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Type I and type III interferons (IFNs) are major components in activating the innate immune response. Common to both are two distinct receptor chains (IFNAR1/IFNAR2 and IFNLR1/IL10R2), which form ternary complexes upon binding their respective ligands. This results in close proximity of the intracellularly associated kinases JAK1 and TYK2, which cross phosphorylate each other, the associated receptor chains, and signal transducer and activator of transcriptions, with the latter activating IFN-stimulated genes. While there are clear similarities in the biological responses toward type I and type III IFNs, differences have been found in their tropism, tuning of activity, and induction of the immune response. Here, we focus on how these differences are embedded in the structure/function relations of these two systems in light of the recent progress that provides in-depth information on the structural assembly of these receptors and their functional implications and how these differ between the mouse and human systems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Natural evolution is driven by random mutations that improve fitness. In vitro evolution mimics this process, however, on a short time-scale and is driven by the given bait. Here, we used directed in vitro evolution of a random mutant library of Uracil glycosylase (eUNG) displayed on yeast surface to select for binding to chaperones GroEL, DnaK + DnaJ + ATP (DnaKJ) or E. coli cell extract (CE), using binding to the eUNG inhibitor Ugi as probe for native fold. The CE selected population was further divided to Ugi binders (+U) or non-binders (-U). The aim here was to evaluate the sequence space and physical state of the evolved protein binding the different baits. We found that GroEL, DnaKJ and CE-U select and enrich for mutations causing eUNG to misfold, with the three being enriched in mutations in buried and conserved positions, with a tendency to increase positive charge. Still, each selection had its own trajectory, with GroEL and CE-U selecting mutants highly sensitive to protease cleavage while DnaKJ selected partially structured misfolded species with a tendency to refold, making them less sensitive to proteases. More general, our results show that GroEL has a higher tendency to purge promiscuous misfolded protein mutants from the system, while DnaKJ binds misfolding-prone mutant species that are, upon chaperone release, more likely to natively refold. CE-U shares some of the properties of GroEL- and DnaKJ-selected populations, while harboring also unique properties that can be explained by the presence of additional chaperones in CE, such as Trigger factor, HtpG and ClpB., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author contributions Oran Melanker: Conceptualization, Investigation, Methodology, Resources, Visualization, Software, Writing. Pierre Goloubinoff: Conceptualization, Investigation, Methodology, Resources, Visualization, Project administration, Funding acquisition, Supervision, Writing. Gideon Schreiber: Conceptualization, Investigation, Methodology, Resources, Visualization, Project administration, Funding acquisition, Supervision, Writing. Classification Protein evolution., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The crowded cellular milieu affect molecular diffusion through hard (occluded space) and soft (weak, non-specific) interactions. Multiple methods have been developed to measure diffusion coefficients at physiological protein concentrations within cells, each with its limitations. Here, we show that Line-FRAP, combined with rigours data analysis, is able to determine diffusion coefficients in a variety of environments, from in vitro to in vivo. The use of Line mode greatly improves time resolution of FRAP data acquisition, from 20-100 Hz in the classical mode to 800 Hz in the line mode. This improves data analysis, as intensity and radius of the bleach at the first post-bleach frame is critical. We evaluated the method on different proteins labelled chemically or fused to YFP in a wide range of environments. The diffusion coefficients measured in HeLa and in E. coli were ~2.5-fold and 15-fold slower than in buffer, and were comparable to previously published data. Increasing the osmotic pressure on E. coli further decreases diffusion, to the point at which proteins virtually stop moving. The method presented here, which requires a confocal microscope equipped with dual scanners, can be applied to study a large range of molecules with different sizes, and provides robust results in a wide range of environments and protein concentrations for fast diffusing molecules., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Type I interferons (IFNs) have a central role in innate and adaptive immunities, proliferation, and cancer surveillance. How IFN binding to its specific receptor, the IFN α and β receptor (IFNAR), can drive such variety of processes is an open question. Here, to systematically and thoroughly investigate the molecular mechanism of IFN signaling, we used a CRISPR/Cas9-based approach in a human cell line (HeLa) to generate knockouts (KOs) of the genes participating in the type 1 IFN signaling cascade. We show that both IFNAR chains (IFNAR1 and IFNAR2) are absolutely required for any IFN-induced signaling. Deletion of either signal transducer and activator of transcription 1 (STAT1) or STAT2 had only a partial effect on IFN-induced antiviral activity or gene induction. However, the deletion of both genes completely abrogated any IFN-induced activity. So did a double STAT2-IFN regulatory factor 1 (IRF1) KO and, to a large extent, a STAT1 KO together with IRF9 knockdown. KO of any of the STATs had no effect on the phosphorylation of other STATs, indicating that they bound IFNAR independently. STAT3 and STAT6 phosphorylations were fully induced by type 1 IFN in the STAT1-STAT2 KO, but did not promote gene induction. Moreover, STAT3 KO did not affect type 1 IFN-induced gene or protein expression. Type 1 IFN also did not activate p38, AKT, or ERK kinase. We conclude that type 1 IFN-induced activities in HeLa cells are mediated by STAT1/STAT2/IRF9, STAT1/STAT1, or STAT2/IRF9 complexes and do not require alternative pathways., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

PurposeIn 2012 we reported in six individuals a clinical condition almost indistinguishable from PLOD1-kyphoscoliotic Ehlers-Danlos syndrome (PLOD1-kEDS), caused by biallelic mutations in FKBP14, and characterized by progressive kyphoscoliosis, myopathy, and hearing loss in addition to connective tissue abnormalities such as joint hypermobility and hyperelastic skin. FKBP14 is an ER-resident protein belonging to the family of FK506-binding peptidyl-prolyl cis-trans isomerases (PPIases); it catalyzes the folding of type III collagen and interacts with type III, type VI, and type X collagens. Only nine affected individuals have been reported to date.MethodsWe report on a cohort of 17 individuals with FKBP14-kEDS and the follow-up of three previously reported patients, and provide an extensive overview of the disorder and its natural history based on clinical, biochemical, and molecular genetics data.ResultsBased on the frequency of the clinical features of 23 patients from the present and previous cohorts, we define major and minor features of FKBP14-kEDS. We show that myopathy is confirmed by histology and muscle imaging only in some patients, and that hearing impairment is predominantly sensorineural and may not be present in all individuals.ConclusionOur data further support the extensive clinical overlap with PLOD1-kEDS and show that vascular complications are rare manifestations of FKBP14-kEDS.

