bluuebunny/crossref_metadata_2025 huggingface dataset:
Object combining: a new aggressive optimization for object intensive programs
Authors: Veldema Ronald , Jacobs Ceriel J. H. , Hofman Rutger F. H. , Bal Henri E.
Published: February 2005

Object combining tries to put objects together that have roughly the same life times in order to reduce strain on the memory manager and to reduce the number of pointer indirections during a program's execution. Object combining works by appending the fields of one object to another, allowing allocation and freeing of multiple objects with a single heap (de)allocation. Unlike object inlining, which will only optimize objects where one has a (unique) pointer to another, our optimization also works if there is no such relation. Object inlining also directly replaces the pointer by the inlined object's fields. Object combining leaves the pointer in place to allow more combining. Elimination of the pointer accesses is implemented in a separate compiler optimization pass. Unlike previous object inlining systems, reference field overwrites are allowed and handled, resulting in much more aggressive optimization. Our object combining heuristics also allow unrelated objects to be combined, for example, those allocated inside a loop; recursive data structures (linked lists, trees) can be allocated several at a time and objects that are always used together can be combined. As Java explicitly permits code to be loaded at runtime and allows the new code to contribute to a running computation, we do not require a closed‐world assumption to enable these optimizations (but it will increase performance). The main focus of object combining in this paper is on reducing object (de)allocation overhead, by reducing both garbage collection work and the number of object allocations. Reduction of memory management overhead causes execution time to be reduced by up to 35%. Indirection removal further reduces execution time by up to 6%. Copyright © 2005 John Wiley & Sons, Ltd.
Ravenscar‐Java: a high‐integrity profile for real‐time Java
Authors: Kwon Jagun , Wellings Andy , King Steve
Published: February 2005

For many, Java is the antithesis of a high‐integrity programming language. Its combination of object‐oriented programming features, its automatic garbage collection, and its poor support for real‐time multi‐threading are all seen as particular impediments. The Real‐Time Specification for Java has introduced many new features that help in the real‐time domain. However, the expressive power of these features means that very complex programming models can be created, necessitating complexity in the supporting real‐time virtual machine. Consequently, Java, with the real‐time extensions as they stand, seems too complex for confident use in high‐integrity systems. This paper presents a Java profile for the development of software‐intensive high‐integrity real‐time systems. This restricted programming model removes language features with high overheads and complex semantics, on which it is hard to perform timing and functional analyses. The profile fits within the J2ME framework and is consistent with well‐known guidelines for high‐integrity software development, such as those defined by the U.S. Nuclear Regulatory Commission. Copyright © 2005 John Wiley & Sons, Ltd.
Improving the memory management performance of RTSJ
Authors: Higuera‐Toledano M. Teresa , Issarny Valérie
Published: February 2005

From a real‐time perspective, the garbage collector (GC) introduces unpredictable pauses that are not tolerated by real‐time tasks. Real‐time collectors eliminate this problem but introduce a high overhead. Another approach is to use memory regions (MRs) within which allocation and deallocation is customized. This facility is supported by the memory model of the Real‐Time Specification for Java (RTSJ). RTSJ imposes strict access and assignment rules to avoid both the dangling inter‐region references and the delays of critical tasks of the GC. A dynamic check solution can incur high overhead, which can be reduced by taking advantage of hardware features. This paper provides an in‐depth analytical investigation of the overhead introduced by dynamic assignments checks in RTSJ, describing and analysing several solutions to reduce the introduced overhead. Copyright © 2005 John Wiley & Sons, Ltd.
Elimination of Java array bounds checks in the presence of indirection
Authors: Luján Mikel , Gurd John R. , Freeman T. L. , Miguel José
Published: February 2005

The Java language specification states that every access to an array needs to be within the bounds of that array, i.e. between 0 and array length − 1. Different techniques for different programming languages have been proposed to eliminate explicit bounds checks. Some of these techniques are implemented in off‐the‐shelf Java Virtual Machines (JVMs). The underlying principle of these techniques is that bounds checks can be removed when a JVM/compiler has enough information to guarantee that a sequence of accesses (e.g. inside a for‐loop) is safe (within the bounds). Most of the techniques for the elimination of array bounds checks have been developed for programming languages that do not support multi‐threading and/or enable dynamic class loading. These two characteristics make most of these techniques unsuitable for Java. Techniques developed specifically for Java have not addressed the elimination of array bounds checks in the presence of indirection; that is, when the index is stored in another array (indirection array). With the objective of optimizing applications with array indirection, this paper proposes and evaluates three implementation strategies, each implemented as a Java class. The classes provide the functionality of Java arrays of type int so that objects of the classes can be used instead of indirection arrays. Each strategy enables JVMs, when examining only one of these classes at a time, to obtain enough information to remove array bounds checks. Copyright © 2005 John Wiley & Sons, Ltd.
Compiling almost‐whole Java programs
Authors: Budimlić Zoran , Kennedy Ken
Published: February 2005

This paper presents a strategy, called almost‐whole‐program compilation, for extending the benefits of whole‐program optimization to large collections of Java components that are packaged as a group after the development phase. This strategy has been implemented in a framework that uses Java visibility and scoping rules to transform a collection of classes into a package that is amenable to whole‐program optimizations, without precluding extensions to the optimized and compiled code. Thus, it enables the Java developer to balance performance against flexibility of the program after the development phase, without compromising the design process. The transformation is shown to incur only modest performance penalties, which are more than compensated for by the interprocedural optimizations it enables. The paper concludes with experimental results showing the benefits that can be achieved using this approach. Copyright © 2005 John Wiley & Sons, Ltd.
Recurrence analysis for effective array prefetching in Java
Authors: Cahoon Brendon , McKinley Kathryn S.
Published: February 2005

