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61.
Bernhard Benkel Sonja Kollers Ruedi Fries Alexei Sazanov Erin Yoshida Edith Valle Jon Davoren Donal Hickey 《Mammalian genome》2005,16(3):194-200
AMP-activated protein kinase (AMPK) represents the mammalian form of the core component of a kinase cascade that is conserved between fungi, plants, and animals. AMPK plays a major role in protecting mammalian cells from metabolic stress by switching off biosynthetic pathways that require ATP and switching on ATP-regenerating pathways. In this report, we describe the isolation and characterization of the gene for the noncatalytic bovine gamma1 subunit of AMPK. The bovine ampkgamma1 (PRKAG1) gene spans in excess of 14 kb and is located at BTA 5q21-q22. It consists of 12 exons ranging in size from 38 b to 166 b, interspersed with 11 introns that range between 97 b and 6753 b in length. The coding region of the bovine gene shares 93% and 90% nucleotide sequence similarity with its human and rat counterparts, and the bovine AMPKgamma1 protein is 98% and 95% identical to its human and rat homologs, respectively, in amino acid sequence. SNP discovery using a cattle DNA panel revealed a number of polymorphisms that may be useful for the evaluation of ampkgamma1 as a candidate gene for energy metabolism-related production traits. 相似文献
62.
It is now well-established that compositional bias in DNA sequences can adversely affect phylogenetic analysis based on those
sequences. Phylogenetic analyses based on protein sequences are generally considered to be more reliable than those derived
from the corresponding DNA sequences because it is believed that the use of encoded protein sequences circumvents the problems
caused by nucleotide compositional biases in the DNA sequences. There exists, however, a correlation between AT/GC bias at
the nucleotide level and content of AT- and GC-rich codons and their corresponding amino acids. Consequently, protein sequences
can also be affected secondarily by nucleotide compositional bias. Here, we report that DNA bias not only may affect phylogenetic
analysis based on DNA sequences, but also drives a protein bias which may affect analyses based on protein sequences. We present
a striking example where common phylogenetic tools fail to recover the correct tree from complete animal mitochondrial protein-coding
sequences. The data set is very extensive, containing several thousand sites per sequence, and the incorrect phylogenetic
trees are statistically very well supported. Additionally, neither the use of the LogDet/paralinear transform nor removal
of positions in the protein alignment with AT- or GC-rich codons allowed recovery of the correct tree. Two taxa with a large
compositional bias continually group together in these analyses, despite a lack of close biological relatedness. We conclude
that even protein-based phylogenetic trees may be misleading, and we advise caution in phylogenetic reconstruction using protein
sequences, especially those that are compositionally biased.
Received: 19 February 1998 / Accepted: 28 August 1998 相似文献
63.
64.
Aerosol delivery to the airways of the human respiratory tract, followed by absorption, constitutes an alternative route of administration for compounds unsuitable for delivery by conventional oral and parenteral routes. The target for aerosol drug delivery is the airways epithelium, i.e. tracheal, bronchial, bronchiolar and alveolar cells, which become the site of drug deposition. These epithelial layers also serve as a barrier to the penetration of inhaled material. An in vitro model for aerosol deposition and transport across epithelia in the human airways may be a good predictor of in vivo disposition. The present preliminary studies begin an investigation that blends the dynamics of aerosol delivery and the basis of an in vitro simulated lung model to evaluate the transport properties of a series of molecular weight marker compounds across human-derived bronchiolar epithelial cell monolayers. An Andersen viable cascade impactor was used as a delivery apparatus for the deposition of size-segregated particles onto monolayers of small airway epithelial cells and Calu-3 cells. It was shown that these cell layers can withstand placement in the impactor, and that permeability can be tested subsequent to removal from the impactor. 相似文献
65.
The purpose of this study was to evaluate the hypothesis that spacer devices have limited effect on the in vitro fine particle
dose emitted from solution metered dose inhalers containing different proportions of HFA134a [1,1,1,2-tetrafluoroethane] propellant.
Two solution formulations (80% and 97.5% wt/wt HFA134a) were tested across the actuator alone, actuator plus Aerochamber,
and Ace holding chamber. Particle size distributions were determined using laser diffraction (LD) and cascade impaction (CI).
