Structural and activity changes in three bioactive anuran peptides when Asp is replaced by isoAsp

The Asp and isoAsp isomers of three bioactive peptides, Crinia angiotensin 11 [APGDRIYHPF(OH)], uperin 1.1 [pEADPNAFYGLM(NH₂)] and citropin 1.1 [GLFDVIKKVASVIGGL(NH₂)] were tested for changes in (i) susceptibility towards proteolytic cleavage, (ii) activity (smooth muscle activity for Crinia angiotensin 11 and uperin 1.1 isomers, and antimicrobial activity for the two isomers of citropin 1.1), and (iii) 3D structures in water, trifluoroethanol-d₃/water (1:1) and DPC micelles as determined by 2D nuclear magnetic resonance spectroscopy. Proteolytic cleavage with trypsin was identical for each pair of Asp/isoAsp isomers. Cleavage with chymotrypsin was the same for the Crinia angiotensin and uperin 1.1 isomeric pairs, but different for the two Asp/isoAsp citropin 1.1 isomers. Chymotrypsin cleaved at Phe3 (adjacent to Asp4) for citropin 1.1, but not at Phe3 (adjacent to isoAsp4) for isoAsp citropin 1.1. The smooth muscle activity of the isoAsp isomer of Crinia angiotensin 11 was less than that of the Asp isomer. The smooth muscle activity of isoAsp3-uperin 1.1 is greater than that of the Asp isomer at low concentration (<10⁻⁹ M) but no different from the Asp isomer at concentrations > 10⁻⁹ M. Citropin 1.1 is a wide-spectrum antibiotic against Gram positive organisms, while the isoAsp isomer is inactive against the test pathogens Staphylococcus aureus and Bacillus subtilis. The observed changes in activity are accompanied by changes in the 3D structures of isomers as determined by 2D nuclear magnetic resonance spectroscopy.     

Muscle mass scaling in primates: an energetic and ecological perspective

Body composition is known to vary dramatically among mammals, even in closely related species, yet this issue has never been systematically investigated. Here, we examine differences in muscle mass scaling among mammals, and explore how primate body composition compares to that of nonprimate mammals. We use a literature-based sample of eutherian and metatherian mammals, and combine this with new dissection-based data on muscularity in a variety of strepsirrhine primates and the haplorhine, Tarsius syrichta. Our results indicate an isometric scaling relationship between total muscle mass and total body mass across mammals. However, we documented substantial variation in muscularity in mammals (21-61% of total body mass), which can be seen both within and between taxonomic groups. We also found that primates are under-muscled when compared to other mammals. This difference in body composition may in part reflect the functional consequences of arboreality, as arboreal species have significantly lower levels of muscularity than terrestrial species.     

Morphological and kinematic basis of the hummingbird flight stroke: scaling of flight muscle transmission ratio

Hummingbirds (Trochilidae) are widely known for their insect-like flight strokes characterized by high wing beat frequency, small muscle strains and a highly supinated wing orientation during upstroke that allows for lift production in both halves of the stroke cycle. Here, we show that hummingbirds achieve these functional traits within the limits imposed by a vertebrate endoskeleton and muscle physiology by accentuating a wing inversion mechanism found in other birds and using long-axis rotational movement of the humerus. In hummingbirds, long-axis rotation of the humerus creates additional wing translational movement, supplementing that produced by the humeral elevation and depression movements of a typical avian flight stroke. This adaptation increases the wing-to-muscle-transmission ratio, and is emblematic of a widespread scaling trend among flying animals whereby wing-to-muscle-transmission ratio varies inversely with mass, allowing animals of vastly different sizes to accommodate aerodynamic, biomechanical and physiological constraints on muscle-powered flapping flight.     

Muscle power attenuation by tendon during energy dissipation

An important function of skeletal muscle is deceleration via active muscle fascicle lengthening, which dissipates movement energy. The mechanical interplay between muscle contraction and tendon elasticity is critical when muscles produce energy. However, the role of tendon elasticity during muscular energy dissipation remains unknown. We tested the hypothesis that tendon elasticity functions as a mechanical buffer, preventing high (and probably damaging) velocities and powers during active muscle fascicle lengthening. We directly measured lateral gastrocnemius muscle force and length in wild turkeys during controlled landings requiring rapid energy dissipation. Muscle-tendon unit (MTU) strain was measured via video kinematics, independent of muscle fascicle strain (measured via sonomicrometry). We found that rapid MTU lengthening immediately following impact involved little or no muscle fascicle lengthening. Therefore, joint flexion had to be accommodated by tendon stretch. After the early contact period, muscle fascicles lengthened and absorbed energy. This late lengthening occurred after most of the joint flexion, and was thus mainly driven by tendon recoil. Temporary tendon energy storage led to a significant reduction in muscle fascicle lengthening velocity and the rate of energy absorption. We conclude that tendons function as power attenuators that probably protect muscles against damage from rapid and forceful lengthening during energy dissipation.     

