Myosin head orientation and mobility during isometric contraction: effects of osmotic compression.

We have correlated the mobility and the generation of force of myosin heads by applying radial compression to isometrically contracting muscle fibers. Osmotic pressure was produced by dextran T-500, and its effect on the orientation and mobility of myosin heads labeled with N-(1-oxy-2,2,5,5-tetramethyl-4-pyperidinyl)maleimide was observed by conventional and saturation-transfer electron paramagnetic resonance methods. A biphasic behavior is spectral changes coinciding with the tension dependence was observed as the fibers were compressed. At diameters above the equilibrium spacing, the large myosin head disorder characteristic during contraction in the absence of compression was largely maintained, whereas the mobility decreased threefold, from tauR approximately 25 microseconds to approximately 80-90 microseconds. The inhibition of fast microsecond motions was not accompanied by tension loss, implying that these motions are not necessary for force generation. At diameters below the equilibrium spacing, both the disorder and the mobility decreased dramatically in parallel with the tension inhibition, suggesting that slower microsecond motions and the disorder of the myosin head are necessary for muscle function.     

The effects of enriched CO2 atmospheres on plant-insect herbivore interactions: growth responses of larvae of the specialist butterfly,Junonia coenia (Lepidoptera: Nymphalidae)

Summary Little is known about the effects of enriched CO₂ environments, which are anticipated to exist in the next century, on natural plant-insect herbivore interactions. To begin to understand such effects on insect growth and survival, I reared both early and penultimate instar larvae of the buckeye, Junonia coenia (Lepidoptera: Nymphalidae), on leaves from one of their major hostplants, plantain, Plantago lanceolata (Plantaginaceae), grown in either ambient (350 PPM) or high (700 PPM) CO₂ atmospheres. Despite consuming more foliage, early instar larvae experienced reduced growth on high CO₂-grown compared to ambient CO₂-grown leaves. However, survivorship of early instar larvae was unaffected by the CO₂ treatment. Larval weight gain was positively correlated with the nitrogen concentration of the plant material and consumption was negatively correlated with foliar nitrogen concentration, whereas neither larval weight gain nor consumption were significantly correlated with foliar water or allelochemical concentrations. In contrast, penultimate instar larvae had similar growth rates on ambient and high CO₂-grown leaves. Significantly higher consumption rates on high CO₂-grown plants enabled penultimate instar larvae to obtain similar amounts of nitrogen in both treatments. These larvae grew at similar rates on foliage from the two CO₂ treatments, despite a reduced efficiency of conversion of ingested food (ECI) on the low nitrogen, high CO₂-grown plants. However, nitrogen utilization efficiencies (NUE) were unaffected by CO₂ treatment. Again, for late instar larvae, consumption rates were negatively correlated with foliar nitrogen concentrations, and ECI was also very highly correlated with leaf nitrogen; foliar water or allelochemical concentrations did not affect either of these parameters. Differences in growth responses of early and late instar larvae to lower nitrogen, high-CO₂ grown foliage may be due to the inability of early instar larvae to efficiently process the increased flow of food through the gut caused by additional consumption of high CO₂ foliage.     

Synthesis and properties of a conformationally restricted spin-labeled analog of ATP and its interaction with myosin and skeletal muscle.

