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1.
Phys Rev Lett ; 132(4): 041001, 2024 Jan 26.
Article in English | MEDLINE | ID: mdl-38335333

ABSTRACT

With excellent energy resolution and ultralow-level radiogenic backgrounds, the high-purity germanium detectors in the Majorana Demonstrator enable searches for several classes of exotic dark matter (DM) models. In this work, we report new experimental limits on keV-scale sterile neutrino DM via the transition magnetic moment from conversion to active neutrinos ν_{s}→ν_{a}. We report new limits on fermionic dark matter absorption (χ+A→ν+A) and sub-GeV DM-nucleus 3→2 scattering (χ+χ+A→ϕ+A), and new exclusion limits for bosonic dark matter (axionlike particles and dark photons). These searches utilize the (1-100)-keV low-energy region of a 37.5-kg y exposure collected by the Demonstrator between May 2016 and November 2019 using a set of ^{76}Ge-enriched detectors whose surface exposure time was carefully controlled, resulting in extremely low levels of cosmogenic activation.

3.
Phys Rev Lett ; 130(6): 062501, 2023 Feb 10.
Article in English | MEDLINE | ID: mdl-36827565

ABSTRACT

The Majorana Demonstrator searched for neutrinoless double-ß decay (0νßß) of ^{76}Ge using modular arrays of high-purity Ge detectors operated in vacuum cryostats in a low-background shield. The arrays operated with up to 40.4 kg of detectors (27.2 kg enriched to ∼88% in ^{76}Ge). From these measurements, the Demonstrator has accumulated 64.5 kg yr of enriched active exposure. With a world-leading energy resolution of 2.52 keV FWHM at the 2039 keV Q_{ßß} (0.12%), we set a half-life limit of 0νßß in ^{76}Ge at T_{1/2}>8.3×10^{25} yr (90% C.L.). This provides a range of upper limits on m_{ßß} of (113-269) meV (90% C.L.), depending on the choice of nuclear matrix elements.

4.
Phys Rev Lett ; 129(8): 080401, 2022 Aug 19.
Article in English | MEDLINE | ID: mdl-36053678

ABSTRACT

The Majorana Demonstrator neutrinoless double-beta decay experiment comprises a 44 kg (30 kg enriched in ^{76}Ge) array of p-type, point-contact germanium detectors. With its unprecedented energy resolution and ultralow backgrounds, Majorana also searches for rare event signatures from beyond standard model physics in the low energy region below 100 keV. In this Letter, we test the continuous spontaneous localization (CSL) model, one of the mathematically well-motivated wave function collapse models aimed at solving the long-standing unresolved quantum mechanical measurement problem. While the CSL predicts the existence of a detectable radiation signature in the x-ray domain, we find no evidence of such radiation in the 19-100 keV range in a 37.5 kg-y enriched germanium exposure collected between December 31, 2015, and November 27, 2019, with the Demonstrator. We explored both the non-mass-proportional (n-m-p) and the mass-proportional (m-p) versions of the CSL with two different assumptions: that only the quasifree electrons can emit the x-ray radiation and that the nucleus can coherently emit an amplified radiation. In all cases, we set the most stringent upper limit to date for the white CSL model on the collapse rate, λ, providing a factor of 40-100 improvement in sensitivity over comparable searches. Our limit is the most stringent for large parts of the allowed parameter space. If the result is interpreted in terms of the Diòsi-Penrose gravitational wave function collapse model, the lower bound with a 95% confidence level is almost an order of magnitude improvement over the previous best limit.

5.
Phys Rev Lett ; 129(8): 081803, 2022 Aug 19.
Article in English | MEDLINE | ID: mdl-36053699

ABSTRACT

Axions were originally proposed to explain the strong-CP problem in QCD. Through axion-photon coupling, the Sun could be a major source of axions, which could be measured in solid state detection experiments with enhancements due to coherent Primakoff-Bragg scattering. The Majorana Demonstrator experiment has searched for solar axions with a set of ^{76}Ge-enriched high purity germanium detectors using a 33 kg-yr exposure collected between January, 2017 and November, 2019. A temporal-energy analysis gives a new limit on the axion-photon coupling as g_{aγ}<1.45×10^{-9} GeV^{-1} (95% confidence level) for axions with mass up to 100 eV/c^{2}. This improves laboratory-based limits between about 1 eV/c^{2} and 100 eV/c^{2}.

