Further studies of the effect of calcium on the time course of action of carbamylcholine at the neuromuscular junction

The rate at which the postjunctional membrane of muscle fibers becomes desensitized to the action of carbamylcholine is increased after the muscle has been soaked in solutions containing increased concentrations of calcium. Some further aspects of this effect of calcium were investigated by measuring changes in the input resistance of single fibers of the frog sartorius during local perfusion of the neuromuscular junction with 2.73 x 10^-3 M carbamylcholine in isolated muscles immersed in 165 mM potassium acetate. It was found that (a) sudden changes in the local concentration of calcium brought about by perfusing fibers with carbamylcholine solutions containing 20 mM calcium, 40 mM oxalate, or 40 mM EDTA were followed within 20 sec by marked changes in the rate of desensitization; (b) prior to 13 sec after the introduction of carbamylcholine, however, no effect on the input resistance could be detected even though the muscle had been presoaked in 10 mM calcium; (c) the ability of high concentrations of calcium to bring about rapid desensitization disappears when a lower concentration of carbamylcholine (0.137 x 10^-3 M) is applied to the muscle fiber. These findings suggest that calcium present in the extracellular fluid can act directly on the postjunctional membrane to promote the desensitization process and that an increased permeability of the membrane to calcium brought about by the presence of carbamylcholine is a factor which contributes to this action.     

The interaction between caffeine and calcium in the desensitization of muscle postjunctional membrane receptors

The interaction between caffeine and calcium on the rate of desensitization of muscle postjunctional membrane (PJM) receptors during the sustained application of 0.27 mM carbamylcholine (CARB) has been studied in vitro on the sartorius muscle of the frog. The rate of PJM repolarization with CARB added to the solution bathing the muscle or the recovery of the effective transmembrane resistance (EMR) during the microperfusion of CARB directly onto the end-plate region of individual fibers was used as an index of the rate of desensitization. Caffeine (1.5 mM) increased the rate of PJM repolarization with bulk application of CARB in a 1.8 or 10 mM calcium Ringer solution but had no effect on PJM repolarization in a calcium-deficient, 4 mM magnesium Ringer solution. For EMR measurements the preparation was rendered mechanically quiescent by repeated challenges with isotonic KCl during an exposure of several hours to a calcium-free, 4 mM magnesium-1 mM EGTA Ringer solution. In these fibers, the microperfusion of 0.27 mM CARB together with 1.8 mM calcium plus 1.5 mM caffeine significantly increased the rate of EMR recovery above that observed in the absence of caffeine. Raising the calcium concentration to 10 mM had a similar effect; however, no additional increase was noted by the inclusion of 1.5 mM caffeine. It is suggested that the major role of caffeine in PJM desensitization is to increase the calcium permeability of the surface membrane. The transmembrane movement of calcium and the consequent intracellular accumulation of calcium is seen as a critical factor in controlling the rate of PJM desensitization.     

The Dual Effect of Lithium Ions on Sodium Efflux in Skeletal Muscle

Sartorius muscle cells from the frog were stored in a K-free Ringer solution at 3°C until their average sodium contents rose to around 23 mM/kg fiber (about 40 mM/liter fiber water). Such muscles, when placed in Ringer''s solution containing 60 mM LiCl and 50 mM NaCl at 20°C, extruded 9.8 mM/kg of sodium and gained an equivalent quantity of lithium in a 2 hr period. The presence of 10^-5 M strophanthidin in the 60 mM LiCl/50 mM NaCl Ringer solution prevented the net extrusion of sodium from the muscles. Lithium ions were found to enter muscles with a lowered internal sodium concentration at a rate about half that for entry into sodium-enriched muscles. When sodium-enriched muscles labeled with radioactive sodium ions were transferred from Ringer''s solution to a sodium-free lithium-substituted Ringer solution, an increase in the rate of tracer sodium output was observed. When the lithium-substituted Ringer solution contained 10^-5 M strophanthidin, a large decrease in the rate of tracer sodium output was observed upon transferring labeled sodium-enriched muscles from Ringer''s solution to the sodium-free medium. It is concluded that lithium ions have a direct stimulating action on the sodium pump in skeletal muscle cells and that a significantly large external sodium-dependent component of sodium efflux is present in muscles with an elevated sodium content. In the sodium-rich muscles, about 23% of the total sodium efflux was due to strophanthidin-insensitive Na-for-Na interchange, about 67% being due to strophanthidin-sensitive sodium pumping.     

