A heterogeneous electrophysiological profile of bone marrow-derived mast cells

Electrophysiological properties of mouse bone marrow-derived mast cells (BMMC) were studied under the whole-cell clamp configuration. About one third of the cells were quiescent, but others expressed either inward or outward currents. Inwardly rectifying (IR) currents were predominant in 14% of the cells, and outwardly rectifying (OR) currents in 24%. The rest (22%) of the cells exhibited both inward and outward currents. The IR currents were eliminated by 1 mm Ba²⁺, and were partially inhibited by 100 μm quinidine. The reversal potential was dependent on extracellular K⁺, thereby indicating that K⁺ mediated the IR currents. The negative conductance region was seen at potentials positive to E _K. The OR currents did not apparently depend on the extracellular K⁺ concentration, but were reduced by lowering the extracellular Cl^? concentration. The OR currents were partially blocked by 1 mm Ba²⁺, and were further blocked by a Cl^? channel blocker, 4,4′-diisothiocyano-2, 2′-stilbenedisulfonate (DIDS). In addition, the reversal potential of the OR currents was positively shifted by decreasing the ratio of external and internal Cl^? concentrations, suggesting that Cl^? was a major ion carrier. In cells exhibiting IR currents, the membrane potential varied among cells and tended to depolarize by elevating the external K⁺ concentration. In cells with OR currents, the resting potential was hyperpolarized in association with an increase in conductance. These results suggest that BMMC have a heterogeneous electrophysiological profile that may underlie a variety of ion channels expressed in different phenotypes of mast cells. Activities of both the inwardly rectifying K⁺ channel and the outwardly rectifying Cl^? channel seem to contribute to the regulation of the membrane potential.     

Tolbutamide-sensitive potassium conductance in the basolateral membrane of A6 cells

K⁺ channels sensitive to intracellular ATP (K_ATP channels) have been described in a number of cell types and are selectively inhibited by sulfonylurea drugs. To look for the presence of this type of K⁺ channel in the basolateral membrane of tight epithelia, we have used an amphibian renal cell line, the A6 cells, grown on filters. After the selective permeabilization of the apical membrane with amphotericin B, the basolateral conductance was studied under voltage-clamp conditions. Tolbutamide inhibited 65.8 ± 6.3% of the barium-sensitive current. The tolbutamide-sensitive conductance had an equilibrium potential of ?83 ± 1 mV and was inward rectifying in spite of the outwardly directed K⁺ gradient. Similar results were obtained with glibenclamide. The half-inhibition constants were 25.7 ± 3.0 μm and 0.114 ± 0.018 μm for tolbutamide and glibenclamide respectively. To study the relation between cellular ATP and the activity of this conductance, A6 cells were treated with glucose (5 mm) and insulin (250 μU/ml). This maneuver significantly increased the cellular ATP and abolished the tolbutamide-sensitive conductance. A sulfonylurea-sensitive K⁺ conductance is present and active in the basolateral membrane of A6 cells. This conductance appears to be modulated by physiological changes of intracellular ATP.     

Protein kinase A-regulated Cl− channel in ML-1 human hematopoietic myeloblasts

Using the inside-out patch clamp technique, we identified a Cl^? channel in patches from the membrane of cultured human hematopoietic myeloblastic leukemia ML-1 cells. The Cl^? channel was not seen at negative membrane potentials in excised patches until the membrane potential was depolarized to greater than +40 mV. The channel was also activated by addition of cAMP-dependent protein kinase (PKA) catalytic subunit at physiological membrane potential (?40 mV). Biophysical studies of the Cl^? channel revealed that the current-voltage (I-V) relationship of the Cl^? channel was outwardly rectifying in symmetrical 142 mm Cl^? solutions. Single channel conductances were 48 pS for the outward current measured at +60 mV and 27 pS for the inward current at ?60 mV. The open time constant of the channel was dependent on the membrane potential and was significantly prolonged at positive membrane potentials. Channels activated by cAMP-dependent protein kinase spent a significantly longer time in the open state compared to those channels activated by depolarization pulses. Pharmacological properties of the Cl^? channel were also studied. Two anion transport inhibitors, anthracene-9-carboxylic acid (9-AC) and 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) caused a flickering block of the channel. Half-inhibitory concentrations (IC⁵⁰) for 9-AC and DIDS were 174 ± 20 and 70±16 μm, respectively. Blockade of the Cl^? channel by 9-AC or DIDS was completely reversible. Our findings suggest that outwardly rectifying Cl^? channels (ORCC) are present in human hematopoietic myeloblasts. The function of ORCC may be involved in hormone-regulated cell growth, cell volume regulation and immune responses.     

