bluuebunny/crossref_metadata_2025 huggingface dataset:
Coexpression of CD25 and CD27 identifies FoxP3+ regulatory T cells in inflamed synovia
Authors: Ruprecht Claudia R. , Gattorno Marco , Ferlito Francesca , Gregorio Andrea , Martini Alberto , Lanzavecchia Antonio , Sallusto Federica
Published: June 2005

A better understanding of the role of CD4+CD25+ regulatory T cells in disease pathogenesis should follow from the discovery of reliable markers capable of discriminating regulatory from activated T cells. We report that the CD4+CD25+ population in synovial fluid of juvenile idiopathic arthritis (JIA) patients comprises both regulatory and effector T cells that can be distinguished by expression of CD27. CD4+CD25+CD27+ cells expressed high amounts of FoxP3 (43% of them being FoxP3+), did not produce interleukin (IL)-2, interferon-γ, or tumor necrosis factor, and suppressed T cell proliferation in vitro, being, on a per cell basis, fourfold more potent than the corresponding peripheral blood population. In contrast, CD4+CD25+CD27− cells expressed low amounts of FoxP3, produced effector cytokines and did not suppress T cell proliferation. After in vitro activation and expansion, regulatory but not conventional T cells maintained high expression of CD27. IL-7 and IL-15 were found to be present in synovial fluid of JIA patients and, when added in vitro, abrogated the suppressive activity of regulatory T cells. Together, these results demonstrate that, when used in conjunction with CD25, CD27 is a useful marker to distinguish regulatory from effector T cells in inflamed tissues and suggest that at these sites IL-7 and IL-15 may interfere with regulatory T cell function.
Cellular immune selection with hepatitis C virus persistence in humans
Authors: Cox Andrea L. , Mosbruger Timothy , Mao Qing , Liu Zhi , Wang Xiao-Hong , Yang Hung-Chih , Sidney John , Sette Alessandro , Pardoll Drew , Thomas David L. , Ray Stuart C.
Published: June 2005

Hepatitis C virus (HCV) infection frequently persists despite substantial virus-specific cellular immune responses. To determine if immunologically driven sequence variation occurs with HCV persistence, we coordinately analyzed sequence evolution and CD8+ T cell responses to epitopes covering the entire HCV polyprotein in subjects who were followed prospectively from before infection to beyond the first year. There were no substitutions in T cell epitopes for a year after infection in a subject who cleared viremia. In contrast, in subjects with persistent viremia and detectable T cell responses, we observed substitutions in 69% of T cell epitopes, and every subject had a substitution in at least one epitope. In addition, amino acid substitutions occurred 13-fold more often within than outside T cell epitopes (P < 0.001, range 5–38). T lymphocyte recognition of 8 of 10 mutant peptides was markedly reduced compared with the initial sequence, indicating viral escape. Of 16 nonenvelope substitutions that occurred outside of known T cell epitopes, 8 represented conversion to consensus (P = 0.015). These findings reveal two distinct mechanisms of sequence evolution involved in HCV persistence: viral escape from CD8+ T cell responses and optimization of replicative capacity.
Divergent and convergent evolution after a common-source outbreak of hepatitis C virus
Authors: Ray Stuart C. , Fanning Liam , Wang Xiao-Hong , Netski Dale M. , Kenny-Walsh Elizabeth , Thomas David L.
Published: June 2005

The genomic sequences of viruses that are highly mutable and cause chronic infection tend to diverge over time. We report that these changes represent both immune-driven selection and, in the absence of immune pressure, reversion toward an ancestral consensus. Sequence changes in hepatitis C virus (HCV) structural and nonstructural genes were studied in a cohort of women accidentally infected with HCV in a rare common-source outbreak. We compared sequences present in serum obtained 18–22 yr after infection to sequences present in the shared inoculum and found that HCV evolved along a distinct path in each woman. Amino acid substitutions in known epitopes were directed away from consensus in persons having the HLA allele associated with that epitope (immune selection), and toward consensus in those lacking the allele (reversion). These data suggest that vaccines for genetically diverse viruses may be more effective if they represent consensus sequence, rather than a human isolate.
Mycobacterium tuberculosisand the host response
Authors: Kaufmann Stefan H.E. , Cole Stewart T. , Mizrahi Valerie , Rubin Eric , Nathan Carl
Published: June 2005

Mycobacterium tuberculosis remains a leading cause of morbidity and mortality worldwide. Advances reported at a recent international meeting highlight insights and controversies in the genetics of M. tuberculosis and the infected host, the nature of protective immune responses, adaptation of the bacillus to host-imposed stresses, animal models, and new techniques.
Modelling ground vibration from railways using wavenumber finite- and boundary-element methods
Authors: Sheng X , Jones C.J.C , Thompson D.J
Published: June 2005

