bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetic Studies on the Hydrolysis of Soluble and Cassava Starches by Maltogenase
Authors: Gaouar Ouassila , Aymard Christian , Zakhia Nadine , Rios Gilbert M.
Published: November 2006

In view of utilizing the commercial enzyme Maltogenase for the production of maltose syrup in a continuous membrane reactor, some kinetic properties of this enzyme were investigated. Preliminary tests were conducted using Zulkowsky soluble starch as substrate. At pH 5.0 and 60°C, the Michaelis and Menten kinetic constants Vmax and KM were respectively 2.47mmol of maltose equivalents·min−1·dm−3 and 9.64g of Zulkowsky soluble starch·dm−3. Initial hydrolysis velocity was reduced at high starch concentrations and in the presence of maltose. When liquefied cassava starch (≈60 to 340g·dm−3) was used as substrate, hydrolysis kinetics was modelled by analyzing whole progress curves. A simple empirical modell was established and allowed satisfactory descriptions of the experimental data for the formation of maltose over time at large enzyme/substrate ratios.
Amino Acids as Plasticizers for Starch‐based Plastics
Authors: Stein Thomas M. , Greene Richard V.
Published: November 2006

Amino acids of differing hydrophobicity were tested as plasticizers in starch‐glycerol (4:1 weight ratio) blends and were compared to the more conventional plasticizers, urea, sucrose and ammonium chloride. In mechanical tests (tensile strength and percent elongation) on extruded ribbons containing up to 20 weight percent plasticizer, glycine and isoleucine hydrochloride were found to be extremely poor plasticizers. However, lysine behaved similarly to sucrose. Proline compared favorably to urea. Moreover, at concentrations of 23 and 29 weight percent, proline was superior to urea in its ability to increase the percent elongation of the starch‐glycerol mixture. The glass transition temperature (Tg) for the standard starch‐glycerol samples was 40°C, as determined by differential scanning calorimetry. Added lysine hydrochloride, sucrose, proline and urea effected the Tg similarly. At 5 weight percent all Tg's increased, while at 20 weight percent they dropped to room temperature or below. Isoleucine, at low or high concentration, yielded a Tg of 60°C, consistent with very brittle material. Biodegradation experiments were conducted on selected formulations by monitoring CO2 evolution after inoculation with Aspergillus niger. Little CO2 accumulated from the standard starch‐glycerol mixture or sucrose plasticized blends, likely due to a lack of combined nitrogen. In contrast, proline and urea‐plasticized blends were rapidly metabolized.
Effect of Moisture Level on the Crystallinity of Wheat Starch Aged at Different Temperatures
Authors: Jang J. K. , Pyun Y. R.
Published: November 2006

Differential Scanning Calorimetry (DSC) was used to study retrogradation of wheat starch. For storage at 4°C, enthalpy (ΔH) values of retrogradation endotherm (recrystallization) of wheat starch containing various moisture levels (25–80%) slowly continued to increase except in samples with moisture level of 50, 60 and 70%. For storage at 26°C and 32°C, most recrystallization of wheat starch occured within one and two weeks of storage time, while recrystallization with a moisture level of 50% at 26°C occurred for 4 weeks. The peak temperatures of retrogradation endotherm at various moisture levels (25–80%) after 4 weeks of storage time increased largely as the storage temperatures increased from 4 to 32°C. The enthalpy values of retrogradation endotherm after 4 weeks of storage occurred to a greater extent at a lower storage temperature. The glass transition temperature and starch recrystallization have a close relationship. In the recrystallization of wheat starch, the nucleation and propagation rate after 4 weeks of storage can be converted into enthalpy and peak temperature of retrogradation endotherm.
High Specific Activity of Raw‐Starch Digesting‐Glucoamylase Producing Rhizopus sp. A‐11 in Liquid Culture
Authors: Morita H. , Fujio Y.
Published: November 2006

Glucoamylase (GA) (EC 3.2.1.3) from Rhizopus sp. A‐11 using metalion regulated liquid medium was purified by ammonium sulfate precipitation and column chromatography on CM‐Sephadex C‐50. Since purification was easy, the enzyme was purified about 5.0 fold with a 72.9% yield. The enzyme was proved to be homogeneous as judged by SDS‐PAGE, and has a molecular weight of about 72.4kDa. The enzyme strongly digested against raw starches. Raw cassava, wheat, and corn starch digesting relative specific activities of the purified enzyme were 15, 13, and 15%, respectively, as compared with soluble starch digesting specific activity (21,360 units/mg protein). Raw cassava, wheat, and corn starch adsorption rates were 99,99, and 99%, respectively. This culture produced little acid or neutral protease. According to the high raw starch digesting activity, high raw starch adsorption rate, low protease activity, and high molecular weight of the GA, it may be classified as GA‐I. Most proteins in the culture were GA. These results suggest a considerable advantage for GA production by liquid cultivation.
Starch and Starch Derivatives Industry in India
Authors: Dias Fausto F. , Mehta Darshan
Published: November 2006

Tapioca and maize are the major sources of starch in India. Tapioca starch is produced in very small units which are not extensively mechanized. This quality of the starch produced is not of a high quality. Maize starch is produced in mechanized units. The liberalization of the economy and growing consumerism bode well for the starch industry in India. Growth of the starch industry would depend on developing applications for starch as well as optimizing usage to derive maximum benefits. The unique properties of maize and tapioca should be considered.
An Attempt to Evaluate the Subsite Structure of Cycloamylose glucanotransferase from Bacillus stearothermophilus: Based on its Transfer Reaction with Substrate Malto‐oligosaccharides
Authors: Ohnishi Matasake , Mitsune Tamaki , Tabata Mihoko , Kubota Michio , Rokushika Souji
Published: November 2006

