Conformational Transitions in Yeast Chorismate Mutase Important for Allosteric Regulation as Identified by Nuclear Magnetic Resonance Spectroscopy

Proteins fluctuate between different conformations in solution, and these conformational fluctuations can be important for protein function and allosteric regulation. The chorismate mutase from Saccharomyces cerevisiae (ScCM), a key enzyme in the biosynthesis of aromatic amino acids, is allosterically activated and inhibited by tryptophan and tyrosine, respectively. It was initially proposed that in the absence of effector, ScCM fluctuates between activated R and inhibited T conformations according to the Monod-Wyman-Changeux (MWC) model, although a more complex regulation pattern was later suggested by mutagenesis and kinetic data. Here we used NMR relaxation dispersion experiments to understand the conformational fluctuations on the microsecond-to-millisecond timescale that occur in ScCM. In the absence of allosteric effectors, ScCM did not exclusively exchange between T and R conformations, suggesting that the two-state MWC model is insufficient to explain conformational dynamics. Addition of tyrosine led to the quenching of much of the motion on this timescale, while new motions were identified in the presence of tryptophan. These new motions are consistent with conformational fluctuations into an alternative conformation that may be important for enzyme activity.     

Ultrastructure of Male Sexual Apparatus of Scutellonema brachyurum

Electron micrographs of serial sections show that the male sexual apparatus of Scutellonema brachyurum includes two morphologically identical spicules. Each is composed of a swollen tubular head, crescentic shaft, and leaf-like blade with membranous velum expanded from the central trunk. The spicules are concave and grooved on the ventral side and convex on the dorsal side near the trunk. The trunk is continuous with the shaft and head. Nerve tissue occupies the core of the spicule and includes a dendritic process which gains access to the exterior via a small pore on the lateral side of the spicule tip. Three protractor and two retractor muscles are associated with each spicule. A sensory accessory piece connects with the tip of the gubernaculum and protrudes from the lower side of the opening of the spicular pouch; it protracts and retracts with the muscularized gubernaculum. The gubernaculum varies from bow-shaped in the distal part to boat-shaped in the mid region. A sac exits beneath the accessory piece as a buffer for its movement. A cuticular guiding bar originating from the dorsal wall of the spicular pouch has a tongue. The ventral surface of the tongue is sclerotized to separate the two spicules. It is mobile by muscles of the protractor gubernaculi, retractor gubernaculi, and seductor gubernaculi.     

Pollen counts statistics and its relevance to precision

In the day to day management of pollen counts from aerobiological samples of national networks, only a small proportion (usually from 12 to 15%) of the daily microscope slide is read. It is generally believed that, otherwise, too much time will be spent reading slides for a minimal increase in precision. Different networks use different slide sampling methods (longitudinal, transverse or at random) and a different number of lines are routinely read. However, the topic of the precision of the different pollen count strategies has seldom been the object of serious investigation. In this study, the precision of different sampling methods of 12 pollen types was investigated by: a) counting pollen grains over the whole slide (3 slides per taxa), b) spatially (i.e. microscope field per microscope field) recording over the 3120 fields found at 400× the location of each pollen grain, c) sub-sampling, by macro procedures, this population by selecting a number of transverse (1 to 48) or longitudinal (1 to 20) lines, or a number of random fields (90 to 2340), so that between 0.96 to 46.15% (transverse), 3 to 66.6% (longitudinal) or 3 to 75% (random) of the whole slide was artificially counted. Between nine and twelve procedures were built per reading strategy. The error found is much higher than what is normally believed, and it was significantly correlated with the abundance of a pollen taxa on the sampled slide. It is only with a total count over 1000 (corresponding to a concentration of above 500 m–3) that the mean error of 4 longitudinal lines (or 13.3% of the slide), the standard protocol of both the Italian Association of Aerobiology (AIA) and the Spanish Aerobiology Network (REA), was always below 30%.     

