Bryan, A. microscopy. The capsule could be guarded against radiation by either of the free radical scavengers ascorbic acid and sorbitol. Sugar composition analysis of polysaccharide removed from the outer and inner parts of the capsule revealed significant differences in glucuronic acid and xylose molar ratios, implying differences in the chemical structure of the constituent polysaccharides. Our results provide compelling evidence for the presence of two zones in the capsule that differ in susceptibility to dimethyl sulfoxide and radiation and, possibly, in packing and composition. The capsule of the human-pathogenic fungus is usually a complex polysaccharide structure that surrounds the cells of this organism in vitro and in vivo. The capsule is composed of several polysaccharides, of which glucuronoxylomannan (GXM) is the major constituent (2, 10). Minor components of the capsule are galactoxylomannan and mannoprotein (9). The structure of the capsule has attracted considerable attention because it is usually a distinctive feature of this organism and an important virulence factor (2, 3, 6, 17). Despite intense interest in capsule-related studies, we know remarkably little about the architecture of Anandamide the assembled capsule because there are relatively few techniques for studying this fragile structure in and other encapsulated microbes. One of the most striking characteristics of the capsule is usually that it changes size depending on the environmental conditions experienced by the fungus. Several factors, such as CO2, limited iron concentration, and contamination in mice, are known to increase capsule size (1, 12, 26). In our laboratories, we have been studying the role of the capsule in complement- and antibody-mediated phagocytosis of (38) and, more recently, described experimental conditions that reliably promote rapid capsular growth in vitro (37, 36) and facilitate studies of capsule growth and architecture. Suspension of encapsulated cells in dimethyl sulfoxide (DMSO) is known to remove a significant proportion of the capsule (21). Recently, Gates et al. used the combination of DMSO capsule removal and diffusion of macromolecules into the capsule to establish that this capsule differs in porosity as a function of radial distance from the cell wall, with the inner layers being more tightly packed (20). That obtaining and the observations that soluble polysaccharide could bind to acapsular strains to form a small capsule and that the capsular polysaccharide was shed into answer during growth suggested that this capsule is usually loosely attached to the fungal cell (24). Consistent with this view, Reese and Doering recently exhibited that this capsular polysaccharide is usually attached to -1,3-glucan in the cell wall through a noncovalent conversation (29). However, the fact that addition of exogenous polysaccharide to acapsular mutants does not reconstitute the full-size capsular structure implies that the naturally assembled structure has significantly greater complexity and is possibly qualitatively different. Three decades ago Dembintzer et al. reported the phenomenon of capsule disappearance after irradiation with large doses of gamma rays (16). We hypothesized that by using much lower doses of radiation, we could use this nonchemical method for removing the capsule gradually and thus study its structure. On the other hand, while carrying out experiments to develop radioimmunotherapy for contamination, we noted that this organism was extremely resistant to external gamma radiation and, conversely, demonstrated a comparatively 1,000-fold Anandamide greater susceptibility to particulate radiation when incubated with radiolabeled capsule binding monoclonal antibody (14). Since monoclonal antibody can alter capsular structure (11, 28) and the capsule protects cells Sdc2 against many insults (6), we also entertained the notion that this capsule may protect the fungus against ionizing radiation and that radiolabeled capsule-binding monoclonal antibodies might have profound effects around the capsule. In this study we have investigated the conversation of the capsule of serotype A with capsule-binding monoclonal antibody 18B7 labeled with rhenium-188 (188Re), external gamma radiation, and the known decapsulating agent DMSO (21) by microscopic, serological, and biochemical techniques. The use of radiation to probe capsule structure provides a new approach for this Anandamide complex problem. The results offer new insights into the architecture of the capsule and indicate that this polysaccharide capsule provides protection against radiation. MATERIALS AND METHODS strains and growth conditions. Capsule induction was performed with the experimental conditions described before (36). Briefly, the serotype A strain H99 was produced.