The strain variation and virulence of granulovirus of diamondback moth (Plutella xylostella Linnaeus, Lep., Yponomeutidae) isolated in Kenya.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
K A Jones
Publications and source records attributed to K A Jones.
Molecular modelling and site-directed mutagenesis of human GALR1 galanin receptor defines determinants of receptor subtype specificity.
Human galanin is a 30 amino acid neuropeptide that elicits a range of biological activities by interaction with G protein-coupled receptors. We have generated a model of the human GALR1 galanin receptor subtype (hGALR1) based on the alpha carbon maps of frog rhodopsin and investigated the significance of potential contact residues suggested by the model using site-directed mutagenesis. Mutation of Phe186 within the second extracellular loop to Ala resulted in a 6-fold decrease in affinity for galanin, representing a change in free energy consistent with hydrophobic interaction. Our model suggests interaction between Phe186 of hGALR1 and Ala7 or Leu11 of galanin. Receptor subtype specificity was investigated by replacement of residues in hGALR1 with the corresponding residues in hGALR2 and use of the hGALR2-specific ligands hGalanin(2-30) and [D-Trp2]hGalanin(1-30). The His267Ile mutant receptor exhibited a pharmacological profile corresponding to that of hGALR1, suggesting that His267 is not involved in a receptor-ligand interaction. The mutation Phe115Ala resulted in a decreased binding affinity for hGalanin and for hGALR2-specific analogues, indicating Phe115 to be of structural importance to the ligand binding pocket of hGALR1 but not involved in direct ligand interaction. Analysis of Glu271Trp suggested that Glu271 of hGALR1 interacts with the N-terminus of galanin and that the Trp residue in the corresponding position in hGALR2 is involved in receptor subtype specificity of binding. Our model supports previous reports of Phe282 of hGALR1 interacting with Trp2 of galanin and His264 of hGALR1 interacting with Tyr9 of galanin.
Amino Acid Substitution↗
Chromatin-specific regulation of LEF-1-beta-catenin transcription activation and inhibition in vitro.
Transcriptional activation of Wnt/Wg-responsive genes requires the stabilization and nuclear accumulation of beta-catenin, a dedicated coactivator of LEF/TCF enhancer-binding proteins. Here we report that recombinant beta-catenin strongly enhances binding and transactivation by LEF-1 on chromatin templates in vitro. Interestingly, different LEF-1 isoforms vary in their ability to bind nucleosomal templates in the absence of beta-catenin, owing to N-terminal residues that repress binding to chromatin, but not nonchromatin, templates. Transcriptional activation in vitro requires both the armadillo (ARM) repeats and the C terminus of beta-catenin, whereas the phosphorylated N terminus is inhibitory to transcription. A fragment spanning the C terminus (CT) and ARM repeats 11 and 12 (CT-ARM), but not the CT alone, functions as a dominant negative inhibitor of LEF-1-beta-cat activity in vitro and can block ATP-dependent binding of the complex to chromatin. LEF-1-beta-cat transactivation in vitro was also repressed by inhibitor of beta-catenin and Tcf-4 (ICAT), a physiological inhibitor of Wnt/Wg signaling that interacts with ARM repeats 11 and 12, and by the nonsteroidal anti-inflammatory compound, sulindac. None of these transcription inhibitors (CT-ARM, ICAT, or sulindac) could disrupt the LEF-1-beta-cat complex after it was stably bound to chromatin. We conclude that the CT-ARM region of beta-catenin functions as a chromatin-specific activation domain, and that several inhibitors of the Wnt/Wg pathway directly modulate LEF-1-beta-cat activity on chromatin.
Amino Acid Sequence↗
Trace amines: identification of a family of mammalian G protein-coupled receptors.
