BACKGROUND

The Brugada syndrome is characterized by marked ST-segment elevation in the right precordial ECG leads and is associated with a high incidence of sudden and unexpected arrhythmic death. Our study examines the cellular basis for this syndrome.

RESULTS

Using arterially perfused wedges of canine right ventricle (RV), we simultaneously recorded transmembrane action potentials from 2 epicardial and 1 endocardial sites, together with unipolar electrograms and a transmural ECG. Loss of the action potential dome in epicardium but not endocardium after exposure to pinacidil (2 to 5 micromol/L), a K(+) channel opener, or the combination of a Na(+) channel blocker (flecainide, 7 micromol/L) and acetylcholine (ACh, 2 to 3 micromol/L) resulted in an abbreviation of epicardial response and a transmural dispersion of repolarization, which caused an ST-segment elevation in the ECG. ACh facilitated loss of the action potential dome, whereas isoproterenol (0.1 to 1 micromol/L) restored the epicardial dome, thus reducing or eliminating the ST-segment elevation. Heterogeneous loss of the dome caused a marked dispersion of repolarization within the epicardium and transmurally, thus giving rise to phase 2 reentrant extrasystole, which precipitated ventricular tachycardia (VT) and ventricular fibrillation (VF). Transient outward current (I(to)) block with 4-aminopyridine (1 to 2 mmol/L) or quinidine (5 micromol/L) restored the dome, normalized the ST segment, and prevented VT/VF. Conclusions-Depression or loss of the action potential dome in RV epicardium creates a transmural voltage gradient that may be responsible for the ST-segment elevation observed in the Brugada syndrome and other syndromes exhibiting similar ECG manifestations. Our results also demonstrate that extrasystolic activity due to phase 2 reentry can arise in the intact wall of the canine RV and serve as the trigger for VT/VF. Our data point to I(to) block (4-aminopyridine, quinidine) as an effective pharmacological treatment.

The axon initial segment (AIS) is a specialized region in neurons where action potentials are initiated. It is commonly assumed that this process requires a high density of voltage-gated sodium (Na(+)) channels. Paradoxically, the results of patch-clamp studies suggest that the Na(+) channel density at the AIS is similar to that at the soma and proximal dendrites. Here we provide data obtained by antibody staining, whole-cell voltage-clamp and Na(+) imaging, together with modeling, which indicate that the Na(+) channel density at the AIS of cortical pyramidal neurons is approximately 50 times that in the proximal dendrites. Anchoring of Na(+) channels to the cytoskeleton can explain this discrepancy, as disruption of the actin cytoskeleton increased the Na(+) current measured in patches from the AIS. Computational models required a high Na(+) channel density (approximately 2,500 pS microm(-2)) at the AIS to account for observations on action potential generation and backpropagation. In conclusion, action potential generation requires a high Na(+) channel density at the AIS, which is maintained by tight anchoring to the actin cytoskeleton.

The influenza A virus M2 protein is expressed abundantly at the cell surface, and in addition to the hemagglutinin (HA) and neuraminidase (NA), is a third virus-specific membrane protein. M2 has an internal hydrophobic membrane anchorage domain and associates with the same cellular membrane fractions as HA and NA. Trypsin treatment of infected cells and immunoprecipitation with site-specific antisera indicate that a minimum of 18 NH2-terminal amino acids of M2 are exposed at the cell surface. Ten NH2-terminal residues are conserved in all strains of influenza A virus for which sequences are available. Antibodies can recognize M2 on the cell surface and therefore it may be an infected-cell surface antigen. We discuss properties of M2 that match it to the elusive major target molecule on influenza A virus-infected cells for cross-reactive cytotoxic T cells.

Recent research has highlighted the fundamental role of the tumour's extracellular metabolic microenvironment in malignant invasion. This microenvironment is acidified primarily by the tumour-cell Na(+)/H(+) exchanger NHE1 and the H(+)/lactate cotransporter, which are activated in cancer cells. NHE1 also regulates formation of invadopodia - cell structures that mediate tumour cell migration and invasion. How do these alterations of the metabolic microenvironment and cell invasiveness contribute to tumour formation and progression?