Small-conductance chloride channels in human peripheral T lymphocytes

During whole-cell patch-clamp recording from normal (nontransformed) human T lymphocytes a chloride current spontaneously activated in >98% of cells (n > 200) in the absence of applied osmotic or pressure gradients. However, some volume sensitivity was observed, as negative pressure pulses reduced the current. With iso-osmotic bath and pipette solutions the peak amplitude built up (time constant ≈23 sec at room temperature), a variable-duration plateau phase followed, then the current ran down spontaneously (time constant ≈280 sec). The anion permeability sequence, calculated from reversal potentials was I^?, Br^? > NO ₃ ^? , Cl^? > CH₃SO ₃ ^? , HCO ₃ ^? > CH₃COO^? > F^? > aspartate, gluconate, SO ₄ ^2? and there was no measurable monovalent cation permeability. The Cl^? current was independent of time during long voltage steps and there was no evidence of voltage-dependent gating; however, the current showed intrinsic outward rectification in symmetrical Cl^? solutions. The conductance of the channels underlying the whole-cell current was calculated from fluctuation analysis, using power-spectral density and variance-vs.-mean analysis. Both methods yielded a single channel conductance of about 0.6 pS at ?70 mV (close to the normal resting potential of T lymphocytes). The power spectral density function was best fit by the sum of two Lorentzian functions, with corner frequencies of 30 and 295 Hz, corresponding to mean open times of 0.54 and 5.13 msec. The pharmacological profile included rapid block by external application of flufenamic acid (50 μm), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 100 μm, [6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5-y1) oxy] acetic acid (IAA-94, 250 μm) or 100 μm 1,9-dideoxyforskolin. The stilbene derivatives DIDS (4,4′-diisothiocyano-2,2′ di-sulphonic acid stilbene, 500 μm) and SITS (4-acetamido-4′-isothiocyano-2, 2′-disulphonic acid stilbene, 500 μm) prevented buildup of Cl^? current after a 30-min preincubation at 500 μm. When tested in a mitogenic assay, DIDS, flufenamic acid, NPPB and IAA-94 all inhibited T-cell proliferation, suggesting a physiological function in addition to the observed volume sensitivity.     

Concentration-dependent modulations of potassium and calcium currents of rat osteoblastic cells by arachidonic acid

We show that the voltage-gated K⁺ and Ca²⁺ currents of rat osteoblastic cells are strongly modulated by arachidonic acid (AA), and that these modulations are very sensitive to the AA concentration. At 2 or 3 μm, AA reduces the amplitude and accelerates the inactivation of the K⁺ current activated by depolarization; at higher concentrations (≥5 μm), AA still blocks this K⁺ current, but also induces a very large noninactivating K⁺ current. At 2 or 3 μm, AA enhances the T-type Ca²⁺ current, close to its threshold of activation, whereas at 10 μm, it blocks that current. AA (1–10 μm) also blocks the dihydropyridine-sensitive L-type Ca²⁺ current. Thus, the effect of AA on Ca²⁺ entry through voltage-gated Ca²⁺ channels can change qualitatively with the AA concentration: at 2 or 3 μm, AA will favor Ca²⁺ entry through T channels, both by lowering the voltage-gated K⁺ conductance and by increasing the T current, whereas at 10 μm, AA will prevent Ca²⁺ entry through voltage-gated Ca²⁺ channels, both by inducing a K⁺ conductance and by blocking Ca²⁺ channels.     

