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Most student work as research assistants occurs at the graduate level of nursing education, and little is known about the role of undergraduate students as research assistants (RAs) in major research projects. Based on our desire to study Mexican American (MA) cancer caregivers, we needed bilingual and bicultural RAs to serve as data collectors with women who spoke Spanish and possessed cultural beliefs that influenced their caregiving. Following successful recruitment, orientation, and mentoring based on Bandura's social learning theory [Bandura, A., 2001. Social learning theory: an agentic perspective. Annual Review of Psychology 52, 1-26] and accepted teaching-learning principles, RAs engaged in various behaviors that facilitated study outcomes. Faculty researchers, RAs, and study participants benefitted greatly from the undergraduate student involvement in this project. This article describes successful student inclusion approaches, ongoing faculty-RA interactions, and lessons learned from the research team experience. Guidelines discussed support the potential for making the undergraduate RA role a useful and unique learning experience. [ABSTRACT FROM AUTHOR]

This grounded theory study report describes the experience of 34 Mexican American (MA) female caregivers who provided care to a family member with cancer. Caregivers identified a process of 'Becoming Stronger' as a result of their caregiver role. The emerging theoretical model of female MA cancer caregiving offers evidence to change current cancer care approaches from patient-focused to family-focused care for this ethnic group. Findings suggest that changes most responsive to cultural values and likely to provide accessible and quality cancer care for MA families are those that involve active partnering with MA caregivers to prioritize the patient's cancer care. [ABSTRACT FROM AUTHOR]

Objective: The study purpose was to generate theory to explain why some hospital staff chose to stay on the job during a prolonged public health crisis., Background: The "great resignation" of 2021 created shortages across the healthcare industry. Why some healthcare staff chose to stay at work when coworkers were leaving in large numbers through retirement, transition to different careers, or perceived suddenly better clinical opportunities was not clear., Methods: Qualitative Grounded Theory methods guided this research study. Sixteen healthcare workers participated in open-ended interviews that provided data to identify major concepts in an emerging model of commitment during crisis., Results: A "Commit to Stay" model emerged showing 4 major influences including sense of personal agency, supportive organization, social connections at work, and external connections and influence., Conclusions: The Commit to Stay conceptual model can help guide nurse leaders as they grapple with supporting those who choose to stay at work in healthcare during intense, sustained healthcare crises., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Dynein-2 is a large multiprotein complex that powers retrograde intraflagellar transport (IFT) of cargoes within cilia/flagella, but the molecular mechanism underlying this function is still emerging. Distinctively, dynein-2 contains two identical force-generating heavy chains that interact with two different intermediate chains (WDR34 and WDR60). Here, we dissect regulation of dynein-2 function by WDR34 and WDR60 using an integrative approach including cryo-electron microscopy and CRISPR/Cas9-enabled cell biology. A 3.9 Å resolution structure shows how WDR34 and WDR60 use surprisingly different interactions to engage equivalent sites of the two heavy chains. We show that cilia can assemble in the absence of either WDR34 or WDR60 individually, but not both subunits. Dynein-2-dependent distribution of cargoes depends more strongly on WDR60, because the unique N-terminal extension of WDR60 facilitates dynein-2 targeting to cilia. Strikingly, this N-terminal extension can be transplanted onto WDR34 and retain function, suggesting it acts as a flexible tether to the IFT "trains" that assemble at the ciliary base. We discuss how use of unstructured tethers represents an emerging theme in IFT train interactions., (© 2024. The Author(s).)

Cys2-His2 zinc finger genes (ZNFs) form the largest family of transcription factors in metazoans. ZNF evolution is highly dynamic and characterized by the rapid expansion and contraction of numerous subfamilies across the animal phylogeny. The forces and mechanisms underlying rapid ZNF evolution remain poorly understood, but there is growing evidence that, in tetrapods, the targeting and repression of lineage-specific transposable elements (TEs) plays a critical role in the evolution of the Krüppel-associated box ZNF (KZNF) subfamily. Currently, it is unknown whether this function and coevolutionary relationship is unique to KZNFs or is a broader feature of metazoan ZNFs. Here, we present evidence that genomic conflict with TEs has been a central driver of the diversification of ZNFs in animals. Sampling from 3221 genome assemblies, we show that the copy number of retroelements correlates with that of ZNFs across at least 750 million years of metazoan evolution. Using computational predictions, we show that ZNFs preferentially bind TEs in diverse animal species. We further investigate the largest ZNF subfamily found in cyprinid fish, which is characterized by a conserved sequence we dubbed the fish N-terminal zinc finger-associated (FiNZ) domain. Zebrafish possess approximately 700 FiNZ-ZNFs, many of which are evolving adaptively under positive selection. Like mammalian KZNFs, most zebrafish FiNZ-ZNFs are expressed at the onset of zygotic genome activation, and blocking their translation using morpholinos during early embryogenesis results in derepression of transcriptionally active TEs. Together, these data suggest that ZNF diversification has been intimately connected to TE expansion throughout animal evolution., (© 2023 Wells et al.; Published by Cold Spring Harbor Laboratory Press.)

Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the non-core IFT-A components are degraded and core components accumulate at the ciliary base. We reveal deep sequence homology of WDR35 and other IFT-A subunits to α and ß' COPI coatomer subunits and demonstrate an accumulation of 'coat-less' vesicles that fail to fuse with Wdr35 mutant cilia. We determine that recombinant non-core IFT-As can bind directly to lipids and provide the first in situ evidence of a novel coat function for WDR35, likely with other IFT-A proteins, in delivering ciliary membrane cargo necessary for cilia elongation., Competing Interests: TQ, JW, EH, NP, WL, PK, JW, LM, MK, JM, EL, GP, PM No competing interests declared, (© 2021, Quidwai et al.)

