Inhibitory Neurotransmission in the Bovine Retractor Penis and the Rat Anococcygeus Muscle

1. The aims of this study were twofold. First to try to detect the non-adrenergic non-cholinergic (NANC) neurotransmitter in the bovine retractor penis by bioassay and secondly to study the effects of various drugs which might reveal its relationship with nitric oxide and endothelium-derived relaxing factor (EDRF). This second objective was pursued in both the bovine retractor penis and rat anococcygeus muscles. 2. The attempt to detect the NANC inhibitory neurotransmitter released by field stimulation from the donor bovine retractor penis muscle and detected by a variety of test tissues (bovine retractor penis, rat anococcygeus and rabbit aortic strip) arranged in several different test systems failed. 3. Superoxide dismutase (10 u./ml. up to 400 u./ml.) had no effect on NANC inhibitory relaxation and had no measurable effect in protecting the NANC neurotransmitter. 4. L-Arginine, a precursor of EDRF, did not potentiate NANC inhibitory relaxation in the BRP either fresh or cold stored up to ten days. 5. L-Monomethyl-N-arginine (L-NMMA, a competitive inhibitor of L-arginine in endothelial cells) and D-monomethyl-N-arginine (D-NMMA) up to 3x10-4M. were without effect on NANC inhibitory relaxation of the bovine retractor penis muscle. But at higher concentration of 10 -3M. L-NMMA inhibited the NANC relaxation and further raised guanethidine-induced tone, an effect partially reversed by L-arginine 10 -3M. 6. L-NMMA (10 -5M. up to 3x10 -4M.), but not D-NMMA (10-5M. up to 3x10-4M.), raised tone and inhibited the NANC inhibitory relaxation on the rat anococcygeus muscle in a dose-dependent manner, an effect competitively reversed by L-arginine (3x10-5M. up to 10-3M.) or by washing out the L-NMMA. 7. L-NMMA up to 6x10 -4M. was without effect on the relaxation induced by nitric oxide on the rat anococcygeus or the rabbit aortic preparations suggesting the inhibition of the response to nerve stimulation was not post-synaptic. 8. L-NMMA, but not D-NMMA, dose-dependently (10-5M. up to 3x10-4M.) inhibited the EDRF relaxation induced by acetylcholine in the rabbit aortic preparations, an effect reversed by L-arginine or washing out L-NMMA. 9. L-Canavanine, an inhibitor of nitric oxide production in macrophages, at concentration up to 3x10 -4M. had no effect on NANC relaxation of the BRP preparation. But at higher concentration of 10 -3M. it inhibited the NANC relaxation, an effect partially reversed by L-arginine 10-3M. 10. L-Canavanine up to 2x10 -3M. exhibited no effect on NANC inhibitory relaxation on the rat anococcygeus preparation

Multiscale Simulations of Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs) lack stable secondary and/or tertiary structures under physiological conditions. The have now been recognized to play important roles in numerous biological processes, particularly cellular signaling and regulation. Mutation of IDPs are frequently associated with human diseases, such as cancers and neuron degenerative diseases. Therefore, it is important to understand the structure, dynamics, and interactions of IDPs, so as to establish the mechanistic basis of how intrinsic disorder mediates versatile functions and how such mechanisms may fail in human diseases. However, the heterogeneous structural ensembles of IDPs are not amenable to high resolution characterization solely through experimental measurements, and molecular modelling and simulation are required to study IDP structures, dynamics, and interactions at the atomistic levels. Here, we first applied the state-of-the-art explicit solvent atomistic simulations to an anti-apoptotic protein Bcl-xL and demonstrated how inherent structural disorder may provide a physical basis of protein regulated unfolding in signaling transduction. We have also constructed a series of efficient coarse-grained models to directly simulate the interactions between IDPs and unveiled how the preexisting structural elements accelerate binding of ACTR to NCBD by promoting efficient folding upon encounter. These studies shed important light on how IDPs perform functions in the cellular regulatory network, but also reveal the necessity of new sampling techniques for more efficient simulations of IDPs. We have thus developed a novel sampling technique, called multiscale enhanced sampling (MSES). MSES couples the atomistic model with coarse-grained ones, to accelerate the sampling of atomistic conformational space. Bias from coupling to a coarse-grained model can be removed using Hamiltonian replica exchange. To achieve the best possible efficiency of MSES simulations, we have developed a new hybrid resolution protein model that could capture the essential features of IDP structures, so as to generate local and long-range fluctuations that are largely consistent with those at the atomistic level. We have also developed an advanced replica exchange protocol, to allow the fast conformational transitions observed in the coupled conditions to be rapidly exchanged to the unbiased limit. Application of these strategies to characterize the structural ensembles of a few non-trivial IDPs shows that faster convergence rate can be achieved, demonstrating the great potential of MSES for atomistic simulations of larger and more complex IDPs

