OLONGAPO, Philippines (AP) — Police in the Philippines say an accident at a northern shipyard has killed five workers and seriously injured eight others.
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New York (AP) — Coffee futures trading on the IntercontinentalExchange (ICE) Wednesday:
(37,500 lbs.; cents per lb.)
| Open | High | Low | Settle | Chg. | |
| COFFEE C | |||||
| Mar | 204.50 | 205.90 | 198.30 | 200.40 | Down 3.80 |
| May | 206.25 | 208.00 | 200.40 | 202.55 | Down 3.60 |
| Jul | 209.45 … |
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Rookie Blake Griffin had career highs of 44 points and …
The Federal Housing Administration is raising fees and tightening lending standards to shore up its strapped finances and avoid a taxpayer bailout.
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Tiny, impoverished East Timor said it was considering the plan at Australia's request, but expressed reservations that it was ready to host such a facility.
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One study seeks people 60 and older who have arthritis in theirlegs or lower back. …
ABSTRACT The anterior byssus retractor muscle of Mytilus edulis was used to characterize the myosin cross-bridge during catch, a state of tonic force maintenance with a very low rate of energy utilization. Addition of MgATP to permeabilized muscles in high force rigor at pCa > 8 results in a rapid loss of some force followed by a very slow rate of relaxation that is characteristic of catch. The fast component is slowed 3-4-fold in the presence of 1 mM MgADP, but the distribution between the fast and slow (catch) components is not dependent on [MgADP]. Phosphorylation of twitchin results in loss of the catch component. Fewer than 4% of the myosin heads have ADP bound in rigor, and the time course (0.2-10 s) of ADP formation following release of ATP from caged ATP is similar whether or not twitchin is phosphorylated. This suggests that MgATP binding to the cross-bridge and subsequent splitting are independent of twitchin phosphorylation, but detachment occurs only if twitchin is phosphorylated. A similar dependence of detachment on twitchin phosphorylation is seen with AMP-PNP and ATP-yS. Single turnover experiments on bound ADP suggest an increase in the rate of release of ADP from the cross-bridge when catch is released by phosphorylation of twitchin. Low [Ca"] and unphosphorylated twitchin appear to cause catch by 1) markedly slowing ADP release from attached cross-bridges and 2) preventing detachment following ATP binding to the rigor cross-bridge.
Abbreviations used: A, actin; AM, actomyosin; AMADP, actomyosin-- ADP; AMATP, actomyosin-ATP; M, myosin.
INTRODUCTION
Some smooth muscles show tonic force maintenance associated with very low rates of energy utilization and very slow shortening velocities. In vertebrate smooth muscles this state of high force output with slow myosin crossbridge cycling is referred to as "latch" (Dillon et al., 1981), but the most extreme example of this type of mechanical behavior is the "catch" state first identified in the early 1900s in invertebrate smooth muscles (for review see Bayliss, 1927). The catch state is characterized by high force maintenance with very little suprabasal energy usage (Baguet and Gillis, 1968; Minihan and Davies, 1966) and an inability of the muscle to actively redevelop force following a quick release (Jewell, 1959). Cholinergic stimulation of a catch muscle such as the anterior byssus retractor muscle (ABRM) of Mytilus edulis results in a rapid increase in force and active shortening if the muscle is released. With time, these properties of the muscle change to the catch state, in which force persists for long periods even though the excitatory stimulus is removed (Jewell, 1959). Serotonergic nerve stimulation results in rapid relaxation of catch force (see Twarog, 1967).
Activation of cross-bridge cycling in molluscan catch muscles results from direct calcium binding to myosin (for review see Szent-Gyorgyi, 1996; Szent-Gyorgyi et al., 1999). When the muscle is stimulated, intracellular [Ca 21] increases and force is generated. [Ca2+] then falls with time, and catch force ensues even though intracellular [Ca2+] approaches resting concentration (Ishii et al., 1989). Relaxation of catch force produced by serotonin is not associated with a change in intracellular [Caz+], but rather results from an increase in [cAMP] and activation of protein kinase A (Cole and Twarog, 1972; for review, see Twarog, 1976).
The fundamental characteristics of catch can be reproduced in permeabilized ABRM (Cornelius, 1980, 1982; Pfitzer and Ruegg, 1982; Castellani and Cohen, 1987; Siegman et al., 1997). An increase in [Ca2+] causes activation of the muscle and fast cross-bridge cycling as evidenced by a high ATPase rate and rapid shortening. The subsequent return to a "resting" [Ca2+] results in a slowly decaying force with very low ATPase and absence of force redevelopment following a quick release. The catch force relaxes rapidly with addition of cAMP (Butler et al., 1998). We have recently reported that the release of catch force is due to a protein kinase A-mediated phosphorylation of twitchin (Siegman et al., 1997, 1998), a mini-titin that is associated with the myosin-containing filament in catch muscles (Vibert et al., 1993).
The mechanisms by which myosin cross-bridges make the transition from force maintenance with rapid cycling to catch force maintenance with slow cycling, and the mechanism of control of relaxation of catch by the phosphorylation of twitchin, are not yet known. In vertebrate smooth muscle, the latch state has been proposed to arise from the formation of slowly detaching cross-bridges that result from dephosphorylation of the myosin regulatory light chain while the cross-bridge is in the high force state (Hai and Murphy, 1988). The accumulation of dephosphorylated, slowly detaching cross-bridges would result in high force maintenance with low cross-bridge cycling rates. Other mechanisms have also been proposed to result in force generation from unphosphorylated cross-bridges with slow cycling rates (Himpens et al., 1988; Vyas et al., 1992; Malmqvist et al., 1997; Haeberle, 1999).
Unphosphorylated, attached cross-bridges in tonic mammalian smooth muscle have a very slow rate of release of ADP (Khromov et al., 1995). Furthermore, such crossbridges have a very high affinity for MgADP (Fuglsang et al., 1993), with the result that relatively small concentrations of MgADP (-100-150 pM) can effectively compete with MgATP (2 mM) for binding to the rigor cross-bridge (Khromov et al., 1995, 1996, 1998). These findings suggest that latch represents an unphosphorylated cross-bridge with ADP bound. Detachment of the cross-bridge would be limited by the slow rate at which ADP comes off and the competition between ADP and ATP binding to the rigor cross-bridge (Khromov et al., 1995). Because the catch cross-bridge represents an extreme of slow cross-bridge cycling, it was of interest to determine whether cross-bridge detachment in catch was limited by a very slow rate of ADP release, and whether ADP affinity of the rigor cross-bridge in catch might exceed that of tonic mammalian smooth muscle. In addition, the rapid relaxation of catch force by phosphorylation of twitchin suggests that these cross-bridge parameters are likely to be highly regulated.
METHODS
Muscle preparation
Mytilus edulis were obtained from Anastasi's Fish Market, Philadelphia, PA. Mussels were housed in an aquarium containing aerated filtered seawater (Instant Ocean, Carolina Biological Supply, Burlington, NC) at 5C. On the day of the experiment, the shell was opened, the anterior byssus retractor muscle (ABRM) was exposed, and the pedal ganglia removed. Muscle bundles (0.2-0.4 mm in diameter and up to 1 cm in length) were mounted on holders and incubated in an artificial seawater solution at 20 deg C until use. The artificial seawater (ASW) contained KCI, 10 mM; MgCl2, 50 mM; CaCl^sub 2^, 10 mM; NaCl, 428 mM; N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), 10 mM at pH 7.4.
Solutions for permeabilized muscles
The compositions of the various solutions are listed in Table 1. A computer program provided by Dr. R. J. Barsotti was used to solve the multiple binding equilibria (see Dantzig et al., 1999). The [Ca"] of the solutions containing EGTA and no added calcium was considered to be pCa > 8. All experiments were performed at 20 deg C.
Muscles were permeabilized by incubation for 30 min in a low EGTA (pCa > 8) rigor solution containing 1% Triton X-100. This was followed by at least three washes of 5 min duration in similar rigor solutions without Triton X-100.
Mechanical measurements
Muscle bundles of -5 turn in length were mounted on a myograph similar to that described previously (Siegman et al., 1984, 1997). Force output was measured with a DSC-6 transducer (Kistler Morse, Bothell, WA) and was recorded on both a strip chart recorder and a digital storage oscilloscope (model 4094 or 310, Nicolet, Madison, WI).
