involved readily available and cheap starting materials and can easily be transposed to large scale manufacturing. The crucial steps of the synthesis included the formation of two different aromatic units (7 and 9) and their coupling reaction. The synthetic prasinic acid exhibited moderate antitumor activity (IC(50) 4.3-9.1 μM) in different lines of cancer cells. PMID:23031589

  14. The design of the TASD (totally active scintillator detector) prototype

    SciTech Connect

    Mefodiev, A. V. Kudenko, Yu. G.

    2015-12-15

    Totally active and magnetic segmented scintillation neutrino detectors are developed for the nextgeneration accelerator neutrino experiments. Such detectors will incorporate scintillation modules with scintillation counters that form X and Y planes. A single counter is a 7 × 10 × 90 mm{sup 3} scintillation bar with gluedin wavelength-shifting fibers and micropixel avalanche photodiodes. The results of measurements of the parameters of these detectors are presented.

  15. Total myrosinase activity estimates in brassica vegetable produce.

    PubMed

    Dosz, Edward B; Ku, Kang-Mo; Juvik, John A; Jeffery, Elizabeth H

    2014-08-13

    Isothiocyanates, generated from the hydrolysis of glucosinolates in plants of the Brassicaceae family, promote health, including anticancer bioactivity. Hydrolysis requires the plant enzyme myrosinase, giving myrosinase a key role in health promotion by brassica vegetables. Myrosinase measurement typically involves isolating crude protein, potentially underestimating activity in whole foods. Myrosinase activity was estimated using unextracted fresh tissues of five broccoli and three kale cultivars, measuring the formation of allyl isothiocyanate (AITC) and/or glucose from exogenous sinigrin. A correlation between AITC and glucose formation was found, although activity was substantially lower measured as glucose release. Using exogenous sinigrin or endogenous glucoraphanin, concentrations of the hydrolysis products AITC and sulforaphane correlated (r = 0.859; p = 0.006), suggesting that broccoli shows no myrosinase selectivity among sinigrin and glucoraphanin. Measurement of AITC formation provides a novel, reliable estimation of myrosinase-dependent isothiocyanate formation suitable for use with whole vegetable food samples. PMID:25051514

  16. The Active Human Gut Microbiota Differs from the Total Microbiota

    PubMed Central

    Peris-Bondia, Francesc; Latorre, Amparo; Artacho, Alejandro; Moya, Andrés; D'Auria, Giuseppe

    2011-01-01

    The human gut microbiota is considered one of the most fascinating reservoirs of microbial diversity hosting between 400 to 1000 bacterial species distributed among nine phyla with Firmicutes, Bacteroidetes and Actinobacteria representing around of the diversity. One of the most intriguing issues relates to understanding which microbial groups are active players in the maintenance of the microbiota homeostasis. Here, we describe the diversity of active microbial fractions compared with the whole community from raw human fecal samples. We studied four healthy volunteers by 16S rDNA gene pyrosequencing. The fractions were obtained by cell sorting based on bacterial RNA concentration. Bacterial families were observed to appear or disappear on applying a cell sorting method in which flow cytometry was used to evaluate the active cells by pyronin-Y staining of RNA. This method was able to detect active bacteria, indicating that the active players differed from that observed in raw fecal material. Generally, observations showed that in the active fractions, the number of reads related to Bacteroidetes decreased whereas several families from Clostridiales (Firmicutes) were more highly represented. Moreover, a huge number of families appeared as part of the active fraction when cell sorting was applied, indicating reads that are simply statistically hidden by the total reads. PMID:21829462

  17. Lactate dehydrogenase X, malate dehydrogenase and total protein in rat spermatozoa during epididymal transit.

    PubMed

    Vermouth, N T; Carriazo, C S; Ponce, R H; Blanco, A

    1986-01-01

    Lactate dehydrogenase isozyme X (LDH X), malate dehydrogenase (MDH) and total soluble protein have been determined in lysates of spermatozoa isolated from caput, corpus and cauda of rat epididymis. Transit of spermatozoa through epididymis is accompanied by a reduction of LDH X, MDH and total protein per cell in sexually rested animals. The profiles of reduction along epididymal segments are different for the three variables studied. Mating with receptive females during the 5 days prior to determinations increases significantly the levels of MDH in spermatozoa from all sections of epididymis and produces increase of total soluble protein in the cells contained in cauda. PMID:3956158

  18. The Behavior of Total Lightning Activity in Severe Florida Thunderstorms

    NASA Technical Reports Server (NTRS)

    Williams, Earle; Boldi, Bob; Matlin, Anne; Weber, Mark; Hodanish, Steve; Sharp, Dave; Goodman, Steve; Raghavan, Ravi; Buechler, Dennis

    1998-01-01

    The development of a new observational system called LISDAD (Lightning Imaging Sensor Demonstration and Display) has enabled a study of severe weather in central Florida. The total flash rates for storms verified to be severe are found to exceed 60 flashes/min, with some values reaching 500 flashes/min. Similar to earlier results for thunderstorm microbursts, the peak flash rate precedes the severe weather at the ground by 5-20 minutes. A distinguishing feature of severe storms is the presence of lightning "jumps"-abrupt increases in flash rate in advance of the maximum rate for the storm. ne systematic total lightning precursor to severe weather of all kinds-wind, hail, tornadoes-is interpreted in terms of the updraft that sows the seeds aloft for severe weather at the surface and simultaneously stimulates the ice microphysics that drives the lightning activity.

  19. Protein Needs of Physically Active Children.

    PubMed

    Volterman, Kimberly A; Atkinson, Stephanie A

    2016-05-01

    Current Dietary Reference Intakes (DRI) for protein for children and youth require revision as they were derived primarily on nitrogen balance data in young children or extrapolated from adult values; did not account for the possible influence of above average physical activity; and did not set an upper tolerable level of intake. Revision of the protein DRIs requires new research that investigates: 1) long-term dose-response to identify protein and essential amino acid requirements of both sexes at various pubertal stages and under differing conditions of physical activity; 2) the acute protein needs (quantity and timing) following a single bout of exercise; 3) the potential adverse effects of chronic high intakes of protein; and 4) new measurement techniques (i.e., IAAO or stable isotope methodologies) to improve accuracy of protein needs. While active individuals may require protein in excess of current DRIs, most active Canadian children and youth have habitual protein intakes that exceed current recommendations. PMID:27137165

  20. Formulation of plant based Rainbow trout feeds on an Ideal Protein Basis can reduce total dietary protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fish meal has been a primary protein source in trout feeds and any changes that can reduce fish meal levels and total costs are beneficial. Replacing fish meal with plant protein is a first step, but amino acid content of plant based diets can be limiting. Amino acids are needed for many metabolic...

  1. Persulfate activation during exertion of total oxidant demand.

    PubMed

    Teel, Amy L; Elloy, Farah C; Watts, Richard J

    2016-09-01

    Total oxidant demand (TOD) is a parameter that is often measured during in situ chemical oxidation (ISCO) treatability studies. The importance of TOD is based on the concept that the oxidant demand created by soil organic matter and other reduced species must be overcome before contaminant oxidation can proceed. TOD testing was originally designed for permanganate ISCO, but has also recently been applied to activated persulfate ISCO. Recent studies have documented that phenoxides activate persulfate; because soil organic matter is rich in phenolic moieties, it may activate persulfate rather than simply exerting TOD. Therefore, the generation of reactive oxygen species was investigated in three soil horizons of varied soil organic carbon content over 5-day TOD testing. Hydroxyl radical may have been generated during TOD exertion, but was likely scavenged by soil organic matter. A high flux of reductants + nucleophiles (e.g. alkyl radicals + superoxide) was generated as TOD was exerted, resulting in the rapid destruction of the probe compound hexachloroethane and the common groundwater contaminant trichloroethylene (TCE). The results of this research document that, unlike permanganate TOD, contaminant destruction does occur as TOD is exerted in persulfate ISCO systems and is promoted by the activation of persulfate by soil organic matter. Future treatability studies for persulfate ISCO should consider contaminant destruction as TOD is exerted, and the potential for persulfate activation by soil organic matter. PMID:27269993

  2. Protein-water dynamics in antifreeze protein III activity

    NASA Astrophysics Data System (ADS)

    Xu, Yao; Bäumer, Alexander; Meister, Konrad; Bischak, Connor G.; DeVries, Arthur L.; Leitner, David M.; Havenith, Martina

    2016-03-01

    We combine Terahertz absorption spectroscopy (THz) and molecular dynamics (MD) simulations to investigate the underlying molecular mechanism for the antifreeze activity of one class of antifreeze protein, antifreeze protein type III (AFP-III) with a focus on the collective water hydrogen bond dynamics near the protein. After summarizing our previous work on AFPs, we present a new investigation of the effects of cosolutes on protein antifreeze activity by adding sodium citrate to the protein solution of AFP-III. Our results reveal that for AFP-III, unlike some other AFPs, the addition of the osmolyte sodium citrate does not affect the hydrogen bond dynamics at the protein surface significantly, as indicated by concentration dependent THz measurements. The present data, in combination with our previous THz measurements and molecular simulations, confirm that while long-range solvent perturbation is a necessary condition for the antifreeze activity of AFP-III, the local binding affinity determines the size of the hysteresis.

  3. Efficient total syntheses and biological activities of two teixobactin analogues.

    PubMed

    Parmar, Anish; Iyer, Abhishek; Vincent, Charlotte S; Van Lysebetten, Dorien; Prior, Stephen H; Madder, Annemieke; Taylor, Edward J; Singh, Ishwar

    2016-04-26

    The discovery of the new antibiotic teixobactin has been timely in the race for unearthing novel antibiotics wherein the emergence of drug resistant bacteria poses a serious threat worldwide. Herein, we present the total syntheses and biological activities of two teixobactin analogues. This approach is simple, efficient and has several advantages: it uses commercially available building blocks (except AllocHN-d-Thr-OH), has a single purification step and a good recovery (22%). By using this approach we have synthesised two teixobactin analogues and established that the d-amino acids are critical for the antimicrobial activity of these analogues. With continuing high expectations from teixobactin, this work can be regarded as a stepping stone towards an in depth study of teixobactin, its analogues and the quest for synthesising similar molecules. PMID:26984316

  4. Total pollen counts do not influence active surface measurements

    NASA Astrophysics Data System (ADS)

    Moshammer, Hanns; Schinko, Herwig; Neuberger, Manfred

    We investigated the temporal association of various aerosol parameters with pollen counts in the pollen season (April 2001) in Linz, Austria. We were especially interested in the relationship between active surface (or Fuchs' surface) because we had shown previously (Atmos. Environ. 37 (2003) 1737-1744) that this parameter during the same observation period was a better predictor for acute respiratory symptoms in school children (like wheezing, shortness of breath, and cough) and reduced lung function on the same day than particle mass (PM 10). While active surface is most sensitive for fine particles with a diameter of less than 100 nm it has no strict upper cut-off regarding particle size and so could eventually be influenced also by larger particles if their numbers were high. All particle mass parameters tested (TSP, PM 10, PM 1) were weakly ( r approximately 0.2) though significantly correlated with pollen counts but neither was active surface nor total particle counts (CPC). The weak association of particle mass and pollen counts was due mainly to similar diurnal variations and a linear trend over time. Only the mass of the coarse fraction (TSP minus PM 10) remained associated with pollen counts significantly after controlling for these general temporal patterns.

  5. Dietary protein and plasma total homocysteine, cysteine concentrations in coronary angiographic subjects

    PubMed Central

    2013-01-01

    Background Dietary patterns are associated with plasma total homocysteine (tHcy) concentrations in healthy populations, but the associations between dietary protein and tHcy, total cysteine (tCys) in high risk populations are unclear. We therefore examined the association between dietary protein and tHcy and tCys concentrations in coronary angiographic subjects. Methods We conducted a cross-sectional study of 1015 Chinese patients who underwent coronary angiography (40–85 y old). With the use of food-frequency questionnaires, we divided the total protein intakes into high animal-protein and high plant-protein diets. Circulating concentrations of tHcy and tCys were simultaneously measured by high-performance liquid chromatography with fluorescence detection. Results We found that high animal-protein diet was positively associated with hyperhomocysteinemia after adjustment for potential confounders, with the subjects in the highest quartile of intake having the greatest increase in risk (OR: 4.14, 95% CI: 2.67-6.43), whereas high plant-protein diet was inversely related to hyperhomocysteinemia, with a higher intake being protective. Compared with the first quartile of intake, the adjusted OR was 0.59 (95% CI: 0.38-0.91) for the fourth quartile. The total protein intake was positively associated with the risk of hypercysteinemia and the participants in highest quartile had significant OR of 1.69 (95% CI: 1.02-2.87) compared with those in lowest quartile. In multivariate linear regression analyses, high animal-protein and total-protein intakes were positively associated with plasma tHcy and tCys concentrations. The plant-protein intake was a negative determinant of plasma tHcy concentrations. Conclusions High animal-protein diet was positively associated with high tHcy concentrations, whereas high plant-protein diet was inversely associated with tHcy concentrations. Furthermore the total protein intake was strongly related to tCys concentrations. PMID:24195518

  6. Changes in total plasma content of electrolytes and proteins with maximal exercise.

    NASA Technical Reports Server (NTRS)

    Van Beaumont, W.; Strand, J. C.; Petrofsky, J. S.; Hipskind, S. G.; Greenleaf, J. E.

    1973-01-01

    To determine to what extent the increases in concentration of plasma proteins and electrolytes with short maximal work were a result of hemoconcentration, the changes in plasma volume and total content of the plasma constituents were simultaneously evaluated. The results obtained from six human subjects indicated that in comparison to preexercise values there was a net decrease in total content of plasma protein, sodium, and chloride in the first 2 min of the postexercise period, due primarily to a significant loss (13-15%) of plasma fluid. The total plasma potassium content was increased immediately after exercise but was significantly below the preexercise plasma content after 2 min of recovery.

  7. Long Wavelength Monitoring of Protein Kinase Activity

    PubMed Central

    Oien, Nathan P.; Nguyen, Luong T.; Jernigan, Finith E.; Priestman, Melanie A.

    2014-01-01

    A family of long wavelength protein kinase fluorescent reporters is described in which the probing wavelength is pre-programmed using readily available fluorophores. These agents can assess protein kinase activity within the optical window of tissue, as exemplified by monitoring endogenous cAMP-dependent protein kinase activity (1) in erythrocyte lysates and (2) in intact erythrocytes using a light-activatable reporter. PMID:24604833

  8. Intestinal mucosa in diabetes: synthesis of total proteins and sucrase-isomaltase

    SciTech Connect

    Olsen, W.A.; Perchellet, E.; Malinowski, R.L.

    1986-06-01

    The effects of insulin deficiency on nitrogen metabolism in muscle and liver have been extensively studied with recent in vivo demonstration of impaired protein synthesis in rats with streptozotocin-induced diabetes. Despite the significant contribution of small intestinal mucosa to overall protein metabolism, the effect of insulin deficiency on intestinal protein synthesis have not been completely defined. The authors studied the effects of streptozotocin-induced diabetes on total protein synthesis by small intestinal mucosa and on synthesis of a single enzyme protein of the enterocyte brush-border membrane sucrase-isomaltase. They used the flood-dose technique to minimize the difficulties of measuring specific radioactivity of precursor phenylalanine and determined incorporation into mucosal proteins and sucrase-isomaltase 20 min after injection of the labeled amino acid. Diabetes did not alter mucosal mass as determined by weight and content of protein and DNA during the 5 days after injection of streptozotocin. Increased rates of sucrase-isomaltase synthesis developed beginning on day 3, and those of total protein developed on day 5. Thus intestinal mucosal protein synthesis is not an insulin-sensitive process.

  9. Activity-Based Protein Profiling of Microbes

    SciTech Connect

    Sadler, Natalie C.; Wright, Aaron T.

    2015-02-01

    Activity-Based Protein Profiling (ABPP) in conjunction with multimodal characterization techniques has yielded impactful findings in microbiology, particularly in pathogen, bioenergy, drug discovery, and environmental research. Using small molecule chemical probes that react irreversibly with specific proteins or protein families in complex systems has provided insights in enzyme functions in central metabolic pathways, drug-protein interactions, and regulatory protein redox, for systems ranging from photoautotrophic cyanobacteria to mycobacteria, and combining live cell or cell extract ABPP with proteomics, molecular biology, modeling, and other techniques has greatly expanded our understanding of these systems. New opportunities for application of ABPP to microbial systems include: enhancing protein annotation, characterizing protein activities in myriad environments, and reveal signal transduction and regulatory mechanisms in microbial systems.

  10. The Characteristics of Total Lightning Activity in Severe Florida Thunderstorms

    NASA Technical Reports Server (NTRS)

    Williams, E.; Goodman, S. J.; Raghavan, R.; Boldi, R.; Matlin, A.; Weber, M.; Hodanish, S.; Sharp, D.

    1997-01-01

    Severe thunderstorms are defined by specific exceedance criteria regarding either wind speed (greater than or equal to 50 kts), hailstone diameter (greater than or equal to 3/4 inch), the occurrence of a tornado, or any combination thereof. Although traditional radar signatures of severe thunderstorms have been well documented, the characteristics of associated total lightning activity (both intracloud and cloud-to-ground) of severe thunderstorms remain poorly established. The reason for this are (1) less than 1% of all storms are actually severe, (2) intracloud lightning, which is typically the dominant form of electrical discharge within thunderstorms, is not routinely measured or recorded, (3) direct visual observations of intracloud lightning are obscured during the daytime, and (4) the migratory nature of many severe thunderstorms can make the accurate detection and mapping of intracloud lightning difficult when using fixed-location sensors. The recent establishment of LISDAD (Lightning Imaging Sensor Data Acquisition and Display - discussed in Goodman et al, this Meeting) has substantially addressed these limitations in east central Florida (ECFL). Analysis of total lightning flash Count histories using the LDAR (Lightning Detection And Ranging) system for known severe thunderstorms (currently irrespective of seasonal aspects and severe storm-type) has revealed flash rates exceeding 1 per second. This appears to be a necessary, but not sufficient,condition for most ECFL severe storm cases. The differences in radar-observed storm structure for high flash rate storms (to include both severe and non-severe categories) will be described together with the timing of peak flash rate vs. the timing of the severe weather manifestation. Comparisons with the satellite-bases OTD (Optical Transient Detector) overhead passes will also be presented when possible.

  11. Study of the effect of total serum protein and albumin concentrations on canine fructosamine concentration.

    PubMed Central

    Loste, A; Marca, M C

    1999-01-01

    The relationship among serum fructosamine concentration and total serum protein and albumin concentrations were evaluated in healthy and sick dogs (diabetics and dogs with insulinoma were not included). Fructosamine was determined using a commercial colorimetric nitroblue tetrazolium method applied to the Technicon RA-500 (Bayer). Serum fructosamine concentration was not correlated to total protein in normoproteinemic (r = 0.03) and hyperproteinemic dogs (r = 0.29), but there was a high correlation (r = 0.73) in hypoproteinemic dogs. Similar comparison between serum fructosamine and albumin concentrations showed middle correlation (r = 0.49) in normoalbuminemic dogs and high degree of correlation (r = 0.67) in hypoalbuminemic dogs. These results showed the importance of recognizing serum glucose concentration as well as total serum protein and albumin concentrations in the assay of canine serum fructosamine concentration. PMID:10369572

  12. Simultaneous serum desalting and total protein determination by macroporous reversed-phase chromatography.

    PubMed

    Boichenko, Alexander; Govorukhina, Natalia; van der Zee, Ate G J; Bischoff, Rainer

    2013-04-01

    Macroporous reversed-phase (mRP) chromatography was successfully used to develop an accurate and precise method for total protein in serum. The limits of detection (0.83 μg, LOD) and quantification (2.51 μg, LOQ) for the mRP method are comparable with those of the widely used micro BCA protein assay. The mRP method can be used to determine the total protein concentration across a wide dynamic range by detecting chromatographic peaks at 215 nm and 280 nm. The method has the added advantage of desalting and denaturing proteins, leading to more complete digestion by trypsin and to better LC-MS-MS identification in shotgun proteomics experiments. PMID:23388688

  13. A flow injection sampling resonance light scattering system for total protein determination in human serum

    NASA Astrophysics Data System (ADS)

    Dong, Lijun; Li, Ying; Zhang, Yaheng; Chen, Xingguo; Hu, Zhide

    2007-04-01

    A novel flow injection method with resonance light scattering detection was developed for the determination of total protein concentrations. This method is based on the enhancement of RLS signals from Methyl Blue (MB) by protein. The enhanced RLS intensities at 333 nm, in a pH 4.1 acidic aqueous solution, were proportional to the protein concentration over the range 2.0-37.3 and 1.0-36.0 μg ml -1 for human serum albumin (HSA) and bovine serum albumin (BSA), respectively. The corresponding limits of detection (3 σ) of 45 ng ml -1 for HSA and 80 ng ml -1 for BSA were attained. The method was successfully applied to the quantification of total proteins in human serum samples, the maximum relative error is less than 1% and the recovery is between 98% and 102%. The sample throughput was 60 h -1.

  14. Dietary protein considerations to support active aging.

    PubMed

    Wall, Benjamin T; Cermak, Naomi M; van Loon, Luc J C

    2014-11-01

    Given our rapidly aging world-wide population, the loss of skeletal muscle mass with healthy aging (sarcopenia) represents an important societal and public health concern. Maintaining or adopting an active lifestyle alleviates age-related muscle loss to a certain extent. Over time, even small losses of muscle tissue can hinder the ability to maintain an active lifestyle and, as such, contribute to the development of frailty and metabolic disease. Considerable research focus has addressed the application of dietary protein supplementation to support exercise-induced gains in muscle mass in younger individuals. In contrast, the role of dietary protein in supporting the maintenance (or gain) of skeletal muscle mass in active older persons has received less attention. Older individuals display a blunted muscle protein synthetic response to dietary protein ingestion. However, this reduced anabolic response can largely be overcome when physical activity is performed in close temporal proximity to protein consumption. Moreover, recent evidence has helped elucidate the optimal type and amount of dietary protein that should be ingested by the older adult throughout the day in order to maximize the skeletal muscle adaptive response to physical activity. Evidence demonstrates that when these principles are adhered to, muscle maintenance or hypertrophy over prolonged periods can be further augmented in active older persons. The present review outlines the current understanding of the role that dietary protein occupies in the lifestyle of active older adults as a means to increase skeletal muscle mass, strength and function, and thus support healthier aging. PMID:25355192

  15. Bioprocess monitoring: minimizing sample matrix effects for total protein quantification with bicinchoninic acid assay.

    PubMed

    Reichelt, Wieland N; Waldschitz, Daniel; Herwig, Christoph; Neutsch, Lukas

    2016-09-01

    Determining total protein content is a routine operation in many laboratories. Despite substantial work on assay optimization interferences, the widely used bicinchoninic acid (BCA) assay remains widely recognized for its robustness. Especially in the field of bioprocess engineering the inaccuracy caused by interfering substances remains hardly predictable and not well understood. Since the introduction of the assay, sample pre-treatment by trichloroacetic acid (TCA) precipitation has been indicated as necessary and sufficient to minimize interferences. However, the sample matrix in cultivation media is not only highly complex but also dynamically changing over process time in terms of qualitative and quantitative composition. A significant misestimation of the total protein concentration of bioprocess samples is often observed when following standard work-up schemes such as TCA precipitation, indicating that this step alone is not an adequate means to avoid measurement bias. Here, we propose a modification of the BCA assay, which is less influenced by sample complexity. The dynamically changing sample matrix composition of bioprocessing samples impairs the conventional approach of compensating for interfering substances via a static offset. Hence, we evaluated the use of a correction factor based on an internal spike measurement for the respective samples. Using protein spikes, the accuracy of the BCA protein quantification could be improved fivefold, taking the BCA protein quantification to a level of accuracy comparable to other, more expensive methods. This will allow reducing expensive iterations in bioprocess development to due inaccurate total protein analytics. PMID:27314233

  16. Modeling the SHG activities of diverse protein crystals

    SciTech Connect

    Haupert, Levi M.; DeWalt, Emma L.; Simpson, Garth J.

    2012-11-01

    The origins of the diversity in the SHG signal from protein crystals are investigated and potential protein-crystal coverage by SHG microscopy is assessed. A symmetry-additive ab initio model for second-harmonic generation (SHG) activity of protein crystals was applied to assess the likely protein-crystal coverage of SHG microscopy. Calculations were performed for 250 proteins in nine point-group symmetries: a total of 2250 crystals. The model suggests that the crystal symmetry and the limit of detection of the instrument are expected to be the strongest predictors of coverage of the factors considered, which also included secondary-structural content and protein size. Much of the diversity in SHG activity is expected to arise primarily from the variability in the intrinsic protein response as well as the orientation within the crystal lattice. Two or more orders-of-magnitude variation in intensity are expected even within protein crystals of the same symmetry. SHG measurements of tetragonal lysozyme crystals confirmed detection, from which a protein coverage of ∼84% was estimated based on the proportion of proteins calculated to produce SHG responses greater than that of tetragonal lysozyme. Good agreement was observed between the measured and calculated ratios of the SHG intensity from lysozyme in tetragonal and monoclinic lattices.

  17. Evidence that the ZNT3 protein controls the total amount of elemental zinc in synaptic vesicles

    USGS Publications Warehouse

    Linkous, D.H.; Flinn, J.M.; Koh, J.Y.; Lanzirotti, A.; Bertsch, P.M.; Jones, B.F.; Giblin, L.J.; Frederickson, C.J.

    2008-01-01

    The ZNT3 protein decorates the presynaptic vesicles of central neurons harboring vesicular zinc, and deletion of this protein removes staining for zinc. However, it has been unclear whether only histochemically reactive zinc is lacking or if, indeed, total elemental zinc is missing from neurons lacking the Slc30a3 gene, which encodes the ZNT3 protein. The limitations of conventional histochemical procedures have contributed to this enigma. However, a novel technique, microprobe synchrotron X-ray fluorescence, reveals that the normal 2- to 3-fold elevation of zinc concentration normally present in the hippocampal mossy fibers is absent in Slc30a3 knockout (ZNT3) mice. Thus, the ZNT3 protein evidently controls not only the "stainability" but also the actual mass of zinc in mossy-fiber synaptic vesicles. This work thus confirms the metal-transporting role of the ZNT3 protein in the brain. ?? The Histochemical Society, Inc.

  18. Evidence That the ZNT3 Protein Controls the Total Amount of Elemental Zinc in Synaptic Vesicles

    SciTech Connect

    Linkous,D.; Flinn, J.; Koh, J.; Lanzirotti, A.; Bertsch, P.; Jones, B.; Giblin, L.; Fredrickson, C.

    2008-01-01

    The ZNT3 protein decorates the presynaptic vesicles of central neurons harboring vesicular zinc, and deletion of this protein removes staining for zinc. However, it has been unclear whether only histochemically reactive zinc is lacking or if, indeed, total elemental zinc is missing from neurons lacking the Slc30a3 gene, which encodes the ZNT3 protein. The limitations of conventional histochemical procedures have contributed to this enigma. However, a novel technique, microprobe synchrotron X-ray fluorescence, reveals that the normal 2- to 3-fold elevation of zinc concentration normally present in the hippocampal mossy fibers is absent in Slc30a3 knockout (ZNT3) mice. Thus, the ZNT3 protein evidently controls not only the 'stainability' but also the actual mass of zinc in mossy-fiber synaptic vesicles. This work thus confirms the metal-transporting role of the ZNT3 protein in the brain.

  19. Age-related changes in total protein and collagen metabolism in rat liver.

    PubMed

    Mays, P K; McAnulty, R; Laurent, G J

    1991-12-01

    Liver collagen levels are determined by a balance between synthesis and degradation, processes known to have rapid rates in growing animals. We report age-related changes in liver collagen synthesis and degradation rates, as well as protein synthesis rates, in rats at five ages from 1 to 24 mo. Fractional collagen synthesis rates were determined after injection of [14C]proline with a flooding dose of unlabeled proline and its incorporation as hydroxy-[14C]proline into proteins. Fractional protein synthesis rates were based on the uptake of [14C]proline into proteins. Fractional collagen degradation rates were calculated from the difference between collagen fractional synthesis and deposition rates. Fractional rates of collagen synthesis were similar between 1 mo (23.0% +/- 4.6%/day) and 24 mo (19.6% +/- 3.4%/day) of age. Collagen deposition into the extracellular matrix was extremely low at every age studied; therefore degradation pathways accounted for the bulk of the collagen synthesized. The mean fractional synthesis rate for the total protein pool was unaltered between 1 mo (105.0% +/- 7.2%/day) and 15 mo (89.9% +/- 6.0%/day) of age, after which it increased to 234.9% +/- 33.0%/day (p less than 0.05) by 24 mo of age. These results indicate that liver collagen and total protein synthesis rates were maintained at relatively high levels during development and maturity but that protein synthesis rates were highest in senescent animals. PMID:1959872

  20. Estimation of Levels of Salivary Mucin, Amylase and Total Protein in Gingivitis and Chronic Periodontitis Patients

    PubMed Central

    Bhandary, Rahul; Thomas, Biju; Kumari, Suchetha

    2014-01-01

    Background: Periodontal diseases are a group of inflammatory conditions resulting from interaction between a pathogenic bacterial biofilm and susceptible host’s inflammatory response eventually leading to the destruction of periodontal structures and subsequent tooth loss. Hence, investigation of salivary proteins in individuals with periodontal diseases may be useful to enhance the knowledge of their roles in these diseases. Materials and Methods: This case-control study was conducted at A.B. Shetty Memorial Institute of Dental Sciences, Mangalore. The study comprised of 90 patients of age between 25-60 years who were clinically examined and divided into three groups of 30 each: namely clinically healthy, gingivitis and chronic periodontitis. These were classified according to the values of gingival index score, clinical attachment loss and probing pocket depth. Unstimulated saliva was collected and salivary mucin, amylase and total protein levels were determined. Statistical analysis: Results obtained were tabulated and statistically analyzed using ANOVA test and Karl pearson’s correlation test. Results: The results of the study showed an increased concentration of salivary mucin, amylase and total protein in gingivitis patients and increased levels of amylase and total protein in saliva of chronic periodontitis patients compared to healthy individuals which were statistically significant. A decrease in mucin concentration was observed in the periodontitis group compared to gingivitis group. A positive correlation was present between salivary mucin, amylase and total protein levels in the three groups. Conclusion: Salivary mucin, amylase and total protein may serve as an important biochemical parameter of inflammation of the periodontium. Also, it can be hypothesized that various enzyme inhibitors might be useful as a part of host modulation therapy in the treatment of periodontal diseases. PMID:25478449

  1. Continuous versus intermittent exercise effects on urinary excretion of albumin and total protein.

    PubMed

    Montelpare, W J; Klentrou, P; Thoden, J

    2002-09-01

    Several studies have reported post-exercise increases of urinary concentrations of plasma proteins. However, under normal conditions, through mechanisms of size and electrical charge selection, the kidney restricts the clearance of molecules as large as albumin. Post-exercise increases in albuminuria occur following the physiological stress of intense exercise, most likely as a result of the exercise induced blood acidity changes which lead to a change in the arrangement of the albumin molecule, and subsequently the filtration characteristics of the glomerular capillary wall. The purpose of the present study was therefore to determine the extent to which different types of exercise could induce a transient condition of post-exercise increases in the urinary output of total protein and albumin. All 14 males, who agreed to participate in the study, performed a continuous and an intermittent cycling protocol on a stationary bicycle ergometer. The results showed that: a) intermittent exercise had a greater influence than continuous exercise on the total output of urine albumin, and of urine total protein; b) concentrations of blood pH and blood lactate, were associated with changes in the clearance of urine albumin and urine total protein. Post-exercise proteinuria response seems to be transient and therefore renal trauma is not suspected at the early stages of observation. Furthermore, these results indicate that the kidney undergoes distinct physiological adjustments during exercise, and that these adjustments are relative to the intensity of the exercise stress. PMID:12413038

  2. Total Protein of Whole Saliva as a Biomarker of Anaerobic Threshold

    ERIC Educational Resources Information Center

    Bortolini, Miguel Junior Sordi; De Agostini, Guilherme Gularte; Reis, Ismair Teodoro; Lamounier, Romeu Paulo Martins Silva; Blumberg, Jeffrey B.; Espindola, Foued Salmen

    2009-01-01

    Saliva provides a convenient and noninvasive matrix for assessing specific physiological parameters, including some biomarkers of exercise. We investigated whether the total protein concentration of whole saliva (TPWS) would reflect the anaerobic threshold during an incremental exercise test. After a warm-up period, 13 nonsmoking men performed a…

  3. Evaluation of body composition and nitrogen content of renal patients on chronic dialysis as determined by total body neutron activation

    SciTech Connect

    Cohn, S.H.; Brennan, B.L.; Yasumura, S.; Vartsky, D.; Vaswani, A.N.; Ellis, K.J.

    1983-07-01

    Total body protein (nitrogen), body cell mass (potassium), fat, and water were measured in 15 renal patients on maintenance hemodialysis (MHD). Total body nitrogen was measured by means of prompt ..gamma.. neutron activation analysis; total body water was determined with tritium labeled water; total body potassium was measured by whole body counting. The extracellular water was determined by a technique utilizing the measurement of total body chloride and plasma chloride. When compared with corresponding values of a control group of the same age, sex, and height, the protein content, body cell mass, and total body fat of the MHD patients were within the normal range. The only significant change was an increase in the extracellular water/body cell mass ratio in the male MHD patients compared to the control. The lack of significant difference of the nitrogen values of the MHD patients compared to matched controls suggests that dialysis minimizes any residual effects of uremic toxicity or protein-calorie malnutrition. These findings further suggest that there is a need to reevaluate the traditional anthropometric and biochemical standards of nutritional status for MHD patients. It was concluded that it is particularly important to measure protein stores of MHD patients with low protein intake to ascertain nutritional status. Finally, in vivo measurement of total body nitrogen and potassium for determination of body composition provides a simple, direct, and accurate assessment of the nutritional status of MHD patients.

  4. Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates

    PubMed Central

    Tanase, Maya; Zolla, Valerio; Clement, Cristina C; Borghi, Francesco; Urbanska, Aleksandra M; Rodriguez-Navarro, Jose Antonio; Roda, Barbara; Zattoni, Andrea; Reschiglian, Pierluigi; Cuervo, Ana Maria; Santambrogio, Laura

    2016-01-01

    Herein we describe a protocol that uses hollow-fiber flow field-flow fractionation (FFF) coupled with multiangle light scattering (MALS) for hydrodynamic size-based separation and characterization of complex protein aggregates. The fractionation method, which requires 1.5 h to run, was successfully modified from the analysis of protein aggregates, as found in simple protein mixtures, to complex aggregates, as found in total cell lysates. In contrast to other related methods (filter assay, analytical ultracentrifugation, gel electrophoresis and size-exclusion chromatography), hollow-fiber flow FFF coupled with MALS allows a flow-based fractionation of highly purified protein aggregates and simultaneous measurement of their molecular weight, r.m.s. radius and molecular conformation (e.g., round, rod-shaped, compact or relaxed). The polyethersulfone hollow fibers used, which have a 0.8-mm inner diameter, allow separation of as little as 20 μg of total cell lysates. In addition, the ability to run the samples in different denaturing and nondenaturing buffer allows defining true aggregates from artifacts, which can form during sample preparation. The protocol was set up using Paraquat-induced carbonylation, a model that induces protein aggregation in cultured cells. This technique will advance the biochemical, proteomic and biophysical characterization of molecular-weight aggregates associated with protein mutations, as found in many CNS degenerative diseases, or chronic oxidative stress, as found in aging, and chronic metabolic and inflammatory conditions. PMID:25521790

  5. Evaluation of Plasma Fibrinogen Degradation Products and Total Serum Protein Concentration in Oral Submucous Fibrosis

    PubMed Central

    B.N.V.S., Satish; B., Maharudrappa; K.M., Prashant; Hugar, Deepa; Allad, Umesh; Prabhu, Prasanth S.

    2014-01-01

    Background: Oral submucous fibrosis (OSMF) is a potentially malignant disorder with a multifactorial etiology. Malnutrition is a major problem for the inhabitants of most countries where OSMF is prevalent. Recently, a new direction in the etiopathogenesis was provided by the identification of fibrinogen degradation products (FDP) in the plasma of OSMF patients. Aims and Objectives: To assess the role of FDP in the etiology of OSMF and to correlate with the nutritional status by evaluating the total serum protein level. The study also determines to evaluate the correlation between the levels of plasma FDP with respect to the staging and grading of OSMF. Correlation between the levels of Total Serum Protein (TSP) with respect to the staging and grading of OSMF was also evaluated. Materials and Methods: The study included 30 cases clinically and histopathologically diagnosed as oral submucous fibrosis. The FDP levels were assessed using both qualitative and semi quantitative method as supplied by ‘Tulip Diagnostics (P) Ltd. Total Serum Protein (TSP) estimation was done by Biuret method using Liquixx Protein kit by Erba, Manheim. Results: The study indicates that in qualitative assessment of FDP only 14 subjects showed the presence of FDP levels>200ng/ml. In semiquantitative assessment there is no significant association between varying clinical stages and histopathological grades and FDP levels. Total serum Protein level showed a marginal increase in all subjects. The study revealed a positive correlation between FDP and TSP in all OSMF subjects. Conclusion: A larger sample size which would be a better representation of the population and the use of different methods which have higher sensitivities and specificities to evaluate FDP level and detailed fractional analysis of protein along with immunoglobulin profiling would facilitate in attaining more conclusive results. PMID:24995245

  6. Cutaneous necrosis in pregnancy secondary to activated protein C resistance in hereditary angioedema.

    PubMed

    Perkins, W; Downie, I; Keefe, M; Chisholm, M

    1995-04-01

    A 26-year-old woman with hereditary angineurotic oedema (HAE) presented at 22 weeks gestation with severe cutaneous necrosis similar to that seen in coumarin skin necrosis. Protein S deficiency secondary to HAE and pregnancy was postulated. Treatment with heparin, C1-inhibitor concentrates, systemic steroids and surgical debridement resulted in a successful outcome for both mother and child. Subsequent investigations revealed normal levels of protein C, antithrombin III, total protein S, free protein S but reduced function protein S activity with evidence of activated protein C resistance. Cutaneous necrosis has not been reported in associated with activated protein C resistance previously and the possible mechanisms are discussed. PMID:7745572

  7. Measurements of long-range interactions between protein-functionalized surfaces by total internal reflection microscopy.

    PubMed

    Wang, Zhaohui; Gong, Xiangjun; Ngai, To

    2015-03-17

    Understanding the interaction between protein-functionalized surfaces is an important subject in a variety of protein-related processes, ranging from coatings for biomedical implants to targeted drug carriers and biosensors. In this work, utilizing a total internal reflection microscope (TIRM), we have directly measured the interactions between micron-sized particles decorated with three types of common proteins concanavalin A (ConA), bovine serum albumin (BSA), lysozyme (LYZ), and glass surface coated with soy proteins (SP). Our results show that the protein adsorption greatly affects the charge property of the surfaces, and the interactions between those protein-functionalized surfaces depend on solution pH values. At pH 7.5-10.0, all these three protein-functionalized particles are highly negatively charged, and they move freely above the negatively charged SP-functionalized surface. The net interaction between protein-functionalized surfaces captured by TIRM was found as a long-range, nonspecific double-layer repulsion. When pH was decreased to 5.0, both protein-functionalized surfaces became neutral and double-layer repulsion was greatly reduced, resulting in adhesion of all three protein-functionalized particles to the SP-functionalized surface due to the hydrophobic attraction. The situation is very different at pH = 4.0: BSA-decorated particles, which are highly charged, can move freely above the SP-functionalized surfaces, while ConA- and LYZ-decorated particles can only move restrictively in a limited range. Our results quantify these nonspecific kT-scale interactions between protein-functionalized surfaces, which will enable the design of surfaces for use in biomedical applications and study of biomolecular interactions. PMID:25719226

  8. DNA-based control of protein activity

    PubMed Central

    Engelen, W.; Janssen, B. M. G.

    2016-01-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  9. DNA-based control of protein activity.

    PubMed

    Engelen, W; Janssen, B M G; Merkx, M

    2016-03-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  10. Why do total-body decay curves of iodine-labeled proteins begin with a delay

    SciTech Connect

    Regoeczi, E.

    1987-09-01

    The initial delay that occurs in total-body radiation curves reaching their single-exponential slopes was analyzed from 106 experiments involving several mammalian species (guinea pig, mouse, rabbit, and rat) and plasma proteins (alpha 1-acid glycoprotein, antithrombin III, fibrinogen, immunoglobulin G, and transferrin) in 14 different combinations. The time interval (Td) between injection and the intercept of the slope with the full-dose value was adopted as a measure of curve nonideality. The overall mean Td was 6.6 h, but individual values showed a significant correlation to protein half-lives, whereby proteins of unequal metabolic properties exhibited different mean Td values. Targeting protein to the liver abolished delay. Choice of the isotope (/sup 125/I or /sup 131/I) and size of the labeled protein had no influence on the magnitude of delay. Whole-body radiation curves of animals that received (/sup 125/I)iodotyrosines, Na/sup 131/I, or /sup 131/I-polyvinylpyrrolidone exhibited no initial delays. These results do not support the earlier notion that delay is caused by a redistribution of the labeled protein in the body to radiometrically more favorable sites. However, they are compatible with the assumption that delayed passage of a protein dose through the extracellular matrix and/or retarded transfer of proteolytic products from extravascular catabolic sites to plasma may be responsible for the phenomenon.

  11. Anthelmintic activity of Leucaena leucocephala protein extracts on Haemonchus contortus.

    PubMed

    Soares, Alexandra Martins dos Santos; de Araújo, Sandra Alves; Lopes, Suzana Gomes; Costa Junior, Livio Martins

    2015-01-01

    The objective of this study was to evaluate the effects of protein extracts obtained from the plant Leucaena leucocephala on the nematode parasite Haemonchus contortus. The seeds, shell and cotyledon of L. leucocephala were separated and their proteins extracted using a sodium phosphate buffer, and named as TE (total seed extract), SE (shell extract) and CE (cotyledon extract). Soluble protein content, protease, protease inhibitory and chitinase activity assays were performed. Exsheathment inhibition of H. contortus larvae were performed at concentrations of 0.6 mg mL-1, and egg hatch assays were conducted at protein concentrations of 0.8, 0.4, 0.2, 0.1 and 0.05 mg mL-1. The effective concentration for 50% hatching inhibition (EC50) was estimated by probit. Different proportions of soluble proteins, protease and chitinase were found in TE and CE. Protease inhibitory activity was detected in all extracts. The EC50 of the CE and TE extracts were 0.48 and 0.33 mg mL-1, respectively. No ovicidal effects on H. contortus were detected in SE extracts, and none of the protein extracts demonstrated larvicidal effects on H. contortus. We therefore conclude that protein extracts of L. leucocephala had a detrimental effect on nematode eggs, which can be correlated with the high protease and chitinase activity of these extracts. PMID:26689178

  12. Total antioxidant and antiproliferative activities of twenty-four Vitis vinifera grapes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The phytochemical profiles of 24 Vitis vinifera grape cultivars, including total phenolics, total flavonoids, total antioxidant activity and antiproliferative activity, were determined. Total phenolic contents in the cultivars ranged from 95.3 to 686.5 mg of gallic acid equivalents/100 g FW, and to...

  13. Elevated total and central adiposity and low physical activity are associated with insulin resistance in children.

    PubMed

    Krekoukia, Maria; Nassis, George P; Psarra, Glykeria; Skenderi, Katerina; Chrousos, George P; Sidossis, Labros S

    2007-02-01

    The aim of this study was 2-fold: (1) to examine insulin resistance, blood lipid levels, and inflammatory markers in 9- to 11.5-year-old obese and lean children and (2) to identify factors that influence insulin resistance in this cohort of youths. Body mass index, skinfold thickness, waist circumference, physical activity (4-day triaxial accelerometer), cardiorespiratory fitness (submaximal bicycle ergometer test), and dietary intake (3-day food records) were evaluated in 27 obese and 27 lean boys and girls. Fasting blood samples were analyzed for insulin, glucose, lipids and lipoproteins, C-reactive protein (CRP), interleukin 6, soluble intercellular adhesion molecule, and soluble vascular cell adhesion molecule. Homeostasis model assessment (HOMA) was used to evaluate insulin resistance (HOMA-IR). Obese children presented higher HOMA-IR, CRP, and blood lipid levels (all P < .01) compared with lean children. Total body fat and waist circumference were positively associated with fasting insulin (r > or = 0.51), HOMA-IR (r > or = 0.56), CRP (r > or = 0.51), and blood triacylglycerol (r > or = 0.38), and were inversely correlated with high-density lipoprotein cholesterol (r > or = -0.39; all P < .01). Cardiorespiratory fitness was inversely associated with HOMA-IR (r = -0.24; P < .05), but this association disappeared when adjusted for age, sex, and fat mass. Waist circumference and total daily physical activity explained 49% of the variance in HOMA-IR in these children. In conclusion, these findings suggest that total and central adiposity are positively associated and physical activity is negatively associated with insulin resistance in children. Interventions to improve glucose metabolism in youth should target at reducing total body and abdominal fat and increasing physical activity. The lack of association between inflammatory markers and HOMA-IR suggests that obesity may precede the elevation of these markers in the evolution of insulin resistance in youth

  14. Effect of altered eating pattern on serum fructosamine: total protein ratio and plasma glucose level.

    PubMed

    Ch'ng, S L; Cheah, S H; Husain, R; Duncan, M T

    1989-05-01

    The effect of alteration of eating pattern during Ramadan on body mass index (BMI), serum fructosamine: total protein ratio (F/TP), and glucose level in 18 healthy male Asiatic Moslems were studied. The results showed a significant decrease (p less than 0.025) in F/TP at the second week of Ramadan in 11 subjects who experienced continuous decrease in BMI throughout Ramadan. The remaining 7 subjects showed no significant changes in BMI and F/TP. No evidence of hypoglycaemia was observed in the subjects during the study. Serum fructosamine: total protein ratio in subjects with altered eating pattern preferably should be interpreted along with the change in body mass index. PMID:2774480

  15. Total protein quantitation using the bicinchoninic acid assay and gradient elution moving boundary electrophoresis.

    PubMed

    Kralj, Jason G; Munson, Matthew S; Ross, David

    2014-07-01

    We investigated the ability of gradient elution moving boundary electrophoresis (GEMBE) with capacitively coupled contactless conductivity detection (C(4) D) to assay total protein concentration using the bicinchoninic acid (BCA) reaction. We chose this format because GEMBE-C(4) D behaves as a concentration dependent detection system, unlike optical methods that also rely on pathlength (due to Beer's law). This system tolerates proteins well compared with other capillary electrophoretic methods, allowing the capillary to be reused without coatings or additional hydroxide wash steps. The typical reaction protocol was modified by reducing the pH slightly from 11.25 to 9.4, which enabled elimination of tartrate from the reagents. We estimated that copper (I) could be detected at approximately 3.0 μmol/L, which agrees with similar GEMBE and CZE systems utilizing C(4) D. Under conditions similar to the BCA "micro method" assay, we determined the LOD for three common proteins (insulin, BSA, and bovine gamma globulin) and found that they agree well with the existing spectroscopic detection methods. Further, we investigated how long reaction times impact the LOD and found that the conversion was proportional to log(time). This indicated that little sensitivity is gained by extending the reaction past 1 h. Hence, GEMBE provides an alternative platform for total protein assays while maintaining the excellent sensitivity of the optical-based methods. PMID:24648165

  16. Active Nuclear Import of Membrane Proteins Revisited

    PubMed Central

    Laba, Justyna K.; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M.

    2015-01-01

    It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker’s yeast. PMID:26473931

  17. Active Nuclear Import of Membrane Proteins Revisited.

    PubMed

    Laba, Justyna K; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M

    2015-01-01

    It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker's yeast. PMID:26473931

  18. Total Participation Techniques: Making Every Student an Active Learner

    ERIC Educational Resources Information Center

    Himmele, Persida; Himmele, William

    2011-01-01

    Yes, there are easy-to-use and incredibly effective alternatives to the "stand and deliver" approach to teaching that causes so many students to tune out--or even drop out. Here's your opportunity to explore dozens of ways to engage K-12 students in active learning and allow them to demonstrate the depth of their knowledge and understanding. The…

  19. Protein kinase activators alter glial cholesterol esterification

    SciTech Connect

    Jeng, I.; Dills, C.; Klemm, N.; Wu, C.

    1986-05-01

    Similar to nonneural tissues, the activity of glial acyl-CoA cholesterol acyltransferase is controlled by a phosphorylation and dephosphorylation mechanism. Manipulation of cyclic AMP content did not alter the cellular cholesterol esterification, suggesting that cyclic AMP is not a bioregulator in this case. Therefore, the authors tested the effect of phorbol-12-myristate 13-acetate (PMA) on cellular cholesterol esterification to determine the involvement of protein kinase C. PMA has a potent effect on cellular cholesterol esterification. PMA depresses cholesterol esterification initially, but cells recover from inhibition and the result was higher cholesterol esterification, suggesting dual effects of protein kinase C. Studies of other phorbol analogues and other protein kinase C activators such as merezein indicate the involvement of protein kinase C. Oleoyl-acetyl glycerol duplicates the effect of PMA. This observation is consistent with a diacyl-glycerol-protein kinase-dependent reaction. Calcium ionophore A23187 was ineffective in promoting the effect of PMA. They concluded that a calcium-independent and protein C-dependent pathway regulated glial cholesterol esterification.

  20. [The antitumour activity of total saponin of Clematis chinensis].

    PubMed

    Qiu, G; Zhang, M; Yang, Y

    1999-07-01

    This paper has shown the cytocidal effects of total saponin of Clermatis chisensis Osbeck (CCS) on experimental tumours EAC cells, S180A cells and HepA cells in vitro, using microculture method 72 hours. The IC50 were 242 micrograms/ml, 193 micrograms/ml and 130 micrograms/ml respectively. In vivo the growth of transplanted mouse tumour(S180) was inhibited by the CCS with inhibitory rates 40.3% (0.25 g/kg), 55.1% (0.5 g/kg), 53.0% (1 g/kg), but CCS(0.2 g/kg, 0.4g/kg, 0.5 g/kg) hasn't effect on survival time of tumour mice(S180A). PMID:12571931

  1. Petunia nectar proteins have ribonuclease activity.

    PubMed

    Hillwig, Melissa S; Liu, Xiaoteng; Liu, Guangyu; Thornburg, Robert W; Macintosh, Gustavo C

    2010-06-01

    Plants requiring an insect pollinator often produce nectar as a reward for the pollinator's visitations. This rich secretion needs mechanisms to inhibit microbial growth. In Nicotiana spp. nectar, anti-microbial activity is due to the production of hydrogen peroxide. In a close relative, Petunia hybrida, limited production of hydrogen peroxide was found; yet petunia nectar still has anti-bacterial properties, suggesting that a different mechanism may exist for this inhibition. The nectar proteins of petunia plants were compared with those of ornamental tobacco and significant differences were found in protein profiles and function between these two closely related species. Among those proteins, RNase activities unique to petunia nectar were identified. The genes corresponding to four RNase T2 proteins from Petunia hybrida that show unique expression patterns in different plant tissues were cloned. Two of these enzymes, RNase Phy3 and RNase Phy4 are unique among the T2 family and contain characteristics similar to both S- and S-like RNases. Analysis of amino acid patterns suggest that these proteins are an intermediate between S- and S-like RNases, and support the hypothesis that S-RNases evolved from defence RNases expressed in floral parts. This is the first report of RNase activities in nectar. PMID:20460362

  2. Petunia nectar proteins have ribonuclease activity

    PubMed Central

    Hillwig, Melissa S.; Liu, Xiaoteng; Liu, Guangyu; Thornburg, Robert W.; MacIntosh, Gustavo C.

    2010-01-01

    Plants requiring an insect pollinator often produce nectar as a reward for the pollinator's visitations. This rich secretion needs mechanisms to inhibit microbial growth. In Nicotiana spp. nectar, anti-microbial activity is due to the production of hydrogen peroxide. In a close relative, Petunia hybrida, limited production of hydrogen peroxide was found; yet petunia nectar still has anti-bacterial properties, suggesting that a different mechanism may exist for this inhibition. The nectar proteins of petunia plants were compared with those of ornamental tobacco and significant differences were found in protein profiles and function between these two closely related species. Among those proteins, RNase activities unique to petunia nectar were identified. The genes corresponding to four RNase T2 proteins from Petunia hybrida that show unique expression patterns in different plant tissues were cloned. Two of these enzymes, RNase Phy3 and RNase Phy4 are unique among the T2 family and contain characteristics similar to both S- and S-like RNases. Analysis of amino acid patterns suggest that these proteins are an intermediate between S- and S-like RNases, and support the hypothesis that S-RNases evolved from defence RNases expressed in floral parts. This is the first report of RNase activities in nectar. PMID:20460362

  3. A Platform for Combined DNA and Protein Microarrays Based on Total Internal Reflection Fluorescence

    PubMed Central

    Asanov, Alexander; Zepeda, Angélica; Vaca, Luis

    2012-01-01

    We have developed a novel microarray technology based on total internal reflection fluorescence (TIRF) in combination with DNA and protein bioassays immobilized at the TIRF surface. Unlike conventional microarrays that exhibit reduced signal-to-background ratio, require several stages of incubation, rinsing and stringency control, and measure only end-point results, our TIRF microarray technology provides several orders of magnitude better signal-to-background ratio, performs analysis rapidly in one step, and measures the entire course of association and dissociation kinetics between target DNA and protein molecules and the bioassays. In many practical cases detection of only DNA or protein markers alone does not provide the necessary accuracy for diagnosing a disease or detecting a pathogen. Here we describe TIRF microarrays that detect DNA and protein markers simultaneously, which reduces the probabilities of false responses. Supersensitive and multiplexed TIRF DNA and protein microarray technology may provide a platform for accurate diagnosis or enhanced research studies. Our TIRF microarray system can be mounted on upright or inverted microscopes or interfaced directly with CCD cameras equipped with a single objective, facilitating the development of portable devices. As proof-of-concept we applied TIRF microarrays for detecting molecular markers from Bacillus anthracis, the pathogen responsible for anthrax. PMID:22438738

  4. Isotopomer distributions in amino acids from a highly expressed protein as a proxy for those from total protein

    SciTech Connect

    Shaikh, Afshan; Shaikh, Afshan S.; Tang, Yinjie; Mukhopadhyay, Aindrila; Keasling, Jay D.

    2008-06-27

    {sup 13}C-based metabolic flux analysis provides valuable information about bacterial physiology. Though many biological processes rely on the synergistic functions of microbial communities, study of individual organisms in a mixed culture using existing flux analysis methods is difficult. Isotopomer-based flux analysis typically relies on hydrolyzed amino acids from a homogeneous biomass. Thus metabolic flux analysis of a given organism in a mixed culture requires its separation from the mixed culture. Swift and efficient cell separation is difficult and a major hurdle for isotopomer-based flux analysis of mixed cultures. Here we demonstrate the use of a single highly-expressed protein to analyze the isotopomer distribution of amino acids from one organism. Using the model organism E. coli expressing a plasmid-borne, his-tagged Green Fluorescent Protein (GFP), we show that induction of GFP does not affect E. coli growth kinetics or the isotopomer distribution in nine key metabolites. Further, the isotopomer labeling patterns of amino acids derived from purified GFP and total cell protein are indistinguishable, indicating that amino acids from a purified protein can be used to infer metabolic fluxes of targeted organisms in a mixed culture. This study provides the foundation to extend isotopomer-based flux analysis to study metabolism of individual strains in microbial communities.

  5. Total Protein Profile and Drug Resistance in Candida albicans Isolated from Clinical Samples

    PubMed Central

    Thawani, Vijay; Mehra, Arti

    2016-01-01

    This study was done to assess the antifungal susceptibility of clinical isolates of Candida albicans and to evaluate its total protein profile based on morphological difference on drug resistance. Hundred and twenty clinical isolates of C. albicans from various clinical specimens were tested for susceptibility against four antifungal agents, namely, fluconazole, itraconazole, amphotericin B, and ketoconazole. A significant increase of drug resistance in clinical isolates of C. albicans was observed. The study showed 50% fluconazole and itraconazole resistance at 32 μg mL−1 with a MIC50 and MIC90 values at 34 and 47 and 36 and 49 μg mL−1, respectively. All isolates were sensitive to amphotericin B and ketoconazole. The SDS-PAGE protein profile showed a prevalent band of ~52.5 kDa, indicating overexpression of gene in 72% strains with fluconazole resistance. Since the opportunistic infections of Candida spp. are increasing along with drug resistance, the total protein profile will help in understanding the evolutionary changes in drug resistance and also to characterize them. PMID:27478638

  6. Total RNA and protein content, Cyclin B1 expression and developmental competence of prepubertal goat oocytes.

    PubMed

    Anguita, Begoña; Paramio, Maria-Teresa; Jiménez-Macedo, Ana R; Morató, Roser; Mogas, Teresa; Izquierdo, Dolors

    2008-01-30

    The aim of this study was to examine the relationship between the developmental competence of oocytes and their total RNA and protein contents, and the level of Cyclin B1 transcription. Ovaries from prepubertal goats were collected from a slaughterhouse. Oocytes were recovered by slicing and those with two or more layers of cumulus cells and homogenous cytoplasm were matured in vitro (20-25 oocytes per drop) for 27 h. Both before and after IVM, samples of oocytes were denuded and categorised into four group treatments by diameter (<110 microm, 110-125 microm, 125-135 microm; >135 microm), separated into sub-groups of 10 oocytes per treatment-replicate and stored in liquid nitrogen until total RNA content analysis by spectophotometry, total protein content analysis by a colorimetric assay and Cyclin B1 transcription analysis by RT-PCR. For the study of developmental competence, the rest of the matured oocytes were fertilised in vitro in groups of 20-25 for 24 h. Presumptive zygotes were denuded, sorted into the four categories of diameter noted above, and placed into culture drops in groups of 18-25 for in vitro culture. Cleavage rate was evaluated at 48 hpi and embryo development at 8 d post-insemination. There were four replicates of each treatment for each assay or evaluation point of the experiment. There were no significant differences between the size categories of oocytes at collection in total RNA content, total protein content and Cyclin B1 mRNA. There were significant differences (P<0.05) in the expression of Cyclin B1 before IVM with oocytes in the >135 mm diameter category having the highest value for this variant. There were no significant differences in these characteristics between the categories of oocyte diameter after IVM except in respect of total RNA content, which was lower for the largest size of oocytes (>135 microm; mean+/-S.D.=12.3+/-1.84 ng/oocyte) than the other three size groups (19.2+/-1.38-22.1+/-4.44 ng/oocyte; P<0.05). Significant

  7. Application of Near Infrared Reflectance Spectroscopy on Determination of Moisture, Total oil and Protein Contents of In-shell Peanuts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Moisture, total oil and protein content of peanuts are important factors in peanut grading. A method that could rapidly and nondestructively measure these parameters for in-shell peanuts would be extremely useful. NIR reflectance spectroscopy was used to analyze the moisture, total oil and protein ...

  8. Antioxidant activity of whey protein hydrolysates in milk beverage system.

    PubMed

    Mann, Bimlesh; Kumari, Anuradha; Kumar, Rajesh; Sharma, Rajan; Prajapati, Kishore; Mahboob, Shaik; Athira, S

    2015-06-01

    The aim of the present study was to evaluate the antioxidant activity of flavoured milk enriched with antioxidative whey protein hydrolysates (WPHs) by radical scavenging method. Whey protein concentrate (WPC) was hydrolyzed by using three commercial proteases; flavouzyme, alcalase and corolase PP and these WPHs were analyzed for degree of hydrolysis and antioxidant activity. The antioxidant activities of these WPHs were evaluated using ABTS method. Trolox equivalent antioxidant activity of all the hydrolysates i.e. flavourzyme (0.81 ± 0.04), alcalase (1.16 ± 0.05) and corolase (1.42 ± 0.12) was higher than the WPC (0.19 ± 0.01). Among these, whey protein hydrolysates prepared using corolase showed maximum antioxidant activity. Total 15 β-lactoglobulin, 1 α-lactoalbumin, and 6 β-casein derived peptide fragments were identified in the WPHs by LC-MS/MS. Due to their size and characteristic amino acid composition, all the identified peptides may contribute for the antioxidant activity. The strawberry and chocolate flavoured milk was supplemented with WPC and WPHs and 2 % addition has shown increase in antioxidant activity upto 42 %. The result suggests that WPH could be used as natural biofunctional ingredients in enhancing antioxidant properties of food products. PMID:26028704

  9. Oxidation of methionine residues in proteins of activated human neutrophils.

    PubMed Central

    Fliss, H; Weissbach, H; Brot, N

    1983-01-01

    A simple assay for the detection of 35S-labeled methionine sulfoxide residues in proteins is described. The assay, which is based on the ability of CNBr to react with methionine but not with methionine sulfoxide, requires the prelabeling of cellular proteins with [35S]methionine. The assay was used to study the extent of methionine oxidation in newly synthesized proteins of both activated and quiescent human neutrophils. In cells undergoing a phorbol 12-myristate 13-acetate-induced respiratory burst, about 66% of all methionine residues in newly synthesized proteins were oxidized to the sulfoxide derivative, as compared with 9% in cells not treated with the phorbol ester. In contrast, quantitation of methionine sulfoxide content in the total cellular protein by means of amino acid analysis showed that only 22% of all methionine residues were oxidized in activated cells as compared with 9% in quiescent cells. It is proposed that methionine residues in nascent polypeptide chains are more susceptible to oxidation than those in completed proteins. PMID:6580633

  10. Macrophage activation and migration in interface tissue around loosening total hip arthroplasty components.

    PubMed

    Ishiguro, N; Kojima, T; Ito, T; Saga, S; Anma, H; Kurokouchi, K; Iwahori, Y; Iwase, T; Iwata, H

    1997-06-01

    The bone-cement interface tissue of failed total hip arthroplasty (THA) has inflammatory characteristics, such as the presence of prostaglandin E2 and interleukin 1 (IL-1). We considered that the bone-cement interface tissue could be the site of granulomatous inflammation caused by a foreign-body reaction. It has been demonstrated that inflammatory cytokines and chemokines have an important role in granulomatous inflammation. Bone-cement interface tissue was obtained at revision from nine patients with failed cemented THA, and the role of macrophages was assessed by immunohistochemistry, electron microscopy, and molecular biological techniques. We used the reverse-transcriptional polymerase chain reaction to examine the expression of mRNA for IL-1 alpha, IL-1 beta, tumor necrosis factor alpha (TNF alpha), macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, IL-8, and monocyte chemoattractant protein. Polyethylene debris surrounded by macrophages and phagocytosis of debris by macrophages was frequently observed in the interface tissue. Macrophage activation and the production of inflammatory cytokines such as IL-1 and TNF alpha might induce the development of interface tissue. Expression of chemokine mRNAs was also commonly seen, suggesting that this led to recruitment of macrophages into the bone-cement interface tissue. Debris released from implants appears to cause activation of macrophages and the production of inflammatory cytokines and chemokines that induce cellular recruitment into interface tissue. This mechanism might form a vicious cycle that aggravates THA loosening. PMID:9138074

  11. Use of liposomal amplifiers in total internal reflection fluorescence fiber-optic biosensors for protein detection.

    PubMed

    Chang, Ying-Feng; Fu, Chen; Chen, Yi-Ting; Fang-Ju Jou, Amily; Chen, Chii-Chang; Chou, Chien; Annie Ho, Ja-An

    2016-03-15

    Evanescent-wave excited fluorescence technology has been demonstrated to enhance sensitivity and reduce matrix effects, making it suitable for biosensor development. In this study, we developed a liposome-based, total internal reflection fluorescence, fiber-optic biosensor (TIRF-FOB) for protein detection, which integrates a liposomal amplifier and sandwich immunoassay format with TIRF-FOB. In addition, the antibody-tagged and fluorophore-entrapped liposomes for heterogeneous detection of target molecules were designed and synthesized. This biosensor successfully detected the target protein (model analyzed here is IgG) with a limit of detection (LOD) of 2.0 attomoles for the target protein (equivalent to 2.0 pg/mL of protein presented in 150 μL of sample solution). The features of this ultra-sensitive liposomal TIRF-FOB are (i) fluorescence is excited via evanescent waves and amplified via liposomes; (ii) the use of two polyclonal antibodies in the sandwich assay format increases the specificity and lowers the cost of our assay. Based on the exceptional detection sensitivity and cost-effectiveness, we believe that the proposed biosensor has great potential as a practical, clinical diagnostic tool in the near future. PMID:26595485

  12. The nutrient composition of European ready meals: protein, fat, total carbohydrates and energy.

    PubMed

    Kanzler, Sonja; Manschein, Martin; Lammer, Guido; Wagner, Karl-Heinz

    2015-04-01

    Despite the increasing social importance of ready meals, only few studies have been conducted on their nutrient composition. Therefore, 32 chilled, frozen and heat-treated ready meals (only main dishes) from the continental European market were analysed for protein, fat, total carbohydrate and energy. Half of the meals were nutritionally imbalanced by providing elevated fat (>30% of energy) and low carbohydrate levels (<50% of energy). Protein was generally above recommendations and ranged from 8.0 to 47.2g per serving. The inter-package variation was high, reaching 19.04 ± 2.90 g/package for fat. After proposing understandable guidelines to improve nutritional quality for the food industry, seven "nutritionally optimised" ready meals were created at the European level and analysed, however success was limited. If product labelling is to be useful for consumers, our results also indicate a need for better quality control to reduce the differences between content and labelling. PMID:25442542

  13. Constrained Total Energy Expenditure and Metabolic Adaptation to Physical Activity in Adult Humans.

    PubMed

    Pontzer, Herman; Durazo-Arvizu, Ramon; Dugas, Lara R; Plange-Rhule, Jacob; Bovet, Pascal; Forrester, Terrence E; Lambert, Estelle V; Cooper, Richard S; Schoeller, Dale A; Luke, Amy

    2016-02-01

    Current obesity prevention strategies recommend increasing daily physical activity, assuming that increased activity will lead to corresponding increases in total energy expenditure and prevent or reverse energy imbalance and weight gain [1-3]. Such Additive total energy expenditure models are supported by exercise intervention and accelerometry studies reporting positive correlations between physical activity and total energy expenditure [4] but are challenged by ecological studies in humans and other species showing that more active populations do not have higher total energy expenditure [5-8]. Here we tested a Constrained total energy expenditure model, in which total energy expenditure increases with physical activity at low activity levels but plateaus at higher activity levels as the body adapts to maintain total energy expenditure within a narrow range. We compared total energy expenditure, measured using doubly labeled water, against physical activity, measured using accelerometry, for a large (n = 332) sample of adults living in five populations [9]. After adjusting for body size and composition, total energy expenditure was positively correlated with physical activity, but the relationship was markedly stronger over the lower range of physical activity. For subjects in the upper range of physical activity, total energy expenditure plateaued, supporting a Constrained total energy expenditure model. Body fat percentage and activity intensity appear to modulate the metabolic response to physical activity. Models of energy balance employed in public health [1-3] should be revised to better reflect the constrained nature of total energy expenditure and the complex effects of physical activity on metabolic physiology. PMID:26832439

  14. The Association between Total Protein and Vegetable Protein Intake and Low Muscle Mass among the Community-Dwelling Elderly Population in Northern Taiwan.

