Comparison of cyclic nucleotide specificity of guanosine 3',5'-monophosphate-dependent protein kinase and adenosine 3',5'-monophosphate-dependent protein kinase.

Guanosine 3',5'-monophosphate-dependent protein kinase (cyclic GMP-dependent protein kinase) and adenosine 3',5'-monophosphate-dependent protein kinase (cyclic AMP-dependent protein kinase) exhibited a high degree of cyclic nucleotide specificity when hormone-sensitive triacylglycerol lipase, phosphorylase kinase, and cardiac troponin were used as substrates. The concentration of cyclic GMP required to activate half-maximally cyclic dependent protein kinase was 1000- to 100-fold less than that of cyclic AMP with these substrates. The opposite was true with cyclic AMP-dependent protein kinase where 1000- to 100-fold less cyclic AMP than cyclic GMP was required for half-maximal enzyme activation. This contrasts with the lower degree of cyclic nucleotide specificity of cyclic GMP-dependent protein kinase of 25-fold when histone H2b was used as a substrate for phosphorylation. Cyclic IMP resembled cyclic AMP in effectiveness in stimulating cyclic GMP-dependent protein kinase but was intermediate between cyclic AMP and cyclic GMP in stimulating cyclic AMP-dependent protein kinase. The effect of cyclic IMP on cyclic GMP-dependent protein kinase was confirmed in studies of autophosphorylation of cyclic GMP-dependent protein kinase where both cyclic AMP and cyclic IMP enhanced autophosphorylation. The high degree of cyclic nucleotide specificity observed suggests that cyclic AMP and cyclic GMP activate only their specific kinase and that crossover to the opposite kinase is unlikely to occur at reported cellular concentrations of cyclic nucleotides.     

Cardioprotection mediated by exosomes is impaired in the setting of type II diabetes but can be rescued by the use of non‐diabetic exosomes in vitro

Many patients with ischaemic heart disease also have diabetes. As myocardial infarction is a major cause of mortality and morbidity in these patients, treatments that increase cell survival in response to ischaemia and reperfusion are needed. Exosomes—nano‐sized, lipid vesicles released from cells—can protect the hearts of non‐diabetic rats. We previously showed that exosomal HSP70 activates a cardioprotective signalling pathway in cardiomyocytes culminating in ERK1/2 and HSP27 phosphorylation. Here, we investigated whether the exosomal cardioprotective pathway remains intact in the setting of type II diabetes. Exosomes were isolated by differential centrifugation from non‐diabetic and type II diabetic patients, from non‐diabetic and Goto Kakizaki type II diabetic rats, and from normoglycaemic and hyperglycaemic endothelial cells. Exosome size and number were not significantly altered by diabetes. CD81 and HSP70 exosome markers were increased in diabetic rat exosomes. However, exosomes from diabetic rats no longer activated the ERK1/2 and HSP27 cardioprotective pathway and were no longer protective in a primary rat cardiomyocytes model of hypoxia and reoxygenation injury. Hyperglycaemic culture conditions were sufficient to impair protection by endothelial exosomes. Importantly, however, exosomes from non‐diabetic rats retained the ability to protect cardiomyocytes from diabetic rats. Exosomes from diabetic plasma have lost the ability to protect cardiomyocytes, but protection can be restored with exosomes from non‐diabetic plasma. These results support the concept that exosomes may be used to protect cardiomyocytes against ischaemia and reperfusion injury, even in the setting of type II diabetes.     

FABMS/derivatisation strategies for the analysis of heparin-derived oligosaccharides

Derivatisation/FABMS strategies applicable to the structure analysis of low microgramme quantities of heparin-derived oligosaccharides are described. Negative and positive FAB data from permethyl derivatives and positive FAB data from the products of subsequent methanolysis are reported for sulfated tetrasaccharides prepared by nitropus acid degradation of heparin. The preparation and FAB behaviour of acetylated derivatives of sulfated oligosaccharides are described for the first time, and the stability of the sulfate groups to base-catalysed acetylation is demonstrated. The acetylation/FABMS methodology, which yields high quality data, shows promise for the characterisation of a wide range of sulfated glycoconjugates.     

Interactions of calcium and other metal ions with caldolysin, the thermostable proteinase from Thermus aquaticus strain T351.

Caldolysin, the extracellular proteinase from the extreme thermophile Thermus aquaticus strain T351, is stabilized by Ca2+. A variety of metal ions were able to substitute for Ca2+. Most were unable to confer as much stability as Ca2+, with the exception of the lanthanide ions, which increased the half-life at 95 degrees C from 1 h to more than 4 h. Results from a variety of separation methods indicated that caldolysin binds 6 Ca2+ ions/molecule of enzyme. The presence of non-linear Ca2+ titration plots, and the removal of 4 Ca2+ ions/molecule by treatment with a cationic ion-exchange gel suggested that caldolysin possesses at least two different types of Ca2+-binding sites, with different affinities. Average binding constants of the two types of binding sites were 2.8 X 10(4)M-1 (for the low-affinity sites) and 7.5 X 10(5) M-1 (for the high-affinity sites). The total Ca2+-binding free energy for caldolysin was shown to be greater than for either thermolysin or Bacillus subtilis neutral proteinase. It appears that the higher thermostability of caldolysin is due to the presence of 6 Ca2+ ions rather than 4 Ca2+ ions/molecule.     

