Sodium Flux in Necturus Proximal Tubule under Voltage Clamp

Na transport and electrical properties of Necturus renal proximal tubules were analyzed, in vivo, by a voltage clamp method which utilizes an axial electrode in the tubule lumen for passage of current and simultaneous determination of net fluid (or Na) flux by the split droplet method. When the average spontaneous transepithelial potential difference of –8 mv (lumen negative) was reduced to zero by current passage, net Na flux doubled from a mean of 107 to 227 pmoles/cm² per sec. The relationship between flux and potential over the range –25 to +10 mv was nonlinear, with flux equilibrium at –15 mv and droplet expansion at more negative values. Calculated Na permeability at flux equilibrium was 7.0 x 10^–6 cm/sec. Voltage transients, similar to those caused by intraepithelial unstirred layers, were observed at the end of clamping periods. Tubular electrical resistance measured by brief square or triangle wave pulses (<100 msec) averaged 43 ohm cm². The epithelial current-voltage relationship was linear over the range –100 to +100 mv, but displayed marked hysteresis during low frequency (<0.04 Hz) triangle wave clamps. The low transepithelial resistance and large opposing unidirectional ion fluxes suggest that passive ionic movements occur across extracellular shunt pathways, while the voltage transients and current-voltage hysteresis are consistent with the development of a local osmotic gradient within epithelium.     

Current- and Voltage-Clamped Studies on Myxicola Giant Axons : Effect of tetrodotoxin

A new dissection procedure for preparing Myxicola giant axons for observation under voltage clamp is described. Preparation time is generally 40–45 min. 65–70% of the preparations attempted may be brought through the entire procedure, including insertion of the long internal electrode, and support an initial action potential amplitude of 100 mv or greater. Mean values for axon diameter, resting membrane potential, action potential amplitude, maximum peak inward transient current, and resting membrane resistance are 560 µ, —66.5 mv, 112 mv, 0.87 ma/cm² and 1.22 KΩ cm ² respectively. Cut branches do not seem to be a problem in this preparation. Behavior under voltage clamp is reasonably stable over several hours. Reductions in maximum inward transient current of 10% and in steady-state current of 5–10% are expected in the absence of any particular treatment. Tetrodotoxin blocks the action potential and both the inward and outward transient current, but has no effect on either the resting membrane potential or the steady-state current. This selective action of tetrodotoxin on the transient current is taken as an indication that this current component is probably carried by Na.     

Electrical Properties and Acetylcholine Sensitivity of Singly and Multiply Innervated Avian Muscle Fibers

Membrane constants and distribution of acetylcholine (ACh) receptors were determined for multiply innervated fibers of the anterior latissimus dorsi (ALD) and singly innervated fibers of the posterior latissimus dorsi (PLD) muscles of 3–6 month old chickens. The values of the various membrane constants were: length constant, 1.78 mm (mean) in ALD, 0.68 mm in PLD; time constant, 35 msec in ALD, 3.7 msec in PLD; transverse membrane resistance, 4388 Ω cm² in ALD, 561 Ω cm² in PLD; and membrane capacitance, 8.2 µF/cm² in ALD, 7.0 µF/cm² in PLD. Peaks of ACh sensitivity occurred at intervals of ca. 740 µ on ALD fibers with a low sensitivity remaining between peaks. Only one peak of ACh sensitivity was detected on PLD fibers. The maximum ACh sensitivity found was 5 ± 4 mv/ncoul for fibers of the ALD and 77 ± 60 mv/ncoul for fibers of the PLD. The distance over which this sensitivity fell to 0.1 was ca. 225 µ in the ALD and 140 µ in the PLD. The membranes of these two muscle fiber types differ widely regarding some electrical properties and the disposition of ACh-sensitive receptor sites.     