New graduates' adjustment to high-acuity specialty areas was evaluated using qualitative methods in a hospital system that uses the Versant New Graduate Residency Program. Subjects were interviewed at baseline in person, answered interview questions at 6 months via computer, and were interviewed at 12 months in person. Twelve themes emerged from the interviews, reflecting intrinsic and extrinsic factors affecting new graduate nurse adjustment. Study results were used to evaluate the program and improve the program implementation., Competing Interests: The authors declare no conflicts of interests., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Transposable elements (TEs) are mobile DNA sequences that propagate within genomes. Through diverse invasion strategies, TEs have come to occupy a substantial fraction of nearly all eukaryotic genomes, and they represent a major source of genetic variation and novelty. Here we review the defining features of each major group of eukaryotic TEs and explore their evolutionary origins and relationships. We discuss how the unique biology of different TEs influences their propagation and distribution within and across genomes. Environmental and genetic factors acting at the level of the host species further modulate the activity, diversification, and fate of TEs, producing the dramatic variation in TE content observed across eukaryotes. We argue that cataloging TE diversity and dissecting the idiosyncratic behavior of individual elements are crucial to expanding our comprehension of their impact on the biology of genomes and the evolution of species.

Cells adjust to nutrient deprivation by reversible translational shutdown. This is accompanied by maintaining inactive ribosomes in a hibernation state, in which they are bound by proteins with inhibitory and protective functions. In eukaryotes, such a function was attributed to suppressor of target of Myb protein 1 (Stm1; SERPINE1 mRNA-binding protein 1 [SERBP1] in mammals), and recently, late-annotated short open reading frame 2 (Lso2; coiled-coil domain containing short open reading frame 124 [CCDC124] in mammals) was found to be involved in translational recovery after starvation from stationary phase. Here, we present cryo-electron microscopy (cryo-EM) structures of translationally inactive yeast and human ribosomes. We found Lso2/CCDC124 accumulating on idle ribosomes in the nonrotated state, in contrast to Stm1/SERBP1-bound ribosomes, which display a rotated state. Lso2/CCDC124 bridges the decoding sites of the small with the GTPase activating center (GAC) of the large subunit. This position allows accommodation of the duplication of multilocus region 34 protein (Dom34)-dependent ribosome recycling system, which splits Lso2-containing, but not Stm1-containing, ribosomes. We propose a model in which Lso2 facilitates rapid translation reactivation by stabilizing the recycling-competent state of inactive ribosomes., Competing Interests: The authors have declared that no competing interests exist.

Purpose: The primary goals of the study were to assess maternal knowledge and attitudes about the newborn screening bloodspot repository program in Texas and to evaluate the effect of a video about the bloodspot repository on the rate of parental permission to store infant bloodspots., Design: This descriptive, comparative study used descriptive statistics, nonparametric tests, and multivariable analyses to describe the sample, compare groups, and determine associations between factors that influenced parents' decisions to permit state storage of infant bloodspots., Results: There were 465 mothers in the study. Maternal knowledge about basic genetics and risks of genetic testing was poor, but mothers reported strong trust in the state to make good decisions about using genetic material for research. Race, ethnicity, and attitude toward research significantly influenced decisions to opt-in to the bloodspot storage program. Postpartum nurses provided the bulk of information about newborn screening and dried bloodspots, although 2/3 of respondents would have preferred to obtain this information prior to birth., Clinical Implications: Although postpartum nurses are the most common source of information about newborn screening, genuine informed consent for bloodspot storage is questionable. Information about newborn screening and bloodspot storage needs to be addressed in community venues prior to birth.

Aneuploidy, a condition characterized by chromosome gains and losses, causes reduced fitness and numerous cellular stresses, including increased protein aggregation. Here, we identify protein complex stoichiometry imbalances as a major cause of protein aggregation in aneuploid cells. Subunits of protein complexes encoded on excess chromosomes aggregate in aneuploid cells, which is suppressed when expression of other subunits is coordinately altered. We further show that excess subunits are either degraded or aggregate and that protein aggregation is nearly as effective as protein degradation at lowering levels of excess proteins. Our study explains why proteotoxic stress is a universal feature of the aneuploid state and reveals protein aggregation as a form of dosage compensation to cope with disproportionate expression of protein complex subunits., (© 2019 Brennan et al.; Published by Cold Spring Harbor Laboratory Press.)

Many human genetic disorders are caused by mutations in protein-coding regions of DNA. Taking protein structure into account has therefore provided key insight into the molecular mechanisms underlying human genetic disease. Although most studies have focused on the intramolecular effects of mutations, the critical role of the assembly of proteins into complexes is being increasingly recognized. Here, we review multiple ways in which consideration of protein complexes can help us to understand and explain the effects of pathogenic mutations. First, we discuss disorders caused by mutations that perturb intersubunit interactions in homomeric and heteromeric complexes. Second, we address how protein complex assembly can facilitate a dominant-negative mechanism, whereby mutated subunits can disrupt the activity of wild-type protein. Third, we show how mutations that change protein expression levels can lead to damaging stoichiometric imbalances. Finally, we review how mutations affecting different subunits of the same heteromeric complex often cause similar diseases, whereas mutations in different interfaces of the same subunit can cause distinct phenotypes., (© 2019 The Protein Society.)

Reconstructing evolutionary relationships in repeat proteins is notoriously difficult due to the high degree of sequence divergence that typically occurs between duplicated repeats. This is complicated further by the fact that proteins with a large number of similar repeats are more likely to produce significant local sequence alignments than proteins with fewer copies of the repeat motif. Furthermore, biologically correct sequence alignments are sometimes impossible to achieve in cases where insertion or translocation events disrupt the order of repeats in one of the sequences being aligned. Combined, these attributes make traditional phylogenetic methods for studying protein families unreliable for repeat proteins, due to the dependence of such methods on accurate sequence alignment.We present here a practical solution to this problem, making use of graph clustering combined with the open-source software package HH-suite, which enables highly sensitive detection of sequence relationships. Carrying out multiple rounds of homology searches via alignment of profile hidden Markov models, large sets of related proteins are generated. By representing the relationships between proteins in these sets as graphs, subsequent clustering with the Markov cluster algorithm enables robust detection of repeat protein subfamilies.

