73,894 research outputs found
Vacuolar ATPase depletion contributes to dysregulation of endocytosis in bloodstream forms of Trypanosoma brucei
BACKGROUND
Vacuolar H-ATPase (V-ATPase) is a highly conserved protein complex which hydrolyzes ATP and pumps protons to acidify vacuolar vesicles. Beyond its role in pH maintenance, the involvement of V-ATPase in endocytosis is well documented in mammals and plants but is less clear in Trypanosoma brucei.
METHODS
In this study, the subcellular localization of V-ATPase subunit B (TbVAB) of T. brucei was assessed via in situ N-terminal YFP-tagging and immunofluorescence assays. Transgenic bloodstream forms (BSF) of T. brucei were generated which comprised either a V-ATPase subunit B (TbVAB) conditional knockout or a V-ATPase subunit A (TbVAA) knockdown. Acridine orange and BCECF-AM were employed to assess the roles of V-ATPase in the pH regulation of BSF T. brucei. The endocytic activities of three markers were also characterized by flow cytometry analyses. Furthermore, trypanosomes were counted from trypanolysis treatment groups (either containing 1% or 5% NHS) and endocytosed trypanosome lytic factor (TLF) was also analyzed by an immunoblotting assay.
RESULTS
TbVAB was found to localize to acidocalcisomes, lysosomes and probably also to endosomes of BSF of T. brucei and was demonstrated to be essential for cell growth. TbVAB depletion neutralized acidic organelles at 24 hours post-tetracycline depletion (hpd), meanwhile the steady state intracellular pH increased from 7.016 ± 0.013 to 7.422 ± 0.058. Trypanosomes with TbVAB depletion at 24 hpd were found to take up more transferrin (2.068 ± 0.277 fold) but less tomato lectin (49.31 ± 22.57%) by endocytosis, while no significant change was detected in dextran uptake. Similar endocytic dysregulated phenotypes were also observed in TbVAA knockdown cells. In addition, TbVAB depleted trypanosomes showed a low uptake of TLF and exhibited less sensitive to lysis in both 1% and 5% NHS treatments.
CONCLUSIONS
TbVAB is a key component of V-ATPase and was found to play a key function in endocytosis as well as exhibiting different effects in a receptor/cargo dependent manner in BSF of T. brucei. Besides vacuolar alkalinization, the dysregulation of endocytosis in TbVAB depleted T. brucei is considered to contribute to the reduced sensitivity to lysis by normal human serum
Brueckner-Hartree-Fock and its renormalized calculations for finite nuclei
We have performed self-consistent Brueckner-Hartree-Fock (BHF) and its
renormalized theory to the structure calculations of finite nuclei. The
-matrix is calculated within the BHF basis, and the exact Pauli exclusion
operator is determined by the BHF spectrum. Self-consistent occupation
probabilities are included in the renormalized Brueckner-Hartree-Fock (RBHF).
Various systematics and convergences are studies. Good results are obtained for
the ground-state energy and radius. RBHF can give a more reasonable
single-particle spectrum and radius. We present a first benchmark calculation
with other {\it ab initio} methods using the same effective Hamiltonian. We
find that the BHF and RBHF results are in good agreement with other
methods
Protocol: optimised electrophyiological analysis of intact guard cells from arabidopsis
Genetic resources available for Arabidopsis thaliana make this species particularly attractive as a model for molecular genetic studies of guard cell homeostasis, transport and signalling, but this facility is not matched by accessible tools for quantitative analysis of transport in the intact cell. We have developed a reliable set of procedures for voltage clamp analysis of guard cells from Arabidopsis leaves. These procedures greatly simplify electrophysiological recordings, extending the duration of measurements and scope for analysis of the predominant K+ and anion channels of intact stomatal guard cells to that achieved previously in work with Vicia and tobacco guard cells
Topological Weyl and Node-Line Semimetals in Ferromagnetic Vanadium-Phosphorous-Oxide -VOPO Compound
We propose that the topological semimetal features can co-exist with
ferromagnetic ground state in vanadium-phosphorous-oxide -VOPO
compound from first-principles calculations. In this magnetic system with
inversion symmetry, the direction of magnetization is able to manipulate the
symmetric protected band structures from a node-line type to a Weyl one in the
presence of spin-orbital-coupling. The node-line semimetal phase is protected
by the mirror symmetry with the reflection-invariant plane perpendicular to
magnetic order. Within mirror symmetry breaking due to the magnetization along
other directions, the gapless node-line loop will degenerate to only one pair
of Weyl points protected by the rotational symmetry along the magnetic axis,
which are largely separated in momentum space. Such Weyl semimetal phase
provides a nice candidate with the minimum number of Weyl points in a condensed
matter system. The results of surface band calculations confirm the non-trivial
topology of this proposed compound. This findings provide a realistic candidate
for the investigation of topological semimetals with time-reversal symmetry
breaking, particularly towards the realization of quantum anomalous Hall effect
in Weyl semimetals.Comment: 5 pages, 4 figure
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