A mathematical model is presented for ground vibration induced by trains, which uses wavenumber finite- and boundary-element methods. The track, tunnel and ground are assumed homogeneous and infinitely long in the track direction (
x
-direction). The models are formulated in terms of the wavenumber in the
x
-direction and discretization in the
yz
-plane. The effect of load motion in the
x
-direction is included. Compared with a conventional, three-dimensional finite- or boundary-element model, this is computationally faster and requires far less memory, even though calculations must be performed for a series of discrete wavenumbers. Thus it becomes practicable to carry out investigative study of train-induced ground vibration. The boundary-element implementation uses a variable transformation to solve the well-known problem of strongly singular integrals in the formulation. A ‘boundary truncation element’ greatly improves accuracy where the infinite surface of the ground is truncated in the boundary-element discretization. Predictions of vibration response on the ground surface due to a unit force applied at the track are performed for two railway tunnels. The results show a substantial difference in the environmental vibration that could be expected from the alternative designs. The effect of a moving load is demonstrated in a surface vibration example in which vibration propagates from an embankment into layered ground.
The optical singularities of bianisotropic crystals
Authors: Berry M.V
Published: June 2005

The electric and magnetic polarization states for plane waves in arbitrary linear crystals, in which each ofDandBis coupled to both ofEandH, can be characterized by their typical singularities in direction space: degeneracies, where two refractive index eigenvalues coincide;CeandCmpoints, where the electric or magnetic field is circularly polarized; andLeandLmlines, where either field is linearly polarized. The well-known 4×4 matrix formalism, expressed in terms of the stereographic projection of directions, enables extensive numerical and visual exploration of the singularities in the general case (which involves 65 crystal parameters), incorporating bianisotropy, natural and Faraday optical activity, and absorption, as well as special cases where one or more effect is absent. For crystals whose anisotropy is weak but which are otherwise general, an unusual perturbation theory leads to a powerful 2×2 formalism capturing all the essential singularity phenomena, including the principal feature of the general case, namely the separation between the electric and magnetic singularities.
Histological Value of Duodenal Biopsies
Authors: Gupta Limci , B. Hamid
Published: June 2005

This study was performed to see the value of histopathological diagnosis in management of patients with duodenal biopsies; to look for correlation of histology and serology in suspected cases of coeliac disease; the reasons for taking duodenal biopsies and whether proper adequate histories are provided on the forms sent with request for histopathological view on duodenal biopsies. Here are the observations of the study followed by the discussion.
The Life Mission Theory VII. Theory of Existential (Antonovsky) Coherence: A Theory of Quality of Life, Health, and Ability for Use in Holistic Medicine
Authors: Ventegodt Søren , Flensborg-Madsen Trine , Andersen Niels Jørgen , Merrick Joav
Published: June 2005

A theoretical framework of existential coherence is presented, explaining how health, quality of life (QOL), and the ability to function were originally created and developed to rehabilitate human life from an existential perspective. The theory is inspired by the work of Aaron Antonovsky and explains our surprising recent empirical findings—that QOL, health, and ability primarily are determined by our consciousness. The theory is a matrix of nine key elements in five layers: (1) coherence; (2) purpose and talent; (3) consciousness, love, and physicality/sexuality; (4) light and joy; and (5) QOL/meaning of life. The layer above causes the layer below, with the layer of QOL again feeding the fundamental layer of coherence. The model holds the person responsible for his or her own degree of reality, happiness, and being present. The model implies that when a person takes responsibility in all nine “dimensions” of life, he or she can improve and develop health, the ability to function, all aspects of QOL, and the meaning of life. The theory of existential coherence integrates a wide range of QOL theories from Jung and Maslow to Frankl and Wilber. It is a nine-ray theory in accordance with Gurjieff's enneagram and the old Indian chakra system. It can be used in the holistic medical clinic and in existential coaching. Love is in the center of the model and rehabilitation of love in its broadest sense is, accordingly, the essence of holistic medicine. To know yourself, your purpose of life (life mission) and talents, and taking these into full use and becoming coherent with life inside and reality outside is what human life is essentially about. The new model has been developed to integrate the existing knowledge in the complex field of holistic medicine. Its strength is that it empowers the holistic physician to treat the patient with even severe diseases and can also be used for existential rehabilitation, holistic psychiatry, and sexology. Its major weakness is that it turns holistic medicine more into an art than into a science because the physician must master intent, which is a poorly understood dimension of existence.
Factors Affecting Placement of a Child with Intellectual Disability
Authors: Kandel Isack , Merrick Joav
Published: June 2005

Parents of disabled children often face the question whether or not to keep the child at home or to place them. The choice between the two alternatives resides with the parents and various factors influence their decision. Several researchers have identified these factors, which include child-related parameters, family and parental attitudes, the influence of the social environment, and the external assistance provided to the family. In a pilot study, we attempted to isolate the main factors involved in the parental decision either to keep the child at home or place the child by examining a sample comprised of 50 parents of children suffering severe intellectual disability studying in a special education school and 48 parents of adults with intellectual disability working in sheltered workshops. Each parent filled out a questionnaire used in a study in the United States and results of the research indicated parental-related factors as the dominant factors that delayed the placement of their child in residential care; guilt feelings were the main factor.
Interaction Between Phosphorus and Zinc on the Biomass Yield and Yield Attributes of the Medicinal Plant Stevia (Stevia rebaudiana)
Authors: Das Kuntal , Dang Raman , Shivananda T. N. , Sur Pintu
Published: June 2005