Protein complexes are assemblies of subunits that have co-evolved to execute one or many coordinated functions in the cellular environment. Functional annotation of mammalian protein complexes is critical to understanding biological processes, as well as disease mechanisms. Here, we used genetic co-essentiality derived from genome-scale RNAi- and CRISPR-Cas9-based fitness screens performed across hundreds of human cancer cell lines to assign measures of functional similarity. From these measures, we systematically built and characterized functional similarity networks that recapitulate known structural and functional features of well-studied protein complexes and resolve novel functional modules within complexes lacking structural resolution, such as the mammalian SWI/SNF complex. Finally, by integrating functional networks with large protein-protein interaction networks, we discovered novel protein complexes involving recently evolved genes of unknown function. Taken together, these findings demonstrate the utility of genetic perturbation screens alone, and in combination with large-scale biophysical data, to enhance our understanding of mammalian protein complexes in normal and disease states., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Experimental methods for the characterization of protein complexes have been instrumental in achieving our current understanding of the protein universe and continue to progress with each year that passes. In this chapter, we review some of the most important tools and techniques in the field, covering the important points in X-ray crystallography, cryo-electron microscopy, NMR spectroscopy, and mass spectrometry. Novel developments are making it possible to study large protein complexes at near-atomic resolutions, and we also now have the ability to study the dynamics and assembly pathways of protein complexes across a range of sizes.

Sequence and structure space are nowadays sufficiently large that we can use computational methods to model the structure of proteins based on sequence similarity alone. Not only useful as a standalone tool, homology modelling has also had a transformative effect on the ease with which we can solve crystal structures and electron density maps. Another technique-molecular dynamics-aims to model protein structures from first principles and, thanks to increases in computational power, is slowly becoming a viable tool for studying protein complexes. Finally, the prediction of protein assembly pathways from three-dimensional structures of complexes is also now becoming possible.

Microorganisms use carboxylase enzymes to form new carbon-carbon bonds by introducing carbon dioxide gas (CO 2 ) or its hydrated form, bicarbonate (HCO 3 - ), into target molecules. Acetone carboxylases (ACs) catalyze the conversion of substrates acetone and HCO 3 - to form the product acetoacetate. Many bicarbonate-incorporating carboxylases rely on the organic cofactor biotin for the activation of bicarbonate. ACs contain metal ions but not organic cofactors, and use ATP to activate substrates through phosphorylation. How the enzyme coordinates these phosphorylation events and new C-C bond formation in the absence of biotin has remained a mystery since these enzymes were discovered. The first structural rationale for acetone carboxylation is presented here, focusing on the 360 kDa (αβγ) 2 heterohexameric AC from Xanthobacter autotrophicus in the ligand-free, AMP-bound, and acetate coordinated states. These structures suggest successive steps in a catalytic cycle revealing that AC undergoes large conformational changes coupled to substrate activation by ATP to perform C-C bond ligation at a distant Mn center. These results illustrate a new chemical strategy for the conversion of CO 2 into biomass, a process of great significance to the global carbon cycle.

Most proteins assemble into complexes, which are involved in almost all cellular processes. Thus it is crucial for cell viability that mechanisms for correct assembly exist. The timing of assembly plays a key role in determining the fate of the protein: if the protein is allowed to diffuse into the crowded cellular milieu, it runs the risk of forming non-specific interactions, potentially leading to aggregation or other deleterious outcomes. It is therefore expected that strong regulatory mechanisms should exist to ensure efficient assembly. In this review we discuss the cotranslational assembly of protein complexes and discuss how it occurs, ways in which it is regulated, potential disadvantages of cotranslational interactions between proteins and the implications for the inheritance of dominant-negative genetic disorders., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Culturally sensitive interventions are needed to assist Mexican American (MA) patients with cancer and their family members in managing their care, navigating the healthcare system, and decreasing disparities in healthcare outcomes for Hispanics with cancer., Objectives: The objectives of this study were to understand the meaning of culturally sensitive care for oncology clinic healthcare providers and to assess the usefulness and feasibility of the role of a promotora de salud to meet caregiver needs., Methods: This study involved focus groups of 18 diverse providers who provided data for qualitative analyses., Findings: The findings (themes) defined the facilitators of and barriers to culturally sensitive care and the perceived role of a promotora de salud to support the healthcare team and improve cancer care provided by MA caregivers. In addition, promotoras de salud can help reduce health costs by decreasing patient clinic visits.

Do young and old protein molecules have the same probability to be degraded? We addressed this question using metabolic pulse-chase labeling and quantitative mass spectrometry to obtain degradation profiles for thousands of proteins. We find that >10% of proteins are degraded non-exponentially. Specifically, proteins are less stable in the first few hours of their life and stabilize with age. Degradation profiles are conserved and similar in two cell types. Many non-exponentially degraded (NED) proteins are subunits of complexes that are produced in super-stoichiometric amounts relative to their exponentially degraded (ED) counterparts. Within complexes, NED proteins have larger interaction interfaces and assemble earlier than ED subunits. Amplifying genes encoding NED proteins increases their initial degradation. Consistently, decay profiles can predict protein level attenuation in aneuploid cells. Together, our data show that non-exponential degradation is common, conserved, and has important consequences for complex formation and regulation of protein abundance., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Tumor lysis syndrome (TLS) is a potentially fatal complication in patients with large, rapidly proliferating tumor cell cancers that may occur after chemotherapy. Patients with TLS are complicated to treat and often have an unpredictable trajectory., Objectives: The purpose of this article is to report two cases with unusual clinical manifestations and unexpected outcomes during cancer treatment and to share best practices for this situation., Methods: The authors described details from two unusual cases and outlined lessons learned. The authors described a newly developed clinical order set (protocol) to support optimal care for patients at risk for TLS., Findings: Implementing best practices, the order set prompts early identification of TLS risk and provides step-by-step guidance to eliminate or control TLS.

