Glutamate-induced increases in intracellular free Mg2+ in cultured cortical neurons.

Intracellular free Mg2+ concentrations ([Mg2+]i) in single rat brain neurons were measured with the Mg(2+)-sensitive fluorescent dye magfura-2. Addition of glutamate with glycine raised [Mg2+]i from 1 to more than 11 mM compared with the resting concentration of 0.5 mM, an effect mediated by N-methyl-D-aspartate receptors. Most of the increase in [Mg2+]i was independent of extracellular Mg2+, but was dependent on extracellular Ca2+. The second component of the increase induced by glutamate was independent of extracellular Ca2+, but required extracellular Mg2+ and was amplified by extracellular Na+ removal. These results indicate that regulation of [Mg2+]i by neurotransmitters such as glutamate may be important in controlling neuronal excitability.

Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases.

Conditional DNA excision between two LoxP sites can be achieved in the mouse using Cre-ER(T), a fusion protein between a mutated ligand binding domain of the human estrogen receptor (ER) and the Cre recombinase, the activity of which can be induced by 4-hydroxy-tamoxifen (OHT), but not natural ER ligands. We have recently characterized a new ligand-dependent recombinase, Cre-ER(T2), which was approximately 4-fold more efficiently induced by OHT than Cre-ER(T) in cultured cells. In order to compare the in vivo efficiency of these two ligand-inducible recombinases to generate temporally-controlled somatic mutations, we have engineered transgenic mice expressing a LoxP-flanked (floxed) transgene reporter and either Cre-ER(T) or Cre-ER(T2) under the control of the bovine keratin 5 promoter that is specifically active in the epidermis basal cell layer. No background recombinase activity could be detected, while recombination was induced in basal keratinocytes upon OHT administration. Interestingly, a dose-response study showed that Cre-ER(T2) was approximately 10-fold more sensitive to OHT induction than Cre-ER(T).

Pharmacological investigation of mitochondrial ca(2+) transport in central neurons: studies with CGP-37157, an inhibitor of the mitochondrial Na(+)-Ca(2+) exchanger.

Mitochondria buffer large changes in [Ca(2+)](i)following an excitotoxic glutamate stimulus. Mitochondrial sequestration of [Ca(2+)](i)can beneficially stimulate oxidative metabolism and ATP production. However, Ca(2+)overload may have deleterious effects on mitochondrial function and cell survival, particularly Ca(2+)-dependent production of reactive oxygen species (ROS) by the mitochondria. We recently demonstrated that the mitochondrial Na(+)-Ca(2+)exchanger in neurons is selectively inhibited by CGP-37157, a benzothiazepine analogue of diltiazem. In the present series of experiments we investigated the effects of CGP-37157 on mitochondrial functions regulated by Ca(2+). Our data showed that 25 microM CGP-37157 quenches DCF fluorescence similar to 100 microM glutamate and this effect was enhanced when the two stimuli were applied together. CGP-37157 did not increase ROS generation and did not alter glutamate or 3mM hydrogen-peroxide-induced increases in ROS as measured by DHE fluorescence. CGP-37157 induces a slight decrease in intracellular pH, much less than that of glutamate. In addition, CGP-37157 does not enhance intracellular acidification induced by glutamate. Although it is possible that CGP-37157 can enhance mitochondrial respiration both by blocking Ca(2+)cycling and by elevating intramitochondrial Ca(2+), we did not observe any changes in ATP levels or toxicity either in the presence or absence of glutamate. Finally, mitochondrial Ca(2+)uptake during an excitotoxic glutamate stimulus was only slightly enhanced by inhibition of mitochondrial Ca(2+)efflux. Thus, although CGP-37157 alters mitochondrial Ca(2+)efflux in neurons, the inhibition of Na(+)-Ca(2+)exchange does not profoundly alter glutamate-mediated changes in mitochondrial function or mitochondrial Ca(2+)content.

Moxalactam kinetics during chronic ambulatory peritoneal dialysis.