Tyramine, beta-phenylethylamine, tryptamine, and octopamine are biogenic amines present in trace levels in mammalian nervous systems. Although some "trace amines" have clearly defined roles as neurotransmitters in invertebrates, the extent to which they function as true neurotransmitters in vertebrates has remained speculative. Using a degenerate PCR approach, we have identified 15 G protein-coupled receptors (GPCR) from human and rodent tissues. Together with the orphan receptor PNR, these receptors form a subfamily of rhodopsin GPCRs distinct from, but related to the classical biogenic amine receptors. We have demonstrated that two of these receptors bind and/or are activated by trace amines. The cloning of mammalian GPCRs for trace amines supports a role for trace amines as neurotransmitters in vertebrates. Three of the four human receptors from this family are present in the amygdala, possibly linking trace amine receptors to affective disorders. The identification of this family of receptors should rekindle the investigation of the roles of trace amines in mammalian nervous systems and may potentially lead to the development of novel therapeutics for a variety of indications.
Amino Acid Sequence↗
Comparison of two murine models of familial hypertrophic cardiomyopathy.
Although sarcomere protein gene mutations cause familial hypertrophic cardiomyopathy (FHC), individuals bearing a mutant cardiac myosin binding protein C (MyBP-C) gene usually have a better prognosis than individuals bearing beta-cardiac myosin heavy chain (MHC) gene mutations. Heterozygous mice bearing a cardiac MHC missense mutation (alphaMHC(403/+) or a cardiac MyBP-C mutation (MyBP-C(t/+)) were constructed as murine FHC models using homologous recombination in embryonic stem cells. We have compared cardiac structure and function of these mouse strains by several methods to further define mechanisms that determine the severity of FHC. Both strains demonstrated progressive left ventricular (LV) hypertrophy; however, by age 30 weeks, alphaMHC(403/+) mice demonstrated considerably more LV hypertrophy than MyBP-C(t/+) mice. In older heterozygous mice, hypertrophy continued to be more severe in the alphaMHC(403/+) mice than in the MyBP-C(t/+) mice. Consistent with this finding, hearts from 50-week-old alphaMHC(403/+) mice demonstrated increased expression of molecular markers of cardiac hypertrophy, but MyBP-C(t/+) hearts did not demonstrate expression of these molecular markers until the mice were >125 weeks old. Electrophysiological evaluation indicated that MyBP-C(t/+) mice are not as likely to have inducible ventricular tachycardia as alphaMHC(403/+) mice. In addition, cardiac function of alphaMHC(403/+) mice is significantly impaired before the development of LV hypertrophy, whereas cardiac function of MyBP-C(t/+) mice is not impaired even after the development of cardiac hypertrophy. Because these murine FHC models mimic their human counterparts, we propose that similar murine models will be useful for predicting the clinical consequences of other FHC-causing mutations. These data suggest that both electrophysiological and cardiac function studies may enable more definitive risk stratification in FHC patients.
Actins↗
Prediction of fracture location in the proximal femur using finite element models.
Finite element (FE) models of the proximal femur are often used to study hip fracture. To interpret the results of these models, it is important to know whether the models accurately predict fracture location and/or type. This study evaluated the ability of automatically generated, CT scan-based linear FE models of the proximal femur to predict fracture location and fracture type. Fracture location was defined as the specific location of the fracture. Fracture type was a categorical variable defined as either a cervical or a trochanteric fracture. FE modeling and mechanical testing of 18 pairs of human femora were performed under two loading conditions, one similar to joint loading during single-limb stance and one simulating impact from a fall. For the stance condition, the predicted and actual fracture locations agreed in 13 of the 18 cases (72% agreement). For the fall condition, the predicted and actual fracture locations agreed in 10 of the 15 cases where the actual fractures could be identified (67% agreement). The FE models correctly predicted that only cervical fractures occurred in the stance configuration. For the fall configuration, FE-predicted and actual fracture types agreed in 11 of the 14 cases that could be compared (9 trochanteric, 2 cervical; 79% agreement). These results provide evidence that CT scan-based FE models of the proximal femur can predict fracture location and fracture type with moderate accuracy.
Aged↗
The effects of ethanol on CA(2+) sensitivity in airway smooth muscle.
