The Na channel voltage sensor associated with inactivation is localized to the external charged residues of domain IV, S4.

Site-3 toxins have been shown to inhibit a component of gating charge (33% of maximum gating charge, Q(max)) in native cardiac Na channels that has been identified with the open-to-inactivated state kinetic transition. To investigate the role of the three outermost arginine amino acid residues in segment 4 domain IV (R1, R2, R3) in gating charge inhibited by site-3 toxins, we recorded ionic and gating currents from human heart Na channels with mutations of the outermost arginines (R1C, R1Q, R2C, and R3C) expressed in fused, mammalian tsA201 cells. All four mutations had ionic currents that activated over the same voltage range with slope factors of their peak conductance-voltage (G-V) relationships similar to those of wild-type channels, although decay of I(Na) was slowest for R1C and R1Q mutant channels and fastest for R3C mutant channels.

Ultra-slow inactivation in mu1 Na+ channels is produced by a structural rearrangement of the outer vestibule.

While studying the adult rat skeletal muscle Na+ channel outer vestibule, we found that certain mutations of the lysine residue in the domain III P region at amino acid position 1237 of the alpha subunit, which is essential for the Na+ selectivity of the channel, produced substantial changes in the inactivation process. When skeletal muscle alpha subunits (micro1) with K1237 mutated to either serine (K1237S) or glutamic acid (K1237E) were expressed in Xenopus oocytes and depolarized for several minutes, the channels entered a state of inactivation from which recovery was very slow, i.e.

Initiation of capnography in a rural EMS system.

Objective: Capnography is an effective means of monitoring cardiopulmonary resuscitation and managing endotracheal intubation. Numerous studies have validated its use in urban settings, but none have described its integration into a rural EMS system. The authors describe the process of integrating capnography training and use in Jackson County, West Virginia.

Temperature dependence of early and late currents in human cardiac wild-type and long Q-T DeltaKPQ Na+ channels.

Na+ current (INa) through wild-type human heart Na+ channels (hH1) is important for normal cardiac excitability and conduction, and it participates in the control of repolarization and refractoriness. INa kinetics depend strongly on temperature, but INa for hH1 has been studied previously only at room temperature. We characterized early INa (the peak and initial decay) and late INa of the wild-type hH1 channel and a mutant channel (DeltaKPQ) associated with congenital long Q-T syndrome.

A specific interaction between the cardiac sodium channel and site-3 toxin anthopleurin B.

The polypeptide neurotoxin anthopleurin B (ApB) isolated from the venom of the sea anemone Anthopleura xanthogrammica is one of a family of toxins that bind to the extracellular face of voltage-dependent sodium channels and retard channel inactivation. Because most regions of the sodium channel known to contribute to inactivation are located intracellularly or within the membrane bilayer, identification of the toxin/channel binding site is of obvious interest. Recently, mutation of a glutamic acid residue on the extracellular face of the fourth domain of the rat neuronal sodium channel (rBr2a) was shown to disrupt toxin/channel binding (Rogers, J.

Acute pulmonary edema caused by ingestion of hydrochlorothiazide.

Hydrochlorothiazide is one of most commonly prescribed antihypertensive diuretics. In this case, an allergic reaction to hydrochlorothiazide resulted in severe pulmonary edema. Hydrochlorothiazide, one of the most commonly prescribed drugs, is a diuretic which is usually well tolerated.

Two human paramyotonia congenita mutations have opposite effects on lidocaine block of Na+ channels expressed in a mammalian cell line.

1. Two mutant human skeletal muscle voltage-gated Na+ channel alpha-subunits (hSkM1), with mutations found in patients with hereditary paramyotonia congenita (T1313M on the III-IV linker and R1448C on the outside of S4 of repeat IV), and wild-type hSkM1 channels were expressed in a human embryonic kidney cell lines (tsA201) using recombinant cDNA. 2.

Optimization of a mammalian expression system for the measurement of sodium channel gating currents.

We describe a new mammalian expression system that optimizes conditions for the measurement of Na channel gating currents (Ig). The small magnitude of Ig limits their study to preparations with high numbers of Na channels to improve signal-to-noise ratios. To increase Na channel Ig signals, single tsA201 cells (approximately 20 microns in diameter) were fused into large, multinucleated cells by treatment with polyethylene glycol.

Cardiac sodium channels expressed in a peripheral neurotumor-derived cell line, RT4-B8.

