bluuebunny/crossref_metadata_2025 huggingface dataset:
One step purification and biochemical characterization of a spermatozoa‐binding protein from porcine oviductal epithelial cells
Authors: Marini Patricia E. , Cabada Marcelo O.
Published: October 2003

In pigs the binding of sperm to oviductal epithelial cells to form a sperm reservoir involves carbohydrate interactions. In the present study, we purify a sperm binding glycoprotein (SBG) from cells from the isthmus of the oviduct using an affinity column. This protein conjugated with FITC is able to bind to the heads of pig sperm. SBG is shown to contain carbohydrates by PAS–silver staining and lectin binding assays. Enzymatic treatment and lectin affinity demonstrate that SBG exposes Galβ1‐3GalNAc disaccharide, which is bound to a serine or a threonin residue by an O‐link. After enzymatic deglycosylation SBG shows an apparent molecular mass of 67.5 kDa, which changes to 85 kDa by reduction with 2‐mercaptoethanol. Both SBG and enzymatically deglycosylated SBG show by isoelectrofocusing two forms of pI 3.6 and pI 3.8. SBG may be involved on sperm–oviduct interaction. Mol. Reprod. Dev. 66: 383–390, 2003. © 2003 Wiley‐Liss, Inc.
Gene expression and in vitro development of inter‐species nuclear transfer embryos
Authors: Arat Sezen , Rzucidlo S. Jacek , Stice Steven L.
Published: October 2003

This study examined the chromatin morphology, in vitro development, and expression of selected genes in cloned embryos produced by transfer of mouse embryonic fibroblasts (MEF) into the bovine ooplasm. After 6 hr of activation, inter‐species nuclear transfer (NT) embryos (MEF‐NT) had one (70%) or two pronuclei (20%), respectively. After 72 hr of culture in vitro, 62.6% of the MEF‐NTs were arrested at the 8‐cell stage, 31.2% reached the 2‐ to 4‐cell stage, and only 6.2% had more than eight blastomeres, but none of these developed to the blastocyst stage. Whereas, 20% of NT embryos derived from bovine embryonic fibroblast fused with bovine ooplasm (BEF‐NT) reached the blastocyst stage. Donor MEF nuclei expressing an Enhanced Green Fluorescent Protein (EGFP) transgene resulted in 1‐ to 8‐cell stage MEF‐NT that expressed EGFP. The expression of selected genes was examined in 8‐cell MEF‐NTs, 8‐cell mouse embryos, enucleated bovine oocytes, and MEFs using RT‐PCR. The mRNA for heat shock protein 70.1 (Hsp 70.1) gene was detected in MEF‐NTs and MEF, but not in mouse embryos. The hydroxy‐phosphoribosyl transferase (HPRT) mRNA was found in normal mouse embryos and MEF but not in MEF‐NTs. Expression of Oct‐4 and embryonic alkaline phospatase (eAP) genes was only detected in normal mouse embryos and not in the inter‐species NT embryos. Abnormal gene expression profiles were associated with an arrest in the development at the 8‐cell stage, but MEF‐NT embryos appeared to have progressed through gross chromatin remodeling, typical of intra‐species NT embryos. Therefore, molecular reprogramming rather than chromatin remodeling may be a better indicator of nuclear reprogramming in inter‐species NT embryos. Mol. Reprod. Dev. 66: 334–342, 2003. © 2003 Wiley‐Liss, Inc.
Changes in JNK1 activity in the frog (Rana esculenta) testis
Authors: Chieffi Paolo
Published: October 2003

Several lines of evidences support a key role of mitogen‐activated protein kinases (MAPK) in male fertility. It has been used as a non‐mammalian vertebrate model, the frog Rana esculenta, to investigate the regulation of Jun amino‐terminal kinase 1 (JNK1) activity in the testis in two different periods of the annual reproductive cycle: winter stasis and breeding season. JNK1 protein was localized by immunohistochemistry in the cytoplasm and nucleus of spermatogonia (SPG) and spermatocytes (SPC) during all the year. Immunoprecipitation analysis of JNK1 clearly shows that this isoform presents a strong phosphorylation status (thr183/Tyr185) in the breeding period, in contrast with only very low activity during the winter stasis. Simultaneously, during the breeding period c‐Jun protein is highly phosphorylated on Ser‐63. These data suggest that the increase of JNK1 activity, with strong c‐Jun phosphorylation (Ser‐63) during the breeding period, could be the signal of increasing transcriptional activity in the frog testis. Mol. Reprod. Dev. 66: 398–402, 2003. © 2003 Wiley‐Liss, Inc.
Feasibility of global gene expression analysis in testicular biopsies from infertile men
Authors: Fox Mark S. , Ares V. Ximena , Turek Paul J. , Haqq Christopher , Reijo Pera Renee A.
Published: October 2003

