bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional domains of APOBEC3G required for antiviral activity
Authors: Li Jinliang , Potash Mary Jane , Volsky David J. 
Published: May 2004

AbstractThe viral protein, Vif, is essential for the production of infectious progeny virions in natural target cells of human immunodeficiency virus type 1 (HIV‐1). Several recent reports indicate that Vif acts by antagonizing the activity of an endogenous human antiviral protein, APOBEC3G. To investigate this route to restrict HIV‐1 infection, we employed mutagenesis to assess APOBEC3G function during HIV‐1 infection including interaction with Vif, localization, and activity in virions. We found that APOBEC3G binds Vif in infected cells and the C′‐terminal region is required for this interaction. APOBEC3G was only incorporated into virions in the absence of Vif and deletion of either the N′‐terminal or C′‐terminal regions of APOBEC3G abrogated virion localization. Assaying endogenous reverse transcription we found that APOBEC3G and its C′‐terminal deletion mutant inhibited full‐length cDNA synthesis, possibly through binding to viral RNA, a function revealed through gel‐shift assays. Taken together, our studies suggest that APOBEC3G inhibits HIV‐1 infection through interference with reverse transcription and that Vif counteracts APOBEC3G by impeding its entry into virions. © 2004 Wiley‐Liss, Inc.
N‐myristoyltransferase inhibitor protein is homologous to heat shock cognate protein 70
Authors: Selvakumar Ponniah , Lakshmikuttyamma Ashakumary , Pasha Mohammed Khysar , King Martin J. , Olson Douglas J.H. , Mori Sumiko , Ross Andrew R.S. , Hayashi Kiyoshi , Dimmock Jonathan R. , Sharma Rajendra K. 
Published: May 2004

AbstractMany of viral and eukaryotic proteins are required for signal transduction and regulatory functions which undergo a lipid modification by the enzyme N‐myristoyltransferase (NMT). In this study, we demonstrated that heat shock cognate protein 70 (HSC70) is homologous to NMT inhibitor protein (NIP71), which was discovered in our laboratory, based on MALDI‐TOF mass spectrometric analysis. The purified bovine cytosolic HSC70 and particulate NIP71 produced a dose‐dependent inhibition of human NMT having half maximal inhibitions of 235 and 230 nM, respectively. Further, Western blot analysis revealed that the purified particulate NIP71 and cytosolic HSC70 cross‐reacted with both anti‐NIP71 and anti‐HSC70 antibodies. The results we obtained imply that molecular chaperones could be involved in the regulation of NMT in normal and cancerous cells. Further studies directed to revealing the role of HSC70 in the regulation of NMT may lead to the development of gene based therapies of colon cancer. © 2004 Wiley‐Liss, Inc.
Platelet‐derived growth factor‐induced arachidonic acid release for enhancement of prostaglandin E2 synthesis in human gingival fibroblasts pretreated with interleukin‐1β
Authors: Nakao Sumi , Ogata Yorimasa , Yamamoto Yoshifumi , Furuyama Shunsuke , Sugiya Hiroshi 
Published: May 2004

AbstractPlatelet‐derived growth factor (PDGF) is a biological mediator for connective tissue cells and plays a critical role in a wide variety of physiological and pathological processes. We here investigated the effect of PDGF on arachidonic acid release and prostaglandin E2 (PGE2) synthesis in human gingival fibroblasts (HGF). PDGF induced arachidonic acid release in a time‐ and dose‐dependent manner, and simultaneously induced a transient increase in intracellular Ca2+ concentration ([Ca2+]i), but less provoked PGE2 release and cyclooxygenase‐2 (COX‐2) mRNA expression. When [Ca2+]i was increased by Ca2+‐mobilizaing reagents, arachidonic acid release was increased. The PDGF‐induced arachidonic acid release and increase in [Ca2+]i were prevented by a tyrosine kinase inhibitor. On the other hand, in the HGF pre‐stimulated with interleukin‐1β (IL‐1β), PDGF clearly increased PGE2 release. The PDGF‐induced PGE2 release was inhibited by a tyrosine kinase inhibitor. In the HGF pretreated with IL‐1β, arachidonic acid strongly enhanced PGE2 release and COX‐2 mRNA expression. These results suggest that PDGF stimulates arachidonic acid release by the increase in [Ca2+]i via tyrosine kinase activation, and which contributes to PGE2 production via COX‐2 expression in HGF primed with IL‐1β. © 2004 Wiley‐Liss, Inc.
Differential effects of PD98059 and U0126 on osteogenesis and adipogenesis
Authors: Dang Zhi‐Chao , Lowik Clemens W.G.M. 
Published: May 2004

