Nek2 localises to the distal portion of the mother centriole/basal body and is required for timely cilium disassembly at the G2/M transition

The NIMA-related kinase Nek2 promotes centrosome separation at the G2/M transition and, consistent with this role, is known to be concentrated at the proximal ends of centrioles. Here, we show by immunofluorescence microscopy that Nek2 also localises to the distal portion of the mother centriole. Its accumulation at this site is cell cycle-dependent and appears to peak in late G2. These findings are consistent with previous data implicating Nek2 in promoting reorganisation of centrosome-anchored microtubules at the G2/M transition, given that microtubules are anchored at the subdistal appendages of the mother centriole in interphase. In addition, we report that siRNA-mediated depletion of Nek2 compromises the ability of cells to resorb primary cilia before the onset of mitosis, while overexpression of catalytically active Nek2A reduces ciliation and cilium length in serum-starved cells. Based on these findings, we propose that Nek2 has a role in promoting cilium disassembly at the onset of mitosis. We also present evidence that recruitment of Nek2 to the proximal ends of centrioles is dependent on one of its substrates, the centrosome cohesion protein C-Nap1.     

The analysis of nanogram levels of free amino acids by reverse-phase high-pressure liquid chromatography.

A simple method and apparatus are described for the efficient recovery of proteins from sodium dodecyl sulfate-polyacrylamide gel systems after electrophoretic resolution. This procedure provides for high yields of proteins which are free of sodium dodecyl sulfate and in certain cases, exhibit significant levels of biological activity.     

Stoichiometry of carbon dioxide release and oxygen uptake during glycine oxidation in mitochondria isolated from spinach (Spinacia oleracea) leaves.

Mitochondria isolated from spinach (Spinacia oleracea) leaves oxidized glycine with a stoichiometry of CO2 evolution to O2 uptake of 2 : 1. In the absence of added substrate, the mitochondria exhibited an extremely low endogenous rate of O2 uptake.     

TRANCE, a TNF family member, activates Akt/PKB through a signaling complex involving TRAF6 and c-Src

TRANCE, a TNF family member, and its receptor, TRANCE-R, are critical regulators of dendritic cell and osteoclast function. Here, we demonstrate that TRANCE activates the antiapoptotic serine/threonine kinase Akt/PKB through a signaling complex involving c-Src and TRAF6. A deficiency in c-Src or addition of Src family kinase inhibitors blocks TRANCE-mediated PKB activation in osteoclasts. c-Src and TRAF6 interact with each other and with TRANCE-R upon receptor engagement. TRAF6, in turn, enhances the kinase activity of c-Src leading to tyrosine phosphorylation of downstream signaling molecules such as c-Cbl. These results define a mechanism by which TRANCE activates Src family kinases and PKB and provide evidence of cross-talk between TRAF proteins and Src family kinases.     

Potent anti-tumor effects of an active site mutant of human manganese-superoxide dismutase. Evolutionary conservation of product inhibition

Mn-SOD serves as the primary cellular defense against oxidative damage by converting superoxide radicals (O(2)(-)) to O(2) and H(2)O(2). A unique characteristic of this mitochondrial anti-oxidant enzyme is the conservation from bacteria to man of a rapidly formed product inhibited state. Using site-directed mutagenesis, we have generated an active site mutant (H30N) of human Mn-SOD, which exhibits significantly reduced product inhibition and increased enzymatic efficiency. Overexpression of the H30N enzyme causes anti-proliferative effects in vitro and anti-tumor effects in vivo. Our results provide a teleological basis for the phylogenetically invariant nature of position His-30 and the evolutionary conservation of product inhibition. These data also provide more direct intracellular evidence for the signaling role associated with H(2)O(2).     

On the nature of the forces controlling selectivity in the high performance capillary electrochromatographic separation of peptides