A precise structural analysis of a fertilization-associated carbohydrate-rich glycopeptide isolated from the fertilized eggs of euryhaline killi fish (Fundulus heteroclitus). Novel penta-antennary N-glycan chains with a bisecting N-acetylglucosaminyl residue

A novel carbohydrate-rich sialoglycopeptide of apparent molecularmass $$$6 kDa was isolated from the fertilized eggs of Fundulusheteroclitus (euryhaline killi fish). This glycopeptide is amember of the L-hyosophorin family, characterized by its highcontent of carbohydrate (80–90% by weight) and formedby depolymerization of the precursor glycopolyprotein (H-hyosophorin)upon fertilization. The structures of the N-glycan chains wereunambiguously established by a combination of compositionalanalysis, methylation analysis, selective chemical degradation(periodate oxidation-Smith degradation and hydrazinolysis-nitrousacid deamination), enzymatic (peptide:N-g]ycosidase F, severalß-galactosidases, (ß-hexosaminidase and     

Chlorotriphenyl(quinolinium-2-carboxylato)tin(IV) monohydrate

The crystal structure of chlorotriphenyl(quinolinium-2-carboxylato)tin(IV) monohydrate is reported. The crystals are monoclinic, space group C2/c with cell parameters a = 20.048(3) Å, b = 11.724(1) Å, c = 23.291(3) Å, ]gb = 113.42(1), Z = 8, refined to R_F = 0.034 on 3331 observed reflections. The tin(IV) atom is five-coordinate, being found to three phenyl groups, the chlorine atom and an oxygen from the quinaldic acid. The geometry around the tin atom is trigonal bipyramidal, with the three phenyl groups occupying the equatorial positions, and the chlorine and quinaldic acid oxygen, the apical ones. The acidic proton of quinaldic acid has shifted position in the complex, and is bound to the heterocyclic nitrogen atom.The acid is thus coordinated in the form of a zwitterion. These trigonal bipyramidal units are linked together as dimers by pairs of water molecules, each of which hydrogen-bonds to the non-coordinated carboxylate oxygen atoms of both quinaldic acid molecules, plus the heterocyclic nitrogen atom of one quinaldic acid molecule. For complex formation with the protonated acid, the heterocyclic nitrogen should be alpha to the carboxylic acid group.     

Cep97 and CP110 suppress a cilia assembly program

Mammalian centrioles play a dynamic role in centrosome function, but they also have the capacity to nucleate the assembly of cilia. Although controls must exist to specify these different fates, the key regulators remain largely undefined. We have purified complexes associated with CP110, a protein that plays an essential role in centrosome duplication and cytokinesis, and have identified a previously uncharacterized protein, Cep97, that recruits CP110 to centrosomes. Depletion of Cep97 or expression of dominant-negative mutants results in CP110 disappearance from centrosomes, spindle defects, and polyploidy. Remarkably, loss of Cep97 or CP110 promotes primary cilia formation in growing cells, and enforced expression of CP110 in quiescent cells suppresses their ability to assemble cilia, suggesting that Cep97 and CP110 collaborate to inhibit a ciliogenesis program. Identification of Cep97 and other genes involved in regulation of cilia assembly may accelerate our understanding of human ciliary diseases, including renal disease and retinal degeneration.     

Analysis of protein-linked glycosylation in a sperm-somatic cell adhesion system

Murine sperm initiate fertilization by binding to the specialized extracellular matrix of their complementary eggs, known as the zona pellucida. On the basis of data reported in this study, mouse sperm also bind to rabbit erythrocytes with higher affinity than they do to murine eggs. This unusual interaction between a germ cell and a somatic cell ("sperm-somatic cell adhesion system") is also carbohydrate dependent based on its sensitivity to mild periodate oxidation. To determine what types of carbohydrate sequences could be involved in this interaction, the protein-linked oligosaccharides of rabbit erythrocytes were sequenced using novel matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry methods that enabled the analysis of individual components up to m/z 9000. The N-glycans are primarily complex biantennary and triantennary types terminated with Galalpha1-3Gal sequences. The majority of these oligosaccharides also possess one antenna consisting of a highly branched polylactosamine-type sequence that is also associated with many glycosphingolipids that coat rabbit erythrocytes. These erythrocytes also express Core 1 and Core 2 O-glycans terminated primarily with Galalpha1-3Gal sequences and to a lesser extent sialic acid. These results confirm that rabbit erythrocytes and mouse eggs present very different types of carbohydrate sequences on their surfaces. However, oligosaccharides terminated with beta1-6-linked N-acetyllactosamine or its alpha1-3 galactosylated analog are expressed on both the mouse zona pellucida and this somatic cell type. The far more abundant presentation of such sequences on rabbit erythrocytes compared with murine eggs could explain why mouse sperm display such exceptional affinity for this somatic cell type.     

Chirality Switching Via Rotation of Bilayer Fourfold Meta-Structure

Chiral effects have been observed from the interaction of chiral plasmonics nanostructures with light. Such nanostructures enhance the chiral response of molecules and provide an ideal platform for biological and chemical sensing. Here, we investigate the chiral switching effects of an array of subwavelength nanostructures with a unit cell composed of four double-layered nanostrips arranged to be rotationally symmetric. We observe chiral switching leading to a change in circular dichroism (CD) signature when the mutual angle between the first and second layer increases from 0° to 90° with respect to each other. This mutual angle can be manipulated to switch the handedness of the nanostructure and cause a change in the outgoing light. We also investigated the field distribution of each mode when circularly polarized light is normally incident into the structure. These modes can be categorized into longitudinal and transverse modes depending on the orientation of their dipole moments. The mode studies clearly show the nature of each plasmonics mode.