    PubMed

    Huang, Ru-Yi; Yang, Kuen-Cheh; Chang, Hao-Hsiang; Lee, Long-Teng; Lu, Chia-Wen; Huang, Kuo-Chin

    2016-01-01

    Sarcopenia, highly linked with fall, frailty, and disease burden, is an emerging problem in aging society. Higher protein intake has been suggested to maintain nitrogen balance. Our objective was to investigate whether pre-sarcopenia status was associated with lower protein intake. A total of 327 community-dwelling elderly people were recruited for a cross-sectional study. We adopted the multivariate nutrient density model to identify associations between low muscle mass and dietary protein intake. The general linear regression models were applied to estimate skeletal muscle mass index across the quartiles of total protein and vegetable protein density. Participants with diets in the lowest quartile of total protein density (<13.2%) were at a higher risk for low muscle mass (odds ratio (OR) 3.03, 95% confidence interval (CI) 1.37-6.72) than those with diets in the highest quartile (≥17.2%). Similarly, participants with diets in the lowest quartile of vegetable protein density (<5.8%) were at a higher risk for low muscle mass (OR 2.34, 95% CI 1.14-4.83) than those with diets in the highest quartile (≥9.4%). Furthermore, the estimated skeletal muscle mass index increased significantly across the quartiles of total protein density (p = 0.023) and vegetable protein density (p = 0.025). Increasing daily intakes of total protein and vegetable protein densities appears to confer protection against pre-sarcopenia status. PMID:27322317

  15. The Association between Total Protein and Vegetable Protein Intake and Low Muscle Mass among the Community-Dwelling Elderly Population in Northern Taiwan

    PubMed Central

    Huang, Ru-Yi; Yang, Kuen-Cheh; Chang, Hao-Hsiang; Lee, Long-Teng; Lu, Chia-Wen; Huang, Kuo-Chin

    2016-01-01

    Sarcopenia, highly linked with fall, frailty, and disease burden, is an emerging problem in aging society. Higher protein intake has been suggested to maintain nitrogen balance. Our objective was to investigate whether pre-sarcopenia status was associated with lower protein intake. A total of 327 community-dwelling elderly people were recruited for a cross-sectional study. We adopted the multivariate nutrient density model to identify associations between low muscle mass and dietary protein intake. The general linear regression models were applied to estimate skeletal muscle mass index across the quartiles of total protein and vegetable protein density. Participants with diets in the lowest quartile of total protein density (<13.2%) were at a higher risk for low muscle mass (odds ratio (OR) 3.03, 95% confidence interval (CI) 1.37–6.72) than those with diets in the highest quartile (≥17.2%). Similarly, participants with diets in the lowest quartile of vegetable protein density (<5.8%) were at a higher risk for low muscle mass (OR 2.34, 95% CI 1.14–4.83) than those with diets in the highest quartile (≥9.4%). Furthermore, the estimated skeletal muscle mass index increased significantly across the quartiles of total protein density (p = 0.023) and vegetable protein density (p = 0.025). Increasing daily intakes of total protein and vegetable protein densities appears to confer protection against pre-sarcopenia status. PMID:27322317

  16. [Mitogen-activated protein kinases in atherosclerosis].

    PubMed

    Bryk, Dorota; Olejarz, Wioletta; Zapolska-Downar, Danuta

    2014-01-01

    Intracellular signalling cascades, in which MAPK (mitogen-activated protein kinases) intermediate, are responsible for a biological response of a cell to an external stimulus. MAP kinases, which include ERK1/2 (extracellular signalling-regulated kinase), JNK (c-Jun N-terminal kinase) and p 38 MAPK, regulate the activity of many proteins, enzymes and transcription factors and thus have a wide spectrum of biological effects. Many basic scientific studies have defined numerous details of their pathway organization and activation. There are also more and more studies suggesting that individual MAP kinases probably play an important role in the pathogenesis of atherosclerosis. They may mediate inflammatory processes, endothelial cell activation, monocyte/macrophage recruitment and activation, smooth muscle cell proliferation and T-lymphocyte differentiation, all of which represent crucial mechanisms involved in pathogenesis of atherosclerosis. The specific inhibition of an activity of the respective MAP kinases may prove a new therapeutic approach to attenuate atherosclerotic plaque formation in the future. In this paper, we review the current state of knowledge concerning MAP kinase-dependent cellular and molecular mechanisms underlying atherosclerosis. PMID:24491891

  17. De Novo Construction of Redox Active Proteins.

    PubMed

    Moser, C C; Sheehan, M M; Ennist, N M; Kodali, G; Bialas, C; Englander, M T; Discher, B M; Dutton, P L

    2016-01-01

    Relatively simple principles can be used to plan and construct de novo proteins that bind redox cofactors and participate in a range of electron-transfer reactions analogous to those seen in natural oxidoreductase proteins. These designed redox proteins are called maquettes. Hydrophobic/hydrophilic binary patterning of heptad repeats of amino acids linked together in a single-chain self-assemble into 4-alpha-helix bundles. These bundles form a robust and adaptable frame for uncovering the default properties of protein embedded cofactors independent of the complexities introduced by generations of natural selection and allow us to better understand what factors can be exploited by man or nature to manipulate the physical chemical properties of these cofactors. Anchoring of redox cofactors such as hemes, light active tetrapyrroles, FeS clusters, and flavins by His and Cys residues allow cofactors to be placed at positions in which electron-tunneling rates between cofactors within or between proteins can be predicted in advance. The modularity of heptad repeat designs facilitates the construction of electron-transfer chains and novel combinations of redox cofactors and new redox cofactor assisted functions. Developing de novo designs that can support cofactor incorporation upon expression in a cell is needed to support a synthetic biology advance that integrates with natural bioenergetic pathways. PMID:27586341

  18. Synaptic Vesicle Proteins and Active Zone Plasticity

    PubMed Central

    Kittel, Robert J.; Heckmann, Manfred

    2016-01-01

    Neurotransmitter is released from synaptic vesicles at the highly specialized presynaptic active zone (AZ). The complex molecular architecture of AZs mediates the speed, precision and plasticity of synaptic transmission. Importantly, structural and functional properties of AZs vary significantly, even for a given connection. Thus, there appear to be distinct AZ states, which fundamentally influence neuronal communication by controlling the positioning and release of synaptic vesicles. Vice versa, recent evidence has revealed that synaptic vesicle components also modulate organizational states of the AZ. The protein-rich cytomatrix at the active zone (CAZ) provides a structural platform for molecular interactions guiding vesicle exocytosis. Studies in Drosophila have now demonstrated that the vesicle proteins Synaptotagmin-1 (Syt1) and Rab3 also regulate glutamate release by shaping differentiation of the CAZ ultrastructure. We review these unexpected findings and discuss mechanistic interpretations of the reciprocal relationship between synaptic vesicles and AZ states, which has heretofore received little attention. PMID:27148040

  19. [Evaluation of the total biological activity and allergenic composition of allergenic extracts].

    PubMed

    Lombardero, M; González, R; Duffort, O; Juan, F; Ayuso, R; Ventas, P; Cortés, C; Carreira, J

    1986-01-01

    In the present study, a complete procedure is presented in order to standardize allergenic extracts, the meaning of which is the measurement of the total allergenic activity and the determination of the allergenic composition. The measurement of the biological activity comprises 2 steps: Preparation of Reference Extracts and determination of their "in vivo" activity. Evaluation of the total allergenic activity of extracts for clinical use. Reference extracts were prepared from the main allergens and their "in vivo" biological activity was determined by a quantitative skin prick test in a sample of at least 30 allergic patients. By definition, the protein concentration of Reference Extract that produces, in the allergic population, a geometric mean wheal of 75 mm.2 has an activity of 100 biological units (BUs). The determination of the biological activity of a problem extract is made by RAST inhibition. The sample is compared with the corresponding Reference Extract by this technique and, from this comparison, it is possible to quantify the activity of the problem extract in biologic units (BUs) with clinical significance. Likewise, different techniques have been used to determine the allergenic composition of extracts. These techniques comprise 2 steps: Separation of the components of the extract. Identification of the components that bind specific human IgE. The separation of the components of the extract has been carried out by isoelectric focusing (IEF) and electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE). In order to identify the allergenic components, an immunoblotting technique has been employed. The separated components in the IEF gel or SDS-PAGE gel are transferred to a nitrocellulose sheet and later on, this membrane is overlaid with a serum pool from allergic patients and a mouse monoclonal anti-human IgE, labelled with 125I. Finally, the autoradiography of the nitrocellulose membrane is obtained. In this way it is possible to compare

  20. Detection of protein C activation in humans.

    PubMed Central

    Bauer, K A; Kass, B L; Beeler, D L; Rosenberg, R D

    1984-01-01

    We have developed a radioimmunoassay (RIA) for the dodecapeptide that is liberated from protein C when this zymogen is activated by thrombin bound to thrombomodulin present on the vascular endothelium. The protein C activation peptide (PCP) was synthesized using the solid-phase method of Merrifield. Antisera were raised in rabbits to the synthetic analogue coupled to bovine serum albumin with glutaraldehyde. The antibody population obtained was used together with a 125I-labeled tyrosinated ligand and various concentrations of unlabeled PCP to construct a double antibody RIA capable of measuring as little as 10 pM of this component. We have established that the synthetic dodecapeptide has the same immunoreactivity as the native peptide and that the reactivity of protein C is less than 1/2,000 that of PCP on a molar basis. The extremely low levels of peptide in normal individuals as well as the nonspecific contributions of plasma constituents to the immunoreactive signal, necessitated the development of a procedure by which the PCP could be reproducibly extracted from plasma and concentrated approximately 20-fold. This methodology permitted us to demonstrate that the plasma PCP levels in 17 normal donors averaged 6.47 pM, and that elevations up to 180 pM were observed in individuals with evidence of disseminated intravascular coagulation. The validity of these measurements of protein C activation is supported by the fact that, in both of these situations, the RIA signal migrates on reverse-phase high pressure liquid chromatography in a manner identical to that of the native dodecapeptide. We have also noted that the mean PCP concentration in seven patients fully anticoagulated with warfarin averaged 2.61 pM. Our studies also show that PCP is cleared from the plasma of primates with a t1/2 of approximately 5 min. Given that the t1/2 of activated protein C is estimated to be 10-15 min, the latter enzyme appears to exert its effects on the activated cofactors of the

  1. Interference of salts used on aqueous two-phase systems on the quantification of total proteins.

    PubMed

    Golunski, Simone Maria; Sala, Luisa; Silva, Marceli Fernandes; Dallago, Rogério Marcos; Mulinari, Jéssica; Mossi, Altemir José; Brandelli, Adriano; Kalil, Susana Juliano; Di Luccio, Marco; Treichel, Helen

    2016-02-01

    In this study the interference of potassium phosphate, sodium citrate, sodium chloride and sodium nitrate salts on protein quantification by Bradford's method was assessed. Potassium phosphate and sodium citrate salts are commonly used in aqueous two-phase systems for enzyme purification. Results showed that the presence of potassium phosphate and sodium citrate salts increase the absorbance of the samples, when compared with the samples without any salt. The increase in absorptivity of the solution induces errors on protein quantification, which are propagated to the calculations of specific enzyme activity and consequently on purification factor. The presence of sodium chloride and sodium nitrate practically did not affect the absorbance of inulinase, probably the metals present in the enzyme extract did not interact with the added salts. PMID:26616454

  2. Assessment of uranium exposure from total activity and 234U:238U activity ratios in urine.

    PubMed

    Nicholas, T; Bingham, D

    2011-03-01

    Radiation workers at Atomic Weapons Establishment (AWE) are monitored for uranium exposure by routine bioassay sampling (primarily urine sampling). However, the interpretation of uranium in urine and faecal results in terms of occupational intakes is difficult because of the presence of uranium due to intakes from environmental (dietary) sources. For uranium in urine data obtained using current analytical techniques at AWE, the mean, median and standard deviation of excreted uranium concentrations were 0.006, 0.002 and 0.012 μg per g creatinine, respectively. These values are consistent with what might be expected from local dietary intakes and the knowledge that occupational exposures at AWE are likely to be very low. However, some samples do exceed derived investigation levels (DILs), which have been set up taking account of the likely contribution from environmental sources. We investigate how the activity and isotopic composition of uranium in the diet affects the sensitivity of uranium in urine monitoring for occupational exposures. We conclude that DILs based on both total uranium in urine activity and also (234)U:(238)U ratios are useful given the likely variation in dietary contribution for AWE workers. Assuming a background excretion rate and that the enrichment of the likely exposure is known, it is possible to assess exposures using (234)U:(238)U ratios and/or total uranium activity. The health implications of internalised uranium, enriched to <5-8 % by mass (235)U, centre on its nephrotoxicity; the DILs for bioassay samples at AWE are an order of magnitude below the conservative recommendations made by the literature. PMID:21036806

  3. Crowding Activates Heat Shock Protein 90.

    PubMed

    Halpin, Jackson C; Huang, Bin; Sun, Ming; Street, Timothy O

    2016-03-18

    Hsp90 is a dimeric ATP-dependent chaperone involved in the folding, maturation, and activation of diverse target proteins. Extensive in vitro structural analysis has led to a working model of Hsp90's ATP-driven conformational cycle. An implicit assumption is that dilute experimental conditions do not significantly perturb Hsp90 structure and function. However, Hsp90 undergoes a dramatic open/closed conformational change, which raises the possibility that this assumption may not be valid for this chaperone. Indeed, here we show that the ATPase activity of Hsp90 is highly sensitive to molecular crowding, whereas the ATPase activities of Hsp60 and Hsp70 chaperones are insensitive to crowding conditions. Polymer crowders activate Hsp90 in a non-saturable manner, with increasing efficacy at increasing concentration. Crowders exhibit a non-linear relationship between their radius of gyration and the extent to which they activate Hsp90. This experimental relationship can be qualitatively recapitulated with simple structure-based volume calculations comparing open/closed configurations of Hsp90. Thermodynamic analysis indicates that crowding activation of Hsp90 is entropically driven, which is consistent with a model in which excluded volume provides a driving force that favors the closed active state of Hsp90. Multiple Hsp90 homologs are activated by crowders, with the endoplasmic reticulum-specific Hsp90, Grp94, exhibiting the highest sensitivity. Finally, we find that crowding activation works by a different mechanism than co-chaperone activation and that these mechanisms are independent. We hypothesize that Hsp90 has a higher intrinsic activity in the cell than in vitro. PMID:26797120

  4. Heat dissipation guides activation in signaling proteins.

    PubMed

    Weber, Jeffrey K; Shukla, Diwakar; Pande, Vijay S

    2015-08-18

    Life is fundamentally a nonequilibrium phenomenon. At the expense of dissipated energy, living things perform irreversible processes that allow them to propagate and reproduce. Within cells, evolution has designed nanoscale machines to do meaningful work with energy harnessed from a continuous flux of heat and particles. As dictated by the Second Law of Thermodynamics and its fluctuation theorem corollaries, irreversibility in nonequilibrium processes can be quantified in terms of how much entropy such dynamics produce. In this work, we seek to address a fundamental question linking biology and nonequilibrium physics: can the evolved dissipative pathways that facilitate biomolecular function be identified by their extent of entropy production in general relaxation processes? We here synthesize massive molecular dynamics simulations, Markov state models (MSMs), and nonequilibrium statistical mechanical theory to probe dissipation in two key classes of signaling proteins: kinases and G-protein-coupled receptors (GPCRs). Applying machinery from large deviation theory, we use MSMs constructed from protein simulations to generate dynamics conforming to positive levels of entropy production. We note the emergence of an array of peaks in the dynamical response (transient analogs of phase transitions) that draw the proteins between distinct levels of dissipation, and we see that the binding of ATP and agonist molecules modifies the observed dissipative landscapes. Overall, we find that dissipation is tightly coupled to activation in these signaling systems: dominant entropy-producing trajectories become localized near important barriers along known biological activation pathways. We go on to classify an array of equilibrium and nonequilibrium molecular switches that harmonize to promote functional dynamics. PMID:26240354

  5. Activation and activities of the p53 tumour suppressor protein

    PubMed Central

    Bálint, É; Vousden, K H

    2001-01-01

    The p53 tumour suppressor protein inhibits malignant progression by mediating cell cycle arrest, apoptosis or repair following cellular stress. One of the major regulators of p53 function is the MDM2 protein, and multiple forms of cellular stress activate p53 by inhibiting the MDM2-mediated degradation of p53. Mutations in p53, or disruption of the pathways that allow activation of p53, seem to be a general feature of all cancers. Here we review recent advances in our understanding of the pathways that regulate p53 and the pathways that are induced by p53, as well as their implications for cancer therapy. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11747320

  6. Determination of total mercury in seafood and other protein-rich products

    SciTech Connect

    Landi, S.; Fagioli, F.; Locatelli, C.

    1992-11-01

    A previously developed wet-digestion method for the determination of total mercury in plants by cold vapor atomic absorption spectroscopy (CVAAS) was extended to the analysis of seafood and other products rich in proteins. Oxidation of matrixes is accomplished by K{sub 2}Cr{sub 2}O{sub 7} in the presence of diluted H{sub 2}SO{sub 4}; a simple air condenser is used to reflux vapors released from the boiling mixture. The original procedure (A) and 2 modifications (B and C), which differ with respect to the mode of acidification and/or digestion time and the types of condensers used, were compared for precision and accuracy by means of National Institute of Standards and Technology Research Material 50 Albacore Tuna and proved to be reliable (Hg present, 0.95{plus_minus}0.1 {mu}g/g; Hg found, 0.97 {plus_minus} 0.029 {mu}g/g [A], 0.98 {plus_minus} 0.018 {mu}g/g [B], and 0.94 {plus_minus} 0.025 {mu}g/g [C]). The modified procedures were tested further in Hg recovery experiments on a variety of biological matrixes with different spiking substances and again showed good analytical characteristics (overall average recoveries = 98 {plus_minus} 5.1% for seafood and 100 {plus_minus} 3.6 for protein-rich baby foods). 22 refs., 1 fig., 5 tabs.

  7. Physicochemical and sensory characteristics of whey protein hydrolysates generated at different total solids levels.

    PubMed

    Spellman, David; O'Cuinn, Gerard; FitzGerald, Richard J

    2005-05-01

    Whey protein hydrolysates were generated at different total solids (TS) levels (50-300 g/l) using the commercially available proteolytic preparation Debitrase HYW20, while enzyme to substrate ratio, pH and temperature were maintained constant. Hydrolysis proceeded at a faster rate at lower TS reaching a degree of hydrolysis (DH) of 16.6% at 300 g TS/l, compared with a DH of 22.7% at 50 g TS/l after 6 h hydrolysis. The slower breakdown of intact whey proteins at high TS was quantified by gel-permeation HPLC. Reversed-phase (RP) HPLC of hydrolysate samples of equivalent DH (approximately 15%) generated at different TS levels indicated that certain hydrophobic peptide peaks were present at higher levels in hydrolysates generated at low TS. Sensory evaluation showed that hydrolysates with equivalent DH values were significantly (P < 0.0005) less bitter when generated at 300 g TS/l (mean bitterness score = 25.4%) than hydrolysates generated at 50 g TS/l (mean bitterness score = 39.9%). A specific hydrophobic peptide peak present at higher concentrations in hydrolysates generated at low TS was isolated and identified as beta-lactoglobulin f(43-57), a fragment having the physical and chemical characteristics of a bitter peptide. PMID:15909678

  8. Total esterase activity in human saliva: Validation of an automated assay, characterization and behaviour after physical stress.

    PubMed

    Tecles, Fernando; Tvarijonaviciute, Asta; De Torre, Carlos; Carrillo, José M; Rubio, Mónica; García, Montserrat; Cugat, Ramón; Cerón, José J

    2016-07-01

    Although saliva has esterase activity, this activity has not been characterized or studied in individuals subjected to physical stress. The aim of this report was to develop and validate an automated spectrophotometric assay for total esterase activity measurement in human saliva, as well as to study the contribution of different enzymes on this activity and its behaviour under physical stress in healthy subjects. The assay used 4-nitrophenyl acetate as substrate and was precise, accurate and provided low limits of detection and quantification. Inhibition with diisopropylfluorophosphate showed that cholinesterase, carboxylesterase and cholesterol esterase contributions not represented more than 20% of total esterase. Addition of standards of lipase and albumin to saliva samples showed that both proteins significantly contributed to esterase activity only when equal or higher than 11.6 IU/L and 250 μg/mL, respectively. Western blot analyses showed absence of paraoxonase-1 and high amount of carbonic anhydrase-VI. The high affinity of purified carbonic anhydrase-VI for the substrate supported a major contribution of this enzyme. Total esterase activity and alpha-amylase was measured in saliva samples from 12 healthy male students before and after participation in an indoor football match. The activity significantly increased after match and positively correlated with salivary alpha-amylase. This method could be used as a biomarker of physical stress in humans, with carbonic anhydrase-VI being the esterase that contributed more to the activity of the assay. PMID:27045801

  9. Biodegradable nanoparticles for protein delivery: analysis of preparation conditions on particle morphology and protein loading, activity and sustained release properties.

    PubMed

    Coleman, Jason; Lowman, Anthony

    2012-01-01

    PLGA particles have been extensively used as a sustained drug-delivery system, but there are multiple drawbacks when delivering proteins. The focus of this work is to address the most significant disadvantages to the W/O/W double emulsion procedure and demonstrate that simple changes to this procedure can have significant changes to particle size and dispersity and considerable improvements to protein loading, activity and sustained active protein release. A systematic approach was taken to analyze the effects of the following variables: solvent miscibility (dichloromethane (DCM), ethyl acetate, acetone), homogenization speed (10 000-25 000 rpm), PLGA concentration (10-30 mg/ml) and additives in both the organic (sucrose acetate isobutyrate (SAIB)) and aqueous (bovine serum albumin (BSA)) phases. Increasing solvent miscibility decreased particle size, dispersity and protein denaturation, while maintaining adequate protein loading. Increasing solvent miscibility also lowered the impact of homogenization on particle size and dispersity and protein activity. Changes to PLGA concentration demonstrated a minimum impact on particle size and dispersity, but showed an inverse relationship between protein encapsulation efficiency and particle protein weight percent. Most particles tested provided sustained release of active protein over 60 days. Increasing solvent miscibility resulted in increases in the percent of active protein released. When subjected to synthesis conditions with DCM as the solvent, BSA as a stabilizer resulted in the maximum stabilization of protein at a concentration of 100 mg/ml. At this concentration, BSA allowed for increases in the total amount of active protein delivered for all three solvents. The benefit of SAIB was primarily increased protein loading. PMID:21639993

  10. Blood count and C-reactive protein evolution in gastric cancer patients with total gastrectomy surgery

    PubMed Central

    CSENDES J., Attila; MUÑOZ Ch., Andrea; BURGOS L., Ana María

    2014-01-01

    Background The complete blood count (CBC) and C-reactive protein (CRP) are useful inflammatory parameters for ruling out acute postoperative inflammatory complications. Aim To determine their changes in gastric cancer patients submitted to total gastrectomy. Methods This is a prospective study, with 36 patients with gastric cancer who were submitted to elective total gastrectomy. On the first, third and fifth postoperative day (POD), blood count and CRP changes were assessed. Patients with postoperative complications were excluded. Results Twenty-one (58%) were men and 15 (42%) women. The mean age was 65 years. The leukocytes peaked on the 1st POD with a mean of 13,826 u/mm³, and decreased to 8,266 u/mm³ by the 5th POD. The bacilliforms peaked on the 1st POD with a maximum value of 1.48%. CRP reached its maximum level on the 3rd POD with a mean of 144.64 mg/l±44.84. Preoperative hematocrit (HCT) was 35% and 33.67% by the 5th POD. Hemoglobin, showed similar values. Conclusions Leukocytes increased during the 1st POD but reached normal values by the 5th POD. CRP peaked on the 3rd POD but did not reach normal values by the 5th POD. PMID:25626929

  11. The Role of Mitogen-Activated Protein Kinase-Activated Protein Kinases (MAPKAPKs) in Inflammation

    PubMed Central

    Moens, Ugo; Kostenko, Sergiy; Sveinbjørnsson, Baldur

    2013-01-01

    Mitogen-activated protein kinase (MAPK) pathways are implicated in several cellular processes including proliferation, differentiation, apoptosis, cell survival, cell motility, metabolism, stress response and inflammation. MAPK pathways transmit and convert a plethora of extracellular signals by three consecutive phosphorylation events involving a MAPK kinase kinase, a MAPK kinase, and a MAPK. In turn MAPKs phosphorylate substrates, including other protein kinases referred to as MAPK-activated protein kinases (MAPKAPKs). Eleven mammalian MAPKAPKs have been identified: ribosomal-S6-kinases (RSK1-4), mitogen- and stress-activated kinases (MSK1-2), MAPK-interacting kinases (MNK1-2), MAPKAPK-2 (MK2), MAPKAPK-3 (MK3), and MAPKAPK-5 (MK5). The role of these MAPKAPKs in inflammation will be reviewed. PMID:24705157

  12. Phospholipases as GTPase activity accelerating proteins (GAPs) in plants.

    PubMed

    Pandey, Sona

    2016-05-01

    GTPase activity accelerating proteins (GAPs) are key regulators of the G-protein signaling cycle. By facilitating effective hydrolysis of the GTP bound on Gα proteins, GAPs control the timing and amplitude of the signaling cycle and ascertain the availability of the inactive heterotrimer for the next round of activation. Until very recently, the studies of GAPs in plants were focused exclusively on the regulator of G-protein signaling (RGS) protein. We now show that phospholipase Dα1 (PLDα1) is also a bona fide GAP in plants and together with the RGS protein controls the level of activeprotein. PMID:27124090

  13. Monooxygenase activity of type 3 copper proteins.

    PubMed

    Itoh, Shinobu; Fukuzumi, Shunichi

    2007-07-01

    The molecular mechanism of the monooxygenase (phenolase) activity of type 3 copper proteins has been examined in detail both in the model systems and in the enzymatic systems. The reaction of a side-on peroxo dicopper(II) model compound ( A) and neutral phenols proceeds via a proton-coupled electron-transfer (PCET) mechanism to generate phenoxyl radical species, which collapse each other to give the corresponding C-C coupling dimer products. In this reaction, a bis(mu-oxo)dicopper(III) complex ( B) generated by O-O bond homolysis of A is suggested to be a real active species. On the other hand, the reaction of lithium phenolates (deprotonated form of phenols) with the same side-on peroxo dicopper(II) complex proceeds via an electrophilic aromatic substitution mechanism to give the oxygenated products (catechols). The mechanistic difference between these two systems has been discussed on the basis of the Marcus theory of electron transfer and Hammett analysis. Mechanistic details of the monooxygenase activity of tyrosinase have also been examined using a simplified enzymatic reaction system to demonstrate that the enzymatic reaction mechanism is virtually the same as that of the model reaction, that is, an electrophilic aromatic substitution mechanism. In addition, the monooxygenase activity of the oxygen carrier protein hemocyanin has been explored for the first time by employing urea as an additive in the reaction system. In this case as well, the ortho-hydroxylation of phenols to catechols has been demonstrated to involve the same ionic mechanism. PMID:17461541

  14. Heat dissipation guides activation in signaling proteins

    PubMed Central

    Weber, Jeffrey K.; Shukla, Diwakar; Pande, Vijay S.

    2015-01-01

    Life is fundamentally a nonequilibrium phenomenon. At the expense of dissipated energy, living things perform irreversible processes that allow them to propagate and reproduce. Within cells, evolution has designed nanoscale machines to do meaningful work with energy harnessed from a continuous flux of heat and particles. As dictated by the Second Law of Thermodynamics and its fluctuation theorem corollaries, irreversibility in nonequilibrium processes can be quantified in terms of how much entropy such dynamics produce. In this work, we seek to address a fundamental question linking biology and nonequilibrium physics: can the evolved dissipative pathways that facilitate biomolecular function be identified by their extent of entropy production in general relaxation processes? We here synthesize massive molecular dynamics simulations, Markov state models (MSMs), and nonequilibrium statistical mechanical theory to probe dissipation in two key classes of signaling proteins: kinases and G-protein–coupled receptors (GPCRs). Applying machinery from large deviation theory, we use MSMs constructed from protein simulations to generate dynamics conforming to positive levels of entropy production. We note the emergence of an array of peaks in the dynamical response (transient analogs of phase transitions) that draw the proteins between distinct levels of dissipation, and we see that the binding of ATP and agonist molecules modifies the observed dissipative landscapes. Overall, we find that dissipation is tightly coupled to activation in these signaling systems: dominant entropy-producing trajectories become localized near important barriers along known biological activation pathways. We go on to classify an array of equilibrium and nonequilibrium molecular switches that harmonize to promote functional dynamics. PMID:26240354

  15. Receptor activity-modifying proteins; multifunctional G protein-coupled receptor accessory proteins.

    PubMed

    Hay, Debbie L; Walker, Christopher S; Gingell, Joseph J; Ladds, Graham; Reynolds, Christopher A; Poyner, David R

    2016-04-15

    Receptor activity-modifying proteins (RAMPs) are single pass membrane proteins initially identified by their ability to determine the pharmacology of the calcitonin receptor-like receptor (CLR), a family B G protein-coupled receptor (GPCR). It is now known that RAMPs can interact with a much wider range of GPCRs. This review considers recent developments on the structure of the complexes formed between the extracellular domains (ECDs) of CLR and RAMP1 or RAMP2 as these provide insights as to how the RAMPs direct ligand binding. The range of RAMP interactions is also considered; RAMPs can interact with numerous family B GPCRs as well as examples of family A and family C GPCRs. They influence receptor expression at the cell surface, trafficking, ligand binding and G protein coupling. The GPCR-RAMP interface offers opportunities for drug targeting, illustrated by examples of drugs developed for migraine. PMID:27068971

  16. Protein-protein interactions in plant mitogen-activated protein kinase cascades.

    PubMed

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2016-02-01

    Mitogen-activated protein kinases (MAPKs) form tightly controlled signaling cascades that play essential roles in plant growth, development, and defense. However, the molecular mechanisms underlying MAPK cascades are still elusive, due largely to our poor understanding of how they relay the signals. Extensive effort has been devoted to characterization of MAPK-substrate interactions to illustrate phosphorylation-based signaling. The diverse MAPK substrates identified also shed light on how spatiotemporal-specific protein-protein interactions function in distinct MAPK cascade-mediated biological processes. This review surveys various technologies used for characterizing MAPK-substrate interactions and presents case studies of MPK4 and MPK6, highlighting the multiple functions of MAPKs. Mass spectrometry-based approaches in identifying MAPK-interacting proteins are emphasized due to their increasing utility and effectiveness. The potential for using MAPKs and their substrates in enhancing plant stress tolerance is also discussed. PMID:26646897

  17. Juvenile GM2 gangliosidosis (AMB variant): inability to activate hexosaminidase A by activator protein.

    PubMed Central

    Inui, K; Grebner, E E; Jackson, L G; Wenger, D A

    1983-01-01

    Two sibling from a consanguineous Puerto Rican marriage were found to have a juvenile-onset type of lipidosis first noted at age 2 1/2 by expressing difficulties with motor function and developmental delay. They continued to deteriorate, showing muscle atrophy, spasticity, and loss of speech, and death occurred at ages 7 and 8. Examination of the brains from these patients revealed that the concentration of GM2 ganglioside was about 56% of the total gangliosides. Hexosaminidase and percent hexosaminidase A (HEX A) and other lysosomal enzymes were normal in cultured skin fibroblasts, liver, and brain. The concentration of the activator protein required for the enzymatic hydrolysis of GM2 ganglioside was in high normal levels in the brain of the patient available. However, the HEX A from the patient's brain and liver as well as from skin fibroblast lysates could not be activated to hydrolyze GM2 ganglioside by the activator protein from a control or himself. The HEX A from a control could be activated by the activator protein from controls or this patient. These patients appear to have a defect in HEX A, which does not affect it heat stability, electrophoretic migration, and activity toward fluorogenic substrates, but may affect the binding of the activator protein required for GM2 ganglioside hydrolysis. We propose to call these patients the AMB variant of GM2 gangliosidosis to denote the mutation in HEX A but with normal levels of HEX A and B with synthetic substrates. This is to distinguish these patients from those missing the activator protein and normal HEX A and B levels. Images Fig. 1 Fig. 2 PMID:6224417

  18. Characterization of phytoconstituents and evaluation of total phenolic content, anthelmintic, and antimicrobial activities of Solanum violaceum Ortega

    PubMed Central

    Raju, Golam Sarwar; Moghal, Mizanur Rahman; Dewan, Syed Masudur Rahman; Amin, Mohammad Nurul; Billah, Mustahsan

    2013-01-01

    Objective: The present study was conducted to detect possible chemicals (phytoconstituents), prove ethno-medicinal value of the plant, and investigate antimicrobial, anthelmintic, and total phenolic content of crude methanolic extract of the Solanum violaceum plant. Materials and Methods: Phytochemical screening was carried out using different chemical group test methods. In anthelmintic activity test (using Pheretima posthuma model), five concentrations (10, 20, 30, 40, 50, and 80 mg/ml in distilled water) of extracts and albendazole as standard were used which involved the vermifuge and vermicidal activity on the worms. For the evaluation of in vitro antimicrobial activity, disc diffusion method, and to determine the total phenolic content, Folin-Ceocalteu method (gallic acid as standard) were used. Results: The phytoconstituent analysis revealed presence of alkaloids, carbohydrate, glycoside, flavonoid, saponin, gum, diterpenes, phenol, protein, and tannin. The crude extract exhibited significant anthelmintic property comparing with the standard. The methanolic extract revealed broad-spectrum antimicrobial activity at the concentration of 400 µg/disc. The results were compared with that of the standard ciprofloxacin. The extract exhibited moderate amount of total phenolic compound (54.67±1.18 mg/gm of gallic acid equivalent). Conclusion: Since S. violaceum have shown antimicrobial, antioxidant, and anthelmintic activities, more studies such as anti-inflammatory, analgesic, antipyretic, and other pharmacological activities should be carried out to justify its traditional use, as the plant is available and used broadly in the rural areas for folkloric remedies. PMID:25050288

  19. Can Specific Protein-Lipid Interactions Stabilize an Active State of the Beta 2 Adrenergic Receptor?

    PubMed

    Neale, Chris; Herce, Henry D; Pomès, Régis; García, Angel E

    2015-10-20

    G-protein-coupled receptors are eukaryotic membrane proteins with broad biological and pharmacological relevance. Like all membrane-embedded proteins, their location and orientation are influenced by lipids, which can also impact protein function via specific interactions. Extensive simulations totaling 0.25 ms reveal a process in which phospholipids from the membrane's cytosolic leaflet enter the empty G-protein binding site of an activated β2 adrenergic receptor and form salt-bridge interactions that inhibit ionic lock formation and prolong active-state residency. Simulations of the receptor embedded in an anionic membrane show increased lipid binding, providing a molecular mechanism for the experimental observation that anionic lipids can enhance receptor activity. Conservation of the arginine component of the ionic lock among Rhodopsin-like G-protein-coupled receptors suggests that intracellular lipid ingression between receptor helices H6 and H7 may be a general mechanism for active-state stabilization. PMID:26488656

  20. 78 FR 14083 - Agency Information Collection Activities: Direct Loan, FFEL, Perkins and TEACH Grant Total and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-04

    ... Agency Information Collection Activities: Direct Loan, FFEL, Perkins and TEACH Grant Total and Permanent... TEACH Grant Total and Permanent Disability Discharge Forms. OMB Control Number: 1845-0065. Type of... Loan, FFEL, or Perkins loan program loans, or TEACH Grant service obligation. The form collects...

  1. Interference of Cerebrospinal Fluid Total Protein Measurement by Povidone-Iodine Contamination

    PubMed Central

    Gounden, Verena; Sacks, David B; Zhao, Zhen

    2014-01-01

    Background A falsely high cerebrospinal fluid (CSF) total protein (TP) result measured by pyrogallol red (PGR) method was suspected to be caused by preparation of the collection site with povidone-iodine (PVP-iodine) solution. Methods CSF TP was evaluated for interference in samples with different final concentrations of PVP-iodine (up to 0.25% PVP and 0.025% iodine) or iodine alone (up to 0.025% iodine) using three methods: PGR, modified biuret and benzethonium chloride (BZTC). Interference exceeding ±20% of the baseline value is considered clinically significant according the criterion defined by College of American Pathologists. Results There was a positive interference with the PGR method and a negative inference for the BZTC method in CSF samples spiked with PVP-iodine. The PVP-iodine (up to 0.25% PVP and 0.025% iodine) did not cause a clinically significant interference with the modified biuret method. PVP alone without iodine caused a positive interference with the PGR method but did not interfere with the modified biuret or the BZTC method. When the samples were spiked with iodine alone, none of the three methods was affected (change < 20%) by iodine concentration up to 0.025%. Conclusions Contamination of CSF specimens with PVP-iodine can lead to interference with CSF TP measurements using PGR or BZTC methods. PMID:25446880

  2. Changes in IgG and total plasma protein glycomes in acute systemic inflammation

    PubMed Central

    Novokmet, Mislav; Lukić, Edita; Vučković, Frano; –Durić, Željko; Keser, Toma; Rajšl, Katarina; Remondini, Daniel; Castellani, Gastone; Gašparović, Hrvoje; Gornik, Olga; Lauc, Gordan

    2014-01-01

    Recovery after cardiac surgery is a complex process that has to compensate for both individual variability and extensive tissue damage in the context of systemic inflammation. Protein glycosylation is essential in many steps of the inflammatory cascade, but due to technological limitations the role of individual variation in glycosylation in systemic inflammation has not been addressed until now. We analysed composition of the total plasma and IgG N-glycomes in 107 patients undergoing cardiac surgery. In nearly all individuals plasma N-glycome underwent the same pattern of changes in the first 72 h, revealing a general mechanism of glycosylation changes. To the contrary, changes in the IgG glycome were very individualized. Bi-clustering analysis revealed the existence of four distinct patterns of changes. One of them, characterized by a rapid increase in galactosylated glycoforms, was associated with nearly double mortality risk measured by EuroSCORE II. Our results indicate that individual variation in IgG glycosylation changes during acute systemic inflammation associates with increased mortality risk and indicates new avenues for the development of personalized diagnostic and therapeutic approach. PMID:24614541

  3. The Effect of Structured Exercise Intervention on Intensity and Volume of Total Physical Activity

    PubMed Central

    Wasenius, Niko; Venojärvi, Mika; Manderoos, Sirpa; Surakka, Jukka; Lindholm, Harri; Heinonen, Olli J.; Aunola, Sirkka; Eriksson, Johan G.; Mälkiä, Esko

    2014-01-01

    This study aimed to investigate the effects of a 12-week structured exercise intervention on total physical activity and its subcategories. Twenty-three overweight or obese middle aged men with impaired glucose regulation were randomized into a 12-week Nordic walking group, a power-type resistance training group, and a non-exercise control group. Physical activity was measured with questionnaires before the intervention (1–4 weeks) and during the intervention (1–12 weeks) and was expressed in metabolic equivalents of task. No significant change in the volume of total physical activity between or within the groups was observed (p > 0.050). The volume of total leisure-time physical activity (structured exercises + non-structured leisure-time physical activity) increased significantly in the Nordic walking group (p < 0.050) but not in the resistance training group (p > 0.050) compared to the control group. In both exercise groups increase in the weekly volume of total leisure-time physical activity was inversely associated with the volume of non-leisure-time physical activities. In conclusion, structured exercise intervention did not increase the volume of total physical activity. Albeit, endurance training can increase the volume of high intensity physical activities, however it is associated with compensatory decrease in lower intensity physical activities. To achieve effective personalized exercise program, individuality in compensatory behavior should be recognised. Key Points Structured NW or RT training does not increase the volume of total physical activity. NW intervention can increase the volume of higher intensity activities. The increased in volume of LTPA induced by the structured NW and RT interventions was associated with the decreased volume of NLTPA. PMID:25435776

  4. Effect of different cooking methods on total phenolic contents and antioxidant activities of four Boletus mushrooms.

    PubMed

    Sun, Liping; Bai, Xue; Zhuang, Yongliang

    2014-11-01

    The influences of cooking methods (steaming, pressure-cooking, microwaving, frying and boiling) on total phenolic contents and antioxidant activities of fruit body of Boletus mushrooms (B. aereus, B. badius, B. pinophilus and B. edulis) have been evaluated. The results showed that microwaving was better in retention of total phenolics than other cooking methods, while boiling significantly decreased the contents of total phenolics in samples under study. Effects of different cooking methods on phenolic acids profiles of Boletus mushrooms showed varieties with both the species of mushroom and the cooking method. Effects of cooking treatments on antioxidant activities of Boletus mushrooms were evaluated by in vitro assays of hydroxyl radical (OH·) -scavenging activity, reducing power and 1, 1-diphenyl-2-picrylhydrazyl radicals (DPPH·) -scavenging activity. Results indicated the changes of antioxidant activities of four Boletus mushrooms were different in five cooking methods. This study could provide some information to encourage food industry to recommend particular cooking methods. PMID:26396332

  5. Estimation of total phenolic content, in-vitro antioxidant and anti-inflammatory activity of flowers of Moringa oleifera

    PubMed Central

    Alhakmani, Fatma; Kumar, Sokindra; Khan, Shah Alam

    2013-01-01

    Objective To evaluate and compare the antioxidant potential and anti-inflammatory activity of ethanolic extract of flowers of Moringa oleifera (M. oleifera) grown in Oman. Methods Flowers of M. oleifera were collected in the month of December 2012 and identified by a botanist. Alcoholic extract of the dry pulverized flowers of M. oleifera were obtained by cold maceration method. The ethanolic flower extract was subjected to preliminary phytochemical screening as the reported methods. Folin-Ciocalteu reagent was used to estimate total phenolic content. DPPH was used to determine in-vitro antioxidant activity and anti-inflammatory activity of flowers was investigated by protein denaturation method. Results Phytochemical analysis of extract showed presence of major classes of phytochemicals such as tannins, alkaloids, flavonoids, cardiac glycosides etc. M. oleifera flowers were found to contain 19.31 mg/g of gallic acid equivalent of total phenolics in dry extract but exhibited moderate antioxidant activity. The anti-inflammatory activity of plant extract was significant and comparable with the standard drug diclofenac sodium. Conclusions The results of our study suggest that flowers of M. oleifera possess potent anti-inflammatory activity and are also a good source of natural antioxidants. Further study is needed to identify the chemical compounds responsible for their anti-inflammatory activity. PMID:23905019

  6. Nucleolar protein B23 has molecular chaperone activities.

    PubMed Central

    Szebeni, A.; Olson, M. O.

    1999-01-01

    Protein B23 is an abundant, multifunctional nucleolar phosphoprotein whose activities are proposed to play a role in ribosome assembly. Szebeni et al. (1997) showed stimulation of nuclear import in vitro by protein B23 and suggested that this effect was due to a molecular chaperone-like activity. Protein B23 was tested for chaperone activities using several protein substrates. The temperature-dependent and -independent aggregation of the HIV-1 Rev protein was measured using a zero angle light scattering (turbidity) assay. Protein B23 inhibited the aggregation of the Rev protein, with the amount of inhibition proportional to the concentration of B23 added. This activity was saturable with nearly complete inhibition when the molar ratio of B23:Rev was slightly above one. Protein B23 also protected liver alcohol dehydrogenase (LADH), carboxypeptidase A, citrate synthase, and rhodanese from aggregation during thermal denaturation and preserved the enzyme activity of LADH under these conditions. In addition, protein B23 was able to promote the restoration of activity of LADH previously denatured with guanidine-HCl. Protein B23 preferentially bound denatured substrates and exposed hydrophobic regions when complexed with denatured proteins. Thus, by several criteria, protein B23 behaves like a molecular chaperone; these activities may be related to its role in ribosome biogenesis. PMID:10211837

  7. Physical Activity in a Total Population of Children and Adolescents with Cerebral Palsy

    ERIC Educational Resources Information Center

    Lauruschkus, Katarina; Westbom, Lena; Hallstrom, Inger; Wagner, Philippe; Nordmark, Eva

    2013-01-01

    The aims of this study were to describe the participation in physical activity of children with cerebral palsy (CP) at school and during leisure time and to identify characteristics associated with physical activity. The frequency of receiving physiotherapeutic interventions were described as a variable of interest. A total population of 364…

  8. Reconstitution of Membrane Proteins into Model Membranes: Seeking Better Ways to Retain Protein Activities

    PubMed Central

    Shen, Hsin-Hui; Lithgow, Trevor; Martin, Lisandra L.

    2013-01-01

    The function of any given biological membrane is determined largely by the specific set of integral membrane proteins embedded in it, and the peripheral membrane proteins attached to the membrane surface. The activity of these proteins, in turn, can be modulated by the phospholipid composition of the membrane. The reconstitution of membrane proteins into a model membrane allows investigation of individual features and activities of a given cell membrane component. However, the activity of membrane proteins is often difficult to sustain following reconstitution, since the composition of the model phospholipid bilayer differs from that of the native cell membrane. This review will discuss the reconstitution of membrane protein activities in four different types of model membrane—monolayers, supported lipid bilayers, liposomes and nanodiscs, comparing their advantages in membrane protein reconstitution. Variation in the surrounding model environments for these four different types of membrane layer can affect the three-dimensional structure of reconstituted proteins and may possibly lead to loss of the proteins activity. We also discuss examples where the same membrane proteins have been successfully reconstituted into two or more model membrane systems with comparison of the observed activity in each system. Understanding of the behavioral changes for proteins in model membrane systems after membrane reconstitution is often a prerequisite to protein research. It is essential to find better solutions for retaining membrane protein activities for measurement and characterization in vitro. PMID:23344058

  9. Fast and selective determination of total protein in milk powder via titration of moving reaction boundary electrophoresis.

    PubMed

    Guo, Cheng-ye; Wang, Hou-yu; Liu, Xiao-ping; Fan, Liu-yin; Zhang, Lei; Cao, Cheng-xi

    2013-05-01

    In this paper, moving reaction boundary titration (MRBT) was developed for rapid and accurate quantification of total protein in infant milk powder, from the concept of moving reaction boundary (MRB) electrophoresis. In the method, the MRB was formed by the hydroxide ions and the acidic residues of milk proteins immobilized via cross-linked polyacrylamide gel (PAG), an acid-base indicator was used to denote the boundary motion. As a proof of concept, we chose five brands of infant milk powders to study the feasibility of MRBT method. The calibration curve of MRB velocity versus logarithmic total protein content of infant milk powder sample was established based on the visual signal of MRB motion as a function of logarithmic milk protein content. Weak influence of nonprotein nitrogen (NPN) reagents (e.g., melamine and urea) on MRBT method was observed, due to the fact that MRB was formed with hydroxide ions and the acidic residues of captured milk proteins, rather than the alkaline residues or the NPN reagents added. The total protein contents in infant milk powder samples detected via the MRBT method were in good agreement with those achieved by the classic Kjeldahl method. In addition, the developed method had much faster measuring speed compared with the Kjeldahl method. PMID:23483553

  10. Designing Mimics of Membrane Active Proteins

    PubMed Central

    Sgolastra, Federica; deRonde, Brittany M.; Sarapas, Joel M.; Som, Abhigyan; Tew, Gregory N.

    2014-01-01

    CONSPECTUS As a semi-permeable barrier that controls the flux of biomolecules in and out the cell, the plasma membrane is critical in cell function and survival. Many proteins interact with the plasma membrane and modulate its physiology. Within this large landscape of membrane-active molecules, researchers have focused significant attention on two specific classes of peptides, antimicrobial peptides (AMPs) and cell penetrating peptides (CPPs) because of their unique properties. In this account, we describe our efforts over the last decade to build and understand synthetic mimics of antimicrobial peptides (SMAMPs). These endeavors represent one specific example of a much larger effort to understand how synthetic molecules interact with and manipulate the plasma membrane. Using both defined molecular weight oligomers and easier to produce, but heterogeneous, polymers, it has been possible to generate scaffolds with biological potency superior to the natural analogs. In one case, a compound has progressed through a phase II clinical trial for pan)staph infections. Modern biophysical assays highlighted the interplay between the synthetic scaffold and lipid composition leading to negative Gaussian curvature, a requirement for both pore formation and endosomal escape. The complexity of this interplay between lipids, bilayer components, and the scaffolds remains to be better resolved, but significant new insight has been provided. It is worthwhile to consider the various aspects of permeation and how these are related to ‘pore formation.’ More recently, our efforts have expanded toward protein transduction domains, or cell penetrating peptide, mimics. The combination of unique molecular scaffolds and guanidinium) rich side chains has produced an array of polymers with robust transduction (and delivery) activity. Being a new area, the fundamental interactions between these new scaffolds and the plasma membrane are just beginning to be understood. Negative Gaussian

  11. Activating AMP-activated protein kinase (AMPK) slows renal cystogenesis.

    PubMed

    Takiar, Vinita; Nishio, Saori; Seo-Mayer, Patricia; King, J Darwin; Li, Hui; Zhang, Li; Karihaloo, Anil; Hallows, Kenneth R; Somlo, Stefan; Caplan, Michael J

    2011-02-01

    Renal cyst development and expansion in autosomal dominant polycystic kidney disease (ADPKD) involves both fluid secretion and abnormal proliferation of cyst-lining epithelial cells. The chloride channel of the cystic fibrosis transmembrane conductance regulator (CFTR) participates in secretion of cyst fluid, and the mammalian target of rapamycin (mTOR) pathway may drive proliferation of cyst epithelial cells. CFTR and mTOR are both negatively regulated by AMP-activated protein kinase (AMPK). Metformin, a drug in wide clinical use, is a pharmacological activator of AMPK. We find that metformin stimulates AMPK, resulting in inhibition of both CFTR and the mTOR pathways. Metformin induces significant arrest of cystic growth in both in vitro and ex vivo models of renal cystogenesis. In addition, metformin administration produces a significant decrease in the cystic index in two mouse models of ADPKD. Our results suggest a possible role for AMPK activation in slowing renal cystogenesis as well as the potential for therapeutic application of metformin in the context of ADPKD. PMID:21262823

  12. Characterization of active and total fungal communities in the atmosphere over the Amazon rainforest

    NASA Astrophysics Data System (ADS)

    Womack, A. M.; Artaxo, P. E.; Ishida, F. Y.; Mueller, R. C.; Saleska, S. R.; Wiedemann, K. T.; Bohannan, B. J. M.; Green, J. L.

    2015-11-01

    Fungi are ubiquitous in the atmosphere and may play an important role in atmospheric processes. We investigated the composition and diversity of fungal communities over the Amazon rainforest canopy and compared these communities to fungal communities found in terrestrial environments. We characterized the total fungal community and the metabolically active portion of the community using high-throughput DNA and RNA sequencing and compared these data to predictions generated by a mass-balance model. We found that the total community was primarily comprised of fungi from the phylum Basidiomycota. In contrast, the active community was primarily composed of members of the phylum Ascomycota and included a high relative abundance of lichen fungi, which were not detected in the total community. The relative abundance of Basidiomycota and Ascomycota in the total and active communities was consistent with our model predictions, suggesting that this result was driven by the relative size and number of spores produced by these groups. When compared to other environments, fungal communities in the atmosphere were most similar to communities found in tropical soils and leaf surfaces. Our results demonstrate that there are significant differences in the composition of the total and active fungal communities in the atmosphere, and that lichen fungi, which have been shown to be efficient ice nucleators, may be abundant members of active atmospheric fungal communities over the forest canopy.

  13. Endoplasmic reticulum stress activates transglutaminase 2 leading to protein aggregation

    PubMed Central

    LEE, JIN-HAENG; JEONG, JAEHO; JEONG, EUI MAN; CHO, SUNG-YUP; KANG, JEONG WOOK; LIM, JISUN; HEO, JINBEOM; KANG, HYUNSOOK; KIM, IN-GYU; SHIN, DONG-MYUNG

    2014-01-01

    Aberrant activation of transglutaminase 2 (TGase2) contributes to a variety of protein conformational disorders such as neurodegenerative diseases and age-related cataracts. The accumulation of improperly folded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), which promotes either repair or degradation of the damaged proteins. Inadequate UPR results in protein aggregation that may contribute to the development of age-related degenerative diseases. TGase2 is a calcium-dependent enzyme that irreversibly modifies proteins by forming cross-linked protein aggregates. Intracellular TGase2 is activated by oxidative stress which generates large quantities of unfolded proteins. However, the relationship between TGase2 activity and UPR has not yet been established. In the present study, we demonstrated that ER stress activated TGase2 in various cell types. TGase2 activation was dependent on the ER stress-induced increase in the intracellular calcium ion concentration but not on the TGase2 protein expression level. Enzyme substrate analysis revealed that TGase2-mediated protein modification promoted protein aggregation concurrently with decreasing water solubility. Moreover, treatment with KCC009, a TGase2 inhibitor, abrogated ER stress-induced TGase2 activation and subsequent protein aggregation. However, TGase2 activation had no effect on ER stress-induced cell death. These results demonstrate that the accumulation of misfolded proteins activates TGase2, which further accelerates the formation of protein aggregates. Therefore, we suggest that inhibition of TGase2 may be a novel strategy by which to prevent the protein aggregation in age-related degenerative diseases. PMID:24481335

  14. Total hip arthroplasty in patients 50 years or less: do we improve activity profiles?

    PubMed

    Kuhn, Margaret; Harris-Hayes, Marcie; Steger-May, Karen; Pashos, Gail; Clohisy, John C

    2013-05-01

    The primary objective of this study was to use step activity monitoring to quantify activity changes after total hip arthroplasty in patients 50 years or less. Secondly, we investigated whether step activity measurements correlated with the Harris hip and UCLA scores. We prospectively analyzed 37 patients (age ≤ 50) treated with primary THA. Patient activity was recorded with a step activity monitor. Harris hip and UCLA scores were analyzed. Total daily stride counts increased by an average of 30.0%. Increases were noted in the percent of daily time spent at high, moderate and low activity. Increases in daily time spent at high activity moderately correlated with the UCLA activity score but did not correlate with the HHS. Both the UCLA score and the HHS did not correlate with mean daily strides. Following THA, patients ≤ 50 years of age increase their activity by taking more daily strides and improve their activity profile by spending more time at higher activity. Improvements in step activity moderately correlate with improvements in UCLA scores. PMID:23499406

  15. Antioxidant Activities and Total Phenolic Content of Aqueous Extract of Pleurotus ostreatus (Cultivated Oyster Mushroom).

    PubMed

    Yim, H S; Chye, F Y; Tan, C T; Ng, Y C; Ho, C W

    2010-08-01

    Pleurotus ostreatus better known as oyster mushroom is widely cultivated and consumed as food in Malaysia. The present study aims to assess the antioxidative potential and total phenolic content of P. ostreatus aqueous extract. The antioxidant activities were evaluated against DPPH and ABTS radical-scavenging activity, ferric-reducing antioxidant power (FRAP) and β-carotene-linoleate bleaching assay, and the Folin-Ciocalteu method for total phenolic content (TPC). The DPPH and ABTS radical-scavenging activity was found to be 63.20% and 87.29% respectively; antioxidant activity using FRAP at 1.45 mM FE/100g and β-carotenelinoleate bleaching assay was 83.51%, while the TPC was found to be 798.55 mg GAE/100g. These antioxidant activities were compared to synthetic antioxidant, BHA and ascorbic acid. Ascorbic acid showed highest scavenging effects on DPPH and ABTS radical, followed by P. ostreatus and BHA (at maximum safety limit). The ferric reducing power of P. ostreatus was significantly higher than BHA and ascorbic acid. The antioxidant activity as assessed in β-carotene-linoleate bleaching assay was found to be higher in BHA compared to P. ostreatus. The aqueous extract of P. ostreatus was found to respond differently in antioxidant assays. The antioxidative activity of the aqueous extract of P. ostreatus correlated with its total phenolic content. Generally, the antioxidant activities of P. ostreatus' aqueous extract are comparable to that of BHA and ascorbic acid to a certain extent. PMID:22691933

  16. Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

    PubMed Central

    Velapatiño, Billie; Zlosnik, James E. A.; Hird, Trevor J.; Speert, David P.

    2013-01-01

    The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining. PMID:24192802

  17. Blood zinc protoporphyrin, serum total protein, and total cholesterol levels in automobile workshop workers in relation to lead toxicity: Our experience.

    PubMed

    Pachathundikandi, Suneesh Kumar; Varghese, Earaly Thomas

    2006-09-01

    Blood zinc protoporphyrin (ZPP), serum total protein (TP), and total cholesterol (TC) levels in automobile workshop workers in relation to lead toxicity were analysed. In the present study, automobile workshop workers (healthy male workers at an age between 28 and 35 from four major automobile workshops in Kottayam, Kerala State, India) and the control (male healthy adults at an age between 28 and 35 residing at Aymanam, a distant village at Kottayam District, Kerala having reduced or no chance of lead exposure) displayed significant difference in blood lead (BPb) and blood ZZP (BZPP) level. The mean value of BPb in automobile workshop workers was 15.76±0.33 μg/dl, while in the control it was 8.20±0.15 μg/dl. In automobile workshop workers, the mean value of BZPP was 34.2±0.62 μg/dl. The control group exhibited a mean of 11.5±0.22 μg/dl. Automobile workshop workers exhibited significant increase in BZPP was corresponding to the increase in BPb level. The total protein levels estimated in automobile workshop workers showed significant decrease compared to control individuals, but was within the reference range of healthy individuals. The mean value of TP level in automobile workshop workers and control was 6.9±0.13 g/dl and 7.71±0.18 g/dl, respectively. There was no significant difference in blood haemoglobin (BHb) level among the automobile workshop workers and control. The serum TC level in automobile workshop workers showed significant decrease compared to the control individuals, but was with in the reference range of healthy individuals. The mean level of serum TC in automobile workshop workers was 162.00±3.44 mg/dl and the same in control was 172.86±4.32 mg/dl. The present study affirms occupational lead toxicity in automobile workshop workers and its effect on serum protein and cholesterol levels. PMID:23105626

  18. Effects of gamma irradiation on total polyphenols, radical scavenging activities and decolourization of Nelumbo nucifera extracts

    NASA Astrophysics Data System (ADS)

    Jeong, Il Yun; Lee, Hyo Jung; Park, Yong Dae; Jin, Chang Hyun; Choi, Dae Seong; Byun, Myung Woo

    2009-07-01

    The ethanolic leaf extract of Nelumbo nucifera (NC) was exposed to γ-irradiation, and its antioxidant activities, total polyphenols and colour characteristics were studied to discern its potential ability as a food or cosmetic materials. The results demonstrated that the radical scavenging activities and total polyphenols of the γ-irradiated leaf extract of NC were not observed to be significantly different. However, γ-irradiation significantly increased the Hunter colour L*-value at doses of 20 and 50 kGy, while the Hunter colour b*-values were decreased under the same conditions.

  19. Protein glycation inhibitory activity and antioxidant capacity of clove extract.

    PubMed

    Suantawee, Tanyawan; Wesarachanon, Krittaporn; Anantsuphasak, Kanokphat; Daenphetploy, Tanuch; Thien-Ngern, Sroshin; Thilavech, Thavaree; Pasukamonset, Porntip; Ngamukote, Sathaporn; Adisakwattana, Sirichai

    2015-06-01

    Syzygium aromaticum (L.) (clove) is one of the most widely cultivated spices in many tropical countries. The aim of this study was to determine the phytochemical content, the antioxidant properties and the antiglycation properties of aqueous extract of clove against fructose-mediated protein glycation and oxidation. The result showed that the content of total phenolics and flavonoids in clove extract was 239.58 ± 0.70 mg gallic acid equivalents/g dried extract and 65.67 ± 0.01 mg catechin equivalents/g dried extract, respectively. In addition, clove exhibited antioxidant properties including DPPH radical scavenging activity (IC50 = 0.29 ± 0.01 mg/ml), Trolox equivalent antioxidant capacity (4.69 ± 0.03 μmol Trolox equivalents/mg dried extract), ferric reducing antioxidant power (20.55 ± 0.11 μmol ascorbic acid equivalents/mg dried extract), Oxygen radical absorbance capacity (31.12 ± 0.21 μmol Trolox equivalents/mg dried extract), hydroxyl radical scavenging activity (0.15 ± 0.04 mg Trolox equivalents/mg dried extract), and superoxide radical scavenging activity (18.82 ± 0.50 mg Trolox equivalents/mg dried extract). The aqueous extract of clove (0.25-1.00 mg/ml) significantly inhibited the formation of fluorescent advanced glycation end products (AGEs) and non-fluorescent AGEs (N(ɛ)-(carboxymethyl) lysine (CML)) in glycated BSA during 4 weeks of incubation. The extract also markedly prevented oxidation-induced protein damage by decreasing protein carbonyl formation and protecting against the loss of protein thiol group. These results clearly demonstrated that a polyphenol enriched clove extract, owing to its antioxidant, was capable to inhibit the formation of AGEs and protein glycation. The findings might lead to the possibility of using the clove extract for targeting diabetic complications. PMID:26028769

  20. [Effects of colchicine on the content of ribonucleic acids and total proteins of cultured nerve cells. Preliminary study].

    PubMed

    Noel-Courtey, B

    1975-03-01

    Nervous cells from chick embryo lumbo-sacral spinal cord have been cultivated in vitro and treated with colchicine. The effects of this alkaloid on the RNA and the total protein contents of nervous cells have been studied by quantitative cytochemical methods. The RNA content has been measured by cytophotometry after DNase digestion and gallocyanine-chromalun staining; the cellular protein content has been measured after Naphtol Yellow S staining. In colchicine treated cultures, the main part of the nervous cell population consists of spheroidal and piriform neuroblasts; some cells are neurocytes with short processes. During the 48 first hours in vitro, the RNA content is approximatively identical in treated cells as in controls. The cellular total protein content is much lower in the treated cells than in the controls. PMID:1222295

  1. Diet-Induced Alterations in Total and Metabolically Active Microbes within the Rumen of Dairy Cows

    PubMed Central

    Lettat, Abderzak; Benchaar, Chaouki

    2013-01-01

    DNA-based techniques are widely used to study microbial populations; however, this approach is not specific to active microbes, because DNA may originate from inactive and/or dead cells. Using cDNA and DNA, respectively, we aimed to discriminate the active microbes from the total microbial community within the rumen of dairy cows fed diets with increasing proportions of corn silage (CS). Nine multiparous lactating Holstein cows fitted with ruminal cannulas were used in a replicated 3×3 Latin square (32-d period; 21-d adaptation) design to investigate diet-induced shifts in microbial populations by targeting the rDNA gene. Cows were fed a total mixed ration with the forage portion being either barley silage (0% CS), a 50∶50 mixture of barley silage and corn silage (50% CS), or corn silage (100% CS). No differences were found for total microbes analyzed by quantitative PCR, but changes were observed within the active ones. Feeding more CS to dairy cows was accompanied by an increase in Prevotella rRNA transcripts (P = 0.10) and a decrease in the protozoal rRNA transcripts (P<0.05). Although they were distributed differently among diets, 78% of the amplicons detected in DNA- and cDNA-based fingerprints were common to total and active bacterial communities. These may represent a bacterial core of abundant and active cells that drive the fermentation processes. In contrast, 10% of amplicons were specific to total bacteria and may represent inactive or dead cells, whereas 12% were only found within the active bacterial community and may constitute slow-growing bacteria with high metabolic activity. It appears that cDNA-based analysis is more discriminative to identify diet-induced shifts within the microbial community. This approach allows the detection of diet-induced changes in the microbial populations as well as particular bacterial amplicons that remained undetected using DNA-based methods. PMID:23593365

  2. Modeling Protein Folding and Applying It to a Relevant Activity

    ERIC Educational Resources Information Center

    Nelson, Allan; Goetze, Jim

    2004-01-01

    The different levels of protein structure that can be easily understood by creating a model that simulates protein folding, which can then be evaluated by applying it to a relevant activity, is presented. The materials required and the procedure for constructing a protein folding model are mentioned.

  3. Equatorial Total Electron Content (TEC) at Low and High Solar Activity

    NASA Astrophysics Data System (ADS)

    Mene, M. N.; Obrou, O. K.; Kobea, A. T.; Zaka, K. Z.

    2007-05-01

    Total Electron Content derived from ionosonde data recorded at Korhogo (Lat=9.33 N, Long =5.43 W, Dip = 0.67 S) are compared to the Internatial Reference Ionosphere (IRI) model predicted TEC for high (1999) and low (1994) solar activity conditions. The result shows that the TEC has a solar activity and seasonal dependence. The IRI predicted values are closer to the observed TEC at high solar activity. However, at low solar activity the IRI overestimates the observed TEC. The deviation is more prominent in equinox during the time range 0900 to 2300 local time. The deviation is estimated to 60% of the observed TEC.

  4. Relation of plasma calcium to total protein and albumin in African grey (Psittacus erithacus) and Amazon (Amazona spp.) parrots.