Endometrial Exosomes/Microvesicles in the Uterine Microenvironment: A New Paradigm for Embryo-Endometrial Cross Talk at Implantation

Exosomes are nanoparticles (∼100 nm diameter) released from cells, which can transfer small RNAs and mRNA via the extracellular environment to cells at distant sites. We hypothesised that exosomes or the slightly larger microvesicles (100–300 nm) are released from the endometrial epithelium into the uterine cavity, and that these contain specific micro (mi)RNA that could be transferred to either the trophectodermal cells of the blastocyst or to endometrial epithelial cells, to promote implantation. The aim of this study was to specifically identify and characterise exosomes/microvesicles (mv) released from endometrial epithelial cells and to determine whether exosomes/mv are present in uterine fluid. Immunostaining demonstrated that the tetraspanins, CD9 and CD63 used as cell surface markers of exosomes are present on the apical surfaces of endometrial epithelial cells in tissue sections taken across the menstrual cycle: CD63 showed cyclical regulation. Exosome/mv pellets were prepared from culture medium of endometrial epithelial cell (ECC1 cells) and from uterine fluid and its associated mucus by sequential ultracentifugation. Exosomes/mv were positively identified in all preparations by FACS and immunofluorescence staining following exosome binding to beads. Size particle analysis confirmed the predominance of particles of 50–150 nm in each of these fluids. MiRNA analysis of the ECC1 cells and their exosomes/mv demonstrated sorting of miRNA into exosomes/mv: 13 of the 227 miRNA were specific to exosomes/mv, while a further 5 were not present in these. The most abundant miRNA in exosomes/mv were hsa-miR-200c, hsa-miR-17 and hsa-miR-106a. Bioinformatic analysis showed that the exosome/mv-specific miRNAs have potential targets in biological pathways highly relevant for embryo implantation. Thus exosomes/mv containing specific miRNA are present in the microenvironment in which embryo implantation occurs and may contribute to the endometrial-embryo cross talk essential for this process.     

AMPHOTERIC BEHAVIOR OF COMPLEX SYSTEMS : IV. NOTE ON THE ISOELECTRIC POINT AND IONIZATION CONSTANTS OF SULFANILIC ACID.

From the solubility minimum the value of the basic ionization constant of sulfanilic acid is shown to lie probably between the values 1.7 x 10^–15 and 3.2 x 10^–15. From solubility measurements the value of this same constant is shown to lie probably between 2.0 and 2.2 x 10^–15, and the isoelectric point of sulfanilic acid is thus at a cH of 0.056 or a pH of 1.25. From conductivity ratios the acid ionization constant of sulfanilic acid is shown to be 7.05 x 10^–4 at room temperature (21°C.). Calculations are made, from data published in preceding papers, of the ionization constants of glycine, K_a being 2.3 x 10^–10, and K_b being 2.2 x 10^–12.     

Differences between Pacemaker and Nonpacemaker Neurons of Aplysia on Voltage Clamping

The responses of pacemaker and nonpacemaker Aplysia neurons to voltage clamp commands of less than 200 msec duration are essentially identical. For moderate depolarizing commands there is an early inward transient current followed by a late outward current and an outward tail current when the membrane is clamped back to resting potential. On long (1–2 sec) commands in pacemakers there is a marked sag in the late current and an inward tail current. E_tail, the potential of the membrane at which there is no net current flow under the conditions prevailing at the end of the clamp, shifts from about -9.0 mv on short commands to +5.0 mv on long commands. In contrast there is no marked sag of the late current or inward tail current on long commands in nonpacemakers, and E_tail is near -9.0 mv for both short and long commands. The current sag and shift in E_tail can be ascribed to a decreased conductance (presumably to K⁺) at the end of the long as compared to the short command in half of the pacemaker neurons. In the remaining cells the essential difference from nonpacemakers appears to be either a greater restricted extracellular space or a more active potential-dependent electrogenic Na⁺ pump in pacemakers.     

