Phase Of Inactivation Research Articles

Influence of polymolecular events on inactivation behavior of xylose isomerase from Thermotoga neapolitana 5068.

The inactivation behavior of the xylose isomerase from Thermotoga neapolitana (TN5068 XI) was examined for both the soluble and immobilized enzyme. Polymolecular events were involved in the deactivation of the soluble enzyme. Inactivation was biphasic at 95 degrees C, pH 7.0 and 7.9, the second phase was concentration-dependent. The enzyme was most stable at low enzyme concentrations, however, the second phase of inactivation was 3- to 30-fold slower than the initial phase. Both phases of inactivation were more rapid at pH 7.9, relative to 7.0. Differential scanning calorimetry of the TN5068 XI revealed two distinct thermal transitions at 99 degrees and 109 degrees C. The relative magnitude of the second transition was dramatically reduced at pH 7.9 relative to pH 7.0. Approximately 24% and 11% activity were recoverable after the first transition at pH 7.0 and 7.9, respectively. When the TN5068 XI was immobilized by covalent attachment to glass beads, inactivation was monophasic with a rate corresponding to the initial phase of inactivation for the soluble enzyme. The immobilized enzyme inactivation rate corresponded closely to the rate of ammonia release, presumably from deamidation of labile asparagine and/or glutamine residues. A second, slower inactivation phase suggests the presence of an unfolding intermediate, which was not observed for the immobilized enzyme. The concentration dependence of the second phase of inactivation suggests that polymolecular events were involved. Formation of a reversible polymolecular aggregate capable of protecting the soluble enzyme from irreversible deactivation appears to be responsible for the second phase of inactivation seen for the soluble enzyme. Whether this characteristic is common to other hyperthermophilic enzymes remains to be seen.

Importance of individual activated protein C cleavage site regions in coagulation Factor V for Factor Va inactivation and for Factor Xa activation

Activated protein C (APC) cleavage of Factor Va (FVa) at residues R506 and R306 correlates with its inactivation. APC resistance and increased thrombotic risk are due to the mutation R506Q in Factor V (FV). To study the effects of individual cleavages in FVa by APC and the importance of regions near the cleavage sites, the following recombinant (r) human FVs were prepared and purified: wild-type, Q306-rFV, Q506-rFV, and Q306Q506-rFV. All had similar time courses for thrombin activation. Q506-rFVa was cleaved by APC at R306 and was moderately resistant to APC in plasma-clotting assays and in prothrombinase assays measuring FVa residual activity, in agreement with studies of purified plasma-derived Q506-FVa. Q306-rFVa was cleaved by APC at R506 and gave a low APC-resistance ratio similar to Q506-rFVa in clotting assays, whereas unactivated Q306-rFV gave a near-normal APC-resistance ratio. When FVa residual activity was measured after long exposure to APC, Q306-rFVa was inactivated by only < or = 40% under conditions where Q506-rFVa was inactivated > 90%, supporting the hypothesis that efficient inactivation of normal FVa by APC requires cleavage at R306. In addition, the heavy chain of Q306-rFVa was cleaved at R506 much more rapidly than activity was lost, suggesting that FVa cleaved at only R506 is partially active. Under the same conditions, Q306Q506-rFVa lost no activity and was not cleaved by APC. Therefore, cleavage at either R506 or R306 appears essential for significant inactivation of FVa by APC. Modest loss of activity, probably due to cleavage at R679, was observed for the single site rFVa mutants, as evidenced by a second phase of inactivation. Q306Q506-rFVa had a low activity-to-antigen ratio of 0.50-0.77, possibly due to abnormal Factor Xa (FXa) binding. Furthermore, Q306Q506-rFV was very resistant to cleavage and activation by FXa. Q306Q506-rFV appeared to bind FXa and inhibit FXa's ability to activate normal FV. Thus, APC may downregulate FV/Va partly by impairing FXa-binding sites upon cleavage at R306 and R506. This study shows that R306 is the most important cleavage site for normal efficient inactivation of FVa by APC and supports other studies suggesting that regions near R306 and R506 provide FXa-binding sites and that FVa cleaved at only R506 retains partial activity.

