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The congenital disorders of glycosylation (CDGs) are recent additions to the repertoire of inherited human genetic diseases. Frequency of CDGs is unknown since most cases are believed to be misdiagnosed or unrecognized. With few patients identified and heterogeneity in disease signs noted, studies of animal models may provide increased understanding of pathogenic mechanisms. However, features of mammalian glycan biosynthesis and species-specific variations in glycan repertoires have cast doubt on whether animal models of human genetic defects in protein glycosylation will reproduce pathogenic events and disease signs. We have introduced a mutation into the mouse germline that recapitulates the glycan biosynthetic defect responsible for human CDG type IIa (CDG-IIa). Mice lacking the Mgat2 gene were deficient in GlcNAcT-II glycosyltransferase activity and complex N-glycans, resulting in severe gastrointestinal, hematologic, and osteogenic abnormalities. With use of a lectin-based diagnostic screen for CDG-IIa, we found that all Mgat2-null mice died in early postnatal development. However, crossing the Mgat2 mutation into a distinct genetic background resulted in a low frequency of survivors. Mice deficient in complex N-glycans exhibited most CDG-IIa disease signs; however, some signs were unique to the aged mouse or are prognostic in human CDG-IIa. Unexpectedly, analyses of N-glycan structures in Mgat2-null mice revealed a novel oligosaccharide branch on the "bisecting" N-acetylglucosamine. These genetic, biochemical, and physiologic studies indicate conserved functions for N-glycan branches produced in the Golgi apparatus among two mammalian species and suggest possible therapeutic approaches to GlcNAcT-II deficiency. Our findings indicate that human genetic disease due to aberrant protein glycosylation can be modeled in the mouse to gain insights into N-glycan-dependent physiology and the pathogenesis of CDG-IIa.

Fibroblast growth factor receptors (FGFR) are widely expressed in many tissues and cell types, and the temporal expression of these receptors and their ligands play important roles in the control of development. There are four FGFR family members, FGFR-1–4, and understanding the ability of these receptors to transduce signals is central to understanding how they function in controlling differentiation and development. We have utilized signal transduction by FGF-1 in PC12 cells to compare the ability of FGFR-1 and FGFR-3 to elicit the neuronal phenotype. In PC12 cells FGFR-1 is much more potent in the induction of neurite outgrowth than FGFR-3. This correlated with the ability of FGFR-1 to induce robust and sustained activation of the Ras-dependent mitogen-activated protein kinase pathways. In contrast, FGFR-3 could not induce strong sustained Ras-dependent signals. In this study, we analyzed the ability of FGFR-3 to induce the expression of sodium channels, peripherin, and Thy-1 in PC12 cells because all three of these proteins are known to be induced via Ras-independent pathways. We determined that FGFR-3 was capable of inducing several Ras-independent gene expression pathways important to the neuronal phenotype to a level equivalent of that induced by FGFR-1. Thus, FGFR-3 elicits phenotypic changes primarily though activation of Ras-independent pathways in the absence of robust Ras-dependent signals.

Sodium channel 6 (NaCh6) is the α‐subunit of a voltage‐gated sodium channel expressed in the rat nervous system. The mRNA for this isoform has been shown to be expressed in both neuronal and glial cells by in situ hybridization. To examine localization of NaCh6 protein, polyclonal antibodies specific for NaCh6 were generated against peptides from two cytoplasmic domains and a fusion protein from an extracellular domain. Affinity‐purified antibodies were used to localize NaCh6 in the brain, spinal cord, peripheral nervous system, and neuromuscular junction. There was widespread labeling of neurons in the brain and spinal cord. NaCh6 was present in both sensory and motor pathways. Radial glial cells in the cerebellum were intensely labeled for both GFAP and NaCh6. At the subcellular level, NaCh6 is found in axons, dendrites, and the cell body. Motor neurons and primary sensory neurons in dorsal root ganglia had strong cytoplasmic and axonal staining. Nodes of Ranvier in peripheral nerve and in the spinal cord were also intensely labeled. Motor neuron axons near the neuromuscular junction were labeled up to, but not including, terminal boutons. Dendrites of pyramidal cells in the cortex, hippocampus, and cerebellum were labeled. NaCh6 is the first NaCh subtype to be localized either at the node of Ranvier or to a dendrite. We conclude that NaCh6 is widely distributed in the central and peripheral nervous systems and is likely to be important for the electrical properties of the axon and dendrite. J. Comp. Neurol. 420:70–83, 2000. © 2000 Wiley‐Liss, Inc.

