bluuebunny/crossref_metadata_2025 huggingface dataset:
Preparation of human heart for laser microdissection and proteomics
Authors: De Souza Ayesha I. , McGregor Emma , Dunn Michael J. , Rose Marlene L.
Published: February 2004

Proteomics generates information on expressed proteins, and laser microdissection (LMD) is a method that allows enrichment of specific cell types from complex heterogeneous tissue. Together they provide a powerful tool for functional genomic research. Here, we have investigated (i) the effects of fixation and staining on cardiac proteins separated by two‐dimensional gel electrophoresis (2‐DE) and (ii) feasibility of using LMD to separately prepare myocytes and blood vessels for 2‐DE gel analysis. This is the first such study of human heart. The effect of fixation (ethanol or acetone), staining with haematoxylin and eosin in the presence and absence of xylene, and antibody staining was investigated. Proteins were separated by 2‐DE and spots detected by silver staining. Quantitative spot analysis showed that contractile proteins were preserved under all conditions, and no significant differences were found when the groups studied were compared with the control group. However, there were differences in the visual quality of the gel patterns. LMD provided enough protein from blood vessels and myocytes to run one large‐format (18×24 cm) 2‐D gel for each subset of cells. Collection of this material took 70 h (≈︁ 2800 blood vessels and 17 000 myocytes) and resulted in tissue‐specific gel patterns for these two structures. In conclusion, the use of haematoxylin and eosin staining without xylene provided the best morphology and did not significantly affect protein spot number.
Mechanisms of hydrazine toxicity in rat liver investigated by proteomics and multivariate data analysis
Authors: Guldberg Klenø Tina , Rønnedal Leonardsen Lise , Ørsted Kjeldal Helle , Møller Laursen Steen , Nørregaard Jensen Ole , Baunsgaard Dorrit
Published: February 2004

A proteomics approach combined with multivariate data analysis was used to examine the hepatotoxic effect of hydrazine in 30 male Sprague Dawley rats, assigned to four treatment groups and two control groups. Liver samples from the individual animals were resolved by two‐dimensional differential gel electrophoresis (2‐D DIGE) and protein patterns from the 2‐D gels were analyzed by principal component analysis (PCA) and partial least squares regression (PLSR). The PCA plot was able to describe the variation in the protein expression related to dose and time, by separation or clustering of different animal groups. PLSR followed by variable selection (Jack‐knifing) was used to select proteins that varied significantly in relation to the dose related response of the hydrazine treatment. The 10 up‐regulated and 10 down‐regulated proteins with highest rank in the PLSR model were identified by mass spectrometry. Hydrazine treatment induced altered expression of proteins related to lipid metabolism, Ca2+ homeostasis, thyroid hormone pathways and stress response. Several of the identified proteins have not previously been implicated in hydrazine toxicity and may thus be regarded as new potential biomarkers of induced liver toxicity.
Proteomics of methylene blue photo‐treated plasma before and after removal of the dye by an absorbent filter
Authors: Crettaz David , Sensebe Luc , Vu Dinh‐Hao , Schneider Philippe , Depasse François , Bienvenut Willy‐Vincent , Quadroni Manfredo , Tissot Jean‐Daniel
Published: February 2004

Methylene blue (MB) and light are used for virus inactivation of plasma for transfusion. However, the presence of MB has been the subject of concern, and efforts have been made to efficiently remove the dye after photo‐treatment. For this study, plasma was collected by apheresis from 10 donors (group A), then treated using the MacoPharma THERAFLEX procedure (MB; 1 μM, and light exposure; 180 J/cm2) (group B), and finally filtered in order to remove the dye (group C). Proteins were analyzed by two‐dimensional electrophoresis, and peptides showing modifications were characterized by mass spectrometry. Clottable and antigenic fibrinogen levels, as well as fibrin polymerization time were measured. Analyses of the gels focused on a region corresponding to pI between 4.5 and 6.5, and Mr from 7000 to 58 000. In this area, 387 ± 47 spots matched, and four of these spots presented significant modifications. They corresponded to changes of the γ‐chain of fibrinogen, of transthyretin, and of apolipoprotein A‐I, respectively. A decrease of clottable fibrinogen and a prolongation of fibrin polymerization time were observed in groups B and C. Removal of MB by filtration was not responsible for additional protein alterations. The effect of over‐treatment of plasma by very high concentrations of MB (50 μM) in association with prolonged light exposure (3 h) was also analyzed, and showed complex alterations of most of the plasma proteins, including fibrinogen γ‐chain, transthyretin, and apolipoprotein A‐I. Our data indicates that MB treatment at high concentration and prolonged illumination severely injure plasma proteins. By contrast, at the MB concentration used to inactivate viruses, damages are apparently very restricted.
Determination of the stoichiometry of protein complexes using liquid chromatography with fluorescence and mass spectrometric detection of fluorescently labeled proteolytic peptides
Authors: Hochleitner Elisabeth O. , Sondermann Peter , Lottspeich Friedrich
Published: February 2004

A method for the determination of the stoichiometry of protein complexes has been developed, which is based on proteolytic digestion of the complex, labeling with a fluorescent reagent, specific for amino or sulfhydryl groups, and separation by liquid chromatography with fluorescence and mass spectrometric detection. The intensity of the fluorescence signal of the labeled peptides resulting from different proteins is directly proportional to the stoichiometry of these proteins in the complex. The performance of the method was evaluated with standard peptides and proteins to ensure that accurate molar ratios can be obtained from the fluorescence chromatogram. Standard deviations of the measured molar ratio from the expected molar ratio were below 10% for both peptides and proteins. The method was finally employed for the determination of the stoichiometry of the 1:1 complex of sFcγRIII and hFc1. Using the described methodology, a stoichiometry of 1:1.1 was measured, which agrees well with a 1:1 complex.
A DSC Analysis of Inverse Salt‐pair Explosive Composition
Authors: Babu E. Suresh , Kaur Sukhminder
Published: February 2004

