Transcriptional activation by hepatocyte nuclear factor-4 in a cell-free system derived from rat liver nuclei

Hepatocyte nuclear factor-4 (HNF4) regulates gene expression by binding to direct repeat motifs of the RG(G/T)TCA sequence separated by one nucleotide (DR1). In this study we demonstrate that endogenous HNF4 present in rat liver nuclear extracts, as well as purified recombinant HNF4, activates transcription from naked DNA templates containing multiple copies of the DR1 element linked to the adenovirus major late promoter. Recombinant HNF4 also activates transcription from the rat cellular retinol binding protein II (CRBPII) promoter in vitro. The region between –105 and –63 bp of this promoter is essential for HNF-mediated transactivation. The addition of a peptide containing the LXXLL motif abolished HNF4-mediated transactivation in vitro suggesting that LXXLL-containing protein factor(s) are involved in HNF4-mediated transactivation in rat liver nuclear extracts. This is the first report on transactivation by HNF4 in a cell-free system derived from rat liver nuclei.

Kinetics of protein import into isolated Xenopus oocyte nuclei

An in vitro assay for nucleocytoplasmic transport was established in which signal-dependent protein import is reproduced faithfully by isolated purified nuclei. The assay permits the precise quantification of import kinetics and the discrimination between translocation through the nuclear envelope and intranuclear transport. Nuclei were manually isolated from Xenopus oocytes and after manual purification incubated with a medium containing a green fluorescent transport substrate, karyopherins α2 and β1, a red fluorescent control substrate, an energy mix and, for keeping an osmotic balance, 20% (wt/vol) BSA. Import of transport substrates into the nucleus and exclusion of the control substrate were monitored simultaneously by two-color confocal microscopy. Two widely differing import substrates were used: the recombinant protein P4K [480 kDa, four nuclear localization sequences (NLSs) per P4K tetramer], and NLS-BSA (90 kDa, 15 NLSs). The measurements suggested that import, at the specific conditions used in this study, consisted of two consecutive processes: (i) the rapid equilibration of the concentration difference across the nuclear envelope, a process involving binding and translocation of substrate by the nuclear pore complex , and (ii) the dissipation of the intranuclear concentration difference by diffusion.

Visualization of oligonucleotide probes and point mutations in interphase nuclei and DNA fibers using rolling circle DNA amplification

Rolling circle amplification (RCA) is a surface-anchored DNA replication reaction that can be exploited to visualize single molecular recognition events. Here we report the use of RCA to visualize target DNA sequences as small as 50 nts in peripheral blood lymphocytes or in stretched DNA fibers. Three unique target sequences within the cystic fibrosis transmembrane conductance regulator gene could be detected simultaneously in interphase nuclei, and could be ordered in a linear map in stretched DNA. Allele-discriminating oligonucleotide probes in conjunction with RCA also were used to discriminate wild-type and mutant alleles in the cystic fibrosis transmembrane conductance regulator, p53, BRCA-1, and Gorlin syndrome genes in the nuclei of cultured cells or in DNA fibers. These observations demonstrate that signal amplification by RCA can be coupled to nucleic acid hybridization and multicolor fluorescence imaging to detect single nucleotide changes in DNA within a cytological context or in single DNA molecules. This provides a means for direct physical haplotyping and the analysis of somatic mutations on a cell-by-cell basis.

Active galactic nuclei

Active galactic nuclei are the most powerful, long-lived objects in the Universe. Recent data confirm the theoretical idea that the power source is accretion into a massive black hole. The common occurrence of obscuration and outflows probably means that the contribution of active galactic nuclei to the power density of the Universe has been generally underestimated.

On the nucleation and growth of amyloid beta-protein fibrils: detection of nuclei and quantitation of rate constants.

