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This paper presents the quasi-static energy absorption of open section monolithic carbon-epoxy specimens for a range of environmental test conditions. Experimental observations showed that the test temperature influenced the failure mode; elevated temperatures (>23 °C) resulted in the specimens exhibiting a splaying failure mode whilst reduced test temperature (−35 °C) resulted in a brittle failure mode. Despite the difference in failure modes, the reduced and elevated test conditions both resulted in lower energy absorption compared with the baseline result at ambient temperature. In addition to the temperature effect, introducing moisture further reduced the energy absorption. The results show that an un-conditioned specimen tested at ambient temperature may not necessarily represent the worst case and care must be taken when using ambient temperature tests to inform the design of crashworthy structures and the influence of environment should be accounted for during the design phase.

This paper reports on the development of accelerated laboratory methods to allow estimation of preservative leaching from pressure-treated wood exposed to precipitation. End-matched lumber specimens were pressure-treated with a boron-copper formulation and exposed to either natural weathering for one year, laboratory immersion protocols, or a laboratory simulated rainfall protocol. The rainfall run-off or immersion water was collected at intervals according to the method used and analyzed for concentrations of copper and boron. Of the laboratory methods evaluated, the simulated rainfall approach resulted in leaching patterns most similar to outdoor exposure, especially in the case of copper. However, this method is relatively complex and not ideally suited for standardized use. Although the immersion methods evaluated initially exaggerated leaching, reasonable approximations of leaching from one year of natural weathering were achieved with accelerated testing. Models were developed to relate hours of immersion to millimeters of precipitation, and used to evaluate how well the immersion methods might predict leaching from natural weathering over many years of exposure. One of the methods produced boron and copper leaching estimates that were within 15% and 7% (respectively) of losses predicted for wood exposed to 5 years of natural weathering. The results indicate that laboratory immersion methods have value in estimating long term preservative leaching from treated wood products exposed to precipitation.

Soils and decayed plant litter contain significant quantities of chlorinated aromatic polymers that have a natural but largely unknown origin. We used cupric oxide ligninolysis coupled with gas chromatography/mass spectrometry to show that Curvularia inaequalis , a widely distributed litter ascomycete, chlorinated the aromatic rings of lignin in wood that it was degrading. In aspen wood decayed for 24 weeks, two chlorolignin fragments, 5-chlorovanillin and 2-chlorosyringaldehyde, were each found at ≈10 μg/g of wood (dry weight). These levels resemble those of similar structures generally found in unpolluted environmental samples. Fractionation of the extractable proteins followed by tandem mass spectrometric analysis showed that the colonized wood contained a previously described C. inaequalis chloroperoxidase that very likely catalyzed lignin chlorination. Chlorolignin produced by this route and humus derived from it are probably significant components of the global chlorine cycle because chloroperoxidase-producing fungi are ubiquitous in decaying lignocellulose and lignin is the earth's most abundant aromatic substance.

The giant glial cell in the neuropil of segmental ganglia of the leech Hirudo medicinalis responds to the activity of the Leydig interneuron and to a peptide of the myomodulin family, the presumed transmitter mediating the Leydig neuron-to-giant glial cell transmission, with a membrane hyperpolarization due to an increased membrane K + conductance [Britz et al. (2002) Glia, 38, 215-227]. We have now studied the second messenger cascade initiated by Leydig neuron stimulation and by the endogenous myomodulin (MMHir) in the voltage-clamped giant glial cell. Glial responses to both stimuli are mediated by a G-protein-coupled receptor linked to adenylyl cyclase by the following criteria: (i) injection of GDP-β-S, but not GDP, resulted in an irreversible decrease of the glial responses to both stimuli; (ii) the responses to both stimuli were reversibly inhibited by the adenylyl cyclase inhibitor SQ22,536; and (3) bath-applied di-butyryl-cyclic AMP, but not di-butyryl-cyclic GMP, elicited an outward current, which reduced the responses elicited by neuronal stimulation or myomodulin. A cocktail of protein kinase (PK) inhibitors (H-8, KT5720), the PKA antagonist Rp-cAMPS, or presumed inhibitors of cyclic nucleotide channels, LY83583 and L-cis-diltiazem, had no effect on the glial responses. Our results suggest that Leydig neuron stimulation and MMHiractivate a cAMP-mediated K + conductance in the glial cell, which appeared neither to be due to the activation of PKA nor of known cyclic nucleotide-gated channels directly.

