Regulation of CFTR Cl− channel gating by ATP binding and hydrolysis

Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is regulated by the interaction of ATP with its two cytoplasmic nucleotide-binding domains (NBD). Although ATP hydrolysis by the NBDs is required for normal gating, the influence of ATP binding versus hydrolysis on specific steps in the gating cycle remains uncertain. Earlier work showed that the absence of Mg2+ prevents hydrolysis. We found that even in the absence of Mg2+, ATP could support channel activity, albeit at a reduced level compared with the presence of Mg2+. Application of ATP with a divalent cation, including the poorly hydrolyzed CaATP complex, increased the rate of opening. Moreover, in CFTR variants with mutations that disrupt hydrolysis, ATP alone opened the channel and Mg2+ further enhanced ATP-dependent opening. These data suggest that ATP alone can open the channel and that divalent cations increase ATP binding. Consistent with this conclusion, when we mutated an aspartate thought to bind Mg2+, divalent cations failed to increase activity compared with ATP alone. Two observations suggested that divalent cations also stabilize the open state. In wild-type CFTR, CaATP generated a long duration open state, whereas ATP alone did not. With a CFTR variant in which hydrolysis was disrupted, MgATP, but not ATP alone, produced long openings. These results suggest a gating cycle for CFTR in which ATP binding opens the channel and either hydrolysis or dissociation leads to channel closure. In addition, the data suggest that ATP binding and hydrolysis by either NBD can gate the channel.

Cleavage of the antithrombin III binding site in heparin by heparinases and its implication in the generation of low molecular weight heparin

Heparin has been used as a clinical anticoagulant for more than 50 years, making it one of the most effective pharmacological agents known. Much of heparin's activity can be traced to its ability to bind antithrombin III (AT-III). Low molecular weight heparin (LMWH), derived from heparin by its controlled breakdown, maintains much of the antithrombotic activity of heparin without many of the serious side effects. The clinical significance of LMWH has highlighted the need to understand and develop chemical or enzymatic means to generate it. The primary enzymatic tools used for the production of LMWH are the heparinases from Flavobacterium heparinum, specifically heparinases I and II. Using pentasaccharide and hexasaccharide model compounds, we show that heparinases I and II, but not heparinase III, cleave the AT-III binding site, leaving only a partially intact site. Furthermore, we show herein that glucosamine 3-O sulfation at the reducing end of a glycosidic linkage imparts resistance to heparinase I, II, and III cleavage. Finally, we examine the biological and pharmacological consequences of a heparin oligosaccharide that contains only a partial AT-III binding site. We show that such an oligosaccharide lacks some of the functional attributes of heparin- and heparan sulfate-like glycosaminoglycans containing an intact AT-III site.

Methyl Jasmonate Induces Lauric Acid ω-Hydroxylase Activity and Accumulation of CYP94A1 Transcripts but Does Not Affect Epoxide Hydrolase Activities in Vicia sativa Seedlings1

Treatment of etiolated Vicia sativa seedlings by the plant hormone methyl jasmonate (MetJA) led to an increase of cytochrome P450 content. Seedlings that were treated for 48 h in a 1 mm solution of MetJA stimulated ω-hydroxylation of 12:0 (lauric acid) 14-fold compared with the control (153 versus 11 pmol min−1 mg−1 protein, respectively). Induction was dose dependent. The increase of activity (2.7-fold) was already detectable after 3 h of treatment. Activity increased as a function of time and reached a steady level after 24 h. Northern-blot analysis revealed that the transcripts coding for CYP94A1, a fatty acid ω-hydroxylase, had already accumulated after 1 h of exposure to MetJA and was maximal between 3 and 6 h. Under the same conditions, a study of the enzymatic hydrolysis of 9,10-epoxystearic acid showed that both microsomal and soluble epoxide hydrolase activities were not affected by MetJA treatment.

