Vascular endothelial growth factor inhibits programmed cell death of endothelial cells induced by clinorotation

The principal aim of this study was to investigate short- and long-term effects of clinorotation on human endothelial cells (EA hy 926 cell line) using a three-dimensional random positioning machine. Moreover, the impact of vascular endothelial growth factor (VEGF) was addressed. Immediately, within one hour and after four and twenty-four hours an increase of apoptotic cells was detected. VEGF significantly inhibited the amount of apoptotic endothelial cells (EC). VEGF reduced the amount of fas-positive EC. Moreover, after 24 hours, proliferating EC grew in form of three-dimensional multicellular spheroids and also as monolayers. The initially formed spheroids (maximum diameter 3 mm) remained stable up to the 15th day of clinorotation. Some spheroids revealed tubular structures. In addition, a clear increase of extracellular matrix proteins such as osteopontin and fibronectin was measured. The three-dimensional clinostat represents an important tool for cell biological experiments. VEGF significantly attenuated the changes of endothelial cells induced by simulated weightlessness in a cell protective manner.     

Invasion of HEp-2 and Other Eukaryotic Cell Lines by Providenciae: Further Evidence Supporting the Role of Providencia alcalifaciens in Bacterial Gastroenteritis

Wild-type strains of Providencia species were evaluated for their ability to invade HEp-2 monolayers based upon microscopic and semi-quantitative assays. Of 14 P. alcalifaciens strains tested, 3 (17%) were found to be highly invasive, 4 (22%) moderately invasive, and the remaining 61% weakly or noninvasive. HEp-2 invasion results were confirmed by thin-section electron microscopy. Invasive capabilities of P. alcalifaciens were greater at higher MOIs (100 to 1000) than at lower inocula (<10 MOI). No strain of P. stuartii or P. rettgeri tested invaded HEp-2 cells. Quantitative assays of Triton X-100-lysed, HEp-2-invaded cells indicated that between 0.001% and 0.013% of the initial bacterial inoculum was gentamicin resistant. Further testing of select strains on various cell lines indicated the efficiency of invasion was Vero > Y1 > INT-407 > HEp-2. Two isolates recovered from a father and son with prolonged diarrhea after returning from Mexico were found to be identical on the basis of biotype, serotype, and genotype. These results provide additional evidence that some P. alcalifaciens strains cause gastroenteritis. Received: 1 December 1997 / Accepted: 25 March 1998     

Identification and Initial Characterization of Elastase Activity Associated with Vibrio cholerae

Strains of Vibrio cholerae O1 (Ogawa, Inaba) and non-O1 serogroups have been found to produce an elastolytic protease that can be detected on 0.3% elastin agar plates or in broth cultures. The elastase enzyme appears to be maximally expressed in late log phase (14–18 h postinoculation) and has optimum activity at a pH range between 7 and 8. Comparative studies indicate that more than 60% of V. cholerae strains analyzed quantitatively produce more elastase in broth (two- to fourfold higher) than other elastase-positive Vibrio species such as Vibrio vulnificus. The V. cholerae elastase enzyme was not inhibited by trypsin, serine-protease, or thiol-protease inhibitors, but was inhibited by phosphoramidon. Ultrafiltration studies indicate the V. cholerae elastase enzyme has a molecular weight >30,000, and a 34K protein with possible elastase activity has been detected by SDS-PAGE for one non-O1 isolate (strain 2396). Cumulative results suggest that the V. cholerae elastase is probably a member of the N-type metalloprotease family and shares similar properties with other elastase enzymes described for pathogenic and nonpathogenic species in this genus. Received: 26 February 1999 / Accepted: 29 March 1999     

Longterm conditions of mimicked weightlessness influences the cytoskeleton in thyroid cells

