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Sample records for denaturing gradient gels

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

    Science.gov (United States)

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

    1999-01-01

    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

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

    Digital Repository Service at National Institute of Oceanography (India)

    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...

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

    NARCIS (Netherlands)

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

    2003-01-01

    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

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    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...

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

    NARCIS (Netherlands)

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

    2004-01-01

    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

  6. Analysis of variations in band positions for normalization in across-gel denaturing gradient gel electrophoresis.

    Science.gov (United States)

    Matsushita, Yuko; Yamamura, Kohji; Morimoto, Sho; Bao, Zhihua; Kurose, Daisuke; Sato, Ikuo; Yoshida, Shigenobu; Tsushima, Seiya

    2015-05-01

    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.

  7. Evaluation of denaturing gradient gel electrophoresis (DGGE) used ...

    African Journals Online (AJOL)

    user1

    2012-08-08

    Aug 8, 2012 ... systems. Therefore, three primer pairs that amplify different variable regions of the 16S rRNA gene (V1, ... ones include the selection of the primer system, the gradient .... some of the strains with sequence heterogeneity in the.

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

    DEFF Research Database (Denmark)

    Christiansen, L; Ged, C; Hombrados, I

    1999-01-01

    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...

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

    Science.gov (United States)

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

    2003-01-01

    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.

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

    Institute of Scientific and Technical Information of China (English)

    R.PASTORELLI; R.PICCOLO; S.SIMONCINI; S.LANDI

    2013-01-01

    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.

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

    Science.gov (United States)

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

    2016-06-28

    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.

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

    NARCIS (Netherlands)

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

    1999-01-01

    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

  13. Application of denaturing gradient gel electrophoresis (DGGE) to the analysis of endodontic infections.

    Science.gov (United States)

    Siqueira, José F; Rôças, Isabela N; Rosado, Alexandre S

    2005-11-01

    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.

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

    Science.gov (United States)

    Endo, Akihito; Okada, Sanae

    2005-03-01

    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.

    Science.gov (United States)

    Li, C; Moe, W M

    2004-05-01

    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. Agarose gel purification of PCR products for denaturing gradient gel electrophoresis results in GC-clamp deletion.

    Science.gov (United States)

    Sun, Guowei; Xiao, Jinzhou; Lu, Man; Wang, Hongming; Chen, Xiaobing; Yu, Yongxin; Pan, Yingjie; Wang, Yongjie

    2015-01-01

    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.

  17. Association of Streptomyces community composition determined by PCR-denaturing gradient gel electrophoresis with indoor mold status

    OpenAIRE

    Johansson, Elisabet; Reponen, Tiina; Meller, Jarek; Vesper, Stephen; Yadav, Jagjit

    2014-01-01

    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...

  18. Denaturing gradient gel electrophoresis of neonatal intestinal microbiota in relation to the development of asthma

    Directory of Open Access Journals (Sweden)

    Desager Kristine N

    2011-04-01

    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.

  19. A PCR-denaturing gradient gel electrophoresis approach to assess Fusarium diversity in asparagus.

    Science.gov (United States)

    Yergeau, E; Filion, M; Vujanovic, V; St-Arnaud, M

    2005-02-01

    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.

  20. Comparative Analysis of Denaturing Gradient Gel Electrophoresis and Temporal Temperature Gradient Gel Electrophoresis Profiles as a Tool for the Differentiation of Candida Species.

    Science.gov (United States)

    Mohammadi, Parisa; Hamidkhani, Aida; Asgarani, Ezat

    2015-10-01

    Candida species are usually opportunistic organisms that cause acute to chronic infections when conditions in the host are favorable. Accurate identification of Candida species is an essential pre-requisite for improved therapeutic strategy. Identification of Candida species by conventional methods is time-consuming with low sensitivity, yet molecular approaches have provided an alternative way for early diagnosis of invasive candidiasis. Denaturing gradient gel electrophoresis (DGGE) and temporal temperature gradient gel electrophoresis (TTGE) are polymerase chain reaction (PCR)-based approaches that are used for studying the community structure of microorganisms. By using these methods, simultaneous identification of multiple yeast species will be possible and reliable results will be obtained quickly. In this study, DGGE and TTGE methods were set up and evaluated for the detection of different Candida species, and their results were compared. Five different Candida species were cultured on potato dextrose agar medium for 24 hours. Next, total DNA was extracted by the phenol-chloroform method. Two sets of primers, ITS3-GC/ITS4 and NL1-GC/LS2 were applied to amplify the desired regions. The amplified fragments were then used to analyze DGGE and TTGE profiles. The results showed that NL1-GC/LS2 primer set could yield species-specific amplicons, which were well distinguished and allowed better species discrimination than that generated by the ITS3-GC/ITS4 primer set, in both DGGE and TTGE profiles. All five Candida species were discriminated by DGGE and TTGE using the NL1-GC/LS2 primer set. Comparison of DGGE and TTGE profiles obtained from NL1-GC/LS2 amplicons exhibited the same patterns. Although both DGGE and TTGE techniques are capable of detecting Candida species, TTGE is recommended because of easier performance and lower costs.

  1. Utilization of denaturing gradient gel electrophoresis for diagnosis of {beta}-thalassemia and ascertainment of new mutations

    Energy Technology Data Exchange (ETDEWEB)

    Ngo, K.Y.; Liu, D.; Lee, J. [Univ. of California, San Diego (United States)] [and others

    1994-09-01

    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.

  2. Comparison of Different Denaturing Gradient Gel Electrophoresis Primer Sets for the Study of Marine Bacterioplankton Communities▿ †

    Science.gov (United States)

    Sánchez, Olga; Gasol, Josep M.; Massana, Ramon; Mas, Jordi; Pedrós-Alió, Carlos

    2007-01-01

    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

  3. Evaluation of mutation screening by heteroduplex analysis in acute intermittent porphyria: comparison with denaturing gradient gel electrophoresis.

    Science.gov (United States)

    Tchernitchko, D; Lamoril, J; Puy, H; Robreau, A M; Bogard, C; Rosipal, R; Gouya, L; Deybach, J C; Nordmann, Y

    1999-01-01

    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.

  4. Comparison of Fecal Methanogenic Archaeal Community Between Erhualian and Landrace Pigs Using Denaturing Gradient Gel Electrophoresis and Real-Time PCR Analysis

    NARCIS (Netherlands)

    Su, Y.; Smidt, H.; Zhu, W.Y.

    2014-01-01

    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

  5. Analysis of bacterial communities in soil by use of denaturing gradient gel electrophoresis and clone libraries, as influenced by different reverse primers

    NARCIS (Netherlands)

    Brons, Jolanda; van Elsas, J.D.

    2008-01-01

    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

  6. Identification and Population Dynamics of Yeasts in Sourdough Fermentation Processes by PCR-Denaturing Gradient Gel Electrophoresis

    Science.gov (United States)

    Meroth, Christiane B.; Hammes, Walter P.; Hertel, Christian

    2003-01-01

    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

  7. Microbial Diversity during Fermentation of Sweet Paste, a Chinese Traditional Seasoning, Using PCR-Denaturing Gradient Gel Electrophoresis.

    Science.gov (United States)

    Mao, Ping; Hu, Yuanliang; Liao, Tingting; Wang, Zhaoting; Zhao, Shumiao; Liang, Yunxiang; Hu, Yongmei

    2017-04-28

    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.

  8. Detection and differentiation of Entamoeba histolytica and Entamoeba dispar in clinical samples through PCR-denaturing gradient gel electrophoresis

    Directory of Open Access Journals (Sweden)

    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.

  9. Analysis of microbial diversity on deli slicers using polymerase chain reaction and denaturing gradient gel electrophoresis technologies.

    Science.gov (United States)

    Koo, O K; Mertz, A W; Akins, E L; Sirsat, S A; Neal, J A; Morawicki, R; Crandall, P G; Ricke, S C

    2013-02-01

    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.

  10. Monitoring the bacterial population dynamics in sourdough fermentation processes by using PCR-denaturing gradient gel electrophoresis.

    Science.gov (United States)

    Meroth, Christiane B; Walter, Jens; Hertel, Christian; Brandt, Markus J; Hammes, Walter P

    2003-01-01

    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.

  11. Detection and differentiation of Entamoeba histolytica and Entamoeba dispar in clinical samples through PCR-denaturing gradient gel electrophoresis.

    Science.gov (United States)

    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

    2017-04-03

    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.

  12. Seven new mutations in hMSH2, an HNPCC Gene, identified by denaturing gradient-gel electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    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)

    1995-05-01

    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.

  13. Variations among Japanese of the factor IX gene (F9) detected by PCR-denaturing gradient gel electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Satoh, Chiyoko; Takahashi, Norio; Asakawa, Junichi; Hiyama, Keiko; Kodaira, Meiko (Radiation Effects Research Foundation, Hiroshima (Japan))

    1993-01-01

    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.

  14. Monitoring of microbial community structure and succession in the biohydrogen production reactor by denaturing gradient gel electrophoresis (DGGE)

    Institute of Scientific and Technical Information of China (English)

    XING; Defeng; REN; Nanqi; GONG; Manli; LI; Jianzheng; LI; Q

    2005-01-01

    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.

  15. Monitoring of microbial community structure and succession in the biohydrogen production reactor by denaturing gradient gel electrophoresis (DGGE).

    Science.gov (United States)

    Xing, Defeng; Ren, Nanqi; Gong, Manli; Li, Jianzheng; Li, Qiubo

    2005-04-01

    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.

  16. Diversity and dynamics of antibiotic-resistant bacteria in cheese as determined by PCR denaturing gradient gel electrophoresis.

    Science.gov (United States)

    Flórez, Ana Belén; Mayo, Baltasar

    2015-12-01

    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.

  17. Dynamic changes of yak ( gut microbiota during growth revealed by polymerase chain reaction-denaturing gradient gel electrophoresis and metagenomics

    Directory of Open Access Journals (Sweden)

    Yuanyang Nie

    2017-07-01

    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

  18. Association of Streptomyces community composition determined by PCR-denaturing gradient gel electrophoresis with indoor mold status.

    Science.gov (United States)

    Johansson, Elisabet; Reponen, Tiina; Meller, Jarek; Vesper, Stephen; Yadav, Jagjit

    2014-12-01

    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.

  19. Assessment of the yeast species composition of cocoa bean fermentations in different cocoa-producing regions using denaturing gradient gel electrophoresis.

    Science.gov (United States)

    Papalexandratou, Zoi; De Vuyst, Luc

    2011-11-01

    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.

  20. Analysis of the insulin receptor gene in noninsulin-dependent diabetes mellitus by denaturing gradient gel blots: A clinical research center study

    Energy Technology Data Exchange (ETDEWEB)

    Magre, J.; Goldfine, A.B.; Warram, J.H. [Harvard Medical School, Boston, MA (United States)] [and others

    1995-06-01

    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.

  1. Denaturing gradient gel electrophoresis (DGGE as a powerful novel alternative for differentiation of epizootic ISA virus variants.

    Directory of Open Access Journals (Sweden)

    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.

  2. A New Standard-Based Polynomial Interpolation (SBPIn) method to address gel-to-gel variability for the comparison of multiple denaturing gradient gel electrophoresis profile matrices.

    Science.gov (United States)

    Valentín-Vargas, Alexis; Chorover, Jon; Maier, Raina M

    2013-02-15

    The Standard-Based Polynomial Interpolation (SBPIn) method is a new simple three-step protocol proposed to address common gel-to-gel variations for the comparison of sample profiles across multiple DGGE gels. The advantages of this method include no requirement for additional software or modification of the standard DGGE protocol.

  3. A New Standard-Based Polynomial Interpolation (SBPIn) Method to Address Gel-to-Gel Variability for the Comparison of Multiple Denaturing Gradient Gel Electrophoresis Profile Matrices

    Science.gov (United States)

    Valentín-Vargas, Alexis; Chorover, Jon; Maier, Raina M.

    2013-01-01

    The Standard-Based Polynomial Interpolation (SBPIn) method is a new simple three-step protocol proposed to address common gel-to-gel variations for the comparison of sample profiles across multiple DGGE gels. The advantages of this method include no requirement for additional software or modification of the standard DGGE protocol. PMID:23234884

  4. 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

    Directory of Open Access Journals (Sweden)

    Meldrum Cliff J

    2003-12-01

    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.

  5. Characterization of bacterial populations in Danish raw milk cheeses made with different starter cultures by denaturating gradient gel electrophoresis and pyrosequencing

    DEFF Research Database (Denmark)

    Masoud, Wafa Mahmoud Hasan; Takamiya, Monica K Wik; Vogensen, Finn Kvist;

    2011-01-01

    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...

  6. Direct detection and identification of lactic acid bacteria in a food processing plant and in meat products using denaturing gradient gel electrophoresis.

    Science.gov (United States)

    Takahashi, Hajime; Kimura, Bon; Yoshikawa, Miwako; Gotou, Seitaro; Watanabe, Itaru; Fujii, Tateo

    2004-11-01

    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.

  7. Analysis of phosphate-accumulating organisms cultivated under different carbon sources with polymerase chain reaction-denaturing gradient gel electrophoresis assay

    Institute of Scientific and Technical Information of China (English)

    YU Shui-li; LIU Ya-nan; JING Guo-lin; ZHAO Bing-jie; GUO Si-yuan

    2005-01-01

    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.

  8. Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

    Science.gov (United States)

    Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Said; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

    2002-01-01

    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.

  9. PCR-denaturing gradient gel electrophoresis analysis of microbial community in soy-daddawa, a Nigerian fermented soybean (Glycine max (L.) Merr.) condiment.

    Science.gov (United States)

    Ezeokoli, Obinna T; Gupta, Arvind K; Mienie, Charlotte; Popoola, Temitope O S; Bezuidenhout, Cornelius C

    2016-03-02

    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.

  10. Denaturing gradient gel electrophoresis fingerprinting of soil bacteria in the vicinity of the Chinese Great Wall Station, King George Island, Antarctica.

    Science.gov (United States)

    Pan, Qi; Wang, Feng; Zhang, Yang; Cai, Minghong; He, Jianfeng; Yang, Haizhen

    2013-08-01

    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.

  11. Denaturing gradient gel electrophoresis fingerprinting of soil bacteria in the vicinity of the Chinese Great Wall Station, King George Island, Antarctica

    Institute of Scientific and Technical Information of China (English)

    Qi Pan; Feng Wang; Yang Zhang; Minghong Cai; Jianfeng He; Haizhen Yang

    2013-01-01

    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.

  12. Comparison of Fecal Methanogenic Archaeal Community Between Erhualian and Landrace Pigs Using Denaturing Gradient Gel Electrophoresis and Real-Time PCR Analysis

    Institute of Scientific and Technical Information of China (English)

    SU Yong; Hauke Smidt; ZHU Wei-Yun

    2014-01-01

    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.

  13. Soil clone library analyses to evaluate specificity and selectivity of PCR primers targeting fungal 18S rDNA for denaturing-gradient gel electrophoresis (DGGE).

    Science.gov (United States)

    Takada Hoshino, Yuko; Morimoto, Sho

    2010-01-01

    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

    Science.gov (United States)

    Li, Baiyuan; Yao, Qing; Zhu, Honghui

    2014-01-01

    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. Comparative microbiota assessment of wilted Italian ryegrass, whole crop corn, and wilted alfalfa silage using denaturing gradient gel electrophoresis and next-generation sequencing.

    Science.gov (United States)

    Ni, Kuikui; Minh, Tang Thuy; Tu, Tran Thi Minh; Tsuruta, Takeshi; Pang, Huili; Nishino, Naoki

    2017-02-01

    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.

  16. Study of microbial community structures in UASB sludge treating municipal wastewater by denaturing gradient gel electrophoresis of 16S rDNA

    Institute of Scientific and Technical Information of China (English)

    ZHANG Ying; Sunny Aiyuk; XU Hui; CHEN Guanxiong; Willy Verstraete

    2005-01-01

    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.

  17. Association of oral streptococci community dynamics with severe early childhood caries as assessed by PCR-denaturing gradient gel electrophoresis targeting the rnpB gene.

    Science.gov (United States)

    Tao, Ye; Zhou, Yan; Ouyang, Yong; Lin, Huan Cai

    2015-08-01

    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.

  18. Characterization of depth-related microbial communities in lake sediment by denaturing gradient gel electrophoresis of amplified 16S rRNA fragments

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    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.

  19. Molecular Fingerprinting by PCR-Denaturing Gradient Gel Electrophoresis Reveals Differences in the Levels of Microbial Diversity for Musty-Earthy Tainted Corks ▿

    Science.gov (United States)

    Prat, Chantal; Ruiz-Rueda, Olaya; Trias, Rosalia; Anticó, Enriqueta; Capone, Dimitra; Sefton, Mark; Bañeras, Lluís

    2009-01-01

    The microbial community structure of cork with marked musty-earthy aromas was analyzed using denaturing gradient gel electrophoresis of amplified ribosomal DNA. Cork stoppers and discs were used for DNA extraction and were analyzed by using selective primers for bacteria and fungi. Stoppers clearly differed from discs harboring a different fungal community. Moreover, musty-earthy samples of both types were shown to have a specific microbiota. The fungi Penicillium glabrum and Neurospora spp. were present in all samples and were assumed to make only a small contribution to off-odor development. In contrast, Penicillium islandicum and Penicillium variabile were found almost exclusively in 2,4,6-trichloroanisole (TCA) tainted discs. Conversely, Rhodotorula minuta and Rhodotorula sloofiae were most common in cork stoppers, where only small amounts of TCA were detected. Alpha- and gammaproteobacteria were the most commonly found bacteria in either control or tainted cork stoppers. Specific Pseudomonas and Actinobacteria were detected in stoppers with low amounts of TCA and 2-methoxy-3,5-dimethylpyrazine. These results are discussed in terms of biological degradation of taint compounds by specific microorganisms. Reliable and straightforward microbial identification methods based on a molecular approach provided useful data to determine and evaluate the risk of taint formation in cork. PMID:19201983

  20. Molecular fingerprinting by PCR-denaturing gradient gel electrophoresis reveals differences in the levels of microbial diversity for musty-earthy tainted corks.

    Science.gov (United States)

    Prat, Chantal; Ruiz-Rueda, Olaya; Trias, Rosalia; Anticó, Enriqueta; Capone, Dimitra; Sefton, Mark; Bañeras, Lluís

    2009-04-01

    The microbial community structure of cork with marked musty-earthy aromas was analyzed using denaturing gradient gel electrophoresis of amplified ribosomal DNA. Cork stoppers and discs were used for DNA extraction and were analyzed by using selective primers for bacteria and fungi. Stoppers clearly differed from discs harboring a different fungal community. Moreover, musty-earthy samples of both types were shown to have a specific microbiota. The fungi Penicillium glabrum and Neurospora spp. were present in all samples and were assumed to make only a small contribution to off-odor development. In contrast, Penicillium islandicum and Penicillium variabile were found almost exclusively in 2,4,6-trichloroanisole (TCA) tainted discs. Conversely, Rhodotorula minuta and Rhodotorula sloofiae were most common in cork stoppers, where only small amounts of TCA were detected. Alpha- and gammaproteobacteria were the most commonly found bacteria in either control or tainted cork stoppers. Specific Pseudomonas and Actinobacteria were detected in stoppers with low amounts of TCA and 2-methoxy-3,5-dimethylpyrazine. These results are discussed in terms of biological degradation of taint compounds by specific microorganisms. Reliable and straightforward microbial identification methods based on a molecular approach provided useful data to determine and evaluate the risk of taint formation in cork.

  1. Diagnosis of cutaneous T-cell lymphoma detecting T-cell receptor gamma chain gene monoclonality by denaturing gradient gel electrophoresis.

    Science.gov (United States)

    Lapière, K; Dhaene, K; Matthieu, L; Hübner, R; Lambert, J; Van Marck, E

    1999-04-01

    Cutaneous T-cell lymphomas represent a group of malignant lymphoproliferative disorders characterised by the occurrence of a monoclonal population of T-lymphocytes. Diagnosis of early stages of this disease is a difficult challenge for both the dermatologist and the dermatopathologist. With the aid of the polymerase chain reaction it is possible to amplify specific regions of the T-cell receptor gamma gene. The amplification products can then be separated by denaturing gradient gel electrophoresis in order to detect a monoclonal population of T-lymphocytes in the infiltrate. We studied 4 patients with the clinicopathologic diagnosis of mycosis fungoides and 2 patients diagnosed as large plaque parapsoriasis. A monoclonal population was detected in 3 of the 4 mycosis fungoides cases and in 1 of the patients with large plaque parapsoriasis. This indicates that our analysis can help us establishing a diagnosis, and it can also help us to identify patients with a possible early stage of the disease, which clinically or histologically is not yet recognised as such.

  2. Co-monitoring bacterial and dinoflagellates communities by denaturing gradient gel electrophoresis (DGGE) and SSU rRNA sequencing during a dinoflagellates bloom

    Institute of Scientific and Technical Information of China (English)

    KANJinjun; CHENFeng

    2004-01-01

    Dinoflagellates are unicellular eukaryotic protists that dominate in all coastal waters, and are also present in oceanic waters. Despite the central importance of dinoflagellates in global primary production, the relationship between dinoflagellates and bacteria are still poorly understood. In order to understand the ecological interaction between bacterial and dinoflageUates communities, denaturing gradient gel electrophoresis (DGGE) and SSU rRNA sequencing were applied to monitoring the population dynamics of bacteria and dinoflagellates from the onset to disappearance of a dinoflagellates bloom occurred in Baltimore Inner Harbor, from April 15 to 24, 2002. Although Prorocentrum minimum was the major bloom forming species under the light microscopy, DGGE method with dinoflagellate specific primers demonstrated that Prorocentrum micans, Gymnodinium galatheanum and Gyrodinium uncatenum were also present during the bloom. Population shifts among the minor dinoflagellate groups were observed. DGGE of PCR-amplified 16S rRNA gene fragments indicated that cyanobacteria, α, β, γ-proteobacteria, FlavobacteriumBacteroides-Cytophaga (FBC), and Planctomcetes were the major components of bacterial assemblages during the bloom. DGGE analysis showed that Cytophagales and α-proteobacteria played important roles at different stages of dinoflagellates bloom. DGGE can be used as a rapid tool to simultaneously monitor population dynamics of both bacterial and dinoflagellates communities in aquatic environments, which is demonstrated here.

  3. Application of nested PCR-DGGE (denaturing gradient gel electrophoresis) for the analysis of ciliate communities in soils.

    Science.gov (United States)

    Shimano, Satoshi; Sambe, Mitsuo; Kasahara, Yasuhiro

    2012-01-01

    Ciliates play important roles as prey and predators in ecosystems. Changes in the ciliate community can affect the composition and population of microfauna and microflora in ecosystems. To investigate the structure of ciliate communities, we developed a nested PCR-DGGE method, which combines a universal eukaryotic-specific primer set in the first PCR step with a ciliate-specific primer set in the second PCR step, to amplify 18S rRNA genes from ciliates. The 300 bp DGGE fragments generated more bands on the gel than the 600 bp DGGE fragments. Prior to bead beating, DNA extraction of ciliates from soil samples was optimized with a combination of freeze-thaw cycles and ultrasonication. We applied this nested PCR-DGGE method to agricultural soils amended with 0, 120, 300, and 600 t ha⁻¹ year⁻¹ of livestock slurry. The results from the DGGE profiles and principal component analysis (PCA) revealed that the supplement of slurry to soils influenced the ciliate communities. From phylogenetic analysis, 108 DGGE bands were assigned to six classes, which included Spirotrichea and Colpodea, of the subphylum Intramacronucleata, and one class of the subphylum Postciliodesmatophora. These results indicated that a wide variety of taxonomic groups were detected by DGGE profiling. Thus, the nested PCR-DGGE method described here could clearly differentiate between ciliate communities within soil samples and allowed for the phylogenetic identification of these ciliates at the class level.

  4. Application of PCR-denaturing-gradient gel electrophoresis (DGGE) method to examine microbial community structure in asparagus fields with growth inhibition due to continuous cropping.

    Science.gov (United States)

    Urashima, Yasufumi; Sonoda, Takahiro; Fujita, Yuko; Uragami, Atsuko

    2012-01-01

    Growth inhibition due to continuous cropping of asparagus is a major problem; the yield of asparagus in replanted fields is low compared to that in new fields, and missing plants occur among young seedlings. Although soil-borne disease and allelochemicals are considered to be involved in this effect, this is still controversial. We aimed to develop a technique for the biological field diagnosis of growth inhibition due to continuous cropping. Therefore, in this study, fungal community structure and Fusarium community structure in continuously cropped fields of asparagus were analyzed by polymerase chain reaction/denaturing-gradient gel electrophoresis (PCR-DGGE). Soil samples were collected from the Aizu region of Fukushima Prefecture, Japan. Soil samples were taken from both continuously cropped fields of asparagus with growth inhibition and healthy neighboring fields of asparagus. The soil samples were collected from the fields of 5 sets in 2008 and 4 sets in 2009. We were able to distinguish between pathogenic and non-pathogenic Fusarium by using Alfie1 and Alfie2GC as the second PCR primers and PCR-DGGE. Fungal community structure was not greatly involved in the growth inhibition of asparagus due to continuous cropping. By contrast, the band ratios of Fusarium oxysporum f. sp. asparagi in growth-inhibited fields were higher than those in neighboring healthy fields. In addition, there was a positive correlation between the band ratios of Fusarium oxysporum f. sp. asparagi and the ratios of missing asparagus plants. We showed the potential of biological field diagnosis of growth inhibition due to continuous cropping of asparagus using PCR-DGGE.

  5. Diversity of lactic acid bacteria from modified atmosphere packaged sliced cooked meat products at sell-by date assessed by PCR-denaturing gradient gel electrophoresis.

    Science.gov (United States)

    Audenaert, Kris; D'Haene, Klaas; Messens, Kathy; Ruyssen, Tony; Vandamme, Peter; Huys, Geert

    2010-02-01

    The predominant lactic acid bacteria (LAB) microbiota associated with three types of modified atmosphere packaged (MAP) sliced cooked meat products (i.e. ham, turkey and chicken) was analyzed at sell-by date using a combination of culturing and molecular population fingerprinting. Likewise routine analyses during industrial MAP production, meat samples were plated on the general heterotrophic Plate Count Agar (PCA) and on the LAB-specific de Man, Rogosa, Sharpe (MRS) agar under different temperature and atmosphere conditions. Subsequently, community DNA extracts were prepared from culturable bacterial fractions harvested from both media and used for PCR targeting the V3 hyper-variable region of the 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) of PCR amplicons (PCR-DGGE). Irrespective of aerobic or anaerobic incubation conditions, V3-16S rDNA DGGE fingerprints of culturable fractions from PCA and MRS medium displayed a high level of similarity indicating that LAB constituted the most dominant group in the culturable bacterial community. Comparison of DGGE profiles of fractions grown at 20, 28 or 37 degrees C indicated that part of the culturable community consisted of psychrotrophs. Four DGGE bands were common among cooked ham, turkey and chicken products, suggesting that these represent the microbiota circulating in the plant where all three MAP product types were sliced and packaged. Based on band sequencing and band position analysis using LAB reference strains, these four bands could be assigned to Lactobacillus sakei and/or the closely related Lactobacillus fuchuensis, Lactobacillus curvatus, Carnobacterium divergens and Leuconostoc carnosum. In conclusion, the PCR-DGGE approach described in this study allows to discriminate, identify and monitor core and occasional LAB microbiota of MAP sliced cooked meat products and provides valuable complementary information to the current plating procedures routinely used in industrial plants.

  6. Detection of Helicobacter species in liver and stomach tissues of patients with chronic liver diseases using polymerase chain reaction-denaturing gradient gel electrophoresis and immunohistochemistry.

    Science.gov (United States)

    Stalke, Piotr; Al-Soud, Waleed Abu; Bielawski, Krzysztof P; Bakowska, Alicja; Trocha, Hanna; Stepinski, Jan; Wadström, Torkel

    2005-09-01

    Helicobacter DNA has been detected in the hepatobiliary tree of patients with chronic liver diseases (CLD). The presence of H. pylori in the stomach compared with in the liver of the same patients with CLD has not been studied, therefore to the aim of this study was to investigate the presence of Helicobacter DNA and antigens in the liver and stomach of Polish patients with chronic liver diseases using molecular and immunological methods. Gastric mucosa and liver tissue samples and sera were collected from 97 Polish patients with CLD. Anti-H. pylori antibodies were detected by enzyme immunoassay (EIA), and H. pylori-like antigens detected by immunohistochemistry. Helicobacter DNA was detected in stomach and liver samples using a semi-nested Helicobacter genus-specific polymerase chain reaction (PCR) assay, and Helicobacter species identified by denaturing gradient gel electrophoresis (DGGE) and sequencing analysis of amplified PCR products. H. pylori was identified by DGGE and sequence analysis in 60/62 (97%) and 25/25 (100%) of the gastric and liver Helicobacter genus-positive samples, respectively, whereas DNA of H. heilmannii was detected in 2/62 (3%) of the Helicobacter genus-positive gastric samples. H. pylori cagA gene was detected in 23/62 (36%) and 3/25 (12%) gastric and liver tissue samples, respectively. H. pylori-like antigens were detected in 61/97 (63%) gastric mucosa and in 40/97 (41%) liver tissue samples. H. pylori-like organisms appeared to dominate the gastric mucosa and liver tissue of Polish patients with CLD. The prevalence of the cagA gene was higher in stomach compared with liver samples, which suggests a possible role of cagA negative H. pylori-like organisms in CLD. On the other hand, no significant correlation was found between the presence of H. pylori-like DNA and antigens in the liver and liver function tests.

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

    NARCIS (Netherlands)

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

    1995-01-01

    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

  8. Analysis of bacterial diversity during the fermentation of inyu, a high-temperature fermented soy sauce, using nested PCR-denaturing gradient gel electrophoresis and the plate count method.

    Science.gov (United States)

    Wei, Chia-Li; Chao, Shiou-Huei; Tsai, Wen-Bin; Lee, Pei-Shan; Tsau, Nai-Hung; Chen, Jhih-Shan; Lai, Wen-Lin; Tu, James Ching-Yueh; Tsai, Ying-Chieh

    2013-04-01

    The diversity of bacteria associated with the fermentation of inyu, also known as black soy sauce, was studied through the nested PCR-denaturing gradient gel electrophoresis (DGGE) of samples collected from the fermentation stages of the inyu production process. The DGGE profiles targeted the bacterial 16S rDNA and revealed the presence of Citrobacter farmeri, Enterobacter cloacae, Enterobacter hormaechei, Enterococcus faecium, Klebsiella pneumoniae, Pantoea agglomerans, Salmonella enterica, Serratia marcescens, Staphylococcus sciuri and Weissella confusa. The bacterial compositions of 4 fermented samples were further elucidated using the plate count method. The bacteria isolated from the koji-making stage exhibited the highest diversity; Brachybacterium rhamnosum, E. hormaechei, K. pneumoniae, Kurthia gibsonii, Pantoea dispersa, Staphylococcus gallinarum, Staphylococcus kloosii and S. sciuri were identified. Koji collected during the preincubation stage presented the largest cell counts, and E. hormaechei, K. pneumoniae, E. cloacae and Enterobacter pulveris were identified. In brine samples aged for 7 and 31 days, the majority of the bacteria isolated belonged to 4 Bacillus species, but 4 Staphylococcus species and Delftia tsuruhatensis were also detected. This study demonstrates the benefits of using a combined approach to obtain a more complete picture of microbial populations and provides useful information for the control or development of bacterial flora during inyu fermentation.

  9. Bacterial communities in Agaricus bisporus compost analysed by denatu-ring gradient gel electrophoresis%双孢蘑菇培养料发酵过程中细菌群落结构分析

    Institute of Scientific and Technical Information of China (English)

    王琳; 李敏; 魏启舜; 张洪海; 周影; 赵荷娟

    2015-01-01

    PCR-denaturing gradient gel electrophoresis ( DGGE) was emloyed to analyze the V3 regions of bacterial 16S ribosomal DNA collected from the compost for Agaricus bisporus cultivation in spring and winter. The dominant DNA were cloned, sequenced and analyzed. Bacillus sp, Flavobacterium sp. , Lysinibacillus sp. , Thermus thermophilus,Pseudomonas fragi, Solibacillus silvestris,Thermobifida fusca, and two kinds of uncultured bacteria were identified in the compost. The bac-teria in spring and winter showed similar changing trend during phase I composting and similar community at the end of com-posting. The bacterial community structure differed at initial composting, phase I composting and phase II composting. The community diversity was environmental temperature-dependant. The bacteria existing in the compost all through the process of composting shared high sequence similarity with that of the bacteria degrading lignocellulose, which was favorable for the com-post to be applied as culture matrix.%为探明双孢蘑菇培养料发酵过程中细菌群落多样性和组成的演变,获得相关优势菌落的信息,本研究利用变性梯度凝胶电泳( Denaturing gradient gel electrophoresis,DGGE)对不同季节不同发酵阶段样品的的细菌进行特异性扩增,并选取主要DNA条带进行克隆、测序和生物信息学分析。结果显示,不同发酵时期带谱差异明显。发酵过程中,培养料中主要有芽孢杆菌属、黄杆菌属、杆菌属、假单胞菌属、Solibacillus、嗜热裂孢菌属、高温双歧菌属和未知分类地位的不可培养细菌。不同季节(春季、冬季)的培养料一次发酵过程中细菌多样性变化趋势相似,且发酵结束时细菌菌落结构相似。双孢蘑菇培养料在发酵过程中细菌群落结构至少经历了3个阶段的演替,即建堆初期、一次发酵阶段和二次发酵阶段。双孢蘑菇培养料发酵过程中细菌种群丰富,并随着发酵的不同阶段发生演

  10. 牙周牙髓联合病变菌群的PCR-DGGE分析%Bacterial analysis of combined periodontal-endodontic lesions using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE)

    Institute of Scientific and Technical Information of China (English)

    夏明慧; 亓庆国

    2012-01-01

    目的 利用变性梯度凝胶电泳(DGGE)技术观察牙周牙髓联合病变患牙牙周组织和根管中原始菌群分布状况及其异同,并通过克隆测序技术来试图探讨两部位可能存在的优势菌.方法 从13例牙周牙髓联合病变患牙分别采集患牙根尖1/3处牙周细菌和根管牙髓细菌,提取细菌总DNA,扩增16S rRNA基因可变区,再进行变性梯度凝胶电泳.应用SPSS17.0软件和Quantity One软件对DGGE图谱菌种条带进行统计学分析和聚类分析.对DGGE凝胶中有代表性的条带进行回收和克隆测序.结果 两取菌部位的菌种条带数间有明显的统计学差异(P<0.01),但二者之间无正相关性.二者间的相似系数为13.1% ~62.5%.牙周牙髓联合病变患牙根尖区1/3处牙周菌属可能有弯曲菌属(Campylobacter)、梭杆菌属(Fusobacterium)、奈瑟菌属(Neisseria)等,该处对应根管中菌属可能有优杆菌属(Mogibacterium)、棒状杆菌属(Corynebacterium)、放线菌属(Actinomyces)等.结论 牙周牙髓联合病变(牙周来源)中牙周组织和邻近根管牙髓组织中菌种在数目和结构上有明显不同.该病变牙周组织和根管中可能存在目前尚未被认知的优势菌种.%Objective To compare the bacterial community profiles present in periodontium and root canals of the same tooth diagnosed as combined periodontal-endodontic lesions by using denaturing gradient gel electrophoresis (DGGE).Methods Samples were collected from 13 extracted teeth with advanced periodontitis,endodontic samples from root tip 1/3 root canal,and periodontal samples from the corresponding neighboring periodontium.Genomic DNA was collected for the following universal bacterial primersPCR.The PCR products were then loaded on the DGGE gels to gain separate bands.The typical DGGE bands were excised,PCR-cloned and sequenced.Results The number of bands,which was indicative of the number of bacterial species,was compared intra-group (periodontal and

  11. Thermal denaturation of type I collagen vitrified gels

    Energy Technology Data Exchange (ETDEWEB)

    Xia, Zhiyong, E-mail: zhiyong.xia@jhuapl.edu [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: morgana.trexler@jhuapl.edu [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)

    2012-01-10

    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.

