bluuebunny/crossref_metadata_2025 huggingface dataset:
Female polyandry under male harassment: the case of the common toad (Bufo bufo)
Authors: Sztatecsny M. , Jehle R. , Burke T. , Hödl W.
Published: June 2006

Several recent studies have demonstrated the occurrence of multiple paternity in anuran amphibians, implying that it is more common than previously thought. However, an adaptive explanation for polyandry in anurans is still lacking. The common toad Bufo bufo is an explosively breeding species that releases its eggs in strings. The operational sex ratio (OSR) is male biased, causing strong scramble competition among males; females can even drown through harassment during multiple amplexi. We used microsatellite markers to determine patterns of paternity in natural B. bufo populations and experimentally mated individuals (females exposed to either two or six males). Thirty per cent of field‐collected and 22% of experimentally produced egg strings were sired by at least two males; all others were sired by a single father. Multiple paternities arose only from multiple amplexi, and we found no indication of fertilization from non‐amplexing males, for example through free‐swimming sperm. Our results suggest that polyandry in B. bufo is likely to occur most often at high population densities, and under the most male‐biased OSRs. Moreover, polyandry might be interpreted as being the consequence of females spawning when amplexed by a few males, to avoid the risk of drowning by amplexus with multiple males.
Mixed‐type noncompetitive inhibition of anthrax lethal factor protease by aminoglycosides
Authors: Kuzmic Petr , Cregar Lynne , Millis Sherri Z. , Goldman Mark
Published: June 2006

We report a detailed kinetic investigation of the aminoglycosides neomycin B and neamine as inhibitors of the lethal factor protease from Bacillus anthracis. Both inhibitors display a mixed‐type, noncompetitive kinetic pattern, which suggests the existence of multiple enzyme–inhibitor binding sites or the involvement of multiple structural binding modes at the same site. Quantitative analysis of the ionic strength effects by using the Debye–Hückel model revealed that the average interionic distance at the point of enzyme–inhibitor attachment is likely to be extremely short, which suggests specific, rather than nonspecific, binding. Only one ion pair seems to be involved in the binding process, which suggests the presence of a single binding site. Combining the results of our substrate competition studies with the ionic strength effects on the apparent inhibition constant, we propose that aminoglycoside inhibitors, such as neomycin B, bind to the lethal factor protease from B. anthracis in two different structural orientations. These results have important implications for the rational design of lethal factor protease inhibitors as possible therapeutic agents against anthrax. The strategies and methods we describe are general and can be employed to investigate in depth the mechanism of inhibition by other bioactive compounds.
Biochemical characterization of recombinant dihydroorotate dehydrogenase from the opportunistic pathogenic yeast Candida albicans
Authors: Zameitat Elke , Gojković Zoran , Knecht Wolfgang , Piškur Jure , Löffler Monika
Published: June 2006

Candida albicans is the most prevalent yeast pathogen in humans, and recently it has become increasingly resistant to the current antifungal agents. In this study we investigated C. albicans dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), which catalyzes the fourth step of de novo pyrimidine synthesis, as a new target for controlling infection. We propose that the enzyme is a member of the DHODH family 2, which comprises mitochondrially bound enzymes, with quinone as the direct electron acceptor and oxygen as the final electron acceptor. Full‐length DHODH and N‐terminally truncated DHODH, which lacks the targeting sequence and the transmembrane domain, were subcloned from C. albicans, recombinantly expressed in Escherichia coli, purified, and characterized for their kinetics and substrate specificity. An inhibitor screening with 28 selected compounds was performed. Only the dianisidine derivative, redoxal, and the biphenyl quinoline‐carboxylic acid derivative, brequinar sodium, which are known to be potent inhibitors of mammalian DHODH, markedly reduced C. albicans DHODH activity. This study provides a background for the development of antipyrimidines with high efficacy for decreasing in situ pyrimidine nucleotide pools in C. albicans.
A Sex Difference by Item Difficulty Interaction in Multiple‐Choice Mathematics Items Administered to National Probability Samples
Authors: Bielinski John , Davison Mark L.
Published: June 2006

A 1998 study by Bielinski and Davison reported a sex difference by item difficulty interaction in which easy items tended to be easier for females than males, and hard items tended to be harder for females than males. To extend their research to nationally representative samples of students, this study used math achievement data from the 1992 NAEP, the TIMSS, and the NELS:88. The data included students in grades 4, 8, 10, and 12. The interaction was assessed by correlating the item difficulty difference (bmale− bfemale) with item difficulty computed on the combined male/female sample. Using only the multiple‐choice mathematics items, the predicted negative correlation was found for all eight populations and was significant in five. An argument is made that this phenomenon may help explain the greater variability in math achievement among males as compared to females and the emergence of higher performance of males in late adolescence.
The Impact of Omitted Responses on the Accuracy of Ability Estimation in Item Response Theory
Authors: De Ayala R. J. , Plake Barbara S. , Impara James C.
Published: June 2006