Alkali nitrates are used as an ingredient in low explosive compositions and pyrotechnics. It has been suggested that alkali nitrates can form inverse salt‐pair explosives with the addition of ammonium chloride. Therefore, the thermal behavior of low explosive compositions containing potassium nitrate mixed with ammonium chloride has been studied using Differential Scanning Calorimetry (DSC). Results provide information about the ion exchange reaction between these two chemical substances and the temperature region at which the formation of a cloud of salt particles of potassium chloride takes place. Furthermore, the addition of ammonium chloride quenches the flame of deflagrating compositions and causes the mixture to undergo explosive decomposition at relatively low temperatures.
Static Electric Sensitivity Characteristics of Zr/BaCrO4 Pyrolants
Authors: Kuwahara Takuo , Kohno Takehiro , Wang Chia Hsiang
Published: February 2004

Zirconium (Zr) easily ignites in air atmosphere and generates high‐energy output. Therefore, it is used as a fuel for pyrolants. Barium chromate (BaCrO4) dose not ignite in air, so it is one of safety materials and it is used as oxidizer. These materials are mixed with viton as coating material/agent, since viton is easily dissolved in acetone. The static electric sensitivity of only‐BaCrO4‐coated material is lower than that of Zr‐coated one. The static electric energy, E, influences the chemical and the physical ignition delay times and they decrease with increasing E. The chemical ignition delay time decreases with increasing concentration of Zr. The chemical and physical ignition delay times of only‐BaCrO4‐coated pyrolant are longer than those of both‐coated material and only‐Zr‐coated, so the pyrolant of only‐BaCrO4‐coated pyrolant is the safer material.
Nanomaterials for Heterogeneous Combustion
Authors: Pivkina Alla , Ulyanova Polina , Frolov Yurii , Zavyalov Sergey , Schoonman Joop
Published: February 2004

Nanophase materials and nanocomposites, characterized by an ultra fine grain size (less than 100 nm) have attracted wide spread interest in recent years by virtue of their unusual mechanical, electrical, optical, magnetic, and energetic properties. Studies have shown that the thermal behavior of nano‐scaled materials is quite different from micron‐sized powders. Nanosized metallic and explosive powders have been used as solid propellant and explosive mixtures to increase efficiency. At the same time recent studies reveal that the presence of nanosized metals in propellants does not necessary translate into an increased burning rate and burning temperature. The reasons of this effect are far from being clear. This paper presents a new approach to the production of nanocomposites of some energetic materials – ammonium nitrite, cyclotrimethylene trinitramine (RDX), and aluminum – by the vacuum co‐deposition technique. The thermal behavior of the synthesized nanopowder and nanocomposites is investigated. A substantial difference in burning rate of RDX nanopowder has been found in comparison to micron‐sized material. Experimental results allow investigating the effects of nanosized materials on the combustion characteristics.
Effects of Nanometer Ni, Cu, Al and NiCu Powders on the Thermal Decomposition of Ammonium Perchlorate
Authors: Liu Leili , Li Fengsheng , Tan Linghua , Ming Li , Yi Yang
Published: February 2004

A study of the decomposition behaviour for Ammonium Perchlorate(AP) was carried out by differential thermal analysis and the two decomposition peaks were observed. The high temperature peak was found to shift to lower temperatures, but the corresponding shift in the low temperature peak was smaller due to the effect of nanometer metal powders. Results shows that Cu and NiCu nanopowders decreased both the high and low decomposition temperature, while Ni and Al nanopowders just decreased the high decomposition temperature and increased the low decomposition temperature. Metal micron‐sized powders show catalytic effects on the thermal decomposition of AP, but their effects are less than that of nanometer metal powders. With the increase in content, nanometer metal powders enhanced their catalytic effect on the high temperature decomposition of AP, however their effect was weakened on the low temperature decomposition.
The Effects of Containment on Detonation Velocity
Authors: Souers P. Clark , Vitello Peter , Esen Sedat , Kruttschnitt Julius , Bilgin H. A.
Published: February 2004

Reactive flow cylinder code runs on six explosives were made with rate constants varying from 0.03 to 70 μs−1. Six unconfined/steel sets of original ANFO and dynamite data are presented. A means of comparing confinement effects both at constant radius and at constant detonation velocity is presented. Calculations show two qualitatively different modes of behavior. For Us/Co≥1.2, where Us is the detonation velocity and Co the zero‐pressure sound speed in steel, we find a sharp shock wave in the metal. The shock passes through the steel and the outer wall has a velocity jump‐off. For Us/Co≤1.04, we find a pressure gradient that moves at the detonation velocity. A precursor pulse drives in the explosive ahead of the detonation front. The outer wall begins to move outward at the same time the shock arrives in the explosive, and the outer wall slowly and continuously increases in velocity. The Us/Co≥1.2 cylinders saturate in detonation velocity for thick walls but the Us/Co<<1.04 case does not. The unconfined cylinder shows an edge lag in the front that approximately equals the reaction zone length, but the highly confined detonation front is straight and contains no reaction zone information. The wall thickness divided by the reaction zone length yields a dimensionless wall thickness, which allows comparison of explosives with different detonation rates. Even so, a rate effect is found in the detonation velocities, which amounts to the inverse 0.15–0.5 power.
Metal/Fluorocarbon Pyrolants: V. Theoretical Evaluation of the Combustion Performance of Metal/Fluorocarbon Pyrolants based on Strained Fluorocarbons
Authors: Koch Ernst‐Christian
Published: February 2004

