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All sourcesNASA NTRSDOE OSTI
NASA NTRS2009

X-ray Interferometry with Transmissive Beam Combiners for Ultra-High Angular Resolution Astronomy

Abstract Interferometry provides one of the possible routes to ultra-high angular resolution for X-ray and gamma-ray astronomy. Sub-micro-arc-second angular resolution, necessary to achieve objectives such as imaging the regions around the event horizon of a super-massive black hole at the center of an active galaxy, can be achieved if beams from parts of the incoming wavefront separated by 100s of meters can be stably and accurately brought together at small angles. One way of achieving this is by using grazing incidence mirrors. We here investigate an alternative approach in which the beams are recombined by optical elements working in transmission. It is shown that the use of diffractive elements is a particularly attractive option. We report experimental results from a simple 2-beam interferometer using a low-cost commercially available profiled film as the diffractive elements. A rotationally symmetric filled (or mostly filled) aperture variant of such an interferometer, equivalent to an X-ray axicon, is shown to offer a much wider bandpass than either a Phase Fresnel Lens (PFL) or a PFL with a refractive lens in an achromatic pair. Simulations of an example system are presented.

Skinner, G. K.↗
NASA NTRS1996

Sequence, overproduction and purification of Vibrio proteolyticus ribosomal protein L18 for in vitro and in vivo studies

A strategy suggested by comparative genomic studies was used to amplify the entire Vibrio proteolyticus (Vp) gene for ribosomal protein L18. Vp L18 and its flanking regions were sequenced and compared with the deduced amino acid (aa) sequences of other known L18 proteins. A 26-aa residue segment at the carboxy terminus contains many strongly conserved residues and may be critical for the L18 interaction with 5S rRNA. This approach should allow rapid characterization of L18 from large numbers of bacteria. Both Vp L18 and Escherichia coli (Ec) L18 were overproduced and purified using a T7 expression vector which fuses an N-terminal peptide segment (His-tag) containing 6 histidine residues to the recombinant protein. The purified fusion proteins, Vp His::L18 and Ec His::L18, were both found to bind to either the Vp 5S or Ec 5S rRNAs in vitro. Vp His::L18 protein was also shown to incorporate into Ec ribosomes in vivo. This His-tag strategy likely will have general applicability for the study of ribosomal proteins in vitro and in vivo.

Non-NASA Center↗
NASA NTRS2002

A model for interphase chromosomes and evaluation of radiation-induced aberrations

We have developed a theoretical model for evaluating radiation-induced chromosomal exchanges by explicitly taking into account interphase (G(0)/G(1)) chromosome structure, nuclear organization of chromosomes, the production of double-strand breaks (DSBs), and the subsequent rejoinings in a faithful or unfaithful manner. Each of the 46 chromosomes for human lymphocytes (40 chromosomes for mouse lymphocytes) is modeled as a random polymer inside a spherical volume. The chromosome spheres are packed randomly inside a spherical nucleus with an allowed overlap controlled by a parameter Omega. The rejoining of DSBs is determined by a Monte Carlo procedure using a Gaussian proximity function with an interaction range parameter sigma. Values of Omega and sigma have been found which yield calculated results of interchromosomal aberration frequencies that agree with a wide range of experimental data. Our preferred solution is one with an interaction range of 0.5 microm coupled with a relatively small overlap parameter of 0.675 microm, which more or less confirms previous estimates. We have used our model with these parameter values and with resolution or detectability limits to calculate yields of translocations and dicentrics for human lymphocytes exposed to low-LET radiation that agree with experiments in the dose range 0.09 to 4 Gy. Five different experimental data sets have been compared with the theoretical results. Essentially all of the experimental data fall between theoretical curves corresponding to resolution limits of 1 Mbp and 20 Mbp, which may reflect the fact that different investigators use different limits for sensitivity or detectability. Translocation yields for mouse lymphocytes have also been calculated and are in good agreement with experimental data from 1 cGy to 10 cGy. There is also good agreement with recent data on complex aberrations. Our model is expected to be applicable to both low- and high-LET radiation, and we include a sample prediction of the yield of interchromosomal rejoining in the dose range 0.22 Gy to 2 Gy of 1000 MeV/nucleon iron particles. This dose range corresponds to average particle traversals per nucleus ranging from 1.0 to 9.12.

