Natural Substrates (natural + substrate)

Distribution by Scientific Domains


Selected Abstracts


Kinetic analysis of hyaluronidase activity using a bioactive MRI contrast agent

CONTRAST MEDIA & MOLECULAR IMAGING, Issue 3 2006
Liora Shiftan
Abstract One of the attractions of molecular imaging using ,smart' bioactive contrast agents is the ability to provide non-invasive data on the spatial and temporal changes in the distribution and expression patterns of specific enzymes. The tools developed for that aim could potentially also be developed for functional imaging of enzyme activity itself, through quantitative analysis of the rapid dynamics of enzymatic conversion of these contrast agents. High molecular weight hyaluronan, the natural substrate of hyaluronidase, is a major antiangiogenic constituent of the extracellular matrix. Degradation by hyaluronidase yields low molecular weight fragments, which are proangiogenic. A novel contrast material, HA-GdDTPA-beads, was designed to provide a substrate analog of hyaluronidase in which relaxivity changes are induced by enzymatic degradation. We show here a first-order kinetic analysis of the time-dependent increase in R2 as a result of hyaluronidase activity. The changes in R2 and the measured relaxivity of intact HA-GdDTPA-beads (r2B) and HA-GdDTPA fragments (r2D) were utilized for derivation of the temporal drop in concentration of GdDTPA in HA-GdDTPA-beads as the consequence of the release of HA-GdDTPA fragments. The rate of dissociation of HA-GdDTPA from the beads showed typical bell-shaped temperature dependence between 7 and 36 °C with peak activity at 25 °C. The tools developed here for quantitative dynamic analysis of hyaluronidase activity by MRI would allow the use of activation of HA-GdDTPA-beads for the determination of the role of hyaluronidase in altering the angiogenic microenvironment of tumor micro metastases. Copyright © 2006 John Wiley & Sons, Ltd. [source]


Salt-Sensitive Hypertension Resulting From Nitric Oxide Synthase Inhibition is Associated with Loss of Regulation of Angiotensin II in the Rat

EXPERIMENTAL PHYSIOLOGY, Issue 1 2002
G. Hodge
In the Dahl salt-sensitive hypertensive rat, a diet containing L-arginine, the natural substrate for nitric oxide synthase, abrogates the hypertension. We postulated that nitric oxide synthase inhibition might induce a salt-sensitive form of hypertension and that this salt sensitivity might be linked to a loss of the regulatory effect of sodium ingestion on angiotensin II (Ang II) and angiotensinogen. Male Wistar-Kyoto rats were randomised to a diet containing 0.008%, 2.2% or 4.4% sodium chloride and to treatment with the NO synthase inhibitor L-NAME (10 mg kg,1 day,1) in the drinking water, or drinking water alone (Controls) for 4 weeks. Blood pressure was measured by tail cuff plethysmography twice weekly. After 4 weeks, the rats were anaesthetised and truncal blood collected for determination of angiotensinogen, renin, angiotensin I (Ang I), Ang II and aldosterone concentrations as well as angiotensin-converting enzyme (ACE) activity. Systolic blood pressure increased with increasing dietary sodium intake in the L-NAME-treated rats (P < 0.05). Plasma renin and aldosterone concentrations decreased with increasing dietary sodium intake in both Control and L-NAME-treated rats. Ang I and ACE activity were unchanged by increasing dietary sodium intake. In contrast, the plasma concentration of Ang II and angiotensinogen increased with increasing dietary sodium (P < 0.05 and P < 0.005, respectively). Treatment with the Ang II receptor blocker, losartan, reversed the blood pressure increase. We conclude that treatment with L-NAME induces an increase in blood pressure that is at least in part salt sensitive. Further, the salt-sensitive component appears to be Ang II-dependent, as it was associated with increasing plasma Ang II levels and could be reversed by treatment with an Ang II receptor antagonist. [source]


First evidence of catalytic mediation by phenolic compounds in the laccase-induced oxidation of lignin models

FEBS JOURNAL, Issue 17 2003
Francesca D'Acunzo
The sulfonephthalein indicator, phenol red, exhibits an unusually slow rate of oxidation by laccase from Poliporus pinsitus, in spite of the fact that it is a phenol and therefore a natural substrate for this phenoloxidase enzyme. Nevertheless, after prolonged exposure to laccase (24 h) phenol red is oxidized by more than 90%. We found that phenol red, which can be oxidatively converted into a resonance-stabilized phenoxy radical, performs as a mediator in the laccase-catalyzed oxidation of a nonphenolic substrate (4-methoxybenzyl alcohol) and also of a hindered phenol (2,4,6-tri- tert -butylphenol). In particular, phenol red was found to be at least 10 times more efficient than 3-hydroxyanthranilate (a reported natural phenolic mediator of laccase) in the oxidation of 4-methoxybenzyl alcohol. Other phenols, which do not bear structural analogies to phenol red, underwent rapid degradation and did not perform as laccase mediators. On the other hand, several variously substituted sulfonephthaleins, of different pK2 values, mediated the laccase catalysis, the most efficient being dichlorophenol red, which has the lowest pK2 of the series. The mediating efficiency of phenol red and dichlorophenol red was found to be pH dependent, as was their oxidation Ep value (determined by cyclic voltammetry). We argue that the relative abundance of the phenoxy anion, which is easier to oxidize than the protonated phenol, may be one of the factors determining the efficiency of a phenolic mediator, together with its ability to form relatively stable oxidized intermediates that react with the desired substrate before being depleted in undesired routes. [source]


