bluuebunny/crossref_metadata_2025 huggingface dataset:
Mono‐ and Bidentate Benzannulated N‐Heterocyclic Germylenes, Stannylenes and Plumbylenes
Authors: Zabula Alexander V. , Hahn F. Ekkehardt
Published: October 2008

The current state of the chemistry of mono‐ and bidentate N‐heterocyclic germylenes, stannylenes, plumbylenes, and related compounds is reviewed with special emphasis placed on benzannulated derivatives. The preparation, electronic structure, aggregation behaviour, and the coordination chemistry of the free benzannulated carbene analogues and their adducts with Lewis bases and complexes with transition metals are discussed. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)
Multivalent 1‐Oxa‐3,5,7‐triazahepta‐1,3,5‐trienes: Synthesis, Structural Properties and Metal Coordination
Authors: Greving Jan‐Bernd , Behrens Henrik , Fröhlich Roland , Würthwein Ernst‐Ulrich
Published: October 2008

Nucleophilic ring opening of bis‐ and tris(1‐oxa‐3,5‐diazinium) salts 1 with n‐propylamine leads to the bis‐ and tris(armed) oligonitrile derivatives 3 and 8 with secondary amino groups in the side chain. Alternatively, treatment of simple 1‐oxa‐3,5‐diazinium salts 2 with primary and secondary di‐ and triamines offers a convenient pathway for the synthesis of bis‐ and tris(armed) oligonitriles 4, 5 and 7. 2,6‐Diaminopyridine furnishes the new pincer‐type ligand 6 upon reaction with 1‐oxa‐3,5‐diazinium salt 2a. Multivalent oligonitriles 4, 6, 7 and 8 containing secondary amino groups are able to form intra‐ and intermolecular hydrogen bonds in the solid state as detected by X‐ray crystallography. The oligonitriles 3a,b, 4b,d,h, 6 and 7a form coordination complexes with various ZnII, CuII, CoII, NiII and PdII salts acting as bidentate ligands to give six‐membered chelates of 1:1, 1:2 and 2:2 ligand/metal ion ratios. More complex structures are obtained from pincer ligand 6 with PdCl2 [compound 6·(PdCl2)2] and from the tridentate ligand 7a in reactions with Ni(ClO4)2 (compound 9). All new complexes were characterized by X‐ray crystallography. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)
Neutral Möbius Aromatics: Derivatives of the Pyrrole Congener Aza[11]annulene as Promising Synthetic Targets
Authors: Mauksch Michael , Tsogoeva Svetlana B.
Published: October 2008

Amino‐ and methyl‐disubstituted derivatives of an aromatic 12π electron pyrrole homologue Möbius 1H‐aza[11]annulene are predicted computationally at the B3LYP/6‐31G* level of theory to be distinctly aromatic and a neutral ground state on its potential surface. The parent annulene has a nonaromatic conformation, which is 6.0 kcal mol–1 lower in energy at the CCSD(T)/D95*//B3LYP/6‐31G* level of theory than a structurally very similar Möbius conformation. After derivatization, however, geometry optimizations of the two lowest‐energy – Möbius and non‐Möbius – heteroannulene conformations both converged two a single Möbius minimum, whereas two (Möbius and non‐Möbius) distinct difluoro‐ substituted species could be located 1.8 kcal mol–1 apart in energy at the B3LYP level. The Möbius minimum is 7.5 kcal mol–1 lower in energy at the coupled cluster level than a “Hückel topology” form derived from a known bridged 1H‐[4,9]methano[11]annulene. Several possible reactive modes are investigated in order to assess the relative stability of the Möbius parent annulene. In addition, we report formal novel examples for charged and neutral Möbius triplet aromatic annulenic and heteroannulenic systems, some of which also exhibit distinct aromatic properties. Further fundamental insights into the phenomenon of “Möbius aromaticity” are suggested. The aromaticity of Möbius annulenes is for the first time linked to wavefunction boundary conditions. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)
Comparative Study of the Chemoselectivity and Yields of the Synthesis of N‐Alkyl‐4‐(trihalomethyl)‐1H‐pyrimidin‐2‐ones
Authors: Zanatta Nilo , Faoro Débora , da S. Fernandes Liana , Brondani Patrícia B. , Flores Darlene C. , Flores Alex F. C. , Bonacorso Helio G. , Martins Marcos A. P.
Published: October 2008

This paper reports a comparative study of the chemoselectivity and yields of the synthesis of N‐alkyl‐4‐(trihalomethyl)‐1H‐pyrimidin‐2‐ones carried out by the cyclocondensation of 4‐alkoxy‐1,1,1‐trihalo‐3‐alken‐2‐ones (enones) with methyl‐ and allylureas with the N‐alkylation of 4‐(trihalomethyl)‐1H‐pyrimidin‐2‐ones with methyl iodide and allyl bromide. To determine the chemoselectivity of the products obtained, all compounds were fully analyzed by 1H and 13C NMR and 2D HMBC spectroscopy. This study has demonstrated that the cyclocondensation reactions give better yields and furnish either N1‐ or N3‐alkylated products depending on both the reaction conditions and the substituents on the enones, whereas the alkylation of pyrimidin‐2‐ones gives lower overall yields and either an N1‐alkylated product or a mixture of N1‐ and O‐alkylated products.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)
Synthesis of Oligosaccharides Related to a Biodynamic Saponin from Calamus Insignis as Their Propargyl Glycosides
Authors: Dasgupta Somnath , Mukhopadhyay Balaram
Published: October 2008

The total synthesis of the carbohydrate portion (penta‐and tetrasaccharides) of steroidal saponins, isolated fromCalamus insignis, is reported. A simple route is followed, starting from commercially available D‐glucose and L‐rhamnose, through high‐yielding protecting group manipulation strategies. Sulfuric acid immobilized on silica (H2SO4/silica) was used as an effective Brönsted acid source in conjunction with N‐iodosuccinimide for activation of thioglycosides or alone for trichloroacetimidate donors in key glycosylation steps. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)
Identification of rat urinary glycoproteome captured by three lectins using gel and LC‐based proteomics
Authors: Moon Pyong‐Gon , Hwang Hyun‐Ho , Boo Yong Chool , Kwon Joseph , Cho Je‐Yoel , Baek Moon‐Chang
Published: October 2008

