Structural basis for coronavirus-mediated membrane fusion. Crystal structure of mouse hepatitis virus spike protein fusion core

The surface transmembrane glycoprotein is responsible for mediating virion attachment to cell and subsequent virus-cell membrane fusion. However, the molecular mechanisms for the viral entry of coronaviruses remain poorly understood. The crystal structure of the fusion core of mouse hepatitis virus S protein, which represents the first fusion core structure of any coronavirus, reveals a central hydrophobic coiled coil trimer surrounded by three helices in an oblique, antiparallel manner. This structure shares significant similarity with both the low pH-induced conformation of influenza hemagglutinin and fusion core of HIV gp41, indicating that the structure represents a fusion-active state formed after several conformational changes. Our results also indicate that the mechanisms for the viral fusion of coronaviruses are similar to those of influenza virus and HIV. The coiled coil structure has unique features, which are different from other viral fusion cores. Highly conserved heptad repeat 1 (HR1) and HR2 regions in coronavirus spike proteins indicate a similar three-dimensional structure among these fusion cores and common mechanisms for the viral fusion. We have proposed the binding regions of HR1 and HR2 of other coronaviruses and a structure model of their fusion core based on our mouse hepatitis virus fusion core structure and sequence alignment. Drug discovery strategies aimed at inhibiting viral entry by blocking hairpin formation may be applied to the inhibition of a number of emerging infectious diseases, including severe acute respiratory syndrome.     

Human MCRS2, a cell-cycle-dependent protein, associates with LPTS/PinX1 and reduces the telomere length

Human LPTS/PinX1 is a telomerase-inhibitory protein, which binds to the telomere protein Pin2/TRF1 and the catalytic subunit hTERT of telomerase. To explore the proteins that might be involved in the telomerase pathway, we performed a yeast two-hybrid screening with LPTS/PinX1 as the bait. A novel gene, MCRS2, encoding for an isoform of MCRS1/p78 and MSP58 was isolated. The expression of MCRS2 protein is cell-cycle dependent, accumulating in the very early S phase. MCRS2 interacts with LPTS/PinX1 in vitro, in vivo and colocalizes with LPTS/PinX1 in cells. MCRS2 and its amino terminus inhibit telomerase activity in vitro and long-term overexpression of MCRS2 in SMMC-7721 cells results in a gradual and progressive shortening of telomeres. Our findings suggest that MCRS2 might be a linker between telomere maintenance and cell-cycle regulation.     

Purification,electrophoretic behavior,and kinetics of iron release of liver ferritin of Dasyatis akajei

From the liver of fish Dasyatis akajei, ferritin has been isolated by thermal denaturation and ammonium sulfate fractionation and then further purified by anion exchange chromatography and gel exclusion chromatography. The molecular weight of the liver ferritin of D. akajei (DALF) was measured to be 400 kDa by PAGE. Moreover, SDS-PAGE experimentation indicates that protein shell of DALF consists of the H and L subunits with molecular weight of 18 and 13 kDa, respectively. Using isoelectric focusing with pH ranging from 5.0 to 6.0, the ferritin purified by the PAGE exhibited three bands with different pI values in the gel slab. Diameters of the protein shell and iron core were also investigated by transmission electron microscope and determined to be 10–12 nm and 5–8 nm, respectively. A kinetic study of DALF reveals that the rate of self-regulation of the protein shell rather than the complex surface of the iron core plays an important role in forming a process for iron release with mixed orders.     

Extremely low frequency magnetic fields affect transposition activity in Escherichia coli

The aim of this study was to verify whether extremely low frequency (ELF) magnetic fields (MF) could affect transposition activity like some environmental stress factors such as heat shock or UV irradiation. Using an Escherichia coli Lac Z(-) strain transformed with a plasmid containing a Tn 10 derivative element expressing beta-galactosidase only after transposition, it was possible to determine the events of transposition evaluating the rate at which the colonies developed dark coloured papillae (Lac Z(+)). We found that those bacteria that had been exposed for a long time (58 h) to a 50 Hz low intensity MF (0.1-1 mT) gave colonies with significantly lower transposition activity compared to sham-exposed bacteria. Such reduction in transposition activity was positively correlated to the intensity of the MF, in a dose-effect manner. This phenomenon was not affected by bacterial cell proliferation, since no significant differences were observed in number, diameter and perimeter between sham-exposed and MF-exposed colonies.     