Identification and Partial Characterization of the Pectin Methyltransferase “Homogalacturonan-Methyltransferase” from Membranes of Tobacco Cell Suspensions1

A membrane preparation from tobacco (Nicotiana tabacum L.) cells contains at least one enzyme that is capable of transferring the methyl group from S-adenosyl-methionine (SAM) to the C6 carboxyl of homogalacturonan present in the membranes. This enzyme is named homogalacturonan-methyltransferase (HGA-MT) to distinguish it from methyltransferases that catalyze methyletherification of the pectic polysaccharides rhamnogalacturonan I or rhamnogalacturonan II. A trichloroacetic acid precipitation assay was used to measure HGA-MT activity, because published procedures to recover pectic polysaccharides via ethanol or chloroform:methanol precipitation lead to high and variable background radioactivity in the product pellet. Attempts to reduce the incorporation of the 14C-methyl group from SAM into pectin by the addition of the alternative methyl donor 5-methyltetrahydrofolate were unsuccessful, supporting the role of SAM as the authentic methyl donor for HGA-MT. The pH optimum for HGA-MT in membranes was 7.8, the apparent Michaelis constant for SAM was 38 μm, and the maximum initial velocity was 0.81 pkat mg−1 protein. At least 59% of the radiolabeled product was judged to be methylesterified homogalacturonan, based on the release of radioactivity from the product after a mild base treatment and via enzymatic hydrolysis by a purified pectin methylesterase. The released radioactivity eluted with a retention time identical to that of methanol upon fractionation over an organic acid column. Cleavage of the radiolabeled product by endopolygalacturonase into fragments that migrated as small oligomers of HGA during thin-layer chromatography, and the fact that HGA-MT activity in the membranes is stimulated by uridine 5′-diphosphate galacturonic acid, a substrate for HGA synthesis, confirms that the bulk of the product recovered from tobacco membranes incubated with SAM is methylesterified HGA.

Características de grãos e amido de diferentes cultivares de quinoa (Chenopodium quinoa Willd.)

Os grãos de quinoa possuem excelente balanço nutricional além das propriedades funcionais, comparativamente superior à dos cereais. A quinoa é cultivada em diversos países e, devido às suas características, têm aumentado o interesse de pesquisadores e consumidores. A quinoa contém pericarpo branco, no entanto, existem grãos com pericarpo vermelho e preto, e todos os tipos são utilizados como alimento em diferentes preparações. Com o objetivo de avaliar as características de grãos de quinoa, amostras de cor branca, preta e vermelha foram analisadas quanto às propriedades físico-químicas e funcionais dos grãos e do amido extraído das diferentes amostras. O amido, extraído pelo método alcalino, foi submetido as análises de teor de amilose, difração de raios X, microscopia eletrônica de varredura (MEV), propriedades térmicas (por DSC-Differential Scanning Calorimeter) e propriedades de pasta (por RVA- Rapid Visco Analyser), além de suscetibilidade à hidrólise enzimática e cor. A composição físico-química dos grãos de quinoa apresentou como principais diferenças o teor de cinzas, fibras e amido. O teor de amilose variou de 13,6% a 21,3%, entre as amostras de amido; os padrões de cristalinidade dos amidos foram de tipo A, típico dos cereais; e, a cristalinidade relativa variou de 25,4 a 29,6 %; as micrografias obtidas por MEV apresentaram as formas poliédricas dos grânulos de amido. Os viscoamilogramas, obtidos para os diferentes amidos, mostraram um comportamento semelhante entre as amostras brancas e pretas. As propriedades térmicas de retrogradação das amostras de quinoa vermelha apresentaram uma menor taxa de retrogradação que variou de 7,5 a 8,5 %; as cultivares brancas apresentaram as maiores taxas de retrogradação de 19,0 a 25,4 %. A hidrólise enzimática dos grânulos de amido, analisada em equivalentes de maltose, variou de 7,2 a 8,7 mg/mL, com uma velocidade maior para a cultivar BSyB, em 60 minutos. O amido extraído das amostras brancas de quinoa apresentou valor de luminosidade de 99,0 e os amidos extraídos das amostras de cor vermelha e preta apresentaram em torno de 97,0. As análises realizadas neste estudo ampliam o conhecimento das características da quinoa de cor branca, vermelha e preta, além de mostrar que a cultivar brasileira (BSyB) apresenta características diferenciadas em vários parâmetros. Devido as suas propriedades todas as amostras analisadas possuem potencial para futuras aplicações tecnológicas.

