Differential effects of prostaglandin E2 on contractions of airway smooth muscle

In an in vitro muscle bath, the active tension generated by strips of canine tracheal smooth muscle responding to cumulative additions of either histamine (10(-8) to 10(-3) M) or acetylcholine (10(-9) to 10(-3) M) was measured in the absence and presence of prostaglandin E2 (PGE2) (10(-6) to 10(-5) M). When contractile responses of equal magnitude were compared, the contractions elicited by acetylcholine were resistant to the inhibitory effects of PGE2, relative to comparable contractions elicited by histamine. To assess the role of adenylate cyclase in determining the different responses to histamine and acetylcholine in the presence of PGE2, we assayed adenylate cyclase activity in membranes prepared from canine tracheal smooth muscle and found that acetylcholine, but not histamine, decreased PGE2-stimulated adenylate cyclase activity by 48 +/- 2% (mean +/- SE; n = 5). However, in other experiments, we found that even large pharmacological increases in tissue adenosine 3',5'-cyclic monophosphate (cAMP) content only partially inhibited muscarinic tone. Also, exogenously applied analogues of cyclic AMP inhibited contractions induced by histamine more effectively than comparable contractions induced by acetylcholine. We concluded that acetylcholine decreased adenylate cyclase activity in membranes prepared from canine tracheal smooth muscle and that this effect may have contributed to, but did not completely account for, the relative resistance of muscarinic contractions to the inhibitory effects of PGE2.     

Two lipocortin-like proteins, endonexin II and anchorin CII, may be alternate splices of the same gene

The annexins are a family of phospholipid- and Ca2+-binding proteins that are structurally related. Two members of this family, human endonexin II and chicken anchorin CII, may arise from the same gene by alternative splicing of two structurally unrelated segments.     

Complement phenotypes in patients with psoriasis

Phenotype frequencies for the complement proteins C4A, C4B, Bf (factor B) and C3 were performed for 49 Caucasian patients with psoriasis. The C4*A6 allele was present in 26.6% of the patients as compared to 5.4% of healthy regional Caucasian controls, p less than 0.001, relative risk = 6.28. The C4*A6 allele is known to be in linkage disequilibrium with the HLA B17 allele and to produce a non-functional gene product when it occurs with the B17 allele. HLA B17 is known to be associated with psoriasis in many Caucasian populations. Additional findings in the present study were a significant reduction in the C4B*2 allele frequency, a non-significant increase in the Bf*F allele frequency and no difference for Bf or C3 phenotype frequencies in the patients with psoriasis as compared to the controls.     

Phospholamban phosphorylation in intact ventricles. Phosphorylation of serine 16 and threonine 17 in response to beta-adrenergic stimulation

Phospholamban is the major membrane protein of the heart phosphorylated in response to beta-adrenergic stimulation. In cell-free systems, cAMP-dependent protein kinase catalyzes exclusive phosphorylation of serine 16 of phospholamban, whereas Ca2+/calmodulin-dependent protein kinase gives exclusive phosphorylation of threonine 17 (Simmerman, H. K. B., Collins, J. H., Theibert, J. L., Wegener, A. D., and Jones, L. R. (1986) J. Biol. Chem. 261, 13333-13341). In this work we have localized the sites of phospholamban phosphorylation in intact ventricles treated with the beta-adrenergic agonist isoproterenol. Isolation of phosphorylated phospholamban from 32P-perfused guinea pig ventricles, followed by partial acid hydrolysis and phosphoamino acid analysis, revealed phosphorylation of both serine and threonine residues. At steady state after isoproterenol exposure, phospholamban contained approximately equimolar amounts of these two phosphoamino acids. Two major tryptic phosphopeptides containing greater than 90% of the incorporated radioactivity were obtained from phospholamban labeled in intact ventricles. The amino acid sequences of these two tryptic peptides corresponded exactly to residues 14-25 and 15-25 of canine cardiac phospholamban, thus localizing the sites of in situ phosphorylation to serine 16 and threonine 17. Phosphorylation of phospholamban at two sites in heart perfused with isoproterenol was supported by detection of 11 distinct mobility forms of the pentameric protein by use of the Western blotting method, consistent with each phospholamban monomer containing two phosphorylation sites, and with each pentamer containing from 0 to 10 incorporated phosphates. Our results localize the sites of in situ phospholamban phosphorylation to serine 16 and threonine 17 and, furthermore, are consistent with the phosphorylations of these 2 residues being catalyzed by cAMP- and Ca2+/calmodulin-dependent protein kinases, respectively.     