Caged ATP experiments
The flash-lamp apparatus for photolysis of caged ATP and the pneumatically driven freeze clamp device used for rapid freezing of muscles at times <10 s after the flash have been described (Vyas et al., 1994). Caged ATP was obtained from Calbiochem (La Jolla, CA) and was treated with apyrase (Grade V, Sigma Chemical Co., St. Louis, MO) before use. Apyrase was removed by centrifugation through a 5000 D cutoff filter. ADP and AT? were not detectable by high performance liquid chromatography (HPLC) analysis after this treatment. Caged 3H-ATP was synthesized and purified as described previously (Vyas et al., 1994). The desired specific activity of the caged 3H-ATP used for individual experiments was obtained by mixing unlabeled caged ATP with the purified caged 3H-ATP having a specific activity of -8 Ci/mmol. Because caged ATP has a lower affinity for Mg2+ than ATP, the free Mg^sup 2+^ of the unphotolyzed solution was adjusted as shown in Table 1.
Muscle freezing and extraction of nucleotides
Muscles were routinely frozen by immersion in liquid NZ except in experiments in which the time course of ADP formation was determined following photolysis of caged 3H-ATP. Muscles were pulverized in frozen 0.5 N HClO^sub 4^, and the acid extract adjusted to pH 7.4 with KOH. The extract was subjected to HPLC on a versapak NH2 column (Alltech Associates, Deerfield, IL) using mobile phases of 0.05-0.5 M NH4H2PO4, 5 mM EDTA, pH 4.0. The [NH4H2PO4] and the mobile phase gradients used for a particular experiment were adjusted to give a good separation of the nucleotides of interest. The column effluent was collected, scintillation fluid added, and radioactivity determined in a liquid scintillation counter. A similar separation technique was used to monitor the purity of the various nucleotides added to the solutions for the permeabilized muscles. Phosphorylation and
thiophosphorylation of twitchin
Twitchin was phosphorylated in the permeabilized muscles by the addition of cAMP (100 /aM) in the presence of MgATP (Siegman et al., 1997; Butler et al., 1998). In some experiments, thiophosphorylation of twitchin was accomplished by incubation of the muscle in a rigor solution containing ATP-/S (100 gM) and cAMP (100 aiM). This was followed by several washes of the muscle in rigor solution. Previous experiments have shown that twitchin is thiophosphorylated by this procedure, and that this thiophosphorylation is resistant to phosphatase activity (Siegman et al., 1997, 1998). Muscles treated in this way were then subjected to the protocols of interest and are designated as "twitchin prethiophosphorylated."
Other materials
Statistics
Data are expressed as mean +/- SEM. Statistical analyses were performed using either the t-test or one-way ANOVA.
RESULTS
Fig. 1 A shows the protocol for the production of catch force in the permeabilized ABRM. When the muscle is transferred to a relaxing solution after activation in pCa 5, there is an initial rapid relaxation followed by a much slower rate of decrease in force. We have previously shown that the force maintained in pCa > 8 is associated with a very low ATPase and a loss of force redevelopment following a rapid decrease in muscle length. The addition of cAMP causes the phosphorylation of twitchin and a rapid decrease of catch force. Also, if twitchin is prethiophosphorylated by treatment of the muscle with ATP-yS and cAMP, catch is prevented and transfer to low [Ca2+] results in rapid relaxation (Siegman et al., 1997).
Relaxation of rigor force with MgATP
In order to study the cross-bridge cycle in catch muscle, we felt that it was appropriate to start with the cross-bridges in a well-defined state such as rigor. The first experimental design addressed the question of whether catch crossbridges in rigor detach following addition of ATP, and whether twitchin phosphorylation plays a role in regulating such detachment. The muscles were put into a high force rigor state by removing ATP during activation in pCa 5. Following treatment with apyrase to minimize [ATP] and [ADPI, the muscle was transferred to the low [Ca2+] solution followed by addition of ATP. Typical force responses are shown in Fig. I B. In muscles in which twitchin was not phosphorylated, addition of ATP resulted in a rapid decrease of -50% of the rigor force with the remaining force declining at a much slower rate. The subsequent addition of cAMP caused a further reduction of force. In muscles in which twitchin was thiophosphorylated before the rigor treatment, the addition of ATP resulted in the rapid loss of almost all of the rigor force with no subsequent effect of cAMP. These results suggest that when twitchin is not phosphorylated, the addition of ATP to muscles in rigor results in some cross-bridges remaining attached to actin in the catch state.
Role of MgADP
Because the affinity of myosin for MgADP can be very high in some smooth muscles (Fuglsang et al., 1993), we carried out several experiments to test whether the persistence of the catch state after rigor treatment was due to ADP remaining on the cross-bridge in the rigor solution or to ADP competing with ATP for binding to the rigor cross-bridge. Total [ADP] in solutions containing 20 mM phosphocreatine, 1 mM MgATP, and creatine phosphokinase was 0.8 0.2 /iM (n = 6), making MgADP -0.5 lkM. Addition of this solution containing very low [MgADP] and an ATP regenerating system to the muscle following rigor treatment caused a similar mechanical response (dotted line, Fig. 1 B). The persistence of catch force is not likely due to ADP competing with ATP for binding to the rigor cross-bridge, unless the myosin in rigor has an extraordinarily high affinity for MgADP.
Another possible mechanism for the persistence of catch force following treatment in rigor solution is that ADP remains bound to the cross-bridge. If so, the catch force after addition of ATP would result from AMADP crossbridges already present. The addition of ATP would have no effect on force output from these cross-bridges until ADP was released and ATP could subsequently bind. In order to investigate this possibility, we measured the ADP content of the muscle in rigor. The procedure involved activation (pCa 5) of the muscle in a solution containing 3H-ATP so that any ADP bound to myosin would contain tritium. The muscles were then subjected to the rigor protocol and frozen. The results are shown in Table 2. Also included are data from muscles that were frozen in a pCa 5 solution containing ATP and phosphocreatine. The latter represents ADP bound during cross-bridge cycling and force maintenance, and has been shown to be a good estimate of myosin SI concentration in mammalian smooth muscle (Butler et al., 1989). Both [ADP] and [ATP] were very low in the rigor muscles. Furthermore, there was no significant effect of thiophosphorylation of twitchin on [ADP] (difference = 0.3 0.5 jiM). We conclude that only a very small fraction (<4%) of myosin retains ADP following the rigor protocol. It is unlikely that a slow ADP release from such a small fraction of myosin could result in the catch force maintenance seen following the addition of ATP to the rigor muscles. Also, the large dependence of mechanical response on the phosphorylation state of twitchin is not matched by a difference in ADP content in the two groups.
Kinetics of relaxation of rigor force following photolysis of caged ATP
In an effort to further characterize the mechanism by which catch force persists following addition of ATP to a rigor muscle, we performed experiments in which a rapid increase in [MgATP] was initiated by photolysis of caged ATP. Muscles were put into high force rigor using a procedure similar to that shown in Fig. 1 B, followed by addition of caged ATP and subsequent flash photolysis. Fig. 2 A shows typical force responses following the flash. When twitchin was prethiophosphorylated, the increase in ATP resulted in a rapid fall in force with little or no further decrease upon addition of cAMP. In contrast, when twitchin was not phosphorylated, there was a much smaller rapid decrease in force and a large subsequent relaxation with cAMP treatment. Fig. 2 B shows the initial time course of the fall in force normalized to the total decrease in force during the 10 s following the flash. Interestingly, the time course of the fast component is independent of the state of twitchin phosphorylation. A simple interpretation of these data is that there are two types of cross-bridges. One type detaches rapidly, and the other type (representing those in catch) detaches very slowly. Twitchin phosphorylation appears to move catch cross-bridges into the fast detaching group.
Effect of MgADP on the kinetics of relaxation of rigor force
The next question that was addressed was whether addition of MgADP could alter the distribution of cross-bridges between the fast and very slowly detaching groups. The experimental design was similar to that used in Fig. 2 except that in some muscles 1 mM MgADP was included in the caged ATP solution. Fig. 3 A shows the force remaining as a fraction of the total change in force that results from addition of ATP and subsequent treatment with cAMP. In the absence of ADP, the half-time for the decrease in force that occurred within 14 s after the flash was similar whether twitchin was phosphorylated or not (0.37 0.05 s and 0.44 0.05 s, respectively, n = 4 in each). However, the fraction of force in the fast component was much larger when twitchin was thiophosphorylated. In the presence of 1 mM MgADP, there is a slower rate of decrease in force following photolysis of caged ATP. The half-time increased to 1.39 0.18 s and 1.24 + 0.29 s (n = 4 in each) in the absence and presence of twitchin thiophosphorylation, respectively. The dramatic effect of MgADP on the early time course (500 ms following photolysis) is shown in Fig. 3 B for muscles in which twitchin was unphosphorylated. In contrast, the force remaining after 2 s is not significantly different in the presence or absence of 1 mM MgADP (see Fig. 3 A). When twitchin is unphosphorylated, it appears that the effect of MgADP is to slow the rapidly detaching cross-bridges, but these cross-bridges still detach much more rapidly than those exhibiting catch. The entire effect of a high concentration of ADP is seen on the fast detaching cross-bridges and is complete in the first several seconds. These results show that the addition of MgATP in the presence of a high concentration of MgADP does not result in a conversion of rapidly detaching cross-bridges into catch cross-bridges that would detach very slowly over a period of several minutes.