K+-dependent 3H-d-glucose transport by hepatopancreatic brush border membrane vesicles of a marine shrimp

The effects of sodium, potassium, sugar inhibitors, and membrane potential on ³H-d-glucose uptake by hepatopancreatic epithelial brush border membrane vesicles (BBMV) of the Atlantic marine shrimp, Litopenaeus setiferus, were investigated. Brush border membrane vesicles were prepared using a MgCl₂/EGTA precipitation method and uptake experiments were conducted using a high speed filtration technique. ³H-d-Glucose uptake was stimulated by both sodium and potassium and these transport rates were almost doubled in the presence of an inside-negative-induced membrane potential. Kinetics of ³H-d-glucose influx were hyperbolic functions of both external Na⁺ or K⁺, and an induced membrane potential increased influx J _max and lowered K_m in both salts. ³H-d-Glucose influx versus [glucose] in both Na⁺ or K⁺ media also displayed Michaelis–Menten properties that were only slightly affected by induced membrane potential. Phloridzin was a poor inhibitor of 0.5 mM ³H-d-glucose influx, requiring at least 5 mM in NaCl and 10 mM in KCl to significantly reduce hexose transport. Several sugars (d-galactose, α-methyl-d-gluco-pyranoside, unlabeled d-glucose, d-fructose, and d-mannose) were used at 75 mM as potential inhibitors of 0.1 mM ³H-d-glucose influx. Only unlabeled d-glucose, d-fructose, and d-mannose significantly (p < 0.05) reduced labeled glucose transport. An additional experiment using increasing concentrations of d-mannose (0, 10, 25, 75, and 100 mM) showed this hexose to be an effective inhibitor of 0.1 mM ³H-d-glucose uptake at concentrations of 75 mM and higher. As a whole these results suggest that ³H-d-glucose transport by hepatopancreatic BBMV occurs by a carrier system that is able to use both Na⁺ and K⁺ as drivers, is enhanced by membrane potential, is relatively refractory to phloridzin, and is only inhibited by itself, d-fructose, and d-mannose. These properties are similar to those exhibited by the mammalian SLC5A9/SGLT4 transporter, suggesting that an invertebrate analogue of this protein may occur in shrimp.     

Cyclic tetrapeptides with –SS– bridging between amino acid side chains for potent histone deacetylases’ inhibition

Cyclic depsipeptide FK228 with an intramolecular disulfide bond is a potent inhibitor of histone deacetylases (HDAC). FK228 is stable in blood because of its prodrug function, whose –SS– bond is reduced within the cell. Here, cyclic peptides with –SS– bridges between a variety of amino acids were synthesized and assayed for HDAC inhibition. Cyclic peptide 3, cyclo(-l-amino acid-l-amino acid-l-Val-d-Pro-), with an –SS– bridge between the first and second amino acids, was found to be a potent HDAC inhibitor. Cyclic peptide 7, cyclo(-l-amino acid-d-amino acid-l-Val-d-Pro-), with an –SS– bridge between the first and second amino acids, was also a potent HDAC inhibitor.     

