Phos-tag-based approach to study protein phosphorylation in the thylakoid membrane

Protein phosphorylation is a fundamental post-translational modification in all organisms. In photoautotrophic organisms, protein phosphorylation is essential for the fine-tuning of photosynthesis. The reversible phosphorylation of the photosystem II (PSII) core and the light-harvesting complex of PSII (LHCII) contribute to the regulation of photosynthetic activities. Besides the phosphorylation of these major proteins, recent phosphoproteomic analyses have revealed that several proteins are phosphorylated in the thylakoid membrane. In this study, we utilized the Phos-tag technology for a comprehensive assessment of protein phosphorylation in the thylakoid membrane of Arabidopsis. Phos-tag SDS-PAGE enables the mobility shift of phosphorylated proteins compared with their non-phosphorylated isoform, thus differentiating phosphorylated proteins from their non-phosphorylated isoforms. We extrapolated this technique to two-dimensional (2D) SDS-PAGE for detecting protein phosphorylation in the thylakoid membrane. Thylakoid proteins were separated in the first dimension by conventional SDS-PAGE and in the second dimension by Phos-tag SDS-PAGE. In addition to the isolation of major phosphorylated photosynthesis-related proteins, 2D Phos-tag SDS-PAGE enabled the detection of several minor phosphorylated proteins in the thylakoid membrane. The analysis of the thylakoid kinase mutants demonstrated that light-dependent protein phosphorylation was mainly restricted to the phosphorylation of the PSII core and LHCII proteins. Furthermore, we assessed the phosphorylation states of the structural domains of the thylakoid membrane, grana core, grana margin, and stroma lamella. Overall, these results demonstrated that Phos-tag SDS-PAGE is a useful biochemical tool for studying in vivo protein phosphorylation in the thylakoid membrane protein

Hypertrophy and Unconventional Cell Division of Hepatocytes Underlie Liver Regeneration

SummaryBackgroundThe size of organs and tissues is basically determined by the number and size of their cells. However, little attention has been paid to this fundamental concept. The liver has a remarkable ability to regenerate after surgical resection (partial hepatectomy [PHx]), and hepatocytes account for about 80% of liver weight, so we investigate how the number and size of hepatocytes contribute to liver regeneration in mice. It has been generally accepted that hepatocytes undergo one or two rounds of cell division after 70% PHx. However, ploidy of hepatocytes is known to increase during regeneration, suggesting an unconventional cell cycle. We therefore examine cell cycle of hepatocytes in detail.ResultsBy developing a method for genetic fate mapping and a high-throughput imaging system of individual hepatocytes, we show that cellular hypertrophy makes the first contribution to liver regeneration; i.e., regeneration after 30% PHx is achieved solely by hypertrophy without cell division, and hypertrophy precedes proliferation after 70% PHx. Proliferation and hypertrophy almost equally contribute to regeneration after 70% PHx. Furthermore, although most hepatocytes enter cell cycle after 70% PHx, not all hepatocytes undergo cell division. In addition, binuclear hepatocytes undergo reductive divisions to generate two mononuclear daughter hepatocytes in some cases.ConclusionsOur findings demonstrate the importance of hypertrophy and the unconventional cell division cycle of hepatocytes in regeneration, prompting a significant revision of the generally accepted model of liver regeneration

Significant suppression of two-magnon scattering in ultrathin Co by controlling the surface magnetic anisotropy at the Co/nonmagnet interfaces

To enable suppression of two-magnon scattering (TMS) in nanometer-thick Co (ultrathin Co) layers and realize low-magnon damping in such layers, the magnon damping in ultrathin Co layers grown on various nonmagnetic seed layers with different surface magnetic anisotropy (SMA) energies are investigated. We verify the significantly weak magnon damping realized by varying the seeding layer species used. Although TMS is enhanced in ultrathin Co on Cu and Al seeding layers, the insertion of a Ti seeding layer below the ultrathin Co greatly suppresses the TMS, which is attributed to suppression of the SMA at the interface between Co and Ti. The Gilbert damping constant of the ultrathin Co layer on Ti (3 nm), 0.020, is comparable to the value for bulk Co, although the Co layer thickness here is only 2 nm. Realization of such weak magnon damping can open the door to tunable magnon excitation, thus enabling coupling of magnons with other quanta such as photons, given that the magnetization of ultrathin ferromagnets can be tuned using an external electric field

c-RET Molecule in Malignant Melanoma from Oncogenic RET-Carrying Transgenic Mice and Human Cell Lines