Histamine inhibits atrial myocytic ANP release via H2 receptor-cAMP-protein kinase signaling

Changes in cyclic nucleotide production and atrial dynamics have been known to modulate atrial natriuretic peptide (ANP) release. Although cardiac atrium expresses histamine receptors and contains histamine, the role of histamine in the regulation of ANP release has to be defined. The purpose of the present study was to define the effect of histamine on the regulation of ANP release in perfused beating rabbit atria. Histamine decreased ANP release concomitantly with increases in cAMP efflux and atrial dynamics in a concentration-dependent manner. Histamine-induced decrease in ANP release was a function of an increase in cAMP production. Blockade of histamine H2 receptor with cimetidine but not of H1 receptor with triprolidine abolished the responses of histamine. Cell-permeable cAMP analog, 8-Br-cAMP, mimicked the effects of histamine, and the responses were dose-dependent and blocked by a protein kinase A (PKA)-selective inhibitor, KT5720. Nifedipine failed to modulate histamine-induced decrease in ANP release. Protein kinase nonselective inhibitor staurosporine blocked histamine-induced changes in a concentration-dependent manner. KT5720 and RP-adenosine 3',5'-cyclic monophosphorothioate, another PKA-selective inhibitor, attenuated histamine-induced changes. These results suggest that histamine decreases atrial ANP release by H2 receptor-cAMP signaling via PKA-dependent and -independent pathways.     

A microbial fuel cell with improved cathode reaction as a low biochemical oxygen demand sensor

Mediator-less microbial fuel cells (MFC) enriched with oligotrophic microbes were optimized through enhancement of cathode reaction and lowering O₂ diffusion into the anode compartment as a low BOD sensor. The optimization of the MFC has greatly improved the maximum current and coulomb yield. The oligotroph-type MFC could be used as a low BOD sensor with high operational stability, good repeatability and reproducibility.     

Xylem-specific expression of a GRP1.8 promoter::4CL gene construct in transgenic tobacco

Lignin is a complex aromatic polymer of vascular plants that provides mechanical strength to the stem and protects cellulose fibres from chemical and biological degradation. 4-Coumarate:CoA ligases (EC 6.2.1.12) are key enzymes for the biosynthetic pathway of monolignols which is an important complex aromatic polymer for lignin biosynthesis and tree growth. Recently, 4-coumarate:CoA ligase has been used as exogenous gene in transgenic plants to genetically modify the lignin biosynthesis pathway. Since most lignin is produced in the vascular cells, a tissue-specific-expressed promoter in the vascular cell would be important and useful to change and modify the content of lignin. Here we report the existence of a promoter of GRP1.8 (the glycine-rich protein 1.8) in Sopho japonica L. (GenBank accession number AF250149) and studies on its function in transgenic tobacco. The promoter activity was analyzed in transgenic tobacco plants by histochemical staining of GUS gene expression driven by a 613-bp sjGRP1.8p promoter sequence. In sjGRP1.8p-GUS transgenic plants, intense GUS staining was detected in the xylem of the stem. To further investigate the regulation of the tissue-specific expression of the 4CL1 gene, we analyzed the activity of the 4CL1 gene which is sense orientated with the sjGRP1.8p promoter in transgenic tobacco. The Pto4CL1 gene was expressed in the stem of transgenic tobacco. The activity of the 4CL1 enzyme was increased 1–2-fold in the stem but not increased in the leaves of transgenic tobacco. In comparison with the control plants, the content of lignin was increased 25% in the stem but there was no increase in the leaves of transgenic tobacco.     

A new algorithm for computational image analysis of deformable motion at high spatial and temporal resolution applied to root growth. Roughly uniform elongation in the meristem and also,after an abrupt acceleration,in the elongation zone

A requirement for understanding morphogenesis is being able to quantify expansion at the cellular scale. Here, we present new software (RootflowRT) for measuring the expansion profile of a growing root at high spatial and temporal resolution. The software implements an image processing algorithm using a novel combination of optical flow methods for deformable motion. The algorithm operates on a stack of nine images with a given time interval between each (usually 10 s) and quantifies velocity confidently at most pixels of the image. The root does not need to be marked. The software calculates components of motion parallel and perpendicular to the local tangent of the root's midline. A variation of the software has been developed that reports the overall root growth rate versus time. Using this software, we find that the growth zone of the root can be divided into two distinct regions, an apical region where the rate of motion, i.e. velocity, rises gradually with position and a subapical region where velocity rises steeply with position. In both zones, velocity increases almost linearly with position, and the transition between zones is abrupt. We observed this pattern for roots of Arabidopsis, tomato (Lycopersicon lycopersicum), lettuce (Lactuca sativa), alyssum (Aurinia saxatilis), and timothy (Phleum pratense). These velocity profiles imply that relative elongation rate is regulated in a step-wise fashion, being low but roughly uniform within the meristem and then becoming high, but again roughly uniform, within the zone of elongation. The executable code for RootflowRT is available from the corresponding author on request.