Three-dimensional electron microscopy reconstructions of native, half-transformed, and transformed ␣ 2 -macroglobulins (␣ 2 Ms) labeled with a monoclonal Fab Fab offer new insight into the mechanism of its proteinase entrapment. Each ␣ 2 M binds four Fabs, two at either end of its dimeric protomers approximately 145 Å apart. In the native structure, the epitopes are near the base of its two chisel-like features, laterally separated by 120 Å, whereas in the methylamine-transformed ␣ 2 M, the epitopes are at the base of its four arms, laterally separated by 160 Å. Upon thiol ester cleavage, the chisels on the native ␣ 2 M appear to split with a separation and rotation to give the four arm-like extensions on transformed ␣ 2 M. Thus, the receptor binding domains previously enclosed within the chisels are exposed. The labeled structures further indicate that the two protomeric strands that constitute the native and transformed molecules are related and reside one on each side of the major axes of these structures. The halftransformed structure shows that the two Fabs at one end of the molecule have an arrangement similar to those on the native ␣ 2 M, whereas on its transformed end, they have rotated. The rotation is associated with a partial untwisting of the strands and an enlargement of the openings to the cavity. We propose that the enlarged openings permit the entrance of the proteinase. Then cleavage of the remaining bait domains by a second proteinase occurs with its entrance into the cavity. This is followed by a retwisting of the strands to encapsulate the proteinases and expose the receptor binding domains associated with the transformed ␣ 2 M.Human ␣ 2 -macroglobulin (␣ 2 M 1 , M r ϭ 720,000) is an essential protein present at a high concentration in the serum (ϳ2 mg/ml) that has the unusual physiological role of a nonspecific proteinase scavenger (1-3). In a presently poorly understood mechanism, native ␣ 2 M irreversibly traps almost all known endoproteinases by undergoing a structural change that involves a large alteration in its shape. Evolutionarily related proteins performing a similar physiological function, termed ␣-macroglobulins, are present in all vertebrates and several invertebrates (1). Recently, an impairment in the ␣ 2 M gene has been implicated in the etiology of Alzheimer's disease (4).␣ 2 M is a glycoprotein assembled from four identical 180-kDa subunits that are disulfide-linked in pairs to form two protomers, which, in turn, are noncovalently associated (1). Each subunit contains an approximately 40-residue-long sequence termed the "bait" region, which displays target sequences for a variety of proteinases (5). Bait region cleavage by a proteinase in turn causes the activation of a functionally important internal thiol ester bond between Cys 949 and Glx 952 of the subunit, which rapidly undergoes a nucleophilic attack (1). Cleavage of the thiol ester moiety triggers a major shape change, aptly termed the "mousetrap mechanism," that causes ␣ 2 M to internally sequester the proteinase, w...

The reconstructions of an intermediate form of human ␣ 2 -macroglobulin (half-transformed ␣ 2 M) in which two of its four bait regions and thiol ester sites were cleaved by chymotrypsin bound to Sepharose were obtained by three-dimensional electron microscopy from stain and frozen-hydrated specimens. The structures show excellent agreement and reveal a structure with approximate dimensions of 195 (length) ؋ 135 (width) and 130 Å (depth) with an internal funnel-shaped cavity. The structure shows that a chisel-shaped body is connected to a broad base at the opposing end by four stands. Four approximately 45 Å diameter large openings in the body of the structure result in a central cavity that is more accessible to the proteinase than those associated with the native or fully transformed structures. The dissimilarity in the shapes between the two ends of ␣ 2 M half-transformed and the similarity between its chisel-shaped body and that of native ␣ 2 M indicate that the chymotrypsin has cleaved both bait regions in the bottom-half of the structure. Consequently, its functional division lies on the minor axis. The structural organization is in accord with biochemical studies, which show that the half-transformed ␣ 2 M migrates on native polyacrylamide gels at a rate intermediate to the native and fully transformed ␣ 2 M and is capable of trapping 1 mol of proteinase. Even though its upper portion is similar to the native molecule, significant differences in their shapes are apparent and these differences may be related to its slower reaction with a proteinase than the native structure. These structural comparisons further support the view that the transformation of ␣ 2 M involves an untwisting of its strands with an opening of the cavity for entrance of the proteinase and a retwisting of the strands around the proteinase resulting in its encapsulation.␣-Macroglobulins (␣Ms) are nonspecific, irreversible inhibitors of endoproteinases found in the circulation of all vertebrates and some invertebrates (for a review, see Ref. 1). Human ␣ 2 -macroglobulin (␣ 2 M), 1 the largest known proteinase inhibitor (M r ϭ 720,000), is a homotetramer formed by two protomeric units, each of which contains two 180-kDa subunits linked by two disulfide bonds. It has a vital role in the clearance of proteinases from the circulation and in regulating their activity in fibrinolysis, coagulation, and complement activation (2, 3). A single ␣ 2 M molecule can entrap two proteinase molecules such as chymotrypsin and trypsin and can therefore be considered to contain two functional domains (1). Each subunit of ␣ 2 M has a bait region with cleavage sites for nearly all known endoproteinases and an internal thiol ester bond. A proteinase cleaves the two bait regions within both functional units, leading to an activation and cleavage of the thiol ester bonds. Consequently, ␣ 2 M undergoes a major structural change resulting in entrapment of the proteinase and its covalent linkage to the molecule (1, 4). The bound proteinase, although inaccessible to...

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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.

Introduction Optical Spectroscopy has been studied for biologic plausisbility, technical efficacy, clinical effectiveness, patient satisfaction and cost-effectiveness. We sought to identify healthcare provider attitudes or practices that might act as barriers or to the dissemination of this new technology. Methods Through an academic-industrial partnership, we conducted a series of focus groups to examine physician barriers to optical diagnosis. The study was conducted in two stages. First, a pilot group of ten physicians (8 obstetrician gynecologists and two family practitioners) was randomly selected from 8 regions of the US and interviewed individually. They were presented with the results of a large trial (N=980) testing the accuracy of a spectroscopy based device in the detection of cervical neoplasia. They were also shown a prototype of the device and were given a period of time to ask questions and receive answers regarding the device. They were also asked to provide feedback of a questionnaire (provided in Appendix A) which was then revised and presented to three larger focus groups (n=13, 15, 17 for a total n=45). The larger focus groups were conducted during national scientific meetings with 20 obstetrician gynecologists and 25 primary care physicians (family practitioners and internists). Results When asked about the dissemination potential of the new cervical screening technology, all study groups tended to rely on established clinical guidelines from their respective professional societies with regard to the screening and diagnosis of cervical cancer. In addition, study participants consistently agreed that real-time spectroscopy would be viewed positively by their patients. Participants were positive about the new technology's potential as an adjunct to colposcopy and agreed that the improved accuracy would result in reduced healthcare costs (due to decreased biopsies and decreased visits). However, while all saw the potential of real-time diagnosis, there were many perceived barriers. These barriers included: changes in scheduling and work-flow, liability, documentation, ease of use, length of training, device cost, and reimbursement by third party payers. Conclusion Barriers exist to the dissemination of optical technologies into physician practice. These will need to be addressed before cervical screening and diagnosis programs can take advantage of spectroscopy-based instruments for cancer control.