5,5'-Dithiobis-(2-nitrobenzoic acid) as a probe for a non-essential cysteine residue at the medium chain acyl-coenzyme A dehydrogenase binding site of the human 'electron transferring flavoprotein' (ETF).

Human 'electron transferring flavoprotein' (ETF) was inactivated by the thiol-specific reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). The kinetic profile showed the reaction followed pseudo-first-order kinetics during the initial phase of inactivation. Monitoring the release of 5-thio-2-nitrobenzoate (TNB) showed that modification of 1 cysteine residue was responsible for the loss of activity. The inactivation of ETF by DTNB could be reversed upon incubation with thiol-containing reagents. The loss of activity was prevented by the inclusion of medium chain acyl-CoA dehydrogenase (MCAD) and octanoyl-CoA. Cyanolysis of the DTNB modified-ETF with KCN led to the release of TNB accompanied presumably by the formation of the thio-cyano enzyme and with almost full recovery of activity. Conservation studies and the lack of 100% inactivation, however, suggested that this cysteine residue is not essential for the interaction with MCAD.

Retardation of thermal and urea induced inactivation of alpha-chymotrypsin by modification with carbohydrate polymers.

Modification of enzymes by means of covalent coupling using soluble polymers results in enzymes which retain high biological activity and display resistance to denaturants, high temperature and chaotropic agents. Alpha-chymotrypsin, which has a potential for use in industrial applications, was covalently modified by reductive alkylation using polymeric sucrose (OSP, molecular weight 70 and 400 kDa), dextran (73 and 250 kDa) and carboxymethyl cellulose (CMC, approximately 12 kDa). The derivatives retained around 50-80% activity depending on the polymer used and the extent of modification. At the same time, they displayed better thermotolerance than their native counterpart with 4-14 degrees C higher T50 values. During thermal inactivation, both the native and modified enzymes showed biphasic inactivation kinetics. Half-life of modified enzymes were 2-66-fold greater for the first phase and 5-250-fold greater than the native for the second phase of inactivation. The activation free energy of inactivation of alpha-chymotrypsin coupled to polymeric sucrose (400 kDa) was 112.85 kJ/mol for the first phase and 114.71 kJ/mol for the second phase, whereas in the case of the native enzyme, the value for the first phase was 101.55 kJ/mol and 103.42 kJ/mol for the second phase. The activation free energy of inactivation (deltaG*), as well as the activation enthalpy values (deltaH*) of all the modified enzymes were greater than those of the native enzyme, which is an indication of stabilization of the protein and a retardation of inactivation that is usually accompanied by unfolding under thermal and chemical stress. The stability of modified alpha-chymotrypsin is in the following order: OSP 400-C > OSP 70-C > CMC-C > Dextran 73-C = Dextran 250-C.

Inhibition of the Fast Na + Current by Taurine in Guinea Pig Ventricular Myocytes

1. Effects of taurine on the fast Na + current ( I Na ) in isolated guinea pig ventricular cardiomyocytes were examined by using the whole-cell voltage-clamp method. Experiments were performed at room temperature (22°C). 2. Test pulses were applied from −60 to +40 mV from a holding potential of −90 mV. Addition of taurine to the bath solution markedly inhibited the I Na in a concentration-dependent manner; at −30 mV, by 39.0±4.1% ( n=8, P<0.01) at 10 mM and by 56.1±4.7% ( n=9, P<0.001) at 20 mM. 3. Simultaneously, the time constant of inactivation phase for I Na decreased to 0.95±0.4 ms ( n=8, P<0.05) at 10 mM and to 1.02±0.3 ms ( n=9, P<0.05) at 20 mM, from 1.29±0.3 ms in normal Tyrode solution. 4. These results indicate that taurine inhibits the I Na current, which would play an important role in the cell functions.