A novel series of N-substituted 4-ureido-5,7-dichloro-quinolines were synthesized to contain pharmacophores directed at voltage-sensitive sodium channels (VSNaCs) and N-methyl-D-aspartate (NMDA) receptors. These compounds were shown to act in a use-dependent manner as antagonists of VSNaCs and to act as selective competitive antagonists at the strychnine-insensitive glycine recognition site of NMDA receptors. These agents had little or no effect on alpha-adrenergic receptors, other glutamate receptors, or sites other than the glycine site on the NMDA receptor, and did not block voltage-sensitive calcium channels in vitro. In vivo, the compounds were active in preventing or reducing the signs and symptoms of neurohyperexcitability and had anxiolytic properties. Unlike benzodiazepines, N-substituted 4-ureido-5, 7-dichloro-quinolines showed little interaction with the sedative effects of ethanol, but were effective in controlling ethanol withdrawal seizures. The combined actions of these compounds on VSNaCs and NMDA receptors also impart properties to these compounds that are important for preventing and reducing excitotoxic neurodegeneration, but these compounds lack the undesirable side effects of other agents used for these purposes.

A variety of complex compounds have been labeled with tritium gas by catalytic exchange in the presence of catalyst precursors [(cod)Ir(dppe)]BF4 or [(cod)Ir(py)(PCy3)]BF4. In most cases, predictable regioselectivity and high specific activities are achieved. These results are compared in some cases to the results of labeling related compounds with [(cod)Ir(PPh3)2]BF4. Prereduction of the catalyst precursors in situ with hydrogen allows the use of smaller quantities of tritium gas and reduces the amount of radioactive waste. Two or more compounds can be labeled simultaneously as mixtures then separated in the HPLC purification step to increase compound throughput. Copyright © 1999 John Wiley & Sons, Ltd.

Summary Using the dialysable detergent CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate), the tetrodotoxin-binding protein from the electroplax of the electric eel has been purified to a high degree of both chemical homogeneity and toxin-binding activity. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the best preparations showed only a single microheterogeneous band atMr approximately 260,000, despite attempts to visualize smaller bands by sample overloading. Upon dialysis, this material became incorporated into the membranes of small unilamellar vesicles, and in this form the purified protein exhibited tetrodotoxin-binding properties similar to the component in the original electroplax membrane. Furthermore, in the presence of activator neurotoxins the vesicles were able to accumulate isotopic sodium in a manner similar to that previously described for less active or less pure preparations of vesicles containing either mammalian or eel electroplax toxinbinding proteins. Quantitative consideration of the isotopic transport activity of this pure material, along with the high degree of purity of the protein, strongly suggests that the 260-kDa glycopeptide from electroplax is necessary and sufficient to account for the sodium channel function seen in these studies, and eliminates the possible involvement of smaller peptides in the channel phenomena observed.