Numerous studies have documented the use of microarray analysis to identify patterns of global gene expression that distinguish normal development from that of the diseased state. Yet, there are no reports that compare global gene expression in the fertile and infertile human testis. Here, we report an initial study of global gene expression in testicular biopsies from several men with different infertility phenotypes. We found that microarray analysis of small biopsy samples was suitable for profiling expression of genes known to function in germ cell development and also identified expression of novel genes. Since it is now common for infertile men with spermatogenic failure to use intracytoplasmic sperm injection (ICSI) to achieve biological paternity, we hypothesize that molecular screening of testicular biopsies with microarrays may be suitable: (1) to categorize the molecular phenoytpes of infertile testes in a manner similar to standard morphologic analysis and (2) to initiate larger studies of gene expression in the infertile testes that may identify genetic signatures from biopsies that allow prediction of outcomes. Mol. Reprod. Dev. 66: 403–421, 2003. © 2003 Wiley‐Liss, Inc.
Accumulation of zinc, copper, and metallothionein mRNA in lizard ovary proceeds without a concomitant increase in metallothionein content
Authors: Riggio Marilisa , Trinchella Francesca , Filosa Silvana , Parisi Elio , Scudiero Rosaria
Published: October 2003

The possible role of metallothionein (MT) in metal homeostasis has been investigated in growing oocytes and eggs of the lizard Podarcis sicula. Chromatographic analysis does not reveal the presence of MT in both ovary and eggs, the only metal‐binding proteins detected being represented by high molecular mass components. De novo synthesis of MT could be observed in the ovary of cadmium‐treated lizards. A cDNA encoding MT was obtained from the liver of P. sicula by RT‐PCR followed by a RACE strategy, using primers designed on consensus motifs of vertebrate MT. In spite of the lack of MT in the ovary of untreated animals, Northern blot analysis demonstrates that the maternal untranslated MT transcript is expressed constitutively in the ovary in all the periods of the ovarian cycle. MT mRNA content increases during the oocyte growth, reaching the highest level in ovulated eggs, concomitantly with the accumulation of zinc and copper. Our findings suggest that maternal MT mRNA accumulates in the egg and is translated sometime during development to cope with the future needs of the growing embryo. The appearance of MT after cadmium treatment suggests that the block that makes the oocytarian MT mRNA untranslatable is removed by the metal. Mol. Reprod. Dev. 66: 374–382, 2003. © 2003 Wiley‐Liss, Inc.
Calgizarrin like gene (Cal) deficient mice undergo normal spermatogenesis
Authors: Mannan Ashraf U. , Nica Gabriela , Nayernia Karim , Mueller Christian , Engel Wolfgang
Published: October 2003

The murine calgizzarin like gene (Cal) encodes for a calcium binding protein, which belongs to the S100 family of EF‐hand proteins. It is specifically expressed in Sertoli cells in the testis and its expression is down‐regulated by unknown factor(s) from spermatocytes/spermatids. In this paper, we show by transfection of a fusion protein of green fluorescent protein and Cal protein into NIH3T3 cells, that the expression of Cal is restricted only in the cytoplasm of the cell. A differentially regulated cytoplasmic expression of the Cal in Sertoli cells during mouse development suggests that Cal might play an important role during spermatogenesis. In order to elucidate the function of the Cal protein in the spermatogenesis, we disrupted the Cal locus in mouse by homologous recombination. In our knockout mouse, we deleted exon 2 and exon 3 of the Cal gene and replaced them with a neomycin cassette, which resulted in a complete loss of the Cal transcript. Male and female Cal4+/− and Cal4−/− mice from genetic backgrounds C57BL/6J × 129X1/SvJ hybrid and 129X1/SvJ inbred exhibited normal phenotype and were fertile. An intensive phenotypic analysis showed no gross abnormalities in testis morphology. The lack of the Cal protein also does not affect the parameters of sperm, as they are able to fertilize the oocytes in a competent manner, which is comparable to wild‐type sperm. Collectively our results demonstrate that Cal is a nonessential protein and it does not play an important role in mouse spermatogenesis or in process of fertilization. Mol. Reprod. Dev. 66: 431–438, 2003. © 2003 Wiley‐Liss, Inc.
Superoxide dismutase content and fatty acid composition in subsets of human spermatozoa from normozoospermic, asthenozoospermic, and polyzoospermic semen samples
Authors: Calamera J. , Buffone M. , Ollero M. , Alvarez J. , Doncel G.F.
Published: October 2003

Human ejaculated sperm comprised discrete subsets of spermatozoa, with different degrees of maturation. These subpopulations can be isolated through density gradient centrifugation. Sperm from the lowest density layer show the highest content of docosahexaenoic acid and sterols, and produce the highest levels of reactive oxygen species. The main objective of this study was to determine the superoxide dismutase (SOD) content and fatty acid composition of subsets of spermatozoa isolated from normozoospermic, asthenozoospermic, and polyzoospermic semen samples. Four sperm fractions (1–4) were obtained using ISolate gradient centrifugation. Morphology, motion parameters, SOD content, and fatty acid composition were assessed in the original samples and their fractions. Overall, sperm from normozoospermic samples had higher SOD content than those of asthenozoospermic or polyzoospermic samples. Once fractionated in subsets, the sperm SOD content decreased significantly (P < 0.0001) from fraction 1 (top) to 4 (bottom) in all three groups of samples. Fatty acid content as well as the oxidation coefficient followed the same pattern, decreasing from fraction 1 to 4 (F1–F4). Normo‐ and polyzoospermic samples showed similar amounts of fatty acids, while asthenozoospermic samples mostly revealed increased levels. Normozoospermic samples displayed the lowest unsaturated fatty acid (UFA)/SOD ratio. Spermatozoa from astheno‐ and polyzoospermic samples, two common seminal pathologies, showed higher UFA and lower SOD content than normal sperm, therefore exhibiting a higher susceptibility to peroxidative damage. F4 from all groups, containing the most mature spermatozoa, displayed the lowest polyunsaturated fatty acid and SOD content of all subsets, suggesting that excessive SOD activity as well as abundant peroxidative targets may both be deleterious to sperm function. Mol. Reprod. Dev. 66: 422–430, 2003. © 2003 Wiley‐Liss, Inc.
Novel purification method for mammalian seminal plasma phospholipid‐binding proteins reveals the presence of a novel member of this family of protein in stallion seminal fluid
Authors: Ménard Martin , Nauc Veronica , Lazure Claude , Vaillancourt Denis , Manjunath Puttaswamy
Published: October 2003