How structural dynamics affects cytokine signaling is under debate. Here, we investigated the dynamics of the type I interferon (IFN) receptor, IFNAR1, and its effect on signaling upon binding IFN and IFNAR2 using a combination of structure-based mechanistic studies, in situ binding, and gene induction assays. Our study reveals that IFNAR1 flexibility modulates ligand-binding affinity, which, in turn, regulates biological signaling. We identified the hinge sites and key interactions implicated in IFNAR1 inter-subdomain (SD1-SD4) movements. We showed that the predicted cooperative movements are essential to accommodate intermolecular interactions. Engineered disulfide bridges, computationally predicted to interfere with IFNAR1 dynamics, were experimentally confirmed. Notably, introducing disulfide bonds between subdomains SD2 and SD3 modulated IFN binding and activity in accordance with the relative attenuation of cooperative movements with varying distance from the hinge center, whereas locking the SD3-SD4 interface flexibility in favor of an extended conformer increased activity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Primiparity has been associated with 3 to 4 mg/dL lower high-density lipoprotein cholesterol concentrations in black and white adult women that persist several years after delivery., Objective: To examine the lasting effects of adolescent pregnancy on blood lipids, an early risk factor for future cardiometabolic diseases., Design: The National Heart Lung and Blood Institute's Growth and Health Study is a multicenter prospective cohort that measured fasting blood lipids for 1013 (513 black, 500 white) participants at baseline (1987-1988) ages 9-10, and again at follow-up (1996-1997) ages 18-19., Methods: Change in fasting plasma total cholesterol, triglycerides, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol, defined as the difference between baseline and follow-up measurements, was compared among 186 (145 black, 41 white) primi- or multiparas, 106 (55 black, 51 white) nulliparous, gravidas versus 721 (313 black, 408 white) nulligravidas. Fully adjusted multiple linear regression models estimated blood lipid changes among these pregnancy groups adjusted for race, age at menarche, baseline lipids, physical inactivity, body mass index, and family sociodemographics., Results: In the 10-year study period, adolescent paras compared with nulligravidas had greater decrements in high-density lipoprotein cholesterol (mg/dL; fully adjusted mean [95% confidence interval] group differences in black -4.3 [-6.7, -2.0]; P < .001 and white: -4.5 [-8.2, -0.7]; P = .016) and greater increments in fasting triglycerides (mg/dL; adjusted mean [95% confidence interval] group differences in black: 10.4 [3.9, 16.8]; P < .001, and white: 11.6 [-3.6, 26.8]; P = .167)., Conclusion: Adolescent pregnancy contributes to pro-atherogenic lipid profiles that persist after delivery. Further research is needed to assess whether adolescent pregnancy has implications for future cardiovascular disease risk in young women., (Copyright © 2012 National Lipid Association. Published by Elsevier Inc. All rights reserved.)

The CAPRI (Critical Assessment of Predicted Interactions) and CASP (Critical Assessment of protein Structure Prediction) experiments have demonstrated the power of community-wide tests of methodology in assessing the current state of the art and spurring progress in the very challenging areas of protein docking and structure prediction. We sought to bring the power of community-wide experiments to bear on a very challenging protein design problem that provides a complementary but equally fundamental test of current understanding of protein-binding thermodynamics. We have generated a number of designed protein-protein interfaces with very favorable computed binding energies but which do not appear to be formed in experiments, suggesting that there may be important physical chemistry missing in the energy calculations. A total of 28 research groups took up the challenge of determining what is missing: we provided structures of 87 designed complexes and 120 naturally occurring complexes and asked participants to identify energetic contributions and/or structural features that distinguish between the two sets. The community found that electrostatics and solvation terms partially distinguish the designs from the natural complexes, largely due to the nonpolar character of the designed interactions. Beyond this polarity difference, the community found that the designed binding surfaces were, on average, structurally less embedded in the designed monomers, suggesting that backbone conformational rigidity at the designed surface is important for realization of the designed function. These results can be used to improve computational design strategies, but there is still much to be learned; for example, one designed complex, which does form in experiments, was classified by all metrics as a nonbinder., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The "4D Biology Workshop for Health and Disease", held on 16-17th of March 2010 in Brussels, aimed at finding the best organising principles for large-scale proteomics, interactomics and structural genomics/biology initiatives, and setting the vision for future high-throughput research and large-scale data gathering in biological and medical science. Major conclusions of the workshop include the following. (i) Development of new technologies and approaches to data analysis is crucial. Biophysical methods should be developed that span a broad range of time/spatial resolution and characterise structures and kinetics of interactions. Mathematics, physics, computational and engineering tools need to be used more in biology and new tools need to be developed. (ii) Database efforts need to focus on improved definitions of ontologies and standards so that system-scale data and associated metadata can be understood and shared efficiently. (iii) Research infrastructures should play a key role in fostering multidisciplinary research, maximising knowledge exchange between disciplines and facilitating access to diverse technologies. (iv) Understanding disease on a molecular level is crucial. System approaches may represent a new paradigm in the search for biomarkers and new targets in human disease. (v) Appropriate education and training should be provided to help efficient exchange of knowledge between theoreticians, experimental biologists and clinicians. These conclusions provide a strong basis for creating major possibilities in advancing research and clinical applications towards personalised medicine., (Copyright © 2010. Published by Elsevier B.V. All rights reserved.)

Cytokines are essential proteins that exert potent control over entire cell populations to fight infections and other pathologies, but can by themselves cause disease. Therefore, cytokine-related drugs act either by stimulating or blocking their activities. Our knowledge of the structures of cytokine-receptor complexes, the biophysical basis of their binding, and their mode of biological activation has substantially increased in recent years. This knowledge has been translated into new drugs and drug candidates. This review summarizes our current understanding of the receptor-mediated activity of cytokines, their relation to health and disease, and the agents in use to activate and block their actions., (2010 Elsevier Ltd. All rights reserved.)

Antizyme (Az) is a highly conserved key regulatory protein bearing a major role in regulating polyamine levels in the cell. It has the ability to bind and inhibit ornithine decarboxylase (ODC), targeting it for degradation. Az inhibitor (AzI) impairs the activity of Az. In this study, we mapped the binding sites of ODC and AzI on Az using Ala scan mutagenesis and generated models of the two complexes by constrained computational docking. In order to scan a large number of mutants in a short time, we developed a workflow combining high-throughput mutagenesis, small-scale parallel partial purification of His-tagged proteins and their immobilization on a tris-nitrilotriacetic-acid-coated surface plasmon resonance chip. This combination of techniques resulted in a significant reduction in time for production and measurement of large numbers of mutant proteins. The data-driven docking results suggest that both proteins occupy the same binding site on Az, with Az binding within a large groove in AzI and ODC. However, single-mutant data provide information concerning the location of the binding sites only, not on their relative orientations. Therefore, we generated a large number of double-mutant cycles between residues on Az and ODC and used the resulting interaction energies to restrict docking. The model of the complex is well defined and accounts for the mutant data generated here, and previously determined biochemical data for this system. Insights on the structure and function of the complexes, as well as general aspects of the method, are discussed.

A new method is presented for the redesign of protein-protein interfaces, resulting in specificity of the designed pair while maintaining high affinity. The design is based on modular interface architecture and was carried out on the interaction between TEM1 beta-lactamase and its inhibitor protein, beta-lactamase inhibitor protein. The interface between these two proteins is composed of several mostly independent modules. We previously showed that it is possible to delete a complete module without affecting the overall structure of the interface. Here, we replace a complete module with structure fragments taken from nonrelated proteins. Nature-optimized fragments were chosen from 10(7) starting templates found in the Protein Data Bank. A procedure was then developed to identify sets of interacting template residues with a backbone arrangement mimicking the original module. This generated a final list of 361 putative replacement modules that were ranked using a novel scoring function based on grouped atom-atom contact surface areas. The top-ranked designed complex exhibited an affinity of at least the wild-type level and a mode of binding that was remarkably specific despite the absence of negative design in the procedure. In retrospect, the combined application of three factors led to the success of the design approach: utilizing the modular construction of the interface, capitalizing on native rather than artificial templates, and ranking with an accurate atom-atom contact surface scoring function.

