Mechano-transduction: from molecules to tissues.

External forces play complex roles in cell organization, fate, and homeostasis. Changes in these forces, or how cells respond to them, can result in abnormal embryonic development and diseases in adults. How cells sense and respond to these mechanical stimuli requires an understanding of the biophysical principles that underlie changes in protein conformation and result in alterations in the organization and function of cells and tissues. Here, we discuss mechano-transduction as it applies to protein conformation, cellular organization, and multi-cell (tissue) function

Probing the heme-thiolate oxygenase domain of inducible nitric oxide synthase with Ru(II) and Re(I) electron tunneling wires

Nitric oxide synthase (NOS) catalyzes the production of nitric oxide from L-arginine and dioxygen at a thiolate-ligated heme active site. Although many of the reaction intermediates are as yet unidentified, it is well established that the catalytic cycle begins with substrate binding and rate-limiting electron transfer to the heme. Here, we show that Ru(II)-diimine and Re(I)-diimine electron tunneling wires trigger nanosecond photoreduction of the active-site heme in the enzyme. Very rapid generation of a reduced thiolate-ligated heme opens the way for direct observation of short-lived intermediates in the NOS reaction cycle

Nanosecond photoreduction of inducible nitric oxide synthase by a Ru-diimine electron tunneling wire bound distant from the active site

A Ru-diimine wire, [(4,4′,5,5′-tetramethylbipyridine)_2Ru(F_9bp)]^(2+) (tmRu-F_9bp, where F_9bp is 4-methyl-4′-methylperfluorobiphenylbipyridine), binds tightly to the oxidase domain of inducible nitric oxide synthase (iNOSoxy). The binding of tmRu-F_9bp is independent of tetrahydrobiopterin, arginine, and imidazole, indicating that the wire resides on the surface of the enzyme, distant from the active-site heme. Photoreduction of an imidazole-bound active-site heme iron in the enzyme-wire conjugate (k_(ET) = 2(1) × 10^7 s^(−1)) is fully seven orders of magnitude faster than the in vivo process

A mutli-technique search for the most primitive CO chondrites

As part of a study to identify the most primitive COs and to look for weakly altered CMs amongst the COs, we have conducted a multi-technique study of 16 Antarctic meteorites that had been classified as primitive COs. For this study, we have determined: (1) the bulk H, C and N abundances and isotopes, (2) bulk O isotopic compositions, (3) bulk modal mineralogies, and (4) for some selected samples the abundances and compositions of their insoluble organic matter (IOM). Two of the 16 meteorites do appear to be CMs – BUC 10943 seems to be a fairly typical CM, while MIL 090073 has probably been heated. Of the COs, DOM 08006 appears to be the most primitive CO identified to date and is quite distinct from the other members of its pairing group. The other COs fall into two groups that are less primitive than DOM 08006 and ALH 77307, the previously most primitive CO. The first group is composed of members of the DOM 08004 pairing group, except DOM 08006. The second group is composed of meteorites belonging to the MIL 03377 and MIL 07099 pairing groups. These two pairing groups should probably be combined. There is a dichotomy in the bulk O isotopes between the primitive (all Antarctic finds) and the more metamorphosed COs (mostly falls). This dichotomy can only partly be explained by the terrestrial weathering experienced by the primitive Antarctic samples. It seems that the more equilibrated samples interacted to a greater extent with 16O-poor material, probably water, than the more primitive meteorites