Specific and reversible immobilization of proteins tagged to the affinity polypeptide C-LytA on functionalized graphite electrodes

We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field.

Hydrothermal Carbons from Hemicellulose-Derived Aqueous Hydrolysis Products as Electrode Materials for Supercapacitors

Acid pretreatment of lignocellulosic biomass, required for bioethanol production, generates large amounts of by-products, such as lignin and hydrolyzed hemicellulose fractions, which have found so far very limited applications. In this work, we demonstrate how the recovered hemicellulose hydrolysis products can be effectively utilized as a precursor for the synthesis of functional carbon materials through hydrothermal carbonization (HTC). The morphology and chemical structure of the synthesized HTC carbons are thoroughly characterized to highlight their similarities with glucose-derived HTC carbons. Furthermore, two routes for introducing porosity within the HTC carbon structure are presented: i) silica nanoparticle hard-templating, which is shown to be a viable method for the synthesis of carbonaceous hollow spheres; and ii) KOH chemical activation. The synthesized activated carbons (ACs) show an extremely high porosity (pore volume≈1.0 cm3 g−1) mostly composed of micropores (90 % of total pore volume). Because of their favorable textural properties, the ACs are further tested as electrodes for supercapacitors, yielding very promising results (300 F g−1 at 250 mA g−1) and confirming the high suitability of KOH-activated HTC carbons derived from spruce and corncob hydrolysis products as materials for electric double layer supercapacitors.

Discovery, structure and biological activities of the cyclotides

The cyclotides are a family of small disulfide rich proteins that have a cyclic peptide backbone and a cystine knot formed by three conserved disulfide bonds. The combination of these two structural motifs contributes to the exceptional chemical, thermal and enzymatic stability of the cyclotides, which retain bioactivity after boiling. They were initially discovered based on native medicine or screening studies associated with some of their various activities, which include uterotonic action, anti-HIV activity, neurotensin antagonism, and cytotoxicity. They are present in plants from the Rubiaceae, Violaceae and Cucurbitaccae families and their natural function in plants appears to be in host defense: they have potent activity against certain insect pests and they also have antimicrobial activity. There are currently around 50 published sequences of cyclotides and their rate of discovery has been increasing over recent years. Ultimately the family may comprise thousands of members. This article describes the background to the discovery of the cyclotides, their structural characterization, chemical synthesis, genetic origin, biological activities and potential applications in the pharmaceutical and agricultural industries. Their unique topological features make them interesting from a protein folding perspective. Because of their highly stable peptide framework they might make useful templates in drug design programs, and their insecticidal activity opens the possibility of applications in crop protection.

The cyclotides and related macrocyclic peptides as scaffolds in drug design

The applicability of linear peptides as drugs is potentially limited by their susceptibility to proteolytic cleavage and poor bioavailability. Cyclotides are macrocyclic cystine-knotted mini-proteins that have a broad range of bioactivities and are exceptionally stable, being resistant to chemical, thermal and enzymatic degradation. The general limitations of peptides as drugs can potentially be overcome by using the cyclotide framework as a scaffold onto which new activities may be engineered. The potential use of cyclotides and related peptide scaffolds for drug design is evaluated herein, with reference to increasing knowledge of the structures and sequence diversity of natural cyclotides and the emergence of new approaches in protein engineering.