We have studied the fibrillogenesis of synthetic amyloid beta-protein-(1-40) fragment (A beta) in 0.1 M HCl. At low pH, A beta formed fibrils at a rate amenable to detailed monitoring by quasi-elastic light-scattering spectroscopy. Examination of the fibrils with circular dichroism spectroscopy and electron microscopy showed them to be highly similar to those found in amyloid plaques. We determined the hydrodynamic radii of A beta aggregates during the entire process of fibril nucleation and growth. Above an A beta concentration of approximately 0.1 mM, the initial rate of elongation and the final size of fibrils were independent of A beta concentration. Below an A beta concentration of 0.1 mM, the initial elongation rate was proportional to the peptide concentration, and the resulting fibrils were significantly longer than those formed at higher concentration. We also found that the surfactant n-dodecylhexaoxyethylene glycol monoether (C12E6) slowed nucleation and elongation of fibrils in a concentration-dependent manner. Our observations are consistent with a model of A beta fibrillogenesis that includes the following key steps: (i) peptide micelles form above a certain critical A beta concentration, (ii) fibrils nucleate within these micelles or on heterogeneous nuclei (seeds), and (iii) fibrils grow by irreversible binding of monomers to fibril ends. Interpretation of our data enabled us to determine the sizes of fibril nuclei and A beta micelles and the rates of fibril nucleation (from micelles) and fibril elongation. Our approach provides a powerful means for the quantitative assay of A beta fibrillogenesis.

Very-long-baseline radio interferometry surveys of the compact structure in active galactic nuclei.

Very-long-baseline radio interferometry (VLBI) imaging surveys have been undertaken since the late 1970s. The sample sizes were initially limited to a few tens of objects but the snapshot technique has now allowed samples containing almost 200 sources to be studied. The overwhelming majority of powerful compact sources are asymmetric corejects of one form or another, most of which exhibit apparent superluminal motion. However 5-10% of powerful flat-spectrum sources are 100-parsec (pc)-scale compact symmetric objects; these appear to form a continuum with the 1-kpc-scale double-lobed compact steep-spectrum sources, which make up 15-20% of lower frequency samples. It is likely that these sub-galactic-size symmetric sources are the precursors to the large-scale classical double sources. There is a surprising peak around 90 degrees in the histogram of misalignments between the dominant source axes on parsec and kiloparsec scales; this seems to be associated with sources exhibiting a high degree of relativistic beaming. VLBI snapshot surveys have great cosmological potential via measurements of both proper motion and angular size vs. redshift as well as searches for gravitational "millilensing."

Optical evidence for the unification of active galactic nuclei and quasi-stellar objects.

There is a variety of optical evidence for some unification of different types of active galactic nuclei and quasi-stellar objects (QSOs). The case is very strong for the unification of at least some Seyfert galaxies, where polarization data show that the type assigned to the Seyfert galaxy must depend on viewing direction. It has been proposed that Fanaroff-Riley type 2 (FR2) radio galaxies are quasars seen in a direction from which the quasar is obscured, and there is some limited direct evidence for this picture. The broad absorption line QSOs may be normal QSOs seen from a special direction. Some of the sources observed to have high luminosities in the far infrared could be obscured QSOs and active nuclei. Mergers and interactions are likely to play an important role in nuclear activity, and active galaxies and QSOs could change their apparent types through these encounters followed by subsequent evolution.

Probing active galactic nuclei with H2O megamasers.