Activation of the human red cell calcium ATPase by calcium pretreatment

Some kinetic parameters of the human red cell Ca²⁺-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37°C. After 30 min treatment with EGTA(1 mm) plus dithioerythritol (1 mm), a V _max of about 0.4 μmol P_i/mg × hr and a K _s of 0.3 μm Ca²⁺ were found. When Mg²⁺ (10 mm) or Ca²⁺(10 μm) were also added during preincubation, V _maxbut not Kwas altered. Ca²⁺ was more effective than Mg²⁺, thus increasing V _max to about 1.3 μmol P_i/mg × hr. The presence of both Ca²⁺ and Mg²⁺ during pretreatment decreasedKto 0.15 μm, while having no apparent effect on V _max. Conversely, addition of ATP (2 mm) with either Ca²⁺ or Ca²⁺ plus Mg²⁺increased V_max without affecting K. Preincubation with Ca²⁺ for periods longer than 30 min further increased V_maxand reduced Kto levels as low as found with calmodulin treatment. The Ca²⁺ activation was not prevented by adding proteinase inhibitors (iodoacetamide, 10 mm; leupeptin, 200 μm; pepstatinA, 100 μm; phenylmethanesulfonyl fluoride, 100 μm). The electrophoretic pattern of membranes preincubated with or without Mg²⁺, Ca²⁺ or Ca²⁺ plus Mg²⁺ did not differ significantly from each other. Moreover, immunodetection of Ca²⁺-ATPase by means of polyclonal antibodiesrevealed no mobility change after the various treatments. The above stimulation was not altered by neomycin (200 μm), washing with EGTA (5 mm) or by both incubating and washing with delipidized serum albumin (1 mg/ml), or omitting dithioerythritol from the preincubation medium. On the other hand, the activation elicited by Ca²⁺ plus ATP in the presence of Mg²⁺ was reduced 25–30% by acridine orange (100 μm), compound 48/80 (100 μm) or leupeptin (200 μm) but not by dithio-bis-nitrobenzoic acid (1 mm). The fluorescence depolarization of 1,6-diphenyl-and l-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating under the different conditions. The results show that proteolysis, fatty acid production, an increased phospholipid metabolism or alteration of membrane fluidity are not involved in the Ca²⁺ effect. Ca²⁺ preincubation may stimulate the Ca²⁺-ATPase activity by stabilizing or promoting the E₁ conformation.     

Effects of membrane potential on just detectable movement in rat skeletal muscle: Effects of denervation

The potential, Vt, at which a brief test depolarization first elicited movement was determined using two-microelectrode point voltage clamp. We expected that inactivation of excitation-contraction coupling at conditioning potentials between ?60 and 0 mV would shift Vt to more positive potentials, and that fibers would become inactivatable with less conditioning depolarization in EDL than soleus. The curve relating Vt to conditioning potential had a negative slope (which was insensitive to addition of 1 mm cobalt or replacement of calcium with 20 mm CaEGTA) between ?60 and ?35 mV and a steep positive slope with further depolarization. Unexpectedly, fibers became inactivatable with less conditioning depolarization in soleus than in EDL when Vt was measured with 50 msec test pulses. However, the positive shift in Vt became less steep as test pulse duration lengthened in soleus fibers. When Vt obtained with test pulses approaching rheobase (10 msec in EDL and 500 msec in soleus) was compared, EDL fibers became inactive with less conditioning depolarization than soleus fibers. The increase in Vt became steeper with 1 mm cobalt or 20 mm CaEGTA and was shifted to more positive potentials by denervation in soleus fibers. We conclude that inactivation (i) does not strongly influence threshold contractions at conditioning potentials between ?60 and ?40 mV and (ii) influences Vt between ?40 and 0 mV in a manner that depends on test pulse duration.     

Characterization of a recombinant l-rhamnose isomerase from Bacillus subtilis and its application on production of l-lyxose and l-mannose

The gene of an l-rhamnose isomerase (RhaA) from Bacillus subtilis was cloned to the pET28a(+) and then expressed in the E. coli ER2566. The expressed enzyme was purified with a specific activity of 3.58 U/mg by His-Trap affinity chromatography. The recombinant enzyme existed as a 194 kDa tetramer and the maximal activity was observed at pH 8.0 and 60°C. The RhaA displayed activity for l-rhamnose, l-lyxose, l-mannose, d-allose, d-gulose, d-ribose, and l-talose, among all aldopentoses and aldohexoses and it showed enzyme activity for l-form monosaccharides such as l-rhamnose, l-lyxose, l-mannose, and l-talose. The catalytic efficiency (k _cat/K _m) of the recombinant enzyme for l-rhamnose, l-lyxose, and l-mannose were 7,460, 1,013, and 258 M/sec. When l-xylulose 100 g/L and l-fructose 100 g/L were used as substrates, the optimum concentrations of RpiB were determined with 6 and 15 U/mL, respectively. The l-lyxose 40 g/L was produced from l-xylulose 100 g/L by the enzyme during 60 min, while l-mannose 25 g/L was produced from l-fructose 100 g/L for 80 min. The results suggest that RhaA from B. subtilis is a potential producer of l-form monosaccharides.     