Physical and chemical factors influencing the germination of Clostridium difficile spores

AIMS: To investigate the influence of chemical and physical factors on the rate and extent of germination of Clostridium difficile spores. METHODS AND RESULTS: Germination of C. difficile spores following exposure to chemical and physical germinants was measured by loss of either heat or ethanol resistance. Sodium taurocholate and chenodeoxycholate initiated germination together with thioglycollate medium at concentrations of 0.1-100 mmol l(-1) and 10-100 mmol l(-1) respectively. Glycine (0.2% w/v) was a co-factor required for germination with sodium taurocholate. There was no significant difference in the rate of germination of C. difficile spores in aerobic and anaerobic conditions (P > 0.05) however, the initial rate of germination was significantly increased at 37 degrees C compared to 20 degrees C (P < 0.05). The optimum pH range for germination was 6.5-7.5, with a decreased rate and extent of germination occurring at pH 5.5 and 8.5. CONCLUSIONS: This study demonstrates that sodium taurocholate and chenodeoxycholate initiate germination of C. difficile spores and is concentration dependant. Temperature and pH influence the rate and extent of germination. SIGNIFICANCE AND IMPACT OF THE STUDY: This manuscript enhances the knowledge of the factors influencing the germination of C. difficile spores. This may be applied to the development of potential novel strategies for the prevention of C. difficile infection.

Site specific growth of metal catalyzed silica nanowires for biological and chemical sensing

In this research the integration of nanostructures and micro-scale devices was investigated using silica nanowires to develop a simple yet robust nanomanufacturing technique for improving the detection parameters of chemical and biological sensors. This has been achieved with the use of a dielectric barrier layer, to restrict nanowire growth to site-specific locations which has removed the need for post growth processing, by making it possible to place nanostructures on pre-pattern substrates. Nanowires were synthesized using the Vapor-Liquid-Solid growth method. Process parameters (temperature and time) and manufacturing aspects (structural integrity and biocompatibility) were investigated. Silica nanowires were observed experimentally to determine how their physical and chemical properties could be tuned for integration into existing sensing structures. Growth kinetic experiments performed using gold and palladium catalysts at 1050°C for 60 minutes in an open-tube furnace yielded dense and consistent silica nanowire growth. This consistent growth led to the development of growth model fitting, through use of the Maximum Likelihood Estimation (MLE) and Bayesian hierarchical modeling. Transmission electron microscopy studies revealed the nanowires to be amorphous and X-ray diffraction confirmed the composition to be SiO2 . Silica nanowires were monitored in epithelial breast cancer media using Impedance spectroscopy, to test biocompatibility, due to potential in vivo use as a diagnostic aid. It was found that palladium catalyzed silica nanowires were toxic to breast cancer cells, however, nanowires were inert at 1μg/mL concentrations. Additionally a method for direct nanowire integration was developed that allowed for silica nanowires to be grown directly into interdigitated sensing structures. This technique eliminates the need for physical nanowire transfer thus preserving nanowire structure and performance integrity and further reduces fabrication cost. Successful nanowire integration was physically verified using Scanning electron microscopy and confirmed electrically using Electrochemical Impedance Spectroscopy of immobilized Prostate Specific Antigens (PSA). The experiments performed above serve as a guideline to addressing the metallurgic challenges in nanoscale integration of materials with varying composition and to understanding the effects of nanomaterials on biological structures that come in contact with the human body.

Annotated record and chemical composition of ferromanganese concretions and crusts sampled from the lakes and streams of Norway

The main objective of the project was to develop a geochemical method for exploration of ores associated with granitic rocks. Fe and Mn oxidates were sampled in streambeds and lakes from 129 localities in Southeastern Norway. 65 of these localities are situated in the northern Oslo Graben. The samples were examined mineralogically and chemically by a variety of methods. Geochemical maps of the element content in oxidates show regional distribution patterns for several elements. Sampling and analysis of oxidates can be used in exploration for mineralizations such as the Skrukkelia Mo-deposit in the northern Oslo Graben. New anomalies (especially for Zn and W) have been detected. Appendix I contains a description of samples, chemical and mineralogical determinations performed on the samples, backscattered electron image-, X-ray image- and scanning electron image pictures of the oxidate preparates. Appendix II contains spectral plots, point analysis with the microprobe, X-ray diffractograms, analytical results, correlation coefficient matrix, scatterplots, frequency distributions and information on data storage. Appendix III containS maps of the element content in oxidates.

