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Query: UNIPROT:P06889 (Mol)
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1. The effect of breathing an anaesthetic aerosol of 5% bupivacaine hydrochloride has been assessed in dog and man. 2. In the dog, the cough reflex was abolished and the Hering-Breuer inflation reflex severely impaired or abolished; breathing became slower and deeper; no pathological changes were found in the lungs of these dogs. 3. In man, no untoward effects resulted from a 10 min period of aerosol inhalation; there were no systematic effects on airway resistance or lung volumes and the cough reflex in response to either tactile or chemical (citric acid aerosol) stimulation was invariably abolished. The Hering-Breuer inflation reflex was impaired, but this was not associated with any change in resting ventilation. The Ve/CO2 response was enhanced after aerosol anaesthesia; subjects felt an exaggerated dyspnoea. The aerosol anaesthesia abolished the afferent pathway of a reflexly elicited bronchoconstriction in one subject. There was no effect on the ability to hold the breath, or on the quality of the associated sensation. 4. Control aerosols of sodium chloride solution or phosphate buffer produced no effects. Control experiments with intravenous infusions of bupivacaine proved that none of the effects could have been produced by systemic effects of the absorbed anaesthetic. 5. Plasma concentrations of bupivacaine in man did not exceed a recognized toxic level. The experiments demonstrate a safe reversible anaesthesia of the airways in man lasting for a period of 10-20 min.
Clin Sci Mol Med 1976 Jun
PMID:The effect of anaesthesia of the airway in dog and man: a study of respiratory reflexes, sensations and lung mechanics. 127 53

To define the heparin-binding site of follistatin, the reduced and S-carboxymethylated recombinant human follistatin containing 288 amino acids was digested by Staphylococcus aureus V8. The digested product was subjected to sulfate cellufine column chromatography and the adsorbed peptide fragments eluted with a stepwise gradient of sodium chloride. The recovered column fractions were further purified by reversed-phase high-performance liquid chromatography (HPLC) and the HPLC peaks subjected to amino-terminal sequence analysis. All of the sulfate cellufine-retarded peptide fragments gave the same N-terminal amino acid sequence, which started at residue-68 of human follistatin, suggested that those fragments starting from residue-68 contain the heparin binding site. The multiple fragments might represent the oxidized, non-glycosylated or glycosylated forms of follistatin(68-113) resulting from the V8 digestion. A synthetic peptide corresponding to the region having the amino acid sequence 72-86 of follistatin was able to bind both heparin and sulfate cellufine, as well as compete with recombinant follistatin for binding to heparin. These findings further define the location of the heparin and heparan sulfate-binding site of follistatin at the basic amino acid-rich region comprising the amino acid sequence Lys75-Lys-Cys-Arg-Met-Asn-Lys-Lys-Asn-Lys-Pro-Arg86.
Mol Cell Endocrinol 1992 Dec
PMID:Localization of the heparin binding site of follistatin. 130 90

The presence of atrial natriuretic peptide (ANP) and the nature of its binding sites were studied in fresh-water (FW)- and seawater (SW)-adapted eels using a heterologous analogue, that of the rat (rANP). Rat ANP-like immunoreactivity was demonstrated in the cardiac atria and ventricles of both FW and SW eels, and electron-dense ANP-like granules were observed. The atria and ventricles of FW eels contained significantly more granules than those of SW animals and, in both types, the atria were more granular than the ventricles. Specific binding sites for rANP were demonstrated by displacement and uptake experiments using labelled rANP in dispersed eel branchial cell preparations, enriched in chloride cells. The concentration of rANP required to produce a 50% inhibition of binding in FW cells was significantly lower than that in SW cells. Scatchard analyses revealed the presence of two classes of binding site in SW eel branchial cells but only a single class of receptor in FW cells. The affinity of the FW receptor was not significantly different from that of the SW high affinity site. Rat ANP stimulated the production of cyclic GMP (cGMP) in a dose-dependent manner, and both basal and stimulated levels of cGMP were significantly greater in SW branchial cells. These studies suggest that ANP is involved in the adaptation of the euryhaline eel to differing environmental salinities; the levels of the peptide in the heart alter with changing salinity, and the nature of the receptors in the sodium chloride-transporting epithelium of the gill changes in response to the need either to eliminate or to absorb sodium chloride.
J Mol Endocrinol 1992 Oct
PMID:Atrial natriuretic peptide in the eel, Anguilla anguilla L.: its cardiac distribution, receptors and actions on isolated branchial cells. 132 3

