Proton NMR of (15)N-choline metabolites enhanced by dynamic nuclear polarization.

Chemical shifts of protons can report on metabolic transformations such as the conversion of choline to phosphocholine. To follow such processes in vivo, magnetization can be enhanced by dynamic nuclear polarization (DNP). We have hyperpolarized in this manner nitrogen-15 spins in (15)N-labeled choline up to 3.3% by irradiating the 94 GHz electron spin resonance of admixed TEMPO nitroxide radicals in a magnetic field of 3.35 T during ca. 3 h at 1.2 K. The sample was subsequently transferred to a high-resolution magnet, and the enhanced polarization was converted from (15)N to methyl- and methylene protons, using the small (2,3)J((1)H,(15)N) couplings in choline. The room-temperature lifetime of nitrogen polarization in choline, T(1)((15)N) approximately 200 s, could be considerably increased by partial deuteration of the molecule. This procedure enables studies of choline metabolites in vitro and in vivo using DNP-enhanced proton NMR.

An Ion Transport-Independent Role for the Cation-Chloride Cotransporter KCC2 in Dendritic Spinogenesis In Vivo.

The neuron-specific K-Cl cotransporter, KCC2, is highly expressed in the vicinity of excitatory synapses in pyramidal neurons, and recent in vitro data suggest that this protein plays a role in the development of dendritic spines. The in vivo relevance of these observations is, however, unknown. Using in utero electroporation combined with post hoc iontophoretic injection of Lucifer Yellow, we show that premature expression of KCC2 induces a highly significant and permanent increase in dendritic spine density of layer 2/3 pyramidal neurons in the somatosensory cortex. Whole-cell recordings revealed that this increased spine density is correlated with an enhanced spontaneous excitatory activity in KCC2-transfected neurons. Precocious expression of the N-terminal deleted form of KCC2, which lacks the chloride transporter function, also increased spine density. In contrast, no effect on spine density was observed following in utero electroporation of a point mutant of KCC2 (KCC2-C568A) where both the cotransporter function and the interaction with the cytoskeleton are disrupted. Transfection of the C-terminal domain of KCC2, a region involved in the interaction with the dendritic cytoskeleton, also increased spine density. Collectively, these results demonstrate a role for KCC2 in excitatory synaptogenesis in vivo through a mechanism that is independent of its ion transport function.

Occupational exposure to diisononyl phthalate (DiNP) in polyvinyl chloride processing operations

PURPOSE: Diisononyl phthalate (DiNP) is primarily used as a plasticizer in polyvinyl chloride (PVC) materials. While information is available on general population exposure to DiNP, occupational exposure data are lacking. We present DiNP metabolite urinary concentrations in PVC processing workers, estimate DiNP daily intake for these workers, and compare worker estimates to other populations. METHODS: We assessed DiNP exposure in participants from two companies that manufactured PVC materials, a PVC film manufacturer (n = 25) and a PVC custom compounder (n = 12). A mid-shift and end-shift urine sample was collected from each participant and analyzed for the DiNP metabolite mono(carboxy-isooctyl) phthalate (MCiOP). Mixed models were used to assess the effect on MCiOP concentrations of a worker being assigned to (1) a task using DiNP and (2) a shift where DiNP was used. A simple pharmacokinetic model was used to estimate DiNP daily intake from the MCiOP concentrations. RESULTS: Creatinine-adjusted MCiOP urinary concentrations ranged from 0.42-80 μg/g in PVC film and from 1.11-13.4 μg/g in PVC compounding. PVC film participants who worked on a task using DiNP (n = 7) had the highest MCiOP geometric mean (GM) end-shift concentration (25.2 μg/g), followed by participants who worked on a shift where DiNP was used (n = 11) (17.7 μg/g) as compared to participants with no task (2.92 μg/g) or shift (2.08 μg/g) exposure to DiNP. The GM end-shift MCiOP concentration in PVC compounding participants (4.80 μg/g) was comparable to PVC film participants with no task or shift exposure to DiNP. Because no PVC compounding participants were assigned to tasks using DINP on the day sampled, DiNP exposure in this company may be underestimated. The highest DiNP intake estimate was 26 μg/kg/day. CONCLUSION: Occupational exposure to DiNP associated with PVC film manufacturing tasks were substantially higher (sixfold to tenfold) than adult general population exposures; however, all daily intake estimates were less than 25% of current United States or European acceptable or tolerable daily intake estimates. Further characterization of DiNP occupational exposures in other industries is recommended.