Moxalactam kinetics in renal failure were followed in eight patients undergoing chronic ambulatory peritoneal dialysis (CAPD) after a single 1-gm IV infusion. Elimination t 1/2 was 16.7 +/- 2.1 hr, with an apparent volume of distribution of 0.21 +/- 0.01 l/kg and plasma clearance of 10.6 +/- 2 ml/min. In 24 hr, 17.4 +/- 3.1% of the dose was present in the dialysis fluids, and 14.6 +/- 5.7% was excreted in the urine. Renal and peritoneal clearance values were thus 2.3 +/- 1.1 and 2.7 +/- 0.5 ml/min. Peritoneal concentrations were high (22.7 +/- 2.2 micrograms/ml). A recommended dosage schedule is proposed on the basis of moxalactam kinetics during CAPD.

Disulfide bridge reorganization induced by proline mutations in maurotoxin.

Maurotoxin (MTX) is a 34-residue toxin that has been isolated from the venom of the chactidae scorpion Scorpio maurus palmatus, and characterized. Together with Pi1 and HsTx1, MTX belongs to a family of short-chain four-disulfide-bridged scorpion toxins acting on potassium channels. However, contrary to other members of this family, MTX exhibits an uncommon disulfide bridge organization of the type C1-C5, C2-C6, C3-C4 and C7-C8, versus C1-C5, C2-C6, C3-C7 and C4-C8 for both Pi1 and HsTx1. Here, we report that the substitution of MTX proline residues located at positions 12 and/or 20, adjacent to C3 (Cys(13)) and C4 (Cys(19)), results in conventional Pi1- and HsTx1-like arrangement of the half-cystine pairings. In this case, this novel disulfide bridge arrangement is without obvious incidence on the overall three-dimensional structure of the toxin. Pharmacological assays of this structural analog, [A(12),A(20)]MTX, reveal that the blocking activities on Shaker B and rat Kv1.2 channels remain potent whereas the peptide becomes inactive on rat Kv1.3. These data indicate, for the first time, that discrete point mutations in MTX can result in a marked reorganization of the half-cystine pairings, accompanied with a novel pharmacological profile for the analog.

Synthesis and antimalarial activity in vitro and in vivo of a new ferrocene-chloroquine analogue.

The antimalarial activities of ferrocenic compounds mimicking chloroquine and active upon chloroquine-resistant strains of Plasmodium falciparum were evaluated. Four 7-chloro-4-[[[2-[(N,N-substituted amino)methyl]ferrocenyl]methyl]amino]quinoline derivatives have been synthesized; one of them, 1a, showed high potent antimalarial activity in vivo on mice infected with Plasmodium berghei N. and Plasmodium yoelii NS. and was 22 times more potent against schizontocides than chloroquine in vitro against a drug-resistant strain of P. falciparum.

Elevated intracellular zinc and altered proton homeostasis in forebrain neurons.

Using the H(+)-sensitive fluorophore 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and microfluorimetry, we investigated how elevated intracellular free zinc ([Zn(2+)](i)) altered intracellular proton concentration (pH(i)) in dissociated cultures of rat forebrain neurons. Neurons exposed to extracellular zinc (3 microM) in the presence of the Zn(2+)-selective ionophore pyrithione (20 microM) underwent intracellular acidification that was not reversed upon washout of the stimulus. Application of a membrane-permeant Zn(2+) chelator, but not an impermeant chelator, partially restored pH(i). Removal of extracellular Ca(2+) greatly inhibited [Zn(2+)](i)-induced acidification, suggesting that acidification was a secondary consequence of Ca(2+) entry. Additional experiments suggested that Ca(2+) entered through the plasma membrane sodium/calcium exchanger (NCE), because a specific inhibitor of reverse mode NCE operation, KB-R7943 (1 microM), significantly inhibited Zn(2+)-induced acidification. In addition to the phenomenon of [Zn(2+)](i)-induced acidification, we found that elevated [Zn(2+)](i) inhibited neuronal recovery from low pH(i). Neurons exposed to a protonophore underwent robust acidification, and pH(i) recovery ensued upon protonophore washout. In contrast, neurons acidified by the protonophore in the presence of Zn(2+) (3 microM) and pyrithione (20 microM) showed no ability to recover from low pH(i). Application of a membrane-permeant Zn(2+) chelator partially restored pH(i) to pre-stimulus values. Experiments designed to elucidate mechanisms responsible for pH(i) regulation revealed that neurons relied primarily on bicarbonate exchange for proton export, suggesting that elevated [Zn(2+)](i) might impede pH(i) by inhibiting proton efflux via bicarbonate exchange. These results provide novel insights into the physiological effects of raising [Zn(2+)](i), and may help illuminate the mechanisms by which Zn(2+) injures neurons.