Electron tunneling through sensitizer wires bound to proteins

We report a quantitative theoretical analysis of long-range electron transfer through sensitizer wires bound in the active-site channel of cytochrome P450cam. Each sensitizer wire consists of a substrate group with high binding affinity for the enzyme active site connected to a ruthenium-diimine through a bridging aliphatic or aromatic chain. Experiments have revealed a dramatic dependence of electron transfer rates on the chemical composition of both the bridging group and the substrate. Using combined molecular dynamics simulations and electronic coupling calculations, we show that electron tunneling through perfluorinated aromatic bridges is promoted by enhanced superexchange coupling through virtual reduced states. In contrast, electron flow through aliphatic bridges occurs by hole-mediated superexchange. We have found that a small number of wire conformations with strong donor–acceptor couplings can account for the observed electron tunneling rates for sensitizer wires terminated with either ethylbenzene or adamantane. In these instances, the rate is dependent not only on electronic coupling of the donor and acceptor but also on the nuclear motion of the sensitizer wire, necessitating the calculation of average rates over the course of a molecular dynamics simulation. These calculations along with related recent findings have made it possible to analyze the results of many other sensitizer-wire experiments that in turn point to new directions in our attempts to observe reactive intermediates in the catalytic cycles of P450 and other heme enzymes

Eaten alive: cannibalism is enhanced by parasites

Cannibalism is ubiquitous in nature and especially pervasive in consumers with stage-specific resource utilization in resource-limited environments. Cannibalism is thus influential in the structure and functioning of biological communities. Parasites are also pervasive in nature and, we hypothesize, might affect cannibalism since infection can alter host foraging behaviour. We investigated the effects of a common parasite, the microsporidian Pleistophora mulleri, on the cannibalism rate of its host, the freshwater amphipod Gammarus duebeni celticus. Parasitic infection increased the rate of cannibalism by adults towards uninfected juvenile conspecifics, as measured by adult functional responses, that is, the rate of resource uptake as a function of resource density. This may reflect the increased metabolic requirements of the host as driven by the parasite. Furthermore, when presented with a choice, uninfected adults preferred to cannibalize uninfected rather than infected juvenile conspecifics, probably reflecting selection pressure to avoid the risk of parasite acquisition. By contrast, infected adults were indiscriminate with respect to infection status of their victims, probably owing to metabolic costs of infection and the lack of risk as the cannibals were already infected. Thus parasitism, by enhancing cannibalism rates, may have previously unrecognized effects on stage structure and population dynamics for cannibalistic species and may also act as a selective pressure leading to changes in resource use

Luminescent Ruthenium(II)− and Rhenium(I)−Diimine Wires Bind Nitric Oxide Synthase

Ru(II)− and Re(I)−diimine wires bind to the oxygenase domain of inducible nitric oxide synthase (iNOSoxy). In the ruthenium wires, [Ru(L)_2L‘]^(2+), L‘ is a perfluorinated biphenyl bridge connecting 4,4‘-dimethylbipyridine to a bulky hydrophobic group (adamantane, 1), a heme ligand (imidazole, 2), or F (3). 2 binds in the active site of the murine iNOSoxy truncation mutants Δ65 and Δ114, as demonstrated by a shift in the heme Soret from 422 to 426 nm. 1 and 3 also bind Δ65 and Δ114, as evidenced by biphasic luminescence decay kinetics. However, the heme absorption spectrum is not altered in the presence of 1 or 3, and Ru−wire binding is not affected by the presence of tetrahydrobiopterin or arginine. These data suggest that 1 and 3 may instead bind to the distal side of the enzyme at the hydrophobic surface patch thought to interact with the NOS reductase module. Complexes with properties similar to those of the Ru−diimine wires may provide an effective means of NOS inhibition by preventing electron transfer from the reductase module to the oxygenase domain. Rhenium−diimine wires, [Re(CO)_3L_1L_1‘]+, where L_1 is 4,7-dimethylphenanthroline and L_1‘ is a perfluorinated biphenyl bridge connecting a rhenium-ligated imidazole to a distal imidazole (F_8bp-im) (4) or F (F_9bp) (5), also form complexes with Δ114. Binding of 4 shifts the Δ114 heme Soret to 426 nm, demonstrating that the terminal imidazole ligates the heme iron. Steady-state luminescence measurements establish that the 4:Δ114 dissociation constant is 100 ± 80 nM. Re−wire 5 binds Δ114 with a K_d of 5 ± 2 μM, causing partial displacement of water from the heme iron. Our finding that both 4 and 5 bind in the NOS active site suggests novel designs for NOS inhibitors. Importantly, we have demonstrated the power of time-resolved FET measurements in the characterization of small molecule:protein interactions that otherwise would be difficult to observe

