Identification of an S-locus glycoprotein allele introgressed from B. napus ssp. rapifera to B. napus ssp. oleifera.

We have previously described a developmentally regulated mRNA in maize that accumulates in mature embryos and is involved in a variety of stress responses in the plant. The sequence of the encoded 16 kDa protein (MA16) predicts that it is an RNA-binding protein, since it possesses a ribonucleoprotein consensus sequence-type RNA-binding domain (CS-RBD). To assess the predicted RNA binding property of the protein and as a starting point to characterize its function we have used ribohomopolymer-binding assays. Here we show that the MA16-encoded protein binds preferentially to uridine- and guanosine-rich RNAs. In light of these results a likely role for this protein in RNA metabolism during late embryogenesis and in the stress response is discussed.     

Molecular evolution of mitochondrial 12S RNA and cytochrome b sequences in the pantherine lineage of Felidae

DNA sequence comparisons of two mitochondrial DNA genes were used to infer phylogenetic relationships among 17 Felidae species, notably 15 in the previously described pantherine lineage. The polymerase chain reaction (PCR) was used to generate sequences of 358 base pairs of the mitochondrial 12S RNA gene and 289 base pairs of the cytochrome b protein coding gene. DNA sequences were compared within and between 17 felid and five nonfelid carnivore species. Evolutionary trees were constructed using phenetic, cladistic, and maximum likelihood algorithms. The combined results suggested several phylogenetic relationships including (1) the recognition of a recently evolved monophyletic genus Panthera consisting of Panthera leo, P. pardus, P. onca, P. uncia, P. tigris, and Neofelis nebulosa; (2) the recent common ancestry of Acinonyx jubatus, the African cheetah, and Puma concolor, the American puma; and (3) two golden cat species, Profelis temmincki and Profelis aurata, are not sister species, and the latter is strongly associated with Caracal caracal. These data add to the growing database of vertebrate mtDNA sequences and, given the relatively recent divergence among the felids represented here (1-10 Myr), allow 12S and cytochrome b sequence evolution to be addressed over a time scale different from those addressed in most work on vertebrate mtDNA.      

The essential role of the Walker A motifs of SUR1 in K-ATP channel activation by Mg-ADP and diazoxide.

The ATP-sensitive K-channel (K-ATP channel) plays a key role in insulin secretion from pancreatic beta-cells. It is closed by glucose metabolism, which stimulates insulin secretion, and opened by the drug diazoxide, which inhibits insulin release. Metabolic regulation is mediated by changes in ATP and Mg-ADP, which inhibit and potentiate channel activity, respectively. The beta-cell K-ATP channel consists of a pore-forming subunit, Kir6.2, and a regulatory subunit, SUR1. We have mutated (independently or together) two lysine residues in the Walker A (W(A)) motifs of the first (K719A) and second (K1384M) nucleotide-binding domains (NBDs) of SUR1. These mutations are expected to inhibit nucleotide hydrolysis. Our results indicate that the W(A) lysine of NBD1 (but not NBD2) is essential for activation of K-ATP currents by diazoxide. The potentiatory effects of Mg-ADP required the presence of the W(A) lysines in both NBDs. Mutant currents were slightly more sensitive to ATP than wild-type currents. Metabolic inhibition led to activation of wild-type and K1384M currents, but not K719A or K719A/K1384M currents, suggesting that there may be a factor in addition to ATP and ADP which regulates K-ATP channel activity.     

Running out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels

K_ATP channels act as key regulators of electrical excitability by coupling metabolic cues—mainly intracellular adenine nucleotide concentrations—to cellular potassium ion efflux. However, their study has been hindered by their rapid loss of activity in excised membrane patches (rundown), and by a second phenomenon, the decline of activation by Mg-nucleotides (DAMN). Degradation of PI(4,5)P₂ and other phosphoinositides is the strongest candidate for the molecular cause of rundown. Broad evidence indicates that most other determinants of rundown (e.g. phosphorylation, intracellular calcium, channel mutations that affect rundown) also act by influencing K_ATP channel regulation by phosphoinositides. Unfortunately, experimental conditions that reproducibly prevent rundown have remained elusive, necessitating post hoc data compensation. Rundown is clearly distinct from DAMN. While the former is associated with pore-forming Kir6.2 subunits, DAMN is generally a slower process involving the regulatory sulfonylurea receptor (SUR) subunits. We speculate that it arises when SUR subunits enter non-physiological conformational states associated with the loss of SUR nucleotide-binding domain dimerization following prolonged exposure to nucleotide-free conditions. This review presents new information on both rundown and DAMN, summarizes our current understanding of these processes and considers their physiological roles.This article is part of the themed issue ‘Evolution brings Ca²⁺ and ATP together to control life and death’.     