Arachidonic acid potentiates the feedback response of mesangial BKCa channels to angiotensin II.

The influence of arachidonic acid (AA) on the feedback regulation of mesangial contraction by large Ca(2+)-activated K+ channels (BKCa) was determined through single-channel analysis using the patch clamp method. The mesangial BKCa is a low-gain negative feedback inhibitor of contraction that is activated in response to agonist-induced Ca2+ transients and membrane depolarization. AA activated BKCa in cell-attached patches in a dose-dependent manner with a maximal effect at 400 nM and a half-maximal response at 49 nM. In inside-out patches, AA directly activated BKCa with a maximal effect at 400 nM. BKCa was activated significantly in response to addition of 100 nM ANG II in the presence but not the absence of AA. Since it was shown previously that fatty acids stimulated both soluble and membrane-bound guanylyl cyclase, we determined whether AA activated BKCa by interfering with cGMP-mediated signal transduction pathways. It was previously shown that 10 microM cGMP, via cGMP-dependent protein kinase, activated BKCa in a biphasic manner with an early increase in probability of a channel existing in an open state (Po) and a subsequent inactivation mediated by protein phosphatase 2A (PP2A). We found that 10 microM dibutyryl-cGMP enhanced BKCa activity in an additive manner with saturating concentrations (400 nM) of AA. Moreover, the inactivation phase mediated by PP2A was not abolished. Thus AA does not affect the phosphorylation/dephosphorylation regulatory cycle for BKCa. It is concluded that AA potentiates the ANG II feedback response of BKCa by a mechanism that is independent of the phosphorylation cycle.

Inactivation Kinetics Study of the Kunitz Soybean Trypsin Inhibitor and the Bowman-Birk Inhibitor.

The inactivation of trypsin inhibitors (TIs) in soy flour exhibits a two-phase inactivation behavior. It is sometimes assumed that this behavior is caused by a difference in the heat stabilities of the Kunitz soybean trypsin inhibitor (KSTI) and the Bowman-Birk inhibitor (BBI). Kinetics studies with KSTI and BBI in soy flour showed that this two-phase inactivation behavior of TIs could not be explained by the difference in the heat stabilities of KSTI and BBI. Inactivation of KSTI and BBI in an aqueous solution and in a starch matrix followed a first-order reaction. KSTI and BBI in a starch matrix with added cysteine showed a two-phase inactivation behavior. The existence of thiols in soy flour seems to be responsible for the two-phase inactivation of TIs in soy flour. It is suggested that TIs in soy flour are inactivated by sulfhydryl-disulfide interchange during the first inactivation phase and by heat during the second phase.

Actions of taurine on the L-type Ca2+ channel current in guinea pig ventricular cardiomyocytes.

Effects of taurine on the L-type channel in isolated guinea pig ventricular cardiomyocytes were examined at different Ca2+ concentrations by using whole-cell and cell-attached voltage-clamp modes. All experiments were performed at 36 degrees C. In whole-cell voltage-clamp experiments, test pulses were applied between -20 to +60 mV from a holding potential of -40 mV. When [Ca]i was pCa 6, addition of 10 and 20 mM taurine to the bath solution reduced the Ca2+ current (I(Ca)) at 0 mV by 14.4 +/- 2.0% (n = 8; p < 0.01) and 31.5 +/- 2.2% (n = 8; p < 0.001), respectively. In contrast, when [Ca]i was pCa 8, I(Ca) at +10 mV was enhanced by 10.1 +/- 2.2% (n = 7; p < 0.05) at 10 mM taurine and by 41.7 +/- 2.1% (n = 7; p < 0.001) at 20 mM taurine. Taurine increased the time constants (tau(f) and tau(s)) of inactivation phase for I(Ca) current at both pCa 8 and 6. In cell-attached voltage-clamp experiments, taurine (20 mM) decreased the open probability of unitary Ba2+ current from 0.63 +/- 0.06 to 0.39 +/- 0.09 (n = 5; p < 0.01) at 5.4 mM [Ca]o, whereas taurine increased it from 0.21 +/- 0.04 to 0.48 +/- 0.07 (n = 4; p < 0.01) at 0.9 mM [Ca]o. Taurine did not affect the channel conductance. In addition, taurine (20 mM) increased the time constants (tau(of) and tau(os)) of the open time and decreased tau(cs) of the closed time at 0.9 mM [Ca]o. At 5.4 mM [Ca]o, the tau(os) and tau(cs) were also increased and decreased, respectively. tau(of) and tau(cf) were unaffected. These results indicate that taurine modulates the open probability of L-type Ca2+ channel dependent on [Ca]i and [Ca]o, thereby maintaining the normal [Ca]i level.

