Structures of the signal recognition particle receptor from the archaeon Pyrococcus furiosus: implications for the targeting step at the membrane

In all organisms, a ribonucleoprotein called the signal recognition particle (SRP) and its receptor (SR) target nascent proteins from the ribosome to the translocon for secretion or membrane insertion. We present the first X-ray structures of an archeal FtsY, the receptor from the hyper-thermophile Pyrococcus furiosus (Pfu), in its free and GDP*magnesium-bound forms. The highly charged N-terminal domain of Pfu-FtsY is distinguished by a long N-terminal helix. The basic charges on the surface of this helix are likely to regulate interactions at the membrane. A peripheral GDP bound near a regulatory motif could indicate a site of interaction between the receptor and ribosomal or SRP RNAs. Small angle X-ray scattering and analytical ultracentrifugation indicate that the crystal structure of Pfu-FtsY correlates well with the average conformation in solution. Based on previous structures of two sub-complexes, we propose a model of the core of archeal and eukaryotic SRP*SR targeting complexes.     

FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells

Autophagy is a membrane-mediated intracellular degradation system. The serine/threonine kinase Atg1 plays an essential role in autophagosome formation. However, the role of the mammalian Atg1 homologues UNC-51-like kinase (ULK) 1 and 2 are not yet well understood. We found that murine ULK1 and 2 localized to autophagic isolation membrane under starvation conditions. Kinase-dead alleles of ULK1 and 2 exerted a dominant-negative effect on autophagosome formation, suggesting that ULK kinase activity is important for autophagy. We next screened for ULK binding proteins and identified the focal adhesion kinase family interacting protein of 200 kD (FIP200), which regulates diverse cellular functions such as cell size, proliferation, and migration. We found that FIP200 was redistributed from the cytoplasm to the isolation membrane under starvation conditions. In FIP200-deficient cells, autophagy induction by various treatments was abolished, and both stability and phosphorylation of ULK1 were impaired. These results suggest that FIP200 is a novel mammalian autophagy factor that functions together with ULKs.     

Degradation of caspase-activated DNase by the ubiquitin-proteasome system

DNA fragmentation is one of the most characteristic features of apoptotic cells and caspase-activated DNase (CAD) is considered to be a major nuclease responsible for DNA fragmentation. CAD forms a complex with its inhibitor (ICAD), which is also required for the functional folding of CAD, leading to CAD stabilization in cells. In this paper, we investigated the involvement of the ubiquitin-proteasome system in CAD stability. The expression and ubiquitination of CAD was remarkably increased by MG132 treatment in the absence of ICAD. These results suggest that CAD protein may be preferentially degraded by the ubiquitin-proteasome system in the absence of ICAD to maintain protein quality control.     

Differentiation of monocytes into multinucleated giant bone-resorbing cells: two-step differentiation induced by nurse-like cells and cytokines

Bone resorption in the joints is the characteristic finding in patients with rheumatoid arthritis (RA). Osteoclast-like cells are present in the synovial tissues and invade the bone of patients with RA. The characteristics of these cells are not completely known. In the work reported here, we generated these cells from peripheral-blood monocytes from healthy individuals. The monocytes were co-cultured with nurse-like cells from synovial tissues of patients with RA (RA-NLCs). Within 5 weeks of culture, the monocytes were activated and differentiated into mononuclear cells positive for CD14 and tartrate-resistant acid phosphatase (TRAP). These mononuclear cells then differentiated into multinucleated giant bone-resorbing cells after stimulation with IL-3, IL-5, IL-7, and/or granulocyte-macrophage-colony-stimulating factor. TRAP-positive cells with similar characteristics were found in synovial fluid from patients with RA. These results indicate that multinucleated giant bone-resorbing cells are generated from monocytes in two steps: first, RA-NLCs induce monocytes to differentiate into TRAP-positive mononuclear cells, which are then induced by cytokines to differentiate into multinucleated giant bone-resorbing cells.     

Fluorometric determination of N-terminal prolyl dipeptides, proline and hydroxyproline in human serum by pre-column high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride

A highly sensitive HPLC method for the determination of prolyl dipeptides, Pro and Hyp in serum was developed. After deproteinization of serum and pretreatment with o-phthalaldehyde, the analytes were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70°C for 10 min. The fluorescent derivatives of prolyl dipeptides, Pro and Hyp, were separated on tandem reversed-phase columns by a gradient elution at 55°C and detected by fluorescence measured at 318 nm (excitation) and 392 nm (emission). The detection limits for prolyl dipeptides were 2–5 fmol/injection (S/N=3). Pro–Hyp, Pro–Gly and Pro–Pro were identified as serum prolyl dipeptides. The within-day and between-day relative standard deviations were 1.5–7.9 and 2.4–10.8%, respectively. The recoveries were in the range of 90.8–97.3%. The concentrations of Pro–Hyp, Pro–Gly, Pro–Pro, Pro and Hyp in normal human serum (n=10) were 0.64±0.35, 0.078±0.047, 0.022±0.016, 177.0±43.0 and 11.1±3.5 μM, respectively. The concentrations of Pro–Hyp and Pro–Pro in serum of a patient with bone metastases of prostatic cancer were about three times and 50 times, respectively, higher than those in normal human serum.     

