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STEAP2 is a member of the Six-Transmembrane Epithelial Antigen of the Prostate (STEAP) protein family that is proposed to function as metalloreductase. While STEAP2 shows a complex subcellular distribution pattern localizing to both secretory and endocytic pathway organelles, how such broad steady-state distribution is maintained is unknown. Similarly, whether STEAP2 undergoes any compartment-specific modulation during intracellular trafficking has not been reported. Leveraging a newly-identified monoclonal antibody that recognizes a conformation-sensitive epitope nested in the second extracellular loop of STEAP2, we demonstrate that the epitope formation was dependent on the cholesterol content of the membrane in which STEAP2 was embedded. Monitoring the STEAP2-dependent internalization of this antibody uncovered STEAP2's rapid internalization from the cell surface and their subsequence trafficking to the Golgi region and endosome-like puncta. Acute inhibition of endocytosis also increased the detectable amount of STEAP2 at the plasma membrane. Collectively, these experiments demonstrate that an intricate balance of membrane flux between the secretory and endocytic pathways underlies the characteristic broad subcellular localization of STEAP2. By using a cell-based assay that detects the metalloreductase functions of cell surface-localizing STEAP4, STEAP2's metalloreductase activities were not detectable, suggesting that its enzymatic function is suppressed at the plasma membrane. The conformational modulation of STEAP2 by the local membrane cholesterol content can therefore serve as a potential mechanism to modulate STEAP2 function in a compartment-restricted manner, by coupling a pre-existing difference in cholesterol content among different cellular membranes to a dynamic trafficking process leading to broad subcellular distribution.

The identification of loss-of-function mutations of the IL36RN gene encoding the interleukin-36 receptor antagonist (IL-36Ra) in generalized pustular psoriasis (GPP) emphasized the key role of this pathway in skin innate immunity and systemic inflammation. It has been previously shown in vitro that removal of the N-terminal amino acid IL36Ra (M1) is critical to its biological activity, but the in vivo contribution of this processing remains unknown. We report herein a new homozygous (c4G>T, pV2F) missense IL36RN mutation segregating in a family with three GPP-affected patients. The V2F mutation does not alter IL-36Ra protein expression but was devoid of any antagonist activity. Mass spectrometry showed that the V2F IL-36Ra mutant retains its first N-terminal methionine. These results provide the first in vivo demonstration that removal of N-terminal methionine of native IL-36Ra is a mandatory step to reach optimal antagonist activity and to prevent sustained skin and systemic inflammation in humans.

In this research, we look at the similarity between frontline service employees’ nonverbal or expressive behavior and customers’ receptivity of nonverbally expressed emotions (i.e., expressive similarity). Supported by evidence from four studies, we demonstrate that expressive similarity between customers and frontline service employees yields positive outcomes for both the employee and the organization under successful service delivery, but it can paradoxically backfire on the organization in service failures. In successful service encounters, higher expressive similarity between customers and employees enhances consumer satisfaction and promotes more direct compliments and positive word of mouth. In contrast, higher expressive similarity increases customer dissatisfaction and intent to engage in negative word of mouth, but it reduces customers’ inclination to lodge direct complaints following a service failure (Study 1). Studies 2 and 3, both field experiments, provide external validation of the key findings on customer satisfaction and voice intentions (Study 2) as well as actual voice behavior (Study 3). Building on these findings, Study 4 reveals that while customer-perceived rapport and trait impressions of the service employee mediate the observed effects of expressive similarity on satisfaction, only rapport significantly explains the effects of expressive similarity on voice intentions. Theoretical and managerial implications, along with suggestions for future research, conclude the paper.