Effects of External Potassium and Strophanthidin on Sodium Fluxes in Frog Striated Muscle

Unidirectional Na fluxes in isolated fibers from the frog''s semitendinosus muscle were measured in the presence of strophanthidin and increased external potassium ion concentrations. Strophanthidin at a concentration of 10^-5 M inhibited about 80 per cent of the resting Na efflux without having any detectable effect on the resting Na influx. From this it is concluded that the major portion of the resting Na efflux is caused by active transport processes. External potassium concentrations from 2.5 to 7.5 mM had little effect on resting Na efflux. Above 7.5 mM and up to 15 mM external K, the Na efflux was markedly stimulated; with 15 mM K the Na influx was 250 to 300 per cent greater than normal. On the other hand, Na influx was unchanged with 15 mM K. The stimulated Na efflux with the higher concentrations was not appreciably reduced when choline or Li was substituted for external Na, but was completely inhibited by 10^-5 M strophanthidin. From these findings it is concluded that the active transport of Na is stimulated by the higher concentrations of K. It is postulated that this effect on the Na "pump" is produced as a result of the depolarization of the muscle membranes and is related to the increased metabolism and heat production found under conditions of high external K.     

Studies of cardiac muscle with a high permeability to calcium produced by treatment with ethylenediaminetetraacetic acid

Thin strips of frog ventricle were isolated and bathed for 15 min in a solution containing 140 mM KCl, 5 mM Na₂ATP, 3 mM EDTA, and 10 mM Tris buffer at pH 7.0. The muscle was then exposed to contracture solutions containing 140 mM KCl, 5 mM Na₂ATP, 1 mM MgCl₂, 10 mM Tris, 3 mM EGTA, and CaCl₂ in amounts to produce concentrations of free calcium from 10^-4.8 M to 10^-9 M. The muscles developed some tension at approximately 10^-8 M, and maximum tension was achieved in 10^-5 M Ca⁺⁺. They relaxed in Ca⁺⁺ concentrations less than 10^-8 M. The development of tension by the EDTA-treated muscles was normalized by comparison with twitch tension at a stimulation rate of 9 per min before exposure to EDTA. In 10^-5 M Ca⁺⁺ tension was always several times the twitch tension and was greater than the contracture tension of a frog ventricular strip in KCl low Na-Ringer. Tension equal to half-maximum was produced at approximately 10^-6.2 M Ca⁺⁺. Intracellular recording of membrane potential indicated that after EDTA treatment the resting potential of cells in Ringer solution with 10^-5 M Ca or less was between 5 and 20 mv. Contracture solutions did not produce tension without prior treatment with EDTA. The high permeability of the membrane produced by EDTA was reversed and the normal resting and action potentials restored in 1 mM Ca-Ringer. Similar studies of EDTA-treated rabbit right ventricular papillary muscle produced a similar tension vs. Ca⁺⁺ concentration relation, and the high permeability state reversed with exposure to normal Krebs solution.     

Glutamate, glutamine, aspartate, asparagine, glucose and ketone-body metabolism in chick intestinal brush-border cells