Many different types of urine proteome studies have been done, but urine glycoprotein studies are insufficient. Therefore, we studied the glycoproteins from rat urine, which could be used to identify biomarkers in an animal model. First, urinary proteins were prepared by using the dialysis and lyophilizing methods from rat urine. Glycoproteins enriched with lectin affinity purification, concanavalin A, jacalin and wheat germ agglutinin from the urinary proteins were separated by means of reverse‐phase fast protein LC (FPLC) or 1‐D PAGE. Each FPLC fraction and 1‐D PAGE gel band were trypsin‐digested and analyzed by means of nanoLC‐MS/MS. LC‐MS/MS analyses were carried out by using linear ion trap MS. A total of 318 rat urinary glycoproteins were identified from the FPLC fractions and gel bands; approximately 90% of identified proteins were confirmed as glycoproteins in Swiss‐Prot. Many glycoproteins, known as biomarkers, including C‐reactive protein, uromodulin, amyloid beta A4 protein, alpha‐1‐inhibitor 3, vitamin D‐binding protein, kallikrein 3 and fetuin‐A were identified in this study. By studying urinary glycoproteins collected from rat, these results may help to assist in identifying urinary biomarkers regarding various types of disease models.
Quantitative assessment of human serum high‐abundance protein depletion
Authors: Stempfer Rene , Kubicek Markus , Lang Irene M. , Christa Noehammer , Gerner Christopher
Published: October 2008

The aim of this study is to quantify the effectivity of the depletion of human high‐abundance serum and plasma proteins for improved protein identification and disease marker candidate discovery and to assess the risk of concomitant removal of relevant marker proteins. 2‐DE and bottom‐up shotgun MS combining 2‐D capillary chromatography with MS/MS were applied in parallel for the analysis of fractions resulting from the depletion procedure. For many proteins the factors of enrichment by the depletion were obvious allowing their enhanced detection and identification upon high‐abundance protein depletion. Nano‐liquid chromatography linked MS allowed the efficient identification of several low‐abundant proteins that were not identified on the 2‐DE gels. Resolving the fractions that were eluted from the matrix upon depletion indicated unspecific binding of disease relevant proteins in plasma samples from acute myocardial infarction patients. The unspecific binding to the depletion matrix of inflammatory markers spiked into the serum was found to depend on the type of capturing agent used. Polyclonal avian antibodies (IgY) displayed the least unspecific binding due to the high immunogenicity of mammalian proteins in avian hosts.
Simultaneous determination of seven pesticides in waters using multi‐walled carbon nanotube SPE and NACE
Authors: Asensio‐Ramos María , Hernández‐Borges Javier , Ravelo‐Pérez Lidia M. , Rodríguez‐Delgado Miguel Ángel
Published: October 2008

In this work, NACE with UV detection is combined with SPE using multi‐walled carbon nanotubes (MWCNT) as stationary phase to determine a group of seven pesticides (pirimicarb, pyrifenox, penconazol, carbendazim, cyromazine, pyrimethanil and cyprodinil) in mineral water samples using ametryn as internal standard. The optimized BGE, consisting of a mixture of MeOH and ACN (1:2 v/v) with 90 mM SDS and 20.5 mM HClO4, was satisfactory to get a good resolution of the seven compounds in less than 13 min. On‐line preconcentration was carried out by electrokinetic injection of the sample dissolved in 78:22 v/v MeOH/ACN, 1.11 mM HClO4. Repeatability was studied for the same day (n=4), for nine different days (n=36) and for four different capillaries. RSD values were appropriate in all cases, i.e. in the range 4.3–9.4% between different capillaries. MWCNT of 10–15 nm od, 2–6 nm id and 0.1–10 μm length were used as SPE materials for the preconcentration of these pesticides from water samples. SPE parameters influencing the enrichment were optimized and the most favorable conditions were as follows: the amount of stationary phase, eluent, sample pH and sample volume were 40 mg MWCNT, 10 mL ACN and 10 mL dichloromethane containing 5% v/v formic acid, pH 8.0, and 750 mL, respectively. Mean recovery values ranged between 53 and 94% for Milli‐Q water and between 47 and 93% for mineral waters (RSD values were in the range 2–16%). The method allowed the determination of these pesticides at concentrations below the maximum residue limits established by the European Union legislation (LOD in the range 27–58 ng/L). When the cost, amount and type of the carbon nanotubes used in this work are compared with those carbon nanotubes previously used in the literature it is clear that the proposed materials can be used as economical stationary phases, even cheaper than conventional SPE cartridges.
SPE and large‐volume sample stacking in MEKC for determination of doxycycline in biological fluids: Comparison of direct injection to SPE‐MEKC
Authors: Injac Rade , Karljikovic‐Rajic Katarina , Strukelj Borut
Published: October 2008

A novel and simple method has been developed for the determination of doxycycline (DOX) in biological fluids. The method is based on SPE, large‐volume sample stacking (LVSS) and MEKC with UV‐DAD detection. Six SPE cartridges have been used in investigation for sample clean up and pre‐concentration (Supelco® LC‐8, LC‐18, LC‐SCX, and LC‐WCX, as well as Strata™‐X and X‐C). DOX was determined on a 56 cm (effective length 50 cm)×50 μm id fused‐silica capillary. The BGE was 20 mM borate buffer, pH 9.3, containing 80 mM SDS and 7.5% v/v of methanol (30 s×50 mbar), and the temperature and voltage were 25°C and 30 kV, respectively. The analytical wavelength was set at 210 nm. Under optimized conditions it is possible to determine DOX in human serum, urine, semen, tears and saliva with recovery of 97.5% (RSD 2.5%). The method was shown to be sensitive (LOD is 1 μg/L) and precise (intra‐day RSD 0.2 and 2.4%; inter‐days 0.4 and 3.5% for migration time and peak area, respectively). Results for developed SPE‐LVSS‐MEKC were compared with LVSS‐MEKC method with direct sample injection. The new LVSS‐MEKC method is presented as a useful technique for rapid determination without extraction procedure of DOX in human urine and serum, using 80 mM of SDS, 10% v/v of methanol and 40 mM borate buffer (pH 9.3; 30 s×50 mbar; 25°C; 30 kV; 350 nm), but not for the other biological fluids, according to lower sensitivity of the method and because of the sample composition.
Dimensioning of data networks: a flow‐level perspective
Authors: Lassila Pasi , Penttinen Aleksi , Virtamo Jorma
Published: October 2008