Based on determination of the transferred products (maltooligosaccharides Gn), the plausible degradation modes of G2, G3, and G4 were analyzed quantitatively, and it was made an attempt to evaluate the binding affinity Ai on some subsites of cycloamylose glucanotransferase (CGT) from Bacillus stearothermophilus. This method can be employed instead of the technique using a radioisotope. The quantities of the substrates (G2–G4) and products Gn were measured by using the HPLC techniques as a function fo time for the CGT‐catalysed reaction.
Surface Modification of Water‐insoluble Drug Particles with Starch
Authors: Rein Hubert , Steffens Klaus‐J.
Published: November 2006

Paranterally applied, water‐insolubile drug‐particles are better tolerated if the

Particle surface is hydrophil enough

Surface‐charge (measured as ζ‐potential) in physiological liquids does not exceed +8mV resp. ‐8mV.

It is possible to coat lipophilic drug‐particles with a thin – hydrophilic – amylosefilm by a new, only temperature‐controlled setback – (=retrogradation) – technique. Thus, it is not necessary to use any unphysiological agents such as monomers or organic solvents. This process requires starches with low set‐back‐temperature (TR), e.g. Schneeapfel – (TR49°C), banana (plantains) – (TR54°C) and chickpea starch (TR55°C) or one of the investigated tapioca starches (Maizena #A6, TR61°C). Starches with higher set‐back‐temperature (cave: particle growth), e.g. lentil‐ and plantains starch (TR94°C) are not recommended. Potato starches (Südstärke, Roquette, Emsland, Klenk) do not retrograde at lowering temperature. Therefore, these starches are not usable for the investigated set‐back‐method.

Amylose coated particles show not only optimal surface‐charge, but also a reduced sedimentation‐velocity, caused by an enlarged hydrodynamic‐diameter.
Inner Structure of Potato Starch Granules
Authors: Tamaki Shinji , Teranishi Katsunori , Hisamatsu Makoto , Yamada Tetsuya
Published: November 2006

A combination treatment (dry‐heat and glucoamylase) was applied on potato starch granules. Many inner granules in the granules after the treatments were observed by SEM. The inner granule located near edge of ellipsoid of granule was partially degraded X‐ray diffraction pattern of starch granules did not change before and after the treatments in spite of over 50% loss by amylase digestion. GPC and iodine reaction of the sample showed the treatments brought about fairly large degradation of both amylopectin and amylose components, but GPC of debranched sample after the combination treatments showed amylose was thoroughly degraded while peaks pattern of amylopectin did not change. Small size granules were more susceptible than large size granules by the treatments.
Säureabbau von Stärke unter semi‐dry Bedingungen
Authors: Klingler Rudolf , Busch Karl‐Georg , Vahedi Bibi
Published: November 2006

Bei der Anwendung des semi‐dry Prozesses zur Säuremodifizierung der Stärke wird die granuläre Substanz in einem Mischer mit Säure in katalytischer Konzentration besprüht, danach kurzzeitig im Mikrowellenfeld gezielt erhitzt, anschließend durch Aufsprühen einer Sodalösung neutralisiert und gleichzeitig gekühlt.

Im Verlauf der milden Säurehydrolyse werden unspezifisch glycosidische Bedingungen der Glucosepolymere gespalten, wovon die Amylopektinfraktion zunächst stärker betroffen ist als die Amylose. Durch die Variation der Modifizierungsbedingungen, insbesondere hinsichtlich der Katalysatormenge und der Erhitzung, ließen sich unterschiedliche Typen dünnkochender Stärke mit variablen Gelbildungseigenschaften bis hin zu reversibel gelbildenden Dextrinen herstellen, die sich gut als Fettreplacer eignen.

Der semi‐dry Prozeß ist besonders umweltfreundlich und bietet gegenüber dem gebräuchlichen Slurry‐Verfahren entscheidende Vorteile: eine kleinere Chemikalienmenge, eine kürzere Reaktionsdauer, kein Auswaschen, kein Abwasser, kein Substanzverlust.
Amylose Conformational Transitions in Binary DMSO/Water Mixtures
Authors: Cheetham Norman W. H. , Tao Leping
Published: November 2006

The effects of percentage water content in DMSO/water mixtures on amylose conformation were measured by following changes in specific optical rotation, limiting viscosity number, and 13C‐NMR chemical shifts. The temperature dependence of specific optical rotation showed differences in amylose conformation for four ratios of dimethyl suphoxide/water. The changes in limiting viscosity of amylose in different proportions of DMSO/water with increasing temperature, and the effect of tetramethylurea on the specific rotation of amylose, indicate that the number of intramolecular hydrogen bonds decreases with increased water content. An amylose conformational change was also deduced from 13C‐NMR chemical shift changes. Thirty‐three percent water appears to be a crossover concentration. above which the helical conformation is progressively lost with increased temperature. With further increase in water conent to 66.6%, transition to a random coil conformation is complete, and iodine complexation may take place. The transition of amylose conformation from helix to loose helix to random coil with increasing water content was deduced from the experimental results.
Rapid Measurement of Potassium Ion in Wheat Flour Using a FIA‐Ion Selective Electrode Based Sensing System
Authors: Haginoya Ryuichi , Nomura Yoko , Sakai Kuniko , Komatsu Takashi , Nagao Seiichi , Sakai Aki , Henmi Hideaki , Ito Satoshi , Asano Yasukazu , Karube Isao
Published: November 2006