Pediatric Drug Safety Surveillance in FDA-AERS: A Description of Adverse Events from GRiP Project

Individual case safety reports (ICSRs) are a cornerstone in drug safety surveillance. The knowledge on using these data specifically for children is limited. We studied characteristics of pediatric ICSRs reported to the US Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS). Public available ICSRs reported in children (0–18 years) to FAERS were downloaded from the FDA-website for the period Jan 2004-Dec 2011. Characteristics of these ICSRs, including the reported drugs and events, were described and stratified by age-groups. We included 106,122 pediatric ICSRs (55% boys and 58% from United States) with a median of 1 drug [range 1–3] and 1 event [1–2] per ICSR. Mean age was 9.1 years. 90% was submitted through expedited (15-days) (65%) or periodic reporting (25%) and 10% by non-manufacturers. The proportion and type of pediatric ICSRs reported were relatively stable over time. Most commonly reported drug classes by decreasing frequency were ‘nervous system drugs’ (58%), ‘antineoplastics’ (32%) and ‘anti-infectives’ (25%). Most commonly reported system organ classes were ‘general’ (13%), ‘nervous system’ (12%) and ‘psychiatric’ (11%) disorders. Duration of use could be calculated for 19.7% of the reported drugs, of which 14.5% concerned drugs being used long-term (>6 months). Knowledge on the distribution of the drug classes and events within FAERS is a key first step in developing pediatric specific methods for drug safety surveillance. Because of several differences in terms of drugs and events among age-categories, drug safety signal detection analysis in children needs to be stratified by each age group.     

Cdc42 antagonizes Rho1 activity at adherens junctions to limit epithelial cell apical tension

In epithelia, cells are arranged in an orderly pattern with a defined orientation and shape. Cadherin containing apical adherens junctions (AJs) and the associated actomyosin cytoskeleton likely contribute to epithelial cell shape by providing apical tension. The Rho guanosine triphosphatases are well known regulators of cell junction formation, maintenance, and function. Specifically, Rho promotes actomyosin activity and cell contractility; however, what controls and localizes this Rho activity as epithelia remodel is unresolved. Using mosaic clonal analysis in the Drosophila melanogaster pupal eye, we find that Cdc42 is critical for limiting apical cell tension by antagonizing Rho activity at AJs. Cdc42 localizes Par6–atypical protein kinase C (aPKC) to AJs, where this complex limits Rho1 activity and thus actomyosin contractility, independent of its effects on Wiskott-Aldrich syndrome protein and p21-activated kinase. Thus, in addition to its role in the establishment and maintenance of apical–basal polarity in forming epithelia, the Cdc42–Par6–aPKC polarity complex is required to limit Rho activity at AJs and thus modulate apical tension so as to shape the final epithelium.     

Electrical and Mechanical Responses in Deep Abdominal Extensor Muscles of Crayfish and Lobster

Electrical and mechanical studies have been made of the deep abdominal extensor muscles, medial (DEAM) and lateral (DEAL), of crayfish and lobster. The medial muscle responds to direct (intracellular) and indirect stimulation with a transient membrane depolarization which exhibits the properties of a propagated non-decremental action potential but does not overshoot the zero level. The amplitude is about 30 mv in crayfish and 50 mv in lobster. It is followed by a fast all-or-none twitch whose duration at 20°C is 30 to 50 msec. and whose developed tension is 500 gm/cm² or about half the tetanic value. Membrane potential is K⁺-dependent and immersion in high K⁺ induces a brief transient tension rise as in other twitch-type muscles. The action potential and twitch are normal even if all external Na⁺ is replaced with sucrose but vary with external Ca⁺⁺, the action potential increasing 8 to 10 mv for a twofold increase in Ca⁺⁺. The lateral muscle (DEAL) is much slower and responds to intracellular stimulation only with an electrotonic or a local response. Mechanical responses and relaxation speeds are slow with minimal duration of contraction of 0.5 to 2 seconds. Immersion in high K solutions induces large maintained tensions. Sarcomere length in the fast DEAM is uniform and about 2 µ at rest, but in the DEAL speed is less and sarcomere length is greater averaging about 4.5 µ but with a mixed population of fibers.     