Traditional network dimensioning formulations have applied the Erlang model where the connections reserve capacity in the network. Until recently, tractable stochastic network models where the connections share the capacity in the network did not exist. The latter are becoming increasingly important as they can be applied to characterise file transfers in current data networks (e.g. IP networks). In particular, they can be utilised for dimensioning of networks with respect to the file transfer performance. To this end, we consider a model where the traffic consists of elastic flows (i.e. file transfers). Flows arrive randomly and share the network resources resulting in stochastically varying transmission rates for flows. Our contribution is to develop efficient methods for capacity planning to meet the performance requirements expressed in terms of the average transmission rate of flows on a given route, i.e. the per‐flow throughput. These methods are validated using ns2 simulations. We discuss also the effects of access rate limitations and how to combine the elastic traffic requirements with those of real‐time traffic. Finally, we outline how the methods can be applied in wireless mesh networks. Our results enable a simple characterisation of the order‐of‐magnitude of the required capacities, which can be utilised as a first step in practical network planning and dimensioning. Copyright © 2008 John Wiley & Sons, Ltd.
Stress differentially regulates the effects of voluntary exercise on cell proliferation in the dentate gyrus of mice
Authors: Kannangara Timal S. , Webber Alina , Gil‐Mohapel Joana , Christie Brian R.
Published: October 2008

It has been well‐established that cell proliferation and neurogenesis in the adult mouse dentate gyrus (DG) can be regulated by voluntary exercise. Recent evidence has suggested that the effects of voluntary exercise can in turn be influenced by environmental factors that regulate the amount of stress an animal is exposed to. In this study, we use bromodeoxyuridine and proliferating cell nuclear antigen immunohistochemistry to show that voluntary exercise produces a significant increase in cell proliferation in the adult mouse DG in both isolated and socially housed mice. This effect on proliferation translates into an increase in neurogenesis and neuronal branching of new neurons in the mice that exercised. Although social condition did not regulate proliferation in young adult mice, an effect of social housing could be observed in mice exposed to acute restraint stress. Surprisingly, only exercising mice housed in isolated conditions showed an increase in cellular proliferation following restraint stress, whereas socially housed, exercising mice, failed to show a significant increase in proliferation. These findings indicate that social housing may increase the effects of any stressful episodes on hippocampal neurogenesis in the mouse DG. © 2008 Wiley‐Liss, Inc.
A role for hilar cells in pattern separation in the dentate gyrus: A computational approach
Authors: Myers Catherine E. , Scharfman Helen E.
Published: October 2008

We present a simple computational model of the dentate gyrus to evaluate the hypothesis that pattern separation, defined as the ability to transform a set of similar input patterns into a less‐similar set of output patterns, is dynamically regulated by hilar neurons. Prior models of the dentate gyrus have generally fallen into two categories: simplified models that have focused on a single granule cell layer and its ability to perform pattern separation, and large‐scale and biophysically realistic models of dentate gyrus, which include hilar cells, but which have not specifically addressed pattern separation. The present model begins to bridge this gap. The model includes two of the major subtypes of hilar cells: excitatory hilar mossy cells and inhibitory hilar interneurons that receive input from and project to the perforant path terminal zone (HIPP cells). In the model, mossy cells and HIPP cells provide a mechanism for dynamic regulation of pattern separation, allowing the system to upregulate and downregulate pattern separation in response to environmental and task demands. Specifically, pattern separation in the model can be strongly decreased by decreasing mossy cell function and/or by increasing HIPP cell function; pattern separation can be increased by the opposite manipulations. We propose that hilar cells may similarly mediate dynamic regulation of pattern separation in the dentate gyrus in vivo, not only because of their connectivity within the dentate gyrus, but also because of their modulation by brainstem inputs and by the axons that “backproject” from area CA3 pyramidal cells. © 2008 Wiley‐Liss, Inc.
Rat hippocampal theta rhythm during sensory mismatch
Authors: Zou D. , Aitake M. , Hori E. , Umeno K. , Fukuda M. , Ono T. , Nishijo H.
Published: October 2008

It has been suggested that sensory mismatch induces motion sickness, but its neural mechanisms remain unclear. To investigate this issue, theta waves in the hippocampal formation (HF) were studied during sensory mismatch by backward translocation in awake rats. A monopolar electrode was implanted into the dentate gyrus in the HF, from which local field potentials were recorded. The rats were placed on a treadmill affixed to a motion stage translocated along a figure 8‐shaped track. The rats were trained to run forward on the treadmill at the same speed as that of forward translocation of the motion stage (a forward condition) before the experimental (recording) sessions. In the experimental sessions, the rats were initially tested in the forward condition, and then tested in a backward (mismatch) condition, in which the motion stage was turned around by 180° before translocation. That is, the rats were moved backward by translocation of the stage although the rats ran forward on the treadmill. The theta (6–9 Hz) power was significantly increased in the backward condition compared with the forward condition. However, the theta power gradually decreased by repeated testing in the backward condition. Furthermore, backward translocation of the stage without locomotion did not increase theta power. These results suggest that the HF might function as a comparator to detect sensory mismatch, and that alteration in HF theta activity might induce motion sickness. © 2008 Wiley‐Liss, Inc.
γ‐Tocopherol‐enriched mixed tocopherol diet inhibits prostate carcinogenesis in TRAMP mice
Authors: Barve Avantika , Khor Tin Oo , Nair Sujit , Reuhl Kenneth , Suh Nanjoo , Reddy Bandaru , Newmark Harold , Kong Ah‐Ng
Published: October 2008

