bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for at least three alternative mechanisms targeting the p16INK4A/cyclin D/Rb pathway in penile carcinoma, one of which is mediated by high‐risk human papillomavirus
Authors: Ferreux Emmanuelle , Lont Anne P , Horenblas Simon , Gallee Maarten PW , Raaphorst Frank M , von Knebel Doeberitz Magnus , Meijer Chris JLM , Snijders Peter JF
Published: August 2003

A comprehensive analysis of 53 penile carcinomas was performed to determine which mechanisms might be involved in the disruption of the p16INK4A/cyclin D/Rb pathway. To that end, human papillomavirus (HPV) presence, p16INK4A expression and promoter methylation, and expression of the BMI‐1 polycomb gene product were studied. Sixteen (30%) of the carcinomas were found to harbour high‐risk HPV DNA, 15 of which contained HPV 16. HPV 16 E6/E7 oncogene transcripts were detected in 13 (87%) of the carcinomas that contained HPV 16. Strong immunostaining for p16INK4A was significantly more frequent in carcinomas that contained high‐risk HPV DNA (p < 0.001) and amongst those with HPV 16 DNA, it was more frequent in lesions in which E6/E7 transcripts were detectable (p = 0.029). This supports an active role for HPV E7 in interfering with the p16INK4A/cyclin D/Rb pathway. Methylation of the p16INK4A promoter or overexpression of the BMI‐1 polycomb gene product may provide alternative modes of interference with this pathway. These phenomena were mutually exclusive and found in the absence of HPV in 15% and 10% of the penile carcinomas, respectively. These data indicate that there are at least three plausible mechanisms by which the p16INK4A/cyclin D/Rb pathway can become disrupted during penile carcinogenesis. Copyright © 2003 John Wiley & Sons, Ltd.
Overexpression of carbonic anhydrase‐related protein VIII in human colorectal cancer
Authors: Miyaji Eiki , Nishimori Isao , Taniuchi Keisuke , Takeuchi Tamotsu , Ohtsuki Yuji , Onishi Saburo
Published: August 2003

Carbonic anhydrase‐related protein (CA‐RP) VIII, which is a member of the CA gene family, has been shown to have no catalytic CA activity and its biological function is still unknown. Recently, overexpression of CAs IX and XII has been reported in certain types of malignancy. To investigate a potential role for CA‐RP VIII in human colorectal epithelial carcinogenesis, colorectal tissue specimens from surgically resected adenocarcinomas (n = 60) and endoscopically polypectomized adenomas (n = 13) were analysed by immunohistochemistry using monoclonal antibodies to CA‐RP VIII and Ki‐67 antigen. Less than 5% of epithelial cells in normal colonic mucosae (n = 73) were CA‐RP VIII‐positive and these were localized to the deep part of the cryptal epithelium. Increased expression of CA‐RP VIII was observed in 78% of colorectal carcinomas. An intense signal was frequently observed at the tumour invasion front and its distribution was completely different from that of Ki‐67 antigen. Colorectal adenomas also showed significant immunopositivity for CA‐RP VIII, but its expression level was much lower than in adenocarcinomas. These findings suggest that CA‐RP VIII plays a role in the process of invasion in colorectal cancer. Copyright © 2003 John Wiley & Sons, Ltd.
Fas ligand and tumour counter‐attack in colorectal cancer stratified according to microsatellite instability status
Authors: Michael‐Robinson Julie M , Pandeya Nirmala , Cummings Margaret C , Walsh Michael D , Young Joanne P , Leggett Barbara A , Purdie David M , Jass Jeremy R , Radford‐Smith Graham L
Published: August 2003

Expression of membrane‐bound Fas ligand (FasL) by colorectal cancer cells may allow the development of an immune‐privileged site by eliminating incoming tumour‐infiltrating lymphocytes (TILs) in a Fas‐mediated counter‐attack. Sporadic colorectal cancer can be subdivided into three groups based on the level of DNA microsatellite instability (MSI). High‐level MSI (MSI‐High) is characterized by the presence of TILs and a favourable prognosis, while microsatellite‐stable (MSS) cancers are TIL‐deficient and low‐level MSI (MSI‐Low) is associated with an intermediate TIL density. The purpose of this study was to establish the relationship between MSI status and FasL expression in primary colorectal adenocarcinoma. Using immunohistochemistry and a selected series of 101 cancers previously classified as 31 MSI‐High, 30 MSI‐Low, and 40 MSS, the present study sought to confirm the hypothesis that increased TIL density in MSI‐High cancers is associated with low or absent membrane‐bound FasL expression, while increased FasL in MSS cancers allows the killing of host TILs. TUNEL/CD3 double staining was also used to determine whether MSS cancers contain higher numbers of apoptotic TILs in vivo than MSI‐High or MSI‐Low cancers. Contrary to the initial hypothesis, it was found that MSI‐High cancers were associated with higher FasL expression (p = 0.04) and a stronger intensity of FasL staining (p = 0.007). In addition, mucinous carcinomas were independently characterized by increased FasL expression (p = 0.03) and staining intensity (p = 0.0005). Higher FasL expression and staining intensity did not correlate with reduced TIL density or increased numbers of apoptotic TILs. However, consistent with the hypothesis that curtailment of the host anti‐tumour immune response contributes to the poor prognosis in MSS cancers, it was found that apoptotic TILs were most abundant in MSS carcinomas and metastatic Dukes' stage C or D tumours (p = 0.004; p = 0.046 respectively). This study therefore suggests that MSS colorectal cancers are killing incoming TILs in an effective tumour counter‐attack, but apparently not via membrane‐bound FasL. Copyright © 2003 John Wiley & Sons, Ltd.
Differential mucosal expression of three superoxide dismutase isoforms in inflammatory bowel disease
Authors: Kruidenier Laurens , Kuiper Ineke , van Duijn Wim , Marklund Stefan L , van Hogezand Ruud A , Lamers Cornelis BHW , Verspaget Hein W
Published: August 2003