Sensory ecology of prey rustling sounds: acoustical features and their classification by wild Grey Mouse Lemurs

FUNCTIONAL ECOLOGY, Issue 1 2007
H. R. GOERLITZ
Summary 1Predatory mammals and birds from several phylogenetic lineages use prey rustling sounds to detect and locate prey. However, it is not known whether these rustling sounds convey information about the prey, such as its size or profitability, and whether predators use them to classify prey accordingly. 2We recorded rustling sounds of insects in Madagascar walking on natural substrate and show a clear correlation between insect mass and several acoustic parameters. 3In subsequent behavioural experiments in the field, we determined whether nocturnal animals, when foraging for insects, evaluate these parameters to classify their prey. We used field-experienced Grey Mouse Lemurs Microcebus murinus in short-term captivity. Mouse Lemurs are generally regarded as a good model for the most ancestral primate condition. They use multimodal sensorial information to find food (mainly fruit, gum, insect secretions and arthropods) in nightly forest. Acoustic cues play a role in detection of insect prey. 4When presented with two simultaneous playbacks of rustling sounds, lemurs spontaneously chose the one higher above their hearing threshold, i.e. they used the rustling sound's amplitude for classification. We were not able, despite attempts in a reinforced paradigm, to persuade lemurs to use cues other than amplitude, e.g. frequency cues, for prey discrimination. 5Our data suggests that Mouse Lemurs, when foraging for insects, use the mass,amplitude correlation of prey-generated rustling sounds to evaluate the average mass of insects and to guide their foraging decisions. [source]


Structural Diversification of Macrolactones by Substrate-Flexible Cytochrome P450 Monooxygenases

ADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 10 2005
Kil Lee
Abstract The substrate flexibilities of several cytochrome P450 monooxygenases involved in macrolide biosynthesis were investigated to test their potential for the generation of novel macrolides. PikC hydroxylase in the pikromycin producer Streptomyces venezuelae accepted oleandomycin as an alternative substrate and introduced a hydroxy group at the C-4 position, which is different from the intrinsic C-12 hydroxylation position in the natural substrate. This is the first report of C-4 hydroxylation activity of cytochrome P450 monooxygenase involved in the biosynthesis of 14-membered macrolides. EryF hydroxylase from the erythromycin biosynthetic pathway of Saccharopolyspora erythraea and OleP oxidase from the oleandomycin biosynthetic pathway of Streptomyces antibioticus also showed a certain degree of plasticity towards alternative substrates. In particular, EryF and OleP were found to oxidize a 12-membered macrolactone as an alternative substrate. These results demonstrate the potential usefulness of these enzymes to diversify macrolactones by post-PKS oxidations. [source]


Bacterial synthesis of poly(hydroxybutyrate- co-hydroxyvalerate) using carbohydrate-rich mahua (Madhuca sp.) flowers

JOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2007
P.K. Anil Kumar
Abstract Aims:, The objective of the present work was to utilize an unrefined natural substrate namely mahua (Madhuca sp.) flowers, as a carbon source for the production of bacterial polyhydroxyalkanoate (PHA) copolymer by Bacillus sp-256. Methods and Results:, In the present work, three bacterial strains were tested for PHA production on mahua flower extract (to impart 20 g l,1 sugar) amongst which, Bacillus sp-256 produced higher concentration of PHA in its biomass (51%) compared with Rhizobium meliloti (31%) or Sphingomonas sp (22%). Biosynthesis of poly(hydroxybutyrate-co-hydroxyvalerate) , P(HB-co-HV) , of 90 : 10 mol% by Bacillus sp-256 was observed by gas chromatographic analysis of the polymer. Major component of the flower is sugars (57% on dry weight basis) and additionally it also contains proteins, vitamins, organic acids and essential oils. The bacterium utilized malic acid present in the substrate as a co-carbon source for the copolymer production. The flowers could be used in the form of aqueous extract or as whole flowers. PHA content of biomass (%) and yield (g l,1) in a 3·0-l stirred tank fermentor after 30 h of fermentation under constant pH (7) and dissolved oxygen content (40%) were 54% and 2·7 g l,1, respectively. Corresponding yields for control fermentation with sucrose as carbon source were 52% and 2·5 g l,1. The polymer was characterized by proton NMR. Conclusions:, Utilization of mahua flowers, a natural substrate for bacterial fermentation aimed at PHA production, had additional advantage, as the sugars and organic acids present in the flowers were metabolized by Bacillus sp-256 to synthesize P(HB-co-HV) copolymer. Significance and Impact of the Study:, Literature reports on utilization of suitable cheaper natural substrate for PHA copolymer production is scanty. Mahua flowers used in the present experiment is a cheaper carbon substrate compared with several commercial substrates and it is rich in main carbon as well as co-carbon sources that can be utilized by bacteria for PHA copolymer production. [source]