A family of bull seminal plasma (BSP) phospholipid‐binding proteins (BSP proteins), potentiate heparin‐ and HDL‐induced capacitation. The homologous proteins have been purified from stallion and boar seminal plasma, and detected in low concentrations in other mammalian seminal plasma. In this study, we developed a new isolation method for mammalian seminal plasma choline phospholipid‐binding proteins wherein they are present in low concentrations. The method is based on the interaction of this family of proteins with egg yolk low‐density lipoprotein fraction (LDF). In order to demonstrate the feasibility of the method, we incubated LDF with alcohol precipitates of bull, boar, and stallion seminal plasma. LDF were re‐isolated by ultracentrifugation along with bound proteins. LDF with associated proteins were dialyzed, lyophilized, and delipidated. BSP homologous proteins were finally purified by p‐aminophenyl phosphorylcholine (PPC)–agarose and/or gelatin–agarose chromatographies, and analyzed by SDS–PAGE. With this new protocol, phospholipid‐binding proteins of bull, boar, and stallion seminal plasma were recovered almost 100%. A new 12 kDa stallion seminal plasma protein of the same family was also isolated and partially sequenced. The radio‐immunoassay (RIA) data showed that 10 mg of LDF can bind all BSP proteins present in 120 mg of alcohol precipitated BSP proteins. These results confirm the efficiency of the method and that the LDF step could be used for the isolation of all BSP proteins homologs from different mammalian species. Mol. Reprod. Dev. 66: 349–357, 2003. © 2003 Wiley‐Liss, Inc.
Experience in calibrating the double‐hardening constitutive model Monot
Authors: A. Hicks M.
Published: October 2003

The Monot double‐hardening soil model has previously been implemented within a general purpose finite element algorithm, and used in the analysis of numerous practical problems. This paper reviews experience gained in calibrating Monot to laboratory data and demonstrates how the calibration process may be simplified without detriment to the range of behaviours modelled. It describes Monot's principal features, important governing equations and various calibration methods, including strategies for overconsolidated, cemented and cohesive soils. Based on a critical review of over 30 previous Monot calibrations, for sands and other geomaterials, trends in parameter values have been identified, enabling parameters to be categorized according to their relative importance. It is shown that, for most practical purposes, a maximum of only 5 parameters is needed; for the remaining parameters, standard default values are suggested. Hence, the advanced stress–strain modelling offered by Monot is attainable with a similar number of parameters as would be needed for some simpler, less versatile, models. Copyright © 2003 John Wiley & Sons, Ltd.
Modelling crack propagation in concrete structures with a two scale approach
Authors: Haidar Khalil , Dubé Jean François , Pijaudier‐Cabot Gilles
Published: October 2003

A simplified computational technique based on a refined global–local method is applied to the failure analysis of concrete structures. The technique distinguishes the scale of the structure, modelled with large size finite elements, from the scale at which material non‐linearity occurs due to progressive cracking and macro‐crack propagation. The finite element solution is split into two parts: a linear elastic analysis on a coarse mesh over the entire structure and a non‐linear analysis over a small part of the structure where a dense finite element grid is employed. In the non‐linear calculation, a non‐local damage model is implemented. These two computations are coupled with the help of an iterative scheme. The size and location of the region where a non‐linear analysis is performed, is adapted to follow the development of the damage zone. Numerical examples of mode I fracture of concrete specimens with straight and curved cracks are presented. Copyright © 2003 John Wiley & Sons, Ltd.
Numerical solution of eddy current problems in ferromagnetic bodies travelling in a transverse magnetic field
Authors: Peterson W.
Published: October 2003

Eddy currents are investigated in a ferromagnetic bar travelling in a transverse magnetic field. Such an open boundary field problem is analysed by a hybrid approach based on Galerkin finite element formulation coupled with a separation of variables. A steady state is considered, introducing time‐periodic boundary conditions. The resultant system of non‐linear equations is solved by an iterative procedure based on Brouwer's fixed‐point theorem. Numerical results are presented for a bar of circular cross‐section made of cast steel or cast iron. Selected examples of the field distribution and characteristics of eddy‐current power losses are enclosed in graphic form. Copyright © 2003 John Wiley & Sons, Ltd.
Oscillatory crack tip triangular elements for finite element analysis of interface cracks
Authors: Liu C. H. , Huang Chunta
Published: October 2003