A greenhouse experiment was conducted at the Indian Institute of Horticultural Research (IIHR), Bangalore to study the interaction effect between phosphorus (P) and zinc (Zn) on the yield and yield attributes of the medicinal plant stevia. The results show that the yield and yield attributes have been found to be significantly affected by different treatments. The total yield in terms of biomass production has been increased significantly with the application of Zn and P in different combinations and methods, being highest (23.34 g fresh biomass) in the treatment where Zn was applied as both soil (10 kg ZnSO4/ha) and foliar spray (0.2% ZnSO4). The results also envisaged that the different yield attributes viz. height, total number of branches, and number of leaves per plant have been found to be varied with treatments, being highest in the treatment where Zn was applied as both soil and foliar spray without the application of P. The results further indicated that the yield and yield attributes of stevia have been found to be decreased in the treatment where Zn was applied as both soil and foliar spray along with P suggesting an antagonistic effect between Zn and P.
Clinical Holistic Medicine: Holistic Treatment of Mental Disorders
Authors: Ventegodt Søren , Andersen Niels Jørgen , Neikrug Shimshon , Kandel Isack , Merrick Joav
Published: June 2005

We believe that holistic medicine can be used for patient's with mental health disorders. With holistic psychiatry, it is possible to help the mentally ill patient to heal existentially. As in holistic medicine, the methods are love or intense care, winning the trust of the patient, getting permission to give support and holding, and daring to be fully at the patient's service. Our clinical experiences have led us to believe that mental health patient's can heal if only you can make him or her feel the existential pain at its full depth, understand what the message of the suffering is, and let go of all the negative attitudes and beliefs connected with the disease. Many mentally ill young people would benefit from a few hours of existential holistic processing in order to confront the core existential pains. To help the mentally ill patient, you must understand the level of responsibility and help process the old traumas that made the patient escape responsibility for his or her own life and destiny. To guide the work, we have developed a responsibility scale going from (1) free perception over (2) emotional pain to (3) psychic death (denial of life purpose) further down to (4) escape and (5) denial to (6) destruction of own perception and (7) hallucination further down to (8) coma, suicide, and unconsciousness. This scale seems to be a valuable tool to understand the state of consciousness and the nature of the process of healing that the patient must go through.
Sublingual Buprenorphine and Methadone Maintenance Treatment: A Three-Year Follow-Up of Quality of Life Assessment
Authors: Giacomuzzi Salvatore M. , Ertl Markus , Kemmler Georg , Riemer Yyvonne , Vigl Alexander
Published: June 2005

This study was conducted to compare long-term outcome effects on the quality of life (QOL) of oral methadone with sublingual buprenorphine maintenance treatment. The QOL status of opioid-dependent patients was assessed using the German version (“Berlin Quality of Life Profile”) of the Lancashire Quality of Life Profile. Physical symptoms were measured using the Opiate Withdrawal Scale (OWS). Urine tests were carried out randomly to detect additional consumption. In the first study period, 53 opioid-dependent subjects were enrolled and 25 could be reached after 3 years. The retention rate was 50% for methadone and 45% for buprenorphine (p= 0.786). Baseline values of the total sample (completers and noncompleters) QOL and somatic complaints did not show significant differences between the two treatment groups. QOL characteristics at 6 months of treatment of the buprenorphine completer and noncompleter groups differed significantly regarding job (p= 0.013), family, and total score of physical symptoms (p= 0.002), in which the completer group showed the more favorable values. Concerning physical symptoms at 36 months, logistic regression revealed significantly less stomach cramps (p= 0.037) and fatigue and tiredness (p= 0.034) in buprenorphine compared to the methadone. Moreover, the buprenorphine-maintained group showed significantly less additional consumption of benzodiazepines (p= 0.015) compared with methadone participants. It is concluded that opioid addicts improved their QOL and health status when treated with methadone or buprenorphine. In summary, regarding QOL and health status, the present data indicate that buprenorphine is also a useful long-term alternative for maintenance treatment of opioid-dependent patients.
Pharmacological Treatment of Major Depressive Disorder in Adolescents
Authors: Farley Rachel L.
Published: June 2005

Major depressive disorder (MDD) affects a significant number of adolescents today. Its consequences (including social isolation, failure to achieve crucial developmental milestones, and suicide) mandate close attention in clinical practice. While tricyclics and monoamine oxidase inhibitors (MAOIs) have been used infrequently and with questionable efficacy, selective serotonin reuptake inhibitors (SSRIs), particularly fluoxetine, consistently have been shown to be of benefit in treating outpatient adolescents with MDD. Despite some success with other drugs in its class, fluoxetine remains the only SSRI that is FDA approved for treatment of children and adolescents with depression. A review of recent studies is presented, including the controversy regarding the relationship of antidepressants and suicidal behavior in this patient population.
Application of a Homogenous Assay for the Detection of 2,4,6-Trinitrotoluene to Environmental Water Samples
Authors: Goldman Ellen R. , Egge Adrienne L. , Medintz Igor L. , Lassman Michael E. , Anderson George P.
Published: June 2005

A homogeneous assay was used to detect 2,4,6-trinitrotoluene (TNT) spiked into environmental water samples. This assay is based on changes in fluorescence emission intensity when TNT competitively displaces a fluorescently labeled, TNT analog bound to an anti-TNT antibody. The effectiveness of the assay was highly dependent on the source of the sample being tested. As no correlation between pH and assay performance was observed, ionic strength was assumed to be the reason for variation in assay results. Addition of 10x phosphate-buffered saline to samples to increase their ionic strength to that of our standard laboratory buffer (about 0.17M) significantly improved the range over which the assay functioned in several river water samples.
Systemic Acquired Tolerance to Virulent Bacterial Pathogens in Tomato
Authors: Block Anna , Schmelz Eric , O'Donnell Phillip J. , Jones Jeffrey B. , Klee Harry J.
Published: June 2005