Synthetic ferrocenic mefloquine and quinine analoguesas potential antimalarial agents.

A few years ago we proposed a strategy for the synthesis of new ferrocene-chloroquine analogues replacing the carbon chain of chloroquine by hydrophobic ferrocenyl moieties. Now, this strategy has been applied to the antimalarial amino-alcohols class to afford new potentially active analogues of mefloquine and quinine bearing a substituted ferrocenic group. The pathway used for the synthesis of the mefloquine analogues includes the coupling of an aminomethyl substituted ferrocene carboxaldehyde with a lithio quinoline compound. On the other hand, the synthesis of quinine analogues was ensured by the 'inverse' reaction of a lithio aminomethyl ferrocene with a quinoline carboxaldehyde. The configurations of each diastereoisomer were unambiguously determined by spectroscopic data. The mechanistic interpretations were fully discussed. Ferrocenyl analogues of mefloquine and quinine exhibited a lower antimalarial activity than mefloquine and quinine themselves. Comparing optical isomers, those isomers dissimilar to ferrocenyl derivatives presented better antimalarial activities than those similar to ferrocenyl.

[Brain-death unit and renal transplantation. Evaluation of 5 years' experience]. / Centre d'accueil de morts cérébrales et transplantation rénale. Bilan de cinq années de fonctionnement.

From 1977 to 1982, 170 potential organ donors were referred to "a brain-death unit". A vast majority of these patients were provided by intensive care units of district general hospitals from Ile-de-France. This fact confirms the dispersion of potential organ donors and the usefulness of an organ-procurement structure based in an University Hospital. Its effectiveness is demonstrated by an harvesting rate of 58%, largely over the already published reports in case of absence of such a center. It is concluded that the adoption of this system by other hospitals would significantly increase the number of cadaver kidney grafts available for transplantation whereas actually the number of kidney grafts remains dramatically low in France.

[Continuous ambulatory peritoneal dialysis before renal transplantation]. / Dialyse péritonéale continue ambulatoire avant transplantation rénale.

In order to evaluate precisely the place of continuous ambulatory peritoneal dialysis in the treatment of chronic renal failure, it is important to find out whether this method may produce complications, mostly infectious, after renal transplantation. From April, 1979 to December, 1983, 419 renal transplantations were performed in our centre; 17 of these patients had previously been treated with peritoneal dialysis over a mean 13.5 months period, with 3.2 peritonitis/patient. The peritoneal catheter was left in situ for 4 to 16 weeks post-graft, so that the patients could easily be dialysed if needed; it was removed during transplantation in the only 3 cases of recent peritonitis. The only complications noted after transplantation were an episode of spontaneously reversible ascites and a peritoneal breach following reintervention on the renal region. This homogeneous series confirms that continuous ambulatory peritoneal dialysis does not constitute a contra-indication, let alone an obstacle, to subsequent renal transplantation. Indeed, it may be regarded as the first-choice method for patients in whom early grafting is envisaged on account of their immune status.

Na+ channel-mediated Ca2+ entry leads to glutamate secretion in mouse neocortical preplate.