γ‐tocopherol (γ‐T) alone or in combination with α‐tocopherol has been shown to suppress biomarkers of oxidative stress in asthamatics and human subjects with metabolic syndrome. Oxidative stress has been implicated as a key event in prostate carcinogenesis. Hence, the purpose of this study was to examine the effects of γ‐tocopherol‐enriched mixed tocopherol diet on prostate carcinogenesis in a murine prostate cancer model (TRAMP). 8 week old TRAMP males were fed 0.1% γ‐T‐enriched mixed tocopherol diet that contained 20‐fold higher levels of γ‐tocopherol, and roughly 3‐fold higher levels of α‐tocopherol. The effect of such diet on tumor and PIN development was observed. The expression of phase II detoxifying, antioxidant enzymes and Nrf2 mRNA and protein were determined by RT‐PCR, immunohistochemistry and western blotting techniques. Treatment with γ‐T‐enriched mixed tocopherols significantly suppressed the incidence of palpable tumor and Prostate Intraepithelial Neoplasia (PIN) development without affecting the expression of the transgene (SV‐40). Tumor progression occurred with a significant suppression of antioxidant enzymes such as catalase, superoxide dismutase, glutathione peroxidase, heme‐oxygenase‐1 and phase II detoxifying enzymes. Treatment with γ‐T‐enriched mixed tocopherol diet upregulated the expression of most detoxifying and antioxidant enzymes. Nrf2—a redox sensitive transcription factor known to mediate the expression of phase II detoxifying enzymes, was also significantly upregulated following treatment with γ‐T‐enriched mixed tocopherol diet. γ‐T‐enriched mixed tocopherols significantly up‐regulated the expression of Nrf2 and its related detoxifying and antioxidant enzymes thereby suppressing PIN and tumor development. © 2008 Wiley‐Liss, Inc.
Tumor‐specific cytotoxic T cell generation and dendritic cell function are differentially regulated by interleukin 27 during development of anti‐tumor immunity
Authors: Shinozaki Yukari , Wang Sen , Miyazaki Yoshiyuki , Miyazaki Kohji , Yamada Hisakata , Yoshikai Yasunobu , Hara Hiromitsu , Yoshida Hiroki
Published: October 2008

Interleukin (IL‐) 27 is a member of IL‐12 cytokine family with Th1‐promoting and anti‐inflammatory effects. IL‐27 has been shown to facilitate tumor‐specific cytotoxic T lymphocyte (CTL) induction against various tumors. However, IL‐27 suppresses cytokine production of lymphocytes and antigen‐presenting function of dendritic cells (DCs). To examine the in vivo role of IL‐27 in generation of anti‐tumor immunity, we examined IL‐27‐mediated antitumor‐effects using WSX‐1 (IL‐27 receptor α chain)‐deficient (WSX‐1−/−) mice. In WSX‐1−/− mice inoculated with B16 melanoma cells, tumor growth was higher than in wild‐type (WT) mice. Accordingly, tumor‐specific CTL generation was lower in WSX‐1−/− mice than in WT mice. CTL induction in WSX‐1−/− mice was not restored by transfer of WT DCs pulsed with TRP2 peptide, indicating that IL‐27 is directly required for generation of tumor‐specific CTLs. However, when transferred into tumor‐bearing mice, WSX‐1−/− DCs pulsed with TRP2 peptide was more potent than WT DCs in tumor growth inhibition and generation of CTLs, indicating suppressive effects of IL‐27 on DC function. Finally, the combination of WT CD8+ T cells and KO DCs is more potent in generation of antigen‐specific CTLs than any other combinations. Expression of perforin gene and percentages of tumor‐specific CD8+ T cells were also the highest in the combination of WT CD8+ T cells and WSX‐1−/− DCs. It was thus revealed that IL‐27 promotes CTL generation while suppressing DC function during generation of tumor immunity. The combination of WT T cells and IL‐27 signal‐defective DCs may have therapeutic potential against tumors. © 2008 Wiley‐Liss, Inc.
Decreased expression of angiogenesis antagonist EFEMP1 in sporadic breast cancer is caused by aberrant promoter methylation and points to an impact of EFEMP1 as molecular biomarker
Authors: Sadr‐Nabavi Ariane , Ramser Juliane , Volkmann Juliane , Naehrig Joerg , Wiesmann Frank , Betz Beate , Hellebrand Heide , Engert Stefanie , Seitz Susanne , Kreutzfeld Rene , Sasaki Takako , Arnold Norbert , Schmutzler Rita , Kiechle Marion , Niederacher Dieter , Harbeck Nadia , Dahl Edgar , Meindl Alfons
Published: October 2008

EGF‐containing fibulin‐like extracellular matrix protein 1 (EFEMP1) was recently described as an antagonist of angiogenesis. Motivated by a strong dependence of tumor growth and metastasis on angiogenesis, we investigated the role of EFEMP1 in human breast cancer. We applied RNA microarray expression analysis and quantitative real‐time PCR (QRT) in a total of 45 sporadic breast cancer tissues and found EFEMP1 down‐regulation in 59% and 61% of the analyzed tissues, respectively. This down‐regulation was confirmed on protein level. Immunohistochemistry in 211 breast cancer tissues resulted in reduced or even abolished EFEMP1 expression in 57–62.5% of the tumors. Bisulphite genomic sequencing in breast cancer cell lines and primary breast cancer tissues revealed promoter methylation as the major cause of this down‐regulation. Furthermore, analysis of 203 clinically well characterized primary breast cancers displayed a significant correlation of reduced EFEMP1 protein expression with poor disease‐free (p = 0.037) and overall survival (p = 0.032), particularly in those node‐positive patients who received adjuvant anthracycline‐based chemotherapy, but not in those treated by either cyclophosphamide‐methotrexate‐5‐fluorouracil (CMF) or Tamoxifen. In summary, the presented data demonstrate for the first time the reduced EFEMP1 expression on RNA and protein level in a substantial number of sporadic breast carcinomas and its correlation with epigenetic alterations. Furthermore, these data point towards a possible predictive impact of EFEMP1 expression in primary breast cancer. © 2008 Wiley‐Liss, Inc.
Combined anti‐PDGFRα and PDGFRβ targeting in non‐small cell lung cancer
Authors: Reinmuth Niels , Liersch Ruediger , Raedel Miriam , Fehrmann Frauke , Fehrmann Nicole , Bayer Michael , Schwoeppe Christian , Kessler Torsten , Berdel Wolfgang , Thomas Michael , Mesters Rolf M.
Published: October 2008