UNLABELLED: Halothane and other volatile anesthetics relax air-way smooth muscle (ASM) in part by decreasing the amount of force produced for a given intracellular Ca(2+) concentration (the Ca(2+) sensitivity) during muscarinic receptor stimulation. To determine whether this is a unique property of the volatile anesthetics, we tested the hypothesis that ethanol, another compound with anesthetic properties, also inhibits calcium sensitization induced by muscarinic stimulation of ASM. A beta-escin permeabilized canine tracheal smooth muscle preparation was used. Ethanol was applied to permeabilized muscles stimulated with calcium in either the absence or presence of acetylcholine. In intact ASM, ethanol produced incomplete relaxation (approximately 40%) at concentrations up to 300 mM. Ethanol significantly increased Ca(2+) sensitivity both in the presence and the absence of muscarinic receptor stimulation. Although ethanol did not affect regulatory myosin light chain (rMLC) phosphorylation during stimulation with Ca(2+) alone, it decreased rMLC phosphorylation by Ca(2+) during muscarinic receptor stimulation. Ethanol, like volatile anesthetics, inhibits increases in rMLC phosphorylation produced by muscarinic receptor stimulation at constant [Ca(2+)](i). However, despite this inhibition, the net effect of ethanol is to increase Ca(2+) sensitivity (defined as the force maintained for a given [Ca(2+)](i)) by a mechanism that is independent of changes in rMLC phosphorylation. IMPLICATIONS: In permeabilized airway smooth muscle, ethanol, like volatile anesthetics, inhibits increases in regulatory protein phosphorylation caused by stimulation of the muscle when intracellular calcium concentration is constant. However, unlike volatile anesthetics, ethanol causes a net increase in force through a process not dependent on protein phosphorylation, an action favoring bronchoconstriction.
Animals↗
Halothane increases smooth muscle protein phosphatase in airway smooth muscle.
BACKGROUND: Halothane relaxes airway smooth muscle, in part, by decreasing the force produced for a given intracellular [Ca(2+)] (i.e., Ca(2+) sensitivity) during muscarinic stimulation, an effect produced by a decrease in regulatory myosin light-chain (rMLC) phosphorylation. The authors tested the hypothesis that halothane reduces rMLC phosphorylation during muscarinic stimulation at constant intracellular [Ca(2+)] by increasing smooth muscle protein phosphatase (SMPP) activity, without changing myosin light-chain kinase (MLCK) activity. METHODS: Enzyme activities were assayed in beta-escin permeabilized strips of canine tracheal smooth muscle. Under conditions of constant intracellular [Ca(2+)], the rate of rMLC phosphorylation was measured by Western blotting during inhibition of SMPP with microcystin-LR (to assay MLCK activity) or during inhibition of MLCK by wortmannin and adenosine triphosphate depletion (to assay SMPP activity). The effect of halothane (0.8 mm) on enzyme activities and isometric force during stimulation with 0.6 microm Ca(2+) and 10 microm acetylcholine was determined. RESULTS: Halothane produced a 14 +/- 8% (mean +/- SD) decrease in isometric force by significantly reducing rMLC phosphorylation (from 32 +/- 9% to 28 +/- 9%). Halothane had no significant effect on any parameter of a monoexponential relation fit to the data for the MLCK activity assay. In contrast, halothane significantly decreased the half-time for rMLC dephosphorylation in the SMPP activity assay (from 0.74 +/- 0.28 min to 0.44 +/- 0.10 min), indicating that it increased SMPP activity. CONCLUSIONS: Halothane decreases Ca(2+) sensitivity and rMLC phosphorylation in airway smooth muscle during muscarinic receptor stimulation by increasing SMPP activity, without affecting MLCK, probably by disrupting receptor G-protein signaling pathways that inhibit SMPP.
Anesthetics, Inhalation↗
Calcium sensitization produced by G protein activation in airway smooth muscle.