The potential of strained fluorocarbons, which can act as oxidizers in metal‐based pyrolant systems, is investigated. The oxidizing performance of fluorocarbons is evaluated by the enthalpy of combustion ΔcH(298K) and the fractional electron transfer ΔN. ΔcH(298K) can be related to hybridisation, molar C/F ratio and strain of the parent carbon skeleton of the fluorocarbon. Considered fluorocarbons are tetrafluorotetrahedrane (CF)4 (3), tetrakis(trifluoromethyl)tetrahedrane C4(CF3)4 (4), hexafluoro[3]prismane (CF)6 (5), hexakis(trifluoromethyl)[3]prismane C6(CF3)6 (6), octafluorocubane (CF)8 (7), octakis(trifluoromethyl)cubane C8(CF3)8 (8), eicosafluorododecahedrane (CF)20 (9), eicosakis(trifluoromethyl)dodecahedrane C20(CF3)20 (10), C60F48 (11) and perfluorofullerane (CF)60 (12). Powerful oxidisers in terms of exothermicity are those possessing both tertiary CF‐units and strained carbon skeletons. Nevertheless the reactivity, which is estimated on basis of ΔN, the fraction of electrons transferred according to Pearson, has been found to be high with the corresponding perfluoromethyl derivatives and maximum with the fluorofulleranes. For part IV see Ref. 17.
Calculation of the Detonation Velocities and Detonation Pressures of Dinitrobiuret (DNB) and Diaminotetrazolium Nitrate (HDAT‐NO3)
Authors: Geith Janna , Klapötke Thomas M. , Weigand Jan , Holl Gerhard
Published: February 2004

The enthalpies of combustion (ΔcombH) of dinitrobiuret (DNB) and diaminotetrazolium nitrate (HDAT‐NO3) were determined experimentally using oxygen bomb calorimetry: ΔcombH(DNB)=5195±200 kJ kg−1, ΔcombH(HDAT‐NO3)=7900±300 kJ kg−1. The standard enthalpies of formation (ΔfH°) of DNB and HDAT‐NO3 were obtained on the basis of quantum chemical computations at the electron‐correlated ab initio MP2 (second order Møller‐Plesset perturbation theory) level of theory using a correlation consistent double‐zeta basis set (cc‐pVTZ): ΔfH°(DNB)=−353 kJ mol−1, −1 829 kJ kg−1; ΔfH°(HDAT‐NO3)=+254 kJ mol−1, +1 558 kJ kg−1. The detonation velocities (D) and detonation pressures (P) of DNB and HDAT‐NO3 were calculated using the empirical equations by Kamlet and Jacobs: D(DNB)=8.66 mm μs−1, P(DNB)=33.9 GPa, D(HDAT‐NO3)=8.77 mm μs−1, P(HDAT‐NO3)=33.3 GPa.
Regulation of prostasin expression and function in the prostate
Authors: Chen Li‐Mei , Zhang Xiaochun , Chai Karl X.
Published: February 2004

BACKGROUND

The invasion suppressor prostasin is down‐regulated in prostate cancer, but the mechanism is unknown. A prostasin‐binding protein (PBP) was found in the seminal vesicles, but its identity remains unclear.

METHODS

Genomic Southern blot analysis using methylation sensitive restriction endonucleases was employed to examine the prostasin gene promoter region in prostate cancer cell lines. RT‐PCR was employed to examine prostasin expression under demethylation, histone deacetylase inhibition, and nerve growth factor (NGF) treatment. Liquid column chromatography was employed to purify the PBP from mouse seminal vesicles. The PBP was further characterized by amino acid sequence analysis, recombinant protein expression, protease inhibition and binding assays. Immunohistochemistry and Western blot analysis were used to evaluate PBP expression in the prostate and prostate cancer cells.

RESULTS

Promoter DNA methylation partly causes the prostasin down‐regulation in DU‐145 and PC‐3 cells, while prostasin expression can be induced by NGF. The PBP is identified to be protease nexin‐1 (PN‐1), a serpin. PN‐1 inhibits prostasin's serine protease activity, is expressed by prostate epithelial cells (PrECs) and prostate cancer cells, and capable of binding to membrane‐anchored prostasin.

CONCLUSIONS

Prostasin's expression and function are regulated by factors in the prostate tissue environment. © 2003 Wiley‐Liss, Inc.
Treatment of epigallocatechin‐3‐gallate inhibits matrix metalloproteinases‐2 and ‐9 via inhibition of activation of mitogen‐activated protein kinases, c‐jun and NF‐κB in human prostate carcinoma DU‐145 cells
Authors: Vayalil Praveen K. , Katiyar Santosh K.
Published: February 2004

BACKGROUND

Matrix metalloproteinases (MMPs) are involved in tumor progression including the carcinoma of the prostate (CaP). Therefore, the effect of (−)‐epigallocatechin‐3‐gallate (EGCG) was determined on the synthesis and activation of tumor invasion‐specific MMP‐2 and MMP‐9 in human prostate carcinoma DU‐145 cells.