Kinetics of Calcium Release from Manganese Peroxidase during Thermal Inactivation

It was previously reported that manganese peroxidase from the white-rot fungusPhanerochaete chrysosporiumwas susceptible to thermal inactivation because it contains relatively labile Ca2+ions required for stability and activity [Sutherland and Aust (1996)Arch. Biochem. Biophys.332, 128–134]. In this work we determined that four Ca2+ions are present in the enzyme as isolated but this was reduced to 2 mol/mol upon treatment with Ca2+-chelating agents or extensive dialysis of dilute enzyme. One of two relatively tightly bound Ca2+remaining in the enzyme was released during thermal inactivation at pH 7.2. Inactive enzyme contained one Ca2+which could be removed in acidic conditions. Inactivation kinetics were biphasic and the rates for the two inactivation steps and the release of Ca2+during inactivation suggested that the first, faster phase of inactivation was coupled to the removal of Ca2+. The weakly associated Ca2+normally present in the enzyme did not affect enzyme activity and did not seem to protect the enzyme from thermal inactivation at submicromolar enzyme concentrations. Excess Ca2+or Mn2+decreased the rate of the thermal inactivation and Mn2+stabilized the enzyme more efficiently than Ca2+at higher temperature. Enzyme stabilization by Mn2+was proposed to be due to binding of Mn2+to the Mn2+substrate binding site. In competition studies, Ca2+was shown to bind to this site with apparent dissociation constants of 10−2and 10−4M at pH 4.5 and 7.2, respectively. Moreover, Ca2+was a poor inhibitor of manganese peroxidase activity at pH 4.5. It is therefore suggested that Ca2+is absent from the substrate site in physiological conditions but can bind to this site at higher pH and therefore may stabilize the enzyme by binding to both the Mn2+site and, as previously proposed, to the distal Ca2+site.

Fast inactivation of Shaker K+ channels is highly temperature dependent.

The energy profile of the interaction between the NH2-terminal inactivation domain and the internal mouth of the Shaker H4 K+ channel has been investigated. Macroscopic currents from channels normally inactivating (Shaker H4) and with the inactivation removed (Shaker H4-IR) were recorded at different temperatures using the cut-open oocyte technique. Changes in temperature had a dramatic effect on the inactivation phase. The following parameters were obtained in Shaker H4, lowering the temperature from 20 degrees C to 5 degrees C: (1) the peak amplitude decreased with the temperature coefficient Q10 equal to 1.51; (2) the activation time constant increased with a Q10 equal to 3.14; (3) the decay time constant increased with a Q10 of 7.20, while the recovery from inactivation was less temperature-dependent (Q10=1.57) than the installation of the inactivation phase. At 0 mV, the ratio between the steady state level and the peak amplitude of the current increased with a Q10 of 2.95. These findings indicate that the installation of a fast inactivation process has a strong temperature dependence, while the recovery phase from inactivation is less temperature dependent. These observations support the idea of an NH2-terminal blocking mechanism for inactivation and flexible conformation of the blocking particle.

