Expression of N-linked sialyl Le(x) determinants and O-glycans in the carbohydrate moiety of human amniotic fluid transferrin during pregnancy

Transferrin, a glycoprotein involved in iron transport in body fluids, was isolated from amniotic fluid of a hydramniospatient by sequential anion-exchange chromatography and gel filtration. The N-glycans of human amniotic fluid transferrin (hAFT) were enzymatically liberated by PNGase-F digestion, isolated by gel filtration and fractionated by (high-pH) anion-exchange chromatography. After alkaline borohydride treatment of native hAFT, the released O-glycans were isolated by gel filtration and fractionated by anion-exchange chroma-tography. Structure elucidation of 14 N- and 2 O-glycans was performed by 500 or 600 MHz1H-NMR spectroscopy. Besides conventional N-glycans established earlier for human serum transferrin (hST), new (alpha1-3)-fucosylated N- glycans were found, representing sialyl Le(x) elements. Furthermore, as compared to hST, a higher degree of (alpha1-6)-fucosylation and an increase in branching from di- to triantennary compounds has been detected. The presence of O-glycans is demonstrated for the first time in transferrin.      

Inhibitors of arachidonic acid metabolism reduce DNA and nuclear fragmentation induced by TNF plus cycloheximide in U937 cells

U937 human myeloid leukemia cells are induced to apoptosis by tumour necrosis factor (TNF) plus cycloheximide (CHX). We have analysed the effect of various inhibitors of the arachidonic acid (AA) metabolism on several features of this process. The formation of high molecular weight and oligonucleosomal DNA fragments as well as nuclear fragmentation were reduced by inhibitors of 5-lipoxygenase (BWA4C and BWB70C), 5-LO activating protein (MK-886), and cytosolic PLA2 (AACOCF3). None of these agents blocked the morphological changes detected by microscopy or flow cytometry, phosphatidylserine exposure on the cell surface or Caspase 3-like activation. AA also induced nuclear fragmentation at a concentration of 1-20 microM. However, the mechanisms by which these inhibitors act, remain unexplained since there was no 5-LO expression in the U937 cells and no AA release followed their stimulation with TNF plus CHX.     

Terminal phase of cytokinesis in D-98S cells

The events leading to the completion of cytokinesis after the formation of the midbody and intercellular bridge in D-98S cells were studied with light and electron microscopy. Pairs of daughter cells corresponding to different stages of cytokineses, as determined previously form time lapse films, were selected from embedded monolayers for serial sectioning. Separation of daughter cells is preceded by the reduction in diameter of the intercellular bridge from 1-1.5 μm to approx. 0.2 μm. Two processes contribute to this reduction: (a) The intercellular bridge becomes gradually thinner after telophase; a progressive breakdown of midbody structures accompanies this change; and (b) the more significant contribution to reduction in bridge diameter occurs through the localized constriction of a segment of the intercellular bridge.. The microtubules within the constricted portion of the bridge are forced closer together, and some microtubules disappear as this narrowing progresses. The plasma membrane over the narrowed segments is thrown into a series of wavelike ripples. Separation of daughter cells is achieved through movements of the cells which stretch and break the diameter-reduced bridge. The midbody is discarded after separation and begins to deteriorate. Occasional pairs of daughter cells were found in which incomplete karyokineses resulted in their nuclei being connected by a strand of nuclear material traversing the bridge and midbody. Such cells do not complete cytokinesis but merge together several hours after telophase. This merging of daughter cells coincides with the nearly complete breakdown of the midbody.