The DnaK-DnaJ-GrpE chaperone system activates inert wild type pi initiator protein of R6K into a form active in replication initiation

The plasmid R6K is an interesting model system for investigating initiation of DNA replication, not only near the primary binding sites of the initiator protein pi but also at a distance, caused by pi -mediated DNA looping. An important milestone in the mechanistic analysis of this replicon was the development of a reconstituted replication system consisting of 22 different highly purified proteins (Abhyankar, M. A., Zzaman, S., and Bastia, D. (2003) J. Biol. Chem. 278, 45476-45484). Although the in vitro reconstituted system promotes ori gamma-specific initiation of replication by a mutant form of the initiator called pi*, the wild type (WT) pi is functionally inert in this system. Here we show that the chaperone DnaK along with its co-chaperone DnaJ and the nucleotide exchange factor GrpE were needed to activate WT pi and caused it to initiate replication in vitro at the correct origin. We show further that the reaction was relatively chaperone-specific and that other chaperones, such as ClpB and ClpX, were incapable of activating WT pi. The molecular mechanism of activation appeared to be a chaperone-catalyzed facilitation of dimeric inert WT pi into iteron-bound monomers. Protein-protein interaction analysis by enzyme-linked immunosorbent assay revealed that, in the absence of ATP, DnaJ directly interacted with pi but its binary interactions with DnaK and GrpE and with ClpB and ClpX were at background levels, suggesting that pi is recruited by protein-protein interaction with DnaJ and then fed into the DnaK chaperone machine to promote initiator activation.     

Pressure effects on the structure and function of human thioredoxin

Thioredoxin is one of the major proteins that catalyze disulfide reduction and defines the thioredoxin superfamily bearing the CXXC structural motif. Human thioredoxin contains only 1 Trp residue proximal to the active site (WCGPC). We are interested in thioredoxin structure-function relationships, in particular, active site hydration and flexibility. Hence, in this study, we used hydrostatic pressure as a perturbation and monitored the conformational changes around the active site of thioredoxin by analyzing Trp fluorescence. The structure of thioredoxin was drastically altered by increasing pressure and did not completely refold after pressure release. The conformation in the active site vicinity was modified at low pressure (less than 100 MPa) and the Trp residue was completely exposed to aqueous medium at pressures above 350 MPa. Upon pressure release, thioredoxin showed no activity, although it folded 80% of the alpha-helical content relative to the native state. According to these results, pressure denaturation induces critical damage for the activity of thioredoxin, indicating extreme fragility of the active site with respect to pressure. This result is in contrast to the pressure effect on protein disulfide isomerase (PDI) which is organized by four thioredoxin-like domains including two WCGHC motifs.     

Promotion of insulin aggregation by protein disulfide isomerase

We examined the aggregation of insulin as a result of reduction of disulfide bonds catalyzed by protein disulfide isomerase (PDI) using various techniques. We demonstrated the kinetic correlation between PDI-catalyzed insulin reduction and the aggregate formation, the relationship between aggregation and amyloid formation, and the structural information on the secondary structure of the aggregates. The initial rate of PDI-catalyzed reduction of insulin, apparent rate constants of aggregate growth for sigmoidal features, and lag times were obtained by changing the PDI concentration, temperature, and insulin concentration. In situ kinetics were studied using the dyes; thioflavin T (ThT) and Congo red (CR) in addition to turbidimetry with the insulin reduction by PDI. The ThT and CR binding assay revealed sigmoidal kinetics, and the spectrum of binding CR showed a red shift against time, suggesting an orderly formation of insulin aggregates. The secondary structure of the PDI-promoted insulin aggregates showed antiparallel beta-sheet conformation by FT-IR measurement.     

Oligomeric initiator protein-mediated DNA looping negatively regulates plasmid replication in vitro by preventing origin melting

Although DNA looping between the initiator binding sites (iterons) of the replication origin (ori) of a plasmid and the iterons located in a cis-acting control sequence called inc has been postulated to promote negative control of plasmid DNA replication, not only was definitive evidence for such looping lacking, but also the detailed molecular mechanism of this control had not been elucidated. Here, we present direct evidence showing that both the monomeric and the dimeric forms of the RepE initiator protein of F factor together promote pairing of incC-oriF sites by DNA looping. By using a reconstituted replication system consisting of 26 purified proteins, we show further that the DNA loop formation negatively regulates plasmid replication by inhibiting the formation of an open complex at the replication origin, thus elucidating a key step of replication control.     

Production of cellulases from alkali-treated bagasse in Trichoderma reesei

Summary Most of the mutants of Trichoderma reesei had good cellulase productivity on Avicel but this was low on alkali-treated bagasse, which could be a most promising cellulosic biomass to use as an inexpensive carbon source for cellulase production. Two T. reesei mutants, PC-3-7 and X-31, in which strong cellulase activity is inducible by l-sorbose, were, however, found to produce cellulase on alkali-treated bagasse. They produced about 100 units of CMCase per ml in 5-1 jar fermentor culture with 4% alkali-treated bagasse as carbon source. They also showed higher cellulase productivity than other mutants on other easily saccharified substrates, such as alkali-treated rice straw and Walseth's cellulose.Production of Ethanol from Biomasses Part IV.Production of Ethanol from Biomasses Part IV.     

Analyse comparative de 3 logiciels de reconstruction itérative de tomoscintigraphie monophotonique myocardique

ObjectiveThis study makes a comparative analysis of the quality reconstruction of three software: 2D ordered subset expectation maximisation (2D OSEM), 3D ordered subsets expectation maximisation (Flash 3D) and Wallis.Patients and methodsThe data from myocardial scintigraphy acquisitions of 50 patients (38 men and 12 women; average age 61 ± 9 years) were successively reconstructed using three myocardial perfusion SPECT algorithms (Flash 3D, OSEM 2D and Wallis). Different combinations of iterations and subsets were considered. For Wallis, only the cut-off frequency was considered. The image's quality was assessed by determining the maximum contrast and the signal to noise ratio.ResultsThe Wallis software provided a higher signal to noise ratio compared to Flash 3D and OSEM 2D at stress and rest. The Wallis signal to noise ratio increased by a factor 1.93 (P = 0.0010) and 2.28 at stress (P = 0.0009); 1.50 (P = 0.0011) and 2.84 at rest (P = 0.0024) compared to respectively Flash 3D and OSEM 2D. Flash 3D provided better signal to noise ratio than OSEM 2D at stress and at rest. The difference in medians and interquartile ranges of the signal-to-noise ratio in post-stress were 22 % and 54 %, respectively between Flash 3D and OSEM 2D. At rest, the difference between the two methods in signal to noise ratio was 32 % ± 0.,29.ConclusionWallis algorithm was improve image quality with better signal to noise ratio compared to the reference method of Siemens Flash 3D. For both Flash 3D and OSEM 2D methods, the combination with 8 subsets and 12 iterations provided the best contrast and signal to noise ratio.     

Induction of mobility in mouse B-lymphocytes by acetylcholine and substances increasing the cellular level of cyclic GMP and calcium

Study of the effects of acetylcholine, exogenous cGMP and Con A on the mobility of B lymphocytes has demonstrated that an increase in both the content of cGMP and calcium in B lymphocytes stimulates their mobility. The measurement of intracellular content of cGMP in B lymphocytes by radioimmunoassay has shown that this content changes rapidly in response to the action of acetylcholine and that it is dependent but negligibly on extracellular calcium. The dependence of the effects of all three substances on trifluoperazine, a calmodulin v blocker, indicates that stimulation of B lymphocyte mobility is ultimately determined by the calcium mechanisms.