Java is an attractive choice for numerical, as well as other, algorithms due to the software engineering benefits of object‐oriented programming. Because numerical programs often use large arrays that do not fit in the cache, they suffer from poor memory performance. To hide memory latency, we describe a new unified compile‐time analysis for software prefetching arrays and linked structures in Java. Our previous work used data‐flow analysis to discover linked data structure accesses. We generalize our prior approach to identify loop induction variables as well, which we call recurrence analysis. Our algorithm schedules prefetches for all array references that contain induction variables. We evaluate our technique using a simulator of an out‐of‐order superscalar processor running a set of array‐based Java programs. Across all of our programs, prefetching reduces execution time by a geometric mean of 23%, and the largest improvement is 58%. We also evaluate prefetching on a PowerPC processor, and we show that prefetching reduces execution time by a geometric mean of 17%. Because our analysis is much simpler and quicker than previous techniques, it is suitable for including in a just‐in‐time compiler. Traditional software prefetching algorithms for C and Fortran use locality analysis and sophisticated loop transformations. We further show that the additional loop transformations and careful scheduling of prefetches from previous work are not always necessary for modern architectures and Java programs. Copyright © 2005 John Wiley & Sons, Ltd.
The Open Runtime Platform: a flexible high‐performance managed runtime environment
Authors: Cierniak Michal , Eng Marsha , Glew Neal , Lewis Brian , Stichnoth James
Published: February 2005

The Open Runtime Platform (ORP) is a high‐performance managed runtime environment (MRTE) that features exact generational garbage collection, fast thread synchronization, and multiple coexisting just‐in‐time compilers (JITs). ORP was designed for flexibility in order to support experiments in dynamic compilation, garbage collection, synchronization, and other technologies. It can be built to run either Java or Common Language Infrastructure (CLI) applications, to run under the Windows or Linux operating systems, and to run on the IA‐32 or Itanium processor family (IPF) architectures. Achieving high performance in a MRTE presents many challenges, particularly when flexibility is a major goal. First, to enable the use of different garbage collectors and JITs, each component must be isolated from the rest of the environment through a well‐defined software interface. Without careful attention, this isolation could easily harm performance. Second, MRTEs have correctness and safety requirements that traditional languages such as C++ lack. These requirements, including null pointer checks, array bounds checks, and type checks, impose additional runtime overhead. Finally, the dynamic nature of MRTEs makes some traditional compiler optimizations, such as devirtualization of method calls, more difficult to implement or more limited in applicability. To get full performance, JITs and the core virtual machine (VM) must cooperate to reduce or eliminate (where possible) these MRTE‐specific overheads. In this paper, we describe the structure of ORP in detail, paying particular attention to how it supports flexibility while preserving high performance. We describe the interfaces between the garbage collector, the JIT, and the core VM; how these interfaces enable multiple garbage collectors and JITs without sacrificing performance; and how they allow the JIT and the core VM to reduce or eliminate MRTE‐specific performance issues. Copyright © 2005 John Wiley & Sons, Ltd.
Experience in integrating Java with C# and .NET
Authors: Bishop Judith , Horspool R. Nigel , Worrall Basil
Published: February 2005

Java programmers cannot help but be aware of the advent of C#, the .NET network environment, and a host of new supporting technologies, such as Web services. Before taking the big step of moving all development to a new environment, programmers will want to know what are the advantages of C# as a language over Java, and whether the new and interesting features of C# and .NET can be incorporated into existing Java software. This paper surveys the advantages of C# and then presents and evaluates experience with connecting it to Java in a variety of ways. The first way provides evidence that Java can be linked to C# at the native code level, albeit through C++ wrappers. The second is a means for retaining the useful applet feature of Java in the server‐side architecture of Web services written in C#. The third is by providing a common XML‐based class for the development of graphical user interfaces (GUIs), which can be incorporated into Java or C#. An added advantage of this system, called Views, is that it can run independently of the resource‐intensive development environment that would otherwise be needed for using C#. A major advantage of the methods described in this paper is that in all cases the Java program is not affected by the fact that it is interfacing with C#. The paper concludes that there are many common shared technologies that bring Java and C# close together, and that innovative ways of using others can open up opportunities not hitherto imagined. Copyright © 2005 John Wiley & Sons, Ltd.
The Mycobacterium tuberculosis cell wall component mycolic acid elicits pathogen‐associated host innate immune responses
Authors: Korf Johanna , Stoltz Anton , Verschoor Jan , De Baetselier Patrick , Grooten Johan
Published: February 2005