    PubMed

    Lumeij, J T

    1990-10-01

    A significant correlation was found between total calcium and albumin concentration in the plasma of 70 African grey parrots (r=0.37; P<0.05). A correlation formula for plasma calcium concentration in the African grey parrot was derived on the basis of the concentration of albumin: Adjusted Ca (mmol/1) = Ca (mmol/1) - 0.015 Albumin (g/1) + 0.4. About 14% of the variability in calcium was attributable to the change in the concentration of plasma albumin concentration (R2=0.137). The correlation between calcium and total protein in African greys and between calcium and albumin and calcium and total protein in Amazons was not significant. PMID:18679980

  5. Normal protein content but abnormally inhibited enzyme activity in muscle carnitine palmitoyltransferase II deficiency.

    PubMed

    Lehmann, Diana; Zierz, Stephan

    2014-04-15

    The biochemical consequences of the disease causing mutations of muscle carnitine palmitoyltransferase II (CPT II) deficiency are still enigmatic. Therefore, CPT II was characterized in muscle biopsies of nine patients with genetically proven muscle CPT II deficiency. Total CPT activity (CPT I+CPT II) of patients was not significantly different from that of controls. Remaining activities upon inhibition by malonyl-CoA and Triton X-100 were significantly reduced in patients. Immunohistochemically CPT II protein was predominantly expressed in type-I-fibers with the same intensity in patients as in controls. Western blot showed the same CPT II staining intensity ratio in patients and controls. CPT I and CPT II protein concentrations estimated by ELISA were not significantly different in patients and in controls. Citrate synthase activity in patients was significantly increased. Total CPT activity significantly correlated with both CPT I and CPT II protein concentrations in patients and controls. This implies (i) that normal total CPT activity in patients with muscle CPT II deficiency is not due to compensatory increase of CPT I activity and that (ii) the mutant CPT II is enzymatically active. The data further support the notion that in muscle CPT II deficiency enzyme activity and protein content are not reduced, but rather abnormally inhibited when fatty acid metabolism is stressed. PMID:24602495

  6. Dissecting the active site of a photoreceptor protein

    NASA Astrophysics Data System (ADS)

    Hoff, Wouter; Hara, Miwa; Ren, Jie; Moghadam, Farzaneh; Xie, Aihua; Kumauchi, Masato

    While enzymes are quite large molecules, functionally important chemical events are often limited to a small region of the protein: the active site. The physical and chemical properties of residues at such active sites are often strongly altered compared to the same groups dissolved in water. Understanding such effects is important for unraveling the mechanisms underlying protein function and for protein engineering, but has proven challenging. Here we report on our ongoing efforts on using photoactive yellow protein (PYP), a bacterial photoreceptor, as a model system for such effects. We will report on the following questions: How many residues affect active site properties? Are these residues in direct physical contact with the active site? Can functionally important residues be recognized in the crystal structure of a protein? What structural resolution is needed to understand active sites? What spectroscopic techniques are most informative? Which weak interactions dominate active site properties?

  7. Global Analysis of Protein Activities Using Proteome Chips

    NASA Astrophysics Data System (ADS)

    Zhu, Heng; Bilgin, Metin; Bangham, Rhonda; Hall, David; Casamayor, Antonio; Bertone, Paul; Lan, Ning; Jansen, Ronald; Bidlingmaier, Scott; Houfek, Thomas; Mitchell, Tom; Miller, Perry; Dean, Ralph A.; Gerstein, Mark; Snyder, Michael

    2001-09-01

    To facilitate studies of the yeast proteome, we cloned 5800 open reading frames and overexpressed and purified their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with proteins and phospholipids. We identified many new calmodulin- and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. The microarrays can also be used to screen protein-drug interactions and to detect posttranslational modifications.

  8. Mass Spectrometry-based Proteomics: Qualitative Identification to Activity-based Protein Profiling

    PubMed Central

    Cardoza, Job D.; Parikh, Jignesh R.; Ficarro, Scott B.; Marto, Jarrod A.

    2011-01-01

    Mass spectrometry has become the method of choice for proteome characterization, including multi-component protein complexes (typically tens to hundreds of proteins) and total protein expression (up to tens of thousands of proteins), in biological samples. Qualitative sequence assignment based on MS/MS spectra is relatively well-defined, while statistical metrics for relative quantification have not completely stabilized. Nonetheless, proteomics studies have progressed to the point whereby various gene-, pathway-, or network-oriented computational frameworks may be used to place mass spectrometry data into biological context. Despite this progress, the dynamic range of protein expression remains a significant hurdle, and impedes comprehensive proteome analysis. Methods designed to enrich specific protein classes have emerged as an effective means to characterize enzymes or other catalytically active proteins that are otherwise difficult to detect in typical discovery mode proteomics experiments. Collectively, these approaches will facilitate identification of biomarkers and pathways relevant to diagnosis and treatment of human disease. PMID:22231900

  9. Total Phenolic Content and Antioxidant Activity of Some Malvaceae Family Species

    PubMed Central

    de Oliveira, Adriana Maria Fernandes; Pinheiro, Lilian Sousa; Pereira, Charlane Kelly Souto; Matias, Wemerson Neves; Gomes, Roosevelt Albuquerque; Chaves, Otemberg Souza; de Souza, Maria de Fátima Vanderlei; de Almeida, Reinaldo Nóbrega; de Assis, Temilce Simões

    2012-01-01

    The antioxidant activity of four species of the Malvaceae family (Sidastrum micranthum (A. St.-Hil.) Fryxell, Wissadula periplocifolia (L.) C. Presl, Sida rhombifolia (L.) E. H. L and Herissantia crispa L. (Brizicky)) were studied using the total phenolic content, DPPH radical scavenging activity and Trolox equivalent antioxidant capacity (TEAC) assays. The antioxidant activity of the crude extract, phases and two isolated flavonoids, kaempferol 3,7-di-O-α-L-rhamnopyranoside (lespedin) and kaempferol 3-O-β-D-(6''-E-p-coumaroil) glucopyranoside (tiliroside) was determined. The results showed that there is a strong correlation between total polyphenol contents and antioxidant activity of the crude extract of Sidastrum micranthum and Wissadula periplocifolia; however, this was not observed between Sida rhombifolia and Herissantia crispa. The ethyl acetate (EaF) phase showed the best antioxidant effect in the total phenolics, DPPH and TEAC assays, followed by the chloroform (CfF) phase, in most species tested. Lespedin, isolated from the EaF phase of W. periplocifolia and H. crispa may not be responsible for the antioxidant activity due to its low antioxidant activity (IC50: DPPH: 1,019.92 ± 68.99 mg/mL; TEAC: 52.70 ± 0.47 mg/mL); whereas tiliroside, isolated from W. periplocifolia, H. crispa and S. micrantum presented a low IC50 value (1.63 ± 0.86 mg/mL) compared to ascorbic acid in the TEAC assay. PMID:26787614

  10. TOTAL PHENOLIC CONTENT, ANTIOXIDANT AND ANTIBACTERIAL ACTIVITIES OF THE EXTRACT OF EPHEDRA PROCERA FISCH. ET MEY.

    PubMed

    Dehkordi, Naser Vahed; Kachouie, Mehrdad Ataie; Pirbalouti, Abdollah Ghasemi; Malekpoor, Fatemeh; Rabei, Mohammad

    2015-01-01

    Ephedra prcera belonging to the family Ephedraceae is a poison and medicinal plant. The main aim of present study was to determine total phenolic content and antioxidant and antibacterial activities of ethanolic extract from the aerial parts of E. procera collected from a natural habitat in Chaharmahal va Bakhtiari province, Southwestern Iran. The total phenolic content of the extract by Folin-Ciocalteu method and the antioxidant activity using DPPH assay were determined. The antibacterial activity, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of the extract were evaluated against five bacteria, including Proteus vulgaris, Pseudomonas aeruginosa, Enteobacter aeogenes, Bacillus ceirus and Staphylococcus aureus. Total phenolic content in the extract of E. procera was 0.718 mg tannic acid/g dry weight extract. The results indicated that the ethanolic extract of E. piocera exhibited radical scavenging activity. In addition, the results of this study confirmed that the ethanolic extract of E. procera exhibited antibacterial activity. In conclusion, the extract of E. piocera could be an important source of phenolic components with antioxidant capacity and antibacterial activity. PMID:26642685

  11. Antioxidant activity and total phenolics in selected cereal grains and their different morphological fractions.

    PubMed

    Zieliński, H; Kozłowska, H

    2000-06-01

    The purpose of this study was to examine the antioxidant properties of water and 80% methanolic extracts of cereal grains and their different morphological fractions. Wheat (Triticum aestivum L.) cv. Almari and cv. Henika, barley (Hordeum vulgare L.) cv. Gregor and cv. Mobek, rye (Secale cereale L.) cv. Dańkowskie Zlote, oat (Avena sativa L.) cv. Slawko and buckwheat (Fagopyrum esculentum Moench) cv. Kora were used. PC (L-alpha-phosphatidylcholine) liposome system and spectrophotometric assay of total antioxidant activity (TAA) were used to evaluate the antioxidative activity of extracts. Among the water extracts, only the one prepared from buckwheat exhibited antioxidant activity at the concentration analyzed. The following hierarchy of antioxidant activity was provided for 80% methanolic extracts originated from whole grain: buckwheat > barley > oat > wheat congruent with rye. The antioxidant activity was observed in extract prepared from separated parts of buckwheat and barley. In respect to hulls, the antioxidant hierarchy was as follows: buckwheat > oat > barley. The correlation coefficient between total phenolic compounds and total antioxidative activity of the extracts was -0.35 for water extracts and 0.96, 0.99, 0.80, and 0.99 for 80% methanolic extracts originated from whole grains, hulls, pericarb with testa fractions and endosperm with embryo fractions, respectively. PMID:10888490

  12. Increasing total and biologically active chromium in wheat grain and spinach by spraying with chromium salts

    SciTech Connect

    Vicini, F.A.; Ellis, B.G.

    1981-06-01

    Recently, chromium has been shown to be necessary for glucose metabolism in man. But most plant species greatly restrict the uptake of Cr. This study was conducted to determine if both total and biologically active Cr could be increased in wheat grain or spinach by spraying the plants with either Cr/sub 2/(SO/sub 4/)/sub 3/ or Cr-EDTA. Concentrations of Cr in wheat grain were about doubled in a greenhouse experiment by spraying with either Cr source. Biologically active Cr (estimated by extraction with ethanol or NH/sub 4/OH) was increased from about 40 to greater than 50% of total Cr when wheat was sprayed with Cr salts. Total Cr in spinach leaves was increased by as much as 10-fold by spraying, with the sulfate source being more effective than the EDTA.

  13. Total synthesis of the cyanobacterial metabolite nostodione A: discovery of its antiparasitic activity against Toxoplasma gondii.

    PubMed

    McNulty, J; Keskar, K; Bordón, C; Yolken, R; Jones-Brando, L

    2014-08-18

    A total synthesis of the cyanobacterial natural product nostodione A is reported involving a convergent, diversity-oriented route. A small assemblage of structural analogues were prepared and their cytotoxicity and anti-invasion activity against the protozoal parasite Toxoplasma gondii is reported for the first time. PMID:24970332

  14. DIETARY PROTEIN AND LACTOSE INCREASE TRANSLATION INITIATION FACTOR ACTIVATION AND TISSUE PROTEIN SYNTHESIS IN NEONATAL PIGS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein synthesis and eukaryotic initiation factor (eIF) activation are increased in muscle and liver of pigs parenterally infused with amino acids and insulin. To examine the effects of enteral protein and carbohydrate on protein synthesis, pigs (n = 42, 1.7 kg body wt) were fed isocaloric milk die...

  15. Development of a new method for determination of total haem protein in fish muscle.

    PubMed

    Chaijan, Manat; Undeland, Ingrid

    2015-04-15

    Using classic haem protein quantification methods, the extraction step in buffer or acid acetone often becomes limiting if muscle is oxidised and/or stored; haem-proteins then tend to bind to muscle components like myofibrils and/or biomembranes. The objective of this study was to develop a new haem protein determination method for fish muscle overcoming such extractability problems. The principle was to homogenise and heat samples in an SDS-containing phosphate buffer to dissolve major muscle components and convert ferrous/ferric haem proteins to hemichromes with a unique absorption peak at 535 nm. Hb-recovery tests with the new and classic methods showed that the new method and Hornsey's method performed significantly better on fresh Hb-enriched cod mince than Brown's and Drabkin's methods; recovery was ⩾98%. However, in highly oxidised samples and in cod protein isolates made with acid pH-shift processing, the new method performed better than Hornsey's method (63% and 87% vs. 50% and 68% recovery). Further, the new method performed well in fish muscle with ⩽30% lipid, <5% NaCl and pH 5.5-7.0; it was also unaffected by freezing/frozen storage. PMID:25466135

  16. The rate of synthesis and decomposition of tissue proteins in hypokinesia and increased muscular activity

    NASA Technical Reports Server (NTRS)

    Fedorov, I. V.; Chernyy, A. V.; Fedorov, A. I.

    1978-01-01

    During hypokinesia and physical loading (swimming) of rats, the radioactivity of skeletal muscle, liver, kidney, heart, and blood proteins was determined after administration of radioactive amino acids. Tissue protein synthesis decreased during hypokinesia, and decomposition increased. Both synthesis and decomposition increased during physical loading, but anabolic processes predominated in the total tissue balance. The weights of the animals decreased in hypokinesia and increased during increased muscle activity.

  17. Decrease of total activity with time at long distances from a nuclear accident or explosion.

    PubMed

    Dolejs, Josef

    2005-05-01

    Two data groups were analyzed: (1) the exposure rate in the former Czechoslovakia after the Chernobyl accident in 1986, and (2) the decrease of beta activity of an atmospheric fallout sample taken in Bratislava during 24 h on 30 May 1965. Both quantities decreased with the first power of time. This pattern of decrease is explained by applying the same mathematical formalism as is also used to describe the decrease in postnatal mortality with age. Following this formalism, the decrease of total activity with the first power of time could be seen as a consequence of a log-normal distribution of decay constants in the fallout. This differs slightly from earlier results that show the total activity decreasing with a power of 1.2 immediately after the nuclear explosion. PMID:15818480

  18. Detecting consciousness in a total locked-in syndrome: an active event-related paradigm.

    PubMed

    Schnakers, Caroline; Perrin, Fabien; Schabus, Manuel; Hustinx, Roland; Majerus, Steve; Moonen, Gustave; Boly, Melanie; Vanhaudenhuyse, Audrey; Bruno, Marie-Aurelie; Laureys, Steven

    2009-08-01

    Total locked-in syndrome is characterized by tetraplegia, anarthria and paralysis of eye motility. In this study, consciousness was detected in a 21-year-old woman who presented a total locked-in syndrome after a basilar artery thrombosis (49 days post-injury) using an active event-related paradigm. The patient was presented sequences of names containing the patient's own name and other names. The patient was instructed to count her own name or to count another target name. Similar to 4 age- and gender-matched healthy controls, the P3 response recorded for the voluntarily counted own name was larger than while passively listening. This P3 response was observed 14 days before the first behavioral signs of consciousness. This study shows that our active event-related paradigm allowed to identify voluntary brain activity in a patient who would behaviorally be diagnosed as comatose. PMID:19241281

  19. "I Feel Totally at One, Totally Alive and Totally Happy": A Psycho-Social Explanation of the Physical Activity and Mental Health Relationship

    ERIC Educational Resources Information Center

    Crone, D.; Smith, A.; Gough, B.

    2005-01-01

    This paper reports findings from a qualitative investigation into the relationship between physical activity and mental health from the experiences of participants on exercise referral schemes. A grounded theory methodology was adopted which used focus groups and semi-structured interviews with participants from three exercise referral schemes in…

  20. Relationship of Circulating Total Homocysteine and C-Reactive Protein to Trabecular Bone in Postmenopausal Women

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Homocysteine (Hcy) and C-reactive protein (CRP) are novel risk factors for osteoporosis. The purpose of this analysis was to determine the relationship of Hcy and CRP to volumetric trabecular bone, but also to assess their relationship to areal composite bone in healthy postmenopausal women (N=184)....

  1. Quiescent and Active Tear Protein Profiles to Predict Vernal Keratoconjunctivitis Reactivation

    PubMed Central

    Micera, Alessandra; Di Zazzo, Antonio; Esposito, Graziana; Sgrulletta, Roberto; Calder, Virginia L.; Bonini, Stefano

    2016-01-01

    Objective. Vernal keratoconjunctivitis (VKC) is a chronic recurrent bilateral inflammation of the conjunctiva associated with atopy. Several inflammatory and tissue remodeling factors contribute to VKC disease. The aim is to provide a chip-based protein analysis in tears from patients suffering from quiescent or active VKC. Methods. This study cohort included 16 consecutive patients with VKC and 10 controls. Participants were subjected to clinical assessment of ocular surface and tear sampling. Total protein quantification, total protein sketch, and protein array (sixty protein candidates) were evaluated. Results. An overall increased Fluorescent Intensity expression was observed in VKC arrays. Particularly, IL1β, IL15, IL21, Eotaxin2, TACE, MIP1α, MIP3α, NCAM1, ICAM2, βNGF, NT4, BDNF, βFGF, SCF, MMP1, and MMP2 were increased in quiescent VKC. Of those candidates, only IL1β, IL15, IL21, βNGF, SCF, MMP2, Eotaxin2, TACE, MIP1α, MIP3α, NCAM1, and ICAM2 were increased in both active and quiescent VKC. Finally, NT4, βFGF, and MMP1 were highly increased in active VKC. Conclusion. A distinct “protein tear-print” characterizes VKC activity, confirming some previously reported factors and highlighting some new candidates common to quiescent and active states. Those candidates expressed in quiescent VKC might be considered as predictive indicators of VKC reactivation and/or exacerbation out-of-season. PMID:26989694

  2. In vivo Prompt Gamma Neutron Activation Analysis Facility for Total Body Nitrogen and Cd

    SciTech Connect

    Munive, Marco; Revilla, Angel; Solis, Jose L.

    2007-10-26

    A Prompt Gamma Neutron Activation Analysis (PGNAA) system has been designed and constructed to measure the total body nitrogen and Cd for in vivo studies. An aqueous solution of KNO{sub 3} was used as phantom for system calibration. The facility has been used to monitor total body nitrogen (TBN) of mice and found that is related to their diet. Some mice swallowed diluted water with Cl{sub 2}Cd, and the presence of Cd was detected in the animals. The minimum Cd concentration that the system can detect was 20 ppm.

  3. Accelerometer-based physical activity: total volume per day and standardized measures.

    PubMed

    Bassett, David R; Troiano, Richard P; McClain, James J; Wolff, Dana L

    2015-04-01

    The use of accelerometers in physical activity (PA) research has increased exponentially over the past 20 yr. The first commercially available accelerometer for assessing PA, the Caltrac, was worn on the waist and estimated PA energy expenditure in kilocalories. Around 1995, the emphasis shifted to measuring minutes of moderate-to-vigorous PA (MVPA), especially for bouts of 10 min or longer. Recent studies, however, show that light-intensity PA and intermittent (nonbout) MVPA also have important health benefits. The total volume of PA performed is an important variable because it takes the frequency, intensity, and duration of activity bouts and condenses them down into a single metric. The total volume of PA is appropriate for many research applications and can enhance comparisons between studies. In the future, machine learning algorithms will provide improved accuracy for activity type recognition and estimation of PA energy expenditure. However, in the current landscape of objectively measured PA, total activity counts per day (TAC/d) is a proxy for the total volume of PA. TAC/d percentiles for age- and gender-specific groups have been developed from the National Health and Nutrition Examination Survey ActiGraph data (2003-2006), providing a novel way to assess PA. The use of TAC/d or standardized units of acceleration could harmonize PA across studies. TAC/d should be viewed as an additional metric, not intended to replace other metrics (e.g., sedentary time, light-intensity PA, moderate PA, and vigorous PA) that may also be related to health. As future refinements to wearable monitors occur, researchers should continue to consider metrics that reflect the total volume of PA in addition to existing PA metrics. PMID:25102292

  4. Poliovirus protein 2C has ATPase and GTPase activities.

    PubMed

    Rodríguez, P L; Carrasco, L

    1993-04-15

    Poliovirus protein 2C belongs to an expanding group of proteins containing a nucleotide binding motif in their sequence. We present evidence that poliovirus 2C has nucleoside triphosphatase (NTPase) activity and binds to RNA. Poliovirus 2C was expressed in Escherichia coli cells as a fusion protein with the maltose binding protein (MBP). The fusion protein MBP-2C is efficiently cut by protease Xa within the 2C region. Thus, the fusion protein as such was used to assay for the putative activities of poliovirus 2C. Deletion mutants were constructed which lacked different portions of the 2C carboxyl terminus: mutant 2C delta 1 lacked the last 169 amino acids, whereas mutant 2C delta 2 had the last 74 amino acids deleted. The fusion proteins MBP-2C, MBP-2BC, and the mutant MBP-2C delta 2 that contained the first 255 amino acids of 2C had NTPase activity. Both ATPase and GTPase activities are inhibited by antibodies directed against the MBP-2C protein. Analysis of the ability of the different proteins to bind to labeled RNA indicates that MBP-2C and MBP-2BC form a complex, whereas none of the mutants interacted with RNA, indicating that the RNA binding domain lies beyond amino acid 255. None of the fusion proteins had detectable helicase activity. We suggest that poliovirus protein 2C shows similarities to the GTPases group involved in vesicular traffic and transports the viral RNA replication complexes. These results provide the first experimental evidence that poliovirus protein 2C is an NTPase and that this protein has affinity for nucleic acids. PMID:8385138

  5. Sports activity after total joint arthroplasty: recommendations for the counseling physician.

    PubMed

    Buza, John A; Fink, Leslie A; Levine, William N

    2013-02-01

    Sports activity after total joint arthroplasty (TJA) has become an increasingly important topic, as many younger patients seeking TJA have higher postoperative expectations with regard to return to athletic activity. Our current knowledge of this area is largely based on retrospective clinical studies and surveys of surgeon recommendations. The decision to participate in sports after TJA depends on the patient's general health, prior athletic experience, type of TJA, and desired sporting activity. Ultimately, patients should discuss these factors with their physician in order to make an educated decision regarding sports activity after TJA. This article summarizes the best available evidence to help guide physicians in their conversation with patients regarding safe and appropriate sports activity after TJA. PMID:23445855

  6. Protein determination in seeds by proton activation

    NASA Astrophysics Data System (ADS)

    Morales, J. R.; Dinator, M. I.; Cerda, P.

    1989-04-01

    A proton beam of 6.6 MeV has been used to produce 11C and 13N in Araucaria Araucana seeds. Their positron decay allows determination of the N/C ratio. In seeds the nitrogen content is associated to proteins while carbon is spread in the organic material. Samples were irradiated for about 10 min with a beam intensity of 5 nA on areas of 1 mm 2. Slices of the seed were radially explored, showing a larger concentration of protein in the center.

  7. Effect of Buddhist meditation on serum cortisol and total protein levels, blood pressure, pulse rate, lung volume and reaction time.

    PubMed

    Sudsuang, R; Chentanez, V; Veluvan, K

    1991-09-01

    Serum cortisol and total protein levels, blood pressure, heart rate, lung volume, and reaction time were studied in 52 males 20-25 years of age practicing Dhammakaya Buddhist meditation, and in 30 males of the same age group not practicing meditation. It was found that after meditation, serum cortisol levels were significantly reduced, serum total protein level significantly increased, and systolic pressure, diastolic pressure and pulse rate significantly reduced. Vital capacity, tidal volume and maximal voluntary ventilation were significantly lower after meditation than before. There were also significant decreases in reaction time after mediation practice. The percentage decrease in reaction time during meditation was 22%, while in subjects untrained in meditation, the percentage decrease was only 7%. Results from these studies indicate that practising Dhammakaya Buddhist meditation produces biochemical and physiological changes and reduces the reaction time. PMID:1801007

  8. Estimation of total phenol and in vitro antioxidant activity of Albizia procera leaves

    PubMed Central

    2013-01-01

    Background Research on natural products has gained a wide popularity due to the potential of discovering active compounds. The antioxidant properties contained in plants have been proposed as one of the mechanisms for the observed beneficial effect. Therefore, the present study investigated the antioxidant activity and total phenolic contents of various solvent extracts of Albizia procera leaves. Methods Antioxidant activity of the methanol extract and its derived fractions petroleum ether (APP), carbon tetrachloride (APC), dichloromethane (APD), ethyl acetate (APE), and residual aqueous fraction (APA) of the leaves of Albizia procera was performed by in vitro chemical analyses. Total phenolic content of the APM and other five fractions were also determined. APM and its derived fractions were also subjected to preliminary phytochemical screening test for various constituents. Results Phytochemical screening revealed the presence of saponins, steroids, tannins, glycosides and flavonoids in the extracts. Amongst the extracts, APE showed the highest total phenolic content (449.18 ± 18.41mg of gallic acid equivalent/g of extract). In DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging test, the IC50 value of APM, APP, APC, APD, APE and APA was 43.43, 63.60, 166.18, 41.15, 11.79, and 63.06 μg/mL, respectively. Therefore, among the APM and its derived fractions, APE showed the highest antioxidant activity which is comparable to that of standard ascorbic acid (AA) (IC50 10.12 μg/mL). The total antioxidant capacity was found to be varied in different fractions. The reducing activity on ferrous ion was ranked as APE > APD > APM > APA > APC. Conclusion The above evidences suggest that APE of A. procera leaf is a potential source of natural antioxidant and can be used to prevent diseases associated with free radicals. PMID:23531304

  9. Monitoring G protein activation in cells with BRET

    PubMed Central

    Masuho, Ikuo; Martemyanov, Kirill A.; Lambert, Nevin A.

    2016-01-01

    Summary Live-cell assays based on fluorescence and luminescence are now indispensable tools for the study of G protein signaling. Assays based on fluorescence and bioluminescence resonance energy transfer (FRET and BRET) have been particularly valuable for monitoring changes in second messengers, protein-protein interactions, and protein conformation. Here we describe a BRET assay that monitors the release of free Gβγ dimers after activation of heterotrimers containing Gα subunits from all four G protein subfamilies. This assay provides useful kinetic and pharmacological information with reasonably high throughput using standard laboratory equipment. PMID:26260597

  10. Why Patients Do Not Participate in Sports Activities After Total Knee Arthroplasty

    PubMed Central

    Chang, Moon Jong; Kang, Yeon Gwi; Chung, Byung June; Chang, Chong Bum; Kim, Tae Kyun

    2015-01-01

    Background It is important to identify the reasons or factors preventing patients from participating in sports activities after total knee arthroplasty (TKA) to improve patient satisfaction and general health that can be gained from regular sports activities. Purpose To determine the reasons for lack of participation in regular sports activities after TKA as perceived by patients and to identify specific factors involved. Study Design Case series; Level of evidence, 4. Methods A total of 369 patients with a follow-up longer than 1 year after TKA were included in this retrospective study. A postal survey regarding sports activities was conducted using a questionnaire gathering information such as sociodemographic data, activity levels, and sports activities after TKA. The reasons perceived by patients for not participating in sports activities were determined. Patients unable to participate in sports activities were compared with patients able to do so in terms of sociodemographic data and pre- and postoperative outcomes. Results Of the 369 patients, 88 (24%) replied that they could not participate in a sports activity. Among the perceived reasons, reasons not related to the replaced knee were more frequent than those related to the replaced knee (76% vs 24%). Symptoms related to the spine or other joints composed 25% of the total perceived reasons. The presence of medical comorbidities accounted for 16%, while symptoms in the nonreplaced knee represented 8% of the reasons. In terms of patient factors, multivariate logistic regression revealed that male sex, a floor-based (ie, non-Western) lifestyle with greater demands on knee flexion, and worse postoperative University of California at Los Angeles activity scale were associated with nonregular sports activity levels. Conclusion The perceived reasons and patient factors hindering regular sports activities after TKA were not restricted to problems with the replaced knee. Nonetheless, orthopaedic surgeons may have a

  11. Validity of physical activity measures in individuals after total knee arthroplasty

    PubMed Central

    Almeida, Gustavo J.; Wert, David M.; Brower, Kelly S.; Piva, Sara R.

    2015-01-01

    OBJECTIVE To determine the concurrent criterion-related validity of two activity monitors in comparison to the criterion method of indirect calorimetry in older adults after total knee arthroplasty (TKA). DESIGN Validation study. SETTING Subjects completed 9 increasingly demanding daily activities in a research laboratory; each activity was performed for 7 minutes, for a total of 80-minutes while the activity monitors and criterion method were used concurrently. PARTICIPANTS Twenty-one subjects, 67% female, mean age 68±7 years old, and BMI 29±4. INTERVENTIONS not applicable. MAIN OUTCOME MEASURE Energy expenditure (EE) in kcal/min measured by accelerometer-based and multisensor-based monitors, and the criterion method. Validity was assessed by paired t-test, intra-class correlation coefficient (ICC), and Bland-Altman plots comparing the measurements from the activity monitors to the criterion method. RESULTS Measurements from the accelerometer-based monitor were significantly lower than those of the criterion method across all walking and non-walking activities. The underestimations ranged from 40% to 100%. The accelerometer-based monitor demonstrated small to moderate agreement compared to the criterion method (ICCs from 0 to 0.38). Measurements from the multisensor-based monitor were significantly lower than the criterion method during several non-walking activities; yet, the differences were minor (2% to 19%). Measurements from the multisensor-based monitor during walking activities were not different compared to the criterion method. The multisensor-based monitor demonstrated moderate to excellent agreement with the criterion method (ICCs from 0.48 to 0.81). CONCLUSION The multisensor-based monitor showed better criterion-related validity than the accelerometer-based monitor, and should be considered as a tool to measure physical activity in individuals after TKA. PMID:25450127

  12. Protein biomarkers for enhancement of radiation dose and injury assessment in nonhuman primate total-body irradiation model.

    PubMed

    Ossetrova, Natalia I; Sandgren, David J; Blakely, William F

    2014-06-01

    Development and validation of early-response radiation injury biomarkers are critical for effective triage and medical management of irradiated individuals. Plasma protein and haematological profiles were evaluated using multivariate linear-regression analysis to provide dose-response calibration curves for photon-radiation dose assessment in 30 rhesus macaques total-body-irradiated to 1-8.5 Gy with (60)Co gamma rays (0.55 Gy min(-1)). Equations for radiation dose received were established based on different combinations of protein biomarkers [i.e. C-reactive protein (CRP), serum amyloid A (SAA), interleukin 6 (IL-6) and Flt3 Ligand (Flt3L)] at samples collection time-points 6 h, 1, 2, 3, 4 and 7 d post-total-body irradiation. Dynamic changes in the levels of CRP, SAA, IL-6 and Flt3L may function as prognostic indicators of the time course and severity of acute radiation sickness (ARS). The combination of protein biomarkers provides greater accuracy for early radiation assessment than any one biomarker alone. PMID:24925901

  13. Speed associated with plasma pH, oxygen content, total protein and urea in an 80 km race.

    PubMed

    Hoffman, R M; Hess, T M; Williams, C A; Kronfeld, D S; Griewe-Crandell, K M; Waldron, J E; Graham-Thiers, P M; Gay, L S; Splan, R K; Saker, K E; Harris, P A

    2002-09-01

    To test the hypothesis that endurance performance may be related quantitatively to changes in blood, we measured selected blood variables then determined their reference ranges and associations with speed during an 80 km race. The plan had 46 horses in a 2 x 2 factorial design testing a potassium-free electrolyte mix and a vitamin supplement. Blood samples were collected before the race, at 21, 37, 56 and 80 km, and 20 min after finishing, for assay of haematocrit, plasma pH, pO2, pCO2, [Na+], [K+], [Ca++], [Mg++], [Cl-], lactate, glucose, urea, cortisol, alpha-tocopherol, ascorbate, creatine kinase, aspartate amino transferase, lipid hydroperoxides, total protein, albumin and creatinine, and erythrocyte glutathione and glutathione peroxidase. Data from 34 finishers were analysed statistically. Reference ranges for resting and running horses were wide and overlapping and, therefore, limiting with respect to evaluation of individual horses. Speed correlations were most repeatable, with variables reflecting blood oxygen transport (enabling exercise), acidity and electrolytes (limiting exercise) and total protein (enabling then, perhaps, limiting). Stepwise regressions also included plasma urea concentration (limiting). The association of speed with less plasma acidity and urea suggests the potential for fat adaptation and protein restriction in endurance horses, as found previously in Arabians performing repeated sprints. Conditioning horses fed fat-fortified and protein-restricted diets may not only improve performance but also avoid grain-associated disorders. PMID:12405657

  14. Estimation of the front-to-total activity ratio for wire screens using CFD simulation.

    PubMed

    Chen, B; Zhuo, W

    2015-11-01

    Wire screens are widely used for sampling radioactive aerosols. The front-to-total activity ratio is a critical factor in describing the self-shielding effect of the wire screens. In this study, computational fluid dynamic method was applied to simulate the deposition of aerosols on the surface of the wire screens. Four different types of screens were investigated for particle size varying from 1 nm to 10 µm. Experimental verification was carried out in a radon chamber. The results showed good agreement between experimental data interception and the simulation. Significant differences on the front-to-total activity ratio for the different types of screens were observed when the size of particle was <20 nm. PMID:25920787

  15. New constitutive latex osmotin-like proteins lacking antifungal activity.

    PubMed

    Freitas, Cleverson D T; Silva, Maria Z R; Bruno-Moreno, Frederico; Monteiro-Moreira, Ana C O; Moreira, Renato A; Ramos, Márcio V

    2015-11-01

    Proteins that share similar primary sequences to the protein originally described in salt-stressed tobacco cells have been named osmotins. So far, only two osmotin-like proteins were purified and characterized of latex fluids. Osmotin from Carica papaya latex is an inducible protein lacking antifungal activity, whereas the Calotropis procera latex osmotin is a constitutive antifungal protein. To get additional insights into this subject, we investigated osmotins in latex fluids of five species. Two potential osmotin-like proteins in Cryptostegia grandiflora and Plumeria rubra latex were detected by immunological cross-reactivity with polyclonal antibodies produced against the C. procera latex osmotin (CpOsm) by ELISA, Dot Blot and Western Blot assays. Osmotin-like proteins were not detected in the latex of Thevetia peruviana, Himatanthus drasticus and healthy Carica papaya fruits. Later, the two new osmotin-like proteins were purified through immunoaffinity chromatography with anti-CpOsm immobilized antibodies. Worth noting the chromatographic efficiency allowed for the purification of the osmotin-like protein belonging to H. drasticus latex, which was not detectable by immunoassays. The identification of the purified proteins was confirmed after MS/MS analyses of their tryptic digests. It is concluded that the constitutive osmotin-like proteins reported here share structural similarities to CpOsm. However, unlike CpOsm, they did not exhibit antifungal activity against Fusarium solani and Colletotrichum gloeosporioides. These results suggest that osmotins of different latex sources may be involved in distinct physiological or defensive events. PMID:26231325

  16. Total antioxidant activity and fiber content of select Florida-grown tropical fruits.

    PubMed

    Mahattanatawee, Kanjana; Manthey, John A; Luzio, Gary; Talcott, Stephen T; Goodner, Kevin; Baldwin, Elizabeth A

    2006-09-20

    Fourteen tropical fruits from south Florida (red guava, white guava, carambola, red pitaya (red dragon), white pitaya (white dragon), mamey sapote, sapodilla, lychee, longan, green mango, ripe mango, green papaya, and ripe papaya) were evaluated for antioxidant activity, total soluble phenolics (TSP), total ascorbic acid (TAA), total dietary fiber (TDF), and pectin. ORAC (oxygen radical absorbance capacity) and DPPH (1,1-diphenyl-2-picrylhydrazyl, radical scavenging activity) assays were used to determine antioxidant activity. The TSP, ORAC, and DPPH ranged from 205.4 to 2316.7 g gallic acid equiv/g puree, <0.1 to 16.7 micromol Trolox equiv/g puree, and 2.1 to 620.2 microg gallic acid equiv/g puree, respectively. The TAA, TDF, and pectin ranged from 7.5 to 188.8 mg/100 g, 0.9 to 7.2 g/100 g, and 0.20 to 1.04 g/100 g, respectively. The antioxidant activities, TSP, TAA, TDF, and pectin were influenced by cultivar (papaya, guava, and dragon fruit) and ripening stage (papaya and/or mango). Antioxidant activity showed high correlations with levels of TSP compounds (r = 0.96) but low correlations with levels of ascorbic acid (r = 0.35 and 0.23 for ORAC and DPPH data, respectively). The antioxidant activities evaluated by both ORAC and DPPH showed similar trends where red guava and carambola exhibited the highest and sapodilla and green papaya exhibited the lowest levels. Guava and mamey sapote exhibited the highest TDF and pectin levels. Many of the tropical fruits were shown to contain an abundance of hydrolyzable tannins, ellagic acid conjugates, and flavone glycosides. Preliminary descriptions are given of the phenols in red/white pitaya (dragonfruit), lychee, and mamey sapote, these fruit being thus far uncharacterized in the literature. PMID:16968105

  17. Total-reflection active-mirror laser with cryogenic Yb:YAG ceramics.

    PubMed

    Furuse, Hiroaki; Kawanaka, Junji; Takeshita, Kenji; Miyanaga, Noriaki; Saiki, Taku; Imasaki, Kazuo; Fujita, Masayuki; Ishii, Shinya

    2009-11-01

    An efficient high-power laser operation has been demonstrated by using a cryogenic Yb:YAG composite ceramic with a total-reflection active-mirror arrangement. The composite ceramic, which had no high-reflection coating and was cooled with liquid nitrogen directly, showed four-level operation even at 67 kW/cm(3) of high pump density. A 273 W cw output power was obtained with 65% optical efficiency and 72% slope efficiency. PMID:19881620

  18. Combined nitrogen limitation and cadmium stress stimulate total carbohydrates, lipids, protein and amino acid accumulation in Chlorella vulgaris (Trebouxiophyceae).

    PubMed

    Chia, Mathias Ahii; Lombardi, Ana Teresa; da Graça Gama Melão, Maria; Parrish, Christopher C

    2015-03-01

    Metals have interactive effects on the uptake and metabolism of nutrients in microalgae. However, the effect of trace metal toxicity on amino acid composition of Chlorella vulgaris as a function of varying nitrogen concentrations is not known. In this research, C. vulgaris was used to investigate the influence of cadmium (10(-7) and 2.0×10(-8)molL(-1) Cd) under varying nitrogen (2.9×10(-6), 1.1×10(-5) and 1.1×10(-3)molL(-1)N) concentrations on its growth rate, biomass and biochemical composition. Total carbohydrates, total proteins, total lipids, as well as individual amino acid proportions were determined. The combination of Cd stress and N limitation significantly inhibited growth rate and cell density of C. vulgaris. However, increasing N limitation and Cd stress stimulated higher dry weight and chlorophyll a production per cell. Furthermore, biomolecules like total proteins, carbohydrates and lipids increased with increasing N limitation and Cd stress. Ketogenic and glucogenic amino acids were accumulated under the stress conditions investigated in the present study. Amino acids involved in metal chelation like proline, histidine and glutamine were significantly increased after exposure to combined Cd stress and N limitation. We conclude that N limitation and Cd stress affects the physiology of C. vulgaris by not only decreasing its growth but also stimulating biomolecule production. PMID:25625522

  19. Protein kinase activity associated with pancreatic zymogen granules.

    PubMed

    Burnham, D B; Munowitz, P; Thorn, N; Williams, J A

    1985-05-01

    Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues. PMID:4004796

  20. Protein kinase activity associated with pancreatic zymogen granules.

    PubMed Central

    Burnham, D B; Munowitz, P; Thorn, N; Williams, J A

    1985-01-01

    Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues. Images Fig. 1. Fig. 2. Fig. 7. Fig. 8. PMID:4004796

  1. The kinetics of ER fusion protein activation in vivo

    PubMed Central

    Wilson, Catherine H.; Gamper, Ivonne; Perfetto, Alessandra; Auw, Jeremy; Littlewood, Trevor D.; Evan, Gerard I.

    2014-01-01

    Reversibly switchable proteins are powerful tools with which to explore protein function in vitro and in vivo. For example, the activity of many proteins fused to the hormone-binding domain of the modified estrogen receptor (ERTAM) can be regulated by provision or removal of 4-hydroxytamoxifen (4-OHT). Despite the widespread use of ERTAM fusions in vivo, inadequate data are available as to the most efficacious routes for systemic tamoxifen delivery. In this study, we have used two well-characterised ERTAM fusion proteins, both reversibly activated by 4-OHT, to compare the effectiveness and kinetics of 4-OHT delivery in mice in vivo by either tamoxifen in food or by intraperitoneal injection. Our data indicate that dietary tamoxifen offers an effective, facile and ethically preferable means for long term activation of ERTAM fusion proteins in vivo. PMID:24662815

  2. In vitro antibacterial, antioxidant activity and total phenolic content of some essential oils.

    PubMed

    Srivastava, Upma; Ojha, Swati; Tripathi, N N; Singh, Pooja

    2015-11-01

    In vitro antibacterial activity of 16 essential oils was investigated by disc diffusion method against two Gram positive bacteria Bacillus subtilis and Staphylococcus aureus and two Gram negative bacteria, Shigella flexneri and Escherichia coli. Oils of Cymbopogon citratus and Ocimum basilicum showed highest antibacterial activity. Gram positive bacteria were found to be more sensitive than Gram negative. Antioxidant activities were tested by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and ABTS radical cation decolourization assay while Folin-Ciocalteu method was used to determine the total phenolic content. In DPPH assay, highest antioxidant activity was observed in 0. basilicum oil followed by Azeratum conyzoides, A. marmelos and C. citratus, with percent inhibition and IC50 value ranging from 66.11-71.93% and 14.10-17.92 µl ml(-1) respectively. In ABTS assay, similar results were obtained but with higher percent inhibition which ranged from 67.48-76.23% and lower IC50 value (12.12-17.21 µ ml(-1)). Moreover, radical scavenging activity of essential oils was lower than that observed for the synthetic antioxidant BHA and BHT. The total phenolic content of the essential oils as GAE in mg 100 µl(-1) of EO was found to be highest in O. basilicum (0.406) oil followed byA. conyzoides (0.322), A. marmelos (0.238) and C. citratus (0.231). The results provide evidence that the oils of C. citratus and O. basilicum can be further commended for treatment of infections caused by these bacterial pathogens and are potential source of natural antioxidants having appreciable amount of total phenolic content. PMID:26688969

  3. XMAP215 activity sets spindle length by controlling the total mass of spindle microtubules.

    PubMed

    Reber, Simone B; Baumgart, Johannes; Widlund, Per O; Pozniakovsky, Andrei; Howard, Jonathon; Hyman, Anthony A; Jülicher, Frank

    2013-09-01

    Metaphase spindles are microtubule-based structures that use a multitude of proteins to modulate their morphology and function. Today, we understand many details of microtubule assembly, the role of microtubule-associated proteins, and the action of molecular motors. Ultimately, the challenge remains to understand how the collective behaviour of these nanometre-scale processes gives rise to a properly sized spindle on the micrometre scale. By systematically engineering the enzymatic activity of XMAP215, a processive microtubule polymerase, we show that Xenopus laevis spindle length increases linearly with microtubule growth velocity, whereas other parameters of spindle organization, such as microtubule density, lifetime and spindle shape, remain constant. We further show that mass balance can be used to link the global property of spindle size to individual microtubule dynamic parameters. We propose that spindle length is set by a balance of non-uniform nucleation and global microtubule disassembly in a liquid-crystal-like arrangement of microtubules. PMID:23974040

  4. Dissociation between Processivity and Total Activity of γ-Secretase: Implications for the Mechanism of Alzheimer-Causing Presenilin Mutations†

    PubMed Central

    Quintero-Monzon, Omar; Martin, Morgan M.; Fernandez, Marty A.; Cappello, Christina A.; Krzysiak, Amanda J.; Osenkowski, Pamela; Wolfe, Michael S.

    2011-01-01

    The amyloid β-peptide (Aβ), strongly implicated in the pathogenesis of Alzheimer’s disease (AD), is produced from the amyloid β-protein precursor (APP) through consecutive proteolysis by β- and γ-secretases. The latter protease contains presenilin as the catalytic component of a membrane-embedded aspartyl protease complex. Missense mutations in presenilin are associated with early-onset familial AD, and these mutations generally both decrease Aβ production and increase the proportion of the aggregation-prone 42-residue form (Aβ42) over the 40-residue form (Aβ40). The connection between these two effects is not understood. Besides Aβ40 and Aβ42, γ-secretase produces a range of Aβ peptides, the result of initial cutting at the ε site to form Aβ48 or Aβ49 and subsequent trimming every 3–4 residues. Thus, γ-secretase displays both overall proteolytic activity (ε cutting) and processivity (trimming) toward its substrate APP. Here we tested whether a decrease in total activity correlates with decreased processivity using wild type and AD-mutant presenilin-containing protease complexes. Changes in pH, temperature and salt concentration that reduced overall activity of the wild type enzyme did not consistently result in increased proportions of longer Aβ peptides. Low salt concentrations and acidic pH were notable exceptions that subtly alter the proportion of individual Aβ peptides, suggesting that the charged state of certain residues may influence processivity. Five different AD-mutant complexes, representing a broad range of effects on overall activity, Aβ42-to-Aβ40 ratios, and ages of disease onset were also tested, revealing again that changes in total activity and processivity can be dissociated. Factors that control initial proteolysis of APP at the ε site apparently differ significantly from factors affecting subsequent trimming and the distribution of Aβ peptides. PMID:21919498

  5. Hemagglutinating activity of proteins from Parkia speciosa seeds.

    PubMed

    Chankhamjon, Kanokwan; Petsom, Amorn; Sawasdipuksa, Narumon; Sangvanich, Polkit

    2010-01-01

    Proteins from Parkia speciosa Hassk. (Fabaceae) seeds were extracted and stepwise precipitated using ammonium sulfate. Proteins precipitated with 25% ammonium sulfate were separated by affinity chromatography on Affi-Gel Blue gel followed by protein liquid chromatography on Superdex 200. The protein Gj, which was identified as a protein similar to putative aristolochene synthase, 3'-partial from Oryza sativa L. (Poaceae), had hemagglutinating activity of 0.39 mug/muL. Moreover, fraction C2 from the proteins precipitated with 60% ammonium sulfate, separated by lectin-specific adsorption chromatography using Con A Sepharose, had hemagglutinating activity of 1.17 mug/muL. Using gel electrophoresis, two proteins C2a and C2b were separated, having molecular weights of 45 kDa and 23 kDa, respectively. From protein identification, C2a was found to be similar to the hypothetical protein B1342F01.11 from Oryza sativa, and C2b was similar to the hypothetical protein At1g51560 from Arabidopsis thaliana (L.) Heynh. (Brassicaceae). PMID:20645760

  6. Essential oils chemical composition, antioxidant activities and total phenols of Astrodaucus persicus

    PubMed Central

    Goodarzi, Saeid; Hadjiakhoondi, Abbas; Yassa, Narguess; Khanavi, Mahnaz; Tofighi, Zahra

    2016-01-01

    Objective(s): Astrodaucus persicus, Apiaceae, is used as vegetable or food additive in some parts of Iran. The essential oils of different parts of Astrodaucus persicus from Kordestan province were analyzed for the first time and compared with other regions. In this study, antioxidant activities and total phenols determination of aerial parts essential oils and root fractions of A. persicus were investigated. Materials and Methods: The essential oils were obtained by hydro-distillation from flowers/fruits, leaves/stems, ripe fruits and roots of plant and analyzed by GC-MS. Crude root extract was fractionated with hexane, chloroform, ethyl acetate and methanol. Antioxidant activities by DPPH and FRAP methods and total phenols by Folin-ciocalteu assay were measured. Results: The abundant compounds of flowers/fruits blue essential oil were α-thujene, β-pinene and α-pinene. The predominant components of blue leaves/stems essential oil were α-thujene, α-pinene and α-fenchene. The major volatiles of ripe fruits blue essential oil were β-pinene, α-thujene and α-pinene. The chief compounds of root yellow essential oil were trans-caryophyllene, bicycogermacrene and germacrene-D. Total root extract and ethyl acetate fraction showed potent antioxidant activities and high amount of total phenols in comparison to other samples. Among volatile oils, the flowers/fruits essential oil showed potent reducing capacity. Conclusion: The major compounds of aerial parts essential oils were hydrocarbon monoterpenes while the chief percentage of roots essential oil constituents were hydrocarbon sesquiterpenes. α-Eudesmol and β-eudesmol were identified as responsible for creation of blue color in aerial parts essential oils. A. persicus was known as a potent antioxidant among Apiaceae. PMID:27081460

  7. Evidence for a receptor protein of activated carcinogen

    PubMed Central

    Mainigi, Kumar D.; Sorof, Sam

    1977-01-01

    During carcinogenesis in rat liver by 3′-methyl-4-dimethylaminoazobenzene, the two moieties of the principal liver carcinogen-protein complex have considerably different turnover rates. With continued ingestion of the azocarcinogen by rats, the bound azo dye in the complex has a half-life of 2.5 ± 0.25 (SD) days, while the protein moiety has a half-life of 8.7 ± 1.6 days (probability of identity <0.001). In addition, the interaction of the azocarcinogen with the principal target protein in vivo appears to extend the half-life of the protein itself from 3.3 ± 0.2 days in normal liver to 8.7 ± 1.6 days (P < 0.001). The slowing of the turnover of the protein by the carcinogen appears to be readily reversible, since soon after the cessation of azocarcinogen feeding, the half-life of the protein returns to that of the target protein in normal liver. The considerable difference in turnover rates of the two moieties of the complex and the reversible effects of the carcinogen in slowing the turnover of the protein moiety suggest that the two moieties of the native azoprotein are noncovalently linked and that they have different biological activities. The native complex appears to contain azo dye in an activated state that is capable of yielding a reactive electrophile, because after protein denaturation the bound azo dye was previously found to have properties that are indicative of covalent linkage to the protein. The retardation in the biological turnover rate of the protein moiety, apparently resulting from interaction with azocarcinogen, is in agreement with the known ligand-induced stabilization in vitro and reduced rate of proteolytic degradation in vivo of other proteins that result from conformational change to a more compact configuration. Our evidence is consistent with the hypothesis that the principal liver carcinogen-protein complex contains hydrophobically bound activated azocarcinogen, whose specificity of reaction with critical macromolecule(s) in

  8. Comparative study of antioxidant activities and total phenolic content of selected edible wild mushrooms.

    PubMed

    Yim, Hip Seng; Chye, Fook Yee; Lee, Mee Yee; Matanjun, Patricia; How, Siew Eng; Ho, Chun Wai

    2011-01-01

    The present study aims to assess the antioxidant activities (AOA) and total phenolic content (TPC) of water extracts of selected edible wild mushrooms: Pleurotus porrigens, Schizophyllum commune, Hygrocybe conica, and Lentinus ciliatus. The AOA were evaluated against DPPH radical and ABTS radical cation scavenging ability, ferric-reducing antioxidant power (FRAP) and beta-carotene-linoleate bleaching (beta-CB) assays, and the Folin-Ciocalteu method for TPC. BHA was used as reference. P. porrigens showed significantly higher (p < 0.05) DPPH* scavenging ability (90.78 +/- 0.30%) and FRAP (6.37 +/- 0.22 mM FE/100g), while Sch. commune showed significantly higher (p < 0.05) ABTS*+ inhibition activity (94.96 +/- 0.70%) and beta-CB inhibition activity (94.18 +/- 0.17%), respectively. TPC was found in a descending order of P. poriggens > L. ciliatus = Pleurotus ostreatus (cultivated) > H. conica = Sch. commune. Positive correlation was observed between the AOA and TPC. When compared to BHA (2 mM), P. porrigens showed significantly higher (p < 0.05) DPPH* scavenging ability and reducing power, while Sch. commune showed comparable DPPH* scavenging ability and ABTS*+ inhibition activity. All the mushrooms have better ABTS*+ inhibition activity than BHA (1 mM). The beta-CB inhibition activity of BHA was significantly higher than those of edible wild mushrooms. The water extracts of edible wild mushrooms showed potent antioxidant activities compared to BHA to a certain extent. PMID:22135876

  9. Antioxidant activity, ascorbic acid and total phenol of exotic fruits occurring in Brazil.

    PubMed

    de Assis, Sandra Aparecida; Vellosa, José Carlos Rebuglio; Brunetti, Iguatemy Lourenço; Khalil, Najeh Maissar; Leite, Kátia Maria da Silva Cerqueira; Martins, Antonio Baldo Geraldo; Oliveira, Olga Maria Mascarenhas de Faria

    2009-08-01

    The antioxidant activity, ascorbic acid and phenolic content were studied in 10 exotic fruits from Brazil: abiu, acerola, wax jambu, cashew, mamey sapote, carambola or star fruit, Surinam cherry, longan, sapodilla and jaboticaba. The ascorbic acid was determined by 2,6-dichloroindophenol titrimetic methods and total phenols were measured colorimetrically using the Folin-Ciocalteu reagent. The antioxidant activity was investigated with three different methods: hypochlorous acid scavenging activity, 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation decolorization assay, and 2,2-diphenyl-1-picrylhydrazyl radical scavenging method. The highest content of vitamin C (1,525.00 mg/100 g pulp) occurred in acerola. The total phenol content was higher in abiu, acerola, Surinam cherry and sapodilla. In relation to antioxidant activity, acerola has showed the great values in all three different methods tested. It was found that the fruits have a significant antioxidant effect when tested by each method, respectively, and these antioxidant capacities are promising. The sample concentration also influenced its antioxidant power. PMID:18785051

  10. Immunological changes following protein losing enteropathy after surgery total cavopulmonary connection (TCPC) by cytomics

    NASA Astrophysics Data System (ADS)

    Bocsi, József; Lenz, Dominik; Mittag, Anja; Sauer, Ursula; Wild, Lena; Hess, John; Schranz, Dietmar; Hambsch, Jörg; Schneider, Peter; Tárnok, Attila

    2008-02-01

    Complex immunophenotyping single-cell analysis are essential for systems biology and cytomics. The application of cytomics in immunology and cardiac research and diagnostics is very broad, ranging from the better understanding of the cardiovascular cell biology to the identification of heart function and immune consequences after surgery. TCPC or Fontan-type circulation is an accepted palliative surgery for patients with a functionally univentricular heart. Protein-losing enteropathy (PLE), the enteric loss of proteins, is a potential late complication after TCPC surgery. PLE etiology is poorly understood, but immunological factors seem to play a role. This study was aimed to gain insight into immune phenotype alterations following post-TCPC PLE. Patients were studied during routine follow-up up to 5yrs after surgery, blood samples of TCPC patients without (n=21, age 6.8+/-2.6 years at surgery; mean+/-SD) and with manifest PLE (n=12, age 12.8+/- 4.5 years at sampling) and age matched healthy children (control, n=22, age 8.6+/-2.5 years) were collected. Routine laboratory, immune phenotype and serological parameters were determined. Following PLE the immune phenotype dramatically changed with signs of acute inflammation (increased neutrophil and monocyte count, CRP, IL-8). In contrast, lymphocyte count (NK-cells, αβTCR +CD4 +, αβTCR +CD8 + cells) decreased (p<0.001). The residual T-cells had elevated CD25 and CD69 expression. In PLE-patients unique cell populations with CD3 +αβ/γδTCR - and αβTCR +CD4 -8 - phenotype were present in increased frequencies. Our studies show dramatically altered leukocyte phenotype after PLE in TCPC patients. These alterations resemble to changes in autoimmune diseases. We conclude that autoimmune processes may play a role in etiology and pathophysiology of PLE.

  11. Cloning of three novel neuronal Cdk5 activator binding proteins.

    PubMed

    Ching, Y P; Qi, Z; Wang, J H

    2000-01-25

    Neuronal Cdc2-like kinase (Nclk) is involved in the regulation of neuronal differentiation and neuro-cytoskeleton dynamics. The active kinase consists of a catalytic subunit, Cdk5, and a 25 kDa activator protein (p25nck5a) derived from a 35 kDa neuronal-specific protein (p35nck5a). As an extension of our previous study (Qi, Z., Tang, D., Zhu, X., Fujita, D.J., Wang, J.H., 1998. Association of neurofilament proteins with neuronal Cdk5 activator. J. Biol. Chem. 270, 2329-2335), which showed that neurofilament is one of the p35nck5a-associated proteins, we now report the isolation of three other novel p35nck5a-associated proteins using the yeast two-hybrid screen. The full-length forms of these three novel proteins, designated C42, C48 and C53, have a molecular mass of 66, 24, and 57 kDa, respectively. Northern analysis indicates that these novel proteins are widely expressed in human tissues, including the heart, brain, skeletal muscle, placenta, lung, liver, kidney and pancreas. The bacterially expressed glutathione S-transferase (GST)-fusion forms of these three proteins were able to co-precipitate p35nck5a complexed with Cdk5 from insect cell lysate. Among these three proteins, only C48 and C53 can be phosphorylated by Nclk, suggesting that they may be the substrates of Nclk. Sequence homology searches have suggested that the C48 protein is marginally related to restin protein, whereas the C42 protein has homologues of unknown function in Caenorhabditis elegans and Arabidopsis thaliana. PMID:10721722

  12. LASERS: Efficient chemical oxygen — iodine laser with a high total pressure of the active medium

    NASA Astrophysics Data System (ADS)

    Zagidullin, M. V.; Nikolaev, V. D.; Svistun, M. I.; Khvatov, N. A.; Heiger, G. D.; Madden, T. J.

    2001-01-01

    A new concept of obtaining a high total pressure of the active medium of a chemical oxygen — iodine laser (OIL) is proposed and verified. The nozzle unit of the laser consists of the alternating vertical arrays of cylindrical nozzles to produce high-pressure nitrogen jets, plane slotted nozzles for the flow of O2(1Δ) oxygen, and vertical arrays of cylindrical nozzles to inject the N2 — I2 mixture between the first two streams. For a molar chlorine flow rate of 39.2 mmol s-1, the output power was 700 W and the chemical efficiency was 19.7 %. The combined use of the ejector nozzle unit proposed to obtain the active medium and a super-sonic diffuser allows a significant simplification of the ejection system for the exhaust active medium of the OIL.

  13. Total synthesis and antiviral activity of indolosesquiterpenoids from the xiamycin and oridamycin families

    PubMed Central

    Meng, Zhanchao; Yu, Haixin; Li, Li; Tao, Wanyin; Chen, Hao; Wan, Ming; Yang, Peng; Edmonds, David J.; Zhong, Jin; Li, Ang

    2015-01-01

    Indolosesquiterpenoids are a growing class of natural products that exhibit a wide range of biological activities. Here, we report the total syntheses of xiamycin A and oridamycins A and B, indolosesquiterpenoids isolated from Streptomyces. Two parallel strategies were exploited to forge the carbazole core: 6π-electrocyclization/aromatization and indole C2–H bond activation/Heck annulation. The construction of their trans-decalin motifs relied on two diastereochemically complementary radical cyclization reactions mediated by Ti(III) and Mn(III), respectively. The C23 hydroxyl of oridamycin B was introduced by an sp3 C–H bond oxidation at a late stage. On the basis of the chemistry developed, the dimeric congener dixiamycin C has been synthesized for the first time. Evaluation of the antiviral activity of these compounds revealed that xiamycin A is a potent agent against herpes simplex virus–1 (HSV-1) in vitro. PMID:25648883

  14. Ethnobotanical study, antifungal activity, phytochemical screening and total phenolic content of Algerian Aristolochia longa

    PubMed Central

    Benarba, Bachir; Meddah, Boumedienne

    2014-01-01

    Aim: Aristolochia longa (from the family Aristolochiaceae) is widely used in Algerian traditional medicine. Here, we document ethnomedicinal uses by local population of Mascara province (West Algeria) and we evaluate the antifungal activity, the phytochemical composition and total phenolic content of aqueous extract (decoction) of the roots of A. longa from Algeria. Materials and Methods: The ethnobotanical investigation was carried out in Mascara Province (West Algeria). Antifungal activity was assessed against Saccharomyces cerevisiae. Total phenolic content was measured using the Folin–Ciocalteu’s reagent. Results: Our results showed that A. longa is widely used to treat several ailments such as cancer (38%), skin infections (14%), and diabetes (11%). Crushed roots are commonly used (89%) mixed with honey, milk, water or other medicinal plants. A. longa aqueous extract induced growth inhibition of S. cerevisiae cells in a dose - and time - dependent manner. An effective suppression of S. cerevisiae (97.06% inhibition of proliferation) was obtained at the 500 µg/mL after 72 h. Results of the phytochemical screening revealed that A. longa aqueous extract contained various bioactive compounds, including polyphenols and flavonoids. Total phenolic content in A. longa aqueous extract was found to be 6.07 ± 0.12 mg (gallic acid equivalents)/g. Conclusion: A. longa may be considered as a promising source of new drugs for treating cancer and as a good antifungal agent. PMID:26401365

  15. Small Increase of Actual Physical Activity 6 Months After Total Hip or Knee Arthroplasty

    PubMed Central

    Bussmann, Hans J.; Stam, Henk J.; Verhaar, Jan A.

    2008-01-01

    Limitation in daily physical activity is one of the reasons for total hip arthroplasty (THA) or total knee arthroplasty (TKA). However, studies of the effects of THA or TKA generally do not determine actual daily activity as part of physical functioning. We determined the effect of THA or TKA on patients’ actual physical activity and body function (pain, stiffness), capacity to perform tasks, and self-reported physical functioning. We also assessed whether there are differences in the effect of the surgery between patients undergoing THA or TKA and whether the improvements vary between these different outcome measures. We recruited patients with long-standing end-stage osteoarthritis of the hip or knee awaiting THA or TKA. Measurements were performed before surgery and 3 and 6 months after surgery. Actual physical activity improved by 0.7%. Patients’ body function, capacity, and self-reported physical functioning also improved. The effects of the surgery on these aspects of physical functioning were similar for THA and TKA. The effect on actual physical activity (8%) was smaller than on body function (80%–167%), capacity (19%–36%), and self-reported physical functioning (87%–112%). Therefore, in contrast to the large effect on pain and stiffness, patients’ capacity, and their self-reported physical functioning, the improvement in actual physical activity of our patients was less than expected 6 months after surgery. Level of Evidence: Level I, prospective study. See the Guidelines for Authors for a complete description of levels of evidence. PMID:18506555

  16. Pestivirus NS3 (p80) protein possesses RNA helicase activity.

    PubMed Central

    Warrener, P; Collett, M S

    1995-01-01

    The pestivirus bovine viral diarrhea virus (BVDV) p80 protein (referred to here as the NS3 protein) contains amino acid sequence motifs predictive of three enzymatic activities: serine proteinase, nucleoside triphosphatase, and RNA helicase. We have previously demonstrated that the former two enzymatic activities are associated with this protein. Here, we show that a purified recombinant BVDV NS3 protein derived from baculovirus-infected insect cells possesses RNA helicase activity. BVDV NS3 RNA helicase activity was specifically inhibited by monoclonal antibodies to the p80 protein. The activity was dependent on the presence of nucleoside triphosphate and divalent cation, with a preference for ATP and Mn2+. Hydrolysis of the nucleoside triphosphate was necessary for strand displacement. The helicase activity required substrates with an un-base-paired region on the template strand 3' of the duplex region. As few as three un-base-paired nucleotides were sufficient for efficient oligonucleotide displacement. However, the enzyme did not act on substrates having a single-stranded region only to the 5' end of the duplex or on substrates lacking single-stranded regions altogether (blunt-ended duplex substrates), suggesting that the directionality of the BVDV RNA helicase was 3' to 5' with respect to the template strand. The BVDV helicase activity was able to displace both RNA and DNA oligonucleotides from RNA template strands but was unable to release oligonucleotides from DNA templates. The possible role of this activity in pestivirus replication is discussed. PMID:7853509

  17. Activation of an Endoribonuclease by Non-intein Protein Splicing.

    PubMed

    Campbell, Stephen J; Stern, David B

    2016-07-29

    The Chlamydomonas reinhardtii chloroplast-localized poly(A)-binding protein RB47 is predicted to contain a non-conserved linker (NCL) sequence flanked by highly conserved N- and C-terminal sequences, based on the corresponding cDNA. RB47 was purified from chloroplasts in association with an endoribonuclease activity; however, protein sequencing failed to detect the NCL. Furthermore, while recombinant RB47 including the NCL did not display endoribonuclease activity in vitro, versions lacking the NCL displayed strong activity. Both full-length and shorter forms of RB47 could be detected in chloroplasts, with conversion to the shorter form occurring in chloroplasts isolated from cells grown in the light. This conversion could be replicated in vitro in chloroplast extracts in a light-dependent manner, where epitope tags and protein sequencing showed that the NCL was excised from a full-length recombinant substrate, together with splicing of the flanking sequences. The requirement for endogenous factors and light differentiates this protein splicing from autocatalytic inteins, and may allow the chloroplast to regulate the activation of RB47 endoribonuclease activity. We speculate that this protein splicing activity arose to post-translationally repair proteins that had been inactivated by deleterious insertions or extensions. PMID:27311716

  18. Iron-chelating activity of chickpea protein hydrolysate peptides.

    PubMed

    Torres-Fuentes, Cristina; Alaiz, Manuel; Vioque, Javier

    2012-10-01

    Chickpea-chelating peptides were purified and analysed for their iron-chelating activity. These peptides were purified after affinity and gel filtration chromatography from a chickpea protein hydrolysate produced with pepsin and pancreatin. Iron-chelating activity was higher in purified peptide fractions than in the original hydrolysate. Histidine contents were positively correlated with the iron-chelating activity. Hence fractions with histidine contents above 20% showed the highest chelating activity. These results show that iron-chelating peptides are generated after chickpea protein hydrolysis with pepsin plus pancreatin. These peptides, through metal chelation, may increase iron solubility and bioavailability and improve iron absorption. PMID:25005984

  19. Production of biologically active human thioredoxin 1 protein in lettuce chloroplasts.

    PubMed

    Lim, Soon; Ashida, Hiroki; Watanabe, Rie; Inai, Koji; Kim, Yun-Soo; Mukougawa, Keiko; Fukuda, Hirokazu; Tomizawa, Ken-ichi; Ushiyama, Kei-ichi; Asao, Hiroshi; Tamoi, Masahiro; Masutani, Hiroshi; Shigeoka, Shigeru; Yodoi, Junji; Yokota, Akiho

    2011-07-01

    The production of human therapeutic proteins in plants provides opportunities for low-cost production, and minimizes the risk of contamination from potential human pathogens. Chloroplast genetic engineering is a particularly promising strategy, because plant chloroplasts can produce large amounts of foreign target proteins. Oxidative stress is a key factor in various human diseases. Human thioredoxin 1 (hTrx1) is a stress-induced protein that functions as an antioxidant against oxidative stress, and overexpression of hTrx1 has been shown to suppress various diseases in mice. Therefore, hTrx1 is a prospective candidate as a new human therapeutic protein. We created transplastomic lettuce expressing hTrx1 under the control of the psbA promoter. Transplastomic plants grew normally and were fertile. The hTrx1 protein accumulated to approximately 1% of total soluble protein in mature leaves. The hTrx1 protein purified from lettuce leaves was functionally active, and reduced insulin disulfides. The purified protein protected mouse insulinoma line 6 cells from damage by hydrogen peroxide, as reported previously for a recombinant hTrx1 expressed in Escherichia coli. This is the first report of expression of the biologically active hTrx1 protein in plant chloroplasts. This research opens up possibilities for plant-based production of hTrx1. Considering that this expression host is an edible crop plant, this transplastomic lettuce may be suitable for oral delivery of hTrx1. PMID:21290168

  20. Inclusion bodies and purification of proteins in biologically active forms.

    PubMed

    Mukhopadhyay, A

    1997-01-01

    Even though recombinant DNA technology has made possible the production of valuable therapeutic proteins, its accumulation in the host cell as inclusion body poses serious problems in the recovery of functionally active proteins. In the last twenty years, alternative techniques have been evolved to purify biologically active proteins from inclusion bodies. Most of these remain only as inventions and very few are commercially exploited. This review summarizes the developments in isolation, refolding and purification of proteins from inclusion bodies that could be used for vaccine and non-vaccine applications. The second section involves a discussion on inclusion bodies, how they are formed, and their physicochemical properties. In vivo protein folding in Escherichia coli and kinetics of in vitro protein folding are the subjects of the third and fourth sections respectively. The next section covers the recovery of bioactive protein from inclusion bodies: it includes isolation of inclusion body from host cell debris, purification in denatured state alternate refolding techniques, and final purification of active molecules. Since purity and safety are two important issues in therapeutic grade proteins, the following three sections are devoted to immunological and biological characterization of biomolecules, nature, and type of impurities normally encountered, and their detection. Lastly, two case studies are discussed to demonstrate the sequence of process steps involved. PMID:8939059

  1. Activators of G Protein Signaling in the Kidney

    PubMed Central

    2015-01-01

    Heterotrimeric G proteins play a crucial role in regulating signal processing to maintain normal cellular homeostasis, and subtle perturbations in its activity can potentially lead to the pathogenesis of renal disorders or diseases. Cell-surface receptors and accessory proteins, which normally modify and organize the coupling of individual G protein subunits, contribute to the regulation of heterotrimeric G protein activity and their convergence and/or divergence of downstream signaling initiated by effector systems. Activators of G protein signaling (AGS) are a family of accessory proteins that intervene at multiple distinct points during the activation–inactivation cycle of G proteins, even in the absence of receptor stimulation. Perturbations in the expression of individual AGS proteins have been reported to modulate signal transduction pathways in a wide array of diseases and disorders within the brain, heart, immune system, and more recently, the kidney. This review will provide an overview of the expression profile, localization, and putative biologic role of the AGS family in the context of normal and diseased states of the kidney. PMID:25628392

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

    SciTech Connect

    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

    2014-12-18

    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.