Mutations in PINK1 cause autosomal recessive Parkinson's disease. PINK1 is a mitochondrial kinase of unknown function. We investigated calcium homeostasis and mitochondrial function in PINK1-deficient mammalian neurons. We demonstrate physiologically that PINK1 regulates calcium efflux from the mitochondria via the mitochondrial Na(+)/Ca(2+) exchanger. PINK1 deficiency causes mitochondrial accumulation of calcium, resulting in mitochondrial calcium overload. We show that calcium overload stimulates reactive oxygen species (ROS) production via NADPH oxidase. ROS production inhibits the glucose transporter, reducing substrate delivery and causing impaired respiration. We demonstrate that impaired respiration may be restored by provision of mitochondrial complex I and II substrates. Taken together, reduced mitochondrial calcium capacity and increased ROS lower the threshold of opening of the mitochondrial permeability transition pore (mPTP) such that physiological calcium stimuli become sufficient to induce mPTP opening in PINK1-deficient cells. Our findings propose a mechanism by which PINK1 dysfunction renders neurons vulnerable to cell death.

Molecular medicine has led to rapid advances in the characterization of hepatobiliary transport systems that determine the uptake and excretion of bile salts and other biliary constituents in the liver and extrahepatic tissues. The bile salt pool undergoes an enterohepatic circulation that is regulated by distinct bile salt transport proteins, including the canalicular bile salt export pump BSEP (ABCB11), the ileal Na(+)-dependent bile salt transporter ISBT (SLC10A2), and the hepatic sinusoidal Na(+)- taurocholate cotransporting polypeptide NTCP (SLC10A1). Other bile salt transporters include the organic anion transporting polypeptides OATPs (SLC21A) and the multidrug resistance-associated proteins 2 and 3 MRP2,3 (ABCC2,3). Bile salt transporters are also present in cholangiocytes, the renal proximal tubule, and the placenta. Expression of these transport proteins is regulated by both transcriptional and posttranscriptional events, with the former involving nuclear hormone receptors where bile salts function as specific ligands. During bile secretory failure (cholestasis), bile salt transport proteins undergo adaptive responses that serve to protect the liver from bile salt retention and which facilitate extrahepatic routes of bile salt excretion. This review is a comprehensive summary of current knowledge of the molecular characterization, function, and regulation of bile salt transporters in normal physiology and in cholestatic liver disease and liver regeneration.

Influenza A and B viruses carry two surface glycoproteins, the haemagglutinin (HA) and the neuraminidase (NA). Both proteins have been found to recognise the same host cell molecule, sialic acid. HA binds to sialic acid-containing receptors on target cells to initiate virus infection, whereas NA cleaves sialic acids from cellular receptors and extracellular inhibitors to facilitate progeny virus release and to promote the spread of the infection to neighbouring cells. Numerous studies performed recently have revealed that an optimal interplay between these receptor-binding and receptor-destroying activities of the surface glycoproteins is required for efficient virus replication. An existing balance between the antagonistic HA and NA functions of individual viruses can be disturbed by various events, such as reassortment, virus transmission to a new host, or therapeutic inhibition of neuraminidase. The resulting decrease in the viral replicative fitness is usually overcome by restoration of the functional balance due to compensatory mutations in HA, NA or both proteins.

New parameter sets of the GROMOS biomolecular force field, 54A7 and 54B7, are introduced. These parameter sets summarise some previously published force field modifications: The 53A6 helical propensities are corrected through new φ/ψ torsional angle terms and a modification of the N-H, C=O repulsion, a new atom type for a charged -CH(3) in the choline moiety is added, the Na(+) and Cl(-) ions are modified to reproduce the free energy of hydration, and additional improper torsional angle types for free energy calculations involving a chirality change are introduced. The new helical propensity modification is tested using the benchmark proteins hen egg-white lysozyme, fox1 RNA binding domain, chorismate mutase and the GCN4-p1 peptide. The stability of the proteins is improved in comparison with the 53A6 force field, and good agreement with a range of primary experimental data is obtained.