Recognition of conserved pathogen‐associated molecular patterns constitutes a crucial step in the initiation of innate immune responses. We studied the contribution to the host‐pathogen interaction of mycolic acid (MA), a major lipid component of the cell envelope of the macrophage intracellular pathogen Mycobacterium tuberculosis and other mycobacteria. MA administered to the peritoneal cavity or to the airways induced a unique macrophage morphotype, similar to the foamy macrophage derivatives observed in tuberculous granulomas and characterized by intracellular accumulation of neutral lipids and entry into mitosis. When assayed for production of inflammatory mediators, a conditioning rather than a direct activation of the MA‐elicited foamy macrophages was observed. MA enabled production of IFN‐γ and myeloperoxidase, enhanced TNF‐α production and suppressed IL‐10 upon renewed exposure to innate triggers. Intratracheal instillation of MA mimicked additional features of the airway response to M. tuberculosis infection, namely a rapid but transient neutrophil influx and IL‐6 production and a chronic IL‐12 production. These MA‐elicited cellular innate defenses and the accompanying formation of foamy macrophages identify for the first time the foamy macrophage morphotype as part of the host response to a pathogen‐associated structure. Furthermore, these results characterize MA as a direct trigger of innate immunity, distinct from Toll‐like receptor ligands.
Long‐term loss of canonical NKT cells following an acute virus infection
Authors: Lin Yinling , Roberts Tonya J. , Wang Chyung‐Ru , Cho Sungyoo , Brutkiewicz Randy R.
Published: February 2005

NKT cell activation plays an important role in regulating innate and adaptive immunity during infection. We have previously found that there is a dramatic reduction in the NKT cell population on day 3 after an acute lymphocytic choriomeningitis virus (LCMV) infection. In this study, we report that this loss continued for at least 3 months and was not simply due to internalization of the TCR. Concomitant with the decrease in NKT cells was an increase in the percentage of Annexin V+ NKT cells that remained in vivo, suggesting that the reduction in NKT cells at these late stages post‐infection occurred by activation‐induced cell death. Interestingly, APC from LCMV‐infected mice could activate NKT cells in vitro at higher levels than those from uninfected mice and was concomitant with an increase in apoptosis in NKT cells. However, this could not be blocked by mAb to murine CD1d, and APC from LCMV‐infected (but not uninfected) CD1d1‐deficient mice could also stimulate NKT cells. Collectively, our data suggest that the activation and subsequent long‐term loss of NKT cells is a normal component of the host's antiviral immune response, and this occurs in a CD1d‐independent manner.
MyD88 is critical for the development of innate and adaptive immunity during acute lymphocytic choriomeningitis virus infection
Authors: Zhou Shenghua , Kurt‐Jones Evelyn A. , Mandell Leisa , Cerny Anna , Chan Melvin , Golenbock Douglas T. , Finberg Robert W.
Published: February 2005

We investigated the roles of Toll‐like receptor 2 (TLR2) and myeloid differentiation factor 88 (MyD88) in the course of a lymphocytic choriomeningitis virus (LCMV) infection and revealed the following: (i) studies of transfected cells and murine peritoneal macrophages demonstrated that TLR2 and MyD88 are essential for the initial pro‐inflammatory cytokine response (human IL‐8, mouse IL‐6) to LCMV; (ii) TLR2 knockout (KO) mice and MyD88 KO mice challenged with LCMV produced less IL‐6 and monocyte chemotactic protein‐1 in the serum than wild‐type mice; (iii) in contrast to inflammatory cytokines, the production of type 1 IFN (IFN‐α) in response to LCMV was MyD88 independent; (iv) MyD88 plays an essential role in antiviral CD8+ T cell responses, CD8+ T cells in MyD88 KO mice were defective in their expression of intracellular antiviral cytokines; and (v) the failure of MyD88 KO mice to activate CD8+ T cells was accompanied by persistent viral infection in MyD88 KO mice. We demonstrate that TLR‐mediated responses are important in the innate immune response to LCMV and that MyD88 is essential for the control of the LCMV infection and the maturation/activation of virus‐specific CD8+ T cells.
Novel method for detection of virus‐specific CD4+ T cells indicates a decreased EBV‐specific CD4+ T cell response in untreated HIV‐infected subjects
Authors: Piriou Erwan R. , van Dort Karel , Nanlohy Nening M. , van Oers Marinus H. , Miedema Frank , van Baarle Debbie
Published: February 2005

A lower function of EBV‐specific CD8+ T cells in HIV‐infected subjects could be related to a lack of specific CD4+ T cell help. Therefore, we studied EBV‐specific CD4+ T cells in both healthy donors and untreated or highly active antiretroviral therapy (HAART)‐treated HIV‐seropositive homosexual men. To this end, PBMC were stimulated with overlapping peptide pools from a latent and a lytic EBV protein, EBV nuclear antigen (EBNA)1 and EBV lytic‐switch protein ZEBRA (BZLF1), respectively. EBV‐specific CD4+ T cell frequencies measured directly ex vivo were low. To measure EBV‐specific memory CD4+ T cells, capable of both expansion and IFN‐γ production upon antigenic challenge, we developed a specific and reproducible assay, combining ex vivo expansion of specific T cells with flow cytometric analysis of IFN‐γ production. Untreated HIV‐infected individuals had a lower CD4+ T cell response to both EBNA1 and BZLF1 as compared to healthy EBV carriers and HAART‐treated HIV‐positive subjects. This suggests loss of EBV‐specific CD4+ T cells due to HIV infection, while HAART might restore this response. In addition, we found an increase in the EBNA1‐specific CD8+ T cell response in HAART‐treated subjects. Interestingly, numbers of EBV‐specific CD4+ and CD8+ T cells were inversely correlated with EBV viral load, suggesting an important role also for EBV‐specific CD4+ T cells in the control of EBV infection.
Endogenous association of Bim BH3‐only protein with Mcl‐1, Bcl‐xL and Bcl‐2 on mitochondria in human B cells
Authors: Gomez‐Bougie Patricia , Bataille Régis , Amiot Martine
Published: February 2005