The ash content of wheat flour is used to evaluate its quality but the conventional method of analysis takes several hours. This paper proposes using the potassium ion concentration of wheat flour samples instead of the ash content since the correlation between the two is very good (the correlation coefficient was 0.99). A flow injection analysis (FIA) system based on a potassium ion selective electrode took less than 10min per measurement with a coefficient of variation less than 3%.
Determination of the Substitution Pattern of Cationic Starch Ethers
Authors: Wilke Olaf , Mischnick Petra
Published: November 2006

Degradation of cationic starches and subsequent permethylation and Hofmann‐elimination of the methyl‐glucosides yields the corresponding enolether derivatives, which can be analyzed by GLC and GLC‐MS. The relative ratio of 2‐, 3‐ and 6‐substitution in the anhydroglucose unit as well as of un‐, mono‐, di‐ and trisubstitution in the anhydroglucose unit as well as of un‐, mono‐, di‐ and trisubstituted monomers can be determined. The experimental results are compared with the data calculated by means of a kinetic model. Only slight deviations from a random distribution in the anhydroglucose unit are observed and discussed. Enzymatic degradation of the cationic starch ethers and measurement of the liberated glucose should give indication to the distribution along the polymer chain.
Characteristics of Canaryseed (Phalaris canariensis L.) Starch
Authors: Abdel‐Aal E.‐S. M. , Hucl P. , Sosulski F. W.
Published: November 2006

Canaryseed starches from three locations were isolated by an alkali steeping process and evaluated for their chemical, pasting and structural properties by amylograph, scanning electron microscope (SEM), differential scanning calorimeter, X‐ray diffractometer and image analysis, in comparison with isolated wheat starch. The average yield of canaryseed starch was 50 ± 2.9g starch per 100g of groat with a mean extraction efficiency of 77%. The starch isolates contained from 93 to 97% starch with amylose contents ranging from 16 to 22%. Canaryseed starch was uniform in appearance, being small polygonal granules having an average diameter of 2.0 μm. The granules were stable when subjected to shear and heat effects as indicated by visco/amylograph response and SEM‐photomicrographs. The transition temperatures (peak and conclusion) and enthalpy of gelatinization of canaryseed starch were higher than those of wheat starch. Canaryseed starch showed major peaks around d‐spacings of 5.9, 5.2, 4.8 and 3.8 Å similar to wheat starch which are characteristics of an A‐type starch. Canaryseed starch was also more susceptible to α‐amylase hydrolysis and more soluble in dimethyl sulfoxide as compared with wheat starch. Based on its ease of extraction and functional properties, canaryseed starch could have potential for food and non‐food uses.
Zur Elektronenstruktur hochsymmetrischer Verbindungen der f‐Elemente. 41 Synthese, Kristall‐, Molekül‐ und Elektronenstruktur eines Bis(cyclohexylisonitril)‐Addukts des Grundkörpers Tris(bis(trimethylsilyl)amido)erbium(III) sowie Elektronenstrukturen ausgewählter Monoaddukte
Authors: Jank Stefan , Guttenberger Clemens , Reddmann Hauke , Hanss Jan , Amberger Hanns‐Dieter
Published: November 2006

Electronic Structures of Highly Symmetrical Compounds of f Elements. 41 Synthesis, Crystal, Molecular and Electronic Structure of a Bis(cyclohexylisonitrile) Adduct Derived from the Tris(bis(trimethylsilyl)amido)erbium(III) Moiety and Electronic Structures of Selected Mono Adducts

The reaction of tris(bis(trimethylsilyl)amido)erbium(III) (Er(btmsa)3) with two equivalents of cyclohexylisonitrile yields the corresponding bis adduct [Er(btmsa)3(CNC6H11)2] (1). Complex 1 crystallizes in the monoclinic space group C2/c with a = 2542.9(11) pm, b = 1208.4(4) pm, c = 1783.0(2) pm, β = 122.39(3)°, V = 4.638(5)·109 pm3, Z = 4 and R = 0.0380. The structure of compound 1 features the five coordinate Er3+ central ion in a nearly exact trigonal bipyramidal environment, with three btmsa ligands in the equatorial and the two cyclohexylisonitrile molecules in the axial positions. On the basis of the absorption spectra of bis adduct 1 and the mono(tetrahydrofuran) as well as the mono(triphenylphosphine oxide) adducts [Er(btmsa)3(THF)] (2) and [Er(btmsa)3(OPPh3)] (3), respectively, the underlying truncated crystal field (CF) splitting patterns of these compounds could be derived, and simulated by fitting the free parameters of a phenomenological Hamiltonian. Reduced r.m.s. deviations of 13.0 cm−1 (42 assignments), 16.0 cm−1 (63 assignments) and 17.5 cm−1 (55 assignments) could be achieved for compounds 1, 2 and 3, respectively. Making use of the phenomenological CF parameters of the fits, the experimentally based non‐relativistic molecular orbital schemes of complexes 1, 2 and 3 were set up, and compared with that of base‐free Er(btmsa)3.
Synthesis and Characterization of [Zn2(4,4′‐bipy)n(AcO)4] – One‐dimensional Chain (n = 1) and One‐dimensional Double‐Chain (n = 2) Coordination Polymers
Authors: Amiri Maryam Ghoreishi , Morsali Ali
Published: November 2006

