Introduction Diffuse alveolar hemorrhage (DAH) is bleeding into the alveolar space of the lungs. Pirfenidone is an antifibrotic agent that is approved for the treatment of idiopathic pulmonary fibrosis (IPF). The most commonly reported side effects include gastrointestinal and skin-related events. We present 3 cases of hemoptysis and DAH among patients on pirfenidone therapy for IPF. Case Summaries An 88-year-old female, a 75-year-old male, and a 73-year-old male all with IPF on pirfenidone presented with hemoptysis and chest computed tomography (CT) findings of usual interstitial pneumonia (UIP) with superimposed opacities. In 2 patients, DAH was confirmed with bronchoscopy. Corticosteroids were initiated and pirfenidone discontinued in all patients, and 2 patients improved while the third continued to deteriorate. Nintedanib was initiated in the remaining 2 patients at follow-up visit with no further issues. Discussion IPF is a chronic, progressive, fibrotic interstitial lung disease (ILD) which appears to be increasing in the United States and has a relatively short survival. Nintedanib and pirfenidone were the first Food and Drug Administration (FDA)-approved agents for the treatment of IPF in October 2014. We present 3 cases of DAH in patients with IPF receiving pirfenidone. Symptoms occurred within 2 months of pirfenidone initiation and resolved with discontinuation of pirfenidone and initiation of systemic corticosteroids in 2 patients; however, one case was complicated by concomitant discontinuation of aspirin. The mechanism by which DAH occurred in our patients remains unclear. Conclusion We report the first cases of possible pirfenidone-induced DAH. Further studies are warranted to explore this reaction, but prescribers should be cognizant of this potential issue when choosing to prescribe pirfenidone.
The effect of ADP and phosphorylation upon the actin binding properties of heavy meromyosin was investigated using three fluorescence methods that monitor the number of heavy meromyosin heads that bind to pyrene-actin: (i) amplitudes of ATP-induced dissociation, (ii) amplitudes of ADP-induced dissociation of the pyrene-actin-heavy meromyosin complex, and (iii) amplitudes of the association of heavy meromyosin with pyrene-actin. Both heads bound to pyrene-actin, irrespective of regulatory light chain phosphorylation or the presence of ADP. This behavior was found for native regulated heavy meromyosin prepared by proteolytic digestion of chicken gizzard myosin with between 5 and 95% heavy chain cleavage at the actin-binding loop, showing that two-head binding is a property of heavy meromyosin with uncleaved heavy chains. These data are in contrast to a previous study using an uncleaved expressed preparation (Berger, C. E., Fagnant, P. M., Heizmann, S., Trybus, K. M., and Geeves, M. A. (2001) J. Biol. Chem. 276, 23240–23245), which showed that one head of the unphosphorylated heavy meromyosin-ADP complex bound to actin and that the partner head either did not bind or bound weakly. Possible explanations for the differences between the two studies are discussed. We have shown that unphosphorylated heavy meromyosin appears to adopt a special state in the presence of ADP based upon analysis of actin-heavy meromyosin association rate constants. Data were consistent with one head binding rapidly and the second head binding more slowly in the presence of ADP. Both heads bound to actin at the same rate for all other states. The effect of ADP and phosphorylation upon the actin binding properties of heavy meromyosin was investigated using three fluorescence methods that monitor the number of heavy meromyosin heads that bind to pyrene-actin: (i) amplitudes of ATP-induced dissociation, (ii) amplitudes of ADP-induced dissociation of the pyrene-actin-heavy meromyosin complex, and (iii) amplitudes of the association of heavy meromyosin with pyrene-actin. Both heads bound to pyrene-actin, irrespective of regulatory light chain phosphorylation or the presence of ADP. This behavior was found for native regulated heavy meromyosin prepared by proteolytic digestion of chicken gizzard myosin with between 5 and 95% heavy chain cleavage at the actin-binding loop, showing that two-head binding is a property of heavy meromyosin