AbstractPD98059 and U0126 are considered as specific inhibitors of the p42/44 mitogen‐activated protein kinases (MAPK) pathway, which affects osteogenesis and adipogenesis. Here, we show unexpected differential effects of PD98059 and U0126 on osteogenesis and adipogenesis as well as on estrogen (E2)‐induced actions in osteoprogenitor KS483 cells. PD98059 dose‐dependently inhibited osteogenesis indicated by cellular alkaline phosphatase (ALP) activity and nodule formation, but stimulated adipogenesis shown by the number of adipocytes. In contrast, U0126 slightly decreased osteogenesis but had no effects on adipogenesis, although it inhibited p42/44 MAPK more potently than PD98059. Furthermore, PD98059, but not U0126, counteracted E2‐induced osteogenesis and adipogenesis. Transfection experiments showed that PD98059, but not U0126, had estrogenic transcriptional activity. Interestingly, both PD98059 and U0126 potentiated E2‐induced estrogenic transcriptional activity in KS483 cells, which is opposite to the response in MCF7 breast cancer cells. Our data indicate that the cross‐talk between growth factors and estrogen receptor (ER)‐mediated pathways in KS483 cells is different from that in MCF7 cells. In summary, the differential effects of PD98059 and U0126 indicate their actions are not exclusively due to an inhibition of MAPK pathway. Caution should be taken in the interpretation of the results obtained using these inhibitors. © 2004 Wiley‐Liss, Inc.
Generation and metabolism of bioactive sphingosine‐1‐phosphate
Authors: Stunff Hervé Le , Milstien Sheldon , Spiegel Sarah 
Published: May 2004

AbstractSphingosine‐1‐phosphate (S1P) is a bioactive lysosphingophospholipid that has been implicated in the regulation of vital biological processes. Abundant evidence indicates that S1P acts as both an intracellular messenger and an extracellular ligand for a family of five specific G protein‐coupled S1P receptors (S1PRs). Cellular levels of S1P are tightly regulated in a spatio‐temporal manner through its synthesis catalyzed by sphingosine kinases (SphKs) and degradation by S1P lyase (SPL) and specific S1P phosphohydrolases. Over the past decade, the identification and cloning of genes encoding S1P metabolizing enzymes has increased rapidly. Overexpression and deletion of these enzymes has provided important insights into the intracellular and the “inside‐out” functions of S1P. The purpose of this review is to summarize the current knowledge of S1P metabolizing enzymes, their enzymatic properties, and their roles in the control of cellular functions by S1P. © 2004 Wiley‐Liss, Inc.
Regulation of biological activity of laminin‐5 by proteolytic processing of γ2 chain
Authors: Ogawa Takashi , Tsubota Yoshiaki , Maeda Masato , Kariya Yoshinobu , Miyazaki Kaoru 
Published: May 2004