6.
Eur Phys J C Part Fields ; 82(3): 226, 2022.
Article in English | MEDLINE | ID: mdl-35310515

ABSTRACT

P-type point contact (PPC) HPGe detectors are a leading technology for rare event searches due to their excellent energy resolution, low thresholds, and multi-site event rejection capabilities. We have characterized a PPC detector's response to α particles incident on the sensitive passivated and p + surfaces, a previously poorly-understood source of background. The detector studied is identical to those in the Majorana Demonstrator experiment, a search for neutrinoless double-beta decay ( 0 ν ß ß ) in 76 Ge. α decays on most of the passivated surface exhibit significant energy loss due to charge trapping, with waveforms exhibiting a delayed charge recovery (DCR) signature caused by the slow collection of a fraction of the trapped charge. The DCR is found to be complementary to existing methods of α identification, reliably identifying α background events on the passivated surface of the detector. We demonstrate effective rejection of all surface α events (to within statistical uncertainty) with a loss of only 0.2% of bulk events by combining the DCR discriminator with previously-used methods. The DCR discriminator has been used to reduce the background rate in the 0 ν ß ß region of interest window by an order of magnitude in the Majorana Demonstrator  and will be used in the upcoming LEGEND-200 experiment.

7.
Minerva Anestesiol ; 76(5): 353-61, 2010 May.
Article in English | MEDLINE | ID: mdl-20395898

ABSTRACT

Primary graft failure (PGF) is one of the major complications that occurs immediately following lung transplantation and greatly contributes to increased morbidity and mortality. The incidence of PGF is correlated with a marked decline in endogenous nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) levels. Therefore, the administration of NO during lung transplantation has been proposed as a possible therapeutic treatment to prevent or attenuate PGF pathogenesis. Despite the initial positive results of experimental and uncontrolled clinical trials, recent randomized clinical trials do not support the prophylactic administration of inhaled nitric oxide (iNO) for the prevention of PGF following lung transplantation under the conditions tested. Nonetheless, there is evidence that iNO administration during PGF can improve oxygenation and reduce pulmonary hypertension without altering systemic vascular resistance. This suggests that iNO may prevent the need for extracorporeal membrane oxygenation (ECMO) during the hypoxic phase of PGF. During the intraoperative phase of transplantation, one-lung ventilation (OLV) and pulmonary artery clamping usually increase PVR, causing decreased right ventricular function and hemodynamic instability. The administration of iNO during these lung transplant procedures could decrease right ventricular dysfunction by reducing PVR and help to avoid the use of cardiopulmonary bypass.


Subject(s)
Bronchodilator Agents/therapeutic use , Lung Transplantation/physiology , Nitric Oxide/therapeutic use , Vasodilator Agents/therapeutic use , Bronchodilator Agents/administration & dosage , Humans , Nitric Oxide/administration & dosage , Pulmonary Circulation , Reperfusion Injury/prevention & control , Respiration, Artificial , Vasodilator Agents/administration & dosage
8.
Phys Med Biol ; 54(7): 2121-30, 2009 Apr 07.
Article in English | MEDLINE | ID: mdl-19287083

ABSTRACT

Non-invasive micro-CT imaging techniques have been developed to investigate lung structure in free-breathing rodents. In this study, we investigate the utility of retrospectively respiratory-gated micro-CT imaging in an emphysema model to determine if anatomical changes could be observed in the image-derived quantitative analysis at two respiratory phases. The emphysema model chosen was a well-characterized, genetically altered model (TIMP-3 knockout mice) that exhibits a homogeneous phenotype. Micro-CT scans of the free-breathing, anaesthetized mice were obtained in 50 s and retrospectively respiratory sorted and reconstructed, providing 3D images representing peak inspiration and end expiration with 0.15 mm isotropic voxel spacing. Anatomical measurements included the volume and CT density of the lungs and the volume of the major airways, along with the diameters of the trachea, left bronchus and right bronchus. From these measurements, functional parameters such as functional residual capacity and tidal volume were calculated. Significant differences between the wild-type and TIMP-3 knockout groups were observed for measurements of CT density over the entire lung, indicating increased air content in the lungs of TIMP-3 knockout mice. These results demonstrate retrospective respiratory-gated micro-CT, providing images at multiple respiratory phases that can be analyzed quantitatively to investigate anatomical changes in murine models of emphysema.


