Kinetochores distinguish GTP from GDP forms of the microtubule lattice

During prometaphase in mitotic cell division, chromosomes attach to the walls of microtubules and subsequently move to microtubule ends, where they stay throughout mitosis. This end-attachment seems to be essential for correct chromosome segregating. However, the mechanism by which kinetochores, the multiprotein complexes that link chromosomes to the microtubules of the mitotic spindle, recognize and stay attached to microtubule ends is not understood. One clue comes from the hydrolysis of GTP that occurs during microtubule polymerization. Although tubulin dimers must contain GTP to polymerize, this GTP is rapidly hydrolysed following the addition of dimers to a growing polymer. This creates a microtubule consisting largely of GDP-tubulin, with a small cap of GTP-tubulin at the end. It is possible that kinetochores distinguish the different structural states of a GTP- versus a GDP-microtubule lattice. We have examined this question in vitro using reconstituted kinetochores from the yeast Saccharomyces cerevisiae. We found that kinetochores in vitro bind preferentially to GTP- rather than GDP-microtubules, and to the plus-end preferentially over the lattice. Our results could explain how kinetochores stay at microtubule ends and thus segregate chromosomes correctly during mitosis in vivo. This result demonstrates that proteins exist that can distinguish the GTP conformation of the microtubule lattice.     

Interaction of prion peptide HuPrP106-126 with nucleic acid

Synthetic prion peptide PrP106-126 has been used as a model to understand prion diseases. The conformation of the peptide depends on the environmental conditions and it forms amyloid in vitro. The potential of this prion peptide to interact with nucleic acids has been studied using a fluorescent labelled nucleic acid by kinetic and equilibrium methods. A decrease in the fluorescence of the labelled DNA induced by the peptide with time is observed which is pH, ionic strength and temperature dependent. The activation energy of the reactions is approximately 100 kJ mol-1. Lysine tripeptide and spermidine, carrying the same number of positive charges as the prion peptide, do not show an appreciable effect on the DNA. The binding constant between the prion peptide and DNA has a value of > 10(6) M-1 in phosphate buffer, pH 8 which is of the same order of magnitude as the binding of a retroviral protein, p10, with model nucleic acids. It is tempting to speculate that this interaction might play a role in the prion diseases.     

Liquid-liquid extraction and separation of total rare earth (RE) metals from polymetallic manganese nodule leaching solution

The study on the solvent extraction for quantitative and selective separation of total rare earth metals from the polymetallic nodule leach liquor was investigated. The typical leach liquor bearing 0. 094 g/L total rare earth, 0. 23 g/L Mn, 0.697 g/L Cu, 0.2 g/L Fe, 0.01 g/L Co and 0.735 g/L Ni was subjected to the removal iron content by precipitation method using Ca(OH)2 at pH 3.95, prior to solvent extraction of rare earth metals. Three different organo-phosphoric acid reagents(D2EHPA, PC88 A, Cyanex 272) were used to ascertain their performances and selectivity towards the loading of rare earth metals in presence of other base metals. Based on the results of eq. pH effect, the performances of above three extractants followed the order as: D2EHPA>PC88A>Cyanex 272. To ensure the absence of extraction of base metals(Cu, Co, Ni), the eq. pH of the solution was optimized at the level of 2.21, though higher rare earth metal extraction efficiency was observed at higher eq. pH with either of the extractants. The complete process flow diagram for substantial recovery of total rare earth was developed using D2 EHPA. Extraction isotherm plot was constructed at A:O=12:1, 3-stages and pHe=2.21, using 0.8 mol/L D2 EHPA and the predicted condition of this study was further confirmed by 6-Cycles Counter Current Simulation(CCS) study. The stripping of total rare earth from loaded organic phase(LO) was conducted using HCl solution. Mc-Cabe Thiele diagram study carried out at A:O=1:5 using 4 mol/L HCl showed that three theoretical stages were needed for quantitative stripping of total rare earth. The subsequent stripped solution resulted thus led to contain total rare earth of 5.6 g/L indicating a very high enrichment of total metals by solvent extraction(SX) process.     

Importance of the Arg-Gly-Asp triplet in human thrombin for maintenance of structure and function

Site-directed mutagenesis was employed to assess the importance of the Arg-Gly-Asp triplet that comprises residues 197 to 199 in the B-chain of thrombin. Properties of the R197E and the D199E variants were compared with those of zeta-thrombin and the inactive S205A variant wherein the active site Ser is replaced by Ala. Relative to zeta-thrombin, the R197E thrombin variant under the assay conditions used exhibits 26% activity toward a small chromogenic substrate, 13% activity in the activation of protein C in the presence of thrombomodulin, < 3% activity in processing fibrinogen, and 1% activity in inducing platelet activation. Thus, the substrate specificity of thrombin was altered by the R197-->E replacement. The D199E variant was essentially inactive. It exhibited only 0.02% of the activity of thrombin toward the chromogenic substrate and its reactivity toward the active site-directed alkylating agent D-Phe-Pro-Arg-CH2Cl was 10,000-fold lower than that of thrombin. Like the inactive S205A thrombin variant, the D199E variant antagonized the interactions of thrombin with hirudin and thrombomodulin, but was a less effective antagonist. The dependence of the antagonism of the thrombin-thrombomodulin interaction on the concentration of D199E thrombin variant provided evidence suggesting the presence of two or more domains in thrombin that independently interact with their counterparts in thrombomodulin. Although the S205A thrombin variant antagonized the action of thrombin on platelets no such activity could be demonstrated for the D199E variant in the concentration range studied (< 800 nm). Comparison of the circular dichroism spectra of zeta-thrombin, the D199E, R197E, and S205A variants indicated that subtle differences in conformation exist between the D199E variant and the other thrombins. These differences in conformation might well account for the altered behavior of the D199E variant with respect to its interactions toward thrombomodulin, hirudin, and platelets.     

Regional mapping panels for chromosomes 3, 4, 5, 11, 15, 17, 18, and X

The erm family of 23S rRNA adenine-N6-methyltransferases confers resistance to all macrolide-lincosamide-streptograminB (MLS) antibiotics, but not all MLS antibiotics induce synthesis of Erm methyltransferase with equal efficiency in a given organism. The induction efficiency of a test panel of MLS antibiotics was studied by using two translational attenuator-lac reporter gene fusion constructs, one based on ermSV from Streptomyces viridochromogenes NRRL 2860 and the other based on ermC from Staphylococcus aureus RN2442. Four types of responses which were correlated with the macrolide ring size were seen, as follows: group 1, both ermSV and ermC were induced by the 14-membered-ring macrolides erythromycin, lankamycin, and matromycin, as well as by the lincosamide celesticetin; group 2, neither ermSV nor ermC was induced by the 12-membered-ring macrolide methymycin or by the lincosamide lincomycin or the streptogramin type B antibiotic ostreogrycin B; group 3, ermSV was selectively induced over ermC by the 16-membered-ring macrolides carbomycin, chalcomycin, cirramycin, kitasamycin, maridomycin, and tylosin; and group 4, ermC was selectively induced over ermSV by the 14-membered-ring macrolide megalomicin. These data suggest that the leader peptide determines the specificity of induction by different classes of MLS antibiotics and that for a given attenuator, a major factor which determines whether a given macrolide induces resistance is its size.