Mucosal tissue damage and dysfunction in chronic inflammatory bowel disease (IBD) are partly caused by an enduring exposure to excessive amounts of reactive oxygen metabolites (ROMs). Although the three human isoforms of superoxide dismutase (SOD), copper/zinc (Cu/Zn)‐SOD, manganese (Mn)‐SOD, and extracellular (EC)‐SOD, form the primary endogenous defence against ROMs, their expression levels and cellular localization in IBD mucosa are largely unknown. The present study used enzyme‐linked immunosorbent assays (ELISAs), spectrophotometric activity assays, and immunohistochemistry to evaluate the protein concentration, enzymatic activity, and distribution of Cu/Zn‐, Mn‐, and EC‐SOD in paired inflamed and non‐inflamed mucosal resection specimens of patients with Crohn's disease (CD) or ulcerative colitis (UC) and compared these with the levels obtained in normal control mucosa. Gut mucosal SOD isoform expression was found to be differentially affected in IBD patients, without major differences between CD and UC. A marked step‐wise increase in Mn‐SOD protein levels was observed in non‐inflamed and inflamed IBD mucosae, whereas the Cu/Zn‐SOD content decreased with inflammation. EC‐SOD was only found in low amounts, which tended to be decreased in IBD patients. Immunohistochemical evaluation confirmed these observations. Mn‐SOD and Cu/Zn‐SOD were both predominantly expressed in intestinal epithelial cells and the percentage of epithelial cells positive for Mn‐SOD was considerably increased in IBD, whereas epithelial Cu/Zn‐SOD expression was much less affected. Within the lamina propria, SOD expression was much lower. Cu/Zn‐SOD and Mn‐SOD were prominently present in neutrophils and macrophages, and EC‐SOD was mainly localized in small vessels, stromal cells, and neutrophils. The percentage of lamina propria cells positive for Cu/Zn‐, Mn‐, or EC‐SOD was not affected by inflammation. Enzyme activity measurements showed consistent results for Cu/Zn‐SOD and EC‐SOD, but the activity of Mn‐SOD did not concordantly increase with the immunological assessments, which may indicate that a proportion of the Mn‐SOD in IBD is present in an enzymatically inactive form. This study reveals remarkable changes in the expression levels of the three SOD isoforms in IBD, particularly in the epithelium. Disturbances in the carefully orchestrated mucosal antioxidant cascade may contribute to the induction and perpetuation of intestinal inflammation in IBD, and may have important implications for the development of antioxidant treatment of IBD patients. Copyright © 2003 John Wiley & Sons, Ltd.
Applications of Fourier transform infrared microspectroscopy in studies of benign prostate and prostate cancer. A pilot study
Authors: Gazi E , Dwyer J , Gardner P , Ghanbari‐Siahkali A , Wade AP , Miyan J , Lockyer NP , Vickerman JC , Clarke NW , Shanks JH , Scott LJ , Hart CA , Brown M
Published: August 2003

Fourier transform infrared (FTIR) microspectroscopy has been applied to a study of prostate cancer cell lines derived from different metastatic sites and to tissue from benign prostate and Gleason‐graded malignant prostate tissue. Paraffin‐embedded tissue samples were analysed by FTIR, after mounting onto a BaF2 plate and subsequent removal of wax using Citroclear followed by acetone. Cell lines were analysed as aliquots of cell suspension held between two BaF2 plates. It was found that the ratio of peak areas at 1030 and 1080 cm−1, corresponding to the glycogen and phosphate vibrations respectively, suggests a potential method for the differentiation of benign from malignant cells. The use of this ratio in association with FTIR spectral imaging provides a basis for estimating areas of malignant tissue within defined regions of a specimen. Initial chemometric treatment of FTIR spectra, using the linear discriminant algorithm, demonstrates a promising method for the classification of benign and malignant tissue and the separation of Gleason‐graded CaP spectra. Using the principle component analysis, this study has achieved for the first time the separation of FTIR spectra of prostate cancer cell lines derived from different metastatic sites. Copyright © 2003 John Wiley & Sons, Ltd.
The clinical relevance of human papillomavirus testing: relationship between analytical and clinical sensitivity
Authors: Snijders Peter JF , van den Brule Adriaan JC , Meijer Chris JLM
Published: August 2003