  12. Denaturing gradient gel electrophoresis analysis of 16S rDNA V3 fragment of bacteria in transgenic wheat soil%转基因小麦田土壤细菌16S rDNA V3片段的DGGE分析

    Institute of Scientific and Technical Information of China (English)

    丁衬衬; 周艳红; 林凡云; 徐剑宏; 吴季荣; 祭芳; 史建荣

    2011-01-01

    为长期监测转基因作物对土壤微生物菌群变化的影响提供可靠的试验方法.以检测转基因小麦田土壤细菌菌群变化为例,利用细菌特异性引物,扩增得到了土壤细菌16S rDNA V3可变区片段;通过变性梯度凝胶电泳(Denaturing gradient gel electrophoresis,DGGE),分析PCR扩增得到的V3可变区片段;根据电泳条带的变化情况评价转基因小麦对土壤环境微生物的安全性.结果表明该方法所提取的DNA,可以不经过纯化直接进行PCR扩增等后续试验;DGGE图谱中条带清晰.该方法可作为长期监测土壤微生物种群变化的试验手段.%This experiment was carried out to establish an effective method for detecting the impact of virus resistant transgenic wheat on its environment bacteria. Variable region fragments of 16S rDNA V3 were amplified by specific and sensitive primers of bacteria, analyzed by denaturing gradient gel electrophoresis ( DGGE) , and applied to evaluate the effect of transgenic wheat on the soil bacterium, according to the change of electrophoretic bands. The results showed that the DNA by this extraction method could be used in PCR without purification. On DGGE profiles, the lighter bands of each lane represented their corresponding dominant microorganisms. The influence of transgenic wheat and non-transgenic wheat on soil microbes by the number and brightness of the bands were obtained, so microorganism population fluctuation could be demonstrated effectively with the method.

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

    Science.gov (United States)

    Sean, David; Slater, Gary W

    2013-03-01

    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.

  14. 利用变性梯度凝胶电泳分析正红菇菌根围土壤真菌群落多样性%Analysis of Fungal Diversity of Russula griseocarnosa Mycorrhizosphere Soil with Denaturing Gradient Gel Electrophoresis

    Institute of Scientific and Technical Information of China (English)

    肖冬来; 陈丽华; 陈宇航; 杨菁; 黄小菁

    2013-01-01

    以正红菇(Russula griseocarnosa)菌根围土壤为研究对象,通过提取土壤基因组DNA,以通用引物扩增真菌18S rRNA基因V1+V2区,将PCR产物进行变性梯度凝胶电泳(Denaturing Gradient Gel Electrophoresis),获得土壤微生物群落的DNA特征指纹图谱,并对图谱中的优势条带回收测序,通过Blast进行同源性比对并构建系统发育树,进而分析正红菇菌根围真菌群落组成及多样性.同源性比对结果表明,在回收测序的19条DGGE条带中,4条为非真菌真核生物序列,系统发育分析显示全部序列可以分为4类菌群,Group Ⅰ主要为担子菌门(Basidiomycota)真菌,GroupⅡ主要为子囊菌门(Ascomycota)真菌,GroupⅢ为未知真菌,GroupⅣ主要为节肢动物门生物(Arthropoda).

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

    NARCIS (Netherlands)

    Lakemond, C.M.M.

    2001-01-01

    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

  16. 利用DGGE分析无龋和有龋儿童牙菌斑细菌组成%Profiling of microbial composition in dental plaque from caries-free and severe early childhood caries children analysed by denaturing gradient gel electrophoresis

    Institute of Scientific and Technical Information of China (English)

    王帅; 黄博; 杜宁; 刘筱娣; 郭丽宏

    2011-01-01

    目的:通过变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)技术分析无龋(caries free,CF)儿童和重型早期婴幼儿龋(severe early childhood caries,SECC)儿童集合牙菌斑内细菌多样性的差异.方法:无龋儿童和SECC儿童各34 例,牙菌斑基因组DNA等量混合后,制备无龋和SECC儿童牙菌斑基因组库,进行全基因组放大.分别以放大前后的基因组为模板,PCR扩增16S rDNA的V2-V3区,DGGE分析,切取SECC样本的特异条带进行克隆、测序、核酸序列比对.结果:基因组在放大前后DGGE图谱一致,SECC组样本的条带数多于CF组的条带数.切取的SECC样本的4 条特异条带测序后证实为3 种未培养微生物和嗜沫嗜血杆菌.结论:利用DGGE技术发现了SECC儿童菌斑与CF儿童菌斑细菌组成的差异,并在SECC样本中发现了区别于CF样本的未培养微生物,这些微生物在致龋过程中发挥的作用还有待研究.%Objective: To analyze the differences of dental plaque microbial composition between caries-free(CF) and severe early childhood caries (SECC) children analysed by denaturing gradient gel electrophoresis (PCR-DGGE). Methods: Genomic DNA was extracted from the dental plaque of CF group (n = 34 ) and SECC group( n = 34) respectively. Each DNA sample of CF and SECC group was mixed equally to construct CF genomic DNA pool and SECC genomic DNA pool, and then the two genomic DNA pools were used as template and amplified by DNA Amplification Kit. V2-V3 region of the 16S rDNA was amplified by PCR and analyzed by DGGE. Four bands in DGGE profile specifically existed in SECC lanes were excised, cloned, sequenced and searched in BLAST to find the closest relatives. Results: After whole genome amplification, DGGE showed the same DNA profile as that without amplification profile, and the bands of SECC lanes were more than those of CF lanes. Three uncultured bacteria and Aggregatibacter aphrophilus were found in SECC group. Conclusion

  17. 发酵乳活性双歧杆菌种类快速识别及定量研究%Isolation of Bifidobacteria from Yoghurts and Assessment of the Bifidobacterial Population by PCR-Denaturing Gradient Gel Electrophoresis and Quantitative Real-Time PCR

    Institute of Scientific and Technical Information of China (English)

    王立平; 刘晓莉; 蔡雪凤; 曹悦; 张帆

    2013-01-01

    In this article, a rapid identification and numberation method for bifidobacterium in fermented milk products was established. Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) technique was used to identify Bifidobacterium present in commercial probiotic yoghurts.Real-Time Polymerase Chain Reaction (Real-Time PCR) technique was used to detect Bifidobacteria. The results showed that PCR-DGGE assays enabled identification of the species of bifidobacterium initially present in commercial fermented milk products with a detection threshold of 105 cells per gram. Real-Time PCR assays enabled numbering Bifidobacterium with a detection threshold of 104 cells per gram of product. Established PCR-DGGE analysis method was suitable for identification and detection of Bifidobacterium in commercial probiotic yoghurts.%本文建立了发酵乳制品中双歧杆菌种类和数量快速测定方法.以发酵乳中双歧杆菌为靶标,采用PCR-DGGE技术快速识别双歧杆菌种类;采用Real-Tine PCR手段测定双歧杆菌数量.研究结果表明,PCR-DGGE能准确、快速鉴别双歧杆菌种类,检出限为105 CFU/g; Real-Tine PCR能准确、快速定量双歧杆菌,检出限为104 CFU/g.该方法可用于发酵乳中双歧杆菌的快速识别和定量.

  18. Use of denaturing gradient gel electrophoresis in screening ...

    African Journals Online (AJOL)

    G. Christopoulos

    2012-08-24

    Aug 24, 2012 ... prevention of the disease through prenatal diagnosis programs. To date, the molecular ... intermedia and 21 b-thalassemia major), one obligate b-thalas- ..... genotyping of beta-thalassemia mutations using DGGE analysis:.

  19. 应用DGGE技术分析自然免耕土与普通耕作土细菌群落的多样性%Analysis of bacterial communities in no-tillage and tillage soils by denaturing gradient gel electrophoresis

    Institute of Scientific and Technical Information of China (English)

    马振刚; 马淑华; 张时恒; 贾俊杰; 李田; 潘国庆; 周泽扬

    2011-01-01

    为探明免耕土壤与普通耕作土壤环境中细菌群落的多样性,获取相关优势菌落信息,该研究利用宏基因组学方法获得免耕土和普通耕作土样品总DNA,利用PCR获得16SrDNAV3片段,并进行变性梯度凝胶电泳(Denaturation gradient gel electrophoresis),通过微生物种群丰富度比较两样品中群落的丰富性,同时选取相关DNA条带进行克隆、测序和生物信息学分析.结果显示:免耕土壤中细菌群落多样性更加丰富;两类型土壤样品间细菌群落组成具有显著差异,证明耕作制度影响了土壤细菌群落结构.BLAST分析与系统发生分析结果表明,免耕土壤中特异存在的细菌群落与具有生物固氮、降解甲苯和倍硫磷等特性的细菌序列相似性较高或进化关系较近,推测其在免耕土壤肥力、有毒物质降解及有机质转变等过程中起作用.%Bacterial population diversity in no-tillage and tillage soils were analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) to get the information on dominant bacteria. The metagenomics technologies were employed to obtain the total DNA, then 16S rDNA V3 region was amplified by PCR. Soil microbial richness was analyzed by 16S rDNA V3-PCR-DGGE. Technologies of cloning and sequencing combined with a phylogeny tree were applied to analyze the evolutional relationship of genes corresponding to several interesting bands involved in this research. The results of DGGE analysis showed that the bacterial population diversity of no-tillage soil was more abundant than that of tillage soils and the UPGMA cluster a-nalysis exhibited a significantly different community composition between no-tillage and tillage soils, demonstrating that the soil tillage system had a remarkable effect on the bacterial communities. Phylogenetic analysis exhibited the distinctive popu-lations specific in no-tillage soil were close to the bacterium chracterized by azotification, methylbenzene and baycid

  20. 利用PCR-DGGE技术监测Lactobacillus plantarum WCFS1对小鼠肠道菌群的动态变化%Monitoring dynamic changes of intestinal microflora in mice by Lactobacillus plantarum WCFS1 using PCR-denaturing gradient gel electrophoresis

    Institute of Scientific and Technical Information of China (English)

    吴青龙; 陈廷涛; 熊顺强; 姜淑英; 李胜杰; 魏华

    2011-01-01

    利用传统培养法和变性梯度凝胶电泳技术(PCR-DGGE)评价Lactobacillus plantarum WCFS1对小鼠肠道微生物数量和种类的影响.结果表明,乳酸杆菌数量在灌胃期和恢复期较空白期显著升高(P<0.05),而肠杆菌数量在灌胃期和恢复期较空白期显著下降(P<0.01);DGGE图谱及多样性分析显示小鼠灌胃期间菌群多样性显著提高(P<0.01),但在停止灌胃7d后与空白期相比无显著性差异(P>0.05).此外,DGGE条带测序表明Lactobacillus plantarum WCFS1在灌胃期间可在大部分小鼠肠道内占据优势地位.%Both traditional culture-dependent method and PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) were used to evaluate the effects of Lactobacillus plantarum WCFS1 on the amount and species of microflora in mice intestinal tract. It is observed that the amount of Lactobacillus in mice intestine during the feeding and recovery stages significantly increased (P<0. 05) when compared with the control stage; while the amount of Enterococcus decreased (P<0. 01). The DGGE pattern and diversity analysis indicated that the diversity of microflora at the feeding stage had great increase (P

  1. A method for easily customizable gradient gel electrophoresis.

    Science.gov (United States)

    Miller, Andrew J; Roman, Brandon; Norstrom, Eric

    2016-09-15

    Gradient polyacrylamide gel electrophoresis is a powerful tool for the resolution of polypeptides by relative mobility. Here, we present a simplified method for generating polyacrylamide gradient gels for routine analysis without the need for specialized mixing equipment. The method allows for easily customizable gradients which can be optimized for specific polypeptide resolution requirements. Moreover, the method eliminates the possibility of buffer cross contamination in mixing equipment, and the time and resources saved with this method in place of traditional gradient mixing, or the purchase of pre-cast gels, are noteworthy given the frequency with which many labs use gradient gel SDS-PAGE. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Protein electrophoretic migration data from custom and commercial gradient gels

    Directory of Open Access Journals (Sweden)

    Andrew J. Miller

    2016-12-01

    Full Text Available This paper presents data related to the article “A method for easily customizable gradient gel electrophoresis” (A.J. Miller, B. Roman, E.M. Norstrom, 2016 [1]. Data is presented on the rate of electrophoretic migration of proteins in both hand-poured and commercially acquired acrylamide gradient gels. For each gel, migration of 9 polypeptides of various masses was measured upon completion of gel electrophoresis. Data are presented on the migration of proteins within separate lanes of the same gel as well as migration rates from multiple gels.

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

    Science.gov (United States)

    Trudel, J; Grenier, J; Asselin, A

    1998-07-01

    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.

  4. 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

    NARCIS (Netherlands)

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

    2002-01-01

    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.

  5. 变性梯度凝胶电泳分析氟化泡沫对牙菌斑微生物的影响%Analysis of the influence of fluoride foam on oral plaque microbiota by PCR-based denaturing gradient gel electrophoresis

    Institute of Scientific and Technical Information of China (English)

    翁金龙; 赵河川; 孙凤; 陈霄迟; 徐韬

    2013-01-01

    genomic DNA of the plaque was isolated.PCR was performed with a set of universal bacterial 16S rDNA primers.The PCR amplified 16S rDNA fragments were separated by denaturing gradient gel electrophoresis (DGGE).The relationship between microflora was assessed by comparing the PCR-DGGE fingerprinting profiles.Results The mean species richness of the bacterial population was 26.9 ± 2.9 at baseline,and fell to 20.1 ± 3.8 after three days of using fluoride foam.The difference was statistically significant (P < 0.05).The overall diversity of plaque samples as measured by the Shannon-Weiner index was 3.18 ±0.31 at baseline and 2.92 ± 0.28 at 3rd day.The difference was statistically significant (P =0.04 by the Mann-Whitney U test).The microbial diversity was changed after the fluoride application by Dice coefficient and clustering analysis.After one month,the microbial diversity was similar to the baseline.Conclusions The results suggest that 1.23% fluoride foam has obviously inhibiting effects on microbial diversity in dental plaque of children during a period of time.The microbial diversity and complexity of the microbial biota in dental plaque were changed after fluoride application and was able to return to original state.

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

    Science.gov (United States)

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

    1993-01-01

    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.

  7. Non-Gradient Blue Native Polyacrylamide Gel Electrophoresis.

    Science.gov (United States)

    Luo, Xiaoting; Wu, Jinzi; Jin, Zhen; Yan, Liang-Jun

    2017-02-02

    Gradient blue native polyacrylamide gel electrophoresis (BN-PAGE) is a well established and widely used technique for activity analysis of high-molecular-weight proteins, protein complexes, and protein-protein interactions. Since its inception in the early 1990s, a variety of minor modifications have been made to this gradient gel analytical method. Here we provide a major modification of the method, which we call non-gradient BN-PAGE. The procedure, similar to that of non-gradient SDS-PAGE, is simple because there is no expensive gradient maker involved. The non-gradient BN-PAGE protocols presented herein provide guidelines on the analysis of mitochondrial protein complexes, in particular, dihydrolipoamide dehydrogenase (DLDH) and those in the electron transport chain. Protocols for the analysis of blood esterases or mitochondrial esterases are also presented. The non-gradient BN-PAGE method may be tailored for analysis of specific proteins according to their molecular weight regardless of whether the target proteins are hydrophobic or hydrophilic. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  8. Impact of Delivery Mode on Intestinal Microbiota in Early Neonates by Polymerase Chain Reaction - Denaturing Gradient Gel Electrophoresis%聚合酶链反应-变性梯度凝胶电泳技术动态分析不同分娩方式对新生儿早期肠道菌群的影响

    Institute of Scientific and Technical Information of China (English)

    刘彩霞; 胡燕; 周东蕊; 蒋犁

    2012-01-01

    Objective To investigate the effects of delivery mode on the dynamic changes of early gut microflora by using polymerase chain reaction - degeneration gradient gel electrophoresis (PCR - DGGE) method. Methods Thirty - two normal term neonates were enrolled in the study (including vaginal delivery group and caesarean section group,each group including 16 neonates) ,and feces samples were collected on the first day( > 12 h) ,3th day and 5th day after birth. After bacterial DNAs were extracted from the feces of the 2 groups,bacterial communities in 2 groups were examined by PCR of 16S rDNA V3 region and DGGE. After DGGE profilings were obtained,the diversity and similarity related indices were used to analyze diversity differences of the gut bacterium structure between 2 groups and the similar degrees between individuals within each group. Results 1. There were few species and quantities in the intestinal microbiota during the first day of life, and the early intestinal bacterial colonization of neonates had a complex individual specificity and presented dynamic changes. 2. The diversity analysis between 2 groups showed that there were no obvious diversity differences between the 2 groups on 3' day after birth. While on 5' day after birth,the Shannon - Weaver Diversity Index(H') and the simpson index(D) in caesarean section group were lower significandy than those in vaginal delivery group(Pa <0.05).3. The similarity analysis showed that the dice similarity coefficient(Cs)between individuals in cesarean section group was higher than that in vaginal delivery group on 3th day after birth, and the Cs lower than (or equal to)0. 30 took up 21.4% of the total Cs in cesarean section group and 76.0%in vaginal delivery group,which had all significant differences between 2 groups (P <0.05). On 5th day after birth,there were no obvious differences between groups in Cs total level and its distribution. Conclusions The intra - group similarity of gut bacterium structure is

  9. A PCR-denaturing gradient gel electrophoresis (DGGE) approach to assess Fusarium diversity in asparagus

    NARCIS (Netherlands)

    Yergeau, E.; Filion, M.; Vujanovic, V.; St-Arnaud, M.

    2005-01-01

    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 org

  10. 7 NEW MUTATIONS IN HMSH2, AN HNPCC GENE, IDENTIFIED BY DENATURING GRADIENT-GEL ELECTROPHORESIS

    NARCIS (Netherlands)

    WIJNEN, J; VASEN, H; KHAN, PM; MENKO, FH; VANDERKLIFT, H; VANLEEUWEN, C; VANDENBROEK, M; VANLEEUWENCORNELISSE, [No Value; NAGENGAST, F; MEIJERSHEIJBOER, A; LINDHOUT, D; GRIFFIOEN, G; CATS, A; KLEIBEUKER, J; VARESCO, L; BERTARIO, L; BISGAARD, ML; MOHR, J; FODDE, R

    1995-01-01

    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 individ

  11. A PCR-denaturing gradient gel electrophoresis (DGGE) approach to assess Fusarium diversity in asparagus

    NARCIS (Netherlands)

    Yergeau, E.; Filion, M.; Vujanovic, V.; St-Arnaud, M.

    2005-01-01

    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

  12. Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis.

    Science.gov (United States)

    Nigg, Patricia E; Pavlovic, Jovan

    2016-10-28

    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.

  13. An improved silver staining procedure for schizodeme analysis in polyacrylamide gradient gels

    Directory of Open Access Journals (Sweden)

    Antonio M. Gonçalves

    1990-03-01

    Full Text Available A simple protocol is described for the silver staining of polyacrylamide gradient gels used for the separation of restriction fragments of kinetoplast DNA [schizodeme analysis of trypanosomatids (Morel et al., 1980]. The method overcomes the problems of non-uniform staining and strong background color which are frequently encountered when conventional protocols for silver staining of linear gels. The method described has proven to be of general applicability for DNA, RNA and protein separations in gradient gels.

  14. Rapid (ten-minute) pore-gradient electrophoresis of proteins and peptides in Micrograd gels.

    Science.gov (United States)

    Wrigley, C W; Margolis, J

    1992-01-01

    Precast gradient gels of short migration length (25 mm) have been developed to provide rapid electrophoretic separation without loss of resolution. These Micrograd gels have been prepared in gel ranges (conventional and unique) to match pore-gradient electrophoresis conditions to proteins/peptides ranging in size from several hundreds to millions. The Hylinx Micrograd gel combines an extreme gel range (6 to 48% polyacrylamide) with a novel crosslinker to provide sieving of polypeptides, and pore-limit electrophoresis of the smallest proteins (e.g. insulin monomer). All gel ranges (such as 3 to 30%) provide zone sharpening in routine analysis of conventional protein mixtures (e.g. serum) within 10 min electrophoresis at 200 to 300 volts. The gels are thin (1 mm) and thus stain quickly, but the gel cassette is of conventional overall width (83 mm), thus fitting many apparatus designs and accommodating 12 samples. The gels are finding valuable use in screening applications, requiring the electrophoretic analysis of many samples, and in cases where a rapid answer is needed, such as monitoring protein purification. The gels have proved particularly useful, in-house, for the latter application in developing Gradipore's new large-scale preparative electrophoresis system, the Gradiflow.

  15. Gradient Index Lenses From Sol-Gel Layering.

    Science.gov (United States)

    2008-02-15

    communications 6 At present, several techniques are used to produce gradient index glassý-10 but most of the processes are limited by the small index...6 8 10 12 14 Depth in mm Figure 6. Refractive index as a funcion of depth for a 1_TiO 2-85SiO 2 GRIN glass #2. A He-Ne laser light beam was used to

  16. Compressive Mechanical Characteristics of Multi-layered Gradient Hydroxyapatite Reinforced Poly (Vinyl Alcohol) Gel Biomaterial

    Institute of Scientific and Technical Information of China (English)

    Yusong Pan; Qianqian Shen; Chengling Pan; Jing Wang

    2013-01-01

    Functional gradient materials provided us a new concept for artificial articular cartilage design with gradient component and gradient structure where one side of the material is high free water content thereby providing excellent lubrication function and the opposite side of the material is high hydroxyapatite content,thereby improving the bioactivity of the material and stimulating cell growth.The goal of the present study was to develop a multi-layered gradient HA/PVA gel biocomposites through layer-by-layer casting method combing with freeze/thaw cycle technology.The various influence factors on the compressive strength and modulus of the multi-layered gradient biocomposites were investigated.The results showed that the compressive mechanical characteristics of the biocomposites were similar to that of natural articular cartilage.Both the compressive strength and modulus of the multi-layered gradient HA/PVA gel biocomposites increased exponentially with the rise of compressive strain ratio.Both the compressive strength and average compressive modulus of the biocomposites improved with the rise of freeze/thaw cycle times and total concentration of HA particles in the biocomposites,but they showed decreasing tendency with the rise of HA concentration difference between adjacent layers.

  17. Gel for simultaneous chemical imaging of anionic and cationic solutes using diffusive gradients in thin films.

    Science.gov (United States)

    Kreuzeder, Andreas; Santner, Jakob; Prohaska, Thomas; Wenzel, Walter W

    2013-12-17

    We report on a novel gel based on diffusive gradients in thin films (DGT) for the simultaneous measurement of cations and anions and its suitability for high resolution chemical imaging by using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS). The new high resolution mixed binding gel (HR-MBG) is based on zirconium-hydroxide and suspended particulate reagent-iminodiacetate (SPR-IDA) as resin materials which are embedded in an ether-based urethane polymer hydrogel. The use of this polymer hydrogel material allows the production of ultrathin, highly stable and tear-proof resin gel layers with superior handling properties compared to existing ultrathin polyacrylamide gels. The gel was characterized regarding its uptake kinetics, the anion and cation capacities, and the effects of pH, ionic strength, and aging on the performance of the HR-MBG. Our results demonstrate the capability of this novel gel for concomitant sampling of anions and cations. The suitability of this new gel type for DGT chemical imaging at submm spatial resolution in soils using LA-ICPMS is shown. 2D images of P, As, Co, Cu, Mn, and Zn distributions around roots of Zea mays L. demonstrate the new opportunities offered by the HR-MBG for high-resolution mapping of solute dynamics in soil and sediment hotspots, such as the rhizosphere, by simultaneous observation of anionic and cationic solute species.

  18. Motion of an Adhesive Gel in a Swelling Gradient a Mechanism for Cell Locomotion

    CERN Document Server

    Joanny, J F; Prost, J; Joanny, Jean-Francois; Julicher, Frank; Prost, Jacques

    2003-01-01

    Motivated by the motion of nematode sperm cells, we present a model for the motion of an adhesive gel on a solid substrate. The gel polymerizes at the leading edge and depolymerizes at the rear. The motion results from a competition between a self-generated swelling gradient and the adhesion on the substrate. The resulting stress provokes the rupture of the adhesion points and allows for the motion. The model predicts an unusual force-velocity relation which depends in significant ways on the point of application of the force.

  19. EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Chevreux, Sylviane; Roudeau, Stephane; Deves, Guillaume; Ortega, Richard [Laboratoire de Chimie Nucleaire Analytique et Bioenvironnementale, CNRS UMR5084, Universite Bordeaux 1, Chemin du Solarium, F-33175 Gradignan cedex (France); Solari, Pier Lorenzo [Synchrotron SOLEIL, L' Orme des Merisiers, BP 48, F-91192 Gif-sur-Yvette cedex, Saint-Aubin (France); Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis, E-mail: ortega@cenbg.in2p3.f [FAME, ESRF, 6 rue Jules Horowitz, BP220, F-38043 Grenoble cedex (France)

    2009-11-15

    Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

  20. EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

    Science.gov (United States)

    Chevreux, Sylviane; Solari, Pier Lorenzo; Roudeau, Stéphane; Deves, Guillaume; Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis; Ortega, Richard

    2009-11-01

    Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

  1. Tris-acetate polyacrylamide gradient gel electrophoresis for the analysis of protein oligomerization.

    Science.gov (United States)

    Cubillos-Rojas, Monica; Schneider, Taiane; Sánchez-Tena, Susana; Bartrons, Ramon; Ventura, Francesc; Rosa, Jose Luis

    2016-02-01

    Here we report a new approach for studying protein oligomerization in cells using a single electrophoresis gel. We combined the use of a crosslinking reagent for sample preparation, such as glutaraldehyde, with the analysis of oligomers by Tris-acetate polyacrylamide gel electrophoresis. The use of a 3-15% Tris-acetate polyacrylamide gradient gel allows for the simultaneous analysis of proteins of masses ranging from 10 to 500 kDa. We showed the usefulness of this method for analyzing endogenous p53 oligomerization with high resolution and sensitivity in human cells. Oligomerization analysis was dependent on the crosslinker concentration used. We also showed that this method could be used to study the regulation of oligomerization. In all experiments, Tris-acetate polyacrylamide gel electrophoresis proved to be a robust, manageable, and cost- and time-efficient method that provided excellent results using a single gel. This approach can be easily extrapolated to the study of other oligomers. All of these features make this method a highly useful tool for the analysis of protein oligomerization.

  2. Association of Stremptomyces community composition determined by PCR-denaturing gradient gel electrophoresis with indoor mold status.

    Science.gov (United States)

    Abstract 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 cultureindependent met...

  3. Assessment of the genotypic diversity of antibiotic-producing Pseudomonas species in the rhizosphere by denaturing gradient gel electrophoresis

    NARCIS (Netherlands)

    Bergsma-Vlami, M.; Prins, M.E.; Staats, M.; Raaijmakers, J.M.

    2005-01-01

    The genotypic diversity of antibiotic-producing Pseudomonas spp. provides an enormous resource for identifying strains that are highly rhizosphere competent and superior for biological control of plant diseases. In this study, a simple and rapid method was developed to determine the presence and gen

  4. High-resolution differentiation of cyanobacteria by using rRNA-Internal Transcribed Spacer denaturing gradient gel electrophoresis

    NARCIS (Netherlands)

    Janse, I.; Meima, M.; Kardinaal, W.E.A.; Zwart, G.

    2003-01-01

    For many ecological studies of cyanobacteria, it is essential that closely related species or strains can be discriminated. Since this is often not possible by using morphological features, cyanobacteria are frequently studied by using DNA-based methods. A powerful method for analysis of the diversi

  5. Association of Stremptomyces community composition determined by PCR-denaturing gradient gel electrophoresis with indoor mold status.

    Science.gov (United States)

    Abstract 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 cultureindependent met...

  6. Complexity of Vaginal Microflora as Analyzed by PCR Denaturing Gradient Gel Electrophoresis in a Patient With Recurrent Bacterial Vaginosis

    Directory of Open Access Journals (Sweden)

    Gregor Reid

    2005-01-01

    Full Text Available Objective:Gardnerella vaginalis has long been the most common pathogen associated with bacterial vaginosis (BV. We aimed to test our hypothesis that symptoms and signs of BV do not necessarily indicate colonization by this organism, and often will not respond to standard metronidazole or clindamycin treatment.

  7. Denaturing gradient electrophoresis (DGE) and single-strand conformation polymorphism (SSCP) molecular fingerprintings revisited by simulation and used as a tool to measure microbial diversity.

    Science.gov (United States)

    Loisel, Patrice; Harmand, Jérôme; Zemb, Olivier; Latrille, Eric; Lobry, Claude; Delgenès, Jean-Philippe; Godon, Jean-Jacques

    2006-04-01

    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.

  8. [Denaturalized psychoanalysts].

    Science.gov (United States)

    Peglau, Andreas

    2011-01-01

    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.

  9. Genotyping BoLA-DRB3 alleles in Brazilian Dairy Gir cattle (Bos indicus) by temperature-gradient gel electrophoresis (TGGE) and direct sequencing.

    Science.gov (United States)

    Da Mota, A F; Martinez, M L; Coutinho, L L

    2004-02-01

    BoLA-DRB3 is a gene of the major histocompatibility complex (MHC) in cattle. The product of the BoLA-DRB3 gene is a beta chain of an MHC class II molecule, a glycoprotein expressed on the surface of antigen-presenting cells (APCs). Responses of CD4+ T lymphocytes to peptides are dependent on the presentation of peptide ligands bound to class II molecules on APCs. Genotyping of the BoLA-DRB3 gene is relatively complex due to the extensive polymorphism of this locus. Current techniques for assignment of genotypes are polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), direct sequencing of PCR products, cloning-sequencing, polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP), and denaturant-gradient gel electrophoresis. These techniques are time-consuming, do not discriminate all possible alleles, or are not readily reproducible. The objective of this study was to genotype BoLA-DRB3 using temperature-gradient gel electrophoresis (TGGE) to separate alleles before sequencing. PCRs using 28 DNA samples from Gir Dairy cattle (a Brazilian breed of Bos indicus) were submitted to TGGE. New PCR products were generated from separated alleles, purified, and sequenced. Allele separation was possible in 21 out of 26 heterozygote samples (81%). Results indicate that two sequence reads (forward and reverse) were sufficient for accurate genotyping of BoLA-DRB3 alleles. Separation of alleles by TGGE provides high-throughput, reliable typing of BoLA-DRB3, which is critical in disease association studies in cattle.

  10. ANALYSIS OF BACTERIAL COMMUNITIES IN SEAGRASS BED SEDIMENTS BY DOUBLE-GRADIENT DENATURING GRADIENT GEL ELECTROPHORESIS OF PCR-AMPLIFIED 16SRRNA GENES

    Science.gov (United States)

    Bacterial communities associated with seagrass bed sediments are not well studied. The work presented here investigated several factors, including the presence or absence of vegetation, depth into sediment, and season, and their impact on bacterial community diversity. Double gra...

  11. ANALYSIS OF BACTERIAL COMMUNITIES IN SEAGRASS BED SEDIMENTS BY DOUBLE-GRADIENT DENATURING GRADIENT GEL ELECTROPHORESIS OF PCR-AMPLIFIED 16SRRNA GENES

    Science.gov (United States)

    Bacterial communities associated with seagrass bed sediments are not well studied. The work presented here investigated several factors, including the presence or absence of vegetation, depth into sediment, and season, and their impact on bacterial community diversity. Double gra...

  12. Metal imaging in non-denaturating 2D electrophoresis gels by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) for the detection of metalloproteins.

    Science.gov (United States)

    Becker, J Susanne; Lobinski, Ryszard; Becker, J Sabine

    2009-01-01

    Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was developed as a powerful analytical technique for metal imaging of 2D gels for the detection of metalloproteins in rat kidney after electrophoretic separation. Protein complexes, extracted with water, were separated in their native state in the first and second dimension by blue native gel electrophoresis (BN-PAGE). Essential and toxic metals, such as zinc, copper, iron, manganese and lead, were monitored by LA-ICP-MS after gel ablation by a focused laser beam in a way that the total surface of a selected fragment of the gel was totally ablated. The metal distribution of this part of the gel was then constructed by plotting the metal (isotope) signal intensity as a function of the x,y (isoelectric point, molecular mass) coordinates of the gel. The proteins at locations rich in metals were cut out, digested with trypsin and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

  13. Population dynamics of two antilisterial cheese surface consortia revealed by temporal temperature gradient gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Hasler Madlen

    2010-03-01

    Full Text Available Abstract Background Surface contamination of smear cheese by Listeria spp. is of major concern for the industry. Complex smear ecosystems have been shown to harbor antilisterial potential but the microorganisms and mechanisms involved in the inhibition mostly remain unclear, and are likely related to complex interactions than to production of single antimicrobial compounds. Bacterial biodiversity and population dynamics of complex smear ecosystems exhibiting antilisterial properties in situ were investigated by Temporal temperature gradient gel electrophoresis (TTGE, a culture independent technique, for two microbial consortia isolated from commercial Raclette type cheeses inoculated with defined commercial ripening cultures (F or produced with an old-young smearing process (M. Results TTGE revealed nine bacterial species common to both F and M consortia, but consortium F exhibited a higher diversity than consortium M, with thirteen and ten species, respectively. Population dynamics were studied after application of the consortia on fresh-produced Raclette cheeses. TTGE analyses revealed a similar sequential development of the nine species common to both consortia. Beside common cheese surface bacteria (Staphylococcus equorum, Corynebacterium spp., Brevibacterium linens, Microbacterium gubbeenense, Agrococcus casei, the two consortia contained marine lactic acid bacteria (Alkalibacterium kapii, Marinilactibacillus psychrotolerans that developed early in ripening (day 14 to 20, shortly after the growth of staphylococci (day 7. A decrease of Listeria counts was observed on cheese surface inoculated at day 7 with 0.1-1 × 102 CFU cm-2, when cheeses were smeared with consortium F or M. Listeria counts went below the detection limit of the method between day 14 and 28 and no subsequent regrowth was detected over 60 to 80 ripening days. In contrast, Listeria grew to high counts (105 CFU cm-2 on cheeses smeared with a defined surface culture

  14. DIFFERENTIATION OF CROATIAN BARLEY VARIETIES BY GRADIENT GEL SDS-PAGE AND ISOELECTRIC FOCUSING OF DRY GRAINS AND GREEN MALT HORDEINS

    Directory of Open Access Journals (Sweden)

    Ivica Strelec

    2011-06-01

    Full Text Available Applicability of polyacrylamide gel electrophoresis and isoelectric focusing of hordeins for discrimination of six two-rowed winter barley varieties (Angora, Sladoran, Rodnik, Rex, Martin and Barun has been investigated. Hordeins extracted from dry grains and green malt of barley varieties were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in gradient gel of 8-18% T, and by isoelectric focusing in pH gradient of 3.5-9.5 and 5.5-8.5, respectively. In all separation experiments better resolution of proteins was achieved with dry grain extracts, than with malt extracts. Angora, Sladoran and Martin variety could be distinguished from other varieties by differences in hordein patterns obtained by gradient gel SDS-PAGE (8-18% T, and Angora, Sladoran, Martin and Rodnik by isoelectric focusing in pH gradient 5.5-8.5.