Human beings have always coveted beautiful objects, but the desire to look good is reaching new heights worldwide. Although the pursuit of beauty appears universal, industry evidence suggests that it is particularly strong in Asia. This research examines the effect of culture on the pursuit of beauty. Three studies provide converging evidence that interdependent self-construal increases the likelihood of using appearance-enhancing products. Study 1 operationalizes culture through nationality and self-construal and shows that Easterners (more interdependent) are more likely to use appearance-enhancing products than Westerners (less interdependent). This use is driven by interdependents’ tendency to conform to societal norms, which in turn leads to heightened self-discrepancy (Study 2). The use of appearance-enhancing tools helps minimize this discrepancy. Study 3 shows that strength of norms moderates the impact of interdependence on the use of appearance-enhancing tools. When norms are loosely defined and adherence is not strictly enforced, interdependents’ appearance enhancement tendency is reduced. This research offers actionable insights into the pursuit of beauty, marketing of beauty brands, policy making, and consumer well-being.

Triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor expressed on the surface of microglia, macrophages, dendritic cells, and osteoclasts. The R47H TREM2 variant is a significant risk factor for late-onset Alzheimer's disease (AD), and the molecular basis of R47H TREM2 loss of function is an emerging area of TREM2 biology. Here, we report three high-resolution structures of the extracellular ligand-binding domains (ECDs) of R47H TREM2, apo-WT, and phosphatidylserine (PS)-bound WT TREM2 at 1.8, 2.2, and 2.2 A, respectively. The structures reveal that Arg47 plays a critical role in maintaining the structural features of the complementarity-determining region 2 (CDR2) loop and the putative positive ligand-interacting surface (PLIS), stabilizing conformations capable of ligand interaction. This is exemplified in the PS-bound structure, in which the CDR2 loop and PLIS drive critical interactions with PS via surfaces that are disrupted in the variant. Together with in vitro and in vivo characterization, our structural findings elucidate the molecular mechanism underlying loss of ligand binding, putative oligomerization, and functional activity of R47H TREM2. They also help unravel how decreased in vitro and in vivo stability of TREM2 contribute to loss of function in disease.

Objective Systemic lupus erythematosus (SLE) is a complex autoimmune disease that is driven in part by chronic B and T lymphocyte hyperresponsiveness to self antigens. A deficiency of interleukin-21 (IL-21) or IL-21 receptor (IL-21R) in mice dramatically reduces inflammation and B and T cell activation in models of autoimmunity, including SLE. However, whether IL-21 is essential for the maintenance and amplification of preestablished inflammation has not been widely examined in various animal models. The purpose of this study was to examine the impact of novel mouse IL-21R neutralizing antibodies on recall responses to antigen challenge and on disease progression in the (NZB × NZW)F1 (NZB/NZW) mouse model of SLE. Methods Humoral and cellular immune responses to immunization with sheep red blood cells (SRBCs) were measured in mice dosed with IL-21R blocking antibodies. Progression of nephritis and markers of immune activation was monitored in NZB/NZW mice following different anti–IL-21R treatment regimens. Results IL-21R blockade specifically inhibited secondary IgG responses to SRBC immunization. In NZB/NZW mice, IL-21R blockade completely inhibited the onset of nephritis, which was associated with dramatic reductions in splenomegaly and in B cell and T cell activation. When administered to mice with preexisting disease, anti–IL-21R antibody halted the disease progression and mortality and reversed the nephritis in a subset of mice. Furthermore, treatment cessation was not followed by rapid reemergence of disease. Conclusion Our results highlight the importance of IL-21 in promoting humoral recall responses and in sustaining autoimmune inflammation.

Online_Appendix for Impact of Culture on the Pursuit of Beauty: Evidence from Five Countries by Shilpa Madan, Shankha Basu, Sharon Ng, and Elison Ai Ching Lim in Journal of International Marketing

Through six studies set in a range of service contexts, we show that consumers perceive the passage of wait time through intrinsic changes in the service experience. Changes, either in service progress speed or in activities within service experiences, consistently influence the perception of wait time. Further, the effects of change on time perception depend on whether the judgment is made prospectively or retrospectively. Results relating to change in progress speeds (Studies 1–3) demonstrate that faster progress during the start of an event shortens prospective time judgments; conversely, quicker progress at the end of a service experience shortens retrospective time judgments. We also demonstrate that prospective time judgments decrease, and retrospective judgments increase, when there is greater change in activities during the service experience (Studies 4–5). Extending this line of inquiry, we explore how within-experience activity change—in terms of the number of segments and how similar the segment activities are—impact retrospective time judgments (Study 6). Taken together, our findings therefore suggest a means (by managing changes in speed or activity within a service experience) of prolonging or quickening time perceptions that can be applied to service contexts to manage consumers’ wait time judgments.