5-Hydroxytryptamine (5-HT), a neurotransmitter and neuromodulator in the central nervous system of the leech Hirudo medicinalis hyperpolarizes the giant glial cell in the neuropil of segmental ganglia at micromolar concentrations. The 5-HT-evoked glial response (EC(50) approximately 2.5 microM) is mediated by a non-desensitizing, G-protein-coupled receptor and due to activation of a Ca(2+)-independent K(+) conductance. The adenylyl cyclase inhibitor SQ22,536 blocks the response to 5-HT; in the presence of 1 mM db-cAMP, but not of 1 mM db-cGMP, the glial response is suppressed. The 5-HT-evoked response is reduced by Ba(2+) with half-maximal inhibition at 50 microM Ba(2+). The results suggest that release of 5-HT from serotonergic neurons, or the maintenance of micromolar levels of extracellular 5-HT in the ganglion, may help to set the glial membrane potential close to the K(+) equilibrium potential.

Background and aims: As reactive oxygen has been demonstrated to participate in immune genes transcription, the aim of this study was to examine the relationship between systemic concentrations of several antioxidants and markers of inflammatory and immune activation in patients with Crohn's disease (CD). Methods: In 26 CD patients and 15 controls we compared plasma selenium and zinc concentrations, erythrocyte glutathione peroxidase (GSHPx) and superoxide dismutase activities, as well as erythrocyte sedimentation rate (ESR), C-reactive protein, tumor necrosis factor-α, interleukin-6, blood neopterin and soluble receptors of interleukin-2 (sIL-2R), and examined the link between these parameters. Results: Selenium concentration and GSHPx activity were decreased in CD patients (54.5 ± 3.2 vs 79 ± 2.2 μg/l, P⪡ 0.05; 28 ± 1.6 vs 38 ± 2.6 IU/g Hb, P⪡ 0.05) and positively correlated to each other's (r= 0.59, P⪡ 0.01). TNF-α was significantly increased in patients (18 ± 2.6 vs 5 ± 0.6 pg/ml;P⪡ 0.001), negatively correlated to GSHPx activity (r= −0.56, P⪡ 0.05) and selenium concentration (r= −0.72, P⪡ 0.001), and positively to neopterin and sIL-2R concentrations. Selenium showed negative correlation with sIL-2R (r= −0.83, P⪡ 0.0001) and ESR. Conclusions: In CD patients low selenium concentration may participate in reduced GSHPx activity facilitating inflammatory and immune activation. In these patients, selenium monitoring and, if needed, supplementation may be of therapeutical interest.

Objective: The aortovenocaval fistular (AVF) rat represents a model of heart failure caused by increased cardiac volume overload and reduced renal function. Both circulating vasoconstrictors like the renin-angiotensin-aldosterone system and vasodilators like atrial and brain natriuretic peptides (ANP and BNP) are activated in this animal model of heart failure. In addition, neutral endopeptidase 24.11 (NEP) in plasma and urine is elevated in AVF rats. In the present investigation we examined the renal and hormonal effects of the NEP inhibitor, ecadotril, in acute and chronic studies in rats with an aortovenocaval fistula (AVF). Methods: Sprague Dawley rats (350–430 g) were prepared by introducing a shunt between abdominal aorta and the vena cava. Results: Acute administration of the neutral endopeptidase inhibitor, ecadotril (30 mg/kg p.o.), significantly improved the reduced renal excretion of sodium in AVF rats (83 ± 10 to 145 ± 14 μmol/kg/h, P < 0.01) but had no significant effect in sham-operated rats. However, neutral endopeptidase activity in urine was significantly decreased after ecadotril in both groups. Plasma ANP was increased after ecadotril only in AVF rats (275 ± 83 to 748 ± 187 pg/ml, P < 0.05), whereas the increase in plasma BNP was not statistically significant. After 4 weeks of observation the ANP and BNP plasma levels, renin activity (PRA), angiotensin I, and neutral endopeptidase activity were significantly higher in AVF rats than in sham-operated rats. Four weeks on ecadotril (30 mg/kg p.o., b.i.d.) increased plasma ANP (245 ± 48 as opposed to 450 ± 77 pg/ml, P < 0.05) and decreased PRA (11.3 ± 1.5 as opposed to 6.8 ± 1.2 ng/ml/h, P < 0.005) in AVF rats. Plasma NEP activity was inhibited in both groups. Ventricle weight was significantly higher in AVF rats than in sham-operated controls, and ecadotril treatment over 4 weeks decreased ventricular hypertrophy to a slight extent. Conclusion: These results indicate that in the AVF rat model of heart failure the neutral endopeptidase inhibitor, ecadotril, improves the reduced kidney function in AVF rats by raising natriuretic peptides in plasma and probably in urine. NEP inhibition with ecadotril could therefore offer useful therapeutic possibilities in the treatment of heart failure.

