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991.
Maria Chiara Colao Carlo Caporale Federica Silvestri Maurizio Ruzzi Vincenzo Buonocore 《The protein journal》2009,28(9-10):375-383
A cDNA encoding a novel laccase from the white-rot fungus Trametes trogii was cloned and expressed in Pichia pastoris. The recombinant protein (Lcc2) exhibited kinetic parameters for both phenolic and non phenolic substrates that were different from the previously described Lcc1, the main laccase isoform expressed by T. trogii; in addition, the pH/activity profiles for phenolic substrates of Lcc2 were shifted upward by 1–1.5 pH units towards neutrality as compared to Lcc1. Comparative modeling of the two laccases (69.2% identity) showed that the overall fold of Lcc2 is very similar to Lcc1 and other laccases. The substrate cavity of Lcc2 contains the Asp residue which is thought to mediate the laccase activity at acidic pHs, whereas two hydrophobic residues (Phe, Ile) on the cavity orifice of Lcc2 replace the two polar residues (Thr, Ser) of Lcc1. These structural differences may be responsible for the unique kinetic performances of Lcc2. 相似文献
992.
Marina Cretich Dr. Gabriele Di Carlo Cinzia Giudici Sven Pokoj Iris Lauer Stephan Scheurer Marcella Chiari 《Proteomics》2009,9(8):2098-2107
Allergen microarrays are under development for a component‐resolved diagnosis of Type I (IgE‐mediated) allergies. Here we report an improved microarray coupled to microfluidics for the detection of allergen specific immunoglobulin E (IgE). The signal intensity for IgE detection in serum has been improved by using glass slides coated with a novel poly[DMA‐co‐NAS] brush copolymer which is able to immobilize allergens in their native conformation and by carrying out the incubation step in dynamic conditions. The assay, fully automated, was performed in a microcell, using a software‐controlled fluidic processor, to bring assay reagents on the surface of the array. Microfluidics turns the binding between serum immunoglobulins and immobilized allergens from a diffusion‐limited to a kinetic‐limited process by ensuring an efficient mixing of serum samples on the surface of the microarray. As a result of this, the binding of high affinity IgE antibodies is enhanced whereas that of low affinity IgG antibodies, which are present at higher concentration, is impaired paving the way to more accurate and sensitive results. 相似文献
993.
Angela Pennacchio Luciana Esposito Adriana Zagari Mosè Rossi Carlo A. Raia 《Extremophiles : life under extreme conditions》2009,13(5):751-761
A mutant of the thermostable NAD+-dependent (S)-stereospecific alcohol dehydrogenase from Sulfolobus solfataricus (SsADH) which has a single substitution, Trp95Leu, located at the substrate binding pocket, was fully characterized to ascertain the role of Trp95 in discriminating between chiral secondary alcohols suggested by the wild-type SsADH crystallographic structure. The Trp95Leu mutant displays no apparent activity with short-chain primary and secondary alcohols and poor activity with aromatic substrates and coenzyme. Moreover, the Trp → Leu substitution affects the structural stability of the archaeal ADH, decreasing its thermal stability without relevant changes in secondary structure. The double mutant Trp95Leu/Asn249Tyr was also purified to assist in crystallographic analysis. This mutant exhibits higher activity but decreased affinity toward aliphatic alcohols, aldehydes as well as NAD+ and NADH compared to the wild-type enzyme. The crystal structure of the Trp95Leu/Asn249Tyr mutant apo form, determined at 2.0 Å resolution, reveals a large local rearrangement of the substrate site with dramatic consequences. The Leu95 side-chain conformation points away from the catalytic metal center and the widening of the substrate site is partially counteracted by a concomitant change of Trp117 side chain conformation. Structural changes at the active site are consistent with the reduced activity on substrates and decreased coenzyme binding. 相似文献
994.