The process of protein-protein association starts with their random collision, which may develop into an encounter complex followed by a transition state and final complex formation. Here we aim to experimentally characterize the nature of the transition state of protein-protein association for three different protein-protein interactions; Barnase-Barstar, TEM1-BLIP and IFNalpha2-IFNAR2, and use the data to model the transition state structures. To model the transition state, we determined inter-protein distance-constraints of the activated complex by using double mutant cycles (DMC) assuming that interacting residues are spatially close. Significant DeltaDeltaG(double dagger)(int) values were obtained only between residues on Barnase and Barstar. However, introducing specific mutations that optimize the charge complementarity between BLIP and TEM1 resulted in the introduction of significant DeltaDeltaG(double dagger)(int) values also between residues of these two proteins. While electrostatic interactions make major contributions towards stabilizing the transition state, we show two examples where steric hindrance exerts an effect on the transition state as well. To model the transition-state structures from the experimental DeltaDeltaG(double dagger)(int) values, we introduced a method for structure perturbation, searching for those inter-protein orientations that best support the experimental DeltaDeltaG(double dagger)(int) values. Two types of transition states were found, specific as observed for Barnase-Barstar and the electrostatically optimized TEM1-BLIP mutants, and diffusive as shown for wild-type TEM1-BLIP and IFNalpha2-IFNAR2. The specific transition states are characterized by defined inter-protein orientations, which cannot be modeled for the diffusive transition states. Mutations introduced through rational design can change the transition state from diffusive to specific. Together, these data provide a structural view of the mechanism allowing rates of association to differ by five orders of magnitude between different protein complexes.

Congenital muscular dystrophy (CMD) due to merosin (laminin alpha2 chain) deficiency is an autosomal recessively inherited disorder characterized by severe muscular weakness and hypotonia from birth on. Brain involvement is the rule and characterized by variable T2 hyperintensities of white matter which appears swollen on cranial MRI. The pathophysiology of these white matter changes is not clear. In five patients with laminin alpha2 deficient CMD we performed short-echo time localized proton MRS with determination of absolute metabolite concentrations in grey and white matter. In affected white matter, a consistent pattern of metabolites was detected comprising reduced concentrations of N-acetylaspartate and N-acetylaspartylglutamate, creatine, and phosphocreatine, and to a milder degree of choline-containing compounds. In contrast, concentrations of myo-inositol were in the normal range. Spectra of cortical and subcortical grey matter were normal. The observed metabolite profile is consistent with white matter edema, that is reduced cellular density, and relative astrocytosis. This interpretation is in line with the hypothesis that laminin alpha2 deficiency results in leakage of fluids across the blood-brain barrier and a histopathological report of astrocytic proliferation in CMD.

Proteins bind one another in aqua's solution to form tight and specific complexes. Previously we have shown that this is achieved through the modular architecture of the interaction network formed by the interface residues, where tight cooperative interactions are found within modules but not between them. Here we extend this study to cover the entire interface of TEM1 beta-lactamase and its protein inhibitor BLIP using an improved method for deriving interaction maps based on REDUCE to add hydrogen atoms and then by evaluating the interactions using modifications of the programs PROBE, NCI and PARE. An extensive mutagenesis study of the interface residues indeed showed that each module is energetically independent on other modules, and that cooperativity is found only within a module. By solving the X-ray structure of two interface mutations affecting two different modules, we demonstrated that protein-protein binding occur via the structural reorganization of the binding modules, either by a "lock and key" or an induced fit mechanism. To explain the cooperativity within a module, we performed multiple-mutant cycle analysis of cluster 2 resulting in a high-resolution energy map of this module. Mutant studies are usually done in reference to alanine, which can be regarded as a deletion of a side-chain. However, from a biological perspective, there is a major interest to understand non-Ala substitutions, as they are most common. Using X-ray crystallography and multiple-mutant cycle analysis we demonstrated the added complexity in understanding non-Ala mutations. Here, a double mutation replacing the wild-type Glu,Tyr to Tyr,Asn on TEM1 (res id 104,105) caused a major backbone structural rearrangement of BLIP, changing the composition of two modules but not of other modules within the interface. This shows the robustness of the modular approach, yet demonstrates the complexity of in silico protein design.

Background: Multidisciplinary clinics have been recommended for the evaluation of patients with lung cancer. Evidence to support this recommendation, however, is limited. A single-center, retrospective review of lung cancer patients at a Veterans Affairs hospital was performed comparing timeliness of diagnostic and treatment decisions during the operation of a multidisciplinary thoracic oncology clinic (MTOC) with a period after it closed (non-MTOC), during which only a weekly multidisciplinary conference was held., Methods: Patients were identified from a tumor registry. Manual chart reviews were performed on all patients. Outcome measures included time from initial presentation to diagnosis (TTD) and time from diagnosis to treatment initiation (TTT)., Results: Three hundred forty-five patients (244 in MTOC, 101 in non-MTOC) diagnosed with lung cancer between 1999 and 2003 were included in the study. Baseline characteristics were similar between the two groups. Median TTD was 48 days (95% confidence interval [CI]: 37-61) and 47 days (95% CI: 39-55) in the MTOC (n = 164) and non-MTOC cohorts (n = 89), respectively (p = 0.09). Median TTT was 22 days (95% CI: 20-27) and 23 days (95% CI: 20-34) in the MTOC (n = 165) and non-MTOC cohorts (n = 89), respectively (p = 0.71). There was no difference in overall survival., Conclusion: Retrospective comparison of sequential cohorts failed to reveal benefit in the timeliness of care measures during the time period of MTOC operation. Potential confounders include the absence of a surgeon in the MTOC setting, an ongoing weekly multidisciplinary conference in the non-MTOC cohort, and existing infrastructures based on previous MTOC experiences and past provider experience. Confirmation of these findings in other health care settings is warranted, preferably in a prospective fashion.

Type I interferons (IFNs) elicit antiviral, antiproliferative and immunomodulatory properties in cells. All of them bind to the same receptor proteins, IFNAR1 and IFNAR2, with different affinities. While the 13 known IFNalphas are highly conserved, the C-terminal unstructured tail was found to have large variation in its net charge, from neutral to +4. This led us to speculate that the tail may have a role in modulation of the IFN biological activity, through fine-tuning the binding to IFNAR2. To evaluate this hypothesis, we replaced the tail of IFNalpha2 with that of IFNalpha8 and IFNbeta tails, or deleted the last five residues of this segment. Mutations to the more positively charged tail of IFNalpha8 resulted in a 20-fold higher affinity to IFNAR2, which results in a higher antiviral and antiproliferative activity. Double and multiple mutant cycle analysis placed the tail near a negatively charged loop on IFNAR2, comprising of residues Glu 132-134. Deleting the tail resulted in only twofold reduction in binding compared to the wild-type. Next, we modeled the location of the tail using a two-step procedure: first we generated 200 models of the tail docked on IFNAR2 using HADDOCK, second the models were scored according to the fit between experimentally determined rates of association of nine mutant complexes, and their calculated rates using the PARE software. From the results we suggest that the unstructured tail of IFNalpha is gaining a specific structure in the bound state, binding to a groove below the 132-134 loop in IFNAR2.