Inhibition of important degradative pathways of atrial natriuretic peptide (ANP) in vivo could be a valuable therapeutic tool for regulating endogenous levels of ANP. The aim was to investigate the in vivo effects of both blockade of atrial natriuretic peptide clearance receptor and inhibition of neutral endopeptidase 24.11, an enzyme shown to be involved in ANP breakdown. Therefore, we infused a specific neutral endopeptidase inhibitor ((S)-thiorphan) and an ANP-C receptor ligand (AP 811) alone or in combination into anaesthetized beagle dogs. Compared with vehicle controls, coadministration of (S)-thiorphan and AP 811 (100 micrograms/kg/min and 10 micrograms/kg/min, resp.) had greater effects on endocrine and renal parameters than administration of either substance alone. Coadministration of both compounds increased urinary excretion of volume and sodium, cGMP and ANP. We found also increased plasma cGMP, plasma ANP and decreased plasma renin activity. No effects were observed with respect to blood pressure, left ventricular pressure or heart rate during the infusion period of 2 h. We conclude from these investigations, that blocking both degrading pathways of ANP with the ANP-C receptor ligand AP 811 and the neutral endopeptidase inhibitor (S)-thiorphan is more effective than inhibition of either system alone. Such a combination might therefore be a useful therapeutic tool in cardiovascular diseases.

The regioselective modification of a snake curaremimetic toxin is described. Toxin-alpha from Naja nigricollis was derivatized with an electron-dense maleimido undecagold cluster 5. The cluster-bound obtained toxin 8 has a very high affinity for the cholinergic binding site (Ki = 70 pM) of Torpedo marmorata nicotinic receptor. It is harboring a compact heavy atom core of about 8 A which makes it very useful for electron microscopy experiments.

A functionalized derivative of the μ opioid agonist carfentanil was synthesized (NH 2 -carfentanil) and showed high specific activity when radiolabeled with iodine. [ 127 I]NH 2 -carfentanil displayed high affinity and pronounced μ-binding selectivity with a δμ selectivity ratio of over 1200. The ability of [ 125 I]NH 2 -carfentanil to interact in vivo with opioid receptors was determined in mouse brain using ex vivo binding techniques. Twenty minutes after intraperitoneal injection, 0.1% of the [ 125 I]NH 2 -carfentanil injected into the mouse was present in the brain. [ 125 I]NH 2 -carfentanil specific binding was inhibited by co-injection of naloxone or morphine while naltrindole, a δ-selective antagonist, was unable to displace the bound radioligand. Autoradiographic experiments revealed a heterogeneous distribution of [ 125 I]NH 2 -carfentanil specific binding sites, maximal binding occurred in areas with high densities of μ receptors. Peripherally administered iodo-NH 2 -carfentanil selectively labelled central μ opioid receptors in mouse indicating great potential for single photon emission computed tomography studies.

Specific photoaffinity labelling of purified acetylcholinesterase from Torpedo marmorata by p-N,N-[3H]dimethylamino benzenediazonium and p-N,N-[3H]dibutylamino benzenediazonium derivatives was demonstrated. This occurred at the active site of the enzyme for lower concentrations of the probes and at the peripheral ammonium binding site for higher concentrations. The affinities and the rate constants of alkylation for each probe on both sites have been established. Specific labelling at the peripheral site of the enzyme with both probes allowed the identification of radio-labelled peptides having the common sequence K270PQELIDVEW. The radioactivity was always associated with the residue Trp279 indicating the preferential ammonium complexation with this aromatic residue.

Binding sites of Torpedo acetylcholinesterase (EC 3.1.1.7) for quaternary ligands were investigated by x-ray crystallography and photoaffinity labeling. Crystal structures of complexes with ligands were determined at 2.8-A resolution. In a complex with edrophonium, and quaternary nitrogen of the ligand interacts with the indole of Trp-84, and its m-hydroxyl displays bifurcated hydrogen bonding to two members of the catalytic triad, Ser-200 and His-440. In a complex with tacrine, the acridine is stacked against the indole of Trp-84. The bisquaternary ligand decamethonium is oriented along the narrow gorge leading to the active site; one quaternary group is apposed to the indole of Trp-84 and the other to that of Trp-279, near the top of the gorge. The only major conformational difference between the three complexes is in the orientation of the phenyl ring of Phe-330. In the decamethonium complex it lies parallel to the surface of the gorge; in the other two complexes it is positioned to make contact with the bound ligand. This close interaction was confirmed by photoaffinity labelling by the photosensitive probe 3H-labeled p-(N,N-dimethylamino)benzenediazonium fluoroborate, which labeled, predominantly, Phe-330 within the active site. Labeling of Trp-279 was also observed. One mole of label is incorporated per mole of AcChoEase inactivated, indicating that labeling of Trp-279 and that of Phe-330 are mutually exclusive. The structural and chemical data, together, show the important role of aromatic groups as binding sites for quaternary ligands, and they provide complementary evidence assigning Trp-84 and Phe-330 to the "anionic" subsite of the active site and Trp-279 to the "peripheral" anionic site.