Does the rigor catch cross-bridge bind and split MgATP?
The observations that addition of ATP to a rigor muscle does not relax catch force and that the amount of catch force that persists following rigor is independent of the [MgADP] raise the possibility that the catch cross-bridge is an attached rigor state that binds neither ATP nor ADP. When ATP is liberated from caged ATP, both skeletal and cardiac muscles in rigor show a rapid burst of ADP formation that is approximately equal to the myosin St concentration under both relaxing and activating conditions (Ferenczi et al., 1984; Barsotti and Ferenczi, 1988). Since the burst of ADP is thought to result from the splitting of ATP on myosin, it should not occur if the rigor cross-bridge does not bind ATP. It should then be possible to detect a decrease in the magnitude of the ADP burst if the rigor cross-bridge in catch does not bind ATP. To test this, we measured the time course of 3H-ADP formation following the photolysis of caged 3H-ATP in rigor muscles whether or not twitchin was prethiophosphorylated. The protocol for these experiments was similar to that shown in Fig. 2 except that radiolabeled caged ATP was used, and the muscles were frozen 0.2 to 10 s after the flash. The results are shown in Fig. 4. There is a rapid burst of ADP formation that is substantially complete by the first measurement at 200 ms. This is followed by a much slower rate of ADP formation. The magnitude of the ADP burst is similar to the magnitude of the 3H-ADP bound in muscles that were frozen in the steady state in a pCa 5 solution containing 3H-ATP and phosphocreatine. As described earlier, this has been shown to be a good estimate of myosin SI concentration in muscle, and the ADP burst in these experiments is thus approximately equal to the myosin SI concentration. Importantly, there is no effect of twitchin thiophosphorylation on the time course of ADP formation, even though there is a large effect of such a thiophosphorylation on the mechanical response following photolytic release of ATP (see Figs. 2 and 3). Therefore, we conclude that there is similar ATP binding to myosin and rapid splitting to bound products, whether there is rapid relaxation when twitchin is thiophosphorylated or whether there is persistence of catch force when twitchin is unphosphorylated. These results suggest that unphosphorylated twitchin prevents the detachment of the rigor catch cross-bridge following ATP binding and that it traps the very slowly cycling catch cross-bridge in an ADP-bound state.
Effect of ATP analogs on relaxation of rigor force
In order to further probe the mechanism responsible for the persistence of catch force maintenance after rigor, we used the ATP analogs AMP-PNP and ATP(gamma)S. These analogs are non- or slowly hydrolyzable and result in weak binding cross-bridge states when bound to myosin (Kraft et al., 1992; Frisbie et al., 1998). In these experiments, muscles were put into a high force rigor followed by the addition of MgAMP-PNP (Fig. 5 A) or MgATP(gamma)S (Fig. 5 B) at pCa > 8. When twitchin was not phosphorylated, the addition of MgAMP-PNP resulted in a relatively small, rapid fall in force followed by a slower rate of relaxation. When twitchin was prethiophosphorylated, the initial rapid fall in force was much larger (Fig. 5 A). The mechanical responses to addition of MgATP-yS showed a similar dependence on twitchin phosphorylation (Fig. 5 B). In the case of MgATP(gamma)S it was also possible to directly show the effect of twitchin phosphorylation by adding cAMP during the slow fall in catch force. When twitchin is thiophosphorylated, there is a large increase in rate of decline in force (Fig. 5 B). These results for both AMP-PNP and ATP-yS are similar to those obtained for the addition of MgATP (see Fig. 1 B). This suggests that the persistence of the catch state following rigor does not require splitting of nucleotide on myosin, and that the effects of twitchin phosphorylation on force output extend to the regulation of the detachment rate of the cross-bridge with nucleoside triphosphate bound.
The results described so far suggest that at low [Ca2+] unphosphorylated twitchin has a major effect on at least two steps of the cross-bridge cycle. Twitchin prevents the detachment of the rigor catch cross-bridge when it binds MgATP. In addition, it appears to trap the cross-bridge in an ADP-bound force-maintaining state. The next series of experiments was designed to investigate the turnover of myosin-bound ADP in the catch state and when catch is released by the cAMP-mediated phosphorylation of twitchin.
Turnover of myosin-bound ADP during catch force maintenance
In order to determine the turnover of myosin-bound ADP in the catch state, muscles were put into high force rigor and subjected to flash photolysis of caged 3H-ATP, as shown by the design in Fig. 6 A. As shown earlier, this results in tritium-labeled ADP being bound to myosin both when twitchin is unphosphorylated and when twitchin is prethiophosphorylated. 14C-ATP containing 20 mM phosphocreatine was added 7 s after the flash and the muscles were frozen 2 min later. In the case when twitchin is unphosphorylated, there is catch force maintenance during the time the muscle is in 14C-ATP, but when twitchin is prethiophosphorylated, catch force is relaxed before 14C-ATP is added. As expected, there was no significant difference in the total exchangeable ADP (the sum of 3H-ADP and 14C-ADP) in the two designs (radiolabeled ADP = 62 3 ttM and 61 + 9 (mu)M for prethiophosphorylated and unphosphorylated twitchin, respectively). However, in muscles maintaining catch force because twitchin was unphosphorylated, 14CADP was a significantly higher fraction of the total ADP (0.325 0.010 versus 0.242 +/- 0.016, for unphosphorylated and thiophosphorylated twitchin, respectively, n = 4 in each). This higher turnover of bound ADP in muscles maintaining catch force suggests that cross-bridges that maintain catch force do indeed cycle and utilize ATP at a rate faster than cross-bridges in muscles in which catch force is not maintained. It is important to note, however, that the extra turnover of bound ADP associated with catch force maintenance is small and is measured over a long time period (2 min). Therefore, it represents a very slow rate of ATPase during catch.
Turnover of myosin-bound ADP during relaxation of catch
The next experiment was designed to determine whether the release of catch force maintenance by cAMP-mediated phosphorylation of twitchin is associated with an increase in the turnover of myosin-bound ADP. This would be expected if the primary cross-bridge state has ADP bound in catch and if the relaxation caused by phosphorylation of twitchin was associated with the release of ADP followed by ATP binding and splitting on myosin. The design is illustrated in Fig. 6 B. Twitchin was not prethiophosphorylated in either design. At 7 s following flash photolysis of caged 3H-ATP, the muscles were placed in a solution containing 14C-ATP and phosphocreatine (20 mM) for 15 s; cAMP was then added to one of the muscles (top trace) which was frozen 30 s later. The control muscle (bottom trace) was treated identically except that cAMP was not added. In paired comparisons, there was a 16 6% (n = 8, p < 0.05) higher ratio of 14C: 3H in ADP in the muscles that have relaxed from catch because of the treatment with cAMP. These results are consistent with the idea that detachment of catch force maintaining cross-bridges is associated with release of ADP from myosin and subsequent binding and splitting of ATP.
DISCUSSION
Addition of ATP to the ABRM in high force rigor at pCa > 8 results in a rapid loss of less than one-half of the force over a time period of several seconds, with the remaining force showing a very slow rate of decay over many minutes. The characteristics of the fast component of force decay are similar to those expected from muscles that do not display catch. The overall time course of relaxation of this component following photolysis of caged ATP (Figs. 2 and 3) is similar to that reported for apyrase-treated rabbit femoral artery (Fuglsang et al., 1993). Our interpretation is that the cross-bridges contributing to this initial rapid relaxation represent a subset of cross-bridges that do not participate in catch force maintenance under the conditions studied. The long-lasting force that persists after addition of ATP is due to catch cross-bridges, which rapidly detach when cAMP is added and twitchin is phosphorylated. The premise that maintenance of catch force results from force-bearing links between myosin and actin raises the question as to how the transition from the rigor state to the catch state in the presence of ATP occurs without apparent detachment of these cross-bridges and loss of force.
Experiments probing the maintenance of tonic force in mammalian smooth muscle have been analyzed using Scheme 1 (Khromov et al., 1995, 1996).
In such a scheme, the long-term persistence of force following addition of ATP to a muscle subjected to rigor treatment could result if 1) a significant fraction of the myosin in the rigor muscle still has ADP bound, and/or 2) there is binding of ADP rather than ATP to the rigor cross-bridge. In the first case, the cross-bridge would not detach until ADP was released, and this could be a very slow process in catch. In the second case, relaxation would be delayed because AMADP is reformed with ADP binding. Both have been shown to contribute to slow detachment of cross-bridges in mammalian smooth muscle (Fuglsang et al., 1993; Khromov et al., 1995, 1996, 1998). The results from the experiments reported here do not support either of these possibilities for the persistence of catch force following addition of ATP to a muscle subjected to a rigor protocol. Measurements of [ADP] show that only a very small fraction of total myosin could have ADP bound in the apyrase-treated muscles (Table 2). Also, there is no apparent difference in the amount of catch force maintained when ATP is added at [MgADP] varying from < 1 pM to 1 mM (see Figs. 1 and 3).