Two new ZnII(μ‐4,4′‐bipy) coordination polymers with acetate anions, [Zn(4,4′‐bipy)(AcO)2] (1) and [Zn2(4,4′‐bipy)(AcO)4] (2), have been synthesized. The compounds were characterized with elemental analysis, IR‐, 1H NMR‐, 13C NMR spectroscopy and studied by thermal analysis, fluorescence measurements and x‐ray crystallography. The structural studies of compound 1 suggest the structure is a coordination polymer of zinc(II) consisting of linear double chains formed by bridging 4,4′‐bipy ligand and connection of the acetate‐bridged centrosymmetric [Zn2(OAc)2]2+ nodes.
Utilizing Program Evaluation as a Strategy to Promote Community Change: Evaluation of a Comprehensive, Community‐Based, Family Violence Initiative
Authors: Kaufman Joy S. , Crusto Cindy A. , Quan Michael , Ross Ellen , Friedman Stacey R. , O'Rielly Kim , Call Stephanie
Published: November 2006

This paper describes the authors' work in a community that received Federal funding for an integrated system of care to reduce the impact and incidence of exposure to violence for children less than six years of age. The paper includes a review of the conceptual framework that guided the work of the authors and provides a brief overview of the issue of family violence, the impact of this violence on young children, and the Federal response to this issue. In addition, a description of the Initiative and the community in which it was based is provided along with some aspects of the evaluation plan. Finally, the authors discuss how their work with this Initiative depicts an approach to facilitating change within communities.
Semiparametric Thurstonian Models for Recurrent Choices: A Bayesian Analysis
Authors: Ansari Asim , Iyengar Raghuram
Published: November 2006

We develop semiparametric Bayesian Thurstonian models for analyzing repeated choice decisions involving multinomial, multivariate binary or multivariate ordinal data. Our modeling framework has multiple components that together yield considerable flexibility in modeling preference utilities, cross-sectional heterogeneity and parameter-driven dynamics. Each component of our model is specified semiparametrically using Dirichlet process (DP) priors. The utility (latent variable) component of our model allows the alternative-specific utility errors to semiparametrically deviate from a normal distribution. This generates a robust alternative to popular Thurstonian specifications that are based on underlying normally distributed latent variables. Our second component focuses on flexibly modeling cross-sectional heterogeneity. The semiparametric specification allows the heterogeneity distribution to mimic either a finite mixture distribution or a continuous distribution such as the normal, whichever is supported by the data. Thus, special features such as multimodality can be readily incorporated without the need to overtly search for the best heterogeneity specification across a series of models. Finally, we allow for parameter-driven dynamics using a semiparametric state-space approach. This specification adds to the literature on robust Kalman filters. The resulting framework is very general and integrates divergent strands of the literatures on flexible choice models, Bayesian nonparametrics and robust time series specifications. Given this generality, we show how several existing Thurstonian models can be obtained as special forms of our model. We describe Markov chain Monte Carlo methods for the inference of model parameters, report results from two simulation studies and apply the model to consumer choice data from a frequently purchased product category. The results from our simulations and application highlight the benefits of using our semiparametric approach.
The inner nuclear membrane protein Lem2 is critical for normal nuclear envelope morphology
Authors: Ulbert Sebastian , Antonin Wolfram , Platani Melpomeni , Mattaj Iain W.
Published: November 2006

The inner nuclear membrane (INM) of eukaryotic cells is characterized by a unique set of transmembrane proteins which interact with chromatin and/or the nuclear lamina. The number of identified INM proteins is steadily increasing, mainly as a result of proteomic and computational approaches. However, despite a link between mutation of several of these proteins and disease, the function of most transmembrane proteins of the INM remains unknown and depletion of many of these proteins from a variety of systems did not produce an obvious phenotype in the affected cells. Here, we report that depletion of the conserved INM protein Lem2 from human cell lines leads to abnormally shaped nuclei and severely reduces cell survival. We suggest that interactions of Lem2 with lamins or chromatin are critical for maintaining the integrity of the nuclear envelope.
Local Anaesthesia for Carpal Tunnel Decompression: A Comparison of Two Techniques
Authors: PATIL S. , RAMAKRISHNAN M. , STOTHARD J.
Published: November 2006

Carpal tunnel decompression is mostly performed as a day case procedure using local anaesthesia. The local anaesthetic is commonly administered by subcutaneous infiltration alone (the Gale technique) or by infiltration of the local anaesthetic into the carpal tunnel in addition to the subcutaneous infiltration (the Altissimi and Mancini technique). The intention of this study was to compare the efficacy of anaesthesia using these two techniques. Twenty patients with bilateral carpal tunnel syndrome were recruited. One side underwent surgery at a time. Patients were randomised to receive anaesthesia by one technique on the first side and the other on the second. Pain was evaluated using the numerical rating pain scale (0–10). Six patients experienced intraoperative pain with the Gale technique, while none had pain with the Altissimi and Mancini technique ( P = 0.02). The postoperative pain was not significantly different between the two groups, although the patients anaesthetised by the Altissimi and Mancini technique required significantly lower numbers of analgesic tablets.
Cis-urocanic acid, a sunlight-induced immunosuppressive factor, activates immune suppression via the 5-HT2Areceptor
Authors: Walterscheid Jeffrey P. , Nghiem Dat X. , Kazimi Nasser , Nutt Leta K. , McConkey David J. , Norval Mary , Ullrich Stephen E.
Published: November 2006