Many scholars and managers have raised concerns that customers' personal loyalty to a salesperson may be a risk to their loyalty to the firm. We tested our theoretical model, which was based on the theories of psychology and organizational behavior, in the context of the direct selling industry, using a sample of 900 salesperson–customer dyads from Amway in China. It was found that customers' personal loyalty toward individuals can work positively and cooperatively toward loyalty to the firm. Specifically, we identified and examined 4 concurrent underlying mechanisms: affect transfer, social influence, behavioral sensemaking, and perceived entitativity. Our findings enrich the marketing literature on customer loyalty and we offer suggestions for managerial strategies and practice.

Crystalline bodies (CBs) can develop in the endoplasmic reticulum (ER) of antibody-producing cells. Although this phenotype is often reported in association with plasma cell dyscrasias and other hematological disorders, the details of CB biogenesis and CB's roles in pathophysiology remain poorly understood. Using an imaging-based screening method, we identified a secretion-competent human IgG2/λ clone that develops spindle-shaped intracellular crystals in transiently-transfected HEK293 cells upon Brefeldin A treatment. When stably overexpressed from CHO cells, the IgG2/λ clone spontaneously produced spindle-shaped CBs in the ER. Some CBs were released to the extracellular space while remaining enclosed by the membranes of secretory pathway origin. Structural modeling on the variable-region did not uncover prominent surface characteristics such as charge clusters. In contrast, alterations to the constant domain-encoded properties revealed their modulatory roles in CB-inducing propensities and CB morphology. For example, deletion of the entire Fc domain changed the morphology of CBs into thin filaments. Elimination of an N-linked glycan by a N297A mutation promoted Russell body biogenesis accompanied by marked reduction in IgG secretion. Isotype class switching from the original IgG2 to IgG1 and IgG4 changed the crystal morphology from spindle-shaped to long needle and acicular shaped, respectively. The IgG3 version, in contrast, suppressed the CB formation. Either the HC or LC alone or the Fc-domain alone did not trigger CB biogenesis. An IgG's in vivo crystal morphology and crystallization propensity can thus be modulated by the properties genetically and biochemically encoded in the HC constant region.

Using an experiment, this study examines how happiness towards, and evaluation of, experiential and material purchases are affected by mindset and comparison alternatives. When primed with a mental subtraction mindset, people became happier with their material purchases and evaluated such purchases more positively; however, those who had acquired experiences did not report significant increases in happiness or purchase evaluations. In contrast, when people did not engage in mental subtraction, recollection of purchase details did not enhance associated happiness for both material and experiential purchases. We also found that, relative to experiences, material purchases induced greater increases in happiness in the presence of inferior alternatives. When comparing purchases with superior alternatives, happiness declined significantly regardless of the type of purchase. Further, such decrements in happiness were more dramatic for material purchases than experiences. Collectively, this research suggests that happiness with material goods can increase over time with the appropriate mental strategies (i.e., mental subtraction and comparison against inferior alternatives).

Pichia pastoris is a highly successful recombinant protein expression system due to its ability to quickly generate large quantities of recombinant proteins in simple media. P. pastoris has been used to successfully generate milligram quantities of many important human membrane proteins, including G-protein coupled receptors, ion channels, and transporters, which are becoming increasingly important therapeutic targets. Despite these successes, protein expression in P. pastoris is still cumbersome due to a need to change growth media from glycerol media to methanol induction media, which minimizes inhibition of the AOX1 promoter by residual glycerol. Taking advantage of this behavior of the AOX1 promoter, we developed Buffered extra-YNB Glycerol Methanol (BYGM) auto-induction media (100 mM potassium phosphate pH 6.0, 2.68% w/v YNB, 0.4% v/v glycerol, 0.5% v/v methanol, and 8 × 10−5% w/v biotin) which not only simplified the protein expression process, but also optimized protein expression levels in P. pastoris. We successfully used this auto-induction method to overexpress the target in both MutS and Mut+ strains. Moreover, we show that this method can facilitate screening high-expressing clones, as well as enable parallel protein production in P. pastoris.