CH4 emissions from rice paddies managed according to farmer's practice in Hunan, China

We measured CH₄ emissions from ricepaddies managed by farmer's practices inChangsha, Hunan Province, China, from 1995 to1997. During the winter season, rice fieldswere left fallow under either drained(C-Fallow) or flooded conditions (C-Flood), andplanted with either Chinese milk vetch (C-GM)or oil-seed rape (C-Rape). The organic manureproduced in the winter (weeds, Chinese milkvetch, or oil-seed rape straw) was incorporatedin situ before the early-ricetransplanting. Both early-rice and late-ricestraws were removed and the soil was notamended with any exogenous organic manure. For1996 to 1997, the average seasonal CH₄emission for the double rice cropping periodwas the highest from the plot that was floodedin the winter (103.5 g CH₄ m^–2) andlowest from the plot planted and incorporatedwith Chinese milk vetch (32.6 gCH₄ m^–2). Precipitation in the winternot only affected growth of green manure, whichwas incorporated in situ, but might alsoaffect CH₄ emissions during the subsequentrice growing period. Therefore, a simplerelationship could not be found between theincorporated amount of green manure andCH₄ emission. In the plots incorporatedwith vetch and oil-seed rape straw CH₄emissions were significantly less during thesubsequent late-rice period than during theearly-rice period. This phenomenon might beattributed to a ``priming effect' of greenmanure, which exhausted soil labile organicmatter. Based on the CH₄ fluxmeasurements, the total CH₄ emissions fromrice fields in Hunan Province during the ricegrowing season were estimated as 1.56 TgCH₄ in 1996 and 1.06 Tg CH₄ in 1997.Large variation of precipitation in the winterwould be an important factor controlling theannual variation of CH₄ emissions from thetreatments.     

MicroRNA-351 Regulates Two-Types of Cell Death,Necrosis and Apoptosis,Induced by 5-fluoro-2'-deoxyuridine

Cell-death can be necrosis and apoptosis. We are investigating the mechanisms regulating the cell death that occurs on treatment of mouse cancer cell-line FM3A with antitumor 5-fluoro-2''-deoxyuridine (FUdR): necrosis occurs for the original clone F28-7, and apoptosis for its variant F28-7-A. Here we report that a microRNA (miR-351) regulates the cell death pattern. The miR-351 is expressed strongly in F28-7-A but only weakly in F28-7. Induction of a higher expression of miR-351 in F28-7 by transfecting an miRNA mimic into F28-7 resulted in a change of the death mode; necrosis to apoptosis. Furthermore, transfection of an miR-351 inhibitor into F28-7-A resulted in the morphology change, apoptosis to necrosis, in this death-by-FUdR. Possible mechanism involving lamin B1 in this miR-351’s regulatory action is discussed.     

A fluorescence temperature-jump study on Ca2(+)-induced conformational changes in calmodulin

Calmodulin has been shown to alter its conformation so as to interact with a number of target proteins upon Ca2+ binding. A Ca2(+)-binding study of calmodulin was performed by monitoring the fluorescence of intrinsic tyrosine residues and the probe 1-anilinonaphthalene-8-sulfonate (ANS). ANS fluorescence was shown to reflect Ca2+ binding to both high- and low-affinity sites. On the one hand, tyrosine fluorescence was sensitive only to the high-affinity Ca2+ binding. Temperature-jump investigation of the ternary complex of Ca2(+)-calmodulin-ANS in combination with monitoring of ANS fluorescence demonstrated the kinetic characteristics of the conformational change. The relaxation process was attributed to Ca2(+)-induced conformational change and the rate constants of this process were evaluated. On the basis of the rate constants of the conformational change, a rapid response of calmodulin in Ca2+ signaling is suggested.     

Collaborative environmental DNA sampling from petal surfaces of flowering cherry <Emphasis Type="Italic">Cerasus</Emphasis> × <Emphasis Type="Italic">yedoensis</Emphasis> ‘Somei-yoshino’ across the Japanese archipelago

Recent studies have shown that environmental DNA is found almost everywhere. Flower petal surfaces are an attractive tissue to use for investigation of the dispersal of environmental DNA in nature as they are isolated from the external environment until the bud opens and only then can the petal surface accumulate environmental DNA. Here, we performed a crowdsourced experiment, the “Ohanami Project”, to obtain environmental DNA samples from petal surfaces of Cerasus?×?yedoensis ‘Somei-yoshino’ across the Japanese archipelago during spring 2015. C. × yedoensis is the most popular garden cherry species in Japan and clones of this cultivar bloom simultaneously every spring. Data collection spanned almost every prefecture and totaled 577 DNA samples from 149 collaborators. Preliminary amplicon-sequencing analysis showed the rapid attachment of environmental DNA onto the petal surfaces. Notably, we found DNA of other common plant species in samples obtained from a wide distribution; this DNA likely originated from the pollen of the Japanese cedar. Our analysis supports our belief that petal surfaces after blossoming are a promising target to reveal the dynamics of environmental DNA in nature. The success of our experiment also shows that crowdsourced environmental DNA analyses have considerable value in ecological studies.