To assess the physiological function of Ca(2+)-dependent protein phosphatase (PP2B) in the yeast Saccharomyces cerevisiae, the phenotypes of PP2B-deficient mutants were investigated. Although PP2B was dispensable for growth under normal conditions, the mutations did, however, cause growth inhibition under certain stress circumstances. The growth of the mutants was inhibited by NaCl and LiCl, but not by KCl, CaCl2, MgCl2 or nonspecific osmotic stresses. Upon shift to high NaCl medium, intracellular Na+ levels of both wild type yeast and the mutants initially increased at a comparable rate. However, internal Na+ in wild type cells started to decline more rapidly than the mutant cells during cultivation in high NaCl medium, indicating that PP2B is important in maintaining a gradient across the membrane. The protection against salt stress was achieved, at least in part, by the stimulation of Na+ export. The maintenance of a high level of internal K+ in high NaCl medium was also PP2B-dependent. In the presence of the immunosuppressant FK506, the growth behaviour and intracellular Na+ and K+ of wild type cells in high NaCl medium became very similar to those of the PP2B-deficient mutant in a manner dependent on the presence of the FK506 binding protein.

A Mn(II)-dependent peroxidase found in the extracellular medium of ligninolytic cultures of the white rot fungus, Phanerochaete chrysosporium, was purified by DEAE-Sepharose ion-exchange chromatography, Blue Agarose chromatography, and gel filtration on Sephadex G-100. Sodium dodecyl sulfate-gel electrophoresis indicated that the homogeneous protein has an Mr of 46,000. The absorption spectrum of the enzyme indicates the presence of a heme prosthetic group. The pyridine hemochrome absorption spectrum indicates that the enzyme contained one molecule of heme as iron protoporphyrin IX. The absorption maximum of the native enzyme (406 nm) shifted to 433 nm in the reduced enzyme and to 423 nm in the reduced-CO complex. Both CN- and N-3 readily bind to the native enzyme, indicating an available coordination site and that the heme iron is high spin. The absorption spectrum of the H2O2 enzyme complex, maximum at 420 nm, is similar to that of horseradish peroxidase compound II. P. chrysosporium peroxidase activity is dependent on Mn(II), with maximal activity attained above 100 microM. The enzyme is also stimulated to varying degrees by alpha-hydroxy acids (e.g., malic, lactic) and protein (e.g., gelatin, albumin). The peroxidase is capable of oxidizing NADH and a wide variety of dyes, including Poly B-411 and Poly R-481. Several of the substrates (indigo trisulfonate, NADH, Poly B-411, variamine blue RT salt, and Poly R-481) are oxidized by this Mn(II)-dependent peroxidase at considerably faster rates than those catalyzed by horseradish peroxidase. The enzyme rapidly oxidizes Mn(II) to Mn(III); the latter was detected by the characteristic absorption spectrum of its pyrophosphate complex. Inhibition of the oxidation of the substrate diammonium 2,2-azino-bis(3-ethyl-6-benzothiazolinesulfonate) (ABTS) by Na-pyrophosphate suggests that Mn(III) plays a role in the enzyme mechanism.

The mechanisms of human mutant superoxide dismutase-1 (mSOD1) toxicity to motor neurons (MNs) are unresolved. We show that MNs in G93A-mSOD1 transgenic mice undergo slow degeneration lacking similarity to apoptosis structurally and biochemically. It is characterized by somal and mitochondrial swelling and formation of DNA single-strand breaks prior to double-strand breaks occurring in nuclear and mitochondrial DNA. p53 and p73 are activated in degenerating MNs, but without nuclear import. The MN death is independent of activation of caspases-1, -3, and -8 or apoptosis-inducing factor within MNs, with a blockade of apoptosis possibly mediated by Aven up-regulation. MN swelling is associated with compromised Na,K-ATPase activity and aggregation. mSOD1 mouse MNs accumulate mitochondria from the axon terminals and generate higher levels of superoxide, nitric oxide, and peroxynitrite than MNs in control mice. Nitrated and aggregated cytochrome c oxidase subunit-I and alpha-synuclein as well as nitrated SOD2 accumulate in mSOD1 mouse spinal cord. Mitochondria in mSOD1 mouse MNs accumulate NADPH diaphorase and inducible nitric oxide synthase (iNOS)-like immunoreactivity, and iNOS gene deletion extends significantly the life span of G93A-mSOD1 mice. Prior to MN loss, spinal interneurons degenerate. These results identify novel mechanisms for mitochondriopathy and MN degeneration in amyotrophic lateral sclerosis (ALS) mice involving blockade of apoptosis, accumulation of MN mitochondria with enhanced toxic potential from distal terminals, NOS localization in MN mitochondria and peroxynitrite damage, and early degeneration of alpha-synuclein(+) interneurons. The data support roles for oxidative stress, protein nitration and aggregation, and excitotoxicity as participants in the process of MN degeneration caused by mSOD1.