AbstractLaminin‐5 (LN5), which regulates both cell adhesion and cell migration, undergoes specific extracellular proteolytic processing at an amino‐terminal region of the γ2 chain as well as at a carboxyl‐terminal region of the α3 chain. To clarify the biological effect of the γ2 chain processing, we prepared a human recombinant LN5 with the 150‐kDa, non‐processed γ2 chain (GAA‐LN5) and natural LN5 with the 105‐kDa, processed γ2 chain (Nat‐LN5). Comparison of their biological activities demonstrated that GAA‐LN5 had an about five‐times higher cell adhesion activity but an about two‐times lower cell migration activity than Nat‐LN5. This implies that the proteolytic processing of LN5 γ2 chain converts the LN5 from the cell adhesion type to the cell migration type. It was also found that human gastric carcinoma cells expressing the LN5 with the non‐processed γ2 chain is more adherent but less migratory than the carcinoma cells expressing a mixture of LN5 forms with the processed γ2 chain and with the unprocessed one. The functional change of LN5 by the proteolytic processing of the γ2 chain may contribute to elevated cell migration under some pathological conditions such as wound healing and tumor invasion. © 2004 Wiley‐Liss, Inc.
Enhancement of immunogenicity of HPV16 E7 oncogene by fusion with E. coli β‐glucuronidase
Authors: Šmahel Michal , Pokorná Dana , Macková Jana , Vlasák Josef 
Published: May 2004

AbstractBackgroundHuman papillomavirus type 16 (HPV16) E7 is an unstable oncoprotein with low immunogenicity. In previous work, we prepared the E7GGG gene containing point mutations resulting in substitution of three amino acids in the pRb‐binding site of the HPV16 E7 protein.Methods and resultsTo increase E7GGG immunogenicity we constructed fusion genes of E. coli β‐glucuronidase (GUS) with one or three copies of E7GGG. Furthermore, a similar construct was prepared with partial E7GGG (E7GGGp, 41 amino acids from the N‐terminus). The expression of the fusion genes was examined in human 293T cells. Quantification of GUS activity and the amount of E7 antigen showed substantially reduced GUS activity of fusion proteins with complete E7GGG that was mainly caused by decrease of their steady‐state level in comparison with GUS or E7GGGpGUS. Still, the steady‐state level of E7GGG.GUS was about 20‐fold higher than that of the E7GGG protein. The immunogenicity of the fusion genes with complete E7GGG was tested by DNA immunisation of C57BL/6 mice with a gene gun. TC‐1 cells and their clone TC‐1/A9 with down‐regulated MHC class I expression were subcutaneously (s.c.) inoculated to induce tumour formation. All mice were protected against challenge with TC‐1 cells and most animals remained tumour‐free in therapeutic‐immunisation experiments with these cells, in contrast to immunisation with unfused E7GGG and the fusion with the lysosome‐associated membrane protein 1 (Sig/E7GGG/LAMP‐1). Significant protection was also recorded against TC‐1/A9 cells. Both tetramer staining and ELISPOT assay showed substantially higher activation of E7‐specific CD8+ lymphocytes in comparison with E7GGG and Sig/E7GGG/LAMP‐1. Deletion of 231 bp in the GUS gene eliminated enzymatic activity, but did not influence the immunogenicity of the E7GGG.GUS gene.ConclusionsThe findings demonstrate the superior immunisation efficacy of the fusion genes of E7GGG with GUS when compared with E7GGG and Sig/E7GGG/LAMP‐1. The E7GGG.GUS‐based DNA vaccine might also be efficient against human tumour cells with reduced MHC class I expression. Copyright © 2004 John Wiley & Sons, Ltd.
Recombinant Sendai virus vectors deleted in both the matrix and the fusion genes: efficient gene transfer with preferable properties
Authors: Inoue Makoto , Tokusumi Yumiko , Ban Hiroshi , Shirakura Masayuki , Kanaya Takumi , Yoshizaki Mariko , Hironaka Takashi , Nagai Yoshiyuki , Iida Akihiro , Hasegawa Mamoru 
Published: May 2004