Chemical composition of Late Cenozoic volcanic and volcaniclastic deposits from the western Woodlark Basin area, SW Pacific

Tephra fallout layers and volcaniclastic deposits, derived from volcanic sources around and on the Papuan Peninsula, form a substantial part of the Woodlark Basin marine sedimentary succession. Sampling by the Ocean Drilling Program Leg 180 in the western Woodlark Basin provides the opportunity to document the distribution of the volcanically-derived components as well as to evaluate their chronology, chemistry, and isotope compositions in order to gain information on the volcanic sources and original magmatic systems. Glass shards selected from 57 volcanogenic layers within the sampled Pliocene-Pleistocene sedimentary sequence show predominantly rhyolitic compositions, with subordinate basaltic andesites, basaltic trachy-andesites, andesites, trachy-andesites, dacites, and phonolites. It was possible to correlate only a few of the volcanogenic layers between sites using geochemical and age information apparently because of the formation of strongly compartmentalised sedimentary realms on this actively rifting margin. In many cases it was possible to correlate Leg 180 volcanic components with their eruption source areas based on chemical and isotope compositions. Likely sources for a considerable number of the volcanogenic deposits are Moresby and Dawson Strait volcanoes (D'Entrecasteaux Islands region) for high-K calc-alkaline glasses. The Dawson Strait volcanoes appear to represent the source for five peralkaline tephra layers. One basaltic andesitic volcaniclastic layer shows affinities to basaltic andesites from the Woodlark spreading tip and Cheshire Seamount. For other layers, a clear identification of the sources proved impossible, although their isotope and chemical signatures suggest similarities to south-west Pacific subduction volcanism, e.g. New Britain and Tonga- Kermadec island arcs. Volcanic islands in the Trobriand Arc (for example, Woodlark Island Amphlett Islands and/or Egum Atoll) are probable sources for several volcaniclastic layers with ages between 1.5 to 3 Ma. The Lusancay Islands can be excluded as a source for the volcanogenic layers found during Leg 180. Generally, the volcanogenic layers indicate much calc-alkaline rhyolitic volcanism in eastern Papua since 3.8 Ma. Starting at 135 ka, however, peralkaline tephra layers appear. This geochemical change in source characteristics might reflect the onset of a change in geotectonic regime, from crustal subduction to spreading, affecting the D'Entrecasteaux Islands region. Initial 143Nd/144Nd ratios as low as 0.5121 and 0.5127 for two of the tephra layers are interpreted as indicating that D'Entrecasteaux Islands volcanism younger than 2.9 Ma occasionally interacted with the Late Archean basement, possibly reflecting the mobilisation of the deep continental crust during active rift propagation.

Bacteria growth, enzyme activities and TEP during KOSMOS 2011 mesocosm experiment in the Raunefjord

