Estrogen receptor (ER) modulators produce distinct tissue-specific biological effects, but within the confines of the established models of ER action it is difficult to understand why. Previous studies have suggested that there might be a relationship between ER structure and activity. Different ER modulators may induce conformational changes in the receptor that result in a specific biological activity. To investigate the possibility of modulator-specific conformational changes, we have applied affinity selection of peptides to identify binding surfaces that are exposed on the apo-ERs ␣ and ␤ and on each receptor complexed with estradiol or 4-OH tamoxifen. These peptides are sensitive probes of receptor conformation. We show here that ER ligands, known to produce distinct biological effects, induce distinct conformational changes in the receptors, providing a strong correlation between ER conformation and biological activity. Furthermore, the ability of some of the peptides to discriminate between different ER ␣ and ER ␤ ligand complexes suggests that the biological effects of ER agonists and antagonists acting through these receptors are likely to be different.

This panel of four serum proteins is valuable in suggesting the diagnosis of lung cancer. These data may be useful for treating patients with an indeterminate pulmonary lesion, and potentially in predicting individuals at high risk for lung cancer.

We demonstrate by using purified bacterial components that the protein kinases that regulate chemotaxis and transcription of nitrogen-regulated genes, CheA and NRII, respectively, have cross-specificities: CheA can phosphorylate the Ntr transcription factor NRI and thereby activate transcription from the nitrogen-regulated ginA promoter, and NRII can phosphorylate CheY. In addition, we find that a high intracellular concentration of a highly active mutant form of NRII can suppress the smooth-swimming phenotype of a cheA mutant. These results argue strongly that sensory transduction in the Ntr and Che systems involves a common protein phosphotransfer mechanism.Bacteria respond to changes in the availability of nutrients such as nitrogen, phosphate, and oxygen; changes in medium osmolarity; and gradients of chemotactic stimuli by means of a family of homologous signal transduction systems (1)(2)(3)(4) 5492The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The phospholipase D (PLD) superfamily includes enzymes of phospholipid metabolism, nucleases, as well as ORFs of unknown function in viruses and pathogenic bacteria. These enzymes are characterized by the invariant sequence motif, H(X)K(X) 4 D. The endonuclease member Nuc of the PLD family was over-expressed in bacteria and purified to homogeneity. Mutation of the conserved histidine to an asparagine in the endonuclease reduced the k cat for hydrolysis by a factor of 10 5 , suggesting that the histidine residue plays a key role in catalysis. In addition to catalyzing hydrolysis, a number of phosphohydrolases will catalyze a phosphate (oxygen)-water exchange reaction. We have taken advantage of this observation and demonstrate that a 32 P-labeled protein could be trapped when the enzyme was incubated with 32 Plabeled inorganic phosphate. The phosphoenzyme intermediate was stable in 1 M NaOH and labile in 1 M HCl and 1 M hydroxylamine, suggesting that the enzyme forms a phosphohistidine intermediate. The pH-stability profile of the phosphoenzyme intermediate was consistent with phosphohistidine and the only radioactive amino acid found after alkaline hydrolysis was phosphohistidine. These results suggest that the enzymes in the PLD superfamily use the conserved histidine for nucleophilic attack on the substrate phosphorus atom and most likely proceed via a common two-step catalytic mechanism.

The Cdc25 cell cycle regulator is a member of the dual-specificity class of protein-tyrosine phosphatases that hydrolyze phosphotyrosine-and phosphothreonine-containing substrates. To study the mechanism of Cdc25B, we have overexpressed and purified the catalytic domain of human Cdc25B (Xu, X., and Burke, S. P. (1996) J. Biol. Chem. 271, 5118 -5124). In the present work, we have analyzed the kinetic properties of the Cdc25B catalytic domain using the artificial substrate 3-O-methylfluorescein phosphate (OMFP). Steady-state kinetic analysis indicated that the k cat /K m for OMFP hydrolysis is almost 3 orders of magnitude greater than that for p-nitrophenyl phosphate hydrolysis. Like other dual-specificity phosphatases, Cdc25 exhibits a two-step catalytic mechanism, characterized by formation and breakdown of a phosphoenzyme intermediate. Presteady-state kinetic analysis of OMFP hydrolysis indicated that formation of the phosphoenzyme intermediate is ϳ20 times faster than subsequent phosphoenzyme breakdown. The resulting burst pattern of product formation allowed us to derive rate constants for enzyme phosphorylation (26 s ؊1) and dephosphorylation (1.5 s ؊1) as well as the dissociation constant for OMFP (0.3 mM). Calculations suggest that OMFP binds with higher affinity and reacts faster with Cdc25B than does p-nitrophenyl phosphate. OMFP is a highly efficient substrate for the dual-specificity protein-tyrosine phosphatases VHR and rVH6, but not for two protein-tyrosine phosphatases, PTP1 and YOP. The ability to observe distinct phases of the reaction mechanism during OMFP hydrolysis will facilitate future analysis of critical catalytic residues in Cdc25 and other dual-specificity phosphatases.