METHODS

MMP‐2 and MMP‐9 were determined by zymography and Western blot analysis. Since fibroblast conditioned medium (FCM) partially mimics in vivo tumor‐host microenvironment, DU145 cells were co‐cultured in FCM.

RESULTS

Treatment of EGCG to DU‐145 cells resulted in dose‐dependent inhibition of FCM‐induced pro and active both forms of MMP‐2 and MMP‐9 concomitant with marked inhibition of phosphorylation of ERK1/2 and p38. In identical conditions, treatment of EGCG or inhibitors of MEK or p38 to DU‐145 cells inhibited FCM‐induced phosphorylation of ERK1/2 and/or p38 concomitant reduction in MMP‐2 and ‐9. EGCG also inhibited androgen‐induced pro‐MMP‐2 expression in LNCaP cells. Further, treatment of EGCG also resulted in inhibition of activation of c‐jun and NF‐κB in in vitro DU‐145 cells.

CONCLUSIONS

The inhibition of MMP‐2 and MMP‐9 in DU145 cells by EGCG is mediated via inhibition of phosphorylation of ERK1/2 and p38 pathways, and inhibition of activation of transcription factors c‐jun and NF‐κB. EGCG may play a role in prevention of invasive metastatic processes of both androgen‐dependent and ‐independent prostate carcinoma. © 2003 Wiley‐Liss, Inc.
Inhibition of cell signaling by the combi‐nitrosourea FD137 in the androgen independent DU145 prostate cancer cell line
Authors: Qiu Qiyu , Dudouit Fabienne , Banerjee Ranjita , McNamee James P. , Jean‐Claude Bertrand J.
Published: February 2004

BACKGROUND

FD137, a nitrosourea appended to a quinazoline ring, was designed to simultaneously block epidermal growth factor receptor (EGFR)‐mediated signaling and damage genomic DNA in refractory EGF‐dependent prostate tumors.

METHODS

The mixed inhibition of cell signaling and DNA damage by FD137 were determined by Western blotting, RT‐PCR, flow cytometry, sulforhodamine B (SRB), and comet assay.

RESULTS

FD137 and its metabolite FD110 induced a dose‐dependent increase in inhibition of EGF‐stimulated EGFR autophosphorylation and this translated into blockade of c‐fos gene expression in DU145 cells. FD137 induced significant levels of DNA damage and showed 150‐fold greater anti‐proliferative activity than BCNU, a classical nitrosourea. In contrast to BCNU, complete inhibition of EGF‐induced cell transition to S‐phase was observed at concentrations of FD137 as low as 3 μM.

CONCLUSION

FD137 could not only damage DNA, but also significantly block downstream EGFR‐mediated signaling. The superior activity of FD137 may be imputable to the combined effect of its mixed EGFR/DNA targeting properties. This novel strategy may well represent a new approach to target nitrosoureas to EGFR‐overexpressing carcinomas of the prostate. © 2004 Wiley‐Liss, Inc.
P‐glycoprotein increases the efflux of the androgen dihydrotestosterone and reduces androgen responsive gene activity in prostate tumor cells
Authors: Fedoruk Matthew N. , Giménez‐Bonafé Pepita , Guns Emma S. , Mayer Lawrence D. , Nelson Colleen C.
Published: February 2004

BACKGROUND

P‐glycoprotein (P‐gp) is commonly associated with multi‐drug resistance (MDR) in cancer cells and the efflux of a broad spectrum of chemicals from the cell, including many chemotherapeutics and certain steroid hormones. The impact of P‐gp and mechanisms involved in androgen transport and cellular accumulation within normal and malignant prostate cells remains unclear.

METHODS

Following incubation of LNCaP, PC‐3, HeLa, and HeLa FLAG–androgen receptor (AR) cells with 3H‐dihydrotestosterone (DHT) alone and in combination with P‐gp inhibitors, PSC‐833 and verapamil, we examined the cellular accumulation and efflux of androgens, as well as gene transcriptional response.

RESULTS

Our data reveal that the cellular transport and accumulation of DHT is dependent on the expression of functional AR and modulated by P‐gp. P‐gp over‐expression by both transient transfection and aspirin treatment in LNCaP cells showed decreased intracellular DHT accumulation, further suggesting DHT efflux is P‐gp regulated.

CONCLUSIONS

Androgen responsiveness may be modulated by P‐gp in prostate cancer cells. The biological consequences of increased P‐gp expression are decreased androgen accumulation and a corresponding decrease in androgen‐regulated transcriptional activity and PSA gene expression. © 2004 Wiley‐Liss, Inc.
Apoptotic impact of α1‐blockers on prostate cancer growth: A myth or an inviting reality?
Authors: Tahmatzopoulos Anastasios , Kyprianou Natasha
Published: February 2004

BACKGROUND

Pharmacological manipulation or genetic targeting of the major apoptosis regulators, such as bcl‐2, caspases, and inhibitors of apoptosis (IAPs), represent clinically attractive avenues towards effective therapeuti strategies for advanced prostate cancer. A wealth of evidence established the α1‐adrenoceptor antagonists to be clinically effective in relieving the symptoms associated with benign prostatic hyperplasia (BPH) by relaxing prostatic smooth muscle tone. This action alone however does not fully account for the long‐term clinical response to these drugs in BPH patients.

METHODS

Experimental and retrospective clinical studies provided new evidence supporting a differential growth‐suppressing function of two α1‐adrenoceptor antagonists against prostate cancer, independent of an α1‐adrenoceptor mechanism.