Subject(s)
Emphysema/diagnostic imaging , Emphysema/pathology , Lung/diagnostic imaging , Lung/pathology , Animals , Disease Models, Animal , Gene Knockout Techniques , Male , Mice , Respiratory-Gated Imaging Techniques , Tissue Inhibitor of Metalloproteinase-3/genetics , Tomography, X-Ray Computed
9.
J Appl Physiol (1985) ; 102(5): 2046-55, 2007 May.
Article in English | MEDLINE | ID: mdl-17255374

ABSTRACT

Lung morphology and function in human subjects can be monitored with computed tomography (CT). Because many human respiratory diseases are routinely modeled in rodents, a means of monitoring the changes in the structure and function of the rodent lung is desired. High-resolution images of the rodent lung can be attained with specialized micro-CT equipment, which provides a means of monitoring rodent models of lung disease noninvasively with a clinically relevant method. Previous studies have shown respiratory-gated images of intubated and respirated mice. Although the image quality and resolution are sufficient in these studies to make quantitative measurements, these measurements of lung structure will depend on the settings of the ventilator and not on the respiratory mechanics of the individual animals. In addition, intubation and ventilation can have unnatural effects on the respiratory dynamics of the animal, because the airway pressure, tidal volume, and respiratory rate are selected by the operator. In these experiments, important information about the symptoms of the respiratory disease being studied may be missed because the respiration is forced to conform to the ventilator settings. In this study, we implement a method of respiratory-gated micro-CT for use with anesthetized free-breathing rodents. From the micro-CT images, quantitative analysis of the structure of the lungs of healthy unconscious mice was performed to obtain airway diameters, lung and airway volumes, and CT densities at end expiration and during inspiration. Because the animals were free breathing, we were able to calculate tidal volume (0.09 +/- 0.03 ml) and functional residual capacity (0.16 +/- 0.03 ml).


Subject(s)
Lung/diagnostic imaging , Lung/physiology , Respiration , Tomography, X-Ray Computed/methods , Anesthesia , Animals , Functional Residual Capacity , Image Processing, Computer-Assisted , Imaging, Three-Dimensional , Lung/anatomy & histology , Lung Volume Measurements , Male , Mice , Mice, Inbred C57BL , Tidal Volume
10.
J Anim Sci ; 80(11): 2996-3004, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12462269

ABSTRACT

The effect of breed and diet on insulin response to glucose challenge and its relation to intramuscular fat deposition was determined in 36 steers with 12 each of greater than 87% Wagyu (referred to as Wagyu), Wagyu x Limousin, and Limousin breeds. Weaned steers were blocked by weight into heavy, medium, and light calves and placed in six pens with two pens per weight type and with two steers of each breed per pen. Three pens with steers from each weightclass were fed backgrounding and finishing diets for 259 d, while the other three pens were fed the same diets where 6% of the barley grain was replaced with sunflower oil. Prior to initiation of the finishing phase of the study the intravenous glucose tolerance test (VGTIT) was conducted in all steers. Once steers were judged as carrying adequate 12th-rib fat, based on weight and days on feed, they were harvested and graded and samples of the longissimus muscle were procured for determination of fat content and fatty acid composition. Dietary oil improved (P = 0.011; 0.06) ADG and feed conversion efficiency of steers during the latter part of backgrounding and only ADG during early part ofthe finishing period. Generally percent kidney, pelvic, and heart fat was the only adiposity assessment increased (P = 0.003) by dietary oil. The IVGTT results indicated that insulin response to intravenous glucose was lower in Limousin steers than in Wagyu steers. Dietary oil decreased (P = 0.052) fasting plasma insulin concentration in Wagyu steers compared with Limousin steers. The correlation coefficients among the IVGTT measures and intramuscular fat content or marbling score were less than 0.4, and only a negative trend existed between fasting insulin and USDA marbling scores. However, the carcasses of the Wagyu steers graded US Choice, and 66% of the Wagyu carcasses graded US Prime, which were substantially better than the quality grades obtained for the carcasses from the other breed types. Dietary oil did not affect muscle fat content but increased (P = 0.01) conjugated linoleic acid (CLA) concentrations by 339%. Results indicated that IVGTT measures were not appropriate indices of marbling potential in cattle and that dietary oil can enhance CLA content of beef.


