Sequence-specific 1H-NMR assignments for the aromatic region of several biologically active, monomeric insulins including native human insulin

The aromatic region of the 1H-FT-NMR spectrum of the biologically fully-potent, monomeric human insulin mutant, B9 Ser----Asp, B27 Thr----Glu has been investigated in D2O. At 1 to 5 mM concentrations, this mutant insulin is monomeric above pH 7.5. Coupling and amino acid classification of all aromatic signals is established via a combination of homonuclear one- and two-dimensional methods, including COSY, multiple quantum filters, selective spin decoupling and pH titrations. By comparisons with other insulin mutants and with chemically modified native insulins, all resonances in the aromatic region are given sequence-specific assignments without any reliance on the various crystal structures reported for insulin. These comparisons also give the sequence-specific assignments of most of the aromatic resonances of the mutant insulins B16 Tyr----Glu, B27 Thr----Glu and B25 Phe----Asp and the chemically modified species des-(B23-B30) insulin and monoiodo-Tyr A14 insulin. Chemical dispersion of the assigned resonances, ring current perturbations and comparisons at high pH have made possible the assignment of the aromatic resonances of human insulin, and these studies indicate that the major structural features of the human insulin monomer (including those critical to biological function) are also present in the monomeric mutant.     

ANKRD13C acts as a molecular chaperone for G protein-coupled receptors

Although the mechanisms that regulate folding and maturation of newly synthesized G protein-coupled receptors are crucial for their function, they remain poorly characterized. By yeast two-hybrid screening, we have isolated ANKRD13C, a protein of unknown function, as an interacting partner for the DP receptor for prostaglandin D(2). In the present study we report the characterization of this novel protein as a regulator of DP biogenesis and trafficking in the biosynthetic pathway. Co-localization by confocal microscopy with an endoplasmic reticulum (ER) marker, subcellular fractionation experiments, and demonstration of the interaction between ANKRD13C and the cytoplasmic C terminus of DP suggest that ANKRD13C is a protein associated with the cytosolic side of ER membranes. Co-expression of ANKRD13C with DP initially increased receptor protein levels, whereas siRNA-mediated knockdown of endogenous ANKRD13C decreased them. Pulse-chase experiments indicated that ANKRD13C can promote the biogenesis of DP by inhibiting the degradation of newly synthesized receptors. However, a prolonged interaction between ANKRD13C and DP resulted in ER retention of misfolded/unassembled forms of the receptor and to their proteasome-mediated degradation. ANKRD13C also regulated the expression of other GPCRs tested (CRTH2, thromboxane A(2) (TPα), and β2-adrenergic receptor), whereas it did not affect the expression of green fluorescent protein, GRK2 (G protein-coupled receptor kinase 2), and VSVG (vesicular stomatitis virus glycoprotein), showing specificity toward G protein-coupled receptors. Altogether, these results suggest that ANKRD13C acts as a molecular chaperone for G protein-coupled receptors, regulating their biogenesis and exit from the ER.     

Exploring QSAR of melatonin receptor ligand benzofuran derivatives using E-state index

Considering the recent challenge to the medicinal chemists for the development of selective melatonin receptor ligands, an attempt has been made to explore physicochemical requirements of benzofuran derivatives for binding with human MT1 and MT2 receptor subtypes and also to explore selectivity requirements. In this study, E-states of different common atoms of the molecules (calculated according to Kier and Hall) and physicochemical parameters (partition coefficient and molar refractivity) were used as independent variables along with suitable dummy parameters. The best equation describing MT1 binding affinity [n = 34, Q2 = 0.670, Ra2 = 0.790, R2 = 0.822, R = 0.907, s = 0.609, F = 25.8 (df 5, 28)] suggests that the binding affinity decreases as the value of n (number of CH2 spacer beside R2) increases while it increases with rise in electrotopological state values of different atoms of the benzofuran ring. Again, presence of methoxy group at R1 and hydrogen, unsubstituted phenyl or fluoro-substituted phenyl group at R2 is conducive to the MT1 binding affinity. The binding affinity decreases if furyl substitution at R3 position is present. The best equation describing MT2 binding affinity [n = 34, Q2 = 0.602, Ra2 = 0.755, R2 = 0.792, R = 0.890, s = 0.584, F = 213 (df 5, 28)] shows that the MT2 binding affinity depends on the similar factors as described for MT1 binding affinity; however, the contributions of the factors for the two affinities are different to some extent as evidenced from the regression coefficients. Among the selectivity relations, the best equation [n = 33, Q2 = 0.496 Ra2 = 0.681, R2 = 0.721, R = 0.849, s = 0.458, F = 18.1(df 4, 28)] suggests that MT2 binding increases with increase in value of n, presence of methoxy group at R1, and E-state values of different atoms of the benzofuran ring, while it decreases in presence of furyl group at R3 position.     