In this paper a special crack tip element has been developed in which displacements and stresses have the same behaviour as those of bi‐material interface cracks with open tips. The element degenerates into a traditional triangular quarter point element in cases of homogeneous cracks. An isoparametric co‐ordinate system (ρ, t) is defined in this study, and numerical techniques using these co‐ordinates to evaluate Jacobian matrices, shape function derivatives, and element stiffness matrices are developed. Also, equations calculating the complex stress intensity factor using displacements are obtained in this study. Numerical results are in good agreement with known analytical solutions in two examples. Copyright © 2003 John Wiley & Sons, Ltd.
A new rigid‐viscoplastic model for simulating thermal strain effects in metal‐forming processes
Authors: Dvorkin Eduardo N. , Toscano Rita G.
Published: October 2003

A new rigid‐viscoplastic model that includes the effect of thermal strains when modelling steady‐state metal‐forming processes was developed. A symmetric approximation to the resulting non‐symmetric stiffness matrix was derived. The thermo‐mechanical flow formulation was implemented using the pseudo‐concentrations technique. The new formulation was numerically tested showing that it provides reliable results. Copyright © 2003 John Wiley & Sons, Ltd.
Studies in elastic fracture mechanics based on the material force method
Authors: Denzer Ralf , Barth Franz Josef , Steinmann Paul
Published: October 2003

The object of this work is to discuss a further improvement of the material force method for non‐linear hyperelastostatic fracture mechanics. We investigate the accuracy of the material force method within a ‘modified boundary layer’‐formulation using a Ramberg–Osgood material type for the sake of comparison. The proposed improvement leads to a reliable and very accurate method to compute the vectorial J‐integral in fracture mechanics. Copyright © 2003 John Wiley & Sons, Ltd.
Earthquake response analysis in the time domain for 2D soil–structure systems using analytical frequency‐dependent infinite elements
Authors: Kim Doo Kie , Yun Chung‐Bang
Published: October 2003

This paper presents a time domain method for soil–structure interaction analysis under seismic excitations. It is based on the finite element formulation incorporating analytical frequency‐dependent infinite elements for the far‐field soil region. Equivalent earthquake input forces are calculated based on the free‐field responses along the interface between the near‐ and far‐field soil regions using the fixed exterior boundary method in the frequency domain. Then, the input forces are transformed into the time domain by using inverse Fourier transform. The dynamic stiffness matrices of the far‐field soil region formulated using the analytical frequency‐dependent infinite elements in the frequency domain can be easily transformed into the corresponding matrices in the time domain. Hence, the response can be analytically computed in the time domain. A recursive procedure is proposed to compute the interaction forces along the interface and the responses of the soil–structure system in the time domain. Earthquake response analyses have been carried out on a multi‐layered half‐space and a tunnel embedded in a layered half‐space, and results are compared with those obtained by the conventional method in the frequency domain. Copyright © 2003 John Wiley & Sons, Ltd.
Tetrahedral mesh generation based on advancing front technique and optimization scheme
Authors: Choi Woo‐Young , Kwak Dae‐Young , Son Il‐Heon , Im Yong‐Taek
Published: October 2003

In recent years, demand for three‐dimensional simulations has continued to grow in the field of computer‐aided engineering. Especially, in the analysis of forming processes a fully automatic and robust mesh generator is necessary for handling complex geometries used in industry. For three‐dimensional analyses, tetrahedral elements are commonly used due to the advantage in dealing with such geometries. In this study, the advancing front technique has been implemented and modified using an optimization scheme. In this optimization scheme, the distortion metric determines ‘when and where’ to smooth, and serves as an objective function. As a result, the performance of the advancing front technique is improved in terms of mesh quality generated. Copyright © 2003 John Wiley & Sons, Ltd.
Work narratives: Gender and race in professional personhood
Authors: Dombeck Mary‐Therese
Published: October 2003

An ethnographic study was conducted to explore how nurses construe and understand their professional culture and their professional personhood. The sample was 36 nurses ranging in age from 26 to 63 (12 African American women, 11 White women, 13 men 12 White and 1 Caribbean Islander). Data were gathered through participant observation, audiotaped individual conversations, a process of seven consecutive group sessions, and short narratives written by the nurses in group sessions. The data were analyzed: (a) by a coding system that focused on the formal and informal roles, rules, and relationships in work and school settings; and (b) by examining the changes in participants' narratives about their professional identity during the process of the group meetings. All the nurses in the sample had been profoundly affected by the socially accepted “feminine” images of nursing. The findings also revealed racial issues in the nursing profession. The implications of this study point to the need for new models of nursing education and nursing leadership to overcome old images and to make nursing attractive to those from diverse backgrounds. © 2003 Wiley Periodicals, Inc. Res Nurs Health 26:351–365, 2003
Validation of measures of middle schoolers' self‐efficacy for physical and emotional health, and academic tasks
Authors: Bray Christell O. , Nash Kathleen , Froman Robin D.
Published: October 2003