The mycotoxin, cyclopiazonic acid (CPA), inhibits the Ca2+-stimulated ATPase (EC 3.6.1.38) and Ca2+ transport activity of sarcoplasmic reticulum (Goeger, D. E., Riley, R. T., Dorner, J. W., and Cole, R. J. (1988) Biochem. Pharmacol. 37, 978-981). We found that at low ATP concentrations (0.5-2 microM) the inhibition of ATPase activity was essentially complete at a CPA concentration of 6-8 nmol/mg protein, indicating stoichiometric reaction of CPA with the Ca2+-ATPase. Cyclopiazonic acid caused similar inhibition of the Ca2+-stimulated ATP hydrolysis in intact sarcoplasmic reticulum and in a purified preparation of Ca2+-ATPase. Cyclopiazonic acid also inhibited the Ca2+-dependent acetylphosphate, p-nitrophenylphosphate and carbamylphosphate hydrolysis by sarcoplasmic reticulum. ATP protected the enzyme in a competitive manner against inhibition by CPA, while a 10(5)-fold change in free Ca2+ concentration had only moderate effect on the extent of inhibition. CPA did not influence the crystallization of Ca2+-ATPase by vanadate or the reaction of fluorescein-5'-isothiocyanate with the Ca2+-ATPase, but it completely blocked at concentrations as low as 1-2 mol of CPA/mol of ATPase the fluorescence changes induced by Ca2+ and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) in FITC-labeled sarcoplasmic reticulum and inhibited the cleavage of Ca2+-ATPase by trypsin at the T2 cleavage site in the presence of EGTA. These observations suggest that CPA interferes with the ATP-induced conformational changes related to Ca2+ transport. The effect of CPA on the sarcoplasmic reticulum Ca2+-ATPase appears to be fairly specific, since the kidney and brain Na+,K+-ATPase (EC 3.6.1.37), the gastric H+,K+-ATPase (EC 3.6.1.36), the mitochondrial F1-ATPase (EC 3.6.1.34), the Ca2+-ATPase of erythrocytes, and the Mg2+-activated ATPase of T-tubules and surface membranes of rat skeletal muscle were not inhibited by CPA, even at concentrations as high as 1000 nmol/mg protein.

In heart failure (HF), Ca(2+)/calmodulin kinase II (CaMKII) expression is increased. Altered Na(+) channel gating is linked to and may promote ventricular tachyarrhythmias (VTs) in HF. Calmodulin regulates Na(+) channel gating, in part perhaps via CaMKII. We investigated effects of adenovirus-mediated (acute) and Tg (chronic) overexpression of cytosolic CaMKIIdelta(C) on Na(+) current (I(Na)) in rabbit and mouse ventricular myocytes, respectively (in whole-cell patch clamp). Both acute and chronic CaMKIIdelta(C) overexpression shifted voltage dependence of Na(+) channel availability by -6 mV (P < 0.05), and the shift was Ca(2+) dependent. CaMKII also enhanced intermediate inactivation and slowed recovery from inactivation (prevented by CaMKII inhibitors autocamtide 2-related inhibitory peptide [AIP] or KN93). CaMKIIdelta(C) markedly increased persistent (late) inward I(Na) and intracellular Na(+) concentration (as measured by the Na(+) indicator sodium-binding benzofuran isophthalate [SBFI]), which was prevented by CaMKII inhibition in the case of acute CaMKIIdelta(C) overexpression. CaMKII coimmunoprecipitates with and phosphorylates Na(+) channels. In vivo, transgenic CaMKIIdelta(C) overexpression prolonged QRS duration and repolarization (QT intervals), decreased effective refractory periods, and increased the propensity to develop VT. We conclude that CaMKII associates with and phosphorylates cardiac Na(+) channels. This alters I(Na) gating to reduce availability at high heart rate, while enhancing late I(Na) (which could prolong action potential duration). In mice, enhanced CaMKIIdelta(C) activity predisposed to VT. Thus, CaMKII-dependent regulation of Na(+) channel function may contribute to arrhythmogenesis in HF.