Atrial natriuretic peptides: reproducibility of renal effects and response of liver blood flow.

To assess the variability of the response to exogenous atrial natriuretic peptide (ANP), it was infused at the rate of 1 microgram/min for 2 h in 6 salt-loaded normal volunteers under controlled conditions on 2 occasions at an interval of 1 week. The effect on solute excretion and the haemodynamic and endocrine actions were highly reproducible. The constant ANP infusion caused a delayed and prolonged excretion of sodium, chloride and calcium, no change in potassium or phosphate excretion or in glomerular filtration rate but a marked decrease in renal plasma flow. Blood pressure, heart rate and the plasma levels of angiotensin II, aldosterone, arginine vasopressin and plasma renin activity were unaltered. The effect of a 2-h infusion of ANP 0.5 microgram/min or its vehicle on apparent hepatic blood flow (HBF) was also studied in 14 normal volunteers by measuring the indocyanine green clearance. A 21% decrease in HBF was observed in subjects who received the ANP infusion (p less than 0.01 vs vehicle). Thus, ANP infused at a dose that did not lower blood pressure decreased both renal and liver blood flow in normotensive volunteers. The renal and endocrine responses to ANP were reproducible over a 1-week interval.

Aggregating Brain Cell Cultures as a Model to Study Ischaemia-induced Neurodegeneration.

Rotation-mediated aggregating brain cell cultures at two different maturational stages (DIV 11 and DIV 20) were subjected for 1 or 2 hours to ischaemic conditions by transient immobilization (arrest of media circulation). During recovery, cell damage was evaluated by measuring changes in cell type-specific enzyme activities and total protein content. It was found that in immature cultures (DIV 11), immobilization for 1 or 2 hours did not affect the parameters measured. By contrast, at DIV 20, ischaemic conditions for 1 hour caused a pronounced decrease in the activities of glutamic acid decarboxylase and choline acetyltransferase. A significant decrease in these neuron-specific enzyme activities was found at post-ischaemic days 1-14, indicating immediate and irreversible neuronal damage. The activity of the astrocyte-specific enzyme, glutamine synthetase, was significantly increased at 4 days post-treatment; equal to control values at 6 days; and significantly decreased at 14 days after the ischaemic insult. Immobilization of DIV 20 cultures for 2 hours caused a drastic reduction in all the parameters measured at post-ischaemic day 6. Generally, the ischaemic conditions appeared to be more detrimental to neurons than to astrocytes, and GABAergic neurons were more affected than cholinergic neurons.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for direct bacterial identification from positive blood culture pellets.

An ammonium chloride erythrocyte-lysing procedure was used to prepare a bacterial pellet from positive blood cultures for direct matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry analysis. Identification was obtained for 78.7% of the pellets tested. Moreover, 99% of the MALDI-TOF identifications were congruent at the species level when considering valid scores. This fast and accurate method is promising.

The twist, writhe and overall shape of supercoiled DNA change during counterion-induced transition from a loosely to a tightly interwound superhelix. Possible implications for DNA structure in vivo.

A cryo-electron microscopy study of supercoiled DNA molecules freely suspended in cryo-vitrified buffer was combined with Monte Carlo simulations and gel electrophoretic analysis to investigate the role of intersegmental electrostatic repulsion in determining the shape of supercoiled DNA molecules. It is demonstrated here that a decrease of DNA-DNA repulsion by increasing concentrations of counterions causes a higher fraction of the linking number deficit to be partitioned into writhe. When counterions reach concentrations likely to be present under in vivo conditions, naturally supercoiled plasmids adopt a tightly interwound conformation. In these tightly supercoiled DNA molecules the opposing segments of interwound superhelix seem to directly contact each other. This form of supercoiling, where two DNA helices interact laterally, may represent an important functional state of DNA. In the particular case of supercoiled minicircles (178 bp) the delta Lk = -2 topoisomers undergo a sharp structural transition from almost planar circles in low salt buffers to strongly writhed "figure-eight" conformations in buffers containing neutralizing concentrations of counterions. Possible implications of this observed structural transition in DNA are discussed.

Differentiation of rat striatal embryonic stem cells in vitro: monolayer culture vs. three-dimensional coculture with differentiated brain cells.

