The Rab5 effector rabankyrin-5 regulates and coordinates different endocytic mechanisms

The small GTPase Rab5 is a key regulator of clathrin-mediated endocytosis. On early endosomes, within a spatially restricted domain enriched in phosphatydilinositol-3-phosphate [PI(3)P], Rab5 coordinates a complex network of effectors that functionally cooperate in membrane tethering, fusion, and organelle motility. Here we discovered a novel PI(3)P-binding Rab5 effector, Rabankyrin-5, which localises to early endosomes and stimulates their fusion activity. In addition to early endosomes, however, Rabankyrin-5 localises to large vacuolar structures that correspond to macropinosomes in epithelial cells and fibroblasts. Overexpression of Rabankyrin-5 increases the number of macropinosomes and stimulates fluid-phase uptake, whereas its downregulation inhibits these processes. In polarised epithelial cells, this function is primarily restricted to the apical membrane. Rabankyrin-5 localises to large pinocytic structures underneath the apical surface of kidney proximal tubule cells, and its overexpression in polarised Madin-Darby canine kidney cells stimulates apical but not basolateral, non-clathrin-mediated pinocytosis. in demonstrating a regulatory role in endosome fusion and (macro) pinocytosis, our studies suggest that Rab5 regulates and coordinates different endocytic mechanisms through its effector Rabankyrin-5. Furthermore, its active role in apical pinocytosis in epithelial cells suggests an important function of Rabankyrin-5 in the physiology of polarised cells.

Fast Structure-Based Assignment of 15N HSQC Spectra of Selectively 15N-Labeled Paramagnetic Proteins

A novel strategy for fast NMR resonance assignment of N-15 HSQC spectra of proteins is presented. It requires the structure coordinates of the protein, a paramagnetic center, and one or more residue-selectively N-15-labeled samples. Comparison of sensitive undecoupled N-15 HSQC spectra recorded of paramagnetic and diamagnetic samples yields data for every cross-peak on pseudocontact shift, paramagnetic relaxation enhancement, cross-correlation between Curie-spin and dipole-dipole relaxation, and residual dipolar coupling. Comparison of these four different paramagnetic quantities with predictions from the three-dimensional structure simultaneously yields the resonance assignment and the anisotropy of the susceptibility tensor of the paramagnetic center. The method is demonstrated with the 30 kDa complex between the N-terminal domain of the epsilon subunit and the theta subunit of Escherichia Coll DNA polymerase III. The program PLATYPUS was developed to perform the assignment, provide a measure of reliability of the assignment, and determine the susceptibility tensor anisotropy.

Magma Flow Instabilities in a Volcanic Conduit: Implications for Long-Period Seismicity

Silicic volcanic eruptions are typically accompanied by repetitive Long-Period (LP) seismicity that originates from a small region of the upper conduit. These signals have the capability to advance eruption prediction, since they commonly precede a change in the eruption vigour. Shear bands forming along the conduit wall, where the shear stresses are highest, have been linked to providing the seismic trigger. However, existing computational models are unable to generate shear bands at the depths where the LP signals originate using simple magma strength models. Presented here is a model in which the magma strength is determined from a constitutive relationship dependent upon crystallinity and pressure. This results in a depth-dependent magma strength, analogous to planetary lithospheres. Hence, in shallow highly-crystalline regions a macroscopically discontinuous brittle type of deformation will prevail, whilst in deeper crystal-poor regions there will be a macroscopically continuous plastic deformation mechanism. This will result in a depth where the brittle-ductile transition occurs, and here shear bands disconnected from the free-surface may develop. We utilize the Finite Element Method and use axi-symmetric coordinates to model magma flow as a viscoplastic material, simulating quasi-static shear bands along the walls of a volcanic conduit. Model results constrained to the Soufrière Hills Volcano, Montserrat, show the generation of two types of shear bands: upper-conduit shear bands that form between the free-surface to a few 100 metres below it and discrete shear bands that form at the depths where LP seismicity is measured to occur corresponding to the brittle-ductile transition and the plastic shear region. It is beyond the limitation of the model to simulate a seismic event, although the modelled viscosity within the discrete shear bands suggests a failure and healing cycle time that supports the observed LP seismicity repeat times. However, due to the paucity of data and large parameter space available these results can only be considered to be qualitative rather than quantitative at this stage.

