Single-channel properties of a stretch-sensitive chloride channel in the human mast cell line HMC-1

A stretch-activated (SA) Cl⁻ channel in the plasma membrane of the human mast cell line HMC-1 was identified in outside-out patch-clamp experiments. SA currents, induced by pressure applied to the pipette, exhibited voltage dependence with strong outward rectification (55.1 pS at +100 mV and an about tenfold lower conductance at −100 mV). The probability of the SA channel being open (P _o) also showed steep outward rectification and pressure dependence. The open-time distribution was fitted with three components with time constants of τ_1o = 755.1 ms, τ_2o = 166.4 ms, and τ_3o = 16.5 ms at +60 mV. The closed-time distribution also required three components with time constants of τ_1c = 661.6 ms, τ_2c = 253.2 ms, and τ_3c = 5.6 ms at +60 mV. Lowering extracellular Cl⁻ concentration reduced the conductance, shifted the reversal potential toward chloride reversal potential, and decreased the P _o at positive potentials. The SA Cl⁻ currents were reversibly blocked by the chloride channel blocker 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) but not by (Z)-1-(p-dimethylaminoethoxyphenyl)-1,2-diphenyl-1-butene (tamoxifen). Furthermore, in HMC-1 cells swelling due to osmotic stress, DIDS could inhibit the increase in intracellular [Ca²⁺] and degranulation. We conclude that in the HMC-1 cell line, the SA outward currents are mediated by Cl⁻ influx. The SA Cl⁻ channel might contribute to mast cell degranulation caused by mechanical stimuli or accelerate membrane fusion during the degranulation process.     

Modulation of the Calmodulin-induced Inhibition of Sarcoplasmic Reticulum Calcium Release Channel (Ryanodine Receptor) by Sulfhydryl Oxidation in Single Channel Current Recordings and [3H]Ryanodine Binding

The modulation of the calmodulin-induced inhibition of the calcium release channel (ryanodine receptor) by two sulfhydryl oxidizing compounds, 4-(chloromercuri)phenyl–sulfonic acid (4-CMPS) and 4,4′-dithiodipyridine (4,4′-DTDP) was determined by single channel current recordings with the purified and reconstituted calcium release channel from rabbit skeletal muscle sarcoplasmic reticulum (HSR) and [³H]ryanodine binding to HSR vesicles. 0.1 μm CaM reduced the open probability (P _o ) of the calcium release channel at maximally activating calcium concentrations (50–100 μm) from 0.502 ± 0.02 to 0.137 ± 0.022 (n= 28), with no effect on unitary conductance. 4-CMPS (10–40 μm) and 4,4′-DTDP (0.1–0.3 mm) induced a concentration dependent increase in P _o (> 0.9) and caused the appearance of longer open states. CaM shifted the activation of the calcium release channel by 4-CMPS or 4,4′-DTDP to higher concentrations in single channel recordings and [³H]ryanodine binding. 40 μm 4-CMPS induced a near maximal (P _o > 0.9) and 0.3 mm 4,4′-DTDP a submaximal (P _o = 0.74) channel opening in the presence of CaM, which was reversed by the specific sulfhydryl reducing agent DTT. Neither 4-CMPS nor 4,4′-DTDP affected Ca-[¹²⁵I]calmodulin binding to HSR. 1 mm MgCl₂ reduced P _o from 0.53 to 0.075 and 20–40 μm 4-CMPS induced a near maximal channel activation (P _o > 0.9). These results demonstrate that the inhibitory effect of CaM or magnesium in a physiological concentration is diminished or abolished at high concentrations of 4-CMPS or 4,4′-DTDP through oxidation of activating sulfhydryls on cysteine residues of the calcium release channel. Received: 22 July 1999/Revised: 15 November 1999     

Activation of the Cardiac Ryanodine Receptor by Sulfhydryl Oxidation is Modified by Mg2+ and ATP