Even though there is a lack of an effect of [MgADP] on the magnitude of catch force following addition of ATP, there was a slowing in the rapid decrease in force in the initial seconds following addition of ATP by photolysis of caged ATP (Fig. 3). The effects of MgADP shown here are similar to those reported by Galler and colleagues (1999) who found that in the ABRM, the force decay following release of ATP was slowed -5-fold in the presence of 0.5 mM MgADP. They also note that even in the presence of high [MgADP], the decrease in force is too rapid to account for catch. The addition of high concentrations of [MgADP] to the rigor muscle is not sufficient to convert the otherwise rapidly detaching cross-bridges to catch cross-bridges. The effect of MgADP on this rapidly detaching subset of crossbridges is as expected using the analysis shown in Scheme 1, and is broadly similar to that seen in experiments on a variety of smooth (Fuglsang et al., 1993), skeletal (Dantzig et al., 1991), and cardiac (Martin and Barsotti, 1994) muscles. An important finding in the ABRM is that no manipulation involving [MgADP] and/or the ratio [MgATP]: [MgADP] changed the fraction of cross-bridges that comprises this rapidly detaching subset. It is the distribution of cross-bridges into those that detach rapidly after the addition of ATP and those that detach with a much slower rate that is controlled only by the phosphorylation state of twitchin.
We also considered the possibility that catch force maintenance after addition of ATP could result from catch being a rigor state (AM) which for some reason binds neither ADP nor ATP. This does not appear to be the case, since the magnitude of the rapid burst of ADP formation following the photolytic release of ATP from caged ATP is similar to the bound ADP present in a contracting muscle at pCa 5 (see Fig. 4). Rather, it appears that most of the myosin binds ATP and splits it to ADP and Pi, whether or not twitchin is phosphorylated. If twitchin is phosphorylated, the crossbridge detaches; if twitchin is not phosphorylated, detachment of the catch cross-bridge does not occur.
The experiments using analogs of ATP (Fig. 5) further delineate the steps in the cross-bridge cycle that are controlled by phosphorylation of twitchin. The effects of twitchin phosphorylation on the mechanical responses to additions of AMP-PNP and ATP-yS to muscles in high-force rigor are very similar to those seen with ATP. The magnitude of the rapid decrease in force following addition of nucleoside triphosphate is much larger when twitchin is thiophosphorylated. X-ray diffraction studies (Frisbie et al., 1998) and mechanical studies (Heizmann et al., 1997) have shown that the complex of AMP-PNP with myosin is similar to the weak binding cross-bridge state normally present in relaxed muscles. Similar results have been reported for the slowly hydrolyzable analog MgATP-yS (Kraft et al., 1992). Our findings of continued maintenance of catch force in the presence of these analogs of ATP suggest that when the catch cross-bridge binds the analog it does not detach from actin. That is, the catch cross-bridge with nucleoside triphosphate bound does not detach from actin until twitchin is phosphorylated. Detachment of the catch cross-bridge is very slow whether ADP or nucleoside triphosphate is bound. Therefore, in the absence of calcium and twitchin phosphorylation, a cross-bridge transition necessary for detachment is prevented whether myosin has nucleoside diphosphate or triphosphate bound.
The persistence of catch force maintenance following rigor in the presence of the non-hydrolyzable analog AMP-- PNP argues against the idea that maintenance of catch force after rigor could result from the cooperative reattachment of cross-bridges that have initially detached from actin. Such a reattachment would have to occur without the splitting of AMP-PNP, and it is unlikely that the cross-bridge could make the transition from the weakly bound to the strongly bound state without such splitting.
These data strongly suggest that the catch cross-bridge in rigor binds MgATP, but does not detach from actin. This inhibition of the detachment of myosin following binding of MgATP to the rigor cross-bridge obviously facilitates the long-term maintenance of force in the catch state. Indeed, no matter what other steps in the cross-bridge cycle are regulated in catch, inhibition of detachment is sufficient to result in catch force maintenance.
We have further investigated the control of the crossbridge cycle in the catch muscle by using single turnover experiments to characterize the turnover of myosin bound ADP. The results in the catch muscle show an extra turnover of bound ADP when 1) the muscle maintains catch force compared to when catch force is not present, and 2) catch force is relaxed by addition of cAMP and phosphorylation of twitchin.
Although the turnover of bound ADP during catch force maintenance was higher than in the absence of catch force, the rate is quite slow. With the simple assumption that all myosin cycles at the same rate, the rate constant for ADP turnover during catch would be 0.2 min-'. This is almost 200-fold slower than the ATPase rate of 0.6 s-1 measured during maximal activation of the permeabilized ABRM (Butler et al., 1998). There have been other reports of small, but measurable, suprabasal ATPases associated with catch force maintenance in both intact (Baguet and Gillis, 1968) and skinned (Butler et al., 1998) ABRM.
The observation that the rigor catch cross-bridge appears to rapidly bind ATP and split it to ADP and Pi (Fig. 4), together with the fact that the ATPase during catch is very slow, suggests that the primary cross-bridge state during catch is one with ADP bound to myosin. A likely scenario for the release of catch following phosphorylation of twitchin would be the release of ADP from the actin-bound cross-bridge followed by ATP binding, detachment of the cross-bridge, and subsequent splitting of ATP on detached myosin. This is essentially the completion of a "normal" cross-bridge cycle. The single turnover experiments reported here show an extra turnover of bound ADP when catch force is released by addition of cAMP and phosphorylation of twitchin. This supports the idea that phosphorylation of twitchin facilitates the release of ADP from the catch cross-bridge. At low [Ca2+], the cross-bridge appears to be "trapped" in an attached, ADP-bound state when twitchin is unphosphorylated. Phosphorylation of twitchin allows the cross-bridge to proceed to the release of ADP and eventual detachment of the cross-bridge.
In summary, the essential elements of the cross-bridge cycle in catch and its regulation by twitchin phosphorylation are twofold. The first is that the cross-bridge in catch is an ADP bound state that releases ADP very slowly. This must result from a dramatic slowing of at least one reaction preceding the release of ADP from actin-bound myosin. This accounts for the very slow ATPase associated with catch and keeps the cross-bridge attached to actin. The second aspect of the catch cross-bridge is inhibition of the detachment of myosin from actin following binding of MgATP to the rigor cross-bridge. This keeps the crossbridge attached to actin in catch even if ADP is released from AMADP. Both ADP release from AMADP and crossbridge detachment following binding of MgATP are increased by phosphorylation of twitchin.
A proposal for the mechanism of catch
Fig. 7 shows a cross-bridge cycle that is consistent with the results from experiments described here. Regulation of the catch state in this cycle is based mainly on the postulate that the unbinding of calcium from an attached force generating cross-bridge traps the cross-bridge in a force-generating state in the absence of twitchin phosphorylation, but not when twitchin is phosphorylated. It is an extension of a model that we have previously described (Butler et al., 1998).
It is assumed that the myosin in ABRM exhibits two force-generating states, as has been described for vertebrate smooth muscle and non-muscle myosins (Whittaker et al., 1995; Jontes et al., 1995; Gollub et al., 1996; Jontes and Milligan, 1997). The first state is designated Ml and the second, which is characterized by an extra swing of the lever arm of myosin, is designated M2 in Fig. 7. We propose that the rate of isomerization of myosin from MI to M2 (reaction 4) is controlled both by calcium binding and phosphorylation of twitchin. When calcium is bound to AM,ADP, the rate constant would be relatively fast and independent of the phosphorylation state of twitchin. When calcium is removed from AM,ADP, the isomerization to AM2ADP is dependent on whether or not twitchin is phosphorylated. When twitchin is phosphorylated, the isomerization proceeds rapidly, but when twitchin is not phosphorylated, isomerization of calcium-free AM1ADP to AM2ADP is essentially prevented. This could occur if the unphosphorylated twitchin interacts with the calcium-free myosin head in such a way as to block the structural rearrangement leading to movement of the lever arm of myosin to its final position. Such an interaction could be analogous to that of cardiac myosin-binding protein C (MyBP-C) and the S2 portion of myosin close to the lever arm (Gruen and Gautel, 1999). The interaction appears to modulate contractility of the muscle (Kunst et al., 2000) and is abolished by phosphorylation of MyBP-C (Gruen et al., 1999).
