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91.
Brizio C Galluccio M Wait R Torchetti EM Bafunno V Accardi R Gianazza E Indiveri C Barile M 《Biochemical and biophysical research communications》2006,344(3):1008-1016
FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl(2), as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8+/-1.3nmol of FAD synthesized/min/mg protein and exhibited a K(M) value for FMN of 1.5+/-0.3microM. This is the first report on characterization of human FADS, and the first cloning and over-expression of FADS from an organism higher than yeast. 相似文献
92.
93.
Scalise M Galluccio M Pochini L Indiveri C 《Biochemical and biophysical research communications》2012,422(1):59-63
pET-21a(+)-mOCTN3-6His was constructed and used for over-expression in Escherichia coli Rosetta(DE3)pLysS. After IPTG induction a protein with apparent molecular mass of 53 kDa was collected in the insoluble fraction of the cell lysate and purified by Ni(2+)-chelating chromatography with a yield of 2mg/l of cell culture. The over-expressed protein was identified with mOCTN3 by anti-His antibody and reconstitution in liposomes. mOCTN3 required peculiar conditions for optimal expression and reconstitution in liposomes. The protein catalyzed a time dependent [(3)H]carnitine uptake which was stimulated by intraliposomal ATP and nearly independent of the pH. The K(m) for carnitine was 36 μM. [(3)H]carnitine transport was inhibited by carnitine analogues and some Cys and NH(2) reagents. This paper represents the first outcome in over-expressing, in active form, the third member of the OCTN sub-family, mOCTN3, in E. coli. 相似文献
94.
Collodel G Cardinali R Moretti E Mattioli S Ruggeri S Castellini C 《Theriogenology》2012,77(5):1021-1026
In the present study, the role of rabbit seminal granules was observed. Their influence on motility, capacitation and acrosome reaction, as well as the presence of apoptosis and the morphology of rabbit sperm, were compared in different conditions. Ejaculated sperm from five mature New Zealand White rabbit bucks during three series of collections were studied, comparing raw semen, Percoll-selected sperm and Percoll-selected sperm plus prostate granules. We observed sperm motility kinetic traits by computer-assisted sperm analyzer (CASA) analysis in each sample. Acrosome status was evaluated by FITC-labeled Pisum sativum Agglutinin staining and chlortetracycline fluorescence assay, phosphatidylserine translocation was determined by AnnexinV/Propidium iodide assay and sperm morphology was studied using transmission electron microscopy (TEM). All traits were observed after 30 min incubation at 37 °C in 5% CO2. Data showed that sperm motility and viability markedly improved in the presence of prostate granules, whereas capacitation, acrosome reaction and phosphatidylserine translocation were lowered. TEM confirmed these results. In conclusion, the role of granules was confirmed in synchronizing sperm capacitation and acrosome reaction with egg availability; indeed, rabbit ovulation occurs only 6 to 10 h after mating. 相似文献
95.
Guignardia bidwellii is the etiological agent of grape black rot, a disease affecting Vitis and other Vitaceae that can cause heavy crop losses in vineyards. Its identification is based mainly on morphological characters and the symptoms on plants but, due to their variability, they may be difficult to interpret to reliably distinguish the pathogen to species. To date, despite the economic importance of G. bidwellii, no molecular investigations have been carried out on Vitis isolates and few sequence data are available for cultures derived from ornamental host plants. We analyzed samples of G. bidwellii collected from grapevine cultivars and ornamental plants of various geographic origins by morphological, molecular and proteomic techniques, including ITS1-ITS2 regions and calmodulin gene sequencing, as well as matrix-assisted laser desorption/ionization analysis by time-of-flight mass spectrometry (MALDI-TOF MS). This polyphasic approach allowed assessing the phylogenetic relationships among the different isolates and suggested the existence of two distinct species. The advantages of a polyphasic approach for the identification of G. bidwellii are highlighted. 相似文献
96.
de Filippo C Bostoen K Stoneking M Pakendorf B 《Proceedings. Biological sciences / The Royal Society》2012,279(1741):3256-3263
The expansion of Bantu languages represents one of the most momentous events in the history of Africa. While it is well accepted that Bantu languages spread from their homeland (Cameroon/Nigeria) approximately 5000 years ago (ya), there is no consensus about the timing and geographical routes underlying this expansion. Two main models of Bantu expansion have been suggested: The 'early-split' model claims that the most recent ancestor of Eastern languages expanded north of the rainforest towards the Great Lakes region approximately 4000 ya, while the 'late-split' model proposes that Eastern languages diversified from Western languages south of the rainforest approximately 2000 ya. Furthermore, it is unclear whether the language dispersal was coupled with the movement of people, raising the question of language shift versus demic diffusion. We use a novel approach taking into account both the spatial and temporal predictions of the two models and formally test these predictions with linguistic and genetic data. Our results show evidence for a demic diffusion in the genetic data, which is confirmed by the correlations between genetic and linguistic distances. While there is little support for the early-split model, the late-split model shows a relatively good fit to the data. Our analyses demonstrate that subsequent contact among languages/populations strongly affected the signal of the initial migration via isolation by distance. 相似文献
97.
98.
Casalino L Magnani D De Falco S Filosa S Minchiotti G Patriarca EJ De Cesare D 《Molecular biotechnology》2012,50(3):171-180
The use of Embryonic Stem Cells (ESCs) holds considerable promise both for drug discovery programs and the treatment of degenerative
disorders in regenerative medicine approaches. Nevertheless, the successful use of ESCs is still limited by the lack of efficient
control of ESC self-renewal and differentiation capabilities. In this context, the possibility to modulate ESC biological
properties and to obtain homogenous populations of correctly specified cells will help developing physiologically relevant
screens, designed for the identification of stem cell modulators. Here, we developed a high throughput screening-suitable
ESC neural differentiation assay by exploiting the Cell
maker robotic platform and demonstrated that neural progenies can be generated from ESCs in complete automation, with high standards
of accuracy and reliability. Moreover, we performed a pilot screening providing proof of concept that this assay allows the
identification of regulators of ESC neural differentiation in full automation. 相似文献
99.
Federico Bernardini Claudio Tuniz Alfredo Coppa Lucia Mancini Diego Dreossi Diane Eichert Gianluca Turco Matteo Biasotto Filippo Terrasi Nicola De Cesare Quan Hua Vladimir Levchenko 《PloS one》2012,7(9)
Evidence of prehistoric dentistry has been limited to a few cases, the most ancient dating back to the Neolithic. Here we report a 6500-year-old human mandible from Slovenia whose left canine crown bears the traces of a filling with beeswax. The use of different analytical techniques, including synchrotron radiation computed micro-tomography (micro-CT), Accelerator Mass Spectrometry (AMS) radiocarbon dating, Infrared (IR) Spectroscopy and Scanning Electron Microscopy (SEM), has shown that the exposed area of dentine resulting from occlusal wear and the upper part of a vertical crack affecting enamel and dentin tissues were filled with beeswax shortly before or after the individual’s death. If the filling was done when the person was still alive, the intervention was likely aimed to relieve tooth sensitivity derived from either exposed dentine and/or the pain resulting from chewing on a cracked tooth: this would provide the earliest known direct evidence of therapeutic-palliative dental filling. 相似文献
100.
Rebeca Sanz-Pamplona Antoni Berenguer David Cordero Samantha Riccadonna Xavier Solé Marta Crous-Bou Elisabet Guinó Xavier Sanjuan Sebastiano Biondo Antonio Soriano Giuseppe Jurman Gabriel Capella Cesare Furlanello Victor Moreno 《PloS one》2012,7(11)