We present an unusual and novel model for initial investigations of a putative role for specifically conformed glycans in cellular interactions. We have used α-and ß-amylase and α-and ß-glucosidase in dose-response experiments evaluating their effects on archenteron organization using the NIH designated sea urchin embryo model. In quantitative dose-response experiments, we show that defined activity levels of α-glucosidase and ß-amylase inhibited archenteron organization in living Lytechinus pictus gastrula embryos, whereas all concentrations of ß-glucosidase and α-amylase were without substantial effects on development. Product inhibition studies suggested that the enzymes were acting by their specific glycosidase activities and polyacrylamide gel electrophoresis suggested that there was no detectable protease contamination in the active enzyme samples. The results provide evidence for a role of glycans in sea urchin embryo cellular interactions with special reference to the possible structural conformation of these glycans based on the differential activities of the α-and ß-glycosidases.

The gene encoding ribosomal protein L25, a primary rRNA-binding protein, was isolated from the protozoan parasite Trypanosoma brucei. Hybridization studies indicate that multiple copies of the gene are present per T. brucei haploid genome. The C-terminal domain of L25 protein from T. brucei is strikingly similar to L23a protein from rat, L25 proteins from fungal species, and L23 proteins from eubacteria, archaebacteria, and chloroplasts. A phylogenetic analysis of L23/25 proteins and the putative binding sites on their respective LSU-rRNAs (large subunit rRNAs) provides a rare opportunity to study molecular co-evolution between an RNA molecule and the protein that binds to it.

SummaryIn Strongylocentrotus purpuratus the hyalins are a set of three to four rather large glycoproteins (hereafter referred to as "hyalin"), which are the major constituents of the hyaline layer, the developing sea urchin embryo's extracellular matrix. Recent research from our laboratories has shown that hyalin is a cell adhesion molecule involved in sea urchin embryo specific cellular interactions. Other laboratories have shown it to consist of 2-3% carbohydrate, and a cloned, sequenced fragment demonstrated repeat domains (HYR) and non-repeat regions. Interest in this molecule has increased because HYR has been identified in organisms as diverse as bacteria, flies, worms, mice and humans, as well as sea urchins. Our laboratories have shown that hyalin appears to mediate a specific cellular interaction that has interested investigators for over a century, archenteron elongation/attachment to the blastocoel roof. We have done this by localizing hyalin on the two components of the cellular interaction and by showing that hyalin and anti-hyalin antibody block the cellular interaction using a quantitative microplate assay. The microplate assay, however, has limitations because it does not directly assess hyalin's effects on the adhesion of the two components of the interaction. Here we have used an elegant direct assay that avoids the limitations, where we microdissected the two components of the adhesive interaction and tested their readhesion to each other, thereby avoiding possible factors in the whole embryos that could confound or confuse results. Using both assays, we found that mild periodate treatment (6 h to 24 h in sodium acetate buffer with 0.2M sodium periodate at 4 °C in the dark) of hyalin eliminates its ability to block the cellular interaction, suggesting that the carbohydrate component(s) may be involved in hyalin's specific adhesive function. This is an important first step in identifying the molecular mechanisms of a well known cellular interaction in the NIH designated sea urchin embryo model, a system that has led to the discovery of scores of physiological mechanisms, including those involved in human health and disease.

The process of Epstein-Barr virus (EBV)-induced transformation of human B lymphocytes results in a cell line that is a mixture of latently and lytically infected cells, with the lytic cells composing roughly 5% to less than 0.0001% of the overall population. A set of nine normal lymphoblastoid cell lines that span a 100to 200-fold range in average EBV DNA content were studied, and the frequency with which these cells entered a lytic phase of viral growth correlated with their EBV DNA copy number (as a population average). However, neither factor correlated with the levels of expression of transcript for the viral genes EBNA-1, EBNA-2, and latent membrane protein, nor did they correlate with the levels of EBNA-2 protein and latent membrane protein. The rate at which a cell line enters into lytic growth spontaneously is therefore not dependent on the overall steady-state levels of expression of these latent-phase genes.

