Customized probability models for severe sepsis: correction and clarification.
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Humans↗
Biomedical subjects
R A Jeffs
Publications and source records attributed to R A Jeffs.
Solubilization of a guanine nucleotide-sensitive form of the P2Y-purinergic receptor.
P2Y-Purinergic receptors were solubilized from turkey erythrocyte plasma membranes with the nonionic detergent digitonin. Adenosine 5'-O-(2-[35S]thiodiphosphate) ([35S]ADP beta S) labeled a single population of soluble high affinity sites (Kd = 12.9 nM; Bmax = 4.5 pmol/mg of protein) in an equilibrium binding assay; adenine nucleotide analogs competitively inhibited [35S]ADP beta S binding with a rank order of potency consistent with that for P2Y-purinergic receptors. Radioligand binding to solubilized P2Y-purinergic receptors was noncompetitively inhibited by guanine nucleotides with a rank order of potency that was in agreement with the potency order observed for guanine nucleotide-mediated inhibition of [35S]ADP beta S binding in purified turkey erythrocyte plasma membranes. The rate constant for dissociation of [35S]ADP beta S from solubilized receptors was increased 2.3-fold by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Plasma membrane P2Y-purinergic receptors were labeled with [35S]ADP beta S or covalently labeled with the photoaffinity probe 3'-O-(4-benzoyl)benzoyl adenosine 5'-[alpha-32P]triphosphate ([alpha-32P]BzATP) before solubilization and gel filtration chromatography on Superose 12. [35S]ADP beta S- or [alpha-32P]BzATP-labeled species eluted as a single peak of radioactivity of apparent Mr greater than or equal to 300,000. Incubation of the Mr greater than or equal to 300,000 protein species with GTP gamma S before rechromatography resulted in loss of labeling of proteins by [35S]ADP beta S and a shift in apparent size of the covalently [alpha-32P]BzATP-labeled species to a single peak of radioactivity of approximate Mr 70,000. These results suggest that a P2Y-purinergic receptor-guanine nucleotide regulatory protein complex is stable to membrane solubilization with digitonin, even in the absence of prebound agonist.
Adenine↗
Evidence that the epidermal growth factor receptor and non-tyrosine kinase hormone receptors stimulate phosphoinositide hydrolysis by independent pathways.
We have shown previously that exposure of a non-transformed continuous line of rat liver epithelial (WB) cells to epidermal growth factor (EGF), adrenaline, angiotensin II or [Arg8]vasopressin results in an accumulation of the inositol phosphates InsP1, InsP2 and InsP3 [Hepler, Earp & Harden (1988) J. Biol. Chem. 263, 7610-7619]. Studies were carried out with WB cells to determine whether the EGF receptor and other, non-tyrosine kinase, hormone receptors stimulate phosphoinositide hydrolysis by common, overlapping or separate pathways. The time courses for accumulation of inositol phosphates in response to angiotensin II and EGF were markedly different. Whereas angiotensin II stimulated a very rapid accumulation of inositol phosphates (maximal by 30 s), increases in the levels of inositol phosphates in response to EGF were measurable only following a 30 s lag period; maximal levels were attained by 7-8 min. Chelation of extracellular Ca2+ with EGTA did not modify this relative difference between angiotensin II and EGF in the time required to attain maximal phospholipase C activation. Under experimental conditions in which agonist-induced desensitization no longer occurred in these cells, the inositol phosphate responses to EGF and angiotensin II were additive, whereas those to angiotensin II and [Arg8]vasopressin were not additive. In crude WB lysates, angiotensin II, [Arg8]vasopressin and adrenaline each stimulated inositol phosphate formation in a guanine-nucleotide-dependent manner. In contrast, EGF failed to stimulate inositol phosphate formation in WB lysates in the presence or absence of guanosine 5'-[gamma-thio]triphosphate (GTP[S]), even though EGF retained the capacity to bind to and stimulate tyrosine phosphorylation of its own receptor. Pertussis toxin, at concentrations that fully ADP-ribosylate and functionally inactivate the inhibitory guanine-nucleotide regulatory protein of adenylate cyclase (Gi), had no effect on the capacity of EGF or hormones to stimulate inositol phosphate accumulation. In intact WB cells, the capacity of EGF, but not angiotensin II, to stimulate inositol phosphate accumulation was correlated with its capacity to stimulate tyrosine phosphorylation of the 148 kDa isoenzyme of phospholipase C. Taken together, these findings suggest that, whereas angiotensin II, [Arg8]vasopressin and alpha 1-adrenergic receptors are linked to activation of one or more phospholipase(s) C by an unidentified G-protein(s), the EGF receptor stimulates phosphoinositide hydrolysis by a different pathway, perhaps as a result of its capacity to stimulate tyrosine phosphorylation of phospholipase C-gamma.
Angiotensin II↗
Biochemical properties of a P2Y-purinergic receptor.
The turkey erythrocyte has substantial value as a model for the study of a receptor that exhibits pharmacological properties very similar to those delineated in mammalian tissues for a P2Y-purinergic receptor. The G protein-dependent coupling of this receptor to phospholipase C can be studied in detail, and the availability of an abundant source of homogeneous cells from which highly purified plasma membranes can be prepared, has led to the development of a radiolabeled, reversibly binding radioligand for a P2Y-purinergic receptor and a photoaffinity covalent radiolabel for this receptor. This source of plasma membranes highly enriched in P2Y-purinergic receptors should also serve as a rich starting material for the eventual purification and structural characterization of this important signaling protein.