This study establishes a mechanism for metabolic hyperalgesia based on the glycolytic metabolite methylglyoxal. We found that concentrations of plasma methylglyoxal above 600 nM discriminate between diabetes-affected individuals with pain and those without pain. Methylglyoxal depolarizes sensory neurons and induces post-translational modifications of the voltage-gated sodium channel Na(v)1.8, which are associated with increased electrical excitability and facilitated firing of nociceptive neurons, whereas it promotes the slow inactivation of Na(v)1.7. In mice, treatment with methylglyoxal reduces nerve conduction velocity, facilitates neurosecretion of calcitonin gene-related peptide, increases cyclooxygenase-2 (COX-2) expression and evokes thermal and mechanical hyperalgesia. This hyperalgesia is reflected by increased blood flow in brain regions that are involved in pain processing. We also found similar changes in streptozotocin-induced and genetic mouse models of diabetes but not in Na(v)1.8 knockout (Scn10(-/-)) mice. Several strategies that include a methylglyoxal scavenger are effective in reducing methylglyoxal- and diabetes-induced hyperalgesia. This previously undescribed concept of metabolically driven hyperalgesia provides a new basis for the design of therapeutic interventions for painful diabetic neuropathy.

K+ secretion by the cortical collecting duct (CCD) is stimulated at high flow rates. Patch-clamp analysis has identified a small-conductance secretory K+ (SK) and a high-conductance Ca(2+)-activated K+ (maxi-K) channel in the apical membrane of the CCD. The SK channel, encoded by ROMK, is believed to mediate baseline K+ secretion. The role of the stretch- and Ca2+-activated maxi-K channel is still uncertain. The purpose of this study was to identify the K+ channel mediating flow-dependent K+ secretion in the CCD. Segments isolated from New Zealand White rabbits were microperfused in the absence and presence of luminal tetraethylammonium (TEA) or charybdotoxin, both inhibitors of maxi-K but not SK channels, or apamin, an inhibitor of small-conductance maxi-K+ channels. Net K+ secretion and Na+ absorption were measured at varying flow rates. In the absence of TEA, net K+ secretion increased from 8.3 +/- 1.0 to 23.4 +/- 4.7 pmol. min(-1). mm(-1) (P < 0.03) as the tubular flow rate was increased from 0.5 to 6 nl. min(-1). mm(-1). Flow stimulation of net K+ secretion was blocked by luminal TEA (8.2 +/- 1.2 vs. 9.9 +/- 2.7 pmol. min(-1). mm(-1) at 0.6 and 6 nl. min(-1). mm(-1) flow rates, respectively) or charybdotoxin (6.8 +/- 1.6 vs. 8.3 +/- 1.6 pmol. min(-1). mm(-1) at 1 and 4 nl. min(-1). mm(-1) flow rates, respectively) but not by apamin. These results suggest that flow-dependent K+ secretion is mediated by a maxi-K channel, whereas baseline K+ secretion occurs through a TEA- and charybdotoxin-insensitive SK (ROMK) channel.

For >20 years, noninvasive transcranial stimulation techniques like repetitive transcranial magnetic stimulation (rTMS) and direct current stimulation (tDCS) have been used to induce neuroplastic-like effects in the human cortex, leading to the activity-dependent modification of synaptic transmission. Here, we introduce a novel method of electrical stimulation: transcranial random noise stimulation (tRNS), whereby a random electrical oscillation spectrum is applied over the motor cortex. tRNS induces consistent excitability increases lasting 60 min after stimulation. These effects have been observed in 80 subjects through both physiological measures and behavioral tasks. Higher frequencies (100-640 Hz) appear to be responsible for generating this excitability increase, an effect that may be attributed to the repeated opening of Na(+) channels. In terms of efficacy tRNS appears to possess at least the same therapeutic potential as rTMS/tDCS in diseases such as depression, while furthermore avoiding the constraint of current flow direction sensitivity characteristic of tDCS.