Given the fact that infection with high‐risk human papillomavirus (HPV) is causally involved in cervical cancer, addition of high‐risk HPV testing to a cervical smear may improve the efficacy of cervical cancer screening programmes, the triage of women with equivocal or borderline Pap smears, and the monitoring of women who have been treated for cervical intraepithelial neoplasia grade 3 (CIN 3). Compared to a cervical smear HPV tests revealed a superior sensitivity (ie clinical sensitivity) for lesions ≥ CIN 3, and a negative predictive value approaching 100%. However, a potential complication is the availability of several HPV testing methods, all displaying a different sensitivity and specificity to detect HPV‐positive women (ie analytical sensitivity and specificity). There is now compelling evidence that the clinical sensitivity and specificity of HPV tests are not simply synonymous to their analytical sensitivity and specificity, respectively. In fact, a distinction between so‐called clinically relevant and irrelevant high‐risk HPV infections should be made when considering HPV tests for primary screening, triage policies, or post‐treatment monitoring. Here, we discuss the potential importance of HPV load in the context of currently widely applied HPV detection methods, to distinguish clinically relevant from irrelevant HPV infections. From this it can be concluded that it is of utmost importance to define criteria, involving viral load threshold and the type of HPV detection method that should be fulfilled by an HPV test before implementation of such a test in clinical practice and population‐based cervical cancer screening programmes. Copyright © 2003 John Wiley & Sons, Ltd.
The need for a literature base for religious giving
Authors: Preissler Scott M.
Published: August 2003

This chapter introduces a research‐based resource that is valuable for capacity building in the nonprofit religious arena.
Strengthening the capacity of cultural organizations to increase participation
Authors: Connolly Paul , Hinand Cady Marcelle
Published: August 2003

This chapter describes how and why more funders are supporting capacity building and how arts and cultural organizations can enhance their effectiveness in this way.
The rest of the world: Capacity building in the international context
Authors: Imdieke Ben
Published: August 2003

Comparative analysis helps reveal that the issue of capacity building is broadly applicable to global civil society in sector, subsector, and organizational terms.
Novel transmittance change of polyelectrolyte hydrogel in DC electric field
Authors: Zhou Xianju , Weng Lihui , Zhang Jianming , Shen Deyan , Xu Jian
Published: August 2003

A novel transmittance change of poly(acrylamide‐co‐acrylic acid) hydrogel in a DC electric field was observed. Transmittance of a specimen increased when the electric field was on and decreased by removing the electric force. In certain cases, the sample was able to switch from white opaque to colorless transparent. The rate and extent of the transition depended on the applied electric potential, ambient temperature, and composition of the specimen. These observations are closely related to the dissociation of hydrogen bonding in the network induced by an external electric field. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 2290–2295, 2003
First‐principles enthalpy landscape analysis of structural recovery in glasses
Authors: Lei Yuning , Cummins Kelly , Lacks Daniel J.
Published: August 2003

The enthalpy landscape is used to derive from first principles the departure functions that quantify structural recovery and aging in glassy materials. The departure functions are identical in form to the phenomenological Kovacs–Aklonis–Hutchinson–Ramos model departure functions, but with the important difference is that the relaxation times do not depend on the instantaneous structure (e.g., volume). This first‐principles derivation elucidates a number of experimental observations in glassy materials, including the asymmetry of approach, the effective relaxation‐rate paradox, and the different time dependences for volume and enthalpy recovery. Example results that qualitatively display these phenomena are obtained with a simple enthalpy landscape. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 2302–2306, 2003
Solubility parameters of poly(sulfonyldiphenylene phenylphosphonate) and its miscibility with poly(ethylene terephthalate)
Authors: Wang Yu‐Zhong
Published: August 2003

The solubility behaviors of poly(sulfonyldiphenylene phenylphosphonate) (PSPPP), a very efficient flame retardant for poly(ethylene terephthalate) (PET), in more than 50 solvents were examined. Its solubility parameters (δ) were determined by the intrinsic viscosity and turbidic titration methods. The two methods obtained consistent results, δ = 21.0–21.6 J1/2/cm3/2 and δ = 21.0 J1/2/cm3/2, and the three‐dimensional solubility parameters were δd = 18.9 J1/2/cm3/2, δp = 8.8 J1/2/cm3/2, and δh = 5.9 J1/2/cm3/2. The miscibility of PSPPP with PET was estimated by the calculation of the heats of mixing, which were related to the difference between the solubility parameters of PSPPP and PET. Fourier transform infrared was used to examine the interactions between PSPPP and PET macromolecules, which were the internal factors of polymer–polymer miscibility. The results showed that PSPPP and PET were miscible within a very wide composition range, especially with less than 15 wt % PSPPP, a composition of interest for the preparation of flame‐retardant PET. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 2296–2301, 2003
Relaxation of liquid‐crystalline polymer fibers in polycarbonate–liquid‐crystalline polymer blend system
Authors: Tan L. P. , Yue C. Y. , Tam K. C. , Lam Y. C. , Hu X. , Nakayama K.
Published: August 2003

The relaxation of liquid‐crystalline polymer (LCP) fibers in the polycarbonate (PC)/LCP blend was examined under various conditions on a hot‐stage microscope. LC5000 is a thermotropic LCP consisting of 80/20 hydroxybenzoic acid and poly(terephthalate). The geometry of the fibers is not an important factor in the relaxation process. Fibers of different aspect ratios and lengths relaxed at the same rate and exhibited identical onset times. Increasing the temperature caused the fibers to relax faster, especially near the nematic‐transition temperature. The fibers relaxed almost immediately when subjected to a temperature of 285 °C. At 280 °C the fibers were stable for 43 min, whereas at 270 °C no noticeable relaxation was evident. Addition of compatibilizer stabilized the fibers by enhancing the interfacial adhesion between the fibers and the PC matrix. Consequently, LCP fibers in the compatibilized system relaxed at a much higher temperature (294 °C) as compared with the uncompatibilized system (275–280 °C). © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 2307–2312, 2003
Hydrogen bonding in polyamide 66/clay nanocomposite
Authors: Lu Yonglai , Zhang Guobao , Feng Meng , Zhang Ying , Yang Mingshu , Shen Deyan
Published: August 2003