Targeting Intrinsically Disordered Proteins through Dynamic Interactions

Intrinsically disordered proteins (IDPs) are over-represented in major disease pathways and have attracted significant interest in understanding if and how they may be targeted using small molecules for therapeutic purposes. While most existing studies have focused on extending the traditional structure-centric drug design strategies and emphasized exploring pre-existing structure features of IDPs for specific binding, several examples have also emerged to suggest that small molecules could achieve specificity in binding IDPs and affect their function through dynamic and transient interactions. These dynamic interactions can modulate the disordered conformational ensemble and often lead to modest compaction to shield functionally important interaction sites. Much work remains to be done on further elucidation of the molecular basis of the dynamic small molecule–IDP interaction and determining how it can be exploited for targeting IDPs in practice. These efforts will rely critically on an integrated experimental and computational framework for disordered protein ensemble characterization. In particular, exciting advances have been made in recent years in enhanced sampling techniques, Graphic Processing Unit (GPU)-computing, and protein force field optimization, which have now allowed rigorous physics-based atomistic simulations to generate reliable structure ensembles for nontrivial IDPs of modest sizes. Such de novo atomistic simulations will play crucial roles in exploring the exciting opportunity of targeting IDPs through dynamic interactions

Atomistic Peptide Folding Simulations Reveal Interplay of Entropy and Long-Range Interactions in Folding Cooperativity

Understanding how proteins fold has remained a problem of great interest in biophysical research. Atomistic computer simulations using physics-based force fields can provide important insights on the interplay of different interactions and energetics and their roles in governing the folding thermodynamics and mechanism. In particular, generalized Born (GB)-based implicit solvent force fields can be optimized to provide an appropriate balance between solvation and intramolecular interactions and successfully recapitulate experimental conformational equilibria for a set of helical and β-hairpin peptides. Here, we further demonstrate that key thermodynamic properties and their temperature dependence obtained from replica exchange molecular dynamics simulations of these peptides are in quantitative agreement with experimental results. Useful lessons can be learned on how the interplay of entropy and sequentially long-range interactions governs the mechanism and cooperativity of folding. These results highlight the great potential of high-quality implicit solvent force fields for studying protein folding and large-scale conformational transitions