Exposure to UV radiation induces skin cancer and suppresses the immune response. To induce immune suppression, the electromagnetic energy of UV radiation must be absorbed by an epidermal photoreceptor and converted into a biologically recognizable signal. Two photoreceptors have been recognized: DNA andtrans-urocanic acid (UCA).Trans-UCA is normally found in the outermost layer of skin and isomerizes to the cis isomer upon exposure to UV radiation. Although UCA was identified as a UV photoreceptor years ago, and many have documented its ability to induce immune suppression, its exact mode of action remains elusive. Particularly vexing has been the identity of the molecular pathway by whichcis-UCA mediates immune suppression. Here we provide evidence thatcis-UCA binds to the serotonin [5-hydroxytryptamine (5-HT)] receptor with relatively high affinity (Kd= 4.6 nM). Anti-cis-UCA antibody precipitates radiolabeled 5-HT, and the binding is inhibited by excess 5-HT and/or excesscis-UCA. Similarly, anti-5-HT antibody precipitates radiolabeledcis-UCA, and the binding is inhibited by excess 5-HT or excesscis-UCA. Calcium mobilization was activated when a mouse fibroblast line, stably transfected with the human 5-HT2Areceptor, was treated withcis-UCA.Cis-UCA-induced calcium mobilization was blocked with a selective 5-HT2Areceptor antagonist. UV- andcis-UCA-induced immune suppression was blocked by antiserotonin antibodies or by treating the mice with 5-HT2Areceptor antagonists. Our findings identifycis-UCA as a serotonin receptor ligand and indicate that the immunosuppressive effects ofcis-UCA and UV radiation are mediated by activation of the 5-HT2Areceptor.
An exceptionally fast actomyosin reaction powers insect flight muscle
Authors: Swank Douglas M. , Vishnudas Vivek K. , Maughan David W.
Published: November 2006

Insects, as a group, have been remarkably successful in adapting to a great range of physical and biological environments, in large part because of their ability to fly. The evolution of flight in small insects was accompanied by striking adaptations of the thoracic musculature that enabled very high wing beat frequencies. At the cellular and protein filament level, a stretch activation mechanism evolved that allowed high-oscillatory work to be achieved at very high frequencies as contraction and nerve stimulus became asynchronous. At the molecular level, critical adaptations occurred within the motor protein myosin II, because its elementary interactions with actin set the speed of sarcomere contraction. Here, we show that the key myosin enzymatic adaptations required for powering the very fast flight muscles in the fruit fly
Drosophila melanogaster
include the highest measured detachment rate of myosin from actin (forward rate constant, 3,698 s
−1
), an exceptionally weak affinity of MgATP for myosin (association constant, 0.2 mM
−1
), and a unique rate-limiting step in the cross-bridge cycle at the point of inorganic phosphate release. The latter adaptations are constraints imposed by the overriding requirement for exceptionally fast release of the hydrolytic product MgADP. Otherwise, as in
Drosophila
embryonic muscle and other slow muscle types, a step associated with MgADP release limits muscle contraction speed by delaying the detachment of myosin from actin.
Structural basis for protein–protein interactions in the 14-3-3 protein family
Authors: Yang Xiaowen , Lee Wen Hwa , Sobott Frank , Papagrigoriou Evangelos , Robinson Carol V. , Grossmann J. Günter , Sundström Michael , Doyle Declan A. , Elkins Jonathan M.
Published: November 2006

The seven members of the human 14-3-3 protein family regulate a diverse range of cell signaling pathways by formation of protein–protein complexes with signaling proteins that contain phosphorylated Ser/Thr residues within specific sequence motifs. Previously, crystal structures of three 14-3-3 isoforms (zeta, sigma, and tau) have been reported, with structural data for two isoforms deposited in the Protein Data Bank (zeta and sigma). In this study, we provide structural detail for five 14-3-3 isoforms bound to ligands, providing structural coverage for all isoforms of a human protein family. A comparative structural analysis of the seven 14-3-3 proteins revealed specificity determinants for binding of phosphopeptides in a specific orientation, target domain interaction surfaces and flexible adaptation of 14-3-3 proteins through domain movements. Specifically, the structures of the beta isoform in its apo and peptide bound forms showed that its binding site can exhibit structural flexibility to facilitate binding of its protein and peptide partners. In addition, the complex of 14-3-3 beta with the exoenzyme S peptide displayed a secondary structural element in the 14-3-3 peptide binding groove. These results show that the 14-3-3 proteins are adaptable structures in which internal flexibility is likely to facilitate recognition and binding of their interaction partners.
Discovery and
in vitro
biosynthesis of haloduracin, a two-component lantibiotic
Authors: McClerren Amanda L. , Cooper Lisa E. , Quan Chao , Thomas Paul M. , Kelleher Neil L. , van der Donk Wilfred A.
Published: November 2006