Biochemical research in mood disorders has focused, along the cascade of events involved in signal transduction, from studies at the level of the monoamine neurotransmitter to the level of the neurotransmitter receptors, and lately to information transduction mechanisms beyond receptors, involving the coupling of receptors with signal transducers. We review findings concerning (a) the involvement of G proteins, in the pathophysiology, diagnosis and treatment of mood disorders; (b) the importance of regulation of receptor-G protein coupling, G protein-coupled receptor kinases (GRKs), beta-arrestins, to the pathophysiology of mood disorders and the mechanism of action of antidepressants. We relate to the special complexity of mental disorders with regards to etiology and pathophysiological diagnosis as well as to the strength and limitations of the 'pharmacological bridge' approach governing studies to unravel the etiology of mental disorders. There are presently no established and reliable, sensitive and specific objective biological diagnostic markers in psychiatry that can serve as 'gold standards'. The future achievement of an objective biochemical differential diagnostic system for major mental disorders that will also enable an objective biological treatment monitoring is expected to be revolutionary for psychiatry with a magnitude similar to the impact of the discovery of psychopharmacological treatments for mental disorders more than 50 years ago.

Type I interferons activate cellular responses by forming a ternary complex with two receptor components, IFNAR1 and IFNAR2. While the binding of the IFNAR2 receptor to interferon is of high affinity and well characterized, the binding to IFNAR1 is weak, transient, and poorly understood. Here, we mapped the complete binding region of IFNAR1 on IFNalpha2 by creating a panel of 21 single alanine mutant proteins, and determined their binding affinities. The IFNAR1 binding site on IFNalpha2 maps to the center of the B and C helices, opposite to the binding site for IFNAR2. No hot spots for binding were found in the interface, with individual mutations having an up to fivefold effect on binding. Of the nine residues that affected binding, three adjacent conserved residues, located on the B helix, conferred an increase in the binding affinity to IFNAR1, as well as an increase in the biological activity of the interferon mutant. This suggests that binding of alpha interferons to the IFNAR1 receptor is sub-optimal. A correlation between binding affinity and biological activity was found, albeit not across the whole range of affinities. In WISH cells, but not DAUDI cells, the anti-proliferative activity was markedly affected by fluctuations in the IFNalpha2 affinity towards the IFNAR1 receptor. On the other hand, the antiviral activity of interferons on WISH cells seems to change in accordance to the binding affinity towards IFNAR1 only as long as the binding affinity is not beyond twofold of the wild-type. In accordance, the biological roles of the two interferon-receptor subunits are discussed.

Is the whole protein surface available for interaction with other proteins, or are specific sites pre-assigned according to their biophysical and structural character? And if so, is it possible to predict the location of the binding site from the surface properties? These questions are answered quantitatively by probing the surfaces of proteins using spheres of radius of 10 A on a database (DB) of 57 unique, non-homologous proteins involved in heteromeric, transient protein-protein interactions for which the structures of both the unbound and bound states were determined. In structural terms, we found the binding site to have a preference for beta-sheets and for relatively long non-structured chains, but not for alpha-helices. Chemically, aromatic side-chains show a clear preference for binding sites. While the hydrophobic and polar content of the interface is similar to the rest of the surface, hydrophobic and polar residues tend to cluster in interfaces. In the crystal, the binding site has more bound water molecules surrounding it, and a lower B-factor already in the unbound protein. The same biophysical properties were found to hold for the unbound and bound DBs. All the significant interface properties were combined into ProMate, an interface prediction program. This was followed by an optimization step to choose the best combination of properties, as many of them are correlated. During optimization and prediction, the tested proteins were not used for data collection, to avoid over-fitting. The prediction algorithm is fully automated, and is used to predict the location of potential binding sites on unbound proteins with known structures. The algorithm is able to successfully predict the location of the interface for about 70% of the proteins. The success rate of the predictor was equal whether applied on the unbound DB or on the disjoint bound DB. A prediction is assumed correct if over half of the predicted continuous interface patch is indeed interface. The ability to predict the location of protein-protein interfaces has far reaching implications both towards our understanding of specificity and kinetics of binding, as well as in assisting in the analysis of the proteome.

Study Objective: To identify factors associated with a misclassification of the true disease stage by comparing the differences between the clinical and pathologic stage of patients with early-stage non-small cell lung cancer (NSCLC)., Design: A prospective cohort study., Setting: A multidisciplinary thoracic oncology clinic at a university-affiliated Veterans Affairs medical center., Patient Population: One hundred nine male veterans with clinical stage I/II NSCLC who had undergone thoracotomy with systematic lymph node dissection., Methods: Prospective data were collected on all patients between September 1997 and April 2002. Logistic regression analysis was used to establish the odds ratio (OR) for predictors of changes in stage., Results: A stage misclassification was found in 35.8% of patients (39 of 109 patients) after thoracotomy with lymph node dissection, and all but one patient were upstaged. Unsuspected nodal involvement (N stage) resulted in the upstaging of 16.5% of the patients, a change in tumor stage (T stage) resulted in the upstaging of 13.8% of the patients, a change in both stages resulted in the upstaging of 2.7% of patients, and the designation of metastatic disease resulted in the upstaging of 1.9% of the patients. The rate of unsuspected mediastinal lymph node involvement (pathologic stage N2) was 8.3% (9 of 109 patients), despite negative mediastinoscopy findings. Complete anatomic resection was performed in all patients. Advanced disease was found in 8.3% of the patients (9 of 109 patients) [stage IIIB or IV]. Having the primary tumor in a lower lobe location was the only statistically significant factor associated with upstaging (OR, 3.56; 95% confidence interval, 1.4 to 9.1). The effect of location was robust after controlling for tumor size and the prior performance of mediastinoscopy. Patient age, smoking history, weight loss, tumor size, and tumor histology were all found not to be associated with upstaging., Conclusion: A lower lobe tumor location in patients with early-stage NSCLC appears to be associated with upstaging after surgery. We conclude that a tumor location in a lower lobe deserves special attention.

