bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel Inhibitors of an Emerging Target in Mycobacterium tuberculosis; Substituted Thiazolidinones as Inhibitors of dTDP‐Rhamnose Synthesis.
Authors: Babaoglu Kerim , Page Mark A. , Jones Victoria C. , McNeil Michael R. , Dong Changjiang , Naismith James H. , Lee Richard E.
Published: June 2005

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Discovery and Evaluation of 3‐(5‐Thien‐3‐ylpyridin‐3‐yl)‐1H‐indoles as a Novel Class of KDR Kinase Inhibitors.
Authors: Fraley Mark E. , et al. et al.
Published: June 2005

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Synthesis and Evaluation of 4‐Anilino‐6,7‐dialkoxy‐3‐quinolinecarbonitriles as Inhibitors of Kinases of the Ras‐MAPK Signaling Cascade.
Authors: Berger Dan , et al. et al.
Published: June 2005

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Preparation of Substituted Quinolinyl and Isoquinolinyl Sulfonyl Chlorides for the Synthesis of Novel Sulfonamides.
Authors: Lai Justine Y. Q. , Ferguson Yvonne , Jones Mark
Published: June 2005

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Imidazo[1,2‐a]pyridines: A Potent and Selective Class of Cyclin‐Dependent Kinase Inhibitors Identified Through Structure‐Based Hybridisation.
Authors: Breault Gloria A. , Thomas Andrew P. , Jewsbury Philip J. , et al. et al.
Published: June 2005

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Structure‐Based Design of 2 ‐Arylamino‐4‐cyclohexylmethyl‐5‐nitroso‐6‐aminopyrimidine Inhibitors of Cyclin‐Dependent Kinases 1 and 2.
Authors: Griffin Roger J. , et al. et al.
Published: June 2005

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Inhibition of Src Kinase Activity by 4‐Anilino‐5,10‐dihydropyrimido[4,5‐b]quinolines.
Authors: Boschelli Diane H. , Powell Dennis , Golas Jennifer M. , Boschelli Frank
Published: June 2005

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Bone‐Targeted 2,6,9‐Trisubstituted Purines: Novel Inhibitors of Src Tyrosine Kinase for the Treatment of Bone Diseases.
Authors: Wang Yihan , et al. et al.
Published: June 2005

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Waste‐Free and Facile Solid‐State Protection of Diamines, Anthranilic Acid, Diols, and Polyols with Phenylboronic Acid.
Authors: Kaupp Gerd , Naimi‐Jamal M. Reza , Stepanenko Vladimir
Published: June 2005

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Cross‐Metathesis of Vinylsilanes Carrying Electron‐Withdrawing Substituents with Olefins in the Presence of the Second‐Generation Grubbs′ Catalyst.
Authors: Pietraszuk Cezary , Marciniec Bogdan , Fischer Helmut
Published: June 2005

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Pyrimidin‐/Thiazol‐/Thiazolin‐ylidenamidothiophosphoric Dichlorides.
Authors: Kabra Vijaya , Gupta Neelima , Jain Sama , Saxena Varsha
Published: June 2005

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Synthesis and Antimicrobial Activity of N‐Substituted N′‐[6‐Methyl‐2‐oxido‐1,3,2‐dioxaphosphinino(5,4‐b)pyridine‐2yl]ureas.
Authors: Reddy P. Vasu Govardhana , Reddy C. Suresh , Venugopal M.
Published: June 2005

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An Efficient Synthesis of 3,4‐Bis(tri‐n‐butylstannyl)furan.
Authors: Mee Simon P. H. , Lee Victor , Baldwin Jack E.
Published: June 2005

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A Xanthone‐Based Neutral Receptor for Zwitterionic Amino Acids.
Authors: Hernandez Jose V. , Muniz Francisco M. , Oliva Ana I. , Simon Luis , Perez Emilio , Moran Joaquin R.
Published: June 2005

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Total Synthesis of the Antipode of Alkaloid 205B.
Authors: Toyooka Naoki , Fukutome Ayako , Shinoda Hiroyuki , Nemoto Hideo
Published: June 2005

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Concise Total Synthesis of the Aporphine Alkaloid 7,7′‐Bisdehydro‐O‐methylisopiline by an InCl3 Mediated Cycloisomerization Reaction.
Authors: Fuerstner Alois , Mamane Victor
Published: June 2005

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Nucleic Acid Related Compounds. Part 120. Protection of the Amino Group of Adenosine and Guanosine Derivatives by Elaboration into a 2,5‐Dimethylpyrrole Moiety.
Authors: Nowak Ireneusz , Robins Morris J.
Published: June 2005

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Construction of Eight‐Membered Ether Rings by Olefin Geometry‐Dependent Internal Alkylation: First Asymmetric Total Syntheses of (+)‐3‐(E)‐ and (+)‐3‐(Z)‐Pinnatifidenyne.
Authors: Kim Hyoungsu , Choi Won Jun , Jung Jaeyoon , Kim Sanghee , Kim Deukjoon
Published: June 2005

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Design and Synthesis of Fluorinated RXR Modulators.
Authors: Gernert D. L. , et al. et al.
Published: June 2005

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Synthesis of (E,Z)‐5‐Bromo‐1,1‐dimethoxy‐5‐trimethylsilyl‐4‐pentene, an Upper Chain Allenic Prostaglandin Building Block.
Authors: Guzman‐Duran Antonio , Guzman Esther , Pannell Keith H. , Lloyd Winston D.
Published: June 2005

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Straightforward Preparation of (2E,4Z)‐2,4‐Heptadien‐1‐ol and (2E,4Z)‐2,4‐Heptadienal.
Authors: Petroski Richard J.
Published: June 2005

