Sample records for maxam-gilbert workup denaturing

  1. Workup for proteinuria.

    Snyder, Samuel; John, Jones Sam


    The kidney has anatomic and physiologic features that prevent protein from reaching the urine. In disease processes, this natural mechanism is disrupted, causing protein to leak into the urine. Proteinuria can be used as a marker for disease and disease progression. The general anatomy of the glomerulus along with preliminary workup to evaluate disease based on history, physical and urinalysis results are reviewed in this article. Examples of commonly encountered diseases in the outpatient setting and relevance of proteinuria in chronic kidney disease along with general complications and treatments are also reviewed. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. [Denaturalized psychoanalysts].

    Peglau, Andreas


    This paper presents hitherto unknown material from the German Foreign Office referring to the denaturalization of Therese Benedek, Bruno Bettelheim, Adolf Storfer and Wilhelm Reich by Nazi Germany. It corroborates the finding that nobody was persecuted by the Nazis solely on the basis of psychoanalytic activities or membership in a psychoanalytic organization.

  3. [Diagnostic workup of fragrance allergy].

    Geier, J; Uter, W


    The diagnostic workup of contact allergy to fragrances must not be limited to patch testing with the two well-established fragrance mixes. False-positive reactions to these mixes occur in up to 50 % of the patch tested patients. For the diagnostic work-up of positive reactions, and in cases of suspected fragrance allergy, patch testing with the single mix components and additional fragrances is mandatory. Frequently sensitizing fragrance materials are the 14 components of the two fragrance mixes and tree moss (Evernia furfuracea), ylang ylang oil (I + II; Cananga odorata), lemongrass oil (Cymbopogon schoenanthus), sandalwood oil (Santalum album), jasmine absolute (Jasminum spp.), and, less frequently, clove oil (Eugenia caryophyllus), cedarwood oil (Cedrus atlantica/deodara, Juniperus virginiana), Neroli oil (Citrus aurantium amara flower oil), salicylaldehyde, narcissus absolute (Narcissus spp.), and patchouli oil (Pogostemon cablin).

  4. Reaction Workup Planning: A Structured Flowchart Approach, Exemplified in Difficult Aqueous Workup of Hydrophilic Products

    Hill, George B.; Sweeney, Joseph B.


    Reaction workup can be a complex problem for those facing novel synthesis of difficult compounds for the first time. Workup problem solving by systematic thinking should be inculcated as mid-graduate-level is reached. A structured approach is proposed, building decision tree flowcharts to analyze challenges, and an exemplar flowchart is presented…

  5. Stability of collagen during denaturation.

    Penkova, R; Goshev, I; Gorinstein, S; Nedkov, P


    The stability of calf skin collagen (CSC) type I during thermal and chemical denaturation in the presence of glycerol was investigated. Thermal denaturation of type I collagen was performed in the presence of glycerol or in combination with urea and sodium chloride. The denaturation curves obtained in the presence of urea or sodium chloride retained their original shape without glycerol. These curves were shifted upward proportionally to the glycerol concentration in the reaction medium. This means that glycerol and the denaturants act independently. The explanation is based on the difference in the mechanism of their action on the collagen molecule.

  6. [Hypercoagulable workup in ophthalmology. When and what].

    Muñoz-Negrete, F J; Casas-Lleras, P; Pérez-López, M; Rebolleda, G


    Most ophthalmologic disorders secondary to hypercoagulabe state are due to the confluence of congenital and adquired factors. A systematic workup is mandatory. Most of congenital coagulation disorders cause venous trombosis and are inherited autosomal dominantly. In order of frequency these are factor V Leiden mutation (activated protein C resistance), G20210A mutation of the prothrombin gen and protein C, protein S, and antithrombin III deficiencies. Sickle cell anemia can determine arerial and venous thrombosis. In relation with arterial occlusion, the markers most frequently involved are homcysteine fasting levels and the markers of antiphospholipid antibody syndrome. Both of them can also determine venous thrombosis. Several acquired factors can lead to hypoercoagulable state, especially hyperhomocysteinemia, antiphospholipid antibody syndrome, hepatic disease, alcohol and tobacco intake, oral contraceptives, immobilization, surgeries and malignancies. In central venous occlusion is only necessary to rule out hyperhomocysteinemia and antiphospholipid antibody syndrome in young patients without known risk factors. In central artery occlusion, hypercoagulable workup is only recommended for patients less than 50 years-old with unknown emboli source. In this cases protein C, protein S, and antithrombin III deficiencies, homocystein, sickle cell disease and antiphospholipid antibody syndrome will ruled out. In non arteritic ischemic optic neuropathy hypercoagulable work up is not necessary. In amaurosis fugax without known emboli source, it is recommended to rule out etiologies of arterial occlusion, especially antithrombin III deficiencies, homocystein, sickle cell disease and antiphospholipid antibody syndrome.

  7. Ictal PET in presurgical workup of refractory extratemporal epilepsy

    Javeria Nooraine


    Full Text Available Ictal Pet in presurgical workup of refractory epilepsy is seldom performed and limited due to technical difficulties. In carefully selected patient subset with frequent extratemporal seizures, ictal PET depicts ′seizure onset zone′ with high spatial resolution even within a widespread pathology. We here depict a four year old with posterior quadrant dysplasia evaluated with ictal PET.

  8. Delay in diagnostic workup and treatment of esophageal cancer

    B.A. Grotenhuis (Brechtje); P. van Hagen (Pieter); B.P.L. Wijnhoven (Bas); V.M.C.W. Spaander (Manon); H.W. Tilanus (Hugo); J.J-B. van Lanschot (Jan)


    textabstractIntroduction: Esophageal cancer should preferably be detected and treated at an early stage, but this may be prohibited by late onset of symptoms and delays in referral, diagnostic workup, and treatment. The aim of this study was to investigate the impact of these delays on outcome in pa

  9. Single DNA denaturation and bubble dynamics

    Metzler, Ralf [Physics Department, Technical University of Munich, James Franck Strasse, 85747 Garching (Germany); Ambjoernsson, Tobias [Chemistry Department, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 (United States); Hanke, Andreas [Department of Physics and Astronomy, University of Texas, 80 Fort Brown, Brownsville (United States); Fogedby, Hans C [Department of Physics and Astronomy, University of Arhus, Ny Munkegade, 8000 Arhus C (Denmark)], E-mail:


    While the Watson-Crick double-strand is the thermodynamically stable state of DNA in a wide range of temperature and salt conditions, even at physiological conditions local denaturation bubbles may open up spontaneously due to thermal activation. By raising the ambient temperature, titration, or by external forces in single molecule setups bubbles proliferate until full denaturation of the DNA occurs. Based on the Poland-Scheraga model we investigate both the equilibrium transition of DNA denaturation and the dynamics of the denaturation bubbles with respect to recent single DNA chain experiments for situations below, at, and above the denaturation transition. We also propose a new single molecule setup based on DNA constructs with two bubble zones to measure the bubble coalescence and extract the physical parameters relevant to DNA breathing. Finally we consider the interplay between denaturation bubbles and selectively single-stranded DNA binding proteins.

  10. Single DNA denaturation and bubble dynamics

    Metzler, Ralf; Ambjörnsson, Tobias; Hanke, Andreas


    for situations below, at, and above the denaturation transition. We also propose a new single molecule setup based on DNA constructs with two bubble zones to measure the bubble coalescence and extract the physical parameters relevant to DNA breathing. Finally we consider the interplay between denaturation......While the Watson-Crick double-strand is the thermodynamically stable state of DNA in a wide range of temperature and salt conditions, even at physiological conditions local denaturation bubbles may open up spontaneously due to thermal activation. By raising the ambient temperature, titration......, or by external forces in single molecule setups bubbles proliferate until full denaturation of the DNA occurs. Based on the Poland-Scheraga model we investigate both the equilibrium transition of DNA denaturation and the dynamics of the denaturation bubbles with respect to recent single DNA chain experiments...

  11. The dynamics of the DNA denaturation transition

    van Erp, Titus S


    The dynamics of the DNA denaturation is studied using the Peyrard-Bishop-Dauxois model. The denaturation rate of double stranded polymers decreases exponentially as function of length below the denaturation temperature. Above Tc, the rate shows a minimum, but then increases as function of length. We also examine the influence of sequence and solvent friction. Molecules having the same number of weak and strong base-pairs can have significantly different opening rates depending on the order of base-pairs.

  12. Bubbles and denaturation in DNA

    Van Erp, T S; Peyrard, M; Erp, Titus S. van; Cuesta-Lopez, Santiago; Peyrard, Michel


    The local opening of DNA is an intriguing phenomenon from a statistical physics point of view, but is also essential for its biological function. For instance, the transcription and replication of our genetic code can not take place without the unwinding of the DNA double helix. Although these biological processes are driven by proteins, there might well be a relation between these biological openings and the spontaneous bubble formation due to thermal fluctuations. Mesoscopic models, like the Peyrard-Bishop-Dauxois model, have fairly accurately reproduced some experimental denaturation curves and the sharp phase transition in the thermodynamic limit. It is, hence, tempting to see whether these models could be used to predict the biological activity of DNA. In a previous study, we introduced a method that allows to obtain very accurate results on this subject, which showed that some previous claims in this direction, based on molecular dynamics studies, were premature. This could either imply that the present...

  13. DNA denaturation in ionic solution

    Maity, Arghya; Singh, Amar; Singh, Navin


    Salt or cations, present in solution play an important role in DNA denaturation and folding kinetics of DNA helix. In this work we study the thermal melting of double stranded DNA (dsDNA) molecule using Peyrard Bishop Dauxois (PBD) model. We modify the potential of H-bonding between the bases of the complimentary strands to introduce the salt and solvent effect. We choose different DNA sequences having different contents of GC pairs and calculate the melting temperatures. The melting temperature increases logarithmically with the salt concentration of the solution. The more GC base pairs in the chain enhance the stability of DNA chain at a fix salt concentration. The obtained results are in good accordance with experimental findings.

  14. Stroke of undetermined cause: workup and secondary prevention.

    Weimar, Christian


    The purpose of this review is to update the reader on current concepts of workup and secondary prevention in patients with stroke of undetermined cause. Clinical research in patients with cryptogenic stroke has been hampered by the lack of standardized, widely accepted diagnostic criteria. The new definition of 'Embolic stroke of undetermined etiology' postulates an embolic mechanism of ischemic stroke. It is based on the exclusion of lacunar infarction by brain imaging, arterial stenosis more than 50% or dissection of the respective brain-supplying artery by computed tomography/magnetic resonance-angiography or ultrasound, atrial fibrillation by at least 24 h EKG monitoring, as well as some rare etiologies such as vasculitis, drug abuse, or coagulopathies. However, it still comprises many patients with atherosclerotic etiologies (but undetermined etiology enabled three ongoing randomized controlled trials which investigate oral anticoagulation versus aspirin for secondary stroke prevention.

  15. Monoclonal gammopathy of undetermined significance: a new proposal of workup.

    Mangiacavalli, Silvia; Cocito, Federica; Pochintesta, Lara; Pascutto, Cristiana; Ferretti, Virginia; Varettoni, Marzia; Zappasodi, Patrizia; Pompa, Alessandra; Landini, Benedetta; Cazzola, Mario; Corso, Alessandro


    Diagnostic criteria for monoclonal gammopathy of undetermined significance (MGUS) require quantification of bone marrow plasma cells (BMPCs) and skeletal survey to discriminate between MGUS and multiple myeloma (MM). By contrast, recent published guidelines suggest that these procedures could be avoided in the presence of serum monoclonal spike (M-spike) of small amount (≤1.5 g/dL). Aim of this study is to better quantify the risk of missing a diagnosis of MM, not performing bone marrow aspirate and skeletal survey in patients with M-spike ≤ 1.5 g/dL asymptomatic for bone pain. We reviewed data of 2282 patients consecutively observed from January 1974 to December 2010 in our single hematology department. We considered eligible for this study 1271 patients with grade <2 NCI bone pain, confirmed to have an MGUS or an MM after extensive standardized diagnostic workup including bone marrow biopsy, skeletal bone survey and laboratory tests. The risk of finding a BMPC infiltration ≥10% in patients with an M-spike ≤ 1.5 g/dL was very low (7.3%), although significantly different according to IgH isotype (4.7% for IgG vs. 20.5% for IgA). The risk of finding bone lesions with M-spike ≤ 1.5 g/dL was negligible (2.5%), regardless of IgH isotype. In asymptomatic patients with M-spike of small amount (≤1.5 g/dL): (i) BMPC evaluation may be reasonably avoided in patients with IgG M-spike, while should always be part of diagnostic workup in the presence of IgA M-spike and (ii) skeletal survey, less predictive for MM, should not be routinely indicated irrespective of IgH isotype. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Chemical shift prediction for denatured proteins

    Prestegard, James H., E-mail:; Sahu, Sarata C.; Nkari, Wendy K.; Morris, Laura C.; Live, David; Gruta, Christian


    While chemical shift prediction has played an important role in aspects of protein NMR that include identification of secondary structure, generation of torsion angle constraints for structure determination, and assignment of resonances in spectra of intrinsically disordered proteins, interest has arisen more recently in using it in alternate assignment strategies for crosspeaks in {sup 1}H-{sup 15}N HSQC spectra of sparsely labeled proteins. One such approach involves correlation of crosspeaks in the spectrum of the native protein with those observed in the spectrum of the denatured protein, followed by assignment of the peaks in the latter spectrum. As in the case of disordered proteins, predicted chemical shifts can aid in these assignments. Some previously developed empirical formulas for chemical shift prediction have depended on basis data sets of 20 pentapeptides. In each case the central residue was varied among the 20 amino common acids, with the flanking residues held constant throughout the given series. However, previous choices of solvent conditions and flanking residues make the parameters in these formulas less than ideal for general application to denatured proteins. Here, we report {sup 1}H and {sup 15}N shifts for a set of alanine based pentapeptides under the low pH urea denaturing conditions that are more appropriate for sparse label assignments. New parameters have been derived and a Perl script was created to facilitate comparison with other parameter sets. A small, but significant, improvement in shift predictions for denatured ubiquitin is demonstrated.

  17. Rousseau's Philosophy of Transformative, "Denaturing" Education

    Riley, Patrick


    Rousseau's political philosophy presents the great legislator as a civic educator who must over time transform naturally self-loving egoists into citizens animated by a general will without destroying freedom. This is an educational process which is "denaturing" but which aims to produce autonomous adults who can ultimately say to their teacher…

  18. Guanidinium-induced denaturation by breaking of salt bridges

    Meuzelaar, H.; Panman, M.R.; Woutersen, S.


    Despite its wide use as a denaturant, the mechanism by which guanidinium (Gdm+) induces protein unfolding remains largely unclear. Herein, we show evidence that Gdm+ can induce denaturation by disrupting salt bridges that stabilize the folded conformation. We study the Gdm+-​induced denaturation of

  19. Hysteresis in pressure-driven DNA denaturation.

    Enrique Hernández-Lemus

    Full Text Available In the past, a great deal of attention has been drawn to thermal driven denaturation processes. In recent years, however, the discovery of stress-induced denaturation, observed at the one-molecule level, has revealed new insights into the complex phenomena involved in the thermo-mechanics of DNA function. Understanding the effect of local pressure variations in DNA stability is thus an appealing topic. Such processes as cellular stress, dehydration, and changes in the ionic strength of the medium could explain local pressure changes that will affect the molecular mechanics of DNA and hence its stability. In this work, a theory that accounts for hysteresis in pressure-driven DNA denaturation is proposed. We here combine an irreversible thermodynamic approach with an equation of state based on the Poisson-Boltzmann cell model. The latter one provides a good description of the osmotic pressure over a wide range of DNA concentrations. The resulting theoretical framework predicts, in general, the process of denaturation and, in particular, hysteresis curves for a DNA sequence in terms of system parameters such as salt concentration, density of DNA molecules and temperature in addition to structural and configurational states of DNA. Furthermore, this formalism can be naturally extended to more complex situations, for example, in cases where the host medium is made up of asymmetric salts or in the description of the (helical-like charge distribution along the DNA molecule. Moreover, since this study incorporates the effect of pressure through a thermodynamic analysis, much of what is known from temperature-driven experiments will shed light on the pressure-induced melting issue.

  20. DSC study of denaturation of β-lactoglobulin B

    王邦宁; 谈夫


    The denaturation of bovine β-lactoglobulin B (β-Lg B) has been studied in phosphate solutions with various concentrations of GuHCl with differential scanning calorimetry The experiments demonstrated that the presence of GuHCl made the β-Lg B undergo both cold denaturation and heat denaturation under the condition of a high concentration of the protein. The enthalpy changes of both kinds of denaturation exhibit opposite signs. Both the cold denaturation and the renaturation of the protein are reproducible, but its heat denaturation is irreversible. The cooperation among monomer molecules of the protein is involved in its heat denaturation The heat denaturation of the protein can be represented by the thermodynamic model Nc D→F. The activation energy of heat denaturation is 285 kJ/mol, which imples that the depression of temperature and enthalpy of heat denaturation of the P-Lg B does not result from decreasing considerably the activation energy by GuHCl As for the cold denaturation of the protein, es

  1. Accuracy and consequences of same-day, invasive lung cancer workup

    Madsen, Kirsten Riis; Høegholm, Asbjørn; Bodtger, Uffe


    pulmonary disease. Tumour located in right upper lobe was associated with need for resampling. DISCUSSION: Our retrospective study suggests that same-day, invasive workup for lung cancer is safe, accurate, and efficacious in reducing time to therapy, even in patients with small lesions and low tumour burden.......BACKGROUND: Though widely used, little is known about accuracy and efficacy of same-day, invasive workup of suspected lung cancer. OBJECTIVE: To evaluate the accuracy and efficacy of same-day, invasive lung cancer workup (diagnosis and mediastinal staging), and to identify differences between...... patients without (Group A) or with (Group B) need for resampling. METHODS: A retrospective study was performed on all consecutive patients referred for surgical treatment for localised lung cancer after invasive diagnostic and staging workup at our unit. Data were extracted from electronic medical files...

  2. Supercoiling induces denaturation bubbles in circular DNA.

    Jeon, Jae-Hyung; Adamcik, Jozef; Dietler, Giovanni; Metzler, Ralf


    We present a theoretical framework for the thermodynamic properties of supercoiling-induced denaturation bubbles in circular double-stranded DNA molecules. We explore how DNA supercoiling, ambient salt concentration, and sequence heterogeneity impact on the bubble occurrence. An analytical derivation of the probability distribution to find multiple bubbles is derived and the relevance for supercoiled DNA discussed. We show that in vivo sustained DNA bubbles are likely to occur due to partial twist release in regions rich in weaker AT base pairs. Single DNA plasmid imaging experiments clearly demonstrate the existence of bubbles in free solution.

  3. 27 CFR 21.151 - List of denaturants authorized for denatured spirits.



  4. 78 FR 38628 - Reclassification of Specially Denatured Spirits and Completely Denatured Alcohol Formulas and...


    ... alcohol. 13-A 10 gallons of ethyl ether. 19 100 gallons of ethyl ether. 23-A 8 gallons of acetone, U.S.P... alcohol. 32 5 gallons of ethyl ether. 35-A 4.25 gallons of ethyl acetate having an ester content of 100... regulations regarding the production, warehousing, denaturing, distribution, sale, export, and use...

  5. Partially folded intermediates during trypsinogen denaturation

    Martins N.F.


    Full Text Available The equilibrium unfolding of bovine trypsinogen was studied by circular dichroism, differential spectra and size exclusion HPLC. The change in free energy of denaturation was = 6.99 ± 1.40 kcal/mol for guanidine hydrochloride and = 6.37 ± 0.57 kcal/mol for urea. Satisfactory fits of equilibrium unfolding transitions required a three-state model involving an intermediate in addition to the native and unfolded forms. Size exclusion HPLC allowed the detection of an intermediate population of trypsinogen whose Stokes radii varied from 24.1 ± 0.4 Å to 26.0 ± 0.3 Å for 1.5 M and 2.5 M guanidine hydrochloride, respectively. During urea denaturation, the range of Stokes radii varied from 23.9 ± 0.3 Å to 25.7 ± 0.6 Å for 4.0 M and 6.0 M urea, respectively. Maximal intrinsic fluorescence was observed at about 3.8 M urea with 8-aniline-1-naphthalene sulfonate (ANS binding. These experimental data indicate that the unfolding of bovine trypsinogen is not a simple transition and suggest that the equilibrium intermediate population comprises one intermediate that may be characterized as a molten globule. To obtain further insight by studying intermediates representing different stages of unfolding, we hope to gain a better understanding of the complex interrelations between protein conformation and energetics.

  6. Post-operative fever in orthopaedic surgery: How effective is the 'fever workup?'

    Ashley, Blair; Spiegel, David A; Cahill, Patrick; Talwar, Divya; Baldwin, Keith D


    Defining the appropriate threshold at which to initiate a fever workup is imperative to promote patient safety, appropriate resource utilization, and antibiotic stewardship. Our group performed a systematic review of the available literature on perioperative fever (POF) workups in orthopaedic patients to evaluate the frequency, timing and utility of blood cultures (BC) and other investigations in the POF workup, to determine the clinical relevance of any infections and to evaluate their cost effectiveness. Studies were identified by searching MEDLINE, EMBASE, Pubmed, Cochrane and Google Scholar for articles through September 2016. Forty-nine articles were retrieved and 22 articles met the pre-determined inclusion criteria. Proportions of positive studies were noted and averaged using random effects analysis. Post-operative pyrexia ranged in prevalence between 8.1% and 87.3%. The studies routinely performed during a fever workup had wide ranges of diagnostic yield, including chest X-rays from 0% to 40%, urinalyses from 8.2% to 38.7%, urine cultures from 0% to 22.4% and BC from 0% to 13.3%. Only two patients with positive BC developed clinical sepsis. Cost per fever evaluation ranged from $350 to $950. The findings of this review suggest that early post-operative fever is an expected event following orthopaedic surgery. Based on the available literature, any kind of workup in the absence of localizing symptoms in the third post-operative day or before is unwarranted and is an inappropriate use of hospital resources.

  7. Can A Denaturant Stabilize DNA? Pyridine Reverses DNA Denaturation in Acidic pH.

    Portella, Guillem; Terrazas, Montserrat; Villegas, Núria; González, Carlos; Orozco, Modesto


    The stability of DNA is highly dependent on the properties of the surrounding solvent, such as ionic strength, pH, and the presence of denaturants and osmolytes. Addition of pyridine is known to unfold DNA by replacing π-π stacking interactions between bases, stabilizing conformations in which the nucleotides are solvent exposed. We show here experimental and theoretical evidences that pyridine can change its role and in fact stabilize the DNA under acidic conditions. NMR spectroscopy and MD simulations demonstrate that the reversal in the denaturing role of pyridine is specific, and is related to its character as pseudo groove binder. The present study sheds light on the nature of DNA stability and on the relationship between DNA and solvent, with clear biotechnological implications.

  8. Kinetically controlled refolding of a heat-denatured hyperthermostable protein

    Koutsopoulos, Sotirios; van der Oost, John; Norde, Willem


    The thermal denaturation of endo-beta-1,3-glucanase from the hyperthermophilic microorganism Pyrococcus furiosus was studied by calorimetry. The calorimetric profile revealed two transitions at 109 and 144 degrees C, corresponding to protein denaturation and complete unfolding, respectively, as

  9. Kinetically controlled refolding of a heat denatured hyperthermostable protein

    Koutsopoulos, S.; Oost, van der J.; Norde, W.


    The thermal denaturation of endo-ß-1,3-glucanase from the hyperthermophilic microorganism Pyrococcus furiosus was studied by calorimetry. The calorimetric profile revealed two transitions at 109 and 144¿°C, corresponding to protein denaturation and complete unfolding, respectively, as shown by

  10. 19 CFR 10.56 - Vegetable oils, denaturing; release.


    ... 19 Customs Duties 1 2010-04-01 2010-04-01 false Vegetable oils, denaturing; release. 10.56 Section... Vegetable Oils § 10.56 Vegetable oils, denaturing; release. (a) Olive, palm-kernel, rapeseed, sunflower, and sesame oil shall be classifiable under subheadings 1509.10.20, 1509.10.40, 1509.90.20, 1509.90.40,...

  11. 27 CFR 19.464 - Denatured spirits inventories.


    ... inventories. 19.464 Section 19.464 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE... of Articles Inventories § 19.464 Denatured spirits inventories. Each proprietor shall take a physical inventory of all denatured spirits in the processing account at the close of each calendar quarter and...

  12. 27 CFR 20.144 - Packages of completely denatured alcohol.


    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Packages of completely denatured alcohol. 20.144 Section 20.144 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS DISTRIBUTION AND USE OF DENATURED ALCOHOL AND RUM...

  13. 27 CFR 20.261 - Records of completely denatured alcohol.


    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Records of completely denatured alcohol. 20.261 Section 20.261 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS DISTRIBUTION AND USE OF DENATURED ALCOHOL AND...

  14. Geriatric work-up in the Nordic countries. The Nordic approach to comprehensive geriatric assessment

    Sletvold, O; Tilvis, R; Jonsson, A


    , the background for developing a Nordic version of geriatric work-up is shared attitudes and principally the same organization of the health care system, and collaboration within geriatrics for many years. Several trials on comprehensive geriatric assessment and management performed in different settings have......, all being modified by extra- and intraindividual factors. Handicap is defined as the disability gap. Different health professionals have varying responsibilities in the geriatric team-work, but all should be dedicated to establish common goals. The geriatric work-up is presented with success factors...

  15. Thermal denaturation of A-DNA

    Valle-Orero, J.; Wildes, A. R.; Theodorakopoulos, N.; Cuesta-López, S.; Garden, J.-L.; Danilkin, S.; Peyrard, M.


    The DNA molecule can take various conformational forms. Investigations focus mainly on the so-called ‘B-form’, schematically drawn in the famous paper by Watson and Crick [1]. This is the usual form of DNA in a biological environment and is the only form that is stable in an aqueous environment. Other forms, however, can teach us much about DNA. They have the same nucleotide base pairs for ‘building blocks’ as B-DNA, but with different relative positions, and studying these forms gives insight into the interactions between elements under conditions far from equilibrium in the B-form. Studying the thermal denaturation is particularly interesting because it provides a direct probe of those interactions which control the growth of the fluctuations when the ‘melting’ temperature is approached. Here we report such a study on the ‘A-form’ using calorimetry and neutron scattering. We show that it can be carried further than a similar study on B-DNA, requiring the improvement of thermodynamic models for DNA.

  16. Growth monitoring and diagnostic work-up of short stature: An International Inventorization

    Grote, F.K.; Oostdijk, W.; Muinck Keizer-Schrama, S.M.P.F. de; Dekker, F.W.; Verkerk, P.H.; Wit, J.M.


    Background/Aims: Growth monitoring is almost universally performed, but few data are available on which referral criteria and diagnostic work-up are used worldwide for children with short stature. Methods: A short questionnaire, containing questions on auxological screening and on diagnostic criteri

  17. Growth monitoring and diagnostic work-up of short stature: An International Inventorization

    Grote, F.K.; Oostdijk, W.; Muinck Keizer-Schrama, S.M.P.F. de; Dekker, F.W.; Verkerk, P.H.; Wit, J.M.


    Background/Aims: Growth monitoring is almost universally performed, but few data are available on which referral criteria and diagnostic work-up are used worldwide for children with short stature. Methods: A short questionnaire, containing questions on auxological screening and on diagnostic

  18. The optimization of the diagnostic work-up in patients with suspected obstructive lung disease

    Visser, F.J.; Vegt, M.J. van der; Wilt, G.J. van der; Janssen, J.P.


    BACKGROUND: Pulmonary function testing is a key procedure in the work-up of patients who are suspected of having asthma and chronic obstructive lung disease (COPD). Therein, clinical visits and pulmonary function tests (PFTs) are the major contributors to the overall financial costs.The aim of this

  19. Should sperm DNA fragmentation testing be included in the male infertility work-up?

    Lewis, Sheena E M


    A response to the editorial 'Are we ready to incorporate sperm DNA fragmentation testing into our male infertility work-up? A plea for more robust studies' by Erma Drobnis and Martin Johnson. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  20. Microwave-enhanced folding and denaturation of globular proteins

    Bohr, Henrik; Bohr, Jakob


    It is shown that microwave irradiation can affect the kinetics of the folding process of some globular proteins, especially beta-lactoglobulin. At low temperature the folding from the cold denatured phase of the protein is enhanced, while at a higher temperature the denaturation of the protein from...... its folded state is enhanced. In the latter case, a negative temperature gradient is needed for the denaturation process, suggesting that the effects of the microwaves are nonthermal. This supports the notion that coherent topological excitations can exist in proteins. The application of microwaves...

  1. Statistical mechanics of thermal denaturation of DNA oligomers

    Navin Singh; Yashwant Singh


    Double stranded DNA chain is known to have non-trivial elasticity. We study the effect of this elasticity on the denaturation profile of DNA oligomer by constraining one base pair at one end of the oligomer to remain in unstretched (or intact) state. The effect of this constraint on the denaturation profile of the oligomer has been calculated using the Peyrard–Bishop Hamiltonian. The denaturation profile is found to be very different from the free (i.e. without the constraint) oligomer. We have also examined how this constraint affects the denaturation profile of the oligomer having a segment of defect sites located at different parts of the chain.

  2. Thermal denaturation of type I collagen vitrified gels

    Xia, Zhiyong, E-mail: [The Johns Hopkins University, Applied Physics Laboratory, Laurel, MD 20723 (United States); Calderon-Colon, Xiomara [The Johns Hopkins University, Applied Physics Laboratory, Laurel, MD 20723 (United States); Trexler, Morgana, E-mail: [The Johns Hopkins University, Applied Physics Laboratory, Laurel, MD 20723 (United States); Elisseeff, Jennifer; Guo, Qiongyu [The Johns Hopkins University, Department of Biomedical Engineering, Baltimore, MD 21231 (United States)


    Highlights: Black-Right-Pointing-Pointer We analyzed the denaturation of vitrigels synthesized under different conditions. Black-Right-Pointing-Pointer Overall denaturation kinetics consisted of both reversible and irreversible steps. Black-Right-Pointing-Pointer More stable vitrigels were formed under high level of vitrification. - Abstract: The denaturation kinetics of type I collagen vitrigels synthesized under different vitrification time and temperature were analyzed by the classical Kissinger approach and the advanced model free kinetics (AMFK) using the Vyazovkin algorithm. The AMFK successfully elucidated the overall denaturation into reversible and irreversible processes. Depending on vitrification conditions, the activation energy for the irreversible process ranged from 100 to 200 kJ/mol, and the reversible enthalpy ranged from 250 to 300 kJ/mol. All of these values increased with the vitrification time and temperature, indicating that a more stable and complex structure formed with increased vitrification. The classical Kissinger method predicted the presence of a critical temperate of approximately 60 Degree-Sign C for the transition between reversible and irreversible processes. Scanning electron microscopy revealed the presence of fibril structures in vitrigels both before and after full denaturation; however the fibrils had became thicker and rougher after denaturation.

  3. Adult Onset Still’s Disease: A Review on Diagnostic Workup and Treatment Options

    Rajesh Gopalarathinam


    Full Text Available Adult onset Still’s disease (AOSD is a rare systemic inflammatory disease of unknown etiology and pathogenesis that presents in 5 to 10% of patients as fever of unknown origin (FUO accompanied by systemic manifestations. We report an interesting case of a 33-year-old African-American male who presented with one-month duration of FUO along with skin rash, sore throat, and arthralgia. After extensive workup, potential differential diagnoses were ruled out and the patient was diagnosed with AOSD based on the Yamaguchi criteria. The case history, incidence, pathogenesis, clinical manifestations, differential diagnoses, diagnostic workup, treatment modalities, and prognosis of AOSD are discussed in this case report.

  4. Self-reported drug allergies and the diagnostic work-up in the surgical population

    Tamayo, E; Álvarez, FJ; Castrodeza, J.; Yánez, J.; Arnaiz, P.; Lajo, C.; Soria, S.


    Producción Científica Objective The diagnostic work-up of a drug hypersensitivity reaction is indeed difficult. In general, medical documentation of allergic reactions in medical reports is usually highly deficient or non-existent. The aim of this study was to analyse the prevalence of selfreported drug allergies in the surgical population as well as the criteria used in the diagnosis of drug hypersensitivity reactions. Methods A prospective study with the consecutive partic...


    Anees eChagpar


    Full Text Available Introduction:Breast cancer is frequently diagnosed, yet variation remains in terms of practice patterns in presurgical workup. We sought to determine factors associated with this variation.Methods:An anonymous web-based survey was distributed to surgeons regarding their practices. Statistical analyses were conducted using SPSS.Results:253 surgeons responded to the survey. 17.0% were in academic practice, 37.5%% were hospital employed, and 41.5% were in private practice. 53.3% claimed that > 50% of their practice was breast-related. Surgeons were asked how often they would use various tests in the workup of an otherwise healthy asymptomatic patients, presenting with a non-palpable mammographic abnormality and a core needle biopsy showing invasive breast cancer. 23.5% stated they always would obtain a breast ultrasound, 17.2% stated they never would. 12.8% stated they never order a breast MRI; 4.1% always would. Workup of patients did not vary significantly based on number of years in practice nor practice setting. However, those whose practice was >50% breast were more likely to state that they would always order a breast ultrasound (32.5% vs. 12.9%, p<0.001, and less likely to state they would never order a breast MRI (3.4% vs. 25.8%, p<0.001. However, the proportions of surgeons who would always order a breast MRI was similar in the two groups (3.4% and 3.2%, respectively. Conclusions:These data highlight the lack of uniformity in the workup of asymptomatic patients presenting with non-palpable breast cancers, pointing to potential areas for improving value by minimizing variability.

  6. Balloon-assisted enteroscopy for suspected Meckel’s diverticulum and indefinite diagnostic imaging workup

    Gomes, Guilherme Francisco; Bonin, Eduardo Aimore; Noda, Rafael William; Cavazzola, Leandro Totti; Bartholomei, Thiago Ferreira


    Meckel’s diverticulum (MD) is estimated to affect 1%-2% of the general population, and it represents a clinically silent finding of a congenital anomaly in up to 85% of the cases. In adults, MD may cause symptoms, such as overt occult lower gastrointestinal bleeding. The diagnostic imaging workup includes computed tomography scan, magnetic resonance imaging enterography, technetium 99m scintigraphy (99mTc) using either labeled red blood cells or pertechnetate (known as the Meckel’s scan) and angiography. The preoperative detection rate of MD in adults is low, and many patients ultimately undergo exploratory laparoscopy. More recently, however, endoscopic identification of MD has been possible with the use of balloon-assisted enteroscopy via direct luminal access, which also provides visualization of the diverticular ostium. The aim of this study was to review the diagnosis by double-balloon enteroscopy of 4 adults with symptomatic MD but who had negative diagnostic imaging workups. These cases indicate that balloon-assisted enteroscopy is a valuable diagnostic method and should be considered in adult patients who have suspected MD and indefinite findings on diagnostic imaging workup, including negative Meckel’s scan. PMID:27803776

  7. 27 CFR 19.57 - Recovery and reuse of denatured spirits in manufacturing processes.


    ... denatured spirits in manufacturing processes. 19.57 Section 19.57 Alcohol, Tobacco Products and Firearms... denatured spirits in manufacturing processes. The following persons are not, by reason of the activities...) Manufacturers who use denatured spirits, or articles or substances containing denatured spirits, in a process...

  8. Dodine as a transparent protein denaturant for circular dichroism and infrared studies.

    Guin, Drishti; Sye, Kori; Dave, Kapil; Gruebele, Martin


    The fungicide dodine combines the cooperative denaturation properties of guanidine with the mM denaturation activity of SDS. It was previously tested only on two small model proteins. Here we show that it can be used as a chemical denaturant for phosphoglycerate kinase (PGK), a much larger two-domain enzyme. In addition to its properties as a chemical denaturant, dodine facilitates thermal denaturation of PGK, and we show for the first time that it also facilitates pressure denaturation of a protein. Much higher quality circular dichroism and amide I' infrared spectra of PGK can be obtained in dodine than in guanidine, opening the possibility for use of dodine as a denaturant when UV or IR detection is desirable. One caution is that dodine denaturation, like other detergent-based denaturants, is less reversible than guanidine denaturation. © 2016 The Protein Society.

  9. Virus inactivation by protein denaturants used in affinity chromatography.

    Roberts, Peter L; Lloyd, David


    Virus inactivation by a number of protein denaturants commonly used in gel affinity chromatography for protein elution and gel recycling has been investigated. The enveloped viruses Sindbis, herpes simplex-1 and vaccinia, and the non-enveloped virus polio-1 were effectively inactivated by 0.5 M sodium hydroxide, 6 M guanidinium thiocyanate, 8 M urea and 70% ethanol. However, pH 2.6, 3 M sodium thiocyanate, 6 M guanidinium chloride and 20% ethanol, while effectively inactivating the enveloped viruses, did not inactivate polio-1. These studies demonstrate that protein denaturants are generally effective for virus inactivation but with the limitation that only some may inactivate non-enveloped viruses. The use of protein denaturants, together with virus reduction steps in the manufacturing process should ensure that viral cross contamination between manufacturing batches of therapeutic biological products is prevented and the safety of the product ensured.

  10. Diagnostic Workup of Paediatric Inflammatory Bowel Disease Patients In Europe: Results of A 5-Year Audit of The EUROKIDS Registry

    de Bie, Charlotte I; Buderus, Stephan; Sandhu, Bhupinder K


    OBJECTIVES:: In 2005, the Inflammatory Bowel Disease (IBD) Working Group of ESPGHAN published consensus guidelines on the diagnostic workup of paediatric IBD, the Porto criteria. According to these guidelines, children suspected of IBD should have an oesophagogastroduodenoscopy (OGD), ileocolonos...

  11. Unusual cold denaturation of a small protein domain.

    Buchner, Ginka S; Shih, Natalie; Reece, Amy E; Niebling, Stephan; Kubelka, Jan


    A thermal unfolding study of the 45-residue α-helical domain UBA(2) using circular dichroism is presented. The protein is highly thermostable and exhibits a clear cold unfolding transition with the onset near 290 K without denaturant. Cold denaturation in proteins is rarely observed in general and is quite unique among small helical protein domains. The cold unfolding was further investigated in urea solutions, and a simple thermodynamic model was used to fit all thermal and urea unfolding data. The resulting thermodynamic parameters are compared to those of other small protein domains. Possible origins of the unusual cold unfolding of UBA(2) are discussed.


    Xin-hua Dai; Zhi-gang Wang; Bo Xiao; Yong-jun Zhang; Chen Wang; Chun-li Bai; Xiao-li Zhang; Jian Xu


    Atomic force microscopy was used to investigate the DNA-cetyltrimethylammonium bromide (CTAB) complexes adsorbed on highly ordered pyrolytic graphite (HOPG). These complexes, at low concentrations, can automatically spread out on the surface of HOPG. The DNA-CTAB complexes display a typically extended structure rather than a globular structure. Partially denaturated DNA produced by binding CTAB to DNA is directly observed by AFM with high resolution.The three-dimensional resolution of partially denaturated DNA obtained by AFM is not available by any other technique at present.

  13. Single-molecule denaturation mapping of DNA in nanofluidic channels

    Reisner, Walter; Larsen, Niels Bent; Silahtaroglu, Asli


    Here we explore the potential power of denaturation mapping as a single-molecule technique. By partially denaturing YOYO (R)-1-labeled DNA in nanofluidic channels with a combination of formamide and local heating, we obtain a sequence-dependent "barcode" corresponding to a series of local dips....... Consequently, the technique is sensitive to sequence variation without requiring enzymatic labeling or a restriction step. This technique may serve as the basis for a new mapping technology ideally suited for investigating the long-range structure of entire genomes extracted from single cells....

  14. Denaturation of membrane proteins and hyperthermic cell killing

    Burgman, Paulus Wilhelmus Johannes Jozef


    Summarizing: heat induced denaturation of membrane proteins is probably related to hyperthermic cell killing. Induced resistance of heat sensitive proteins seems to be involved in the development of thermotolerance. Although many questions remain still to be answered, it appears that HSP72, when

  15. Urea Induced Denaturation of Pre-Q1 Riboswitch

    Yoon, Jeseong; Thirumalai, Devarajan; Hyeon, Changbong


    Urea, a polar molecule with a large dipole moment, not only destabilizes the folded RNA structures, but can also enhance the folding rates of large ribozymes. Unlike the mechanism of urea-induced unfolding of proteins, which is well understood, the action of urea on RNA has barely been explored. We performed extensive all atom molecular dynamics (MD) simulations to determine the molecular underpinnings of urea-induced RNA denaturation. Urea displays its denaturing power in both secondary and tertiary motifs of the riboswitch (RS) structure. Our simulations reveal that the denaturation of RNA structures is mainly driven by the hydrogen bonds and stacking interactions of urea with the bases. Through detailed studies of the simulation trajectories, we found that geminate pairs between urea and bases due to hydrogen bonds and stacks persist only ~ (0.1-1) ns, which suggests that urea-base interaction is highly dynamic. Most importantly, the early stage of base pair disruption is triggered by penetration of water molecules into the hydrophobic domain between the RNA bases. The infiltration of water into the narrow space between base pairs is critical in increasing the accessibility of urea to transiently disrupted bases, thus allowing urea to displace inter base hydrogen bonds. This mechanism, water-induced disruption of base-pairs resulting in the formation of a "wet" destabilized RNA followed by solvation by urea, is the exact opposite of the two-stage denaturation of proteins by urea. In the latter case, initial urea penetration creates a dry-globule, which is subsequently solvated by water penetration leading to global protein unfolding. Our work shows that the ability to interact with both water and polar, non-polar components of nucleotides makes urea a powerful chemical denaturant for nucleic acids.

  16. Non-traumatic cortical subarachnoid haemorrhage: diagnostic work-up and aetiological background

    Spitzer, C.; Kosinski, C.M. [University Hospital of RWTH Aachen, Department of Neurology, Aachen (Germany); Mull, M. [University Hospital of RWTH Aachen, Department of Neuroradiology, Aachen (Germany); Rohde, V. [University Hospital of RWTH Aachen, Department of Neurosurgery, Aachen (Germany)


    Only 15% of all subarachnoid haemorrhages (SAHs) are not of aneurysmal origin. Among those, circumscribed SAHs along the cortical convexity are rare and have only been described in singular case reports so far. Here, we present a collection of 12 cases of SAH along the convexity, of non-traumatic origin. Over a period of 10 years, 12 cases of circumscribed SAH along the convexity were identified at our clinic. The clinical presentations, neuroradiological SAH characteristics, further diagnostic work-up to identify the underlying aetiologies, the therapy and clinical outcome were analysed. The patients' chief complaints were unspecific cephalgia, focal or generalised seizures and focal neurological deficits. Typical signs of basal SAH, such as nuchal rigidity, thunderclap-headache or alteration of consciousness, were rare. Magnetic resonance imaging (MRI) and digital subtraction angiography (DSA) revealed different aetiologies, namely postpartal posterior encephalopathy (three), cerebral vasculitis (two), dural sinus thrombosis (two), cortical venous thrombosis (one), intracerebral abscesses (one) and cerebral cavernoma (one). Two cases remained unresolved. Treatment of the underlying disease and symptomatic medication led to good clinical outcome in almost all cases. On the basis of these findings, we demonstrate that the clinical presentation, localisation and aetiology of cortical SAH differ clearly from other SAHs. A diagnostic work-up with MRI and eventually DSA is essential. Mostly, the causative disease can be identified, and specific treatment allows a favourable outcome. (orig.)

  17. CT virtual reality in the preoperative workup of malunited distal radius fractures: preliminary results

    Rieger, Michael; Gruber, Hannes; Jaschke, Werner R. [Innsbruck University Hospital, Department of Radiology I, Innsbruck (Austria); Gabl, Markus [Innsbruck University Hospital, Department of Trauma Surgery, Innsbruck (Austria); Mallouhi, Ammar [Innsbruck University Hospital, Department of Radiology II, Innsbruck (Austria)


    Our objective was to evaluate the usefulness of CT virtual preoperative planning in the surgical repositioning of malunited distal radius fracture. Eleven patients with malunited distal radius fracture underwent multislice CT of both wrists. A preoperative workup was performed in a virtual reality environment created from the CT data sets. Virtual planning comprised three main procedures, carrying out the virtual osteotomy of the radius, prediction of the final position of the distal radius after osteotomy and computer-assisted manufacturing of a repositioning device, which was later placed at the surgical osteotomy site to reposition objectively the distal radius fragment before fixation with the osteosynthesis. All patients tolerated the surgical procedure well. During surgery, the orthopedic surgeons were not required in any of the cases to alter the position of the distal radius that was determined by the repositioning device. At postoperative follow-up, the anatomic relationship of the distal radius was restored (radial inclination, 21.4 ; volar tilt, 10.3 ; ulnar variance, 0.5 mm). Clinically, a significant improvement of pronation (P=0.012), supination (P=0.01), flexion (P=0.001) and extension (P=0.006) was achieved. Pain decreased from 54 to 7 points. CT virtual reality is a valuable adjunct for the preoperative workup and surgical reposition of malunited distal radius fractures. (orig.)

  18. Denaturing and non-denaturing gel electrophoresis as methods for the detection ofjunctional diversity in rearranged T cell receptor sequences

    Offermans, M.T.C.; Sonneveld, R.D.; Bakker, E.; Deutz-Terlouw, P.P.; Geus, B. de; Rozing, J.


    Two nucleic acid gel electrophoresis techniques were tested as a possible tool for analyzing junctional diversity in rearranged T cell receptor (TcR) sequences in order to define the extent of T cell heterogeneity. For this purpose denaturing gradient gel electrophoresis (DGGE) as well as

  19. Clinical Indicators of Late-Onset Sepsis Workup in Very Low-Birth-Weight Infants in the Neonatal Intensive Care Unit.

    Das, Anirudha; Shukla, Sonia; Rahman, Nazia; Gunzler, Douglas; Abughali, Nazha


    Background Late-onset sepsis (LOS) in very low-birth-weight (VLBW) infants is associated with significant morbidity and mortality. Objectives To determine the incidence of LOS workup, association, and predictive value of clinical indicators leading to culture-positive versus culture-negative sepsis workup. Methods All sepsis workups performed after 7 days of life, in neonates with birth weight of birth weight, corrected gestational age, and chronological age, at the time of workup. The clinical indicators leading to the performance of sepsis workup were compared between cases and controls. Results The incidence of culture-positive workup was 87/345 (25.2%) and that of LOS was 84/279 (30.1%). Among various clinical indicators, hypothermia and apnea were significantly associated with culture-positive sepsis workup (p = 0.015 and 0.004, respectively), with a positive predictive value of 81.2 and 71.4%, respectively. Conclusion In VLBW infants, one-fourth of sepsis workups resulted in a positive culture. Apnea and hypothermia were the most significant predictors of culture-positive workup after matching for GA, birth weight, chronological age, and corrected GA at the time of the workup.

  20. Theoretical Study on Effects of Salt and Temperature on Denaturation Transition of Double-stranded DNA

    DONG Rui-Xin; YAN Xun-Ling; PANG Xiao-Feng; JIANG Shan; LIU Sheng-Gang


    We investigate the statistical mechanics properties of a nonlinear dynamics model of the denaturation of the DNA double-helix and study the effects of salt concentration and temperature on denaturation transition of DNA. The specific heat, entropy, and denaturation temperature of the system versus salt concentration are obtained. These results show that the denaturation of DNA not only depends on the temperature but also is influenced by the salt concentration in the solution of DNA, which are in agreement with experimental measurement.

  1. Rheological properties of sweet potato starch before and after denaturalization

    肖华西; 林亲录; 夏新剑; 李丽辉; 林利忠; 吴卫国


    Based on the sweet potato starch,cationic starch,acetic starch and cationic-acetic compoundedly modified starch were made through chemical denaturalization.The above three kinds of static rheological parameter and dynamic rheological parameter were measured,respectively.The experimental result reveals that the thermal stability of starchy viscosity increases after chemical denaturalization.Under the condition of identical shearing rate,the shear stress of cationic-acetic ester compoundedly modified sweet potato starch paste is the largest among these kinds of sweet potato starch.This attributes to a phenomenon of shearing thinning.Furthermore,raw sweet potato starch has a larger gel intensity than that of modified starch.

  2. How water contributes to pressure and cold denaturation of proteins

    Bianco, Valentino


    The mechanisms of cold- and pressure-denaturation of proteins are matter of debate and are commonly understood as due to water-mediated interactions. Here we study several cases of proteins, with or without a unique native state, with or without hydrophilic residues, by means of a coarse-grain protein model in explicit solvent. We show, using Monte Carlo simulations, that taking into account how water at the protein interface changes its hydrogen bond properties and its density fluctuations is enough to predict protein stability regions with elliptic shapes in the temperature-pressure plane, consistent with previous theories. Our results clearly identify the different mechanisms with which water participates to denaturation and open the perspective to develop advanced computational design tools for protein engineering.

  3. Influence of Ficoll on urea induced denaturation of fibrinogen

    Sankaranarayanan, Kamatchi; Meenakshisundaram, N.


    Ficoll is a neutral, highly branched polymer used as a molecular crowder in the study of proteins. Ficoll is also part of Ficoll-Paque used in biology laboratories to separate blood to its components (erythrocytes, leukocytes etc.,). Role of Ficoll in the urea induced denaturation of protein Fibrinogen (Fg) has been analyzed using fluorescence, circular dichroism, molecular docking and interfacial studies. Fluorescence studies show that Ficoll prevents quenching of Fg in the presence of urea. From the circular dichroism spectra, Fg shows conformational transition to random coil with urea of 6 M concentration. Ficoll helps to shift this denaturation concentration to 8 M and thus constraints by shielding Fg during the process. Molecular docking studies indicate that Ficoll interacts favorably with the protein than urea. The surface tension and shear viscosity analysis shows clearly that the protein is shielded by Ficoll.

  4. Incidental prostate cancer prevalence at radical cystoprostatectomy--importance of the histopathological work-up.

    Wetterauer, C; Weibel, M; Gsponer, J R; Vlajnic, T; Zellweger, T; Bütikofer, S; Müller, G; Püschel, H; Bachmann, A; Gasser, T C; Bubendorf, L; Rentsch, C A


    The reported incidental prostate cancer prevalence rates at radical cystoprostatectomy cover a range from 4 to 60 %. We investigated the influence of the histopathological work-up on prostate cancer prevalence rates. We identified 114 patients who had undergone cystoprostatectomy for bladder cancer between 2000 and 2012. Complete histopathological assessment was defined as follows: (i) complete embedding of the prostate gland, (ii) sectioning of 15 or more prostate sections, and (iii) processing as whole mount slides. Prostate cancer prevalence rates derived from complete and incomplete histopathological assessments were compared. The overall prostate cancer prevalence rate was 59.6 %. A mean of 14.4 macroscopic tissue sections (thickness 3-5 mm) were sectioned. Sectioning ≥15 sections resulted in a prostate cancer detection rate of 75 %, compared to 42.6 % when sectioning prevalence rates at cystoprostatectomy (CP). The high proportion of significant prostate cancer found in our series calls for a careful surgical approach to the prostate during CP.

  5. Moving Beyond Metabolic Encephalopathy: An Update on Delirium Prevention, Workup, and Management.

    Brown, Ethan G; Douglas, Vanja C


    Delirium is a condition that frequently complicates hospitalization and consists of an acute decline in orientation and attention, often accompanied by other cognitive changes. Delirium is tied to multiple detrimental outcomes both in the short and long term, including cognitive and functional decline, inpatient complications, and mortality. Postoperative, elderly medical, and critical care patients have been identified as populations at particular risk. In this review, the authors discuss current theories on pathophysiology, recommended workup, and evidence-based prevention and management of inpatient delirium. In general, instituting a system of active screening of at-risk populations and nonpharmacologic interventions for prevention and treatment seems to be the most effective method of addressing delirium. More research is needed to clarify the etiology of delirium and develop safe therapeutic options that address the underlying pathophysiology.

  6. In the workup of patients with obscure gastrointestinal bleed, does 64-slice MDCT have a role?

    Chinmay Kulkarni


    Full Text Available Purpose: The purpose was to prospectively determine the sensitivity of 64-slice MDCT in detecting and diagnosing the cause of obscure gastrointestinal bleed (OGIB. Materials and Methods: Our study included 50 patients (male 30, female 20 in the age range of 3-82 years (average age: 58.52 years who were referred to our radiology department as part of their workup for clinically evident gastrointestinal (GI bleed or as part of workup for anemia (with and without positive fecal occult blood test. All patients underwent conventional upper endoscopy and colonoscopy before undergoing CT scan. Following a noncontrast scan, all patients underwent triple-phase contrast CT scan using a 64-slice CT scan system. The diagnostic performance of 64-slice MDCT was compared to the results of capsule endoscopy, 99m-technetium-labeled red blood cell scintigraphy (99mTc-RBC scintigraphy, digital subtraction angiography, and surgery whenever available. Results: CT scan showed positive findings in 32 of 50 patients. The sensitivity, specificity, positive predictive value, and negative predictive values of MDCT for detection of bleed were 72.2%, 42.8%, 81.2%, and 44.4%, respectively. Capsule endoscopy was done in 15 patients and was positive in 10 patients; it had a sensitivity of 71.4%. Eleven patients had undergone 99mTc-RBC scintigraphy prior to CT scan, and the result was positive in seven patients (sensitivity 70%. Digital subtraction angiography was performed in only eight patients and among them all except one patient showed findings consistent with the lesions detected on MDCT. Conclusion: MDCT is a sensitive and noninvasive tool that allows rapid detection and localization of OGIB. It can be used as the first-line investigation in patients with negative endoscopy and colonoscopy studies. MDCT and capsule endoscopy have complementary roles in the evaluation of OGIB.

  7. How to tackle tremor – systematic review of the literature and diagnostic work-up

    Arthur W.G. Buijink


    Full Text Available BackgroundTremor is the most prevalent movement disorder in clinical practice. It is defined as involuntary, rhythmic, oscillatory movements. The diagnostic process of patients with tremor can be laborious and challenging, and a clear, systematic overview of available diagnostic techniques is lacking. Tremor can be a symptom of many diseases, but can also represent a distinct disease entity.ObjectiveThe objective of this review is to give a clear, systematic and step-wise overview of the diagnostic work-up of a patient with tremor. The clinical relevance and value of available laboratory tests in patients with tremor will be explored.MethodsWe systematically searched through EMBASE. The retrieved articles were supplemented by articles containing relevant data or provided important background information. Studies that were included investigated the value and/or usability of diagnostic tests for tremor.ResultsIn most patients, history and clinical examination by an experienced movement disorders neurologist are sufficient to establish a correct diagnosis, and further ancillary examinations will not be needed. Ancillary investigation should always be guided by tremor type(s present and other associated signs and symptoms. The main ancillary examination techniques currently are electromyography and SPECT imaging. Unfortunately, many techniques have not been studied in large prospective, diagnostic studies to be able to determine important variables like sensitivity and specificity.ConclusionWhen encountering a patient with tremor, history and careful clinical examination should guide the diagnostic process. Adherence to the diagnostic work-up provided in this review will help the diagnostic process of these patients.

  8. Temperature induced denaturation of collagen in acidic solution.

    Mu, Changdao; Li, Defu; Lin, Wei; Ding, Yanwei; Zhang, Guangzhao


    The denaturation of collagen solution in acetic acid has been investigated by using ultra-sensitive differential scanning calorimetry (US-DSC), circular dichroism (CD), and laser light scattering (LLS). US-DSC measurements reveal that the collagen exhibits a bimodal transition, i.e., there exists a shoulder transition before the major transition. Such a shoulder transition can recover from a cooling when the collagen is heated to a temperature below 35 degrees C. However, when the heating temperature is above 37 degrees C, both the shoulder and major transitions are irreversible. CD measurements demonstrate the content of triple helix slowly decreases with temperature at a temperature below 35 degrees C, but it drastically decreases at a higher temperature. Our experiments suggest that the shoulder transition and major transition arise from the defibrillation and denaturation of collagen, respectively. LLS measurements show the average hydrodynamic radius R(h), radius of gyration R(g)of the collagen gradually decrease before a sharp decrease at a higher temperature. Meanwhile, the ratio R(g)/R(h) gradually increases at a temperature below approximately 34 degrees C and drastically increases in the range 34-40 degrees C, further indicating the defibrillation of collagen before the denaturation.

  9. [DNA degradation during standard alkaline of thermal denaturation].

    Drozhdeniuk, A P; Sulimova, G E; Vaniushin, B F


    Essential degradation 8 DNA (up to 10 per cent) with liberation of acid-soluble fragments takes place on the standard alkaline (0,01 M sodium phosphate, pH 12, 60 degrees, 15 min) or thermal (0.06 M sodium phosphate buffer, pH 6.8, 102 degrees C, 15 min) denaturation. This degradation is more or less selective: fraction of low molecular weight fragments, isolated by hydroxyapatite cromatography and eluted by 0.06 M sodium phosphate buffer, pH 6.8 is rich in adenine and thymine and contains about 2 times less 5-methylcytosine than the total wheat germ DNA. The degree of degradation of DNA on thermal denaturation is higher than on alkaline degradation. Therefore while studying reassociation of various DNA, one and the same standard method of DNA denaturation should be used. Besides, both the level of DNA degradation and the nature of the resulting products (fragments) should be taken into account.

  10. Protein's native state stability in a chemically induced denaturation mechanism.

    Olivares-Quiroz, L; Garcia-Colin, L S


    In this work, we present a generalization of Zwanzig's protein unfolding analysis [Zwanzig, R., 1997. Two-state models of protein folding kinetics. Proc. Natl Acad. Sci. USA 94, 148-150; Zwanzig, R., 1995. Simple model of protein folding kinetics. Proc. Natl Acad. Sci. USA 92, 9801], in order to calculate the free energy change Delta(N)(D)F between the protein's native state N and its unfolded state D in a chemically induced denaturation. This Extended Zwanzig Model (EZM) is both based on an equilibrium statistical mechanics approach and the inclusion of experimental denaturation curves. It enables us to construct a suitable partition function Z and to derive an analytical formula for Delta(N)(D)F in terms of the number K of residues of the macromolecule, the average number nu of accessible states for each single amino acid and the concentration C(1/2) where the midpoint of the ND transition occurs. The results of the EZM for proteins where chemical denaturation follows a sigmoidal-type profile, as it occurs for the case of the T70N human variant of lysozyme (PDB code: T70N) [Esposito, G., et al., 2003. J. Biol. Chem. 278, 25910-25918], can be splitted into two lines. First, EZM shows that for sigmoidal denaturation profiles, the internal degrees of freedom of the chain play an outstanding role in the stability of the native state. On the other hand, that under certain conditions DeltaF can be written as a quadratic polynomial on concentration C(1/2), i.e., DeltaF approximately aC(1/2)(2)+bC(1/2)+c, where a,b,c are constant coefficients directly linked to protein's size K and the averaged number of non-native conformations nu. Such functional form for DeltaF has been widely known to fit experimental measures in chemically induced protein denaturation [Yagi, M., et al., 2003. J. Biol. Chem. 278, 47009-47015; Asgeirsson, B., Guojonsdottir, K., 2006. Biochim. Biophys. Acta 1764, 190-198; Sharma, S., et al., 2006. Protein Pept. Lett. 13(4), 323-329; Salem, M., et al

  11. Retrograde-viewing device improves adenoma detection rate in colonoscopies for surveillance and diagnostic workup

    Peter D Siersema; Amit Rastogi; Anke M Leufkens; Paul A Akerman; Kassem Azzouzi; Richard I Rothstein; Frank P Vleggaar


    AIM:To determine which patients might benefit most from retrograde viewing during colonoscopy through subset analysis of randomized,controlled trial data.METHODS:The Third Eye(R) Retroscope(R) Randomized Clinical Evaluation (TERRACE) was a randomized,controlled,multicenter trial designed to evaluate the efficacy of a retrograde-viewing auxiliary imaging device that is used during colonoscopy to provide a second video image which allows viewing of areas on the proximal aspect of haustral folds and flexures that are difficult to see with the colonoscope's forward view.We performed a post-hoc analysis of the TER-RACE data to determine whether certain subsets of the patient population would gain more benefit than others from use of the device.Subjects were patients scheduled for colonoscopy for screening,surveillance or diagnostic workup,and each underwent same-day tandem examinations with standard colonoscopy (SC)and Third Eye colonoscopy (TEC),randomized to SC followed by TEC or vice versa.RESULTS:Indication for colonoscopy was screening in 176/345 subjects (51.0%),surveillance after previous polypectomy in 87 (25.2%) and diagnostic workup in 82 (23.8%).In 4 subjects no indication was specified.Previously reported overall results had shown a net additional adenoma detection rate (ADR) with TEC of 23.2% compared to SC.Relative risk (RR) of missing adenomas with SC vs TEC as the initial procedure was 1.92 (P =0.029).Post-hoc subset analysis shows additional ADRs for TEC compared to SC were 4.4% for screening,35.7% for surveillance,55.4% for diagnostic and 40.7% for surveillance and diagnostic combined.The RR of missing adenomas with SC vs TEC was 1.11 (P =0.815) for screening,3.15 (P =0.014) for surveillance,8.64 (P =0.039) for diagnostic and 3.34(P =0.003) for surveillance and diagnostic combined.Although a multivariate Poisson regression suggested gender as a possibly significant factor,subset analysis showed that the difference between genders was

  12. Liver CT for vascular mapping during radioembolisation workup : comparison of an early and late arterial phase protocol

    van den Hoven, Andor F; Braat, Manon N G J A; Prince, Jip F; van Doormaal, Pieter J; van Leeuwen, Maarten S; Lam, Marnix G E H; van den Bosch, Maurice A A J


    OBJECTIVES: To compare right gastric (RGA) and segment 4 artery (A4) origin detection rates during radioembolisation workup between early and late arterial phase liver CT protocols. METHODS: 100 consecutive patients who underwent liver CT between May 2012-January 2015 with early or late arterial pha

  13. No Value for Routine Chest Radiography in the Work-Up of Early Stage Cervical Cancer Patients

    Hoogendam, Jacob P.; Zweemer, Ronald P.; Verkooijen, Helena M.; de Jong, Pim A.; van den Bosch, Maurice A. A. J.; Verheijen, Rene H. M.; Veldhuis, WB


    Aim Evidence supporting the recommendation to include chest radiography in the work-up of all cervical cancer patients is limited. We investigated the diagnostic value of routine chest radiography in cervical cancer staging. Methods All consecutive cervical cancer patients who presented at our terti

  14. Anorectal function testing and anal endosonography in the diagnostic work-up of patients with primary pelvic organ prolapse

    A.G. Groenendijk (Annette); E. Birnie (Erwin); G.E. Boeckxstaens (Guy); J-P.W. Roovers (Jan-Paul); G.J. Bonsel (Gouke)


    textabstractAIM: To study the pathophysiology of defecation disorders in patients with primary pelvic organ prolapse (POP) and the diagnostic potential of anorectal function testing (AFT) including endosonography in the work-up of these patients. METHODS: 59 Patients were evaluated with a validated

  15. Anorectal function testing and anal endosonography in the diagnostic work-up of patients with primary pelvic organ prolapse

    A.G. Groenendijk (Annette); E. Birnie (Erwin); G.E. Boeckxstaens (Guy); J-P.W. Roovers (Jan-Paul); G.J. Bonsel (Gouke)


    textabstractAIM: To study the pathophysiology of defecation disorders in patients with primary pelvic organ prolapse (POP) and the diagnostic potential of anorectal function testing (AFT) including endosonography in the work-up of these patients. METHODS: 59 Patients were evaluated with a validated

  16. Collagen thermal denaturation study for thermal angioplasty based on modified kinetic model: relation between the artery mechanical properties and collagen denaturation rate

    Shimazaki, N.; Hayashi, T.; Kunio, M.; Arai, T.


    We have been developing the novel short-term heating angioplasty in which sufficient artery lumen-dilatation was attained with thermal softening of collagen fiber in artery wall. In the present study, we investigated on the relation between the mechanical properties of heated artery and thermal denaturation fractures of arterial collagen in ex vivo. We employed Lumry-Eyring model to estimate temperature- and time-dependent thermal denaturation fractures of arterial collagen fiber during heating. We made a kinetic model of arterial collagen thermal denaturation by adjustment of K and k in this model, those were the equilibrium constant of reversible denaturation and the rate constant of irreversible denaturation. Meanwhile we demonstrated that the change of reduced scattering coefficient of whole artery wall during heating reflected the reversible denaturation of the collagen in artery wall. Based on this phenomenon, the K was determined experimentally by backscattered light intensity measurement (at 633nm) of extracted porcine carotid artery during temperature elevation and descending (25°C-->80°C-->25°C). We employed the value of according to our earlier report in which the time-and temperature- dependent irreversible denaturation amount of the artery collagen fiber that was assessed by the artery birefringence. Then, the time- and temperature- dependent reversible (irreversible) denaturation fraction defined as the reversible ((irreversible) denatured collagen amount) / (total collagen amount) was calculated by the model. Thermo-mechanical analysis of artery wall was performed to compare the arterial mechanical behaviors (softening, shrinkage) during heating with the calculated denaturation fraction with the model. In any artery temperature condition in 70-80°, the irreversible denaturation fraction at which the artery thermal shrinkage started was estimated to be around 20%. On the other hand, the calculated irreversible denaturation fraction remained below

  17. Denatured ethanol release into gasoline residuals, Part 1: Source behaviour

    Freitas, Juliana G.; Barker, James F.


    With the increasing use of ethanol in fuels, it is important to evaluate its fate when released into the environment. While ethanol is less toxic than other organic compounds present in fuels, one of the concerns is the impact ethanol might have on the fate of gasoline hydrocarbons in groundwater. One possible concern is the spill of denatured ethanol (E95: ethanol containing 5% denaturants, usually hydrocarbons) in sites with pre-existing gasoline contamination. In that scenario, ethanol is expected to increase the mobility of the NAPL phase by acting as a cosolvent and decreasing interfacial tension. To evaluate the E95 behaviour and its impacts on pre-existing gasoline, a field test was performed at the CFB-Borden aquifer. Initially gasoline contamination was created releasing 200 L of E10 (gasoline with 10% ethanol) into the unsaturated zone. One year later, 184 L of E95 was released on top of the gasoline contamination. The site was monitored using soil cores, multilevel wells and one glass access tube. At the end of the test, the source zone was excavated and the compounds remaining were quantified. E95 ethanol accumulated and remained within the capillary fringe and unsaturated zone for more than 200 days, despite ~ 1 m oscillations in the water table. The gasoline mobility increased and it was redistributed in the source zone. Gasoline NAPL saturations in the soil increased two fold in the source zone. However, water table oscillations caused a separation between the NAPL and ethanol: NAPL was smeared and remained in deeper positions while ethanol moved upwards following the water table rise. Similarly, the E95 denaturants that initially were within the ethanol-rich phase became separated from ethanol after the water table oscillation, remaining below the ethanol rich zone. The separation between ethanol and hydrocarbons in the source after water table oscillation indicates that ethanol's impact on hydrocarbon residuals is likely limited to early times.

  18. Strategies for denaturing the weapons-grade plutonium stockpile

    Buckner, M.R.; Parks, P.B.


    In the next few years, approximately 50 metric tons of weapons-grade plutonium and 150 metric tons of highly-enriched uranium (HEU) may be removed from nuclear weapons in the US and declared excess. These materials represent a significant energy resource that could substantially contribute to our national energy requirements. HEU can be used as fuel in naval reactors, or diluted with depleted uranium for use as fuel in commercial reactors. This paper proposes to use the weapons-grade plutonium as fuel in light water reactors. The first such reactor would demonstrate the dual objectives of producing electrical power and denaturing the plutonium to prevent use in nuclear weapons.

  19. RNA Denaturation: Excluded Volume, Pseudoknots, and Transition Scenarios

    Baiesi, M.; Orlandini, E.; Stella, A. L.


    A lattice model of RNA denaturation which fully accounts for the excluded volume effects among nucleotides is proposed. A numerical study shows that interactions forming pseudoknots must be included in order to get a sharp continuous transition. Otherwise a smooth crossover occurs from the swollen linear polymer behavior to highly ramified, almost compact conformations with secondary structures. In the latter scenario, which is appropriate when these structures are much more stable than pseudoknot links, probability distributions for the lengths of both loops and main branches obey scaling with nonclassical exponents.

  20. DSC study of cold and heat denaturation processes of β-lactoglobulin A with guanidine hydrochloride

    王邦宁; 谈夫


    The cold and heat denaturations of bovine β-lactoglobuhn A (β-lg A) has been studied in solutions of guanidine hydrochloride (GuHCl) by differential scanning calorimelry (DSC) The experimental results are presented and discussed.It is shown that the number of protons bound by the monomeric molecules of β-lg A was unchanged before and after its heat denaturation below pH 3,and that the activation energy of the heat denaturation was depressed owing to the presence of GuHCl.In the solutions with 2.50 and 3.06 mol/L of GuHCl,both the cold and heat denat-urations of β-lg A were observed.In comparison with the heat denaturation,the activation energy of cold denaturation was far lower and the number of GuHCl molecules bound by the unfolded polypeptide chains after cold denaturation increased a lot.The absolute value of the enthalpy of cold denaturation was larger than that of heat denaturation It was found by the analysis that the contribution to the total denaturational enthalpy of conformational change i

  1. Inclusion of educational messages in laboratory reports aids to complete the diagnostic workup of hyperglycemia.

    Pérez-Jáuregui, José; González-Cardel, Ana María; Olay-Fuentes, Gabriela; Reza-Albarrán, Alfredo; Mehta, Roopa; Aguilar-Salinas, Carlos A


    To evaluate whether educational messages regarding oral glucose tolerance test (OGTT) indications in laboratory reports increase the number of OGTTs appropriately requested. The following message was printed on the lab reports of individuals with a fasting plasma glucose (FPG) concentration between 5.5 and 6.9 mmol/l: "A FPG between 5.5 and 6.9 mmol/l is considered abnormal by the American Diabetes Association (impaired fasting glucose). An OGTT is recommended if the patient does not have a diagnosis of diabetes and suffers from conditions associated with an increased risk for having type 2 diabetes (i.e., overweight, high blood pressure, abnormal plasma lipids or family history of diabetes)." The number of educational messages printed was 81,099. The intervention resulted in a significant increase in the number of OGTTs requested, from 78 +/- 19 to 268 +/- 48 tests per month. It also resulted in a greater proportion of case subjects that had an abnormal OGTT result. Educational messages in laboratory reports aid in the diagnostic workup of hyperglycemia.

  2. [Which is the method of choice for evaluating uterine cavity in infertility workup?].

    Ait Benkaddour, Y; Gervaise, A; Fernandez, H


    Uterine factors represent only 2 to 3 % of infertility, but intra-uterine lesions are much more common in infertile women (40-50 %). These lesions can interfere with spontaneous fertility and can compromise pregnancy rates in assisted reproduction. Exploration of the uterine cavity is actually one of the basic explorations in infertility workup. Classically, hysterosalpingography and transvaginal sonography are most communally used for this purpose. Hysteroscopy, with the development and miniaturization of equipment, is currently simple, outpatient cost-effective exploration and it is considered the gold standard for diagnosis of intrauterine lesions. However, the benefit of the systematic use of hysteroscopy in the initial assessment of infertility remains unclear and the exploration of the uterine cavity in the initial assessment of infertility should be based on hysterosalpingography or hysterosonography. Systematic hysteroscopy before IVF is widely accepted practice that is supposed to improve pregnancy rates but still lacks scientific evidence. After repeated implantation failure in IVF cycles, uterine cavity should be reevaluated by hysteroscopy and this practice has been demonstrated to improve pregnancy rates.

  3. Diagnostic workup in carotid stenosis - a neurologist's perspective

    Rosenkranz, Michael; Gerloff, Christian [University Medical Center Hamburg-Eppendorf, Department of Neurology, Hamburg (Germany)


    Carotid artery stenosis is associated with the risk of stroke, myocardial infarction, and vascular death. In selected patients, revascularization of carotid narrowing by endarterectomy may reduce the risk of stroke distal to the stenosis. Carotid artery stenting has evolved as a potential alternative to endarterectomy. Four randomized clinical trials comparing safety and efficacy of endarterectomy versus stenting of symptomatic carotid stenosis have been published in recent years, but there remains some uncertainty about the implications of these trials for clinical routine. Both carotid stenting and endarterectomy are based on different treatment strategies which may result in different specific risk factors associated with each procedure. Hence, the procedural risk of either modality varies not only with the skills of the surgeon or the interventionalist but may depend on patient characteristics. It appears that the most important question is not whether one revascularization modality is superior but for which patient one modality is better than the other. A comprehensive diagnostic workup of patients with carotid stenosis based on a broad panel of covariates that affect the risk of vascular events may improve selection of patients for carotid revascularization and may help to decide for whom one revascularization modality is likely to be better than the other. (orig.)

