Effect of L-arginine on Na+,K+-ATPase activity in rat aorta endothelium

The effect of L-arginine on the Na⁺,K⁺-ATPase activity in rat aorta endothelium was studied at its physiological concentrations in the range of 10^–6-10^–3 M. The enzyme activity was 35.5% increased by low concentrations of L-arginine (10^–5 M) and its activity was 32.3-37.1% decreased at the L-arginine concentrations of 10^–4-10^–3 M. A similar inhibition (by 34.5-42.8%) was also found in the presence of a NO-donor nitroglycerol (10^–4-10^–3 M). An optical isomer of L-arginine, D-arginine, at the concentrations of 10^–5 M also increased the enzyme activity by 37.1%, but its inhibiting effect was much less pronounced and was 15.7% at the D-arginine concentration of 10^–3 M. An inhibitor of NO-synthase, L-NAME (N^G-nitroarginine, methyl ester), failed to inhibit Na⁺,K⁺-ATPase. However, the presence of L-NAME abolished the inhibition of Na⁺,K⁺-ATPase by high concentrations of L-arginine. Thus, the effect of L-arginine on the endothelial Na⁺-pump depended on its concentration, and it is suggested that the enzyme inhibition by high concentrations of L-arginine should be associated with activation of the endogenous synthesis of NO.     

Changes in orientation of actin during contraction of muscle

It is well documented that muscle contraction results from cyclic rotations of actin-bound myosin cross-bridges. The role of actin is hypothesized to be limited to accelerating phosphate release from myosin and to serving as a rigid substrate for cross-bridge rotations. To test this hypothesis, we have measured actin rotations during contraction of a skeletal muscle. Actin filaments of rabbit psoas fiber were labeled with rhodamine-phalloidin. Muscle contraction was induced by a pulse of ATP photogenerated from caged precursor. ATP induced a single turnover of cross-bridges. The rotations were measured by anisotropy of fluorescence originating from a small volume defined by a narrow aperture of a confocal microscope. The anisotropy of phalloidin-actin changed rapidly at first and was followed by a slow relaxation to a steady-state value. The kinetics of orientation changes of actin and myosin were the same. Extracting myosin abolished anisotropy changes. To test whether the rotation of actin was imposed by cross-bridges or whether it reflected hydrolytic activity of actin itself, we labeled actin with fluorescent ADP. The time-course of anisotropy change of fluorescent nucleotide was similar to that of phalloidin-actin. These results suggest that orientation changes of actin are caused by dissociation and rebinding of myosin cross-bridges, and that during contraction, nucleotide does not dissociate from actin.     

Rotation of the lever arm of Myosin in contracting skeletal muscle fiber measured by two-photon anisotropy

The rotation of the lever arm of myosin cross-bridges is believed to be responsible for muscle contraction. To resolve details of this rotation, it is necessary to observe a single cross-bridge. It is still impossible to do so in muscle fiber, but it is possible to investigate a small population of cross-bridges by simultaneously activating myosin in a femtoliter volume by rapid release of caged ATP. In earlier work, in which the number of observed cross-bridges was limited to approximately 600 by confocal microscopy, we were able to measure the rates of cross-bridge detachment and rebinding. However, we were unable to resolve the power stroke. We speculated that the reason for this was that the number of observed cross-bridges was too large. In an attempt to decrease this number, we used two-photon microscopy which permitted observation of approximately 1/2 as many cross-bridges as before with the same signal/noise ratio. With the two-photon excitation, the number of cross-bridges was small enough to resolve the beginning of the power stroke. The results indicated that the power stroke begins approximately 170 ms after the rigor cross-bridge first binds ATP.     

Rotations of a few cross-bridges in muscle by confocal total internal reflection microscopy

In order to measure the cycling of a few ( approximately 6) myosin heads in contracting skeletal muscle, myofibrils were illuminated by Total Internal Reflection and observed through a confocal aperture. Myosin heads rotated at a rate approximately equal to the ATPase rate, suggesting that bulk ATPase of a whole muscle reflects the cycle frequency of individual heads.     

Statistical modeling of biomedical corpora: mining the Caenorhabditis Genetic Center Bibliography for genes related to life span

Background  

The statistical modeling of biomedical corpora could yield integrated, coarse-to-fine views of biological phenomena that complement discoveries made from analysis of molecular sequence and profiling data. Here, the potential of such modeling is demonstrated by examining the 5,225 free-text items in the Caenorhabditis Genetic Center (CGC) Bibliography using techniques from statistical information retrieval. Items in the CGC biomedical text corpus were modeled using the Latent Dirichlet Allocation (LDA) model. LDA is a hierarchical Bayesian model which represents a document as a random mixture over latent topics; each topic is characterized by a distribution over words.     

