bluuebunny/crossref_metadata_2025 huggingface dataset:
A Simple Synthesis of Brominated Analogues of 3‐Chloro‐4‐(dichloromethyl)‐5‐hydroxy‐2(5H)‐furanone (MX).
Authors: Lumbard Keith W. , Nixon Neil S. , Scheinmann Feodor
Published: June 2005

For Abstract see ChemInform Abstract in Full Text.
Synthesis of (.+‐.)‐trans‐2‐ [3‐Methoxy‐4‐(4‐chlorophenylthioethoxy)‐5‐ (N‐methyl‐N‐hydroxyureidyl)methylphenyl] ‐5‐(3,4,5‐trimethoxyphenyl)tetrahydrofuran.
Authors: Ram B. , Balram B. , Ram S. Raghu
Published: June 2005

For Abstract see ChemInform Abstract in Full Text.
Asymmetric Synthesis of 5‐Arylmethylpyrrolidin‐2‐ones and 2‐Arylmethylpyrrolidines.
Authors: Lebrun Stephane , Couture Axel , Deniau Eric , Grandclaudon Pierre
Published: June 2005

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A Direct Entry to the 1‐Methoxyindole Skeleton and to the Corresponding Indoles by a Novel Rearrangement: General Syntheses of Substituted 1‐Methoxyindoles.
Authors: Selvakumar N. , Reddy B. Yadi , Azhagan A. Malar , Khera Manoj Kumar , Babu J. Moses , Iqbal Javed
Published: June 2005

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Palladium‐Catalyzed Synthesis of 2‐Aminoindoles by a Heteroannulation Reaction.
Authors: Witulski Bernhard , Alayrac Carole , Tevzadze‐Saeftel Lali
Published: June 2005

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1‐N‐Substituted Thiocarbamoyl‐3‐phenyl‐5‐thienyl‐2‐pyrazolines: Synthesis and Evaluation as MAO Inhibitors.
Authors: Goekhan Nesrin , Yesilada Akguel , Ucar Guelberk , Erol Kevser , Bilgin A. Altan
Published: June 2005

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Synthesis and Biological Activity of Triazolides
Authors: Selezneva E. S. , Belousova Z. P. , Ten'gaev E. I. , Kadatskaya A. V. , Zvereva I. A. , Poletaeva E. N.
Published: June 2005

For Abstract see ChemInform Abstract in Full Text.
Titanium(IV)‐Promoted Mukaiyama Aldol—Prins Cyclizations.
Authors: Patterson Brian , Marumoto Shinji , Rychnovsky Scott D.
Published: June 2005

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[4 + 2]‐Cyclodimer of Sabinone: Formation, Crystal Structure, and NMR Spectra.
Authors: Suleimenov E. M. , Raldugin V. A. , Shakirov M. M. , Bagryanskaya I. Yu. , Gatilov Yu. V. , Kulyjiasov A. T. , Adekenov S. M.
Published: June 2005

For Abstract see ChemInform Abstract in Full Text.
Synthesis of Some Antibacterial Compounds from 4‐Hydroxycoumarin.
Authors: Mulwad V. V. , Pawar Rupesh B.
Published: June 2005

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Enantioselective Synthesis of cis‐ and trans‐3,5‐Disubstituted Piperidines. Synthesis of 20S‐ and 20R‐Dihydrocleavamine.
Authors: Amat Mercedes , Escolano Carmen , Lozano Oscar , Llor Nuria , Bosch Joan
Published: June 2005

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Microwave‐Assisted Rapid Synthesis of Neurotransmitter Release Enhancer Linopiridine and Its New Analogues.
Authors: Yadav J. S. , Reddy B. V. S.
Published: June 2005

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Highly Diastereoselective Synthesis of New, Carbostyril‐Based Type of Conformationally‐Constrained β‐Phenylserines.
Authors: Ueki Hisanori , Ellis Trevor K. , Khan Masood A. , Soloshonok Vadim A.
Published: June 2005

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A Convenient Catalytic Procedure for the Highly Enantioselective Aldol Condensation of O‐Silyldienolates.
Authors: De Rosa Margherita , Acocella Maria Rosaria , Villano Rosaria , Soriente Annunziata , Scettri Arrigo
Published: June 2005

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The Reaction of Thioesters with Nitriles. A New Synthetic Approach to the Preparation of Substituted 4‐Alkylthio‐ and 4‐Arylthiopyrimidine Derivatives.
Authors: Herrera Antonio , Martinez‐Alvarez Roberto , Ramiro Pedro
Published: June 2005

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Newer Potential Quinazolinones as Hypotensive Agents.
Authors: Kumar Ashok , Tyagi Mirdula , Srivastava V. K.
Published: June 2005

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Selenium‐Promoted Synthesis of Enantiomerically Pure Substituted Morpholines Starting from Alkenes and Chiral Aminoalcohols.
Authors: Tiecco Marcello , Testaferri Lorenzo , Marini Francesca , Sternativo Silvia , Santi Claudio , Bagnoli Luana , Temperini Andrea
Published: June 2005

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New Polyamide Cryptand for Anion Binding.
Authors: Kang Sung Ok , Llinares Jose M. , Powell Douglas , VanderVelde David , Bowman‐James Kristin
Published: June 2005

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One‐Pot Synthesis of Aryldiazepinothiophenones.
Authors: Pozarentzi Minothora , Stephanidou‐Stephanatou Julia , Tsoleridis Constantinos A.
Published: June 2005

