RIDME spectroscopy on high-spin Mn2+ centers.

Pulsed EPR dipolar spectroscopy is a powerful tool for determining the structure and conformational dynamics of biological macromolecules, as it allows precise measurements of distances in the range of 1.5-10 nm. Utilization of high-spin Mn2+ species as spin probes for distance measurements is of significant interest, because they are biologically compatible and endogenous in numerous biological systems. However, to date dipolar spectroscopy experiments with this kind of species have been underexplored. Here we present pulsed electron electron double resonance (PELDOR also called DEER) and relaxation-induced dipolar modulation enhancement (RIDME) experiments, which have been performed at W-band (94 GHz) and J-band frequencies (263 GHz) on a bis-MnDOTA (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate) model system. The distances obtained from these experiments are in good agreement with predictions. RIDME experiments reveal a significantly higher modulation depth compared to PELDOR, which is an important consideration for biological samples. These experiments also feature higher harmonics of the dipolar coupling frequency due to effective multiple-quantum relaxation of high-spin Mn2+ as well as the multiple-component background function. Harmonics of the dipolar coupling frequency were taken into account by including additional terms in the kernel function of Tikhonov regularization analysis.

The effect of gamma-ray irradiation on the Mn(II) speciation in Deinococcus radiodurans and the potential role of Mn(II)-orthophosphates.

D. radiodurans accumulates large quantities of Mn(II), which is believed to form low molecular weight complexes with phosphate and metabolites that protect D. radiodurans from radiation damage. The concentration of Mn(II) species in D. radiodurans during the exponential and stationary phase was determined using high-field EPR and biochemical techniques. In the exponential growth phase cells a large fraction of the manganese was in the form of Mn(II)-orthophosphate complexes. By contrast, the intracellular concentration of these compounds in stationary phase cells was less than 16 µM, while that of Mn superoxide dismutase was 320 µM and that of another, yet unidentified, Mn(II) protein was 250 µM. Stationary cells were found to be equally resistant to irradiation as the exponential cells in spite of having significant lower Mn(II)-orthophosphate concentrations. Gamma irradiation induced no changes in the Mn(II) speciation. During stationary growth phase D. radiodurans favours the production of the two Mn-proteins over low molecular weight complexes suggesting that the latter were not essential for radio-resistance at this stage of growth.

The HERC1 E3 Ubiquitin Ligase is essential for normal development and for neurotransmission at the mouse neuromuscular junction.

The ubiquitin-proteasome system (UPS) plays a fundamental role in protein degradation in neurons, and there is strong evidence that it fulfills a key role in synaptic transmission. The aim of the present work was to study the implication of one component of the UPS, the HERC1 E3 Ubiquitin Ligase, in motor function and neuromuscular transmission. The tambaleante (tbl) mutant mouse carries a spontaneous mutation in HERC1 E3 Ubiquitin Ligase, provoking an ataxic phenotype that develops in the second month of life. Our results show that motor performance in mutant mice is altered at postnatal day 30, before the cerebellar neurodegeneration takes place. This defect is associated with by: (a) a reduction of the motor end-plate area, (b) less efficient neuromuscular activity in vivo, and (c) an impaired evoked neurotransmitter release. Together, these data suggest that the HERC1 E3 Ubiquitin Ligase is fundamental for normal muscle function and that it is essential for neurotransmitter release at the mouse neuromuscular junction.

Variations in Mn(II) speciation among organisms: what makes D. radiodurans different.

The manganese(II) speciation in intact cells of D. radiodurans, E. coli, S. cerevisiae and Arabidopsis thaliana seeds was measured using high-field electron paramagnetic resonance techniques. The majority of the Mn(II) ions in these organisms were six-coordinate, bound predominately by water, phosphates and nitrogen-based molecules. The relative distribution of the different phosphates in bacteria and S. cerevisiae was the same and dominated by monophosphate monoesters. Mn(II) was also found bound to the phosphate backbone of nucleic acids in these organisms. Phosphate ligation in Arabidopsis seeds was dominated by phytate. The extent of nitrogen ligation in the four organisms was also determined. On average, the Mn(II) in D. radiodurans had the most nitrogen ligands followed by E. coli. This was attributed to higher concentrations of Mn(II) bound to proteins in these species. Although constitutively expressed in all four organisms, MnSOD was only detected in D. radiodurans. As previously reported, D. radiodurans also accumulates a second abundant Mn containing protein species. The high concentration of proteinaceous Mn(II) is a unique feature of D. radiodurans.