Practitioners typically face situations in which examinees have not responded to all test items. This study investigated the effect on an examinee's ability estimate when an examinee is presented an item, has ample time to answer, but decides not to respond to the item. Three approaches to ability estimation (biweight estimation, expected a posteriori, and maximum likelihood estimation) were examined. A Monte Carlo study was performed and the effect of different levels of omissions on the simulee's ability estimates was determined. Results showed that the worst estimation occurred when omits were treated as incorrect. In contrast, substitution of 0.5 for omitted responses resulted in ability estimates that were almost as accurate as those using complete data. Implications for practitioners are discussed.
The New Faces of Fairness
Authors: Cole Nancy S. , Zieky Michael J.
Published: June 2006

For most of the 20th century, measurement professionals paid little interest to item and test fairness. A confluence of events in the late 1960s and early 1970s led to an intense interest in fairness issues among measurement professionals. In spite of more than 30 years of effort, there is still no generally accepted definition of fairness with respect to testing and no measure that can prove or disprove the fairness of a test. To advance the fairness of tests, measurement professionals must pay more attention to reducing group differences at the design stage of test development, to providing all examinees an opportunity to demonstrate their knowledge and skills, to deterring test misuse, and to accommodating differences among individuals.
Increasing the Reliability of Ability‐Achievement Difference Scores: An Example Using the Kaufman Assessment Battery for Children
Authors: Caruso John C. , Witkiewitz Katie
Published: June 2006

In this study, we focused on increasing the reliability of ability‐achievement difference scores using the Kaufman Assessment Battery for Children (KABC) as an example. Ability‐achievement difference scores are often used as indicators of learning disabilities, but when they are derived from traditional equally weighted ability and achievement scores, they have suboptimal psychometric properties because of the high correlations between the scores. As an alternative to equally weighted difference scores, we examined an orthogonal reliable component analysis, (RCA) solution and an oblique principal component analysis (PCA) solution for the standardization sample of the KABC (among 5‐ to 12‐year‐olds). The components were easily identifiable as the simultaneous processing, sequential processing, and achievement constructs assessed by the KABC. As judged via the score intercorrelations, all three types of scores had adequate convergent validity, while the orthogonal RCA scores had superior discriminant validity, followed by the oblique PCA scores. Differences between the orthogonal RCA scores were more reliable than differences between the oblique PCA scores, which were in turn more reliable than differences between the traditional equally weighted scores. The increased reliability with which the KABC differences are assessed with the orthogonal RCA method has important practical implications, including narrower confidence intervals around difference scores used in individual administrations of the KABC.
Inferences About Variance Components and Reliability‐Generalizability Coefficients in the Absence of Random Sampling
Authors: Kane Michael
Published: June 2006

Generalizability theory (G theory) employs random‐effects ANOVA to estimate the variance components included in generalizability coefficients, standard errors, and other indices of precision. The ANOVA models depend on random sampling assumptions, and the variance‐component estimates are likely to be sensitive to violations of these assumptions. Yet, generalizability studies do not typically sample randomly. This kind of inconsistency between assumptions in statistical models and actual data collection procedures is not uncommon in science, but it does raise fundamental questions about the substantive inferences based on the statistical analyses. This article reviews criticisms of sampling assumptions in G theory (and in reliability theory) and examines the feasibility of using representative sampling, stratification, homogeneity assumptions, and replications to address these criticisms.
Identifying Content and Cognitive Skills that Produce Gender Differences in Mathematics: A Demonstration of the Multidimensionality‐Based DIF Analysis Paradigm
Authors: Gierl Mark J. , Bisanz Jeffrey , Bisanz Gay L. , Boughton Keith A.
Published: June 2006

Progress has been made in developing statistical methods for identifying DIF items, but procedures to aid with the substantive interpretations of these items have lagged behind. To overcome this problem, Roussos and Stout (1996) proposed a multidimensionality‐based DIF analysis paradigm. We illustrate and evaluate an application of this framework as it applied to the study of gender differences in mathematics. Four characteristics distinguish this study from previous research: the substantive analysis was guided by past research on the content and cognitive‐related sources of gender differences in mathematics achievement, as presented in the taxonomy by Gallagher, De Lisi, Holst, McGillicuddy‐De Lisi, Morely, and Cahalan (2000); the substantive analysis was conducted by reviewers who were highly knowledgeable about the cognitive strategies students use to solve math problems; three statistical methods were used to test hypotheses about gender differences, including SIBTEST, DIMTEST, and multiple linear regression; and the data were from a curriculum‐based achievement test developed with the goal of minimizing obvious, content‐related gender differences. We show that the framework can lead to clearly interpretable results and we highlight both the strengths and weaknesses of applying the Roussos and Stout framework to the study of group differences.
A Method for Maintaining Scale Stability in the Presence of Test Speededness
Authors: Wollack James A. , Cohen Allan S. , Wells Craig S.
Published: June 2006

Administering tests under time constraints may result in poorly estimated item parameters, particularly for items at the end of the test (Douglas, Kim, Habing, & Gao, 1998; Oshima, 1994). Bolt, Cohen, and Wollack (2002) developed an item response theory mixture model to identify a latent group of examinees for whom a test is overly speeded, and found that item parameter estimates for end‐of‐test items in the nonspeeded group were similar to estimates for those same items when administered earlier in the test. In this study, we used the Bolt et al. (2002) method to study the effect of removing speeded examinees on the stability of a score scale over an II‐year period. Results indicated that using only the nonspeeded examinees for equating and estimating item parameters provided a more unidimensional scale, smaller effects of item parameter drift (including fewer drifting items), and less scale drift (i.e., bias) and variability (i.e., root mean squared errors) when compared to the total group of examinees.
Population Invariance in Equating and Linking: Concept and History
Authors: Kolen Michael J.
Published: June 2006