AbstractBackgroundSendai virus (SeV) is a new type of cytoplasmic RNA vector, which infects and replicates in most mammalian cells, directs high‐level expression of the genes on its genome and is free from genotoxicity. In order to improve this vector, both the matrix (M) and fusion (F) genes were deleted from its genome.MethodsFor the recovery of the M and F genes‐deleted SeV (SeV/ΔMΔF), the packaging cell line was established by using a Cre/loxP induction system. SeV/ΔMΔF was characterized and compared with wild‐type and F or M gene‐deleted SeV vectors in terms of transduction ability, particle formation, transmissible property and cytotoxicity.ResultsSeV/ΔMΔF was propagated in high titers from the packaging cell line. When this vector was administered into the lateral ventricle and the respiratory tissue, many of the ependymal and epithelial cells were transduced, respectively, as in the case of wild‐type SeV. F gene‐deletion made the SeV vector non‐transmissible, and M gene‐deletion worked well to inhibit formation of the particles from infected cells. Simultaneous deletions of these two genes in the same genome resulted in combining both advantages. That is, both virus maturation into particles and transmissible property were almost completely abolished in cells infected with SeV/ΔMΔF. Further, the cytopathic effect of SeV/ΔMΔF was significantly attenuated rather than that of wild type in vitro and in vivo.ConclusionsSeV/ΔMΔF is an advanced type of cytoplasmic RNA vector, which retains efficient gene transfer, gains non‐transmissible properties and loses particle formation with less cytopathic effect. Copyright © 2004 John Wiley & Sons, Ltd.
Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer
Authors: Boeckle Sabine , von Gersdorff Katharina , van der Piepen Silke , Culmsee Carsten , Wagner Ernst , Ogris Manfred 
Published: May 2004

AbstractBackgroundNonviral vectors based on polyethylenimine (PEI) usually contain an excess of PEI that is not complexed to DNA. Since unbound PEI contributes to cellular and systemic toxicity, purification of polyplexes from unbound PEI is desirable.MethodsSize exclusion chromatography (SEC) was used to purify PEI polyplexes of free PEI. Transfection properties of purified polyplexes and the effect of free PEI on gene delivery were studied in vitro and in vivo after systemic application into mice.ResultsSEC did not change the size and zeta‐potential of polyplexes. Independent of the amount of PEI used for complex formation, purified PEI polyplexes had the same final PEI nitrogen/DNA phosphate ratio of 2.5. Notably, purified PEI polyplexes demonstrated low cellular and systemic toxicity. High transfection efficiency was achieved with purified polyplexes at high DNA concentrations (8–15 µg/ml). At low DNA concentrations (2–4 µg/ml) gene transfer with purified particles was less efficient than with polyplexes containing free PEI both in vitro and in vivo. Mechanistic studies showed that free PEI partly blocked cellular association of DNA complexes but was essential for the following intracellular gene delivery. Adding free PEI to cells treated with purified particles with a delay of up to 4 h resulted in significantly enhanced transfection efficiency compared with non‐purified particles or purified particles without free PEI.ConclusionsThis study presents an efficient method to remove free PEI from PEI polyplexes by SEC. Our results from transfection experiments demonstrate that free PEI substantially contributes to efficient gene expression but also mediates toxic effects in a dose‐dependent manner. Purified polyplexes without free PEI have to be applied at increased concentrations to achieve high transfection levels, but exhibit a greatly improved toxicity profile. Copyright © 2004 John Wiley & Sons, Ltd.
Lentiviral vectors for treating and modeling human CNS disorders
Authors: Azzouz Mimoun , Kingsman Susan M. , Mazarakis Nicholas D. 
Published: May 2004

AbstractVectors based on lentiviruses efficiently deliver genes into many different types of primary neurons from a broad range of species including man and the resulting gene expression is long term. These vectors are opening up new approaches for the treatment of neurological diseases such as Parkinson's disease (PD), Huntington's disease (HD), and motor neuron diseases (MNDs). Numerous animal studies have now been undertaken with these vectors and correction of disease models has been obtained. Lentiviral vectors also provide a new strategy for in vivo modeling of human diseases; for example, the lentiviral‐mediated overexpression of mutated human α‐synuclein or huntingtin genes in basal ganglia induces neuronal pathology in animals resembling PD and HD in man. These vectors have been refined to a very high level and can be produced safely for the clinic. This review will describe the general features of lentiviral vectors with particular emphasis on vectors derived from the non‐primate lentivirus, equine infectious anemia virus (EIAV). It will then describe some key examples of genetic correction and generation of genetic animal models of neurological diseases. The prospects for clinical application of lentiviral vectors for the treatment of PD and MNDs will also be outlined. Copyright © 2004 John Wiley & Sons, Ltd.
One‐pot synthesis of [14C]arsenobetaine bromide
Authors: Bernardo Martha , Francesconi Kevin , Kollenz Gert 
Published: May 2004

