Close Relationships Between the PSII Repair Cycle and Thylakoid Membrane Dynamics

In chloroplasts, a three-dimensional network of thylakoid membranes is formed by stacked grana and interconnecting stroma thylakoids. The grana are crowded with photosynthetic proteins, where PSII-light harvesting complex II (LHCII) supercomplexes often show semi-crystalline arrays for efficient energy trapping, transfer and use. Although light is essential for photosynthesis, PSII is damaged by reactive oxygen species that are generated from primary photochemical reactions when plants are exposed to excess light. Because PSII complexes are embedded in the lipid bilayers of thylakoid membranes, their functions are affected by the conditions of the lipids. Electron paramagnetic resonance (EPR) spin trapping measurements showed that singlet oxygen was formed through peroxidation of thylakoid lipids, suggesting that lipid peroxidation can damage proteins, including the D1 protein. After photodamage, PSII is restored by a specific repair system in thylakoid membranes. In the PSII repair cycle, phosphorylation and dephosphorylation of the PSII proteins control the timing of PSII disassembly and subsequent degradation of the D1 protein. Under light stress, stacked grana turn into unstacked thylakoids with bent grana margins. These structural changes may be closely linked to the mechanisms of the PSII repair cycle because PSII can move more easily from the grana core to the stroma thylakoids through an expanded stromal gap between each thylakoid. Thus, plants modulate the structure of thylakoid membranes under high light to carry out efficient PSII repair. This review focuses on the behavior of the PSII complex and the active role of structural changes to thylakoid membranes under light stress

Quality control of photosystem II: The molecular basis for the action of FtsH protease and the dynamics of the thylakoid membranes

Abstract The reaction center-binding D1 protein of Photosystem II is damaged by excessive light, which leads to photoinhibition of Photosystem II. The damaged D1 protein is removed immediately by specific proteases, and a metalloprotease FtsH located in the thylakoid membranes is involved in the proteolytic process. According to recent studies on the distribution and organization of the protein complexes/supercomplexes in the thylakoid membranes, the grana of higher plant chloroplasts are crowded with Photosystem II complexes and light-harvesting complexes. For the repair of the photodamaged D1 protein, the majority of the active hexameric FtsH proteases should be localized in close proximity to the Photosystem II complexes. The unstacking of the grana may increase the area of the grana margin and facilitate easier access of the FtsH proteases to the damaged D1 protein. These results suggest that the structural changes of the thylakoid membranes by light stress increase the mobility of the membrane proteins and support the quality control of Photosystem II. (159 words

Evidence for efficient phosphorylation of EGFR and rapid endocytosis of phosphorylated EGFR via the early/late endocytic pathway in a gefitinib-sensitive non-small cell lung cancer cell line

Gefitinib (Iressa)–a specific inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase–has been shown to suppress the activation of EGFR signaling required for cell survival and proliferation in non-small cell lung cancer (NSCLC) cell lines. We recently provided novel evidence that gefitinib-sensitive PC9 cells show normal endocytosis of EGFR: internalized EGF-EGFR complexes were transported to late endosomes/lysosomes 15 min after EGF stimulation, and then degraded within the lysosomes. However, gefitinib-resistant QG56 cells showed internalized EGFR accumulation in early endosomes after 60 min of internalization, instead of its trafficking to lysosomes, indicating an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/lysosomes. Therefore, we postulate that impairment in some steps of EGF-EGFR trafficking from early endosomes to late endosomes/lysosomes might confer gefitinib-resistance in NSCLC cell lines. To further substantiate the detailed internalization mechanism of gefitinib-sensitive and gefitinib-resistant cells, using confocal immunofluorescence microscopy, we examined the endocytic trafficking of phosphorylated EGFR (pEGFR) in the absence or presence of gefitinib. In PC9 and QG56 cells without EGF stimulation, a large number of pEGFR-positive small vesicular structures not colocalized with late endosomes/lysosomes were spread throughout the cytoplasm, and some pEGFR staining was distributed in the nucleus. This implies a novel intracellular trafficking pathway for pEGFR from cytoplasmic vesicles to the nucleus. Furthermore, an aggregated vesicular structure of early endosomes was observed in the perinuclear region of QG56 cells; it was revealed to be associated with SNX1, originally identified as a protein that interacts with EGFR. Therefore, we confirmed our previous data that an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/lysosomes occurs in QG56 cells. Furthermore, in PC9 cells, efficient phosphorylation of EGFR and rapid internalization of pEGFR was observed at 3 min after EGF stimulation; these internalized pEGFR-positive vesicles were trafficked to late endosomes at 15 min, indicating rapid trafficking of EGF-pEGFR complexes from early to late endosomes in PC9 cells. Gefitinib treatment strongly reduced the phosphorylation level of EGFR, and subsequent endocytosis of EGFR was significantly suppressed in PC9 cells. In contrast, in QG56 cells, EGFR trafficking via the early endocytic pathway was basically impaired; therefore, gefitinib appeared to slightly suppress the internalization of pEGFR. Collectively, our data provide novel evidence that extensive impairment in pEGFR endocytosis via the early endocytic pathway might confer gefitinib-resistance in QG56 cells