The concept of invariance in equating and linking is traced from the 1950s to the present. A number of research studies that examined population invariance are reviewed. Theory and research suggest that linkings other than equatings are population dependent. Theory also indicates that equatings are population dependent, although when test forms are built to detailed tables of content and statistical specifications and alternate forms are very similar to one another, the research suggests that equatings might be approximately population invariant. Suggestions are made about further research that should be conducted on methodology for examining population invariance and on empirical research to better understand the conditions under which equatings are sufficiently population invariant for practical purposes.
Using Subpopulation Invariance to Assess Test Score Equity
Authors: Dorans Neil J.
Published: June 2006

Score equity assessment (SEA) is introduced, and placed within a fair assessment context that includes differential prediction or fair selection and differential item functioning. The notion of subpopulation invariance of linking functions is central to the assessment of score equity, just as it has been for differential item functioning and differential prediction. Advanced Placement (AP) data are used for illustrative purposes. The use of multiple‐choice and constructed response items in AP provides an opportunity to observe a case where subpopulation invariance of linking functions does not hold (U.S. History), and a case in which it does hold (Calculus AB). The lack of invariance for U.S. History might be attributed to several sources. The role of SEA in assessing the fairness of test assembly processes is discussed.
Identification of Novel Small‐molecule Inducers of Fetal Hemoglobin Using Pharmacophore and ‘PSEUDO’ Receptor Models
Authors: Bohacek Regine , Boosalis Michael S. , McMartin Colin , Faller Douglas V. , Perrine Susan P.
Published: June 2006

Pharmacologic reinduction of the developmentally silenced fetal (γ) globin genes has been achieved in hemoglobinopathy patients using short chain fatty acid derivatives, with therapeutic effects. However, higher‐potency inducers than are available in currently identified short chain fatty acid derivatives are desirable for long‐term use. Using several short‐chain fatty acids with established γ‐globin induction activity, a pharmacophore template was constructed with the TFIT module of the flo software and used to select several new candidate compounds, three of which exhibited significant activity in a γ‐globin gene reporter transcriptional assay which detects only strong inducers. The data were used to construct a new pharmacophore and a ‘pseudo’ receptor around it. Six hundred and thirty low‐molecular weight compounds were docked into this receptor model. Of 26 compounds selected and tested in functional assays, two compounds showed activity >500% over control levels and two had activity 200% over control range, significantly more active than previously identified short chain fatty acid derivative fetal globin gene inducers. Three compounds had less activity; the remainder showed moderate activity. These findings demonstrate the feasibility of using iterative construction of pharmacophores, pseudo‐binding site modeling, and virtual screening to identify small molecules with the ability to induce transcription of specific target genes, for potential therapeutics.
Making lawyers moral? Ethical codes and moral character
Authors: Nicolson Donald
Published: June 2006

This article argues that professional codes of conduct cannot perform the important task of ensuring that lawyers uphold high ethical standards. Instead, moral behaviour by lawyers requires the development of fixed behavioural attributes relevant to legal practice - what may be called a lawyer's professional moral character. At the same time, however, along with other factors, professional codes are important in that they can either contribute to or detract from the successful development of professional moral character. If so, it is argued that in order to have the best chance of assisting the character development of lawyers, codes should neither take the form of highly detailed or extremely vague, aspirational norms, but should instead guide ethical decision-making by requiring them to consider a wide range of contextual factors when resolving ethical dilemmas.
Measurement of Forces Inside a Three-Dimensional Pile of Frictionless Droplets
Authors: Zhou J. , Long S. , Wang Q. , Dinsmore A. D.
Published: June 2006

We present systematic and detailed measurements of interparticle contact forces inside three-dimensional piles of frictionless liquid droplets. We measured long-range chainlike correlations of the directions and magnitudes of large forces, thereby establishing the presence of force chains in three dimensions. Our correlation definition provides a chain persistence length of 10 mean droplet diameters, decreasing as load is applied to the pile. We also measured the angles between contacts and showed that the chainlike arrangement arises from the balance of forces. Moreover, we found that piles whose height was comparable to the chain persistence length exhibited substantially greater strain hardening than did tall piles, which we attributed to the force chains. Together, the results establish a connection between the microscopic force network and the elastic response of meso- or macroscopic granular piles. The conclusions drawn here should be relevant in jammed systems generally, including concentrated emulsions and piles of sand or other heavy particles.
Neuronal Pathway from the Liver Modulates Energy Expenditure and Systemic Insulin Sensitivity
Authors: Uno Kenji , Katagiri Hideki , Yamada Tetsuya , Ishigaki Yasushi , Ogihara Takehide , Imai Junta , Hasegawa Yutaka , Gao Junhong , Kaneko Keizo , Iwasaki Hiroko , Ishihara Hisamitsu , Sasano Hironobu , Inukai Kouichi , Mizuguchi Hiroyuki , Asano Tomoichiro , Shiota Masakazu , Nakazato Masamitsu , Oka Yoshitomo
Published: June 2006