RT4-B is one of several cell lines derived from a multipotent stem cell line, RT4-AC, which originated from a rat peripheral neurotumor. Based on Northern blot and ribonuclease protection experiments, RT4-B8 cells have been proposed to express rat cardiac Na channel mRNA as the major isoform. We report here direct electrophysiological evidence that the expressed voltage-gated Na channels in the RT4-B8 cell line are of the cardiac phenotype with no evidence for subpopulations expressing other Na channel isoforms.

Coexpression of beta 1 with cardiac sodium channel alpha subunits in oocytes decreases lidocaine block.

Coexpression of the rat beta 1 subunit with rat brain and skeletal muscle sodium channel alpha subunits in Xenopus oocytes normalizes currents by accelerating sodium current decay kinetics, shifting steady state availability relationships, and accelerating recovery from inactivation. Unlike brain and skeletal muscle, the heart alpha subunit expressed without beta 1 has native-like decay kinetics in oocytes. Messenger RNA for beta 1 has been found in heart, but whether and how it affects cardiac sodium channel function are unclear.

Purification, properties, and immunological characterization of GalT-3 (UDP-galactose: GM2 ganglioside, beta 1-3 galactosyltransferase) from embryonic chicken brain.

A beta 1-3 galactosyltransferase (GalT-3; UDP-Gal; GM2 beta 1-3 galactosyltransferase) was purified over 5100-fold from 19-day-old embryonic chicken brain homogenate employing detergent solubilization, alpha-lactalbumin Sepharose, Q-Sepharose, UDP-hexanolamine Sepharose, and GalNAc beta 1-4Gal beta-Synsorb column chromatography. The purified enzyme was resolved into two bands on reducing gels with apparent molecular weights of 62 kDa and 65 kDa, respectively. GalT-3 activity was also localized in the same regions by activity gel analysis and sucrose-density gradient centrifugation of a detergent-solubilized extract of 19-day-old embryonic chicken brain.

TGF-beta modulates the synthesis of proteoglycans by myocardial fibroblasts in culture.

In this study we examined the production of proteoglycans by fibroblasts cultured from the left ventricular myocardium of normal adult rats. Various molecular species of proteoglycan were detected, either by labeling glycosaminoglycan chains with 35SO4 or by labeling the proteoglycan core protein with [35S]methionine. The medium of the cell cultures, which contained quantitatively most of the proteoglycans, appeared to consist mainly of biglycan, lesser amounts of decorin and proteoglycans of higher molecular weight.

High level expression and export of beta-glucuronidase from murine mucopolysaccharidosis VII cells corrected by a double-copy retrovirus vector.

Retrovirus vectors were constructed to transfer and express the cDNA of the human lysosomal acid hydrolase beta-glucuronidase (GUSB) under control of the human GUSB promoter. Expression of the transcription unit (minigene) was evaluated in a GUSB-negative cell line established from a mouse with the lysosomal storage disease mucopolysaccharidosis (MPS) type VII. A vector designed to transfer single copies of the minigene (N2H beta H) expressed normal levels of GUSB activity in the deficient cells.

The saxitoxin/tetrodotoxin binding site on cloned rat brain IIa Na channels is in the transmembrane electric field.

The rat brain IIa (BrIIa) Na channel alpha-subunit and the brain beta 1 subunit were coexpressed in Xenopus oocytes, and peak whole-oocyte Na current (INa) was measured at a test potential of -10 mV. Hyperpolarization of the holding potential resulted in an increased affinity of STX and TTX rested-state block of BrIIa Na channels. The apparent half-block concentration (ED50) for STX of BrIIa current decreased with hyperpolarizing holding potentials (Vhold).

Enzyme replacement therapy for murine mucopolysaccharidosis type VII.

Recombinant mouse beta-glucuronidase administered intravenously to newborn mice with mucopolysaccharidosis type VII (MPS VII) is rapidly cleared from the circulation and localized in many tissues. Here we determine the tissue distribution of injected enzyme and describe its effects on the histopathology in 6-wk-old MPS VII mice that received either one injection of 28,000 U recombinant beta-glucuronidase at 5 wk of age or received six injections of 28,000 U given at weekly intervals beginning at birth. These mice were compared with untreated 6-wk-old MPS VII mice.

Post-repolarization block of cloned sodium channels by saxitoxin: the contribution of pore-region amino acids.

Sodium channels expressed in oocytes exhibited isoform differences in phasic block by saxitoxin (STX). Neuronal channels (rat IIa co-expressed with beta 1 subunit, Br2a + beta 1) had slower kinetics of phasic block for pulse trains than cardiac channels (RHI). After the membrane was repolarized from a single brief depolarizing step, a test pulse at increasing intervals showed first a decrease in current (post-repolarization block) then eventual recovery in the presence of STX.

Exercise-induced pulmonary syndromes.