Subject(s)
Blood Glucose/metabolism , Cattle/physiology , Insulin/blood , Meat/standards , Muscle, Skeletal/metabolism , Plant Oils/pharmacology , Adipose Tissue/metabolism , Animal Feed , Animal Husbandry/methods , Animal Nutritional Physiological Phenomena , Animals , Area Under Curve , Breeding , Cattle/growth & development , Cattle/metabolism , Dietary Fats , Fatty Acids/analysis , Glucose/administration & dosage , Glucose Tolerance Test/veterinary , Insulin/metabolism , Linoleic Acid/metabolism , Male , Muscle, Skeletal/chemistry , Plant Oils/metabolism , Sunflower Oil
11.
Dev Biol ; 236(1): 46-63, 2001 Aug 01.
Article in English | MEDLINE | ID: mdl-11456443

ABSTRACT

The CyIIa gene of the sea urchin embryo is a model for study of cis-regulation downstream of cell-type specification, as CyIIa transcription follows the specification and initial differentiation of the embryonic domains in which it is expressed. These are the skeletogenic and secondary mesenchyme and gut. We carried out a detailed structural and functional analysis of a cis-regulatory region of this gene, extending 780 bp upstream and 125 bp downstream of the transcription start site, that had been shown earlier to reproduce faithfully the complex and dynamic CyIIa pattern of expression. This analysis revealed that the overall pattern of expression of the CyIIa gene appears to be governed mainly by two independent sets of DNA elements, which are target sites for specific proteins present in blastula-stage nuclear extract. One type of element, which controls a dynamic program of expression in both skeletogenic and secondary mesenchyme cells, contains the consensus-binding site for a member of the ets transcription factor family. The other, which is responsible for the terminal or permanent phase of CyIIa expression in the gut, shares homologies with the late module of the endoderm-specific Endo16 gene (endo16 Module B). Oligonucleotides containing replicas of these two target sites fused upstream of a sea urchin basal promoter are sufficient to confer accurate mesenchyme and late gut expression of an injected GFP construct. The finding of a single protein target site that recapitulates CyIIa expression in both primary and secondary mesenchyme cells suggests the existence of a pan-mesodermal gene expression program in the sea urchin embryo.


Subject(s)
Actins/genetics , Actins/physiology , Gene Expression Regulation, Developmental , Mesoderm/metabolism , Transcription, Genetic , Animals , Base Sequence , Binding Sites , Binding, Competitive , Biological Evolution , Cell Nucleus/metabolism , DNA/metabolism , Gastrula/metabolism , Genes, Reporter , Green Fluorescent Proteins , Luminescent Proteins/metabolism , Models, Genetic , Molecular Sequence Data , Protein Binding , Recombinant Fusion Proteins/metabolism , Sea Urchins , Sequence Homology, Nucleic Acid , Transcription Factors/metabolism
12.
J Basic Microbiol ; 41(2): 97-104, 2001.
Article in English | MEDLINE | ID: mdl-11441464

ABSTRACT

The presence of adenine in the L-alanine defined medium substantially inhibited the growth of the moderately halophilic eubacterium Halomonas elongata. Extensive attempts to reverse the adenine toxicity for growth were made using a variety of purine and pyrimidine compounds, vitamins, and amino acids. Of the compounds tested, only cytosine was found to reverse the adenine growth inhibition. This indicates a mechanism similar to that found for some strains of Escherichia coli in which the presence of exogenous purines (e.g. adenine) was found to stop purine de novo synthesis and repress the synthesis of the pyrimidine salvage enzyme cytosine deaminase. H. elongata was found to possess an active adenine uptake system that was sodium dependent with only lithium having a considerable capacity to replace the sodium. A competition study indicated that the adenine transport system was quite specific. This paper represents the initial study of purine and pyrimidine salvage pathways and adenine uptake for the moderately halophilic eubacteria.