Orientational changes of crossbridges during single turnover of ATP

Muscle contraction results from rotation of actin-bound myosin crossbridges. Crossbridges consist of the globular N-terminal catalytic domain and the alpha-helical C-terminal regulatory domain containing the essential and regulatory light chains. The essential light chain exists in two isoforms, of which the larger one has a 41-amino acid extension piece added at the N-terminus. The catalytic domain is responsible for binding to actin and for setting the stage for the main force-generating event, which is a "swing" of the regulatory domain. We measured the kinetics of the swing associated with the turnover of a single molecule of ATP. Muscle was labeled at the regulatory domain by replacing native essential or regulatory light chain with fluorescent adducts. The rotations were measured by the anisotropy of fluorescence originating from approximately 400 crossbridges residing in a small volume defined by a confocal aperture of a microscope. The crossbridges were synchronized by rapid photogeneration of a stoichiometric amount of ATP. The rotations reflected dissociation from thin filaments followed by a slow reattachment. The dissociation was the same for each light chain (halftime approximately 120 ms) but the rate of reattachment depended on the type of light chain. The halftimes were 920 +/- 50 ms and 660 +/- 100 ms for isoforms 1 and 3 of the essential light chain, respectively. The reason that the lifetimes were so long was creation of a small amount of ATP, enough only for a single turnover of crossbridges. A model was constructed that quantified this effect. After accounting for the slowdown, the halftimes of dissociation and attachment were 34 and 200 ms, respectively.     

Effect of sodium nitroprusside on Na+, K+-ATPase of rat myocardium and renal cortex

The influence of NO donor, sodium nitroprusside (SNP) in concentrations of 10(-5)-10(-3) M, on the Na-pump activity in membrane preparations (microsomes) of rat miocardium and renal cortex was studied. A correlation has been observed between Na-pump activation by 31.4% and 73.6% and increase in thiol group content by 45.0% and 94.0% in native microsomes of myocardium and renal cortex by low concentrations of SNP (10(-5)-10(-4) M). The Na-pump activation as well as the increase in the number of DTNB-reactive thiol groups was abolished after the detergent treatment (0.025% sodium dodecylsulphate); however, the detergent by itself exerted similar influence on Na-pump activity and thiol group content in microsomes. A comparable effect of other oxidants (hydrogen peroxide and nitroglycerine) on SH-group content in microsomes of renal cortex was also shown. A conclusion was made that non-specific oxidative action of SNP on some membrane structures in microsome preparations led to their exposure and activation of the Na-pump by demasking of its latent activity.     

Regulatory and essential light chains of myosin rotate equally during contraction of skeletal muscle

Myosin head consists of a globular catalytic domain and a long alpha-helical regulatory domain. The catalytic domain is responsible for binding to actin and for setting the stage for the main force-generating event, which is a "swing" of the regulatory domain. The proximal end of the regulatory domain contains the essential light chain 1 (LC1). This light chain can interact through the N and C termini with actin and myosin heavy chain. The interactions may inhibit the motion of the proximal end. In consequence the motion of the distal end (containing regulatory light chain, RLC) may be different from the motion of the proximal end. To test this possibility, the angular motion of LC1 and RLC was measured simultaneously during muscle contraction. Engineered LC1 and RLC were labeled with red and green fluorescent probes, respectively, and exchanged with native light chains of striated muscle. The confocal microscope was modified to measure the anisotropy from 0.3 microm(3) volume containing approximately 600 fluorescent cross-bridges. Static measurements revealed that the magnitude of the angular change associated with transition from rigor to relaxation was less than 5 degrees for both light chains. Cross-bridges were activated by a precise delivery of ATP from a caged precursor. The time course of the angular change consisted of a fast phase followed by a slow phase and was the same for both light chains. These results suggest that the interactions of LC1 do not inhibit the angular motion of the proximal end of the regulatory domain and that the whole domain rotates as a rigid body.     

Decrease of mitochondrial sensitivity to Ca2+-induced pore opening during long-term incubation

It is shown that mitochondria of rat myocardium exhibit high sensitivity to Ca2+-induced permeability transition pore (PTP) during an hour after their isolation. The threshold Ca2+ concentrations necessary to induce PTP are as low as 25-50 nmol/mg of protein. Apparent K(Ca) constant of Ca2+-dependent PTP activation is 40 microM. Incubation of mitochondria during 2, 3 and 4 hours after their isolation leads to gradual increase in K(Ca) values up to 0.4 microM, which is accompanied by simultaneous decrease in sensitivity of mitochondria to pore opening. A correlation is supposed between changes of kinetic parameters of Ca2+-uniporter and changes in sensitivity of mitochondria to Ca2+-induced permeability transition.