Recent studies on the interactions between plants and pathogenic microorganisms indicate that the processes of disease symptom development and pathogen growth can be uncoupled. Thus, in many instances, the symptoms associated with disease represent an active host response to the presence of a pathogen. These host responses are frequently mediated by phytohormones. For example, ethylene and salicylic acid (SA) mediate symptom development but do not influence bacterial growth in the interaction between tomato (Lycopersicon esculentum) and virulent Xanthomonas campestris pv vesicatoria (Xcv). It is not apparent why extensive tissue death is integral to a defense response if it does not have the effect of limiting pathogen proliferation. One possible function for this hormone-mediated response is to induce a systemic defense response. We therefore assessed the systemic responses of tomato to Xcv. SA- and ethylene-deficient transgenic lines were used to investigate the roles of these phytohormones in systemic signaling. Virulent and avirulent Xcv did induce a systemic response as evidenced by expression of defense-associated pathogenesis-related genes in an ethylene- and SA-dependent manner. This systemic response reduced cell death but not bacterial growth during subsequent challenge with virulent Xcv. This systemic acquired tolerance (SAT) consists of reduced tissue damage in response to secondary challenge with a virulent pathogen with no effect upon pathogen growth. SAT was associated with a rapid ethylene and pathogenesis-related gene induction upon challenge. SAT was also induced by infection with Pseudomonas syringae pv tomato. These data show that SAT resembles systemic acquired resistance without inhibition of pathogen growth.
CINNAMYL ALCOHOL DEHYDROGENASE-C and -D Are the Primary Genes Involved in Lignin Biosynthesis in the Floral Stem of Arabidopsis
Authors: Sibout Richard , Eudes Aymerick , Mouille Gregory , Pollet Brigitte , Lapierre Catherine , Jouanin Lise , Séguin Armand
Published: June 2005

During lignin biosynthesis in angiosperms, coniferyl and sinapyl aldehydes are believed to be converted into their corresponding alcohols by cinnamyl alcohol dehydrogenase (CAD) and by sinapyl alcohol dehydrogenase (SAD), respectively. This work clearly shows that CAD-C and CAD-D act as the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis thaliana by supplying both coniferyl and sinapyl alcohols. An Arabidopsis CAD double mutant (cad-c cad-d) resulted in a phenotype with a limp floral stem at maturity as well as modifications in the pattern of lignin staining. Lignin content of the mutant stem was reduced by 40%, with a 94% reduction, relative to the wild type, in conventional β-O-4–linked guaiacyl and syringyl units and incorportion of coniferyl and sinapyl aldehydes. Fourier transform infrared spectroscopy demonstrated that both xylem vessels and fibers were affected. GeneChip data and real-time PCR analysis revealed that transcription of CAD homologs and other genes mainly involved in cell wall integrity were also altered in the double mutant. In addition, molecular complementation of the double mutant by tissue-specific expression of CAD derived from various species suggests different abilities of these genes/proteins to produce syringyl-lignin moieties but does not indicate a requirement for any specific SAD gene.
The Structure of a Cyanobacterial Sucrose-Phosphatase Reveals the Sugar Tongs That Release Free Sucrose in the Cell
Authors: Fieulaine Sonia , Lunn John E. , Borel Franck , Ferrer Jean-Luc
Published: June 2005

Sucrose-phosphatase (SPP) catalyzes the final step in the pathway of sucrose biosynthesis in both plants and cyanobacteria, and the SPPs from these two groups of organisms are closely related. We have crystallized the enzyme from the cyanobacterium Synechocystis sp PCC 6803 and determined its crystal structure alone and in complex with various ligands. The protein consists of a core domain containing the catalytic site and a smaller cap domain that contains a glucose binding site. Two flexible hinge loops link the two domains, forming a structure that resembles a pair of sugar tongs. The glucose binding site plays a major role in determining the enzyme's remarkable substrate specificity and is also important for its inhibition by sucrose and glucose. It is proposed that the catalytic reaction is initiated by nucleophilic attack on the substrate by Asp9 and involves formation of a covalent phospho-Asp9-enzyme intermediate. From modeling based on the SPP structure, we predict that the noncatalytic SPP-like domain of the Synechocystis sucrose-phosphate synthase could bind sucrose-6F-phosphate and propose that this domain might be involved in metabolite channeling between the last two enzymes in the pathway of sucrose synthesis.
Transcriptional Profile of the Arabidopsis Root Quiescent Center
Authors: Nawy Tal , Lee Ji-Young , Colinas Juliette , Wang Jean Y. , Thongrod Sumena C. , Malamy Jocelyn E. , Birnbaum Kenneth , Benfey Philip N.
Published: June 2005