Preparation and properties of creatine kinase from the breast muscle of normal and dystrophic chicken (Gallus domesticus)

1. The purification of creatine kinase from normal and genetically dystrophic chicken breast muscle is described. Enzyme recovery was significantly lower from dystrophic muscle. 2. Both enzymes had the same number of reactive and total thiol groups and had similar specific activities and similar amino acid compositions. 3. No significant differences were observed in sedimentation, electrophoretic or kinetic properties. 4. Peptide ;maps' showed no significant differences, and electrophoresis of partial acid hydrolysates of the labelled enzymes suggested that corresponding amino acid sequences around all the thiol groups were very similar. 5. The enzymes showed identical temperature stabilities. 6. No significant differences between the enzymes from normal and dystrophic muscle were observed.     

Fertilization triggers localized activation of Src-family protein kinases in the zebrafish egg

Fertilization triggers activation of Src-family kinases in eggs of various species including marine invertebrates and lower vertebrates. While immunofluorescence studies have localized Src-family kinases to the plasma membrane or cortical cytoplasm, no information is available regarding the extent to which these kinases are activated in different regions of the zygote. The objective of the present study was to detect the subcellular distribution of activated Src-family kinases in the fertilized zebrafish egg. An antibody specific for the active, non-phosphorylated form of Src-family PTKs was used to detect these activated kinases by immunofluorescence. The results demonstrate that Fyn, and possibly other Src family members are activated by dephosphorylation of the C-terminal tyrosine at fertilization. The activated Src-family kinases are asymmetrically distributed around the egg cortex with an area of higher kinase activity localized adjacent to the micropyle near the presumptive animal pole. Fertilization initially caused elevation of kinase activity in the cytoplasm underlying the micropyle, but this quickly spread to involve the entire zygote cortex. Later, during egg activation, formation of the blastodisc involved concentration of active Src-family kinase in the blastodisc cortex. As cytokinesis began, activated Src-family kinases were no longer limited to the cortex, but became more evenly distributed in the clear apical cytoplasm of the blastomeres. The results demonstrate that the cortex of the zebrafish egg is functionally differentiated and that fertilization triggers localized activation of Src-family kinases at the point of sperm entry, which subsequently progresses through the entire egg cortex.     

Spatial repartition and genetic relationship of green and albino individuals in mixed populations of Cephalanthera orchids

Several green orchids of the Neottieae tribe acquire organic carbon both from their mycorrhizal fungi and from photosynthesis. This strategy may represent an intermediate evolutionary step towards mycoheterotrophy of some non‐photosynthetic (albino) orchids. Mixed populations of green and albino individuals possibly represent a transient evolutionary stage offering opportunities to understand the evolution of mycoheterotrophy. In order to understand the emergence of albinos, we investigated patterns of spatial and genetic relationships among green and albino individuals in three mixed populations of Cephalanthera damasonium and one of C. longifolia using spatial repartition and Amplified fragment length polymorphism (AFLP) markers. Two of these populations were monitored over two consecutive flowering seasons. In spatial repartition analyses, albino individuals did not aggregate more than green individuals. Genetic analyses revealed that, in all sampled populations, albino individuals did not represent a unique lineage, and that albinos were often closer related to green individuals than to other albinos from the same population. Genetic and spatial comparison of genets from the 2‐year monitoring revealed that: (i) albinos had lower survival than green individuals; (ii) accordingly, albinos detected in the first year did not correspond to the those sampled in the second year; and (iii) with one possible exception, all examined albinos did not belong to any green genet from the same and/or from the previous year, and vice versa. Our results support a scenario of repeated insurgence of the albino phenotypes within the populations, but unsuccessful transition between the two contrasting phenotypes. Future studies should try to unravel the genetic and ecological basis of the two phenotypes.     

The purification and properties of Factor X from pig serum and its role in hypercoagulability in vivo

The molecular weights or shapes of Factor X preparations determined by gel filtration were dependent on the density of the BaSO(4) used for the initial adsorption from serum. One form obtained with BaSO(4) of density 2g/ml behaved as if it had a molecular weight of 39000 and possessed preformed clotting activity (Factor Xa), whereas that of the form adsorbed with BaSO(4) of density 1g/ml had a molecular weight of 69000 and consisted of inactive Factor X precursor. Thus degradation accompanied by activation seems to occur as a result of surface adsorption on high-density BaSO(4) and is associated with an interchange of protein between the two bands observed electrophoretically. The clotting and esterase activities measurable in vitro after complete activation were not matched by a corresponding ability to induce thrombus formation and ;lethality' in vivo. The most effective preparations of Factor X in this respect possessed preformed activity, which was enhanced in the presence of phospholipid. Factor X lost activity more rapidly in dilute solution, and its concentration at the surface of phospholipid micelles probably decreases loss by dilution in circulating blood.