The Spanish influenza pandemic of 1918 to 1919 swept the globe and resulted in the deaths of at least 20 million people. The basis of the pulmonary damage and high lethality caused by the 1918 H1N1 influenza virus remains largely unknown. Recombinant influenza viruses bearing the 1918 influenza virus hemagglutinin (HA) and neuraminidase (NA) glycoproteins were rescued in the genetic background of the human A/Texas/36/91 (H1N1) (1918 HA/NA:Tx/91) virus. Pathogenesis experiments revealed that the 1918 HA/NA:Tx/91 virus was lethal for BALB/c mice without the prior adaptation that is usually required for human influenza A H1N1 viruses. The increased mortality of 1918 HA/NA:Tx/91-infected mice was accompanied by (i) increased (>200-fold) viral replication, (ii) greater influx of neutrophils into the lung, (iii) increased numbers of alveolar macrophages (AMs), and (iv) increased protein expression of cytokines and chemokines in lung tissues compared with the levels seen for control Tx/91 virus-infected mice. Because pathological changes in AMs and neutrophil migration correlated with lung inflammation, we assessed the role of these cells in the pathogenesis associated with 1918 HA/NA:Tx/91 virus infection. Neutrophil and/or AM depletion initiated 3 or 5 days after infection did not have a significant effect on the disease outcome following a lethal 1918 HA/NA:Tx/91 virus infection. By contrast, depletion of these cells before a sublethal infection with 1918 HA/NA:Tx/91 virus resulted in uncontrolled virus growth and mortality in mice. In addition, neutrophil and/or AM depletion was associated with decreased expression of cytokines and chemokines. These results indicate that a human influenza H1N1 virus possessing the 1918 HA and NA glycoproteins can induce severe lung inflammation consisting of AMs and neutrophils, which play a role in controlling the replication and spread of 1918 HA/NA:Tx/91 virus after intranasal infection of mice.

Febrile seizures affect approximately 3% of all children under six years of age and are by far the most common seizure disorder. A small proportion of children with febrile seizures later develop ongoing epilepsy with afebrile seizures. Segregation analysis suggests the majority of cases have complex inheritance but rare families show apparent autosomal dominant inheritance. Two putative loci have been mapped (FEB1 and FEB2), but specific genes have not yet been identified. We recently described a clinical subset, termed generalized epilepsy with febrile seizures plus (GEFS+), in which many family members have seizures with fever that may persist beyond six years of age or be associated with afebrile generalized seizures. We now report linkage, in another large GEFS+ family, to chromosome region 19q13.1 and identification of a mutation in the voltage-gated sodium (Na+)-channel beta1 subunit gene (SCN1B). The mutation changes a conserved cysteine residue disrupting a putative disulfide bridge which normally maintains an extracellular immunoglobulin-like fold. Co-expression of the mutant beta1 subunit with a brain Na+-channel alpha subunit in Xenopus laevis oocytes demonstrates that the mutation interferes with the ability of the subunit to modulate channel-gating kinetics consistent with a loss-of-function allele. This observation develops the theme that idiopathic epilepsies are a family of channelopathies and raises the possibility of involvement of other Na+-channel subunit genes in febrile seizures and generalized epilepsies with complex inheritance patterns.

A cDNA was isolated from rat C6 glioma cells by expression cloning which encodes a novel Na+-independent neutral amino acid transporter designated LAT1. For functional expression in Xenopus oocytes, LAT1 required the heavy chain of 4F2 cell surface antigen (CD98), a type II membrane glycoprotein. When co-expressed with 4F2 heavy chain, LAT1 transported neutral amino acids with branched or aromatic side chains and did not accept basic amino acids or acidic amino acids. The transport via LAT1 was Na+-independent and sensitive to a system L-specific inhibitor 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid. These functional properties correspond to those of the classically characterized amino acid transport system L, a major nutrient transporter. In in vitro translation, LAT1 was shown to be a nonglycosylated membrane protein consistent with the property of 4F2 light chain, suggesting LAT1 is at least one of the proteins formerly referred to as 4F2 light chain. LAT1 exhibits relatively low but significant amino acid sequence similarity to mammalian cationic amino acid transporters and amino acid permeases of bacteria and yeasts, indicating LAT1 is a new member of the APC superfamily. Because of highly regulated nature and high level of expression in tumor cell lines, LAT1 is thought to be up-regulated to support the high protein synthesis for cell growth and cell activation. The cloning of LAT1 is expected to facilitate the research on the protein-protein interaction in the transporter field and to provide a clue to the search for still unidentified transporters.