Anticoagulation intensity and outcomes among patients prescribed oral anticoagulant therapy: a systematic review and meta-analysis

Background

Patients taking oral anticoagulant therapy balance the risks of hemorrhage and thromboembolism. We sought to determine the association between anticoagulation intensity and the risk of hemorrhagic and thromboembolic events. We also sought to determine how under-or overanticoagulation would influence patient outcomes.

Methods

We reviewed the MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials and CINAHL databases to identify studies involving patients taking anticoagulants that reported person-years of observation and the number of hemorrhages or thromboemboli in 3 or more discrete ranges of international normalized ratios. We estimated the overall relative and absolute risks of events specific to anticoagulation intensity.

Results

We included 19 studies. The risk of hemorrhage increased significantly at high international normalized ratios. Compared with the therapeutic ratio of 2–3, the relative risk (RR) of hemorrhage (and 95% confidence intervals [CIs]) were 2.7 (1.8–3.9; p < 0.01) at a ratio of 3–5 and 21.8 (12.1–39.4; p < 0.01) at a ratio greater than 5. The risk of thromboemboli increased significantly at ratios less than 2, with a relative risk of 3.5 (95% CI 2.8–4.4; p < 0.01). The risk of hemorrhagic or thromboembolic events was lower at ratios of 3–5 (RR 1.8, 95% CI 1.2–2.6) than at ratios of less than 2 (RR 2.4, 95% CI 1.9–3.1; p = 0.10). We found that a ratio of 2–3 had the lowest absolute risk (AR) of events (AR 4.3%/yr, 95% CI 3.0%–6.3%).

Conclusions

The risks of hemorrhage and thromboemboli are minimized at international normalized ratios of 2–3. Ratios that are moderately higher than this therapeutic range appear safe and more effective than subtherapeutic ratios.Oral anticoagulant therapy is essential for the treatment and prevention of many thromboembolic disorders. Since anticoagulants can cause serious adverse events,^1–3 physicians monitor the international normalized ratios of patients taking these drugs to ensure that their ratios fall within a target range.An international normalized ratio of 2–3 is the most common target range. Results of previous studies revealed an increased risk of bleeding among patients whose ratios exceeded 4, an increased risk of stroke among patients whose ratios were 1.5–2 and a decreased risk of stroke at a ratio of 2.4.^4,5 However, the evidence supporting the range of 2–3 has some deficiencies. We sought to determine whether the risk of hemorrhagic and thromboembolic events is minimized at an international normalized ratio of 2–3 among patients taking anticoagulants. In addition, it has been observed that patients spend more time with a ratio below 2 than above 3.^6,7 The impact of such systematic underanticoagulation on patient outcomes is unknown. We sought to determine the effect of under-or overanticoagulation on the risk of thromboemboli and hemorrhage.     

THE STRUCTURE OF THE COLLODION MEMBRANE AND ITS ELECTRICAL BEHAVIOR : XII. THE PREPARATION AND PROPERTIES OF "MEGAPERMSELECTIVE" PROTAMINE COLLODION MEMBRANES COMBINING HIGH IONIC SELECTIVITY WITH HIGH PERMEABILITY

The technique of Abrams and Sollner for the preparation of electropositive dried protamine collodion membranes has been improved. Porous collodion membranes cast on the outside of rotating tubes are treated for 48 hours with a solution of 2 per cent protamine sulfate buffered at pH 11. After being washed thoroughly the membranes are dried in air for several hours, soaked in water for several hours, and removed from the tubes. Further drying in air but without support shrinks the membranes slightly. The resulting membranes are designated "permselective" or "megapermselective" protamine collodion membranes. These membranes regularly give characteristic concentration potentials of –52 to –53 mv. and (in 0.1 M KCl) resistance of 0.5 to 15 ohms per membrane of 50 cm.² area. This resistance is several orders of magnitude smaller than that of the conventional dyestuff- and alkaloid-impregnated positive membranes. The megapermselective protamine collodion membranes can be kept either dry or in water for prolonged periods without detectable deterioration. They are quite smooth, have a regular shape, and stand considerable handling without breakage. The megapermselective protamine collodion membranes are the electropositive analogues of the electronegative megapermselective collodion membranes described by Carr and Sollner.     