To facilitate efficient allelic exchange of genetic information into a wild-type strain background, we improved upon and merged approaches using a temperature-sensitive plasmid and a counter-selectable marker in the chromosome. We first constructed intermediate strains of Escherichia coli K12 in which we replaced wild-type chromosomal sequences, at either the fimB-A or lacZ-A loci, with a newly constituted DNA cassette. The cassette consists of the sacB gene from Bacillus subtilis and the neomycin (kanamycin) resistance gene of Tn5, but, unlike another similar cassette, it lacks IS1 sequences. We found that sucrose sensitivity was highly dependent on incubation temperature and sodium chloride concentration. The DNA to be exchanged into the chromosome was first cloned into derivatives of plasmid pMAK705, a temperature-sensitive pSC101 replicon. The exchanges were carried out in two steps, first selecting for plasmid integration by standard techniques. In the second step, we grew the plasmid integrates under non-selective conditions at 42 degrees C, and then in the presence of sucrose at 30 degrees C, allowing positive selection for both plasmid excision and curing. Despite marked locus-specific strain differences in sucrose sensitivity and in the growth retardation due to the integrated plasmids, the protocol permitted highly efficient exchange of cloned DNA into either the fim or lac chromosomal loci. This procedure should allow the exchange of any DNA segment, in addition to the original or mutant allelic DNA, into any non-essential parts of the E. coli chromosome.
Mol Microbiol 1991 Jun
PMID:Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon. 168 93

Human sperm nuclei were isolated with mixed alkyltrimethylammonium bromide and dithiothreitol (MATAB/DTT) and decondensed by treatments with lithium diiodosalicylate (LIS), sodium chloride, or Tris salts. Concentrations as low as 1 mM LIS induced measurable nuclear swelling compared to 600 mM required for the other two salts. As measured by image analyses, the projected nuclear area increased linearly up to approximately fivefold with LIS concentrations up to 10 mM. Swollen nuclei also maintained the elliptical shapes characteristic of the human sperm head. Expanded sperm nuclei of three men were hybridized with a fluorescently labeled 3.4 kb Y chromosome-specific repetitive DNA probe; 50.1% of the nuclei of each semen sample showed fluorescent labeling over a part of the nucleus indicating presence of the Y chromosome. In comparison, unswollen sperm did not yield reliable hybridization signals. This procedure is suitable for determining the proportion of human sperm with Y chromosomes and can be used to evaluate sperm separation techniques. The availability of probes specific for most human chromosomes suggests that this procedure may find general application in studies of sperm chromosomal constitution.
Mol Reprod Dev 1990 Nov
PMID:Fluorescence in situ hybridization to Y chromosomes in decondensed human sperm nuclei. 207 35

1. The measurement of cellular mRNA content by quantitative in situ hybridization is a valuable approach to the study of gene expression in brain since this tissue exhibits a high degree of phenotypic heterogeneity. 2. The cellular content of vasopressin and oxytocin mRNA in hypothalamo-neurohypophysial system neurons was altered by maintaining rats for 24 hr on 2% sodium chloride water. 3. Statistical and graphical techniques were then used to analyze cell by cell how mRNA levels were altered as a result of osmotic stimulation. We propose that the negative binomial probability distribution is a suitable model to describe how mRNA content varies across a defined cell population. For both measures of oxytocin and vasopressin mRNA levels, maximum-likelihood estimation indicated that this model adequately described empirical findings obtained from rats drinking tap water or salt water. 4. Both graphical and statistical analyses suggested how the defined neural system responds to osmotic stimulation: mRNA content was altered as a multiplicative function of "initial state." The utility and limitations of the quantitative approach are discussed.
Cell Mol Neurobiol 1990 Mar
PMID:Quantitative in situ hybridization to measure single-cell changes in vasopressin and oxytocin mRNA levels after osmotic stimulation. 233 46

