111Indium antimyosin antibody imaging of primary myocardial involvement in systemic diseases. (57/5414)

OBJECTIVE: The diagnosis of primary myocardial involvement in systemic diseases is clinically relevant but difficult in the absence of specific criteria. Whatever the underlying disease, myocytes degeneration is observed during the active phase of myocardial damage. The aim of this study was to assess the diagnostic value of scintigraphic imaging with 111Indium antimyosin antibody (AM), a specific marker of the damaged myocyte, for ongoing myocardial damage related to systemic diseases. METHODS: 40 patients with histologically confirmed systemic diseases were studied. They were classified into two groups according to the presence (group 1, n = 30), or the absence (group 2, n = 10) of clinical, electrocardiographic (ECG) or echocardiographic signs suggestive of myocardial involvement. Planar and tomographic acquisitions were obtained 48 hours after injection of AM (90 MBq). Rest 201thallium (T1) scintigraphy was also performed to assess myocardial perfusion and scarring. Clinical, ECG, and echocardiographic +/- scintigraphic evaluations were repeated during follow up (17 +/- 19 months) in 36 of 40 patients. RESULTS: In group 1, 13 of 30 patients (43%) showed diffuse significant AM uptake throughout the left ventricle (LV), and no or mild T1 abnormality. Two of these were asymptomatic, four had normal ECG, and two had no clinical or echographic LV dysfunction. All patients in group 2 had negative AMA scintigraphy and normal T1 scintigraphy. During follow up of 12 AM positive patients, cardiac status improved after immunosuppressive treatment was intensified in nine cases, worsened in two cases, and remained stable in one. During follow up of 24 AM negative patients, cardiac status remained stable in 23 cases despite treatment not being increased in 20, including two patients with sequellary myocardial involvement. The last patient developed mild LV dysfunction after 36 months. CONCLUSION: AM scintigraphy allows detection of active myocardial damage related to systemic diseases, with increased specificity compared with conventional methods, and increased sensitivity in some cases. Further studies are needed to assess the potential value of AM scintigraphy as a therapeutic guide.  (+info)

Functional significance of the conserved residues in the flexible hinge region of the myosin motor domain. (58/5414)

Analysis of the three-dimensional crystal structure of the Dictyostelium myosin motor domain revealed that the myosin head is required to bend at residues Ile-455 and Gly-457 to produce the conformation changes observed in the ternary complexes that resemble the pre- and post-hydrolysis states (Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M., and Rayment, I. (1995) Biochemistry 34, 8960-8972). Asp-454, Ile-455, and Gly-457 of smooth muscle myosin were substituted by Ala, Met, and Ala, respectively, and the mechano-enzymatic activities were determined to study the role of these residues in myosin motor function. Whereas the basal steady-state Mg2+-ATPase activity of D454A was higher than that of the wild type, the rate of the hydrolytic step is reduced approximately 2,000-fold and becomes rate-limiting. M-ATP rather than M-ADP-P is the predominant steady-state intermediate, and the initial Pi burst and the ATP-induced enhancement of intrinsic tryptophan fluorescence are absent in D454A. D454A binds actin in the absence of ATP but is not dissociated from actin by ATP. Moreover, actin inhibits rather than activates the ATPase activity; consequently, D454A does not support actin translocating activity. I455M has normal actin-activated ATPase activity, Pi burst, and ATP-induced enhancement of intrinsic tryptophan fluorescence, suggesting that the enzymatic properties are normal. However, the actin translocating activity was completely inhibited. This suggests that the side chain at Ile-455 is critical for myosin motor activity but not for relatively normal enzymatic function, which indicates an apparent uncoupling between enzymatic activity and motile function. Although G457A has normal ATP-dependent actin dissociation, ATP hydrolytic step is reduced by approximately 10(5)-fold in the presence or absence of actin; consequently, G457A does not have actin translocating activity. These results indicate the importance of these conserved residues at the hinge region for normal myosin motor function.  (+info)

Inhibitory effect of the catalytic domain of myosin light chain kinase on actin-myosin interaction: insight into the mode of inhibition. (59/5414)