The reactive disulfide 4,4′-dithiodipyridine (4,4′DTDP) was added to single cardiac ryanodine receptors (RyRs) in lipid bilayers. The activity of native RyRs, with cytoplasmic (cis) [Ca²⁺] of 10⁻⁷ m (in the absence of Mg²⁺ and ATP), increased within ∼1 min of addition of 1 mm 4,4′-DTDP, and then irreversibly ceased 5 to 6 min after the addition. Channels, inhibited by either 1 mm cis Mg²⁺ (10⁻⁷ m cis Ca²⁺) or by 10 mm cis Mg²⁺ (10⁻³ m cis Ca²⁺), or activated by 4 mm ATP (10⁻⁷ m cis Ca²⁺), also responded to 1 mm cis 4,4′-DTDP with activation and then loss of activity. P _o and mean open time (T _o ) of the maximally activated channels were lower in the presence of Mg²⁺ than in its absence, and the number of openings within the long time constant components of the open time distribution was reduced. In contrast to the reduced activation by 1 mm 4,4′-DTDP in channels inhibited by Mg²⁺, and the previously reported enhanced activation by 4,4′-DTDP in channels activated by Ca²⁺ or caffeine (Eager et al., 1997), the activation produced by 1 mm cis 4,4′-DTDP was the same in the presence and absence of ATP. These results suggest that there is a physical interaction between the ATP binding domain of the cardiac RyR and the SH groups whose oxidation leads to channel activation. Received: 8 September 1997/Revised: 20 January 1998     

Effects of physiological integration on photosynthetic efficiency of <Emphasis Type="Italic">Trifolium repens</Emphasis> in response to heterogeneous UV-B radiation

Several studies have found the photosynthetic integration in clonal plants to response to resource heterogeneity, while little is known how it responses to heterogeneity of UV-B radiation. In this study, the effects of heterogeneous UV-B radiation (280–315 nm) on gas exchange and chlorophyll fluorescence of a clonal plant Trifolium repens were evaluated. Pairs of connected and severed ramets of the stoloniferous herb T. repens were grown under the homogeneity (both of ramets received only natural background radiation, ca. 0.6 kJ m⁻² d⁻¹) and heterogeneity of UV-B radiation (one of the ramet received only natural background radiation and the other was exposed to supplemental UV-B radiation, 2.54 kJ m⁻² d⁻¹) for seven days. Stomatal conductance (g _s), intercellular CO₂ concentration (C _i) and transpiration rate (E) showed no significant differences in connected and severed ramets under homogenous and heterogeneous UV-B radiation, however, net photosynthetic rate (P _N) and maximum photosynthetic rate (P _max) of ramets suffered from supplemental increased UV-B radiation and that of its connected sister ramet decreased significantly. Moreover, additive UV-B radiation resulted in a notable decrease of the minimal fluorescence of dark-adapted state (F_o), the electron transport rate (ETR) and photochemical quenching coefficient (q_P) and an increase of nonphotochemical quenching (NPQ) under supplemental UV-B radiation, while physiological connection reverse the results. In all, UV-B stressed ramets could benefit from unstressed ramets by physiological integration in photosynthetic efficiency, and clonal plants are able to optimize the efficiency to maintain their presence in less favourable sites.     

Purification and characterization of UDP-glucose: anthocyanin 3′,5′-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase from <Emphasis Type="Italic">Clitoria ternatea</Emphasis>

A UDP-glucose: anthocyanin 3′,5′-O-glucosyltransferase (UA3′5′GT) (EC 2.4.1.-) was purified from the petals of Clitoria ternatea L. (Phaseoleae), which accumulate polyacylated anthocyanins named ternatins. In the biosynthesis of ternatins, delphinidin 3-O-(6″-O-malonyl)-β-glucoside (1) is first converted to delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′-O-β-glucoside (2). Then 2 is converted to ternatin C5 (3), which is delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′,5′-di-O-β-glucoside. UA3′5′GT is responsible for these two steps by transferring two glucosyl groups in a stepwise manner. Its substrate specificity revealed the regioselectivity to the anthocyanin′s 3′- or 5′-OH groups. Its kinetic properties showed comparable k _cat values for 1 and 2, suggesting the subequality of these anthocyanins as substrates. However, the apparent K _m value for 1 (3.89 × 10⁻⁵ M), which is lower than that for 2 (1.38 × 10⁻⁴ M), renders the k _cat/K _m value for 1 smaller, making 1 catalytically more efficient than 2. Although the apparent K _m value for UDP-glucose (6.18 × 10⁻³ M) with saturated 2 is larger than that for UDP-glucose (1.49 × 10⁻³ M) with saturated 1, the k _cat values are almost the same, suggesting the UDP-glucose binding inhibition by 2 as a product. UA3′5′GT turns the product 2 into a substrate possibly by reversing the B-ring of 2 along the C2-C1′ single bond axis so that the 5′-OH group of 2 can point toward the catalytic center. K. Kogawa, N. Kato, K. Kazuma, and N. Noda contributed equally to this work.     