Analysis of proteomic changes in roots of soybean seedlings during recovery after flooding

A proteomic approach was used to identify proteins involved in post-flooding recovery in soybean roots. Two-day-old soybean seedlings were flooded with water for up to 3 days. After the flooding treatment, seedlings were grown until 7 days after sowing and root proteins were then extracted and separated using two-dimensional polyacrylamide gel electrophoresis (2-DE). Comparative analysis of 2-D gels of control and 3 day flooding-experienced soybean root samples revealed 70 differentially expressed protein spots, from which 80 proteins were identified. Many of the differentially expressed proteins are involved in protein destination/storage and metabolic processes. Clustering analysis based on the expression profiles of the 70 differentially expressed protein spots revealed that 3 days of flooding causes significant changes in protein expression, even during post-flooding recovery. Three days of flooding resulted in downregulation of ion transport-related proteins and upregulation of proteins involved in cytoskeletal reorganization, cell expansion, and programmed cell death. Furthermore, 7 proteins involved in cell wall modification and S-adenosylmethionine synthesis were identified in roots from seedlings recovering from 1 day of flooding. These results suggest that alteration of cell structure through changes in cell wall metabolism and cytoskeletal organization may be involved in post-flooding recovery processes in soybean seedlings.     

Novel tyrosine phosphorylation sites in rat skeletal muscle revealed by phosphopeptide enrichment and HPLC-ESI-MS/MS

Tyrosine phosphorylation plays a fundamental role in many cellular processes including differentiation, growth and insulin signaling. In insulin resistant muscle, aberrant tyrosine phosphorylation of several proteins has been detected. However, due to the low abundance of tyrosine phosphorylation (<1% of total protein phosphorylation), only a few tyrosine phosphorylation sites have been identified in mammalian skeletal muscle to date. Here, we used immunoprecipitation of phosphotyrosine peptides prior to HPLC-ESI-MS/MS analysis to improve the discovery of tyrosine phosphorylation in relatively small skeletal muscle biopsies from rats. This resulted in the identification of 87 distinctly localized tyrosine phosphorylation sites in 46 muscle proteins. Among them, 31 appear to be novel. The tyrosine phosphorylated proteins included major enzymes in the glycolytic pathway and glycogen metabolism, sarcomeric proteins, and proteins involved in Ca(2+) homeostasis and phosphocreatine resynthesis. Among proteins regulated by insulin, we found tyrosine phosphorylation sites in glycogen synthase, and two of its inhibitors, GSK-3α and DYRK1A. Moreover, tyrosine phosphorylation sites were identified in several MAP kinases and a protein tyrosine phosphatase, SHPTP2. These results provide the largest catalogue of mammalian skeletal muscle tyrosine phosphorylation sites to date and provide novel targets for the investigation of human skeletal muscle phosphoproteins in various disease states.     

Myosin light chain 3f attenuates age-induced decline in contractile velocity in MHC type II single muscle fibers

Aging is characterized by a progressive loss of muscle mass and impaired contractility (e.g., decline in force, velocity, and power). Although the slowing of contraction speed in aging muscle is well described, the underlying molecular mechanisms responsible for the decrement in speed are unknown. Myosin heavy chain (MHC) isoforms are the primary molecules determining contractile velocity; however, the contraction speed of single fibers within a given MHC isoform type is variable. Recent evidence proposes that the decline in shortening velocity (Vo) with aging is associated with a decrease in the relative content of essential myosin light chain 3f (MLC(3f) ) isoform. In the current study, we first evaluated the relative content of MLC(3f) isoform and Vo in adult and old rats. We then used recombinant adenovirus (rAd) gene transfer technology to increase MLC(3f) protein content in the MHC type II semimembranosus muscle (SM). We hypothesized that (i) aging would decrease the relative MLC(3f) content and Vo in type II fibers, and (ii) increasing the MLC(3f) content would restore the age-induced decline in Vo. We found that there was an age-related decrement in relative MLC(3f) content and Vo in MHC type II fibers. Increasing MLC(3f) content, as indicated by greater % MLC(3f) and MLC(3f) /MLC(2f) ratio, provided significant protection against age-induced decline in Vo without influencing fiber diameter, force generation, MHC isoform distribution, or causing cellular damage. To the best of our knowledge, these are the first data to demonstrate positive effects of MLC(3f) against slowing of contractile function in aged skeletal muscle.     

Bupivacaine inhibits large conductance, voltage- and Ca2+- activated K+ channels in human umbilical artery smooth muscle cells

Bupivacaine is a local anesthetic compound belonging to the amino amide group. Its anesthetic effect is commonly related to its inhibitory effect on voltage-gated sodium channels. However, several studies have shown that this drug can also inhibit voltage-operated K(+) channels by a different blocking mechanism. This could explain the observed contractile effects of bupivacaine on blood vessels. Up to now, there were no previous reports in the literature about bupivacaine effects on large conductance voltage- and Ca(2+) -activated K(+) channels (BK(Ca)). Using the patch-clamp technique, it is shown that bupivacaine inhibits single-channel and whole-cell K(+) currents carried by BK(Ca) channels in smooth muscle cells isolated from human umbilical artery (HUA). At the single-channel level bupivacaine produced, in a concentration- and voltage-dependent manner (IC(50) 324 μM at +80 mV), a reduction of single-channel current amplitude and induced a flickery mode of the open channel state. Bupivacaine (300 μM) can also block whole-cell K(+) currents (~45% blockage) in which, under our working conditions, BK(Ca) is the main component. This study presents a new inhibitory effect of bupivacaine on an ion channel involved in different cell functions. Hence, the inhibitory effect of bupivacaine on BK(Ca) channel activity could affect different physiological functions where these channels are involved. Since bupivacaine is commonly used during labor and delivery, its effects on umbilical arteries, where this channel is highly expressed, should be taken into account.