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

    DOE PAGESBeta

    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.; et al

    2014-12-18

    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.more » 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.« less

  4. GPS-Total electron content variability in Brazil during low solar activity

    NASA Astrophysics Data System (ADS)

    Mendes da Costa, A.; Vilas Boas, J.; Fonseca Junior, E.

    Variations of ionospheric Total Electron Content (TEC) have been calculated using GPS data obtained over a low latitude station Presidente Prudente, Brazil (22.1o S; 51.4o W ) in 1997, a period of low solar activity. Two hourly TEC averages are presented for the period. Diurnal, seasonal, solar activity variations and the Equatorial Anomaly effects are discussed. TEC diurnal means compared with IRI-95 predictions for equinoxes and solstices months showed that IRI-95 systematically overestimates the observed values. Pre-midnight TEC enhancements were observed all over the year, except in May and June. The TEC values measured reproduce the same general trend of the TEC observations over Cachoeira Paulista, Brazil (22.5o S 45o W). The "fountain" effect seems to be more effective at Presidente Prudente because of its lower magnetic dip latitude. A correlation between TEC experimental values and low solar flux (62 < F10.7 < 116 flux units) emphasizes the inadequacy of IRI to model low latitude TEC values at low solar activity periods. These are the first results obtained using TEC-GPS technique for total electron content measureme n t s at low latitudes in Brazil.

  5. Anti-pseudomonas activity of essential oil, total extract, and proanthocyanidins of Pinus eldarica Medw. bark

    PubMed Central

    Sadeghi, Masoud; Zolfaghari, Behzad; Jahanian-Najafabadi, Ali; Abtahi, Seyed Reza

    2016-01-01

    Pinus eldarica Medw. (Iranian pine) is native to Transcaucasian region and has been vastly planted in Iran, Afghanistan, and Pakistan. Various parts of this plant have been widely used in traditional medicine for the treatment of various diseases including infectious conditions (e.g. infectious wounds). In this study we aimed to investigate the antibacterial activity of P. eldarica bark extract, essential oil and proanthocyanidins on three important bacteria, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Antibacterial analysis was performed using standard disk diffusion method with different concentrations of essential oil, bark total hydroalcoholic extract, and bark proanthocyanidins (0.5, 1, 2 and 3 mg/ml). After incubation at 37°C for 24 h, the antibacterial activity was assessed by measuring the zone of growth inhibition surrounding the disks. The results indicated that the essential oil, total hydroalcoholic extract, and proanthocyanidins of the bark of the P. eldarica were effective against the gram negative bacteria, P. aeruginosa, and significantly inhibited its growth in disk diffusion method (P<0.001) of which the essential oil had the most potent inhibitory effect. However, none of the bark preparations could significantly inhibit the growth of S. aureus or E. coli. Our findings showed that P. eldarica bark components have significant anti-pseudomonas activity having potentials for new sources of antibacterial agents or antibacterial herbal preparations. PMID:27051433

  6. V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

    PubMed Central

    Posch, Anton; Kohn, Jonathan; Oh, Kenneth; Hammond, Matt; Liu, Ning

    2013-01-01

    The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a "black box" because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as β-actin, β-tubulin, GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative and reliable. PMID:24429481

  7. Determination of secondary structural changes in gluten proteins during mixing using Fourier transform horizontal attenuated total reflectance spectroscopy.

    PubMed

    Seabourn, Bradford W; Chung, Okkyung K; Seib, Paul A; Mathewson, Paul R

    2008-06-11

    Fourier transform horizontal attenuated total reflectance (FT-HATR) was used to examine changes in the secondary structure of gluten proteins in a flour-water dough system during mixing. Midinfrared spectra of mixed dough revealed changes in four bands in the amide III region associated with secondary structure in proteins: 1317 (alpha-helix), 1285 (beta-turn), 1265 (random coil), and 1242 cm (-1) (beta-sheet). The largest band, which also showed the greatest change in second derivative band area (SDBA) during mixing, was located at 1242 cm (-1). The bands at 1317 and 1285 cm (-1) also showed an increase in SDBA over time. Conversely, the band at 1265 cm (-1) showed a corresponding decrease over time as the doughs were mixed. All bands reached an optimum corresponding to the minimum mobility of the dough as determined by the mixograph. Increases in alpha-helix, beta-turn, and beta-sheet secondary structures during mixing suggest that the dough proteins assume a more ordered conformation. These results demonstrate that it is possible, using infrared spectroscopic techniques, to relate the rheological behavior of developing dough in a mixograph directly to changes in the structure of the gluten protein system. PMID:18489117

  8. Antioxidant potential, cytotoxic activity and total phenolic content of Alpinia pahangensis rhizomes

    PubMed Central

    2013-01-01

    Background Alpinia pahangensis, a wild ginger distributed in the lowlands of Pahang, Malaysia, is used by the locals to treat flatulence. In this study, the antioxidant and cytotoxic activities of the crude aqueous methanol and fractionated extracts of Alpinia pahangensis against five different cancer and one normal cell lines were investigated. The total phenolic content of each extract and its fractions were also quantified. This is the first report on the antioxidant and cytotoxic activities of Alpinia pahangensis extract. Methods In the current study, the crude methanol and fractionated extract of the rhizomes of Alpinia pahangensis were investigated for their antioxidant activity using four different assays namely, the DPPH scavenging activity, superoxide anion scavenging, β-carotene bleaching and reducing power assays whilst their phenolic contents were measured by the Folin-Ciocalteu’s method. In vitro neutral red cytotoxicity assay was employed to evaluate the cytotoxic activity against five different cancer cell lines, colon cancer (HCT 116 and HT-29), cervical cancer (Ca Ski), breast cancer (MCF7) and lung cancer (A549) cell lines, and one normal cell line (MRC-5). The extract that showed high cytotoxic activity was further investigated for its chemical constituents by GC-MS (gas chromatography–mass spectrometry) analysis. Results The ethyl acetate fraction showed the strongest DPPH radical scavenging (0.35 ± 0.094 mg/ml) and SOD activities (51.77 ± 4.9%) whilst the methanol extract showed the highest reducing power and also the strongest antioxidant activity in the β-carotene bleaching assays in comparison to other fractions. The highest phenolic content was found in the ethyl acetate fraction, followed by the crude methanol extract, hexane and water fractions. The results showed a positive correlation between total phenolic content with DPPH radical scavenging capacities and SOD activities. The hexane fraction showed potent cytotoxic

  9. Contributions of non-occupational activities to total noise exposure of construction workers.

    PubMed

    Neitzel, Richard; Seixas, Noah; Goldman, Bryan; Daniell, William

    2004-07-01

    This paper describes how exposures received during routine and episodic non-occupational activities contribute to total noise exposure in a group of occupationally exposed workers. Two-hundred and sixty-six construction apprentices enrolled in a longitudinal hearing loss study and completed questionnaires at 1 yr of follow-up to determine their episodic activities (e.g. concert attendance, power tool use, firearms exposure). Noise exposure levels for these episodic exposures were determined from the published literature. Routine activities were assessed using activity cards filled out over 530 subject-days, along with noise dosimetry measurements made over 124 subject-days of measurement. Equivalent Leq exposure levels were then calculated for specific activities. These activity-specific Leq values were combined into estimated individual annual Leq exposure levels for the 6760 nominal annual non-occupational hours in a year (LAeq6760h), which were then transformed into equivalent levels for a 2000 h exposure period (LA2000hn) for comparison with occupational noise exposure risk criteria. The mean non-occupational LAeq6760h exposure values for the cohort ranged from 56 to 87 dBA (equivalent LA2000hn 62-93 dBA). At the mid range of the routine and episodic activity exposure level distribution, the mean LAeq6760h was 73 dBA (LA2000hn 78 dBA). Nineteen percent of the LA2000hn non-occupational exposures exceeded 85 dBA, the generally recommended occupational limit for a 2000 h workyear, at the mid-range of exposure levels. Due to a lack of available data, firearms use could not be incorporated into the total noise exposure estimates. However, firearms users reported more exposure to other noisy non-occupational activities and had statistically significantly higher estimated exposure levels even without including their firearms exposure than did non-shooters. When compared with the high levels of occupational noise found in construction, non-occupational noise exposures

  10. Notum deacylates Wnt proteins to suppress signalling activity.

    PubMed

    Kakugawa, Satoshi; Langton, Paul F; Zebisch, Matthias; Howell, Steven A; Chang, Tao-Hsin; Liu, Yan; Feizi, Ten; Bineva, Ganka; O'Reilly, Nicola; Snijders, Ambrosius P; Jones, E Yvonne; Vincent, Jean-Paul

    2015-03-12

    Signalling by Wnt proteins is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnt proteins from the cell surface. However, this view fails to explain specificity, as glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which probably help Notum to co-localize with Wnt proteins. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnt proteins and thus constitutes the first known extracellular protein deacylase. PMID:25731175

  11. Effect of Cell Phone Use on Salivary Total Protein, Enzymes and Oxidative Stress Markers in Young Adults: A Pilot Study

    PubMed Central

    Joy, Jasmi; Sunitha, Venkatesh; Rai, Manoj P.; Rao, Suresh; Nambranathayil, Shafeeque; Baliga, Manjeshwar Shrinath

    2015-01-01

    Introduction: The present study aimed to assess the levels of salivary enzymes, protein and oxidant-antioxidant system in young college-going cell phone users. Materials and Methods: The cell users (students) were categorized in to two groups – less mobile users and high mobile users, based on the duration and frequency of cell use. Unstimulated whole saliva samples of the volunteers were analysed for amylase, lactate dehydrogenase (LDH), malondialdehdye (MDA) and glutathione (GSH). Results: High mobile users had significantly higher levels of amylase (p = 0.001), LDH (p = 0.002) and MDA (p = 0.002) in saliva, when compared to less mobile users. The marginal decrease in salivary total proteins, GSH and flow rate were statistically not significant (p >0.05). Conclusion: Significant changes in salivary enzymes and MDA suggest adverse effect of high use of cell phones on cell health. PMID:25859446

  12. Antioxidant activity and total phenolic content of Boerhavia elegans (choisy) grown in Baluchestan, Iran

    PubMed Central

    Sadeghi, Zahra; Valizadeh, Jafar; Azyzian Shermeh, Omid; Akaberi, Maryam

    2015-01-01

    Objective: Boerhaavia elegans L. (Nyctaginaceae) is a medicinal plant used for the treatment of kidney disorders, urinary tract disorders and blood purification in Baluch tribe. The aim of present study is to evaluate the antioxidant property of B. elegans species for the first time. Materials and Methods: Different parts (leaf, stem and fruit) of the plant were extracted by using various solvents (water, methanol, chloroform and ethyl acetate) and evaluated for their antioxidant activity using DPPH (2, 2-diphenyl-1 picryl hydrazyl) and FRAP (ferric reducing antioxidant power) methods. In addition, total phenolic content was determined by Folin–Ciocalteu reagent. Results: Antioxidant results were expressed as IC50. The antioxidant power in DPPH and FRAP assay were evaluated as shown in decreasing order: Methanolic extract > Aqueous extract > Ethyl acetate extract > Chloroform extract, for all parts of the plant. In both methods of antioxidant assay and Folin-Ciocalteu method, methanolic extract of leaf exhibited the highest activity and the most phenolic content IC50= 6.85 ppm and 16.41 mg GA/g d w respectively. Total phenolic content had a positive relationship with antioxidant capacity in extracts and there was a high correlation (r=1.00, p<0.01) between antioxidant activities as determined by both antioxidant assays for various parts. Conclusion: The results of the experiments showed that B. elegans extract had significant antioxidant effects. This high antioxidant activity may be linked to phenolic contents of the plant but complementary investigations are suggested in order to determine active elements. PMID:25767751

  13. Effect of Drought Stress on Total Phenolic, Lipid Peroxidation, and Antioxidant Activity of Achillea Species.

    PubMed

    Gharibi, Shima; Tabatabaei, Badraldin Ebrahim Sayed; Saeidi, Ghodratollah; Goli, Sayed Amir Hossein

    2016-02-01

    The changes in total phenolic content (TPC), total flavonoid content (TFC), proline, malondialdehyde (MDA), H2O2, and antioxidant activity were assessed based on three model systems in three Achillea species (Achillea millefolium, A. nobilis, and A. filipendulina) growing under four irrigation regimes, including 100% FC (field capacity as normal irrigation) 75% FC (low stress), 50% FC (moderate stress), and 25% FC (severe stress) conditions. The highest TPC (47.13 mg tannic acid/g DW) and TFC (20.86 mg quercetin/g W) were obtained in A. filipendulina under moderate and severe stress conditions. In 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, the highest and the lowest antioxidant activity was obtained for A. millefolium (70.28%) and A. filipendulina (53.21%), respectively, while in the FTC model system A. nobilis revealed the highest antioxidant activity (1.934) in severe drought condition. In the linoleic model system, the highest antioxidant activity was observed under low drought stress condition in A. nobilis. MDA and H2O2 content were increased due to both low (75% FC) and moderate (50% FC) drought stress, but they were decreased under severe stress condition (25% FC). Furthermore, A. millefolium revealed the lowest H2O2 (4.96 nm/g FW) and MDA content (176.32 μmol/g). Investigation of the relationship among different metabolites showed a strong positive correlation with TPC and TFC. Finally, the moderate drought stress treatment (50% FC) was introduced as the optimum condition to obtain appreciable TPC and TFC,, while the highest antioxidant activity was obtained in severe stress condition (25%FC). PMID:26541161

  14. Fluorogen Activating Proteins in Flow Cytometry for the Study of Surface Molecules and Receptors

    PubMed Central

    Saunders, Matthew J.; Szent-Gyorgyi, Christopher; Fisher, Gregory W.; Jarvik, Jonathan W.; Bruchez, Marcel P.; Waggoner, Alan S.

    2012-01-01

    The use of fluorescent proteins, particularly when genetically fused to proteins of biological interest, have greatly advanced many flow cytometry research applications. However, there remains a major limitation to this methodology in that only total cellular fluorescence is measured. Commonly used fluorescent proteins (e.g. EGFP and its variants) are fluorescent whether the fusion protein exists on the surface or in sub-cellular compartments. A flow cytometer cannot distinguish between these separate sources of fluorescence. This can be of great concern when using flow cytometry, plate readers or microscopy to quantify cell surface receptors or other surface proteins genetically fused to fluorescent proteins. Recently developed fluorogen activating proteins (FAPs) solve many of these issues by allowing the selective visualization of only those cell surface proteins that are exposed to the extra cellular milieu. FAPs are GFP-sized single chain antibodies that specifically bind to and generate fluorescence from otherwise non-fluorescent dyes (‘activate the fluorogen’). Like the fluorescent proteins, FAPs can be genetically fused to proteins of interest. When exogenously added fluorogens bind FAPs, fluorescence immediately increases by as much as 20,000 fold, rendering the FAP fusion proteins highly fluorescent. Moreover, since fluorogens can be made membrane impermeant, fluorescence can be limited to only those receptors expressed on the cell surface. Using cells expressing beta-2 adrenergic receptor (β2AR) fused at its N-terminus to a FAP, flow cytometry based receptor internalization assays have been developed and characterized. The fluorogen/FAP system is ideally suited to the study of cell surface proteins by fluorescence and avoids drawbacks of using receptor/fluorescent protein fusions, such as internal accumulation. We also briefly comment on extending FAP-based technologies to the study of events occurring inside of the cell as well. PMID:22366230

  15. Determination of phytochemicals, antioxidant activity and total phenolic content in Andrographis paniculata using chromatographic methods.

    PubMed

    Kurzawa, Marzanna; Filipiak-Szok, Anna; Kłodzińska, Ewa; Szłyk, Edward

    2015-07-15

    Antioxidant activity, total phenolics content and selected phytochemicals (alkaloids and andrographolides) were determined in Andrographis paniculata and in dietary supplements containing this plant. Antioxidant activity was measured by FRAP, CUPRAC and DPPH procedures and ranged from 503.36 to 6164.09μmol TE/100g d.m. depending on methods, part of plant and kind of dietary supplement. The total phenolics (175.13-1723.79mg GAE/100g) and andrographolides content (19.44-85.13mg/g) in the studied samples were correlated with antioxidant activities determined by CUPRAC, FRAP and DPPH (r>0.95, p<0.05 level). Purine alkaloids: caffeine, theobromine, theophylline and indole alkaloids: harmine, harmane, harmol, yohimbine, brucine and strychnine were detected in the studied samples by different chromatographic techniques (HPLC-DAD, LC-MS/MS, GC-MS). The total alkaloids content in APs-roots and APs-leaves varies from 50.71±0.36mg/g d.m. to 78.71±0.48mg/g d.m., respectively, whereas for dietary supplements (Pn and DK) TAC was found between 19.52±0.15mg/g and 22.18±0.15mg/g d.m.. The highest concentration of andrographolides was found in A. paniculata leaves, whereas the lowest in dietary supplement Pn. Moreover principal component analysis, cluster analysis and one-way ANOVA follow by Duncan's tests were also performed. PMID:26050926

  16. Evaluation of antibacterial and anthelmintic activities with total phenolic contents of Piper betel leaves

    PubMed Central

    Akter, Kazi Nahid; Karmakar, Palash; Das, Abhijit; Anonna, Shamima Nasrin; Shoma, Sharmin Akter; Sattar, Mohammad Mafruhi

    2014-01-01

    Objective: The study was conducted to investigate the antibacterial and anthelmintic activities and to determine total phenolic contents of methanolic extract of Piper betel leaves. Materials and Methods: The extract was subjected to assay for antibacterial activity using both gram positive and gram negative bacterial strains through disc diffusion method; anthelmintic activity with the determination of paralysis and death time using earthworm (Pheritima posthuma) at five different concentrations and the determination of total phenolic contents using the Folin-ciocalteau method. Results: The extract showed significant (p<0.01) zone of inhibitions against gram positive Staphylococcus aureus [(6.77±0.25) mm] and Gram negative Escherichia coli [(8.53±0.25) mm], Salmonella typhi [(5.20±0.26) mm], Shigella dysenteriae [(11.20±0.26) mm] compared to positive control Azithromycin (ranging from 20.10±0.17 to 25.20±0.35 mm) while no zone inhibitory activity was found for both the extract and the standard drug against Gram positive Bacillus cereus. The extract also showed potent anthelmintic activity requiring less time for paralysis and death compared to the standard drug albendazole (10 mg/ml). At concentrations 10, 20, 40, 60 and 80 mg/ml, leaves extract showed paralysis at mean time of 9.83±0.60, 8.50±0.29, 6.60±0.17, 6.20±0.44 and 4.16±0.60; death at 11.33±0.88, 9.67±0.33, 7.83±0.17, 7.16±0.60 and 5.16±0.72 minutes, respectively. Whereas the standard drug showed paralysis and death at 19.33±0.71 and 51.00±0.23 minutes respectively. The extract confirmed the higher concentration of phenolic contents (124.42±0.14 mg of GAE /g of extract) when screened for total phenolic compounds. Conclusion: As results confirmed potential antibacterial and anthelmintic activities of Piper betel leaves extract, therefore it may be processed for further drug research. PMID:25386394

  17. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    NASA Astrophysics Data System (ADS)

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  18. Regulatory Crosstalk by Protein Kinases on CFTR Trafficking and Activity

    PubMed Central

    Farinha, Carlos M.; Swiatecka-Urban, Agnieszka; Brautigan, David L.; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e., channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease. PMID:26835446

  19. Activation of a human chromosomal replication origin by protein tethering

    PubMed Central

    Chen, Xiaomi; Liu, Guoqi; Leffak, Michael

    2013-01-01

    The specification of mammalian chromosomal replication origins is incompletely understood. To analyze the assembly and activation of prereplicative complexes (pre-RCs), we tested the effects of tethered binding of chromatin acetyltransferases and replication proteins on chromosomal c-myc origin deletion mutants containing a GAL4-binding cassette. GAL4DBD (DNA binding domain) fusions with Orc2, Cdt1, E2F1 or HBO1 coordinated the recruitment of the Mcm7 helicase subunit, the DNA unwinding element (DUE)-binding protein DUE-B and the minichromosome maintenance (MCM) helicase activator Cdc45 to the replicator, and restored origin activity. In contrast, replication protein binding and origin activity were not stimulated by fusion protein binding in the absence of flanking c-myc DNA. Substitution of the GAL4-binding site for the c-myc replicator DUE allowed Orc2 and Mcm7 binding, but eliminated origin activity, indicating that the DUE is essential for pre-RC activation. Additionally, tethering of DUE-B was not sufficient to recruit Cdc45 or activate pre-RCs formed in the absence of a DUE. These results show directly in a chromosomal background that chromatin acetylation, Orc2 or Cdt1 suffice to recruit all downstream replication initiation activities to a prospective origin, and that chromosomal origin activity requires singular DNA sequences. PMID:23658226

  20. Total Synthesis, Stereochemical Assignment, and Biological Activity of All Known (−)-Trigonoliimines

    PubMed Central

    Han, Sunkyu; Morrison, Karen C.; Hergenrother, Paul J.; Movassaghi, Mohammad

    2013-01-01

    A full account of our concise and enantioselective total syntheses of all known (−)-trigonoliimine alkaloids is described. Our retrobiosynthetic analysis of these natural products enabled identification of a single bistryptamine precursor as a precursor to all known trigonoliimines through a sequence of transformations involving asymmetric oxidation and reorganization. Our enantioselective syntheses of these alkaloids enabled the revision of the absolute stereochemistry of (−)-trigonoliimines A, B, and C. We report that trigonoliimines A, B, C, and structurally related compounds showed weak anticancer activities against HeLa and U-937 cells. PMID:24127681

  1. Total synthesis of leopolic acid A, a natural 2,3-pyrrolidinedione with antimicrobial activity.

    PubMed

    Dhavan, Atul A; Kaduskar, Rahul D; Musso, Loana; Scaglioni, Leonardo; Martino, Piera Anna; Dallavalle, Sabrina

    2016-01-01

    The first total synthesis of leopolic acid A, a fungal metabolite with a rare 2,3-pyrrolidinedione nucleus linked to an ureido dipeptide, was designed and carried out. Crucial steps for the strategy include a Dieckmann cyclization to obtain the 2,3-pyrrolidinedione ring and a Wittig olefination to install the polymethylene chain. An oxazolidinone-containing leopolic acid A analogue was also synthesized. The antibacterial activity showed by both compounds suggests that they could be considered as promising candidates for future developments. PMID:27559415

  2. Total synthesis of leopolic acid A, a natural 2,3-pyrrolidinedione with antimicrobial activity

    PubMed Central

    Dhavan, Atul A; Kaduskar, Rahul D; Musso, Loana; Scaglioni, Leonardo; Martino, Piera Anna

    2016-01-01

    Summary The first total synthesis of leopolic acid A, a fungal metabolite with a rare 2,3-pyrrolidinedione nucleus linked to an ureido dipeptide, was designed and carried out. Crucial steps for the strategy include a Dieckmann cyclization to obtain the 2,3-pyrrolidinedione ring and a Wittig olefination to install the polymethylene chain. An oxazolidinone-containing leopolic acid A analogue was also synthesized. The antibacterial activity showed by both compounds suggests that they could be considered as promising candidates for future developments. PMID:27559415

  3. A reliable non-separation fluorescence quenching assay for total glycated serum protein: a simple alternative to nitroblue tetrazolium reduction.

    PubMed

    Blincko, S; Colbert, D; John, W G; Edwards, R

    2000-05-01

    A simple non-separation assay for the measurement of total glycated serum protein is described. It was found that the fluorescence intensity of a solution of a fluorescein-boronic acid derivative was quenched in proportion to the amount of serum added. This led to the development of an assay in which 10 microL of serum is added to 4 mL of a solution of the fluorescein-boronic acid derivative and the fluorescence intensity is measured after 15 min. The results, as measured by drop in fluorescence intensity, calibrated by a single standard, were compared with the results for nitroblue tetrazolium (NBT) reduction of fructosamine and showed good correlation (r=0.936, n=114). The intra-assay precision (seven samples each measured 10 times) was less than 2.1% (concentration range 190-660 micromol/L); inter-assay precision for seven samples in 10 assays was less than 2.5% (over the same concentration range). Dilution of serum that had a high concentration of total glycated protein showed the assay to be linear. Serum samples (with low, medium and high total glycated protein concentrations) showed less than 2.1% difference from base results with added glucose (up to 60 mmol/L), less than 9.7% difference with added bilirubin (up to 250 micromol/L) and less than 6.9% with added triglycerides (up to 50 mmol/L). Addition of haemoglobin (up to 0.9 g/dL) with high glycation (11.7% HbA1c) to plasma (298 micromol/L total glycated protein) showed less than 10% difference from the base result. Assays performed over a range of temperatures (12-34 degrees C) showed no significant differences in the results. The assay gives similar results to the currently used NTB method but with significantly less susceptibility to interferences. As such the method should be a useful aid in the management of diabetes. PMID:10817254

  4. A New Method To Image Heme-Fe, Total Fe, and Aggregated Protein Levels after Intracerebral Hemorrhage

    PubMed Central

    Hackett, Mark J.; DeSouza, Mauren; Caine, Sally; Bewer, Brian; Nichol, Helen; Paterson, Phyllis G.; Colbourne, Frederick

    2015-01-01

    An intracerebral hemorrhage (ICH) is a devastating stroke that results in high mortality and significant disability in survivors. Unfortunately, the underlying mechanisms of this injury are not yet fully understood. After the primary (mechanical) trauma, secondary degenerative events contribute to ongoing cell death in the peri-hematoma region. Oxidative stress is thought to be a key reason for this delayed injury, which is likely due to free-Fe-catalyzed free radical reactions. Unfortunately, this is difficult to prove with conventional biochemical assays that fail to differentiate between alterations that occur within the hematoma and peri-hematoma zone. This is a critical limitation, as the hematoma contains tissue severely damaged by the initial hemorrhage and is unsalvageable, whereas the peri-hematoma region is less damaged but at risk from secondary degenerative events. Such events include oxidative stress mediated by free Fe presumed to originate from hemoglobin breakdown. Therefore, minimizing the damage caused by oxidative stress following hemoglobin breakdown and Fe release is a major therapeutic target. However, the extent to which free Fe contributes to the pathogenesis of ICH remains unknown. This investigation used a novel imaging approach that employed resonance Raman spectroscopic mapping of hemoglobin, X-ray fluorescence microscopic mapping of total Fe, and Fourier transform infrared spectroscopic imaging of aggregated protein following ICH in rats. This multimodal spectroscopic approach was used to accurately define the hematoma/peri-hematoma boundary and quantify the Fe concentration and the relative aggregated protein content, as a marker of oxidative stress, within each region. The results revealed total Fe is substantially increased in the hematoma (0.90 μg cm−2), and a subtle but significant increase in Fe that is not in the chemical form of hemoglobin is present within the peri-hematoma zone (0.32 μg cm−2) within 1 day of ICH

  5. A method for determination of unoxidized and total methionine in protein concentrates, with special reference to fish meals.

    PubMed

    Njaa, L R

    1980-03-01

    1. An automated colorimetric method for determination of methionine using an iodoplatinate reagent is described. Methionine sulphoxide does not react under the chosen conditions. 2. The method may be used to distinguish between unoxidized and total methionine by doing one determination without and one determination with previous reduction of a portion of the sample with titanium trichloride. Methionine sulphoxide is then obtained by difference. 3. The method has been used with protein concentrates, mainly fish meals, after hydrolysis with barium hydroxide. Interference from cysteine-cystine is eliminated by adding a small amount of cadmium acetate to the sample before hydrolysis. 4. Results obtained for total methionine and for methionine sulphoxide by independent methods show good agreement with results obtained with the iodoplatinate method. PMID:7378341

  6. Antioxidant activity and total phenolic content of 24 Lamiaceae species growing in Iran.

    PubMed

    Firuzi, Omidreza; Javidnia, Katayoun; Gholami, Maryam; Soltani, Mohammad; Miri, Ramin

    2010-02-01

    The antioxidant activities of the methanolic extracts of 9 Salvia species and 15 other Lamiaceae plants growing in Iran were evaluated using ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assays. FRAP values ranged form 8.5 to 79.0 microM quercetin equivalents/g dry weight, and IC50 values in the DPPH assay from 115.7 to 1350.2 microg dry weight/mL. Salvia species showed the highest antioxidant activities. S. santolinifolia, S. eremophila and S. palestina, which have not been studied before, were the most active plants. These were more active than the previously studied species from this family, such as S. multicaulis and Marrubium vulgare. S. hydrangea and Gontscharovia popovii also showed high antioxidant activities. FRAP and DPPH assay results showed good correlations with the total phenolic contents of the plants, measured by the Folin-Ciocalteau assay (r2 = 0.925 and 0.799, respectively, p < 0.0001). In conclusion, our study shows that some Lamiaceae plants growing in Iran represent good potential sources of natural antioxidants useful for either prevention or treatment of oxidative stress-related diseases. PMID:20334140

  7. Cellular reprogramming through mitogen-activated protein kinases

    PubMed Central

    Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression—including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes. PMID:26579181

  8. Cognitive Stimulation Modulates Platelet Total Phospholipases A2 Activity in Subjects with Mild Cognitive Impairment

    PubMed Central

    Balietti, Marta; Giuli, Cinzia; Fattoretti, Patrizia; Fabbietti, Paolo; Postacchini, Demetrio; Conti, Fiorenzo

    2016-01-01

    We evaluated the effect of cognitive stimulation (CS) on platelet total phospholipases A2 activity (tPLA2A) in patients with mild cognitive impairment (MCI_P). At baseline, tPLA2A negatively correlated with Mini-Mental State Examination score (MMSE_s): patients with MMSE_s <26 (Subgroup 1) had significantly higher activity than those with MMSE_s ≥26 (Subgroup 2), who had values similar to the healthy elderly. Regarding CS effect, Subgroup 1 had a significant tPLA2A reduction, whereas Subgroup 2 did not significantly changes after training. Our results showed for the first time that tPLA2A correlates with the cognitive conditions of MCI_P, and that CS acts selectively on subjects with a dysregulated tPLA2A. PMID:26836161

  9. G Protein Activation without a GEF in the Plant Kingdom

    PubMed Central

    Wang, Hao; Matthews, Melissa; Bradford, William; Bennetzen, Jeffrey L.; Jones, Alan M.

    2012-01-01

    Animal heterotrimeric G proteins are activated by guanine nucleotide exchange factors (GEF), typically seven transmembrane receptors that trigger GDP release and subsequent GTP binding. In contrast, the Arabidopsis thaliana G protein (AtGPA1) rapidly activates itself without a GEF and is instead regulated by a seven transmembrane Regulator of G protein Signaling (7TM-RGS) protein that promotes GTP hydrolysis to reset the inactive (GDP-bound) state. It is not known if this unusual activation is a major and constraining part of the evolutionary history of G signaling in eukaryotes. In particular, it is not known if this is an ancestral form or if this mechanism is maintained, and therefore constrained, within the plant kingdom. To determine if this mode of signal regulation is conserved throughout the plant kingdom, we analyzed available plant genomes for G protein signaling components, and we purified individually the plant components encoded in an informative set of plant genomes in order to determine their activation properties in vitro. While the subunits of the heterotrimeric G protein complex are encoded in vascular plant genomes, the 7TM-RGS genes were lost in all investigated grasses. Despite the absence of a Gα-inactivating protein in grasses, all vascular plant Gα proteins examined rapidly released GDP without a receptor and slowly hydrolyzed GTP, indicating that these Gα are self-activating. We showed further that a single amino acid substitution found naturally in grass Gα proteins reduced the Gα-RGS interaction, and this amino acid substitution occurred before the loss of the RGS gene in the grass lineage. Like grasses, non-vascular plants also appear to lack RGS proteins. However, unlike grasses, one representative non-vascular plant Gα showed rapid GTP hydrolysis, likely compensating for the loss of the RGS gene. Our findings, the loss of a regulatory gene and the retention of the “self-activating” trait, indicate the existence of divergent

  10. Oxidative Stress Impairs the Stimulatory Effect of S100 Proteins on Protein Phosphatase 5 Activity.

    PubMed

    Yamaguchi, Fuminori; Tsuchiya, Mitsumasa; Shimamoto, Seiko; Fujimoto, Tomohito; Tokumitsu, Hiroshi; Tokuda, Masaaki; Kobayashi, Ryoji

    2016-01-01

    Oxidative stress is the consequence of an imbalance between the production of harmful reactive oxygen species and the cellular antioxidant system for neutralization, and it activates multiple intracellular signaling pathways, including apoptosis signal-regulating kinase 1 (ASK1). Protein phosphatase 5 (PP5) is a serine/threonine phosphatase involved in oxidative stress responses. Previously, we reported that S100 proteins activate PP5 in a calcium-dependent manner. S100 proteins belong to a family of small EF-hand calcium-binding proteins involved in many processes such as cell proliferation, differentiation, apoptosis, and inflammation. Therefore, we investigated the effects of oxidative stress on S100 proteins, their interaction with PP5, and PP5 enzyme activity. Recombinant S100A2 was easily air-oxidized or Cu-oxidized, and oxidized S100A2 formed cross-linked dimers and higher molecular-mass complexes. The binding of oxidized S100A2 to PP5 was reduced, resulting in decreased PP5 activation in vitro. Oxidation also impaired S100A1, S100A6, S100B, and S100P to activate PP5, although the low dose of oxidized S100 proteins still activated PP5. Hydrogen peroxide (H2O2) induced S100A2 oxidation in human keratinocytes (HaCaT) and human hepatocellular carcinoma (Huh-7) cells. Furthermore, H2O2 reduced the binding of S100A2 to PP5 and decreased PP5 activation in HaCaT and Huh-7 cells. Importantly, even the low dose of S100A2 achieved by knocking down increased dephosphorylation of ASK1 and reduced caspase 3/7 activity in Huh-7 cells treated with H2O2. These results indicate that oxidative stress impairs the ability of S100 proteins to bind and activate PP5, which in turn modulates the ASK1-mediated signaling cascades involved in apoptosis. PMID:27600583

  11. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    PubMed

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins. PMID:27080133

  12. Clinical Outcomes of Anatomical Total Shoulder Arthroplasty in a Young, Active Population.

    PubMed

    Kusnezov, Nicholas; Dunn, John C; Parada, Stephen A; Kilcoyne, Kelly; Waterman, Brian R

    2016-01-01

    Glenohumeral arthritis in young, active patients poses many treatment challenges, and significant concerns about component loosening and failure limit the available surgical options. We conducted a study of the clinical outcomes of total shoulder arthroplasty (TSA) for glenohumeral arthritis in a young, high-demand population. We searched the Military Health System Management Analysis and Reporting Tool database to retrospectively review the cases of all US military service members who had undergone anatomical TSA (Current Procedural Terminology code 23472) between 2007 and 2014. Demographic information, occupational parameters, and clinical outcomes were extracted from electronic medical records. Twenty-four service members (26 shoulders) met the inclusion criteria. The cohort was predominantly male (n = 25). Mean age was 45.8 years (range, 35-54 years). The most common etiology of glenohumeral arthritis was post-instability arthropathy (50.0%). At mean follow-up of 41 months, 9 patients had a total of 12 complications (46.2%), including 6 component failures caused by neurologic injury (2 cases), adhesive capsulitis (2), and venous thrombosis (2). The reoperation rate for all component failures was 23.1% (6 cases, 5 patients). Ten patients (41.7%) remained on active duty at 2 years, and 5 (20.8%) were subsequently deployed. Ultimately, 9 patients (37.5%) underwent medical discharge for persistent shoulder disability. TSA in young, active patients provides reliable improvements in range of motion and pain. However, roughly one-third of patients in this study were unable to continue high-demand activities by 2 years after surgery. The short-term complication profile (46.2%) and reoperation rate for component failure (23.1%) should be emphasized during preoperative counseling. PMID:27552465

  13. Biologically active protein fragments containing specific binding regions of serum albumin or related proteins

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor)

    1998-01-01

    In accordance with the present invention, biologically active protein fragments can be constructed which contain only those specific portions of the serum albumin family of proteins such as regions known as subdomains IIA and IIIA which are primarily responsible for the binding properties of the serum albumins. The artificial serums that can be prepared from these biologically active protein fragments are advantageous in that they can be produced much more easily than serums containing the whole albumin, yet still retain all or most of the original binding potential of the full albumin proteins. In addition, since the protein fragment serums of the present invention can be made from non-natural sources using conventional recombinant DNA techniques, they are far safer than serums containing natural albumin because they do not carry the potentially harmful viruses and other contaminants that will be found in the natural substances.

  14. Thymic Stromal Lymphopoietin Promotes Fibrosis and Activates Mitogen-Activated Protein Kinases in MRC-5 Cells

    PubMed Central

    Li, Li; Tang, Su; Tang, Xiaodong

    2016-01-01

    Background Acute lung injury (ALI) is a life-threatening hypoxemic respiratory disorder with high incidence and mortality. ALI usually manifests as widespread inflammation and lung fibrosis with the accumulation of pro-inflammatory and pro-fibrotic factors and collagen. Thymic stromal lymphopoietin (TSLP) has a significant role in regulation of inflammation but little is known about its roles in lung fibrosis or ALI. This study aimed to define the role and possible regulatory mechanism of TSLP in lung fibrosis. Material/Methods We cultured human lung fibroblast MRC-5 cells and overexpressed or inhibited TSLP by the vector or small interfering RNA transfection. Then, the pro-fibrotic factors skeletal muscle actin alpha (α-SMA) and collagen I, and the 4 mitogen-activated protein kinases (MAPKs) – MAPK7, p38, extracellular signal-regulated kinase 1 (ERK1), and c-Jun N-terminal kinase 1 (JNK1) – were detected by Western blot. Results Results showed that TSLP promoted the production of α-SMA and collagen I (P<0.001), suggesting that it can accelerate MRC-5 cell fibrosis. It also activated the expression of MAPK7, p-p38, p-ERK1, and p-JNK1, but the total MAPK7, p-38, ERK1, and JNK1 protein levels were mostly unchanged, indicating the activated MAPK pathways that might contribute to the promotion of cell fibrosis. Conclusions This study shows the pro-fibrotic role of TSLP in MRC-5 cells, suggesting TSLP is a potential therapeutic target for treating lung fibrosis in ALI. It possibly functions via activating MAPKs. These findings add to our understanding of the mechanism of fibrosis. PMID:27385084

  15. Thymic Stromal Lymphopoietin Promotes Fibrosis and Activates Mitogen-Activated Protein Kinases in MRC-5 Cells.

    PubMed

    Li, Li; Tang, Su; Tang, Xiaodong

    2016-01-01

    BACKGROUND Acute lung injury (ALI) is a life-threatening hypoxemic respiratory disorder with high incidence and mortality. ALI usually manifests as widespread inflammation and lung fibrosis with the accumulation of pro-inflammatory and pro-fibrotic factors and collagen. Thymic stromal lymphopoietin (TSLP) has a significant role in regulation of inflammation but little is known about its roles in lung fibrosis or ALI. This study aimed to define the role and possible regulatory mechanism of TSLP in lung fibrosis. MATERIAL AND METHODS We cultured human lung fibroblast MRC-5 cells and overexpressed or inhibited TSLP by the vector or small interfering RNA transfection. Then, the pro-fibrotic factors skeletal muscle actin alpha (α-SMA) and collagen I, and the 4 mitogen-activated protein kinases (MAPKs) - MAPK7, p38, extracellular signal-regulated kinase 1 (ERK1), and c-Jun N-terminal kinase 1 (JNK1) - were detected by Western blot. RESULTS Results showed that TSLP promoted the production of α-SMA and collagen I (P<0.001), suggesting that it can accelerate MRC-5 cell fibrosis. It also activated the expression of MAPK7, p-p38, p-ERK1, and p-JNK1, but the total MAPK7, p-38, ERK1, and JNK1 protein levels were mostly unchanged, indicating the activated MAPK pathways that might contribute to the promotion of cell fibrosis. CONCLUSIONS This study shows the pro-fibrotic role of TSLP in MRC-5 cells, suggesting TSLP is a potential therapeutic target for treating lung fibrosis in ALI. It possibly functions via activating MAPKs. These findings add to our understanding of the mechanism of fibrosis. PMID:27385084

  16. Liposomal packaging generates Wnt protein with in vivo biological activity.

    PubMed

    Morrell, Nathan T; Leucht, Philipp; Zhao, Ludan; Kim, Jae-Beom; ten Berge, Derk; Ponnusamy, Karthik; Carre, A Lyonel; Dudek, Henryk; Zachlederova, Marie; McElhaney, Michael; Brunton, Shirley; Gunzner, Janet; Callow, Marinella; Polakis, Paul; Costa, Mike; Zhang, Xiaoyan M; Helms, Jill A; Nusse, Roel

    2008-01-01

    Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context. PMID:18698373

  17. Antimicrobial activities of rhizomes of Polygonatum verticillatum: attributed to its total flavonoidal and phenolic contents.

    PubMed

    Khan, Haroon; Saeed, Muhammad; Muhammad, Naveed; Ghaffar, Rukhsana; Khan, Saeed Ahmad; Hassan, Sohail

    2012-04-01

    The current study was undertaken to evaluate the rhizomes of Polygonatum verticillatum against various pathogenic bacteria and fungi. Broad spectrum antibacterial activity was demonstrated by the crude extract of the plant and its subsequent solvent fractions; predominantly against Gram-negative bacteria. MICs of the extracts against Escherchia coli, Salmonella typhi and Shigella flexeneri were in the range of 1.5-40 μg/ml, 03-06 μg/ml and 03-40 μg/ml, respectively. The only sensitive Gram-positive bacterium was Staphylococcus aureus with MICs in the range of 75-80 μg/ml. The fungicidal activity was limited to Microspoum canis and Fusarium solani and the MICs were in the range of 350-360 μg/ml and 190-290 μg/ml respectively. The various fractions of rhizomes contained significant concentration of total flavonoidal and total phenolic contents that could be responsible for the current findings. PMID:22459478

  18. Activation of protein kinase C induces mitogen-activated protein kinase dephosphorylation and pronucleus formation in rat oocytes.

    PubMed

    Lu, Qing; Smith, Gary D; Chen, Da-Yuan; Han, Zhi-Ming; Sun, Qing-Yuan

    2002-07-01

    Mammalian oocytes are arrested at metaphase of the second meiotic division (MII) before fertilization. When oocytes are stimulated by spermatozoa, they exit MII stage and complete meiosis. It has been suggested that an immediate increase in intracellular free calcium concentration and inactivation of maturation promoting factor (MPF) are required for oocyte activation. However, the underlying mechanism is still unclear. In the present study, we investigated the role of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase, and their interplay in rat oocyte activation. We found that MAP kinase became dephosphorylated in correlation with pronucleus formation after fertilization. Protein kinase C activators, phorbol 12-myriatate 13-acetate (PMA) and 1,2-dioctanoyl-rac-glycerol (diC8), triggered dephosphorylation of MAP kinase and pronucleus formation in a dose-dependent and time-dependent manner. Dephosphorylation of MAP kinase was also correlated with pronucleus formation when oocytes were treated with PKC activators. Effects of PKC activators were abolished by the PKC inhibitors, calphostin C and staurosporine, as well as a protein phosphatase blocker, okadaic acid (OA). These results suggest that PKC activation may cause rat oocyte pronucleus formation via MAP kinase dephosphorylation, which is probably mediated by OA-sensitive protein phosphatases. We also provide evidence supporting the involvement of such a process in fertilization. PMID:12080000

  19. Comparison of total protein and enzyme levels in successive regenerations of venom from individual coralsnakes.

    PubMed

    Kopper, Randall A; Harper, George R; Occidental, Michael; Gamalie, Vlad; Spradley, Ples

    2015-07-01

    Coralsnakes produce highly potent neurotoxic venoms, but little is known about variations in specific enzyme components within a species or from one replenishment of venom to the next within the same animal. Since published studies are often conducted using venom pools from multiple snakes, individual differences are masked and variations among individual snakes and between subsequent venom regenerations from the same snake have rarely been documented. This study involves the analysis and comparison of four successive venom collections from each of nine individual coralsnakes in order to detect these differences. Significant variation was found within the successive re-synthesis of venom components. Even greater differences were observed between the venoms from similar individual snakes. Since studies of variation in enzymatic activity would be significant only if they were above these normal variations, it is important to be aware of these differences. These results suggest the importance of understanding the variations present within and between individuals of the same species when interpreting the potential significance of differences found as the result of genetic, environmental or ecological factors. PMID:25935458

  20. Energy transfer at the active sites of heme proteins

    SciTech Connect

    Dlott, D.D.; Hill, J.R.

    1995-12-31

    Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes.

  1. Variability of total electron content over an equatorial West African station during low solar activity

    NASA Astrophysics Data System (ADS)

    Bolaji, O. S.; Adeniyi, J. O.; Radicella, S. M.; Doherty, P. H.

    2012-01-01

    Slant total electron content (STEC) data measured by the Global Positioning System receiver at Ilorin, Nigeria, with geographical coordinates 8.47°N, 4.68°E for the year 2009 (a low-activity year) was used to study the diurnal, monthly standard deviation and monthly median value of total electron content (TEC). The vertical total electron content (VTEC) values are estimated from the STEC data. The thin shell approximation with an ionospheric shell height of 350 km was used for the analysis. The diurnal variation of VTEC (DTEC) and its corresponding monthly median variation (MTEC) shows a minimum at presunrise between the hours of 05:00 and 06:00 LT. The DTEC values show a maximum variation range from ˜24 to ˜34 total electron content unit (TECU). The daytime maximum TEC values observed in all the months were broad with a slight daytime depression in May, June, July, and November. The maximum variation of MTEC after slight daytime depression is greater than its variation before the slight daytime depression in the months affected with the month of July as exception. The slight daytime depression was lowest in the month of May and has a value of 0.99 TECU. A postsunset decrease at 20:00 LT with corresponding enhancement 2 h later was observed in the month of March. This post sunset decrease and enhancement in the month of March could be a strong indicator of the abrupt onset of scintillations, plasma bubbles, and spread F phenomenon. The monthly standard deviation depicts summary behavior of all the diurnal variations in each month. Annual and seasonal variations were also investigated.

  2. Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

    PubMed

    Nagasawa, Hideko; Uto, Yoshihiro; Sasaki, Hideyuki; Okamura, Natsuko; Murakami, Aya; Kubo, Shinichi; Kirk, Kenneth L; Hori, Hitoshi

    2005-01-01

    The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research. PMID:16302727

  3. Counteracting Protein Kinase Activity in the Heart: The Multiple Roles of Protein Phosphatases

    PubMed Central

    Weber, Silvio; Meyer-Roxlau, Stefanie; Wagner, Michael; Dobrev, Dobromir; El-Armouche, Ali

    2015-01-01

    Decades of cardiovascular research have shown that variable and flexible levels of protein phosphorylation are necessary to maintain cardiac function. A delicate balance between phosphorylated and dephosphorylated states of proteins is guaranteed by a complex interplay of protein kinases (PKs) and phosphatases. Serine/threonine phosphatases, in particular members of the protein phosphatase (PP) family govern dephosphorylation of the majority of these cardiac proteins. Recent findings have however shown that PPs do not only dephosphorylate previously phosphorylated proteins as a passive control mechanism but are capable to actively control PK activity via different direct and indirect signaling pathways. These control mechanisms can take place on (epi-)genetic, (post-)transcriptional, and (post-)translational levels. In addition PPs themselves are targets of a plethora of proteinaceous interaction partner regulating their endogenous activity, thus adding another level of complexity and feedback control toward this system. Finally, novel approaches are underway to achieve spatiotemporal pharmacologic control of PPs which in turn can be used to fine-tune misleaded PK activity in heart disease. Taken together, this review comprehensively summarizes the major aspects of PP-mediated PK regulation and discusses the subsequent consequences of deregulated PP activity for cardiovascular diseases in depth. PMID:26617522

  4. Carotenoids, total polyphenols and antioxidant activity of grapes (Vitis vinifera) cultivated in organic and conventional systems

    PubMed Central

    2012-01-01

    Background Organic agriculture involve plants which are cultivated without using synthetic pesticides, herbicides or fertilizers and promotes biodiversity, biological cycles and improve the product quality. The carotenoids, total polyphenols and the antioxidant activity from skins of some wine and table grapes cultivated in organic and conventional agriculture were studied. Results The main carotenoids identified using high performance liquid chromatography were lutein and ß-carotene. Muscat Ottonel variety has the highest ß-carotene concentration 504.9 μg/kg for organic and 593.2 μg/kg for conventional grapes. For the organic farming, the total polyphenols content were in the range of 163.23 – 1341.37 mg GAE/kg fresh weight (FW) and 148.47 – 1231.38 mg GAE/kg FW for the conventional grapes. The highest ORAC values were obtained for blue-black variety Napoca in both farming system (43.5 ± 0.95 μmol TE/g organic; 40.4 ± 0.5 μmol TE/g conventional) and lowest for Aromat de Iaşi (16.8 ± 0.6 μmol TE/g organic; 14.7 ± 1.6 μmol TE/g conventional). Napoca variety showed also the highest antioxidant activity measured by DPPH method in both cultivated system. Conclusion Nine grape varieties cultivated in organic and conventional systems were compared regarding the carotenoids, total polyphenols and antioxidant activity. The white grape varieties have a higher carotenoids content compared with the blue-black cultivars while the blue-black varieties contain higher TPC and exhibit higher antioxidant activity (except for Muscat Hamburg-ORAC). Vitis vinifera grape skins originating from wine or table grape varieties can be used as a potential source of natural antioxidants. PMID:22762349

  5. Activities of the Sex-lethal protein in RNA binding and protein:protein interactions.

    PubMed Central

    Samuels, M; Deshpande, G; Schedl, P

    1998-01-01

    The Drosophila sex determination gene Sex-lethal (Sxl) controls its own expression, and the expression of downstream target genes such as transformer , by regulating pre-mRNA splicing and mRNA translation. Sxl codes an RNA-binding protein that consists of an N-terminus of approximately 100 amino acids, two 90 amino acid RRM domains, R1 and R2, and an 80 amino acid C-terminus. In the studies reported here we have examined the functional properties of the different Sxl protein domains in RNA binding and in protein:protein interactions. The two RRM domains are responsible for RNA binding. Specificity in the recognition of target RNAs requires both RRM domains, and proteins which consist of the single domains or duplicated domains have anomalous RNA recognition properties. Moreover, the length of the linker between domains can affect RNA recognition properties. Our results indicate that the two RRM domains mediate Sxl:Sxl protein interactions, and that these interactions probably occur both in cis and trans. We speculate that cis interactions between R1 and R2 play a role in RNA recognition by the Sxl protein, while trans interactions stabilize complex formation on target RNAs that contain two or more closely spaced binding sites. Finally, we show that the interaction of Sxl with the snRNP protein Snf is mediated by the R1 RRM domain. PMID:9592147

  6. Heated Proteins are Still Active in a Functionalized Nanoporous Support

    SciTech Connect

    Chen, Baowei; Qi, Wen N.; Li, Xiaolin; Lei, Chenghong; Liu, Jun

    2013-07-08

    We report that even under the heated condition, the conformation and activity of a protein can be hoarded in a functionalized nanoporous support via non-covalent interaction, although the hoarded protein was not exhibiting the full protein activity, the protein released subsequently still maintained its native conformation and activity. Glucose oxidase (GOX) was spontaneously and largely entrapped in aminopropyl-functionalized mesoporous silica (NH2-FMS) at 20 oC via a dominant electrostatic interaction. Although FMS-GOX displayed 45% activity of the free enzyme in solution, the GOX released from FMS exhibited its 100% activity prior to the entrapment. Surprisingly, the released GOX from FMS still maintained 89% of its initial activity prior to the entrapment after FMS-GOX was incubated at 60 oC for 1 h prior to release, while the free GOX in solution lost nearly all activity under the same incubation. Intrinsic fluorescence emission of GOX and native electrophoresis demonstrated that the heating resulted in significant conformational changes and oligomeric structures of the free GOX, but FMS efficiently maintained the thermal stability of GOX therein and resisted the thermal denaturation and oligomeric aggregation.

  7. Probing heterotrimeric G protein activation: applications to biased ligands

    PubMed Central

    Denis, Colette; Saulière, Aude; Galandrin, Ségolène; Sénard, Jean-Michel; Galés, Céline

    2012-01-01

    Cell surface G protein-coupled receptors (GPCRs) drive numerous signaling pathways involved in the regulation of a broad range of physiologic processes. Today, they represent the largest target for modern drugs development with potential application in all clinical fields. Recently, the concept of “ligand-directed trafficking” has led to a conceptual revolution in pharmacological theory, thus opening new avenues for drug discovery. Accordingly, GPCRs do not function as simple on-off switch but rather as filters capable of selecting activation of specific signals and thus generating textured responses to ligands, a phenomenon often referred to as ligand-biased signaling. Also, one challenging task today remains optimization of pharmacological assays with increased sensitivity so to better appreciate the inherent texture of ligand responses. However, considering that a single receptor has pleiotropic signalling properties and that each signal can crosstalk at different levels, biased activity remains thus difficult to evaluate. One strategy to overcome these limitations would be examining the initial steps following receptor activation. Even if some G protein-independent functions have been recently described, heterotrimeric G protein activation remains a general hallmark for all GPCRs families and the first cellular event subsequent to agonist binding to the receptor. Herein, we review the different methodologies classically used or recently developed to monitor G protein activation and discuss them in the context of G protein biased -ligands. PMID:22229559

  8. Bryostatins activate protein kinase C in intact human platelets

    SciTech Connect

    Smith, J.B.; Tallant, E.A.; Pettit, G.R.; Wallace, R.W.

    1986-05-01

    Bryostatins, macrocyclic lactones isolated from a marine bryozoan, have antineoplastic activity in the P388 lymphocytic leukemia system. These compounds also stimulate growth in Swiss 3T3 cells, induce secretion in leukocytes, inhibit phorbol dibutyrate binding to a high affinity receptor, and activate the C-kinase in vitro. In human platelets, phorbol esters induce aggregation and activate protein kinase C, resulting in phosphorylation of a 47K protein and the 20K myosin light chain. The authors now show that bryostatin 7 (B-7) triggers platelet aggregation to the same rate and extent as phorbol 12-myristate 13-acetate (PMA). B-7 also causes the in vivo activation of the C-kinase, resulting in phosphorylation of both the 47K and the 20K proteins; the time courses and dose-responses of these B-7-induced phosphorylations were similar to those found with PMA. In addition, B-7 increases the level of /sup 32/P-incorporation into the platelet polyphosphoinositides, which also occurs in response to PMA. Bryostatin 3 (B-3), which has been shown to be much less potent than B-7 in mimicking other PMA effects, was much less effective than PMA or B-7 in inducing platelet aggregation and in stimulating /sup 32/P-incorporation into both proteins and the phosphoinositides. These results demonstrate that, intact human platelets, bryostatins mimic the phorbol esters tumor promoters and directly activate protein kinase C.

  9. Probing heterotrimeric G protein activation: applications to biased ligands.

    PubMed

    Denis, Colette; Saulière, Aude; Galandrin, Segolene; Sénard, Jean-Michel; Galés, Céline

    2012-01-01

    Cell surface G protein-coupled receptors (GPCRs) drive numerous signaling pathways involved in the regulation of a broad range of physiologic processes. Today, they represent the largest target for modern drugs development with potential application in all clinical fields. Recently, the concept of "ligand-directed trafficking" has led to a conceptual revolution in pharmacological theory, thus opening new avenues for drug discovery. Accordingly, GPCRs do not function as simple on-off switch but rather as filters capable of selecting the activation of specific signals and thus generating texture responses to ligands, a phenomenon often referred to as ligand-biased signaling. Also, one challenging task today remains optimization of pharmacological assays with increased sensitivity so to better appreciate the inherent texture of ligands. However, considering that a single receptor has pleiotropic signaling properties and that each signal can crosstalk at different levels, biased activity remains thus difficult to evaluate. One strategy to overcome these limitations would be examining the initial steps following receptor activation. Even, if some G protein independent functions have been recently described, heterotrimeric G protein activation remains a general hallmark for all GPCRs families and the first cellular event subsequent to agonist binding to the receptor. Herein, we review the different methodologies classically used or recently developed to monitor G protein activation and discussed them in the context of G protein biased-ligands. PMID:22229559

  10. Determination of fungal activity in modified wood by means of micro-calorimetry and determination of total esterase activity

    PubMed Central

    Verma, Pradeep; Dyckmans, Jens; Militz, Holger

    2008-01-01

    Beech and pine wood blocks were treated with 1,3-dimethylol-4,5-dihydroxyethylen urea (DMDHEU) to increasing weight percent gains (WPG). The resistance of the treated specimens against Trametes versicolor and Coniophora puteana, determined as mass loss, increased with increasing WPG of DMDHEU. Metabolic activity of the fungi in the wood blocks was assessed as total esterase activity (TEA) based on the hydrolysis of fluorescein diacetate and as heat or energy production determined by isothermal micro-calorimetry. Both methods revealed that the fungal activity was related with the WPG and the mass loss caused by the fungi. Still, fungal activity was detected even in wood blocks of the highest WPG and showed that the treatment was not toxic to the fungi. Energy production showed a higher consistency with the mass loss after decay than TEA; higher mass loss was more stringently reflected by higher heat production rate. Heat production did not proceed linearly, possibly due to the inhibition of fungal activity by an excess of carbon dioxide. PMID:18542949

  11. Subcellular and single-molecule imaging of plant fluorescent proteins using total internal reflection fluorescence microscopy (TIRFM)

    PubMed Central

    Vizcay-Barrena, Gema; Webb, Stephen E. D.; Martin-Fernandez, Marisa L.; Wilson, Zoe A.

    2011-01-01

    Total internal reflection fluorescence microscopy (TIRFM) has been proven to be an extremely powerful technique in animal cell research for generating high contrast images and dynamic protein conformation information. However, there has long been a perception that TIRFM is not feasible in plant cells because the cell wall would restrict the penetration of the evanescent field and lead to scattering of illumination. By comparative analysis of epifluorescence and TIRF in root cells, it is demonstrated that TIRFM can generate high contrast images, superior to other approaches, from intact plant cells. It is also shown that TIRF imaging is possible not only at the plasma membrane level, but also in organelles, for example the nucleus, due to the presence of the central vacuole. Importantly, it is demonstrated for the first time that this is TIRF excitation, and not TIRF-like excitation described as variable-angle epifluorescence microscopy (VAEM), and it is shown how to distinguish the two techniques in practical microscopy. These TIRF images show the highest signal-to-background ratio, and it is demonstrated that they can be used for single-molecule microscopy. Rare protein events, which would otherwise be masked by the average molecular behaviour, can therefore be detected, including the conformations and oligomerization states of interacting proteins and signalling networks in vivo. The demonstration of the application of TIRFM and single-molecule analysis to plant cells therefore opens up a new range of possibilities for plant cell imaging. PMID:21865179

  12. Determination of total protein content in white wines by solid phase spectrometry in a SI-LOV system.

    PubMed

    Vidigal, Susana S M P; Tóth, Ildikó V; Rangel, António O S S

    2012-07-15

    Although present at low concentration in wine samples, proteins, have considerable technological importance, due to their capability of haze formation. The present work presents a methodology for the quantification of total protein in white wine in a sequential injection lab-on-valve system, exploiting the bead injection concept for solid phase extraction with spectrophotometric detection. The method is based on the retention of the proteins in the solid support, NTA (nitrilotriacetic acid) superflow beads, charged by Cu(2+). The change in the absorbance is monitored at 500nm at the surface of the beads after addition of the Folin-Ciocalteu's reagent (FCr). The developed method presented a sample consumption of 400μL per assay and a consumption of FCr and Cu(2+) solution of 25μL and 100μL per assay, respectively. It was possible to achieve a linear range up to 0.30g/L with a limit of detection and quantification of 0.03 and 0.10g/L, respectively. The proposed method was successfully applied to white wine samples. PMID:22817935

  13. NALP3 inflammasome activation in protein misfolding diseases.

    PubMed

    Shi, Fushan; Kouadir, Mohammed; Yang, Yang

    2015-08-15

    Protein-misfolding diseases, such as Alzheimer's disease, type 2 diabetes, Prion diseases, and Parkinson's disease, are characterized by inflammatory reactions. In all these diseases, IL-1β (Interlukine-1β) has been shown to be an important regulator, and the misfolded proteins are proved to be triggers of the release of IL-1β. Recently, several reports demonstrated that the inflammasome activation is involved in the progress of the misfolded protein diseases, and that the inflammasome can recognize pathogenic proteins leading to the release of IL-1β. In this review, we discuss the role of inflammasome in the pathogenesis of misfolded protein diseases and the potential of inflammasome-targeting therapeutic interventions in the management of these diseases. PMID:26037399

  14. Effect of storage time and temperature on the total protein concentration and electrophoretic fractions in equine serum

    PubMed Central

    Alberghina, Daniela; Casella, Stefania; Giannetto, Claudia; Marafioti, Simona; Piccione, Giuseppe

    2013-01-01

    Serum protein electrophoresis (SPE) is a technique that could be considered one of the most useful diagnostic aids available to the clinician. The effect of storage time and temperature on the total proteins and electrophoretic fractions (albumin, α1-, α2-, β1-, β2-, and γ-globulins) was assessed in 24 healthy horses. All samples, collected by jugular vein puncture, were centrifuged and divided into 4 aliquots. The 1st aliquot was analyzed within 3 h from collection (time 0), the 2nd was refrigerated at +4°C for 24 h, the 3rd was refrigerated at +4°C for 48 h, and the last was frozen at −20°C for 48 h. One-way repeated-measures analysis of variance (ANOVA) showed a significant effect (P < 0.05) of the different storage conditions on the concentrations of all the parameters studied and significant variations in the percentages of albumin, α1-globulins, α2-globulins, and γ-globulins. Compared with time 0 the total protein concentration increased significantly after 48 h at −20°C, the albumin percentage decreased after 48 h at −20°C, the α1-globulin percentage increased after 24 h at +4°C, the α2-globulin percentage increased after 48 h at +4°C and at −20°C, and the γ-globulin percentage increased after 48 h at −20°C. The results should help veterinary practitioners handle and store equine serum samples appropriately. Further investigations at different storage times and temperatures could be useful. PMID:24124272

  15. Alterations in lower limb multimuscle activation patterns during stair climbing in female total knee arthroplasty patients.