Lantibiotics are ribosomally synthesized peptides that undergo posttranslational modifications to their mature, antimicrobial form. They are characterized by the unique amino acids lanthionine and methyllanthionine, introduced by means of dehydration of Ser/Thr residues followed by reaction of the resulting dehydro amino acids with cysteines to form thioether linkages. Two-component lantibiotics use two peptides that are each posttranslationally modified to yield two functionally distinct products that act in synergy to provide bactericidal activity. By using genetic data instead of isolation, a two-component lantibiotic, haloduracin, was identified in the genome of the Gram-positive alkaliphilic bacterium
Bacillus halodurans
C-125. We show that heterologously expressed and purified precursor peptides HalA1 and HalA2 are processed by the purified modification enzymes HalM1 and HalM2 in an
in vitro
reconstitution of the biosynthesis of a two-component lantibiotic. The activity of each HalM enzyme is substrate-specific, and the assay products exhibit antimicrobial activity after removal of their leader sequences at an engineered Factor Xa cleavage site, indicating that correct thioether formation has occurred. Haloduracin's biological activity depends on the presence of both modified peptides. The structures of the two mature haloduracin peptides Halα and Halβ were investigated, indicating that they have similarities as well as some distinct differences compared with other two-component lantibiotics.
Recent history of artificial outcrossing facilitates whole-genome association mapping in elite inbred crop varieties
Authors: Rostoks Nils , Ramsay Luke , MacKenzie Katrin , Cardle Linda , Bhat Prasanna R. , Roose Mikeal L. , Svensson Jan T. , Stein Nils , Varshney Rajeev K. , Marshall David F. , Graner Andreas , Close Timothy J. , Waugh Robbie
Published: November 2006

Genomewide association studies depend on the extent of linkage disequilibrium (LD), the number and distribution of markers, and the underlying structure in populations under study. Outbreeding species generally exhibit limited LD, and consequently, a very large number of markers are required for effective whole-genome association genetic scans. In contrast, several of the world's major food crops are self-fertilizing inbreeding species with narrow genetic bases and theoretically extensive LD. Together these are predicted to result in a combination of low resolution and a high frequency of spurious associations in LD-based studies. However, inbred elite plant varieties represent a unique human-induced pseudooutbreeding population that has been subjected to strong selection for advantageous alleles. By assaying 1,524 genomewide SNPs we demonstrate that, after accounting for population substructure, the level of LD exhibited in elite northwest European barley, a typical inbred cereal crop, can be effectively exploited to map traits by using whole-genome association scans with several hundred to thousands of biallelic SNPs.
Two modes of microtubule sliding driven by cytoplasmic dynein
Authors: Shima Tomohiro , Kon Takahide , Imamula Kenji , Ohkura Reiko , Sutoh Kazuo
Published: November 2006

Dynein is a huge multisubunit microtubule (MT)-based motor, whose motor domain resides in the heavy chain. The heavy chain comprises a ring of six AAA (ATPases associated with diverse cellular activities) modules with two slender protruding domains, the tail and stalk. It has been proposed that during the ATP hydrolysis cycle, this tail domain swings against the AAA ring as a lever arm to generate the power stroke. However, there is currently no direct evidence to support the model that the tail swing is tightly linked to dynein motility. To address the question of whether the power stroke of the tail drives MT sliding, we devised an
in vitro
motility assay using genetically biotinylated cytoplasmic dyneins anchored on a glass surface in the desired orientation with a biotin–streptavidin linkage. Assays on the dyneins with the site-directed biotin tag at eight different locations provided evidence that robust MT sliding is driven by the power stroke of the tail. Furthermore, the assays revealed slow MT sliding independent of dynein orientation on the glass surface, which is mechanically distinct from the sliding driven by the power stroke of the tail.
Small dsRNAs induce transcriptional activation in human cells
Authors: Li Long-Cheng , Okino Steven T. , Zhao Hong , Pookot Deepa , Place Robert F. , Urakami Shinji , Enokida Hideki , Dahiya Rajvir
Published: November 2006

Recent studies have shown that small noncoding RNAs, such as microRNAs and siRNAs, regulate gene expression at multiple levels including chromatin architecture, transcription, RNA editing, RNA stability, and translation. Each form of RNA-dependent regulation has been generally found to silence homologous sequences and collectively called RNAi. To further study the regulatory role of small RNAs at the transcriptional level, we designed and synthesized 21-nt dsRNAs targeting selected promoter regions of human genes E-cadherin, p21
WAF1/CIP1
(p21), and VEGF. Surprisingly, transfection of these dsRNAs into human cell lines caused long-lasting and sequence-specific induction of targeted genes. dsRNA mutation studies reveal that the 5′ end of the antisense strand, or “seed” sequence, is critical for activity. Mechanistically, the dsRNA-induced gene activation requires the Argonaute 2 (Ago2) protein and is associated with a loss of lysine-9 methylation on histone 3 at dsRNA-target sites. In conclusion, we have identified several dsRNAs that activate gene expression by targeting noncoding regulatory regions in gene promoters. These findings reveal a more diverse role for small RNA molecules in the regulation of gene expression than previously recognized and identify a potential therapeutic use for dsRNA in targeted gene activation.
Blood flow velocity effects and role of activation delay time on growth and form of platelet thrombi
Authors: Pivkin Igor V. , Richardson Peter D. , Karniadakis George
Published: November 2006