Function of aspartic acid residues in optimum pH control of l-arabinose isomerase from Lactobacillus fermentum

l-Arabinose isomerase (l-AI) catalyzes the isomerization of l-arabinose to l-ribulose and d-galactose to d-tagatose. Most reported l-AIs exhibit neutral or alkaline optimum pH, which is less beneficial than acidophilic ones in industrial d-tagatose production. Lactobacillus fermentum l-AI (LFAI) is a thermostable enzyme that can achieve a high conversion rate for d-galactose isomerization. However, its biocatalytic activity at acidic conditions can still be further improved. In this study, we report the single- and multiple-site mutagenesis on LFAI targeting three aspartic acid residues (D268, D269, and D299). Some of the lysine mutants, especially D268K/D269K/D299K, exhibited significant optimum pH shifts (from 6.5 to 5.0) and enhancement of pH stability (half-life time increased from 30 to 62 h at pH 6.0), which are more favorable for industrial applications. With the addition of borate, d-galactose was isomerized into d-tagatose by D268K/D269K/D299K at pH 5.0, resulting in a high conversion rate of 62 %. Based on the obtained 3.2-Å crystal structure of LFAI, the three aspartic acid residues were found to be distant from the active site and possibly did not participate in substrate catalysis. However, they were proven to possess similar optimum pH control ability in other l-AI, such as that derived from Escherichia coli. This study sheds light on the essential residues of l-AIs that can be modified for desired optimum pH and better pH stability, which are useful in d-tagatose bioproduction.     

Uptake of amino acids by actidione-treated yeast cells

The active uptake ofl-aspartic acid, glycine andl-lysine by actidione-treated cells ofSaccharomyces cerevisiae was found to be inhibited by anaerobic conditions in the absence of a source of energy, only facilitated diffusion persisting. Similarly, metabolic inhibitors (iodoacetamide, sodium fluoride and potassium sorbate) inhibited the uptake very substantially. 2,4-Dinitrophenol and sodium azide appeared to inhibit the movement of the transport carrier itself, while uranyl ions showed a complex interaction pattern, ranging from inhibition at concentrations of 10^?6–10^?4 m, to stimulation at concentrations of 3×10^?4–10^?3 m, to pronounced inhibition at higher concentrations. The uptake was pH-dependent with optima forl-aspartic acid near pH 4, for glycine near pH 5, forl-lysine near pH 6.5.     

Karyological and taxonomic observations onDracocephalum foetidum Bunge andKoenigia islandica L.

Chromosome numbers are reported for two Mongolian species,Dracocephalum foetidum Bunge (2n=12) andKoenigia islandica L. (2n=14). The relationship ofD. foetidum toD. moldavica L. (2n=10) and some patterns of phenotypic variation inK. islandica are briefly discussed. The following new combinations are proposed:K. cyanadra (Diels) Měsí?ek etSoják,K. forrestii (Diels) Měsí?ek etSoják,K. hubertii (Lingelsh.) Měsí?ek etSoják, andK. nummularifolia (Meisn.) Měsí?ek etSoják.     

Effects of Tryptophan and 5-Hydroxytryptophan on the Hepatic Cell Membrane Rigidity Due to Oxidative Stress

The ability of several indoleamines to scavenge free radicals is well documented. Our aim was to evaluate the ability of 0.01–3 mm tryptophan (Trp) and 0.1–5 mm 5-hydroxytryptophan (5-OH-Trp) to protect hepatic cell membranes against 0.1 mm FeCl₃ plus 0.1 mm ascorbic acid–induced lipid peroxidation and increases in membrane rigidity. Membrane fluidity was evaluated using fluorescence spectroscopy. Lipid and protein oxidation were estimated by quantifying malondialdehyde (MDA) plus 4-hydroxyalkenals (4-HDA) concentrations and carbonyl group content, respectively. Exposure to FeCl₃ plus ascorbic acid increased hepatic cell membrane rigidity, MDA + 4-HDA and carbonyl content. The presence of 5-OH-Trp, but not Trp, attenuated these changes. In the absence of oxidative stress, neither indoleamine modified fluidity, MDA + 4-HDA or carbonylation. These results suggest that C5 hydroxylation determines the ability of Trp to preserve membrane fluidity in the presence of oxidative stress.     