We describe the characteristics of the rapidly rotating molecular disk in the nucleus of the mildly active galaxy NGC4258. The morphology and kinematics of the disk are delineated by the point-like watervapor emission sources at 1.35-cm wavelength. High angular resolution [200 microas where as is arcsec, corresponding to 0.006 parsec (pc) at 6.4 million pc] and high spectral resolution (0.2 km.s-1 or nu/Deltanu = 1.4 x 10(6)) with the Very-Long-Baseline Array allow precise definition of the disk. The disk is very thin, but slightly warped, and is viewed nearly edge-on. The masers show that the disk is in nearly perfect Keplerian rotation within the observable range of radii of 0.13-0.26 pc. The approximately random deviations from the Keplerian rotation curve among the high-velocity masers are approximately 3.5 km.s-1 (rms). These deviations may be due to the masers lying off the midline by about +/-4 degrees or variations in the inclination of the disk by +/-4 degrees. Lack of systematic deviations indicates that the disk has a mass of <4 x 10(6) solar mass (M[symbol: see text]). The gravitational binding mass is 3.5 x 10(7) M[symbol: see text], which must lie within the inner radius of the disk and requires that the mass density be >4 x 10(9) M[symbol: see text].pc-3. If the central mass were in the form of a star cluster with a density distribution such as a Plummer model, then the central mass density would be 4 x 10(12) M[symbol: see text].pc-3. The lifetime of such a cluster would be short with respect to the age of the galaxy [Maoz, E. (1995) Astrophys. J. Lett. 447, L91-L94]. Therefore, the central mass may be a black hole. The disk as traced by the systemic velocity features is unresolved in the vertical direction, indicating that its scale height is <0.0003 pc (hence the ratio of thickness to radius, H/R, is <0.0025). For a disk in hydrostatic equilibrium the quadrature sum of the sound speed and Alfven velocity is <2.5 km.s-1, so that the temperature of the disk must be <1000 K and the toroidal magnetic field component must be <250 mG. If the molecular mass density in the disk is 10(10) cm-3, then the disk mass is approximately 10(4) M[symbol: see text], and the disk is marginally stable as defined by the Toomre stability parameter Q (Q = 6 at the inner edge and 1 at the outer edge). The inward drift velocity is predicted to be <0.007 km.s-1, for a viscosity parameter of 0.1, and the accretion rate is <7 x 10(-5) M[symbol: see text].yr-1. At this value the accretion would be sufficient to power the nuclear x-ray source of 4 x 10(40) ergs-1 (1 erg = 0.1 microJ). The volume of individual maser components may be as large as 10(46) cm3, based on the velocity gradients, which is sufficient to supply the observed luminosity. The pump power undoubtedly comes from the nucleus, perhaps in the form of x-rays. The warp may allow the pump radiation to penetrate the disk obliquely [Neufeld, D. A. & Maloney, P. R. (1995) Astrophys. J. Lett. 447, L17-L19]. A total of 15 H2O megamasers have been identified out of >250 galaxies searched. Galaxy NGC4258 may be the only case where conditions are optimal to reveal a well-defined nuclear disk. Future measurement of proper motions and accelerations for NGC4258 will yield an accurate distance and a more precise definition of the dynamics of the disk

Gravitational lensing of active galactic nuclei.

Most of the known cases of strong gravitational lensing involve multiple imaging of an active galactic nucleus. The properties of lensed active galactic nuclei make them promising systems for astrophysical applications of gravitational lensing; in particular, they show structure on scales of milliseconds of arc to tens of seconds of arc, they are variable, and they are polarized. More than 20 cases of strong gravitational lenses are now known, and about half of them are radio sources. High-resolution radio imaging is making possible the development of well-constrained lens models. Variability studies at radio and optical wavelengths are beginning to yield results of astrophysical interest, such as an independent measure of the distance scale and limits on source sizes.

The vesicular monoamine transporter 2 is present in small synaptic vesicles and preferentially localizes to large dense core vesicles in rat solitary tract nuclei.

In central neurons, monamine neurotransmitters are taken up and stored within two distinct classes of regulated secretory vesicles: small synaptic vesicles and large dense core vesicles (DCVs). Biochemical and pharmacological evidence has shown that this uptake is mediated by specific vesicular monamine transporters (VMATs). Recent molecular cloning techniques have identified the vesicular monoamine transporter (VMAT2) that is expressed in brain. This transporter determines the sites of intracellular storage of monoamines and has been implicated in both the modulation of normal monoaminergic neurotransmission and the pathogenesis of related neuropsychiatric disease. We used an antiserum against VMAT2 to examine its ultrastructural distribution in rat solitary tract nuclei, a region that contains a dense and heterogeneous population of monoaminergic neurons. We find that both immunoperoxidase and immunogold labeling for VMAT2 localize to DCVs and small synaptic vesicles in axon terminals, the trans-Golgi network of neuronal perikarya, tubulovesicles of smooth endoplasmic reticulum, and potential sites of vesicular membrane recycling. In axon terminals, immunogold labeling for VMAT2 was preferentially associated with DCVs at sites distant from typical synaptic junctions. The results provide direct evidence that a single VMAT is expressed in two morphologically distinct types of regulated secretory vesicles in central monoaminergic neurons.