Sheep red blood cells sensitized by 7S, but not by 19S rabbit anti-Forssman antibodies, adhere and form rosettes on mouse macrophages and on a few monocytes and polymorphonuclear cells (PMN). When, however, C' factors from mouse serum are added to the antigen-19S antibody complex (EAC'), rosettes are formed on most mouse peritoneal macrophages and PMN and on a few monocytes. In addition EAC' also adheres to 10-25% of lymph node lymphocytes but not to thymus lymphocytes. EAC' prepared with 7S anti-Forssman antibodies has identical properties. The adherence of red cells induces an increase in the membrane activity of the leukocytes and causes injury to the red cells which rapidly become deformed and fragmented. Adherence of EAC' occurs at 37 degrees C and is minimal at 4 degrees C. Probably only the first four C' components are involved in this phenomenon as mouse serum deficient in C'5 or rabbit serum, deficient in C'6 can be used as a source of C' components. Treatment of EAC' with EDTA does not modify its leukocyte-adherence properties. The adherence of EAC' to the leukocytes is not inhibited in the presence of serum. The receptors for C' on macrophages, PMN, and monocytes differ from those found on lymphocytes. Rosette formation by EAC' on macrophages, PMN, and monocytes depends on divalent cations (Mg(++)) and can be reversed by Na(3)H EDTA, while adherence to lymphocytes is independent of these ions and occurs in the presence of 0.01 M Na(3)H EDTA. Both types of receptors for C' components are destroyed by trypsin treatment of the leukocytes, in contrast with the receptors for 7S antibodies on the same cells which persist after enzyme treatment.

The hypothesis advanced in this article requires further validation. Undoubtedly it will require modification as our knowledge of biochemical control increases. Nevertheless, it should prove useful in focusing attention on the apparent similarity in the response of a large number of specific cell types to particular stimuli. Emphasis has been placed on a few common and apparently key elements in these responses. It is recognized that other factors are undoubtedly involved. Specifically, the changes in membrane potentials indicate the likelihood of widespread changes in the properties of the cell membrane, for example, changes in Na(+) and K(+) transport and distribution. These aspects of cellular responses may eventually prove to be of equal or greater importance than those common aspects of the system already identified.

We describe a novel and very effective single-cell labeling method with unique advantages for revealing the axonal and dendritic fields of any extracellularly recorded neuron. This procedure involves the use of fine glass micro-pipettes (tip diameter: approximately 1 micron), which contain biocytin or Neurobiotin dissolved in a salt solution, for the simultaneous juxtacellular recording and tracer iontophoresis. Once a neuron is well-isolated and identified, low intensity (< 10 nA) positive-current pulses are injected by way of the micro-electrode such as to modulate its firing. Juxtacellular tracer iontophoresis may last as long as the cell electrophysiologically remains in good health, while determining some of its physiological properties. Control experiments, including the selective killing of previously injected cells, provide convincing evidence that it is the stained unit that was recorded and 'tickled' by the juxtamembranous iontophoretic pulses. Electrophysiological and histochemical data further show that neuronal filling could occur during an electrically induced, transient, physical micro-damage of a somatic or dendritic membrane patch. This simple, single-cell staining method has been used to label several types of rat brain neurons, including projection neurons and interneurons. Its success rate (>> 86%) far exceeds that obtained by direct intracellular injections of tracers as shown by the labeling of a large sample of 100 individual cells (from 115 attempts) in the thalamic reticular (Rt) nucleus of 33 rats. We thereby demonstrate that Rt cells project to restricted regions of a single thalamic nucleus, including anterior thalamic nuclei, and that the thalamus and Rt complex have reciprocal connections. The juxtacellular procedure thus represents an ideal directed single-cell labeling tool for determination of functional properties, for subsequent identification, for delineation of overall neuronal architecture and for tracing neuronal pathways, provided care is taken to avoid the possible drawbacks and pitfalls that are illustrated and discussed in the present paper.