Non-NASA Center↗
NASA NTRS1999

Mitochondria mediate tumor necrosis factor-alpha/NF-kappaB signaling in skeletal muscle myotubes

Tumor necrosis factor-alpha (TNF-alpha) is implicated in muscle atrophy and weakness associated with a variety of chronic diseases. Recently, we reported that TNF-alpha directly induces muscle protein degradation in differentiated skeletal muscle myotubes, where it rapidly activates nuclear factor kappaB (NF-kappaB). We also have found that protein loss induced by TNF-alpha is NF-kappaB dependent. In the present study, we analyzed the signaling pathway by which TNF-alpha activates NF-kappaB in myotubes differentiated from C2C12 and rat primary myoblasts. We found that activation of NF-kappaB by TNF-alpha was blocked by rotenone or amytal, inhibitors of complex I of the mitochondrial respiratory chain. On the other hand, antimycin A, an inhibitor of complex III, enhanced TNF-alpha activation of NK-kappaB. These results suggest a key role of mitochondria-derived reactive oxygen species (ROS) in mediating NF-kappaB activation in muscle. In addition, we found that TNF-alpha stimulated protein kinase C (PKC) activity. However, other signal transduction mediators including ceramide, Ca2+, phospholipase A2 (PLA2), and nitric oxide (NO) do not appear to be involved in the activation of NF-kappaB.

Non-NASA Center↗
NASA NTRS2000

A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats

Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

NASA Discipline Plant Biology↗
NASA NTRS2000

Role of hypoxia-inducible factor-1 in transcriptional activation of ceruloplasmin by iron deficiency

A role of the copper protein ceruloplasmin (Cp) in iron metabolism is suggested by its ferroxidase activity and by the tissue iron overload in hereditary Cp deficiency patients. In addition, plasma Cp increases markedly in several conditions of anemia, e.g. iron deficiency, hemorrhage, renal failure, sickle cell disease, pregnancy, and inflammation. However, little is known about the cellular and molecular mechanism(s) involved. We have reported that iron chelators increase Cp mRNA expression and protein synthesis in human hepatocarcinoma HepG2 cells. Furthermore, we have shown that the increase in Cp mRNA is due to increased rate of transcription. We here report the results of new studies designed to elucidate the molecular mechanism underlying transcriptional activation of Cp by iron deficiency. The 5'-flanking region of the Cp gene was cloned from a human genomic library. A 4774-base pair segment of the Cp promoter/enhancer driving a luciferase reporter was transfected into HepG2 or Hep3B cells. Iron deficiency or hypoxia increased luciferase activity by 5-10-fold compared with untreated cells. Examination of the sequence showed three pairs of consensus hypoxia-responsive elements (HREs). Deletion and mutation analysis showed that a single HRE was necessary and sufficient for gene activation. The involvement of hypoxia-inducible factor-1 (HIF-1) was shown by gel-shift and supershift experiments that showed HIF-1alpha and HIF-1beta binding to a radiolabeled oligonucleotide containing the Cp promoter HRE. Furthermore, iron deficiency (and hypoxia) did not activate Cp gene expression in Hepa c4 hepatoma cells deficient in HIF-1beta, as shown functionally by the inactivity of a transfected Cp promoter-luciferase construct and by the failure of HIF-1 to bind the Cp HRE in nuclear extracts from these cells. These results are consistent with in vivo findings that iron deficiency increases plasma Cp and provides a molecular mechanism that may help to understand these observations.

NASA Discipline Cardiopulmonary↗
NASA NTRS1996

Yeast respond to hypotonic shock with a calcium pulse

We have used the transgenic AEQUORIN calcium reporter system to monitor the cytosolic calcium ([Ca2+]cyt) response of Saccharomyces cerevisiae to hypotonic shock. Such a shock generates an almost immediate and transient rise in [Ca2+]cyt which is eliminated by gadolinium, a blocker of stretch-activated channels. In addition, this transient rise in [Ca2+]cyt is initially insensitive to 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), an extracellular calcium chelator. However, BAPTA abruptly attenuates the maintenance of that transient rise. These data show that hypotonic shock generates a stretch-activated channel-dependent calcium pulse in yeast. They also suggest that the immediate calcium influx is primarily generated from intracellular stores, and that a sustained increase in [Ca2+]cyt depends upon extracellular calcium.