Immunohistochemical identification of proliferating cells in organ culture using bromodeoxyuridine and a monoclonal antibody

The ability to measure cell proliferation is important in the study of cancer biology. The usual technique for quantitating proliferating cells in tissue explant and organ culture by detection of [3H]-thymidine incorporation into DNA by autoradiography is tedious and time-consuming. We have developed a technique for identification and quantitation of bromodeoxyuridine (an analogue of thymidine) in cultured tissue explants. Fetal mouse colon explants were exposed in vitro to bromodeoxyuridine (BUdR) or [3H]-thymidine for 3 to 72 hr and then for various periods to unlabeled thymidine. The tissues were stained with a monoclonal anti-bromodeoxyuridine antibody and in parallel [3H]-thymidine incorporation was detected by autoradiography. Incorporation of BUdR was measured by quantitating the amount of pigment deposited over nuclei after immunohistochemical staining, using an optical data digitizer. It was found that both techniques identified proliferating cells. Dividing cells were present both in crypts and in the surrounding stroma in Day 14 fetal mouse colon cultures. The immunohistochemical technique was more rapid and less cumbersome than autoradiography.     

Binding of fluoresceinated epidermal growth factor to A431 cell sub-populations studied using a model-independent analysis of flow cytometric fluorescence data.

A method is developed for determining ligand-cell association parameters from a model-free analysis of data obtained with a flow cytometer. The method requires measurement of the average fluorescence per cell as a function of ligand and cell concentration. The analysis is applied to data obtained for the binding of fluoresceinated epidermal growth factor to a human epidermoid carcinoma cell line, A431. The results indicate that the growth factor binds to two classes of sites on A431 cells: 4 X 10(4) sites with a dissociation constant (KD) of less than or equal to 20 pM, and 1.5 X 10(6) sites with a KD of 3.7 nM. A derived plot of the average fluorescence per cell versus the average number of bound ligands per cell is used to construct binding isotherms for four sub-populations of A431 cells fractionated on the basis of low-angle light scatter. The four sub-populations bind the ligand with equal affinity but differ substantially in terms of the number of binding sites per cell. We also use this new analysis to critically evaluate the use of 'Fluorotrol' as a calibration standard in flow cytometry.     

Interrelationship of islet metabolism, adenosine triphosphate content and insulin release

The oxidation of some exogenous substrates and their effects on ATP content and insulin release in mouse pancreatic islets were measured. The ATP concentration of islets incubated without exogenous substrate shows a gradual decrease, which can be prevented by glucose or mannose (20mm) or leucine (2.5mm); d-glyceraldehyde (5mm) is as effective as glucose (5mm); fructose or N-acetylglucosamine (20mm), pyruvate (10mm) and dl-3-hydroxybutyrate (2mm) are less effective; galactose (20mm), acetate (10mm), octanoate (2mm) and succinate (10mm) have no ATP-maintaining ability. Islets oxidize glucose, mannose, glyceraldehyde, leucine and, less readily, N-acetylglucosamine and glucosamine; galactose, however, is poorly metabolized. Mannoheptulose inhibits the oxidation of glucose but not of glyceraldehyde. Insulin release, measured over a 2h incubation, is stimulated by glucose, mannose, leucine, glyceraldehyde or glucosamine but not by fructose or N-acetylglucosamine. The latter, however, potentiates the effects of glucose or glyceraldehyde (5mm) or leucine (2.5mm) on release; the potentiating effects are inhibited by mannoheptulose, which also blocks glucose-, but not glyceraldehyde- or leucine-stimulated release. In the presence of glucose (20mm), metabolic inhibitors depress insulin release and islet ATP content in parallel. However, rates of insulin release and ATP content measured after incubation with various combinations of exogenous substrates do not appear to be correlated. Sulphonylureas stimulate insulin release but decrease islet ATP concentrations. These results provide further evidence of a close association between the metabolic activity of exogenous substrates and their ability to initiate insulin release. Glucoreceptor models are formulated in the light of these observations and discussed.     

