An ENU-induced splicing mutation reveals a role for Unc93b1 in early immune cell activation following influenza A H1N1 infection.

Genetic and immunological analysis of host-pathogen interactions can reveal fundamental mechanisms of susceptibility and resistance to infection. Modeling human infectious diseases among inbred mouse strains is a proven approach but is limited by naturally occurring genetic diversity. Using N-ethyl-N-nitrosourea mutagenesis, we created a recessive loss-of-function point mutation in Unc93b1 (unc-93 homolog B1 (C. elegans)), a chaperone for endosomal Toll-like receptors (TLR)3, TLR7 and TLR9, which we termed Letr for 'loss of endosomal TLR response'. We used Unc93b1(Letr/Letr) mice to study the role of Unc93b1 in the immune response to influenza A/PR/8/34 (H1N1), an important global respiratory pathogen. During the early phase of infection, Unc93b1(Letr/Letr) mice had fewer activated exudate macrophages and decreased expression of CXCL10, interferon (IFN)-γ and type I IFN. Mutation of Unc93b1 also led to reduced expression of the CD69 activation marker and a concomitant increase in the CD62L naive marker on CD4(+) and CD8(+) T cells in infected lungs. Finally, loss of endosomal TLR signaling resulted in delayed viral clearance that coincided with increased tissue pathology during infection. Taken together, these findings establish a role for Unc93b1 and endosomal TLRs in the activation of both myeloid and lymphoid cells during the innate immune response to influenza.

The evolution of eProtocols that enable reproducible clinical research and care methods.

Unnecessary variation in clinical care and clinical research reduces our ability to determine what healthcare interventions are effective. Reducing this unnecessary variation could lead to further healthcare quality improvement and more effective clinical research. We have developed and used electronic decision support tools (eProtocols) to reduce unnecessary variation. Our eProtocols have progressed from a locally developed mainframe computer application in one clinical site (LDS Hospital) to web-based applications available in multiple languages and used internationally. We use eProtocol-insulin as an example to illustrate this evolution. We initially developed eProtocol-insulin as a local quality improvement effort to manage stress hyperglycemia in the adult intensive care unit (ICU). We extended eProtocol-insulin use to translate our quality improvement results into usual clinical care at Intermountain Healthcare ICUs. We exported eProtocol-insulin to support research in other US and international institutions, and extended our work to the pediatric ICU. We iteratively refined eProtocol-insulin throughout these transitions, and incorporated new knowledge about managing stress hyperglycemia in the ICU. Based on our experience in the development and clinical use of eProtocols, we outline remaining challenges to eProtocol development, widespread distribution and use, and suggest a process for eProtocol development. Technical and regulatory issues, as well as standardization of protocol development, validation and maintenance, need to be addressed. Resolution of these issues should facilitate general use of eProtocols to improve patient care.

Induction of airway mucus production By T helper 2 (Th2) cells: a critical role for interleukin 4 in cell recruitment but not mucus production.

Airway inflammation is believed to stimulate mucus production in asthmatic patients. Increased mucus secretion is an important clinical symptom and contributes to airway obstruction in asthma. Activated CD4 Th1 and Th2 cells have both been identified in airway biopsies of asthmatics but their role in mucus production is not clear. Using CD4 T cells from mice transgenic for the OVA-specific TCR, we studied the role of Th1 and Th2 cells in airway inflammation and mucus production. Airway inflammation induced by Th2 cells was comprised of eosinophils and lymphocytes; features found in asthmatic patients. Additionally, there was a marked increase in mucus production in mice that received Th2 cells and inhaled OVA, but not in mice that received Th1 cells. However, OVA-specific Th2 cells from IL-4-deficient mice were not recruited to the lung and did not induce mucus production. When this defect in homing was overcome by administration of TNF-alpha, IL-4 -/- Th2 cells induced mucus as effectively as IL-4 +/+ Th2 cells. These studies establish a role for Th2 cells in mucus production and dissect the effector functions of IL-4 in these processes. These data suggest that IL-4 is crucial for Th2 cell recruitment to the lung and for induction of inflammation, but has no direct role in mucus production.

T helper 1 cells and interferon gamma regulate allergic airway inflammation and mucus production.