NASA Discipline Plant Biology↗
NASA NTRS1996

Promoter-dependent and -independent activation of insulin-like growth factor binding protein-5 gene expression by prostaglandin E2 in primary rat osteoblasts

Insulin-like growth factor (IGF) action is mediated by high affinity cell surface IGF receptors and modulated by a family of secreted IGF binding proteins (IGFBPs). IGFBP-5, the most conserved of six IGFBPs characterized to date, uniquely potentiates the anabolic actions of IGF-I for skeletal cells. In osteoblasts, IGFBP-5 production is stimulated by prostaglandin E2 (PGE2), a local factor that mediates certain effects induced by parathyroid hormone, cytokines such as interleukin-1 and transforming growth factor-beta, and mechanical strain. In this study, we show that transcriptional and post-transcriptional events initiated by PGE2 collaborate to enhance IGFBP-5 gene expression in primary fetal rat osteoblast cultures. PGE2 treatment stimulated up to a 7-fold rise in steady-state levels of IGFBP-5 mRNA throughout 32 h of incubation. Analysis of nascent IGFBP-5 mRNA suggested that PGE2 had only a modest stimulatory effect on IGFBP-5 gene transcription, and transient transfection studies with IGFBP-5 promoter-reporter genes confirmed that PGE2 enhanced promoter activity by approximately 2-fold. Similar stimulatory effects were seen with forskolin. A DNA fragment with only 51 base pairs of the 5'-flanking sequence retained hormonal responsiveness, which may be mediated by a binding site for transcription factor AP-2 located at positions -44 to -36 in the proximal IGFBP-5 promoter. Incubation of osteoblasts with the mRNA transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that PGE2 enhanced IGFBP-5 mRNA stability by 2-fold, increasing the t1/2 from 9 to 18 h. The effects of PGE2 on steady-state IGFBP-5 transcripts were abrogated by preincubating cells with cycloheximide, indicating that the effects of PGE2 on both gene transcription and mRNA stability required ongoing protein synthesis. Therefore, both promoter-dependent and -independent pathways converge to enhance IGFBP-5 gene expression in response to PGE2 in osteoblasts.

Non-NASA Center↗
NASA NTRS1995

Circadian oscillations of cytosolic and chloroplastic free calcium in plants

Tobacco and Arabidopsis plants, expressing a transgene for the calcium-sensitive luminescent protein apoaequorin, revealed circadian oscillations in free cytosolic calcium that can be phase-shifted by light-dark signals. When apoaequorin was targeted to the chloroplast, circadian chloroplast calcium rhythms were likewise observed after transfer of the seedlings to constant darkness. Circadian oscillations in free calcium concentrations can be expected to control many calcium-dependent enzymes and processes accounting for circadian outputs. Regulation of calcium flux is therefore fundamental to the organization of circadian systems.

NASA Discipline Plant Biology↗
DOE OSTI2020

Time-variable Radio Recombination Line Emission in W49A

We present new Jansky Very Large Array (VLA) images of the central region of the W49A star-forming region at 3.6 cm and at 7 mm at resolutions of 0.″15 (1650 au) and 0.″04 (440 au), respectively. The 3.6 cm data reveal new morphological detail in the ultracompact H ii region population, as well as several previously unknown and unresolved sources. In particular, source A shows elongated, edge-brightened bipolar lobes, indicative of a collimated outflow, and source E is resolved into three spherical components. We also present VLA observations of radio recombination lines at 3.6 cm and 7 mm, and IRAM Northern Extended Millimeter Array (NOEMA) observations at 1.2 mm. Three of the smallest ultracompact H ii regions (sources A, B2, and G2) all show broad kinematic linewidths, with ΔV {sub FWHM} ≳ 40 km s{sup −1}. A multi-line analysis indicates that broad linewidths remain after correcting for pressure broadening effects, suggesting the presence of supersonic flows. Substantial changes in linewidth over the 21 yr time baseline at both 3.6 cm and 7 mm are found for source G2. At 3.6 cm, the linewidth of G2 changed from 31.7 ± 1.8 km s{sup −1} to 55.6 ± 2.7 km s{sup −1}, an increase of +23.9 ± 3.4 km s{sup −1}. The G2 source was previously reported to have shown a 3.6 cm continuum flux density decrease of 40% between 1994 and 2015. This source sits near the center of a very young bipolar outflow whose variability may have produced these changes.