The self-renewal characteristics of stem cells render them vital engines of development. To better understand the molecular mechanisms that determine the properties of stem cells, transcript profiling was conducted on quiescent center (QC) cells from the Arabidopsis thaliana root meristem. The AGAMOUS-LIKE 42 (AGL42) gene, which encodes a MADS box transcription factor whose expression is enriched in the QC, was used to mark these cells. RNA was isolated from sorted cells, labeled, and hybridized to Affymetrix microarrays. Comparisons with digital in situ expression profiles of surrounding tissues identified a set of genes enriched in the QC. Promoter regions from a subset of transcription factors identified as enriched in the QC conferred expression in the QC. These studies demonstrated that it is possible to successfully isolate and profile a rare cell type in the plant. Mutations in all enriched transcription factor genes including AGL42 exhibited no detectable root phenotype, raising the possibility of a high degree of functional redundancy in the QC.
Lipid Transfer Proteins Enhance Cell Wall Extension in Tobacco
Authors: Nieuwland Jeroen , Feron Richard , Huisman Bastiaan A.H. , Fasolino Annalisa , Hilbers Cornelis W. , Derksen Jan , Mariani Celestina
Published: June 2005

Plant cells are enclosed by a rigid cell wall that counteracts the internal osmotic pressure of the vacuole and limits the rate and direction of cell enlargement. When developmental or physiological cues induce cell extension, plant cells increase wall plasticity by a process called loosening. It was demonstrated previously that a class of proteins known as expansins are mediators of wall loosening. Here, we report a type of cell wall–loosening protein that does not share any homology with expansins but is a member of the lipid transfer proteins (LTPs). LTPs are known to bind a large range of lipid molecules to their hydrophobic cavity, and we show here that this cavity is essential for the cell wall–loosening activity of LTP. Furthermore, we show that LTP-enhanced wall extension can be described by a logarithmic time function. We hypothesize that LTP associates with hydrophobic wall compounds, causing nonhydrolytic disruption of the cell wall and subsequently facilitating wall extension.
Functional Hammerhead Ribozymes Naturally Encoded in the Genome of Arabidopsis thaliana
Authors: Przybilski Rita , Gräf Stefan , Lescoute Aurélie , Nellen Wolfgang , Westhof Eric , Steger Gerhard , Hammann Christian
Published: June 2005

The hammerhead ribozyme (HHRz) is an autocatalytic RNA motif found in subviral plant pathogens and transcripts of repetitive DNA sequences in animals. Here, we report the discovery and characterization of unique HHRzs encoded in a plant genome. Two novel sequences were identified on chromosome IV of Arabidopsis thaliana in a database search, which took into account recently defined structural requirements. The HHRzs are expressed in several tissues and coexist in vivo as both cleaved and noncleaved species. In vitro, both sequences cleave efficiently at physiological Mg2+ concentrations, indicative of functional loop–loop interactions. Kinetic analysis of loop nucleotide variants was used to determine a three-dimensional model of these tertiary interactions. Based on these results, on the lack of infectivity of hammerhead-carrying viroids in Arabidopsis, and on extensive sequence comparisons, we propose that the ribozyme sequences did not invade this plant by horizontal transfer but have evolved independently to perform a specific, yet unidentified, biological function.
Visualization of Diffuse Centromeres with Centromere-Specific Histone H3 in the Holocentric PlantLuzula nivea
Authors: Nagaki Kiyotaka , Kashihara Kazunari , Murata Minoru
Published: June 2005

Although holocentric species are scattered throughout the plant and animal kingdoms, only holocentric chromosomes of the nematode worm Caenorhabditis elegans have been analyzed with centromeric protein markers. In an effort to determine the holocentric structure in plants, we investigated the snowy woodrush Luzula nivea. From the young roots, a cDNA encoding a putative centromere-specific histone H3 (LnCENH3) was successfully isolated based on sequence similarity among plant CENH3s. The deduced amino acid sequence was then used to raise an anti-LnCENH3 antibody. Immunostaining clearly revealed the diffuse centromere-like structure that appears in the linear shape at prophase to telophase. Furthermore, it was shown that the amount of LnCENH3 decreased significantly at interphase. The polar side positioning on each chromatid at metaphase to anaphase also confirmed that LnCENH3 represents one of the centromere-specific proteins in L. nivea. These data from L. nivea are compared with those from C. elegans, and common features of holocentric chromosomes are discussed.
Assessing strategies for improved superfamily recognition
Authors: Sillitoe Ian , Dibley Mark , Bray James , Addou Sarah , Orengo Christine
Published: June 2005

There are more than 200 completed genomes and over 1 million nonredundant sequences in public repositories. Although the structural data are more sparse (∼13,000 nonredundant structures solved to date), several powerful sequence‐based methodologies now allow these structures to be mapped onto related regions in a significant proportion of genome sequences. We review a number of publicly available strategies for providing structural annotations for genome sequences, and we describe the protocol adopted to provide CATH structural annotations for completed genomes. In particular, we assess the performance of several sequence‐based protocols employing Hidden Markov model (HMM) technologies for superfamily recognition, including a new approach (SAMOSA [sequence augmented models of structure alignments]) that exploits multiple structural alignments from the CATH domain structure database when building the models. Using a data set of remote homologs detected by structure comparison and manually validated in CATH, a single‐seed HMM library was able to recognize 76% of the data set. Including the SAMOSA models in the HMM library showed little gain in homolog recognition, although a slight improvement in alignment quality was observed for very remote homologs. However, using an expanded 1D‐HMM library, CATH‐ISL increased the coverage to 86%. The single‐seed HMM library has been used to annotate the protein sequences of 120 genomes from all three major kingdoms, allowing up to 70% of the genes or partial genes to be assigned to CATH superfamilies. It has also been used to recruit sequences from Swiss‐Prot and TrEMBL into CATH domain superfamilies, expanding the CATH database eightfold.
The aggregation kinetics of Alzheimer's β‐amyloid peptide is controlled by stochastic nucleation
Authors: Hortschansky Peter , Schroeckh Volker , Christopeit Tony , Zandomeneghi Giorgia , Fändrich Marcus
Published: June 2005