Sodium and Potassium Ion Effluxes from Squid Axons under Voltage Clamp Conditions

Squid giant axons loaded with Na²⁴ were subjected to short duration (0.5 msec.) clamped depolarizations of about 100 mv at frequencies of 20/sec. and 60/sec. while in choline sea water. Under such conditions the early outward current was just about maximal at the time of termination of the clamping pulse. An integration of the early current versus time record gave 1.2 μcoulomb/cm² pulse, while a measurement of the extra Na²⁴ efflux resulting from repetitive pulsing gave a charge transfer of 1.4 μcoulomb/cm² pulse. In sodium-containing sea water and with pulses 50-75 mv more positive than E_Na the Na²⁴ efflux is about 3 times the measured charge transfer. The efflux of K⁴² from a previously loaded axon into normal sea water is only 50 per cent of the measured charge transfer when the membrane is held for about 5 msec. at a potential such that there is no early current, and such pulses are at 10-20/sec. The experiments appear to confirm the suggestion that the early current during bioelectric activity is sodium but provide unsatisfactory support for the identification of the delayed but sustained current solely with potassium ions. Resting Na⁺ efflux is 0.6 pmole/cm² sec. mmole [Na]₁, while the apparent K⁺ efflux is about 250 pmole/cm² sec. and is little affected by hyperpolarization.     

The Effect of N Fertilizer Placement on the Fate of Urea-15N and Yield of Winter Wheat in Southeast China

A field micro-plot experiment using nitrogen isotope (¹⁵N) labeling was conducted to determine the effects of placement methods (broadcast and band) and N rates (60, 150 and 240 kg ha^–1) on the fate of urea-¹⁵N in the wheat–soil system in Guangde County of Anhui Province, China. N fertilizer applied in bands increased grain yield by 15% compared with broadcast application. The N fertilizer application rate had a significant effect on grain yield, straw yield and aboveground biomass, as well as on N uptake and N concentration of wheat. The recovery of urea-¹⁵N was a little higher for broadcast (34.0–39.0%) than for band treatment (31.2–38.2%). Most of the soil residual N was retained in the 0–20 cm soil layer. At the N rates of 60 and 240 kg ha^–1, the residual ¹⁵N was higher for band (34.4 and 108.7 kg ha^–1, respectively) than for broadcast application (29.6 and 88.4 kg ha^–1, respectively). Compared with broadcast treatment, banded placement of N fertilizer decreased the N loss in the wheat–soil system. Band application one time is an alternative N management practice for winter wheat in this region.     

Cellular responses to increasing Cd concentrations in the freshwater crab,<Emphasis Type="Italic"> Potamonautes warreni</Emphasis>, harbouring microbial gill infestations

We investigated the uptake, transport, storage and defence mechanisms in the freshwater crab, Potamonautes warreni, harbouring microbial gill infestations and exposed to increasing chronic (0.2, 0.5, 1.0 mg l^–1) and acute (2.0 mg l^–1) cadmium (Cd) concentrations under controlled laboratory conditions over a period of 21 days. Transmission electron microscopy and X-ray microanalysis revealed that the microbial gill fauna was eliminated on exposure to 0.2 mg Cd²⁺ l^–1 and that Cd became increasingly adsorbed and incorporated into lamellar crystal deposits and permeated the cuticle of the gills of P. warreni. Degeneration of the apical membrane infoldings and vacuolation of epithelial cells occurred concurrently with pinocytosis, endocytosis and pronounced phagocytotic activity in the epithelia and haemal canal of the gills. Elevated Cd exposures (0.5 or 1.0 mg l^–1) resulted in the swelling and dissociation of mitochondrial outer membranes together with an increase in transport of Cu, Cl and S by haemocytes in the haemal canal to epithelial tissues depleted in these elements. Cd also accumulated in tightly coiled concentric membrane whorls in the haemal canal, whereas the highest concentrations of Cd were found within aggregates of lysosome-like bodies in cuticulin-secreting cells of the gill stem. Chronic exposure to Cd induced increased fatigue and mild uncoordinated motor activity. In contrast, at an acute exposure of 2.0 mg l^–1 over 48 h, P. warreni showed a time-specific rapid loss of motor function, although only mild cellular lesions occurred in the gill tissues. The significance of cellular changes in the gill epithelia and altered motor activity of P. warreni with increased waterborne Cd are discussed as potential biomarker responses in monitoring aquatic pollution.     

