Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid.

Reaction conditions for conjugation of two fluorescent ortho-substituted aniline derivatives, 2-amino benzamide (2-AB) and 2-anthranilic acid (2-AA), to N- and O-glycans have been investigated. Conjugation conditions for attaching 2-AB and 2-AA to core-fucosylated and non-fucosylated glycans were developed using complex N-glycans radiolabeled at the nonreducing terminus with [3H]C6-galactose. Optimal conditions for each of the following reaction parameters were experimentally defined: [glycans], [2-AB] or [2-AA], solvent and acid composition, temperature and time of Schiff's base formation, nature of reductant, and temperature and time of reduction. Using the optimized reaction conditions it has been shown with several standard glycans and glycoprotein-derived glycan libraries that (i) molar labeling efficiencies are high and essentially independent of the amount of glycans; (ii) negligible (< 2 mol%) desialylation occurs during conjugation; (iii) glycan labeling is nonselective, i.e., independent of glycan structure; and (iv) insignificant fluorescent or chemical "blank" is recovered during the glycan-labeling and purification protocol. Labeling glycan pools with 2-AB or 2-AA therefore allows representative glycan profiles to be obtained and also allows relative molar quantitation of individual glycans in a pool. The 2-AB label is compatible with several chromatographic means for separation of carbohydrates including Bio Gel P4 gel permeation, high-performance anion-exchange chromatography with fluorescence detection, and a variety of HPLC procedures, as well as with mass spectrometric methods including matrix-assisted laser desorption-mass spectrometry and electrospray-mass spectrometry. The 2-AA label is particularly well-suited for electrophoretic separations by polyacrylamide gel electrophoresis. These fluorophores show high intrinsic sensitivity and thus facilitate very sensitive analysis of protein glycosylation.

Immunoreactive S-antigen in cerebrospinal fluid: a marker of pineal parenchymal tumors?

This investigation evaluated the possibility that the occurrence of S-antigen in cerebrospinal fluid (CSF) might be used as a preoperative marker of pineal parenchymal tumors (pineoblastoma and pineocytoma). Such a marker could provide a means of preoperatively differentiating these neoplasms from pineal region tumors of other origin. The S-antigen, also known as the 48-kD protein or arrestin, is a highly antigenic protein originally found in the retina and pineal gland. In the retinal photoreceptors and submammalian pineal photoreceptors the protein is thought to be involved in phototransduction; its function in the mammalian pinealocyte is unknown. S-Antigen immunoreactivity also occurs in certain neoplastic cells of retinoblastomas, pineocytomas, pineoblastomas, and cerebellar medulloblastomas. This study included a group of 13 patients with tumors of the pineal region. Samples of CSF were obtained preoperatively and analyzed for the S-antigen using western blot technology. Tumor biopsy material was classified according to conventional neurohistological criteria and was also examined by immunocytochemical techniques for the presence of the S-antigen. S-Antigen immunoreactivity was found in the preoperative CSF of the one patient found to have pineocytoma; tumor tissue removed from this patient was the only neoplastic tissue examined in this study which contained S-Antigen immunoreactive tumor cells. Furthermore, hydroxyindole-O-methyltransferase activity was detectable in the pineocytoma but not in three other pineal tumors, and melatonin levels in the CSF of the pineocytoma patient were the highest in the patient group examined. These preliminary results suggest that testing for S-antigen in CSF might be useful in characterizing and treating tumors of the pineal region and, when identified in conjunction with other markers, it might also help to better define pineal parenchymal tumors. This study needs confirmation with a larger number of patients. If this approach is eventually found to be a reliable predictor of pineal cell tumors, it may supplant the need for surgical biopsies before initiating appropriate adjunctive therapy.

Synthesis and characterization of a photosensitive interface for hydrogen generation: Chemically modified p-type semiconducting silicon photocathodes.

p-Si photocathodes functionalized first with an N,N'-dialkyl-4,4'-bipyridinium redox reagent, (PQ(2+/+-))(surf), and then with a Pt precursor, PtCl(6) (2-), give significant efficiency (up to 5%) for photoelectrochemical H(2) generation with 632.8-nm light. Naked p-Si photocathodes give nearly zero efficiency, owing to poor H(2) evolution kinetics that are improved by the (PQ(2+/+-))(surf)/Pt modification. The mechanism of H(2) evolution from p-Si/(PQ(2+/+-))(surf)/Pt is first photoexcitation of electrons to the conduction band of Si followed by (PQ(2+))(surf) --> (PQ(+-))(surf) reduction. The dispersion of Pt then catalyzes H(2)O reduction to give H(2) and regeneration of (PQ(2+))(surf). The overall energy conversion efficiency rivals the best direct optical to chemical conversion systems reported to date.