Activation of the platelet‐derived growth factor (PDGF)‐receptors is critically involved into various stromal cell functions including recruitment of stromal cells and vascular endothelial growth factor (VEGF) induction in tumor and perivascular cells. To evaluate the effects of combined PDGFRα and ‐β inhibition in a non‐small cell lung cancer model, we stably transfected A549 lung cancer cells with the PDGF‐A mutant PDGF‐0. PDGF‐0 has been generated by substituting amino acids in the binding region of PDGF‐A with the corresponding VEGF‐A region, leading to a decreased receptor‐binding affinity and activation. Compared with control vector transfected cells, transfection with PDGF‐0 had no impact on monolayer growth and apoptosis in vitro, but significantly impaired the number of colony formation in soft agar. After subcutaneous injections, all mice developed tumors within 5 days. While control vector transfected A549 cells were characterized by constant tumor growth, PDGF‐0 transfected A549 revealed a reduced tumor mass (p < 0.001) with no further growth beyond 14 days (2 months observation time) and complete regressions in 7 of 13 cases. Immunohistochemical analyses revealed that PDGF‐0 transfected tumors demonstrated decreased recruitment of periendothelial cells, while the tumor invasion zone was similar to control vector transfectants. Similarly, conditioned medium from PDGF‐0 transfected cells induced significantly less migration of smooth muscle cells and fibroblasts in vitro. Interestingly, in PDGF‐0 transfectants, neither total vessel count nor VEGF expression were significantly altered. These studies demonstrate that combined inhibition of PDGFRα and ‐β results in markedly decreased tumor growth in vivo because of impaired recruitment of periendothelial cells. © 2008 Wiley‐Liss, Inc.
Down‐modulation of keratin 8 phosphorylation levels by PRL‐3 contributes to colorectal carcinoma progression
Authors: Mizuuchi Eri , Semba Shuho , Kodama Yoshinori , Yokozaki Hiroshi
Published: October 2008

Phosphatase of regenerating liver‐3 (PRL‐3) is a member of the PRL protein tyrosine phosphatase family and has been proposed to promote the invasiveness and metastastic capability of colorectal cancers (CRCs); however, the underlying mechanisms and target molecules of PRL‐3 protein remain unknown. On the basis of the biological significance of PRL‐3 phosphatase activity confirmed by the catalytically inactive PRL‐3 mutant (C104S) and a PRL‐3 inhibitor in CRC‐derived SW480 cells, we performed protein expression profiling to search for PRL‐3‐mediated effector proteins. By a comparative study of phosphorylated proteins that differentially expressed in wild type and C104S mutant PRL‐3‐transfected SW480 cells; the cytoskeletal intermediate filament keratin 8 (KRT8) was identified as a physiological PRL‐3‐interacting protein. Indeed, treatment with the PRL‐3 inhibitor effectively suppressed the phosphorylation of KRT8 at S73 and S431. Moreover, we detected the physiological interaction between PRL‐3 and KRT8 and their colocalization at cellular lamellipodias and ruffles in vivo. In CRC tissue samples, tumor cells with high PRL‐3 expression showed reduction or loss of phosphorylated KRT8 expression, particularly at the invasive front and in the liver metastases. In conclusion, our results indicate that PRL‐3 may play an important role for the promotion of CRC cell migration and metastatic potential through direct KRT8 dephosphorylation. © 2008 Wiley‐Liss, Inc.
A‐FABP, a candidate progression marker of human transitional cell carcinoma of the bladder, is differentially regulated by PPAR in urothelial cancer cells
Authors: Boiteux Guillaume , Lascombe Isabelle , Roche Emmanuelle , Plissonnier Marie‐Laure , Clairotte Anne , Bittard Hugues , Fauconnet Sylvie
Published: October 2008

Superficial pT1 bladder tumors are characterized by a high risk of recurrence and progression in grade and stage. Few studies provided evidence that loss of adipocyte‐fatty acid binding protein (A‐FABP) expression was associated with bladder cancer progression. A‐FABP is a lipid binding protein playing a role in intracellular lipid transport and metabolism, as well as in signal transduction. We reported from bladder tumors that decrease of A‐FABP transcript level significantly correlated to tumor stage and to histologic grade (p < 0.05). Namely, in poor prognosis high grade pT1 tumors there was a loss of A‐FABP expression compared to good prognosis tumors suggesting that re‐expression of A‐FABP could be a therapeutic approach in early stage bladder cancer to prevent disease progression. We demonstrated for the first time that this marker is upregulated by Peroxisome Proliferator‐Activated Receptor (PPAR) α, β and γ in T24 cells (derived from an undifferentiated grade III carcinoma) and only by PPARβ in RT4 cells (derived from a well differentiated grade I papillary tumor). This effect occurred through a PPAR‐dependent transcriptional mechanism without modifying mRNA stability and interestingly required de novo protein synthesis. Data as a whole suggest a prognostic significance of A‐FABP in bladder cancer outcome and the potential utility of overexpression of this protein by PPAR agonists open up new perspectives in the treatment of bladder cancer. © 2008 Wiley‐Liss, Inc.
Mechanism of metabolic activation and DNA adduct formation by the human carcinogen diethylstilbestrol: The defining link to natural estrogens
Authors: Saeed Muhammad , Rogan Eleanor , Cavalieri Ercole
Published: October 2008

