Deletion of microsomal prostaglandin E synthase-1 protects neuronal cells from cytotoxic effects of β-amyloid peptide fragment 31-35

Epidemiological studies have suggested that the long-term use of nonsteroidal anti-inflammatory drugs that inhibit cyclooxygenase (COX) activity moderates the onset or progression of Alzheimer's disease (AD). Thus it has been suggested that prostaglandin E(2) (PGE(2)), a major end-product of COX, may play a pathogenic role in AD, but the involvement of PGE synthase (PGES), a terminal enzyme downstream from COX, has not been fully elucidated. To examine the involvement in AD pathology of microsomal PGES-1 (mPGES-1), a PGES enzyme, we here prepared primary cerebral neuronal cells from the cerebri of wild-type and mPGES-1-deficient mice and then treated them with β-amyloid (Aβ) fragment 31-35 (Aβ(31-35)), which represents the shortest sequence of native Aβ peptide required for neurotoxicity. Treatment of wild-type neuronal cells with Aβ(31-35) induced mPGES-1 gene expression and PGE(2) production, followed by significant apoptotic cell death, but apoptosis was not induced in mPGES-1-deficient cells. Furthermore, the combined treatment of Aβ(31-35) and PGE(2) induced apoptosis in mPGES-1-deficient neuronal cells. These results indicated that mPGES-1 is induced during Aβ-mediated neuronal cell death and is involved in Aβ-induced neurotoxicity associated with AD pathology.     

Adaptive behavior of bacterial mechanosensitive channels is coupled to membrane mechanics

Mechanosensitive channel of small conductance (MscS), a tension-driven osmolyte release valve residing in the inner membrane of Escherichia coli, exhibits a complex adaptive behavior, whereas its functional counterpart, mechanosensitive channel of large conductance (MscL), was generally considered nonadaptive. In this study, we show that both channels exhibit similar adaptation in excised patches, a process that is completely separable from inactivation prominent only in MscS. When a membrane patch is held under constant pressure, adaptation of both channels is manifested as a reversible current decline. Their dose–response curves recorded with 1–10-s ramps of pressure are shifted toward higher tension relative to the curves measured with series of pulses, indicating decreased tension sensitivity. Prolonged exposure of excised patches to subthreshold tensions further shifts activation curves for both MscS and MscL toward higher tension with similar magnitude and time course. Whole spheroplast MscS recordings performed with simultaneous imaging reveal activation curves with a midpoint tension of 7.8 mN/m and the slope corresponding to ∼15-nm² in-plane expansion. Inactivation was retained in whole spheroplast mode, but no adaptation was observed. Similarly, whole spheroplast recordings of MscL (V23T mutant) indicated no adaptation, which was present in excised patches. MscS activities tried in spheroplast-attached mode showed no adaptation when the spheroplasts were intact, but permeabilized spheroplasts showed delayed adaptation, suggesting that the presence of membrane breaks or edges causes adaptation. We interpret this in the framework of the mechanics of the bilayer couple linking adaptation of channels in excised patches to the relaxation of the inner leaflet that is not in contact with the glass pipette. Relaxation of one leaflet results in asymmetric redistribution of tension in the bilayer that is less favorable for channel opening.     

2,2,2-Trifluoroethanol changes the transition kinetics and subunit interactions in the small bacterial mechanosensitive channel MscS

2,2,2-Trifluoroethanol (TFE), a low-dielectric solvent, has recently been used as a promising tool to probe the strength of intersubunit interactions in membrane proteins. An analysis of inner membrane proteins of Escherichia coli has identified several SDS-resistant protein complexes that separate into subunits upon exposure to TFE. One of these was the homo-heptameric stretch-activated mechanosensitive channel of small conductance (MscS), a ubiquitous component of the bacterial turgor-regulation system. Here we show that a substantial fraction of MscS retains its oligomeric state in cold lithium-dodecyl-sulfate gel electrophoresis. Exposure of MscS complexes to 10-15 vol % TFE in native membranes or nonionic detergent micelles before lithium-dodecyl-sulfate electrophoresis results in a complete dissociation into monomers, suggesting that at these concentrations TFE by itself disrupts or critically compromises intersubunit interactions. Patch-clamp analysis of giant E. coli spheroplasts expressing MscS shows that exposure to TFE in lower concentrations (0.5-5.0 vol %) causes leftward shifts of the dose-response curves when applied extracellularly, and rightward shifts when added from the cytoplasmic side. In the latter case, TFE increases the rate of tension-dependent inactivation and lengthens the process of recovery to the resting state. MscS responses to pressure ramps of different speeds indicate that in the presence of TFE most channels reside in the resting state and only at tensions near the activation threshold does TFE dramatically speed up inactivation. The effect of TFE is reversible as normal channel activity returns 15-30 min after a TFE washout. We interpret the observed midpoint shifts in terms of asymmetric partitioning of TFE into the membrane and distortion of the bilayer lateral pressure profile. We also relate the increased rate of inactivation and subunit separation with the capacity of TFE to perturb buried interhelical contacts in proteins and discuss these effects in the framework of the proposed gating mechanism of MscS.     