Identification of a new σ B-controlled gene, csbX, in Bacillus subtilis

We have identified a new member, csbX, of the general stress regulon controlled by σ B in Bacillus subtilis. As with other members of the σ B regulon csbX is expressed during the stationary phase of cell growth and inactivation of this gene produces no obvious phenotype during cell growth or early development. csbX lies uspstream from the sporulation gene bofC which is co-transcribed during stationary phase.

K+ channel inactivation mediated by the concerted action of the cytoplasmic N- and C-terminal domains

We have examined the molecular mechanism of rapid inactivation gating in a mouse Shal K+ channel (mKv4.1). The results showed that inactivation of these channels follows a complex time course that is well approximated by the sum of three exponential terms. Truncation of an amphipathic region at the N-terminus (residues 2-71) abolished the rapid phase of inactivation (r = 16 ms) and altered voltage-dependent gating. Surprisingly, these effects could be mimicked by deletions affecting the hydrophilic C-terminus. The sum of two exponential terms was sufficient to describe the inactivation of deletion mutants. In fact, the time constants corresponded closely to those of the intermediate and slow phases of inactivation observed with wild-type channels. Further analysis revealed that several basic amino acids at the N-terminus do not influence inactivation, but a positively charged domain at the C-terminus (amino acids 420-550) is necessary to support rapid inactivation. Thus, the amphipathic N-terminus and the hydrophilic C-terminus of mKv4.1 are essential determinants of inactivation gating and may interact with each other to maintain the N-terminal inactivation gate near the inner mouth of the channel. Furthermore, this inactivation gate may not behave like a simple open-channel blocker because channel blockade by internal tetraethylammonium was not associated with slower current decay and an elevated external K+ concentration retarded recovery from inactivation.

Effects of nicotine on spontaneous activity and underlying ionic currents in rabbit sinoatrial nodal cells

1. 1. Effects of nicotine on the spontaneous action potentials and the underlying ionic currents in rabbit sinoatrial (SA) nodal cells were investigated using current-clamp and whole-cell voltage-clamp modes. 2. 2. Nicotine (30 μM to 1 mM) produced a negative chronotropic effect in a concentration-dependent manner (at 1 mM by 10.6±2.8%, n=9, p<0.01). Nicotine at 300 μM significantly decreased the maximum rate of depolarization by 9.8±1.3% ( n=9, p<0.05). Other action potential parameters were not affected to any significant extent. 3. 3. Pretreatment with atropine (1 μM) and hexamethonium (1 mM) did not modify the nicotine-induced effects. After washout, these responses were reversible. 4. 4. Isoprenaline decreased the responses induced by nicotine, but ACh increased them. 5. 5. Nicotine at 100 μM did not affect the L-type Ca 2+ current ( I Ca), but at 300 μM inhibited it at +10 mV by 21.6±2.9% ( n=6, p<0.05). The fast time constant ( τ f ) of the inactivation phase for I Ca was not affected, but the slow one ( τ 5) significantly increased from 36.8±1.9 ms to 41.2±2.8 ms ( n=6) at 300 μM nicotine. The activation and inactivation kinetics ( d ∞ and f ∞) for I Ca were not modified. 6. 6. Nicotine also did not affect the delayed rectifier K + current ( I K) and its activation kinetic ( P ∞) 7. 7. These results suggest that nicotine depresses the action potentials and causes a negative chronotropic effect due to inhibitions of the ionic currents in the SA nodal cells.

Angiotensin II activation of protein kinase C decreases delayed rectifier K+ current in rabbit vascular myocytes.