    PubMed

    Kuntze, G; von Tscharner, V; Hutchison, C; Ronsky, J L

    2015-11-01

    Total knee arthroplasty (TKA) patients commonly experience neuromuscular adaptations that may affect stair climbing competence. This study identified multimuscle pattern (MMP) changes in postoperative female TKA patients during stair climbing with a support vector machine (SVM). It was hypothesized that TKA patients adopt temporal and spectral muscle activation characteristics indicative of muscle atrophy and cocontraction strategies. Nineteen female subjects [10 unilateral sex-specific TKAs, 62.2 ± 8.6 yr, body mass index (BMI) 28.2 ± 5.4 kg/m(2); 9 healthy control subjects, 61.4 ± 7.4 yr, BMI 25.6 ± 2.4 kg/m(2)] were recruited. Surface electromyograms (EMGs) were obtained for seven lower limb muscles of the affected limb of TKA subjects and a randomly assigned limb for control subjects during stair climbing. Stance phase (±30%) EMG data were wavelet transformed and normalized to total power. Data across all muscles were combined to form MMPs and analyzed with a SVM. Statistical analysis was performed with binomial tests, independent group t-tests, or independent group Mann-Whitney U-tests in SPSS (P < 0.05). SVM results indicated significantly altered muscle activation patterns in the TKA group for biceps femoris (recognition rate 84.2%), semitendinosus (recognition rate 73.7%), gastrocnemius (recognition rate 68.4%), and tibialis anterior (recognition rate 68.4%). Further analysis identified no significant differences in spectral activation characteristics between groups. Temporal adaptations, indicative of cocontraction strategies, were, however, evident in TKA MMPs. This approach may provide a valuable tool for clinical neuromuscular function assessment and rehabilitation monitoring. PMID:26354313

  16. Peptides and proteins with antimicrobial activity

    PubMed Central

    Coutinho, Henrique Douglas Melo; Lôbo, Katiuscia Menezes; Bezerra, Denise Aline Casimiro; Lôbo, Inalzuir

    2008-01-01

    The increase of microbial resistance to antibiotics has led to a continuing search for newer and more effective drugs. Antimicrobial peptides are generally found in animals, plants, and microorganisms and are of great interest to medicine, pharmacology, and the food industry. These peptides are capable of inhibiting pathogenic microorganisms. They can attack parasites, while causing little or no harm to the host cells. The defensins are peptides found in granules in the polymorphonuclear neutrophils (PMNs) and are responsible for the defense of the organism. Several animal defensins, like dermaseptin, antileukoprotease, protegrin, and others, have had their activities and efficacy tested and been shown to be effective against bacteria, fungi, and protists; there are also specific defensins from invertebrates, e.g., drosomycin and heliomicin; from plants, e.g., the types A and B; and the bacteriocins, e.g., acrocin, marcescin, etc. The aim of the present work was to compile a comprehensive bibliographic review of the diverse potentially antimicrobial peptides in an effort to systematize the current knowledge on these substances as a contribution for further researches. The currently available bibliography does not give a holistic approach on this subject. The present work intends to show that the mechanism of defense represented by defensins is promising from the perspective of its application in the treatment of infectious diseases in human, animals and plants. PMID:21264153

  17. Enhancer-specific modulation of E protein activity.

    PubMed

    Markus, Maurice; Du, Zhimei; Benezra, Robert

    2002-02-22

    Homodimeric complexes of members of the E protein family of basic helix-loop-helix (bHLH) transcription factors are important for tissue-specific activation of genes in B lymphocytes (Bain, G., Gruenwald, S., and Murre, C. (1993) Mol. Cell Biol. 13, 3522-3529; Shen, C. P., and Kadesch, T. (1995) Mol. Cell Biol. 15, 4518-4524; Jacobs, Y., et al. (1994) Mol. Cell Biol. 14, 4087-4096; Wilson, R. B., et al. (1991) Mol. Cell Biol. 11, 6185-6191). These homodimers, however, have little activity on myogenic enhancers (Weintraub, H., Genetta, T., and Kadesch, T. (1994) Genes Dev. 8, 2203-2211). We report here the identification of a novel cis-acting transcriptional repression domain in the E protein family of bHLH transcription factors. This domain, the Rep domain, is present in each of the known vertebrate E proteins. Extensive mapping analysis demonstrates that this domain is an acidic region of 30 amino acids with a predicted loop structure. Fusion studies indicate that the Rep domain can repress both of the E protein transactivation domains (AD1 and AD2). Physiologically, the Rep domain plays a key role in maintaining E protein homodimers in an inactive state on myogenic enhancers. In addition, we demonstrate that Rep domain mediated repression of AD1 is a necessary for the function of MyoD-E protein heterodimeric complexes. These studies demonstrate that the Rep domain is important for modulating the transcriptional activity of E proteins and provide key insights into both the selectivity and mechanism of action of E protein containing bHLH protein complexes. PMID:11724804

  18. Chemical labelling of active serum thioester proteins for quantification.

    PubMed

    Holm, Lotta; Ackland, Gareth L; Edwards, Mark R; Breckenridge, Ross A; Sim, Robert B; Offer, John

    2012-02-01

    The complement serum proteins C3 and C4 and the protease inhibitor α-2 macroglobulin are all members of the C3/α-2M thioester protein family, an evolutionarily ancient and conserved family that contains an intrachain thioester bond. The chemistry of the thioester bond is a key to the function of the thioester proteins. All these proteins function by covalently linking to their target by acyl transfer of the protein via the thioester moiety. We show that the signature thioester bond can be targeted with nucleophiles linked to a bioreporter molecule, site-specifically modifying the whole, intact thioester protein. Conditions were optimised to label selectively and efficiently pull-down unprocessed thioester-containing proteins from serum. We demonstrated pull-down of full-length C3, α-2M and C4 from sera in high salt, using a biotinylated nucleophile and streptavidin-coated resin, confirmed by MALDI-TOF MS identification of the gel bands. The potential for the development of a quantitative method for measuring active C3 in serum was investigated in patient sera pre and post operation. Quantifying active C3 in clinical assays using current methods is difficult. Methods based on antibody detection (e.g. nephelometry) do not distinguish between active C3 and inactive breakdown products. C3-specific haemolytic assays can be used, but these require use of relatively unstable reagents. The current work represents a promising robust, enzyme- and antibody-free chemical method for detecting active thioester proteins in blood, plasma or serum. PMID:21852021

  19. Endocytosis of Seven-Transmembrane RGS Protein Activates G- protein Coupled Signaling in Arabidopsis

    PubMed Central

    Urano, Daisuke; Phan, Nguyen; Jones, Janice C.; Yang, Jing; Huang, Jirong; Grigston, Jeffrey; Taylor, J. Philip; Jones, Alan M.

    2012-01-01

    Signal transduction typically begins by ligand-dependent activation of a concomitant partner which is otherwise in its resting state. However, in cases where signal activation is constitutive by default, the mechanism of regulation is unknown. The Arabidopsis thaliana heterotrimeric Gα protein self-activates without accessory proteins, and is kept in its resting state by the negative regulator, AtRGS1 (Regulator of G protein Signaling 1), which is the prototype of a seven transmembrane receptor fused with an RGS domain. Endocytosis of AtRGS1 by ligand-dependent endocytosis physically uncouples the GTPase accelerating activity of AtRGS1 from the Gα protein, permitting sustained activation. Phosphorylation of AtRGS1 by AtWNK8 kinase causes AtRGS1 endocytosis, required both for G protein-mediated sugar signaling and cell proliferation. In animals, receptor endocytosis results in signal desensitization, whereas in plants, endocytosis results in signal activation. These findings reveal how different organisms rearrange a regulatory system to result in opposite outcomes using similar phosphorylation-dependent endocytosis. PMID:22940907

  20. Determination of Radical Scavenging Activity and Total Phenols of Wine and Spices: A Randomized Study.

    PubMed

    Lugemwa, Fulgentius Nelson; Snyder, Amanda L; Shaikh, Koonj

    2013-01-01

    Thirty eight bottles of red wine (Carbanet Sauvignon) were randomly selected based on vintage, region, price, and age (number of months in a barrel). The total phenolic content of each wine was determined using Folin-Ciocalteau assay. The radical scavenging activity was evaluated using 2,2-diphenyl-1-picryhydrazyl (DPPH) assay. Apart from a few bottles that exhibited above average radical scavenging activity and phenolic content, there was no good correlation of those two quantities with region, price or vintage. The average phenolic amount was 2874 mg/L. The lowest phenolic content was found to be 1648 mg/L for an eight dollar wine. Wine with the highest amount of phenol of 4495 mg/L was a 2007, nine dollar bottle from South America. High amount of phenols did not translate into high radical scavenging activity. Barrel-aging did not increase the amount of phenols or the radical scavenging activity of wine. In order to discover new and potent sources of antioxidants from plants, the following spices were studied: ginger, cilantro, cumin, anise, linden, eucalyptus, marjoram, oregano, sage, thyme and rosemary. Whole spices were crushed and extracted for 96 h at room temperature using a combination of ethyl acetate, ethyl alcohol and water in the ratio of 4.5:4.5:1 (v/v/v). The radical scavenging activity of extracts was evaluated using 2,2-diphenyl-1-picryhydrazyl (DPPH) assay. The total phenolic content of each spice was also determined using the Folin-Ciocalteau assay. Eucalyptus was found to be the most potent antioxidant with an LC50 of 324.1 mg of phenol/L, followed by marjoram with an LC50 of 407.5 mg of phenol/L, and rosemary with an LC50 of 414.0 mg/L. The least potent antioxidants were ginger and cilantro with LC50 of 7604 mg/L of phenol and 7876 mg of phenol/L, respectively. PMID:26784340

  1. Determination of Radical Scavenging Activity and Total Phenols of Wine and Spices: A Randomized Study

    PubMed Central

    Lugemwa, Fulgentius Nelson; Snyder, Amanda L.; Shaikh, Koonj

    2013-01-01

    Thirty eight bottles of red wine (Carbanet Sauvignon) were randomly selected based on vintage, region, price, and age (number of months in a barrel). The total phenolic content of each wine was determined using Folin-Ciocalteau assay. The radical scavenging activity was evaluated using 2,2-diphenyl-1-picryhydrazyl (DPPH) assay. Apart from a few bottles that exhibited above average radical scavenging activity and phenolic content, there was no good correlation of those two quantities with region, price or vintage. The average phenolic amount was 2874 mg/L. The lowest phenolic content was found to be 1648 mg/L for an eight dollar wine. Wine with the highest amount of phenol of 4495 mg/L was a 2007, nine dollar bottle from South America. High amount of phenols did not translate into high radical scavenging activity. Barrel-aging did not increase the amount of phenols or the radical scavenging activity of wine. In order to discover new and potent sources of antioxidants from plants, the following spices were studied: ginger, cilantro, cumin, anise, linden, eucalyptus, marjoram, oregano, sage, thyme and rosemary. Whole spices were crushed and extracted for 96 h at room temperature using a combination of ethyl acetate, ethyl alcohol and water in the ratio of 4.5:4.5:1 (v/v/v). The radical scavenging activity of extracts was evaluated using 2,2-diphenyl-1-picryhydrazyl (DPPH) assay. The total phenolic content of each spice was also determined using the Folin-Ciocalteau assay. Eucalyptus was found to be the most potent antioxidant with an LC50 of 324.1 mg of phenol/L, followed by marjoram with an LC50 of 407.5 mg of phenol/L, and rosemary with an LC50 of 414.0 mg/L. The least potent antioxidants were ginger and cilantro with LC50 of 7604 mg/L of phenol and 7876 mg of phenol/L, respectively. PMID:26784340

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

    PubMed

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

    2012-10-01

    Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers ((13)C-lysine, (15)N-glycine, ²H5-phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g · kg(-1) · day(-1) compared to 0.8 g · kg(-1) · day(-1) in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h. PMID:22666780

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

    PubMed Central

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

    2012-01-01

    Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers (13C-lysine, 15N-glycine, 2H5-phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g·kg−1·day−1 compared to 0.8 g·kg−1·day−1 in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h. PMID:22666780

  4. Circulating vitamin D binding protein, total, free and bioavailable 25-hydroxyvitamin D and risk of colorectal cancer

    PubMed Central

    Ying, Hou-Qun; Sun, Hui-Ling; He, Bang-Shun; Pan, Yu-Qin; Wang, Feng; Deng, Qi-Wen; Chen, Jie; Liu, Xian; Wang, Shu-Kui

    2015-01-01

    Epidemiological investigation have suggested that there is a significantly inverse association between circulating 25-hydroxyvitamin D (25(OH)D) and the risk for developing colorectal cancer (CRC) in humans. However, little is known about the role of vitamin D binding protein (VDBP) in colorectal carcinogenesis. Blood samples were collected from 212 CRC patients and 212 controls matched with age, gender and blood collection time. We used logistic regression to calculate the odds ratios and 95% confidence intervals for further estimation of the association of the quartiles of VDBP, total, free and bioavailable 25(OH)D with CRC risk. The results revealed that there was no significant association between circulating VDBP concentrations and CRC in the present study, and that a negative association existed between total 25(OH)D and the risk of CRC, which was unchanged after adjustment for VDBP. Higher levels of free and bioavailable 25(OH)D were significantly associated with decreased risk of CRC. After stratifying by VDBP, high levels of total, free and bioavailable 25(OH)D were associated significantly with decreased CRC risk among participants with circulating VDBP below the median. These findings indicate that VDBP is not directly associated with the risk of CRC, but it modulates circulating free and bioavailable 25(OH)D concentration. PMID:25609140

  5. Cultivation of a bacterial consortium with the potential to degrade total petroleum hydrocarbon using waste activated sludge.

    PubMed

    Sivakumar, S; Song, Y C; Kim, S H; Jang, S H

    2015-11-01

    Waste activated sludge was aerobically treated to demonstrate multiple uses such as cultivating an oil degrading bacterial consortium; studying the influence of a bulking agent (peat moss) and total petroleum hydrocarbon concentration on bacterial growth and producing a soil conditioner using waste activated sludge. After 30 days of incubation, the concentration of oil-degrading bacteria was 4.3 x 10(8) CFU g(-1) and 4.5 x 10(8) CFU g(-1) for 5 and 10 g of total petroleum hydrocarbon, respectively, in a mixture of waste activated sludge (1 kg) and peat moss (0.1 kg). This accounts for approximately 88.4 and 91.1%, respectively, of the total heterotrophic bacteria (total-HB). The addition of bulking agent enhanced total-HB population and total petroleum hydrocarbon-degrading bacterial population. Over 90% of total petroleum hydrocarbon degradation was achieved by the mixture of waste activated sludge, bulking agent and total petroleum hydrocarbon. The results of physico-chemical parameters of the compost (waste activated sludge with and without added peat moss compost) and a substantial reduction in E. coli showed that the use of this final product did not exhibit risk when used as soil conditioner. Finally, the present study demonstrated that cultivation of total petroleum hydrocarbon-degrading bacterial consortium and production of compost from waste activated sludge by aerobic treatment was feasible. PMID:26688976

  6. Enzymatic activation of cellulose acetate membrane for reducing of protein fouling.

    PubMed

    Koseoglu-Imer, Derya Y; Dizge, Nadir; Koyuncu, Ismail

    2012-04-01

    In this study, the surface of cellulose acetate (CA) ultrafiltration membrane was activated with serine protease (Savinase) enzyme to reduce protein fouling. Enzyme molecules were covalently immobilized with glutaraldehyde (cross-linking agent) onto the surface of CA membranes. The membrane activation was verified using filtration experiments and morphological analysis. Scanning electron microscopy (SEM) images and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy of the activated membrane when compared with raw membrane were confirmed that the enzyme was immobilized onto the membrane surface. The immobilization efficiencies changed from 13.2 to 41.2% according to the enzyme ratios from 2.5 to 10.0 mg/mL. However, the permeability values decreased from 232±6 to 121±4 L/m(2) h bar with increasing enzyme concentration from 2.5 to 10.0 mg/mL. In fouling experiments, bovine serum albumin (BSA) was used as the protein model solution and activated sludge was used as the model biological sludge. Enzyme-activated membranes exhibited good filtration performances and protein rejection efficiencies were compared with raw CA membrane. Also the relative flux reduction (RFR) ratios of membranes were calculated as 97% and 88% for raw CA and enzyme-activated membranes (5 mg/mL savinase), respectively. The membrane activated with Savinase enzyme could be proposed as a surface treatment method before filtration to mitigate protein fouling. PMID:22218336

  7. Evaluation of anti-fatigue activity of total saponins of Radix notoginseng

    PubMed Central

    Yong-xin, Xu; Jian-jun, Zhang

    2013-01-01

    Background & objectives: Several biological activities of total saponins of Radix notoginseng (TSRN), a trational Chinese medicine have been reported. The present study was carried out to investigate anti-fatigue activity of TSRN in male Kunming mice. Methods: Mice were divided into four groups. The first group designated as control group was administered with distilled water by gavage every day. The second, third and fourth groups designated as TSRN treatment groups were administered with TSRN of 20, 40 and 80 mg/kg body weight/day, respectively. The treatment continued for 28 days. Exhaustive swimming time, blood lactate and tissue glycogen contents of mice after swimming were determined. Results: TSRN extended exhaustive swimming time of mice, effectively delayed the increase of lactate in the blood, as well as increased the tissue glycogen contents. Interpretation & conclusions: TSRN showed promising anti-fatigue activity in animal model. However, further study is needed to elucidate the mechanism of the effect of TSRN on fatigue. PMID:23481065

  8. The relationship between total cholinesterase activity and mortality in four butterfly species

    USGS Publications Warehouse

    Bargar, Timothy A.

    2012-01-01

    The relationship between total cholinesterase activity (TChE) and mortality in four butterfly species (great southern white [Ascia monuste], common buckeye [Junonia coenia], painted lady [Vanessa cardui], and julia butterflies [Dryas julia]) was investigated. Acute contact toxicity studies were conducted to evaluate the response (median lethal dose [LD50] and TChE) of the four species following exposure to the organophosphate insecticide naled. The LD50 for these butterflies ranged from 2.3 to 7.6 μg/g. The average level of TChE inhibition associated with significant mortality ranged from 26 to 67%, depending on the species. The lower bounds of normal TChE activity (2 standard deviations less than the average TChE for reference butterflies) ranged from 8.4 to 12.3 μM/min/g. As a percentage of the average reference TChE activity for the respective species, the lower bounds were similar to the inhibition levels associated with significant mortality, indicating there was little difference between the dose resulting in significant TChE inhibition and that resulting in mortality.

  9. The relationship between total cholinesterase activity and mortality in four butterfly species.

    PubMed

    Bargar, Timothy A

    2012-09-01

    The relationship between total cholinesterase activity (TChE) and mortality in four butterfly species (great southern white [Ascia monuste], common buckeye [Junonia coenia], painted lady [Vanessa cardui], and julia butterflies [Dryas julia]) was investigated. Acute contact toxicity studies were conducted to evaluate the response (median lethal dose [LD50] and TChE) of the four species following exposure to the organophosphate insecticide naled. The LD50 for these butterflies ranged from 2.3 to 7.6 µg/g. The average level of TChE inhibition associated with significant mortality ranged from 26 to 67%, depending on the species. The lower bounds of normal TChE activity (2 standard deviations less than the average TChE for reference butterflies) ranged from 8.4 to 12.3 µM/min/g. As a percentage of the average reference TChE activity for the respective species, the lower bounds were similar to the inhibition levels associated with significant mortality, indicating there was little difference between the dose resulting in significant TChE inhibition and that resulting in mortality. PMID:22740147

  10. Enhancing anaerobic digestion of waste activated sludge by pretreatment: effect of volatile to total solids.

    PubMed

    Wang, Xiao; Duan, Xu; Chen, Jianguang; Fang, Kuo; Feng, Leiyu; Yan, Yuanyuan; Zhou, Qi

    2016-06-01

    In this study the effect of volatile to total solids (VS/TS) on anaerobic digestion of waste activated sludge (WAS) pretreated by alkaline, thermal and thermal-alkaline strategies was studied. Experimental results showed that the production of methane from sludge was increased with VS/TS. When anaerobic digesters were fed with sludge pretreated by the thermal-alkaline method, the average methane yield was improved from 2.8 L/d at VS/TS 0.35 to 4.7 L/d at VS/TS 0.56. Also, the efficiency of VS reduction during sludge anaerobic digestion varied between 18.9% and 45.6%, and increased gradually with VS/TS. Mechanism investigation of VS/TS on WAS anaerobic digestion suggested that the general activities of anaerobic microorganisms, activities of key enzymes related to sludge hydrolysis, acidification and methanogenesis, and the ratio of Archaea to Bacteria were all increased with VS/TS, showing good agreement with methane production. PMID:26698921

  11. Evaluation of Antioxidant Activity, Total Flavonoids, Tannins and Phenolic Compounds in Psychotria Leaf Extracts

    PubMed Central

    Formagio, Anelise Samara Nazari; Volobuff, Carla Roberta Ferreira; Santiago, Matheus; Cardoso, Claudia Andrea Lima; Vieira, Maria do Carmo; Pereira, Zefa Valdevina

    2014-01-01

    The antioxidant activity of Psychotria carthagenensis, P. leiocarpa, P. capillacea and P. deflexa (Rubiaceae) extracts were investigated, and the concentrations of total phenolics, flavonoids, condensed tannins and flavonols were determined. The chemical compositions of the extracts were investigated using the high performance liquid chromatography (HPLC/PAD) method. We used 1,1-diphenyl-1-picrylhydrazyl free radical (DPPH), β-Carotene bleaching and 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cations to determine antioxidant activity. The ability to scavenge radical was measured in these experiments by the discoloration of the solution. Concentrations of constituents were measured spectrophotometrically. P. carthagenensis and P. capillacea exhibited the highest antioxidant activity, in the DPPH test, β-carotene bleaching and ABTS system. The highest phenolic, flavonoid, condensed tannin and flavonol concentration was found in P. carthagenensis and P. capillacea extracts. HPLC-PDA analysis of P. carthagenensis and P. capillacea revealed hydroxycinnamic acid (p-coumaric acid). This is the first report on the antioxidant properties and constituent analysis of these Psychotria extracts. PMID:26785238

  12. Cyclic AMP-dependent protein kinase activity in Trypanosoma cruzi.

    PubMed Central

    Ulloa, R M; Mesri, E; Esteva, M; Torres, H N; Téllez-Iñón, M T

    1988-01-01

    A cyclic AMP-dependent protein kinase activity from epimastigote forms of Trypanosoma cruzi was characterized. Cytosolic extracts were chromatographed on DEAE-cellulose columns, giving two peaks of kinase activity, which were eluted at 0.15 M- and 0.32 M-NaCl respectively. The second activity peak was stimulated by nanomolar concentrations of cyclic AMP. In addition, a cyclic AMP-binding protein co-eluted with the second kinase activity peak. Cyclic AMP-dependent protein kinase activity was further purified by gel filtration, affinity chromatography on histone-agarose and cyclic AMP-agarose, as well as by chromatography on CM-Sephadex. The enzyme ('holoenzyme') could be partially dissociated into two different components: 'catalytic' and 'regulatory'. The 'regulatory' component had specific binding for cyclic AMP, and it inhibited phosphotransferase activity of the homologous 'catalytic component' or of the 'catalytic subunit' from bovine heart. Cyclic AMP reversed these inhibitions. A 'holoenzyme preparation' was phosphorylated in the absence of exogenous phosphate acceptor and analysed by polyacrylamide-gel electrophoresis. A 56 kDa band was phosphorylated. The same preparation was analysed by Western blotting, by using polyclonal antibodies to the regulatory subunits of protein kinases type I or II. Both antibodies reacted with the 56 kDa band. Images Fig. 7. Fig. 8. PMID:2848508

  13. MAPK-Activated Protein Kinases (MKs): Novel Insights and Challenges

    PubMed Central

    Gaestel, Matthias

    2016-01-01

    Downstream of MAPKs, such as classical/atypical ERKs and p38 MAPKs, but not of JNKs, signaling is often mediated by protein kinases which are phosphorylated and activated by MAPKs and, therefore, designated MAPK-activated protein kinases (MAPKAPKs). Recently, novel insights into the specificity of the assembly of MAPK/MAPKAPK hetero-dimeric protein kinase signaling complexes have been gained. In addition, new functional aspects of MKs have been described and established functions have been challenged. This short review will summarize recent developments including the linear motif (LM) in MKs, the ERK-independent activation of RSK, the RSK-independent effects of some RSK-inhibitors and the challenged role of MK5/PRAK in tumor suppression. PMID:26779481

  14. Hydrodynamic collective effects of active proteins in biological membranes.

    PubMed

    Koyano, Yuki; Kitahata, Hiroyuki; Mikhailov, Alexander S

    2016-08-01

    Lipid bilayers forming biological membranes are known to behave as viscous two-dimensional fluids on submicrometer scales; usually they contain a large number of active protein inclusions. Recently, it was shown [A. S. Mikhailov and R. Kapral, Proc. Natl. Acad. Sci. USA 112, E3639 (2015)PNASA60027-842410.1073/pnas.1506825112] that such active proteins should induce nonthermal fluctuating lipid flows leading to diffusion enhancement and chemotaxislike drift for passive inclusions in biomembranes. Here, a detailed analytical and numerical investigation of such effects is performed. The attention is focused on the situations when proteins are concentrated within lipid rafts. We demonstrate that passive particles tend to become attracted by active rafts and are accumulated inside them. PMID:27627343

  15. MAPK-Activated Protein Kinases (MKs): Novel Insights and Challenges.

    PubMed

    Gaestel, Matthias

    2015-01-01

    Downstream of MAPKs, such as classical/atypical ERKs and p38 MAPKs, but not of JNKs, signaling is often mediated by protein kinases which are phosphorylated and activated by MAPKs and, therefore, designated MAPK-activated protein kinases (MAPKAPKs). Recently, novel insights into the specificity of the assembly of MAPK/MAPKAPK hetero-dimeric protein kinase signaling complexes have been gained. In addition, new functional aspects of MKs have been described and established functions have been challenged. This short review will summarize recent developments including the linear motif (LM) in MKs, the ERK-independent activation of RSK, the RSK-independent effects of some RSK-inhibitors and the challenged role of MK5/PRAK in tumor suppression. PMID:26779481

  16. Totally synthetic peptide-based immunocontraceptive vaccines show activity in dogs of different breeds.

    PubMed

    Walker, John; Ghosh, Souravi; Pagnon, Joanne; Colantoni, Caterina; Newbold, Andrea; Zeng, Weiguang; Jackson, David C

    2007-10-10

    In this study we examine the immunogenicity of totally synthetic peptide-based immunocontraceptive vaccines in dogs. Seven individual epitope-based vaccines were assembled in which a different T helper (T(H)) cell epitope derived from the sequence of F protein of canine distemper virus was synthesized in tandem with a peptide representing luteinising hormone releasing hormone (LHRH). Each of the individual T(H)-LHRH peptide vaccines was inoculated subcutaneously into dogs. The results demonstrate that five of the seven peptide vaccines were able to elicit strong anti-LHRH antibody responses in beagle foxhounds accompanied by a concomitant suppression in the levels of the hormones testosterone and progesterone in the majority of the animals. A pool of these five peptides was then used to inoculate five different breeds of dogs. All animals responded with high levels of anti-LHRH antibody. An investigation of the proliferative responses of peripheral blood mononuclear cells (PBMC) obtained from inoculated dogs showed that the majority of breeds responded to each of the individual T helper cell epitope tested. The results provide a strategy for development of an immunocontraceptive vaccine for use in multiple breeds of dogs. PMID:17825958

  17. Proteins of the ETS family with transcriptional repressor activity.

    PubMed

    Mavrothalassitis, G; Ghysdael, J

    2000-12-18

    ETS proteins form one of the largest families of signal-dependent transcriptional regulators, mediating cellular proliferation, differentiation and tumorigenesis. Most of the known ETS proteins have been shown to activate transcription. However, four ETS proteins (YAN, ERF, NET and TEL) can act as transcriptional repressors. In three cases (ERF, NET and TEL) distinct repression domains have been identified and there are indications that NET and TEL may mediate transcription via Histone Deacetylase recruitment. All four proteins appear to be regulated by MAPKs, though for YAN and ERF this regulation seems to be restricted to ERKs. YAN, ERF and TEL have been implicated in cellular proliferation although there are indications suggesting a possible involvement of YAN and TEL in differentiation as well. Other ETS-domain proteins have been shown to repress transcription in a context specific manner, and there are suggestions that the ETS DNA-binding domain may act as a transcriptional repressor. Transcriptional repression by ETS domain proteins adds an other level in the orchestrated regulation by this diverse family of transcription factors that often recognize similar if not identical binding sites on DNA and are believed to regulate critical genes in a variety of biological processes. Definitive assessment of the importance of this novel regulatory level will require the identification of ETS proteins target genes and the further analysis of transcriptional control and biological function of these proteins in defined pathways. PMID:11175368

  18. Signal peptides are allosteric activators of the protein translocase.

    PubMed

    Gouridis, Giorgos; Karamanou, Spyridoula; Gelis, Ioannis; Kalodimos, Charalampos G; Economou, Anastassios

    2009-11-19

    Extra-cytoplasmic polypeptides are usually synthesized as 'preproteins' carrying amino-terminal, cleavable signal peptides and secreted across membranes by translocases. The main bacterial translocase comprises the SecYEG protein-conducting channel and the peripheral ATPase motor SecA. Most proteins destined for the periplasm and beyond are exported post-translationally by SecA. Preprotein targeting to SecA is thought to involve signal peptides and chaperones like SecB. Here we show that signal peptides have a new role beyond targeting: they are essential allosteric activators of the translocase. On docking on their binding groove on SecA, signal peptides act in trans to drive three successive states: first, 'triggering' that drives the translocase to a lower activation energy state; second, 'trapping' that engages non-native preprotein mature domains docked with high affinity on the secretion apparatus; and third, 'secretion' during which trapped mature domains undergo several turnovers of translocation in segments. A significant contribution by mature domains renders signal peptides less critical in bacterial secretory protein targeting than currently assumed. Rather, it is their function as allosteric activators of the translocase that renders signal peptides essential for protein secretion. A role for signal peptides and targeting sequences as allosteric activators may be universal in protein translocases. PMID:19924216

  19. Clonorchis sinensis-derived total protein attenuates airway inflammation in murine asthma model by inducing regulatory T cells and modulating dendritic cell functions

    SciTech Connect

    Jeong, Young-Il; Kim, Seung Hyun; Ju, Jung Won; Cho, Shin Hyeong; Lee, Won Ja; Park, Jin Wook; Park, Yeong-Min; Lee, Sang Eun

    2011-04-22

    Highlights: {yields} Treatment with Clonorchis sinensis-derived total protein attenuates OVA-induced airway inflammation and AHR to methacholine. {yields} Induction of CD4{sup +}CD25{sup +}Foxp3{sup +} T cells and IL-10 along with suppression of splenocyte proliferation by C. sinensis-derived total protein. {yields} C. sinensis-derived total protein interferes with the expression of co-stimulatory molecules in DCs. -- Abstract: Asthma is characterized by Th2-mediated inflammation, resulting in airway hyperresponsiveness (AHR) through airway remodeling. Recent epidemiological and experimental reports have suggested an inverse relationship between the development of allergy and helminth infections. Infection by Clonorchis sinensis, a liver fluke that resides in the bile duct of humans, is endemic predominantly in Asia including Korea and China. Using a murine model for asthma, we investigated the effects of C. sinensis-derived total protein (Cs-TP) on allergen-induced airway inflammation and the mechanism underlying the protective effects of Cs-TP administration on asthma. Treatment with Cs-TP attenuated OVA-induced airway inflammation and methacholine-induced AHR, as well as eosinophilia development, lymphocyte infiltration into the lung, and goblet cell metaplasia. This protective effect of Cs-TP is associated with markedly reduced OVA-specific IgE and Th1/Th2 cytokine production. Moreover, Cs-TP increased the number of CD4{sup +}CD25{sup +}Foxp3{sup +} regulatory T (Treg) cells as well as their suppressive activity. In fact, proliferation of OVA-restimulated splenocytes was suppressed significantly. Cs-TP also inhibited the expression of such co-stimulatory molecules as CD80, CD86, and CD40 in LPS- or OVA-stimulated dendritic cells (DCs), suggesting that Cs-TP could interfere with the capacity of airway DCs to prime naive T cells. These data demonstrate the capacity of C. sinensis to ameliorate allergic asthma and broaden our understanding of the paradoxical

  20. G Protein Activation Stimulates Phospholipase D Signaling in Plants.

    PubMed Central

    Munnik, T.; Arisz, S. A.; De Vrije, T.; Musgrave, A.

    1995-01-01

    We provide direct evidence for phospholipase D (PLD) signaling in plants by showing that this enzyme is stimulated by the G protein activators mastoparan, ethanol, and cholera toxin. An in vivo assay for PLD activity in plant cells was developed based on the use of a "reporter alcohol" rather than water as a transphosphatidylation substrate. The product was a phosphatidyl alcohol, which, in contrast to the normal product phosphatidic acid, is a specific measure of PLD activity. When 32P-labeled cells were treated with 0.1% n-butanol, 32P-phosphatidyl butanol (32P-PtdBut) was formed in a time-dependent manner. In cells treated with any of the three G protein activators, the production of 32P-PtdBut was increased in a dose-dependent manner. The G protein involved was pertussis toxin insensitive. Ethanol could activate PLD but was itself consumed by PLD as transphosphatidylation substrate. In contrast, secondary alcohols (e.g., sec-butyl alcohol) activated PLD but did not function as substrate, whereas tertiary alcohols did neither. Although most of the experiments were performed with the green alga Chlamydomonas eugametos, the relevance for higher plants was demonstrated by showing that PLD in carnation petals could also be activated by mastoparan. The results indicate that PLD activation must be considered as a potential signal transduction mechanism in plants, just as in animals. PMID:12242371

  1. A Theileria parva type 1 protein phosphatase activity.

    PubMed

    Cayla, X; Garcia, A; Baumgartner, M; Ozon, R; Langsley, G

    2000-09-01

    The protozoan parasite Theileria (spp. parva and annulata) infects bovine leukocytes and provokes a leukaemia-like disease in vivo. In this study, we have detected a type 1 serine/threonine phosphatase activity with phosphorylase a as a substrate, in protein extracts of parasites purified from infected B lymphocytes. In contrast to this type 1 activity, dose response experiments with okadaic acid (OA), a well characterised inhibitor of type 1 and 2A protein phosphatases, indicated that type 2A is the predominant activity detected in host B cells. Furthermore, consistent with polycation-specific activation of the type 2A phosphatase, protamine failed to activate the parasite-associated phosphorylase a phosphatase activity. Moreover, inhibition of phosphorylase a dephosphorylation by phospho-DARPP-32, a specific type 1 inhibitor, clearly demonstrated that a type 1 phosphatase is specifically associated with the parasite, while the type 2A is predominantly expressed in the host lymphocyte. Since an antibody against bovine catalytic protein phosphatase 1 (PP1) subunit only recognised the PP1 in B cells, but not in parasite extracts, we conclude that in parasites the PP1 activity is of parasitic origin. Intriguingly, since type 1 OA-sensitive phosphatase activity has been recently described in Plasmodium falciparum, we can conclude that these medically important parasites produce their one PP1. PMID:10989153

  2. Antioxidant, Cytotoxic, and Antiproliferative Activities and Total Polyphenol Contents of the Extracts of Geissospermum reticulatum Bark

    PubMed Central

    Barnes, Nicholas M.; Wawer, Iwona; Paradowska, Katarzyna

    2016-01-01

    Geissospermum species are medically important plants due to their health-promoting effects. The objective of this study was to determine the antioxidant ability and antiproliferative and cytotoxic effects of infusions, tinctures, and ethanolic extracts of Geissospermum reticulatum barks in relation to the contents of total phenolics and flavonoids. Seven samples of barks were collected in various regions of Peruvian Amazonia. We found that the amount of total phenolics in the studied products varied from 212.40 ± 0.69 to 1253.92 ± 11.20 mg GAE/kg. In our study there is a correlation (R2 = 0.7947) between the results of antioxidants assays: FRAP and ORAC for tinctures, infusions, and ethanolic extracts of G. reticulatum barks. We have also observed antiproliferative activities of the ethanolic extracts on normal T-cells. These extracts have caused death on malignant cell lines (THP-1 and HL-60) and this data correlates well with their antioxidant capacity measured by ORAC method. Interestingly, the highest concentration of the ethanolic extract was not toxic in the zebrafish embryo developmental assay. Our results indicate that G. reticulatum is rich in antioxidants and have cytotoxic and antiproliferative properties. The data suggests potential immunosuppressive role of the extracts. This is the first study presenting the results of chemical and biological analysis of multiple preparations from G. reticulatum.

  3. Volatile flavor compounds, total polyphenolic contents and antioxidant activities of a China gingko wine.

    PubMed

    Wang, Xu; Xie, Kelin; Zhuang, Haining; Ye, Ran; Fang, Zhongxiang; Feng, Tao

    2015-09-01

    The volatile compounds in gingko wine, a novel functional wine, were extracted by head-space solid phase micro-extraction (SPME) and analyzed by gas chromatography-mass spectrometry (GC-MS) coupled with odor activity value (OAV) and relative odor contribution (ROC) analyses. In addition, the total polyphenolic content of gingko wine was determined using the Folin-Ciocalteu reagent, and its antioxidant capacity was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. Fifty-eight compounds were tentatively identified, including 13 esters, 10 alcohols, 11 acids, 12 carbonyl compounds, 2 lactones, 2 phenols, and 8 hydrocarbons. Ethyl hexanoate, ethyl pentanoate, nonanal, ethyl butyrate and ethyl heptanoate were the major contributors to the gingko wine aroma based on the results of OAV and ROC. The total phenols content of the gingko wine was 456 mg/L gallic acid equivalents, and its antioxidant capacity was higher than those of typical Chinese liquors analyzed in this paper. PMID:25842306

  4. Total Synthesis and Antifungal Activity of Palmarumycin CP17 and Its Methoxy Analogues.

    PubMed

    Wang, Ruina; Liu, Guoyue; Yang, Mingyan; Wang, Mingan; Zhou, Ligang

    2016-01-01

    Total synthesis of naturally occurring spirobisnaphthalene palmarumycin CP17 and its methoxy analogues was first achieved through Friedel-Crafts acylation, Wolff-Kishner reduction, intramolecular cyclization, ketalization, benzylic oxidation, and demethylation using the inexpensive and readily available methoxybenzene, 1,2-dimethoxybenzene and 1,4-dimethoxybenzene and 1,8-dihydroxynaphthalene as raw materials. Demethylation with (CH₃)₃SiI at ambient temperature resulted in ring A aromatization and acetal cleavage to give rise to binaphthyl ethers. The antifungal activities of these spirobisnaphthalene derivatives were evaluated, and the results revealed that 5 and 9b exhibit EC50 values of 9.34 µg/mL and 12.35 µg/mL, respectively, against P. piricola. PMID:27164077

  5. Total Synthesis and Selective Activity of a NewClass of Conformationally Restrained Epothilones

    PubMed Central

    Alhamadsheh, Mamoun M.; Gupta, Shuchi; Hudson, Richard A.; Perera, Lalith

    2009-01-01

    Stereoselective total syntheses of two novel conformationally restrained epothilone analogues are described. Evans asymmetric alkylation, Brown allylation, and a diastereoselective aldol reaction served as the key steps in the stereoselective synthesis of one of the two key fragments of the convergent synthetic approach.Enzyme resolution was employed to obtain the second fragment as a single enantiomer. The molecules were assembled by esterification, followed by ring-closing metathesis. In preliminary cytotoxicity studies, one of the analogues showed strong and selective growth inhibitory activity against two leukemia cell lines over solid human tumor cell lines. The precise biological mechanism of action and high degree of selectivity of this analogue remain to be examined. PMID:17955508

  6. Patient-level improvements in pain and activities of daily living after total knee arthroplasty

    PubMed Central

    Lewallen, David G.

    2014-01-01

    Objective. To study patient-level improvements in pain and limitations of key activities of daily living (ADLs) after primary or revision total knee arthroplasty (TKA). Methods. We analysed prospectively collected data from the Mayo Clinic Total Joint Registry for improvements in index knee pain severity and limitations in three key ADLs (walking, climbing stairs and rising from a chair) from pre-operative to 2 and 5 years post-TKA. Results. The primary TKA cohort consisted of 7229 responders pre-operatively, 7139 at 2 years and 4234 at 5 years post-operatively. The revision TKA cohort consisted of 1206 responders pre-operatively, 1533 at 2 years and 881 at 5 years post-operatively. In the primary TKA cohort, important pain reduction to mild or no knee pain at 2 years was reported by 92% with moderate pre-operative pain and 93% with severe pre-operative pain; respective proportions were 91% and 91% at 5 years follow-up. For revision TKA, respective proportions were 71% and 66% at 2 years and 68% and 74% at 5 years. Three per cent with no/mild pre-operative overall limitation and 19% with moderate/severe pre-operative overall limitation had moderate/severe overall activity limitation 2 years post-operatively; at 5 years the respective proportions were 4% and 22%. Respective proportions for revision TKA were up to 3% and 32% at 2 years and 4% and 34% at 5 years. Conclusion. Our study provides comprehensive data for patient-level improvements in pain and key ADLs. These data can be used to inform patients pre-operatively of expected outcomes, based on pre-operative status, which may further help patients set realistic goals for improvements after TKA. PMID:24162150

  7. Contractions Activate Hormone-Sensitive Lipase in Rat Muscle by Protein Kinase C and Mitogen-Activated Protein Kinase

    PubMed Central

    Donsmark, Morten; Langfort, Jozef; Holm, Cecilia; Ploug, Thorkil; Galbo, Henrik

    2003-01-01

    Intramuscular triacylglycerol is an important energy store and is also related to insulin resistance. The mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by both adrenaline and contractions. Adrenaline acts via cAMP-dependent protein kinase (PKA). The signalling mediating the effect of contractions is unknown and was explored in this study. Incubated soleus muscles from 70 g male rats were electrically stimulated to perform repeated tetanic contractions for 5 min. The contraction-induced activation of HSL was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and calphostin C and reduced 50 % by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also completely blocked extracellular signal-regulated kinase (ERK) 1 and 2 phosphorylation. None of the inhibitors reduced adrenaline-induced HSL activation in soleus muscle. Both phorbol-12-myristate-13-acetate (PMA), which activates PKC and, in turn, ERK, and caffeine, which increases intracellular Ca2+ without eliciting contraction, increased HSL activity. Activated ERK increased HSL activity in supernatant from basal but not from electrically stimulated muscle. In conclusion, in muscle, PKC can stimulate HSL through ERK. Contractions and adrenaline enhance muscle HSL activity by different signalling mechanisms. The effect of contractions is mediated by PKC, at least partly via the ERK pathway. PMID:12794177

  8. The effects of different preservation processes on the total protein and growth factor content in a new biological product developed from human amniotic membrane.

    PubMed

    Russo, Alessandra; Bonci, Paola; Bonci, Paolo

    2012-06-01

    The aim of this work is to quantify the total protein and growth factors content in a tissue-suspension obtained from processed human amniotic membrane (hAM). hAM was collected, frozen, freeze dried, powdered and sterilized by γ-irradiation. At each step of the process, samples were characterized for the total protein amounts by a Bradford protein assay and for the growth factor concentrations by ELISA test of the tissue suspensions. Frozen-hAM samples show higher release of total proteins and specific growth factors in the tissue suspension in comparison with freeze-dried hAM. We observed that even if the protein extraction is hindered once the tissue is dried, the powdering process allows a greater release in the tissue suspension of total proteins and growth factors after tissue re-solubilization in comparison with only the freeze-drying process (+91 ± 13% for EGF, +16 ± 4% for HGF, +11 ± 5% for FGF, +16 ± 9% for TGF-β1), and a greater release of EGF (85 ± 10%) in comparison with only the freezing process, because proteins become much readily solubilized in the solution. According with these results, we describe a protocol to obtain a new sterile biological product from hAM tissue, with well-known effects of thermal, mechanical and physical processes on the total protein and grow factors contents. PMID:21681392

  9. Agglutinating activity of alcohol-soluble proteins from quinoa seed flour in celiac disease.

    PubMed

    De Vincenzi, M; Silano, M; Luchetti, R; Carratù, B; Boniglia, C; Pogna, N E

    1999-01-01

    The edible seeds of the quinoa plant contain small quantities of alcohol-soluble protein which, after peptic-tryptic digestion, are unable to agglutinate K562(s) cells. When separated by affinity chromatography on sepharose-6B coupled with mannan, peptic-tryptic digest separated in two fractions. Fraction B peptides (about 1% of total protein) were shown to agglutinate K562(s) cells at a very low concentration, whereas peptides in fraction A and in the mixed fraction A+B were inactive, suggesting that fraction A contains protective peptides that interfere with the agglutinating activity of toxic peptides in fraction B. PMID:10646556

  10. Total Phenolic Content and Antioxidant Activity of Different Types of Chocolate, Milk, Semisweet, Dark, and Soy, in Cerebral Cortex, Hippocampus, and Cerebellum of Wistar Rats

    PubMed Central

    da Silva Medeiros, Niara; Koslowsky Marder, Roberta; Farias Wohlenberg, Mariane; Funchal, Cláudia; Dani, Caroline

    2015-01-01

    Chocolate is a product consumed worldwide and it stands out for presenting an important amount of phenolic compounds. In this study, the total phenolic content and antioxidant activity in the cerebral cortex, hippocampus, and cerebellum of male Wistar rats when consuming different types of chocolate, including milk, semisweet, dark, and soy, was evaluated. The total polyphenols concentration and antioxidant activity in vitro by the method of DPPH radical-scavenging test were evaluated in chocolate samples. Lipid peroxidation (TBARS), protein oxidation (carbonyl), sulfhydryl groups, and activity of SOD enzyme in cerebral cortex, hippocampus, and cerebellum of rats treated or not with hydrogen peroxide and/or chocolate were also evaluated. The dark chocolate demonstrated higher phenolic content and antioxidant activity, followed by semisweet, soy, and milk chocolates. The addition of chocolate in the diet of the rats reduced lipid peroxidation and protein oxidation caused by hydrogen peroxide. In the sulfhydryl assay, we observed that the levels of nonenzymatic defenses only increased with the chocolate treatments The SOD enzyme activity was modulated in the tissues treated with the chocolates. We observed in the samples of chocolate a significant polyphenol content and an important antioxidant activity; however, additional studies with different chocolates and other tissues are necessary to further such findings. PMID:26649198

  11. Evaluation of antioxidant activities of zein protein fractions.

    PubMed

    Tang, Ning; Zhuang, Hong

    2014-11-01

    Zein protein was extracted from the by-product corn gluten meal. The obtained zein protein was 1st hydrolyzed by 4 different proteases. The antioxidant activities of the hydrolysates or peptides were evaluated by free radical scavenging activity, metal ion chelating activity, and lipid peroxidation inhibitory capacity. Among hydrolysates produced, alkaline protease hydrolysates exhibited the highest antioxidant activity. A regression model was established by uniform design to optimize the alkaline protease hydrolysis conditions. The hydrolysates with molecular weight < 3 kDa obtained from ultrafiltration showed the highest antioxidant activities in all relevant assays. The hydrolysates with molecular weight <3 kDa were subsequently purified by gel filtration chromatography, and fraction F3 exhibited the highest antioxidant activities. Two peptides were identified from fraction F3 using LC-ESI-Q-TOF MS/MS as Pro-Phe (263.13 Da) and Leu-Pro-Phe (375.46 Da). These peptides exhibited good free radical scavenging activity and lipid peroxidation inhibitory effect. The results clearly indicated that zein protein fractions are good sources for the development of natural antioxidants for the food industry. PMID:25350353

  12. Assessing the impact of long term frozen storage of faecal samples on protein concentration and protease activity

    PubMed Central

    Morris, Laura S.; Marchesi, Julian R.

    2016-01-01

    Background The proteome is the second axis of the microbiome:host interactome and proteases are a significant aspect in this interaction. They interact with a large variety of host proteins and structures and in many situations are implicated in pathogenesis. Furthermore faecal samples are commonly collected and stored frozen so they can be analysed at a later date. So we were interested to know whether long term storage affected the integrity of proteases and total protein and whether historical native faecal samples were still a viable option for answering research questions around the functional proteome. Methods Faecal samples were collected from 3 healthy volunteers (3 biological replicates) and processed in order to be stored at both − 20 °C and − 80 °C and in a variety of storage buffers. Protein extraction, protein content and protease activity were assessed at the time of collection, after 24 h, 1 week, 1 month, 3 months 6 months and finally 1 year. Results Beadbeating impacted the quantity of protein extracted, while sodium azide did not impact protease assays. Long term storage of extracted proteins showed that both total protein and protease activity were affected when they were stored as extracted protein. Intact faecal samples were shown to maintain both protein levels and protease activity regardless of time and temperature. Conclusions Beadbeating increases the protein and protease activity when extracting from a faecal sample, however, the extracted protein is not stable and activity is lost, even with a suitable storage buffer. The most robust solution is to store the proteins in an intact frozen native faecal matrix and extract at the time of assay or analysis, this approach was shown to be suitable for samples in which, there are low levels of protease activity and which had been frozen for a year. PMID:26853125

  13. Total Phenolic Content and Antimicrobial Activity of Different Lithuanian Propolis Solutions

    PubMed Central

    Ramanauskienė, Kristina; Inkėnienė, Asta Marija; Petrikaitė, Vilma; Briedis, Vitalis

    2013-01-01

    The manufacture of ethanol-free propolis solutions offers a broader application. A few trials with Lithuanian propolis have been conducted. The aims of the study are to manufacture propolis water and water-free solutions and evaluate the quality and antimicrobial activity of these solutions. The studied solutions containing 2.5%, 5%, and 10% propolis are prepared. As solvents, purified water, 70% v/v ethanol, 96.3% v/v ethanol, propylene glycol, and their systems were used. Determination of total levels of phenolic compounds (FAE mg/g) is based on colour oxidation-reduction reaction using Folin-Ciocalteu reagent under alkaline conditions and performed at 765 nm wavelength using UV spectrophotometer. The highest content of phenolic compounds was determined in solutions containing 10% propolis extracts, and the lowest amounts in 2.5% propolis extracts. The water extracted the lowest amount of phenolic compounds from crude propolis, ethanol extracted the highest amount, and propylene glycol ranked the middle position. It is determined that technological parameters (stirring, temperature) contribute to content of phenolic compounds. During microbiological study, MICs were determined. The studies showed that water extracted propolis solutions and solvents mixture did not inhibit the growth of the studied microorganisms, and propolis solutions in propylene glycol were found to have antimicrobial activity. PMID:23573156

  14. Phytochemistry, antioxidant capacity, total phenolic content and anti-inflammatory activity of Hibiscus sabdariffa leaves.

    PubMed

    Zhen, Jing; Villani, Thomas S; Guo, Yue; Qi, Yadong; Chin, Kit; Pan, Min-Hsiung; Ho, Chi-Tang; Simon, James E; Wu, Qingli

    2016-01-01

    A liquid chromatography-mass spectrometry method was developed for the simultaneous separation, and determination of natural compounds including phenolic acids and flavonoids in the leaves of Hibiscus sabdariffa. By analyzing the UV and MS data, and comparison with authenticated standards, 10 polyphenols including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, quercetin, kaempferol and their glycosides were identified together with 5-(hydroxymethyl)furfural. Major constituents in the leaves of 25 different populations from worldwide accessions were quantified and compared with each other. The total phenolic content of each accession was determined using Folin-Ciocalteu assay, ranging from 18.98 ± 2.7 to 29.9 ± 0.5 mg GAE/g. Their in vitro antioxidant activities were measured by ABTS radical cation decolorization assay, varying from 17.5 to 152.5 ± 18.8 μmol Trolox/g. After the treatment of H. sabdariffa leaf extract, the reduction of LPS-induced NO production dose-dependently in RAW 264.7 cell indicates the extract's potential anti-inflammatory activity. PMID:26213025

  15. Comparative Antioxidant Activity and Total Flavonoid Content of Persian Pomegranate (Punica granatum L.) Cultivars.

    PubMed

    Shams Ardekani, Mohammad Reza; Hajimahmoodi, Mannan; Oveisi, Mohammad Reza; Sadeghi, Naficeh; Jannat, Behrooz; Ranjbar, Ali Mohammad; Gholam, Narges; Moridi, Tahereh

    2011-01-01

    Pomegranate (Punica granatum L.), Lythraceae, is mainly grown in Mediterranean region. It is one of the major cultivated productions of Iran, which have been used in folk medicine for many centuries. It has been proved that pomegranate has a high antioxidant activity and is effective in the prevention of atherosclerosis. This study compares the antioxidant activity, total phenolic and flavonoid contents of nine different pomegranate cultivars grown in Iran. Aqueous solutions of known Fe(+2) concentration, vitamin E, vitamin C, gallic acid and catechin were used for calibration. The results showed that Sour summer pulp cultivar had the most antioxidant effect with significant difference with the other cultivar (p < 0.05) which can be introduced as a potent source of natural antioxidants, and the peel of three cultivars (Sweet saveh malas, Sour summer and Black peel) as a suitable source for extraction and purification of phenolic and flavonoid compound. The antioxidant capacity of pomegranate peel extract is 10 times higher than the pulp extract. PMID:24250384

  16. Comparative Antioxidant Activity and Total Flavonoid Content of Persian Pomegranate (Punica granatum L.) Cultivars

    PubMed Central

    Shams Ardekani, Mohammad Reza; Hajimahmoodi, Mannan; Oveisi, Mohammad Reza; Sadeghi, Naficeh; Jannat, Behrooz; Ranjbar, Ali Mohammad; Gholam, Narges; Moridi, Tahereh

    2011-01-01

    Pomegranate (Punica granatum L.), Lythraceae, is mainly grown in Mediterranean region. It is one of the major cultivated productions of Iran, which have been used in folk medicine for many centuries. It has been proved that pomegranate has a high antioxidant activity and is effective in the prevention of atherosclerosis. This study compares the antioxidant activity, total phenolic and flavonoid contents of nine different pomegranate cultivars grown in Iran. Aqueous solutions of known Fe+2 concentration, vitamin E, vitamin C, gallic acid and catechin were used for calibration. The results showed that Sour summer pulp cultivar had the most antioxidant effect with significant difference with the other cultivar (p < 0.05) which can be introduced as a potent source of natural antioxidants, and the peel of three cultivars (Sweet saveh malas, Sour summer and Black peel) as a suitable source for extraction and purification of phenolic and flavonoid compound. The antioxidant capacity of pomegranate peel extract is 10 times higher than the pulp extract. PMID:24250384

  17. DNA binding specificity and sequence of Xanthomonas campestris catabolite gene activator protein-like protein.

    PubMed Central

    Dong, Q; Ebright, R H

    1992-01-01

    The Xanthomonas campestris catabolite gene activator protein-like protein (CLP) can substitute for the Escherichia coli catabolite gene activator protein (CAP) in transcription activation at the lac promoter (V. de Crecy-Lagard, P. Glaser, P. Lejeune, O. Sismeiro, C. Barber, M. Daniels, and A. Danchin, J. Bacteriol. 172:5877-5883, 1990). We show that CLP has the same DNA binding specificity as CAP at positions 5, 6, and 7 of the DNA half site. In addition, we show that the amino acids at positions 1 and 2 of the recognition helix of CLP are identical to the amino acids at positions 1 and 2 of the recognition helix of CAP:i.e., Arg at position 1 and Glu at position 2. PMID:1322886

  18. Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis.

    PubMed

    Kohansal-Nodehi, Mahdokht; Chua, John Je; Urlaub, Henning; Jahn, Reinhard; Czernik, Dominika

    2016-01-01

    Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes associated with presynaptic activity and provides a foundation for further functional analysis of key phosphosites involved in presynaptic plasticity. PMID:27115346

  19. Methods to distinguish various types of protein phosphatase activity

    SciTech Connect

    Brautigan, D.L.; Shriner, C.L.

    1988-01-01

    To distinguish the action of protein Tyr(P) and protein Ser(P)/Thr(P) phosphatases on /sup 32/P-labeled phosphoproteins in subcellular fractions different inhibitors and activators are utilized. Comparison of the effects of added compounds provides a convenient, indirect method to characterize dephosphorylation reactions. Protein Tyr(P) phosphatases are specifically inhibited by micromolar Zn2+ or vanadate, and show maximal activity in the presence of EDTA. The other class of cellular phosphatases, specific for protein Ser(P) and Thr(P) residues, are inhibited by fluoride and EDTA. In this class of enzymes two major functional types can be distinguished: those sensitive to inhibition by the heat-stable protein inhibitor-2 and not stimulated by polycations, and those not sensitive to inhibition and stimulated by polycations. Preparation of /sup 32/P-labeled Tyr(P) and Ser(P) phosphoproteins also is presented for the direct measurement of phosphatase activities in preparations by the release of acid-soluble (/sup 32/P)phosphate.

  20. Higher Total Protein Intake and Change in Total Protein Intake Affect Body Composition but Not Metabolic Syndrome Indexes in Middle-Aged Overweight and Obese Adults Who Perform Resistance and Aerobic Exercise for 36 Weeks123

    PubMed Central

    Campbell, Wayne W; Kim, Jung Eun; Amankwaah, Akua F; Gordon, Susannah L; Weinheimer-Haus, Eileen M

    2015-01-01

    Background: Studies assessing the effects of protein supplementation on changes in body composition (BC) and health rarely consider the impact of total protein intake (TPro) or the change in TPro (CTPro) from participants’ usual diets. Objective: This secondary data analysis assessed the impact of TPro and CTPro on changes in BC and metabolic syndrome (MetS) indexes in overweight and obese middle-aged adults who participated in an exercise training program. Methods: Men and women [n = 117; age: 50 ± 0.7 y, body mass index (BMI; in kg/m2): 30.1 ± 0.3; means ± SEs] performed resistance exercise 2 d/wk and aerobic exercise 1 d/wk and consumed an unrestricted diet along with 200-kcal supplements (0, 10, 20, or 30 g whey protein) twice daily for 36 wk. Protein intake was assessed via 4-d food records. Multiple linear regression model and stratified analysis were applied for data analyses. Results: Among all subjects, TPro and CTPro were inversely associated (P < 0.05) with changes in body mass, fat mass (FM), and BMI. Changes in BC were different (P < 0.05) among groups that consumed <1.0 (n = 43) vs. ≥1.0 to <1.2 (n = 29) vs. ≥1.2 g · kg−1 · d−1 (n = 45). The TPro group with ≥1.0 to <1.2 g · kg−1 · d−1 reduced FM and %FM and increased percentage of LM (%LM) compared with the lowest TPro group, whereas the TPro group with ≥1.2 g · kg−1 · d−1 presented intermediate responses on changes in FM, %FM, and %LM. The gain in LM was not different among groups. In addition, MetS indexes were not influenced by TPro and CTPro. Conclusions: In conjunction with exercise training, higher TPro promoted positive changes in BC but not in MetS indexes in overweight and obese middle-aged adults. Changes in TPro from before to during the intervention also influenced BC responses and should be considered in future research when different TPro is achieved via diet or supplements. This trial was registered at clinicaltrials.gov as NCT00812409. PMID:26246322

  1. Ubiquitously expressed transcript is a novel interacting protein of protein inhibitor of activated signal transducer and activator of transcription 2

    PubMed Central

    KONG, XIANG; MA, SHIKUN; GUO, JIAQIAN; MA, YAN; HU, YANQIU; WANG, JIANJUN; ZHENG, YING

    2015-01-01

    Protein inhibitor of activated signal transducer and activator of transcription 2 (PIAS2) is a member of the PIAS protein family. This protein family modulates the activity of several transcription factors and acts as an E3 ubiquitin ligase in the sumoylation pathway. To improve understanding of the physiological roles of PIAS2, the current study used a yeast two-hybrid system to screen mouse stem cell cDNA libraries for proteins that interact with PIAS2. The screening identified an interaction between PIAS2 and ubiquitously expressed transcript (UXT). UXT, also termed androgen receptor trapped clone-27, is an α-class prefoldin-type chaperone that acts as a coregulator for various transcription factors, including nuclear factor-κB and androgen receptor (AR). A direct interaction between PIAS2 and UXT was confirmed by direct yeast two-hybrid analysis. In vitro evidence of the association of UXT with PIAS2 was obtained by co-immunoprecipitation. Colocalization between PIAS2 and UXT was identified in the nucleus and cytoplasm of HEK 293T and human cervical carcinoma HeLa cells. The results of the current study suggested that UXT is a binding protein of PIAS2, and interaction between PIAS2 and UXT may be important for the transcriptional activation of AR. PMID:25434787

  2. Detergent activation of the binding protein in the folate radioassay

    SciTech Connect

    Hansen, S.I.; Holm, J.; Lyngbye, J.

    1982-01-01

    A minor cow's whey protein associated with ..beta..-lactoglobulin is used as binding protein in the competitive radioassay for serum and erythrocyte folate. Seeking to optimize the assay, we tested the performance of binder solutions of increasing purity. The folate binding protein was isolated from cow's whey by means of CM-Sepharose CL-6B cation-exchange chromatography, and further purified on a methotrexate-AH-Sepharose 4B affinity matrix. In contrast to ..beta..-lactoglobulin, the purified protein did not bind folate unless the detergents cetyltrimethylammonium (10 mmol/Ll) or Triton X-100 (1 g/L) were present. Such detergent activation was not needed in the presence of serum. There seems to be a striking analogy between these phenomena and the well-known reactivation of certain purified membrane-derived enzymes by surfactants (lipids/detergents).

  3. Activities at the Universal Protein Resource (UniProt)

    PubMed Central

    2014-01-01

    The mission of the Universal Protein Resource (UniProt) (http://www.uniprot.org) is to provide the scientific community with a comprehensive, high-quality and freely accessible resource of protein sequences and functional annotation. It integrates, interprets and standardizes data from literature and numerous resources to achieve the most comprehensive catalog possible of protein information. The central activities are the biocuration of the UniProt Knowledgebase and the dissemination of these data through our Web site and web services. UniProt is produced by the UniProt Consortium, which consists of groups from the European Bioinformatics Institute (EBI), the SIB Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR). UniProt is updated and distributed every 4 weeks and can be accessed online for searches or downloads. PMID:24253303

  4. Steroidogenic Acute Regulatory Protein Overexpression Correlates with Protein Kinase A Activation in Adrenocortical Adenoma.

    PubMed

    Zhou, Weiwei; Wu, Luming; Xie, Jing; Su, Tingwei; Jiang, Lei; Jiang, Yiran; Cao, Yanan; Liu, Jianmin; Ning, Guang; Wang, Weiqing

    2016-01-01

    The association of pathological features of cortisol-producing adrenocortical adenomas (ACAs) with somatic driver mutations and their molecular classification remain unclear. In this study, we explored the association between steroidogenic acute regulatory protein (StAR) expression and the driver mutations activating cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling to identify the pathological markers of ACAs. Immunohistochemical staining for StAR and mutations in the protein kinase cAMP-activated catalytic subunit alpha (PRKACA), protein kinase cAMP-dependent type I regulatory subunit alpha (PRKAR1A) and guanine nucleotide binding protein, alpha stimulating (GNAS) genes were examined in 97 ACAs. The association of StAR expression with the clinical and mutational features of the ACAs was analyzed. ACAs with mutations in PRKACA, GNAS, and PRKAR1A showed strong immunopositive staining for StAR. The concordance between high StAR expression and mutations activating cAMP/PKA signaling in the ACAs was 99.0%. ACAs with high expression of StAR had significantly smaller tumor volume (P < 0.001) and higher urinary cortisol per tumor volume (P = 0.032) than those with low expression of StAR. Our findings suggest that immunohistochemical staining for StAR is a reliable pathological approach for the diagnosis and classification of ACAs with cAMP/PKA signaling-activating mutations. PMID:27606678

  5. Continuous monitoring of a large active earth flow using an integrated GPS - automatic total station approach

    NASA Astrophysics Data System (ADS)

    Corsini, A.