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Total Synthesis of seco‐Lateriflorone.
Authors: Tisdale Eric J. , Vong Binh G. , Li Hongmei , Kim Sun Hee , Chowdhury Chinmay , Theodorakis Emmanuel A.
Published: June 2005

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Enantioselective Total Syntheses of (+)‐Decursin and Related Natural Compounds Using Catalytic Asymmetric Epoxidation of an Enone.
Authors: Nemoto Tetsuhiro , Ohshima Takashi , Shibasaki Masakatsu
Published: June 2005

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Total Synthesis of the Ubiquitin‐Activating Enzyme Inhibitor (+)‐Panepophenanthrin.
Authors: Lei Xiaoguang , Johnson Richard P. , Porco John A. Jr.
Published: June 2005

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Solid State Dye‐Sensitized Solar Cells — An Alternative Route Towards Low‐Cost Photovoltaic Devices
Authors: Bach Udo
Published: June 2005

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Potassium Fluorotantalate in Solid, Dissolved and Molten Conditions
Authors: Agulyansky Anatoly
Published: June 2005

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Bulky Amido Ligands in Rare Earth Chemistry — Syntheses, Structures, and Catalysis
Authors: Roesky Peter W.
Published: June 2005

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Ligand Design in Coordination Chemistry: Approaches to New Catalysts, New Materials, and a More Sustainable Environment
Authors: Elsevier Cornelis J. , Reedijk Jan , Walton Paul H. , Ward Michael D.
Published: June 2005

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Complexity with Simplicity: A Steric Continuum of Chelating Diamines with Copper(I) and Dioxygen
Authors: Stack T. Daniel P.
Published: June 2005

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Recent Developments in the Catalytic Asymmetric Synthesis of α‐ and β‐Amino Acids
Authors: Ma Jun‐An
Published: June 2005

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Metal‐Assisted Cyclizations: Cascade and Domino Reactions
Authors: de Meijere Armin , Schelper Michael
Published: June 2005

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Recent Advances in Asymmetric Synthesis with Chiral Imide Auxiliaries
Authors: Evans David A. , Shaw Jared T.
Published: June 2005

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Dynamic Kinetic Resolution
Authors: Pellissier Helene
Published: June 2005

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Short Syntheses of Melatonin
Authors: Huegel Helmut M. , Nurlawis Faizul
Published: June 2005

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Allenylplumbanes
Authors: Eagle P. A. C.
Published: June 2005

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Alkynylplumbanes
Authors: Eagle P. A. C.
Published: June 2005

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Polyhydroxyalkanoate (PHA) Homeostasis: The Role of the PHA Synthase
Authors: Stubbe JoAnne , Tian Jiamin
Published: June 2005

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Arene Hydrogenation by Homogeneous Catalysts: Fact or Fiction?
Authors: Dyson Paul J.
Published: June 2005

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Oxidation Electronics: Bond—Band—Barrier Correlation and Its Applications
Authors: Sun Chang Q.
Published: June 2005

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Basic Study for Next‐Generation Gene Therapy Products
Authors: Mizuguchi Hiroyuki
Published: June 2005

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Benzonitriles: Survey of Their Importance and Scaling of Their Vibrational Frequencies
Authors: Palafox M. Alcolea , Rastogi V. K. , Mittal Lalit
Published: June 2005

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Valence Bond and Molecular Orbital Studies of the A‐F Bond Lengths in Some AFn Type Molecules and Their Fluorinated Cations
Authors: Harcourt Richard D. , Klapoetke Thomas M.
Published: June 2005

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Novel ABO3 Oxides Related to Perovskite and YAlO3 Structure Types in the La—B—V—O (B: Ni, Cu) Systems.
Authors: Goenen Z. Serpil , Mandal Tapas Kumar , Gopalakrishnan J. , Eichhorn Bryan W. , Greene Richard L.
Published: June 2005

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Luminescent Blinking from Silver Nanostructures.
Authors: Geddes Chris D. , Parfenov Alex , Gryczynski Ignacy , Lakowicz Joseph R.
Published: June 2005

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Synthesis and Characterization of Gadolinium Phosphate Neutron Absorber.
Authors: Lessing Paul A. , Erickson Arnold W.
Published: June 2005

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Synthesis and Structure of UO2I2 (OH2)2×4Et2O: First Structurally Characterized U(VI)—I Bond and Lightest Missing Member of the UO2X2 (X: Halide) Series.
Authors: Crawford Margaret‐Jane , Ellern Arkady , Noeth Heinrich , Suter Max
Published: June 2005

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Gold(I) and Mercury(II) Xenon Complexes.
Authors: Hwang In‐Chul , Seidel Stefan , Seppelt Konrad
Published: June 2005

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3D Porous and 3D Interpenetrating Triple Framework Structures Constructed by Aurophilicity—Coordination Interplay in {Mn[Au(CN)2]2(H2O)2}n and {KFe[Au(CN)2]3}n.
Authors: Dong Wen , Zhu Li‐Na , Sun Ya‐Qiu , Liang Mao , Liu Zhan‐Quan , Liao Dai‐Zheng , Jiang Zong‐Hui , Yan Shi‐Ping , Cheng Peng
Published: June 2005

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On the Stereochemistry of β‐Elimination of β‐Silyl Azides.
Authors: Chabaud Laurent , Landais Yannick
Published: June 2005

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An Easy Method for the Generation of Amides from Ketones by a Beckmann Type Rearrangement Mediated by Microwave.
Authors: Eshghi Hossein , Gordi Zinat
Published: June 2005

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Phase Transfer Catalyzed Asymmetric Alkylations of Imine Glycinamides.
Authors: Kumar Sanjeev , Ramachandran Uma
Published: June 2005

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Lanthanum Chloride Mediated‐Regeneration of Carbonyl Compounds from Oximes in Water.
Authors: Narsaiah A. Venkat , Nagaiah K.
Published: June 2005