Hydrogen bonding in polyamide 66/clay nanocomposite (PA66CN) was first investigated with temperature Fourier transform infrared (FTIR), the results of which were compared with that of pristine polyamide 66 (PA66) with the same thermal history. FTIR spectra at room temperature revealed that there is essentially 100% hydrogen bonding in both PA66CN and PA66, and the difference in hydrogen‐bonding status between them is tiny. Additionally, DSC showed that the crystalline degrees and melting temperatures of PA66CN and PA66 prepared by melt quenching are similar. However, the changes of hydrogen bonding with temperature in PA66CN and PA66 are different. As the temperature rose, the hydrogen bonding in PA66CN attenuated and dissociated considerably at a smaller rate than PA66. According to transmission electron microscopic morphology of PA66CN, we analyzed the effect of nanodispersion clay layers on the motion of a polymer chain and the thermal expansion of crystalline lamella for interpreting the observed phenomenon. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 2313–2321, 2003
Morphology of poly(butylene terephthalate)–poly(ethylene terephthalate‐co‐isophthalate‐co‐sebacate) segmented copolyesters
Authors: Zhao Yun Hui , Xu Gen Hui , Jia Hong Tao , Sheng Jing
Published: August 2003

Segmented copolyesters, namely, poly(butylene terephthalate)–poly(ethylene terephthalate‐co‐isophthalate‐co‐sebacate) (PBT‐PETIS), were synthesized with the melting transesterification processing in vacuo condition involving bulk polyester produced on a large scale (PBT) and ternary amorphous random copolyester (PETIS). Investigations on the morphology of segmented copolyesters were undertaken. The two‐phase morphology model was confirmed by transmission electron microscopy and dynamic mechanical thermal analysis. One of the phases was composed of crystallizable PBT, and the other was a homogeneous mixture of PETIS and noncrystallizable PBT. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 2257–2263, 2003
Supertoughness and critical interparticle distance dependence in poly(butylene terephthalate) and poly(ethylene‐co‐glycidyl methacrylate) blends
Authors: Aróstegui A. , Nazábal J.
Published: August 2003

Supertough poly(butylene terephthalate) (PBT)‐based blends were obtained by the melt blending of PBT with 0–30 wt % poly(ethylene‐co‐glycidyl methacrylate) (EGMA). The reaction between PBT and EGMA was detected by torque measurements. The particle size was almost constant with increasing EGMA content, and this indicated that compatibilization occurred. The minimum EGMA content for achieving supertoughness (i.e., an impact strength 16 times greater than that of PBT) was 20 wt %. The interparticle distance was the parameter controlling toughness in these PBT/EGMA blends. The dependence of the critical interparticle distance (τc) on the modulus of the dispersed phase appeared only at low τc values, and the primary dependence of τc on the ratio of the modulus of the matrix to the modulus of the rubbery dispersed phase was proposed. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 2236–2247, 2003
Impact‐modified polypropylene/vermiculite nanocomposites
Authors: Tjong S. C. , Meng Y. Z.
Published: August 2003

Impact‐modified polypropylene (PP)/vermiculite (VMT) nanocomposites toughened with maleated styrene–ethylene butylene–styrene (SEBS‐g‐MA) were compounded in a twin‐screw extruder and injection‐molded. VMT was treated with maleic anhydride, which acted both as a compatibilizer for the polymeric matrices and as a swelling agent for VMT in the nanocomposites. The effects of the impact modifier on the morphology and the impact, static, and dynamic mechanical properties of the PP/VMT nanocomposites were investigated. Transmission electron microscopy revealed that an exfoliated VMT silicate layer structure was formed in ternary (PP–SEBS‐g‐MA)/VMT nanocomposites. Tensile tests showed that the styrene–ethylene butylene–styrene additions improved the tensile ductility of the (PP–SEBS‐g‐MA)/VMT ternary nanocomposites at the expense of their tensile stiffness and strength. Moreover, Izod impact measurements indicated that the SEBS‐g‐MA addition led to a significant improvement in the impact strength of the nanocomposites. The SEBS‐g‐MA elastomer was found to be very effective at converting brittle PP/VMT organoclay composites into tough nanocomposites. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 2332–2341, 2003
Effect of a silicate filler on the crystal morphology of poly(trimethylene terephthalate)/clay nanocomposites
Authors: Hu Xianbo , Lesser Alan J.
Published: August 2003

A series of intercalated poly(trimethylene terephthalate) (PTT)/clay nanocomposites were prepared in a twin‐screw extruder by the melt mixing of PTT with either quaternary or ternary ammonium salt‐modified clays. The morphology and structure, along with the crystallization and melting behavior, and the dynamic mechanical behavior of the composites were characterized by X‐ray diffraction, transmission electron microscopy, differential scanning calorimetry, and dynamic mechanical thermal analysis. The results showed that the PTT chains could undergo center‐mass transport from the polymer melt into the silicate galleries successfully during the blending and extrusion process. More coherent stacking of the silicate layers was reserved at higher clay concentrations and shorter blend times. Compared with conventionally compounded composites, the nanoscale‐dispersed organophilic clays were more effective as crystal nucleation agents. The influence of the nanosilicates on the crystallization and melting behavior of PTT became distinct when the concentration of clay was around 3 wt %. The changes in the crystallization behavior of the polymer/clay nanocomposites depended not only on the size of the silicates but also on the intrinsic crystallization characteristics of the polymers. The resulting nanocomposites showed an increase in the dynamic modulus of PTT and a decrease in the relaxation intensity (both in loss modulus and loss tangent magnitude). © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 2275–2289, 2003
Wetting and dynamics of structured liquid films
Authors: Green Peter F.
Published: August 2003

