The KCAL VERA 22 GHz calibrator survey

We observed at 22 GHz with the VLBI array VERA a sample of 1536 sources with correlated flux densities brighter than 200 mJy at 8 GHz. One half of target sources has been detected. The detection limit was around 200 mJy. We derived the correlated flux densities of 877 detected sources in three ranges of projected baseline lengths. The objective of these observations was to determine the suitability of given sources as phase calibrators for dual-beam and phase-referencing observations at high frequencies. Preliminary results indicate that the number of compact extragalactic sources at 22 GHz brighter than a given correlated flux density level is twice less than at 8 GHz.Comment: Accepted for publication by the Astronomical Journal. 6 pages, 3 figures, 3 table. The machine readable catalogue file, kcal_cat.txt can be extracted from the source of this submissio

Protein Conformational Changes in the Bacteriorhodopsin Photocycle: Comparison of Findings from Electron and X-Ray Crystallographic Analyses

Light-driven conformational changes in the membrane protein bacteriorhodopsin have been studied extensively using X-ray and electron crystallography, resulting in the deposition of >30 sets of coordinates describing structural changes at various stages of proton transport. Using projection difference Fourier maps, we show that coordinates reported by different groups for the same photocycle intermediates vary considerably in the extent and nature of conformational changes. The different structures reported for the same intermediate cannot be reconciled in terms of differing extents of change on a single conformational trajectory. New measurements of image phases obtained by cryo-electron microscopy of the D96G/F171C/F219L triple mutant provide independent validation for the description of the large protein conformational change derived at 3.2 Å resolution by electron crystallography of 2D crystals, but do not support atomic models for light-driven conformational changes derived using X-ray crystallography of 3D crystals. Our findings suggest that independent determination of phase information from 2D crystals can be an important tool for testing the accuracy of atomic models for membrane protein conformational changes

Rotational Alignment Altered by Source Position Correlations

In the construction of modern Celestial Reference Frames (CRFs) the overall rotational alignment is only weakly constrained by the data. Therefore, common practice has been to apply a 3-dimensional No-Net-Rotation (NNR) constraint in order to align an under-construction frame to the ICRF. We present evidence that correlations amongst source position parameters must be accounted for in order to properly align a CRF at the 5-10 (mu)as level of uncertainty found in current work. Failure to do so creates errors at the 10-40 (mu)as level

Co-expression and purification of the RadA recombinase with the RadB paralog from Haloferax volcanii yields heteromeric ring-like structures

The study of archaeal proteins and the processes to which they contribute poses particular challenges due to the often extreme environments in which they function. DNA recombination, replication and repair proteins of the halophilic euryarchaeon, Haloferax volcanii (Hvo) are of particular interest as they tend to resemble eukaryotic counterparts in both structure and activity, and genetic tools are available to facilitate their analysis. In the present study, we show using bioinformatics approaches that the Hvo RecA-like protein RadA is structurally similar to other recombinases although is distinguished by a unique acidic insertion loop. To facilitate expression of Hvo RadA a co-expression approach was used, providing its lone paralog, RadB, as a binding partner. At present, structural and biochemical characterization of Hvo RadA is lacking. Here, we describe for the first time co-expression of Hvo RadA with RadB and purification of these proteins as a complex under in vitro conditions. Purification procedures were performed under high salt concentration (>1 M sodium chloride) to maintain the solubility of the proteins. Quantitative densitometry analysis of the co-expressed and co-purified RadAB complex estimated the ratio of RadA to RadB to be 4 : 1, which suggests that the proteins interact with a specific stoichiometry. Based on a combination of analyses, including size exclusion chromatography, Western blot and electron microscopy observations, we suggest that RadA multimerizes into a ring-like structure in the absence of DNA and nucleoside co-factor

Identification of acetyltransferase genes (HAT1 and KAT8) regulating HBV replication by RNAi screening

Background: The initiation of hepatitis B virus (HBV) replication involves the formation of covalently closed circular DNA (cccDNA) and its transcription into pregenomic RNA (pgRNA) in hepatocyte nuclei. The regulatory mechanism of HBV replication by acetyltransferase is thus far not well understood, but human acetyltransferase has been reported as being involved in the regulation of HBV replication. Results: Depletion of KAT8 or HAT1 via RNA interference (RNAi) markedly down-regulated HBV-DNA and pgRNA levels in HepG2.2.15 cells, with KAT8 knockdown reducing both HBsAg and HBeAg more than HAT1 knockdown. Con-sistent with these observations, HBV replication regulators hepatocyte nuclear factor-4-α (HNF4α) and peroxisome proliferator-activated receptor gamma coactivator- (PPARGC-) 1-α were decreased following knockdown of HAT1 or KAT8. Conclusions: These data suggest that KAT8 or HAT1 regulate HBV replication and may be potential drug targets of anti-HBV therapy

Active Membrane Fluctuations Studied by Micropipet Aspiration

We present a detailed analysis of the micropipet experiments recently reported in J-B. Manneville et al., Phys. Rev. Lett. 82, 4356--4359 (1999), including a derivation of the expected behaviour of the membrane tension as a function of the areal strain in the case of an active membrane, i.e., containing a nonequilibrium noise source. We give a general expression, which takes into account the effect of active centers both directly on the membrane, and on the embedding fluid dynamics, keeping track of the coupling between the density of active centers and the membrane curvature. The data of the micropipet experiments are well reproduced by the new expressions. In particular, we show that a natural choice of the parameters quantifying the strength of the active noise explains both the large amplitude of the observed effects and its remarkable insensitivity to the active-center density in the investigated range. [Submitted to Phys Rev E, 22 March 2001]Comment: 14 pages, 5 encapsulated Postscript figure