79 ASTRONOMY AND ASTROPHYSICS↗
NASA NTRS2005

Eta Carinae: What We have learned recently with HST/STIS, VLT/UVES and VLTI: What We Might Learn with VLT/VLTI across the 2008/9 Minimum

Eta Carinae, the LBV that we have learned to love and hate, has revealed many clues of its character over the past seven years by studies with HST and VLT. Based upon X-Ray, optical and IR observations, Eta Carinae is convincingly a massive binary system that uniquely has major nebular ejecta that are connected with historical outbursts. We have successhlly followed the stellar and nebular changes over the 5.5 year cycle, especially with STIS and RXTE, and across the spectroscopic minimum in 2003.5 with STIS, CHANDRA, RXTE, FUSE, and VLT/UVES. The HST/STIS high spatial resolution, combined with appropriate spectral resolving powers from 1150 to 10300 A, has revealed much about the Central Source and especially the spatially resolved extended stellar atmosphere and the ejecta, known as the Homunculus. Indeed the neutral, dusty outer Homunculus, ejected in the 1840s, envelops the newly discovered ionized Little Homunculus, ejected in the 1890s. In line of sight, multiple hot clumps, both ionized and neutral, are seen in absorption and provide much information on the physical conditions of these massive ejecta. Against the nebular-scattered starlight, wind and nebular absorptions provide views at different angles from line of sight. The VLT/UVES studies from 2002 through 2004 provided very important time-sampled spectra of both the star as seen directly and as seen by the SE lobe viewing the polar region of the star. The VLTI 2.2 micron measures of the central source are consistent with a prolate spheroid with its axis extending along the axis of the Homunculus. This is consistent with the noticeably larger wind mass and higher terminal velocity along the axis of the Homunculus compared to that measured in line of sight at about 45 degrees from the polar axes. We understand the system to be a massive primary with an O or WR companion in a very highly elliptical orbit. The spectroscopic minimum occurs during periastron, when the greatly extended primary atmosphere and wind bottles up the ionizing uv radiation of the companion star. This transient drop in uv photons leads to recombination of much of the wind structure and of the nearby ejecta. Doubly-ionized elements (iron, neon, argon, vanadium) recombine to singly ionized forms and extended structures on the scales of below 0.03 arcseconds to an arcsecond change, appear, or disappear. With the loss of the STIS on HST, ground-based observations, especially with high spatial-resolution facilities, including the VLT and VLTI will be key to further studies across the minimum centered on 2009.0. Now is the time to plan for these studies.

Gull, Theodore R.↗
NASA NTRS1996

Activation of a Ca(2+)-dependent protein kinase involves intramolecular binding of a calmodulin-like regulatory domain

Ca(2+)-dependent protein kinases (CDPKs) are regulated by a C-terminal calmodulin-like domain (CaM-LD). The CaM-LD is connected to the kinase by a short junction sequence which contains a pseudosubstrate autoinhibitor. To understand how the CaM-LD regulates a CDPK, a recombinant CDPK (isoform CPK-1 from Arabidopsis, accession no. L14771) was made as a fusion protein in Escherichia coli. We show here that a truncated CDPK lacking a CaM-LD (e.g. mutant delta NC-26H) can be activated by exogenous calmodulin or an isolated CaM-LD (Kact approximately 2 microM). We propose that Ca2+ activation of a CDPK normally occurs through intramolecular binding of the CaM-LD to the junction. When the junction and CaM-LD are made as two separate polypeptides, the CaM-LD can bind the junction in a Ca(2+)-dependent fashion with a dissociation constant (KD) of 6 x 10(-6) M, as determined by kinetic binding analyses. When the junction and CaM-LD are tethered in a single polypeptide (e.g. in protein JC-1), their ability to engage in bimolecular binding is suppressed (e.g. the tethered CaM-LD cannot bind a separate junction). A mutation which disrupts the putative CaM-LD binding sequence (e.g. substitution LRV-1444 to DLPG) appears to block intramolecular binding, as indicated by the restored ability of a tethered CaM-LD to engage in bimolecular binding. This mutation, in the context of a full-length enzyme (mutant KJM46H), appears to block Ca2+ activation. Thus, a disruption of intramolecular binding correlates with a disruption of the Ca2+ activation mechanism. CDPKs provide the first example of a member of the calmodulin superfamily where a target binding sequence is located within the same polypeptide.