1. The effect of angiotension II (Ang) on delayed rectifier K+ current (IK(V)) was studied in isolated rabbit portal vein smooth muscle cells using standard whole-cell voltage clamp technique. The effect of 100 nM Ang on macroscopic, whole-cell IK(V) was assessed in myocytes dialysed with 10 mM BAPTA, 5 mM ATP and 1 mM GTP either at room temperature or at 30 degrees C. 2. Application of Ang caused a decline in IK(V) which was reversed upon washout of the drug. Tail current recorded after 250 ms pulses to +30 mV and repolarization to -40 mV was reduced from 3.9 +/- 0.7 to 2.5 +/- 0.5 pA pF-1 at 20 degrees C (n = 6) and from 4.5 +/- 0.5 to 3.13 +/- 0.4 pA pF-1 at 30 degrees C(n = 17). 3. Ang had no effect on outward current in the presence of an AT1 selective antagonist, losartan (1 microM), which alone had no direct effect on the amplitude of IK(V). Substitution of extracellular Ca2+ with Mg2+ in the presence of 10 microM intracellular BAPTA did not affect the suppression of IK(V) by Ang. 4. Ang induced a decrease in time constant for the rapid phase of inactivation of the macroscopic current (tau 1 reduced from 377 +/- 32 to 245 +/- 11 ms; tau 2 unchanged, n = 17). Neither the voltage dependence of activation nor inactivation were affected by Ang. 5. The inhibition of IK(V) by Ang was abolished by intracellular dialysis with the selective PKC inhibitors, calphostin C (1 microM) and chelerythrine (50 microM). These data provide strong evidence that the decline in IK(V) due to Ang treatment is due to PKC activation. 6. The pattern of expression of PKC isoforms was examined in rabbit portal vein using isoenzyme-specific antibodies: alpha, epsilon and zeta isoenzymes were detected, but beta, gamma, delta and eta isoenzymes were not. 7. The lack of requirement for Ca2+, as well as the sensitivity of the Ang response to chelerythrine, suggest the involvement of the Ca(2+)-independent PKC isoenzyme epsilon in the signal transduction pathway responsible for IK(V) inhibition by Ang.

Sea Anemone Toxin (ATX II) Modulation of Heart and Skeletal Muscle Sodium Channel α-Subunits Expressed in tsA201 Cells

We have expressed recombinant alpha-subunits of hH1 (human heart subtype 1), rSkM1 (rat skeletal muscle subtype 1) and hSkM1 (human skeletal muscle) sodium channels in human embryonic kidney cell line, namely the tsA201 cells and compared the effects of ATX II on these sodium channel subtypes. ATX II slows the inactivation phase of hH1 with little or no effect on activation. At intermediate concentrations of ATX II the time course of inactivation is biexponential due to the mixture of free (fast component, taufasth) and toxin-bound (slow component, tauslowh) channels. The relative amplitude of tauslowh allows an estimate of the IC50 values approximately 11 nM. The slowing of inactivation in the presence of ATX II is consistent with destabilization of the inactivated state by toxin binding. Further evidence for this conclusion is: (i) The voltage-dependence of the current decay time constants (tauh) is lost or possibly reversed (time constants plateau or increase at more positive voltages in contrast to these of untreated channels). (ii) The single channel mean open times are increased by a factor of two in the presence of ATX II. (iii) The recovery from inactivation is faster in the presence of ATX II. Similar effects of ATX II on rSkM1 channel behavior occur, but only at higher concentrations of toxin (IC50 = 51 nM). The slowing of inactivation on hSkM1 is comparable to the one seen with rSkM1. A residual or window current appears in the presence of ATX II that is similar to that observed in channels containing mutations associated with some of the familial periodic paralyses.

Orthophosphate salts induce calcium current recovery from blockade by gadolinium in isolated guinea-pig ventricular myocytes.