Summary The voltage-dependent sodium channel from the eel electroplax was purified and reconstituted into vesicles of varying lipid composition. Isotopic sodium uptake experiments were conducted with vesicles at zero membrane potential, using veratridine to activate channels and tetrodotoxin to block them. Under these conditions, channel-dependent uptake of isotopic sodium by the vesicles was observed, demonstrating that a certain fraction of the reconstituted protein was capable of mediating ion fluxes. In addition, vesicles untreated with veratridine showed significant background uptake of sodium; a considerable proportion of this flux was blocked by tetrodotoxin. Thus these measurements showed that a significant subpopulation of channels was present that could mediate ionic fluxes in the absence of activating toxins. The proportion of channels exhibiting this behavior was dependent on the lipid composition of the vesicles and the temperature at which the uptake was measured; furthermore, the effect of temperature was reversible. However, the phenomenon was not affected by the degree of purification of the protein used for reconstitution, and channels in resealed electroplax membrane fragments or reconstituted, solely into native eel lipids did not show this behavior. The kinetics of vesicular uptake through these spontaneously-opening channels was slow, and we attribute this behavior to a modification of sodium channel inactivation.

The axonal distribution of voltage‐dependent Na+channels was determined during inflammatory demyelinating disease of the peripheral nervous system. Experimental allergic neuritis was induced in Lewis rats by active immunization. In diseased spinal roots Na+channel immunofluorescence at many nodes of Ranvier changed from a highly focal ring to a more diffuse pattern and, as the disease progressed, eventually became undetectable. The loss of nodal channels corresponded closely with the development of clinical signs. Electrophysiological measurements and computations showed that a lateral spread of nodal Na+channels could contribute significantly to temperature sensitivity and conduction block. During recovery new clusters of Na+channels were seen. In fibers with large‐scale demyelination, the new aggregates formed at the edges of adhering Schwann cells and appeared to fuse to form new nodes. At nodes with demyelination limited to paranodal retraction, Na+channels were often found divided into two symmetric highly focal clusters. These results suggest that reorganization of Na+channels plays an important role in the pathogenesis of demyelinating neuropathies. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:1019–1032, 1998.

1.A new method of deasphalting reduced crudes with a moving bed of adsorbent has been investigated; this process gives efficient separation of the basic chemical groups and a high yield of valuable oil components.2.The primary deasphalted oils obtained from a series of Volgograd, Perm', Bulgarian and Zhirnovsk crudes are thoroughly deresined products (1.5–3% resins) with high viscosities (30–45 cSt at 100°C) and carbon residues (2–4%), as a result of increased recovery of high-viscosity paraffinic, naphthenic, and aromatic hydrocarbons and compounds from the reduced crudes.3.When the adsorption-deasphalted oils are further treated with a moving bed of adsorbent, light-colored deresined residual oils can be obtained in various viscosity levels (from aviation oils up to heavy bright stocks).

1. We have examined the binding of tritium‐labelled and unlabelled tetrodotoxin to frog twitch muscle. Bio‐assay as well as radioisotope experiments show a saturable component of tetrodotoxin binding with a binding capacity of about 22 p‐mole/g wet wt., and a dissociation constant of about 5 nM. 2. If the observed uptake of tetrodotoxin by muscles represents one‐to‐one binding of the drug to sodium channels, the channel density is about 380 channels/mum2 of a muscle fibre's surface membrane. On the basis of this result and electrical measurements of sodium conductance in frog muscle, we calculate that the conductance of a single sodium channel is of the order of 10(−12) reciprocal ohms. This is one to two orders of magnitude less than previous estimates. 3. We have looked for an effect of membrane depolarization on saturable tetrodotoxin binding, and have found none. This suggests that there is little molecular interaction between the “gating” portion of the sodium channel molecule, and that which binds tetrodotoxin.

Several calcium-entry blockers, i.e., verapamil, nifedipine, flunarizine and diltiazem, were evaluated for their effects in models of immediate hypersensitivity disease. Verapamil, flunarizine and diltiazem were all effective in inhibiting antigen-induced bronchospasm in the guinea pig; however, the effects seen were at relatively high doses compared to the doses known to cause cardiovascular effects. Nifedipine caused no significant inhibition of resistance or compliance changes induced by antigen. Flunarizine, verapamil and diltiazem were ineffective in inhibiting antigen-induced histamine release from rat peritoneal mast cells in vitro. Although these compounds were active inhibitors of 5-D-[5,6,8,9,H,12,14,15-3H(N)]-hydroxy-6,8,11,14-eicosatetraenoic acid production in rat basophilic leukemia-1 cells, only flunarizine and verapamil showed effects on the 5-lipoxygenase enzyme when assayed directly. Also, these compounds were ineffective on SRS-A mediated bronchospasm in vivo. These data suggest that the currently available calcium entry blockers have little potential use in immediate hypersensitivity reactions.