SUMMARY Some patients with cancer never develop metastasis, and their host response may provide cues for innovative treatment strategies. We previously reported an association between autoantibodies against complement factor H (CFH) and early stage lung cancer. CFH prevents complement-mediated cytotoxicity (CDC) by inhibiting formation of cell-lytic membrane attack complexes on self-surfaces. In an effort to translate these findings into a biologic therapy for cancer, we isolated and expressed DNA sequences encoding high affinity human CFH antibodies directly from single, sorted B cells obtained from patients with the antibody. The co-crystal structure of a CFH antibody-target complex shows a conformational change in the target relative to the native structure. This recombinant CFH antibody causes complement activation, release of anaphylatoxins, and promotes CDC of tumor cell lines, and inhibits tumor growth in vivo. The isolation of anti-tumor antibodies derived from single human B cells represents an alternative paradigm in antibody drug discovery.

Ascending pathways to the telencephalon from the secondary gustatory nucleus (SGN), preglomerular tertiary gustatory nucleus (pTGN), and medial preglomerular nucleus (PGm) were examined by tract‐tracing experiments in goldfish Carassius auratus. Tracer injections to the SGN suggest the presence of direct ascending pathways to the supracommissural and the dorsal parts of the ventral telencephalic area, and the medial part of the dorsal telencephalic area (Dm), restricted to its ventral region. The SGN experiments also suggest projections to the pTGN and PGm, and several neuronal types in the primary gustatory centers were newly found to give rise to ascending fibers to the SGN. Injections to the pTGN suggest reciprocal connections of the nucleus with the dorsal region of the Dm (dDm). Injections to the PGm resulted in labeled cells in the dorsal part of the SGN, the secondary general visceral nucleus, and the posterior part of the dorsal telencephalic area, suggesting that this preglomerular nucleus receives gustatory, general visceral, and olfactory inputs. Fibers labeled from the PGm terminated in the central part of the dorsal telencephalic area and the dDm; the latter region contained many labeled somata. The terminal zone of PGm fibers in the dDm is located laterally adjacent to that from the pTGN. Injection experiments to the pTGN and PGm also suggest connections of these nuclei with the inferior lobar nuclei and torus lateralis. Based on the results of the present as well as recent studies, an updated map is provided that shows by and large distinct sensory representation within the goldfish dorsal telencephalic area. J. Comp. Neurol. 520:2475–2499, 2012. © 2012 Wiley Periodicals, Inc.

Characterization of the humoral immune response in selected patients with cancer who uniformly do well may lead to the development of novel therapeutic strategies. We have previously shown an association between patients with early-stage nonmetastatic lung cancer and autoantibodies to complement factor H (CFH). CFH protects normal and tumor cells from destruction by the alternative complement pathway by inactivating C3b, a protein that is essential for formation of a lytic complex on the cell surface. Here, we show that CFH autoantibodies in lung cancer patients recognize a conformationally distinct form of CFH in vitro, are IgG3 subclass, and epitope map to a crucial functional domain of CFH known to interact with C3b. Purified CFH autoantibodies inhibited binding of CFH to A549 lung tumor cells, increased C3b deposition, and caused complement-dependent tumor cell lysis. This work demonstrates that CFH autoantibodies isolated from patients with lung cancer can kill tumor cells in vitro, suggesting that they may perform this function in vivo as well. Development of specific antibodies to the conformationally distinct epitope of CFH may lead to a useful biologic therapy for lung cancer.