Protein folding, topogenesis and intracellular targeting of G protein-coupled receptors (GPCRs) must be precisely coordinated to ensure correct receptor localization. To elucidate how different steps of GPCR biosynthesis work together, we investigated the process of membrane topology determination and how it relates to the acquisition of cell surface trafficking competence in human GPR34. By monitoring a fused FLAG-tag and a conformation-sensitive native epitope during the expression of GPR34 mutant panel, a tri-basic motif in the first intracellular loop was identified as the key topogenic signal that dictates the orientation of transmembrane domain-1 (TM1). Charge disruption of the motif perturbed topogenic processes and resulted in the conformational epitope loss, post-translational processing alteration, and trafficking arrest in the Golgi. The placement of a cleavable N-terminal signal sequence as a surrogate topogenic determinant overcame the effects of tri-basic motif mutations and rectified the TM1 orientation; thereby restored the conformational epitope, post-translational modifications, and cell surface trafficking altogether. Progressive N-tail truncation and site-directed mutagenesis revealed that a proline-rich segment of the N-tail and all four cysteines individually located in the four separate extracellular regions must simultaneously reside in the ER lumen to muster the conformational epitope. Oxidation of all four cysteines was necessary for the epitope formation, but the cysteine residues themselves were not required for the trafficking event. The underlying biochemical properties of the conformational epitope was therefore the key to understand mechanistic processes propelled by positive-inside rule that simultaneously regulate the topogenesis and intracellular trafficking of GPR34.

In three studies, the authors introduce and probe the role of emotional receptivity in consumer–marketer interactions. Emotional receptivity refers to a person's disposition toward experiencing a preferred level of emotional intensity. The results from the first two studies conducted in the laboratory and the field collectively demonstrate that consumers feel greater enjoyment and enhanced liking for the marketer when there is a close match between their emotional receptivity and the level of emotional intensity displayed by the marketer. In the third study, the authors show that consumers’ emotional receptivity can be temporarily increased or decreased using advertisements that depict more or less expressive social interactions, respectively. The findings are similar to those observed in the first two studies. In addition, a preliminary study establishes the discriminant validity of the proposed battery of emotional receptivity items from existing related constructs. The authors discuss the theoretical and managerial implications and offer suggestions for further research.

Using three idiom characteristics (familiarity, literality, and compositionality), an idiom-processing model is advanced and applied to an advertising setting. Specifically, we propose that idioms of varying characteristics lead to one of six processing outcomes that can be differentiated based on processing time and comprehension. Further, we posit that consumers will exhibit different recall and attitude responses for advertisements using different types of idioms. Results from two studies provide some preliminary support for these predictions. Implications of the proposed framework for idiom research as well as for marketing theory and practice are discussed, and future research directions suggested.

This empirical study predicts and finds that effectiveness of ad humor is jointly determined by the humor process (i.e., incongruity resolution and arousal safety) employed in the ad and the cultural orientation (i.e., uncertainty avoidance and individualism-collectivism) of the ad viewer. In particular, attitudinal effects elicited by the cognitive/affective uncertainty elements in ad humor are more pronounced among consumers who have higher uncertainty avoidance, as well as among those with more collectivistic orientations. The theoretical and managerial implications stemming from the cultural boundaries of ad humor are considered. Research avenues suggested by the findings are also discussed.

Desire for status or control may motivate some consumers to own certain types of pets. In the light of recent debates surrounding “designer pets,” this article examines the dark side of pet ownership through interpretive interviews with dog owners. The findings compare and contrast two types of ownership motivation — pets as companions to love versus pets as toys, status markers, and brands. This latter category forms part of the dark side of pet ownership. Owners differ in their motivation for ownership, their appreciation of the pet, the nature of human–animal interaction, breed choice, and the purchase of pet-related paraphernalia.