The voltage- and frequency-dependent interactions of pentobarbital with voltage-gated sodium channels were examined in whole-cell patch-clamp recordings. Using rat brain IIA and rat muscle rSkM1 sodium channels expressed in stably transfected Chinese hamster ovary cell lines, it was found that pentobarbital reduced peak inward sodium currents with IC50 values of 1.2 mM (brain) and 1.0 mM (muscle). Analysis of steady state channel availability curves revealed two distinct effects of pentobarbital on both channel isoforms, i.e., a voltage-independent current reduction and an additional hyperpolarizing shift in the voltage dependence of channel availability. The latter effect leads to a voltage dependence of pentobarbital potency. Pentobarbital was also found to slow channel recovery after depolarization, yielding an additional use-dependent component of current suppression. Use-dependent block was enhanced by higher stimulation frequencies, longer pulse durations, and more depolarized holding and pulse potentials. All effects were identical for both channels. These findings can be explained in terms of the modulated receptor hypothesis and are consistent with a preferential interaction of pentobarbital with the inactivated channel state. As a consequence, actual pentobarbital potency would depend largely on experimental conditions or, in vivo, on the physiological parameters of a particular cell.

The axonal distribution of voltage-dependent Na⁺ channels was determined during inflammatory demyelinating disease of the peripheral nervous system. Experimental allergic neuritis was induced in Lewis rats by active immunization. In diseased spinal roots Na⁺ channel immunofluorescence at many nodes of Ranvier changed from a highly focal ring to a more diffuse pattern and, as the disease progressed, eventually became undetectable. The loss of nodal channels corresponded closely with the development of clinical signs. Electrophysiological measurements and computations showed that a lateral spread of nodal Na⁺ channels could contribute significantly to temperature sensitivity and conduction block. During recovery new clusters of Na⁺ channels were seen. In fibers with large-scale demyelination, the new aggregates formed at the edges of adhering Schwann cells and appeared to fuse to form new nodes. At nodes with demyelination limited to paranodal retraction, Na⁺ channels were often found divided into two symmetric highly focal clusters. These results suggest that reorganization of Na⁺ channels plays an important role in the pathogenesis of demyelinating neuropathies. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:1019–1032, 1998.

After a polyethylene glycol extraction which removes monomeric immunoglobulin G (IgG), circulating immune complexes were assayed by the FIAX fluorescence assay (Whittaker M. A. Bioproducts, Walkersville, Md.). The extraction portion of the procedure can be completed within a few hours after an overnight precipitation step, and the fluorescence assay takes about 0.5 h. The fluorescence assay is similar to the routine FIAX method for measuring IgG in cerebrospinal fluid except that a 10-microliter sample from sera is substituted for a 50-microliter sample. Good parallelism between endogenous immune complexes, monomeric IgG, and aggregated human globulin, along with good between-run precision (coefficients of variation, less than 10%), indicates that monomeric IgG calibrators from the kit could be used to standardize the assay. This standardization eliminates the need for aggregated human globulin, which is unstable and difficult to prepare. A reference range of 9 to 63 mg/liter followed a gaussian distribution. Correlation data indicate that the test provides information similar to the C1q-binding test (rho = 0.87; n = 30) but has better diagnostic sensitivity and better discrimination in the high range. Because of its simplicity, good reproducibility, and accessibility to equipment available in many laboratories, the method described here may be a preferred technique for measuring circulating immune complexes.