Subject(s)
Adenine/metabolism , Adenine/pharmacology , Halomonas/drug effects , Halomonas/metabolism , Biological Transport, Active , Culture Media , Cytosine/metabolism , Halomonas/growth & development , Purines/metabolism , Pyrimidines/metabolism , Sodium Chloride
13.
Appl Environ Microbiol ; 66(12): 5206-12, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11097891

ABSTRACT

Reactivation of UV-C-inactivated Pseudomonas aeruginosa bacteriophages D3C3, F116, G101, and UNL-1 was quantified in host cells infected during the exponential phase, during the stationary phase, and after starvation (1 day, 1 and 5 weeks) under conditions designed to detect dark repair and photoreactivation. Our experiments revealed that while the photoreactivation capacity of stationary-phase or starved cells remained about the same as that of exponential-phase cells, in some cases their capacity to support dark repair of UV-inactivated bacteriophages increased over 10-fold. This enhanced reactivation capacity was correlated with the ca. 30-fold-greater UV-C resistance of P. aeruginosa host cells that were in the stationary phase or exposed to starvation conditions prior to irradiation. The dark repair capacity of P. aeruginosa cells that were infected while they were starved for prolonged periods depended on the bacteriophage examined. For bacteriophage D3C3 this dark repair capacity declined with prolonged starvation, while for bacteriophage G101 the dark repair capacity continued to increase when cells were starved for 24 h or 1 week prior to infection. For G101, the reactivation potentials were 16-, 18-, 10-, and 3-fold at starvation intervals of 1 day, 1 week, 5 weeks, and 1. 5 years, respectively. Exclusive use of exponential-phase cells to quantify bacteriophage reactivation should detect only a fraction of the true phage reactivation potential.


Subject(s)
Escherichia coli Proteins , Pseudomonas Phages/growth & development , Pseudomonas aeruginosa/growth & development , Pseudomonas aeruginosa/virology , Adenosine Triphosphatases/genetics , Bacterial Proteins/genetics , DNA-Binding Proteins/genetics , Genes, Bacterial , Photobiology , Pseudomonas Phages/radiation effects , Pseudomonas aeruginosa/genetics , Radiation Tolerance , Ultraviolet Rays , Virus Activation
14.
Science ; 290(5489): 103-7, 2000 Oct 06.
Article in English | MEDLINE | ID: mdl-11021788

ABSTRACT

We present the discovery by optical and near-infrared imaging of an extremely red, low-luminosity population of isolated objects in the young, nearby stellar cluster around the multiple, massive star final sigma Orionis. The proximity (352 parsecs), youth (1 million to 5 million years), and low internal extinction make this cluster an ideal site to explore the substellar domain from the hydrogen mass limit down to a few Jupiter masses. Optical and near-infrared low-resolution spectroscopy of three of these objects confirms the very cool spectral energy distribution (atmospheric effective temperatures of 1700 to 2200 kelvin) expected for cluster members with masses in the range 5 to 15 times that of Jupiter. Like the planets of the solar system, these objects are unable to sustain stable nuclear burning in their interiors, but in contrast they are not bound to stars. This new kind of isolated giant planet, which apparently forms on time scales of less than a few million years, offers a challenge to our understanding of the formation processes of planetary mass objects.