We determined whether activation of G proteins can affect the force developed for a given intracellular Ca(2+) concentration ([Ca(2+)]; i.e., the Ca(2+) sensitivity) by mechanisms in addition to changes in regulatory myosin light chain (rMLC) phosphorylation. Responses in alpha-toxin-permeabilized canine tracheal smooth muscle were determined with Ca(2+) alone or in the presence of ACh, endothelin-1 (ET-1), or aluminum fluoride (AlF; acute or 1-h exposure). Acute exposure to each compound increased Ca(2+) sensitivity without changing the response to high [Ca(2+)] (maximal force). However, chronic exposure to AlF, but not to chronic ACh or ET-1, increased maximal force by increasing the force produced for a given rMLC phosphorylation. Studies employing thiophosphorylation of rMLC showed that the increase in force produced by chronic AlF exposure required Ca(2+) during activation to be manifest. Unlike the acute response to receptor agonists, which is mediated solely by increases in rMLC phosphorylation, chronic direct activation of G proteins further increases Ca(2+) sensitivity in airways by additional mechanisms that are independent of rMLC phosphorylation.
Acetylcholine↗
A neural network predicts progression for men with gleason score 3+4 versus 4+3 tumors after radical prostatectomy.
OBJECTIVES: To determine the significance of Gleason scores 3+4 (GS3+4) versus 4+3 (GS4+3) with respect to biochemical recurrence in a retrospective review of a series of men with clinically localized prostate cancer who underwent radical retropubic prostatectomy (RRP) and to develop and test an artificial neural network (ANN) to predict the biochemical recurrence after surgery for this group of men using the pathologic and clinical data. METHODS: From 1982 to 1998, 600 men had pathologic Gleason score 7 disease without lymph node or seminal vesicle involvement. We analyzed the freedom from biochemical (prostate-specific antigen) progression after RRP on 564 of these men on the basis of their GS3+4 versus GS4+3 (Gleason 7) status. The Cox proportional hazards model was used to determine the importance of Gleason 7 status as an independent predictor of progression. In addition, an ANN was developed using randomly selected training and validation sets for predicting biochemical recurrence at 3 or 5 years. Different input variable subsets, with or without Gleason 7 status, were compared for the ability of the ANN to maximize the prediction of progression. Standard logistic regression was used concurrently on the same random patient population sets to calculate progression risk. RESULTS: A significant recurrence-free survival advantage was found in men who underwent RRP for GS3+4 compared with those with GS4+3 disease (P <0.0001). The ANN, logistic regression, and proportion hazard models demonstrated the importance of Gleason 7 status in predicting patient outcome. The ANN was better than logistic regression in predicting patient outcome, in terms of prostate-specific antigen progression, at 3 and 5 years. CONCLUSIONS: A simple modification of the Gleason scoring system for men with Gleason 7 disease revealed a difference in the patient outcome after RRP. ANN models can be developed and used to better predict patient outcome when pathologic and clinical features are known.
Disease Progression↗
Identification and characterization of two G protein-coupled receptors for neuropeptide FF.
The central nervous system octapeptide, neuropeptide FF (NPFF), is believed to play a role in pain modulation and opiate tolerance. Two G protein-coupled receptors, NPFF1 and NPFF2, were isolated from human and rat central nervous system tissues. NPFF specifically bound to NPFF1 (K(d) = 1.13 nm) and NPFF2 (K(d) = 0.37 nm), and both receptors were activated by NPFF in a variety of heterologous expression systems. The localization of mRNA and binding sites of these receptors in the dorsal horn of the spinal cord, the lateral hypothalamus, the spinal trigeminal nuclei, and the thalamic nuclei supports a role for NPFF in pain modulation. Among the receptors with the highest amino acid sequence homology to NPFF1 and NPFF2 are members of the orexin, NPY, and cholecystokinin families, which have been implicated in feeding. These similarities together with the finding that BIBP3226, an anorexigenic Y1 receptor ligand, also binds to NPFF1 suggest a potential role for NPFF1 in feeding. The identification of NPFF1 and NPFF2 will help delineate their roles in these and other physiological functions.
Amino Acid Sequence↗
HIV-1 TAR RNA enhances the interaction between Tat and cyclin T1.