Before synaptogenesis, early excitability implicating voltage-dependent and transmitter-activated channels is known to be crucial for neuronal development. We previously showed that preplate (PP) neurons of the mouse neocortex express functional Na(+) channels as early as embryonic day 12. In this study, we investigated the role of these Na(+) channels in signaling during early development. In the neocortex of embryonic-day-13 mice, activation of Na(+) channels with veratridine induced a large Ca(2+) response throughout the neocortex, even in cell populations that lack the Na(+) channel. This Na(+)-dependent Ca(2+) activity requires external Ca(2+) and is completely blocked by inhibitors of Na(+)/Ca(2+) exchangers. Moreover, veratridine-induced Ca(2+) increase coincides with a burst of exocytosis in the PP. In parallel, we show that Na(+) channel stimulation enhances glutamate secretion in the neocortical wall. Released glutamate triggers further Ca(2+) response in PP and ventricular zone, as indicated by the decreased response to veratridine in the presence of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor and NMDA-receptor inhibitors. Therefore, the combined activation of the Na(+) channel and the Na(+)/Ca(2+) exchanger triggers Ca(2+) signaling in the PP neurons, leading to glutamate secretion, which amplifies the signal and serves as an autocrine/paracrine transmitter before functional synapses are formed in the neocortex. Membrane depolarization induced by glycine receptors activation could be one physiological activator of this Na(+) channel-dependent pathway.

Two novel RXR alpha isoforms from mouse testis.

We report the isolation from mouse testis cDNA of two novel RXR alpha isoforms, mRXR alpha 2 and mRXR alpha 3, with distinct sequences upstream of exon 2. These two isoforms encode a similar protein (mRXR alpha 2/3) which lacks that 28 N-terminal amino acid residues of the major RXR alpha isoform, mRXR alpha 1. The N-terminal activation function (AF-1) of mRXR alpha 2/3 appears altered when compared to that of mRXR alpha 1. mRXR alpha 2 and mRXR alpha 3 are specifically expressed in the testis, and their expression is strongly upregulated in this tissue at puberty. These observations increase the molecular complexity of RXRs, and indicate that RXR alpha may play a specific function during spermatogenesis.

Quantitative evaluation of mitochondrial calcium content in rat cortical neurones following a glutamate stimulus.

1. Recent observations showed that a mitochondrial Ca2+ increase is necessary for an NMDA receptor stimulus to be toxic to cortical neurones. In an attempt to determine the magnitude of the Ca2+ fluxes involved in this phenomenon, we used carbonylcyanide-p-(trifluoromethoxy)phenylhydrazone (FCCP), a mitochondrial proton gradient uncoupler, to release mitochondrial free calcium ([Ca2+]m) during and following a glutamate stimulus, and magfura-2 to monitor cytoplasmic free calcium ([Ca2+]c). 2. FCCP treatment of previously unstimulated neurones barely changed [Ca2+]c whereas when added after a glutamate stimulus it elevated [Ca2+]c to a much greater extent than did exposure to glutamate, suggesting a very large accumulation of Ca2+ in the mitochondria. 3. Mitochondrial Ca2+ uptake was dependent on glutamate concentration, whereas the changes in the overall quantity of Ca2+ entering the cell, obtained by simultaneously treating neurones with glutamate and FCCP, showed a response that was essentially all-or-none. 4. Mitochondrial Ca2+ uptake was also dependent on the nature and duration of a given stimulus as shown by comparing [Ca2+]m associated with depolarization and treatment with kainate, NMDA or glutamate. Large mitochondrial Ca2+ accumulation only occurred after a glutamate or NMDA stimulus. 5. These studies provide a method of estimating the accumulation of Ca2+ in the mitochondria of neurones, and suggest that millimolar concentrations of Ca2+ may be reached following intense glutamate stimulation. It was shown that substantially more Ca2+ enters neurones following glutamate receptor activation than is reflected by [Ca2+]c increases.

Relationship between the degree of unsaturation of dietary fatty acids and adipose tissue fatty acids assessed by natural-abundance 13C magnetic resonance spectroscopy in man.