    2009-04-01

    Landslide monitoring has evolved as a crucial tool in civil protection to mitigate and prevent disasters. The research presents an approach to continuous monitoring of a large-scale active earth flow using a system that integrates surface measurements obtained by a GPS and an automatic total station. With the data obtained from the system the landslide can be monitored in near-real-time and surface displacements can be directly utilized to provide early warning of slope movements and to study the behavior of the landslide, e.g. to predict timing and mechanisms of future failure. The Valoria landslide located in the northern Apennines of Italy was reactivated in 2001, 2005 and 2007 damaging roads and endangering houses. A monitoring system was installed in 2007-2008 in the frame of a civil protection plan aimed at risk mitigation. The system consists of an automatic total station measuring about 40 prisms located in the landslide to a maximum distance of 1.800 km; one double-frequency GPS receiver connects in streaming by wireless communication with 4 single-frequency GPS in side the flow. Until December 2007 the monitoring network was operated with periodic static surveying followed by the data post-processing. From September 2007 until March 2008 the landslide deformation was evaluated by periodic surveys with the total station and the GPS system. This first measure showed that the displacements were influenced by the rainfall events and by the snow melting. The total displacements measured vary from centimeter scale in the crown zone, where retrogressive movements were in progress, to over 50 m in the flow track zone. Starting in March 2008 data acquisition by the total station system and GPS were automated in order to allow continuous and near-real-time data processing. The displacement data collected in one and a half year of continuous operation show different acceleration and deceleration phases as a result of the pore water pressure distribution inside the

  6. Installing hydrolytic activity into a completely de novo protein framework.

    PubMed

    Burton, Antony J; Thomson, Andrew R; Dawson, William M; Brady, R Leo; Woolfson, Derek N

    2016-09-01

    The design of enzyme-like catalysts tests our understanding of sequence-to-structure/function relationships in proteins. Here we install hydrolytic activity predictably into a completely de novo and thermostable α-helical barrel, which comprises seven helices arranged around an accessible channel. We show that the lumen of the barrel accepts 21 mutations to functional polar residues. The resulting variant, which has cysteine-histidine-glutamic acid triads on each helix, hydrolyses p-nitrophenyl acetate with catalytic efficiencies that match the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the first report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of our system also allows the facile incorporation of unnatural side chains to improve activity and probe the catalytic mechanism. Such a predictable and robust construction of truly de novo biocatalysts holds promise for applications in chemical and biochemical synthesis. PMID:27554410

  7. Streptococcal Surface Proteins Activate the Contact System and Control Its Antibacterial Activity*

    PubMed Central

    Wollein Waldetoft, Kristofer; Svensson, Lisbeth; Mörgelin, Matthias; Olin, Anders I.; Nitsche-Schmitz, D. Patric; Björck, Lars; Frick, Inga-Maria

    2012-01-01

    Group G streptococci (GGS) are important bacterial pathogens in humans. Here, we investigated the interactions between GGS and the contact system, a procoagulant and proinflammatory proteolytic cascade that, upon activation, also generates antibacterial peptides. Two surface proteins of GGS, protein FOG and protein G (PG), were found to bind contact system proteins. Experiments utilizing contact protein-deficient human plasma and isogenic GGS mutant strains lacking FOG or PG showed that FOG and PG both activate the procoagulant branch of the contact system. In contrast, only FOG induced cleavage of high molecular weight kininogen, generating the proinflammatory bradykinin peptide and additional high molecular weight kininogen fragments containing the antimicrobial peptide NAT-26. On the other hand, PG protected the bacteria against the antibacterial effect of NAT-26. These findings underline the significance of the contact system in innate immunity and demonstrate that GGS have evolved surface proteins to exploit and modulate its effects. PMID:22648411

  8. Gi/o proteins: expression for direct activation enquiry.

    PubMed

    Di Cesare Mannelli, Lorenzo; Pacini, Alessandra; Toscano, Annarita; Fortini, Martina; Berti, Debora; Ghelardini, Carla; Galeotti, Nicoletta; Baglioni, Piero; Bartolini, Alessandro

    2006-05-01

    G protein-mediated pathways are fundamental mechanisms of cell signaling. In this paper, the expression and the characterization of the alphai1, alphai3, alphao1, beta1, and gamma2 subunits of the human G protein are described. This approach was developed to evaluate the G protein activation profile of new compounds. pCR-TOPO T7 vectors, engineered to contain the target sequences, were used to transform Escherichia coli competent cells. Subunits were over-expressed in a preparative scale as fusion proteins with a six-histidine tag, and subsequently purified by metal chelate chromatography. Afterward, the His-tag was removed by enterokinase digestion, and the secondary structures of the recombinant subunits were analyzed by circular dichroism. To assess the functionality of the subunits, the rate of GTP hydrolysis and GTPgammaS binding were evaluated both in the absence and in the presence of two modulators: the peptidic activator Mastoparan and the non-peptidic activator N-dodecyl-lysinamide (ML250). Tests were conducted on isolated alpha-subunit and on heterotrimeric alphabetagamma complex, alone or reconstituted in phospholipidic vesicles. Our results show that recombinant subunits are stable, properly folded and, fully active, which makes them suitable candidates for functional studies. PMID:16364655

  9. Protein kinase A activity and Hedgehog signaling pathway.

    PubMed

    Kotani, Tomoya

    2012-01-01

    Protein kinase A (PKA) is a well-known kinase that plays fundamental roles in a variety of biological processes. In Hedgehog-responsive cells, PKA plays key roles in proliferation and fate specification by modulating the transduction of Hedgehog signaling. In the absence of Hedgehog, a basal level of PKA activity represses the transcription of Hedgehog target genes. The main substrates of PKA in this process are the Ci/Gli family of bipotential transcription factors, which activate and repress Hedgehog target gene expression. PKA phosphorylates Ci/Gli, promoting the production of the repressor forms of Ci/Gli and thus repressing Hedgehog target gene expression. In contrast, the activation of Hedgehog signaling in response to Hedgehog increases the active forms of Ci/Gli, resulting in Hedgehog target gene expression. Because both decreased and increased levels of PKA activity cause abnormal cell proliferation and alter cell fate specification, the basal level of PKA activity in Hedgehog-responsive cells should be precisely regulated. However, the mechanism by which PKA activity is regulated remains obscure and appears to vary between cell types, tissues, and organisms. To date, two mechanisms have been proposed. One is a classical mechanism in which PKA activity is regulated by a small second messenger, cAMP; the other is a novel mechanism in which PKA activity is regulated by a protein, Misty somites. PMID:22391308

  10. Protein synthesis inhibitors reveal differential regulation of mitogen-activated protein kinase and stress-activated protein kinase pathways that converge on Elk-1.

    PubMed Central

    Zinck, R; Cahill, M A; Kracht, M; Sachsenmaier, C; Hipskind, R A; Nordheim, A

    1995-01-01

    Inhibitors of protein synthesis, such as anisomycin and cycloheximide, lead to superinduction of immediate-early genes. We demonstrate that these two drugs activate intracellular signaling pathways involving both the mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK) cascades. The activation of either pathway correlates with phosphorylation of the c-fos regulatory transcription factor Elk-1. In HeLa cells, anisomycin stabilizes c-fos mRNA when protein synthesis is inhibited to only 50%. Under these conditions, anisomycin, in contrast to cycloheximide, rapidly induces kinase activation and efficient Elk-1 phosphorylation. However, full inhibition of translation by either drug leads to prolonged activation of SAPK activity, while MAPK induction is transient. This correlates with prolonged Elk-1 phosphorylation and c-fos transcription. Elk-1 induction and c-fos activation are also observed in KB cells, in which anisomycin strongly induces SAPKs but not MAPKs. Purified p54 SAPK alpha efficiently phosphorylates the Elk-1 C-terminal domain in vitro and comigrates with anisomycin-activated kinases in in-gel kinase assays. Thus, Elk-1 provides a potential convergence point for the MAPK and SAPK signaling pathways. The activation of signal cascades and control of transcription factor function therefore represent prominent processes in immediate-early gene superinduction. PMID:7651411

  11. Solar activity variability in the IRI at high latitudes: Comparisons with GPS total electron content

    NASA Astrophysics Data System (ADS)

    Themens, David R.; Jayachandran, P. T.

    2016-04-01

    Total electron content (TEC) measurements from 10 dual-frequency GPS receivers in the Canadian High Arctic Ionospheric Network (CHAIN) are used to evaluate the performance of International Reference Ionosphere (IRI)-2007 within the Canadian sector. Throughout the region, we see systematic underestimation of daytime TEC, particularly at solar maximum, where summer and equinox root-mean-square errors reach as high as 14 total electron content units, 1 TECU = 1016 el m-2 (TECU). It is also shown that the use of a monthly IG index, in place of the IRI's standard IG12 index, leads to an improvement in TEC specification by up to 3 TECU in the polar cap and up to 6 TECU in the subauroral region during periods of short-term, large amplitude changes in solar activity. On diurnal timescales, variability in TEC is found to be underestimated by the IRI, during equinox periods, by up to 40% at subauroral latitudes and up to 70% in the polar cap region. During the winter, diurnal variations are overestimated by up to 40% in the subauroral region and are underestimated within the polar cap by up to 80%. Using collocated ionosonde data, we find IRI bottomside TEC to be within 1 TECU of observation with errors largest during the equinoxes. For the topside we find good agreement during the winter but significant underestimation of topside TEC by the IRI during summer and equinox periods, exceeding 6 TECU at times. By ingesting measured NmF2 into the IRI, we show that the topside thickness parameterization is the source of the bulk of the observed TEC errors.

  12. Immersion freezing of ice nucleating active protein complexes

    NASA Astrophysics Data System (ADS)

    Hartmann, S.; Augustin, S.; Clauss, T.; Voigtländer, J.; Niedermeier, D.; Wex, H.; Stratmann, F.

    2012-08-01

    Biological particles, e.g. bacteria and their Ice Nucleating Active (INA) protein complexes, might play an important role for the ice formation in atmospheric mixed-phase clouds. Therefore, the immersion freezing behavior of INA protein complexes generated from a SnomaxTM solution/suspension was investigated as function of temperature in a range of -5 °C to -38 °C at the Leipzig Aerosol Cloud Interaction Simulator (LACIS). The immersion freezing of droplets containing small numbers of INA protein complexes occurs in a temperature range of -7 °C and -10 °C. The experiments performed in the lower temperature range, where all droplets freeze which contain at least one INA protein complex, are used to determine the average number of INA protein complexes present, assuming that the INA protein complexes are Poisson distributed over the droplet ensemble. Knowing the average number of INA protein complexes, the heterogeneous ice nucleation rate and rate coefficient of a single INA protein complex is determined by using the newly-developed CHESS model (stoCHastic model of idEntical poiSSon distributed ice nuclei). Therefore, we assume the ice nucleation process to be of stochastic nature, and a parameterization of the INA protein complex's nucleation rate. Analyzing the results of immersion freezing experiments from literature (SnomaxTM and Pseudomonas syringae bacteria), to results gained in this study, demonstrates that first, a similar temperature dependence of the heterogeneous ice nucleation rate for a single INA protein complex was found in all experiments, second, the shift of the ice fraction curves to higher temperatures can be explained consistently by a higher average number of INA protein complexes being present in the droplet ensemble, and finally the heterogeneous ice nucleation rate of one single INA protein complex might be also applicable for intact Pseudomonas syringae bacteria cells. The results obtained in this study allow a new perspective on the

  13. Does Physical Activity Increase After Total Hip or Knee Arthroplasty for Osteoarthritis? A Systematic Review.

    PubMed

    Arnold, John B; Walters, Julie L; Ferrar, Katia E

    2016-06-01

    Study Design Systematic review. Background Despite improvements in self-reported symptoms and perceived functional ability after total hip arthroplasty (THA) and total knee arthroplasty (TKA), it is unclear whether changes in objectively measured physical activity (PA) occur after surgery. Objective To determine if objectively measured PA increases after THA and TKA in adults with osteoarthritis. Methods Five electronic databases were searched from inception to March 3, 2015. All study designs objectively measuring PA before and after THA or TKA were eligible, including randomized controlled trials, cohort studies, and case-control studies. Two reviewers independently screened abstracts and full texts and extracted study demographic, PA, and clinical outcome data. Standardized mean differences (SMDs) and 95% confidence intervals were calculated for accelerometer- and pedometer-derived estimates of PA. Risk of methodological bias was assessed with Critical Appraisal Skills Programme checklists. Results Eight studies with a total of 373 participants (238 TKA, 135 THA) were included. Findings were mixed regarding improvement in objectively measured PA at 6 months after THA (SMDs, -0.20 to 1.80) and TKA (SMDs, -0.36 to 0.63). Larger improvements from 2 studies at 1 year postsurgery were generally observed after THA (SMDs, 0.39 to 0.79) and TKA (SMDs, 0.10 to 0.85). However, at 1 year, PA levels were still considerably lower than those of healthy controls (THA SMDs, -0.25 to -0.77; TKA SMDs, -1.46 to -1.80). Risk-of-bias scores ranged from 3 to 9 out of 11 (27%-82%) for cohort studies, and from 3 to 8 out of 10 (30%-80%) for case-control studies. Conclusion The best available evidence indicates negligible changes in PA at 6 months after THA or TKA, with limited evidence for larger changes at 1 year after surgery. In the 4 studies that reported control-group data, postoperative PA levels were still considerably less than those of healthy controls. Improved perioperative

  14. Estrogen receptor α inhibitor activates the unfolded protein response, blocks protein synthesis, and induces tumor regression.

    PubMed

    Andruska, Neal D; Zheng, Xiaobin; Yang, Xujuan; Mao, Chengjian; Cherian, Mathew M; Mahapatra, Lily; Helferich, William G; Shapiro, David J

    2015-04-14

    Recurrent estrogen receptor α (ERα)-positive breast and ovarian cancers are often therapy resistant. Using screening and functional validation, we identified BHPI, a potent noncompetitive small molecule ERα biomodulator that selectively blocks proliferation of drug-resistant ERα-positive breast and ovarian cancer cells. In a mouse xenograft model of breast cancer, BHPI induced rapid and substantial tumor regression. Whereas BHPI potently inhibits nuclear estrogen-ERα-regulated gene expression, BHPI is effective because it elicits sustained ERα-dependent activation of the endoplasmic reticulum (EnR) stress sensor, the unfolded protein response (UPR), and persistent inhibition of protein synthesis. BHPI distorts a newly described action of estrogen-ERα: mild and transient UPR activation. In contrast, BHPI elicits massive and sustained UPR activation, converting the UPR from protective to toxic. In ERα(+) cancer cells, BHPI rapidly hyperactivates plasma membrane PLCγ, generating inositol 1,4,5-triphosphate (IP3), which opens EnR IP3R calcium channels, rapidly depleting EnR Ca(2+) stores. This leads to activation of all three arms of the UPR. Activation of the PERK arm stimulates phosphorylation of eukaryotic initiation factor 2α (eIF2α), resulting in rapid inhibition of protein synthesis. The cell attempts to restore EnR Ca(2+) levels, but the open EnR IP3R calcium channel leads to an ATP-depleting futile cycle, resulting in activation of the energy sensor AMP-activated protein kinase and phosphorylation of eukaryotic elongation factor 2 (eEF2). eEF2 phosphorylation inhibits protein synthesis at a second site. BHPI's novel mode of action, high potency, and effectiveness in therapy-resistant tumor cells make it an exceptional candidate for further mechanistic and therapeutic exploration. PMID:25825714

  15. Design, Synthesis, and Investigation of Protein Kinase C Inhibitors: Total Syntheses of (+)-Calphostin D, (+)- Phleichrome, Cercosporin and New Photoactive Perylenequinones

    PubMed Central

    Morgan, Barbara J.; Dey, Sangeeta; Johnson, Steven W.; Kozlowski, Marisa C.

    2010-01-01

    The total syntheses of the PKC inhibitors (+)-calphostin D, (+)-phleichrome, cercosporin, and 10 novel perylenequinones are detailed. The highly convergent and flexible strategy developed employed an enantioselective oxidative biaryl coupling and a double cuprate epoxide opening, allowing the selective syntheses of all the possible stereoisomers in pure form. In addition, this strategy permitted rapid access to a broad range of analogs, including those not accessible from the natural products. These compounds provided a powerful means for evaluation of the perylenequinones structural features necessary to PKC activity. Simpler analogs were discovered with superior PKC inhibitory properties and superior photopotentiation in cancer cell lines relative to the more complex natural products. PMID:19489582

  16. Activation of immobilized, biotinylated choleragen AI protein by a 19-kilodalton guanine nucleotide-binding protein.

    PubMed

    Noda, M; Tsai, S C; Adamik, R; Bobak, D A; Moss, J; Vaughan, M

    1989-09-19

    Cholera toxin catalyzes the ADP-ribosylation that results in activation of the stimulatory guanine nucleotide-binding protein of the adenylyl cyclase system, known as Gs. The toxin also ADP-ribosylates other proteins and simple guanidino compounds and auto-ADP-ribosylates its AI protein (CTA1). All of the ADP-ribosyltransferase activities of CTAI are enhanced by 19-21-kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors, or ARFs. CTAI contains a single cysteine located near the carboxy terminus. CTAI was immobilized through this cysteine by reaction with iodoacetyl-N-biotinyl-hexylenediamine and binding of the resulting biotinylated protein to avidin-agarose. Immobilized CTAI catalyzed the ARF-stimulated ADP-ribosylation of agmatine. The reaction was enhanced by detergents and phospholipid, but the fold stimulation by purified sARF-II from bovine brain was considerably less than that observed with free CTA. ADP-ribosylation of Gsa by immobilized CTAI, which was somewhat enhanced by sARF-II, was much less than predicted on the basis of the NAD:agmatine ADP-ribosyltransferase activity. Immobilized CTAI catalyzed its own auto-ADP-ribosylation as well as the ADP-ribosylation of the immobilized avidin and CTA2, with relatively little stimulation by sARF-II. ADP-ribosylation of CTA2 by free CTAI is minimal. These observations are consistent with the conclusion that the cysteine near the carboxy terminus of the toxin is not critical for ADP-ribosyltransferase activity or for its regulation by sARF-II. Biotinylation and immobilization of the toxin through this cysteine may, however, limit accessibility to Gsa or SARF-II, or perhaps otherwise reduce interaction with these proteins whether as substrates or activator. PMID:2514798

  17. Reassessing the Potential Activities of Plant CGI-58 Protein.

    PubMed

    Khatib, Abdallah; Arhab, Yani; Bentebibel, Assia; Abousalham, Abdelkarim; Noiriel, Alexandre

    2016-01-01

    Comparative Gene Identification-58 (CGI-58) is a widespread protein found in animals and plants. This protein has been shown to participate in lipolysis in mice and humans by activating Adipose triglyceride lipase (ATGL), the initial enzyme responsible for the triacylglycerol (TAG) catabolism cascade. Human mutation of CGI-58 is the cause of Chanarin-Dorfman syndrome, an orphan disease characterized by a systemic accumulation of TAG which engenders tissue disorders. The CGI-58 protein has also been shown to participate in neutral lipid metabolism in plants and, in this case, a mutation again provokes TAG accumulation. Although its roles as an ATGL coactivator and in lipid metabolism are quite clear, the catalytic activity of CGI-58 is still in question. The acyltransferase activities of CGI-58 have been speculated about, reported or even dismissed and experimental evidence that CGI-58 expressed in E. coli possesses an unambiguous catalytic activity is still lacking. To address this problem, we developed a new set of plasmids and site-directed mutants to elucidate the in vivo effects of CGI-58 expression on lipid metabolism in E. coli. By analyzing the lipid composition in selected E. coli strains expressing CGI-58 proteins, and by reinvestigating enzymatic tests with adequate controls, we show here that recombinant plant CGI-58 has none of the proposed activities previously described. Recombinant plant and mouse CGI-58 both lack acyltransferase activity towards either lysophosphatidylglycerol or lysophosphatidic acid to form phosphatidylglycerol or phosphatidic acid and recombinant plant CGI-58 does not catalyze TAG or phospholipid hydrolysis. However, expression of recombinant plant CGI-58, but not mouse CGI-58, led to a decrease in phosphatidylglycerol in all strains of E. coli tested, and a mutation of the putative catalytic residues restored a wild-type phenotype. The potential activities of plant CGI-58 are subsequently discussed. PMID:26745266

  18. Reassessing the Potential Activities of Plant CGI-58 Protein

    PubMed Central

    Khatib, Abdallah; Arhab, Yani; Bentebibel, Assia; Abousalham, Abdelkarim; Noiriel, Alexandre

    2016-01-01

    Comparative Gene Identification-58 (CGI-58) is a widespread protein found in animals and plants. This protein has been shown to participate in lipolysis in mice and humans by activating Adipose triglyceride lipase (ATGL), the initial enzyme responsible for the triacylglycerol (TAG) catabolism cascade. Human mutation of CGI-58 is the cause of Chanarin-Dorfman syndrome, an orphan disease characterized by a systemic accumulation of TAG which engenders tissue disorders. The CGI-58 protein has also been shown to participate in neutral lipid metabolism in plants and, in this case, a mutation again provokes TAG accumulation. Although its roles as an ATGL coactivator and in lipid metabolism are quite clear, the catalytic activity of CGI-58 is still in question. The acyltransferase activities of CGI-58 have been speculated about, reported or even dismissed and experimental evidence that CGI-58 expressed in E. coli possesses an unambiguous catalytic activity is still lacking. To address this problem, we developed a new set of plasmids and site-directed mutants to elucidate the in vivo effects of CGI-58 expression on lipid metabolism in E. coli. By analyzing the lipid composition in selected E. coli strains expressing CGI-58 proteins, and by reinvestigating enzymatic tests with adequate controls, we show here that recombinant plant CGI-58 has none of the proposed activities previously described. Recombinant plant and mouse CGI-58 both lack acyltransferase activity towards either lysophosphatidylglycerol or lysophosphatidic acid to form phosphatidylglycerol or phosphatidic acid and recombinant plant CGI-58 does not catalyze TAG or phospholipid hydrolysis. However, expression of recombinant plant CGI-58, but not mouse CGI-58, led to a decrease in phosphatidylglycerol in all strains of E. coli tested, and a mutation of the putative catalytic residues restored a wild-type phenotype. The potential activities of plant CGI-58 are subsequently discussed. PMID:26745266

  19. In vitro antithrombotic activities of peanut protein hydrolysates.

    PubMed

    Zhang, Shao Bing

    2016-07-01

    The antithrombotic activities of peanut protein hydrolysates were investigated using a microplates assay. When peanut proteins were hydrolyzed to a limited extent by various enzymes, their thrombin inhibitory abilities were significantly enhanced. However, the resultant hydrolysates showed significantly different activities even at the same degrees of hydrolysis. The hydrolysates generated by Alcalase 2.4L displayed the best antithrombotic activities and the hydrolysis process was further optimized by response surface methodology. The antithrombotic activities were increased to 86% based on a protein concentration of 50mg/ml under the optimal conditions: pH 8.5, enzyme concentration of 5000IU/g of peanut proteins, and 2h hydrolysis time at 50°C. The Alcalase 2.4L crude hydrolysates were then fractionated successively by preparative and semi-preparative reverse-phase high-performance liquid chromatography (RP-HPLC). The peptide fraction collected inhibited thrombin-catalyzed coagulation of fibrinogen completely at a concentration of 0.4mg/ml, with an antithrombotic activity close to that of heparin at quite a low concentration (0.2mg/ml). This peptide fraction was further analyzed by online reverse-phase ultra-performance liquid chromatography (RP-UPLC) coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and three new peptides were identified as Ser-Trp-Ala-Gln-Leu, Gly-Asn-His-Glu-Ala-Gly-Glu and Cys-Phe-Asn-Glu-Tyr-Glu, respectively. This research provided an effective way to produce antithrombotic peptides from peanut proteins, and also helped to elucidate the structure-function relationships of peanut peptides. PMID:26920259

  20. Association of total daily physical activity with disability in community-dwelling older persons: a prospective cohort study

    PubMed Central

    2012-01-01

    Background Based on findings primarily using self-report measures, physical activity has been recommended to reduce disability in old age. Collecting objective measures of total daily physical activity in community-dwelling older adults is uncommon, but might enhance the understanding of the relationship of physical activity and disability. We examined whether greater total daily physical activity was associated with less report of disability in the elderly. Methods Data were from the Rush Memory and Aging Project, a longitudinal prospective cohort study of common, age-related, chronic conditions. Total daily physical activity was measured in community-dwelling participants with an average age of 82 using actigraphy for approximately 9 days. Disability was measured via self-reported basic activities of daily living (ADL). The odds ratio and 95% Confidence Interval (CI) were determined for the baseline association of total daily physical activity and ADL disability using a logistic regression model adjusted for age, education level, gender and self-report physical activity. In participants without initial report of ADL disability, the hazard ratio and 95% CI were determined for the relationship of baseline total daily physical activity and the development of ADL disability using a discrete time Cox proportional hazard model adjusted for demographics and self-report physical activity. Results In 870 participants, the mean total daily physical activity was 2. 9 × 105 counts/day (range in 105 counts/day = 0.16, 13. 6) and the mean hours/week of self-reported physical activity was 3.2 (SD = 3.6). At baseline, 718 (82.5%) participants reported being independent in all ADLs. At baseline, total daily physical activity was protective against disability (OR per 105 counts/day difference = 0.55; 95% CI = 0.47, 0.65). Of the participants without baseline disability, 584 were followed for 3.4 years on average. Each 105 counts/day additional total daily physical activity

  1. Correlation of diacylglycerol level and protein kinase C activity in rat retina to retinal circulation.

    PubMed

    Shiba, T; Inoguchi, T; Sportsman, J R; Heath, W F; Bursell, S; King, G L

    1993-11-01

    The increases in diacylglycerol (DAG) level and protein kinase C (PKC) activity have been characterized biochemically and functionally in the retina and the brain of diabetic rats as well as in cultured vascular cells. PKC specific activities were increased in the membraneous fraction of retina from streptozotocin (STZ)-induced diabetic rats and the genetically determined diabetic BB rats, respectively, after 1 or 2 wk of diabetes, compared with control. The ratio of total PKC activities from membraneous and cytosol fractions was also increased in the retina of diabetic rats. With diabetes, all the isoenzymes and the total DAG level were increased in the rat retina, whereas no changes were found in the rat brain. Insulin treatment normalized plasma glucose levels and partially prevented the increases in the membraneous PKC activity and all the isoenzymes in the retina. In the retinal endothelial cells, the total DAG level and PKC specific activities are increased by 36 and 22%, respectively, in the membraneous pool when the glucose levels are changed from 5.5 to 22 mM. Activation of PKC activity and isoform beta II by the vitreal injection of phorbol dibutyrate mimicked the abnormal retinal blood circulation observed in diabetic rats (2.22 +/- 0.24 vs. 1.83 +/- 0.40 s). Thus diabetes and elevated glucose levels will increase DAG level and PKC activities and its isoenzyme specifically in vascular cells and may affect retinal hemodynamics. PMID:8238505

  2. Drivers shaping the diversity and biogeography of total and active bacterial communities in the South China Sea

    PubMed Central

    Zhang, Yao; Zhao, Zihao; Dai, Minhan; Jiao, Nianzhi; Herndl, Gerhard J

    2014-01-01

    To test the hypothesis that different drivers shape the diversity and biogeography of the total and active bacterial community, we examined the bacterial community composition along two transects, one from the inner Pearl River estuary to the open waters of the South China Sea (SCS) and the other from the Luzon Strait to the SCS basin, using 454 pyrosequencing of the 16S rRNA and 16S rRNA gene (V1-3 regions) and thereby characterizing the active and total bacterial community, respectively. The diversity and biogeographic patterns differed substantially between the active and total bacterial communities. Although the composition of both the total and active bacterial community was strongly correlated with environmental factors and weakly correlated with geographic distance, the active bacterial community displayed higher environmental sensitivity than the total community and particularly a greater distance effect largely caused by the active assemblage from deep waters. The 16S rRNA vs. rDNA relationships indicated that the active bacteria were low in relative abundance in the SCS. This might be due to a high competition between active bacterial taxa as indicated by our community network models. Based on these analyses, we speculate that high competition could cause some dispersal limitation of the active bacterial community resulting in a distinct distance-decay relationship. Altogether, our results indicated that the biogeographic distribution of bacteria in the SCS is the result of both environmental control and distance decay. PMID:24684298

  3. Dietary Intake of Total, Animal, and Vegetable Protein and Risk of Type 2 Diabetes in the European Prospective Investigation into Cancer and Nutrition (EPIC)-NL Study

    PubMed Central

    Sluijs, Ivonne; Beulens, Joline W.J.; van der A, Daphne L.; Spijkerman, Annemieke M.W.; Grobbee, Diederick E.; van der Schouw, Yvonne T.

    2010-01-01

    OBJECTIVE Dietary recommendations are focused mainly on relative dietary fat and carbohydrate content in relation to diabetes risk. Meanwhile, high-protein diets may contribute to disturbance of glucose metabolism, but evidence from prospective studies is scarce. We examined the association among dietary total, vegetable, and animal protein intake and diabetes incidence and whether consuming 5 energy % from protein at the expense of 5 energy % from either carbohydrates or fat was associated with diabetes risk. RESEARCH DESIGN AND METHODS A prospective cohort study was conducted among 38,094 participants of the European Prospective Investigation into Cancer and Nutrition (EPIC)-NL study. Dietary protein intake was measured with a validated food frequency questionnaire. Incident diabetes was verified against medical records. RESULTS During 10 years of follow-up, 918 incident cases of diabetes were documented. Diabetes risk increased with higher total protein (hazard ratio 2.15 [95% CI 1.77–2.60] highest vs. lowest quartile) and animal protein (2.18 [1.80–2.63]) intake. Adjustment for confounders did not materially change these results. Further adjustment for adiposity measures attenuated the associations. Vegetable protein was not related to diabetes. Consuming 5 energy % from total or animal protein at the expense of 5 energy % from carbohydrates or fat increased diabetes risk. CONCLUSIONS Diets high in animal protein are associated with an increased diabetes risk. Our findings also suggest a similar association for total protein itself instead of only animal sources. Consumption of energy from protein at the expense of energy from either carbohydrates or fat may similarly increase diabetes risk. This finding indicates that accounting for protein content in dietary recommendations for diabetes prevention may be useful. PMID:19825820

  4. Pivotal Role of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 in Inflammatory Pulmonary Diseases

    PubMed Central

    Qian, Feng; Deng, Jing; Wang, Gang; Ye, Richard D.; Christman, John W.

    2016-01-01

    Mitogen-activated protein kinase (MAPK)-activated protein kinase (MK2) is exclusively regulated by p38 MAPK in vivo. Upon activation of p38 MAPK, MK2 binds with p38 MAPK, leading to phosphorylation of TTP, Hsp27, Akt and Cdc25 that are involved in regulation of various essential cellular functions. In this review, we discuss current knowledge about molecular mechanisms of MK2 in regulation of TNF-α production, NADPH oxidase activation, neutrophil migration, and DNA-damage-induced cell cycle arrest which are involved in the molecular pathogenesis of acute lung injury, pulmonary fibrosis, and non-small-cell lung cancer. Collectively current and emerging new information indicate that developing MK2 inhibitors and blocking MK2-mediated signal pathways is a potential therapeutic strategy for treatment of inflammatory and fibrotic lung diseases and lung cancer. PMID:26119506

  5. Design of a Split Intein with Exceptional Protein Splicing Activity.

    PubMed

    Stevens, Adam J; Brown, Zachary Z; Shah, Neel H; Sekar, Giridhar; Cowburn, David; Muir, Tom W

    2016-02-24

    Protein trans-splicing (PTS) by split inteins has found widespread use in chemical biology and biotechnology. Herein, we describe the use of a consensus design approach to engineer a split intein with enhanced stability and activity that make it more robust than any known PTS system. Using batch mutagenesis, we first conduct a detailed analysis of the difference in splicing rates between the Npu (fast) and Ssp (slow) split inteins of the DnaE family and find that most impactful residues lie on the second shell of the protein, directly adjacent to the active site. These residues are then used to generate an alignment of 73 naturally occurring DnaE inteins that are predicted to be fast. The consensus sequence from this alignment (Cfa) demonstrates both rapid protein splicing and unprecedented thermal and chaotropic stability. Moreover, when fused to various proteins including antibody heavy chains, the N-terminal fragment of Cfa exhibits increased expression levels relative to other N-intein fusions. The durability and efficiency of Cfa should improve current intein based technologies and may provide a platform for the development of new protein chemistry techniques. PMID:26854538

  6. A Novel Method for Assessing the Chaperone Activity of Proteins.

    PubMed

    Hristozova, Nevena; Tompa, Peter; Kovacs, Denes

    2016-01-01

    Protein chaperones are molecular machines which function both during homeostasis and stress conditions in all living organisms. Depending on their specific function, molecular chaperones are involved in a plethora of cellular processes by playing key roles in nascent protein chain folding, transport and quality control. Among stress protein families-molecules expressed during adverse conditions, infection, and diseases-chaperones are highly abundant. Their molecular functions range from stabilizing stress-susceptible molecules and membranes to assisting the refolding of stress-damaged proteins, thereby acting as protective barriers against cellular damage. Here we propose a novel technique to test and measure the capability for protective activity of known and putative chaperones in a semi-high throughput manner on a plate reader. The current state of the art does not allow the in vitro measurements of chaperone activity in a highly parallel manner with high accuracy or high reproducibility, thus we believe that the method we report will be of significant benefit in this direction. The use of this method may lead to a considerable increase in the number of experimentally verified proteins with such functions, and may also allow the dissection of their molecular mechanism for a better understanding of their function. PMID:27564234

  7. A Novel Method for Assessing the Chaperone Activity of Proteins

    PubMed Central

    Hristozova, Nevena; Tompa, Peter; Kovacs, Denes

    2016-01-01

    Protein chaperones are molecular machines which function both during homeostasis and stress conditions in all living organisms. Depending on their specific function, molecular chaperones are involved in a plethora of cellular processes by playing key roles in nascent protein chain folding, transport and quality control. Among stress protein families–molecules expressed during adverse conditions, infection, and diseases–chaperones are highly abundant. Their molecular functions range from stabilizing stress-susceptible molecules and membranes to assisting the refolding of stress-damaged proteins, thereby acting as protective barriers against cellular damage. Here we propose a novel technique to test and measure the capability for protective activity of known and putative chaperones in a semi-high throughput manner on a plate reader. The current state of the art does not allow the in vitro measurements of chaperone activity in a highly parallel manner with high accuracy or high reproducibility, thus we believe that the method we report will be of significant benefit in this direction. The use of this method may lead to a considerable increase in the number of experimentally verified proteins with such functions, and may also allow the dissection of their molecular mechanism for a better understanding of their function. PMID:27564234

  8. Protein kinase domain of twitchin has protein kinase activity and an autoinhibitory region.

    PubMed

    Lei, J; Tang, X; Chambers, T C; Pohl, J; Benian, G M

    1994-08-19

    Twitchin is a 753-kDa polypeptide located in the muscle A-bands of the nematode, Caenorhabditis elegans. It consists of multiple copies of both fibronectin III and immunoglobulin C2 domains and, near the C terminus, a protein kinase domain with greatest homology to the catalytic domains of myosin light chain kinases. We have expressed and purified from Escherichia coli twitchin's protein kinase catalytic core and flanking sequences that do not include fibronectin III and immunoglobulin C2 domains. The protein was shown to phosphorylate a model substrate and to undergo autophosphorylation. The autophosphorylation occurs at a slow rate, attaining a maximum at 3 h with a stoichiometry of about 1.0 mol of phosphate/mol of protein, probably through an intramolecular mechanism. Sequence analysis of proteolytically derived phosphopeptides revealed that autophosphorylation occurred N-terminal to the catalytic core, predominantly at Thr-5910, with possible minor sites at Ser5912 and/or Ser-5913. This portion of twitchin (residues 5890-6268) was also phosphorylated in vitro by protein kinase C in the absence of calcium and phosphotidylserine, but not by cAMP-dependent protein kinase. By comparing the activities of three twitchin segments, the enzyme appears to be inhibited by the 60-amino acid residues lying just C-terminal to the kinase catalytic core. Thus, like a number of other protein kinases including myosin light chain kinases, the twitchin kinase appears to be autoregulated. PMID:8063727

  9. NRIP, a novel calmodulin binding protein, activates calcineurin to dephosphorylate human papillomavirus E2 protein.

    PubMed

    Chang, Szu-Wei; Tsao, Yeou-Ping; Lin, Chia-Yi; Chen, Show-Li

    2011-07-01

    Previously, we found a gene named nuclear receptor interaction protein (NRIP) (or DCAF6 or IQWD1). We demonstrate that NRIP is a novel binding protein for human papillomavirus 16 (HPV-16) E2 protein. HPV-16 E2 and NRIP can directly associate into a complex in vivo and in vitro, and the N-terminal domain of NRIP interacts with the transactivation domain of HPV-16 E2. Only full-length NRIP can stabilize E2 protein and induce HPV gene expression, and NRIP silenced by two designed small interfering RNAs (siRNAs) decreases E2 protein levels and E2-driven gene expression. We found that NRIP can directly bind with calmodulin in the presence of calcium through its IQ domain, resulting in decreased E2 ubiquitination and increased E2 protein stability. Complex formation between NRIP and calcium/calmodulin activates the phosphatase calcineurin to dephosphorylate E2 and increase E2 protein stability. We present evidences for E2 phosphorylation in vivo and show that NRIP acts as a scaffold to recruit E2 and calcium/calmodulin to prevent polyubiquitination and degradation of E2, enhancing E2 stability and E2-driven gene expression. PMID:21543494

  10. Redox Control of Protein Arginine Methyltransferase 1 (PRMT1) Activity.

    PubMed

    Morales, Yalemi; Nitzel, Damon V; Price, Owen M; Gui, Shanying; Li, Jun; Qu, Jun; Hevel, Joan M

    2015-06-12

    Elevated levels of asymmetric dimethylarginine (ADMA) correlate with risk factors for cardiovascular disease. ADMA is generated by the catabolism of proteins methylated on arginine residues by protein arginine methyltransferases (PRMTs) and is degraded by dimethylarginine dimethylaminohydrolase. Reports have shown that dimethylarginine dimethylaminohydrolase activity is down-regulated and PRMT1 protein expression is up-regulated under oxidative stress conditions, leading many to conclude that ADMA accumulation occurs via increased synthesis by PRMTs and decreased degradation. However, we now report that the methyltransferase activity of PRMT1, the major PRMT isoform in humans, is impaired under oxidative conditions. Oxidized PRMT1 displays decreased activity, which can be rescued by reduction. This oxidation event involves one or more cysteine residues that become oxidized to sulfenic acid (-SOH). We demonstrate a hydrogen peroxide concentration-dependent inhibition of PRMT1 activity that is readily reversed under physiological H2O2 concentrations. Our results challenge the unilateral view that increased PRMT1 expression necessarily results in increased ADMA synthesis and demonstrate that enzymatic activity can be regulated in a redox-sensitive manner. PMID:25911106

  11. Mitogen Activated Protein kinase signal transduction pathways in the prostate

    PubMed Central

    Maroni, Paul D; Koul, Sweaty; Meacham, Randall B; Koul, Hari K

    2004-01-01

    The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy. PMID:15219238

  12. Activation of autophagy by unfolded proteins during endoplasmic reticulum stress.

    PubMed

    Yang, Xiaochen; Srivastava, Renu; Howell, Stephen H; Bassham, Diane C

    2016-01-01

    Endoplasmic reticulum stress is defined as the accumulation of unfolded proteins in the endoplasmic reticulum, and is caused by conditions such as heat or agents that cause endoplasmic reticulum stress, including tunicamycin and dithiothreitol. Autophagy, a major pathway for degradation of macromolecules in the vacuole, is activated by these stress agents in a manner dependent on inositol-requiring enzyme 1b (IRE1b), and delivers endoplasmic reticulum fragments to the vacuole for degradation. In this study, we examined the mechanism for activation of autophagy during endoplasmic reticulum stress in Arabidopsis thaliana. The chemical chaperones sodium 4-phenylbutyrate and tauroursodeoxycholic acid were found to reduce tunicamycin- or dithiothreitol-induced autophagy, but not autophagy caused by unrelated stresses. Similarly, over-expression of BINDING IMMUNOGLOBULIN PROTEIN (BIP), encoding a heat shock protein 70 (HSP70) molecular chaperone, reduced autophagy. Autophagy activated by heat stress was also found to be partially dependent on IRE1b and to be inhibited by sodium 4-phenylbutyrate, suggesting that heat-induced autophagy is due to accumulation of unfolded proteins in the endoplasmic reticulum. Expression in Arabidopsis of the misfolded protein mimics zeolin or a mutated form of carboxypeptidase Y (CPY*) also induced autophagy in an IRE1b-dependent manner. Moreover, zeolin and CPY* partially co-localized with the autophagic body marker GFP-ATG8e, indicating delivery to the vacuole by autophagy. We conclude that accumulation of unfolded proteins in the endoplasmic reticulum is a trigger for autophagy under conditions that cause endoplasmic reticulum stress. PMID:26616142

  13. A GTPase-activating protein for the G protein Galphaz. Identification, purification, and mechanism of action.

    PubMed

    Wang, J; Tu, Y; Woodson, J; Song, X; Ross, E M

    1997-02-28

    A GTPase-activating protein (GAP) specific for Galphaz was identified in brain, spleen, retina, platelet, C6 glioma cells, and several other tissues and cells. Gz GAP from bovine brain is a membrane protein that is refractory to solubilization with most detergents but was solubilized with warm Triton X-100 and purified up to 50,000-fold. Activity is associated with at least two separate proteins of Mr approximately 22,000 and 28,000, both of which have similar specific activities. In an assay that measures the rate of hydrolysis of GTP pre-bound to detergent-soluble Galphaz, the GAP accelerates hydrolysis over 200-fold, from 0.014 to 3 min -1 at 15 degrees C, or to >/=20 min-1 at 30 degrees C. It does not alter rates of nucleotide association or dissociation. When co-reconstituted into phospholipid vesicles with trimeric Gz and m2 muscarinic receptor, Gz GAP accelerates agonist-stimulated steady-state GTP hydrolysis as predicted by its effect on the hydrolytic reaction. In the single turnover assay, the Km of the GAP for Galphaz-GTP is 2 nM. Its activity is inhibited by Galphaz-guanosine 5'-O-thiotriphosphate (Galphaz-GTPgammaS) or by Galphaz-GDP/AlF4 with Ki approximately 1.5 nM for both species; Galphaz-GDP does not inhibit. G protein betagamma subunits inhibit Gz GAP activity, apparently by forming a GTP-Galphazbetagamma complex that is a poor GAP substrate. Gz GAP displays little GAP activity toward Galphai1 or Galphao, but its activity with Galphaz is competitively inhibited by both Galphai1 and Galphao at nanomolar concentrations when they are bound to GTPgammaS but not to GDP. Neither phospholipase C-beta1 (a Gq GAP) nor several adenylyl cyclase isoforms display Gz GAP activity. PMID:9038185

  14. Current activities of the Yersinia effector protein YopM.

    PubMed

    Höfling, Sabrina; Grabowski, Benjamin; Norkowski, Stefanie; Schmidt, M Alexander; Rüter, Christian

    2015-05-01

    Yersinia outer protein M (YopM) belongs to the group of Yop effector proteins, which are highly conserved among pathogenic Yersinia species. During infection, the effectors are delivered into the host cell cytoplasm via the type 3 secretion system to subvert the host immune response and support the survival of Yersinia. In contrast to the other Yop effectors, YopM does not possess a known enzymatic activity and its molecular mechanism(s) of action remain(s) poorly understood. However, YopM was shown to promote colonization and dissemination of Yersinia, thus being crucial for the pathogen's virulence in vivo. Moreover, YopM interacts with several host cell proteins and might utilize them to execute its anti-inflammatory activities. The results obtained so far indicate that YopM is a multifunctional protein that counteracts the host immune defense by multiple activities, which are at least partially independent of each other. Finally, its functions seem to be also influenced by differences between the specific YopM isoforms expressed by Yersinia subspecies. In this review, we focus on the global as well as more specific contribution of YopM to virulence of Yersinia during infection and point out the various extra- and intracellular molecular functions of YopM. In addition, the novel cell-penetrating ability of recombinant YopM and its potential applications as a self-delivering immunomodulatory therapeutic will be discussed. PMID:25865799

  15. Evaluation of anti-oxidant activities and total phenolic content of Chromolaena odorata.

    PubMed

    Srinivasa Rao, K; Chaudhury, Pradeep Kumar; Pradhan, Anshuman

    2010-02-01

    The aim of this study was to assess the in vitro potential of chloroform extract of Chromolaena odorata leaves. The DPPH activity of the extract (0.1-5 mg/ml) was increased in a dose dependent manner, which was found in the range of 23.48-91.61% as compared to ascorbic acid (33.69-94.10%). The IC50 values of chloroform extract in DPPH radical, hydroxyl radical, nitric oxide, ABTS radical were obtained to be 0.31, 0.43, 0.28 and 1.32 mg/ml, respectively. However, the IC50 values for the standard ascorbic acid were noted to be 0.24, 0.41, 0.23 and 1 mg/ml, respectively. Measurement of total phenolic content of the chloroform extract of C. odorata was achieved using Folin-Ciocalteau reagent containing 242.2 mg/g of phenolic content, which was found significantly higher when compared to reference standard gallic acid. The results obtained in this study clearly indicate that C. odorata has a significant potential to use as a natural anti-oxidant agent. PMID:20026159

  16. Decreased Total Antioxidant Activity in Major Depressive Disorder Patients Non-Responsive to Antidepressant Treatment

    PubMed Central

    Baek, Song-Eun; Lee, Gyoung-Ja; Rhee, Chang-Kyu; Rho, Dae-Young; Kim, Do-Hoon; Huh, Sun

    2016-01-01

    Objective This study aimed to evaluate the total antioxidant activity (TAA) in patients with major depressive disorder (MDD) and the effect of antidepressants on TAA using a novel potentiometric method. Methods Twenty-eight patients with MDD and thirty-one healthy controls were enrolled in this study. The control group comprised 31 healthy individuals matched for gender, drinking and smoking status. We assessed symptoms of depression using the Hamilton Depression Rating Scale (HAMD) and the Beck Depression Inventory (BDI). We measured TAA using potentiometry. All measurements were made at baseline and four and eight weeks later. Results There was a significant negative correlation between BDI scores and TAA. TAA was significantly lower in the MDD group than in controls. When the MDD group was subdivided into those who showed clinical response to antidepressant therapy (response group) and those who did not (non-response group), only the non-response group showed lower TAA, while the response group showed no significant difference to controls at baseline. After eight weeks of antidepressant treatment, TAA in both the response and non-response groups was similar, and there was no significant difference among the three groups. Conclusion These results suggest that the response to antidepressant treatment in MDD patients might be predicted by measuring TAA. PMID:27081384

  17. Assessment of bacterial growth and total organic carbon removal on granular activated carbon contactors.

    PubMed Central

    Bancroft, K; Maloney, S W; McElhaney, J; Suffet, I H; Pipes, W O

    1983-01-01

    The overall growth rate of bacteria on granular activated carbon (GAC) contactors at the Philadelphia Torresdale Water Treatment Pilot Plant facility was found to decrease until steady state was reached. The growth rate was found to fluctuate between 6.94 X 10(-3) and 8.68 X 10(-4) doublings per h. The microbiological removal of total organic carbon (TOC) was calculated by considering the GAC contactors as semiclosed continuous culture systems and using growth yield factors determined in laboratory experiments. After ozonation, the average TOC entering the contactors was 1,488 micrograms/liter, and the average effluent TOC was 497 micrograms/liter. Microbiological TOC removal was found to average 240 micrograms/liter on GAC contactors, which was not significantly different from microbiological TOC (220 micrograms/liter) removal across a parallel sand contactor where no adsorption took place. Thus, GAC did not appear to enhance biological TOC removal. Bacterial growth and maintenance was responsible for approximately 24% of the TOC removal on GAC under the conditions of this study. PMID:6639023

  18. Activation Energies and Dissipation in Biased Quantum Hall Bilayer Systems at Total Filling Factor ν=1.

    NASA Astrophysics Data System (ADS)

    Roostaei, Bahman; Fertig, H. A.; Mullen, K. J.; Simon, Steven

    2007-03-01

    Electrons in a closely spaced bilayer semiconductor structure, such as a double quantum well, are thought to form an interlayer coherent state when a perpendicular magnetic field is applied such that the total Landau level filling factor ν is 1. When the Zeeman energy is sufficiently large to polarize electron spins, the low energy excitations are thought to be topological pseudospin meron-antimeron pairs[1]. These objects carry charge ±e/2,vorticity, and electric dipole moments perpendicular to the layers. Disorder is likely to unbind merons from antimerons and allow them to diffuse through the system independently[2]. Due to their different dipole moments, the various types of merons and antimerons may then in principle be distinguished in transport activation energies by an interlayer bias potential. We report on estimates of these energy differences in various circumstances, and discuss the connection of our results with recent experiments[3].[1]K.Moon,et. al., PRB 51,5138(1995). [2]H.A.Fertig,G.Murthy, PRL 95,156802(2005). [3]R.D.Wiersma,et.al., PRL 93,266805(2004).

  19. In vitro antioxidant activity and total phenolic content of ethanolic leaf extract of Stevia rebaudiana Bert.

    PubMed

    Shukla, Shruti; Mehta, Archana; Bajpai, Vivek K; Shukla, Savita

    2009-09-01

    The aim of this study was to assess the in vitro potential of ethanolic leaf extract of Stevia rebaudiana as a natural antioxidant. The DPPH activity of the extract (20, 40, 50, 100 and 200 microg/ml) was increased in a dose dependent manner, which was found in the range of 36.93-68.76% as compared to ascorbic acid 64.26-82.58%. The IC(50) values of ethanolic extract and ascorbic acid in DPPH radical scavenging assay were obtained to be 93.46 and 26.75 microg/ml, respectively. The ethanolic extract was also found to scavenge the superoxide generated by EDTA/NBT system. Measurement of total phenolic content of the ethanolic extract of S. rebaudiana was achieved using Folin-Ciocalteau reagent containing 61.50 mg/g of phenolic content, which was found significantly higher when compared to reference standard gallic acid. The ethanolic extract also inhibited the hydroxyl radical, nitric oxide, superoxide anions with IC(50) values of 93.46, 132.05 and 81.08 microg/ml, respectively. However, the IC(50) values for the standard ascorbic acid were noted to be 26.75, 66.01 and 71.41 microg/ml respectively. The results obtained in this study clearly indicate that S. rebaudiana has a significant potential to use as a natural antioxidant agent. PMID:19540900

  20. Pharmacological activities in thermal proteins: relationships in molecular evolution

    NASA Technical Reports Server (NTRS)

    Fox, S. W.; Hefti, F.; Hartikka, J.; Junard, E.; Przybylski, A. T.; Vaughan, G.

    1987-01-01

    The model of protobiological events that has been presented in these pages has increasing relevance to pharmacological research. The thermal proteins that function as key substances in the proteinoid theory have recently been found to prolong the survival of rat forebrain neurons in culture and to stimulate the growth of neurites. A search for such activity in thermal proteins added to cultures of modern neurons was suggested by the fact that some of the microspheres assembled from proteinoids rich in hydrophobic amino acids themselves generate fibrous outgrowths.

  1. Novel condensation products having high activity to insolubilize proteins and protein-insolubilized products

    SciTech Connect

    Krasnobajew, V.; Boeniger, R.

    1980-01-01

    According to the invention a substantially more active product with respect to the fixing or insolubilization pf proteins, including enzymes, is obtained when 1,3 phenylenediamine is condensed with glutardialdehyde. One application of the process is the enzymatic hydrolysis of lactose in milk products by lactase.

  2. Protein Kinase Cδ Mediates Neurogenic but Not Mitogenic Activation of Mitogen-Activated Protein Kinase in Neuronal Cells

    PubMed Central

    Corbit, Kevin C.; Foster, David A.; Rosner, Marsha Rich

    1999-01-01

    In several neuronal cell systems, fibroblast-derived growth factor (FGF) and nerve growth factor (NGF) act as neurogenic agents, whereas epidermal growth factor (EGF) acts as a mitogen. The mechanisms responsible for these different cellular fates are unclear. We report here that although FGF, NGF, and EGF all activate mitogen-activated protein (MAP) kinase (extracellular signal-related kinase [ERK]) in rat hippocampal (H19-7) and pheochromocytoma (PC12) cells, the activation of ERK by the neurogenic agents FGF and NGF is dependent upon protein kinase Cδ (PKCδ), whereas ERK activation in response to the mitogenic EGF is independent of PKCδ. Antisense PKCδ oligonucleotides or the PKCδ-specific inhibitor rottlerin inhibited FGF- and NGF-induced, but not EGF-induced, ERK activation. In contrast, EGF-induced ERK activation was inhibited by the phosphatidylinositol-3-kinase inhibitor wortmannin, which had no effect upon FGF-induced ERK activation. Rottlerin also inhibited the activation of MAP kinase kinase (MEK) in response to activated Raf, but had no effect upon c-Raf activity or ERK activation by activated MEK. These results indicate that PKCδ functions either downstream from or in parallel with c-Raf, but upstream of MEK. Inhibition of PKCδ also blocked neurite outgrowth induced by FGF and NGF in PC12 cells and by activated Raf in H19-7 cells, indicating a role for PKCδ in the neurogenic effects of FGF, NGF, and Raf. Interestingly, the PKCδ requirement is apparently cell type specific, since FGF-induced ERK activation was independent of PKCδ in NIH 3T3 murine fibroblasts, in which FGF is a mitogen. These data demonstrate that PKCδ contributes to growth factor specificity and response in neuronal cells and may also promote cell-type-specific differences in growth factor signaling. PMID:10330161

  3. Pharmacokinetics of activated protein C in guinea pigs

    SciTech Connect

    Berger, H. Jr.; Kirstein, C.G.; Orthner, C.L. )

    1991-05-15

    Protein C is a vitamin K-dependent zymogen of the serine protease, activated protein C (APC), an important regulatory enzyme in hemostasis. In view of the potential of human APC as an anticoagulant and profibrinolytic agent, the pharmacokinetics and tissue distribution of APC were studied in guinea pigs. The plasma elimination of a trace dose of {sup 125}I-APC was biphasic following an initial rapid elimination of approximately 15% of the injected dose within 1 to 2 minutes. This rapid removal of {sup 125}I-APC from the circulation was found to be a result of an association with the liver regardless of the route of injection. Essentially identical results were obtained with active site-blocked forms of APC generated with either diisopropylfluorophosphate or D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone, which indicates that the active site was not essential for the liver association. Accumulation of all three forms of APC in the liver peaked at 30 minutes and then declined as increasing amounts of degraded radiolabeled material appeared in the gastrointestinal tract and urine. Removal of the gamma-carboxyglutamic acid (gla) domain of diisopropylphosphoryl-APC resulted in a 50% reduction in the association with liver and an accumulation in the kidneys. Protein C and protein S were cleared from the circulation at rates approximately one-half and one-fourth, respectively, that of APC. Both in vitro and in vivo, APC was found to form complexes with protease inhibitors present in guinea pig plasma. Complex formation resulted in a more rapid disappearance of the enzymatic activity of APC than elimination of the protein moiety. These findings indicate two distinct mechanisms for the elimination of APC. One mechanism involves reaction with plasma protease inhibitors and subsequent elimination by specific hepatic receptors. (Abstract Truncated)

  4. Functions of AMP-activated protein kinase in adipose tissue

    PubMed Central

    Daval, Marie; Foufelle, Fabienne; Ferré, Pascal

    2006-01-01

    AMP-activated protein kinase (AMPK) is involved in cellular energy homeostasis. Its functions have been extensively studied in muscles and liver. AMPK stimulates pathways which increase energy production (glucose transport, fatty acid oxidation) and switches off pathways which consume energy (lipogenesis, protein synthesis, gluconeogenesis). This has led to the concept that AMPK has an interesting pharmaceutical potential in situations of insulin resistance and it is indeed the target of existing drugs and hormones which improve insulin sensitivity. Adipose tissue is a key player in energy metabolism through the release of substrates and hormones involved in metabolism and insulin sensitivity. Activation of AMPK in adipose tissue can be achieved through situations such as fasting and exercise. Leptin and adiponectin as well as hypoglycaemic drugs are activators of adipose tissue AMPK. This activation probably involves changes in the AMP/ATP ratio and the upstream kinase LKB1. When activated, AMPK limits fatty acid efflux from adipocytes and favours local fatty acid oxidation. Since fatty acids have a key role in insulin resistance, especially in muscles, activating AMPK in adipose tissue might be found to be beneficial in insulin-resistant states, particularly as AMPK activation also reduces cytokine secretion in adipocytes. PMID:16709632

  5. Sexual Activity after Total Hip Replacement in Korean Patients: How They Do, What They Want, and How to Improve

    PubMed Central

    Yoon, Byung-Ho; Lee, Kyung-Hag; Noh, Serae; Ha, Yong-Chan; Koo, Kyung-Hoi

    2013-01-01

    Background Concerns of patients on sexual activity after total hip arthroplasty have not been well studied in Asian patients. This study aimed to determine the following: (1) what are the concerns of patients related to sexual activity after total hip arthroplasty? (2) what are the changes in sexual activity after total hip replacement in Korean patients? Methods Details of sexual activity and concerns were obtained using a questionnaire designed specifically for the study. The questionnaire was administered to 64 patients in a face-to-face interview at an outpatient clinic. Results Preoperatively, 53.1% of patients experienced difficulties, primarily due to hip pathology and limitations of motion. The median time to the resumption of sexual activity was 3 months postoperatively, and most patients had no increase in the frequency of sexual activity after the total hip replacement. In 39.1% of patients were seen having difficulties with leg positioning following total hip replacement, and they were likely to change coital positions. The most common concern regarding sexual activity of patients was the fear of dislocation. Furthermore, patients with a higher stress level had lower satisfaction rates. Most patients were unable to obtain information on sexual activity following the total hip arthroplasty, and they did not consult with a physician due to the private nature of the topic. Conclusions Dislocation was the most common concern of patients during sexual activity following a total hip arthroplasty, and a higher stress level was found to be associated with a lower satisfaction rate. Because most patients were unprepared to consult a physician, the provision of appropriate information before a consultation might be beneficial. PMID:24340146

  6. Circadian Regulation of Sucrose Phosphate Synthase Activity in Tomato by Protein Phosphatase Activity.

    PubMed Central

    Jones, T. L.; Ort, D. R.

    1997-01-01

    Sucrose phosphate synthase (SPS), a key enzyme in sucrose biosynthesis, is regulated by protein phosphorylation and shows a circadian pattern of activity in tomato. SPS is most active in its dephosphorylated state, which normally coincides with daytime. Applying okadaic acid, a potent protein phosphatase inhibitor, prevents SPS activation. More interesting is that a brief treatment with cycloheximide, a cytoplasmic translation inhibitor, also prevents the light activation of SPS without any effect on the amount of SPS protein. Cordycepin, an inhibitor of transcript synthesis and processing, has the same effect. Both of these inhibitors also prevent the activation phase of the circadian rhythm in SPS activity. Conversely, cycloheximide and cordycepin do not prevent the decline in circadian SPS activity that normally occurs at night. These observations indicate that SPS phosphatase activity but not SPS kinase activity is controlled, directly or indirectly, at the level of gene expression. Taken together, these data imply that there is a circadian rhythm controlling the transcription of a protein phosphatase that subsequently dictates the circadian rhythm in SPS activity via effects on this enzyme's phosphorylation state. PMID:12223667

  7. Unexpected depletion of plasma arachidonate and total protein in cats fed a low arachidonic acid diet due to peroxidation.

    PubMed

    Chamberlin, Amy; Mitsuhashi, Yuka; Bigley, Karen; Bauer, John E

    2011-10-01

    An opportunity to investigate a low-arachidonic acid (AA) feline diet possibly related to elevated peroxide value (PV) during storage on plasma phospholipid (PL) and reproductive tissue fatty acid (FA) profiles presented itself in the present study. Cats (nine animals per group) had been fed one of three dry extruded, complete and balanced diets for 300 d before spaying. The diets contained adequate AA (0.3 g/kg), similar concentration of antioxidants and were stored at ambient temperature, but differed in FA composition. The diets were designated as follows: diet A (high linoleic acid), diet B (high γ-linolenic acid) and diet C (adequate linoleic acid). Diet samples that were obtained the week before spaying revealed an elevated PV of diet A v. diets B and C (135 v. 5.80 and 2.12 meq/kg fat, respectively). Records revealed decreased food consumption of diet A cats beginning at 240 d but without weight loss; thus an opportunity presented to investigate diet PV effects. Total plasma protein and PL-AA concentrations in group A were significantly decreased at 140 and 300 d. Uterine and ovarian tissues collected at surgery revealed modest decrements of AA. Diet A was below minimum standards at 0.015 % (minimum 0.02 %), probably due to oxidation. The time at which diet A became unacceptable may have occurred between 60 and 140 d because plasma PL-AA was within our normal colony range (approximately 4-7 % relative) after 56 d of feeding. High-linoleic acid-containing diets may be more likely to be oxidised requiring additional antioxidants. The findings suggest that reduced plasma protein in combination with plasma AA concentrations may serve as biomarkers of diet peroxidation in cats before feed refusal, weight loss or tissue depletion. PMID:22005409

  8. Protein immobilization on epoxy-activated thin polymer films: effect of surface wettability and enzyme loading.

    PubMed

    Chen, Bo; Pernodet, Nadine; Rafailovich, Miriam H; Bakhtina, Asya; Gross, Richard A

    2008-12-01

    A series of epoxy-activated polymer films composed of poly(glycidyl methacrylate/butyl methacrylate/hydroxyethyl methacrylate) were prepared. Variation in comonomer composition allowed exploration of relationships between surface wettability and Candida antartica lipase B (CALB) binding to surfaces. By changing solvents and polymer concentrations, suitable conditions were developed for preparation by spin-coating of uniform thin films. Film roughness determined by AFM after incubation in PBS buffer for 2 days was less than 1 nm. The occurrence of single CALB molecules and CALB aggregates at surfaces was determined by AFM imaging and measurements of volume. Absolute numbers of protein monomers and multimers at surfaces were used to determine values of CALB specific activity. Increased film wettability, as the water contact angle of films increased from 420 to 550, resulted in a decreased total number of immobilized CALB molecules. With further increases in the water contact angle of films from 55 degrees to 63 degrees, there was an increased tendency of CALB molecules to form aggregates on surfaces. On all flat surfaces, two height populations, differing by more than 30%, were observed from height distribution curves. They are attributed to changes in protein conformation and/or orientation caused by protein-surface and protein-protein interactions. The fraction of molecules in these populations changed as a function of film water contact angle. The enzyme activity of immobilized films was determined by measuring CALB-catalyzed hydrolysis of p-nitrophenyl butyrate. Total enzyme specific activity decreased by decreasing film hydrophobicity. PMID:18991420

  9. Site–Specific Monoubiquitination Activates Ras by Impeding GTPase Activating Protein Function

    PubMed Central

    Baker, Rachael; Lewis, Steven M.; Sasaki, Atsuo T.; Wilkerson, Emily M.; Locasale, Jason W.; Cantley, Lewis C.; Kuhlman, Brian; Dohlman, Henrik G.; Campbell, Sharon L.

    2012-01-01

    SUMMARY Cell growth and differentiation are controlled by growth factor receptors coupled to the GTPase Ras. Oncogenic mutations disrupt GTPase activity leading to persistent Ras signaling and cancer progression. Recent evidence indicates that monoubiquitination of Ras leads to Ras activation. Mutation of the primary site of monoubiquitination impairs the ability of activated K–Ras to promote tumor growth. To determine the mechanism of human Ras activation we chemically ubiquitinated the protein and analyzed its function by NMR, computational modeling, and biochemical activity measurements. We established that monoubiquitination has little effect on Ras GTP binding, GTP hydrolysis, or exchange factor activation, but severely abrogates the response to GTPase activating proteins in a site–specific manner. These findings reveal a new mechanism by which Ras can trigger persistent signaling in the absence of receptor activation or an oncogenic mutation. PMID:23178454

  10. Effects of total solids content on waste activated sludge thermophilic anaerobic digestion and its sludge dewaterability.

    PubMed

    Wang, Tianfeng; Chen, Jie; Shen, Honglang; An, Dong

    2016-10-01

    The role of total solids content on sludge thermophilic anaerobic digestion was investigated in batch reactors. A range of total solids content from 2% to 10% was evaluated with two replicates. The lowest inhibitory concentration for free ammonia and total ammonia of sludge thermophilic anaerobic digestion was 110.9-171.4mg/L and 1313.1-1806.7mg/L, respectively. The volumetric biogas production rate increased with increasing of total solids content, but the corresponding biogas yield per gram volatile solid decreased. The result of normalized capillary suction time indicated that the dewaterability of digested sludge at high total solids content was poor, while solid content of sediment obtained by centrifuging sludge at 2000g for 10min increased with increasing of total solids content of sludge. The results suggest that thickened sludge mixed with dewatered sludge at an appropriate ratio could get high organic loading rate, high biogas yield and adequate dewatering effort. PMID:26897469

  11. L-Alanylglutamine inhibits signaling proteins that activate protein degradation, but does not affect proteins that activate protein synthesis after an acute resistance exercise.

    PubMed

    Wang, Wanyi; Choi, Ran Hee; Solares, Geoffrey J; Tseng, Hung-Min; Ding, Zhenping; Kim, Kyoungrae; Ivy, John L

    2015-07-01

    Sustamine™ (SUS) is a dipeptide composed of alanine and glutamine (AlaGln). Glutamine has been suggested to increase muscle protein accretion; however, the underlying molecular mechanisms of glutamine on muscle protein metabolism following resistance exercise have not been fully addressed. In the present study, 2-month-old rats climbed a ladder 10 times with a weight equal to 75 % of their body mass attached at the tail. Rats were then orally administered one of four solutions: placebo (PLA-glycine = 0.52 g/kg), whey protein (WP = 0.4 g/kg), low dose of SUS (LSUS = 0.1 g/kg), or high dose of SUS (HSUS = 0.5 g/kg). An additional group of sedentary (SED) rats was intubated with glycine (0.52 g/kg) at the same time as the ladder-climbing rats. Blood samples were collected immediately after exercise and at either 20 or 40 min after recovery. The flexor hallucis longus (FHL), a muscle used for climbing, was excised at 20 or 40 min post exercise and analyzed for proteins regulating protein synthesis and degradation. All supplements elevated the phosphorylation of FOXO3A above SED at 20 min post exercise, but only the SUS supplements significantly reduced the phosphorylation of AMPK and NF-kB p65. SUS supplements had no effect on mTOR signaling, but WP supplementation yielded a greater phosphorylation of mTOR, p70S6k, and rpS6 compared with PLA at 20 min post exercise. However, by 40 min post exercise, phosphorylation of mTOR and rpS6 in PLA had risen to levels not different than WP. These results suggest that SUS blocks the activation of intracellular signals for MPB, whereas WP accelerates mRNA translation. PMID:25837301

  12. Analysis of antifreeze protein activity using colorimetric gold nanosensors

    NASA Astrophysics Data System (ADS)

    Jing, Xu; Choi, Ho-seok; Park, Ji-In; Kim, Young-Pil

    2015-07-01

    High activity and long stability of antifreeze proteins (AFPs), also known as ice-binding proteins (IBPs), are necessary for exerting their physiological functions in biotechnology and cryomedicine. Here we report a simple analysis of antifreeze protein activity and stability based on self-assembly of gold nanoparticles (AuNPs) via freezing and thawing cycles. While the mercaptosuccinic acid-capped AuNP (MSA-AuNP) was easily self-assembled after a freezing/thawing cycle, due to the mechanical attack of ice crystal on the MSA-AuNP surface, the presence of AFP impeded the self-assembly of MSA-AuNP via the interaction of AFP with ice crystals via freezing and thawing cycles, which led to a strong color in the MSA-AuNP solution. As a result, the aggregation parameter (E520/E650) of MSA-AuNP showed the rapid detection of both activity and stability of AFPs. We suggest that our newly developed method is very suitable for measuring antifreeze activity and stability in a simple and rapid manner with reliable quantification.