The development and psychometric study of a measure of middle schoolers' perceptions of self‐efficacy for physical, emotional, and academic behaviors appropriate to their age and developmental tasks are described. The validation of the Middle School Self‐Efficacy Questionnaire (MISSE) included the adaptation of existing items and the generation of new ones. Items were subjected to judges' review by two expert panels and were tested with a large, geographically diverse sample of youngsters aged 11–14. Statistical analyses included factor analysis, internal consistency estimations,ttests, and analyses of variance for hypothesis testing related to construct validity. Results showed support for the expected factors and provided evidence of the validity of the MISSE. © 2003 Wiley Periodicals, Inc. Res Nurs Health 26:376–386, 2003
A new methodological approach in nursing research: An actor, partner, and interaction effect model for family outcomes
Authors: Rayens Mary Kay , Svavarsdottir Erla Kolbrun
Published: October 2003

Methodological and statistical challenges have been identified in family research when the parental dyad is under study. The purposes of this article are to review the literature on methodological and statistical procedures in research on families, and to introduce an analysis technique that is new to the nursing literature, the actor–partner interdependence model (APIM). The use and interpretation of the APIM method is illustrated through an example of families of young children with asthma. In the APIM an actor effect is the impact a person has on his or her own outcome, whereas a partner effect is the impact a member of a dyad has on his or her partner's outcome. The main findings from the data example indicate that family adaptation is influenced by the actor effect of resiliency factors (sense of coherence and family hardiness), the partner effect of caregiving demands, as well as by both the actor and partner effects of family demands. The APIM method gives new insight into how parental data can be analyzed by taking both the individual and the partner contribution into account when studying family phenomena. Knowing how outcomes may be influenced not only by individuals (via an actor effect) but also by their partners (through a partner effect) enriches our understanding of how families function. © 2003 Wiley Perioicals, Inc. Res Nurs Health 26:409–419, 2003
Alzheimer's disease caregiving information and skills. Part I: Care recipient issues and concerns
Authors: Farran Carol J. , Loukissa Dimitra , Perraud Suzanne , Paun Olimpia
Published: October 2003

Increasing attention has been given to testing clinical trials with family caregivers of the elderly. More recent intervention studies indicated that caregiver skill‐building interventions may be more effective than information/support interventions. Researchers have given considerable attention to the content and support needed by family caregivers, but we know less about how this content and support translates into caregiver skills. This is the first in a series of three articles on a study in which qualitative methods were used to analyze summaries from the group component of a larger caregiver clinical trial. In this article we identify content and skills that dementia family caregivers need in addressing three major care recipient issues and concerns: (a) difficult behaviors and emotional responses, (b) personal and instrumental activities of daily living, and (c) cognitive decline. © 2003 Wiley Periodicals, Inc. Res Nurs Health 26:366–375, 2003
Smoking and adolescence: Narratives of identity
Authors: Johnson Joy L. , Lovato Chris Y. , Maggi Stefania , Ratner Pamela A. , Shoveller Jean , Baillie Lynne , Kalaw Cecilia
Published: October 2003

The purpose of this study was to understand the identities that youth hold in relation to smoking, as revealed in narrative accounts of their smoking experiences. The analysis was a narrative inquiry, a qualitative approach based on the propensity of people to narrate or tell stories about the experiences in their daily lives. A purposeful sample of 35 youths ages 14–18 years with a variety of smoking histories (all had tried smoking) participated in in‐depth interviews. A detailed analysis of the transcripts revealed the key identities communicated by the youths including theconfident nonsmoker, thevulnerable nonsmoker, theardent nonsmoker, theaccepting nonsmoker, thein‐control smoker, theconfirmed smoker, and thecontrite smoker. Tobacco control interventions for youth must be designed to respond to and incorporate multiple smoking identities. © 2003 Wiley Periodicals, Inc. Res Nurs Health 26:387–397, 2003
Adverse events in acute care: An integrative literature review
Authors: Kellogg Victoria A. , Havens Donna Sullivan
Published: October 2003

An integrative literature review was conducted to investigate studies on adverse events reported in medical, health services, and nursing literature. The review was guided by the method proposed by Jackson (1980) and Ganong (1987). Three questions shaped the review: (a) What terms are used to denote adverse events? (b) What purposes drive adverse events research? and (c) What data sources are used to study adverse events? Adverse events was the dominant term, the study of adverse events as an outcome variable was the prevailing research purpose, and monitoring or screening the patient clinical record and self‐reported incidents by health care professionals were the main data sources. Future research is recommended to conceptualize and study adverse events. © 2003 Wiley Periodicals, Inc. Res Nurs Health 26:398–408, 2003
Unfolding of the cold shock protein studied with biased molecular dynamics
Authors: Morra Giulia , Hodoscek Milan , Knapp Ernst‐Walter
Published: October 2003

