Here we examine the kinetics of alpha methyl mannose induced dissociation of yeast (Saccharomyces cerevisiae) that have been previously allowed to bind to concanavalin A (Con A) derivatized agarose beads, a model for eluting glycans from Con A beads in purification protocols and for releasing pathogens from human cells. This study tests the statistical reliability of this model using one glycan concentration with hundreds of replicate experiments. Future dose‐response studies will precisely identify the reagents that are most effective at the lowest concentrations. In 963 trials, yeast release from the Con A beads was measured by counting yeast remaining on single yeast bound beads in the presence and absence of 1.5M alpha methyl mannose over a 60 min time course. Two‐ sample t tests showed significant differences in the amount of yeast remaining bound in the sugar versus control (no sugar) samples after 20 min (p less than 0.0001), 40 min (p less than 0.0001) and 60 min (p less than 0.0001), where the sugar caused substantially more dissociation than in its absence. Repeated measures‐ANOVA indicated a significant effect of the glycan treatment on the number of bound yeast (F= 624, p less than 0.0001), as well as a significant interaction between the treatment and time elapsed (F= 339, p less than 0.0001). This is therefore a reliable, simple, precise and inexpensive model for dissociation kinetic studies. Two other studies in these proceeding using different reagents support this conclusion (Supported by NIH NIGMS SCORE (S0648680), RISE, MARC, the Joseph Drown Foundation and the Sidney Stern Memorial Trust).

Using a model system of washed yeast (Saccharomyces cerevisiae) that possess mannose‐rich cell surfaces and washed concanavalin A derivatized agarose beads that preferentially bind to glycans containing mannose/glucose residues, we tested 4 concentrations of alpha methyl mannose and the same 4 concentrations of D‐melezitose (0.025 mM – 25 mM) and a 50/50 mixture of these 2 sugars at the same final concentrations, over a 30 minute time course, assaying how much yeast remained bound to the beads at 10 minute intervals with and without sugar at each sugar concentration tested. In 15,379 replicate experiments, averaging about 300 trials for each sugar at each time tested, alpha methyl mannose and D‐melezitose caused dissociation of yeast from the Con A beads in a concentration dependent manner with an almost linear time course with little dissociation occurring immediately and incrementally increased dissociation occurring after 10,20 and 30 minutes. The highest sugar concentration (25 mM) tested here was most effective in causing dissociation and the 50/50 mixture of sugars was less effective than the single sugars in causing dissociation at the lower concentrations (less than 25 mM). The results suggest that careful attention should be paid to sugar concentration and incubation times in experiments using lectin derivatized beads in molecular purification protocols and in development of carbohydrate‐based drugs and diagnostic tests (Supported by NIH NIGMS SCORE (S06‐48680, NIH MBRS RISE, NIH MARC and the Joseph Drown Foundation).

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Adhesion between the archenteron tip and blastocoel roof in the NIH designated sea urchin embryo model is a cellular interaction that has interested investigators for over a century, but its molecular basis is not understood. Here we microdissect the two components of this cellular interaction and use alpha methyl mannose to map molecular groups that may be involved in mediating the cellular interaction. Whole 48–54 hour fixed Lytechinus pictus embryos were washed three times and then dissected in pH 8.0 artificial sea water (ASW), 15°C. The roofs of the blastocoel and the tips of the archenteron were put together and observed to stick together. In over 50 separate trials using bound sets of roof and archenterons attached together, either 0.2 M or 1.0 M alpha methyl mannose was added to the pieces. The pieces remained together. However in 100% of the cases, manually separating the pieces in alpha methyl mannose at either molarity resulted in blocking the rebinding of the blastocoel roofs and archenteron tips.Re‐binding occurred in the absence of the sugar. The results suggest that alpha methyl mannose binding receptors are involved in mediating the cellular interaction. These studies offer a novel approach to map glycans and glycan binding partners that may be functionally important. By microdissecting the components of cellular interactions out of the embryo proper, these components can be probed in pristine media away from factors in intact embryos that could confuse results (Supported by NIH NIGMS SCORE S0648680, MARC, RISE, the Joseph Drown Foundation, the Sidney Stern Memorial Trust, and CSU Northridge Biology Full Immersion Research Experience (FIRE) course funding).

Past studies have examined the inhibition of binding of mannose‐rich yeast to immobilized concanavalin A (Con A) in the presence and absence of specific saccharides. This is a model system for testing potential drugs that could block pathogen binding to human cells. Here 2.0M, 0.2 M and 0.02 M NaCl and KCl were tested in a much more extensive study than in the past, for their ability to inhibit binding of yeast (Saccharomyces cerevisiae) to immobilized Con A over a 30 min time course. In about 15,000 replicate experiments, both salts inhibited binding in a nearly identical way, in a concentration dependent manner, ranging from about 20% to about 60% reduced binding over controls. The salt effects reached a plateau at 20‐30 minutes, with 2.0 M salt showing the greatest inhibitory effects. These results are similar to those obtained in studies with specific saccharides, suggesting that salt effectively can block lectin‐saccharide binding, with possible implications in pathogen binding to human cells (Supported by NIH NIGMS SCORE (S0648680), MARC, RISE, the Joseph Drown Foundation and Sidney Stern Memorial Trust.