Note: Limitations of the optoelectronic control for carbon nanoparticles synthesis via arc-discharge in solution.

Submerged electric arc discharge in liquids has shown to be a promising method for synthesizing a wide variety of nanomaterials. However, it requires an accurate current stability control to ensure the desired purity and structure of the products. The discharge stability control through light emission has been previously studied, but still requires further investigation to clarify the influence of some parameters. The present work has studied the solution's transmittance variation over time, the correlation between the arc light emission and the arc current, and the feasibility of controlling the arc current by using a specific wavelength of the arc light spectrum. Several limitations of the optoelectronic control were found at low currents (I < 50 A).

α-Synuclein A30P decreases neurodegeneration and increases synaptic vesicle release probability in CSPα-null mice.

α-Synuclein and Cysteine-string protein-α (CSPα) are presynaptic proteins that participate in the maintenance of synaptic function. Mutations or overexpression of the wild type form of α-synuclein have been related to Parkinson's disease, and CSPα mutations cause one type of neuronal ceroid lipofuscinosis. Both are adult-onset neurodegenerative diseases characterized by neuronal protein aggregations. Strikingly, while in mouse the lack of CSPα produces defective neurotransmission and neurodegeneration of motor terminals, blindness and early lethality, the moderate overexpression of wild-type α-synuclein fully rescues the CSPα-null phenotype. Contrarily, the overexpression of the mutated human α-synuclein A30P (α-synuclein(hA30P)) has much less effect in CSPα KO mice. To explore how the A30P mutation affects the neuroprotective function of α-synuclein we investigated synaptic structure and neurotransmission in motor nerve terminals of wild-type and CSPα-null mice transgenic for α-synuclein(hA30P). We found that although α-synuclein(hA30P) did not fully prevent neurodegeneration, it significantly improved synaptic organization and function in CSPα-null mice by enhancing quantal content, release probability, synaptic vesicle content, active zone number, postsynaptic area, and microtubule appearance. These results demonstrate that α-synuclein(hA30P) is able to ameliorate synapse degeneration, despite its apparent lack of functionality and its long-term pathogenic effects in neurons. These findings may help to understand better the dual function of α-synuclein regarding neurodegeneration. This article is part of the Special Issue entitled 'The Synaptic Basis of Neurodegenerative Disorders'.

Cysteine string protein-alpha is essential for the high calcium sensitivity of exocytosis in a vertebrate synapse.

Cysteine string protein (CSPalpha) is a synaptic vesicle protein present in most central and peripheral nervous system synapses. Previous studies demonstrated that the deletion of CSPalpha results in postnatal sensorial and motor impairment and premature lethality. To understand the participation of CSPalpha in neural function in vertebrates, we have studied the properties of synaptic transmission of motor terminals in wild-type and CSPalpha knockout mice. Our results demonstrate that, in the absence of CSPalpha, fast Ca2+-triggered release was not affected at postnatal day (P)14 but was dramatically reduced at P18 and P30 without a change in release kinetics. Although mutant terminals also exhibited a reduction in functional vesicle pool size by P30, further analysis showed that neurotransmission could be 'rescued' by high extracellular [Ca2+] or by the presence of a phorbol ester, suggesting that an impairment in the fusion machinery, or in vesicle recycling, was not the primary cause of the dysfunction of this synapse. The specific shift to the right of the Ca2+ dependence of synchronous release, and the lineal dependence of secretion on extracellular [Ca2+] in mutant terminals after P18, suggests that CSPalpha is indispensable for a normal Ca2+ sensitivity of exocytosis in vertebrate mature synapses.

Relationship between fusion pore opening and release during mast cell exocytosis studied with patch amperometry.