Application of Deuterated A-Tocopherols to the Biokinetics and Bioavailability of Vitamin E

-Tocopherol, a superior chain-breaking, peroxyl radical-trapping antioxidant and the most active component of vitamin E, is elevated in liver tumor cells, contributing to their greater resistance towards lipid peroxidation compared to cells from normal tissues. Also, in regenerating rat liver the level of vitamin E has been found to fluctuate in phase with the rate of cell division. In order to study the biokinetcis and mechanisms of the distribution of vitamin E in organs and within tissues of animals, deuterated forms of -tocopherol have been synthesized and their uptake into blood and tissues has been measured by gas chromatography-mass spectrometry. Measurement of the competitive uptake from a mixture of the RRR-and SRR--tocopherol stereoisomers labelled with different amounts of deuterium shows that the liver exerts a strong preference for secretion of the natural (RRR) stereoisomer into the plasma. It is suggested that a tocopherol-binding protein plays a key role in this process.     

Metabolism of [U-13C5]Glutamine in Cultured Astrocytes Studied by NMR Spectroscopy: First Evidence of Astrocytic Pyruvate Recycling

Abstract: Metabolism of [U-¹³C₅]glutamine was studied in primary cultures of cerebral cortical astrocytes in the presence or absence of extracellular glutamate. Perchloric acid extracts of the cells as well as redissolved lyophilized media were subjected to nuclear magnetic resonance and mass spectrometry to identify ¹³C-labeled metabolites. Label from glutamine was found in glutamate and to a lesser extent in lactate and alanine. In the presence of unlabeled glutamate, label was also observed in aspartate. It could be clearly demonstrated that some [U-¹³C₅]glutamine is metabolized through the tricarboxylic acid cycle, although to a much smaller extent than previously shown for [U-¹³C₅]glutamate. Lactate formation from tricarboxylic acid cycle intermediates has previously been demonstrated. It has, however, not been demonstrated that pyruvate, formed from glutamate or glutamine, may reenter the tricarboxylic acid cycle after conversion to acetyl-CoA. The present work demonstrates that this pathway is active, because [4,5-¹³C₂]glutamate was observed in astrocytes incubated with [U-¹³C₅]glutamine in the additional presence of unlabeled glutamate. Furthermore, using mass spectrometry, mono-labeled alanine, glutamate, and glutamine were detected. This isotopomer could be derived via the action of pyruvate carboxylase using ¹³CO₂ produced within the mitochondria or from labeled intermediates that had stayed in the tricarboxylic acid cycle for more than one turn.     

Recolonisation and recruitment of fishes to intertidal rockpools at Wellington, New Zealand

Synopsis A study of recolonisation of rockpools by intertidal fishes on the Wellington south coast, New Zealand, found the assemblage to be resilient and seasonally stable. A total of 26 species from nine families were recorded, dominated by the Tripterygiidae (triplefins) and Gobiesocidae (clingfishes). A pattern of alternating species dominance occurred, with the triplefinsBellapiscis medius andForsterygion lapillum being numerically dominant in summer, but becoming less common in winter and replaced as dominants by the clingfishesTrachelochismus pinnulatus andGastroscyphus hectoris. Juvenile recruits of eleven species occurred in the samples from spring to early summer, however only the aforementioned four species recruit to the intertidal zone in large numbers. The speed of rockpool recolonisation by fishes after extractive sampling is seasonally dependent, being quicker in the summer than winter. In general, recolonisation takes at least one month, but probably fewer than three. While stochastic factors influence assemblage composition in the short term, overall regulation of the fish assemblage of rockpools appears to be primarily deterministic, resulting in an essentially predictable taxonomic structure.     

An essential E box in the promoter of the gene encoding the mRNA cap-binding protein (eukaryotic initiation factor 4E) is a target for activation by c-myc.

The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF4E]) binds the m7 GpppN cap on mRNA, thereby initiating translation. eIF4E is essential and rate limiting for protein synthesis. Overexpression of eIF4E transforms cells, and mutations in eIF4E arrest cells in G, in cdc33 mutants. In this work, we identified the promoter region of the gene encoding eIF4E, because we previously identified eIF4E as a potential myc-regulated gene. In support of our previous data, a minimal, functional, 403-nucleotide promoter region of eIF4E was found to contain CACGTG E box repeats, and this core eIF4E promoter was myc responsive in cotransfections with c-myc. A direct role for myc in activating the eIF4E promoter was demonstrated by cotransfections with two dominant negative mutants of c-myc (MycdeltaTAD and MycdeltaBR) which equally suppressed promoter function. Furthermore, electrophoretic mobility shift assays demonstrated quantitative binding to the E box motifs that correlated with myc levels in the electrophoretic mobility shift assay extracts; supershift assays demonstrated max and USF binding to the same motif. cis mutations in the core or flank of the eIF4E E box simultaneously altered myc-max and USF binding and inactivated the promoter. Indeed, mutations of this E box inactivated the promoter in all cells tested, suggesting it is essential for expression of eIF4E. Furthermore, the GGCCACGTG(A/T)C(C/G) sequence is shared with other in vivo targets for c-myc, but unlike other targets, it is located in the immediate promoter region. Its critical function in the eIF4E promoter coupled with the known functional significance of eIF4E in growth regulation makes it a particularly interesting target for c-myc regulation.