  4. Sex differentiation disorders (SDD) prenatal sonographic diagnosis, genetic and hormonal work-up.

    Katorza, Eldad; Pinhas-Hamiel, Orit; Mazkereth, Ram; Gilboa, Yinon; Achiron, Reuven


    Gender is determined by the genetic, gonadal and hormonal/ phenotypic sex. Genetic sex is determined at conception. The establishment of the gonadal sex (ovary/testis) and the phenotypic sex (external and internal genitalia) is a complicated multistep process which is determined during fetal life mainly during the first trimester. Recently more genes have been found to be involved in this process. Prenatal diagnosis of fetal gender can be made using ultrasound technology, genetic and hormonal examinations. Nowadays using a vaginal and abdominal transducer for US examination recognition of external and internal genitalia of both genders is possible. The determination of gender during fetal life is important not only as a matter of curiosity; in some cases of ambiguity (for example congenital adrenal hyperplasia) prenatal treatment can change the natural history of the disease. Prenatal diagnosis can also subtype the ambiguity, and its severity can be established. In this review we describe our experience in prenatal diagnosis and establishment of the fetal gender, the subtypes of ambiguity and our suggestion for the process of diagnostic work-up.

  5. Unnecessary Workup of Asymptomatic Neonates in the Era of Group B Streptococcus Prophylaxis

    Brad Buckler


    Full Text Available Asymptomatic term neonates born to mothers who are Group B Streptococcus (GBS unknown or GBS positive but “inadequately” treated prior to delivery do not require invasive laboratory evaluation. We conducted a retrospective cohort study of mother/baby dyads born from January 1, 2005 until September 30, 2007 at the Medical College of Georgia. Their current protocol is to obtain a Complete Blood Count with Differential (CBC with D, Blood Culture (BC, and C-reactive protein (CRP after birth. Mother/baby dyads (=242 that met inclusion criteria were reviewed. Of these 242 babies 25 (10% were started on antibiotics after the initial lab values were known. None of the blood cultures were positive and the CRP's were normal. The 2002 GBS guidelines call for laboratory evaluation of “at-risk” neonates, but the workup of these babies is not only costly, it does not provide any advantage over old fashioned clinical observation for the evaluation and treatment of early onset GBS sepsis.

  6. The Use of Cardiac Magnetic Resonance Imaging in the Diagnostic Workup and Treatment of Atrial Fibrillation

    Peter Haemers


    Full Text Available Atrial fibrillation (AF is the most common cardiac arrhythmia and imposes a huge clinical and economic burden. AF is correlated with an increased morbidity and mortality, mainly due to stroke and heart failure. Cardiovascular imaging modalities, including echocardiography, computed tomography (CT, and cardiovascular magnetic resonance (CMR, play a central role in the workup and treatment of AF. One of the major advantages of CMR is the high contrast to noise ratio combined with good spatial and temporal resolution, without any radiation burden. This allows a detailed assessment of the structure and function of the left atrium (LA. Of particular interest is the ability to visualize the extent of LA wall injury. We provide a focused review of the value of CMR in identifying the underlying pathophysiological mechanisms of AF, its role in stroke prevention and in the guidance of radiofrequency catheter ablation. CMR is a promising technique that could add valuable information for therapeutic decision making in specific subpopulations with AF.

  7. Calorimetric Study of Thermal Denaturation of Superoxide Dismutase

    王邦宁; 谈夫


    The thermal denaturation of superoxide dismutase (SOD) from bovine erythrocytes was studied at various pH values of different buffers and at various concentrations of solutions of two neutral salts by differential scanning calorimetry. The experiments performed indicate that the PIPES is a buffer non-coordinating with the SOD, and that the binding of the anions studied influences more or less the thermal denaturation of SOD, but the effect on the oxidation form of SOD is more apparent. A new conformer of SOD with lower thermostability was discovered by the experiments performed in different buffers at certain pH values higher than the isoelectric point of SOD, or at higher concentrations of neutral salt solutions. The new conformer may be converted irreversibly into the usual conformer with high thermostability during heating. Based on the thermodynamic parameters obtained in distilled water and by thermodynamic analysis using the Ooi’s model, it is revealed that the large enthalpy △Hdc contributed by

  8. [Characterization of thermal denaturation process of proteinase K by spectrometry].

    Zhang, Qi-Bing; Na, Xin-Zhu; Yin, Zong-Ning


    The effect of different temperatures on the activity and conformational changes of proteinase K was studied. Methods Proteinase K was treated with different temperatures, then denatured natural substrate casein was used to assay enzyme activity, steady-state and time-resolved fluorescence spectroscopy was used to study tertiary structure, and circular dichroism was used to study secondary structure. Results show with the temperature rising from 25 to 65 degrees C, the enzyme activity and half-life of proteinase K dropped, maximum emission wavelength red shifted from 335 to 354 nm with fluorescence intensity decreasing. Synchronous fluorescence intensity of tryptophan residues decreased and that of tyrosine residues increased. Fluorescence lifetime of tryptophan residues reduced from 4. 427 1 to 4. 032 4 ns and the fraction of alpha-helix dropped. It was concluded that it is simple and accurate to use steady-state/time-resolved fluorescence spectroscopy and circular dichroism to investigate thermal stability of proteinase K. Thermal denaturation of proteinase K followed a three-state process. Fluorescence intensity of proteinase K was affected by fluorescence resonance energy transfer from tyrosine to tryptophan residues. The alpha-helix was the main structure to maintain conformational stability of enzyme active site of proteinase K.

  9. Denatured Thermodynamics of Proteins in Weak Cation-exchange Chromatography

    LI Rong; CHEN Guo-Liang


    The thermostability of some proteins in weak cation-exchange chromatography was investigated at 20-80 ℃. The results show that there is a fixed thermal denaturation transition temperature for each protein. The appearance of the thermal transition temperature indicates that the conformations of the proteins are destroyed seriously. The thermal behavior of the proteins in weak cation-exchange and hydrophobic interaction chromatographies were compared in a wide temperature range. It was found that the proteins have a higher thermostability in a weak cation-exchange chromatography system. The thermodynamic parameters(ΔH0, ΔS0) of those proteins were determined by means of Vant Hoff relationship(lnk-1/T). According to standard entropy change(ΔS0), the conformational change of the proteins was judged in the chromatographic process. The linear relationships between ΔH0 and ΔS0 can be used to evaluate "compensation temperature"(β) at the protein denaturation and identify the identity of the protein retention mechanism in weak cation-exchange chromatography.

  10. Optical real-time measurement of collagen denaturation

    Sankaran, Vanitha; Walsh, Joseph T., Jr.


    Linear birefringence is a property of collagenous tissue that results from both its composition and structure. Previous investigations have shown that birefringence provides an indication of structural changes in collagen during slow heating. We now report the birefringent response of both mature and young rat tail tendon to laser-heating. The results indicate that denaturation of collagen from mature rats induced by a 200-microsecond(s) -long Ho:YAG laser pulse may not be described accurately by kinetic parameters. Several second-long pulses of CO2 laser pulse may not be described from young rats fit an Arrhenius model with Ea equals 12.1 kcal/mol and A equals e18.03 s-1. Typically, for slow-heating of collagen, Ea equals kcal/mol and A equals e120 s-1. Thus, it seems likely that the temperature and energy needed to initiate collagen denaturation is lower in young collagen, possibly due to its decreased hydroxyproline content and consequent decreased thermal stability.

  11. Second-harmonic generation investigation of collagen thermal denaturation

    Chen, Wei-Liang; Sun, Yen; Lin, Sung-Jan; Jee, Shiou-Hwa; Chen, Yang-Fang; Lin, Ling-Chih; So, Peter T. C.; Dong, Chen-Yuan


    Using the technique of second-harmonic generation (SHG) microscopy we obtained large area image of type I collagen from rat tail tendon as it is heated from 40°C to 70°C for 0 to 180 minutes. The high resolution images allowed us to investigate the collagen structural change. We observed that heating the tendon below the temperature of 54°C does not produce any change in the averaged SHG intensity. At the heating temperature of 54°C and above, we find that increasing the heating temperature and time leads to decreasing SHG intensity. As the tendon is heated above 54°C, a decrease in the SHG signal occurs uniformly throughout the tendon, but the regions where the SHG signal vanishes form a tiger-tail like pattern. By comparing the relative SHG intensities in small and large areas, we found that the denaturation process responsible for forming the tiger-tail like pattern occurs at a higher rate than the global denaturation process occurring throughout the tendon. Our results show that second-harmonic generation microscopy is effective in monitoring the thermal damage to collagen and has potential applications in biomedicine.

  12. Heterogeneity of mitochondrial DNA from Saccharomyces carlsbergensis. Denaturation mapping by electron microscopy.

    Christiansen, Gunna; Christiansen, C; Bak, AL


    Electronmicroscopic observation of the denaturation pattern of 130 partially denaturated linear mitochondrial DNA molecules from Saccharomyces carlsbergensis was used to investigate the distribution of AT-rich sequences within the mitochondrial genome. The molecules were observed after heating...... denaturated sequences in the mitochondrial DNA. These sequences which presumably correspond to the very AT-rich regions, known to exist in the yeast mitochondrial DNA, were found at intervals of about 0.5 - 3 mum on the map....

  13. 27 CFR 21.92 - Denaturants listed as U.S.P. or N.F.


    ....P. or N.F. 21.92 Section 21.92 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND... for Denaturants § 21.92 Denaturants listed as U.S.P. or N.F. Denaturing materials and products listed in this part as “U.S.P.” or “N.F.” shall meet the specifications set forth in the current...

  14. Electrochemical behaviour of denatured ethanol for use in direct ethanol fuel cells

    Bayer, Domnik; Kintzel, Birgit; Joos, Martin; Cremers, Carsten; Tuebke, Jens [Fraunhofer-Institut fuer Chemische Technologie (ICT), Pfinztal (Germany)


    In the present study, two denaturing agents, an adjuvant to a denaturing agent and a mixture of a denaturing agend and the adjuvant were tested with regard to their fuel cell compatibility. Therefore, various electrochemical tests including cyclic voltammetry, chronoamperometry and differential electrochemical mass spectroscopy have been conducted at platinum as model catalyst in acidic and alkaline medium. In addition, the most promising denaturing agent, a mixture of tert-butyl ethyl ether (ETBE) with the adjuvant Bitrex {sup registered}, has also been tested at commercial fuel cell catalysts in both acidic and alkaline media. (orig.)

  15. Visualization of early events in acetic acid denaturation of HIV-1 protease: a molecular dynamics study.

    Aditi Narendra Borkar

    Full Text Available Protein denaturation plays a crucial role in cellular processes. In this study, denaturation of HIV-1 Protease (PR was investigated by all-atom MD simulations in explicit solvent. The PR dimer and monomer were simulated separately in 9 M acetic acid (9 M AcOH solution and water to study the denaturation process of PR in acetic acid environment. Direct visualization of the denaturation dynamics that is readily available from such simulations has been presented. Our simulations in 9 M AcOH reveal that the PR denaturation begins by separation of dimer into intact monomers and it is only after this separation that the monomer units start denaturing. The denaturation of the monomers is flagged off by the loss of crucial interactions between the α-helix at C-terminal and surrounding β-strands. This causes the structure to transit from the equilibrium dynamics to random non-equilibrating dynamics. Residence time calculations indicate that denaturation occurs via direct interaction of the acetic acid molecules with certain regions of the protein in 9 M AcOH. All these observations have helped to decipher a picture of the early events in acetic acid denaturation of PR and have illustrated that the α-helix and the β-sheet at the C-terminus of a native and functional PR dimer should maintain both the stability and the function of the enzyme and thus present newer targets for blocking PR function.

  16. Thoracic splenosis. Report of a case and review of the diagnostic workup.

    Bugiantella, Walter; Crusco, Federico; Avenia, Nicola; Fabio, Rondelli


    L’impianto di tessuto splenico in siti ectopici dopo un trauma della milza viene definito splenosi. Sebbene si verifichi più comunemente in addome, essa può localizzarsi anche nel torace in caso di rottura del diaframma. La splenosi toracica (TS) è spesso asintomatica ed è diagnosticata nel corso di indagini radiologiche del torace. Descriviamo il caso di un uomo, con storia di pregresso trauma toraco-addominale, che ha eseguito una radiografia del torace per routine con evidenza di immagini radioopache in adiacenza dell’arco inferiore sinistro dell’ombra cardiaca, che sono risultate essere noduli di TS confermate dalla CT con mezzo di contrasto. La CT o la MRI unite ad una anamnesi accurata sono di solito sufficiente per diagnosticare la TS, altrimenti è necessaria la scintigrafia con 99mTc. La biopsia guidata dall’imaging e la toracoscopia dovrebbero essere eseguite solo quando la scintigrafia non è disponibile o i risultati delle altre metodiche non sono risolutivi. Normalmente non è necessario rimuovere la TS perché il tessuto splenico ha una crescita lenta, non invasiva e benigna. Raramente la chirurgia può essere necessaria quando la TS è sintomatica (emottisi, tosse, dolore di tipo pleuritico). La TS può essere difficile da diagnosticare, specialmente se l’aspetto dei noduli non è univoco e l’anamnesi non è nota. Ciò può portare a un iter diagnostico eccessivo e a procedure non necessarie (biopsia, toracoscopia fino alla toracotomia). Nel work-up dei noduli toracici la TS dovrebbe essere considerata nei pazienti con storia di trauma e rottura della milza.

  17. Y Choromosomal Microdeletion Screening in The Workup of Male Infertility and Its Current Status in India

    Ramaswamy Suganthi


    Full Text Available Spermatogenesis is an essential stage in human male gamete development, which is regulated by many Y chromosome specific genes. Most of these genes are centred in a specific region located on the long arm of the human Y chromosome known as the azoospermia factor region (AZF. Deletion events are common in Y chromosome because of its peculiar structural organization. Astonishingly, among the several known genetic causes of male infertility, Y chromosomal microdeletions emerged as the most frequent structural chromosome anomaly associated with the quantitative reduction of sperm. The development of assisted reproductive techniques (ART like intra-cytoplasmic sperm injection (ICSI and testicular sperm extraction (TESE helps to bypass the natural barriers of fertilization, but it increases the concern about the transmission of genetic defects. Experimental evidence suggested that the men with Y chromosomal microdeletions vertically transmitted their deletion as well as related fertility disorders to their offspring via these ART techniques. In India, infertility is on alarming rise. ART centres have opened up in virtually every state but still most of the infertility centres in India do not choose to perform Y chromosomal microdeletion diagnosis because of some advanced theoretical reasons. Moreover, there is no consensus among the clinicians about the diagnosis and management of Y chromosomal microdeletion defects. The current review discusses thoroughly the role of Y chromosome microdeletion screening in the workup of male infertility, its significance as a diagnostic test, novel approaches for screening Y deletions and finally a systematic review on the current status of Y chromosome microdeletion deletion screening in India.

  18. International survey and surgeon’s preferences in diagnostic work-up towards treatment of anterior shoulder instability

    Weel, Hanneke; Tromp, Wouter; Krekel, Peter R; Randelli, Pietro; van den Bekerom, Michel P. J.; van Deurzen, Derek F. P.


    Purpose Recurrent anterior shoulder instability after surgical treatment can be caused by bony defects. Several diagnostic tools have been designed to measure the extent of these bony lesions. Currently, there is no consensus which measurement tool to use and decide which type of surgery is most appropriate. We therefore performed an evaluation of agreement in surgeons’ preference of diagnostic work-up and surgical treatment of anterior shoulder instability. Methods An international survey wa...

  19. Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods.

    Borgia, Alessandro; Zheng, Wenwei; Buholzer, Karin; Borgia, Madeleine B; Schüler, Anja; Hofmann, Hagen; Soranno, Andrea; Nettels, Daniel; Gast, Klaus; Grishaev, Alexander; Best, Robert B; Schuler, Benjamin


    There has been a long-standing controversy regarding the effect of chemical denaturants on the dimensions of unfolded and intrinsically disordered proteins: A wide range of experimental techniques suggest that polypeptide chains expand with increasing denaturant concentration, but several studies using small-angle X-ray scattering (SAXS) have reported no such increase of the radius of gyration (Rg). This inconsistency challenges our current understanding of the mechanism of chemical denaturants, which are widely employed to investigate protein folding and stability. Here, we use a combination of single-molecule Förster resonance energy transfer (FRET), SAXS, dynamic light scattering (DLS), and two-focus fluorescence correlation spectroscopy (2f-FCS) to characterize the denaturant dependence of the unfolded state of the spectrin domain R17 and the intrinsically disordered protein ACTR in two different denaturants. Standard analysis of the primary data clearly indicates an expansion of the unfolded state with increasing denaturant concentration irrespective of the protein, denaturant, or experimental method used. This is the first case in which SAXS and FRET have yielded even qualitatively consistent results regarding expansion in denaturant when applied to the same proteins. To more directly illustrate this self-consistency, we used both SAXS and FRET data in a Bayesian procedure to refine structural ensembles representative of the observed unfolded state. This analysis demonstrates that both of these experimental probes are compatible with a common ensemble of protein configurations for each denaturant concentration. Furthermore, the resulting ensembles reproduce the trend of increasing hydrodynamic radius with denaturant concentration obtained by 2f-FCS and DLS. We were thus able to reconcile the results from all four experimental techniques quantitatively, to obtain a comprehensive structural picture of denaturant-induced unfolded state expansion, and to

  20. Irreversible denaturation of maltodextrin glucosidase studied by differential scanning calorimetry, circular dichroism, and turbidity measurements.

    Goyal, Megha; Chaudhuri, Tapan K; Kuwajima, Kunihiro


    Thermal denaturation of Escherichia coli maltodextrin glucosidase was studied by differential scanning calorimetry, circular dichroism (230 nm), and UV-absorption measurements (340 nm), which were respectively used to monitor heat absorption, conformational unfolding, and the production of solution turbidity. The denaturation was irreversible, and the thermal transition recorded at scan rates of 0.5-1.5 K/min was significantly scan-rate dependent, indicating that the thermal denaturation was kinetically controlled. The absence of a protein-concentration effect on the thermal transition indicated that the denaturation was rate-limited by a mono-molecular process. From the analysis of the calorimetric thermograms, a one-step irreversible model well represented the thermal denaturation of the protein. The calorimetrically observed thermal transitions showed excellent coincidence with the turbidity transitions monitored by UV-absorption as well as with the unfolding transitions monitored by circular dichroism. The thermal denaturation of the protein was thus rate-limited by conformational unfolding, which was followed by a rapid irreversible formation of aggregates that produced the solution turbidity. It is thus important to note that the absence of the protein-concentration effect on the irreversible thermal denaturation does not necessarily means the absence of protein aggregation itself. The turbidity measurements together with differential scanning calorimetry in the irreversible thermal denaturation of the protein provided a very effective approach for understanding the mechanisms of the irreversible denaturation. The Arrhenius-equation parameters obtained from analysis of the thermal denaturation were compared with those of other proteins that have been reported to show the one-step irreversible thermal denaturation. Maltodextrin glucosidase had sufficiently high kinetic stability with a half-life of 68 days at a physiological temperature (37°C).

  1. Feasibility of dynamic MR-hysterosalpingography for the diagnostic work-up of infertile women

    Winter, Leopold; Gluecker, Thomas; Steinbrich, Wolfgang; Pegios, Wassilios (Dept. of Radiology, Univ. Hospital Basel (Switzerland)), e-mail:; Steimann, Sabine; De Geyter, Christian (Women' s Hospital, Univ. Hospital Basel (Switzerland)); Froehlich, Johannes M. (Guerbet AG, Zuerich (Switzerland))


    Background: Tubal disturbances often contribute to infertility. Conventional hysterosalpingography (HSG) is considered as standard in the assessment of the patency of the fallopian tubes, but requires ionizing radiation and is restricted to the imaging of endoluminal structures. Purpose: To evaluate dynamic magnetic resonance-HSG (dMR-HSG) in the diagnostic work-up in patients with infertility. Material and Methods: Thirty-seven consecutive infertile women underwent dMR-HSG: 20 ml of gadolinium-polyvidone solution (18.4 mM Dotarem 1:20 with polyvidone) were injected intracervically through a 5-Charriere balloon catheter while acquiring five consecutive flash-3D T1-weighted MR sequences with fat saturation. Two experienced readers assessed image quality and anatomic-pathologic correlations prospectively. The relevance of results was evaluated in the clinical context of each patient. Patient comfort was evaluated with a standardized questionnaire. Results: dMR-HSG was successfully completed in 33/37 patient with an average study time of 45 min. In 4 of 37 patients the catheter became dislodged during the examination, resulting in two complete diagnostic failures. Failure in another two patients was due to preliminary termination because of excessive pain and discomfort during the application of the contrast solution. The uterine cavity was completely visualized and bilateral fallopian tube patency was confirmed by dMR-HSG in 27 of 33 patients. Bilateral tubal occlusion was diagnosed in one of the remaining six patients and was confirmed by laparoscopy. Successful selective tubal catheterization was performed in one additional patient with unilateral and one patient with bilateral fallopian tube occlusion. In three cases, the catheter became dislocated at the end of the examination without demonstration of tubal patency. Since all three patients refused diagnostic laparoscopy and conventional HSG, possible bilateral occlusions of the fallopian tubes could not be

  2. Residual ordered structure in denatured proteins and the problem of protein folding.

    Basharov, Mahmud A


    Structural characteristics of numerous globular proteins in the denatured state have been reviewed using literature data. Recent more precise experiments show that in contrast to the conventional standpoint, proteins under strongly denaturing conditions do not unfold completely and adopt a random coil state, but contain significant residual ordered structure. These results cast doubt on the basis of the conventional approach representing the process of protein folding as a spontaneous transition of a polypeptide chain from the random coil state to the unique globular structure. The denaturation of proteins is explained in terms of the physical properties of proteins such as stability, conformational change, elasticity, irreversible denaturation, etc. The spontaneous renaturation of some denatured proteins most probably is merely the manifestation of the physical properties (e.g., the elasticity) of the proteins per se, caused by the residual structure present in the denatured state. The pieces of the ordered structure might be the centers of the initiation of renaturation, where the restoration of the initial native conformation of denatured proteins begins. Studies on the denaturation of proteins hardly clarify how the proteins fold into the native conformation during the successive residue-by-residue elongation of the polypeptide chain on the ribosome.

  3. The generation of denatured reactor plutonium by different options of the fuel cycle

    Broeders, C.H.M.; Kessler, G. [Inst. for Neutron Physics and Reactor Technology, Research Center Karlsruhe (Germany)


    Denatured (proliferation resistant) reactor plutonium can be generated in a number of different fuel cycle options. First denatured reactor plutonium can be obtained if, instead of low enriched U-235 PWR fuel, re-enriched U-235/U-236 from reprocessed uranium is used (fuel type A). Also the envisaged existing 2,500 t of reactor plutonium (being generated world wide up to the year 2010), mostly stored in intermediate fuel storage facilities at present, could be converted during a transition phase into denatured reactor plutonium by the options fuel type B and D. Denatured reactor plutonium could have the same safeguards standard as present low enriched (<20% U-235) LWR fuel. It could be incinerated by recycling once or twice in PWRs and subsequently by multi-recycling in FRs (CAPRA type or IFRs). Once denatured, such reactor plutonium could remain denatured during multiple recycling. In a PWR, e.g., denatured reactor plutonium could be destroyed at a rate of about 250 kg/GWey. While denatured reactor plutonium could be recycled and incinerated under relieved IAEA safeguards, neptunium would still have to be monitored by the IAEA in future for all cases in which considerable amounts of neptunium are produced. (orig.)

  4. In Vitro Reassembly of Tobacco Ribulose-1,5-bisphosphate Carboxylase/ Oxygenase from Fully Denatured Subunits

    Zhen-Hua YONG; Gen-Yun CHEN; Jiao-Nai SHI; Da-Quan XU


    It has been generally proved impossible to reassemble ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from fully denatured subunits in vitro in higher plant, because large subunit of fully denatured Rubisco is liable to precipitate when the denaturant is removed by common methods of direct dilution and one-step dialysis. In our experiment, the problem of precipitation was resolved by an improved gradual dialysis method, which gradually decreased the concentration of denaturant. However, fully denatured Rubisco subunits still could not be reassembled into holoenzyme using gradual dialysis unless chaperonin 60was added. The restored activity of reassembled Rubisco was approximately 8% of natural enzyme. The quantity of reassembled Rubisco increased greatly when heat shock protein 70 was present in the reassembly process. ATP and Mg2+ were unnecessary for in vitro reassembly of Rubisco, and Mg2+ inhibited the reassembly process. The reassembly was weakened when ATP, Mg2+ and K+ existed together in the reassembly process.

  5. Dynamic light scattering study of peanut agglutinin: Size, shape and urea denaturation

    Sagarika Dev; Avadhesha Surolia


    Peanut agglutinin (PNA) is a homotetrameric protein with a unique open quaternary structure. PNA shows non-two state profile in chaotrope induced denaturation. It passes through a monomeric molten globule like state before complete denaturation (Reddy et al 1999). This denaturation profile is associated with the change in hydrodynamic radius of the native protein. Though the molten globule-like state is monomeric in nature it expands in size due to partial denaturation. The size and shape of the native PNA as well as the change in hydrodynamic radius of the protein during denaturation has been studied by dynamic light scattering (DLS). The generation of two species is evident from the profile of hydrodynamic radii. This study also reveals the extent of compactness of the intermediate state.

  6. In situ observation of collagen thermal denaturation by second harmonic generation microscopy

    Liao, C.-S.; Zhuo, Z.-Y.; Yu, J.-Y.; Chao, P.-H. G.; Chu, S.-W.


    Collagen denaturation is of fundamental importance for clinical treatment. Conventionally, the denaturation process is quantified by the shrinkage of collagen fibers, but the underlying molecular origin has not been fully understood. Since second harmonic generation (SHG) is related to the molecular packing of the triple helix in collagen fibers, this nonlinear signal provides an insight of molecular dynamics during thermal denaturation. With the aid of SHG microscopy, we found a new step in collagen thermal denaturation process, de-crimp. During the de-crimp step, the characteristic crimp pattern of collagen fascicles disappeared due to the breakage of interconnecting bonds between collagen fibrils, while SHG intensity remained unchanged, suggesting the intactness of the triple helical molecules. At higher temperature, shrinkage is observed with strongly reduced SHG intensity, indicating denaturation at the molecular level.

  7. Refolding of detergent-denatured lysozyme using β-cyclodextrin-assisted ion exchange chromatography.

    Zhang, Li; Zhang, Qinming; Wang, Chaozhan


    Chromatography-based protein refolding is widely used. Detergent is increasingly used for protein solubilization from inclusion bodies. Therefore, it is necessary to develop a refolding method for detergent-denatured/solubilized proteins based on liquid chromatography. In the present work, sarkosyl-denatured/dithiothreitol-reduced lysozyme was used as a model, and a refolding method based on ion exchange chromatography, assisted by β-cyclodextrin, was developed for refolding detergent-denatured proteins. Many factors affecting the refolding, such as concentration of urea, concentration of β-cyclodextrin, pH and flow rate of mobile phases, were investigated to optimize the refolding conditions for sarkosyl-denatured lysozymes. The results showed that the sarkosyl-denatured lysozyme could be successfully refolded using β-cyclodextrin-assisted ion exchange chromatography.

  8. Dissociative mechanism of F-actin thermal denaturation.

    Mikhailova, V V; Kurganov, B I; Pivovarova, A V; Levitsky, D I


    We have applied differential scanning calorimetry to investigate thermal unfolding of F-actin. It has been shown that the thermal stability of F-actin strongly depends on ADP concentration. The transition temperature, T(m), increases with increasing ADP concentration up to 1 mM. The T(m) value also depends on the concentration of F-actin: it increases by almost 3 degrees C as the F-actin concentration is increased from 0.5 to 2.0 mg/ml. Similar dependence of the T(m) value on protein concentration was demonstrated for F-actin stabilized by phalloidin, whereas it was much less pronounced in the presence of AlF4(-). However, T(m) was independent of protein concentration in the case of monomeric G-actin. The results suggest that at least two reversible stages precede irreversible thermal denaturation of F-actin; one of them is dissociation of ADP from actin subunits, and another is dissociation of subunits from the ends of actin filaments. The model explains why unfolding of F-actin depends on both ADP and protein concentration.

  9. Role of 3.0 Tesla magnetic resonance hysterosalpingography in the diagnostic work-up of female infertility.

    Cipolla, Valentina; Guerrieri, Daniele; Pietrangeli, Daniela; Santucci, Domiziana; Argirò, Renato; de Felice, Carlo


    Imaging evaluation plays a crucial role in the diagnostic work-up of female infertility. In recent years, the possibility to evaluate tubal patency using 1.5 Tesla magnetic resonance (1.5T MR) has been studied. To assess the feasibility of 3.0 Tesla magnetic resonance (3.0T MR) hysterosalpingography and its role in the diagnostic work-up of female infertility and to evaluate if this fast "one-stop-shop" imaging approach should be proposed as a first-line examination. A total of 116 infertile women were enrolled in this prospective study; all underwent 3.0T MR hysterosalpingography. After standard imaging of the pelvis, tubal patency was assessed by acquiring 3D dynamic time-resolved T1-weighted (T1W) sequences during manual injection of 4-5 mL of contrast solution consisting of gadolinium and normal sterile saline. Images were evaluated by two radiologists with different experience in MR imaging (MRI). The examination was successfully completed in 96.5% of cases, failure rate was 3.5%. Dynamic sequences showed bilateral tubal patency in 64.3%, unilateral tubal patency in 25.9%, and bilateral tubal occlusion in 9.8%. Extratubal abnormalities were found in 69.9% of patients. Comprehensive analysis of morphological and dynamic sequences showed extratubal abnormalities in 43.1% of patients with bilateral tubal patency. 3.0T MR hysterosalpingography is a feasible, simple, fast, safe, and well-tolerated examination, which allows evaluation of tubal patency and other pelvic causes of female infertility in a single session, and it may thus represent a "one-stop-shop" solution in female infertility diagnostic work-up. © The Foundation Acta Radiologica 2015.

  10. Advanced radiological work-up as an adjunct to decision in early reconstructive surgery in brachial plexus injuries

    Björkman Anders


    Full Text Available Abstract Background As neurophysiologic tests may not reveal the extent of brachial plexus injury at the early stage, the role of early radiological work-up has become increasingly important. The aim of the study was to evaluate the concordance between the radiological and clinical findings with the intraoperative findings in adult patients with brachial plexus injuries. Methods Seven consecutive male patients (median age 33; range 15-61 with brachial plexus injuries, caused by motor cycle accidents in 5/7 patients, who underwent extensive radiological work-up with magnetic resonance imaging (MRI, computed tomography myelography (CT-M or both were included in this retrospective study. A total of 34 spinal nerve roots were evaluated by neuroradiologists at two different occasions. The degree of agreement between the radiological findings of every individual nerve root and the intraoperative findings was estimated by calculation of kappa coefficient (К-value. Using the operative findings as a gold standard, the accuracy, sensitivity, specificity, positive predictive value (PPV and negative predictive value (NPV of the clinical findings and the radiological findings were estimated. Results The diagnostic accuracy of radiological findings was 88% compared with 65% for the clinical findings. The concordance between the radiological findings and the intraoperative findings was substantial (К = 0.76 compared with only fair (К = 0.34 for the clinical findings. There were two false positive and two false negative radiological findings (sensitivity and PPV of 0.90; specificity and NPV of 0.87. Conclusions The advanced optimized radiological work-up used showed high reliability and substantial agreement with the intraoperative findings in adult patients with brachial plexus injury.

  11. Nonisothermal denaturation kinetics of human hair and the effects of oxidation.

    Wortmann, F-J; Popescu, C; Sendelbach, G


    Human hair as alpha-keratin fiber exhibits a complex morphology, which for the context of this investigation is considered as a filament/matrix-composite, comprising the intermediate filaments (IF) and a variety of amorphous protein components as matrix. Differential scanning calorimetry (DSC) under aqueous conditions was used to analyze the denaturation of the alpha-helical material in the IFs and to assess the changes imparted by repeated, oxidative bleaching processes. The DSC curves were submitted to kinetic analysis by applying the Friedman method and assuming first order kinetics. It was found that the course of the denaturation process remains largely unchanged through oxidation, despite the fact that pronounced decreases of denaturation temperature as well as of enthalpy occur. In parallel, the reaction rate constant at the denaturation temperature, k(TD), increases with repeated treatments, that is with cumulative chemical modification. However, this effect is in fact small compared to the overall change of k(T) through the denaturation process. This leads to conclude that once the temperature rise in combination with the chemical change has induced a suitable drop of the viscosity of the matrix around the IFs, denaturation of the remaining helical material occurs along a pathway that is largely independent of temperature and of the pretreatment history. This emphasizes the kinetic control of the matrix over the denaturation process of the helical segments in the filament/matrix composite. Copyright 2006 Wiley Periodicals, Inc.

  12. Studies on the refolding of the reduced-denatured insulin with size exclusion chromatography

    BAI Quan; KONG Yu; DONG Cuihua; GENG Xindu


    The refolding of the reduced-denatured insulin from bovine pancreas was investigated with the size exclusion chromatography (SEC). It was shown that the reduced-denatured insulin originally denatured with 7.0 mol·L-1 guanidine hydrochloride (GuHCl) or 8.0 mol·L-1 urea could not be refolded with a non-oxidized mobile phase. Although the oxidized and reduced glutathione (GSSG and GSH) were employed in the oxidized mobile phase, the reduced-denatured insulin still could not be renatured. However, in the presence of 2.0 mol·L-1 urea in the oxidized mobile phase employed, the reduced-denatured insulin can be refolded with SEC, and the aggregation of denatured insulin can be diminished by urea. In addition, the disulfide exchange of reduced-denatured insulin also can be accelerated with GSSG/GSH in the oxidized mobile phase. The three disulfide bridges of insulin were formed correctly and the reduced-unfolded insulin can be renatured completely. The results were further tested with reversed-phase liquid chromatography (RPLC) and hydrophobic interaction chromatography (HIC).

  13. The ageing and myasthenic thymus: a morphometric study validating a standard procedure in the histological workup of thymic specimens.

    Ströbel, Philipp; Moritz, Regina; Leite, Maria Isabel; Willcox, Nick; Chuang, Wen-Yu; Gold, Ralf; Nix, Wilfred; Schalke, Berthold; Kiefer, Reinhard; Müller-Hermelink, Hans-Konrad; Jaretzki Iii, Alfred; Newsom-Davis, John; Marx, Alexander


    The thymus is believed to play an important role in the pathogenesis of myasthenia gravis (MG). The 80% of MG patients with anti-acetylcholine receptor autoantibodies fall into three clinical subgroups: 1) thymoma; 2) early-onset MG (workup and reporting system used in this trial.

  14. Assessment of collagen crosslinking and denaturation for the design of regenerative scaffolds.

    Madaghiele, Marta; Calò, Emanuela; Salvatore, Luca; Bonfrate, Valentina; Pedone, Deborah; Frigione, Mariaenrica; Sannino, Alessandro


    Crosslinking and denaturation were two variables that deeply affected the performance of collagen-based scaffolds designed for tissue regeneration. If crosslinking enhances the mechanical properties and the enzymatic resistance of collagen, while masking or reducing the available cell binding sites, denaturation has very opposite effects, as it impairs the mechanical and the enzymatic stability of collagen, but increases the number of exposed cell adhesive domains. The quantification of both crosslinking and denaturation was thus fundamental to the design of collagen-based scaffolds for selected applications. The aim of this work was to investigate the extents of crosslinking and denaturation of collagen-based films upon dehydrothermal (DHT) treatment, that is, one of the most commonly employed methods for zero-length crosslinking that shows the unique ability to induce partial denaturation. Swelling measurements, differential scanning calorimetry, Fourier transform infrared spectroscopy, colorimetric assays for the quantification of primary amines, and mechanical tests were performed to analyze the effect of the DHT temperature on crosslinking and denaturation. In particular, chemically effective and elastically effective crosslink densities were evaluated. Both crosslinking and denaturation were found to increase with the DHT temperature, although according to different trends. The results also showed that DHT treatments performed at temperatures up to 120°C maintained the extent of denaturation under 25%. Coupling a mild DHT treatment with further crosslinking may thus be very useful not only to modulate the crosslink density, but also to induce a limited amount of denaturation, which shows potential to partially compensate the loss of cell binding sites caused by crosslinking.

  15. Urea-temperature phase diagrams capture the thermodynamics of denatured state expansion that accompany protein unfolding.

    Tischer, Alexander; Auton, Matthew


    We have analyzed the thermodynamic properties of the von Willebrand factor (VWF) A3 domain using urea-induced unfolding at variable temperature and thermal unfolding at variable urea concentrations to generate a phase diagram that quantitatively describes the equilibrium between native and denatured states. From this analysis, we were able to determine consistent thermodynamic parameters with various spectroscopic and calorimetric methods that define the urea-temperature parameter plane from cold denaturation to heat denaturation. Urea and thermal denaturation are experimentally reversible and independent of the thermal scan rate indicating that all transitions are at equilibrium and the van't Hoff and calorimetric enthalpies obtained from analysis of individual thermal transitions are equivalent demonstrating two-state character. Global analysis of the urea-temperature phase diagram results in a significantly higher enthalpy of unfolding than obtained from analysis of individual thermal transitions and significant cross correlations describing the urea dependence of ΔH0 and ΔCP0 that define a complex temperature dependence of the m-value. Circular dichroism (CD) spectroscopy illustrates a large increase in secondary structure content of the urea-denatured state as temperature increases and a loss of secondary structure in the thermally denatured state upon addition of urea. These structural changes in the denatured ensemble make up ∼40% of the total ellipticity change indicating a highly compact thermally denatured state. The difference between the thermodynamic parameters obtained from phase diagram analysis and those obtained from analysis of individual thermal transitions illustrates that phase diagrams capture both contributions to unfolding and denatured state expansion and by comparison are able to decipher these contributions.

  16. Relaxation rate for an ultrafast folding protein is independent of chemical denaturant concentration.

    Cellmer, Troy; Henry, Eric R; Kubelka, Jan; Hofrichter, James; Eaton, William A


    The connection between free-energy surfaces and chevron plots has been investigated in a laser temperature jump kinetic study of a small ultrafast folding protein, the 35-residue subdomain from the villin headpiece. Unlike all other proteins that have been studied so far, no measurable dependence of the unfolding/refolding relaxation rate on denaturant concentration was observed over a wide range of guanidinium chloride concentration. Analysis with a simple Ising-like theoretical model shows that this denaturant-invariant relaxation rate can be explained by a large movement of the major free energy barrier, together with a denaturant- and reaction coordinate-dependent diffusion coefficient.

  17. Heat Denaturation of Protein Structures and Chlorophyll States in PSII Membranes

    李冬海; 阮翔; 许强; 王可玢; 公衍道; 匡廷云; 赵南明


    Heat denaturation is an important technique in the study of the structure and function of photosynthetic proteins. Heat denaturation of photosystem II (PSII) membrane was studied using circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC) and oxygen electrode. Complete loss of oxygen-evolving activity of the PSII membrane was observed at temperatures below 45℃. The decrease of excitonic interaction between chlorophyll molecules occurred more rapidly than the change of the protein secondary structure of the PSII membrane at temperatures above 45℃. The results indicate that the protein secondary structure of the membrane proteins in PSII membranes is more stable than the excitonic interaction between chlorophyll molecules during heat denaturation.

  18. Protein denaturation and functional properties of Lenient Steam Injection heat treated whey protein concentrate

    Dickow, Jonatan Ahrens; Kaufmann, Niels; Wiking, Lars


    Whey protein concentrate (WPC) was heat treated by use of the novel heat treatment method of Lenient Steam Injection (LSI) to elucidate new functional properties in relation to heat-induced gelation of heat treated WPC. Denaturation was measured by both DSC and FPLC, and the results of the two...... methods were highly correlated. Temperatures of up to 90 °C were applicable using LSI, whereas only 68 °C could be reached by plate heat exchange before coagulation/fouling. Denaturation of whey proteins increased with increasing heat treatment temperature up to a degree of 30–35% denaturation at 90 °C...

  19. On the Effect of Sodium Chloride and Sodium Sulfate on Cold Denaturation.

    Andrea Pica

    Full Text Available Both sodium chloride and sodium sulfate are able to stabilize yeast frataxin, causing an overall increase of its thermodynamic stability curve, with a decrease in the cold denaturation temperature and an increase in the hot denaturation one. The influence of low concentrations of these two salts on yeast frataxin stability can be assessed by the application of a theoretical model based on scaled particle theory. First developed to figure out the mechanism underlying cold denaturation in water, this model is able to predict the stabilization of globular proteins provided by these two salts. The densities of the salt solutions and their temperature dependence play a fundamental role.

  20. The influence of applied heat treatments on whey protein denaturation

    Fetahagić Safet


    . Distribution of nitrogen matter from milk 8%+3%DWP heat treated at 85ºC/10 min, 90ºC/10 min and 95ºC/10 min to sera samples were 9.64%, 8.66% and 8.67%, respectively. Whey protein denaturation increased with increasing of the temperature of the applied heat treatment. Denaturation was the most significant for milk sample 11%.

  1. The Appropriate Use of Neuroimaging in the Diagnostic Work-Up of Dementia


    Background Diagnosis of dementia is challenging and requires both ruling out potentially treatable underlying causes and ruling in a diagnosis of dementia subtype to manage patients and suitably plan for the future. Objectives This analysis sought to determine the appropriate use of neuroimaging during the diagnostic work-up of dementia, including indications for neuroimaging and comparative accuracy of alternative technologies. Data Sources A literature search was performed using Ovid MEDLINE, Ovid MEDLINE In-Process and Other Non-Indexed Citations, Ovid Embase, the Wiley Cochrane Library, and the Centre for Reviews and Dissemination database, for studies published between 2000 and 2013. Review Methods Data on diagnostic accuracy and impact on clinical decision making were abstracted from included studies. Quality of evidence was assessed using GRADE. Results The search yielded 5,374 citations and 15 studies were included. Approximately 10% of dementia cases are potentially treatable, though less than 1% reverse partially or fully. Neither prediction rules nor clinical indications reliably select the subset of patients who will likely benefit from neuroimaging. Clinical utility is highest in ambiguous cases or where dementia may be mixed, and lowest for clinically diagnosed Alzheimer disease or clinically excluded vascular dementia. There is a lack of evidence that MRI is superior to CT in detecting a vascular component to dementia. Accuracy of structural imaging is moderate to high for discriminating different types of dementia. Limitations There was significant heterogeneity in estimates of diagnostic accuracy, which often prohibited a statistical summary of findings. The quality of data reported by studies prohibited calculation of likelihood ratios in the present analysis. No studies from primary care were found; thus, generalizability beyond tertiary care settings may be limited. Conclusions A diagnosis of reversible dementia is rare. Imaging has the most

  2. Protein folding by distributed computing and the denatured state ensemble.

    Marianayagam, Neelan J; Fawzi, Nicolas L; Head-Gordon, Teresa


    The distributed computing (DC) paradigm in conjunction with the folding@home (FH) client server has been used to study the folding kinetics of small peptides and proteins, giving excellent agreement with experimentally measured folding rates, although pathways sampled in these simulations are not always consistent with the folding mechanism. In this study, we use a coarse-grain model of protein L, whose two-state kinetics have been characterized in detail by using long-time equilibrium simulations, to rigorously test a FH protocol using approximately 10,000 short-time, uncoupled folding simulations starting from an extended state of the protein. We show that the FH results give non-Poisson distributions and early folding events that are unphysical, whereas longer folding events experience a correct barrier to folding but are not representative of the equilibrium folding ensemble. Using short-time, uncoupled folding simulations started from an equilibrated denatured state ensemble (DSE), we also do not get agreement with the equilibrium two-state kinetics because of overrepresented folding events arising from higher energy subpopulations in the DSE. The DC approach using uncoupled short trajectories can make contact with traditionally measured experimental rates and folding mechanism when starting from an equilibrated DSE, when the simulation time is long enough to sample the lowest energy states of the unfolded basin and the simulated free-energy surface is correct. However, the DC paradigm, together with faster time-resolved and single-molecule experiments, can also reveal the breakdown in the two-state approximation due to observation of folding events from higher energy subpopulations in the DSE.

  3. Impact of magnetic resonance urography on preoperative diagnostic workup in children affected by hydronephrosis: should IVU be replaced?

    Leppert, A; Nadalin, S; Schirg, E; Petersen, C; Kardorff, R; Galanski, M; Fuchs, J


    The aim of this study was to determine the role of magnetic resonance urography (MRU) in preoperative diagnostic workup of children with hydronephrosis in a prospective clinical study with comparison of MRU, standard diagnostic investigations, and intraoperative findings. Thirty-one children with hydronephrosis secondary to different causes underwent ultrasound scan (US), intravenous urography (IVU), micturation cysto-urethrography (MCU), isotope nephrography (ING) and MRU. For MRU the authors performed sagittal and coronal halve-Fourier SSFSE scans in a 1.5 Tesla MR system. T1- and T2-weighted sequences were used in axial orientation to improve morphologic information. In 24 patients, preoperative data were compared with intraoperative findings. Comparison of the different imaging modalities proved MRU to be able to provide more detailed information about the correct localization of stenoses along the urinary tract and the morphology of renal parenchyma. MRU showed the highest concordance of all imaging modalities with intraoperative findings. As a reliable investigation, MRU has the potentials to replace IVU in preoperative diagnostic workup of hydronephrosis in childhood. Copyright 2002, Elsevier Science (USA). All rights reserved.

  4. Limited diagnostic workup for deep vein thrombosis after major joint surgery: findings from a prospective, multicentre, cohort study.

    Monreal, Manuel; Peidro, Luis; Resines, Carlos; Garcés, Carlos; Fernández, José Luís; Garagorri, Eduardo; González, Juan Carlos


    While deep vein thrombosis (DVT) may be clinically suspected at several time points after major orthopedic surgery, clinical examination is often unreliable, and compression ultrasonography (CUS) screening at discharge is of limited value. A prospective cohort study was carried out in 1,033 consecutive patients who had undergone major hip or knee surgery, aimed to assess the accuracy of a strategy consisting of clinical investigation followed by CUS in the detection of proximal DVT before discharge. The circumferences of both legs were measured in all patients; those exhibiting >2 cm difference between them were considered to have suspected DVT, and underwent bilateral CUS. The same diagnostic workup was repeated on days 45 and 90 after surgery. Three patients developed pulmonary embolism (PE) during admission (one died). Five additional patients died before discharge. Routine clinical evaluation before discharge was done in 1,025 patients, and 105 (10%) had suspected DVT. CUS confirmed the diagnosis in 24 (2.3% of the overall series). After discharge, 59 patients had suspected DVT on day 45, 53 on day 90. DVT diagnosis was confirmed by CUS in 27 (26%). Three additional patients developed PE (1 fatal). This translates into a sensitivity of the routine examinations at discharge of 44%. A limited diagnostic workup for DVT before discharge has the capacity to identify 44% of those patients who will become symptomatic afterwards.

  5. The role of PET in initial work-up and evaluation after therapy in patients with carcinoma of unknown primary

    Ryoo, Baek Yeol; Kang, Yoon Koo


    The carcinoma of unknown primary occupied 5 - 10 % of all malignancies. It is heterogenous in origin and has poor prognosis. The indentification of primary site and definition of involved area are more helpful in the management. The efficacy of positron emission tomography (PET) with fluorine-18- fluorodeoxyglucose (F18-FDG) positron emission tomography (PET) with fluorine-18-fluorodeoxyglucose (F18-FDG) was evaluated in several tumors such as breast, pancreas and head and neck cancers. In carcinoma of unknown primary, it was reported that the concentration of FDG was increased in tumor tissues, and that PET with F18-FDG may be much helpful in identifying primary site and defining involved area. The authors evaluated the usefulness of PET with F18-FDG in initial work-up and in evaluation after radical therapy for the patients with carcinoma of unknown primary. The visual analysis of FDG-PET would be helpful in identifying primary site and defining involved area. In detecting recurrent of residual lesions, FDG-PET seemed to be less helpful than conventional diagnostic work-up. But more studies with larger number of cases and longer follow-up were required. The results of this study can be bases for the direction of future studies for the usefulness of PET in carcinoma of unknown primary.

  6. Denaturing gradient gel electrophoresis profiling of bacterial communities composition in Arabian Sea

    Singh, S.K.; Ramaiah, N.

    Denaturing gradient gel electrophoresis (DGGE) was used to elucidate spatial and temporal variations in bacterial community composition (BCC) from four locations along the central west coast of India. DNA extracts from 36 water samples collected...

  7. Streptokinase Recovery by Cross-Flow Microfiltration: Study of Enzyme Denaturation

    HERNANDEZ-PINZON, Inmaculada; MILLAN, Francisco; BAUTISTA, Juan


    ...% remained in the retentate. Immunological experiments using polyclonal antibodies against SK have demonstrated that SK activity loss during CFMF processes could be related to denaturation of SK, forming molecules of lower or no activity...

  8. Single molecule study of the DNA denaturation phase transition in the force-torsion space

    Salerno, D; Mai, I; Brogioli, D; Ziano, R; Cassina, V; Mantegazza, F


    We use the "magnetic tweezers" technique to reveal the structural transitions that DNA undergoes in the force-torsion space. In particular, we focus on regions corresponding to negative supercoiling. These regions are characterized by the formation of so-called denaturation bubbles, which have an essential role in the replication and transcription of DNA. We experimentally map the region of the force-torsion space where the denaturation takes place. We observe that large fluctuations in DNA extension occur at one of the boundaries of this region, i.e., when the formation of denaturation bubbles and of plectonemes are competing. To describe the experiments, we introduce a suitable extension of the classical model. The model correctly describes the position of the denaturation regions, the transition boundaries, and the measured values of the DNA extension fluctuations.

  9. How Chain Length and Charge Affect Surfactant Denaturation of Acyl Coenzyme A Binding Protein (ACBP)

    Andersen, Kell Kleiner; Otzen, Daniel


    Using intrinsic tryptophan fluorescence, equilibria and kinetics of unfolding of acyl coenzyme A binding protein (ACBP) have been investigated in sodium alkyl sulfate surfactants of different chain length (8-16 carbon atoms) and with different proportions of the nonionic surfactant dodecyl...... maltoside (DDM). The aim has been to determine how surfactant chain length and micellar charge affect the denaturation mechanism. ACBP denatures in two steps irrespective of surfactant chain length, but with increasing chain length, the potency of the denaturant rises more rapidly than the critical micelle...... constants increases linearly with denaturant concentration below the cmc but declines at higher concentrations. Both shortening chain length and decreasing micellar charge reduce the overall kinetics of unfolding and makes the dependence of unfolding rate constants on surfactant concentration more complex...

  10. Effect of mechanical denaturation on surface free energy of protein powders

    Mohammad, Mohammad Amin; Grimsey, Ian M.; Forbes, Robert T.; Blagbrough, Ian S; Barbara R. Conway


    Globular proteins are important both as therapeutic agents and excipients. However, their fragile native\\ud conformations can be denatured during pharmaceutical processing, which leads to modification of\\ud the surface energy of their powders and hence their performance. Lyophilized powders of hen eggwhite\\ud lysozyme and �-galactosidase from Aspergillus oryzae were used as models to study the effects\\ud of mechanical denaturation on the surface energies of basic and acidic protein powders, r...

  11. Avoiding adsorption of DNA to polypropylene tubes and denaturation of short DNA fragments

    Gaillard, Claire; Strauss, Francois


    Two problems can arise when working with small quantities of DNA in polypropylene tubes: first, significant amounts of DNA can become lost by sticking to the tube walls; second, short DNA fragments tend to denature when binding to polypropylene. In addition, DNA also tends to denature upon dehydration. We have found that a simple way to solve these problems is by using polyallomer tubes instead of polypropylene and by avoiding certain salts, such as sodium acetate, when drying DNA.

  12. Thermal denaturation behavior of collagen fibrils in wet and dry environment.

    Suwa, Yosuke; Nam, Kwangwoo; Ozeki, Kazuhide; Kimura, Tsuyoshi; Kishida, Akio; Masuzawa, Toru


    We have developed a new minimally invasive technique--integrated low-level energy adhesion technique (ILEAT)--which uses heat, pressure, and low-frequency vibrations for binding living tissues. Because the adhesion mechanism of the living tissues is not fully understood, we investigated the effect of thermal energy on the collagen structure in living tissues using ILEAT. To study the effect of thermal energy and heating time on the structure of the collagen fibril, samples were divided in two categories-wet and dry. Further, atomic force microscopy was used to analyze the collagen fibril structure before and after heating. Results showed that collagen fibrils in water denatured after 1 minute at temperatures higher than 80 °C, while partial denaturation was observed at temperatures of 80 °C and a heating time of 1 min. Furthermore, complete denaturation was achieved after 90 min, suggesting that the denaturation rate is temperature and time dependent. Moreover, the collagen fibrils in dry condition maintained their native structure even after being heated to 120 °C for 90 min in the absence of water, which specifically suppressed denaturation. However, partial denaturation of collagen fibrils could not be prevented, because this determines the adhesion between the collagen molecules, and stabilizes tissue bonding.

  13. Interim assessment of the denatured /sup 233/U fuel cycle: feasibility and nonproliferation characteristics

    Abbott, L.S.; Bartine, D.E.; Burns, T.J. (eds.)


    A fuel cycle that employs /sup 233/U denatured with /sup 238/U and mixed with thorium fertile material is examined with respect to its proliferation-resistance characteristics and its technical and economic feasibility. The rationale for considering the denatured /sup 233/U fuel cycle is presented, and the impact of the denatured fuel on the performance of Light-Water Reactors, Spectral-Shift-Controlled Reactors, Gas-Cooled Reactors, Heavy-Water Reactors, and Fast Breeder Reactors is discussed. The scope of the R, D and D programs to commercialize these reactors and their associated fuel cycles is also summarized and the resource requirements and economics of denatured /sup 233/U cycles are compared to those of the conventional Pu/U cycle. In addition, several nuclear power systems that employ denatured /sup 233/U fuel and are based on the energy center concept are evaluated. Under this concept, dispersed power reactors fueled with denatured or low-enriched uranium fuel are supported by secure energy centers in which sensitive activities of the nuclear cycle are performed. These activities include /sup 233/U production by Pu-fueled transmuters (thermal or fast reactors) and reprocessing. A summary chapter presents the most significant conclusions from the study and recommends areas for future work.

  14. Effects of protein and phosphate buffer concentrations on thermal denaturation of lysozyme analyzed by isoconversional method.

    Cao, X M; Tian, Y; Wang, Z Y; Liu, Y W; Wang, C X


    Thermal denaturation of lysozymes was studied as a function of protein concentration, phosphate buffer concentration, and scan rate using differential scanning calorimetry (DSC), which was then analyzed by the isoconversional method. The results showed that lysozyme thermal denaturation was only slightly affected by the protein concentration and scan rate. When the protein concentration and scan rate increased, the denaturation temperature (Tm) also increased accordingly. On the contrary, the Tm decreased with the increase of phosphate buffer concentration. The denaturation process of lysozymes was accelatated and the thermal stability was reduced with the increase of phosphate concentration. One part of degeneration process was not reversible where the aggregation occurred. The other part was reversible. The apparent activation energy (Ea) was computed by the isoconversional method. It decreased with the increase of the conversion ratio (α). The observed denaturation process could not be described by a simple reaction mechanism. It was not a process involving 2 standard reversible states, but a multi-step process. The new opportunities for investigating the kinetics process of protein denaturation can be supplied by this novel isoconversional method.

  15. Effect of mechanical denaturation on surface free energy of protein powders.

    Mohammad, Mohammad Amin; Grimsey, Ian M; Forbes, Robert T; Blagbrough, Ian S; Conway, Barbara R


    Globular proteins are important both as therapeutic agents and excipients. However, their fragile native conformations can be denatured during pharmaceutical processing, which leads to modification of the surface energy of their powders and hence their performance. Lyophilized powders of hen egg-white lysozyme and β-galactosidase from Aspergillus oryzae were used as models to study the effects of mechanical denaturation on the surface energies of basic and acidic protein powders, respectively. Their mechanical denaturation upon milling was confirmed by the absence of their thermal unfolding transition phases and by the changes in their secondary and tertiary structures. Inverse gas chromatography detected differences between both unprocessed protein powders and the changes induced by their mechanical denaturation. The surfaces of the acidic and basic protein powders were relatively basic, however the surface acidity of β-galactosidase was higher than that of lysozyme. Also, the surface of β-galactosidase powder had a higher dispersive energy compared to lysozyme. The mechanical denaturation decreased the dispersive energy and the basicity of the surfaces of both protein powders. The amino acid composition and molecular conformation of the proteins explained the surface energy data measured by inverse gas chromatography. The biological activity of mechanically denatured protein powders can either be reversible (lysozyme) or irreversible (β-galactosidase) upon hydration. Our surface data can be exploited to understand and predict the performance of protein powders within pharmaceutical dosage forms. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Biophysical analysis of phaseolin denaturation induced by urea, guanidinium chloride, pH, and temperature.

    Dyer, J M; Nelson, J W; Murai, N


    The structural stability of phaseolin was determined by using absorbance, circular dichroism (CD), fluorescence emission, and fluorescence polarization anisotropy to monitor denaturation induced by urea, guanidinium chloride (GdmCl), pH changes, increasing temperature, or a combination thereof. Initial results indicated that phaseolin remained folded to a similar extent in the presence or absence of 6.0 M urea or GdmCl at room temperature. In 6.0 M GdmCl, phaseolin denatures at approximately 65 degrees C when probed with absorbance, CD, and fluorescence polarization anisotropy. The transition occurs at lower temperatures by decreasing pH. Kinetic measurements of denaturation using CD indicated that the denaturation is slow below 55 degrees C and is associated with an activation energy of 52 kcal/mol in 6.0 M GdmCl. In addition, kinetic measurement using fluorescence emission indicated that the single tryptophan residue was sensitive to at least two steps of the denaturation process. The fluorescence emission appeared to reflect some other structural perturbation than protein denaturation, as fluorescence inflection occurred approximately 5 degrees C prior to the changes observed in absorbance, CD, and fluorescence polarization anisotropy.

  17. Calcium Binding and Disulfide Bonds Regulate the Stability of Secretagogin towards Thermal and Urea Denaturation

    Weiffert, Tanja; Ní Mhurchú, Niamh; O’Connell, David; Linse, Sara


    Secretagogin is a calcium-sensor protein with six EF-hands. It is widely expressed in neurons and neuro-endocrine cells of a broad range of vertebrates including mammals, fishes and amphibia. The protein plays a role in secretion and interacts with several vesicle-associated proteins. In this work, we have studied the contribution of calcium binding and disulfide-bond formation to the stability of the secretagogin structure towards thermal and urea denaturation. SDS-PAGE analysis of secretagogin in reducing and non-reducing conditions identified a tendency of the protein to form dimers in a redox-dependent manner. The denaturation of apo and Calcium-loaded secretagogin was studied by circular dichroism and fluorescence spectroscopy under conditions favoring monomer or dimer or a 1:1 monomer: dimer ratio. This analysis reveals significantly higher stability towards urea denaturation of Calcium-loaded secretagogin compared to the apo protein. The secondary and tertiary structure of the Calcium-loaded form is not completely denatured in the presence of 10 M urea. Reduced and Calcium-loaded secretagogin is found to refold reversibly after heating to 95°C, while both oxidized and reduced apo secretagogin is irreversibly denatured at this temperature. Thus, calcium binding greatly stabilizes the structure of secretagogin towards chemical and heat denaturation. PMID:27812162

  18. Tissue sealing device associated thermal spread: a comparison of histologic methods for detecting adventitial collagen denaturation

    Jones, Ryan M.; Grisez, Brian T.; Thomas, Aaron C.; Livengood, Ryan H.; Coad, James E.


    Thermal spread (thermal tissue damage) results from heat conduction through the tissues immediately adjacent to a hyperthermic tissue sealing device. The extent of such heat conduction can be assessed by the detection of adventitial collagen denaturation. Several histologic methods have been reported to measure adventitial collagen denaturation as a marker of thermal spread. This study compared hematoxylin and eosin staining, Gomori trichrome staining and loss of collagen birefringence for the detection of collagen denaturation. Twenty-eight ex vivo porcine carotid arteries were sealed with a commercially available, FDA-approved tissue sealing device. Following formalin fixation and paraffin embedding, two 5-micron tissue sections were hematoxylin and eosin and Gomori trichrome stained. The hematoxylin and eosin-stained section was evaluated by routine bright field microscopy and under polarized light. The trichromestained section was evaluated by routine bright field microscopy. Radial and midline adventitial collagen denaturation measurements were made for both the top and bottom jaw sides of each seal. The adventitial collagen denaturation lengths were determined using these three methods and statistically compared. The results showed that thermal spread, as represented by histologically detected collagen denaturation, is technique dependent. In this study, the trichrome staining method detected significantly less thermal spread than the hematoxylin and eosin staining and birefringence methods. Of the three methods, hematoxylin and eosin staining provided the most representative results for true thermal spread along the adjacent artery.

  19. Problem-Solving Test: Pyrosequencing

    Szeberenyi, Jozsef


    Terms to be familiar with before you start to solve the test: Maxam-Gilbert sequencing, Sanger sequencing, gel electrophoresis, DNA synthesis reaction, polymerase chain reaction, template, primer, DNA polymerase, deoxyribonucleoside triphosphates, orthophosphate, pyrophosphate, nucleoside monophosphates, luminescence, acid anhydride bond,…

  20. Denaturant-Dependent Conformational Changes in a [beta]-Trefoil Protein: Global and Residue-Specific Aspects of an Equilibrium Denaturation Process

    Latypov, Ramil F.; Liu, Dingjiang; Jacob, Jaby; Harvey, Timothy S.; Bondarenko, Pavel V.; Kleemann, Gerd R.; Brems, David N.; Raibekas, Andrei A.; (Amgen)


    Conformational properties of the folded and unfolded ensembles of human interleukin-1 receptor antagonist (IL-1ra) are strongly denaturant-dependent as evidenced by high-resolution two-dimensional nuclear magnetic resonance (NMR), limited proteolysis, and small-angle X-ray scattering (SAXS). The folded ensemble was characterized in detail in the presence of different urea concentrations by 1H-15N HSQC NMR. The {beta}-trefoil fold characteristic of native IL-1ra was preserved until the unfolding transition region beginning at 4 M urea. At the same time, a subset of native resonances disappeared gradually starting at low denaturant concentrations, indicating noncooperative changes in the folded state. Additional evidence of structural perturbations came from the chemical shift analysis, nonuniform and bell-shaped peak intensity profiles, and limited proteolysis. In particular, the following nearby regions of the tertiary structure became progressively destabilized with increasing urea concentrations: the {beta}-hairpin interface of trefoils 1 and 2 and the H2a-H2 helical region. These regions underwent small-scale perturbations within the native baseline region in the absence of populated molten globule-like states. Similar regions were affected by elevated temperatures known to induce irreversible aggregation of IL-1ra. Further evidence of structural transitions invoking near-native conformations came from an optical spectroscopy analysis of its single-tryptophan variant W17A. The increase in the radius of gyration was associated with a single equilibrium unfolding transition in the case of two different denaturants, urea and guanidine hydrochloride (GuHCl). However, the compactness of urea- and GuHCl-unfolded molecules was comparable only at high denaturant concentrations and deviated under less denaturing conditions. Our results identified the role of conformational flexibility in IL-1ra aggregation and shed light on the nature of structural transitions within the

  1. Pragmatic approach to the clinical work-up of patients with putative allergic disease to metallic orthopaedic implants before and after surgery

    Thyssen, J P; Menné, T; Schalock, P C;


    on in the work-up of patients with putative allergic complications following surgery. Few studies have investigated whether subjects with metal contact allergy have increased risk of developing complications following orthopaedic implant insertion. Metal allergy might in a minority increase the risk......, and as surgeons may refer patients with complications following total joint arthroplasty for diagnostic work-up, there is a continuous need for updated guidelines. This review presents published evidence for patch testing prior to surgery and proposes tentative diagnostic criteria which clinicians can rely...... testing prior to surgery unless the patient has already had implant surgery with complications suspected to be allergic or has a history of clinical metal intolerance of sufficient magnitude to be of concern to the patient or a health provider. The clinical work-up of a patient suspected of having...

  2. Correlated parameter fit of arrhenius model for thermal denaturation of proteins and cells.

    Qin, Zhenpeng; Balasubramanian, Saravana Kumar; Wolkers, Willem F; Pearce, John A; Bischof, John C


    Thermal denaturation of proteins is critical to cell injury, food science and other biomaterial processing. For example protein denaturation correlates strongly with cell death by heating, and is increasingly of interest in focal thermal therapies of cancer and other diseases at temperatures which often exceed 50 °C. The Arrhenius model is a simple yet widely used model for both protein denaturation and cell injury. To establish the utility of the Arrhenius model for protein denaturation at 50 °C and above its sensitivities to the kinetic parameters (activation energy E a and frequency factor A) were carefully examined. We propose a simplified correlated parameter fit to the Arrhenius model by treating E a, as an independent fitting parameter and allowing A to follow dependently. The utility of the correlated parameter fit is demonstrated on thermal denaturation of proteins and cells from the literature as a validation, and new experimental measurements in our lab using FTIR spectroscopy to demonstrate broad applicability of this method. Finally, we demonstrate that the end-temperature within which the denaturation is measured is important and changes the kinetics. Specifically, higher E a and A parameters were found at low end-temperature (50 °C) and reduce as end-temperatures increase to 70 °C. This trend is consistent with Arrhenius parameters for cell injury in the literature that are significantly higher for clonogenics (45-50 °C) vs. membrane dye assays (60-70 °C). Future opportunities to monitor cell injury by spectroscopic measurement of protein denaturation are discussed.

  3. Development of novel short-term heating angioplasty: thermal denaturation dynamics of collagen in artery wall

    Shimazaki, N.; Tokunaga, H.; Katou, Y.; Hayashi, T.; Arai, T.


    We have studied to develop the new thermal angioplasty methodology, photo-thermo dynamic balloon angioplasty (PTDBA), which provides artery dilatation with short-term (collagen in artery media may be the important factor to attain sufficient artery dilatation for the PTDBA. In order to predict the optimum heating condition i.e. the balloon temperature and heating duration, we investigated the thermal denaturation dynamics of artery collagen in ex vivo. The extracted fresh porcine carotid artery was used. The temperature-dependent light scattering property and mechanical property of the artery specimen were simultaneously measured during artery temperature rising by specially made setup to assess the denaturation of arterial collagen. The change rate of the backscattered light intensity from the artery specimen; I(T)/I0 with 633nm was measured to evaluate the artery scattering property change with the thermal denaturation. The artery specimen was heated from 25°C to 80°C with constant temperature rising rate of 3°C/min. The measured I(T)/I0 was suddenly increased over 48°C. This boundary temperature might be the initiation temperature of the arterial collagen denaturation. We defined the variation of the I(T)/I0 as the collagen denaturation ratio, and calculated the reactive enthalpy by the chemical equilibrium theory. Since the calculated enthalpy was similar to the enthalpy in literature report, the variety of I(T)/I0 during the temperature rising might be attributed to the collagen conformational change due to the denaturation. In terms of the artery internal force measurement, the artery force was decreased with increasing of the artery temperature up to 65°C (i.e. softening), and increased over 65°C (i.e. shrinkage). We confirmed that the changes of the backscattered light (at 633nm in wavelength) from the artery might represent the artery collagen thermal denaturation degree.

  4. Effect of high-pressure treatment on denaturation of bovine lactoferrin and lactoperoxidase.

    Mazri, C; Sánchez, L; Ramos, S J; Calvo, M; Pérez, M D


    Lactoferrin and lactoperoxidase are whey proteins with biological properties that may provide health benefits to consumers. These properties are vulnerable to potentially denaturing conditions during processing. High-pressure treatment is an appealing alternative to the traditional heat processing of foods because it exerts an antimicrobial effect without changing the sensory and nutritional quality of foods. In this work, the effect of high-pressure treatment on the denaturation of lactoferrin and lactoperoxidase present in skim milk and whey, and as isolated proteins in buffer, was studied over a pressure range of 450 to 700 MPa at 20°C. Denaturation of lactoferrin was measured by the loss of reactivity with their specific antibodies using a sandwich ELISA. Denaturation of lactoperoxidase was determined by measuring the loss of enzymatic activity using a spectrophotometric technique. No substantial inactivation of lactoperoxidase was observed in any treatment assayed. The concentration of the residual immunoreactive lactoferrin after each pressure treatment was determined, and the data were subjected to kinetic analysis to obtain D and Z values. Denaturation of lactoferrin increased with pressure and holding time, and D values were lower when lactoferrin was treated in whey than in milk, and lower in both whey and milk than in phosphate buffer. Thus, protein is denatured more slowly in buffer and in milk than in whey. Denaturation of lactoferrin in the 3 media was found to follow a reaction order of n=1.5. Volumes of activation of about -34.77, -24.35, and -24.09 mL/mol were obtained for lactoferrin treated in skim milk, whey, and buffer, respectively, indicating a decrease in protein volume under pressure.

  5. MicroRNA-23b Inhibits the Proliferation and Migration of Heat-Denatured Fibroblasts by Targeting Smad3.

    Xipeng Zhang

    Full Text Available Skin grafting with the preservation of denatured dermis is a novel strategy for the treatment of burn-injured skin. Denatured dermis has the ability to restore to the morphology and function of normal skin, but the underlying molecular mechanism is elusive. MicroRNAs (miRNA are small noncoding RNAs and regulate normal physiology as well as disease development. In this study, we assessed the potential role of miRNA-23b (miR-23b in the regulation of cell proliferation and migration of heat-denatured fibroblasts and identified the underlying mechanism.The expression of miR-23b in denatured dermis and heat-denatured fibroblasts was detected by quantitative real-time polymerase chain reaction (RT-PCR. The effects of miR-23b on cell proliferation and migration of heat-denatured fibroblasts were assessed by transient transfection of miR-23b mimics and inhibitor. The target gene of miR-23b and the downstream pathway were further investigated.miR-23b was downregulated in denatured dermis and heat-denatured fibroblasts. Downregulation of miR-23b dramatically promoted the proliferation and migration of heat-denatured fibroblasts. Subsequent analyses demonstrated that Smad3 was a direct and functional target of miR-23b in heat-denatured fibroblasts, which was validated by the dual luciferase reporter assay. Moreover, immunohistochemistry analysis showed that denatured dermis from rats displayed enhanced staining of Smad3. In addition, miR-23b modulated denatured dermis by activating the Notch1 and TGF-β signaling pathways.Our findings suggest that downregulation of miR-23b contributes to the recovery of denatured dermis, which may be valuable for treatment of skin burns.

  6. Incidence of malignancy in patients with pleural effusion referred for workup by pulmonologists: Diagnostic yield of thoracentesis, and use of other investigational procedures

    Lindegaard, Dennis V.; Reuter, Simon; Laursen, Christian B.


    Introduction Pleural effusion (PE) is a common condition. Malignancy accounts for lt;25% in general populations. The proportion is unknown in patients referred to pulmonologists for workup.Finding malignant cells in PE by thoracentesis suggests metastatic and incurable disease making further tests...... and staging procedures superfluous.Objectives In patients with PE referred to pulmonologists for workup, we wanted to ascertain A) risk of malignancy; B)diagnostic yield of a single thoracentesis; C)time from PE to diagnosis; and D)impact of finding malignant cells in PE on use of additional tests...... immunohistochemistry, or same-day investigational procedures might be underlying causes for this discrepancy....

  7. Alcohol-induced structural transitions in the acid-denatured Bacillus licheniformis α-amylase

    Adyani Azizah Abd Halim


    Full Text Available Alcohol-induced structural changes in the acid-denatured Bacillus licheniformis α-amylase (BLA at pH 2.0 were studied by far-ultra violet circular dichroism, intrinsic, three-dimensional and 8-anilino-1-naphthalene sulfonic acid (ANS fluorescence, acrylamide quenching and thermal denaturation. All the alcohols used in this study produced partial refolding in the acid-denatured BLA as evident from the increased mean residue ellipticity at 222 nm, increased intrinsic fluorescence and decreased ANS fluorescence. The order of effectiveness of these alcohols to induce a partially folded state of BLA was found to be: 2,2,2-trifluoroethanol/tert-butanol > 1-propanol/2-propanol > 2-chloroethanol > ethanol > methanol. Three-dimensional fluorescence and acrylamide quenching results obtained in the presence of 5.5 M tert-butanol also suggested formation of a partially folded state in the acid-denatured BLA. However, 5.5 M tert-butanol-induced state of BLA showed a non-cooperative thermal transition. All these results suggested formation of a partially folded state of the acid-denatured BLA in the presence of these alcohols. Furthermore, their effectiveness was found to be guided by their chain length, position of methyl groups and presence of the substituents.