Forty-three patients allergic to mites and suffering mainly from asthma were recruited: 25 were mite-monosensitive and 18 were polysensitive, as determined by skin tests and specific serum IgE determinations with various allergens. In vitro spontaneous histamine release (SHR) by washed blood basophils was measured once or several times for each patient. Throughout this study, the mean periods of high and low mite-allergen exposure were defined on the basis of relative indoor humidity and temperature data. For the mite-monosensitive patients, there was a significant increase in mean SHR during the season of high mite-allergen exposure as compared to the months of lower mite-allergen presence (P < 0.002). No significant difference between mean SHR values was observed when comparing the monosensitive group during the season of high mite-allergen exposure with polysensitive patients (allergic to mite and pollen) during the period of exposure to both allergens. Differences in mean SHR reported here emphasize the positive relationship between intense allergen exposure and the in vitro SHR increase in blood basophils of mite-allergic patients.

m-Sulfonate benzene diazonium chloride (MSBD) was used to affinity-label the gamma-aminobutyric acid (GABA) binding site from rat brain membranes. To assess the irreversibility of the labeling reaction, we used an efficient ligand dissociation procedure combined to a rapid [3H]muscimol binding assay, both steps being performed on filter-adsorbed membranes. Inactivation of specific [3H]-muscimol binding sites by MSBD and its prevention by GABA were both time- and concentration-dependent. The time course of MSBD labeling was shortened as the pH of the incubation medium was increased from 6.2 to 8. These data suggest that MSBD can efficiently label the GABA binding site through alkylation of a residue having an apparent dissociation constant around neutrality.

The synthesis of the radiolabelled heterobifunctional reagent N-methyl-N-chloroformyl-N′-terbutoxycarbonyl-para-phenylenediamine A (fig. 1) is presented. In particular, the use of tri-tritiated methyl iodide in defined reaction conditions allowed the synthesis of this probe B (fig. 1) with a high specific radioactivity (77 Ci/mmol).

Nα[(tert-Butoxy)carbonyl]-2-diazo-L-histidine methyl ester 1 was synthesized starting from the corresponding L-histidine derivative. The physico-chemical properties of this new photoactivatable amino-acid derivative were established. The synthetic precursor of 1, 2-amino-L-histidine derivative 3, was best isolated and characterized as 2-amino-Nα-[(tert-butoxy)carbonyl]-Nτ-tosyl-L-histidine methyl ester (4). Selective deprotections of 4 (Nα-Boc, Nα-Tos, COOMe) were achieved, thus allowing the use of the corresponding products in peptide synthesis. The optically active dipeptides 8 and 9 were synthesized by coupling 2-amino-Nτ-tosyl-L-histidine methyl ester (5) with N-[(tert-butoxy)carbonyl]-L-alanine and Nα-[(tert-butoxy)carbonyl]-Nτ-tosyl-L-histidine (6) with L-alanine methyl ester, respectively. The question of selective diazotization of a 2-aminohistidine residue in a synthetic peptide was studied using competitive diazotizations between 2-amino-1H-imidazole and several amino-acid derivatives susceptible to undergo nitrosylation. The results show that synthetic photoactivatable peptides incorporating a 2-diazohistidine residue might become useful photoaffinity probes.

Glycerol-induced acute renal failure (ARF) is characterized by an increase in serum creatinine, urea, and phosphate concentration, and severe impairment of creatinine clearance. Secondary hyperparathyroidism develops rapidly during ARF. The calcium antagonist nisoldipine clearly improves renal function, which becomes evident by an improvement of creatinine clearance and attenuation of the increase of serum creatinine, urea, and phosphate concentrations. Further secondary hyperparathyroidism is ameliorated by nisoldipine treatment. In spite of normalization of the hypocalcemia in ARF by nisoldipine, weak hyperparathyroidism persists, suggesting that hypocalcemia is not exclusively responsible for elevated parathyroid hormone serum levels in ARF.

SUMMARY Glial cells can be activated by neurotransmitters viametabotropic, G protein-coupled receptors. We have studied the effects of`global' G protein activation by GTP-γ-S on the membrane potential,membrane conductance, intracellular Ca2+ and Na+ of the giant glial cell in isolated ganglia of the leech Hirudo medicinalis. Uncaging GTP-γ-S (injected into a giant glial cell as caged compound) by moderate UV illumination hyperpolarized the membrane due to an increase in K+ conductance. Uncaging GTP-γ-S also evoked rises in cytosolic Ca2+ and Na+, both of which were suppressed after depleting the intracellular Ca2+ stores with cyclopiazonic acid (20 μmol l–1). Uncaging inositol-trisphosphate evoked a transient rise in cytosolic Ca2+ and Na+ but no change in membrane potential. Injection of the fast Ca2+ chelator BAPTA or depletion of intracellular Ca2+ stores did not suppress the membrane hyperpolarization induced by uncaging GTP-γ-S. Our results suggest that global activation of G proteins in the leech giant glial cell results in a rise of Ca2+-independent membrane K+conductance, a rise of cytosolic Ca2+, due to release from intracellular stores, and a rise of cytosolic Na+, presumably due to increased Na+/Ca2+ exchange.