Genetic disruption of mineralocorticoid receptor leads to impaired neurogenesis and granule cell degeneration in the hippocampus of adult mice

To dissect the effects of corticosteroids mediated by the mineralocorticoid (MR) and the glucocorticoid receptor (GR) in the central nervous system, we compared MR^–/– mice, whose salt loss syndrome was corrected by exogenous NaCl administration, with GR^–/– mice having a brain-specific disruption of the GR gene generated by the Cre/loxP-recombination system. Neuropathological analyses revealed a decreased density of granule cells in the hippocampus of adult MR^–/– mice but not in mice with disruption of GR. Furthermore, adult MR^–/– mice exhibited a significant reduction of granule cell neurogenesis to 65% of control levels, possibly mediated by GR due to elevated corticosterone plasma levels. Neurogenesis was unaltered in adult mice with disruption of GR. Thus, we could attribute long-term trophic effects of adrenal steroids on dentate granule cells to MR. These MR-related alterations may participate in the pathogenesis of hippocampal changes observed in ageing, chronic stress and affective disorders.     

EPSPs of masseter motoneurons evoked by stimulation of low-threshold infraorbital nerve afferents in cat

Stimulation of the infraorbital nerve at strengths 1.4–2.5 times higer than the threshold of excitation of A fibers in cats anesthetized with chloralose and pentobarbital evoked EPSPs with an amplitude up to 3.0 mV and a duration of 9–15 msec in 69% of masseter motoneurons after 1.5–3.0 msec. These EPSPs were complex and formed by summation of simpler short-latency and long-latency EPSPs. The short-latency EPSPs appeared in response to infraorbital nerve stimulation at 1.1–1.5 thresholds and had a slow rate of rise (2.5–4.5 msec, mean 3.7±0.4 msec), low amplitude (under 2.0 mV), and short duration (5–6 msec). Their latent period varied from 1.5 to 3.0 msec (mean 2.1±0.2 msec). The shortness of the latent period and its constancy during stimulation of the nerve at increasing strength, and also the character of development of facilitation and inhibition of the EPSP during high-frequency stimulation suggests that these EPSPs are monosynaptic. The slow rate of rise suggested that these EPSPs arise on distal dendrites of the motoneurons. Long-latency EPSPs appeared 7–9 msec after stimulation of the infraorbital nerve at 1.1–1.5 thresholds. Their amplitude reached 1.5–2.0 mV and their duration 7–9 msec. The long duration of the latent period combined with low ability to reproduce high-frequency stimulation (up to 30/sec) points to the polysynaptic origin of these EPSPs.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 9, No. 6, pp. 583–591, November–December, 1977.     

Galectin-3, a Novel Centrosome-associated Protein,Required for Epithelial Morphogenesis

Galectin-3 is a β-galactoside–binding protein widely expressed in all epithelia where it is involved in tissue homeostasis and cancer progression. We recently reported unique abnormalities in the identity of membrane domains in galectin-3 null mutant mice, suggesting that galectin-3 may participate in epithelial polarity program. We investigated the potential role of galectin-3 on early events in polarization of epithelial renal cells, using three-dimensional cultures of MDCK cells and also galectin-3 null mutant mouse kidneys. We show that depletion in galectin-3 systematically leads to severe perturbations of microtubular network associated with defects in membrane compartimentation, both in vitro and in vivo. Moreover, the absence of galectin-3 impinges on the morphology of the primary cilium, which is three times longer and unusually shaped. By immunological and biochemical approaches, we could demonstrate that endogenous galectin-3 is normally associated with basal bodies and centrosomes, where it closely interacts with core proteins, such as centrin-2. However, this association transiently occurs during the process of epithelial polarization. Interestingly, galectin-3–depleted cells contain numerous centrosome-like structures, demonstrating an unexpected function of this protein in the formation and/or stability of the centrosomes. Collectively, these data establish galectin-3 as a key determinant in epithelial morphogenesis via its effect on centrosome biology.     