The model shows the following scenario for a catch contraction. An increase in intracellular [Ca 21] leads to calcium binding to myosin, cross-bridge attachment and fast cycling as shown in reactions 1-8. When [Ca2+] decreases and unbinding of Ca2+ from myosin occurs, some crossbridges are trapped in AM^sub 1^ADP (the catch state) since the isomerization of AM,ADP to AM2ADP is inhibited. Phosphorylation of twitchin allows the isomerization (reaction 4) to proceed with completion of the cycle. This results in the relaxation of force and the turnover of myosin bound ADP associated with reactions 4-8.
There are several results from the experiments described here that suggest that the scheme involving steps 1- 8 in Fig. 7 needs to be expanded. This is based mainly on the finding that catch force persists after rigor treatment if twitchin is not phosphorylated. Under conditions where [Ca2+] is low and twitchin is not phosphorylated, there are rigor crossbridges that do not detach upon addition of ATP, even though ATP binds and is split to ADP and Pi. This suggests that the structural conformation of myosin that is associated with the catch state persists under rigor conditions. There is also evidence that both the intermediate and final force-- generating conformations of myosin exist in rigor conditions in vertebrate smooth muscle fibers (Gollub et al., 1999). We have therefore included a rigor state designated AM, to the scheme shown in Fig. 7. The next question is what happens when AM, binds ATP. Our data suggest that if twitchin is not phosphorylated, ATP is split without detachment of the cross-bridge (reaction 9), and the muscle remains in catch. If twitchin is phosphorylated, it is likely that AM^sub 1^ATP makes the transition to AM2ATP (reaction 10) followed by detachment, etc. Of course, ATP binding to AM2 would result in rapid detachment of the cross-bridge, and this could account for the initial rapid relaxation of some of the force seen upon addition of ATP to the rigor muscle.
The model is also consistent with the observed effects of MgADP. The cross-bridge in the AM, state stays attached (in catch) whether it binds MgATP or MgADP. The force response of the catch cross-bridge is therefore independent of any competition between MgATP and MgADP binding to the rigor cross-bridge. However, addition of MgADP to AM2 would delay the decrease in force seen upon addition of MgATP, but does not cause myosin to revert to AM,. This would account for the slowing of the initial relaxation by MgADP as shown in Fig. 3 without an effect on the fraction of cross-bridges in catch. The muscle appears to enter into the catch state independently of the ADP concentration, and also to relax catch force even at high ADP concentrations when twitchin is phosphorylated. Such a mechanism is well suited to the effective functioning of a muscle, which must maintain high force and resistance to stretch for very long periods of time and must be able to relax when required.
This work was supported by National Institutes of Health Grant AR 42758 (to M.J.S.).
[Reference]
REFERENCES
[Reference]
Axelson, J. T., J. W. Bodley, and T. F. Walseth. 1981. A volatile liquid chromatography system for nucleotides. Anal. Biochem. 116:357-360.
Baguet, F., and J. M. Gillis. 1968. Energy cost of tonic contraction in lamellibranch catch muscle. J. Physiol. 198:127-143.
Barsotti, R. J., and M. A. Ferenczi. 1988. Kinetics of ATP hydrolysis and tension production in skinned cardiac muscle of the guinea pig. J. Biol. Chem. 263:16750-16756.
Bayliss, W. M. 1927. Principles of General Physiology. Longmans, Green and Co., New York.
Butler, T. M., S. U. Mooers, C. Li, S. Narayan, and M. J. Siegman. 1998. Regulation of catch muscle by twitchin phosphorylation: effects on force, ATPase, and shortening. Biophys. J. 75:1904-1914.
Butler, T. M., D. S. Pacifico, and M. J. Siegman. 1989. ADP release from myosin in permeabilized smooth muscle. Am. J. Physiol. Cell Physiol. 256:C59-66.
Castellani, L., and C. Cohen. 1987. Myosin rod phosphorylation and the catch state of molluscan muscles. Science. 235:334-337.
Cole, R. A., and B. M. Twarog. 1972. Relaxation of catch in a molluscan smooth muscle I. Effects of drugs which act on the adenyl cyclase system. Comp. Biochem. Physiol. 43:321-336.
Cornelius, F. 1980. The regulation of tension in a chemically skinned molluscan smooth muscle. Effect of Mgz+ on the Caz+-activated tension generation. J. Gen. Physiol. 75:709-727.
Cornelius, F. 1982. Tonic contraction and the control of relaxation in a chemically skinned molluscan smooth muscle. J. Gen. Physiol. 79: 821-834.
[Reference]
Dantzig, J. A., R. J. Barsotti, S. Manz, H. L. Sweeney, and Y. E. Goldman. 1999. The ADP release step of the smooth muscle cross-bridge cycle is not directly associated with force generation. Biophys. J. 77:386-397.
Dantzig, J. A., M. G. Hibberd, D. R. Trentham, and Y. E. Goldman. 1991. Cross-bridge kinetics in the presence of MgADP investigated by photolysis of caged ATP in rabbit psoas muscle fibres. J. Physiol. 432: 639-680.
Dillon, P. F., M. 0. Aksoy, S. P. Driska, and R. A. Murphy. 1981. Myosin phosphorylation and crossbridge cycle in arterial smooth muscle. Science. 211:495-497.
Ferenczi, M. A., E. Homsher, and D. R. Trentham. 1984. The kinetics of magnesium adenosine triphosphate cleavage in skinned muscle fibres of the rabbit. J. Physiol. 352:575-599.
Frisbie, S. M., S. Xu, J. M. Chalovich, and L. C. Yu. 1998. Characterizations of cross-bridges in the presence of saturating concentrations of MgAMP-PNP in rabbit permeabilized psoas muscle. Biophys. J. 74: 3072-3082.
[Reference]
Fuglsang, A., A. Khromov, K. Tor&k, A. V. Somlyo, and A. P. Somlyo. 1993. Flash photolysis studies of relaxation and cross-bridge detachment: higher sensitivity of tonic than phasic smooth muscle to MgADP. J. Muscle Res. Cell Motil. 14:666-677.
Galley, S., H. Kogler, M. Ivemeyer, and J. C. Ruegg. 1999. Force responses of skinned molluscan catch muscle following photoliberation of ATP. Pflugers Arch.-Eur. J. Physiol. 438:525-530.
Gollub, J., C. R. Cremo, and R. Cooke. 1996. ADP release produces a rotation of the neck region of smooth myosin but not skeletal myosin. Nat. Struct. Biol. 3:796-802.
Gollub, J., C. R. Cremo, and R. Cooke. 1999. Phosphorylation regulates the ADP-induced rotation of the light chain domain of smooth muscle myosin. Biochemistry. 38:10107-10118.
Green, M., and M. Gautel. 1999. Mutations in beta-myosin S2 that cause familial hypertrophic cardiomyopathy (FHC) abolish the interaction with the regulatory domain of myosin-binding protein-C. J. Mol. Biol. 286:933-949.
Green, M., H. Prinz, and M. Gautel. 1999. cAPK-phosphorylation controls the interaction of the regulatory domain of cardiac myosin binding protein C with myosin-S2 in an on-off fashion. FEBS Lett. 453: 254-259.
Haeberle, J. R. 1999. Thin-filament linked regulation of smooth muscle myosin. J. Muscle Res. Cell Motil. 20:363-370.
[Reference]
Hai, C. M., and R. A. Murphy. 1988. Cross-bridge phosphorylation and regulation of latch state in smooth muscle. Am. J. Physiol. Cell Physiol. 254:C99-06.
Heizmann, S., T. Kraft, and B. Brenner. 1997. AMP-PNP: an analog for weak-binding cross-bridge states. Biophys. J. 72:380a. (Abstr.). Himpens, B., G. Matthijs, A. V. Somlyo, T. M. Butler, and A. P. Somlyo.
1988. Cytoplasmic free calcium, myosin light chain phosphorylation, and force in phasic and tonic smooth muscle. J. Gen. Physiol. 92: 713-729.
Ishii, N., A. W. M. Simpson, and C. C. Ashley. 1989. Free calcium at rest during "catch" in single smooth muscle cells. Science. 243:1367-1368. Jewell, B. R. 1959. The nature of the phasic and the tonic responses of the
anterior byssal retractor muscle of Mytilus. J. Physiol. 149:154-177. Jontes, J. D., and R. A. Milligan. 1997. Brush border myosin-I structure and ADP-dependent conformational changes revealed by cryoelectron microscopy and image analysis. J. Cell Biol. 139:683-693.
Jontes, J. D., E. M. Wilson-Kubalek, and R. A. Milligan. 1995. A 32 degree tail swing in brush border myosin I on ADP release. Nature. 378: 751-753.
[Reference]
Khromov, A. S., A. V. Somlyo, and A. P. Somlyo. 1996. Nucleotide binding by actomyosin as a determinant of relaxation kinetics of rabbit phasic and tonic smooth muscle. J. Physiol 492:669-673.