The clearance of synaptically released glutamate is one of the pivotal functions of glial cells. We have studied the role of 5-hydroxytryptamine (5-HT, 30 microM), a neurotransmitter and neurohormone in the leech central nervous system with a versatile action spectrum, on the efficacy of glial glutamate uptake. The activity of the glutamate uptake carrier in the giant glial cell in isolated ganglia of Hirudo medicinalis was monitored by measuring the membrane current and the change in the intracellular Na(+) concentration (Na(+) (i)) as induced by the glutamate carrier substrate D-aspartate (D-asp, 1 mM). 5-HT increased the D-asp-induced current (EC(50) at 5 microM) and rise in Na(+) (i), an effect which was mimicked by the membrane-permeable cyclic nucleotide analogue dibutyryl-cyclic AMP (db-cAMP). The adenylyl cyclase inhibitor SQ 22,536 and the protein kinase A antagonist Rp-cAMP inhibited the effect of 5-HT. Blocking the G protein in the giant glial cell by injecting GDP-beta-S suppressed the effect of 5-HT, but not the effect of db-cAMP, on the D-asp-induced current. Our results suggest that 5-HT enhances the glial uptake of glutamate via cAMP- and PKA-mediated pathway.

The giant glial cell in the neuropil of segmental ganglia of the leech Hirudo medicinalis responds to the activity of the Leydig interneuron and to a peptide of the myomodulin family, the presumed transmitter mediating the Leydig neuron-to-giant glial cell transmission, with a membrane hyperpolarization due to an increased membrane K+ conductance [Britz et al. (2002) Glia, 38, 215-227]. We have now studied the second messenger cascade initiated by Leydig neuron stimulation and by the endogenous myomodulin (MMHir) in the voltage-clamped giant glial cell. Glial responses to both stimuli are mediated by a G-protein-coupled receptor linked to adenylyl cyclase by the following criteria: (i) injection of GDP-beta-S, but not GDP, resulted in an irreversible decrease of the glial responses to both stimuli; (ii) the responses to both stimuli were reversibly inhibited by the adenylyl cyclase inhibitor SQ22,536; and (3) bath-applied di-butyryl-cyclic AMP, but not di-butyryl-cyclic GMP, elicited an outward current, which reduced the responses elicited by neuronal stimulation or myomodulin. A cocktail of protein kinase (PK) inhibitors (H-8, KT5720), the PKA antagonist Rp-cAMPS, or presumed inhibitors of cyclic nucleotide channels, LY83583 and l-cis-diltiazem, had no effect on the glial responses. Our results suggest that Leydig neuron stimulation and MMHir activate a cAMP-mediated K+ conductance in the glial cell, which appeared neither to be due to the activation of PKA nor of known cyclic nucleotide-gated channels directly.

The cardiovascular consequences of endothelin (ET) blockade with the ETA receptor antagonist FR 139317 were evaluated by determining long-term effects of the drug on hemodynamic, hormonal, and structural parameters in stroke-prone spontaneously hypertensive rats (SHR-SP). Young SHR-SP on a high-sodium diet develop malignant hypertension accompanied by renovascular and cerebrovascular lesions. In control SHR-SPs the systolic blood pressure increased from 196 +/- 3 to 260 +/- 4 mm Hg, whereas in animals treated with FR 139317 (20 mg/kg, i.p., b.i.d.) blood pressure increased only from 196 +/- 4 to 212 +/- 3 mm Hg during a treatment period of 6 weeks. The increase in heart weight was also delayed. At the end of the experiment, the plasma levels of ANP and BNP were significantly lower in the group treated with FR 139317 than in the controls. The plasma ET levels were significantly higher and the plasma renin activity was lower in the group treated with the ET receptor antagonist. These data indicate that ET is involved in the maintenance of high blood pressure and cardiac hypertrophy in malignant hypertension, as exemplified by an SHR-SP rat model.

Pheochromocytoma and neuroblastoma are distinct tumours, but their biological diagnosis is based on secretion increase of one or several catecholamines. Assays have to be very sensible and specific for an early diagnosis. 24 hours urinary catecholamines and metabolites are currently measured, but technical improvements permit plasma metanephrine assay, an excellent indicator of pheochromocytoma. HPLC coupled to electrochemical detection represents the most efficient methodology. After a review of urinary and plasma assay methods, the authors show usual values of catecholamines, metanephrines, HVA and VMA, according to ages, and give examples of results encountered in classical or not tumours and in falsely positive cases. Urinary metanephrine assay is the most sensible and specific in biological diagnosis of pheochromocytoma, while catecholamines and VMA assays lack of sensibility. Results have to be given by 24 hours and by creatinine ratio. Metanephrine assay can be performed also in plasma and exhibits the same interest. However, in urine as in plasma, in case of renal failure, results cannot be interpreted. Neuroblastoma biological diagnosis is based classically on HVA, VMA, and dopamine assays, nowadays only in 24 hours urine (or in urinary micturition for screening), and results are also expressed as creatinine ratio. But even if several assays are advisable, 5% of the neuroblastoma cases do not produce increased catecholamine values. In some cases, metanephrine assay could be of interest. After the age of 12 months, clinical expression of neuroblastoma is dramatic in 70% of cases. So, a biological screening has been experimented in several countries including France. A French translation of the consensus conference report (1998) is appended, which shows the complexity of neuroblastoma screening. Now, there is no evidence that early tumour detection by screening lessens the mortality rate, but a weak benefit is not excluded.