Compiling a GIS database of tree farms on Leyte Island

As part of ACIAR project ASEM/2003/052, Improving Financial Returns to Smallholder Tree Farmers in the Philippines, plantations of timber trees in Leyte Island, the Philippines were located using a systematic survey of the island. The survey was undertaken in order to compile a database of plantations which could be used to guide the planning of project activities. In addition to recording a range of qualitative and quantitative information for each plantation, the survey spatially referenced each site using a Global Positioning System (GPS) to electronic maps of the island which were held in a Geographical Information System (GIS). Microsoft Excel and Mapsource® software were used as the software links between GPS coordinates and the GIS. Mapping of farm positions was complicated by different datums being used for maps of Leyte Island and this caused GPS positions to be displaced from equivalent positions on the map. Photos of the sites were hyperlinked to their map positions in the GIS in order to assist staff to recall site characteristics.

The critical power function is dependent on the duration of the predictive exercise tests chosen

The linear relationship between work accomplished (W-lim) and time to exhaustion (t(lim)) can be described by the equation: W-lim = a + CP.t(lim). Critical power (CP) is the slope of this line and is thought to represent a maximum rate of ATP synthesis without exhaustion, presumably an inherent characteristic of the aerobic energy system. The present investigation determined whether the choice of predictive tests would elicit significant differences in the estimated CP. Ten female physical education students completed, in random order and on consecutive days, five art-out predictive tests at preselected constant-power outputs. Predictive tests were performed on an electrically-braked cycle ergometer and power loadings were individually chosen so as to induce fatigue within approximately 1-10 mins. CP was derived by fitting the linear W-lim-t(lim) regression and calculated three ways: 1) using the first, third and fifth W-lim-t(lim) coordinates (I-135), 2) using coordinates from the three highest power outputs (I-123; mean t(lim) = 68-193 s) and 3) using coordinates from the lowest power outputs (I-345; mean t(lim) = 193-485 s). Repeated measures ANOVA revealed that CPI123 (201.0 +/- 37.9W) > CPI135 (176.1 +/- 27.6W) > CPI345 (164.0 +/- 22.8W) (P < 0.05). When the three sets of data were used to fit the hyperbolic Power-t(lim) regression, statistically significant differences between each CP were also found (P < 0.05). The shorter the predictive trials, the greater the slope of the W-lim-t(lim) regression; possibly because of the greater influence of 'aerobic inertia' on these trials. This may explain why CP has failed to represent a maximal, sustainable work rate. The present findings suggest that if CP is to represent the highest power output that an individual can maintain for a very long time without fatigue then CP should be calculated over a range of predictive tests in which the influence of aerobic inertia is minimised.

Observations of QSO J2233-606 in the Southern Hubble Deep Field

The Hubble Deep Field South (HDF-S) Hubble Space Telescope (HST) observations are expected to begin in 1998 October. We present a composite spectrum of the QSO in the HDF-S held covering UV/optical/near-IR wavelengths, obtained by combining data from the Australian National University 2.3 m telescope with STIS on the HST.(1) This intermediate-resolution spectrum covers the range 1600-10000 Angstrom and allows us to derive some basic information on the intervening absorption systems which will be important in planning future higher resolution studies of this QSO. The QSO J2233 - 606 coordinates are alpha = 22(h)33(m)37(s).6, delta = -60 degrees 33'29 (J2000), the magnitude is B = 17.5, and its redshift is z(em) = 2.238, derived by simultaneously fitting several emission lines. The spectral index is alpha = -0.7 +/- 0.1, measured between the Ly alpha and Mg II emission lines. Many absorption systems are present, including systems with metal lines redward of the Ly alpha emission line at z(abs) 2.204, 1.942, 1.870, 1.787 and a few very strong Ly alpha features at z(abs) = 2.077, 1.928, without similarly strong metal lines. There is a conspicuous Lyman limit (LL) absorption system that is most likely associated with the z(abs) = 1.942 system with a neutral hydrogen column density of N-HI = (3.1 +/- 1.0) x 10(17) cm(-2). There is some evidence for the presence of a second LL absorber just to the blue of the conspicuous system at z = 1.870. We have employed a new technique, based on an analysis of the shape of the observed spectrum in the region of the LL absorption, to explore the properties of the gas. We tentatively conclude that this system might have suitable characteristics for measuring the deuterium-to-hydrogen (D/H) ratio.

