The specific binding of N-acetylneuraminic acid to wheat-germ agglutinin is based on configurational similarities between N-acetylneuraminic acid and N-acetylglucosamine. The N-acetamido group and an adjacent hydroxyl group, both in an equatorial position are shown to be the main determinants. The N-acetylneuraminic acid -wheat-germ agglutinin interaction is increased by the removal of the last two carbons Cs and C9. The interaction between wheat-germ agglutinin and glycoconjugates containing N-acetylneuraminic acid is shown to be dependent on a charge effect and on an avidity effect. Succinylated wheat-germ agglutinin which is negatively charged at physiological pH, in contrast with wheat-germ agglutinin which is positively charged, does not bind cell surface glycoconjugates containing N-acetylneuraminic acid but does bind cell surface glycoconjugates containing N-acetylglucosamine. The use of wheat-germ agglutinin and of SUCcinylated wheat-germ agglutinin leads to the determination of the number of cell surface receptors containing N-acetylneuramipic acid Wheat-germ agglutinin is a plant lectin which agglutinates various types of animal cells, of malignant cells and of protease-treated cells [l-31. The agglutination of cells is inhibited by N-acetylglucosamine and its /3l-4 oligomers [2,4]. In addition, wheat-germ agglutinin has been claimed to bind N-acetylneuraminic acid on the basis of equilibrium dialysis [5] and nuclear magnetic resonance [6] studies. Furthermore, the agglutination of neuraminidasetreated cells usually requires a higher concentration of wheat-germ agglutinin than required for untreated cells [2,7] although in some instances the neuraminidase treatment does not lower the cell agglutinability by the lectin [8]. Immobilized wheat germ agglutinin binds membrane sialoglycoproteins [9,10] but fails to bind the related sialoglycopeptides [9]. In the present paper, we wish to report several data showing that N-acetylneuraminic acid, gangliosides and glycoAbbreviations. Glc, glucose; Gal, galactose, NeuAc, N-acetylneuraminic acid ; NeuGc, N-glycoloylneuraminic acid ; Cer, ceramide; GM3, NeuAca2+3Gal~l+4GlcCer; GMz, GalNAc(3tZa-NeuAc)~l+4Gal~1+4GlcCer; GMI, Ga1/31+ 3GalNAc(3+2a-N e u A c )~l + 4 G a l~I -4GlcCer; GDI,, NeuAca2-3GalP1+3-GalNAc(3t2ctNeuAc)fll+4Gal~1-r4GlcCer; GDlb, Galpl -r 3-GalNAc(3+2aNeuAcXt2aNeuAc)~l+4Gal~I + 4GlcCer; GTl, NeuAcn2+ 3Gal/1'1+ 3GalNAc(3 + 2aNeuAcX t 2aNeuAc)flI -j 4-Gal81 +4GlcCer, Nph, p-nitrophenyl-.