with uncleaved heavy chains. These data are in contrast to a previous study using an uncleaved expressed preparation (Berger, C. E., Fagnant, P. M., Heizmann, S., Trybus, K. M., and Geeves, M. A. (2001) J. Biol. Chem. 276, 23240–23245), which showed that one head of the unphosphorylated heavy meromyosin-ADP complex bound to actin and that the partner head either did not bind or bound weakly. Possible explanations for the differences between the two studies are discussed. We have shown that unphosphorylated heavy meromyosin appears to adopt a special state in the presence of ADP based upon analysis of actin-heavy meromyosin association rate constants. Data were consistent with one head binding rapidly and the second head binding more slowly in the presence of ADP. Both heads bound to actin at the same rate for all other states. Smooth muscle myosin (SMM), 1The abbreviations used are: SMM, smooth muscle myosin; HMM, heavy meromyosin; u-HMM, unphosphorylated HMM; tp-HMM, thiophosphorylated HMM; RLC, regulatory light chain; ELC, essential light chain; S1, subfragment 1 of myosin; MLCK, myosin light chain kinase; FTP, formycin triphosphate; mant-ATP, 2′(3)-O-(N-methylanthraniloyl)-ATP; DTT, dithiothreitol; AP5A, P 1,P 5-di(adenosine 5′)-pentaphosphate; ATPγS, adenosine-5′-O-(3-thiophosphate); MOPS, 4-morpholinepropanesulfonic acid 1The abbreviations used are: SMM, smooth muscle myosin; HMM, heavy meromyosin; u-HMM, unphosphorylated HMM; tp-HMM, thiophosphorylated HMM; RLC, regulatory light chain; ELC, essential light chain; S1, subfragment 1 of myosin; MLCK, myosin light chain kinase; FTP, formycin triphosphate; mant-ATP, 2′(3)-O-(N-methylanthraniloyl)-ATP; DTT, dithiothreitol; AP5A, P 1,P 5-di(adenosine 5′)-pentaphosphate; ATPγS, adenosine-5′-O-(3-thiophosphate); MOPS, 4-morpholinepropanesulfonic acid like other members of the myosin II family, has two heads connected by a coiled-coil tail. SMM and the double-headed subfragment HMM are regulated by phosphorylation of the two regulatory light chains, one on each head (1Sellers J.R. Curr. Opin. Cell Biol. 1991; 3: 98-104Crossref PubMed Scopus (172) Google Scholar, 2Sellers J.R. Goodson H. Motor Protein 2: Myosin.in: Sheterline P. Protein Profile. 2. Academic Press Limited, London1995Google Scholar, 3Hartshorne D.J. Biochemistry of the Contractile Process in Smooth Muscle.in: Johnson L.R. Physiology of the Gastrointestinal Tract. Second Ed. Raven Press, New York1987: 423-481Google Scholar). In contrast, single-headed SMM (4Cremo C.R. Sellers J.R. Facemyer K.C. J. Biol. Chem. 1995; 270: 2171-2175Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 5Konishi K. Katoh T. Morita F. Yazawa M. J. Biochem. (Tokyo). 1998; 124: 163-170Crossref PubMed Scopus (14) Google Scholar) and the single-headed S1 (6Ikebe M. Hartshorne D.J. Biochemistry. 1985; 24: 2380-2387Crossref PubMed Scopus (127) Google Scholar, 7Sellers J.R. Eisenberg E. Adelstein R.S. J. Biol. Chem. 1982; 257: 12880-12883Abstract Full Text PDF Google Scholar, 8Konishi K. Kojima S. Katoh T. Yazawa M. Kato K. Fujiwara K. Onishi H. J. Biochem. (Tokyo). 2001; 129: 365-372Crossref PubMed Scopus (23) Google Scholar) are not regulated by phosphorylation. Non-muscle HMM IIB is also regulated by phosphorylation, and constructs lacking one motor domain have been shown to be unregulated (9Cremo C.R. Wang F. Facemyer K. Sellers J.R. J. Biol. Chem. 2001; 276: 41465-41472Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Therefore, two motor domains are required for regulation.Structural differences between unphosphorylated and phosphorylated HMM have been demonstrated by a number of studies. Reconstruction of images of expressed unphosphorylated HMM in the presence of ATP in two-dimensional crystalline arrays (10Wendt T. Taylor D. Messier T. Trybus K.M. Taylor K.A. J. Cell Biol. 1999; 147: 1385-1390Crossref PubMed Scopus (94) Google Scholar, 11Wendt T. Taylor D. Trybus K.M. Taylor K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4361-4366Crossref PubMed Scopus (242) Google Scholar) shows an asymmetrical structure with the converter domain of one head bound to the actin-binding site of the other head. No interaction was seen between the motor domains of phosphorylated HMM. This model is supported by data from