Physiological media constitutes a crowded environment that serves as the field of action for protein-protein interaction in vivo. Measuring protein-protein interaction in crowded solutions can mimic this environment. In this work we follow the process of protein-protein association and its rate constants (k(on)) of the beta-lactamase (TEM)-beta-lactamase inhibitor protein (BLIP) complex in crowded solution using both low and high molecular mass crowding agents. In all crowded solutions (0-40% (w/w) of ethylene glycol (EG), poly(ethylene glycol) (PEG) 200, 1000, 3350, 8000 Da Ficoll-70 and Haemaccel the measured absolute k(on), but not k(off) values, were found to be slower as compared to buffer. However, there is a fundamental difference between low and high mass crowding agents. In the presence of low mass crowding agents and Haemaccel k(on) depends inversely on the solution viscosity. In high mass polymer solutions k(on) changes only slightly, even at viscosities 12-fold higher than water. The border between low and high molecular mass polymers is sharp and is dictated by the ratio between the polymer length (L) and its persistence length (Lp). Polymers that are long enough to form a flexible coil (L/Lp > 2) behave as high molecular mass polymers and those who are unable to do so (L/Lp < 2) behave as low molecular mass polymers. We concluded that although polymers solution are crowded, this property is not uniform; i.e. there are areas in the solution that contain bulk water, and in these areas proteins can diffuse and associate almost as if they were in diluted environment. This porous medium may be taken as mimicking some aspects of the cellular environment, where many of the macromolecules are organized along membranes and the cytoskeleton. To determine the contribution of electrostatic attraction between proteins in crowded milieu, we followed k(on) of wt-TEM and three BLIP analogs with up to 100-fold increased values of k(on) due to electrostatic steering. Faster associating BLIP variants keep their relative advantage in all crowded solutions, including Haemaccel. This result suggests that faster associating protein complexes keep their advantage also in complex environment.

Study Objectives: To provide an evidence-based background for developing the American College of Chest Physicians (ACCP) lung cancer guidelines, a systematic review of the literature was performed to identify published lung cancer guidelines and evaluate their quality., Design, Setting, and Participants: A systematic search was performed for relevant literature from MEDLINE, Cancerlit, CINAHL, HealthStar, the Cochrane Library, and the National Guidelines Clearinghouse published from January 1989 to July 2001., Measurement and Results: From 369 citations, 51 relevant guidelines were identified. Each guideline was evaluated by at least four reviewers using the Appraisal of Guidelines for Research and Evaluation (AGREE) instrument and was coded for clinical topics covered. The recommendations included in each guideline also were abstracted. Of the 51 guidelines evaluated, 27 (53%) were evidence-based. Clinical topics identified by the ACCP for their guideline effort each were represented by at least one existing guideline. Of the 880 clinical recommendations abstracted from the guidelines, only 253 (29%) were evidence-based. The AGREE instrument rates guidelines along six domains. As a group, the guidelines performed well in the scope and purpose domain, with only six guidelines (12%) scoring < 50%. For the remaining domains, however, the guidelines did not perform as well, as follows: for stakeholder involvement, 41 guidelines (80%) scored < 50%; for rigor of development, 29 guidelines (57%) scored < 50%; for clarity and presentation, 17 guidelines (33%) scored < 50%; for applicability, 46 guidelines (90%) scored < 50%; and for editorial independence, 47 guidelines (92%) scored < 50%. After considering the domain scores, the reviewers recommended only 19 of the guidelines (37%)., Conclusions: All major clinical lung cancer topics are covered by at least one guideline, but no single guideline addresses all areas. Furthermore, although existing guidelines may accurately reflect clinical practice, most performed poorly when evaluated for quality. Future guideline efforts that address each item of the AGREE instrument would add substantially to the literature.

Study Objectives: To determine the test performance characteristics of various modalities for the diagnosis of suspected lung cancer., Design, Setting, and Participants: A systematic search of MEDLINE, HealthStar, and Cochrane Library databases to July 2001 and print bibliographies was performed to identify studies comparing the results of sputum cytology, bronchoscopy, transthoracic needle aspirate (TTNA), or biopsy with histologic reference standard diagnoses among at least 50 patients with suspected lung cancer., Measurement and Results: For sputum cytology, the pooled specificity was 0.99 and the pooled sensitivity was 0.66, but sensitivity was higher for central lesions than for peripheral lesions (0.71 vs 0.49, respectively). Studies on bronchoscopic procedures provided data only on diagnostic yield (sensitivity). The diagnosis of endobronchial disease by bronchoscopy in 30 studies showed the highest sensitivity for endobronchial biopsy (0.74), followed by cytobrushing (0.59) and washing (0.48). The sensitivity for all modalities combined was 0.88. Thirty studies reported on peripheral lesions. Cytobrushing demonstrated the highest sensitivity (0.52), followed by transbronchial biopsy (0.46) and BAL/washing (0.43). The overall sensitivity for all modalities was 0.69. Peripheral lesions < 2 cm or > 2 cm in diameter showed sensitivities of 0.33 and 0.62, respectively. Updating a previous meta-analysis with 19 studies revealed a pooled sensitivity of 0.90 for TTNA. A trend toward lower sensitivity was noted for lesions that were < 2 cm in diameter. The accuracy in differentiating between small cell and non-small cell cytology for the various diagnostic modalities was 0.98, with individual studies ranging from 0.94 to 1.0. The average false-positive and false-negative rates were 0.09 and 0.02, respectively., Conclusions: The sensitivity of bronchoscopy is high for endobronchial disease and poor for peripheral lesions that are < 2 cm in diameter. The sensitivity of TTNA is excellent for malignant disease. The distinction between small cell lung cancer and non-small cell lung cancer by cytology appears to be accurate.

Association of a protein complex follows a two step reaction mechanism, with the first step being the formation of an encounter complex which evolves into the final complex. Here we present new experimental data for the association of the bacterial ribonuclease barnase and its polypeptide inhibitor barstar which shed light on the thermodynamics and structure of the transition state and preceding encounter complex of association at diminishing electrostatic attraction. We show that the activation entropy at the transition state is close to zero, with the activation enthalpy being equal to the free energy of binding. This observation was independent of the magnitude of the mutual electrostatic attraction, which were altered by mutagenesis or by addition of salt. The low activation entropy implies that the transition state is mostly solvated at all ionic strengths. The structure of the transition state was probed by measuring pairwise interaction energies using double-mutant-cycles. While at low ionic strength all proximal charge-pairs form contacts, at high salt only a subset of these interactions are maintained. More specifically, charge-charge interactions between partially buried residues are lost, while exposed charged residues maintain their ability to form specific interactions even at the highest salt concentration. Uncharged residues do not interact at any ionic strength. The results presented here suggest that the barnase-barstar binding sites are correctly aligned during the transition state even at diminishing electrostatic attraction, although specific short range interactions of uncharged residues are not yet formed. Furthermore, most of the interface desolvation (which contributes to the entropy of the system) has not yet occurred. This picture seems to be valid at low and high salt. However, at high salt, interactions of the activated complex are limited to a more restricted set of residues which are easier approached during diffusion, prior to final docking. This suggest that the steering region at high salt is more limited, albeit maintaining its specificity., (Copyright 2001 Academic Press.)