  8. Drying and denaturation characteristics of whey protein isolate in the presence of lactose and trehalose.

    Haque, M Amdadul; Chen, Jie; Aldred, Peter; Adhikari, Benu


    The denaturation kinetics of whey protein isolate (WPI), in the presence and absence of lactose and trehalose, was quantified in a convective air-drying environment. Single droplets of WPI, WPI-lactose and WPI-trehalose were dried in conditioned air (2.5% RH, 0.5m/s air velocity) at two temperatures (65°C and 80°C) for 500s. The initial solid concentration of these solutions was 10% (w/v) in all the samples. Approximately 68% of WPI was denatured when it was dried in the absence of sugars. Addition of 20% trehalose prevented the irreversible denaturation of WPI at both temperatures. Thirty percent lactose was required to prevent denaturation of WPI at 65°C and the same amount of lactose protected only 70% of WPI from denaturation at 80°C. The secondary structures of WPI were found to be altered by the drying-induced stresses, even in the presence of 20% trehalose and 30% lactose.

  9. Denaturation of milk proteins and their influence on the yield of fresh cheese

    Ana Mejía-López


    Full Text Available To determine the denaturation of milk proteins by the effects of heat treatment on pasteurization and to establish their influence on the yield of the fresh cheese manufactured, 20 laboratory- scale controlled trials and 40 plant productions were made. Crude and treated milk was used at 65 ° C for 30 minutes, 72 ° C for 15 seconds and boiled for 2 seconds, and the protein was quantified in milk to calculate percent denaturation. In the cheese the moisture content was determined and the amount of cheese obtained was quantified. The data were processed by Tukey's mean analysis (p> 0.05. The results at the laboratory level showed that the increase in temperature caused higher denaturation of the proteins, a higher yield and an increase in moisture in the cheese compared to that obtained with raw milk. However, statistically the results showed that the heat treatment does influence the denaturation of the proteins but not the performance of the cheese. The results obtained in the factory investigation revealed that at 65 and 72 ° C the yield decreases relative to the production with raw milk, but statistically does not present significant differences in the yield, concluding that the pasteurization at different temperatures denature the protein But does not influence the performance of fresh processed cheese.

  10. A versatile protein microarray platform enabling antibody profiling against denatured proteins.

    Wang, Jie; Barker, Kristi; Steel, Jason; Park, Jin; Saul, Justin; Festa, Fernanda; Wallstrom, Garrick; Yu, Xiaobo; Bian, Xiaofang; Anderson, Karen S; Figueroa, Jonine D; LaBaer, Joshua; Qiu, Ji


    We aim to develop a protein microarray platform capable of presenting both natural and denatured forms of proteins for antibody biomarker discovery. We will further optimize plasma screening protocols to improve detection. We developed a new covalent capture protein microarray chemistry using HaloTag fusion proteins and ligand. To enhance protein yield, we used HeLa cell lysate as an in vitro transcription translation (IVTT) system. Escherichia coli lysates were added to the plasma blocking buffer to reduce nonspecific background. These protein microarrays were probed with plasma samples and autoantibody responses were quantified and compared with or without denaturing buffer treatment. We demonstrated that protein microarrays using the covalent attachment chemistry endured denaturing conditions. Blocking with E. coli lysates greatly reduced the background signals and expression with IVTT based on HeLa cell lysates significantly improved the antibody signals on protein microarrays probed with plasma samples. Plasma samples probed on denatured protein arrays produced autoantibody profiles distinct from those probed on natively displayed proteins. This versatile protein microarray platform allows the display of both natural and denatured proteins, offers a new dimension to search for disease-specific antibodies, broadens the repertoire of potential biomarkers, and will potentially yield clinical diagnostics with greater performance. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Comparison of chemical and thermal protein denaturation by combination of computational and experimental approaches. II

    Wang, Qian; Christiansen, Alexander; Samiotakis, Antonios; Wittung-Stafshede, Pernilla; Cheung, Margaret S.


    Chemical and thermal denaturation methods have been widely used to investigate folding processes of proteins in vitro. However, a molecular understanding of the relationship between these two perturbation methods is lacking. Here, we combined computational and experimental approaches to investigate denaturing effects on three structurally different proteins. We derived a linear relationship between thermal denaturation at temperature Tb and chemical denaturation at another temperature Tu using the stability change of a protein (ΔG). For this, we related the dependence of ΔG on temperature, in the Gibbs-Helmholtz equation, to that of ΔG on urea concentration in the linear extrapolation method, assuming that there is a temperature pair from the urea (Tu) and the aqueous (Tb) ensembles that produces the same protein structures. We tested this relationship on apoazurin, cytochrome c, and apoflavodoxin using coarse-grained molecular simulations. We found a linear correlation between the temperature for a particular structural ensemble in the absence of urea, Tb, and the temperature of the same structural ensemble at a specific urea concentration, Tu. The in silico results agreed with in vitro far-UV circular dichroism data on apoazurin and cytochrome c. We conclude that chemical and thermal unfolding processes correlate in terms of thermodynamics and structural ensembles at most conditions; however, deviations were found at high concentrations of denaturant.

  12. Polar or apolar--the role of polarity for urea-induced protein denaturation.

    Martin C Stumpe


    Full Text Available Urea-induced protein denaturation is widely used to study protein folding and stability; however, the molecular mechanism and driving forces of this process are not yet fully understood. In particular, it is unclear whether either hydrophobic or polar interactions between urea molecules and residues at the protein surface drive denaturation. To address this question, here, many molecular dynamics simulations totalling ca. 7 micros of the CI2 protein in aqueous solution served to perform a computational thought experiment, in which we varied the polarity of urea. For apolar driving forces, hypopolar urea should show increased denaturation power; for polar driving forces, hyperpolar urea should be the stronger denaturant. Indeed, protein unfolding was observed in all simulations with decreased urea polarity. Hyperpolar urea, in contrast, turned out to stabilize the native state. Moreover, the differential interaction preferences between urea and the 20 amino acids turned out to be enhanced for hypopolar urea and suppressed (or even inverted for hyperpolar urea. These results strongly suggest that apolar urea-protein interactions, and not polar interactions, are the dominant driving force for denaturation. Further, the observed interactions provide a detailed picture of the underlying molecular driving forces. Our simulations finally allowed characterization of CI2 unfolding pathways. Unfolding proceeds sequentially with alternating loss of secondary or tertiary structure. After the transition state, unfolding pathways show large structural heterogeneity.

  13. Experimental and Modelling Study of the Denaturation of Milk Protein by Heat Treatment

    Qian, Fang; Sun, Jiayue; Cao, Di; Tuo, Yanfeng; Jiang, Shujuan; Mu, Guangqing


    Heat treatment of milk aims to inhibit the growth of microbes, extend the shelf-life of products and improve the quality of the products. Heat treatment also leads to denaturation of whey protein and the formation of whey protein-casein polymer, which has negative effects on milk product. Hence the milk heat treatment conditions should be controlled in milk processing. In this study, the denaturation degree of whey protein and the combination degree of whey protein and casein when undergoing heat treatment were also determined by using the Native-PAGE and SDS-PAGE analysis. The results showed that the denaturation degree of whey protein and the combination degree of whey protein with casein extended with the increase of the heat-treated temperature and time. The effects of the heat-treated temperature and heat-treated time on the denaturation degree of whey protein and on the combination degree of whey protein and casein were well described using the quadratic regression equation. The analysis strategy used in this study reveals an intuitive and effective measure of the denaturation degree of whey protein, and the changes of milk protein under different heat treatment conditions efficiently and accurately in the dairy industry. It can be of great significance for dairy product proteins following processing treatments applied for dairy product manufacturing. PMID:28316470

  14. Antibodies with specificity for native and denatured forms of ovalbumin differ in reactivity between enzyme-linked immunosorbent assays

    Holm, B.E.; Bergmann, A.C.; Hansen, Paul Robert


    In this study, polyclonal and monoclonal antibodies to native and denatured chicken ovalbumin (OVA) were produced to compare their dependency on continuous and three-dimensional epitopes. These antibodies were characterized with respect to reactivity to native and denatured OVA by enzyme...... to native OVA reacted strongly with native and denatured OVA in both assays, but did not react with the overlapping peptides. Polyclonal antibodies to denatured OVA reacted strongly with both OVA forms and with several of the overlapping peptides. Monoclonal antibodies to native OVA reacted preferentially...... with three-dimensional epitopes on native OVA and not with denatured OVA. Monoclonal antibodies to denatured OVA showed reactivity to both OVA forms. Two of these monoclonal antibodies, HYB 94-06 and 94-07, showed reactivity to overlapping peptides and their epitopes were identified as flexible structures...

  15. Inhibition of thermal induced protein denaturation of extract/fractions of Withania somnifera and isolated withanolides.

    Khan, Murad Ali; Khan, Haroon; Rauf, Abdul; Ben Hadda, Taibi


    This study describes the in vitro inhibition of protein denaturation of extract/fractions of Withania somnifera and isolated withanolides including 20β hydroxy-1-oxo(22R)-witha-2,5,24 trienolide (1), (20R,22R-14α,20α)-dihydroxy-1-oxowitha-2,5,16,24 tetraenolide (2). The results showed that the extract/fractions of the plant evoked profound inhibitory effect on thermal-induced protein denaturation. The chloroform fraction caused the most dominant attenuation of 68% at 500 μg/mL. The bioactivity-guided isolation from chloroform fraction led to the isolation of compounds 1 and 2 that showed profound protein inhibition with 78.05% and 80.43% effect at 500 μg/mL and thus strongly complimented the activity of extract/fractions. In conclusion, extract/fractions of W. somnifera possessed strong inhibition of protein denaturation that can be attributed to these isolated withanolides.

  16. Nonsurgical transurethral radiofrequency collagen denaturation: results at three years after treatment.

    Elser, Denise M; Mitchell, Gretchen K; Miklos, John R; Nickell, Kevin G; Cline, Kevin; Winkler, Harvey; Wells, W Glen


    Objective. To assess treatment efficacy and quality of life in women with stress urinary incontinence 3 years after treatment with nonsurgical transurethral radiofrequency collagen denaturation. Methods. This prospective study included 139 women with stress urinary incontinence due to bladder outlet hypermobility. Radiofrequency collagen denaturation was performed using local anesthesia in an office setting. Assessments included incontinence quality of life (I-QOL) and urogenital distress inventory (UDI-6) instruments. Results. In total, 139 women were enrolled and 136 women were treated (mean age, 47 years). At 36 months, intent-to-treat analysis (n = 139) revealed significant improvements in quality of life. Mean I-QOL score improved 17 points from baseline (P = .0004), while mean UDI-6 score improved (decreased) 19 points (P = .0005). Conclusions. Transurethral collagen denaturation is a low-risk, office-based procedure that results in durable quality-of-life improvements in a significant proportion of women for as long as 3 years.

  17. Stabilizing Effect of Various Polyols on the Native and the Denatured States of Glucoamylase

    Mohammed Suleiman Zaroog


    Full Text Available Different spectral probes were employed to study the stabilizing effect of various polyols, such as, ethylene glycol (EG, glycerol (GLY, glucose (GLC and trehalose (TRE on the native (N, the acid-denatured (AD and the thermal-denatured (TD states of Aspergillus niger glucoamylase (GA. Polyols induced both secondary and tertiary structural changes in the AD state of enzyme as reflected from altered circular dichroism (CD, tryptophan (Trp, and 1-anilinonaphthalene-8-sulfonic acid (ANS fluorescence characteristics. Thermodynamic analysis of the thermal denaturation curve of native GA suggested significant increase in enzyme stability in the presence of GLC, TRE, and GLY (in decreasing order while EG destabilized it. Furthermore, CD and fluorescence characteristics of the TD state at 71°C in the presence of polyols showed greater effectiveness of both GLC and TRE in inducing native-like secondary and tertiary structures compared to GLY and EG.

  18. Gel electrophoresis of DNA partially denatured at the ends: what are the dominant conformations?

    Sean, David; Slater, Gary W


    Gel electrophoresis of a partially denatured dsDNA fragment is studied using Langevin Dynamics computer simulations. For simplicity, the denatured ssDNA sections are placed at the ends of the fragment in a symmetrical fashion. A squid-like conformation is found to sometimes cause the fragment to completely block in the gel. In fact, this conformation is the principal cause of the steep reduction in mobility observed in the simulations. As the field is increased, it is found that the occurrence of this conformation dominates the migration dynamics. Although the squid conformation seems to be more stable at high fields, the field can eventually force the fragments to thread through the gel pores regardless. We qualitatively explore the behavior of this squid-like conformation across a range of fields and degrees of denaturation, and we discuss the relevance of our findings for TGGE. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


    Heriberto Molina-Pérez


    Full Text Available This work presents an approach to match the foaling thickness of dairy food and the concentration of denatured β-lg (β-lactoglobuline by a mathematical model. This includes, on the one hand, the dynamic simulation of fouling and, on the other hand, the generation of denatured β-lg under a kinetic model. In both cases a transient energy balance is developed, including the rigorous calculation of the global coefficient, and the properties by the Choi-Okos model. The solution was obtained by a fourth order Runge-Kutta written in Excel’s Macro language Visual Basic. The equivalence concluded with a model obtained by a non-linear multiple regression that relates the concentration of denatured β-lg and the foaling thickness. This methodology is applicable to analysis equipment cleaning in which kinetic cleaning has equality by reduction of the foaling thickness.

  20. Statistical mechanics of the denatured state of a protein using replica-averaged metadynamics.

    Camilloni, Carlo; Vendruscolo, Michele


    The characterization of denatured states of proteins is challenging because the lack of permanent structure in these states makes it difficult to apply to them standard methods of structural biology. In this work we use all-atom replica-averaged metadynamics (RAM) simulations with NMR chemical shift restraints to determine an ensemble of structures representing an acid-denatured state of the 86-residue protein ACBP. This approach has enabled us to reach convergence in the free energy landscape calculations, obtaining an ensemble of structures in relatively accurate agreement with independent experimental data used for validation. By observing at atomistic resolution the transient formation of native and non-native structures in this acid-denatured state of ACBP, we rationalize the effects of single-point mutations on the folding rate, stability, and transition-state structures of this protein, thus characterizing the role of the unfolded state in determining the folding process.

  1. Nonsurgical Transurethral Radiofrequency Collagen Denaturation: Results at Three Years after Treatment

    Denise M. Elser


    Full Text Available Objective. To assess treatment efficacy and quality of life in women with stress urinary incontinence 3 years after treatment with nonsurgical transurethral radiofrequency collagen denaturation. Methods. This prospective study included 139 women with stress urinary incontinence due to bladder outlet hypermobility. Radiofrequency collagen denaturation was performed using local anesthesia in an office setting. Assessments included incontinence quality of life (I-QOL and urogenital distress inventory (UDI-6 instruments. Results. In total, 139 women were enrolled and 136 women were treated (mean age, 47 years. At 36 months, intent-to-treat analysis (n=139 revealed significant improvements in quality of life. Mean I-QOL score improved 17 points from baseline (P=.0004, while mean UDI-6 score improved (decreased 19 points (P=.0005. Conclusions. Transurethral collagen denaturation is a low-risk, office-based procedure that results in durable quality-of-life improvements in a significant proportion of women for as long as 3 years.

  2. Effects of the protein denaturant guanidinium chloride on aqueous hydrophobic contact-pair interactions.

    Macdonald, Ryan D; Khajehpour, Mazdak


    Guanidinium chloride (GdmCl) is one of the most common protein denaturants. Although GdmCl is well known in the field of protein folding, the mechanism by which it denatures proteins is not well understood. In fact, there are few studies looking at its effects on hydrophobic interactions. In this work the effect of GdmCl on hydrophobic interactions has been studied by observing how the denaturant influences model systems of phenyl and alkyl hydrophobic contact pairs. Contact pair formation is monitored through the use of fluorescence spectroscopy, i.e., measuring the intrinsic phenol fluorescence being quenched by carboxylate ions. Hydrophobic interactions are isolated from other interactions through a previously developed methodology. The results show that GdmCl does not significantly affect hydrophobic interactions between small moieties such as methyl groups and phenol; while on the other hand, the interaction of larger hydrophobes such as hexyl and heptyl groups with phenol is significantly destabilized.

  3. Comparison of a gene expression profiling strategy to standard clinical work-up for determination of tumour origin in cancer of unknown primary (CUP)

    Ades, Felipe; de Azambuja, Evandro; Daugaard, Gedske


    CupPrint® is a genomic signature able to identify 47 different cancer types. The aim of our study was to compare the accuracy of this genomic signature to that of a full clinical work-up in diagnosing the primary tumour site. Patients with newly diagnosed, untreated metastatic tumours were eligib...

  4. Implementation of flow cytometry in the diagnostic work-up of myelodysplastic syndromes in a multicenter approach : Report from the Dutch Working Party on Flow Cytometry in MDS

    Westers, Theresia M.; van der Velden, Vincent H. J.; Alhan, Canan; Bekkema, Roelof; Bijkerk, Andre; Brooimans, Rik A.; Cali, Claudia; Drager, Angelika M.; de Haas, Valerie; Homburg, Christa; Kuiper-Kramer, P. (Ellen) A.; Leenders, Marije; Lommerse, Ingrid; Marvelde, Jeroen G. te; van der Molen-Sinke, Joke K.; Moshaver, Bijan; Mulder, Andre B.; Preijers, Frank W. M. B.; Schindhelm, Roger K.; van der Sluijs, Alita; van Wering, Elisabeth R.; Westra, August H.; van de Loosdrecht, Arjan A.; de Jong, A.


    Flow cytometry (FC) is recognized as an important tool in the diagnosis of myelodysplastic syndromes (MDS) especially when standard criteria fail. A working group within the Dutch Society of Cytometry aimed to implement FC in the diagnostic work-up of MDS. Hereto, guidelines for data acquisition, an

  5. Non-invasive diagnostic workup of patients with suspected stable angina by combined computed tomography coronary angiography and magnetic resonance perfusion imaging

    S.W.M. Kirschbaum (Sharon); K. Nieman (Koen); T. Springeling (Tirza); A.C. Weustink (Annick); S. Ramcharitar (Steve); C.A.G. van Mieghem (Carlos); A.G. Rossi (Adriano); E. Duckers (Eric); P.W.J.C. Serruys (Patrick); H. Boersma (Eric); P.J. de Feyter (Pim); R.J.M. van Geuns (Robert Jan)


    textabstractBackground: To evaluate additional adenosine magnetic resonance perfusion (MRP) imaging in the diagnostic workup of patients with suspected stable angina with computed tomography coronary angiography (CTCA) as first-line diagnostic modality. Methods and Results: Two hundred and thirty sy

  6. Impact of organic modifier and temperature on protein denaturation in hydrophobic interaction chromatography.

    Bobaly, Balázs; Beck, Alain; Veuthey, Jean-Luc; Guillarme, Davy; Fekete, Szabolcs


    The goal of this study was to better understand the chromatographic conditions in which monoclonal antibodies (mAbs) of broad hydrophobicity scale and a cysteine conjugated antibody-drug conjugate (ADCs), namely brentuximab-vedotin, could denaturate. For this purpose, some experiments were carried out in HIC conditions using various organic modifier in natures and proportions, different mobile phase temperatures and also different pHs. Indeed, improper analytical conditions in hydrophobic interaction chromatography (HIC) may create reversed-phase (RP) like harsh conditions and therefore protein denaturation. In terms of organic solvents, acetonitrile (ACN) and isopropanol (IPA) were tested with proportions ranging from 0 to 40%. It appeared that IPA was a less denaturating solvent than ACN, but should be used in a reasonable range (10-15%). Temperature should also be kept reasonable (below 40°C), to limit denaturation under HIC conditions. However, the combined increase of temperature and organic content induced denaturation of protein biopharmaceuticals in all cases. Indeed, above 30-40°C and 10-15% organic modifier in mobile phase B, heavy chain (HC) and light chain (LC) fragments dissociated. Mobile phase pH was also particularly critical and denaturation was significant even under moderately acidic conditions (pH of 5.4). Today, HIC is widely used for measuring drug-to-antibody ratio (DAR) of ADCs, which is a critical quality attribute of such samples. Here, we demonstrated that the estimation of average DAR can be dependent on the amount of organic modifier in the mobile phase under HIC conditions, due to the better recovery of the most hydrophobic proteins in presence of organic solvent (IPA). So, special care should be taken when measuring the average DAR of ADCs in HIC. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Children presenting in delayed fashion after minor head trauma with scalp swelling: do they require further workup?

    Sellin, Jonathan N; Moreno, Amee; Ryan, Sheila L; Lam, Sandi K; Donaruma-Kwoh, Marcella; Luerssen, Thomas G; Jea, Andrew


    It is common to evaluate children who have sustained minor head trauma with computed tomography (CT) of the head. Scalp swelling, in particular, has been associated with intracranial injury. A subset of patients, however, present in delayed fashion, often days after the head trauma, as soft tissue edema progresses and their caregiver notices scalp swelling. We explore the value of further workup in this setting. We conducted a retrospective review of a prospectively collected cohort of children ≤24 months of age presenting to the Texas Children's Hospital with scalp swelling more than 24 h following a head trauma. Cases were collected over a 2-year study period from June 1, 2014 to May 31, 2016. Seventy-six patients comprising 78 patient encounters were included in our study. The mean age at presentation was 8.8 months (range 3 days-24 months). All patients had noncontrast CT of the head as part of their evaluation by emergency medicine, as well as screening for nonaccidental trauma (NAT) by the Child Protection Team. The most common finding on CT head was a linear/nondisplaced skull fracture (SF) with associated extra-axial hemorrhage (epidural or subdural hematoma), which was found in 31/78 patient encounters (40%). Of all 78 patient encounters, 43 patients (55%) were discharged from the emergency room (ER), 17 patients (22%) were admitted for neurologic monitoring, and 18 patients (23%) were admitted solely to allow further NAT evaluation. Of those patients admitted, none experienced a neurologic decline and all had nonfocal neurologic exams on discharge. No patient returned to the ER in delayed fashion for a neurologic decline. Of all the patient encounters, no patient required surgery. Pediatric patients ≤24 months of age presenting to the ER in delayed fashion with scalp swelling after minor head trauma-who were otherwise nonfocal on examination-did not require surgical intervention and did not experience any neurologic decline. Further radiographic

  8. Effects of Glucides on Thermal Denaturation and Coagulation of Whey Proteins Studied by Ultraviolet Spectroscopy

    Mongo Antoine, Etou; Abena, A. A.; Gbeassor, M.; Chaveron, H.

    The thermal coagulation of whey proteins concentrates was inhibited by various glucides. The disaccharides, saccharose and lactose, were most effective and the amino sugar, glucosamine, least effective in this respect. Ultraviolet absorption and light-scattering measurements on thermal denaturation and coagulation of both unfractionated and individual whey proteins (α-lactalbumin, ß-lactoglobulin and bovine serum albumin) showed that saccharose promotes the denaturation of these proteins but inhibits their subsequent coagulation. These results are interpreted in terms of the effect of saccharose on the hydrophobic interactions between solvent and protein.

  9. Surprisingly high stability of barley lipid transfer protein, LTP1, towards denaturant, heat and proteases

    Lindorff-Larsen, Kresten; Winther, J R


    Barley LTP1 belongs to a large family of plant proteins termed non-specific lipid transfer proteins. The in vivo function of these proteins is unknown, but it has been suggested that they are involved in responses towards stresses such as pathogens, drought, heat, cold and salt. Also, the proteins...... have been suggested as transporters of monomers for cutin synthesis. We have analysed the stability of LTP1 towards denaturant, heat and proteases and found it to be a highly stable protein, which apparently does not denature at temperatures up to 100 degrees C. This high stability may be important...

  10. Refolding of Denatured/Reduced Lysozyme Using Weak-Cation Exchange Chromatography

    Yan WANG; Bo Lin GONG; Xin Du GENG


    Oxidative refolding of the denatured/reduced lysozyme was investigated by using weak-cation exchange chromatography (WCX). The stationary phase of WCX binds to the reduced lysozyme and prevented it from forming intermolecular aggregates. At the same time urea and ammonium sulfate were added to the mobile phase to increase the elution strength for lysozyme. Ammonium sulfate can more stabilize the native protein than a common eluting agent, sodium chloride. Refolding of lysozyme by using this WCX is successfully. It was simply carried out to obtain a completely and correctly refolding of the denatured lysozyme at high concentration of 20.0 mg/mL.

  11. Effect of ethanol denaturant on gasoline RVP (revised). Topical report, June 21, 1993--December 31, 1993

    Wu, L.; Timpe, R.C.


    The Clean Air Act (CAA) Amendments of 1990 require further reduction in gasoline Reid vapor pressure (RVP) to reduce pollution. This research focused on characterizing the effect of ethanol denaturant and water on the RVP of the final ethanol-blended fuel. Anectdotal stories tell of up to a 0.5-psi effect of ethanol denaturant on the RVP of the finished ethanol-blended gasoline. Additionally, earlier Energy & Environmental Research Center (EERC) data indicated water could have a significant effect on the RVP. It was necessary to scientifically verify these effects using acceptable laboratory protocols.

  12. Prevalence and predictors of early cardiovascular events after kidney transplantation: evaluation of pre-transplant cardiovascular work-up.

    Marianne Delville

    Full Text Available Cardiovascular disease is the leading cause of mortality after renal transplantation. The purpose of this study was to analyze cardiovascular risk factors at transplantation, occurrence of cardiovascular events in the first year after transplantation and evaluate pre-transplant work-up.In total, 244 renal transplant recipients older than 50 years were included. The results of pre-transplant work-up, including clinical evaluation, electrocardiogram, echocardiography, myocardial perfusion testing and coronary angiography were analyzed.Patients had multiple risk factors at inclusion on renal transplantation waiting list as high blood pressure (94.7%, dyslipidemia (81.1%, smoking (45.3%, diabetes (23.6%, past history of cardiovascular disease (21.3% and obesity (12.7%. Following transplantation, 15.5% (n = 38 of patients experienced a cardiovascular event, including 2.8% (n = 7 acute coronary syndrome, 5.8% (n = 14 isolated increase in troponin level and 5.3% (n = 13 new onset atrial fibrillation. The pre-transplant parameters associated with a cardiovascular event were a past medical history of cardiovascular disease (HR = 2.06 [1.06-4.03], p = 0.03, echocardiographic left ventricular hypertrophy (HR = 2.04 [1.04-3.98], p = 0.037 and abnormal myocardial perfusion testing (HR = 2.25 [1.09 -5.96], p = 0.03. Pre-transplantation evaluation allowed the diagnosis of unknown coronary artery lesions in 8.9% of patients.

  13. Opportunities to deter transplant tourism exist before referral for transplantation and during the workup and management of transplant candidates.

    Gill, Jagbir; Diec, Olivier; Landsberg, David N; Rose, Caren; Johnston, Olwyn; Keown, Paul A; Gill, John S


    Transplant tourism is a global issue, and physicians in the developed world may be in a position to actively deter this practice. To examine such opportunities, we identified 93 residents of British Columbia, Canada who had a kidney graft through tourism and determined their previous interactions with our transplant programs. These patients were mainly ethnic minorities (90%) who traveled to their country of origin for transplantation. Many tourists were transplanted early in their disease course, with 27 having a preemptive transplant. Among the 65 tourists referred for transplant, 33 failed to complete the evaluation. All tourists who completed an evaluation were placed on a waiting list in British Columbia and, after waiting a median of 2 years, pursued tourism. Most of these patients (62%) had a potential living donor, but none had an approved donor, with 13 donors found medically unsuitable, 8 ABO incompatible, and 12 who did not complete their evaluation. Thus, strategies to deter tourism should start before the development of end-stage renal disease and should be part of pretransplant workup and wait-list management, focusing on patients not progressing through their evaluation, those with a declined living donor, and those facing longer wait times, as these groups appear to be at higher risks for transplant tourism. Further studies are needed to identify individuals at risk for transplant tourism and to define effective strategies to deter these individuals.

  14. Liver CT for vascular mapping during radioembolisation workup: comparison of an early and late arterial phase protocol

    Hoven, Andor F. van den; Braat, Manon N.G.J.A.; Prince, Jip F.; Doormaal, Pieter J. van; Leeuwen, Maarten S. van; Lam, Marnix G.E.H.; Bosch, Maurice A.A.J. van den [University Medical Center Utrecht, Department of Radiology and Nuclear Medicine, Utrecht (Netherlands)


    To compare right gastric (RGA) and segment 4 artery (A4) origin detection rates during radioembolisation workup between early and late arterial phase liver CT protocols. 100 consecutive patients who underwent liver CT between May 2012-January 2015 with early or late arterial phase protocol (n = 50 each, 10- vs. 20-s post-threshold delay) were included. RGA/A4 origin detection rates, assessed by two raters, and contrast-to-noise ratio (CNR) of the hepatic artery relative to the portal vein were compared between the protocols. The first-second rater scored the RGA origin as visible in 58-65 % (specific proportion of agreement 82 %, κ = 0.62); A4 origin in 96-89 % (94 %, κ = 0.54). Thirty-six percent of RGA origins not detectable by DSA were identified on CT. Origin detection rates were not significantly different for early/late arterial phases. Mean CNR was higher in the early arterial phase protocol (1.7 vs. 1.2, p < 0.001). A 10-s delay arterial phase CT protocol does not significantly improve detection of small intra- and extrahepatic branches. RGA origin detection requires further optimization, whereas A4/MHA origin detection is adequate, with good inter-rater reproducibility. CT remains important for preprocedural planning, because it may reveal arterial anatomy not discernible on DSA. (orig.)

  15. Breast cancer: determining the genetic profile from ultrasound-guided percutaneous biopsy specimens obtained during the diagnostic workups.

    López Ruiz, J A; Zabalza Estévez, I; Mieza Arana, J A


    To evaluate the possibility of determining the genetic profile of primary malignant tumors of the breast from specimens obtained by ultrasound-guided percutaneous biopsies during the diagnostic imaging workup. This is a retrospective study in 13 consecutive patients diagnosed with invasive breast cancer by B-mode ultrasound-guided 12 G core needle biopsy. After clinical indication, the pathologist decided whether the paraffin block specimens seemed suitable (on the basis of tumor size, validity of the sample, and percentage of tumor cells) before sending them for genetic analysis with the MammaPrint® platform. The size of the tumors on ultrasound ranged from 0.6cm to 5cm. In 11 patients the preserved specimen was considered valid and suitable for use in determining the genetic profile. In 1 patient (with a 1cm tumor) the pathologist decided that it was necessary to repeat the core biopsy to obtain additional samples. In 1 patient (with a 5cm tumor) the specimen was not considered valid by the genetic laboratory. The percentage of tumor cells in the samples ranged from 60% to 70%. In 11/13 cases (84.62%) it was possible to do the genetic analysis on the previously diagnosed samples. In most cases, regardless of tumor size, it is possible to obtain the genetic profile from tissue specimens obtained with ultrasound-guided 12 G core biopsy preserved in paraffin blocks. Copyright © 2015 SERAM. Published by Elsevier España, S.L.U. All rights reserved.

  16. Diagnostic work-up of neurological syndromes in a rural African setting: knowledge, attitudes and practices of health care providers.

    Alain Mpanya

    Full Text Available BACKGROUND: Neurological disorders of infectious origin are common in rural sub-Saharan Africa and usually have serious consequences. Unfortunately, these syndromes are often poorly documented for lack of diagnostic tools. Clinical management of these diseases is a major challenge in under-equipped rural health centers and hospitals. We documented health care provider knowledge, attitudes and practices related to this syndrome in two rural health zones in Bandundu Province, Democratic Republic of Congo. METHODS: We used a qualitative research approach combining observation, in-depth interviews and focus group discussions. We observed 20 patient-provider contacts related to a neurological syndrome, conducted 12 individual interviews and 4 focus group discussions with care providers. All interviews were audiotaped and the transcripts were analyzed with the software ATLAS.ti. RESULTS: Care providers in this region usually limit their diagnostic work-up to clinical examination primarily because of the financial hurdles in this entirely out-of-pocket payment system. The patients prefer to purchase drugs rather than diagnostic tests. Moreover the general lack of diagnostic tools and the representation of the clinician as a "diviner" do not enhance any use of laboratory or other diagnostic methods. CONCLUSION: Innovation in diagnostic technology for neurological disorders is badly needed in Central-Africa, but its uptake in clinical practice will only be a success if tools are simple, affordable and embedded in a patient-centered approach.

  17. Heat denaturation of soy glycinin. Structural characteristics in relation to aggregation and gel formation.

    Lakemond, C.M.M.


    key words: soy protein; glycinin; thermal stability; pH; ionic strength;genetic variant; solubility; gelationThe main aim of this thesis was to study structural changes of soy glycinin at different conditions (pH and ionic strength) during thermal denaturation and their effect on aggregation and gel

  18. Joule Heating and Thermal Denaturation of Proteins in Nano-ESI Theta Tips

    Zhao, Feifei; Matt, Sarah M.; Bu, Jiexun; Rehrauer, Owen G.; Ben-Amotz, Dor; McLuckey, Scott A.


    Electro-osmotically induced Joule heating in theta tips and its effect on protein denaturation were investigated. Myoglobin, equine cytochrome c, bovine cytochrome c, and carbonic anhydrase II solutions were subjected to electro-osmosis in a theta tip and all of the proteins were denatured during the process. The extent of protein denaturation was found to increase with the applied square wave voltage and electrolyte concentration. The solution temperature at the end of a theta tip was measured directly by Raman spectroscopy and shown to increase with the square wave voltage, thereby demonstrating the effect of Joule heating through an independent method. The electro-osmosis of a solution comprised of myoglobin, bovine cytochrome c, and ubiquitin demonstrated that the magnitude of Joule heating that causes protein denaturation is positively correlated with protein melting temperature. This allows for a quick determination of a protein's relative thermal stability. This work establishes a fast, novel method for protein conformation manipulation prior to MS analysis and provides a temperature-controllable platform for the study of processes that take place in solution with direct coupling to mass spectrometry. [Figure not available: see fulltext.

  19. Manifestations of native topology in the denatured state ensemble of Rhodopseudomonas palustris cytochrome c'.

    Dar, Tanveer A; Schaeffer, R Dustin; Daggett, Valerie; Bowler, Bruce E


    To provide insight into the role of local sequence in the nonrandom coil behavior of the denatured state, we have extended our measurements of histidine-heme loop formation equilibria for cytochrome c' to 6 M guanidine hydrochloride. We observe that there is some reduction in the scatter about the best fit line of loop stability versus loop size data in 6 M versus 3 M guanidine hydrochloride, but the scatter is not eliminated. The scaling exponent, ν(3), of 2.5 ± 0.2 is also similar to that found previously in 3 M guanidine hydrochloride (2.6 ± 0.3). Rates of histidine-heme loop breakage in the denatured state of cytochrome c' show that some histidine-heme loops are significantly more persistent than others at both 3 and 6 M guanidine hydrochloride. Rates of histidine-heme loop formation more closely approximate random coil behavior. This observation indicates that heterogeneity in the denatured state ensemble results mainly from contact persistence. When mapped onto the structure of cytochrome c', the histidine-heme loops with slow breakage rates coincide with chain reversals between helices 1 and 2 and between helices 2 and 3. Molecular dynamics simulations of the unfolding of cytochrome c' at 498 K show that these reverse turns persist in the unfolded state. Thus, these portions of the primary structure of cytochrome c' set up the topology of cytochrome c' in the denatured state, predisposing the protein to fold efficiently to its native structure.

  20. ASP53, a thermostable protein from Acacia erioloba seeds that protects target proteins against thermal denaturation

    Mtwisha, L


    Full Text Available stages of protein thermal denaturation. ASP53 decreased the rate of loss of alcohol dehydrogenase activity at 55°C, decreased the rate of temperature-dependent loss of secondary structure of haemoglobin and completely inhibited the temperature...

  1. Protecting role of cosolvents in protein denaturation by SDS: a structural study

    Wouters Johan


    Full Text Available Abstract Background Recently, we reported a unique approach to preserve the activity of some proteins in the presence of the denaturing agent, Sodium Dodecyl Sulfate (SDS. This was made possible by addition of the amphipathic solvent 2,4-Methyl-2-PentaneDiol (MPD, used as protecting but also as refolding agent for these proteins. Although the persistence of the protein activity in the SDS/MPD mixture was clearly established, preservation of their structure was only speculative until now. Results In this paper, a detailed X-ray study addresses the pending question. Crystals of hen egg-white lysozyme were grown for the first time in the presence of MPD and denaturing concentrations of SDS. Depending on crystallization conditions, tetragonal crystals in complex with either SDS or MPD were collected. The conformation of both structures was very similar to the native lysozyme and the obtained complexes of SDS-lysozyme and MPD-lysozyme give some insights in the interplay of protein-SDS and protein-MPD interactions. Conclusion This study clearly established the preservation of the enzyme structure in a SDS/MPD mixture. It is hypothesized that high concentrations of MPD would change the properties of SDS and lower or avoid interactions between the denaturant and the protein. These structural data therefore support the hypothesis that MPD avoids disruption of the enzyme structure by SDS and can protect proteins from SDS denaturation.

  2. Denaturing gradient gel electrophoresis analysis to study bacterial community structure in pockets of periodontitis patients

    Zijnge, V.; Harmsen, H.J.M.; Kleinfelder, J.W.; Rest, M.E. van der; Degener, J.E.; Welling, G.W.


    Bacteria are involved in the onset and progression of periodontitis. A promising molecular technique, denaturing gradient gel electrophoresis (DGGE), to study microbial population dynamics in the subgingival pocket is presented. Twenty-three samples were taken from the subgingival pockets of nine pa

  3. Investigating the fermentation of cocoa by correlating denaturing gradient gel electrophoresis profiles and near infrared spectra

    Nielsen, Dennis Sandris; Snitkjær, Pia; van der Berg, Franciscus Winfried J


    of the beans and the chemical processes inside the beans have been carried out previously. Recently it has been shown that Denaturing Gradient Gel Electrophoresis (DGGE) offers an efficient tool for monitoring the microbiological changes taking place during the fermentation of cocoa. Near Infrared (NIR...

  4. A simple remedy against artifactual double bands in denaturing gradient gel electrophoresis

    Janse, I.; Bok, J.M.; Zwart, G.


    Denaturant gradient gel electrophoresis (DGGE) is a widely used method for mutation analysis and for studies of microbial diversity. Particular combinations of target gene fragments and primers may give rise to erroneous DGGE profiles. We report on a very straightforward means to eliminate the

  5. Thermal denaturation of protein studied by terahertz time-domain spectroscopy

    Fu, Xiuhua; Li, Xiangjun; Liu, Jianjun; Du, Yong; Hong, Zhi


    In this study, the absorption spectra of native or thermal protein were measured in 0.2-1.4THz using terahertz time-domain spectroscopy (THz-TDS) system at room temperature, their absorption spectra and the refractive spectra were obtained. Experimental results indicate that protein both has strong absorption but their characteristics were not distinct in the THz region, and the absorption decreased during thermal denatured state. In order to prove protein had been denatured, we used Differential scanning calorimeter (DSC) measured their denatured temperature, from their DSC heating traces, collagen Td=101℃, Bovine serum albumin Td=97℃. While we also combined the Fourier transform infrared spectrometer (FTIR) to investigate their secondary and tertiary structure before and after denatuation, but the results did not have the distinct changes. We turned the absorption spectra and the refractive spectra to the dielectric spectra, and used the one-stage Debye model simulated the terahertz dielectric spectra of protein before and after denaturation. This research proved that the terahertz spectrum technology is feasible in testing protein that were affected by temperature or other factors which can provide theoretical foundation in the further study about the THz spectrum of protein and peptide temperature stability.

  6. Mesoscopic modeling of DNA denaturation rates: Sequence dependence and experimental comparison

    Dahlen, Oda, E-mail:; Erp, Titus S. van, E-mail: [Department of Chemistry, Norwegian University of Science and Technology (NTNU), Høgskoleringen 5, Realfagbygget D3-117 7491 Trondheim (Norway)


    Using rare event simulation techniques, we calculated DNA denaturation rate constants for a range of sequences and temperatures for the Peyrard-Bishop-Dauxois (PBD) model with two different parameter sets. We studied a larger variety of sequences compared to previous studies that only consider DNA homopolymers and DNA sequences containing an equal amount of weak AT- and strong GC-base pairs. Our results show that, contrary to previous findings, an even distribution of the strong GC-base pairs does not always result in the fastest possible denaturation. In addition, we applied an adaptation of the PBD model to study hairpin denaturation for which experimental data are available. This is the first quantitative study in which dynamical results from the mesoscopic PBD model have been compared with experiments. Our results show that present parameterized models, although giving good results regarding thermodynamic properties, overestimate denaturation rates by orders of magnitude. We believe that our dynamical approach is, therefore, an important tool for verifying DNA models and for developing next generation models that have higher predictive power than present ones.

  7. Affinity chromatography of chaperones based on denatured proteins: Analysis of cell lysates of different origin.

    Marchenko, N Yu; Sikorskaya, E V; Marchenkov, V V; Kashparov, I A; Semisotnov, G V


    Molecular chaperones are involved in folding, oligomerization, transport, and degradation of numerous cellular proteins. Most of chaperones are heat-shock proteins (HSPs). A number of diseases of various organisms are accompanied by changes in the structure and functional activity of chaperones, thereby revealing their vital importance. One of the fundamental properties of chaperones is their ability to bind polypeptides lacking a rigid spatial structure. Here, we demonstrate that affinity chromatography using sorbents with covalently attached denatured proteins allows effective purification and quantitative assessment of their bound protein partners. Using pure Escherichia coli chaperone GroEL (Hsp60), the capacity of denatured pepsin or lysozyme-based affinity sorbents was evaluated as 1 mg and 1.4 mg of GroEL per 1 ml of sorbent, respectively. Cell lysates of bacteria (E. coli, Thermus thermophilus, and Yersinia pseudotuberculosis), archaea (Halorubrum lacusprofundi) as well as the lysate of rat liver mitochondria were analyzed using affinity carrier with denatured lysozyme. It was found that, apart from Hsp60, other proteins with a molecular weight of about 100, 50, 40, and 20 kDa are able to interact with denatured lysozyme.

  8. Reversible Dimerization of Acid-Denatured ACBP Controlled by Helix A4

    Fieber, Wolfgang; Kragelund, Birthe Brandt; Meldal, Morten Peter;


    of dimers and revealed a cooperative stabilization of helix A4 in this process. This emphasizes its special role in the structure formation in the denatured state of ACBP. No dimers are formed in the presence of guanidine hydrochloride, which underlines the fundamental difference between the nature...

  9. Sufficient minimal model for DNA denaturation: Integration of harmonic scalar elasticity and bond energies.

    Singh, Amit Raj; Granek, Rony


    We study DNA denaturation by integrating elasticity - as described by the Gaussian network model - with bond binding energies, distinguishing between different base pairs and stacking energies. We use exact calculation, within the model, of the Helmholtz free-energy of any partial denaturation state, which implies that the entropy of all formed "bubbles" ("loops") is accounted for. Considering base pair bond removal single events, the bond designated for opening is chosen by minimizing the free-energy difference for the process, over all remaining base pair bonds. Despite of its great simplicity, for several known DNA sequences our results are in accord with available theoretical and experimental studies. Moreover, we report free-energy profiles along the denaturation pathway, which allow to detect stable or meta-stable partial denaturation states, composed of bubble, as local free-energy minima separated by barriers. Our approach allows to study very long DNA strands with commonly available computational power, as we demonstrate for a few random sequences in the range 200-800 base-pairs. For the latter, we also elucidate the self-averaging property of the system. Implications for the well known breathing dynamics of DNA are elucidated.

  10. Denaturing Effects of Urea and Guanidine Hydrochloride on Hyperthermophilic Esterase from Aeropyrum pernix K1


    The changes in the activity and the conformation of the hyperthermophilic esterase derived from aerobic thermophilic Aeropyrumpernix K1 (APE1547) were studied during denaturation by guanidine hydrochloride (GdnHCl)and urea. The denaturation course of APE1547 was followed by the steady-state and time resolved fluorescence methods. An increase in the denaturant concentration in the denatured system can significantly enhance the inactivation and unfolding of APE1547. The enzyme can be completely inactivated with a urea concentration of 2. 7 mol/L or a GdnHCl concentration of 7.5 mol/L. The fluorescence emission maximum of the enzyme protein red shifts in magnitude to a maximum value(355 nm) when the concentration of GdnHCl is 5.1 mol/L. The experimental results indicate that APE1547 has a high resistance to urea. Unfolding of APE1547 in GdnHCl(4.2-6.0 mol/L) was shown to be an irreversible process. The present results indicate that the ion pairs in this protein may be a key factor for the stability of this esterase.

  11. Sufficient minimal model for DNA denaturation: Integration of harmonic scalar elasticity and bond energies

    Singh, Amit Raj; Granek, Rony


    We study DNA denaturation by integrating elasticity — as described by the Gaussian network model — with bond binding energies, distinguishing between different base pairs and stacking energies. We use exact calculation, within the model, of the Helmholtz free-energy of any partial denaturation state, which implies that the entropy of all formed "bubbles" ("loops") is accounted for. Considering base pair bond removal single events, the bond designated for opening is chosen by minimizing the free-energy difference for the process, over all remaining base pair bonds. Despite of its great simplicity, for several known DNA sequences our results are in accord with available theoretical and experimental studies. Moreover, we report free-energy profiles along the denaturation pathway, which allow to detect stable or meta-stable partial denaturation states, composed of bubble, as local free-energy minima separated by barriers. Our approach allows to study very long DNA strands with commonly available computational power, as we demonstrate for a few random sequences in the range 200-800 base-pairs. For the latter, we also elucidate the self-averaging property of the system. Implications for the well known breathing dynamics of DNA are elucidated.

  12. Lysozyme Thermal Denaturation and Self-Interaction: Four Integrated Thermodynamic Experiments for the Physical Chemistry Laboratory

    Schwinefus, Jeffrey J.; Schaefle, Nathaniel J.; Muth, Gregory W.; Miessler, Gary L.; Clark, Christopher A.


    As part of an effort to infuse our physical chemistry laboratory with biologically relevant, investigative experiments, we detail four integrated thermodynamic experiments that characterize the denaturation (or unfolding) and self-interaction of hen egg white lysozyme as a function of pH and ionic strength. Students first use Protein Explorer to…

  13. Triggered isothermal PCR by denaturation bubble-mediated strand exchange amplification.

    Shi, Chao; Shang, Fanjin; Zhou, Meiling; Zhang, Pansong; Wang, Yifan; Ma, Cuiping


    Here, we introduced the concept of strand exchange amplification (SEA) mediated by denaturation bubbles. Similar to traditional PCR, it only employed a DNA polymerase and a pair of common primers to realize a three-step cycle process, but the entire SEA reaction was performed at a single temperature.

  14. Comparison of the fine structure of mitochondrial DNA from Saccharomyces cerevisiae and S. carlsbergensis: electron microscopy of partially denatured molecules

    Christiansen, Gunna; Christiansen, C


    Denaturation-maps of mitochondrial DNA from Saccharomyces cerevisiae and S. carlsbergensis have been derived from electron microscopic observations of partially denatured complete circular molecules and large fragments of these circles. The mitochondrial DNA from the two species differ by 6...

  15. Reversible denaturation of Brazil nut 2S albumin (Ber e1) and implication of structural destabilization on digestion by pepsin

    Koppelman, S.J.; Nieuwenhuizen, W.F.; Gaspari, M.; Knippels, L.M.J.; Penninks, A.H.; Knol, E.F.; Hefle, S.L.; Jongh,


    The high resistance of Brazil nut 2S albumin, previously identified as an allergen, against proteolysis by pepsin was examined in this work. Although the denaturation temperature of this protein exceeds the 110 °C at neutral pH, at low pH a fully reversible thermal denaturation was observed at ∼82 °

  16. Molecular assessment of collagen denaturation in decellularized tissues using a collagen hybridizing peptide.

    Hwang, Jeongmin; San, Boi Hoa; Turner, Neill J; White, Lisa J; Faulk, Denver M; Badylak, Stephen F; Li, Yang; Yu, S Michael


    Decellularized extracellular matrix (ECM) derived from tissues and organs are emerging as important scaffold materials for regenerative medicine. Many believe that preservation of the native ECM structure during decellularization is highly desirable. However, because effective techniques to assess the structural damage in ECM are lacking, the disruptive effects of a decellularization method and the impact of the associated structural damage upon the scaffold's regenerative capacity are often debated. Using a novel collagen hybridizing peptide (CHP) that specifically binds to unfolded collagen chains, we investigated the molecular denaturation of collagen in the ECM decellularized by four commonly used cell-removing detergents: sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), sodium deoxycholate (SD), and Triton X-100. Staining of the detergent-treated porcine ligament and urinary bladder matrix with carboxyfluorescein-labeled CHP demonstrated that SDS and Triton X-100 denature the triple helical collagen molecule while CHAPS and SD do not, although second harmonic generation imaging and transmission electron microscopy (TEM) revealed that all four detergents disrupt collagen fibrils. Our findings from the CHP staining were further confirmed by the circular dichroism spectra of intact triple helical collagen molecules in CHAPS and SD solutions, and the TEM images of CHP-conjugated gold nanoparticles binding only to the SDS and Triton X-100 treated collagen fibrils. CHP is a powerful new tool for direct and reliable measurement of denatured collagen molecules in decellularized tissues. It is expected to have wide applications in the development and standardization of the tissue/organ decellularization technology. Preservation of the native ECM structure in decellularized tissues is highly desirable, since denaturation of ECM molecules (e.g., collagen) during decellularization can strongly influence the cellular response

  17. Structural properties of cyanase. Denaturation, renaturation, and role of sulfhydryls and oligomeric structure in catalytic activity.

    Little, R M; Anderson, P M


    Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to give ammonia and bicarbonate. The enzyme is composed of 8-10 identical subunits (Mr = 17,008). The objective of this study was to clarify some of the structural properties of cyanase for the purpose of understanding the relationship between oligomeric structure and catalytic activity. Circular dichroism studies showed that cyanase has a significant amount of alpha-helix and beta-sheet structure. The one sulfhydryl group per subunit does not react with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) unless cyanase is denatured. Denaturation is apparently complete in 10 M urea or 6 M guanidine hydrochloride, but is significantly reduced in 10 M urea by the presence of azide (analog of cyanate) and is incomplete in 8 M urea. Denatured cyanase could be renatured and reactivated (greater than 85%) by removal of denaturants. Reactivation was greatly facilitated by the presence of certain anions, particularly bicarbonate, and by high ionic strength and protein concentration. The catalytic activity of renatured cyanase was associated only with oligomer. Cyanase that had been denatured in the presence of DTNB to give a cyanase-DTNB derivative could also be renatured at 26 degrees C to give active cyanase-DTNB oligomer. The active oligomeric form of the cyanase-DTNB derivative could be converted reversibly to inactive dimer by lowering the temperature to 4 degrees C or by reduction of the ionic strength and removal of monoanions. These results provide evidence that free sulfhydryl groups are not required for catalytic activity and that catalytic activity may be dependent upon oligomeric structure.

  18. [Some properties of complexes formed by small heat shock proteins with denatured actin].

    Pivovarova, A V; Chebotareva, N A; Guseev, N B; Levitskiĭ, D I


    We applied different methods to analyze the effects of the recombinant wild-type small heat shock protein with an apparent molecular mass of 27 kD (Hsp27-wt) and its S15,78,82D mutant (Hsp27-3D), which mimics the naturally occurring phosphorylation of this protein, on the thermal denaturation and aggregation of F-actin. It has been shown that, at the weight ratio of Hsp27/actin equal to 1/4, both Hsp27-wt and Hsp27-3D do not affect the thermal unfolding of F-actin but effectively prevent the aggregation of F-actin by forming soluble complexes with denatured actin. The formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. It is known that Hsp27-wt forms high-molecular-mass oligomers, whereas Hsp27-3D forms small dimers or tetramers. However, the complexes formed by Hsp27-wt and Hsp27-3D with denatured actin did not differ in their size, as measured by dynamic light scattering, and demonstrated the same hydrodynamic radius of 17-18 nm. On the other hand, the sedimentation coefficients of these complexes were distributed within the range 10-45 S in the case of Hsp27-3D and 18-60 S in the case of Hsp27-wt. Thus, the ability of Hsp27 to form soluble complexes with denatured actin does not significantly depend on the initial oligomeric state of Hsp27.

  19. Detection of the End Point Temperature of Thermal Denatured Protein in Fish and Chicken Meat Through SDS-PAGE Electrophoresis

    GAO Hongwei; MAO Mao; LIANG Chengzhu; LIN Chao; XIANG Jianhai


    Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was applied in the detection of the end point temperature (EPT) of thermal denatured protein in fish and meat in this study. It was also used in studying the thermal denatured temperature range of proteins in salmon and chicken meat. The results show that the temperature ranges of denatured proteins were from 65℃ to 75℃, and these temperature ranges were influenced by the processing methods. Through SDS-PAGE, the features of repeated heating thermal denatured proteins under the same temperature and processing time were studied. The electrophoresis pat-terns of thermal denatured proteins determined through repeated heating at the same temperature did not exhibit any change. For the detection of cooked fish and meat samples, they were subjected to applying the SDS-PAGE method, which revealed an EPT ranging from 60℃ to 80℃.

  20. Male infertility workup needs additional testing of expressed prostatic secretion and/or post-massage urine.

    Margus Punab

    Full Text Available The male factor accounts for almost 50% of infertility cases. Inflammation may reduce semen quality via several pathways, including oxidative stress (OxS. As male infertility routinely is assessed using semen analysis only, the possible presence of non-leukocytospermic asymptomatic inflammatory prostatitis may be overlooked. We compared local and systemic OxS levels in male partners of infertile couples with different inflammation patterns in their genital tract and/or oligospermia. Subjects (n=143 were grouped according to inflammation in their semen, expressed prostatic secretion (EPS, and/or post-massage urine (post-M. Systemic (8-isoprostanes in urine and local (diene conjugates and total antioxidant capacity in seminal plasma OxS was measured The levels of OxS markers were significantly elevated in both severe inflammation groups--leukocytospermic men and subjects whose inflammation was limited only to EPS and/or post-M. Comparison between oligospermic and non-oligospermic men with genital tract inflammation, and oligozoospermic men with or without inflammation in the genital tract indicated that inflammation but not oligospermia status had significant impact on the measured OxS markers. Hence, a high leukocyte count in prostate-specific materials (EPS, post-M, even in absence of clear leukocytopsermia, is an important source of local and systemic OxS that may be associated with male infertility and affect general health. We suggest including the tests for detection of inflammation of the prostate into the workup of infertile men as was suggested in the WHO 1993 recommendation.

  1. The use of 4D-CTA in the diagnostic work-up of brain arteriovenous malformations

    Willems, Peter W.A. [Toronto Western Hospital, UHN, Division of Neuroradiology, Department of Medical Imaging, Toronto, Ontario (Canada); Leiden University Medical Center, Department of Radiology, Leiden (Netherlands); Taeshineetanakul, Patamintita; Terbrugge, Karel G.; Krings, Timo [Toronto Western Hospital, UHN, Division of Neuroradiology, Department of Medical Imaging, Toronto, Ontario (Canada); Schenk, Barry; Brouwer, Patrick A. [Leiden University Medical Center, Department of Radiology, Leiden (Netherlands)


    We aimed to evaluate the use of time-resolved whole-head CT angiography (4D-CTA) in patients with an untreated arteriovenous malformation of the brain (bAVM), as demonstrated by catheter angiography (DSA). Seventeen patients with a DSA-proven bAVM were enrolled. These were subjected to 4D-CTA imaging using a 320 detector row CT scanner. Using a standardized scoring sheet, all studies were analyzed by a panel of three readers. This panel was blind to the DSA results at the time of reading the 4D-CTA. 4D-CTA detected all bAVMs. With regard to the Spetzler-Martin grade, 4D-CTA disagreed with DSA in only one case, where deep venous drainage was missed. Further discrepancies between 4D-CTA and DSA analyses included underestimation of the nidus size in small lesions (four cases), misinterpretation of a feeding vessel (one case), misinterpretation of indirect feeding through pial collaterals (three cases) and oversight of mild arterial enlargement (two cases). 4D-CTA correctly distinguished low-flow from high-flow lesions and detected dural/transosseous feeding (one case), venous narrowing (one case) and venous pouches (nine cases). In this series, 4D-CTA was able to detect all bAVMs. Although some angioarchitectural details were missed or misinterpreted when compared to DSA, 4D-CTA evaluation was sufficiently accurate to diagnose the shunt and classify it. Moreover, 4D-CTA adds cross-sectional imaging and perfusion maps, helpful in treatment planning. 4D-CTA appears to be a valuable new adjunct in the non-invasive diagnostic work-up of bAVMs and their follow-up when managed conservatively. (orig.)

  2. The value of FDG-PET/CT in the diagnostic work-up of extra cardiac infectious manifestations in infectious endocarditis

    Ozcan, C; Asmar, A; Gill, S;


    Infectious endocarditis (IE) is a serious condition with a high morbidity and mortality. The optimal management of IE depends not only on correct antibiotic therapy and surgery when needed, but involves identification of the portal of entry and detection of extracardiac infectious manifestations....... To discover the latter an (18)F-fluorodeoxyglucose positron emission tomography ((18)F-FDG-PET)/CT examination has been proposed. However, the diagnostic value of a PET/CT in this setting remains unresolved. Thus, we wished to assess the usefulness of a PET/CT study in patients with IE as a supplemental...... method to standard work-up in evaluating primary and distant infective foci. A retrospective cohort study of 72 IE patients admitted from 2008 to 2010, which had an (18)F-FDG-PET/CT performed. Findings were assessed in relation to the routine work-up, which served as the "gold standard". One hundred...

  3. Isothermal DNA origami folding: avoiding denaturing conditions for one-pot, hybrid-component annealing

    Kopielski, Andreas; Schneider, Anne; Csáki, Andrea; Fritzsche, Wolfgang


    The DNA origami technique offers great potential for nanotechnology. Using biomolecular self-assembly, defined 2D and 3D nanoscale DNA structures can be realized. DNA origami allows the positioning of proteins, fluorophores or nanoparticles with an accuracy of a few nanometers and enables thereby novel nanoscale devices. Origami assembly usually includes a thermal denaturation step at 90 °C. Additional components used for nanoscale assembly (such as proteins) are often thermosensitive, and possibly damaged by such harsh conditions. They have therefore to be attached in an extra second step to avoid defects. To enable a streamlined one-step nanoscale synthesis - a so called one-pot folding - an adaptation of the folding procedures is required. Here we present a thermal optimization of this process for a 2D DNA rectangle-shaped origami resulting in an isothermal assembly protocol below 60 °C without thermal denaturation. Moreover, a room temperature protocol is presented using the chemical additive betaine, which is biocompatible in contrast to chemical denaturing approaches reported previously.The DNA origami technique offers great potential for nanotechnology. Using biomolecular self-assembly, defined 2D and 3D nanoscale DNA structures can be realized. DNA origami allows the positioning of proteins, fluorophores or nanoparticles with an accuracy of a few nanometers and enables thereby novel nanoscale devices. Origami assembly usually includes a thermal denaturation step at 90 °C. Additional components used for nanoscale assembly (such as proteins) are often thermosensitive, and possibly damaged by such harsh conditions. They have therefore to be attached in an extra second step to avoid defects. To enable a streamlined one-step nanoscale synthesis - a so called one-pot folding - an adaptation of the folding procedures is required. Here we present a thermal optimization of this process for a 2D DNA rectangle-shaped origami resulting in an isothermal assembly

  4. Audit of the association between length of time spent on diagnostic work-up and tumour stage in patients with symptomatic colon cancer.

    Tiong, Jimmy; Gray, Andrew; Jackson, Christopher; Thompson-Fawcett, Mark; Schultz, Michael


    Colorectal cancer is one of the most incident cancers in New Zealand. Due to resource limitations, some patients experienced protracted wait times before reaching a definitive diagnosis. We analysed the relationship between time to diagnosis and clinical stage and reviewed the length of time for components of the diagnostic work-up to identify priority areas for service improvement. We benchmarked our timeliness against introduced standards. This retrospective study included all patients with colonic (not rectal) cancer between October 2007 and September 2009. Patients were stratified into an early and advanced group. Types of delay were calculated from the onset of symptoms to the administration of treatment. The compliance with target waiting times was assessed. Fifty-eight patients were included in the early group and 83 patients in the advanced group. There were no significant differences in demographics or symptoms. The work-up was longer than international benchmarks, but with wide variations. There was no statistical difference between lengths of work-up in the groups. The advanced group had increased utilization of private and emergency investigations. Forty-four per cent met the diagnostic colonoscopy target waiting time of 42 days with a trend in favour of the advanced group and 21% received treatment within 62 days (non-significant). Current systems are not sophisticated enough to predict the stage of colon cancer. Here, long waiting times were not associated with cancer stage in symptomatic patients. Resources need to be directed to diagnostic colonic imaging. © 2014 Royal Australasian College of Surgeons.

  5. Clinical impact of 18F-FDG-PET/CT in the extra cardiac work-up of patients with infective endocarditis

    Asmar, Ali; Ozcan, Cengiz; Diederichsen, Axel C P;


    OBJECTIVE: The purpose of this study was to assess the clinical importance of (18)F-FDG-PET/CT used in the extra cardiac work-up of patients with infective endocarditis (IE). BACKGROUND: IE is a serious condition with a significant mortality. Besides the degree of valvular involvement, the progno...... lesions of clinical importance in one of seven IE patients and may be a substantial imaging technique for tracing peripheral infectious embolism due to IE. Thus, (18)F-FDG-PET/CT may help to guide adequate therapy and thereby improve the prognosis of patients with IE.......OBJECTIVE: The purpose of this study was to assess the clinical importance of (18)F-FDG-PET/CT used in the extra cardiac work-up of patients with infective endocarditis (IE). BACKGROUND: IE is a serious condition with a significant mortality. Besides the degree of valvular involvement......, the prognosis relies crucially on the presence of systemic infectious embolism. METHODS: Seventy-two patients (71% males and mean age 63 ± 17 years) with IE were evaluated with (18)F-FDG-PET/CT in addition to standard work-up including patient history, physical examination, conventional imaging modalities...

  6. Strategies for selecting optimal sampling and work-up procedures for analysing alkylphenol polyethoxylates in effluents from non-activated sludge biofilm reactors.

    Stenholm, Ake; Holmström, Sara; Hjärthag, Sandra; Lind, Ola


    Trace-level analysis of alkylphenol polyethoxylates (APEOs) in wastewater containing sludge requires the prior removal of contaminants and preconcentration. In this study, the effects on optimal work-up procedures of the types of alkylphenols present, their degree of ethoxylation, the biofilm wastewater treatment and the sample matrix were investigated for these purposes. The sampling spot for APEO-containing specimens from an industrial wastewater treatment plant was optimized, including a box that surrounded the tubing outlet carrying the wastewater, to prevent sedimented sludge contaminating the collected samples. Following these changes, the sampling precision (in terms of dry matter content) at a point just under the tubing leading from the biofilm reactors was 0.7% RSD. The findings were applied to develop a work-up procedure for use prior to a high-performance liquid chromatography-fluorescence detection analysis method capable of quantifying nonylphenol polyethoxylates (NPEOs) and poorly investigated dinonylphenol polyethoxylates (DNPEOs) at low microg L(-1) concentrations in effluents from non-activated sludge biofilm reactors. The selected multi-step work-up procedure includes lyophilization and pressurized fluid extraction (PFE) followed by strong ion exchange solid phase extraction (SPE). The yields of the combined procedure, according to tests with NP10EO-spiked effluent from a wastewater treatment plant, were in the 62-78% range.

  7. Effects of urea denatureation and pH on the ability of porcine myoglobin to undergo reduction.

    Zhu, L G; Brewer, M S


    To determine the effects of globin moiety denaturation and pH on the ability of metmyoglobin (MetMb) to undergo reduction, MetMb isolated from porcine hearts was denatured in 8.5M urea. Both native and denatured MetMb solutions were serially reduced with Na(2)S(2)O(4) (0, 7.5, 15, 18.75, 22.5, 26.25, 30, 30.75, and 45 umol). Reduction was conducted at pH 5, 5.2, 5.4, 5.6, 6, 6.2, 6.4, 6.6, and 7. After reduction, absorbance was determined at 635 nm and the percent of the original MetMb which was reduced was calculated. The average percent MetMb reduced from the native and denatured forms was 35 and 25%, respectively. pH significantly influenced the percentage of MetMb reduced, especially when pH was <6. If the MetMb was denatured prior to reduction, the influence of pH on its ability to undergo reduction was slight. The percentage of denatured MetMb reduced was higher at pH 7 than at all other pHs. High pH enhanced the ability of MetMb to undergo reduction; while low pH decreases it. Low pH may have denatured the native globin moiety.

  8. On the influence of the mixture of denaturants on protein structure stability: A molecular dynamics study

    Shao, Qiang; Wang, Jinan; Zhu, Weiliang


    Mixtures of osmolytes and/or inorganic salts are present in the cell. Therefore, the understanding of the interplay of mixed osmolyte molecules and inorganic salts and their combined effects on protein structure is of fundamental importance. A novel test is presented to investigate the combined effects of urea and a chaotropic inorganic salt, potassium iodide (KI), on protein structure by using molecular dynamics simulation. It is found that the coexistence of KI and urea does not affect their respective distribution in solution. The solvation of KI salt in urea solution makes the electrostatic interactions of urea more favorable, promoting the hydrogen bonding between urea (and water) to protein backbone. The interactions from K+ and hydrogen bonding from urea and water to protein backbone work as the driving force for protein denaturation. The collaborative behavior of urea and KI salt thus enhances the denaturing ability of urea and KI mixed solution.

  9. Protective role of salt in catalysis and maintaining structure of halophilic proteins against denaturation

    Rajeshwari eSinha


    Full Text Available Search for new enzymes of industrial relevance, bestowed with novel properties continues to be a desirable pursuit in enzyme research. Halophilism is the unusual existence of life in saline/ hypersaline habitats and haloenzymes, are the proteins from such origin, naturally endowed with unique structural features which enable them to sustain functionality under high salt. Driven by industrial requirements, halophilic enzymes have been explored for their stability and catalytic abilities under harsh operational conditions. These have been documented to withstand high temperature, pH, organic solvents, and chaotropic agents. However, this stability is modulated by salt. Understanding the basis of salt mediated protection amidst a denaturing milieu will add significantly to the existing knowledge about structure function relationships in halophilic proteins. Exploring their protein architecture may provide template for rationale design of stable enzymes. The article encompasses the current level of understanding about haloadaptations in halophiles and structural basis of their stability against classical denaturants.

  10. pH-sensitive polymer-assisted refolding of urea-denatured fibroblast growth factor

    Zhi Feng Huang; Shan Shan Wang; Chun Yan Ni; Shu Lin Yang; Xiao Kun Li; Susanna S.J.Leong


    A pH-responsive polymer Eudragit S-100 has been found to assist in correct folding of FGF-2(fibroblast growth factor-2)denatured with 8 mol/L urea and 10 mmol/L dithiothreitol at pH 7.2.The refolding of FGF-2 was performed by directly diluting denatured FGF-2 into a refolding buffer containing Eudragit S-100.The ability of Eudragit S-100 to enhance protein refolding level was investigated using MTT method,fluorescence emission spectroscopy and reversc phase HPLC.On the other hand,the result shows the ability of Eudragit S-100 to enhance the refolding level of protein is due to the interaction between Eudragit S-100 and positively charged FGF-2.

  11. Theoretical aspects of pressure and solute denaturation of proteins: A Kirkwood-buff-theory approach

    Ben-Naim, Arieh


    A new approach to the problem of pressure-denaturation (PD) and solute-denaturation (SD) of proteins is presented. The problem is formulated in terms of Le Chatelier principle, and a solution is sought in terms of the Kirkwood-Buff theory of solutions. It is found that both problems have one factor in common; the excluded volumes of the folded and the unfolded forms with respect to the solvent molecules. It is shown that solvent-induced effects operating on hydrophilic groups along the protein are probably the main reason for PD. On the other hand, the SD depends on the preferential solvation of the folded and the unfolded forms with respect to solvent and co-solvent molecules.

  12. ZnO nanoparticles assist the refolding of denatured green fluorescent protein.

    Pandurangan, Muthuraman; Zamany, Ahmad Jawid; Kim, Doo Hwan


    Proteins are essential for cellular and biological processes. Proteins are synthesized and fold into the native structure to become active. The inability of a protein molecule to remain in its native conformation is called as protein misfolding, and this is due to several environmental factors. Protein misfolding and aggregation handle several human diseases. Protein misfolding is believed to be one of the causes of several disorders such as cancer, degenerative diseases, and metabolic pathologies. The zinc oxide (ZnO) nanoparticle was significantly promoted refolding of thermally denatured green fluorescent protein (GFP). In the present study, ZnO nanoparticles interaction with GFP was investigated by ultraviolet-visible spectrophotometer, fluorescence spectrophotometer, and dynamic light scattering. Results suggest that the ZnO nanoparticles significantly assist the refolding of denatured GFP. Copyright © 2015 John Wiley & Sons, Ltd.

  13. Small angle neutron scattering studies on protein denaturation induced by different methods

    S Chodankar; V K Aswal; J Kohlbrecher; R Vavrin; A G Wagh


    Small angle neutron scattering (SANS) has been used to study conformational changes in protein bovine serum albumin (BSA) as induced by varying temperature and in the presence of protein denaturating agents urea and surfactant. BSA has pro-late ellipsoidal shape and is found to be stable up to 60°C above which it denaturates and subsequently leads to aggregation. The protein solution exhibits a fractal structure at temperatures above 64°C, with fractal dimension increasing with temperature. BSA protein is found to unfold in the presence of urea at concentrations greater than 4 M and acquires a random coil Gaussian chain conformation. The conformation of the unfolded protein in the presence of surfactant has been determined directly using contrast variation SANS measurements by contrast matching surfactant molecules. The protein acquires a random coil Gaussian conformation on unfolding with its radius of gyration increasing with increase in surfactant concentration

  14. Teaching what one does not know: strangeness and denaturation in (autobiographical narrations

    Jorge Luiz da Cunha


    Full Text Available The thematic focus in this text are the estrangement/denaturation processes in (autobiographical narrations. The aim of this study was to reflect on the possibility to promote estrangement/denatura - tion in (autobiographical writings made by teenagers in the space/ time of the classroom environment. The methodological proposal consisted on developing (autobiographical writings by students from sociology classes in High School. A total of 138 teenagers from a public school, attending the first school trimester in the year 2013, have participated in the study. The concepts of estrangement/de - naturation are located in the anthropology field and, the work with (autobiographical narrations is located in the socio-clinic perspec - tives and of biographization processes. The results indicate that (autobiographical narrations provide estrangements/denaturation and go towards teaching what one does not know. We can, then, conclude that this possibility, as an educational act, may generate knowledge suspension to self-inventiveness.


    Md. Imtaiyaz Hassan


    Full Text Available Zinc α2-glycoprotein (ZAG and Prolactin induced protein (PIP are considered as important elements for fertility and biomarker for prostate and breast carcinomas. The stabilities of ZAG alone and its naturally occurring complex with PIP were compared. A significant difference in CD signal was recorded for native ZAG and ZAG-PIP complex against pH-, GdnHCl- and temperature-induced denaturation. These finding suggests that PIP plays a protective role for ZAG against several denaturants. PIP contributes to the hydrophobic as well as electrostatic interactions on ZAG for the complex formation. Moreover, the observed changes in far-UV spectra between ZAG and ZAG-PIP complex in the presence of PEG support the hydrophobic nature of the forces governing the formation of complex. This pH dependent study provides evidence that formation of the complex is a natural event required for physiological function.

