Ultramicroanalysis of reducing carbohydrates by capillary electrophoresis with laser-induced fluorescence detection as 7-nitro-2,1,3-benzoxadiazole-tagged N-methylglycamine derivatives

A method for ultramicroanalysis of carbohydrates using capillary electrophoresis with laser-induced fluorescence detection was developed, based on precapillary conversion to 7-nitro-2,1, 3-benzoxadiazole (NBD)-tagged N-methylglycamines. Although the derivatization involves two-step reactions, i.e., reductive N-methylamination followed by condensation with NBD-F, they can be carried out in a one-pot fashion and proceed quantitatively within ca. 50 min in total. Since the reaction conditions are mild, it does not cause desialylation. The derivatives can be well separated by capillary electrophoresis and sensitively detected by argon laser-induced fluorescence. It allowed detection of monosaccharides of down to nanomolar concentrations for analytical sample solution, which correspond to the attomole injected amounts, and good linearity was observed over a wide range. It was also successfully applied to analysis of N-glycans in a microgram quantity of a glycoprotein. Studies on the cleanup of derivatized product are also described in relation to N-glycan analysis.     

Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization

A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method.     

IL-4, but not vitamin D(3), induces monoblastic cell line UG3 to differentiate into multinucleated giant cells on osteoclast lineage

The formation of multinucleated giant cells (MGCs) from monocytes/macrophages is controlled by various cytokines, the roles of which are not fully understood. Both interleukin (IL)-4 and 1alpha,25(OH)(2) vitamin D(3) (D(3)) are known to induce MGC formation from monocytes/macrophages. D(3) is also known as a stimulator of osteoclast formation in the presence of stroma cells, and IL-4 as an inhibitor. Previously, we showed that IL-4-induced MGCs from monocytes/macrophages expressed tartrate resistant acid phosphatase (TRAP) activity and hydroxyapatite-resorptive activity in the presence of M-CSF without stroma cells. In this study, we examined the effects of D(3) and/or IL-4 on MGC formation and the characteristics of these MGCs using a monoblastic cell line (UG3), to elucidate the involvement of these factors in osteoclast development without stroma cells. D(3)-induced MGCs showed none of the markers of osteoclasts, such as TRAP activity, calcitonin receptor (cal-R) expression, hydroxyapatite-resorptive activity, and bone-resorptive activity. A low concentration of D(3) synergistically stimulated IL-4-induced TRAP-positive MGC formation, whereas a high concentration of D(3) inhibited it. When IL-4 was added on day 7 of the 2-week culture with D(3), TRAP positivity reached maximum. On the other hand, delayed addition of D(3) on day 7 of culture did not increase the TRAP positivity. Although the fusion rate increased during the first week of the 2-week culture in the presence of D(3), it increased further in the second week following the addition of IL-4 on day 7. Furthermore, IL-4-induced, or IL-4- and D(3)-induced MGCs differentiated into functional osteoclasts with bone-resorptive activity following coculture with osteoblastic cells, whereas D(3)-induced MGCs did not acquire bone-resorptive activity even after coculture with osteoblastic cells in the presence of D(3). These findings suggest that IL-4 initiates osteoclast development of UG3 cells, although stroma cells were necessary for development of functional osteoclasts. On the other hand, D(3) had only a "supportive" effect on this differentiation. IL-4 and direct contact with stroma cells may regulate different stages in the multistep process of osteoclastogenesis of UG3 cells.     

Attachment of Nontypable Haemophilus influenzae to Human Pharyngeal Epithelial Cells Mediated by a Ganglioside Receptor

Nontypable Haemophilus influenzae (NTHI) is one of the major pathogens of human respiratory infections and has the ability to attach to pharyngeal epithelial cells. We characterized the epithelial cell receptor to which NTHI bind. Neuraminidase pretreatment of pharyngeal epithelial cells resulted in a significant decrease in NTHI attachment, suggesting sialic acid as an important component of the receptor. The attachment was not decreased in NTHI pretreated with 1,000 μg/ml of fucose, N-acetyl-neuraminic acid, N-acetyl-glucosamine, N-acetyl-galactosamine, acetyl-salicylic acid and colominic acid. Only treatment with gangliosides D1a, D1b and D2 at a concentration of 12.5 μg/ml significantly decreased the attachment. On the other hand, treatment with gangliosides M1, M2, M3, D3, T1b and asialoganglioside M1 did not decrease the attachment of NTHI. Only ganglioside D2 inhibited the attachment significantly at a concentration of 12.5 ng/ml. Other isolates of NTHI showed a decrease in attachment after treatment with ganglioside D2. Treatment of cells with anti-human GD2 monoclonal antibody also decreased the attachment of NTHI in a dose-dependent manner. This study indicates that sialic acid glycoconjugate, GD2, is one of the receptors of NTHI on human pharyngeal epithelial cells.