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A New Reaction of Sulfur Ylides. Imination of Dimethylsulfonium Ketoylides with Tosyl Isocyanate.
Authors: Gololobov Yu. G. , Golding I. R. , Petrovskii P. V. , Klemenkova Z. S.
Published: June 2005

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A Facile Reduction of Nitroarenes to Anilines Using FeCl3×6H2O/Indium.
Authors: Yoo Byung Woo , Choi Jin Woo , Hwang Sun Kyun , Kim Dong Yoon , Baek Heung Soo , Choi Kyung Il , Kim Joong Hyup
Published: June 2005

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A Novel Reaction of Ketone Arylhydrazones with Nitric Oxide.
Authors: Yang Desuo , Lei Liandi , Liu Zhongquan , Wu Longmin
Published: June 2005

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Regioselective Reaction of Epoxides with Disulfides Using Zn/AlCl3 System: A Simple Synthesis of β‐Hydroxy Sulfides.
Authors: Movassagh B. , Sobhani S. , Kheirdoush F. , Fadaei Z.
Published: June 2005

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A New Chiral Diol Derived from Tetralone for the Complexation of Lewis Acids.
Authors: Coogan Michael P. , Haigh Robert , Hall Adrian , Harris Lisa D. , Hibbs David E. , Jenkins Robert L. , Jones Claire L. , Tomkinson Nicholas C. O.
Published: June 2005

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Reactions of Ethyl Methylsulfonylpyruvate and Its Sodium Salt with a Mixture of Aromatic Aldehyde and Arylamine.
Authors: Aliev Z. G. , Atovmyan L. O. , Gein V. L. , Gein L. F. , Kataeva A. V.
Published: June 2005

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Synthesis of 3‐Substituted Isoindolin‐1‐ones via a Palladium‐Catalyzed 3‐Component Carbonylation/Amination/Michael Addition Process.
Authors: Gai Xinjie , Grigg Ronald , Khamnaen Tossapol , Rajviroongit Shuleewan , Sridharan Visuvanathar , Zhang Lixin , Collard Simon , Keep Ann
Published: June 2005

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Sodium Carbonate as Carbon Dioxide Source for the Synthesis of Cyclic Carbonates Containing the Trifluoromethyl Group.
Authors: Jiang Zhong‐Xiang , Qing Feng‐Ling
Published: June 2005

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Design and Novel Synthesis of Aryl‐heteroaryl‐imidazole MAP Kinase Inhibitors.
Authors: Dobler Markus R.
Published: June 2005

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Synthesis and Evaluation of Antiinflammatory and Analgesic Activity of Some New 1,3‐Diphenyl‐2‐aryltetrahydroimidazoles.
Authors: Khan M. S. Y. , Gupta Monica
Published: June 2005

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Synthesis and Analgesic and Antiinflammatory Activity of Some New 6‐Acyl‐2‐benzoxazolinone and 6‐Acyl‐2‐benzothiazolinone Derivatives with Acetic Acid and Propanoic Acid Residues.
Authors: Uenlue Serdar , Oenkol Tijen , Duendar Yasemin , Oekcelik Berna , Kuepeli Esra , Yesilada Erdem , Noyanalpan Ningur , Sahin M. Fethi
Published: June 2005

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Synthesis and Antiinflammatory Activity of Alkyl/Arylidene‐2‐aminobenzothiazoles and 1‐Benzothiazol‐2‐yl‐3‐chloro‐4‐substituted‐azetidin‐2‐ones.
Authors: Khedekar Pramod B. , Bahekar Rajesh H. , Chopade Rajendra S. , Umathe Sudhir N. , Rao Akkinpalli Raghu Ram , Bhusari Kishore P.
Published: June 2005

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A Convenient Regiospecific Synthesis of New Conjugated Tetrazole Derivatives via the Reaction of Dienones with the Tetrachlorosilane‐Sodium Azide Reagent and Their NMR Structural Assignment.
Authors: Salama Tarek A. , El‐Ahl Abdel‐Aziz S. , Khalil Abdel‐Galil M. , Girges Margret M. , Lackner Bernd , Steindl Christian , Elmorsy Saad S.
Published: June 2005

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Synthesis of Coumarin by Yb(OTf)3 Catalyzed Pechmann Reaction under Solvent‐Free Conditions.
Authors: Wang Limin , Xia Jianjun , Tian He , Qian Changtao , Ma Yun
Published: June 2005

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Synthesis of Some 3‐Aryloxymethyl‐3,4‐dihydroisocoumarins
Authors: Brahmbhatt D. I. , Pandya Urvish R.
Published: June 2005

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Substituted Amides and Hydrazides of Dicarboxylic Acids. Part 18. Hypertensive Effect of 3(6)‐Methyl‐2‐pyridylamides of Several Dicarboxylic Acids.
Authors: Dolzhenko A. V. , Syropyatov B. Ya. , Koz'minykh V. O. , Kolotova N. V. , Zakhmatov A. V. , Borodin A. Yu.
Published: June 2005

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Syntheses of 1‐Alkyl‐2‐(substituted‐2‐pyridyl)benzimidazoles.
Authors: Dubey P. K. , Kumar C. Ravi , Reddy P. V. V. Prasada
Published: June 2005

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Vilsmeier—Haack Reaction on Quinaldines.
Authors: Kumar R. Nandha , Suresh T. , Mohan P. S.
Published: June 2005

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Synthesis of Carbo‐ and Heterocycles via a Palladium‐Catalyzed Allene Insertion—Nucleophile Incorporation—Michael Addition Cascade.
Authors: Gai Xinjie , Grigg Ronald , Koeppen Ines , Marchbank John , Sridharan Visuvanathar
Published: June 2005