The tetrodotoxin-binding component associated with the voltage-sensitive sodium channel from electroplax membranes of Electrophorus electricus has been purified. The toxin-binding site could be efficiently solubilized with Lubrol-PX, resulting in an extract of high initial specific activity. Purification was facilitated by the development of a rapid, quantitative binding assay. The binding component was stabilized during purification by the use of mixed lipid/detergent micelles of defined composition, and by the saturation of the site with tetrodotoxin. The purification was achieved by means of a highly selective adsorption of the toxin-binding component to DEASE-Sephadex A-25, followed by desorption at high ionic strength and chromatography over Sepharose 6B. Final peak specific activities were at least 50% of the specific activity expected for a pure, undenatured toxin-binding componenet of 230,000 molecular weight. The purified material exhibited a sedimentation coefficient of approximately 8 S and an unusual Stokes radius of 95 A. Purified material showed a relatively simple pattern on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, being comprised of only three polypeptides.

To probe the structure-function relationships of voltage-dependent sodium channels, we have been examining the mechanisms of channel modification by batrachotoxin (BTX), veratridine (VTD), and grayanotoxin-I (GTX), investigating the unifying mechanisms that underlie the diverse modifications of this class of neurotoxins. In this paper, highly purified sodium channel polypeptides from the electric organ of the electric eel were incorporated into planar lipid bilayers in the presence of GTX for comparison with our previous studies of BTX (Recio-Pinto, E., D. S. Duch, S. R. Levinson, and B. W. Urban. 1987. J. Gen. Physiol. 90:375-395) and VTD (Duch, D. S., E. Recio-Pinto, C. Frenkel, S. R. Levinson, and B. W. Urban. 1989. J. Gen. Physiol. 94:813-831) modifications. GTX-modified channels had a single channel conductance of 16 pS. An additional large GTX-modified open state (40-55 pS) was found which occurred in bursts correlated with channel openings and closings. Two voltage-dependent processes controlling the open time of these modified channels were characterized: (a) a concentration-dependent removal of inactivation analogous to VTD-modified channels, and (b) activation gating similar to BTX-modified channels, but occurring at more hyperpolarized potentials. The voltage dependence of removal of inactivation correlated with parallel voltage-dependent changes in the estimated K1/2 of VTD and GTX modifications. Ranking either the single channel conductances or the depolarization required for 50% activation, the same sequence is obtained: unmodified > BTX > GTX > VTD. The efficacy of the toxins as activators follows the same ranking (Catterall, W. A. 1977. J. Biol. Chem. 252:8669-8676).

Abstract: Angiographic examination in a patient with sciatic-like pain on the right side and a firm, pulsatile, non-tender mass in the right buttock revealed a large sciatic artery aneurysm. The aneurysm was successfully resected at surgery. The primitive sciatic artery is the main arterial supply to the lower extremities in the 9-mm embryo. Its persistence, while very rare, is of clinical significance because of the tendency for aneurysms to develop in the artery. Surgical resection is indicated in sciatic artery aneurysms because of the danger of rupture or embolic occlusion of arteries distal to the aneurysm.

Associations between blood pressures and three indices commonly used to assess relative obesity (weight (kg)/height (m)2, weight (kg)/height (m)3, and weight (kg)/height (m)p) were examined in a multiracial sample of children attending private schools in Chicago, Illinois. The 1,723 boys and 1,878 girls were 5-10 years old at the time of the survey (1975-1978). Inclusion of white, black, Hispanic, and Asian children permitted comparison of the relation between anthropometric variables and blood pressures across racial groups. The three weight-for-height indices used as estimates of relative obesity yielded nearly identical regression models, suggesting that, in spite of ongoing debate about the most appropriate index for describing relative obesity in children, it makes little difference which index is used.