  15. Correlation of acidic and basic carrier ampholyte and immobilized pH gradient two-dimensional gel electrophoresis patterns based on mass spectrometric protein identification

    DEFF Research Database (Denmark)

    Nawrocki, A; Larsen, Martin Røssel; Podtelejnikov, A V

    1998-01-01

    Separation of proteins on either carrier ampholyte-based or immobilized pH gradient-based two-dimensional (2-D) gels gives rise to electrophoretic patterns that are difficult to compare visually. In this paper we have used matrix-assisted laser desorption/ionization mass spectrometry (MALDI......-references demonstrated that there is no obvious pattern by which the mobility of a protein in one gel system can be used to predict its mobility in the other. Thus, as laboratories adopt the immobilized pH gradient-based 2-D gel systems, the only reliable means of translating the data gained with the carrier ampholyte......-MS) to determine the identities of 335 protein spots in these two 2-D gel systems, including a substantial number of basic proteins which had never been identified before. Proteins that were identified in both gel systems allowed us to cross-reference the gel patterns. Vector analysis of these cross...

  16. Mapping and identification of interferon gamma-regulated HeLa cell proteins separated by immobilized pH gradient two-dimensional gel electrophoresis

    DEFF Research Database (Denmark)

    Shaw, AC; Rossel Larsen, M; Roepstorff, P

    1999-01-01

    magnitude of IFN-gamma responsive genes has been reported previously. Our goal is to identify and map IFN-gamma-regulated HeLa cell proteins to the two-dimensional polyacrylamide gel electrophoresis with the immobilized pH gradient (IPG) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) system...

  17. Efficient separation of semiconducting single-wall carbon nanotubes by surfactant-composition gradient in gel filtration

    Science.gov (United States)

    Thendie, Boanerges; Omachi, Haruka; Miyata, Yasumitsu; Shinohara, Hisanori

    2017-01-01

    Gel filtration is a powerful method of separating and purifying semiconducting single-wall carbon nanotubes (s-SWCNTs) from their metallic (m-) counterpart. However, a small amount of m-SWCNTs usually remains, thus reducing the purity of the s-SWCNTs obtained. We have investigated the effect of elution with a gradient concentration of the surfactant on the separation and purity of s-SWCNTs. By utilizing the controlled low-gradient elution (CLGE) that we have developed, the purity of s-SWCNTs is improved to 94% from the 90% obtained with the conventional separation. Furthermore, CLGE simultaneously allows diameter-based separation of small-diameter s-SWCNTs, which indicates a promising utilization of CLGE for s-SWCNT separation.

  18. Role of pH gradients in the actuation of electro-responsive polyelectrolyte gels

    NARCIS (Netherlands)

    Glazer, P.J.; van Erp, M.; Embrechts, A.; Lemay, Serge Joseph Guy; Mendes, E.

    2012-01-01

    Polyelectrolyte gels are able to mimic artificial muscles, swelling, shrinking or bending in response to environmental stimuli. Mechanical response is also observed in the presence of an electric field, in which case electrical energy is directly converted into mechanical energy. Although several

  19. Fabrication and Crystal Structure of Sol-Gel Deposited BST Thin Films with Compositional Gradient

    Directory of Open Access Journals (Sweden)

    Czekaj D.

    2017-06-01

    Full Text Available In the present research technology of compositionally graded barium strontium titanate Ba1-xSrxTiO3 thin films deposited on stainless steel substrates by sol-gel spin coating followed with thermal annealing at T = 650°C is reported. Results of thermal behavior of the sol-gel derived powders with compositions used for fabrication of graded structure (i.e. with Sr mole fraction x = 0.5, 0.4 and 0.3 are described. X-ray diffraction studies of the phase composition and crystal structure of such complex thin film configuration are given. It was found that gel powders exhibited a large total weight loss of about Δm ≈ 44-47%. Three stages of weight loss took place at temperature ranges: below T ≈ 300°C, at ΔT ≈ 300-500°C and between T = 600°C and T = 800°C. Phase analysis has shown that the dominating phase is Ba0.67Sr0.33TiO3 compound while the second phase is Ba0.7Sr0.3TiO3 or Ba0.5Sr0.5TiO3 for “up-graded” and “down-graded” structure, respectively.

  20. Preparation of polyacrylamide gel-filled capillaries with step gradients and low UV-detection background

    Institute of Scientific and Technical Information of China (English)

    陈义

    1997-01-01

    Polyacrylamide- filled capillaries with step gradients were designed and prepared with a newly established method,which is also suitable for producing other sorts of capillaries.The resulting capillaries allow the use of any UV light to approach the most sensitive detection and have the features of fast running speed and high separation efficiency In addition,the capillaries can he used continuously for more than two weeks.

  1. "Leucine aminopeptidase" (neutral arylamidase) in sheep sera: improved resolution with gradient gel electrophoresis.

    Science.gov (United States)

    Manwell, C; Baker, C M

    1986-01-01

    Electrophoretic resolution of the heterogeneity of sheep serum "leucine aminopeptidase" is greatly improved by the use of gradients of acrylamide polymer, together with enzyme localisation involving L-alanyl beta-naphthylamide and cobaltous ion. The improved resolution contradicts an earlier claim of the existence of only two patterns of individual variation in the heterogeneity of sheep serum "leucine aminopeptidase", with one pattern completely dominant to the other. While the sheep enzyme is unusual among mammalian serum "leucine aminopeptidases" in its complex heterogeneity, it does conform to the typical mammalian pattern of codominant individual variation. The complexity of sheep serum "leucine aminopeptidase" is useful in the study of sheep evolution.

  2. Using the polymerase chain reaction coupled with denaturing gradient gel electrophoresis to investigate the association between bacterial translocation and systemic inflammatory response syndrome in predicted acute severe pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Callum B Pearce; Vitaly Zinkevich; Iwona Beech; Viera Funjika; Ana Garcia Ruiz; Afraa Aladawi; Hamish D Duncan

    2005-01-01

    AIM: To investigate the use of PCR and DGGE to investigate the association between bacterial translocation and systemic inflammatory response syndrome in predicted severe AP.METHODS: Patients with biochemical and clinical evidence of acute pancreatitis and an APACHE Ⅱ score ≥8 were enrolled. PCR and DGGE were employed to detect bacterial translocation in blood samples collected on d1,3, and 8 after the admission. Standard microbial blood cultures were taken when there was clinical evidence of sepsis or when felt to be clinically indicated by the supervising team.RESULTS: Six patients were included. Of all the patients investigated, only one developed septic complications;the others had uneventful illness. Bacteria were detected using PCR in 4 of the 17 collected blood samples. The patient with sepsis was PCR-positive in two samples (taken on d 1 and 3), despite three negative blood cultures. Using DGGE and specific primers, the bacteria in all blood specimens which tested positive for the presence of bacterial DNA were identified as E coli.CONCLUSION: Our study confirmed thatunlike traditional microbiological techniques, PCR can detect the presence of bacteria in the blood of patients with severe AP. Therefore, this latter method in conjunction with DGGE is potentially an extremely useful tool in predicting septic morbidity and evaluating patients with the disease. Further research using increased numbers of patients, in particular those patients with necrosis and sepsis, is required to assess the reliability of PCR and DGGE in the rapid diagnosis of infection in AP.

  3. The impact of different DNA extraction methods on the analysis of microbial diversity of oral saliva from healthy youths by polymerase chain reaction-denaturing gradient gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Ming Chen

    2016-03-01

    Conclusion: PCR-DGGE was more accurate in assessing oral microbial diversity by QIAamp DNA Micro Kit. Different individuals had large differences in oral microbial diversity but also had some common microbial dominant communities.

  4. Influences of Different DNA Polymerase for Denaturing Gradient Gel Electrophoresis(DGGE)%不同DNA聚合酶对PCR-DGGE技术的影响

    Institute of Scientific and Technical Information of China (English)

    张敏; 蔡俊鹏

    2011-01-01

    [目的]研究不同DNA聚合酶对PCR-DGGE技术的影响.[方法]选用3种不同的DNA聚合酶对单一的7株菌进行PCR扩增,通过DGGE图谱分析探讨不同的DNA聚合酶对PCR-DGGE技术的影响.[结果]用相同的DNA聚合酶1时,上游引物使用357F1比357F2的效果要好.当357F1作为上游引物时,DNA聚合酶2明显好于DNA聚合酶1,但仍有干扰结果出现;当357F2作为上游引物时,使用DNA聚合酶1和DNA聚合酶2都不能使各菌株分离开来.使用高质量的DNA聚合酶3时,上游引物所加入的无论是40 bp还是只有18 bp的"GC"夹,都能够使不同的DNA片段分离开来.[结论]不同的DNA聚合酶对PCR-DGGE技术的影响是很大的,选择合适的DNA聚合酶是至关重要的.%[ Objective ] The research aimed to study the influences of different DNA polymerase for DGGE. [ Method ] 7 strains were PCR amplified by using three different DNA polymerase ,through the analysis of profiles to compare the influence of different DNA polymerase for DGGE.[ Result] When using the same DNA polymerase 1, the effects of forward primer 357F1 was better than 357F2. When the forward primer was 357F1 ,the effects of DNA polymerase 2 was.better DNA polymerase l ,but still had interference. When the forward primer was 357F2,DNA polymerase 1 and DNA polymerase 2 could both separate 7 strains. When high quality DNA polymerase 3 was used,whatever the forward primer was 40 bp and 18 bp GC-clamp the DNA fragment could all be separated. [Conclusion] Different DNA polymerase has big influence on PCR- DGGE,and it is important to selecting suitable DNA polymerase.

  5. Detection and characterization of fungal infections of Ammophila arenaria (marram grass) roots by denaturing gradient gel electrophoresis of specifically amplified 18S rDNA

    NARCIS (Netherlands)

    Kowalchuk, G.A.; Gerards, S.; Woldendorp, J.W.

    1997-01-01

    Marram grass (Ammophila arenaria L.), a sand stabilizing plant species in coastal dune areas, is affected by a specific pathosystem thought to include both plant-pathogenic fungi and nematodes, To study the fungal component of this pathosystem, we developed a method for the cultivation-independent d

  6. 16S rRNA gene-based identification of bacteria in postoperative endophthalmitis by PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) fingerprinting

    OpenAIRE

    Yendi Navarro-Noya; César Hernández-Rodríguez; Zenteno, Juan C.; Beatriz Buentello-Volante; Cancino-Díaz, Mario E.; Janet Jan-Roblero; Juan C. Cancino-Díaz

    2012-01-01

    Conventional microbiological culture techniques are frequently insufficient to confirm endophthalmitis clinical cases which could require urgent medical attention because it could lead to permanent vision loss. We are proposing PCR-DGGE and 16S rRNA gene libraries as an alternative to improve the detection and identification rate of bacterial species from endophthalmitis cases.

  7. Analysis of Fusarium populations in a soybean field under different fertilization management by real-time quantitative PCR and denaturing gradient gel electrophoresis

    Science.gov (United States)

    The abundance and population structure of Fusarium spp. in field soils were assessed to determine the effect of different fertilization treatments on soil microbial community and potential role in disease management. The field was under soybean-wheat-corn rotation located in the black soil (Udic Mo...

  8. Analysis of endophytic bacterial communities of potato by plating and denaturing gradient gel electrophoresis (DGGE) of 16S rDNA based PCR fragments

    NARCIS (Netherlands)

    Garbeva, P.; Overbeek, van L.S.; Vuurde, van J.W.L.; Elsas, van J.D.

    2001-01-01

    The diversity of endophytic bacterial populations of potato (Solanum tuberosum cv Desiree) was assessed using a combination of dilution plating of plant macerates followed by isolation and characterization of isolates, and direct PCR-DGGE on the basis of DNA extracted from plants. The culturable end

  9. Denaturing Gradient Gel Electrophoretic Analysis of Ammonia-Oxidizing Bacterial Community Structure in the Lower Seine River: Impact of Paris Wastewater Effluents

    NARCIS (Netherlands)

    Cébron, A.; Coci, M.; Garnier, J.; Laanbroek, H.J.

    2004-01-01

    The Seine River is strongly affected by the effluents from the Achères wastewater treatment plant (WWTP) downstream of the city of Paris. We have shown that the effluents introduce large amounts of ammonia and inoculate the receiving medium with nitrifying bacteria. The aim of the present study was

  10. Denaturing gradient gel electrophoretic analysis of ammonia-oxidizing bacterial community structure in the lower Seine River: Impact of Paris wastewater effluents

    NARCIS (Netherlands)

    Cebron, A.; Coci, M.; Garnier, J.; Laanbroek, H.J.

    2004-01-01

    The Seine River is strongly affected by the effluents from the Acheres wastewater treatment plant (WWTP) downstream of the city of Paris. We have shown that the effluents introduce large amounts of ammonia and inoculate the receiving medium with nitrifying bacteria. The aim of the present study was

  11. Denaturing Gradient Gel Electrophoretic Analysis of Ammonia-Oxidizing Bacterial Community Structure in the Lower Seine River: Impact of Paris Wastewater Effluents

    NARCIS (Netherlands)

    Cébron, A.; Coci, M.; Garnier, J.; Laanbroek, H.J.

    2004-01-01

    The Seine River is strongly affected by the effluents from the Achères wastewater treatment plant (WWTP) downstream of the city of Paris. We have shown that the effluents introduce large amounts of ammonia and inoculate the receiving medium with nitrifying bacteria. The aim of the present study

  12. Design and evaluation of nematode 18S rDNA primers for PCR and denaturing gradient gel electrophoresis (DGGE) of soil community DNA

    NARCIS (Netherlands)

    Waite, I.S.; O'Donnell, A.G.; Harrison, A.; Davies, J.T.; Colvan, S.R.; Ekschmitt, K.; Dogan, H.; Wolters, V.; Bongers, A.M.T.; Bongers, M.; Bakonyi, G.; Nagy, P.; Papatheodorou, E.M.; Stamou, G.P.; Boström, S.

    2003-01-01

    Consensus nematode 185 ribosomal DNA primers were designed by aligning available 185 sequences and identifying a variable region flanked by highly conserved regions. These primers were then used to amplify nematode 18S rDNA from whole soil community DNA extracted from a range of European grassland t

  13. DENATURING GRADIENT GEL-ELECTROPHORESIS FOR RAPID DETECTION OF LATENT CARRIERS OF A SUBTYPE OF ACUTE INTERMITTENT PORPHYRIA WITH NORMAL ERYTHROCYTE PORPHOBILINOGEN DEAMINASE ACTIVITY

    NARCIS (Netherlands)

    BOURGEOIS, F; GU, XF; DEYBACH, JC; VELDE, MPT; DEROOIJ, F; NORDMANN, Y; GRANDCHAMP, B

    1992-01-01

    Acute intermittent porphyria is an autosomal dominant disorder defined by a partial deficiency of porphobilinogen deaminase (EC 4.3.1.8). Clinical manifestations of the disease are characterized by acute attacks of neurological dysfunction often linked to environmental factors. Early diagnosis of ge

  14. Mapping and identification of HeLa cell proteins separated by immobilized pH-gradient two-dimensional gel electrophoresis and construction of a two-dimensional polyacrylamide gel electrophoresis database

    DEFF Research Database (Denmark)

    Shaw, AC; Rossel Larsen, M; Roepstorff, P

    1999-01-01

    improvements of two-dimensional gel electrophoresis with immobilized pH gradients (IPG) compared to isoelectric focusing with carrier ampholytes, a highly reproducible method for examining global changes in HeLa cell protein expression due to different stimuli is now available. Therefore, we have initiated...

  15. Mapping and identification of HeLa cell proteins separated by immobilized pH-gradient two-dimensional gel electrophoresis and construction of a two-dimensional polyacrylamide gel electrophoresis database

    DEFF Research Database (Denmark)

    Shaw, AC; Rossel Larsen, M; Roepstorff, P

    1999-01-01

    improvements of two-dimensional gel electrophoresis with immobilized pH gradients (IPG) compared to isoelectric focusing with carrier ampholytes, a highly reproducible method for examining global changes in HeLa cell protein expression due to different stimuli is now available. Therefore, we have initiated...

  16. Comparison between Gradient Gel Electrophoresis and Nuclear Magnetic Resonance Spectroscopy in Estimating Coronary Heart Disease Risk Associated with LDL and HDL Particle Size

    NARCIS (Netherlands)

    B.J. Arsenault; I. Lemieux; J.P. Després; N.J. Wareham; E.S.G. Stroes; J.J.P. Kastelein; K.T. Khaw; S.M. Boekholdt

    2010-01-01

    BACKGROUND: Gradient gel electrophoresis (GGE) and nuclear magnetic resonance (NMR) spectroscopy are both widely accepted methods for measuring LDL and HDL particle size. However, whether or not GGE- or NMR-measured LDL or HDL particle size predicts coronary heart disease (CHD) risk to a similar ext

  17. Microbial Biogeography along an Estuarine Salinity Gradient: Combined Influences of Bacterial Growth and Residence Time

    OpenAIRE

    Crump, Byron C.; Hopkinson, Charles S.; Sogin, Mitchell L.; Hobbie, John E

    2004-01-01

    Shifts in bacterioplankton community composition along the salinity gradient of the Parker River estuary and Plum Island Sound, in northeastern Massachusetts, were related to residence time and bacterial community doubling time in spring, summer, and fall seasons. Bacterial community composition was characterized with denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA. Average community doubling time was calculated from bacterial production ([14C]leucine incorpo...

  18. Application of temperature gradient gel electrophoresis to the study of yeast diversity in the estuary of the Tagus river, Portugal.

    Science.gov (United States)

    Gadanho, Mário; Sampaio, José Paulo

    2004-12-01

    Temperature gradient gel electrophoresis (TGGE) was employed for the assessment of yeast diversity in the estuary of the Tagus river (Portugal). The molecular detection of yeasts was carried out directly from water samples and, in parallel, a cultivation approach by means of an enrichment step was employed. A nested PCR was employed to obtain a fungal amplicon containing the D2 domain of the 26S rRNA gene. For identification the TGGE bands were extracted, re-amplified, and sequenced. Fourteen fungal taxa were detected and all except one were yeasts. Most yeast sequences corresponded to members of the Ascomycota and only three belonged to the Basidiomycota. Five yeasts (four ascomycetes and one basidiomycete) could not be identified to the species level due to the uniqueness of their sequences. The number of species detected after enrichment was higher than the number of taxa found using the direct detection method. This suggests that some yeast populations are present in densities that are below the detection threshold of the method. With respect to the analysis of the yeast community structure, our results indicate that the dominant populations belong to Debaryomyces hansenii, Rhodotorula mucilaginosa, Cryptococcus longus, and to an uncultured basidiomycetous yeast phylogenetically close to Cr. longus. The combined analysis of direct detection and cultivation approaches indicates a similar community structure at the two sampled sites since nine species were present at both localities.

  19. Mapping and identification of interferon gamma-regulated HeLa cell proteins separated by immobilized pH gradient two-dimensional gel electrophoresis

    DEFF Research Database (Denmark)

    Shaw, AC; Rossel Larsen, M; Roepstorff, P

    1999-01-01

    magnitude of IFN-gamma responsive genes has been reported previously. Our goal is to identify and map IFN-gamma-regulated HeLa cell proteins to the two-dimensional polyacrylamide gel electrophoresis with the immobilized pH gradient (IPG) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) system....... A semiconfluent layer of HeLa cells was grown on tissue culture plates, and changes in protein expression due to 100 U/mL IFN-gamma were investigated at different periods after treatment, using pulse labeling with [35S]methionine/cysteine in combination with 2-D PAGE (IPG). The identity of eight protein spots...

  20. Two dimensional gel electrophoresis using narrow pH 3-5.6 immobilised pH gradient strips identifies potential novel disease biomarkers in plasma or serum

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Bevin Gangadharan & Nicole Zitzmann ### Abstract Two-dimensional gel electrophoresis (2-DE) is a protein separation technique often used to separate plasma or serum proteins in an attempt to identify novel biomarkers. This protocol describes how to run 2-DE gels using narrow pH 3-5.6 immobilised pH gradient strips to separate 2 mg of serum proteins. pH 3-6 ampholytes are used to enhance the solubility of proteins in this pH range before the serum proteins are separated in...

  1. A Gel Probe Equilibrium Sampler for Measuring Arsenic Porewater Profiles And Sorption Gradients in Sediments: I. Laboratory Development

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, K.M.; Root, R.; O' Day, P.A.; Hering, J.G.

    2009-05-14

    A gel probe equilibrium sampler has been developed to study arsenic (As) geochemistry and sorption behavior in sediment porewater. The gels consist of a hydrated polyacrylamide polymer, which has a 92% water content. Two types of gels were used in this study. Undoped (clear) gels were used to measure concentrations of As and other elements in sediment porewater. The polyacrylamide gel was also doped with hydrous ferric oxide (HFO), an amorphous iron (Fe) oxyhydroxide. When deployed in the field, HFO-doped gels introduce a fresh sorbent into the subsurface thus allowing assessment of in situ sorption. In this study, clear and HFO-doped gels were tested under laboratory conditions to constrain the gel behavior prior to field deployment. Both types of gels were allowed to equilibrate with solutions of varying composition and re-equilibrated in acid for analysis. Clear gels accurately measured solution concentrations ({+-}1%), and As was completely recovered from HFO-doped gels ({+-}4%). Arsenic speciation was determined in clear gels through chromatographic separation of the re-equilibrated solution. For comparison to speciation in solution, mixtures of As(III) and As(V) adsorbed on HFO embedded in gel were measured in situ using X-ray absorption spectroscopy (XAS). Sorption densities for As(III) and As(V) on HFO embedded in gel were obtained from sorption isotherms at pH 7.1. When As and phosphate were simultaneously equilibrated (in up to 50-fold excess of As) with HFO-doped gels, phosphate inhibited As sorption by up to 85% and had a stronger inhibitory effect on As(V) than As(III). Natural organic matter (>200 ppm) decreased As adsorption by up to 50%, and had similar effects on As(V) and As(III). The laboratory results provide a basis for interpreting results obtained by deploying the gel probe in the field and elucidating the mechanisms controlling As partitioning between solid and dissolved phases in the environment.

  2. The gel-forming behaviour of dextran in the presence of KCl: a quantitative 13C and pulsed field gradient (PFG) NMR study.

    Science.gov (United States)

    Naji, L; Schiller, J; Kaufmann, J; Stallmach, F; Kärger, J; Arnold, K

    2003-05-01

    Although the gel forming ability of certain polysaccharides in the presence of ions is a well-known phenomenon, detailed physicochemical mechanisms of such processes are still unknown. In this investigation high resolution 13C NMR as well as 1H pulsed field gradient (PFG) NMR were used to investigate the mobility of dextran in the sol and in the gel state. Gel-formation of dextran can be easily induced by the addition of large amounts of potassium chloride. No major differences in the T(1) relaxation times of dextran in the sol and in the gel state could be observed. Accordingly, the analysis of the 13C NMR spectroscopic data did not provide any indication of an observable line-broadening upon gel-formation. However, a KCl concentration dependent decrease of signal intensity in comparison to an internal standard was detected. On the other hand, the PFG NMR studies clearly indicated a gradual diminution of the self-diffusion coefficient of the dextran with increasing molecular weight as well as in the presence of potassium chloride. These measurements revealed in agreement with spectroscopic data that at least one potassium ion per monomer subunit (i.e. one glycopyranose residue) is necessary for gel formation.

  3. A gel probe equilibrium sampler for measuring arsenic porewater profiles and sorption gradients in sediments: I. Laboratory development

    Science.gov (United States)

    Campbell, K.M.; Root, R.; O'Day, P. A.; Hering, J.G.

    2008-01-01

    A gel probe equilibrium sampler has been developed to study arsenic (As) geochemistry and sorption behavior in sediment porewater. The gels consist of a hydrated polyacrylamide polymer, which has a 92% water content. Two types of gels were used in this study. Undoped (clear) gels were used to measure concentrations of As and other elements in sediment porewater. The polyacrylamide gel was also doped with hydrous ferric oxide (HFO), an amorphous iron (Fe) oxyhydroxide. When deployed in the field, HFO-doped gels introduce a fresh sorbent into the subsurface thus allowing assessment of in situ sorption. In this study, clear and HFO-doped gels were tested under laboratory conditions to constrain the gel behavior prior to field deployment. Both types of gels were allowed to equilibrate with solutions of varying composition and re-equilibrated in acid for analysis. Clear gels accurately measured solution concentrations (??1%), and As was completely recovered from HFO-doped gels (??4%). Arsenic speciation was determined in clear gels through chromatographic separation of the re-equilibrated solution. For comparison to speciation in solution, mixtures of As(III) and As(V) adsorbed on HFO embedded in gel were measured in situ using X-ray absorption spectroscopy (XAS). Sorption densities for As(III) and As(V) on HFO embedded in gel were obtained from sorption isotherms at pH 7.1. When As and phosphate were simultaneously equilibrated (in up to 50-fold excess of As) with HFO-doped gels, phosphate inhibited As sorption by up to 85% and had a stronger inhibitory effect on As(V) than As(III). Natural organic matter (>200 ppm) decreased As adsorption by up to 50%, and had similar effects on As(V) and As(III). The laboratory results provide a basis for interpreting results obtained by deploying the gel probe in the field and elucidating the mechanisms controlling As partitioning between solid and dissolved phases in the environment. ?? 2008 American Chemical Society.

  4. Microwave-enhanced folding and denaturation of globular proteins

    DEFF Research Database (Denmark)

    Bohr, Henrik; Bohr, Jakob

    2000-01-01

    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...

  5. Detection of bacteriophage phi 6 minus-strand RNA and novel mRNA isoconformers synthesized in vivo and in vitro, by strand-separating agarose gels

    Energy Technology Data Exchange (ETDEWEB)

    Pagratis, N.; Revel, H.R. (Univ. of Chicago, IL (USA))

    1990-07-01

    Two urea-free agarose gel protocols that resolve the six individual strands of bacteriophage phi 6 dsRNA were developed and used to analyze phage RNA synthesis in vivo and in vitro. Citrate gels separate strands of the large and medium chromosomes while Tris-borate-EDTA (TBE) gels resolve the medium and small dsRNA segments. Minus strands migrate faster than plus strands on citrate gels but are retarded on TBE gels. A study of electrophoretic conditions showed that pH affects strand resolution on citrate gels, and that voltage gradient, agarose concentration, and ethidium bromide significantly alter strand migration on TBE gels. Analysis of native phi 6 RNA synthesized in vivo and in vitro showed that the large and medium message RNAs comigrate with the corresponding plus strands of denatured virion dsRNA. The small messenger RNA is exceptional. Native small mRNA was detected as three isoconformers in vivo and in vitro. The isoconformers were converted by heat denaturation to a single RNA species that comigrates with the virion s+ strand. Minus strands labeled in vivo were detected only after heat denaturation. Minus strand synthesis was detected also in heat-denatured samples from in vitro phi 6 nucleocapsid RNA polymerase reactions at pH values suboptimal for transcription.

  6. Comparison of diazo-coupling, formazan, and silver staining techniques for visualizing alkaline phosphatase isoenzymes after electrophoresis in homogeneous-pore and gradient-pore polyacrylamide gels.

    Science.gov (United States)

    Hodson, A W; Skillen, A W

    1988-03-01

    Three techniques for visualization of alkaline phosphatase after polyacrylamide-gel electrophoresis are compared. These are diazo-dye simultaneous coupling with the substrate sodium naphthyl phosphate and 5-chloro-2-toluene diazonium chloride; formazan precipitation with the substrate 5-bromo-4-chloro-3-indolyl phosphate and 3-[4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; and silver staining with the substrate sodium glycerophosphate. Each staining technique was tested with gradient-pore and homogeneous-pore acrylamide-gel electrophoresis. The main factors assessed are sensitivity; separation of the human serum alkaline phosphatase isoenzymes of the liver, bone, and intestinal types; and differences in substrate affinity, as well as the complexity of each technique. Using the three techniques only minor differences in substrate affinity are evident. There is some nonspecific staining with the diazo-coupling technique but not with the formazan and silver staining techniques. The differences, in the mobility of the liver, bone, and intestinal isoenzymes achieved by homogeneous-pore gel electrophoresis are sufficient to allow them to be clearly distinguished. However, only very small differences in mobility are found with gradient-pore gel electrophoresis, but the sharper bands in this medium allow much smaller amounts of activity to be detected. As little as 160 microU of enzyme can be visualized by the diazo technique. Silver staining gives an approximately fourfold increase in sensitivity over the formazan technique, which in turn gives a fourfold increase over the diazo technique. An important aspect of the silver staining technique is the potential of increasing sensitivity much further by improvements in the photographic physical development stage.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Analysis of polyacrylamide gels for trace metals using diffusive gradients in thin films and laser ablation inductively coupled plasma mass spectrometry.

    Science.gov (United States)

    Warnken, Kent W; Zhang, Hao; Davison, William

    2004-10-15

    A simple method for the analysis of polyacrylamide diffusive gradients in thin film (DGT) gels by laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS), employing a novel use of (115)In internal standardization, has been developed. This method allows the determination of Co, Ni, Cu, Zn, Cd, and Pb concentrations (at the DGT filter face) or fluxes in sediments at a spatial resolution of 100 microm. Single-layered gels, using an optimized laser defocus of 4000 microm at 400 mJ power, showed high precision (generally approximately 10%) and a linear response during solution deployment. Of the elements Sc, In, Ba, La, Ce, and Tb, Ba most closely tracked variations in laser energy and showed the highest analytical precision but could not be used as an internal standard due to its elevated presence in natural sediments. Therefore, internal standardization, necessary to normalize data collected on different days, was carried out using (115)In contained within a second layer of backing gel and dried along with the analyte layer as a dual-gel disk. This multilayered gel standard required a laser defocus setting of 1000 microm and a laser power of approximately 800 mJ. Analytical precision for a 64-spot ablation grid at 100-microm spacing was approximately 10%. Verification of this method was carried out on DGT sediment probes deployed in Priest Pot (English Lake District). Results obtained by conventional slicing techniques and aqueous elution agreed with laser ablation results when the different sampling areas were considered. The elution results varied by a factor of laser ablation technique showed a variability of approximately 4, indicating localized elevated concentrations of Co. This higher resolution LA-ICPMS method could ultimately lead to an improved understanding of the geochemical processes responsible for metal uptake and release in sediments.

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

    Science.gov (United States)

    Roberts, Peter L; Lloyd, David

    2007-10-01

    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.

  9. Stability of collagen during denaturation.

    Science.gov (United States)

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

    1999-05-01

    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.

  10. Direct measurement of tubulin and bulk message distributions on polysomes of growing, starved and deciliated Tetrahymena using RNA gel blots of sucrose gradients containing acrylamide.

    Science.gov (United States)

    Calzone, F J; Callahan, R; Gorovsky, M A

    1988-10-25

    A method was developed using sucrose gradients containing acrylamide which greatly simplifies the measurement of the polysomal distribution of messages. After centrifugation, the acrylamide was polymerized, forming a "polysome gel". RNA gel blots of polysome gels were used to determine the polysomal distributions of alpha-tubulin and total polyadenylated mRNA in growing, starved (nongrowing) and starved-deciliated Tetrahymena and the number of messages loaded onto polysomes was calculated. These measurements indicated that the translational efficiencies of alpha-tubulin mRNA and total polyadenylated mRNA are largely unaffected when the rates of tubulin and total protein synthesis vary dramatically. Thus, differential regulation of alpha-tubulin mRNA translation initiation does not contribute to the greater than 100-fold induction of tubulin synthesis observed during cilia regeneration and in growing cells. The major translation-level process regulating tubulin synthesis in Tetrahymena appears to be a change in message loading mediated by a non-specific message recruitment or unmasking factor.

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

    Science.gov (United States)

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

    2007-01-01

    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.

  12. Analysis of metal-binding proteins separated by non-denaturating gel electrophoresis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS).

    Science.gov (United States)

    Becker, J Susanne; Mounicou, Sandra; Zoriy, Miroslav V; Becker, J Sabine; Lobinski, Ryszard

    2008-09-15

    Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) have become established as very efficient and sensitive biopolymer and elemental mass spectrometric techniques for studying metal-binding proteins (metalloproteins) in life sciences. Protein complexes present in rat tissues (liver and kidney) were separated in their native state in the first dimension by blue native gel electrophoresis (BN-PAGE). Essential and toxic metals, such as zinc, copper, iron, nickel, chromium, cadmium and lead, were detected by scanning the gel bands using quadrupole LA-ICP-MS with and without collision cell as a microanalytical technique. Several proteins were identified by using MALDI-TOF-MS together with a database search. For example, on one protein band cut from the BN-PAGE gel and digested with the enzyme trypsin, two different proteins - protein FAM44B and cathepsin B precursor - were identified. By combining biomolecular and elemental mass spectrometry, it was possible to characterize and identify selected metal-binding rat liver and kidney tissue proteins.

  13. Separation of single-walled carbon nanotubes by gel-based chromatography using surfactant step-gradient techniques and development of new instrumentation for studying SWCNT reaction processes

    Science.gov (United States)

    Breindel, Leonard M.

    Single-walled carbon nanotube (SWCNT) synthesis methods such as CoMoCATTM, HiPcoTM, pulsed laser vaporization (PLV), and catalytic chemical vapor deposition (CCVD) produce several different distributions of (n,m) SWCNT structures, where ( n,m) defines the nanotube diameter and chiral wrapping angle. Post-synthesis processing such as functionalization and/or separations must therefore be employed to yield high purity electronic or single (n,m) samples. Through the use of a surfactant gradient across a gel-based chromatographic column, separations of single (n,m) species can be achieved. Anionic surfactants such as SDS, SDBS, and AOT display different separation effectiveness for single (n,m) species. Results of near-infrared optical absorption for separated SWCNT surfactant suspensions will be discussed, leading to a broader understanding of the important factors necessary for the gel chromatography separation technique. In particular, the effects of SWCNT/surfactant micelle structure are found to be key to achieving fast, simple SWCNT electronic type separations. Additionally, development of new instrumentation for the near-infrared spectrofluorimetric analysis (NIR-SFA) of SWCNTs is useful to the advancement of fundamental SWCNT research and applications. NIR-SFA, for instance, allows for the (n,m) structures of a sample to be identified and monitored during the progress of a chemical reaction or separation experiment. Seeking to achieve the time resolutions necessary for such experiments, the design and optimizations of a system utilizing single-wavelength excitation by diode lasers coupled with a fast NIR detection system are presented.