The synthesis is described of a spin-labeled analog of ATP, 2',3'-O-(1-oxy-2,2,6,6-tetramethyl-4-piperidylidene)adenosine 5'-triphosphate (SL-ATP). The spin-label moiety is attached by two bonds to the ribose ring as a spiroketal and hence has restricted conformational mobility relative to the ribose moiety of ATP. The synthesis proceeds via an acid-catalyzed addition of adenosine 5'-monophosphate to 1-acetoxy-4-methoxy-2,2,6,6-tetramethyl-1,2,5,6-tetrahydropyridine in acetonitrile. The spiroketal product is pyrophosphorylated, and alkaline hydrolysis with concomitant aerial oxidation gives the required product. The spin-labeled moiety probably takes up two rapidly interconverting conformations with respect to the ribose ring on the basis of the 1H NMR spectra of its precursors and related uridine derivatives [Alessi et al. (1991) J. Chem. Soc., Perkin Trans.1,2243-2247]. SL-ATP is a substrate for myosin and actomyosin with similar kinetic parameters to ATP during triphosphatase activity. SL-ATP supports muscle contraction and permits relaxation of permeabilized rabbit skeletal muscle fibers. SL-ADP is a substrate for yeast 3-phosphoglycerate kinase, thus permitting regeneration of SL-ATP from SL-ADP within muscle fibers. Electron paramagnetic resonance (EPR) studies of SL-ADP bound to myosin filaments and to myofibrils show a degree of nanosecond motion independent of that of the protein, which may be due to conformational flexibility of the ribose moiety of ATP bound to myosin's active site. This nanosecond motion is more restricted in myofibrils than in myosin filaments, suggesting that the binding of actin affects the ribose binding site in myosin. EPR studies on SL-ADP bound to rigor cross-bridges in muscle fiber bundles showed the nucleotide to be highly oriented with respect to the fiber axis.     

Rotational dynamics of phospholamban determined by multifrequency electron paramagnetic resonance

We have used multifrequency electron paramagnetic resonance to define the multistate structural dynamics of an integral membrane protein, phospholamban (PLB), in a lipid bilayer. PLB is a key regulator of cardiac calcium transport, and its function requires transitions between distinct states of intramolecular dynamics. Monomeric PLB was synthesized with the TOAC spin label at positions 11 (in the cytoplasmic domain) and 46 (in the transmembrane domain) and reconstituted into lipid bilayers. Unlike other protein spin labels, TOAC reports directly the motion of the peptide backbone, so quantitative analysis of its dynamics is worthwhile. Electron paramagnetic resonance spectra at 9.4 GHz (X-band) and 94 GHz (W-band) were analyzed in terms of anisotropic rotational diffusion of the two domains. Motion of the transmembrane domain is highly restricted, while the cytoplasmic domain exhibits two distinct conformations, a major one with moderately restricted nanosecond dynamics (T) and another with nearly unrestricted subnanosecond motion (R). The global analysis of spectra at two frequencies yielded values for the rotational correlation times and order parameters that were much more precisely determined than at either frequency alone. Multifrequency EPR is a powerful approach for analysis of complex rotational dynamics of proteins.     

Unusual presentation of choriocarcinoma

Background  

Choriocarcinoma is an aggressive neoplasm arising in the body of the uterus. The disease normally spreads to lung and brain.     

Differential expression level of cytokeratin 8 in cells of the bovine nucleus pulposus complicates the search for specific intervertebral disc cell markers

Introduction  

Development of cell therapies for repairing the intervertebral disc is limited by the lack of a source of healthy human disc cells. Stem cells, particularly mesenchymal stem cells, are seen as a potential source but differentiation strategies are limited by the lack of specific markers that can distinguish disc cells from articular chondrocytes.     

Bergs Methode zur Bestimmung von Vermahlungseigenschaften an einzelnen Weizenpflanzen

Summary The micro-method of Berg gives a good varietal characteristic of milling quality of wheat.10 g of wheat of normal water content is ground to a meal, which is then sifted for 10 minutes on a sieving machine with 3 bolting cloths; No 9, 12 and 15 (Silk cloth XX) representing a mesh-width of 150, 105 and 75 respectively.The flour fraction in g under cloth 15 gives the unit of Berg and has proved very useful for determination of the grittiness of flour from different varieties of wheat. Therefore it has been used to a great extent in the wheat breeding work at Weibullsholm, Sweden.The method could be modified, if it should be desirable from local points of view.     