The physicochemical and binding properties of succinylated wheat germ agglutinin are described in comparison with these of unmodified wheat germ agglutinin. Succinylated wheat germ agglutinin is an acidic protein with a p l of 4.0 0.2 while the native lectin is basic, p l of 8.5. The solubility of succinylated wheat germ agglutinin is about 100 times higher than that of the unmodified lectin at neutral pH. Both lectins are dimeric at pH down to 5, and the dissociation occurs at pH lower than 4.5. The binding of oligosaccharides of N-acetylglucosamine to both lectins is very similar on the basis of fluorescence and phosphorescence studies. The minimal concentration required to agglutinate rabbit red blood cells is about 2 pg/ml with both lectins and the concentrations of AT-acetylglucosamine and di-N-acetylchitobiose which inhibit agglutination are similar with both lectins.The number of succinylated wheat germ agglutinin molecules bound to the surface of mouse thymocytes was ten times lower than that of the unmodified lectin although the apparent binding constant was only slightly different between the two lectins.The dramatic decrease of the apparent number of cell surface receptors upon succinylation of the lectin is discussed on the basis of the decrease of the isoelectric point and of the acidic properties of the cell surface.Wheat germ agglutinin is a plant lectin which is capable of agglutinating erythrocytes and some other types of cells [l] (for recent reviews see [2,3]).Agglutination is inhibited by N-acetylglucosamine and its (gl-4)oligomers [4-61. Wheat germ agglutinin is a basic protein with an isoelectric point of 8.7 & 0.3 [7]. At neutral pH, wheat germ agglutinin is a dimer; in acidic medium, it dissociates into two subunits of M , 18000 [8,9].Subunit dissociation also occurs upon acetylation [7] or tryptophan oxidation with N-bromosuccinimide [lo]; this dissociation is concommitent with a total loss of agglutination properties. However upon succinylation, neither subunit dissociation nor loss of A+ human red-blood-cell agglutinating activity occur [7]. In the present paper, the physicochemical properties and the sugar binding properties of succinylated wheat germ agglutinin are compared with those of the unmodified lectin. MATERIALS AND METHODS MaterialsWheat germ agglutinin prepared as in [ l l ] was purchased from Pharmindustrie-IBF-Reactifs, Clichy (France). Tri-N-acetylchitotriose and tetra-N-acetylchitotetraose were prepared by acetolysis of chitin, isolation of the per-0,N-acetylated oligosaccharides by silica gel column chromatography [12], de-0-acetylation by catalytic methanolysis with 0.01 M sodium methoxide in methanol at 4°C for 24 h. The tri-Nacetylchitotriose and the tetra-N-acetylchitotetraose were obtained by crystallization from their methanolic solution. Biogel P-60 was purchased from Biorad. Ultrogel ACA 54, Ultrogel A4 and HA-Ultrogel (hydroxyapatite) were purchased from Pharmindustrie-IBF-Reactifs. Fluorescein isothiocyanate was obtained from Research Organics Inc. (Clevela...

Aims-To investigate the eVects of slide storage on immunohistochemical staining, since recent reports have indicated that storage of unstained paraYn slides for up to 12 weeks may lead to false negative immunostaining of tumour markers. Methods-11 antibodies (anti-cytokeratin, epithelial membrane antigen (EMA), vimentin, smooth muscle actin, PS100, chromogranin, CD45, CD20, CD3, CD30, and oestrogen receptor (OR)) were tested on unstained paraYn slides of breast carcinomas, lymphomas, and neuroendocrine tumours that had been stored for three to 10 years. All the paraYn blocks were recut less than one week before immunostaining. Immunostainings of years old slides were compared with those of recent slides in at least five cases for each antibody. For three antibodies (antichromogranin, anti-CD3, and anti-OR) we also tested one year old and three months old slides. Results-Intensity of staining on years old slides was strikingly reduced for chromogranin and CD3 in several cases and was slightly stronger for vimentin. In some cases a significant decrease of OR positivity was observed after three months storage, and a complete loss of OR immunostaining after 12 months. No significant diVerence was noted with the other antibodies. Conclusions-Immunohistochemical detection of some antigens located either in the nucleus, in the cytoplasm, or on the cytoplasmic membrane could be impaired by storage of paraYn slides as short a time as three months. One should be cautious of doing retrospective immunohistochemical studies on stored unstained slides.

Ad CFTR, a replication-deficient adenovirus expressing the human cystic fibrosis transmembrane conductance regulator (CFTR), was administered by aerosolization in a single escalating dose to three pairs (cohorts) of cystic fibrosis (CF) patients. Buffer only was administered to the nose and lungs 9-14 days before nasal instillation of virus followed the day after by aerosolization of Ad CFTR to the lung. Nasal doses (defined in terms of viral plaque forming units, pfu) were 10(5), 10(7), and 4 x 10(8), whereas aerosolized doses were 10(7), 10(8), 5.4 x 10(8) for each cohort, respectively. No acute toxic effects were observed in the first 4 weeks after virus treatment. Shedding of infectious Ad CFTR was never detected, whereas detection of vector DNA sequences and CFTR expression demonstrated DNA transfer to the nose and airways of patients. No significant deviations in immunological and inflammatory parameters were observed in serum and in bronchoalveolar lavage (BAL). Importantly, for all patients, the serum anti-adenovirus antibody levels did not change significantly from baseline and no antibodies against adenovirus were found in BAL.