Isolation and structural characterization of fucosylated gangliosides with linear poly-N-acetyllactosaminyl chains from human granulocytes

The isolation and structural characterization of fucosylatedneolacto-series gangliosides with linear poly N-acetyllactosaminylchains from normal human granulocytes is described. Gangliosideswere purified by consecutive use of anion exchange HPLC on FractogelTMAE-650(S), adsorption and reversed phase HPLC on Nucleosil50-7 and Nucleosil 7C₁₈ columns, respectively. TLC immunostainingwith carbohydrate specific monoclonal antibodies, fast atombombardment-mass spectrometry (FAB-MS) of the permethylatedderivatives and gas chromatography-electron impact mass spectrometry(GC-EIMS) of partially methylated alditol acetates were usedfor structure elucidations. One ganglioside was identified assialyl Lewis antigen with nLcOse₆Cer core, Neu5-Ac     

Meiofauna of silty sediments in the coastal area of the North Adriatic,with special reference to sampling methods

At five coastal silty sediment stations ranging in depth from 8 to 30 m, the abundance and composition of meiofauna were investigated. Three methods of sampling were used, i.e. Pfleger corer, Van Veen grab and SCUBA divers. Four samples per station were taken. The mean density of total meiofauna was 660 ± 109 ind. 10 cm². The main meiofauna group was Nematoda, the second abundant was Copepoda, and third was Kinorhyncha. Statistical tests showed significant differences in meiofaunal abundance between corer and grab samples, and between corer and divers samples.Differences in meiofauna abundance between stations were found.     

A sequencing strategy for the localization of O-glycosylation sites of MUC1 tandem repeats by PSD-MALDI mass spectrometry

It is demonstrated with glycopeptides of the polymorphic epithelialmucin (MUC1) that post-source decay matrix-assisted laser desorptionionization (PSD-MALDI) is a fast, highly sensitive, and reproduciblemethod for the localization of O-glycosylation sites by reflectrontime-of-flight (TOF) mass spectrometry. We have analyzed GalNAc-carryingpeptides of up to 25 amino acids, and could distinguish evenneighboring glycosylation sites. This method was also able tolocalize and characterize disaccharides (e.g., the Thomsen-Friedenreichdisaccharide) on MUC1 derived peptides. PSD-MALDI-MS fragmention patterns were recorded in the positive ion mode from thesynthetic peptide TAP25 [(T^1aAPPAHGVT⁹S¹⁰APDT¹⁴RPAPGS²⁰) T^1bAPPA],an overlapping sequence of MUC1 tandem repeats, which was glycosylatedwith GalNAc in vitro. The glycosylation sites found were eitherThr9 or Thr1b in the monoglycosylated, Thr9 and Thr1b in thediglycosylated, and Thr9, Thr1b, and Ser20 in the triglycosylatedpeptide. A single PSD-MALDI-MS spectrum of the underivatizedand uncleaved di- or triglycosylated TAP25 peptide was sufficientto identify the glycosylation sites, thereby distinguishingsix potential, partly adjacent, glycosylation sites. The monoglycosylatedfraction was found to consist of a mixture of two glycosylatedspecies with the same molecular weight. This was shown by theanalysis of proteolytic digests. PSD-MALDI-MS of the resultingpeptides right out of the digestion probe was sufficient toidentify the GalNAc-glycosylation sites as either Thr9 or Thr1b,respectively. Beyond the methodical aspects the results revealedthat in vitro glycosylation of the TAP25 peptide with a transferasesystem from human milk differs from that obtained with a breastcancer cell transferase system. glycosylation sites O-glycosylation PSD-MALDI-MS MUC1 mucins     

Different binding capacities of influenza A and Sendai viruses to gangliosides from human granulocytes