RESULTS

The quinazoline‐based antagonists, doxazosin and terazosin, induce apoptosis, inhibit cell adhesion to the extracellular matrix (by activating anoikis), and prevent cell invasion and migration of prostate tumor epithelial cells and vascular endothelial cells. Tamsulosin, a sulphonamide‐based, clinically effective α1‐adrenoceptor antagonist for BPH treatment, fails to exert a similar apoptotic action against prostate cells. Furthermore, at pharmacologically relevant doses, doxazosin suppresses benign and malignant prostate growth in in vivo experimental models. The effect is characterized by three intriguing features: (a) it is mediated by an α1‐adrenoceptor‐independent action, (possibly related to the quinazoline nucleus); (b) it is targeted at the apoptotic process without affecting cell proliferation; and (c) the elevated apoptotic index correlated with symptom score improvement in BPH patients.

CONCLUSIONS

This evidence challenges conventional knowledge of the mechanism of action of α1‐adrenoceptor antagonists, and points to a new therapeutic value for these drugs by providing a differential molecular basis for their anti‐tumor efficacy. The present review focuses on the characterization of the apoptotic/anti‐angiogenic effect of quinazoline‐based α1‐adrenoceptor antagonists against prostate cancer cells and discusses the clinical significance of this action in the prevention and treatment of prostate cancer. © 2003 Wiley‐Liss, Inc.
Expression of bone morphogenetic protein‐7 (BMP‐7) in human prostate
Authors: Masuda Hiroshi , Fukabori Yoshitatsu , Nakano Katsuya , Shimizu Nobuaki , Yamanaka Hidetoshi
Published: February 2004

BACKGROUND

It is known that BMP is a factor that strongly induces bone formation in muscle tissue. However, the expression of BMP‐7 is not detected in normal bone tissues. Recently, we reported that the level of BMP‐7 expression was significantly higher in metastatic bone lesions than in normal bone and that the high expression of BMP‐7 in metastatic bone lesions was related to osteoblastic metastasis. The aim of this study was to investigate the level of BMP‐7 gene expression in normal prostate glandular tissues, newly diagnosed prostate cancer tissues, and local recurrent prostate cancer tissues.

METHODS

Total RNA was extracted from 23 samples of normal prostate glandular tissues, 28 samples of newly diagnosed prostate cancer tissues, and 23 samples of local recurrent prostate cancer tissues. We also examined the expression of BMP‐7 in human prostatic epithelial cells (HPECs) under conditions of androgen replacement. After reverse transcription of the RNA samples, the expression of BMP‐7 was measured by real‐time quantitative polymerase chain reaction (PCR). Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal standard.

RESULTS

The level of BMP‐7 expression was highest in the normal prostate glandular tissue (P < 0.05). The expression of BMP‐7 tended to be lower during the development and progression of prostate cancer. The expression of BMP‐7 in prostate epithelial cells treated with 10−7 M dihydrotestosterone (DHT) was significantly higher than in the DHT‐free control (P = 0.0285).

CONCLUSIONS

It has been reported that the BMP‐7 mRNA level is androgen‐dependent in the mouse prostate. The present results suggest that the BMP‐7 mRNA level is androgen‐dependent in the human prostate, since the expression of BMP‐7 was decreased in local recurrent prostate cancer tissues after hormonal therapy and was increased by DHT. © 2004 Wiley‐Liss, Inc.
Free energy simulations and MM–PBSA analyses on the affinity and specificity of steroid binding to antiestradiol antibody
Authors: Laitinen Tuomo , Kankare Jussi A. , Peräkylä Mikael
Published: February 2004

Antiestradiol antibody 57‐2 binds 17β‐estradiol (E2) with moderately high affinity (Ka = 5 × 108 M−1). The structurally related natural estrogens estrone and estriol as well synthetic 17‐deoxy‐estradiol and 17α‐estradiol are bound to the antibody with 3.7–4.9 kcal mol−1 lower binding free energies than E2. Free energy perturbation (FEP) simulations and the molecular mechanics–Poisson–Boltzmann surface area (MM–PBSA) method were applied to investigate the factors responsible for the relatively low cross‐reactivity of the antibody with these four steroids, differing from E2 by the substituents of the steroid D‐ring. In addition, computational alanine scanning of the binding site residues was carried out with the MM–PBSA method. Both the FEP and MM–PBSA methods reproduced the experimental relative affinities of the five steroids in good agreement with experiment. On the basis of FEP simulations, the number of hydrogen bonds formed between the antibody and steroids, which varied from 0 to 3 in the steroids studied, determined directly the magnitude of the steroid–antibody interaction free energies. One hydrogen bond was calculated to contribute about 3 kcal mol−1 to the interaction energy. Because the relative binding free energies of estrone (two antibody‐steroid hydrogen bonds), estriol (three hydrogen bonds), 17‐deoxy‐estradiol (no hydrogen bonds), and 17α‐estradiol (two hydrogen bonds) are close to each other and clearly lower than that of E2 (three hydrogen bonds), the water–steroid interactions lost upon binding to the antibody make an important contribution to the binding free energies. The MM–PBSA calculations showed that the binding of steroids to the antiestradiol antibody is driven by van der Waals interactions, whereas specificity is solely due to electrostatic interactions. In addition, binding of steroids to the antiestradiol antibody 57‐2 was compared to the binding to the antiprogesterone antibody DB3 and antitestosterone antibody 3‐C4F5, studied earlier with the MM–PBSA method. Proteins 2004. © 2004 Wiley‐Liss, Inc.
Probing the α‐complementing domain of E. coli β‐galactosidase with use of an insertional pentapeptide mutagenesis strategy based on Mu in vitro DNA transposition
Authors: Poussu Eini , Vihinen Mauno , Paulin Lars , Savilahti Harri
Published: February 2004