Effect of floating nest platforms on the breeding performance of Black Terns

JOURNAL OF FIELD ORNITHOLOGY, Issue 2 2006
David A. Shealer
ABSTRACT In 2003 and 2004, we placed 41 floating nest platforms on Grassy Lake in southeastern Wisconsin (USA) to test the hypothesis that reproductive success of Black Terns (Chlidonias niger) is limited by the quality of suitable nesting habitat. Extreme differences in water levels between these 2 yr provided a natural experiment to evaluate the effectiveness of the nest platforms during a drought year (2003) when natural nesting substrate was abundant, and a flood year (2004) when natural substrate was limited during the peak nesting period. Terns nested on 27 of 41 (66%) of the platforms in 2003 and 26 of 41 (63%) in 2004. No difference in the occupancy rate of platforms and natural nests was evident in 2003, but the pattern of clutch initiations early in the season in 2004 indicated that platforms were preferred over natural substrates. In 2003, nest survival rates did not differ between nests placed on platforms and those placed on natural substrates, but platform nests had significantly higher hatching success and nest survival rates in 2004. Both the Kaplan-Meier and Apparent Nest Success methods of calculating nest survival provided similar estimates. In both years, eggs laid on platforms were significantly larger than those laid on natural substrates, suggesting that platforms were occupied by high-quality birds. Our study indicates that floating nest platforms can be an effective management tool to enhance nesting habitat for Black Terns and other aquatic birds that construct floating nests, primarily because platforms provide nest sites when natural sites are not available due to flooding. Nest platforms also may be useful for addressing questions concerning habitat selection and parental quality. SINOPSIS De 2003-2004 colocamos 41 plataformas flotantes para poner aprueba la hipótesis que el éxito reproductivo de la gaviota Chlidonias níger, esta limitado por la calidad y lo apropiado del hábitat de anidamiento. El estudio se llevó a cabo en el Grassy Lake, Wisconsin. Extremos (bajos y altos) en los niveles de agua, causados por sequía (2203) y fuertes lluvias (2004) proveyeron el escenario adecuado para evaluar la efectividad de las plataformas. Las gaviotas anidaron en 27 (66%) de las 41 plataformas en el 2003 y en 26 (63%) de las 41 en el 2004. No se encontraron diferencias en la tasa de uso de las plataformas y áreas naturales en el 2003, pero el inicio de las camadas temprano en la temporada durante el 2004, dieron a indicar que las plataformas fueron preferidas a los lugares naturales. Durante el 2003, no hubo diferencia en la tasa de supervivencia de nidos en plataformas y o en áreas naturales. Sin embargo, durante el 2004 el éxito de eclosionamiento, de los nidos y la supervivencia fue mas alto en las plataformas. Tanto el método Kaplan-Meier como el "Aparente Éxito de Anidamiento", utilizados para calcular la supervivencia de los nidos, ofrecieron resultados similares. En ambos años el tamaño de los huevos fue mayor en las plataformas que en áreas naturales, lo que implica que las primeras fueron utilizadas por aves sumamente vigorosas o de alta calidad. El estudio indica que las plataformas flotantes pueden ser una herramienta de manejo adecuada para mejorar el hábitat de anidamiento de la gaviota estudiada, al igual que otras aves que construyen nidos flotantes. Las plataformas proveen de lugares de anidamientos cuando no hay disponible lugares naturales debido a inundaciones. Las plataformas de anidamiento muy bien pudieran proveer de información útil con respecto a la selección de hábitat y la calidad del cuidado parental. [source]


The active site of hydroxynitrile lyase from Prunus amygdalus: Modeling studies provide new insights into the mechanism of cyanogenesis

PROTEIN SCIENCE, Issue 2 2002
Ingrid Dreveny
Abstract The FAD-dependent hydroxynitrile lyase from almond (Prunus amygdalus, PaHNL) catalyzes the cleavage of R -mandelonitrile into benzaldehyde and hydrocyanic acid. Catalysis of the reverse reaction,the enantiospecific formation of ,-hydroxynitriles,is now widely utilized in organic syntheses as one of the few industrially relevant examples of enzyme-mediated C,C bond formation. Starting from the recently determined X-ray crystal structure, systematic docking calculations with the natural substrate were used to locate the active site of the enzyme and to identify amino acid residues involved in substrate binding and catalysis. Analysis of the modeled substrate complexes supports an enzymatic mechanism that includes the flavin cofactor as a mere "spectator" of the reaction and relies on general acid/base catalysis by the conserved His-497. Stabilization of the negative charge of the cyanide ion is accomplished by a pronounced positive electrostatic potential at the binding site. PaHNL activity requires the FAD cofactor to be bound in its oxidized form, and calculations of the pKa of enzyme-bound HCN showed that the observed inactivation upon cofactor reduction is largely caused by the reversal of the electrostatic potential within the active site. The suggested mechanism closely resembles the one proposed for the FAD-independent, and structurally unrelated HNL from Hevea brasiliensis. Although the actual amino acid residues involved in the catalytic cycle are completely different in the two enzymes, a common motif for the mechanism of cyanogenesis (general acid/base catalysis plus electrostatic stabilization of the cyanide ion) becomes evident. [source]


6,-Methyl- B -norandrostenedione

ACTA CRYSTALLOGRAPHICA SECTION C, Issue 4 2010
L. C. R. Andrade
The title compound, C19H26O2, a B -norandrogen with a 6,-methyl group, is a recently identified and experimentally tested potent new aromatase inhibitor. It shares structural and physicochemical similarities both with the natural substrate of the enzyme, androstenedione, and with exemestane, another potent aromatase inhibitor having a 6-methylidene group. X-ray diffraction results indicate that the B -nor molecule and exemestane have nearly the same oxygen,oxygen and methyl,methyl separations, though they have distinct configurations of the hydrophobic groups at the 6-position. These structural comparisons allow correlations to be inferred between the active site geometry of the molecules and the aromatase inhibition power of the studied compound. [source]


Near-atomic resolution structures of urate oxidase complexed with its substrate and analogues: the protonation state of the ligand