Purpose: To examine the validity of maturation self-assessments and to investigate the association between race and validity of self-assessments among young black and white girls., Methods: Self-assessments and examiner-assessments of areolar and pubic hair development using line drawings were compared at three visits among a cohort of 11- to 14-year-old girls enrolled in the National Heart, Lung, and Blood Institute's Growth and Health Study. Accuracy rates and kappa coefficients were calculated to measure the agreement between girls and examiners. Logistic regression models were used to assess the racial differences in the accuracy of self-assessments while adjusting for possible confounders., Results: Fair to moderate agreement was found between self- and examiner-assessments (areolar self-assessments: adjusted accuracy rates: 60.7-69.9%, kappa: 0.32-0.51; pubic hair self-assessments: adjusted accuracy rates: 57.9-70.7%, kappa: 0.36-0.55). While there were indications of racial differences in the ability to perform self-assessment with black girls tending to self-assess less accurately, most of the differences disappeared after adjusting for nurse-assessed stage., Conclusions: These findings suggest that self-assessment can substitute for examiner evaluations only when crude estimates of maturation are needed. However, when accurate maturation stage data are required, examiner-assessments are necessary. Because black girls are usually more pubertally advanced and tend to underestimate their development, the value of self-assessment is questionable for assessing populations with young black and white girls. Use of self-assessment might present biased estimates of maturation and confound research findings.

Investigating protein-protein interactions by mutational analysis requires practical techniques for quantifying rate constants and equilibrium constants over several orders of magnitude with reasonably high sample throughput. We have employed spectroscopic interferometry for label-free monitoring of the interaction between the cytokine interferon alpha2 (IFNalpha2) and the extracellular domain of its receptor ifnar2 (ifnar2-EC). We implemented a versatile surface chemistry for the glass substrate of this transducer for covalent immobilization of proteins. Affinity capturing with a monoclonal anti-ifnar2-EC antibody (mAb) followed by crosslinking with a second, noncompetitive mAb provided stable, but still reversible, immobilization of ifnar2-EC. We measured kinetics and affinity of numerous of mutants of IFNalpha2 and ifnar2-EC. Dissociation rate constants up to 0.3 s(-1) and association rate constants up to 3 x 10(6) M(-)1 s(-1) were resolved by the system. Dissociation constants down to 200 microM were measured with protein concentrations up to 50 microM without no background signal or nonspecific binding. The instrument detection limit is approximately 10 pm without the need for temperature stabilization or referencing channels. The system proved effective for large-scale mutational analysis involving alanine scanning mutagenesis and double mutant cycles., (Copyright 2001 Academic Press.)

An experimental approach to evaluate the net binding free energy of buried hydrogen bonds and salt bridges is presented. The approach, which involves a modified multiple-mutant cycle protocol, was applied to selected interactions between TEM-1-beta-lactamase and its protein inhibitor, BLIP. The selected interactions (two salt bridges and two hydrogen bonds) all involving BLIP-D49, define a distinct binding unit. The penta mutant, where all side-chains constructing the binding unit were mutated to Ala, was used as a reference state to which combinations of side-chains were introduced. At first, pairs of interacting residues were added allowing the determination of interaction energies in the absence of neighbors, using double mutant cycles. Addition of neighboring residues allowed the evaluation of their cooperative effects on the interaction. The two isolated salt bridges were either neutral or repulsive whereas the two hydrogen bonds contribute 0.3 kcal mol(-1 )each. Conversely, a double mutant cycle analysis of these interactions in their native environment showed that they all stabilize the complex by 1-1.5 kcal mol(-1). Examination of the effects of neighboring residues on each of the interactions revealed that the formation of a salt bridge triad, which involves two connected salt bridges, had a strong cooperative effect on stabilizing the complex independent of the presence or absence of additional neighbors. These results demonstrate the importance of forming net-works of buried salt bridges. We present theoretical electrostatic calculations which predict the observed mode of cooperativity, and suggest that the cooperative networking effect results from the favorable contribution of the protein to the interaction. Furthermore, a good correlation between calculated and experimentally determined interaction energies for the two salt bridges, and to a lesser extent for the two hydrogen bonds, is shown. The data analysis was performed on values of DeltaDeltaG(double dagger)K(d) which reflect the strength of short range interactions, while DeltaDeltaG(o)K(D) values which include the effects of long range electrostatic forces that alter specifically DeltaDeltaG(double dagger)k(a) were treated separately., (Copyright 2000 Academic Press.)

Type I interferons (IFN) exert pleiotropic activities through binding to two cell surface receptors, ifnar1 and ifnar2. We are investigating the biophysical basis of IFN signaling by characterizing the complex of the extra-cellular domain of ifnar2 (ifnar2-EC) with IFNs on the level of purified recombinant proteins in vitro. Here, we present a detailed mutational study on the functional epitopes on both IFN and ifnar2. Kinetic and thermodynamic parameters were determined by label-free heterogeneous phase detection. On IFNalpha2, a relatively small functional epitope comprising ten amino acid residues was localized, which is nearly entirely formed by residues on the AB loop. Two hot-spot residues, L30 and R33, account for two-thirds of the total interaction energy. Comparing the anti-viral potency of the various mutants to the binding affinity towards ifnar2 revealed a proportional correlation between the two, suggesting a rate-limiting role of ifnar2 binding in IFN signaling. On ifnar2, residues T46, I47 and M48 were identified as hot-spots in the interaction with IFNalpha2. For another ten residues on ifnar2, significant contribution of interaction energy was determined. Based on these data, the functional epitope on ifnar2 was defined according to a homology model based on other members of the class II hCR family in good agreement with the complementary functional epitope on IFNalpha2. Although IFNalpha2 and IFNbeta bind competitively to the same functional epitope, mutational analysis revealed distinct centers of binding for these IFNs on ifnar2. This small shift of the binding site may result in different angular orientation, which can be critically coupled to cytoplasmic signaling., (Copyright 1999 Academic Press.)

Type I interferons are cytokines which activate an anti-viral response by binding to two specific cell surface receptors, ifnar1 and ifnar2. Here, we report purification and refolding of the extracellular part of human ifnar2 (ifnar2-EC) expressed in Escherichia coli and its characterization with respect to its interaction with interferon alpha2 (IFNalpha2). The 25 kDa, non-glycosylated ifnar2-EC is a stable, fully active protein, which inhibits antiviral activity of IFNalpha2. The stoichiometry of binding IFNalpha2 is 1:1, as determined by gel filtration, chemical cross-linking and solid-phase detection. The affinity of this interaction is 10 nM, which is similar to the affinity measured for the cell surface-bound ifnar2 receptor. No difference in affinity was found throughout various assays using optical detection as BIAcore or reflectometric interference spectorscopy. However, the binding kinetics as measured in homogeneous phase by fluorescence de-quenching was about three times faster than that measured on a sensor surface. The rate of complex formation is relatively high compared to other cytokine-receptor interactions. The salt dependence of the association kinetics suggest a limited but significant contribution of electrostatic forces towards the rate of complex formation. The dissociation constant increases with decreasing pH according to the protonation of a base with a pKa of 6.7. The surface properties of the IFNalpha2 binding surface on ifnar2 were interpreted according to the pH and salt dependence of the interaction., (Copyright 1999 Academic Press.)