Diethylstilbestrol (DES) is a human carcinogen, based on sufficient epidemiological evidence. DES is mainly metabolized to its catechol, 3′‐hydroxyDES (3′‐OH‐DES), which can further oxidize to DES‐3′,4′‐quinone (DES‐3′,4′‐Q). Similarly to estradiol‐3,4‐quinone, the reaction of DES‐3′,4′‐Q with DNA would form the depurinating 3′‐OH‐DES‐6′‐N3Ade and 3′‐OH‐DES‐6′‐N7Gua adducts. To prove this hypothesis, synthesis of DES‐3′,4′‐Q by oxidation of 3′‐OH‐DES with Ag2O was tried; this failed due to instantaneous formation of a spiro‐quinone. Oxidation of 3′‐OH‐DES by lactoperoxidase or tyrosinase in the presence of DNA led to the formation of 3′‐OH‐DES‐6′‐N3Ade and 3′‐OH‐DES‐6′‐N7Gua adducts. These adducts were tentatively identified by LC‐MS/MS as 3′‐OH‐DES‐6′‐N3Ade, m/z = 418 [M+H]+, and 3′‐OH‐DES‐6′‐N7Gua, m/z = 434 [M+H]+. Demonstration of their structures derived from their oxidation by MnO2 to the DES quinone adducts and subsequent tautomerization to the dienestrol (DIES) catechol adducts, which are identical to the standard 3′‐OH‐DIES‐6′‐N3Ade, m/z = 416 [M+H]+, and 3′‐OH‐DIES‐6′‐N7Gua, m/z = 432 [M+H]+, adducts. The reaction of DIES‐3′,4′‐Q or lactoperoxidase‐activated 3′‐OH‐DIES with DNA did not produce any depurinating adducts, due to the dienic chain being perpendicular to the phenyl planes, which impedes the intercalation of DIES into the DNA. Enzymic oxidation of 3′‐OH‐DES suggests that the catechol of DES intercalates into DNA and is then oxidized to its quinone to yield N3Ade and N7Gua adducts. These results suggest that the common denominator of tumor initiation by the synthetic estrogen DES and the natural estrogen estradiol is formation of their catechol quinones, which react with DNA to afford the depurinating N3Ade and N7Gua adducts. © 2008 Wiley‐Liss, Inc.
An intron 4 VNTR polymorphism of the endothelial nitric oxide synthase gene is associated with early‐onset colorectal cancer
Authors: Yeh Chih‐Ching , Santella Regina M. , Hsieh Ling‐Ling , Sung Fung‐Chang , Tang Reiping
Published: October 2008

Endothelial‐derived nitric oxide, which is produced by endothelial nitric oxide synthase (eNOS), may play an important role in colorectal carcinogenesis. However, the putative contribution of commoneNOSgenetic polymorphisms to colorectal cancer risk remains unknown. We genotyped 3 polymorphisms ofeNOS(T‐786C, G894T, and intron4b/a) in 727 colorectal adenocarcinoma cases and 736 age‐ and sex‐matched healthy controls in Taiwan. Genotypes of the T‐786C and G894T polymorphisms were determined by fluorescence polarization assays and the 27‐bp variable number of tandem repeat (VNTR) polymorphism in intron 4 (intron4b/a) was analyzed by PCR. Logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs). Among younger participants (≤60 yrs), the intron4a variant genotype was associated with a significantly increased risk of colorectal cancer, compared with the intron4bb genotype (OR = 1.60, 95% CI = 1.04–2.46). In addition, those young individuals bearing a greater number of high‐risk genotypes (OR > 1,i.e., CT+TT for T‐786C, ba+aa for intron4b/a, and GG for G894T) ofeNOShad a higher colorectal cancer risk (ptrend= 0.039). Compared with younger individuals without any putative high‐risk genotypes, those with 3 high‐risk genotypes had a significantly greater cancer risk (OR = 1.89, 95% CI = 1.04–3.43). Our results suggest that theeNOSintron4b/a polymorphism may contribute to early‐onset colorectal cancer risk in the Taiwanese population. © 2008 Wiley‐Liss, Inc.
HAI‐2 is epigenetically downregulated in human hepatocellular carcinoma, and its Kunitz domain type 1 is critical for anti‐invasive functions
Authors: Tung Edmund Kwok‐Kwan , Wong Chun‐Ming , Yau Tai‐On , Lee Joyce Man‐Fong , Ching Yick‐Pang , Ng Irene Oi‐Lin
Published: October 2008

Pharmacological demethylation‐based gene expression profile analysis is a useful tool to identify epigenetically silenced tumour suppressor genes. HGF activator inhibitor 2 (HAI‐2), a serine protease inhibitor, has been identified as one of the candidate tumour suppressor genes in human hepatocellular carcinoma (HCC) with this technique. In this study, we aimed to characterise the epigenetic status and tumour suppressive function of HAI‐2 in HCC. We validated that HAI‐2 expression was either absent or low in most of the HCC cell lines tested, and 5‐Aza‐2′‐deoxycytidine treatment significantly restored its expression in 9 (75%) of these 12 cell lines. HAI‐2 was found to be frequently underexpressed in human HCCs (p < 0.001). With bisulphite DNA sequencing and methylation‐specific PCR, we found that the promoter of the HAI‐2 gene was frequently hypermethylated in both HCC cell lines and human HCCs. Ectopic expression of HAI‐2 significantly inhibited cell migration and invasiveness of HCC cells in vitro and suppressed tumourigenicity in vivo. In addition, we also provided the first evidence that HAI‐2 mediated its tumour suppressor function via the Kunitz domain 1 (KD‐1), as KD‐1 but not KD‐2 inactivating mutant abolished its anti‐tumour invasiveness in vitro. Our findings suggest that HAI‐2 is a candidate tumour suppressor gene that is frequently hypermethylated and underexpressed in human HCCs, and the KD‐1 domain of HAI‐2 is the key region responsible for its anti‐invasive function. © 2008 Wiley‐Liss, Inc.
Prevalence and type distribution of human papillomavirus in carcinoma and intraepithelial neoplasia of the vulva, vagina and anus: A meta‐analysis
Authors: De Vuyst Hugo , Clifford Gary M. , Nascimento Maria Claudia , Madeleine Margaret M. , Franceschi Silvia
Published: October 2008