Several groups have demonstrated the existence of self-renewing stem cells in embryonic and adult mouse brain. In vitro, these cells proliferate in response to epidermal growth factor, forming clusters of nestin-positive cells that may be dissociated and subcultured repetitively. Here we show that, in stem cell clusters derived from rat embryonic striatum, cell proliferation decreased with increasing number of passages and in response to elevated concentrations of potassium (30 mM KCl). In monolayer culture, the appearance of microtubule-associated protein type-5-immunoreactive (MAP-5(+)) cells (presumptive neurons) in response to basic fibroblast growth factor (bFGF) was reduced at low cell density and with increasing number of passages. In the presence of bFGF, elevated potassium caused a more differentiated neuronal phenotype, characterized by an increased proportion of MAP-5(+) cells, extensive neuritic branching, and higher specific activity of glutamic acid decarboxylase. Dissociated stem cells were able to invade cultured brain cell aggregates containing different proportions of neurons and glial cells, whereas they required the presence of a considerable proportion of glial cells in the host cultures to become neurofilament H-positive. The latter observation supports the view that astrocyte-derived factors influence early differentiation of the neuronal cell lineage.

The human CFTR protein expressed in CHO cells activates aquaporin-3 in a cAMP-dependent pathway: study by digital holographic microscopy.

The transmembrane water movements during cellular processes and their relationship to ionic channel activity remain largely unknown. As an example, in epithelial cells it was proposed that the movement of water could be directly linked to cystic fibrosis transmembrane conductance regulator (CFTR) protein activity through a cAMP-stimulated aqueous pore, or be dependent on aquaporin. Here, we used digital holographic microscopy (DHM) an interferometric technique to quantify in situ the transmembrane water fluxes during the activity of the epithelial chloride channel, CFTR, measured by patch-clamp and iodide efflux techniques. We showed that the water transport measured by DHM is fully inhibited by the selective CFTR blocker CFTRinh172 and is absent in cells lacking CFTR. Of note, in cells expressing the mutated version of CFTR (F508del-CFTR), which mimics the most common genetic alteration encountered in cystic fibrosis, we also show that the water movement is profoundly altered but restored by pharmacological manipulation of F508del-CFTR-defective trafficking. Importantly, whereas activation of this endogenous water channel required a cAMP-dependent stimulation of CFTR, activation of CFTR or F508del-CFTR by two cAMP-independent CFTR activators, genistein and MPB91, failed to trigger water movements. Finally, using a specific small-interfering RNA against the endogenous aquaporin AQP3, the water transport accompanying CFTR activity decreased. We conclude that water fluxes accompanying CFTR activity are linked to AQP3 but not to a cAMP-stimulated aqueous pore in the CFTR protein.

Potassium restores vasorelaxation of resistance arterioles in non-hypertensive DOCA/salt fed mice.

BACKGROUND: Potassium-enriched diets exert renal and cardiovascular protective effects, but the underlying mechanisms are largely unknown. METHODS: Using the dorsal skinfold chamber model for intravital microscopy, we examined endothelium-dependent vasorelaxation of precapillary resistance arterioles in response to acetylcholine or the NO donor SNAP in awake mice. Experiments were performed in uni-nephrectomized one renin gene (Ren-1c) C57BL/6 mice (control group) and in mice having received a continuous administration of deoxycorticosterone acetate and a dietary supplementation of 1% sodium chloride for 8weeks (DOCA/salt group). An additional group of DOCA/salt treated animals received a dietary supplement of 0.4% KCl for 3weeks prior to the experiments (DOCA/salt + potassium group). RESULTS: DOCA/salt treatment for 8weeks resulted in hypokalemia, but blood pressure remained unchanged. In DOCA/salt mice, relaxation of resistance arterioles was blunted in response to acetylcholine, and to a lesser extent to SNAP, suggesting endothelial dysfunction. Endothelium-dependent vasorelaxation was restored by the potassium-enriched diet. CONCLUSION: This study is the first to demonstrate a protective effect of potassium on endothelium-dependent vasorelaxation in the absence of confounding anti-hypertensive effects, as observed in most animal models and the clinical situation. We propose that the known cardio- and nephro-protective effects of potassium might - at least in part - be mediated by the salutary effects on endothelium-dependent arteriolar relaxation.

