Dynamics of Kv1 Channel Transport in Axons

Concerted actions of various ion channels that are precisely targeted along axons are crucial for action potential initiation and propagation, and neurotransmitter release. However, the dynamics of channel protein transport in axons remain unknown. Here, using time-lapse imaging, we found fluorescently tagged Kv1.2 voltage-gated K+ channels (YFP-Kv1.2) moved bi-directionally in discrete puncta along hippocampal axons. Expressing Kvβ2, a Kv1 accessory subunit, markedly increased the velocity, the travel distance, and the percentage of moving time of these puncta in both anterograde and retrograde directions. Suppressing the Kvβ2-associated protein, plus-end binding protein EB1 or kinesin II/KIF3A, by siRNA, significantly decreased the velocity of YFP-Kv1.2 moving puncta in both directions. Kvβ2 mutants with disrupted either Kv1.2-Kvβ2 binding or Kvβ2-EB1 binding failed to increase the velocity of YFP-Kv1.2 puncta, confirming a central role of Kvβ2. Furthermore, fluorescently tagged Kv1.2 and Kvβ2 co-moved along axons. Surprisingly, when co-moving with Kv1.2 and Kvβ2, EB1 appeared to travel markedly faster than its plus-end tracking. Finally, using fission yeast S. pombe expressing YFP-fusion proteins as reference standards to calibrate our microscope, we estimated the numbers of YFP-Kv1.2 tetramers in axonal puncta. Taken together, our results suggest that proper amounts of Kv1 channels and their associated proteins are required for efficient transport of Kv1 channel proteins along axons

Interaction of microtubules and actin during the post-fusion phase of exocytosis

Exocytosis is the intracellular trafficking step where a secretory vesicle fuses with the plasma membrane to release vesicle content. Actin and microtubules both play a role in exocytosis; however, their interplay is not understood. Here we study the interaction of actin and microtubules during exocytosis in lung alveolar type II (ATII) cells that secrete surfactant from large secretory vesicles. Surfactant extrusion is facilitated by an actin coat that forms on the vesicle shortly after fusion pore opening. Actin coat compression allows hydrophobic surfactant to be released from the vesicle. We show that microtubules are localized close to actin coats and stay close to the coats during their compression. Inhibition of microtubule polymerization by colchicine and nocodazole affected the kinetics of actin coat formation and the extent of actin polymerisation on fused vesicles. In addition, microtubule and actin cross-linking protein IQGAP1 localized to fused secretory vesicles and IQGAP1 silencing influenced actin polymerisation after vesicle fusion. This study demonstrates that microtubules can influence actin coat formation and actin polymerization on secretory vesicles during exocytosis

DNA damage by lipid peroxidation products: implications in cancer, inflammation and autoimmunity

Oxidative stress and lipid peroxidation (LPO) induced by inflammation, excess metal storage and excess caloric intake cause generalized DNA damage, producing genotoxic and mutagenic effects. The consequent deregulation of cell homeostasis is implicated in the pathogenesis of a number of malignancies and degenerative diseases. Reactive aldehydes produced by LPO, such as malondialdehyde, acrolein, crotonaldehyde and 4-hydroxy-2-nonenal, react with DNA bases, generating promutagenic exocyclic DNA adducts, which likely contribute to the mutagenic and carcinogenic effects associated with oxidative stress-induced LPO. However, reactive aldehydes, when added to tumor cells, can exert an anticancerous effect. They act, analogously to other chemotherapeutic drugs, by forming DNA adducts and, in this way, they drive the tumor cells toward apoptosis. The aldehyde-DNA adducts, which can be observed during inflammation, play an important role by inducing epigenetic changes which, in turn, can modulate the inflammatory process. The pathogenic role of the adducts formed by the products of LPO with biological macromolecules in the breaking of immunological tolerance to self antigens and in the development of autoimmunity has been supported by a wealth of evidence. The instrumental role of the adducts of reactive LPO products with self protein antigens in the sensitization of autoreactive cells to the respective unmodified proteins and in the intermolecular spreading of the autoimmune responses to aldehyde-modified and native DNA is well documented. In contrast, further investigation is required in order to establish whether the formation of adducts of LPO products with DNA might incite substantial immune responsivity and might be instrumental for the spreading of the immunological responses from aldehyde-modified DNA to native DNA and similarly modified, unmodified and/or structurally analogous self protein antigens, thus leading to autoimmunity

Apical sealing of root canal fillings performed with five different endodontic sealers: analysis by fluid filtration

OBJECTIVES: To evaluate the sealing ability of five root canal sealers, including two experimental cements (MBP and MTA-Obtura) using the fluid filtration method. MATERIAL AND METHODS: Teeth were divided into 5 study groups: G1-AH Plus; G2-Acroseal; G3Sealapex; G4-MBP; G5-MTA-Obtura; and two controls. Chemical-mechanical preparation was performed with ProFile rotary nickel-titanium instruments 1 mm short of the apical foramen. The sealing ability was evaluated by fluid filtration at 15, 30, and 60 days. RESULTS: The statistical analysis showed significant difference between the materials at different periods (p<0.05). AH Plus and MBP had similar leakage values at 15 and 60 days, alternating with significant reduction at 30 days, while the other materials showed progressive increase in leakage values. Acroseal and Sealapex presented the best results at 15 days and the worst at 60 days. CONCLUSIONS: All sealers evaluated presented fluid leakage, with AH Plus and MBP showing the best results at the end of the experimental period. Acroseal, Sealapex, and MTA-Obtura presented increase in leakage values at longer observation periods