We have studied exocytosis in rat peritoneal mast cells by cell-attached patch amperometry. Step increases in capacitance were accompanied by typical amperometric spikes due to the release of 5-hydroxytryptamine (serotonin), indicating exocytosis of typical mast cell granules. We have measured the time course of fusion pore expansion, and correlated it with release from the granule matrix. The fusion pore of mast cell granules grows in three stages. The initial expansion of the pore occurred at a rate of 5 nS/s, and in many cases an observable amperometric foot was detected. A second, rapid expansion phase occurred with a rate as high as 1000 nS/s, coinciding with the upstroke of the amperometric spike. A third, slower phase, with a rate of 5 nS/s, completed the final expansion of the fusion pore. These data reveal the very late stages in the exocytotic process, and demonstrate that the size of the fusion pore does not limit release during the upstroke of the amperometric spike or during the final, slow expansion that occurs during for the decay of the amperometric spike.

Dopamine induces intracellular Ca2+ signals mediated by alpha1B-adrenoceptors in rat pineal cells.

We have studied the functional interaction of dopamine with alpha1-adrenoceptor subtypes by measuring intracellular Ca2+ levels in pineal cells, a cell type where adrenoceptors are well characterized. We show that dopamine induces transient intracellular Ca2+ signals in only 70% of cells responding to phenylephrine. Dopamine-induced Ca2+ signals desensitise faster than Ca2+ transients elicited with phenylephrine and are selectively blocked by desipramine, imipramine, and alpha1B-adrenoceptor antagonists. These results suggest that dopamine induced Ca2+ signals are mainly due to the activation of one subtype of alpha-adrenoceptor, the alpha1B.

Exocytosis of catecholamine (CA)-containing and CA-free granules in chromaffin cells.

Recent evidence suggests that endocytosis in neuroendocrine cells and neurons can be tightly coupled to exocytosis, allowing rapid retrieval from the plasma membrane of fused vesicles for future use. This can be a much faster mechanism for membrane recycling than classical clathrin-mediated endocytosis. During a fast exo-endocytotic cycle, the vesicle membrane does not fully collapse into the plasma membrane; nevertheless, it releases the vesicular contents through the fusion pore. Once the vesicle is depleted of transmitter, its membrane is recovered without renouncing its identity. In this report, we show that chromaffin cells contain catecholamine-free granules that retain their ability to fuse with the plasma membrane. These catecholamine-free granules represent 7% of the total population of fused vesicles, but they contributed to 47% of the fusion events when the cells were treated with reserpine for several hours. We propose that rat chromaffin granules that transiently fuse with the plasma membrane preserve their exocytotic machinery, allowing another round of exocytosis.

Estrogen modulates alpha(1)/beta-adrenoceptor- induced signaling and melatonin production in female rat pinealocytes.

Nocturnal rise in pineal melatonin output is due to the night-induced acceleration of noradrenergic transmission and alpha(1)- and beta-adrenoceptor activation. In addition, in female animals, cyclic oscillations in circulating levels of sex steroid hormones are accompanied by changes in the rate of pineal melatonin secretion. To investigate whether estrogen directly affects pineal adrenoceptor responsiveness, pinealocytes from 21-day-old ovariectomized rats were exposed to physiological concentrations of 17beta-estradiol (17beta-E(2)) and treated with noradrenergic agonists. Direct exposure to 17beta-E(2) reduced alpha(1)/beta-adrenoceptor-induced stimulation of melatonin synthesis and release. This effect was mediated by an estrogen-dependent inhibition of both beta-adrenoceptor-induced accumulation of cAMP and alpha(1)-adrenoceptor-induced phosphoinositide hydrolysis. Furthermore, estrogen reduced transient Ca(2+) signals elicited in single pinealocytes by alpha(1)-adrenoceptor activation or by potassium-induced depolarization. In the case of beta-adrenoceptor responsiveness, neither forskolin- nor cholera toxin-induced accumulation of cAMP were affected by previous exposure to 17beta-E(2). This indicates that estrogen effects must be exerted upstream from adenylylcyclase activation, and independent of modifications in G protein expression, therefore suggesting changes in either adrenoceptor expression or receptor-effector coupling mechanisms. Since estrogen effects upon adrenoceptor responsiveness in pineal cells was not mimicked by 17beta-E(2) coupled to bovine serum albumin and showed a latency of 48 h, this effect could be compatible with a genomic action mechanism. This is also consistent with the presence of two estrogen receptor proteins, alpha- and beta-subtypes, in female rat pinealocytes under the present experimental conditions.