  14. Speciation of lead in seawater and river water by using Saccharomyces cerevisiae immobilized in agarose gel as a binding agent in the diffusive gradients in thin films technique

    Energy Technology Data Exchange (ETDEWEB)

    Pescim, Guilherme Favoreto; Marrach, Gabriela; Vannuci-Silva, Monizze; Souza, Lais Alves; Menegario, Amauri Antonio [Universidade Estadual Paulista, Centro de Estudos Ambientais, Rio Claro, SP (Brazil)

    2012-09-15

    Saccharomyces cerevisiae immobilized in agarose gel is proposed as a binding agent for the diffusive gradients in thin films (DGT) technique for determination of Pb in river water and seawater. DGT samplers were assembled with the proposed binding agent (25-mm disk containing 20 %, m/v, S. cerevisiae and 3.0 %, m/v, agarose) and a diffusive layer of cellulose (3MM Chr chromatography paper of 25-mm diameter). The effects of some DGT parameters (e.g., immersion time, ionic strength, and pH) were evaluated. Elution of Pb from the binding agent was effectively done with 1.75 mol L{sup -1} HNO{sub 3}. The deployment curve (between 2 and 24 h) was characterized by a significant uptake of Pb (346 ng Pb h{sup -1}) and good linear regression (R {sup 2} = 0.9757). The experimental results are in excellent agreement with the predicted theoretical curve for mass uptake. Consistent results were found for solutions with ionic strengths of 0.005 mol L{sup -1} or greater and within a pH range of 4.5-8.5. Interferences from Cu (20:1), Mn (20:1), Fe (20:1), Zn (20:1), Ca (250:1), and Mg (250:1) in Pb retention were negligible. Determination of Pb in spiked river water samples (from the Corumbatai and Piracicaba rivers) performed using the proposed device was in agreement with total dissolved Pb, whereas measurements in seawater suggest that of the various species of Pb present in the samples, only cationic Pb species are adsorbed by the agarose-yeast gel disks. The in situ concentration of Pb obtained at two different sites of the Rio Claro stream (Corumbatai basin) were 1.13 {+-} 0.01 and 1.34 {+-} 0.04 {mu}g L{sup -1}. For 72-h deployments, a detection limit of 0.75 {mu}g L{sup -1} was calculated. The combination of inductively coupled plasma optical emission spectroscopy and in situ deployments of DGT samplers during the 72-h period makes possible the determination of labile Pb in river water. (orig.)

  15. Development and validation of PCR primers to assess the diversity of Clostridium spp. in cheese by temporal temperature gradient gel electrophoresis.

    Science.gov (United States)

    Le Bourhis, Anne-Gaëlle; Saunier, Katiana; Doré, Joël; Carlier, Jean-Philippe; Chamba, Jean-François; Popoff, Michel-Robert; Tholozan, Jean-Luc

    2005-01-01

    A nested-PCR temporal temperature gradient gel electrophoresis (TTGE) approach was developed for the detection of bacteria belonging to phylogenetic cluster I of the genus Clostridium (the largest clostridial group, which represents 25% of the currently cultured clostridial species) in cheese suspected of late blowing. Primers were designed based on the 16S rRNA gene sequence, and the specificity was confirmed in PCRs performed with DNAs from cluster I and non-cluster I species as the templates. TTGE profiles of the PCR products, comprising the V5-V6 region of the 16S rRNA gene, allowed us to distinguish the majority of cluster I species. PCR-TTGE was applied to analyze commercial cheeses with defects. All cheeses gave a signal after nested PCR, and on the basis of band comigration with TTGE profiles of reference strains, all the bands could be assigned to a clostridial species. The direct identification of Clostridium spp. was confirmed by sequencing of excised bands. C. tyrobutyricum and C. beijerinckii contaminated 15 and 14 of the 20 cheese samples tested, respectively, and C. butyricum and C. sporogenes were detected in one cheese sample. Most-probable-number counts and volatile fatty acid were determined for comparison purposes. Results obtained were in agreement, but only two species, C. tyrobutyricum and C. sporogenes, could be isolated by the plating method. In all cheeses with a high amount of butyric acid (>100 mg/100 g), the presence of C. tyrobutyricum DNA was confirmed by PCR-TTGE, suggesting the involvement of this species in butyric acid fermentation. These results demonstrated the efficacy of the PCR-TTGE method to identify Clostridium in cheeses. The sensitivity of the method was estimated to be 100 CFU/g.

  16. Genetic diversity of the biofilm covering Montacuta ferruginosa (Mollusca, bivalvia) as evaluated by denaturing gradient gel electrophoresis analysis and cloning of PCR-Amplified gene fragments coding for 16S rRNA

    NARCIS (Netherlands)

    Gillan, D.C.; Speksnijder, Arjen; Zwart, G.; De Ridder, C.

    1998-01-01

    The shell of the bivalve Montacuta ferruginosa, a symbiont living in the burrow of an echinoid, is covered with a rust- colored biofilm, This biofilm includes different morphotypes of bacteria that are encrusted with a mineral rich in ferric ion and phosphate, The aim of this research was to determi

  17. PCR-denaturing gradient gel electrophoresis profiling of the inter- and intra-species 18S rRNA gene sequence heterogeneity is an accurate and sensitive method to assess species diversity of arbuscular mycorrhizal fungi of the genus Gigaspora

    NARCIS (Netherlands)

    De Souza, F.A.; Kowalchuk, G.A.; Leeflang, P.; Van Veen, J.A.

    2004-01-01

    Despite the importance of arbuscular mycorrhizal fungi in the majority of terrestrial ecosystems, their ecology, genetics, and evolution are poorly understood, partly due to difficulties associated with detecting and identifying species. We explored the inter- and intraspecies variations of the 18S

  18. Genetic diversity of the biofilm covering Montacuta ferruginosa (Mollusca, bivalvia) as evaluated by denaturing gradient gel electrophoresis analysis and cloning of PCR-Amplified gene fragments coding for 16S rRNA

    NARCIS (Netherlands)

    Gillan, D.C.; Speksnijder, Arjen; Zwart, G.; De Ridder, C.

    1998-01-01

    The shell of the bivalve Montacuta ferruginosa, a symbiont living in the burrow of an echinoid, is covered with a rust- colored biofilm, This biofilm includes different morphotypes of bacteria that are encrusted with a mineral rich in ferric ion and phosphate, The aim of this research was to

  19. 中华蜜蜂肠道细菌群落的PCR-DGGE分析%Denaturing Gradient Gel Electrophoresis Analysis of 16S Ribosomal DNA in Apis cerana cerana Gut Bacterial Communities

    Institute of Scientific and Technical Information of China (English)

    丁进; 张国只; 苏丽娟

    2015-01-01

    肠道微生物与中华蜜蜂的生长发育密切相关,在为宿主提供营养、抵抗病原菌侵袭等方面起重要作用。为了探究中华蜜蜂在封盖一日龄蛹、破巢幼蜂和采集蜂三个重要发育阶段肠道细菌群落的结构组成及差异变化,我们对细菌16S rDNA的V6-V8可变区进行PCR-DGGE和克隆测序,并计算封盖一日龄蛹、破巣蜂和采集蜂阶段中华蜜蜂肠道菌群的多样性指数和相似性系数,结果表明:采集蜂肠道内细菌多样性指数最大,而封盖一日龄蛹和破巣幼蜂之间的肠道菌群相似性较高;测序结果初步得到了Gilliamella、Snodgrassella、Carnobacterium、Neisseriaceae、Frischella、Janthinobacterium、Pseudomonas、Lactobacillus、Lactococcus和Leuconostoc十种菌属,其中封盖一日龄蛹和破巢幼蜂的优势菌属为Snodgrassella和Pseudomonas;采集蜂的优势菌属为Gilliamella和Snodgrassella 。本研究旨在为提高中华蜜蜂的环境适应性和病虫害的防治提供依据。%Gut bacterial communities are closely related to the growth and development of Apis cerana cerana,and such communities play important roles both in the insect nutrition and colonization resistance against invasion of exotic microbes. The purpose of this study is to investigate the diversities of gut bacterial communities in different developmental stages(first instar pupa,pharate adult bee and forager bee)of A. cerana cerana and the structural differences among them. We amplified the V6-V8 region of the 16S rDNA gene of samples using universal primers and separated by DGGE. Then the separated DGGE bands were extracted,cloned and sequenced. The diversity index and similarity coefficient were calculated to reveal the constituents and dynamic changes of gut bacter ial communities in the three developmental stages of A. cerana cerana. The results showed that the microbial diversity of forager bee was significantly higher than that of first instar pupa and pharate adult bee. Compared with forager bee ,the similarities of intestinal microbes in instar pupa and pharate adult bee were much higher. The dominant bands of DGGE could be grouped into ten genera:Gilliamella,Snodgrassella,Carnobacterium,Neisseriaceae,Frischella,Janthinobacterium, Pseudomonas,Lactobacillus,Lactococcus and Leuconostoc. First instar pupa and pharate adult bee showed very similar bacterial community compositions in which Snodgrassella and Pseudomonas were the most important groups. However, the dominant genera in forager bee were Gilliamella and Snodgrassella. These results supply basic information for improving bee’s adaptability to environment,and controlling its diseases and insect pests.

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

    Institute of Scientific and Technical Information of China (English)

    肖华西; 林亲录; 夏新剑; 李丽辉; 林利忠; 吴卫国

    2008-01-01

    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.

  1. Single DNA denaturation and bubble dynamics

    Energy Technology Data Exchange (ETDEWEB)

    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: metz@ph.tum.de

    2009-01-21

    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.

  2. Single DNA denaturation and bubble dynamics

    DEFF Research Database (Denmark)

    Metzler, Ralf; Ambjörnsson, Tobias; Hanke, Andreas

    2009-01-01

    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...

  3. The dynamics of the DNA denaturation transition

    CERN Document Server

    van Erp, Titus S

    2012-01-01

    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.

  4. Coastal microbial mat diversity along a natural salinity gradient.

    Directory of Open Access Journals (Sweden)

    Henk Bolhuis

    Full Text Available The North Sea coast of the Dutch barrier island of Schiermonnikoog is covered by microbial mats that initiate a succession of plant communities that eventually results in the development of a densely vegetated salt marsh. The North Sea beach has a natural elevation running from the low water mark to the dunes resulting in gradients of environmental factors perpendicular to the beach. These gradients are due to the input of seawater at the low water mark and of freshwater from upwelling groundwater at the dunes and rainfall. The result is a natural and dynamic salinity gradient depending on the tide, rainfall and wind. We studied the microbial community composition in thirty three samples taken every ten meters along this natural salinity gradient by using denaturing gradient gel electrophoresis (DGGE of rRNA gene fragments. We looked at representatives from each Domain of life (Bacteria, Archaea and Eukarya and with a particular emphasis on Cyanobacteria. Analysis of the DGGE fingerprints together with pigment composition revealed three distinct microbial mat communities, a marine community dominated by diatoms as primary producers, an intermediate brackish community dominated by Cyanobacteria as primary producers and a freshwater community with Cyanobacteria and freshwater green algae.

  5. Bubbles and denaturation in DNA

    CERN Document Server

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

    2006-01-01

    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...

  6. DNA denaturation in ionic solution

    Science.gov (United States)

    Maity, Arghya; Singh, Amar; Singh, Navin

    2016-05-01

    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.

  7. Estudio de comunidades bacterianas naturales: diferenciación de las actinobacterias mediante electroforesis en doble gel en gradiente de desnaturalización (DDGGE)

    OpenAIRE

    Ortiz Martínez, Alberto

    2005-01-01

    El primer objetivo, en esta Tesis, es la optimización de las condiciones a emplear para el estudio de comunidades bacterianas naturales. Esto se conseguirá mejorando los gradientes empleados en los geles en condiciones desnaturalizantes. En lo referente a la optimización de los gradientes, hay que tener en cuenta que en las muestras naturales las comunidades presentes son heterogéneas, lo que hace necesaria una optimización de los gradientes de desnaturalización, con el fin de mejorar la sepa...

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

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

    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℃.

  9. A gel probe equilibrium sampler for measuring arsenic porewater profiles and sorption gradients in sediments: II. Field application to Haiwee reservoir sediment

    Science.gov (United States)

    Campbell, K.M.; Root, R.; O'Day, P. A.; Hering, J.G.

    2008-01-01

    Arsenic (As) geochemistry and sorption behavior were measured in As- and iron (Fe)-rich sediments of Haiwee Reservoir by deploying undoped (clear) polyacrylamide gels and hydrous ferric oxide (HFO)-doped gels in a gel probe equilibrium sampler, which is a novel technique for directly measuring the effects of porewater composition on As adsorption to Fe oxides phases in situ. Arsenic is deposited at the sediment surface as As(V) and is reduced to As(III) in the upper layers of the sediment (0-8 cm), but the reduction of As(V) does not cause mobilization into the porewater. Dissolved As and Fe concentrations increased at depth in the sediment column driven by the reductive dissolution of amorphous Fe(III) oxyhydroxides and conversion to a mixed Fe(II, III) green rust-type phase. Adsorption of As and phosphorous (P) onto HFO-doped gels was inhibited at intermediate depths (10-20 cm), possibly due to dissolved organic or inorganic carbon, indicating that dissolved As concentrations were at least partially controlled by porewater composition rather than surface site availability. In sediments that had been recently exposed to air, the region of sorption inhibition was not observed, suggesting that prior exposure to air affected the extent of reductive dissolution, porewater chemistry, and As adsorption behavior. Arsenic adsorption onto the HFO-doped gels increased at depths >20 cm, and the extent of adsorption was most likely controlled by the competitive effects of dissolved phosphate. Sediment As adsorption capacity appeared to be controlled by changes in porewater composition and competitive effects at shallower depths, and by reductive dissolution and availability of sorption sites at greater burial depths. ?? 2008 American Chemical Society.

  10. A Gel Probe Equilibrium Sampler for Measuring Arsenic Porewater Profiles And Sorption Gradients in Sediments: Ii. Field Application to Haiwee Reservoir Sediment

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, K.M.; Root, R.; O' Day, P.A.; Hering, J.G.

    2009-05-12

    Arsenic (As) geochemistry and sorption behavior were measured in As- and iron (Fe)-rich sediments of Haiwee Reservoir by deploying undoped (clear) polyacrylamide gels and hydrous ferric oxide (HFO)-doped gels in a gel probe equilibrium sampler, which is a novel technique for directly measuring the effects of porewater composition on As adsorption to Fe oxides phases in situ. Arsenic is deposited at the sediment surface as As(V) and is reduced to As(III) in the upper layers of the sediment (0-8 cm), but the reduction of As(V) does not cause mobilization into the porewater. Dissolved As and Fe concentrations increased at depth in the sediment column driven by the reductive dissolution of amorphous Fe(III) oxyhydroxides and conversion to a mixed Fe(II, III) green rust-type phase. Adsorption of As and phosphorous (P) onto HFO-doped gels was inhibited at intermediate depths (10-20 cm), possibly due to dissolved organic or inorganic carbon, indicating that dissolved As concentrations were at least partially controlled by porewater composition rather than surface site availability. In sediments that had been recently exposed to air, the region of sorption inhibition was not observed, suggesting that prior exposure to air affected the extent of reductive dissolution, porewater chemistry, and As adsorption behavior. Arsenic adsorption onto the HFO-doped gels increased at depths >20 cm, and the extent of adsorption was most likely controlled by the competitive effects of dissolved phosphate. Sediment As adsorption capacity appeared to be controlled by changes in porewater composition and competitive effects at shallower depths, and by reductive dissolution and availability of sorption sites at greater burial depths.

  11. Down-regulation of triose phosphate isomerase in Vineristine-resistant gastric cancer SGC7901 cell line identified by immobilized pH gradient two-dimensional gel electrophoresis and mierosequencing

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective:To exkplore new multidrug-resistance-related proteins in gastric SC7901 cells and clarify their mechanisms.Methods:Two-dimensional(2-D) polyacrylamide gel electrophoresis with immobilized pH gradients(IPG) was applied to compare the differential expression of multidrug-resistance-related proteins in gastric cancer SGC7901 cells and Vineristine-resistant SGC7901 cells (SGC7901/VCR) induced by vincristine sulfate.The 2-D gels were silver-stained.Then,preparative 2-D PAGE was performed.The differential proteins of PVDF membranes were cxcised and identified by N-terminal microsequencing.The mRNA expressions of differential proteins were detected in SGC 7901 cells and SGC7901/VCR cells by RT-PCR.Results:Approximatedly 680 protein sports were resolved on each 2-D gel by silver staining.Most protein spots showed no difference in composition,shape or density.25 proteins differed in abundance (6 higher in SGC7901/VCR cells;19 higher in 7901 cells);5 proteins were unique to one kind of cell or the othe(3 in SGC7901/VRC cells,2 in 7901 cells).One drug-resistance-related protein,which was down-regulated in SGC7901/VCR cells,was identified as trisephosphate isomerase(TPI),a glycolytic pathway enzyme.Conclusions:the results suggest that these differential proteins including TPI may be related to the Vincristine-resistant mechanism in human gastric cancer SGC7901/VCR cell line.

  12. Genotypic distribution of an indigenous model microorganism along an estuarine gradient.

    Science.gov (United States)

    Hawkins, Robert J; Purdy, Kevin J

    2007-11-01

    Using the genus Desulfobulbus as an indigenous model, microbial distribution along an estuarine gradient was investigated. Denaturing gradient gel electrophoresis analysis indicated a sequential change in Desulfobulbus genotypes along the estuary with marine, brackish and freshwater genotypes detected. This differential distribution was significantly correlated with porewater concentrations of chloride, sulphate and nitrate. Temporal analysis indicated that there was a shift in the whole Desulfobulbus community towards to marine end of the estuary by the end of the winter compared with the late summer. This distribution pattern is similar to those seen with other estuarine organisms and, given the correlation with chloride and sulphate, is indicative of classic niche separation within this genus driven by gross environmental factors such as salinity. These results will help in developing a truly ecological description of microbial biodiversity.

  13. Transport Phenomena in Gel

    Directory of Open Access Journals (Sweden)

    Masayuki Tokita

    2016-05-01

    Full Text Available Gel becomes an important class of soft materials since it can be seen in a wide variety of the chemical and the biological systems. The unique properties of gel arise from the structure, namely, the three-dimensional polymer network that is swollen by a huge amount of solvent. Despite the small volume fraction of the polymer network, which is usually only a few percent or less, gel shows the typical properties that belong to solids such as the elasticity. Gel is, therefore, regarded as a dilute solid because its elasticity is much smaller than that of typical solids. Because of the diluted structure, small molecules can pass along the open space of the polymer network. In addition to the viscous resistance of gel fluid, however, the substance experiences resistance due to the polymer network of gel during the transport process. It is, therefore, of importance to study the diffusion of the small molecules in gel as well as the flow of gel fluid itself through the polymer network of gel. It may be natural to assume that the effects of the resistance due to the polymer network of gel depends strongly on the network structure. Therefore, detailed study on the transport processes in and through gel may open a new insight into the relationship between the structure and the transport properties of gel. The two typical transport processes in and through gel, that is, the diffusion of small molecules due to the thermal fluctuations and the flow of gel fluid that is caused by the mechanical pressure gradient will be reviewed.

  14. In situ selective determination of methylmercury in river water by diffusive gradient in thin films technique (DGT) using baker's yeast (Saccharomyces cerevisiae) immobilized in agarose gel as binding phase

    Energy Technology Data Exchange (ETDEWEB)

    Tafurt-Cardona, Makenly [Programa de Pós-graduação em Geociências e Meio Ambiente, Instituto de Geociências e Ciências Exatas, UNESP – Univ. Estadual Paulista, Av. 24-A, 1515, CEP: 13506-900, Rio Claro, SP (Brazil); Centro de Estudos Ambientais, UNESP – Univ. Estadual Paulista, Av. 24-A, 1515, CEP: 13506-900, Rio Claro, SP (Brazil); Eismann, Carlos Eduardo; Suárez, Carlos Alfredo [Centro de Estudos Ambientais, UNESP – Univ. Estadual Paulista, Av. 24-A, 1515, CEP: 13506-900, Rio Claro, SP (Brazil); Menegário, Amauri Antonio, E-mail: amenega@rc.unesp.br [Programa de Pós-graduação em Geociências e Meio Ambiente, Instituto de Geociências e Ciências Exatas, UNESP – Univ. Estadual Paulista, Av. 24-A, 1515, CEP: 13506-900, Rio Claro, SP (Brazil); Centro de Estudos Ambientais, UNESP – Univ. Estadual Paulista, Av. 24-A, 1515, CEP: 13506-900, Rio Claro, SP (Brazil); Silva Luko, Karen [Programa de Pós-graduação em Geociências e Meio Ambiente, Instituto de Geociências e Ciências Exatas, UNESP – Univ. Estadual Paulista, Av. 24-A, 1515, CEP: 13506-900, Rio Claro, SP (Brazil); Centro de Estudos Ambientais, UNESP – Univ. Estadual Paulista, Av. 24-A, 1515, CEP: 13506-900, Rio Claro, SP (Brazil); and others

    2015-08-05

    Saccharomyces cerevisiae immobilized in agarose gel as binding phase and polyacrylamide as diffusive layer in the diffusive gradient in thin films technique (DGT) was used for selective determination of methylmercury (MeHg). Deployment tests showed good linearity in mass uptake up to 48 h (3276 ng). When coupling the DGT technique with Cold Vapor Atomic Fluorescence Spectrometry, the method has a limit of detection of 0.44 ng L{sup −1} (pre concentration factor of 11 for 48 h deployment). Diffusion coefficient of 7.03 ± 0.77 × 10{sup −6} cm{sup 2} s{sup −1} at 23 °C in polyacrylamide gel (pH = 5.5 and ionic strength = 0.05 mol L{sup −1} NaCl) was obtained. Influence of ionic strength (from 0.0005 mol L{sup −1} to 0.1 mol L{sup −1} NaCl) and pH (from 3.5 to 8.5) on MeHg uptake were evaluated. For these range, recoveries of 84–105% and 84–98% were obtained for ionic strength and pH respectively. Potential interference due to presence of Cu, Fe, Mn, Zn was also assessed showing good recoveries (70–87%). The selectivity of the proposed approach was tested by deployments in solutions containing MeHg and Hg(II). Results obtained showed recoveries of 102–115 % for MeHg, while the uptake of Hg(II) was insignificant. The proposed approach was successfully employed for in situ measurements in the Negro River (Manaus-AM, Brazil). - Highlights: • A method for in situ selective determination of MeHg by DGT technique is proposed. • Saccharomyces cerevisiae immobilized in agarose gel was used as binding agent. • Effects of pH, ionic strength and concomitant ions on uptake of MeHg were evaluated. • DGT device containing polyacrylamide gel as diffusive layer showed better selectivity. • The proposed approach was successfully applied for analysis of river water.

  15. Chemical shift prediction for denatured proteins

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-02-15

    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.

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

    Science.gov (United States)

    Riley, Patrick

    2011-01-01

    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…

  17. Speciation analysis of inorganic arsenic in river water by Amberlite IRA 910 resin immobilized in a polyacrylamide gel as a selective binding agent for As(V) in diffusive gradient thin film technique.

    Science.gov (United States)

    Rolisola, Ana M C M; Suárez, Carlos A; Menegário, Amauri A; Gastmans, Didier; Kiang, Chang H; Colaço, Camila D; Garcez, Daniel L; Santelli, Ricardo E

    2014-09-07

    In this study, a method is proposed for the selective retention of As(V) using diffusive gradient in thin film (DGT) samplers containing a strongly basic anion exchange resin (Amberlite IRA 910) supported on a polyacrylamide gel. In addition, the total arsenic content is determined by ferrihydrite gel discs. Subsequently, the concentration of As(III) was obtained by determining the difference between the total As and As(V). DGT experiments showed linear accumulation of As(V) (up to 280 ng) until a deployment time of 8 h deployment (R(2) > 0.99). The retention of As(V) was appropriate (97.9-112.3%) between pH 5 and 9. For a solution with an ionic strength ranging from 0.001 to 0.05 mol L(-1), the As(V) uptake ranged from 90-120%. The proposed method was applied for the speciation of arsenic in river water. For the analysis of spiked samples collected at the Furnas stream, the recoveries of total arsenic content ranged between 103.9% and 118.8%. However, the recoveries of As(III) and As(V) were 43.3-75.2% and 147.3-153.4%, respectively. These differences were probably because of the oxidation of As(III) to As(V) during deployments. For spiked samples collected at the Ribeirão Claro, the recoveries of dissolved As(III), As(V) and As(T) were 103.1%, 108.0% and 106.3%, respectively. Thus, the DGT technique with Amberlite IRA 910 resin as the binding phase can be employed for the in situ redox speciation of inorganic arsenic.

  18. CONFORMANCE IMPROVEMENT USING GELS

    Energy Technology Data Exchange (ETDEWEB)

    Randall S. Seright

    2003-09-01

    This report describes work performed during the second year of the project, ''Conformance Improvement Using Gels.'' The project has two objectives. The first objective is to identify gel compositions and conditions that substantially reduce flow through fractures that allow direct channeling between wells, while leaving secondary fractures open so that high fluid injection and production rates can be maintained. The second objective is to optimize treatments in fractured production wells, where the gel must reduce permeability to water much more than that to oil. Pore-level images from X-ray computed microtomography were re-examined for Berea sandstone and porous polyethylene. This analysis suggests that oil penetration through gel-filled pores occurs by a gel-dehydration mechanism, rather than a gel-ripping mechanism. This finding helps to explain why aqueous gels can reduce permeability to water more than to oil. We analyzed a Cr(III)-acetate-HPAM gel treatment in a production well in the Arbuckle formation. The availability of accurate pressure data before, during, and after the treatment was critical for the analysis. After the gel treatment, water productivity was fairly constant at about 20% of the pre-treatment value. However, oil productivity was stimulated by a factor of 18 immediately after the treatment. During the six months after the treatment, oil productivity gradually decreased to approach the pre-treatment value. To explain this behavior, we proposed that the fracture area open to oil flow was increased substantially by the gel treatment, followed by a gradual closing of the fractures during subsequent production. For a conventional Cr(III)-acetate-HPAM gel, the delay between gelant preparation and injection into a fracture impacts the placement, leakoff, and permeability reduction behavior. Formulations placed as partially formed gels showed relatively low pressure gradients during placement, and yet substantially reduced the

  19. Guanidinium-induced denaturation by breaking of salt bridges

    NARCIS (Netherlands)

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

    2015-01-01

    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

  20. Hysteresis in pressure-driven DNA denaturation.

    Directory of Open Access Journals (Sweden)

    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.

  1. Biodiversity patterns of soil ciliates along salinity gradients.

    Science.gov (United States)

    Zhao, Feng; Xu, Kuidong

    2016-04-01

    We evaluated ciliate diversity in saline soils with a salinity range from 6.5 to 65 psu by the morphological method of the Ludox-quantitative protargol stain (QPS) and the molecular techniques of ciliate-specific clone library and denaturing gradient gel electrophoresis. No active ciliates could be detected with the Ludox-QPS method, while high molecular diversity of ciliates was found. The highest ciliate molecular diversity was obtained from the soil at salinity of 8.9 psu, moderate diversity was found at salinity of 6.5 psu, and the diversity sharply decreased at salinity of 50.5 psu. By contrast, the number of ciliate classes clearly decreased with increasing soil salinity: six, five, four and two classes from sites with salinity of 6.5 psu, 8.9 psu, 29.5 psu and 50.5 psu, respectively. Ciliate diversity pattern is different from that of bacteria, whose diversity is also high in extremely saline environments. Meanwhile, the composition of ciliate community was significantly different along salinity gradient. Colpodea and Oligohymenophorea were diverse in soils at salinity less than 29.5 psu, while absent in soils with salinity above 50.5 psu. BIOENV analysis indicated soil salinity and water content were the main factors regulating the distribution of ciliates in saline soils.

  2. DSC study of denaturation of β-lactoglobulin B

    Institute of Scientific and Technical Information of China (English)

    王邦宁; 谈夫

    1995-01-01

    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

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

    Science.gov (United States)

    Raikos, Vassilios; Duthie, Garry; Ranawana, Viren

    2015-09-01

    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.

  4. Supercoiling induces denaturation bubbles in circular DNA.

    Science.gov (United States)

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

    2010-11-12

    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.

  5. 27 CFR 21.151 - List of denaturants authorized for denatured spirits.

    Science.gov (United States)

    2010-04-01

    ... TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM... (Glycerol), U.S.P S.D.A. 31-A. Green soap, U.S.P S.D.A. 27-B. Guaiacol, N.F.X S.D.A. 38-B. Heptane C.D.A....

  6. 78 FR 38628 - Reclassification of Specially Denatured Spirits and Completely Denatured Alcohol Formulas and...

    Science.gov (United States)

    2013-06-27

    ... 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...

  7. Partially folded intermediates during trypsinogen denaturation

    Directory of Open Access Journals (Sweden)

    Martins N.F.

    1999-01-01

    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.

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

    Science.gov (United States)

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

    2015-09-01

    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.

  9. Synthesis and characterization of erbium-doped SiO{sub 2}-TiO{sub 2} thin films prepared by sol-gel and dip-coating techniques onto commercial glass substrates as a route for obtaining active GRadient-INdex materials

    Energy Technology Data Exchange (ETDEWEB)

    Gómez-Varela, Ana I. [Microoptics and GRIN Optics Group, Department of Applied Physics, Faculty of Optics and Optometry and Faculty of Physics, Universidade de Santiago de Compostela, Campus Vida s/n, Santiago de Compostela E-15782 (Spain); Castro, Yolanda, E-mail: castro@icv.csic.es [Instituto de Cerámica y Vidrio (CSIC), Kelsen 5, Campus de Cantoblanco, Madrid 28049 (Spain); Durán, Alicia [Instituto de Cerámica y Vidrio (CSIC), Kelsen 5, Campus de Cantoblanco, Madrid 28049 (Spain); De Beule, Pieter A.A. [Applied Nano-Optics Laboratory, International Iberian Nanotechnology Laboratory, Braga 4715-330 (Portugal); Flores-Arias, María T. [Microoptics and GRIN Optics Group, Department of Applied Physics, Faculty of Optics and Optometry and Faculty of Physics, Universidade de Santiago de Compostela, Campus Vida s/n, Santiago de Compostela E-15782 (Spain); Bao-Varela, Carmen, E-mail: carmen.bao@usc.es [Microoptics and GRIN Optics Group, Department of Applied Physics, Faculty of Optics and Optometry and Faculty of Physics, Universidade de Santiago de Compostela, Campus Vida s/n, Santiago de Compostela E-15782 (Spain)

    2015-05-29

    In this work, SiO{sub 2}-TiO{sub 2} films doped with erbium were prepared by dip-coating sol-gel process onto commercial glass substrates. The surface morphology of the films was characterized using atomic force microscopy, while thickness, refractive index, extinction coefficient and porosity of the films were determined by ellipsometric measurements in a wavelength region of 400-1000 nm. Optical constants and porosity were found to vary with erbium concentration. The proof of principle presented in this paper is applicable to systems of different nature by tailoring the sol-gel precursors in such a way that active GRadient-INdex media described by a complex, parabolic-like refractive index distribution for beam shaping purposes is obtained. - Highlights: • Sol-gel route for preparation of active GRadient-INdex materials is proposed. • SiO{sub 2}-TiO{sub 2} films doped with erbium were prepared by dipping onto commercial glasses. • Morphological and optical characterization of the samples was performed. • Optical constants and porosity were found to vary with erbium concentration. • Refractive index diminishes with dopant content; the contrary occurs for porosity.

  10. CONFORMANCE IMPROVEMENT USING GELS

    Energy Technology Data Exchange (ETDEWEB)

    Randall S. Seright

    2004-09-30

    , (3) using partially formed gels, (4) using combinations of high and low molecular weight (Mw) polymers, (5) using secondary crosslinking reactions, (6) injecting un-hydrated polymer particles, and (7) incorporating particulates. All of these methods showed promise in some aspects, but required performance improvements in other aspects. All materials investigated to date showed significant performance variations with fracture width. High pressure gradients and limited distance of penetration are common problems in tight fractures. Gravity segregation and low resistance to breaching are common problems in wide fractures. These will be key issues to address in future work. Although gels can exhibit disproportionate permeability reduction in fractures, the levels of permeability reduction for oil flow are too high to allow practical exploitation in most circumstances. In contrast, disproportionate permeability reduction provided by gels that form in porous rock (adjacent to the fractures) has considerable potential in fractured systems.

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

    NARCIS (Netherlands)

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

    2007-01-01

    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

  12. Kinetically controlled refolding of a heat denatured hyperthermostable protein

    NARCIS (Netherlands)

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

    2007-01-01

    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

  13. 19 CFR 10.56 - Vegetable oils, denaturing; release.

    Science.gov (United States)

    2010-04-01

    ... 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,...

  14. 27 CFR 19.464 - Denatured spirits inventories.

    Science.gov (United States)

    2010-04-01

    ... 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...

  15. 27 CFR 20.144 - Packages of completely denatured alcohol.

    Science.gov (United States)

    2010-04-01

    ... 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...

  16. 27 CFR 20.261 - Records of completely denatured alcohol.

    Science.gov (United States)

    2010-04-01

    ... 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...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2003-05-15

    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.

  18. Reducing the stiffness of concentrated whey protein isolate (WPI) gels by using WPI microparticles

    NARCIS (Netherlands)

    Purwanti, N.; Moerkens, A.; Goot, van der A.J.; Boom, R.M.

    2012-01-01

    Concentrated protein gels were prepared using native whey protein isolate (WPI) and WPI based microparticles. WPI microparticles were produced by making gel pieces from a concentrated WPI suspension (40% w/w), which were dried and milled. The protein within the microparticles was denatured and the

  19. Stability of capillary gels for automated sequencing of DNA.

    Science.gov (United States)

    Swerdlow, H; Dew-Jager, K E; Brady, K; Grey, R; Dovichi, N J; Gesteland, R

    1992-08-01

    Recent interest in capillary gel electrophoresis has been fueled by the Human Genome Project and other large-scale sequencing projects. Advances in gel polymerization techniques and detector design have enabled sequencing of DNA directly in capillaries. Efforts to exploit this technology have been hampered by problems with the reproducibility and stability of gels. Gel instability manifests itself during electrophoresis as a decrease in the current passing through the capillary under a constant voltage. Upon subsequent microscopic examination, bubbles are often visible at or near the injection (cathodic) end of the capillary gel. Gels have been prepared with the polyacrylamide matrix covalently attached to the silica walls of the capillary. These gels, although more stable, still suffer from problems with bubbles. The use of actual DNA sequencing samples also adversely affects gel stability. We examined the mechanisms underlying these disruptive processes by employing polyacrylamide gel-filled capillaries in which the gel was not attached to the capillary wall. Three sources of gel instability were identified. Bubbles occurring in the absence of sample introduction were attributed to electroosmotic force; replacing the denaturant urea with formamide was shown to reduce the frequency of these bubbles. The slow, steady decline in current through capillary sequencing gels interferes with the ability to detect other gel problems. This phenomenon was shown to be a result of ionic depletion at the gel-liquid interface. The decline was ameliorated by adding denaturant and acrylamide monomers to the buffer reservoirs. Sample-induced problems were shown to be due to the presence of template DNA; elimination of the template allowed sample loading to occur without complications.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Thermal denaturation of A-DNA

    Science.gov (United States)

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

    2014-11-01

    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.