CD4 T helper (Th) type 1 and Th2 cells have been identified in the airways of asthmatic patients. Th2 cells are believed to contribute to pathogenesis of the disease, but the role of Th1 cells is not well defined. In a mouse model, we previously reported that transferred T cell receptor-transgenic Th2 cells activated in the respiratory tract led to airway inflammation with many of the pathologic features of asthma, including airway eosinophilia and mucus production. Th1 cells caused inflammation with none of the pathology associated with asthma. In this report, we investigate the role of Th1 cells in regulating airway inflammation. When Th1 and Th2 cells are transferred together into recipient mice, there is a marked reduction in airway eosinophilia and mucus staining. To address the precise role of Th1 cells, we asked (i), Are Th2-induced responses inhibited by interferon (IFN)-gamma? and (ii) Can Th1 cells induce eosinophilia and mucus in the absence of IFN-gamma? In IFN-gamma receptor(-/-) recipient mice exposed to inhaled antigen, the inhibitory effects of Th1 cells on both airway eosinophilia and mucus production were abolished. In the absence of IFN-gamma receptor signaling, Th1 cells induced mucus but not eosinophilia. Thus, we have identified new regulatory pathways for mucus production; mucus can be induced by Th2 and non-Th2 inflammatory responses in the lung, both of which are inhibited by IFN-gamma. The blockade of eosinophilia and mucus production by IFN-gamma likely occurs through different inhibitory pathways that are activated downstream of Th2 cytokine secretion and require IFN-gamma signaling in tissue of recipient mice.

Essential role of nuclear factor kappaB in the induction of eosinophilia in allergic airway inflammation.

The molecular mechanisms that contribute to an eosinophil-rich airway inflammation in asthma are unclear. A predominantly T helper 2 (Th2)-type cell response has been documented in allergic asthma. Here we show that mice deficient in the p50 subunit of nuclear factor (NF)- kappaB are incapable of mounting eosinophilic airway inflammation compared with wild-type mice. This deficiency was not due to a block in T cell priming or proliferation in the p50(-/-) mice, nor was it due to a defect in the expression of the cell adhesion molecules VCAM-1 and ICAM-1 that are required for the extravasation of eosinophils into the airways. The major defects in the p50(-/-) mice were the lack of production of the Th2 cytokine interleukin 5 and the chemokine eotaxin, which are crucial for proliferation and for differentiation and recruitment, respectively, of eosinophils into the asthmatic airway. Additionally, the p50(-/-) mice were deficient in the production of the chemokines macrophage inflammatory protein (MIP)-1alpha and MIP-1beta that have been implicated in T cell recruitment to sites of inflammation. These results demonstrate a crucial role for NF-kappaB in vivo in the expression of important molecules that have been implicated in the pathogenesis of asthma.

Interferon gamma induction of pulmonary emphysema in the adult murine lung.

Chronic inflammation containing CD8(+) lymphocytes, neutrophils, and macrophages, and pulmonary emphysema coexist in lungs from patients with chronic obstructive pulmonary disease. Although this inflammatory response is believed to cause the remodeling that is seen in these tissues, the mechanism(s) by which inflammation causes emphysema have not been defined. Here we demonstrate that interferon gamma (IFN-gamma), a prominent product of CD8(+) cells, causes emphysema with alveolar enlargement, enhanced lung volumes, enhanced pulmonary compliance, and macrophage- and neutrophil-rich inflammation when inducibly targeted, in a transgenic fashion, to the adult murine lung. Prominent protease and antiprotease alterations were also noted in these mice. They included the induction and activation of matrix metalloproteinase (MMP)-12 and cathepsins B, H, D, S, and L, the elaboration of MMP-9, and the selective inhibition of secretory leukocyte proteinase inhibitor. IFN-gamma causes emphysema and alterations in pulmonary protease/antiprotease balance when expressed in pulmonary tissues.

Interleukin-13 induces tissue fibrosis by selectively stimulating and activating transforming growth factor beta(1).