A hair seeding technique has been developed to obtain diffraction quality crystals of yeast (Saccharomyces cerevisiae) iso-2-cytochrome c, a model for studies of protein folding and biological electron transfer reactions. Deep red crystals of this protein were obtained from 88 to 92% saturated solutions of ammonium sulfate containing 20 mg protein/ml, 0.1 M-sodium phoshate, 0.3 M-sodium chloride, 0.04 M-dithiothreitol and adjusted to phosphate, 0.3 M-sodium chloride, 0.04 M-dithiothreitol and adjusted to pH 6.0. Rapid crystal growth was observed, but only along the path of the seeding hair stroke. The space group is P4(3)2(1)2 (or P4(1)2(1)2) with a = b = 36.4 A, c = 137.8 A (1 A = 0.1 nm) and Z = 8. Crystals are stable in the X-ray beam for more than 10 days and diffract to at least 2.5 A resolution. The same hair seeding methodology has proven useful in obtaining crystals of specifically designed mutant iso-2 proteins and in other protein systems where consistent crystal growth had previously proven difficult to attain.
J Mol Biol 1989 Apr 20
PMID:Crystallization of yeast iso-2-cytochrome c using a novel hair seeding technique. 254 32

Escherichia coli glycerol kinase, a major regulatory enzyme which catalyzes the reversible MgATP-dependent phosphorylation of glycerol has been crystallized by the hanging drop vapor diffusion method at room temperature. Three different crystal forms have been obtained in the presence of glycerol and appear to be suitable for X-ray crystallographic studies. Vapor diffusion against 55% ammonium sulfate and 1% beta-octyl glucoside (pH 7.0) yields rhombohedral crystals with space group R32, a = b = 277.1 A, c = 78.7 A (hexagonal indexing) containing a dimer of Mr 112,000 in the asymmetric unit (Vm = 2.64 A3/dalton). Vapor diffusion against sodium chloride in the presence of 10% (w/v) polyethylene glycol (pH 6.5 to 7.0) yields two different crystal forms, both with space group P2(1). The first form has a = 88.1 A, b = 99.3 A, c = 114.6 A, beta = 119 degrees, the second form has a = 92.5 A, b = 117.6 A, c = 108.3 A, beta = 93.64 degrees. Addition of ADP enhances growth of the monoclinic forms. These forms appear to contain an entire tetramer of Mr 224,000 in the asymmetric unit and have Vm values of 2.28 and 2.65 A3/dalton, respectively. All forms diffract to better than 3.0 A resolution while the second monoclinic form diffracts to approximately 1.8 A.
J Mol Biol 1989 Jun 05
PMID:Crystallization and preliminary X-ray studies of Escherichia coli glycerol kinase. 254 69

Crystals of alcohol oxidase purified from Pichia pastoris were grown in microdialysis buttons in a solution of polyethylene glycol, sodium chloride and sodium azide. The crystals were stratified along the major axis and up to 3 mm in length. X-ray diffraction experiments indicated a space group of P2(1) and unit cell dimensions of a = 157.3 A, b = 171.5 A and c = 231.6 A. Crystals diffract to beyond 2.7 A and are suitable for X-ray structure analysis.
J Mol Biol 1989 Jul 05
PMID:Crystallization of alcohol oxidase from Pichia pastoris. 267 87

Single crystals of glycosylated recombinant human renin have been obtained using the hanging-drop vapor diffusion method with polyethylene glycol and sodium chloride as coprecipitants. The crystals belong to the cubic space group P2(1)3 with a = 143.0 A and contain two molecules of renin in the asymmetric unit. A self-rotation function study using 5.5 A data shows the orientation of a non-crystallographic 2-fold axis relating these two monomers.
J Mol Biol 1989 Nov 05
PMID:Preliminary crystallographic study of glycosylated recombinant human renin. 268 30


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