Different O-glycosylation of respiratory mucin glycopeptides from a patient with cystic fibrosis

The O-linked oligosaccharides from three fractions of highly glycosylated mucin glycopeptides obtained from sputum of a patient with cystic fibrosis were characterized and compared regarding size, composition, sequence and when possible linkage positions. Neutral and sialic acid-containing glycans were permethylated and analyzed by high-temperature GC-MS and MALDI-MS, showing more than 60 different oligosaccharides with a size of up to 15 monosaccharide units. Some of the observed oligosaccharides are novel for respiratory secretions, one being a trifucosylated heptasaccharide with the proposed structure: Fuc-Gal-4(Fuc-3)GlcNAc-(Fuc-)Gal-3GalNAcol. The glycosylation of two of the glycopeptide fractions was similar with regard to the neutral and sialylated oligosaccharides despite their different origins from the sol or gel phase. Analysis of the sulfated oligosaccharides by FAB-MS/MS indicated that the gel fraction contained C-6 linked sulfate groups while the two sol fractions also contained C-3 linked sulfate. The results suggest the presence of different glycosylated mucin domains, probably originating from different mucin glycoforms and/or apoproteins in the airway of cystic fibrosis patients.     

Expression of sialyltransferase is not required for interaction of Neisseria gonorrhoeae with human epithelial cells and human neutrophils

Sialyltransferase (Stase) in Neisseria gonorrhoeae organisms (gonococci [GC]) transfers sialic acid (N-acetylneuraminic acid [NANA]) from cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NANA) mainly to the terminal galactose (Gal) residue in the Gal beta-1,4 N-acetylglucosamine (Gal-GlcNAc)-R lipooligosaccharide (LOS) structure. Sialylated GC resist killing by normal human serum, sometimes show reduced invasion of epithelial cells, and have reduced adhesion to and stimulation of human neutrophils. We questioned whether Stase itself modulates the interactions of GC with human epithelial cells and neutrophils in the absence of exogenous CMP-NANA. To that end, we treated strain F62 with ethyl methanesulfonate and grew approximately 175,000 colonies on CMP-NANA plates, and screened them with monoclonal antibody 1B2-1B7 (MAb 1B2). MAb 1B2 is specific for Gal-GlcNAc and reacts only with asialylated GC. We isolated 13 MAb 1B2-reactive mutants, including five null mutants, that had Stase activities ranging from barely detectable to fivefold less than that of wild-type (WT) F62. The LOS phenotype of Stase null mutants was identical to that of WT F62, yet the mutants could not sialylate their LOS when grown with CMP-NANA. The Stase null phenotype was rescuable to Stase+ by transformation with chromosomal DNA from WT F62. Stase null mutants remained serum sensitive even when grown with CMP-NANA. One Stase null mutant, ST94A, adhered to and invaded the human cervical epithelial cell line ME-180 at levels indistinguishable from that of WT F62 in the absence of CMP-NANA. In human neutrophil studies, ST94A stimulated the oxidative burst in and adhered to human neutrophils at levels similar to those of WT F62. ST94A and WT F62 were also phagocytically killed by neutrophils at similar levels. These results indicate that expression of Stase activity is not required for interaction of GC with human cells.     

Voraussage der Bitterkeit von Peptiden aus deren Aminos?urezu-sammensetzung

Zusammenfassung Es wind eine neue Methode beschrieben, die es gestattet, anhand der Aminosäurezusammensetzung eines Peptides vorauszusagen, ob theses bitter ist. Dazu wird aus Energie-Daten für die relativen Löslichkeiten der einzelnen Aminosäuren ein WertQ errechnet. Peptide mitQ < 1300 sind nicht bitter. Peptide mitQ > 1400 haben einen bitteren Geschmack. Alle bisher beschriebenen synthetischen Peptide mit definiertem Geschmack (Di- bis Eicosapeptide, 21 selbst synthetisiert, 37 aus der Literatur) entsprechen unserer Regel. Auch 8 in der Literatur beschriebene Oligopeptide aus Spinat mit bekannter Aminosdurezusammensetzung passen in die Regel.

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Prediction of bitterness of peptides from their amino acid composition

Summary A method is described permitting to predict whether a peptide is bitter or not. The method is based on the amino composition, whereby a valueQ is calculated from the solubility data of the individual amino acids. Peptides withQ < 1300 are not bitter, peptides withQ > 1400 show a bitter taste.All synthetic peptides with defined taste described so far (di- to eicosapeptides, 21 synthesised ourselves, 37 from the literature) do comply with our rule. Eight oligopeptides from spinach with known amino acid composition described in the literature fit also in this scheme.