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Synthesis of Substituted 2‐Amino‐3‐cyano‐7,9‐dimethyl‐4H‐pyrano[2′,3′:4,5]thieno [2,3‐b]pyridines.
Authors: Shestopalov A. M. , Naumov O. A.
Published: June 2005

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Cycloaddition Reactions of 2,4‐Diphenyl‐1,3‐diazabuta‐1,3‐dienes with Isocyanates and Isothiocyanates.
Authors: Abbiati Giorgio , de Carvalho Alessandro Cirrincione , Rossi Elisabetta
Published: June 2005

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Chemical Constituents from the Roots of Schnabelia tetradonta.
Authors: Dou Hui , Liao Xun , Peng Shu‐Lin , Pan Yuan‐Jiang , Ding Li‐Sheng
Published: June 2005

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Direct Peptide Coupling of Novel Amino Acid Derivatives Produced by Rearrangement of Catalytically Generated Ammonium Ylides.
Authors: Clark J. Stephen , Middleton Mark D.
Published: June 2005

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A New Co(0) Complex Mediated Synthesis of C‐Glycoside Analogues
Authors: Orsini Fulvia , Di Teodoro Emanuela
Published: June 2005

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A New Efficient Synthetic Process for the Construction of the Pentacyclic Core of Marine Alkaloid Ecteinascidins.
Authors: Tang Ye‐Feng , Liu Zhan‐Zhu , Chen Shi‐Zhi
Published: June 2005

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New Stereoselective Entry to Azaspirocyclic Nucleus of Halichlorine and Pinnaic Acids by Radical Translocation/Cyclization Reaction.
Authors: Takasu Kiyosei , Ohsato Hiroshi , Ihara Masataka
Published: June 2005

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Two New Insecticidal Amides and a New Alcoholic Amide from Piper nigrum Linn.
Authors: Siddiqui Bina S. , Gulzar Tahsin , Begum Sabira , Rasheed Munawwer , Sattar Fouzia A. , Afshan Farhana
Published: June 2005

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New Haliangicin Isomers, Potent Antifungal Metabolites Produced by a Marine Myxobacterium.
Authors: Kundim Bangi A. , Itou Yu , Sakagami Youji , Fudou Ryosuke , Iizuka Takashi , Yamanaka Shigeru , Ojika Makoto
Published: June 2005

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Chemical Constituents of the Flowers of Azadirachta indica.
Authors: Siddiqui Bina S. , Ali Syed Tariq , Rasheed Munawwer , Kardar Muhammad Nadeem
Published: June 2005

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Organometallics: Introduction
Authors: Imamoto T.
Published: June 2005

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Ferrocenes
Authors: Perseghini M. , Togni A.
Published: June 2005

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Antagonists and Agonists at the Glycine Site of the NMDA Receptor for Therapeutic Interventions
Authors: Jansen Michaela , Dannhardt Gerd
Published: June 2005

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Triorganolead Cyanides and Triorganolead Cyanates
Authors: Eagle P. A. C.
Published: June 2005

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Catalytic Enantioselective Allylic Oxidation of Olefins
Authors: Kamble Rajesh M. , Sekar G. , Chandrasekhar M. , Singh Vinod K.
Published: June 2005

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Effect of Molecular Chirality on the Morphology of Biomimetic Langmuir Monolayers
Authors: Nandi Nilashis , Vollhardt Dieter
Published: June 2005

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Natural Dyes as Neutralization Indicators — A Short Review
Authors: Indrayan A. K.
Published: June 2005

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Drug Binding Analysis of Human α1‐Acid Glycoprotein Using Capillary Electrophoresis
Authors: Kuroda Yukihiro , Shibukawa Akimasa , Nakagawa Terumichi
Published: June 2005

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Sensitive method for measuring apoptosis and cell surface phenotype in human thymocytes by flow cytometry
Authors: Schmid Ingrid , Uittenbogaart Christel H. , Giorgi Janis V.
Published: June 2005

A rapid, gentle, and sensitive method for quantification of cells undergoing apoptosis is presented. The method allows the simultaneous determination of dual‐color cell surface immunofluorescence. Cells are stained for 7 min with the vital dye Hoechst 33342 (HO342) for identification of live and apoptotic cells. 7‐amino‐actinomycin D (7‐AAD) is added to distinguish cells that have lost membrane integrity from apoptotic and live cells. Due to its spectral properties 7‐AAD can be utilized on cells that are dual‐surface labelled with fluoresceinisothiocyanate (FITC) and phycoerythrin (PE). The value of the method is demonstrated on human thymocytes, which constitutively undergo programmed cell death and which show an increase in the rate of apoptosis after exposure to the glucocorticoid dexamethasone (DEX). Vital staining with HO342 permits earlier detection of apoptotic changes compared to a staining technique in which cells are treated with a hypotonic citrate solution containing propidium iodide (PI) and the apoptotic cells are represented in a hypodiploid, “sub‐G1” peak. The HO342/7AAD method may be particularly applicable to studies of programmed cell death in cells in which DNA fragmentation is difficult to detect by decreased DNA stainability. © 1994 Wiley‐Liss, Inc.
Improved flow‐cytometric detection of low P‐glycoprotein expression in leukaemic blasts by histogram subtraction analysis
Authors: Müller M. R. , Lennartz K. , Nowrousian M. R. , Dux R. , Tsuruo T. , Rajewsky M. F. , Seeber S.
Published: June 2005