Directed evolution of a far-red fluorescent rhodopsin

Microbial rhodopsins are a diverse group of photoactive transmembrane proteins found in all three domains of life. A member of this protein family, Archaerhodopsin-3 (Arch) of halobacterium Halorubrum sodomense, was recently shown to function as a fluorescent indicator of membrane potential when expressed in mammalian neurons. Arch fluorescence, however, is very dim and is not optimal for applications in live-cell imaging. We used directed evolution to identify mutations that dramatically improve the absolute brightness of Arch, as confirmed biochemically and with live-cell imaging (in Escherichia coli and human embryonic kidney 293 cells). In some fluorescent Arch variants, the pK_a of the protonated Schiff-base linkage to retinal is near neutral pH, a useful feature for voltage-sensing applications. These bright Arch variants enable labeling of biological membranes in the far-red/infrared and exhibit the furthest red-shifted fluorescence emission thus far reported for a fluorescent protein (maximal excitation/emission at ∼620 nm/730 nm)

Attractant and Repellent Signaling Conformers of Sensory Rhodopsin−Transducer Complexes†

ABSTRACT: Attractant and repellent signaling conformers of the dual-signaling phototaxis receptor sensory rhodopsin I and its transducer subunit (SRI-HtrI) have recently been distinguished experimentally by the opposite connection of their retinylidene protonated Schiff bases to the outwardly located periplasmic side and inwardly located cytoplasmic side. Here we show that the pKa of the outwardly located Asp76 counterion in the outwardly connected conformer is lowered by ∼1.5 units from that of the inwardly connected conformer. The pK a difference enables quantitative determination of the relative amounts of the two conformers in wild-type cells and behavioral mutants prior to photoexcitation, comparison of their absorption spectra, and determination of their relative signaling efficiency. We have shown that the onephoton excitation of the SRI-HtrI attractant conformer causes a Schiff base connectivity switch from inwardly connected to outwardly connected states in the attractant signaling photoreaction. Conversely, a second near-UV photon drives the complex back to the inwardly connected conformer in the repellent signaling photoreaction. The results suggest a model of the color-discriminating dual-signaling mechanism in which phototaxis responses (his-kinase modulation) result from the photointerconversion of the two oppositely connected SRI-HtrI conformers by one-photon and two-photon activation. Furthermore, we find that the related repellent phototaxis SRII-HtrII receptor complex has an outwardly connecte

Estimation and analysis of multi-GNSS differential code biases using a hardware signal simulator

In ionospheric modeling, the differential code biases (DCBs) are a non-negligible error source, which are routinely estimated by the different analysis centers of the International GNSS Service (IGS) as a by-product of their global ionospheric analysis. These are, however, estimated only for the IGS station receivers and for all the satellites of the different GNSS constellations. A technique is proposed for estimating the receiver and satellites DCBs in a global or regional network by first estimating the DCB of one receiver set as reference. This receiver DCB is then used as a ‘known’ parameter to constrain the global ionospheric solution, where the receiver and satellite DCBs are estimated for the entire network. This is in contrast to the constraint used by the IGS, which assumes that the involved satellites DCBs have a zero mean. The ‘known’ receiver DCB is obtained by simulating signals that are free of the ionospheric, tropospheric and other group delays using a hardware signal simulator. When applying the proposed technique for Global Positioning System legacy signals, mean offsets in the order of 3 ns for satellites and receivers were found to exist between the estimated DCBs and the IGS published DCBs. It was shown that these estimated DCBs are fairly stable in time, especially for the legacy signals. When the proposed technique is applied for the DCBs estimation using the newer Galileo signals, an agreement at the level of 1–2 ns was found between the estimated DCBs and the manufacturer’s measured DCBs, as published by the European Space Agency, for the three still operational Galileo in-orbit validation satellites

VLBI for Gravity Probe B. III. A Limit on the Proper Motion of the "Core" of the Quasar 3C 454.3

We made VLBI observations at 8.4 GHz between 1997 and 2005 to estimate the coordinates of the "core" component of the superluminal quasar, 3C 454.3, the ultimate reference point in the distant universe for the NASA/Stanford Gyroscope Relativity Mission, Gravity Probe B. These coordinates are determined relative to those of the brightness peaks of two other compact extragalactic sources, B2250+194 and B2252+172, nearby on the sky, and within a celestial reference frame (CRF), defined by a large suite of compact extragalactic radio sources, and nearly identical to the International Celestial Reference Frame 2 (ICRF2). We find that B2250+194 and B2252+172 are stationary relative to each other, and also in the CRF, to within 1-sigma upper limits of 15 and 30 micro-arcsec/yr in RA and decl., respectively. The core of 3C 454.3 appears to jitter in its position along the jet direction over ~0.2 mas, likely due to activity close to the putative supermassive black hole nearby, but on average is stationary in the CRF within 1-sigma upper limits on its proper motion of 39 micro-arcsec/yr (1.0c) and 30 micro-arcsec/yr (0.8c) in RA and decl., respectively, for the period 2002 - 2005. Our corresponding limit over the longer interval, 1998 - 2005, of more importance to GP-B, is 46 and 56 micro-arcsec/yr in RA and decl., respectively. Some of 3C 454.3's jet components show significantly superluminal motion with speeds of up to ~200 micro-arcsec/yr or 5c in the CRF. The core of 3C 454.3 thus provides for Gravity Probe B a sufficiently stable reference in the distant universe.Comment: Accepted for publication in the Astrophysical Journal Supplement Serie