AbstractA one‐pot synthesis of [14C]arsenobetaine from 2‐dimethylarsinoylacetic acid is described. Cation‐ and anion‐exchange columns are used in the purification of the compound and its conversion to the bromide salt. Copyright © 2004 John Wiley & Sons, Ltd.
Ultrasonic particle size reduction of radiolabeled pharmaceuticals
Authors: Schenk David J. , Jones Allen N. , Braun Matthew P. , Yao Dan , Wallace Michael A. , Marques Rosemary , Jenkins Herbert J. , Chang Anson , Jia Xiujuan , Crocker Louis S. , Dean Dennis C. , Melillo David G. 
Published: May 2004

AbstractHigh‐intensity ultrasonic treatment of MeOH/H2O, IPA/H2O, EtOH or H2O suspensions of C‐14 labeled and unlabeled pharmaceuticals successfully reduced the average particle diameter from a range of 8–260 to 4–15 µm as determined by optical microscopy. Copyright © 2004 John Wiley & Sons, Ltd.
Synthesis of tritiated Cyclosporin A and FK‐506 by metal‐catalyzed hydrogen isotope exchange
Authors: Shevchenko V. P. , Nagaev I. Yu. , Myasoedov N. F. , Andres H. , Moenius T. , Susan A. 
Published: May 2004

AbstractTritium‐labeled Cyclosporin A (I) and FK‐506 (II) have been prepared using a metal‐catalysed hydrogen isotope exchange procedure and high specific activity tritiated water. Specific activities of the labeled compounds were 0.15 and 0.59 TBq/mmol, respectively. Copyright © 2004 John Wiley & Sons, Ltd.
First no‐carrier‐added radioselenation of an adenosine‐A1 receptor ligand
Authors: Blum Till , Ermert Johannes , Wutz Walter , Bier Dirk , Coenen Heinz H. 
Published: May 2004

AbstractThe precursor synthesis and the no‐carrier‐added (n.c.a.) radiosynthesis of the adenosine‐A1 receptor ligand 5′‐(methyl[75Se]seleno)‐N6‐cyclopentyladenosine ([75Se]1) are described in this report. A method was developed starting from elemental n.c.a. selenium‐75, followed by a three‐step polymer‐supported radioselenation and deprotection which gave the radioligand with a radiochemical yield of 30%, a radiochemical purity of > 99% and a specific radioactivity of > 300 GBq/mmol (8 Ci/mmol). Preparation time was 40 min. The nonradioactive compound 5′‐(methylseleno)‐N6‐cyclopentyladenosine (1) was pharmacologically evaluated in vitro and showed high affinity and selectivity for the adenosine‐A1 receptor. These preliminary results suggest that this compound could be a useful radioligand for the noninvasive imaging of the brain adenosine‐A1 receptors using positron emission tomography (PET) when labelled with the positron emitter selenium‐73 (half‐life: 7.1 h). Copyright © 2004 John Wiley & Sons, Ltd.
Comparison of pathways to the versatile synthon of no‐carrier‐added 1‐bromo‐4‐[18F]fluorobenzene
Authors: Ermert Johannes , Hocke Carsten , Ludwig Thomas , Gail René , Coenen Heinz H. 
Published: May 2004

AbstractThe availability of no‐carrier‐added (n.c.a.) 1‐bromo‐4‐[18F]fluorobenzene with high radiochemical yields is important for 18F‐arylation reactions using metallo‐organic 4‐[18F]fluorophenyl compounds (e.g. of lithium or magnesium) or Pd‐catalyzed coupling. In this study, different methods for the preparation of 1‐bromo‐4‐[18F]fluorobenzene by nucleophilic aromatic substitution reactions using n.c.a. [18F]fluoride were examined. Of six pathways compared, symmetrical bis‐(4‐bromphenyl)iodonium bromide proved most useful to achieve the title compound in a direct, one‐step nucleophilic substitution with a radiochemical yield (RCY) of 65% within 10 min. Copyright © 2004 John Wiley & Sons, Ltd.
Simultaneous determination of mono‐, di‐ and tributyltin in environmental samples using isotope dilution gas chromatography mass spectrometry
Authors: Centineo Giuseppe , Rodríguez‐González Pablo , González Elisa Blanco , García Alonso J. Ignacio , Sanz‐Medel Alfredo 
Published: May 2004