Mural thrombi are composed dominantly of platelets and develop under a blood flow. Portions can break off and are carried in the blood flow as emboli. Thrombus growth rates are affected by the velocity of the blood flow, but they do not simply increase with it, they exhibit a maximum, with subsequent decrease. Whereas this variation indicates an interaction of biochemical and physical processes, studies have concentrated widely on understanding only the biochemical processes. Here we show results of simulation of thrombus formation in 3D flows by accounting for the movements of individual platelets. Each platelet follows prescribed rules for interactions while the local flow around the thrombus continuously adjusts to the growing structure of the thrombus, also when embolization occurs. With an activation delay time assigned to each platelet we demonstrate the dependence of thrombus growth rate on blood velocity as found experimentally by Begent and Born [Begent N, Born GV (1970)
Nature
227:926–930]. With activated platelets having mutual tensile action sustainable up to a prescribed distance we achieve thrombus growth faster than with shorter maximum distances that make a thrombus less porous; when the prescribed maximum distance is large enough the thrombus shape is not like a “hill” but like a “carpet.” We find that thrombus growth rate is enhanced by modest pulsatility but less so when pulsations are amplified in part because of more embolization.
Direct interaction between DNMT1 and G9a coordinates DNA and histone methylation during replication
Authors: Estève Pierre-Olivier , Chin Hang Gyeong , Smallwood Andrea , Feehery George R. , Gangisetty Omkaram , Karpf Adam R. , Carey Michael F. , Pradhan Sriharsa
Published: November 2006

Chromatin methylation is necessary for stable repression of gene expression during mammalian development. During cell division, DNMT1 maintains the DNA methylation pattern of the newly synthesized daughter strand, while G9a methylates H3K9. Here, DNMT1 is shown to directly bind G9a both in vivo and in vitro and to colocalize in the nucleus during DNA replication. The complex of DNMT1 and G9a colocalizes with dimethylated H3K9 (H3K9me2) at replication foci. Similarly, another H3K9 histone methyltransferase, SUV39H1, colocalizes with DNMT1 on heterochromatic regions of the nucleoli exclusively before cell division. Both DNMT1 and G9a are loaded onto the chromatin simultaneously in a ternary complex with loading factor PCNA during chromatin replication. Small interfering RNA (siRNA) knockdown of DNMT1 impairs DNA methylation, G9a loading, and H3K9 methylation on chromatin and rDNA repeats, confirming DNMT1 as the primary loading factor. Additionally, the complex of DNMT1 and G9a led to enhanced DNA and histone methylation of in vitro assembled chromatin substrates. Thus, direct cooperation between DNMT1 and G9a provides a mechanism of coordinated DNA and H3K9 methylation during cell division.
Endotoxic shock inAUF1knockout mice mediated by failure to degrade proinflammatory cytokine mRNAs
Authors: Lu Jin-Yu , Sadri Navid , Schneider Robert J.
Published: November 2006

Excessive production of proinflammatory cytokines, particularly tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β), plays a critical role in septic shock induced by bacterial endotoxin (endotoxemia). Precise control of cytokine expression depends on rapid degradation of cytokine mRNAs, mediated by an AU-rich element (ARE) in the 3′ noncoding region and by interacting ARE-binding proteins, which control the systemic inflammatory response. To understand the function of the ARE-binding protein AUF1, we developed anAUF1knockout mouse. We show that AUF1 normally functions to protect against the lethal progression of endotoxemia. Upon endotoxin challenge,AUF1knockout mice display symptoms of severe endotoxic shock, including vascular hemorrhage, intravascular coagulation, and high mortality, resulting from overproduction of TNFα and IL-1β. Overexpression of these two cytokines is specific, and shown to result from an inability to rapidly degrade these mRNAs in macrophages following induction. Neutralizing antibodies to TNFα and IL-1β protectAUF1knockout mice against lethal endotoxic shock. These and other data describe a novel post-transcriptional mechanism whereby AUF1 acts as a crucial attenuator of the inflammatory response, promoting the rapid decay of selective proinflammatory cytokine mRNAs following endotoxin activation. Defects in the AUF1 post-transcriptionally controlled pathway may be involved in human inflammatory disease.
DTL/CDT2 is essential for both CDT1 regulation and the early G2/M checkpoint
Authors: Sansam Christopher L. , Shepard Jennifer L. , Lai Kevin , Ianari Alessandra , Danielian Paul S. , Amsterdam Adam , Hopkins Nancy , Lees Jacqueline A.
Published: November 2006

Checkpoint genes maintain genomic stability by arresting cells after DNA damage. Many of these genes also control cell cycle events in unperturbed cells. By conducting a screen for checkpoint genes in zebrafish, we found that dtl/cdt2 is an essential component of the early, radiation-induced G2/M checkpoint. We subsequently found that dtl/cdt2 is required for normal cell cycle control, primarily to prevent rereplication. Both the checkpoint and replication roles are conserved in human DTL. Our data indicate that the rereplication reflects a requirement for DTL in regulating CDT1, a protein required for prereplication complex formation. CDT1 is degraded in S phase to prevent rereplication, and following DNA damage to prevent origin firing. We show that DTL associates with the CUL4–DDB1 E3 ubiquitin ligase and is required for CDT1 down-regulation in unperturbed cells and following DNA damage. The cell cycle defects of Dtl-deficient zebrafish are suppressed by reducing Cdt1 levels. In contrast, the early G2/M checkpoint defect appears to be Cdt1-independent. Thus, DTL promotes genomic stability through two distinct mechanisms. First, it is an essential component of the CUL4–DDB1 complex that controls CDT1 levels, thereby preventing rereplication. Second, it is required for the early G2/M checkpoint.
Genome-Wide Analysis of the Arabidopsis Leaf Transcriptome Reveals Interaction of Phosphate and Sugar Metabolism
Authors: Müller Renate , Morant Marc , Jarmer Hanne , Nilsson Lena , Nielsen Tom Hamborg
Published: November 2006