Interleukin (IL)-13 is a key mediator of tissue fibrosis caused by T helper cell type 2 inflammation. We hypothesized that the fibrogenic effects of IL-13 are mediated by transforming growth factor (TGF)-beta. To test this hypothesis we compared the regulation of TGF-beta in lungs from wild-type mice and CC10-IL-13 mice in which IL-13 overexpression causes pulmonary fibrosis. IL-13 selectively stimulated TGF-beta(1) production in transgenic animals and macrophages were the major site of TGF-beta(1) production and deposition in these tissues. IL-13 also activated TGF-beta(1) in vivo. This activation was associated with decreased levels of mRNA encoding latent TGF-beta-binding protein-1 and increased mRNA encoding urinary plasminogen activator, matrix metalloproteinase (MMP)-9, and CD44. TGF-beta(1) activation was abrogated by the plasmin/serine protease antagonist aprotinin. It was also decreased in progeny of crosses of CC10-IL-13 mice and MMP-9 null mice but was not altered in crosses with CD44 null animals. IL-13-induced fibrosis was also significantly ameliorated by treatment with the TGF-beta antagonist soluble TGFbetaR-Fc (sTGFbetaR-Fc). These studies demonstrate that IL-13 is a potent stimulator and activator of TGF-beta(1) in vivo. They also demonstrate that this activation is mediated by a plasmin/serine protease- and MMP-9-dependent and CD44-independent mechanism(s) and that the fibrogenic effects of IL-13 are mediated, in great extent, by this TGF-beta pathway.

Granulomatous bronchiolitis of Crohn's disease successfully treated with inhaled budesonide.

A 39-year-old white woman with longstanding Crohn's disease presented with the rare complication of granulomatous bronchiolitis. Rapid resolution after inhaled budesonide is highlighted, as this is the first case described in the literature successfully treated without the need for systemic therapy. This less toxic approach to therapy is warranted in granulomatous bronchiolitis of Crohn's disease to avoid unwanted side effects of steroids and infliximab.

Clinicians' perceptions about use of computerized protocols: a multicenter study.

PURPOSE: Implementation of evidence-based techniques, such as explicit computerized protocols, has achieved limited success among clinicians. In this study, we describe the development and validation of an instrument for assessing clinicians' perceptions about use of explicit computerized protocols. METHODS: Qualitative assessment of semi-structured interviews with clinicians gave rise to a cognitive model evaluating the factors that motivate clinicians to use explicit computerized protocols. Using these constructs we developed a 35-item instrument which was administered to 240 clinicians (132 nurses, 53 physicians and 55 respiratory therapists), in three health-care institutions. RESULTS: Factor analysis identified nine factors that accounted for 66% of the total variance cumulatively. Factors identified were: Beliefs regarding Self-Efficacy, Environmental Support, Role Relevance, Work Importance, Beliefs regarding Control, Attitude towards Information Quality, Social Pressure, Culture, and Behavioral Intention. The strongest predictor was Beliefs regarding Self-Efficacy, which accounted for 26% of the total variance of intention to use explicit computerized protocols. Results supported the reliability and construct validity of the instrument. CONCLUSIONS: Clinicians' perceptions play a critical role in determining their intention to use explicit computerized protocols in routine clinical practice. Behavioral theories will help us understand factors predicting clinicians' intention to use explicit computerized protocols and recognize the implications of these factors in the design and implementation of these protocols.

Regulated overexpression of interleukin 11 in the lung. Use to dissociate development-dependent and -independent phenotypes.

Standard overexpression transgenic approaches are limited in their ability to model waxing and waning diseases and frequently superimpose development-dependent and -independent phenotypic manifestations. We used the clara cell 10-kD protein (CC10) promoter and the reverse tetracycline transactivator (rtTA) to create a lung-specific, externally regulatable, overexpression transgenic system and used this system to express human interleukin 11 (IL-11) in respiratory structures. Gene induction could be achieved in utero, in neonates and in adult animals. Moreover, gene expression could be turned off by removal of the inducing stimulus. When gene activation was initiated in utero and continued into adulthood, subepithelial airway fibrosis, peribronchiolar mononuclear nodules, and alveolar enlargement (emphysema) were noted. Induction in the mature lung caused airway remodeling and peribronchiolar nodules, but alveolar enlargement was not appreciated. In contrast, induction in utero and during the first 14 d of life caused alveolar enlargement without airway remodeling or peribronchiolar nodules. Thus, IL-11 overexpression causes abnormalities that are dependent (large alveoli) and independent (airway remodeling, peribronchiolar nodules) of lung growth and development, and the CC10-rtTA system can be used to differentiate among these effector functions. The CC10-rtTA transgenic system can be used to model waxing and waning, childhood and growth and development-related biologic processes with enhanced fidelity.

Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production.

