Binding and reactivity at the "glucose" site of galactosyl-beta-galactosidase (Escherichia coli)

A large number of sugars and alcohols were tested to see how well they bound and how readily they reacted at the "glucose" site of the galactosyl form of beta-galactosidase. Two classes of compounds were found to bind well to the galactosyl form of the enzyme. One class contained sugars and alcohols similar in structure to D-glucose in its pyranose ring form, and the other class was composed of relatively hydrophobic sugars and alcohols. On the other hand, several factors seemed to control k4. Large k4 values were found for straight-chain alcohols as compared to the values for the corresponding ring sugars. Also, if the acceptors had hydroxyl groups at the end of the molecule, the reactivity (k4) was greater than if hydroxyl groups were only in the middle of the molecule. In addition, if there was a hydroxyl at an asymmetric carbon next to a terminal hydroxymethyl group, it was necessary that it be in the same orientation as the D configuration of glucose; otherwise, the k4 was low. Overall, the results showed that it is the binding effect, more than the reactivity, which is responsible for the specificity at the "glucose" site. More specifically, these studies showed that the reason glucose is such an ideal molecule for transgalactosylation is that it leaves the galactosyl form of the enzyme very slowly, that is, k-a is relatively small. Thus, glucose remains attached to the galactosyl form of beta-galactosidase for a sufficient time to allow transgalactosylation to occur, while other acceptors, despite being as reactive (or more reactive) in terms of their k4 values, dissociate from the "glucose" site of the galactosyl form of the enzyme very readily and thus are poor acceptors.     

Antigens on rat spermatozoa with a potential role in fertilization

Eight monoclonal antibodies (McAbs), directed against antigens on rat cauda epididymal spermatozoa, were tested for their capacity to interfere with fertilization in vitro as a means of identifying molecules with a potential role in sperm-egg recognition and fusion. Antigens recognized by the McAbs were visualized on live spermatozoa by indirect immunofluorescence (IIF) and characterized by immunoblotting. Five McAbs (designated 1B5, 2C4, 4B5, 5B1, and 8C4) recognized antigens specifically on the sperm acrosome and three (designated 2B1, 2D6, and 6B2) bound to the flagellum. Of the eight McAbs investigated, three (2B1, 2C4, and 6B2) were effective in blocking fertilization in vitro when added as culture supernatants to mixtures of sperm and eggs. McAb 6B2 was inhibitory due to its ability to agglutinate spermatozoa. McAbs 2B1 and 2C4 did not agglutinate capacitated spermatozoa, had no observable effect on motility, and yet blocked fertilization in a dose-dependent manner. McAb 2C4 did not give a reaction on immunoblots, but the 2B1 antigen was identified as an Mr 40 kD glycoprotein. McAb 2B1 appeared to block fertilization at the level of zona binding, whereas the effects of 2C4 were directed more against zona penetration and/or fusion with the vitellus. When sperm-egg complexes were stained with 2C4 or 2B1 McAbs and viewed by IIF, all spermatozoa that were attached to the zona showed fluorescence on the head. These results suggest that different antigens on the rat sperm head participate in different aspects of the fertilization process and that during capacitation there is either exposure of these antigens or else they migrate to their site of action from the flagellum.     

MIDAS: software for analysis and visualisation of interallelic disequilibrium between multiallelic markers

Background  

Various software tools are available for the display of pairwise linkage disequilibrium across multiple single nucleotide polymorphisms. The HapMap project also presents these graphics within their website. However, these approaches are limited in their use of data from multiallelic markers and provide limited information in a graphical form.     

Microwave-assisted synthesis of galacto-oligosaccharides from lactose with immobilized beta-galactosidase from Kluyveromyces lactis

Galacto-oligosaccharides (GOS) were synthesized from lactose by immobilized and free -galactosidase from Kluyveromyces lactis (Lactozym 3000 L HP-G) using either focused microwave irradiation or conventional heating. Immobilization of the -galactosidase on to Duolite A-568 increased the synthesis of GOS. GOS selectivity (GOS synthesis/lactose hydrolysis ratio) increased when the water activity of the media was reduced, notably with a high initial lactose concentration but also by using co-solvents in the media. The advantage of microwave heating on GOS formation was also examined. Addition of solvent and carrying out the reaction under microwave irradiation resulted an increase in the production of GOS. The selectivity for GOS synthesis can be increased by 217-fold under microwave irradiation, using immobilized -glucosidase and with added co-solvents such as hexanol.     

Vertebrate caudal gene expression gradients investigated by use of chick cdx-A/lacZ and mouse cdx-1/lacZ reporters in transgenic mouse embryos: evidence for an intron enhancer

The vertebrate caudal proteins, being upstream regulators of the Hox genes, play a role in establishment of the body plan. We describe analysis of two orthologous caudal genes (chick cdx-A and mouse cdx-1) by use of lacZ reporters expressed in transgenic mouse embryos. The expression patterns show many similarities to the expression of endogenous mouse cdx-1. At 8.7 days, cdx/lacZ activity within neurectoderm and mesoderm forms posterior-to-anterior gradients, and we discuss the possibility that similar gradients of cdx gene expression may function as morphogen gradients for the establishment of Hox gene expression boundaries. Our observations suggest that gradients form by decay of cdx/lacZ activity in cells that have moved anterior to the vicinity of the node. The cdx-A/lacZ expression pattern requires an intron enhancer that includes two functional control elements: a DR2-type retinoic acid response element and a Tcf/beta-catenin binding motif. These motifs are structurally conserved in mouse cdx-1.     

cdx4/lacZ and cdx2/lacZ protein gradients formed by decay during gastrulation in the mouse

Expression of the mouse caudal genes cdx4 and cdx2 is examined by use of lacZ reporter constructs expressed in transgenic mouse embryos. During early gastrulation, up to at least 8.5 days of development, reporter mRNA distributions are apparently similar to those of endogenous cdx mRNAs. By 8.25 to 8.8 days, cdx/lacZ protein activities have become distributed as posterior-to-anterior gradients along the neural and mesoderm tissues. The gradients form by decay of activity as cells become distanced from the regressing tailbud. In situ hybridization studies indicate that the decay is primarily in cdx/lacZ protein activities rather than mRNAs. As gastrulation proceeds, the locations of the gradients regress progressively posteriorly along the growing axis. Our results indicate how cdx4 and cdx2 protein gradients might be generated by decay during normal development. The smoothness of the gradients that we detect shows that there cannot be extensive mixing of cells once they leave the tailbud to contribute to the growing axis. An enhancer element located in the first intron of the cdx4 gene is essential for correct transgene expression.