Coordinated control of energy metabolism and glucose homeostasis requires communication between organs and tissues. We identified a neuronal pathway that participates in the cross talk between the liver and adipose tissue. By studying a mouse model, we showed that adenovirus-mediated expression of peroxisome proliferator–activated receptor (PPAR)–g2 in the liver induces acute hepatic steatosis while markedly decreasing peripheral adiposity. These changes were accompanied by increased energy expenditure and improved systemic insulin sensitivity. Hepatic vagotomy and selective afferent blockage of the hepatic vagus revealed that the effects on peripheral tissues involve the afferent vagal nerve. Furthermore, an antidiabetic thiazolidinedione, a PPARg agonist, enhanced this pathway. This neuronal pathway from the liver may function to protect against metabolic perturbation induced by excessive energy storage.
Obligatory Requirement for Antibody in Recovery from a Primary Poxvirus Infection
Authors: Chaudhri Geeta , Panchanathan Vijay , Bluethmann Horst , Karupiah Gunasegaran
Published: June 2006

To understand the correlates of protective immunity against primary variola virus infection in humans, we have used the well-characterized mousepox model. This is an excellent surrogate small-animal model for smallpox in which the disease is caused by infection with the closely related orthopoxvirus, ectromelia virus. Similarities between the two infections include virus replication and transmission, aspects of pathology, and development of pock lesions. Previous studies using ectromelia virus have established critical roles for cytokines and effector functions of CD8 T cells in the control of acute stages of poxvirus infection. Here, we have used mice deficient in B cells to demonstrate that B-cell function is also obligatory for complete virus clearance and recovery of the host. In the absence of B cells, virus persists and the host succumbs to infection, despite the generation of CD8 T-cell responses. Intriguingly, transfer of naive B cells or ectromelia virus-immune serum to B-cell-deficient mice with established infection allowed these animals to clear virus and fully recover. In contrast, transfer of ectromelia virus-immune CD8 T cells was ineffective. Our data show that mice deficient in CD8 T-cell function die early in infection, whereas those deficient in B cells or antibody production die much later, indicating that B-cell function becomes critical after the effector phase of the CD8 T-cell response to infection subsides. Strikingly, our results show that antibody prevents virus from seeding the skin and forming pock lesions, which are important for virus transmission between hosts.
Cyclooxygenase 2 Induced by Kaposi's Sarcoma-Associated Herpesvirus Early during In Vitro Infection of Target Cells Plays a Role in the Maintenance of Latent Viral Gene Expression
Authors: Sharma-Walia Neelam , Raghu Hari , Sadagopan Sathish , Sivakumar Ramu , Veettil Mohanan Valiya , Naranatt Pramod P. , Smith Marilyn M. , Chandran Bala
Published: June 2006

Infection of human dermal microvascular endothelial (HMVEC-d) cells and human foreskin fibroblast (HFF) cells in vitro by Kaposi's sarcoma-associated herpesvirus (KSHV) provides an excellent in vitro model system to study viral latency. KSHV infection is characterized by the induction of preexisting host signal cascades; sustained expression of the latency-associated open reading frame 73 (ORF73) (LANA-1), ORF72, and K13 genes; transient expression of a limited number of lytic genes, including the lytic cycle switch ORF50 (replication and transcription activator) gene; and reprogramming of host transcriptional machinery regulating a variety of cellular processes, including several proinflammatory responses. The cyclooxygenase 2 (COX-2) gene was one of the host cell genes that was highly up-regulated at 2 and 4 h postinfection (p.i.) of HMVEC-d and HFF cells (P. P. Naranatt, H. H. Krishnan, S. R. Svojanovsky, C. Bloomer, S. Mathur, and B. Chandran, Cancer Res. 64:72-84, 2004). Since COX-2 is an important mediator of inflammatory and angiogenic responses, here, using real-time PCR, Western blot, and immunofluorescence assays, we characterized the COX-2 stimulation and its role in KSHV infection. KSHV induced a robust COX-2 expression, which reached a maximum at 2 h p.i. in HMVEC-d cells and at 8 h p.i. in HFF cells, and significantly higher levels were continuously detected for up to 72 h p.i. Constitutive COX-1 protein levels were not modulated by KSHV infection. Moderate levels of COX-2 were also induced by UV-irradiated KSHV and by envelope glycoproteins gB and gpK8.1A; however, viral gene expression appears to be essential for the increased COX-2 induction. High levels of prostaglandin E
2
(PGE
2
), a COX-2 product, were released in the culture supernatant medium of infected cells. PGE
2
synthase, catalyzing the biosynthesis of PGE
2
, also increased upon infection and inhibition of COX-2 by NS-398, and indomethacin drastically reduced the levels of PGE
2
and PGE
2
synthase. COX-2 inhibition did not affect KSHV binding, internalization of virus, or the trafficking to the infected cell nuclei. However, latent ORF73 gene expression and ORF73 promoter activity were significantly reduced by COX-2 inhibitors, and this inhibition was relieved by exogenous supplementation with PGE
2
. In contrast, lytic ORF50 gene expression and ORF50 promoter activity were unaffected. These studies demonstrate that COX-2 and PGE
2
play roles in facilitating latent viral gene expression and the establishment and maintenance of latency and suggest that KSHV has evolved to utilize the inflammatory responses induced during infection of endothelial cells for the maintenance of viral latent gene expression.
Intracellular Activated Notch1 Is Critical for Proliferation of Kaposi's Sarcoma-Associated Herpesvirus-Associated B-Lymphoma Cell Lines In Vitro
Authors: Lan Ke , Choudhuri Tathagata , Murakami Masanao , Kuppers Daniel A. , Robertson Erle S.
Published: June 2006