High calcium concentrations shift the mode of exocytosis to the kiss-and-run mechanism.

Exocytosis, the fusion of secretory vesicles with the plasma membrane to allow release of the contents of the vesicles into the extracellular environment, and endocytosis, the internalization of these vesicles to allow another round of secretion, are coupled. It is, however, uncertain whether exocytosis and endocytosis are tightly coupled, such that secretory vesicles fuse only transiently with the plasma membrane before being internalized (the 'kiss-and-run' mechanism), or whether endocytosis occurs by an independent process following complete incorporation of the secretory vesicle into the plasma membrane. Here we investigate the fate of single secretory vesicles after fusion with the plasma membrane by measuring capacitance changes and transmitter release in rat chromaffin cells using the cell-attached patch-amperometry technique. We show that raised concentrations of extracellular calcium ions shift the preferred mode of exocytosis to the kiss-and-run mechanism in a calcium-concentration-dependent manner. We propose that, during secretion of neurotransmitters at synapses, the mode of exocytosis is modulated by calcium to attain optimal conditions for coupled exocytosis and endocytosis according to synaptic activity.

Intracellular calcium release mediated by noradrenaline and acetylcholine in mammalian pineal cells.

The effects of noradrenergic and cholinergic receptor agonists on intracellular Ca2+ concentration ([Ca2+]i) in single dissociated rat pineal cells were investigated by microfluorimetric measurements in Fura-2 acetoxymethyl ester (Fura-2/AM) loaded cells. Noradrenaline (NA) evoked characteristic biphasic increments of intracellular Ca2+ consisting of one or more leading spikes followed by a plateau, resulting from the release of Ca2+ from intracellular stores and from the influx of Ca2+ from the external medium, respectively. This response was reproduced by the alpha 1-adrenoceptor agonist, phenylephrine (PE), in the presence of the beta-adrenoceptor antagonist, propranolol, and was abolished when NA or PE was applied in conjunction with the alpha 1-adrenoceptor antagonist, prazosin. The curve relating the peak amplitude of the Ca2+ increments to different PE concentrations (0.5-10 microM) showed a half-maximum response at 0.6 microM PE, and saturation at concentrations greater than 2 microM. Acetylcholine (ACh) also elicited transient Ca2+ increments consisting of an abrupt rise to a maximum value which decayed exponentially to the basal Ca2+ level. A half-maximum response was achieved at 59 microM ACh. The muscarinic cholinergic receptor agonist, carbachol (CCh), similarly activated Ca2+ increments while the muscarinic antagonist, atropine, abolished them. In the absence of extracellular Ca2+, repetitive stimuli with either alpha 1-adrenergic and muscarinic agonists produced a progressive decrement in the amplitude of the Ca2+ signals because of the depletion of intracellular stores. However, extinction of the response to muscarinic agonists did not preclude a response to adrenergic agonists, while the contrary was not true. These results suggest that these agonists liberate Ca2+ from two functionally distinct, caffeine-insensitive, Ca2+ intracellular stores.

[Acute systemic nocardiosis. Renal abscess]. / Nocardiosis sistémica aguda. Abceso renal.

OBJECTIVES: Nocardiosis is an infection caused by the Nocardia genus that rarely involves the genitourinary system. The microbiological characteristics of Nocardia asteroides and the pathogenesis, possible symptoms, diagnosis and treatment of nocardiosis are reviewed. METHODS/RESULTS: We describe a patient with lung involvement and intraparenchymal abscess in the left kidney that required nephrectomy. Nocardia asteroides was isolated in the culture. CONCLUSIONS: Systemic nocardiosis is a serious infectious disease characterized by infection of the brain or two nonadjacent organs. Specific genitourinary involvement is rare and involvement of the renal parenchyma usually occurs during the aggressive course of acute systemic nocardiosis. The fatal outcome of our case conforms to the high rate of mortality described in the literature, particularly in acute systemic nocardiosis with neurological involvement, and emphasizes the importance of early diagnosis and appropriate antibiotic therapy.