Characterization of an ecto-ATPase activity in Fonsecaea pedrosoi

In this work, we characterized an ecto-ATPase activity in intact mycelial forms of Fonsecaea pedrosoi, the primary causative agent of chromoblastomycosis. In the presence of 1 mM EDTA, fungal cells hydrolyzed adenosine-5′-triphosphate (ATP) at a rate of 84.6 ± 11.3 nmol Pi h⁻¹ mg⁻¹ mycelial dry weight. The ecto-ATPase activity was increased at about five times (498.3 ± 27.6 nmol Pi h⁻¹ mg⁻¹) in the presence of 5 mM MgCl₂, with values of V _max and apparent K _m for Mg-ATP²⁻corresponding to 541.9 ± 48.6 nmol Pi h⁻¹ mg⁻¹ cellular dry weight and 1.9 ± 0.2 mM, respectively. The Mg²⁺-stimulated ecto-ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A₁ (V-ATPases)_, and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate, and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The surface of the Mg²⁺-stimulated ATPase in F. pedrosoi was confirmed by assays in which 4,4′-diisothiocyanostylbene-2,2′-disulfonic acid (DIDS), a membrane impermeant inhibitor, and suramin, an inhibitor of ecto-ATPase and antagonist of P₂ purinoreceptors. Based on the differential expression of ecto-ATPases in the different morphological stages of F. pedrosoi, the putative role of this enzyme in fungal biology is discussed.     

Ethidium bromide binding to native and denatured poly(dA)poly(dT)

Isotherms of the EtBr adsorption on native and denatured poly(dA)poly(dT) in the temperature interval 20–70°C were obtained. The EtBr binding constants and the number of binding sites were determined. The thermodynamic parameters of the EtBr intercalation complex upon changes of solution temperature 20–48°C were calculated: 1.0·10⁶ M⁻¹≤K≤1.4·10⁶ M⁻¹, free energy ΔG ^o=−8.7±0.3 kcal/mol, enthalpy ΔH ^o≅0, and entropy ΔS ^o=28±0.5 cal/(mol deg). UV melting has shown that the melting temperature (T _m) of EtBr-poly(dA)poly(dT) complexes (μ=0.022,4.16·10⁻⁵ M EtBr) increased by 17°C as compared with the ΔT _m of free homopolymer, whereas the half-width of the transition (T _m) is not changed. It was shown for the first time that EtBr forms complexes of two types on single-stranded regions of poly(dA)poly(dT) denatured at 70°C: strong (K ₁=1.7·10⁵ M⁻¹; ΔG ^o=−8.10±0.03 kcal/mol) and weak (K ₂=2.9·10³ M⁻¹; ΔG ^o=−6.0±0.3 kcal/mol).The ΔG ^o of the strong and weak complexes was independent of the solution ionic strength, 0.0022≤μ≤0.022. A model of EtBr binding with single-stranded regions of poly(dA)poly(dT) is discussed.     

Chloride Currents Activated by Calcitonin and cAMP in Primary Cultures of Rabbit Distal Convoluted Tubule

