Synthesis of delta4-beta-D-glucopyranosiduronic acids as mimetics of 2,3-unsaturated sialic acids for sialidase inhibition.

Mimetics of Neu5Ac2en and KDN2en, based on delta4-beta-delta-glucopyranosiduronic acids, have been synthesised. The Neu5Ac2en mimetic 5 showed inhibition of both bacterial and viral sialidases, with inhibition of the viral sialidase being comparable to that of Neu5Ac2en itself.

The synthesis and evaluation of novel sialic acid analogues bound to matrices for the purification of sialic acid-recognising proteins.

A novel N-acetylneuraminic acid analogue, 2-S-(5'-aminopentyl) 5-acetamido-3,5-dideoxy-2-thio-D-glycero-alpha-D-galacto-2- nonulopyranosidonic acid, as well as the thiosialoside 2-S-(2'-aminoethyl) 5-acetamido-3,5-dideoxy-2-thio-D-glycero-alpha-D-galacto-2- nonulopyranosidonic acid, have been synthesised and successfully coupled to CNBr-activated Sepharose 4B through the terminal amino group. The resultant affinity resins have proved efficient in purifying a number of sialic acid-recognising proteins such as Vibrio cholerae sialidase, sialidase-L from leech, trans-sialidase from Trypanosoma cruzi, and sialyltransferases from rat liver, all in high yield.

Insulin-responsive tissues contain the core complex protein SNAP-25 (synaptosomal-associated protein 25) A and B isoforms in addition to syntaxin 4 and synaptobrevins 1 and 2.

SNAP-25 (synaptosomal-associated protein 25), syntaxin and synaptobrevin are the three SNARE [soluble NSF attachment protein receptor (where NSF = N-ethylmaleimide-sensitive fusion protein)] proteins that form the core complex involved in synaptic vesicle docking and subsequent fusion with the target membrane. The present study is aimed at understanding the mechanisms of fusion of vesicles carrying glucose transporter proteins with the plasma membrane in human insulin-responsive tissues. It describes the isolation and characterization of cDNA molecules encoding SNAP-25 A and B isoforms, syntaxin 4 and synaptobrevins (also known as vehicle-associated membrane proteins) from two major human insulin-responsive tissues, skeletal muscle and fat. The DNA and deduced amino acid sequences of SNAP-25 revealed perfect identity with the previously reported human neural SNAP-25 A and B isoforms. Our results indicate the presence of both isoforms both in insulin-responsive tissues and in in vitro cultured 3T3-L1 cells, but suggest a differential pattern of gene expression: isoform A is the major species in adipose tissue, and isoform B is the major species in skeletal muscle. The presence of SNAP-25 protein in 3T3-L1 cells was demonstrated by immunofluorescence microscopy using an anti-SNAP-25 monoclonal antibody. Immunoprecipitation experiments using the same monoclonal antibody also revealed the presence of SNAP-25 protein in plasma membrane fractions from rat epididymal fat pads. The syntaxin 4-encoding region from skeletal muscle contains five nucleotide differences from the previously reported placental cDNA sequence, two of which result in amino acid changes: Asp-174 to Glu and Val-269 to Ala. The synaptobrevin 1 cDNA from skeletal muscle contains two nucleotide differences when compared with the corresponding clone from neural tissues, one of which is silent and the other resulting in the amino acid change Thr-102 to Ala. The cDNA sequence of the protein from fat is identical with that of human synaptobrevin 1 from neural tissues. Furthermore, we have confirmed the presence of syntaxin 4 in fat and of synaptobrevin 2 in skeletal muscle by PCR amplification and Southern hybridization analysis. Using the yeast two-hybrid system, an interaction was observed between the full-length cytoplasmic domains of syntaxin 4 and synaptobrevin 2, a vesicle membrane SNARE previously shown by others to be associated with vesicles carrying the GLUT4 glucose transporter protein, but no interaction was seen with synaptobrevin 1. Flow cytometry of low-density microsomes isolated from fat cells was used to demonstrate the binding of syntaxin 4 to a subset of vesicles carrying GLUT4 protein; whereas SNAP-25 on its own bound poorly to these vesicles, the syntaxin 4-SNAP-25 complex gave a strong interaction.

Preparation of highly purified human myelin oligodendrocyte glycoprotein in quantities sufficient for encephalitogenicity and immunogenicity studies.

Myelin oligodendrocyte glycoprotein, (MOG), a quantitatively minor central nervous system (CNS) myelin component, is a candidate target antigen for autoimmune-mediated demyelination. It is a highly hydrophobic protein present in very small amounts in CNS tissue and thereby difficult to purify. Our aim was to devise a purification procedure that would yield sufficient quantities of highly purified MOG to subsequently test its potential encephalitogenic activity, as well as investigate the humoral and cell-mediated responses to this antigen in naturally occurring and experimentally induced autoimmune demyelinating diseases. MOG was purified from human CNS white matter using immunoaffinity chromatography, a procedure that gave a final yield of MOG corresponding to 0.02% total white matter protein. The final product, which migrated as two bands of molecular weight 28 kDa and 58 kDa, was highly pure as shown also by specific reactivity with monoclonal anti-MOG antibodies on immunoblots in the absence of any detectable reactivity with antibodies specific for myelin basic protein, proteolipid protein and myelin-associated glycoprotein. Partial amino acid sequence was obtained from both MOG bands separated by SDS-PAGE and electroblotted onto PVDF. The sequence of the first 17 N-terminal amino acids had approximately 55% homology with the reported rat MOG sequence deduced from the cloned cDNA sequence; small internal sequences obtained showed also very high homology. Our purified MOG preparations have been used to investigate T cell response to MOG by peripheral blood lymphocytes of multiple sclerosis patients and to induce a relapsing remitting demyelinating disease in Lewis rats.