Human immunodeficiency virus, type 1 (HIV-1), Tat activates elongation of RNA polymerase II transcription at the HIV-1 promoter through interaction with the cyclin T1 (CycT1) subunit of the positive transcription elongation factor complex, P-TEFb. Binding of Tat to CycT1 induces cooperative binding of the P-TEFb complex onto nascent HIV-1 TAR RNA. Here the specific interaction between Tat protein, human cyclin T1, and HIV-1 TAR RNA was analyzed by fluorescence resonance energy transfer, using fluorescein-labeled TAR RNA and a rhodamine-labeled Tat protein synthesized through solid-phase chemistry. We find that CycT1 remodels the structure of Tat to enhance its affinity for TAR RNA and that TAR RNA further enhances the interaction between Tat and CycT1. We conclude that TAR RNA nucleates the formation of the Tat.P-TEFb complex through an induced fit mechanism.
Base Sequence↗
Functional selectivity of recombinant mammalian SWI/SNF subunits.
The SWI/SNF family of chromatin-remodeling complexes plays a key role in facilitating the binding of specific transcription factors to nucleosomal DNA in diverse organisms from yeast to man. Yet the process by which SWI/SNF and other chromatin-remodeling complexes activate specific subsets of genes is poorly understood. We show that mammalian SWI/SNF regulates transcription from chromatin-assembled genes in a factor-specific manner in vitro. The DNA-binding domains (DBDs) of several zinc finger proteins, including EKLF, interact directly with SWI/SNF to generate DNase I hypersensitivity within the chromatin-assembled beta-globin promoter. Interestingly, we find that two SWI/SNF subunits (BRG1 and BAF155) are necessary and sufficient for targeted chromatin remodeling and transcriptional activation by EKLF in vitro. Remodeling is achieved with only the BRG1-BAF155 minimal complex and the EKLF zinc finger DBD, whereas transcription requires, in addition, an activation domain. In contrast, the BRG1-BAF155 complex does not interact or function with two unrelated transcription factors, TFE3 and NF-kappaB. We conclude that specific domains of certain transcription factors differentially target SWI/SNF complexes to chromatin in a gene-selective manner and that individual SWI/SNF subunits play unique roles in transcription factor-directed nucleosome remodeling.
Adenosine Triphosphatases↗
Exploring the transcription-chromatin interface.
Explore the source record for details and available documents.
Adenosine Triphosphate↗
CK-MB isoforms for early risk stratification of emergency department patients.
The potential clinical utility of single sample CK-MB isoforms measurement for early risk stratification of Emergency Department (ED) patients with possible myocardial ischemia was evaluated among 405 patients presenting to two urban EDs. Clinical and serologic data were prospectively collected and the occurrence of adverse events (AEs) and myocardial infarction (MI) during the 14-day outcome period was recorded and utilized to calculate and compare relative risks (RR) and predictive values of isoforms and CK-MB alone. Among the 405 patients, 67 accrued 105 AEs. Both isoforms and CK-MB alone were predictive of AEs with RR of 3.32 (2.09, 5.27) and 6.28 (4.64, 8.52), respectively. Isoforms had higher sensitivity for AEs compared to CK-MB (65.7% [54.3, 77.0] vs. 14.9% [6.4, 23.5]; p<0. 01) but lower specificity (69.2% [64.3, 74.2] vs. 99.7% [99.1,100. 0]; p<0.01). Isoforms' superior sensitivity allowed identification of many high risk patients missed by CK-MB alone. Further, for the prediction of MI, isoforms had superior diagnostic sensitivity and equivalent specificity. This investigation supports the emergency department use of early, single sample CK-MB isoform testing.
Adult↗
Signal transduction by GABA(B) receptor heterodimers.
GABA(B) receptors are G-protein-coupled receptors that mediate inhibition throughout the central and peripheral nervous systems. A single cloned receptor, GABA(B)R1, which has at least three alternatively spliced forms, appears to account for the vast majority of binding sites in the brain for high-affinity antagonists. In heterologous expression systems GABA(B)R1 is poorly expressed on the plasma membrane and largely fails to couple to ion channels. A second gene, GABA(B)R2, which exhibits moderately low homology to GABA(B)R1, permits surface expression of GABA(B)R1 and the appearance of baclofen-sensitive K(+) and Ca(+1) currents. We review the data that supports a model of the native GABA(B) receptor as a heterodimer composed of GABA(B)R1 and GABA(B)R2 proteins. New data from mutagenesis experiments are presented that point to amino acid residues on GABA(B)R1 critical for ligand activation of the heterodimer. The possible role of GABA(B)R2 in signal transduction is also discussed. The interdependent nature of the two subunits for receptor function makes the GABA(B) receptor a useful model to explore the larger significance of GPCR dimerization for G-protein activation.