Multimodal particle size distributions were identified using LD. CI analyses were characterized by a major mode located at
≈0.5 μm. The fine particle dose emitted from the inhaler spacer combinations containing 97.5% HFA134a was independent of the
device setup used. Fine particle doses were influenced by spacer setup in 80% HFA134a formulations, indicating different plume
dynamics of low vapor pressure formulations. Sampling inlet deposition was approximately O when spacer devices were used with
either formulation. When spacers were not used, sampling inlet deposition was increased significantly. However, inlet deposition
with the 97.5% HFA134a formulation was significantly less than that of the 80% HFA134a formulation (≈25% of emitted dose compared
with 69% respectively). Thus, high propellant concentration formulations appear to have more robust in vitro performance.
This is particularly important given the preponderance of poor patient compliance that is associated with spacer use. High
propellant concentrations had the advantage of fine particle doses that were independent of the device setup and significantly
lowered sampling inlet deposition when no spacer was used. 相似文献
66.
Critical role of the alpha 4 integrin/VCAM-1 pathway in cerebral leukocyte trafficking in lupus-prone MRL/fas(lpr) mice 总被引:1,自引:0,他引:1
MRL/fas(lpr) mice are affected by a systemic autoimmune disease that results in leukocyte recruitment to a wide range of vascular beds, including the cerebral microvasculature. The mechanisms responsible for the leukocyte trafficking to the brain in these animals are not known. Therefore, the aim of this study was to directly examine the cerebral microvasculature in MRL/fas(lpr) mice and determine the molecular mechanisms responsible for this leukocyte recruitment. Intravital microscopy was used to assess leukocyte-endothelial cell interactions (rolling, adhesion) in the pial microcirculation of MRL(+/+) (control) and MRL/fas(lpr) mice at 8, 12, and 16 wk of age. Leukocyte rolling and adhesion were rarely observed in MRL(+/+) mice of any age. MRL/fas(lpr) mice displayed similar results at 8 and 12 wk. However, at 16 wk, significant increases in leukocyte rolling and adhesion were observed in these mice. Histological analysis revealed that the interacting cells were exclusively mononuclear. Leukocyte rolling was reduced, but not eliminated in P-selectin(-/-)-MRL/fas(lpr) mice. However, leukocyte adhesion was not reduced in these mice, indicating that P-selectin-dependent rolling was not required for leukocyte recruitment to the cerebral vasculature in this model of systemic inflammation. E-selectin blockade also had no effect on leukocyte rolling. In contrast, blockade of either the alpha4 integrin or VCAM-1 eliminated P-selectin-independent leukocyte rolling. alpha4 Integrin blockade also significantly inhibited leukocyte adhesion. These studies demonstrate that the systemic inflammatory response that affects MRL/fas(lpr) mice results in leukocyte rolling and adhesion in the cerebral microcirculation, and that the alpha4 integrin/VCAM-1 pathway plays a central role in mediating these interactions. 相似文献
67.
Wells RM Lu J Hickey AJ Jeffs AG 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2001,130(3):339-347
The larval life of the spiny lobster Jasus edwardsii is one of the longest and most complex of any marine organism and is poorly understood due to the difficulty of studying cryptic, pelagic organisms. Hence, the capacity for active swimming in the phyllosoma, puerulus and juvenile stages and the use of possible metabolic fuel reserves was inferred from a number of enzyme activities, including citrate synthase, lactate dehydrogenase, and HOAD. High activities of CS and LDH in abdominal tissues of Stage 11 phyllosoma and pueruli are consistent with a capacity to commence active on-shore movement. The activities of LDH and HOAD showed positive allometry while CS was independent of body mass. The body mass dependence of LDH activity may reflect the developing ability of the lobster to initiate brief escape manoeuvres, and the scaling of HOAD reflects an increased use of lipid fuel reserves. Aerobic enzyme activities were higher in abdominal tissues than in cephalic tissues of pelagic pueruli, but high activities appear in the cephalic tissues of juveniles. These changes mirror a developmental shift in activity from pelagic oceanic swimming to a benthic existence on the seabed of the near shore. The low LDH activity in pueruli confirmed previous findings that they have limited feeding capacity, with carbohydrate contributing little towards the major energy reserves. The highest LDH activities occur in the abdominal muscles of juveniles and correlate with rapid tail-flicking escape behaviour. The activities of HOAD increased throughout development, and in the abdominal tissues of juveniles, may reflect lipid transformation and accumulation as an energy reserve. Enzyme activities, therefore, provide useful information concerning migratory behaviour that is presently unavailable from ecological studies. 相似文献
68.