Natural-abundance 13C magnetic resonance spectroscopy was used for determining noninvasively the relative concentration of mono- and polyunsaturated fatty acids of adipose tissue in two groups of volunteers. The first consisted of subjects who had followed a fat-reduced diet for at least half a year before the 13C measurements. The second were control subjects who were on a usual high-fat diet. The ratio of unsaturated to total fatty acids in adipose tissue determined by 13C MRS correlated significantly with the same ratio in fat of the diet composition estimated by a dietician according to food records. The results indicate that 13C MRS is capable of assessing the degree of unsaturation of dietary fatty acids consumed during the preceding months.

A chimeric Cre recombinase inducible by synthetic,but not by natural ligands of the glucocorticoid receptor.

We have developed a new ligand-dependent chimeric recombinase (Cre-GRdex) by fusing the site-specific Cre recombinase to the ligand binding domain (LBD) of a mutant human glucocorticoid receptor (GRdex). The synthetic glucocorticoid receptor (GR) ligands dexamethasone, triamcinolone acetonide and RU38486efficiently induce recombinase activity in F9 murine embryonal carcinoma cells expressing constitutively Cre-GRdex. In contrast, no recombinase activity was detected in the absence of ligand or in the presence of the natural GR ligands corticosterone, cortisol or aldosterone. Moreover, physiological concentrations of these natural GR ligands do not affect Cre-GRdexrecombinase activity induced by dexamethasone. Thus, as previously shown using Cre-oestrogen receptor (ER) fusion proteins, Cre-GRdexmight be useful for achieving loxP site-directed mutagenesis in cultured cells and spatio-temporally controlled somatic cell mutagenesis in transgenic mice.

[Effects and side effects of a 1-year treatment of primary hypercholesterolemia with simvastatin]. / Wirkungen und Nebenwirkungen einer einjährigen Behandlung der primären Hypercholesterinämie mit Simvastatin.

The HMG-CoA-reductase inhibitors lovastatin, pravastatin and simvastatin (statins) represent a new group of drugs for the treatment of hypercholesterolemia. The present study was performed with simvastatin, the first statin introduced in Switzerland, and involved 46 patients with primary hypercholesterolemia during one year. The dose of simvastatin was adjusted according to the serum cholesterol level; during treatment with 10, 20 and 40 mg, total cholesterol was lowered by 18, 26 and 27% respectively and LDL cholesterol by 27, 38 and 35% respectively, after one year. A combination of 40 mg simvastatin with a bile acid sequestrant or a nicotinic acid derivative resulted in a cholesterol lowering of 38% and an LDL lowering of 48% respectively. The serum triglycerides decreased only at a dose of 10 mg. The changes in LDL and HDL cholesterol were accompanied by parallel alterations in apoprotein B and A1 concentrations. Although none of the patients showed a significant increase (greater than 3 x UNL) in serum ASAT, ALAT or CK, all these values increased slightly but significantly. Tolerance of the drug was otherwise excellent; gastrointestinal side effects were only rarely reported. Therefore, simvastatin is an effective and well tolerated cholesterol-lowering drug. Whether prevention of coronary heart disease is possible, however, is not yet proven.

Ligand-activated site-specific recombination in mice.

Current mouse gene targeting technology is unable to introduce somatic mutations at a chosen time and/or in a given tissue. We report here that conditional site-specific recombination can be achieved in mice using a new version of the Cre/lox system. The Cre recombinase has been fused to a mutated ligand-binding domain of the human estrogen receptor (ER) resulting in a tamoxifen-dependent Cre recombinase, Cre-ERT, which is activated by tamoxifen, but not by estradiol. Transgenic mice were generated expressing Cre-ERT under the control of a cytomegalovirus promoter. We show that excision of a chromosomally integrated gene flanked by loxP sites can be induced by administration of tamoxifen to these transgenic mice, whereas no excision could be detected in untreated animals. This conditional site-specific recombination system should allow the analysis of knockout phenotypes that cannot be addressed by conventional gene targeting.