A critical role for cystathionine-β-synthase in hydrogen sulfide-mediated hypoxic relaxation of the coronary artery

Hypoxia-induced coronary artery vasodilatation protects the heart by increasing blood flow under ischemic conditions, however its mechanism is not fully elucidated. Hydrogen sulfide (H2S) is reported to be an oxygen sensor/transducer in the vasculature. The present study aimed to identify and characterise the role of H2S in the hypoxic response of the coronary artery, and to define the H2S synthetic enzymes involved. Immunoblotting and immunohistochemistry showed expression of all three H2S-producing enzymes, cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST), in porcine coronary artery. Artery segments were mounted for isometric tension recording; hypoxia caused a transient endothelium-dependent contraction followed by prolonged endothelium-independent relaxation. The CBS inhibitor amino-oxyacetate (AOAA) reduced both phases of the hypoxic response. The CSE inhibitor dl-propargylglycine (PPG) and aspartate (limits MPST) had no effect alone, but when applied together with AOAA the hypoxic relaxation response was further reduced. Exogenous H2S (Na2S and NaHS) produced concentration-dependent contraction followed by prolonged relaxation. Responses to both hypoxia and exogenous H2S were dependent on the endothelium, NO, cGMP, K+ channels and Cl−/HCO3 − exchange. H2S production in coronary arteries was blocked by CBS inhibition (AOAA), but not by CSE inhibition (PPG). These data show that H2S is an endogenous mediator of the hypoxic response in coronary arteries. Of the three H2S-producing enzymes, CBS, expressed in the vascular smooth muscle, appears to be the most important for H2S generated during hypoxic relaxation of the coronary artery. A contribution from other H2S-producing enzymes only becomes apparent when CBS activity is inhibited

Particle Energies and Filling Fractions of Radio Bubbles in Cluster Cores

Using Chandra images of cluster cores with clear radio bubbles, we have determined k, which is the ratio of the total particle energy to that of the electrons radiating between 10 MHz and 10 GHz. Radiative and dynamical constraints on the bubbles indicate that the ratio of the energy factor, k, to the volume filling factor, f, lies within the range 1 < k/f < 1000. Assuming pressure equilibrium between the radio-emitting plasma and the surrounding X-ray gas, none of the lobes have equipartition between relativistic particles and magnetic field. There is no evidence for any dependence of the upper limit of the k/f ratio on any physical parameter of the cluster or the radio source. The distribution of the upper limit on k/f appears to be bimodal, the value for some clusters being ~3 and for the others ~300. We show that this is may due to the composition of the jet which forms the bubbles, the variation in the volume filling fraction or variation in the amount of re-acceleration occurring in the bubble.Comment: 12 pages, 9 figures; accepted for publication in MNRA

X-ray Bright Active Galactic Nuclei in Massive Galaxy Clusters I: Number Counts and Spatial Distribution

We present an analysis of the X-ray bright point source population in 43 massive clusters of galaxies observed with the Chandra X-ray Observatory. We have constructed a catalog of 4210 rigorously selected X-ray point sources in these fields, which span a survey area of 4.2 square degrees. This catalog reveals a clear excess of sources when compared to deep blank-field surveys, which amounts to roughly 1 additional source per cluster, likely Active Galactic Nuclei (AGN) associated with the clusters. The excess sources are concentrated within the virial radii of the clusters, with the largest excess observed near the cluster centers. The average radial profile of the excess X-ray sources of the cluster are well described by a power law (N(r) ~ r^\beta) with an index of \beta ~ -0.5. An initial analysis using literature results on the mean profile of member galaxies in massive X-ray selected clusters indicates that the fraction of galaxies hosting X-ray AGN rises with increasing clustercentric radius, being approximately 5 to 10 times higher near the virial radius than in the central regions. This trend is qualitatively similar to that observed for star formation in cluster member galaxies.Comment: 18 Pages, 10 Figures, Submitted to MNRAS. Please contact Steven Ehlert ([email protected]) for higher resolution figures. Updated to reflect small changes requested by referee. This version has been accepted into MNRA