Cholera toxin B-subunit incorporation into synaptic vesicles of the neuromuscular junction of the rat

The B-subunit of cholera toxin, a nontoxic macromolecule which binds specifically to GM1 ganglioside, was conjugated to colloidal gold and injected into skeletal muscle of the rat. It was taken up rapidly in vesicles in the terminal axons at the neuromuscular junctions. Injection of albumin-colloidal gold conjugates resulted in an insignificant uptake. The results indicate that uptake of extracellular macromolecules into the terminal axon of the neuromuscular junction may be greatly enhanced by binding to gangliosides at the presynaptic membrane, and that it may occur without association with vesicular recycling related to transmitter release.     

Reduction of high affinity glutamate uptake in rat hippocampus by two polyamine-like toxins isolated from the venom of the predatory wasp Philanthus triangulum F

Two components of the venom of the predatory wasp Philanthus triangulum F. significantly reduce--to a greater or less extent--the high affinity uptake of glutamate in rat hippocampus. A concentration of 10 microM delta-PTX caused a reduction of 74%, while the other component, beta-PTX, at the same concentration, caused a reduction of 18%. Hence the effect of delta-PTX on high affinity glutamate uptake in the hippocampus is comparable with its effect on high affinity glutamate uptake in insect neuromuscular junctions. Contrary to our previous findings that beta-PTX has no effect on high affinity glutamate uptake in insect glutamatergic terminal axons, however, beta-PTX significantly reduces high affinity glutamate uptake in the hippocampus, albeit less effectively than delta-PTX.     

Reduction of high affinity glutamate uptake in rat hippocampus by two polyamine-like toxins isolated from the venom of the predatory waspphilanthus triangulum F

Summary Two components of the venom of the predatory waspPhilanthus triangulum F. significantly reduce — to a greater or less extent — the high affinity uptake of glutamate in rat hippocampus. A concentration of 10 M -PTX caused a reduction of 74%, while the other component, -PTX, at the same concentration, caused a reduction of 18%. Hence the effect of -PTX on high affinity glutamate uptake in the hippocampus is comparable with its effect on high affinity glutamate uptake in insect neuromuscular junctions. Contrary to our previous findings that -PTX has no effect on high affinity glutamate uptake in insect glutamatergic terminal axons, however, -PTX significantly reduces high affinity glutamate uptake in the hippocampus, albeit less effectively than -PTX.     

Metaraminol uptake by human thrombocytes: A poor model for neuronal noradrenaline uptake

Summary The IC₅₀ of a number of antidepressants and related drugs on the uptake of 1-metaraminol and serotonin into human thrombocytes, and of noradrenaline and serotonin into rat midbrain synaptosomes were compared. In accordance with previous reports, it was found that platelets, provide a good model for the study of neuronal uptake of serotonin. Platelet uptake of 1-metaraminol, although correlated to some extent with noradrenaline uptake into synaptosomes, seems to be an unsatisfactory model for the neuronal uptake of the latter amine.     

Actions d'un inhibiteur sélectif des acétylcholinestérases,le 3318 CT,sur la transmission neuromusculaire du chat

Summary 3318 CT [bis (piperidinomethylcoumaranyl-5) cetone dimethiodide], a specific inhibitor of acetylcholinesterase, antagonizes, in small dosages, the neuromuscular block induced by Flaxedil in the cat; in larger dosages, 3318 CT blocks the transmission: this paralysing action is not related to an accumulation of acetylcholine, as acetylcholine itself antagonizes it; decamethonium also restores, partially at least, the indirect contractions. Prostigmine and m-hydroxyphenyltrimethylammonium have no action. The neuromuscular block by 3318 CT thus appears as a competitive curarization in an animal with inhibited acetylcholinesterases. There was no indication that pseudo-cholinesterase can play a part in the neuromuscular transmission; whereas in the case of the vagal transmission such a possibility could not be ruled out.     

Dantrolene and the effect of temperature on the spontaneous release of transmitter at the frog neuromuscular junction

Summary The characteristic effect of temperature on m.e.p.p. frequency at the amphibian neuromuscular junction is unaltered by the presence of Dantrolene (an agent that is believed to reduce the efflux of Ca²⁺ from intracellular stores) or by changes in [Ca²⁺]_o. It is concluded that temperature affects the release system directly, with a transition temperature at about 16°C.     