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Copper(I)‐Catalyzed Arylselenylation of Aryl Bromides and Iodides.
Authors: Beletskaya Irina P. , Sigeev Alexander S. , Peregudov Alexander S. , Petrovskii Pavel V.
Published: June 2005

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Practical Synthetic Route to Functionalized Rhodamine Dyes.
Authors: Nguyen Trung , Francis Matthew B.
Published: June 2005

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Novel NLO‐phores with Proaromatic Donor and Acceptor Groups.
Authors: Andreu Raquel , Garin Javier , Orduna Jesus , Alcala Rafael , Villacampa Belen
Published: June 2005

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Studies Towards the Total Synthesis of Novel Dolabellane‐Type Diterpenoids: Construction of the 5,11‐Fused Bicyclic Framework.
Authors: Mehta Goverdhan , Umarye Jayant D.
Published: June 2005

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A Concise and Stereoselective Synthesis of Squalamine.
Authors: Zhang Dong‐Hui , Cai Feng , Zhou Xiang‐Dong , Zhou Wei‐Shan
Published: June 2005

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New Stereoselective Synthesis of the Peptidic Aminopeptidase Inhibitors Bestatin, Phebestin and Probestin.
Authors: Righi Giuliana , D'Achille Claudia , Pescatore Giovanna , Bonini Carlo
Published: June 2005

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Novel Stereoselective Synthesis of 1‐(β‐D‐ and α‐L‐Glycopyranosyl)‐5‐methyl‐1H‐1,2,4‐triazoles.
Authors: Yu Jianxin , Li Zhongjun , Lu Wenjie , Zhang Suna , Cai Mengshen
Published: June 2005

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Approaches to Intramolecular Sialylation. Part 1. Synthesis of Pseudodisaccharide Neu5Ac‐(1‐2)‐Gal with Ester‐Linked Monosaccharide Residues.
Authors: Kononov L. O. , Volodin D. A. , Magnusson G.
Published: June 2005

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Indoloquinazoline Alkaloids from Araliopsis tabouensis.
Authors: Christopher Ezugwu , Bedir Erdal , Dunbar Chuck , Khan Ikhlas A. , Okunji Christopher O. , Schuster Brian M. , Iwu Maurice M.
Published: June 2005

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Synthetic Analogues of the Antibiotic Pestalone.
Authors: Kaiser Florian , Schmalz Hans‐Guenther
Published: June 2005

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Structural Features of Solid Phases and Topology of Phase Diagrams
Authors: Khaldoyanidi K. A.
Published: June 2005

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Some Applications of Silica Aerogels
Authors: Pajonk Gerard Marcel
Published: June 2005

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Lithium Lanthanum Titanates: A Review
Authors: Stramare S. , Thangadurai V. , Weppner W.
Published: June 2005

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Organometallic Complexes of Platinum
Authors: Ogawa A. , Hirao T.
Published: June 2005

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Organometallic Complexes of Palladium— Product Subclass 1: Palladium—Diene Complexes.
Authors: Takacs J. M. , Jiang X. , Vayalakkada S.
Published: June 2005

For Abstract see ChemInform Abstract in Full Text.
Metallocyclodextrins and Related Species
Authors: Sliwa Wanda , Girek Tomasz
Published: June 2005

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Resorcinarenes
Authors: Sliwa W. , Zujewska T. , Bachowska B.
Published: June 2005

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Recent Developments in Asymmetric Reduction of Ketones with Biocatalysts
Authors: Nakamura Kaoru , Yamanaka Rio , Matsuda Tomoko , Harada Tadao
Published: June 2005

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Recent Progress in the Synthesis and Chemistry of Azetidinones
Authors: Singh G. S.
Published: June 2005

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Plumbyl Enol Ethers
Authors: Guiry P. J. , McCormack P. J.
Published: June 2005

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Tin Selenides and Tellurides
Authors: Jousseaume B.
Published: June 2005

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Comprehensive Survey of Combinatorial Library Synthesis: 2002
Authors: Dolle Roland E.
Published: June 2005

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Structure and Function of “Metalloantibiotics”
Authors: Ming Li‐June
Published: June 2005

For Abstract see ChemInform Abstract in Full Text.
Structural Changes Accompanying Intramolecular Electron Transfer: Focus on Twisted Intramolecular Charge‐Transfer States and Structures
Authors: Grabowski Zbigniew R. , Rotkiewicz Krystyna , Rettig Wolfgang
Published: June 2005

For Abstract see ChemInform Abstract in Full Text.
Variation in modes of chemotherapy administration for breast carcinoma and association with hospitalization for chemotherapy‐related toxicity
Authors: Du Xianglin L. , Chan Wenyaw , Giordano Sharon , Geraci Jane M. , Delclos George L. , Burau Keith , Fang Shenying
Published: June 2005

BACKGROUND

To the authors' knowledge, few studies to date have addressed the patterns of how chemotherapy was administered (administration modes) over time. In the current study, the goal of the authors was to describe how chemotherapy for breast carcinoma was administered and to determine whether chemotherapy administration modes were associated with toxicity in a community‐based large cohort.