This study investigates the effects of cultural conditioning, product type, and benefit claim type on attitudes and brand personality perceptions among consumers from a society that is more culturally conditioned towards utilitarian consumption (Shanghai, China) and an economy that is less culturally conditioned towards utilitarian consumption (Singapore). Our findings reveal that consumers in Shanghai preferred ads promoting utilitarian rather than hedonic products. They also rated brands of utilitarian products as more sophisticated, competent, exciting, and sincere than hedonic products. No such difference was observed among Singaporeans. These consumers preferred hedonic over utilitarian products but did not perceive them as being different from utilitarian products in terms of brand personality. Theoretical and managerial implications are forwarded, together with directions for future research.

We examine the role of in-store music-brand "fit" in reinforcing brand position using in-depth interviews. Fit is particularly important for stores wishing to attract new consumers without prior brand experience or knowledge because these consumers view music as an important signaling cue to the brand's position, image and quality. We also identified the effect of misfit. Misfit had two contrasting brand effects: misfit resulted in counterfactual thinking about the brand, leading to a decline in the consumer-brand relationship, and misfit could be used strategically as part of a repositioning strategy. These positive effects of fit were moderated by music volume. Copyright 2006 Elsevier Inc. All rights reserved.

This study investigates whether metaphors in advertising have a synergistic or compensatory effect on brand personality perceptions of utilitarian and symbolic products. The effects of metaphoric headlines versus pictures are also compared. In general, brands using metaphors in ads were perceived to be more sophisticated and exciting, but less sincere and competent than those using literal headlines or pictures. Ad attitudes, brand attitudes, and purchase intention were also enhanced with metaphoric advertising. In addition, compared with utilitarian products, symbolic products were perceived to be more sophisticated and exciting, but less sincere and competent. It is interesting to note that when metaphors were used for utilitarian products, perceptions of sophistication and excitement were enhanced, whereas sincerity was diluted. The results suggest that metaphors can be strategically used to influence brand personality perceptions, particularly for utilitarian products. Theoretical and managerial impli...

Cryoprecipitation of immunoglobulins is often reported in association with B-cell lymphoproliferative disorders and plasma cell dyscrasias. However, the biochemical basis of such cryoglobulin behaviors is not well understood because of a general lack of suitable experimental systems. Here, we report the identification and characterization of a single-chain antibody (scFv-Fc) that recapitulates cryoglobulin-like properties. When model scFv-Fc protein was engineered to multimerize, by appending the secretory tailpiece (stp) of human immunoglobulin μ-chain to the C terminus, the resulting oligomeric scFv-Fc-stp protein acquired two unexpected properties: the induction of a morular cell phenotype during protein biosynthesis and the cryoprecipitation of secreted proteins in harvested cell culture media. The turbidity of the culture media and the inclusion bodies that gave morular appearances were attributed to microscopic spherical protein droplet formation, a hallmark characteristic of liquid-liquid phase separation (LLPS) event. Mutagenesis approaches revealed that these two phenomena were independent of covalent protein oligomerization induced by stp. Disruption of the N-linked glycosylation motif in the stp region enhanced morular phenotype propensity but reduced protein secretion. Intermolecular disulfide bonds that stabilize Fc dimers and oligomers were necessary for efficient induction of LLPS, but their simultaneous elimination could not abrogate the LLPS propensity completely. Noncovalent protein-protein interactions between scFv-Fc-stp chains sufficiently established a basis for LLPS induction. Morular cell phenotypes and cryoprecipitation were clearly underpinned by intrinsic physicochemical properties embedded in the overexpressed cargo protein. Overproduction of condensation-prone secretory proteins that culminate in LLPS in the endoplasmic reticulum therefore serves as a path to produce morular Russell body phenotype.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease that is driven in part by chronic B and T lymphocyte hyperresponsiveness to self antigens. A deficiency of interleukin-21 (IL-21) or IL-21 receptor (IL-21R) in mice dramatically reduces inflammation and B and T cell activation in models of autoimmunity, including SLE. However, whether IL-21 is essential for the maintenance and amplification of preestablished inflammation has not been widely examined in various animal models. The purpose of this study was to examine the impact of novel mouse IL-21R neutralizing antibodies on recall responses to antigen challenge and on disease progression in the (NZB × NZW)F1 (NZB/NZW) mouse model of SLE.Humoral and cellular immune responses to immunization with sheep red blood cells (SRBCs) were measured in mice dosed with IL-21R blocking antibodies. Progression of nephritis and markers of immune activation was monitored in NZB/NZW mice following different anti-IL-21R treatment regimens.IL-21R blockade specifically inhibited secondary IgG responses to SRBC immunization. In NZB/NZW mice, IL-21R blockade completely inhibited the onset of nephritis, which was associated with dramatic reductions in splenomegaly and in B cell and T cell activation. When administered to mice with preexisting disease, anti-IL-21R antibody halted the disease progression and mortality and reversed the nephritis in a subset of mice. Furthermore, treatment cessation was not followed by rapid reemergence of disease.Our results highlight the importance of IL-21 in promoting humoral recall responses and in sustaining autoimmune inflammation.