The structures of gangliosides from human granulocytes were elucidated by fast atom bombardment mass spectrometry and by gas chromatography/mass spectrometry as their partially methylated alditol acetates. In human granulocytes besides G_M3 (II³Neu5Ac-LacCer), neolacto-series gangliosides (IV³Neu5Ac-nLcOse₄Cer, IV⁶Neu5Ac-nLcOse₄Cer and VI³Neu5Ac-nLcOse₆Cer) containing C_24:1, and to some extent C_22:0; and C_16:0 fatty acid in their respective ceramide portions, were identified as major components. In this study we demonstrate that gangliosides from human granulocytes, the second most abundant cells in peripheral blood, can serve as receptors for influenza viruses A/PR/8/34 (H1N1), A/X-31 (H3N2), and a parainfluenza virus Sendai virus (HNF1, Z-strain). Viruses were found to exhibit specific adhesion to terminal Neu5Ac2-3Gal and/or Neu5Ac2-6Gal sequences as well as depending on the chain length of ganglioside carbohydrate backbones from human granulocytes, these important effector cells which represent the first line of defence in immunologically mediated reactions. Abbreviations: FAB-MS, fast atom bombardment mass spectrometry; GC/EIMS, gas chromatography/electron impact mass spectrometry; GSL(s) glycosphingolipids; HPTLC, high performance thin-layer chromatography; Neu5Ac,N-acetylneuraminic acid [26], PFU, plaque forming unit. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations, and the ganglioside nomenclature system of Svennerholm was used. LacCer or lactosylceramide, Gal1-4Glc1-1Cer gangliotetraosylceramide or GgOse₄Cer, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; lacto-N-tetraosylceramide or nLcOse₄Cer, Gal1-4GlcNAc1-3Gal1-4-Glc1-1Cer; lacto-N-norhexaosylceramide or nLcOse₆Cer, Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal 1-4-Glc1-1Cer; G_M3, II³Neu5Ac-LacCer; G_M1, II³Neu5Ac-GgOse₄Cer; G_D1a, IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer; G_D1b, II³(Neu5Ac)₂-GgOse₄Cer; G_T1b, IV³Neu5Ac, II³(Neu5Ac)₂-GgOse₄Cer; G_Q1b, IV³(Neu5Ac)₂, II³(Neu5Ac)₂-GgOse₄Cer; sialyllacto-N-tetraosylceramide, IV³Neu5Ac/IV⁶Neu5Ac-nLcOse₄Cer; sialyllacto-N-norhexaosylceramide or i-active ganglioside, VI³Neu5Ac-nLcOse₆Cer.     

Monoclonal antibodies raised against membrane glycoproteins from mouse brain recognize N-linked oligomannosidic glycans

Monoclonal L3 and L4 antibodies have been shown to recognizecarbohydrate epitopes on several neural cell adhesion molecules;these epitopes can be released by treatment with endoglycosidaseH. In the present study, we have identified the oligosaccharidesreleased by endoglycosidase H from the cell adhesion moleculesAMOG and L1 by fast-atom bombardment mass spectrometry as beingsolely of the oligomannosidic type. Using neoglycolipids ofoligomannosidc glycans, we also report that both antibodiesshow the highest reactivity with the     

Attachment of β2-glycoprotein I to negatively charged liposomes may prevent the release of daughter vesicles from the parent membrane

The temperature-induced budding of POPC-cardiolipin-cholesterol, POPC-POPS-cholesterol and POPC-POPG-cholesterol giant lipid vesicles in the presence of beta(2)-glycoprotein I (beta(2)-GPI) in the outer solution was studied experimentally and theoretically. The observed budding transition of vesicles was continuous which can be explained by taking into account the orientational ordering and direct interactions between oriented lipids. The attachment of positively charged beta(2)-GPI to the negatively charged outer surface of POPC-cardiolipin-cholesterol, POPC-POPS-cholesterol and POPC-POPG-cholesterol giant vesicles caused coalescence of the spheroidal membrane bud with the parent vesicle before the bud could detach from the parent vesicle, i.e. vesiculate. Theoretically, the protein-mediated attraction between the membrane of a bud and the parent membrane was described as an interaction between two electric double layers. It was shown that the specific spatial distribution of charge within beta(2)-GPI molecules attached to the negatively charged membrane surface may explain the observed attraction between like-charged membrane surfaces.     