Computation of bifurcation boundaries for power systems: A new Delta-plane method

This paper is devoted to the problems of finding the load flow feasibility, saddle node, and Hopf bifurcation boundaries in the space of power system parameters. The first part contains a review of the existing relevant approaches including not-so-well-known contributions from Russia. The second part presents a new robust method for finding the power system load flow feasibility boundary on the plane defined by any three vectors of dependent variables (nodal voltages), called the Delta plane. The method exploits some quadratic and linear properties of the load now equations and state matrices written in rectangular coordinates. An advantage of the method is that it does not require an iterative solution of nonlinear equations (except the eigenvalue problem). In addition to benefits for visualization, the method is a useful tool for topological studies of power system multiple solution structures and stability domains. Although the power system application is developed, the method can be equally efficient for any quadratic algebraic problem.

Field quantization, photons and non-Hermitean modes

Field quantization in unstable optical systems is treated by expanding the vector potential in terms of non-Hermitean (Fox-Li) modes. We define non-Hermitean modes and their adjoints in both the cavity and external regions and make use of the important bi-orthogonality relationships that exist within each mode set. We employ a standard canonical quantization procedure involving the introduction of generalized coordinates and momenta for the electromagnetic (EM) field. Three-dimensional systems are treated, making use of the paraxial and monochromaticity approximations for the cavity non-Hermitean modes. We show that the quantum EM field is equivalent to a set of quantum harmonic oscillators (QHOs), associated with either the cavity or the external region non-Hermitean modes, and thus confirming the validity of the photon model in unstable optical systems. Unlike in the conventional (Hermitean mode) case, the annihilation and creation operators we define for each QHO are not Hermitean adjoints. It is shown that the quantum Hamiltonian for the EM field is the sum of non-commuting cavity and external region contributions, each of which can be expressed as a sum of independent QHO Hamiltonians for each non-Hermitean mode, except that the external field Hamiltonian also includes a coupling term responsible for external non-Hermitean mode photon exchange processes. The non-commutativity of certain cavity and external region annihilation and creation operators is associated with cavity energy gain and loss processes, and may be described in terms of surface integrals involving cavity and external region non-Hermitean mode functions on the cavity-external region boundary. Using the essential states approach and the rotating wave approximation, our results are applied to the spontaneous decay of a two-level atom inside an unstable cavity. We find that atomic transitions leading to cavity non-Hermitean mode photon absorption are associated with a different coupling constant to that for transitions leading to photon emission, a feature consequent on the use of non-Hermitean mode functions. We show that under certain conditions the spontaneous decay rate is enhanced by the Petermann factor.

Propagation of nonstationary curved and stretched premixed flames with multistep reaction mechanisms

The propagation speed of a thin premixed flame disturbed by an unsteady fluid flow of a larger scale is considered. The flame may also have a general shape but the reaction zone is assumed to be thin compared to the flame thickness. Unlike in preceding publications, the presented asymptotic analysis is performed for a general multistep reaction mechanism and, at the same time, the flame front is curved by the fluid flow. The resulting equations define the propagation speed of disturbed flames in terms of the properties of undisturbed planar flames and the flame stretch. Special attention is paid to the near-equidiffusion limit. In this case, the flame propagation speed is shown to depend on the effective Zeldovich number Z(f) , and the flame stretch. Unlike the conventional Zeldovich number, the effective Zeldovich number is not necessarily linked directly to the activation energies of the reactions. Several examples of determining the effective Zeldovich number for reduced combustion mechanisms are given while, for realistic reactions, the effective Zeldovich number is determined from experiments. Another feature of the present approach is represented by the relatively simple asymptotic technique based on the adaptive generalized curvilinear system of coordinates attached to the flame (i.e., intrinsic disturbed flame equations [IDFE]).