Volunteers experimentally infected with influenza A/Texas/36/91 (H1N1) virus and treated with the neuraminidase (NA) inhibitor oseltamivir were monitored for the emergence of drug-resistant variants. Two (4%) of 54 resistant viruses were detected by NA inhibition assay among last-day isolates recovered from 54 drug recipients. They bore a substitution His274Tyr in the NA. Hemagglutinin (HA) variants detected in the placebo group differed from the egg-adapted inoculum virus by virtue of amino acid substitutions at residues 137, 225, or both. These variants had a higher affinity for Neu5Ac(alpha2-6)Gal-containing receptors, which are characteristic of human respiratory epithelium, than for Neu5Ac(alpha2-3)Gal-containing receptors, which are typical of chicken egg allantoic membrane. Although appearing to be more sensitive to oseltamivir in humans, the variants with increased affinity for Neu5Ac(alpha2-6)Gal receptors were less sensitive than the Neu5Ac(alpha2-3)Gal-binding variants in Madin-Darby canine kidney cells. Thus, HA affinity for receptors is an essential feature of influenza virus susceptibility to NA inhibitors, both in cell culture and in humans.

This article examines the pathophysiology of lesions caused by focal cerebral ischemia. Ischemia due to middle cerebral artery occlusion encompasses a densely ischemic focus and a less densely ischemic penumbral zone. Cells in the focus are usually doomed unless reperfusion is quickly instituted. In contrast, although the penumbra contains cells "at risk," these may remain viable for at least 4 to 8 hours. Cells in the penumbra may be salvaged by reperfusion or by drugs that prevent an extension of the infarction into the penumbral zone. Factors responsible for such an extension probably include acidosis, edema, K+/Ca++ transients, and inhibition of protein synthesis. Central to any discussion of the pathophysiology of ischemic lesions is energy depletion. This is because failure to maintain cellular adenosine triphosphate (ATP) levels leads to degradation of macromolecules of key importance to membrane and cytoskeletal integrity, to loss of ion homeostasis, involving cellular accumulation of Ca++, Na+, and Cl-, with osmotically obligated water, and to production of metabolic acids with a resulting decrease in intra- and extracellular pH. In all probability, loss of cellular calcium homeostasis plays an important role in the pathogenesis of ischemic cell damage. The resulting rise in the free cytosolic intracellular calcium concentration (Ca++) depends on both the loss of calcium pump function (due to ATP depletion), and the rise in membrane permeability to calcium. In ischemia, calcium influx occurs via multiple pathways. Some of the most important routes depend on activation of receptors by glutamate and associated excitatory amino acids released from depolarized presynaptic endings. However, ischemia also interfers with the intracellular sequestration and binding of calcium, thereby contributing to the rise in intracellular Ca++. A second key event in the ischemic tissue is activation of anaerobic glucolysis. The main reason for this activation is inhibition of mitochondrial metabolism by lack of oxygen; however, other factors probably contribute. For example, there is a complex interplay between loss of cellular calcium homeostasis and acidosis. On the one hand, a rise in intracellular Ca++ is apt to cause mitochondrial accumulation of calcium. This must interfere with ATP production and enhance anaerobic glucolysis. On the other hand, acidosis must interfere with calcium binding, thereby contributing to the rise in intracellular Ca++.

