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Biomedical subjects

A E Johnson

Publications and source records attributed to A E Johnson.

At least 163 records · Page 9Linked to original sources
PubMed1985

Distribution of cholinergic muscarinic binding sites in guinea-pig brain as determined by in vitro autoradiography of [3H]N-methyl scopolamine binding.

The distribution of muscarinic binding sites was analyzed in regions of the guinea-pig brain with semi-quantitative densitometry of [3H]N-methyl scopolamine binding, a muscarinic antagonist. In the rostral forebrain, high levels of binding were detected in the caudate putamen, nucleus accumbens and olfactory tubercle while intermediate levels of binding were observed in the medial and lateral septum, bed nucleus, and vertical and horizontal limbs of the diagonal band. The hypothalamus displayed binding that ranged from low levels in the preoptic area to intermediate levels in the mammillary nucleus. In limbic areas such as the thalamus, amygdala and hippocampus, a heterogeneous pattern of binding was evident in various subregions which tended to correspond with known innervation by cholinergic afferents. In the midbrain, binding was high in the superficial layer of the superior colliculus and the medial geniculate while intermediate binding was recorded in the lateral geniculate and the lateral aspect of the central gray. The pattern of muscarinic binding observed in the brain of the guinea-pig is similar to distributions of this binding site previously reported in the rat brain and the human brain.

Animals↗
PubMed1985

Tritium-sensitive film autoradiography of guinea pig brain alpha 2-noradrenergic receptors.

Tritium-sensitive film autoradiography was used to determine the distribution of alpha2-noradrenergic receptors (i.e. [3H]p-aminoclonidine binding sites) in guinea pig forebrain. Alpha 2-Receptors are heterogeneously distributed throughout the forebrain. Many limbic system structures, such as bed nucleus of stria terminalis, medial preoptic area, medial amygdaloid nucleus and lacunosum molecular layer in hippocampus were heavily labeled. We did not quantify receptor density in areas containing principally white matter but the optical density in those areas was similar to film background suggesting a very low receptor density. Low receptor concentrations were also found in areas that do not contain a high percentage of white matter, such as lateral septum and ventromedial hypothalamic nucleus.

Animals↗
PubMed1985

Estradiol modulation of alpha 2-noradrenergic receptors in guinea pig brain assessed by tritium-sensitive film autoradiography.

In this experiment, we examined the influence of estradiol on alpha 2-noradrenergic receptor binding in the female guinea pig brain with tritium-sensitive film autoradiography. Treatment of ovariectomized guinea pigs with estradiol increased alpha 2-receptor binding in regions of the preoptic area and decreased binding in the ventromedial nucleus of the hypothalamus. There was no effect of estradiol on alpha 2-receptor concentrations in other estradiol concentrating regions or in brain areas which do not concentrate estradiol.

Animals↗
PubMed1985

Direct determination of the association constant between elongation factor Tu X GTP and aminoacyl-tRNA using fluorescence.

We have investigated the formation of the aa-tRNA X EF-Tu X GTP ternary complex spectroscopically by monitoring a fluorescence change that accompanies the association of EF-Tu X GTP with Phe-tRNAPhe-F8, a functionally active analogue of Phe-tRNAPhe with a fluorescein moiety covalently attached to the s4U-8 base. With this approach, the protein-nucleic acid interaction could be examined by direct means and at equilibrium. The fluorescence emission intensity of each Phe-tRNAPhe-F8 increased by 36-55% upon association with EF-Tu X GTP, depending on the solvent conditions. Thus, when Phe-tRNAPhe-F8 was titrated with EF-Tu X GTP, the extent of ternary complex formation was determined from the increase in emission intensity. A nonlinear least-squares analysis of the titration data yielded a dissociation constant of 0.85 nM for the ternary complex in 50 mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (pH 7.6), 10 mM MgCl2, and 50 mM NH4Cl, at 6 degrees C. The delta H degrees of this interaction, determined by the temperature dependence of Kd, was -16 kcal/mol; the delta S degrees was therefore -16 cal mol-1 deg-1 at 6 degrees C in this buffer. In a more physiological polycation-containing solvent ("polymix"), the Kd was 4.7 nM. The ionic strength dependence of ternary complex formation showed that a minimum of two salt bridges and a substantial nonelectrostatic contribution are involved in the binding of aa-tRNA to EF-Tu. The affinities of unmodified aa-tRNAs for EF-Tu X GTP were determined by their abilities to compete with the fluorescent aa-tRNA for binding to the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli↗
PubMed1985

Toxicity of Riddell's groundsel (Senecio riddellii) to cattle.