The rate of association of proteins is dictated by diffusion, but can be enhanced by favorable electrostatic forces. Here the relationship between the electrostatic energy of interaction, and the kinetics of protein-complex formation was analyzed for the protein pairs of: hirudin-thrombin, acetylcholinesterase-fasciculin and barnase-barstar, and for a panel of point mutants of these proteins. Electrostatic energies of interaction were calculated as the difference between the electrostatic energy of the complex and the sum of the energies of the two individual proteins, using the computer simulation package DelPhi. Calculated electrostatic energies of interaction were compared to experimentally determined rates of association. One kcal/mol of Coulombic interaction energy increased the rate of association by a factor of 2.8, independent of the protein-complex or mutant analyzed. Electrostatic energies of interaction were also determined from the salt dependence of the association rate constant, using the same basic equation as for the theoretical calculation. A Brönsted analysis of the electrostatic energies of interactions plotted versus experimentally determined ln(rate)s of association shows a linear relation between the two, with a beta value close to 1. This is interpreted as the energy of the transition state varies according to the electrostatic interaction energy, fitting a two state model for the association reaction. Calculating electrostatic rate enhancement from the electrostatic interaction energy can be used as a powerful tool to design protein complexes with altered rates of association and affinities., (Copyright 1999 Academic Press.)

Purpose: We examined changes in self-esteem and feelings of competence with physical appearance and social acceptance over approximately 5 years in 1166 white and 1213 black girls, aged 9 and 10 years at baseline., Methods: Maturation stage and body mass index (BMI) were assessed annually. Biennially girls completed Harter's Self-Perception Profile for children. Changes were analyzed in the context of race, sexual maturation, BMI, and household income. Longitudinal regression models were used to compare trends with age in global self-worth, physical appearance, and social acceptance., Results: Mean global self-worth showed little change over ages 9-14 years in blacks (p = 0.09) but decreased in whites (p < 0.001). Mean physical appearance scores for both races declined between ages 9 and 14 years (blacks, p < 0.001; whites, p < 0.001). Mean social acceptance scores increased for both races between ages 9 and 14 years (blacks, p < 0.001; whites, p < 0.001). For all three scores, these changes differed between blacks and whites (all three p values, < or = 0.002). Adjustment for maturation stage, BMI, and household income did not alter the significance or direction of racial differences in the changes with age in global self-worth and physical appearance scores. Self-worth, physical appearance, and social acceptance scores decreased with increasing BMI. Decreases in physical appearance and social acceptance scores with increasing BMI were smaller in blacks than in whites (p < 0.05). After adjustment for maturation stage and household income, racial differences in social acceptance scores depended on BMI (p < 0.05) but not on age (p = 0.008)., Conclusions: This article reports the first data on self-esteem scores by age for a large population of black girls aged 9 and 14 years and concludes that self-esteem does not follow the same developmental pattern in black as in white girls. A reason for black girls' higher and more stable self-worth and their greater satisfaction with their physical appearance compared to white girls may be racial differences in attitudes toward physical appearance and obesity.

The electrostatic enhancement of the association rate of barnase and barstar is calculated using a transition-state theory like expression and atomic-detail modeling of the protein molecules. This expression predicts that the rate enhancement is simply the average Boltzmann factor in the region of configurational space where association occurs instantaneously in the diffusion-controlled limit. Based on experimental evidence, this "transition state" is defined by configurations in which, relative to the stereospecifically bound complex, the two proteins are shifted apart by approximately 8 A (so a layer of water can be accommodated in the interface) and the two binding surfaces are rotated away by 0 degrees to 3 degrees. The values of the average Boltzmann factor, calculated by solving the Poisson-Boltzmann equation, for the wild-type complex and 16 complexes with single mutations are found to correlate well with experimental results for the electrostatic rate enhancement. The predicted rate enhancement is found to be somewhat insensitive to the precise definition of the transition state, due to the long-range nature of electrostatic interactions. The experimental ionic strength dependence of the rate enhancement is also reasonably reproduced., (Copyright 1998 Academic Press Limited.)

Purpose: Obesity is assumed to have a negative impact on self-esteem because of the associated social stigmatization in Western society. Studies of the psychological effect of obesity in children are inconclusive and limited, particularly pertaining to minority populations. Most studies have assessed global rather than domain-specific measures of self-esteem and hence, may have lacked specificity to detect impairment of certain aspects of self-esteem most closely associated with obesity. The purpose of this study is to examine the effect of adiposity and other environmental factors on measures of perceived competence and self-adequacy in 2205 black and white girls aged 9-10 years., Methods: Domain-specific measures of self-esteem were studied by race and degree of adiposity, using Harter's "Self-Perception Profile for Children". Three Harter scales deemed more relevant to obesity (social acceptance (SA), physical appearance (PA), and global self-worth (GSW)) were selected for univariate and multivariate linear regression models to examine relationships between self-esteem level and adiposity (measured by the sum of triceps, subscapular, and suprailiac skinfolds (SSF)), race, pubertal maturation, and parental education. The relationship between adiposity and Harter scores was further examined with LOESS curves and also by comparing the mean scores of each quintile of SSF by race, as well as inter-quintile differences within race., Results: Adiposity in general impacted negatively on the scores of all three selected Harter scales. There was also racial variation in the relationship between the scores and adiposity, with the magnitude of the effect somewhat less in black girls. White girls exhibited a significant inverse relationship between SSF and SA scores while, in striking contrast, there was no variation in scores in black girls across all ranges of adiposity. Although there was a significant inverse relationship between adiposity and PA and GSW in both groups, the slope was steeper in white girls, particularly at higher ranges of SSF. Non-linearity in the relationship between SSF and the scores was seen in SA and PA scales., Conclusions: The present study demonstrated a significant negative association between adiposity and the level of self-esteem in girls as young as 9 to 10 years. There were also intriguing racial differences in the selected domains of esteem. These results may help better understand cultural differences regarding the psychological impact of obesity and could be used to formulate appropriate strategies for public health policy.

The association of sociodemographic and family composition data with obesity was studied in 1213 black and 1166 white girls, ages 9 and 10, enrolled in the National Heart, Lung, and Blood Institute's Growth and Health Study. Obesity was defined as body mass index at or greater than age- and sex-specific 85th percentile as outlined in the Second National Health and Nutrition Examination Survey. The prevalence of obesity was higher for pubertal girls than for prepubertal girls and for girls with older mothers/female guardians. As odds ratio of 1.14 was observed for each 5-year increase in maternal age. Obesity was less common for girls with more siblings; the odds for obesity decreased by 14% for each additional sibling in the household. In blacks, the prevalence of obesity was not related to parental employment or to parental education. In whites, the odds of obesity were higher for girls with no employed parent/guardian in the household and for girls with parents or guardians with lower levels of educational attainment. Examining the associations between sociodemographic factors and risk of childhood obesity provides important clues for understanding racial differences in obesity, a major risk factor for coronary heart disease.

