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101.
Jonathan Walklate Carlos Vera Marieke J. Bloemink Michael A. Geeves Leslie Leinwand 《The Journal of biological chemistry》2016,291(19):10318-10331
The embryonic myosin isoform is expressed during fetal development and rapidly down-regulated after birth. Freeman-Sheldon syndrome (FSS) is a disease associated with missense mutations in the motor domain of this myosin. It is the most severe form of distal arthrogryposis, leading to overcontraction of the hands, feet, and orofacial muscles and other joints of the body. Availability of human embryonic muscle tissue has been a limiting factor in investigating the properties of this isoform and its mutations. Using a recombinant expression system, we have studied homogeneous samples of human motors for the WT and three of the most common FSS mutants: R672H, R672C, and T178I. Our data suggest that the WT embryonic myosin motor is similar in contractile speed to the slow type I/β cardiac based on the rate constant for ADP release and ADP affinity for actin-myosin. All three FSS mutations show dramatic changes in kinetic properties, most notably the slowing of the apparent ATP hydrolysis step (reduced 5–9-fold), leading to a longer lived detached state and a slowed Vmax of the ATPase (2–35-fold), indicating a slower cycling time. These mutations therefore seriously disrupt myosin function. 相似文献
102.
103.
Marieke van Eijk 《Culture, medicine and psychiatry》2017,41(4):590-608
What is a clinician to do when people needing medical care do not have access to consistent or sufficient health insurance coverage and cannot pay for care privately? Analyzing ethnographically how clinicians at a university-based transgender clinic in the United States responded to this challenge, I examine the U.S. health insurance system, insurance paperwork, and administrative procedures that shape transgender care delivery. To buffer the impact of the system’s failure to provide sufficient health insurance coverage for transgender care, clinicians blended administrative routines with psychological therapy, counseled people’s minds and finances, and leveraged the prestige of their clinic in attempts to create space for gender nonconforming embodiments in gender conservative insurance policies. My analysis demonstrates that in a market-based health insurance system with multiple payers and gender binary insurance rules, health care may be unaffordable, or remain financially challenging, even for transgender people with health insurance. Moreover, insurance carriers’ “reliance” on clinicians’ insurance-related labor is problematic as it exacerbates existing insurance barriers to the accessibility and affordability of transgender care and obscures the workings of a financial payment model that prioritizes economic expediency over gender nonconforming health. 相似文献
104.
105.
αβ T cells and γδ T cells perform nonoverlapping immune functions. In mammalian species with a high percentage of very diverse
γδ T cells, like ruminants and pigs, it is often assumed that αβ T cells are less diverse than γδ T cells. Based on the bovine
genome, we have created a map of the bovine TRA/TRD locus and show that, in cattle, in addition to the anticipated >100 TRDV
genes, there are also >300 TRAV or TRAV/DV genes. Among the V genes in the TRA/TRD locus, there are several genes that lack
a CDR2 and are functionally rearranged and transcribed and, in some cases, have an extended CDR1. The number of bovine V genes
is a multiple of the number in mice and humans and may encode T cell receptors that use a novel way of interacting with antigen.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
106.
Marieke J. Bloemink Aileen F. Knowles Michael A. Geeves Sanford I. Bernstein 《Journal of molecular biology》2009,389(4):707-1122
We investigated the biochemical and biophysical properties of one of the four alternative regions within the Drosophila myosin catalytic domain: the relay domain encoded by exon 9. This domain of the myosin head transmits conformational changes in the nucleotide-binding pocket to the converter domain, which is crucial to coupling catalytic activity with mechanical movement of the lever arm. To study the function of this region, we used chimeric myosins (IFI-9b and EMB-9a), which were generated by exchange of the exon 9-encoded domains between the native embryonic body wall (EMB) and indirect flight muscle isoforms (IFI). Kinetic measurements show that exchange of the exon 9-encoded region alters the kinetic properties of the myosin S1 head. This is reflected in reduced values for ATP-induced actomyosin dissociation rate constant (K1k+2) and ADP affinity (KAD), measured for the chimeric constructs IFI-9b and EMB-9a, compared to wild-type IFI and EMB values. Homology models indicate that, in addition to affecting the communication pathway between the nucleotide-binding pocket and the converter domain, exchange of the relay domains between IFI and EMB affects the communication pathway between the nucleotide-binding pocket and the actin-binding site in the lower 50-kDa domain (loop 2). These results suggest an important role of the relay domain in the regulation of actomyosin cross-bridge kinetics. 相似文献
107.