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2010
Laure Gabison
Urate oxidase (uricase; EC 1.7.3.3; UOX) from Aspergillus flavus catalyzes the oxidation of uric acid in the presence of molecular oxygen to 5-hydroxyisourate in the degradation cascade of purines; intriguingly, catalysis proceeds using neither a metal ion (Fe, Cu etc.) nor a redox cofactor. UOX is a tetrameric enzyme with four active sites located at the interface of two subunits; its structure was refined at atomic resolution (1,Å) using new crystal data in the presence of xanthine and at near-atomic resolution (1.3,1.7,Å) in complexes with the natural substrate (urate) and two inhibitors: 8-nitroxanthine and 8-thiouric acid. Three new features of the structural and mechanistic behaviour of the enzyme were addressed. Firstly, the high resolution of the UOX,xanthine structure allowed the solution of an old structural problem at a contact zone within the tetramer; secondly, the protonation state of the substrate was determined from both a halochromic inhibitor complex (UOX,8-nitroxanthine) and from the H-atom distribution in the active site, using the structures of the UOX,xanthine and the UOX,uric acid complexes; and thirdly, it was possible to extend the general base system, characterized by the conserved catalytic triad Thr,Lys,His, to a large water network that is able to buffer and shuttle protons back and forth between the substrate and the peroxo hole along the reaction pathway. [source]


Structures of potent selective peptide mimetics bound to carboxypeptidase B

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2008
Marc Adler
This article reports the crystal structures of inhibitors of the functional form of thrombin-activatable fibrinolysis inhibitor (TAFIa). In vivo experiments indicate that selective inhibitors of TAFIa would be useful in the treatment of heart attacks. Since TAFIa rapidly degrades in solution, the homologous protein porcine pancreatic carboxypeptidase B (pp-CpB) was used in these crystallography studies. Both TAFIa and pp-CpB are zinc-based exopeptidases that are specific for basic residues. The final development candidate, BX 528, is a potent inhibitor of TAFIa (2,nM) and has almost no measurable effect on the major selectivity target, carboxypeptidase N. BX 528 was designed to mimic the tripeptide Phe-Val-Lys. A sulfonamide replaces the Phe-Val amide bond and a phosphinate connects the Val and Lys groups. The phosphinate also chelates the active-site zinc. The electrostatic interactions with the protein mimic those of the natural substrate. The primary amine in BX 528 forms a salt bridge to Asp255 at the base of the S1, pocket. The carboxylic acid interacts with Arg145 and the sulfonamide is hydrogen bonded to Arg71. Isopropyl and phenyl groups replace the side chains of Val and Phe, respectively. A series of structures are presented here that illustrate the evolution of BX 528 from thiol-based inhibitors that mimic a free C-terminal arginine. The first step in development was the replacement of the thiol with a phosphinate. This caused a precipitous drop in binding affinity. Potency was reclaimed by extending the inhibitors into the downstream binding sites for the natural substrate. [source]


Extracellular matrix alters the relationship between tritiated thymidine incorporation and proliferation of MC3T3-E1 cells during osteogenesis in vitro

CELL PROLIFERATION, Issue 1 2002
W. J. Peterson
Bone cells in vivo exist in direct contact with extracellular matrix, which regulates their basic biological processes including metabolism, development, growth and differentiation. Thus, the in vitro activity of cells cultured on tissue culture treated plastic could be different from the activity of cells cultured on their natural substrate. We selected MC3T3-E1 pre-osteoblastic cells to study the effect of extracellular matrix on cell proliferation because these cells undergo a progressive developmental sequence of proliferation and differentiation. MC3T3-E1 cells were cultured on plastic or plastic coated with ECM, fibronectin, collagen type I, BSA or poly l -lysine and their ability to proliferate was assessed by incorporation of [3H]dT or by enumeration of cells. Our results show that (1) ECM inhibits incorporation of [3H]dT by MC3T3-E1 cells; (2) collagen type I, but not BSA, poly l -lysine or fibronectin also inhibits incorporation of [3H]dT; (3) the level of ECM inhibition of [3H]dT incorporation is directly related to the number of cells cultured, but unrelated to the cell cycle distribution or endogenous thymidine content; (4) the kinetic profile of [3H]dT uptake suggest that ECM inhibits transport of [3H]dT from the extracellular medium, and (5) cell counts are similar in cultures whether cells are grown on plastic or ECM. These results suggest that decreased incorporation of [3H]dT by cells cultured on ECM is not reflective of bone cell proliferation. [source]


Recombinant human glucose-6-phosphate dehydrogenase

FEBS JOURNAL, Issue 14 2002
Evidence for a rapid-equilibrium random-order mechanism
Cloning and over-expression of human glucose 6-phosphate dehydrogenase (Glc6P dehydrogenase) has for the first time allowed a detailed kinetic study of a preparation that is genetically homogeneous and in which all the protein molecules are of identical age. The steady-state kinetics of the recombinant enzyme, studied by fluorimetric initial-rate measurements, gave converging linear Lineweaver,Burk plots as expected for a ternary-complex mechanism. Patterns of product and dead-end inhibition indicated that the enzyme can bind NADP+ and Glc6P separately to form binary complexes, suggesting a random-order mechanism. The Kd value for the binding of NADP+ measured by titration of protein fluorescence is 8.0 µm, close to the value of 6.8 µm calculated from the kinetic data on the assumption of a rapid-equilibrium random-order mechanism. Strong evidence for this mechanism and against either of the compulsory-order possibilities is provided by repeating the kinetic analysis with each of the natural substrates replaced in turn by structural analogues. A full kinetic analysis was carried out with deaminoNADP+ and with deoxyglucose 6-phosphate as the alternative substrates. In each case the calculated dissociation constant upon switching a substrate in a random-order mechanism (e.g. that for NADP+ upon changing the sugar phosphate) was indeed constant within experimental error as expected. The calculated rate constants for binding of the leading substrate in a compulsory-order mechanism, however, did not remain constant when the putative second substrate was changed. Previous workers, using enzyme from pooled blood, have variously proposed either compulsory-order or random-order mechanisms. Our study appears to provide unambiguous evidence for the latter pattern of substrate binding. [source]


