SummaryOne hypothesis for the mechanism of chlamydial interaction with its eukaryotic host cell invokes a trimolecular mechanism, whereby a Chlamydia-derived glycosaminoglycan bridges a chlamydial acceptor molecule and a host receptor enabling attachment and invasion. We show that a heparan sulphate-speci®c monoclonal antibody speci®cally binds a glycosaminoglycan localized to the surface of the chlamydial organism and effectively neutralizes infectivity of both C. trachomatis and C. pneumoniae. In addition to the ability of this antibody to neutralize infectivity, direct visualization using immuno¯uorescence demonstrated staining of chlamydial organisms localized to the intracellular vacuole. The chlamydial-associated glycosaminoglycan was speci®cally labelled with [ 14 C]-glucosamine, and the labelled compound was immunoprecipitated and resolved by gel electrophoresis. The chlamydial-associated glycosaminoglycan is a high-molecular-weight compound similar in size to heparin or heparan sulphate and was sensitive to cleavage by heparan sulphate lyase. These data demonstrate that a glucosamine-containing sulphated polysaccharide is produced within the intracellular vacuole containing chlamydiae and is a target for antibody-mediated neutralization of infectivity.

Using polystyrene microspheres coated with heparin or heparan sulfate, it was shown that coated microspheres specifically bound eukaryotic cells and were endocytosed by nonprofessional phagocytic cells. Coated microspheres displayed properties of binding to eukaryotic cells that were similar to those of chlamydiae, and the microspheres were competitively inhibited by chlamydial organisms. Endocytosis of heparin-coated beads resulted in the tyrosine phosphorylation of a similar set of host proteins as did endocytosis of chlamydiae; however, unlike viable chlamydial organisms, which prevent phagolysosomal fusion, endocytosed beads were trafficked to a lysosomal compartment. These findings suggest that heparin-coated beads and Chlamydia trachomatis enter eukaryotic cells by similar pathways.Numerous bacterial pathogens enter eukaryotic cells and use this intracellular site for replication and for persistence in their human hosts. The molecular mechanism of attachment and entry in terms of the bacterial ligand and host cell receptor has been described for only a few bacteria (12). It is nevertheless apparent that microbial strategies for mammalian cell attachment and entry are intimately coupled to natural biological functions of the eukaryotic cell hosts. Thus, understanding the molecular basis of entry into nonprofessional phagocytic cells, such as epithelial cells, should provide important mechanistic insights into fundamental biological properties of both the pathogen and the eukaryotic cell.Chlamydia trachomatis is an obligate intracellular bacterium that causes a wide spectrum of human disease affecting hundreds of millions of people (15). C. trachomatis infections are the most common cause of sexually transmitted diseases, often resulting in severe pathology in women and newborns (33). C. trachomatis is the most prevalent reported infection in the United States and may increase the risk of human immunodeficiency virus infection (22). These organisms are also the cause of trachoma, the leading cause of preventable blindness in the world (24). Chlamydiae attach to, and enter, eukaryotic epithelial cells of mucosal surfaces and grow within host cell membrane-bound vacuoles that do not fuse with lysosomes (26). Entry of chlamydiae into host cells is thought to be by receptor-mediated endocytosis (40), although little is known about the receptor on eukaryotic cells to which chlamydiae bind. Binding to the receptor is saturable (39) and sensitive to trypsin treatment of host cells (5). In addition, the host cell interaction with chlamydiae is inhibitable by exogenous heparin or heparan sulfate (21, 42).It has previously been proposed that heparin and heparan sulfate are structural and functional analogs of the C. trachomatis LGV biovar attachment ligand, because attachment and consequent infectivity can be (i) competitively inhibited by heparin or heparan sulfate, (ii) abolished following treatment of chlamydiae with a heparan sulfate-specific lyase, and (iii) rescued by coating heparan sulfate lyase-treated chlamy...

There is a general need for additional selectable marker genes for plant transformation. Only a few have been reported in wheat (Triticum aestivum L.) transformation experiments, some of which are under patent restriction or have other disadvantages. A new selectable marker gene was identified which can be used to select resistant callus in tissue culture and regenerate transgenic wheat plants. A gene from the soil fungus Myrothecium verrucaria (Albertini & Schwein.) Ditmar:Fr., coding for the enzyme cyanamide hydratase which converts cyanamide into urea, was previously described. In our wheat transformation experiments, the gene conferred resistance to cyanamide at a tissue culture level and therefore cyanamide could be used to select for transformants. At the whole plant level, progeny of transformed wheat plants showed resistance to cyanamide, whereas sensitive plants expressed a lethal necrosis and yellowing when cyanamide was applied. This gene has several potential advantages when compared with other selectable marker genes. Transformed wheat plants can be selected at the tissue culture level and may be able to convert cyanamide into a useful nitrogen compound (fertilizer). The selectable marker gene could be introduced with other genes for value‐added traits in wheat and might also be applicable in other transformation systems.