Chloride (Cl⁻) conductances were studied in primary cultures of the bright part of rabbit distal convoluted tubule (DCTb) by the whole cell patch clamp technique. The bath solution (33°C) contained (in mm): 140 NaCl, 1 CaCl₂, 10 N-2-hydroxy-ethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4 and the pipette solution 140 N-methyl-d-glucamine (NMDG)-Cl, 5 MgATP, 1 ethylene-glycol-bis(b-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 10 HEPES, pH 7.4. We identified a Cl⁻ current activated by 10⁻⁵ m forskolin, 10⁻³ m 8-bromo adenosine 3′,5′-cyclic monophophosphate (8 Br-cAMP), 10⁻⁶ m phorbol 12-myristate 13-acetate (PMA), 10⁻³ m intracellular adenosine 3′,5′-cyclic monophophosphate (cAMP) and 10⁻⁷ m calcitonin. The current-voltage relationship was linear and the relative ion selectivity was Br⁻ > Cl⁻≫ I⁻ > glutamate. This current was inhibited by 10⁻³ m diphenylamine-2-carboxylate (DPC) and 10⁻⁴ m 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and was insensitive to 10⁻³ m 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). These characteristics are similar to those described for the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ conductance. In a few cases, forskolin and calcitonin induced an outwardly rectifying Cl⁻ current blocked by DIDS. To determine the exact location of the Cl⁻ conductance 6-methoxy-1-(3-sulfonatopropyl) quinolinium (SPQ) fluorescence experiments were carried out. Cultures seeded on collagen-coated permeable filters were loaded overnight with 5 mm SPQ and the emitted fluorescence analyzed by laser-scan cytometry. Cl⁻ removal from the apical solution induced a Cl⁻ efflux which was stimulated by 10⁻⁵ m forskolin, 10⁻⁷ calcitonin and inhibited by 10⁻⁵ m NPPB. In 140 mm NaBr, forskolin stimulated an apical Br⁻ influx through the Cl⁻ pathway. Forskolin and calcitonin had no effect on the basolateral Cl⁻ permeability. Thus in DCTb cultured cells, exposure to calcitonin activates a Cl⁻ conductance in the apical membrane through a cAMP-dependent mechanism. Received: 5 July 1995/Revised: 21 December 1995     

Entropy budgets of deciduous plant leaves and a theorem of oscillating entropy production

From an energy budget of a deciduous plant leaf in moderate conditions, entropy fluxes into or out of the leaf due to solar radiation, infrared radiation, evaporation of water and heat conduction are calculated. Net entropy flow into the leaf is negative. On the assumption that the entropy in the leaf is in a steady state, the entropy production in the typical deciduous leaf in moderate conditions [the solar energy absorbed by both sides of the leaf isE _solar=0.0602 (J cm⁻² s⁻¹)] becomesS _prod=1.8×10⁻⁴ (J cm⁻² s⁻¹ K⁻¹). The positiveness of the entropy production shows that the Second Law of Thermodynamics certainly holds in the plant leaf. Entropy productions in other conditions are also calculated. The entropy production in the leafS _prod becomes a linear function of the solar energy absorbed by the leafE _solar:S _prod≈-(29.5E _solar)×10⁻⁴. A theorem is presented: the entropy production in plant leaves oscillates during the period of one day, paralleling the daily solar energy absorbed by leaves.     

Effects of different irradiances on the photosynthetic process during ex-vitro acclimation of Anoectochilus plantlets

Six months old in vitro-grown Anoectochilus formosanus plantlets were transferred to ex-vitro acclimation under low irradiance, LI [60 μmol(photon) m⁻² s⁻¹], intermediate irradiance, II [180 μmol(photon) m⁻² s⁻¹], and high irradiance, HI [300 μmol(photon) m⁻² s⁻¹] for 30 d. Imposition of II led to a significant increase of chlorophyll (Chl) b content, rates of net photosynthesis (P _N) and transpiration (E), stomatal conductance (g _s), electron transfer rate (ETR), quantum yield of electron transport from water through photosystem 2 (Φ_PS2), and activity of ribulose-1,5-bisphosphate carboxylase/ oxygenase (RuBPCO, EC 4.1.1.39). This indicates that Anoectochilus was better acclimated at II compared to LI treatment. On the other hand, HI acclimation led to a significant reduction of Chl a and b, P _N, E, g _s, photochemical quenching, dark-adapted quantum efficiency of open PS2 centres (F_v/F_m), probability of an absorbed photon reaching an open PS2 reaction centre (F_v′/F_m′), ETR, Φ_PS2, and energy efficiency of CO₂ fixation (Φ_CO2/Φ_PS2). This indicates that HI treatment considerably exceeded the photo-protective capacity and Anoectochilus suffered HI induced damage to the photosynthetic apparatus. Imposition of HI significantly increased the contents of antheraxanthin and zeaxanthin (ZEA), non-photochemical quenching, and conversion of violaxanthin to ZEA. Thus Anoectochilus modifies its system to dissipate excess excitation energy and to protect the photosynthetic machinery.     