In 1975-1978, the Chicago Department of Health conducted a screening program that included measurements of blood pressure, heart rate, height, weight, triceps skinfold thickness, and arm circumference, and calculation of body mass index and muscle circumference for non-public school children. Based on data on 4,086 boys and girls aged 5-10 years from the program, this study examined the ethnic differences in blood pressure and heart rate among children of white, black, Latino, and Oriental ethnicity. Mean levels for both systolic and diastolic blood pressure were higher for Oriental and black children than for white and Latino children. These differences were independent of age, height, weight, and skinfold thickness. The black children had a much lower mean heart rate than the other children. A seasonal variation was observed for systolic blood pressure, i.e., with each sex group, the mean systolic blood pressure adjusted for age, skinfold thickness, and height tended to be higher in spring than in fall and winter. (Note-- no child was screened during the summer because of summer break.) With control for season, ethnic differences in systolic blood pressure disappeared, but not the ethnic differences in diastolic blood pressure and heart rate.

1.On the basis of a mechanistic study of the transport of fine-grained absorbent in vessels with a sectionalized fluid bed, a mathematical model of such a vessel has been constructed.2.Analysis of the mathematical model has shown the inadvisability of combining in a single vessel two types of downcomers, and has furnished grounds for recommending the use of bottom-drain downcomers only.

Summary: Rat sciatic nerve fibres were demyelinated by injection of lysolecithin and examined at several stages as Schwann cells proliferated, adhered, and initiated remyelination. Immunoperoxidase EM has been used to follow the clustering of Na+ channels that represents an early step in the formation of new nodes of Ranvier. At the peak of demyelination, 1 week postinjection, only isolated sites, suggestive of the original nodes, were labelled. As Schwann cells adhered and extended processes along the axons, regions of axonal Na+ channel immunoreactivity were often found just beyond their leading edges. These channel aggregates were associated only with the axolemma and Na+ channels were not detected on glial membranes. Sites with more than one cluster in close proximity and broadly labelled aggregates between Schwann cells suggested that new nodes of Ranvier formed as neighbouring Na+ channel groups merged. Schwann cells thus seem to play a major role in ion channel distributions in the axolemma. In all of these stages Na+ channel label was found primarily just outside the region of close contact between axon and Schwann cell. This suggests that Schwann cell adherence acts in part to exclude Na+ channels, or that diffusible substances are involved and can act some distance from regions of direct contact.

Summary The distributions of (Na+ + K+)ATPase and sodium channels in skeletal muscle fibres and electrocytes were determined by immunofluorescent and immunoelectron microscopic techniques using antibodies against rat and eel (Na+ + K+)ATPase and the eel electric organ sodium channel. The extrajunctional sarcolemma of skeletal muscle was uniformly stained by polyclonal antibodies against (Na+ + K+)ATPase and the sodium channel. The T-tubule system of skeletal muscle was also labelled heavily for both (Na+ + K+)ATPase and the sodium channel. The terminal cisternae of the sarcoplasmic reticulum was stained for (Na+ + K+)ATPase but not sodium channels. At the motor endplate, (Na+ + K+)ATPase-like immunoreactivity was present along the plasmalemma of motor nerve terminals but not along the postsynaptic junctional sarcolemma. Paradoxically, a monoclonal antibody that binds to the a form of the catalytic subunit of (Na+ + K+)ATPase from rat hepatocytes and renal tubule cells did not label the enzyme in rat skeletal muscle. In electrocytes, (Na+ + K+)ATPase-like irnmunoreactivity was concentrated primarily along the plasmalemma and calveolae of the non-innervated face. In contrast, sodium channel-like immunoreactivity was concentrated along the plasmalemma of the innervated face except in the clefts of the postsynaptic membrane. Thus, we conclude that at endplates both the (Na+ + K+)ATPase of rat skeletal muscle and sodium channels of eel electrocytes are not concentrated in the juxtaneuronal postsynaptic membrane. We also interpret the failure of the monoclonal anti-a (Na+ + K+)ATPase antibodies to bind to the enzyme in muscle to indicate that the catalytic subunit of skeletal muscle (Na+ + K+)ATPase displays different epitopes than does the a subunit of kidney and liver.