  1. Gradient networks

    Science.gov (United States)

    Toroczkai, Zoltán; Kozma, Balázs; Bassler, Kevin E.; Hengartner, N. W.; Korniss, G.

    2008-04-01

    Gradient networks are defined (Toroczkai and Bassler 2004 Nature 428 716) as directed graphs formed by local gradients of a scalar field distributed on the nodes of a substrate network G. We present the derivation for some of the general properties of gradient graphs and give an exact expression for the in-degree distribution R(l) of the gradient network when the substrate is a binomial (Erd{\\;\\kern -0.10em \\raise -0.35ex \\{{^{^{\\prime\\prime}}}}\\kern -0.57em \\o} s-Rényi) random graph, G_{N,p} , and the scalars are independent identically distributed (i.i.d.) random variables. We show that in the limit N \\to \\infty, p \\to 0, z = pN = \\mbox{const} \\gg 1, R(l)\\propto l^{-1} for l Bassler (2004 Nature 428 716).

  2. Effect of nitrite and nitrate on in situ sulfide production in an activated sludge immobilized agar gel film as determined by use of microelectrodes.

    Science.gov (United States)

    Okabe, Satoshi; Santegoeds, Cecilia M; De Beer, Dirk

    2003-03-01

    Microelectrode, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE) analyses were used to investigate the effect of nitrite and nitrate on in situ sulfide production in an activated sludge immobilized agar gel film. Microelectrode measurements of O(2), H(2)S, NO(3)(-), NO(2)(-), and pH revealed that the addition of NO(2)(-) and NO(3)(-) forced sulfate reduction zones deeper in the agar gel and significantly reduced the in situ sulfide production levels. The sulfate reduction zone was consequently separated from O(2) and NO(2)(-) or NO(3)(-) respiration zones with increasing the concentrations of NO(2)(-) and NO(3)(-). These NO(2)(-) and NO(3)(-) treatments had only a transient effect on sulfide production. The in situ sulfide production quickly recovered to the previous levels when NO(2)(-) and NO(3)(-) were removed. The PCR-DGGE and FISH analyses revealed that 2-day-continuous addition of 500 microM NO(3)(-) did not change the metabolically active sulfate-reducing bacterial (SRB) community. On the basis of these data, it could be concluded that the addition of NO(2)(-) and NO(3)(-) did not kill SRB, but induced the interspecies competition for common carbon source (i.e., acetate) between nitrate-reducing heterotrophic bacteria and SRB and enhanced the oxidation of the produced sulfide, which were main possible causes of the suppression of in situ sulfide production in the agar gel.

  3. Statistical mechanics of thermal denaturation of DNA oligomers

    Indian Academy of Sciences (India)

    Navin Singh; Yashwant Singh

    2003-08-01

    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.

  4. Simultaneous immobilization of dehydrogenases on polyvinylidene difluoride resin after separation by non-denaturing two-dimensional electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    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: yoji@dpc.ehime-u.ac.jp; Kadota, Mariko [Faculty of Science, Ehime University, Matsuyama (Japan)

    2008-06-16

    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.

  5. pH-responsive polymer-assisted refolding of urea- and organic solvent-denatured alpha-chymotrypsin.

    Science.gov (United States)

    Roy, I; Gupta, M N

    2003-12-01

    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.

  6. Mutation analysis of APC and p53 genes in colorectal cancer by denaturing gradient gel electrophoresis%变性梯度凝胶电泳分析检测结直肠癌APC和p53基因突变

    Institute of Scientific and Technical Information of China (English)

    王艳; 李岩

    2007-01-01

    目的: 探讨APC和p53基因突变在结直肠癌中的意义.方法:采用变性梯度凝胶电泳(DGGE), DNA测序法分析15例正常人和60例散发性结直肠癌标本的APC基因15外显子和p53基因第5, 7外显子的基因突变.结果: 在结直肠癌组检出14例APC和16例p53基因突变, 测序证实其中13/14例APC发生在突变集中区域;9/16例p53基因突变位于第5外显子, 7/16例p53基因突变位于第7外显子, 2例同时检出了APC基因和p53基因突变.结论:DGGE是一种快速、简便、高效、灵敏的突变检测技术. 同时也证明APC基因突变和p53基因突变均参与了结直肠癌的发生、发展过程.

  7. Analysis of bacterial diversity of kefir grains by denaturing gradient gel electrophoresis and 16S rDNA sequencing%应用变性梯度凝胶电泳和16S rDNA序列分析对kefir粒中细菌多样性的研究

    Institute of Scientific and Technical Information of China (English)

    王荫榆; 李会荣; 贾士芳; 吴正钧; 郭本恒

    2006-01-01

    以开菲尔(Kefir)粒为材料,经过DNA抽提和16S rDNA V3区PCR扩增,扩增产物经变性梯度凝胶电泳(DGGE)分离并切割电泳条带进行序列测定,并与现有的数据库进行了比较,对Kefir粒的细菌多样性进行分析.结 果表明,DGGE图谱中可检测到的8条带的16S rDNA基因序列中有7个基因序列与GenBank数据库登录的相关序列的相似性大于98%,余下的1个基因序列的相似性也大于96%.相似性大于98%的7个克隆中,有3个属于鞘氨醇杆菌属(Sphingobacterium),2个属于乳杆菌属(Lactobacillus),其它2个分别属于肠杆菌属(Enterobacter)和不动杆菌属(Acinetobacter).首次报道了鞘氨醇杆菌作为优势菌群存在开菲尔Kefir粒中.

  8. DNA Preparation for Analyzing Petroleum Contaminated Soil Microorganism by Denaturing Gradient Gel Electrophoresis (DGGE)%适用变性梯度凝胶电泳分析的石油污染土壤微生物DNA模板制备的研究

    Institute of Scientific and Technical Information of China (English)

    韦超英; 杨滨银; 方新湘; 娄恺; 晁群芳

    2011-01-01

    [目的]为了快速、简便和经济地获得理化性质不同的石油污染土壤中微生物总DNA,以用于后续微生物群落结构分析及动态监测.[方法]采用3种提取方法对石油污染土壤中细菌总DNA进行提取,并通过DNA产量、纯度、片段大小、基因组完整性等对提取得到的DNA质量进行评价;并对16S rDNA V3可变区的PCR扩增产物进行DGGE分析.[结果]采用3种方法均能从石油污染土壤中提取得到相应的细菌DNA片段,且针对同一种提取方法,土壤理化性质的差异对DNA提取效果影响不明显,但不同方法提取得到的DNA在浓度和纯度上存在明显差异.采用3种方法提取得到的DNA量分别可达98.3、79.9和43.8 ng/μl.[结论]选取方法1提取基因组DNA并进一步纯化,纯化后的DNA分别采用引物F27/R1492和F357/R518进行16S rDNA及116S rDNA V3可变区的扩增,获得了条带清晰、浓度高、无污染的DNA目的片段;对其PCR扩增产物进行DGGE分析,得到的DGGE图谱可直观反应石油污染土壤中微生物的多样性及优势种群.%[ Objective] In order to obtain total DNA in a rapid high-efficient and economic way from microorganism in petroleum contaminated soil with different properties. The total DNA can be used in the microbial community structural analysis and dynamic monitoring. [Methods] Three DNA extraction methods were used for isolation of total DNA form petroleum contaminated soil and the evaluation of DNA quality was based on the DNA yield, purity, fragment size and genome integrity. The amplification of 16S rDNA V3 variable region and the subsequent a-nalysis of amplified products by DGGE. [Result] The results showed that three methods can extract corresponding DNA fragment from samples , and for the same extraction method , different properties soil had no obvious effect on the DNA extraction; but different methods had obvious distinction in the concentration and purity of extracted DNA. The extracted DNA yield of three methods are up to 98. 3,79. 9 and 48.3 ng/μl, respectively. [Conclusion]So the genomic DNA was extracted and further purified by the first method, the 16S rDNA and 16S rDNA V3 were amplified with universal primes F27/R1492 and F357/R518. The amplified DNA fragment were separated by parallel DGGE. The DGGE profiles can directly reflect the petroleum contaminated soil microbial diversity and dominant species.

  9. Characteristics of polyacrylamide gel with THPC and Turnbull Blue gel dosimeters evaluated using optical tomography

    Science.gov (United States)

    Pilařová (Vávrů), Kateřina; Kozubíková, Petra; Šolc, Jaroslav; Spěváček, Václav

    2014-11-01

    The purpose of this study was to compare characteristics of radiochromic gel - Turnbull Blue gel (TB gel) with polymer gel - polyacrylamide gel and tetrakis hydroxymethyl phosphonium chloride (PAGAT) using optical tomography. Both types of gels were examined in terms of dose sensitivity, dose response linearity and background value of spectrophotometric absorbance. The calibration curve was obtained for 60Co irradiation performed on Gammacell 220 at predefined gamma dose levels between 0 and 140 Gy for TBG and 0-15 Gy for PAGAT. To measure relative dose distributions from stereotactic irradiation, dosimeters were irradiated on Leksell Gamma Knife Perfexion. The cylindrical glass housings filled with gel were attached to the stereotactic frame. They were exposed with single shot and 16 mm collimator by 65 Gy to a 50% prescription isodose for TB gel and 4 Gy to a 50% prescription isodose for PAGAT. Evaluations of dosimeters were performed on an UV-vis Spectrophotometer Helios β and an optical cone beam homemade tomography scanner with a 16-bit astronomy CCD camera with a set of color filters. The advantages and potential disadvantages for both types of gel dosimeters were summarized. Dose distribution in central slice and measured profiles of 16 mm shot shows excellent correspondence with treatment planning system Leksell GammaPlan® for both PAGAT and Turnbull Blue gels. Gel dosimeters are suitable for steep dose gradient verification. An optical tomography evaluation method is successful. Dose response characteristics of TB gel and PAGAT gel are presented.

  10. 27 CFR 19.57 - Recovery and reuse of denatured spirits in manufacturing processes.

    Science.gov (United States)

    2010-04-01

    ... 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...

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

    Science.gov (United States)

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

    2016-05-01

    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.

  12. Combination of native and denaturing PAGE for the detection of protein binding regions in long fragments of genomic DNA

    Directory of Open Access Journals (Sweden)

    Metsis Madis

    2008-06-01

    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

  13. Two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimension (IPG-Dalt): the state of the art and the controversy of vertical versus horizontal systems.

    Science.gov (United States)

    Görg, A; Boguth, G; Obermaier, C; Posch, A; Weiss, W

    1995-07-01

    After having established the basic protocol of two-dimensional electrophoresis with immobilized pH gradients in the first dimension (IPG-Dalt) in 1988 (A. Görg et al., Electrophoresis 1988, 9, 531-546), some critical parameters of the actual IPG-Dalt protocols as well as the results obtained with horizontal and vertical second-dimensional sodium dodecyl sulfate-electrophoresis are demonstrated and discussed.

  14. RET and GDNF gene scanning in Hirschprung patients using two dual denaturing gel systems

    NARCIS (Netherlands)

    Hofstra, RMW; Wu, Y; Stulp, RP; Elfferich, P; Osinga, J; Maas, SM; Siderius, L; Brooks, AS; Von der Ende, JJ; Heydendael, VMR; Severijnen, RSVM; Bax, KMA; Meijers, C; Buys, CHCM

    2000-01-01

    Hirschsprung disease (HSCR) is a congenital disorder characterised by intestinal obstruction due to an absence of intramural ganglia along variable lengths of the intestine. RET is the major gene involved in HSCR. Mutations in the GDNF gene, and encoding one of the RET ligands, either alone or in co

  15. An assessment of the use of native and denatured forms of okra seed proteins as coagulants in drinking water treatment.

    Science.gov (United States)

    Jones, Alfred Ndahi; Bridgeman, John

    2016-10-01

    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.

  16. Heat-denatured lysozyme aggregation and gelation as revealed by combined dielectric relaxation spectroscopy and light scattering measurements.

    Science.gov (United States)

    Giugliarelli, A; Sassi, P; Paolantoni, M; Onori, G; Cametti, C

    2012-09-06

    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.

  17. Unusual cold denaturation of a small protein domain.

    Science.gov (United States)

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

    2012-08-21

    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.

  18. OBSERVATION OF DNA PARTIAL DENATURATION BY ATOMIC FORCE MICROSCOPY

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

    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.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    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....

  20. Thermoresponsive Gels

    Directory of Open Access Journals (Sweden)

    M. Joan Taylor

    2017-01-01

    Full Text Available Thermoresponsive gelling materials constructed from natural and synthetic polymers can be used to provide triggered action and therefore customised products such as drug delivery and regenerative medicine types as well as for other industries. Some materials give Arrhenius-type viscosity changes based on coil to globule transitions. Others produce more counterintuitive responses to temperature change because of agglomeration induced by enthalpic or entropic drivers. Extensive covalent crosslinking superimposes complexity of response and the upper and lower critical solution temperatures can translate to critical volume temperatures for these swellable but insoluble gels. Their structure and volume response confer advantages for actuation though they lack robustness. Dynamic covalent bonding has created an intermediate category where shape moulding and self-healing variants are useful for several platforms. Developing synthesis methodology—for example, Reversible Addition Fragmentation chain Transfer (RAFT and Atomic Transfer Radical Polymerisation (ATRP—provides an almost infinite range of materials that can be used for many of these gelling systems. For those that self-assemble into micelle systems that can gel, the upper and lower critical solution temperatures (UCST and LCST are analogous to those for simpler dispersible polymers. However, the tuned hydrophobic-hydrophilic balance plus the introduction of additional pH-sensitivity and, for instance, thermochromic response, open the potential for coupled mechanisms to create complex drug targeting effects at the cellular level.

  1. Succession of Denitrifying Community Composition in Coastal Wetland Soils Along a Salinity Gradient

    Institute of Scientific and Technical Information of China (English)

    PIAO Zhe; ZHANG Wen-Wen; MA Shuai; LI Yu-Min; YIN Shi-Xue

    2012-01-01

    To better understand the effect of salinity on denitrification communities,soils along a salinity gradient (ranging from 7.32 to 1.70 mS cm-1) in a wetland along the Yellow Sea coastline in Jiangsu Province,China,were studied using both culture-dependent and -independent methods.Culture efforts yielded 82 isolates in total,81.7% of which were close relatives of Bacillus sp.based on partial sequences of their 16S rRNA genes.Denaturing gradient gel electrophoresis (DGGE) analysis based on 16S rRNA sequences suggested possible existence of bacterial community succession along the salinity gradient.Clone library analysis based on nosZ gene sequences (coding nitrous oxide reductase) showed that operational taxonomic units (OTUs) associated with α-proteobacteria dominated in all three soils,whereas those associated with β-and γ-subdivisions showed a clear succession.In the high salinity soil,only the OTUs associated with α-subdivision were found.In the medium salinity soil,small proportions ofβ- (6.5%) and γ-associated (19.6%) OTUs were found.In the low salinity soil,the proportions were further increased to 33% and 25% for β- and γ-Proteobacteria,respectively.Statistic analysis using Unifrac P test showed that nosZ-communities in different saline soils were significantly different from each other.It could be concluded that α-subdivision of nosZ-community tended to be sustained in high salinity environments whereas β- and γ-subdivisions,especially the former,tended to be sustained in low salinity environments.Salinity was the key determinant of nosZ-community composition in the environment.

  2. Denaturation of membrane proteins and hyperthermic cell killing

    NARCIS (Netherlands)

    Burgman, Paulus Wilhelmus Johannes Jozef

    1993-01-01

    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

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

    Science.gov (United States)

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

    2016-02-01

    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.

  4. Genotypic distribution of a specialist model microorganism, Methanosaeta, along an estuarine gradient: does metabolic restriction limit niche differentiation potential?

    Science.gov (United States)

    Carbonero, Franck; Oakley, Brian B; Hawkins, Robert J; Purdy, Kevin J

    2012-05-01

    A reductionist ecological approach of using a model genus was adopted in order to understand how microbial community structure is driven by metabolic properties. The distribution along an estuarine gradient of the highly specialised genus Methanosaeta was investigated and compared to the previously determined distribution of the more metabolically flexible Desulfobulbus. Methanosaeta genotypic distribution along the Colne estuary (Essex, UK) was determined by DNA- and RNA-based denaturing gradient gel electrophoresis and 16S rRNA gene sequence analyses. Methanosaeta distribution was monotonic, with a consistently diverse community and no apparent niche partitioning either in DNA or RNA analyses. This distribution pattern contrasts markedly with the previously described niche partitioning and sympatric differentiation of the model generalist, Desulfobulbus. To explain this difference, it is hypothesised that Methanosaeta's strict metabolic needs limit its adaptation potential, thus populations do not partition into spatially distinct groups and so do not appear to be constrained by gross environmental factors such as salinity. Thus, at least for these two model genera, it appears that metabolic flexibility may be an important factor in spatial distribution and this may be applicable to other microbes.

  5. Urea Induced Denaturation of Pre-Q1 Riboswitch

    Science.gov (United States)

    Yoon, Jeseong; Thirumalai, Devarajan; Hyeon, Changbong

    2013-01-01

    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.

  6. Effect of electron irradiation on the gel properties of Collichthys lucidus surimi

    Science.gov (United States)

    Deng, Siyao; Lv, Liangyu; Yang, Wenge; Xu, Dalun; Lou, Qiaoming; Zhang, Jinjie

    2017-01-01

    This study evaluated the effect of electron irradiation on the gel properties of Collichthys lucidus surimi. The results showed that irradiation decreased the trichloroacetic acid-soluble peptide content of the surimi gel. At 5 kGy, a more compact and ordered gel network structure was achieved, resulting in a higher gel strength, whiteness, and water-holding capacity than non-irradiated surimi gel. During heat-induced formation of the gel, the α-helix content decreased, whilst the β-sheet and β-turn content increased. Irradiation treatments also decreased the α-helix content and increased β-sheet content, and this transformation is beneficial for the protein denaturation and gel formation. Collectively, the results suggest that electron irradiation, at an optimal dose of 5 kGy, could be an effective method for application in the surimi manufacturing industry

  7. One-dimensional SDS-polyacrylamide gel electrophoresis (1D SDS-PAGE).

    Science.gov (United States)

    Brunelle, Julie L; Green, Rachel

    2014-01-01

    This protocol describes a denaturing polyacrylamide gel system utilizing sodium dodecyl sulfate (SDS) to separate protein molecules based on size as first described by Laemmli (1970). SDS-PAGE can be used to monitor protein purifications, check the purity of samples, and to estimate molecular weights for unknown proteins. © 2014 Elsevier Inc. All rights reserved.

  8. Rheological properties of patatin gels compared with ß-lactoglobulin, ovalbumin, and glycinin

    NARCIS (Netherlands)

    Creusot, N.P.; Wierenga, P.A.; Laus, M.C.; Giuseppin, M.L.F.; Gruppen, H.

    2011-01-01

    BACKGROUND: The thermal unfolding and rheological properties of patatin gels were compared with those of commonly used proteins (ß-lactoglobulin, ovalbumin, glycinin). RESULTS: A significant difference between these proteins was observed in both the denaturation temperature (59 °C for patatin; about

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

    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.

  10. How it all began: a personal history of gel electrophoresis.

    Science.gov (United States)

    Smithies, Oliver

    2012-01-01

    Arne Tiselius' moving boundary electrophoresis method was still in general use in 1951 when this personal history begins, although zonal electrophoresis with a variety of supporting media (e.g., filter paper or starch grains) was beginning to replace it. This chapter is an account of 10 years of experiments carried out by the author during which molecular sieving gel electrophoresis was developed and common genetic variants of two proteins, haptoglobin and transferrin, were discovered in normal individuals. Most of the figures are images of pages from the author's laboratory notebooks, which are still available, so that some of the excitement of the time and the humorous moments are perhaps apparent. Alkaline gels, acidic gels with and without denaturants, vertical gels, two-dimensional gels, and gels with differences in starch concentration are presented. The subtle details that can be discerned in these various gels played an indispensable role in determining the nature of the change in the haptoglobin gene (Hp) that leads to the polymeric series characteristic of Hp ( 2 ) /Hp ( 2 ) homozygotes. Where possible, the names of scientific friends who made this saga of gel electrophoresis so memorable and enjoyable are gratefully included.

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

    CERN Document Server

    Bianco, Valentino

    2015-01-01

    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.

  12. Influence of Ficoll on urea induced denaturation of fibrinogen

    Science.gov (United States)

    Sankaranarayanan, Kamatchi; Meenakshisundaram, N.

    2016-03-01

    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.

  13. Temperature induced denaturation of collagen in acidic solution.

    Science.gov (United States)

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

    2007-07-01

    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.

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

    Science.gov (United States)

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

    1976-01-01

    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.

  15. NADP-specific isocitrate dehydrogenase of Escherichia coli. IV. Purification by chromatography on Affi-Gel Blue.

    Science.gov (United States)

    Vasquez, B; Reeves, H C

    1979-05-23

    Affinity chromatography on Affi-Gel Blue has been used to purify the NADP-specific isocitrate dehydrogenase (EC 1.1.1.42) from Escherichia coli. The protocol permits rapid purification of the enzyme in milligram quantities with a yield of 50% and is carried out almost entirely at room temperature. The preparation was judged to be homogeneous by non-denaturing electrophoresis at pH 7.5 and denaturing electrophoresis in the presence of sodium dodecyl sulfate. The subunit molecular weight of 53 000, determined by sodium dodecyl sulfate gel electrophoresis, is in reasonable agreement with the value of 46 900 estimated from the amino acid composition data.

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

    Science.gov (United States)

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

    2002-08-15

    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.

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

    Science.gov (United States)

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

    2007-05-21

    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

  18. Establishment of in vitro models of denatured collagen%变性胶原体外培养模型的建立

    Institute of Scientific and Technical Information of China (English)

    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

    2013-01-01

    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. Silver and Cyanine Staining of Oligonucleotides in Polyacrylamide Gel.

    Directory of Open Access Journals (Sweden)

    Weizhong Tang

    Full Text Available To explore why some oligonucleotides in denaturing polyacrylamide gel could not be silver-stained, 134 different oligonucleotides were analyzed using denaturing polyacrylamide gel electrophoresis stained with silver and asymmetric cyanine. As a result, we found that the sensitivity of oligos (dA, (dC, (dG and (dT to silver staining could be ranged as (dA > (dG > (dC > (dT from high to low. It was unexpected that oligo (dT was hard to be silver-stained. Moreover, the silver staining of an oligonucleotide containing base T could be partially or completely inhibited by base T. The inhibition of silver staining by base T was a competitive inhibition which could be affected by the amounts of the argyrophil nucleobase and base T, the cis-distance between the argyrophil nucleobase and base T, and the gel concentration. The changes of the intensity of an oligonucleotide band caused by the changes of DNA base composition were diverse and interesting. The intensity of some oligonucleotide bands would significantly change when the changes of DNA base composition accumulated to a certain extent (usually ≥ 4 nt. The sensitivity of cyanine staining of ≤ 11-nt long oligonucleotides could be enhanced about 250-fold by fixing the gels with methanol fixing solution.

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

    Science.gov (United States)

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

    2010-02-01

    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

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

    Science.gov (United States)

    Freitas, Juliana G.; Barker, James F.

    2013-05-01

    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.

  2. Strategies for denaturing the weapons-grade plutonium stockpile

    Energy Technology Data Exchange (ETDEWEB)

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

    1992-10-01

    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.

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

    Science.gov (United States)

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

    2003-11-01

    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.

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

    Institute of Scientific and Technical Information of China (English)

    王邦宁; 谈夫

    1997-01-01

    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

  5. Testosterone Nasal Gel

    Science.gov (United States)

    Testosterone nasal gel is used to treat symptoms of low testosterone in men who have hypogonadism (a condition in which the ... does not produce enough natural testosterone). Testosterone nasal gel is used only for men with low testosterone ...

  6. Calorimetric Study of Thermal Denaturation of Superoxide Dismutase

    Institute of Scientific and Technical Information of China (English)

    王邦宁; 谈夫

    1994-01-01

    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

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

    Science.gov (United States)

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

    2013-07-01

    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.

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

    Institute of Scientific and Technical Information of China (English)

    LI Rong; CHEN Guo-Liang

    2003-01-01

    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.

  9. Optical real-time measurement of collagen denaturation

    Science.gov (United States)

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

    1997-06-01

    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.

  10. Second-harmonic generation investigation of collagen thermal denaturation

    Science.gov (United States)

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

    2007-02-01

    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.

  11. Gradient chromatofocusing high-performance liquid chromatography. II. Theoretical aspects.

    Science.gov (United States)

    Liu, Y; Anderson, D J

    1997-02-21

    This article is Part II of a series describing a newly-developed gradient chromatofocusing high-performance liquid chromatography (HPLC) technique. Theoretical aspects of the technique are discussed. In gradient chromatofocusing, the column pH gradient with respect to column distance can be varied without necessarily affecting the outlet pH gradient with respect to time. Factors influencing the value of the slope of the column pH gradient are identified through derived equations and a computer simulation model. A newly-identified parameter is introduced, column travel time, which can be uniquely varied in gradient chromatofocusing. Experiments show increased conversion of fibrinogen to denatured forms with increased column travel time. Another unique aspect of gradient chromatofocusing is that the mobile phase buffer concentration can be manipulated without necessarily affecting the outlet pH gradient slope, giving the technique expanded versatility for optimizing the separation. In the present work, the pIapparent for fibrinogen is found to increase with increased mobile phase buffer concentration.

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

    DEFF Research Database (Denmark)

    Christiansen, Gunna; Christiansen, C; Bak, AL

    1975-01-01

    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.

    Science.gov (United States)

    2010-04-01

    ....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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-07-01

    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.

    Directory of Open Access Journals (Sweden)

    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. Gradient Gel Electrophoresis in a Patient With Recurrent Bacterial Vaginosis

    Directory of Open Access Journals (Sweden)

    Estelle Devillard

    2005-01-01

    Full Text Available Objective:Gardnerella vaginalis has long been the most common pathogen associated with bacterial vaginosis (BV. We aimed to test our hypothesis that symptoms and signs of BV do not necessarily indicate colonization by this organism, and often will not respond to standard metronidazole or clindamycin treatment.

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

    Science.gov (United States)

    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

    2016-09-14

    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

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

    Science.gov (United States)

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

    2014-01-01

    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).

  19. Sol-Gel Glasses

    Science.gov (United States)

    Mukherjee, S. P.

    1985-01-01

    Multicomponent homogeneous, ultrapure noncrystalline gels/gel derived glasses are promising batch materials for the containerless glass melting experiments in microgravity. Hence, ultrapure, homogeneous gel precursors could be used to: (1) investigate the effect of the container induced nucleation on the glass forming ability of marginally glass forming compositions; and (2) investigate the influence of gravity on the phase separation and coarsening behavior of gel derived glasses in the liquid-liquid immiscibility zone of the nonsilicate systems having a high density phase. The structure and crystallization behavior of gels in the SiO2-GeO2 as a function of gel chemistry and thermal treatment were investigated. As are the chemical principles involved in the distribution of a second network former in silica gel matrix being investigated. The procedures for synthesizing noncrystalline gels/gel-monoliths in the SiO2-GeO2, GeO2-PbO systems were developed. Preliminary investigations on the levitation and thermal treatment of germania silicate gel-monoliths in the Pressure Facility Acoustic Levitator were done.

  20. Gel-aided sample preparation (GASP)--a simplified method for gel-assisted proteomic sample generation from protein extracts and intact cells.

    Science.gov (United States)

    Fischer, Roman; Kessler, Benedikt M

    2015-04-01

    We describe a "gel-assisted" proteomic sample preparation method for MS analysis. Solubilized protein extracts or intact cells are copolymerized with acrylamide, facilitating denaturation, reduction, quantitative cysteine alkylation, and matrix formation. Gel-aided sample preparation has been optimized to be highly flexible, scalable, and to allow reproducible sample generation from 50 cells to milligrams of protein extracts. This methodology is fast, sensitive, easy-to-use on a wide range of sample types, and accessible to nonspecialists. © 2014 The Authors. PROTEOMICS published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Science.gov (United States)

    Basharov, Mahmud A

    2012-02-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2006-11-15

    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.)

  3. Pulse Field Gel Electrophoresis.

    Science.gov (United States)

    Sharma-Kuinkel, Batu K; Rude, Thomas H; Fowler, Vance G

    2016-01-01

    Pulse Field Gel Electrophoresis (PFGE) is a powerful genotyping technique used for the separation of large DNA molecules (entire genomic DNA) after digesting it with unique restriction enzymes and applying to a gel matrix under the electric field that periodically changes direction. PFGE is a variation of agarose gel electrophoresis that permits analysis of bacterial DNA fragments over an order of magnitude larger than that with conventional restriction enzyme analysis. It provides a good representation of the entire bacterial chromosome in a single gel with a highly reproducible restriction profile, providing clearly distinct and well-resolved DNA fragments.

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

    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

    Indian Academy of Sciences (India)

    Sagarika Dev; Avadhesha Surolia

    2006-12-01

    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

    Science.gov (United States)

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

    2010-02-01

    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.

    Science.gov (United States)

    Zhang, Li; Zhang, Qinming; Wang, Chaozhan

    2013-03-01

    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. Improved high-throughput DNA fragment analyzer employing horizontal ultrathin gel electrophoresis

    Science.gov (United States)

    Brumley, Robert L., Jr.; Luckey, John A.

    1996-04-01

    We are currently developing a significantly improved gel electrophoresis and detection system that will allow more than an order of magnitude enhancement in the speed of DNA fragment analysis. This system is based upon the technique of horizontal ultrathin gel electrophoresis (HUGE) which employs denaturing polyacrylamide gels that are 75 microns thick. Because of the thinness of the gel, very high electric field strengths may be applied without deleterious thermal effects on resolution. Our proprietary fluorescence detector that scans the gel during electrophoresis allows for the simultaneous detection of up to four fluorophores. Because of the efficiency of the system of light collection, the gel can be scanned at speeds fast enough to generate high resolution gel images despite the high speed of separations. In addition, we are able to increase sample density by collecting 500 datapoints across the width of the gel. The resulting instrument has the capability to separate and resolve single-stranded DNA molecules that are between 25 and 300 bases in length from each of 60 lanes in less than 45 minutes. With the advent of 96 lane gels and attendant automation, this instrument will have the ability to analyze 18,432 genotypes per day.

  9. Dissociative mechanism of F-actin thermal denaturation.

    Science.gov (United States)

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

    2006-11-01

    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.

  10. GelTouch

    DEFF Research Database (Denmark)

    Miruchna, Viktor; Walter, Robert; Lindlbauer, David

    2015-01-01

    We present GelTouch, a gel-based layer that can selectively transition between soft and stiff to provide tactile multi-touch feedback. It is flexible, transparent when not activated, and contains no mechanical, electromagnetic, or hydraulic components, resulting in a compact form factor (a 2mm thin...... touchscreen layer for our prototype). The activated areas can be morphed freely and continuously, without being limited to fixed, predefined shapes. GelTouch consists of a poly(N-isopropylacrylamide) gel layer which alters its viscoelasticity when activated by applying heat (>32 C). We present three different...... a tablet with 6x4 tactile areas, enabling a tactile numpad, slider, and thumbstick. We show that the gel is up to 25 times stiffer when activated and that users detect tactile features reliably (94.8%)....

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

    Science.gov (United States)

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

    2006-12-15

    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

    Institute of Scientific and Technical Information of China (English)

    BAI Quan; KONG Yu; DONG Cuihua; GENG Xindu

    2005-01-01

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

    Science.gov (United States)

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

    2016-01-01

    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.

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

    Science.gov (United States)

    Tischer, Alexander; Auton, Matthew

    2013-09-01

    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.

  15. Function, structure, and stability of enzymes confined in agarose gels.

    Directory of Open Access Journals (Sweden)

    Jeffrey Kunkel

    Full Text Available Research over the past few decades has attempted to answer how proteins behave in molecularly confined or crowded environments when compared to dilute buffer solutions. This information is vital to understanding in vivo protein behavior, as the average spacing between macromolecules in the cell cytosol is much smaller than the size of the macromolecules themselves. In our study, we attempt to address this question using three structurally and functionally different model enzymes encapsulated in agarose gels of different porosities. Our studies reveal that under standard buffer conditions, the initial reaction rates of the agarose-encapsulated enzymes are lower than that of the solution phase enzymes. However, the encapsulated enzymes retain a higher percentage of their activity in the presence of denaturants. Moreover, the concentration of agarose used for encapsulation had a significant effect on the enzyme functional stability; enzymes encapsulated in higher percentages of agarose were more stable than the enzymes encapsulated in lower percentages of agarose. Similar results were observed through structural measurements of enzyme denaturation using an 8-anilinonaphthalene-1-sulfonic acid fluorescence assay. Our work demonstrates the utility of hydrogels to study protein behavior in highly confined environments similar to those present in vivo; furthermore, the enhanced stability of gel-encapsulated enzymes may find use in the delivery of therapeutic proteins, as well as the design of novel strategies for biohybrid medical devices.

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

    Science.gov (United States)

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

    2007-11-28

    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

    Institute of Scientific and Technical Information of China (English)

    李冬海; 阮翔; 许强; 王可玢; 公衍道; 匡廷云; 赵南明

    2002-01-01

    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

    DEFF Research Database (Denmark)

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

    2012-01-01

    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.

    Directory of Open Access Journals (Sweden)

    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. Fabrication of Janus hydrogels with stiffness gradient using drop coalescence

    Science.gov (United States)

    Lee, Donghee; Golden, Kale; Ryu, Sangjin

    2016-11-01

    The stiffness of the extracellular matrix (ECM) regulates cellular behaviors, and polyacrylamide (PAAM) gels with stiffness gradient have been used to simulate inhomogeneous ECM and to study the effects of the ECM stiffness on cells. Such hydrogel substrates with stiffness gradient can be fabricated with relatively complicated methods using microfluidics and moving masks. In our study, we develop a simpler method for fabricating Janus hydrogel which has a gradient of stiffness. Two prepolymer solutions were prepared for soft and stiff gel compositions, respectively, and one drop of each solution was placed on a hydrophobic patterned glass. Then, these two drops were gently squeezed by another glass being slowly lowered until coalescence, and gel polymerization was initiated after a certain time period for mixing. The motion of the drops was guided by the hydrophobic pattern. AFM nano-indentation showed that the fabricated Janus PAAM gels have a stiffness gradient which could be controlled by increasing mixing time. This study was supported by Bioengineering for Human Health Grant from UNL and UNMC.

