The effects of GDP and of aurodox (N-methylkirromycin) on the affinity of elongation factor Tu (EF-Tu) for aminoacyl-tRNA (aa-tRNA) have been quantified spectroscopically by using Phe-tRNA(Phe)-Fl8, ...a functionally active analogue of Phe-tRNA(Phe) with a fluorescein dye convalently attached to the s4U-8 base. The association of EF-Tu.GDP with Phe-tRNA(Phe)-Fl8 resulted in an average increase of 33% in fluorescein emission intensity. This spectral change was used to monitor the extent of ternary complex formation as a function of EF-Tu.GDP concentration, and hence to obtain a dissociation constant, directly and at equilibrium, for the EF-Tu.GDP-containing ternary complex. The Kd for the Phe-tRNA(Phe)-Fl8.EF-Tu.GDP complex was found to average 28.5 microM, more than 33,000-fold greater than the Kd of the Phe-tRNA(Phe)-Fl8.EF-Tu.GTP complex under the same conditions. In terms of free energy, the delta G degree for ternary complex formation at 6 degrees C was -11.5 kcal/mol with GTP and -5.8 kcal/mol with GDP. Thus, the hydrolysis of the ternary complex GTP results in a dramatic decrease in the affinity of EF-Tu for aa-tRNA, thereby facilitating the release of EF-Tu.GDP from the aa-tRNA on the ribosome. Aurodox (200 microM) decreased the Kd of the GDP complex by nearly 20-fold, to 1.46 microM, and increased the Kd of the GTP complex by at least 6-fold. The binding of aurodox to EF-Tu therefore both considerably strengthens EF-Tu.GDP affinity for aa-tRNA and also weakens EF-Tu.GTP affinity for aa-tRNA.
Summary
We investigated whether vitamin E supplementation and supplemental energy sources (fat or starch) influenced plasma and milk levels of vitamin E, and reproductive and other parameters in 28 ...Italian Friesian multiparous dry cows. From 14 days before expected calving to 7 days after, the animals were assigned to either basal diet (containing 1000 IU/day of vitamin E) or an extra 1000 IU/day of vitamin E (total 2000 IU). In addition they received either 0.5 kg/day of corn or 0.2 kg/day of calcium soaps. Plasma samples were collected 4 days before expected calving and 4 days after calving and analysed for α‐tocopherol and cholesterol. Milk yield as well as the composition, somatic cell count (SCC) and α‐tocopherol of milk were determined 7 and 14 days after calving. Milk yield and composition were unaffected by treatments. SCC was significantly lower in (SCC Log 4.62 versus Log 5.1, P < 0.01) 2000 IU/day animals than in the 1000 IU/day group. Milk α‐tocopherol was higher (P < 0.001) in animals receiving 2000 IU/day (1.11 vs. 0.65 µg/ml, P < 0.01). Plasma α‐tocopherol in animals receiving 2000 IU/day was also higher (P < 0.001) than in cows receiving 1000 IU/day (4.85 vs. 3.25 µg/ml), but was not affected by dietary energy source. Number of services and days to conception were lower (P < 0.01) in the 2000 IU vitamin E supplemented cows. To conclude, dietary vitamin E supplementation to periparturient dairy cows increased plasma and milk vitamin E, decreased SCC in milk, and improved fertility but different energy sources had no effect on any measured variable.
The endocytosis of alpha-galactosidase A was studied in cultured fibroblasts from patients with Fabry disease. Alpha-galactosidase A was purified from human placenta by chromatography on concanavalin ...A-Sepharose, DEAE-cellulose, and N-epsilon-aminocaproyl-alpha-D-galactosylamine-Sepharose. Separation of the high-uptake form of the enzyme from the low-uptake form was accomplished by chromatography on ECTEOLA-cellulose. With the high-uptake form of the enzyme, the uptake was linear at low concentrations of enzyme and had a Kuptake of 0.01 U/ml of medium that corresponds to a Km of 5.0 x 10(-9) M. At high concentrations of enzyme, it became saturated. The high-uptake form could be converted to the low-uptake form by treatment with acid phosphatase. Mannose-6-P strongly inhibited the active uptake of the enzyme. Once taken up into the lysosomes of Fabry disease fibroblasts, alpha-galactosidase A activity was rapidly lost in the first 2 days of incubation at 37 degrees C, but was fairly stable for the next 6 days. The half-life of internalized alpha-galactosidase A activity was calculated to be 4 days. Crosslinking of the enzyme with hexamethylene diisocyanate did not increase the intracellular stability of alpha-galactosidase A activity.
Verocytotoxic Escherichia coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral ...administration in transgenic seeds could be an effiient system to stimulate local immunity. this study was conducted to transform tobacco plants for the ssed- specific expression of antigenic proteins from a porcine vercytotoxic E. coli strain.
EST projects in aquaculture: sea bass, red tuna and perch [Expressed Sequence Tag] Gornati, R. (Università dell´Insubria, Varese (Italy). Dipartimento di Biotecnologie e Scienze Molecolari); Chini, V. (Università dell´Insubria, Varese (Italy). Dipartimento di Biotecnologie e Scienze Molecolari); Rimoldi, S. (Università dell´Insubria, Varese (Italy). Dipartimento di Biotecnologie e Scienze Molecolari) ...
Italian journal of animal science,
01/2005, Letnik:
4, Številka:
sup2
Journal Article
Recenzirano
Odprti dostop
These three species are arousing more and more interest. Therefore, a program aimed at evaluating fish behaviour and possible problems in different phases of biologic cycle was carried out, studying, ...particularly, the expression of genes involved in gamete maturation and in fertilization and/or genes which play a role in biologic response to environmental stressors. To this end, we started a project by setting up a cDNA library of sea bass liver and sequencing some hundreds of clones; 55% of them resulted singlets, whereas the other were grouped in contigs. At the same time a study on red tuna gonads and perch liver is in progress. The objective of this project is the creation of libraries of cDNA sequences of these commercially interesting species. From such libraries about 5,000 clones will be sequenced; the obtained sequences, recorded in data base, will be available through Internet for all people interested to research in this area of interest.
Queste tre specie stanno richiamando un sempre maggior interesse. Si è reso quindi necessario effettuare un programma che permetta di valutare le abitudini comportamentali dei pesci di allevamento e gli eventuali problemi nelle varie fasi del ciclo biologico indagando, per esempio, l´espressione di geni coinvolti nella maturazione dei gameti e nella fecondazione e/o quelli che hanno un ruolo nella risposta di tipo biologico agli stressori ambientali. A tal fine abbiamo dato il via a questo progetto preparando una library di cDNA di fegato di branzino e sequenziando alcune centinaia di cloni il 55% dei quali risultano essere sequenze singole, mentre i restanti vanno a formare dei contig. Contemporaneamente è in corso uno studio sulle gonadi di tonno rosso e sul fegato di persico. L´ obiettivo di questo progetto è quello di creare database di sequenze di cDNA di queste tre specie di pesci ad interesse commerciale. Di tali library verranno sequenziati circa 5.000 cloni; le sequenze ottenute, depositate in banca dati, saranno disponibili via Internet a tutta la comunità scientifica, che le potrà utilizzare per le ricerche più disparate
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