The assembly of heteromeric protein complexes is an inherently stochastic process in which multiple genes are expressed separately into proteins, which must then somehow find each other within the cell. Here, we considered one of the ways by which prokaryotic organisms have attempted to maximize the efficiency of protein complex assembly: the organization of subunit-encoding genes into operons. Using structure-based assembly predictions, we show that operon gene order has been optimized to match the order in which protein subunits assemble. Exceptions to this are almost entirely highly expressed proteins for which assembly is less stochastic and for which precisely ordered translation offers less benefit. Overall, these results show that ordered protein complex assembly pathways are of significant biological importance and represent a major evolutionary constraint on operon gene organization., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The interaction of biological macromolecules is a fundamental attribute of cellular life. Proteins, in particular, often form stable complexes with one another. Although the importance of protein complexes is widely recognized, we still have only a very limited understanding of the mechanisms underlying their assembly within cells. In this article, we review the available evidence for one such mechanism, namely the coupling of protein complex assembly to translation at the polysome. We discuss research showing that co-translational assembly can occur in both prokaryotic and eukaryotic organisms and can have important implications for the correct functioning of the complexes that result. Co-translational assembly can occur for both homomeric and heteromeric protein complexes and for both proteins that are translated directly into the cytoplasm and those that are translated into or across membranes. Finally, we discuss the properties of proteins that are most likely to be associated with co-translational assembly., (© 2015 Authors; published by Portland Press Limited.)

Background: Nurses have an essential role in implementing evidence-based practices (EBP) that contribute to high-quality outcomes. It remains unknown how healthcare facilities can increase nurse engagement in EBP., Purpose: To determine whether individual or organizational qualities could be identified that were related to registered nurses' (RNs') readiness for EBP as measured by their reported EBP barriers, ability, desire, and frequency of behaviors., Methods: A descriptive cross-sectional survey was used in which a convenience sample of 2,441 nurses within one United States healthcare system completed a modified version of the Information Literacy for Evidence-Based Nursing questionnaire. Descriptive statistics, t tests, one-way ANOVA, and regression modeling were used to analyze the data., Results: RNs employed by facilities designated by the American Nurses Credentialing Center (ANCC) as Magnet® or Pathway to Excellence® reported significantly fewer barriers to EBP than those RNs employed by non designated facilities. RNs in Magnet organizations had higher desire for EBP than Pathway to Excellence or non designated facilities. RNs educated at the baccalaureate level or higher reported significantly fewer barriers to EBP than nurses with less education; they also had higher EBP ability, desire, and frequency of behaviors. A predictive model found higher EBP readiness scores among RNs who participated in research, had specialty certifications, and engaged in a clinical career development program., Linking Evidence to Action: Education, research, and certification standards promoted by the Magnet program may provide a nursing workforce that is better prepared for EBP. Organizations should continue structural supports that increase professional development and research opportunities so nurses are empowered to practice at their full capacity., (© 2015 Sigma Theta Tau International.)

Spanish-speaking Mexican Americans (MAs) need representation in cancer research studies to provide an empirical base for developing culturally relevant health care interventions. One factor that limits research with MAs is the lack of Spanish language measurement tools. Bilingual, bicultural student research assistants (RAs), working with faculty researchers and translation consultants, adapted the English version Distress Thermometer and Problem List (DT-PL) tool into the Spanish language. Additionally, RAs assessed tool feasibility with five MA women to determine its usefulness for a later study. The translation process resulted in a distress assessment instrument suitable for use in a low-literacy, Spanish-speaking population. RAs can enhance a process of adapting a measurement tool for use in research. Health care researchers should now pilot the Spanish DT-PL tool to assess its reliability and validity.

Female family caregivers of various global cultures provide basic care in health, social, emotional, and financial domains for family members with cancer and may sacrifice their own health to do so. To learn about role-related mood, health status self-perceptions, and burden of one cultural group, we used qualitative and quantitative approaches to study 34 Mexican American (MA) women who provided care for an ill family member with cancer. We report quantitative data on study variables and make comparisons with caregiver qualitative reports. Implications for health planning, service delivery, and future research with underserved, minority female caregivers are presented.

Hispanic and Anglo women differ in their practice of breast health behavior. A likely factor is differences in purpose-in-life (PIL) that influence motivation to achieve goals. To determine the relationship between PIL and breast health behavior, the PIL Test was modified and translated into Spanish, and the Breast Health Behavior Questionnaire (BHBQ) was generated. Both Spanish and English versions of the PIL Test and the BHBQ were measured in 40 Spanish and 40 Anglo women ages 20 to 49. Cronbach's alpha for the PIL Test were .86 for the English version and .72 for the Spanish; Cronbach's alpha for the BHBQ were .78 for the English and .70 for the Spanish version. There was a significant relationship between PIL and breast health behaviors in Anglo women but not in Hispanic women. Findings suggest further study of PIL in Hispanic women and may indicate a need for teaching the benefits of self-regulation to maintain health.