     

Significance of local extradural cooling on cervical spinal cord (experimental study)

The PQ-interval was measured while resting supine before exercise testing, in the erect position on the bicycle before starting exercise, and resting supine after exercise in 68 men 6--8 weeks after acute myocardial infarction. During a 6-year follow-up period the death was non-sudden (greater than 1 hour) in 25 of these patients. In this group the PQ-time was significantly shorter (p less than 0.001) during somatomotor activation on the bicycle before exercise than resting supine. The same directional change (p less than 0.01) was seen in the sudden death (less than 1 hour) group (N = 21), but not in the patients who survived. PQ-time at supine rest before exercise testing, however, was significantly shorter (p less than 0.02) in surviving patients than in the non-sudden death group. The possible mechanisms of these, and of previously reported changes in the R-wave amplitudes and QT-times, are discussed.     

Biochemical properties and cellular localization of the Drosophila DG1 cGMP-dependent protein kinase

The protein encoded by the Drosophila cGMP-dependent protein kinase gene, DG1, was expressed in Sf9 cells. cGMP (10 microM) stimulated histone H2B phosphorylation by the DG1 protein kinase 20-fold. Maximal activity was observed at 40-50 mM Mg2+. The concentrations of cGMP, cAMP, cIMP, 8-bromo-cGMP, and 8-bromo-cAMP that gave 50% activation were 0.19 +/- 0.06, 11.7 +/- 2.8, 5.3 +/- 1.5, 0.04 +/- 0. 01, and 0.62 +/- 0.06 microM, respectively. cGMP activation was cooperative with a Hill coefficient (nH) of 1.28 +/- 0.10, whereas activation by cAMP was not cooperative. DG1 kinase expressed in Sf9 cells was found to be a dimer with an amino-terminal dimerization domain. It also autophosphorylated in a reaction stimulated by cGMP and cAMP. Immunoadsorbed DG1 protein from fly extracts was also capable of autophosphorylation, and this assay was used to quantitate the DG1 kinase in extracts from heads and bodies of adults and whole embryos. Activity was highest in heads of either sex and male bodies, intermediate in female bodies, and lowest in embryos. These results were in accord with DG1 mRNA abundance. Tissue distribution of the DG1 kinase was investigated by immunohistochemistry. In embryos, specific immunoreactivity was observed in large cells scattered along the anterior-posterior axis at stage 13. Prominent staining of adult heads was restricted to the proximal level of the lamina cortex.     

Inhibitory effects of interleukin-1 blockers on corneal fibroblast proliferation

The regulation of would healing is one of the most important fields of research in ophthalmology today. Rabbit corneal fibroblast cultures were used to study the effects of interleukin-1 (IL-1) blockers on the proliferation of fibroblasts which is closely related to the wound healing. It was found that IL-1 blockers, such as CK-17, CK-101A, CK-2 and CK-103A, suppress fibroblast proliferation in a concentration-dependent manner. DNA synthesis was significantly inhibited by CK-17 and CK-103A but not by CK-101A and CK1-102. Although the synthesis of mRNA was reduced by all CK-compounds at most concentration tested, the synthesis of protein was only slightly reduced or unaffected. These results indicate that CK-compounds are potent fibroblast inhibitors but not cytolytic agents.     

Ab initio calculation of the BCC Fe–Al–Mo (Iron–Aluminum–Molybdenum) phase diagram: Implications for the nature of the phase

The metastable phase diagram of the BCC-based ordering equilibria in the Fe–Al–Mo system has been calculated via a truncated cluster expansion, through the combination of Full-Potential-Linear augmented Plane Wave (FP-LAPW) electronic structure calculations and of Cluster Variation Method (CVM) thermodynamic calculations in the irregular tetrahedron approximation. Four isothermal sections at 1750 K, 2000 K, 2250 K and 2500 K are calculated and correlated with recently published experimental data on the system. The results confirm that the critical temperature for the order–disorder equilibrium between Fe₃Al–D0₃ and FeAl–B2 is increased by Mo additions, while the critical temperature for the FeAl–B2/A2 equilibrium is kept approximately invariant with increasing Mo contents. The stabilization of the Al-rich A2 phase in equilibrium with overstoichiometric B2–(Fe,Mo)Al is also consistent with the attribution of the A2 structure to the τ₂ phase, stable at high temperatures in overstoichiometric B2–FeAl.