Interleukin (IL)-13 is a pleiotropic cytokine produced in large quantities by activated CD4(+) Th2 lymphocytes. To define further its potential in vivo effector functions, the Clara cell 10-kDa protein promoter was used to express IL-13 selectively in the lung, and the phenotype of the resulting transgenic mice was characterized. In contrast to transgene-negative littermates, the lungs of transgene-positive mice contained an inflammatory response around small and large airways and in the surrounding parenchyma. It was mononuclear in nature and contained significant numbers of eosinophils and enlarged and occasionally multinucleated macrophages. Airway epithelial cell hypertrophy, mucus cell metaplasia, the hyperproduction of neutral and acidic mucus, the deposition of Charcot-Leyden-like crystals, and subepithelial airway fibrosis were also prominently noted. Eotaxin protein and mRNA were also present in large quantities in the lungs of the transgene-positive, but not the transgene-negative, mice. IL-4, IL-5, granulocyte-macrophage colony-stimulating factor, and monocyte chemoattractant protein-5 were not similarly detected. Physiological evaluations revealed significant increases in baseline airways resistance and airways hyperresponsiveness (AHR) to methacholine in transgene-positive animals. Thus, the targeted pulmonary expression of IL-13 causes a mononuclear and eosinophilic inflammatory response, mucus cell metaplasia, the deposition of Charcot-Leyden-like crystals, airway fibrosis, eotaxin production, airways obstruction, and nonspecific AHR. IL-13 may play an important role in the pathogenesis of similar responses in asthma or other Th2-polarized tissue responses.

Targeted expression of IL-11 in the murine airway causes lymphocytic inflammation, bronchial remodeling, and airways obstruction.

Interleukin-11 is a pleotropic cytokine produced by lung stromal cells in response to respiratory viruses, cytokines, and histamine. To further define its potential effector functions, the Clara cell 10-kD protein promoter was used to express IL-11 and the airways of the resulting transgene mice were characterized. In contrast to transgene (-) littermates, the airways of IL-11 transgene (+) animals manifest nodular peribronchiolar mononuclear cell infiltrates and impressive airways remodeling with subepithelial fibrosis. The inflammatory foci contained large numbers of B220(+) and MHC Class II(+) cells and lesser numbers of CD3(+), CD4(+), and CD8(+) cells. The fibrotic response contained increased amounts of types III and I collagen, increased numbers of alpha smooth muscle actin and desmin-containing cells and a spectrum of stromal elements including fibroblasts, myofibroblasts, and smooth muscle cells. Physiologic evaluation also demonstrated that 2-mo-old transgene (+) mice had increased airways resistance and non-specific airways hyperresponsiveness to methacholine when compared with their transgene (-) littermates. These studies demonstrate that the targeted expression of IL-11 in the mouse airway causes a B and T cell-predominant inflammatory response, airway remodeling with increased types III and I collagen, the local accumulation of fibroblasts, myofibroblasts, and myocytes, and obstructive physiologic dysregulation. IL-11 may play an important role in the inflammatory and fibrotic responses in viral and/or nonviral human airway disorders.

Inducible targeting of IL-13 to the adult lung causes matrix metalloproteinase- and cathepsin-dependent emphysema.

Cigarette smoke exposure is the major cause of chronic obstructive pulmonary disease (COPD). However, only a minority of smokers develop significant COPD, and patients with asthma or asthma-like airway hyperresponsiveness or eosinophilia experience accelerated loss of lung function after cigarette smoke exposure. Pulmonary inflammation is a characteristic feature of lungs from patients with COPD. Surprisingly, the mediators of this inflammation and their contributions to the pathogenesis and varied natural history of COPD are not well defined. Here we show that IL-13, a critical cytokine in asthma, causes emphysema with enhanced lung volumes and compliance, mucus metaplasia, and inflammation, when inducibly overexpressed in the adult murine lung. MMP-2, -9, -12, -13, and -14 and cathepsins B, S, L, H, and K were induced by IL-13 in this setting. In addition, treatment with MMP or cysteine proteinase antagonists significantly decreased the emphysema and inflammation, but not the mucus in these animals. These studies demonstrate that IL-13 is a potent stimulator of MMP and cathepsin-based proteolytic pathways in the lung. They also demonstrate that IL-13 causes emphysema via a MMP- and cathepsin-dependent mechanism(s) and highlight common mechanisms that may underlie COPD and asthma.

IL-13 stimulates vascular endothelial cell growth factor and protects against hyperoxic acute lung injury.