Lead into gold: the science of finding nothing

Scientists are always doing experiments or making observations that disappoint them. Most negative experiments are consigned to the file drawer. But in physics, lead is regularly transmuted into gold by treating a negative result as an upper limit—an observation of the maximum strength of the phenomenon under investigation. The logic and sociology of upper limits and the logic and sociology of positive results are different. I explore the difference through a case study in the physical sciences. In the conclusion I ask why social sciences only rarely translate their negative findings into successes.     

Fatigue in neuromuscular disorders: focus on Guillain–Barré syndrome and Pompe disease

Fatigue accounts for an important part of the burden experienced by patients with neuromuscular disorders. Substantial high prevalence rates of fatigue are reported in a wide range of neuromuscular disorders, such as Guillain–Barré syndrome and Pompe disease. Fatigue can be subdivided into experienced fatigue and physiological fatigue. Physiological fatigue in turn can be of central or peripheral origin. Peripheral fatigue is an important contributor to fatigue in neuromuscular disorders, but in reaction to neuromuscular disease fatigue of central origin can be an important protective mechanism to restrict further damage. In most cases, severity of fatigue seems to be related with disease severity, possibly with the exception of fatigue occurring in a monophasic disorder like Guillain–Barré syndrome. Treatment of fatigue in neuromuscular disease starts with symptomatic treatment of the underlying disease. When symptoms of fatigue persist, non-pharmacological interventions, such as exercise and cognitive behavioral therapy, can be initiated.     

Activity increases quantal size at the frog neuromuscular junction

Two h of nerve stimulation at 10 Hz or of elevated spontaneous release in hypertonic solution increased the size of miniature end-plate potentials (m.e.p.p.'s) and currents at the frog neuromuscular junction, probably by increasing the amount of acetylcholine in a quantum. Increases in quantal size may modulate synaptic transmission.     

Ectoprotein kinase-mediated phosphorylation of FAT/CD36 regulates palmitate uptake by human platelets

Glycoprotein IV (FAT/CD36) has been shown to be phosphorylated by a cAMP-dependent, platelet membrane-bound ectokinase. In this study, we demonstrate that ectophosphorylation of FAT/CD36 regulates initial palmitate uptake. This is the first time that short-term regulation of the activity of a long-chain fatty acid carrier could be shown. Phosphorylation of FAT/CD36 was paralleled by a significant decrease in initial palmitate uptake by morphologically and functionally intact platelets. Maximum inhibition of palmitate uptake was achieved at 0.5 nM extracellular ATP, being significantly decreased to 72% compared to the control. Inhibition of palmitate uptake was abolished by co-incubation with the specific protein kinase A inhibitor peptide PKI or with β,γ-methylene-ATP, and was reversible upon addition of alkaline phosphatase. An extracellular ATP concentration above 5 μM completely prevented the ectophosphorylation-mediated inhibition of palmitate uptake. We conclude that FAT/CD36-mediated palmitate uptake by human platelets is short-term regulated via cAMP-dependent ectophosphorylation of FAT/CD36. Received 18 July 2002; received after revision 29 August 2002; accepted 19 September 2002 RID="*" ID="*"Corresponding author.     

Quantitative differences in the pharmacological effects of (+)- and (−)-cathinone

Summary The optically pure isomers of cathinone were prepared by separating synthetic cathinone racemate and used to study central and peripheral effects of these indirect sympathomimetics in rats and guinea pigs. The (–)-isomer was significantly more potent than the (+)-isomer in stimulating locomotor activity whereas no difference was observed with respect to their cardiac effects. In analogy to observations with (+)- and (–)-amphetamine such variable isomer discrimination may be due to different stereoselectivities of amine uptake mechanisms in the target tissues.     

Proliferation and cell loss of human leukemic cell subpopulations in liquid culture

Summary A kinetic study was performed on leukemic blasts from patients with acute myeloid leukemia, separated into 2 subpopulations by a specific density gradient. The growth curve and the [³H]-thymidine uptake were simultaneously analyzed. While cumulative nucleotide uptake fitted with the growth kinetics in the low-density fraction, such a concordance was not found in the high-density subpopulation. That indicated the occurrence of simultaneous growth and loss in the high density fraction, which could not be evaluated by a simple numerical determination.     