Berger et al. (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), who demonstrated that only one of the heads of an unphosphorylated expressed smooth muscle HMM-ADP complex binds to actin. Either the binding of the first head prevented the binding of the second head or the second head bound weakly such that no signal was observed for its binding. Both heads bound to actin in the phosphorylated state. These data are inconsistent with two studies of the effect of ADP and phosphorylation in intact gizzard muscle, which are consistent with binding of both heads of myosin to actin irrespective of ADP or phosphorylation (13Gollub J. Cremo C.R. Cooke R. Biochemistry. 1999; 38: 10107-10118Crossref PubMed Scopus (22) Google Scholar, 14Dantzig J.A. Barsotti R.J. Manz S. Sweeney H.L. Goldman Y.E. Biophys. J. 1999; 77: 386-397Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar).We investigated the actin binding properties of HMM derived from chicken gizzards. Digestion of SMM by Staphylococcus aureusV8 protease or chymotrypsin generates HMM with varying degrees of internal cleavage at loop 2 (the actin-binding loop), but the cleavage products remain associated under non-denaturing conditions (6Ikebe M. Hartshorne D.J. Biochemistry. 1985; 24: 2380-2387Crossref PubMed Scopus (127) Google Scholar, 15Ikebe M. Hartshorne D.J. Biochemistry. 1986; 25: 6177-6185Crossref PubMed Scopus (19) Google Scholar, 16Bonet A. Mornet D. Audemard E. Derancourt J. Bertrand R. Kassab R. J. Biol. Chem. 1987; 262: 16524-16530Abstract Full Text PDF PubMed Google Scholar, 17Sellers J.R. Myosins.in: Sheterline P. Protein Profiles. 2nd Ed. Oxford University Press, Oxford1999Google Scholar, 18Seidel J.C. J. Biol. Chem. 1980; 255: 4355-4361Abstract Full Text PDF PubMed Google Scholar, 19Ikebe M. Mitra S. Hartshorne D.J. J. Biol. Chem. 1993; 268: 25948-25951Abstract Full Text PDF PubMed Google Scholar). Based upon the model discussed previously, it was possible that the extent of internal heavy chain cleavage at the actin-binding loop could alter the actin binding behavior. Therefore, we produced HMM with between 5 and 95% heavy chain cleavage for this study. Measurements of the fluorescence changes upon binding of HMM to pyrene-actin and upon ATP-induced dissociation from pyrene-actin were used to determine the stoichiometry of HMM binding to actin in the unphosphorylated and thiophosphorylated states. We show that both heads of tissue-derived HMM bind to actin. This two-headed binding was observed irrespective of the extent of internal heavy chain cleavage, the presence or absence of ADP, or the phosphorylation state of the RLC. These experiments were consistent with the fact that ADP did not induce dissociation of the pyrene-actin HMM complex irrespective of the phosphorylation state. As these data contrast those for an expressed HMM construct (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), explanations for the differences between the two protein preparations are presented.We also measured the actin-activated ATPase activity of unphosphorylated and thiophosphorylated HMM by single turnover assays with both ATP and FTP. All HMM preparations used in this study were found to be fully regulated as defined by a slow turnover rate in the presence of actin for the unphosphorylated protein. Therefore, we have shown that the native tissue-derived unphosphorylated HMM-ADP complex binds to actin with two heads. The one-headed actin binding mode of an unphosphorylated HMM-ADP complex predicted by the model of Wendtet al. (10Wendt T. Taylor D. Messier T. Trybus K.M. Taylor K.A. J. Cell Biol. 1999; 147: 1385-1390Crossref PubMed Scopus (94) Google Scholar, 11Wendt T. Taylor D. Trybus K.M. Taylor K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4361-4366Crossref PubMed Scopus (242) Google Scholar) is not a property required for down-regulation.DISCUSSIONWe have demonstrated that both heads of tissue-derived HMM and HMM-ADP bind to actin, irrespective of the phosphorylation state of the RLC. These findings are consistent with the effects of ADP and phosphorylation upon measurements of RLC mobility (13Gollub J. Cremo C.R. Cooke R. Biochemistry. 