Using the whole-cell configuration of the patch-clamp technique, we studied the incomplete recovery of the high voltage-activated calcium current (ICa,L) from a complete blockade by 10 microM gadolinium. The study was performed on isolated guinea-pig ventricular myocytes. ICa,L recovery depended on the duration of application of Gd3+: for a mean application time of 1.63 +/- 0.45 min, a recovery to 67 +/- 22% of the initial current amplitude (n = 12 cells) was observed; when the application lasted 5.1 +/- 1.2 min ICa,L recovered to 27 +/- 13% (n = 14 cells). The partial recovery of ICa,L was accompanied by a slowing of the inactivation phase of the current. IF, during the incomplete recovery, cells were exposed to a solution containing 330 microM of an orthophosphate salt, such as NaH2PO4 or KH2PO4, the current amplitude increased to 80 +/- 13% of the initial current (n = 10 cells) in a reversible manner. However, the slowing of the inactivation phase was maintained. Our results show that the partial recovery of ICa,L from blockade by gadolinium is due to an interaction of the blocker with an extracellular part of the channel, possibly one involved in voltage-dependent inactivation.

Halothane and isoflurane preferentially depress a slowly inactivating component of Ca2+ channel current in guinea-pig myocytes.

1. The effects of the inhalational anaesthetics halothane and isoflurane on the high-voltage-activated Ca2+ channels were determined in isolated guinea-pig ventricular myocytes using the patch-clamp technique. 2. Recording solutions were equilibrated with inhalational anaesthetic vapour delivered from a calibrated vaporizer set at clinically relevant ranges of partial pressure. Anaesthetic concentrations in solution were determined using gas chromatography. 3. Halothane (0.9 mM in solution) and isoflurane (0.8 mM in solution) decreased peak whole-cell CA2+ current (ICa) by approximately 40 and approximately 20%, respectively, while increasing the apparent rate of inactivation. 4. The sum of fast and slow exponential decay functions was required to fit the inactivation phase of ICa. The anaesthetics preferentially affected the slow component of inactivation while also increasing the rate of slow inactivation. The physiological significance of these effects was addressed by examining ICa evoked by a ventricular action potential waveform. 5. Measurement of the current carried by Ba2+ through Ca2+ channels (IBa) permitted the isolation of the slow component of inactivation. Halothane and isoflurane diminished peak IBa at 0 mV by approximately 45 and approximately 20% respectively, with similar changes in rate and magnitude of the slowly inactivating component as with ICa. 6. Cell-attached patch-clamp measurements of Ca2+ channel activity revealed that halothane did not alter single-channel conductance. Instead, the anaesthetic reduced channel open probability to the same extent as observed during the whole-cell recording, an effect partially due to an increase in null sweeps. In patches with a single channel present, the open-time distribution, fitted by a single exponential, showed a decrease in mean open time. The closed-time distribution, fitted by the sum of slow and fast exponential components, revealed an anaesthetic-induced increase in the duration of the slow component with no effect on the fast component. Results are presented in terms of a channel-gating model, and model predictions are examined with a computer simulation.

Characterization of the GABA response on identified dialysed Lymnaea neurons

1. 1. The effect of γ-aminobutyric acid has been studied on identified, internally perfused dialysed neurons of Lymnaea stagnalis L. (Pulmonata, Basommatophora). It was shown that: On the majority of neurons GABA (10 −8-10 −3 M) depolarized the membrane with a decrease in input resistance and activated a Cl- dependent inward current with − 20 ± 4 mV E GABA. In some cells, the outward current with − 67 ± 8 mV E GABA was also recorded. 2. 2. The GABA-induced inward current was fast (1.5 ± 0.3 sec, n=4) or slow (3.2 ± 0.2 sec, n=3) peaking in a voltage-independent manner. The inactivation phase could be fitted by one or two exponentials characterized with fast ( τ = 0.7 sec) and slow ( τ = 3.6 sec) time constants. The outward current component was slow and activated at more positive V h( − 30-20 mV). 3. 3. The agonist effects (GABA and muscimol) indicated the involvement of GABAA receptors and Cl-permeability changes in activating inward current. Picrotoxin (10 −5-10 −4 M) and Cd 2+ completely inhibited the GABA-activated inward current also affecting E GABA. Furosemide was without effect on the peak value of GABA-induced inward current, but slightly modified the slope of inactivation. 4. 4. High concentrations of Ca-ions and their substitution with Ba-ions in extracellular saline failed to alter the GABA-induced inward current. However, omission of Cl-ions from extracellular media shifted E GABA to the right by 18 ± 8 mV ( n = 4). 5. 5. Omission of Cl-ions from intracellular saline led to inhibition of the fast component of GABA-induced inward current. Full recovery followed readdition of Cl-ions. 6. 6. The results are in agreement with the data obtained on cloned Lymnaea GABA receptors.