1. Suspensions of isolated chick jejunal columnar absorptive (brush-border) cells respired on endogenous substrates at a rate 40% higher than that shown by rat brush-border cells. 2. Added d-glucose (5 or 10mm), l-glutamine (2.5mm) and l-glutamate (2.5mm) were the only individual substrates which stimulated respiration by chick cells; l-aspartate (2.5 or 6.7mm), glutamate (6.7mm), glutamine (6.7mm), l-alanine (1 or 10mm), pyruvate (1 or 2mm), l-lactate (5 or 10mm), butyrate (10mm) and oleate (1mm) did not stimulate chick cell respiration; l-asparagine (6.7mm) inhibited slightly; glucose (5mm) stimulated more than did 10mm-glucose. 3. Acetoacetate (10mm) and d-3-hydroxybutyrate (10mm) were rapidly consumed but, in contrast to rat brush-border cells, did not stimulate respiration. 4. Glucose (10mm) was consumed more slowly than 5mm-glucose; the dominant product of glucose metabolism during vigorous respiration was lactate; the proportion of glucose converted to lactate was greater with 10mm- than with 5mm-glucose. 5. Glutamate and aspartate consumption rates decreased, and alanine and glutamine consumption rates increased when their initial concentrations were raised from 2.5 to 6.7 or 10mm. 6. The metabolic fate of glucose was little affected by concomitant metabolism of any one of aspartate, glutamate or glutamine except for an increased production of alanine; the glucose-stimulated respiration rate was unaffected by concomitant metabolism of these individual amino acids. 7. Chick cells produced very little alanine from aspartate and, in contrast to rat cells, likewise produced very little alanine from glutamate or glutamine; in chick cells alanine appeared to be predominantly a product of transmination of pyruvate derived from glucose metabolism. 8. In chick cells, glutamate and glutamine were formed from aspartate (2.5 or 6.7mm); aspartate and glutamine were formed from glutamate (2.5mm) but only aspartate from 6.7mm-glutamate; glutamate was the dominant product formed from glutamine (6.7mm) but aspartate only was formed from 2.5mm-glutamine. 9. Chick brush-border cells can thus both catabolize and synthesize glutamine; glutamine synthesis is always diminished by concomitant metabolism of glucose, presumably by allosteric inhibition of glutamine synthetase by alanine. 10. Proline was formed from glutamine (2.5mm) but not from glutamine (2.5mm)+glucose (5mm) and not from 2.5mm-glutamate; ornithine was formed from glutamine (2.5mm)+glucose (5.0mm) but not from glutamine alone; serine was formed from glutamine (2.5mm)+glucose (5mm) and from these two substrates plus aspartate (2.5mm). 11. Total intracellular adenine nucleotides (22μmol/g dry wt.) remained unchanged during incubation of chick cells with glucose. 12. Intracellular glutathione (0.7–0.8mm) was depleted by 40% during incubation of respiring chick cells without added substrates for 75min at 37°C; partial restoration of the lost glutathione was achieved by incubating cells with l-glutamate+l-cysteine+glycine.     

Effects of Sodium Azide on Sodium Fluxes in Frog Striated Muscle

Unidirectional Na fluxes from frog''s striated muscle were measured in the presence of 0 to 5 mM sodium azide. With azide concentrations of 2 and 5 mM the Na efflux was markedly stimulated; the Na efflux with 5 mM azide was about 300 per cent greater than normal. A similar increase was present when all but the 5.0 mM sodium added with azide was replaced by choline. 10^-5 M strophanthidin abolished the azide effect on Na²⁴ efflux. Concentrations of azide of 1.0 mM or less had no effect on Na efflux. The Na influx, on the other hand, was only increased by 41 per cent in the presence of 5 mM NaN₃. From these findings it is concluded that the active transport of Na is stimulated by the higher concentrations of azide. The hypothesis is advanced that the active transport of Na is controlled by the transmembrane potential and that the stimulation of Na efflux is produced as a consequence of the membrane depolarization caused by the azide.     

Calcium efflux from frog twich muscle fibers

An apparatus is described which collects the effluent from the center 0.7 cm of a single muscle fiber or bundle of muscle fibers. It was used to study the efflux of ⁴⁵Ca from twitch muscle fibers. The efflux can be described by three time constants 18 ± 2 min, 300 ± 40 min, and 882 ± 172 min. These kinetics have been interpreted as those of a three-compartment system. The fastest is thought to be on the surface membrane of the muscle and of the T system. It contains 0.07 ± 0.03 mM Ca/liter of fiber and the Ca efflux is 0.11 ± 0.04 pM Ca/cm². sec. The intermediate rate compartment is thought to represent the Ca in the longitudinal reticulum. It contains approximately 0.77 mM Ca/liter. Only the efflux from this compartment increases during stimulation. The most slowly exchanging compartment is poorly defined. Neither Ca-free nor Ni-Ringer solutions alter the rate of loss from the fastest exchanging compartment. Ni apparently alters the rate of loss from the slowest compartment.     