Khromov, A., A. V. Somlyo, and A. P. Son-Ayo. 1998. MgADP promotes a catch-like state developed through force-calcium hysteresis in tonic smooth muscle. Biophys. J. 75:1926-1934.
Khromov, A., A. V. Somlyo, D. R. Trentham, B. Zimmermann, and A. P. Somlyo. 1995. The role of MgADP in force maintenance by dephosphorylated crossbridges in smooth muscle: a flash photolysis study. Biophys. J. 69:2611-2622.
Kraft, T., L. C. Yu, H. J. Kuhn, and B. Brenner. 1992. Effect of Ca 21 on weak cross-bridge interaction with actin in the presence of adenosine 5'-[gamma-thio]triphosphate. Proc. Natl. Acad. Sci. U.S.A. 89: 11362-11366.
Kunst, G., K. R. Kress, M. Green, D. Uttenweiler, M. Gautel, and R. H. Fink. 2000. Myosin binding protein C, a phosphorylation-dependent force regulator in muscle that controls the attachment of myosin heads by its interaction with myosin S2. Circ. Res. 86:51-58.
Malmqvist, U., K. M. Trybus, S. Yagi, J. Carmichael, and F. S. Fay. 1997. Slow cycling of unphosphorylated myosin is inhibited by calponin, thus keeping smooth muscle relaxed. Proc. Natl. Acad. Sci. U.S.A. 94: 7655-7660.
Martin, H., and R. J. Barsotti. 1994. Relaxation from rigor of skinned trabeculae of the guinea pig induced by laser photolysis of caged ATP. Biophys. J. 66:1115-1128.
[Reference]
Minihan, K., and R. E. Davies. 1966. Changes in inorganic phosphate and arginine during the development, maintenance and loss of tension in the anterior byssus retractor muscle of Mytilus edulis. Biochem. Z. 345: 173-187.
Pfitzer, G., and J. C. Ruegg. 1982. Molluscan catch muscle. Regulation and mechanics in living and skinned anterior byssus retractor muscle of Mytilus edulis. J. Comp. Physiol. 147:137-142.
Siegman, M. J., T. M. Butler, S. U. Mooers, and A. Michalek. 1984. Ca" can affect V._ without changes in myosin light chain phosphorylation in smooth muscle. Pflugers Arch. 401:385-390.
Siegman, M. J., D. Funabara, S. Kinoshita, S. Watabe, D. J. Hartshorne, and T. M. Butler. 1998. Phosphorylation of a twitchin-related protein controls catch and calcium sensitivity of force production in invertebrate smooth muscle. Proc. Natl. Acad. Sci. U.S.A. 95:5383-5388.
Siegman, M. J., S. U. Mooers, C. Li, S. Narayan, L. Trinkle-Mulcahy, S. Watabe, D. J. Hartshorne, and T. M. Butler. 1997. Phosphorylation of a high molecular weight (-600 kDa) protein regulates catch in invertebrate smooth muscle. J. Muscle Res. Cell Motil. 18:655-670.
Szent-Gyorgyi, A. G. 1996. Regulation of contraction by calcium binding myosins. Biophys Chem. 59:357-363.
Szent-Gyorgyi, A. G., V. N. Kalabokis, and C. L. Perreault-Micale. 1999. Regulation by molluscan myosins. Mol. Cell Biochem 190:55-62.
Twarog, B. M. 1967. The regulation of catch in molluscan muscle. J. Gem Physiol. 50:157-169.
Twarog, B. M. 1976. Aspects of smooth muscle function in molluscan catch muscle. Physiol. Rev. 56:829-838.
Vibert, P., S. M. Edelstein, L. Castellani, and B. W. Elliott. 1993. Minititins in striated and smooth molluscan muscles: structure, location and immunological crossreactivity. J. Muscle Res. Cell Motil. 14:598 - 607.
Vyas, T. B., S. U. Mooers, S. R. Narayan, M. I Siegman, and T. M. Butler. 1994. Cross-bridge cycling at rest and during activation: turnover of myosin-bound ADP in permeabilized smooth muscle. J. Biol. Chem. 269:7316-7322.
Vyas, T. B., S. U. Mooers, S. R. Narayan, J. C. Witherell, M. J. Siegman, and T. M. Butler. 1992. Cooperative activation of myosin by light chain phosphorylation in permeabilized smooth muscle. Am. J. Physiol. Cell Physiol. 263:C210-C219.
Whittaker, M., E. M. Wilson-Kubalek, J. E. Smith, L. Faust, R. A. Milligan, and H. L. Sweeney. 1995. A 35-A movement of smooth muscle myosin on ADP release. Nature. 378:748-751.
[Author Affiliation]
Thomas M. Butler,* Srinivasa R. Narayan,* Susan U. Mooers,* David J. Hartshorne,t and Marion J. Siegman* *Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, and
^Muscle Biology Group, University of Arizona, Tucson, Arizona 85721 USA
[Author Affiliation]
Address reprint requests to Thomas M. Butler, Ph.D., Department of Physiology, Jefferson Medical College, 1020 Locust St., Philadelphia, PA 19107. Tel.: 215-503-6583; Fax: 215-503-2073; E-mail: tom.butler@ mail. tju.edu.
Japan's stock market fell to its lowest level in 2 1/2 years in since September 2005 amid renewed concern about a slowdown in the U.S. economy.
The Nikkei 225 stock average tumbled 250.67 points, or 1.96 percent, to 12,532.13 points on the Tokyo Stock Exchange, its lowest since Sept. 1, 2005. The index lost 3.27 percent Friday.
The selling came after a worse-than-expected U.S. jobs report on Friday stoked fears of a recession in the United States, a vital export market for Japan.
Market watchers said that the drop in Japan might have been worse if Japan's machinery orders _ often used to gauge the outlook of business investment _ had been weaker. Core machinery orders jumped 19.6 percent in January from February, the biggest gain in seven years, the Cabinet Office said Monday.
"The stronger-than-expected machinery data made investors hesitate to sell aggressively," said Yasuyoshi Shizuma, senior sales person at global execution services at BNP Paribas.
Mining shares fell after a stumble in metal prices Friday. Sumitomo Metal Mining sank 11 percent to 1,989 yen, while Toho Zinc dropped 10 percent to 588 yen.
Also reflecting worry about the global economy, a number of trading companies were lower. Itochu lost 8.6 percent to 1,026 yen and Marubeni shed 8.2 percent to 801 yen.
The Topix index of all the Tokyo Stock Exchange First Section issues fell 23.38 points, or 1.87 percent, to 1,224.39.
In currencies, the dollar fell to 102.00 yen, from 103.09 yen late Friday afternoon in New York. The euro rose to US$1.5382, from US$1.5335.
FRONT ROW: New film offerings from Israel
ONLY INFREQUENTLY DO ISRAELI FILMS make it onto screens in the Chicago area. Thus the upcoming Israel Film Festival is a welcome opportunity to view feature films, television series and documentaries that are being made today by Israeli filmmakers.
Included in the festival, which runs from Apr. 28 to May 3, are comedies ("Grandma Operation"), dramas ("Time of Favor") and thrillers ("Facing the Forest"), and documentaries that range from a portrait of a suicidal teen ("Liar Is The One Who is Happy"), an examination of love ("5 Love Stories") to a look at Jews who aided the Nazis in the ghettoes and camps ("Kapos").
All films will be shown at the Landmark Century Centre Cinemas, 2828 N. Clark, in Chicago. For listings and schedule call 773-248-7744.
The festival opens Sat. evening, Apr. 28, at 7:30 p.m., with the feature, "Yellow Asphalt", directed by Danny Verete and shot in the Judaean desert using Bedouin actors.
It will be followed at 9:30 p.m. by "Clean Sweep", a romantic thriller and battle of the sexes directed by Oden Davidoff.
"Time of Favor", Israel's entry for an Academy Award nomination and winner of numerous awards in Israel (reviewed in the Jewish Star, Feb. 9, 2001), can be seen on four different days of the festival.
Written and directed by Joseph Cedar, the film is based on a true story.
Set in a West Bank religious settlement, the film revolves around a charismatic yeshiva head, his vision for a Temple Mount that is in Jewish hands, and the student who determines to carry out the vision by blowing up the Dome of the Rock mosque.
One Israeli filmmaker who may be familiar to Chicago audiences is Amos Gitai, whose "Kadosh", an unflattering look at ultra-Orthodox life, played locally and is available on video (reviewed May 5, 2000).
His newest film, "Kippur", based on his experiences during the 1973 Yom Kippur War, is an anti-war film that has been shown at Cannes, Toronto and New York film festivals.
Three episodes of a popular Israeli television series have been combined into "Reaching for Heaven", directed by Yankul Goldwasser and Ron Ninio.
The father of a Tel Aviv family adopts Orthodox religious practices, and the series looks at the effects on his family, his friends and his work.