The antiasthmatic drug enprofylline was the first known selective, though not potent, A(2B) antagonist. On the basis of structure-activity relationships (SARs) of xanthine derivatives, we designed a novel selective adenosine A(2B) receptor antagonist, 3-isobutyl-8-pyrrolidinoxanthine (IPDX), with potency greater than that of enprofylline. IPDX displaced [3H]ZM241385 ([3H]4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a]-[1,3,5]triazin-5-ylamino]ethyl)phenol) from human A(2B) adenosine receptors with a K(i) value of 470 +/- 2 nM and inhibited A(2B)-dependent cyclic AMP (cAMP) accumulation in human erythroleukemia (HEL) cells with a K(B) value of 625 +/- 71 nM. We found that IPDX was more selective than enprofylline toward human A(2B) receptors. It was 38-, 55-, and 82-fold more selective for human A(2B) than for human A(1) (K(i) value of 24 +/- 8 microM), human A(2A) (K(B) value of 36 +/- 8 microM), and human A(3) (K(i) value of 53 +/- 10 microM) adenosine receptors, respectively. IPDX inhibited NECA (5'-N-ethylcarboxamidoadenosine)-induced interleukin-8 secretion in human mast cells (HMC-1) with a potency close to that determined for A(2B)-mediated cAMP accumulation in HEL cells, thus confirming the role of A(2B) adenosine receptors in mediating human mast cell activation. Since adenosine triggers bronchoconstriction in asthmatic patients through human mast cell activation, IPDX may become a basis for the development of new antiasthmatic drugs with improved properties compared with those of enprofylline. Our data demonstrate that IPDX can be used as a tool to differentiate between A(2B) and other adenosine receptor-mediated responses.

Fatigue is the most common side effect of cancer treatment, yet is sometimes ignored by healthcare professionals. Home care and hospice nurses work closely with patients and families experiencing the overwhelming impact fatigue has on a patient's quality of life. This article provides a comprehensive discussion of the causes of cancer-related fatigue and the interventions to be considered.

The purpose of the study was to test the psychometric properties of a culturally sensitive and theory-based instrument: the Breast Health Behavior Questionnaire. This instrument was translated into Spanish and back-translated at a third- to fourth-grade reading level. The pilot group consisted of 70 Hispanic women who attended a class at a local church. Subsequent to pilot testing, another 40 Hispanic women who attended a class at the local health department comprised the study sample. The participants responded to the 15-item questionnaire, which is formatted as a Likert scale. Content validity of the Breast Health Behavior Questionnaire was determined by a panel of experts. A factor analysis of this instrument showed five separate dimensions accounting for 71.82% of the instrument's variance. The three major components of self-regulation theory (schema, coping, and appraisal criteria) were found clustered within the first three dimensions after three items were discarded. The Breast Health Behavior Questionnaire demonstrated an internal consistency reliability coefficient of .7172. The psychometric properties of the Spanish version of this questionnaire warrant further research. The instrument may support a better understanding of the Hispanic woman's practice of breast health behavior. Eventually, the Breast Health Behavior Questionnaire may assist nurses in the formulation of culturally grounded interventions.

The substituted-cysteine accessibility method (SCAM) was applied to transmembrane span seven of the human A(1) adenosine receptor (hA(1)AR) to reveal a subset of amino acids that are exposed to the ligand-binding crevice. The SCAM approach involved a systematic probe of receptor structure by individual substitutions of residues K265 (7.30) to R296 (7.61) with cysteine. In most cases, hA(1)AR substituted-cysteine mutant membranes displayed antagonist dissociation binding constants that did not differ significantly from wild-type (WT). Radioligand binding assays were used to compare cell membranes that were treated with hydrophilic, sulfhydryl-specific methanethiosulfonate derivatives with control cell membranes. Position H278 was previously reported to be required for A(1)AR ligand binding; however, that report did not establish that H278 represents a contact point for ligands. Cysteine-substitution at H278 yields membrane preparations with greatly decreased receptor density compared with WT membranes from cells in the same transfection experiment. However, H278C membranes retain a measurable fraction of antagonist binding. This observation allows for the investigation of binding-crevice accessibility at position 278 and suggests that H278 may not be required for binding of antagonist ligands. Our data reveal the binding-crevice accessibility of residues T270 (7.35), A273 (7.38), I274 (7.39), T277 (7.42), H278 (7.43), N284 (7.49), and Y288 (7.53) in the hA(1)AR. These data are consistent with the high-resolution structure of bovine rhodopsin that features three alpha-helical turns in this region that are interrupted by an elongated, nonhelical structure from positions 7.43 to 7.48 in the primary amino acid sequence.

To examine the importance of the nine native cysteine residues in the human A(1) adenosine receptor, each cysteine was individually mutated to both serine and alanine. Saturation binding with the A(1) selective antagonist [(3)H]DPCPX [8-cyclopentyl-1,3-di(2, 3-(3)H-propyl)xanthine] resulted in a wild-type K(d) value of 0.92 nM. All serine and alanine mutants had similar K(d) values with the exception of serine/alanine mutations at Cys80 and Cys169. These two cysteine residues, which are highly conserved in G protein-coupled receptors and hypothesized to be linked through a disulfide bridge, demonstrated no detectable binding with [(3)H]DPCPX. Both serine and alanine mutations at residues Cys80 and Cys169 resulted in receptors that were not detectable at the cell surface, as visualized by immunostaining. The serine/alanine mutants that did bind [(3)H]DPCPX were characterized further through competition binding with the antagonist theophylline and the agonists NECA (5'-N-ethylcarboxamidoadenosine) and R-PIA [(R)N(6)-phenylisopropyl adenosine]. The wild-type theophylline K(i) value was 2.41 microM, with the serine/alanine mutants having similar values. Wild-type NECA and R-PIA K(i) values were 0.74 microM and 97.0 nM, respectively. All mutants had K(i) values similar to wild-type with the exception of the Cys85Ser mutant, which had NECA and R-PIA values of 9.30 microM and 387.3 nM, respectively. These data show that Cys80 and Cys169 are absolutely required for delivery of the receptor to the plasma membrane. The Cys85Ser data indicate that although a cysteine is not required at this position, this residue may have an important role in ligand binding or for the structure of the receptor.