BACKGROUND

On March 30, 2013, a novel avian influenza A H7N9 virus that infects human beings was identified. This virus had been detected in six provinces and municipal cities in China as of April 18, 2013. We correlated genomic sequences from avian influenza viruses with ecological information and did phylogenetic and coalescent analyses to extrapolate the potential origins of the virus and possible routes of reassortment events.

METHODS

We downloaded H7N9 virus genome sequences from the Global Initiative on Sharing Avian Influenza Data (GISAID) database and public sequences used from the Influenza Virus Resource. We constructed phylogenetic trees and did 1000 bootstrap replicates for each tree. Two rounds of phylogenetic analyses were done. We used at least 100 closely related sequences for each gene to infer the overall topology, removed suspicious sequences from the trees, and focused on the closest clades to the novel H7N9 viruses. We compared our tree topologies with those from a bayesian evolutionary analysis by sampling trees (BEAST) analysis. We used the bayesian Markov chain Monte Carlo method to jointly estimate phylogenies, divergence times, and other evolutionary parameters for all eight gene fragments. We used sequence alignment and homology-modelling methods to study specific mutations regarding phenotypes, specifically addressing the human receptor binding properties.

RESULTS

The novel avian influenza A H7N9 virus originated from multiple reassortment events. The HA gene might have originated from avian influenza viruses of duck origin, and the NA gene might have transferred from migratory birds infected with avian influenza viruses along the east Asian flyway. The six internal genes of this virus probably originated from two different groups of H9N2 avian influenza viruses, which were isolated from chickens. Detailed analyses also showed that ducks and chickens probably acted as the intermediate hosts leading to the emergence of this virulent H7N9 virus. Genotypic and potential phenotypic differences imply that the isolates causing this outbreak form two separate subclades.

CONCLUSIONS

The novel avian influenza A H7N9 virus might have evolved from at least four origins. Diversity among isolates implies that the H7N9 virus has evolved into at least two different lineages. Unknown intermediate hosts involved might be implicated, extensive global surveillance is needed, and domestic-poultry-to-person transmission should be closely watched in the future.

BACKGROUND

China Ministry of Science and Technology Project 973, National Natural Science Foundation of China, China Health and Family Planning Commission, Chinese Academy of Sciences.

The distal end of the axon initial segment (AIS) is the preferred site for action potential initiation in cortical pyramidal neurons because of its high Na(+) channel density. However, it is not clear why action potentials are not initiated at the proximal AIS, which has a similarly high Na(+) channel density. We found that low-threshold Na(v)1.6 and high-threshold Na(v)1.2 channels preferentially accumulate at the distal and proximal AIS, respectively, and have distinct functions in action potential initiation and backpropagation. Patch-clamp recording from the axon cut end of pyramidal neurons in the rat prefrontal cortex revealed a high density of Na(+) current and a progressive reduction in the half-activation voltage (up to 14 mV) with increasing distance from the soma at the AIS. Further modeling studies and simultaneous somatic and axonal recordings showed that distal Na(v)1.6 promotes action potential initiation, whereas proximal Na(v)1.2 promotes its backpropagation to the soma.

Organic substrates (sugars, amino acids, carboxylic acids and neutrotransmitters) are actively transported into eukaryotic cells by Na+ co-transport. Some of the transport proteins have been identified--for example, intestinal brush border Na+/glucose and Na+/proline transporters and the brain Na+/CI-/GABA transporter--and progress has been made in locating their active sites and probing their conformational states. The archetypical Na+-driven transporter is the intestinal brush border Na+/glucose co-transporter (see ref. 8), and a defect in the co-transporter is the origin of the congenital glucose-galactose malabsorption syndrome. Here we describe cloning of this co-transporter by a method new to membrane proteins. We have sequenced the cloned DNA and have found no homology between the Na+/glucose co-transporter and either the mammalian facilitated glucose carrier or the bacterial sugar transport proteins. This suggests that the mammalian Na+-driven transporter has no evolutionary relationship to the other sugar transporters.

