Cryo-electron microscopy reveals how acetogenins inhibit mitochondrial respiratory complex I

Mitochondrial complex I (NADH:ubiquinone oxidoreductase), a crucial enzyme in energy metabolism, captures the redox potential energy from NADH oxidation/ubiquinone reduction to create the proton motive force used to drive ATP synthesis in oxidative phosphorylation. High-resolution single-particle electron cryo-EM analyses have provided detailed structural knowledge of the catalytic machinery of complex I, but not of the molecular principles of its energy transduction mechanism. Although ubiquinone is considered to bind in a long channel at the interface of the membrane-embedded and hydrophilic domains, with channel residues likely involved in coupling substrate reduction to proton translocation, no structures with the channel fully occupied have yet been described. Here, we report the structure (determined by cryo-EM) of mouse complex I with a tight-binding natural product acetogenin inhibitor, which resembles the native substrate, bound along the full length of the expected ubiquinone-binding channel. Our structure reveals the mode of acetogenin binding and the molecular basis for structure–activity relationships within the acetogenin family. It also shows that acetogenins are such potent inhibitors because they are highly hydrophobic molecules that contain two specific hydrophilic moieties spaced to lock into two hydrophilic regions of the otherwise hydrophobic channel. The central hydrophilic section of the channel does not favor binding of the isoprenoid chain when the native substrate is fully bound but stabilizes the ubiquinone/ubiquinol headgroup as it transits to/from the active site. Therefore, the amphipathic nature of the channel supports both tight binding of the amphipathic inhibitor and rapid exchange of the ubiquinone/ubiquinol substrate and product.     

A potential mechanism for the impairment of nitric oxide formation caused by prolonged oral exposure to arsenate in rabbits

We have recently found evidence for impairment of nitric oxide (NO) formation and induction of oxidative stress in residents of an endemic area of chronic arsenic poisoning in Inner Mongolia, China. To investigate the underlying mechanisms responsible for these phenomena, a subchronic animal experiment was conducted using male New Zealand White rabbits. After 18 weeks of continuous exposure of rabbits to 5 mg/l of arsenate in drinking water, a significant decrease in systemic NO production occurred, as shown by significantly reduced plasma NO metabolites levels (76% of control) and a tendency towards decreased serum cGMP levels (81.4% of control). On the other hand, increased oxidative stress, as shown by significantly increased urinary hydrogen peroxide (H(2)O(2)) (120% of control), was observed in arsenate-exposed rabbits. In additional experiments measuring aortic tension, the addition of either the calcium ionophore A23187 or acethylcholine (ACh) induced a transient vasoconstriction of aortic rings prepared from arsenate-exposed rabbits, but not in those prepared from control animals. This calcium-dependent contractility action observed in aorta rings from arsenate-exposed rabbits was markedly attenuated by the superoxide (O2(.-)) scavenging enzyme Cu, Zn-SOD, as well as diphenyleneiodonium (DPI) or N(G)-nitro-L-arginine methyl ester (L-NAME), which are inhibitors for nitric oxide synthase (NOS). However, the cyclooxygenase inhibitor indomethacin or the xanthine oxidase blocker allopurinol had no effect on this vasoconstriction. These results suggest that arsenate-mediated reduction of systemic NO may be associated with the enzymatic uncoupling reaction of NOS with a subsequent enhancement of reactive oxygen species such as O2(.-), an endothelium-derived vasoconstricting factor. Furthermore, hepatic levels of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH(4)), a cofactor for NOS, were markedly reduced in arsenate-exposed rabbits to 62% of control, while no significant change occurred in cardiac L-arginine levels. These results suggest that prolonged exposure of rabbits to oral arsenate may impair the bioavailability of BH(4) in endothelial cells and, as a consequence, disrupt the balance between NO and O2(.-) produced from endothelial NOS, such that enhanced free radicals are produced at the expense of NO.     

A physical model for galvanotaxis of Paramecium cell

We propose a qualitative physical model of galvanotaxis of Paramecium cells using a bottom-up approach to link the microscopic ciliary motion and the macroscopic behavior of the cells. From the characteristic pattern of ciliary motion called the Ludloff phenomenon, the torque that orients the cell toward the cathode is derived mathematically. Dynamical equations of motion are derived and their stability is discussed. In numerical simulations using our model, cells exhibit realistic behavior, such as U-turns, like real cells.     

Altered localization of amyloid precursor protein under endoplasmic reticulum stress

Recent reports have shown that the endoplasmic reticulum (ER) stress is relevant to the pathogenesis of Alzheimer disease. Following the amyloid cascade hypothesis, we therefore attempted to investigate the effects of ER stress on amyloid-beta peptide (Abeta) generation. In this study, we found that ER stress altered the localization of amyloid precursor protein (APP) from late compartments to early compartments of the secretory pathway, and decreased the level of Abeta 40 and Abeta 42 release by beta- and gamma-cutting. Transient transfection with BiP/GRP78 also caused a shift of APP and a reduction in Abeta secretion. It was revealed that the ER stress response facilitated binding of BiP/GRP78 to APP, thereby causing it to be retained in the early compartments apart from a location suitable for the cleavages of Abeta. These findings suggest that induction of BiP/GRP78 during ER stress may be one of the regulatory mechanisms of Abeta generation.     