The cGMP-binding cGMP-specific phosphodiesterase (PDE-5) contains distinct catalytic and allosteric binding sites, and each is cGMP-specific. Cyclic nucleotide phosphodiesterase inhibitors, such as 3-isobutyl-1-methylxanthine (IBMX), are believed to compete with cyclic nucleotides at the catalytic sites of these enzymes, but the portion of PDE-5 that accounts for interaction of either of these inhibitors of the substrates themselves with the catalytic domain of the enzymes has not been identified. IBMX was derivatized to yield the photoaffinity probe 8([3-125I,-4-azido]-benzyl)-IBMX, which is referred to as 8(125IAB)-IBMX. This probe was incubated with partially purified recombinant bovine PDE-5. After UV irradiation and SDS-PAGE, a single radiolabeled band that coincided with the position of PDE-5 was visualized on the gel, and the photoaffinity labeling of PDE-5 was linear with increasing concentration of the 8(125IAB)-IBMX. Prominent Coomassie blue-stained bands other than PDE-5 were not labeled significantly. The photoaffinity labeling was progressively blocked by cGMP at concentrations higher than 10 microM, whereas cAMP or 5'-GMP exhibited only weak inhibitory effects. Other compounds that are believed to interact with the PDE-5 catalytic site, including IBMX, cIMP, and beta-phenyl-1,N2-etheno-cGMP (PET-cGMP), also inhibited the photoaffinity labeling in a concentration-dependent manner. The IC50 of PET-cGMP for inhibition of photoaffinity labeling was 10 microM, which compared favorably with an IC50 of 5 microM for inhibition of PDE-5 catalytic activity by this compound. It is concluded that the interaction of this photoaffinity probe with PDE-5 is highly specific for the catalytic site over the allosteric binding sites of PDE-5 and could prove useful in studies to map the catalytic site of PDE-5.

Structure-based design of subtype-selective ligands for the A1 adenosine receptor will require a reliable model of the ligand-binding pocket. It should be possible to develop a reliable model based on the results of affinity labeling experiments that provide atomic coordinates for the ligand in relation to predicted receptor helices. A high affinity, A1-selective xanthine antagonist photoaffinity probe, 125l-3-(4-azidophenethyl)-1-propyl-B-cyclopentylxanthine, was used to covalently modify the A1 receptor. Chemical or enzymatic fragmentation experiments were performed to localize the region or regions of incorporation within the receptor. The fragmentation profiles for radiolabeled A1 receptor obtained with endoproteinase Glu-C, endoproteinase Lys-C, cyanogen bromide, and hydroxylamine were consistent with the interpretation that the covalent linkage was within the first four predicted transmembrane regions. This interpretation was confirmed by the demonstration that the radioactive endoproteinase Glu-C fragment derived from an A1 receptor that contains an amino-terminal FLAG epitope was recognized by an anti-FLAG monoclonal antibody. Sequential digestion with endoproteinase Glu-C/endoproteinase Lys-C limited the possible labeling to the first three predicted transmembrane spans, and endoproteinase Glu-C/trypsin digestion refined this prediction to include only transmembrane spans III and IV. Taken together, our findings suggest that the adenosine antagonist 125l-3-(4-azidophenethyl)-1-propyl-8-cyclopentyl-xanthine covalently modifies transmembrane III of the A1 receptor because this was the only receptor region common to all radiolabeled fragments.

The alpha 2A adrenergic receptor (alpha 2AAR) previously was shown to be directly delivered to and retained on the lateral subdomain of renal epithelial cells. The present studies demonstrate that, in contrast, wild-type and epitope-tagged canine A1 adenosine receptors (A1AdoR) are apically enriched (65-83%) in Madin-Darby canine kidney (MDCKII) and porcine renal epithelial (LLC-PKI) cells, based on surface biotinylation strategies detecting photoaffinity-labeled A1AdoR. Confocal microscopy corroborated the apical enrichment of the epitopetagged A1AdoR. Metabolic labeling studies revealed that this steady-state polarization is achieved by direct delivery to both the apical (60-75%) and basolateral surface. Growth of A1AdoR-expressing cells as monolayers presence of A1AdoR antagonists, which decreased cell growth, suggesting that A1AdoR elicit MDCKII cell proliferation. The preferential apical but detectable basolateral localization of A1AdoR provides a molecular understanding of published reports that functional responses can be elicited following apical as well as basolateral delivery of adenosine agonists in varying renal preparations. These findings also suggest that receptor chimeras derived from the Gi/Go-protein-coupled alpha 2AAR and A1AdoR will be informative in revealing structural features critical for basolateral versus apical targeting.

Isometric tension responses of isolated porcine coronary artery rings were studied in the presence of concentrations of propranolol higher than those necessary to block effects mediated by beta-adrenergic receptors. Propranolol (50-300 microM) inhibited contractions induced by 30 mM KCl and by histamine, norepinephrine, and acetylcholine in a concentration-dependent, noncompetitive fashion. The (+) propranolol isomer and the racemic mixture were equipotent inhibitors of contraction. Propranolol inhibition was partly reversed by increased extracellular Ca++. These effects of propranolol thus appeared to be independent of beta-blockade and could be relevant to some of the drug's observed but still unexplained in vivo actions.

We have shown previously that 125I-2-[4-[2-[2-[(4-azidophenyl)methylcarbonylamino] ethylaminocarbonyl]ethyl]phenyl] ethylamino-5'-N-ethylcarboxamidoadenosine (125I-azido-PAPA-APEC) specifically and selectively photolabels RDC8 A2a adenosine receptors that have been overexpressed in COS M6 cells. Glycosylated, 125I-azido-PAPA-APEC-labeled, wild-type (412 residues; 45,031 Da) and carboxyl-terminally truncated (315 residues; 35,427 Da) receptors migrate with apparent molecular masses of > 40 and 31.5 kDa, respectively, whereas unglycosylated or deglycosylated wild-type and truncated A2a receptors migrate with apparent molecular masses of 40 and 28.5 kDa, respectively. Because nonspecific photoincorporation is not a complication, the present peptide mapping studies of the full length and truncated canine A2a adenosine receptors were carried out on unpurified COS M6 membrane preparations. After partial proteolysis it became clear that glycosylation increased the apparent molecular mass of either the wild-type or mutant A2a receptor by approximately 3 kDa. Although the A2a receptor was readily cleaved by a variety of chemical reagents and proteases, trypsin and endoproteinase Glu-C generated the most reproducible and, in the case of trypsin, the most complete fragmentation patterns. Radiolabeled peptides were identified by their apparent molecular masses, (in)abilities to be recognized by an antipeptide antibody to amino acids Tyr155-Val172 of the presumed second extracellular loop of the receptor, and (in)sensitivities to endoglycosidase F and tunicamycin treatments. A prominent, 7-kDa, radiolabeled peptide that was generated by trypsin digestion implicated putative alpha-helix V in the binding of 125I-azido-PAPA-APEC.