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

    Directory of Open Access Journals (Sweden)

    Fetahagić Safet

    2002-01-01

    . 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%.

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

    Science.gov (United States)

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

    2005-11-15

    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. Heat-induced whey protein gels: protein-protein interactions and functional properties.

    Science.gov (United States)

    Havea, Palatasa; Watkinson, Philip; Kuhn-Sherlock, Barbara

    2009-02-25

    Heat-induced gelation (80 degrees C for 30 min or 85 degrees C for 60 min) of whey protein concentrate (WPC) solutions was studied using small deformation dynamic rheology, small and large deformation compression, and polyacrylamide gel electrophoresis (PAGE). The WPC solutions (15% w/w, pH 6.9) were prepared by dispersing WPC powder in water (control), 1% (w/w) sodium dodecyl sulfate (SDS) solution, and N-ethylmaleimide (NEM) solution at a protein/NEM molar ratio of 1:1 or in 10 mM dithiothreitol (DTT) solution. PAGE analyses showed that the heat treatment of control solutions contained both disulfide and non-covalent linkages between denatured protein molecules. Only disulfide linkages were formed in heated SDS-WPC solutions, whereas only non-covalent linkages were formed in DTT-WPC and NEM-WPC solutions during heating. In heated NEM-WPC solutions, the pre-existing disulfide linkages remained unaltered. Small deformation rheology measurements showed that the storage modulus (G') values, compared with those of the control WPC gels (approximately 14000 Pa), were 3 times less for the SDS-WPC gels (approximately 4000 Pa), double for the NEM-WPC gels (approximately 24000 Pa), and even higher for the DTT-WPC gels (approximately 30000 Pa). Compression tests suggested that the rubberiness (fracture strain) of the WPC gels increased as the degree of disulfide linkages within the gels increased, whereas the stiffness (modulus) of the gels increased as the degree of non-covalent associations among the denatured protein molecules increased.

  4. Gradients are shaping up.

    Science.gov (United States)

    Bollenbach, Tobias; Heisenberg, Carl-Philipp

    2015-04-23

    In animal embryos, morphogen gradients determine tissue patterning and morphogenesis. Shyer et al. provide evidence that, during vertebrate gut formation, tissue folding generates graded activity of signals required for subsequent steps of gut growth and differentiation, thereby revealing an intriguing link between tissue morphogenesis and morphogen gradient formation.

  5. Fast Distributed Gradient Methods

    CERN Document Server

    Jakovetic, Dusan; Moura, Jose M F

    2011-01-01

    The paper proposes new fast distributed optimization gradient methods and proves convergence to the exact solution at rate O(\\log k/k), much faster than existing distributed optimization (sub)gradient methods with convergence O(1/\\sqrt{k}), while incurring practically no additional communication nor computation cost overhead per iteration. We achieve this for convex (with at least one strongly convex,) coercive, three times differentiable and with Lipschitz continuous first derivative (private) cost functions. Our work recovers for distributed optimization similar convergence rate gains obtained by centralized Nesterov gradient and fast iterative shrinkage-thresholding algorithm (FISTA) methods over ordinary centralized gradient methods. We also present a constant step size distributed fast gradient algorithm for composite non-differentiable costs. A simulation illustrates the effectiveness of our distributed methods.

  6. Mechanically induced gel formation

    NARCIS (Netherlands)

    van Herpt, Jochem T.; Stuart, Marc C. A.; Browne, Wesley R.; Feringa, Ben L.

    2013-01-01

    Mechanical triggering of gelation of an organic solution by a carbazole-based bisurea organogelator is described. Both the duration of the mechanical stimulation and the gelator concentration control the gelation process and the characteristics of the gel obtained.

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

    National Research Council Canada - National Science Library

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

    1997-01-01

    ...% 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

    CERN Document Server

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

    2012-01-01

    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)

    DEFF Research Database (Denmark)

    Andersen, Kell Kleiner; Otzen, Daniel

    2009-01-01

    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. Conformance Improvement Using Gels

    Energy Technology Data Exchange (ETDEWEB)

    Seright, Randall S.; Schrader, Richard; II Hagstrom, John; Wang, Ying; Al-Dahfeeri, Abdullah; Gary, Raven; Marin; Amaury; Lindquist, Brent

    2002-09-26

    This research project had two objectives. The first objective was to identify gel compositions and conditions that substantially reduce flow through fractures that allow direct channeling between wells, while leaving secondary fractures open so that high fluid injection and production rates can be maintained. The second objective was to optimize treatments in fractured production wells, where the gel must reduce permeability to water much more than that to oil.

  11. Crystallization from Gels

    Science.gov (United States)

    Narayana Kalkura, S.; Natarajan, Subramanian

    Among the various crystallization techniques, crystallization in gels has found wide applications in the fields of biomineralization and macromolecular crystallization in addition to crystallizing materials having nonlinear optical, ferroelectric, ferromagnetic, and other properties. Furthermore, by using this method it is possible to grow single crystals with very high perfection that are difficult to grow by other techniques. The gel method of crystallization provides an ideal technique to study crystal deposition diseases, which could lead to better understanding of their etiology. This chapter focuses on crystallization in gels of compounds that are responsible for crystal deposition diseases. The introduction is followed by a description of the various gels used, the mechanism of gelling, and the fascinating phenomenon of Liesegang ring formation, along with various gel growth techniques. The importance and scope of study on crystal deposition diseases and the need for crystal growth experiments using gel media are stressed. The various crystal deposition diseases, viz. (1) urolithiasis, (2) gout or arthritis, (3) cholelithiasis and atherosclerosis, and (4) pancreatitis and details regarding the constituents of the crystal deposits responsible for the pathological mineralization are discussed. Brief accounts of the theories of the formation of urinary stones and gallstones and the role of trace elements in urinary stone formation are also given. The crystallization in gels of (1) the urinary stone constituents, viz. calcium oxalate, calcium phosphates, uric acid, cystine, etc., (2) the constituents of the gallstones, viz. cholesterol, calcium carbonate, etc., (3) the major constituent of the pancreatic calculi, viz., calcium carbonate, and (4) cholic acid, a steroidal hormone are presented. The effect of various organic and inorganic ions, trace elements, and extracts from cereals, herbs, and fruits on the crystallization of major urinary stone and gallstone

  12. Preparation of chitosan gel

    Directory of Open Access Journals (Sweden)

    Lagerge S.

    2012-06-01

    Full Text Available Aerogel conditioning of the chitosan makes it possible to prepare porous solids of significant specific surface. The increase in the chitosan concentration or the degree of acetylation decreases the specific surface of the synthesized chitosan gel. Whereas drying with supercritical CO2 more effectively makes it possible to preserve the volume of the spheres of gel and to have a more significant specific surface in comparison with evaporative drying.

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

    OpenAIRE

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

    2016-01-01

    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...

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

    OpenAIRE

    Gaillard, Claire; Strauss, Francois

    1998-01-01

    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.

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

    Science.gov (United States)

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

    2016-04-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1978-12-01

    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.

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

    Science.gov (United States)

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

    2016-07-03

    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.

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

    Science.gov (United States)

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

    2016-10-01

    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.

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

    Science.gov (United States)

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

    1992-06-01

    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.

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

    Science.gov (United States)

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

    2016-01-01

    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

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

    Science.gov (United States)

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

    2013-02-01

    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.

  2. Electrophoretic analysis of proteins from Mycoplasma hominis strains detected by SDS-PAGE, two-dimensional gel electrophoresis and immunoblotting

    DEFF Research Database (Denmark)

    Andersen, H; Birkelund, Svend; Christiansen, Gunna

    1987-01-01

    The proteins of 14 strains of Mycoplasma hominis were compared by SDS-PAGE in gradient gels, by two-dimensional (2D) gel electrophoresis of extracts of 35S-labelled cells and by immunoblot analysis of cell proteins. The strains examined included the M. hominis type strain PG21 and 13 others...... isolated variously from genital tract, mouth, blood, upper urinary tract and a wound. These 14 strains shared 76-99% of proteins in SDS-gradient gel analysis and 41-72% in the 2D gels. As expected, the immunoblot analysis likewise revealed the existence of an extensive common protein pattern in M. hominis...

  3. Modifying the Cold Gelation Properties of Quinoa Protein Isolate: Influence of Heat-Denaturation pH in the Alkaline Range.

    Science.gov (United States)

    Mäkinen, Outi E; Zannini, Emanuele; Arendt, Elke K

    2015-09-01

    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.

  4. Design of Autonomous Gel Actuators

    Directory of Open Access Journals (Sweden)

    Shuji Hashimoto

    2011-01-01

    Full Text Available In this paper, we introduce autonomous gel actuators driven by chemical energy. The polymer gels prepared here have cyclic chemical reaction networks. With a cyclic reaction, the polymer gels generate periodical motion. The periodic motion of the gel is produced by the chemical energy of the oscillatory Belouzov-Zhabotinsky (BZ reaction. We have succeeded in making synthetic polymer gel move autonomously like a living organism. This experimental fact represents the great possibility of the chemical robot.

  5. Laser textured surface gradients

    Science.gov (United States)

    Ta, Van Duong; Dunn, Andrew; Wasley, Thomas J.; Li, Ji; Kay, Robert W.; Stringer, Jonathan; Smith, Patrick J.; Esenturk, Emre; Connaughton, Colm; Shephard, Jonathan D.

    2016-05-01

    This work demonstrates a novel technique for fabricating surfaces with roughness and wettability gradients and their subsequent applications for chemical sensors. Surface roughness gradients on brass sheets are obtained directly by nanosecond laser texturing. When these structured surfaces are exposed to air, their wettability decreases with time (up to 20 days) achieving both spatial and temporal wettability gradients. The surfaces are responsive to organic solvents. Contact angles of a series of dilute isopropanol solutions decay exponentially with concentration. In particular, a fall of 132° in contact angle is observed on a surface gradient, one order of magnitude higher than the 14° observed for the unprocessed surface, when the isopropanol concentration increased from 0 to 15.6 wt%. As the wettability changes gradually over the surface, contact angle also changes correspondingly. This effect offers multi-sensitivity at different zones on the surface and is useful for accurate measurement of chemical concentration.

  6. Second gradient poromechanics

    CERN Document Server

    Sciarra, Giulio; Coussy, Olivier

    2010-01-01

    Second gradient theories have been developed in mechanics for treating different phenomena as capillarity in fluids, plasticity and friction in granular materials or shear band deformations. Here, there is an attempt of formulating a second gradient Biot like model for porous materials. In particular the interest is focused in describing the local dilatant behaviour of a porous material induced by pore opening elastic and capillary interaction phenomena among neighbouring pores and related micro-filtration phenomena by means of a continuum microstructured model. The main idea is to extend the classical macroscopic Biot model by including in the description second gradient effects. This is done by assuming that the surface contribution to the external work rate functional depends on the normal derivative of the velocity or equivalently assuming that the strain work rate functional depends on the porosity and strain gradients. According to classical thermodynamics suitable restrictions for stresses and second g...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-01-12

    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

  8. Heat-denaturation and aggregation of quinoa (Chenopodium quinoa) globulins as affected by the pH value.

    Science.gov (United States)

    Mäkinen, Outi E; Zannini, Emanuele; Koehler, Peter; Arendt, Elke K

    2016-04-01

    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.

  9. Efficacy of whey protein gel networks as potential viability-enhancing scaffolds for cell immobilization of Lactobacillus rhamnosus GG.

    Science.gov (United States)

    Doherty, S B; Gee, V L; Ross, R P; Stanton, C; Fitzgerald, G F; Brodkorb, A

    2010-03-01

    This study investigated cell immobilization of Lactobacillus rhamnosus GG in three separate protein products: native, denatured and hydrolysed whey protein isolate (WPI). Treatments were assessed for their ability to enhance probiotic survival during storage, heat stress and ex vivo gastric incubation. Spatial distribution of probiotic cells within immobilized treatments was evaluated by atomic force and confocal scanning laser microscopy, while cell viability was enumerated by plate count and flow cytometry (FACS). Microscopic analysis of denatured treatments revealed an oasis of immobilized cells, phase-separated from the surrounding protein matrix; an environmental characteristic analogous to hydrolysed networks. Cell immobilization in hydrolysed and denatured WPI enhanced survival by 6.1+/-0.1 and 5.8+/-0.1 log10 cycles, respectively, following 14 day storage at 37 degrees C and both treatments generated thermal protection at 57 degrees C (7.3+/-0.1 and 6.5+/-0.1 log(10) cfu/ml). Furthermore, denatured WPI enhanced probiotic protection (8.9+/-0.2 log(10) cfu/ml) following 3h gastric incubation at 37 degrees C. In conclusion, hydrolysed or denatured WPI were the most suitable matrices for cell immobilization, while native protein provided the weakest safeguard against thermal and acid stress, thus making it possible to envision whey protein gel networks as protective substrates for cell immobilization applications.

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

    Science.gov (United States)

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

    2014-12-01

    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.

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

    Science.gov (United States)

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

    2009-02-01

    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.

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

    Science.gov (United States)

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

    2012-02-01

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  14. Gel electrolytes and electrodes

    Energy Technology Data Exchange (ETDEWEB)

    Fleischmann, Sven; Bunte, Christine; Mikhaylik, Yuriy V.; Viner, Veronika G.

    2017-09-05

    Gel electrolytes, especially gel electrolytes for electrochemical cells, are generally described. In some embodiments, the gel electrolyte layers comprise components a) to c). Component a) may be at least one layer of at least one polymer comprising polymerized units of: a1) at least one monomer containing an ethylenically unsaturated unit and an amido group and a2) at least one crosslinker. Component b) may be at least one conducting salt and component c) may be at least one solvent. Electrodes may comprise the components a), d) and e), wherein component a) may be at least one layer of at least one polymer as described herein. Component d) may be at least one electroactive layer and component e) may be at least one ceramic layer. Furthermore, electrochemical cells comprising component a) which may be at least one layer of at least one polymer as described herein, are also provided.

  15. Active Polymer Gel Actuators

    Directory of Open Access Journals (Sweden)

    Shuji Hashimoto

    2010-01-01

    Full Text Available Many kinds of stimuli-responsive polymer and gels have been developed and applied to biomimetic actuators or artificial muscles. Electroactive polymers that change shape when stimulated electrically seem to be particularly promising. In all cases, however, the mechanical motion is driven by external stimuli, for example, reversing the direction of electric field. On the other hand, many living organisms can generate an autonomous motion without external driving stimuli like self-beating of heart muscles. Here we show a novel biomimetic gel actuator that can walk spontaneously with a wormlike motion without switching of external stimuli. The self-oscillating motion is produced by dissipating chemical energy of oscillating reaction. Although the gel is completely composed of synthetic polymer, it shows autonomous motion as if it were alive.

  16. Hydrolytic Activity of Esterase-Antibody Complexes Retained Within Gel Capsules After Complex Isolation.

    Science.gov (United States)

    Shimazaki, Youji; Miyatsuka, Rino

    2017-01-09

    Delipidation in biological samples is important for some diagnostic tests and protein analyses. Lipids in the samples can be hydrolyzed by native esterases (ESs) within gel capsules after ES, and ES-antibody complexes are specifically trapped, extracted, and separated. Acrylamide and agarose gel capsules containing complexes of ES antibody were produced after the complexes were extracted using protein A-immobilized membranes, separated by non-denaturing electrophoresis, and stained by colloidal silver using glucose as a reductant. ES activity of ES-antibody complexes within the gel capsule was significantly higher than that in the complexes with the control antibodies upon isolation, separation, and detection of the complex. In addition, lipids bound to human serum albumin decreased after human plasma was treated with gel capsules containing ES-antibody complexes. We demonstrate that the gel capsule containing ES-antibody complexes can be successfully isolated using techniques described in this study. Furthermore, delipidation of human plasma is obtained by incubation with the gel capsule. These results indicate that surplus materials such as lipids in biological samples can be removed or reduced by gel capsule containing enzymes.

  17. Terahertz spectroscopy of dry, hydrated, and thermally denatured biological macromolecules

    Science.gov (United States)

    Lipscomb, Dawn; Echchgadda, Ibtissam; Ibey, Bennett L.; Beier, Hope; Thomas, Robert J.; Peralta, Xomalin; Wilmink, Gerald J.

    2012-03-01

    Terahertz time-domain spectroscopy (THz-TDS) is an effective technique to probe the intermolecular and collective vibrational modes of biological macromolecules at THz frequencies. To date, the vast majority of spectroscopic studies have been performed on dehydrated biomolecular samples. Given the fact that all biochemical processes occur in aqueous environments and water is required for proper protein folding and function, we hypothesize that valuable information can be gained from spectroscopic studies performed on hydrated biomolecules in their native conformation. In this study, we used a THz-TDS system that exploits photoconductive techniques for THz pulse generation and freespace electro-optical sampling approaches for detection. We used the THz spectrometer to measure the time-dependent electric field of THz waves upon interaction with water, phosphate buffered saline (PBS), and collagen gels. By comparing these waveforms with references, we simultaneously determined each sample's index of refraction (n) and absorption coefficients (μa) as a function of frequency. Our data show that the properties we measure for the water, PBS and collagen are comparable to those reported in the literature. In the future, we plan to examine the effect that both temperature and pH have on the optical properties of other biological macromolecules. Studies will also be performed to compare our results to those generated using molecular dynamics simulations.

  18. Temperature Gradient Approach for Rapidly Assessing Sensor Binding Kinetics and Thermodynamics.

    Science.gov (United States)

    Wagner, Caleb E; Macedo, Lucyano J A; Opdahl, Aric

    2015-08-01

    We report a highly resolved approach for quantitatively measuring the temperature dependence of molecular binding in a sensor format. The method is based on surface plasmon resonance (SPR) imaging measurements made across a spatial temperature gradient. Simultaneous recording of sensor response over the range of temperatures spanned by the gradient avoids many of the complications that arise in the analysis of SPR measurements where temperature is varied. In addition to simplifying quantitative analysis of binding interactions, the method allows the temperature dependence of binding to be monitored as a function of time, and provides a straightforward route for calibrating how temperature varies across the gradient. Using DNA hybridization as an example, we show how the gradient approach can be used to measure the temperature dependence of binding kinetics and thermodynamics (e.g., melt/denaturation profile) in a single experiment.

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

    Directory of Open Access Journals (Sweden)

    Adyani Azizah Abd Halim

    2017-01-01

    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.

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

    Science.gov (United States)

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

    2015-06-15

    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.

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

    Directory of Open Access Journals (Sweden)

    Ana Mejía-López

    2017-03-01

    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.

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

    Science.gov (United States)

    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

    2013-06-01

    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.

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

    Science.gov (United States)

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

    2011-11-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Martin C Stumpe

    2008-11-01

    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.

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

    Science.gov (United States)

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

    2017-01-01

    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

  6. High Gradient Accelerator Research

    Energy Technology Data Exchange (ETDEWEB)

    Temkin, Richard [Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States). Dept. of Physics. Plasma Science and Fusion Center

    2016-07-12

    The goal of the MIT program of research on high gradient acceleration is the development of advanced acceleration concepts that lead to a practical and affordable next generation linear collider at the TeV energy level. Other applications, which are more near-term, include accelerators for materials processing; medicine; defense; mining; security; and inspection. The specific goals of the MIT program are: • Pioneering theoretical research on advanced structures for high gradient acceleration, including photonic structures and metamaterial structures; evaluation of the wakefields in these advanced structures • Experimental research to demonstrate the properties of advanced structures both in low-power microwave cold test and high-power, high-gradient test at megawatt power levels • Experimental research on microwave breakdown at high gradient including studies of breakdown phenomena induced by RF electric fields and RF magnetic fields; development of new diagnostics of the breakdown process • Theoretical research on the physics and engineering features of RF vacuum breakdown • Maintaining and improving the Haimson / MIT 17 GHz accelerator, the highest frequency operational accelerator in the world, a unique facility for accelerator research • Providing the Haimson / MIT 17 GHz accelerator facility as a facility for outside users • Active participation in the US DOE program of High Gradient Collaboration, including joint work with SLAC and with Los Alamos National Laboratory; participation of MIT students in research at the national laboratories • Training the next generation of Ph. D. students in the field of accelerator physics.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    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...

  8. Symbiodinium (Dinophyceae) diversity in reef-invertebrates along an offshore to inshore reef gradient near Lizard Island, Great Barrier Reef.

    Science.gov (United States)

    Tonk, Linda; Sampayo, Eugenia M; LaJeunesse, Todd C; Schrameyer, Verena; Hoegh-Guldberg, Ove

    2014-06-01

    Despite extensive work on the genetic diversity of reef invertebrate-dinoflagellate symbioses on the Great Barrier Reef (GBR; Australia), large information gaps exist from northern and inshore regions. Therefore, a broad survey was done comparing the community of inshore, mid-shelf and outer reefs at the latitude of Lizard Island. Symbiodinium (Freudenthal) diversity was characterized using denaturing gradient gel electrophoresis fingerprinting and sequencing of the ITS2 region of the ribosomal DNA. Thirty-nine distinct Symbiodinium types were identified from four subgeneric clades (B, C, D, and G). Several Symbiodinium types originally characterized from the Indian Ocean were discovered as well as eight novel types (C1kk, C1LL, C3nn, C26b, C161a, C162, C165, C166). Multivariate analyses on the Symbiodinium species diversity data showed a strong link with host identity, consistent with previous findings. Of the four environmental variables tested, mean austral winter sea surface temperature (SST) influenced Symbiodinium distribution across shelves most significantly. A similar result was found when the analysis was performed on Symbiodinium diversity data of genera with an open symbiont transmission mode separately with chl a and PAR explaining additional variation. This study underscores the importance of SST and water quality related variables as factors driving Symbiodinium distribution on cross-shelf scales. Furthermore, this study expands our knowledge on Symbiodinium species diversity, ecological partitioning (including host-specificity) and geographic ranges across the GBR. The accelerating rate of environmental change experienced by coral reef ecosystems emphasizes the need to comprehend the full complexity of cnidarian symbioses, including the biotic and abiotic factors that shape their current distributions.

  9. gonarthrosis; therapy; Karmolis gel.

    Directory of Open Access Journals (Sweden)

    B. V. Zavodovsky

    2014-01-01

    Full Text Available Objective: to evaluate the efficacy, tolerance, and safety of Carmolis topical gel in patients with gonarthrosis. Subjects and methods. The investigation enrolled 60 patients with knee osteoarthrosis (OA who were divided into two groups: 1 40 patients received Carmolis topical gel in addition to nonsteroidal anti-inflammatory drugs (NSAIDs; 2 20 patients took NSAIDS only (a control group. The treatment duration was 2 weeks. In both groups, therapeutic effectiveness was evaluated from changes in the WOMAC index, pain intensity at rest and during movement by the visual analog scale (VAS. The disease activity was also assessed by a physician and a patient (a Likert scale, local swelling and hyperthermia of the affected joint, the efficiency of treatment, and daily needs for NSAIDs were deter- mined. Results. The performed treatment in both patent groups showed positive clinical changes. Combination therapy involving Carmolis gel displayed greater reductions in WOMAC pain and resting and movement pain than in the con- trol group (as assessed by VAS. On completion of the investigation, considerable improvement was, in the physicians' opinion, noted in 38 (95% patients using Carmolis, which coincided with self-evaluations of the patients. During Carmolis application, the starting dose of NSAIDs could be reduced in 18 (45% patients. Adverse reactions occurred infrequently and required no therapy discontinuation. Conclusion. Carmolis topical gel is effective in relieving clinical symptoms in patients with gonarthrosis, well tolerated, and safe, which can recommend its use in the combination treatment of knee OA.

  10. Effects of ascorbic acid and sugars on solubility, thermal, and mechanical properties of egg white protein gels.

    Science.gov (United States)

    Mohammadi Nafchi, Abdorreza; Tabatabaei, Ramin H; Pashania, Bita; Rajabi, Hadiseh Z; Karim, A A

    2013-11-01

    The effects of reducing sugars (fructose, glucose, ribose, and arabinose), sucrose, and ascorbic acid were studied on thermo-mechanical properties and crosslinking of egg white proteins (EWP) through Maillard reaction. Sugars (0%, 1%, 5%, and 10%) and ascorbic acid (0%, 0.25%, 0.5%, and 2.5%) were added to EWP solutions. Thermal denaturation and crosslinking of EWP were characterized by differential scanning calorimetry (DSC). Mechanical properties (failure strength, failure strain and Young's modulus) of modified and unmodified EWP gels were evaluated by texture analyzer. Ascorbic acid decreased thermal denaturation temperature of EWP, but the reducing sugars increased the denaturation temperature. DSC thermograms of EWP showed that ascorbic acid exhibited an exothermic transition (≈110 °C) which was attributed to Maillard crosslinking of the protein. The reduction in pH (from 7.21 to ≈6) and protein solubility of egg white protein gel (from ≈70% to ≈10%) provides further evidence of the formation of Maillard cross-linking. Reactive sugars (ribose and arabinose) increased the mechanical properties of EWP gels, whereas ascorbic acid decreased the mechanical properties. Generally, the effect of ascorbic acid was more pronounced than that of various reducing sugars on the thermal and mechanical properties of egg white proteins.

  11. Uniform gradient expansions

    Directory of Open Access Journals (Sweden)

    Massimo Giovannini

    2015-06-01

    Full Text Available Cosmological singularities are often discussed by means of a gradient expansion that can also describe, during a quasi-de Sitter phase, the progressive suppression of curvature inhomogeneities. While the inflationary event horizon is being formed the two mentioned regimes coexist and a uniform expansion can be conceived and applied to the evolution of spatial gradients across the protoinflationary boundary. It is argued that conventional arguments addressing the preinflationary initial conditions are necessary but generally not sufficient to guarantee a homogeneous onset of the conventional inflationary stage.

  12. Uniform gradient expansions

    Energy Technology Data Exchange (ETDEWEB)

    Giovannini, Massimo, E-mail: massimo.giovannini@cern.ch [Department of Physics, Theory Division, CERN, 1211 Geneva 23 (Switzerland); INFN, Section of Milan-Bicocca, 20126 Milan (Italy)

    2015-06-30

    Cosmological singularities are often discussed by means of a gradient expansion that can also describe, during a quasi-de Sitter phase, the progressive suppression of curvature inhomogeneities. While the inflationary event horizon is being formed the two mentioned regimes coexist and a uniform expansion can be conceived and applied to the evolution of spatial gradients across the protoinflationary boundary. It is argued that conventional arguments addressing the preinflationary initial conditions are necessary but generally not sufficient to guarantee a homogeneous onset of the conventional inflationary stage.

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

    Science.gov (United States)

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

    2015-01-01

    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.

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

    Science.gov (United States)

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

    2011-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Mohammed Suleiman Zaroog

    2013-01-01

    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.

  16. EQUIVALENCY OF FOULING THICKNESS WITH DENATURED Β-LG IN HEATING OF MILK

    Directory of Open Access Journals (Sweden)

    Heriberto Molina-Pérez

    2015-03-01

    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.

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

    Science.gov (United States)

    Camilloni, Carlo; Vendruscolo, Michele

    2014-06-25

    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.

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

    Directory of Open Access Journals (Sweden)

    Denise M. Elser

    2011-01-01

    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.

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

    Science.gov (United States)

    Macdonald, Ryan D; Khajehpour, Mazdak

    2015-01-01

    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.

  20. Gradient systems and mechanical systems

    Institute of Scientific and Technical Information of China (English)

    Fengxiang Mei; Huibin Wu

    2016-01-01

    All types of gradient systems and their properties are discussed. Two problems connected with gradient sys-tems and mechanical systems are studied. One is the direct problem of transforming a mechanical system into a gradi-ent system, and the other is the inverse problem, which is transforming a gradient system into a mechanical system.

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

    Science.gov (United States)

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

    2016-11-30

    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.

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

    Science.gov (United States)

    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.

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

    DEFF Research Database (Denmark)

    Lindorff-Larsen, Kresten; Winther, J R

    2001-01-01

    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...

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

    Institute of Scientific and Technical Information of China (English)

    Yan WANG; Bo Lin GONG; Xin Du GENG

    2003-01-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Wu, L.; Timpe, R.C.

    1993-12-01

    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.

  6. Fabrication of high-density collagen fibril matrix gels by renaturation of triple-helix collagen from gelatin.

    Science.gov (United States)

    Ohyabu, Yoshimi; Yunoki, Shunji; Hatayama, Hirosuke; Teranishi, Yoshikazu

    2013-11-01

    Collagen-based 3-D hydrogels often lack sufficient mechanical strength for tissue engineering. We developed a method for fabrication of high-density collagen fibril matrix (CFM) gels from concentrated solutions of uncleaved gelatin (UCG). Denatured random-coil UCG exhibited more rapid and efficient renaturation into collagen triple-helix than cleaved gelatin (CG) over a broad range of setting temperatures. The UCG solution formed opaque gels with high-density reconstituted collagen fibrils at 28-32 °C and transparent gels similar to CG at 5%) and elasticity (1.28 ± 0.15 kPa at 5% and 4.82 ± 0.38 kPa at 8%) with minimal cell loss. The elastic modulus of these gels was higher than that of conventional CFM containing 0.5% collagen. High-strength CFM may provide more durable hydrogels for tissue engineering and regenerative medicine.

  7. Agarose gel electrophoresis for the separation of DNA fragments.

    Science.gov (United States)

    Lee, Pei Yun; Costumbrado, John; Hsu, Chih-Yuan; Kim, Yong Hoon

    2012-04-20

    Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb(1). Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits(2). During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight(3). The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along(4). The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation(5); 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: Understand the mechanism by which DNA fragments are separated within a gel matrix Understand how conformation of the DNA molecule will determine its mobility through a gel matrix Identify an agarose solution of appropriate

  8. Diffusion from gel in brain: modelisation and identification.

    Science.gov (United States)

    Bellagoun, A; Meulemans, A; Cherruault, Y

    1992-03-01

    A mathematical model is proposed for describing the mechanism of diffusion from gel (Tissucol) into the extracellular space. After diffusion of the antibiotic in one dimension, the gradient concentration was determined with microvoltametric electrodes. These microelectrodes measure the free diffusible form of electroactive antibiotics in the extracellular brain space. The aim of this study was to find simultaneously the coefficient of diffusion and extraction of some antibiotics (in our case the Fotemustin) using the Alienor Algorithm. These coefficients are useful for predicting the concentration gradient into abscesses, fibrin, etc.

  9. Active Polymer Gel Actuators

    OpenAIRE

    Shuji Hashimoto; Ryo Yoshida; Yusuke Hara; Shingo Maeda

    2010-01-01

    Many kinds of stimuli-responsive polymer and gels have been developed and applied to biomimetic actuators or artificial muscles. Electroactive polymers that change shape when stimulated electrically seem to be particularly promising. In all cases, however, the mechanical motion is driven by external stimuli, for example, reversing the direction of electric field. On the other hand, many living organisms can generate an autonomous motion without external driving stimuli like self-beating of he...

  10. Evaluation of the impact of six different DNA extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel-based DNA profiling method.

    Science.gov (United States)

    Dilhari, Ayomi; Sampath, Asanga; Gunasekara, Chinthika; Fernando, Neluka; Weerasekara, Deepaka; Sissons, Chris; McBain, Andrew; Weerasekera, Manjula

    2017-09-19

    Infected chronic wounds are polymicrobial in nature which include a diverse group of aerobic and anaerobic microorganisms. Majority of these communal microorganisms are difficult to grow in vitro. DNA fingerprinting methods such as polymerase chain reaction-denaturation gradient gel electrophoresis (PCR-DGGE) facilitate the microbial profiling of complex ecosystems including infected chronic wounds. Six different DNA extraction methods were compared for profiling of the microbial community associated with chronic wound infections using PCR-DGGE. Tissue debris obtained from chronic wound ulcers of ten patients were used for DNA extraction. Total nucleic acid was extracted from each specimen using six DNA extraction methods. The yield, purity and quality of DNA was measured and used for PCR amplification targeting V2-V3 region of eubacterial 16S rRNA gene. QIAGEN DNeasy Blood and Tissue Kit (K method) produced good quality genomic DNA compared to the other five DNA extraction methods and gave a broad diversity of bacterial communities in chronic wounds. Among the five conventional methods, bead beater/phenol-chloroform based DNA extraction method with STES buffer (BP1 method) gave a yield of DNA with a high purity and resulted in a higher DGGE band diversity. Although DNA extraction using heat and NaOH had the lowest purity, DGGE revealed a higher bacterial diversity. The findings suggest that the quality and the yield of genomic DNA are influenced by the DNA extraction protocol, thus a method should be carefully selected in profiling a complex microbial community.

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

    Science.gov (United States)

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

    2017-10-01

    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.

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

    Science.gov (United States)

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

    2011-02-15

    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.

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

    CSIR Research Space (South Africa)

    Mtwisha, L

    2007-02-01

    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...

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

    Directory of Open Access Journals (Sweden)

    Wouters Johan

    2008-06-01

    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.

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

    Science.gov (United States)

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

    2012-12-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Dahlen, Oda, E-mail: oda.dahlen@ntnu.no; Erp, Titus S. van, E-mail: titus.van.erp@ntnu.no [Department of Chemistry, Norwegian University of Science and Technology (NTNU), Høgskoleringen 5, Realfagbygget D3-117 7491 Trondheim (Norway)

    2015-06-21

    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.

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

    Science.gov (United States)

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

    2016-03-01

    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.

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    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...

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

    Science.gov (United States)

    Singh, Amit Raj; Granek, Rony

    2016-10-14

    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.

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

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    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.

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

    Science.gov (United States)

    Singh, Amit Raj; Granek, Rony

    2016-10-01

    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.

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

    Science.gov (United States)

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

    2008-01-01

    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…

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

    Science.gov (United States)

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

    2016-10-04

    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.

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

    DEFF Research Database (Denmark)

    Christiansen, Gunna; Christiansen, C

    1976-01-01

    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...

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

    NARCIS (Netherlands)

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

    2005-01-01

    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 °

  6. Bigravity from gradient expansion

    Energy Technology Data Exchange (ETDEWEB)

    Yamashita, Yasuho [Yukawa Institute for Theoretical Physics, Kyoto University,606-8502, Kyoto (Japan); Tanaka, Takahiro [Yukawa Institute for Theoretical Physics, Kyoto University,606-8502, Kyoto (Japan); Department of Physics, Kyoto University,606-8502, Kyoto (Japan)

    2016-05-04

    We discuss how the ghost-free bigravity coupled with a single scalar field can be derived from a braneworld setup. We consider DGP two-brane model without radion stabilization. The bulk configuration is solved for given boundary metrics, and it is substituted back into the action to obtain the effective four-dimensional action. In order to obtain the ghost-free bigravity, we consider the gradient expansion in which the brane separation is supposed to be sufficiently small so that two boundary metrics are almost identical. The obtained effective theory is shown to be ghost free as expected, however, the interaction between two gravitons takes the Fierz-Pauli form at the leading order of the gradient expansion, even though we do not use the approximation of linear perturbation. We also find that the radion remains as a scalar field in the four-dimensional effective theory, but its coupling to the metrics is non-trivial.