1999; 38: 10107-10118Crossref PubMed Scopus (22) Google Scholar) and tension (14Dantzig J.A. Barsotti R.J. Manz S. Sweeney H.L. Goldman Y.E. Biophys. J. 1999; 77: 386-397Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) in intact smooth muscle. In contrast, Berger et al. (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) found that only one head of expressed smooth muscle u-HMM-ADP bound to actin, whereas tp-HMM, tp-HMM-ADP, and u-HMM bound with two heads. The buffer conditions and protein concentrations used in this study and the Berger et al. (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) study were similar. Berger et al. (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) used the same ATP-induced dissociation method that we used here. Therefore, it appears that the HMM preparations used in the two studies are different.One obvious difference between the HMM preparations is the presence of internal heavy chain cleavage at the actin-binding loop in tissue-derived HMM. In our HMM preparation with 5% heavy chain cleavage, a maximum of 10% of all molecules would contain at least one cleaved head with at least 90% of all molecules containing two uncleaved heads. If these uncleaved u-HMM-ADP molecules behaved in a manner similar to the expressed u-HMM-ADP, the ΔF max/F final (Table I) or ΔF max/F initial(Table II) for u-HMM-ADP would be 45% lower than that for u-HMM. Our data for u-HMM with 5% heavy chain cleavage show no significant differences between maximal fluorescence amplitude changes obtained in the presence or absence of ADP using two different methods (Fig. 3 and Table I, Fig. 5 and Table II). We conclude that the extent of heavy chain cleavage at the actin-binding loop is not the reason for the differences between expressed and tissue-derived HMM.In addition to the actin-binding loop cleavage, chymotrypsin and V8 protease cleave a small number of residues from the N terminus of the heavy chain. The V8 protease preparation was missing only 9 residues and is unlikely to explain the different actin binding behavior. For 5% cleaved chymotryptic HMM, 27 residues are cleaved, but we estimated that only 5–10% of the N terminus is cleaved. This suggests that N-terminal cleavage is not likely to explain the differences in actin binding behavior by the same reasoning described above for the actin-binding loop.Our tissue-derived HMM was not frozen at any stage of the preparation, whereas the expressed HMM was frozen in liquid nitrogen in the presence of sucrose and stored at −80 °C. We have found that freezing tissue-derived HMM in this manner causes loss of regulation. However, both Berger et al. (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) and this study (Figs. 6 and 7) showed that HMM preparations were regulated using single turnover approaches.Berger et al. (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) found that dissociation of u-HMM heads from pyrene-actin by ATP resulted in a ΔF max/F final of ∼0.4 in the absence of ADP and ∼0.2 in the presence of ADP. We wondered why they did not observe a ΔF max/F final of ∼0.8 in the absence of ADP, as would be expected from previous studies with tissue-derived smooth muscle S1 (28Cremo C.R. Geeves M.A. Biochemistry. 1998; 37: 1969-1978Crossref PubMed Scopus (148) Google Scholar), and consequently ∼0.4 in the presence of ADP. A lower than expected pyrene-actin quenching by expressed HMM might be due to the following. First, it could be an inherent property of the molecule, although the amino acid sequence of the expressed HMM is identical to that of tissue-derived HMM except for a FLAG tag at the C terminus. Second, it is possible that there are unknown post-translational modifications specific to the tissue-derived HMM. Third, a significant population of “dead heads” or “rigor heads” (heads that bind irreversibly to actin) (21Ellison P.A. Sellers J.R. Cremo C.R. J. Biol. Chem. 2000; 275: 15142-15151Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 31Dash P.K. Hackney D.D. Biochem. Int. 1991; 25: 1013-1022PubMed Google Scholar) would lower the ΔF max/F finalwithout altering the observed stoichiometry. Berger et al.