Type I and II classical cadherins help to determine the adhesive specificities of animal cells. Crystal-structure determination of ectodomain regions from three type II cadherins reveals adhesive dimers formed by exchange of N-terminal beta strands between partner extracellular cadherin-1 (EC1) domains. These interfaces have two conserved tryptophan side chains that anchor each swapped strand, compared with one in type I cadherins, and include large hydrophobic regions unique to type II interfaces. The EC1 domains of type I and type II cadherins appear to encode cell adhesive specificity in vitro. Moreover, perturbation of motor neuron segregation with chimeric cadherins depends on EC1 domain identity, suggesting that this region, which includes the structurally defined adhesive interface, encodes type II cadherin functional specificity in vivo.

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Cadherins constitute a family of cell-surface proteins that mediate intercellular adhesion through the association of protomers presented from juxtaposed cells. Differential cadherin expression leads to highly specific intercellular interactions in vivo. This cellcell specificity is difficult to understand at the molecular level because individual cadherins within a given subfamily are highly similar to each other both in sequence and structure, and they dimerize with remarkably low binding affinities. Here, we provide a molecular model that accounts for these apparently contradictory observations. The model is based in part on the fact that cadherins bind to one another by ''swapping'' the N-terminal ␤-strands of their adhesive domains. An inherent feature of strand swapping (or, more generally, the domain swapping phenomenon) is that ''closed'' monomeric conformations act as competitive inhibitors of dimer formation, thus lowering affinities even when the dimer interface has the characteristics of high-affinity complexes. The model describes quantitatively how small affinity differences between low-affinity cadherin dimers are amplified by multiple cadherin interactions to establish large specificity effects at the cellular level. It is shown that cellular specificity would not be observed if cadherins bound with high affinities, thus emphasizing the crucial role of strand swapping in cell-cell adhesion. Numerical estimates demonstrate that the strength of cellular adhesion is extremely sensitive to the concentration of cadherins expressed at the cell surface. We suggest that the domain swapping mechanism is used by a variety of cell-adhesion proteins and that related mechanisms to control affinity and specificity are exploited in other systems.binding affinity ͉ domain swapping ͉ binding specificity ͉ protein interfaces A dhesive interactions between cadherin family members presented on cell surfaces is thought to provide a key driving force in the development of tissue architecture (1-3). Morphogenetic changes often correlate directly with changes in expression of individual cadherin family members, and genetic deletion of individual cadherins in vivo, or misexpression by transgenesis, interferes with tissue development in characteristic ways for each cadherin family member. Such data have been interpreted as evidence of the presence of highly specific homophilic interactions in the molecular recognition properties of individual cadherins. However, cell-sorting experiments in vitro often fail to reveal adhesive specificity in the binding behavior of cells expressing different cadherin types (4, 5). Moreover, analysis of cadherin sequences and structures do not clearly reveal why homodimer formation should be substantially preferred over the formation of heterodimers (6). Thus, cell-cell adhesion specificity is not simply correlated with molecular-binding specificity within the cadherin family. Indeed, it has been suggested that cellular binding specificity arises primarily from differences in overall cadheri...

Methods that predict membrane helices have become increasingly useful in the context of analyzing entire proteomes, as well as in everyday sequence analysis. Here, we analyzed 27 advanced and simple methods in detail. To resolve contradictions in previous works and to reevaluate transmembrane helix prediction algorithms, we introduced an analysis that distinguished between performance on redundancy-reduced highand low-resolution data sets, established thresholds for significant differences in performance, and implemented both per-segment and per-residue analysis of membrane helix predictions. Although some of the advanced methods performed better than others, we showed in a thorough bootstrapping experiment based on various measures of accuracy that no method performed consistently best. In contrast, most simple hydrophobicity scale-based methods were significantly less accurate than any advanced method as they overpredicted membrane helices and confused membrane helices with hydrophobic regions outside of membranes. In contrast, the advanced methods usually distinguished correctly between membrane-helical and other proteins. Nonetheless, few methods reliably distinguished between signal peptides and membrane helices. We could not verify a significant difference in performance between eukaryotic and prokaryotic proteins. Surprisingly, we found that proteins with more than five helices were predicted at a significantly lower accuracy than proteins with five or fewer. The important implication is that structurally unsolved multispanning membrane proteins, which are often important drug targets, will remain problematic for transmembrane helix prediction algorithms. Overall, by establishing a standardized methodology for transmembrane helix prediction evaluation, we have resolved differences among previous works and presented novel trends that may impact the analysis of entire proteomes.