Nuria Albet-Torres Marieke J. Bloemink Tom Barman Robin Candau Kerstin Fr?lander Michael A. Geeves Kerstin Golker Christian Herrmann Corinne Lionne Claudia Piperio Stephan Schmitz Claudia Veigel Alf M?nsson 《The Journal of biological chemistry》2009,284(34):22926-22937
Amrinone is a bipyridine compound with characteristic effects on the force-velocity relationship of fast skeletal muscle, including a reduction in the maximum shortening velocity and increased maximum isometric force. Here we performed experiments to elucidate the molecular mechanisms for these effects, with the additional aim to gain insight into the molecular mechanisms underlying the force-velocity relationship. In vitro motility assays established that amrinone reduces the sliding velocity of heavy meromyosin-propelled actin filaments by 30% at different ionic strengths of the assay solution. Stopped-flow studies of myofibrils, heavy meromyosin and myosin subfragment 1, showed that the effects on sliding speed were not because of a reduced rate of ATP-induced actomyosin dissociation because the rate of this process was increased by amrinone. Moreover, optical tweezers studies could not detect any amrinone-induced changes in the working stroke length. In contrast, the ADP affinity of acto-heavy meromyosin was increased about 2-fold by 1 mm amrinone. Similar effects were not observed for acto-subfragment 1. Together with the other findings, this suggests that the amrinone-induced reduction in sliding velocity is attributed to inhibition of a strain-dependent ADP release step. Modeling results show that such an effect may account for the amrinone-induced changes of the force-velocity relationship. The data emphasize the importance of the rate of a strain-dependent ADP release step in influencing the maximum sliding velocity in fast skeletal muscle. The data also lead us to discuss the possible importance of cooperative interactions between the two myosin heads in muscle contraction.Muscle contraction, as well as several other aspects of cell motility, results from cyclic interactions between myosin II motors and actin filaments. These force-generating interactions are driven by the hydrolysis of ATP at the myosin active site as outlined in Scheme 1 (1–3). In the absence of actin, the Pi and ADP release steps (k4 and k5) are rate-limiting for the entire cycle at high (>12 °C) and low temperatures, respectively (4–6). In the presence of actin, the rate of Pi release increases significantly, and the overall cycle is accelerated more than 2 orders of magnitude. The sliding velocity of myosin-propelled motors is generally believed to be rate-limited by actomyosin dissociation (rate constant k′5, k′6, or k′2 in Scheme 1) (7). Alternatively, some studies (8, 9) have suggested that the sliding velocity is determined by the fraction of myosin heads in the weak-binding states, AM4 ATP and AM ADP Pi. However, it is worth emphasizing that KT is very low under physiological conditions (1, 3) with low population of these states. For the same reason, the rate of dissociation of the AM complex is governed by K′1 and k′2.Open in a separate windowSCHEME 1.Simplified kinetics scheme for MgATP turnover by myosin (lower row) and actomyosin (upper row). Inorganic phosphate is denoted by Pi; MgATP is denoted by ATP, and MgADP is denoted by ADP; myosin is denoted by M. The states AM*ADP and AM ADP correspond to myosin heads with their nucleotide binding pocket in a partially closed and open conformation, respectively (7, 52). Rate constants are indicated by lowercase letters (rightward transitions, k2 − k5 and k′2 − k′5, or leftward transitions, k−2 − k−5 and k′−2 − k′−5) and equilibrium constants by uppercase letters (K1, K′1, KT, K3, K′3, K6, k′6, and KDP). The equilibrium constants are association constants except for simple bimolecular reactions where they are defined as ki/k−i.For the study of contractile mechanisms in both muscle and other types of cells, drugs may be useful as pharmacological tools affecting different transitions or states in the force-generating cycle. Whereas the use of drugs as tools may be less specific than site-directed mutagenesis, it also has advantages. The motor protein function may be studied in vivo, with maintained ordering of the protein components, e.g. as in the muscle sarcomere, allowing more insight into the relationship between specific molecular events and contractile properties of muscle. A drug that has been used quite extensively in this context is butanedione monoxime. The usefulness of this drug is based on firm characterization of its effect on actomyosin function on the molecular level (3, 10–13). More recently other drugs, like N-benzyl-p-toluene sulfonamide (14, 15) and blebbistatin (16), have been found to affect myosin function, and their effects at the molecular level have also been elucidated in some detail (14, 15, 17, 18). Both these drugs appear to