The amiloride-sensitive epithelial sodium channel, ENaC, is a heteromultimeric protein made up of three homologous subunits (alpha, beta and gamma) (1,2). In vitro, assembly and expression of functional active sodium channels in the Xenopus oocyte is strictly dependent on alpha-ENaC--the beta and gamma subunits by themselves are unable to induce an amiloride-sensitive sodium current in this heterologous expression system (2). In vivo, ENaC constitutes the limiting step for sodium absorption in epithelial cells that line the distal renal tubule, distal colon and the duct of several exocrine glands. The adult lung expresses alpha, beta and gamma ENaC (3,4), and an amiloride-sensitive electrogenic sodium reabsorption has been documented in upper and lower airways (3-7), but it is not established whether this sodium transport is mediated by ENaC in vivo. We inactivated the mouse alpha-ENaC gene by gene targeting. Amiloride-sensitive electrogenic Na+ transport was abolished in airway epithelia from alpha-ENaC(-/-) mice. Alpha-ENaC(-/-) neonates developed respiratory distress and died within 40 h of birth from failure to clear their lungs of liquid. This study shows that ENaC plays a critical role in the adaptation of the newborn lung to air breathing.

Interneurons in lateral part of neostriatum were studied in isolated slices from juvenile rats (16-20 d postnatal) by whole-cell, current-clamp recording at 33-34 degrees C, followed by intracellular staining with biocytin and double immunocytochemical or histochemical staining for parvalbumin, ChAT, and NADPH diaphorase. Medium-sized spiny neurons (MS cells) had distal dendrites with many spines and were likely projection cells, while interneurons had dendrites with fewer spines. The neostriatal interneurons could be further divided into three classes by physiological, chemical, and morphological criteria. The first class of interneurons (fast-spiking cells, FS cells) fired very short-duration action potentials with short-duration afterhyperpolarizations at constant spike frequency during depolarizing current pulses. FS cells had more negative resting potentials and lower input resistances than the other two classes. At depolarized potentials, FS cells fired repetitive spikes in response to synaptic excitation. FS cells were immunoreactive for parvalbumin. As all parvalbumin-immunoreactive cells in the neostriatum were also immunoreactive for GABA, FS cells were considered to be GABAergic. FS cells were further divided into two morphological types: FS cells with local dendritic fields and FS cells with extended dendritic fields. The axons of both types of FS cells had their densest collateralization within or near their dendritic fields. The other two classes of interneuron, PLTS cells (persistent and low-threshold spike cells) and LA cells (long-lasting afterhyperpolarization cells), were distinguished from FS cells by longer-duration action potentials and larger input resistances, had less negative resting potentials, and had longer-lasting afterhyperpolarizations. Afterhyperpolarizations of PLTS cells had a shorter time to peak than those of LA cells. PLTS cells fired both Na(+)-dependent, persistent depolarization spikes and Ca(2+)-dependent, low-threshold spikes in addition to fast spikes. Low-threshold spikes in PLTS cells were induced only from hyperpolarized potentials. Both persistent depolarizations and low-threshold spikes could also be evoked by synaptic activation. PLTS cells were histochemically identified as NADPH diaphorase-positive cells. As all NADPH diaphorase-positive cells in the same tissue were immunoreactive for nitric oxide (NO) synthase, PLTS cells were considered to release NO. PLTS cells had the largest axonal fields. Some PLTS cells appeared to have two axonal origins from the somata and dendrites. LA cells were mostly large aspiny cells with Ca(2+)-dependent long-lasting afterhyperpolarizations and strong time-dependent hyperpolarizing rectification. As this slowly occurring anomalous rectification was blocked by 2 mM cesium, this potential was considered to be due to activation of Ih.(ABSTRACT TRUNCATED AT 400 WORDS)