Protein structure‐function relationships can be studied by using linker insertion mutagenesis, which efficiently identifies essential regions in target proteins. Bacteriophage Mu in vitro DNA transposition was used to generate an extensive library of pentapeptide insertion mutants within the α‐complementing domain 1 of Escherichia coli β‐galactosidase, yielding mutants at 100% efficiency. Each mutant contained an accurate 15‐bp insertion that translated to five additional amino acids within the protein, and the insertions were distributed essentially randomly along the target sequence. Individual mutants (α‐donors) were analyzed for their ability to restore (by α‐complementation) β‐galactosidase activity of the M15 deletion mutant (α‐acceptor), and the data were correlated to the structure of the β‐galactosidase tetramer. Most of the insertions were well tolerated, including many of those disrupting secondary structural elements even within the protein's interior. Nevertheless, certain sites were sensitive to mutations, indicating both known and previously unknown regions of functional importance. Inhibitory insertions within the N‐terminus and loop regions most likely influenced protein tetramerization via direct local effects on protein–protein interactions. Within the domain 1 core, the insertions probably caused either lateral shifting of the polypeptide chain toward the protein's exterior or produced more pronounced structural distortions. Six percent of the mutant proteins exhibited temperature sensitivity, in general suggesting the method's usefulness for generation of conditional phenotypes. The method should be applicable to any cloned protein‐encoding gene. Proteins 2004. © 2004 Wiley‐Liss, Inc.
Protein conformer selection by sequence‐dependent packing contacts in crystals of 3‐phosphoglycerate kinase
Authors: Kovári Zoltán , Vas Mária
Published: February 2004

In several crystal structures of 3‐phosphoglycerate kinase (PGK), the two domains occupy different relative positions. It is intriguing that the two extreme (open and closed) conformations have never been observed for the enzyme from the same species. Furthermore, in certain cases, these different crystalline conformations represent the enzyme‐ligand complex of the same composition, such as the ternary complex containing either the substrate 3‐phosphoglycerate (3‐PG) and β,γ‐imido‐adenosine‐5′‐triphosphate (AMP‐PNP), an analogue of the substrate MgATP, or 3‐PG and the product MgADP. Thus, the protein conformation in the crystal is apparently determined by the origin of the isolated enzyme: PGK from pig muscle has only been crystallized in open conformation, whereas PGK from either Thermotoga maritima or Trypanosoma brucei has only been reported in closed conformations. A systematic analysis of the underlying sequence differences at the crucial hinge regions of the molecule and in the protein–protein contact surfaces in the crystal, in two independent pairs of open and closed states, have revealed that 1) sequential differences around the molecular hinges do not explain the appearance of fundamentally different conformations and 2) the species‐specific intermolecular contacts between the nonconserved residues are responsible for stabilizing one conformation over the other in the crystalline state. A direct relationship between the steric position of the contacts in the three‐dimensional structure and the conformational state of the protein has been demonstrated. Proteins 2004. © 2004 Wiley‐Liss, Inc.
Funnel‐like organization in sequence space determines the distributions of protein stability and folding rate preferred by evolution
Authors: Xia Yu , Levitt Michael
Published: February 2004

To understand the physical and evolutionary determinants of protein folding, we map out the complete organization of thermodynamic and kinetic properties for protein sequences that share the same fold. The exhaustive nature of our study necessitates using simplified models of protein folding. We obtain a stability map and a folding rate map in sequence space. Comparison of the two maps reveals a common organizational principle: optimality decreases more or less uniformly with distance from the optimal sequence in the sequence space. This gives a funnel‐shaped optimality surface. Evolutionary dynamics of a sequence population on these two maps reveal how the simple organization of sequence space affects the distributions of stability and folding rate preferred by evolution. Proteins 2004. © 2004 Wiley‐Liss, Inc.
Prediction of α‐turns in proteins using PSI‐BLAST profiles and secondary structure information
Authors: Kaur Harpreet , Raghava G.P.S.
Published: February 2004

In this paper a systematic attempt has been made to develop a better method for predicting α‐turns in proteins. Most of the commonly used approaches in the field of protein structure prediction have been tried in this study, which includes statistical approach “Sequence Coupled Model” and machine learning approaches; i) artificial neural network (ANN); ii) Weka (Waikato Environment for Knowledge Analysis) Classifiers and iii) Parallel Exemplar Based Learning (PEBLS). We have also used multiple sequence alignment obtained from PSIBLAST and secondary structure information predicted by PSIPRED. The training and testing of all methods has been performed on a data set of 193 non‐homologous protein X‐ray structures using five‐fold cross‐validation. It has been observed that ANN with multiple sequence alignment and predicted secondary structure information outperforms other methods. Based on our observations we have developed an ANN‐based method for predicting α‐turns in proteins. The main components of the method are two feed‐forward back‐propagation networks with a single hidden layer. The first sequence‐structure network is trained with the multiple sequence alignment in the form of PSI‐BLAST‐generated position specific scoring matrices. The initial predictions obtained from the first network and PSIPRED predicted secondary structure are used as input to the second structure–structure network to refine the predictions obtained from the first net. The final network yields an overall prediction accuracy of 78.0% and MCC of 0.16. A web server AlphaPred (http://www.imtech.res.in/raghava/alphapred/) has been developed based on this approach. Proteins 2004. © 2004 Wiley‐Liss, Inc.
The role of carbon‐donor hydrogen bonds in stabilizing tryptophan conformations
Authors: Petrella Robert J. , Karplus Martin
Published: February 2004