(12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) showed that ADP could dissociate ∼40% of the heads from an acto-u-HMM complex but not from an acto-tp-HMM complex. The rate of this process for the acto-u-HMM complex was much slower than the rate of ADP binding and thus was consistent with a rearrangement. This suggested that these ADP heads initially bound to actin but eventually found a thermodynamically more stable place to bind or somehow lost their normal tight actin binding properties (K d < 40 nm). This result would be obtained if dead heads were abundant, as we suggested previously, and if the surface of dead heads had an extremely tight binding site for the actin-binding site of a functional partner ADP head (perhaps in a structure similar to that proposed by Wendt et al. (10Wendt T. Taylor D. Messier T. Trybus K.M. Taylor K.A. J. Cell Biol. 1999; 147: 1385-1390Crossref PubMed Scopus (94) Google Scholar,11Wendt T. Taylor D. Trybus K.M. Taylor K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4361-4366Crossref PubMed Scopus (242) Google Scholar)). Formation of this nonphysiological structure might be expected to be slow, as Berger et al. (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) observed, as it would involve dissociation of a functional ADP head from actin followed by binding (nearly irreversibly) to the partner dead head. The lack of such a motor-motor domain interaction in a tp-HMM-ADP preparation containing dead heads would be compatible with the structural data of Wendt et al. (10Wendt T. Taylor D. Messier T. Trybus K.M. Taylor K.A. J. Cell Biol. 1999; 147: 1385-1390Crossref PubMed Scopus (94) Google Scholar, 11Wendt T. Taylor D. Trybus K.M. Taylor K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4361-4366Crossref PubMed Scopus (242) Google Scholar) showing no interaction between motor domains in tp-HMM. Our results, under identical conditions to Bergeret al. (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), showed that ADP could not dissociate heads from the acto-HMM complex regardless of the phosphorylation state (Table I). Therefore, the results from both studies are internally consistent, suggesting that the HMM preparations are different.Berger et al. (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) reported that ∼25% of u-HMM heads bound ADP with an affinity of 2 μm, ∼25% bound ADP with a much weaker affinity, and the remaining ∼50% did not respond to ATP at maximal ADP concentrations. These nonresponsive heads may be attributed to dead heads behaving as described above. Nevertheless, their data strongly suggest that functional HMM binds to ADP with two different affinities. We are currently characterizing the ADP binding properties of our preparations.A test for the presence of dead heads is to compare the ΔF max/F initial and ΔF max/F final values for association and dissociation experiments, respectively. These values should be the same in the absence of dead heads. The ΔF max/F initial from an association experiment should not be affected by the presence of dead heads, whereas the ΔF max/F final from a dissociation experiment would be lowered. In our study, both dissociation (Table I) and association (Table II) of HMM heads from/to pyrene-actin resulted in maximal fluorescence changes consistent with earlier studies with tissue-derived smooth S1 (28Cremo C.R. Geeves M.A. Biochemistry. 1998; 37: 1969-1978Crossref PubMed Scopus (148) Google Scholar). This agreement between dissociation and association data is strong evidence that our preparations do not contain a significant fraction of dead heads. Furthermore, the single turnover measurements in Fig. 6 are consistent with previous steady-state measurements from our laboratory (21Ellison P.A. Sellers J.R. Cremo C.R. J. Biol. Chem. 2000; 275: 15142-15151Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Dash and Hackney (31Dash P.K. Hackney D.D. Biochem. Int. 1991; 25: 1013-1022PubMed Google Scholar) estimated that the V8-cleaved tissue-derived preparation contains ∼8% dead heads, consistent with the study of Ellison et al. (21Ellison P.A. Sellers J.R. Cremo C.R. J. Biol. Chem. 2000; 275: 15142-15151Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar).Our data do not rule out the possibility that the tissue-derived u-HMM-ADP complex can adopt a conformation like that described by Wendtet al. (10Wendt T. Taylor D. Messier T. Trybus K.M. Taylor K.A. J. Cell Biol. 1999; 147: 