The amyloid beta-peptide (A beta) that accumulates as insoluble plaques in the brain in Alzheimer's disease can be directly neurotoxic and can increase neuronal vulnerability to excitotoxic insults. The mechanism of A beta toxicity is unclear but is believed to involve generation of reactive oxygen species (ROS) and loss of calcium homeostasis. We now report that exposure of cultured rat hippocampal neurons to A beta 1-40 or A beta 25-35 causes a selective reduction in Na+/K(+)-ATPase activity which precedes loss of calcium homeostasis and cell degeneration. Na+/K(+)-ATPase activity was reduced within 30 min of exposure to A beta 25-35 and declined to less than 40% of basal level by 3 hr. A beta did not impair other Mg(2+)-dependent ATPase activities or Na+/Ca2+ exchange. Experiments with ouabain, a specific inhibitor of the Na+/K(+)-ATPase, demonstrated that impairment of this enzyme was sufficient to induce an elevation of [Ca2+]i and neuronal injury. Impairment of Na+/K(+)-ATPase activity appeared to be causally involved in the elevation of [Ca2+]i and neurotoxicity since suppression of Na+ influx significantly reduced A beta- and ouabain-induced [Ca2+]i elevation and neuronal death. Neuronal degeneration induced by ouabain appeared to be of an apoptotic form as indicated by nuclear condensation and DNA fragmentation. The antioxidant free radical scavengers vitamin E and propylgallate significantly attenuated A beta-induced impairment of Na+/K(+)-ATPase activity, elevation of [Ca2+]i and neurotoxicity, suggesting a role for ROS. Finally, exposure of synaptosomes from postmortem human hippocampus to A beta resulted in a significant and specific reduction in Na+/K(+)-ATPase and Ca(2+)-ATPase activities, without affecting other Mg(2+)-dependent ATPase activities or Na+/Ca2+ exchange. These data suggest that impairment of ion-motive ATPases may play a role in the pathogenesis of neuronal injury in Alzheimer's disease.

The Na(+)/H(+) exchanger (NHE) is involved in intracellular pH homeostasis of many mammalian cell types. To date seven NHE isoforms (NHE1-NHE7) have been identified. NHE1 is the most predominant isoform expressed in heart where it contributes to cardiomyocyte pH homeostasis. Although the NHE activation is essential for the restoration of physiological pH, hyperactivation of NHE1 during ischemia-reperfusion episodes disrupts the intracellular ion balance, leading to cardiac dysfunction and damage. Beside its ability to inhibit a conductive Na(+) channel and the Na(+)/Ca(++) exchanger, amiloride was the first drug described as NHE inhibitor. Double substitution of the nitrogen of the 5-amino group of amiloride gave DMA, EIPA, MIBA and HMA. Later, several acylguanidines were prepared to selectively inhibit NHE1. The replacement of the pyrazine ring of amiloride by a pyridine ring or by a phenyl increased the potency and the NHE selectivity. The simultaneous replacement of the pyrazine ring by a phenyl, of the 6-chloro by a sulfomethyl led to drugs such as HOE-694, cariporide, eniporide and BIIB-513 which also selectively inhibited NHE1. In the last decade several bicyclic guanidines were prepared: zoniporide, MS-31038, SM-20220, SM-20550, SMP-300, KB-R9032, BMS-284640, T-162559, TY-12533, S-3226 or SL-591227. Extensive pre-clinical studies indicated that NHE inhibitors afford substantial protection in different animal models of myocardial ischemia (MI) and reperfusion, but the results of clinical trials involving eniporide and cariporide were mixed.

Machado-Joseph disease (MJD; also called spinocerebellar ataxia type 3) is a dominantly inherited late-onset neurodegenerative disorder caused by expansion of polyglutamine (polyQ)-encoding CAG repeats in the MJD1 gene (also known as ATXN3). Proteolytic liberation of highly aggregation-prone polyQ fragments from the protective sequence of the MJD1 gene product ataxin 3 (ATXN3) has been proposed to trigger the formation of ATXN3-containing aggregates, the neuropathological hallmark of MJD. ATXN3 fragments are detected in brain tissue of MJD patients and transgenic mice expressing mutant human ATXN3(Q71), and their amount increases with disease severity, supporting a relationship between ATXN3 processing and disease progression. The formation of early aggregation intermediates is thought to have a critical role in disease initiation, but the precise pathogenic mechanism operating in MJD has remained elusive. Here we show that L-glutamate-induced excitation of patient-specific induced pluripotent stem cell (iPSC)-derived neurons initiates Ca(2+)-dependent proteolysis of ATXN3 followed by the formation of SDS-insoluble aggregates. This phenotype could be abolished by calpain inhibition, confirming a key role of this protease in ATXN3 aggregation. Aggregate formation was further dependent on functional Na(+) and K(+) channels as well as ionotropic and voltage-gated Ca(2+) channels, and was not observed in iPSCs, fibroblasts or glia, thereby providing an explanation for the neuron-specific phenotype of this disease. Our data illustrate that iPSCs enable the study of aberrant protein processing associated with late-onset neurodegenerative disorders in patient-specific neurons.