Bim is an essential regulator of lymphoid system homeostasis and appears essential for B cell apoptosis induction. The mechanism by which Bim isoforms are held in an inactive form remains poorly documented in normal B cells. In the current study, we demonstrated that in normal tonsil B cells the three major Bim isoforms are strongly associated with the anti‐apoptotic Bcl‐2 family members Mcl‐1, Bcl‐2 and Bcl‐xL. On the other hand, only a weak association of BimEL and L with the dynein LC8 chain has been found. In addition, there is no free Bim in normal B cells. Moreover, subcellular fractionation demonstrated that Bim and the anti‐apoptotic counterparts are localized preferentially in the mitochondria‐rich fraction. The fact that most Bim was found in this fraction supports the hypothesis that it is sequestered by anti‐apoptotic molecules in mitochondria where its pro‐apoptotic activity is controlled. Of interest, BimS is essentially complexed to Mcl‐1 and the Mcl‐1/Bim complex is the most abundant among the three types of complexes. This supports the idea that this complex is critical for the control of B cell death. In conclusion, these results favor a model in which Bim release from anti‐apoptotic proteins is a critical event for initiation of apoptosis.
Definitive radiotherapy with interstitial implant boost for squamous cell carcinoma of the tongue base
Authors: Karakoyun‐Celik Omur , Norris Charles M. , Tishler Roy , Mahadevan Anand , Clark John R. , Goldberg Saveli , Devlin Phillip , Busse Paul M.
Published: February 2005

Background.

The purpose of this study was to examine the long‐term outcome of a cohort of patients with unresected base of tongue carcinoma who received interstitial brachytherapy after comprehensive external beam radiation therapy.

Methods.

Between 1983 and 2000, 122 patients with primary or recurrent squamous cell carcinoma of the oropharynx or oral cavity received interstitial brachytherapy as part of their overall management. Forty patients had primary, unresected carcinoma of the base of tongue and are the subjects of this analysis. The median age was 54 years. Fifty‐four percent had T3 or T4 disease, and 70% had clinical or radiographic lymphadenopathy. Twenty‐four (60%) received two to three cycles of neoadjuvant chemotherapy. The oropharynx, bilateral neck, and supraclavicular fossae were comprehensively irradiated, and the tongue base received a median external beam dose of 61.2 Gy (50–72 Gy). The primary site was then boosted with an interstitial 192Iridium implant by use of a gold‐button single‐strand technique and three‐dimensional treatment planning. The dose rate was prescribed at 0.4 to 0.5 Gy/hr. The median implant dose was 17.4 Gy (9.6–24 Gy) and adjusted to reach a total dose to the primary tumor of 80 Gy. N2 to 3 disease was managed by a planned neck dissection performed at the time of the implant.

Results.

The median follow‐up for all patients was 56 months, and the overall survival rates were 62% at 5 years and 27% at 10 years. The actuarial primary site control was 78% at 5 years and 70% at 10 years. The overall survival and primary site control were independent of T classification, N status, or overall stage. Systemic therapy was associated with an improvement in overall survival (p = .04) and a trend toward increased primary site control with greater clinical response. There were seven documented late effects, the most frequent being grade 3 osteonecrosis (n = 2), grade 2 swallowing dysfunction (n = 2), trismus (n = 2), and chronic throat pain (n = 1).

Conclusions.

In an era of greatly improved dose distributions made possible by three‐dimensional treatment planning and intensity‐modulated radiation therapy, brachytherapy allows a highly conformal dose to be delivered in sites such as the oropharynx. If done properly, the procedure is safe and delivers a dose that is higher than what can be achieved by external beam radiation alone with the expected biologic advantages. The long‐term data presented here support an approach of treating advanced tongue base lesions that includes interstitial brachytherapy as part of the overall management plan. This approach has led to a 78% rate of organ preservation at 5 years, with a 5% incidence of significant late morbidity (osteonecrosis) that has required medical management. © 2005 Wiley Periodicals, Inc. Head Neck 27: XXX–XXX, 2005
In vitro drug release studies of 2‐hydroxyethyl acrylate or 2‐hydroxypropyl methacrylate‐4‐{(1E,4E)‐5‐[4‐(acryloyloxy)phenyl]‐3‐oxopenta‐1,4‐dienyl}phenyl acrylate copolymer beads
Authors: Arun A. , Reddy B.S.R.
Published: February 2005

4‐{(1E,4E)‐5‐[4‐(Acryloyloxy)phenyl]‐3‐oxopenta‐1,4‐dienyl}phenyl acrylate (APPA) was synthesized by reacting (1E,4E)‐1,5‐bis(4‐hydroxyphenyl)penta‐1,4‐dien‐3‐one (HPD) and acryloyl chloride using triethylamine as a base. 2′,4‐Dichloro‐5′‐fluoro‐1‐ene‐2‐(4‐hydroxyphenyl)phenone (EHP) (Arun A, Reddy BSR, Rajkumar M. Polymeric drug for antimicrobial activity studies: Synthesis and characterization. J Bioact Compat Polym 2003;18:219) was used for the controlled release studies. Two types of porous hydrogels were prepared by copolymerizing 2‐hydroxyethyl acrylate (HEA) or 2‐hydroxypropyl methacrylate (HPMA) with APPA (as a crosslinker) by employing the suspension polymerization technique. The morphology of the hydrogels were characterized by the optical microscopy (OM) and scanning electron microscopy (SEM). Different variations were employed in the preparation of hydrogels to study the effect of crosslinker percentage and drug the loading percentage. Totally 18 types of hydrogels were prepared. The drug‐releasing pattern was monitored over a period of 4 weeks using the UV spectroscopic technique by measuring the absorptions at 330.5 nm. In vitro release experiments were carried out at pH 7.4 and pH 9.2. In total, 36 releasing experiments were conducted. The results showed that the controlled release of the drug was dependent on the monomer (HEA or HPMA), crosslinker percentage (CLP), drug‐loading percentage (DLP), and pH. The release rate was higher for HEA‐based hydrogels when compared with HPMA‐based hydrogels in all compositions. The release rate was noticeably higher in pH 9.2 than pH 7.4. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater
Finite subgraphs of uncountably chromatic graphs
Authors: Komjáth Péter , Shelah Saharon
Published: February 2005