Amino Acid Sequence↗
Effects of intravenous anesthetics on Ca2+ sensitivity in canine tracheal smooth muscle.
BACKGROUND: Halothane and other volatile anesthetics relax airway smooth muscle in part by decreasing the amount of force produced for a particular intracellular calcium concentration (the Ca2+ sensitivity) during muscarinic receptor stimulation. In this study, ketamine, propofol, and midazolam were evaluated to determine whether the inhibitory effect of volatile anesthetics on this signal transduction pathway is a general property of other types of anesthetic drugs. METHODS: A beta-escin permeabilized canine tracheal smooth muscle preparation was used. Ketamine, propofol, and midazolam, in concentrations producing near-maximal relaxation in intact airway smooth muscle (200 microM, 270 microM, and 100 microM, respectively), were applied to permeabilized muscles stimulated with calcium in either the absence or the presence of muscarinic receptor stimulation provided by acetylcholine. The effect of halothane also was evaluated. RESULTS: Confirming previous studies, halothane (0.75 mM) decreased calcium sensitivity during muscarinic receptor stimulation. None of the intravenous anesthetics studied affected Ca2+ sensitivity, either in the absence or the presence of muscarinic receptor stimulation. CONCLUSIONS: Intravenous anesthetics in high concentrations directly relax canine tracheal smooth muscle without affecting Ca2+ sensitivity. The inhibition of agonist-induced increases in Ca2+ sensitivity of canine tracheal smooth is not a common property of anesthetics, but is unique to volatile agents.
Anesthetics, Inhalation↗
CDK9 autophosphorylation regulates high-affinity binding of the human immunodeficiency virus type 1 tat-P-TEFb complex to TAR RNA.
Human immunodeficiency virus type 1 (HIV-1) Tat interacts with cyclin T1 (CycT1), a regulatory partner of CDK9 in the positive transcription elongation factor (P-TEFb) complex, and binds cooperatively with CycT1 to TAR RNA to recruit P-TEFb and promote transcription elongation. We show here that Tat also stimulates phosphorylation of affinity-purified core RNA polymerase II and glutathione S-transferase-C-terminal-domain substrates by CycT1-CDK9, but not CycH-CDK7, in vitro. Interestingly, incubation of recombinant Tat-P-TEFb complexes with ATP enhanced binding to TAR RNA dramatically, and the C-terminal half of CycT1 masked binding of Tat to TAR RNA in the absence of ATP. ATP incubation lead to autophosphorylation of CDK9 at multiple C-terminal Ser and Thr residues, and full-length CycT1 (amino acids 728) [CycT1(1-728)], but not truncated CycT1(1-303), was also phosphorylated by CDK9. P-TEFb complexes containing a catalytically inactive CDK9 mutant (D167N) bound TAR RNA weakly and independently of ATP, as did a C-terminal truncated CDK9 mutant that was catalytically active but unable to undergo autophosphorylation. Analysis of different Tat proteins revealed that the 101-amino-acid SF2 HIV-1 Tat was unable to bind TAR with CycT1(1-303) in the absence of phosphorylated CDK9, whereas unphosphorylated CDK9 strongly blocked binding of HIV-2 Tat to TAR RNA in a manner that was reversed upon autophosphorylation. Replacement of CDK9 phosphorylation sites with negatively charged residues restored binding of CycT1(1-303)-D167N-Tat, and rendered D167N a more potent inhibitor of transcription in vitro. Taken together, these results demonstrate that CDK9 phosphorylation is required for high-affinity binding of Tat-P-TEFb to TAR RNA and that the state of P-TEFb phosphorylation may regulate Tat transactivation in vivo.
Adenosine Triphosphate↗