Invasive ant (<Emphasis Type="Italic">Anoplolepis gracilipes</Emphasis>) disrupts pollination in pumpkin

Yellow crazy ant (Anoplolepis gracilipes (F. Smith); “YCA”) is known for its aggressive predatory ability and ability to exert exploitation competition on both native and other invasive ants via floral nectar. We argue that YCA invasion can exert both interference and exploitation competition on legitimate pollinators. In pumpkin fields (Cucurbita maxima L.) of south India, YCA infested the flowers, particularly the pistillate flowers, for nectar foraging. Pumpkin is a honey bee-mediated cross-pollinated monoecious plant that produces disproportionately very few pistillate flowers. We hypothesize that YCA presence in the flowers can affect the visitation rate and foraging time of honey bees in the flowers, the fruit set in pumpkins, and can exert predatory pressure on the honey bees if the bees linger in ant-colonized flowers. Both YCA and honey bees preferred to forage on the limited pistillate flowers in the plants. After colonizing the flowers, YCA did not retreat for hours, even upon disturbance by competitors, such as honey bees. Both the visitation frequency and the foraging time of honey bees were drastically reduced in ant-colonized flowers, and none of the ant-colonized flowers developed into fruits, suggesting that the YCA exert both an ecological and evolutionary pressure on pumpkin. The ants preyed upon about 17% of the honey bees that lingered in ant-colonized flowers, and the time the bees spent foraging predicted the fate of the bees. Exploitation competition exerted by the YCA on pumpkin may have far-reaching consequences for the pollination and productivity of this cash crop.     

Global Analysis of the Hortaea werneckii proteome: studying steroid response in yeast

The response of the halophilic black yeast Hortaea werneckii to the steroid hormone progesterone has been studied at the protein level using fluorescent two-dimensional differential gel electrophoresis (2D-DIGE) technology in combination with mass spectrometry. Data on protein identification from this study reveal molecular mechanisms of the response to progesterone. In particular, the overexpression of Pck2 and Pac2 in the stimulated cells indicates the interactions of progesterone with the cell growth and reproduction signaling pathways.     

Risk of pneumonia recurrence in patients previously hospitalized for pneumonia--a retrospective study (1998-2000)

Although elderly hospitalized patients, irrespective of the cause of hospitalization, are known to be at a high risk of subsequent development of pneumonia, some studies suggest the risk to be even higher in those hospitalized for pneumonia than in those hospitalized for other diseases. The aim of this retrospective study was to determine the association of hospitalization for pneumonia and some other diseases with subsequent pneumonia morbidity and mortality. The risk of recurrent pneumonia in patients hospitalized for pneumonia was investigated. Rehospitalization of pneumonia patients previously hospitalized for the same disease was followed-up and compared with rehospitalization of patients hospitalized for other diseases during the same study period. The study included patients aged overl8, initially hospitalized in 1998 for pneumonia (J12-J18), or for some particular gastrointestinal (K20-K31) and urogenital diseases (N10-N12, N30-N39). All rehospitalizations for pneumonia in nine Zagreb hospitals were followed-up during a 3-year study period (1998-2000). Out of 975 patients followed-up for rehospitalization, 227 (23.3%) had initially been hospitalized for pneumonia, and 748 (76.7%) for other diagnoses. During the 3-year period, 30 patients were rehospitalized for pneumonia, out of which number 22 had initially been hospitalized for pneumonia, yielding a statistically significant difference between the two study groups (chi2 = 34.780, p < 0.001). The mortality directly caused by pneumonia was also significantly higher in the group of patients with the initial diagnosis of pneumonia than in the group of patients with other diagnoses (chi2 = 15.82, p < 0.001).