It is consistent that for every function f:ω → ω there is a graph with size and chromatic number ℵ1 in which every n‐chromatic subgraph contains at least f(n) vertices (n ≥ 3). This solves a $ 250 problem of Erdős. It is consistent that there is a graph X with Chr(X)=|X|=ℵ1 such that if Y is a graph all whose finite subgraphs occur in X then Chr(Y)≤ℵ2 (so the Taylor conjecture may fail). It is also consistent that if X is a graph with chromatic number at least ℵ2 then for every cardinal λ there exists a graph Y with Chr(Y)≥λ all whose finite subgraphs are induced subgraphs of X. © 2005 Wiley Periodicals, Inc. J Graph Theory 49: 28–38, 2005
Semistrong edge coloring of graphs
Authors: Gyárfás András , Hubenko Alice
Published: February 2005

Weakening the notion of a strong (induced) matching of graphs, in this paper, we introduce the notion of a semistrong matching. A matching M of a graph G is called semistrong if each edge of M has a vertex, which is of degree one in the induced subgraph G[M]. We strengthen earlier results by showing that for the subset graphs and for the Kneser graphs the sizes of the maxima of the strong and semistrong matchings are equal and so are the strong and semistrong chromatic indices. Similar properties are conjectured for the n‐dimensional cube. © 2005 Wiley Periodicals, Inc. J Graph Theory 49: 39–47, 2005
Thin‐layer chromatography/electrospray ionization triple‐quadrupole linear ion trap mass spectrometry system: analysis of rhodamine dyes separated on reversed‐phase C8 plates
Authors: Ford Michael J. , Kertesz Vilmos , Van Berkel Gary J.
Published: February 2005

The direct analysis of separated rhodamine dyes on reversed‐phase C8 thin‐layer chromatography plates using a surface sampling/electrospray emitter probe coupled with a triple‐quadrupole linear ion trap mass spectrometer is presented. This report represents continuing work to advance the performance metrics and utility of this basic surface sampling electrospray mass spectrometry system for the analysis of thin‐layer chromatography plates. Experimental results examining the role of sampling probe spray end configuration on liquid aspiration rate and gas‐phase ion signal generated are discussed. The detection figures‐of‐merit afforded by full‐scan, automated product ion and selected reaction monitoring modes of operation were examined. The effect of different eluting solvents on mass spectrum signal levels with the reversed‐phase C8 plate was investigated. The combined effect of eluting solvent flow‐rate and development lane surface scan rate on preservation of chromatographic resolution was also studied. Analysis of chromatographically separated red pen ink extracts from eight different pens using selected reaction monitoring demonstrated the potential of this surface sampling electrospray mass spectrometry system for targeted compound analysis with real samples. Copyright © 2005 John Wiley & Sons, Ltd.
Aberrations in the progesterone receptor gene and the risk of recurrent endometrial carcinoma
Authors: Pijnenborg JMA , Romano A , Dam‐de Veen GC , Dunselman GAJ , Fischer D‐C , Groothuis PG , Kieback DG
Published: February 2005

A case–control study was performed in order to determine whether expression of the progesterone receptor (PR) and/or aberrations of the PR gene contribute to the development of recurrent endometrial carcinoma. Primary tumours from 44 patients with recurrence of stage I endometrial carcinoma (patients) within 3 years after initial treatment were compared with tumours from 44 matched patients who were free of recurrence for a minimum of 3 years (controls). Paraffin wax‐embedded primary tumours (n = 88) and recurrent tumours (n = 32) were analysed immunohistochemically for PR expression. A staining index (SI = 0–9) based on the staining intensity and the number of stained cells was calculated. DNA extracted from paraffin wax‐embedded tissues was subjected to PCR–restriction fragment length polymorphism analysis (PCR–RFLP) for determination of the PROGINS DNA sequence alterations and the +331G/A‐promoter polymorphism. Low PR expression (SI < 1.0) was observed in 7% of primary tumours derived from controls, 25% of primary tumours from patients with recurrence, and 38% of recurrent tumours. The expression of PR was significantly lower in primary tumours from patients with recurrence (SI = 4.0 ± 0.5) than in the tumours in the control group (SI = 5.6 ± 0.5) (T‐test for paired analysis, p < 0.05). The PROGINS and +331G/A‐promoter polymorphism were not related to age at diagnosis, tumour grade or myometrial invasion. The +331G/A‐promoter polymorphism was present in 14% of primary tumours from patients without recurrence, compared with 17% of patients with recurrence. The PROGINS polymorphism was observed in 16% of primary tumours from patients without, and in 34% of patients with, recurrence (OR 2.6; 95% CI: 0.9–7.6). Most interestingly, patients who carried the PROGINS variant and in whom a PR‐expressing tumour was diagnosed were at significantly enhanced risk of relapse (OR 4.7; 95% CI: 1.3–17.1). In conclusion, low PR expression tended to be associated with recurrent disease, and PR expression in tumours from patients carrying the PROGINS allele was predictive of the risk of recurrence. Copyright © 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Aberrant expression of transforming growth factor β‐1 (TGFβ‐1) per se does not discriminate fibrotic from non‐fibrotic chronic myeloproliferative disorders
Authors: Bock Oliver , Loch Gero , Schade Ulrika , von Wasielewski Reinhard , Schlué Jerome , Kreipe Hans
Published: February 2005