The stability of a sufficiently thin, supported, homopolymer film against the development of local thickness fluctuations which can become amplified, eventually leading to structural destabilization of the film, is typically determined by long and short‐range intermolecular forces. In A‐B diblock copolymers, the connectivity between the blocks, the preferential attraction of one block to an external interface, combined with an incompatibility between the A‐B segments, the situation is very different. Two cases, largely dictated by χN, wher χ is the Flory‐Huggins interaction parameter andNis the degree of polymerization, can arise in thin copolyme films. When χNis large, thin films exhibit comparatively stable topographical structures, where the dimensions of the topographies normal to the substrate reflect a natural length‐scale associated with phase separation in the material. In the other situation, where χNis sufficiently small, the copolymer bulk structure is homogeneous. An ordered structure can be induced into the otherwise compositionally homogeneous structure in the vicinity of a substrate. Here, depending on film thickness, a series of transient and stable topographies can develop. Wetting, early stage structural destabilization dynamics leading to the formation of droplets, and late stage coarsening of the droplets are discussed. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 2219–2235, 2003
Prostate‐specific antigen‐derived epitopes capable of inducing cellular and humoral responses in HLA‐A24+ prostate cancer patients
Authors: Harada Mamoru , Kobayashi Kazuhiko , Matsueda Satoko , Nakagawa Masami , Noguchi Masanori , Itoh Kyogo
Published: August 2003

BACKGROUND

We tried to identify prostate‐specific antigen (PSA)‐derived epitopes immunogenic in HLA‐A24+ prostate cancer patients.

METHODS

Peripheral blood mononuclear cells (PBMCs) were in vitro stimulated with each of four different PSA peptides carrying the HLA‐A24 binding motif, and their HLA‐A24‐restricted anti‐tumor responses were examined using a parental HLA‐A24‐negative prostate cancer cell line (PC93) and its HLA‐A24‐expressing transfectant line (PC93‐A24). Serum levels of immunoglobulin G (IgG) against PSA peptides were measured by enzyme‐linked immunosorbent assay (ELISA).

RESULTS

PBMCs, which were in vitro stimulated with either the PSA152–160 or PSA248–257 peptide, showed higher levels of IFN‐γ production and cytotoxicity against the PC93‐A24 than against the PC93. IgG against the PSA248–257 peptide was detected in half of the prostate cancer patients tested.

CONCLUSIONS

The PSA152–160 and PSA248–257 peptides could be appropriate target molecules in use for specific immunotherapy of HLA‐A24+ prostate cancer patients. Prostate 57: 152–159, 2003. © 2003 Wiley‐Liss, Inc.
Transgenic mouse with high cre recombinase activity in all prostate lobes, seminal vesicle, and ductus deferens
Authors: Jin Chengliu , McKeehan Kerstin , Wang Fen
Published: August 2003

BACKGROUND

Prostate‐specific gene ablation provides a powerful tool for functional characterization of genes that have impact on embryonic development or on other organs, specifically in the prostate. Uniform expression of Cre with high recombinase activity in the prostate is needed for prostate‐specific gene ablation based on Cre‐loxP recombinations. Currently, available strains of Cre transgenic mice only express Cre recombinase adequately in certain lobes of the prostate. In other lobes, the expression is low and mosaic. Additional strains of transgenic mice expressing high levels of prostate‐specific Cre in all prostate lobes would be useful to study the impact of genome manipulation in all prostate lobes.

METHODS

The ARR2PB composite promoter with improved capacity to drive androgen‐responsive gene expression was used to initiate expression of a transgene bearing the cDNA encoding a recently modified Cre recombinase with improved recombination activity. In addition, an insulator element from the chicken globin locus that minimized negative effect on transcription of the transgene imposed by chromosome structure was employed. The derived transgenic founders were crossed with the Z/AP reporter mouse and Fgfr2f/f mice bearing loxP flanking the FGFR2 locus. Immunochemical and mRNA analyses were employed to test expression and efficacy of the Cre recombinase in the prostate and other tissues.

RESULTS

The ARR2PBi‐Cre transgenic mouse specifically and uniformly expressed Cre recombinase in the dorsal, lateral, ventral, and anterior lobes of the prostate, seminal vesicles, and ductus deferens. The Cre recombinase in these tissues effectively excised loxP flanked DNA fragments in the Z/AP reporter that triggered expression of β‐galactosidase, and the loxP–flanked FGFR2f/f locus resulting in specific ablation of FGFR2 in the prostate.

CONCLUSIONS

Compared with the currently available prostate‐specific Cre strains, the new ARR2PBi‐Cre strain exhibited higher and more uniform expression of Cre recombinase in the prostate as well as in seminal vesicles and ductus deferens. This provides an additional tool for efficient hormone‐dependent gene targeting in epithelial cells of all lobes of the adult prostate, seminal vesicle, and ductus deferens. Prostate 57: 160–164, 2003. © 2003 Wiley‐Liss, Inc.
Laterally directed biopsies detect more clinically threatening prostate cancer: Computer simulated results
Authors: Kawata Nozomu , Miller Gary J. , Crawford E. David , Torkko Kathleen C. , Stewart J. Scott , Lucia M. Scott , Miller Heidi L. , Hirano Daisaku , Werahera Priya N.
Published: August 2003

BACKGROUND

The purpose of this study was to determine, whether a modified fan‐shaped biopsy (MFSB) technique which utilizes six laterally directed biopsies would lead to higher detection rates of clinically threatening prostatic carcinoma than the six random systematic core biopsy (SRSCB) method.

