Synthesis and in situ insertion of a site-specific fluorescently labeled membrane protein into cell-sized liposomes

The integral membrane protein bacteriorhodopsin, containing a fluorescent amino acid at a specific position, was synthesized in the presence of hydrated lipid films using an in vitro translation system expanded with a four-base codon/anticodon pair. Cell-sized liposomes with the labeled protein inserted into the liposome membranes were generated after the translation reaction. This study also demonstrated that this labeling method could be used to analyze the dynamic properties of membrane proteins in situ by fluorescence correlation spectroscopy.     

Mechanism of amyloid β-protein aggregation mediated by GM1 ganglioside clusters

It is widely accepted that the conversion of the soluble, nontoxic amyloid β-protein (Aβ) monomer to aggregated toxic Aβ rich in β-sheet structures is central to the development of Alzheimer's disease. However, the mechanism of the abnormal aggregation of Aβ in vivo is not well understood. We have proposed that ganglioside clusters in lipid rafts mediate the formation of amyloid fibrils by Aβ, the toxicity and physicochemical properties of which are different from those of amyloids formed in solution. In this paper, the mechanism by which Aβ-(1-40) fibrillizes in raftlike lipid bilayers composed of monosialoganglioside GM1, cholesterol, and sphingomyelin was investigated in detail on the basis of singular-value decomposition of circular dichroism data and analysis of fibrillization kinetics. At lower protein densities in the membrane (Aβ:GM1 ratio of less than ～0.013), only the helical species exists. At intermediate protein densities (Aβ:GM1 ratio between ～0.013 and ～0.044), the helical species and aggregated β-sheets (～15-mer) coexist. However, the β-structure is stable and does not form larger aggregates. At Aβ:GM1 ratios above ～0.044, the β-structure is converted to a second, seed-prone β-structure. The seed recruits monomers from the aqueous phase to form amyloid fibrils. These results will shed light on a molecular mechanism for the pathogenesis of the disease.     

Mathematical modeling of cell cycle regulation in response to DNA damage: exploring mechanisms of cell-fate determination

After DNA damage, cells activate p53, a tumor suppressor gene, and select a cell fate (e.g., DNA repair, cell cycle arrest, or apoptosis). Recently, a p53 oscillatory behavior was observed following DNA damage. However, the relationship between this p53 oscillation and cell-fate selection is unclear. Here, we present a novel model of the DNA damage signaling pathway that includes p53 and whole cell cycle regulation and explore the relationship between p53 oscillation and cell fate selection. The simulation run without DNA damage qualitatively realized experimentally observed data from several cell cycle regulators, indicating that our model was biologically appropriate. Moreover, the comprehensive sensitivity analysis for the proposed model was implemented by changing the values of all kinetic parameters, which revealed that the cell cycle regulation system based on the proposed model has robustness on a fluctuation of reaction rate in each process. Simulations run with four different intensities of DNA damage, i.e. Low-damage, Medium-damage, High-damage, and Excess-damage, realized cell cycle arrest in all cases. Low-damage, Medium-damage, High-damage, and Excess-damage corresponded to the DNA damage caused by 100, 200, 400, and 800 J/m² doses of UV-irradiation, respectively, based on expression of p21, which plays a crucial role in cell cycle arrest. In simulations run with High-damage and Excess-damage, the length of the cell cycle arrest was shortened despite the severe DNA damage, and p53 began to oscillate. Cells initiated apoptosis and were killed at 400 and 800 J/m² doses of UV-irradiation, corresponding to High-damage and Excess-damage, respectively. Therefore, our model indicated that the oscillatory mode of p53 profoundly affects cell fate selection.     

Effect of muscle contraction levels on the force-length relationship of the human Achilles tendon during lengthening of the triceps surae muscle-tendon unit

Findings from animal experiments are sometimes contradictory to the idea that the tendon structure is a simple elastic spring in series with muscle fibers, and suggest influence of muscle contraction on the tendon mechanical properties. The purpose of the present study was to investigate the influence of muscle contraction levels on the force-length relationship of the human Achilles tendon during lengthening of the triceps surae muscle-tendon unit. For seven subjects, ankle dorsiflexion was performed without (passive condition) and with contraction of plantar flexor muscles (eccentric conditions, at 3 contraction levels) on an isokinetic dynamometer. Deformation of the Achilles tendon during each trial was measured using ultrasonography. The Achilles tendon force corresponding to the tendon elongation of 10mm in the passive condition was significantly smaller than those in the eccentric conditions (p<0.05 or p<0.01). Within the eccentric conditions, the Achilles tendon force corresponding to the tendon elongation of 10mm was significantly greater in the maximal contraction level than those in submaximal eccentric conditions (p<0.05 or p<0.01). In addition, the tendon stiffness was greater in higher contraction levels (p<0.05 or p<0.01). Present results suggest that the human tendon structure is not a simple elastic spring in series with muscle fibers.     

