We applied the quick-freezing technique to investigate precise temporal coincidence between onset of quantal secretion and appearance fusions synaptic vesicles with prejunctional membrane. Frog cutaneous pectoris nerve-muscle preparations were soaked in modified Ringer's solution 1 mM 4-aminopyridine, 10 Ca2+, 10(-4) M d-Tubocurarine quick-frozen 1-10 ms after a single supramaximal shock. The frozen muscles then either freeze-fractured or cryosubstituted acetone 13% OsO4 processed for thin...

Black widow spider venom (BWSV) was applied to frog nerve-muscle preparations bathed in Ca2+-containing, or Ca2+-free, solutions and the neuromuscular junctions were studied by freeze-fracture technique. When BWSV for short periods (10-15 min) presence of Ca2+, numerous dimples (P face) protuberances (E appeared on presynaptive membrane approximately 86% located immediately adjacent double rows large intramembrane particles that line active zones. 1 h nerve terminals depleted vesicles, few...

Frog cutaneous pectoris nerve muscle preparations were studied by the freeze-fracture technique under following conditions: (a) during repetitive indirect stimulation for 20 min, 10/s; (b) recovery from this stimulation; and (c) treatment with mM K+. Indirect causes numerous dimples or protuberances to appear on presynaptic membrane of terminal, most are located near active zones. Deep infoldings axolemma often develop between Neither number nor distribution dimples, protuberances, changes...

Synapsin I is a synaptic vesicle-associated protein which inhibits neurotransmitter release, an effect abolished upon its phosphorylation by Ca2+/calmodulin-dependent kinase II (CaM II). Based on indirect evidence, it was suggested that this release may be achieved the reversible anchoring of vesicles to actin cytoskeleton nerve terminal. Using video-enhanced microscopy, we have now obtained experimental evidence in support model: presence dephosphorylated synapsin necessary for bind actin;...

Release of Ca2+ from intracellular stores was studied in the parent PC12 cell line and recently isolated clones sensitive or insensitive to caffeine. In caffeine-sensitive cells cytosolic free concentration ([Ca2+]i) responses by xanthine drug stimulants receptors coupled inositol 1,4,5-trisphosphate (Ins-P3) generation (bradykinin, ATP) depend on separate pathways because 1) caffeine does not stimulate hydrolysis phosphatidylinositol 4,5-bisphosphate 2) Ca(2+)-induced release, process...

Astrocytes play a crucial role in proper iron handling within the central nervous system. This competence can be fundamental, particularly during neuroinflammation, and neurodegenerative processes, where an increase content favor oxidative stress, thereby worsening disease progression. Under these pathological conditions, astrocytes undergo process of activation that confers them either beneficial or detrimental on neuronal survival. Our work investigates mechanisms entry cultures quiescent...

The characterization of iron handling in neurons is still lacking, with contradictory and incomplete results. In particular, the relevance non-transferrin-bound (NTBI), under physiologic conditions, during aging neurodegenerative disorders, undetermined. This study investigates mechanisms underlying NTBI entry into primary hippocampal evaluates consequence elevation on neuronal viability. Fluorescence-based single cell analysis revealed that an increase extracellular free Fe(2+) (the main...

The calcium pools segregated within the endoplasmic reticulum, Golgi complex, exocytic, and other organelles are believed to participate in regulation of a variety cell functions. Until now, however, precise intracellular distribution element had not been established. Here, we report about first high-resolution mapping obtained neurosecretory PC12 cells by imaging mode electron energy loss spectroscopy technique. preparation procedure used included quick freezing monolayers, followed...

We employed induced pluripotent stem cell (iPSC)-derived neurons obtained from Friedreich ataxia (FRDA) patients and healthy subjects, FRDA CT neurons, respectively, to unveil phenotypic alterations related frataxin (FXN) deficiency investigate if they can be reversed by treatments that upregulate FXN. control iPSCs were equally capable of differentiating into a neuronal or astrocytic phenotype. showed lower levels iron–sulfur (Fe–S) lipoic acid-containing proteins, higher labile iron pool...

BackgroundMutations of the mitochondrial protein paraplegin cause hereditary spastic paraplegia type 7 (SPG7), a so-far untreatable degenerative disease upper motoneuron with still undefined pathomechanism.The intermittent permeability transition pore (mPTP) opening, called flickering, is an essential process that operates to maintain homeostasis by reducing intra-matrix Ca2+ and reactive oxygen species (ROS) concentration, critical for efficient synaptic function.MethodsWe use...

As the main beta-secretase of central nervous system, BACE-1 is a key protein in pathogenesis Alzheimer's disease. Excessive expression might cause an overproduction neurotoxic beta-amyloid peptide. Therefore, tight regulation expected vivo. In addition to possible transcriptional control, transcript leader contains features that constitute mechanisms translational expression. Moreover, recent work has revealed increase and activity some disease patients, although corresponding not been...