Non-NASA Center↗
NASA NTRS2004

Removing Spectral Diagnostics of Galactic and Stellar X-Ray Emission from Charged Exchange Recombination

Our research uses the electron beam ion trap (EBIT) at the Lawrence Livermore National Laboratory to study X-ray emission from the charge exchange (CX) of highly charged ions with neutral gases. The resulting data help to fill a void in existing experimental and theoretical understanding of this atomic physics process, and are needed to explain all or part of the observed X-ray emission from the soft X-ray background, stellar winds, the Galactic Center and Galactic Ridge, supernova ejecta, and photoionized nebulae. Appreciation of the astrophysical relevance of our work continues to grow with the publication of roughly a dozen papers in the past four years describing Chandra and XMM observations of geocoronal and heliospheric CX emission, the temporal variation of such emission and correlation with X-ray emission enhancements observed by ROSAT, the theoretical spatial distribution of that emission, and CX emission around other stars. A similar number of papers were also published during that time describing CX emission from planets and comets. We expect that the launch of ASTRSE2, with its second-generation XRS microcalo- (with 6-eV resolution), will reveal even more clearly the contributions of CX to astrophysical emission. In our EBIT work we collected CX spectra from such ions as H-like and He-like Ne, Ar, and Fe. Our early measurements were made with a high-purity Ge detector, but during the second year we began operation of the first-generation XRS microcalorimeter (a twin of the XRS on ASTRO-E) and greatly improved the resolution of our measurements from roughly 150 eV (FWHM) with the Ge detectors to 10 eV with the XRS. We found that saturation of the XRS counting apparatus, which we described in our proposal as a potential concern, is not a problem for studying CX. During the course of our research, we expanded the number of injection gases permitted by the LLNL safety team, purchased and eventually operated an atomic H source, and clearly demonstrated the feasibility of our longer-range plan. For example, we successfully injected He into EBIT (not a small feat because of the difficulty of maintaining a good vacuum with He and avoiding electrical breakdown) to collect a H-like oxygen CX spectrum. The highest energy CX spectrum recorded with the XRS to date is that of the Ar K-shell emission. These measurements provided the first observation of the relative intensity ratios of resolved He-like singlet and triplet n=2->1 lines. We also carried out measurements of He-like Ne as a function of collision energy (i.e., ion temperature). Significant differences in the resulting x-ray spectra were noted. In all cases, the intensity of high-n H-like Lyman lines is significantly higher than current theoretical CX models predict.

Wargelin, Brad↗
NASA NTRS1998

Interstellar Extinction in the Vicinity of the Galactic Center

We present J (1.2 microns) H (1-6 microns) K' (2.11 microns) and Br(gamma) (2.166 microns) images from four large regions within the central 40 pc of the Galaxy. Localized variations in the extinction, as determined by observations of the stellar population, are examined using the median H-K' color as a function of position within each region. As the value of the derived extinction from the stars is dependent upon the intrinsic magnitude of the assumed stellar type, the J-H vs. H-K' diagrams are first used to investigate the distribution of stellar types in the four regions. We find that there is a distinct OB population, contrary to earlier assumptions, with the ratio of K and M giants and supergiants to OB stars approximately twice that of the solar neighborhood. Although the on the scale of approx. 1 min. fluctuations in the extinction are on the order of A(sub V) approx. greater than 2, throughout the entire region the extinction varies from A(sub V) approx. greater than 25 to A(sub V) approx. less than 40. We also examine whether there is any variation in the extinction and stellar number density relative to the unusual radio features in these regions and do not find a significant correlation. Spectral imaging in Br(gamma) 2.166 microns emission shows a strong morphological correspondence between the 6 cm radio images and the diffuse Br(gamma) emission. By comparing the theoretical Br(gamma) flux derived from the radio flux using recombination theory, with our measured Br(gamma) flux, we obtain a second, independent, estimate of the extinction. We compare the two data sets and find that the extinction as derived from the stars is consistently greater, sometimes by a factor of two, than the value of the extinction derived from the Br(gamma) images. The derived extinction in various regions, however, is insufficient for any of these regions to be located behind the molecular clouds as previously observed in the Galactic Center, consistent with the theory that the observed radio emission is produced on the foreground surface of these clouds.