The toxicity of Riddell's groundsel (Senecio riddellii) fed to calves in gelatin capsules, by gavage, or mixed in their hay ration was determined. Dosages were varied according to the pyrrolizidine alkaloid (PA) content of the plant, and the calves' responses were measured by clinical signs, serum enzyme changes, survival time, and histopathologic changes. Calves fed S riddellii to provide 10 mg of PA/kg of body weight/day in capsules or by gavage for 20 consecutive days did not develop clinical signs of seneciosis and did not have meaningful serum enzyme changes. However, feedings of the plant that provided 15 to 20 mg of PA/kg/day or more (gavaged or fed in capsules for the same time period) resulted in high mortality. The Senecio plant mixed in calves' hay ration was eaten slowly and reluctantly and was tolerated at dosages greater than 20 mg/kg/day, emphasizing that the toxicity was dependent on the rate at which the dosage was consumed and that mortality was not necessarily dependent on the cumulative dosage. Because of its high PA content, S riddellii presents a great hazard to cattle managed under conditions where they can consume quantities of the plant in short periods of time.

Animals↗
PubMed1984

Derivatives of blood coagulation factor IX contain a high affinity Ca2+-binding site that lacks gamma-carboxyglutamic acid.

We have examined the calcium-binding properties and metal ion-dependent conformational changes of proteolytically modified derivatives of factor IX that lack gamma-carboxyglutamic acid (Gla) residues. Equilibrium dialysis experiments demonstrated that a Gla-domainless factor IX species retained a single high affinity calcium ion-binding site (Kd = 85 +/- 5 microM). Ca2+ binding to this site was accompanied by a decrease in intrinsic fluorescence emission intensity (Kd = 63 +/- 15 microM). These spectral changes were reversed upon the addition of EDTA. Titration with Sr2+ resulted in little change in fluorescence intensity below 1 mM, while titration with Tb3+ caused fluorescence changes similar to those observed with Ca2+. Tb3+ and Ca2+ appear to bind to the same site because tryptophan-dependent terbium emission was reduced by the addition of Ca2+. Similar results were obtained with a Gla-domainless factor IX species lacking the activation peptide. Gla domain-containing factor IX species exhibited fluorescence changes similar to those of the Gla-domainless proteins at low Ca2+, but an additional structural transition was found at higher Ca2+ concentrations (apparent Kd greater than 0.8 mM). Thus, the conformations of factor IX proteins are nucleated and/or stabilized by calcium binding to a high affinity site which does not contain Gla residues. The binding of Ca2+ to lower affinity Gla domain-dependent metal ion-binding sites elicits an additional conformational change. The strong similarities between these results and those obtained with protein C (Johnson, A. E., Esmon, N. L., Laue, T. M. & Esmon, C. T. (1983) J. Biol. Chem. 258, 5554-5560), coupled with the remarkable sequence homologies of the vitamin K-dependent proteins, suggest that the high affinity Gla-independent Ca2+-binding site may be a common feature of vitamin K-dependent proteins.

1-Carboxyglutamic Acid↗
PubMed1984

Structure of bovine blood coagulation factor Va. Determination of the subunit associations, molecular weights, and asymmetries by analytical ultracentrifugation.

Thrombin-activated bovine factor V (factor Va), an essential component of the blood clotting cascade pro thrombinase complex, is composed of two nonidentical subunits (Vl and Vh) and Ca2+ in tight association. We have examined Vl, Vh, and factor Va using analytical ultracentrifugation. At pH 7.65 in 50 mM tris(hydroxymethyl)aminomethane, 0.1 M NaCl, 1 mM benzamidine, and 10 mM Ca2+, the Vl subunit has a molecular weight (Mr) of 82 500, an S0(20) ,w = 5. 0(2)S , and, assuming a model of a prolate ellipsoid with 0.3 g of H2O/g of protein, an axial ratio of 5:1. The corresponding values for the Vh subunit are an Mr of 92 300, an S0(20) ,w = 5.2(9) S, and an axial ratio of 5:1. We found these same values for Vl and for Vh in a buffer that contained 2 mM ethylenediaminetetraacetate (EDTA) rather than the 10 mM Ca2+. The Vl subunit undergoes a weak, reversible self-association at 9 degrees C with an apparent monomer-dimer association constant of 5.6 X 10(3) M-1 in the presence of 2 mM EDTA and 2.3 X 10(3) M-1 in the presence of 10 mM Ca2+. Our data indicate that the Vl self-association includes dimer and higher oligomers. Factor Va, examined in the presence of 10 mM Ca2+ and at 20 degrees C, has an Mr of 174 000, and S0(20) ,w = 8.1(8)S, an axial ratio of 5:1, and an apparent Vl-Vh association constant of at least 2.7 X 10(8) M-1. Our results suggest that factor Va self-associates to form higher multimers. When solutions of Va are dialyzed against a buffer that contains no Ca2+ and 2 mM EDTA, the apparent Vl-Vh subunit association constant is reduced to 9.4 X 10(3) M-1. Our hydrodynamic data indicate that there is a substantial decrease in molecular asymmetry when factor V is proteolytically activated by thrombin to form factor Va and that Vl and Vh are arranged "side by side" rather than "end to end" in factor Va.