affect the actomyosin interaction in a similar way as butanedione monoxime by inhibiting a step before (or very early in) the myosin power stroke, leading to the inhibition of actomyosin cross-bridge formation and force production.In contrast to the reduced isometric force, caused by the above mentioned drugs, the bipyridine compound amrinone (Fig. 1A) has been found to increase the isometric force production of fast intact skeletal muscles of the frog (19, 20) and mouse (21) and also of fast (but much less slow) skinned muscle fibers of the rat (22). In all the fast myosin preparations, the effect of about 1 mm amrinone on isometric force was associated with characteristic changes of the force-velocity relationship (Fig. 1B), including a reduced maximum velocity of shortening (19–22) and a reduced curvature of the force-velocity relationship (19–22). The latter effect was accompanied (20, 21) by a less pronounced deviation of the force-velocity relationship from the hyperbolic shape (23) at high loads. There have been different interpretations of the drug effects. It has been proposed (20–22) that amrinone might competitively inhibit the MgATP binding by myosin. However, more recently, results from in vitro motility assay experiments (24) challenged this idea. These results showed that amrinone reduces the sliding velocity (Vmax) at saturating MgATP concentrations but not at MgATP concentrations close to, or below, the Km value for the hyperbolic relationship between MgATP concentration and sliding velocity. Such a combination of effects is consistent with a reduced MgADP release rate (24) but not with competitive inhibition of substrate binding. However, effects of amrinone on the MgADP release rate have not been directly demonstrated. Additionally, in view of the uncertainty about what step actually determines the sliding velocity at saturating [MgATP] (see above and Refs. 7–9), it is of interest to consider other possible drug effects that could account for the data of Klinth et al. (24). These include the following: 1) an increased drag force, e.g. because of enhancement of weak actomyosin interactions; 2) a reduced step length; and 3) effects of the drug on the rate of MgATP-induced dissociation of actomyosin.Open in a separate windowFIGURE 1.A, structure of amrinone. B, experimental force-velocity data obtained in the presence (filled symbols) and absence (open symbols) of 1.1 mm amrinone. The data, from intact single frog muscle fibers, were obtained at 2 °C and fitted by Hill''s (42) hyperbola (lines) for data truncated at 80% of the maximum isometric force. Filled line, equation fitted to control data, a/P0* = 0.185; P0*/P0 = 1.196. Dashed line, amrinone, a/P0* = 0.347; P0*/P0 = 1.009. Force-velocity data were obtained in collaboration with Professor K. A. P. Edman. Same data as in Fig. 8 of Ref. 20. Note a decrease in maximum sliding velocity and curvature of the force-velocity relationship at low force, in response to amrinone. Also note that amrinone caused increased isometric force and a reduced deviation of the force-velocity relationship from the Hill''s hyperbola at high force. All changes of the force-velocity relationship were statistically significant (20), and similar changes were later also observed in intact mouse muscle and skinned rat muscle fibers. Data in Fig. 1 are published by agreement with Professor K. A. P. Edman.To differentiate between these hypotheses for the amrinone effects, and to gain more general insight into fundamental aspects of muscle function (e.g. mechanisms underlying the force-velocity relationship), we here study the molecular effects of amrinone on fast skeletal muscle myosin preparations in the presence and absence of actin.In vitro motility assay studies at different ionic strengths suggest that drag forces, caused by increased fraction of myosin heads in weak binding states, are not important for the effect of amrinone on sliding velocity. Likewise, optical tweezers studies showed no effect of the drug on the myosin step length. Finally, ideas that amrinone should reduce sliding velocity by reduced rate of MgATP-induced dissociation could be discarded because the drug actually increased the rate of this process. Instead, we found an amrinone-induced increase in the MgADP affinity of heavy meromyosin (HMM) in the presence of actin. Interestingly, similar effects of amrinone were not observed using myosin S1. As discussed below, this result and other results point to an amrinone-induced reduction in the rate of a strain-dependent MgADP release step. Simulations, using a model modified from that of Edman et al. (25), support this proposed mechanism of action. The results are discussed in relation to fundamental mechanisms underlying the force-velocity relationship of fast skeletal muscle, including which step determines shortening velocity and the possible importance of inter-head cooperativity. 相似文献
108.