Adenosine Triphosphate↗
Effects of low extracellular chloride on dopamine release and the dopamine transporter.
Removal of chloride (Cl-) from the superfusion medium results in increased spontaneous efflux of dopamine (DA) but not acetylcholine from rabbit striatal slices prelabeled with [3H]DA and [14C]choline. Cl- was substituted to varying degrees with the impermeant anion, isethionate (IS-), or the permeant anion, nitrate (NO3-). The magnitude of low Cl(-)-induced DA efflux was inversely related to the external [Cl-] and was greater with IS- than with NO3-. Analysis of the composition of the 3H efflux in terms of DA and its metabolites revealed an increase in [3H]DA with decreasing Cl- concentration. Reduction of external Ca++ from 1.3 to 0.13 mM increased low Cl(-)-induced DA efflux. In slices depleted of vesicular DA by reserpine pretreatment and subsequently labeled and superfused in the presence of monoamine oxidase and catechol-O-methyltransferase inhibitors, the same inverse relationship between [Cl-] and spontaneous DA efflux was observed. Neuronal DA uptake inhibitors, nomifensine, mazindol, GBR-12909 and cocaine, all increased the rate of low Cl(-)-induced DA efflux in the reserpinized preparation. Cl(-)-induced DA efflux in untreated and reserpinized preparations was not blocked by tetrodotoxin, amiloride, furosemide, picrotoxin or 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid). Low Cl- inhibited initial rates of [3H]DA uptake. At Cl- concentrations producing significantly different efflux rates (0 and 7.4 mM Cl-, IS- and NO3- substitution), DA uptake was inhibited in all cases by greater than 90%. Additionally, the binding of [3H]mazindol, one of the uptake inhibitors, to striatal membranes was unaffected by removal of Cl-. In summary, low Cl- produces a nonexocytotic rapid outward transport of DA. Extracellular Cl- is not required for the binding to transporter sites or for the inhibition of neuronal uptake produced by neuronal uptake inhibitors.
Acetylcholine↗
Phorbol diester treatment promotes enhanced adenylate cyclase activity in frog erythrocytes.
Incubation of intact frog erythrocytes with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a tumor-promoting phorbol diester which activates protein kinase C, results in an approximate two- to threefold increase in subsequently tested beta-adrenergic agonist-stimulated adenylate cyclase activity. This increase is due to an elevation in the Vmax of the enzyme rather than to a change in affinity for the agonist. TPA treatment of frog erythrocytes does not alter the affinity (KD) or the binding capacity (Bmax) for the beta-adrenergic antagonist [125I]cyanopindolol. In addition, agonist/[125I]cyanopindolol competition curves are not affected by TPA pretreatment nor is their sensitivity to guanine nucleotides. Incubation of frog erythrocyte membranes alone with TPA does not promote sensitization or activation of adenylate cyclase activity. Pretreatment of intact frog erythrocytes with TPA also produces approximately two- to threefold increases in basal, guanine nucleotide-, prostaglandin E1-, forskolin-, NaF-, and MnCl2-stimulated adenylate cyclase activities in frog erythrocyte membranes. This enhancement of adenylate cyclase activity by TPA is induced rapidly (t1/2 approximately equal to 5 min) and with an EC50 of about 10(-7) to 10(-6) M. Other tumor-promoting phorbol diesters or phorbol diester-like compounds including 4 beta-phorbol 12,13-dibutyrate, 4 beta-phorbol 12,13-didecanoate, and mezerein are effective in promoting enhanced adenylate cyclase activity. In contrast, phorbols such as 4 beta-phorbol, 4 alpha-phorbol 12,13-didecanoate, and 4-O-methylphorbol 12-myristate 13-acetate, which are inactive in tumor promotion and which do not activate protein kinase C, do not affect frog erythrocyte adenylate cyclase activity. These data are suggestive of a protein kinase C-mediated phosphorylation of one of the adenylate cyclase components that is distal to the receptor, i.e., the nucleotide regulatory and/or catalytic components.
Adenylyl Cyclases↗
The influence of indol-3yl acetic acid and sugar on the pattern of induced differentiation in plant tissue culture.
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Arabinose↗
Experimental induction of vascular tissue in an undifferentiated plant callus.
1. By the implantation of wedges containing indol-3-ylacetic acid and sucrose into blocks of undifferentiated bean-callus tissue it has been possible to induce the formation of xylem and phloem cells. 2. The differentiation has been investigated cytologically and measured chemically. 3. The optimum concentrations of the nutrients in the wedge, which gave differentiation closely resembling the vascular development found in the stem of the intact plant, was 0.1mg. of indol-3-ylacetic acid/l. and 2% sucrose. 4. The ratios of the xylose/arabinose concentrations of the tissues increased in the differentiated callus tissue compared with those of the undifferentiated tissue. A similar increase has been found for the ratios determined for xylem tissue compared with those for cambium. 5. The lignin content of the differentiated tissue compared with the undifferentiated tissue was greater in both the callus and stem tissue. 6. Chemical analysis of lignin showed that in the differentiated callus tissue it consisted of sub-units based on p-hydroxybenzaldehyde and vanillin. This was compared with the lignin obtained from undifferentiated callus tissue and that obtained from the tissues of the intact stem. 7. The results of the investigation have been discussed with reference to the problems of cell growth and differentiation and related to the changing patterns of the ultrastructure of the cell during its development.
Arabinose↗