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.     

Multiple factors determine the selection of the ectodomain cleavage site of human cell surface macrophage colony-stimulating factor

Human cell surface macrophage colony-stimulating factor (CSF-1256, M-CSF alpha) is converted to a soluble growth factor by a regulated proteolytic cleavage process at amino acid residues 157-159. We have previously shown that multiple factors specified by the juxtamembrane region determine the cleavage efficiency [Deng, P., Rettenmier, C. W., and Pattengale, P. K. (1996) J. Biol. Chem. 271, 16338-16343]. In the present paper, we studied the effect of various deletion, insertion, and substitution mutations at or near the cleavage site on both the number and size of cleaved CSF-1(256) products to identify the mechanisms by which the cleavage sites are selected. Deletion of regions 161-162 or 163-165, C-terminal to the cleavage site, as well as deletion of region 150-156, N-terminal to the cleavage site, each yielded a single cleavage product that was smaller than that derived from the wild type (WT). In these experiments cleavage apparently occurred at a specific distance from the transmembrane domain. Insertion of three additional residues between the normal cleavage site and the transmembrane domain yielded one major product that was the same size as the processed form of WT CSF-1(256). In this case the selection of the cleavage site was apparently determined by the amino acid sequence of the juxtamembrane region rather than by the distance from the transmembrane domain. Other amino acid substitutions at the cleavage site caused changes in cleavage site selection, providing additional evidence for the role of amino acid sequence in cleavage site selection. Finally, a comparison of cleavage site selection in the presence and absence of tunicamycin treatment showed that N-glycosylation of certain mutant forms of CSF-1(256) sterically interfered with protease accessibility, which in turn had an effect on the selection of the site used for cleavage. Taken together, these results indicate that cleavage site selection is determined by the amino acid sequence of the juxtamembrane region, the distance of the site from the transmembrane domain, and steric accessibility of the protease.     

Some factors affecting the transverse strength of repaired denture acrylic resin

The reappearance of strongyle eggs in faeces after treatment with ivermectin or pyrantel embonate was investigated in 22 foals, 36 yearlings, and 45 adult horses on five Dutch horse farms. The results confirmed earlier studies which showed an egg reappearance period of 9 and 6 weeks after ivermectin and pyrantel treatment, respectively. There were no differences between the egg reappearance periods of foals, yearlings, and adult horses. The mean egg counts of the yearlings were, however, consistently higher than the mean egg counts of the adult horses and foals in both ivermectin- and pyrantel-treated animals. It is concluded that shorter treatment intervals in foals and yearlings are not obligatory. However, longer intervals must be prevented in yearlings because their contribution to pasture contamination is relatively high.     

Effects of some chelating agents on urinary copper excretion by the rat

In order to estimate the potential advantages of new chelating agents which can enhance copper excretion in the chronic copper intoxication arising in Wilson's disease, the relative ability of none chelating agents to induce the urinary excretion of copper was compared with that of D-penicillamine (DPA) and triethylenetetramine.2HCl (TRIEN), all given ip at 1 mmol/kg to male Sprague-Dawley rats. The compounds examined were as follows: tris(2-aminoethyl)-amine.3HCl (TREN), tetraethylenepentamine.5HCl (TETREN), pentaethylenehexamine.6HCl (PENTEN), 1,4,7,11-tetraazaundecane.4HCl (TAUD), 1,5,8,12-tetraazadodecane.4HCl (TADD), 1-N-benzyltriethylenetetramine.4HCl (BzTT), 4,7,10,13-tetraazatridecanoic acid.2H2SO4 (TTPA), 1,10-bis(2-pyridylmethyl)-1,4,7,10-tetraazadecane.4HCl (BPTETA), and N,N-bis(2-pyridylmethyl)-4-(aminomethyl)benzoic acid (4ABA). Of these, BzTT, TTPA, and 4ABA are new chelating agents not previously reported. The factors by which these chelating agents enhanced copper excretion over control (untreated) levels were as follows: DPA, 7.2; TREN, 1.6; TRIEN, 4.0; TETREN, 10.1; PENTEN, 7.8; TAUD, 7.8; TADD, 2.6; TTPA, 5.6; BzTT, 1.8; and 4ABA, 5.5. The results indicate that it may well be possible to develop additional chelating agents which are equal or superior to those now used in the treatment of Wilson's disease, as well as structural types whose immunological properties may be significantly different from DPA or TRIEN, the compounds currently used in the clinic for this disorder.     

Glia, cytokines, and neurotoxicity

Activated glial cells (microglia and astrocytes) are a hallmark of several neurodegenerative disorders. A growing body of evidence supports the hypothesis that activation of glial cells by cytokines contributes to neurotoxicity. Although the precise mechanisms underlying glia-mediated neurotoxicity are unclear, it has been proposed that the generation of toxic free radicals or other neurotoxins is involved. This review focuses on the role of immune mediators released by activated glial cells in causing or preventing neuronal injury. Also, techniques used to assess neurotoxicity are discussed. It is hoped that research in this field will yield insights that will result in new therapies for neurodegenerative disorders.