Genetic diversity and distribution of periphytic Synechococcus spp. in biofilms and picoplankton of Lake Constance

FEMS MICROBIOLOGY ECOLOGY, Issue 2 2004
Sven Becker
Abstract In various water depths of the littoral zone of Lake Constance (Bodensee) cyanobacteria of the Synechococcus -type were isolated from biofilms (periphyton) on three natural substrates and an artificial one (unglazed tiles). From one tile three strains of phycoerythrin (PE)-rich Synechococcus spp. were isolated, the first examples of these organisms in the epibenthos. Phylogenetic inference based on the 16S,23S rRNA intergenic spacer (ITS-1) assigned all periphytic isolates to two clusters of the picophytoplankton clade (evolutionary lineage VI of cyanobacteria). The sequence divergence in the ITS-1 was used to design specific PCR primers to allow direct, culture-independent detection and quantification of isolated Synechococcus strains in natural periphytic and pelagic samples. Denaturing gradient gel electrophoresis (DGGE) analysis revealed depth-related differences of Synechococcus spp. distribution on tiles placed in the littoral zone. Synechococcus genotypes were observed which occurred in both the periphyton (on tiles) and in the pelagic picoplankton. A strain with one of these genotypes, Synechococcus sp. BO 8805, was isolated from the pelagic zone in 1988. Its genotype was found on tiles that had been exposed at different water depths in the littoral zone in spring and autumn of the year 2000. Quantitative analysis with a genotype-specific TaqMan probe and real-time Taq nuclease assays (TNA) confirmed its presence in the pelagic zone, although appearance of this and related genotypes was highly irregular and exhibited strong differences between consecutive years. Our results show that the ability to form significant subpopulations in pelagic and periphytic communities exists in three out of four phylogenetic clusters of Synechococcus spp. in Lake Constance. This versatility may be a key feature in the ubiquity of the evolutionary lineage VI of cyanobacteria. [source]


The use of the green fluorescent protein as a biomarker for sapstain fungi

FOREST PATHOLOGY, Issue 3 2002
S. LEE
To understand wood colonization by sapstain fungi and their potential biocontrol agents, it is necessary to differentiate these organisms directly on their natural substrates. In the present study the feasibility of transforming with the green fluorescent protein (GFP), the sapstain fungus Ophiostoma piceae and a potential biocontrol agent Cartapip®, an Ophiostoma piliferum albino strain was assessed. Transformants of the two fungal species were screened by polymerase chain reaction and Southern blot analyses. The GFP was expressed in spores, synnemata and mycelia of the transformants grown in artificial media or wood. The growth, pigmentation and wood colonization of the transformants were similar to that of the non-transformants, suggesting that the presence of the gfp gene had no negative effect on the biology of the transformants. Using fluorescence and confocal microscopy, the GFP-expressing fungi were easily differentiated from the wild-type strains and other fungal species in wood, even 4 months after inoculation. The results show that the use of the GFP system is feasible to monitor Ophiostoma fungi in wood. Utilisation de la protéine fluorescente verte (GFP) comme marqueur biologique des champignons de bleuissement du bois Pour comprendre la colonisation du bois par les champignons de bleuissement et par les agents de lutte biologique potentiels, il est nécessaire de distinguer ces organismes directement dans leur substrat naturel. Nous avons évalué la possibilité de transformation par la protéine fluorescente verte (GFP) du champignon de bleuissement Ophiostoma piceae et d'une souche albinos de Ophiostoma piliferum, agent de lutte biologique potentiel Cartapip®. Des transformants des deux espèces fongiques ont été triés par analyses PCR et Southern blot. La GFP a été exprimée dans les spores, les synnemas et le mycélium des transformants cultivés sur milieux artificiels et sur bois. Avec les transformants, la croissance, la pigmentation et la colonisation du bois étaient semblables à celles des non transformants, ce qui suggère que la présence du gène gfp n'a pas d'effet négatif sur la biologie des transformants. Par microscopie confocale à fluorescence, les champignons exprimant la GFP ont été facilement distingués des souches de type sauvage et d'autres espèces fongiques dans le bois, même 4 mois après inoculation. Nos résultats montrent que l'utilisation de la GFP est possible pour suivre les Ophiostoma dans le bois. Verwendung des Grünen Fluoreszenzproteins als Biomarker für Bläuepilze Um die Besiedelung von Holz durch Bläuepilze und ihre möglichen Antagonisten zu verstehen, muss man diese Organismen direkt auf ihrem natürlichen Substrat unterscheiden können. Es wurde überprüft, ob sich der Bläuepilze Ophiostoma piceae und der mögliche Antagonist Cartapip®, ein Albinostamm von Ophiostoma piliferum, mit dem Grünen Fluoreszenzprotein (GFP) transformieren lassen. Transformierte Stämme der beiden Pilzarten wurden mit PCR und Southern Blot Analysen untersucht. Das GFP wurde in Sporen, Synnemata und Myzelien der transformierten Stämme exprimiert. Dies war auf künstlichen Medien ebenso wie auf Holz der Fall. Wachstum, Pigmentierung und Holzbesiedelung waren bei den transformierten Stämmen ähnlich wie bei den nichttransformierten; somit dürfte die Präsenz des gfpGens keine negativen Auswirkungen auf die Biologie der transformierten Stämme haben. Mit Hilfe der Fluoreszenz- und Konfokal-Mikroskopie konnten die GFP exprimierenden Pilze leicht von den Wildtyp-Stämmen und anderen Pilzarten auf Holz unterschieden werden. Dies war auch noch vier Monate nach der Inokulation der Fall. Die Ergebnisse zeigen, dass das GFP-System zur Beobachtung von Ophiostoma -Arten im Holz geeignet ist. [source]