Reactive oxygen species (ROS), including H2O2, cause intracellular calcium overload and ischemia-reperfusion damage. The objective of this study was to examine the hypothesis that H2O2-induced arrhythmic activity and contractile dysfunction are the results of an effect of H2O2 to increase the magnitude of the late sodium current (late INa). Guinea pig and rabbit isolated ventricular myocytes were exposed to 200 microM H2O2. Transmembrane voltages and currents and twitch shortening were measured using the whole-cell patch-clamp technique and video edge detection, respectively. [Na+]i and [Ca2+]i were determined by fluorescence measurements. H2O2 caused a persistent late INa that was almost completely inhibited by 10 microM tetrodotoxin (TTX). H2O2 prolonged the action potential duration (APD), slowed the relaxation rate of cell contraction, and induced early afterdepolarizations (EADs) and aftercontractions. H2O2 also caused increases of [Na+]i and [Ca2+]i. Ranolazine (10 microM), a novel inhibitor of late INa, attenuated H2O2-induced late INa by 51+/-9%. TTX (2 microM) or 10 microM ranolazine attenuated H2O2-induced APD prolongation and suppressed EADs. Ranolazine accelerated the twitch relaxation rate in the presence of H2O2 and abolished H2O2-induced aftercontractions. Pretreatment of myocytes with ranolazine delayed and reduced the increases of APD, [Na+]i, and [Ca2+]i caused by H2O2. In conclusion, the results confirm the hypothesis that an increase in late INa during exposure of ventricular myocytes to H2O2 contributes to electrical and contractile dysfunction and suggest that inhibition of late INa may offer protection against ROS-induced Na+ and Ca2+ overload.

To identify genetic factors influencing cardiac conduction, we carried out a genome-wide association study of electrocardiographic time intervals in 6,543 Indian Asians. We identified association of a nonsynonymous SNP, rs6795970, in SCN10A (P = 2.8 x 10(-15)) with PR interval, a marker of cardiac atrioventricular conduction. Replication testing among 6,243 Indian Asians and 5,370 Europeans confirmed that rs6795970 (G>A) is associated with prolonged cardiac conduction (longer P-wave duration, PR interval and QRS duration, P = 10(-5) to 10(-20)). SCN10A encodes Na(V)1.8, a sodium channel. We show that SCN10A is expressed in mouse and human heart tissue and that PR interval is shorter in Scn10a(-/-) mice than in wild-type mice. We also find that rs6795970 is associated with a higher risk of heart block (P < 0.05) and a lower risk of ventricular fibrillation (P = 0.01). Our findings provide new insight into the pathogenesis of cardiac conduction, heart block and ventricular fibrillation.

Influenza virus is a highly infectious respiratory pathogen that results in severe morbidity and mortality. The current licensed trivalent vaccine formulations in the U.S. are made from virus grown in allantoic fluid from infected hen eggs that is then chemically inactivated and split into subunit components. These vaccines elicit antibodies, primarily to the viral hemagglutinin (HA), which are efficacious in healthy adults, but are limited in protecting high risk individuals, such as the elderly and immunocompromised. To address the need for improved influenza vaccines and the limitations of egg-based manufacturing, we have engineered an influenza virus-like particle (VLP) as a new generation of non-egg or non-mammalian cell culture-based candidate vaccine against influenza infection. VLPs, based on the A/Fujian/411/2002 (H3N2) isolate, were purified from the supernatants of Spodoptera frugiperda Sf9 insect cells following infection of baculovirus vectors encoding an expression cassette comprised of only three influenza virus structural proteins, hemagglutinin (HA), neuraminidase (NA), and matrix (M1). Mice or ferrets were vaccinated intramuscularly with VLPs in a dose sparing experiment, based on HA concentration (3 microg-24 ng), and the immune responses were compared to responses elicited in animals vaccinated with recombinant HA (rHA) or inactivated whole influenza virions (WIV). All vaccinated animals had high titer anti-HA antibodies regardless of the vaccine immunogen and animals vaccinated with the highest doses of VLPs (3 microg and 600 ng) also had antibodies against NA. Purified rHA elicited primarily IgG1 antibodies, which is indicative of a T helper (Th) type 2 response, whereas mice vaccinated with the VLPs or WIV were associated with a dominant Th1 immune response (IgG2a and IgG2b). Interestingly, VLPs elicited antibodies that recognized a broader panel of antigenically distinct H3N2 viral isolates compared to rHA or WIV in a hemagglutination-inhibition (HAI) assay.

