Identification of an alpha3-fucosyltransferase and a novel alpha2- fucosyltransferase activity in cercariae of the schistosome Trichobilharzia ocellata: biosynthesis of the Fucalpha1-->2Fucalpha1-- >3[Gal(NAc)beta1-->4]GlcNAc sequence

Fucose is a major constituent of the protein- and lipid-linked glycans of the various life-cycle stages of schistosomes. These fucosylated glycans are highly antigenic and seem to play a role in the pathology of schistosomiasis. In this article we describe the identification and characterization of two fucosyltransferases (FucTs) in cercariae of the avian schistosome Trichobilharzia ocellata, a GDP-Fuc:[Galbeta1-- >4]GlcNAcbeta-R alpha1-->3-FucT and a novel GDP-Fuc:Fucalpha-R alpha1-- >2-FucT. Triton X-100 extracts of cercariae were assayed for FucT activity using a variety of acceptor substrates. Type 1 chain (Galbeta1- ->3GlcNAc) based compounds were poor acceptors, whereas those based on a type 2 chain (Galbeta1-->4GlcNAc), whether alpha2'-fucosylated, alpha3'-sialylated, or unsubstituted, and whether present as oligosaccharide or contained in a glycopeptide or glycoprotein, all served as acceptor substrates. In this respect the schistosomal alpha3- FucT resembles human FucT V and VI rather than other known FucTs. N- ethylmaleimide, an inhibitor of several human FucTs, had no effect on the activity of the schistosomal alpha3-FucT, whereas GDP-beta-S was strongly inhibitory. Large scale incubations were carried out with Galbeta1-->4GlcNAc, GalNAcbeta1-->4GlcNAcbeta-O -(CH2)8COOCH3 and Fucalpha1-->3GlcNAcbeta1-->2Man as acceptor substrates and the products of the incubations were isolated using a sequence of chromatographic techniques. By methylation analysis and 2D-TOCSY and ROESY1H-NMR spectroscopy the products formed were shown to be Galbeta1-- >4[Fucalpha1-->2Fucalpha1-->3]GlcNAc, GalNAcbeta1-->4[Fucalpha1-- >2Fucalpha1-->3]GlcNAcbe ta-O-(CH2)8COOCH3, and Fucalpha1-->2Fucalpha1-- >3GlcNAcbeta1-->2Man, respectively. It is concluded that the alpha2- FucT and alpha3-FucT are involved in the biosynthesis of the (oligomeric) Lewisx sequences and the Fucalpha1-->2Fucalpha1-->3GlcNAc structural element that have been described on schistosomal glycoconjugates.      

An autoradiographic evaluation of the regeneration of corneal epithelium in Bufo marinus.

Epithelial regeneration following scraping of a central portion of corneal epithelium of the toad, Bufo marinus, was studied. Wound closure occurred by migration of a single layer of epithelial cells from adjacent noninjured areas. Autoradiography indicated that following wound closure at two days after scraping, DNA synthesis began in these epithelial cells covering the former scraped area, and was even more pronounced three days after scraping. At four days the former denuded area was covered by an epithelium two to four cells thick and by the fifth day regeneration was complete. These studies also indicated that mitotic time in this organism is rapid.     

Metabolism and function in animal tissues of agmatine,a biogenic amine formed from arginine

Summary. Recently agmatine, decarboxylated arginine, has been shown to be an important biological compound in several animal tissues. This paper summarizes the known information regarding the transport of arginine, its decarboxylation and the effects of the agmatine formed mainly on NO and polyamine synthesis.     

Understanding the mechanism of action of poly(amidoamine)s as endosomolytic polymers: correlation of physicochemical and biological properties

Bioresponsive poly(amidoamine)s (PAA)s are currently under development as endosomolytic polymers for intracellular delivery of proteins and genes. Here for the first time, small-angle neutron scattering (SANS) is used to systematically investigate the pH-dependent conformational change of an endosomolytic polymer, the PAA ISA 23. The radius of gyration of the ISA23 was determined as a function of pH and counterion, the aim being to correlate changes in polymer conformation with membrane activity assessed using a rat red blood cell haemolysis assay. With decreasing pH, the ISA23 radius of gyration increased to a maximum (R(g) approximately 80 A) around pH = 3, before subsequently decreasing once more. At high pH and therefore high ionic strengths, the polymer is negatively charged and adopts a rather compact structure (R(g) approximately 20 A), presumably with the dissociated carboxylic groups on the exterior of the polymer coil. At low pH, the coil again collapses (R(g) < 20 A), presumably due to the effects of the high ionic strength. It is concluded that the nature of the salt form has no direct bearing on the size of the polymer coil, but it does indirectly determine the prevailing pH and, hence, polymer conformation. Pulsed-gradient spin-echo NMR measurements were in good agreement with the SANS estimates of the radius of gyration, although ISA23 polydispersity does complicate the data interpretation/comparison. These results support the proposed mode of action of PAAs, namely a coil expansion on passing from a neutral pH (extracellular) to an acidic pH (endosomal and lysosomal) environments. The results do, however, suggest that the charge on the polymer shows a closer correlation with the haemolysis activity rather than the polymer conformation.     

The DNA-binding activity of protein disulfide isomerase ERp57 is associated with the a(') domain

ERp57 belongs to the protein disulfide isomerases, a family of homologous proteins mainly localized in the endoplasmic reticulum and characterized by the presence of a thioredoxin-like folding domain. ERp57 is a protein chaperone with thiol-dependent protein disulfide isomerase and additional activities and recently it has been shown to be involved, in cooperation with calnexin or with calreticulin, in the correct folding of glycoproteins. However, we have demonstrated that the same protein is also present in the nucleus, mainly associated with the internal nuclear matrix fraction. In vitro studies have shown that ERp57 has DNA-binding properties which are strongly dependent on its redox state, the oxidized form being the competent one. A comparison study on a recombinant form of ERp57 and several deletion mutants, obtained as fusion proteins and expressed in Escherichia coli, allowed us to identify the C-terminal a(') domain as directly involved in the DNA-binding activity of ERp57.