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus expressing latent antigens critical for pathogenesis. The mechanism by which KSHV mediates oncogenesis has not been fully elucidated. Notch signaling is an evolutionarily conserved pathway controlling diverse events related to development, proliferation, and tissue homeostasis. Deregulation of Notch signaling has also been shown to be highly correlated with oncogenesis. Here we show that the activated intracellular domain of Notch1 (ICN) is aberrantly accumulated in latently KSHV-infected pleural effusion lymphoma cells and results in increased proliferation. Specifically, growth of the infected cells was dramatically inhibited at the G1phase by treatment with a γ-secretase inhibitor which specifically blocks the production of ICN. Increased ICN also up-regulated the cyclin D1 cell cycle regulator. Taken together, these studies define an important mechanism directly linking latent KSHV infection to induction of oncogenesis through dysregulation of the conserved Notch signaling pathway.
Poxvirus CD8+T-Cell Determinants and Cross-Reactivity in BALB/c Mice
Authors: Tscharke David C. , Woo Wai-Ping , Sakala Isaac G. , Sidney John , Sette Alessandro , Moss Denis J. , Bennink Jack R. , Karupiah Gunasegaran , Yewdell Jonathan W.
Published: June 2006

Mouse models of orthopoxvirus disease provide great promise for probing basic questions regarding host responses to this group of pathogens, which includes the causative agents of monkeypox and smallpox. However, some essential tools for their study that are taken for granted with other mouse models are not available for these viruses. Here we map and characterize the initial CD8+T-cell determinants for poxviruses inH-2d-haplotype mice. CD8+T cells recognizing these three determinants make up around 40% of the total responses to vaccinia virus during and after resolution of infection. We then use these determinants to test if predicted conservation across orthopoxvirus species matches experimental observation and find an unexpectedly cross-reactive variant peptide encoded by ectromelia (mousepox) virus.
Suppression of the Interferon-Mediated Innate Immune Response by Pseudorabies Virus
Authors: Brukman Alla , Enquist L. W.
Published: June 2006

Pseudorabies virus (PRV) is an alphaherpesvirus related to the human pathogens herpes simplex virus type 1 (HSV-1) and varicella-zoster virus. PRV is capable of infecting and killing a wide variety of mammals. How it avoids innate immune defenses in so many hosts is not understood. While the anti-interferon (IFN) strategies of HSV-1 have been studied, little is known about how PRV evades the IFN-mediated immune response. In this study, we determined if wild-type PRV infection can overcome the establishment of a beta interferon (IFN-β)-induced antiviral state in primary rat fibroblasts. Using microarray technology, we found that the expression of a subset of genes normally induced by IFN-β in these cells was not induced when the cells were simultaneously infected with a wild-type PRV strain. Expression of transcripts associated with major histocompatibility complex class I antigen presentation and NK cell activation was reduced, while transcripts associated with inflammation either were unaffected or were induced by viral infection. This suppression of IFN-stimulated gene expression occurred because IFN signal transduction, in particular the phosphorylation of STAT1, became less effective in PRV-infected cells. At least one virion-associated protein is involved in inhibition of STAT1 tyrosine phosphorylation. This ability to disarm the IFN-β response offers an explanation for the uniform lethality of virulent PRV infection of nonnatural hosts.
A Highly Restricted T-Cell Receptor Dominates the CD8+T-Cell Response to Parvovirus B19 Infection in HLA-A*2402-Positive Individuals
Authors: Kasprowicz V. , Isa A. , Jeffery K. , Broliden K. , Tolfvenstam T. , Klenerman P. , Bowness P.
Published: June 2006

Six of seven HLA-A*2402-positive individuals with acute parvovirus B19 infections made vigorous CD8-positive cytotoxic T-cell (CTL) responses to the viral epitope FYTPLADQF. All responders showed highly focused T-cell receptor (TCR) usage, using almost exclusively BV5.1. The BV5.1 TCR dominated the acute response, was maintained over time, and was also used by a remotely infected individual. Nine CTL clones and two oligoclonal lines obtained from three unrelated individuals used BV5.1, BJ2.1, and a conserved TCR CDR3 of nine amino acids. This commonly recognized epitope is likely important in long-term protective immunity and should be included in vaccine design.
Semi-quantitative scoring of potentially predictive markers for endocrine treatment of breast cancer: a comparison between whole sections and tissue microarrays
Authors: Henriksen Katrine L , Rasmussen Birgitte B , Lykkesfeldt Anne E , Møller Susann , Ejlertsen Bent , Mouridsen Henning T
Published: June 2006

Aim:To assess whether immunohistochemically stained tissue microarrays (TMA) of 2 mm cores from paraffin embedded tumour tissue may replace whole sections in semi-quantitative evaluation of selected potential markers for endocrine treatment.

