Biomedical subjects

Denong Wang

Publications and source records attributed to Denong Wang.

8 recordsLinked to original sources

PubMed2006

Photochemical micropatterning of carbohydrates on a surface.

In this report, we demonstrate a versatile method for the immobilization and patterning of unmodified carbohydrates onto glass substrates. The method employs a novel self-assembled monolayer to present photoactive phthalimide chromophores at the air-monolayer interface. Upon exposure to UV radiation, the phthalimide end-groups graft to surface-adsorbed carbohydrates, presumably by a hydrogen abstraction mechanism followed by radical recombination to form a covalent bond. Immobilized carbohydrate thin films are evidenced by fluorescence, ellipsometry and contact-angle measurements. Surface micropatterns of mono-, oligo-, and polysaccharides are generated by exposure through a contact photomask and are visualized by condensing water onto the surface. The efficiency of covalent coupling is dependent on the thermodynamic state of the surface. The amount of surface-grafted carbohydrate is enhanced when carbohydrate surface interactions are increased by the incorporation of amine-terminated molecules into the monolayer. Glass substrates modified with mixed monolayers of this nature are used to construct carbohydrate microarrays by spotting the carbohydrates with a robot and subsequently illuminating them with UV light to covalently link the carbohydrates. Surface-immobilized polysaccharides display well-defined antigenic determinants for antibody recognition. We demonstrate, therefore, that this novel technology combines the ability to create carbohydrate microarrays using the current state-of-the-art technology of robotic microspotting and the ability to control the shape of immobilized carbohydrate patterns with a spatial resolution defined by the UV wavelength and a shape defined by a photomask.

Carbohydrates↗

PubMed2005

PRDM1/Blimp-1 is expressed in human B-lymphocytes committed to the plasma cell lineage.

PRDM1/Blimp-1 (in human and mouse, respectively) has a central role in determining and shaping the secretory arm of mature B-cell differentiation. In this study, a mouse monoclonal antibody that recognizes PRDM1 was used to detail its distribution in normal human lymphoid tissue and in lymphoid neoplasms that correspond to different stages of B-cell differentiation. PRDM1 was expressed in germinal centre blasts that co-express Pax5, CD19, CD20, and CD10, but not BCL6 or MTA-3. Pax5 was downregulated and full plasma cell morphology and phenotype were acquired by PRDM1+, nuclear cREL-, pre-plasma cells upon exit from the germinal centre. Activated extrafollicular B-cells (CD30+, Pax5+) were largely PRDM1-. PRDM1 was also absent in tissue histiocytes and the majority of resting T-cells and S-100+ antigen-presenting cells. PRDM1 and CD138 were expressed simultaneously in human lymphomas with plasma cell differentiation, but not in marginal zone lymphomas or chronic lymphocytic leukaemias. A minority of diffuse large B-cell lymphomas expressed PRDM1 and Hodgkin lymphomas were largely PRDM1-. Infiltrating T-cells in PRDM1- B-cell lymphomas expressed PRDM1. In conclusion, PRDM1 staining is a reliable and informative assay to define plasma cell commitment and differentiation in human normal and neoplastic B-cell lineages.

Animals↗

PubMed2005

A practical protocol for carbohydrate microarrays.

We have established a high-throughput biochip platform for constructing carbohydrate microarrays. Using this technology, carbohydrate-containing macromolecules of diverse structures, including polysaccharides, natural glycoconjugates, and mono- and oligosaccharides coupled to carrier molecules, can be stably immobilized on a glass chip without chemical modification. Here, we describe a practical protocol for this technology. We hope that anyone who has access to a standard cDNA microarray facility will be able to explore this technology for his or her own research interest. We also provide an example to illustrate that the carbohydrate microarray is also a discovery tool; this is particularly useful for identifying immunologic sugar moieties, including complex carbohydrates of cancer cells and sugar signatures of previously unrecognized microbial pathogens.

Animals↗

PubMed2005

Production of dextran in transgenic potato plants.

The production of dextran in potato tubers and its effect on starch biosynthesis were investigated. The mature dextransucrase (DsrS) gene from Leuconostoc mesenteroides was fused to the chloroplastic ferredoxin signal peptide (FD) enabling amyloplast entry, which was driven by the highly tuber-expressed patatin promoter. After transformation of two potato genotypes (cv. Kardal and the amylose-free (amf) mutant), dextrans were detected by enzyme-linked immunosorbent assay (ELISA) in tuber juices of Kardal and amf transformants. The dextran concentration appeared two times higher in the Kardal (about 1.7 mg/g FW) than in the amf transformants. No dextran was detected by ELISA inside the starch granule. Interestingly, starch granule morphology was affected, which might be explained by the accumulation of dextran in tuber juices. In spite of that, no significant changes of the physicochemical properties of the starches were detected. Furthermore, we have observed no clear changes in chain length distributions, despite the known high acceptor efficiency of DSRS.