When respiratory distress occurs in the exercise arena, the clinician must differentiate between a potential serious bout of EIA or the commoner EIB. The physician's game day medical kit should include epinephrine for initial treatment in suspected EIA. Sports medicine personnel need to maintain a high index of suspicion for EIB in athletes at risk and confirm the diagnosis with a treadmill exercise challenge test.

Mucopolysaccharidosis VII: postmortem biochemical and pathological findings in a young adult with beta-glucuronidase deficiency.

The postmortem biochemical and pathological findings in the first patient reported with mucopolysaccharidosis VII are described. Clinical, radiographic, and biochemical features of this 19-yr-old black man were initially reported in 1973 when, at age 2 1/2 yr his enzymatic defect, deficiency of beta-glucuronidase, was identified. The autopsy findings are now described with biochemical data further characterizing the enzyme deficiency and resultant glycosaminoglycan accumulation.

Regulation of expression of cell surface neolacto-glycolipids and cloning of embryonic chicken brain GalT-4 (UDP-Gal: GlcNAc-R beta 1-4 galactosyltransferase).

The biosynthesis of GM1 ganglioside (Gal beta 1-3GalNAc beta 1-4 (NeuAc alpha 2-3) Gal beta 1-4Glc-Cer) and nLcOse4Cer is catalyzed by two different beta-galactosyltransferases GalT-3 (UDP-Gal: GM2 beta 1-3GalT) and GalT-4 (UDP-Gal: Lc3 beta 1-4GalT) respectively. Solubilized GalT-3 and GalT-4 have been purified 3,000-fold and 22,000-fold, respectively, from 11-19-day-old embryonic chicken brain. The purified GalT-3 transfers galactose to GM2 very actively (Km 33 microM), whereas acetyl GM2 is not an active substrate (Km 350 microM), GalT-3 and GalT-4 are classified as HYCARS (hydrophobic recognition site) and CARS (carbohydrate recognition sites), respectively.

Photoreceptor degeneration and altered distribution of interphotoreceptor matrix proteoglycans in the mucopolysaccharidosis VII mouse.

Recent studies suggest that chondroitin sulfate proteoglycans in the retinal interphotoreceptor matrix (IPM) play a role in maintaining photoreceptor viability. We report herein studies on the retinas from mice with mucopolysaccharidosis type VII (MPS VII), a storage disorder resulting from virtual absence of beta-glucuronidase, an enzyme that is involved in the lysosomal degradation of chondroitin sulfate and other beta-glucuronide-containing proteoglycans. The distribution of IPM chondroitin sulfate proteoglycans was examined by immunohistochemistry and lectin-based histochemistry, and compared with the morphology of photoreceptor and retinal pigmented epithelial (RPE) cells at various ages in MPS VII-affected mice.

Two types of colorectal carcinoma in Crohn's disease.

Fourteen examples of carcinoma developing in the colon or rectum were encountered in a series of 1008 patients with Crohn's disease treated in Aberdeen during the 34-year period, 1955-1988. Seven patients were under 55 years of age at the time of onset of Crohn's disease, and had a long history of the disease before carcinoma supervened. Older patients had short histories and a better prognosis.

The cloned cardiac Na channel alpha-subunit expressed in Xenopus oocytes show gating and blocking properties of native channels.

The neonatal rat cardiac Na channel alpha-subunit directed currents in oocytes show characteristic cardiac relative resistance to tetrodotoxin (TTX) block. TTX-sensitive currents obtained by expression in Xenopus oocytes of the alpha-subunits of the rat brain (BrnIIa) and adult skeletal muscle (microI) Na channels show abnormally slow decay kinetics. In order to determine if currents directed by the cardiac alpha-subunit (RHI) exhibit kinetics in oocytes like native currents, we compared RHI-directed currents in oocytes to Na currents in freshly isolated neonatal rat myocytes.

Crohn's disease in the northeastern and northern Isles of Scotland: an epidemiological review.

An epidemiological study of Crohn's disease has been conducted in northeastern Scotland since 1955 and in the Orkney and Shetland Islands since 1966 through the end of 1988. There were 856 verified cases on the mainland and 58 in the northern isles. There has been no change in the sex ratio on the mainland in recent years, with 63% of patients female.

Exercise-induced bronchospasm in the young athlete: guidelines for routine screening and initial management.

Exercise induced bronchospasm (EIB) commonly occurs several minutes into or following an exercise event. Respiratory heat loss and respiratory water loss have been suspected as the precursor to exercise-induced bronchospasm. Obstructive EIB has been reported in elite Olympic athletes as well as the recreational athlete.