Mathematical studies of the interaction of respiratory gases with whole blood I. O2 absorption

Some progress has been made on the problem of the interaction of respiratory gases with whole blood. A practical working model for oxygen absorption in and interaction with whole blood is developed by assuming that oxygen molecules compete with protons for binding sites on the hemoglobin molecule and by invoking the Wyman-Allen (Jour. of Polymer Science,5, 499–518, 1951) hypothesis that two oxygen molecules go on the hemoglobin at one time. Extensive tests of this model against saturation measurements on blood from humans, horses, oxen and sheep are made. Values for the equilibrium constants are calculated and compared. In addition a second working model has been developed in an attempt to explain why O₂ saturation measurements when expressed as (100 percent — percent saturation) are an exponential function of oxygen partial pressure. Considerations which make plausible the following expression for saturation, [1−2e ^−γx/h1/2/(1+(1/20)(β′/h ^1/2+h ^1/2/β′))] are presented. Herex denotes oxygen tension,h denotes hydrogen ion concentration and β′ and γ are parameters.     

Large Conductance Ca2+-activated K+ Channels in the Soma of Rat Motoneurones

Properties of large conductance Ca²⁺-activated K⁺ channels were studied in the soma of motoneurones visually identified in thin slices of neonatal rat spinal cord. The channels had a conductance of 82 ± 5 pS in external Ringer solution (5.6 mm K⁺ _o //155 mm K⁺ _i ) and 231 ± 4 pS in external high-K _o solution (155 mm K⁺ _o //155 mm K⁺ _i ). The channels were activated by depolarization and by an increase in internal Ca²⁺ concentration. Potentials of half-maximum channel activation (E₅₀) were −13, −34, −64 and −85 mV in the presence of 10⁻⁶, 10⁻⁵, 10⁻⁴ and 10⁻³ m internal Ca²⁺, respectively. Using an internal solution containing 10⁻⁴ m Ca²⁺, averaged K_Ca currents showed fast activation within 2–3 msec after a voltage step to +50 mV. Averaged K_Ca currents did not inactivate during 400 msec voltage pulses. External TEA reduced the apparent single-channel amplitude with a 50% blocking concentration (IC₅₀) of 0.17 ± 0.02 mm. K_Ca channels were completely suppressed by externally applied 100 mm charybdotoxin. It is concluded that K_Ca channels activated by Ca²⁺ entry during the action potential play an important role in the excitability of motoneurones. Received: 7 November 1996/Revised: 29 October 1997     

The acid tolerance response of Bacillus cereus ATCC14579 is dependent on culture pH, growth rate and intracellular pH

The food pathogen Bacillus cereus is likely to encounter acidic environments (i) in food when organic acids are added for preservation purposes, and (ii) during the stomachal transit of aliments. In order to characterise the acid stress response of B. cereus ATCC14579, cells were grown in chemostat at different pH values (pH_o from 9.0 to 5.5) and different growth rates (μ from 0.1 to 0.8 h⁻¹), and were submitted to acid shock at pH 4.0. Cells grown at low pH_o were adapted to acid media and induced a significant acid tolerance response (ATR). The ATR induced was modulated by both pH_o and μ, and the μ effect was more marked at pH_o 5.5. Intracellular pH (pH_i) was affected by both pH_o and μ. At a pH_o above 6, the pH_i decreased with the decrease of pH_o and the increase of μ. At pH_o 5.5, pH_i was higher compared to pH_o 6.0, suggesting that mechanisms of pH_i homeostasis were induced. The acid survival of B. cereus required protein neo-synthesis and the capacity of cells to maintain their pH_i and ΔpH (pH_i - pH_o). Haemolysin BL and non-haemolytic enterotoxin production were both influenced by pH_o and μ.     

Synthesis and characterization of new optically active poly(azo-ester-imide)s via interfacial polycondensation

N,N′-Pyromelliticdiimido-di-l-amino acids (1a–1d) were prepared from the reaction of pyromellitic dianhydride with the corresponding l-amino acids in a solution of glacial acetic acid/pyridine (3:2) at refluxing temperature. 4,4′-sulfonyl bis(4,1-phenylene) bis(diazene-2,1-diyl) diphenol, 4,4′-oxy bis(4,1-phenylene) bis(diazene-2,1-diyl) diphenol and 4,4′-methylene bis(4,1-phenylene) bis(diazene-2,1-diyl) diphenol, were prepared from 4,4′-diamino diphenyl sulfone, 4,4′-diamino diphenyl ether, 4,4′-diamino diphenyl methane, sodium nitrite and phenol following the general procedure of diazo coupling. Interfacial polycondensation method was used to prepare the corresponding poly(azo-ester-imid)s (PAEI _1–12 ) in biphasic solution of water/dichloromethane. The resulting polymers (PAEIs) have been obtained in high yields having good inherent viscosities (0.32–0.57 dl g⁻¹), optical activities and thermal stabilities.     