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

    Science.gov (United States)

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

    2017-04-15

    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

  8. Increasing SLEDed Linac Gradient

    Energy Technology Data Exchange (ETDEWEB)

    Farkas, Zoltan D

    2001-11-08

    This note will show how to increase the SLED [1] gradient by varying Q{sub e}, the external Q of the SLED cavity, by increasing its Q{sub 0} and by increasing the compression ratio. If varying the external Q is to be effective, then the copper losses should be small so that Q{sub 0} >> Q{sub e}. Methods of varying Q{sub e} will be indicated but no experimental data will be presented. If we increase the klystron pulse width from 3.5 to 5 {micro}S and increase Q{sub 0} from the present 100000 to 300000, then the gradient increases by 19% and the beam energy increases from 50 to 60 GeV. This note will also discuss SLED operation at 11424 MHz, the NLC frequency. Without Q{sub e} switching, using SLED at 11424 MHz increases the SLAC gradient from 21 MV/m to 34 MV/m, and at the same repetition rate, uses about 1/5 of rf average power. If we also double the compression ratio, we reach 47 MV/m and over 100 GeV beam energy.

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

    Science.gov (United States)

    Little, R M; Anderson, P M

    1987-07-25

    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.

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

    Science.gov (United States)

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

    2008-01-01

    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.

  11. Studies on supramolecular gel formation using DOSY NMR.

    Science.gov (United States)

    Nonappa; Šaman, David; Kolehmainen, Erkki

    2015-04-01

    Herein, we present the results obtained from our studies on supramolecular self-assembly and molecular mobility of low-molecular-weight gelators (LMWGs) in organic solvents using pulsed field gradient (PFG) diffusion ordered spectroscopy (DOSY) NMR. A series of concentration-dependent DOSY NMR experiments were performed on selected LMWGs to determine the critical gelation concentration (CGC) as well as to understand the behaviour of the gelator molecules in the gel state. In addition, variable-temperature DOSY NMR experiments were performed to determine the gel-to-sol transition. The PFG NMR experiments performed as a function of gradient strength were further analyzed using monoexponential DOSY processing, and the results were compared with the automated Bayesian DOSY transformation to obtain 2D plots. Our results provide useful information on the stepwise self-assembly of small molecules leading to gelation. We believe that the results obtained from these experiments are applicable in determining the CGC and gel melting temperatures of supramolecular gels.

  12. Factors influencing alginate gel biocompatibility.

    Science.gov (United States)

    Tam, Susan K; Dusseault, Julie; Bilodeau, Stéphanie; Langlois, Geneviève; Hallé, Jean-Pierre; Yahia, L'Hocine

    2011-07-01

    Alginate remains the most popular polymer used for cell encapsulation, yet its biocompatibility is inconsistent. Two commercially available alginates were compared, one with 71% guluronate (HiG), and the other with 44% (IntG). Both alginates were purified, and their purities were verified. After 2 days in the peritoneal cavity of C57BL/6J mice, barium (Ba)-gel and calcium (Ca)-gel beads of IntG alginate were clean, while host cells were adhered to beads of HiG alginate. IntG gel beads, however, showed fragmentation in vivo while HiG gel beads stayed firm. The physicochemical properties of the sodium alginates and their gels were thoroughly characterized. The intrinsic viscosity of IntG alginate was 2.5-fold higher than that of HiG alginate, suggesting a greater molecular mass. X-ray photoelectron spectroscopy indicated that both alginates were similar in elemental composition, including low levels of counterions in all gels. The wettabilities of the alginates and gels were also identical, as measured by contact angles of water on dry films. Ba-gel beads of HiG alginate resisted swelling and degradation when immersed in water, much more than the other gel beads. These results suggest that the main factors contributing to the biocompatibility of gels of purified alginate are the mannuronate/guluronate content and/or intrinsic viscosity.

  13. Reinforcement Learning by Value Gradients

    CERN Document Server

    Fairbank, Michael

    2008-01-01

    The concept of the value-gradient is introduced and developed in the context of reinforcement learning. It is shown that by learning the value-gradients exploration or stochastic behaviour is no longer needed to find locally optimal trajectories. This is the main motivation for using value-gradients, and it is argued that learning value-gradients is the actual objective of any value-function learning algorithm for control problems. It is also argued that learning value-gradients is significantly more efficient than learning just the values, and this argument is supported in experiments by efficiency gains of several orders of magnitude, in several problem domains. Once value-gradients are introduced into learning, several analyses become possible. For example, a surprising equivalence between a value-gradient learning algorithm and a policy-gradient learning algorithm is proven, and this provides a robust convergence proof for control problems using a value function with a general function approximator.

  14. Time-varied magnetic field enhances transport of magnetic nanoparticles in viscous gel.

    Science.gov (United States)

    MacDonald, Cristin; Friedman, Gary; Alamia, John; Barbee, Kenneth; Polyak, Boris

    2010-01-01

    The potential of magnetic nanoparticles (MNPs) to deliver various forms of therapy has not been fully realized, in part due to difficulties in transporting the carriers through soft tissue to different target sites. The aim of this study was to demonstrate that transport of MNPs through a viscous gel can be controlled by a combined AC (time-varying) magnetic field and static field gradient. MNP velocity and transport efficiency were measured in a viscous gel at various settings of magnetic field and magnetite loadings. Combined application of an AC magnetic field with the static field gradient resulted in a nearly 30-fold increase in MNP transport efficiency in viscous gel for 30% (w/w) magnetite-loaded particles as compared with static field conditions. The 'oscillating' effect of an AC magnetic field greatly improves the ability to transport MNPs within soft media by decreasing the effective viscosity of the gel.

  15. Combining high-throughput MALDI-TOF mass spectrometry and isoelectric focusing gel electrophoresis for virtual 2D gel-based proteomics.

    Science.gov (United States)

    Lohnes, Karen; Quebbemann, Neil R; Liu, Kate; Kobzeff, Fred; Loo, Joseph A; Ogorzalek Loo, Rachel R

    2016-07-15

    The virtual two-dimensional gel electrophoresis/mass spectrometry (virtual 2D gel/MS) technology combines the premier, high-resolution capabilities of 2D gel electrophoresis with the sensitivity and high mass accuracy of mass spectrometry (MS). Intact proteins separated by isoelectric focusing (IEF) gel electrophoresis are imaged from immobilized pH gradient (IPG) polyacrylamide gels (the first dimension of classic 2D-PAGE) by matrix-assisted laser desorption/ionization (MALDI) MS. Obtaining accurate intact masses from sub-picomole-level proteins embedded in 2D-PAGE gels or in IPG strips is desirable to elucidate how the protein of one spot identified as protein 'A' on a 2D gel differs from the protein of another spot identified as the same protein, whenever tryptic peptide maps fail to resolve the issue. This task, however, has been extremely challenging. Virtual 2D gel/MS provides access to these intact masses. Modifications to our matrix deposition procedure improve the reliability with which IPG gels can be prepared; the new procedure is described. Development of this MALDI MS imaging (MSI) method for high-throughput MS with integrated 'top-down' MS to elucidate protein isoforms from complex biological samples is described and it is demonstrated that a 4-cm IPG gel segment can now be imaged in approximately 5min. Gel-wide chemical and enzymatic methods with further interrogation by MALDI MS/MS provide identifications, sequence-related information, and post-translational/transcriptional modification information. The MSI-based virtual 2D gel/MS platform may potentially link the benefits of 'top-down' and 'bottom-up' proteomics.

  16. THE PROPERTIES OF CARRAGEENAN GELS

    Directory of Open Access Journals (Sweden)

    Grubnik I.M., Gladukh Ye.V., Chernyaev S.V.

    2012-04-01

    Full Text Available The article presents the results of studies on the functional properties of carrageenan, depending on the concentration of sodium chloride and xanthan in gels. It is established that the main factors in the syneresis of carrageenan gels are its concentration, the presence of ions and gums in solution. If using sodium chloride there is a change in the structure of mesh of the resulting gel, which leads to an increase in syneresis.

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

    Science.gov (United States)

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

    2015-01-01

    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

  18. Gel polymer electrolytes for batteries

    Science.gov (United States)

    Balsara, Nitash Pervez; Eitouni, Hany Basam; Gur, Ilan; Singh, Mohit; Hudson, William

    2014-11-18

    Nanostructured gel polymer electrolytes that have both high ionic conductivity and high mechanical strength are disclosed. The electrolytes have at least two domains--one domain contains an ionically-conductive gel polymer and the other domain contains a rigid polymer that provides structure for the electrolyte. The domains are formed by block copolymers. The first block provides a polymer matrix that may or may not be conductive on by itself, but that can soak up a liquid electrolyte, thereby making a gel. An exemplary nanostructured gel polymer electrolyte has an ionic conductivity of at least 1.times.10.sup.-4 S cm.sup.-1 at 25.degree. C.

  19. Energy in density gradient

    CERN Document Server

    Vranjes, J

    2015-01-01

    Inhomogeneous plasmas and fluids contain energy stored in inhomogeneity and they naturally tend to relax into lower energy states by developing instabilities or by diffusion. But the actual amount of energy in such inhomogeneities has remained unknown. In the present work the amount of energy stored in a density gradient is calculated for several specific density profiles in a cylindric configuration. This is of practical importance for drift wave instability in various plasmas, and in particular in its application in models dealing with the heating of solar corona because the instability is accompanied with stochastic heating, so the energy contained in inhomogeneity is effectively transformed into heat. It is shown that even for a rather moderate increase of the density at the axis in magnetic structures in the corona by a factor 1.5 or 3, the amount of excess energy per unit volume stored in such a density gradient becomes several orders of magnitude greater than the amount of total energy losses per unit ...

  20. Energy in density gradient

    Energy Technology Data Exchange (ETDEWEB)

    Vranjes, J., E-mail: jvranjes@yahoo.com [Instituto de Astrofísica de Canarias, 38205 La Laguna, Tenerife (Spain); Departamento de Astrofísica, Universidad de La Laguna, 38205 La Laguna, Tenerife (Spain); Kono, M., E-mail: kono@fps.chuo-u.ac.jp [Faculty of Policy Studies, Chuo University, Tokyo (Japan)

    2015-01-15

    Inhomogeneous plasmas and fluids contain energy stored in inhomogeneity and they naturally tend to relax into lower energy states by developing instabilities or by diffusion. But the actual amount of energy in such inhomogeneities has remained unknown. In the present work, the amount of energy stored in a density gradient is calculated for several specific density profiles in a cylindrical configuration. This is of practical importance for drift wave instability in various plasmas, and, in particular, in its application in models dealing with the heating of solar corona because the instability is accompanied with stochastic heating, so the energy contained in inhomogeneity is effectively transformed into heat. It is shown that even for a rather moderate increase of the density at the axis in magnetic structures in the corona by a factor 1.5 or 3, the amount of excess energy per unit volume stored in such a density gradient becomes several orders of magnitude greater than the amount of total energy losses per unit volume (per second) in quiet regions in the corona. Consequently, within the life-time of a magnetic structure such energy losses can easily be compensated by the stochastic drift wave heating.

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

    Science.gov (United States)

    Zhu, L G; Brewer, M S

    2003-04-01

    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.

  2. Transient, nonlinear rheology of reversible colloidal gels by dynamic simulation

    Science.gov (United States)

    Landrum, Benjamin; Russel, William; Zia, Roseanna

    2014-11-01

    We study the nonlinear rheology of reversible colloidal gels via dynamic simulation as they undergo age- and flow-induced structural evolution, with a view toward understanding and predicting transient behaviors such as multi-step and delayed yield. The gel is formed from 750,000 Brownian spheres interacting via hard-sphere repulsion and O(kT) short-range attraction, where thermal fluctuations are strong enough to allow continued structural rearrangement in the absence of flow. During startup of imposed strain rate, the transition to steady state is characterized by one or more ``overshoots'' in the stress which suggest initial yield, formation of a stronger gel, and subsequent yield of the new gel. When subjected to step-shear stress, the microstructure undergoes limited creep, followed by viscous flow. This macroscopic ``delayed flow'' is consistent with previously proposed models of competition between breakage and formation of particle bonds among static load-bearing structures. Our findings suggest, however, that the load-bearing structures evolve, and that the gel's resistance to delayed failure depends upon this structural evolution and reinforcement. We put forth a micro-mechanical model of stress gradient-driven particle transport that captures this macroscopic behavior.

  3. Sol-gel precursors and products thereof

    Science.gov (United States)

    Warren, Scott C.; DiSalvo, Jr., Francis J.; Weisner, Ulrich B.

    2017-02-14

    The present invention provides a generalizable single-source sol-gel precursor capable of introducing a wide range of functionalities to metal oxides such as silica. The sol-gel precursor facilitates a one-molecule, one-step approach to the synthesis of metal-silica hybrids with combinations of biological, catalytic, magnetic, and optical functionalities. The single-source precursor also provides a flexible route for simultaneously incorporating functional species of many different types. The ligands employed for functionalizing the metal oxides are derived from a library of amino acids, hydroxy acids, or peptides and a silicon alkoxide, allowing many biological functionalities to be built into silica hybrids. The ligands can coordinate with a wide range of metals via a carboxylic acid, thereby allowing direct incorporation of inorganic functionalities from across the periodic table. Using the single-source precursor a wide range of functionalized nanostructures such as monolith structures, mesostructures, multiple metal gradient mesostructures and Stober-type nanoparticles can be synthesized. ##STR00001##

  4. Sol-gel precursors and products thereof

    Energy Technology Data Exchange (ETDEWEB)

    Warren, Scott C.; DiSalvo, Jr., Francis J.; Weisner, Ulrich B.

    2017-02-14

    The present invention provides a generalizable single-source sol-gel precursor capable of introducing a wide range of functionalities to metal oxides such as silica. The sol-gel precursor facilitates a one-molecule, one-step approach to the synthesis of metal-silica hybrids with combinations of biological, catalytic, magnetic, and optical functionalities. The single-source precursor also provides a flexible route for simultaneously incorporating functional species of many different types. The ligands employed for functionalizing the metal oxides are derived from a library of amino acids, hydroxy acids, or peptides and a silicon alkoxide, allowing many biological functionalities to be built into silica hybrids. The ligands can coordinate with a wide range of metals via a carboxylic acid, thereby allowing direct incorporation of inorganic functionalities from across the periodic table. Using the single-source precursor a wide range of functionalized nanostructures such as monolith structures, mesostructures, multiple metal gradient mesostructures and Stober-type nanoparticles can be synthesized. ##STR00001##

  5. Microfluidic dielectrophoretic sorter using gel vertical electrodes

    Science.gov (United States)

    Luo, Jason; Nelson, Edward L.; Li, G. P.; Bachman, Mark

    2014-01-01

    We report the development and results of a two-step method for sorting cells and small particles in a microfluidic device. This approach uses a single microfluidic channel that has (1) a microfabricated sieve which efficiently focuses particles into a thin stream, followed by (2) a dielectrophoresis (DEP) section consisting of electrodes along the channel walls for efficient continuous sorting based on dielectric properties of the particles. For our demonstration, the device was constructed of polydimethylsiloxane, bonded to a glass surface, and conductive agarose gel electrodes. Gold traces were used to make electrical connections to the conductive gel. The device had several novel features that aided performance of the sorting. These included a sieving structure that performed continuous displacement of particles into a single stream within the microfluidic channel (improving the performance of downstream DEP, and avoiding the need for additional focusing flow inlets), and DEP electrodes that were the full height of the microfluidic walls (“vertical electrodes”), allowing for improved formation and control of electric field gradients in the microfluidic device. The device was used to sort polymer particles and HeLa cells, demonstrating that this unique combination provides improved capability for continuous DEP sorting of particles in a microfluidic device. PMID:24926390

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

    Science.gov (United States)

    Shao, Qiang; Wang, Jinan; Zhu, Weiliang

    2014-09-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Rajeshwari eSinha

    2014-04-01

    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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

    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.

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

    Science.gov (United States)

    Ben-Naim, Arieh

    2012-12-01

    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.

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

    Science.gov (United States)

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

    2016-04-01

    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.

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

    Indian Academy of Sciences (India)

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

    2008-11-01

    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

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

    Directory of Open Access Journals (Sweden)

    Jorge Luiz da Cunha

    2014-01-01

    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.

  13. PROTECTIVE EFFECT OF PROLACTIN INDUCED PROTEIN ON ZINC α2-GLYCOPROTEIN AGAINST VARIOUS DENATURANTS

    Directory of Open Access Journals (Sweden)

    Md. Imtaiyaz Hassan

    2012-12-01

    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.

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

    Science.gov (United States)

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

    2008-01-01

    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

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

    Directory of Open Access Journals (Sweden)

    Kaikai Wang

    2015-12-01

    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.

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

    Institute of Scientific and Technical Information of China (English)

    王洪睿; 王希成; 张彤; 周海梦

    1995-01-01

    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.

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    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...

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

    Science.gov (United States)

    Butzloff, Peter R.

    2011-01-01

    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

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

    Science.gov (United States)

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

    1990-12-11

    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.

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

    Science.gov (United States)

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

    2012-10-01

    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.

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

    OpenAIRE

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

    2014-01-01

    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...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2006-06-15

    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.

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

    OpenAIRE

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

    2008-01-01

    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)...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2004-02-09

    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.

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

    Directory of Open Access Journals (Sweden)

    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

  6. Effects of oxidative modification on gel properties of isolated porcine myofibrillar protein by peroxyl radicals.

    Science.gov (United States)

    Zhou, Feibai; Zhao, Mouming; Zhao, Haifeng; Sun, Weizheng; Cui, Chun

    2014-04-01

    AAPH-derived (2,2'-azobis (2-amidinopropane) dihydrochloride) peroxyl radicals were selected as representative free radicals of lipid peroxidation to investigate the effects of oxidative modifications on isolated porcine myofibrillar protein structures as well as their rheological and gelling properties. Incubation of myofibrillar protein with increasing concentrations of AAPH resulted in a gradual increase (p3 mM) concentrations of AAPH induced aggregation of myosin and denaturation of myosin, troponin and tropomyosin, respectively. These structural changes resulted in changes on gelation of myofibrillar protein. Low level protein oxidation (AAPH≤0.5 mM) had no remarkable effect (p>0.05) on the viscoelastic pattern of myofibrillar protein gelation. Moderate oxidative modification (AAPH~1mM) enhanced the water-holding capacity (WHC) and texture properties of gels, while further oxidation (AAPH>3mM) significantly reduced the gel quality.

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

    Science.gov (United States)

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

    2013-05-01

    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.

  8. DENATURATION OF NATIVE AND DEGLYCOSYLATED α-GALACTOSIDASES FROM Penicillium canescens BY GUANIDINE HYDROCHLORIDE

    Directory of Open Access Journals (Sweden)

    Borzova N. V.

    2015-08-01

    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.

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

    CERN Document Server

    Florescu, Ana-Maria; 10.1063/1.3626870

    2011-01-01

    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...

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

    Science.gov (United States)

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

    2010-01-27

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1996-01-05

    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.

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

    Science.gov (United States)

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

    2017-08-15

    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.

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

    Science.gov (United States)

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

    2015-01-01

    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.

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

    Science.gov (United States)

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

    2015-01-01

    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.

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

    Science.gov (United States)

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

    2013-06-01

    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.

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

    Science.gov (United States)

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

    2016-10-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Marwa Khaldi

    2015-01-01

    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.

  18. Rheology and structure of milk protein gels

    NARCIS (Netherlands)

    Vliet, van T.; Lakemond, C.M.M.; Visschers, R.W.

    2004-01-01

    Recent studies on gel formation and rheology of milk gels are reviewed. A distinction is made between gels formed by aggregated casein, gels of `pure` whey proteins and gels in which both casein and whey proteins contribute to their properties. For casein' whey protein mixtures, it has been shown th

  19. Rheology and structure of milk protein gels

    NARCIS (Netherlands)

    Vliet, van T.; Lakemond, C.M.M.; Visschers, R.W.

    2004-01-01

    Recent studies on gel formation and rheology of milk gels are reviewed. A distinction is made between gels formed by aggregated casein, gels of `pure` whey proteins and gels in which both casein and whey proteins contribute to their properties. For casein' whey protein mixtures, it has been shown

  20. Nanocrystal/sol-gel nanocomposites

    Science.gov (United States)

    Petruska, Melissa A.; Klimov, Victor L.

    2007-06-05

    The present invention is directed to solid composites including colloidal nanocrystals within a sol-gel host or matrix and to processes of forming such solid composites. The present invention is further directed to alcohol soluble colloidal nanocrystals useful in formation of sol-gel based solid composites.

  1. Generalized conjugate gradient squared

    Energy Technology Data Exchange (ETDEWEB)

    Fokkema, D.R.; Sleijpen, G.L.G. [Utrecht Univ. (Netherlands)

    1994-12-31

    In order to solve non-symmetric linear systems of equations, the Conjugate Gradient Squared (CGS) is a well-known and widely used iterative method. In practice the method converges fast, often twice as fast as the Bi-Conjugate Gradient method. This is what you may expect, since CGS uses the square of the BiCG polynomial. However, CGS may suffer from its erratic convergence behavior. The method may diverge or the approximate solution may be inaccurate. BiCGSTAB uses the BiCG polynomial and a product of linear factors in an attempt to smoothen the convergence. In many cases, this has proven to be very effective. Unfortunately, the convergence of BiCGSTAB may stall when a linear factor (nearly) degenerates. BiCGstab({ell}) is designed to overcome this degeneration of linear factors. It generalizes BiCGSTAB and uses both the BiCG polynomial and a product of higher order factors. Still, CGS may converge faster than BiCGSTAB or BiCGstab({ell}). So instead of using a product of linear or higher order factors, it may be worthwhile to look for other polynomials. Since the BiCG polynomial is based on a three term recursion, a natural choice would be a polynomial based on another three term recursion. Possibly, a suitable choice of recursion coefficients would result in method that converges faster or as fast as CGS, but less erratic. It turns out that an algorithm for such a method can easily be formulated. One particular choice for the recursion coefficients leads to CGS. Therefore one could call this algorithm generalized CGS. Another choice for the recursion coefficients leads to BiCGSTAB. It is therefore possible to mix linear factors and some polynomial based on a three term recursion. This way one may get the best of both worlds. The authors will report on their findings.

  2. Sucrose release from polysaccharide gels.

    Science.gov (United States)

    Nishinari, Katsuyoshi; Fang, Yapeng

    2016-05-18

    Sucrose release from polysaccharide gels has been studied extensively because it is expected to be useful in understanding flavour release from solid foods and to find a new processing method which produces more palatable and healthier foods. We provide an overview of the release of sucrose and other sugars from gels of agar and related polysaccharides. The addition of sucrose to agar solutions leads to the increase in transparency of the resulting gels and the decrease in syneresis, which is attributed to the decrease in mesh size in gels. The syneresis occurring in the quiescent condition and fluid release induced by compression is discussed. The relationship between the sugar release and the structural, rheological and thermal properties of gels is also discussed. Finally, the future research direction is proposed.

  3. Electrochemical Light-Emitting Gel

    Directory of Open Access Journals (Sweden)

    Nobuyuki Itoh

    2010-06-01

    Full Text Available Light-emitting gel, a gel state electroluminescence material, is reported. It is composed of a ruthenium complex as the emitter, an ionic liquid as the electrolyte, and oxide nanoparticles as the gelation filler. Emitted light was produced via electrogenerated chemiluminescence. The light-emitting gel operated at low voltage when an alternating current was passed through it, regardless of its structure, which is quite thick. The luminescence property of the gel is strongly affected by nanoparticle materials. TiO2 nanoparticles were a better gelation filler than silica or ZnO was, with respect to luminescence stability, thus indicating a catalytic effect. It is demonstrated that the light-emitting gel device, with quite a simple fabrication process, flashes with the application of voltage.

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

    Science.gov (United States)

    Pikal-Cleland, K A; Carpenter, J F

    2001-09-01

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    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)

    2012-07-01

    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

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    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...

  7. Rectified cell migration on saw-like micro-elastically patterned hydrogels with asymmetric gradient ratchet teeth.

    Directory of Open Access Journals (Sweden)

    Satoru Kidoaki

    Full Text Available To control cell motility is one of the essential technologies for biomedical engineering. To establish a methodology of the surface design of elastic substrate to control the long-range cell movements, here we report a sophisticated cell culture hydrogel with a micro-elastically patterned surface that allows long-range durotaxis. This hydrogel has a saw-like pattern with asymmetric gradient ratchet teeth, and rectifies random cell movements. Durotaxis only occurs at boundaries in which the gradient strength of elasticity is above a threshold level. Consequently, in gels with unit teeth patterns, durotaxis should only occur at the sides of the teeth in which the gradient strength of elasticity is above this threshold level. Therefore, such gels are expected to support the long-range biased movement of cells via a mechanism similar to the Feynman-Smoluchowski ratchet, i.e., rectified cell migration. The present study verifies this working hypothesis by using photolithographic microelasticity patterning of photocurable gelatin gels. Gels in which each teeth unit was 100-120 µm wide with a ratio of ascending:descending elasticity gradient of 1:2 and a peak elasticity of ca. 100 kPa supported the efficient rectified migration of 3T3 fibroblast cells. In addition, long-range cell migration was most efficient when soft lanes were introduced perpendicular to the saw-like patterns. This study demonstrates that asymmetric elasticity gradient patterning of cell culture gels is a versatile means of manipulating cell motility.

  8. sol-gel

    Directory of Open Access Journals (Sweden)

    Humberto A. Monreal

    2005-01-01

    Full Text Available En este trabajo sintetizamos nanocilindros de dióxido de titanio de 30 a 400 nm por medio de ADN del plásmido pBR322 de 4,362 pares de bases y el uso de isopropóxido de titanio como precursor por medio del proceso sol-gel. Los geles resultantes fueron calcinados y los polvos caracterizados por medio de Microscopio Electrónico de Barrido (MEB, Espectroscopía de Energía Dispersiva, Microscopio Electrónico de Transmisión (MET y Difracción de Rayos X. Los resultados muestran que la síntesis in vitro de nanorods en presencia de ADN, puede ser activada. Muchas otras moléculas sintéticas pueden producirse por medio del uso de sistemas orgánicos, es así como reportamos la síntesis de híbridos hechos de ácidos nucleicos en materiales inorgánicos que pueden tener diversas aplicaciones en sistemas catalíticos, biomateriales y materiales nanoestructurados.

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

    Science.gov (United States)

    Sun, Yao; O'Neill, Brian

    2013-03-10

    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.

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

    Science.gov (United States)

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

    2008-09-19

    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.

  11. Mechanical Failure in Colloidal Gels

    Science.gov (United States)

    Kodger, Thomas Edward

    When colloidal particles in a dispersion are made attractive, they aggregate into fractal clusters which grow to form a space-spanning network, or gel, even at low volume fractions. These gels are crucial to the rheological behavior of many personal care, food products and dispersion-based paints. The mechanical stability of these products relies on the stability of the colloidal gel network which acts as a scaffold to provide these products with desired mechanical properties and to prevent gravitational sedimentation of the dispersed components. Understanding the mechanical stability of such colloidal gels is thus of crucial importance to predict and control the properties of many soft solids. Once a colloidal gel forms, the heterogeneous structure bonded through weak physical interactions, is immediately subject to body forces, such as gravity, surface forces, such as adhesion to a container walls and shear forces; the interplay of these forces acting on the gel determines its stability. Even in the absence of external stresses, colloidal gels undergo internal rearrangements within the network that may cause the network structure to evolve gradually, in processes known as aging or coarsening or fail catastrophically, in a mechanical instability known as syneresis. Studying gel stability in the laboratory requires model colloidal system which may be tuned to eliminate these body or endogenous forces systematically. Using existing chemistry, I developed several systems to study delayed yielding by eliminating gravitational stresses through density matching and cyclic heating to induce attraction; and to study syneresis by eliminating adhesion to the container walls, altering the contact forces between colloids, and again, inducing gelation through heating. These results elucidate the varied yet concomitant mechanisms by which colloidal gels may locally or globally yield, but then reform due to the nature of the physical, or non-covalent, interactions which form

  12. Surface grafted chitosan gels. Part II. Gel formation and characterization

    DEFF Research Database (Denmark)

    Liu, Chao; Thormann, Esben; Claesson, Per M.

    2014-01-01

    Responsive biomaterial hydrogels attract significant attention due to their biocompatibility and degradability. In order to make chitosan based gels, we first graft one layer of chitosan to silica, and then build a chitosan/poly(acrylic acid) multilayer using the layer-by-layer approach. After...... cross-linking the chitosan present in the polyelectrolyte multilayer, poly(acrylic acid) is partly removed by exposing the multilayer structure to a concentrated carbonate buffer solution at a high pH, leaving a surface-grafted cross-linked gel. Chemical cross-linking enhances the gel stability against......-linking density. The amount of poly(acrylic acid) trapped inside the surface grafted films was found to decrease with decreasing cross-linking density, as confirmed in situ using TIRR, and ex situ by Fourier transform infrared (FTIR) measurements on dried films. The responsiveness of the chitosan-based gels...

  13. Dynamic partitioning of nanoparticulate metal species between gel layers and aqueous media

    NARCIS (Netherlands)

    Veeken, van der P.L.R.

    2010-01-01

    This thesis deals with several aspects of the use of Diffusive Gradient in Thin film (DGT) and Diffusive Equilibriation in Thin film (DET) in dynamic metal speciation analysis. It has a clear focus on the properties of the diffusive gel layer, and their possible impact on metal speciation measuremen

  14. Dynamic partitioning of nanoparticulate metal species between gel layers and aqueous media

    NARCIS (Netherlands)

    Veeken, van der P.L.R.

    2010-01-01

    This thesis deals with several aspects of the use of Diffusive Gradient in Thin film (DGT) and Diffusive Equilibriation in Thin film (DET) in dynamic metal speciation analysis. It has a clear focus on the properties of the diffusive gel layer, and their possible impact on metal speciation

  15. Sobolev gradients and differential equations

    CERN Document Server

    Neuberger, J W

    2010-01-01

    A Sobolev gradient of a real-valued functional on a Hilbert space is a gradient of that functional taken relative to an underlying Sobolev norm. This book shows how descent methods using such gradients allow a unified treatment of a wide variety of problems in differential equations. For discrete versions of partial differential equations, corresponding Sobolev gradients are seen to be vastly more efficient than ordinary gradients. In fact, descent methods with these gradients generally scale linearly with the number of grid points, in sharp contrast with the use of ordinary gradients. Aside from the first edition of this work, this is the only known account of Sobolev gradients in book form. Most of the applications in this book have emerged since the first edition was published some twelve years ago. What remains of the first edition has been extensively revised. There are a number of plots of results from calculations and a sample MatLab code is included for a simple problem. Those working through a fair p...

  16. Fluoride Rinses, Gels and Foams

    DEFF Research Database (Denmark)

    Twetman, Svante; Keller, Mette K

    2016-01-01

    AIM: The aim of this conference paper was to systematically review the quality of evidence and summarize the findings of clinical trials published after 2002 using fluoride mouth rinses, fluoride gels or foams for the prevention of dental caries. METHODS: Relevant papers were selected after...... (6 on fluoride mouth rinse, 10 on fluoride gel and 3 on fluoride foam); 6 had a low risk of bias while 2 had a moderate risk. All fluoride measures appeared to be beneficial in preventing crown caries and reversing root caries, but the quality of evidence was graded as low for fluoride mouth rinse......, moderate for fluoride gel and very low for acidulated fluoride foam. No conclusions could be drawn on the cost-effectiveness. CONCLUSIONS: This review, covering the recent decade, has further substantiated the evidence for a caries-preventive effect of fluoride mouth rinse, fluoride gel and foam...

  17. Colloidal gels: Clay goes patchy

    Science.gov (United States)

    Kegel, Willem K.; Lekkerkerker, Henk N. W.

    2011-01-01

    Empty liquids and equilibrium gels have so far been only theoretical possibilities, predicted for colloids with patchy interactions. But evidence of both has now been found in Laponite, a widely studied clay.

  18. Janus Gel Fabrication Using Liquid Drop Coalescence and Limited Mixing in the Hele-Shaw Geometry

    Science.gov (United States)

    Gonzalez, Brittany; Moran, Alexis; Lee, Donghee; Ryu, Sangjin

    2015-11-01

    Hydrogel substrates of tunable stiffness have been actively utilized for in vitro cell mechanobiology study. Here we present a new method to fabricate Janus polyacrylamide gel based on limited mixing between liquid drops coalescing in the Hele-Shaw geometry. Two pre-polymer drops with different concentrations were sandwiched and squeezed between two parallel glass surfaces. Once the drops coalesced in the decreased gap between the surfaces, gelation was initiated by UV light exposure with various time delays. AFM nano-indentation was utilized to map the Young's modulus of obtained gels. Fabricated Janus gels had two regions of different Young's moduli interfaced by the stiffness gradient zone, and the width of the gradient zone increased with the delay time. We acknowledge support from Bioengineering for Human Health grant from UNL and UNMC, and NSF REU grant for UNL.

  19. Silica reinforced triblock copolymer gels

    DEFF Research Database (Denmark)

    Theunissen, E.; Overbergh, N.; Reynaers, H.

    2004-01-01

    The effect of silica and polymer coated silica particles as reinforcing agents on the structural and mechanical properties of polystyrene-poly(ethylene/butylene)-polystyrene (PS-PEB-PS) triblock gel has been investigated. Different types of chemically modified silica have been compared in order...... a viscoclastic rubber to a plastic fluid and from a plastic fluid to a viscoelastic liquid are shifted to more elevated temperatures when silica is added to the triblock copolymer gel. (C) 2004 Elsevier Ltd. All rights reserved....

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

    Science.gov (United States)

    Hung, H C; Chang, G G

    2001-12-01

    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

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

    Science.gov (United States)

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

    2005-09-01

    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.

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

    Science.gov (United States)

    Lighezan, Liliana; Georgieva, Ralitsa; Neagu, Adrian

    2012-09-01

    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.

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

    Science.gov (United States)

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

    2016-01-01

    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.

  4. Gradient zone boundary control in salt gradient solar ponds

    Energy Technology Data Exchange (ETDEWEB)

    Hull, John R. (Downers Grove, IL)

    1984-01-01

    A method and apparatus for suppressing zone boundary migration in a salt gradient solar pond includes extending perforated membranes across the pond at the boundaries, between the convective and non-convective zones, the perforations being small enough in size to prevent individual turbulence disturbances from penetrating the hole, but being large enough to allow easy molecular diffusion of salt thereby preventing the formation of convective zones in the gradient layer. The total area of the perforations is a sizable fraction of the membrane area to allow sufficient salt diffusion while preventing turbulent entrainment into the gradient zone.

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

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    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.

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

    Science.gov (United States)

    Zhu, Chunli; Li, Jing

    2016-09-01

    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.

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

    DEFF Research Database (Denmark)

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

    2017-01-01

    . 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...

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

    OpenAIRE

    Bünemann, H

    1982-01-01

    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...

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

    Science.gov (United States)

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

    2012-04-12

    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.

  10. Behavior of Heat-Denatured Whey: Buttermilk Protein Aggregates during the Yogurt-Making Process and Their Influence on Set-Type Yogurt Properties.