Hyperoxia is an important cause of acute lung injury. To determine whether IL-13 is protective in hyperoxia, we compared the survival in 100% O(2) of transgenic mice that overexpress IL-13 in the lung and of nontransgenic littermate controls. IL-13 enhanced survival in 100% O(2). One hundred percent of nontransgenic mice died in 4-5 days, whereas 100% of IL-13-overexpressing mice lived for more than 7 days, and many lived 10-14 days. IL-13 also stimulated VEGF accumulation in mice breathing room air, and it interacted with 100% (2) to increase VEGF accumulation further. The 164-amino acid isoform was the major VEGF moiety in bronchoalveolar lavage from transgenic mice in room air, whereas the 120- and 188-amino acid isoforms accumulated in these mice during hyperoxia. In addition, antibody neutralization of VEGF decreased the survival of IL-13-overexpressing mice in 100% (2). These studies demonstrate that IL-13 has protective effects in hyperoxic acute lung injury. They also demonstrate that IL-13, alone and in combination with 100% (2), stimulates pulmonary VEGF accumulation, that this stimulation is isoform-specific, and that the protective effects of IL-13 are mediated, in part, by VEGF.

Targeted lung expression of interleukin-11 enhances murine tolerance of 100% oxygen and diminishes hyperoxia-induced DNA fragmentation.

Acute lung injury is a frequent and treatment-limiting consequence of therapy with hyperoxic gas mixtures. To determine if IL-11 is protective in oxygen toxicity, we compared the effects of 100% O2 on transgenic mice that overexpress IL-11 in the lung and transgene (-) controls. IL-11 markedly enhanced survival in 100% O2 with 100% of transgene (-) animals dying within 72-96 h and > 90% of transgene (+) animals surviving for more than 10 d. This protection was associated with markedly diminished alveolar-capillary protein leak, endothelial and epithelial membrane injury, lipid peroxidation, and pulmonary neutrophil recruitment. Significant differences in copper zinc superoxide dismutase and catalase activities were not noted and the levels of total, reduced and oxidized glutathione were similar in transgene (+) and (-) animals. Glutathione reductase, glutathione peroxidase, and manganese superoxide dismutase activities were slightly higher in transgene (+) as versus (-) mice after 100% O2 exposure, and IL-11 diminished hyperoxia-induced expression of IL-1 and TNF. Hyperoxia also caused cell death with DNA fragmentation in the lungs of transgene (-) animals and IL-11 markedly diminished this cell death response. These studies demonstrate that IL-11 markedly diminishes hyperoxic lung injury. They also demonstrate this protection is associated with small changes in lung antioxidants, diminished hyperoxia-induced IL-1 and TNF production, and markedly suppressed hyperoxia-induced DNA fragmentation.

Molar incisor hypomineralisation: experience and perceived challenges among dentists specialising in paediatric dentistry and a group of general dental practitioners in the UK.

AIM: To assess the views and experience of the UK dentists specialising in paediatric dentistry (trainees) about molar incisor hypomineralisation (MIH) and compare the findings with the responses from a group of UK general dental practitioners. METHOD: A web-based questionnaire was sent to dentists undergoing specialist training in paediatric dentistry. The same questionnaire was completed by a group of general dentists who stated an interest in treating children, with various levels of experience. The questionnaire sought information on clinical experience and the views of the dentists on the impact of MIH on children and families. RESULTS: Specialty trainees (37) from different paediatric dental departments in the UK completed the online survey, giving a total response rate of 71%. The questionnaire was also completed by 31 general dental practitioners. There was difficulty in distinguishing MIH from other conditions for both groups. Increased sensitivity of affected teeth was the most frequently encountered problem with 51% of the trainees and 76% of the dentists saying this was often or always a challenge. The trainees were particularly concerned about the pain children experienced and about the appearance of the condition. Both groups felt that parental anxiety occurred in almost all cases. CONCLUSIONS: Both groups felt that MIH presents several clinical challenges and has a negative effect on the quality of life of the affected children and their families. There were significant differences in the views and perceptions between the two groups.

An investigation of the electronic and steric environments of tyrosyl residues in ribonuclease A and Erwinia carotovora L-asparaginase through fluorescence quenching by caesium, iodide and phosphate ions.