Voltage-gated Ca2+ channels link changes in membrane potential to the delivery of Ca2+, a key second messenger for many cellular responses. Ca2+ channels show selectivity for Ca2+ over more plentiful ions such as Na+ or K+ by virtue of their high-affinity binding of Ca2+ within the pore. It has been suggested that this binding involves four conserved glutamate residues in equivalent positions in the putative pore-lining regions of repeats I-IV in the Ca2+ channel a1 subunit. We have carried out a systematic series of single amino-acid substitutions in each of these positions and find that all four glutamates participate in high-affinity binding of Ca2+ or Cd2+. Each glutamate carboxylate makes a distinct contribution to ion binding, with the carboxylate in repeat III having the strongest effect. Some single glutamate-to-lysine mutations completely abolish micromolar Ca2+ block, indicating that the pore does not possess any high-affinity binding site that acts independently of the four glutamate residues. The prevailing model of Ca2+ permeation must thus be modified to allow binding of two Ca2+ ions in close proximity, within the sphere of influence of the four glutamates. The functional inequality of the glutamates may be advantageous in allowing simultaneous interactions with multiple Ca2+ ions moving single-file within the pore. Competition among Ca2+ ions for individual glutamates, together with repulsive ion-ion electrostatic interaction, may help achieve rapid flux rates through the channel.

Heat shock proteins (HSPs) are detected in all cells, prokaryotic and eukaryotic. In vivo and in vitro studies have shown that various stressors transiently increase production of HSPs as protection against harmful insults. Increased levels of HSPs occur after environmental stresses, infection, normal physiological processes, and gene transfer. Although the mechanisms by which HSPs protect cells are not clearly understood, their expression can be modulated by cell signal transducers, such as changes in intracellular pH, cyclic AMP, Ca2+, Na+, inositol trisphosphate, protein kinase C, and protein phosphatases. Most of the HSPs interact with other proteins in cells and alter their function. These and other protein-protein interactions may mediate the little understood effects of HSPs on various cell functions. In this review, we focus on the structure of the HSP-70 family (HSP-70s), regulation of HSP-70 gene expression, their cytoprotective effects, and the possibility of regulating HSP-70 expression through modulation of signal transduction pathways. The clinical importance and therapeutic potential of HSPs are discussed.

Studies assessing mechanisms of proximal tubular cell (PTC) physiology and pathophysiology increasingly utilize cell culture systems to avoid the complexity of whole organ/whole animal experiments. However, no well-differentiated PTC line derived from adult human kidney currently exists. Therefore, the goal of this research was to establish such a line by transduction with human papilloma virus (HPV 16) E6/E7 genes. A primary PTC culture from normal adult human renal cortex was exposed to a recombinant retrovirus containing the HPV 16 E6/E7 genes, resulting in a cell line designated HK-2 (human kidney-2) which has grown continuously in serum free media for more than one year. HK-2 cell growth is epidermal growth factor dependent and the cells retain a phenotype indicative of well-differentiated PTCs (positive for alkaline phosphatase, gamma glutamyltranspeptidase, leucine aminopeptidase, acid phosphatase, cytokeratin, alpha 3 beta 1 integrin, fibronectin; negative for factor VIII-related antigen, 6.19 antigen and CALLA endopeptidase). Furthermore, HK-2 cells retain functional characteristics of proximal tubular epithelium (Na+ dependent/phlorizin sensitive sugar transport; adenylate cyclase responsiveness to parathyroid, but not to antidiuretic, hormone). The E6/E7 genes are present in the HK-2 genome, as determined by PCR. To assess its potential usefulness as a tool for studying injury and repair, HK-2 cells were exposed to a toxic concentration of H2O2 +/- iron chelation (deferoxamine) or hydroxyl radical scavenger (Na benzoate) therapy. Only the former blocked H2O2 cytotoxicity, reproducing results previously obtained with freshly isolated rat proximal tubular segments. In conclusion, an immortalized adult human PTC line has been established by transduction with HPV 16 E6/E7 genes. It appears to be well-differentiated on the basis of its histochemical, immune cytochemical, and functional characteristics, and it can reproduce experimental results obtained with freshly isolated PTCs. Thus, this new PTC line could have substantial research application.