This study investigates protein glycosylation in the asexual intraerythrocytic stage of the malaria parasite, Plasmodium ,fakiparum, and the presence in the infected erythrocyte of the respective precursors.In in vitro cultures, P. fakiparum can be metabolically labeled with radioactive sugars, and its multiplication can be affected by glycosylation inhibitors, suggesting the capability of the parasite to perform protein-glycosylation reactions. Gel-filtration analysis of sugar-labeled malarial proteins before and after specific cleavage of N-glycans or 0-glycans, respectively, revealed the majority of the protein-bound sugar label to be incorporated into 0-glycans, but only little (7-12% of the glucosamine label) or no N-glycans were found. Analysis of the nucleotide sugar and sugar-phosphate fraction showed that radioactive galactose, glucosamine, fucose and ethanolamine were converted to their activated derivatives required for incorporation into protein. Mannose was mainly recovered as a bisphosphate, whereas the level of radiolabeled GDP-mannose was below the detection limit. The analysis of organic-solvent extracts of sugar-labeled cultures showed no evidence for the formation by the parasite of dolichol cycle intermediates, the dedicated precursors in protein N-glycosylation. Consistently, the amount of UDP-N-acetylglucosamine formed did not seem to be affected by the presence of tunicamycin in the culture. Oligosaccharyl-transferase activity was not detectable in a lysate of P. fakiparum, using exogenous glycosyl donors and acceptors.Our studies show that 0-glycosylation is the major form of protein glycosylation in intrderythrocytic P. julciparum, whereas there is little or no protein N-glycosylation. A part of these studies has been published in abstract form [Dieckmann-Schuppert, A,, Hensel, J. and Schwarz, R. T. (1991) Biol. Chem. Hoppe-Seyler 372,6451.Plasmodium fakiparum is the causative agent of human malignant malaria tropica. Despite huge efforts in vaccine and chemotherapy development, today this disease still causes the death of several million people/year (World Health Organization, 1989). A more thorough understanding of the biochemistry and cell biology of this parasite is required in order to develop better chemotherapy and vaccination strategies. One of the neglected areas of malaria biochemistry is the glycobiology of the parasite. Very little is known to date about the biological significance of oligosaccharides in P. julcipurum, be they linked to lipids or to proteins. Glycolipids may be membrane components and as such be potential antigens, or be involved in the formation of glycoproteins, e. g. dolichol

To evaluate shear stress-induced effects on cultured cells we have extended the mechanical setup of a multichannel in vitro rheological system and developed software allowing entire processing control and image data analysis. The values of cell motility, degree of orientation (alignment), and cell elongation were correlated as a function of time (morphodynamics). Collective and individual endothelial cells within confluent cultures displayed a shear stress-dependent characteristic phase behavior of the following time course: resting conditions (phase I), change of motility (phase II), onset of alignment (phase III), and finally cell elongation (phase IV). Especially cell motility was characterized by a randomized zigzag movement around mean trajectories (fluctuations) together with mean cell locomotion. Onset of shear stress caused a down-regulation of fluctuations of 30% within <10 min and simultaneously increased locomotion velocities preferring the flow direction (phase II). After a lag period of 10 to 20 min cells orientated in the direction of flow (phase III) without significant cell elongation, which finally occurs within hours (phase IV). These data provide first evidence that cells within confluent endothelial monolayers respond to shear stress with a characteristic phase behavior.

Actin-binding proteins are essential for linear and branched actin filament dynamics that control shape change, cell migration, and cell junction remodeling in vascular endothelium (endothelial cells [ECs]). The epithelial protein lost in neoplasm (EPLIN) is an actin-binding protein, expressed as EPLIN-a and EPLIN-b by alternative promoters; however, the isoform-specific functions are not yet understood. Aortic compared to cava vein ECs and shear stress-exposed cultured ECs express increased EPLIN-b levels that stabilize stress fibers. In contrast, EPLIN-a expression is increased in growing and migrating ECs, is targeted to membrane protrusions, and terminates their growth via interaction with the Arp2/3 complex. The data indicate that EPLIN-a controls protrusion dynamics while EPLIN-b has an actin filament stabilizing role, which is consistent with FRAP analyses demonstrating a lower EPLIN-b turnover rate compared to EPLIN-a. Together, EPLIN isoforms differentially control actin dynamics in ECs, essential in shear stress responses, cell migration, and barrier function.

The glycosylation pattern of human vascular endothelial cadherin (VE-cadherin), purified from cultured human umbilical cord vein endothelial cells, was analyzed. VE-cadherin was metabolically radiolabeled with D-[6-3 H]glucosamine, isolated by immunoprecipitation, purified by SDS-PAGE and in-gel digested with endoproteinase Asp N. Oligosaccharides were sequentially released from resulting glycopeptides and analyzed by chromatographic profiling. The results revealed that VE-cadherin carries predominantly sialylated diantennary and hybrid-type glycans in addition to some triantennary and high mannose-type species. Highly branched, tetraantennary oligosaccharides were found in trace amounts only. Immunohistochemical labeling of VE-cadherin and sialic acids displayed a codistribution along the intercellular junctions in endothelial cells of human umbilical arteries, veins, and cultured endothelial monolayers. Ca 2+ -depletion, performed on cultured endothelial cells, resulted in a reversible complete disappearance of VE-cadherin and of almost all sialic acid staining from the junctions. Sialidase treatment of whole cells caused a change of VE-cadherin immunofluorescence from a continuous and netlike superstructural organization to a scattered inconsistent one. Hence, cell surface sialic acids might play a role in VE-cadherin organization.

Aims Oscillatory shear stress (OSS) is an atheroprone haemodynamic force that occurs in areas of vessel irregularities and is implicated in the pathogenesis of atherosclerosis. Changes in signalling and transcriptional programme in response to OSS have been vigorously studied; however, the underlying changes in the chromatin landscape controlling transcription remain to be elucidated. Here, we investigated the changes in the regulatory element (RE) landscape of endothelial cells under atheroprone OSS conditions in an in vitro model. Methods and results Analyses of H3K27ac chromatin immunoprecipitation-Seq enrichment and RNA-Seq in primary human umbilical vein endothelial cells 6 h after onset of OSS identified 2806 differential responsive REs and 33 differentially expressed genes compared with control cells kept under static conditions. Furthermore, gene ontology analyses of putative RE-associated genes uncovered enrichment of WNT/HIPPO pathway and cytoskeleton reorganization signatures. Transcription factor (TF) binding motif analysis within RE sequences identified over-representation of ETS, Zinc finger, and activator protein 1 TF families that regulate cell cycle, proliferation, and apoptosis, implicating them in the development of atherosclerosis. Importantly, we confirmed the activation of EGR1 as well as the YAP/TAZ complex early (6 h) after onset of OSS in both cultured human vein and artery endothelial cells and, by undertaking luciferase assays, functionally verified their role in RE activation in response to OSS. Conclusions Based on the identification and verification of specific responsive REs early upon OSS exposure, we propose an expanded mechanism of how OSS might contribute to the development of atherosclerosis.