The fluorescence lifetimes and relative quantum yields of several derivatives of tyrosine are reported. The quenching of the fluorescence of these compounds by phosphate, caesium and iodide ions has been investigated; the encounter rate constants, calculated from the quenching parameters and lifetimes, show a clear dependence on the charges borne by the quenchers and fluorophores. The ratio of the Stern-Volmer constants of iodide and caesium, ions of similar size, defines an electrostatic parameter sensitive to the charge of the fluorophore which can be evaluated without knowledge of the fluorescent lifetimes. The mean of the encounter rate constants for caesium and iodide ions defines a rate constant which is largely charge-independent and is used to establish a steric parameter. The two parameters are used to investigate the tyrosine environment in bovine ribonuclease A (EC 3.1.4.23) and Erwinia carotovora L-asparaginase (EC 3.5.1.1). The quantum yield of L-asparaginase (0.12) is very high for a class A protein and may be associated with the absence of disulphide bridges. There was no evidence for more than one type of tyrosine residue from the quenching experiments with either enzyme, an observation which is attributed to efficient energy transfer amongst tyrosine residues. At pH values close to the isoelectric points of the enzymes the electrostatic parameter suggests that the environment of the quenchable tyrosines in L-asparaginase is somewhat more positive than in ribonuclease. In 1% sodium dodecyl sulphate the tyrosine environment of L-asparaginase becomes markedly negative as expected. The steric parameter indicates a lower accessibility of the tyrosine residues in L-asparaginase than in ribonuclease; an illustrative calculation is provided linking the steric parameter with the number of exposed tyrosine residues by taking into account the greater collision frequency of the larger protein molecules and the encounter distance for quenching determined from charge effects on the quenching of the model compounds. The calculation suggests that three tyrosyl residues are accessible in ribonuclease, in good agreement with other studies, but in L-asparaginase the number increases from 0.4 at pH 5.73 to 0.8 at pH 9.16 suggesting a loosening of the enzyme structure at high pH.

A pH-dependent conformational change in the coat protein subunits from potato virus X.

Both the circular dichroism and fluorescence spectra of the dissociated coat protein subunits from potato virus X changed substantially over the pH range 8 to 4, irreversible changes resulted below pH 4, with tyrosyl and tryptophanyl residues affected most. The titration curves show a pKa of about 5.6 and do not require cooperative interactions between the coat protein subunits, thus they are in marked contrast to titrations of tobacco mosaic virus A-protein. The spectra of the intact virus were little changed between pH 8 and 4 and suggested that the coat protein was locked into a conformation similar to that of the subunits in solution at pH 7. It is proposed that the pH induced conformational change is responsible for determining the acidic branch of the pH profile for reconstitution of potato virus X from its dissociated coat protein subunits and RNA.

An investigation of an unusual histidyl residue in Escherichia coli B L-asparaginase through fluorescence quenching.

The tryptophanyl fluorescence of Escherichia coli B L-asparaginase is partially quenched by the protonated form of a base with pKa 6.0 at 25 degrees C, mu = 0.1. This base has been identified as a histidyl residue through the effect of ionic strength and solvent polarity on the pKa. In addition diethylpyrocarbonate which modifies two histidyl residues in the enzyme abolishes the fluorescenc titration and reduces enzymic activity by 90%. The temperature dependence of the histidine pKa is unusual, showing a minimum at 25 degrees C, a thermodynamic analysis of the data shows this to be due to a large negative delta Cp term associated with the ionisation. This is interpreted in terms of the movement of hydrophobic residues into the enzyme on deprotonation of the histidyl residue. The quantum yield of L-asparaginase and its temperature dependence have been measured. The quantum yield is high and there is a low activation energy for radiationless deactivation of the excited state both of which are consistent with a tryptophanyl environment remote from the solvent.

Circular dichroism and fluorescence studies on potato virus X and its structural components.

Circular dichroism (DC) measurements of the coat protein subunits of potato virus X show that native subunits that can reassemble with RNA to form infectious virus particles have appreciable alpha-helical structure. The CD of intact potato virus X was less intense below and more intense above 250 nm, and the maxima and minima were at longer wavelengths, than those of a CD spectrum computed from the individual contributions of the coat protein and RNA. The differences between the measured and computed spectra below 250 nm were attributed to the effects of differential light scattering and absorption flattening on measurements of the virus particle CD. The differences at longer wavelengths, were the CD contribution of the nucleic acid predominates, probably reflect the difference between a base-paired conformation of the RNA in solution and the more rigid single-stranded conformation imposed by the structure of the virus. The CD evidence suggests that the tertiary structure and potato virus X coat protein subunits in solution and in intact virus particles is similar. Both CD and fluorescence emission results indicate differences between the tryptophan environment in dissociated protein subunits and that in intact virus. These are attributed to local differences in subunit conformation or to the occurrence of intersubunit interactions involving tryptophan in the intact virus.