Expression of the drug efflux pump P‐glycoprotein (PGP) was determined by flow cytometry in human lung cancer cell lines and in leukaemic blasts derived from 60 patients with acute myeloid leukaemia (AML). Cells from the PGP‐negative parent cell line H69/P and the multidrug resistant (MDR)‐variant H69/LX4 could be clearly distinguished by immunostaining with the anti‐PGP monoclonal antibody MRK16. In leukaemic blasts, the differences in fluorescence intensities between samples incubated with the idiotypic nonspecific (control sample) and specific antibody (test sample) were small, resulting in nondisjunct distributions. Only in a few leukaemia specimens were PGP‐expressing cells detectable by simple subtraction of histograms using a threshold. Therefore, an improved histogram subtraction analysis, based on curve fitting and a statistical test, was applied to distinguish antigen‐positive from antigen‐negative cells. Moreover, a multiparametric staining procedure employing propidium iodide (PI) and Hoechst 33342 was used to reduce staining artefacts. By this approach, leukaemic cells with low expression of PGP were detected in 39 out of 60 cases. Subpopulations with strong PGP expression, resulting in disjunct fluorescence distributions, were not observed. Only in 5 out of 60 specimens were PGP expressing cells detected by a conventional subtraction of histograms using a threshold. Comparison of data obtained with or without the multiparametric gating procedure indicated that the increase in sensitivity was mainly due to the application of the data analysis. However, exclusion of cell debris using PI and Hoechst staining properties reduced the deviation of data from mean values. No relation between PGP expression and cell cycle position was observed in either cell lines or in leukaemic blasts. In relation to clinical status, the median fraction of PGP‐positive blasts was elevated 3.5‐fold in relapsed. patients (n=28) in comparison to patients at first presentation (n=29). In newly diagnosed patients, the median fraction of PGP expressing blasts was 4‐fold higher in specimens from patients who failed to reach complete remission (n=11) in comparison to responsive patients (n=18). Refined histogram subtraction analysis thus facilitates the detection of clinically relevant, low‐level PGP expression in subpopulations of leukaemic blasts. © 1994 Wiley‐Liss, Inc.
Flow‐cytometric detection of the RIα subunit of type I cAMP‐dependent protein kinase in human cells
Authors: Pepe Stefano , Ruggiero Angela , Tortora Giampaolo , Ciardiello Fortunato , Garbi Corrado , Yokozaki Hiroshi , Cho‐Chung Yoon S. , Clair Timothy , Skalhegg Bjorn S. , Bianco Angelo R.
Published: June 2005

cAMP‐dependent protein kinase (PKA) is composed of two genetically distinct catalytic (C) and regulatory (R) subunits. There are two different classes of PKA designated as type I and type II, which contain distinct R subunits (RI or RII, respectively) but share a common C subunit. Enhanced expression of type I PKA has been correlated with cell proliferation and neoplastic transformation. Detection of the different PKA subunits is usually performed by photoaffinity labeling with 8–N3‐32P‐cAMP or by radioimmunolabeling techniques. Both techniques are time, consuming and require a high number of cells and the use of radioactive reagents. Using the MCF‐10A normal human mammary cell line infected with a recombinant retroviral vector contaming the human RIa gene (MCF‐10A RIα), we have developed a flow‐cytometric assay to detect the intracellular content of Met protein in human cells. MCF‐10A and MCF‐10A RIα cells were fixed in 1.5% paraformaldehyde at 37°C for 15 min and permeabilized by methanol and acetone (1:1) at ‐20°C for 5 min before staining with a specific IgG2a MoAb followed by a FITC‐conjugate rabbit‐anti mouse IgG. This procedure was also successfully utilized to recognize RIα protein content in human peripheral blood lymphocytes. Flow‐cytometric detection of the RIa subunit in human cells is feasible and allows the study of the role of type I PKA in cell growth and neoplastic transformation. © 1994 Wiley‐Liss, Inc.
Practical aspects of fixatives in high resolution nuclear image analysis
Authors: Makkus A. C. F. , Van't Hof‐Grootenboer A. E. , Pahlplatz M. M. M. , de Wilde P. C. M. , Gemmink A. J. , Cuypers V. M. J. I. , Vooijs G. P.
Published: June 2005

Three different methods of fixation (ethanol/Carbowax, formaldehyde, and Carnoy) and four different protocols (without Böhm post‐fixation on an uncoated slide, and with Böhm post‐fixation on Poly‐L‐Lysine coated slide, an uncoated slide and a previously Papanicolaou stained slide) were evaluated for their application in high resolution image analysis of Feulgen stained nuclei. The aim of the study was to find a combination with the best reproducibility and the least variance under normal laboratory conditions. Care was taken not to exclude any “normal” laboratory variability. The combinations were evaluated for densitometric, geometric, as well as texture features. Selected features were determined on a CAS‐100 using the Cell Measurement Program (Cell Analysis Systems, Inc. Lombard, IL).

Diploid and tetraploid rat liver nuclei were used. Ethanol/Carbowax fixation‐with Böhm post‐fixation proved most stable. This fixation method also gave feature values for previously Papanicolaou stained slides that were comparable to direct Feulgen stained nuclei. Acceptable results were achieved with all three fixatives and the various combinations if one adhered strictly to protocol. In routine practice this usually does not happen. Therefore Ethanol/Carbowax fixation with Böhm post‐fixation was considered most suited for routine determination of feature values on the CAS‐100. © 1994 Wiley‐Liss, Inc.
Changes in actin organization during the cytotoxic process
Authors: Radošsević Katarina , van Leeuwen Anne Marie T. , Segers‐Nolten Ine M. J. , Figdor Carl G. , de Grooth Bart G. , Greve Jan
Published: June 2005

