Search

The sericin 1 primary transcript of the silkworm Bombyx mori is differentially spliced via a tissue- and developmentally-regulated process. From a middle silk gland cDNA library, we have elucidated the sequence of one of the four mRNAs, the 4.0 kb Ser1B mRNA. Determination of alternative or constitutive exons and intron-exon boundaries allowed us to establish the nine exon-eight intron structure of the Ser1 gene. From these and previous data, it was possible to deduce the sequence of the sericins 1 and to predict the secondary structure and physiochemical properties of the different regions of the proteins.

Varying length cDNAs encoding the N-terminal nucleotide-binding domain (NBD1) from mouse mdr1 P-glyco- protein were prepared on the basis of structure predictions. Corresponding recombinant proteins were overexpressed in Escherichia coli, and the shortest one containing amino acids 395-581 exhibited the highest solubility. Insertion of an N-terminal hexahistidine tag allowed domain purification by nickel-chelate affinity chromatography. NBD1 efficiently interacted with nucleotides. Fluorescence methods showed that ATP bound at millimolar concentrations and its 2',3'-O-(2,4,6-trinitrophenyl) derivative at micromolar concentrations, while the 2'(3')-N-methylanthraniloyl derivative had intermediate affinity. Photoaffinity labeling was achieved upon irradiation with 8-azido-ATP. The domain exhibited ATPase activity with a Km for MgATP in the millimolar range, and ATP hydrolysis was competitively inhibited by micromolar 2',3'-O-(2,4,6-trinitrophenyl)-ATP. NBD1 contained a single cysteine residue, at position 430, that was derivatized with radiolabeled N-ethylmaleimide. Cysteine modification increased 6-fold the Kd for 2'(3')-N-methylanthraniloyl-ATP and prevented 8-azido-ATP photolabeling. ATPase activity was inhibited with a 5-fold increase in the Km for MgATP. The results suggest that chemical modification of Cys-430 is involved in the N-ethylmaleimide inhibition of whole P-glycoprotein by altering substrate interaction.

The secondary structure of a conserved non-collagenous module in alpha 1(V), alpha 1(XI), alpha 1(IX), alpha 1(XII), alpha 1(XIV) and alpha 1(XVI) collagen chains and in proline- and arginine-rich protein was analyzed using different algorithms. The results predict that a common anti-parallel beta-sheet structure composed of nine consensus beta-strands is present in these non-collagenous modules. A model for the packing of these beta-sheets is proposed which suggests that the predicted beta-sheet structure may be involved in molecular recognition functions.

a single tertiary center. The date and cause of death were recorded and checked using the national death registry. Results: Baseline characteristics of 361 included patients were: age: 60.0±9.8 yrs, male: 71.7%, Child Pugh score: 6.1±1.8 (A: 69.7%, B: 23.3%, C: 7.0%), Meld score: 11.3±4.5, follow-up: 3.1±1.4 yrs, death rate: 28.8% (liver related death in 62.5%). The 144 patients with non-selective BB were compared to the 190 controls without BB; 27 patients with selective BB were excluded. At baseline, there were higher frequencies of esophageal (p < 0.001) or gastric varices (p = 0.019) and alcohol withdrawal (p = 0.001) in the BB group. Child–Pugh (p =0.56) and Meld (p =0.25) scores did not differ. The independent predictors of overall survival were Meld (p < 0.001) and age (p =0.028) by Cox model. The independent predictors of liver related survival included Meld (p < 0.001) and gastric varices (p = 0.026), and a significant interaction between Meld and BB (p =0.035). Therefore, we assessed the BB effect according to baseline Meld score: in the lowest tertile, BB group had an improved overall survival as compared to controls (logrank: p =0.071); a beneficial BB effect was also observed in the second tertile until 4 years of follow-up; in contrast, in the highest tertile, patients treated with BB had decreased survival beyond 2 years (p = 0.095). Liver survival showed the same trends in the first (p = 0.140) and second (p =0.306) tertiles; in the highest tertile, the poorer survival in BB group was more pronounced than for overall survival (p = 0.019). Conclusion: The BB effect on survival differed according to liver disease severity in alcoholic cirrhosis: in good condition patients, BB tended to improve survival during the first years whereas there was a significantly decreased liver survival in the most severe patients.

Resume Connaitre la sequence des proteines constitue une premiere etape indispensable, mais insuffisante, pour comprendre leur role dans l'organisme. Afin d'avancer dans cette voie, encore faut-il determiner leur structure dans l'espace. La bio-informatique a un role a jouer, en proposant une plethore de methodes.

Pig heart mitochondrial membranes depleted of F1 and OSCP by various treatments were analyzed for their content in alpha and beta subunits of F1 and in OSCP using monoclonal antibodies. Membrane treatments and conditions of rebinding of F1 and OSCP were optimized to reconstitute efficient NADH- and ATP-dependent proton fluxes, ATP synthesis and oligomycin-sensitive ATPase activity. F1 and OSCP can be rebound independently to depleted membranes but to avoid unspecific binding of F1 to depleted membranes (ASUA) which is not efficient for ATP synthesis, F1 must be rebound before the addition of OSCP. The rebinding of OSCP to depleted membranes reconstituted with F1 inhibits the ATPase activity of rebound F1, while it restores the ATP-driven proton flux measured by the quenching of ACMA fluorescence. The rebinding of OSCP also renders the ATPase activity of bound F1 sensitive to uncouplers. The rebinding of OSCP alone or F1 alone, does not modify the NADH-dependent proton flux, while the rebinding of both F1 and OSCP controls this flux, inducing an inhibition of the rate of NADH oxidation. Similarly, oligomycin, which seals the F0 channel even in the absence of F1 and OSCP, inhibits the rate of NADH oxidation. OSCP is required to adjust the fitting of F1 to F0 for a correct channelling of protons efficient for ATP synthesis. All reconstituted energy-transfer reactions reach their optimal value for the same amount of OSCP. This amount is consistent with a stoichiometry of two OSCP per F1 in the F0-F1 complex.