METHODS

The authors studied 5256 women who were diagnosed with breast carcinoma at age 65 years or older between 1992–1999 and received chemotherapy. The patients were identified from the Surveillance, Epidemiology, and End Results (SEER)Program–Medicare linked databases. Chemotherapy drugs and modes of administration were determined through procedure codes in Medicare claims.

RESULTS

Of the 5256 patients who received chemotherapy, 33% received it through an intravenous infusion for less than 1 hour; 39% through an intravenous infusion lasting 1–8 hours; 15% through an intravenous infusion lasting longer than 8 hours and requiring a pump; 12% through an intravenous push technique; and 1% through a subcutaneous, intramuscular, or intralesional injection. These modes varied substantially across the 11 SEER areas. The risks of hospitalization for chemotherapy‐related toxicities (neutropenia, fever, thrombocytopenia, and adverse effects of systemic therapy) were not found to be significantly associated with different modes of chemotherapy after adjusting for other factors. Compared with patients receiving 5‐flurouracil using an intravenous infusion for longer than 8 hours, the risk of toxicity was determined to be 0.96 (95% confidence interval [95% CI], 0.63–1.47) for patients treated with an intravenous infusion lasting 1–8 hours; 0.94 (95% CI, 0.62–1.41) for patients treated with an intravenous infusion lasting less than 1 hour; and 0.66 (95% CI, 0.38–1.08) for patients treated with subcutaneous, intramuscular, or intralesional injection or an intravenous push technique.

CONCLUSIONS

There were substantial geographic variations noted in the modes of administering chemotherapy; however, these variations did not appear to be associated with the risk of toxicities (neutropenia, fever, thrombocytopenia, and adverse effects of systemic therapy). Cancer 2005. © 2005 American Cancer Society.
Effect of electric field pulses on the viability and on the membrane‐bound immunoglobulins of LPS‐activated murine B‐lymphocytes: Correlation with the cell cycle
Authors: Djuzenova Cholpon S. , Sukhorukov Vladimir L. , Klöck Gerd , Arnold W. Michael , Zimmermann Ulrich
Published: June 2005

The effects, of microsecond electropulses (1–kV/cm) on the viability of murine B lymphocytes and on their binding of antibodies by surface immunoglobulin (Ig) were studied in relation to the cell cycle. Before electropulsing, cultures given 48 h mitogenic stimulation showed at least two cell subpopulations, which were distinguishable by their levels of surface‐Ig expression as assessed with FITC‐labelled antibodies against mouse Ig. The immunofluorescence intensity of cells in S and G2/M phases was higher than that of G0/G1 cells.

After exposure of the mitogen‐stimulated lymphocytes to three exponentially decaying (time constantτ=5–40 μs) electric field pulses, dye exclusion assay showed that pulsing at 1 or 2 kV/cm (at 4°C or 20°C) did not cause permeabilization. Field strengths of 3, 4, or 5 kV/cm resulted in 20%, 45%, or 70% of dye‐permeable cells, respectively, if the pulsed cells were transferred to phosphate‐buffered saline on ice for 30 min. Incubation in full medium at 37°C for 30 min (“resealing”) significantly decreased the percentage of permeabilized cells. Electropulsed G0/G1 cells were not only more resistant to direct electric exposure (tolerated higher field strengths) than S+G2/M cells but also responded better to resealing.

The surface Ig of lymphocytes pulsed at higher fields and low temperature (4 or 5 kV/cm, τ =5 μs, three pulses, 4°C) was less easily immunostained than in controls or in cells pulsed at 2 kV/cm or less. At 5 kV/cm those cells that were not permeabilized showed a greater reduction in immunostaining, especially if resealed. © 1994 Wiley‐Liss, Inc.
Evaluation of testicular germ cell tumor proliferation using DNA flow cytometry and nucleolar organizer silver staining
Authors: Hasegawa Soichiro , Okamura Kikuo , Shimoji Toshio , Miyake Koji , Yamakawa Yoshinori , Sakamoto Hirofumi
Published: June 2005

We studied 41 testicular germ cell tumors (TGCT) using DNA flow cytometry and argyrophilic (silver staining) nucleolar organizer region (Ag‐NOR) quantitation to evaluate the proliferative characteristics of TGCT and the significance of those analytical techniques. In nonseminomatous germ cell tumor (NSGCT), the percentage of S + G2M phase cells (% S + G2M) in DNA aneuploid tumor was higher than in DNA of diploid tumors. Likewise, in NSGCT the aneuploid Ag‐NOR sites per nucleus (Ag‐NOR count) were significantly higher in DNA aneuploid than in DNA diploid tumors. There was also a significant linear correlation between % S + G2M phase cells and the Ag‐NOR count in TGCT. However, the DNA ploidy pattern and the % S + G2M phase cells, and Ag‐NOR counts, did not correlate significantly with the patient survival rate or clinical stage. In conclusion, our study suggests that Ag‐NOR enumeration is an effective technique for assessing cellular proliferative activity that is somewhat less expensive than DNA flow cytometry. © 1994 Wiley‐Liss, Inc.
Adsorption of aerosil on erythrocyte surface by flow cytometry measurements
Authors: Gerashchenko Bogdan I. , Gerashchenko Igor I. , Bogomaz Valery I. , Pantazis Cooley G.
Published: June 2005