Global gene expression was analyzed in Arabidopsis (Arabidopsis thaliana) by microarrays comprising 21,500 genes. Leaf segments derived from phosphorus (P)-starved and P-replenished plants were incubated with or without sucrose (Suc) to obtain tissues with contrasting combinations of P and carbohydrate levels. Transcript profiling revealed the influence of the two factors individually and the interactions between P- and sugar-dependent gene regulation. A large number of gene transcripts changed more than 2-fold: In response to P starvation, 171 genes were induced and 16 repressed, whereas Suc incubation resulted in 337 induced and 307 repressed genes. A number of new candidate genes involved in P acquisition were discovered. In addition, several putative transcription factors and signaling proteins of P sensing were disclosed. Several genes previously identified to be sugar responsive were also regulated by P starvation and known P-responsive genes were sugar inducible. Nearly 150 genes were synergistically or antagonistically regulated by the two factors. These genes exhibit more prominent or contrasting regulation in response to Suc and P in combination than expected from the effect of the two factors individually. The genes exhibiting interactions form three main clusters with different response patterns and functionality of genes. One cluster (cluster 1) most likely represents a regulatory program to support increased growth and development when both P and carbohydrates are ample. Another cluster (cluster 3) represents genes induced to alleviate P starvation and these are further induced by carbohydrate accumulation. Thus, interactions between P and Suc reveal two different signaling programs and novel interactions in gene regulation in response to environmental factors. cis-Regulatory elements were analyzed for each factor and for interaction clusters. PHR1 binding sites were more frequent in promoters of P-regulated genes as compared to the entire Arabidopsis genome, and E2F and PHR1 binding sites were more frequent in interaction clusters 1 and 3, respectively.
Time course of the renal functional response to partial nephrectomy: measurements in conscious rats
Authors: Chamberlain R. M. , Shirley D. G.
Published: November 2006

Previous investigations into the functional responses of the surviving nephrons following reductions in renal mass have been performed largely in anaesthetized animals and have taken little account of how the compensatory changes develop with time. The present study has assessed a method for determining glomerular filtration rate (GFR) in unrestrained, uncatheterized, conscious rats (plasma disappearance of 99mTc‐diethylenetriamene pentaacetic acid (DTPA)) and has used this method to document the time course of the changes in GFR over a 32 day period following uninephrectomy or 5/6 nephrectomy. Concurrent measurements of excretion rates and of the clearance of lithium (the latter being an index of end‐proximal fluid delivery) provided information on changes in overall tubular function and segmental reabsorption. After uninephrectomy, the GFR of the remaining kidney (compared with that of a single kidney of sham‐operated animals) increased maximally (by ∼50%) within 8 days; after 5/6 nephrectomy, the increase in the GFR of the remnant kidney was maximal (at ∼300%) within 16 days. Overall excretion rates of sodium and potassium were well maintained in partially nephrectomized animals throughout the period of study, while the excretion of water increased (by ∼30% after uninephrectomy and by ∼120% after 5/6 nephrectomy), partly as a result of the compensatory increases in GFR but mainly as a consequence of moderate (after uninephrectomy) or marked (after 5/6 nephrectomy) reductions in fractional reabsorption. During the early period after 5/6 nephrectomy, potassium excretion sometimes exceeded the filtered load, indicating net secretion. Lithium clearance data indicated that the changes in tubular function after 5/6 nephrectomy include a reduction in fractional reabsorption in the proximal tubule, whereas after uninephrectomy any such effect on the proximal tubule is minor and transient.
Increased sympathetic activity in rats submitted to chronic intermittent hypoxia
Authors: Zoccal Daniel B. , Bonagamba Leni G. H. , Oliveira Fabíola R. T. , Antunes‐Rodrigues José , Machado Benedito H.
Published: November 2006

Long‐term exposure to intermittent hypoxia may lead to important cardiovascular dysfunctions, such as hypertension. Rodent models of chronic intermittent hypoxia (CIH) have been used to study the mechanisms underlying the increase in mean arterial pressure (MAP) observed after exposure to CIH. Several studies suggest that the hypertension of rats submitted to CIH is associated with an increase in sympathetic activity. However, there are no studies documenting the direct measurement of sympathetic activity in conscious freely moving rats exposed to CIH. Therefore, the present study aimed to evaluate whether or not the increase of MAP in rats exposed to CIH is associated with an increase in sympathetic activity. To reach this goal, we analysed the effect of ganglionic blockade on baseline MAP as well as the plasma levels of catecholamines. Rats submitted to CIH (fractional inspired O2 of 6%, for 40 s in every 9 min, 8 h day−1) for 35 days (n= 31) exhibited a significant increase in MAP compared with control rats (n= 28) maintained under normoxia (112 ± 2 versus 103 ± 1 mmHg, P= 0.0003). The injection of the ganglionic blocker hexamethonium resulted in a similar fall in MAP in CIH and control groups (−46 ± 2 versus−41 ± 3 mmHg). However, hexamethonium after previous antagonism of the angiotensin II type 1 (AT1) receptors with losartan produced a larger decrease in MAP in the CIH than in the control group (−58 ± 2 versus−50 ± 2 mmHg, P= 0.0165). The injection of losartan itself produced no major changes in the baseline MAP in both groups. The measurement of plasma catecholamines showed an increase in plasma noradrenaline (10.12 ± 0.90 versus 4.74 ± 0.32 ng ml−1, P= 0.0042) in rats exposed to CIH compared with control rats. These data provide strong evidence to support the concept that rats submitted to CIH exhibit an increase in sympathetic activity, which seems to be determinant in the maintenance of hypertension in this experimental model.