Effects of ATP-sensitive Potassium Channel Regulators on Chloride Channels in the Sarcoplasmic Reticulum Vesicles from Rabbit Skeletal Muscle

The lipid bilayer technique was used to examine the effects of the ATP-sensitive K⁺ channel inhibitor (glibenclamide) and openers (diazoxide, minoxidil and cromakalim) and Cl⁻ channel activators (GABA and diazepam) on two types of chloride channels in the sarcoplasmic reticulum (SR) from rabbit skeletal muscle. Neither diazepam at 100 μm nor GABA at 150 μm had any significant effect on the conductance and kinetics of the 75 pS small chloride (SCl) channel. Unlike the 150 pS channel, the SCl channel is sensitive to cytoplasmic glibenclamide with K _i ∼ 30 μm. Glibenclamide induced reversible decline in the values of current (maximal current amplitude, I _max and average mean current, I′) and kinetic parameters (frequency of opening F _o , probability of the channel being open P _o and mean open time, T _o , of the SCl channel. Glibenclamide increased mean closed time, T _c , and was a more potent blocker from the cytoplasmic side (cis) than from the luminal side (trans) of the channel. Diazoxide increased I′, P _o , and T _o in the absence of ATP and Mg²⁺ but it had no effect on I _max and also failed to activate or remove the glibenclamide- and ATP-induced inhibition of the SCl channel. Minoxidil induced a transient increase in I′ followed by an inhibition of I _max, whereas cromakalim reduced P _o and I′ by increasing channel transitions to the closed state and reducing T _o without affecting I _max. The presence of diazoxide, minoxidil or cromakalim on the cytoplasmic side of the channel did not prevent [ATP] _cis or [glibenclamide] _cis from blocking the channel. The data suggest that the action(s) of these drugs are not due to their effects on the phosphorylation of the channel protein. The glibenclamide- and cromakalim-induced effects on the SCl channel are mediated via a ``flicker' type block mechanism. Modulation of the SCl channel by [diazoxide] _cis and [glibenclamide] _cis highlights the therapeutic potential of these drugs in regulating the Ca²⁺-counter current through this channel. Received: 2 September 1997/Revised: 20 March 1998     

Biosorption of organochlorine pesticides using fungal biomass

Cladosporium strain AJR³18,501 was tested for its ability to sorb the organochlorine pesticide (OCP) p,p′-DDT from aqueous media. When p,p′-DDT was added to distilled water, ethanol or 1-propanol solutions in excess of its solubility, p,p′-DDT was sorbed onto the fungal biomass. Increasing the amount of p,p′-DDT in solution by changing the medium composition increased sorbent uptake: p,p′-DDT uptake by the fungal biomass was 2.5 times greater in 25% 1-propanol (17 mg of p,p′-DDT g⁻¹ dry weight fungal biomass) than in distilled water. When p,p′-DDT was dissolved in 25% 1-propanol (12 mg l⁻¹), rapid p,p′-DDT sorption occurred during the first 60 min of incubation. p,p′-DDT in solution was reduced to 2.5 mg l⁻¹ with the remaining p,p′-DDT recovered from the fungal biomass. A number of environmental parameters were tested to determine their effect on p,p′-DDT biosorption. As arsenic (As) is prevalent at DDT-contaminated cattle dip sites, its effect on p,p′-DDT uptake was determined. The presence of As [As(III) or As(V) up to 50 mg l⁻¹] did not inhibit p,p′-DDT uptake and neither As species could be sorbed by the fungal biomass. Changing the pH of the medium from pH 3 to 10 had a small effect on p,p′-DDT sorption at low pH indicating that an ion exchange process is not the major mechanism for p,p′-DDT sorption. Other mechanisms such as Van der Waals forces, chemical binding, hydrogen bonding or ligand exchange may be involved in p,p′-DDT uptake by Cladosporium strain AJR³18,501. Journal of Industrial Microbiology & Biotechnology (2002) 29, 163–169 doi:10.1038/sj.jim.7000280 Received 15 January 2002/ Accepted in revised form 18 May 2002     