Concentration of adenosine 3':5'-cyclic monophosphate in mouse pancreatic islets measured by a protein-binding radioassay

A protein-binding radioassay for cyclic AMP was modified to detect less than 0.025pmol of the nucleotide. The method was applied to the measurement of cyclic AMP in small numbers of mouse pancreatic islets (as little as 25μg of tissue) by use of barium acetate–H₂SO₄ for deproteinization. The concentration of cyclic AMP in mouse islets incubated in media containing 3.3 or 20mm-glucose was 0.016pmol/10 islets (approx. 1μm in intracellular water). Glucose concentration (3.3 or 20mm) had no detectable effect on islet concentrations of cyclic AMP with periods of incubation or perifusion ranging from 0.5 to 60min, although insulin release rate was rapidly increased by 20mm-glucose. Caffeine (5mm) or 3-isobutyl-1-methylxanthine (1mm), which are known inhibitors of islet cyclic AMP phosphodiesterase, produced marked and rapid increases in islet cyclic AMP concentration at 3.3 or 20mm-glucose, but only enhanced the insulin release rate at the higher glucose concentration. The role of cyclic AMP in insulin release induced by glucose is discussed.     

Cyclic AMP-dependent protein phosphorylation and insulin secretion in intact islets of Langerhans.

Effects on insulin release, cyclic AMP content and protein phosphorylation of agents modifying cyclic AMP levels have been tested in intact rat islets of Langerhans. Insulin release induced by glucose was potentiated by dibutyryl cyclic AMP, glucagon, cholera toxin and 3-isobutyl-1-methylxanthine (IBMX); the calmodulin antagonist trifluoperazine reversed these potentiatory effects. Inhibition by trifluoperazine of IBMX-potentiated release was, however, confined to concentrations of IBMX below 50 microM; higher concentrations, up to 1 mM, were resistant to inhibition by trifluoperazine. IBMX-potentiated insulin release was also inhibited by 2-deoxyadenosine, an inhibitor of adenylate cyclase. In the absence of glucose, IBMX at concentrations up to 1 mM did not stimulate insulin release and in the presence of 3.3 mM-glucose IBMX was effective only at a concentration of 1 mM; under the latter conditions trifluoperazine again did not inhibit insulin secretion. The maximum effect on insulin release was achieved with 25 microM-IBMX. Islet [cyclic AMP] was increased by IBMX, with the maximum rise occurring with 100 microM-IBMX. The increase in [cyclic AMP] elicited by IBMX was more rapid than that induced by cholera toxin. Trifluoperazine did not significantly affect islet cyclic AMP levels under any of the conditions tested. When islets were incubated with [32P]Pi, radioactivity was incorporated into islet ATP predominantly in the gamma-position. The rate of equilibration of label was dependent on medium Pi and glucose concentration and at optimal concentrations of these 100% equilibration of internal [32P]ATP with external [32P]Pi required a period of 3h. Radioactivity was incorporated into islet protein and, in response to an increase in islet [cyclic AMP], the major effect was on a protein of Mr 15 000 on sodium dodecyl sulphate/polyacrylamide gels. The extent of phosphorylation of the Mr-15 000 protein was correlated with the level of cyclic AMP: phosphorylation in response to IBMX was inhibited by 2-deoxyadenosine but not by trifluoperazine. Fractionation of islets suggested that the Mr-15 000 protein was of nuclear origin: the protein co-migrated with histone H3 on acetic acid/urea/Triton gels. In the islet cytosol a number of proteins were phosphorylated in response to elevation of islet [cyclic AMP]: the major species had Mr values of 18 000, 25 000, 34 000, 38 000 and 48 000. Culture of islets with IBMX increased the rate of [3H]-thymidine incorporation.(ABSTRACT TRUNCATED AT 400 WORDS)