Effects of caffeine on crayfish muscle fibers. I. Activation of contraction and induction of Ca spike electrogenesis

Contractions are evoked in single muscle fibers of crayfish by intracellular as well as extracellular applications of caffeine. Responses to external applications in concentrations above 2 mM could be induced indefinitely. With concentrations above 5 mM the caffeine-induced responses were highly repeatable. Tensions were transient even when the caffeine remained in the bath. There was no change in resting potential, but during the contraction the effective resistance decreased about 10%. A number of factors (change in pH, Ca, K, and Cl) modified the responses. The time course of the tension was greatly prolonged when the transverse tubular system (TTS) was s swollen and was again shortened when the TTS was caused to shrink. An increased permeability to Ca induced by caffeine was evidenced by the transformation of the normally graded electrical responses to Ca spikes, which are insensitive to tetrodotoxin. The overshoot is a function of both external Ca and caffeine. A 10-fold change in Ca changed the overshoot by 19 mv in the presence of 10 mM caffeine and by 29 mv in 80 mM caffeine. The role of the increased permeability to Ca for caffeine-induced contractions will be analyzed in the accompanying paper.     

The elastic fiber. I. The separation and partial characterization of its macromolecular components

The two morphologically different constituents of the mature elastic fiber, the central amorphous and the peripheral microfibrillar components, have been separated and partially characterized. A pure preparation of elastic fibers was obtained from fetal bovine ligamentum nuchae by extraction of the homogenized ligament with 5 M guanidine followed by digestion with collagenase. The resultant preparation consisted of elastic fibers which were morphologically identical with those seen in vivo. The microfibrillar components of these elastic fibers were removed either by proteolytic enzymes or by reduction of disulfide bonds with dithioerythritol in 5 M guanidine. The microfibrils solubilized by both methods were rich in polar, hydroxy, and sulfur-containing amino acids and contained less glycine, valine, and proline than the amorphous component of the elastic fiber. In contrast, the amino acid composition of the amorphous component was identical with that previously described for elastin. This component demonstrated selective susceptibility to elastase digestion, but was relatively resistant to the action of other proteolytic enzymes and to reduction. These observations establish that the microfibrils consist of a different connective tissue protein (or proteins) that is neither collagen nor elastin. During embryologic development the microfibrils form an aggregate structure before the amorphous component is secreted. These microfibrils may therefore play a primary role in the morphogenesis of the elastic fiber.     

Amino Acid and Sugar Transport in Rabbit Ileum

L-Alanine and 3-O-methyl-D-glucose accumulation by mucosal strips from rabbit ileum has been investigated with particular emphasis on the interaction between Na and these transport processes. L-Alanine is rapidly accumulated by mucosal tissue and intracellular concentrations of approximately 50 mM are reached within 30 min when extracellular L-alanine concentration is 5 mM. Evidence is presented that intracellular alanine exists in an unbound, osmotically active form and that accumulation is an active transport process. In the absence of extracellular Na, the final ratio of intracellular to extracellular L-alanine does not differ significantly from unity and the rate of net uptake is markedly inhibited. Amino acid accumulation is also inhibited by 5 x 10^-5 M ouabain. 3-O-methyl-D-glucose accumulation by this preparation is similarly affected by ouabain and by incubation in a Na-free medium. The effects of amino acid accumulation, of ouabain, and of incubation in a Na-free medium on cell water content and intracellular Na and K concentrations have also been investigated. These results are discussed with reference to the two hypotheses which have been suggested to explain the interaction between Na and intestinal nonelectrolyte transport.     

The effect of calcium on the desensitization of membrane receptors at the neuromuscular junction

Desensitization, as represented by the progressive decline in the electromotive effects of depolarizing agents at the neuromuscular junction, was studied by observing the time course of changes in effective transmembrane resistance during the prolonged application of 0.27 mM carbamylcholine to the postjunctional region of frog skeletal muscle fibers. The effective transmembrane resistance was measured by means of two intracellular microelectrodes implanted in the junctional region of single muscle fibers. When carbamylcholine was applied to the muscle there was an immediate decrease in the effective membrane resistance followed by a slower return toward control values which was identified as the phase of desensitization. When the calcium concentration was increased from 0 to 10 mM there was an approximately sevenfold increase in the rate of desensitization. On the other hand, an increase in the concentration of sodium from 28 to 120 mM caused a slowing of the rate of desensitization. Even in muscles depolarized by potassium sulfate, calcium increased the rate of desensitization while high concentrations of potassium tended to prolong the process. Some mechanisms by which calcium might exert these effects are discussed.     

