383 research outputs found
The light chain of tetanus toxin inhibits calcium-dependent vasopressin release from permeabilized nerve endings
The effects of tetanus toxin and its light and heavy chain subunits on vasopressin release were investigated in digitonin-permeabilized neurosecretory nerve terminals isolated from the neural lobe of the rat pituitary gland. Exocytosis was induced by challenging the permeabilized nerve endings with micromolar calcium concentrations. Tetanus toxin inhibited vasopressin release only in the presence of the reducing agent dithiothreitol. This effect was irreversible. The purified light chain of tetanus toxin strongly inhibited exocytosis in a dose-dependent manner with half-maximal effect at c. 10 nM. The action of the light chain was observed after only 2.5 min of preincubation. Separated heavy chain subunit had no effect on hormone secretion. Inhibition of vasopressin release could be prevented by preincubating the light chain of tetanus toxin with an immune serum against tetanus toxin.
The data clearly demonstrate that in mammalian neurosecretory nerve endings tetanus toxin acts at a step downstream from the activation by Ca2+ of the exocytotic machinery and that the functional domain of this toxin is confined to its light chain
Synaptobrevin cleavage by the tetanus toxin light chain is linked to the inhibition of exocytosis in chromaffin cells
Exocytosis of secretory granules by adrenal chromaffin cells is blocked by the tetanus toxin light chain in a zinc specific manner. Here we show that cellular synaptobrevin is almost completely degraded by the tetanus toxin light chain within 15 min. We used highly purified adrenal secretory granules to show that synaptobrevin, which can be cleaved by the tetanus toxin light chain, is localized in the vesicular membrane. Proteolysis of synaptobrevin in cells and in secretory granules is reversibly inhibited by the zinc chelating agent dipicolinic acid. Moreover, cleavage of synaptobrevin present in secretory granules by the tetanus toxin light chain is blocked by the zinc peptidase inhibitor captopril and by synaptobrevin derived peptides. Our data indicate that the tetanus toxin light chain acts as a zinc dependent protease that cleaves synaptobrevin of secretory granules, an essential component of the exocytosis machinery in adrenal chromaffin cells
Introduction of Macromolecules into Bovine Adrenal Medullary Chromaffin Cells and Rat Pheochromocytoma Cells (PC12) by Permeabilization with Streptolysin O: Inhibitory Effect of Tetanus Toxin on Catecholamine Secretion
Conditions are described for controlled plasma membrane permeabilization of rat pheochromocytoma cells (PC12) and cultured bovine adrenal chromaffin cells by Streptolysin O (SLO). The transmembrane pores created by SLO invoke rapid efflux of intracellular 86Rb+ and ATP, and also permit passive diffusion of proteins, including immunoglobulins, into the cells. SLO-permeabilized PC12 cells release [3H]dopamine in response to micromolar concentrations of free Ca2+. Permeabilized adrenal chromaffin cells present a similar exocytotic response to Ca2+ in the presence of Mg2+/ ATP. Permeabilized PC12 cells accumulate antibodies against synaptophysin and calmodulin, but neither antibody reduces the Ca2+-dependent secretory response. Reduced tetanus toxin, although ineffective when applied to intact chromaffin cells, inhibits Ca2+-induced exocytosis by both types of permeabilized cells studied. Omission of dithiothreitol, toxin inactivation by boiling, or preincubation with neutralizing antibodies abolishes the inhibitory effect. The data indicate that plasma membrane permeabilization by Streptolysin O is a useful tool to probe and define cellular components that are involved in the final steps of exocytosis
GTP and Ca2+ Modulate the Inositol 1,4,5-Trisphosphate-Dependent Ca2+ Release in Streptolysin O-Permeabilized Bovine Adrenal Chromaffin Cells
The inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release was studied using streptolysin O-permeabilized bovine adrenal chromaffin cells. The IP3-induced Ca2+ release was followed by Ca2+ reuptake into intracellular compartments. The IP3-induced Ca2+ release diminished after sequential applications of the same amount of IP3. Addition of 20 μM GTP fully restored the sensitivity to IP3. Guanosine 5'-O-(3-thio)triphosphate (GTPγS) could not replace GTP but prevented the action of GTP. The effects of GTP and GTPγS were reversible. Neither GTP nor GTPγS induced release of Ca2+ in the absence of IP3. The amount of Ca2+ whose release was induced by IP3 depended on the free Ca2+ concentration of the medium. At 0.3 μM free Ca2+, a half-maximal Ca2+ release was elicited with ∼0.1 μM IP3. At 1 μM free Ca2+, no Ca2+ release was observed with 0.1 μM IP3; at this Ca2+ concentration, higher concentrations of IP3 (0.25 μM) were required to evoke Ca2+ release. At 8 μM free Ca2+, even 0.25 μM IP3 failed to induce release of Ca2+ from the store. The IP3-induced Ca2+ release at constant low (0.2 μM) free Ca2+ concentrations correlated directly with the amount of stored Ca2+. Depending on the filling state of the intracellular compartment, 1 mol of IP3 induced release of between 5 and 30 mol of Ca2+
Further Characterization of Dopamine Release by Permeabilized PC 12 Cells
Rat pheochromocytoma cells (PC 12) permeabilized with staphylococcal α-toxin release [3H]dopamine after addition of micromolar Ca2+. This does not require additional Mg2+-ATP (in contrast to bovine adrenal medullary chromaffin cells). We also observed Ca2+-dependent [3H]-dopamine release from digitonin-permeabilized PC 12 cells. Permeabilization with α-toxin or digitonin and stimulation of the cells were done consecutively to wash out endogenous Mg2+-ATP. During permeabilization, ATP was removed effectively from the cytoplasm by both agents but the cells released [3H]dopamine in response to micromolar Ca2+ alone. Replacement by chloride of glutamate, which could sustain mitochondrial ATP production in permeabilized cells, does not significantly alter catecholamine release induced by Ca2+. However, Mg2+ without ATP augments the Ca2+-induced release. The release was unaltered by thiol-, hydroxyl-, or calmodulin-interfering substances. Thus Mg2+-ATP, calmodulin, or proteins containing -SH or -OH groups are not necessary for exocytosis in permeabilized PC 12 cells