Transforming growth factor β‐1 (TGFβ‐1) is a potent inducer of fibrosis and has been shown to be essential for the development of bone marrow fibrosis in an animal model of idiopathic myelofibrosis (IMF). IMF belongs to the Philadelphia chromosome negative chronic myeloproliferative disorders (Ph− CMPD). Megakaryocytes and platelets have been suggested as the major cellular source of TGFβ‐1 in IMF. The osteoclastogenesis inhibitory factor osteoprotegerin (OPG) seems to be regulated by TGFβ‐1 and substantial involvement of OPG expression in the process of osteosclerosis in IMF has recently been suggested. In order to determine TGFβ‐1 expression in IMF and other Ph− CMPD, total bone marrow cells as well as laser‐microdissected megakaryocytes were quantitatively analysed by real‐time RT‐PCR. OPG mRNA expression in fibrotic IMF was correlated with TGFβ‐1 mRNA expression in a case‐specific manner. Both OPG and TGFβ‐1 were detected immunohistochemically in order to delineate cellular origin. When total bone marrow cells were investigated, TGFβ‐1 mRNA expression was increased in some but not all cases of IMF (n = 21), with highest values in fibrotic cases. Unexpectedly, increased values were also observed in essential thrombocythaemia (ET, n = 11) when compared to non‐neoplastic haematopoiesis (n = 38). Megakaryocytes isolated by laser microdissection displayed elevated TGFβ‐1 mRNA levels in most of the CMPD samples with no significant differences discernible between fibrotic IMF, polycythaemia vera (PV) and ET. TGFβ‐1 protein was predominantly expressed by the myeloid lineage in Ph− CMPD and non‐neoplastic haematopoiesis, which, however, displayed lower expression. IMF cases with advanced fibrosis concomitantly overexpressed TGFβ‐1 and OPG. Immunohistochemically, OPG expression was found in different stromal cells and a subfraction of megakaryocytes. In conclusion, enhanced TGFβ‐1 expression occurs in megakaryocytes as well as myeloid cells in Ph− CMPD. TGFβ‐1 may be necessary, but is not sufficient, to induce bone marrow fibrosis in IMF because non‐fibrotic Ph− CMPD entities share this feature with IMF and cannot be discriminated from each other on the basis of TGFβ‐1 expression. Copyright © 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Block balance in hydrogenated polybutadiene‐b‐polymethylmethacrylate diblock copolymer for efficient interfacial activity in low‐density polyethylene/polymethylmethacrylate blend: Phase morphology development
Authors: Harrats C. , Jérôme R.
Published: February 2005

The objective of the present study was to determine the best molecular balance between the two hydrogenated polybutadiene (HPB) and polymethylmethacrylate (PMMA) blocks that promotes an HPB‐b‐PMMA diblock copolymer with efficient compatibilization activity in a low‐density polyethylene (LDPE)/PMMA immiscible blend. The model blend selected, LDPE/PMMA, is “more immiscible” than the LDPE/polystyrene pair largely reported in open literature. The blends having a composition of 80LDPE/20PMMA exhibit a droplet‐in‐matrix phase morphology whereas in 20LDPE/80PMMA a co‐continuous phase morphology was developed. In the droplet‐in‐matrix phase morphology, the emulsifying efficiency of the copolymer was evaluated based on the maximum reduction of the PMMA droplet size it is able to promote. Whereas, in the co‐continuous phase morphology, the copolymer was evaluated based on its ability to stabilize the maximum phase co‐continuity. The sequences of the best emulsifying copolymer revealed are not symmetrical. An HPB‐b‐PMMA where the ratio of molar mass of the blocks, $\overline{M_{\rm n}}$ HPB/$\overline{M_{\rm n}}$ PMMA, is within 1.8–1.95 exhibits a much better interfacial activity in LDPE/PMMA blends than a copolymer of much lower ratio (longer PMMA block). This is ascribed to the much higher interactions (cohesive energy density) encountered in PMMA (PMMA of the copolymer and PMMA phase of the blend) compared with the LDPE side (HPB of the copolymer and LDPE phase of the blend). © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 837–848, 2005
Volume transition and internal structures of small poly(N‐isopropylacrylamide) microgels
Authors: Arleth Lise , Xia Xiaohu , Hjelm Rex P. , Wu Jianzhong , Hu Zhibing
Published: February 2005