Barnase, a small extracellular ribonuclease from Bacillus amyloliquefaciens and its intracellular inhibitor barstar have co-evolved to bind tightly and rapidly. Barnase has also evolved to be catalytically active. The active site of barnase and its binding site for barstar use the same subset of amino acids. The exception is Glu73 (the general base in catalysis), which although located at the centre of the binding site, is separated by three ordered water molecules from barstar. We examined in this work the contribution of Glu73 to both catalysis and barstar binding. Truncation mutants of the general base (Glu73 --> Ala or Ser) retain a residual RNase activity of about 0.3% while mutants with larger hydrophobic replacements (Glu 73 --> Trp or Phe) have virtually no catalytic activity. This, and binding data of 3'-GMP with the different barnase mutants suggest that the loss in activity results from the elimination of the general base, which can be substituted to some extent by water or other polar side-chains in truncation mutants. All of the Glu73 mutations lead to a weakening of the free energy of complex formation with barstar by 1.4 to 3.0 kcal/mol (including Gln). This is surprising, since Glu73 does not interact directly with barstar and there is an electrostatic repulsion between Glu73 on barnase and the negatively charged binding surface of barstar. A newly developed method of constructing double mutant cycles between multiple mutations at the same site appears to pinpoint a favourable interaction between Glu73 and one of its nearest neighbours in barstar, Asp39. The coupling energy between those residues is presumably indirect: the carboxylate of Glu73 organizes neighbouring positively charged groups in barnase, Lys27, Arg83, and Arg87 to interact with Asp39 in barstar. This emphasizes that an apparent interaction between a pair of residues as measured with double mutant cycles is the sum of their direct and indirect interactions.

G28-5 sFv-PE40 is a single-chain immunotoxin targeted to CD40, which is highly expressed on human hematologic malignancies, including non-Hodgkin's lymphoma, B-lineage leukemias, multiple myeloma, and Hodgkin's disease, as well as certain carcinomas. In vitro analysis showed that this monovalent immunotoxin had a binding affinity of 3 nmol/L, within 15-fold of the bivalent parental monoclonal antibody. G28-5 sFv-PE40 was stable when incubated in mouse serum at 37 degrees C for 6 hours and cleared from the circulation of mice with a half-life of 16.7 minutes. This immunotoxin was effective in treating human Burkitt's lymphoma xenografted SCID mice with complete responses, defined by an asymptomatic phenotype for greater than 120 days, obtained at doses of 0.13 to 0.26 mg/kg. The efficacy of treatment was dependent on the schedule used, with every three days for five injections being the most effective tested. The toxicity of G28-5 sFv-PE40 was examined in SCID mice, rats, and monkeys, with the maximum tolerated dose being 0.48, 1.0, and 1.67 mg/kg, respectively. Comparative immunohistology showed that the G28-5 specificity was qualitatively similar between human and monkey tissue. In summary, G28-5 sFv-PE40 was effective at inducing complete antitumor responses in lymphoma xenografted mice at doses that were well tolerated in mice, rats, and monkeys.

Escherichia coli ribonuclease P (RNase P), a ribonucleoprotein complex which primarily functions in tRNA biosynthesis, is composed of a catalytic RNA subunit, M1 RNA, and a protein cofactor, C5 protein. The fluorescence emission spectrum of the single tryptophan residue-containing C5 protein exhibits maxima at 318 nm and 332 nm. Based on a comparison of the emission spectra of wild-type C5 protein and some of its mutant derivatives, we have determined that the 318 nm maximum could be the result of a complex formed in the excited state as a result of hydrophobic interactions between Trp109, Phe18 and Phe73. The analogous tryptophan fluorescence emission spectra of wild-type C5 protein and the barstar mutant W38F/W44F, taken together with the detailed structural information available for barstar, provide a possible explanation for the unusual emission spectrum of C5 protein.

We have studied the thermodynamics of the interaction between the ribonuclease barnase and its natural polypeptide inhibitor barstar. The contribution of specific residues and interactions within the barnase-barstar interface to the enthalpy of binding has been examined using isothermal titration calorimetry and protein engineering. The enthalpy of association of the wild-type proteins is -18.9 (+/-0.1) kcal/mol at pH 8 and at 25 degrees C. The enthalpy of binding remains favourable for 31 different combinations of mutations in the interface. The effects on the binding enthalpy upon replacing a side-chain involved in the interaction of barnase and barstar are, however, always unfavourable and in most cases larger than the effects on the free energy of binding. Interaction enthalpies calculated by double mutant cycle analysis are in some cases much larger than the interaction free energies. The interaction enthalpies for complexes between different barnase mutants with amino acid substitutions of the general base residue glutamic acid 73 and a barstar variant (D39A) vary by as much as 8.3 kcal/mol while the coupling free energies differ only by 1 kcal/mol. The use of enthalpies for the analysis of structure-activity relationships appears to be complicated by enthalpy-entropy compensation of weak intermolecular interactions. These tend to cancel out in measurements of free energy, which is thus the preferred quantity for simple analysis of interactions.

Purpose: To determine whether there are racial differences in the frequency with which black and white girls engaged in eating practices commonly targeted for modification in weight reduction programs., Methods: This is part of the NHLBI Growth and Health Study, a longitudinal study of preadolescent girls designed to examine the factors associated with development of obesity, and its later effects on cardiovascular risk factors. Black and white girls ages 9-10 years at entry (n = 2,379) were recruited at three clinical sites. Racial differences were examined in 11 "weight-related" eating practices such as eating with TV, eating while doing homework, and skipping meals. Multiple logistic regression analyses were then conducted for each of the dependent variables., Results: Black girls were more than twice as likely as white girls to frequently engage in the targeted weight-related eating practices. The odds of a study girl frequently engaging in most of these eating practices decreased with an increase in parents' income and education level. However, even when controlling for socioeconomic and demographic effects, black girls remained more likely to engage in these eating practices than white girls. For most of the behaviors, girls who frequently practiced a behavior had higher energy intakes compared to those who practiced it infrequently., Conclusions: The finding that black girls at an early age more frequently engage in eating practices associated with weight gain may have significant implications for obesity development. For both young black and white girls, early education efforts may be necessary in helping develop good eating habits. Since it appears that black girls have a higher risk of developing adverse weight-related eating practices, culturally appropriate education materials may be required.

The purpose of this investigation was to determine whether measures of socioeconomic status (SES) are inversely associated with obesity in 9- to 10-year-old black and white girls and their parents. Subjects were participants in the Growth and Health Study (NGHS) of the National Heart, Lung, and Blood Institute. Extensive SES, anthropometric, and dietary data were collected at baseline on 2379 NGHS participants. The prevalence of obesity was examined in the NGHS girls and parents in relation to SES and selected environmental factors. Less obesity was observed at higher levels of household income and parental education in white girls but not in black girls. Among the mothers of the NGHS participants who were seen, lower prevalence of obesity was observed with higher levels of income and education for white mothers, but no consistent patterns were seen in black mothers. Univariate logistic models indicated that the prevalence of obesity was significantly and inversely associated with parental income and education and number of parents in the household in white girls whereas caloric intake and TV viewing were significantly and positively associated with obesity. Among black girls, only TV viewing was significantly and positively associated with the prevalence of obesity. Multivariate logistic regression models revealed that lower parental educational attainment, one-parent household, and increased caloric intake were significantly associated with the prevalence of obesity in white girls; for black girls, only increased hours of TV viewing were significant in these models. It is concluded that socioeconomic status, as measured by education and income, was related to the prevalence of obesity in girls, with racial variation in these associations. A lower prevalence of obesity was seen at higher levels of socioeconomic status in white girls, whereas no clear relationship was detected in black girls. These findings raise new questions regarding the correlates of obesity in black girls.