Force generation and work production by covalently cross-linked actin-myosin cross-bridges in rabbit muscle fibers.

To separate a fraction of the myosin cross-bridges that are attached to the thin filaments and that participate in the mechanical responses, muscle fibers were cross-linked with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and then immersed in high-salt relaxing solution (HSRS) of 0.6 M ionic strength for detaching the unlinked myosin heads. The mechanical properties and force-generating ability of the cross-linked cross-bridges were tested with step length changes (L-steps) and temperature jumps (T-jumps) from 6-10 degrees C to 30-40 degrees C. After partial cross-linking, when instantaneous stiffness in HSRS was 25-40% of that in rigor, the mechanical behavior of the fibers was similar to that during active contraction. The kinetics of the T-jump-induced tension transients as well as the rate of the fast phase of tension recovery after length steps were close to those in unlinked fibers during activation. Under feedback force control, the T-jump initiated fiber shortening by up to 4 nm/half-sarcomere. Work produced by a cross-linked myosin head after the T-jump was up to 30 x 10(-21) J. When the extent of cross-linking was increased and fiber stiffness in HSRS approached that in rigor, the fibers lost their viscoelastic properties and ability to generate force with a rise in temperature.     

High-throughput genotyping assays for identification of glycophorin B deletion variants in population studies

Glycophorins are the most abundant sialoglycoproteins on the surface of human erythrocyte membranes. Genetic variation in glycophorin region of human chromosome 4 (containing GYPA, GYPB, and GYPE genes) is of interest because the gene products serve as receptors for pathogens of major public health interest, including Plasmodium sp., Babesia sp., Influenza virus, Vibrio cholerae El Tor Hemolysin, and Escherichia coli. A large structural rearrangement and hybrid glycophorin variant, known as Dantu, which was identified in East African populations, has been linked with a 40% reduction in risk for severe malaria. Apart from Dantu, other large structural variants exist, with the most common being deletion of the whole GYPB gene and its surrounding region, resulting in multiple different deletion forms. In West Africa particularly, these deletions are estimated to account for between 5 and 15% of the variation in different populations, mostly attributed to the forms known as DEL1 and DEL2. Due to the lack of specific variant assays, little is known of the distribution of these variants. Here, we report a modification of a previous GYPB DEL1 assay and the development of a novel GYPB DEL2 assay as high-throughput PCR-RFLP assays, as well as the identification of the crossover/breakpoint for GYPB DEL2. Using 393 samples from three study sites in Ghana as well as samples from HapMap and 1000 G projects for validation, we show that our assays are sensitive and reliable for genotyping GYPB DEL1 and DEL2. To the best of our knowledge, this is the first report of such high-throughput genotyping assays by PCR-RFLP for identifying specific GYPB deletion types in populations. These assays will enable better identification of GYPB deletions for large genetic association studies and functional experiments to understand the role of this gene cluster region in susceptibility to malaria and other diseases.     

Effects of cardiac myosin binding protein-C on the regulation of interaction of cardiac myosin with thin filament in an in vitro motility assay

Modulatory role of whole cardiac myosin binding protein-C (сMyBP-C) in regulation of cardiac muscle contractility was studied in the in vitro motility assay with rabbit cardiac myosin as a motor protein. The effects of cMyBP-C on the interaction of cardiac myosin with regulated thin filament were tested in both in vitro motility and ATPase assays. We demonstrate that the addition of cMyBP-C increases calcium regulated Mg-ATPase activity of cardiac myosin at submaximal calcium. The Hill coefficient for ‘pCa-velocity’ relation in the in vitro motility assay decreased and the calcium sensitivity increased when сMyBP-C was added. Results of our experiments testifies in favor of the hypothesis that сMyBP-C slows down cross-bridge kinetics when binding to actin.     

Structural changes in the actin-myosin cross-bridges associated with force generation induced by temperature jump in permeabilized frog muscle fibers.