Changes in organization of F‐actin during the cytotoxic process between NK and K562 cells have been observed and studied using confpcal laser scanning microscopy and quantitative fluorescence microscopy. An increase in F‐actin content and orientation of F‐actin towards the target cell have been observed in, conjugated NK cells. The increase in F‐actin content probably reflects activation of the NK cell for the killing process. An increase in F‐actin content in the conjugated K562 cell, occurring simultaneously with the appearance of filamentous actin structures that often originated/ended at the contact place with the NK cell, was also observed. These changes were delayed compared to the increase in F‐actin content in the NK cell and were accompanied by increasing cytotoxic activity. This indicates that they were results of the interaction of the K562 cell with the activated NK cell. The possible role of target cell microfilaments in the cytotoxic process is addressed. © 1994 Wiley‐Liss, Inc.
Immunofluorescent quantification of tyrosine phosphorylation of cellular proteins in whole cells by flow cytometry
Authors: Far Dariush Farahi , Peyron Jean‐François , Imbert Veronique , Rossi Bernard
Published: June 2005

Tyrosine phosphorylation of proteins, a major event in the transduction of mitogenic signals, was analysed by flow cytometry with a fluorescent antiphosphotyrosine monoclonal antibody, on formaldehyde‐fixed, permeabilized cells. We have used this method (PY‐Facs) to study activation of normal human T lymphocytes and cells of a leukemic T‐cell line: Jurkat. In contrast to normal T cells, Jurkat cells as well as three other leukemic cell lines display a higher constitutive level of tyrosine phosphorylation. This level of tyrosine phosphorylation results from an equilibrium that can be up‐regulated by the tyrosine phosphatase inhibitor, vanadate peroxyde and down‐regulated, by the tyrosine kinase inhibitors, genistein and staurosporine. We have also observed an increased tyrosine phosphorylation of proteins after mitogenic stimulation of Jurkat cells via T‐cell receptor triggering. In addition, the entry of normal purified T cells from G0 phase into the cell cycle after co‐stimulation with a phorbol ester and an antireceptor antibody is correlated with a pronounced increase in tyrosine phosphorylation. We thus confirmed that this biochemical event was tightly associated with the activation status of the cells. The rapidity and sensitivity of the method we describe here make it particularly convenient for routine use and processing of a large number of samples, e.g., during analysis of human tumors. Moreover, because it retains sufficiently the integrity of treated cells and does not alter expression of membrane antigens, this method is suitable for multiparametric analysis, particularly for simultaneous studies associating the measure of tyrosine phosphorylation levels with possible modifications of membrane or intracellular structures as well as with cell cycle status. The PY‐Facs method appears to be an interesting approach to the study of signal transduction events leading to cell activation and proliferation. © 1994 Wiley‐Liss, Inc.
Simultaneous measurement of five‐cell surface antigens by five‐colour immunofluorescence
Authors: Beavis Andrew J. , Pennline Kenneth J.
Published: June 2005

Multicolour immunofluorescence and flow cytometry were used for simultaneous measurement of five‐cell surface antigens on murine spleen cells. We have been able to quantitate T‐cells, T‐cell subsets, B cells, and expression of the activation marker I‐Ad from a single sample using four directly conjugated monoclonal antibodies LYT2‐APC, LM‐PE, B220‐RED613, I‐Ad‐FITC and one indirect step THY1.2‐biotin/streptavidin‐Cascade Blue Three excitation wavelengths were used (488 nm, 647 nm, and U. V. 351–364 nm) for fluorescence measurements. The combination of fluorochromes used provided good resolution such that all five fluorescence signals were spectrally resolved. The percentage of cells positive for expression of a specific cell surface marker were almost identical for singlecolour samples and the five‐colour analysis, differing by only 0.3–1.5 percentage points. © 1994 Wiley‐Liss, Inc.
Application of flow cytometric methods for the routine detection ofCryptosporidiumandGiardiain water
Authors: Vesey Graham , Hutton Primrose , Champion Alan , Ashbolt Nicholas , Williams Keith L. , Warton Andrzej , Veal Duncan
Published: June 2005

CryptosporidiumandGiardiaare common causes of waterborne disease. The currently used methods of detecting these organisms in water rely on filtration capture, immunofluorescence labelling, and epifluorescence microscopy. These methods are inefficient, labour intensive, and require a highly skilled microscopist. We describe an alternative technique using flocculation concentration, followed by flow cytometry with fluorescence activated cell sorting. Environmental samples were analysed, and protozoan‐like particles were sorted and collected before confirmation with epifluorescence microscopy. The technique was found to be significantly more sensitive and considerably faster than the conventional methods. © 1994 Wiley‐Liss, Inc.
Classification of chromosomes using a probabilistic neural network
Authors: Sweeney Walter P. , Musavi Mohamad T. , Guidi John N.
Published: June 2005

This paper describes the application of a probabilistic neural network (PNN) to the classification of normal human chromosomes. The inputs to the network are 30 different features extracted from each chromosome in digitized images of meta‐phase spreads. The output is 1 of 24 different classes of chromosomes (the 22 autosomes plus the sex chromosomes X and Y). An updating procedure was implemented to take advantage of the fact that in a normal somatic cell only two chromosomes can be assigned to each class. The network has been tested using the Copenhagen, Edinburgh, and Philadelphia databases of digitized images of human chromosomes. The recognition rates achieved in this study are superior to those reported using either the maximum likelihood or back propagation neural network techniques. © 1994 Wiley‐Liss, Inc.
Measurement by flow cytometry of genomic AT/GC ratio and genome size
Authors: Vinogradov Alexander E.
Published: June 2005