Animals↗
PubMed1984

Glycerol, sucrose, and other diol-containing reagents are not inert components in in vitro incubations containing aminoacyl-tRNA.

The addition of glycerol, sucrose, or other diol-containing reagents to solutions of aminoacyl-tRNA (aa-tRNA) substantially increased the rate of hydrolysis of the aminoacyl ester bond. Glycerol at 4.9% (v/v) doubled the rate of deacylation for several aa-tRNAs and peptidyl-tRNAs, including fMet-tRNAMetf, while 1% (v/v) glycerol increased the deacylation rate by 20%. This effect was not caused by a nuclease contamination, and tRNA deacylated in the presence of glycerol could be fully recharged. The deacylation of aa-tRNA was accelerated by glycerol and sucrose even in the presence of EF-Tu X GTP. In addition, the extent of tRNA aminoacylation was reduced when glycerol was present at concentrations above 2% (v/v). Thus, glycerol and sucrose are not necessarily inert or neutral additions to an in vitro incubation.

Animals↗
PubMed1984

Hypothalamic and preoptic area alpha 1-receptor concentrations decrease, and cerebral cortical alpha 1-receptor concentrations increase during postnatal maturation of male guinea pigs.

alpha 1-Receptors were measured in intact male guinea pigs during the course of postnatal development. Receptor concentrations were significantly higher at day 1 in hypothalamus and at days 1 and 14 in preoptic area than at day 60. Conversely, in cerebral cortex, receptor concentrations were significantly lower in neonatal animals than in 60-day-old animals. There was no change in affinity of the receptor during development. Castration has no apparent effect on alpha 1-receptor concentration in any brain area.

Animals↗
PubMed1984

Toxicity of threadleaf groundsel (Senecio douglasii var longilobus) to cattle.

Threadleaf groundsel (Senecio douglasii var longilobus), a common plant on much of the rangeland of southwestern United States, was experimentally fed to 55 cattle by gastric gavage, mixed in alfalfa hay, or pelleted in feed. Since the pyrrolizidine alkaloid (PA) content of threadleaf groundsel fluctuates greatly, dosage was determined by the plant PA content. Animals that died survived up to 506 days. Whether they were acutely or chronically affected they had typical signs of seneciosis. Major gross and histopathologic changes are reported. The estimated minimum lethal plant dose by gavage was sufficient plant to provide 13 mg of PA/kg of calf body weight/day for 15 days or a total of approximately 200 mg of PA/kg in a 15-day period. Cattle that consumed up to 600 mg of PA/kg total in 20- to 100-day periods in hay or pellets were unaffected or minimally affected. These findings emphasize the importance of the time-dose relationship in PA toxicosis in cattle and indicate that there is a threshold level of PA that must be exceeded for the toxicosis to develop.

Animals↗
PubMed1983

Changes in aminoacyl transfer ribonucleic acid conformation upon association with elongation factor Tu-guanosine 5'-triphosphate. fluorescence studies of ternary complex conformation and topology.

The association of aminoacyl-tRNA (aa-tRNA) with elongation factor Tu.GTP to form an aa-tRNA.EF-Tu.GTP ternary complex was investigated by using two different fluorescent probes, both of which monitored structural changes near the juncture of the two arms of the L-shaped tRNA. Aminoacylation of tRNAPhe-F8, a functionally active analogue of tRNAPhe with a fluorescein moiety covalently attached to the s4U-8 base, did not cause a change in the fluorescence emission intensity. However, when EF-Tu.GTP bound to Phe-tRNAPhe-F8, the emission intensity increased by approximately 30%, depending upon the solvent conditions. About half of this increase in fluorescence was due to an increase in the molar absorptivity of the fluorescein dye. Ternary complex formation did not alter the rate of iodide ion quenching of the Phe-tRNAPhe-F8 fluorescence. Since solvent access to fluorescein was not reduced when EF-Tu.GTP was bound to Phe-tRNAPhe-F8, the fluorescence intensity change noted above was not caused by a direct interaction between fluorescein and EF-Tu. Instead, the binding of EF-Tu.GTP to the aa-tRNA resulted in a conformational change in the aa-tRNA near s4U-8. Ternary complex formation also altered the nature of the single strong binding site for ethidium in unfractionated and unmodified aa-tRNA. However, ethidium binding to its strong site was not blocked. These results indicate that only the acceptor-T psi C arm of aa-tRNA interacts directly with EF-Tu.GTP and that the anticodon-D arm is available for direct interaction with the ribosome during recognition. Our data also suggest that EF-Tu facilitates protein biosynthesis by ensuring that every aa-tRNA is in a particular (possibly the same) conformation prior to initiation of the recognition process at the ribosomal complex.