Cross-priming utilizes antigen not available to the direct presentation pathway

IMMUNOLOGY, Issue 1 2006
Keri B. Donohue
Summary CD8+ T cells play a crucial role in protective immunity to viruses and tumours. Antiviral CD8+ T cells are initially activated by professional antigen presenting cells (pAPCs) that are directly infected by viruses (direct-priming) or following uptake of exogenous antigen transferred from virus-infected or tumour cells (cross-priming). In order to efficiently target each of these antigen-processing pathways during vaccine design, it is necessary to delineate the properties of the natural substrates for either of these antigen-processing pathways. In this study, we utilized a novel T-cell receptor (TCR) transgenic mouse to examine the requirement for both antigen synthesis and synthesis of other cellular factors during direct or cross-priming. We found that direct presentation required ongoing synthesis of antigen, but that cross-priming favoured long-lived antigens and did not require ongoing antigen production. Even after prolonged blockade of protein synthesis in the donor cell, cross-priming was unaffected. In contrast, direct-presentation was almost undetectable in the absence of antigen neosynthesis and required ongoing protein synthesis. This suggests that the direct- and cross-priming pathways may utilize differing pools of antigen, an observation that has far-reaching implications for the rational design of vaccines aimed at the generation of protective CD8+ T cells. [source]


Caddisfly diapause aggregations facilitate benthic invertebrate colonization

JOURNAL OF ANIMAL ECOLOGY, Issue 6 2003
Declan J. McCabe
Summary 1We used natural and manipulative field experiments to examine the effects of caddisfly (Trichoptera) diapause aggregations on benthic macroinvertebrates communities in a Vermont river. 2Natural substrates with aggregations of Neophylax and Brachycentrus (Trichoptera: Uenoidae and Brachycentridae) had higher species richness than did substrates lacking aggregations. Aggregations of caddisfly cases added to artificial substrates (bricks) also accumulated greater abundance, species density (number of species per unit area), and species richness (number of species per standard number of individuals) than did control bricks. 3Low-density, uniformly spaced, Brachycentrus cases accumulated higher species density and species richness than did an equivalent density of clumped cases. Similarly, empty Neophylax cases accumulated higher diversity than did cases still occupied by Neophylax pupae. 4Although natural substrates had higher species richness than artificial substrates, substrate type did not change qualitatively the effect of caddisfly aggregations on species richness. 5We subsampled individuals randomly from aggregations and control surfaces to provide an estimate of species richness unbiased by abundance. Expected species richness was higher in aggregations than on control surfaces. These results suggest that caddisfly aggregations increase species density by altering the shape of the species,abundance distribution as well as by accumulating individuals and species passively. 6We conclude that caddisfly diapause aggregations increase habitat complexity and facilitate colonization of other benthic species. [source]


Production of gliotoxin on natural substrates by Trichoderma virens

JOURNAL OF BASIC MICROBIOLOGY, Issue 1 2005
R. Anitha Dr.
Gliotoxin, an epithiodiketopiperazine toxin produced by the ,Q' strain of Trichoderma virens is essential for curtailing growth and multiplication of phytopathogens (Howellet al. 1993, Fravel 1988). Three isolates (Gv, Gv-A and Gv-V) of Trichoderma virens were grown on natural substrates such as bengal gram hull, gingelly cake, green gram hull, rice bran, soya meal, sugarcane bagasse, soyameal + tapioca, tapioca powder, tapioca peel and wheat bran). It was evident from this study that maximum gliotoxin (64 mg/l) was produced on tapioca powder by the alien isolate Gv. However sugarcane bagasse significantly enhanced gliotoxin production (36 mg/l) in the native isolate Gv-A, when compared to other substrates like greengram hull and rice bran. So far, studies on production of gliotoxin on synthetic media has been reported. We report the production of gliotoxin by T. virens on natural substrates "in vitro" for the first time. (© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source]


Enzyme Replacement Therapy for Murine Hypophosphatasia,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2008
José Luis Millán PhD
Abstract Introduction: Hypophosphatasia (HPP) is the inborn error of metabolism that features rickets or osteomalacia caused by loss-of-function mutation(s) within the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNALP). Consequently, natural substrates for this ectoenzyme accumulate extracellulary including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5,-phosphate (PLP), a co-factor form of vitamin B6. Babies with the infantile form of HPP often die with severe rickets and sometimes hypercalcemia and vitamin B6 -dependent seizures. There is no established medical treatment. Materials and Methods: Human TNALP was bioengineered with the C terminus extended by the Fc region of human IgG for one-step purification and a deca-aspartate sequence (D10) for targeting to mineralizing tissue (sALP-FcD10). TNALP-null mice (Akp2,/,), an excellent model for infantile HPP, were treated from birth using sALP-FcD10. Short-term and long-term efficacy studies consisted of once daily subcutaneous injections of 1, 2, or 8.2 mg/kg sALP-FcD10 for 15, 19, and 15 or 52 days, respectively. We assessed survival and growth rates, circulating levels of sALP-FcD10 activity, calcium, PPi, and pyridoxal, as well as skeletal and dental manifestations using radiography, ,CT, and histomorphometry. Results:Akp2,/, mice receiving high-dose sALP-FcD10 grew normally and appeared well without skeletal or dental disease or epilepsy. Plasma calcium, PPi, and pyridoxal concentrations remained in their normal ranges. We found no evidence of significant skeletal or dental disease. Conclusions: Enzyme replacement using a bone-targeted, recombinant form of human TNALP prevents infantile HPP in Akp2,/, mice. [source]