Effects of the Intracellular Ca Ion Concentration upon the Excitability of the Muscle Fiber Membrane of a Barnacle

The membrane excitability and contraction were examined in single barnacle muscle fibers with different internal Ca⁺⁺ concentrations by using buffer solutions made up with EGTA and Ca-gluconate in various proportions. During the passage of dc currents the membrane shows all-or-none spike potentials for internal Ca⁺⁺ concentrations below about 8 x 10^-8 M, oscillatory potential changes in the range between 8 x 10^-8 to 5 x 10^-7 M, but neither oscillatory nor spike potentials were seen for concentrations above 5 x 10^-7 M. All-or-none spike potentials were suppressed when the internal Mg⁺⁺ concentration exceeded 5 mM. The suppression threshold of the internal Ca⁺⁺ concentration for the Sr spike is much higher than that for the Ca spike. The threshold concentration of internal Ca⁺⁺ for contraction was about 8 x 10^-7 M.     

THE DIRECT ISOLATION OF THE MITOTIC APPARATUS

A method for isolating the mitotic apparatus from dividing sea urchin eggs without the use of ethyl alcohol or of detergents is described. In the present method, the eggs are dispersed directly in a medium containing 1 M (to 1.15 M) sucrose, 0.15 M dithiodiglycol, and 0.001 M Versene at pH 6, releasing the visibly intact mitotic apparatus. The method is designed for studies of enzyme activities, lipid components, and the variables affecting the stability of the apparatus.     

Ribosome crystallization in chicken embryos. I. Isolation, characterization, and in vitro activity of ribosome tetramers

Isolated tetrameric particles (166S) derived from the crystalline lattices known to appear in hypothermic chicken embryos consist of mature 80S ribosomes which contain all species of ribosomal RNA and a complete set of ribosomal proteins. Ribosome tetramers are not a special type of polysomes since in solutions of high ionic strengths (500 mM KCl and 50 nM triethanolamine-HCl buffer) containing 5 mM MgCl₂ they dissociate into 40S and 60S ribosomal subunits, without the need of puromycin, and at a concentration of Mg⁺⁺ higher than 3 mM they are not disassembled by mild RNase treatment. Tetramers spontaneously disassemble into 80S monomers when the Mg⁺⁺ concentration is lowered to 1 mM at relatively low ionic strength. Tetramers failed to couple in vitro puromycin-³H into an acid-insoluble product, indicating the lack of nascent polypeptide chains. Although tetramers have no endogenous messenger RNA activity, they can be programmed in vitro with polyuridylic acid (poly U) to synthesize polyphenylalanine. All ribosomes within a tetramer can accept poly U, without the need of disassembly of the tetramers into monomers or subunits.     

The Conserved miR-51 microRNA Family Is Redundantly Required for Embryonic Development and Pharynx Attachment in Caenorhabditis elegans

