Passive transfer of Lambert-Eaton myasthenic syndrome induces dihydropyridine sensitivity of ICa in mouse motor nerve terminals

Mice were injected for 30 days with plasma from three patients with Lambert-Eaton Myasthenic Syndrome (LEMS). Recordings were made from the perineurial sheath of motor axon terminals of triangularis sterni muscle preparations. The objective was to characterize pharmacologically the identity of kinetically distinct, defined potential changes associated with motor nerve terminal Ca2+ currents (ICa) that were affected by LEMS autoantibodies. ICa elicited at 0.01 Hz were significantly reduced in amplitude by approximately 35% of control in LEMS-treated nerve terminals. During 10-Hz stimulation, ICa amplitude was unchanged in LEMS-treated motor nerve terminals, but was depressed in control. During 20- or 100-Hz trains, facilitation of ICa occurred in LEMS-treated nerve terminals whereas in control, no facilitation occurred during the trains at 20 Hz and marked depression occurred at 100 Hz. Saturation for amplitude and duration of ICa in control terminals occurred at 2 and 4-6 mM extracellular Ca2+, respectively; in LEMS-treated terminals, the extracellular Ca2+ concentration had to increase by two to three times of control to cause saturation. Amplitude of the two components of ICa observed when the preparation was exposed to 50 microM 3,4-diaminopyridine and 1 mM tetraethylammonium were both reduced by LEMS plasma treatment. The fast component (ICa,s) was reduced by 35%, whereas the slow component (ICa, s) was reduced by 37%. omega-Agatoxin IVA (omega-Aga-IVA; 0.15 microM) and omega-conotoxin-MVIIC (omega-CTx-MVIIC; 5 microM) completely blocked ICa in control motor nerve terminals. The same concentrations of toxins were 20-30% less effective in blocking ICa in LEMS-treated terminals. The residual ICa remaining after treatment with omega-Aga-IVA or omega-CTx-MVIIC was blocked by 10 microM nifedipine and 10 microM Cd2+. Thus LEMS plasma appears to downregulate omega-Aga-IVA-sensitive (P-type) and/or omega-CTx-MVIIC-sensitive (Q-type) Ca2+ channels in murine motor nerve terminals, whereas dihydropyridine (DHP)-sensitive (L-type) Ca2+ channels are unmasked in these terminals. Acute exposure (90 min) of rat forebrain synaptosomes to LEMS immunoglobulins (Igs; 4 mg/ml) did not alter the binding of [3H]-nitrendipine or [125I]-omega-conotoxin-GVIA (-omega-CgTx GVIA) when compared with synaptosomes incubated with an equivalent concentration of control Igs. Conversely, LEMS Igs significantly decreased the Bmax for [3H]-verapamil to approximately 45% of control. The apparent affinity of verapamil (KD) for the remaining receptors was not significantly altered. Thus acute exposure of isolated central nerve terminals to LEMS Igs does not increase DHP sensitivity, whereas it reduces the number of binding sites for verapamil but not for nitrendipine or omega-CgTx-GVIA. These results suggest that chronic but not acute exposure to LEMS Igs either upregulates or unmasks DHP-sensitive Ca2+ channels in motor nerve endings.     

Distinct morphological characteristics of touch, temperature, and mechanical nociceptive neurons in the crotaline trigeminal ganglia

Intrasomal recording and horseradish peroxidase injection techniques were employed in vivo to determine the morphological characteristics of touch, temperature, and mechanical nociceptive neurons in the trigeminal ganglia of crotaline snakes. The touch neurons, with a peripheral axon conducting at the A-beta range, could be subdivided into tactile and vibrotactile neurons according to their response properties, but there were no morphological differences between them. These neurons exhibited a large and oval soma and possessed a set of large stem, peripheral, and central axons which were all myelinated and equal in diameter with a constriction at the bifurcation. The temperature neurons, which conducted peripherally at the A-delta range, were physiologically separated into thermosensitive and thermo-mechanosensitive neurons, which were also morphologically indistinguishable. The temperature neurons had a round soma of medium size and a set of medium axons with varied axonal bifurcation patterns. All axons of these neurons were myelinated, but the central axon was thinner than the stem and peripheral axons. The mechanical nociceptive neurons, which had a peripheral axon conducting at the A-delta range, were morphologically heterogeneous based on their conduction velocities. The neurons conducting at the fast A-delta range were morphologically similar to the temperature neurons in the ganglion excepting their thinner central axons, whereas those at the slow A-delta range had a thinner myelinated stem axon that gave rise to a thinner myelinated peripheral axon and an unmyelinated stem axon with a bifurcation of either a triangular expansion at the bifurcating point or a central axon arising straightforwardly from the constant stem and peripheral axons. This study revealed that distinct morphological characteristics do exist for the touch and temperature neurons and the subtypes of mechanical nociceptive neurons in the trigeminal ganglion, but not for the subfunctional types of touch neurons or temperature neurons.     