These studies were designed to investigate the protective effects of the new angiotensin II receptors antagonist BAY 10-6734 (6-n-butyl-4-methoxycarbonyl-2-oxo-1[(2'-(1H-tetrazol-5-yl) -3-fluorobiphenyl-4-yl)methyl] 1,2-dihydropyridine, CAS 156001-18-2) on haemodynamic, hormonal, renal, and structural parameters in renin transgenic rats (TGR(mRen2)27), salt-loaded Dahl S and R rats, and salt-loaded stroke-prone spontaneously hypertensive rats (SHR-SP) in long-term trials. Study 1: In SHR-SP the development of blood pressure, cardiac hypertrophy, and the deleterious effects of salt loading on kidney structure and kidney function was prevented by BAY 10-6734. Study 2: In salt-loaded Dahl S rats with a suppressed plasma renin activity treatment with BAY 10-6734 did not delay the increase in blood pressure but prevented cardiac hypertrophy and the increase in plasma ANP (Atrial natriuretic peptide). Study 3: TGR develop malignant hypertension associated with cardiac hypertrophy, elevated left-ventricular end-diastolic pressure and increased plasma ANP. After 6 weeks of treatment with BAY 10-6734 (30 mg/kg p.o. bid) cardiac pump function was improved and cardiac hypertrophy was reversed in this angiotension dependent form of hypertension. The beneficial effects of BAY 10-6734 in these different animal hypertension models are also emphasized by a reduction in mortality.

It has been shown that near-infrared spectroscopy (NIRS) permits the assessment of functional brain activation.8,9,14,21 Four studies were dedicated to the visual system which partly yielded conflicting results.10,11,13,21 Though all studies showed an increase in [oxy-Hb] during visual stimulation using different stimulation paradigms, the changes observed in [deoxy-Hb] were not uniform. Meek et al.13 reported on a decrease in [deoxy-Hb] in 4 of 10 subjects but an increase in the other 6 subjects, while Kato et al.11 and Hoshi et al.10 using photic stimulation of 8 Hz and 10 Hz found an increase in [deoxy-Hb] in their studies. On the other hand, Villringer et al.21 observed a decrease in [deoxy-Hb] during photic stimulation and picture observation, but this included only 3 subjects lacking therefore statistical evaluation. All NIRS-investigators10,11,13,21 localised the optical probes only with respect to external bony landmarks without knowledge of the exact spatial relation of these landmarks to the anatomy of the brain. This could have led to inaccurate positions of the optical probes over the visual cortex as there is a great variability of the calcarine fissure concerning these external landmarks.18 Localising the optical probes individually according to previously acquired 3D MRI, the aim of the current study was to investigate whether there is a consistent, statistically evident pattern of changes in [oxy-Hb] and [deoxy-Hb] during visual stimulation. Furthermore, we investigated if NIRS is able to detect oxygenation changes due to activation of a secondary or prestriate visual area known from studies both in monkeys and humans as area V5 or MT to be sensitive to the submodality “visual motion”.12,19,22,24

The influence of different lengths of the pre-stimulation resting period on the magnitude of a hemodynamic response evoked by motor stimulation was examined in 10 subjects by means of near-infrared spectroscopy (NIRS). A motor stimulus was used which has been previously established as a model for functional activation studies with NIRS. Subjects performed a 20 s finger opposition task in the hand contralateral to NIRS probe localization over left sensorimotor area (C3', according to the 10-20 system). The duration of the pre-stimulation resting period was varied from 10s to 50s and response magnitude was assessed for each of the interstimulus intervals (10 s, 20 s, 30 s, 40 s and 50 s). Data analysis showed that response magnitude in oxygenated and deoxygenated haemoglobin concentration changed with different interstimulation intervals. Interestingly the greatest NIRS response was obtained with resting period 30 s prior to stimulation; shorter and longer resting periods resulted in smaller responses. The time course and the dependence of response magnitude on interstimulus interval differed between [oxy-Hb] and [deoxy-Hb] changes. For [oxy-Hb] the previously described fast initial increase ('overshoot') and the post-stimulation undershoot was more clearly seen with long prestimulation resting periods. Cytochromeoxidase oxygenation changes did not change significantly with different interstimulus intervals. We conclude that comparisons between different functional activation studies with techniques relying on stimulus evoked changes in cerebral hemodynamics must take into account not only the quality of the experimental paradigm and the length of the stimulation period, but also that the resting period between repetitive stimulations is important for response amplitude and its time course.