Additive Nonparametric Reconstruction of Dynamical Systems from Time Series
We present a nonparametric way to retrieve a system of differential equations
in embedding space from a single time series. These equations can be treated
with dynamical systems theory and allow for long term predictions. We
demonstrate the potential of our approach for a modified chaotic Chua
oscillator.Comment: accepted for Phys. Rev. E, Rapid Com
Functional characterization of the catalytic site of the tetanus toxin light chain using permeabilized adrenal chromaffin cells
The molecular events underlying the inhibition of exocytosis by tetanus toxin were investigated in permeabilized adrenal chromaffin cells. We found that replacement of amino acid residues within the putative zinc binding domain of the tetanus toxin light chain such as of histidine (position 233) by cysteine or valine, or of glutamate (position 234) by glutamine completely abolished the effect of the light chains on Ca2+ induced catecholamine release. Dipicolinic acid, a strong chelating agent for zinc, also prevented the effect of the tetanus toxin light chain. Zn2+ and, less potently Cu2+ and Ni2+, but not Cd2+ and Co2+, restored the activity of the neurotoxin. These data show that zinc and the putative zinc binding domain constitute the active site of the tetanus toxin light chain. Neither captopril, an inhibitor of synaptobrevin cleavage nor peptides spanning the site of synaptobrevins cleaved by the tetanus toxin in neurons, prevented the inhibition of Ca2+ induced catecholamine release by the tetanus toxin light chain. This suggests that synaptobrevins are not a major target of tetanus toxin in adrenal chromaffin cells
Self-assembly, modularity and physical complexity
We present a quantitative measure of physical complexity, based on the amount
of information required to build a given physical structure through
self-assembly. Our procedure can be adapted to any given geometry, and thus to
any given type of physical system. We illustrate our approach using
self-assembling polyominoes, and demonstrate the breadth of its potential
applications by quantifying the physical complexity of molecules and protein
complexes. This measure is particularly well suited for the detection of
symmetry and modularity in the underlying structure, and allows for a
quantitative definition of structural modularity. Furthermore we use our
approach to show that symmetric and modular structures are favoured in
biological self-assembly, for example of protein complexes. Lastly, we also
introduce the notions of joint, mutual and conditional complexity, which
provide a useful distance measure between physical structures.Comment: 9 pages, submitted for publicatio
Molecular Aspects of Secretory Granule Exocytosis by Neurons and Endocrine Cells
Neuronal communication and endocrine signaling are fundamental for integrating
the function of tissues and cells in the body. Hormones released by endocrine
cells are transported to the target cells through the circulation. By contrast, transmitter
release from neurons occurs at specialized intercellular junctions, the synapses.
Nevertheless, the mechanisms by which signal molecules are synthesized,
stored, and eventually secreted by neurons and endocrine cells are very similar.
Neurons and endocrine cells have in common two different types of secretory
organelles, indicating the presence of two distinct secretory pathways. The synaptic
vesicles of neurons contain excitatory or inhibitory neurotransmitters, whereas the
secretory granules (also referred to as dense core vesicles, because of their electron
dense content) are filled with neuropeptides and amines. In endocrine cells, peptide
hormones and amines predominate in secretory granules. The function and content
of vesicles, which share antigens with synaptic vesicles, are unknown for most
endocrine cells. However, in B cells of the pancreatic islet, these vesicles contain
GABA, which may be involved in intrainsular signaling.'
Exocytosis of both synaptic vesicles and secretory granules is controlled by
cytoplasmic calcium. However, the precise mechanisms of the subsequent steps,
such as docking of vesicles and fusion of their membranes with the plasma membrane,
are still incompletely understood. This contribution summarizes recent observations
that elucidate components in neurons and endocrine cells involved in
exocytosis. Emphasis is put on the intracellular aspects of the release of secretory
granules that recently have been analyzed in detail
Genetic-Algorithm-based Light Curve Optimization Applied to Observations of the W UMa star BH Cas
I have developed a procedure utilizing a Genetic-Algorithm-based optimization
scheme to fit the observed light curves of an eclipsing binary star with a
model produced by the Wilson-Devinney code. The principal advantages of this
approach are the global search capability and the objectivity of the final
result. Although this method can be more efficient than some other comparably
global search techniques, the computational requirements of the code are still
considerable. I have applied this fitting procedure to my observations of the W
UMa type eclipsing binary BH Cassiopeiae. An analysis of V-band CCD data
obtained in 1994/95 from Steward Observatory and U- and B-band photoelectric
data obtained in 1996 from McDonald Observatory provided three complete light
curves to constrain the fit. In addition, radial velocity curves obtained in
1997 from McDonald Observatory provided a direct measurement of the system mass
ratio to restrict the search. The results of the GA-based fit are in excellent
agreement with the final orbital solution obtained with the standard
differential corrections procedure in the Wilson-Devinney code.Comment: 9 pages, 2 figures, 2 tables, uses emulateapj.st
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