METHODS

We reconstructed 3‐dimensional solid computer models of 86 autopsy prostates and 40 radical prostatatectomy specimens. Simulations of SRSCB and MFSB were then performed using the same biopsy sites except that the biopsy probe was rotated 45° toward posterolateral peripheral zone for MFSB. When the Gleason sum was less than 7, clinically threatening cancers were defined as having a tumor volume ≥0.25 cc or ≥0.5 cc.

RESULTS

When the cut off volume was 0.25 cc, MFSB detected significantly more threatening carcinomas in autopsy prostates than did SRSCB (P < 0.0082). This was also true for the surgical prostates (P < 0.0047) as well as for a sub‐group of non‐palpable carcinomas (P < 0.0047). When the cut off volume was increased to 0.5 cc, MFSB detected significantly more threatening carcinomas in the radical series (P < 0.0047) and for the non‐palpable carcinomas (P < 0.0082), but not in the autopsy series.

CONCLUSIONS

The MFSB technique, which utilizes laterally directed biopsies, appears to be an effective approach to improve the detection of clinically threatening prostatic carcinoma. Prostate 57: 118–128, 2003. © 2003 Wiley‐Liss, Inc.
The origin and extent of coarse‐grained regularities in protein internal packing
Authors: Bagci Zerrin , Kloczkowski Andrzej , Jernigan Robert L. , Bahar Ivet
Published: August 2003

Despite the suitability of various lattice geometries for coarse‐grained modeling of proteins, the actual packing geometry of residues in folded structures has remained largely unexplored. A strong tendency to assume a regular packing geometry is shown here by optimally reorienting and superimposing clusters of neighboring residues from databank structures examined on a coarse‐grained (single‐site‐per‐residue) scale. The orientation function (or order parameter) of the examined coordination clusters with respect to fcc lattice directions is found to be 0.82. The observed geometry, which may be termed an incomplete distorted face‐centered cubic (fcc) packing, is apparently favored by the drive to maximize packing density, in a fashion analogous to the way identical spheres pack densely and follow fcc geometry. About 2/3 of all residues obey this packing geometry, while the remainder occupy other context‐dependent positions. The preferred coordination directions show relatively small variations over the various amino acid types, consistent with uniform residue viewpoint. Both the extremes of solvent‐exposed and completely buried residue neighborhoods approximate the same generic packing, the only difference being in the numbers (and not the orientations) of coordination sites that are occupied (or left void for solvent occupancy). We observe the prevalence of a rather uniform (tight) residue packing density throughout the structure, including even the residues packed near solvent‐exposed regions. The observed orientation distribution reveals an underlying, intrinsic orientation lattice for proteins. Proteins 2003. © 2003 Wiley‐Liss, Inc.
Anatomy of specific interactions between λ repressor and operator DNA
Authors: Oobatake Motohisa , Kono Hidetoshi , Wang Yifei , Sarai Akinori
Published: August 2003

Recognition of specific DNA sequences by proteins is essential for regulation of gene expression. To fully understand the recognition mechanism, it is necessary to understand not only the structure of the specific protein–DNA interactions but also the energetics. We therefore performed a computer analysis in which a phage DNA‐binding protein, λ repressor, was used to examine the changes in binding free energy (ΔΔG) and its energy components caused by single base mutations. We then determined which of the calculated energy components best correlated with the experimental data. The experimental ΔΔG values were well reproduced by the calculations. Component analysis revealed that the electrostatic and hydrogen bond energies were most strongly correlated with the experimental data. Among the 51 single base‐substitution mutants examined, positive ΔΔG values, corresponding to weakened binding, were caused by the loss of favorable electrostatic interactions and hydrogen bonds, the introduction of steric collisions and electrostatic repulsion, the loss of favorable interactions with a thymine methyl group, and the increase of unfavorable hydration energy from isolated DNA. This analysis also showed distinct patterns of recognition at A‐T and G‐C positions, as different combinations of energy components were involved in ΔΔG caused by the two substitution types. We have thus been able to identify the energy components that most strongly correlate with sequence‐dependent ΔΔG and determine their contribution to the specificity of DNA sequence recognition by the λ repressor. Application of this method to other systems should provide additional insight into the molecular mechanism of protein–DNA recognition. Proteins 2003. © 2003 Wiley‐Liss, Inc.
Evolutionary analysis reveals collective properties and specificity in the C‐type lectin and lectin‐like domain superfamily
Authors: Ebner Sharon , Sharon Nathan , Ben‐Tal Nir
Published: August 2003