Flow cytometry with the AT‐specific fluorochrome Hoechst 33258 (HO) and the GC‐specific fluorochrome olivomycin (OM) was used for measurement of base pair specific DNA content in 20 species of vertebrates. The results were found to be in good correlation with the biochemical literature on base pair frequencies (r = 0.972, P < 1 × 10−8). This correlation allows one to determine the percent of GC/AT‐pairs and genome size from flow cytometric data. The genome sizes obtained were compared with the literature data on flow cytometric genome size values determined with the use of propidium iodide (PI) that is usually believed to be non‐base pair specific. The results were found to be in general agreement; however, the previously reported slight GC‐preference of PI is confirmed. The optimal conditions for flow cytometry of AT/GC ratio and genome size with the use of OM and HO are discussed. The approach can be useful for research in ecology, fisheries science, species conservation, and other environmental studies as a tool for rapid survey of a vast array of specimens. © 1994 Wiley‐Liss, Inc.
Quantification of low frequency white blood cells expressing human cytomegalovirus antigen by image cytometry
Authors: Jiwa N. M. , Mesker W. E. , Ploem‐Zaaijer J. J. , van Dorp W. , The T. H. , Raap A. K.
Published: June 2005

Human cytomegalovirus (HCMV) infections cause considerable morbidity and mortality in immunocompromized patients. During HCMV infection, leukocytes appear in the circulation in low frequencies that express the HCMV pp65 protein antigen. Since there is evidence that changes in the frequency of antigen‐positive cells in the early phase of the infection have prognostic value, we applied automated image cytometry to quantify these antigen‐positive cells. For this purpose weekly peripheral blood leukocyte samples of 80 kidney transplant recipients were visually examined for the presence of antigen‐positive cells using an immunocytochemical detection method. Seventeen patients, who reacted positive with this assay, were identified. Next, automated image cytometry was applied to quantitate the frequency of antigen‐positive cells in sequential blood samples from the 17 patients. Patients who developed a period of HCMV viremia had a significantly longer antigenemic period and a significantly higher frequency of antigen‐positive cells than patients with a HCMV infection who remained non‐viremic. Therefore, automated image immunocytometry based screening can be used to distinguish patients at risk for the development of a HCMV viremia. Moreover, automated quantitation reveals prognostic information about the HCMV infection at a more sensitive level than other HCMV detection techniques. © 1994 Wiley‐Liss, Inc.
Bovine papillomavirus E1 protein affects the host cell cycle phase fractions
Authors: Belyavskyi Michail , Miller Jane , Wilson Van
Published: June 2005

C127 murine fibroblast cells were electroporated with a bovine papillomavirus E1 protein expression vector and examined by flow cytometry. E1 expressing cells (E1+) within the total cell population were distinguished from nonexpressing cells (E1−) by immunofluorescent staining with anti‐E1 serum and a fluorescein‐conjugated second antibody. Under conditions of saturation with the first and second antibodies, the specific green fluorescence reflected the level of intracellular E1 protein. Simultaneous staining with a DNA‐specific dye, propidium iodide (PI), enabled the cell cycle distributions for the E1+ and E1− cell populations to be determined. It was found that the E1+ subpopulation had a reduced percentage of cells in G1 phase and an increased percentage of G2+M phase cells, compared to the E1− subpopulation. There was no significant difference in overall doubling time or percentage of noncycling cells in the E1+ vs. E1− populations, indicating that the change in cell cycle distribution was not due to a general activation or inhibition of cell growth by E1. Direct measurement of cell cycle phase fractions confirmed that the G1 phase was decreased and the G2+M phase was increased in E1 expressing cells. As these observations were made in the absence of other viral proteins or viral DNA replication, it suggests that the E1 protein exerts an effect on the host cell independent of its direct role in viral DNA replication. Thus, E1 may interact directly with the host cell cycle regulatory machinery. © 1994 Wiley‐Liss, Inc.
Simultaneous analysis of c‐erbB‐2 expression and DNA content in breast cancer using flow cytometry
Authors: Stål Olle , Sullivan Siw , Sun Xiao‐Feng , Wingren Sten , Nordenskjöld Bo
Published: June 2005

The c‐erbB‐2 oncogene is frequently amplified and overexpressed in human breast cancer. We have studied the c‐erbB‐2protein in conjunction with DNA content in frozen samples from breast cancers using flow cytometry. The cell suspensions were obtained by mechanical disaggregation followed by a short fixation in 1% paraformaldehyde. The level of c‐erbB‐2 expression was calculated as a fluorescence index, taking into account the relative amount of total cellular fluorescence compared to non‐specific fluorescence. The flow cytometric value correlated with immunohistochemical results obtained with the same monoclonal antibody (c‐neu, clone 9G6). Overexpression of c‐erbB‐2 was significantly more frequent in DNA aneuploid tumors than in DNA diploid ones and correlated with increasing S‐phase fraction and estrogen receptor negativity. In 10 DNA multiploid tumors, the different aneuploid stemlines uniformly expressed c‐erbB‐2, supporting the hypothesis that overexpression is an early event in breast cancer. Of the 172 tumors, the 37 (22%) judged as positive with immunohistochemistry showed a somewhat higher rate of distant recurrence than others (P= 0.14). The fluorescence index was significantly associated with prognosis (P= 0.0012), as it was also among the immunohistochemically positive cases. If the degree of overexpression is important, then flow cytometry could be a feasible technique for classification. © 1994 Wiley‐Liss, Inc.
Bivariate flow karyotyping with air‐cooled lasers
Authors: Frey Tom , Houck David W. , Shenker Bruce J. , Hoffman Robert A.
Published: June 2005

An experimental flow cytometer was constructed using a quartz flow cell optically coupled to a 1.22 NA lens. A pair of crossed cylindrical quartz lenses allowed multilaser excitation. Two helium‐cadmium (HeCd) lasers, emitting 16 mW at 442 nm and 35 mW at 325 nm, were used to excite chromomycin A3 and Hoechst 33258 fluorescence, respectively. Bivariate flow karyotypes from normal human male chromosomes and from the Daudi cell line were obtained and were compared to those from a standard instrument using dual water‐cooled lasers. The new experimental instrument exhibited comparable resolution to that from the standard instrument. In further experiments with Daudi chromosomes, the 35 mW HeCd laser was replaced with a 10 mW HeCd laser, and the system still gave good, though slightly decreased, resolution. © 1994 Wiley‐Liss, Inc.
Quantitative precision of an automated image cytometric system for the measurement of DNA content and distribution in cells labeled with fluorescent nucleic acid stains
Authors: Poulin N. , Harrison A. , Palcic B.
Published: June 2005