1385-1390Crossref PubMed Scopus (94) Google Scholar, 11Wendt T. Taylor D. Trybus K.M. Taylor K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4361-4366Crossref PubMed Scopus (242) Google Scholar). Indeed, our association rate data are consistent with the idea that the heads interact in some manner in the presence of ADP but not in its absence. Our association rate data are in agreement with a previous study by Rosenfeld et al. (32Rosenfeld S.S. Xing J. Cheung H.C. Brown F. Kar S. Sweeney H.L. J. Biol. Chem. 1998; 273: 28682-28690Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). They measured the rates of pyrene-actin binding of tissue-derived u-HMM and tp-HMM with and without ADP at high actin/HMM ratios. The binding was monophasic except for the u-HMM-ADP complex, which bound in a biphasic manner with two phases of similar amplitude. They interpreted these results to indicate that both heads of the u-HMM-ADP complex bound to actin but that an interaction between the heads slowed the binding of the second head. Under conditions similar to theirs (at the highest actin/HMM ratios of Fig. 5), we made the same observations. Therefore, our data, like those of Rosenfeld et al. (32Rosenfeld S.S. Xing J. Cheung H.C. Brown F. Kar S. Sweeney H.L. J. Biol. Chem. 1998; 273: 28682-28690Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), are consistent with an interaction between the two heads of the u-HMM-ADP complex, which must be broken to allow the second head to bind to actin. It is possible that this interaction is between the two motor domains, as described in the model of Wendt et al.(10Wendt T. Taylor D. Messier T. Trybus K.M. Taylor K.A. J. Cell Biol. 1999; 147: 1385-1390Crossref PubMed Scopus (94) Google Scholar, 11Wendt T. Taylor D. Trybus K.M. Taylor K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4361-4366Crossref PubMed Scopus (242) Google Scholar), but our data do not address this structural issue. Our data suggest that for tissue-derived HMM, if such an interaction is occurring, it is not strong enough to compete with actin to prevent binding of both heads to actin. We have also shown that one-headed actin binding behavior for u-HMM-ADP is not a requirement for down-regulation of smooth muscle myosin and that two-headed actin binding in the presence of ADP is a property of the native, undamaged, fully regulated molecule. Smooth muscle myosin (SMM), 1The abbreviations used are: SMM, smooth muscle myosin; HMM, heavy meromyosin; u-HMM, unphosphorylated HMM; tp-HMM, thiophosphorylated HMM; RLC, regulatory light chain; ELC, essential light chain; S1, subfragment 1 of myosin; MLCK, myosin light chain kinase; FTP, formycin triphosphate; mant-ATP, 2′(3)-O-(N-methylanthraniloyl)-ATP; DTT, dithiothreitol; AP5A, P 1,P 5-di(adenosine 5′)-pentaphosphate; ATPγS, adenosine-5′-O-(3-thiophosphate); MOPS, 4-morpholinepropanesulfonic acid 1The abbreviations used are: SMM, smooth muscle myosin; HMM, heavy meromyosin; u-HMM, unphosphorylated HMM; tp-HMM, thiophosphorylated HMM; RLC, regulatory light chain; ELC, essential light chain; S1, subfragment 1 of myosin; MLCK, myosin light chain kinase; FTP, formycin triphosphate; mant-ATP, 2′(3)-O-(N-methylanthraniloyl)-ATP; DTT, dithiothreitol; AP5A, P 1,P 5-di(adenosine 5′)-pentaphosphate; ATPγS, adenosine-5′-O-(3-thiophosphate); MOPS, 4-morpholinepropanesulfonic acid like other members of the myosin II family, has two heads connected by a coiled-coil tail. SMM and the double-headed subfragment HMM are regulated by phosphorylation of the two regulatory light chains, one on each head (1Sellers J.R. Curr. Opin. Cell Biol. 1991; 3: 98-104Crossref PubMed Scopus (172) Google Scholar, 2Sellers J.R. Goodson H. Motor Protein 2: Myosin.in: Sheterline P. Protein Profile. 2. Academic Press Limited, London1995Google Scholar, 3Hartshorne D.J. Biochemistry of the Contractile Process in Smooth Muscle.in: Johnson L.R. Physiology of the Gastrointestinal Tract. Second Ed. Raven Press, New York1987: 423-481Google Scholar). In contrast, single-headed SMM (4Cremo C.R. Sellers J.R. Facemyer K.C. J. Biol. Chem. 1995; 270: 2171-2175Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 5Konishi K. Katoh T. Morita F. Yazawa M. J. Biochem. (Tokyo). 