We have constructed a full-length rat brain Na+ channel alpha subunit cDNA that differs from the previously reported alpha subunit of Noda et al. at 6 amino acid positions. Transcription of the cDNA in vitro and injection into Xenopus oocytes resulted in the synthesis of functional Na+ channels. Although the single-channel conductance of the channels resulting from cloned cDNA was the same as that of channels resulting from injection of rat brain RNA, we observed two significant differences in the gating properties of the channels. The Na+ currents from cloned cDNA displayed much slower macroscopic inactivation compared with those from rat brain mRNA. In addition, the current-voltage relationship for currents from cloned cDNA was shifted 20-25 mV in the depolarizing direction compared with currents from rat brain RNA. Coinjection of low MW rat brain RNA restored normal inactivation of the channels indicating the presence of a component, either a structural subunit of the channel complex or a modifying enzyme, necessary for normal gating of the channel.

BACKGROUND

Hyperlactataemia during septic shock is often viewed as evidence of tissue hypoxia. However, this blood disorder is not usually correlated with indicators of perfusion or diminished with increased oxygen delivery. Muscles can generate lactate under aerobic conditions in a process linking glycolytic ATP supply to stimulation of Na+K+ ATPase. Using in-vivo microdialysis, we tested whether inhibition of Na+K+ ATPase can reduce muscle lactate.

METHODS

In 14 patients with septic shock, two microdialysis probes were inserted into the quadriceps muscles and infused with lactate-free Ringer's solution in the absence or presence of 10(-7) mol/L ouabain, a specific inhibitor of Na+K+ ATPase. We measured lactate and pyruvate concentrations in both the dialysate fluid and arterial blood samples.

RESULTS

All patients had increased blood lactate concentrations (mean 4.0 mmol/L; SD 2.1). Lactate and pyruvate concentrations were consistently higher in muscle than in arteries during the study period, with a mean positive gradient of 1.98 mmol/L (SD 0.2; p=0.001) and 230 micromol/L (30; p=0.01), respectively. Ouabain infusion stopped over production of muscle lactate and pyruvate (p=0.0001). Muscle lactate to pyruvate ratios remained unchanged during ouabain infusion with no differences between blood and muscle.

CONCLUSIONS

Skeletal muscle could be a leading source of lactate formation as a result of exaggerated aerobic glycolysis through Na+K+ ATPase stimulation during septic shock. Lactate clearance as an end-point of resuscitation could therefore prove useful.

CONCLUSIONS

In patients with septic shock, a high lactate concentration should be interpreted as a marker of disease, portending a bad outcome. The presence of hyperlactataemia in resuscitated septic patients should not be taken as proof of oxygen debt needing increases in systemic or regional oxygen transport to supranormal values. Lactate, instead of being regarded only as a marker of hypoxia, might be an important metabolic signal.

The presence of a detectable Ca current during the excitation of a cardiac fiber implies that the Ca lost during the resting interval of the duty cycle must also be detectable. Ca outward movement appears to be effected by Na/Ca exchange when more Na enters than Ca leaves per cycle, thus making the mechanism electrogenic. Since Na/Ca exchange can move Ca either inward or outward depending on the direction of the electrochemical gradient for Na, a potential exists where there is no electric current generated by the Na/Ca exchange mechanism, i.e., a reversal potential ER. Cardiac fibers appear to have a reversal potential that is about midway between their resting membrane potential and their plateau. Carrier currents both inward and outward are therefore generated during cardiac action potentials. The implications of the conditions stated above are explored.