Cotera, Angela S.↗
NASA NTRS2000

Interstellar Extinction in the Vicinity of the Galactic Center

We present J (1.2 microns), H (1-6 microns), K' (2.11 microns) and Br(gamma) (2.166 microns) images from four large regions within the central 40 pc of the Galaxy. Localized variations in the extinction, as determined by observations of the stellar population, are examined using the median H-K' color as a function of position within each region. As the value of the derived extinction from the stars is dependent upon the intrinsic magnitude of the assumed stellar type, the J-H vs. H-K' diagrams are first used to investigate the distribution of stellar types in the four regions. We find that there is a distinct OB population, contrary to earlier assumptions, with the ratio of K and M giants and supergiants to OB stars approximately twice that of the solar neighborhood. Although the on the scale of approx. l' fluctuations in the extinction are on the order of A(sub V) approx. 2, throughout the entire region the extinction varies from A(sub V) approx. greater than 25 to A(sub V) approx. less than 40. We also examine whether there is any variation in the extinction and stellar number density relative to the usual radio features in these regions and do not find a significant correlation. Spectral imaging in Br(gamma) 2.166 microns emission shows a strong morphological correspondence between the 6 cm radio images and the diffuse Br(gamma) emission. By comparing the theoretical Br(gamma) flux derived from the radio flux using recombination theory, with our measured Br(gamma) flux, we obtain a second, independent, estimate of the extinction. We compare the two data sets and find that the extinction as derived from the stars is consistently greater, sometimes by a factor of two, than the value of the extinction derived from the Br(gamma) images. The derived extinction in various regions, however, is insufficient for any of these regions-to be located behind the molecular clouds as previously observed in the Galactic Center, consistent with the theory that the observed radio emission is produced on the foreground surface of these clouds.

Cotera, Angela S.↗
NASA NTRS1975

The rocket-ultraviolet spectrum of the planetary nebula NGC 7027

An ultraviolet spectrum of NGC 7027, obtained with a rocket-borne telescope, is analyzed. Absolute values are presented for the observed fluxes, and upper limits are given for the strongest predicted lines that were not observed. The results are corrected for interstellar extinction using the observed and calculated line ratios between H-beta and the hydrogenic recombination line of He II at 1640 A. The corrected C IV resonance line at 1549 A is found to be in good agreement with the intensity calculated from models, but the intercombination line of C III at 1909 A is found to be too bright by a factor of 10. This discrepancy is reduced to a factor of 4 by taking dielectric recombination into account and is eliminated by using the solar carbon abundance, which implies attenuation in the C IV line. It is shown that no appreciable number of absorbing grains can exist in the C IV-producing region of the nebula since the optical depth is of the order of 10,000 at the line center.

Bohlin, R. C.↗
NASA NTRS1993

Mutations in the putative calcium-binding domain of polyomavirus VP1 affect capsid assembly

Calcium ions appear to play a major role in maintaining the structural integrity of the polyomavirus and are likely involved in the processes of viral uncoating and assembly. Previous studies demonstrated that a VP1 fragment extending from Pro-232 to Asp-364 has calcium-binding capabilities. This fragment contains an amino acid stretch from Asp-266 to Glu-277 which is quite similar in sequence to the amino acids that make up the calcium-binding EF hand structures found in many proteins. To assess the contribution of this domain to polyomavirus structural integrity, the effects of mutations in this region were examined by transfecting mutated viral DNA into susceptible cells. Immunofluorescence studies indicated that although viral protein synthesis occurred normally, infective viral progeny were not produced in cells transfected with polyomavirus genomes encoding either a VP1 molecule lacking amino acids Thr-262 through Gly-276 or a VP1 molecule containing a mutation of Asp-266 to Ala. VP1 molecules containing the deletion mutation were unable to bind 45Ca in an in vitro assay. Upon expression in Escherichia coli and purification by immunoaffinity chromatography, wild-type VP1 was isolated as pentameric, capsomere-like structures which could be induced to form capsid-like structures upon addition of CaCl2, consistent with previous studies. However, although VP1 containing the point mutation was isolated as pentamers which were indistinguishable from wild-type VP1 pentamers, addition of CaCl2 did not result in their assembly into capsid-like structures. Immunogold labeling and electron microscopy studies of transfected mammalian cells provided in vivo evidence that a mutation in this region affects the process of viral assembly.

Non-NASA Center↗