The cold shock protein from Bacillus caldolyticus is a small β‐barrel protein that folds in a two‐state mechanism. For the native protein and for several mutants, a wealth of experimental data are available on stability and folding, so that it is an optimal system to study this process. We compare data from unfolding simulations (trajectories of 5 and up to 12 ns) obtained with a bias potential at room temperature and from unbiased thermal unfolding simulations with experimental data. The unfolding patterns derived from the trajectories starting from different native‐like conformations and subject to different unfolding conditions agree. The transition state found in the simulations of unfolding is close to the native structure in agreement with experiment. Moreover, a lower value of the free energy barrier of unfolding was found for the mutant R3E than for the mutant E46A and the native protein, as indicated by experimental data. The first unfolding event involves the three‐stranded β‐sheet whose decomposition corresponds to the transition state. In contrast to conclusions drawn from experiments, we found that the two‐stranded β‐strand forms the most stable substructure, which decomposes very late in the unfolding process. However, assuming that this structure forms very early in the folding process, our findings would not contradict the experiments but require a different interpretation of them. Proteins 2003;53:000–000. © 2003 Wiley‐Liss, Inc.
Importance of sequence specificity for predicting protein folding pathways: Perturbed Gaussian chain model
Authors: Kameda Tomoshi
Published: October 2003

Recent experimental and theoretical studies suggest that rates and pathways of protein folding are largely decided by topology of the native structures, at least for small proteins. However, some exceptions are known; for example, protein L and protein G have the same topology, but exhibit different characteristics of the TSE. Thus, folding pathways of some proteins are critically affected by detailed information on amino acid sequences. To investigate the sequence specificity, we calculate folding pathways of 20 small proteins using the perturbed Gaussian chain model developed by Portman et al. (Phys Rev Lett 1998;81:5237–5240; J Chem Phys 2001;114:5069–5081). Characteristics of the TSE predicted by the model are in good agreement with experimental ϕ‐value data for many proteins at coarse‐grained level. Especially, estimation of folding TSE for protein G and protein L based on both topology and additional sequence information are consistent with experimental ϕ‐value data. With only topology information, however, the model predicts the TSE of protein G incorrectly. Moreover, the model that uses topology and sequence information describes free energy profiles of two‐state and three‐state folders consistently with experiment, whereas the topology only model predicts free energy profiles of some proteins incorrectly. This indicates that sequence specificity also has critical roles in determining the folding pathways for some proteins. Proteins 2003. © 2003 Wiley‐Liss, Inc.
Atomic contact vectors in protein‐protein recognition
Authors: Mintseris Julian , Weng Zhiping
Published: October 2003

The ability to analyze and compare protein‐protein interactions on the structural level is critical to our understanding of various aspects of molecular recognition and the functional interplay of components of biochemical networks. In this study, we introduce atomic contact vectors (ACVs) as an intuitive way to represent the physico‐chemical characteristics of a protein‐protein interface as well as a way to compare interfaces to each other. We test the utility of ACVs in classification by using them to distinguish between homodimers and crystal contacts. Our results compare favorably with those reported by other authors. We then apply ACVs to mine the PDB for all known protein‐protein complexes and separate transient recognition complexes from permanent oligomeric ones. Getting at the basis of this difference is important for our understanding of recognition and we achieved a success rate of 91% for distinguishing these two classes of complexes. Although accessible surface area of the interface is a major discriminating feature, we also show that there are distinct differences in the contact preferences between the two kinds of complexes. Illustrating the superiority of ACVs as a basic comparison measure over a sequence‐based approach, we derive a general rule of thumb to determine whether two protein‐protein interfaces are redundant. With this method, we arrive at a nonredundant set of 209 recognition complexes—the largest set reported so far. Proteins 2003. © 2003 Wiley‐Liss, Inc.
The tissue factor/factor VIIa/factor Xa complex: A model built by docking and site‐directed mutagenesis
Authors: Norledge Brian V. , Petrovan Ramona J. , Ruf Wolfram , Olson Arthur J.
Published: October 2003

Factor X is activated to factor Xa (fXa) in the extrinsic coagulation pathway by the tissue factor (TF)/factor VIIa (fVIIa) complex. Upon activation, the fXa molecule remains associated with the TF/fVIIa complex, and this ternary complex is known to activate protease‐activated receptors (PARs) 1 and 2. Activation of fVII in the TF complex by fXa is also seen at physiologic concentrations. The ternary complexes TF/fVII/fXa, TF/fVIIa/fX, and TF/fVIIa/fXa are therefore all physiologically relevant and of interest as targets for inhibition of both coagulation and cell‐signaling pathways that are important in cardiovascular disease and inflammation. We therefore present a model of the TF/fVIIa/fXa complex, built with the use of the available structures of the TF/fVIIa complex and fXa by protein–protein docking calculations with the program Surfdock. The fXa model has an extended conformation, similar to that of fVIIa in the TF/fVIIa complex, with extensive interactions with TF and the protease domain of fVIIa. All four domains of fXa are involved in the interaction. The gamma‐carboxyglutamate (Gla) and epithelial growth factor (EGF1 and EGF2) domains of fVIIa are not significantly involved in the interaction. Docking of the Gla domain of fXa to TF/fVIIa has been reported previously. The docking results identify potential interface residues, allowing rational selection of target residues for site‐directed mutagenesis. This combination of docking and mutagenesis confirms that residues Glu51 and Asn57 in the EGF1 domain, Asp92 and Asp95 in the EGF2 domain, and Asp 185a, Lys 186, and Lys134 in the protease domain of factor Xa are involved in the interaction with TF/fVIIa. Other fX protease domain residues predicted to be involved in the interaction come from the 160s loop and the N‐terminus of the fX protease domain, which is oriented in such a way that activation of both fVII by fXa, and the reciprocal fX activation by fVIIa, is possible. Proteins 2003;53:000–000. © 2003 Wiley‐Liss, Inc.
Automatic annotation of protein function based on family identification
Authors: Abascal Federico , Valencia Alfonso
Published: October 2003