A major question about cytokinesis concerns the role of the septin proteins, which localize to the division site in all animal and fungal cells but are essential for cytokinesis only in some cell types. For example, in Schizosaccharomyces pombe, four septins localize to the division site, but deletion of the four genes produces only a modest delay in cell separation. To ask if the S. pombe septins function redundantly in cytokinesis, we conducted a synthetic-lethal screen in a septin-deficient strain and identified seven mutations. One mutation affects Cdc4, a myosin light chain that is an essential component of the cytokinetic actomyosin ring. Five others cause frequent cell lysis during cell separation and map to two loci. These mutations and their dosage suppressors define a signaling pathway (including Rho1 and a novel arrestin) for repairing cell-wall damage. The seventh mutation affects the poorly understood RNA-binding protein Scw1 and severely delays cell separation when combined either with a septin mutation or with a mutation affecting the septin-interacting, anillin-like protein Mid2, suggesting that Scw1 functions in a pathway parallel to that of the septins. Taken together, our results suggest that the S. pombe septins participate redundantly in one or more pathways that cooperate with the actomyosin ring during cytokinesis and that a septin defect causes septum defects that can be repaired effectively only when the cell-integrity pathway is intact.THE fission yeast Schizosaccharomyces pombe provides an outstanding model system for studies of cytokinesis (McCollum and Gould 2001; Balasubramanian et al. 2004; Pollard and Wu 2010). As in most animal cells, successful cytokinesis in S. pombe requires an actomyosin ring (AMR). The AMR begins to assemble at the G₂/M transition and involves the type II myosin heavy chains Myo2 and Myp2 and the light chains Cdc4 and Rlc1 (Wu et al. 2003). Myo2 and Cdc4 are essential for cytokinesis under all known conditions, Rlc1 is important at all temperatures but essential only at low temperatures, and Myp2 is essential only under stress conditions. As the AMR constricts, a septum of cell wall is formed between the daughter cells. The primary septum is sandwiched by secondary septa and subsequently digested to allow cell separation (Humbel et al. 2001; Sipiczki 2007). Because of the internal turgor pressure of the cells, the proper assembly and structural integrity of the septal layers are essential for cell survival.Septum formation involves the β-glucan synthases Bgs1/Cps1/Drc1, Bgs3, and Bgs4 (Ishiguro et al. 1997; Le Goff et al. 1999; Liu et al. 1999, 2002; Martín et al. 2003; Cortés et al. 2005) and the α-glucan synthase Ags1/Mok1 (Hochstenbach et al. 1998; Katayama et al. 1999). These synthases are regulated by the Rho GTPases Rho1 and Rho2 and the protein kinase C isoforms Pck1 and Pck2 (Arellano et al. 1996, 1997, 1999; Nakano et al. 1997; Hirata et al. 1998; Calonge et al. 2000; Sayers et al. 2000; Ma et al. 2006; Barba et al. 2008; García et al. 2009b). The Rho GTPases themselves appear to be regulated by both GTPase-activating proteins (GAPs) and guanine-nucleotide-exchange factors (GEFs) (Nakano et al. 2001; Calonge et al. 2003; Iwaki et al. 2003; Tajadura et al. 2004; Morrell-Falvey et al. 2005; Mutoh et al. 2005; García et al. 2006, 2009a,b). In addition, septum formation and AMR function appear to be interdependent. In the absence of a normal AMR, cells form aberrant septa and/or deposit septal materials at random locations, whereas a mutant defective in septum formation (bgs1) is also defective in AMR constriction (Gould and Simanis 1997; Le Goff et al. 1999; Liu et al. 1999, 2000). Both AMR constriction and septum formation also depend on the septation initiation network involving the small GTPase Spg1 (McCollum and Gould 2001; Krapp and Simanis 2008). Despite this considerable progress, many questions remain about the mechanisms and regulation of septum formation and its relationships to the function of the AMR.One major question concerns the role(s) of the septins. Proteins of this family are ubiquitous in fungal and animal cells and typically localize to the cell cortex, where they appear to serve as scaffolds and diffusion barriers for other proteins that participate in a wide variety of cellular processes (Longtine et al. 1996; Gladfelter et al. 2001; Hall et al. 2008; Caudron and Barral 2009). Despite the recent progress in elucidating the mechanisms of septin assembly (John et al. 2007; Sirajuddin et al. 2007; Bertin et al. 2008; McMurray and Thorner 2008), the details of septin function remain obscure. However, one prominent role of the septins and associated proteins is in cytokinesis. Septins concentrate at the division site in every cell type that has been examined, and in Saccharomyces cerevisiae (Hartwell 1971; Longtine et al. 1996; Lippincott et al. 2001; Dobbelaere and Barral 2004) and at least some Drosophila (Neufeld and Rubin 1994; Adam et al. 2000) and mammalian (Kinoshita et al. 1997; Surka et al. 2002) cell types, the septins are essential for cytokinesis. In S. cerevisiae, the septins are required for formation of the AMR (Bi et al. 1998; Lippincott and Li 1998). However, this cannot be their only role, because the AMR itself is not essential for cytokinesis in this organism (Bi et al. 1998; Korinek et al. 2000; Schmidt et al. 2002). Moreover, there is no evidence that the septins are necessary for AMR formation or function in any other organism. A further complication is that in some cell types, including most Caenorhabditis elegans cells (Nguyen et al. 2000; Maddox et al. 2007) and some Drosophila cells (Adam et al. 2000; Field et al. 2008), the septins do not appear to be essential for cytokinesis even though they localize to the division site.S. pombe has seven septins, four of which (Spn1, Spn2, Spn3, and Spn4) are expressed in vegetative cells and localize to the division site shortly before AMR constriction and septum formation (Longtine et al. 1996; Berlin et al. 2003; Tasto et al. 2003; Wu et al. 2003; An et al. 2004; Petit et al. 2005; Pan et al. 2007; Onishi et al. 2010). Spn1 and Spn4 appear to be the core members of the septin complex (An et al. 2004; McMurray and Thorner 2008), and mutants lacking either of these proteins do not assemble the others at the division site. Assembly of a normal septin ring also depends on the anillin-like protein Mid2, which colocalizes with the septins (Berlin et al. 2003; Tasto et al. 2003). Surprisingly, mutants lacking the septins are viable and form seemingly complete septa with approximately normal timing. These mutants do, however, display a variable delay in separation of the daughter cells, suggesting that the septins play some role(s) in the proper completion of the septum or in subsequent processes necessary for cell separation (Longtine et al. 1996; An et al. 2004; Martín-Cuadrado et al. 2005).It is possible that the septins localize to the division site and yet are nonessential for division in some cell types because their role is redundant with that of some other protein(s) or pathway(s). To explore this possibility in S. pombe, we screened for mutations that were lethal in combination with a lack of septins. The results suggest that the septins cooperate with the AMR during cytokinesis and that, in the absence of septin function, the septum is not formed properly, so that an intact system for recognizing and repairing cell-wall damage becomes critical for cell survival.     