Monodispersed poly(N‐isopropylacrylamide) (PNIPAM) nanoparticles, with hydrodynamic radius less than 50 nm at room temperature, have been synthesized in the presence of a large amount of emulsifiers. These microgel particles undergo a swollen–collapsed volume transition in an aqueous solution when the temperature is raised to around 34 °C. The volume transition and structure changes of the microgel particles as a function of temperature are probed using laser light scattering and small angle neutron scattering (SANS) with the objective of determining the small particle internal structure and particle–particle interactions. Apart from random fluctuations in the crosslinker density below the transition temperature, we find that, within the resolution of the experiments, these particles have a uniform radial crosslinker density on either side of the transition temperature. This result is in contrast to previous reports on the heterogeneous structures of larger PNIPAM microgel particles, but in good agreement with recent reports based on computer simulations of smaller microgels. The particle interactions change across the transition temperature. At temperatures below the transition, the interactions are described by a repulsive interaction far larger than that expected for a hard sphere contact potential. Above the volume transition temperature, the potential is best described by a small, attractive interaction. Comparison of the osmotic second virial coefficient from static laser light scattering at low concentrations with similar values determined from SANS at 250‐time greater concentration suggests a strong concentration dependence of the interaction potential. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 849–860, 2005
Functional analysis of CYP2D6.31 variant: Homology modeling suggests possible disruption of redox partner interaction by Arg440His substitution
Authors: Allorge Delphine , Bréant Didier , Harlow Jacky , Chowdry Joey , Lo‐Guidice Jean‐Marc , Chevalier Dany , Cauffiez Christelle , Lhermitte Michel , Blaney Frank E. , Tucker Geoffrey T. , Broly Franck , Ellis S. Wynne
Published: February 2005

Cytochrome P450 2D6 (CYP2D6) is an important human drug‐metabolizing enzyme that exhibits a marked genetic polymorphism. Numerous CYP2D6 alleles have been characterized at a functional level, although the consequences for expression and/or catalytic activity of a substantial number of rare variants remain to be investigated. One such allele, CYP2D6*31, is characterized by mutations encoding three amino acid substitutions: Arg296Cys, Arg440His and Ser486Thr. The identification of this allele in an individual with an apparent in vivo poor metabolizer phenotype prompted us to analyze the functional consequence of these substitutions on enzyme activity using yeast as a heterologous expression system. We demonstrated that the Arg440His substitution, alone or in combination with Arg296Cys and/or Ser486Thr, altered the respective kinetic parameters [Km (μM) and kcat (min−1)] of debrisoquine 4‐hydroxylation (wild‐type, 25; 0.92; variants, 43–68; 0.05–0.11) and dextromethorphan O‐demethylation (wild‐type, 1; 4.72; variants, 12–23; 0.64‐1.43), such that their specificity constants (kcat/Km) were decreased by more than 95% compared to those observed with the wild‐type enzyme. The rates of oxidation of rac‐metoprolol at single substrate concentrations of 40 and 400 μM were also markedly decreased by approximately 90% with each CYP2D6 variant containing the Arg440His substitution. These in vitro data confirm that the CYP2D6*31 allele encodes an enzyme with a severely impaired but residual catalytic activity and, furthermore, that the Arg440His exchange alone is the inactivating mutation. A homology model of CYP2D6 based on the crystal structure of rabbit CYP2C5 locates Arg440 on the proximal surface of the protein. Docking the structure of the FMN domain of human cytochrome P450 reductase to the CYP2D6 model suggests that Arg440 is a key member of a cluster of basic amino acid residues important for reductase binding. Proteins 2005. © 2005 Wiley‐Liss, Inc.
Solution structure of a tobacco lipid transfer protein exhibiting new biophysical and biological features
Authors: Da Silva Pedro , Landon Céline , Industri Benoit , Marais Antoine , Marion Didier , Ponchet Michel , Vovelle Françoise
Published: February 2005

Plant lipid transfer proteins are small soluble extracellular proteins that are able to bind and transfer a variety of lipidsin vitro. Recently, it has been proposed that lipid transfer proteins may play a key role in plant defence mechanisms, especially during the induction of systemic acquired resistance. However, very little is known about the proteins expressed in developing plants and tissues, since almost all the biophysical and structural data available to date on lipid transfer proteins originate from proteins present in storage tissues of monocot cereal seeds. In this paper, we report the structural and functional characteristics of a lipid transfer protein (named LTP1_1) constitutively expressed in young aerial organs ofNicotiana tabacum(common tobacco). The unlabelled and uniformly labelled proteins were produced in the yeastPichia pastoris, and we determined the three‐dimensional (3D) structure of LTP1_1 using nuclear magnetic resonance (NMR) spectroscopy and molecular modeling techniques. The global fold of LTP1_1 is very close to the previously published structures of LTP1 extracted from cereal seeds, including an internal cavity. However, the chemical shift variations of several NMR signals upon lipid binding show that tobacco LTP1_1 is able to bind only one LysoMyristoylPhosphatidylCholine (LMPC), while wheat and maize LTPs can bind either one or two. Titration experiments using intrinsic tyrosine fluorescence confirm this result not only with LMPC but also with two fatty acids. These differences can be explained by the presence in tobacco LTP1_1 of a hydrophobic cluster closing the second possible access to the protein cavity. This result suggests that LTP1 lipid binding properties could be modulated by subtle changes in a conserved global structure. The biological significance of this finding is discussed in the light of the signalling properties of the tobacco LTP1_1–jasmonate complex described elsewhere.1Proteins 2005. © 2005 Wiley‐Liss, Inc.
Evolution and similarity evaluation of protein structures in contact map space
Authors: Gupta Nitin , Mangal Nitin , Biswas Somenath
Published: February 2005