A class of single-stranded telomeric DNA-binding proteins required for Rap1p localization in yeast nuclei.

We have identified a class of proteins that bind single-stranded telomeric DNA and are required for the nuclear organization of telomeres and/or telomere-associated proteins. Rlf6p was identified by its sequence similarity to Gbp1p, a single-stranded telomeric DNA-binding protein from Chlamydomonas reinhardtii. Rlf6p and Gbp1p bind yeast single-stranded G-strand telomeric DNA. Both proteins include at least two RNA recognition motifs, which are found in many proteins that interact with single-stranded nucleic acids. Disruption of RLF6 alters the distribution of repressor/activator protein 1 (Rap1p), a telomere-associated protein. In wild-type yeast cells, Rap1p localizes to a small number of perinuclear spots, while in rlf6 cells Rap1p appears diffuse and nuclear. Interestingly, telomere position effect and telomere length control, which require RAP1, are unaffected by rlf6 mutations, demonstrating that Rap1p localization can be uncoupled from other Rap1p-dependent telomere functions. In addition, expression of Chlamydomonas GBP1 restores perinuclear, punctate Rap1p localization in rlf6 mutant cells. The functional complementation of a fungal gene by an algal gene suggests that Rlf6p and Gbp1p are members of a conserved class of single-stranded telomeric DNA-binding proteins that influence nuclear organization. Furthermore, it demonstrates that, despite their unusual codon bias, C. reinhardtii genes can be efficiently translated in Saccharomyces cerevisiae cells.

Plasmid DNA entry into postmitotic nuclei of primary rat myotubes.

These studies were initiated to elucidate the mechanism of DNA nuclear transport in mammalian cells. Biotin- or gold-labeled plasmid and plasmid DNA expression vectors for Escherichia coli beta-galactosidase or firefly luciferase were microinjected into the cytoplasm of primary rat myotubes in culture. Plasmid DNA was expressed in up to 70% of the injected myotubes, which indicates that it entered intact, postmitotic nuclei. The nuclear transport of plasmid DNA occurred through the nuclear pore by a process common to other large karyophilic macromolecules. The majority of the injected plasmid DNA was sequestered by cytoplasmic elements. This understanding of plasmid DNA nuclear transport provides a basis for increasing the efficiency of gene transfer.

Fluorescent Biological Aerosol Particles in Coastal Canada and the Link to Atmospheric Ice Nuclei

Bioaerosols are a subgroup of atmospheric aerosols and are often linked to the spread of human, animal and plant diseases. Bioaerosols also may play an indirect effect on environmental processes, including the formation of precipitation and alteration of the global climate through their role as nuclei for cloud droplet formation. Several types of biological organisms (e.g., fungi and bacteria) have been shown to be effective ice nuclei (IN) and cloud condensation nuclei (CCN). During 21 days in August 2013 we participated in a collaborative international campaign at a rural, coastal site near the village of Ucluelet on the west coast of Vancouver Island, British Columbia, Canada. The experiments were conducted as part of the NETCARE project (the NETwork on Climate and Aerosols: Addressing Key Uncertainties in Remote Canadian Environments), in part to examine cloud nuclei properties of marine aerosol. The study was conducted from a mobile trailer located approximately 100 m from the coast. A suite of aerosol instrumentation was operated for approximately one month. Key instruments utilized as a part of this thesis include the wideband integrated bioaerosol sensor (WIBS-4A) and the multiple orifice uniform deposition impactor (MOUDI) coupled with an off-line droplet freezing technique (DFT) for the measurement of ice nucleation activity of particles in immersion mode. The WIBS measures the concentration and properties of individual fluorescent particles suspended in the air, which can serve as a proxy for airborne biological particle content. Particles shown to be fluorescent by the WIBS instrument were divided into seven categories based on the pattern of fluorescence each particle exhibited in the three fluorescent channels. Results of the WIBS analysis show that the fluorescent particle concentration in the region correlated well with IN number. The fluorescent particle concentration correlated well with the number of particles shown to be ice active as a function of both particle size and freezing temperature. Correlations involving marine aerosols and marine biological activity indicate that the majority of IN measured at the coastal site likely are not from have marine sources.