  13. Methods of measuring Protein Disulfide Isomerase activity: a critical overview

    NASA Astrophysics Data System (ADS)

    Watanabe, Monica; Laurindo, Francisco; Fernandes, Denise

    2014-09-01

    Protein disulfide isomerase is an essential redox chaperone from the endoplasmic reticulum (ER) and is responsible for correct disulfide bond formation in nascent proteins. PDI is also found in other cellular locations in the cell, particularly the cell surface. Overall, PDI contributes to ER and global cell redox homeostasis and signaling. The knowledge about PDI structure and function progressed substantially based on in vitro studies using recombinant PDI and chimeric proteins. In these experimental scenarios, PDI reductase and chaperone activities are readily approachable. In contrast, assays to measure PDI isomerase activity, the hallmark of PDI family, are more complex. Assessment of PDI roles in cells and tissues mainly relies on gain- or loss-of-function studies. However, there is limited information regarding correlation of experimental readouts with the distinct types of PDI activities. In this mini-review, we evaluate the main methods described for measuring the different kinds of PDI activity: thiol reductase, thiol oxidase, thiol isomerase and chaperone. We emphasize the need to use appropriate controls and the role of critical interferents (e.g., detergent, presence of reducing agents). We also discuss the translation of results from in vitro studies with purified recombinant PDI to cellular and tissue samples, with critical comments on the interpretation of results.

  14. Unwinding activity of cold shock proteins and RNA metabolism.

    PubMed

    Phadtare, Sangita

    2011-01-01

    Temperature downshift from 37 °C to 15 °C results in the exertion of cold shock response in Escherichia coli, which induces cold shock proteins, such as CsdA. Previously, we showed that the helicase activity of CsdA is critical for its function in the cold acclimation of cells and its primary role is mRNA degradation. Only RhlE (helicase), CspA (RNA chaperone) and RNase R (exoribonuclease) were found to complement the cold shock function of CsdA. RNase R has two independent activities, helicase and ribonuclease, only helicase being essential for the functional complementation of CsdA. Here, we discuss the significance of above findings as these emphasize the importance of the unwinding activity of cold-shock-inducible proteins in the RNA metabolism at low temperature, which may be different than that at 37 °C. It requires assistance of proteins to destabilize the secondary structures in mRNAs that are stabilized upon temperature downshift, hindering the activity of ribonucleases. PMID:21445001

  15. Protein kinase activity associated with simian virus 40 T antigen.

    PubMed Central

    Griffin, J D; Spangler, G; Livingston, D M

    1979-01-01

    Incubation of simian virus 40 (SV40) tumor (T) antigen-containing immunoprecipitates with [gamma-32P]ATP results in the incorporation of radioactive phosphate into large T antigen. Highly purified preparations of large T antigen from a SV40-transformed cell line, SV80, are able to catalyze the phosphorylation of a known phosphate acceptor, casein. The kinase activity migrates with large T antigen through multiple purification steps. Sedimentation analysis under non-T-antigen-aggregating conditions reveals that kinase activity and the immunoreactive protein comigrate as a 6S structure. The kinase activity of purified preparations of large T antigen can be specifically adsorbed to solid-phase anti-T IgG, and partially purified T antigen from a SV40 tsA transformation is thermolabile in its ability to phosphorylate casein when compared to comparably purified wild-type T antigen. These observations indicate that the SV40 large T antigen is closely associated with protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) activity. Images PMID:223152

  16. Dynamical Theory of Activated Processes in Globular Proteins

    NASA Astrophysics Data System (ADS)

    Northrup, Scott H.; Pear, Michael R.; Lee, Chyuan-Yih; McCammon, J. Andrew; Karplus, Martin

    1982-07-01

    A methos is described for calculating the reaction rate in globular proteins of activated processes such as ligand binding or enzymatic catalysis. The method is based on the determination of the probability that the system is in the transition state and of the magnitude of the reactive flux for transition-state systems. An ``umbrella sampling'' simulation procedure is outlined for evaluating the transition-state probability. The reactive flux is obtained from an approach described previously for calculating the dynamics of transition-state trajectories. An application to the rotational isomerization of an aromatic ring in the bovine pancreatic trypsin inhibitor is presented. The results demonstrate the feasibility of calculating rate constants for reactions in proteins and point to the importance of solvent effects for reactions that occur near the protein surface.

  17. Receptor Activity-Modifying Proteins (RAMPs): New Insights and Roles.

    PubMed

    Hay, Debbie L; Pioszak, Augen A

    2016-01-01

    It is now recognized that G protein-coupled receptors (GPCRs), once considered largely independent functional units, have a far more diverse molecular architecture. Receptor activity-modifying proteins (RAMPs) provide an important example of proteins that interact with GPCRs to modify their function. RAMPs are able to act as pharmacological switches and chaperones, and they can regulate signaling and/or trafficking in a receptor-dependent manner. This review covers recent discoveries in the RAMP field and summarizes the known GPCR partners and functions of RAMPs. We also discuss the first peptide-bound structures of RAMP-GPCR complexes, which give insight into the molecular mechanisms that enable RAMPs to alter the pharmacology and signaling of GPCRs. PMID:26514202

  18. Dynamical theory of activated processes in globular proteins.

    PubMed Central

    Northrup, S H; Pear, M R; Lee, C Y; McCammon, J A; Karplus, M

    1982-01-01

    A method is described for calculating the reaction rate in globular proteins of activated processes such as ligand binding or enzymatic catalysis. The method is based on the determination of the probability that the system is in the transition state and of the magnitude of the reactive flux for transition-state systems. An "umbrella sampling" simulation procedure is outlined for evaluating the transition-state probability. The reactive flux is obtained from an approach described previously for calculating the dynamics of transition-state trajectories. An application to the rotational isomerization of an aromatic ring in the bovine pancreatic trypsin inhibitor is presented. The results demonstrate the feasibility of calculating rate constants for reactions in proteins and point to the importance of solvent effects for reactions that occur near the protein surface. PMID:6955788

  19. Antibacterial activity of a lectin-like Burkholderia cenocepacia protein.

    PubMed

    Ghequire, Maarten G K; De Canck, Evelien; Wattiau, Pierre; Van Winge, Iris; Loris, Remy; Coenye, Tom; De Mot, René

    2013-08-01

    Bacteriocins of the LlpA family have previously been characterized in the γ-proteobacteria Pseudomonas and Xanthomonas. These proteins are composed of two MMBL (monocot mannose-binding lectin) domains, a module predominantly and abundantly found in lectins from monocot plants. Genes encoding four different types of LlpA-like proteins were identified in genomes from strains belonging to the Burkholderia cepacia complex (Bcc) and the Burkholderia pseudomallei group. A selected recombinant LlpA-like protein from the human isolate Burkholderia cenocepacia AU1054 displayed narrow-spectrum genus-specific antibacterial activity, thus representing the first functionally characterized bacteriocin within this β-proteobacterial genus. Strain-specific killing was confined to other members of the Bcc, with mostly Burkholderia ambifaria strains being susceptible. In addition to killing planktonic cells, this bacteriocin also acted as an antibiofilm agent. PMID:23737242

  20. Molecular architecture of the active mitochondrial protein gate.

    PubMed

    Shiota, Takuya; Imai, Kenichiro; Qiu, Jian; Hewitt, Victoria L; Tan, Khershing; Shen, Hsin-Hui; Sakiyama, Noriyuki; Fukasawa, Yoshinori; Hayat, Sikander; Kamiya, Megumi; Elofsson, Arne; Tomii, Kentaro; Horton, Paul; Wiedemann, Nils; Pfanner, Nikolaus; Lithgow, Trevor; Endo, Toshiya

    2015-09-25

    Mitochondria fulfill central functions in cellular energetics, metabolism, and signaling. The outer membrane translocator complex (the TOM complex) imports most mitochondrial proteins, but its architecture is unknown. Using a cross-linking approach, we mapped the active translocator down to single amino acid residues, revealing different transport paths for preproteins through the Tom40 channel. An N-terminal segment of Tom40 passes from the cytosol through the channel to recruit chaperones from the intermembrane space that guide the transfer of hydrophobic preproteins. The translocator contains three Tom40 β-barrel channels sandwiched between a central α-helical Tom22 receptor cluster and external regulatory Tom proteins. The preprotein-translocating trimeric complex exchanges with a dimeric isoform to assemble new TOM complexes. Dynamic coupling of α-helical receptors, β-barrel channels, and chaperones generates a versatile machinery that transports about 1000 different proteins. PMID:26404837

  1. Dietary total antioxidant capacity from different assays in relation to serum C-reactive protein among young Japanese women

    PubMed Central

    2012-01-01

    Background The association between dietary total antioxidant capacity (TAC) from different assays and serum C-reactive protein (CRP) has not been assessed in non-Western populations. We examined the association between dietary TAC and serum CRP concentration in young Japanese women using different four TAC assays. Methods The subjects were 443 young Japanese women aged 18–22 years. Dietary TAC was assessed with a self-administered diet history questionnaire and the TAC value of each food using the following four assays: ferric reducing ability of plasma (FRAP); oxygen radical absorbance capacity (ORAC); Trolox equivalent antioxidant capacity (TEAC); and total radical-trapping antioxidant parameter (TRAP). Serum CRP concentrations were measured by highly sensitive nephelometry. Results The major contributor to dietary TAC was green, barley, and oolong tea (FRAP: 53%, ORAC: 45%, TEAC: 36%, and TRAP: 44%). The prevalence of elevated CRP concentrations (≥ 1 mg/L) was 5.6%. TAC from FRAP was inversely associated with serum CRP concentrations (adjusted odds ratio [OR] for elevated CRP concentration in high [compared with low] dietary TAC group: 0.39 [95% confidence interval (CI): 0.16-0.98]; P = 0.04). TAC from ORAC was inversely associated with CRP, although the association was not significant (OR: 0.48 [95% CI: 0.20-1.14]; P = 0.10). TAC from TEAC was inversely associated with CRP (OR: 0.32 [95% CI: 0.12-0.82]; P = 0.02), as was TAC from TRAP (OR: 0.31 [95% CI: 0.12-0.81]; P = 0.02). Conclusions Dietary TAC was inversely associated with serum CRP concentration in young Japanese women regardless of assay. Further studies are needed in other populations to confirm these results. PMID:23110638

  2. Modulation of the protein kinase activity of mTOR.

    PubMed

    Lawrence, J C; Lin, T A; McMahon, L P; Choi, K M

    2004-01-01

    mTOR is a founding member of a family of protein kinases having catalytic domains homologous to those in phosphatidylinositol 3-OH kinase. mTOR participates in the control by insulin of the phosphorylation of lipin, which is required for adipocyte differentiation, and the two translational regulators, p70S6K and PHAS-I. The phosphorylation of mTOR, itself, is stimulated by insulin in Ser2448, a site that is also phosphorylated by protein kinase B (PKB) in vitro and in response to activation of PKB activity in vivo. Ser2448 is located in a short stretch of amino acids not found in the two TOR proteins in yeast. A mutant mTOR lacking this stretch exhibited increased activity, and binding of the antibody, mTAb-1, to this region markedly increased mTOR activity. In contrast, rapamycin-FKBP12 inhibited mTOR activity towards both PHAS-I and p70S6K, although this complex inhibited the phosphorylation of some sites more than that of others. Mutating Ser2035 to Ile in the FKBP12-rapamycin binding domain rendered mTOR resistant to inhibition by rapamycin. Unexpectedly, this mutation markedly decreased the ability of mTOR to phosphorylate certain sites in both PHAS-I and p70S6K. The results support the hypotheses that rapamycin disrupts substrate recognition instead of directly inhibiting phosphotransferase activity and that mTOR activity in cells is controlled by the phosphorylation of an inhibitory regulatory domain containing the mTAb-1 epitope. PMID:14560959

  3. Incorporation of single dinitrophenyl-modified proteins in to the 30S subunit of Escherichia coli ribosomes by total reconstitution for localization by immune electron microscopy

    SciTech Connect

    Olah, T.V.

    1989-01-01

    The ribosome is a structurally defined organelle whose function is central to the existence of all organisms. It is the unique site of protein biosynthesis in all cells. A detailed understanding of ribosome structure is essential in understanding the process of translation. This thesis represents a new approach to the systematic localization of individual proteins contained in the small subunit of Escherichia coli ribosomes using immunoelectron microscopy. All 30S proteins were purified using high performance liquid chromatography (HPLC) and eight isolated proteins (S12,S21,S14,S19,S18,S17,S16 and S13) were derivatized with 2,4-(3,5-{sup 3}H)dinitrofluorobenzene (DNFB). The extent of modification of these proteins was estimated by both radioactivity and integrated peak areas, using dual wavelength monitoring at 214nm to detect protein and 360nm (to detect dinitrophenyl groups). Each dinitrophenylated protein was introduced in place of the corresponding unmodified protein into totally reconstituted 30S subunits. Antibodies raised against the DNP-hapten bound effectively to such reconstituted subunits and did not cause dissociation of the modified protein from the subunit. Electron microscopy of the immune complexes was used to localize the modified protein on the subunit surface. Incorporation of any of the DNP-modified proteins, with the exception of DNP-S18, does not interfere with the functionality of the ribosome as measure by the binding of Phe-tRNA{sup Phe} or the synthesis of poly(Phe) in a poly(U)-dependent manner. Results show that unmodified protein competes with DNP-protein and that DNP-protein can function, as its native counterpart, in stimulating uptake of specific proteins during reconstitution. This data provides evidence that each DNP-protein occupies the same position in 30S subunits as does the corresponding unmodified protein.

  4. Novel mechanisms for activated protein C cytoprotective activities involving noncanonical activation of protease-activated receptor 3

    PubMed Central

    Burnier, Laurent

    2013-01-01

    The direct cytoprotective activities of activated protein C (APC) on cells convey therapeutic, relevant, beneficial effects in injury and disease models in vivo and require the endothelial protein C receptor (EPCR) and protease activated receptor 1 (PAR1). Thrombin also activates PAR1, but its effects on cells contrast APC’s cytoprotective effects. To gain insights into mechanisms for these contrasting cellular effects, protease activated receptor 3 (PAR3) activation by APC and thrombin was studied. APC cleaved PAR3 on transfected and endothelial cells in the presence of EPCR. Remarkably, APC cleaved a synthetic PAR3 N-terminal peptide at Arg41, whereas thrombin cleaved at Lys38. On cells, APC failed to cleave R41Q-PAR3, whereas K38Q-PAR3 was still cleaved by APC but not by thrombin. PAR3 tethered-ligand peptides beginning at amino acid 42, but not those beginning at amino acid 39, conveyed endothelial barrier-protective effects. In vivo, the APC-derived PAR3 tethered-ligand peptide, but not the thrombin-derived PAR3 peptide, blunted vascular endothelial growth factor (VEGF)-induced vascular permeability. These data indicate that PAR3 cleavage by APC at Arg41 can initiate distinctive APC-like cytoprotective effects. These novel insights help explain the differentiation of APC’s cytoprotective versus thrombin’s proinflammatory effects on cells and suggest a unique contributory role for PAR3 in the complex mechanisms underlying APC cytoprotective effects. PMID:23788139

  5. Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins

    PubMed Central

    Vallejo, Luis Felipe; Rinas, Ursula

    2004-01-01

    Recent advances in generating active proteins through refolding of bacterial inclusion body proteins are summarized in conjunction with a short overview on inclusion body isolation and solubilization procedures. In particular, the pros and cons of well-established robust refolding techniques such as direct dilution as well as less common ones such as diafiltration or chromatographic processes including size exclusion chromatography, matrix- or affinity-based techniques and hydrophobic interaction chromatography are discussed. Moreover, the effect of physical variables (temperature and pressure) as well as the presence of buffer additives on the refolding process is elucidated. In particular, the impact of protein stabilizing or destabilizing low- and high-molecular weight additives as well as micellar and liposomal systems on protein refolding is illustrated. Also, techniques mimicking the principles encountered during in vivo folding such as processes based on natural and artificial chaperones and propeptide-assisted protein refolding are presented. Moreover, the special requirements for the generation of disulfide bonded proteins and the specific problems and solutions, which arise during process integration are discussed. Finally, the different strategies are examined regarding their applicability for large-scale production processes or high-throughput screening procedures. PMID:15345063

  6. Natural fermentation of lentils. Influence of time, concentration and temperature on protein content, trypsin inhibitor activity and phenolic compound content.

    PubMed

    Tabera, J; Frias, J; Estrella, I; Villa, R; Vidal-Valverde, C

    1995-12-01

    Lentil (Lens culinaris var. vulgaris) flour was naturally fermented for 4 days at different temperatures (28 degrees C, 35 degrees C and 42 degrees C) and concentrations (79 milligrams, 150 milligrams and 221 milligrams). Samples were analysed to establish the changes of total protein content and in vitro protein digestibility, trypsin inhibitor activity (TIA) and phenolic compound content during natural fermentation of lentils. The preparation of lentil flour suspensions to be fermented caused a slight increase in total protein and in vitro protein digestibility content, a decrease of TIA and a sharp decrease the tannin/catechin ratio. During the whole fermentation procedure, the minimum initial lentil concentration and temperature used (79 milligrams, 28 degrees C) achieved the maximum protein content and the lowest tannin/catechin ratio. The TIA was more affected by temperature than by concentration, and a 62.5% reduction was observed at 42 degrees C and 79 milligrams. PMID:8585337

  7. Hepatitis B virus X protein activates transcription factor NF-kappa B without a requirement for protein kinase C.

    PubMed Central

    Lucito, R; Schneider, R J

    1992-01-01

    The hepatitis B virus X protein stimulates transcription from a variety of promoter elements, including those activated by transcription factor NF-kappa B. A diverse group of extra- and intracellular agents, including growth factors and the human immunodeficiency virus tat protein, have been shown to require a functional protein kinase C (PKC) system to achieve activation of NF-kappa B. In this study we have investigated the molecular mechanism by which X protein activates NF-kappa B. We demonstrate that in hepatocytes, X protein induces a maximal activation of NF-kappa B corresponding to the sequestered pool of factor, which is also activated by phorbol esters. To determine whether X protein requires activation of PKC to stimulate transcription by NF-kappa B, we attempted to prevent transactivation by X protein in the presence of the PKC inhibitors calphostin C and H7. We show that PKC inhibitors do not block X protein activation of NF-kappa B, whereas they largely impair activation by phorbol esters. In addition, activation of PKC is correlated with its translocation from the cytoplasm to the plasma membrane. The subcellular distribution of PKC was investigated by introducing X protein from a replication-defective adenovirus vector, followed by immunochemical detection of PKC in cell fractions. These data also indicate that X protein stimulates transcription by NF-kappa B without the activation and translocation of PKC. Images PMID:1309924

  8. Localization microscopy using noncovalent fluorogen activation by genetically encoded fluorogen activating proteins

    PubMed Central

    Maji, Suvrajit; Huang, Fang; Szent-Gyorgyi, Chris; Lidke, Diane S.; Lidke, Keith A.; Bruchez, Marcel P.

    2014-01-01

    The noncovalent equilibrium activation of a fluorogenic malachite green dye and its cognate fluorogen activating protein has been exploited to produce a sparse labeling distribution of densely tagged genetically encoded proteins, enabling single molecule detection and superresolution imaging in fixed and living cells. These sparse labeling conditions are achieved by control of the dye concentration in the milieu, and do not require any photoswitching or photoactivation. The labeling is achieved using physiological buffers and cellular media, and does not require additives or switching buffer to obtain superresolution images. We evaluate superresolution properties and images obtained from a selected fluorogen activating protein clone fused to actin, and show that the photon counts per object fall between those typically reported for fluorescent proteins and switching dye-pairs, resulting in 10-30 nm localization precision per object. This labeling strategy complements existing approaches, and may simplify multicolor labeling of cellular structures. PMID:24194371

  9. Activation of G Proteins by Guanine Nucleotide Exchange Factors Relies on GTPase Activity

    PubMed Central

    Stanley, Rob J.; Thomas, Geraint M. H.

    2016-01-01

    G proteins are an important family of signalling molecules controlled by guanine nucleotide exchange and GTPase activity in what is commonly called an ‘activation/inactivation cycle’. The molecular mechanism by which guanine nucleotide exchange factors (GEFs) catalyse the activation of monomeric G proteins is well-established, however the complete reversibility of this mechanism is often overlooked. Here, we use a theoretical approach to prove that GEFs are unable to positively control G protein systems at steady-state in the absence of GTPase activity. Instead, positive regulation of G proteins must be seen as a product of the competition between guanine nucleotide exchange and GTPase activity—emphasising a central role for GTPase activity beyond merely signal termination. We conclude that a more accurate description of the regulation of G proteins via these processes is as a ‘balance/imbalance’ mechanism. This result has implications for the understanding of intracellular signalling processes, and for experimental strategies that rely on modulating G protein systems. PMID:26986850

  10. Mitogen-activated protein kinase cascades in Vitis vinifera

    PubMed Central

    Çakır, Birsen; Kılıçkaya, Ozan

    2015-01-01

    Protein phosphorylation is one of the most important mechanisms to control cellular functions in response to external and endogenous signals. Mitogen-activated protein kinases (MAPK) are universal signaling molecules in eukaryotes that mediate the intracellular transmission of extracellular signals resulting in the induction of appropriate cellular responses. MAPK cascades are composed of four protein kinase modules: MAPKKK kinases (MAPKKKKs), MAPKK kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In plants, MAPKs are activated in response to abiotic stresses, wounding, and hormones, and during plant pathogen interactions and cell division. In this report, we performed a complete inventory of MAPK cascades genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with MAPK, MAPK kinases, MAPK kinase kinases and MAPK kinase kinase kinase kinase members of Arabidopsis thaliana, we revealed the existence of 14 MAPKs, 5 MAPKKs, 62 MAPKKKs, and 7 MAPKKKKs in Vitis vinifera. We identified orthologs of V. vinifera putative MAPKs in different species, and ESTs corresponding to members of MAPK cascades in various tissues. This work represents the first complete inventory of MAPK cascades in V. vinifera and could help elucidate the biological and physiological functions of these proteins in V. vinifera. PMID:26257761

  11. [Total Synthesis of Biologically Active Natural Products toward Elucidation of the Mode of Action].

    PubMed

    Yoshida, Masahito

    2015-01-01

    Total synthesis of biologically active cyclodepsipeptide destruxin E using solid- and solution-phase synthesis is described. The solid-phase synthesis of destruxin E was initially investigated for the efficient synthesis of destruxin analogues. Peptide elongation from polymer-supported β-alanine was efficiently performed using DIC/HOBt or PyBroP/DIEA, and subsequent cleavage from the polymer-support under weakly acidic conditions furnished a cyclization precursor in moderate yield. Macrolactonization of the cyclization precursor was smoothly performed using 2-methyl-6-nitrobenzoic anhydride (MNBA)/4-(dimethylamino)pyridine N-oxide (DMAPO) to afford macrolactone in moderate yield. Finally, formation of the epoxide in the side chain via three steps provided destruxin E, and the stereochemistry of the epoxide was determined to be S. Its diastereomer, epi-destruxin E, was also synthesized in the same manner used to synthesize the natural product. The stereochemistry of the epoxide was critical for the V-ATPase inhibition; natural product destruxin E exhibited 10-fold more potent V-ATPase inhibition than epi-destruxin E. Next, the scalable synthesis of destruxin E for in vivo study was also performed via solution-phase synthesis. The scalable synthesis of a key component, (S)-HA-Pro-OH, was achieved using osmium-catalyzed diastereoselective dihydroxylation with (DHQD)2PHAL as a chiral ligand; peptide synthesis using Cbz-protected amino acid derivatives furnished the cyclization precursor on a gram-scale. Macrolactonization smoothly provided the macrolactone without forming a dimerized product, even at 6 mM, and the synthesis of destruxin E was achieved via three steps on a gram scale in high purity (>98%). PMID:26423864

  12. Complement activation and cytokine response by BioProtein, a bacterial single cell protein.

    PubMed

    Sikkeland, L I B; Thorgersen, E B; Haug, T; Mollnes, T E

    2007-04-01

    The bacterial single cell protein (BSCP), BioProtein, is dried bacterial mass derived from fermentation of the gram negative bacteria Methylococcus capsulatus, used for animal and fish feed. Workers in this industry suffer frequently from pulmonary and systemic symptoms which may be induced by an inflammatory reaction. The aim of the present study was to examine the effect of BSCP on inflammation in vitro as evaluated by complement activation and cytokine production. Human serum was incubated with BSCP and complement activation products specific for all pathways were detected by enzyme-linked immunosorbent assay (ELISA). Human whole blood anti-coagulated with lepirudin was incubated with BSCP and a panel of 27 biological mediators was measured using multiplex technology. BSCP induced a dose-dependent complement activation as revealed by a pronounced increase in alternative and terminal pathway activation (fivefold and 20-fold, respectively) at doses from 1 microg BSCP/ml serum and a similar, but less extensive (two- to fourfold) increase in activation of the lectin and classical pathways at doses from 100 and 1000 microg BSCP/ml serum, respectively. Similarly, BSCP induced a dose-dependent production of a number of cytokines, chemokines and growth factors in human whole blood. At doses as low as 0 x 05-0 x 5 microg BSCP/ml blood a substantial increase was seen for tumour necrosis factor (TNF)-alpha, interleukin (IL)-1-beta, IL-6, interferon (IFN)-gamma, IL-8, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, IL-4, IL-9, IL-17, IL-1Ra, granulocyte-colony-stimulating factor (G-CSF) and vascular endothelial growth factor (VEGF). Thus, BSCP induced a substantial activation of all three initial complement pathways as well as a pronounced cytokine response in vitro, indicating a potent inflammatory property of this agent. PMID:17302729

  13. Interaction of receptor-activity-modifying protein1 with tubulin.

    PubMed

    Kunz, Thomas H; Mueller-Steiner, Sarah; Schwerdtfeger, Kerstin; Kleinert, Peter; Troxler, Heinz; Kelm, Jens M; Ittner, Lars M; Fischer, Jan A; Born, Walter

    2007-08-01

    Receptor-activity-modifying protein (RAMP) 1 is an accessory protein of the G protein-coupled calcitonin receptor-like receptor (CLR). The CLR/RAMP1 heterodimer defines a receptor for the potent vasodilatory calcitonin gene-related peptide. A wider tissue distribution of RAMP1, as compared to that of the CLR, is consistent with additional biological functions. Here, glutathione S-transferase (GST) pull-down, coimmunoprecipitation and yeast two-hybrid experiments identified beta-tubulin as a novel RAMP1-interacting protein. GST pull-down experiments indicated interactions between the N- and C-terminal domains of RAMP1 and beta-tubulin. Yeast two-hybrid experiments confirmed the interaction between the N-terminal region of RAMP1 and beta-tubulin. Interestingly, alpha-tubulin was co-extracted with beta-tubulin in pull-down experiments and immunoprecipitation of RAMP1 coprecipitated alpha- and beta-tubulin. Confocal microscopy indicated colocalization of RAMP1 and tubulin predominantly in axon-like processes of neuronal differentiated human SH-SY5Y neuroblastoma cells. In conclusion, the findings point to biological roles of RAMP1 beyond its established interaction with G protein-coupled receptors. PMID:17493758

  14. Regulation of Orange Carotenoid Protein Activity in Cyanobacterial Photoprotection.

    PubMed

    Thurotte, Adrien; Lopez-Igual, Rocio; Wilson, Adjélé; Comolet, Léa; Bourcier de Carbon, Céline; Xiao, Fugui; Kirilovsky, Diana

    2015-09-01

    Plants, algae, and cyanobacteria have developed mechanisms to decrease the energy arriving at reaction centers to protect themselves from high irradiance. In cyanobacteria, the photoactive Orange Carotenoid Protein (OCP) and the Fluorescence Recovery Protein are essential elements in this mechanism. Absorption of strong blue-green light by the OCP induces carotenoid and protein conformational changes converting the orange (inactive) OCP into a red (active) OCP. Only the red orange carotenoid protein (OCP(r)) is able to bind to phycobilisomes, the cyanobacterial antenna, and to quench excess energy. In this work, we have constructed and characterized several OCP mutants and focused on the role of the OCP N-terminal arm in photoactivation and excitation energy dissipation. The N-terminal arm largely stabilizes the closed orange OCP structure by interacting with its C-terminal domain. This avoids photoactivation at low irradiance. In addition, it slows the OCP detachment from phycobilisomes by hindering fluorescence recovery protein interaction with bound OCP(r). This maintains thermal dissipation of excess energy for a longer time. Pro-22, at the beginning of the N-terminal arm, has a key role in the correct positioning of the arm in OCP(r), enabling strong OCP binding to phycobilisomes, but is not essential for photoactivation. Our results also show that the opening of the OCP during photoactivation is caused by the movement of the C-terminal domain with respect to the N-terminal domain and the N-terminal arm. PMID:26195570

  15. Screening and appraisal for immunological adjuvant-active fractions from Platycodon grandiflorum total saponins.

    PubMed

    Ouyang, Ke; Chen, Liqing; Sun, Hongxiang; Du, Jing; Shi, Minghua

    2012-02-01

    In this study, the total saponins from the root of Platycodon grandiflorum (PGS(t)) was subjected to D101 macroreticular resin column chromatography to afford four fractions (PGS₃₀, PGS₅₀, PGS₇₅ and PGS₉₅). PGS(t) and its four fractions were evaluated and compared for the haemolytic activities and adjuvant potentials on the specific cellular and humoral immune responses of ICR mice against recombinant hepatitis B surface antigen (HBsAg). PGS(t), PGS₃₀, PGS₅₀, PGS₇₅, and PGS₇₅ showed a slight haemolytic effect, with their concentration inducing 50% of the maximum haemolysis (HD₅₀) being 16.13 ± 0.81, >200, 17.53 ± 0.24, 20.16 ± 0.76, 76.31 ± 2.20 μg/mL against 0.5% rabbit red blood cell, respectively. PGS(t), PGS₅₀, and PGS₇₅ significantly not only enhanced the Con A-, lipopolysaccharide-, and HBsAg-induced splenocyte proliferation, but promoted the killing activities of natural killer (NK) cells from splenocytes in HBsAg-immunized mice (P < 0.01 or P < 0.001). HBsAg-specific IgG, IgG1, IgG2a, and IgG2b antibody levels in serum were also significantly enhanced by PGS(t), PGS₅₀, and PGS₇₅ compared with HBsAg control group (P < 0.05, P < 0.01, or P < 0.001). Moreover, the adjuvant effects of PGS₅₀ and PGS₇₅ on the cellular immune responses and HBsAg-specific IgG2a and IgG2b antibody responses were more significant than those of Alum, PGS₃₀, and PGS₉₅. The results indicated that PGS₅₀ and PGS₇₅ could improve both cellular and humoral immune responses, and elicit a balanced Th1/Th2 response to HBsAg in mice, and that PGS₇₅ may be developed as an ideal candidate adjuvant for hepatitis B vaccine. PMID:21682667

  16. Physical activity, sedentary behavior and total wellness changes among sedentary adults: a 4-week randomized controlled trial

    PubMed Central

    2013-01-01

    Background The construct of total wellness includes a holistic approach to the body, mind and spirit components of life. While the health benefits of reducing sedentary behavior and increasing physical activity are well documented, little is known about the influence on total wellness of an internet-based physical activity monitor designed to help people to achieve higher physical activity levels. Purpose The purpose of this four-week, personal activity monitor-based intervention program was to reduce sedentary behavior and increase physical activity levels in daily living for sedentary adults and to determine if these changes would also be associated with improvement in total wellness. Methods Twenty-two men and 11 women (27 years ± 4.0) were randomly assigned to either an intervention (n = 18) or control group (n = 15). The intervention group interacted with an online personal activity monitor (Gruve Solution™) designed to reduce sedentary time and increase physical activity during activities of daily living. The control group did not interact with the monitor, as they were asked to follow their normal daily physical activities and sedentary behavior routines. The Wellness Evaluation of Lifestyle (WEL) inventory was used to assess total wellness. Sedentary time, light, walking, moderate and vigorous intensity physical activities were assessed for both intervention and control groups at baseline and at week-4 by the 7-day Sedentary and Light Intensity Physical Activity Log (7-day SLIPA Log) and the International Physical Activity Questionnaire (IPAQ). Results Significant increases in pre-post total wellness scores (from 64% ± 5.7 to 75% ± 8.5) (t (17) = -6.5, p < 0.001) were observed in the intervention group by the end of week four. Intervention participants decreased their sedentary time (21%, 2.3 hours/day) and increased their light (36.7%, 2.5 hours/day), walking (65%, 1057 MET-min/week), moderate (67%, 455 MET-min/week) and

  17. Protein A-like activity and streptococcal cross-reactions.

    PubMed Central

    Kingston, D

    1981-01-01

    Recognition of the protein A-like activity of some strains of group A streptococci has thrown doubt on much previous work suggesting antigenic cross-reactions between these streptococci and mammalian tissues. The strains used in our previous studies have now been examined by the mixed reverse passive antiglobulin reaction (MRPAH) for the 'non-specific' absorption of purified Fc portion of human IgG. They were found to have only traces of activity. The strain of Staphylococcus aureus used to control 'non-specific' absorption by bacterial cell walls was strongly positive. Protein A-like material as detected in this way was not therefore responsible for our earlier results. PMID:7039880

  18. Purification, characterization and activities of two hemolytic and antibacterial proteins from coelomic fluid of the annelid Eisenia fetida andrei.

    PubMed

    Milochau, A; Lassègues, M; Valembois, P

    1997-01-01

    The coelomic fluid of the earthworm Eisenia fetida andrei exhibits antibacterial, hemolytic and hemagglutinating activities. These activities are mainly mediated by two proteins, named fetidins, of apparent molecular mass 40 kDa and 45 kDa, respectively. For the first time, the two proteins have been purified to homogeneity from dialysed coelomic fluid by means of anion-exchange chromatography. Three peaks had hemolytic activity. The first fraction was found to correspond to the 40 kDa fetidin, the second to mixed 40 and 45 kDa fetidins, the last one to the 45 kDa fetidin. Both purified proteins still exhibited their hemolytic and antibacterial activities as dialysed coelomic fluid did. In this study, the amino-acid sequence of purified proteins is compared to the amino-acid sequence predicted by cDNA. This cDNA was isolated by screening an expression cDNA library from earthworm total tissues (unpublished data). PMID:9003444

  19. Change in active travel and changes in recreational and total physical activity in adults: longitudinal findings from the iConnect study

    PubMed Central

    2013-01-01

    Background To better understand the health benefits of promoting active travel, it is important to understand the relationship between a change in active travel and changes in recreational and total physical activity. Methods These analyses, carried out in April 2012, use longitudinal data from 1628 adult respondents (mean age 54 years; 47% male) in the UK-based iConnect study. Travel and recreational physical activity were measured using detailed seven-day recall instruments. Adjusted linear regression models were fitted with change in active travel defined as ‘decreased’ (<−15 min/week), ‘maintained’ (±15 min/week) or ‘increased’ (>15 min/week) as the primary exposure variable and changes in (a) recreational and (b) total physical activity (min/week) as the primary outcome variables. Results Active travel increased in 32% (n=529), was maintained in 33% (n=534) and decreased in 35% (n=565) of respondents. Recreational physical activity decreased in all groups but this decrease was not greater in those whose active travel increased. Conversely, changes in active travel were associated with commensurate changes in total physical activity. Compared with those whose active travel remained unchanged, total physical activity decreased by 176.9 min/week in those whose active travel had decreased (adjusted regression coefficient −154.9, 95% CI −195.3 to −114.5) and was 112.2 min/week greater among those whose active travel had increased (adjusted regression coefficient 135.1, 95% CI 94.3 to 175.9). Conclusion An increase in active travel was associated with a commensurate increase in total physical activity and not a decrease in recreational physical activity. PMID:23445724

  20. Effects of Total Ginsenosides on the Feeding Behavior and Two Enzymes Activities of Mythimna separata (Walker) Larvae.

    PubMed

    Zhang, Ai-Hua; Tan, Shi-Qiang; Zhao, Yan; Lei, Feng-Jie; Zhang, Lian-Xue

    2015-01-01

    Ginsenosides, the main effective components of Panax ginseng C.A. Meyer and Panax quinquefolius L., are important allelochemicals of ginseng. Although many studies have targeted the pharmacological, chemical, and clinical properties of ginsenosides, little is known about their ecological role in ginseng population adaptation and evolution. Pests rarely feed on ginseng, and it is not known why. This study investigated the effects of total ginsenosides on feeding behavior and activities of acetylcholinesterase (AChE) and glutathione s-transferase (GST) in Mythimna separata (Walker) larvae. The results showed that the total ginsenosides had significant antifeeding activity against M. separata larvae, determined by nonselective and selective antifeeding bioassays. In addition, the total ginsenosides had inhibitory effects on the activities of GST and AChE. The antifeeding ratio was the highest at 8 h, then decreased, and was the lowest at 16 h. Both GST and AChE activities decreased from 0 h to 48 h in all total ginsenosides treatments but increased at 72 h. Total ginsenosides had antifeeding activity against M. separata larvae and inhibitory effects on the activities of GST and AChE. PMID:26074991

  1. Intact and total insulin-like growth factor-binding protein-3 (IGFBP-3) levels in relation to breast cancer risk factors: a cross-sectional study

    PubMed Central

    Diorio, Caroline; Brisson, Jacques; Bérubé, Sylvie; Pollak, Michael

    2008-01-01

    Introduction Levels of insulin-like growth factor (IGF)-I and its main binding protein (IGFBP-3) have been associated with breast cancer risk among premenopausal women. However, associations of IGFBP-3 levels with breast cancer risk have been inconsistent, possibly due to the different predominant forms of circulating IGFBP-3 (intact versus fragmented) that were measured in these studies. Here, we examine the association of breast cancer risk factors with intact and total IGFBP-3 levels. Methods This cross-sectional study includes 737 premenopausal women recruited at screening mammography. Plasma intact and total IGFBP-3 and IGF-I levels were measured by enzyme-linked immunosorbent assay methods. Percent and absolute breast density were estimated using a computer-assisted method. The associations were evaluated using generalized linear models and Pearson (r) or Spearman (rs) partial correlation coefficients. Results Means ± standard deviations of intact and total IGFBP-3 levels (ng/mL) were 1,044 ± 234 and 4,806 ± 910, respectively. Intact and total IGFBP-3 levels were correlated with age and smoking. Levels of intact IGFBP-3 were negatively correlated with waist-to-hip ratio (WHR) (r = -0.128; P = 0.0005), parity (rs = -0.078; P = 0.04), and alcohol intake (r = -0.137; P = 0.0002) and positively correlated with energy intake (r = 0.075; P = 0.04). In contrast, total IGFBP-3 levels were positively correlated with WHR (r = 0.115; P = 0.002), parity (rs = 0.089; P = 0.02), body mass index (BMI) (r = 0.115; P = 0.002), physical activity (r = 0.118; P = 0.002), and IGF-I levels (r = 0.588; P < 0.0001) and negatively correlated with percent or absolute breast density (r = -0.095; P = 0.01 and r = -0.075; P = 0.04, respectively). Conclusion Our data show that associations of some breast cancer risk factors with intact levels of IGFBP-3 are different from those with total (intact and fragmented) IGFBP-3 levels. These findings suggest that different molecular forms of

  2. AMP-activated Protein Kinase Is Activated as a Consequence of Lipolysis in the Adipocyte

    Technology Transfer Automated Retrieval System (TEKTRAN)

    AMP-activated protein kinase (AMPK) is activated in adipocytes during exercise and other states in which lipolysis is stimulated. However, the mechanism(s) responsible for this effect and its physiological relevance are unclear. To examine these questions, 3T3-L1 adipocytes were treated with agents...

  3. Wounding systemically activates a mitogen-activated protein kinase in forage and turf grasses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Forage and turf grasses are continually cut and grazed by livestock, however very little is known concerning the perception or molecular responses to wounding. Mechanical wounding rapidly activated a 46 kDa and a 44 kDa mitogen-activated protein kinase (MAPK) in six different grass species. In the m...

  4. Cell-free protein synthesis enables high yielding synthesis of an active multicopper oxidase.

    PubMed

    Li, Jian; Lawton, Thomas J; Kostecki, Jan S; Nisthal, Alex; Fang, Jia; Mayo, Stephen L; Rosenzweig, Amy C; Jewett, Michael C

    2016-02-01

    Multicopper oxidases (MCOs) are broadly distributed in all kingdoms of life and perform a variety of important oxidative reactions. These enzymes have potential biotechnological applications; however, the applications are impeded by low expression yields in traditional recombinant hosts, solubility issues, and poor copper cofactor assembly. As an alternative to traditional recombinant protein expression, we show the ability to use cell-free protein synthesis (CFPS) to produce complex MCO proteins with high soluble titers. Specifically, we report the production of MCOs in an Escherichia coli-based cell-free transcription-translation system. Total yields as high as 1.2 mg mL(-1) were observed after a 20-h batch reaction. More than 95% of the protein was soluble and activity was obtained by simple post-CFPS addition of copper ions in the form of CuSO4 . Scale-up reactions were achieved from 15 to 100 µL without a decrease in productivity and solubility. CFPS titers were higher than in vivo expression titers and more soluble, avoiding the formation of inclusion bodies. Our work extends the utility of the cell-free platform to the production of active proteins containing copper cofactors and demonstrates a simple method for producing MCOs. PMID:26356243

  5. Protein inhibitor of activated STAT3 inhibits adipogenic gene expression

    SciTech Connect

    Deng Jianbei; Hua Kunjie; Caveney, Erica J.; Takahashi, Nobuyuki; Harp, Joyce B. . E-mail: jharp@unc.edu

    2006-01-20

    Protein inhibitor of activated STAT3 (PIAS3), a cytokine-induced repressor of signal transducer and activator of transcription 3 (STAT3) and a modulator of a broad array of nuclear proteins, is expressed in white adipose tissue, but its role in adipogenesis is not known. Here, we determined that PIAS3 was constitutively expressed in 3T3-L1 cells at all stages of adipogenesis. However, it translocated from the nucleus to the cytoplasm 4 days after induction of differentiation by isobutylmethylxanthine, dexamethasone, and insulin (MDI). In ob/ob mice, PIAS3 expression was increased in white adipose tissue depots compared to lean mice and was found in the cytoplasm of adipocytes. Overexpression of PIAS3 in differentiating preadipocytes, which localized primarily to the nucleus, inhibited mRNA level gene expression of adipogenic transcription factors C/EBP{alpha} and PPAR{gamma}, as well as their downstream target genes aP2 and adiponectin. PIAS3 also inhibited C/EBP{alpha} promoter activation mediated specifically by insulin, but not dexamethasone or isobutylmethylxanthine. Taken together, these data suggest that PIAS3 may play an inhibitory role in adipogenesis by modulating insulin-activated transcriptional activation events. Increased PIAS3 expression in adipose tissue may play a role in the metabolic disturbances of obesity.

  6. Pokeweed Antiviral Protein, a Ribosome Inactivating Protein: Activity, Inhibition and Prospects

    PubMed Central

    Domashevskiy, Artem V.; Goss, Dixie J.

    2015-01-01

    Viruses employ an array of elaborate strategies to overcome plant defense mechanisms and must adapt to the requirements of the host translational systems. Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome inactivating protein (RIP) and is an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin (S/R) loop of large rRNA, arresting protein synthesis at the translocation step. PAP is thought to play an important role in the plant’s defense mechanism against foreign pathogens. This review focuses on the structure, function, and the relationship of PAP to other RIPs, discusses molecular aspects of PAP antiviral activity, the novel inhibition of this plant toxin by a virus counteraction—a peptide linked to the viral genome (VPg), and possible applications of RIP-conjugated immunotoxins in cancer therapeutics. PMID:25635465

  7. Crystal Structure of the Protein Kinase Domain of Yeast AMP-Activated Protein Kinase Snf1

    SciTech Connect

    Rudolph,M.; Amodeo, G.; Bai, Y.; Tong, L.

    2005-01-01

    AMP-activated protein kinase (AMPK) is a master metabolic regulator, and is an important target for drug development against diabetes, obesity, and other diseases. AMPK is a hetero-trimeric enzyme, with a catalytic ({alpha}) subunit, and two regulatory ({beta} and {gamma}) subunits. Here we report the crystal structure at 2.2 Angstrom resolution of the protein kinase domain (KD) of the catalytic subunit of yeast AMPK (commonly known as SNF1). The Snf1-KD structure shares strong similarity to other protein kinases, with a small N-terminal lobe and a large C-terminal lobe. Two negative surface patches in the structure may be important for the recognition of the substrates of this kinase.

  8. [Virucidal activity of disinfectants. Influence of the serum protein upon the virucidal activity of disinfectants].

    PubMed

    Noda, M; Matsuda, S; Kobayashi, M

    2000-08-01

    Five disinfectants were tested for virucidal activity on three DNA viruses and three RNA viruses in the presence or absence of serum protein. Disinfectants of the aldehyde and halogen groups had a virucidal activity on human herpes virus, bovine rhabdo virus, human immunodeficiency virus, human adeno virus, porcine parvo virus, and polio virus. Disinfectants of the invert and amphoteric soap groups, and biganide group had a destructive effect on RNA and DNA viruses possessing an envelope. The presence of serum protein exerted great influence upon the virucidal activity of disinfectants of the invert and amphoteric soap groups. PMID:11019515

  9. Proapoptotic Activities of Protein Disulfide Isomerase (PDI) and PDIA3 Protein, a Role of the Bcl-2 Protein Bak*

    PubMed Central

    Zhao, Guoping; Lu, Huayi; Li, Chi

    2015-01-01

    Protein disulfide isomerase (PDI) family proteins are classified as enzymatic chaperones for reconstructing misfolded proteins. Previous studies have shown that several PDI members possess potential proapoptotic functions. However, the detailed molecular mechanisms of PDI-mediated apoptosis are not completely known. In this study, we investigated how two members of PDI family, PDI and PDIA3, modulate apoptotic signaling. Inhibiting PDI and PDIA3 activities pharmacologically alleviates apoptosis induced by various apoptotic stimuli. Although a decrease of PDIA3 expression alleviates apoptotic responses, overexpression of PDIA3 exacerbates apoptotic signaling. Importantly, Bak, but not Bax, is essential for PDIA3-induced proapoptotic signaling. Furthermore, both purified PDI and PDIA3 proteins induce Bak-dependent, but not Bax-dependent, mitochondrial outer membrane permeabilization in vitro, probably through triggering Bak oligomerization on mitochondria. Our results suggest that both of PDI and PDIA3 possess Bak-dependent proapoptotic function through inducing mitochondrial outer membrane permeabilization, which provides a new mechanism linking ER chaperone proteins and apoptotic signaling. PMID:25697356

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

    SciTech Connect

    Ganguly, C.; Chelladurai, M.; Ganguly, P.

    1986-05-01

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

  11. Antioxidant, Antimicrobial Activity and Total Phenolic Content within the Aerial Parts of Artemisia absinthum, Artemisia santonicum and Saponaria officinalis

    PubMed Central

    Sengul, Memnune; Ercisli, Sezai; Yildiz, Hilal; Gungor, Neva; Kavaz, Arzu; Çetin, Bülent

    2011-01-01

    Three native Turkish medicinal and aromatic plants (Artemisia absinthum, Artemisia santonicum and Saponaria officinalis) were investigated to analyze their antioxidant activity, total phenolic content and antimicrobial activity. Their total antioxidant activity was determined by using a β-carotene bleaching assay and their antimicrobial activity was determined by utilizing an agar disc diffusion assay. Methanol extracts of the three species analyzed showed high antioxidant activity and among them Artemisia absinthum possessed the highest quantity (71.78%). The total phenolic content (Folin-Ciocalteu assay) was shown to be between 6.57 μgGAE/mg dry weight basis (Saponaria officinalis) and 8.86 μgGAE/mg dry weight basis (Artemisia absinthum). There was a positive correlation (R = 0.819) between the total phenolic content and the antioxidant activity measured in the plant samples. The aqueous and methanol extracts of the aerial parts of the species showed antibacterial activities against a number of microorganisms. The methanol extracts were found to inhibit the growth of microorganisms more than the aqueous extracts. These findings suggest that the methanol extracts of the plants tested contain compounds with antimicrobial properties. These exhibited properties propose that such plant extracts can possibly be used as natural preservatives in the food and pharmaceutical industries. PMID:24363680

  12. Fast calcium sensor proteins for monitoring neural activity

    PubMed Central

    Badura, Aleksandra; Sun, Xiaonan Richard; Giovannucci, Andrea; Lynch, Laura A.; Wang, Samuel S.-H.

    2014-01-01

    Abstract. A major goal of the BRAIN Initiative is the development of technologies to monitor neuronal network activity during active information processing. Toward this goal, genetically encoded calcium indicator proteins have become widely used for reporting activity in preparations ranging from invertebrates to awake mammals. However, slow response times, the narrow sensitivity range of Ca2+ and in some cases, poor signal-to-noise ratio still limit their usefulness. Here, we review recent improvements in the field of neural activity-sensitive probe design with a focus on the GCaMP family of calcium indicator proteins. In this context, we present our newly developed Fast-GCaMPs, which have up to 4-fold accelerated off-responses compared with the next-fastest GCaMP, GCaMP6f. Fast-GCaMPs were designed by destabilizing the association of the hydrophobic pocket of calcium-bound calmodulin with the RS20 binding domain, an intramolecular interaction that protects the green fluorescent protein chromophore. Fast-GCaMP6f-RS06 and Fast-GCaMP6f-RS09 have rapid off-responses in stopped-flow fluorimetry, in neocortical brain slices, and in the intact cerebellum in vivo. Fast-GCaMP6f variants should be useful for tracking action potentials closely spaced in time, and for following neural activity in fast-changing compartments, such as axons and dendrites. Finally, we discuss strategies that may allow tracking of a wider range of neuronal firing rates and improve spike detection. PMID:25558464

  13. Circulating FGF21 proteolytic processing mediated by fibroblast activation protein

    PubMed Central

    Zhen, Eugene Y.; Jin, Zhaoyan; Ackermann, Bradley L.; Thomas, Melissa K.; Gutierrez, Jesus A.

    2015-01-01

    Fibroblast growth factor 21 (FGF21), a hormone implicated in the regulation of glucose homoeostasis, insulin sensitivity, lipid metabolism and body weight, is considered to be a promising therapeutic target for the treatment of metabolic disorders. Despite observations that FGF21 is rapidly proteolysed in circulation rending it potentially inactive, little is known regarding mechanisms by which FGF21 protein levels are regulated. We systematically investigated human FGF21 protein processing using mass spectrometry. In agreement with previous reports, circulating human FGF21 was found to be cleaved primarily after three proline residues at positions 2, 4 and 171. The extent of FGF21 processing was quantified in a small cohort of healthy human volunteers. Relative abundance of FGF21 proteins cleaved after Pro-2, Pro-4 and Pro-171 ranged from 16 to 30%, 10 to 25% and 10 to 34%, respectively. Dipeptidyl peptidase IV (DPP-IV) was found to be the primary protease responsible for N-terminal cleavages after residues Pro-2 and Pro-4. Importantly, fibroblast activation protein (FAP) was implicated as the protease responsible for C-terminal cleavage after Pro-171, rendering the protein inactive. The requirement of FAP for FGF21 proteolysis at the C-terminus was independently demonstrated by in vitro digestion, immunodepletion of FAP in human plasma, administration of an FAP-specific inhibitor and by human FGF21 protein processing patterns in FAP knockout mouse plasma. The discovery that FAP is responsible for FGF21 inactivation extends the FGF21 signalling pathway and may enable novel approaches to augment FGF21 actions for therapeutic applications. PMID:26635356

  14. Positive feedback of protein kinase C proteolytic activation during apoptosis.

    PubMed Central

    Leverrier, Sabrina; Vallentin, Alice; Joubert, Dominique

    2002-01-01

    In contrast with protein kinase Calpha (PKCalpha) and PKCepsilon, which are better known for promoting cell survival, PKCdelta is known for its pro-apoptotic function, which is exerted mainly through a caspase-3-dependent proteolytic activation pathway. In the present study, we used the rat GH3B6 pituitary adenoma cell line to show that PKCalpha and PKCepsilon are activated and relocalized together with PKCdelta when apoptosis is induced by a genotoxic stress. Proteolytic activation is a crucial step used by the three isoforms since: (1) the catalytic domains of the PKCalpha, PKCepsilon or PKCdelta isoforms (CDalpha, CDepsilon and CDdelta respectively) accumulated, and this accumulation was dependent on the activity of both calpain and caspase; and (2) transient expression of CDalpha, CDepsilon or CDdelta sufficed to induce apoptosis. However, following this initial step of proteolytic activation, the pathways diverge; cytochrome c release and caspase-3 activation are induced by CDepsilon and CDdelta, but not by CDalpha. Another interesting finding of the present study is the proteolysis of PKCdelta induced by CDepsilon expression that revealed the existence of a cross-talk between PKC isoforms during apoptosis. Hence the PKC family may participate in the apoptotic process of pituitary adenoma cells at two levels: downstream of caspase and calpain, and via retro-activation of caspase-3, resulting in the amplification of its own proteolytic activation. PMID:12238950

  15. Noise exposure immediately activates cochlear mitogen-activated protein kinase signaling

    PubMed Central

    Alagramam, Kumar N.; Stepanyan, Ruben; Jamesdaniel, Samson; Chen, Daniel H.-C.; Davis, Rickie R.

    2015-01-01

    Noise-induced hearing loss (NIHL) is a major public health issue worldwide. Uncovering the early molecular events associated with NIHL would reveal mechanisms leading to the hearing loss. Our aim is to investigate the immediate molecular responses after different levels of noise exposure and identify the common and distinct pathways that mediate NIHL. Previous work showed mice exposed to 116 decibels sound pressure level (dB SPL) broadband noise for 1 h had greater threshold shifts than the mice exposed to 110 dB SPL broadband noise, hence we used these two noise levels in this study. Groups of 4–8-week-old CBA/CaJ mice were exposed to no noise (control) or to broadband noise for 1 h, followed by transcriptome analysis of total cochlear RNA isolated immediately after noise exposure. Previously identified and novel genes were found in all data sets. Following exposure to noise at 116 dB SPL, the earliest responses included up-regulation of 243 genes and down-regulation of 61 genes, while a similar exposure at 110 dB SPL up-regulated 155 genes and down-regulated 221 genes. Bioinformatics analysis indicated that mitogen-activated protein kinase (MAPK) signaling was the major pathway in both levels of noise exposure. Nevertheless, both qualitative and quantitative differences were noticed in some MAPK signaling genes, after exposure to different noise levels. Cacna1b, Cacna1g, and Pla2g6, related to calcium signaling were down-regulated after 110 dB SPL exposure, while the fold increase in the expression of Fos was relatively lower than what was observed after 116 dB SPL exposure. These subtle variations provide insight on the factors that may contribute to the differences in NIHL despite the activation of a common pathway. PMID:25387536

  16. Antioxidative activity of protein hydrolysates prepared from alkaline-aided channel catfish protein isolates.

    PubMed

    Theodore, Ann E; Raghavan, Sivakumar; Kristinsson, Hordur G

    2008-08-27

    Antioxidative activity of hydrolyzed protein prepared from alkali-solubilized catfish protein isolates was studied. The isolates were hydrolyzed to 5, 15, and 30% degree of hydrolysis using the protease enzyme, Protamex. Hydrolyzed protein was separated into hydrolysates and soluble supernatants, and both of these fractions were studied for their metal chelating ability, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability, ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC), and their ability to inhibit the formation of thiobarbituric acid reactive substances (TBARS) in washed tilapia muscle containing tilapia hemolysate. Both hydrolysates and supernatants were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results showed that DPPH radical scavenging ability and reducing power of catfish protein hydrolysates decreased, whereas the ORAC value, metal chelating ability, and ability to inhibit TBARS increased, with an increase in the degree of hydrolysis. Hydrolysate samples showed higher DPPH radical scavenging ability and Fe(3+) reducing ability, and supernatant samples had higher metal chelating ability. In general, low molecular weight (MW) peptides had high ORAC values and high metal chelating ability, and high MW peptides had a higher reducing power (FRAP) and were more effective in scavenging DPPH radicals. In a washed muscle model system, the ability of catfish protein hydrolysates and their corresponding supernatants to inhibit the formation of TBARS increased with an increase in the degree of hydrolysis. PMID:18662014

  17. Activation of G protein by opioid receptors: role of receptor number and G-protein concentration.

    PubMed

    Remmers, A E; Clark, M J; Alt, A; Medzihradsky, F; Woods, J H; Traynor, J R

    2000-05-19

    The collision-coupling model for receptor-G-protein interaction predicts that the rate of G-protein activation is dependent on receptor density, but not G-protein levels. C6 cells expressing mu- or delta-opioid receptors, or SH-SY5Y cells, were treated with beta-funaltrexamine (mu) or naltrindole-5'-isothiocyanate (delta) to decrease receptor number. The time course of full or partial agonist-stimulated ¿35SGTPgammaS binding did not vary in C6 cell membranes containing <1-25 pmol/mg mu-opioid receptor, or 1. 4-4.3 pmol/mg delta-opioid receptor, or in SHSY5Y cells containing 0. 16-0.39 pmol/mg receptor. The association of ¿35SGTPgammaS binding was faster in membranes from C6mu cells than from C6delta cells. A 10-fold reduction in functional G-protein, following pertussis toxin treatment, lowered the maximal level of ¿35SGTPgammaS binding but not the association rate. These data indicate a compartmentalization of opioid receptors and G protein at the cell membrane. PMID:10822058

  18. A high correlation indicating for an evaluation of antioxidant activity and total phenolics content of various chilli varieties.

    PubMed

    Sricharoen, Phitchan; Techawongstein, Suchila; Chanthai, Saksit

    2015-12-01

    Use of dimethyl sulfoxide (DMSO) as a suitable extraction solvent under the optimum conditions of microwave assisted extraction (MAE) prior to total phenolics determination and antioxidant activity assay was conducted. The MAE method was done with 0.05 g sample in 10 mL DMSO at 500 W within 5 min. The effects of DMSO on various antioxidant activities using DPPH(·+), DMPD(·+), ABTS(·+) and FRAP, and Folin-Ciocalteu reagent were investigated. From the results, it is clearly demonstrated that the DMSO itself shows no effect on any of those antioxidant assays including total phenolics content. The DMSO extracts of 14 local chilli varieties gave their antioxidant activities in the following ranges: DPPH, 3.07-20.0; DMPD, 1.52-6.61; ABTS, 20.4-56.0; FRAP, 8.98-42.1 mg GA/g DW. Their total phenolics contents were found in the range of 53.7-200 mg GA/g DW. This study demonstrates that DMSO was found as the most suitable extraction solvent for antioxidants and phenolics from chilli. In addition, analysis of the data obtained among four antioxidant activity assays with respect to total phenolics shows a highly significant and positive regression coefficient (r > 0.92), indicating the total phenolics are primarily responsible for their antioxidant activity of the chilli extract. PMID:26604380

  19. Caspase processing activates atypical protein kinase C zeta by relieving autoinhibition and destabilizes the protein.

    PubMed Central

    Smith, Lucinda; Wang, Zhi; Smith, Jeffrey B

    2003-01-01

    Treatment of HeLa cells with tumour necrosis factor alpha (TNFalpha) induced caspase processing of ectopic PKC (protein kinase C) zeta, which converted most of the holoenzyme into the freed kinase domain and increased immune-complex kinase activity. The goal of the present study was to determine the basis for the increased kinase activity that is associated with caspase processing of PKC zeta. Atypical PKC iota is largely identical with PKC zeta, except for a 60-amino-acid segment that lacks the caspase-processing sites of the zeta isoform. Replacement of this segment of PKC zeta with the corresponding segment of PKC iota prevented caspase processing and activation of the kinase function. Processing of purified recombinant PKC zeta by caspase 3 in vitro markedly increased its kinase activity. Caspase processing activated PKC zeta in vitro or intracellularly without increasing the phosphorylation of Thr410 of PKC zeta, which is required for catalytic competency. The freed kinase domain of PKC zeta had a much shorter half-life than the holoenzyme in transfected HeLa cells and in non-transfected kidney epithelial cells. Treatment with TNF-alpha shortened the half-life of the kinase domain protein, and proteasome blockade stabilized the protein. Studies of kinase-domain mutants indicate that a lack of negative charge at Thr410 can shorten the half-life of the freed kinase domain. The present findings indicate that the freed kinase domain has substantially higher kinase activity and a much shorter half-life than the holoenzyme because of accelerated degradation by the ubiquitin-proteasome system. PMID:12887331

  20. Centromeric binding and activity of Protein Phosphatase 4

    PubMed Central

    Lipinszki, Zoltan; Lefevre, Stephane; Savoian, Matthew S.; Singleton, Martin R.; Glover, David M.; Przewloka, Marcin R.

    2015-01-01

    The cell division cycle requires tight coupling between protein phosphorylation and dephosphorylation. However, understanding the cell cycle roles of multimeric protein phosphatases has been limited by the lack of knowledge of how their diverse regulatory subunits target highly conserved catalytic subunits to their sites of action. Phosphoprotein phosphatase 4 (PP4) has been recently shown to participate in the regulation of cell cycle progression. We now find that the EVH1 domain of the regulatory subunit 3 of Drosophila PP4, Falafel (Flfl), directly interacts with the centromeric protein C (CENP-C). Unlike other EVH1 domains that interact with proline-rich ligands, the crystal structure of the Flfl amino-terminal EVH1 domain bound to a CENP-C peptide reveals a new target-recognition mode for the phosphatase subunit. We also show that binding of Flfl to CENP-C is required to bring PP4 activity to centromeres to maintain CENP-C and attached core kinetochore proteins at chromosomes during mitosis. PMID:25562660

  1. Covalent agonists for studying G protein-coupled receptor activation

    PubMed Central

    Weichert, Dietmar; Kruse, Andrew C.; Manglik, Aashish; Hiller, Christine; Zhang, Cheng; Hübner, Harald; Kobilka, Brian K.; Gmeiner, Peter

    2014-01-01

    Structural studies on G protein-coupled receptors (GPCRs) provide important insights into the architecture and function of these important drug targets. However, the crystallization of GPCRs in active states is particularly challenging, requiring the formation of stable and conformationally homogeneous ligand-receptor complexes. Native hormones, neurotransmitters, and synthetic agonists that bind with low affinity are ineffective at stabilizing an active state for crystallogenesis. To promote structural studies on the pharmacologically highly relevant class of aminergic GPCRs, we here present the development of covalently binding molecular tools activating Gs-, Gi-, and Gq-coupled receptors. The covalent agonists are derived from the monoamine neurotransmitters noradrenaline, dopamine, serotonin, and histamine, and they were accessed using a general and versatile synthetic strategy. We demonstrate that the tool compounds presented herein display an efficient covalent binding mode and that the respective covalent ligand-receptor complexes activate G proteins comparable to the natural neurotransmitters. A crystal structure of the β2-adrenoreceptor in complex with a covalent noradrenaline analog and a conformationally selective antibody (nanobody) verified that these agonists can be used to facilitate crystallogenesis. PMID:25006259

  2. Superoxide dismutase activity of Cu-bound prion protein

    NASA Astrophysics Data System (ADS)

    Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry

    2009-03-01

    Misfolding of the prion protein, PrP, has been linked to a group of neurodegenerative diseases, including the mad cow disease in cattle and the Creutzfeldt-Jakob disease in humans. The normal function of PrP is still unknown, but it was found that the PrP can efficiently bind Cu(II) ions. Early experiments suggested that Cu-PrP complex possesses significant superoxide dismutase (SOD) activity, but later experiments failed to confirm it and at present this issue remains unresolved. Using a recently developed hybrid DFT/DFT method, which combines Kohn-Sham DFT for the solute and its first solvation shells with orbital-free DFT for the remainder of the solvent, we have investigated SOD activity of PrP. The PrP is capable of incorporating Cu(II) ions in several binding modes and our calculations find that each mode has a different SOD activity. The highest activity found is comparable to those of well-known SOD proteins, suggesting that the conflicting experimental results may be due to different bindings of Cu(II) in those experiments.

  3. [National evaluation of the diagnosis of activated protein C resistance].

    PubMed

    Montiel-Manzano, Guadalupe; de la Peña-Díaz, Aurora; Majluf-Cruz, Abraham; Cesarman-Maus, Gabriela; Corona-de la Peña, Norma; Cruz-Cruz, Donají; Gaminio, Elizabeth; Martínez-Murillo, Carlos; Mayagoitia, Teresa; Miranda-Peralta, Enrique; Poblete, Teresita; Quintana-Martínez, Sandra; Ramírez, Raúl; Razo, Daniel; Ruiz de Chávez-Ochoa, Adriana; Reyes-Núñez, Virginia Adriana; Salazar, Rosario; Vicencio-Santiago, Guadalupe Virginia; Villa, Rosario; Reyes-Núñez, Aurelia Virginia

    2003-01-01

    Thrombophilia or prothrombotic state appears when activation of blood hemostatic mechanisms overcomes the physiological anticoagulant capacity allowing a thrombotic event. Thrombosis is the leading worldwide mortality cause and due to its high associated morbidity and mortality, it should be insisted in the opportune identification of a thrombophilic state. The study of thrombophilia identifies individuals at high risk for thrombosis. This meeting was conceived first to analyze the current status of the diagnosis of thrombophilia in Mexico and second to create the base for a national consensus for thrombophilia screening and for the establishment of a national center for laboratory reference and quality control for thrombophilia. Since searching of activated protein C resistance (APCR) and FV Leiden seem to have priority either in the clinical setting and in public health services, it was decided to start with these two abnormalities as a model to analyze the current status of thrombophilia diagnosis in the clinical laboratory. At this time, several thrombophilic abnormalities have been described however, APCR remains the most important cause of thrombophilia, accounting for as much as 20% to 60% of all venous thrombosis. APCR is a consequence of the resistance of activated FV to be inactivated by activated protein C. Procoagulant activity of activated FV increases the risk of thrombosis. Hereditary APCR is almost always due to a point mutation at the nucleotide 1691 of the FV gen inducing an Arg506Glu substitution in FV molecule. This mutation is better known as FV Leiden. Heterocygous carriers of FV Leiden have a thrombotic risk 5 to 10 times higher than general population while the risk for the homocygote state is increased 50 to 100-fold. When activated PC is added to plasma from patients with FV Leiden, this last resists the anticoagulant effect of activated PC. Therefore, thrombin production is not inhibited. This phenomenon is called APCR. The functional

  4. Rapid fluorescent monitoring of total protein patterns on sodium dodecyl sulfate-polyacrylamide gels and western blots before immunodetection and sequencing.