Influence of hypoxia on contractility and calcium uptake in rabbit aorta

Summary The influence of hypoxia on noradrenaline (NA)-induced contractions and⁴⁵Ca uptake has been studied on isolated rabbit aortae. Hypoxia significantly decreased the contractility of aortic strips. NA stimulation resulted in increased or decreased⁴⁵Ca uptake by normoxic or hypoxic specimens, respectively. Relating⁴⁵Ca movement with mechanical activity, the results suggest that decrease in Ca⁺⁺ uptake may be mechanism for hypoxic relaxation of aortic smooth muscle.The author acknowledges facilities at the Wellcome Surgical Research Institute, University of Glasgow, U.K. Much of this work was in collaboration with Drs Sheila Jennett and J.D. Pickard.     

Variation of the time constant (tau) of the decay of end-plate current during focalized transmitter release]

The variations of the time constant tau of the end-plate current (epc) have been studied at the clamped neuromuscular junction of the frog while transmitter release was focalised by local iontophoretic application of Ca++. In the absence of any anticholinesterasic drug, tau varies according to the variations of the intensity of epc (Iepc) due to pre or postsynaptic experimental procedure. It is suggested that focalisation of transmitter release induces local acetylcholinesterase inhibition by an excess of substrate; then the transmitter life time in the synaptic cleft is momentarily increased, allowing repeated binding of Ach to postsynaptic receptors.     

Drosophila Nesprin-1 controls glutamate receptor density at neuromuscular junctions

Nesprin-1 is a core component of a protein complex connecting nuclei to cytoskeleton termed LINC (linker of nucleoskeleton and cytoskeleton). Nesprin-1 is anchored to the nuclear envelope by its C-terminal KASH domain, the disruption of which has been associated with neuronal and neuromuscular pathologies, including autosomal recessive cerebellar ataxia and Emery–Dreifuss muscular dystrophy. Here, we describe a new and unexpected role of Drosophila Nesprin-1, Msp-300, in neuromuscular junction. We show that larvae carrying a deletion of Msp-300 KASH domain (Msp-300 ^?KASH ) present a locomotion defect suggestive of a myasthenia, and demonstrate the importance of muscle Msp-300 for this phenotype, using tissue-specific RNAi knock-down. We show that Msp-300 ^?KASH mutants display abnormal neurotransmission at the larval neuromuscular junction, as well as an imbalance in postsynaptic glutamate receptor composition with a decreased percentage of GluRIIA-containing receptors. We could rescue Msp-300 ^?KASH locomotion phenotypes by GluRIIA overexpression, suggesting that the locomotion impairment associated with the KASH domain deletion is due to a reduction in junctional GluRIIA. In summary, we found that Msp-300 controls GluRIIA density at the neuromuscular junction. Our results suggest that Drosophila is a valuable model for further deciphering how Nesprin-1 and LINC disruption may lead to neuronal and neuromuscular pathologies.     

In vivo and in vitro differences between leukocytic uptake of oligodeoxynucleotides

Development and application of therapeutic oligonucleotides rely on proper analysis of binding and uptake. We have used several model oligodeoxynucleotides (ODNs) to analyze binding/uptake by rat and human leukocytes. Here we describe: (1) differences between in vivo and in vitro uptake of ODNs to rat leukocytes, (2) differences after injection of lipopolysaccharide (LPS), (3) large in vitro differences between primary mononuclear cells in PBS, plasma and blood, and (4) differences of ODN uptake between rat and human leukocytes. Our data show that ODN uptake by primary blood cells was different in PBS, plasma and blood. In addition, LPS treatment increased ODN uptake by leukocytes in blood, indicating that pathological conditions may influence ODN uptake. Furthermore, ODN uptake in rat and human blood is also different, suggesting that preclinical ODN uptake data from rat blood cannot easily be extrapolated to the human condition. Received 17 December 2007; received after revision 16 January 2008; accepted 5 February 2008     