The following are brief notes on some of the films we previewed:
* "The Investigation Must Go On" is a police drama directed by Marek Rozenbaum. Initially very confusing, with jumpy camera work, it begins as an investigation of an armed robbery and murder, in which the prime suspect is a well-known singer.
Although it seems clear he is not the killer, the police investigator falls for the suspect's wife and becomes obsessed by the case, determined that the investigation will go on. He'll get a confession, he insists.
There's plenty of police brutality, and what began as an almost caper-like story turns deadly and sinister.
At the same time, there is little character development, the police are buffoons and worse, and we never have much reason to care about what happens.
* "Farewell My Cousin", a television drama directed by Sharon Amrani, is the story of a family torn apart by a spy from within their midst.
It is never clear why, but Tzion, an army officer, sold Israeli military secrets to Iran. He was caught, and sentenced to 30 years in prison.
The film opens when, after 10 years in jail, Tzion escapes and returns to his family to retrieve the money he has hidden and then to leave the country.
The family, ashamed by his actions, distressed by his escape, nonetheless remains loyal and loving.
His young cousin, however, himself about to enter the army, is thrown into turmoil. The two had once been very close, but Tzion is a traitor and action must be taken against him. No matter which way they turn, the family is devastated.
* Israel's first nomination for an Academy Award (there have only ever been four) was Ephraim Kishon's "Sallah", a 1963 comedy about a North African immigrant family and their problems in adjusting to life in Israel. Its satiric spoof on tourists and Jewish National Fund tree-planting became a classic scene.
This was probably the first Israeli film many North Americans saw, and it introduced a future star -- Topol. It is the only old film to be shown at the festival.
At an opening reception on Sun., Apr. 29, the Israel Film Festival will present Chicago film director Chuck Olin with its Lifetime Visionary Award for his documentary work.
His latest documentary, "Is Jerusalem Burning? Myth, Memory and the Battle of Latrun" will be screened April 30 (see review, this page).
Photo (Israeli actors Alon Abutbul and Ido Mouseri in Sharon Amrani's television drama, "Farewell My Cousin")
DUBLIN, Ireland - Former President Jimmy Carter accused the U.S., Israel and the European Union on Tuesday of seeking to divide the Palestinian people by reopening aid to President Mahmoud Abbas' new government in the West Bank while denying the same to the Hamas-controlled Gaza Strip.
Carter, a Nobel Peace Prize winner who was addressing a human rights conference in Ireland, also said the Bush administration's refusal to accept Hamas' 2006 election victory was "criminal."
Carter said Hamas, besides winning a fair and democratic mandate that should have entitled it to lead the Palestinian government, had proven itself to be far more organized in its political and military showdowns with Abbas' moderate Fatah movement.
Hamas fighters routed Fatah in their violent takeover of the Gaza Strip last week. The split prompted Abbas to dissolve the power-sharing government with his rivals in Hamas and set up a Fatah-led administration to govern the West Bank.
Carter said the consensus of the U.S., Israel and the EU to start funneling aid to Abbas' new government in the West Bank but continue blocking Hamas in the Gaza Strip represented an "effort to divide Palestinians into two peoples."
"All efforts of the international community should be to reconcile the two, but there's no effort from the outside to bring the two together," he said.
The U.S. and European countries cut off the Hamas-led government last year because of the Islamic militant group's refusal to renounce violence and recognize Israel. They have continued to send humanitarian aid to Gaza through the United Nations and other organizations.
In the latest crisis, the U.S., Israel and much of the West have been trying to shore up Abbas in hopes that the West Bank can be made into a democratic example that would bring along Gaza.
During his speech to Ireland's annual Forum on Human Rights, the 83-year-old former president said monitors from his Carter Center observed the 2006 election that Hamas won. He said the vote was "orderly and fair" and Hamas triumphed, in part, because it was "shrewd in selecting candidates," whereas a divided, corrupt Fatah ran multiple candidates for single seats.
Far from encouraging Hamas' move into parliamentary politics, Carter said the U.S. and Israel, with European Union acquiescence, sought to subvert the outcome by shunning Hamas and helping Abbas to keep the reins of political and military power.
"That action was criminal," he said in a news conference after his speech.
"The United States and Israel decided to punish all the people in Palestine and did everything they could to deter a compromise between Hamas and Fatah," he said.
Carter said the U.S. and others supplied the Fatah-controlled security forces in Gaza with vastly superior weaponry in hopes they would "conquer Hamas in Gaza" - but Hamas routed Fatah in the fighting last week because of its "superior skills and discipline."
Japan's retail sales in June rose for the sixth straight month, but the pace of growth is slowing.
The government said Thursday that June retail sales climbed 3.2 percent from a year earlier. While the June result was in line with economists' projection, the pace of growth was slower than March and April when retail sales jumped almost 5 percent.
"Growth in retail sales was modest. By sectors, we saw strong growth in auto sales, but that was mainly due to government incentives," said Hideki Matsumura, economist at Japan Research Institute, referring to tax breaks and government subsidies for the purchase of energy-efficient vehicles.
Auto sales in June jumped 12.4 percent year-on-year, while household machinery sales increased 4.9 percent.
Government stimulus measures to support auto sales will expire in September. Matsumura said auto demand is likely to slacken after October amid Japan's feeble economic recovery.
Sales at large retail stores fell 3.0 percent in June, down for the 27th consecutive month, underlining a deep slump in Japan's consumer demand.
NEW DELHI (AP) — Indian field hockey teams found success in past decades with deft stick work, nimble feet and artistic forays. Now, in the modern era of fast synthetic turfs, they have been reduced to "also rans."
Field hockey, widely considered India's national game, is in desperate need of a standout performance that fans hope will come at this year's Olympic Games.
But to get to London, the men's team must first qualify from an ongoing tournament in New Delhi, which fans hope will not end like the disastrous 2-0 loss to Britain in the final the Beijing 2008 qualifying competition in Chile four years ago that deprived India of an Olympic berth for the first time.
India has a rich field hockey tradition with eight Olympic gold medals, but the decline in standards since the advent of artificial surfaces in the 1970s has left it lagging far behind the national obsession of cricket.
Indian hockey teams are regularly overpowered by the likes of Australia, Germany, the Netherlands and South Korea due to a lack of power and stamina, and often given up early advantages even to less fancied teams.
India captain Bharat Chhetri acknowledges that a lack of competitiveness has been an issue in recent years.
"We're addressing problems that we face toward the end of games," Chhetri told reporters ahead of the Delhi qualifiers. "We will go into the game with the idea of attacking and setting up the game early, so that we do not have much to do toward the end.
"We'll not only qualify for the Olympics but also do well there," he said.
Australian Michael Nobbs, who took over from Spaniard Jose Brasa as India's national coach, says the team needs stronger players and must develop the ruthlessness of Australia to do well at the global level.
"We're working toward getting well-built and physically fit players in the team," Nobbs said in New Delhi as India prepared for the qualifiers. "Today Australians are successful because they also have some physically fit players in the team. Such players give you lot of advantages. They can quickly recover the ball after losing it and can also break into any attack and defense with ease."
Organizers of an upcoming club-based World Series Hockey, that does not enjoy the sanction of world body FIH, are also banking on India winning the qualifications final on Feb. 26 to provide a good promotion for their own controversial tournament that starts three days later.
The game has been in the news for all the wrong reasons since the 2010 World Cup, organized by Hockey India, which runs the game in the country.
The body organizing the WSH is the original national body — Indian Hockey Federation — which was de-registered several years ago by the FIH and the Indian Olympic Association over corruption charges and the inability to unify the men's and women's bodies.
In such a scenario, will a failure to qualify for the Olympic Games sound the death knell for the game?
"I do not subscribe to the view that Indian hockey will die if we fail to qualify for the Olympics yet again," Brasa wrote in a column in The Times of India. "I think people in India really love their national team."
India won six consecutive Olympic gold medals starting from 1928 in Amsterdam, but slumped in the world rankings since winning the 1975 World Cup and clinched the last of its eight Olympic gold medals at the boycott-marred 1980 Moscow Olympics.
Indian teams have shown glimpses of their former glory, but have failed to qualify for the semifinals of eight successive World Cups and six consecutive Olympic Games before altogether missing out on reaching Beijing.
A police chief in northern Iraq says a fire engulfed a five-story hotel killing 29 people, including foreigners.
Police Brig. Gen. Najim-al-Din Qadir of the northern Iraqi city of Sulaimaniyah says an electrical short caused the fire. The victims include Bangladeshis, several from African countries and at least one Canadian, he added Friday.
Another 22 people were injured.
He said the fire in the Suma hotel erupted late Thursday.
Sulaimaniyah is located 160 miles (260 kilometers) northeast of Baghdad. It's the second biggest city in Iraq's Kurdish autonomous region.