Role of S-Nitrosothiols in Non-Adrenergic Non-Cholinergic Neurotransmission

1. The aim of this study was to examine the possibility that S-nitrosocysteine or S- nitrosoglutathione rather than nitric oxide functions as the inhibitory non- adrenergic, non-cholinergic (NANC) neurotransmitter in the bovine retractor penis (BRP) muscle. This was investigated firstly, by examining whether free sulfhydryl groups are required for NANC relaxation to take place upon nerve stimulation in the BRP muscle and secondly, by examining the properties of the new relaxant produced when nitric oxide is reacted with a range of sulfhydryl compounds. 2. Treatment of BRP muscle with the sulfhydryl oxidising agent, diamide (1 mM), inhibited NANC relaxation induced by nerve stimulation. This effect was completely prevented and almost completely reversed by treating the tissue with the sulfhydryl compounds, L-cysteine (3 mM), L-glutathione (3 mM) or dithiothreitol (3 mM). The inhibition was not specific, however, since the oxidising agent also inhibited the relaxant actions of glyceryl trinitrate (0.01-0.1 muM) and isoprenaline (0.01-1 muM). 3. Treatment of BRP muscle with the sulfhydryl alkylating agent, N-ethylmaleimide (0.3 mM), inhibited NANC relaxation induced by nerve stimulation. This effect was completely prevented but not reversed by treating the tissue with the sulfhydryl compounds, L-cysteine (3 mM), L-glutathione (3 mM) or dithiothreitol (3 mM). As with diamide, inhibition was not specific, however, since the alkylating agent also inhibited the relaxant actions of glyceryl trinitrate (0.01-0.1 muM) and isoprenaline (0.01-1 muM). 4. The weak vasodilator activity of sodium nitrite (10 muM) was greatly enhanced by acidification. The optimal pH for enhancement of vasodilator activity was pH 1-2. Deoxygenation before acidification further enhanced and bubbling to saturation with oxygen for 10 minutes before acidification inhibited relaxant activity. Increases in relaxant activity upon acidification were associated with decreases in nitrite content. Neutralisation of acidified samples led to a rapid loss of relaxant activity. These results are consistent with formation of nitric oxide upon acidification of nitrite and this is destroyed by oxygen and protected in an oxygen-free environment. 5,The first means adopted to form an S-nitrosothiol was to acidify nitrite (10 mM) in the presence of L-cysteine (1.5 M). The relaxant activity, assessed both in magnitude and duration, greatly exceeded that of equivalent solutions of acidified nitrite. Again maximum generation of relaxant activity occurred at pH 2, but unlike solutions of acidified nitrite, these solutions retained their relaxant activity upon neutralisation. These solutions were pink in colour and their relaxant activity was destroyed by the nitric oxide-binding substance, haemoglobin, but was unaffected by the inhibitor of nitric oxide synthase, NG-nitro-L-arginine. 6, In the spectrophotometer at 190-900 nm, nitric oxide in the gas phase produced several narrow absorption bands at wavelengths less than 230 nm, whereas in aqueous solution it produced only a single peak at 190 nm. Upon admission of oxygen, nitrogen dioxide was rapidly formed in both the gas and liquid phases assessed by formation of its characteristic absorption peaks at 300-400 nm. 7, A second means adopted for the formation of S-nitrosothiols was by reacting nitric oxide gas with L-cysteine. In phenylephrine (0.3 muM)-contracted rabbit aortic rings denuded of endothelium and in BRP strips, nitric oxide (1-1,000 nM) alone induced transient relaxation in a concentration-dependent manner, whereas L-cysteine (0.15-4.5 mM) was without effect. When aqueous solutions of L-cysteine (15 mM) were reacted with nitric oxide (5 mM) in nominally oxygen-free conditions at pH 3 for 10 minutes followed by purging with oxygen-free nitrogen to remove free nitric oxide and neutralisation and added to achieve bath concentrations equal to 10 nM nitric oxide and 30 muM L-cysteine, however, more powerful and prolonged relaxation was produced than could be attributed to nitric oxide alone. 8. In the HPLC, aqueous solutions of L-cysteine (10 mM) at pH 3.0 produced two peaks corresponding to L-cystine (-0.85 %, peak 1) and L-cysteine C(-99.15 %, peak 2), respectively. After saturating this solution with nitric oxide gas for 10 minutes under nominal oxygen-free conditions, the solution produced three peaks in the HPLC corresponding to L-cystine (-0.85 %), L-cysteine (- 98 %) and a new substance (-1.15%, peak 3) which was entirely responsible for relaxant activity. Re-run of the peaks in the HPLC revealed that L-cystine was stable, whereas L-cysteine and the new relaxant decayed slowly to form L-cystine.9. Using a series of structural analogues of L-cysteine (all at 15 mM) it was found that removal of the carboxyl group (L-cysteamine), replacement of the carboxyl with an ester function (L-cysteine methyl ester) or substitution at the amino group (N-acetyl-L-cysteine) had no effect on the ability to generate new relaxant activity upon reaction with nitric oxide (0.1 mM). (Abstract shortened by ProQuest.)

Study on barrel vibration characteristics of typical sniper rifle

The firing accuracy of sniper rifle is significantly affected by the barrel vibration induced by shooting load. The barrel vibration is an important parameter needing to be precisely controlled, whose measurement is less studied before. The vertical vibration characteristics of sniper rifle barrel is obtained for the duration of 500 ms with high speed photography system. Both the averaged vibration frequency and maximum amplitude of the measured muzzle are gained. It is found from the test that after the appearance of the maximum value, the vibration amplitude of the muzzle reduces with nearly fixed vibration frequency. In addition, the finite element model is conducted for the barrel vibration regarding sniper rifle. And then the natural frequency as well as the vibration reflection of the barrel under the effort of shooting load is calculated with the model. The model accuracy is well validated for the numerical results are substantially in line with the measurements