Jiang HY Hickey RJ Abdel-Aziz W Tom TD Wills PW Liu J Malkas LH 《Journal of cellular biochemistry》2002,85(4):762-774
A discrete high molecular weight multiprotein complex containing DNA polymerase alpha has been identified by a native Western blotting technique. An enrichment of this complex was seen at each step in its purification. Further purification of this complex by ion-exchange chromatography indicates that the peak of DNA polymerase alpha activity co-purifies with the peak of in vitro SV40 DNA replication activity eluting from the column. The complex has a sedimentation coefficient of 18S in sucrose density gradients. We have designated this complex as the DNA synthesome. We further purified the DNA synthesome by electroeluting this complex from a native polyacrylamide gel. The eluted complex retains in vitro DNA synthetic activity, and by Western blot analysis, contains DNA polymerase delta, proliferating cell nuclear antigen, and replication protein A. Enzymatic analysis of the electroeluted DNA synthesome indicates that the synthesome contains topoisomerase I and II activities, and SDS-PAGE analysis of the electroeluted DNA synthesome revealed the presence of at least 25 major polypeptides with molecular weights ranging from 20 to 240 kDa. Taken together, our evidence suggests that the DNA synthesome may represent the minimal DNA replication unit of the human cell. 相似文献
69.
Fox H Hickey PC Fernández-Abalos JM Lunness P Read ND Doonan JH 《Molecular microbiology》2002,45(5):1219-1230
Mutation of bimG, the major protein phosphatase 1 gene in Aspergillus nidulans, causes multiple cell cycle and hyphal growth defects that are associated with overphosphorylation of subcellular components. We have used functional translational fusions with the green fluorescent protein (GFP) to show that BIMG has at least four discrete locations within growing hyphae. Three of these locations, the hyphal tip, the spindle pole body and the nucleus, correlate with previously known requirements for bimG(PP1) in mitosis and hyphal growth and are highly dynamic. BIMG-GFP in the hyphal tip seemed to be associated with the plasma membrane and formed a collar of fluorescence within the apical dome. The distribution of nuclear BIMG-GFP varied depending on nutritional conditions; on poor medium, it concentrated more in the nucleolus than in the nucleoplasm, whereas on rich medium, it was more evenly distributed between the two nuclear regions. The association of BIMG-GFP with developing septa was transient, and we present evidence that BIMG phosphatase plays a direct role in septum formation, distinct from its role in mitosis. We conclude that, by being physically present at several sites, the BIMG phosphatase has roles in multiple cellular processes. 相似文献
70.
The purpose of this study was to determine if aerosol delivery of drug loaded microparticles to lungs infected withMycobacterium tuberculosis may be achieved by predicting dispersion of dry powders through knowledge of particle surface properties. Particle sizes
of rifampicin-loaded poly(lactide-co-glycolide) microparticles (R-PLGA), rifampicin alone, and lactose and maltodextrin carrier
particles (bulk and 75-125-μm sieved fractions) were determined by electron microscopy for the projected area diameter (Dp) and laser diffraction for the volume diameter (Dv). Surface energies (Y) of R-PLGA, rifampicin alone, lactose, and maltodextrin were obtained by inverse phase gas chromatography,
surface areas (Sa) by N2 adsorption, and cohesive energy densities by calculation. Particle dispersion was evaluated (Andersen nonviable impactor)
for 10% blends of R-PLGA and rifampicin alone with bulk and sieved fractions of the carriers. Dp for R-PLGA and rifampicin alone was 3.02 and 2.83 μm, respectively. Dv was 13±1 and 2±1 μm for R-PLGA and rifampicin alone, respectively, indicating that R-PLGA was more aggregated. This was evident
in Y of 35±1 and 19±6 mJ/m2 for R-PLGA and rifampicin alone. Dp for lactose and maltodextrin (sieved and bulk) was approximately 40 mm. Bulk maltodextrin (Dv=119±6 mm) was more aggregated than bulk lactose (Dv=54±2 mm). This was a result of the higher Sa for maltodextrin (0.54 m2/g) than for lactose (0.21 m2/g). The Y of bulk lactose and maltodextrin was 40±4 and 60±6 mJ/m2 and of sieved lactose and maltodextrin was 39±1 and 50±1 mJ/m2. Impaction studies yielded higher fine particle fractions of R-PLGA from sieved lactose, 13%±3%, than from sieved maltodextrin,
7%±1%, at 90 L/min. An expression, based on these data, is proposed as a predictor of drug dispersion from carrier particles.
Delivery of dry powder formulations can be achieved by characterizing particle surfaces and predicting impact on dispersion. 相似文献