Effect of floating nest platforms on the breeding performance of Black Terns

JOURNAL OF FIELD ORNITHOLOGY, Issue 2 2006
David A. Shealer
ABSTRACT In 2003 and 2004, we placed 41 floating nest platforms on Grassy Lake in southeastern Wisconsin (USA) to test the hypothesis that reproductive success of Black Terns (Chlidonias niger) is limited by the quality of suitable nesting habitat. Extreme differences in water levels between these 2 yr provided a natural experiment to evaluate the effectiveness of the nest platforms during a drought year (2003) when natural nesting substrate was abundant, and a flood year (2004) when natural substrate was limited during the peak nesting period. Terns nested on 27 of 41 (66%) of the platforms in 2003 and 26 of 41 (63%) in 2004. No difference in the occupancy rate of platforms and natural nests was evident in 2003, but the pattern of clutch initiations early in the season in 2004 indicated that platforms were preferred over natural substrates. In 2003, nest survival rates did not differ between nests placed on platforms and those placed on natural substrates, but platform nests had significantly higher hatching success and nest survival rates in 2004. Both the Kaplan-Meier and Apparent Nest Success methods of calculating nest survival provided similar estimates. In both years, eggs laid on platforms were significantly larger than those laid on natural substrates, suggesting that platforms were occupied by high-quality birds. Our study indicates that floating nest platforms can be an effective management tool to enhance nesting habitat for Black Terns and other aquatic birds that construct floating nests, primarily because platforms provide nest sites when natural sites are not available due to flooding. Nest platforms also may be useful for addressing questions concerning habitat selection and parental quality. SINOPSIS De 2003-2004 colocamos 41 plataformas flotantes para poner aprueba la hipótesis que el éxito reproductivo de la gaviota Chlidonias níger, esta limitado por la calidad y lo apropiado del hábitat de anidamiento. El estudio se llevó a cabo en el Grassy Lake, Wisconsin. Extremos (bajos y altos) en los niveles de agua, causados por sequía (2203) y fuertes lluvias (2004) proveyeron el escenario adecuado para evaluar la efectividad de las plataformas. Las gaviotas anidaron en 27 (66%) de las 41 plataformas en el 2003 y en 26 (63%) de las 41 en el 2004. No se encontraron diferencias en la tasa de uso de las plataformas y áreas naturales en el 2003, pero el inicio de las camadas temprano en la temporada durante el 2004, dieron a indicar que las plataformas fueron preferidas a los lugares naturales. Durante el 2003, no hubo diferencia en la tasa de supervivencia de nidos en plataformas y o en áreas naturales. Sin embargo, durante el 2004 el éxito de eclosionamiento, de los nidos y la supervivencia fue mas alto en las plataformas. Tanto el método Kaplan-Meier como el "Aparente Éxito de Anidamiento", utilizados para calcular la supervivencia de los nidos, ofrecieron resultados similares. En ambos años el tamaño de los huevos fue mayor en las plataformas que en áreas naturales, lo que implica que las primeras fueron utilizadas por aves sumamente vigorosas o de alta calidad. El estudio indica que las plataformas flotantes pueden ser una herramienta de manejo adecuada para mejorar el hábitat de anidamiento de la gaviota estudiada, al igual que otras aves que construyen nidos flotantes. Las plataformas proveen de lugares de anidamientos cuando no hay disponible lugares naturales debido a inundaciones. Las plataformas de anidamiento muy bien pudieran proveer de información útil con respecto a la selección de hábitat y la calidad del cuidado parental. [source]


Heterologous production of the Piromyces equi cinnamoyl esterase in Trichoderma reesei for biotechnological applications

LETTERS IN APPLIED MICROBIOLOGY, Issue 6 2009
L. Poidevin
Abstract Aims:, The objective of the study was to produce and characterize the cinnamoyl esterase EstA from the anaerobic fungus Piromyces equi for potential industrial applications. Methods and Results:, The catalytic domain EstA was produced in Trichoderma reesei. Because the two fungi displayed different genome features, including different codon usage and GC content, a synthetic gene was designed and expressed, leading to the production of the corresponding protein at around 33 mg per litre in the T. reesei culture medium. After the recombinant protein was purified, biochemical characterization showed that EstA presents peak activity at pH 6·5 and at 50,60°C. Furthermore, EstA remained stable at pH 6,8 and below 50°C. EstA was compared to cinnamoyl esterases FaeA and FaeB from Aspergillus niger in terms of ferulic acid (FA) release from wheat bran (WB), maize bran (MB) and sugar beet pulp (SBP). Conclusion:, The synthetic gene was successfully cloned and overexpressed in T. reesei. EstA from P. equi was demonstrated to efficiently release FA from various natural substrates. Significance and Impact of the Study:, Recombinant EstA produced in an industrial enzyme producer, T. reesei, was biochemically characterized, and its capacity to release an aromatic compound (FA) for biotechnological applications was demonstrated. [source]