Chicago is changing in a way no one expected. For decades, thiswas the city of smokestacks. Now it's emerging as a world-rankedcity of arts.
Think about it. Chicago has become a great place togallery-hop, listen to classical music, drop in on a blues club or afamous museum, listen to poetry, sit in on a festival or see a playin your neighborhood.
For the big picture, take a long look at our report on theastonishing state of the arts in Chicago.
PITTSBURGH (AP) — Andrew McCutchen toned down his walk-off home run celebration over the past two years.
When McCutchen hit a game-ending homer as a rookie two years ago, the then-22-year-old centerfielder leaped high into the air with his arms held out wide as he landed on home plate to meet a mob of teammates.
This time, as McCutchen approached home after homering in the 12th inning to give the Pirates a 3-2 win over the Arizona Diamondbacks, he neatly set his helmet on the ground and nonchalantly stepped on the plate as teammates surrounded him.
"It's different now since Kendry Morales got hurt last season," McCutchen said, referring to the Los Angeles Angels' first baseman who hasn't played since breaking his leg leaping to the plate celebrating last May.
"I don't think you'll ever see me go leaping into home plate anymore like I did that night. It's too dangerous. Plus, I was tired. It was a long, hot night and I was just glad it was over and we got the win."
McCutchen led off the 12th by taking a 3-2 pitch from Zach Kroenke (0-1) high and just inside the left-field foul pole for his team-leading 10th home run.
The Pirates evened their record this late in the season for the first time since also being 30-30 on June 11, 2005. Pittsburgh has won four of five and six of eight.
"We're .500 and that's about the significance of it to us," manager Clint Hurdle said. "It will have more significance later in season when we're 500. I understand the importance of it to the fans, though, when I'm walking the streets, having lunch, having dinner, doing things around the 'Burgh. I'm glad people are excited about it, but we don't want to be satisfied with being .500 in June."
Arizona has lost three consecutive since going on a 18-4 run.
Daniel McCutchen (2-1) worked out of jams in both the 11th and 12th to earn the win in a game in which each team blew a save in the late innings.
"We had a chance to win the game," Diamondbacks manager Kirk Gibson said. "We had several opportunities in extra innings — we just didn't get it done."
McCutchen had three hits, scored twice and had two RBIs. Jose Tabata had four hits for the second time in his career and scored Pittsburgh's first run.
The Diamondbacks' Stephen Drew entered the game in the eighth as a pinch-hitter and twice drove in Kelly Johnson with singles to account for the tying and go-ahead runs.
The usual cleanup hitter, Drew was not in the lineup against left-handed starter Paul Maholm. But he extended his hitting streak to five games when he lined a single in the eighth off of Jose Veras to tie the game at 1.
Two innings later, Johnson — another regular who was given the beginning of the night off but pinch hit in the eighth — again led off by reaching base, again was sacrificed to second and again scored on a single to center by Drew.
"The first at bat, I was like, 'Just get a pitch and put it out in the middle and hit it to left-center," Drew said. "I wasn't trying to do too much. Second at bat, the same thing with Johnson on. I wasn't trying to do too much, and it worked out."
But it didn't work out for Arizona in the bottom of the inning, as J.J. Putz blew his second save in 19 opportunities when Neil Walker's one-out single scored McCutchen.
McCutchen started that rally by doubling down the right-field line.
Good friends and fellow left-handed starters Zach Duke and Maholm both had strong outings but were given no-decisions when a win would have meant either would be the all-time victories leader at PNC Park.
The Diamondbacks managed only one hit against Maholm in six shutout innings, and former Pirates teammate Duke allowed one run on nine hits in seven innings.
Maholm was in line to break a tie with Duke with 31 wins at the Pirates' 11-year-old ballpark.
"It was a good game because we both pitched well," Maholm said. "Zach did what he does. He gave up some hits but he also found ways to make big pitches to get out of some jams."
Making his third start with the Diamondbacks after spending his first six seasons with Pittsburgh, Duke allowed 11 baserunners. Only one would cross home — on a McCutchen sacrifice fly in the third.
Notes: Pirates C Chris Snyder left the game due to low back pain after sliding awkwardly at second trying to stretch a single into a double in the second inning. He was replaced by Dusty Brown and will be evaluated Thursday. ... Duke received a mixture of cheers and boos when he was announced to bat. ... Diamondbacks 1B Juan Miranda extended fully to make a diving catch on a line drive hit down the line by Walker in the eighth. ... Overbay hit his 300th double. ... Pittsburgh 3B Pedro Alvarez has experienced a re-aggravation of the tightness in his right quadriceps that has him on the disabled list. That has pushed back the schedule of when he could go on a rehabilitation assignment and ultimately return to the majors.
JOB CUTS REVERSAL?
Things were looking better in June, when the number of job cutshit 59,715, a 31-month low. But they jumped up to 85,117 in July,according to Chicago outplacement firm Challenger, Gray & Christmas.Here are the top 10 sectors for July job losses:
Consumer products 15,665
Transportation 9,820
Government 9,369
Services 7,605
Industrial goods 6,753
Health care 6,271
Aerospace/Defense 5,159
Telecommunications 4,357
Automotive 4,091
Computer 2,799
In the Midwest, 18,155 jobs were lost. Illinois led the way:
Illinois 8,438
Michigan 3,495
Missouri 2,340
Indiana 1,378
Iowa 642
Minnesota 630
Ohio 549
Wisconsin 528
Kansas 109
Nebraska 46
My friends and I continue to be perplexed by the reluctance toexpand gaming in Illinois. Are we the only residents concerned thatbillions of dollars cross the Illinois borders to the casinos inWisconsin, Michigan and Iowa?
A recent mailing from Potawatomi Bingo Casino, stated, "Theexcitement is building!" in relation to a major expansion.Certainly, a large portion of the construction cost is funded byplayers from Illinois. My understanding is Michigan and Wisconsinare both building new casinos closer to Illinois. Does anyone wonderwhy? Aren't we taking the "good neighbor policy" a little too far?
What's the reluctance to having slot machines at Illinois racetracks? They rightly deserve something after providing tax dollarsto Illinois for decades. It certainly would be cost-effective. Thebuildings are there, the people are present and as soon as themachines were installed the tax dollars would start flowing. Talkabout immediate gratification.
The rebuttal to the opposition is simple -- if you don't believein gambling, don't gamble. If you don't believe in alcohol, don'tdrink. Why the focus on a possible Chicago casino? How about everyIllinois county having a piece of the pie?
It's so American to have competition and beneficial for everyone,too.
Marilyn C. Urso,
Earlville
Restructuring seminar delivered by (a church leader who has done it and lived to tell the tale'
"If all you ever do is all you've ever done, then all you'll ever get is all you ever got." This old proverb was on the overhead as the participants entered a seminar for congregational leaders recently.
Leading the seminar was Cam Shapansky, the last congregational chair at Wanner Mennonite Church, near Kitchener, Ont., who currently works for the Californiabased Blue North Strategies organization. The church-restructuring seminar was billed as "a real life example from a church leader who has done it and lived to tell the tale!"
Shapansky became the leader of Wanner's first ministries team, a four-person group that replaced the much larger church council. Besides the church council, all the committees were reshaped into ministry teams, moving from more than 80 elected positions to only 16.
He described the process Wanner followed to restructure after the church had developed a new congregational vision, priorities and guiding principles after discovering that there were many people in the congregation who were nearly burned out and ready to leave after years of service.
On the other hand, he said that others who wanted to serve couldn't find ways of using their gifts. One woman who wanted to decorate the sanctuary for worship was shunted from one committee to another, to no avail. Under the new structure, though, Shapansky said she was blessed to go and work.
The new teams were given job descriptions with blank areas "to write their own ideas and tasks," he explained, noting that terms were shortened and leaders were tasked with finding their own replacements, a responsibility not for the faint of heart.
Quoting John Kotter, he underlined the need for a "burning platform," a term used in business management and change. "The congregation needs to have come to the place where the status quo is not an option," he said.
When challenged on this "negativity," he noted that Wanner had begun by looking at its human assets and then worked towards finding ways to free these people for service. Many times during the implementation process leaders had wondered about going back to the old structure, he admitted. He reported, though, that now over a year into the change process, people are beginning to find the new structures freeing.
The seminar was offered by Associates Resourcing the Church and Conrad Grebel University College, Waterloo, Ont., as part of a course leading to a conflict management and congregational leadership certificate.
[Sidebar]
Ross Shantz of Wilmot Mennonite Church, New Hamburg, Ont, discusses church restructuring issues with keynote speaker Cam Shapansky at a church restructuring seminar at Conrad Grebel University College.
[Author Affiliation]
STORY AND PHOTO BY DAVE ROGALSKY
Eastern Canada Correspondent
WATERLOO, ONT.