Quality control of Photosystem II: The molecular basis for the action of FtsH protease and the dynamics of the thylakoid membranes

The reaction center-binding D1 protein of Photosystem II is damaged by excessive light, which leads to photoinhibition of Photosystem II. The damaged D1 protein is removed immediately by specific proteases, and a metalloprotease FtsH located in the thylakoid membranes is involved in the proteolytic process. According to recent studies on the distribution and organization of the protein complexes/supercomplexes in the thylakoid membranes, the grana of higher plant chloroplasts are crowded with Photosystem II complexes and light-harvesting complexes. For the repair of the photodamaged D1 protein, the majority of the active hexameric FtsH proteases should be localized in close proximity to the Photosystem II complexes. The unstacking of the grana may increase the area of the grana margin and facilitate easier access of the FtsH proteases to the damaged D1 protein. These results suggest that the structural changes of the thylakoid membranes by light stress increase the mobility of the membrane proteins and support the quality control of Photosystem II

The Relationship between Inferior Vena Cava Distensibility and Arterial Blood Pressure

The purpose of this study was to clarify the relationship between the inferior vena cava distensibility and blood pressure. Six Japanese healthy males volunteered to participate in this study. We measured the cross-sectional area of inferior vena cava (CSAivc) and blood pressure at rest and during passive leg raising in supine position. We calculated the change rates of each parameter, based on the value at rest in supine position. We observed a negative correlation between the change rate of CSAivc and systolic blood pressure (P<0.05). These results suggest that the inferior vena cava distensibility affects partially systolic blood pressure

Toxicity of Titanate Nanosheets on Human Immune Cells

Titanium oxide is regarded as a bio-inert material, but studies concerning the toxic effects of titanium dioxide (TiO2), particularly nano-scaled TiO2 particles, have been accumulating that indicate nano-scaled TiO2 particles show more harm and cause greater alteration of immune functions compared with large particles. Inorganic nanosheets have been the focus of increasing interest because of their ultrathin structure, as well as diversity of compounds and structures leading to various functions. Oxide nanosheets are included in the group comprising inorganic nanosheets, and titanate nanosheets (TiNSs) represent a form of oxide nanosheets. We therefore examined the toxicity of nano-scaled 2D materials of TiNSs on human immune cells. Our study revealed that TiNSs have the potential to cause harm through caspase-dependent apoptosis of human peripheral blood mononuclear cells (PBMCs) to the same degree as asbestos. Furthermore, isolated monocytes developed marked vacuoles prior to cell death upon exposure to TiNSs, which were found in the vacuoles and indicated engulfment of TiNSs. A consideration of these findings with the co-localization of vacuoles with endocytosed fluorescence-labeled dextran indicates that TiNSs entered the endosomal pathway, leading to the formation of vacuoles in monocytes and subsequent cell death. TiNSs might therefore affect immune functions through interference of endo-lysosomal functions