Cloning and analysis of biochemical and catalytic properties of DNA methyltransferase M1.BspACI

DNA methyltransferases genes of the BspACI restriction-modification system from Bacillus psychrodurans AC have been cloned in E. coli cells. Analysis of amino acid sequences of the proteins showed that both of these genes belong to C5 DNA methyltransferases. Gene M1.BspACI has been subcloned in pJW2 vector. A high-purity recombinant enzyme has been obtained using chromatography on different carriers. It has been shown that M1.BspACI modifies the first cytosine residue in the sequence 5′-CCGC-3′. Kinetic parameters of DNA methylation by the enzyme have been determined. Catalytic constant appears to be 0.095 ± 0.002 min⁻¹. K _{mphage is λ DNA}—0.053 ± 0.007 μM, and K _mSAM is 5.1 ± 0.3 μM.     

A Patch-Clamp Study of Ion Channels in Protoplasts Prepared from the Marine Alga Valonia utricularis

The giant marine alga Valonia utricularis is a classical model system for studying the electrophysiology and water relations of plant cells by using microelectrode and pressure probe techniques. The recent finding that protoplasts can be prepared from the giant ``mother cells' (Wang, J., Sukhorukov, V.L., Djuzenova, C.S., Zimmermann, U., Müller, T., Fuhr, G., 1997, Protoplasma 196:123–134) allowed the use of the patch-clamp technique to examine ion channel activity in the plasmalemma of this species. Outside-out and cell-attached experiments displayed three different types of voltage-gated Cl<sup>−</sup> channels (VAC1, VAC2, VAC3, Valonia Anion Channel 1,2,3), one voltage-gated K<sup>+</sup> channel (VKC1, Valonia K <sup>+</sup> Channel 1) as well as stretch-activated channels. In symmetrical 150 mm Cl<sup>−</sup> media, VAC1 was most frequently observed and had a single channel conductance of 36 ± 7 pS (n= 4) in the outside-out and 33 ± 5 pS (n= 10) in the cell-attached configuration. The reversal potential of the corresponding current-voltage curves was within 0 ± 4 mV (n= 4, outside-out) and 9 ± 7 mV (n= 10, cell-attached) close to the Nernst potential of Cl<sup>−</sup> and shifted towards more negative values when cell-attached experiments were performed in asymmetrical 50:150 mm Cl<sup>−</sup> media (bath/pipette; E <sub>Cl−</sub> −20 ± 7 mV (n= 4); Nernst potential −28 mV). Consistent with a selectivity for Cl<sup>−</sup>, VAC1 was inhibited by 100 μM DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid). VAC1 was activated by a hyperpolarization of the patch. Boltzmann fits of the channel activity under symmetrical 150 mm Cl<sup>−</sup> conditions yielded a midpoint potential of −12 ± 5 mV (n= 4, outside-out) and −3 ± 6 mV (n= 9, cell-attached) and corresponding apparent minimum gating charges of 15 ± 3 (n= 4) and 18 ± 5 (n= 9). The midpoint potential shifted to more negative values in the presence of a Cl<sup>−</sup> gradient. VAC2 was activated by voltages more negative than E <sub>Cl−</sub> and was always observed together with VAC1, but less frequently. It showed a ``flickering' gating. The single channel conductance was 99 ± 10 pS (n= 6). VAC3 was activated by membrane depolarization and frequently exhibited several subconductance states. The single channel conductance of the main conductance state was 36 ± 5 pS (n= 5). VKC1 was also activated by positive clamped voltages. Up to three conductance states occurred whereby the main conductance state had a single channel conductance of 124 ± 27 pS (n= 6). In the light of the above results it seems to be likely that VAC1 contributes mainly to the Cl⁻ conductance of the plasmalemma of the turgescent ``mother cells' and that this channel (as well as VAC2) can operate in the physiological membrane potential range. The physiological significance of VAC3 and VKC1 is unknown, but may be related (as the stretch-activated channels) to processes involved in turgor regulation. Received: 24 June 1999/Revised: 2 September 1999     