Structure-activity relationships of several cardiotonic steroids with respect to inhibition of ion transport in frog muscle

Cesium uptake by sodium-loaded frog sartorius muscles was inhibited 100% by 10^-6 M ouabain and 10^-6 M cymarin. The doses for 50% inhibition of cesium uptake by five cardiotonic aglycones were 1.5 x 10^-6 M for strophanthidin, 2 x 10^-7 M for telocinobufagin, 1.6 x 10^-6 for digitoxigenin, 2.4 x 10^-6 M for periplogenin, and 6.3 x 10^-6 M for uzarigenin. Because of the limited solubility of sarmentogenin the maximum concentration studied was 2 x 10^-6 M which inhibited cesium uptake about 36%. Inhibition of cesium uptake by cymarin was not reversed during a 3.5 hr incubation in fresh solution while the muscles treated with ouabain and strophanthidin recovered partly during this time. Cymarin was a more potent inhibitor of sodium efflux than strophanthidin and periplogenin was less potent. Increased cesium ion concentration in the external solution decreased the strophanthidin inhibition of cesium uptake but 25 mM cesium did not overcome the inhibition by 10^-8-10^-6 M strophanthidin. Increased potassium ion concentration in the external solution decreased but did not completely overcome inhibition of sodium efflux by strophanthidin. It is concluded that potassium or cesium ions do not compete with these drugs for a particular site on the ion transport complex. The same structural features of the drugs are necessary for inhibition of ion transport in frog muscle as are required for inhibition of ion transport in other tissues, inhibition of sodium-potassium-stimulated ATPases, and toxicity to animals.     

Electrical properties and excitation-contraction coupling in skeletal muscle treated with ethylene glycol

The contractility of the frog sartorius muscle was suppressed after treatment with a Ringer solution added with ethylene glycol (EGR). No contraction was elicited by nerve stimulation when the muscle was brought back to normal Ringer solution after having been soaked in 876 mM EGR for 4 hr or in 1095 mM EGR for 2 hr. However, the action potential of normal amplitude was generated and followed by a depolarizing afterpotential. The resting membrane potential was slightly decreased from the mean normal value of –91.1 mv to –78.8 mv when 1095 mM EGR was used, and to –82.3 mv when 876 mM EGR was used, but remained almost constant for as long as 2 hr. The afterpotential that follows a train of impulses and a slow change in membrane potential produced by a step hyperpolarizing current (so-called "creep") were suppressed after treatment with ethylene glycol. The specific membrane capacity decreased to about 50% of the control values while the specific membrane resistance increased to about twice the control values Therefore, the membrane time constant remained essentially unchanged. The water content of the muscle decreased by about 30% during a 2 hr immersion in 1095 mM EGR, and increased by about 30% beyond the original control level after bringing the muscle back to normal Ringer. The intracellular potassium content did not change significantly during these procedures. Some differences between the present results and those obtained with glycerol are discussed.