Nasopharyngeal perforation: an exceptional accident during digestive endoscopy]

We describe a case of systemic cytomegalovirus (CMV) infection in an ulcerative colitis patient admitted to the hospital for an acute flare-up of his colitis. He was treated with combination immunosuppressive therapy including i.v. cyclosporine and corticosteroids and PO azathioprine. Severe bilateral stabbing knee pain was the only manifestation of CMV disease, which quickly responded to adequate antiviral therapy.     

Photoaffinity labeling of oxidosqualene cyclase and squalene cyclase by a benzophenone-containing inhibitor

A new orally active oxidosqualene:lanosterol cyclase (OSLC) inhibitor (Ro48-8071; Morand, O. H. et al. (1997) J. Lipid Res. 38, 373-390) showed potent noncompetitive inhibition of bacterial squalene:hopene cyclase (SHC) from Alicyclobacillus acidocaldarius (IC50 = 9.0 nM, KI = 6.6 nM) and OSLC (IC50 = 40 nM, KI = 22 nM for homogeneous rat liver OSLC). A tritium-labeled isotopomer (18.8 Ci/mmol) of this nonterpenoid inhibitor, which possesses a benzophenone (BP) photophore, was chemically synthesized as a photoaffinity label. Specific, efficient covalent modification of both OSLC and SHC enzymes was observed after UV irradiation at 360 nm. Labeling of both OSLC and SHC by [3H]Ro48-8071 was competitively displaced by coincubation with a 1000-fold molar excess of 18-thia-2, 3-oxidosqualene or the nonterpenoid inhibitor BIBX79. Displacement of labeling of OSLC was also achieved with the suicide substrate (3S)-29-methylidene-2,3-oxidosqualene. Thus, the nonsubstrate Ro48-8071 and both terpenoid and nonterpenoid inhibitors of these enzymes appear to share a common binding site.     

Biphenylsulfonamide endothelin antagonists: structure-activity relationships of a series of mono- and disubstituted analogues and pharmacology of the orally active endothelin antagonist 2'-amino-N- (3,4-dimethyl-5-isoxazolyl)-4'-(2-methylpropyl)[1, 1'-biphenyl]-2-sulfonamide (BMS-187308)

Substitution at the ortho position of N-(3,4-dimethyl-5-isoxazolyl) benzenesulfonamide led to the identification of the biphenylsulfonamides as a novel series of endothelin-A (ETA) selective antagonists. Appropriate substitutions on the pendant phenyl ring led to improved binding as well as functional activity. A hydrophobic group such as isobutyl or isopropoxyl was found to be optimal at the 4'-position. Introduction of an amino group at the 2'-position also led to improved analogues. Combination of the optimal 4'-isobutyl substituent with the 2'-amino function afforded an analogue (20, BMS-187308) with improved ETA binding affinity and functional activity. Compound 20 also has good oral activity in inhibiting the pressor effect caused by an ET-1 infusion in rats. Doses of 10 and 30 micromol/kg iv 20 attenuated the pressor responses due to the administration of exogenous ET-1 to conscious monkeys, indicating that the compound inhibits the in vivo activity of endothelin-1 in nonhuman primates.     