  16. Surface characterization of proteins using multi-fractal property of heat-denatured aggregates

    Lahiri, Tapobrata; Mishra, Hrishikesh; Sarkar, Subrata; Misra, Krishna


    Multi-fractal property of heat-denatured protein aggregates (HDPA) is characteristic of its individual form. The visual similarity between digitally generated microscopic images of HDPA with that of surface-image of its individual X-ray structures in protein databank (PDB) displayed using Visual Molecular Dynamics (VMD) viewer is the basis of the study. We deigned experiments to view the fractal nature of proteins at different aggregate scales. Intensity based multi-fractal dimensions (ILMFD) extracted from various planes of digital microscopic images of protein aggregates were used to characterize HDPA into different classes. Moreover, the ILMFD parameters extracted from aggregates show similar classification pattern to digital images of protein surface displayed by VMD viewer using PDB entry. We discuss the use of irregular patterns of heat-denatured aggregate proteins to understand various surface properties in native proteins. PMID:18795110

  17. Acid Denaturation Inducing Self-Assembly of Curcumin-Loaded Hemoglobin Nanoparticles

    Kaikai Wang


    Full Text Available Hemoglobin is a promising drug carrier but lacks extensive investigation. The chemical conjugation of hemoglobin and drugs is costly and complex, so we have developed curcumin-loaded hemoglobin nanoparticles (CCM-Hb-NPs via self-assembly for the first time. Using the acid-denaturing method, we avoid introducing denaturants and organic solvents. The nanoparticles are stable with uniform size. We have conducted a series of experiments to examine the interaction of hemoglobin and CCM, including hydrophobic characterization, SDS-PAGE. These experiments substantiate that this self-assembly process is mainly driven by hydrophobic forces. Our nanoparticles achieve much higher cell uptake efficiency and cytotoxicity than free CCM solution in vitro. The uptake inhibition experiments also demonstrate that our nanoparticles were incorporated via the classic clathrin-mediated endocytosis pathway. These results indicate that hemoglobin nanoparticles formed by self-assembly are a promising drug delivery system for cancer therapy.

  18. Comparison between conformational change and inactivation rates of aminoacylase during denaturation in urea solutions

    王洪睿; 王希成; 张彤; 周海梦


    The kinetic method of the substrate reaction in the presence of mactivator previously described by Tsou has been applied to the determination of inactivation rates of aminoacylase during denaturation in urea solutions. The protective effect of substrate on the inactivation of aminoacylase by urea has been investigated. Simultaneously, the comparison between conformational change and inactivation rates of enzyme in the urea solutions of different concentrations has been studied. Results obtained show that the inactivation rate constants of the enzyme are larger than the rate constants of conformational changes. The present results show that the active site of metal enzyme-aminoacylase is also located in a limited and flexible region of the molecule that is more sensitive to denaturants than the enzyme as a whole.

  19. Should there be a standardised approach to the diagnostic workup of suspected adult encephalitis? a case series from Australia

    Williams David


    assessment is necessary to exclude treatable agents and identify pathogens warranting public health interventions, such as those transmitted by mosquitoes and those that are vaccine preventable. An algorithm and guidelines for the diagnostic workup of encephalitis cases would assist in optimising laboratory testing so that clinical management can be best tailored to the pathogen, and appropriate public health measures implemented.

  20. Formation of Native and Non-native Interactions in Ensembles of Denatured ACBP Molecules from Paramagnetic Relaxation Enhancement Studies

    Kristjansdottir, S.; Lindorff-Larsen, Kresten; Fieber, W.;


    in the denatured states with those in the transition state for folding we also provided new insights into the mechanism of formation of the native state of this protein. Keywords: protein folding; denatured state; NMR; molecular dynamics; structural studies Abbreviations: ACBP, acyl coenzyme A binding protein; Gu...... of the residual structure in the denatured state of ACBP under these different conditions has enabled us to infer that regions in the N and C-terminal parts of the protein sequence have a high tendency to interact in the unfolded state under physiological conditions. By comparing the structural features...

  1. Micro-CT Imaging of Denatured Chitin by Silver to Explore Honey Bee and Insect Pathologies

    Butzloff, Peter R.


    Background Chitin and cuticle coatings are important to the environmental and immune defense of honey bees and insect pollinators. Pesticides or environmental effects may target the biochemistry of insect chitin and cuticle coating. Denaturing of chitin involves a combination of deacetylation, intercalation, oxidation, Schweiger-peeling, and the formation of amine hydrochloride salt. The term “denatured chitin” calls attention to structural and property changes to the internal membranes and external carapace of organisms so that some properties affecting biological activities are diminished. Methodology/Principal Findings A case study was performed on honey bees using silver staining and microscopic computer-tomographic x-ray radiography (micro-CT). Silver nitrate formed counter-ion complexes with labile ammonium cations and reacted with amine hydrochloride. Silver was concentrated in the peritrophic membrane, on the abdomen, in the glossa, at intersegmental joints (tarsi), at wing attachments, and in tracheal air sacs. Imaged mono-esters and fatty acids from cuticle coating on external surfaces were apparently reduced by an alcohol pretreatment. Conclusions/Significance The technique provides 3-dimensional and sectional images of individual honey bees consistent with the chemistries of silver reaction and complex formation with denatured chitin. Environmental exposures and influences such as gaseous nitric oxide intercalant, trace oxidants such as ozone gas, oligosachharide salt conversion, exposure to acid rain, and chemical or biochemical denaturing by pesticides may be studied using this technique. Peritrophic membranes, which protect against food abrasion, microorganisms, and permit efficient digestion, were imaged. Apparent surface damage to the corneal lenses of compound eyes by dilute acid exposure consistent with chitin amine hydrochloride formation was imaged. The technique can contribute to existing insect pathology research, and may provide an

  2. Thermal denaturation of beta-galactosidase and of two site-specific mutants.

    Edwards, R A; Jacobson, A L; Huber, R E


    The thermal denaturation of wild-type beta-galactosidase and two beta-galactosidases with substitutions at the active site was studied by kinetics, differential scanning calorimetry, electrophoresis, molecular exclusion chromatography, and circular dichroism. From the results, a model is developed for thermal denaturation of beta-galactosidase which includes the reversible dissociation of ligands, reversible formation of an inactive tetramer, irreversible dissociation of the inactive tetramer to inactive monomers, and subsequent aggregation of inactive monomers to dimers and larger aggregates. Under some conditions, partial reversibility of the activity loss could be demonstrated, and several intermediates in the thermal denaturation process were trapped by quenching and observed by electrophoresis and molecular exclusion chromatography. The ligands Mg2+ and phenylethyl thio-beta-D-galactoside increase the stability of beta-galactosidase to heat denaturation by shifting the ligand binding equilibrium according to Le Chatelier's principle, thus decreasing the concentration of the ligand-free tetramer which can proceed to subsequent steps. Circular dichroism results indicated that beta-galactosidase is dominated by beta-sheet with lower amounts of alpha-helix. Large changes in secondary structure begin to occur only after activity has been lost. Single amino acid changes at the active site can have significant effects on thermal stability of beta-galactosidases. Some of the effects result from increased thermal stability of the ligand-free enzyme itself. Other effects result from changes in ligand binding, but the magnitude of the resulting changes in stability is not related to the strength of ligand binding in a simple fashion.

  3. Detection of Rifampin Resistance in Mycobacterium tuberculosis by Double Gradient-Denaturing Gradient Gel Electrophoresis

    Scarpellini, Paolo; Braglia, Sergio; Carrera, Paola; Cedri, Maura; Cichero, Paola; Colombo, Alessia; Crucianelli, Rosella; Gori, Andrea; Ferrari, Maurizio; Lazzarin, Adriano


    We applied double gradient-denaturing gradient gel electrophoresis (DG-DGGE) for the rapid detection of rifampin (RMP) resistance from rpoB PCR products of Mycobacterium tuberculosis isolates and clinical samples. The results of this method were fully concordant with those of DNA sequencing and susceptibility testing analyses. DG-DGGE is a valid alternative to the other methods of detecting mutations for predicting RMP resistance. PMID:10508043

  4. A kinetic study of jack-bean urease denaturation by a new dithiocarbamate bismuth compound

    Menezes, D. C.; Borges, E.; Torres, M. F.; Braga, J. P.


    A kinetic study concerning enzymatic inhibitory effect of a new bismuth dithiocarbamate complex on jack-bean urease is reported. A neural network approach is used to solve the ill-posed inverse problem arising from numerical treatment of the subject. A reaction mechanism for the urease denaturation process is proposed and the rate constants, relaxation time constants, equilibrium constants, activation Gibbs free energies for each reaction step and Gibbs free energies for the transition species are determined.

  5. Combining an Optical Resonance Biosensor with Enzyme Activity Kinetics to Understand Protein Adsorption and Denaturation

    Wilson, Kerry A.; Finch, Craig A.; Anderson, Phillip; Vollmer, Frank; Hickman, James J.


    Understanding protein adsorption and resultant conformation changes on modified and unmodified silicon dioxide surfaces is a subject of keen interest in biosensors, microfluidic systems and for medical diagnostics. However, it has been proven difficult to investigate the kinetics of the adsorption process on these surfaces as well as understand the topic of the denaturation of proteins and its effect on enzyme activity. A highly sensitive optical whispering gallery mode (WGM) resonator was us...

  6. Multifocal peritoneal splenosis in Tc-99m-labeled heat-denatured red blood cell scintigraphy

    Yoon, Min Ki; Hwang, Kyung Hoon; Choe, Won Sick [Gachon University Gil Medical Center, Incheon (Korea, Republic of)


    A 44-year-old man with a past medical history of splenectomy came to hospital because of epigastric pain abdominopelvic computed tomography(CT) showed a soft tissue mass and multifocal variable-sized nodules as well as finding suggestive of cholecystitis. Subsequently, he underwent Tc-99m-labeled heat- denatured red blood cell(RBC) scintigraphy to evaluate the mass and nodules. The scintigraphy confirmed multifocal peritoneal splenosis in the abdominopelvic cavity.

  7. Surface characterization of proteins using multi-fractal property of heat-denatured aggregates

    Lahiri, Tapobrata; Mishra, Hrishikesh; Sarkar, Subrata; Misra, Krishna


    Multi-fractal property of heat-denatured protein aggregates (HDPA) is characteristic of its individual form. The visual similarity between digitally generated microscopic images of HDPA with that of surface-image of its individual X-ray structures in protein databank (PDB) displayed using Visual Molecular Dynamics (VMD) viewer is the basis of the study. We deigned experiments to view the fractal nature of proteins at different aggregate scales. Intensity based multi-fractal dimensions (ILMFD)...

  8. Thermal stability of chemically denatured green fluorescent protein (GFP) A preliminary study

    Nagy, Attila; Malnasi-Csizmadia, Andras; Somogyi, Bela; Lorinczy, Denes


    Green fluorescent protein (GFP) is a light emitter in the bioluminescence reaction of the jellyfish Aequorea victoria. The protein consist of 238 amino acids and produces green fluorescent light ({lambda}{sub max}=508 nm), when irradiated with near ultraviolet light. The fluorescence is due to the presence of chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide -Ser{sup 65}-Tyr{sup 66}-Gly{sup 67}-, which buried into {beta}-barrel. GFP is extremely compact and heat stable molecule. In this work, we present data for the effect of chemical denaturing agent on the thermal stability of GFP. When denaturing agent is applied, global thermal stability and the melting point of the molecule is decreases, that can be monitored with differential scanning calorimetry. The results indicate, that in 1-6 M range of GuHCl the melting temperature is decreasing continuously from 83 to 38 deg. C. Interesting finding, that the calculated calorimetric enthalpy decreases with GuHCl concentration up to 3 M (5.6-0.2 kJ mol{sup -1}), but at 4 M it jumps to 8.4 and at greater concentration it is falling down to 1.1 kJ mol{sup -1}. First phenomena, i.e. the decrease of melting point with increasing GuHCl concentration can be easily explained by the effect of the extended chemical denaturation, when less and less amount of heat required to diminish the remaining hydrogen bonds in {beta}-barrel. The surprising increase of calorimetric enthalpy at 4 M concentration of GuHCl could be the consequence of a dimerization or a formation of stable complex between GFP and denaturing agent as well as a precipitation at an extreme GuHCl concentration. We are planning further experiments to elucidate fluorescent consequence of these processes.

  9. Micro-CT imaging of denatured chitin by silver to explore honey bee and insect pathologies.

    Peter R Butzloff

    Full Text Available BACKGROUND: Chitin and cuticle coatings are important to the environmental and immune defense of honey bees and insect pollinators. Pesticides or environmental effects may target the biochemistry of insect chitin and cuticle coating. Denaturing of chitin involves a combination of deacetylation, intercalation, oxidation, Schweiger-peeling, and the formation of amine hydrochloride salt. The term "denatured chitin" calls attention to structural and property changes to the internal membranes and external carapace of organisms so that some properties affecting biological activities are diminished. METHODOLOGY/PRINCIPAL FINDINGS: A case study was performed on honey bees using silver staining and microscopic computer-tomographic x-ray radiography (micro-CT. Silver nitrate formed counter-ion complexes with labile ammonium cations and reacted with amine hydrochloride. Silver was concentrated in the peritrophic membrane, on the abdomen, in the glossa, at intersegmental joints (tarsi, at wing attachments, and in tracheal air sacs. Imaged mono-esters and fatty acids from cuticle coating on external surfaces were apparently reduced by an alcohol pretreatment. CONCLUSIONS/SIGNIFICANCE: The technique provides 3-dimensional and sectional images of individual honey bees consistent with the chemistries of silver reaction and complex formation with denatured chitin. Environmental exposures and influences such as gaseous nitric oxide intercalant, trace oxidants such as ozone gas, oligosachharide salt conversion, exposure to acid rain, and chemical or biochemical denaturing by pesticides may be studied using this technique. Peritrophic membranes, which protect against food abrasion, microorganisms, and permit efficient digestion, were imaged. Apparent surface damage to the corneal lenses of compound eyes by dilute acid exposure consistent with chitin amine hydrochloride formation was imaged. The technique can contribute to existing insect pathology research, and may

  10. Comparison of membrane electroporation and protein denature in response to pulsed electric field with different durations.

    Huang, Feiran; Fang, Zhihui; Mast, Jason; Chen, Wei


    In this paper, we compared the minimum potential differences in the electroporation of membrane lipid bilayers and the denaturation of membrane proteins in response to an intensive pulsed electric field with various pulse durations. Single skeletal muscle fibers were exposed to a pulsed external electric field. The field-induced changes in the membrane integrity (leakage current) and the Na channel currents were monitored to identify the minimum electric field needed to damage the membrane lipid bilayer and the membrane proteins, respectively. We found that in response to a relatively long pulsed electric shock (longer than the membrane intrinsic time constant), a lower membrane potential was needed to electroporate the cell membrane than for denaturing the membrane proteins, while for a short pulse a higher membrane potential was needed. In other words, phospholipid bilayers are more sensitive to the electric field than the membrane proteins for a long pulsed shock, while for a short pulse the proteins become more vulnerable. We can predict that for a short or ultrashort pulsed electric shock, the minimum membrane potential required to start to denature the protein functions in the cell plasma membrane is lower than that which starts to reduce the membrane integrity.


    Borzova N. V.


    Full Text Available The aim of this work was the study of native and galactosidases from Penicillium canescens under denaturing conditions caused by guanidine hydrochloride. Calculation of kinetics and constants of enzymes inactivation was carried out on using experimental kinetic curves of enzyme denaturation. We observed significant differences in the kinetics of inactivation of native and deglycosylated α-galactosidases from P. canescens caused by guanidine hydrochloride. Native enzyme was stable within the selected range of guanidine hydrochloride concentrations (from 0.1 to 3.0 M, retaining no less than 50% of the initial enzyme activity for 3 days. Deglycosylated enzyme preparations were less stable and they lost their activity within 5–30 minutes, when they were treated with guanidine hydrochloride in concentrations above 1 M. Dissociation rate constant of native and deglycosylated forms of the enzyme differed by 10 to 100 folds. It was shown that subunit interactions play a major role in the process of inactivation of the enzyme, and the carbohydrate component is essential for stabilizing of subunit bonds and maintaining conformational stability of the enzyme under denaturing conditions of chemical agents.

  12. Thermal and mechanical denaturation properties of a DNA model with three sites per nucleotide

    Florescu, Ana-Maria; 10.1063/1.3626870


    In this paper, we show that the coarse grain model for DNA, which has been proposed recently by Knotts, Rathore, Schwartz and de Pablo (J. Chem. Phys. 126, 084901 (2007)), can be adapted to describe the thermal and mechanical denaturation of long DNA sequences by adjusting slightly the base pairing contribution. The adjusted model leads to (i) critical temperatures for long homogeneous sequences that are in good agreement with both experimental ones and those obtained from statistical models, (ii) a realistic step-like denaturation behaviour for long inhomogeneous sequences, and (iii) critical forces at ambient temperature of the order of 10 pN, close to measured values. The adjusted model furthermore supports the conclusion that the thermal denaturation of long homogeneous sequences corresponds to a first-order phase transition and yields a critical exponent for the critical force equal to sigma=0.70. This model is both geometrically and energetically realistic, in the sense that the helical structure and th...

  13. Single domain antibodies are specially suited for quantitative determination of gliadins under denaturing conditions.

    Doña, Vanina; Urrutia, Mariela; Bayardo, Mariela; Alzogaray, Vanina; Goldbaum, Fernando Alberto; Chirdo, Fernando G


    Food intended for celiac patients' consumption must be analyzed for the presence of toxic prolamins using high detectability tests. Though 60% ethanol is the most commonly used solvent for prolamins extraction, 2-mercaptoethanol (2-ME) and guanidinium chloride (GuHCl) can be added to increase protein recovery. However, ethanol and denaturing agents interfere with antigen recognition when conventional antibodies are used. In the present work, a new method for gliadins quantification is shown. The method is based on the selection of llama single domain antibody fragments able to operate under denaturing conditions. Six out of 28 VHH-phages obtained retained their binding capacity in 15% ethanol. Selected clones presented a long CDR3 region containing two additional cysteines that could be responsible for the higher stability. One of the clones (named VHH26) was fully operative in the presence of 15% ethanol, 0.5% 2-ME, and 0.5 M GuHCl. Capture ELISA using VHH26 was able to detect gliadins in samples shown as negatives by conventional ELISA. Therefore, this new strategy appears as an excellent platform for quantitative determination of proteins or any other immunogenic compound, in the presence of denaturing agents, when specific recognition units with high stability are required.

  14. Remarkable activation of enzymes in nonaqueous media by denaturing organic cosolvents

    Almarsson, O.; Klibanov, A.M. [Massachusetts Inst. of Tech., Cambridge, MA (United States). Dept. of Chemistry


    The rates of transesterification reactions catalyzed by the protease subtilisin Carlsberg suspended in various anhydrous solvents at 30 C can be increased more than 100-fold by the addition of denaturing organic cosolvents (dimethyl sulfoxide or formamide); in water, the same cosolvents exert no enzyme activation. At 4 C, the activation effect on the lyophilized protease is even higher, reaching 1,000-fold. Marked enhancement of enzymatic activity in anhydrous solvents by formamide is also observed for two other enzymes, {alpha}-chymotrypsin and Rhizomucor miehei lipase, and is manifested in two transesterification reactions. In addition to lyophilized subtilisin, crosslinked crystals of subtilisin are also amenable to the dramatic activation by the denaturing cosolvents. In contrast, subtilisin solubilized in anhydrous media by covalent modification with poly(ethylene glycol) exhibits only modest activation. These observations are rationalized in terms of a mechanistic hypothesis based on an enhanced protein flexibility in anhydrous milieu brought about by the denaturing organic cosolvents. The latter exert their lubricating effect largely at the interfaces between enzyme molecules in a solid preparation, thus easing the flexibility constraints imposed by protein-protein contacts.

  15. Effects of high pressure freezing (HPF) on denaturation of natural actomyosin extracted from prawn (Metapenaeus ensis).

    Cheng, Lina; Sun, Da-Wen; Zhu, Zhiwei; Zhang, Zhihang


    Effects of protein denaturation caused by high pressure freezing, involving Pressure-Factors (pressure, time) and Freezing-Factors (temperature, phase transition, recrystallization, ice crystal types), are complicated. In the current study, the conformation and functional changes of natural actomyosin (NAM) under pressure assisted freezing (PAF, (100,150,300,400,500MPa)P-20°C/25min), pressure shift freezing (PSF, (200MPa)P-20°C/25min), and immersion freezing ((0.1MPa)P-20°C/5min) after pressure was released to 0.1MPa, as compared to normal immersion freezing process (IF, (0.1MPa)P-20°C/30min). Results indicated that PSF ((200MPa)P-20°C/30min) could reduce the denaturation of frozen NAM and a pressure of 300MPa was the critical point to induce such a denaturation. During the periods of B→D in PSF or B→C→D in PAF, the generation and growth of ice crystals played an important role on changing the secondary and tertiary structure of the treated NAM. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Fourier transform infrared spectroscopy provides an evidence of papain denaturation and aggregation during cold storage.

    Rašković, Brankica; Popović, Milica; Ostojić, Sanja; Anđelković, Boban; Tešević, Vele; Polović, Natalija


    Papain is a cysteine protease with wide substrate specificity and many applications. Despite its widespread applications, cold stability of papain has never been studied. Here, we used differential spectroscopy to monitor thermal denaturation process. Papain was the most stabile from 45 °C to 60 °C with ΔG°321 of 13.9±0.3 kJ/mol and Tm value of 84±1 °C. After cold storage, papain lost parts of its native secondary structures elements which gave an increase of 40% of intermolecular β-sheet content (band maximum detected at frequency of 1621 cm(-1) in Fourier transform infrared (FT-IR) spectrum) indicating the presence of secondary structures necessary for aggregation. The presence of protein aggregates after cold storage was also proven by analytical size exclusion chromatography. After six freeze-thaw cycles around 75% of starting enzyme activity of papain was lost due to cold denaturation and aggregation of unfolded protein. Autoproteolysis of papain did not cause significant loss of the protein activity. Upon the cold storage, papain underwent structural rearrangements and aggregation that correspond to other cold denatured proteins, rather than autoproteolysis which could have the commercial importance for the growing polypeptide based industry.

  17. Stabilization of Human Serum Albumin against Urea Denaturation by Diazepam and Ketoprofen.

    Manoharan, Pralad; Wong, Yin H; Tayyab, Saad


    Stabilizing effect of diazepam and ketoprofen, Sudlow's site II markers on human serum albumin (HSA) against urea denaturation was studied using fluorescence spectroscopy. The two-step, three-state urea transition of HSA was transformed into a single-step, two-state transition with the abolishment of the intermediate state along with a shift of the transition curve towards higher urea concentrations in the presence of diazepam or ketoprofen. Interestingly, a greater shift in the transition curve of HSA was observed in the presence of ketoprofen compared to diazepam. A comparison of the intrinsic fluorescence and three-dimensional fluorescence spectra of HSA and partially-denatured HSAs, obtained in the absence and the presence of diazepam or ketoprofen suggested significant retention of native-like conformation in the partially-denatured states of HSA in the presence of Sudlow's site II markers. Taken together, all these results suggested stabilization of HSA in the presence of diazepam or ketoprofen, being greater in the presence of ketoprofen.

  18. Evaluation of gasoline-denatured ethanol as a carbon source for denitrification.

    Kazasi, Anna; Boardman, Gregory D; Bott, Charles B


    In this study concerning denitrification, the performance of three carbon sources, methanol (MeOH), ethanol (EtOH) and gasoline-denatured ethanol (dEtOH), was compared and evaluated on the basis of treatment efficiency, inhibition potential and cost. The gasoline denaturant considered here contained mostly aliphatic compounds and little of the components that typically boost the octane rating, such as benzene, toluene, ethylbenzene and xylenes. Results were obtained using three lab-scale SBRs operated at SRT of 12.0 +/- 0.9 days. After biomass was acclimated, denitrification rates with dEtOH were similar to those of EtOH (201 +/- 50 and 197 +/- 28 NO3-N/g MLVSS x d, respectively), and higher than those of MeOH (165 +/- 49 mg NO3-N/g MLVSS x d). The denaturant did not affect biomass production, nitrification or denitrification. Effluent soluble COD concentrations were always less than the analytical detection limit. Although the cost of dEtOH ($2.00/kg nitrate removed) was somewhat higher than that of methanol ($1.63/kg nitrate removed), the use of dEtOH is very promising and utilities will have to decide if it is worth paying a little extra to take advantage of its benefits.

  19. AFM visualization at a single-molecule level of denaturated states of proteins on graphite.

    Barinov, Nikolay A; Prokhorov, Valery V; Dubrovin, Evgeniy V; Klinov, Dmitry V


    Different graphitic materials are either already used or believed to be advantageous in biomedical and biotechnological applications, e.g., as biomaterials or substrates for sensors. Most of these applications or associated important issues, such as biocompatibility, address the problem of adsorption of protein molecules and, in particular the conformational state of the adsorbed protein molecule on graphite. High-resolution AFM demonstrates highly oriented pyrolytic graphite (HOPG) induced denaturation of four proteins of blood plasma, such as ferritin, fibrinogen, human serum albumin (HSA) and immunoglobulin G (IgG), at a single molecule level. Protein denaturation is accompanied by the decrease of the heights of protein globules and spreading of the denatured protein fraction on the surface. In contrast, the modification of HOPG with the amphiphilic oligoglycine-hydrocarbon derivative monolayer preserves the native-like conformation and provides even more mild conditions for the protein adsorption than typically used mica. Protein unfolding on HOPG may have universal character for "soft" globular proteins. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Denaturation Kinetics of Whey Protein Isolate Solutions and Fouling Mass Distribution in a Plate Heat Exchanger

    Marwa Khaldi


    Full Text Available Few investigations have attempted to connect the mechanism of dairy fouling to the chemical reaction of denaturation (unfolding and aggregation occurring in the bulk. The objective of this study is to contribute to this aspect in order to propose innovative controls to limit fouling deposit formation. Experimental investigations have been carried out to observe the relationship between the deposit mass distribution generated in plate heat exchangers (PHE by a whey protein isolate (WPI mainly composed of β-lactoglobulin (β-Lg and the ratio between the unfolding and aggregation rate constants. Experiments using a PHE were carried out at a pilot scale to identify the deposit distribution of a model fouling solution with different calcium contents. In parallel, laboratory experiments were performed to determine the unfolding/aggregation rate constants. Data analysis showed that (i β-Lg denaturation is highly dependent on the calcium content, (ii for each fouling solution, irrespective of the imposed temperature profile, the deposit mass in each channel and the ratio between the unfolding and aggregation rate constants seem to be well correlated. This study demonstrates that both the knowledge of the thermal profile and the β-Lg denaturation rate constants are required in order to predict accurately the deposit distribution along the PHE.

  1. Lyophilization-induced protein denaturation in phosphate buffer systems: monomeric and tetrameric beta-galactosidase.

    Pikal-Cleland, K A; Carpenter, J F


    During freezing in phosphate buffers, selective precipitation of a less soluble buffer component and subsequent pH shifts may induce protein denaturation. Previous reports indicate significantly more inactivation and secondary structural perturbation of monomeric and tetrameric beta-galactosidase (beta-gal) during freeze-thawing in sodium phosphate (NaP) buffer as compared with potassium phosphate (KP) buffer. This observation was attributed to the significant pH shifts (from 7.0 to as low as 3.8) observed during freezing in the NaP buffer (1). In the current study, we investigated the impact of the additional stress of dehydration after freezing on the recovery of active protein on reconstitution and the retention of the native structure in the dried state. Freeze-drying monomeric and tetrameric beta-gal in either NaP or KP buffer resulted in significant secondary structural perturbations, which were greatest for the NaP samples. However, similar recoveries of active monomeric protein were observed after freeze-thawing and freeze-drying, indicating that most dehydration-induced unfolding was reversible on reconstitution of the freeze-dried protein. In contrast, the tetrameric protein was more susceptible to dehydration-induced denaturation as seen by the greater loss in activity after reconstitution of the freeze-dried samples relative to that measured after freeze-thawing. To ensure optimal protein stability during freeze-drying, the protein must be protected from both freezing and dehydration stresses. Although poly(ethylene glycol) and dextran are preferentially excluded solutes and should confer protection during freezing, they were unable to prevent lyophilization-induced denaturation. In addition, Tween did not foster maintenance of native protein during freeze-drying. However, sucrose, which hydrogen bonds to dried protein in the place of lost water, greatly reduced freezing- and drying-induced denaturation, as observed by the high retention of native

  2. Protein denaturation due to the action of surfactants: a study by SAXS and ITC

    Oseliero Filho, Pedro Leonidas; Oliveira, Cristiano Luis Pinto de [Universidade de Sao Paulo (USP), SP (Brazil); Pedersen, Jan Skov; Otzen, Daniel Erik [University of Aarhus (Denmark)


    Full text: Proteins are the major constituent of biological systems along with carbohydrates, lipids and nucleic acids (DNA and RNA). According to their structure and composition, proteins perform several functions in the organism, starting from the macroscopic level, with participation on the olfaction of animals, down to the cellular level, allocated in the membrane and making the connection between extra and intracellular environment. The function of a protein (which may be enzymatic, hormonal, structural, energetic, transport etc) is related to several factors including its structure (primary, secondary, tertiary or quaternary). Denaturation occurs when the secondary structure and/or tertiary is lost, which is almost always followed by the loss of the associated biological function. Temperature, pH and the action of surfactants influence the process of the denaturation. The influence of surfactants to the protein structure and function is the aim of this work. Therefore we are using an isolated protein, {alpha}-lactalbumin, that is found in the milk and whose function is related to the synthesis of galactose. The purpose is to characterize, in a thermodynamic-structural point of view, the denaturation of alpha-lactalbumin in the presence of surfactants anionic (sodium dodecyl sulfate - SDS), cationic (tetradecyltrimethylammonium bromide - TTAB), zwitterionic (2-diheptanoyl-sn-glycero-3- phosphocholine - DHPC) and nonionic (decyl-{beta}-D-Maltopyranoside - DM). The isothermal titration calorimetry (ITC) technique, which provides information of structural changes from changes in energy, represents the starting point for the study, while the technique of small angle X-ray scattering (SAXS) provides information about the structural characteristics of surfactant-protein complexes formed at each step of the denaturation process. The data analysis is in the initial stage, but it was possible to obtain general parameters related to the complex formed from the

  3. Antibodies with specificity for native and denatured forms of ovalbumin differ in reactivity between enzyme-linked immunosorbent assays

    Holm, B. E.; Bergmann, Ann Christina; Hansen, Paul Robert


    In this study, polyclonal and monoclonal antibodies to native and denatured chicken ovalbumin (OVA) were produced to compare their dependency on continuous and three-dimensional epitopes. These antibodies were characterized with respect to reactivity to native and denatured OVA by enzyme......-linked immunosorbent assay (ELISA) employing surface-bound OVA and streptavidin-capture ELISA to determine whether effects of different coating influence antibody specificity and with respect to epitope specificity by peptide ELISA, using overlapping peptides, covering the complete OVA sequence. Polyclonal antibodies...... to native OVA reacted strongly with native and denatured OVA in both assays, but did not react with the overlapping peptides. Polyclonal antibodies to denatured OVA reacted strongly with both OVA forms and with several of the overlapping peptides. Monoclonal antibodies to native OVA reacted preferentially...

  4. Imaging high-intensity focused ultrasound-induced tissue denaturation by multispectral photoacoustic method: an ex vivo study.

    Sun, Yao; O'Neill, Brian


    We present an ex vivo study for the first time, to the best of our knowledge, in multispectral photoacoustic imaging (PAI) of tissue denaturation induced by high-intensity focused ultrasound (HIFU) in this paper. Tissue of bovine muscle was thermally treated in a heated water bath and by HIFU, and then was imaged using a multispectral photoacoustic approach. Light at multiple optical wavelengths between 700 and 900 nm was delivered to the treated bovine muscle tissue to excite the photoacoustic signal. Apparent tissue denaturation has been observed in multispectral photoacoustic images after being treated in a water bath and by HIFU. It is interesting that the denaturation is more striking at shorter optical wavelength photoacoustic images than at longer optical wavelength photoacoustic images. Multispectral photoacoustic images of the tissue denaturation were further analyzed and the photoacoustic spectrums of the denaturized tissue were calculated in this paper. This study suggests that a multispectral PAI approach might be a promising tool to evaluate tissue denaturation induced by HIFU treatment.

  5. Denatured-state energy landscapes of a protein structural database reveal the energetic determinants of a framework model for folding.

    Wang, Suwei; Gu, Jenny; Larson, Scott A; Whitten, Steven T; Hilser, Vincent J


    Position-specific denatured-state thermodynamics were determined for a database of human proteins by use of an ensemble-based model of protein structure. The results of modeling denatured protein in this manner reveal important sequence-dependent thermodynamic properties in the denatured ensembles as well as fundamental differences between the denatured and native ensembles in overall thermodynamic character. The generality and robustness of these results were validated by performing fold-recognition experiments, whereby sequences were matched with their respective folds based on amino acid propensities for the different energetic environments in the protein, as determined through cluster analysis. Correlation analysis between structure and energetic information revealed that sequence segments destined for beta-sheet in the final native fold are energetically more predisposed to a broader repertoire of states than are sequence segments destined for alpha-helix. These results suggest that within the subensemble of mostly unstructured states, the energy landscapes are dominated by states in which parts of helices adopt structure, whereas structure formation for sequences destined for beta-strand is far less probable. These results support a framework model of folding, which suggests that, in general, the denatured state has evolutionarily evolved to avoid low-energy conformations in sequences that ultimately adopt beta-strand. Instead, the denatured state evolved so that sequence segments that ultimately adopt alpha-helix and coil will have a high intrinsic structure formation capability, thus serving as potential nucleation sites.

  6. Multiple unfolding intermediates of human placental alkaline phosphatase in equilibrium urea denaturation.

    Hung, H C; Chang, G G


    Alkaline phosphatase is an enzyme with a typical alpha/beta hydrolase fold. The conformational stability of the human placental alkaline phosphatase was examined with the chemical denaturant urea. The red shifts of fluorescence spectra show a complex unfolding process involving multiple equilibrium intermediates indicating differential stability of the subdomains of the enzyme. None of these unfolding intermediates were observed in the presence of 83 mM NaCl, indicating the importance of ionic interactions in the stabilization of the unfolding intermediates. Guanidinium chloride, on the other hand, could stabilize one of the unfolding intermediates, which is not a salt effect. Some of the unfolding intermediates were also observed in circular dichroism spectroscopy, which clearly indicates steady loss of helical structure during unfolding, but very little change was observed for the beta strand content until the late stage of the unfolding process. The enzyme does not lose its phosphate-binding ability after substantial tertiary structure changes, suggesting that the substrate-binding region is more resistant to chemical denaturant than the other structural domains. Global analysis of the fluorescence spectral change demonstrated the following folding-unfolding process of the enzyme: N I(1) I(2) I(3) I(4) I(5) D. These discrete intermediates are stable at urea concentrations of 2.6, 4.1, 4.7, 5.5, 6.6, and 7.7 M, respectively. These intermediates are further characterized by acrylamide and/or potassium iodide quenching of the intrinsic fluorescence of the enzyme and by the hydrophobic probes, 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid. The stepwise unfolding process was interpreted by the folding energy landscape in terms of the unique structure of the enzyme. The rigid central beta-strand domain is surrounded by the peripheral alpha-helical and coil structures, which are marginally stable toward a chemical

  7. Brownian dynamics simulations of sequence-dependent duplex denaturation in dynamically superhelical DNA

    Mielke, Steven P.; Grønbech-Jensen, Niels; Krishnan, V. V.; Fink, William H.; Benham, Craig J.


    The topological state of DNA in vivo is dynamically regulated by a number of processes that involve interactions with bound proteins. In one such process, the tracking of RNA polymerase along the double helix during transcription, restriction of rotational motion of the polymerase and associated structures, generates waves of overtwist downstream and undertwist upstream from the site of transcription. The resulting superhelical stress is often sufficient to drive double-stranded DNA into a denatured state at locations such as promoters and origins of replication, where sequence-specific duplex opening is a prerequisite for biological function. In this way, transcription and other events that actively supercoil the DNA provide a mechanism for dynamically coupling genetic activity with regulatory and other cellular processes. Although computer modeling has provided insight into the equilibrium dynamics of DNA supercoiling, to date no model has appeared for simulating sequence-dependent DNA strand separation under the nonequilibrium conditions imposed by the dynamic introduction of torsional stress. Here, we introduce such a model and present results from an initial set of computer simulations in which the sequences of dynamically superhelical, 147 base pair DNA circles were systematically altered in order to probe the accuracy with which the model can predict location, extent, and time of stress-induced duplex denaturation. The results agree both with well-tested statistical mechanical calculations and with available experimental information. Additionally, we find that sites susceptible to denaturation show a propensity for localizing to supercoil apices, suggesting that base sequence determines locations of strand separation not only through the energetics of interstrand interactions, but also by influencing the geometry of supercoiling.

  8. A study of the thermal denaturation of the S-layer protein from Lactobacillus salivarius

    Lighezan, Liliana; Georgieva, Ralitsa; Neagu, Adrian


    Surface layer (S-layer) proteins display an intrinsic self-assembly property, forming monomolecular crystalline arrays, identified in outermost structures of the cell envelope in many organisms, such as bacteria and archaea. Isolated S-layer proteins also possess the ability to recrystallize into regular lattices, being used in biotechnological applications, such as controlling the architecture of biomimetic surfaces. To this end, the stability of the S-layer proteins under high-temperature conditions is very important. In this study, the S-layer protein has been isolated from Lactobacillus salivarius 16 strain of human origin, and purified by cation-exchange chromatography. Using circular dichroism (CD) spectroscopy, we have investigated the thermal denaturation of the S-layer protein. The far- and near-UV CD spectra have been collected, and the temperature dependence of the CD signal in these spectral domains has been analyzed. The variable temperature results show that the secondary and tertiary structures of the S-layer protein change irreversibly due to the heating of the sample. After the cooling of the heated protein, the secondary and tertiary structures are partially recovered. The denaturation curves show that the protein unfolding depends on the sample concentration and on the heating rate. The secondary and tertiary structures of the protein suffer changes in the same temperature range. We have also detected an intermediate state in the protein denaturation pathway. Our results on the thermal behavior of the S-layer protein may be important for the use of S-layer proteins in biotechnological applications, as well as for a better understanding of the structure and function of S-layer proteins.

  9. Stabilizing effect of biochar on soil extracellular enzymes after a denaturing stress.

    Elzobair, Khalid A; Stromberger, Mary E; Ippolito, James A


    Stabilizing extracellular enzymes may maintain enzymatic activity while protecting enzymes from proteolysis and denaturation. A study determined whether a fast pyrolysis hardwood biochar (CQuest™) would reduce evaporative losses, subsequently stabilizing soil extracellular enzymes and prohibiting potential enzymatic activity loss following a denaturing stress (microwaving). Soil was incubated in the presence of biochar (0%, 1%, 2%, 5%, or 10% by wt.) for 36 days and then exposed to microwave energies (0, 400, 800, 1600, or 3200 J g(-1) soil). Soil enzymes (β-glucosidase, β-d-cellobiosidase, N-acetyl-β-glucosaminidase, phosphatase, leucine aminopeptidase, β-xylosidase) were analyzed by fluorescence-based assays. Biochar amendment reduced leucine aminopeptidase and β-xylosidase potential activity after the incubation period and prior to stress exposure. The 10% biochar rate reduced soil water loss at the lowest stress level (400 J microwave energy g(-1) soil). Enzyme stabilization was demonstrated for β-xylosidase; intermediate biochar application rates prevented a complete loss of this enzyme's potential activity after soil was exposed to 400 (1% biochar treatment) or 1600 (5% biochar treatment) J microwave energy g(-1) soil. Remaining enzyme potential activities were not affected by biochar, and activities decreased with increasing stress levels. We concluded that biochar has the potential to reduce evaporative soil water losses and stabilize certain extracellular enzymes where activity is maintained after a denaturing stress; this effect was biochar rate and enzyme dependent. While biochar may reduce the potential activity of certain soil extracellular enzymes, this phenomenon was not universal as the majority of enzymes assayed in this study were unaffected by exposure to biochar.

  10. Detection of enzymes active on various beta-1,3-glucans after denaturing polyacrylamide gel electrophoresis.

    Trudel, J; Grenier, J; Asselin, A


    Enzymes were assayed for glucanase activity after denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in gels containing beta-1,3-glucans embedded as substrate. Lentinan, curdlan, paramylon, baker's yeast alkali-insoluble glucan, baker's yeast alkali-soluble glucan and carboxymethyl (CM)-pachyman were compared to oligomeric laminarin, which is the usual substrate for assaying beta-1,3-glucanase activities. Detecting enzyme activities by aniline blue fluorescent staining was also compared with the staining of released reducing sugars by 2,3,5-triphenyltetrazolium chloride (TTC). For the nonreduced proteins, the Driselase extract exhibited one major band at 32.5 kDa and one less intense band at 23 kDa for most substrates with the two detection procedures. No Lyticase enzyme was detected in either detection procedures for all tested substrates. For barley enzymes, no activity was revealed after aniline blue staining while one undescribed 19 kDa glucanase activity was best shown after TTC staining with curdlan, paramylon and CM-pachyman as substrates. In the case of reduced proteins, the Lyticase extract yielded three bands (33, 36 and 46 kDa) on several substrates with both detection procedures. This was the same for the barley leaf extract (32, 36 and 39 kDa). The Driselase extract showed one 42 kDa band. Many enzymes active on beta-1,3-glucans are thus best revealed when proteins are denatured and reduced and when protein renaturation after SDS-PAGE involves a pH 8.0 treatment and the inclusion of 1 mM cysteine in buffers. However, some enzymes are only detected when proteins are denatured without reduction. Finally, the use of various polymeric beta-1,3-glucan substrates different from oligomeric laminarin is necessary to detect new types of enzymes such as the 19 kDa barley glucanase.

  11. Screening for mutations in the uroporphyrinogen decarboxylase gene using denaturing gradient gel electrophoresis

    Christiansen, L; Ged, C; Hombrados, I


    The two porphyrias, familial porphyria cutanea tarda (fPCT) and hepatoerythropoietic porphyria (HEP), are associated with mutations in the gene encoding the enzyme uroporphyrinogen decarboxylase (UROD). Several mutations, most of which are private, have been identified in HEP and fPCT patients......, confirming the heterogeneity of the underlying genetic defects of these diseases. We have established a denaturing gradient gel electrophoresis (DGGE) assay for mutation detection in the UROD gene, enabling the simultaneous screening for known and unknown mutations. The established assay has proved able...

  12. Light and Heat Induced Denaturation of Photosystem Ⅱ Core Antenna Complex CP47


    Light and heat induced denaturation of CP47, the core antenna complex of photosystem Ⅱ purified from spinach, were investigated using absorption and circular dichroism spectra.Light caused the destruction of chlorophyll a and excitonic interaction of chlorophyll a in CP47, while the protein secondary structure was not apparently changed.Heat induced the destruction of protein secondary structure and excitonic interaction of chlorophyll a, but the chlorophyll a molecule was not damaged.The results suggest that both the chlorophyll a molecular structure and the protein native conformation are necessary for excitonic interaction of chlorophyll a and the energy transfer function of the chlorophyll a binding protein.

  13. Optical Tweezers Analysis of Double-Stranded DNA Denaturation in the Presence of Urea

    Zhu, Chunli; Li, Jing


    Urea is a kind of denaturant prone to form hydrogen bonds with the electronegative centers of the nitrogenous bases, threatening the stability of hydrogen bonds between DNA base pairs. In this paper, the stability and stiffness of DNA double helix influenced by urea are investigated at single-molecule level using optical tweezers. Experimental results show that DNA's double helix stability and stiffness both decrease with increasing urea concentration. In addition, the re-forming of ruptured hydrogen bonds between the base pairs is blocked by urea as the tension on DNA is released.

  14. Two-dimensional salt and temperature DNA denaturation analysis using a magnetoresistive sensor

    Rizzi, Giovanni; Dufva, Martin; Hansen, Mikkel Fougt


    . The results demonstrate that concentration melting provides an attractive alternative to temperature melting in on-chip DNA denaturation experiments and further show that the magnetoresistive platform is attractive due to its low cross-sensitivity to temperature and liquid composition....... temperature as well as salt concentration. The salt concentration melting curves were found to be more reliable than temperature melting curves. We performed a two-dimensional mapping of the melting profiles of a target to probes targeting its wild type (WT) and mutant type (MT) variants in the temperature...

  15. Immobilization of denatured DNA to macroporous supports: II. Steric and kinetic parameters of heterogeneous hybridization reactions.

    Bünemann, H


    The accessibility of immobilized DNA has been shown to depend more crucially on the method of immobilization than on the type of support used for fixation. When sonicated denatured DNA is coupled via diazotization or via cyanogen bromide reaction to solid Sephadex G-25 and Cellex 410 or to macroporous Sephacryl S-500 and Sepharose C1-6B its accessibility varies from 100 to 24 percent. Generally the loss of accessibility is linked to a depression of the melting temperature of DNA helices forme...

  16. Glassy behavior of denatured DNA films studied by differential scanning calorimetry.

    Valle-Orero, Jessica; Garden, Jean-Luc; Richard, Jacques; Wildes, Andrew; Peyrard, Michel


    We use differential scanning calorimetry (DSC) to study the properties of DNA films, made of oriented fibers, heated above the thermal denaturation temperature of the double helical form. The films show glassy properties that we investigate in two series of experiments, a slow cooling at different rates followed by a DSC scan upon heating and aging at a temperature below the glass transition. Introducing the fictive temperature to characterize the glass allows us to derive quantitative information on the relaxations of the DNA films, in particular to evaluate their enthalpy barrier. A comparison with similar aging studies on PVAc highlights some specificities of the DNA samples.

  17. Effect of heating strategies on whey protein denaturation--Revisited by liquid chromatography quadrupole time-of-flight mass spectrometry.

    Akkerman, M; Rauh, V M; Christensen, M; Johansen, L B; Hammershøj, M; Larsen, L B


    Previous standards in the area of effect of heat treatment processes on milk protein denaturation were based primarily on laboratory-scale analysis and determination of denaturation degrees by, for example, electrophoresis. In this study, whey protein denaturation was revisited by pilot-scale heating strategies and liquid chromatography quadrupole time-of-flight mass spectrometer (LC/MC Q-TOF) analysis. Skim milk was heat treated by the use of 3 heating strategies, namely plate heat exchanger (PHE), tubular heat exchanger (THE), and direct steam injection (DSI), under various heating temperatures (T) and holding times. The effect of heating strategy on the degree of denaturation of β-lactoglobulin and α-lactalbumin was determined using LC/MC Q-TOF of pH 4.5-soluble whey proteins. Furthermore, effect of heating strategy on the rennet-induced coagulation properties was studied by oscillatory rheometry. In addition, rennet-induced coagulation of heat-treated micellar casein concentrate subjected to PHE was studied. For skim milk, the whey protein denaturation increased significantly as T and holding time increased, regardless of heating method. High denaturation degrees were obtained for T >100°C using PHE and THE, whereas DSI resulted in significantly lower denaturation degrees, compared with PHE and THE. Rennet coagulation properties were impaired by increased T and holding time regardless of heating method, although DSI resulted in less impairment compared with PHE and THE. No significant difference was found between THE and PHE for effect on rennet coagulation time, whereas the curd firming rate was significantly larger for THE compared with PHE. Micellar casein concentrate possessed improved rennet coagulation properties compared with skim milk receiving equal heat treatment.

  18. Comparative study of denaturation of whey protein isolate (WPI) in convective air drying and isothermal heat treatment processes.

    Haque, M Amdadul; Aldred, Peter; Chen, Jie; Barrow, Colin J; Adhikari, Benu


    The extent and nature of denaturation of whey protein isolate (WPI) in convective air drying environments was measured and analysed using single droplet drying. A custom-built, single droplet drying instrument was used for this purpose. Single droplets having 5±0.1μl volume (initial droplet diameter 1.5±0.1mm) containing 10% (w/v) WPI were dried at air temperatures of 45, 65 and 80°C for 600s at constant air velocity of 0.5m/s. The extent and nature of denaturation of WPI in isothermal heat treatment processes was measured at 65 and 80°C for 600s and compared with those obtained from convective air drying. The extent of denaturation of WPI in a high hydrostatic pressure environment (600MPa for 600s) was also determined. The results showed that at the end of 600s of convective drying at 65°C the denaturation of WPI was 68.3%, while it was only 10.8% during isothermal heat treatment at the same medium temperature. When the medium temperature was maintained at 80°C, the denaturation loss of WPI was 90.0% and 68.7% during isothermal heat treatment and convective drying, respectively. The bovine serum albumin (BSA) fraction of WPI was found to be more stable in the convective drying conditions than β-lactoglobulin and α-lactalbumin, especially at longer drying times. The extent of denaturation of WPI in convective air drying (65 and 80°C) and isotheral heat treatment (80°C) for 600s was found to be higher than its denaturation in a high hydrostatic pressure environment at ambient temperature (600MPa for 600s).

  19. Air leak seal for lung dissection plane with diode laser irradiation: monitoring heat-denature with auto-fluorescence

    Gotoh, Maya; Arai, Tsunenori


    We studied the monitoring of heat-denature by autofluorescence spectrum from lung dissection plane during laser air leak sealing procedure. In order to seal the air leakage from lung in thoracotomy, we proposed novel laser sealing method with the combination of the diode laser (810nm wavelength) irradiation and indocyanine green staining (peak absorption wavelength: 805 nm). This sealing method is expected to preserve the postoperative ventilatory capacity and achieve minimally invasive surgery. We previously reported that this laser sealing only requires thin sealing margin (less than 300 μm in thickness) compared with that of the suturing or stapling. The most serious issue on the laser air leak sealing might be re-air-leakage due to rigid surface layer caused by excessive heat-denature, such as carbonization. We should achieve laser air leak sealing minimizing the degree of heat denature. Dissection planes of isolated porcine lung with /without the diode laser irradiation were prepared as samples. We measured the auto-fluorescence from these samples using a spectrometer. When the diode laser was irradiated with 400J/cm2, the surface of diode laser irradiated lung was fully carbonized. The ration of auto-fluorescence emission of 450nm / 500 nm, with 280 nm excitation wavelength was decreased less tha 50 % of initial value. That of 600 nm / 500 nm was increased over 700 % of initial value. The decreasing of the 450 nm auto-fluorescence intensity might be attributed to the heat-denaturing of the interstitial collagen in lung. However, increasing of the 600 nm didn't specify the origins, we suppose it might be originated from heat-denature substance, like carbonization. We could establish the useful monitoring for lung heat-denaturing with simple methodology. We think the auto-fluorescence measurement can be helpful not only for understanding the sealing mechanism, but also for controlling the degree of heat-denaturing during the procedure.

  20. International recommendation for a comprehensive neuropathologic workup of epilepsy surgery brain tissue: A consensus Task Force report from the ILAE Commission on Diagnostic Methods.

    Blümcke, Ingmar; Aronica, Eleonora; Miyata, Hajime; Sarnat, Harvey B; Thom, Maria; Roessler, Karl; Rydenhag, Bertil; Jehi, Lara; Krsek, Pavel; Wiebe, Samuel; Spreafico, Roberto


    Epilepsy surgery is an effective treatment in many patients with drug-resistant focal epilepsies. An early decision for surgical therapy is facilitated by a magnetic resonance imaging (MRI)-visible brain lesion congruent with the electrophysiologically abnormal brain region. Recent advances in the pathologic diagnosis and classification of epileptogenic brain lesions are helpful for clinical correlation, outcome stratification, and patient management. However, application of international consensus classification systems to common epileptic pathologies (e.g., focal cortical dysplasia [FCD] and hippocampal sclerosis [HS]) necessitates standardized protocols for neuropathologic workup of epilepsy surgery specimens. To this end, the Task Force of Neuropathology from the International League Against Epilepsy (ILAE) Commission on Diagnostic Methods developed a consensus standard operational procedure for tissue inspection, distribution, and processing. The aims are to provide a systematic framework for histopathologic workup, meeting minimal standards and maximizing current and future opportunities for morphofunctional correlations and molecular studies for both clinical care and research. Whenever feasible, anatomically intact surgical specimens are desirable to enable systematic analysis in selective hippocampectomies, temporal lobe resections, and lesional or nonlesional neocortical samples. Correct orientation of sample and the sample's relation to neurophysiologically aberrant sites requires good communication between pathology and neurosurgical teams. Systematic tissue sampling of 5-mm slabs along a defined anatomic axis and application of a limited immunohistochemical panel will ensure a reliable differential diagnosis of main pathologies encountered in epilepsy surgery.

  1. Molecular monitoring of the intestinal flora by denaturing high performance liquid chromatography.

    Goldenberg, Oliver; Herrmann, Stefanie; Marjoram, Gina; Noyer-Weidner, Mario; Hong, George; Bereswill, Stefan; Göbel, Ulf B


    Gut flora analysis is hampered by the complexity of the intestinal microbiota and by inherent limitations of culture-based approaches. Therefore, culture-independent molecular methods based upon 16S rRNA gene analysis were applied successfully for the analysis of complex microbial communities. However, generally accepted and validated profiling methods such as denaturing and temperature gradient gel electrophoresis (DGGE/TGGE) are still laborious and time consuming. Thus, we adapted the separation of amplified bacterial 16S rRNA gene fragments by denaturing high performance liquid chromatography (DHPLC) using the WAVE Microbial Analysis System as a rapid and convenient means to display complex intestinal bacterial communities and to monitor changes in the gut flora. The separation of 16S rRNA gene fragments amplified from reference strains representing main gut bacterial populations and from human stool samples revealed that DHPLC analysis effectively detects bacterial groups predominant in the human gut flora. The investigation of faecal samples from hospitalized patients before, during and after antibiotic therapy showed that PCR-based DHPLC can be used to monitor gut flora changes. Results from DHPLC analysis were comparable with DGGE profiles generated from the same samples, demonstrating that the adapted DHPLC protocol is well suited for the analysis of complex microbial communities.

  2. Effect of sulfoxides on the thermal denaturation of hen lysozyme: A calorimetric and Raman study

    Torreggiani, A.; Di Foggia, M.; Manco, I.; De Maio, A.; Markarian, S. A.; Bonora, S.


    A multidisciplinary study of the thermal denaturation of lysozyme in the presence of three sulfoxides with different length in hydrocarbon chain (DMSO, DESO, and DPSO) was carried out by means of DSC, Raman spectroscopy, and SDS-PAGE techniques. In particular, the Td and Δ H values obtained from the calorimetric measurements showed that lysozyme is partially unfolded by sulfoxides but most of the conformation holds native state. The sulfoxide denaturing ability increases in the order DPSO > DESO > DMSO. Moreover, only DMSO and DESO have a real effect in preventing the heat-induced inactivation of the protein and their maximum heat-protective ability is reached when the DMSO and DESO amount is ⩾25% w/w. The sulfoxide ability to act as effective protective agents against the heat-induced inactivation was confirmed by the protein analysis. The enzymatic activity, as well as the SDS-PAGE analysis, suggested that DESO, having a low hydrophobic character and a great ability to stabilise the three-dimensional water structure, is the most heat-protective sulfoxide. An accurate evaluation of the heat-induced conformational changes of the lysozyme structure before and after sulfoxide addition was obtained by the analysis of the Raman spectra. The addition of DMSO or DESO in low concentration resulted to sensitively decrease the heat-induced structural modifications of the protein.

  3. Two conformational states in D-shaped DNA: Effects of local denaturation

    Lee, O.-Chul; Kim, Cheolhee; Kim, Jae-Yeol; Lee, Nam Ki; Sung, Wokyung


    The bending of double-stranded(ds) DNA on the nano-meter scale plays a key role in many cellular processes such as nucleosome packing, transcription-control, and viral-genome packing. In our recent study, a nanometer-sized dsDNA bent into a D shape was formed by hybridizing a circular single-stranded(ss) DNA and a complementary linear ssDNA. Our fluorescence resonance energy transfer (FRET) measurement of D-DNA revealed two types of conformational states: a less-bent state and a kinked state, which can transform into each other. To understand the origin of the two deformed states of D-DNA, here we study the presence of open base-pairs in the ds portion by using the breathing-DNA model to simulate the system. We provide strong evidence that the two states are due to the emergence of local denaturation, i.e., a bubble in the middle and two forks at ends of the dsDNA portion. We also study the system analytically and find that the free-energy landscape is bistable with two minima representative of the two states. The kink and fork sizes estimated by the analytical calculation are also in excellent agreement with the results of the simulation. Thus, this combined experimental-simulation-analytical study corroborates that highly bent D-DNA reduces bending stress via local denaturation.

  4. Ultrafast folding of WW domains without structured aromatic clusters in the denatured state.

    Ferguson, N; Johnson, C M; Macias, M; Oschkinat, H; Fersht, A


    Ultrafast-folding proteins are important for combining experiment and simulation to give complete descriptions of folding pathways. The WW domain family comprises small proteins with a three-stranded antiparallel beta-sheet topology. Previous studies on the 57-residue YAP 65 WW domain indicate the presence of residual structure in the chemically denatured state. Here we analyze three minimal core WW domains of 38-44 residues. There was little spectroscopic or thermodynamic evidence for residual structure in either their chemically or thermally denatured states. Folding and unfolding kinetics, studied by using rapid temperature-jump and continuous-flow techniques, show that each domain folds and unfolds very rapidly in a two-state transition through a highly compact transition state. Folding half-times were as short as 17 micros at 25 degrees C, within an order of magnitude of the predicted maximal rate of loop formation. The small size and topological simplicity of these domains, in conjunction with their very rapid two-state folding, may allow us to reduce the difference in time scale between experiment and theoretical simulation.

  5. Increasing protein stability: Importance of ΔCp and the denatured state

    Fu, Hailong; Grimsley, Gerald; Scholtz, J Martin; Pace, C Nick


    Increasing the conformational stability of proteins is an important goal for both basic research and industrial applications. In vitro selection has been used successfully to increase protein stability, but more often site-directed mutagenesis is used to optimize the various forces that contribute to protein stability. In previous studies, we showed that improving electrostatic interactions on the protein surface and improving the β-turn sequences were good general strategies for increasing protein stability, and used them to increase the stability of RNase Sa. By incorporating seven of these mutations in RNase Sa, we increased the stability by 5.3 kcal/mol. Adding one more mutation, D79F, gave a total increase in stability of 7.7 kcal/mol, and a melting temperature 28°C higher than the wild-type enzyme. Surprisingly, the D79F mutation lowers the change in heat capacity for folding, ΔCp, by 0.6 kcal/mol/K. This suggests that this mutation stabilizes structure in the denatured state ensemble. We made other mutants that give some insight into the structure present in the denatured state. Finally, the thermodynamics of folding of these stabilized variants of RNase Sa are compared with those observed for proteins from thermophiles. PMID:20340133

  6. Culture-independent analysis of probiotic products by denaturing gradient gel electrophoresis.

    Temmerman, R; Scheirlinck, I; Huys, G; Swings, J


    In order to obtain functional and safe probiotic products for human consumption, fast and reliable quality control of these products is crucial. Currently, analysis of most probiotics is still based on culture-dependent methods involving the use of specific isolation media and identification of a limited number of isolates, which makes this approach relatively insensitive, laborious, and time-consuming. In this study, a collection of 10 probiotic products, including four dairy products, one fruit drink, and five freeze-dried products, were subjected to microbial analysis by using a culture-independent approach, and the results were compared with the results of a conventional culture-dependent analysis. The culture-independent approach involved extraction of total bacterial DNA directly from the product, PCR amplification of the V3 region of the 16S ribosomal DNA, and separation of the amplicons on a denaturing gradient gel. Digital capturing and processing of denaturing gradient gel electrophoresis (DGGE) band patterns allowed direct identification of the amplicons at the species level. This whole culture-independent approach can be performed in less than 30 h. Compared with culture-dependent analysis, the DGGE approach was found to have a much higher sensitivity for detection of microbial strains in probiotic products in a fast, reliable, and reproducible manner. Unfortunately, as reported in previous studies in which the culture-dependent approach was used, a rather high percentage of probiotic products suffered from incorrect labeling and yielded low bacterial counts, which may decrease their probiotic potential.

  7. Immobilization of denatured DNA to macroporous supports: II. Steric and kinetic parameters of heterogeneous hybridization reactions.

    Bünemann, H


    The accessibility of immobilized DNA has been shown to depend more crucially on the method of immobilization than on the type of support used for fixation. When sonicated denatured DNA is coupled via diazotization or via cyanogen bromide reaction to solid Sephadex G-25 and Cellex 410 or to macroporous Sephacryl S-500 and Sepharose C1-6B its accessibility varies from 100 to 24 percent. Generally the loss of accessibility is linked to a depression of the melting temperature of DNA helices formed on the support. This correlation shows a characteristic course for a particular coupling method. DNA coupled under denaturing conditions may become totally inaccessible when only 3 percent of its bases are involved in the covalent linkage. Kinetic experiments with sonicated E.coli DNA have shown that the rate constants for renaturation or hybridization reactions are very similar for DNA immobilized by different methods to solid or macroporous supports. Generally the second order rate constant for a heterogeneous reaction (between mobile and immobilized DNA) is about one order of magnitude smaller than that of the analogous homogeneous reaction (in solution).

  8. Urea-induced denaturation of apolipoprotein serum amyloid A reveals marginal stability of hexamer

    Wang, Limin; Colón, Wilfredo


    Serum Amyloid A (SAA) is an acute phase reactant protein that is predominantly found bound to high-density lipoprotein in plasma. Upon inflammation, the plasma concentration of SAA can increase dramatically, occasionally leading to the development of amyloid A (AA) amyloidosis, which involves the deposition of SAA amyloid fibrils in major organs. We previously found that the murine isoform SAA2.2 exists in aqueous solution as a hexamer containing a central channel. Here we show using various biophysical and biochemical techniques that the SAA2.2 hexamer can be totally dissociated into monomer by ~2 M urea, with the concerted loss of its α-helical structure. However, limited trypsin proteolysis experiments in urea showed a conserved digestion profile, suggesting the preservation of major backbone topological features in the urea-denatured state of SAA2.2. The marginal stability of hexameric SAA2.2 and the presence of residual structure in the denatured monomeric protein suggest that both forms may interconvert in vivo to exert different functions to meet the various needs during normal physiological conditions and in response to inflammatory stimuli. PMID:15937280

  9. Urea denatured state ensembles contain extensive secondary structure that is increased in hydrophobic proteins

    Nick Pace, C; Huyghues-Despointes, Beatrice M P; Fu, Hailong; Takano, Kazufumi; Scholtz, J Martin; Grimsley, Gerald R


    The goal of this article is to gain a better understanding of the denatured state ensemble (DSE) of proteins through an experimental and computational study of their denaturation by urea. Proteins unfold to different extents in urea and the most hydrophobic proteins have the most compact DSE and contain almost as much secondary structure as folded proteins. Proteins that unfold to the greatest extent near pH 7 still contain substantial amounts of secondary structure. At low pH, the DSE expands due to charge–charge interactions and when the net charge per residue is high, most of the secondary structure is disrupted. The proteins in the DSE appear to contain substantial amounts of polyproline II conformation at high urea concentrations. In all cases considered, including staph nuclease, the extent of unfolding by urea can be accounted for using the data and approach developed in the laboratory of Wayne Bolen (Auton et al., Proc Natl Acad Sci 2007; 104:15317–15323). PMID:20198681

  10. Approach to analysis of single nucleotide polymorphisms by automated constant denaturant capillary electrophoresis

    Bjoerheim, Jens; Abrahamsen, Torveig Weum; Kristensen, Annette Torgunrud; Gaudernack, Gustav; Ekstroem, Per O


    Melting gel techniques have proven to be amenable and powerful tools in point mutation and single nucleotide polymorphism (SNP) analysis. With the introduction of commercially available capillary electrophoresis instruments, a partly automated platform for denaturant capillary electrophoresis with potential for routine screening of selected target sequences has been established. The aim of this article is to demonstrate the use of automated constant denaturant capillary electrophoresis (ACDCE) in single nucleotide polymorphism analysis of various target sequences. Optimal analysis conditions for different single nucleotide polymorphisms on ACDCE are evaluated with the Poland algorithm. Laboratory procedures include only PCR and electrophoresis. For direct genotyping of individual SNPs, the samples are analyzed with an internal standard and the alleles are identified by co-migration of sample and standard peaks. In conclusion, SNPs suitable for melting gel analysis based on theoretical thermodynamics were separated by ACDCE under appropriate conditions. With this instrumentation (ABI 310 Genetic Analyzer), 48 samples could be analyzed without any intervention. Several institutions have capillary instrumentation in-house, thus making this SNP analysis method accessible to large groups of researchers without any need for instrument modification.

  11. Exact Solution to the Extended Zwanzig Model for Quasi-Sigmoidal Chemically Induced Denaturation Profiles: Specific Heat and Configurational Entropy

    G. E. Aguilar-Pineda


    Full Text Available Temperature and chemically induced denaturation comprise two of the most characteristic mechanisms to achieve the passage from the native state N to any of the unstructured states Dj in the denatured ensemble in proteins and peptides. In this work we present a full analytical solution for the configurational partition function qs of a homopolymer chain poly-X in the extended Zwanzig model (EZM for a quasisigmoidal denaturation profile. This solution is built up from an EZM exact solution in the case where the fraction α of native contacts follows exact linear dependence on denaturant’s concentration ζ; thus an analytical solution for L in the case of an exact linear denaturation profile is also provided. A recently established connection between the number ν of potential nonnative conformations per residue and temperature-independent helical propensity ω complements the model in order to identify specific proteinogenic poly-X chains, where X represents any of the twenty naturally occurring aminoacid residues. From qs, equilibrium thermodynamic potentials like entropy and average internal energy 〈E〉 and thermodynamic susceptibilities like specific heat C are calculated for poly-valine (poly-V and poly-alanine (poly-A chains. The influence of the rate at which native contacts denature as function of ζ on thermodynamic stability is also discussed.

  12. Effects of thermally induced denaturation on technological-functional properties of whey protein isolate-based films.

    Schmid, M; Krimmel, B; Grupa, U; Noller, K


    This study examined how and to what extent the degree of denaturation affected the technological-functional properties of whey protein isolate (WPI)-based coatings. It was observed that denaturation affected the material properties of WPI-coated films significantly. Surface energy decreased by approximately 20% compared with native coatings. Because the surface energy of a coating should be lower than that of the substrate, this might result in enhanced wettability characteristics between WPI-based solution and substrate surface. Water vapor barrier properties increased by about 35% and oxygen barrier properties increased by approximately 33%. However, significant differences were mainly observed between coatings made of fully native WPI and ones with a degree of denaturation of 25%. Higher degrees of denaturation did not lead to further improvement of material properties. This observation offers cost-saving potential: a major share of denatured whey proteins may be replaced by fully native ones that are not exposed to energy-intensive heat treatment. Furthermore, native WPI solutions can be produced with higher dry matter content without gelatinizing. Hence, less moisture has to be removed through drying, resulting in reduced energy consumption.

  13. Denaturation and Oxidative Stability of Hemp Seed (Cannabis sativa L.) Protein Isolate as Affected by Heat Treatment.

    Raikos, Vassilios; Duthie, Garry; Ranawana, Viren


    The present study investigated the impact of heat treatments on the denaturation and oxidative stability of hemp seed protein during simulated gastrointestinal digestion (GID). Heat-denatured hemp protein isolate (HPI) solutions were prepared by heating HPI (2 mg/ml, pH 6.8) to 40, 60, 80 and 100 °C for 10 min. Heat-induced denaturation of the protein isolates was monitored by polyacrylamide gel electrophoresis. Heating HPI at temperatures above 80 °C significantly reduced solubility and led to the formation of large protein aggregates. The isolates were then subjected to in vitro GID and the oxidative stability of the generated peptides was investigated. Heating did not significantly affect the formation of oxidation products during GID. The results suggest that heat treatments should ideally remain below 80 °C if heat stability and solubility of HPI are to be preserved.

  14. Achieving continuous manufacturing: technologies and approaches for synthesis, workup, and isolation of drug substance. May 20-21, 2014 Continuous Manufacturing Symposium.

    Baxendale, Ian R; Braatz, Richard D; Hodnett, Benjamin K; Jensen, Klavs F; Johnson, Martin D; Sharratt, Paul; Sherlock, Jon-Paul; Florence, Alastair J


    This whitepaper highlights current challenges and opportunities associated with continuous synthesis, workup, and crystallization of active pharmaceutical ingredients (drug substances). We describe the technologies and requirements at each stage and emphasize the different considerations for developing continuous processes compared with batch. In addition to the specific sequence of operations required to deliver the necessary chemical and physical transformations for continuous drug substance manufacture, consideration is also given to how adoption of continuous technologies may impact different manufacturing stages in development from discovery, process development, through scale-up and into full scale production. The impact of continuous manufacture on drug substance quality and the associated challenges for control and for process safety are also emphasized. In addition to the technology and operational considerations necessary for the adoption of continuous manufacturing (CM), this whitepaper also addresses the cultural, as well as skills and training, challenges that will need to be met by support from organizations in order to accommodate the new work flows. Specific action items for industry leaders are: Develop flow chemistry toolboxes, exploiting the advantages of flow processing and including highly selective chemistries that allow use of simple and effective continuous workup technologies. Availability of modular or plug and play type equipment especially for workup to assist in straightforward deployment in the laboratory. As with learning from other industries, standardization is highly desirable and will require cooperation across industry and academia to develop and implement. Implement and exploit process analytical technologies (PAT) for real-time dynamic control of continuous processes. Develop modeling and simulation techniques to support continuous process development and control. Progress is required in multiphase systems such as

  15. Biological function evaluation and effects of laser micro-pore burn-denatured acellular dermal matrix.

    Zhang, Youlai; Zeng, Yuanlin; Xin, Guohua; Zou, Lijin; Ding, Yuewei; Duyin, Jiang


    In the field of burns repairs, many problems exist in the shortage of donor skin, the expense of allograft or xenograft skin, temporary substitution and unsatisfactory extremity function after wound healing. Previous studies showed that burn-denatured skin could return to normal dermis formation and function. This study investigates the application of laser micro-pore burn-denatured acellular dermis matrix (DADM) from an escharotomy in the repair of burn wounds and evaluates the biological properties and wound repair effects of DADM in implantation experiments in Kunming mice. Specific-pathogen-free (SPF) Kunming mice were used in this study. A deep II° burn wound was created on the dorsum of the mice by an electric heated water bath. The full-thickness wound tissue was harvested. The necrotic tissue and subcutaneous tissue were removed. The denatured dermis was preserved and treated with 0.25% trypsin, 0.5% Triton X-100. The DADM was drilled by laser micro-pore. The biological properties and grafting effects of laser micro-pore burn-DADM were evaluated by morphology, cytokine expression levels and subcutaneous implantation experiments in Kunming mice. We found statistical significance (Plaser micro-pore burn-DADM (experimental group) compared to the control group (no laser micro-pore burn-DADM). Cytokine expression level was different in the dermal matrixes harvested at various time points after burn (24h, 48h, 72h and infected wound group). Comparing the dermal matrix from 24h burn tissue to infected wound tissue, the expression level of IL-6, MMP-24, VE-cadherin and VEGF were decreased. We found no inflammatory cells infiltration in the dermal matrix were observed in both experimental and control groups (24h burn group), while the obviously vascular infiltration and fiber fusion were observed in the experimental group after subcutaneous implantation experiments. There was better bio-performance, low immunogenicity and better dermal incorporation after treated

  16. The Challenge of Prenatal Diagnostic Work-Up of Maternally Inherited X-Linked Opitz G/BBB: Case Report and Literature Review.

    Spinelli, Marialuigia; Sica, Carmine; Dallapiccola, Bruno; Novelli, Antonio; Di Meglio, Letizia; Martinelli, Pasquale


    Background. Prenatal diagnosis of Optiz G/BBB syndrome (OS) is challenging because the characteristic clinical features, such as facial and genitourinary anomalies, may be subtle at sonography and rather unspecific. Furthermore, molecular testing of the disease gene is not routinely performed, unless a specific diagnosis is suggested. Method. Both familial and ultrasound data were used to achieve the diagnosis of X-linked OS (XLOS), which was confirmed by molecular testing of MID1 gene (Xp22.3) at birth. Results. Sequencing of MID1 gene disclosed the nucleotide change c.1285 +1 G>T, previously associated with XLOS. Conclusions. This case illustrates current challenges of the prenatal diagnostic work-up of XLOS and exemplifies how clinical investigation, including family history, and accurate US foetal investigations can lead to the correct diagnosis.