AbstractThe development of a rapid, precise and accurate speciation method for the simultaneous determination of mono‐, di‐ and tributyltin in environmental samples is described. The method is based on using isotope dilution gas chromatography/mass spectrometry (GC/MS) with electron ionization, a widely used technique in routine testing laboratories. A mixed spike containing 119Sn‐enriched monobutyltin (MBT), dibutyltin (DBT) and tributyltin (TBT) was used for the isotope dilution of the samples. Five molecular ions were monitored for each analyte, corresponding to the 116Sn, 117Sn, 118Sn, 119Sn and 120Sn isotopes. The detection at masses corresponding to 116Sn and 117Sn were used to correct for m + 1 and m + 2 contributions of 13C from the organic groups attached to the tin atom on the 118Sn, 119Sn and 120Sn masses with simple mathematical equations and the concentrations of the butyltin compounds were calculated based on the corrected 118Sn/119Sn and 120Sn/119Sn isotope ratios. The 119Sn‐enriched multispecies spike was applied with satisfactory results to the simultaneous determination of MBT, DBT and TBT in three certified reference materials: two sediments, PACS‐2 and BCR 646, and the mussel tissue CRM 477. The method was compared with a previously published GC/inductively coupled plasma MS isotope dilution procedure, developed in our laboratory, by injecting the same samples into both instruments. Comparable analytical results in terms of precision and accuracy are demonstrated for both atomic and molecular mass spectrometric detectors. Thus, reliable quantitative organotin speciation analysis can be achieved using the more widespread and inexpensive GC/MS instrument. Copyright © 2004 John Wiley & Sons, Ltd.
Simultaneous quantitative analysis of isobars by tandem mass spectrometry from unresolved chromatographic peaks
Authors: Kushnir Mark M. , Rockwood Alan L. , Nelson Gordon J. 
Published: May 2004

AbstractA method was developed for the simultaneous quantitation of isobars from unresolved chromatographic peaks. The method is based on differences in branching ratios of ion abundances in their tandem mass spectra and an assumption that the product ion mass spectra of a mixture can be considered as a linear combination of the spectra of individual constituents. We present analytical equations and a matrix‐based approach for deconvoluting the concentration of individual components from the total peak intensity for two and three isobars and also a matrix‐based generalization to any number of compounds. The feasibility of the simultaneous analysis of mixtures containing two compounds was assessed. The approach was evaluated for the analysis of structural isomers of methylmalonic and succinic acids in human plasma and urine samples for a group of 270 samples. The linear regression equation, standard error and correlation coefficient for the agreement with a traditional method utilizing chromatographic separation of the isomers were y = 0.999x − 0.005, 0.024 µmol l−1, and 0.985, respectively. The utility of a spectral contrast angle as a predictor of analysis feasibility was evaluated. Copyright © 2004 John Wiley & Sons, Ltd.
Mass spectrometry of bisquinolizidine alkaloids: 2‐ and 17‐cyano‐substituted derivatives of sparteine and lupanine
Authors: Jasiewicz Beata , Boczoń Władysław , Wyrzykiewicz Elżbieta 
Published: May 2004

AbstractThe mass spectral fragmentations of 2‐cyano‐2‐methylsparteine (1), 2‐cyano‐2‐phenylsparteine (2), 2‐cyano‐2‐(p‐tolyl)sparteine (3), 17‐cyanosparteine (4) and 17‐cyanolupanine (5) were investigated. Fragmentation pathways, whose identification was assisted by accurate mass measurements and a correlation between the abundances of the M+• and the selected fragment ions of the investigated compounds, are discussed. The data obtained create the basis for distinguishing the structural metamers. Copyright © 2004 John Wiley & Sons, Ltd.