Three-dimensional visualization and morphometry of small airways from microfocal X-ray computed tomography

Physiological morphometry is a critical factor in the flow dynamics in small airways. In this study, we visualized and analyzed the three-dimensional structure of the small airways without dehydration and fixation. We developed a two-step method to visualize small airways in detail by staining the lung tissue with a radiopaque solution and then visualizing the tissue with a cone-beam microfocal X-ray computed tomographic (CT) system. To verify the applicability of this staining and CT imaging (SCT) method, we used the method to visualize small airways in excised rat lungs. By using the SCT method to obtain continuous CT images, three-dimensional branching and merging bronchi ranging from 500 to 150 microm (the airway generation=8-16) were successfully reconstructed. The morphometry of the small airways (diameter, length, branching angle and gravity angle between the gravity direction and airway vector) was analyzed using the three-dimensional thinning algorithm. The diameter and length exponentially decreased with the airway generation. The asymmetry of the bifurcation decreased with generation and one branching angle decided the other pair branching angle. The SCT method is the first reported method that yields faithful high-resolution images of soft tissue geometry without fixation and the three-dimensional morphometry of small airways is useful for studying the biomechanical dynamics in small airways.     

Functional reconstitution and characterization of the Arabidopsis Mg(2+) transporter AtMRS2-10 in proteoliposomes

Magnesium (Mg(2+)) plays critical role in many physiological processes. The mechanism of Mg(2+) transport has been well documented in bacteria; however, less is known about Mg(2+) transporters in eukaryotes. The AtMRS2 family, which consists of 10 Arabidopsis genes, belongs to a eukaryotic subset of the CorA superfamily proteins. Proteins in this superfamily have been identified by a universally conserved GlyMetAsn motif and have been characterized as Mg(2+) transporters. Some members of the AtMRS2 family, including AtMRS2-10, may complement bacterial mutants or yeast mutants that lack Mg(2+) transport capabilities. Here, we report the purification and functional reconstitution of AtMRS2-10 into liposomes. AtMRS2-10, which contains an N-terminal His-tag, was expressed in Escherichia coli and solubilized with sarcosyl. The purified AtMRS2-10 protein was reconstituted into liposomes. AtMRS2-10 was inserted into liposomes in a unidirectional orientation. Direct measurement of Mg(2+) uptake into proteoliposomes revealed that reconstituted AtMRS2-10 transported Mg(2+) without any accessory proteins. Mutation in the GMN motif, M400 to I, inactivated Mg(2+) uptake. The AtMRS2-10-mediated Mg(2+) influx was blocked by Co(III)hexamine, and was independent of the external pH from 5 to 9. The activity of AtMRS2-10 was inhibited by Co(2+) and Ni(2+); however, it was not inhibited by Ca(2+), Fe(2+), or Fe(3+). While these results indicate that AtMRS2-10 has similar properties to the bacterial CorA proteins, unlike bacterial CorA proteins, AtMRS2-10 was potently inhibited by Al(3+). These studies demonstrate the functional capability of the AtMRS2 proteins in proteoliposomes to study structure-function relationships.     

Computer simulation of trabecular remodeling in human proximal femur using large-scale voxel FE models: Approach to understanding Wolff's law

Ever since Julius Wolff proposed the law of bone transformation in the 19th century, it has been widely known that the trabecular structure of cancellous bone adapts functionally to the loading environment. To understand the mechanism of Wolff's law, a three-dimensional (3D) computer simulation of trabecular structural changes due to surface remodeling was performed for a human proximal femur. A large-scale voxel finite element model was constructed to simulate the structural changes of individual trabeculae over the entire cancellous region. As a simple remodeling model that considers bone cellular activities regulated by the local mechanical environment, nonuniformity of local stress was assumed to drive the trabecular surface remodeling to seek a uniform stress state. Simulation results demonstrated that cell-scale (～10 μm) remodeling in response to mechanical stimulation created complex 3D trabecular structures of the entire bone-scale (～10 cm), as illustrated in the reference of Wolff. The bone remodeling reproduced the characteristic anisotropic structure in the coronal cross section and the isotropic structures in other cross sections. The principal values and axes of a structure characterized by fabric ellipsoids corresponded to those of the apparent stress of the structure. The proposed large-scale computer simulation indicates that in a complex mechanical environment of a hierarchical bone structure of over 10⁴ length scale (from ～10 μm to ～10 cm), a simple remodeling at the cellular/trabecular levels creates a highly complex and functional trabecular structure, as characterized by bone density and orientation.     

Dissolved oxygen concentration in river sediment of the Lake Biwa tributaries, Japan

The dissolved oxygen concentration in the sediment pore water downstream of rivers in the Lake Biwa basin was measured, and the factors affecting the dissolved oxygen concentration were analyzed. In August 2003, nine rivers (Sakai, Nakanoi, Hebisuna, Anziki, Yasu, Echi, Ane, Oh, and Ohura) were surveyed. The dissolved oxygen was depleted in the sediment pore water of the rivers with a high proportion of particles less than 250 μm in size. For these rivers, the difference between the dissolved oxygen concentrations of the river surface water and the pore water was large, ranging from −9.54 to −5.26 mg L⁻¹. It was found that the proportion of land turned to paddy fields has an effect on the percentage of the particles below 250 μm (standard partial regression coefficient = 0.807, p = 0.023). These results suggest that, in the Lake Biwa basin, the sedimentation of the fine particles released from paddy fields results in poor dissolved oxygen in the river sediment downstream. In addition, the water flow conditions in small- and medium-scale rivers without headwaters also affect the sedimentation of suspended particles.