Light scattering properties of the erythrocytes in less than 0. 01% aerosil suspension were measured. It was shown that light scattering properties of intact cells and cells associated with silica (aerosil) are different. It was also established that the aerosil particles are characterized by more intensive autofluorescence in the channel of green fluorescence than the erythrocytes. We propose that this observation allows a facile means of studying the interaction of purified silica and cell membranes. Hemolysis, as a result of interaction between the aerosil particles and the erythrocytes, was confirmed by photometric measurement of hemoglobin in the supernatant. © 1994 Wiley‐Liss, Inc.
Propidium iodide and the thiol‐specific reagent dacm as a dye pair for fluorescence resonance energy transfer analysis: An application to mouse sperm chromatin
Authors: Bottiroli G. , Croce A. C. , Pellicciari C. , Ramponi R.
Published: June 2005

The dyes N‐(‐7‐dimethylamino‐4‐methyl‐coumarinyl) maleimide and propidium iodide, specific for the thiol group and DNA, respectively, were considered as a donor‐acceptor couple suitable for investigating “in situ” the relative spatial distribution of DNA and protamines in mouse spermatozoa chromatin. The two dyes are characterized by favourable spectral properties, so that a simplified analytical procedure, based on the measurement of both donor and acceptor emission in double‐stained samples, can be applied to evaluate the relative efficiency of the energy transfer process and its topological distribution. The results obtained indicate that during the maturation process: 1) the basic arrangement of protamine‐DNA complex does not undergo structure changes, and 2) the oxidation of sulfhydryl to disulfide groups, resulting in chromatin stabilization, first involves the protamine thiols spatially closer to DNA. Fluorescence energy transfer imaging suggests that chromatin stabilization starts in the midportion of the sperm head, then spreads towards the periphery. © 1994 Wiley‐Liss, Inc.
TOTO and YOYO: New very bright fluorochromes for DNA content analyses by flow cytometry
Authors: Hirons G. T. , Fawcett J. J. , Crissman H. A.
Published: June 2005

Flow cytometric (FCM) studies were performed on nuclei, ethanol‐fixed CHO cells, and isolated human GM130 chromosomes stained with two new cyanine dyes, TOTO and YOYO. These fluorochromes, which are dimers of thiazole orange and oxazole yellow, respectively, have high quantum efficiencies and exhibit specificities for both DNA and RNA. Bound to dsDNA in solution, TOTO and YOYO emit at 530 and 510 nm, respectively, when excited at 488 nm and 457 nm, wavelengths available from most lasers employed in FCM. RNase‐treated CHO nuclei, stained with either TOTO or YOYO, provided DNA histograms, with low coefficients of variation, that were as good as or better than those obtained with nuclei stained with propidium iodide (PI) or mithramycin (MI). In addition, by comparison on an equimolar basis, nuclei stained with YOYO fluoresced over 1,000 times more intensely than nuclei stained with MI. Fluorescence ratio analyses of nuclei stained with both YOYO and Hoechst 33258 showed that the ratio of YOYO to Hoechst fluorescence remained relatively constant for G1 and S phase cells, but decreased significantly for cells in G2/M. These results indicate that the cyanine dyes may be useful in examining specific changes in chromatin structure during G2/M phases of the cell cycle. Ethanol‐fixed CHO cells stained with TOTO or YOYO did not yield reproducible DNA histograms of good quality, presumably because of the poor accessibility of DNA to these large fluorochromes. However, bivariate analyses of human GM130 chromosomes stained with TOTO or YOYO alone and excited sequentially with uv and visible wavelengths showed resolution of many individual chromosome peaks similar to results obtained for chromosomes stained with HO and chromomycin A3. Collectively, these studies show potential advantages for the use of these new cyanine dyes in FCM studies that require the sensitive detection of DNA. © 1994 Wiley‐Liss, Inc.
Discrimination of bromodeoxyuridine labelled and unlabelled mitotic cells in flow cytometric bromodeoxyuridine/DNA analysis
Authors: Jensen Peter Østrup , Larsen Jørgen K. , Christensen Ib Jarle , van Erp Piet E. J.
Published: June 2005

Bromodeoxyuridine (BrdUrd) labelled and unlabelled mitotic cells, respectively, can be discriminated from interphase cells using a new method, based on immunocytochemical staining of BrdUrd and flow cytometric four‐parameter analysis of DNA content, BrdUrd incorporation, and forward and orthogonal light scatter. The method was optimized using the human leukemia cell lines HL‐60 and K‐562. Samples of 105 ethanolfixed cells were treated with pepsin/HCl and stained as a nuclear suspension with anti‐BrdUrd antibody, FITC‐conjugated secondary antibody, and propidium iodine. Labelled mitoses could be discriminated from unlabelled mitoses, and from labelled and unlabelled G2 cells, by their intermediate log FITC fluorescence intensity. In addition, mitoses and G2 nuclei differed in forward and orthogonal light scattering, but had equal intensity of propidium iodide fluorescence. This method for discrimination of labelled mitoses was also tested on cultured normal adult human keratinocytes labelled with iododeoxyuridine (IdUrd). In keratinocytes, where the cell structure was preserved after pepsin/HCl, IdUrd labelled mitotic cells were similarly discriminated in the log FITC/propidium iodide fluorescence distribution. This interpretation was supported by experiments using mitotic arrest, fluorescence activated cell sorting and microscopy, and comparison with an alternative flow cytometric method for discrimination of mitoses. © 1994 Wiley‐Liss, Inc.
Detection of genetic changes in Barrett's adenocarcirioma and Barrett's esophagus by DNA in situ hybridization and immunohistochemistry
Authors: Krishnadath K. K. , Tilanus H. W. , Alers J. C. , Mulder A. H. , van Dekken H.
Published: June 2005