House cleaning, a part of good housekeeping

MOLECULAR MICROBIOLOGY, Issue 1 2006
Michael Y. Galperin
Summary Cellular metabolism constantly generates by-products that are wasteful or even harmful. Such compounds are excreted from the cell or are removed through hydrolysis to normal cellular metabolites by various ,house-cleaning' enzymes. Some of the most important contaminants are non-canonical nucleoside triphosphates (NTPs) whose incorporation into the nascent DNA leads to increased mutagenesis and DNA damage. Enzymes intercepting abnormal NTPs from incorporation by DNA polymerases work in parallel with DNA repair enzymes that remove lesions produced by modified nucleotides. House-cleaning NTP pyrophosphatases targeting non-canonical NTPs belong to at least four structural superfamilies: MutT-related (Nudix) hydrolases, dUTPase, ITPase (Maf/HAM1) and all-, NTP pyrophosphatases (MazG). These enzymes have high affinity (Km's in the micromolar range) for their natural substrates (8-oxo-dGTP, dUTP, dITP, 2-oxo-dATP), which allows them to select these substrates from a mixture containing a ,1000-fold excess of canonical NTPs. To date, many house-cleaning NTPases have been identified only on the basis of their side activity towards canonical NTPs and NDP derivatives. Integration of growing structural and biochemical data on these superfamilies suggests that their new family members cleanse the nucleotide pool of the products of oxidative damage and inappropriate methylation. House-cleaning enzymes, such as 6-phosphogluconolactonase, are also part of normal intermediary metabolism. Genomic data suggest that house-cleaning systems are more abundant than previously thought and include numerous analogous enzymes with overlapping functions. We discuss the structural diversity of these enzymes, their phylogenetic distribution, substrate specificity and the problem of identifying their true substrates. [source]


Identifying natural substrates for chaperonins using a sequence-based approach

PROTEIN SCIENCE, Issue 1 2005
George Stan
Abstract The Escherichia coli chaperonin machinery, GroEL, assists the folding of a number of proteins. We describe a sequence-based approach to identify the natural substrate proteins (SPs) for GroEL. Our method is based on the hypothesis that natural SPs are those that contain patterns of residues similar to those found in either GroES mobile loop and/or strongly binding peptide in complex with GroEL. The method is validated by comparing the predicted results with experimentally determined natural SPs for GroEL. We have searched for such patterns in five genomes. In the E. coli genome, we identify 1422 (about one-third) sequences that are putative natural SPs. In Saccharomyces cerevisiae, 2885 (32%) of sequences can be natural substrates for Hsp60, which is the analog of GroEL. The precise number of natural SPs is shown to be a function of the number of contacts an SP makes with the apical domain (NC) and the number of binding sites (NB) in the oligomer with which it interacts. For known SPs for GroEL, we find ,4 < NC < 5 and 2 , NB , 4. A limited analysis of the predicted binding sequences shows that they do not adopt any preferred secondary structure. Our method also predicts the putative binding regions in the identified SPs. The results of our study show that a variety of SPs, associated with diverse functions, can interact with GroEL. [source]


Structure of human semicarbazide-sensitive amine oxidase/vascular adhesion protein-­1

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2005
Joakim Nilsson
Semicarbazide-sensitive amine oxidase (SSAO) belongs to a ubiquitous family of copper-containing amine oxidases (CuAOs). SSAO is also known as vascular adhesion protein-­1 (VAP-1) and has been identified as one of the adhesion molecules involved in the leukocyte-extravasation process. The structure of a truncated soluble form of human SSAO has been solved and refined to 2.5,Å. As expected, SSAO is a homodimer with a fold typical of the CuAO family. The topaquinone (TPQ) cofactor and a copper ion characteristic of CuAOs are present in the active site, with the TPQ in the active `off-copper' conformation. The structure reveals that a leucine residue (Leu469) located adjacent to the active site could function as a gate controlling its accessibility. An RGD motif is displayed on the surface, where it could be involved in integrin binding and possibly play a role in the shedding of SSAO from the membrane. Carbohydrate moieties are observed at five of six potential N-glycosylation sites. Carbohydrates attached to Asn232 flank the active-site entrance and might influence substrate specificity. The structure of an adduct of SSAO and the irreversible inhibitor 2-hydrazinopyridine has been solved and refined to 2.9,Å resolution. Together, these structures will aid efforts to identify natural substrates, provide valuable information for the design of specific inhibitors and direct further studies. [source]


High-Throughput Substrate Specificity Studies of Sialidases by Using Chemoenzymatically Synthesized Sialoside Libraries

CHEMBIOCHEM, Issue 2 2007
Harshal A. Chokhawala
Abstract Sialidases, or neuraminidases, are enzymes that cleave terminal sialic acid (Sia) residues from complex sialic acid-containing structures. They have been found in many animals and microorganisms and are important in various physiological and pathological processes. In order to understand the biological significance of diverse sialidases, it is important to study in detail the structural determinants of their natural substrates. Here, we report the synthesis of sialoside libraries containing para -nitrophenol-tagged sialosides with different naturally occurring sialic acid forms, different sialyl linkages, and different penultimate monosaccharides using a highly efficient one-pot three-enzyme chemoenzymatic approach. By using these compounds in a 96-well plate-based colorimetric high-throughput screening platform, the diversity of substrate preference is shown for seven bacterial sialidases. The sialoside libraries and the screening method are convenient tools for unravelling the substrate specificity and the biological function of sialidases. [source]