Activation of the Xenopus cyclin degradation machinery by full-length cyclin A.

The entry into mitosis is dependent on the activation of mitotic forms of cdc2 kinase. In many cell types, cyclin A-associated kinase activity peaks just prior to that of cyclin B, although the precise role of cyclin A-associated kinase in the entry into mitosis is still unclear. Previous work has suggested that while cyclin B is capable of triggering cyclin destruction in Xenopus cell-free systems, cyclin A-associated kinase is not able to support this function. Here we have expressed a full-length human cyclin A in Escherichia coli and purified the protein to homogeneity by virtue of an N-terminal histidine tag. We have found that when added to Xenopus cell-free extracts free of cyclin B and incapable of protein synthesis, the temporal pattern of cyclin A-associated cdc2 kinase activity showed distinct differences that were dependent on the concentration of cyclin A added. When cyclin A was added to a concentration that generated levels of cdc2 kinase activity capable of inducing nuclear envelope breakdown, the histone H1 kinase activity profile was bi-phasic, consisting of an activation phase followed by an inactivation phase. Inactivation was found to be due to cyclin destruction, which was prevented by mos protein. Cyclin destruction was followed by nuclear reassembly and an additional round of DNA replication, indicating that there is no protein synthesis requirement for DNA replication in this embryonic system. It has been suggested that the evolutionary recruitment of cyclin A into an S phase function may have necessitated the loss of an original mitotic ability to activate the cyclin destruction pathway. The results presented here indicate that cyclin A has not lost the ability to activate its own destruction and that cyclin A-mediated activation of the cyclin destruction pathway permitted destruction of cyclin B1 as well as cyclin A, indicating that there are not distinct cyclin A and cyclin B destruction pathways. Thus the ordered progression of the cell cycle requires the careful titration of cyclin. A concentration in order to avoid activation of the cyclin destruction pathway before sufficient active cyclin B/cdc2 kinase has accumulated.

T cell major histocompatibility complex class II molecules down‐regulate CD4+ T cell clone responses following LAG‐3 binding

T cell response to its antigen requires recognition by the T cell receptor together with a co-receptor molecule, either CD4 or CD8. Additional molecules have been identified that are capable of delivering the co-stimulatory signals provided by APC. Following T cell priming, a number of T cell activation antigens are expressed that may play a role in the inactivation phase of the T cell response. The lymphocyte activation gene (LAG)-3 protein and its counter-receptors, the major histocompatibility complex (MHC) class II molecules, are such activation antigens whose interaction may result in the down-regulation of the ongoing immune response. To investigate the role of LAG-3/class II molecule interaction, we produced a soluble form of LAG-3 by fusing the extracellular Ig domains of this membrane protein to the constant region of human IgG1 (LAG-3Ig). Here, we show a direct and specific binding of LAG-3Ig to class II molecules on the cell surface. In addition, we show that LAG-3/class II molecule interaction leads to the down-regulation of CD4+ Ag-specific T cell clone proliferation and cytokine secretion. This inhibitory effect is observed at the level of the effector cells and not the APC and is also found with anti-CD3 mAb, PHA + PMA or low-dose IL-2 driven stimulation in the absence of APC. These functional studies indicate that T cell MHC class II molecules down-regulate T cell proliferation following LAG-3 binding and suggest a role for LAG-3 in the control of the CD4+ T cell response.