We have investigated the occurrence of chromosomal DNA and cell cycle‐related protein changes in Barrett's epithelium and adenocarcinoma. The presence of numerical chromosomal aberrations was studied by applying nonisotopic in situ hybridization (ISH) with (peri‐)centromeric DNA probes, specific for chromosomes 7, 8, 17, and Y, to routinely processed tissue sections of five cases (4 male, 1 female) of Barrett's adenocarcinoma and adjacent Barrett's epithelium. Cell cycle‐related protein expression was studied by immunohistochemistry (IHC) for p53 protein and the Ki‐67 antigen (Mib‐1) in subsequent sections. P53 protein overexpression was found in 3 of the 5 tumors. Overrepresentation of chromosome 8 and loss of chromosome 17 were found in 2 adenocarcinomas, both also negative for p53 protein overexpression. Y‐loss, mostly clonal, was detected in 3 of the 4 male adenocarcinomas and 2 cases of adjacent Barrett's epithelium. One tumor had both areas of overrepresentation and loss of the Y chromosome. All Barrett's adenocarcinomas appeared to contain aneuploid cell populations. No relation was found between cell proliferation characteristics and chromosomal aberrations. We conclude that ISH with chromosome specific DNA probes can be applied for the assessment of potentially important numerical chromosome changes in Barrett's esophagus. Further, the combination of IHC and ISH is useful for evaluation of specific genetic events. © 1994 Wiley‐Liss, Inc.
Automated image cytometry for detection of rare, viral antigen‐positive cells in peripheral blood
Authors: Ploem‐Zaaijer Joke J. , Mesker Wilma E. , Boland Greet J. , Sloos Willem C. R. , van de Rijke Frans M. , Jiwa Mehdi , Raap Anton K.
Published: June 2005

A cell detection method based upon automated screening is described for recognition of low frequencies (1 in 100,000) of immuno‐enzymatically labelled white blood cells in human peripheral blood. The used image cytometry instrumentation (LEYTAS) includes a wide‐field, fully automated microscope (Autoplan) and a modular image analysis computer (MIAC), both from Leica, Wetzlar, Germany. The MIAC contains image boards for optimum use of mathematical morphology algorithms. Communication with the MIAC is via a personal computer. Programs for automated cell analysis have been written in C language. Main features of the system are fast analysis of, large microscope fields including a count of all cells, selection of objects of interest (alarms), and display of digitally stored images of these alarms. We tested this system for the detection of white blood cells expressing antigen of cytomegalovirus (pp65) in 50 human blood smears from kidney transplant recipients. Immuno‐enzymatic (peroxidase) staining was performed with DAB and counterstaining with hematoxylin. For determination of the sensitivity, a series of dilutions of a positive sample with a negative sample was performed. The lowest frequency detected was 1 antigenpositive cell / 3 × 105 antigen‐negative cells. Screening time was about 60 min for one million cells. © 1994 Wiley‐Liss, Inc.
Flow cytometric analysis of bioluminescence emitted by recombinant baculovirus‐infected insect cells
Authors: Lindqvist Christer , Karp Matti , Åkerman Karl , Oker‐Blom Christian
Published: June 2005

Five recombinant baculoviruses, each containing a different insect luciferase gene encoding a protein with characteristic light emission properties, namely, luc GR (546 nm), luc FF (556 nm), luc YG (560 nm), luc YE (578 nm), and luc OR (593 nm) were constructed. All genes were inserted under the transcriptional control of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed in Spodoptera frugiperda insect cells during viral infection. The biological activity of the different luciferases was characterized by using intact recombinant baculovirus infected cells. Addition of the substrate, D‐luciferin, immediately prior to the analysis allowed monitoring of light emission by flow cytometry. Also, the kinetics of the light emission of lucGR was analyzed with the flow cytometer. The emission peaks of the infected cells were clearly separated by wavelength scanning. Especially, the firefly luciferase (lucFF) had a broad peak and transient luminescence. The highest maximal intensity values in vivo were recorded for luc GR and luc YG. SDSPAGE analysis showed that the major protein expressed had a molecular weight similar to authentic luciferase. Flow cytometry and insect luciferases with clearly separated emission spectra appear to be of value for sensitive in vivo analysis of gene promoter activity. © 1994 Wiley‐Liss, Inc.
Flow cytometric analysis of entire microbial colonies
Authors: Sahar Eli , Nir Raphael , Lamed Raphael
Published: June 2005

Much could be gained if the scope of flow cytometry could be broadened to the study of entire cell colonies, rather than to populations of single, separate cells. This can be achieved by encapsulating single microbial cells in small spheres in a way that allows each cell to multiply and form a colony within its respective microbead, which is then amenable to analysis by flow cytometry. Methods for performing the encapsulation within beads of appropriate size and homogeneity have been developed (Nir et al., Appl. Environ. Microbiol. 56:2870–2875, 1990). We describe here how a variety of properties of the entrapped colonies, such as mass, growth rate, enzymatic activity, and expression of specific antigens, can be measured, and we discuss how these constructs can be utilized to select microbial mutants. © 1994 Wiley‐Liss, Inc.
Further flow cytometric studies of the effects of triethylenemelamine on somatic and testicular tissues of the rat
Authors: Bickham John W. , Sawin Virginia L. , McBee Karen , Smolen Michael J. , Derr James N.
Published: June 2005