Chloride channels in the nuclear membrane.

Chloride-selective ion channels were measured from isolated rat liver nuclei. Single ion channel currents were recorded in both "nuclear-attached" and in excised patches in the inside-out configuration of the patch-clamp technique. Two types of chloride conductance were defined, a large conductance (150 pS; iCl,N) channel with complex kinetics and multiple substates, and a second smaller conductance (58 pS;ICln) channel sensitive to block by ATP. The channels were inhibited by pharmacological agents known to block chloride channels and were insensitive to internal and external changes in calcium and magnesium. Presumably the channels reside in the external membrane of the nuclear double membrane and may mediate charge balance in the release and uptake of calcium from the perinuclear space.

Properties of calcium and potassium currents of clonal adrenocortical cells.

The ionic currents of clonal Y-1 adrenocortical cells were studied using the whole-cell variant of the patch-clamp technique. These cells had two major current components: a large outward current carried by K ions, and a small inward Ca current. The Ca current depended on the activity of two populations of Ca channels, slow (SD) and fast (FD) deactivating, that could be separated by their different closing time constants (at -80 mV, SD, 3.8 ms, and FD, 0.13 ms). These two kinds of channels also differed in (a) activation threshold (SD, approximately -50 mV; FD, approximately -20 mV), (b) half-maximal activation (SD, between -15 and -10 mV; FD between +10 and +15 mV), and (c) inactivation time course (SD, fast; FD, slow). The total amplitude of the Ca current and the proportion of SD and FD channels varied from cell to cell. The amplitude of the K current was strongly dependent on the internal [Ca2+] and was almost abolished when internal [Ca2+] was less than 0.001 microM. The K current appeared to be independent, or only slightly dependent, of Ca influx. With an internal [Ca2+] of 0.1 microM, the activation threshold was -20 mV, and at +40 mV the half-time of activation was 9 ms. With 73 mM external K the closing time constant at -70 mV was approximately 3 ms. The outward current was also modulated by internal pH and Mg. At a constant pCa gamma a decrease of pH reduced the current amplitude, whereas the activation kinetics were not much altered. Removal of internal Mg produced a drastic decrease in the amplitude of the Ca-activated K current. It was also found that with internal [Ca2+] over 0.1 microM the K current underwent a time-dependent transformation characterized by a large increase in amplitude and in activation kinetics.

Calcium action potentials in cultured adrenocortical cells.

Membrane potential has been recorded in Y-1 adrenocortical cells with intracellular microelectrodes. Resting potential averaged -68 +/- 13 mV (n = 63). Cells were silent at rest but depolarization by current pulses evoked repetitive action potentials of 80-120 mV amplitude and variable duration (between 10-300 ms). Action potentials were unaffected by removal of external Na or addition of TTX, however they were completely abolished by substitution of external Ca by Co. In Ca-free solutions with 2.4 mM Ba, action potentials had a lower threshold and lasted for the whole duration of the current pulse.

Effects of membrane depolarization and divalent cations on anaphylactic histamine secretion.

The effects of membrane depolarization and divalent cations on histamine release have been studied in sensitized mast cells. Membrane potential of these cells has been measured with intracellular microelectrodes. Our results show that mast cells have a large resting potential (-61 +/- 12 mV) however they do not generate active membrane electrical responses when are depolarized by passing current through the recording microelectrode. High external K+ does not increase histamine release. Histamine secretion is supported by alkali-earth divalent cations (Ca2+ greater than Sr2+ greater than Ba2+) but strongly inhibited by transition metals. Ca2+ concentrations above 1 mM inhibit histamine release, however, this effect is not mimicked by Sr2+ and Ba2+.