  17. Multidetector computed tomography angiography in clinically suspected hyperacute ischemic stroke in the anterior circulation: an etiological workup in a cohort of Brazilian patients

    Felipe Torres Pacheco


    Full Text Available Objective The potential of computed tomography angiography (CTA was assessed for early determination of stroke subtypes in a Brazilian cohort of patients with stroke. Method From July 2011 to July 2013, we selected patients with suspected hyperacute stroke (< 6 hours. Intracranial and cervical arteries were scrutinized on CTA and their imaging features were correlated with concurrent subtype of stroke. Results Stroke was documented in 50/106 selected patients (47.2% based on both clinical grounds and imaging follow-up (stroke group, with statistically significant arterial stenosis and vulnerable plaques on CTA. Intracranial large artery disease was demonstrated in 34% of patients in the stroke group. Partial territorial infarct prevailed (86% while artery-to-artery embolization was the most common stroke mechanism (52%. Conclusion Multidetector CTA was useful for the etiologic work-up of hyperacute ischemic stroke and facilitated the knowledge about the topographic pattern of brain infarct in accordance with its causative mechanism.

  18. The Challenge of Prenatal Diagnostic Work-Up of Maternally Inherited X-Linked Opitz G/BBB: Case Report and Literature Review

    Marialuigia Spinelli


    Full Text Available Background. Prenatal diagnosis of Optiz G/BBB syndrome (OS is challenging because the characteristic clinical features, such as facial and genitourinary anomalies, may be subtle at sonography and rather unspecific. Furthermore, molecular testing of the disease gene is not routinely performed, unless a specific diagnosis is suggested. Method. Both familial and ultrasound data were used to achieve the diagnosis of X-linked OS (XLOS, which was confirmed by molecular testing of MID1 gene (Xp22.3 at birth. Results. Sequencing of MID1 gene disclosed the nucleotide change c.1285 +1 G>T, previously associated with XLOS. Conclusions. This case illustrates current challenges of the prenatal diagnostic work-up of XLOS and exemplifies how clinical investigation, including family history, and accurate US foetal investigations can lead to the correct diagnosis.

  19. Can value for money be improved by changing the sequence of our donor work-up in the living kidney donor programme?

    Larsen, J.; Sorensen, S.S.; Feldt-Rasmussen, B.


    (range 22-69). Sixty-four participants were rejected as donors. Abdominal CT-scan with angiography and urography ruled out 22 of the above 64 potential organ donors; thus, 48% of the volunteers for living kidney donation were unsuited for donation. Abdominal CT-scan with angiography and urography......The aim of the study was to identify procedures of maximum importance for acceptance or rejection of kidney donation from a living donor as well as making the process more cost-effective. We identified all potential living related donors who were examined during the period between January 2002...... was the procedure identifying most subjects who were unsuited for kidney donation. A rearrangement of the present donor work-up programme could potentially reduce the costs from euro6911 to euro5292 per donor--saving 23% of the costs. By changing the sequence of examinations, it might be possible to cut down...

  20. Light flux density threshold at which protein denaturation is induced by synchrotron radiation circular dichroism beamlines.

    Miles, A J; Janes, Robert W; Brown, A; Clarke, D T; Sutherland, J C; Tao, Y; Wallace, B A; Hoffmann, S V


    New high-flux synchrotron radiation circular dichroism (SRCD) beamlines are providing important information for structural biology, but can potentially cause denaturation of the protein samples under investigation. This effect has been studied at the new CD1 dedicated SRCD beamline at ISA in Denmark, where radiation-induced thermal damage effects were observed, depending not only on the radiation flux but also on the focal spot size of the light. Comparisons with similar studies at other SRCD facilities worldwide has lead to the estimation of a flux density threshold under which SRCD beamlines should be operated when samples are to be exposed to low-wavelength vacuum ultraviolet radiation for extended periods of time.

  1. Third-strand in situ hybridization (TISH) to non-denatured metaphase spreads and interphase nuclei.

    Johnson, M D; Fresco, J R


    A methodology has been developed for binding oligodeoxyribonucleotide 'third strands' to duplex DNA targets in fixed but not additionally denatured metaphase spreads and interphase nuclei under conditions found to be optimal in solution. Third-strand in situ hybridization (TISH) at pH 6.0 of a psoralen- and biotin-modified 16-nucleotide homopyrimidine third strand to a unique multicopy target sequence in human chromosome 17 alpha-satellite (D17Z1 locus) is described. UVA-photofixed third strands, rendered fluorescent by fluorescein isothiocyanate-labeled avidin, are reproducibly centromere-specific for chromosome 17, and visible without amplification of the signal in lymphocyte and somatic cell hybrid spreads and interphase nuclei. Two third-strand-specific D17Z1 haplotypes were identified. TISH has potential diagnostic, biochemical, and flow cytometric applicability to native metaphase and interphase chromatin.

  2. Fuel utilization improvement in PWRs using the denatured /sup 233/U-Th cycle

    Jones, H.M.; Schwenk, G.A.; Toops, E.C.; Yotinen, V.O.


    A number of changes in PWR core design and/or operating strategy were evaluated to assess the fuel utilization improvement achievable by their implementation in a PWR using thorium-based fuel and operating in a recycle mode. The reference PWR for this study was identical to the B and W Standard Plant except that the fuel pellets were of denatured (/sup 233/U//sup 238/U-Th)O/sub 2/. An initial scoping study identified the three most promising improvement concepts as (1) a very tight lattice, (2) thorium blankets, and (3) ThO/sub 2/ rods placed in available guide tubes. A conceptual core design incorporating these changes was then developed, and the fuel utilization of this modified design was compared with that of the reference case.

  3. Denatured G-protein coupled receptors as immunogens to generate highly specific antibodies.

    Talmont, Franck; Moulédous, Lionel; Boué, Jérôme; Mollereau, Catherine; Dietrich, Gilles


    G-protein coupled receptors (GPCRs) play a major role in a number of physiological and pathological processes. Thus, GPCRs have become the most frequent targets for development of new therapeutic drugs. In this context, the availability of highly specific antibodies may be decisive to obtain reliable findings on localization, function and medical relevance of GPCRs. However, the rapid and easy generation of highly selective anti-GPCR antibodies is still a challenge. Herein, we report that highly specific antibodies suitable for detection of GPCRs in native and unfolded forms can be elicited by immunizing animals against purified full length denatured recombinant GPCRs. Contrasting with the currently admitted postulate, our study shows that an active and well-folded GPCR is not required for the production of specific anti-GPCR antibodies. This new immunizing strategy validated with three different human GPCR (μ-opioid, κ-opioid, neuropeptide FF2 receptors) might be generalized to other members of the GPCR family.

  4. New Primers for Denaturing Gradient Gel Electrophoresis Analysis of Nitrate-Reducing Bacterial Community in Soil



    The narG gene is frequently used as a molecular marker for bacterial nitrate-reducing community analysis.In this study,a new set of primers targeting the narG gene was designed and applied to semi-nested polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) assay.The potential of the new primers was verified on DNA directly extracted from soils from five different experimental sites distributed in Central and Southern Italy.Specificity of the primers was determined by excision,amplification,and sequencing of bands resolved by DGGE.A phylogenetic analysis showed the correlation between the sequences retrieved from the soils studied and the narG sequences from β and γ-Proteobacteria.These primers expanded the existing molecular tools for ecological study on the size and diversity of nitrate-reducing bacterial community in soil.

  5. An approach for protein to be completely reversible to thermal denaturation even at autoclave temperatures.

    Iwakura, M; Nakamura, D; Takenawa, T; Mitsuishi, Y


    Reversibility of protein denaturation is a prerequisite for all applications that depend on reliable enzyme catalysis, particularly, for using steam to sterilize enzyme reactors or enzyme sensor tips, and for developing protein-based devices that perform on-off switching of the protein function such as enzymatic activity, ligand binding and so on. In this study, we have successfully constructed an immobilized protein that retains full enzymatic activity even after thermal treatments as high as 120 degrees C. The key for the complete reversibility was the development of a new reaction that allowed a protein to be covalently attached to a surface through its C-terminus and the protein engineering approach that was used to make the protein compatible with the new attachment chemistry.

  6. Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions.

    Cao, Enhong; Chen, Yahuei; Cui, Zhanfeng; Foster, Peter R


    The freeze denaturation of model proteins, LDH, ADH, and catalase, was investigated in absence of cryoprotectants using a microcryostage under well-controlled freezing and thawing rates. Most of the experimental data were obtained from a study using a dilute solution with an enzyme concentration of 0.025 g/l. The dependence of activity recovery of proteins on the freezing and thawing rates showed a reciprocal and independent effect, that is, slow freezing (at a freezing rate about 1 degrees C/min) and fast thawing (at a thawing rate >10 degrees C/min) produced higher activity recovery, whereas fast freezing with slow thawing resulted in more severe damage to proteins. With minimizing the freezing concentration and pH change of buffer solution by using a potassium phosphate buffer, this phenomenon could be ascribed to surface-induced denaturation during freezing and thawing process. Upon the fast freezing (e.g., when the freezing rate >20 degrees C/min), small ice crystals and a relatively large surface area of ice-liquid interface are formed, which increases the exposure of protein molecules to the ice-liquid interface and hence increases the damage to the proteins. During thawing, additional damage to proteins is caused by recrystallization process. Recrystallization exerts additional interfacial tension or shear on the entrapped proteins and hence causes additional damage to the latter. When buffer solutes participated during freezing, the activity recovery of proteins after freezing and thawing decreased due to the change of buffer solution pH during freezing. However, the patterns of the dependence on freezing and thawing rates of activity recovery did not change except for that at extreme low freezing rates (solutions could be reduced by changing the buffer type and composition and by optimizing the freezing-thawing protocol.

  7. Microbial Community Structure of Korean Cabbage Kimchi and Ingredients with Denaturing Gradient Gel Electrophoresis.

    Hong, Sung Wook; Choi, Yun-Jeong; Lee, Hae-Won; Yang, Ji-Hee; Lee, Mi-Ai


    Kimchi is a traditional Korean fermented vegetable food, the production of which involves brining of Korean cabbage, blending with various other ingredients (red pepper powder, garlic, ginger, salt-pickled seafood, etc.), and fermentation. Recently, kimchi has also become popular in the Western world because of its unique taste and beneficial properties such as antioxidant and antimutagenic activities, which are derived from the various raw materials and secondary metabolites of the fermentative microorganisms used during production. Despite these useful activities, analysis of the microbial community present in kimchi has received relatively little attention. The objective of this study was to evaluate the bacterial community structure from the raw materials, additives, and final kimchi product using the culture-independent method. Specifically, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze the 16S rRNA partial sequences of the microflora. One primer set for bacteria, 341F(GC)-518R, reliably produced amplicons from kimchi and its raw materials, and these bands were clearly separated on a 35-65% denaturing gradient gel. Overall, 117 16S rRNA fragments were identified by PCR-DGGE analysis. Pediococcus pentosaceus, Leuconostoc citreum, Leuconostoc gelidum, and Leuconostoc mesenteroides were the dominant bacteria in kimchi. The other strains identified were Tetragenococcus, Pseudomonas, Weissella, and uncultured bacterium. Comprehensive analysis of these microorganisms could provide a more detailed understanding of the biologically active components of kimchi and help improve its quality. PCR-DGGE analysis can be successfully applied to a fermented food to detect unculturable or other species.

  8. Size is a major determinant of dissociation and denaturation behaviour of reconstituted high-density lipoproteins.

    Gianazza, Elisabetta; Eberini, Ivano; Sirtori, Cesare R; Franceschini, Guido; Calabresi, Laura


    Lipid-free apolipoprotein A-I (apoA-I) and A-I(Milano) (A-I(M)) were compared for their denaturation behaviour by running across transverse gradients of a chaotrope, urea, and of a ionic detergent, SDS. For both apo A-I and monomeric apoA-I(M) in the presence of increasing concentrations of urea the transition from high to low mobility had a sigmoidal course, whereas for dimeric A-I(M)/A-I(M) a non-sigmoidal shape was observed. The co-operativity of the unfolding process was lower for dimeric A-I(M)/A-I(M) than for apoA-I or for monomeric apoA-I(M). A slightly higher susceptibility to denaturation was observed for dimeric A-I(M)/A-I(M) than for monomeric apoA-I(M). A similar behaviour of A-I(M)/A-IM versus apoA-I(M) was observed in CD experiments. Large- (12.7/12.5 nm) and small- (7.8 nm) sized reconstituted high-density lipoproteins (rHDL) containing either apoA-I or A-I(M)/A-I(M) were compared with respect to their protein-lipid dissociation behaviour by subjecting them to electrophoresis in the presence of urea, of SDS and of a non-ionic detergent, Nonidet P40. A higher susceptibility to dissociation of small-sized versus large-sized rHDL, regardless of the apolipoprotein component, was observed in all three instances. Our data demonstrate that the differential plasticity of the various classes of rHDL is a function of their size; the higher stability of 12.5/12.7 nm rHDL is likely connected to the higher number of protein-lipid and lipid-lipid interactions in larger as compared with smaller rHDL.

  9. Thermal, chemical and pH induced unfolding of turmeric root lectin: modes of denaturation.

    Himadri Biswas

    Full Text Available Curcuma longa rhizome lectin, of non-seed origin having antifungal, antibacterial and α-glucosidase inhibitory activities, forms a homodimer with high thermal stability as well as acid tolerance. Size exclusion chromatography and dynamic light scattering show it to be a dimer at pH 7, but it converts to a monomer near pH 2. Circular dichroism spectra and fluorescence emission maxima are virtually indistinguishable from pH 7 to 2, indicating secondary and tertiary structures remain the same in dimer and monomer within experimental error. The tryptophan environment as probed by acrylamide quenching data yielded very similar data at pH 2 and pH 7, implying very similar folding for monomer and dimer. Differential scanning calorimetry shows a transition at 350.3 K for dimer and at 327.0 K for monomer. Thermal unfolding and chemical unfolding induced by guanidinium chloride for dimer are both reversible and can be described by two-state models. The temperatures and the denaturant concentrations at which one-half of the protein molecules are unfolded, are protein concentration-dependent for dimer but protein concentration-independent for monomer. The free energy of unfolding at 298 K was found to be 5.23 Kcal mol-1 and 14.90 Kcal mol-1 for the monomer and dimer respectively. The value of change in excess heat capacity upon protein denaturation (ΔCp is 3.42 Kcal mol-1 K-1 for dimer. The small ΔCp for unfolding of CLA reflects a buried hydrophobic core in the folded dimeric protein. These unfolding experiments, temperature dependent circular dichroism and dynamic light scattering for the dimer at pH 7 indicate its higher stability than for the monomer at pH 2. This difference in stability of dimeric and monomeric forms highlights the contribution of inter-subunit interactions in the former.

  10. Aqueous Solutions of the Ionic Liquid 1-butyl-3-methylimidazolium Chloride Denature Proteins

    Baker, Gary A [ORNL; Heller, William T [ORNL


    As we advance our understanding, ionic liquids (ILs) are finding ever broader scope within the chemical sciences including, most recently, pharmaceutical, enzymatic, and bioanalytical applications. With examples of enzymatic activity reported in both neat ILs and in IL/water mixtures, enzymes are frequently assumed to adopt a quasi-native conformation, even if little work has been carried out to date toward characterizing the conformation, dynamics, active-site perturbation, cooperativity of unfolding transitions, free energy of stabilization, or aggregation/oligomerization state of enzymes in the presence of an IL solvent component. In this study, human serum albumin and equine heart cytochrome c were characterized in aqueous solutions of the fully water-miscible IL 1-butyl-3-methylimidazolium chloride, [bmim]Cl, by small-angle neutron and X-ray scattering. At [bmim]Cl concentrations up to 25 vol.%, these two proteins were found to largely retain their higher-order structures whereas both proteins become highly denatured at the highest IL concentration studied here (i.e., 50 vol.% [bmim]Cl). The response of these proteins to [bmim]Cl is analogous to their behavior in the widely studied denaturants guanidine hydrochloride and urea which similarly lead to random coil conformations at excessive molar concentrations. Interestingly, human serum albumin dimerizes in response to [bmim]Cl, whereas cytochrome c remains predominantly in monomeric form. These results have important implications for enzymatic studies in aqueous IL media, as they suggest a facile pathway through which biocatalytic activity can be altered in these nascent and potentially green electrolyte systems.

  11. Comprehensive TP53-denaturing gradient gel electrophoresis mutation detection assay also applicable to archival paraffin-embedded tissue

    Hayes, V M; Bleeker, W; Verlind, E; Timmer, T; Karrenbeld, A; Plukker, J T; Marx, M P; Hofstra, R M; Buys, C H


    A comprehensive mutation detection assay is described for the entire coding region and all splice site junctions of TP53. The assay is based on denaturing gradient gel electrophoresis, which follows either multiplex polymerase chain reaction (PCR) applied to DNA extracted from fresh or frozen tissue

  12. Denaturation and in Vitro Gastric Digestion of Heat-Treated Quinoa Protein Isolates Obtained at Various Extraction pH

    Ruiz, Geraldine Avila; Opazo-Navarrete, Mauricio; Meurs, Marlon; Minor, Marcel; Sala, Guido; Boekel, van Martinus; Stieger, Markus; Janssen, Anja E.M.


    The aim of this study was to determine the influence of heat processing on denaturation and digestibility properties of protein isolates obtained from sweet quinoa (Chenopodium quinoa Willd) at various extraction pH values (8, 9, 10 and 11). Pretreatment of suspensions of protein isolates at 60,

  13. Heat-induced denaturation and aggregation of ovalbumin at neutral pH described by irreversible first-order kinetics

    Weijers, M.; Barneveld, P.A.; Cohen Stuart, M.A.; Visschers, R.W.


    The heat-induced denaturation kinetics of two different sources of ovalbumin at pH 7 was studied by chromatography and differential scanning calorimetry. The kinetics was found to be independent of protein concentration and salt concentration, but was strongly dependent on temperature. For highly

  14. Easily denaturing nucleic acids derived from intercalating nucleic acids: thermal stability studies, dual duplex invasion and inhibition of transcription start

    Filichev, Vyacheslav V; Vester, Birte; Hansen, Lykke Haastrup;


    The bulged insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (monomer P) in two complementary 8mer DNA strands (intercalating nucleic acids) opposite to each other resulted in the formation of an easily denaturing duplex, which had lower thermal stability (21.0 degrees C) than the wild-type double...

  15. Denaturation and in Vitro Gastric Digestion of Heat-Treated Quinoa Protein Isolates Obtained at Various Extraction pH

    Ruiz, Geraldine Avila; Opazo-Navarrete, Mauricio; Meurs, Marlon; Minor, Marcel; Sala, Guido; Boekel, van Tiny; Stieger, Markus; Janssen, Anja E.M.


    The aim of this study was to determine the influence of heat processing on denaturation and digestibility properties of protein isolates obtained from sweet quinoa (Chenopodium quinoa Willd) at various extraction pH values (8, 9, 10 and 11). Pretreatment of suspensions of protein isolates at 60,

  16. Reaction of Native and Denatured Brucella abortus (S19 Proteins with Antibody Using Affinity Chromatography and Immunoblotting

    R. Karimi


    Full Text Available Western blotting or immunoblotting commonly use for study of reaction between antigens and antibodies. Denaturation of many proteins in immunoblotting can affect greatly the reactivity of antibodies and outcome of the procedure.In this study proteins of Brucella abortus (S19 was extracted by a mild method and reaction of the extracted proteins with serum of infected human and goat and immunized rabbit compared by affinity chromatography and immunoblotting. Gamma globulin (mostly IgG fraction of the sera was precipitated by half saturation of ammonium sulfate and linked to activated sepharose 4B. The extracted proteins were loaded on the affinity column. Attached proteins was eluted by low pH and analyzed by SDS-PAGE. Reaction of the total extract and eluted fractions with IgG fraction of sera was evaluated by Western blotting.Upon the results of affinity chromatography and immunoblotting, Brucella proteins can be classified in four groups: 1- The proteins that adsorbed to the affinity column and react with IgG in westernblotting. 2- Proteins that react with IgG in native state but no in denatured state. 3- Proteins that do not react with IgG in native state but react in denatured state. 4- Proteins that do not react with IgG in native and denatured state.

  17. High-intensity focused ultrasound monitoring using harmonic motion imaging for focused ultrasound (HMIFU) under boiling or slow denaturation conditions.

    Hou, Gary Y; Marquet, Fabrice; Wang, Shutao; Apostolakis, Iason-Zacharias; Konofagou, Elisa E


    Harmonic motion imaging for focused ultrasound (HMIFU) is a recently developed high-intensity focused ultrasound (HIFU) treatment monitoring method that utilizes an amplitude-modulated therapeutic ultrasound beam to induce an oscillatory radiation force at the HIFU focus and estimates the focal tissue displacement to monitor the HIFU thermal treatment. In this study, the performance of HMIFU under acoustic, thermal, and mechanical effects was investigated. The performance of HMIFU was assessed in ex vivo canine liver specimens (n = 13) under slow denaturation or boiling regimes. A passive cavitation detector (PCD) was used to assess the acoustic cavitation activity, and a bare-wire thermocouple was used to monitor the focal temperature change. During lesioning with slow denaturation, high quality displacements (correlation coefficient above 0.97) were observed under minimum cavitation noise, indicating the tissue initial-softening-then- stiffening property change. During HIFU with boiling, HMIFU monitored a consistent change in lesion-to-background displacement contrast (0.46 ± 0.37) despite the presence of strong cavitation noise due to boiling during lesion formation. Therefore, HMIFU effectively monitored softening-then-stiffening during lesioning under slow denaturation, and detected lesioning under boiling with a distinct change in displacement contrast under boiling in the presence of cavitation. In conclusion, HMIFU was shown under both boiling and slow denaturation regimes to be effective in HIFU monitoring and lesioning identification without being significantly affected by cavitation noise.

  18. Evaluation of several techniques to modify denatured muscle tissue to obtain a scaffold for peripheral nerve regeneration

    Meek, MF; den Dunnen, WFA; Schakenraad, JM; Robinson, PH

    The aim of this study was to (1) evaluate the effect of several preparation techniques of denatured muscle tissue to obtain an open three-dimensional structure, and (2) test if this scaffold is suitable for peripheral nerve regeneration. Four samples (A-D) of muscle tissue specimens were evaluated

  19. The unfolding/denaturation of immunogammaglobulin of isotype 2b and its F-ab and F-c fragments

    Vermeer, AWP; Norde, W; van Amerongen, A


    The unfolding and further denaturation of IgG and its F-ab and F-c fragments were studied both on a macroscopic and molecular level, using differential scanning calorimetry and circular dichroism spectroscopy, respectively. It was shown that the structural integrity of the F-ab and F-c units was ret

  20. Sequential Events in the Irreversible Thermal Denaturation of Human Brain-Type Creatine Kinase by Spectroscopic Methods

    Yan-Song Gao


    Full Text Available The non-cooperative or sequential events which occur during protein thermal denaturation are closely correlated with protein folding, stability, and physiological functions. In this research, the sequential events of human brain-type creatine kinase (hBBCK thermal denaturation were studied by differential scanning calorimetry (DSC, CD, and intrinsic fluorescence spectroscopy. DSC experiments revealed that the thermal denaturation of hBBCK was calorimetrically irreversible. The existence of several endothermic peaks suggested that the denaturation involved stepwise conformational changes, which were further verified by the discrepancy in the transition curves obtained from various spectroscopic probes. During heating, the disruption of the active site structure occurred prior to the secondary and tertiary structural changes. The thermal unfolding and aggregation of hBBCK was found to occur through sequential events. This is quite different from that of muscle-type CK (MMCK. The results herein suggest that BBCK and MMCK undergo quite dissimilar thermal unfolding pathways, although they are highly conserved in the primary and tertiary structures. A minor difference in structure might endow the isoenzymes dissimilar local stabilities in structure, which further contribute to isoenzyme-specific thermal stabilities.

  1. Evaluation of several techniques to modify denatured muscle tissue to obtain a scaffold for peripheral nerve regeneration

    Meek, MF; den Dunnen, WFA; Schakenraad, JM; Robinson, PH


    The aim of this study was to (1) evaluate the effect of several preparation techniques of denatured muscle tissue to obtain an open three-dimensional structure, and (2) test if this scaffold is suitable for peripheral nerve regeneration. Four samples (A-D) of muscle tissue specimens were evaluated u

  2. Influence of pH and Ionic Strength on Heat-Induced Formation and Rheological Properties of Soy Protein Gels in Relation to Denaturation and Their Protein Compositions

    Renkema, J.M.S.; Gruppen, H.; Vliet, van T.


    The influence of pH and ionic strength on gel formation and gel properties of soy protein isolate (SPI) in relation to denaturation and protein aggregation/precipitation was studied. Denaturation proved to be a prerequisite for gel formation under all conditions of pH and ionic strength studied.

  3. Should PET/CT be implemented in the routine imaging work-up of locally advanced head and neck squamous cell carcinoma? A prospective analysis.

    Cacicedo, Jon; Fernandez, Iratxe; Del Hoyo, Olga; Dolado, Ainara; Gómez-Suarez, Javier; Hortelano, Eduardo; Sancho, Aintzane; Pijoan, Jose I; Alvarez, Julio; Espinosa, Jose M; Gaafar, Ayman; Bilbao, Pedro


    The objective of this study was to determine the incremental staging information provided by positron emission tomography/computed tomography (PET/CT) and its impact on management plans in patients with untreated stage III-IV head and neck squamous cell carcinoma (HNSCC). We prospectively studied, between September 2011 and February 2013, 84 consecutive patients [median age 63.5 years (39-84); 73 men] with histologically confirmed HNSCC. First, based on a conventional work-up (physical examination, CT imaging of the head, neck and chest), the multidisciplinary Head and Neck Tumour Board documented the TNM stage and a management plan for each patient, outlining the modalities to be used, including surgery, radiation therapy (RT), chemotherapy or a combination. After release of the PET/CT results, new TNM staging and management plans were agreed on by the multidisciplinary Tumour Board. Any changes in stage or intended management due to the PET/CT findings were then analysed. The impact on patient management was classified as: low (treatment modality, delivery and intent unchanged), moderate (change within the same treatment modality: type of surgery, radiation technique/dose) or high (change in treatment intent and/or treatment modality → curative to palliative, or surgery to chemoradiation or detection of unknown primary tumour or a synchronous second primary tumour). TNM stage was validated by histopathological analysis, additional imaging or follow-up. Accuracy of the conventional and PET/CT-based staging was compared using McNemar's test. Conventional and PET/CT stages were discordant in 32/84 (38 %) cases: the T stage in 2/32 (6.2 %), the N stage in 21/32 (65.7 %) and the M stage 9/32 (28.1 %). Patient management was altered in 22/84 (26 %) patients, with a moderate impact in 8 (9.5 %) patients and high impact in 14 (16.6 %) patients. PET/CT TNM classification was significantly more accurate (92.5 vs 73.7 %) than conventional staging with a p value < 0

  4. Should PET/CT be implemented in the routine imaging work-up of locally advanced head and neck squamous cell carcinoma? A prospective analysis

    Cacicedo, Jon; Bilbao, Pedro [Cruces University Hospital, Radiation Oncology Department, Barakaldo, Bizkaia (Basque Country) (Spain); BioCruces Health Research Institute, Bizkaia, Basque Country (Spain); Fernandez, Iratxe [Cruces University Hospital, Nuclear Medicine Department, Barakaldo (Spain); Hoyo, Olga del; Hortelano, Eduardo [Cruces University Hospital, Radiation Oncology Department, Barakaldo, Bizkaia (Basque Country) (Spain); Dolado, Ainara [Cruces University Hospital, Radiodiagnostic and Medical Imaging Department, Barakaldo (Spain); Gomez-Suarez, Javier [Cruces University Hospital, Otolaryngology Department, Barakaldo (Spain); Sancho, Aintzane [Cruces University Hospital, Medical Oncology Department, Barakaldo (Spain); Pijoan, Jose I. [BioCruces Health Research Institute, Bizkaia, Basque Country (Spain); Cruces University Hospital, Clinical Epidemiology Unit, Barakaldo (Spain); CIBER de Epidemiologia y Salud Publica (CIBERESP), Madrid (Spain); Alvarez, Julio [Cruces University Hospital, Oral and Maxillofacial Surgery Department, Barakaldo (Spain); Espinosa, Jose M. [Cruces University Hospital, Medical Physics Department, Barakaldo (Spain); Gaafar, Ayman [Cruces University Hospital, Department of Pathology, Barakaldo (Spain)


    The objective of this study was to determine the incremental staging information provided by positron emission tomography/computed tomography (PET/CT) and its impact on management plans in patients with untreated stage III-IV head and neck squamous cell carcinoma (HNSCC). We prospectively studied, between September 2011 and February 2013, 84 consecutive patients [median age 63.5 years (39-84); 73 men] with histologically confirmed HNSCC. First, based on a conventional work-up (physical examination, CT imaging of the head, neck and chest), the multidisciplinary Head and Neck Tumour Board documented the TNM stage and a management plan for each patient, outlining the modalities to be used, including surgery, radiation therapy (RT), chemotherapy or a combination. After release of the PET/CT results, new TNM staging and management plans were agreed on by the multidisciplinary Tumour Board. Any changes in stage or intended management due to the PET/CT findings were then analysed. The impact on patient management was classified as: low (treatment modality, delivery and intent unchanged), moderate (change within the same treatment modality: type of surgery, radiation technique/dose) or high (change in treatment intent and/or treatment modality → curative to palliative, or surgery to chemoradiation or detection of unknown primary tumour or a synchronous second primary tumour). TNM stage was validated by histopathological analysis, additional imaging or follow-up. Accuracy of the conventional and PET/CT-based staging was compared using McNemar's test. Conventional and PET/CT stages were discordant in 32/84 (38 %) cases: the T stage in 2/32 (6.2 %), the N stage in 21/32 (65.7 %) and the M stage 9/32 (28.1 %). Patient management was altered in 22/84 (26 %) patients, with a moderate impact in 8 (9.5 %) patients and high impact in 14 (16.6 %) patients. PET/CT TNM classification was significantly more accurate (92.5 vs 73.7 %) than conventional staging with a p value < 0

  5. A survey about further work-up for cases with positive sputum cytology during lung cancer mass screening in Ishikawa Prefecture, Japan: a retrospective analysis about quality assurance of lung cancer screening.

    Sagawa, Motoyasu; Kobayashi, Takeshi; Uotani, Chika; Kibe, Yoshinori; Tanaka, Makoto; Machida, Yuichiro; Motono, Nozomu; Maeda, Sumiko; Usuda, Katsuo


    In cancer screening programs, performing appropriate further work-up is essential. In order to elucidate whether the further work-up for the subjects with positive screening results by sputum cytology was performed appropriately, the present study was conducted as the first large-scale thorough survey in Japan. All of the lung cancer screening records from 2007 to 2012 in Ishikawa Prefecture were reviewed. Additional investigations about the further work-up were performed. In total, 2 234 984 people were invited to undergo lung cancer screening, and 494 424 people participated in the screening. Of these, 25 264 people underwent sputum cytology, and 68 positive cases were identified. Three of these 68 cases did not undergo further work-up, and another three cases had already been diagnosed to have lung cancer. Forty-five of the remaining 62 cases did not have suspicious chest shadows, and bronchoscopic examinations were performed in 36 cases. Seventeen of these 36 cases were diagnosed as having cancer, whereas none of the nine cases who did not receive the examination was diagnosed (P = 0.038). A bronchoscopic examination was not performed due to other medical conditions in three cases, due to the patient's refusal in another three cases and in the remaining three cases, the reasons were unknown. The participation rate for further work-up was very high. However, there are some issues to be resolved regarding the transmission of information. With our new registered hospital system, the quality assurance of our screening program will be improved. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email:

  6. Intermolecular hydrogen bonds: From temperature-driven proton transfer in molecular crystals to denaturation of DNA

    Mark Johnson


    We have combined neutron scattering and a range of numerical simulations to study hydrogen bonds in condensed matter. Two examples from a recent thesis will be presented. The first concerns proton transfer with increasing temperature in short inter-molecular hydrogen bonds [1,2]. These bonds have unique physical and chemical properties and are thought to play a fundamental role in processes like enzymatic catalysis. By combining elastic and inelastic neutron scattering results with ab initio, lattice dynamics and molecular dynamics simulations, low frequency lattice modes are identified which modulate the potential energy surface of the hydrogen bond proton and drive proton transfer. The second example concerns base-pair opening in DNA which is the fundamental physical process underlying biological processes like denaturation and transcription. We have used an emprical force field and a large scale, all-atom phonon calculation to gain insight into the base-pair opening modes and the apparent `energy gap' between the accepted frequencies for these modes (∼ 100 cm-1 or ∼ 140 K) and the temperature of the biological processes (room temperature to 100° C) [3]. Inelastic neutron scattering spectra on aligned, highly crystalline DNA samples, produced at the ILL, provide the reference data for evaluating the precision of these simulation results.

  7. The quaternary structure of the recombinant bovine odorant-binding protein is modulated by chemical denaturants.

    Olga V Stepanenko

    Full Text Available A large group of odorant-binding proteins (OBPs has attracted great scientific interest as promising building blocks in constructing optical biosensors for dangerous substances, such as toxic and explosive molecules. Native tissue-extracted bovine OBP (bOBP has a unique dimer folding pattern that involves crossing the α-helical domain in each monomer over the other monomer's β-barrel. In contrast, recombinant bOBP maintaining the high level of stability inherent to native tissue bOBP is produced in a stable native-like state with a decreased tendency for dimerization and is a mixture of monomers and dimers in a buffered solution. This work is focused on the study of the quaternary structure and the folding-unfolding processes of the recombinant bOBP in the absence and in the presence of guanidine hydrochloride (GdnHCl. Our results show that the recombinant bOBP native dimer is only formed at elevated GdnHCl concentrations (1.5 M. This process requires re-organizing the protein structure by progressing through the formation of an intermediate state. The bOBP dimerization process appears to be irreversible and it occurs before the protein unfolds. Though the observed structural changes for recombinant bOBP at pre-denaturing GdnHCl concentrations show a local character and the overall protein structure is maintained, such changes should be considered where the protein is used as a sensitive element in a biosensor system.

  8. Thermodynamic denaturation of {beta}-lactoglobulin in the presence of cetylpyridinium chloride

    Sahihi, M. [Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111 (Iran, Islamic Republic of); Bordbar, A.K., E-mail: [Department of Chemistry, University of Isfahan, Isfahan 81746-73441 (Iran, Islamic Republic of); Ghayeb, Y. [Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111 (Iran, Islamic Republic of)


    In this work, we determined the stability parameters of bovine {beta}-lactoglobulin, variant A, (BLG-A), in relation to their transition curves induced by cetylpyridinium chloride (CPC) as a cationic surfactant. The experiments took place over the temperature range of 298 K to 358 K. For each transition curve at any specific temperature, the conventional method of analysis, which assumes a linear concentration dependence of the pre- and post-transition base lines, gave the most realistic values for {Delta}G{sub D}(H{sub 2}O). Results show that the minimum value of {Delta}G{sub D}(H{sub 2}O) occurs at T = 328 K. Using the Gibbs-Helmholtz equation, the values of enthalpy, {Delta}H{sub D}, and entropy, {Delta}S{sub D}, of denaturation have been calculated considering temperature dependence of {Delta}G{sub D} at any specified concentration of CPC. The values of 12.05 kJ . mol{sup -1}, 18.54 kJ . mol{sup -1}, and 18.32 J . mol{sup -1} . K{sup -1}, were obtained for {Delta}G{sub D}(H{sub 2}O), {Delta}H{sub D}(H{sub 2}O), and {Delta}S{sub D}(H{sub 2}O), respectively. The results show that the enthalpy term dominates the entropy term.

  9. Screening for TP53 mutations in osteosarcomas using constant denaturant gel electrophoresis (CDGE).

    Smith-Sørensen, B; Gebhardt, M C; Kloen, P; McIntyre, J; Aguilar, F; Cerutti, P; Børresen, A L


    We have previously developed conditions to screen for TP53 point mutations inside the conserved domains II-V of the gene by using constant denaturant gel electrophoresis (CDGE). The present study reports conditions for screening more of the codons in the frequently mutated region exon 5-8 and for detecting mutations in sequences encoding functional domains in the N- and C-terminal part of the protein. The ability of the CDGE technique to detect mutations was studied using controls with known sequence deviations. The resolution power of the technique to separate different types of mutations was tested by using seven different single base pair mutants all residing in a stretch of four base pairs. All mutants were separated from the wild type. The established CDGE screening strategy was then used to look for mutations in DNA from 28 osteosarcomas. Six (21.5%) of the samples were shown to have a TP53 mutation, and the exact characterization was performed by direct sequencing. All of these were within the frequently reported mutated region exon 5-8.

  10. Amaltheys: A fluorescence-based analyzer to assess cheese milk denatured whey proteins.

    Lacotte, Pierre; Gomez, Franck; Bardeau, Floriane; Muller, Sabine; Acharid, Abdelhaq; Quervel, Xavier; Trossat, Philippe; Birlouez-Aragon, Inès


    The cheese industry faces many challenges to optimize cheese yield and quality. A very precise standardization of the cheese milk is needed, which is achieved by a fine control of the process and milk composition. Thorough analysis of protein composition is important to determine the amount of protein that will be retained in the curd or lost in the whey. The fluorescence-based Amaltheys analyzer (Spectralys Innovation, Romainville, France) was developed to assess pH 4.6-soluble heat-sensitive whey proteins (sWP*) in 5 min. These proteins are those that can be denatured upon heat-treatment and further retained in the curd after coagulation. Monitoring of sWP* in milk and subsequent adaptation of the process is a reliable solution to achieve stable cheese yield and quality. Performance of the method was evaluated by an accredited laboratory on a 0 to 7 g/L range. Accuracy compared with the reference Kjeldahl method is also provided with a standard error of 0.25 g/L. Finally, a 4-mo industrial trial in a cheese plant is described, where Amaltheys was used as a process analytical technology to monitor sWP* content in ingredients and final cheese milk. Calibration models over quality parameters of final cheese were also built from near-infrared and fluorescence spectroscopic data. The Amaltheys analyzer was found to be a rapid, compact, and accurate device to help implementation of standardization procedures in the dairy industry.

  11. Protein denaturation of whey protein isolates (WPIs) induced by high intensity ultrasound during heat gelation.

    Frydenberg, Rikke P; Hammershøj, Marianne; Andersen, Ulf; Greve, Marie T; Wiking, Lars


    In this study, the impact of high intensity ultrasound (HIU) on proteins in whey protein isolates was examined. Effects on thermal behavior, secondary structure and nature of intra- and intermolecular bonds during heat-induced gelling were investigated. Ultrasonication (24 kHz, 300 W/cm(2), 2078 J/mL) significantly reduced denaturation enthalpies, whereas no change in secondary structure was detected by circular dichroism. The thiol-blocking agent N-ethylmaleimide was applied in order to inhibit formation of disulfide bonds during gel formation. Results showed that increased contents of α-lactalbumin (α-La) were associated with increased sensitivity to ultrasonication. The α-La:β-lactoglobulin (β-Lg) ratio greatly affected the nature of the interactions formed during gelation, where higher amounts of α-La lead to a gel more dependent on disulfide bonds. These results contribute to clarifying the mechanisms mediating the effects of HIU on whey proteins on the molecular level, thus moving further toward implementing HIU in the processing chain in the food industry. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Preliminary studies on the renaturation of denatured catfish (Clarias gariepinus) glutathione transferase.

    Ojopagogo, Yetunde Adedolapo; Adewale, Isaac Olusanjo; Afolayan, Adeyinka


    Purified juvenile catfish (Clarias gariepinus) glutathione transferase (cgGST) was denatured in vitro and renatured in the absence and presence of different concentrations of endogenous or xenobiotic model substrates. Protein transitions during unfolding and refolding were monitored by activity measurement as well as changes in protein conformation using UV difference spectra at 230 nm. Gdn-HCl at 0.22 M caused 50 % inactivation of the enzyme and at 1.1 M, the enzyme was completely unfolded. Refolding of cgGST main isozyme was not completely reversible at higher concentrations of Gdn-HCl and is dependent on protein concentration. An enzyme concentration of 30 μg/ml yielded 40 % percentage residual activity in the presence of glutathione (GSH), regardless of the concentration that was present as opposed to 30 % obtained in its absence. The xenobiotic model substrate, lindane, appears to have no effect on the refolding of the enzyme. In summary, our results show that GSH assists in the refolding of cgGST in a concentration-independent manner and may be involved in the same function in vivo whereas the xenobiotic model substrate does not.

  13. Thermal denaturation studies of collagen by microthermal analysis and atomic force microscopy.

    Bozec, Laurent; Odlyha, Marianne


    The structural properties of collagen have been the subject of numerous studies over past decades, but with the arrival of new technologies, such as the atomic force microscope and related techniques, a new era of research has emerged. Using microthermal analysis, it is now possible to image samples as well as performing localized thermal measurements without damaging or destroying the sample itself. This technique was successfully applied to characterize the thermal response between native collagen fibrils and their denatured form, gelatin. Thermal transitions identified at (150 ± 10)°C and (220 ± 10)°C can be related to the process of gelatinization of the collagen fibrils, whereas at higher temperatures, both the gelatin and collagen samples underwent two-stage transitions with a common initial degradation temperature at (300 ± 10)°C and a secondary degradation temperature of (340 ± 10)°C for the collagen and of (420 ± 10)°C for the gelatin, respectively. The broadening and shift in the secondary degradation temperature was linked to the spread of thermal degradation within the gelatin and collagen fibrils matrix further away from the point of contact between probe and sample. Finally, similar measurements were performed inside a bone resorption lacuna, suggesting that microthermal analysis is a viable technique for investigating the thermomechanical response of collagen for in situ samples that would be, otherwise, too challenging or not possible using bulk techniques.

  14. Monitoring the lactic acid bacterial diversity during shochu fermentation by PCR-denaturing gradient gel electrophoresis.

    Endo, Akihito; Okada, Sanae


    The presence of lactic acid bacteria (LAB) during shochu fermentation was monitored by PCR-denaturing gradient gel electrophoresis (DGGE) and by bacteriological culturing. No LAB were detected from fermented mashes by PCR-DGGE using a universal bacterial PCR primer set. However, PCR-DGGE using a new primer specific for the 16S rDNA of Lactococcus, Streptococcus, Tetragenococcus, Enterococcus, and Vagococcus and two primers specific for the 16S rDNA of Lactobacillus, Pediococcus, Leuconostoc, and Weissella revealed that Enterococcus faecium, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus nagelii, Lactobacillus plantarum, Lactococcus lactis, Leuconostoc citreum, Leuconostoc mesenteroides, and Weissella cibaria inhabited in shochu mashes. It was also found that the LAB community composition during shochu fermentation changed after the main ingredient and water were added during the fermentation process. Therefore, we confirmed that PCR-DGGE using all three primers specific for groups of LAB together was well suited to the study of the LAB diversity in shochu mashes. The results of DGGE profiles were similar to the results of bacteriological culturing. In conclusion, LAB are present during shochu fermentation but not dominant.

  15. Assessment of microbial populations in methyl ethyl ketone degrading biofilters by denaturing gradient gel electrophoresis.

    Li, C; Moe, W M


    Denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction-amplified genes coding for 16S rRNA was used to assess differences in bacterial community structure as a function of spatial location along the height of two biofilters used to treat a model waste gas stream containing methyl ethyl ketone (MEK). One of the laboratory-scale biofilters was operated as a conventional continuous-flow biofilter (CFB) and the other was operated as a sequencing batch biofilter (SBB). Both biofilters, inoculated with an identical starting culture and operated over a period lasting more than 300 days, received the same influent MEK concentration and same mass of MEK on a daily basis. The systems differed, however, in terms of the fraction of time during which contaminated air was supplied and the overall operating strategy employed. DGGE analysis indicated that microbial community structures differed as a function of height in each of the biofilters. The DGGE banding patterns also differed between the two biofilters, suggesting that operating strategies imposed on the biofilters imparted a sufficiently large selective pressure to influence microbial community structures. This may explain, in part, the superior performance of the SBB over the CFB during model transient loading conditions, and it may open new possibilities for purposely manipulating the microbial populations in biofilters treating gas-phase contaminants in a manner that leads to more favorable treatment performance.

  16. Salt-specific stability and denaturation of a short salt-bridge forming alpha-helix

    Dzubiella, Joachim


    The structure of a single alanine-based Ace-AEAAAKEAAAKA-Nme peptide in explicit aqueous electrolyte solutions (NaCl, KCl, NaI, and KF) at large salt concentrations (3-4 M) is investigated using 1 microsecond molecular dynamics (MD) computer simulations. The peptide displays 71 alpha-helical structure without salt and destabilizes with the addition of NaCl in agreement with experiments of a somewhat longer version. It is mainly stabilized by direct and indirect (i+4)EK salt bridges between the Lys and Glu side chains and a concomitant backbone shielding mechanism. NaI is found to be a stronger denaturant than NaCl, while the potassium salts hardly show influence. Investigation of the molecular structures reveals that consistent with recent experiments Na+ has a much stronger affinity to side chain carboxylates and backbone carbonyls than K+, thereby weakening salt bridges and secondary structure hydrogen bonds. At the same time the large I- has a considerable affinity to the nonpolar alanine in line with rece...

  17. Cold denaturation induces inversion of dipole and spin transfer in chiral peptide monolayers

    Eckshtain-Levi, Meital; Capua, Eyal; Refaely-Abramson, Sivan; Sarkar, Soumyajit; Gavrilov, Yulian; Mathew, Shinto P.; Paltiel, Yossi; Levy, Yaakov; Kronik, Leeor; Naaman, Ron


    Chirality-induced spin selectivity is a recently-discovered effect, which results in spin selectivity for electrons transmitted through chiral peptide monolayers. Here, we use this spin selectivity to probe the organization of self-assembled α-helix peptide monolayers and examine the relation between structural and spin transfer phenomena. We show that the α-helix structure of oligopeptides based on alanine and aminoisobutyric acid is transformed to a more linear one upon cooling. This process is similar to the known cold denaturation in peptides, but here the self-assembled monolayer plays the role of the solvent. The structural change results in a flip in the direction of the electrical dipole moment of the adsorbed molecules. The dipole flip is accompanied by a concomitant change in the spin that is preferred in electron transfer through the molecules, observed via a new solid-state hybrid organic-inorganic device that is based on the Hall effect, but operates with no external magnetic field or magnetic material.

  18. Urea denaturation of barnase: pH dependence and characterization of the unfolded state.

    Pace, C N; Laurents, D V; Erickson, R E


    To investigate the pH dependence of the conformational stability of barnase, urea denaturation curves were determined over the pH range 2-10. The maximum conformational stability of barnase is 9 kcal mol-1 and occurs between pH 5 and 6. The dependence of delta G on urea concentration increases from 1850 cal mol-1 M-1 at high pH to about 3000 cal mol-1 M-1 near pH 3. This suggests that the unfolded conformations of barnase become more accessible to urea as the net charge on the molecule increases. Previous studies suggested that in 8 M urea barnase unfolds more completely than ribonuclease T1, even with the disulfide bonds broken [Pace, C.N., Laurents, D. V., & Thomson, J.A. (1990) Biochemistry 29, 2564-2572]. In support of this, solvent perturbation difference spectroscopy showed that in 8 M urea the Trp and Tyr residues in barnase are more accessible to perturbation by dimethyl sulfoxide than in ribonuclease T1 with the disulfide bonds broken.

  19. Environmental-induced acquisition of nuptial plumage expression: a role of denaturation of feather carotenoproteins?

    Blanco, Guillermo; Frías, Oscar; Garrido-Fernández, Juan; Hornero-Méndez, Dámaso


    Several avian species show a bright carotenoid-based coloration during spring and following a period of duller coloration during the previous winter, despite carotenoids presumably being fully deposited in feathers during the autumn moult. Carotenoid-based breast feathers of male linnets (Carduelis cannabina) increased in hue (redness), saturation and brightness after exposing them to outdoor conditions from winter to spring. This represents the first experimental evidence showing that carotenoid-based plumage coloration may increase towards a colourful expression due to biotic or abiotic environmental factors acting directly on full-grown feathers when carotenoids may be fully functional. Sunlight ultraviolet (UV) irradiation was hypothesized to denature keratin and other proteins that might protect pigments from degradation by this and other environmental factors, suggesting that sunlight UV irradiation is a major factor in the colour increase from winter to spring. Feather proteins and other binding molecules, if existing in the follicles, may be linked to carotenoids since their deposition into feathers to protect colourful features of associated carotenoids during the non-breeding season when its main signalling function may be relaxed. Progress towards uncovering the significance of concealment and subsequent display of colour expression should consider the potential binding and protecting nature of feather proteins associated with carotenoids. PMID:16191594

  20. Numerical study of the disordered Poland Scheraga model of DNA denaturation

    Garel, Thomas; Monthus, Cécile


    We numerically study the binary disordered Poland-Scheraga model of DNA denaturation, in the regime where the pure model displays a first-order transition (loop exponent c = 2.15>2). We use a Fixman-Freire scheme for the entropy of loops and consider chain length up to N = 4 × 105, with averages over 104 samples. We present in parallel the results of various observables for two boundary conditions, namely bound-bound (bb) and bound-unbound (bu), because they present very different finite-size behaviours, both in the pure case and in the disordered case. Our main conclusion is that the transition remains first order in the disordered case: in the (bu) case, the disorder averaged energy and contact densities present crossings for different values of N without rescaling. In addition, we obtain that these disorder averaged observables do not satisfy finite-size scaling, as a consequence of strong sample to sample fluctuations of the pseudo-critical temperature. For a given sample, we propose a procedure to identify its pseudo-critical temperature, and show that this sample then obeys first order transition finite-size scaling behaviour. Finally, we obtain that the disorder averaged critical loop distribution is still governed by P(l)~1/lc in the regime l \\ll N , as in the pure case.

  1. Oligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradation

    Ponnuswamy, Nandhini; Bastings, Maartje M. C.; Nathwani, Bhavik; Ryu, Ju Hee; Chou, Leo Y. T.; Vinther, Mathias; Li, Weiwei Aileen; Anastassacos, Frances M.; Mooney, David J.; Shih, William M.


    DNA nanostructures have evoked great interest as potential therapeutics and diagnostics due to ease and robustness of programming their shapes, site-specific functionalizations and responsive behaviours. However, their utility in biological fluids can be compromised through denaturation induced by physiological salt concentrations and degradation mediated by nucleases. Here we demonstrate that DNA nanostructures coated by oligolysines to 0.5:1 N:P (ratio of nitrogen in lysine to phosphorus in DNA), are stable in low salt and up to tenfold more resistant to DNase I digestion than when uncoated. Higher N:P ratios can lead to aggregation, but this can be circumvented by coating instead with an oligolysine-PEG copolymer, enabling up to a 1,000-fold protection against digestion by serum nucleases. Oligolysine-PEG-stabilized DNA nanostructures survive uptake into endosomal compartments and, in a mouse model, exhibit a modest increase in pharmacokinetic bioavailability. Thus, oligolysine-PEG is a one-step, structure-independent approach that provides low-cost and effective protection of DNA nanostructures for in vivo applications.

  2. Binding of DNA by a dinitro-diester calix[4]arene: denaturation and condensation of DNA.

    Ostos, F J; Lebron, J A; Moyá, M L; Deasy, M; López-Cornejo, P


    A study of a dinitro-diester calix[4]arene (5,17-(3-nitrobenzylideneamino)-11,23-di-tert-butyl-25,27-diethoxycarbonyl methyleneoxy-26,28-dihydroxycalix[4]arene) interaction with calf-thymus DNA was carried out using several techniques. The measurements were done at various molar ratios X=[calixarene]/[DNA]. Results show diverse changes in the DNA conformation depending on the X value. Thus, at low macrocycle concentrations, the calixarene binds to the polynucleotide. This interaction, mainly in groove mode, weakens the hydrogen bonds between base pairs of the helix inducing denaturation of the double strands, as well as condensation of the macromolecule, from an extended coil state to a globular state. An opposite effect is observed at X molar ratios higher than 0.07. The de-condensation of DNA happens, that is, the transition from a compact state to a more extended conformation, probably due to the stacking of calixarene molecules in the solution. Results also show the importance of making a proper choice of the system under consideration.

  3. Cyclitols protect glutamine synthetase and malate dehydrogenase against heat induced deactivation and thermal denaturation.

    Jaindl, Martina; Popp, Marianne


    The accumulation of cyclitols in plants is a widespread response that provides protection against various environmental stresses. The capacity of myo-Inositol, pinitol, quercitol, and other compatible solutes (i.e., sorbitol, proline, and glycinebetaine) to protect proteins against thermally induced denaturation and deactivation was examined. Enzymatic activity measurements of L-glutamine synthetase from Escherichia coli and Hordeum vulgare showed that the presence of cyclitols during heat treatment resulted in a significantly higher percentage of residual activity. CD spectroscopy experiments were used to study thermal stabilities of protein secondary structures upon the addition of myo-Inositol, pinitol, and glucose. 0.4 M myo-Inositol was observed to raise the melting temperature (Tm) of GS from E. coli by 3.9 degrees C and MDH from pig heart by 3.4 degrees C, respectively. Pinitol showed an increase in Tm of MDH by 3.8 degrees C, whereas glucose was not effective. Our results show a great potential of stabilizing proteins by the addition of cyclitols.

  4. Structural and dynamical study about denatured states of yeast phosphoglycerate kinase by neutrons scattering and X-rays; Etude structurale et dynamique des etats denatures de la phosphoglycerate kinase de levure par diffusion des neutrons et des rayons X

    Receveur, V


    During a long time, the neutron scattering and X-rays techniques have not been used for the studies bearing on the folding of proteins. The compactness and the globularness of a protein are two structural characteristics describing the denatured states and the intermediate states of folding, and the neutrons and x-rays scattering are probably the two techniques the most appropriate to give this kind of information; they are sensible to the spatial extent and to the molecules compactness, and to their general shape. For these three or four last years, the works using these techniques are increasing, giving precious knowledge on the different steps of folding and on the interactions stabilizing the denatured or intermediate states. This thesis falls into this category. (N.C.).

  5. NT-proBNP <95 ng/l can exclude pulmonary hypertension on echocardiography at diagnostic workup in patients with interstitial lung disease

    Charlotte Andersen


    Full Text Available Background: Pulmonary hypertension (PH is a serious complication to interstitial lung disease (ILD and has a poor prognosis. PH is often diagnosed by screening with echocardiography followed by right heart catheterisation. A previous study has shown that a value of NT-pro-brain natriuretic peptide (NT-proBNP 95 ng/l for signs of PH on echocardiography were calculated. The study was approved by the Danish Health Authority. Results: In 118 patients, data from both echocardiography and measurements of NT-proBNP were available. Eleven of these were screened positive for PH on echocardiography. Sensitivity, specificity, NPV and PPV of NT-proBNP <95 ng/l for PH were 100, 44, 16 and 100%, respectively. Furthermore, no patients with left heart failure as the cause of dyspnoea were missed using this cut-off value. Conclusion: NT-proBNP <95 ng/l precludes a positive echocardiographic screen for PH in ILD patients at referral for diagnostic workup.

  6. High resolution MRI for preoperative work-up of neonates with an anorectal malformation: a direct comparison with distal pressure colostography/fistulography

    Thomeer, Maarten G. [Erasmus MC, Department of Radiology, Rotterdam (Netherlands); Devos, Annick; Lequin, Maarten; Graaf, Nanko de; Meradji, Morteza [Erasmus MC, Department of Pediatric Radiology, Rotterdam (Netherlands); Meeussen, Conny J.H.M.; Blaauw, Ivo de; Sloots, Cornelius E.J. [Erasmus MC, Department of Pediatric Surgery, Rotterdam (Netherlands)


    To compare MRI and colostography/fistulography in neonates with anorectal malformations (ARM), using surgery as reference standard. Thirty-three neonates (22 boys) with ARM were included. All patients underwent both preoperative high-resolution MRI (without sedation or contrast instillation) and colostography/fistulography. The Krickenbeck classification was used to classify anorectal malformations, and the level of the rectal ending in relation to the levator muscle was evaluated. Subjects included nine patients with a bulbar recto-urethral fistula, six with a prostatic recto-urethral fistula, five with a vestibular fistula, five with a cloacal malformation, four without fistula, one with a H-type fistula, one with anal stenosis, one with a rectoperineal fistula and one with a bladderneck fistula. MRI and colostography/fistulography predicted anatomy in 88 % (29/33) and 61 % (20/33) of cases, respectively (p = 0.012). The distal end of the rectal pouch was correctly predicted in 88 % (29/33) and 67 % (22/33) of cases, respectively (p = 0.065). The length of the common channel in cloacal malformation was predicted with MRI in all (100 %, 5/5) and in 80 % of cases (4/5) with colostography/fistulography. Two bowel perforations occurred during colostography/fistulography. MRI provides the most accurate evaluation of ARM and should be considered a serious alternative to colostography/fistulography during preoperative work-up. (orig.)

  7. Diagnostic Workup for Disorders of Bone and Mineral Metabolism in Patients with Chronic Kidney Disease in the Era of KDIGO Guidelines

    Luigi Francesco Morrone


    Full Text Available KDIGO (Kidney Disease: Improving Global Outcomes is an international nonprofit organization devoted to “improve the care and outcomes of kidney disease patients worldwide through promoting coordination, collaboration, and integration of initiatives to develop and implement clinical practice guidelines.” The mineral and bone disorder (MBD in patients with chronic kidney disease (CKD has been the first area of interest of KDIGO international initiative. KDIGO guidelines on CKD-MBD were published in 2009 with the intent to modify the previous KDOQI guidelines that had failed to consistently change the global outcome of CKD patients. After the publication of KDOQI guidelines for bone metabolism and disease in 2003, a large number of observational data emerged in literature linking disordered mineral metabolism with adverse clinical outcomes. Notwithstanding this large body of observational data, a paucity of evidence from high-quality clinical trials was available for the development of KDIGO guidelines. Herein, a summary will be provided of the most important findings of KDIGO guidelines regarding the diagnostic workup and clinical monitoring of CKD-MBD patients.

  8. Can the Diagnostics of Triangular Fibrocartilage Complex Lesions Be Improved by MRI-Based Soft-Tissue Reconstruction? An Imaging-Based Workup and Case Presentation

    Niels Hammer


    Full Text Available Introduction. The triangular fibrocartilage complex (TFCC provides both mobility and stability of the radiocarpal joint. TFCC lesions are difficult to diagnose due to the complex anatomy. The standard treatment for TFCC lesions is arthroscopy, posing surgery-related risks onto the patients. This feasibility study aimed at developing a workup for soft-tissue reconstruction using clinical imaging, to verify these results in retrospective patient data. Methods. Microcomputed tomography (μ-CT, 3 T magnetic resonance imaging (MRI, and plastination were used to visualize the TFCC in cadaveric specimens applying segmentation-based 3D reconstruction. This approach further trialed the MRI dataset of a patient with minor radiological TFCC alterations but persistent pain. Results. TFCC reconstruction was impossible using μ-CT only but feasible using MRI, resulting in an appreciation of its substructures, as seen in the plastinates. Applying this approach allowed for visualizing a Palmer 2C lesion in a patient, confirming ex postum the arthroscopy findings, being markedly different from MRI (Palmer 1B. Discussion. This preliminary study showed that image-based TFCC reconstruction may help to identify pathologies invisible in standard MRI. The combined approach of μ-CT, MRI, and plastination allowed for a three-dimensional appreciation of the TFCC. Image quality and time expenditure limit the approach’s usefulness as a diagnostic tool.

  9. A Comparison Between Denaturing Gradient Gel Electrophoresis and Denaturing High Performance Liquid Chromatography in Detecting Mutations in Genes Associated with Hereditary Non-Polyposis Colorectal Cancer (HNPCC and the Identification of 9 New Mutations Previously Unidentified by DGGE

    Meldrum Cliff J


    Full Text Available Abstract Denaturing high performance liquid chromatography is a relatively new method by which heteroduplex structures formed during the PCR amplification of heterozygote samples can be rapidly identified. The use of this technology for mutation detection in hereditary non-polyposis colorectal cancer (HNPCC has the potential to appreciably shorten the time it takes to analyze genes associated with this disorder. Prior to acceptance of this method for screening genes associated with HNPCC, assessment of the reliability of this method should be performed. In this report we have compared mutation and polymorphism detection by denaturing gradient gel electrophoresis (DGGE with denaturing high performance liquid chromatography (DHPLC in a set of 130 families. All mutations/polymorphisms representing base substitutions, deletions, insertions and a 23 base pair inversion were detected by DHPLC whereas DGGE failed to identify four single base substitutions and a single base pair deletion. In addition, we show that DHPLC has been used for the identification of 5 different mutations in exon 7 of hMSH2 that could not be detected by DGGE. From this study we conclude that DHPLC is a more effective and rapid alternative to the detection of mutations in hMSH2 and hMLH1 with the same or better accuracy than DGGE. Furthermore, this technique offers opportunities for automation, which have not been realised for the majority of other methods of gene analysis.

  10. The guanidine hydrochloride-induced denaturation of CP43 and CP47 studied by terahertz time-domain spectroscopy

    QU YuanGang; CHEN Hua; QIN XiaoChun; WANG Li; LI LiangBi; KUANG TingYun


    Terahertz time-domain spectroscopy (THz-TDS) is a new technique in studying the conformational state of a molecule in recent years. In this work, we reported the first use of THz-TDS to examine the denaturation of two photosynthesis membrane proteins: CP43 and CP47. THz-TDS was proven to be useful in discriminating the different conformational states of given proteins with similar structure and in monitoring the denaturation process of proteins. Upon treatment with guanidine hydrochloride (GuHCI),a 1.8 THz peak appeared for CP47 and free chlorophyll a (Chi a). This peak was deemed to originate from the interaction between Chli a and GuHCI molecules. The Chl a molecules in CP47 interacted with GuHCI more easily than those in CP43.

  11. Study of dynamical process of heat denaturation in optically trapped single microorganisms by near-infrared Raman spectroscopy

    Xie, Changan; Li, Yong-qing; Tang, Wei; Newton, Ronald J.


    The development of laser traps has made it possible to investigate single cells and record real-time Raman spectra during a heat-denaturation process when the temperature of the surrounding medium is increased. Large changes in the phenylalanine band (1004 cm-1) of near-infrared spectra between living and heat-treated cells were observed in yeast and Escerichia coli and Enterobacter aerogenes bacteria. This change appears to reflect the change in environment of phenylalanine as proteins within the cells unfold as a result of increasing temperatures. As a comparison, we measured Raman spectra of native and heat-denatured solutions of bovine serum albumin proteins, and a similar change in the phenylalanine band of spectra was observed. In addition, we measured Raman spectra of native and heat-treated solutions of pure phenylalanine molecules; no observable difference in vibrational spectra was observed. These findings may make it possible to study conformational changes in proteins within single cells.

  12. Simultaneous immobilization of dehydrogenases on polyvinylidene difluoride resin after separation by non-denaturing two-dimensional electrophoresis

    Shimazaki, Youji [Graduate School of Science and Engineering (Science Section) and Venture Business Laboratory, Ehime University, Bunkyo-cho 2-5, Matsuyama City 790-8577 (Japan)], E-mail:; Kadota, Mariko [Faculty of Science, Ehime University, Matsuyama (Japan)


    We detected mouse liver malate, sorbitol and aldehyde dehydrogenases by negative staining, analysis of malate and sorbitol dehydrogenase activities using each substrate, and electron transfers including nicotinamide adenine dinucleotide (NAD) and nitroblue tetrazolium in non-denaturing two-dimensional electrophoresis (2-DE) gel. Dehydrogenases were also identified by electrospray ionization tandem mass spectrometry (ESI-MS/MS) after 2-DE separation and protein detection by negative staining. Spots of dehydrogenases separated by 2-DE were excised, and simultaneously transferred and immobilized on polyvinylidene difuoride (PVDF) resin by electrophoresis. The dehydrogenase activities remained intact after immobilization. In conclusion, resin-immobilized dehydrogenases can be simultaneously obtained after separation by non-denaturing 2-DE, detection by negative staining and transferring to resins.

  13. Electrochemically-driven large amplitude pH cycling for acid-base driven DNA denaturation and renaturation.

    Wang, Yong-Chun; Lin, Cong-Bin; Su, Jian-Jia; Ru, Ying-Ming; Wu, Qiao; Chen, Zhao-Bin; Mao, Bing-Wei; Tian, Zhao-Wu


    In this paper, we present an electrochemically driven large amplitude pH alteration method based on a serial electrolytic cell involving a hydrogen permeable bifacial working electrode such as Pd thin foil. The method allows solution pH to be changed periodically up to ±4~5 units without additional alteration of concentration and/or composition of the system. Application to the acid-base driven cyclic denaturation and renaturation of 290 bp DNA fragments is successfully demonstrated with in situ real-time UV spectroscopic characterization. Electrophoretic analysis confirms that the denaturation and renaturation processes are reversible without degradation of the DNA. The serial electrolytic cell based electrochemical pH alteration method presented in this work would promote investigations of a wide variety of potential-dependent processes and techniques.

  14. pH-responsive polymer-assisted refolding of urea- and organic solvent-denatured alpha-chymotrypsin.

    Roy, I; Gupta, M N


    A pH-responsive polymer Eudragit S-100 has been found to assist in correct folding of alpha-chymotrypsin denatured with 8 M urea and 100 mM dithiothreitol at pH 8.2. The complete activity could be regained within 10 min during refolding. Both native and refolded enzymes showed emission of intrinsic fluorescence with lambda(max) of 342 nm. Gel electrophoresis showed that the presence of Eudragit S-100 led to dissociation of multimers followed by the appearance of a band at the monomer position. The unfolding (by 8 M urea) and folding (assisted by the polymer) also led to complete renaturation of alpha-chymotrypsin initially denatured by 90% dioxane. The implications of the data in recovery of enzyme activity from inclusion bodies and the interesting possibility in the in vivo context of reversing protein aggregation in amyloid-based diseases have been discussed.

  15. Mercury(II) binds to both of chymotrypsin's histidines, causing inhibition followed by irreversible denaturation/aggregation.

    Stratton, Amanda; Ericksen, Matthew; Harris, Travis V; Symmonds, Nick; Silverstein, Todd P


    The toxicity of mercury is often attributed to its tight binding to cysteine thiolate anions in vital enzymes. To test our hypothesis that Hg(II) binding to histidine could be a significant factor in mercury's toxic effects, we studied the enzyme chymotrypsin, which lacks free cysteine thiols; we found that chymotrypsin is not only inhibited, but also denatured by Hg(II). We followed the aggregation of denatured enzyme by the increase in visible absorbance due to light scattering. Hg(II)-induced chymotrypsin precipitation increased dramatically above pH 6.5, and free imidazole inhibited this precipitation, implicating histidine-Hg(II) binding in the process of chymotrypsin denaturation/aggregation. Diethylpyrocarbonate (DEPC) blocked chymotrypsin's two histidines (his40 and his57 ) quickly and completely, with an IC50 of 35 ± 6 µM. DEPC at 350 µM reduced the hydrolytic activity of chymotrypsin by 90%, suggesting that low concentrations of DEPC react with his57 at the active site catalytic triad; furthermore, DEPC below 400 µM enhanced the Hg(II)-induced precipitation of chymotrypsin. We conclude that his57 reacts readily with DEPC, causing enzyme inhibition and enhancement of Hg(II)-induced aggregation. Above 500 µM, DEPC inhibited Hg(II)-induced precipitation, and [DEPC] >2.5 mM completely protected chymotrypsin against precipitation. This suggests that his40 reacts less readily with DEPC, and that chymotrypsin denaturation is caused by Hg(II) binding specifically to the his40 residue. Finally, we show that Hg(II)-histidine binding may trigger hemoglobin aggregation as well. Because of results with these two enzymes, we suggest that metal-histidine binding may be key to understanding all heavy metal-induced protein aggregation.

  16. The regulatory subunit of PKA-I remains partially structured and undergoes β-aggregation upon thermal denaturation.

    Khanh K Dao

    Full Text Available BACKGROUND: The regulatory subunit (R of cAMP-dependent protein kinase (PKA is a modular flexible protein that responds with large conformational changes to the binding of the effector cAMP. Considering its highly dynamic nature, the protein is rather stable. We studied the thermal denaturation of full-length RIα and a truncated RIα(92-381 that contains the tandem cyclic nucleotide binding (CNB domains A and B. METHODOLOGY/PRINCIPAL FINDINGS: As revealed by circular dichroism (CD and differential scanning calorimetry, both RIα proteins contain significant residual structure in the heat-denatured state. As evidenced by CD, the predominantly α-helical spectrum at 25°C with double negative peaks at 209 and 222 nm changes to a spectrum with a single negative peak at 212-216 nm, characteristic of β-structure. A similar α→β transition occurs at higher temperature in the presence of cAMP. Thioflavin T fluorescence and atomic force microscopy studies support the notion that the structural transition is associated with cross-β-intermolecular aggregation and formation of non-fibrillar oligomers. CONCLUSIONS/SIGNIFICANCE: Thermal denaturation of RIα leads to partial loss of native packing with exposure of aggregation-prone motifs, such as the B' helices in the phosphate-binding cassettes of both CNB domains. The topology of the β-sandwiches in these domains favors inter-molecular β-aggregation, which is suppressed in the ligand-bound states of RIα under physiological conditions. Moreover, our results reveal that the CNB domains persist as structural cores through heat-denaturation.

  17. Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions.

    Monera, O. D.; Kay, C. M.; Hodges, R. S.


    The objective of this study was to address the question of whether or not urea and guanidine hydrochloride (GdnHCl) give the same estimates of the stability of a particular protein. We previously suspected that the estimates of protein stability from GdnHCl and urea denaturation data might differ depending on the electrostatic interactions stabilizing the proteins. Therefore, 4 coiled-coil analogs were designed, where the number of intrachain and interchain electrostatic attractions (A) were systematically changed to repulsions (R): 20A, 15A5R, 10A10R, and 20R. The GdnHCl denaturation data showed that the 4 coiled-coil analogs, which had electrostatic interactions ranging from 20 attractions to 20 repulsions, had very similar [GdnHCl]1/2 values (average of congruent to 3.5 M) and, as well, their delta delta Gu values were very close to 0 (0.2 kcal/mol). In contrast, urea denaturation showed that the [urea]1/2 values proportionately decreased with the stepwise change from 20 electrostatic attractions to 20 repulsions (20A, 7.4 M; 15A5R, 5.4 M; 10A10R, 3.2 M; and 20R, 1.4 M), and the delta delta Gu values correspondingly increased with the increasing differences in electrostatic interactions (20A-15A5R, 1.5 kcal/mol; 20A-10A10R, 3.7 kcal/mol; and 20A-20R, 5.8 kcal/mol). These results indicate that the ionic nature of GdnHCl masks electrostatic interactions in these model proteins, a phenomenon that was absent when the unchanged urea was used. Thus, GdnHCl and urea denaturations may give vastly different estimates of protein stability, depending on how important electrostatic interactions are to the protein. PMID:7703845

  18. Association of Streptomyces community composition determined by PCR-denaturing gradient gel electrophoresis with indoor mold status

    Johansson, Elisabet; Reponen, Tiina; Meller, Jarek; Vesper, Stephen; Yadav, Jagjit


    Both Streptomyces species and mold species have previously been isolated from moisture-damaged building materials; however, an association between these two groups of microorganisms in indoor environments is not clear. In this study we used a culture-independent method, PCR denaturing gradient gel electrophoresis (PCR-DGGE) to investigate the composition of the Streptomyces community in house dust. Twenty-three dust samples each from two sets of homes categorized as high-mold and low-mold bas...

  19. Selection for Protein Kinetic Stability Connects Denaturation Temperatures to Organismal Temperatures and Provides Clues to Archaean Life

    Romero-Romero, M. Luisa; Risso, Valeria A.; Martinez-Rodriguez, Sergio; Gaucher, Eric A.; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.