15.
Can J Microbiol ; 46(2): 180-7, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10721487

ABSTRACT

Both the moderately halophilic bacterium, Halomonas elongata, and the extremely halophilic archaea, Halobacterium salinarum, can be found in hypersaline environments (e.g., salterns). On complex media, H. elongata grows over a salt range of 0.05-5.2 M, whereas, H. salinarum multiplies over a salt range of 2.5-5.2 M. The purpose of this study was to illustrate the effect that solar (UV-A and UV-B) and germicidal radiation (UV-C) had on the growth patterns of these bacteria at varied salt concentrations. Halomonas elongata grown on a complex medium at 0.05, 1.37, and 4.3 M NaCl was found to be more sensitive to UV-A and UV-B radiation, as the salt concentration of the medium increased. Halobacterium salinarum grown on a complex medium at 3.0 and 4.3 M NaCl did not show a significant drop in viability after 39.3 kJ.m-2 of UV-A and UV-B exposure. When exposed to UV-C, H. elongata exhibited substantially more sensitivity than H. salinarum. In H. elongata, differential sensitivity to UV-C was observed. At 0.05 M NaCl, H. elongata was less sensitive to UV-C than at 1.37 and 4.3 M NaCl. Both bacteria showed some photoreactivation when incubated under visible light following both UV-A, UV-B, and UV-C exposure. Mutagenesis following UV-C exposure was demonstrated by both organisms.


Subject(s)
Halobacterium salinarum/radiation effects , Halomonas/radiation effects , Ultraviolet Rays , Anti-Bacterial Agents/pharmacology , DNA Repair , Drug Resistance, Microbial , Halobacterium salinarum/drug effects , Halobacterium salinarum/growth & development , Halomonas/drug effects , Halomonas/growth & development , Mutagenesis , Novobiocin/pharmacology , Rifampin/pharmacology , Sodium Chloride/pharmacology
16.
Appl Environ Microbiol ; 65(6): 2606-13, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10347050

ABSTRACT

UNL-1, a lytic virus of Pseudomonas aeruginosa, was observed to express a novel inducible DNA damage reactivation activity in UV-A-irradiated P. aeruginosa host cells. The expression of bacteriophage reactivation was quantified in hosts exposed to either UV-C or UV-A radiation. While reactivation of UV-C-damaged UNL-1 was not inducible in UV-C-irradiated host cells, an approximately 13-fold induction was observed in UV-A-irradiated host cells. When host cells were exposed to sunlight, reactivation of damaged UNL-1 virus increased eightfold. The UV-A induction of UNL-1 DNA damage reactivation was supported in hosts lacking recA gene function. This report is the first description of a recA-independent, UV-inducible virus DNA damage repair system. Our findings suggest that a combination of both host and virus DNA repair processes contribute to the persistence and sustained replication of some bacterial viruses in aquatic environments.


Subject(s)
DNA Repair , Pseudomonas Phages/physiology , Pseudomonas aeruginosa/virology , Ultraviolet Rays , Virus Activation , Base Composition , Blotting, Southern , DNA Damage , DNA, Viral/chemistry , DNA, Viral/genetics , Pseudomonas Phages/genetics , Pseudomonas Phages/isolation & purification , Pseudomonas Phages/radiation effects , Pseudomonas aeruginosa/radiation effects , Sunlight , Transduction, Genetic
17.
Hum Factors ; 41(3): 495-506; discussion 507-10, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10665216

ABSTRACT

Immediate feedback was given to correct observers' estimates of distance in an experiment in which those estimates were made outdoors at night while observers wore night vision goggles (NVGs). Initially observers made unguided estimates of distances between marked positions in an open field. Those distances ranged from 7.6 m (25 ft) to 64 m (210 ft). Later the same observers made more estimates. After each of these they were told the measured distance between the positions. During this training, the observers' height from the ground plane was either at a standing position or at an elevated position raised 2.3 m (7 ft 7 in) from standing position. After the training--either immediately after, a week later, or at both times--observers made unguided estimates of distance for a second time. These latter estimates of ground distance made with the NVGs were improved. Average improvement of the observers' estimates persisted for at least one week after training. This training can be applied to improve clearance estimates and estimates of hover height for pilots of rotary-wing aircraft.