Behavioral Sequence Leading to Sexual Isolation Between <Emphasis Type="Italic">Drosophila ananassae</Emphasis> and <Emphasis Type="Italic">D. pallidosa</Emphasis>

Drosophila ananassae and D. pallidosa are closely related, sympatric species that lack postmating isolation. Sexual isolation has been considered important in maintaining them as independent species. To clarify the behavioral processes leading to sexual isolation, we analyzed behavioral sequences and examined the effect of courtship song on mating success and on behaviors of both sexes by surgically removing male wings (song generators), female aristae (song receivers), or female wings (means of fluttering). We found that heterospecific courtship songs evoked female wing fluttering, whereas conspecific courtship song did not. Furthermore, female wing fluttering made courting males discontinue courtship. These findings suggest that strong sexual isolation is achieved through the following behavioral sequence: heterospecific song→female wing fluttering→courtship discontinuation.     

Effects of sediment removal on nitrogen uptake by riparian plants in the higher floodplain of the Chikuma River, Japan

Riparian plants can use nitrogen (N) from soil and river water, but the use of river water N might be limited in higher floodplain environments of the Chikuma River. The purpose of this study is to reveal the relationship between N uptake by riparian plants and the floodplain topography (relative height and distance from a river channel). We examined the hypothesis that surface sediment removal from the higher floodplain increases river water N uptake by riparian plants by using a stable isotope analysis. The δ¹⁵N value of river water samples (ca. 8‰) were significantly higher than those of the soil extracts (ca. 3‰) in the study area. The δ¹⁵N value of riparian plants increased from +3.0‰ (standard deviation, SD ±2.1‰) before sediment removal to +9.6‰ (±2.1‰) after sediment removal, although there was no significant change in the δ¹⁵N value in N sources of soil and river water. The sediment removal enhanced frequency of flood disturbance, relative ground water level, and river water N uptake by riparian plants on the floodplain.     

Intramembrane processing by signal peptide peptidase regulates the membrane localization of hepatitis C virus core protein and viral propagation

Hepatitis C virus (HCV) core protein has shown to be localized in the detergent-resistant membrane (DRM), which is distinct from the classical raft fraction including caveolin, although the biological significance of the DRM localization of the core protein has not been determined. The HCV core protein is cleaved off from a precursor polyprotein at the lumen side of Ala(191) by signal peptidase and is then further processed by signal peptide peptidase (SPP) within the transmembrane region. In this study, we examined the role of SPP in the localization of the HCV core protein in the DRM and in viral propagation. The C terminus of the HCV core protein cleaved by SPP in 293T cells was identified as Phe(177) by mass spectrometry. Mutations introduced into two residues (Ile(176) and Phe(177)) upstream of the cleavage site of the core protein abrogated processing by SPP and localization in the DRM fraction. Expression of a dominant-negative SPP or treatment with an SPP inhibitor, L685,458, resulted in reductions in the levels of processed core protein localized in the DRM fraction. The production of HCV RNA in cells persistently infected with strain JFH-1 was impaired by treatment with the SPP inhibitor. Furthermore, mutant JFH-1 viruses bearing SPP-resistant mutations in the core protein failed to propagate in a permissive cell line. These results suggest that intramembrane processing of HCV core protein by SPP is required for the localization of the HCV core protein in the DRM and for viral propagation.     

A region of calpastatin domain L that reprimes cardiac L-type Ca2+ channels

Calpastatin, an endogenous inhibitor of calpain, is composed of domain L and four repetitive homologous domains 1-4. Domains 1-4 inhibit calpain, whereas domain L partially reprimes L-type Ca2+ channels for voltage-gated activation. In the present study, the effects on Ca2+ channel activity of four isoforms and a series of fragments of calpastatin domain L were investigated in guinea-pig ventricular myocytes with the patch-clamp method. With one exception, all the isoforms and fragment peptides that contained amino acid residues 54-64 of domain L reprimed the Ca2+ channels to comparable levels (9-15% of control activity) to those observed previously with a full-length form of calpastatin. These results suggest that the region containing amino acid residues 54-64 (EGKPKEHTEPK) is responsible for the Ca2+ channel repriming function of calpastatin domain L.     

Cerebral Blood Flow Threshold and Regional Heterogeneity of Heat Shock Protein 72 Induction Following Transient Forebrain Ischemia in Rats

Heat shock proteins (HSPs) induced by brain ischemia may play an important role in neuroprotection from neuronal degeneration. In this study, we examined the cerebral blood flow (CBF) threshold to produce regional differences in HSP72 induction after transient forebrain ischemia in spontaneously hypertensive rats (SHRs). Female SHRs were subjected to 20 min of cerebral ischemia induced by bilateral carotid artery occlusion. The CBF was measured by laser Doppler flowmetry. At forty-eight hours after cerebral ischemia and reperfusion, the rats were decapitated and the brains were removed. Specific areas (hippocampal CA1, CA2-3, dentate gyrus, dorsolateral and ventromedial striatum, and parietal cortex) were thereafter dissected from the brain. The amounts of HSP72 in these samples were determined using Western blot analysis. In the hippocampus, HSP72 was induced when the CBF decreased to less than 18–25% of the resting level. The mean values of HSP72 produced in the CA1 area, CA2-3 area, and the dentate gyrus following ischemia and reperfusion treatment were 4.44 ± 1.43 (±SD) ng/g prtein, 3.51 ± 0.72 ng/g protein and 3.77 ± 1.05 ng/g protein, respectively. In the parietal cortex, the amount of HSP72 induction was less pronounced (2.55 ± 0.40 ng/g protein), while HSP72 was hardly detected at all in the striatum, even under conditions of very severe CBF reduction and reperfusion. We demonstrated the existence of both a CBF threshold (i.e., approximately 20% of the resting level) for HSP72 induction and regional heterogeneity for the induction of HSP72 protein.