    The relationship between the denaturation temperatures of proteins (Tm values) and the living temperatures of their host organisms (environmental temperatures: TENV values) is poorly understood. Since different proteins in the same organism may show widely different Tm’s, no simple universal relationship between Tm and TENV should hold, other than Tm≥TENV. Yet, when analyzing a set of homologous proteins from different hosts, Tm’s are oftentimes found to correlate with TENV’s but this correlation is shifted upward on the Tm axis. Supporting this trend, we recently reported Tm’s for resurrected Precambrian thioredoxins that mirror a proposed environmental cooling over long geological time, while remaining a shocking ~50°C above the proposed ancestral ocean temperatures. Here, we show that natural selection for protein kinetic stability (denaturation rate) can produce a Tm↔TENV correlation with a large upward shift in Tm. A model for protein stability evolution suggests a link between the Tm shift and the in vivo lifetime of a protein and, more specifically, allows us to estimate ancestral environmental temperatures from experimental denaturation rates for resurrected Precambrian thioredoxins. The TENV values thus obtained match the proposed ancestral ocean cooling, support comparatively high Archaean temperatures, and are consistent with a recent proposal for the environmental temperature (above 75°C) that hosted the last universal common ancestor. More generally, this work provides a framework for understanding how features of protein stability reflect the environmental temperatures of the host organisms. PMID:27253436

  20. The N-terminal domain of a TonB-dependent transporter undergoes a reversible stepwise denaturation.

    Flores Jiménez, Ricardo H; Cafiso, David S


    Gram-negative bacteria contain a family of outer membrane transport proteins that function in the uptake of rare nutrients, such as iron and vitamin B(12). These proteins are termed TonB-dependent because transport requires an interaction with the inner-membrane protein TonB. Using a combination of site-directed spin labeling and chemical denaturation, we examined the site-specific unfolding of regions of the Escherichia coli vitamin B(12) transporter, BtuB. The data indicate that a portion of the N-terminal region of the protein, which occupies the lumen of the BtuB barrel, denatures prior to the unfolding of the barrel and that the free energy of folding for the N-terminus is smaller than that typically seen for globular proteins. Moreover, the data indicate that the N-terminal domain does not unfold in a single event but unfolds in a series of independent steps. The unfolding of the N-terminus is reversible, and removal of denaturant restores the native fold of the protein. These data are consistent with proposed transport mechanisms that involve a transient rearrangement or unfolding of the N-terminus of the protein, and they provide evidence of a specific protein conformation that might be an intermediate accessed during transport.

  1. Thermal, chemical and pH induced denaturation of a multimeric β-galactosidase reveals multiple unfolding pathways.

    Devesh Kishore

    Full Text Available BACKGROUND: In this case study, we analysed the properties of unfolded states and pathways leading to complete denaturation of a multimeric chick pea β-galactosidase (CpGAL, as obtained from treatment with guanidium hydrochloride, urea, elevated temperature and extreme pH. METHODOLOGY/PRINCIPAL FINDINGS: CpGAL, a heterodimeric protein with native molecular mass of 85 kDa, belongs to α+β class of protein. The conformational stability and thermodynamic parameters of CpGAL unfolding in different states were estimated and interpreted using circular dichroism and fluorescence spectroscopic measurements. The enzyme was found to be structurally and functionally stable in the entire pH range and upto 50 °C temperature. Further increase in temperature induces unfolding followed by aggregation. Chemical induced denaturation was found to be cooperative and transitions were irreversible, non-coincidental and sigmoidal. Free energy of protein unfolding (ΔG(0 and unfolding constant (K(obs were also calculated for chemically denatured CpGAL. SIGNIFICANCE: The protein seems to use different pathways for unfolding in different environments and is a classical example of how the environment dictates the path a protein might take to fold while its amino acid sequence only defines its final three-dimensional conformation. The knowledge accumulated could be of immense biotechnological significance as well.

  2. An assessment of the use of native and denatured forms of okra seed proteins as coagulants in drinking water treatment.

    Jones, Alfred Ndahi; Bridgeman, John


    The effects of temperature, storage time and water pH on the coagulation performance of okra seed protein in water treatment were assessed. In a jar test experiment, okra salt extract achieved a notable improvement in treatment efficiency with storage time and showed good performance in quality after thermal treatment at 60, 97 and 140 °C temperatures for 6, 4 and 2 hours, respectively. The performance improvement of more than 8% is considered to be due to the denaturation and subsequent removal of coagulation-hindering proteins in okra seed. Furthermore, the results of a sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis show two distinctive bands of protein responsible for the coagulation process after denaturation. It was further shown that at optimal coagulant dose, the pH of the treated water remained unaffected as a result of the protein's buffering capability during coagulation. Therefore, denatured okra seed exhibited improved performance compared to the native crude extract and offers clear benefits as a water treatment coagulant.

  3. Heat-denatured lysozyme aggregation and gelation as revealed by combined dielectric relaxation spectroscopy and light scattering measurements.

    Giugliarelli, A; Sassi, P; Paolantoni, M; Onori, G; Cametti, C


    The dielectric behavior of native and heat-denatured lysozyme in ethanol-water solutions was examined in the frequency range from 1 MHz to 2 GHz, using frequency-domain dielectric relaxation spectroscopy. Because of the conformational changes on unfolding, dielectric methods provide information on the denaturation process of the protein and, at protein concentration high enough, on the subsequent aggregation and gelation. Moreover, the time evolution of the protein aggregation and gelation was monitored measuring, by means of dynamic light scattering methods, the diffusion coefficient of micro-sized polystyrene particles, deliberately added to the protein solution, which act as a probe of the viscosity of the microenvironment close to the particle surface. All together, our measurements indicate that heat-induced denaturation favors, at high concentrations, a protein aggregation process which evolves up to the full gelation of the system. These findings have a direct support from IR measurements of the absorbance of the amide I band that, because of the unfolding, indicate that proteins entangle each other, producing a network structure which evolves, in long time limit, in the gel.

  4. Selection for Protein Kinetic Stability Connects Denaturation Temperatures to Organismal Temperatures and Provides Clues to Archaean Life.

    M Luisa Romero-Romero

    Full Text Available The relationship between the denaturation temperatures of proteins (Tm values and the living temperatures of their host organisms (environmental temperatures: TENV values is poorly understood. Since different proteins in the same organism may show widely different Tm's, no simple universal relationship between Tm and TENV should hold, other than Tm≥TENV. Yet, when analyzing a set of homologous proteins from different hosts, Tm's are oftentimes found to correlate with TENV's but this correlation is shifted upward on the Tm axis. Supporting this trend, we recently reported Tm's for resurrected Precambrian thioredoxins that mirror a proposed environmental cooling over long geological time, while remaining a shocking ~50°C above the proposed ancestral ocean temperatures. Here, we show that natural selection for protein kinetic stability (denaturation rate can produce a Tm↔TENV correlation with a large upward shift in Tm. A model for protein stability evolution suggests a link between the Tm shift and the in vivo lifetime of a protein and, more specifically, allows us to estimate ancestral environmental temperatures from experimental denaturation rates for resurrected Precambrian thioredoxins. The TENV values thus obtained match the proposed ancestral ocean cooling, support comparatively high Archaean temperatures, and are consistent with a recent proposal for the environmental temperature (above 75°C that hosted the last universal common ancestor. More generally, this work provides a framework for understanding how features of protein stability reflect the environmental temperatures of the host organisms.

  5. A Proposed Mechanism for the Thermal Denaturation of a Recombinant Bacillus Halmapalus Alpha-amylase - the Effect of Calcium Ions

    Nielsen, Anders D.; Pusey, Marc L.; Fuglsang, Claus C.; Westh, Peter


    The thermal stability of a recombinant alpha-amylase from Bacillus halmapalus alpha-amylase (BHA) has been investigated using circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). This alpha-amylase is homologous to other Bacillus alpha-amylases where previous crystallographic studies have identified the existence of 3 calcium binding sites in the structure. Denaturation of BHA is irreversible with a Tm of approximately 89 C, and DSC thermograms can be described using a one-step irreversible model. A 5 C increase in T(sub m) in the presence of 10 fold excess CaCl2 was observed. However, a concomitant increase in the tendency to aggregate was also observed. The presence of 30-40 fold excess calcium chelator (EDTA or EGTA) results in a large destabilization of BHA corresponding to about 40 C lower T(sub m), as determined by both CD and DSC. Ten fold excess EGTA reveals complex DSC thermograms corresponding to both reversible and irreversible transitions, which possibly originate from different populations of BHA:calcium complexes. The observations in the present study have, in combination with structural information of homologous alpha-amylases, provided the basis for the proposal of a simple denaturation mechanism of BHA. The proposed mechanism describes the irreversible thermal denaturation of different BHA:calcium complexes and the calcium binding equilibrium involved. Furthermore, the model accounts for a temperature induced reversible structural change associated with calcium binding.

  6. High Intensity Focused Ultrasound Monitoring using Harmonic Motion Imaging for Focused Ultrasound (HMIFU) under boiling or slow denaturation conditions

    Hou, Gary Y.; Marquet, Fabrice; Wang, Shutao; Apostolakis, Iason-Zacharias; Konofagou, Elisa E.


    Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a recently developed High-Intensity Focused Ultrasound (HIFU) treatment monitoring method that utilizes an amplitude-modulated therapeutic ultrasound beam to induce an oscillatory radiation force at the HIFU focus and estimates the focal tissue displacement to monitor the HIFU thermal treatment. In this study, the performance of HMIFU under acoustic, thermal and mechanical effects were investigated. The performance of HMIFU was assessed in ex vivo canine liver specimens (n=13) under slow denaturation or boiling regimes. Passive Cavitation Detector (PCD) was used to assess the acoustic cavitation activity while a bare-wire thermocouple was used to monitor the focal temperature change. During lesioning with slow denaturation, high quality displacements (correlation coefficient above 0.97) were observed under minimum cavitation noise, indicating tissue the initial-softening-then-stiffening property change. During HIFU with boiling, HMIFU monitored a consistent change in lesion-to-background displacement contrast (0.46±0.37) despite the presence of strong cavitation noise due to boiling during lesion formation. Therefore, HMIFU effectively monitored softening-then-stiffening during lesioning under slow denaturation, and detected lesioning under boiling with a distinct change in displacement contrast under boiling in the presence of cavitation. In conclusion, HMIFU was shown effective in HIFU monitoring and lesioning identification without being significantly affected by cavitation noise. PMID:26168177

  7. Preparation of denatured protein bone sterilized with gamma radiation; Preparacion de hueso desproteinizado esterilizado con radiacion gamma

    Luna Z, D. [ININ, 52045 Ocoyoacac, Estado de Mexico (Mexico)]. e-mail:


    The bone is one of the tissues more transplanted in the entire world by that the bone necessity for transplant every day becomes bigger. In the Bank of tissues Radio sterilized of the ININ the amnion and the pig skin are routinely processed. The tissue with which will be continued is with bone. Due to that in our country it doesn't have enough bone of human origin for the necessities required in the bone transplant, an option is the bone of bovine. Of this bone one can obtain denatured protein bone, with the same characteristics of the denatured protein human bone, the one which has been proven that it has good acceptance and incorporation in the human body when is transplanted. The method for the obtaining of the denatured protein bone of bovine, with the confirmation of the final product by means of X-ray diffraction is described. The radiosterilization of this bone with gamma rays and the determination of the lead content. (Author)

  8. [Singultus - Diagnostic Workup and Therapy].

    Marcus, Ohlrich; Royl, Georg


    A hiccup is a reflex movement with diffusely distributed afferents and efferents in the thorax; its functional relevance is controversial. In its physiological form, it is mostly a minor complaint that stops spontaneously and rarely leads to medical consultation. However, prolonged agonizing hiccups represent serious deterioration of quality of life. Chronic hiccups by definition last for more than 48 h, with gastroesophageal reflux being the frequent underlying disease. Various other causes affect multiple organ systems, some with serious underlying diseases. A hiccup may be the only symptom at the first manifestation of some neurological disorders. In neuroimaging a lesion of the medulla oblongata is often seen. A NMO and an ischemic stroke with Wallenberg syndrome are 2 frequently underlying neurological diseases, but other inflammatory and vascular diseases and tumors of the central nervous system may be present. No optimal evidencebased recommendations for diagnosis and management of chronic hiccups are available. The search for the underlying disease often requires an interdisciplinary approach by internists, neurologists, and otolaryngologists. Symptomatic treatment may be necessary even before diagnosis. Persistent hiccups, a common problem in oncological palliative care, are often challenging. Proton pump inhibitor or prokinetics are used for treating underlying gastroesophageal reflux and baclofen with or without gabapentin in other cases. Anticonvulsants, antipsychotics, antidepressants, and calcium channel blockers represent other alternative treatment possibilities. In therapy-refractory cases, invasive procedures such as the selective phrenic nerve block are available. More studies are needed to help deal with the diagnostic and therapeutic challenge that hiccups present for neurologists. © Georg Thieme Verlag KG Stuttgart · New York.

  9. Denaturing gradient gel electrophoresis of neonatal intestinal microbiota in relation to the development of asthma

    Desager Kristine N


    Full Text Available Abstract Background The extended 'hygiene hypothesis' suggests that the initial composition of the infant gut microbiota is a key determinant in the development of atopic disease. Several studies have demonstrated that the microbiota of allergic and non-allergic infants are different even before the development of symptoms, with a critical time window during the first 6 months of life. The aim of the study was to investigate the association between early intestinal colonisation and the development of asthma in the first 3 years of life using DGGE (denaturing gradient gel electrophoresis. Methods In a prospective birth cohort, 110 children were classified according to the API (Asthma Predictive Index. A positive index included wheezing during the first three years of life combined with eczema in the child in the first years of life or with a parental history of asthma. A fecal sample was taken at the age of 3 weeks and analysed with DGGE using universal and genus specific primers. Results The Asthma Predictive Index was positive in 24/110 (22% of the children. Using universal V3 primers a band corresponding to a Clostridum coccoides XIVa species was significantly associated with a positive API. A Bacteroides fragilis subgroup band was also significantly associated with a positive API. A final DGGE model, including both bands, allowed correct classification of 73% (80/110 of the cases. Conclusion Fecal colonisation at age 3 weeks with either a Bacteroides fragilis subgroup or a Clostridium coccoides subcluster XIVa species is an early indicator of possible asthma later in life. These findings need to be confirmed in a new longitudinal follow-up study.

  10. DNA-based diagnosis of isolated sulfite oxidase deficiency by denaturing high-performance liquid chromatography.

    Lam, Ching-Wan; Li, Chi-Keung; Lai, Chi-Kong; Tong, Sui-Fan; Chan, Kwok-Yin; Ng, Grace Sui-Fun; Yuen, Yuet-Ping; Cheng, Anna Wai-Fun; Chan, Yan-Wo


    Isolated sulfite oxidase deficiency is a rare autosomal recessive disease, characterized by severe neurological abnormalities, seizures, mental retardation, and dislocation of the ocular lenses, that often leads to death in infancy. There is a special demand for prenatal diagnosis, since no effective treatment is available for isolated sulfite oxidase deficiency. Until now, the cDNA sequence of the sulfite oxidase (SUOX) gene has been available, but the genomic sequence of the SUOX gene has not been published. In this study, we have performed a DNA-based diagnosis of isolated sulfite oxidase deficiency in a Chinese patient. To do so, we designed oligonucleotide primers for amplification of the predicted exons and intron-exon boundaries of the SUOX gene obtained from the completed draft version of the human genome. Using overlapping PCR products, we confirmed the flanking intronic sequences of the coding exons and that the entire 466-residue mature peptide is encoded by the last exon of the gene. We then performed mutation detection using denaturing high-performance liquid chromatography (DHPLC). The DHPLC chromatogram of exon 2b showed the presence of heteroduplex peaks only after mixing of the mutant DNA with the wild-type DNA, indicating the presence of a homozygous mutation. Direct DNA sequencing showed a homozygous base substitution at codon 160, changing the codon from CGG to CAG, which changes the amino acid from arginine to glutamine, i.e., R160Q. The DNA-based diagnosis of isolated sulfite oxidase deficiency will enable us to make an accurate determination of carrier status and to perform prenatal diagnosis of this disease. The availability of the genomic sequences of human genes from the completed draft human genome sequence will simplify the development of molecular genetic diagnoses of human diseases from peripheral blood DNA.

  11. Application of denaturing gradient gel electrophoresis (DGGE) to the analysis of endodontic infections.

    Siqueira, José F; Rôças, Isabela N; Rosado, Alexandre S


    The recent expanding use of cultivation-independent techniques for bacterial identification is reliant on the lack of knowledge of the conditions under which most bacteria are growing in their natural habitat and the difficulty to develop culture media that accurately reproduce these conditions. A molecular method that has been recently used in several areas to examine the bacterial diversity living in diverse environments is the denaturing gradient gel electrophoresis (DGGE). In DGGE, polymerase chain reaction (PCR)-generated DNA fragments of the same length but with different base-pair sequences can be separated. Separation is based on electrophorectic mobility of a partially melted double-strand DNA molecule in polyacrylamide gels, which is decreased when compared with that of the completely helical form of the molecule. Molecules with different sequences may have a different melting behavior and will therefore stop migrating at different positions in the gel. Application of the PCR-DGGE method in endodontic research has revealed that there are significant differences in the predominant bacterial composition between asymptomatic and symptomatic cases. This suggests that the structure of the bacterial community can play a role in the development of symptoms. In addition, new bacterial phylotypes have been disclosed in primary endodontic infections. PCR-DGGE has also confirmed that intra-radicular infections are a common finding in root-filled teeth associated with persistent periradicular lesions. The microbiota in failed cases significantly vary from teeth to teeth, with a mean number of species far higher than previously shown by culturing approaches. Application of the PCR-DGGE technique in endodontic microbiology research has the potential to shed light on several aspects of the different types of endodontic infection as well as on the effects of treatment procedures with regard to infection control.

  12. Protein thermal denaturation is modulated by central residues in the protein structure network.

    Souza, Valquiria P; Ikegami, Cecília M; Arantes, Guilherme M; Marana, Sandro R


    Network structural analysis, known as residue interaction networks or graphs (RIN or RIG, respectively) or protein structural networks or graphs (PSN or PSG, respectively), comprises a useful tool for detecting important residues for protein function, stability, folding and allostery. In RIN, the tertiary structure is represented by a network in which residues (nodes) are connected by interactions (edges). Such structural networks have consistently presented a few central residues that are important for shortening the pathways linking any two residues in a protein structure. To experimentally demonstrate that central residues effectively participate in protein properties, mutations were directed to seven central residues of the β-glucosidase Sfβgly (β-D-glucoside glucohydrolase; EC These mutations reduced the thermal stability of the enzyme, as evaluated by changes in transition temperature (Tm ) and the denaturation rate at 45 °C. Moreover, mutations directed to the vicinity of a central residue also caused significant decreases in the Tm of Sfβgly and clearly increased the unfolding rate constant at 45 °C. However, mutations at noncentral residues or at surrounding residues did not affect the thermal stability of Sfβgly. Therefore, the data reported in the present study suggest that the perturbation of the central residues reduced the stability of the native structure of Sfβgly. These results are in agreement with previous findings showing that networks are robust, whereas attacks on central nodes cause network failure. Finally, the present study demonstrates that central residues underlie the functional properties of proteins.

  13. Analysis of variations in band positions for normalization in across-gel denaturing gradient gel electrophoresis.

    Matsushita, Yuko; Yamamura, Kohji; Morimoto, Sho; Bao, Zhihua; Kurose, Daisuke; Sato, Ikuo; Yoshida, Shigenobu; Tsushima, Seiya


    Variation in band position between gels is a well-known problem in denaturing gradient gel electrophoresis (DGGE). However, few reports have evaluated the degree of variation in detail. In this study, we investigated the variation in band positions of DNA samples extracted from soil, normalized using reference positions within marker lanes for DGGE in three organismal (bacterial, fungal, and nematode) conditions. For sample lanes, marker DNA (as a control) and sample DNA were used. The test for normality of distribution showed that the position data of a large percentage of bands were normally distributed but not for certain bands. For the normally-distributed data, their variations [standard deviation of marker bands (SDM) and standard deviation of sample bands (SDS), respectively] were assessed. For all organismal conditions, the degree of within-gel variation were similar between SDMs and SDSs, while between-gel variations in SDSs were larger than those in SDMs. Due to the large effect of between-gel variations, the total variations in SDSs were more varied between sample bands, and the mean variations of all sample bands were higher than those in the markers. We found that the total variation in the fungal and nematode SDSs decreased when the intervals between marker bands were narrowed, suggesting that band interval is important for reducing total variation in normalized band positions. For the non-normally distributed data, the distribution was examined in detail. This study provided detailed information on the variation of band positions, which could help to optimize markers for reducing band position variation, and could aid in the accurate identification of bands in across-gel DGGE analyses. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Nonlinear acoustic properties of ex vivo bovine liver and the effects of temperature and denaturation.

    Jackson, E J; Coussios, C-C; Cleveland, R O


    Thermal ablation by high intensity focused ultrasound (HIFU) has a great potential for the non-invasive treatment of solid tumours. Due to the high pressure amplitudes involved, nonlinear acoustic effects must be understood and the relevant medium property is the parameter of nonlinearity B/A. Here, B/A was measured in ex vivo bovine liver, over a heating/cooling cycle replicating temperatures reached during HIFU ablation, adapting a finite amplitude insertion technique, which also allowed for measurement of sound-speed and attenuation. The method measures the nonlinear progression of a plane wave through liver and B/A was chosen so that numerical simulations matched the measured waveforms. To create plane-wave conditions, sinusoidal bursts were transmitted by a 100 mm diameter 1.125 MHz unfocused transducer and measured using a 15 mm diameter 2.25 MHz broadband transducer in the near field. Attenuation and sound-speed were calculated using a reflected pulse from the smaller transducer using the larger transducer as the reflecting interface. Results showed that attenuation initially decreased with heating then increased after denaturation, the sound-speed initially increased with temperature and then decreased, and B/A showed an increase with temperature but no significant post-heating change. The B/A data disagree with other reports that show a significant change and we suggest that any nonlinear enhancement in the received ultrasound signal post-treatment is likely due to acoustic cavitation rather than changes in tissue nonlinearity.

  15. Influence of denatured and intermediate states of folding on protein aggregation.

    Fawzi, Nicolas L; Chubukov, Victor; Clark, Louis A; Brown, Scott; Head-Gordon, Teresa


    We simulate the aggregation thermodynamics and kinetics of proteins L and G, each of which self-assembles to the same alpha/beta [corrected] topology through distinct folding mechanisms. We find that the aggregation kinetics of both proteins at an experimentally relevant concentration exhibit both fast and slow aggregation pathways, although a greater proportion of protein G aggregation events are slow relative to those of found for protein L. These kinetic differences are correlated with the amount and distribution of intrachain contacts formed in the denatured state ensemble (DSE), or an intermediate state ensemble (ISE) if it exists, as well as the folding timescales of the two proteins. Protein G aggregates more slowly than protein L due to its rapidly formed folding intermediate, which exhibits native intrachain contacts spread across the protein, suggesting that certain early folding intermediates may be selected for by evolution due to their protective role against unwanted aggregation. Protein L shows only localized native structure in the DSE with timescales of folding that are commensurate with the aggregation timescale, leaving it vulnerable to domain swapping or nonnative interactions with other chains that increase the aggregation rate. Folding experiments that characterize the structural signatures of the DSE, ISE, or the transition state ensemble (TSE) under nonaggregating conditions should be able to predict regions where interchain contacts will be made in the aggregate, and to predict slower aggregation rates for proteins with contacts that are dispersed across the fold. Since proteins L and G can both form amyloid fibrils, this work also provides mechanistic and structural insight into the formation of prefibrillar species.

  16. Denatured ethanol release into gasoline residuals, Part 2: Fate and transport

    Freitas, Juliana G.; Barker, James F.


    When denatured ethanol (E95) is spilled in a site with previous gasoline contamination, it modifies the source distribution (Part 1). But it can also impact the transport and fate of hydrocarbons in the groundwater. Ethanol could cause an increase in dissolved concentrations and more persistent plumes due to cosolvency and decreased hydrocarbon biodegradation rates. To investigate these possibilities, two controlled releases were performed: first of E10 (gasoline with 10% ethanol) and one year later of E95 on top of the gasoline. Groundwater concentrations were monitored above and below the water table in multilevel wells. Soil cores and vapor samples were also collected over a period of approximately 400 days. Surprisingly, ethanol transport was very limited; at wells located 2.3 m downgradient from the mid-point of the release trench, the maximum concentration measured was around 2400 mg/L. After 392 days, only 3% of the ethanol released migrated past 2.3 m, and no ethanol remained in the source. The processes that caused ethanol loss were likely volatilization, aerobic biodegradation in the unsaturated zone, and anaerobic biodegradation. Evidence that biodegradation was significant in the source zone includes increased CO2 concentrations in the vapor and the presence of biodegradation products (acetate concentrations up to 2300 mg/L). The position of the dissolved hydrocarbon plumes was slightly shifted, but the concentrations and mass flux remained within the same range as before the spill, indicating that cosolvency was not significant. Hydrocarbons in the groundwater were significantly biodegraded, with more than 63% of the mass being removed in 7.5 m, even when ethanol was present in the groundwater. The impacts of ethanol on the hydrocarbon transport and fate were minimal, largely due to the separation of ethanol and hydrocarbons in the source (Part 1).

  17. A PCR-denaturing gradient gel electrophoresis approach to assess Fusarium diversity in asparagus.

    Yergeau, E; Filion, M; Vujanovic, V; St-Arnaud, M


    In North America, asparagus (Asparagus officinalis) production suffers from a crown and root rot disease mainly caused by Fusarium oxysporum f. sp. asparagi and F. proliferatum. Many other Fusarium species are also found in asparagus fields, whereas accurate detection and identification of these organisms, especially when processing numerous samples, is usually difficult and time consuming. In this study, a PCR-denaturing gradient gel electrophoresis (DGGE) method was developed to assess Fusarium species diversity in asparagus plant samples. Fusarium-specific PCR primers targeting a partial region of the translation elongation factor-1 alpha (EF-1 alpha) gene were designed, and their specificity was tested against genomic DNA extracted from a large collection of closely and distantly related organisms isolated from multiple environments. Amplicons of 450 bp were obtained from all Fusarium isolates, while no PCR product was obtained from non-Fusarium organisms. The ability of DGGE to discriminate between Fusarium taxa was tested over 19 different Fusarium species represented by 39 isolates, including most species previously reported from asparagus fields worldwide. The technique was effective to visually discriminate between the majority of Fusarium species and/or isolates tested in pure culture, while a further sequencing step permitted to distinguish between the few species showing similar migration patterns. Total genomic DNA was extracted from field-grown asparagus plants naturally infested with different Fusarium species, submitted to PCR amplification, DGGE analysis and sequencing. The two to four bands observed for each plant sample were all affiliated with F. oxysporum, F. proliferatum or F. solani, clearly supporting the reliability, sensitivity and specificity of this approach for the study of Fusarium diversity from asparagus plants samples.

  18. Hydrophobic collapse and cold denaturation in the Jagla model of water

    Buldyrev, Sergey V; Weiner, Saul [Department of Physics, Yeshiva University, 500 West 185th Street, New York, NY 10033 (United States); Kumar, Pradeep [Center for Studies in Physics and Biology, University of Texas at Austin, Austin, TX 78712-1167 (United States); Sastry, Srikanth [Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, Karnataka (India); Eugene Stanley, H [Center for Polymer Studies and Department of Physics, Boston University, Boston, MA 02215 (United States)


    The Jagla model is a coarse-grained model of water which describes interactions between groups of water molecules by a spherically symmetric potential characterized by a hard core, a linear repulsive ramp and a long-range attractive ramp. The Jagla model qualitatively reproduces the thermodynamics and dynamics of liquid water including density and diffusion anomalies as well as certain chemical properties such the increase of solubility of small hydrophobic particles upon cooling. We examine, via molecular dynamics simulation, the behavior of the bead-on-a-string polymers of various lengths in the Jagla model. We find that such polymers exhibit swelling upon cooling similar to cold denaturation of proteins in water. We show that while for short polymers the swelling is gradual, longer polymers exhibit a first-order-like phase transition between a globular phase at high temperatures to a random coil state at cold temperatures. This transition is associated with the formation of a liquid-polymer phase boundary surrounding the globule and complete dewetting of the central parts of the globule at high temperatures. We study thermodynamics of this transition and find that the entropy, volume, and potential energy of the solvent-random coil system is lower than those of the globule-solvent system. Accordingly the slope of the coil-globule transition line on a PT plane has positive slope. We present simple thermodynamic considerations similar to classical nucleation theory, which relate the temperature of the cold swelling transition to polymer length and relate the dewetting of the globule to its diameter and to the Egelstaff-Widom length scale.

  19. Hydrophobic collapse and cold denaturation in the Jagla model of water

    Buldyrev, Sergey V.; Kumar, Pradeep; Sastry, Srikanth; Stanley, H. Eugene; Weiner, Saul


    The Jagla model is a coarse-grained model of water which describes interactions between groups of water molecules by a spherically symmetric potential characterized by a hard core, a linear repulsive ramp and a long-range attractive ramp. The Jagla model qualitatively reproduces the thermodynamics and dynamics of liquid water including density and diffusion anomalies as well as certain chemical properties such the increase of solubility of small hydrophobic particles upon cooling. We examine, via molecular dynamics simulation, the behavior of the bead-on-a-string polymers of various lengths in the Jagla model. We find that such polymers exhibit swelling upon cooling similar to cold denaturation of proteins in water. We show that while for short polymers the swelling is gradual, longer polymers exhibit a first-order-like phase transition between a globular phase at high temperatures to a random coil state at cold temperatures. This transition is associated with the formation of a liquid-polymer phase boundary surrounding the globule and complete dewetting of the central parts of the globule at high temperatures. We study thermodynamics of this transition and find that the entropy, volume, and potential energy of the solvent-random coil system is lower than those of the globule-solvent system. Accordingly the slope of the coil-globule transition line on a PT plane has positive slope. We present simple thermodynamic considerations similar to classical nucleation theory, which relate the temperature of the cold swelling transition to polymer length and relate the dewetting of the globule to its diameter and to the Egelstaff-Widom length scale.

  20. Positive predictive value of CEA and Ca19-9 as tumor markers for recurrent colorectal cancer in cases where conventional work-up fail to localize disease.

    Yana Bocheva


    Full Text Available Introduction: Routine surveillance of colorectal cancer includes serial measurements of CEA levels. Although not routinely indicated Ca 19-9 is also a tool for recurrence. When any of these serum markers is elevated during follow up, this could represent a recurrence. The management of elevated tumor marker levels include clinical exams, endoscopy and conventional imaging –ultrasound, CT, MRI.Objective: To evaluate the positive predictive value of CEA and Ca19-9 as tumor markers for recurrent colorectal cancer in cases where conventional imaging and endoscopic studies fail to localize disease.Materials and methods: A total of 75 patients with elevated CEA and/or Ca19-9 serum levels and negative endoscopic exam as well as negative abdominal CT and Chest X-ray were included in the study. CEA levels were tested in 50 patients. Ca 19-9 was tested in 65 patients. 34 of the patients had both markers tested. All patients underwent whole body 18F-FDG PET/CT. Patients with negative of equivocal PET scan were further followed up (10 to 24 months.Results: Based on the reference standard – the results from PET/CT, if positive and the results from follow-up in cases of negative or equivocal scans, the positive predictive value of Ca 19-9 was 84% and that of CEA -83%. There was no significant difference in the PPV of Ca19-9 and CEA.Conclusion: Elevated CEA and Ca 19-9 levels in patients under active surveillance after operation for colorectal cancer have high positive predictive value for recurrence, even in cases where conventional work-up – endoscopy and CT don’t localize disease.

  1. Mandatory imaging cuts costs and reduces the rate of unnecessary surgeries in the diagnostic work-up of patients suspected of having appendicitis

    Lahaye, M.J.; Lambregts, D.M.J.; Mutsaers, E.; Beets-Tan, R.G.H. [Maastricht University Medical Centre, Department of Radiology, Maastricht (Netherlands); Essers, B.A.B. [Maastricht University Medical Centre, Department of Epidemiology and Medical Technology, Maastricht (Netherlands); Breukink, S.; Beets, G.L. [Maastricht University Medical Centre, Department of Surgery, Maastricht (Netherlands); Cappendijk, V.C. [Jeroen Bosch Hospital, Department of Radiology, ' s Hertogenbosch (Netherlands)


    To evaluate whether mandatory imaging is an effective strategy in suspected appendicitis for reducing unnecessary surgery and costs. In 2010, guidelines were implemented in The Netherlands recommending the mandatory use of preoperative imaging to confirm/refute clinically suspected appendicitis. This retrospective study included 1,556 consecutive patients with clinically suspected appendicitis in 2008-2009 (756 patients/group I) and 2011-2012 (800 patients/group II). Imaging use (none/US/CT and/or MRI) was recorded. Additional parameters were: complications, medical costs, surgical and histopathological findings. The primary study endpoint was the number of unnecessary surgeries before and after guideline implementation. After clinical examination by a surgeon, 509/756 patients in group I and 540/800 patients in group II were still suspected of having appendicitis. In group I, 58.5% received preoperative imaging (42% US/12.8% CT/3.7% both), compared with 98.7% after the guidelines (61.6% US/4.4% CT/ 32.6% both). The percentage of unnecessary surgeries before the guidelines was 22.9%. After implementation, it dropped significantly to 6.2% (p<0.001). The surgical complication rate dropped from 19.9% to 14.2%. The average cost-per-patient decreased by 594 EUR from 2,482 to 1,888 EUR (CL:-1081; -143). Increased use of imaging in the diagnostic work-up of patients with clinically suspected appendicitis reduced the rate of negative appendectomies, surgical complications and costs. (orig.)

  2. Further development of a robust workup process for solution-phase high-throughput library synthesis to address environmental and sample tracking issues.

    Kuroda, Noritaka; Hird, Nick; Cork, David G


    During further improvement of a high-throughput, solution-phase synthesis system, new workup tools and apparatus for parallel liquid-liquid extraction and evaporation have been developed. A combination of in-house design and collaboration with external manufacturers has been used to address (1) environmental issues concerning solvent emissions and (2) sample tracking errors arising from manual intervention. A parallel liquid-liquid extraction unit, containing miniature high-speed magnetic stirrers for efficient mixing of organic and aqueous phases, has been developed for use on a multichannel liquid handler. Separation of the phases is achieved by dispensing them into a newly patented filter tube containing a vertical hydrophobic porous membrane, which allows only the organic phase to pass into collection vials positioned below. The vertical positioning of the membrane overcomes the hitherto dependence on the use of heavier-than-water, bottom-phase, organic solvents such as dichloromethane, which are restricted due to environmental concerns. Both small (6-mL) and large (60-mL) filter tubes were developed for parallel phase separation in library and template synthesis, respectively. In addition, an apparatus for parallel solvent evaporation was developed to (1) remove solvent from the above samples with highly efficient recovery and (2) avoid the movement of individual samples between their collection on a liquid handler and registration to prevent sample identification errors. The apparatus uses a diaphragm pump to achieve a dynamic circulating closed system with a heating block for the rack of 96 sample vials and an efficient condenser to trap the solvents. Solvent recovery is typically >98%, and convenient operation and monitoring has made the apparatus the first choice for removal of volatile solvents.

  3. High resolution MRI for preoperative work-up of neonates with an anorectal malformation: a direct comparison with distal pressure colostography/fistulography.

    Thomeer, Maarten G; Devos, Annick; Lequin, Maarten; De Graaf, Nanko; Meeussen, Conny J H M; Meradji, Morteza; De Blaauw, Ivo; Sloots, Cornelius E J


    To compare MRI and colostography/fistulography in neonates with anorectal malformations (ARM), using surgery as reference standard. Thirty-three neonates (22 boys) with ARM were included. All patients underwent both preoperative high-resolution MRI (without sedation or contrast instillation) and colostography/fistulography. The Krickenbeck classification was used to classify anorectal malformations, and the level of the rectal ending in relation to the levator muscle was evaluated. Subjects included nine patients with a bulbar recto-urethral fistula, six with a prostatic recto-urethral fistula, five with a vestibular fistula, five with a cloacal malformation, four without fistula, one with a H-type fistula, one with anal stenosis, one with a rectoperineal fistula and one with a bladderneck fistula. MRI and colostography/fistulography predicted anatomy in 88 % (29/33) and 61 % (20/33) of cases, respectively (p = 0.012). The distal end of the rectal pouch was correctly predicted in 88 % (29/33) and 67 % (22/33) of cases, respectively (p = 0.065). The length of the common channel in cloacal malformation was predicted with MRI in all (100 %, 5/5) and in 80 % of cases (4/5) with colostography/fistulography. Two bowel perforations occurred during colostography/fistulography. MRI provides the most accurate evaluation of ARM and should be considered a serious alternative to colostography/fistulography during preoperative work-up. • High-resolution MRI is feasible without the use of sedation or anaesthesia. • MRI is more accurate than colostography/fistulography in visualising the type of ARM. • MRI is as reliable as colostography/fistulography in predicting the level of the rectal pouch. • Colostography/fistulography can be complicated by bowel perforation.

  4. Diagnostic imaging work-up for disease relapse after radical treatment for prostate cancer: how to differentiate local from systemic disease? The urologist point of view.

    Schiavina, R; Brunocilla, E; Borghesi, M; Vagnoni, V; Castellucci, P; Nanni, C; Ceci, F; Gacci, M; Martorana, G; Fanti, S


    About 40% of all patients undergoing radical treatment for localized prostate cancer (PCa) develop biochemical relapse (BCR) during lifetime but only 10-20% of them will show clinically detectable recurrences. Prostatic bed, pelvic or retroperitoneal lymph nodes (LN) and bones (especially the spine) are the sites where we must focus our attention in the early phase of PSA relapse. Time to PSA relapse, PSA kinetics, pathological Gleason score and pathological stage are the main factors related to the likelihood of local vs. distant relapse. Before an extensive diagnostic work-up in patients with BCR, is mandatory to understand if there is a therapeutic consequence or not for the patient. Current imaging techniques have some potential but many limits are yet encountered in the diagnosis of disease relapse. Transrectal ultrasound (TRUS) and Multiparametric Magnetic Resonance Imaging (MRI) have low accuracy in the detection of the recurrence. Today, Choline PET/CT may visualize the site of recurrence earlier, with better accuracy than conventional imaging, in a single step and even in the presence of low PSA level. In recent years, the new radiotracer (18)F-FACBC has been proposed as a possible alternative radiopharmaceutical to detect PCa relapse. From a clinical point of view, first clinical studies showed very promising and reproducible results with an improvement in sensitivity is about 20-25% with respect to Choline PET/CT, rendering the FACBC the possible radiotracer of the future for PCa. In conclusion, many improvements have been recently achieved in imaging techniques for PCa restaging, essentially in Nuclear Medicine and MRI, but negative results remain in many cases. Low sensitivity, costs, availability of technologies and confirmation of the results remain the major limitations in most cases.

  5. Contemporary update on pathology-related issues on routine workup of prostate biopsy: sectioning, tumor extent measurement, specimen orientation, and immunohistochemistry.

    Montironi, Rodolfo; Lopez-Beltran, Antonio; Mazzucchelli, Roberta; Scarpelli, Marina; Galosi, Andrea B; Cheng, Liang


    While the prime goal of the needle biopsy is to diagnose prostatic adenocarcinoma (PCa), once PCa is detected further descriptive information regarding the type of cancer, amount of tumor, and grade in prostate needle cores forms the cornerstone for contemporary management of the patient and to assess the potential for local cure and the risk for distant metastasis. This review gives an update on selected pathology-related issues on routine workup of prostate biopsy with special references to adequate histologic sectioning necessary to maximize cancer yield, tumor extent measurements and methodologies, specimen orientation, and the role of immunohistochemistry in the evaluation of the prostate. Multiple factors influence the diagnostic yield of prostate biopsies. Many of these factors are fixed and uncontrollable. Other factors are controlled by the urologist, including number of cores obtained, method and location of biopsy, and amount of tissue obtained. The yield of cancer is also controlled by the pathologist and histotechnologist. It is necessary to report the number of cores submitted and the number of positive cores, thereby giving the fraction of positive cores. The percentage involvement by carcinoma with or without the linear extent of carcinoma of the single core with the greatest amount of tumor should also be provided. Using the marking technique, we can add a new pathological parameter: pathological orientation. Cancer or atypical lesions can be accurately located within the biopsy specimen and integrated to biopsy approach. Probably the most common use of immunohistochemistry in the evaluation of the prostate is for the identification of basal cells, which are absent with rare exception in adenocarcinoma of the prostate and in general positive in mimickers of prostate cancer. If a case is still considered atypical by a uropathology expert after negative basal cell staining, positive staining for alpha-methylacyl-CoA-racemase can help establish in 50

  6. Diagnostic work-up for detection of paroxysmal atrial fibrillation after acute ischemic stroke: cross-sectional survey on German stroke units.

    Rizos, Timolaos; Quilitzsch, Anika; Busse, Otto; Haeusler, Karl Georg; Endres, Matthias; Heuschmann, Peter; Veltkamp, Roland


    Multiple methods to detect paroxysmal atrial fibrillation (pAF) in patients with acute stroke are available. However, it is unknown which approaches are currently used in clinical routine and guidelines remain vague to the extent of cardiac monitoring. We characterize diagnostic efforts for pAF detection on German stroke units (SU). A standardized anonymous questionnaire was sent to all clinical leads of certified SUs in Germany. The questionnaire focused on basic characteristics of SUs, procedures to detect AF, and estimates on AF detection. One hundred seventy-nine SU leads participated (response rate 71.6%). All patients undergo continuous bedside ECG monitoring. A percentage of 77.6 SUs initiate additional 24-hour Holter ECG in >50% of patients without known AF. Patients with transient ischemic attack are monitored significantly shorter than patients with ischemic stroke. Independent of SU type or size, 67.6% of leads assumed to fail detecting pAF in 5% to 20% of patients. In cryptogenic stroke, additional ECG monitoring is recommended by 90.2% but only 13.8% of SUs perform routine ECG follow-up visits. The use of implanted event recorders is low (1-10 patients/y by 60.7% of SUs; 28.1%: no use). A percentage of 83.9 do not use external event recorders. Our survey demonstrates substantial heterogeneity among German SUs on diagnostic work-up for pAF. Future prospective multicenter studies should systematically evaluate the impact of different methods to uncover pAF. © 2015 American Heart Association, Inc.

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

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


    Although the thermodynamics of protein denaturation at temperatures over 100 °C is essential for the rational design of highly stable proteins, it is not understood well because of the associated technical difficulties...

  8. A comparison of the pH, urea, and temperature-denatured states of barnase by heteronuclear NMR: implications for the initiation of protein folding.

    Arcus, V L; Vuilleumier, S; Freund, S M; Bycroft, M; Fersht, A R


    The denatured states of barnase that are induced by urea, acid, and high temperature and acid have been assigned and characterised by high resolution heteronuclear NMR. The assignment was completed using a combination of triple-resonance and magnetisation-transfer methods. The latter was facilitated by selecting a suitable mutant of barnase (Ile-->Val51) which has an appropriate rate of interconversion between native and denatured states in urea. 3J NH-C alpha H coupling constants were determined for pH and urea-denatured barnase and intrinsic "random coil" coupling constants are shown to be different for different residue types. All the denatured states are highly unfolded. But, a consistent series of weak correlations in chemical shift, NOESY and coupling constant data provides evidence that the acid-denatured state has some residual structure in regions that form the first and second helices and the central strands of beta-sheet in the native protein. The acid/temperature-denatured states has less structure in these regions, and the urea-denatured state, less still. These observations may be combined with detailed analyses of the folding pathway of barnase from kinetic studies to illuminate the relevance of residual structure in the denatured states of proteins to the mechanism of protein folding. First, the folding of barnase is known to proceed in its later stages through structures in which the first helix and centre of the beta-sheet are extensively formed. Thus, embryonic initiation sites for these do exist in the denatured states and so could well develop into true nuclei. Second, it has been clearly established that the second helix is unfolded in these later states, and so residual structure in this region of the protein is non-productive. These data fit a model of protein folding in which local nucleation sites are latent in the denatured state and develop only when they make interactions elsewhere in the protein that stabilise them during the folding

  9. Differential Scanning Calorimetry Analysis of the Effects of Heat and Pressure on Protein Denaturation in Soy Flour Mixed with Various Types of Plasticizers.

    Kweon, Meera; Slade, Louise; Levine, Harry


    The effects of heat and pressure on protein denaturation in soy flour were explored by an experimental design that used pressure (atmospheric to 600 MPa), temperature (room to 90 °C), time (1 to 60 min), and type of aqueous plasticizer (NaCl, sucrose, betaine, and lactobionic acid (LBA)) as factors. When 50% (w/w) soy flour-water paste was high hydrostatic pressure (HHP)-treated for 20 min at 25 °C, the treatment at 200 MPa showed a small effect on denaturation of only the 7S soy globulin, but the treatment at 600 MPa showed a significant effect on denaturation of both the 7S and 11S soy globulins. The treatment at 60 °C showed a less-pronounced effect on denaturation of the 11S globulin, even at 600 MPa, but that at 90 °C showed a similar extent of denaturation of the 11S globulin at 600 MPa to that at 25 °C. Chaotropic 2N NaCl, 50% sucrose-, 50% betaine-, or 50% LBA-water solutions showed protective effects on protein denaturation during HHP treatment at 25 °C. Although LBA enhanced the extent of thermostability of soy protein less than did 2N NaCl, LBA exhibited better stabilization against pressure. The results from DSC analysis demonstrated that thermostable soy proteins were not always barostable. © 2017 Institute of Food Technologists®.

  10. Detection of ligation products of DNA linkers with 5'-OH ends by denaturing PAGE silver stain.

    Feng Gao

    Full Text Available To explore if DNA linkers with 5'-hydroxyl (OH ends could be joined by commercial T4 and E. coli DNA ligase, these linkers were synthesized by using the solid-phase phosphoramidite method and joined by using commercial T4 and E. coli DNA ligases. The ligation products were detected by using denaturing PAGE silver stain and PCR method. About 0.5-1% of linkers A-B and E-F, and 0.13-0.5% of linkers C-D could be joined by T4 DNA ligases. About 0.25-0.77% of linkers A-B and E-F, and 0.06-0.39% of linkers C-D could be joined by E. coli DNA ligases. A 1-base deletion (-G and a 5-base deletion (-GGAGC could be found at the ligation junctions of the linkers. But about 80% of the ligation products purified with a PCR product purification kit did not contain these base deletions, meaning that some linkers had been correctly joined by T4 and E. coli DNA ligases. In addition, about 0.025-0.1% of oligo 11 could be phosphorylated by commercial T4 DNA ligase. The phosphorylation products could be increased when the phosphorylation reaction was extended from 1 hr to 2 hrs. We speculated that perhaps the linkers with 5'-OH ends could be joined by T4 or E. coli DNA ligase in 2 different manners: (i about 0.025-0.1% of linkers could be phosphorylated by commercial T4 DNA ligase, and then these phosphorylated linkers could be joined to the 3'-OH ends of other linkers; and (ii the linkers could delete one or more nucleotide(s at their 5'-ends and thereby generated some 5'-phosphate ends, and then these 5'-phosphate ends could be joined to the 3'-OH ends of other linkers at a low efficiency. Our findings may probably indicate that some DNA nicks with 5'-OH ends can be joined by commercial T4 or E. coli DNA ligase even in the absence of PNK.

  11. Whole-body-MR imaging including DWIBS in the work-up of patients with head and neck squamous cell carcinoma: A feasibility study

    Noij, Daniel P., E-mail: [Department of Radiology and Nuclear Medicine, VU University Medical Center, De Boelelaan 1117, PO Box 7057, 1007 MB Amsterdam (Netherlands); Boerhout, Els J., E-mail: [Department of Radiology and Nuclear Medicine, VU University Medical Center, De Boelelaan 1117, PO Box 7057, 1007 MB Amsterdam (Netherlands); Pieters-van den Bos, Indra C., E-mail: [Department of Radiology and Nuclear Medicine, VU University Medical Center, De Boelelaan 1117, PO Box 7057, 1007 MB Amsterdam (Netherlands); Comans, Emile F., E-mail: [Department of Radiology and Nuclear Medicine, VU University Medical Center, De Boelelaan 1117, PO Box 7057, 1007 MB Amsterdam (Netherlands); Oprea-Lager, Daniela, E-mail: [Department of Radiology and Nuclear Medicine, VU University Medical Center, De Boelelaan 1117, PO Box 7057, 1007 MB Amsterdam (Netherlands); Reinhard, Rinze, E-mail: [Department of Radiology and Nuclear Medicine, VU University Medical Center, De Boelelaan 1117, PO Box 7057, 1007 MB Amsterdam (Netherlands); Hoekstra, Otto S., E-mail: [Department of Radiology and Nuclear Medicine, VU University Medical Center, De Boelelaan 1117, PO Box 7057, 1007 MB Amsterdam (Netherlands); Bree, Remco de, E-mail: [Department Otolaryngology/Head and Neck Surgery, VU University Medical Center, De Boelelaan 1117, PO Box 7057, 1007 MB Amsterdam (Netherlands); Graaf, Pim de, E-mail: [Department of Radiology and Nuclear Medicine, VU University Medical Center, De Boelelaan 1117, PO Box 7057, 1007 MB Amsterdam (Netherlands); Castelijns, Jonas A., E-mail: [Department of Radiology and Nuclear Medicine, VU University Medical Center, De Boelelaan 1117, PO Box 7057, 1007 MB Amsterdam (Netherlands)


    Objectives: To assess the feasibility of whole-body magnetic resonance imaging (WB-MRI) including diffusion-weighted whole-body imaging with background-body-signal-suppression (DWIBS) for the evaluation of distant malignancies in head and neck squamous cell carcinoma (HNSCC); and to compare WB-MRI findings with {sup 18}F-fluorodeoxyglucose positron emission tomography/computed tomography ({sup 18}F-FDG-PET/CT) and chest-CT. Methods: Thirty-three patients with high risk for metastatic spread (26 males; range 48–79 years, mean age 63 ± 7.9 years (mean ± standard deviation) years) were prospectively included with a follow-up of six months. WB-MRI protocol included short-TI inversion recovery and T1-weighted sequences in the coronal plane and half-fourier acquisition single-shot turbo spin-echo T2 and contrast-enhanced-T1-weighted sequences in the axial plane. Axial DWIBS was reformatted in the coronal plane. Interobserver variability was assessed using weighted kappa and the proportion specific agreement (PA). Results: Two second primary tumors and one metastasis were detected on WB-MRI. WB-MRI yielded seven clinically indeterminate lesions which did not progress at follow-up. The metastasis and one second primary tumor were found when combining {sup 18}F-FDG-PET/CT and chest-CT findings. Interobserver variability for WB-MRI was κ = 0.91 with PA ranging from 0.82 to 1.00. For {sup 18}F-FDG-PET/CT κ could not be calculated due to a constant variable in the table and PA ranged from 0.40 to 0.99. Conclusions: Our WB-MRI protocol with DWIBS is feasible in the work-up of HNSCC patients for detection and characterization of distant pathology. WB-MRI can be complementary to {sup 18}F-FDG-PET/CT, especially in the detection of non {sup 18}F-FDG avid second primary tumors.

  12. Conformational diversity of acid-denatured cytochrome c studied by a matrix analysis of far-UV CD spectra.

    Konno, T


    The singular value decomposition (SVD) analysis was applied to a large set of far-ultraviolet circular dichroism (far-UV CD) spectra (100-400 spectra) of horse heart cytochrome c (cyt c). The spectra were collected at pH 1.7-5.0 in (NH4)2SO4, sorbitol and 2,2,2-trifluoroethanol (TFE) solutions. The present purpose is to develop a rigorous matrix method applied to far-UV CD spectra to resolve in details conformational properties of proteins in the non-native (or denatured) regions. The analysis established that three basis spectral components are contained in a data set of difference spectra (referred to the spectrum of the native state) used here. By a further matrix transformation, any observed spectrum could be decomposed into fractions of the native (N), the molten-globule (MG), the highly denatured (D), and the alcohol-induced helical (H) spectral forms. This method could determine fractional transition curves of each conformer as a function of solution conditions, which gave the results consistent with denaturation curves of cyt c monitored by other spectroscopic methods. The results in sorbitol solutions, for example, suggested that the preferential hydration effect of the co-solvent stabilizes the MG conformer of cyt c. This report has found that the systematic SVD analysis of the far-UV CD spectra is a powerful tool for the conformational analysis of the non-native species of a protein when it is suitably supplemented with other experimental methods.

  13. The non-uniform early structural response of globular proteins to cold denaturing conditions: A case study with Yfh1

    Chatterjee, Prathit; Bagchi, Sayan, E-mail:, E-mail:; Sengupta, Neelanjana, E-mail:, E-mail: [Physical Chemistry Division, CSIR-National Chemical Laboratory, Pune 411008 (India)


    The mechanism of cold denaturation in proteins is often incompletely understood due to limitations in accessing the denatured states at extremely low temperatures. Using atomistic molecular dynamics simulations, we have compared early (nanosecond timescale) structural and solvation properties of yeast frataxin (Yfh1) at its temperature of maximum stability, 292 K (T{sub s}), and the experimentally observed temperature of complete unfolding, 268 K (T{sub c}). Within the simulated timescales, discernible “global” level structural loss at T{sub c} is correlated with a distinct increase in surface hydration. However, the hydration and the unfolding events do not occur uniformly over the entire protein surface, but are sensitive to local structural propensity and hydrophobicity. Calculated infrared absorption spectra in the amide-I region of the whole protein show a distinct red shift at T{sub c} in comparison to T{sub s}. Domain specific calculations of IR spectra indicate that the red shift primarily arises from the beta strands. This is commensurate with a marked increase in solvent accessible surface area per residue for the beta-sheets at T{sub c}. Detailed analyses of structure and dynamics of hydration water around the hydrophobic residues of the beta-sheets show a more bulk water like behavior at T{sub c} due to preferential disruption of the hydrophobic effects around these domains. Our results indicate that in this protein, the surface exposed beta-sheet domains are more susceptible to cold denaturing conditions, in qualitative agreement with solution NMR experimental results.

  14. Decrimping: The first stage of collagen thermal denaturation unraveled by in situ second-harmonic-generation imaging

    Liao, Chien-Sheng; Zhuo, Zong-Yan; Yu, Jiun-Yann; Tzeng, Yu-Yi; Chu, Shi-Wei; Yu, Shih-Fan; Chao, Pen-Hsiu Grace


    With polarized and time-lapsed second-harmonic-generation (SHG) imaging, three distinct thermodynamic stages are revealed during heating of collagenous tissue. In the first "decrimping" stage, SHG intensity remains unchanged while the characteristic crimp pattern of collagen fiber disappears. The intactness of underlying fibrils is confirmed by unaffected second-order susceptibility, suggesting decrimping is related to the breakage of cross-linking between collagen fibrils. In the latter stages, significant SHG decrease is observed, providing quantification to collagen thermal denaturation. This study manifests the benefits of adopting SHG for understanding the thermal response of collagen, and will be useful toward better thermal therapy design.

  15. Combination of native and denaturing PAGE for the detection of protein binding regions in long fragments of genomic DNA

    Metsis Madis


    Full Text Available Abstract Background In a traditional electrophoresis mobility shift assay (EMSA a 32P-labeled double-stranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE in nondenaturing conditions. An extension of this method uses the large population of fragments derived from long genomic regions (approximately 600 kb for the identification of fragments containing protein binding regions. With this method, genomic DNA is fragmented by restriction enzymes, fragments are amplified by PCR, radiolabeled, incubated with nuclear proteins and the resulting DNA-protein complexes are separated by two-dimensional PAGE. Shifted DNA fragments containing protein binding sites are identified by using additional procedures, i. e. gel elution, PCR amplification, cloning and sequencing. Although the method allows simultaneous analysis of a large population of fragments, it is relatively laborious and can be used to detect only high affinity protein binding sites. Here we propose an alternative and straightforward strategy which is based on a combination of native and denaturing PAGE. This strategy allows the identification of DNA fragments containing low as well as high affinity protein binding regions, derived from genomic DNA ( Results We have combined an EMSA-based selection step with subsequent denaturing PAGE for the localization of protein binding regions in long (up to10 kb fragments of genomic DNA. Our strategy consists of the following steps: digestion of genomic DNA with a 4-cutter restriction enzyme (AluI, BsuRI, TruI, etc, separation of low and high molecular weight fractions of resultant DNA fragments, 32P-labeling with Klenow polymerase, traditional EMSA, gel elution and identification of the shifted bands (or smear by denaturing PAGE. The identification of DNA fragments containing protein binding sites is carried out by running the gel-eluted fragments alongside

  16. Determination of the folding of proteins as a function of denaturants, osmolytes or ligands using circular dichroism

    Greenfield, Norma J.


    Circular dichroism (CD) is an excellent tool for examining the interactions and stability of proteins. This protocol covers methods to obtain and analyze circular dichroism spectra to measure changes in the folding of proteins as a function of denaturants, osmolytes or ligands. Applications include determination of the free energy of folding of a protein, the effects of mutations on protein stability and the estimation of binding constants for the interactions of proteins with other proteins, DNA or ligands, such as substrates or inhibitors. The experiments take 2-5 h. PMID:17406529

  17. Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance

    Vogtt K.


    Full Text Available COSY proton nuclear magnetic resonance was used to measure the exchange rates of amide protons of hen egg white lysozyme (HEWL in the pressure-assisted cold-denatured state and in the heat-denatured state. After dissolving lysozyme in deuterium oxide buffer, labile protons exchange for deuterons in such a way that exposed protons are substituted rapidly, whereas "protected" protons within structured parts of the protein are substituted slowly. The exchange rates k obs were determined for HEWL under heat treatment (80ºC and under high pressure conditions at low temperature (3.75 kbar, -13ºC. Moreover, the influence of co-solvents (sorbitol, urea on the exchange rate was examined under pressure-assisted cold denaturation conditions, and the corresponding protection factors, P, were determined. The exchange kinetics upon heat treatment was found to be a two-step process with initial slow exchange followed by a fast one, showing residual protection in the slow-exchange state and P-factors in the random-coil-like range for the final temperature-denatured state. Addition of sorbitol (500 mM led to an increase of P-factors for the pressure-assisted cold denatured state, but not for the heat-denatured state. The presence of 2 M urea resulted in a drastic decrease of the P-factors of the pressure-assisted cold denatured state. For both types of co-solvents, the effect they exert appears to be cooperative, i.e., no particular regions within the protein can be identified with significantly diverse changes of P-factors.

  18. Sorbitol counteracts temperature- and chemical-induced denaturation of a recombinant α-amylase from alkaliphilic Bacillus sp. TS-23.

    Chi, Meng-Chun; Wu, Tai-Jung; Chen, Hsing-Ling; Lo, Huei-Fen; Lin, Long-Liu


    Enzymes are highly complex systems with a substantial degree of structural variability in their folded state. In the presence of cosolvents, fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways; alternatively, certain conformations can be stabilized or destabilized. To understand the contribution of osmolytes to the stabilization of structural changes and enzymatic activity of a truncated Bacillus sp. TS-23 α-amylase (BACΔNC), we monitored amylolytic activity, circular dichroism, and fluorescence as a function of osmolytes. In the presence of trimethylamine N-oxide (TMAO) and sorbitol, BACΔNC activity was retained significantly at elevated temperatures. As compared to the control, the secondary structures of this enzyme were essentially conserved upon the addition of these two kinds of osmolytes. Fluorescence results revealed that the temperature-induced conformational change of BACΔNC was prevented by TMAO and sorbitol. However, glycerol did not provide profound protection against thermal denaturation of the enzyme. Sorbitol was further found to counteract guanidine hydrochloride- and SDS-induced denaturation of BACΔNC. Thus, some well-known naturally occurring osmolytes make a dominant contribution to the stabilization of BACΔNC.

  19. Counteraction of Trehalose on N, N-Dimethylformamide-Induced Candida rugosa Lipase Denaturation: Spectroscopic Insight and Molecular Dynamic Simulation.

    Xin Yang

    Full Text Available Candida rugosa lipase (CRL has been widely used as a biocatalyst for non-aqueous synthesis in biotechnological applications, which, however, often suffers significant loss of activity in organic solvent. Experimental results show that trehalose could actively counteract the organic-solvent-induced protein denaturation, while the molecular mechanisms still don't unclear. Herein, CRL was used as a model enzyme to explore the effects of trehalose on the retention of enzymatic activity upon incubation in N,N-dimethylformamide (DMF. Results showed that both catalytic activity and conformation changes of CRL influenced by DMF solvent were inhibited by trehalose in a dose-dependent fashion. The simulations further indicated that the CRL protein unfolded in binary DMF solution, but retained the native state in the ternary DMF/trehalose system. Trehalose as the second osmolyte added into binary DMF solution decreased DMF-CRL hydrogen bonds efficiently, whereas increased the intermolecular hydrogen bondings between DMF and trehalose. Thus, the origin of its denaturing effects of DMF on protein is thought to be due to the preferential exclusion of trehalose as well as the intermolecular hydrogen bondings between trehalose and DMF. These findings suggest that trehalose protect the CRL protein from DMF-induced unfolding via both indirect and direct interactions.

  20. Partially unfolded species populated during equilibrium denaturation of the beta-sheet protein Y74W apo-pseudoazurin.

    Jones, S; Reader, J S; Healy, M; Capaldi, A P; Ashcroft, A E; Kalverda, A P; Smith, D A; Radford, S E


    Apo-pseudoazurin is a single domain cupredoxin. We have engineered a mutant in which a unique tryptophan replaces the tyrosine residue found in the tyrosine corner of this Greek key protein, a region that has been proposed to have an important role in folding. Equilibrium denaturation of Y74W apo-pseudoazurin demonstrated multistate unfolding in urea (pH 7.0, 0.5 M Na(2)SO(4) at 15 degrees C), in which one or more partially folded species are populated in 4. 3 M urea. Using a variety of biophysical techniques, we show that these species, on average, have lost a substantial portion of the native secondary structure, lack fixed tertiary packing involving tryptophan and tyrosine residues, are less compact than the native state as determined by fluorescence lifetimes and time-resolved anisotropy, but retain significant residual structure involving the trytophan residue. Peptides ranging in length from 11 to 30 residues encompassing this region, however, did not contain detectable nonrandom structure, suggesting that long-range interactions are important for stabilizing the equilibrium partially unfolded species in the intact protein. On the basis of these results, we suggest that the equilibrium denaturation of Y74W apo-pseudoazurin generates one or more partially unfolded species that are globally collapsed and retain elements of the native structure involving the newly introduced tryptophan residue. We speculate on the role of such intermediates in the generation of the complex Greek key fold.

  1. Purification of inclusion bodies using PEG precipitation under denaturing conditions to produce recombinant therapeutic proteins from Escherichia coli.

    Chen, Huanhuan; Li, Ninghuan; Xie, Yueqing; Jiang, Hua; Yang, Xiaoyi; Cagliero, Cedric; Shi, Siwei; Zhu, Chencen; Luo, Han; Chen, Junsheng; Zhang, Lei; Zhao, Menglin; Feng, Lei; Lu, Huili; Zhu, Jianwei


    It has been documented that the purification of inclusion bodies from Escherichia coli by size exclusion chromatography (SEC) may benefit subsequent refolding and recovery of recombinant proteins. However, loading volume and the high cost of the column limits its application in large-scale manufacturing of biopharmaceutical proteins. We report a novel process using polyethylene glycol (PEG) precipitation under denaturing conditions to replace SEC for rapid purification of inclusion bodies containing recombinant therapeutic proteins. Using recombinant human interleukin 15 (rhIL-15) as an example, inclusion bodies of rhIL-15 were solubilized in 7 M guanidine hydrochloride, and rhIL-15 was precipitated by the addition of PEG 6000. A final concentration of 5% (w/v) PEG 6000 was found to be optimal to precipitate target proteins and enhance recovery and purity. Compared to the previously reported S-200 size exclusion purification method, PEG precipitation was easier to scale up and achieved the same protein yields and quality of the product. PEG precipitation also reduced manufacturing time by about 50 and 95% of material costs. After refolding and further purification, the rhIL-15 product was highly pure and demonstrated a comparable bioactivity with a rhIL-15 reference standard. Our studies demonstrated that PEG precipitation of inclusion bodies under denaturing conditions holds significant potential as a manufacturing process for biopharmaceuticals from E. coli protein expression systems.

  2. Evidence of β-sheet structure induced kinetic stability of papain upon thermal and sodium dodecyl sulphate denaturation

    Rašković Brankica


    Full Text Available Papain is a protease that consists of α-helical and β-sheet domains which unfold almost independently. Both, papain considerable thermal stability and sodium dodecyl sulphate (SDS resistance have been shown. However, the ability of each domain to unfold upon thermal and SDS denaturation has never been studied. This work shows that fruit papain has slightly higher thermal inactivation resistance when it is compared to stem papain with rather high activation energy (Ea of 223 ± 16 kJmol-1 and Tm50 value of 79 ± 2 °C. SDS resistance of fruit papain was estimated by SDS-PAGE analysis and activity staining. It has been noted that, in the presence of SDS, unless heat energy was applied in order to unfold papain, the protein remained active. Furthermore, it has been proven via Fourier transform infrared spectroscopy (FT-IR that α-helical domain of fruit papain is more prone to unfolding at elevated temperatures and in the presence of SDS then β-sheet rich domain. Thermal denaturation of papain without detergent present led to accelerated formation of aggregation specific intermolecular β-sheets as compared to native protein. Presented results are both, of fundamental and application importance. [Projekat Ministarstva nauke Republike Srbije, br. 172049

  3. Yeast frataxin is stabilized by low salt concentrations: cold denaturation disentangles ionic strength effects from specific interactions.

    Sanfelice, Domenico; Puglisi, Rita; Martin, Stephen R; Di Bari, Lorenzo; Pastore, Annalisa; Temussi, Piero Andrea


    Frataxins are a family of metal binding proteins associated with the human Friedreich's ataxia disease. Here, we have addressed the effect of non-specifically binding salts on the stability of the yeast ortholog Yfh1. This protein is a sensitive model since its stability is strongly dependent on the environment, in particular on ionic strength. Yfh1 also offers the unique advantage that its cold denaturation can be observed above the freezing point of water, thus allowing the facile construction of the whole protein stability curve and hence the measurement of accurate thermodynamic parameters for unfolding. We systematically measured the effect of several cations and, as a control, of different anions. We show that, while strongly susceptible to ionic strength, as it would be in the cellular environment, Yfh1 stability is sensitive not only to divalent cations, which bind specifically, but also to monovalent cations. We pinpoint the structural bases of the stability and hypothesize that the destabilization induced by an unusual cluster of negatively charged residues favours the entrance of water molecules into the hydrophobic core, consistent with the generally accepted mechanism of cold denaturation.

  4. Using Denatured Egg White as a Macroscopic Model for Teaching Protein Structure and Introducing Protein Synthesis for High School Students

    Correia, Paulo R. M.; Torres, Bayardo B.


    The success of teaching molecular and atomic phenomena depends on the didactical strategy and the media selection adopted, in consideration of the level of abstraction of the subject to be taught and the students' capability to deal with abstract operations. Dale's cone of experience was employed to plan three 50-minute classes to discuss protein denaturation from a chemical point of view. Only low abstraction level activities were selected: (i) two demonstrations showing the denaturation of albumin by heating and by changing the solvent, (ii) the assembly of a macroscopic model representing the protein molecule, and (iii) a role-play for simulating glucagon synthesis. A student-centered approach and collaborative learning were used throughout the classes. The use of macroscopic models is a powerful didactical strategy to represent molecular and atomic events. They can convert microscopic entities into touchable objects, reducing the abstraction level required to discuss chemistry with high school students. Thus, interesting topics involving molecules and their behavior can take place efficiently when mediated by concrete experiences.

  5. Conformational distributions of denatured and unstructured proteins are similar to those of 20 Multiplication-Sign 20 blocked dipeptides

    Oh, Kwang-Im [Korea University, Department of Chemistry (Korea, Republic of); Jung, Young-Sang; Hwang, Geum-Sook, E-mail: [Korea Basic Science Institute (Korea, Republic of); Cho, Minhaeng, E-mail: [Korea University, Department of Chemistry (Korea, Republic of)


    Understanding intrinsic conformational preferences of amino-acids in unfolded proteins is important for elucidating the underlying principles of their stability and re-folding on biological timescales. Here, to investigate the neighbor interaction effects on the conformational propensities of amino-acids, we carried out {sup 1}H NMR experiments for a comprehensive set of blocked dipeptides and measured the scalar coupling constants between alpha protons and amide protons as well as their chemical shifts. Detailed inspection of these NMR properties shows that, irrespective of amino-acid side-chain properties, the distributions of the measured coupling constants and chemical shifts of the dipeptides are comparatively narrow, indicating small variances of their conformation distributions. They are further compared with those of blocked amino-acids (Ac-X-NHMe), oligopeptides (Ac-GGXGG-NH{sub 2}), and native (lysozyme), denatured (lysozyme and outer membrane protein X from Escherichia coli), unstructured (Domain 2 of the protein 5A of Hepatitis C virus), and intrinsically disordered (hNlg3cyt: intracellular domain of human NL3) proteins. These comparative investigations suggest that the conformational preferences and local solvation environments of the blocked dipeptides are quite similar to not only those of other short oligopeptides but also those of denatured and natively unfolded proteins.

  6. Spectroscopic characterization of the interaction of phenosafranin and safranin O with double stranded, heat denatured and single stranded calf thymus DNA.

    Saha, Ishita; Kumar, Gopinatha Suresh


    Interaction of phenosafranin and safranin O with double stranded, heat denatured and single stranded calf thymus DNA has been studied by fluorescence, absorbance and circular dichroic techniques. Binding to the double stranded and heat denatured DNA conformations induced strong quenching in the fluorescence spectra of both dyes. Linear Scatchard plots indicated the binding to be of one type and the affinity evaluated to be of the order of 10(5) M(-1) with double stranded and heat denatured DNAs. Fluorescence quenching was much weaker with the single stranded DNA and the binding affinity was one order lower. Ferrocyanide quenching studies revealed that the fluorescence emission of the dye molecules bound to the double stranded and heat denatured DNAs was quenched much less compared to that bound to the single stranded DNA. Further, there was significant emission polarization for the bound dyes and strong energy transfer from the DNA base pairs to the dye molecules indicating intercalative binding. Salt dependence of the binding phenomenon revealed that electrostatic forces have significant role in the binding process. The intercalation of these molecules to double stranded and heat denatured DNA and simple stacking to single strands was proved by these fluorescence techniques. Support to the fluorescence results have been derived from absorption and circular dichroic results. Phenosafranin was revealed to be a stronger binding species compared to safranin O.

  7. Testing the dependence of stabilizing effect of osmolytes on the fractional increase in the accessible surface area on thermal and chemical denaturations of proteins.

    Rahman, Safikur; Ali, Syed Ausaf; Islam, Asimul; Hassan, Md Imtaiyaz; Ahmad, Faizan


    Here we have generated two different denatured states using heat- and guanidinium chloride (GdmCl)-induced denaturations of three disulfide bond free proteins (barstar, cytochrome-c and myoglobin). We have observed that these two denatured states of barstar and myoglobin are structurally and energetically different, for, heat-induced denatured state contains many un-melted residual structure that has a significant amount of secondary and tertiary interactions. We show that structural properties of the denatured state determine the magnitude of the protein stabilization in terms of Gibbs free energy change (ΔGD°) induced by an osmolyte, i.e., the greater the exposed surface area, the greater is the stabilization. Furthermore, we predicted the m-values (ability of osmolyte to fold or unfold proteins) using Tanford's transfer-free energy model for the transfer of proteins to osmolyte solutions. We observed that, for each protein, m-value is comparable with our experimental data in cases of TMAO (trimethylamine-N-oxide) and sarcosine. However, a significant discrepancy between predicted and experimental m-values were observed in the case of glycine-betaine.