The catalytic domain of myosin light chain kinase (MLCK) not only exerts kinase activity to phosphorylate the 20 kDa light chain but also inhibits the actin-myosin interaction. The site of action of this novel role of the domain has been suggested to be myosin [Okagaki et al. (1999) J. Biochem. 125, 619-626]. In this study, we have analyzed the amino acid sequences of MLCK and myosin that are involved in the inhibition. The ATP-binding peptide of Gly526-Lys548 of chicken gizzard MLCK exerted the inhibitory effect on the movement of actin filaments on a myosin-coated glass surface. However, the peptide that neighbors the sequence failed to inhibit the movement. The inhibition of the ATP-binding peptide was confirmed by measuring ATPase activities of the myosin. The inhibition by parent MLCK of the movement was relieved by the 20 kDa light chain, but not by the 17 kDa myosin light chain. The peptide of the 20 kDa light chain sequence of Ser1-Glu29 also relieved the inhibition. Thus, the interaction of the ATP-binding sequence with the 20 kDa light chain sequence should cause the inhibition of the actin-myosin interaction. Concerning the regulation of the inhibition, calmodulin relieved the inhibitory effect of MLCK on the movement of actin filaments. The calmodulin-binding peptide (Ala796 Ser815) prevented the relief, suggesting the involvement of this sequence. Thus, the mode of regulation by Ca2+ and calmodulin of the novel role of the catalytic domain is similar, but not identical, to the mode of regulation of the kinase activity of the domain.  (+info)

De novo autoimmunity to cardiac myosin after heart transplantation and its contribution to the rejection process. (60/5414)

Allograft rejection is initiated by an immune response to donor MHC proteins. We recently reported that this response can result in breakdown of immune tolerance to a recipient self Ag. However, the contribution of this autoimmune response to graft rejection has yet to be determined. Here, we found that after mouse allogeneic heart transplantation, de novo CD4+ T cell and B cell autoimmune response to cardiac myosin (CM), a major contractile protein of cardiac muscle, is elicited in recipients. Importantly, CM is the autoantigen that causes autoimmune myocarditis, a heart autoimmune disease whose histopathological features resemble those observed in rejected cardiac transplants. Furthermore, T cell responses directed to CM peptide myhcalpha 334-352, a known myocarditogenic determinant, were detected in heart-transplanted mice. No responses to CM were observed in mice that had received an allogeneic skin graft or a syngeneic heart transplant, demonstrating that this response is tissue specific and that allogeneic response is necessary to break tolerance to CM. Next, we showed that sensitization of recipient mice with CM markedly accelerates the rejection of allogeneic heart. Therefore, posttransplant autoimmune response to CM is relevant to the rejection process. We conclude that transplantation-induced autoimmune response to CM represents a new mechanism that may play a significant role in cardiac transplant rejection.  (+info)

Interactions of actin, myosin, and a new actin-binding protein of rabbit pulmonary macrophages. II. Role in cytoplasmic movement and phagocytosis. (61/5414)

Actin and myosin of rabbit pulmonary macrophages are influenced by two other proteins. A protein cofactor is required for the actin activation of macrophage myosin Mg2 ATPase activity, and a high molecular weight actin-binding protein aggregates actin filaments (Stossel T.P., and J.H. Hartwig. 1975. J. Biol. Chem. 250:5706-5711)9 When warmed in 0.34 M sucrose solution containing Mg2-ATP and dithiothreitol, these four proteins interact cooperatively. Acin-binding protein in the presence of actin causes the actin to form a gel, which liquifies when cooled. The myosin contracts the gel into an aggregate, and the rate of aggregation is accelerated by the cofactor. Therefore, we believe that these four proteins also effec the temperature-dependent gelation and aggregation of crude sucrose extracts pulmonary macrophages containing Mg2-ATP and dithiothreitol. The gelled extracts are composed of tangled filaments. Relative to homogenates of resting macrophages, the distribution of actin-binding protein in homogenates of phagocytizing macrophages is altered such that 2-6 times more actin-binding protein is soluble. Sucrose extracts of phagocytizing macrophages gel more rapidly than extracts of resting macrophages. Phagocytosis by pulmonary macrophages involves the formation of peripheral pseudopods containing filaments. The findings suggest that the actin-binding protein initiates a cooperative interaction of contractile proteins to generate cytoplasmic gelation, and that phagocytosis influences the behavior of the actin-binding protein.  (+info)