Sigma-1 receptor stimulation by dehydroepiandrosterone ameliorates cognitive impairment through activation of CaM kinase II, protein kinase C and extracellular signal-regulated kinase in olfactory bulbectomized mice

Dehydroepiandrosterone (DHEA) is one of the most abundant neurosteroids synthesized de novo in the CNS. We here found that sigma-1 receptor stimulation by DHEA improves cognitive function through phosphorylation of synaptic proteins in olfactory bulbectomized (OBX) mouse hippocampus. We have previously reported that calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) were impaired in OBX mouse hippocampus. OBX mice were administered once a day for 7-8 days with DHEA (30 or 60 mg/kg p.o.) 10 days after operation. The spatial, cognitive and conditioned fear memories in OBX mice were significantly improved as assessed by Y-maze, novel object recognition and passive avoidance task, respectively. DHEA also improved impaired hippocampal long-term potentiation in OBX mice. Notably, DHEA treatment restored PKCα (Ser-657) autophosphorylation and NR1 (Ser-896) and myristoylated alanine-rich protein kinase C substrate (Ser-152/156) phosphorylation to the control levels in the hippocampal CA1 region. Likewise, DHEA treatment improved CaMKIIα (Thr-286) autophosphorylation and GluR1 (Ser-831) phosphorylation to the control levels in the CA1 region. Furthermore, DHEA treatment improved ERK and cAMP-responsive element-binding protein (Ser-133) phosphorylation to the control levels. Finally, NE-100, sigma-1 receptor antagonist, significantly inhibited the DHEA-induced improvement of memory-related behaviors and CaMKII, PKC and ERK phosphorylation in CA1 region. Taken together, sigma-1 receptor stimulation by DHEA ameliorates OBX-induced impairment in memory-related behaviors and long-term potentiation in the hippocampal CA1 region through activation of CaMKII, PKC and ERK.     

Structural insights into differences in drug-binding selectivity between two forms of human alpha1-acid glycoprotein genetic variants, the A and F1*S forms

Human α(1)-acid glycoprotein (hAGP) in serum functions as a carrier of basic drugs. In most individuals, hAGP exists as a mixture of two genetic variants, the F1*S and A variants, which bind drugs with different selectivities. We prepared a mutant of the A variant, C149R, and showed that its drug-binding properties were indistinguishable from those of the wild type. In this study, we determined the crystal structures of this mutant hAGP alone and complexed with disopyramide (DSP), amitriptyline (AMT), and the nonspecific drug chlorpromazine (CPZ). The crystal structures revealed that the drug-binding pocket on the A variant is located within an eight-stranded β-barrel, similar to that found in the F1*S variant and other lipocalin family proteins. However, the binding region of the A variant is narrower than that of the F1*S variant. In the crystal structures of complexes with DSP and AMT, the two aromatic rings of each drug interact with Phe-49 and Phe-112 at the bottom of the binding pocket. Although the structure of CPZ is similar to those of DSP and AMT, its fused aromatic ring system, which is extended in length by the addition of a chlorine atom, appears to dictate an alternative mode of binding, which explains its nonselective binding to the F1*S and A variant hAGPs. Modeling experiments based on the co-crystal structures suggest that, in complexes of DSP, AMT, or CPZ with the F1*S variant, Phe-114 sterically hinders interactions with DSP and AMT, but not CPZ.     

Methyl pyruvate rescues mitochondrial damage caused by SIGMAR1 mutation related to amyotrophic lateral sclerosis

Background

Amyotrophic lateral sclerosis (ALS) is a disease caused by motor neuron degeneration. Recently, a novel SIGMAR1 gene variant (p.E102Q) was discovered in some familial ALS patients.

Methods

We address mechanisms underlying neurodegeneration caused by the mutation using Neuro2A cells overexpressing σ₁R^E102Q, a protein of a SIGMAR1 gene variant (p.E102Q) and evaluate potential amelioration by ATP production via methyl pyruvate (MP) treatment.

Results

σ₁R^E102Q overexpression promoted dissociation of the protein from the endoplasmic reticulum (ER) membrane and cytoplasmic aggregation, which in turn impaired mitochondrial ATP production and proteasome activity. Under ER stress conditions, overexpression of wild-type σ₁R suppressed ER stress-induced mitochondrial injury, whereas σ₁R^E102Q overexpression aggravated mitochondrial damage and induced autophagic cell death. Moreover, σ₁R^E102Q-overexpressing cells showed aberrant extra-nuclear localization of the TAR DNA-binding protein (TDP-43), a condition exacerbated by ER stress. Treatment of cells with the mitochondrial Ca^2 + transporter inhibitor Ru360 mimicked the effects of σ₁R^E102Q overexpression, indicating that aberrant σ₁R-mediated mitochondrial Ca^2 + transport likely underlies TDP-43 extra-nuclear localization, segregation in inclusion bodies, and ubiquitination. Finally, enhanced ATP production promoted by methyl pyruvate (MP) treatment rescued proteasome impairment and TDP-43 extra-nuclear localization caused by σ₁R^E102Q overexpression.