Exposure to the mutagen triethylenemelamine on rat bone marrow, blood, and testis was studied using flow cytometry of DAPI‐stained nuclei. Increased coefficients of variation (CVs) of the G1 peaks were observed in bone marrow and blood after both 1 d and 5 d exposures. After 5 d exposure and 7 d recovery both tissues had recovered, in some cases to significantly lower CVs. Increased CVs of the 1C peak of testis were observed only after 5 d exposure to the high dose with, no subsequently observed recovery. Bone marrow cells also were stained with, Hoechst 33258 and Propidium Iodide. No differences among dyes were observed indicating that increased CVs likely are due to DNA damage resulting from interactions with the mutagen rather than differences in how the dyes bind to DNA relative to mutagen binding. This study demonstrates that differences occur among tissues in how quickly they respond and recover from mutagen exposure. Increased CVs, cell cycle alterations, and decreased CVs after recovery are all potentially useful biomarkers of effect for laboratory and field studies in environmental toxicology. © 1994 Wiley‐Liss, Inc.
Detection of human papillomavirus type 16/18 DNA in cervicovaginal cells by fluorescence based in situ hybridization and automated image cytometry
Authors: Siadat‐Pajouh Majid , Periasamy Ammasi , Ayscue Andrea H. , Moscicki Anna B. , Palefsky Joel M. , Walton Leslie , Demars Leslie R. , Power Jon D. , Herman Brian , Lockett Stephen J.
Published: June 2005

Automatic fluorescence image cytometry (AFIC) is a fast, sensitive, and reliable approach for screening slide‐based clinical specimens. In this study, we applied AFIC to identify cancer‐associated human papillomavirus (HPV) genotypes 16 and 18 in individual cells of cervical smears using a sensitive fluorescence based in situ hybridization (FISH) assay. HPV sequences were labeled by FISH and the cells imaged using an epi‐fluorescence microscope coupled to a low‐light color CCD camera. Before application to clinical specimens, AFIC was assessed using fluorescent calibration beads and cervical cancer cell lines containing known numbers of integrated HPV genomes per nucleus. Assessment showed that our AFIC had a linear response, was quantitatively accurate, and had the sensitivity to detect one HPV genome per nucleus: After acquisition of images, computer algorithms identified every cell nucleus (via a fluorescent DNA counterstain) and quantified the FISH signal per nucleus. AFIC was employed to screen 27 patient specimens for HPV 16/18, of which 12 were positive. The HPV status of the specimens positively correlated with the pathological diagnosis, and since AFIC automatically and correctly located every cell, it was possible to directly compare morphology and HPV status in the same cell. In conclusion, the combination of FISH and AFIC is a sensitive and quantitative method to detect high risk HPV sequences in cervical smears. © 1994 Wiley‐Liss, Inc.
Lymphocyte subset analysis by boolean algebra: A phenotypic approach using a cocktail of 5 antibodies and 3 color immunofluorescence
Authors: Hunter Stephen D. , Peters Lyndsay E. , Wotherspoon John S. , Crowe Suzanne M.
Published: June 2005

Commercial reagent kits for the evaluation of leukocyte subsets involve the staining of a panel of up to six tubes using combinations of pre‐mixed fluorescein isothiocyanate (FITC) and R‐phycoerythrin (PE) conjugated, monoclonal antibodies. We describe a rapid method whereby total CD3+ T‐cells, CD4+ T‐cells (CD3+ CD4+), CD8+ T‐cells (CD3+CD8+), putative γδ‐receptor‐T‐cells (CD3+CD4−CD8−), and T‐cells that are CD3+ CD4+ CD8+ as well as B‐lymphocytes and NK‐cells can be enumerated after staining in a single tube. Whole blood specimens are labelled with a mixture of antibodies: FITC‐conjugated antibodies to CD4 and CD19, PE‐conjugated antibodies to CD8 and CD16, and either peridinin chlorophyll protein (PerCP) or allophycocyanin (APC) labelling for antibodies to CD3. After recording 20,000 events the data were analysed on the Consort 32 computer system and LYSYS‐II (Becton Dickinson, San Jose, CA) and all of the lymphocyte subset values were determined by Boolean algebra using a technique we refer to as Boolean gate analysis (BGA). Our study has shown that BGA is statistically equivalent to SimulSET lymphocyte subset analysis. Furthermore, the procedure reduces the number of tubes required to two with consequential saving in reagents, consumables, and time. © 1994 Wiley‐Liss, Inc.
Visible diode lasers can be used for flow cytometric immunofluorescence and DNA analysis
Authors: Doornbos Richard M. P. , de Grooth Bart G. , Kraan Yvonne M. , van der Poel Carel J. , Greve Jan
Published: June 2005