Wound-induced assembly and closure of an actomyosin purse string in Xenopus oocytes. (62/5414)

BACKGROUND: Both single cells and multicellular systems rapidly heal physical insults but are thought to do so by distinctly different mechanisms. Wounds in single cells heal by calcium-dependent membrane fusion, whereas multicellular wounds heal by a variety of different mechanisms, including circumferential contraction of an actomyosin 'purse string' that assembles around wound borders and is dependent upon the small GTPase Rho. RESULTS: We investigated healing of puncture wounds made in Xenopus oocytes, a single-cell system. Oocyte wounds rapidly assumed a circular morphology and constricted circumferentially, coincident with the recruitment of filamentous actin (F-actin) and myosin-II to the wound borders. Surprisingly, recruitment of myosin-II to wound borders occurred before that of F-actin. Further, experimental disruption of F-actin prevented healing but did not prevent myosin-II recruitment. Actomyosin purse-string assembly and closure was dependent on Rho GTPases and extracellular calcium. Wounding resulted in reorganization of microtubules into an array similar to that which forms during cytokinesis in Xenopus embryos. Experimental perturbation of oocyte microtubules before wounding inhibited actomyosin recruitment and wound closure, whereas depolymerization of microtubules after wounding accelerated wound closure. CONCLUSIONS: We conclude the following: actomyosin purse strings can close single-cell wounds; myosin-II is recruited to wound borders independently of F-actin; purse-string assembly is dependent on a Rho GTPase; and purse-string assembly and closure are controlled by microtubules. More generally, the results indicate that actomyosin purse strings have been co-opted through evolution to dispatch a broad variety of single-cell and multicellular processes, including wound healing, cytokinesis and morphogenesis.  (+info)

The force exerted by a molecular motor. (63/5414)

The stochastic driving force exerted by a single molecular motor (e. g., a kinesin, or myosin) moving on a periodic molecular track (microtubule, actin filament, etc.) is discussed from a general viewpoint open to experimental test. An elementary "barometric" relation for the driving force is introduced that (i) applies to a range of kinetic and stochastic models, (ii) is consistent with more elaborate expressions entailing explicit representations of externally applied loads, and (iii) sufficiently close to thermal equlibrium, satisfies an Einstein-type relation in terms of the velocity and diffusion coefficient of the (load-free) motor. Even in the simplest two-state models, the velocity-vs.-load plots exhibit a variety of contrasting shapes (including nonmonotonic behavior). Previously suggested bounds on the driving force are shown to be inapplicable in general by analyzing discrete jump models with waiting time distributions.  (+info)

Myosin light-chain kinase of smooth muscle stimulates myosin ATPase activity without phosphorylating myosin light chain. (64/5414)

Myosin light-chain kinase (MLCK) of smooth muscle is multifunctional, being composed of N-terminal actin-binding domain, central kinase domain, and C-terminal myosin-binding domain. The kinase domain is the best characterized; this domain activates the interaction of smooth-muscle myosin with actin by phosphorylating the myosin light chain. We have recently shown that the Met-1-Pro-41 sequence of MLCK binds to actin to inhibit this interaction. However, it is not known whether the myosin-binding domain modifies the actin-myosin interaction. We designed MLCK.cDNA to overexpress the Asp-777-Glu-972 sequence in Escherichia coli. The purified Asp-777-Glu-972 fragment, although devoid of the kinase activity, exerted a stimulatory effect on the ATPase activity of dephosphorylated myosin (Vmax = 7.36 +/- 0.44-fold, Km = 1.06 +/- 0. 20 microM, n = 4). When the N-terminal 39 residues of the fragment were deleted from the fragment, the resultant fragment, Met-816-Glu-972, lost the stimulatory activity. We synthesized the Ala-777-Ser-815 peptide that was deleted from the fragment and confirmed its stimulatory effect of the peptide (Vmax = 3.03 +/- 0. 22-fold, Km = 6.93 +/- 1.61 microM, n = 3). When this peptide was further divided into Asp-777-Met-795 and Ala-796-Ser-815 peptides, the stimulatory activity was found in the latter. We confirmed that the myosin phosphorylation did not occur during the experiments with the above fragments and peptides. Therefore, we suggest that phosphorylation is not obligatory for smooth-muscle myosin not to be active.  (+info)