Although the collecting duct is regarded as the primary site at which mineralocorticoids regulate renal sodium transport in the kidney, recent evidence points to the distal convoluted tubule as a possible site of mineralocorticoid action. To investigate whether mineralocorticoids regulate the expression of the thiazide-sensitive Na-Cl cotransporter (TSC), the chief apical sodium entry pathway of distal convoluted tubule cells, we prepared an affinity-purified, peptide-directed antibody to TSC. On immunoblots, the antibody recognized a prominent 165-kDa band in membrane fractions from the renal cortex but not from the renal medulla. Immunofluorescence immunocytochemistry showed TSC labeling only in distal convoluted tubule cells. Semiquantitative immunoblotting studies demonstrated a large increase in TSC expression in the renal cortex of rats on a low-NaCl diet (207 +/- 21% of control diet). Immunofluorescence localization in tissue sections confirmed the strong increase in TSC expression. Treatment of rats for 10 days with a continuous subcutaneous infusion of aldosterone also increased TSC expression (380 +/- 58% of controls). Furthermore, 7-day treatment of rats with an orally administered mineralocorticoid, fludrocortisone, increased TSC expression (656 +/- 114% of controls). We conclude that the distal convoluted tubule is an important site of action of the mineralocorticoid aldosterone, which strongly up-regulates the expression of TSC.

We provide physiological, pharmacological, and structural evidence that axons of hippocampal principal cells are electrically coupled, with prepotentials or spikelets forming the physiological substrate of electrical coupling as observed in cell somata. Antidromic activation of neighboring axons induced somatic spikelet potentials in neurons of CA3, CA1, and dentate gyrus areas of rat hippocampal slices. Somatic invasion by these spikelets was dependent on the activation of fast Na(+) channels in the postjunctional neuron. Antidromically elicited spikelets were suppressed by gap junction blockers and low intracellular pH. Paired axo-somatic and somato-dendritic recordings revealed that the coupling potentials appeared in the axon before invading the soma and the dendrite. Using confocal laser scanning microscopy we found that putative axons of principal cells were dye coupled. Our data thus suggest that hippocampal neurons are coupled by axo-axonal junctions, providing a novel mechanism for very fast electrical communication.

The structure of [Val1]gramicidin A incorporated into sodium dodecyl-d25 sulphate micelles has been studied by two-dimensional proton NMR spectroscopy. Analysis of nuclear Overhauser effects, spin-spin couplings and solvent accessibility of NH groups show that the conformation of the Na+ complex of gramicidin A in detergent micelles, which in many ways mimic the phospholipid bilayer of biomembranes, is an N-terminal to N-terminal (head-to-head) dimer (Formula: see text) formed by two right-handed, single-stranded beta 6.3 helices with 6.3 residues per turn, differing from Urry's structure by handedness of the helices.

1. Calcium currents were recorded with whole-cell voltage-clamp procedures in relay neurones of the rat thalamus which had been acutely isolated by an enzymatic dissociation procedure. 2. Low-threshold and high-threshold Ca2+ currents were elicited by depolarizing voltage steps from holding potentials more negative than -60 mV. A transient current, analogous to the T-current in sensory neurones, was activated at low threshold near -65 mV and was completely inactivating at command steps up to -35 mV. Voltage steps to more depolarized levels activated a high-threshold current that inactivated slowly and incompletely during a 200 ms step depolarization. 3. The high-threshold current contained both non-inactivating and slowly inactivating components which were insensitive and sensitive to holding potential, respectively. 4. A 'T-type' current was prominent in relay neurones, in both absolute terms (350 pA peak current average) and in relation to high-threshold currents. The average ratio of maximum transient to maximum sustained current was greater than 2. 5. T-current could be modelled in a manner analogous to that employed for the fast Na+ current underlying action potential generation, using the m3h format. The rate of activation of T-current was voltage dependent, with a time constant (tau m) varying between 8 and 2 ms at command potentials of -60 to -10 mV at 23 degrees C. The rate of inactivation was also voltage dependent, and the time constant tau h varied between 50 and 20 ms over the same voltage range. With command potentials more positive than -35 mV, the inactivation of Ca2+ current could no longer be fitted by a single exponential. 6. Steady-state inactivation of T-current could be well fitted by a Boltzman equation with slope factor of 6.3 and half-inactivated voltage of -83.5 mV. 7. Recovery from inactivation of T-current was not exponential. The major component of recovery (70-80% of total) was not very voltage sensitive at potentials more negative than -90 mV, with tau r of 251 ms at -92 mV and 23 degrees C, compared to 225 ms at -112 mV. A smaller, voltage-sensitive component accounted for the remainder of recovery. 8. All kinetic properties, including rates of activation, inactivation, and recovery from inactivation, as well as the amplitude of T-current, were temperature sensitive with Q10 (temperature coefficient) values of greater than 2.5.(ABSTRACT TRUNCATED AT 400 WORDS)