Methods:Whole sections and 2 mm cores on TMA were used for immunohistochemical staining of potential markers for endocrine treatment. The Allred scoring system was used for the markers with nuclear localisation: the oestrogen receptor, the progesterone receptor, p27 and the oestrogen receptor co-regulator amplified in breast cancer 1 (AIB1). The Allred scoring system was also used for the non-nuclear markers Bcl-2, pS2 and cyclooxygenase 2 (COX-2); the membrane receptors HER-2, insulin-like growth factor I receptor (IGF-IR) and epidermal growth factor receptor were quantified according to the guidelines for the Herceptest.

Results:The data and statistical analyses showed that the semi-quantitative evaluation of oestrogen receptor, progesterone receptor, AIB1, COX-2, HER-2 and IGF-IR on TMA blocks was comparable with analysis on whole sections.

Conclusions:This study shows that semi-quantitative scoring of 2 mm cores on TMA is feasible for several potential markers for endocrine therapy. Considering the small size of many breast tumours, the speed and cost-effectiveness of immunohistochemistry on TMA compared with whole sections, and the importance of the expression level of the proteins, semi-quantitative scoring on TMA has great potential in both retrospective and prospective studies aiming at improving the prediction of response to endocrine treatment.
Overexpression of cellular inhibitor of apoptosis protein 2 is an early event in the progression of pancreatic cancer
Authors: Esposito Irene , Kleeff Jörg , Abiatari Ivane , Shi Xined , Giese Nathalia , Bergmann Frank , Roth Wilfried , Friess Helmut , Schirmacher Peter
Published: June 2006

Aim:To determine the role of two antiapoptotic proteins of the inhibitor of apoptosis protein family, cellular inhibitor of apoptosis protein 1 (cIAP1) and cellular inhibitor of apoptosis protein 2 (cIAP2), in human pancreatic carcinogenesis.

Methods:mRNA levels were measured in pancreatic tissues and pancreatic cancer cell lines by quantitative reverse transcriptase PCR. Protein expression was assessed in pancreatic cancer cell lines by immunoblotting and in pancreatic tissues by immunohistochemistry, and correlated with pathological and survival data.

Results:cIAP1 expression was constantly high in non-neoplastic pancreatic tissues, in pancreatic intraepithelial neoplasia (PanIN) lesions, as well as in a subset of primary and metastatic pancreatic ductal adenocarcinomas (PDAC), and a preferential cytoplasmatic localisation was observed in the tumour tissues. cIAP1 expression was rare in a cohort of cystic tumours. cIAP2 mRNA levels were significantly higher (2.4 fold) in PDAC than in normal tissues. cIAP2 protein was overexpressed in PDAC, and was detectable in low- and high-grade PanIN lesions. Moreover, cIAP2 was often expressed in pancreatic cystic tumours. cIAP1 and cIAP2 mRNA and protein were detected in all the examined cell lines. Survival analysis revealed a shorter survival in patients with cIAP1/cIAP2-positive tumours.

Conclusions:cIAP1 might contribute to the regulation of the apoptotic process in the normal and in the neoplastic pancreas, depending on its subcellular localisation. Overexpression of cIAP2 is a common and early event in the progression of pancreatic cancer, and could therefore potentially influence the important pathophysiological aspects of PDAC, such as anoikis or chemoresistance.
STUDY OF TWO BIOACTIVE PEPTIDES IN VACUUM AND SOLVENT BY MOLECULAR MODELING
Authors: YAŞAR F. , DEMIR K.
Published: June 2006

The thermodynamic and structural properties of Tyrosine-Glycine-Leusine-Phenylalanine (YGLF, in a one letter code) and Lysine-Valine-Leusine-Proline-Valine-Proline-Glutamine (KVLPVPQ) peptide sequences were studied by three-dimensional molecular modeling in vacuum and solution. All the three-dimensional conformations of each peptide sequences were obtained by multicanonical simulations with using ECEPP/2 force field and each simulation started from completely random initial conformation. Solvation contributions are included by a term that is proportional to solvent-accessible surface areas of peptides. In the present study, we calculated the average values of total energy, specific heat, fourth-order cumulant and end-to-end distance for two peptide sequences of milk protein as a function of temperature. With using major advantage of this simulation technique, Ramachandran plots were prepared and analysed to predict the relative occurrence probabilities of β-turn, γ-turn and helical structures. Although structural predictions of these sequences indicate both the presence of high level of γ-turns and low level of β-turns in vacuum and solvent, it was observed that these probabilities in vacuum were higher than the ones in solvent model.
Emergence of Action Potential Generation and Synaptic Transmission in Vestibular Nucleus Neurons
Authors: Shao Mei , Hirsch June C. , Peusner Kenna D.
Published: June 2006