High frequency shoot regeneration from Rhynchostylis gigantea (orchidaceae) using thin cell layers

Often regeneration in orchids is only achieved through protocorms, i.e., the juvenile stage. In order to produce directly shoots via bud regeneration both rapidly and with a high frequency, a transverse thin cell layer (tTCL) method is extended to Rhynchostylis gigantea. Transverse thin cell layer (tTCL) explants (0.3--0.5 mm) excised along the stem from the basis to the shoot tip of one-year-old plantlets were cultured on Murashige and Skoog (1962) medium supplemented with different combinations of benzyladenine (BAP), 1-naphthaleneacetic acid (NAA), thidiazuron (TDZ) and 3% sucrose. The optimal combination for maximal bud regeneration was 3 µM BAP and 3 µM TDZ, giving rise to 11.7 buds per tTCL. Roots were obtained with 10 µM forchlofenuron (CPPU) and 1% sucrose. The in vitro plants (> 3 cm long) obtained 4 to 6 weeks after the tTCLs culture were transferred to the greenhouse; their morphology was normal. Efficient micropropagation of direct production of shoots without passing through protocorm stage of orchid species can be achieved using the thin cell layer (TCL) method.     

白及假鳞茎薄片诱导不定芽及快繁体系的构建

以白及(Bletilla striata)假鳞茎为外植体,根据上、中、下部位切取薄片,探索假鳞茎不同部位BA、NAA和TDZ对假鳞茎薄片诱导不定芽的影响,比较假鳞茎薄片不同厚度对褐化率和出芽数的影响,采用正交试验,研究了BA和NAA对不定芽增殖的效果,并对组培苗进行壮苗、生根和移栽。结果表明:假鳞茎的部位对诱导不定芽作用极显著,下部的出芽率显著高于上部和中部,BA和TDZ对诱导不定芽作用显著,NAA对诱导不定芽作用不显著。最佳诱导不定芽的方式为假鳞茎下部薄片在基本培养基+2.0 mg·L^-1BA+1.0 mg·L^-1TDZ的培养基上培养4周,出芽率为93.3%,出芽数为15个,厚度为1.6~2.0 mm的假鳞茎薄片其褐化率最低。最佳的增殖培养基为基本培养基+1.5 mg·L^-1BA+0.3 mg·L^-1NAA,增殖系数达4.3,平均苗高为7.8 cm。本研究成功建立了白及假鳞茎薄片诱导芽为关键技术的快繁技术体系,为白及种质资源创新奠定了基础。     

Validation of an Allosteric Binding Site of Src Kinase Identified by Unbiased Ligand Binding Simulations

Allostery plays a primary role in regulating protein activity, making it an important mechanism in human disease and drug discovery. Identifying allosteric regulatory sites to explore their biological significance and therapeutic potential is invaluable to drug discovery; however, identification remains a challenge. Allosteric sites are often “cryptic” without clear geometric or chemical features. Since allosteric regulatory sites are often less conserved in protein kinases than the orthosteric ATP binding site, allosteric ligands are commonly more specific than ATP competitive inhibitors. We present a generalizable computational protocol to predict allosteric ligand binding sites based on unbiased ligand binding simulation trajectories. We demonstrate the feasibility of this protocol by revisiting our previously published ligand binding simulations using the first identified viral proto-oncogene, Src kinase, as a model system. The binding paths for kinase inhibitor PP1 uncovered three metastable intermediate states before binding the high-affinity ATP-binding pocket, revealing two previously known allosteric sites and one novel site. Herein, we validate the novel site using a combination of virtual screening and experimental assays to identify a V-type allosteric small-molecule inhibitor that targets this novel site with specificity for Src over closely related kinases. This study provides a proof-of-concept for employing unbiased ligand binding simulations to identify cryptic allosteric binding sites and is widely applicable to other protein–ligand systems.     

Ion conductances of the surface and transverse tubular membranes of skeletal muscle

Summary A combination voltage clamp and admittance analysis of single skeletal muscle fibers showed that moderate depolarizations activated a steady-state negative sodium conductance in both the surface and transverse tubular membranes. The density of the voltage-dependent channels was similar for the surface and tubular conductances. The relaxation times associated with the negative conductance were in the millisecond range and markedly potential dependent. The negative tubular conductance has the consequence of increasing the apparent steady-state radial space constant to large values. This occurs because the positiv conductance is counterbalanced by the maintained inward-going sodium current. The enhancement of the space constant by a negative conductance provides a means for the nearly simultaneous activation of excitation-contraction coupling.     

Crimping in rat tail tendon collagen: morphology and transverse mechanical anisotropy

Examination of rat tail tendon units in scanning electron microscopy (SEM) after the removal of the endotendinium by the use of swelling agents and in comparison with controls confirms and extends our knowledge of a substantially planar crimping along the fibre axis. Polarizing optical microscopy of intact units subjected to lateral compression of controlled direction indicates a definite transverse mechanical anisotropy directly related to the morphological defined crimp plane, sensitive to shear disruption but capable of reconstitution on low strain cyclin.