Conclusions

Our observations suggest that neurodegeneration seen in some forms of ALS are due in part to aberrant mitochondrial ATP production and proteasome activity as well as TDP-43 mislocalization resulting from the SIGMAR1 mutation.

General significance

ATP supplementation by MP represents a potential therapeutic strategy to treat ALS caused by SIGMAR1 mutation.     

Evaluation of the lower protein limit in the budding yeast Saccharomyces cerevisiae using TIPI-gTOW

Background

Identifying permissible limits of intracellular parameters such as protein expression provides important information for examining robustness. In this study, we used the TEV protease-mediated induction of protein instability (TIPI) in combination with the genetic Tug-of-War (gTOW) to develop a method to measure the lower limit of protein level. We first tested the feasibility of this method using ADE2 as a marker and then analyzed some cell cycle regulators to reveal genetic interactions.

Results

Using TIPI-gTOW, we successfully constructed a strain in which GFP-^TDegFAde2 was expressed at the lower limit, just sufficient to support cellular growth under the -Ade condition by accelerating degradation by TEV protease. We also succeeded in constructing a strain in which the minimal level of GFP-^TDegFCdc20 was expressed by TIPI-gTOW. Using this strain, we studied genetic interactions between cell cycle regulators and CDC20, and the result was highly consistent with the previously identified interactions. Comparison of the experimental data with predictions of a mathematical model revealed some interactions that were not implemented into the current model.

Conclusions

TIPI-gTOW is useful for estimating changes in the lower limit of a protein under different conditions, such as different genetic backgrounds and environments. TIPI-gTOW is also useful for analyzing genetic interactions of essential genes whose deletion mutants cannot be obtained.     

Effects of Adding Alkaline Material on the Heavy Metal Chemical Fractions in Soil under Flooded and Non-Flooded Conditions

We investigated the effect of adding an alkaline material (containing calcium carbonate and gypsum) on the immobilization of heavy metals (Cd, Cu, Pb, and Zn) in a paddy soil slightly contaminated with Cd and Zn under flooded and non-flooded conditions in the laboratory. Adding the alkaline material increased the soil pH and significantly decreased the exchangeable fraction of all of the metals, especially for Cd (>75% decrease) and Zn (ca. 90% decrease), under both flooded and non-flooded conditions. Drying the flooded soil samples increased the ratio of exchangeable fraction to the total fraction, particularly for Cd. The exchangeable fraction ratio was lower in the dried, previously flooded samples that contained the alkaline material than in the samples that did not contain the alkaline material, indicating that adding the alkaline material would be an effective way of immobilizing heavy metals during the oxidation of anoxic soils. These results show that the alkaline material can be used to immobilize heavy metals under both anoxic and oxic conditions, and that the effects of flooding and amending a paddy soil with alkaline material on the chemical forms will be different between heavy metals.     

Airway Wall Area Derived from 3-Dimensional Computed Tomography Analysis Differs among Lung Lobes in Male Smokers

Background

It is time-consuming to obtain the square root of airway wall area of the hypothetical airway with an internal perimeter of 10 mm (√Aaw at Pi10), a comparable index of airway dimensions in chronic obstructive pulmonary disease (COPD), from all airways of the whole lungs using 3-dimensional computed tomography (CT) analysis. We hypothesized that √Aaw at Pi10 differs among the five lung lobes and √Aaw at Pi10 derived from one certain lung lobe has a high level of agreement with that derived from the whole lungs in smokers.

Methods

Pulmonary function tests and chest volumetric CTs were performed in 157 male smokers (102 COPD, 55 non-COPD). All visible bronchial segments from the 3^rd to 5^th generations were segmented and measured using commercially available 3-dimensional CT analysis software. √Aaw at Pi10 of each lung lobe was estimated from all measurable bronchial segments of that lobe.

Results

Using a mixed-effects model, √Aaw at Pi10 differed significantly among the five lung lobes (R² = 0.78, P<0.0001). The Bland-Altman plots show that √Aaw at Pi10 derived from the right or left upper lobe had a high level of agreement with that derived from the whole lungs, while √Aaw at Pi10 derived from the right or left lower lobe did not.

Conclusion

In male smokers, CT-derived airway wall area differs among the five lung lobes, and airway wall area derived from the right or left upper lobe is representative of the whole lungs.