The enzymatic activities of commercially prepared glycosidases were verified by direct chemical assays using defined substrates and fixed and live sea urchin (Lytechinus pictus) embryos to determine if a model cellular interaction of interest to developmental biologists for over a century (interaction of archenteron tip and roof of the blastocoel) was mediated by glycans. Glycosidases (active and denatured) were incubated with microdissected archenterons and blastocoel roofs in a direct assay to learn if their enzymatic activities could prevent the normal adhesive interaction. Of the five glycosidases tested only β-amylase (an exoglycosidase) immediately inhibited the interaction at relatively low unit activity. α-Amylase (an endoglycosidase) had no measurable effect, while other glycosidases (α-glucosidase, β-glucosidase, β-galactosidase) only substantially inhibited adhesion after a 12-h incubation. We demonstrated that the five glycosidases were active (not inhibited) in the presence of embryo materials, and that cleaved sugars could be detected directly after incubation of some enzymes with the embryos. The biochemical purity of the enzymes was examined using gel electrophoresis under denaturing conditions, and the absence of contaminating proteases was confirmed using Azocoll™ substrate. As we cannot entirely rule out the presence of minor contaminating enzymatic activities, only inhibitions of adhesion after very short incubations with enzyme were considered significant and biologically relevant. Although glycans in indirect experiments have been implicated in mediating the interaction of the tip of the archenteron and roof of the blastocoel, to our knowledge, this is the first study that directly implicates polyglucans with terminal 1,4-linked glucose residues in this adhesive event.

In a search for trypanosome DNA sequences that permit replication and stable maintenance of extrachromosomal elements, a 1-kilobase-pair (kbp) fragment from a m ondrial kinetlst DNA (kDNA) micircie of Trypanosoma and conserved sequence characteristics such as purine/ pyrimidine strand biases. A specific UMS-binding protein has been identified in kinetoplastids (9); however, it is not known whether it has a role in kDNA replication.It has recently become possible to transfect Trypanosoma brucei with either transient expression vectors or through homologous recombination (10); however, we wished to increase the genetic capabilities of the organism by developing a shuttle vector that would allow the maintenance of multiple extrachromosomal copies ofa transfected sequence.By introducing random fiagments oftotal T. brucei DNA into a vector carrying a hygromycin-resistance gene, a mitochondrial DNA element was identifiedt that permits plasmid replication and maintenance in the nuclei of T. brucei. The plasmid is maintained stably under continuous drug selection as a supercoiled head-to-tail concatemer composed of approximately eight monomer units. A second kDNA minicircle element, chosen at random and similarly tested, also permits autonomous replication. These findings suggest that minicircles may have the interesting capability of engendering DNA replication in both the mitochondrion and nucleus. MATERIALS AND METHODSTrnfection of Cels. T. brucei (subspecies brucei, strain IsTat 1.1) were cultured in Cunningham's medium (11) supplemented with 10%o (vol/vol) fetal bovine serum. Electroporation and transfection of T. brucei were performed by the method ofKapler (12) with 10-50 pg ofplasmid DNA isolated from Escherichia coli (SURE strain, Stratagene) by Qiagen (Chatsworth, CA) column chromatography. After electroporation, cells were cultured for 24-48 hr without selection before the addition of 100 pg of hygromycin B (Calbiochem) per ml.Purification of DNA from T. brucei. Total DNA was prepared from T. bracei by the method of Milhausen et al. (13) or White et al. (14) and dialyzed against TE (10 mM Tris chloride/i mM EDTA, pH 7.5). Supercoiled DNA molecules were fractionated by equilibrium centrifugation (15)

Alkaline lysis of Escherichia coli is usually the method of choice for plasmid preparation, but ''ghost bands" of denatured supercoiled DNA can result if the pH is too high or the period of lysis is too long. By replacing the usual sodium hydroxide lysis solution with an arginine buffer prepared in the range of pH 11.4 to 12.0, we were able to stabilize the pH during lysis and obtain plasmid that is suitably pure for restriction digestion and DNA sequencing.

The sea urchin embryo is recognized as a model system to reveal developmental mechanisms involved in human health and disease. In Part I of this series, six carbohydrates were tested for their effects on gastrulation in embryos of the sea urchin Lytechinus pictus. Only l-rhamnose caused dramatic increases in the numbers of unattached archenterons and exogastrulated archenterons in living, swimming embryos. It was found that at 30 h post-fertilization the l-rhamnose had an unusual inverse dose-dependent effect, with low concentrations (1-3 mM) interfering with development and higher concentrations (30 mM) having little to no effect on normal development. In this study, embryos were examined for inhibition of archenteron development after treatment with α-l-rhamnosidase, an endoglycosidase that removes terminal l-rhamnose sugars from glycans. It was observed that the enzyme had profound effects on gastrulation, an effect that could be suppressed by addition of l-rhamnose as a competitive inhibitor. The involvement of l-rhamnose-containing glycans in sea urchin gastrulation was unexpected, since there are no characterized biosynthetic pathways for rhamnose utilization in animals. It is possible there exists a novel l-rhamnose-containing glycan in sea urchins, or that the enzyme and sugar interfere with the function of rhamnose-binding lectins, which are components of the innate immune system in many vertebrate and invertebrate species.