We constructed a library of >10(12) unique, covalently coupled mRNA-protein molecules by randomizing three exposed loops of an immunoglobulin-like protein, the tenth fibronectin type III domain (10Fn3). The antibody mimics that bound TNF-alpha were isolated from the library using mRNA display. Ten rounds of selection produced 10Fn3 variants that bound TNF-alpha with dissociation constants (K(d)) between 1 and 24 nM. After affinity maturation, the lowest K(d) measured was 20 pM. Selected antibody mimics were shown to capture TNF-alpha when immobilized in a protein microarray. 10Fn3-based scaffold libraries and mRNA-display allow the isolation of high-affinity, specific antigen binding proteins; potential applications of such binding proteins include diagnostic protein microarrays and protein therapeutics.

The underlying reasons for why some mAb (monoclonal antibody) clones are much more inclined to induce a Russell body (RB) phenotype during immunoglobulin biosynthesis remain elusive. Although RBs are morphologically understood as enlarged globular aggregates of immunoglobulins deposited in the endoplasmic reticulum (ER), little is known about the properties of the RB-inducing mAb clones as secretory cargo and their physical behaviors in the extracellular space. To elucidate how RB-inducing propensities, secretion outputs, and the intrinsic physicochemical properties of individual mAb clones are interrelated, we used HEK293 cells to study the biosynthesis of 5 human IgG mAbs for which prominent solution behavior problems were known a priori. All 5 model mAbs with inherently high condensation propensities induced RB phenotypes both at steady state and under ER-to-Golgi transport block, and resulted in low secretion titer. By contrast, one reference mAb that readily crystallized at neutral pH in vitro produced rod-shaped crystalline bodies in the ER without inducing RBs. Another reference mAb without notable solution behavior issues did not induce RBs and was secreted abundantly. Intrinsic physicochemical properties of individual IgG clones thus directly affected the biosynthetic steps in the ER, and thereby produced distinctive cellular phenotypes and influenced IgG secretion output. The findings implicated that RB formation represents a phase separation event or a loss of colloidal stability in the secretory pathway organelles. The process of RB induction allows the cell to preemptively reduce the extracellular concentration of potentially pathogenic, highly aggregation-prone IgG clones by selectively storing them in the ER.

Examines consumers’ motivation for buying counterfeits or pirated goods. The findings indicated that, compared to those who did not buy pirated music CDs, those who bought the counterfeits viewed such purchases as less risky, and trusted stores that sell counterfeits more. They did not see counterfeits as too unfair to singers or the music industry, thought they benefit the society more, and did not see people who buy them as unethical. We also investigated the influence of social, personality, and demographic factors on consumers’ attitude towards piracy. We found that the more value‐conscious and less normatively susceptible one was, and the less integrity one had, the more favorable was one’s attitude towards piracy. Demographic characteristics were also significant in influencing attitude. Males and those from lower income groups held more favorable attitudes. Finally, attitude towards piracy was significant in influencing purchase intention.