    Science.gov (United States)

    Saffon, Maxime; Richard, Véronique; Jiménez-Flores, Rafael; Gauthier, Sylvie F; Britten, Michel; Pouliot, Yves

    2013-09-30

    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.

  11. Behavior of Heat-Denatured Whey: Buttermilk Protein Aggregates during the Yogurt-Making Process and Their Influence on Set-Type Yogurt Properties

    Directory of Open Access Journals (Sweden)

    Maxime Saffon

    2013-09-01

    Full Text Available 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.

  12. Surface grafted chitosan gels. Part II. Gel formation and characterization.

    Science.gov (United States)

    Liu, Chao; Thormann, Esben; Claesson, Per M; Tyrode, Eric

    2014-07-29

    Responsive biomaterial hydrogels attract significant attention due to their biocompatibility and degradability. In order to make chitosan based gels, we first graft one layer of chitosan to silica, and then build a chitosan/poly(acrylic acid) multilayer using the layer-by-layer approach. After cross-linking the chitosan present in the polyelectrolyte multilayer, poly(acrylic acid) is partly removed by exposing the multilayer structure to a concentrated carbonate buffer solution at a high pH, leaving a surface-grafted cross-linked gel. Chemical cross-linking enhances the gel stability against detachment and decomposition. The chemical reaction between gluteraldehyde, the cross-linking agent, and chitosan was followed in situ using total internal reflection Raman (TIRR) spectroscopy, which provided a molecular insight into the complex reaction mechanism, as well as the means to quantify the cross-linking density. The amount of poly(acrylic acid) trapped inside the surface grafted films was found to decrease with decreasing cross-linking density, as confirmed in situ using TIRR, and ex situ by Fourier transform infrared (FTIR) measurements on dried films. The responsiveness of the chitosan-based gels with respect to pH changes was probed by quartz crystal microbalance with dissipation (QCM-D) and TIRR. Highly cross-linked gels show a small and fully reversible behavior when the solution pH is switched between pH 2.7 and 5.7. In contrast, low cross-linked gels are more responsive to pH changes, but the response is fully reversible only after the first exposure to the acidic solution, once an internal restructuring of the gel has taken place. Two distinct pKa's for both chitosan and poly(acrylic acid), were determined for the cross-linked structure using TIRR. They are associated with populations of chargeable groups displaying either a bulk like dissociation behavior or forming ionic complexes inside the hydrogel film.

  13. Electrolyte transport in neutral polymer gels embedded with charged inclusions

    Science.gov (United States)

    Hill, Reghan

    2005-11-01

    Ion permeable membranes are the basis of a variety of molecular separation technologies, including ion exchange, gel electrophoresis and dialysis. This work presents a theoretical model of electrolyte transport in membranes comprised of a continuous polymer gel embedded with charged spherical inclusions, e.g., biological cells and synthetic colloids. The microstructure mimics immobilized cell cultures, where electric fields have been used to promote nutrient transport. Because several important characteristics can, in principle, be carefully controlled, the theory provides a quantitative framework to help tailor the bulk properties for enhanced molecular transport, microfluidic pumping, and physicochemical sensing applications. This talk focuses on the electroosmotic flow driven by weak electric fields and electrolyte concentration gradients. Also of importance is the influence of charge on the effective ion diffusion coefficients, bulk electrical conductivity, and membrane diffusion potential.

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

    Science.gov (United States)

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

    2016-01-01

    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.

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

    Science.gov (United States)

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

    2013-11-15

    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).

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

    Science.gov (United States)

    Gotoh, Maya; Arai, Tsunenori

    2008-02-01

    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.

  17. Feasibility of radiochromic gels for 3D dosimetry of brachytherapy sources

    Science.gov (United States)

    Šolc, Jaroslav; Sochor, Vladimír

    2012-10-01

    Two radiochromic gel dosimeters, Fricke-xylenol orange (FXO) gel and Turnbull Blue (TB) gel, were studied in the scope of the iMERA+ project ‘Increasing cancer treatment efficacy using 3D brachytherapy’ for their feasibility for the determination of relative 3D dose distribution of brachytherapy (BT) sources. Initially, the dose, dose rate and energy dependence of the gels were investigated. Subsequently, the gels were irradiated by a point low-dose-rate source IsoSeed I25.S16 (125I) and a high-dose-rate source GammaMed+ (192Ir) and scanned using optical computed tomography. Optical transmission images of irradiated gels were processed to obtain detailed 3D optical density maps inside the gels with voxel dimensions of 0.25 × 0.25 × 0.25 mm3. The radial dose function between 1.5 mm and 35 mm from the source and the anisotropy function at 10 mm radius were determined and compared with Monte Carlo calculations and TG-43 data, showing agreement mostly within the measurement uncertainty. Results revealed that the TB gel is feasible for measurements of the relative 3D dose distributions very close to the point BT source because it conserves sharp dose gradients as this gel does not suffer diffusion of dye created upon irradiation. On the other hand, FXO gel underestimates doses closer than 5 mm from the source due to diffusion effects, but it has a significantly higher sensitivity which enables convenient measurement of relative doses up to 35 mm from the source. Further development, especially on gel composition and corrections to optical CT images, is desirable.

  18. Analysis of spatial diffusion of ferric ions in PVA-GTA gel dosimeters through magnetic resonance imaging

    Science.gov (United States)

    Marrale, Maurizio; Collura, Giorgio; Gallo, Salvatore; Nici, Stefania; Tranchina, Luigi; Abbate, Boris Federico; Marineo, Sandra; Caracappa, Santo; d'Errico, Francesco

    2017-04-01

    This work focused on the analysis of the temporal diffusion of ferric ions through PVA-GTA gel dosimeters. PVA-GTA gel samples, partly exposed with 6 MV X-rays in order to create an initial steep gradient, were mapped using magnetic resonance imaging on a 7T MRI scanner for small animals. Multiple images of the gels were acquired over several hours after irradiation and were analyzed to quantitatively extract the signal profile. The spatial resolution achieved is 200 μm and this makes this technique particularly suitable for the analysis of steep gradients of ferric ion concentration. The results obtained with PVA-GTA gels were compared with those achieved with agarose gels, which is a standard dosimetric gel formulation. The analysis showed that the diffusion process is much slower (more than five times) for PVA-GTA gels than for agarose ones. Furthermore, it is noteworthy that the diffusion coefficient value obtained through MRI analysis is significantly consistent with that obtained in separate study Marini et al. (Submitted for publication) using a totally independent method such as spectrophotometry. This is a valuable result highlighting that the good dosimetric features of this gel matrix not only can be reproduced but also can be measured through independent experimental techniques based on different physical principles.

  19. Superoxide dismutase isozyme detection using two-dimensional gel electrophoresis zymograms.

    Science.gov (United States)

    Niyomploy, Ploypat; Srisomsap, Chantragan; Chokchaichamnankit, Daranee; Vinayavekhin, Nawaporn; Karnchanatat, Aphichart; Sangvanich, Polkit

    2014-03-01

    Superoxide dismutases (SODs) are ubiquitous antioxidant enzymes involved in cell protection from reactive oxygen species. Their antioxidant activities make them of interest to applied biotechnology industries and are usually sourced from plants. SODs are also involved in stress signaling responses in plants, and can be used as indicators of these responses. In this article, a suitable method for the separation of different SOD isoforms using two-dimensional-gel electrophoresis (2D-GE) zymograms is reported. The method was developed with a SOD standard from bovine erythrocytes and later applied to extracts from Stemona tuberosa. The first (non-denaturing isoelectric focusing) and second (denaturing sodium dodecylsulphate-polyacrylamide gel electrophoresis) dimensions of duplicate 2D-GE gels were stained with either Coomassie brilliant blue G-250 for total protein visualization, or SOD activity (zymogram) using riboflavin/nitroblue tetrazolium. For confirmation, putative SOD activity positive spots were subject to trypsin digestion and nano-liquid chromatography tandem mass spectrometry, followed by searching the MASCOT database for potential identification. The method could separate different SOD isoforms from a plant extract and at least partially maintain or allow renaturation to the native forms of the enzyme. Peptide sequencing of the 2D-GE suggested that the SODs were resolved correctly, identifying the control CuZn-SOD from bovine erythrocytes. The two SODs from S. tuberosa tubers were found to be likely homologous of a CuZn-SOD. SOD detection and isoform separation by 2D-GE zymograms was efficient and reliable. The method is likely applicable to SOD detection from plants or other organisms. Moreover, a similar approach could be developed for detection of other important enzymes in the future. Copyright © 2013 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2008-11-01

    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.

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

    Science.gov (United States)

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

    2016-06-01

    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.

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

    Science.gov (United States)

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

    2001-11-06

    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.

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

    Science.gov (United States)

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

    2010-01-01

    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

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

    Science.gov (United States)

    Bünemann, H

    1982-11-25

    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).

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

    Science.gov (United States)

    Wang, Limin; Colón, Wilfredo

    2005-01-01

    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

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

    Science.gov (United States)

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

    2010-01-01

    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

  7. Thixotropic gel for vadose zone remediation

    Science.gov (United States)

    Riha, Brian D.

    2012-07-03

    A thixotropic gel suitable for use in subsurface bioremediation is provided along with a process of using the gel. The thixotropic gel provides a non-migrating injectable substrate that can provide below ground barrier properties. In addition, the gel components provide for a favorable environment in which certain contaminants are preferentially sequestered in the gel and subsequently remediated by either indigenous or introduced microorganisms.

  8. Thixotropic gel for vadose zone remediation

    Science.gov (United States)

    Rhia, Brian D.

    2011-03-01

    A thixotropic gel suitable for use in subsurface bioremediation is provided along with a process of using the gel. The thixotropic gel provides a non-migrating injectable substrate that can provide below ground barrier properties. In addition, the gel components provide for a favorable environment in which certain contaminants are preferentially sequestered in the gel and subsequently remediated by either indigenous or introduced microorganisms.

  9. Thixotropic gel for vadose zone remediation

    Energy Technology Data Exchange (ETDEWEB)

    Riha, Brian D.; Looney, Brian B.

    2015-10-27

    A thixotropic gel suitable for use in subsurface bioremediation is provided along with a process of using the gel. The thixotropic gel provides a non-migrating injectable substrate that can provide below ground barrier properties. In addition, the gel components provide for a favorable environment in which certain contaminants are preferentially sequestered in the gel and subsequently remediated by either indigenous or introduced microorganisms.

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

    Directory of Open Access Journals (Sweden)

    G. E. Aguilar-Pineda

    2014-01-01

    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.

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

    Science.gov (United States)

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

    2014-09-01

    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.

  12. Sobolev gradients and differential equations

    CERN Document Server

    Neuberger, John William

    1997-01-01

    A Sobolev gradient of a real-valued functional is a gradient of that functional taken relative to the underlying Sobolev norm. This book shows how descent methods using such gradients allow a unified treatment of a wide variety of problems in differential equations. Equal emphasis is placed on numerical and theoretical matters. Several concrete applications are made to illustrate the method. These applications include (1) Ginzburg-Landau functionals of superconductivity, (2) problems of transonic flow in which type depends locally on nonlinearities, and (3) minimal surface problems. Sobolev gradient constructions rely on a study of orthogonal projections onto graphs of closed densely defined linear transformations from one Hilbert space to another. These developments use work of Weyl, von Neumann and Beurling.

  13. Conjugate Gradient with Subspace Optimization

    CERN Document Server

    Karimi, Sahar

    2012-01-01

    In this paper we present a variant of the conjugate gradient (CG) algorithm in which we invoke a subspace minimization subproblem on each iteration. We call this algorithm CGSO for "conjugate gradient with subspace optimization". It is related to earlier work by Nemirovsky and Yudin. We apply the algorithm to solve unconstrained strictly convex problems. As with other CG algorithms, the update step on each iteration is a linear combination of the last gradient and last update. Unlike some other conjugate gradient methods, our algorithm attains a theoretical complexity bound of $O(\\sqrt{L/l} \\log(1/\\epsilon))$, where the ratio $L/l$ characterizes the strong convexity of the objective function. In practice, CGSO competes with other CG-type algorithms by incorporating some second order information in each iteration.

  14. Flame Propagation Through Concentration Gradient

    Institute of Scientific and Technical Information of China (English)

    JunyaIINO; MitsuakiTANABE; 等

    2000-01-01

    The experiment was carried out in homogeneous propane-air mixture and in several concentration gradient of mixture.Igniter is put on the upper side of the combustion chamber,In concentration gradient experiment.ixture was ignited from lean side.An experimental study was conducted in a combustion chamber.The combustion chamber has glass windows for optical measurements at any side.For the measurement of distribution of fuel concentration,infraed absorption method using 3.39μm He-Ne laser was used,and for the observation of proagating flams,Schlieren method was employed.As a measurment result of flame propagation velocity and flammable limit,for a mixture of an identical local equivalence ratio.flame propagation velocity in concentration gradient is faster than that in homogeneous mixture,and rich flammable limit in concentration gradient shows a tendency to be higher than that in homogeneous mixture.

  15. Engineering functionality gradients by dip coating process in acceleration mode.

    Science.gov (United States)

    Faustini, Marco; Ceratti, Davide R; Louis, Benjamin; Boudot, Mickael; Albouy, Pierre-Antoine; Boissière, Cédric; Grosso, David

    2014-10-08

    In this work, unique functional devices exhibiting controlled gradients of properties are fabricated by dip-coating process in acceleration mode. Through this new approach, thin films with "on-demand" thickness graded profiles at the submillimeter scale are prepared in an easy and versatile way, compatible for large-scale production. The technique is adapted to several relevant materials, including sol-gel dense and mesoporous metal oxides, block copolymers, metal-organic framework colloids, and commercial photoresists. In the first part of the Article, an investigation on the effect of the dip coating speed variation on the thickness profiles is reported together with the critical roles played by the evaporation rate and by the viscosity on the fluid draining-induced film formation. In the second part, dip-coating in acceleration mode is used to induce controlled variation of functionalities by playing on structural, chemical, or dimensional variations in nano- and microsystems. In order to demonstrate the full potentiality and versatility of the technique, original graded functional devices are made including optical interferometry mirrors with bidirectional gradients, one-dimensional photonic crystals with a stop-band gradient, graded microfluidic channels, and wetting gradient to induce droplet motion.

  16. Ionic conductivity of polymer gels deriving from alkali metal ionic liquids and negatively charged polyelectrolytes

    Energy Technology Data Exchange (ETDEWEB)

    Ogihara, Wataru; Yoshizawa, Masahiro; Ohno Hiroyuki [Tokyo University of Agriculture and Technology (Japan). Dept. of Biotechnology; Sun, Jiazeng; Forsyth, M. [Monash University, Clayton (Australia). School of Materials Engineering; MacFarlane, D.R. [Monash University, Clayton (Australia). School of Chemistry

    2004-04-30

    We have prepared polymer gel electrolytes with alkali metal ionic liquids (AMILs) that inherently contain alkali metal ions. The AMIL consisted of sulfate anion, imidazolium cation, and alkali metal cation. AMILs were mixed directly with poly(3-sulfopropyl acrylate) lithium salt or poly(2-acrylamido-2-methylpropanesulfonic acid) lithium salt to form polymer gels. The ionic conductivity of these gels decreased with increasing polymer fraction, as in general ionic liquid/polymer mixed systems. At low polymer concentrations, these gels displayed excellent ionic conductivity of 10{sup -4} to 10{sup -3} S cm{sup -1} at room temperature. Gelation was found to cause little change in the {sup 7}Li diffusion coefficient of the ionic liquid, as measured by pulse-field-gradient NMR. These data strongly suggest that the lithium cation migrates in successive pathways provided by the ionic liquids. (author)

  17. Microchannel gel electrophoretic separation systems and methods for preparing and using

    Science.gov (United States)

    Herr, Amy; Singh, Anup K; Throckmorton, Daniel J

    2013-09-03

    A micro-analytical platform for performing electrophoresis-based immunoassays was developed by integrating photopolymerized cross-linked polyacrylamide gels within a microfluidic device. The microfluidic immunoassays are performed by gel electrophoretic separation and quantifying analyte concentration based upon conventional polyacrylamide gel electrophoresis (PAGE). To retain biological activity of proteins and maintain intact immune complexes, native PAGE conditions were employed. Both direct (non-competitive) and competitive immunoassay formats are demonstrated in microchips for detecting toxins and biomarkers (cytokines, c-reactive protein) in bodily fluids (serum, saliva, oral fluids). Further, a description of gradient gels fabrication is included, in an effort to describe methods we have developed for further optimization of on-chip PAGE immunoassays. The described chip-based PAGE immunoassay method enables immunoassays that are fast (minutes) and require very small amounts of sample (less than a few microliters). Use of microfabricated chips as a platform enables integration, parallel assays, automation and development of portable devices.

  18. Microchannel gel electrophoretic separation systems and methods for preparing and using

    Energy Technology Data Exchange (ETDEWEB)

    Herr, Amy E; Singh, Anup K; Throckmorton, Daniel J

    2015-02-24

    A micro-analytical platform for performing electrophoresis-based immunoassays was developed by integrating photopolymerized cross-linked polyacrylamide gels within a microfluidic device. The microfluidic immunoassays are performed by gel electrophoretic separation and quantifying analyte concentration based upon conventional polyacrylamide gel electrophoresis (PAGE). To retain biological activity of proteins and maintain intact immune complexes, native PAGE conditions were employed. Both direct (non-competitive) and competitive immunoassay formats are demonstrated in microchips for detecting toxins and biomarkers (cytokines, c-reactive protein) in bodily fluids (serum, saliva, oral fluids). Further, a description of gradient gels fabrication is included, in an effort to describe methods we have developed for further optimization of on-chip PAGE immunoassays. The described chip-based PAGE immunoassay method enables immunoassays that are fast (minutes) and require very small amounts of sample (less than a few microliters). Use of microfabricated chips as a platform enables integration, parallel assays, automation and development of portable devices.

  19. Microchannel gel electrophoretic separation systems and methods for preparing and using

    Energy Technology Data Exchange (ETDEWEB)

    Herr, Amy; Singh, Anup K; Throckmorton, Daniel J

    2013-09-03

    A micro-analytical platform for performing electrophoresis-based immunoassays was developed by integrating photopolymerized cross-linked polyacrylamide gels within a microfluidic device. The microfluidic immunoassays are performed by gel electrophoretic separation and quantifying analyte concentration based upon conventional polyacrylamide gel electrophoresis (PAGE). To retain biological activity of proteins and maintain intact immune complexes, native PAGE conditions were employed. Both direct (non-competitive) and competitive immunoassay formats are demonstrated in microchips for detecting toxins and biomarkers (cytokines, c-reactive protein) in bodily fluids (serum, saliva, oral fluids). Further, a description of gradient gels fabrication is included, in an effort to describe methods we have developed for further optimization of on-chip PAGE immunoassays. The described chip-based PAGE immunoassay method enables immunoassays that are fast (minutes) and require very small amounts of sample (less than a few microliters). Use of microfabricated chips as a platform enables integration, parallel assays, automation and development of portable devices.

  20. Long pendulums in gravitational gradients

    Energy Technology Data Exchange (ETDEWEB)

    Suits, B H [Physics Department, Michigan Technological University, Houghton, MI 49931 (United States)

    2006-03-01

    Previous results for long pendulums above a spherical Earth are generalized for arbitrary non-uniform gravitational fields in the limit of small oscillation. As is the case for the previous results, gravitational gradients are multiplied by the length of the string even though the string is assumed massless. The effect is shown to arise from the constraint on the motion imposed by the string. The significance of these results for real gradients is discussed. (letters and comments)

  1. Low-gradient aortic stenosis.

    Science.gov (United States)

    Clavel, Marie-Annick; Magne, Julien; Pibarot, Philippe

    2016-09-07

    An important proportion of patients with aortic stenosis (AS) have a 'low-gradient' AS, i.e. a small aortic valve area (AVA <1.0 cm(2)) consistent with severe AS but a low mean transvalvular gradient (<40 mmHg) consistent with non-severe AS. The management of this subset of patients is particularly challenging because the AVA-gradient discrepancy raises uncertainty about the actual stenosis severity and thus about the indication for aortic valve replacement (AVR) if the patient has symptoms and/or left ventricular (LV) systolic dysfunction. The most frequent cause of low-gradient (LG) AS is the presence of a low LV outflow state, which may occur with reduced left ventricular ejection fraction (LVEF), i.e. classical low-flow, low-gradient (LF-LG), or preserved LVEF, i.e. paradoxical LF-LG. Furthermore, a substantial proportion of patients with AS may have a normal-flow, low-gradient (NF-LG) AS: i.e. a small AVA-low-gradient combination but with a normal flow. One of the most important clinical challenges in these three categories of patients with LG AS (classical LF-LG, paradoxical LF-LG, and NF-LG) is to differentiate a true-severe AS that generally benefits from AVR vs. a pseudo-severe AS that should be managed conservatively. A low-dose dobutamine stress echocardiography may be used for this purpose in patients with classical LF-LG AS, whereas aortic valve calcium scoring by multi-detector computed tomography is the preferred modality in those with paradoxical LF-LG or NF-LG AS. Although patients with LF-LG severe AS have worse outcomes than those with high-gradient AS following AVR, they nonetheless display an important survival benefit with this intervention. Some studies suggest that transcatheter AVR may be superior to surgical AVR in patients with LF-LG AS.

  2. Bacterioplankton Diversity in Xiamen Houxi River Watershed Along an Urban-rural Gradient and Its Relation to Environmental Factors%厦门后溪流域沿城乡梯度浮游细菌多样性及其与环境因子的关系

    Institute of Scientific and Technical Information of China (English)

    刘乐冕; 杨军; 余小青; 余正; 张永雨; 田野; 张冬红

    2012-01-01

    为研究城市化对河流浮游细菌的影响,选取厦门后溪流域沿城乡梯度采集样品,并利用变性梯度凝胶电泳(Denaturing gradient gel electrophoresis,DGGE)和多元统计方法对浮游细菌群落空间分布格局进行分析.结果表明,Shannon-Weiner多样性指数和DGGE条带数随着采样站点和城市中心距离的减小呈现先升高后下降的趋势.聚类分析(Cluster)和多维尺度分析(Multidimensional scaling,MDS)将12个站点的浮游细菌群落分为4组,测序结果显示β-变形菌( Betaproteobacteria)是优势类群,占40.0%.统计分析表明,pH和总氮(Total nitrogen,TN)是影响后溪浮游细菌群落组成最主要的环境因子,他们共同解释了47.6%的群落组成变化.总之,沿城乡梯度随着水体理化指标的变化,浮游细菌群落也发生明显转变,表明后溪浮游细菌受到城市化的强烈影响此外,结果也提示DGGE可以作为监测与评价河流生态系统健康的重要工具.%To investigate the urbanization effect on riverine bacterioplankton, surface water samples were collected along an urban - rural gradient in the Houxi River Watershed, Xiamen, southeast China. Denaturing gradient gel electrophoresis (DGGE) and nvultivariable statistical approaches were used to characterize the spatial patterns of change in the bacterioplankton communities. The overall trend of Shannon-Weiner index and DGGE bands increased from site 1 to site 5 and then decreased from site 6 to site 12 with the decreasing distance between the sampling sites and downtown Xiamen. The cluster and multidimensional scaling analysis (MDS) resulted in four identical groups from the 12 sites, and Betaproteobacteria (40,0%) was the most dominant taxon derived from DGGE bands. Redundancy analysis (RDA) revealed that pH and total nitrogen (TN) were the main factors influencing bacterioplankton community composition in the Houxi River Watershed. Further, both pH and TN explained 47.6% of the variation

  3. A chemotactic gradient sequestered on endothelial heparan sulfate induces directional intraluminal crawling of neutrophils.

    Science.gov (United States)

    Massena, Sara; Christoffersson, Gustaf; Hjertström, Elina; Zcharia, Eyal; Vlodavsky, Israel; Ausmees, Nora; Rolny, Charlotte; Li, Jin-Ping; Phillipson, Mia

    2010-09-16

    During infection, chemokines sequestered on endothelium induce recruitment of circulating leukocytes into the tissue where they chemotax along chemokine gradients toward the afflicted site. The aim of this in vivo study was to determine whether a chemokine gradient was formed intravascularly and influenced intraluminal neutrophil crawling and transmigration. A chemokine gradient was induced by placing a macrophage inflammatory protein-2 (MIP-2)-containing (CXCL2) gel on the cremaster muscle of anesthetized wild-type mice or heparanase-overexpressing transgenic mice (hpa-tg) with truncated heparan sulfate (HS) side chains. Neutrophil-endothelial interactions were visualized by intravital microscopy and chemokine gradients detected by confocal microscopy. Localized extravascular chemokine release (MIP-2 gel) induced directed neutrophil crawling along a chemotactic gradient immobilized on the endothelium and accelerated their recruitment into the target tissue compared with homogeneous extravascular chemokine concentration (MIP-2 superfusion). Endothelial chemokine sequestration occurred exclusively in venules and was HS-dependent, and neutrophils in hpa-tg mice exhibited random crawling. Despite similar numbers of adherent neutrophils in hpa-tg and wild-type mice, the altered crawling in hpa-tg mice was translated into decreased number of emigrated neutrophils and ultimately decreased the ability to clear bacterial infections. In conclusion, an intravascular chemokine gradient sequestered by endothelial HS effectively directs crawling leukocytes toward transmigration loci close to the infection site.

  4. Graded/Gradient Porous Biomaterials

    Directory of Open Access Journals (Sweden)

    Xigeng Miao

    2009-12-01

    Full Text Available Biomaterials include bioceramics, biometals, biopolymers and biocomposites and they play important roles in the replacement and regeneration of human tissues. However, dense bioceramics and dense biometals pose the problem of stress shielding due to their high Young’s moduli compared to those of bones. On the other hand, porous biomaterials exhibit the potential of bone ingrowth, which will depend on porous parameters such as pore size, pore interconnectivity, and porosity. Unfortunately, a highly porous biomaterial results in poor mechanical properties. To optimise the mechanical and the biological properties, porous biomaterials with graded/gradient porosity, pores size, and/or composition have been developed. Graded/gradient porous biomaterials have many advantages over graded/gradient dense biomaterials and uniform or homogenous porous biomaterials. The internal pore surfaces of graded/gradient porous biomaterials can be modified with organic, inorganic, or biological coatings and the internal pores themselves can also be filled with biocompatible and biodegradable materials or living cells. However, graded/gradient porous biomaterials are generally more difficult to fabricate than uniform or homogenous porous biomaterials. With the development of cost-effective processing techniques, graded/gradient porous biomaterials can find wide applications in bone defect filling, implant fixation, bone replacement, drug delivery, and tissue engineering.

  5. Sol-gel derived sorbents

    Science.gov (United States)

    Sigman, Michael E.; Dindal, Amy B.

    2003-11-11

    Described is a method for producing copolymerized sol-gel derived sorbent particles for the production of copolymerized sol-gel derived sorbent material. The method for producing copolymerized sol-gel derived sorbent particles comprises adding a basic solution to an aqueous metal alkoxide mixture for a pH.ltoreq.8 to hydrolyze the metal alkoxides. Then, allowing the mixture to react at room temperature for a precalculated period of time for the mixture to undergo an increased in viscosity to obtain a desired pore size and surface area. The copolymerized mixture is then added to an immiscible, nonpolar solvent that has been heated to a sufficient temperature wherein the copolymerized mixture forms a solid upon the addition. The solid is recovered from the mixture, and is ready for use in an active sampling trap or activated for use in a passive sampling trap.

  6. Copolymers For Capillary Gel Electrophoresis

    Science.gov (United States)

    Liu, Changsheng; Li, Qingbo

    2005-08-09

    This invention relates to an electrophoresis separation medium having a gel matrix of at least one random, linear copolymer comprising a primary comonomer and at least one secondary comonomer, wherein the comonomers are randomly distributed along the copolymer chain. The primary comonomer is an acrylamide or an acrylamide derivative that provides the primary physical, chemical, and sieving properties of the gel matrix. The at least one secondary comonomer imparts an inherent physical, chemical, or sieving property to the copolymer chain. The primary and secondary comonomers are present in a ratio sufficient to induce desired properties that optimize electrophoresis performance. The invention also relates to a method of separating a mixture of biological molecules using this gel matrix, a method of preparing the novel electrophoresis separation medium, and a capillary tube filled with the electrophoresis separation medium.

  7. Intraprostatic injection of alcohol gel for the treatment of benign prostatic hyperplasia: preliminary clinical results.

    Science.gov (United States)

    Larson, Benjamin T; Netto, Nelson; Huidobro, Christian; de Lima, Marcelo Lopez; Matheus, Wagner; Acevedo, Cristian; Larson, Thayne R

    2006-09-06

    Benign prostatic hyperplasia (BPH) is one of the most common diseases ailing older men. Office-based procedures offer the advantage of being more effective than medications, while limiting the adverse effects, cost, and recovery of surgery. This study presents preliminary data on a new procedure that utilizes intraprostatic alcohol gel injection to ablate prostatic tissue. The purpose of this study is to evaluate the feasibility of using this gel as a treatment for BPH. A total of 65 patients with lower urinary tract symptoms (LUTS) due to BPH were treated with intraprostatic injections of alcohol gel. The gel is composed of 97% denatured alcohol and a patented polymer to cause viscosity. Three different methods of injection were utilized: transrectal (TR) injections (8), transurethral (TU) injections (36), and transperineal (TP) injections guided by biplaned ultrasound (21). Each method provided easy access to the center of the prostate, where a volume of gel, approximately 20-30% of the prostatic volume, was injected. Follow-up was based on changes in peak urinary flow (Qmax), IPSS scores, quality of life scores (QoL), adverse effects, and failures. Data are available at 3 and 12 months. The procedure was well tolerated with only local or no anesthesia in the TR and TP groups; the TU group received spinal anesthesia. All groups showed statistically significant (p gel injected was 8.05 ml, representing 21.56% of the prostatic volume. Qmax increased from a baseline mean of 8.50 to 12.01 ml/s at 3 months, and to 11.29 ml/s at 12 months. IPSS scores improved from a baseline mean of 21.12 to 10.00 at 3 months, and to 11.84 at 12 months. QoL scores were only available for 55 patients. QoL scores improved from a baseline of 3.93 to 1.98 at 3 months, and to 2.18 at 12 months. No extraprostatic injury or adverse effects were reported due to treatment. This preliminary study presents significant results showing that intraprostatic injection of alcohol gel could be an

  8. Trehalose-induced destabilization of interdigitated gel phase in dihexadecylphosphatidylcholine.

    Science.gov (United States)

    Takahashi, H; Ohmae, H; Hatta, I

    1997-01-01

    Trehalose is believed to have the ability to protect some organisms against low temperatures. To clarify the cryoprotective mechanism of trehalose, the structure and the phase behavior of fully hydrated dihexadecylphosphatidylcholine (DHPC) membranes in the presence of various concentrations of trehalose were studied by means of differential scanning calorimetry (DSC), static x-ray diffraction, and simultaneous x-ray diffraction and DSC measurements. The temperature of the interdigitated gel (Lbeta(i))-to-ripple (Pbeta') phase transition of DHPC decreases with a rise in trehalose concentration up to approximately 1.0 M. Above a trehalose concentration of approximately 1.0 M, no Lbeta(i) phase is observed. In this connection, the electron density profile calculated from the lamellar diffraction data in the presence of 1.6 M trehalose indicates that DHPC forms noninterdigitated bilayers below the P beta' phase. It was concluded that trehalose destabilizes the Lbeta(i) phase of DHPC bilayers. This suggests that trehalose reduces the area at the interface between the lipid and water. The relation between this effect of trehalose and a low temperature tolerance was discussed from the viewpoint of cold-induced denaturation of proteins. PMID:9414217

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

    Science.gov (United States)

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

    2017-08-18

    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

  10. Screening effect on nanostructure of charged gel

    DEFF Research Database (Denmark)

    Sugiyama, M; Annaka, M; Hino, M

    2004-01-01

    Charge screening effects on nanostructures of N-isopropylacrylamide-sodium acrylate (NIPA-SA) and -acrylic acid (NIPA-AAc) gels are investigated with small-angle neutron scattering. The NIPA-SA and NIPA-AAc gels with low water content exhibit microphase separations with different dimensions....... The dehydrated NIPA-SA gel also makes the microphase separation but the dehydrated NIPA-AAc gel does not. These results indicate that ionic circumstance around charged bases strongly affects the nanostructures both of the dehydrated gel and the gel with low water content. (C) 2004 Elsevier B. V. All rights...

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

    Science.gov (United States)

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

    2008-07-01

    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.

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

    Science.gov (United States)

    Johnson, M D; Fresco, J R

    1999-07-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1980-06-01

    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.

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

    Science.gov (United States)

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

    2012-01-01

    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.

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

    Science.gov (United States)

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

    2001-08-01

    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.

  16. Studies on the formation mechanism and the structure of the anisotropic collagen gel prepared by dialysis-induced anisotropic gelation.

    Science.gov (United States)

    Furusawa, Kazuya; Sato, Shoichi; Masumoto, Jyun-ichi; Hanazaki, Yohei; Maki, Yasuyuki; Dobashi, Toshiaki; Yamamoto, Takao; Fukui, Akimasa; Sasaki, Naoki

    2012-01-09

    We have found that dialysis of 5 mg/mL collagen solution into the phosphate solution with a pH of 7.1 and an ionic strength of 151 mM [corrected] at 25 °C results in a collagen gel with a birefringence and tubular pores aligned parallel to the growth direction of the gel. The time course of averaged diameter of tubular pores during the anisotropic gelation was expressed by a power law with an exponent of 1/3, suggesting that the formation of tubular pores is attributed to a spinodal decomposition-like phase separation. Small angle light scattering patterns and high resolution confocal laser scanning microscope images of the anisotropic collagen gel suggested that the collagen fibrils are aligned perpendicular to the growth direction of the gel. The positional dependence of the order parameter of the collagen fibrils showed that the anisotropic collagen gel has an orientation gradient.

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

    Science.gov (United States)

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

    2003-06-20

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-01-01

    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.

  20. Physicochemical behaviour of chitin gels.

    Science.gov (United States)

    Vachoud, L; Zydowicz, N; Domard, A

    2000-06-30

    Syneresis of chitin gels formed in the course of N-acetylation of chitosan in hydroalcoholic media has been studied. A critical cross-linking density related to a critical acetylation degree for which the gel undergoes weak syneresis and swells in water was shown (degree of acetylation (DA) 88%). Above this value, the weight loss during syneresis increases with DA. Conversely, syneresis decreases on increasing the polymer concentration, but disappears at a macroscopic level for a polymer concentration close to the critical concentration of entanglement in the initial solution. An increase in temperature favours the formation of hydrophobic interactions and new inter- and intramolecular hydrogen bondings. Due to the weak polyelectrolyte character of chitin, the weight of the gel depends on the pH and ionic strength of the media. Swelling-deswelling experiments show that the swelling of the gel is not fully reversible in relation with the formation of new cross-links during the depletion of the network. Our results reveals that the balance between segment-segment and segment-solvent interactions as well as the molecular mobility play the major role.