Although most side‐chain torsion angles correspond to low‐energy rotameric positions, deviations occur with significant frequency. One striking example arises in Trp residues, which have an important role in stabilizing protein structures because of their size and mixed hydrophobic/hydrophilic character. Ten percent of Trp side‐chains have unexplained conformations with χ2 near 0° instead of the expected 90°. The current work is a structural and energetic analysis of these conformations. It is shown that many Trp residues with these orientations are stabilized by three‐center carbon‐donor hydrogen bonds of the form CH…X…HC, where X is a polar hydrogen‐bond acceptor in the environment of the side‐chain. The bridging hydrogen bonds occur both within the Trp side‐chain and between the side‐chain and the local protein backbone. Free energy maps of an isolated Trp residue in an explicit water environment show a minimum corresponding to the off‐rotamer peak observed in the crystallographic data. Bridging carbon‐donor hydrogen bonds are also shown to stabilize on‐rotamer Trp conformations, and similar bridging hydrogen bonds also stabilize some off‐rotamer Asp conformations. The present results suggest a previously unrecognized role for three‐center carbon‐donor hydrogen bonds in protein structures and support the view that the off‐rotamer Trp side‐chain orientations are real rather than artifacts of crystallographic refinements. Certain of the off‐rotamer Trp conformations appear to have a functional role. Proteins 2004;54:000–000. © 2004 Wiley‐Liss, Inc.
Vicinal disulfide bridge conformers by experimental methods and by ab initio and DFT molecular computations
Authors: Hudáky Ilona , Gáspári Zoltán , Carugo Oliviero , Čemažar Maša , Pongor Sándor , Perczel András
Published: February 2004

A systematic comparison is made between experimental and computational data gained on vicinal disulfide bridges in proteins and peptides. Structural and stability data of ab initio and density functional theory (DFT) calculations on the model compound 4,5‐ditiaheptano‐7‐lactam and the model peptide HCOox‐[CysCys]NH2 at RHF/3‐21G*, B3LYP/6‐31+G(d), and B3LYP/6‐311++G(d,p) levels of theory are presented. The data on XxxCysCysYyy type amino acid sequence units retrieved from PDB SELECT, along with data on sequence units that have vicinal disulfide bridge, taken from the Brookhaven Protein Data Bank, are conformationally characterized. Amino acid backbone conformations, cis‐trans isomerism of the amide bond between the two cysteine residues, and ring puckering are studied. Ring puckers are characterized by their relation to the conformers of the parent 4,5‐ditiaheptano‐7‐lactam. Computational precision and accuracy are proved by frequency calculation and solvent model optimization on selected conformers. It is found that the ox‐[CysCys] unit is able to accept types I, II, VIa, VIb, and VIII β‐turn structures. Proteins 2004. © 2003 Wiley‐Liss, Inc.
Squeezed exponential kinetics to describe a nonglassy downhill folding as observed in a lattice protein model
Authors: Nakamura Hironori K. , Sasai Masaki , Takano Mitsunori
Published: February 2004

We previously studied the so‐called strange kinetics in the two‐dimensional lattice HP model. To further study the strange kinetics, folding processes of a 27‐mer cubic lattice protein model with Gō potential were investigated by simulating how the bundle of folding trajectories, consisting of a number of independent Monte Carlo simulations, evolves as the folding reaction proceeds, covering a wide range of temperature. Three realms of folding kinetics were observed depending on temperature. Although at temperatures where folding was two‐state‐like, the kinetics was conventional single exponential, we found that the time course data were well represented by a squeezed (or “shrunken”) exponential function, exp [−(t/τ)β] with β > 1, at temperatures lower than the folding temperature, where folding was fastest and of a nonglassy downhill type. The squeezed exponential kinetics was found to pertain to the subdiffusion on the nonglassy downhill free energy surface and presents a marked contrast both to the single exponential kinetics and to the stretched exponential kinetics that was observed at lower temperatures where folding was also downhill but topological frustration came into effect. The observed temperature dependence of the folding kinetics suggests that some small single‐domain proteins may follow the squeezed exponential kinetics at about the room temperature. Proteins 2004;55:99–106. © 2004 Wiley‐Liss, Inc.
A structural and dynamic model for the interaction of interleukin‐8 and glycosaminoglycans: Support from isothermal fluorescence titrations
Authors: Krieger Elmar , Geretti Elena , Brandner Barbara , Goger Birgit , Wells Timothy N. , Kungl Andreas J.
Published: February 2004