  8. Diagnostic work-up of pulmonary nodules. Management of pulmonary nodules detected with low-dose CT screening; Abklaerung von Lungenrundherden. Management durch Frueherkennungsuntersuchungen detektierter pulmonaler Rundherde

    Wormanns, D. [Evangelische Lungenklinik Berlin, Berlin (Germany)


    Pulmonary nodules are the most frequent pathological finding in low-dose computed tomography (CT) scanning for early detection of lung cancer. Early stages of lung cancer are often manifested as pulmonary nodules; however, the very commonly occurring small nodules are predominantly benign. These benign nodules are responsible for the high percentage of false positive test results in screening studies. Appropriate diagnostic algorithms are necessary to reduce false positive screening results and to improve the specificity of lung cancer screening. Such algorithms are based on some of the basic principles comprehensively described in this article. Firstly, the diameter of nodules allows a differentiation between large (>8 mm) probably malignant and small (<8 mm) probably benign nodules. Secondly, some morphological features of pulmonary nodules in CT can prove their benign nature. Thirdly, growth of small nodules is the best non-invasive predictor of malignancy and is utilized as a trigger for further diagnostic work-up. Non-invasive testing using positron emission tomography (PET) and contrast enhancement as well as invasive diagnostic tests (e.g. various procedures for cytological and histological diagnostics) are briefly described in this article. Different nodule morphology using CT (e.g. solid and semisolid nodules) is associated with different biological behavior and different algorithms for follow-up are required. Currently, no obligatory algorithm is available in German-speaking countries for the management of pulmonary nodules, which reflects the current state of knowledge. The main features of some international and American recommendations are briefly presented in this article from which conclusions for the daily clinical use are derived. (orig.) [German] Lungenrundherde sind die haeufigsten pathologischen Befunde bei Untersuchungen mit der Niedrigdosis-CT zur Lungenkrebsfrueherkennung. Fruehstadien des Lungenkarzinoms manifestieren sich meist als Rundherd

  9. Extension work-up for non-small-cell lung cancer; Thorax - cancer broncho-pulmonaire. Bilan d'extension des tumeurs malignes primitives broncho-pulmonaires non a petites cellules

    Ait-Ameur, A.; El Fikri, A.; Teriitehau, C.; Minvielle, F.; Le Bivic, T.; Jeanbourquin, D. [Hopital d' Instruction des Armees Percy, Service d' Imagerie, 92 - Clamart (France)


    Surgery is the only potentially effective treatment of the non-small-cell lung neoplasm. Patient selection for surgery requires a rigorous protocol. In order to optimize the therapeutic strategy, the extension work-up must be designed to identify correctly locoregional spread and any contraindications as well as metastasis with assessment of mediastino-pulmonary lymph nodes. Radiology, particularly computed tomography, is highly contributive. Plain chest X-ray, ultrasound, magnetic resonance imaging (MRI) and possibly positron omission tomography (PET) may also be required. (authors)

  10. Small heat shock protein Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin.

    Pivovarova, Anastasia V; Chebotareva, Natalia A; Chernik, Ivan S; Gusev, Nikolai B; Levitsky, Dmitrii I


    Previously, we have shown that the small heat shock protein with apparent molecular mass 27 kDa (Hsp27) does not affect the thermal unfolding of F-actin, but effectively prevents aggregation of thermally denatured F-actin [Pivovarova AV, Mikhailova VV, Chernik IS, Chebotareva NA, Levitsky DI & Gusev NB (2005) Biochem Biophys Res Commun331, 1548-1553], and supposed that Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin. In the present work, we applied dynamic light scattering, analytical ultracentrifugation and size exclusion chromatography to examine the properties of complexes formed by denatured actin with a recombinant human Hsp27 mutant (Hsp27-3D) mimicking the naturally occurring phosphorylation of this protein at Ser15, Ser78, and Ser82. Our results show that formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. All the methods show that the size of actin-Hsp27-3D complexes decreases with increasing Hsp27-3D concentration in the incubation mixture and that saturation occurs at approximately equimolar concentrations of Hsp27-3D and actin. Under these conditions, the complexes exhibit a hydrodynamic radius of approximately 16 nm, a sedimentation coefficient of 17-20 S, and a molecular mass of about 2 MDa. It is supposed that Hsp27-3D binds to denatured actin monomers or short oligomers dissociated from actin filaments upon heating and protects them from aggregation by forming relatively small and highly soluble complexes. This mechanism might explain how small heat shock proteins prevent aggregation of denatured actin and by this means protect the cytoskeleton and the whole cell from damage caused by accumulation of large insoluble aggregates under heat shock conditions.

  11. Unassisted refolding of urea-denatured arginine kinase from shrimp Feneropenaeus chinensis: evidence for two equilibrium intermediates in the refolding pathway.

    Pan, Ji-Cheng; Yu, Zhenhang; Su, Xiao-Yang; Sun, Ye-Qing; Rao, Xue-Ming; Zhou, Hai-Meng


    The refolding process and the equilibrium intermediates of urea-denatured arginine kinase (AK) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) intrinsic fluorescence, far-UV circular dichroism (CD), size-exclusion chromatography (SEC), and enzymatic activity. In dilute denaturant, two equilibrium refolding intermediates (I and N') were discovered, and a refolding scheme of urea-denatured AK was proposed. During the refolding of urea-denatured AK, the fluorescence intensity increased remarkably, accompanied by a significant blue shift of the emission maximum and a pronounced increase in molar ellipticity of CD at 222 nm. The first folding intermediate (I) was inactive in urea solution ranging between 2.4 and 3.0 M. The second (N') existed between a 0.4- and 0.8-M urea solution, with slightly increased activity. Neither the blue shift emission maximum nor the molar ellipticity of CD at 222 nm showed significant changes in these two regions. The two intermediates were characterized by monitoring the ANS binding ability in various residual urea solutions, and two peaks of the emission intensity were observed in urea solutions of 0.6 and 2.8 M, respectively. The SEC results indicated that a distribution coefficient (K(D)) platform existed in urea solutions ranging between 2.4 and 3.0 M urea, suggesting that there was a similarly apparent protein profile and size in the urea solution region. The refolding kinetics showed that the urea-denatured AK was in two-phase refolding. Proline isomerization occurred in the unfolding process of AK, which blocked the slow phase of refolding. These results suggested that the refolding process of urea-denatured AK contained at the least two equilibrium refolding intermediates.

  12. Mapping between the order of thermal denaturation and the shape of the critical line of mechanical unzipping in 1-dimensional DNA models

    Buyukdagli, Sahin; 10.1016/j.cplett.2009.11.061


    In this Letter, we investigate the link between thermal denaturation and mechanical unzipping for two models of DNA, namely the Dauxois-Peyrard-Bishop model and a variant thereof we proposed recently. We show that the critical line that separates zipped from unzipped DNA sequences in mechanical unzipping experiments is a power-law in the temperature-force plane. We also prove that for the investigated models the corresponding critical exponent is proportional to the critical exponent alpha, which characterizes the behaviour of the specific heat in the neighbourhood of the critical temperature for thermal denaturation.

  13. The guanidine hydrochloride-induced denaturation of CP43 and CP47 studied by terahertz time-domain spectroscopy


    Terahertz time-domain spectroscopy (THz-TDS) is a new technique in studying the conformational state of a molecule in recent years. In this work, we reported the first use of THz-TDS to examine the dena- turation of two photosynthesis membrane proteins: CP43 and CP47. THz-TDS was proven to be useful in discriminating the different conformational states of given proteins with similar structure and in monitoring the denaturation process of proteins. Upon treatment with guanidine hydrochloride (GuHCl), a 1.8 THz peak appeared for CP47 and free chlorophyll a (Chl a). This peak was deemed to originate from the interaction between Chl a and GuHCl molecules. The Chl a molecules in CP47 interacted with GuHCl more easily than those in CP43.

  14. Comparison of Different Denaturing Gradient Gel Electrophoresis Primer Sets for the Study of Marine Bacterioplankton Communities▿ †

    Sánchez, Olga; Gasol, Josep M.; Massana, Ramon; Mas, Jordi; Pedrós-Alió, Carlos


    An annual seasonal cycle of composition of a bacterioplankton community in an oligotrophic coastal system was studied by denaturing gradient gel electrophoresis (DGGE) using five different primer sets. Analysis of DGGE fingerprints showed that primer set 357fGC-907rM grouped samples according to seasons. Additionally, we used the set of 16S rRNA genes archived in the RDPII database to check the percentage of perfect matches of each primer for the most abundant bacterial groups inhabiting coastal plankton communities. Overall, primer set 357fGC-907rM was the most suitable for the routine use of PCR-DGGE analyses in this environment. PMID:17660308

  15. Analysis of microsatellite instability in stool DNA of patients with colorectal cancer using denaturing high performance liquid chromatography

    Seok-Byung Lim; Yong Shin; Sang-Geun Jang; Jae-Hyun Park; Jae-Gahb Park; Seung-Yong Jeong; Il-Jin Kim; Dae Yong Kim; Kyung Hae Jung; Hee Jin Chang; Hyo Seong Choi; Dae Kyung Sohn; Hio Chung Kang


    AIM: To evaluate the usefulness of denaturing high performance liquid chromatography (DHPLC) for analyzing microsatellite instability (MSI) status in stool DNA of patients with colorectal cancer.METHODS: A total of 80 cancer tissues from patients with primary sporadic colorectal tumor (proximal cancer:27, distal cancer: 53) and matched stool (which were employed for comparison with the tissues) were analyzed for MSI status in BAT 26. DNA samples extracted from stool were evaluated by nested polymerase chain reaction (PCR) and DHPLC for MSI analysis.RESULTS: Six cases (7.5%) of MSI were identified in BAT 26 from 80 cancer tissues. All the stool DNA samples from patients whose cancer tissue showed MSI also displayed MSI in BAT 26.CONCLUSION: As MSI is one of the established fecal DNA markers to screen colorectal cancer, we propose to use DHPLC for the MSI analysis in fecal DNA.

  16. Rapid Mutation Scanning of Genes Associated with Familial Cancer Syndromes Using Denaturing High-Performance Liquid Chromatography

    Deborah J. Marsh


    Full Text Available Germline mutations in tumor suppressor genes, or less frequently oncogenes, have been identified in up to 19 familial cancer syndromes including Li-Fraumeni syndrome, familial paraganglioma, familial adenomatous polyposis coli and breast and ovarian cancers. Multiple genes have been associated with some syndromes as approximately 26 genes have been linked to the development of these familial cancers. With this increased knowledge of the molecular determinants of familial cancer comes an equal expectation for efficient genetic screening programs. We have trialled denaturing highperformance liquid chromatography (dHPLC as a tool for rapid germline mutation scanning of genes implicated in three familial cancer syndromes - Cowden syndrome (PTEN mutation, multiple endocrine neoplasia type 2 (RET mutation and von Hippel-Lindau disease (VHL mutation. Thirty-two mutations, including 21 in PTEN, 9 in RET plus a polymorphism, and 2 in VHL, were analyzed using the WAVE DNA fragment analysis system with 100% detection efficiency. In the case of the tumor suppressor gene PTEN, mutations were scattered along most of the gene. However, mutations in the RET proto-oncogene associated with multiple endocrine neoplasia type 2 were limited to specific clusters or “hot spots”. The use of GC-clamped primers to scan for mutations scattered along PTEN exons was shown to greatly enhance the sensitivity of detection of mutant hetero- and homoduplex peaks at a single denaturation temperature compared to fragments generated using non-GC-clamped primers. Thus, when scanning tumor suppressor genes for germline mutation using dHPLC, the incorporation of appropriate GCclamped primers will likely increase the efficiency of mutation detection.

  17. Utilization of denaturing gradient gel electrophoresis for diagnosis of {beta}-thalassemia and ascertainment of new mutations

    Ngo, K.Y.; Liu, D.; Lee, J. [Univ. of California, San Diego (United States)] [and others


    During the past two years we have tested 2,300 Southeast Asians for alpha- and beta-thaleassemia mutations. We found the incidence of hemoglobin E ({beta}{sup 26}) to be 47% among Laotians and 38% among Cambodians. The incidence of beta thalassemia trait is 9% for Laotians and 6% for Cambodians. Thus, the risk for hemoglobin E/{beta}{sup 26} thalassemia, a transfusion-dependent disorder, is increased in these two population groups. Denaturing gradient gel electrophoresis (DGGE) has proven to be useful in testing for beta-thalassemia carriers and identifying new mutations in the beta globin gene. DNA was extracted from venous blood obtained from patients with elevated Hgb A2 (>4%). Five DNA fragments, encompassing the beta globin gene cluster, were amplified by PCR and analyzed, along with known beta gene mutations as controls, by DGGE using different denaturing gradient concentrations. Different mutations at the same nucleotide position can be distinguished by migration pattern on the DGGE (e.g., in IVS-I-1, G{r_arrow}A and T). Compound heterozygotes for {beta}-thalassemia can be detected on the same gel (e.g., HbE/mutation codon 17). New mutations are identified by their migration pattern compared with controls and determined by subsequent sequencing. We have identified three new mutations: codon 82 CAA{r_arrow}AAA in one Cambodian patient; IVS-II-667, T{r_arrow}C and IVS-II-672, A{r_arrow}C in two Laotian patients. When the parent`s genotypes are known, prenatal diagnosis can be obtained within 24 hours. Thus, PCR/DGGE combination is a rapid and reliable diagnostic approach to clinically significant {beta}-thalassemia. The most important steps are carefully designed primers and predetermined gradient concentrations for DGGE.

  18. p53 Amino-terminus region (1-125 stabilizes and restores heat denatured p53 wild phenotype.

    Anuj Kumar Sharma

    Full Text Available BACKGROUND: The intrinsically disordered N-ter domain (NTD of p53 encompasses approximately hundred amino acids that contain a transactivation domain (1-73 and a proline-rich domain (64-92 and is responsible for transactivation function and apoptosis. It also possesses an auto-inhibitory function as its removal results in remarkable reduction in dissociation of p53 from DNA. PRINCIPAL FINDINGS/METHODOLOGY: In this report, we have discovered that p53-NTD spanning amino acid residues 1-125 (NTD125 interacted with WT p53 and stabilized its wild type conformation under physiological and elevated temperatures, both in vitro and in cellular systems. NTD125 prevented irreversible thermal aggregation of heat denatured p53, enhanced p21-5'-DBS binding and further restored DBS binding activity of heat-denatured p53, in vitro, in a dose-dependent manner. In vivo ELISA and immunoprecipitation analysis of NTD125-transfected cells revealed that NTD125 shifted equilibrium from p53 mutant to wild type under heat stress conditions. Further, NTD125 initiated nuclear translocation of cytoplasmic p53 in transcriptionally active state in order to activate p53 downstream genes such as p21, Bax, PUMA, Noxa and SUMO. CONCLUSION/SIGNIFICANCE: Here, we showed that a novel chaperone-like activity resides in p53-N-ter region. This study might have significance in understanding the role of p53-NTD in p53 stabilization, conformational activation and apoptosis under heat-stress conditions.

  19. pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1.

    Pace, C N; Laurents, D V; Thomson, J A


    To investigate the pH dependence of the conformational stability of ribonucleases A and T1, urea and guanidine hydrochloride denaturation curves have been determined over the pH range 2-10. The maximum conformational stability of both proteins is about 9 kcal/mol and occurs near pH 4.5 for ribonuclease T1 and between pH 7 and 9 for ribonuclease A. The pH dependence suggests that electrostatic interactions among the charged groups make a relatively small contribution to the conformational stability of these proteins. The dependence of delta G on urea concentration increases from about 1200 cal mol-1 M-1 at high pH to about 2400 cal mol-1 M-1 at low pH for ribonuclease A. This suggests that the unfolded conformations of RNase A become more accessible to urea as the net charge on the molecule increases. For RNase T1, the dependence of delta G on urea concentration is minimal near pH 6 and increases at both higher and lower pH. An analysis of information of this type for several proteins in terms of a model developed by Tanford [Tanford, C. (1964) J. Am. Chem. Soc. 86, 2050-2059] suggests that the unfolded states of proteins in urea and GdnHCl solutions may differ significantly in the extent of their interaction with denaturants. Thus, the conformations assumed by unfolded proteins may depend to at least some extent on the amino acid sequence of the protein.

  20. The effects of urea and n-propanol on collagen denaturation: using DSC, circular dicroism and viscosity

    Usha, R.; Ramasami, T


    The effect of urea and n-propanol on circular dichroism (CD) and viscosity of purified type1 collagen solution at various temperatures and differential scanning calorimetry (DSC) of rat-tail tendon (RTT) collagen fibre have been studied. CD reveals a spectrum with a positive peak at around 220 nm and a negative peak at 200 nm characteristics of collagen triple helix. The molar ellipticity decreases as the concentration of urea increases up to particular concentration (collagen solution treated with 265 {mu}M of urea) and after that it increases (collagen solution treated with 500 {mu}M of urea). There is a linear decrease in molar ellipticity as the concentration of n-propanol increases. Denaturation temperature of urea and n-propanol treated with purified collagen solution has been studied using viscosity method. Additives such as urea and n-propanol decrease the thermal stability of collagen triple helix in solution and in RTT collagen fibre. Thermal helix to coil transition of urea and n-propanol treated collagen depends on the degree of hydration and the concentration of these additives. Thermodynamic parameters such as the peak temperature, enthalpy of activation, and energy of activation for collagen-gelatin transition for native, urea and n-propanol treated RTT collagen fibre has been calculated using DSC. The change in the thermodynamic parameters has been observed for native, urea and n-propanol treated RTT collagen fibres. The experimental results show that the change in the water structure, dehydration and desolvation induced by different additives such as urea and n-propanol on RTT may vary with the type of denaturation.

  1. The recognition of adsorbed and denatured proteins of different topographies by β2 integrins and effects on leukocyte adhesion and activation

    Brevig, T.; Holst, B.; Ademovic, Z.


    Leukocyte beta(2) integrins Mac-1 and p150,95 are promiscuous cell-surface receptors that recognise and mediate cell adhesion to a variety of adsorbed and denatured proteins. We used albumin as a model protein to study whether leukocyte adhesion and activation depended on the nm-scale topography ...

  2. Electronmicroscopical evaluation of short-term nerve regeneration through a thin-walled biodegradable poly(DLLA-epsilon-CL) nerve guide filled with modified denatured muscle tissue

    Meek, MF; Robinson, PH; Stokroos, [No Value; Blaauw, EH; Kors, G; den Dunnen, WFA


    The aim of this study was to evaluate short-term peripheral nerve regeneration across a 15-mm gap in the sciatic nerve of the rat, using a thin-walled biodegradable poly(DL-lactide-epsilon -caprolactone) nerve guide filled with modified denatured muscle tissue (MDMT). The evaluation was performed us

  3. Comparison of Fecal Methanogenic Archaeal Community Between Erhualian and Landrace Pigs Using Denaturing Gradient Gel Electrophoresis and Real-Time PCR Analysis

    Su, Y.; Smidt, H.; Zhu, W.Y.


    Erhualian and Landrace breeds are typical genetically obese and lean pigs, respectively. To compare the fecal methanogenic Archaeal community between these two pig breeds, fecal samples from different growth phase pigs were collected and used for PCR-denaturing gradient gel electrophoresis (DGGE) wi

  4. Evaluation of functional nerve recovery after reconstruction with a poly (DL-lactide-epsilon-caprolactone) nerve guide, filled with modified denatured muscle tissue

    Meek, MF; Den Dunnen, WFA; Schakenraad, JM; Robinson, PH


    The aim of this study was to compare the speed of functional nerve recovery after reconstruction with a biodegradable p(DLLA-epsilon -CL) nerve guide, as filled with either modified denatured muscle tissue (MDMT) or phosphate-buffered saline (PBS). To evaluate both motor and sensory nerve recovery,

  5. Characterization of the native and denatured herceptin by enzyme linked immunosorbent assay and quartz crystal microbalance using a high-affinity single chain fragment variable recombinant antibody.

    Shang, Yuqin; Mernaugh, Ray; Zeng, Xiangqun


    Herceptin/Trastuzumab is a humanized IgG1κ light chain antibody used to treat some forms of breast cancer. A phage-displayed recombinant antibody library was used to obtain a single chain fragment variable (scFv, designated 2B4) to a linear synthetic peptide representing Herceptin's heavy chain CDR3. Enzyme linked immunosorbent assays (ELISAs) and piezoimmunosensor/quartz crystal microbalance (QCM) assays were used to characterize 2B4-binding activity to both native and heat denatured Herceptin. The 2B4 scFv specifically bound to heat denatured Herceptin in a concentration dependent manner over a wide (35-220.5 nM) dynamic range. Herceptin denatures and forms significant amounts of aggregates when heated. UV-vis characterization confirms that Herceptin forms aggregates as the temperature used to heat Herceptin increases. QCM affinity assay shows that binding stoichiometry between 2B4 scFv and Herceptin follows a 1:2 relationship proving that 2B4 scFv binds strongly to the dimers of heat denatured Herceptin aggregates and exhibits an affinity constant of 7.17 × 10(13) M(-2). The 2B4-based QCM assay was more sensitive than the corresponding ELISA. Combining QCM with ELISA can be used to more fully characterize nonspecific binding events in assays. The potential theoretical and clinical implications of these results and the advantages of the use of QCM to characterize human therapeutic antibodies in samples are also discussed.

  6. Analysis of bacterial communities in soil by use of denaturing gradient gel electrophoresis and clone libraries, as influenced by different reverse primers

    Brons, Jolanda; van Elsas, J.D.


    To assess soil bacterial diversity, PCR systems consisting of several slightly different reverse primers together with forward primer F968-GC were used along with subsequent denaturing gradient gel electrophoresis (DGGE) or clone library analyses. In this study, a set of 13 previously used and novel

  7. Utility of birefringence changes due to collagen thermal denaturation rate process analysis: vessel wall temperature estimation for new short term heating balloon angioplasty

    Kaneko, Kenji; Shimazaki, Natsumi; Gotoh, Maya; Nakatani, Eriko; Arai, Tsunenori


    Our photo thermal reaction heating architecture balloon realizes less than 10 s short term heating that can soften vessel wall collagen without damaging surrounding tissue thermally. New thermal balloon angioplasty, photo-thermo dynamic balloon angioplasty (PTDBA) has experimentally shown sufficient opening with 2 atm low pressure dilation and prevention of chronic phase restenosis and acute phase thrombus in vivo. Even though PTDBA has high therapeutic potential, the most efficient heating condition is still under study, because relationship of treatment and thermal dose to vessel wall is not clarified yet. To study and set the most efficient heating condition, we have been working on establishment of temperature history estimation method from our previous experimental results. Heating target of PTDBA, collagen, thermally denatures following rate process. Denaturation is able to be quantified with measured collagen birefringence value. To express the denaturation with equation of rate process, the following ex vivo experiments were performed. Porcine extracted carotid artery was soaked in two different temperature saline baths to enforce constant temperature heating. Higher temperature bath was set to 40 to 80 degree Celsius and soaking duration was 5 to 40 s. Samples were observed by a polarizing microscope and a scanning electron microscope. The birefringence was measured by polarizing microscopic system using Brace-Koehler compensator 1/30 wavelength. The measured birefringence showed temperature dependency and quite fit with the rate process equation. We think vessel wall temperature is able to be estimated using the birefringence changes due to thermal denaturation.

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

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


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

  9. Electronmicroscopical evaluation of short-term nerve regeneration through a thin-walled biodegradable poly(DLLA-epsilon-CL) nerve guide filled with modified denatured muscle tissue

    Meek, MF; Robinson, PH; Stokroos, [No Value; Blaauw, EH; Kors, G; den Dunnen, WFA


    The aim of this study was to evaluate short-term peripheral nerve regeneration across a 15-mm gap in the sciatic nerve of the rat, using a thin-walled biodegradable poly(DL-lactide-epsilon -caprolactone) nerve guide filled with modified denatured muscle tissue (MDMT). The evaluation was performed us

  10. Detection of activity and mass spectrometric identification of mouse liver carboxylesterase and aldehyde dehydrogenase separated by non-denaturing two-dimensional electrophoresis after extraction with detergents.

    Shimazaki, Youji; Manabe, Takashi


    To examine the activities and identity of enzymes associated with organelles such as microsomes and mitochondria, proteins from mouse liver were extracted using the non-ionic detergents Nonidet P-40 (NP-40), polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene isooctylphenyl ester (Triton X), n-octyl beta-D-glucoside (octyl glycoside) or anionic detergent sodium dodecylsulfate (SDS) after the removal of cytosolic proteins. The proteins extracted by detergents were separated by non-denaturing two-dimensional electrophoresis (2-DE). The activities of esterase and aldehyde dehydrogenase were retained by non-denaturing 2-DE after treatment with each non-ionic detergent, but the activities were reduced or lost when the proteins were extracted with more than 0.5% SDS. For proteomic analysis of the organelle-associated proteins in mouse liver, proteins were separated by non-denaturing 2-DE and were identified using electrospray ionization tandem mass spectrometry (ESI-MS/MS) after the proteins were solubilized by octyl glycoside, NP-40 and 0.1% SDS. Several organelle-associated proteins such as carboxylesterase, aldehyde dehydrogenase, glucose regulated protein and HSP60 were identified. These results indicate that the activities and identity of detergent-soluble enzymes can be examined by this non-denaturing 2-DE and mass spectrometry.

  11. Role of XIST/miR-29a/LIN28A pathway in denatured dermis and human skin fibroblasts (HSFs) after thermal injury.

    Guo, Le; Huang, Xu; Liang, Pengfei; Zhang, Pihong; Zhang, Minghua; Ren, Licheng; Zeng, Jizhang; Cui, Xv; Huang, Xiaoyuan


    Denatured dermis is a part of the dermis in deep burn wound and has the ability to restore normal morphology and function. In our previous study, we revealed that miR-29a downregulation in denatured dermis may help burn wound healing in the later phase, and further enhance type I collagen synthesis. LIN28A, a highly-conserved RNA binding protein expressed during embryogenesis, plays roles in development, pluripotency, metabolism, as well as tissue repair in adults. In the present study, we investigated the functional roles of LIN28A in human skin fibroblasts (HSFs) and extracellular matrix (ECM), and the interaction between miR-29a and LIN28A. In recent years, long non-coding RNAs have been reported to play a key role in normal development and physiology, as well as in disease development. By using online tools, we screened out several candidate lncRNAs of miR-29a, among which XIST was inversely regulated by miR-29a. XIST, one of the first found cancer-associated lncRNAs, has been frequently reported to play major role in several biological processes. Further, we evaluated the roles and mechanism of XIST in HSF proliferation, migration, and ECM synthesis. Through regulation of miR-29a/LIN28A, XIST knockdown suppressed HSF proliferation, migration, and ECM synthesis. In denatured dermis tissues, XIST, and LIN28A expression was upregulated, miR-29a expression was downregulated. Taken together, promoting XIST expression in denatured dermis, thus to inhibit miR-29a and promote LIN28A expression, further promote HSF proliferation, migration, and ECM synthesis presents a promising strategy for denatured dermis repair. © 2017 Wiley Periodicals, Inc.

  12. Numerical study of DNA denaturation with self-avoidance: pseudo-critical temperatures and finite size behaviour

    Coluzzi, Barbara; Yeramian, Edouard


    We perform an extensive numerical study of the disordered Poland-Scheraga (PS) model for DNA denaturation in which self-avoidance is completely taken into account. To complement to our previous work, we focus here on the finite size scaling in terms of pseudo-critical temperatures. Notably, we find that the mean value and the fluctuations of the pseudo-T c scale with the same exponent, the correlation length exponent {ν\\text{r}} (for which we provide the refined evaluation {ν\\text{r}}=2.9+/- 0.4 ). This result (coherent with the typical picture that describes random ferromagnets when disorder is relevant) is at variance with the numerical results reported in the literature for the PS model with self-avoidance, leading to an alternative scenario with a pseudo-first-order transition. We moreover introduce a crossover chain length N *, which we evaluate, appropriate for characterizing the approach to the asymptotic regime in this model. Essentially, below N *, the behaviour of the model in our study could also agree with such an alternative scenario. Based on an approximate prediction of the dependence of N * on the parameters of the model, we show that following the choice of such parameters it would not be possible to reach the asymptotic regime in practice. In such a context it becomes then possible to reconcile the apparently contradictory numerical studies.

  13. Microbial Diversity during Fermentation of Sweet Paste, a Chinese Traditional Seasoning, Using PCR-Denaturing Gradient Gel Electrophoresis.

    Mao, Ping; Hu, Yuanliang; Liao, Tingting; Wang, Zhaoting; Zhao, Shumiao; Liang, Yunxiang; Hu, Yongmei


    The aim of this study was to elucidate the changes in the microbial community and biochemical properties of a traditional sweet paste during fermentation. PCR-denaturing gradient gel electrophoresis (DGGE) analysis showed that Aspergillus oryzae was the predominant species in the koji (the fungal mixture), and the majority of the fungi isolated belonged to two Zygosaccharomyces species in the mash. The bacterial DGGE profiles revealed the presence of Bacillus subtilis during fermentation, and Lactobacillus acidipiscis, Lactobacillus pubuzihii, Lactobacillus sp., Staphylococcus kloosi, and several uncultured bacteria were also detected in the mash after 14 days of main fermentation. Additionally, during main fermentation, amino-type nitrogen and total acid increased gradually to a maximum of 6.77 ± 0.25 g/kg and 19.10 ± 0.58 g/kg (30 days) respectively, and the concentration of reducing sugar increased to 337.41 ± 3.99 g/kg (7 days). The 180-day fermented sweet paste contained 261.46 ± 19.49 g/kg reducing sugar and its pH value remained at around 4.65. This study has used the PCR-DGGE technique to demonstrate the microbial community (including bacteria and fungi) in sweet paste and provides useful information (biochemical properties) about the assessment of the quality of sweet paste throughout fermentation.

  14. Stable and pH-sensitive protein nanogels made by self-assembly of heat denatured soy protein.

    Chen, Nannan; Lin, Lianzhu; Sun, Weizheng; Zhao, Mouming


    In this study, we examined the possibility of preparing stable soy protein nanogels by simply heating homogeneous soy protein dispersion. The protein nanogels formed were characterized by z-average hydrodynamic diameter, polydispersity index, turbidity, ζ-potential, morphology, and their stability to pH and ionic strength change. Soy protein dispersion (1% w/v) was homogeneous around pH 5.9 where it had the lowest polydispersity index (∼0.1). Stable and spherical nanogels were formed by heating soy protein dispersion at pH 5.9 under 95 °C. They sustained constantly low polydispersity index (∼0.1) in the investigated pH range of 6.06-7.0 and 2.6-3.0. The nanogels were pH-sensitive and would swell with pH change. They were stable at 0-200 mM NaCl concentration. Denaturation of soy glycinin was the prerequisite for the formation of stable nanogels. Soy protein nanogels had a core-shell structure with basic polypeptides and β subunits interacting together as the hydrophobic core; and acid polypeptides, α', and α subunits locating outside the core as hydrophilic shell. The inner structure of soy protein nanogels was mainly stabilized by disulfide bonds cross-linked network and hydrophobic interaction. Soy protein nanogels made in this study would be useful as functional ingredients in biotechnological, pharmaceutical, and food industries.

  15. Strong disorder renewal approach to DNA denaturation and wetting: typical and large deviation properties of the free energy

    Monthus, Cécile


    For the DNA denaturation transition in the presence of random contact energies, or equivalently the disordered wetting transition, we introduce a strong disorder renewal approach to construct the optimal contacts in each disordered sample of size L. The transition is found to be of infinite order, with a correlation length diverging with the essential singularity \\ln ξ (T)\\propto |T-{{T}\\text{c}}{{|}-1} . In the critical region, we analyze the statistics over samples of the free-energy density f L and of the contact density, which is the order parameter of the transition. At the critical point, both decay as a power-law of the length L but remain distributed, in agreement with the general phenomenon of lack of self-averaging at random critical points. We also obtain that for any real q  >  0, the moment \\overline{ZLq} of order q of the partition function at the critical point is dominated by some exponentially rare samples displaying a finite free-energy density, i.e. by the large deviation sector of the probability distribution of the free-energy density.

  16. Detection and differentiation of Entamoeba histolytica and Entamoeba dispar in clinical samples through PCR-denaturing gradient gel electrophoresis

    P. López-López

    Full Text Available Amebiasis is one of the twenty major causes of disease in Mexico; however, the diagnosis is difficult due to limitations of conventional microscopy-based techniques. In this study, we analyzed stool samples using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE to differentiate between Entamoeba histolytica (pathogenic and E. dispar (non-pathogenic. The target for the PCR amplification was a small region (228 bp of the adh112 gene selected to increase the sensitivity of the test. The study involved 62 stool samples that were collected from individuals with complaints of gastrointestinal discomfort. Of the 62 samples, 10 (16.1% were positive for E. histolytica while 52 (83.9% were negative. No sample was positive for E. dispar. These results were validated by nested PCR-RFLP (restriction fragment length polymorphism and suggest that PCR-DGGE is a promising tool to differentiate among Entamoeba infections, contributing to determine the specific treatment for patients infected with E. histolytica, and therefore, avoiding unnecessary treatment of patients infected with the non-pathogenic E. dispar.

  17. Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis.

    Nigg, Patricia E; Pavlovic, Jovan


    The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

  18. Analysis of microbial diversity on deli slicers using polymerase chain reaction and denaturing gradient gel electrophoresis technologies.

    Koo, O K; Mertz, A W; Akins, E L; Sirsat, S A; Neal, J A; Morawicki, R; Crandall, P G; Ricke, S C


    Cross-contamination of pathogenic and spoilage bacteria from food-contact surfaces to food products is a serious public health issue. Bacteria may survive and attach to food-contact surfaces by residual food components and/or background bacteria which may subsequently transfer to other food products. Deli slicers, generally used for slicing ready-to-eat products, can serve as potential sources for considerable bacterial transfer. The objective of this study was to assess the extent and distribution of microbial diversity of deli slicers by identification of pathogenic and background bacteria. Slicer-swab samples were collected from restaurants in Arkansas and Texas in the United States. Ten surface areas for each slicer were swabbed using sterile sponges. Denaturing gradient gel electrophoresis (DGGE) was applied to investigate the fingerprint of samples, and each band was further identified by sequence analysis. Pseudomonads were identified as the dominant bacteria followed by Enterobacteriaceae family, and lactic acid bacteria such as Lactococcus lactis and Streptococcus thermophilus were also found. Bacterial distribution was similar for all surface areas, while the blade guard exhibited the greatest diversity. This study provides a profile of the microbial ecology of slicers using DGGE to develop more specific sanitation practices and to reduce cross-contamination during slicing.

  19. Rapid genetic diagnosis and prenatal diagnosis of spinal muscular atrophy by denaturing high-performance liquid chromatography

    ZHU Hai-yan; WU Ling-qian; PAN Qian; TANG Bei-sha; LIANG De-sheng; LONG Zhi-gao; DAI He-ping; XIA Kun; XIA Jia-hui


    @@ Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder1 (1in 6000 to 10 000 births) caused by mutations in the SMN1 gene at 5q13. More than 90%-98% of SMA patients show homozygous deletion of SMN1,2which has proved to be useful in the diagnosis of SMA. But it is hampered because of the existence of a highly homologous gene, SMN2.3 Based on nucleotide mismatches between SMN1 and SMN2,the following two DNA tests are usually performed:single-strand conformational polymorphism (SSCP)3and polymerase chain reaction (PCR) followed by a restriction enzyme digestion.4,5 In this study we developed a new method for rapid genetic diagnosis of SMA by denaturing high-performance liquid chromatography (DHPLC), which is based on different retention of homoduplexes and heteroduplexes in detecting the homozygous deletion of SMN1. Both genetic and prenatal diagnoses were performed successfully for a SMA family by DHPLC, which was confirmed as a rapid and effective technique for detecting the deletion of SMN1.

  20. Monitoring the bacterial population dynamics in sourdough fermentation processes by using PCR-denaturing gradient gel electrophoresis.

    Meroth, Christiane B; Walter, Jens; Hertel, Christian; Brandt, Markus J; Hammes, Walter P


    Four sourdoughs (A to D) were produced under practical conditions by using a starter mixture of three commercially available sourdough starters and a baker's yeast constitutively containing various species of lactic acid bacteria (LAB). The sourdoughs were continuously propagated until the composition of the LAB flora remained stable. Two LAB-specific PCR-denaturing gradient gel electrophoresis (DGGE) systems were established and used to monitor the development of the microflora. Depending on the prevailing ecological conditions in the different sourdough fermentations, only a few Lactobacillus species were found to be competitive and became dominant. In sourdough A (traditional process with rye flour), Lactobacillus sanfranciscensis and a new species, L. mindensis, were detected. In rye flour sourdoughs B and C, which differed in the process temperature, exclusively L. crispatus and L. pontis became the predominant species in sourdough B and L. crispatus, L. panis, and L. frumenti became the predominant species in sourdough C. On the other hand, in sourdough D (corresponding to sourdough C but produced with rye bran), L. johnsonii and L. reuteri were found. The results of PCR-DGGE were consistent with those obtained by culturing, except for sourdough B, in which L. fermentum was also detected. Isolates of the species L. sanfranciscensis and L. fermentum were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively.

  1. Detection and differentiation of Entamoeba histolytica and Entamoeba dispar in clinical samples through PCR-denaturing gradient gel electrophoresis.

    López-López, P; Martínez-López, M C; Boldo-León, X M; Hernández-Díaz, Y; González-Castro, T B; Tovilla-Zárate, C A; Luna-Arias, J P


    Amebiasis is one of the twenty major causes of disease in Mexico; however, the diagnosis is difficult due to limitations of conventional microscopy-based techniques. In this study, we analyzed stool samples using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to differentiate between Entamoeba histolytica (pathogenic) and E. dispar (non-pathogenic). The target for the PCR amplification was a small region (228 bp) of the adh112 gene selected to increase the sensitivity of the test. The study involved 62 stool samples that were collected from individuals with complaints of gastrointestinal discomfort. Of the 62 samples, 10 (16.1%) were positive for E. histolytica while 52 (83.9%) were negative. No sample was positive for E. dispar. These results were validated by nested PCR-RFLP (restriction fragment length polymorphism) and suggest that PCR-DGGE is a promising tool to differentiate among Entamoeba infections, contributing to determine the specific treatment for patients infected with E. histolytica, and therefore, avoiding unnecessary treatment of patients infected with the non-pathogenic E. dispar.

  2. Design of an automated multicapillary instrument with fraction collection for DNA mutation discovery by constant denaturant capillary electrophoresis (CDCE).

    Li, Qingbo; Deka, Chiranjit; Glassner, Brian J; Arnold, Kevin; Li-Sucholeiki, Xiao-Cheng; Tomita-Mitchell, Aoy; Thilly, William G; Karger, Barry L


    A fundamental goal ingenomics is the discovery of genetic variation that contributes to disease states or to differential drug responses. Single nucleotide polymorphism (SNP) detection has been the focus of much attention in the study of genetic variation over the last decade. These SNPs typically occur at a frequency greater than 1% in the human genome. Recently, low-frequency alleles are also being increasingly recognized as critical to obtain an improved understanding of the correlation between genetic variation and disease. Although many methods have been reported for the discovery and scoringof SNPs, sensitive, automated, and cost-effective methods and platforms for the discovery of low-frequency alleles are not yet readily available. We describe here an automated multicapillary instrument for high-throughput detection of low-frequency alleles from pooled samples using constant denaturant capillary electrophoresis. The instrument features high optical sensitivity (1 x 10(-12) M fluorescein detection limit), precise and stable temperature control (+/- 0.01degrees C), and automation for sample delivery, injection, matrix replacement, and fraction collection. The capillary array is divided into six groups of four capillaries, each of which can be independently set at any temperature ranging from room temperature to 90 degrees C. The key performance characteristics of the instrument are reported.

  3. Robust Denaturation of Villin Headpiece by MoS2 Nanosheet: Potential Molecular Origin of the Nanotoxicity.

    Gu, Zonglin; Yang, Zaixing; Kang, Seung-Gu; Yang, Jerry R; Luo, Judong; Zhou, Ruhong


    MoS2 nanosheet, a new two-dimensional transition metal dichalcogenides nanomaterial, has attracted significant attentions lately due to many potential promising biomedical applications. Meanwhile, there is also a growing concern on its biocompatibility, with little known on its interactions with various biomolecules such as proteins. In this study, we use all-atom molecular dynamics simulations to investigate the interaction of a MoS2 nanosheet with Villin Headpiece (HP35), a model protein widely used in protein folding studies. We find that MoS2 exhibits robust denaturing capability to HP35, with its secondary structures severely destroyed within hundreds of nanosecond simulations. Both aromatic and basic residues are critical for the protein anchoring onto MoS2 surface, which then triggers the successive protein unfolding process. The main driving force behind the adsorption process is the dispersion interaction between protein and MoS2 monolayer. Moreover, water molecules at the interface between some key hydrophobic residues (e.g. Trp-64) and MoS2 surface also help to accelerate the process driven by nanoscale drying, which provides a strong hydrophobic force. These findings might have shed new light on the potential nanotoxicity of MoS2 to proteins with atomic details, which should be helpful in guiding future biomedical applications of MoS2 with its nanotoxicity mitigated.

  4. A simple two-step, 'hit and fix' method to generate subtle mutations in BACs using short denatured PCR fragments.

    Yang, Yongping; Sharan, Shyam K


    The bacteriophage lambda recombination system has proven to be a valuable tool for engineering bacterial artificial chromosomes (BAC). Due to its high efficiency, subtle alterations in the BACs can be generated using oligonucleotides as targeting vectors. Since no selection marker is used, recombinant clones are identified utilizing a selective PCR screening method. However, occasionally the selective PCR screening is not feasible. We describe here a two-step 'hit and fix' method that can be reliably used for generating any subtle alteration in BACs using short denatured PCR fragments as targeting vectors. In the first step of this method, 6-20 nucleotides are changed around the base where the mutation has to be generated. In the second step, these altered nucleotides are reverted to the original sequence and simultaneously a subtle alteration is introduced. Since in each step several nucleotides are changed, PCR primers specific for such alterations can be designed. This two-step method provides a simple and efficient tool for generating subtle alterations in BACs that can be very valuable for functional analysis of genes.

  5. Cowpea Vicilins: Fractionation of Urea Denatured Sub-Units and Effects on Callosobruchus maculatus F. (Coleoptera: Bruchidae Development

    Antônio Chagas Mota


    Full Text Available Vicilins (7S storage globulins isolated from cowpea (Vigna unguiculata L. seeds which were susceptible (S and resistant (R to the cowpea weevil (Callosobruchus maculatus F., Coleoptera: Bruchidae were denatured by urea and fractionated by ion-exchange chromatography. Isolated fractions were incorporated in artificial seeds for assessment of their toxicity to C. maculatus. The most acidic fractions of both susceptible (CE-31 cultivar and resistant (IT81D-1045 line seeds were shown to affect development and survival of the bruchid. Results indicated that vicilin polypeptides of toxic nature were expressed in both types of storage globulins although at different levels.Vicilinas (globulinas de reserva 7S isoladas de sementes de feijão-de-corda (Vigna unguiculata L., susceptíveis (S e resistentes (R ao caruncho/gorgulho (Callosobruchus maculatus F., Coleoptera: Bruchidae foram desnaturadas por uréia e fracionadas por cromatografia de troca iônica. As frações isoladas foram incorporadas em sementes artificiais para avaliação de sua toxicidade a C. maculatus. As fracões mais ácidas de ambas vicilinas afetaram o desenvolvimento e a sobrevivência do bruquídeo. Sugerimos que polipeptídeos de vicilinas de natureza tóxica são expressos em ambos tipos de globulinas de reserva, embora em níveis diferentes.

  6. Denaturing gradient gel electrophoresis (DGGE as a powerful novel alternative for differentiation of epizootic ISA virus variants.

    Marisela Carmona

    Full Text Available Infectious Salmon Anemia is a devastating disease critically affecting world-wide salmon production. Chile has been particularly stricken by this disease which in all cases has been directly related with its causative agent, a novel orthomyxovirus which presents specific and distinctive infective features. Among these, two molecular markers have been directly associated with pathogenicity in two of the eight RNA sub genomic coding units of the virus: an insertion hot spot region present in viral segment 5 and a Highly Polymorphic Region (HPR located in viral segment 6. Here we report the successful adaptation of a PCR-dependent denaturing gel electrophoresis technique (DGGE, which enables differentiation of selected reported HPR epizootic variants detected in Chile. At the same time, the technique allows us to distinguish one nucleotide differences in sequences associated with the intriguing, and still not well-understood, insertion events which tend to occur on RNA Segment 5. Thus, the versatility of the technique opens new opportunities for improved understanding of the complex biology of all ISA variants as well as possible applications to other highly variable pathogens.

  7. Tungsten-induced denaturation and aggregation of epoetin alfa during primary packaging as a cause of immunogenicity.

    Seidl, Andreas; Hainzl, Otmar; Richter, Marleen; Fischer, Robert; Böhm, Stephan; Deutel, Britta; Hartinger, Martin; Windisch, Jörg; Casadevall, Nicole; London, Gerard Michel; Macdougall, Iain


    Following two cases of neutralizing antibodies to epoetin alfa in an investigational clinical study, a small number of individual syringes of two drug product batches were found to contain unusually high levels of aggregation at the end of the clinical trial. We undertook an extensive analytical approach to determine the root-cause of the increased aggregation in the affected batches. Soluble tungsten was found in the syringes, most likely derived from the pins used to manufacture the syringes. Spiking of epoetin alfa with sodium polytungstate or an extract of tungsten pins used to manufacture the syringes induced the formation of aggregates, both dimers that appeared to be covalently linked by disulphide bonds as well as higher-order aggregates. Sodium polytungstate had also a strong denaturing effect on the protein. We propose tungsten-mediated unfolding and aggregation of epoetin alfa in pre-filled syringes as a potential root cause for increased immunogenicity. This finding may be more broadly applicable to this and other classes of therapeutic proteins.

  8. Seven new mutations in hMSH2, an HNPCC Gene, identified by denaturing gradient-gel electrophoresis

    Wijnen, J.; Vasen, H.; Khan, P.M.; Klift, H. van der; Leeuwen, C. van; Broek, M. van den; Leeuwen-Cornelisse, I. van; Fodde, R.; Menko, F.H. [Univ. Medical Center, Leiden (Netherlands); Nagengast, F. [Free Univ. Hospital, Amsterdam (Netherlands)


    Hereditary nonpolyposis colorectal cancer (HNPCC) is a relatively common autosomal dominant cancer-susceptibility condition. The recent isolation of the DNA mismatch repair genes (hMSH2, hMLH1, hPMS1, and hPMS2) responsible for HNPCC has allowed the search for germ-line mutations in affected individuals. In this study we used denaturing gradient-gel electrophoresis to screen for mutations in the hMSH2 gene. Analysis of all the 16 exons of HMSH2, in 34 unrelated HNPCC kindreds, has revealed seven novel pathogenic germ-line mutations resulting in stop codons either directly or through frameshifts. Additionally, nucleotide substitutions giving rise to one missense, two silent, and one useful polymorphism have been identified. The proportion of families in which hMSH2 mutations were found is 21%. Although the spectrum of mutations spread at the hMSH2 gene among HNPCC patients appears extremely heterogeneous, we were not able to establish any correlation between the site of the individual mutations and the corresponding tumor spectrum. Our results indicate that, given the genomic size and organization of the hMSH2 gene and the heterogeneity of its mutation spectrum, a rapid and efficient mutation detection procedure is necessary for routine molecular diagnosis and presymptomatic detection of the disease in a clinical setup. 34 refs., 2 figs., 3 tabs.

  9. Variations among Japanese of the factor IX gene (F9) detected by PCR-denaturing gradient gel electrophoresis

    Satoh, Chiyoko; Takahashi, Norio; Asakawa, Junichi; Hiyama, Keiko; Kodaira, Meiko (Radiation Effects Research Foundation, Hiroshima (Japan))


    In the course of feasibility studies to examine the efficiencies and practicalities of various techniques for screening for genetic variations, the human coagulation factor IX (F9) genes of 63 Japanese families were examined by PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Four target sequences with lengths of 983-2,891 bp from the F9 genes of 126 unrelated individuals from Hiroshima and their 100 children were amplified by PCR, digested with restriction enzymes to approximately 500-bp fragments, and examined by DGGE - a total of 6,724 bp being examined per individual. GC-rich sequences (GC-clamps) of 40 bp were attached to both ends of the target sequences, as far as was feasible. Eleven types of new nucleotide substitutions were detected in the population, none of which produced RFLPs or caused hemophilia B. By examining two target sequences in a single lane, approximately 8,000 bp in a diploid individual could be examined. This approach is very effective for the detection of variations in DNA and is applicable to large-scale population studies. 46 refs., 3 figs., 1 tab.

  10. Heat-denaturation and aggregation of quinoa (Chenopodium quinoa) globulins as affected by the pH value.

    Mäkinen, Outi E; Zannini, Emanuele; Koehler, Peter; Arendt, Elke K


    The influence of heating (100 °C; 0-15 min) on the relative molecular mass, protein unfolding and secondary structure of quinoa globulins was studied at pH 6.5 (low solubility), 8.5 and 10.5 (high solubility). The patterns of denaturation and aggregation varied with pH. Heating triggered the disruption of the disulfide bonds connecting the acidic and basic chains of the chenopodin subunits at pH 8.5 and 10.5, but not at pH 6.5. Large aggregates unable to enter a 4% SDS-PAGE gel were formed at pH 6.5 and 8.5, which became soluble under reducing conditions. Heating at pH 10.5 lead to a rapid dissociation of the native chenopodin and to the disruption of the subunits, but no SDS-insoluble aggregates were formed. No major changes in secondary structure occurred during a 15 min heating, but an increase in hydrophobicity indicated unfolding of the tertiary structure in all samples.

  11. Employing the fluorescence anisotropy and quenching kinetics of tryptophan to hunt for residual structures in denatured proteins

    Satish Kumar; Rajaram Swaminathan


    Residual structures in denatured proteins have acquired importance in recent years owing to their role as protein-folding initiation sites. Locating these structures in proteins has proved quite formidable, requiring techniques like NMR. Here in this report, we take advantage of the ubiquitous presence of tryptophan residues in residual structures to hunt for their presence using steady-state fluorescence spectroscopy. The surface accessibility and rotational dynamics of tryptophan in putative residual structures among ten different proteins, namely glucagon, melittin, subtilisin carlsberg, myelin basic protein, ribonuclease T1, human serum albumin, barstar mutant, bovine serum albumin, lysozyme and Trp-Met-Asp-Phe-NH2 peptide, was studied using steady state fluorescence quenching and anisotropy, respectively. Five proteins, namely ribonuclease T1, bovine serum albumin, melittin, barstar and hen egg white lysozyme appear likely to possess tryptophan(s) in hydrophobic clusters based on their reduced bimolecular quenching rates and higher steady-state anisotropy in proportion to their chain length. We also show that the fluorescence emission maximum of tryptophan is insensitive to the presence of residual structures.

  12. Effect of heat, pH, ultrasonication and ethanol on the denaturation of whey protein isolate using a newly developed approach in the analysis of difference-UV spectra.

    Nikolaidis, Athanasios; Andreadis, Marios; Moschakis, Thomas


    A newly developed method of analysis of difference-UV spectra was successfully implemented in the study of the effect of heat, pH, ultrasonication and ethanol on the denaturation of whey protein isolate. It was found that whey proteins exhibit their highest stability against heat denaturation at pH 3.75. At very low pH values, i.e. 2.5, they exhibited considerable cold denaturation, while after heating at this pH value, the supplementary heat denaturation rate was lower compared to that at neutral pH. The highest heat denaturation rates were observed at pH values higher than neutral. High power sonication on whey proteins, previously heated at 90°C for 30min, resulted in a rather small reduction of the fraction of the heat denatured protein aggregates. Finally, when ethanol was used as a cosolvent in the concentration range 20-50%, a sharp increase in the degree of denaturation, compared to the native protein solution, was observed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. 1H, 15N and 13C assignments of domain 5 of Dictyostelium discoideum gelation factor (ABP-120) in its native and 8M urea-denatured states.

    Hsu, Shang-Te Danny; Cabrita, Lisa D; Christodoulou, John; Dobson, Christopher M


    The gelation factor from Dictyostelium discoideum (ABP-120) is an actin binding protein consisting of six immunoglobulin (Ig) domains in the C-terminal rod domain. We have recently used the pair of domains 5 and 6 of ABP-120 as a model system for studying multi-domain nascent chain folding on the ribosome. Here we present the NMR assignments of domain 5 in its native and 8M urea-denatured states.

  14. Role of N-terminal residues on folding and stability of C-phycoerythrin: simulation and urea-induced denaturation studies.

    Anwer, Khalid; Sonani, Ravi; Madamwar, Datta; Singh, Parvesh; Khan, Faez; Bisetty, Krishna; Ahmad, Faizan; Hassan, Md Imtaiyaz


    The conformational state of biliproteins can be determined by optical properties of the covalently linked chromophores. Recently determined crystal structure of truncated form of α-subunit of cyanobacterial phycoerythrin (αC-PE) from Phormidium tenue provides a new insight into the structure-function relationship of αC-PE. To compare their stabilities, we have measured urea-induced denaturation transitions of the full length αC-PE (FL-αC-PE) and truncated αC-PE (Tr-αC-PE) followed by observing changes in absorbance at 565 nm, fluorescence at 350 and 573 nm, and circular dichroism at 222 nm as a function of [urea], the molar concentration of urea. The transition curve of each protein was analyzed for ΔG(D)(0), the value of Gibbs free energy change on denaturation (ΔG(D)) in the absence of urea; m, the slope (=∂∆G(D)/∂[urea]), and C(m), the midpoint of the denaturation curve, i.e. [urea] at which ΔG(D) = 0. A difference of about 10% in ΔG(D)(0) observed between FL-αC-PE and Tr-αC-PE, suggests that the two proteins are almost equally stable, and the natural deletion of 31 residues from the N-terminal side of the full length protein does not alter its stability. Furthermore, normalization of probes shows that the urea-induced denaturation of both the proteins is a two-state process. Folding of both structural variants (Tr-αC-PE and FL-αC-PE) of P. tenue were also studied using molecular dynamics simulations at 300 K. The results show clearly that the stability of the proteins is evenly distributed over the whole structure indicating no significant role of N-terminal residues in the stability of both proteins.

  15. Human Papillomavirus Genotyping After Denaturation of Specimens for Hybrid Capture 2 Testing: Feasibility Study for the HPV Persistence and Progression Cohort†


    Human papillomavirus (HPV) genotyping could be clinically useful, depending on the results of large, prospective studies like the HPV Persistence and Progression cohort. The cohort is based on genotyping and follow-up of Hybrid Capture-positive women at Kaiser Permanente, Northern California. HPV DNA testing by Hybrid Capture 2 requires denaturation with alkali, possibly damaging the DNA for optimal PCR-based genotyping. A feasibility study was conducted on paired aliquots of anonymized speci...

  16. Effect of enzymatic fish frame protein hydrolysate on freeze-induced denaturation of myofibrillar protein from bighead carp fish (Aristichthys nobilis) during frozen storage

    Xue Yong; Xue Changhu; Zhang Yongqin; Li Zhaojie; Gao Ruichang; Gao Xin


    Three kinds of fish frame protein hydrolysates (PPH, APH and FPH) were prepared from fish frame of red drum (Sciaenops ocellatus) by papain, alkaline proteinase and flavorzyme treatment. The hydrolysates were mainly composed of peptide (83.5%-84.6%) and displayed different molecular weight distribution pattern. The protective effects of hydrolysates on the freeze-induced denaturation of myofibrillar protein (Mf) from bighead carp (Aristichthys nobilis) mince during storage at -20℃ for 12 weeks were investigated. The hydrolysate (5% dried weight/wet weight) reduced the freeze-induced denaturation of Mf as evidenced by the lowered decrease in Ca-ATPase activity and reactive sulfhydryl contents as well as the impeded increase in surface hydrophobicity. Microscopic photographs indicated that the hydrolysates inhibited the growth of ice crystal in fish mince, and then prevented the aggregation of Mf during frozen storage. The protective effects of hydrolysates on freeze-induced denaturation of Mf were influenced by the molecular weight distribution. PPH had strongest cryoprotective ability among three hydrolysates.

  17. Anti-tumour-promoting and thermal-induced protein denaturation inhibitory activities of β-sitosterol and lupeol isolated from Diospyros lotus L.

    Rauf, Abdur; Uddin, Ghias; Khan, Haroon; Raza, Muslim; Zafar, Muhammad; Tokuda, Harukuni


    In this study, the anti-tumour-promoting and thermal-induced protein denaturation inhibitory activities of β-sitosterol (1) and lupeol (2), isolated from Diospyros lotus L., were explored. Compound 1 showed a marked concentration-dependent inhibition against 12-O-tetradecanoylphorbol-13-acetate (20 ng/32 pmol)-induced Epstein-Barr virus early antigen activation in Raji cells with IC50 of 270 μg/ml, without significant toxicity (70% viability). Compound 2 showed significant anti-tumour-promoting effect with IC50 of 412 μg/ml, without significant toxicity (60% viability). In heat-induced protein denaturation assay, compound 1 exhibited a concentration-dependent attenuation with a maximum effect of 73.5% at 500 μg/ml with EC50 of 117 μg/ml, while compound 2 exhibited a maximum effect of 59.2% at 500 μg/ml with EC50 of 355 μg/ml. Moreover, in silico docking studies against the phosphoinositide 3-kinase enzyme also show the inhibitory potency of these compounds. In short, both the compounds exhibited a marked anti-tumour-promoting and potent inhibitory effect on thermal-induced protein denaturation.

  18. Establishment of in vitro models of denatured collagen%变性胶原体外培养模型的建立

    苏荣家; 王志勇; 刘英开; 原博; 王西樵; 董叫云; 宋菲; 姜育智; 陆树良


    目的 探讨不同温度对Ⅰ型胶原分子二级结构的影响,确定合适的胶原变性温度,研究热变性后胶原纤维排列及三维凝胶性质的改变,比较胶原变性后不同培养环境成纤维细胞形态差异,以建立变性胶原-细胞体外培养模型. 方法 Ⅰ型胶原蛋白溶液在不同温度作用后通过蛋白质圆二色光谱仪分析胶原分子二级结构改变.扫描探针显微镜观察胶原变性后纤维结构的改变.制备不同种类三维胶原凝胶并通过气相压力仪检测胶原凝胶断裂模量.将变性后的胶原进行二维包被和三维胶原凝胶制作,倒置相差显微镜及光镜下观察不同培养环境下细胞形态变化. 结果 温度达到50℃时,Ⅰ型胶原分子二级结构发生明显改变,在二维胶原包被时可见胶原纤维凝集成团,含变性胶原的三维凝胶断裂模量明显下降.在变性胶原存在环境中培养成纤维细胞,细胞形态均有显著改变. 结论 经50℃作用后Ⅰ型胶原分子二级结构发生明显改变,含变性胶原的三维凝胶断裂模量明显下降,Ⅰ型胶原包被及三维凝胶模型培养的成纤维细胞形态明显不同,可作为变性胶原影响细胞生物学活性的体外模型.%Objective To investigate influence of different temperatures on secondary structure of type Ⅰ collagen,determine the proper temperature for collagen denaturation,observe changes of collagen fibre arrangement and three dimensional collagen gel properties after thermal denaturation,compare morphological variation of fibroblasts seeded in mediums with denatured collagen and therefore establish a standardized culture model with denatured collagen in vitro.Methods Changes of the secondary structure of type Ⅰ collagen was measured by circular dichroism spectrameter after the collagen solution had been treated with different temperatures.Changes of the fibre structure after collagen denaturation were observed by scanning probe

  19. Analysis of Thermally Denatured Depth in Laser Vaporization for Benign Prostatic Hyperplasia using a Simulation of Light Propagation and Heat Transfer (secondary publication).

    Takada, Junya; Honda, Norihiro; Hazama, Hisanao; Ioritani, Naomasa; Awazu, Kunio


    Background and Aims: Laser vaporization of the prostate is expected as a less invasive treatment for benign prostatic hyperplasia (BPH), via the photothermal effect. In order to develop safer and more effective laser vaporization of the prostate, it is essential to set optimal irradiation parameters based on quantitative evaluation of temperature distribution and thermally denatured depth in prostate tissue. Method: A simulation model was therefore devised with light propagation and heat transfer calculation, and the vaporized and thermally denatured depths were estimated by the simulation model. Results: The results of the simulation were compared with those of an ex vivo experiment and clinical trial. Based on the accumulated data, the vaporized depth strongly depended on the distance between the optical fiber and the prostate tissue, and it was suggested that contact laser irradiation could vaporize the prostate tissue most effectively. Additionally, it was suggested by analyzing thermally denatured depth comprehensively that laser irradiation at the distance of 3 mm between the optical fiber and the prostate tissue was useful for hemostasis. Conclusions: This study enabled quantitative and reproducible analysis of laser vaporization for BPH and will play a role in clarification of the safety and efficacy of this treatment.

  20. Establishment of intein-mediated protein ligation under denaturing conditions: C-terminal labeling of a single-chain antibody for biochip screening.

    Sydor, Jens R; Mariano, Maria; Sideris, Steve; Nock, Steffen


    Intein-mediated protein ligation is a recently developed method that enables the C-terminal labeling of proteins. This technique requires a correctly folded intein mutant that is fused to the C-terminus of a target protein to create a thioester, which allows the ligation of a peptide with an N-terminal cysteine (1, 2). Here we describe the establishment of this method for the labeling, under denaturing conditions, of target proteins that are expressed insolubly as intein fusion proteins. A GFPuv fusion protein with the Mycobacterium xenopi gyrA intein was expressed in inclusion bodies in Escherichia coli and initially used as a model protein to verify intein cleavage activity under different refolding conditions. The intein showed activity after refolding in nondenaturing and slightly denaturing conditions. A construct of the same intein with an anti-neutravidin single-chain antibody was also expressed in an insoluble form. The intein-mediated ligation was established for this single chain antibody-intein fusion protein under denaturing conditions in 4 M urea to prevent significant precipitation of the fusion protein during the first refolding step. Under optimized conditions, the single-chain antibody was labeled with a fluorescent peptide and used for antigen screening on a biochip after final refolding. This screening procedure allowed the determination of binding characteristics of the scFv for avidin proteins in a miniaturized format.

  1. Structural and denaturation studies of two mutants of a cold adapted superoxide dismutase point to the importance of electrostatic interactions in protein stability.

    Merlino, Antonello; Russo Krauss, Irene; Castellano, Immacolata; Ruocco, Maria Rosaria; Capasso, Alessandra; De Vendittis, Emmanuele; Rossi, Bianca; Sica, Filomena


    A peculiar feature of the psychrophilic iron superoxide dismutase from Pseudoalteromonas haloplanktis (PhSOD) is the presence in its amino acid sequence of a reactive cysteine (Cys57). To define the role of this residue, a structural characterization of the effect of two PhSOD mutations, C57S and C57R, was performed. Thermal and denaturant-induced unfolding of wild type and mutant PhSOD followed by circular dichroism and fluorescence studies revealed that C→R substitution alters the thermal stability and the resistance against denaturants of the enzyme, whereas C57S only alters the stability of the protein against urea. The crystallographic data on the C57R mutation suggest an involvement of the Arg side chain in the formation of salt bridges on protein surface. These findings support the hypothesis that the thermal resistance of PhSOD relies on optimization of charge-charge interactions on its surface. Our study contributes to a deeper understanding of the denaturation mechanism of superoxide dismutases, suggesting the presence of a structural dimeric intermediate between the native state and the unfolded state. This hypothesis is supported by the crystalline and solution data on the reduced form of the enzyme. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Human papillomavirus genotyping after denaturation of specimens for Hybrid Capture 2 testing: feasibility study for the HPV persistence and progression cohort.

    LaMere, Brandon J; Kornegay, Janet; Fetterman, Barbara; Sadorra, Mark; Shieh, Jen; Castle, Philip E


    Human papillomavirus (HPV) genotyping could be clinically useful, depending on the results of large, prospective studies like the HPV persistence and progression (PaP) cohort. The cohort is based on genotyping and follow-up of Hybrid Capture-positive women at Kaiser Permanente, Northern California. HPV DNA testing by Hybrid Capture 2 requires denaturation with alkali, possibly damaging the DNA for optimal PCR-based genotyping. A feasibility study was conducted on paired aliquots of anonymized specimens from 100 women with low-grade intraepithelial lesion cytology. Test aliquots were left in denaturant for 10 or 18h at 4 degrees C and then neutralized; comparison aliquots were not denatured but diluted to match the timing, temperature, concentration and salt conditions of the treated specimens. The masked aliquots were tested using a commercialized PCR-based assay that detects of 37 HPV genotypes. There was no overall effect of treatment on test positivity or number of types. HPV16 was marginally more likely to be detected in untreated versus treated aliquots (P=0.09) but HPV45 was marginally more likely to be detected in treated than untreated aliquots (P=0.07), suggesting that these differences represented chance (intra-test variability). It can be concluded that residual Hybrid Capture-positive specimens can be genotyped by PCR after Hybrid Capture 2 processing.

  3. Weak and saturable protein-surfactant interactions in the denaturation of apo-alpha-lactalbumin by acidic and lactonic sophorolipid

    Kell K Andersen


    Full Text Available Biosurfactants are of growing interest as sustainable alternatives to fossil-fuel-derived chemical surfactants, particularly for the detergent industry. To realize this potential, it is necessary to understand how they affect proteins which they may encounter in their applications. However knowledge of such interactions is limited. Here we present a study of the interactions between the model protein apo-alpha-lactalbumin and the biosurfactant sophorolipid (SL produced by the yeast Starmerella bombicola. SL occurs both as an acidic and a lactonic form; the lactonic form (lactSL is sparingly soluble and has a lower critical micelle concentration than the acidic form (acidSL. We show that acidSL affects apo-aLA in a similar way to the related glycolipid biosurfactant rhamnolipid (RL, with the important difference that RL is also active below the cmc in contrast to acidSL. Using isothermal titration calorimetry data, we show that acidSL has weak and saturable interactions with apo-aLA at low concentrations; due to the relatively low cmc of acidSL (which means that the monomer concentration is limited to ca. 0-1 mM SL, it is only possible to observe interactions with monomeric acidSL at high apo-aLA concentrations. However, the denaturation kinetics of apo-aLA in the presence of acidSL are consistent with a collaboration between monomeric and micellar surfactant species, similar to RL and nonionic or zwitterionic surfactants. Inclusion of lactSL as mixed micelles with acidSL lowers the cmc and this effectively reduces the rate of unfolding, emphasizing that SL like other biosurfactants is a gentle anionic surfactant. Our data highlight the potential of these biosurfactants for future use in the detergent industry.

  4. Agarose gel purification of PCR products for denaturing gradient gel electrophoresis results in GC-clamp deletion.

    Sun, Guowei; Xiao, Jinzhou; Lu, Man; Wang, Hongming; Chen, Xiaobing; Yu, Yongxin; Pan, Yingjie; Wang, Yongjie


    The 16S ribosomal RNA (rRNA) gene of marine archaeal samples was amplified using a nested PCR approach, and the V3 region of 16S rRNA gene of crab gut microbiota (CGM) was amplified using the V3 universal primer pair with a guanine and cytosine (GC)-clamp. Unpurified PCR products (UPPs), products purified from reaction solution (PPFSs), and products purified from gel (PPFGs) of above two DNA samples were used for denaturing gradient gel electrophoresis (DGGE) analysis, respectively. In contrast to almost identical band patterns shared by both the UPP and PPFS, the PPFGs were barely observed on the DGGE gel for both the marine archaea and CGM samples. Both PPFS and PPFG of CGM V3 regions were subjected to cloning. A small amount of positive clones was obtained for PPFS, but no positive clones were observed for PPFG. The melt curve and direct sequencing analysis of PPFS and PPFG of E. coli V3 region indicated that the Tm value of PPFG (82.35 ± 0.19 °C) was less than that of PPFS (83.81 ± 0.11 °C), and the number of shorter GC-clamps was significant higher in PPFG than in PPFS. The ultraviolet exposure experiment indicated that the ultraviolet was not responsible for the deletion of the GC-clamps. We conclude that the gel purification method is not suitable for DGGE PCR products or even other GC-rich DNA samples.

  5. Physical origin of hydrophobicity studied in terms of cold denaturation of proteins: comparison between water and simple fluids.

    Yoshidome, Takashi; Kinoshita, Masahiro


    A clue to the physical origin of the hydrophobicity is in the experimental observations, which show that it is weakened at low temperatures. By considering a solvophobic model protein immersed in water and three species of simple solvents, we analyze the temperature dependence of the changes in free energy, energy, and entropy of the solvent upon protein unfolding. The angle-dependent and radial-symmetric integral equation theories and the morphometric approach are employed in the analysis. Each of the changes is decomposed into two terms, which depend on the excluded volume and on the area and curvature of the solvent-accessible surface, respectively. The excluded-volume term of the entropy change is further decomposed into two components representing the protein-solvent pair correlation and the protein-solvent-solvent triplet and higher-order correlation, respectively. We show that water crowding in the system becomes more serious upon protein unfolding but this effect becomes weaker as the temperature is lowered. If the hydrophobicity originated from the water structuring near a nonpolar solute, it would be strengthened upon lowering of the temperature. Among the three species of simple solvents, considerable weakening of the solvophobicity at low temperatures is observed only for the solvent where the particles interact through a strong attractive potential and the particle size is as small as that of water. Even in the case of this solvent, however, cold denaturation of a protein cannot be reproduced. It would be reproducible if the attractive potential was substantially enhanced, but such enhancement causes the appearance of the metastability limit for a single liquid phase.

  6. Monitoring of microbial community structure and succession in the biohydrogen production reactor by denaturing gradient gel electrophoresis (DGGE)

    XING; Defeng; REN; Nanqi; GONG; Manli; LI; Jianzheng; LI; Q


    To study the structure of microbial communities in the biological hydrogen production reactor and determine the ecological function of hydrogen producing bacteria, anaerobic sludge was obtained from the continuous stirred tank reactor (CSTR) in different periods of time, and the diversity and dynamics of microbial communities were investigated by denaturing gradient gel electrophoresis (DGGE). The results of DGGE demonstrated that an obvious shift of microbial population happened from the beginning of star-up to the 28th day, and the ethanol type fermentation was established. After 28 days the structure of microbial community became stable, and the climax community was formed. Comparative analysis of 16S rDNA sequences from reamplifying and sequencing the prominent bands indicated that the dominant population belonged to low G+C Gram-positive bacteria (Clostridium sp. And Ethanologenbacterium sp.), β- proteobacteria (Acidovorax sp.), γ-proteobacteria (Kluyvera sp.), Bacteroides (uncultured bacterium SJA-168), and Spirochaetes (uncultured eubacterium E1-K13), respectively. The hydrogen production rate increased obviously with the increase of Ethanologenbacterium sp., Clostridium sp. And uncultured Spirochaetes after 21 days, meanwhile the succession of ethanol type fermentation was formed. Throughout the succession the microbial diversity increased however it decreased after 21 days. Some types of Clostridium sp. Acidovorax sp., Kluyvera sp., and Bacteroides were dominant populations during all periods of time. These special populations were essential for the construction of climax community. Hydrogen production efficiency was dependent on both hydrogen producing bacteria and other populations. It implied that the co-metabolism of microbial community played a great role of biohydrogen production in the reactors.

  7. Evaluation of mutation screening by heteroduplex analysis in acute intermittent porphyria: comparison with denaturing gradient gel electrophoresis.

    Tchernitchko, D; Lamoril, J; Puy, H; Robreau, A M; Bogard, C; Rosipal, R; Gouya, L; Deybach, J C; Nordmann, Y


    Acute intermittent porphyria is the major autosomal dominant form of acute hepatic porphyrias. The disease is due to mutations in the gene encoding for porphobilinogen deaminase (PBGD). Many different strategies have been developed to screen for mutations. However the high prevalence (0.6 per thousand) of PBGD gene defect, the large allelic heterogeneity of mutations (n = 130), and the limitations of the PBGD enzymatic assay for asymptomatic patients' detection, require for diagnosis an efficient and easy to handle strategy for locating mutations within the PBGD gene. In a recent study the sensitivity of the denaturing gradient gel electrophoresis (DGGE) technique was 100%. However DGGE requires the preparation of gradient gels and the use of primers with long GC-clamps; thus alternative methods should be preferable in the clinical laboratory. We have compared the detection rate of DGGE with heteroduplex analysis (HA) using 16 characterized PBGD gene mutations. Six different HA conditions were used to determine the efficiency of the method, including: (1) MDE (mutation detection enhancement) gel concentration; (2) addition of urea and sodium dodecyl sulfate (SDS); (3) radioactive labelling. The sensitivity of each HA condition varied from 31 to 81% vs. 100% in DGGE analysis. HA using 1 x MDE with 15% urea with or without 0.55% SDS was the most sensitive condition. This first comparative study of DGGE and HA mutation screening methods suggests that DGGE is a more sensitive screening assay than optimized HA. However, because of its simplicity HA should be considered as an efficient alternative mutation screening method.

  8. Diagnosis of Russell-Silver syndrome by the combined bisulfite restriction analysis-denaturing high-performance liquid chromatography assay.