Tjisse van der Heide Egbert H. van Nes Marieke M. van Katwijk Marten Scheffer A. Jan Hendriks Alfons J. P. Smolders 《Ecosystems》2010,13(6):841-850
Many populations are exposed to naturally occurring or synthetic toxicants. An increasing number of studies demonstrate that
the toxicity of such compounds is not only dependent on the concentration or load, but also on the biomass or density of exposed
organisms. At high biomass, organisms may be able to alleviate adverse effects of the toxicant by actively lowering ambient
concentrations through either a joint detoxification mechanism or growth dilution. We show in a conceptual model that this
mechanism may potentially lead to alternative stable states if the toxicant is lethal at low densities of organisms, whereas
a high density is able to reduce the toxicant concentrations to sub-lethal levels. We show in an example that this effect
may be relevant in real ecosystems. In an earlier published experimental laboratory study, we demonstrated that ammonia toxicity
in eelgrass is highly dependent on the eelgrass shoot density. Here, we used the results of these experiments to construct
a model describing the complex interactions between the temperate seagrass Zostera marina and potentially lethal ammonia. Analyses of the model show that alternative stable states are indeed present over wide ranges
of key-parameter settings, suggesting that the mechanism might be important especially in sheltered, eutrophicated estuaries
where mixing of the water layer is poor. We argue that the same mechanism could cause alternative stable states in other biological
systems as well. 相似文献
109.
Marieke Koopmans Dirk Martens Rene H. Wijffels 《Marine biotechnology (New York, N.Y.)》2010,12(3):340-349
To obtain more knowledge about carbon requirements for growth by sponges, the growth rate, respiration rate, and clearance
rate was measured in situ in Haliclona oculata. We found that only 34% of the particulate carbon pumped through the sponge was used for both respiration and growth. The
net growth efficiency, being the ratio of carbon incorporated in biomass and the total carbon used by the sponge for respiration
and growth, was found to be 0.099 ± 0.013. Thus, about 10% of the total used carbon was fixed in biomass, and over 90% was
used for generating energy for growth, maintenance, reproduction, and pumping. H. oculata had 2.5 μmol C available for every micromole O2 consumed. A value of 0.75 for respiratory quotient (RQ in micromole CO2 micromole O2−1) was used for H. oculata, which is the average value reported in literature for different marine invertebrates. Thus, carbon was available
in excess to meet the respiratory demand. Oxygen was found not to be the limiting factor for growth, since only 3.3% of the
oxygen pumped through the sponge body was used. Our results indicate that both oxygen and carbon availability are not limiting.
The low growth efficiency agrees with the low growth rates found for the species used in this study. 相似文献
110.
Rheumatoid arthritis (RA) is a chronic autoimmune disease affecting 0.5–1% of the population worldwide. The disease has a
heterogeneous character, including clinical subsets of anti-citrullinated protein antibody (ACPA)-positive and APCA-negative
disease. Although the pathogenesis of RA is poorly understood, progress has been made in identifying genetic factors that
contribute to the disease. The most important genetic risk factor for RA is found in the human leukocyte antigen (HLA) locus.
In particular, the HLA molecules carrying the amino acid sequence QKRAA, QRRAA, or RRRAA at positions 70–74 of the DRβ1 chain
are associated with the disease. The HLA molecules carrying these “shared epitope” sequences only predispose for ACPA-positive
disease. More than two decades after the discovery of HLA-DRB1 as a genetic risk factor, the second genetic risk factor for
RA was identified in 2003. The introduction of new techniques, such as methods to perform genome-wide association has led
to the identification of more than 20 additional genetic risk factors within the last 4 years, with most of these factors
being located near genes implicated in immunological pathways. These findings underscore the role of the immune system in
RA pathogenesis and may provide valuable insight into the specific pathways that cause RA. 相似文献