The amino acids that comprise the ligand binding sites of adenosine receptors have not been identified. Adenosine and its agonist analogues differ from ligands for the well studied biogenic amine receptors and rhodopsin in that the adenosine receptor agonists are larger, contain a ribose moiety, and are uncharged at physiological pH. Thus, the locations of the ligand binding pockets of the adenosine receptors could differ significantly from those of the biogenic amine receptors. This report describes the characterization of a purification-amenable truncated mutant of the canine A2a adenosine receptor and demonstrates that neither the long carboxyl-terminal tail nor the glycosidic moiety appears to be required for ligand binding. The dog thyroid A2a adenosine receptor cDNA (RDC8) was subcloned into the mammalian expression vector pCMV4. A mutant A2a construct, in which six histidines replaced residues 310-412 as the carboxyl terminus of the protein, also was prepared. When overexpressed transiently in COS M6 cells, the wild-type and mutant A2a receptors exhibited similar 2-[p-(2-[3H]carboxyethyl)phenylethylamino]-5'-N- ethylcarboxamidoadenosine saturation binding and competition curve profiles. The following biochemical techniques confirmed that the COS M6 cells were transcribing and translating A2a receptors of the expected molecular masses: (a) immunoblotting with an antipeptide antibody directed against the putative carboxyl-terminal side of the second extracellular loop (Tyr155-Val172) of the canine A2a adenosine receptor, (b) photoaffinity labeling with the A2a-selective agonist 125I-2-[4-[2-[2-[(4-azidophenyl)methylcarbonylamino] ethylaminocarbonyl]ethyl]phenyl]ethylamino-5'-N-ethylcarboxamidoad enosine (125I-azido-PAPA-APEC), and (c) partial purification of the hexahistidine-tagged receptor on Ni2+.nitrilotriacetic acid resin. A presumed A2a receptor (44 kDa) from rabbit striatal membranes also was detected with the antisera against amino acids Tyr155-Val172 of the RDC8 receptor. Not only could the mutant A2a receptor be photolabeled specifically with 125I-azido-PAPA-APEC but so too could unglycosylated A2a receptors (i.e., from tunicamycin-treated COS M6 cells), either full length or truncated. In all of these cases, photolabeling was attenuated by both agonist and antagonist competitors.

Smooth muscle preparations of human aorta or pig coronary arteries contain nearly equal amounts of cGMP-dependent protein kinase isozymes (cGMP kinase I alpha and I beta). In order to understand the roles of these isozymes in relaxing vascular smooth muscle, several new cGMP analogs were synthesized and tested for potencies in activating each enzyme and in relaxing pig coronary arteries. Analogs modified with a derivatized phenylthio group at the 8-position were as much as 72-fold more potent in activating purified cGMP kinase I alpha than cGMP kinase I beta. Electron-donating substituents, such as hydroxy, amino, and methoxy, on the phenyl ring enhanced the potencies of these analogs in activating cGMP kinase I alpha. The most potent of these cGMP analogs [8-(4-hydroxyphenylthio)-cGMP] was 17 times more potent (EC50 = 1.1 microM) as a muscle relaxant than the most efficacious analog tested previously. Among derivatives with an 8-halo group, 8-iodo-cGMP was the most potent compound (Ka = 9 nM for I alpha and 122 nM for I beta) for both I alpha and I beta. Analogs modified at the 1,N2-position or at both the 1,N2-and 8-positions of cGMP were highly potent for activating both isozymes. Within this group, 8-I-beta-phenyl-1,N2-etheno-cGMP had Ka values of 22 nM and 17 nM for cGMP kinase I alpha and I beta, respectively, whereas the Ka values of cGMP were 110 nM and 250 nM for the two isozymes. 8-I-beta-phenyl-1,N2-etheno-cGMP was the most potent muscle relaxant tested, with EC50 of 0.4 microM. For all cGMP analogs tested, there was a positive correlation between potency for activation of cGMP kinase I alpha and that for relaxation of pig coronary arteries. Assuming that the kinase assay conditions yielded a cyclic nucleotide specificity similar to that which would exist in intact cells, it was concluded that the cGMP kinase I alpha isozyme mediates the relaxation of pig coronary artery smooth muscle caused by cGMP elevation. However, an additional role for cGMP kinase I beta in the relaxation process could not be ruled out.