We report here a recombinant expression system that allows production of large quantities of Alzheimer's Aβ(1–40) peptide. The material is competent to dissolve in water solutions with “random‐coil properties,” although its conformation and factual oligomerization state are determined by the physico‐chemical solution conditions. When dissolved in 50 mM sodium phosphate buffer (pH 7.4) at 37°C, the peptide is able to undergo a nucleated polymerization reaction. The aggregation profile is characteristically bipartite, consisting of lag and growth phase. From these curves we determined the lag time as well as the rate of aggregation. Both values were found to depend on peptide concentration and addition or formation of seeds. Moreover, they can vary considerably between apparently identical samples. These data imply that the nucleation event is under influence of a stochastic factor that can manifest itself in profound macroscopic differences in the aggregation kinetics of otherwise indistinguishable samples.
Crystal structure of methylenetetrahydromethanopterin reductase (Mer) in complex with coenzyme F420: Architecture of the F420/FMN binding site of enzymes within the nonprolyl cis‐peptide containing bacterial luciferase family
Authors: Aufhammer Stephan W. , Warkentin Eberhard , Ermler Ulrich , Hagemeier Christoph H. , Thauer Rudolf K. , Shima Seigo
Published: June 2005

Methylenetetratetrahydromethanopterin reductase (Mer) is involved in CO2 reduction to methane in methanogenic archaea and catalyses the reversible reduction of methylenetetrahydromethanopterin (methylene‐H4MPT) to methyl‐H4MPT with coenzyme F420H2, which is a reduced 5′‐deazaflavin. Mer was recently established as a TIM barrel structure containing a nonprolyl cis‐peptide bond but the binding site of the substrates remained elusive. We report here on the crystal structure of Mer in complex with F420 at 2.6 Å resolution. The isoalloxazine ring is present in a pronounced butterfly conformation, being induced from the Re‐face of F420 by a bulge that contains the non‐prolyl cis‐peptide bond. The bindingmode of F420 is very similar to that in F420‐dependent alcohol dehydrogenase Adf despite the low sequence identity of 21%. Moreover, binding of F420 to the apoenzyme was only associated with minor conformational changes of the polypeptide chain. These findings allowed us to build an improved model of FMN into its binding site in bacterial luciferase, which belongs to the same structural family as Mer and Adf and also contains a nonprolyl cis‐peptide bond in an equivalent position.
Structural analysis of an HIV‐1 protease I47A mutant resistant to the protease inhibitor lopinavir
Authors: Kagan Ron M. , Shenderovich Mark D. , Heseltine Peter N.R. , Ramnarayan Kal
Published: June 2005

We have identified a rare HIV‐1 protease (PR) mutation, I47A, associated with a high level of resistance to the protease inhibitor lopinavir (LPV) and with hypersusceptibility to the protease inhibitor saquinavir (SQV). The I47A mutation was found in 99 of 112,198 clinical specimens genotyped after LPV became available in late 2000, but in none of 24,426 clinical samples genotyped from 1998 to October 2000. Phenotypic data obtained for five I47A mutants showed unexpected resistance to LPV (86‐ to >110‐fold) and hypersusceptibility to SQV (0.1‐ to 0.7‐fold). Molecular modeling and energy calculations for these mutants using our structural phenotyping methodology showed an increase in the binding energy of LPV by 1.9–3.1 kcal/mol with respect to the wild type complex, corresponding to a 20‐ to >100‐fold decrease in binding affinity, consistent with the observed high levels of LPV resistance. In the WT PR–LPV complex, the Ile 47 side chain is positioned close to the phenoxyacetyl moiety of LPV and its van der Waals interactions contribute significantly to the ligand binding. These interactions are lost for the smaller Ala 47 residue. Calculated binding energy changes for SQV ranged from −0.4 to −1.2 kcal/mol. In the mutant I47A PR–SQV complexes, the PR flaps are packed more tightly around SQV than in the WT complex, resulting in the formation of additional hydrogen bonds that increase binding affinity of SQV consistent with phenotypic hypersusceptibility. The emergence of mutations at PR residue 47 strongly correlates with increasing prescriptions of LPV (Spearman correlation rs=0.96, P<.0001).
DOM‐fold: A structure with crossing loops found in DmpA, ornithine acetyltransferase, and molybdenum cofactor‐binding domain
Authors: Cheng Hua , Grishin Nick V.
Published: June 2005