An automated image cytometric system is described for the measurement of DNA content and distribution in cells stained with fluorescent DNA binding or intercalating compounds. The quantitative precision of integrated optical intensity (IOI) measurements using this system was estimated to be 2.0%, based on the coefficient of variation (cv) of the IOI of DNA check beads. Using peripheral blood lymphocytes stained with 4′‐6‐diamidino‐2‐phenylindole (DAPI), the cv of IOI was found to be 3.5%. Slide to slide variability of the IOI for peripheral blood lymphocytes (cv over 10 slides of the mean IOI) was found to be 2%. The most important sources of error in these measurements were glare, illumination instability, image calibration instability, and staining non‐uniformity. These errors were investigated and minimized. © 1994 Wiley‐Liss, Inc.
Flow cytometric analysis of the cell cycle in polyamine‐depleted cells
Authors: Pohjanpelto Pirkko , Nordling Stig , Knuutila Sakari
Published: June 2005

Polyamines are found in all cells but their function is not fully understood. We have studied the effect of polyamines on the passage of cells through the cell cycle using a polyamine auxotrophic mutant, CHO‐P22, which has no detectable ornithine decarboxylase activity. The ability of these cells to grow without serum allows efficient polyamine depletion. A flow cytometric analysis of DNA content and bromodeoxyuridine labeling showed that without added polyamines the cells accumulated in S‐phase, the rate of DNA synthesis was retarded, and the entry into mitosis was blocked. Addition of polyamines to cultures deprived of polyamines induced cells in all phases of the cell cycle to reinitiate cycling. Earlier studies have shown that cells with damaged DNA are blocked from entering into mitosis but caffeine can partly overcome this block and induce premature chromosome condensation. Polyamine‐depleted CHO‐P22 cells responded to caffeine in the same way as cells with damaged DNA. These results show that polyamine depletion in CHO‐P22 cells primarily affects DNA synthesis. The finding that polyamine‐starved cells continuously take up bromodeoxyuridine without a corresponding increase in the amount of DNA is compatible with extensive repair of erroneous and/or damaged DNA. Polyamine auxotrophic Chinese hamster ovary (CHO) cells might be useful in studies on the regulation of mitosis in mammalian cells. © 1994 Wiley‐Liss, Inc.
Insulin binding to human cultured lymphocytes measured by flow cytometry using three ligands
Authors: Ziegler O. , Cantin C. , Germain L. , Dupuis M. , Sekaly R. P. , Drouin P. , Chiasson J. L.
Published: June 2005

The binding of insulin to cultured IM‐9 human lymphocytes was studied by flow cytometry using FITC‐insulin and biotinylated insulins coupled to streptavidin‐phycoerythrin (NαB1‐biotinylinsulin (B‐insulin) and NαB1‐(biotinyl‐ϵ‐aminocaproyl)insulin (NBC‐insulin)). The reference methods were 125I‐insulin binding and the insulin‐antiinsulin antibody complexes for flow cytometry. There was a close correlation between 125I‐insulin binding and increase in fluorescence for B‐insulin, NBC‐insulin, and insulin‐antiinsulin antibody complexes, but not for FITC‐insulin. NBC‐insulin gave the largest increase in fluorescence (79 ± 9 channels) and the the insulin‐antiinsulin antibody complexes the smallest (34 ± 2 channels) (P < 0.05). FITC‐insulin and B‐insulin gave similar results: 47 ± 6 and 59 ± 6 channels. The concentration reducing 125I‐insulin binding by 50% was 1.1 × 10−9 M for native insulin, 2.7 × 10−9 M for B‐insulin, 3.3 × 10−9 M for NBC‐insulin, and 6.6 × 10−9 M for FITC‐insulin (P < 0.05). Nonspecific binding was low for B‐insulin and NBC‐insulin but reached 75% for 10−6 M FITC‐insulin. These results suggest that B‐insulin and NBC‐insulin are suitable ligands for insulin binding studies using flow cytometry. This two‐step procedure is easier than the insulin‐antiinsulin antibody complex technique. Its poor affinity, specificity, and sensitivity make FITC‐insulin less suitable. © 1994 Wiley‐Liss, Inc.
Evaluation of interspecific DNA content variations and sex identification in Falconiformes and Strigiformes by flow cytometric analysis
Authors: De Vita R. , Cavallo D. , Eleuteri P. , Dell'Omo G.
Published: June 2005

A high interspecific karyotype variability has been evidenced in birds especially in Falconiformes and Strigiformes. Avian cytogenetic analysis, conventionally used for this study, presents several difficulties. We used flow cytometric analysis in order to obtain further information on the DNA patterns of different species of birds belonging to the above‐mentioned orders. Our study was performed on blood samples while chicken erythrocytes and human lymphocytes, with known cytometric DNA content, were used as reference cells. The blood samples of the birds under study were stained, simultaneously to the reference cell, with a lysis‐staining buffer containing propidium iodide. The nuclear DNA content of the bird samples was calculated as DNA index in relation to reference cells, and was expressed as nuclear DNA mass in picograms (pg) with respect to the standard value of 7.0 pg per human lymphocyte nucleus. The results obtained showed an interspecific variability of DNA content and evidenced the usefulness of FCM analysis as a rapid and easy tool for studying the DNA pattern of different species of birds. Moreover, our results have confirmed and extended the possibility of sex identification in species of birds characterized by sexual monomorphism by evaluating the small DNA content difference which exists between males and females. © 1994 Wiley‐Liss, Inc.