M. Manuela Rigano Carmela Manna Anna Giulini Emanuela Pedrazzini Maria Capobianchi Concetta Castilletti Antonino Di Caro Giuseppe Ippolito Paola Beggio Carlo De Giuli Morghen Luigi Monti Alessandro Vitale Teodoro Cardi 《Plant biotechnology journal》2009,7(6):577-591
Orthopoxviruses (OPVs) have recently received increasing attention because of their potential use in bioterrorism and the occurrence of zoonotic OPV outbreaks, highlighting the need for the development of safe and cost‐effective vaccines against smallpox and related viruses. In this respect, the production of subunit protein‐based vaccines in transgenic plants is an attractive approach. For this purpose, the A27L immunogenic protein of vaccinia virus was expressed in tobacco using stable transformation of the nuclear or plastid genome. The vaccinia virus protein was expressed in the stroma of transplastomic plants in soluble form and accumulated to about 18% of total soluble protein (equivalent to approximately 1.7 mg/g fresh weight). This level of A27L accumulation was 500‐fold higher than that in nuclear transformed plants, and did not decline during leaf development. Transplastomic plants showed a partial reduction in growth and were chlorotic, but reached maturity and set fertile seeds. Analysis by immunofluorescence microscopy indicated altered chlorophyll distribution. Chloroplast‐synthesized A27L formed oligomers, suggesting correct folding and quaternary structure, and was recognized by serum from a patient recently infected by a zoonotic OPV. Taken together, these results demonstrate that chloroplasts are an attractive production vehicle for the expression of OPV subunit vaccines. 相似文献
995.
996.
Cross talk between the KNOX and ethylene pathways is mediated by intron-binding transcription factors in barley 总被引:1,自引:0,他引:1
Osnato M Stile MR Wang Y Meynard D Curiale S Guiderdoni E Liu Y Horner DS Ouwerkerk PB Pozzi C Müller KJ Salamini F Rossini L 《Plant physiology》2010,154(4):1616-1632
In the barley (Hordeum vulgare) Hooded (Kap) mutant, the duplication of a 305-bp intron sequence leads to the overexpression of the Barley knox3 (Bkn3) gene, resulting in the development of an extra flower in the spikelet. We used a one-hybrid screen to identify four proteins that bind the intron-located regulatory element (Kap intron-binding proteins). Three of these, Barley Ethylene Response Factor1 (BERF1), Barley Ethylene Insensitive Like1 (BEIL1), and Barley Growth Regulating Factor1 (BGRF1), were characterized and their in vitro DNA-binding capacities verified. Given the homology of BERF1 and BEIL1 to ethylene signaling proteins, we investigated if these factors might play a dual role in intron-mediated regulation and ethylene response. In transgenic rice (Oryza sativa), constitutive expression of the corresponding genes produced phenotypic alterations consistent with perturbations in ethylene levels and variations in the expression of a key gene of ethylene biosynthesis. In barley, ethylene treatment results in partial suppression of the Kap phenotype, accompanied by up-regulation of BERF1 and BEIL1 expression, followed by down-regulation of Bkn3 mRNA levels. In rice protoplasts, BEIL1 activates the expression of a reporter gene driven by the 305-bp intron element, while BERF1 can counteract this activation. Thus, BEIL1 and BERF1, likely in association with other Kap intron-binding proteins, should mediate the fine-tuning of Bkn3 expression by ethylene. We propose a hypothesis for the cross talk between the KNOX and ethylene pathways. 相似文献
997.
Feinan Wu Nancy T. Eannetta Yimin Xu Jörg Plieske Martin Ganal Carlo Pozzi Nicolas Bakaher Steven D. Tanksley 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2010,120(4):809-827
Using single-copy conserved ortholog set (COSII) and simple sequence repeat (SSR) markers, we have constructed two genetic
maps for diploid Nicotiana species, N. tomentosiformis and N. acuminata, respectively. N. acuminata is phylogenetically closer to N. sylvestris than to N. tomentosiformis, the latter two of which are thought to contribute the S-genome and T-genome, respectively, to the allotetraploid tobacco
(N. tabacum L., 2n = 48). A comparison of the two maps revealed a minimum of seven inversions and one translocation subsequent to the divergence
of these two diploid species. Further, comparing the diploid maps with a dense tobacco map revealed that the tobacco genome
experienced chromosomal rearrangements more frequently than its diploid relatives, supporting the notion of accelerated genome
evolution in allotetraploids. Mapped COSII markers permitted the investigation of Nicotiana–tomato syntenic relationships. A minimum of 3 (and up to 10) inversions and 11 reciprocal translocations differentiate the
tomato genome from that of the last common ancestor of N. tomentosiformis and N. acuminata. Nevertheless, the marker/gene order is well preserved in 25 conserved syntenic segments. Molecular dating based on COSII
sequences suggested that tobacco was formed 1.0MYA or later. In conclusion, these COSII and SSR markers link the cultivated
tobacco map to those of wild diploid Nicotiana species and tomato, thus providing a platform for cross-reference of genetic and genomic information among them as well as
other solanaceous species including potato, eggplant, pepper and the closely allied coffee (Rubiaceae). Therefore they will
facilitate genetic research in the genus Nicotiana. 相似文献
998.
