Sialic acid incorporation into gangliosides and glycoproteins of the fish brain

—The concentration of lipid- and non-lipid-bound sialic acid in the optic nerve tract and tectum and in whole brain of fish was estimated. The incorporation of sialic acid into gangliosides and non-lipid components was studied in fish by intracranial or intraocular application of N-[³H]acetylmannosamine or N-[³H]acetylglucosamine. After intracranial injection of N-[³H]acetylmannosamine autoradiography showed lipid- and non-lipid-bound radioactivity in the tectum opticum evenly distributed over regions of nerve fibres or perikarya indicating an ubiquitous incorporation of label. Sialic acid incorporation into glycoproteins after intracranial injection of N-acetylmannosamine always exceeded that into gangliosides. TCA-precipitable non-lipid material is labelled from intracranially applied N-acetylmannosamine in the sialic acid portion and also in nonsialic acid components, whereby the percentage of label in sialic acid increases reaching 90 per cent of the total radioactivity after 90 min. After intraocular application of N-[³H]acetylmannosamine, sialic acid in gangliosides was generally found to be more highly labelled than in glycoproteins. The ratio of radioactivity in gangliosides and glycoproteins increased with time of incubation and the distance from the eye. TCA-soluble radioactivity was translocated by fast axonal transport. Cycloheximide inhibited incorporation of N-acetylmannosamine-derived radioactivity into gangliosides and proteins but not the transport of TCA-soluble material, which accumulates in the tectum. After intraocular application of N-[³H]acetylglucosamine, TCA-soluble label arrives later in the optic tectum than radioactivity of high molecular weight components. The ratio of lipid to non-lipid-bound radioactivity does not change considerably with the time after injection or the distance from the eye. There was no accumulation of TCA-soluble radioactivity after the inhibition of incorporation into high molecular weight components.     

The sugar part of kappa-caseins from cow milk and colostrum and its microheterogeneity.

Cow kappa-casein contains only three different sugars (Gal, GalNAc, NeuNAc). However detailed analyses achieved mainly by gas liquid chromatography suggested a microheterogeneity at the sugar level. After alkaline borohydride treatment, filtration on Bio-Gel P4 and paper chromatography, different carbohydrate parts were obtained. The two main compounds had the following molar compositions: GalNAc (1), Gal(1) and NeuNAc (1) and GalNAc (1), Gal(1) and NeuNAc (2). From these data and our previous sequence studies, some formulae of the polysaccharide part were proposed. One of them was closely related to the sugar sequence of a glycopeptide with MN activity which was in agreement with our observation concerning a cross antigenic reactivity between the N blood group substances and the caseinoglycopeptides. All the polysaccharide parts isolated from colostrum caseinoglycopeptide were much more complex than those obtained from the normal glycopeptide, confirming an evolution of the sugar part as a function of time after parturition.     

Sialic acid activation.

Cytidine 5'-monophosphosialic acid (CMP-sialic acid) is the activated form of sialic acid which is required for the biosynthesis of sialic acid-containing complex carbohydrates. Its discovery over 30 years ago by the laboratory of Dr Saul Roseman was a landmark in research dealing with the biosynthesis of these compounds. A review is presented of the salient features concerning this molecule: its discovery, chemistry, biosynthesis, subcellular location, enzymatic cleavage, transport and molecular biology. This report does not deal with its utilization by the sialyltransferases.     

Solid-phase assembly of cow colostrum trypsin inhibitor gene.

A gene for cow colostrum trypsin inhibitor (CTI) was constructed from synthetic oligonucleotides using a novel method of solid-phase gene assembly. In the first step an anchor oligonucleotide was covalently bound to the CNBr-activated Sephacryl S-500 support. Next, triads or tetrads of separately annealed oligonucleotides were stepwise hybridized to the immobilized complementary sequence, with washing after each step. In the last step a linearized vector molecule was ligated to the assembled gene. The whole construct was released from the solid support with a restriction enzyme, circularized, and used for transformation, with a high yield of recombinant clones being obtained. The method represents a generally applicable approach to rapid and efficient assembly of extended DNA duplexes.