1998; 124: 163-170Crossref PubMed Scopus (14) Google Scholar) and the single-headed S1 (6Ikebe M. Hartshorne D.J. Biochemistry. 1985; 24: 2380-2387Crossref PubMed Scopus (127) Google Scholar, 7Sellers J.R. Eisenberg E. Adelstein R.S. J. Biol. Chem. 1982; 257: 12880-12883Abstract Full Text PDF Google Scholar, 8Konishi K. Kojima S. Katoh T. Yazawa M. Kato K. Fujiwara K. Onishi H. J. Biochem. (Tokyo). 2001; 129: 365-372Crossref PubMed Scopus (23) Google Scholar) are not regulated by phosphorylation. Non-muscle HMM IIB is also regulated by phosphorylation, and constructs lacking one motor domain have been shown to be unregulated (9Cremo C.R. Wang F. Facemyer K. Sellers J.R. J. Biol. Chem. 2001; 276: 41465-41472Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Therefore, two motor domains are required for regulation. Structural differences between unphosphorylated and phosphorylated HMM have been demonstrated by a number of studies. Reconstruction of images of expressed unphosphorylated HMM in the presence of ATP in two-dimensional crystalline arrays (10Wendt T. Taylor D. Messier T. Trybus K.M. Taylor K.A. J. Cell Biol. 1999; 147: 1385-1390Crossref PubMed Scopus (94) Google Scholar, 11Wendt T. Taylor D. Trybus K.M. Taylor K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4361-4366Crossref PubMed Scopus (242) Google Scholar) shows an asymmetrical structure with the converter domain of one head bound to the actin-binding site of the other head. No interaction was seen between the motor domains of phosphorylated HMM. This model is supported by data from Berger et al. (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), who demonstrated that only one of the heads of an unphosphorylated expressed smooth muscle HMM-ADP complex binds to actin. Either the binding of the first head prevented the binding of the second head or the second head bound weakly such that no signal was observed for its binding. Both heads bound to actin in the phosphorylated state. These data are inconsistent with two studies of the effect of ADP and phosphorylation in intact gizzard muscle, which are consistent with binding of both heads of myosin to actin irrespective of ADP or phosphorylation (13Gollub J. Cremo C.R. Cooke R. Biochemistry. 1999; 38: 10107-10118Crossref PubMed Scopus (22) Google Scholar, 14Dantzig J.A. Barsotti R.J. Manz S. Sweeney H.L. Goldman Y.E. Biophys. J. 1999; 77: 386-397Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). We investigated the actin binding properties of HMM derived from chicken gizzards. Digestion of SMM by Staphylococcus aureusV8 protease or chymotrypsin generates HMM with varying degrees of internal cleavage at loop 2 (the actin-binding loop), but the cleavage products remain associated under non-denaturing conditions (6Ikebe M. Hartshorne D.J. Biochemistry. 1985; 24: 2380-2387Crossref PubMed Scopus (127) Google Scholar, 15Ikebe M. Hartshorne D.J. 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Measurements of the fluorescence changes upon binding of HMM to pyrene-actin and upon ATP-induced dissociation from pyrene-actin were used to determine the stoichiometry of HMM binding to actin in the unphosphorylated and thiophosphorylated states. We show that both heads of tissue-derived HMM bind to actin. This two-headed binding was observed irrespective of the extent of internal heavy chain cleavage, the presence or absence of ADP, or the phosphorylation state of the RLC. These experiments were consistent with the fact that ADP did not induce dissociation of the pyrene-actin HMM complex irrespective of the phosphorylation state. As these data contrast those for an expressed HMM construct (12Berger C.E. Fagnant P.M. Heizmann S. Trybus K.M. Geeves M.A. J. Biol. Chem. 2001; 276: 23240-23245Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), explanations for the differences between the two protein preparations