The cystic fibrosis transmembrane conductance regulator (CFTR) inhibits ENaC through an increase in the intracellular Cl- concentration

Activation of the CFTR Cl^– channel inhibits epithelial Na⁺ channels (ENaC), according to studies on epithelial cells and overexpressing recombinant cells. Here we demonstrate that ENaC is inhibited during stimulation of the cystic fibrosis transmembrance conductance regulator (CFTR) in Xenopus oocytes, independent of the experimental set-up and the magnitude of the whole-cell current. Inhibition of ENaC is augmented at higher CFTR Cl^– currents. Similar to CFTR, ClC-0 Cl^– currents also inhibit ENaC, as well as high extracellular Na⁺ and Cl^– in partially permeabilized oocytes. Thus, inhibition of ENaC is not specific to CFTR and seems to be mediated by Cl^–.     

Potassium Exchange and Afterpotentials in Frog Sartorius Muscles Treated with Glycerol

The potassium exchange properties of glycerol-treated sartorius muscles of the frog were determined. Potassium (⁴²K) uptake, efflux, and net flux were measured in the presence of glycerol and at various times after exposure to glycerol and return to isotonic Ringer solution. Potassium uptake was not altered by the presence of glycerol but was reduced on the average 53% after glycerol treatment. Efflux transiently increased in the presence of glycerol and was reduced 37% after glycerol removal. Consequently, there was a net loss of intracellular potassium as well as a gain of sodium. In contrast to the irreversible alterations of potassium exchange induced by glycerol treatment, action potentials with normal negative afterpotentials (N.A.P.) were elicited 4–5 hr after glycerol removal. The reappearance of the N.A.P. was associated with a return of the membrane potential to normal values (90 ± 2 mv). However, the response of these muscles to reduced extracellular potassium was anomalous. In K⁺-free Ringer solution the average resting membrane potential was 74 ± 3 mv and a positive afterpotential of 11 ± 3 mv was associated with the action potential.     

NMR Structural Studies of Antimicrobial Peptides: LPcin Analogs

Lactophoricin (LPcin), a component of proteose peptone (113–135) isolated from bovine milk, is a cationic amphipathic antimicrobial peptide consisting of 23 amino acids. We designed a series of N- or C-terminal truncated variants, mutated analogs, and truncated mutated analogs using peptide-engineering techniques. Then, we selected three LPcin analogs of LPcin-C8 (LPcin-YK1), LPcin-T2WT6W (LPcin-YK2), and LPcin-T2WT6W-C8 (LPcin-YK3), which may have better antimicrobial activities than LPcin, and successfully expressed them in E. coli with high yield. We elucidated the 3D structures and topologies of the three LPcin analogs in membrane environments by conducting NMR structural studies. We investigated the purity of the LPcin analogs and the α-helical secondary structures by performing ¹H-¹⁵N 2D HSQC and HMQC-NOESY liquid-state NMR spectroscopy using protein-containing micelle samples. We measured the 3D structures and tilt angles in membranes by conducting ¹⁵N 1D and 2D ¹H-¹⁵N SAMMY type solid-state NMR spectroscopy with an 800 MHz in-house-built ¹H-¹⁵N double-resonance solid-state NMR probe with a strip-shield coil, using protein-containing large bicelle samples aligned and confirmed by molecular-dynamics simulations. The three LPcin analogs were found to be curved α-helical structures, with tilt angles of 55–75° for normal membrane bilayers, and their enhanced activities may be correlated with these topologies.