RNA pur•pur-pyr and pyr•pur-pyr (pur = purine, pyr = pyrimidine) triple helices consisting of a Watson−Crick base-paired 28mer hairpin duplex and a Hoogsteen base-paired purine or pyrimidine 12mer are targeted with photoactivated cleavage by the metal complex Rh(phen)_2phi^(3+) (phen = phenanthroline, phi = 9,10-phenanthrenequinone diimine). The metal complex interacts with these triple helices in a structure-specific manner. Different cleavage patterns are seen with the pyr•pur-pyr and pur•pur-pyr motifs. Cleavage is seen on both of the Watson−Crick strands of the former motif and primarily on the purine Watson−Crick strand of the latter motif. Little cleavage is seen on the Hoogsteen strand for either motif. Importantly, the metal complex shows no detectable cleavage on the A-form RNA duplex in the absence of the third Hoogsteen strand. The cleavage patterns are consistent with an intercalated model for the metal complex in the triple helix. Similar cleavage is seen on DNA triple helices, but over a background of duplex cleavage. Targeting of synthetic RNA triple helices, but not duplex regions, by Rh(phen)_2phi^(3+) provides a basis for the chemical probing of triply bonded sites in folded RNA molecules.

The Tat-binding site of the bovine immunodeficiency virus TAR RNA hairpin has been targeted by Rh(phen)_2phi^(3+) (phen = phenanthroline, phi = 9,10-phenanthrenequinone diimine), a photochemical probe of RNA tertiary structure. The primary site cleaved by the rhodium complex, upon photoactivation, is U24, a base which participates in the novel base triple (with bases A13 and U10) characteristic of this folded RNA. Δ-Rh(phen)_2phi^(3+) binds to this site with an affinity of 2 × 10^6M^(−1). Upon mutation of U24 and A13 to A24 and U13, respectively, so that the RNA oligomer is unable to form the base triple, site-specific cleavage by the rhodium complex is abolished. Moreover, as determined through rhodium photocleavage, at a concentration of 20 μM, Rh(phen)_2phi^(3+) inhibits specific binding of BIV-Tat peptide (2 μM) to its target site. Thus the rhodium complex, in matching its shape to the opened major groove of the properly folded RNA, specifically targets its site and is able to compete for its target with the BIV-Tat peptide.

Stimulation of red cell production through agonism of the erythropoietin receptor (EpoR) has historically been accomplished through administration of erythropoietin (EPO), the native ligand. The short half-life of EPO has led to the development of a variety of other agonists, including antibodies. It is of considerable interest to understand how these agents might activate the EpoR and whether or not it is important to bind in a manner similar to the native ligand. The binding epitopes of a panel of eight agonistic, single-chain antibody (scFv-Fc) constructs were determined through scanning alanine mutagenesis as well as more limited arginine mutagenesis of the receptor. It was found that while some of these constructs bound to receptor epitopes shared by the ligand, others bound in completely unique ways. The use of a panel of agonists and scanning mutagenesis can define the critical binding regions for signaling; in the case of the EpoR, these regions were remarkably broad.