    Hattori, Mitsu; Torii, Chiharu; Yagihashi, Tatsuhiko; Izumi, Kosuke; Suda, Naoto; Ohyama, Kimie; Takahashi, Takao; Moriyama, Keiji; Kosaki, Kenjiro


    Russell-Silver syndrome (RSS) is characterized by prenatal and postnatal growth retardation, triangular facies, and fifth-finger clinodactyly. Half of all patients with RSS have hypomethylation of the differentially methylated region of the H19 gene on chromosome 11p15.5. Hence, a quantitative methylation analysis of this region can be useful for the molecular diagnosis of RSS. However, conventional assays based on bisulfite clone sequencing are rather time and labor consuming and are not suitable for clinical use. In the present study, we investigated a possible method of quantitatively determining H19 hypomethylation in RSS patients using a combined bisulfite restriction analysis (COBRA)-denaturing high-performance liquid chromatography (DHPLC) assay; in this combined assay, polymerase chain reaction products amplified from the H19 differentially methylated region of bisulfite-treated genomic DNA were analyzed using a COBRA assay, which detects methylation-dependent sequence differences in the bisulfite-treated genomic DNA using a restriction enzyme analysis. We designed the assay so that a restriction enzyme (HinfI) would cut the methylated, but not the unmethylated, template. The molar ratio between the cut and uncut fragments was measured using DHPLC, and the construction of a calibration curve enabled the methylation index for the original genomic DNA to be estimated. An analysis of seven RSS patients using the COBRA-DHPLC assay demonstrated that three of the seven RSS patients had a low methylation index of around 10%. A comparison of the methylation indices obtained using COBRA-DHPLC and conventional bisulfite clone sequencing revealed an excellent intermethod agreement. In summary, we have developed a robust, rapid, and cost-effective COBRA-DHPLC-based screening system for RSS.

  9. Identification of protein secondary structures by laser induced autofluorescence: A study of urea and GnHCl induced protein denaturation

    Siddaramaiah, Manjunath; Satyamoorthy, Kapaettu; Rao, Bola Sadashiva Satish; Roy, Suparna; Chandra, Subhash; Mahato, Krishna Kishore


    In the present study an attempt has been made to interrogate the bulk secondary structures of some selected proteins (BSA, HSA, lysozyme, trypsin and ribonuclease A) under urea and GnHCl denaturation using laser induced autofluorescence. The proteins were treated with different concentrations of urea (3 M, 6 M, 9 M) and GnHCl (2 M, 4 M, 6 M) and the corresponding steady state autofluorescence spectra were recorded at 281 nm pulsed laser excitations. The recorded fluorescence spectra of proteins were then interpreted based on the existing PDB structures of the proteins and the Trp solvent accessibility (calculated using "Scratch protein predictor" at 30% threshold). Further, the influence of rigidity and conformation of the indole ring (caused by protein secondary structures) on the intrinsic fluorescence properties of proteins were also evaluated using fluorescence of ANS-HSA complexes, CD spectroscopy as well as with trypsin digestion experiments. The outcomes obtained clearly demonstrated GnHCl preferably disrupt helix as compared to the beta β-sheets whereas, urea found was more effective in disrupting β-sheets as compared to the helices. The other way round the proteins which have shown detectable change in the intrinsic fluorescence at lower concentrations of GnHCl were rich in helices whereas, the proteins which showed detectable change in the intrinsic fluorescence at lower concentrations of urea were rich in β-sheets. Since high salt concentrations like GnHCl and urea interfere in the secondary structure analysis by circular dichroism Spectrometry, the present method of analyzing secondary structures using laser induced autofluorescence will be highly advantageous over existing tools for the same.

  10. Monitoring of microbial community structure and succession in the biohydrogen production reactor by denaturing gradient gel electrophoresis (DGGE).

    Xing, Defeng; Ren, Nanqi; Gong, Manli; Li, Jianzheng; Li, Qiubo


    To study the structure of microbial communities in the biological hydrogen production reactor and determine the ecological function of hydrogen producing bacteria, anaerobic sludge was obtained from the continuous stirred tank reactor (CSTR) in different periods of time, and the diversity and dynamics of microbial communities were investigated by denaturing gradient gel electrophoresis (DGGE). The results of DGGE demonstrated that an obvious shift of microbial population happened from the beginning of star-up to the 28th day, and the ethanol type fermentation was established. After 28 days the structure of microbial community became stable, and the climax community was formed. Comparative analysis of 16S rDNA sequences from reamplifying and sequencing the prominent bands indicated that the dominant population belonged to low G+C Gram-positive bacteria (Clostridium sp. and Ethanologenbacterium sp.), beta-proteobacteria (Acidovorax sp.), gamma-proteobacteria (Kluyvera sp.), Bacteroides (uncultured bacterium SJA-168), and Spirochaetes (uncultured eubacterium E1-K13), respectively. The hydrogen production rate increased obviously with the increase of Ethanologenbacterium sp., Clostridium sp. and uncultured Spirochaetes after 21 days, meanwhile the succession of ethanol type fermentation was formed. Throughout the succession the microbial diversity increased however it decreased after 21 days. Some types of Clostridium sp. Acidovorax sp., Kluyvera sp., and Bacteroides were dominant populations during all periods of time. These special populations were essential for the construction of climax community. Hydrogen production efficiency was dependent on both hydrogen producing bacteria and other populations. It implied that the co-metabolism of microbial community played a great role of biohydrogen production in the reactors.

  11. Diversity and dynamics of antibiotic-resistant bacteria in cheese as determined by PCR denaturing gradient gel electrophoresis.

    Flórez, Ana Belén; Mayo, Baltasar


    This work reports the composition and succession of tetracycline- and erythromycin-resistant bacterial communities in a model cheese, monitored by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Bacterial 16S rRNA genes were examined using this technique to detect structural changes in the cheese microbiota over manufacturing and ripening. Total bacterial genomic DNA, used as a template, was extracted from cultivable bacteria grown without and with tetracycline or erythromycin (both at 25 μg ml(-1)) on a non-selective medium used for enumeration of total and viable cells (Plate Count agar with Milk; PCA-M), and from those grown on selective and/or differential agar media used for counting various bacterial groups; i.e., lactic acid bacteria (de Man, Rogosa and Sharpe agar; MRSA), micrococci and staphylococci (Baird-Parker agar; BPA), and enterobacteria (Violet Red Bile Glucose agar; VRBGA). Large numbers of tetracycline- and erythromycin-resistant bacteria were detected in cheese samples at all stages of ripening. Counts of antibiotic-resistant bacteria varied widely depending on the microbial group and the point of sampling. In general, resistant bacteria were 0.5-1.0 Log10 units fewer in number than the corresponding susceptible bacteria. The PCR-DGGE profiles obtained with DNA isolated from the plates for total bacteria and the different bacterial groups suggested Escherichia coli, Lactococcus lactis, Enterococcus faecalis and Staphylococcus spp. as the microbial types resistant to both antibiotics tested. This study shows the suitability of the PCR-DGGE technique for rapidly identifying and tracking antibiotic resistant populations in cheese and, by extension, in other foods.

  12. Weak and Saturable Protein–Surfactant Interactions in the Denaturation of Apo-α-Lactalbumin by Acidic and Lactonic Sophorolipid

    Andersen, Kell K.; Vad, Brian S.; Roelants, Sophie; van Bogaert, Inge N. A.; Otzen, Daniel E.


    Biosurfactants are of growing interest as sustainable alternatives to fossil-fuel-derived chemical surfactants, particularly for the detergent industry. To realize this potential, it is necessary to understand how they affect proteins which they may encounter in their applications. However, knowledge of such interactions is limited. Here, we present a study of the interactions between the model protein apo-α-lactalbumin (apo-aLA) and the biosurfactant sophorolipid (SL) produced by the yeast Starmerella bombicola. SL occurs both as an acidic and a lactonic form; the lactonic form (lactSL) is sparingly soluble and has a lower critical micelle concentration (cmc) than the acidic form [non-acetylated acidic sophorolipid (acidSL)]. We show that acidSL affects apo-aLA in a similar way to the related glycolipid biosurfactant rhamnolipid (RL), with the important difference that RL is also active below the cmc in contrast to acidSL. Using isothermal titration calorimetry data, we show that acidSL has weak and saturable interactions with apo-aLA at low concentrations; due to the relatively low cmc of acidSL (which means that the monomer concentration is limited to ca. 0–1 mM SL), it is only possible to observe interactions with monomeric acidSL at high apo-aLA concentrations. However, the denaturation kinetics of apo-aLA in the presence of acidSL are consistent with a collaboration between monomeric and micellar surfactant species, similar to RL and non-ionic or zwitterionic surfactants. Inclusion of diacetylated lactonic sophorolipid (lactSL) as mixed micelles with acidSL lowers the cmc and this effectively reduces the rate of unfolding, emphasizing that SL like other biosurfactants is a gentle anionic surfactant. Our data highlight the potential of these biosurfactants for future use in the detergent and pharmaceutical industry. PMID:27877155

  13. Identification and Population Dynamics of Yeasts in Sourdough Fermentation Processes by PCR-Denaturing Gradient Gel Electrophoresis

    Meroth, Christiane B.; Hammes, Walter P.; Hertel, Christian


    Four sourdoughs (A to D) were produced under practical conditions, using a starter obtained from a mixture of three commercially available sourdough starters and baker's yeast. The doughs were continuously propagated until the composition of the microbiota remained stable. A fungi-specific PCR-denaturing gradient gel electrophoresis (DGGE) system was established to monitor the development of the yeast biota. The analysis of the starter mixture revealed the presence of Candida humilis, Debaryomyces hansenii, Saccharomyces cerevisiae, and Saccharomyces uvarum. In sourdough A (traditional process with rye flour), C. humilis dominated under the prevailing fermentation conditions. In rye flour sourdoughs B and C, fermented at 30 and 40°C, respectively, S. cerevisiae became predominant in sourdough B, whereas in sourdough C the yeast counts decreased within a few propagation steps below the detection limit. In sourdough D, which corresponded to sourdough C in temperature but was produced with rye bran, Candida krusei became dominant. Isolates identified as C. humilis and S. cerevisiae were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively. The yeast species isolated from the sourdoughs were also detected by PCR-DGGE. However, in the gel, additional bands were visible. Because sequencing of these PCR fragments from the gel failed, cloning experiments with 28S rRNA amplicons obtained from rye flour were performed, which revealed Cladosporium sp., Saccharomyces servazii, S. uvarum, an unculturable ascomycete, Dekkera bruxellensis, Epicoccum nigrum, and S. cerevisiae. The last four species were also detected in sourdoughs A, B, and C. PMID:14660398

  14. Dynamic changes of yak ( gut microbiota during growth revealed by polymerase chain reaction-denaturing gradient gel electrophoresis and metagenomics

    Yuanyang Nie


    Full Text Available Objective To understand the dynamic structure, function, and influence on nutrient metabolism in hosts, it was crucial to assess the genetic potential of gut microbial community in yaks of different ages. Methods The denaturing gradient gel electrophoresis (DGGE profiles and Illumina-based metagenomic sequencing on colon contents of 15 semi-domestic yaks were investigated. Unweighted pairwise grouping method with mathematical averages (UPGMA clustering and principal component analysis (PCA were used to analyze the DGGE fingerprint. The Illumina sequences were assembled, predicted to genes and functionally annotated, and then classified by querying protein sequences of the genes against the Kyoto encyclopedia of genes and genomes (KEGG database. Results Metagenomic sequencing showed that more than 85% of ribosomal RNA (rRNA gene sequences belonged to the phylum Firmicutes and Bacteroidetes, indicating that the family Ruminococcaceae (46.5%, Rikenellaceae (11.3%, Lachnospiraceae (10.0%, and Bacteroidaceae (6.3% were dominant gut microbes. Over 50% of non-rRNA gene sequences represented the metabolic pathways of amino acids (14.4%, proteins (12.3%, sugars (11.9%, nucleotides (6.8%, lipids (1.7%, xenobiotics (1.4%, coenzymes, and vitamins (3.6%. Gene functional classification showed that most of enzyme-coding genes were related to cellulose digestion and amino acids metabolic pathways. Conclusion Yaks’ age had a substantial effect on gut microbial composition. Comparative metagenomics of gut microbiota in 0.5-, 1.5-, and 2.5-year-old yaks revealed that the abundance of the class Clostridia, Bacteroidia, and Lentisphaeria, as well as the phylum Firmicutes, Bacteroidetes, Lentisphaerae, Tenericutes, and Cyanobacteria, varied more greatly during yaks’ growth, especially in young animals (0.5 and 1.5 years old. Gut microbes, including Bacteroides, Clostridium, and Lentisphaeria, make a contribution to the energy metabolism and synthesis of amino

  15. Association of Streptomyces community composition determined by PCR-denaturing gradient gel electrophoresis with indoor mold status.

    Johansson, Elisabet; Reponen, Tiina; Meller, Jarek; Vesper, Stephen; Yadav, Jagjit


    Both Streptomyces species and mold species have previously been isolated from moisture-damaged building materials; however, an association between these two groups of microorganisms in indoor environments is not clear. In this study, we used a culture-independent method, PCR-denaturing gradient gel electrophoresis (PCR-DGGE), to investigate the composition of the Streptomyces community in house dust. Twenty-three dust samples each from two sets of homes categorized as high-mold and low-mold based on mold-specific quantitative PCR analysis were used in the study. Taxonomic identification of prominent bands was performed by cloning and sequencing. Associations between DGGE amplicon band intensities and home mold status were assessed using univariate analyses as well as multivariate recursive partitioning (decision trees) to test the predictive value of combinations of bands intensities. In the final classification tree, a combination of two bands was significantly associated with mold status of the home (p = 0.001). The sequence corresponding to one of the bands in the final decision tree matched a group of Streptomyces species that included Streptomyces coelicolor and Streptomyces sampsonii, both of which have been isolated from moisture-damaged buildings previously. The closest match for the majority of sequences corresponding to a second band consisted of a group of Streptomyces species that included Streptomyces hygroscopicus, an important producer of antibiotics and immunosuppressors. Taken together, the study showed that DGGE can be a useful tool for identifying bacterial species that may be more prevalent in mold-damaged buildings.

  16. Guanidinium chloride and urea denaturations of beta-lactoglobulin A at pH 2.0 and 25 degrees C: the equilibrium intermediate contains non-native structures (helix, tryptophan and hydrophobic patches).

    Dar, Tanveer Ali; Singh, Laishram Rajendrakumar; Islam, Asimul; Anjum, Farah; Moosavi-Movahedi, Ali Akbar; Ahmad, Faizan


    We have carried out guanidinium chloride (GdmCl) and urea denaturations of bovine beta-lactoglobulin A (beta-lgA) at pH 2.0 and 25 degrees C, using far-UV and near-UV circular dichroism, near-UV absorption and tryptophan fluorescence spectroscopies. The stable intermediate state that occurs during GdmCl denaturation has been characterized by the far- and near-UV circular dichroism, tryptophan difference absorption, tryptophan fluorescence and 8-anilino-1-naphthalene sulphonic acid binding measurements. Following conclusions have been reached. (a) Urea-induced denaturation is not a two-state process. (b) GdmCl-induced denaturation is composed of two distinct two-state processes. (c) alpha-Helical content, burial of tryptophan residues and burial of hydrophobic surface area are more in the GdmCl-induced stable intermediate than those originally present in the native protein.

  17. Computed tomography workup of patients suspected of acute ischemic stroke: perfusion computed tomography adds value compared with clinical evaluation, noncontrast computed tomography, and computed tomography angiogram in terms of predicting outcome.

    Zhu, Guangming; Michel, Patrik; Aghaebrahim, Amin; Patrie, James T; Xin, Wenjun; Eskandari, Ashraf; Zhang, Weiwei; Wintermark, Max


    To determine whether perfusion computed tomography (PCT) adds value to noncontrast head CT (NCT), CT angiogram (CTA), and clinical assessment in patients suspected of acute ischemic stroke. We retrospectively reviewed 165 patients with acute ischemic stroke. PCT was used to calculate the volumes of infarct core and ischemic penumbra on admission. Other imaging data included Alberta Score Program Early CT Score, site of occlusion, and collateral flow. Clinical data included age, time, National Institutes of Health Stroke Scale at baseline, treatment type, and modified Rankin score (mRS) at 90 days. Recanalization status was assessed on follow-up imaging. In a first multivariate regression analysis, we assessed whether volumes of PCT penumbra and infarct core could be predicted from clinical variables, NCT, or CTA, or whether they represented independent information. In a second multivariate regression analysis, we used mRS at 90 days as outcome and determined which variables predicted it best. Of 165 patients identified, 76 had a mRS score of 0 to 2 at 90 days, 89 had a mRS score >2. PCT infarct could be predicted by clinical data, NCT, CTA, and combinations of this data (Pstroke CT workup, including NCT and CTA.

  18. Modifying the Cold Gelation Properties of Quinoa Protein Isolate: Influence of Heat-Denaturation pH in the Alkaline Range.

    Mäkinen, Outi E; Zannini, Emanuele; Arendt, Elke K


    Heat-denaturation of quinoa protein isolate (QPI) at alkali pH and its influence on the physicochemical and cold gelation properties was investigated. Heating QPI at pH 8.5 led to increased surface hydrophobicity and decreases in free and bound sulfhydryl group contents. Heating at pH 10.5 caused a lesser degree of changes in sulfhydryl groups and surface hydrophobicity, and the resulting solutions showed drastically increased solubility. SDS PAGE revealed the presence of large aggregates only in the sample heated at pH 8.5, suggesting that any aggregates present in the sample heated at pH 10.5 were non-covalently bound and disintegrated in the presence of SDS. Reducing conditions partially dissolved the aggregates in the pH 8.5 heated sample indicating the occurrence of disulphide bonding, but caused no major alterations in the separation pattern of the pH 10.5 heated sample. Denaturation pH influenced the cold gelation properties greatly. Solutions heated at pH 8.5 formed a coarse coagulum with maximum G' of 5 Pa. Heat-denaturation at 10.5 enabled the proteins to form a finer and regularly structured gel with a maximum G' of 1140 Pa. Particle size analysis showed that the pH 10.5 heated sample contained a higher level of very small particles (0.1-2 μm), and these readily aggregated into large particles (30-200 μm) when pH was lowered to 5.5. Differences in the nature of aggregates formed during heating may explain the large variation in gelation properties.

  19. Determination of malic Acid using a malate dehydrogenase reactor after purification and immobilization in non-denaturing conditions and staining with ponceau S.

    Shimazaki, Youji; Sakikawa, Takahiro


    Mouse liver cytosolic malate dehydrogenase was separated by non-denaturing two-dimensional electrophoresis and identified. Furthermore, the activity of the enzyme was preserved even after separation, electroblotting onto a membrane and staining with Ponceau S in acidic buffer solution (pH 5.1). Using the membrane-immobilized enzyme, the malic acid content was estimated by measuring absorbance changes due to the conversion of nicotinamide adenine dinucleotide (NAD) to NADH. These results indicate that enzyme reactors can be systematically produced after purification, immobilization and staining with Ponceau S.

  20. Effect of ligand structure of stationary phase of high per-formance hydrophobic interaction chromatography on re-naturation efficiency of GuHCl-denatured α-chymotrypsin

    SHEN; Yehua; WANG; Haibo; BAI; Quan; GENG; Xindu


    The renaturation of the denatured α-chymotrypsin (α-Chy) with 1.7 mol · L-1 guanidine hydrochloride (GuHCI) by three kinds of stationary phase of high performance hydrophobic interaction chromatography (STHIC) with a comparable hydrophobicity but different ligand structures was investigated. The obtained result indicates that the ligand structures of the three STHIC contribute to the renaturation efficiency of α-Chy in the order of the end ligands PEG-600< phenyl group < tetrahydrofurfuryl alcohol (THFA).

  1. Purification of correctly oxidized MHC class I heavy-chain molecules under denaturing conditions: a novel strategy exploiting disulfide assisted protein folding

    Ferré, Henrik; Ruffet, Emmanuel; Blicher, Thomas


    conditions for disulfide bond formation ( approximately pH 8) and peptide binding ( approximately pH 6.6) are far from complementary. Here we present a two-step strategy, which relies on uncoupling the events of disulfide bond formation and peptide binding. In the first phase, heavy-chain molecules...... with correct disulfide bonding are formed under non-reducing denaturing conditions and separated from scrambled disulfide bond forms by hydrophobic interaction chromatography. In the second step, rapid refolding of the oxidized heavy chains is afforded by disulfide bond-assisted folding in the presence of beta...

  2. Essential roles of protein-solvent many-body correlation in solvent-entropy effect on protein folding and denaturation: comparison between hard-sphere solvent and water.

    Oshima, Hiraku; Kinoshita, Masahiro


    In earlier works, we showed that the entropic effect originating from the translational displacement of water molecules plays the pivotal role in protein folding and denaturation. The two different solvent models, hard-sphere solvent and model water, were employed in theoretical methods wherein the entropic effect was treated as an essential factor. However, there were similarities and differences in the results obtained from the two solvent models. In the present work, to unveil the physical origins of the similarities and differences, we simultaneously consider structural transition, cold denaturation, and pressure denaturation for the same protein by employing the two solvent models and considering three different thermodynamic states for each solvent model. The solvent-entropy change upon protein folding/unfolding is decomposed into the protein-solvent pair (PA) and many-body (MB) correlation components using the integral equation theories. Each component is further decomposed into the excluded-volume (EV) and solvent-accessible surface (SAS) terms by applying the morphometric approach. The four physically insightful constituents, (PA, EV), (PA, SAS), (MB, EV), and (MB, SAS), are thus obtained. Moreover, (MB, SAS) is discussed by dividing it into two factors. This all-inclusive investigation leads to the following results: (1) the protein-water many-body correlation always plays critical roles in a variety of folding/unfolding processes; (2) the hard-sphere solvent model fails when it does not correctly reproduce the protein-water many-body correlation; (3) the hard-sphere solvent model becomes problematic when the dependence of the many-body correlation on the solvent number density and temperature is essential: it is not quite suited to studies on cold and pressure denaturating of a protein; (4) when the temperature and solvent number density are limited to the ambient values, the hard-sphere solvent model is usually successful; and (5) even at the ambient

  3. Diagnostic work-up in cardiomyopathies

    Rapezzi, Claudio; Arbustini, Eloisa; Caforio, Alida L P


    In 2008, The ESC Working Group on Myocardial and Pericardial Diseases proposed an updated classification of cardiomyopathies based on morphological and functional phenotypes and subcategories of familial/genetic and non-familial/non-genetic disease. In this position statement, we propose a framew...

  4. Denaturing high-performance liquid chromatography to diagnose cerebral autosomal dominant arteriopathy in Chinese patients with subcortical infarcts and leukoencephalopathy

    Xiaomei Tang; Biao Chen


    BACKGROUND: Notch3 mutations are the molecular genetic foundation for cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Of all currently available detection methods, direct sequencing or restriction enzymes are frequently used, but the cost is relatively high, because the Notch3 gene is composed of many exons and mutational sites are widely distributed. Denaturing high-performance liquid chromatography (DHPLC) exhibits high efficiency and specificity and has been applied to gene detection. To date, there has no report regarding DHPLC in gene detection of large-scale CADASIL families in China. OBJECTIVE: To explore the application and value of DHPLC in the diagnosis of CADASIL by a mutation screening for Notch3 gene in CADASIL probands and their family members. DESIGN, TIME AND SETTING: A comparative observation was performed at the Genetic Diagnosis Laboratory of Institute of Geriatrics, Xuanwu Hospital of Capital Medical University and the Key Laboratory for Neurodegenerative Disease of the Ministry of Education between August 2003 and May 2004. PARTICIPANTS: Fourteen CADASIL patients and their family members, comprising eight males and six females, aged 38-62 years, were included. Their key features included recurrent sub-cortical ischemic events and vascular dementia. In addition, 100 healthy physical examinees were selected as controls, including 52 males and 48 females, aged 56-72 years, who had no neurodegenerative disease or psychosis, and no history or high risk for cerebrovascular disease. METHODS: DNA was extracted from white blood cells. Ten hotspots of the Notch3 gene for sequence variation were first amplified by PCR, and the products were detected using DHPLC. Exons exhibiting a variant in the DHPLC profile underwent another PCR amplification, followed by DNA sequencing to identify the mutation type. In addition, patients with normal DHPLC peak profiles underwent PCR amplification for the remaining

  5. Clinical utility of (18)F-FDG positron emission tomography/computed tomography scan vs. (99m)Tc-HMPAO white blood cell single-photon emission computed tomography in extra-cardiac work-up of infective endocarditis.

    Lauridsen, Trine K; Iversen, Kasper K; Ihlemann, Nikolaj; Hasbak, Philip; Loft, Annika; Berthelsen, Anne K; Dahl, Anders; Dejanovic, Danijela; Albrecht-Beste, Elisabeth; Mortensen, Jann; Kjær, Andreas; Bundgaard, Henning; Bruun, Niels Eske


    The extra-cardiac work-up in infective endocarditis (IE) comprises a search for primary and secondary infective foci. Whether (18)FDG-PET/CT or WBC-SPECT/CT is superior in detection of clinically relevant extra-cardiac manifestations in IE is unexplored. The objectives of this study were to identify the numbers of positive findings detected by each imaging modality, to evaluate the clinical relevance of these findings and to define the reproducibility for extra-cardiac foci in patients with definite IE. Each modality was evaluated for numbers and location of positive extra-cardiac foci in patients with definite IE. A team of 2 × 2 cardiologists evaluated each finding to determine clinical relevance. Clinical utility was determined by 4 criteria converted into an ordinal scale. Using the manifestation with highest clinical utility rating in each patient, the clinical impact of the two imaging modalities was expressed in a clinical utility score. To evaluate reproducibility for each modality, an imaging core laboratory reviewed all findings. In 55 IE patients, 91 pathological foci were found by FDG-PET/CT and 37 foci were identified by WBC-SPECT/CT (p < 0.001). The clinical utility of FDG-PET/CT was significantly higher than that of WBC-SPECT/CT when comparing clinical utility score (2.06 vs. 1.17; p = 0.01). In assessment of extra-cardiac diagnostics in IE, inter-observer reproducibility was substantial for WBC-SPECT/CT (k 0.69, 95% CI 0.49-0.89) and substantial to excellent for FDG-PET/CT (k 0.79, 95% CI 0.61-0.98). FDG-PET/CT has a significantly higher clinical utility score than WBC SPECT/CT and is potentially superior to WBC-SPECT/CT in detection of extra-cardiac pathology in patients with IE.

  6. Role of Combined 68Ga-DOTATOC and 18F-FDG Positron Emission Tomography/Computed Tomography in the Diagnostic Workup of Pancreas Neuroendocrine Tumors: Implications for Managing Surgical Decisions.

    Cingarlini, Sara; Ortolani, Silvia; Salgarello, Matteo; Butturini, Giovanni; Malpaga, Anna; Malfatti, Veronica; DʼOnofrio, Mirko; Davì, Maria Vittoria; Vallerio, Paola; Ruzzenente, Andrea; Capelli, Paola; Citton, Elia; Grego, Elisabetta; Trentin, Chiara; De Robertis, Riccardo; Scarpa, Aldo; Bassi, Claudio; Tortora, Giampaolo


    Ga-DOTATOC (Ga) positron emission tomography (PET)/computed tomography (CT) is recommended in the workup of pancreas neuroendocrine tumors (PanNETs); evidence suggests that F-FDG (F) PET/CT can also provide prognostic information. Aims of this study were to assess the role of combined Ga- and F-PET/CT in the evaluation of grade (G) 1-2 PanNETs and to test the correlation between F-PET/CT positivity and tumor grade. Preoperative Ga- and F-PET/CT of 35 patients with surgically resected G1-2 PanNETs were evaluated. For grading, the 2010 World Health Organization Classification was used; an ancillary analysis with Ki67 cutoffs at 5% to 20% was conducted. Correlation between F-PET/CT positivity (SUVmax > 3.5) and grade was assessed. Of 35 PanNETs, 28.6% and 71.4% were G1 and G2 as per World Health Organization. Ga-PET/CT showed high sensitivity (94.3%) in detecting G1-2 PanNETs. F-PET/CT was positive in 20% and 76% G1 and G2 tumors (P = 0.002). F-PET/CT identified G2 PanNETs with high positive predictive value (PPV, 90.5%). F-PET/CT correlated with tumor grade also in the ancillary analysis (P = 0.009). The high sensitivity of Ga-PET/CT in NET detection is known. The high PPV of F-PET/CT in the identification of G2 forms suggests its potential role in PanNETs prognostication and risk stratification.

  7. Assessment of the yeast species composition of cocoa bean fermentations in different cocoa-producing regions using denaturing gradient gel electrophoresis.

    Papalexandratou, Zoi; De Vuyst, Luc


    The yeast species composition of 12 cocoa bean fermentations carried out in Brazil, Ecuador, Ivory Coast and Malaysia was investigated culture-independently. Denaturing gradient gel electrophoresis of 26S rRNA gene fragments, obtained through polymerase chain reaction with universal eukaryotic primers, was carried out with two different commercial apparatus (the DCode and CBS systems). In general, this molecular method allowed a rapid monitoring of the yeast species prevailing during fermentation. Under similar and optimal denaturing gradient gel electrophoresis conditions, the CBS system allowed a better separated band pattern than the DCode system and an unambiguous detection of the prevailing species present in the fermentation samples. The most frequent yeast species were Hanseniaspora sp., followed by Pichia kudriavzevii and Saccharomyces cerevisiae, independent of the origin of the cocoa. This indicates a restricted yeast species composition of the cocoa bean fermentation process. Exceptionally, the Ivorian cocoa bean box fermentation samples showed a wider yeast species composition, with Hyphopichia burtonii and Meyerozyma caribbica among the main representatives. Yeasts were not detected in the samples when the temperature inside the fermenting cocoa pulp-bean mass reached values higher than 45 °C or under early acetic acid production conditions.

  8. Analysis of the insulin receptor gene in noninsulin-dependent diabetes mellitus by denaturing gradient gel blots: A clinical research center study

    Magre, J.; Goldfine, A.B.; Warram, J.H. [Harvard Medical School, Boston, MA (United States)] [and others


    We have used a new technique of denaturing gradient gel blotting to determine the prevalence of alterations in the intracellular domain of the insulin receptor in normal individuals and subjects with non-insulin-dependent diabetes mellitus (NIDDM). This method detects DNA sequence differences as restriction fragment melting polymorphisms (RFMP) and is sensitive to changes in sequence at both restriction sites and within the fragments themselves. Using restriction digests with AluI, HaeIII, HinfI, RsaI, Sau3A, and Sau96, 12 RFMPs were found to localize to the region of the {beta}-subunit of the insulin receptor gene. Using exon-specific probes, these RFMPs could be localized to specific regions surrounding individual exons, including exons, 14, 15, 16, 18, 20, and 22. In general, linkage disequilibrium between polymorphisms was inversely related to their distance in the gene structure, although there was a {open_quotes}hot spot{close_quotes} for recombination between exons 19 and 20. No difference in melting temperatures or allele frequency was observed between NIDDM patients and controls. These data indicate that the region of the insulin receptor gene coding for the intracellular portion of the {beta}-subunit is highly polymorphic and that polymorphisms surrounding specific exons can be identified by denaturing gradient gel blotting, but there is no evidence that variation at this locus contributes to NIDDM susceptibility in most individuals. 36 refs., 3 figs., 3 tabs.

  9. Preparation of Fat Substitute using Enzymatic Denatured Potato Starch%酶解马铃薯淀粉制备脂肪模拟品的研究

    丛美娟; 郭华


    利用马铃薯淀粉制备酶变性淀粉,在单因素试验的基础上选择了淀粉浆浓度、酶解温度、酶解时间进行三因素三水平的正交试验,确定了较优的淀粉酶解工艺条件,使酶解淀粉的DE值稍大于2.再确定酶变性淀粉的糊化温度和糊化时间,制备用来模拟油脂的变性淀粉,以减少食用者的热量摄入,更加有利于人体健康.%Potato starch was used to prepare enzymatic denatured starch. On the basis of single factor experiments, three factors( chose concentration of starch slurry, enzymatic hydrolysis temperature and enzyme hydrolysis time) were chosen for orthogonal experiment to determine the optimum process conditions of amylase solution to keep DE value being of higher than 2. And then the pasting temperature and pasting time of enzymatic denatured starch were determined. Using enzymatic modified starch to simulate the oil in food can reduce the intake of calories and was more benefit health.

  10. Polymeric complements to the Alzheimer's disease biomarker β-amyloid isoforms Aβ1-40 and Aβ1-42 for blood serum analysis under denaturing conditions.

    Urraca, Javier L; Aureliano, Carla S A; Schillinger, Eric; Esselmann, Hermann; Wiltfang, Jens; Sellergren, Börje


    Treatment of Alzheimer's disease (AD) is plagued by a lack of practical and reliable methods allowing early diagnosis of the disease. We here demonstrate that robust receptors prepared by molecular imprinting successfully address current limitations of biologically derived receptors in displaying affinity for hydrophobic peptide biomarkers for AD under denaturing conditions. C-terminal epitope-imprinted polymers showing enhanced binding affinity for Aβ1-42 were first identified from a 96-polymer combinatorial library. This information was then used to synthesize molecularly imprinted polymers for both of the β-amyloid (Aβ) isoforms and a corresponding nonimprinted polymer. A solid-phase extraction method was developed to be compatible with sample loading under conditions of complete protein denaturation. This resulted in a method capable of quantitatively and selectively enriching a shorter C-terminal peptide corresponding to the sequences Aβ33-40 and Aβ33-42 as well as the full-length sequence Aβ1-40 and Aβ1-42 from a 4 M guanidinum chloride solution. Application of the method to serum allowed selective, high-recovery extraction of both biomarkers at spiking levels marginally higher than clinically relevant concentrations found in cerebrospinal fluid.

  11. Temperature-mediated heteroduplex analysis for the detection of drug-resistant gene mutations in clinical isolates of Mycobacterium tuberculosis by denaturing HPLC, SURVEYOR nuclease.

    Shi, Ruiru; Otomo, Koji; Yamada, Hiroyuki; Tatsumi, Taiga; Sugawara, Isamu


    Denaturing high-performance liquid chromatography (DHPLC) is a relatively new technique, which utilizes heteroduplex formation between wild-type and mutated DNA strands to identify point mutations. Heteroduplex molecules are separated from homoduplex molecules by ion-pair, reverse-phase liquid chromatography on a special column matrix with partial heat denaturation of the DNA strands. In order to investigate the application of this method for point mutation detection in drug-resistant genes of Mycobacterium tuberculosis, katG, rpoB, embB, gyrA, pncA and rpsL genes, which are responsible for isoniazid, rifampicin, ethambutol, fluoroquinolone, pyrazinamide and streptomycin resistance, respectively, were detected by temperature-mediated DHPLC in 10 multidrug-resistant and 10 drug-susceptible clinical isolates. The DHPLC data were compared with those from a conventional MIC test. The results show that DHPLC is cost-effective with high capacity and accuracy, and is potentially useful for genotypic screening for mutations associated with anti-tuberculosis drug resistance.

  12. Preoperative workup to assess indication for laparoscopic treatment in gastroesophageal reflux disease Aseguramiento preoperatorio en la indicación de tratamiento laparoscópico en la enfermedad por reflujo gastroesofágico

    S. Pérez-Holanda


    Full Text Available Introduction and objectives: antireflux surgery performed by an experienced surgeon is a maintenance option for patients with well-documented gastroesophageal reflux disease (GERD. Well-documented GERD is difficult to find, as GERD is a multifactorial disease in which the gastroesophageal junction, with its special anatomical and functional components, is important. In order to examine patient preoperative workups, and their indication for surgical treatment in GERD, we retrospectively studied patients who underwent a laparoscopic antireflux procedure. Methods: preoperative workups in patients from our health care area who underwent a laparoscopic antireflux procedure from December 1997 to February 2007 were retrospectively analyzed. Data related to epidemiological findings, symptoms, morphologic and functional evaluation, medical therapy, and indication for surgical treatment were recorded and statistically analyzed by means of a bivariate test. Differences were significant when the p value was equal to or less than 0.05. Results: 100 patients (50 % female, 51.31 ± 13.53 years of age underwent a laparoscopic antireflux surgery after 56.47 ± 61.33 months with symptoms. Ninety-five percent of patients had an anatomical abnormality. The pH monitoring test diagnosed three quarters of cases. The most frequent indication for GERD treatment was persistent or recurrent esophagitis despite adequate medical treatment (52 cases. Conclusions: based on our preoperative workup, as described, 100 percent of subjects were well documented and diagnosed with GERD (both non-erosive reflux disease and erosive reflux disease, and their indication for laparoscopic treatment was retrospectively assessed in 94% of cases.Introducción y objetivos: la cirugía antirreflujo realizada por un cirujano experto es una opción para el tratamiento de mantenimiento para el paciente con enfermedad por reflujo gastroesofágico (ERGE bien documentada. La "buena documentaci

  13. Coamplification at lower denaturation temperature polymerase chain reaction enables selective identification of K-Ras mutations in formalin-fixed, paraffin-embedded tumor tissues without tumor-cell enrichment.

    Yu, Shaorong; Xie, Li; Hou, Zhibo; Qian, Xiaoping; Yu, Lixia; Wei, Jia; Ding, Yitao; Liu, Baorui


    Conventional polymerase chain reaction-based Sanger sequencing is the standard assay for the detection of K-Ras mutations. However, this method is deficient in identifying small numbers of mutation-bearing cells, and tumor-cell enrichment methods such as microdissection or macrodissection are labor intensive and not always achievable. We applied the recently described coamplification at lower denaturation temperature polymerase chain reaction, which amplifies minority alleles selectively, to detect K-Ras mutations directly in 29 formalin-fixed, paraffin-embedded pancreatic specimens and compared the results with those of conventional polymerase chain reaction. To avoid a false-negative result from the coamplification at lower denaturation temperature polymerase chain reaction assay, we applied a more sensitive peptide nucleic acid polymerase chain reaction method as the gold standard. Dilution experiments indicated an approximately 5-fold improvement in sensitivity with coamplification at lower denaturation temperature polymerase chain reaction-based Sanger sequencing. Conventional polymerase chain reaction detected K-Ras mutations in 11 formalin-fixed, paraffin-embedded pancreatic specimens (37.9%), whereas coamplification at lower denaturation temperature polymerase chain reaction could identify all of those mutations as well as mutations in 10 additional samples, for a total of 21 (72.4%, P = .002) of 29. Unlike peptide nucleic acid polymerase chain reaction, coamplification at lower denaturation temperature polymerase chain reaction identified all K-Ras mutations in specimens in which tumor cells accounted for at least 20% of the total. Adoption of coamplification at lower denaturation temperature polymerase chain reaction is straightforward and requires no additional reagents or instruments. The technique is a good strategy to detect K-Ras mutations selectively in formalin-fixed, paraffin-embedded tissues without tumor-cell enrichment.

  14. Retardation of Protein Dynamics by Trehalose in Dehydrated Systems of Photosynthetic Reaction Centers. Insights from Electron Transfer and Thermal Denaturation Kinetics.

    Malferrari, Marco; Francia, Francesco; Venturoli, Giovanni


    Conformational protein dynamics is known to be hampered in amorphous matrixes upon dehydration, both in the absence and in the presence of glass forming disaccharides, like trehalose, resulting in enhanced protein thermal stability. To shed light on such matrix effects, we have compared the retardation of protein dynamics in photosynthetic bacterial reaction centers (RC) dehydrated at controlled relative humidity in the absence (RC films) or in the presence of trehalose (RC-trehalose glasses). Small scale RC dynamics, associated with the relaxation from the dark-adapted to the light-adapted conformation, have been probed up to the second time scale by analyzing the kinetics of electron transfer from the photoreduced quinone acceptor (QA(-)) to the photoxidized primary donor (P(+)) as a function of the duration of photoexcitation from 7 ns (laser pulse) to 20 s. A more severe inhibition of dynamics is found in RC-trehalose glasses than in RC films: only in the latter system does a complete relaxation to the light-adapted conformation occur even at extreme dehydration, although strongly retarded. To gain insight into the large scale RC dynamics up to the time scale of days, the kinetics of thermal denaturation have been studied at 44 °C by spectral analysis of the Qx and Qy bands of the RC bacteriochlorin cofactors, as a function of the sugar/protein molar ratio, m, varied between 0 and 10(4). Upon increasing m, denaturation is slowed progressively, and above m ∼ 500 the RC is stable at least for several days. The stronger retardation of RC relaxation and dynamics induced by trehalose is discussed in the light of a recent molecular dynamics simulation study performed in matrixes of the model protein lysozyme with and without trehalose. We suggest that the efficiency of trehalose in retarding RC dynamics and preventing thermal denaturation stems mainly from its propensity to form and stabilize extended networks of hydrogen bonds involving sugar, residual water, and

  15. Characterization of bacterial populations in Danish raw milk cheeses made with different starter cultures by denaturating gradient gel electrophoresis and pyrosequencing

    Masoud, Wafa Mahmoud Hasan; Takamiya, Monica K Wik; Vogensen, Finn Kvist;


    The bacterial populations in Danish raw milk cheeses were identified using denaturating gradient gel electrophoresis (DGGE) of PCR amplicons of the V3 region of the 16S rRNA gene and pyrosequencing of tagged amplicons of the V3 and V4 regions of the 16S rRNA gene. Both DNA and RNA extracted from...... cheeses were studied in order to determine the metabolically active bacteria. The main bacteria, which included Lactococcus, Lactobacillus and Streptococcus, were detected by pyrosequencing and DGGE in both 16S rDNA and cDNA obtained from cheeses indicating their viability and contribution to cheese...... ripening. Other bacteria like Corynebacterium, Halomonas, Pediococcus, Micrococcus and Staphylococcus, which were encountered in some cheese samples at low percentages compared with the total bacterial populations, were only detected by pyrosequencing. 16S rRNA gene pyrosequencing is an efficient method...

  16. Direct detection and identification of lactic acid bacteria in a food processing plant and in meat products using denaturing gradient gel electrophoresis.

    Takahashi, Hajime; Kimura, Bon; Yoshikawa, Miwako; Gotou, Seitaro; Watanabe, Itaru; Fujii, Tateo


    We established a novel system using denaturing gradient gel electrophoresis (DGGE) to quickly identify bacteria known to be responsible for spoilage in meat processing plants and meat products. We extracted bacterial DNA from swabbed samples at various locations in the plant and from meat products and performed PCR amplification, targeting 16S rDNA from the dominant organisms. The amplification products were subjected to DGGE, and the contaminating bacteria in the meat products and the plant were analyzed. This analysis indicated that lactic acid bacteria and spoilage-causing bacteria are widely distributed within the meat processing plant. We developed molecular size markers to identify the dominant organisms obtained from the plant and meat products. The establishment of the present method allows quick and simple identification of bacteria causing the possible deterioration of products and contamination and thus permits constant monitoring of any harmful bacteria within meat processing plants.

  17. High pressure inactivation of relevant target microorganisms in poultry meat products and the evaluation of pressure-induced protein denaturation of marinated poultry under different high pressure treatments

    Schmidgall, Johanna; Hertel, Christian; Bindrich, Ute; Heinz, Volker; Toepfl, Stefan


    In this study, the possibility of extending shelf life of marinated poultry meat products by high pressure processing was evaluated. Relevant spoilage and pathogenic strains were selected and used as target microorganisms (MOs) for challenge experiments. Meat and brine were inoculated with MOs and treated at 450 MPa, 4 °C for 3 min. The results of inactivation show a decreasing pressure tolerance in the series Lactobacillus > Arcobacter > Carnobacterium > Bacillus cereus > Brochothrix thermosphacta > Listeria monocytogenes. Leuconostoc gelidum exhibited the highest pressure tolerance in meat. A protective effect of poultry meat was found for L. sakei and L. gelidum. In parallel, the influence of different marinade formulations (pH, carbonates, citrates) on protein structure changes during a pressure treatment was investigated. Addition of sodium carbonate shows a protection against denaturation of myofibrillar proteins and provides a maximum water-holding capacity. Caustic marinades allowed a higher retention of product characteristics than low-pH marinades.

  18. Conformity assessment of multicomponent materials or objects: Risk of false decisions due to measurement uncertainty - A case study of denatured alcohols.

    Kuselman, Ilya; Pennecchi, Francesca; da Silva, Ricardo J N B; Brynn Hibbert, D


    Risk of a false decision on conformity of a multicomponent material or object due to measurement uncertainty is discussed. Even if conformity assessment for each component of a material sample is successful, the total probability of a false decision (total consumer's risk or producer's risk) concerning the sample as a whole might still be significant. A model of the total probability of such false decisions is formulated based on the law (theorem) of total probability. It is shown that the total risk can be evaluated as a combination of the particular risks of conformity assessment of sample components. For a more complicated task, i.e. for a larger number of components of a sample under control, the total risk is greater. As a case study, the total probability of false conforming (total consumer's risk) is evaluated for customs control of completely denatured alcohols, where conformity assessment is performed by comparison of chemical analytical test results with the regulatory limits.

  19. Analysis of phosphate-accumulating organisms cultivated under different carbon sources with polymerase chain reaction-denaturing gradient gel electrophoresis assay

    YU Shui-li; LIU Ya-nan; JING Guo-lin; ZHAO Bing-jie; GUO Si-yuan


    To investigate the microbial communities of microorganisms cultivated under different carbon sources, three sequencing batch reactors were operated. They were supplied with sewage, glucose and sodium acetate as carbon sources respectively and showed high phosphorus removal performance. The results of denaturing gradient gel electrophoresis(DGGE) of polymerase chain reaction-amplified (PCR) 16S rDNA fragments demonstrated that β-protebacteria, Actinomyces sp. and γ-protebacteria only exited in 1 # reactor. The microbiological diversity of 1 # reactor exceeded the other two reactors. Flavobacterium, Bacillales, Actinomyces, Actinobacteridae and uncultured bacteria(AF527584, AF502204, AY592749, AB076862, AJ619051, AF495454 and AY133070) could be detected in the biological phosphorus removal reactors.

  20. On scaling laws at the phase transition of systems with divergent order parameter and/or internal length the example of DNA denaturation

    Buyukdagli, S; Buyukdagli, Sahin; Joyeux, Marc


    We used the Transfer-Integral method to compute, with an uncertainty smaller than 5%, the six fundamental characteristic exponents of two dynamical models for DNA thermal denaturation and investigate the validity of the scaling laws. Doubts concerning this point arise because the investigated systems (i) have a divergent internal length, (ii) are described by a divergent order parameter, (iii) are of dimension 1. We found that the assumption that the free energy can be described by a single homogeneous function is robust, despite the divergence of the order parameter, so that Rushbrooke's and Widom's identities are valid relations. Josephson's identity is instead not satisfied. This is probably due to the divergence of the internal length, which invalidates the assumption that the correlation length is solely responsible for singular contributions to thermodynamic quantities. Fisher's identity is even wronger. We showed that this is due to the d=1 dimensionality and obtained an alternative law, which is well ...

  1. In vitro xanthine oxidase and albumin denaturation inhibition assay of Barringtonia racemosa L. and total phenolic content analysis for potential anti-inflammatory use in gouty arthritis

    Osman, Nurul Izzati; Sidik, Norrizah Jaafar; Awal, Asmah; Adam, Nurul Athirah Mohamad; Rezali, Nur Inani


    Aim: This study was conducted to evaluate the in vitro anti-inflammatory activities and total phenolic content (TPC) of methanolic extracts of infloresence axes, endosperms, leaves, and pericarps of Barringtonia racemosa L. Methods: The anti-inflammatory study was conducted by assessing the potential through xanthine oxidase (XO) and albumin denaturation inhibition assays. Meanwhile, the TPC in the extracts were assessed by Folin-Ciocalteu assay. Results: In the XO inhibition assay, the infloresence axes extract was found to exert the highest inhibition capacity at 0.1% (w/v) with 59.54 ± 0.001% inhibition followed by leaves (58.82 ± 0.001%), pericarps (57.99 ± 0.003%), and endosperms (57.20 ± 0.003%) extracts. Similarly in the albumin denaturation inhibition assay, the infloresence axes extract had shown the greatest inhibition capacity with 70.58 ± 0.004% inhibition followed by endosperms (66.80 ± 0.024%), leaves (65.29 ± 0.006%), and pericarps extracts (43.33 ± 0.002%). Meanwhile, for TPC analysis, leaves extract was found to have the highest phenolic content (53.94 ± 0.000 mg gallic acid equivalent [GAE]/g DW) followed by infloresence axes (31.54 ± 0.001 mg GAE/g DW), endosperms (22.63 ± 0.001 mg GAE/g DW), and the least was found in pericarps (15.54 ± 0.001 mg GAE/g DW). Conclusion: The results indeed verified the in vitro anti-inflammatory activities of B. racemosa and supported its potential to be used in alleviating gouty arthritis and XO-related diseases. PMID:27757263

  2. A Simple Test Method of Whey Protein Denaturation and its Application%快速测定乳清蛋白变性程度方法的应用

    曲冬梅; 许明向; 冯玉红; 姜胡兵


    采用一种较为简单的方法测定牛奶中乳清蛋白变性程度,探讨了不同的预热处理条件和UHT杀菌条件对乳清蛋白变性程度的影响,并测试了市售的全脂奶粉的乳清蛋白变性情况。同时,对比了不同热处理程度的奶粉在经过一定处理后罐装杀菌后的状态。结果表明,牛奶经过低热处理后,乳清蛋白变性程度只有0.15%;经过90℃/30s,110℃/15s,124℃/15s和135℃/15s等模拟鲜奶加工成奶粉过程中不同程度预热处理后,变性程度分别是16%,62%,75%和80%;鲜奶经过中性乳制品常用的不同UHT杀菌条件处理后,乳清蛋白变性程度基本在70%~80%之间。从检测情况来看,市售全脂奶粉一般为高热奶粉。乳清蛋白变性程度对奶粉的应用会有一定程度的影响。%A simple method for testing the denaturation level of whey proteinswas used to investigate the influence of preheat treatment and UHT treatment on the denaturation of whey protein. The denaturation of whey proteins in bovine milk after heat treatment and whey proteins in whole milk powder was measured using this method. The results showed that the whey proteins denatured about 16%,62%,75%,and 80% after heating at 90℃ for 30s,110℃ for 15s,124℃ for 15s,and 130℃for 15s respectively. The denaturation of whey proteins was between 70%and 80% after UHT treatment. In sum,intensive heat treatment has influence on the denaturation of whey proteins. This also indicates that the denaturation level of whey proteins may influence the application of whole milk powder as whole milk powder is normally produced with intensive heat treatment.

  3. Denaturing gradient electrophoresis (DGE) and single-strand conformation polymorphism (SSCP) molecular fingerprintings revisited by simulation and used as a tool to measure microbial diversity.

    Loisel, Patrice; Harmand, Jérôme; Zemb, Olivier; Latrille, Eric; Lobry, Claude; Delgenès, Jean-Philippe; Godon, Jean-Jacques


    The exact extent of microbial diversity remains unknowable. Nevertheless, fingerprinting patterns [denaturing gradient electrophoresis (DGE), single-strand conformation polymorphism (SSCP)] provide an image of a microbial ecosystem and contain diversity data. We generated numerical simulation fingerprinting patterns based on three types of distribution (uniform, geometric and lognormal) with a range of units from 10 to 500,000. First, simulated patterns containing a diversity of around 1000 units or more gave patterns similar to those obtained in experiments. Second, the number of bands or peaks saturated quickly to about 35 and were unrelated to the degree of diversity. Finally, assuming lognormal distribution, we used an estimator of diversity on in silico and experimental fingerprinting patterns. Results on in silico patterns corresponded to the simulation inputs. Diversity results in experimental patterns were in the same range as those obtained from the same DNA sample in molecular inventories. Thus, fingerprinting patterns contain extractable data about diversity although not on the basis of a number of bands or peaks, as is generally assumed to be the case.

  4. Combination of multiplex PCR with denaturing high-performance liquid chromatography for rapid detection of Mycobacterium genus and simultaneous identification of the Mycobacterium tuberculosis complex.

    Chen, Ru; Gao, Xiao-Bo; Liu, Zhi-Hui; Shen, Xiao-Bing; Guo, Ai-Zhen; Duan, Yan-Yu; Liu, Zhi-Ling; Wu, Xiao-Wei; Zhu, Dao-Zhong


    A new assay with the combination of multiplex polymerase chain reaction and denaturing high-performance liquid chromatography analysis was developed for simultaneous detection of Mycobacterium genus and identification of the Mycobacterium tuberculosis complex (MTC). Targeting at genus-specific 16S rRNA sequence of Mycobacterium and specific insertion elements IS6110 and IS1081 of MTC, the assay was validated with 84 strains covering 23 mycobacteria species and 30 strains of non-mycobacteria species. No cross reactivity was observed. Clinical application was carried out on 198 specimens (155 human sputum and 43 bovine tissue samples) and compared with culture. The multiplex assay detected all culture-positive (36 in number) and 35.2% (57/162) culture-negative specimens. The molecular assay was fast that could be completed within 1 h on purified DNA, with the limit of detection as 0.8-1.6 pg per reaction on DNA template. This work provided a useful laboratory tool for rapid identification of Mycobacterium and differentiation of MTC and nontuberculous mycobacteria.

  5. Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

    Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Said; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.


    A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

  6. PCR-denaturing gradient gel electrophoresis analysis of microbial community in soy-daddawa, a Nigerian fermented soybean (Glycine max (L.) Merr.) condiment.

    Ezeokoli, Obinna T; Gupta, Arvind K; Mienie, Charlotte; Popoola, Temitope O S; Bezuidenhout, Cornelius C


    Soy-daddawa, a fermented soybean (Glycine max (L.) Merr.) condiment, plays a significant role in the culinary practice of West Africa. It is essential to understand the microbial community of soy-daddawa for a successful starter culture application. This study investigated the microbial community structure of soy-daddawa samples collected from Nigerian markets, by PCR-denaturing gradient gel electrophoresis (DGGE) targeting the V3-V5 region of the 16S rRNA gene of bacteria and internal transcribed spacer 2 (ITS2) region of fungi. Six bacterial and 16 fungal (nine yeasts and seven molds) operational taxonomic units (OTUs)/species were obtained at 97% sequence similarity. Taxonomic assignments revealed that bacterial OTUs belonged to the phyla Firmicutes and Actinobacteria, and included species from the genera Atopostipes, Bacillus, Brevibacterium and Nosocomiicoccus. Densitometric analysis of DGGE image/bands revealed that Bacillus spp. were the dominant OTU/species in terms of population numbers. Fungal OTUs belonged to the phyla Ascomycota and Zygomycota, and included species from the genera, Alternaria, Aspergillus, Candida, Cladosporium, Dokmaia, Issatchenkia, Kodamaea, Lecythophora, Phoma, Pichia, Rhizopus, Saccharomyces and Starmerella. The majority of fungal species have not been previously reported in soy-daddawa. Potential opportunistic human pathogens such as Atopostipes suicloacalis, Candida rugosa, Candida tropicalis, and Kodamaea ohmeri were detected. Variation in soy-daddawa microbial communities amongst samples and presence of potential opportunistic pathogens emphasises the need for starter culture employment and good handling practices in soy-daddawa processing.

  7. Denaturing gradient gel electrophoresis fingerprinting of soil bacteria in the vicinity of the Chinese Great Wall Station, King George Island, Antarctica.

    Pan, Qi; Wang, Feng; Zhang, Yang; Cai, Minghong; He, Jianfeng; Yang, Haizhen


    Bacterial diversity was investigated in soil samples collected from 13 sites around the Great Wall Station, Fildes Peninsula, King George Island, Antarctica, using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes. The classes alpha-, beta-, and gamma-Proteobacteria, as well as the phylum Actinobacteria, were found to be the dominant bacteria in the soils around the Great Wall Station. Although the selected samples were not contaminated by oil, a relationship between soil parameters, microbial biodiversity, and human impact was still seen. Sample sites in human impacted areas showed lower bacterial biodiversity (average H' = 2.65) when compared to non-impacted sites (average H' = 3.05). There was no statistically significant correlation between soil bacterial diversity and total organic carbon (TOC), total nitrogen, or total phosphorus contents of the soil. Canonical correlation analysis showed that TOC content was the most important factor determining bacterial community profiles among the measured soil parameters. In conclusion, microbial biodiversity and community characteristics within relatively small scales (1.5 km) were determined as a function of local environment parameters and anthropogenic impact.

  8. Denaturing gradient gel electrophoresis fingerprinting of soil bacteria in the vicinity of the Chinese Great Wall Station, King George Island, Antarctica

    Qi Pan; Feng Wang; Yang Zhang; Minghong Cai; Jianfeng He; Haizhen Yang


    Bacterial diversity was investigated in soil samples collected from 13 sites around the Great Wall Station,Fildes Peninsula,King George Island,Antarctica,using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes.The classes α-,β-,and γ-Proteobacteria,as well as the phylum Actinobacteria,were found to be the dominant bacteria in the soils around the Great Wall Station.Although the selected samples were not contaminated by oil,a relationship between soil parameters,microbial biodiversity,and human impact was still seen.Sample sites in human impacted areas showed lower bacterial biodiversity (average H' =2.65) when compared to non-impacted sites (average H' =3.05).There was no statistically significant correlation between soil bacterial diversity and total organic carbon (TOC),total nitrogen,or total phosphorus contents of the soil.Canonical correlation analysis showed that TOC content was the most important factor determining bacterial community profiles among the measured soil parameters.In conclusion,microbial biodiversity and community characteristics within relatively small scales (1.5 km) were determined as a function of local environment parameters and anthropogenic impact.

  9. Efficient detection of factor IX mutations by denaturing high-performance liquid chromatography in Taiwanese hemophilia B patients, and the identification of two novel mutations

    Pei-Chin Lin


    Full Text Available Hemophilia B (HB is an X-linked recessive disorder characterized by mutations in the clotting factor IX (FIX gene that result in FIX deficiency. Previous studies have shown a wide variation of FIX gene mutations in HB. Although the quality of life in HB has greatly improved mainly because of prophylactic replacement therapy with FIX concentrates, there exists a significant burden on affected families and the medical care system. Accurate detection of FIX gene mutations is critical for genetic counseling and disease prevention in HB. In this study, we used denaturing high-performance liquid chromatography (DHPLC, which has proved to be a highly informative and practical means of detecting mutations, for the molecular diagnosis of our patients with HB. Ten Taiwanese families affected by HB were enrolled. We used the DHPLC technique followed by direct sequencing of suspected segments to detect FIX gene mutations. In all, 11 FIX gene mutations (8 point mutations, 2 small deletions/insertions, and 1 large deletion, including two novel mutations (exon6 c.687–695, del 9 mer and c.460–461, ins T were found. According to the HB pedigrees, 25% and 75% of our patients were defined as familial and sporadic HB cases, respectively. We show that DHPLC is a highly sensitive and cost-effective method for FIX gene analysis and can be used as a convenient system for disease prevention.

  10. Comparison of Fecal Methanogenic Archaeal Community Between Erhualian and Landrace Pigs Using Denaturing Gradient Gel Electrophoresis and Real-Time PCR Analysis

    SU Yong; Hauke Smidt; ZHU Wei-Yun


    Erhualian and Landrace breeds are typical genetically obese and lean pigs, respectively. To compare the fecal methanogenic Archaeal community between these two pig breeds, fecal samples from different growth phase pigs were collected and used for PCR-denaturing gradient gel electrophoresis (DGGE) with two primer pairs (344fGC/519r and 519f/915rGC) and real-time PCR analysis. Results showed that a better separation and higher quality of bands pattern were obtained in DGGE proifles using primers 344fGC/519r as compared with primers 519f/915rGC. Sequencing of DGGE bands showed that the predominant methanogens in the feces of Erhualian and Landrace pigs belonged to Methanobrevibacter spp. and Methanosphaera spp. Real-time PCR analysis revealed that there was no signiifcant difference in the numbers of fecal total methanogens between Erhualian and Landrace pigs;however, pig growth phase affected the numbers of 16S rRNA genes of total methanogens and Methanobrevibacter smithii. Dissociation curves of methyl coenzyme-M reductase subunit A (mcrA) gene fragments ampliifed with real-time PCR showed all samples possessed a single peak at 82°C, which might be associated with M. smithii. Samples from the same growth phase of each breed showed good replicative dissociation curves. The results suggest that the growth phase (including diet factor) other than genotype of pig may affect the fecal methanogenic Archaeal community of pigs.

  11. Evidence of alpha fluctuations in myoglobin's denaturation in the high temperature region: Average relaxation time from an Adam-Gibbs perspective.

    Olivares-Quiroz, Luis; Garcia-Colin, Leopoldo S


    In this work, we derive an analytical expression for the relaxation time tau as a function of temperature T for myoglobin protein (Mb, PDB:1MBN) in the high temperature limit (T>T(g)=200K). The method is based on a modified version of the Adam-Gibbs theory (AG theory) for the glass transition in supercooled liquids and an implementation of differential geometry techniques. This modified version of the AG theory takes into account that the entropic component in protein's denaturation has two major sources: a configurational contribution DeltaS(c) due to the unfolding of the highly ordered native state N and a hydration contribution DeltaS(hyd) arising from the exposure of non-polar residues to direct contact with solvent polar molecules. Our results show that the configurational contribution DeltaS(c) is temperature-independent and one order of magnitude smaller than its hydration counterpart DeltaS(hyd) in the temperature range considered. The profile obtained for log tau(T) from T=200 K to T=300 K exhibits a non-Arrhenius behavior characteristic of alpha relaxation mechanisms in hydrated proteins and glassy systems. This result is in agreement with recent dielectric spectroscopy data obtained for hydrated myoglobin, where at least two fast relaxation processes in the high temperature limit have been observed. The connection between the relaxation process calculated here and the experimental results is outlined.

  12. Is dynamic heterogeneity of water in presence of a protein denaturing agent different from that in presence of a protein stabilizer? A molecular dynamics simulation study



    Rotational and translational dynamic heterogeneities (DHs) of ambient aqueous solutions of trimethylamine-N-oxide (TMAO) and tetramethylurea (TMU) at several solute concentrations have been investigated and compared. Motional characteristics of water molecules at solute interfaces and in bulk solutionshave been thoroughly examined for the search of slow dynamics. Note, TMAO possesses zwitterionic structure and is a protein stabilizer whereas TMU is a neutral dipolar molecule and a strong denaturant. Results suggest that water-TMAO solutions possess stronger DH than water-TMU solutions with the solute concentration dependence being stronger for TMAO than for TMU. Diffusive dynamics slows down near the solute surface for both the solutes. Solvation structure shows TMAO-water interaction is stronger than TMU-waterinteraction, producing longer H-bond fluctuation timescale in TMAO solutions. In short, this paper presents, for the first time, a systematic and comparative study of motional features and inter-species interactions between aqueous solutions containing solutes that differ in their individual impacts on protein stability.

  13. Soil clone library analyses to evaluate specificity and selectivity of PCR primers targeting fungal 18S rDNA for denaturing-gradient gel electrophoresis (DGGE).

    Takada Hoshino, Yuko; Morimoto, Sho


    We evaluated the fungal specificity and detection bias of four fungal 18S rRNA gene (18S rDNA) primer sets for denaturing-gradient gel electrophoresis (DGGE). We constructed and compared clone libraries amplified from upland and paddy field soils with each primer set (1, NS1/GCFung; 2, FF390/FR1-GC; 3, NS1/FR1-GC; and 4, NS1/EF3 for the first PCR and NS1/FR1-GC for the second PCR). Primer set 4 (for nested PCR) showed the highest specificity for fungi but biased specific sequences. Sets 1, 2, and 3 (for single PCR) amplified non-fungal eukaryotic sequences (from 7 to 16% for upland soil and from 20 to 31% for paddy field soil) and produced libraries with similar distributions of fungal 18S rDNA sequences at both the phylum and the class level. Set 2 tended to amplify more diverse fungal sequences, maintaining higher specificity for fungi. In addition, clone analyses revealed differences among primer sets in the frequency of chimeras. In upland field soil, the libraries amplified with primer sets 3 and 4, which targeted long fragments, contained many chimeric 18S rDNA sequences (18% and 48%, respectively), while the libraries obtained with sets 1 and 2, which targeted short fragments, contained fewer chimeras (5% and 10%, respectively).

  14. Approach to Analyze the Diversity of Myxobacteria in Soil by Semi-Nested PCR-Denaturing Gradient Gel Electrophoresis (DGGE) Based on Taxon-Specific Gene

    Li, Baiyuan; Yao, Qing; Zhu, Honghui


    The genotypic diversity of insoluble macromolecules degraded myxobacteria, provided an opportunity to discover new bacterial resources and find new ecological functions. In this study, we developed a semi-nested-PCR-denaturing gradient gel electrophoresis (DGGE) strategy to determine the presence and genotypic diversity of myxobacteria in soil. After two rounds of PCR with myxobacteria-specific primers, an 194 bp fragment of mglA, a key gene involved in gliding motility, suitable for DGGE was obtained. A large number of bands were observed in DGGE patterns, indicating diverse myxobacteria inhabiting in soils. Furthermore, sequencing and BLAST revealed that most of the bands belonged to the myxobacteria-group, and only three of the twenty-eight bands belonged to other group, i.e., Deinococcus maricopensis. The results verified that myxobacterial strains with discrepant sequence compositions of gene mglA could be discriminated by DGGE with myxobacteria-specific primers. Collectively, the developed semi-nested-PCR-DGGE strategy is a useful tool for studying the diversity of myxobacteria. PMID:25280065

  15. Behavior of Heat-Denatured Whey: Buttermilk Protein Aggregates during the Yogurt-Making Process and Their Influence on Set-Type Yogurt Properties.

    Saffon, Maxime; Richard, Véronique; Jiménez-Flores, Rafael; Gauthier, Sylvie F; Britten, Michel; Pouliot, Yves


    The objective of this study was to assess the impact of using heat-denatured whey:buttermilk protein aggregate in acid-set type yogurt production. Whey and buttermilk (25:75) protein concentrate was adjusted to pH 4.6, heated at 90 °C for 5 min, homogenized and freeze-dried. Set-type yogurts were prepared from skim milk standardized to 15% (w/v) total solids and 4.2% (w/v) protein using different levels of powdered skim milk or freeze-dried protein aggregate. The use of the protein aggregate significantly modified yogurt texture, but did not affect the water-holding capacity of the gel. Confocal laser-scanning microscope images showed the presence of large particles in milk enriched with protein aggregate, which directly affected the homogeneity of the clusters within the protein matrix. Thiol groups were freed during heating of the protein aggregate suspended in water, suggesting that the aggregates could interact with milk proteins during heating.

  16. Comparative microbiota assessment of wilted Italian ryegrass, whole crop corn, and wilted alfalfa silage using denaturing gradient gel electrophoresis and next-generation sequencing.

    Ni, Kuikui; Minh, Tang Thuy; Tu, Tran Thi Minh; Tsuruta, Takeshi; Pang, Huili; Nishino, Naoki


    The microbiota of pre-ensiled crop and silage were examined using denaturing gradient gel electrophoresis (DGGE) and next-generation sequencing (NGS). Wilted Italian ryegrass (IR), whole crop corn (WC), and wilted alfalfa (AL) silages stored for 2 months were examined. All silages contained lactic acid as a predominant fermentation product. Across the three crop species, DGGE detected 36 and 28 bands, and NGS identified 253 and 259 genera in the pre-ensiled crops and silages, respectively. The NGS demonstrated that, although lactic acid bacteria (LAB) became prevalent in all silages after 2 months of storage, the major groups were different between crops: Leuconostoc spp. and Pediococcus spp. for IR silage, Lactobacillus spp. for WC silage, and Enterococcus spp. for AL silage. The predominant silage LAB genera were also detected by DGGE, but the presence of diverse non-LAB species in pre-ensiled crops was far better detected by NGS. Likewise, good survival of Agrobacterium spp., Methylobacterium spp., and Sphingomonas spp. in IR and AL silages was demonstrated by NGS. The diversity of the microbiota described by principal coordinate analysis was similar between DGGE and NGS. Our finding that analysis of pre-ensiled crop microbiota did not help predict silage microbiota was true for both DGGE and NGS.

  17. Study of microbial community structures in UASB sludge treating municipal wastewater by denaturing gradient gel electrophoresis of 16S rDNA

    ZHANG Ying; Sunny Aiyuk; XU Hui; CHEN Guanxiong; Willy Verstraete


    The structures of microbial communities in lab-scale upflow anaerobic sludge blanket (UASB) reactors for treating municipal wastewater with different ratios of COD soluble/COD total were studied using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes.The microbial structure of the inoculum sludge obtained from a full-scale UASB reactor of treating potato processing wastewater was compared with the structures of sludges collected from three lab-scale UASB reactors after eight months feeding with raw municipal wastewater, with CEPS (chemically enhanced primary sedimentation) pretreated municipal wastewater, and with a synthetic municipal sewage, respectively. Computer-aided numerical analysis of the DGGE fingerprints showed that the bacterial community underwent major changes. The sludges for treating raw and CEPS pretreated wastewater had very similar bacterial and archaeal communities (82%and 96% similarity) but were different from that for treating the synthetic sewage. Hence, despite similar % COD in the particulate form in the synthetic and the real wastewater, the two wastewaters were selected for different microbial communities. Prominent DGGE bands of Bacteria and Archaea were purified and sequenced. The 16S rRNA gene sequences of the dominant archaeal bands found in the inoculum, and UASB sludge fed with raw sewage, CEPS pretreated wastewater, and synthetic sewage were closely associated with Methanosaeta concilii. In the UASB sludge fed with synthetic sewage, another dominant band associated with an uncultured archaeon 39-2 was found together with M. concilii.

  18. Detection of CpG methylations in human mismatch repair gene hMLH1 promoter by denaturing high-performance liquid chromatography (DHPLC)


    Objectives: To develop a novel method to detect CpG methylation by DHPLC. Methods: After DNA was treated with sodium bisulfite, mismatch repair gene hMLH1 promoter was amplified by polymerase chain reaction (PCR). DHPLC was used to separate the PCR products at their partially denaturing temperatures. BstUI digestion assay was also used for comparison study. Results: A 294bp band was obtained by PCR from each DNA samples of colon cancer cell line RKO and gastric cancer cell line PACM82. These two bands could be separated completely by DHPLC at 53° C (retention time 6.7 min for RKO vs. 6.2 min for PACM82). We concluded that the hMLH1 promoter in RKO cells is methylated, while PACM82 is not methylated, since methylation can protect the conversion of C to T and keep higher C/G content after bisulfite treatment, leading to the delayed time. These results consistent with those from BstUI digestion assay. Conclusion: Methylation in CpG islands of hMLH1 could be detected conveniently by DHPLC after bisulfite modification.

  19. Association of oral streptococci community dynamics with severe early childhood caries as assessed by PCR-denaturing gradient gel electrophoresis targeting the rnpB gene.

    Tao, Ye; Zhou, Yan; Ouyang, Yong; Lin, Huan Cai


    This study sought to investigate the possible association between the dynamics of oral streptococci community profiles and severe early childhood caries (S-ECC) development, compared with caries-free (CF) controls. Supragingival plaque samples were evaluated from 8-32-month-old children who had previously been assessed for overall profiles of their oral microbial community. Twelve children were in each group. Bacterial genomic DNA was extracted and amplified using rnpB-specific primers for streptococci; the products were then subjected to denaturing gradient gel electrophoresis (DGGE) and sequence analysis. We observed that the mean values for species richness (N) and diversity of oral streptococci (H') were significantly lower in the S-ECC group than in the CF group (N = 1.25 ± 4.14 vs 14.92 ± 2.84; H' = 1.41 ± 0.29 vs 1.64 ± 0.18) at 32  months of age (P diversity of oral streptococci, that the detection rates of S. sanguinis and S. gordonii have negative correlations with S-ECC; and that there are high levels of intra-individual similarity for the oral streptococci community over time.

  20. Characterization of depth-related microbial communities in lake sediment by denaturing gradient gel electrophoresis of amplified 16S rRNA fragments


    The characterization of microbial communities of different depth sediment samples was examined by a culture-independent method and compared with physicochemical parameters, those are organic matter (OM), total nitrogen (TN), total phosphorus (TP), pH and redox potential (Eh). Total genomic DNA was extracted from samples derived from different depths. After they were amplified with the GC-341f/907r primer sets of partial bacterial 16S rRNA genes, the products were separated by denaturing gradient gel electrophoresis (DGGE). The profile of DGGE fingerprints of different depth sediment samples revealed that the community structure remained relatively stable along the entire 45 cm sediment core, however, principal-component analysis of DGGE patterns revealed that at greater sediment depths, successional shifts in community structure were evident. The principle coordinates analysis suggested that the bacterial communities along the sediment core could be separated into two groups, which were located 0-20 cm and 21-45 cm, respectively. The sequencing dominant bands demonstrated that the major phylogenetic groups identified by DGGE belonged to Bacillus, Bacterium, Brevibacillus, Exiguobacterium, γ-Proteobacterium, Acinetobacter sp. And some uncultured or unidentified bacteria. The results indicated the existence of highly diverse bacterial community in the lake sediment core.