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VE-cadherin is an adhesion molecule localized at the adherens junctions of endothelial cells. It is crucial for the proper assembly of vascular structures during angiogenesis and maintaining vascular integrity. We have studied 3 monoclonal antibodies (mAbs) against murine VEcadherin that inhibit angiogenesis and tumor growth. Two of these, BV13 and 10G4, also disrupted normal vessels, resulting in severe vascular leakage, whereas the third, E4G10, did not. The goal of the current report was to identify the epitope of E4G10 and distinguish it from those of the disruptive mAbs. We mapped the epitope of E4G10 to within the first 10 amino acids of mature VEcadherin and demonstrated that conserved tryptophan residues in this sequence are required for VE-cadherinmediated trans-adhesion. The disruptive mAbs target a different epitope within amino acids 45 to 56, which structural homology modeling suggests is not involved in trans-adhesion. From our studies, we hypothesize that E4G10 can only bind the neovasculature, where VEcadherin has not yet engaged in transadhesion and its epitope is fully exposed. Thus, E4G10 can inhibit junction formation and angiogenesis but is unable to target normal vasculature because its epitope is masked. In contrast, BV13 and 10G4 bind an epitope that is accessible regardless of VE-cadherin interactions, leading to the disruption of adherens junctions. Our findings establish the immediate N-terminal region of VE-cadherin as a novel target for inhibiting angiogenesis. IntroductionCadherins are a large family of adhesion molecules involved in the formation of specific cell-cell contacts. 1 In humans, more than 80 members of the cadherin superfamily have been identified and are classified into subfamilies according to the presence of conserved domains and sequence motifs. 2 Cadherins are single-pass transmembrane glycoproteins defined by distinctive extracellular cadherin domains (ECDs) of about 110 amino acids. They mediate calciumdependent homophilic interactions and are responsible for selective cell-cell recognition and adhesion. These processes play an important role during embryonic morphogenesis and maintenance of tissue architecture. 2,3 Cadherins are subdivided according to specific sequence features; subfamilies include the type I (eg, N-, E-, and C-) and type II (eg, VE-and MN-) cadherins. Both type I and type II cadherins consist of 5 ECDs (ECD1-5) and are anchored to the actin cytoskeleton through their cytoplasmic tail. 4 The determinants of adhesion and adhesive specificity among the type I classic cadherins, specifically the N-, E-and C-cadherins, have been extensively studied. [5][6][7][8][9][10] In particular, the 3-dimensional structures of the N-terminal domains of N-and E-cadherins as well as the entire 5 ECDs of C-cadherin have been solved. These data indicate that the crucial adhesive determinants reside in the N-terminal ECD1 with the central feature being a conserved tryptophan residue (W2) that inserts into the hydrophobic core of the partner cadherin molecule pres...

We have developed a practice procedure for prostate lymphoscintigraphy using SPECT/CT and filtered 99m Tc-sulfur nanocolloid, as an alternative to the proprietary product 99m Tc-Nanocoll, which is not approved in the United States. Methods: Ten patients were enrolled for this study, and all received radiotracer prepared using a 100-nm membrane filter at a commercial radiopharmacy. Whole-body scans and SPECT/CT studies were performed within 1.5-3 h after the radiotracer had been administered directly into 6 locations of the prostate gland under transrectal ultrasound guidance. The radiation dose was estimated from the first 3 patients. Lymphatic drainage mapping was performed, and lymph nodes were identified. Results: The estimated radiation dose ranged from 3.9 to 5.2 mSv/MBq. The locations of lymph nodes draining the prostate gland were similar to those found using the proprietary product. Conclusion: When the proprietary radiolabeled nanocolloid indicated for lymphoscintigraphy is not available, prostate lymph node mapping and identification are still feasible using filtered 99m Tc-sulfur nanocolloid.

Although widespread PSA screening has inevitably led to increased diagnosis of lower risk prostate cancer, the number of patients with nodal involvement at baseline remains high (nearly 40% of high risk patients initially staged cN0). These rates probably do not reflect the true incidence of prostate cancer with lymph node involvement among patients selected for external beam radiotherapy (EBRT), as patients selected for surgery often have more favorable prognostic features. At many institutions, radical treatment directed only at the prostate is considered standard and patients known to have regional disease are often managed palliatively with androgen deprivation therapy (ADT) for presumed systemic disease. New imaging tools such as MR lymphangiography, choline-based PET imaging or combined SPECT/CT now allow surgeons and radiation oncologists to identify and target nodal metastasis and/or lymph nodes with a high risk of occult involvement. Recent advances in the field of surgery including the advent of extended nodal dissection and sentinel node procedures have suggested that cancer-specific survival might be improved for lymph-node positive patients with a low burden of nodal involvement when managed with aggressive interventions. These new imaging tools can provide radiation oncologists with maps to guide delivery of high dose conformal radiation to a target volume while minimizing radiation toxicity to non-target normal tissue. This review highlights advances in imaging and reports how they may help to define a new paradigm to manage node-positive prostate cancer patients with a curative-intent.