Ethidium↗
PubMed1983

The role of the Gla domain in the activation of bovine coagulation factor X by the snake venom protein XCP.

The activation by XCP of coagulation factor X and a factor X species lacking the Gla-domain was studied in the presence and absence of Ca2+. Both proteins could be activated at low rates in the absence of Ca2+. The activation of the unmodified factor X was stimulated by the addition of Ca2+, whereas GD factor X activation was insensitive to Ca2+. The stimulatory effect of Ca2+ seen with the unmodified factor X correlated strongly with a calcium-dependent change in intrinsic protein fluorescence. This conformational change required the Gla-domain as the fluorescence emission of GD factor X was the same with or without Ca2+. Fluorescence changes which accompanied activation were the same for both factor X and GD factor X. This suggests that the Gla-domain does not participate in the structural changes which accompany activation.

1-Carboxyglutamic Acid↗
PubMed1983

Burkitt cell leukemia with abnormality of chromosome No. 1.

A case of L3 (Burkitt cell) leukemia with chromosome 1 abnormality but no detectable abnormalities of chromosomes 8 or 14 is reported. This is the first time that L3 leukemia has been shown to be associated with a primary abnormality of chromosome 1. The implications of these findings relative to the development of B-cell ALL and the role of chromosome 1 in human neoplasia is discussed.

Chromosome Aberrations↗
PubMed1983

Effects on cattle and their calves of tansy ragwort (Senecio jacobaea) fed in early gestation.

Tansy ragwort (Senecio jacobaea) fed to pregnant cows on gestation days 15 through 30 in lethal and near-lethal doses did not cause detectable damage to 100-day-old or to full-term fetuses as determined by gross examinations, serum enzyme tests chosen to detect hepatic changes, and by histopathologic examinations. Some cows that appeared normal during gestation expired soon after parturition from typical seneciosis apparently triggered by parturition stress. Pregnant cows fed 12% smaller tansy ragwort doses on gestation days 30 through 45 had only minimal serum enzyme changes and gave birth to normal calves that had normal serum enzymes up to 64 days after parturition.

Animals↗
PubMed1982

Studies on avian erythrocyte metabolism. XII. The synthesis and degradation of inositol pentakis (dihydrogen phosphate).

The pathway(s) of synthesis and degradation of inositol 1, 3, 4, 5, 6 pentakis (dihydrogen phosphate) (inositol-P5), found predominantly in the avian erythrocyte, are unknown. Myo-inositol (inositol), D-glucose, inosine, and phosphate have been studied as potential precursors of inositol-P5 synthesis in chicken erythrocytes. Whole blood from chickens at several ages has been incubated for prolonged periods and the concentration of inositol-P5 measured to determine the ability of the avian erythrocyte to catabolize inositol-P5. Incubation of erythrocyte suspensions from 5-day chicks with [U-14 C]myo-inositol (inositol) for 17 hr lead to the appearance of 19.8% of the radioactivity in the aqueous acid-soluble extract of the erythrocytes (RBC). Fractionation of this extract on an anion exchange column yielded five major radioactive peaks, three of which represent 1) free myo-inositol, accounting for 63.5% of the radioactivity; 2) myo-inositol monokis (dihydrogen phosphate) (inositol-P), representing 1.6% of the radioactivity; and 3) inositol 1, 3, 4, 5, 6 pentakis (dihydrogen phosphate) (inositol-P5), representing 27.0% of the label in the erythrocyte. Sodium fluoride did not inhibit the incorporation of [U-14 C]myo-inositol into inositol-P5. Similar incubations with D-[U-14 C]-glucose and [U-14 C]-inosine yielded no incorporation of radioactivity into inositol-P5. These data are consistent with the interpretation that inositol is the major precursor for synthesis of inositol-P5 in chick red cells. Incubation of whole blood from 1-, 5-, and 42-day chicks and mature birds at 40 C for as long as 72 hr resulted in increases in RBC inorganic phosphate (Pi) thought due primarily to depletion of adenosine triphosphate (ATP). Inositol-P5 content of RBC of 5- and 42-day birds decreased 28 and 20%, respectively, after 72 hr, but no change was noted in inositol-P5 levels of RBC from mature birds. The red cells of the mature bird appear to be unable to catabolize inositol-P5 at significant rates. The apparent inability of the chicken erythrocyte to alter readily its concentration of inositol-P5 suggests that the hemoglobin oxygen delivery system of birds may be less adaptable to changing oxygen requirements by regulating the concentration of hemoglobin modulator than is that of the mammals.

Adenosine Triphosphate↗