Malignant melanoma is one of the most aggressive cancers and its incidence worldwide has been increasing at a greater rate than that of any other cancer. We previously reported that constitutively activated RFP-RET-carrying transgenic mice (RET-mice) spontaneously develop malignant melanoma. In this study, we showed that expression levels of intrinsic c-Ret, glial cell line-derived neurotrophic factor (Gdnf) and Gdnf receptor alpha 1 (Gfra1) transcripts in malignant melanomas from RET-transgenic mice were significantly upregulated compared with those in benign melanocytic tumors. These results suggest that not only introduced oncogenic RET but also intrinsic c-Ret/Gdnf are involved in murine melanomagenesis in RET-mice. We then showed that c-RET and GDNF transcript expression levels in human malignant melanoma cell lines (HM3KO and MNT-1) were higher than those in primary cultured normal human epithelial melanocytes (NHEM), while GFRa1 transcript expression levels were comparable among NHEM, HM3KO and MNT-1. We next showed c-RET and GFRa1 protein expression in HM3KO cells and GDNF-mediated increased levels of their phosphorylated c-RET tyrosine kinase and signal transduction molecules (ERK and AKT) sited potentially downstream of c-RET. Taken together with the finding of augmented proliferation of HM3KO cells after GDNF stimulation, our results suggest that GDNF-mediated c-RET kinase activation is associated with the pathogenesis of malignant melanoma

Myeloperoxidase-Antineutrophil Cytoplasmic Antibody-Negative Microscopic Polyangiitis with Pulmonary Haemorrhage and IgA Nephropathy

Aim: To report a case of a patient with myeloperoxidase-antineutrophil cytoplasmic antibody (ANCA)-negative microscopic polyangiitis (MPA) and IgA nephropathy associated with severe pulmonary haemorrhage. Case Report: A 59-year-old man presented with ANCA-negative systemic vasculitis accompanied by purpura, nephritis and pulmonary haemorrhage. A skin biopsy specimen revealed pandermal leucocytoclastic vasculitis without IgA deposition and a kidney biopsy showed mesangial nephritis with IgA deposition. Considering these findings, the patient was diagnosed as having MPA with IgA nephropathy. Discussion: In most cases, MPA presents with rapidly progressive necrotizing glomerulonephritis and sometimes lung haemorrhage, while IgA nephropathy is less common among MPA cases. As recent research suggested that in MPA immunoglobulin deposition in the kidney may be an exacerbating factor for renal dysfunction and poor prognosis, close observation is required in these cases

Influence of lifestyle choices on risks of CYP1B1 polymorphisms for prostate cancer.

Cytochrome P450 1B1 (CYP1B1) converts xenobiotics to carcinogens and how lifestyle choices may interact with CYP1B1 polymorphisms and affect prostate cancer risk was assessed. Blood genomic DNA from a Caucasian population was analysed at polymorphic sites of the 5' untranslated region of CYP1B1 using TaqMan genotyping assays. Overall, drinker status and minor alleles at rs2551188, rs2567206 and rs10175368 were associated with prostate cancer. Linkage was observed between rs2551188, rs2567206, rs2567207 and rs10175368, and the G-C-T-G haplotype (major allele at respective sites) was decreased in cancer. Interestingly when classified by lifestyle factors, no associations of genotypes were found for non-smokers and non-drinkers, whereas on the contrary, minor type at rs2567206 and rs10175368 increased and major G-C-T-G decreased risk for cancer among smokers and drinkers. Interestingly, rs2551188, rs2567206 and rs10175368 minor genotypes correlated with increased tissue CYP1B1 as determined by immunohistochemistry. Further, rs10175368 enhanced luciferase activity and mobility shift show stronger binding of nuclear factor for the minor allele. These results demonstrate smoking and alcohol consumption to modify the risks of CYP1B1 polymorphisms for prostate cancer which may be through rs10175368, and this is of importance in understanding their role in the pathogenesis and as a biomarker for this disease

Differential expression of miR-34b and androgen receptor pathway regulate prostate cancer aggressiveness between African-Americans and Caucasians.

African-Americans are diagnosed with more aggressive prostate cancers and have worse survival than Caucasians, however a comprehensive understanding of this health disparity remains unclear. To clarify the mechanisms leading to this disparity, we analyzed the potential involvement of miR-34b expression in African-Americans and Caucasians. miR-34b functions as a tumor suppressor and has a multi-functional role, through regulation of cell proliferation, cell cycle and apoptosis. We found that miR-34b expression is lower in human prostate cancer tissues from African-Americans compared to Caucasians. DNA hypermethylation of the miR-34b-3p promoter region showed significantly higher methylation in prostate cancer compared to normal samples. We then sequenced the promoter region of miR-34b-3p and found a chromosomal deletion in miR-34b in African-American prostate cancer cell line (MDA-PCA-2b) and not in Caucasian cell line (DU-145). We found that AR and ETV1 genes are differentially expressed in MDA-PCa-2b and DU-145 cells after overexpression of miR-34b. Direct interaction of miR-34b with the 3' untranslated region of AR and ETV1 was validated by luciferase reporter assay. We found that miR-34b downregulation in African-Americans is inversely correlated with high AR levels that lead to increased cell proliferation. Overexpression of miR-34b in cell lines showed higher inhibition of cell proliferation, apoptosis and G1 arrest in the African-American cells (MDA-PCa-2b) compared to Caucasian cell line (DU-145). Taken together, our results show that differential expression of miR-34b and AR are associated with prostate cancer aggressiveness in African-Americans