Ca/calmodulin-sensitive cyclic nucleotide phosphodiesterase (CaM-PDE) is an important enzyme regulating cGMP levels and relaxation of vascular smooth muscle. This modification study was conducted mostly with bovine brain CaM-PDE to identify essential functional groups involved in catalysis. The effect of pH on Vmax/Km indicates two essential residues with pKa values of 6.4 and 8.2. Diethyl pyrocarbonate (DEP), a histidine-modifying agent, inhibits CaM-PDE with a second-order rate constant of 130 M-1 min-1 at pH 7.0 and 30 degrees C. Activity is restored by NH2OH. The pH dependence of inactivation reveals that the essential residue modified by DEP has an apparent pKa of 6.5. The difference spectrum of the intact and DEP-treated enzyme shows a maximum between 230 and 240 nm, suggesting formation of carbethoxy derivatives of histidine. The enzyme is also inactivated by N-ethylmaleimide (NEM) and 5,5'-dithiobis-(2-nitrobenzoic acid), both sulfhydryl-modifying agents, with the latter effect reversed by dithiothreitol, which suggests inactivation resulting from modification of cysteine residue(s). Partial inactivation of the enzyme by DEP or NEM results in an apparent decrease in the Vmax without a change in the Km or the extent of CaM stimulation. The rate of inactivation by DEP is greater in the presence than in the absence of Ca/CaM. A substrate analogue, Br-cGMP, and the competitive inhibitor 3-isobutyl-1-methylxanthine partially protect the enzyme against inactivation by DEP or NEM, suggesting that the modification of histidine and cysteine residues occurs at or near the active site. DEP also inactivated porcine brain CaM-PDE.(ABSTRACT TRUNCATED AT 250 WORDS)

We have studied the effects of a synthetic peptide that is based on the autoinhibitory domain of protein kinase C on tension development in detergent-permeabilized coronary artery smooth muscle. This peptide inhibited two forms of protein kinase C that were isolated from the coronary artery media layer by hydroxylapatite chromatography, with apparent Ki values in the 5-8 microM range. Contractions induced by calcium in the permeabilized arteries were not affected by the peptide (30 microM). Potentiation of calcium-induced contractions by 1 microM phorbol-12,13-dibutyrate was partially inhibited by 10 microM peptide and was completely abolished by 30 microM peptide. These results indicate that phorbol-12,13-dibutyrate potentiates calcium-induced contractions of permeabilized coronary arteries by activation of protein kinase C, but activated protein kinase C is not a requirement for the induction of contractions by calcium alone.

Rat platelets and adipocytes were used as models to investigate alterations of the A2- and of the A1-adenosine receptor-adenylate cyclase system of peripheral cells caused by chronic caffeine administration. The maximum effects of 5'-N-ethylcarboxamidoadenosine (NECA) to stimulate adenylate cyclase activity in suspensions of platelet membranes and to inhibit aggregation were significantly greater with platelets from caffeine-treated rats than from control rats. The effects of 1 to 100 nM prostaglandin E1 to inhibit platelet aggregation and to stimulate adenylate cyclase activity in platelet membranes were also significantly greater with caffeine-treated than with control rats. These data suggest that the increased ability of NECA to inhibit platelet aggregation after chronic caffeine ingestion was a result of increased cyclic AMP accumulation induced by this agonist. The increased stimulatory effect of NECA on adenylate cyclase in platelet membranes could be due to an increased A2-adenosine receptor number and/or an increased functional coupling between A2-adenosine receptor and stimulatory guanine nucleotide binding proteins. In contrast, although A1-receptor number was 37% higher in fat cell membranes from caffeine-treated rats than in those from control rats, increased A1-adenosine receptor-mediated inhibition of lipolysis and of adenylate cyclase was not detected. Thus, chronic caffeine consumption causes alterations in the response of some but not all peripheral cell types to agonists of adenosine receptors.

8-Substituted xanthines currently represent the most potent class of adenosine-receptor antagonists. A series of 8-substituted 1,3-dipropylxanthines was prepared and their potency as antagonists of A1 and A2 adenosine receptors of human platelets and rat adipocytes, respectively, were determined. No agents studied were as potent as 8-cyclopentyl-1,3-dipropylxanthine as antagonists of the A1 adenosine receptor, but 8-(2-methylcyclopropyl)-1,3-dipropylxanthine was at least 1000-fold more potent as an antagonist of A1 than of A2 adenosine receptors. While most substitutions on the 8-cycloalkyl moiety caused decreased potency to inhibit both A1 and A2 adenosine receptors, 8-[trans-4-(acetamidomethyl)cyclohexyl]-1,3-dipropylxanthine was nearly equipotent as an antagonist of the two receptors and appeared to be the most potent antagonist of A2 adenosine receptors reported to date.

The purpose of this study was to test the hypothesis that endogenous adenosine functions to restrain the renin release response to pharmacological and pathophysiological stimuli. To achieve this objective, we examined the effects of an adenosine receptor antagonist, 1,3-dipropyl-8-(p-sulfophenyl)xanthine (DPSPX), on the renin release response induced by acute administration of hydralazine or by chronic clipping of the left renal artery (renovascular hypertensive rats). In conscious, unrestrained rats, DPSPX significantly increased plasma renin activity (PRA) in control rats, in rats treated with hydralazine, and in renovascular hypertensive rats. The effect of DPSPX on PRA was significantly greater in rats treated with hydralazine or in renovascular hypertensive rats compared with control rats. DPSPX did not influence arterial blood pressure in any group, did not affect the measurement of PRA, and did not alter the elimination of renin activity from the circulation. Additional experiments were performed in the in situ autoperfused kidney so that the effects of DPSPX on renal hemodynamics and renal excretory function could be assessed. In this experimental model, DPSPX also increased PRA in hydralazine-treated rats and in renovascular hypertensive rats without affecting arterial pressure, renal blood flow, or sodium excretion. In a final set of studies in conscious, unrestrained rats, adenosine deaminase increased PRA in a dose-dependent manner in hydralazine-treated rats and significantly increased the slope of the relation between PRA and the depressor response to hydralazine. We conclude: 1) Although the kidney has both A1 and A2 adenosine receptors mediating inhibitory and stimulatory actions, respectively, on renin release, the dominant effect of endogenous adenosine on renin release is inhibitory. 2) Even under basal physiological conditions, endogenous adenosine tonically inhibits renin release. 3) This inhibitory effect is augmented whenever the renin-angiotensin system is stimulated regardless of the approach used to activate renin release. 4) Endogenous adenosine negatively modulates renin release by a direct effect on juxtaglomerular cells.