Understanding relationships between sequence, structure, and evolution is important for functional characterization of proteins. Here, we define a novel DOM‐fold as a consensus structure of the domains inDmpA (L‐aminopeptidase D‐Ala‐esterase/amidase),OAT (ornithine acetyltransferase), andMocoBD (molybdenum cofactor‐binding domain), and discuss possible evolutionary scenarios of its origin. As shown by a comprehensive structure similarity search, DOM‐fold distinguished by a two‐layered β/α architecture of a particular topology with unusual crossing loops is unique to those three protein families. DmpA and OAT are evolutionarily related as indicated by their sequence, structural, and functional similarities. Structural similarity between the DmpA/OAT superfamily and the MocoBD domains has not been reported before. Contrary to previous reports, we conclude that functional similarities between DmpA/OAT proteins and N‐terminal nucleophile (Ntn) hydrolases are convergent and are unlikely to be inherited from a common ancestor.
Characterization of calretinin I–II as an EF‐hand, Ca2+, H+‐sensing domain
Authors: Palczewska Małgorzata , Batta Gyula , Groves Patrick , Linse Sara , Kuźnicki Jacek
Published: June 2005

Calretinin, a neuronal protein with well‐defined calcium‐binding properties, has a poorly defined function. The pH dependent properties of calretinin (CR), the N‐terminal (CR I–II), and C‐terminal (CR III–VI) domains were investigated. A drop in pH within the intracellular range (from pH 7.5 to pH 6.5) leads to an increased hydrophobicity of calcium‐bound CR and its domains as reported by fluorescence spectroscopy with the hydrophobic probe 2‐(p‐toluidino)‐6‐naphthalenesulfonic acid (TNS). The TNS data for the N‐ and C‐terminal domains of CR are additive, providing further support for their independence within the full‐length protein. Our work concentrated on CR I–II, which was found to have hydrophobic properties similar to calmodulin at lower pH. The elution of CR I–II from a phenyl‐Sepharose column was consistent with the TNS data. The pH‐dependent structural changes were further localized to residues 13–28 and 44–51 using nuclear magnetic resonance spectroscopy chemical shift analysis, and there appear to be no large changes in secondary structure. Protonation of His 12 and/or His 27 side chains, coupled with calcium chelation, appears to lead to the organization of a hydrophobic pocket in the N‐terminal domain. CR may sense and respond to calcium, proton, and other signals, contributing to conflicting data on the proteins role as a calcium sensor or calcium buffer.
Urea‐induced denaturation of apolipoprotein serum amyloid A reveals marginal stability of hexamer
Authors: Wang Limin , Colón Wilfredo
Published: June 2005

Serum Amyloid A (SAA) is an acute phase reactant protein that is predominantly found bound to high‐density lipoprotein in plasma. Upon inflammation, the plasma concentration of SAA can increase dramatically, occasionally leading to the development of amyloid A (AA) amyloidosis, which involves the deposition of SAA amyloid fibrils in major organs. We previously found that the murine isoform SAA2.2 exists in aqueous solution as a hexamer containing a central channel. Here we show using various biophysical and biochemical techniques that the SAA2.2 hexamer can be totally dissociated into monomer by ∼2 M urea, with the concerted loss of its α‐helical structure. However, limited trypsin proteolysis experiments in urea showed a conserved digestion profile, suggesting the preservation of major backbone topological features in the urea‐denatured state of SAA2.2. The marginal stability of hexameric SAA2.2 and the presence of residual structure in the denatured monomeric protein suggest that both forms may interconvert in vivo to exert different functions to meet the various needs during normal physiological conditions and in response to inflammatory stimuli.
Protein dynamics control proton transfer from bulk solvent to protein interior: A case study with a green fluorescent protein
Authors: Saxena Anoop M. , Udgaonkar Jayant B. , Krishnamoorthy Guruswamy
Published: June 2005

The kinetics of proton transfer in Green Fluorescent Protein (GFP) have been studied as a model system for characterizing the correlation between dynamics and function of proteins in general. The kinetics in EGFP (a variant of GFP) were monitored by using a laser‐induced pH jump method. The pH was jumped from 8 to 5 by nanosecond flash photolysis of the “caged proton,” o‐nitrobenzaldehyde, and subsequent proton transfer was monitored by following the decrease in fluorescence intensity. The modulation of proton transfer kinetics by external perturbants such as viscosity, pH, and subdenaturing concentrations of GdnHCl as well as of salts was studied. The rate of proton transfer was inversely proportional to solvent viscosity, suggesting that the rate‐limiting step is the transfer of protons through the protein matrix. The rate is accelerated at lower pH values, and measurements of the fluorescence properties of tryptophan 57 suggest that the enhancement in rate is associated with an enhancement in protein dynamics. The rate of proton transfer is nearly independent of temperature, unlike the rate of the reverse process. When the stability of the protein was either decreased or increased by the addition of co‐solutes, including the salts KCl, KNO3, and K2SO4, a significant decrease in the rate of proton transfer was observed in all cases. The lack of correlation between the rate of proton transfer and the stability of the protein suggests that the structure is tuned to ensure maximum efficiency of the dynamics that control the proton transfer function of the protein.
Charge–charge interactions in the denatured state influence the folding kinetics of ribonuclease Sa
Authors: Trefethen Jared M. , Pace C. Nick , Scholtz J. Martin , Brems David N.
Published: June 2005