Inhibition of galectin-3 mediated cellular interactions by pectic polysaccharides from dietary sources

Pectic polysaccharides from dietary sources such as Decalepis hamiltonii—swallow root (SRPP), Hemidesmus indicus (HPP), Nigella sativa—black cumin (BCPP), Andrographis serpyllifolia—(APP), Zingiber officinale—ginger (GRPP) and, citrus pectin (CPP) were examined for galectin inhibitory activity. Inhibition of (a) galectin-3 of MDA-MB-231 cells induced hemagglutination of red blood cells; (b) galectin-3 mediated interaction between normal/metastatic human buccal cells (NBC)/(MBC) and; (c) invasion of MDA-MB-231 and MBC in the invasive chamber was assessed. Results indicated that SRPP inhibited hemagglutination at Minimum Inhibitory Concentration (MIC) of 1.86 μg ml⁻¹ equivalent of carbohydrate as apposed to those of BCPP (130 μg ml⁻¹), APP (40 μg ml⁻¹), HPP (40 μg ml⁻¹) and CPP (25 μg ml⁻¹). GRPP even at concentration >1–6 mg ml⁻¹ did not inhibit agglutination. Also SRPP showed ∼15 and 2 fold potent anti hemagglutination activity relative to that of galectin-3 specific sugars—galactose (MIC-27.1 μg ml⁻¹) and lactose (MIC-4.16 μg ml⁻¹) respectively. Further, SRPP at 10 μg ml⁻¹ inhibited agglutination of NBC by galectin-3 of MDA-MB-231 cells. Modified swallow root pectic polysaccharide (MSRPP) of 50 kDa retained anti hemagglutination activity (MIC of 1.03 μg ml⁻¹) and inhibited MDA-MB-231 and MBC invasion by 73 and 50% with an IC₅₀ of 136 and 200 μg ml⁻¹ respectively. Both SRPP and MSRPP induced apoptosis up to 80% at 100 μg ml⁻¹ concentration by activating ∼2 and 8 folds of Caspase-3 activity. Sugar composition analysis and its correlation with the galectin inhibitory property indicated that pectic polysaccharides with higher arabinose and galactose content—arabinogalactan inhibited hemagglutination significantly.     

A Novel,Volume-correlated Cl− Conductance in Marginal Cells Dissociated from the Stria Vascularis of Gerbils

Using the whole-cell patch-clamp technique, we examined Cl⁻-selective currents manifested by strial marginal cells isolated from the inner ear of gerbils. A large Cl⁻-selective conductance of ∼18 nS/pF was found from nonswollen cells in isotonic buffer containing 150 mm Cl⁻. Under a quasi-symmetrical Cl⁻ condition, the `instantaneous' current-voltage relation was close to linear, while the current-voltage relation obtained at the end of command pulses of duration 400 msec showed weak outward rectification. The permeability sequence for anionic currents was as SCN⁻ > Br⁻≅ Cl⁻ > F⁻ > NO⁻ ₃≅ I⁻ > gluconate⁻, corresponding to Eisenmann's sequence V. When whole-cell voltage clamped in isotonic bathing solutions, the cells exhibited volume changes that were accounted for by the Cl⁻ currents driven by the imposed electrochemical potential gradients. The volume change was elicited by lowered extracellular Cl⁻ concentration, anion substitution and altered holding potentials. The Cl⁻ conductance varied in parallel with cell volume when challenged by bath anisotonicity. The whole-cell Cl⁻ current was only partially blocked by both 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 0.5 mm) and diphenylamine-2-carboxylic acid (DPC, 1.0 mm), but 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid (SITS, 0.5 mm) was without effect. The properties of the present whole-cell Cl⁻ current resembled those of the single Cl⁻ channel previously found in the basolateral membrane of the marginal cell (Takeuchi et al., Hearing Res. 83:89–100, 1995), suggesting that the volume-correlated Cl⁻ conductance could be ascribed predominantly to the basolateral membrane. This Cl⁻ conductance may function not only in cell volume regulation but also for the transport of Cl⁻ and the setting of membrane potential in marginal cells under physiological conditions. Received: 15 August 1995/Revised: 3 November 1995