This meta‐analysis investigated human papillomavirus (HPV) prevalence in vulvar, vaginal and anal intraepithelial neoplasia (VIN, VAIN, AIN) grades 1–3 and carcinoma from 93 studies conducted in 4 continents and using PCR assays. Overall HPV prevalence was 67.8%, 85.3% and 40.4% among 90 VIN1, 1,061 VIN2/3 and 1,873 vulvar carcinomas; 100%, 90.1% and 69.9% among 107 VAIN1, 191 VAIN2/3 and 136 vaginal carcinomas; and 91.5%, 93.9% and 84.3% among 671 AIN1, 609 AIN2/3 and 955 anal carcinomas, respectively. HPV16 was found more frequently (>75%) and HPV18 less frequently (<10%) in HPV‐positive vulvar, vaginal and anal carcinomas than in cervical carcinoma. HPV6 and 11 were common in VIN1 and AIN1, but not in VAIN1. HPV prevalence in vulvar carcinoma varied most by histological type (69.4% in warty‐basaloid and 13.2% in keratinized type) and was also higher in women 60 years or younger and in studies carried out in North America. HPV prevalence in anal carcinoma was higher among women (90.8%) than men (74.9%), but no difference by gender emerged in North America. The majority of AIN2/3 derived from studies of HIV‐positive individuals and/or men who have sex with men. Among AIN2/3, HIV infection was associated with higher HPV prevalence, more multiple‐type infections and a relative under‐representation of HPV16. In conclusion, ∼40% of vulvar, 60% of vaginal and 80% of anal carcinoma may be avoided by prophylactic vaccines against HPV16/18. This proportion would be similar for the corresponding high‐grade lesions of the vagina and anus, but higher for VIN2/3 (75%) than for vulvar carcinoma. © 2008 Wiley‐Liss, Inc.
Diminished IFN‐γ and IL‐10 and elevated Foxp3 mRNA expression in the cervix are associated with CIN 2 or 3
Authors: Scott Mark E. , Ma Yifei , Kuzmich Lili , Moscicki Anna‐Barbara
Published: October 2008

Cervical mucosal expression of cytokines involved in mediating cellular immunity is believed to influence the persistence of human papillomavirus (HPV) infection, a necessary prerequisite for the development of cervical intraepithelial neoplasia (CIN). Additionally, regulatory T (Treg) cells are increasingly understood to be important modulators of cellular immunity. Using quantitative RT‐PCR, we measured, in cross‐sectional design, the cervical mRNA expression of IFN‐γ, IL‐10, and IL‐12, as well as the Treg transcription factor Forkhead box P3 (Foxp3), in a cohort of young women representing CIN 1, 2, and 3, as well as benign histology. Higher levels of IFN‐γ and IL‐10 were significantly (p ≤ 0.05) associated with decreased odds of having high‐grade cervical disease (CIN 2 or 3) in multivariate logistic regression models. In contrast, higher levels of mucosal Foxp3 expression were associated with increased odds of having CIN 2 or 3 (p = 0.004). In a multivariate model including cervical infection with HPV16 and/or another high‐risk HPV type, Foxp3 remained higher in the CIN 2/3 group, but the difference was notably less significant (p = 0.05). These findings support a model in which diminished cellular immunity in the cervical mucosa and mucosal enrichment of Treg cells both contribute to the development of high‐grade lesions. © 2008 Wiley‐Liss, Inc.
Downregulation of vascular endothelial growth factor and induction of tumor dormancy by 15‐lipoxygenase‐2 in prostate cancer
Authors: Tang Yong , Wang Man‐Tzu , Chen Yakun , Yang Dianer , Che Mingxin , Honn Kenneth V. , Akers Gregory D. , Johnson Stephen R. , Nie Daotai
Published: October 2008

The enzyme 15‐lipoxygenase‐2 (15‐LOX‐2) utilizes arachidonic acid, a polyunsaturated fatty acid, to synthesize 15(S)‐hydroxyeicosatetraenoic acid. Abundantly expressed in normal prostate epithelium but frequently suppressed in the cancerous tissues, 15‐LOX‐2 has been suggested as a functional suppressor of prostate cancer, but the mechanism(s) involved remains unknown. To study the functional role of 15‐LOX‐2 in prostate cancer, we expressed 15‐LOX‐2 as a fusion protein with GFP in DU145 and PC‐3 cells and found that 15‐LOX‐2 increased cell cycle arrest at G0/G1 phase. When injected into athymic nu/nu mice, prostate cancer cells with 15‐LOX‐2 expression could still form palpable tumors without significant changes in tumorigenicity. But, the tumors with 15‐LOX‐2 expression grew significantly slower than those derived from vector controls and were kept dormant for a long period of time. Histological evaluation revealed an increase in cell death in tumors derived from prostate cancer cells with 15‐LOX‐2 expression, while in vitro cell culture conditions, no such increase in apoptosis was observed. Further studies found that the expression of vascular endothelial growth factor A (VEGF‐A) was significantly reduced in prostate cancer cells with 15‐LOX‐2 expression restored. Our studies suggest that 15‐LOX‐2 suppresses VEGF gene expression and sustains tumor dormancy in prostate cancer. Loss of 15‐LOX‐2 functionalities, therefore, represents a key step for prostate cancer cells to exit from dormancy and embark on malignant progression in vivo. © 2008 Wiley‐Liss, Inc.
Optical knock out of stem cells with extremely ultrashort femtosecond laser pulses
Authors: Uchugonova Aisada , Isemann Andreas , Gorjup Erwin , Tempea Gabriel , Bückle Rainer , Watanabe Wataru , König Karsten
Published: October 2008