During performance of a sequential finger opposition task we measured changes in regional cerebral blood oxygenation (rCBO) over the motor cortex and blood flow velocity changes (CBFV) in the middle cerebral artery in a combined near-infrared spectroscopy (NIRS) and transcranial Doppler Sonography (TCD) study. Stimulus duration was 60 followed by a 90 s rest period. During performance of the motor task we observed an increase in [oxy-Hb] a decrease in [deoxy-Hb] and an increase in MCA flow velocity. These changes were significantly more pronounced contralaterally than ipsilaterally to the moving hand. The time course of changes in [oxy-Hb] and CBFV were strikingly similar, showing a pronounced initial over-shoot. This study proves the feasibility of a simultaneous assessment of microcirculatory hemodynamics and cerebral oxygenation at high temporal resolution.

The nicotinic acetylcholine receptor (AChR) exhibits at least four different conformational states varying in affinity for agonists such as acetylcholine (ACh). Photoaffinity labeling has been previously used to elucidate the topography of the AChR. However, to date, the photosensitive probes used to explore the cholinergic binding site photolabeled only closed or desensitized states of the receptor. To identify the structural modifications occurring at the ACh binding site on allosteric transition associated with receptor activation, we have investigated novel photoactivatable 4-diazocyclohexa-2,5-dienone derivatives as putative cholinergic agonists. Such compounds are fairly stable in the dark and generate highly reactive carbenic species on irradiation. In binding experiments using AChRs from Torpedo marmorata, these ligands had affinities for the ACh binding site in the micromolar range and did not interact with the noncompetitive blocker site (greater than millimolar affinity). Irreversible photoinactivation of ACh binding sites was obtained with the ligand 1b (up to 42% at 500 microM) in a protectable manner. In patch-clamp studies, 1b was shown to be a functional agonist of peripheral AChR in TE 671 cells, with the interesting property of exhibiting no or very little desensitization even at high concentrations.

Laboratory investigation of catecholamines is useful for the clinician in the diagnosis and management of phaeochromocytoma and neuroblastoma. This work summarizes current knowledge on catecholamines and their metabolites and discusses the main indications for their determination. It also examines the most practical methods for studying the secretion of these hormones, ie extraction, separation and quantification using high performance liquid chromatography coupled with electrochemical detection. The workshops attended by the members of the catecholamines working party of the French Clinical Biology Society and phaeochromocytoma and neuroblastoma specialists enabled the role of such determinations in the diagnosis and management of these diseases to be clarified.

It has been found that opiate abusers once detoxified are afflicted with personality disorders. An attempt was made to test the origin of drug abuse by comparing a group of drug abusers with personality disorders diagnosed as borderline or schizotypal with a group of people suffering from similar personality disorders but who did not use drugs. The results of the test are discussed.

p-Butyroxybenzenediazonium fluoroborate 1 was shown to be a substrate of both acetylcholinesterase (AcChE) and butyrylcholinesterase (BuChE) with Michaelis constants of 6·10−5 M and 1.3·10−4 M, respectively. Upon incubation in the dark, 1 was able to discriminate between the two enzymes AcChE was efficiently inactivated in a time-dependent manner while BuChE remained unaffected. Kinetic analysis of the inactivation of AcChE (i) by various concentrations of 1 indicated that it behaves as an affinity label, (ii) at three different pH levels suggested that the pKa of the labelled residue was higher than 7 and (iii) in the presence of different selective ligands for either the active site (edrophonium) or the peripheral site (propidium) indicated that 1 alkylated the active site rather than the peripheral one. Differences of reactivity between AcChE and BuChE suggest a different positioning and/or a different chemical environment of the substrate within the two active sites.

We have synthesized a new thyrotropin releasing hormone (TRH) analog, pGlu-2-diazo-His-Pro-NH2 5, which is a potential photoaffinity label for the TRH receptor. Its precursor, pGlu-2-amino-His-Pro-NH2 4, has been synthesized through successive coupling of a 2-aminohistidine derivative 1 with proline and pyroglutamic acid derivatives. Diazotization of the fully deprotected tripeptide 4 gave the photoactivatable TRH analog 5. Compounds 4 and 5 exhibit IC50 values of 2 and 8 microM respectively, to be compared to the 0.02 microM value for native TRH.