This report describes a feasibility study concerning the use of a visible diode laser for two important fluorescence applications in a flow cytometer. With a 3 mW 635 nm. diode laser, we performed immunofluorescence measurements using the fluorophore allophycocyanin (APC). We have measured CD8 positive lymphocytes with a two‐step labeling procedure and the resulting histograms showed good separation between the negative cells and the dim and the bright fluorescent subpopulations. As a second fluorescence application, we chose DNA analysis with the recently developed DNA/ RNA stains TOTO‐3 and TO‐PRO‐3. In our setup TO‐PRO‐3 yielded the best results with a CV of 3.4%. Our results indicate that a few milliwatts of 635 nm light from a visible diode laser is sufficient to do single color immunofluorescence measurements with allophycocyanin and DNA analysis with TO‐PRO‐3. The major advantages of using a diode laser in a flow cytometer are the small size, the low price, the high efficiency, and the long lifetime. © 1994 Wiley‐Liss, Inc.
Simultaneous cytokinetic measurement of aneuploid tumors and associated diploid cells following continuous labelling with chlorodeoxyuridine
Authors: White R. Allen , Pollack Alan , Terry Nicholas H. A.
Published: June 2005

Cells from a murine tumor, MCa‐K, were continuously labelled with the thymidine analogue chlorodeoxyuridine (CldUrd) and analyzed by bivariate flow cytometry in order to measure the growth fraction (GF) and potential doubling time (T pot) of both the DNA‐aneuploid tumor cells and the associated DNA‐diploid cells. MCa‐K has a DNA index of 1.7, rendering two, partially overlapping, populations observable with labelled and unlabelled cells in each population. The data from these tumors may be divided into three regions of differing DNA content, with one region containing a pure DNA‐diploid population, a second region with both cell types, and a third region including only DNA‐aneuploid cells. Equations are presented to characterize the fractions of labelled cells in each region as a function of labelling time and cell type, thereby permitting estimation of the proliferative properties of the populations. These equations include the possibility that DNA‐aneuploid cells cease cycling both in G1 and in S phase to account for the observed numbers of unlabelled cells with S phase contents. The estimated value of Tpot of the DNA‐diploid cells is 126.0 h with a GF of 42%, while that of the DNA‐aneuploid cells is 36.9 h with a GF of 69%. It is also estimated that between 2% and 6% of all DNA‐aneuploid cells starting DNA synthesis cease cycling, leading to 25% of the cells having an S‐phase DNA content being noncycling. © 1994 Wiley‐Liss, Inc.
Evaluation of methods for measuring cellular glutathione content using flow cytometry
Authors: Hedley David W. , Chow Sue
Published: June 2005

The currently available flow cytometric stains for cellular glutathione were evaluated, examining the labelling of both human and rodent cell lines under various conditions of concentration, time, and temperature. Procedures were used that depleted, glutathione (GSH) while having a minimal effect on other cellular sulphydryls in order to estimate linearity and the extent of background staining. As previously reported, monochlorobimane was highly specific for GSH in rodent cells but failed to label human cells adequately because of its low affinity for human glutathione S‐transferases. Higher concentrations of monochlorobimane achieved more complete labelling of, the human cellular GSH pool but gave increased background fluorescence due to non‐GSH binding. The analogue monobromobimane, which binds nonenzymatically to sulphydryls, reacted, more readily with GSH than with protein sulphydryls and, provided that stain concentration and incubation time were controlled, gave reproducible staining of human cells with 20% of total fluorescence due to background staining. Of the currently available stains for measuring GSH in human cells, monobromobimane is the agent of choice. Mercury orange also binds more readily to GSH than to protein, giving a degree of specificity, and it has the additional advantage of being excited at 488 nm. However, the reproducibility of staining with mercury orange was less consistent than that using monobromobimane, and a higher background fluorescence was seen. Two additional stains, O‐phthaldialdehyde and chloromethyl fluorescein, could also be used to label cellular GSH, but both gave an unacceptably high level of background staining. It is recommended that flow cytometric GSH assays should routinely include a sample of cells that have been depleted of GSH in order to determine the extent of background labelling. © 1994 Wiley‐Liss, Inc.
Detection of intracellular HIV in lymphocytes by flow cytometry
Authors: Gadol Nancy , Crutcher Gillian J. , Busch Michael P.
Published: June 2005

Various permeabilization procedures and defined gating techniques were tested in order to optimize existing flow cytometric assays and devise a specific assay for the direct detection of intracellular HIV‐1 antigens in clinical blood specimens. In our optimal procedure, blood lysed with Orthomune Lysing Reagent was fixed with 3.7% formaldehyde for 10 min at room temperature and then permeabilized with 0.2% Tween 20 for 15 min at room, temperature. Cells from whole blood were labeled with either FITC‐anti‐p18 or FITC‐anti‐p24 monoclonal antibodies and PE‐anti‐Leu M9 (CD33) in order to exclude monocytes and granulocytes from the lymphocyte gate. The assay demonstrated that mean percentages of HIV p24 antigen positive cells were increased in patients with advanced disease. The assay in its present form is useful for monitoring disease progression and for monitoring the effects of antiviral therapy in individuals, but it is not currently sensitive enough to detect consistently the low levels of HIV infected peripheral blood cells in asymptomatic individuals. © 1994 Wiley‐Liss, Inc.
Automatic fluorescence metaphase finder speeds translocation scoring in FISH painted chromosomes
Authors: Piper J. , Poggensee M. , Hill W. , Jensen R. , Ji L. , Poole I. , Stark M. , Sudar D.
Published: June 2005