Covalent inhibition of hAChE by organophosphates causes homodimer dissociation through long-range allosteric effects

Acetylcholinesterase (EC 3.1.1.7), a key acetylcholine-hydrolyzing enzyme in cholinergic neurotransmission, is present in a variety of states in situ, including monomers, C-terminally disulfide-linked homodimers, homotetramers, and up to three tetramers covalently attached to structural subunits. Could oligomerization that ensures high local concentrations of catalytic sites necessary for efficient neurotransmission be affected by environmental factors? Using small-angle X-ray scattering (SAXS) and cryo-EM, we demonstrate that homodimerization of recombinant monomeric human acetylcholinesterase (hAChE) in solution occurs through a C-terminal four-helix bundle at micromolar concentrations. We show that diethylphosphorylation of the active serine in the catalytic gorge or isopropylmethylphosphonylation by the R_P enantiomer of sarin promotes a 10-fold increase in homodimer dissociation. We also demonstrate the dissociation of organophosphate (OP)-conjugated dimers is reversed by structurally diverse oximes 2PAM, HI6, or RS194B, as demonstrated by SAXS of diethylphosphoryl-hAChE. However, binding of oximes to the native ligand-free hAChE, binding of high-affinity reversible ligands, or formation of an S_P-sarin-hAChE conjugate had no effect on homodimerization. Dissociation monitored by time-resolved SAXS occurs in milliseconds, consistent with rates of hAChE covalent inhibition. OP-induced dissociation was not observed in the SAXS profiles of the double-mutant Y337A/F338A, where the active center gorge volume is larger than in wildtype hAChE. These observations suggest a key role of the tightly packed acyl pocket in allosterically triggered OP-induced dimer dissociation, with the potential for local reduction of acetylcholine-hydrolytic power in situ. Computational models predict allosteric correlated motions extending from the acyl pocket toward the four-helix bundle dimerization interface 25 Å away.     

Frequent Seizures Are Associated with a Network of Gray Matter Atrophy in Temporal Lobe Epilepsy with or without Hippocampal Sclerosis

Objective

Patients with temporal lobe epilepsy (TLE) with hippocampal sclerosis (HS) have diffuse subtle gray matter (GM) atrophy detectable by MRI quantification analyses. However, it is not clear whether the etiology and seizure frequency are associated with this atrophy. We aimed to evaluate the occurrence of GM atrophy and the influence of seizure frequency in patients with TLE and either normal MRI (TLE-NL) or MRI signs of HS (TLE-HS).

Methods

We evaluated a group of 172 consecutive patients with unilateral TLE-HS or TLE-NL as defined by hippocampal volumetry and signal quantification (122 TLE-HS and 50 TLE-NL) plus a group of 82 healthy individuals. Voxel-based morphometry was performed with VBM8/SPM8 in 3T MRIs. Patients with up to three complex partial seizures and no generalized tonic-clonic seizures in the previous year were considered to have infrequent seizures. Those who did not fulfill these criteria were considered to have frequent seizures.

Results

Patients with TLE-HS had more pronounced GM atrophy, including the ipsilateral mesial temporal structures, temporal lobe, bilateral thalami and pre/post-central gyri. Patients with TLE-NL had more subtle GM atrophy, including the ipsilateral orbitofrontal cortex, bilateral thalami and pre/post-central gyri. Both TLE-HS and TLE-NL showed increased GM volume in the contralateral pons. TLE-HS patients with frequent seizures had more pronounced GM atrophy in extra-temporal regions than TLE-HS with infrequent seizures. Patients with TLE-NL and infrequent seizures had no detectable GM atrophy. In both TLE-HS and TLE-NL, the duration of epilepsy correlated with GM atrophy in extra-hippocampal regions.

Conclusion

Although a diffuse network GM atrophy occurs in both TLE-HS and TLE-NL, this is strikingly more evident in TLE-HS and in patients with frequent seizures. These findings suggest that neocortical atrophy in TLE is related to the ongoing seizures and epilepsy duration, while thalamic atrophy is more probably related to the original epileptogenic process.