It is commonly acknowledged that calcium antagonists inhibit transmembrane calcium influx in arteriolar smooth muscle cells and thus reduce total peripheral resistance. This mechanism of action is supposed to be the prime basis of the antihypertensive action of these agents. However, despite their action as arteriolar vasodilators, therapeutically, calcium antagonists differ from vasodilators with other mechanisms of action (e.g., hydralazine or minoxidil) in having a marked natriuretic and diuretic action [6,8,17] and have been shown to preserve structure and function of heart, kidney, and mesenteric arterioles of hypertensive rats [2–4,10,13,18,24–26]. Therefore, tissue-protective effects of dihydropyridine calcium antagonists which might be unrelated to their hemodynamic actions, might add significantly to their long-term therapeutic efficacy in hypertension. We investigated the antihypertensive and tissue-protective effects of dihydropyridine calcium antagonists in long-term experiments: (1) nisoldipine in salt-loaded Dahl S and R rats in a preventive and therapeutic experiment, (2) nitrendipine in spontaneously hypertensive rats and Wistar-Kyoto rats in comparison to a classical vasodilator, and (3) nimodipine in stroke-prone spontaneously hypertensive rats in comparison to parathyroidectomy.

We have defined the molecular environment of a snake neurotoxin interacting with the high- and low-affinity binding sites of the nicotinic acetylcholine receptor (AcChoR). This was done by photocoupling reactions using three toxin derivatives with photoactivatable moieties on Lys-15, Lys-47, and Lys-51. Competition data showed that Lys-47 belongs to the toxin-AcChoR interacting domain whereas the other two residues are excluded from it. We first tentatively determined the threshold of covalent coupling, indicative of the proximity between the photoactivatable probes and subunits, by quantifying the coupling occurring between the same derivatives and a model compound (i.e., a toxin-specific monoclonal antibody). We then (i) quantified the coupling yields occurring when both binding sites of AcChoR were occupied by the toxin derivatives, (ii) discriminately quantified the coupling yields at the high-affinity binding site, and (iii) deduced the coupling yields at the low-affinity binding site. In the high-affinity site, the probes on Lys-15 and Lys-47 predominantly reacted with the high-affinity site of the AcChoR alpha subunit whereas the probe on Lys-51 reacted with the delta subunit. In the low-affinity site, the probe on Lys-47 predominantly reacted with the low-affinity site of the alpha chain and the beta chain whereas those on Lys-15 and Lys-51 reacted with the gamma and delta chains, respectively. A three-dimensional model showing a unique organization of AcChoR bound to two toxin molecules is presented.

Elucidation of the role of (elevated) endogenous atrial natriuretic factor (ANF) in chronic heart failure has been hampered by a lack of specific inhibitors. We used a newly developed monoclonal antibody that has been shown to specifically block both exogenously and endogenously released ANF in vivo. For assessment of the vasodilatory action of ANF in chronic heart failure, either this antibody against ANF or ascites (control serum) was injected in rats with myocardial infarction and failure and in sham animals. Ascites did not alter central hemodynamics in either the sham or infarcted group. Antibody significantly increased right atrial pressure, left ventricular end-diastolic pressure, and systemic vascular resistance (SVR) in the infarction group but did not affect these variables in the sham group. Because renal blood flow, as measured by radioactive microspheres, decreased significantly in all four groups, probably due to nonspecific renal vasoconstrictor effects of the ascites, a separate group of infarcted animals was treated with purified ANF antibody (devoid of nonspecific effects) or mouse IgG as a control injection. In these animals, right atrial pressure increased from 1.1 +/- 0.7 to 2.6 +/- 0.7 mm Hg (p less than 0.001). Although SVR, renal blood flow velocity (measured by Doppler probe), and renal vascular resistance did not change in the infarcted animals after administration of purified ANF antibody, a significant correlation was found between baseline plasma ANF values and the change in SVR exerted by purified ANF antibody (r = 0.758, p less than 0.02, n = 9); that is, SVR increased in rats with high baseline plasma ANF (greater than 350 pg/ml), but decreased in animals with plasma ANF less than 200 pg/ml. These results suggest that moderately elevated endogenous plasma ANF levels in chronic heart failure do affect central hemodynamics, primarily by reducing venous pressure (e.g., by decreasing intravascular volume or by venous dilation). Arterial vasodilation, however, appears to emerge when plasma ANF is greatly increased.

In our attempt to characterize an opioid receptor at the molecular level, we constructed a random primed cDNA expression library from NG 108-15 cells RNA. Pools of plasmidic DNA were transfected into COS cells, which were screened for their ability to bind [3H] DTLET. A cDNA was isolated which encodes a 372 amino acid residue protein presenting all the structural characteristics of receptors that interact with guanine nucleotide-binding proteins. When expressed in COS cells, the receptor exhibits pharmacological properties similar to that of the native receptor and is negatively coupled to adenylate cyclase. A 8.5 kb transcript is detected in NG 108-15 cells as well as in mouse brain by northern analysis