Binding of interleukin‐8 (IL‐8) to glycosaminoglycans (GAGs) on the surface of endothelial cells is crucial for the recruitment of neutrophils to an inflammatory site. Deriving structural knowledge about this interaction from in silico docking experiments has proved difficult because of the high flexibility and the size of GAGs. Therefore, we developed a docking method that takes into account ligand and protein flexibility by running ∼15,000 molecular dynamics simulations of the docking event with different initial orientations of the binding partners. The method was shown to successfully reproduce the residues of basic fibroblast growth factor involved in GAG binding. Docking of a heparin hexasaccharide to IL‐8 gave an interaction interface involving the basic residues His18, Lys20, Arg60, Lys64, Lys67, and Arg68. By subjecting IL‐8 single‐site mutants, in which these amino acids were replaced by alanine, to isothermal fluorescence titrations, the affinities for heparin were determined to be wtIL‐8 > IL‐8(H18A) ≫ IL‐8(R68A) > IL‐8(K67A) ≫ IL‐8(K20A) > IL‐8(R60A) ≫ IL‐8(K64A). A comparison with the binding energies calculated from the model revealed high values for wtIL‐8 and the H18A mutant and significantly lower but similar energies for the remaining mutants. Connecting the two fully sulfated hexasaccharides bound to each of the two IL‐8 monomers in the dimeric chemokine by an N‐acetylated dodecasaccharide gave a complex structure in which the GAG molecule aligned in a parallel fashion to the N‐terminal α‐helices of IL‐8 like a horseshoe. A 5‐ns molecular dynamics simulation of this complex confirmed its structural stability and revealed a reorientation in both binding sites where a disaccharide became the central binding unit. Isothermal fluorescence titration experiments using differently sulfated heparin disaccharides confirmed that a single disaccharide can indeed bind IL‐8 with high affinity. Proteins 2004;54:000–000. © 2004 Wiley‐Liss, Inc.
Crystal structure of the YGR205w protein from Saccharomyces cerevisiae: Close structural resemblance to E. coli pantothenate kinase
Authors: Li de La Sierra‐Gallay Ines , Collinet Bruno , Graille Marc , Quevillon‐Cheruel Sophie , Liger Dominique , Minard Philippe , Blondeau Karine , Henckes Gilles , Aufrère Robert , Leulliot Nicolas , Zhou Cong‐Zhao , Sorel Isabelle , Ferrer Jean‐Luc , Poupon Anne , Janin Joël , van Tilbeurgh Herman
Published: February 2004

The protein product of the YGR205w gene of Saccharomyces cerevisiae was targeted as part of our yeast structural genomics project. YGR205w codes for a small (290 amino acids) protein with unknown structure and function. The only recognizable sequence feature is the presence of a Walker A motif (P loop) indicating a possible nucleotide binding/converting function. We determined the three‐dimensional crystal structure of Se‐methionine substituted protein using multiple anomalous diffraction. The structure revealed a well known mononucleotide fold and strong resemblance to the structure of small metabolite phosphorylating enzymes such as pantothenate and phosphoribulo kinase. Biochemical experiments show that YGR205w binds specifically ATP and, less tightly, ADP. The structure also revealed the presence of two bound sulphate ions, occupying opposite niches in a canyon that corresponds to the active site of the protein. One sulphate is bound to the P‐loop in a position that corresponds to the position of β‐phosphate in mononucleotide protein ATP complex, suggesting the protein is indeed a kinase. The nature of the phosphate accepting substrate remains to be determined. Proteins 2004;54:000–000. © 2004 Wiley‐Liss, Inc.
Immunoglobulin kappa light chain and its amyloidogenic mutants: A molecular dynamics study
Authors: Nowak Mateusz
Published: February 2004

AL amyloidosis and LCDD are pathological conditions caused by extracellural deposition of monoclonal Ig light chain variable domains. In the former case, deposits have a form of amyloid fibrils, in the latter, amorphous aggregates. 1REI κ light chain variable domain and its two point mutants, R61N and D82I, were chosen for the analysis in this work. Wild 1REI does not create deposits in vitro, while R61N aggregates as amyloid fibrils and D82I creates amorphous aggregates. Both mutated residues create a conserved salt bridge; thus, substitution of any of them should decrease VL domain stability. For these three proteins, 5 ns MD simulations were conducted in temperatures of 300 K and 400 K, with protonated and unprotonated acidic residues, mimicking acidic and neutral experimental pH conditions (3 sets: N300, N400, and A400). The analysis of trajectories focused on characterization of changes in conformational behavior and stability of Ig κ light chain variable domain caused by single aminoacid substitutions that were experimentally proved to enhance aggregation propensity, both in the form of amyloid and amorphous aggregates. Residue D82 turns out to be involved not only in R61–D82 but also in K45–D82 interaction, which was not observed in the X‐ray structure, but frequently populated simulations of 1REI. The substitution D82I excludes both interactions, resulting in substantial destabilization (i.e., easier aggregation). Examination of behavior of edge regions of VL β‐sandwich reveals significant alterations in D82I mutant compared to wild 1REI, while relatively small changes occur in R61N. This suggests that mild and slow destabilization is the reason of the conversion of VL to partially folded amyloidogenic intermediate structure. Proteins 2004. © 2004 Wiley‐Liss, Inc.
Characterization of the amino acid contribution to the folding degree of proteins
Authors: Estrada Ernesto
Published: February 2004

The folding degree index (Estrada, Bioinformatics 2002;18:697–704) is extended to account for the contribution of amino acids to folding. First, the mathematical formalism for extending the folding degree index is presented. Then, the amino acid contributions to folding degree of several proteins are used to analyze its relation to secondary structure. The possibilities of using these contributions in helping or checking the assignation of secondary structure to amino acids are also introduced. The influence of external factors to the amino acids contribution to folding degree is studied through the temperature effect on ribonuclease A. Finally, the analysis of 3D protein similarity through the use of amino acid contributions to folding degree is studied by selecting a series of lysozymes. These results are compared to that obtained by sequence alignment (2D similarity) and 3D superposition of the structures, showing the uniqueness of the current approach. Proteins 2004;54:000–000. © 2004 Wiley‐Liss, Inc.