Principal cells of the chick tangential nucleus are vestibular nucleus neurons in the hindbrain. Although detailed information is available on the morphogenesis of principal cells and synaptogenesis of primary vestibular fibers, this is the first study of their early functional development, when vestibular terminals emerge at embryonic days 10 and 13 (E10 and E13). At E10, 60% of principal cells generated spikes on depolarization, whereas 50% exhibited excitatory postsynaptic currents (EPSCs) on vestibular-nerve stimulation. The frequency was 0.2 Hz for glutamatergic spontaneous EPSCs (sEPSCs) at −60 mV, and 0.6 Hz for spontaneous inhibitory postsynaptic current (sIPSC) at +10 mV and completely GABAergic. All of these synaptic events were TTX-insensitive, miniature events. At E13, 50% of principal cells generated spikes on depolarization and 82% exhibited EPSCs on vestibular-nerve stimulation. The frequency was 0.7 Hz for sEPSCs at −60 mV, and 0.8 Hz for sIPSCs at +10 mV. Most principal cells had sIPSCs composed of both GABAergic (75%) and glycinergic (25%) events, but a few cells had only GABAergic sIPSCs. TTX decreased the frequency of EPSCs by 12%, and the IPSCs by 17%. In summary, at E10, some principal cells generated immature spikes on depolarization and EPSCs on vestibular-nerve stimulation. At E10, GABAergic events predominated, AMPA events had low frequencies, and glycinergic activity was absent. By E13, glycinergic events first appeared. This data were compared systematically to that obtained from the late-term embryo and hatchling to reveal the long-term sequence of changes in synaptic events and excitability and offer a broader understanding of how the vestibular system is assembled during development.
Rotation of Motoneurons During Prolonged Isometric Contractions in Humans
Authors: Bawa Parveen , Pang Marco Y. , Olesen Kari A. , Calancie Blair
Published: June 2006

Prolonged and weak isometric contractions can result in neuromuscular fatigue. Alternation of discharge of motor units with similar thresholds (termed rotation) could be useful to minimize neuromuscular fatigue by providing periods for metabolic recovery of the contractile elements. In the present study, we investigated the prevalence of motoneuron rotation during prolonged contractions of distal limb muscles. Electromyographic (EMG; needle and surface) was recorded from muscles of the forearm and distal leg. The subject made a slowly increasing isometric contraction to recruit and discharge a motor unit ( 1) for a prolonged period of time (>30 min). Sometimes an additional motor unit ( 2) was recruited in which case the subject relaxed the contraction slightly so that only one motor unit remained tonic. Often it was this newly recruited motor unit (i.e., unit 2) that continued discharging, while motor unit 1 fell silent. Continued contraction would then lead to the resumption of tonic discharge of unit 1 and silence of unit 2. This would complete a rotation between motor units 1 and 2. During prolonged contractions, rotation was observed in ∼80% of the motor-unit pairs examined. There was no difference in rotation incidence by muscle type. For the unit pairs showing rotation, surface EMG values were significantly higher immediately prior to rotation than after rotation had occurred. Our findings show that rotation of motor units with similar recruitment thresholds during such contractions is common in distal muscles of the arm and leg and may help offset neuromuscular fatigue.
Activation and Deactivation of Voltage-Dependent K+ Channels During Synaptically Driven Action Potentials in the MNTB
Authors: Klug Achim , Trussell Laurence O.
Published: June 2006

K+ channels shape individual action potentials and determine their pattern of firing. In auditory relays, both high- and low-voltage–activated K+ channels (HVA and LVA) are critical for preservation of auditory timing cues. We examined how these channels participate in firing in the medial nucleus of the trapezoid body. Principal cells at physiological temperature were voltage clamped using spike waveforms previously recorded in response to calyceal firing. Current components were isolated by digital subtraction of traces recorded in the channel antagonists dendrotoxin-I or tetraethylammonium. During orthodromic spikes delivered at 300 and 600 Hz, both currents activated with a slight delay, peaking just after the crest of the spike. The decay of HVA was sufficiently fast to match the time course of the spike. By contrast, with 300-Hz stimuli, LVA continued to decay after the spikes reached a stable interspike potential. Although LVA currents partially inactivate during prolonged voltage steps, their peak amplitudes remained stable or increased during trains of spikelike stimuli. At 600 Hz, LVA did not fully deactivate between the spikes and therefore generated a leak current. To determine the effect of blocking LVA channels on spiking, prerecorded postsynaptic conductances were injected, with and without dendrotoxin-I. After block of LVA channels, strong synaptic conductances produced broader spikes, greater spike jitter, and prolonged depolarized states. HVA blockade with tetraethylammonium also broadened spikes but led to less error in timing. These results reveal multiple roles for LVA channels in spike repolarization and timing during synaptic activity.
Melanoma-Reactive T Cells in the Bone Marrow of Melanoma Patients: Association with Disease Stage and Disease Duration
Authors: Müller-Berghaus Jan , Ehlert Katrin , Ugurel Selma , Umansky Viktor , Bucur Mariana , Schirrmacher Volker , Beckhove Philipp , Schadendorf Dirk
Published: June 2006