Diffusion of d-glucose measured in the cytosol of a single astrocyte

Astrocytes interact with neurons and endothelial cells and may mediate exchange of metabolites between capillaries and nerve terminals. In the present study, we investigated intracellular glucose diffusion in purified astrocytes after local glucose uptake. We used a fluorescence resonance energy transfer (FRET)-based nano sensor to monitor the time dependence of the intracellular glucose concentration at specific positions within the cell. We observed a delay in onset and kinetics in regions away from the glucose uptake compared with the region where we locally super-fused astrocytes with the d-glucose-rich solution. We propose a mathematical model of glucose diffusion in astrocytes. The analysis showed that after gradual uptake of glucose, the locally increased intracellular glucose concentration is rapidly spread throughout the cytosol with an apparent diffusion coefficient (D _app) of (2.38 ± 0.41) × 10^?10 m² s^?1 (at 22–24 °C). Considering that the diffusion coefficient of d-glucose in water is D = 6.7 × 10^?10 m² s^?1 (at 24 °C), D _app determined in astrocytes indicates that the cytosolic tortuosity, which hinders glucose molecules, is approximately three times higher than in aqueous solution. We conclude that the value of D _app for glucose measured in purified rat astrocytes is consistent with the view that cytosolic diffusion may allow glucose and glucose metabolites to traverse from the endothelial cells at the blood–brain barrier to neurons and neighboring astrocytes.     

Low-density lipoprotein receptor-mediated endocytosis of PEGylated nanoparticles in rat brain endothelial cells

Poly(methoxypolyethyleneglycol cyanoacrylate-co-hexadecylcyanoacrylate) (PEG-PHDCA) nanoparticles have demonstrated their capacity to diffuse through the blood-brain barrier after intravenous administration. However, the mechanism of transport of these nanoparticles into brain has not yet been clearly elucidated. The development of a model of rat brain endothelial cells (RBEC) in culture has allowed investigations into this mechanism. A study of the intracellular trafficking of nanoparticles by cell fractionation and confocal microscopy showed that nanoparticles are internalized by the endocytic pathway. Inhibition of the caveolae-mediated pathway by preincubation with filipin and nystatin did not modify the cellular uptake of the nanoparticles. In contrast, chlorpromazine and NaN₃ pretreatment, which interferes with clathrin and energy-dependent endocytosis, caused a significant decrease of nanoparticle internalization. Furthermore, cellular uptake experiments with nanoparticles preincubated with apolipoprotein E and blocking of low-density lipoprotein receptors (LDLR) clearly suggested that the LDLR-mediated pathway was involved in the endocytosis of PEGPHDCA nanoparticles by RBEC. Received 1 September 2006; received after revision 4 December 2006; accepted 18 December 2006     

Dantrolene and the effect of temperature on the spontaneous release of transmitter at the frog neuromuscular junction

The characteristic effect of temperature on m.e.p.p. frequency at the amphibian neuromuscular junction is unaltered by the presence of Dantrolene (an agent that is believed to reduce the efflux of Ca2+ from intracellular stores) or by changes in [Ca2+)o. It is concluded that temperature affects the release system directly, with a transition temperature at about 16 degrees C.     

Long-chain fatty acid uptake by skeletal myocytes: a confocal laser scanning microscopy study

Studies of regulation of free fatty acid (FFA) utilization by skeletal muscles have focused on plasma FFA delivery and on intracellular factors affecting FFA metabolism. The present study was conducted to directly analyse the uptake process of fatty acids into single myocytes. Cells were isolated from the rat flexor digitorum brevis muscle. Confocal laser scanning microscopy was utilized to analyse the uptake of the fluorescent fatty acid derivative 12-NBD-stearate, which is not metabolized by muscle tissue. Uptake represented a saturable function of the unbound fatty acid concentration in the medium (K _m 366 ± 118 nM, V _max 2.1 ± 0.3 AU/s) and depended on the medium sodium concentration. Reduced buffer pH increased initial uptake rates, whereas lactate (10 mM) had no effect. Membrane hyper- and depolarization decreased uptake rates. This study demonstrates for the first time kinetic data from isolated myocytes with evidence for a carrier-mediated transport mechanism for long-chain fatty acids. Received 31 March 1998; accepted 8 May 1998