Members of the C‐type lectin/C‐type lectin‐like domain (CTL/CTLD) superfamily share a common fold and are involved in a variety of functions, such as generalized defense mechanisms against foreign agents, discrimination between healthy and pathogen‐infected cells, and endocytosis and blood coagulation. In this work we used ConSurf, a computer program recently developed in our lab, to perform an evolutionary analysis of this superfamily in order to further identify characteristics of all or part of its members. Given a set of homologous proteins in the form of multiple sequence alignment (MSA) and an inferred phylogenetic tree, ConSurf calculates the conservation score in every alignment position, taking into account the relationships between the sequences and the physicochemical similarity between the amino acids. The scores are then color‐coded onto the three‐dimensional structure of one of the homologous proteins. We provide here and at http://ashtoret.tau.ac.il/∼sharon a detailed analysis of the conservation pattern obtained for the entire superfamily and for two subgroups of proteins: (a) 21 CTLs and (b) 11 heterodimeric CTLD toxins. We show that, in general, proteins of the superfamily have one face that is constructed mostly of conserved residues and another that is not, and we suggest that the former face is involved in binding to other proteins or domains. In the CTLs examined we detected a region of highly conserved residues, corresponding to the known calcium‐ and carbohydrate‐binding site of the family, which is not conserved throughout the entire superfamily, and in the CTLD toxins we found a patch of highly conserved residues, corresponding to the known dimerization region of these proteins. Our analysis also detected patches of conserved residues with yet unknown function(s). Proteins 2003. © 2003 Wiley‐Liss, Inc.
An improved protein decoy set for testing energy functions for protein structure prediction
Authors: Tsai Jerry , Bonneau Richard , Morozov Alexandre V. , Kuhlman Brian , Rohl Carol A. , Baker David
Published: August 2003

We have improved the original Rosetta centroid/backbone decoy set by increasing the number of proteins and frequency of near native models and by building on sidechains and minimizing clashes. The new set consists of 1,400 model structures for 78 different and diverse protein targets and provides a challenging set for the testing and evaluation of scoring functions. We evaluated the extent to which a variety of all‐atom energy functions could identify the native and close‐to‐native structures in the new decoy sets. Of various implicit solvent models, we found that a solvent‐accessible surface area–based solvation provided the best enrichment and discrimination of close‐to‐native decoys. The combination of this solvation treatment with Lennard Jones terms and the original Rosetta energy provided better enrichment and discrimination than any of the individual terms. The results also highlight the differences in accuracy of NMR and X‐ray crystal structures: a large energy gap was observed between native and non‐native conformations for X‐ray structures but not for NMR structures. Proteins 2003. © 2003 Wiley‐Liss, Inc.
Subtle functional collective motions in pancreatic‐like ribonucleases: From ribonuclease A to angiogenin
Authors: Merlino Antonello , Vitagliano Luigi , Ceruso Marc Antoine , Mazzarella Lelio
Published: August 2003

The analysis of the dynamic behavior of enzymes is fundamental to structural biology. A direct relationship between protein flexibility and biological function has been shown for bovine pancreatic ribonuclease (RNase A) (Rasmussen et al., Nature 1992;357:423–424). More recently, crystallographic studies have shown that functional motions in RNase A involve the enzyme β‐sheet regions that move concertedly on substrate binding and release (Vitagliano et al., Proteins 2002;46:97–104). These motions have been shown to correspond to intrinsic dynamic properties of the native enzyme by molecular dynamics (MD) simulations. To unveil the occurrence of these collective motions in other members of pancreatic‐like superfamily, we carried out MD simulations on human angiogenin (Ang). Essential dynamics (ED) analyses performed on the trajectories reveal that Ang exhibits collective motions similar to RNase A, despite the limited sequence identity (33%) of the two proteins. Furthermore, we show that these collective motions are also present in ensembles of experimentally determined structures of both Ang and RNase A. Finally, these subtle concerted β‐sheet motions were also observed for other two members of the pancreatic‐like superfamily by comparing the ligand‐bound and ligand‐free structures of these enzymes. Taken together, these findings suggest that pancreatic‐like ribonucleases share an evolutionary conserved dynamic behavior consisting of subtle β‐sheet motions, which are essential for substrate binding and release. Proteins 2003. © 2003 Wiley‐Liss, Inc.
Host‐guest scale of left‐handed polyproline II helix formation
Authors: Rucker Adam L. , Pager Cara T. , Campbell Margaret N. , Qualls Joseph E. , Creamer Trevor P.
Published: August 2003

Despite the clear importance of the left‐handed polyproline II (PPII) helical conformation in many physiologically important processes as well as its potential significance in protein unfolded states, little is known about the physical determinants of this conformation. We present here a scale of relative PPII helix‐forming propensities measured for all residues, except tyrosine and tryptophan, in a proline‐based host peptide system. Proline has the highest measured propensity in this system, a result of strong steric interactions that occur between adjacent prolyl rings. The other measured propensities are consistent with backbone solvation being an important component in PPII helix formation. Side chain to backbone hydrogen bonding may also play a role in stabilizing this conformation. The PPII helix‐forming propensity scale will prove useful in future studies of the conformational properties of proline‐rich sequences as well as provide insights into the prevalence of PPII helices in protein unfolded states. Proteins 2003. © 2003 Wiley‐Liss, Inc.
Conformational interconversion in compstatin probed with molecular dynamics simulations
Authors: Mallik Buddhadeb , Lambris John D. , Morikis Dimitrios
Published: August 2003