Although genomes are being sequenced at an impressive rate, the information generated tells us little about protein function, which is slow to characterize by traditional methods. Automatic protein function annotation based on computational methods has alleviated this imbalance. The most powerful current approach for inferring the function of new proteins is by studying the annotations of their homologues, since their common origin is assumed to be reflected in their structure and function. Unfortunately, as proteins evolve they acquire new functions, so annotation based on homology must be carried out in the context of orthologues or subfamilies. Evolution adds new complications through domain shuffling: homology (or orthology) frequently corresponds to domains rather than complete proteins. Moreover, the function of a protein may be seen as the result of combining the functions of its domains. Additionally, automatic annotation has to deal with problems related to the annotations in the databases: errors (which are likely to be propagated), inconsistencies, or different degrees of function specification. We describe a method that addresses these difficulties for the annotation of protein function. Sequence relationships are detected and measured to obtain a map of the sequence space, which is searched for differentiated groups of proteins (similar to islands on the map), which are expected to have a common function and correspond to groups of orthologues or subfamilies. This mapmaking is done by applying a clustering algorithm based on Normalized cuts in graphs. The domain problem is addressed in a simple way: pairwise local alignments are analyzed to determine the extent to which they cover the entire sequence lengths of the two proteins. This analysis determines both what homologues are preferred for functional inheritance and the level of confidence of the annotation. To alleviate the problems associated with database annotations, the information on all the homologues that are grouped together with the query protein are taken into account to select the most representative functional descriptors. This method has been applied for the annotation of the genome of Buchnera aphidicola (specific host Baizongia pistaciae). Human inspection of the annotations allowed an estimation of accuracy of 94%; the different kinds of error that may appear when using this approach are described. Results can be accessed at http://www.pdg.cnb.uam.es/funcut.html. The programs are available upon request, although installation in other systems may be complicated. Proteins 2003;53:000–000. © 2003 Wiley‐Liss, Inc.
Characterization of the active site of DNA polymerase β by molecular dynamics and quantum chemical calculation
Authors: Rittenhouse Robert C. , Apostoluk Wlodzimierz K. , Miller John H. , Straatsma T. P.
Published: October 2003

It is well established that the fully formed polymerase active site of the DNA repair enzyme, polymerase β (pol β), including two bound Mg2+ cations and the nucleoside triphosphate (dNTP) substrate, exists at only one point in the catalytic cycle just prior to the chemical nucleotidyl transfer step. The structure of the active conformation has been the subject of much interest as it relates to the mechanism of the chemical step and also to the question of fidelity assurance. Although crystal structures of ternary pol β–(primer‐template) DNA–dNTP complexes have provided the main structural features of the active site, they are necessarily incomplete due to intentional alterations (e.g., removal of the 3′OH groups from primer and substrate) needed to obtain a structure from midcycle. Working from the crystal structure closest to the fully formed active site [Protein Data Bank (PDB) code: 1bpy], two molecular dynamics (MD) simulations of the solvated ternary complex were performed: one with the missing 3′OHs restored, via modeling, to the primer and substrate, and the other without restoration of the 3′OHs. The results of the simulations, together with ab initio optimizations on simplified active‐site models, indicate that the missing primer 3′OH in the crystal structure is responsible for a significant perturbation in the coordination sphere of the catalytic cation and allow us to suggest several corrections and additions to the active‐site structure as observed by crystallography. In addition, the calculations help to resolve questions raised regarding the protonation states of coordinating ligands. Proteins 2003;53:000–000. © 2003 Wiley‐Liss, Inc.
A second binding site revealed by C‐terminal truncation of calpain small subunit, a penta‐EF‐hand protein
Authors: Leinala E.K. , Arthur J.S.C. , Grochulski P. , Davies P.L. , Elce J.S. , Jia Z.
Published: October 2003

The subunits in calpain and in the related penta‐EF‐hand (PEF) proteins are bound through contacts between the unpaired EF‐hand 5 from each subunit. To study subunit binding further, a tetra‐EF‐hand 18 kDa N‐ and C‐terminally truncated form of the calpain small subunit was prepared (18k). This protein does not combine with the calpain large subunit to form active calpain, but forms homodimers in solution, as shown by ultracentrifugation. The X‐ray structure of the 18k protein in the presence of cadmium was solved to a resolution of 2.0 Å. The structure of the monomer is almost identical to the known structure of the calpain small subunit, but the 18k protein forms an oligomer in the crystal by the use of two binding sites. One of these sites is an artefact arising from the C‐terminal truncation, but the other is a naturally occurring site that is fully exposed to water in intact purified calpain. The characteristics of this site suggest that it may be important in binding other protein modulators involved in the regulation of calpain and of PEF proteins. Proteins 2003. © 2003 Wiley‐Liss, Inc.
RDOCK: Refinement of rigid‐body protein docking predictions
Authors: Li Li , Chen Rong , Weng Zhiping
Published: October 2003