Weightlessness influences the human immune and hormone system, reduces bone mass, leads to muscle atrophy and cardiac atrophy. Effects on control mechanisms for proliferation, programmed cell death and differentiation are well documented. The principal aim of this study was to investigate changes of the cytoskeleton in thyroid cells cultured in vector-averaged gravity under clinostat rotation. After 12 hours the formation of multicellular spheroids started. An increase of extracellular matrix proteins and beta 1-integrin was observed. Laser scanning confocal microscopy of ML-1 follicular thyroid carcinoma cells and normal thyroid HTU-5 cells immunostained with anti-cytokeratin to demonstrate these intermediate filaments revealed that cytokeratin filaments extended from centers, were thickened, coalesced and shortened as compared to control cells. Moreover, vimentin was highly disorganized. The vimentin network formed a coiled aggregate closely associated with the nucleus. Western blot analyses of talin, alpha- and beta-tubulin showed a clear increase of these proteins in cells cultured under simulated 0 g. Our data suggest that the effects of microgravity on cultured human thyroid cells are accompanied by noticeable functional cellular changes. Future studies to clarify the pathway that regulate the observed integrin activation and the mechanisms by which they function have to be performed.     

Human serum IgA1 is substituted with up to six O-glycans as shown by matrix assisted laser desorption ionisation time-of-flight mass spectrometry

The micro-heterogeneity of human serum IgA1 results from variable O-glycan substitutions in the 'hinge region' of the molecule and this O-glycosylation may be altered in a number of medical conditions. This micro-heterogeneity has been monitored by analysis of IgA1-derived tryptic O-glycopeptides using matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-ToF-MS) analysis. With ammonium citrate-trihydroxyacetophenone matrix, individual compositional glycoforms have been baseline resolved in more than 70 samples and these spectra revealed for the first time that, in addition to expected substitution with 3,4 and 5 GalNAcs, a sixth GalNAc substitution was also present in the hinge region of the molecule. The spectra obtained from subsequent exoglycosidase-treated samples confirmed hexa-O-substitution. Following endoprotease digestions of the exoglycosidase treated samples, possible locations for the sixth GalNAc were indicated from further MALDI-ToF-MS analysis. Hexa-substitution accounts for around 5-10% the glycoforms. This is, we believe, the first report of hexa-O-substitution with GalNAc of human serum IgA1.     

Identification and characterization of maize pathogenesis-related proteins. Four maize PR proteins are chitinases

Eight pathogenesis-related proteins extractable at pH 2.8 were found to accumulate in maize leaves after mercuric chloride treatment or brome mosaic virus infection. These proteins were called PRm (pathogenesis-related maize) proteins. Seven PRm proteins were purified to homogeneity by preparative polyacrylamide gel electrophoresis and their amino acid compositions determined. Estimated molecular weights in SDS-containing gels were: PRm 1 14.2 kDa; Prm 2 16.5 kDa; PRm 3 and PRm 4 25 kDa; PRm 6b 30.5 kDa; PRm 6a 32 kDa; PRm 7 34.5 kDa. Antisera raised against either PRm 3 or PRm 4 reacted specifically each with PRm 3 or PRm 4. Antisera raised against PRm 6b reacted with PRm 6b as well as with PRm 6a and antisera against PRm 7 reacted with PRm 7 and PRm 5. Tobacco anti-PR 1b antisera reacted with maize PRm 2.Chitinase (poly[1,4-(N-acetyl--D-glucosamide)]glycanhydrolase, EC 3.2.1.14) activity was found for PRm 3, PRm 4, PRm 5, and PRm 7.     

Integration of pro- and anti-angiogenic signals by endothelial cells

Angiogenesis or neovascularization is a complex multi-step physiological process that occurs throughout life both in normal tissues and in disease. It is tightly regulated by the balance between pro-angiogenic and anti-angiogenic factors. The angiogenic switch has been identified as the key step during tumor progression in which the balance between pro-angiogenic and anti-angiogenic factors leans toward pro-angiogenic stimuli promoting the progression of tumors from dormancy to dysplasia and ultimately malignancy. This event can be described as either the outcome of a genetic event occurring in cancer cells themselves, or the positive and negative cross-talk between tumor-associated endothelial cells and other cellular components of the tumor microenvironment. In recent years, the mechanisms underlying the angiogenic switch have been extensively investigated in particular to identify therapeutic targets that can lead to development of effective therapies. In this review, we will discuss the current findings on the regulatory pathways in endothelial cells that are involved in the angiogenic switch with an emphasis on the role of anti-angiogenic protein, thrombospondin-1 (TSP-1) and pro-angiogenic factor, vascular endothelial growth factor (VEGF).