Strategies adopted by parasitoids and kleptoparasites co-evolve with the defensive adaptations of their hosts, and vice-versa.
Hedychrum rutilans and Hedychrum nobile are brood parasites of, respectively, Philanthus triangulum and Cerceris arenaria, two digger wasps that share most aspects of their nesting biology (solitary females dig aggregated nests in the ground and
mass-provision the brood with paralyzed insects). We tested the hypothesis that similarity in the hosts’ nesting habits corresponds
to similar defensive strategies against these cuckoo wasps. Peak provisioning activity by P. triangulum occurred in late afternoon (and early morning in 1 year) while peak H. rutilans activity was in early afternoon. In contrast, peak provisioning by C. arenaria and peak H. nobile activity occurred in early afternoon. Thus, P. triangulum (as previously found in other populations) appears to have timed its provisioning to avoid its brood parasite whereas C. arenaria did not, rejecting our hypothesis. The daily activity of both chrysidid wasps was positively correlated to air temperature.
Host nest density positively affected only H. rutilans activity, in agreement with previous reports for other populations of this species, whereas the daily pattern of activity
of the host was the key correlate with the activity of H. nobile. Mortality due to cuckoo wasps was low for both digger wasps, although it was somewhat higher at sites with many nests of
P. triangulum. We suggest that perhaps the degree to which digger wasps time their activity to avoid cuckoo wasps is related to the degree
of specialization in host choice by their brood parasite. H. rutilans is more highly dependent on P. triangulum, which may have had a greater effect on the timing of provision by its host. 相似文献
999.
Ralf Frederik Claas Michael ter Braak Bianca Hegen Verena Hardel Carlo Angioni Helmut Schmidt Karl H. Jakobs Paul P. Van Veldhoven Dagmar Meyer zu Heringdorf 《Cellular signalling》2010,22(3):476-483
Sphingosine-1-phosphate (S1P) regulates cell growth and survival, migration and adhesion in many cell types. S1P is generated by sphingosine kinases (SphKs), and dephosphorylated by phosphatases or cleaved by S1P lyase. Extracellular S1P activates specific G protein-coupled receptors while intracellular S1P can mobilize Ca2+ from thapsigargin-sensitive stores. Here, we have studied Ca2+ signalling in mouse embryonic fibroblasts (MEFs) deficient in S1P lyase. In these cells, S1P and sphingosine concentrations were elevated about 6-fold and 2-fold, respectively, as measured by liquid chromatography/tandem mass spectrometry. Measurements with fura-2-loaded cells in suspension revealed that resting [Ca2+]i was elevated and agonist-induced [Ca2+]i increases were augmented in S1P lyase-deficient MEFs both in the presence and absence of extracellular Ca2+. Importantly, [Ca2+]i increases and Ca2+ mobilization induced by the SERCA inhibitor, thapsigargin, were augmented, indicating enhanced Ca2+ storage in S1P lyase-deficient MEFs. Measurements with single cells expressing the calmodulin-based Ca2+ sensor, cameleon, revealed that at least two cell types could be distinguished in both MEF cell populations, one with a rapid and transient [Ca2+]i increase and the other with a slower and prolonged [Ca2+]i elevation upon stimulation with thapsigargin. The area under the time course of thapsigargin-induced [Ca2+]i increases, reflecting overall Ca2+ release, was significantly increased by more than 50% in both rapidly and slowly responding S1P lyase-deficient cells. It is concluded that elevated concentrations of S1P and/or sphingosine lead to enhanced Ca2+ storage and elevated basal [Ca2+]i. S1P metabolism thus plays a role not only in acute Ca2+ mobilization but also in long-term regulation of Ca2+ homeostasis. 相似文献
1000.
Bergamini Carlo M. Dondi Alessia Lanzara Vincenzo Squerzanti Monica Cervellati Carlo Montin Katy Mischiati Carlo Tasco Gianluca Collighan Russel Griffin Martin Casadio Rita 《Amino acids》2010,39(1):297-304
Amino Acids - The transamidating activity of tissue transglutaminase is regulated by the ligands calcium and GTP, via conformational changes which facilitate or interfere with interaction with the... 相似文献