Prediction of fold from amino acid sequence of a protein has been an active area of research in the past few years, but the limited accuracy of existing techniques emphasizes the need to develop newer approaches to tackle this task. In this study, we use contact map prediction as an intermediate step in fold prediction from sequence. Contact map is a reduced graph‐theoretic representation of proteins that models the local and global inter‐residue contacts in the structure. We start with a population of random contact maps for the protein sequence and “evolve” the population to a “high‐feasibility” configuration using a genetic algorithm. A neural network is employed to assess the feasibility of contact maps based on their 4 physically relevant properties. We also introduce 5 parameters, based on algebraic graph theory and physical considerations, that can be used to judge the structural similarity between proteins through contact maps. To predict the fold of a given amino acid sequence, we predict a contact map that will sufficiently approximate the structure of the corresponding protein. Then we assess the similarity of this contact map with the representative contact map of each fold; the fold that corresponds to the closest match is our predicted fold for the input sequence. We have found that our feasibility measure is able to differentiate between feasible and infeasible contact maps. Further, this novel approach is able to predict the folds from sequences significantly better than a random predictor. Proteins 2005. © 2005 Wiley‐Liss, Inc.
Solution structure of two insect‐specific spider toxins and their pharmacological interaction with the insect voltage‐gated Na+ channel
Authors: Ferrat G. , Bosmans F. , Tytgat J. , Pimentel C. , Chagot B. , Gilles N. , Nakajima T. , Darbon H. , Corzo G.
Published: February 2005

δ‐PaluIT1 and δ‐paluIT2 are toxins purified from the venom of the spider Paracoelotes luctuosus. Similar in sequence to μ‐agatoxins from Agelenopsis aperta, their pharmacological target is the voltage‐gated insect sodium channel, of which they alter the inactivation properties in a way similar to α‐scorpion toxins, but they bind on site 4 in a way similar to β‐scorpion toxins. We determined the solution structure of the two toxins by use of two‐dimensional nuclear magnetic resonance (NMR) techniques followed by distance geometry and molecular dynamics. The structures of δ‐paluIT1 and δ‐paluIT2 belong to the inhibitory cystine knot structural family, i.e. a compact disulfide‐bonded core from which four loops emerge. δ‐PaluIT1 and δ‐paluIT2 contain respectively two‐ and three‐stranded anti‐parallel β‐sheets as unique secondary structure. We compare the structure and the electrostatic anisotropy of those peptides to other sodium and calcium channel toxins, analyze the topological juxtaposition of key functional residues, and conclude that the recognition of insect voltage‐gated sodium channels by these toxins involves the β‐sheet, in addition to loops I and IV. Besides the position of culprit residues on the molecular surface, difference in dipolar moment orientation is another determinant of receptor binding and biological activity differences. We also demonstrate by electrophysiological experiments on the cloned insect voltage‐gated sodium channel, para, heterologuously co‐expressed with the tipE subunit in Xenopus laevis oocytes, that δ‐paluIT1 and δ‐paluIT2 procure an increase of Na+ current. δ‐PaluIT1‐OH seems to have less effect when the same concentrations are used. Proteins 2005. © 2005 Wiley‐Liss, Inc.
Hydrophobic tendency of polar group hydration as a major force in type I antifreeze protein recognition
Authors: Yang Cheng , Sharp Kim A.
Published: February 2005

The random network model of water quantitatively describes the different hydration heat capacities of polar and apolar solutes in terms of distortions of the water–water hydrogen bonding angle in the first hydration shell (Gallagher and Sharp, JACS 2003;125:9853). The distribution of this angle in pure water is bimodal, with a low‐angle population and high‐angle population. Polar solutes increase the high‐angle population while apolar solutes increase the low‐angle population. The ratio of the two populations quantifies the hydrophobicity of the solute and provides a sensitive measure of water structural distortions. This method of analysis is applied to study hydration of type I thermal hysteresis protein (THP) from winter flounder and three quadruple mutants of four threonine residues at positions 2, 13, 24, and 35. Wild‐type and two mutants (VVVV and AAAA) have antifreeze (thermal hysteresis) activity, while the other mutant (SSSS) has no activity. The analysis reveals significant differences in the hydration structure of the ice‐binding site. For the SSSS mutant, polar groups have a typical polar‐like hydration, that is, more high‐angle H‐bonds than bulk water. For the wild‐type and active mutants, polar groups have unusual, very apolar‐like hydration, that is, more low‐angle H‐bonds than bulk water. This pattern of hydration was seen previously in the structurally distinct type III THPs (Yang & Sharp Biophys Chem 2004;109:137), suggesting for the first time a general mechanism for different THP classes. The specific shape, residue size, and clustering of both polar and apoler groups are essential for an active ice binding surface. Proteins 2005. © 2005 Wiley‐Liss, Inc.
Determination of the mobility ratio in InSb at the temperature of the conductivity type conversion
Authors: Somogyi K.
Published: February 2005

The temperature dependence of magneto‐transport in bulk p‐type InSb was investigated, and a special approach was applied for the precise determination of the temperature dependence. Such measurements also allow the determination of the mobility ratio (b) which is known to be large in InSb. Although this type of investigation is standard, in this work an improved method of carrying out temperature‐dependent Hall measurements is reported and improved calculations for the determination of large b values in InSb are presented. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)