Cerebellar Purkinje cells are known to receive strong excitatory input from two major pathways originating outside the cerebellum and inhibitory input from two types of neurons in the cerebellar cortex. The functions and synaptic strengths of these pathways are only partially known. We have used the patch-clamp technique applied to Purkinje cells in thin slices of rat cerebellum to measure directly the postsynaptic currents arising from the two major excitatory pathways and one of the inhibitory inputs. Inhibitory synaptic currents occur spontaneously with high frequency and are variable in amplitude, ranging, in our recording conditions with high internal Cl-, from less than 100 pA to more than 1 nA. These currents are blocked by the gamma-aminobutyrate type A antagonist bicuculline. One of the excitatory inputs is all or none. For threshold stimulation, the synaptic current is either full amplitude, when the presynaptic fiber is successfully stimulated, or completely absent. This synaptic current is often larger than 1 nA and is virtually eliminated by 2 microM 6-cyano-7-nitroquinoxaline-2,3-dione, a blocking agent thought to be specific for glutamate receptors that are not of the N-methyl-D-aspartate type. Its all-or-none character identifies it as arising from a climbing-fiber synapse. The other excitatory input produces a synaptic current that is smoothly graded as a function of stimulus intensity. This response we believe arises from the stimulation of mossy fibers or granule cells. The synaptic current associated with this input is also largely eliminated by 2 microM 6-cyano-7-nitroquinoxaline-2,3-dione.

Dendrimers are routinely synthesized as tuneable nanostructures that may be designed and regulated as a function of their size, shape, surface chemistry and interior void space. They are obtained with structural control approaching that of traditional biomacromolecules such as DNA/RNA or proteins and are distinguished by their precise nanoscale scaffolding and nanocontainer properties. As such, these important properties are expected to play an important role in the emerging field of nanomedicine. This review will describe progress on the use of these features for both targeted diagnostic imaging and drug-delivery applications. Recent efforts have focused on the synthesis and pre-clinical evaluation of a multipurpose STARBURST PAMAM (polyamidoamine) dendrimer prototype that exhibits properties suitable for use as: (i) targeted, diagnostic MRI (magnetic resonance imaging)/NIR (near-IR) contrast agents, (ii) and/or for controlled delivery of cancer therapies. Special emphasis will be placed on the lead candidate, namely [core: 1,4-diaminobutane; G (generation)=4.5], [dendri-PAMAM(CO(2)Na)(64)]. This dendritic nanostructure (i.e. approximately 5.0 nm diameter) was selected on the basis of a very favourable biocompatibility profile [The Nanotechnology Characterization Laboratory (NCL), an affiliate of the National Cancer Institute (NCI), has completed extensive in vitro studies on the lead compound and have found it to be very benign, non-immunogenic and highly biocompatible], the expectation that it will exhibit desirable mammalian kidney excretion properties and demonstrated targeting features.

We have developed a method, exon amplification, for fast and efficient isolation of coding sequences from complex mammalian genomic DNA. This method is based on the selection of RNA sequences, exons, which are flanked by functional 5' and 3' splice sites. Fragments of cloned genomic DNA are inserted into an intron, which is flanked by 5' and 3' splice sites of the human immunodeficiency virus 1 tat gene contained within the plasmid pSPL1. COS-7 cells are transfected with these constructs, and the resulting RNA transcripts are processed in vivo. Splice sites of exons contained within the inserted genomic fragment are paired with splice sites of the flanking tat intron. The resulting mature RNA contains the previously unidentified exons, which can then be amplified via RNA-based PCR and cloned. Using this method, we have isolated exon sequences from cloned genomic fragments of the murine Na,K-ATPase alpha 1-subunit gene. We have also screened randomly selected genomic clones known to be derived from a segment of human chromosome 19 and have isolated exon sequences of the DNA repair gene ERCC1. The sensitivity and ease of the exon amplification method permit screening of 20-40 kilobase pairs of genomic DNA in a single transfection. This approach will be extremely useful for rapid identification of mammalian exons and the genes from which they are derived as well as for the generation of chromosomal transcription maps.