Calcitonin gene related peptide (CGRP) and adrenomedullin are potent biologically active peptides that have been proposed to play an important role in vascular inflammatory diseases. Their function the central nervous system is still unclear since they as either pro-inflammatory or neuroprotective factors. We investigated effects of two on astrocytes microglia, cells exert a strong modulatory activity neuroinflammatory processes. In particular, we studied ability CGRP modulate microglia...

Neuroferritinopathy is a rare genetic disease with dominant autosomal transmission caused by mutations of the ferritin light chain gene (FTL). It belongs to Neurodegeneration Brain Iron Accumulation, group disorders where iron dysregulation tightly associated neurodegeneration. We studied 498-499InsTC mutation which causes substitution last 9 amino acids and an elongation extra 16 at C-terminus L-ferritin peptide. An analysis cyclic voltammetry on purified protein showed that this structural...

Conventional protein kinase C (PKC) isoforms are abundant neuronal signaling proteins with important roles in regulating synaptic plasticity and other processes. Here, we investigate the role of ionotropic metabotropic glutamate receptor (iGluR mGluR, respectively) activation on generation Ca 2+ diacylglycerol (DAG) signals subsequent neuron-specific PKCγ isoform hippocampal neurons. By combining imaging total internal reflection microscopy analysis specific biosensors, show that elevation...

BACE1 is the protease responsible for production of amyloid-β peptides that accumulate in brain Alzheimer's disease (AD) patients. expression regulated at transcriptional, as well post-transcriptional level. Very high mRNA levels have been observed pancreas, but protein and activity were found mainly brain. An up-regulation has described some AD patients without a change transcript levels. The features 5′ untranslated region (5 ′ UTR), such length, GC content, evolutionary conservation...

1. Electrophysiology and morphology have been combined to investigate the time course of exocytosis quanta neurotransmitter induced by elevated concentrations K+ at frog neuromuscular junction. 2. Replicas freeze‐fractured resting nerve terminals fixed in presence 20 mM‐K+ showed images fusion synaptic vesicles with presynaptic axolemma which were closely associated active zones. After 1 min nM‐K+ fusions appeared also outside zones, 5 they became uniformly distributed over membrane. 3. The...

The divalent metal transporter 1 (DMT1) is the best characterized Fe²⁺ involved in cellular iron uptake mammals. Four possible isoforms have been identified as a result of alternative promoter (DMT1-1A and DMT1-1B) splicing involving C-terminus producing transcripts with or without an responsive element [DMT1-IRE⁺ DMT1-IRE⁻, respectively]. Despite general importance DMT1 controlling homeostasis, distribution role CNS still controversial. In this study, we characterize expression hippocampal...

Within pancreatic islet cells, rhythmic changes in the cytosolic Ca2+ concentration have been reported to occur response stimulatory glucose concentrations and be synchronous with pulsatile release of insulin. We explored possible mechanisms responsible for signal propagation within particular regard gap junction communication, pathway widely credited being coordination secretory activity. Using fura-2 imaging, we found that multiple control signaling cells. Gap blockade by 18...

Regulated exocytosis triggered by the photolysis of a caged Ca 2+ compound, DM-nitrophen, was investigated patch-clamp capacitance measurements in two clones PC12, first wild-type and second (PC12-27) defective both types classical secretory vesicles together with neuronal-type receptors for attachment proteins N-ethylmaleimide-sensitive fusion protein, so called SNAREs. Moreover, electrophysiological data were correlated ultrastructure resting quick-frozen–freeze-dried cells clones....

The small protein Bv8, secreted by the skin of frog Bombina variegata , belongs to a novel family proteins whose orthologues have been identified in snakes (MIT) and mammals (prokineticins (PKs)). A characteristic feature this is same N‐terminal sequence, AVITGA, presence 10 cysteines with identical spacing C‐terminal domain. Two closely related G protein‐coupled receptors that mediate signal transduction Bv8/PKs cloned (PK‐R1 PK‐R2). In mammals, Bv8/PK involved number biological activities...

Fura-2 imaging microscopy was used to study [Ca2+]i in nerve growth factor-differentiated PC12 cells exposed agonists (bradykinin, carbamylcholine, and ATP) binding receptors coupled polyphosphoinositide hydrolysis. With all the treatments employed, response an individual agonist often incomplete, i.e., composed of either release from intracellular stores or influx only. In responses were closely similar when only one same markedly heterogeneous, with considerable variation release/influx...

Abstract Background Astrocytes respond to local insults within the brain and spinal cord with important changes in their phenotype. This process, overall known as “activation”, is observed upon proinflammatory stimulation leads astrocytes acquire either a detrimental phenotype, thereby contributing neurodegenerative or protective thus supporting neuronal survival. Within mechanisms responsible for inflammatory neurodegeneration, oxidative stress plays major role has recently been recognized...