Spatial distribution of lipid headgroups and water molecules at membrane/water interfaces visualized by three-dimensional scanning force microscopy

At biological interfaces, flexible surface structures and mobile water interact with each other to present non-uniform three-dimensional (3D) distributions. In spite of their impact on biological functions, molecular-scale understanding of such phenomena has remained elusive. Here we show direct visualization of such interfacial structures with subnanometer-scale resolution by 3D scanning force microscopy (3D-SFM). We measured a 3D force distribution at an interface between a model biological membrane and buffer solution by scanning a sharp tip within the 3D interfacial space. We found that vertical cross sections of the 3D image taken along a specific lateral direction show characteristic molecular-scale contrasts tilted at 30° to the membrane surface. Detailed analysis of the 3D image reveals that the tilted contrast corresponds to the time-averaged conformation of fluctuating lipid headgroups. On the basis of the obtained results, we discuss the relationships among the hydration structure, headgroup fluctuation, molecular fluidity, and mechanical strength of the membrane. The results demonstrate that 3D-SFM is capable of visualizing averaged 3D distribution of fluctuating surface structures as well as that of mobile water (i.e., hydration structure) at interfaces between biological systems and water. © 2012 American Chemical Society

Ureterolithotripsy for a Ureteral Calculus at the Ureteroureterostomy of a Renal-transplant Recipient

We describe a 40-year-old living-donor renal-transplant recipient who underwent successful ureterolithotripsy. He had been on hemodialysis for >15 years pre-transplant and underwent ureteroureterostomy along with the surgery. One year post-transplant, ultrasound examination demonstrated hydronephrosis, and CT showed a 6-mm ureteral calculus at the ureteroureterostomy site. No pain and no elevated serum creatinine were present. As the ureter was easily accessed, we performed a ureterolithotripsy, which would confirm whether a suture caused the calculus. Despite ureteral tortuosity, laser stone fragmentation succeeded. The calculus was completely removed with an antegrade guidewire. Mild postoperative ureteral stenosis resolved with a temporary ureteral stent without balloon dilation. Ureterolithotripsy is effective even in renal transplant recipients with ureteroureterostomy

Spatiotemporal T790M Heterogeneity in Individual Patients with EGFR-Mutant Non–Small-Cell Lung Cancer after Acquired Resistance to EGFR-TKI

IntroductionEpidermal growth factor receptor (EGFR) mutation T790M accounts for approximately half of acquired resistances to EGFR-tyrosine kinase inhibitor (TKI). Because T790M is mediated by TKI exposure, its penetration and “on–off” may affect T790M status.MethodsWe retrospectively reviewed T790M status and clinical course of patients who had undergone multiple rebiopsies after acquired resistance to EGFR-TKI.ResultsOf 145 patients with EGFR-mutant NSCLC receiving rebiopsy after acquired resistance, 30 underwent multiple site rebiopsies, and 24 received repeated rebiopsies at the same lesion. In 22 patients who underwent rebiopsies from both central nervous system (CNS; 20 cerebrospinal fluids [CSF] and 2 brain tumoral tissues) and thoracic lesions (7 lung tissues, 14 pleural effusions, and 1 lymph node), 12 were thoracic-T790M-positive. Of these 12 patients, 10 were CNS-T790M-negative, despite exhibiting thoracic-T790M-positive. All 10 thoracic-T790M-negatives were CNS-T790M-negative. Three patients revealed a spatial heterogeneous T790M status among their thoracic lesions. In 24 patients receiving repeated rebiopsies at the same lesion (12 lung tissues, 6 CSFs, and 6 pleural effusions), T790M status of lung lesions varied in five patients after TKI-free interval. In all five patients whose T790M status changed from positive to negative, EGFR-TKI rechallenge was effective. In three of these five patients, after further TKI exposure, T790M status changed from negative to positive again. There was also a patient whose CSF T790M status changed from negative to positive after high-dose erlotinib therapy.ConclusionsT790M status in an individual patient can be spatiotemporally heterogeneous because of selective pressure from EGFR-TKI