Novel ultracompact multiphoton sub‐20 femtosecond near infrared 85 MHz laser scanning microscopes and conventional 250 fs laser microscopes have been used to perform high spatial resolution two‐photon imaging of stem cell clusters as well as selective intracellular nanoprocessing and knock out of living single stem cells within an 3D microenvironment without any collateral damage. Also lethal cell exposure of large parts of cell clusters was successfully probed while maintaining single cells of interest alive. The mean power could be kept in the milliwatt range for 3D nanoprocessing and even in the microwatt range for two‐photon imaging. Ultracompact low power sub‐20 fs laser systems may become interesting tools for optical nanobiotechnology such as optical cleaning of stem cell clusters as well as optical transfection. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
The titanium binding protein of Rhodococcus ruber GIN1 (NCIMB 40340) is a cell‐surface homolog of the cytosolic enzyme dihydrolipoamide dehydrogenase
Authors: Siegmann Ari , Komarska Avital , Betzalel Yifaat , Brudo Irene , Jindou Sadanari , Mor Gil , Fleminger Gideon
Published: October 2008

Rhodococcus ruber GIN1 (formally Rh. strain GIN1) was previously isolated on the basis of its strong adherence to coal fly ash (CFA) and titanium dioxide particles from CFA sedimentation ponds of an electrical power plant in Israel. The interaction of the bacterium with oxides has been shown to be mediated by a cell surface protein designated TiBP (titanium binding protein) involving primarily strong, non‐electrostatic forces. In this work, we set forward to identify this unique exocellular protein. Sequence analysis of the purified protein by mass spectrometry (LC/MS/MS) following trypsinization revealed 11 peptides. All of them showed >90% amino acid residues identity with sequences of one of the orthologs (dldh1) of the cytosolic enzyme dihydrolipoamide dehydrogenase (DLDH), based on the genome sequence of Rhodococcus strain RHA1. This genome was selected as a reference since currently it is the only sequenced Rhodococcal genome. Altogether, these peptides covered over 25% of the 52 kDa protein molecule. N‐ and C‐termini primers were prepared and used to sequence the paralog gene from Rh. ruber GIN1 after polymerase chain reaction (PCR) amplification. All 11 peptides showed 100% identity with the sequence of this gene. The homology of TiBP with the supposedly cytosolic DLDH raised the question of whether the exocellular TiBP possesses DLDH activity. Indeed, intact late logarithmic phase Rh. ruber GIN1 cells, previously shown to express TiBP, were found to possess such activity, while very low activity was associated with stationary phase cells which possess diminished TiBP expression on their surface. Further evidence for the exocellular location of TiBP/DLDH was achieved using specific anti‐TiBP polyclonal antibodies by whole cell and protein enzyme‐linked immunosorbent assay (ELISA), showing high reactivity of the logarithmic phase cell surface and substantially lower reactivity with the stationary phase cells. As expected, logarithmic phase spheroplasts were not recognized by these antibodies. Similar results were obtained by fluorescence and scanning electron microscopy. Our postulation that DLDH is located on the surface of Rh. ruber GIN1, serving as a TiO2 binding protein, is in accordance with literary evidence on DLDH in other organisms, Bacteria, Archea, and Eukaryots that suggests it is associated with the outer membranes or cell surfaces. As an exocellular protein DLDH assumes various tasks which are not related to its classical role as a 2‐oxoacid dehydrogenase, including serving as an adhesion/binding protein in certain bacteria. Copyright © 2008 John Wiley & Sons, Ltd.
Understanding SERS of bacteria
Authors: Efrima S. , Zeiri L.
Published: October 2008

Surface‐enhanced Raman spectroscopy (SERS) has been suggested as a powerful tool to identify bacteria, drawing from its high fingerprint (vibrational) information content, its extreme sensitivity (down to the single molecule level) and its obliviousness to the aqueous environment intrinsic to biological systems. We review here in a comparative manner the various studies that attempted to utilize SERS for this important goal in light of the work carried out by our own group over the past 10 years or so. We show that SERS has an additional major advantage, namely, it introduces a new dimension of selectivity, which, on the one hand, makes it even more suitable as an analytical tool, but on the other hand, it requires gaining control of the precise manner in which the SERS‐active metal centers are produced and brought into contact with the micro‐organism. Our emphasis in this review is on understanding the spectra in terms of the nature of the SERS‐active centers and their placement within the bacterium. On the interpretation and assignment of the spectra, we constantly keep in mind the final goal of bacteria identification. Copyright © 2008 John Wiley & Sons, Ltd.
Simultaneous enantioseparation of four β2‐agonists by capillary electrophoresis with cyclodextrin additives. Study of the enantioselective mechanism
Authors: Yang Jing , Lu Xiumei , Pan Li , Jiang Kun , Cheng Maosheng , Li Famei
Published: October 2008

The simultaneous capillary electrophoretic enantioseparation of adrenergic β2‐agonists enantiomers (trantinterol, mabuterol, clenbuterol, bambuterol) was studied with β‐cyclodextrin, ethyl‐β‐CD, methyl‐β‐CD, hydroxypropyl‐β‐CD, and hydroxyethyl‐β‐CD as chiral selector. The type and concentration of the chiral selector and buffer pH played a very important role in the enantioseparation of the analyzed compounds. Hydroxypropyl‐β‐CD was found to be the most effective complexing agent and allowed excellent chiral/achiral resolutions compared to the other CDs. The simultaneous enantioseparation of four β2‐agonists was achieved using 100 mM citric acid–10 mM Na2HPO4 buffer at pH 2.5 containing 120 mM hydroxypropyl‐β‐CD with an applied voltage of 20 kV. Method validation in terms of repeatability, linearity, and limits of detection and quantification was performed. The effect of structural features of analytes on Rs and tm was studied. Complexation binding constants for the interactions between the four compounds and three different CDs were evaluated for elucidating the enantioseparation mechanism. It was found that very small differences in the chemical structure of the analytes resulted in significant changes in stereoselective recognition.