Direct visualization of Ras proteins in spatially distinct cell surface microdomains

Localization of signaling complexes to specific micro-domains coordinates signal transduction at the plasma membrane. Using immunogold electron microscopy of plasma membrane sheets coupled with spatial point pattern analysis, we have visualized morphologically featureless microdomains including lipid rafts, in situ and at high resolution. We find that an inner-plasma membrane lipid raft marker displays cholesterol-dependent clustering in microdomains with a mean diameter of 44 nm that occupy 35% of the cell surface. Cross-linking an outer-leaflet raft protein results in the redistribution of inner leaflet rafts, but they retain their modular structure. Analysis of Ras microlocalization shows that inactive H-ras is distributed between lipid rafts and a cholesterol-independent micro-domain. Conversely, activated H-ras and K-ras reside predominantly in nonoverlapping, cholesterol-independent microdomains. Galectin-1 stabilizes the association of activated H-ras with these nonraft microdomains, whereas K-ras clustering is supported by farnesylation, but not geranylgeranylation. These results illustrate that the inner plasma membrane comprises a complex mosaic of discrete microdomains. Differential spatial localization within this framework can likely account for the distinct signal outputs from the highly homologous Ras proteins.

Chk1 regulates the S phase checkpoint by coupling the physiological turnover and ionizing radiation-induced accelerated proteolysis of Cdc25A

Chk1 kinase coordinates cell cycle progression and preserves genome integrity. Here, we show that chemical or genetic ablation of human Chk1 triggered supraphysiological accumulation of the S phase-promoting Cdc25A phosphatase, prevented ionizing radiation (IR)-induced degradation of Cdc25A, and caused radioresistant DNA synthesis (RDS). The basal turnover of Cdc25A operating in unperturbed S phase required Chk1-dependent phosphorylation of serines 123, 178, 278, and 292. IR-induced acceleration of Cdc25A proteolysis correlated with increased phosphate incorporation into these residues generated by a combined action of Chk1 and Chk2 kinases. Finally, phosphorylation of Chk1 by ATM was required to fully accelerate the IR-induced degradation of Cdc25A. Our results provide evidence that the mammalian S phase checkpoint functions via amplification of physiologically operating, Chk1-dependent mechanisms.

The preparation of zinc(II) and cadmium(II) complexes of the pentadentate N3S2 ligand formed from 2,6-diacetylpyridine and S-benzyldithiocarbazate (H2SNNNS) and the X-ray crystal structure of the novel dimeric [Zn2(SNNNS)2] complex

The pentadentate chelating agent, 2,6-diacetylpyridinebis(S-benzyldithiocarbazate) (H2SNNNS) reacts with zinc(II) and cadmium(II) ions forming stable complexes of empirical formula, [M(SNNNS)] (M=Zn2+, Cd2+; SNNNS2 =doubly deprotonated anionic form of the Schiff base). These complexes have been characterized by a variety of physico-chemical techniques. IR and H-1 NMR spectral evidence indicate that the Schiff base coordinates to the zinc(II) and cadmium(II) ions via the pyridine nitrogen atoms, the azomethine nitrogen atoms and the mercaptide sulfur atoms. The crystal and molecular structure of the zinc(II) complex has been determined by X-ray diffraction. The complex is a dimer in which the pyridine nitrogen atom,the azomethine nitrogen atom and the thiolate sulfur atom from one ligand coordinate to one of the zinc(II) ions whereas the azomethine and thiolate sulfur atoms from another ligand complete pentacoordination around the zinc(II) ion, the ligands being coordinated in their deprotonated forms. The coordination geometry about each zinc(II) can be considered as intermediate between a square-pyramid and trigonal-bipyramid. The cadmium(II) complex is also assigned with a dimeric structure. (C) 2003 Elsevier Ltd. All rights reserved.