Chromosome counts of some Cuban Angiosperms

Chromosome numbers are reported for 14 species collected in Cuba. The first chromosome records are reported forAlbizzia cubana Britton etWilson (2n=26),Atkinsia cubensis (Britton etWilson)Howard (2n=26),Caesalpinia violacea (Mill.)Standl. (2n=24),Colubrina ferruginosa Brongn. (2n=24). Chromosome numbers of the following species were confirmed:Albizzia lebbeck (L.)Benth. (2n=26),Canavalia maritima (Aubl.)Thouars (2n=22),Casuarina equisetifolia Forst. (2n=18),Cedrela mexicana M.J. Roem. (2n=56),Delonix regia (Bojer)Raf. (2n=28),Guazuma tomentosa H.B.K. (2n=16),Lysiloma bahamense Benth. (2n=26),L. latisiliqua (L.)Benth. (2n=26),Samanea saman (Jacq.)Merrill (2n=26),Thespesia populnea (L.)Soland (2n=26).     

Simultaneous electrochemical determination of l-cysteine and l-cysteine disulfide at carbon ionic liquid electrode

A linear sweep voltammetric method is used for direct simultaneous determination of l-cysteine and l-cysteine disulfide (cystine) based on carbon ionic liquid electrode. With carbon ionic liquid electrode as a high performance electrode, two oxidation peaks for l-cysteine (0.62 V) and l-cysteine disulfide (1.3 V) were observed with a significant separation of about 680 mV (vs. Ag/AgCl) in phosphate buffer solution (pH 6.0). The linear ranges were obtained as 1.0–450 and 5.0–700 μM and detection limits were estimated to be 0.298 and 4.258 μM for l-cysteine and l-cysteine disulfide, respectively. This composite electrode was applied for simultaneous determination of l-cysteine and l-cysteine disulfide in two real samples, artificial urine and nutrient broth. Satisfactory results were obtained which clearly indicate the applicability of the proposed electrode for simultaneous determination of these compounds in complex matrices.     

Notes on some African hepatic genera 1–5

The present study deals with five genera of hepatics in Africa, Isotachis Mitt., Anastrophyllum (Spruce) Steph., Tritomaria Schiffn. ex Loeske, Gymnocoleopsis (Schust.) Schust. and Lophozia (Dum.) Dum. All African populations of the genus Isotachis Mitt. are considered to be one species, I. aubertii (Schwaegr.) Mitt. Four species of Anastrophyllum (Spruce) Steph. (s.l.), A. auritum (Lehm.) Steph., A. piligerum (Nees) Spruce, A. subcomplicatum (Lehm. et Lindenb.) Steph. and A. minutum (Schreb.) Schust., and two species of Tritomaria Schiffn. et Loeske, T. camerunensis S. Arnell and T. exsecta (Schrad.) Schiffn. ex Loeske occur in Africa. Gymmocoleopsis multiflora (Steph.) Schust. represents a genus and species hitherto unreported for the African flora. Finally, five Lophozia (Dum.) Dum. species, L. argentina (Steph.) Schust., L. capensis S. Arnell, L. decolorans (Limpr.) Steph., L. hedbergii S. Arnell and L. tristaniana (S. Arnell) Váňa, are reported from central and southern Africa; two of these (L. argentina (Steph.) Schust. and L. decolorans (Limpr.) Steph.) represent the first reports from Africa.     

Tres Lichenes novi Hawaiienses

Diagnoses of three corticolous species of Discolichenes from Hawaii are presented:Bacidia violascens K. Kalb etVězda sp. n.,Dimerella degeneri K. Kalb etVězda sp. n. andD. frederici K. Kalb sp. n. The isotypes will be distributed in fasc. No. 70 of the “Lichenes selecti exsiccati” (issued by the Botanical Institute, Czechoslovak Academy of Sciences, Pr?honice near Praha).