Gaining a better understanding of the denatured state ensemble of proteins is important for understanding protein stability and the mechanism of protein folding. We studied the folding kinetics of ribonuclease Sa (RNase Sa) and a charge‐reversal variant (D17R). The refolding kinetics are similar, but the unfolding rate constant is 10‐fold greater for the variant. This suggests that charge–charge interactions in the denatured state and the transition state ensembles are more favorable in the variant than in RNase Sa, and shows that charge–charge interactions can influence the kinetics and mechanism of protein folding.
Solution structure and backbone dynamics of Calsensin, an invertebrate neuronal calcium‐binding protein
Authors: Venkitaramani Deepa V. , Fulton D. Bruce , Andreotti Amy H. , Johansen Kristen M. , Johansen Jørgen
Published: June 2005

Calsensin is an EF‐hand calcium‐binding protein expressed by a subset of peripheral sensory neurons that fasciculate into a single tract in the leech central nervous system. Calsensin is a 9‐kD protein with two EF‐hand calcium‐binding motifs. Using multidimensional NMR spectroscopy we have determined the solution structure and backbone dynamics of calcium‐bound Calsensin. Calsensin consists of four helices forming a unicornate‐type four‐helix bundle. The residues in the third helix undergo slow conformational exchange indicating that the motion of this helix is associated with calciumbinding. The backbone dynamics of the protein as measured by15N relaxation rates and heteronuclear NOEs correlate well with the three‐dimensional structure. Furthermore, comparison of the structure of Calsensin with other members of the EF‐hand calcium‐binding protein family provides insight into plausible mechanisms of calcium and target protein binding.
Investigation of protein refolding using a fractional factorial screen: A study of reagent effects and interactions
Authors: Willis Melissa Swope , Hogan James K. , Prabhakar Prakash , Liu Xun , Tsai Kuenhi , Wei Yunyi , Fox Ted
Published: June 2005

A recurring obstacle for structural genomics is the expression of insoluble, aggregated proteins. In these cases, the use of alternative salvage strategies, like in vitro refolding, is hindered by the lack of a universal refolding method. To overcome this obstacle, fractional factorial screens have been introduced as a systematic and rapid method to identify refolding conditions. However, methodical analyses of the effectiveness of refolding reagents on large sets of proteins remain limited. In this study, we address this void by designing a fractional factorial screen to rapidly explore the effect of 14 different reagents on the refolding of 33 structurally and functionally diverse proteins. The refolding data was analyzed using statistical methods to determine the effect of each refolding additive. The screen has been miniaturized for automation resulting in reduced protein requirements and increased throughput. Our results show that the choice of pH and reducing agent had the largest impact on protein refolding. Bis‐mercaptoacetamide cyclohexane (BMC) and tris (2‐carboxyethylphosphine) (TCEP) were superior reductants when compared to others in the screen. BMC was particularly effective in refolding disulfide‐containing proteins, while TCEP was better for nondisulfide‐containing proteins. From the screen, we successfully identified a positive synergistic interaction between nondetergent sulfobetaine 201 (NDSB 201) and BMC on Cdc25A refolding. The soluble protein resulting from this interaction crystallized and yielded a 2.2 Å structure. Our method, which combines a fractional factorial screen with statistical analysis of the data, provides a powerful approach for the identification of optimal refolding reagents in a general refolding screen.
Stereo‐selectivity of HIV‐1 reverse transcriptase toward isomers of thymidine‐5′‐O‐1‐thiotriphosphate
Authors: Radzio Jessica , Sluis‐Cremer Nicolas
Published: June 2005

The first pre‐steady‐state kinetic analysis of the stereo‐selective incorporation of Rp‐ and Sp‐isomers of thymidine‐5′‐O‐1‐thiotriphosphate (TTPαS) by HIV‐1 reverse transcriptase (RT) is reported. Rates of polymerization (kpol), apparent dissociation constants (Kd), and substrate specificities (kpol/Kd) were measured for TTP, Rp‐TTPαS, and Sp‐TTPαS in the presence of Mg2+, Mn2+, and Co2+. HIV‐1 RT exhibits a strong preference to incorporate Sp‐TTPαS over Rp‐TTPαS in the presence of Mg2+; however, this stereo‐selective preference was decreased when Mg2+ was replaced with Mn2+ and Co2+. Furthermore, HIV‐1 RT exhibited no phosphorothioate elemental effects for the incorporation of Sp‐TTPαS, but large elemental effects were calculated for Rp‐TTPαS for each of the metals tested. These results are discussed in relation to our current understanding of the RT active‐site structure and the mechanism of DNA synthesis.
Do all backbone polar groups in proteins form hydrogen bonds?
Authors: Fleming Patrick J. , Rose George D.
Published: June 2005

Evidence from proteins and peptides supports the conclusion that intrapeptide hydrogen bonds stabilize the folded form of proteins. Paradoxically, evidence from small molecules supports the opposite conclusion, that intrapeptide hydrogen bonds are less favorable than peptide–water hydrogen bonds. A related issue—often lost in this debate about comparing peptide–peptide to peptide– water hydrogen bonds—involves the energetic cost of an unsatisfied hydrogen bond. Here, experiment and theory agree that breaking a hydrogen bond costs between 5 and 6 kcal/mol. Accordingly, the likelihood of finding an unsatisfied hydrogen bond in a protein is insignificant. This realization establishes a powerful rule for evaluating protein conformations.