We present a simple and effective algorithm RDOCK for refining unbound predictions generated by a rigid‐body docking algorithm ZDOCK, which has been developed earlier by our group. The main component of RDOCK is a three‐stage energy minimization scheme, followed by the evaluation of electrostatic and desolvation energies. Ionic side chains are kept neutral in the first two stages of minimization, and reverted to their full charge states in the last stage of brief minimization. Without side chain conformational search or filtering/clustering of resulting structures, RDOCK represents the simplest approach toward refining unbound docking predictions. Despite its simplicity, RDOCK makes substantial improvement upon the top predictions by ZDOCK with all three scoring functions and the improvement is observed across all three categories of test cases in a large benchmark of 49 non‐redundant unbound test cases. RDOCK makes the most powerful combination with ZDOCK2.1, which uses pairwise shape complementarity as the scoring function. Collectively, they rank a near‐native structure as the number‐one prediction for 18 test cases (37% of the benchmark), and within the top 4 predictions for 24 test cases (49% of the benchmark). To various degrees, funnel‐like energy landscapes are observed for these 24 test cases. To the best of our knowledge, this is the first report of binding funnels starting from global searches for a broad range of test cases. These results are particularly exciting, given that we have not used any biological information that is specific to individual test cases and the whole process is entirely automated. Among three categories of test cases, the best results are seen for enzyme/inhibitor, with a near‐native structure ranked as the number‐one prediction for 48% test cases, and within the top 10 predictions for 78% test cases. RDOCK is freely available to academic users at http://zlab.bu.edu/∼rong/dock. Proteins 2003. © 2003 Wiley‐Liss, Inc.
Dissecting subunit interfaces in homodimeric proteins
Authors: Bahadur Ranjit Prasad , Chakrabarti Pinak , Rodier Francis , Janin Joël
Published: October 2003

The subunit interfaces of 122 homodimers of known three‐dimensional structure are analyzed and dissected into sets of surface patches by clustering atoms at the interface; 70 interfaces are single‐patch, the others have up to six patches, often contributed by different structural domains. The average interface buries 1,940 Å2 of the surface of each monomer, contains one or two patches burying 600–1,600 Å2, is 65% nonpolar and includes 18 hydrogen bonds. However, the range of size and of hydrophobicity is wide among the 122 interfaces. Each interface has a core made of residues with atoms buried in the dimer, surrounded by a rim of residues with atoms that remain accessible to solvent. The core, which constitutes 77% of the interface on average, has an amino acid composition that resembles the protein interior except for the presence of arginine residues, whereas the rim is more like the protein surface. These properties of the interfaces in homodimers, which are permanent assemblies, are compared to those of protein‐protein complexes where the components associate after they have independently folded. On average, subunit interfaces in homodimers are twice larger than in complexes, and much less polar due to the large fraction belonging to the core, although the amino acid compositions of the cores are similar in the two types of interfaces. Proteins 2003. © 2003 Wiley‐Liss, Inc.
Elucidation of the disulfide‐folding pathway of hirudin by a topology‐based approach
Authors: Micheletti C. , De Filippis V. , Maritan A. , Seno F.
Published: October 2003

A theoretical model for the folding of proteins containing disulfide bonds is introduced. The model exploits the knowledge of the native state to favor the progressive establishment of native interactions. At variance with traditional approaches based on native topology, not all native bonds are treated in the same way; in particular, a suitable energy term is introduced to account for the special strength of disulfide bonds, as well as their ability to undergo intramolecular reshuffling. The model thus possesses the minimal ingredients necessary to investigate the much debated issue of whether the refolding process occurs through partially structured intermediates with native or non‐native disulfide bonds. This strategy is applied to a context of particular interest, the refolding process of hirudin, a thrombin‐specific protease inhibitor, for which conflicting folding pathways have been proposed. We show that the only two parameters in the model (temperature and disulfide strength) can be tuned to reproduce well a set of experimental transitions between species with different number of formed disulfides. This model is then used to provide a characterization of the folding process and a detailed description of the species involved in the rate‐limiting step of hirudin refolding. Proteins 2003;53:000–000. © 2003 Wiley‐Liss, Inc.
Many residues in cytochrome c populate alternative states under equilibrium conditions
Authors: Williamson Michael P.
Published: October 2003

A curved temperature dependence of an amide proton NMR chemical shift indicates that it explores discrete alternative conformations at least 1% of the time; that is, it accesses conformations that lie within 5 kcal/mol−1 of the ground state. The simulations presented show how curvature varies with the nature of the alternative state, and are compared to experimental results. From studies in different denaturant concentrations, it is concluded that at least 25% of residues in reduced horse cytochrome c, covering most of the protein, with the exception of the center of the N‐ and C‐terminal helices, visit alternative states under equilibrium conditions. The conformational ensemble of the protein therefore has high structural entropy. The density of alternative states is particularly high near the heme ligand Met80, which is of interest because both redox change and the first identified stage in unfolding are associated with change in Met80 ligation. By combining theoretical and experimental approaches, it is concluded that the alternative states each comprise approximately five residues, have in general less structure than the native state, and are accessed independently. They are therefore locally unfolded structures. The locations of the alternative states match what is known of the global unfolding pathway of cytochrome c, suggesting that they may determine the pathway. Proteins 2003;53:000–000. © 2003 Wiley‐Liss, Inc.