Compstatin is a 13‐residue cyclic peptide that has the potential to become a therapeutic agent against unregulated complement activation. In our effort to understand the structural and dynamic characteristics of compstatin that form the basis for rational and combinatorial optimization of structure and activity, we performed 1‐ns molecular dynamics (MD) simulations. We used as input in the MD simulations the ensemble of 21 lowest energy NMR structures, the average minimized structure, and a global optimization structure. At the end of the MD simulations we identified five conformations, with populations ranging between 9% and 44%. These conformations are as follows: 1) coil with αR‐αR β‐turn, as was the conformation of the initial ensemble of NMR structures; 2) β‐hairpin with ϵ‐αR β‐turn; 3) β‐hairpin with αR‐αR β‐turn; 4) β‐hairpin with αR‐β β‐turn; and 5) α‐helical. Conformational switch was possible with small amplitude backbone motions of the order of 0.1–0.4 Å and free energy barrier crossing of 2–11 kcal/mol. All of the 21 MD structures corresponding to the NMR ensemble possessed a β‐turn, with 14 structures retaining the αR‐αR β‐turn type, but the average minimized structure and the global optimization structures were converted to α‐helical conformations. Overall, the MD simulations have aided to gain insight into the conformational space sampled by compstatin and have provided a measure of conformational interconversion. The calculated conformers will be useful as structural and possibly dynamic templates for optimization in the design of compstatin using structure‐activity relations (SAR) or dynamics‐activity relations (DAR). Proteins 2003. © 2003 Wiley‐Liss, Inc.
Relationship between protein structures and disulfide‐bonding patterns
Authors: Chuang Chao‐Chun , Chen Chun‐Yin , Yang Jinn‐Moon , Lyu Ping‐Chiang , Hwang Jenn‐Kang
Published: August 2003

We found that that disulfide‐bonding patterns can be used to discriminate structure similarity. Our method, based on the hierarchical clustering scheme, is applicable to proteins with two or more disulfide bonds and is able to detect the structural similarities of proteins of low sequence identities (<25%). Our results show the surprisingly close relationship between disulfide‐bonding patterns and proteins structures. Our findings should be useful in protein structure modeling. Proteins 2003. © 2003 Wiley‐Liss, Inc.
Relative stability of protein structures determined by X‐ray crystallography or NMR spectroscopy: A molecular dynamics simulation study
Authors: Fan Hao , Mark Alan E.
Published: August 2003

The relative stability of protein structures determined by either X‐ray crystallography or nuclear magnetic resonance (NMR) spectroscopy has been investigated by using molecular dynamics simulation techniques. Published structures of 34 proteins containing between 50 and 100 residues have been evaluated. The proteins selected represent a mixture of secondary structure types including all α, all β, and α/β. The proteins selected do not contain cysteine–cysteine bridges. In addition, any crystallographic waters, metal ions, cofactors, or bound ligands were removed before the systems were simulated. The stability of the structures was evaluated by simulating, under identical conditions, each of the proteins for at least 5 ns in explicit solvent. It is found that not only do NMR‐derived structures have, on average, higher internal strain than structures determined by X‐ray crystallography but that a significant proportion of the structures are unstable and rapidly diverge in simulations. Proteins 2003. © 2003 Wiley‐Liss, Inc.
Crystallographic analysis of an “anticalin” with tailored specificity for fluorescein reveals high structural plasticity of the lipocalin loop region
Authors: Korndörfer Ingo P. , Beste Gerald , Skerra Arne
Published: August 2003

The artificial lipocalin FluA with novel specificity toward fluorescein was derived via combinatorial engineering from the bilin‐binding protein, BBP by exchange of 16 amino acids in the ligand pocket. Here, we describe the crystal structure of FluA at 2.0 Å resolution in the space group P21 with two protein–ligand complexes in the asymmetric unit. In both molecules, the characteristic β‐barrel architecture with the attached α‐helix is well preserved. In contrast, the four loops at one end of the β‐barrel that form the entrance to the binding site exhibit large conformational deviations from the wild‐type protein, which can be attributed to the sidechain replacements. Specificity for the new ligand is furnished by hydrophobic packing, charged sidechain environment, and hydrogen bonds with its hydroxyl groups. Unexpectedly, fluorescein is bound in a much deeper cavity than biliverdin IXγ in the natural lipocalin. Triggered by the substituted residues, unmutated sidechains at the bottom of the binding site adopt conformations that are quite different from those observed in the BBP, illustrating that not only the loop region but also the hydrophobic interior of the β‐barrel can be reshaped for molecular recognition. Particularly, Trp 129 participates in a tight stacking interaction with the xanthenolone moiety, which may explain the ultrafast electron transfer that occurs on light excitation of the bound fluorescein. These structural findings support our concept of using lipocalins as a scaffold for the engineering of so‐called “anticalins” directed against prescribed targets as an alternative to recombinant antibody fragments. Proteins 2003. © 2003 Wiley‐Liss, Inc.
Dispersion of parameters characterizing the Landau levelsin crystals with cubic symmetry
Authors: Olszewski S. , Baszczak M. , Kozak R.
Published: August 2003

Broadening of the Landau levels due to the presence of a crystal cubic lattice is examined on a semi‐classical basis for not too strong magnetic fields. The energies of the Landau levels remain discrete also in case of the presence of the lattice potential, but an essential broadening occurs in the momentum space of the electron motion when this motion is limited to a plane being transversal to the applied field. The broadening in the momentum space, attained without any electron–electron interaction admitted for the Landau levels, is described in terms of the band structure parameters characteristic for the presence of the magnetic field. Moreover, for a magnetic field tilted to the crystallographic axes, the broadening is dependent on the inclination angle of the field. Different crystal lattices exhibit different kinds of dispersion. In particular, for the Landau levels of tightly‐bound s‐electrons in a simple cubic and body‐centred cubic lattice there is no dispersion in the electron gyration frequency and degeneracy number of the level area over the value of the wavevector component parallel to the magnetic field. On the other hand, this kind of dispersion exists in the case of the face‐centred cubic lattice.