Using IFN-γ enzyme-linked immunospot, we investigated reactivity of T cells from bone marrow and peripheral blood to melanoma lysate-pulsed autologous dendritic cells in 40 melanoma patients. Melanoma-reactive T cells were present in the bone marrow of seven patients and in peripheral blood of four patients. In the bone marrow, melanoma-reactive T cells were present in 6 of 21 stage IV patients and in 1 of 10 stage III patients, whereas none were detected in stage I to II patients (0 of 9). The occurrence of tumor-reactive bone marrow T cells in melanoma patients was associated with advanced disease stage, disease duration and tumor load, and independent of treatment. These findings provide new insights into the generation of T-cell responses in melanoma patients. (Cancer Res 2006; 66(12): 5997-6001)
Haploinsufficiency of RAD51B Causes Centrosome Fragmentation and Aneuploidy in Human Cells
Authors: Date Osamu , Katsura Mari , Ishida Mari , Yoshihara Takashi , Kinomura Aiko , Sueda Taijiro , Miyagawa Kiyoshi
Published: June 2006

The Rad51-like proteins, Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3, have been shown to form two distinct complexes and seem to assist Rad51 in the early stages of homologous recombination. Although these proteins share sequence similarity with Rad51, they do not show functional redundancy. Among them, Rad51B is unique in that the gene maps to the human chromosome 14q23-24, the region frequently involved in balanced chromosome translocations in benign tumors particularly in uterine leiomyomas. Despite accumulating descriptive evidence of altered Rad51B function in these tumors, the biological significance of this aberration is still unknown. To assess the significance of reduced Rad51B function, we deleted the gene in the human colon cancer cell line HCT116 by gene targeting. Here, we show that haploinsufficiency of RAD51B causes mild hypersensitivity to DNA-damaging agents, a mild reduction in sister chromatid exchange, impaired Rad51 focus formation, and an increase in chromosome aberrations. Remarkably, haploinsufficiency of RAD51B leads to centrosome fragmentation and aneuploidy. In addition, an ∼50% reduction in RAD51B mRNA levels by RNA interference also leads to centrosome fragmentation in the human fibrosarcoma cell line HT1080. These findings suggest that the proper biallelic expression of RAD51B is required for the maintenance of chromosome integrity in human cells. (Cancer Res 2006; 66(12): 6018-24)
Osteopontin Is an Oncogenic Vav1– but not Wild-type Vav1–Responsive Gene: Implications for Fibroblast Transformation
Authors: Schapira Vered , Lazer Galit , Katzav Shulamit
Published: June 2006

Mammalian wild-type Vav1 (wtVav1) encodes a specific GDP/GTP nucleotide exchange factor that is exclusively expressed in the hematopoietic system. Despite numerous studies, the mechanism underlying transformation of fibroblasts by oncogenic Vav1 (oncVav1) is not well defined. We identified osteopontin, a marker for tumor aggressiveness, as an oncVav1-inducible gene. Osteopontin is highly expressed in oncVav1-transformed NIH3T3 cells (NIH/oncVav1) but is barely detected in NIH3T3 expressing wtVav1 (NIH/wtVav1) even following epidermal growth factor stimulation, which normally induces osteopontin. Depleting oncVav1 in NIH/oncVav1 using small interfering RNA led to a considerable decrease in osteopontin, whereas reducing osteopontin expression did not affect oncVav1 expression, suggesting that oncVav1 operates upstream of osteopontin. Vav1-depleted NIH/oncVav1 cells, but not osteopontin-depleted NIH/oncVav1 cells, exhibited impaired extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase phosphorylation. Inhibition of ERK phosphorylation in NIH/oncVav1 cells led to a decrease in osteopontin expression, implying that the elevated osteopontin expression in these cells is dependent on ERK phosphorylation. Vav1-depleted or osteopontin-depleted NIH/oncVav1 cells lost their tumorigenic properties as judged by the soft agar and invasion assays, although loss of osteopontin expression had a less dramatic effect. Suppression of Vav1 expression in NIH/oncVav1 cells led to reversion to “normal” morphology, whereas when only osteopontin expression was diminished cells retained their transformed morphology. This work strongly supports a role for oncVav1 as a master oncogene and provides clues to the molecular mechanism underlying oncVav1 transformation. (Cancer Res 2006; 66(12): 6183-91)
Characterization of Endogenous Human Promyelocytic Leukemia Isoforms
Authors: Condemine Wilfried , Takahashi Yuki , Zhu Jun , Puvion-Dutilleul Francine , Guegan Sarah , Janin Anne , de Thé Hugues
Published: June 2006

Promyelocytic leukemia (PML) has been implicated in a variety of functions, including control of TP53 function and modulation of cellular senescence. Sumolated PML is the organizer of mature PML bodies, recruiting a variety of proteins onto these nuclear domains. The PML gene is predicted to encode a variety of protein isoforms. Overexpression of only one of them, PML-IV, promotes senescence in human diploid fibroblasts, whereas PML-III was proposed to specifically interact with the centrosome. We show that all PML isoform proteins are expressed in cell lines or primary cells. Unexpectedly, we found that PML-III, PML-IV, and PML-V are quantitatively minor isoforms compared with PML-I/II and could not confirm the centrosomal targeting of PML-III. Stable expression of each isoform, in a pml-null background, yields distinct subcellular localization patterns, suggesting that, like in other RBCC/TRIM proteins, the COOH-terminal domains of PML are involved in interactions with specific cellular components. Only the isoform-specific sequences of PML-I and PML-V are highly conserved between man and mouse. That PML-I contains all conserved exons and is more abundantly expressed than PML-IV suggests that it is a critical contributor to PML function(s). (Cancer Res 2006; 66(12): 6192-8)