Marine bacteria are the main consumers of freshly produced organic matter. Many enzymatic processes involved in the bacterial digestion of organic compounds were shown to be pH sensitive in previous studies. Due to the continuous rise in atmospheric CO2 concentration, seawater pH is presently decreasing at a rate unprecedented during the last 300 million years but the consequences for microbial physiology, organic matter cycling and marine biogeochemistry are still unresolved. We studied the effects of elevated seawater pCO2 on a natural plankton community during a large-scale mesocosm study in a Norwegian fjord. Nine Kiel Off-Shore Mesocosms for Future Ocean Simulations (KOSMOS) were adjusted to different pCO2 levels ranging initially from ca. 280 to 3000 µatm and sampled every second day for 34 days. The first phytoplankton bloom developed around day 5. On day 14, inorganic nutrients were added to the enclosed, nutrient-poor waters to stimulate a second phytoplankton bloom, which occurred around day 20. Our results indicate that marine bacteria benefit directly and indirectly from decreasing seawater pH. During the first phytoplankton bloom, 5-10% more transparent exopolymer particles were formed in the high pCO2 mesocosms. Simultaneously, the efficiency of the protein-degrading enzyme leucine aminopeptidase increased with decreasing pH resulting in up to three times higher values in the highest pCO2/lowest pH mesocosm compared to the controls. In general, total and cell-specific aminopeptidase activities were elevated under low pH conditions. The combination of enhanced enzymatic hydrolysis of organic matter and increased availability of gel particles as substrate supported up to 28% higher bacterial abundance in the high pCO2 treatments. We conclude that ocean acidification has the potential to stimulate the bacterial community and facilitate the microbial recycling of freshly produced organic matter, thus strengthening the role of the microbial loop in the surface ocean.

Avaliação da influência de surfactantes químico e biológico na hidrólise enzimática de casca de coco verde após pré-tratamento ácido/alcalino e com peróxido de hidrogênio alcalino

In Brazil many types of bioproducts and agroindustrial waste are generated currently, such as cacashew apple bagasse and coconut husk, for example. The final disposal of these wastes causes serious environmental issues. In this sense, waste lignocellulosic content, as the shell of the coconut is a renewable and abundant raw material in which its use has an increased interest mainly for the 2nd generation ethanol production. The hydrolysis of cellulose to reducing sugars such as glucose and xylose is catalysed by a group of enzymes called cellulases. However, the main bottleneck in the enzymatic hydrolysis of cellulose is the significant deactivation of the enzyme that shows irreversible adsorption mechanism leading to reduction of the cellulose adsorption onto cellulose. Studies have shown that the use of surfactants can modify the surface property of the cellulose therefore minimizing the irreversible binding. The main objective of the present study was to evaluate the influence of chemical and biological surfactants during the hydrolysis of coconut husk which was subjected to two pre-treatment in order to improve the accessibility of the enzymes to the cellulose, removing this way, part of the lignin and hemicellulose present in the structure of the material. The pre-treatments applied to coconut bagasse were: Acid/Alkaline using 0.6M H2SO4 followed by 1M NaOH, and the one with Alkaline Hydrogen Peroxide at a concentration of 7.35% (v/v) and pH 11.5. Both the material no treatment and pretreated were characterized using analysis of diffraction X-ray (XRD), Scanning Electron Microscopy (SEM) and methods established by NREL. The influence of both surfactants, chemical and biological, was used at concentrations below the critical micelle concentration (CMC), and the concentrations equal to the CMC. The application of pre-treatment with coconut residue was efficient for the conversion to glucose, as well as for the production of total reducing sugars, it was possible to observe that the pretreatment fragmented the structure as well as disordered the fibers. Regarding XRD analysis, a significant increase in crystallinity index was observed for pretreated bagasse acid/alkali (51.1%) compared to the no treatment (31.7%), while that for that treated with PHA, the crystallinity index was slightly lower, around 29%. In terms of total reducing sugars it was not possible to observe a significant difference between the hydrolysis carried out without the use of surfactant compared to the addition of Triton and rhamnolipid. However, by observing the conversions achieved during the hydrolysis, it was noted that the best conversion was using the rhamnolipíd for the husk pretreated with acid/alkali, reaching a value of 33%, whereas using Triton the higher conversion was 23.8%. The coconut husk is a residue which can present a high potential to the 2nd generation ethanol production, being the rhamonolipid a very efficient biosurfactant for use as an adjuvant in the enzymatic process in order to act on the material structure reducing its recalcitrance and therefore improving the conditions of access for enzymes to the substrate increasing thus the conversion of cellulose to glucose.