Carbohydrate Sequence↗

PubMed2004

Glycan arrays lead to the discovery of autoimmunogenic activity of SARS-CoV.

Using carbohydrate microarrays, we characterized the carbohydrate binding activity of SARS-CoV neutralizing antibodies elicited by an inactivated SARS-CoV vaccine. In these antibodies, we detected undesired autoantibody reactivity specific for the carbohydrate moieties of an abundant human serum glycoprotein asialo-orosomucoid (ASOR). This observation provides important clues for the selection of specific immunologic probes to examine whether SARS-CoV expresses antigenic structures that mimic the host glycan. We found that lectin PHA-L (Phaseolus vulgaris L.), which is specific for a defined complex carbohydrate of ASOR, stained the SARS-CoV-infected cells specifically and intensively. Taken together, we present immunologic evidence that a carbohydrate structure of SARS-CoV shares antigenic similarity with host glycan complex carbohydrates. The experimental approaches we applied in this study are likely applicable for the identification of immunologic targets of other viral pathogens.

Antibodies, Viral↗

PubMed2003

Protein synthesis at synapse versus cell body: enhanced but transient expression of long-term facilitation at isolated synapses.

Protein synthesis at synaptic terminals contributes to LTP in hippocampus and to the formation of new synaptic connections by sensory neurons (SNs) of Aplysia. Here we report that after removal of the SN cell body, isolated SN synapses of Aplysia in culture express protein-synthesis dependent long-term facilitation (LTF) produced by 5-HT that decays rapidly. Changes in expression of a SN-specific neuropeptide sensorin in isolated SN varicosities parallel the changes in synaptic efficacy. At 24 h after 5-HT the magnitude of LTF produced at isolated SN synapses was significantly greater than that produced when SN cell bodies were present. LTF was maintained at 48 h at connections with SN cell bodies, but not at isolated SN synapses. The increase in synaptic efficacy at isolated SN synapses at 24 h was blocked by the protein synthesis inhibitor anisomycin. LTF was accompanied by changes in expression of sensorin. The increase in sensorin level at isolated SN varicosities with 5-HT was blocked by anisomycin or was reversed 48 h after 5-HT treatment alone. The results suggest that, as is the case for initial synapse formation between SNs and L7, changes in protein synthesis at synaptic terminals may contribute directly to LTF of stable synapses. Changes in expression within the cell body provide additional contributions for long-term maintenance of the new level of synaptic efficacy that was initiated directly by local changes in protein synthesis at or near synaptic terminals.

Animals↗

PubMed2003

Carbohydrate microarrays.

Sugar chains are abundantly expressed on the outer surfaces of the vast majority of viral, bacterial, protozoan and fungal pathogens, as well as on the membranes of mammalian cells. This class of carbohydrate molecule is without peer in structural diversity and is characteristically suitable for storing and displaying biological signals for molecular and cellular recognition. Exploring the biological information contained in sugar chains is an important topic of current postgenomic research. To facilitate these investigations, we have focused on the establishment of a carbohydrate-based microarray technology. Recently, we reported that a large panel of carbohydrate-containing macromolecules, including polysaccharides, natural glycoconjugates, and the mono- and oligosaccharides coupled to carrier molecules, can be stably immobilized on a microglass slide to produce a large-scale carbohydrate microarray. In this review, we attempt to summarize our recent progress in using this technology to uncover the carbohydrate-based biological signals that are recognized by the human and animal immune systems. We also discuss the potential of various platforms of carbohydrate microarrays that were recently established and analyze the challenges to future development of carbohydrate microarray technologies and their applications.

Animals↗

PubMed2002

Carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells.

We describe here the development of a carbohydrate-based microarray to extend the scope of biomedical research on carbohydrate-mediated molecular recognition and anti-infection responses. We have demonstrated that microbial polysaccharides can be immobilized on a surface-modified glass slide without chemical conjugation. With this procedure, a large repertoire of microbial antigens (approximately 20,000 spots) can be patterned on a single micro-glass slide, reaching the capacity to include most common pathogens. Glycoconjugates of different structural characteristics are shown here to be applicable for microarray fabrication, extending the repertoires of diversity and complexity of carbohydrate microarrays. The printed microarrays can be air-dried and stably stored at room temperature for long periods of time. In addition, the system is highly sensitive, allowing simultaneous detection of a broad spectrum of antibody specificities with as little as a few microliters of serum specimen. Finally, the potential of carbohydrate microarrays is demonstrated by the discovery of previously undescribed cellular markers, Dex-Ids.

Animals↗