Subject(s)
Aerospace Medicine , Dark Adaptation , Distance Perception , Eyeglasses , Aviation , Feedback , Female , Humans , Linear Models , Male , Task Performance and Analysis
18.
Development ; 125(8): 1381-95, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9502720

ABSTRACT

CyIIa, a cytoskeletal actin gene of Strongylocentrotus purpuratus, is expressed specifically though transiently in the embryonic skeletogenic and secondary mesenchyme and, later in development, is permanently activated in the hindgut and midgut. CyIIa transcription follows, and is therefore downstream of, the initial specification of these embryonic domains. A detailed functional analysis of the cis-regulatory system governing the rate and the location of CyIIa expression during development was carried out using GFP expression constructs. About 4.4 kb of CyIIa sequence including a leader intron were examined for cis-regulatory function. Distal elements scattered over several kb account for 60% of the quantitative output of the expression construct and a strong amplifier of expression is located within the leader intron. However, the complex spatial pattern of CyIIa expression is completely reproduced by a compact upstream regulatory element <450 bp in length. We found no evidence anywhere in the 4.4 kb sequence examined for negative regulators required to repress ectopic expression. The specific site that mediates CyIIa expression in the midgut in late embryos and larvae was identified. This site is the same as that necessary and sufficient for midgut expression of the Endo16 gene late in development, and was shown to bind the same transcription factor. Except for some temporal and quantitative features, the S. purpuratus expression construct is expressed accurately and specifically in the same diverse cell types when introduced into embryos of Lytechinus pictus, which belongs to a different echinoid order. No ectopic expression was observed, in contrast to the result of a similar interspecific gene transfer experiment carried out earlier on a different cytoskeletal actin gene that is expressed much earlier in development. Presentation of the set of transcription factors that activate CyIIa in the differentiated cells in which it is expressed is apparently a conserved feature of these cell types.


Subject(s)
Actins/biosynthesis , Embryo, Nonmammalian/physiology , Embryonic Induction/physiology , Gene Expression Regulation, Developmental , Sea Urchins/embryology , Actins/genetics , Animals , Base Sequence , Binding Sites , Body Patterning , Cytoskeleton/physiology , DNA Primers , Digestive System/embryology , Digestive System/metabolism , Genes, Reporter , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/biosynthesis , Recombinant Fusion Proteins , Regulatory Sequences, Nucleic Acid , Restriction Mapping , Sequence Alignment , Sequence Homology, Nucleic Acid , Transcription, Genetic
19.
J Biol Chem ; 271(1): 361-6, 1996 Jan 05.
Article in English | MEDLINE | ID: mdl-8550587

ABSTRACT

The C terminus of the G protein alpha subunit represents an important site of interaction between heterotrimeric G proteins and their cognate receptors. We have screened a combinatorial peptide library based on the C terminus of the alpha subunit of Gt (340-350) and have identified unique sequences that bind rhodopsin with high affinity. Six of these sequences, as both fusion proteins and synthetic peptides, were significantly more potent than the parent sequence in binding to and stabilization of metarhodopsin II. These sequences provide information about which residues are required for appropriate receptor interaction. We observed that in all the high affinity sequences, a positively charged residue at position 341 was changed to a neutral one. Thus, it appears that the receptor-G protein interaction was designed to be low affinity to ensure efficient catalysis of G protein activation. We also observed Cys-347 and Gly-348 to be invariant, and hydrophobic residues were always located at positions 340, 344, 349, and 350, demonstrating the critical nature of these residues. A composite of the structures of the high affinity sequences was modeled based upon the structure of rhodopsin-bound trNOESY NMR of this region of Gt alpha (Dratz, E. D., Fursteneau, J. E., Lambert, C. G., Thireault, D. L., Rarick, H., Schepers, T., Pakhlevaniants, S., and Hamm, H. E. (1993) Nature 363, 276-280) and provides insight into the complementary G protein-binding surface of the receptor.


Subject(s)
GTP-Binding Proteins/metabolism , Peptides/metabolism , Receptors, Cell Surface/metabolism , Amino Acid Sequence , Base Sequence , Catalysis , Molecular Sequence Data , Protein Conformation , Receptors, Cell Surface/chemistry , Rhodopsin/analogs & derivatives , Rhodopsin/metabolism , Sequence Homology, Amino Acid
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