Myotonic dystrophy transgenic mice exhibit pathologic abnormalities in diaphragm neuromuscular junctions and phrenic nerves

Myotonic dystrophy Type 1 (DM-1) is caused by abnormal expansion of a (CTG) repeat located in the DM protein kinase gene. Respiratory problems have long been recognized to be a major feature of this disorder. Because respiratory failure can be associated with dysfunction of phrenic nerves and diaphragm muscle, we examined the diaphragm and respiratory neural network in transgenic mice carrying the human genomic DM-1 region with expanded repeats of more than 300 CTG, a valid model of the human disease. Morphologic and morphometric analyses revealed distal denervation of diaphragm neuromuscular junctions in DM-1 transgenic mice indicated by a decrease in the size and shape complexity of end-plates and a reduction in the concentration of acetyl choline receptors on the postsynaptic membrane. More importantly, there was a significant reduction in numbers of unmyelinated, but not of myelinated, fibers in DM-1 phrenic nerves; no morphologic alternations of the nerves or loss of neuronal cells were detected in medullary respiratory centers or cervical phrenic motor neurons. Because neuromuscular junctions are involved in action potential transmission and the afferent phrenic unmyelinated fibers control the inspiratory activity, our results suggest that the respiratory impairment associated with DM-1 may be partially due to pathologic alterations in neuromuscular junctions and phrenic nerves.

Swiss survey on salt intake: main results

[Contents] 1. Executive summary. 2. Introduction. 3. Methods. 4. Main results. 4.1. Participants. 4.2. Estimation of dietary salt intake using 24-hour urine collection. 4.3. Blood pressure and hypertension. 4.4. Anthropometric data (Body weight, height and body mass index BMI; prevalence of overweight and obesity; waist circumference;...). 4.5. Knowledge and behaviors towards salt. 5. Discussion.

Cholinergic neurons of fetal rat telencephalon in aggregating cell culture respond to NGF as well as to protein kinase C-activating tumor promoters.

Serum-free aggregating cell cultures of fetal rat telencephalon treated with the potent tumor promoter phorbol 12-myristate 13-acetate (PMA) showed a dose-dependent, persistent stimulation of the enzymes choline acetyltransferase (ChAT), glutamic acid decarboxylase and glutamine synthetase. After elimination of the proliferating cells by treatment of the cultures with Ara-C (0.4 microM) only the cholinergic marker enzyme, ChAT, could be stimulated by tumor promoters. The non-promoting phorbol ester, 4 alpha-phorbol 12,13-didecanoate proved to be inactive in these cultures, whereas the potent non-phorbol tumor promoter, mezerein, produced an even greater stimulatory effect than PMA. Since PMA and mezerein are potent and specific activators of protein kinase C, the present results suggest a role for this second messenger in the development of cholinergic telencephalon neurons. Stimulation of ChAT required prolonged exposure (48 h) of the cultures to PMA and the responsiveness of the cholinergic neurons to the tumor promoters decreased with progressive cellular maturation. The cholinergic telencephalon neurons showed the same pattern of responsiveness for tumor promoters as for nerve growth factor (NGF). However, the combined treatment with NGF and either PMA or mezerein produced an additive stimulatory effect, suggesting somewhat different mechanisms of action.

Muscimol-induced death of GABAergic neurons in rat brain aggregating cell cultures.

During brain development, spontaneous neuronal activity has been shown to play a crucial role in the maturation of neuronal circuitries. Activity-related signals may cause selective neuronal cell death and/or rearrangement of neuronal connectivity. To study the effects of sustained inhibitory activity on developing inhibitory (GABAergic) neurons, three-dimensional primary cell cultures of fetal rat telencephalon were used. In relatively immature cultures, muscimol (10 microns), a GABAA receptor agonist, induced a transient increase in apoptotic cell death, as evidenced by a cycloheximide-sensitive increase of free nucleosomes and an increased frequency of DNA double strand breaks (TUNEL labeling). Furthermore, muscimol caused an irreversible reduction of glutamic acid decarboxylase activity, indicating a loss of GABAergic neurons. The muscimol-induced death of GABAergic neurons was attenuated by the GABAA receptor blockers bicuculline (100 microns) and picrotoxin (100 microns), by depolarizing potassium concentrations (30 mM KCl) and by the L-type calcium channel activator BAY K8644 (2 microns). As compared to the cholinergic marker (choline acetyltransferase activity), glutamic acid decarboxylase activity was significantly more affected by various agents known to inhibit neuronal activity, including tetrodotoxin (1 micron), flunarizine (5 microns), MK 801 (50 microns) and propofol (40 microns). The present results suggest that the survival of a subpopulation of immature GABAergic neurons is dependent on sustained neuronal activity and that these neurons may undergo apoptotic cell death in response to GABAA autoreceptor activation.