In this minireview, the nature of the forces controlling selectivity in the high performance capillary electrochromatographic (HP-CEC) separation of peptides has been examined. For uncharged and charged peptides, a synergistic interplay occurs in HP-CEC systems between adsorptive/partitioning events and electrokinetically driven motion. Moreover, at high field strengths, both bulk electrophoretic migration and surface electrodiffusion occur. Thus, the migration behavior of peptides in different HP-CEC systems can be rationalized in terms of the combined consequences of these various processes. Moreover, in HP-CEC, the buffer electrolyte interacts with both the peptide analytes and the sorbent as bulk phenomena. These buffer-mediated processes control the solvational characteristics, ionization status and conformational behavior of the peptides as well as regulate the double-layer properties of the sorbent, and the ion flux and electro-osmotic flow characteristics of the HP-CEC system per se. These buffer electrolyte effects mediate mutual interactions between the peptide and the sorbent, irrespective of whether the interaction occurs at the surface of microparticles packed into a capillary, at the surface of a contiguous monolithic structure formed or inserted within the capillary or at the walls of the capillary as is the case with open tubular HP-CEC. Diverse molecular and submolecular forces thus coalesce to provide the basis for the different experimental modes under which HP-CEC can be carried out. As a consequence of this interplay, experimental parameters governing the separation of peptides in HP-CEC can be varied over a wide range of conditions, ensuring numerous options for enhanced selectivity, speed, and resolution of peptides. The focus of the peptide separation examples presented in this minireview has been deliberately restricted to the use of HP-CEC capillaries packed with n-alkyl-bonded silicas or mixed-mode strong ion exchange sorbents, although other types of sorbent chemistries can be employed. From these examples, several conclusions have been drawn related to the use of HP-CEC in the peptide sciences. These observations confirm that variation of a specific parameter, such as the pH or the content of the organic solvent modifier in the buffer electrolyte, simultaneously influences all other physicochemical aspects of the specific HP-CEC separation. Peptide selectivity in HP-CEC thus cannot be fine-tuned solely through the use of single parameter optimization methods. In this context, HP-CEC differs significantly from the analogous reverse phase high performance liquid chromatography (RP-HPLC) procedures with peptides. Rather, more sophisticated multiparameter optimization procedures, involving knowledge of (a) the field strength polarity, (b) its contour and flux characteristics, (c) effects of buffer electrolyte composition and pH, (e) the influence of the temperature, and (f) the impact of the sorbent characteristics, are required if the full capabilities offered by HP-CEC procedures are to be exploited. In this minireview, the HP-CEC migration behavior of several different sets of synthetic peptides has been examined, and general guidelines elaborated from these fundamental considerations to facilitate the interpretation and modulation of peptide selectivity in HP-CEC.     

Humoral immune response in aspergillosis: an immunodominant glycoprotein of 35 kDa from Aspergillus flavus

A glycoprotein preparation containing 70% carbohydrates and 30% proteins was isolated from the mycelium of two strains of Aspergillus flavus and fractionated by ConA-Sepharose affinity chromatography. An immunodominant 35-kDa antigen was detected in a ConA-bound fraction (B fraction). It contained mannose and galactose in a 1.4:1.0 ratio. This antigen seems to be able to elicit an antibody response in patients with aspergillosis and in rabbits immunized with A. flavus whole cells. The carbohydrate units of the BF fraction appeared to be responsible for the antigenicity, since treatment with periodate removed most of the antibody binding capacity.     

The uracil transporter Fur4p associates with lipid rafts

Sphingolipids are abundant components of eucaryotic membranes, where they perform essential functions. To uncover new roles for sphingolipids, we studied Saccharomyces cerevisiae lcb1-100 cells, which have a temperature-sensitive block in the first step in sphingolipid synthesis. We find that the level of all five species of the sphingoid long chain base intermediates is reduced 2-7-fold in cells grown at a permissive temperature, and the level of complex sphingolipids is reduced 50%. In addition, lcb1-100 cells make no detectable phosphorylated sphingoid bases. After transfer to a restrictive temperature (a heat shock), the level of the major sphingoid bases drops rather than transiently rising, as in wild type cells. These changes affect lcb1-100 cells in multiple ways. Basal uracil transport by Fur4p is reduced 25%, and when cells are heat-shocked, uracil transport activity falls rapidly and is not restored as it is in wild type cells. Restoration requires a functional secretory pathway and synthesis of complex sphingolipids, leading us to hypothesize that Fur4p associates with lipid rafts. The finding that Fur4p is insoluble in TritonX-100 at 4 degrees C and behaves like a raft-associated protein on a density gradient supports this hypothesis. Raft association may be essential for regulating breakdown of Fur4p in response to stresses and other factors that govern uracil transport activity. Our results show that long chain bases do not contribute to the inactivation of Fur4p transport activity after heat stress, but they are essential for some later, but unknown, process that leads to degradation of the protein. Further studies using lcb1-100 cells should reveal new roles of sphingolipids in nutrient uptake and other membrane-dependent processes.