CT-322 is a new anti-angiogenic therapeutic agent based on an engineered variant of the tenth type III domain of human fibronectin, i.e., an Adnectin™, designed to inhibit vascular endothelial growth factor receptor (VEGFR)-2. This PE Gylated Adnectin was developed using an mRNA display technology. CT-322 bound human VEGFR-2 with high affinity (K(D), 11 nM), but did not bind VEGFR-1 or VEGFR-3 at concentrations up to 100 nM, as determined by surface plasmon resonance studies. Western blot analysis showed that CT-322 blocked VEGF-induced phosphorylation of VEGFR-2 and mitogen-activated protein kinase in human umbilical vascular endothelial cells. CT-322 significantly inhibited the growth of human tumor xenograft models of colon carcinoma and glioblastoma at doses of 15-60 mg/kg administered 3 times/week. Anti-tumor effects of CT-322 were comparable to those of sorafenib or sunitinib, which inhibit multiple kinases, in a colon carcinoma xenograft model, although CT-322 caused less overt adverse effects than the kinase inhibitors. CT-322 also enhanced the anti-tumor activity of the chemotherapeutic agent temsirolimus in the colon carcinoma model. The high affinity and specificity of CT-322 binding to VEGFR-2 and its anti-tumor activities establish CT-322 as a promising anti-angiogenic therapeutic agent. Our results further suggest that Adnectins are an important new class of targeted biologics that can be developed as potential treatments for a wide variety of diseases.

Glass cDNA microarrays can be used to profile the expression of thousands of gene targets in a single experiment. However, the potential for hybridization cross-reactivity needs to be considered when interpreting the results. Here, we describe hybridization experiments with a model array representing four distinct functional classes (families): chemokines, cytochrome P-450 isozymes, G proteins, and proteases. The cDNA clones selected for this array exhibited pairwise sequence identities ranging from 55% to 100%, as determined by a homology scoring algorithm (LALIGN). Targets for microarraying were amplified by PCR and spotted in 4-fold replication for signal averaging. One designated target from each family was further amplified by PCR to incorporate a T7 promoter sequence for the production of synthetic RNA transcripts. These transcripts were used to generate fluorescent hybridization probes by reverse transcription at varying input concentrations. As expected, hybridization signals were highest at the matching target elements. Targets containing less than 80% sequence identity relative to the hybridization probe sequences showed cross-reactivities ranging from 0.6% to 12%. Targets containing greater than 80% identity showed higher cross-reactivities (26%–57%). These cross-reactive signals were analyzed for statistical correlation with the length of sequence overlap, percent sequence identity, and homology score determined by LALIGN. Overall, percent sequence identity was the best predictor of hybridization cross-reactivity. These results provide useful guidelines for interpreting glass cDNA microarray data.

The three-dimensional folding of tDNA^(Phe) has been examined and compared to native tRNA^(Phe) using a series of shape-selective transition metal complexes as chemical probes of nucleic acid structure. Rh(phen)_2phi^(3+) (phen = phenanthroline, phi = 9,10-phenanthrenequinonediimine), which targets sites of tertiary interaction in tRNA^(Phe), cleaves specifically at similar sites on tDNA^(Phe). However, this rhodium complex also targets 5'-pyr-pyr-pur-3' sites within the acceptor and anticodon stems of tDNA; Rh(phen)_2phi^(3+)generally targets 5'-pyr-pyr-pur-3' sites in B-form duplex DNA. On tRNA^(Phe), Rh(DIP)_3^(3+) (DIP = 4,7- diphenyl-1,10-phenanthroline) specifically cleaves C70, which neighbors a GU mismatch, and targets ψ55 as well, within the hydrophobic region of tRNA^(Phe). On tDNA^(Phe) no specific cleavage by Rh(DIP)_3^(3+) is observed. The cleavage studies, taken together, indicate that globally the tertiary folding of tDNA^(Phe) resembles that of tRNA^(Phe). However, the double helical regions of the DNA analog differ from tRNA^(Phe), likely in adopting a more B-like conformation. As a consequence, the GT mismatch within the acceptor stem of tDNA does not present the same recognition elements as in tRNA, and the GT mismatch is no longer recognized by the shape-selective rhodium complex. The present work underscores the utility of applying DNA analogs to studies of RNA structure and function, since the general folding characteristics of the two polymers are likely to be similar. However, structural probing with transition metal complexes offers a valuable companion to such experiments, since the shape-selective probes, with sensitivity, may be used to delineate locally on the polymer those regions which may differ in structure.

A set of examples that vividly demonstrate the power of 13C NMR spectroscopy to solve stereochemical problems unambiguously and to differentiate pairs of carbon atoms that are homotopic or enantiotopic from pairs which are diastereotopic.