Effect of mannitol crystallization on the stability and aerosol performance of a spray-dried pharmaceutical protein, recombinant humanized anti-IgE monoclonal antibody

We have examined the stability and aerosol performance of the pharmaceutical protein recombinant humanized anti-IgE monoclonal antibody (rhuMAbE25) spray dried with mannitol. The aerosol performance was measured by the fine particle fraction (FPF), and stability was assessed by the formation of soluble aggregates. When mannitol was added to the spray-dried rhuMAbE25 formulation, its ability to stabilize the protein leveled off above about 20% (w/w, dry basis). The FPF of the spray-dried formulations was stable during storage for rhuMAbE25 containing 10% and 20% mannitol, but the 30% formulation exhibited a dramatic decrease upon storage at both 5 degreesC and 30 degreesC, due to mannitol crystallization. We tested the addition of sodium phosphate to a 60:40 rhuMAbE25:mannitol (w:w) mixture, which otherwise crystallized upon spray drying and yielded a nonrespirable powder. The presence of sodium phosphate was successful in inhibiting mannitol crystallization upon spray drying and dramatically lowering the rate of solid-state aggregation. However, over long-term storage some crystallization was observed even for the phosphate-containing samples, concomitantly with increased particle size and decreased suitability for aerosol delivery. Therefore, the physical state of mannitol (i.e., amorphous or crystalline) plays a role both in maintaining protein stability and providing suitable aerosol performance when used as an excipient for spray-dried powders. Agents which retard mannitol crystallization, e.g., sodium phosphate, may be useful in extending the utility of mannitol as an excipient in spray-dried protein formulations.     

Subclavian mycotic aneurysm presenting as mediastinal abscess

A 60-year-old man was admitted to a hospital for evaluation of intermittent fever, dysphagia, hoarseness, and general chest discomfort. Great vessel mycotic aneurysm was suspected when antibiotic trials failed and chest X-ray showed paraaortic mass with pleural effusion mimicking mediastinitis. Although the correct diagnosis of mycotic aneurysm of innominate artery was made thereafter and vigorous treatment was initiated immediately, this patient succumbed to overwhelming sepsis, probably due to a 2-week delay in another hospital. This case is reported to remind readers of the possibility of this unusual location of mycotic aneurysm. A high index of suspicion should be maintained to make an earlier diagnosis and obtain better prognosis. Computed tomography and 3D magnetic resonance angiography also significantly improve the diagnosis when mycotic aneurysm location is unusual and presentation is equivocal.     

Sequence of the luxD gene encoding acyltransferase of the lux operon from Photobacterium leiognathi

The nucleotide sequence of luxD (EMBL accession No. X65611), encoding acyltransferase (ACT), of the lux operon from Photobacterium leiognathi PL741 was determined, and the amino acid (aa) sequence was deduced. ACT is a component of the fatty acid reductase complex, which is responsible for converting fatty acid to aldehyde that serves as the substrate in the luciferase-catalyzed bioluminescent reactions. The protein has a calculated M(r) of 34,384 and comprises 305 aa residues. Alignment and comparison of the ACT of P. leiognathi with that of Vibrio fischeri ATCC7744, V. harveyi B392 and Xenorhabdus luminescens Hm shows that there is 66%, 59% and 61% aa identity, respectively.     

Hydrogen peroxide production by monoamine oxidase during ischemia-reperfusion in the rat brain

Monoamine oxidase (MAO) as a source of hydrogen peroxide (H2O2) was evaluated during ischemia-reperfusion in vivo in the rat brain. H2O2 production was assessed with and without inhibition of MAO during and after 15 min of ischemia. Metabolism of H2O2 by catalase during ischemia and reperfusion was measured in forebrain homogenates using aminotriazole (ATZ), an irreversible H2O2-dependent inhibitor of catalase. Catecholamine and glutathione concentrations in forebrain were measured with and without MAO inhibitors. During ischemia, forebrain blood flow was reduced to 8% of baseline and H2O2 production decreased as measured at the microperoxisome. During reperfusion, a rapid increase in H2O2 generation occurred within 5 min as measured by a threefold increase in oxidized glutathione (GSSG). The H2O2-dependent rates of ATZ inactivation of catalase between control and ischemia-reperfusion were similar, indicating that H2O2 was more available to glutathione peroxidase than to catalase in this model. MAO inhibitors eliminated the biochemical indications of increased H2O2 production and increased the catecholamine concentrations. Mortality was 67% at 48 h after ischemia-reperfusion, and there was no improvement in survival after inhibition of MAO. We conclude that MAO is an important source of H2O2 generation early in brain reperfusion, but inhibition of the enzyme does not improve survival in this model despite ablating H2O2 production.