Structural changes induced by Joule temperature jumps (T-jumps) in frog muscle fibers were monitored using time-resolved x-ray diffraction. Experiments made use of single, permeabilized fibers that were fully activated after slight cross-linking with 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide to preserve their structural order. After T-jumps from 5-6 to approximately 17 degrees C and then on to approximately 30 degrees C, tension increased by a factor of 1.51 and 1.84, respectively, whereas fiber stiffness did not change with temperature. The tension rise was accompanied by a decrease in the intensity of the (1, 0) equatorial x-ray reflection by 15 and 26% (at approximately 17 and approximately 30 degrees C) and by an increase in the intensity of the M3 myosin reflection by 20% and 41%, respectively. The intensity of the (1,1) equatorial reflection increased slightly. The peak of the intensity on the 6th actin layer line shifted toward the meridian with temperature. The intensity of the 1st actin layer line increased from 12% (of its rigor value) at 5-6 degrees C to 36% at approximately 30 degrees C, so that the fraction of the cross-bridges labeling the actin helix estimated from this intensity increased proportionally to tension from approximately 35% at 5-6 degrees C to approximately 60% at approximately 30 degrees C. This suggests that force is generated during a transition of nonstereo-specifically attached myosin cross-bridges to a stereo-specific binding state.     

小麦植株内糖类的昼夜变化

1.田间条件下灌浆期的小麦,叶子、叶鞘、及节间里的含糖量有着昼夜的变化,其中主要是蔗糖含量的变化。在日间下午3—6时含糖量最高,而晚上含糖量降低。2.小麦叶子、叶鞘及节间含糖量的变化受到环境因素的影响。这里主要是光线及植物社会的影响。3.在阴天只有单行植株第1叶和第2叶里的蔗糖有显著的昼夜变化;叶子、叶鞘变化很小,还维持了原来的水平。中行植株的叶子、叶鞘及节间的含糖量一般是降低的。4.在晴天,不问单行植株、中行植株,叶子、叶鞘和节间的含糖量都有昼夜的变化。但含糖量变化的进程在单行植株和中行植株之间有着很明显的差异。单行植株的含糖水/平高于中行植株。它们之间差异的幅度,越是在下部的器官越是显著。单行植株叶子、叶鞘的蔗糖最高含量在下午6时到达,而中行植株叶子、叶鞘里的蔗糖最高含量早在下午3时就达到了。这种差异的形成由于中行植株被左右边行的植株所荫蔽,在光线条件上处于不利的状态。在阴天中行植株的光线条件更差,以至阻碍了光合作用的进行。5.小麦在灌浆期,叶、叶鞘、节间糖的分布是这样的:叶子里的糖浓度小于叶鞘,叶鞘里的糖浓度小于节间的。但当阳光充足的时候,叶子里的浓度有时高于叶鞘中的浓度。此外第2节间的糖浓度高于第1节...     

ENVIRONMENTAL CONDITIONS, GROWTH AND PRODUCTION OF ANADARA SENILIS (LINNAEUS, 1758) IN A SENEGAL LAGOON

Two populations of Anadara senilis were sampled periodicallyin a lagoon of Senegal. Spatfall occurs in summer during therainy season. Population density decreases from about 50, 000.m^–2,two months after spatfall, down to 6, 750 one year later and750 after 28 months. The average growth, measured on the surfaceof the shell, following the umbo rim axis is 0.8 mm per month.The main reduction in growth rate occurs in winter and endsby the deposition of a growth ring. A second growth reductiontakes place during the rainy season and sometimes ends by aweak growth ring. The microgrowth increments are conspicuousand widest during spring equinox. They are not directly relatedto the tidal cycle, but it was impossible to determine if theyhave a tidal or a daily periodicity. (Received 11 February 1990; accepted 16 July 1993)     

Optical trap as a tool for studying motor proteins

The review briefs the optical trap method, a modern experimental tool based on the recently discovered ability of light to trap and hold micron and submicron particles in a focused beam. The physical principle underlying the optical trap and the opportunities that it provides for studying the molecular nature of biological motility are considered. Several studies into the physical characteristics and functions of single motor protein molecules performed using the optical trap and recording nanometer displacements and piconewton forces are analyzed.     

X-ray interferometry of the axial movement of myosin heads during muscle force generation initiated by T-Jump

The interference fine structure of the M3 reflection in the low-angle X-ray diffraction patterns of muscle fibers is used for the measurements of axial movements of myosin heads with a precision of 0.1–0.2 nm. We have measured changes in the M3 interference profile during tension rise induced by a 5°C to 30°C temperature jump in thin bundles of contracting fibers from rabbit skeletal muscle. Interpreting the data with a point diffractor model gives an estimate for the axial movement of the myosin heads during force rise of less than 0.6 nm. Modifications of the point diffractor model are discussed. We show that our experimental data can be explained by a model where myosin heads bind actin in a number of structurally different states either stereoor non-stereo-specifically.