    PubMed

    Alba, F J; Daban, J R

    1998-10-01

    The fluorogenic dye 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) has been used for the detection of total protein patterns on polyvinylidene difluoride (PVDF) membranes. Fluorescent staining of protein bands on membranes with this covalent dye is completed in 20 min. Wet membranes are translucent, allowing protein visualization by transillumination with ultraviolet light. The resulting images can be recorded using Polaroid film or a charge-coupled device camera. Electrophoretic bands containing 5-10 ng of protein can be detected on the MDPF-stained Western blot. When proteins are directly transferred to the membrane using a slot blotting device, as little as 0.5 ng of protein can be detected. Previous visualization of protein bands on sodium dodecyl sulfate-polyacrylamide gels with the noncovalent fluorescent dye Nile red (Alba et al., BioTechniques, 1996, 21, 625-626) does not interfere with further MDPF staining and fluorescent detection of these bands transferred to PVDF membranes. Thus, Nile red and MDPF staining can be performed sequentially, allowing the rapid monitoring of total protein patterns on both the electrophoretic gel and Western blot. Using the conditions described in this study, MDPF staining does not preclude further N-terminal microsequencing and immunodetection of specific bands with polyclonal antibodies. PMID:9820958

  5. Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis

    PubMed Central

    Kohansal-Nodehi, Mahdokht; Chua, John JE; Urlaub, Henning; Jahn, Reinhard; Czernik, Dominika

    2016-01-01

    Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes associated with presynaptic activity and provides a foundation for further functional analysis of key phosphosites involved in presynaptic plasticity. DOI: http://dx.doi.org/10.7554/eLife.14530.001 PMID:27115346

  6. Fluctuation driven active molecular transport in passive channel proteins

    NASA Astrophysics Data System (ADS)

    Kosztin, Ioan

    2006-03-01

    Living cells interact with their extracellular environment through the cell membrane, which acts as a protective permeability barrier for preserving the internal integrity of the cell. However, cell metabolism requires controlled molecular transport across the cell membrane, a function that is fulfilled by a wide variety of transmembrane proteins, acting as either passive or active transporters. In this talk it is argued that, contrary to the general belief, in active cell membranes passive and spatially asymmetric channel proteins can act as active transporters by consuming energy from nonequilibrium fluctuations fueled by cell metabolism. This assertion is demonstrated in the case of the E. coli aquaglyceroporin GlpF channel protein, whose high resolution crystal structure is manifestly asymmetric. By calculating the glycerol flux through GlpF within the framework of a stochastic model, it is found that, as a result of channel asymmetry, glycerol uptake driven by a concentration gradient is enhanced significantly in the presence of non-equilibrium fluctuations. Furthermore, the enhancement caused by a ratchet-like mechanism is larger for the outward, i.e., from the cytoplasm to the periplasm, flux than for the inward one, suggesting that the same non-equilibrium fluctuations also play an important role in protecting the interior of the cell against poisoning by excess uptake of glycerol. Preliminary data on water and sugar transport through aquaporin and maltoporin channels, respectively, are indicative of the universality of the proposed nonequilibrium-fluctuation-driven active transport mechanism. This work was supported by grants from the Univ. of Missouri Research Board, the Institute for Theoretical Sciences and the Department of Energy (DOE Contract W-7405-ENG-36), and the National Science Foundation (FIBR-0526854).

  7. Totally Tree-mendous Activities: Projects To Discover the Beauty and Benefits of Trees.

    ERIC Educational Resources Information Center

    Hollister, Sarah

    This teacher's guide supplies information and hands-on activities to teach about trees from several disciplines. Activities are grouped into six areas that cover botany, social studies, arts and literature (aesthetics), and trees as a resource. Sections include: (1) Tree Identification, which defines trees and leaves and presents activities that…

  8. Mitogen-activated protein kinase (MAPK) in cardiac tissues.

    PubMed

    Page, C; Doubell, A F

    Mitogen-activated protein kinase (MAPK) has recently emerged as a prominent role player in intracellular signalling in the ventricular myocyte with attention being focussed on its possible role in the development of ventricular hypertrophy. It is becoming clear that MAPK is also active in other cells of cardiac origin such as cardiac fibroblasts and possible functions of this signalling pathway in the heart have yet to be explored. In this report the mammalian MAPK pathway is briefly outlined, before reviewing current knowledge of the MAPK pathway in cardiac tissue (ventricular myocytes, vascular smooth muscle cells and cardiac fibroblasts). New data is also presented on the presence and activity of MAPK in two additional cardiac celltypes namely atrial myocytes and vascular endothelial cells from the coronary microcirculation. PMID:8739228

  9. Expression of the vertebrate Gli proteins in Drosophila reveals a distribution of activator and repressor activities.

    PubMed

    Aza-Blanc, P; Lin, H Y; Ruiz i Altaba, A; Kornberg, T B

    2000-10-01

    The Cubitus interruptus (Ci) and Gli proteins are transcription factors that mediate responses to Hedgehog proteins (Hh) in flies and vertebrates, respectively. During development of the Drosophila wing, Ci transduces the Hh signal and regulates transcription of different target genes at different locations. In vertebrates, the three Gli proteins are expressed in overlapping domains and are partially redundant. To assess how the vertebrate Glis correlate with Drosophila Ci, we expressed each in Drosophila and monitored their behaviors and activities. We found that each Gli has distinct activities that are equivalent to portions of the regulatory arsenal of Ci. Gli2 and Gli1 have activator functions that depend on Hh. Gli2 and Gli3 are proteolyzed to produce a repressor form able to inhibit hh expression. However, while Gli3 repressor activity is regulated by Hh, Gli2 repressor activity is not. These observations suggest that the separate activator and repressor functions of Ci are unevenly partitioned among the three Glis, yielding proteins with related yet distinct properties. PMID:10976059

  10. Mycobacteriophage putative GTPase-activating protein can potentiate antibiotics.

    PubMed

    Yan, Shuangquan; Xu, Mengmeng; Duan, Xiangke; Yu, Zhaoxiao; Li, Qiming; Xie, Longxiang; Fan, Xiangyu; Xie, Jianping

    2016-09-01

    The soaring incidences of infection by antimicrobial resistant (AR) pathogens and shortage of effective antibiotics with new mechanisms of action have renewed interest in phage therapy. This scenario is exemplified by resistant tuberculosis (TB), caused by resistant Mycobacterium tuberculosis. Mycobacteriophage SWU1 A321_gp67 encodes a putative GTPase-activating protein. Mycobacterium smegmatis with gp67 overexpression showed changed colony formation and biofilm morphology and supports the efficacy of streptomycin and capreomycin against Mycobacterium. gp67 down-regulated the transcription of genes involved in cell wall and biofilm development. To our knowledge, this is the first report to show that phage protein in addition to lysin or recombination components can synergize with existing antibiotics. Phage components might represent a promising new clue for better antibiotic potentiators. PMID:27345061

  11. Peptide biosensors for the electrochemical measurement of protein kinase activity.

    PubMed

    Kerman, Kagan; Song, Haifeng; Duncan, James S; Litchfield, David W; Kraatz, Heinz-Bernhard

    2008-12-15

    The kinase activities are elucidated using the novel redox-active cosubstrate adenosine 5'-[gamma-ferrocene] triphosphate (Fc-ATP), which enables the kinase-catalyzed transfer of a redox active gamma-phosphate-Fc to a hydroxyamino acid. In this report, a versatile electrochemical biosensor is developed for monitoring the activity and inhibition of a serine/threonine kinase, casein kinase 2 (CK2), and protein tyrosine kinases, Abl1-T315I and HER2, in buffered solutions and in cell lysates. The method is based on the labeling of a specific phosphorylation event with Fc, followed by electrochemical detection. The electrochemical response obtained from the "ferrocenylated" peptides enables monitoring the activity of the kinase and its substrate, as well as the inhibition of small molecule inhibitors on protein phosphorylation. Kinetic information was extracted from the electrochemical measurements for the determination of K(m) and V(m) values, which were in agreement with those previously reported. Kinase reactions were also performed in the presence of well-defined inhibitors of CK2, 4,5,6,7-tetrabromo-2-azabenzimidazole, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, and E-3-(2,3,4,5-tetrabromophenyl)acrylic acid as well as the nonspecific kinase inhibitors, staurosporine and N-benzoylstaurosporine. On the basis of the dependency of the Fc signal on inhibitor concentration, K(i) of the inhibitors was estimated, which were also in agreement with the literature values. The performance of the biosensor was optimized including the kinase reaction, incubation with Fc-ATP, and the small molecule inhibitors. Peptide modified electrochemical biosensors are promising candidates for cost-effective in vitro kinase activity and inhibitor screening assays. PMID:18989981

  12. On the Total Energy Deposition Between Periodically Occurring Activations of the Aurora

    NASA Technical Reports Server (NTRS)

    Spann, James F., Jr.; Germany, G. A.; Parks, G. K.; Brittnacher, M. J.; Winglee, R. W.

    1998-01-01

    Total energy deposition in the northern latitudes is used in models to determine the state of the magnetosphere. It is known that on occasion, a series of intensifications of the aurora occur that are regularly spaced. The energy profile of the total energy deposited reflects this occurance. What can be said of the state of the magnetosphere based on these profiles. We present the result of a study which looks at several of these periods when a series of intensifications occur. Conclusions as to what the magnetosphere may be doing are presented.

  13. S100A9 is a Biliary Protein Marker of Disease Activity in Primary Sclerosing Cholangitis

    PubMed Central

    Ruppert, Thomas; Giese, Thomas; Flechtenmacher, Christa; Weiss, Karl Heinz; Kloeters-Plachky, Petra; Stremmel, Wolfgang; Schirmacher, Peter; Sauer, Peter; Gotthardt, Daniel Nils

    2012-01-01

    Background and Aims Bile analysis has the potential to serve as a surrogate marker for inflammatory and neoplastic disorders of the biliary epithelium and may provide insight into biliary pathophysiology and possible diagnostic markers. We aimed to identify biliary protein markers of patients with primary sclerosing cholangitis (PSC) by a proteomic approach. Methods Bile duct-derived bile samples were collected from PSC patients (n = 45) or patients with choledocholithiasis (n = 24, the control group). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to analyse the proteins, 2-D-gel patterns were compared by densitometry, and brush cytology specimens were analysed by RT-PCR. Results A reference bile-duct bile proteome was established in the control group without signs of inflammation or maligancy comprising a total of 379 non-redundant biliary proteins; 21% were of unknown function and 24% had been previously described in serum. In PSC patients, the biliary S100A9 expression was elevated 95-fold (p<0.005), serum protein expression was decreased, and pancreatic enzyme expression was unchanged compared to controls. The S100A9 expression was 2-fold higher in PSC patients with high disease activity than in those with low activity (p<0.05). The brush cytology specimens from the PSC patients with high disease activity showed marked inflammatory activity and leukocyte infiltration compared to the patients with low activity, which correlated with S100A9 mRNA expression (p<0.05). Conclusions The bile-duct bile proteome is complex and its analysis might enhance the understanding of cholestatic liver disease. Biliary S100A9 levels may be a useful marker for PSC activity, and its implication in inflammation and carcinogenesis warrants further investigation. PMID:22253789

  14. Recombinant Human Peptidoglycan Recognition Proteins Reveal Antichlamydial Activity.

    PubMed

    Bobrovsky, Pavel; Manuvera, Valentin; Polina, Nadezhda; Podgorny, Oleg; Prusakov, Kirill; Govorun, Vadim; Lazarev, Vassili

    2016-07-01

    Peptidoglycan recognition proteins (PGLYRPs) are innate immune components that recognize the peptidoglycan and lipopolysaccharides of bacteria and exhibit antibacterial activity. Recently, the obligate intracellular parasite Chlamydia trachomatis was shown to have peptidoglycan. However, the antichlamydial activity of PGLYRPs has not yet been demonstrated. The aim of our study was to test whether PGLYRPs exhibit antibacterial activity against C. trachomatis Thus, we cloned the regions containing the human Pglyrp1, Pglyrp2, Pglyrp3, and Pglyrp4 genes for subsequent expression in human cell lines. We obtained stable HeLa cell lines that secrete recombinant human PGLYRPs into culture medium. We also generated purified recombinant PGLYRP1, -2, and -4 and confirmed their activities against Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. Furthermore, we examined the activities of recombinant PGLYRPs against C. trachomatis and determined their MICs. We also observed a decrease in the infectious ability of chlamydial elementary bodies in the next generation after a single exposure to PGLYRPs. Finally, we demonstrated that PGLYRPs attach to C. trachomatis elementary bodies and activate the expression of the chlamydial two-component stress response system. Thus, PGLYRPs inhibit the development of chlamydial infection. PMID:27160295

  15. The unfolded protein response selectively targets active smoothened mutants.

    PubMed

    Marada, Suresh; Stewart, Daniel P; Bodeen, William J; Han, Young-Goo; Ogden, Stacey K

    2013-06-01

    The Hedgehog signaling pathway, an essential regulator of developmental patterning, has been implicated in playing causative and survival roles in a range of human cancers. The signal-transducing component of the pathway, Smoothened, has revealed itself to be an efficacious therapeutic target in combating oncogenic signaling. However, therapeutic challenges remain in cases where tumors acquire resistance to Smoothened antagonists, and also in cases where signaling is driven by active Smoothened mutants that exhibit reduced sensitivity to these compounds. We previously demonstrated that active Smoothened mutants are subjected to prolonged endoplasmic reticulum (ER) retention, likely due to their mutations triggering conformation shifts that are detected by ER quality control. We attempted to exploit this biology and demonstrate that deregulated Hedgehog signaling driven by active Smoothened mutants is specifically attenuated by ER stressors that induce the unfolded protein response (UPR). Upon UPR induction, active Smoothened mutants are targeted by ER-associated degradation, resulting in attenuation of inappropriate pathway activity. Accordingly, we found that the UPR agonist thapsigargin attenuated mutant Smoothened-induced phenotypes in vivo in Drosophila melanogaster. Wild-type Smoothened and physiological Hedgehog patterning were not affected, suggesting that UPR modulation may provide a novel therapeutic window to be evaluated for targeting active Smoothened mutants in disease. PMID:23572559

  16. Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID

    PubMed Central

    Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inês CR; Willige, Björn C; Fastner, Astrid; Streit, Verena; Port, Sarah A; Colcombet, Jean; de la Fuente van Bentem, Sergio; Hirt, Heribert; Kuster, Bernhard; Schulze, Waltraud X; Hammes, Ulrich Z; Schwechheimer, Claus

    2014-01-01

    The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the—in many cells—asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant. DOI: http://dx.doi.org/10.7554/eLife.02860.001 PMID:24948515

  17. Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID.

    PubMed

    Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inês C R; Willige, Björn C; Fastner, Astrid; Streit, Verena; Port, Sarah A; Colcombet, Jean; de la Fuente van Bentem, Sergio; Hirt, Heribert; Kuster, Bernhard; Schulze, Waltraud X; Hammes, Ulrich Z; Schwechheimer, Claus

    2014-01-01

    The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the--in many cells--asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant. PMID:24948515

  18. Monoclonal antibodies against a specific nonhistone chromosomal protein of Drosophila associated with active genes.

    PubMed

    Howard, G C; Abmayr, S M; Shinefeld, L A; Sato, V L; Elgin, S C

    1981-01-01

    Hybridomas secreting monoclonal antibodies have been produced by fusion of NS-1 mouse myeloma cells with the spleen cells of mice inoculated with a 60-65,000-mol wt fraction of proteins released from Drosophila embryo nuclei treated with DNase I. The antibodies secreted by the hybridomas were examined with polytene chromosomes of formaldehyde-fixed salivary gland squashes by an immunofluorescence assay. Most of the clonal antibodies obtained resulted in specific staining of the chromosomes relative to the cytoplasmic debris. In the case of clone 28, the antibodies showed a preferential association with sites of gene activity, both puffs and loci identified as puffing at some time during the third instar and prepupal period. In larvae that were heat shocked (exposed to 35 degrees C for 15 min before removal and fixation of the glands), the antibodies of clone 28 stained preferentially the induced heat-shock loci while continuing to stain most of the normal set of loci. The antigen for clone 28 was identified as a single protein of approximately 62,000 mol wt by using the antibodies followed by 125I-rabbit anti-mouse Ig to stain nitrocellulose replicas of SDS polyacrylamide gels of total chromosomal proteins. This study demonstrates that monoclonal antibodies can be used successfully in immunofluorescence staining of formaldehyde-fixed polytene chromosomes. The results verify the hypothesis that a specific nonhistone chromosomal protein is preferentially associated with the set of loci that includes both active sites and those scheduled to be active at some time in this developmental program. Such proteins may play a general role in the mechanisms of cell determination and gene activation. PMID:6782108

  19. Ribosomal protein S14 negatively regulates c-Myc activity.

    PubMed

    Zhou, Xiang; Hao, Qian; Liao, Jun-Ming; Liao, Peng; Lu, Hua

    2013-07-26

    The ribosomal gene RPS14 is associated with the cancer-prone 5q-syndrome, which is caused by an interstitial deletion of the long arm of human chromosome 5. Previously, we found that ribosomal protein S14 (RPS14) binds to and inactivates MDM2, consequently leading to p53-dependent cell-cycle arrest and growth inhibition. However, it remains elusive whether RPS14 regulates cell proliferation in a p53-independent manner. Here, we show that RPS14 interacts with the Myc homology box II (MBII) and the C-terminal basic helix-loop-helix leucine zipper (bHLH-LZ) domains of the oncoprotein c-Myc. Further, RPS14 inhibited c-Myc transcriptional activity by preventing the recruitment of c-Myc and its cofactor, TRRAP, to the target gene promoters, as thus suppressing c-Myc-induced cell proliferation. Also, siRNA-mediated RPS14 depletion elevated c-Myc transcriptional activity determined by its target gene, Nucleolin, expression. Interestingly, RPS14 depletion also resulted in the induction of c-Myc mRNA and subsequent protein levels. Consistent with this, RPS14 promoted c-Myc mRNA turnover through an Argonaute 2 (Ago2)- and microRNA-mediated pathway. Taken together, our study demonstrates that RPS14 negates c-Myc functions by directly inhibiting its transcriptional activity and mediating its mRNA degradation via miRNA. PMID:23775087

  20. Coq7p relevant residues for protein activity and stability.

    PubMed

    Busso, Cleverson; Ferreira-Júnior, José Ribamar; Paulela, Janaina A; Bleicher, Lucas; Demasi, Marilene; Barros, Mario H

    2015-12-01

    Coenzyme Q (Q) is an isoprenylated benzoquinone electron carrier required for electronic transport in the mitochondrial respiratory chain, shuttling electrons from complexes I and II to complex III. Q synthesis requires proteins termed Coq (Coq1-Coq11). Coq7p is part of the multimeric complex involved in Q synthesis catalyzing the hydroxylation of demethoxy-Q6 (DMQ6), the last monooxygenase step in Q synthesis with a catalytic center containing a carboxylate-bridged di-iron at the active site of the enzyme. Here we indicate a group of Coq7p residues that modulate protein activity: D53, R57, V111 and S114. R57, V111 and S114 are very conserved residues; V111 and S114 are present in separated communities of amino acid correlation analysis. The coq7 double mutant V111G/S114A and S114E show respiratory deficiency at non permissive temperature, DMQ6 accumulation and lower content of Q6. Therefore we conclude that phosphomimetic S114E inhibit Coq7p activity, and propose that S114 phosphorylation is required to move a non-structured loop of 25 amino acids between helix 2 and 3, and that affects the di-iron coordination in Coq7p catalytic center. PMID:26497406

  1. Egg Activation at Fertilization by a Soluble Sperm Protein.

    PubMed

    Swann, Karl; Lai, F Anthony

    2016-01-01

    The most fundamental unresolved issue of fertilization is to define how the sperm activates the egg to begin embryo development. Egg activation at fertilization in all species thus far examined is caused by some form of transient increase in the cytoplasmic free Ca(2+) concentration. What has not been clear, however, is precisely how the sperm triggers the large changes in Ca(2+) observed within the egg cytoplasm. Here, we review the studies indicating that the fertilizing sperm stimulates a cytosolic Ca(2+) increase in the egg specifically by delivering a soluble factor that diffuses into the cytosolic space of the egg upon gamete membrane fusion. Evidence is primarily considered in species of eggs where the sperm has been shown to elicit a cytosolic Ca(2+) increase by initiating Ca(2+) release from intracellular Ca(2+) stores. We suggest that our best understanding of these signaling events is in mammals, where the sperm triggers a prolonged series of intracellular Ca(2+) oscillations. The strongest empirical studies to date suggest that mammalian sperm-triggered Ca(2+) oscillations are caused by the introduction of a sperm-specific protein, called phospholipase C-zeta (PLCζ) that generates inositol trisphosphate within the egg. We will discuss the role and mechanism of action of PLCζ in detail at a molecular and cellular level. We will also consider some of the evidence that a soluble sperm protein might be involved in egg activation in nonmammalian species. PMID:26631595

  2. Mitogen activated protein kinase at the nuclear pore complex

    PubMed Central

    Faustino, Randolph S; Maddaford, Thane G; Pierce, Grant N

    2011-01-01

    Abstract Mitogen activated protein (MAP) kinases control eukaryotic proliferation, and import of kinases into the nucleus through the nuclear pore complex (NPC) can influence gene expression to affect cellular growth, cell viability and homeostatic function. The NPC is a critical regulatory checkpoint for nucleocytoplasmic traffic that regulates gene expression and cell growth, and MAP kinases may be physically associated with the NPC to modulate transport. In the present study, highly enriched NPC fractions were isolated and investigated for associated kinases and/or activity. Endogenous kinase activity was identified within the NPC fraction, which phosphorylated a 30 kD nuclear pore protein. Phosphomodification of this nucleoporin, here termed Nup30, was inhibited by apigenin and PD-98059, two MAP kinase antagonists as well as with SB-202190, a pharmacological blocker of p38. Furthermore, high throughput profiling of enriched NPCs revealed constitutive presence of all members of the MAP kinase family, extracellular regulated kinases (ERK), p38 and Jun N-terminal kinase. The NPC thus contains a spectrum of associated MAP kinases that suggests an intimate role for ERK and p38 in regulation of nuclear pore function. PMID:20497490

  3. Genome activation by raspberry bushy dwarf virus coat protein.

    PubMed

    Macfarlane, Stuart A; McGavin, Wendy J

    2009-03-01

    Two sets of infectious cDNA clones of raspberry bushy dwarf virus (RBDV) have been constructed, enabling either the synthesis of infectious RNA transcripts or the delivery of infectious binary plasmid DNA by infiltration of Agrobacterium tumefaciens. In whole plants and in protoplasts, inoculation of RBDV RNA1 and RNA2 transcripts led to a low level of infection, which was greatly increased by the addition of RNA3, a subgenomic RNA coding for the RBDV coat protein (CP). Agroinfiltration of RNA1 and RNA2 constructs did not produce a detectable infection but, again, inclusion of a construct encoding the CP led to high levels of infection. Thus, RBDV replication is greatly stimulated by the presence of the CP, a mechanism that also operates with ilarviruses and alfalfa mosaic virus, where it is referred to as genome activation. Mutation to remove amino acids from the N terminus of the CP showed that the first 15 RBDV CP residues are not required for genome activation. Other experiments, in which overlapping regions at the CP N terminus were fused to the monomeric red fluorescent protein, showed that sequences downstream of the first 48 aa are not absolutely required for genome activation. PMID:19218221

  4. Ileal and total tract nutrient digestibilities and fecal characteristics of dogs as affected by soybean protein inclusion in dry, extruded diets.

    PubMed

    Clapper, G M; Grieshop, C M; Merchen, N R; Russett, J C; Brent, J L; Fahey, G C

    2001-06-01

    Plant-based protein sources are generally less variable in chemical composition than animal-based protein sources. However, relatively few data are available on the nutrient digestibilities of plant-based protein sources by companion animals. The effects of including selected soybean protein sources in dog diets on nutrient digestion at the ileum and in the total tract, as well as on fecal characteristics, were evaluated. Six protein sources were used: soybean meal (SBM), Soyafluff 200W (soy flour), Profine F (traditional aqueous-alcohol extracted soy protein concentrate [SPC 1]), Profine E (extruded SPC [SPC 2]), Soyarich I (modified molecular weight SPC [SPC 3]), and poultry meal (PM). All diets were extruded and kibbled. Test ingredients varied in CP and fat contents; however, diets were formulated to be isonitrogenous and isocaloric. Nutrient intakes were similar, except for total dietary fiber (TDF), which was lower (P < 0.01) for dogs fed the PM diet. Apparent ileal digestibilities of DM, OM, fat, and TDF were not different among treatments; however, CP digestibility at the terminal ileum was higher (P < 0.01) for diets containing soy protein sources than for PM. Total tract CP digestibility was greater (P < 0.01) for soy protein-containing diets than for PM. Apparent total tract digestibilities of DM, OM, fat, and TDF were not different among treatments. Apparent amino acid digestibilities at the terminal ileum, excluding methionine, threonine, alanine, and glycine, were higher (P < 0.01) for soy protein-containing diets than for PM. Dogs fed SPC diets had lower (P < 0.01) fecal outputs (g asis feces/g DMI) than dogs fed the SF diet, and dogs fed SBM tended (P < 0.11) to have lower fecal outputs than dogs fed the SF diet. However, dogs fed the PM diet had lower (P < 0.03) fecal outputs than dogs fed SPC-containing diets. Fecal outputs and scores reflected the TDF and nonstructural carbohydrate contents of the soy protein fraction. Soy protein sources are

  5. Small glutamine-rich protein/viral protein U–binding protein is a novel cochaperone that affects heat shock protein 70 activity

    PubMed Central

    Angeletti, Peter C.; Walker, Doriann; Panganiban, Antonito T.

    2002-01-01

    Molecular chaperone complexes containing heat shock protein (Hsp) 70 and Hsp90 are regulated by cochaperones, including a subclass of regulators, such as Hsp70 interacting protein (Hip), C-terminus of Hsp70 interacting protein (CHIP), and Hsp70-Hsp90 organizing factor (Hop), that contain tetratricopeptide repeats (TPRs), where Hsp70 refers to Hsp70 and its nearly identical constitutive counterpart, Hsc70, together. These proteins interact with the Hsp70 to regulate adenosine triphosphatase (ATPase) and folding activities or to generate the chaperone complex. Here we provide evidence that small glutamine-rich protein/viral protein U–binding protein (SGT/UBP) is a cochaperone that negatively regulates Hsp70. By “Far-Western” and pull-down assays, SGT/UBP was shown to interact directly with Hsp70 and weakly with Hsp90. The interaction of SGT/UBP with both these protein chaperones was mapped to 3 TPRs in SGT/UBP (amino acids 95–195) that are flanked by charged residues. Moreover, SGT/UBP caused an approximately 30% reduction in both the intrinsic ATPase activity of Hsc70 and the ability of Hsc70 to refold denatured luciferase in vitro. This negative effect of SGT/UBP on Hsc70 is similar in magnitude to that observed for the cochaperone CHIP. A role for SGT/UBP in protein folding is also supported by evidence that a yeast strain containing a deletion in the yeast homolog to SGT/UBP (ΔSGT/UBP) displays a 50-fold reduction in recovery from heat shock compared with the wild type parent. Together, these results are consistent with a regulatory role for SGT/UBP in the chaperone complex. PMID:12482202

  6. Ahnak protein activates protein kinase C (PKC) through dissociation of the PKC-protein phosphatase 2A complex.

    PubMed

    Lee, In Hye; Lim, Hee Jung; Yoon, Suhyeon; Seong, Je Kyung; Bae, Duk Soo; Rhee, Sue Goo; Bae, Yun Soo

    2008-03-01

    We have previously reported that central repeated units (CRUs) of Ahnak act as a scaffolding protein networking phospholipase Cgamma and protein kinase C (PKC). Here, we demonstrate that an Ahnak derivative consisting of four central repeated units binds and activates PKC-alpha in a phosphatidylserine/1,2-dioleoyl-sn-glycerol-independent manner. Moreover, NIH3T3 cells expressing the 4 CRUs of Ahnak showed enhanced c-Raf, MEK, and Erk phosphorylation in response to phorbol 12-myristate 13-acetate (PMA) compared with parental cells. To evaluate the effect of loss-of-function of Ahnak in cell signaling, we investigated PKC activation and Raf phosphorylation in embryonic fibroblast cells (MEFs) of the Ahnak knock-out (Ahnak(-/-)) mouse. Membrane translocation of PKC-alpha and phosphorylation of Raf in response to PMA or platelet-derived growth factor were decreased in Ahnak null MEF cells compared with wild type MEFs. Several lines of evidence suggest that PKC-alpha activity is regulated through association with protein phosphatase 2A (PP2A). A co-immunoprecipitation assay indicated that the association of PKC-alpha with PP2A was disrupted in NIH3T3 cells expressing 4 CRUs of Ahnak in response to PMA. Consistently, Ahnak null MEF cells stimulated by PMA showed enhanced PKC-PP2A complex formation, and add-back expression of Ahnak into Ahnak null MEF cells abolished the PKC-PP2A complex formation in response to PMA. These data indicate that Ahnak potentiates PKC activation through inhibiting the interaction of PKC with PP2A. PMID:18174170

  7. Activation of protein kinase C by lysophosphatidic acid: dependence on composition of phospholipid vesicles.

    PubMed Central

    Sando, J J; Chertihin, O I

    1996-01-01

    Lysophosphatidic acid (LPA) has attracted recent attention as a major serum-derived regulator implicated in responses to vascular injury and inflammation, in tumour invasiveness and in neuronal signalling and remodelling. Although the possibility of a specific G-protein-coupled LPA receptor protein has been suggested, characterization of such a receptor is lacking. Since LPA can activate protein kinase C (PKC) pathways in many cells and PKC activators mimic many LPA effects, the possibility of more direct LPA effects on PKC was investigated. Phosphatidylcholine (PC)/phosphatidylserine (PS)/diacylglycerol (DAG) lipid vesicles of defined acyl chain composition were used to activate the enzyme. At total concentrations of saturated PC/PS + DAG vesicles (2-3 mM) that provided maximal PKC activation, 1-10 mol % [18:1]-LPA led to a further approx. 2-fold activation of PKC alpha. At lower lipid concentrations, a greater increase was observed with LPA concentrations up to 16-20 mol %. Higher concentrations of LPA were inhibitory. The LPA activation of PKC was dependent on the presence of DAG, PS and Ca2+. [18:1]-Lysophosphatidylcholine produced similar PKC activation in PC/PS/DAG vesicles. [14:0]-LPA was less effective, and longer-chain saturated lysolipids were ineffective. In unsaturated PC/PS vesicles, very little to no effect of LPA was discernable. These results suggest that physiologically or pathologically relevant concentrations of LPA can contribute to PKC activation depending on the composition of the lipid membrane. We hypothesize that LPA may affect the formation of lipid domains that are recognized by the enzyme. PMID:8713089

  8. Immersion freezing of ice nucleation active protein complexes

    NASA Astrophysics Data System (ADS)

    Hartmann, S.; Augustin, S.; Clauss, T.; Wex, H.; Šantl-Temkiv, T.; Voigtländer, J.; Niedermeier, D.; Stratmann, F.

    2013-06-01

    Utilising the Leipzig Aerosol Cloud Interaction Simulator (LACIS), the immersion freezing behaviour of droplet ensembles containing monodisperse particles, generated from a Snomax™ solution/suspension, was investigated. Thereto ice fractions were measured in the temperature range between -5 °C to -38 °C. Snomax™ is an industrial product applied for artificial snow production and contains Pseudomonas syringae} bacteria which have long been used as model organism for atmospheric relevant ice nucleation active (INA) bacteria. The ice nucleation activity of such bacteria is controlled by INA protein complexes in their outer membrane. In our experiments, ice fractions increased steeply in the temperature range from about -6 °C to about -10 °C and then levelled off at ice fractions smaller than one. The plateau implies that not all examined droplets contained an INA protein complex. Assuming the INA protein complexes to be Poisson distributed over the investigated droplet populations, we developed the CHESS model (stoCHastic modEl of similar and poiSSon distributed ice nuclei) which allows for the calculation of ice fractions as function of temperature and time for a given nucleation rate. Matching calculated and measured ice fractions, we determined and parameterised the nucleation rate of INA protein complexes exhibiting class III ice nucleation behaviour. Utilising the CHESS model, together with the determined nucleation rate, we compared predictions from the model to experimental data from the literature and found good agreement. We found that (a) the heterogeneous ice nucleation rate expression quantifying the ice nucleation behaviour of the INA protein complex is capable of describing the ice nucleation behaviour observed in various experiments for both, Snomax™ and P. syringae bacteria, (b) the ice nucleation rate, and its temperature dependence, seem to be very similar regardless of whether the INA protein complexes inducing ice nucleation are attached

  9. The bovine salivary proteins BSP30a and BSP30b are independently expressed BPI-like proteins with anti-Pseudomonas activity.

    PubMed

    Haigh, B; Hood, K; Broadhurst, M; Medele, S; Callaghan, M; Smolenski, G; Dines, M; Wheeler, T

    2008-04-01

    The family of BPI-like proteins are thought to play a role in innate immunity of the airways and oral cavity. They have similarities with bactericidal/permeability-increasing protein (BPI), an important host defence molecule in mammals, in the nucleotide sequence of their mRNAs, organisation of exons and their predicted protein structure. We compared the expression and function of 2 of the 13 known bovine BPI-like proteins, BSP30a and b, which together constitute 30% of the total protein in bovine saliva. Despite their recent divergence, we found that the two proteins have unique expression patterns, considerable inter- and intra-animal variation in abundance and differ in their glycosylation status. Recombinant and native BSP30a and b exhibited growth suppression activity against Pseudomonas aeruginosa and Streptococcus pneumoniae. Native BSP30a and b had no significant lipopolysaccharide-binding activity. These data provide functional evidence supporting a role for the BPI-like proteins in host defence and suggest that BSP30a and b contribute to the growth suppression function of bovine saliva through a mechanism independent of LPS opsonisation. PMID:18055015

  10. Total joint replacement: A multiple risk factor analysis of physical activity level 1-2 years postoperatively.

    PubMed

    Paxton, Elizabeth W; Torres, Andy; Love, Rebecca M; Barber, Thomas C; Sheth, Dhiren S; Inacio, Maria C S

    2016-07-01

    Background and purpose - The effect of total joint arthroplasty (TJA) on physical activity is not fully understood. We investigated the change in physical activity after TJA and patient factors associated with change. Patients and methods - Using a total joint replacement registry, primary total hip arthroplasty (THA) patients (n = 5,678) and knee arthroplasty (TKA) patients (n = 11,084) between January 1, 2010 and December 31, 2012 were identified. Median age at THA was 68 and median age at TKA was 67. Change in self-reported physical activity (minutes per week) from before TJA (within 1 year of surgery) to after TJA (1-2 years) was the outcome of interest. Patient demographics and comorbidities were evaluated as risk factors. Multiple linear regression was used. Results - Median physical activity before surgery was 50 min/week (IQR: 0-140) for THA patients and 58 (IQR: 3-143) for TKA patients. Median physical activity after surgery was 150 min/week (IQR: 60-280) for both THA patients and TKA patients. Following TJA, 50% of patients met CDC/WHO physical activity guideline criteria. Higher body mass index was associated with lower change in physical activity (THA: -7.1 min/week; TKA: -5.9 min/week). Females had lower change than males (THA: -11 min/week; TKA: -9.1 min/week). In TKA patients, renal failure was associated with lower change (-17 min/week), as were neurological disorders (-30 min/week). Interpretation - Self-reported minutes of physical activity increased from before to after TJA, but 50% of TJA patients did not meet recommended physical activity guideline criteria. Higher body mass index, female sex, and specific comorbidities were found to be associated with low change in physical activity. Patient education on the benefits of physical activity should concentrate on these subgroups of patients. PMID:27299567

  11. Keap1-Independent Regulation of Nrf2 Activity by Protein Acetylation and a BET Bromodomain Protein

    PubMed Central

    Chatterjee, Nirmalya; Tian, Min; Spirohn, Kerstin; Boutros, Michael; Bohmann, Dirk

    2016-01-01

    Mammalian BET proteins comprise a family of bromodomain-containing epigenetic regulators with complex functions in chromatin organization and gene regulation. We identified the sole member of the BET protein family in Drosophila, Fs(1)h, as an inhibitor of the stress responsive transcription factor CncC, the fly ortholog of Nrf2. Fs(1)h physically interacts with CncC in a manner that requires the function of its bromodomains and the acetylation of CncC. Treatment of cultured Drosophila cells or adult flies with fs(1)h RNAi or with the BET protein inhibitor JQ1 de-represses CncC transcriptional activity and engages protective gene expression programs. The mechanism by which Fs(1)h inhibits CncC function is distinct from the canonical mechanism that stimulates Nrf2 function by abrogating Keap1-dependent proteasomal degradation. Consistent with the independent modes of CncC regulation by Keap1 and Fs(1)h, combinations of drugs that can specifically target these pathways cause a strong synergistic and specific activation of protective CncC- dependent gene expression and boosts oxidative stress resistance. This synergism might be exploitable for the design of combinatorial therapies to target diseases associated with oxidative stress or inflammation. PMID:27233051

  12. Effects of Different Drying Methods and Storage Time on Free Radical Scavenging Activity and Total Phenolic Content of Cosmos caudatus

    PubMed Central

    Mediani, Ahmed; Abas, Faridah; Tan, Chin Ping; Khatib, Alfi

    2014-01-01

    The present study was conducted to determine the effect of air (AD), oven (OD) and freeze drying (FD) on the free radical scavenging activity and total phenolic content (TPC) of Cosmos caudatus and the effect of storage time by the comparison with a fresh sample (FS). Among the three drying methods that were used, AD resulted in the highest free radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) (IC50 = 0.0223 mg/mL) and total phenolic content (27.4 g GAE/100 g), whereas OD produced the lowest scavenging activity and TPC value. After three months of storage, the dried samples showed a high and consistent free radical scavenging activity when compared to stored fresh material. The drying methods could preserve the quality of C. caudatus during storage and the stability of its bioactive components can be maintained. PMID:26784876

  13. In Plant Activation: An Inducible, Hyperexpression Platform for Recombinant Protein Production in Plants[W][OPEN

    PubMed Central

    Dugdale, Benjamin; Mortimer, Cara L.; Kato, Maiko; James, Tess A.; Harding, Robert M.; Dale, James L.

    2013-01-01

    In this study, we describe a novel protein production platform that provides both activation and amplification of transgene expression in planta. The In Plant Activation (INPACT) system is based on the replication machinery of tobacco yellow dwarf mastrevirus (TYDV) and is essentially transient gene expression from a stably transformed plant, thus combining the advantages of both means of expression. The INPACT cassette is uniquely arranged such that the gene of interest is split and only reconstituted in the presence of the TYDV-encoded Rep/RepA proteins. Rep/RepA expression is placed under the control of the AlcA:AlcR gene switch, which is responsive to trace levels of ethanol. Transgenic tobacco (Nicotiana tabacum cv Samsun) plants containing an INPACT cassette encoding the β-glucuronidase (GUS) reporter had negligible background expression but accumulated very high GUS levels (up to 10% total soluble protein) throughout the plant, within 3 d of a 1% ethanol application. The GUS reporter was replaced with a gene encoding a lethal ribonuclease, barnase, demonstrating that the INPACT system provides exquisite control of transgene expression and can be adapted to potentially toxic or inhibitory compounds. The INPACT gene expression platform is scalable, not host-limited, and has been used to express both a therapeutic and an industrial protein. PMID:23839786

  14. Mutations that alter the ability of the Escherichia coli cyclic AMP receptor protein to activate transcription.

    PubMed

    Bell, A; Gaston, K; Williams, R; Chapman, K; Kolb, A; Buc, H; Minchin, S; Williams, J; Busby, S

    1990-12-25

    The effects of a number of mutations in the E. coli cyclic AMP receptor protein (CRP) have been determined by monitoring the in vivo expression and in vitro open complex formation at two semi-synthetic promoters that are totally CRP-dependent. At one promoter the CRP-binding site is centered around 41.5 base pairs upstream from the transcription start whilst at the other promoter it is 61.5 base pairs upstream. The CRP mutation E171K reduces expression from both promoters whilst H159L renders CRP totally inactive: neither mutation stops CRP binding at either promoter. The mutations K52N and K52Q reverse the effect of H159L and 'reeducate' CRP to activate transcription. CRP carrying both H159L and K52N activates transcription from the promoter with the CRP site at -41.5 better than wild type CRP. In sharp contrast, this doubly changed CRP is totally inactive with respect to the activation of transcription from the promoter carrying the CRP site at -61.5. Our results suggest that CRP can use different contacts and/or conformations during transcription activation at promoters with different architectures. PMID:2259621

  15. Mutations that alter the ability of the Escherichia coli cyclic AMP receptor protein to activate transcription.

    PubMed Central

    Bell, A; Gaston, K; Williams, R; Chapman, K; Kolb, A; Buc, H; Minchin, S; Williams, J; Busby, S

    1990-01-01

    The effects of a number of mutations in the E. coli cyclic AMP receptor protein (CRP) have been determined by monitoring the in vivo expression and in vitro open complex formation at two semi-synthetic promoters that are totally CRP-dependent. At one promoter the CRP-binding site is centered around 41.5 base pairs upstream from the transcription start whilst at the other promoter it is 61.5 base pairs upstream. The CRP mutation E171K reduces expression from both promoters whilst H159L renders CRP totally inactive: neither mutation stops CRP binding at either promoter. The mutations K52N and K52Q reverse the effect of H159L and 'reeducate' CRP to activate transcription. CRP carrying both H159L and K52N activates transcription from the promoter with the CRP site at -41.5 better than wild type CRP. In sharp contrast, this doubly changed CRP is totally inactive with respect to the activation of transcription from the promoter carrying the CRP site at -61.5. Our results suggest that CRP can use different contacts and/or conformations during transcription activation at promoters with different architectures. Images PMID:2259621

  16. Total Phenolics and Antioxidant Activity of Extracts from Distillers' Dried Grains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Corn is an abundant source of phenolic acids; therefore, distillers’ dried grains (DDG), may exhibit important market value for its phenolic content and antioxidant activity. The purpose of this study was to determine the efficacy of extracting phenolic compounds with antioxidant activity from DDG ...

  17. Coupling of total hemoglobin concentration, oxygenation, and neural activity in rat somatosensory cortex.

    PubMed

    Devor, Anna; Dunn, Andrew K; Andermann, Mark L; Ulbert, Istvan; Boas, David A; Dale, Anders M

    2003-07-17

    Recent advances in brain imaging techniques, including functional magnetic resonance imaging (fMRI), offer great promise for noninvasive mapping of brain function. However, the indirect nature of the imaging signals to the underlying neural activity limits the interpretation of the resulting maps. The present report represents the first systematic study with sufficient statistical power to quantitatively characterize the relationship between changes in blood oxygen content and the neural spiking and synaptic activity. Using two-dimensional optical measurements of hemodynamic signals, simultaneous recordings of neural activity, and an event-related stimulus paradigm, we demonstrate that (1) there is a strongly nonlinear relationship between electrophysiological measures of neuronal activity and the hemodynamic response, (2) the hemodynamic response continues to grow beyond the saturation of electrical activity, and (3) the initial increase in deoxyhemoglobin that precedes an increase in blood volume is counterbalanced by an equal initial decrease in oxyhemoglobin. PMID:12873390

  18. Apoptosis Induction by the Total Flavonoids from Arachniodes exilis in HepG2 Cells through Reactive Oxygen Species-Mediated Mitochondrial Dysfunction Involving MAPK Activation

    PubMed Central

    Chen, Jing; Xiong, Chaomei; Wei, Han; Yin, Changchang; Ruan, Jinlan

    2014-01-01

    Arachniodes exilis is used as a folk medicine in China and proved to have antibacterial, anti-inflammatory, and sedative activities. In the present study, the antitumor effect of the total flavonoids of A. exilis (TFAE) against HepG2 cells was evaluated. The results showed that TFAE inhibited the growth of HepG2 cells in a dosage- and time-dependent manner. Flow cytometry and Hoechst 33342 fluorescence staining results showed that TFAE could significantly increase the apoptosis ratio of HepG2 cells, which is accompanied with increased intracellular reactive oxygen species (ROS) production and decreased mitochondrial membrane potential (ΔΨm). Western blotting indicated that TFAE downregulated the ratio of Bcl-2/Bax, increased cytochrome c release, and activated the caspases-3 and -9. Further analysis showed that TFAE stimulated the mitogen-activated protein kinase (MAPK). However, treatment with NAC (reactive oxygen species scavenger) and MAPK-specific inhibitors (SP600125 and SB203580) could reverse the changes of these apoptotic-related proteins. These results suggested that TFAE possessed potential anticancer activity in HepG2 cells through ROS-mediated mitochondrial dysfunction involving MAPK pathway. PMID:24976852

  19. Impacts of Activation of the Mitogen-Activated Protein Kinase Pathway in Pancreatic Cancer

    PubMed Central

    Furukawa, Toru

    2015-01-01

    Pancreatic cancer is characterized by constitutive activation of the mitogen-activated protein kinase (MAPK) pathway. Mutations of KRAS or BRAF and epigenetic abrogation of DUSP6 contribute synergistically to the constitutive activation of MAPK. Active MAPK induces the expression of a variety of genes that are thought to play roles in malignant phenotypes of pancreatic cancer. By blocking the functions of such induced genes, it is possible to attenuate the malignant phenotypes. The development of drugs targeting genes downstream of MAPK may provide a novel therapeutic option for pancreatic cancer. PMID:25699241

  20. Contribution of Particle-Bound Bacteria to Total Microheterotrophic Activity in Five Ponds and Two Marshes

    PubMed Central

    Kirchman, David; Mitchell, Ralph

    1982-01-01

    We examined the abundance and heterotrophic uptake of bacteria attached to particulate matter suspended in five coastal ponds and two marshes near Woods Hole, Mass. Although the number of particle-bound bacteria was low (<10%), these bacteria incorporated a large proportion (>40%) of [14C]glucose and [14C]glutamate in selected aquatic systems. The uptake per cell was significantly higher for epibacteria than for unattached bacteria in all systems. Two groups of the aquatic environments sampled were statistically different in the contribution made by particle-bound bacteria to total bacterial abundance and to total assimilation of [14C]glucose and [14C]glutamate. Particle-bound bacteria were more important in those waters with a high particle concentration and not flushed regularly by tides than in waters with a low particle concentration and flushed regularly. PMID:16345921

  1. Thermally activated charge transport in microbial protein nanowires

    PubMed Central

    Lampa-Pastirk, Sanela; Veazey, Joshua P.; Walsh, Kathleen A.; Feliciano, Gustavo T.; Steidl, Rebecca J.; Tessmer, Stuart H.; Reguera, Gemma

    2016-01-01

    The bacterium Geobacter sulfurreducens requires the expression of conductive protein filaments or pili to respire extracellular electron acceptors such as iron oxides and uranium and to wire electroactive biofilms, but the contribution of the protein fiber to charge transport has remained elusive. Here we demonstrate efficient long-range charge transport along individual pili purified free of metal and redox organic cofactors at rates high enough to satisfy the respiratory rates of the cell. Carrier characteristics were within the orders reported for organic semiconductors (mobility) and inorganic nanowires (concentration), and resistivity was within the lower ranges reported for moderately doped silicon nanowires. However, the pilus conductance and the carrier mobility decreased when one of the tyrosines of the predicted axial multistep hopping path was replaced with an alanine. Furthermore, low temperature scanning tunneling microscopy demonstrated the thermal dependence of the differential conductance at the low voltages that operate in biological systems. The results thus provide evidence for thermally activated multistep hopping as the mechanism that allows Geobacter pili to function as protein nanowires between the cell and extracellular electron acceptors. PMID:27009596

  2. Thermally activated charge transport in microbial protein nanowires.

    PubMed

    Lampa-Pastirk, Sanela; Veazey, Joshua P; Walsh, Kathleen A; Feliciano, Gustavo T; Steidl, Rebecca J; Tessmer, Stuart H; Reguera, Gemma

    2016-01-01

    The bacterium Geobacter sulfurreducens requires the expression of conductive protein filaments or pili to respire extracellular electron acceptors such as iron oxides and uranium and to wire electroactive biofilms, but the contribution of the protein fiber to charge transport has remained elusive. Here we demonstrate efficient long-range charge transport along individual pili purified free of metal and redox organic cofactors at rates high enough to satisfy the respiratory rates of the cell. Carrier characteristics were within the orders reported for organic semiconductors (mobility) and inorganic nanowires (concentration), and resistivity was within the lower ranges reported for moderately doped silicon nanowires. However, the pilus conductance and the carrier mobility decreased when one of the tyrosines of the predicted axial multistep hopping path was replaced with an alanine. Furthermore, low temperature scanning tunneling microscopy demonstrated the thermal dependence of the differential conductance at the low voltages that operate in biological systems. The results thus provide evidence for thermally activated multistep hopping as the mechanism that allows Geobacter pili to function as protein nanowires between the cell and extracellular electron acceptors. PMID:27009596

  3. Physical activity and high-sensitivity C-reactive protein.

    PubMed

    Plaisance, Eric P; Grandjean, Peter W

    2006-01-01

    Cardiovascular disease (CVD) remains one of the leading causes of death and disability in developed countries around the world despite the documented success of lifestyle and pharmacological interventions. This illustrates the multifactorial nature of atherosclerosis and the use of novel inflammatory markers as an adjunct to risk factor reduction strategies. As evidence continues to accumulate that inflammation is involved in all stages of the development and progression of atherosclerosis, markers of inflammation such as high-sensitivity C-reactive protein (CRP) may provide additional information regarding the biological status of the atherosclerotic lesion. Recent investigations suggest that physical activity reduces CRP levels. Higher levels of physical activity and cardiorespiratory fitness are consistently associated with 6-35% lower CRP levels. Longitudinal training studies that have demonstrated reductions in CRP concentrations range from 16% to 41%, an effect that may be independent of baseline levels of CRP, body composition or weight loss. The average change in CRP associated with physical activity appears to be at least as good, if not better, than currently prescribed pharmacological interventions in similar populations. The primary purpose of this review will be to present evidence from both cross-sectional and longitudinal investigations that physical activity lowers CRP levels in a dose-response manner. Finally, this review will examine factors such as body composition, sex, blood sample timing, diet and smoking, which may influence the CRP response to physical activity. PMID:16646631

  4. Perivascular fat, AMP-activated protein kinase and vascular diseases

    PubMed Central

    Almabrouk, T A M; Ewart, M A; Salt, I P; Kennedy, S

    2014-01-01

    Perivascular adipose tissue (PVAT) is an active endocrine and paracrine organ that modulates vascular function, with implications for the pathophysiology of cardiovascular disease (CVD). Adipocytes and stromal cells contained within PVAT produce mediators (adipokines, cytokines, reactive oxygen species and gaseous compounds) with a range of paracrine effects modulating vascular smooth muscle cell contraction, proliferation and migration. However, the modulatory effect of PVAT on the vascular system in diseases, such as obesity, hypertension and atherosclerosis, remains poorly characterized. AMP-activated protein kinase (AMPK) regulates adipocyte metabolism, adipose biology and vascular function, and hence may be a potential therapeutic target for metabolic disorders such as type 2 diabetes mellitus (T2DM) and the vascular complications associated with obesity and T2DM. The role of AMPK in PVAT or the actions of PVAT have yet to be established, however. Activation of AMPK by pharmacological agents, such as metformin and thiazolidinediones, may modulate the activity of PVAT surrounding blood vessels and thereby contribute to their beneficial effect in cardiometabolic diseases. This review will provide a current perspective on how PVAT may influence vascular function via AMPK. We will also attempt to demonstrate how modulating AMPK activity using pharmacological agents could be exploited therapeutically to treat cardiometabolic diseases. PMID:24490856

  5. Quantitation of yeast total proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer for uniform loading.

    PubMed

    Sheen, Hyukho

    2016-04-01

    Proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer are difficult to quantitate due to SDS and reducing agents being in the buffer. Although acetone precipitation has long been used to clean up proteins from detergents and salts, previous studies showed that protein recovery from acetone precipitation varies from 50 to 100% depending on the samples tested. Here, this article shows that acetone precipitates proteins highly efficiently from SDS-PAGE sample buffer and that quantitative recovery is achieved in 5 min at room temperature. Moreover, precipitated proteins are resolubilized with urea/guanidine, rather than with SDS. Thus, the resolubilized samples are readily quantifiable with Bradford reagent without using SDS-compatible assays. PMID:26796977

  6. Mycobacterium tuberculosis Rv1096 protein: gene cloning, protein expression, and peptidoglycan deacetylase activity

    PubMed Central

    2014-01-01

    Background Many bacteria modulate and evade the immune defenses of their hosts through peptidoglycan (PG) deacetylation. The PG deacetylases from Streptococcus pneumonia, Listeria monocytogenes and Lactococcus lactis have been characterized. However, thus far, the PG deacetylase of Mycobacterium tuberculosis has not been identified. Results In this study, we cloned the Rv1096 gene from the M. tuberculosis H37Rv strain and expressed Rv1096 protein in both Escherichia coli and M. smegmatis. The results showed that the purified Rv1096 protein possessed metallo-dependent PG deacetylase activity, which increased in the presence of Co2+. The kinetic parameters of the PG deacetylase towards M. smegmatis PG as a substrate were as follows: Km, 0.910 ± 0.007 mM; Vmax, 0.514 ± 0.038 μMmin-1; and Kcat = 0.099 ± 0.007 (S-1). Additionally, the viability of M. smegmatis in the presence of over-expressed Rv1096 protein was 109-fold higher than that of wild-type M. smegmatis after lysozyme treatment. Additionally, light microscopy and scanning electron microscopy showed that in the presence of over-expressed Rv1096 protein, M. smegmatis kept its regular shape, with an undamaged cell wall and smooth surface. These results indicate that Rv1096 caused deacetylation of cell wall PG, leading to lysozyme resistance in M. smegmatis. Conclusion We have determined that M. tuberculosis Rv1096 is a PG deacetylase. The PG deacetylase activity of Rv1096 contributed to lysozyme resistance in M. smegmatis. Our findings suggest that deacetylation of cell wall PG may be involved in evasion of host immune defenses by M. tuberculosis. PMID:24975018

  7. Structure and Activity of Tryptophan-rich TSPO Translocator Proteins

    PubMed Central

    Guo, Youzhong; Kalathur, Ravi C.; Liu, Qun; Kloss, Brian; Bruni, Renato; Ginter, Christopher; Kloppmann, Edda; Rost, Burkhard; Hendrickson, Wayne A.

    2015-01-01

    TSPO translocator proteins bind steroids and porphyrins, and they are implicated in many human diseases, for which they serve as biomarkers and therapeutic targets. TSPOs have tryptophan-rich sequences that are fhighly conserved from bacteria to mammals. We report crystal structures for Bacillus cereus TSPO (BcTSPO) down to 1.7Å resolution, including a complex with the benzodiazepine-like inhibitor PK11195. We also describe BcTSPO-mediated protoporphyrin IX (PpIX) reactions, including catalytic degradation to a previously undescribed heme derivative. We used structure-inspired mutations to investigate reaction mechanisms, and we showed that TSPOs from Xenopus and man have similar PpIX-directed activities. Although TSPOs have been regarded as transporters, the catalytic activity in PpIX degradation suggests physiological importance for TSPOs in protection against oxidative stress. PMID:25635100

  8. Mitogen-activated protein kinases in male reproductive function

    PubMed Central

    Li, Michelle W.M.; Mruk, Dolores D.; Cheng, C. Yan

    2009-01-01

    Recent studies have shown that male reproductive function is modulated via the mitogen-activated protein kinase (MAPK) cascade. The MAPK cascade is involved in numerous male reproductive processes, including spermatogenesis, sperm maturation and activation, capacitation and acrosome reaction, before fertilization of the oocyte. In this review, we discuss the latest findings in this rapidly developing field regarding the role of MAPK in male reproduction in animal models and in human spermatozoa in vitro. This research will facilitate the design of future studies in humans, although much work is needed before this information can be used to manage male infertility and environmental toxicant-induced testicular injury in men, such as blood–testis-barrier disruption. PMID:19303360

  9. Small Molecule Inhibitors Targeting Activator Protein 1 (AP-1)

    PubMed Central

    2015-01-01

    Activator protein 1 (AP-1) is a pivotal transcription factor that regulates a wide range of cellular processes including proliferation, apoptosis, differentiation, survival, cell migration, and transformation. Accumulating evidence supports that AP-1 plays an important role in several severe disorders including cancer, fibrosis, and organ injury, as well as inflammatory disorders such as asthma, psoriasis, and rheumatoid arthritis. AP-1 has emerged as an actively pursued drug discovery target over the past decade. Excitingly, a selective AP-1 inhibitor T-5224 (51) has been investigated in phase II human clinical trials. Nevertheless, no effective AP-1 inhibitors have yet been approved for clinical use. Despite significant advances achieved in understanding AP-1 biology and function, as well as the identification of small molecules modulating AP-1 associated signaling pathways, medicinal chemistry efforts remain an urgent need to yield selective and efficacious AP-1 inhibitors as a viable therapeutic strategy for human diseases. PMID:24831826

  10. Analysis of refractometry for determining total plasma protein in hybrid striped bass(Morone chrysops x M. saxatilis) at various salinities

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Total plasma protein (TPP) is a broad clinical indicator of health, stress, and well being. A simple and rapid technique for determining TPP is refractometry, which measures the refractive index of all dissolved materials in solution. It was hypothesized plasma dissolved solids in fish held at inc...

  11. Fe3O4NPs mediated nonenzymatic electrochemical immunosensor for the total protein of Nosema bombycis detection without addition of substrate.

    PubMed

    Xie, Hua; Zhang, Qiqi; Wang, Qin; Chai, Yaqin; Yuan, Yali; Yuan, Ruo

    2015-04-28

    In this work, we proposed a novel electrochemical immunosensor for sensitive detection of the total protein of Nosema bombycis based on Fe3O4 nanoparticles (Fe3O4NPs) as catalyst to electrocatalyze the reduction of methylene blue (MB) with the aid of Fe3O4NPs-DNA dendrimers for the signal amplification. PMID:25806964

  12. Evaluation of plant and animal protein sources as partial or total replacement of fish meal in diets for juvenile Nile tilapia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A feeding trial was conducted in a closed system with Nile tilapia (Oreochromis niloticus) juveniles (mean weight, 2.84 g) to examine the effects of total replacement of fish meal (FM), with and without supplementation of DL-methionine (Met) and L-lysine (Lys), by plant protein sources. Fish were f...

  13. Bryostatins: potent, new activators of protein kinase C

    SciTech Connect

    Smith, L.; Pettit, G.R.; Smith, J.B.

    1986-03-01

    Bryostatins (B) are a class of 17 macrocyclic lactones that have antineoplastic activity in the murine P388 lymphocytic leukemia system. Bryostatin-1 (B-1) is a potent co-mitogen for the Swiss 3T3 line of murine fibroblasts that have been arrested in G/sub 1//G/sub 0/. B-1 and insulin synergistically increase entry into the S phase of the cell cycle measured autoradiographically as % nuclei labeled with (/sup 3/H)thymidine. A prior treatment of the cells with phorbol 13-myristate 12-acetate (PMA) selectively eliminated the mitogenic response to B-1 or PMA. Conversely, a prior treatment of the cells with B-1 eliminated the mitogenic response to PMA or B-1. Five other B are approximately equipotent to B-1, but B-3 is 5 to 10 times less potent than B-1 as a mitogen. B-1 inhibits the binding of (/sup 3/H)phorbol dibutyrate ((/sup 3/H)PDB) at 4/sup 0/C to a high affinity receptor in the cells. B-3 was also less potent than B-1 as an inhibitor of (/sup 3/H)PDB binding. B-3 differs from B-1 in the diacylglycerol-like component of the molecule. In vitro B-1 and PMA are similarly potent activators of protein kinase C from bovine brain. Further comparisons of the relative activities of the various B are needed to define the structural features that are critical for the activation of protein kinase C which may help in the design of tumor promoter antagonists.

  14. Antistaphylococcal activity of bacteriophage derived chimeric protein P128

    PubMed Central

    2012-01-01

    Background Bacterial drug resistance is one of the most significant challenges to human health today. In particular, effective antibacterial agents against methicillin-resistant Staphylococcus aureus (MRSA) are urgently needed. A causal relationship between nasal commensal S. aureus and infection has been reported. Accordingly, elimination of nasal S. aureus reduces the risk of infection. Enzymes that degrade bacterial cell walls show promise as antibacterial agents. Bacteriophage-encoded bacterial cell wall-degrading enzymes exhibit intrinsic bactericidal activity. P128 is a chimeric protein that combines the lethal activity of the phage tail-associated muralytic enzyme of Phage K and the staphylococcal cell wall targeting-domain (SH3b) of lysostaphin. Here we report results of in vitro studies evaluating the susceptibility of staphylococcal strains to this novel protein. Results Using the broth microdilution method adapted for lysostaphin, we found that P128 is effective against S. aureus clinical strains including MRSA, methicillin-sensitive S. aureus (MSSA), and a mupirocin-resistant S. aureus. Minimum bactericidal concentrations and minimum inhibitory concentrations of P128 (1-64 μg/mL) were similar across the 32 S. aureus strains tested, demonstrating its bactericidal nature. In time-kill assays, P128 reduced colony-forming units by 99.99% within 1 h and inhibited growth up to 24 h. In an assay simulating topical application of P128 to skin or other biological surfaces, P128 hydrogel was efficacious when layered on cells seeded on solid media. P128 hydrogel was lethal to Staphylococci recovered from nares of healthy people and treated without any processing or culturing steps, indicating its in situ efficacy. This methodology used for in vitro assessment of P128 as an agent for eradicating nasal carriage is unique. Conclusions The novel chimeric protein P128 is a staphylococcal cell wall-degrading enzyme under development for clearance of S. aureus nasal

  15. Proteins with RNA Chaperone Activity: A World of Diverse Proteins with a Common Task—Impediment of RNA Misfolding

    PubMed Central

    Semrad, Katharina

    2011-01-01

    Proteins with RNA chaperone activity are ubiquitous proteins that play important roles in cellular mechanisms. They prevent RNA from misfolding by loosening misfolded structures without ATP consumption. RNA chaperone activity is studied in vitro and in vivo using oligonucleotide- or ribozyme-based assays. Due to their functional as well as structural diversity, a common chaperoning mechanism or universal motif has not yet been identified. A growing database of proteins with RNA chaperone activity has been established based on evaluation of chaperone activity via the described assays. Although the exact mechanism is not yet understood, it is more and more believed that disordered regions within proteins play an important role. This possible mechanism and which proteins were found to possess RNA chaperone activity are discussed here. PMID:21234377

  16. Inflammatory Eicosanoids Increase Amyloid Precursor Protein Expression via Activation of Multiple Neuronal Receptors

    PubMed Central

    Herbst-Robinson, Katie J.; Liu, Li; James, Michael; Yao, Yuemang; Xie, Sharon X.; Brunden, Kurt R.

    2015-01-01

    Senile plaques comprised of Aβ peptides are a hallmark of Alzheimer’s disease (AD) brain, as are activated glia that release inflammatory molecules, including eicosanoids. Previous studies have demonstrated that amyloid precursor protein (APP) and Aβ levels can be increased through activation of thromboxane A2-prostanoid (TP) receptors on neurons. We demonstrate that TP receptor regulation of APP expression depends on Gαq-signaling and conventional protein kinase C isoforms. Importantly, we discovered that Gαq-linked prostaglandin E2 and leukotriene D4 receptors also regulate APP expression. Prostaglandin E2 and thromboxane A2, as well as total APP levels, were found to be elevated in the brains of aged 5XFAD transgenic mice harboring Aβ plaques and activated glia, suggesting that increased APP expression resulted from eicosanoid binding to Gαq-linked neuronal receptors. Notably, inhibition of eicosanoid synthesis significantly lowered brain APP protein levels in aged 5XFAD mice. These results provide new insights into potential AD therapeutic strategies. PMID:26672557

  17. The Effect of Fish Oil, Vitamin D and Protein on URTI Incidence in Young Active People.

    PubMed

    Da Boit, M; Gabriel, B M; Gray, P; Gray, S R

    2015-05-01

    Upper respiratory tract infections (URTI) are a frequent illness among athletes. We investigated the effect of a multi-nutrient supplement (vitamin D, fish oil and protein) on the occurrence of URTI in young active people. 42 young recreational athletes were randomly assigned to receive either supplementation (550 mg DHA, 550 mg EPA, 10 µg vitamin D3 and 8 g whey protein) or placebo for 16 weeks. Unstimulated saliva samples were collected by passive drool. Samples were analysed for IgA (sIgA) concentration and the secretion rate extrapolated by multiplying concentration by saliva flow rate. Physical activity levels and URTI incidence were monitored by questionnaire. Training status was not different between the 2 groups. There were no differences in the incidence, severity and duration of URTI. However the number of symptom days was lower in the supplemented compared to the control group (1.72±1.67 vs. 2.79±1.76; P<0.05). sIgA concentration and secretion rate did not differ between groups. This study demonstrates that 16 weeks of supplementation with fish oil, vitamin D and protein did not modify the incidence, severity and duration of URTI, although the total number of symptom days was reduced, in a healthy active population. PMID:25664999

  18. Bioinsecticidal activity of Murraya koenigii miraculin-like protein against Helicoverpa armigera and