Idiopathic subglottic stenosis (ISS) is a rare type of airway stenosis of unclear etiology. Open resection, while effective, remains a complex surgery and requires a hospital stay. Endoscopic management is often preferred but has historically been associated with a high recurrence rate. We aimed to analyze our experience, consisting of a standardized endoscopic approach combined with an empiric medical treatment.Retrospective cohort study.All patients with ISS managed with standardized endoscopic treatment at our institution between 1987 and 2012 were identified, and their electronic medical records were reviewed. The treatment consisted of CO2 laser resection without dilatation and local infiltration with steroids and application of mitomycin C. Patients were also treated with antireflux medications, inhaled corticosteroids, and occasionally trimethoprim-sulfamethoxazole. The influence of medical management on annual recurrence rate was analyzed using negative binomial logistic regression.A total of 110 patients treated with standardized endoscopic management were included in our analysis. The procedure was well tolerated by all patients without complications. Recurrences were observed in approximately 60% of patients at 5 years. There was a trend suggesting an association between aggressive medical treatment and a reduction in the rate of recurrence/person/year (relative risk = 0.52, P = 0.051).A standardized endoscopic management of ISS consisting of CO2 laser vaporization of the fibrotic scar appears effective in symptom control, with 40% of patients not requiring retreatment in the follow-up period, and with recurrence noted in a majority of patients. Aggressive medical treatment may have a role, but further prospective studies are required to confirm these findings.4.
cheal tube positioned several centimeters above the main carina.We have found that the flexible bronchoscope can easily pass through the endotracheal tube and past the deflated endobronchial blocker en route to the site of biopsy.The cryoprobe is then introduced through the working channel of the bronchoscope, and biopsies are obtained under fluoroscopic guidance as previously described [1][2][3][4] .The endobronchial blocker remains deflated unless needed to assist in controlling bleeding.Upon completion of the procedure, the endobronchial blocker is removed en bloc with the endotracheal tube when the patient is clinically ready for extubation.Cryobiopsies show promise in the diagnosis of diffuse parenchymal lung diseases.While multiple small studies have reported no increased risk for significant bleeding [1][2][3][4]6] , experience with transbronchial cryobiopsy is still limited, and concerns regarding potential complications remain.Use of an endobronchial blocker is helpful to manage procedurally related hemorrhage.Being able to utilize the endobronchial blocker without the use of rigid bronchoscopy increases its availability to bronchoscopists without significant rigid bronchoscopy experience and allows for safer performance of peripheral cryobiopsies.
Objectively measured severe physical inactivity (SPI) has been reported as the strongest independent predictor of mortality in patients with chronic obstructive pulmonary disease (COPD). Activity monitoring is not feasible in routine clinical practice; therefore, we set out to determine the utility of simple clinical measures for predicting SPI in patients with COPD. A total of 165 patients with COPD wore an activity monitor for 5 days to define the presence or absence of SPI. Logistic models were generated including the modified Medical Research Council (MMRC) dyspnea grade, spirometry and the age–dyspnea–airflow obstruction (ADO) index. Physical Activity Scale for the Elderly (PASE) and Stanford Brief Activity Scale (SBAS) were also tested for validity and reliability in a subgroup of 67 patients. The MMRC dyspnea grade, PASE score, ADO index and SBAS score were associated with SPI, but general self-efficacy and spirometry were not. An MMRC dyspnea grade ≥3 was the best independent predictor of SPI (AUC: 0.74; PPV: 0.83; NPV: 0.68) followed closely by a PASE score of <111. The combination of MMRC dyspnea grade and PASE score provided the most robust model (AUC: 0.83; Positive Predictive Value (PPV): 0.95; Negative Predictive Value (NPV): 0.63). The results were confirmed using 5000 bootstrapped models from the cohort of 165 patients. MMRC dyspnea grade ≥3 may be the best triage tool for SPI in patients with COPD. The combination of the MMRC and PASE score provided the most robust prediction. Our results may have significant practical applicability for clinicians caring for patients with COPD.