A fluorescence metaphase finder was constructed with commercially available hardware and a standard Unix workstation. Its accuracy was measured in terms of the number of false positive and false negative detected metaphases on a variety of different slide preparations. The metaphase finder was used in a translocation scoring experiment in which metaphase preparations of human peripheral blood lymphocytes were hybridized with whole chromosome probes to chromosomes #1, #2, and #4. The automatic finder presented metaphases to the cytogeneticist, centered in the eyepieces at × 63. The cytogeneticist's scores of analyzable metaphases and of painted chromosomes involved in rearrangements were recorded. The time for the analysis was recorded and compared to the time to analyze a similar number of cells in a purely visual experiment in which the cytogeneticist scanned for cells and analyzed them, both at × 63. The results showed that, neglecting the machine time spent scanning unattended, the amount of time required for the analysis was reduced by a factor of three. Furthermore, in this experiment the metaphase finder found more scorable metaphases than the cytogeneticist found by visual scanning. Machine‐assisted scoring had additional, less quantifiable, benefits; notably that digital images of metaphases sometimes assisted the analysis of chromosome rearrangements, that cells could be revisited easily, and that the analysis was much less fatiguing. © 1994 Wiley‐Liss, Inc.
Flow‐cytometric analysis of apoptotic and nonapoptotic T‐cell receptor‐transgenic thymocytes following in vitro presentation of antigen
Authors: Curnow S. J. , Barad M. , Brun‐Roubereau N. , Schmitt‐Verhulst A.‐M.
Published: June 2005

The use of flow cytometry to detect apoptotic thymocytes is now well established. We have further developed the technique of Hoechst 33342 vital staining to identify discrete stages of murine thymocyte apoptosis (induced by 37°C culture), in conjunction with propidium iodide (PI), cell scatter profile, and surface marker analysis. The first detectable stage was an increase in Hoechst fluorescence without any change in plasma membrane permeability (measured by PI staining). At this early stage thymocytes had already reduced in size, fragmented their DNA, and for the predominant CD4+ CD8+ double positive population, reduced expression of CD4 and CD8. Subsequent to this stage thymocytes continued to reduce in size and decrease expression of CD4 and CD8, though this was accompanied by an increase in membrane permeability. This technique was applied to an in vitro antigen‐specific deletion system, where apoptosis of T cell‐receptor‐transgenic thymocytes was induced upon presentation of self‐antigen. Although self‐antigen‐induced apoptotic thymocytes showed similar characteristics to those undergoing spontaneous apoptosis, there was a significant population of nonapoptotic CD4+8+ thymocytes that also had reduced expression of CD4 and CD8. Therefore, we have been able to show that the reduced expression of CD4 and CD8 is not limited to apoptotic thymocytes. © 1994 Wiley‐Liss, Inc.
Tumor cell kinetics using two labels and flow cytometry
Authors: Ritter Mark A. , Fowler John F. , Kim Youngmi J. , Gilchrist Kennedy W. , Morrissey Lawrence W. , Kinsella Timothy J.
Published: June 2005

A flow cytometry based, single sampling method employing sequential bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd) labeling with an intervening interval is examined. BrdUrd‐specific and nonspecific monoclonal antibodies and flow cytometry are used to estimate the duration of DNA synthesis and the potential doubling time in tumor cell populations using a single sampling or biopsy. A correction for labeled, divided cells allows postlabel incubation intervals to exceed the length of the G2/M phase of the cell cycle. The method yields reliable results when tested in experimental tumor systems in vitro and in situ, as well as in nine human tumors labeled in situ. It uses a somewhat simpler analysis than the alternative relative movement method, requiring only labeling indices, rather than both a labeling index and relative movement, but does require the administration of two, rather than one, label. It provides an independent verification of and a useful single sampling alternative to the relative movement method for the estimation of the length of S phase and, from this, the potential doubling time in experimental or clinical human tumors. © 1994 Wiley‐Liss, Inc.
Slit‐scanning technique using standard cell sorter instruments for analyzing and sorting nonacrocentric human chromosomes, including small ones
Authors: Rens W. , Van Oven C. H. , Stap J. , Jakobs M. E. , Aten J. A.
Published: June 2005

We have investigated the performance of two types of standard flow cell sorter instruments, a System 50 Cytofluorograph and a FACSTar PLUS cell sorter, for the on‐line centromeric index (CI) analysis of human chromosomes. To optimize the results, we improved the detection efficiency for centromeres in two ways. A higher efficiency was obtained first by elongation of the chromosomes and second by introducing a high resolution lens system for laser beam focusing. In the two‐parameter flow karyotype of CI and DNA content of human chromosomes, distinct peaks are produced not only by the larger chromosomes 1‐‐8 and X, but by the smaller nonacrocentric chromosomes 9–12 and 16–20 as well. As the chromosomes 9–12 cannot be distinguished by other flow karyotyping methods, we discriminated and sorted chromosomes 12 and 10 from 9 and 11 to investigate the capacity for the separation of chromosomes in this group. A purity of at least 90% was achieved; in the isolated population the fraction chromosomes 12 was 55%; the remaining 45% were chromosomes 10 (40%) and unidentifiable chromosomes (5%). © 1994 Wiley‐Liss, Inc.
Influence of sampling time on assessment of potential doubling time
Authors: Høyer Morten , Bentzen Søren M. , Salling Lisbeth N. , Overgaard Jens
Published: June 2005

Potential doubling time (Tpot), S‐phase transit time (Ts), and labeling index were determined in three experimental rodent tumors by in vivo incorporation of bromodeoxyuridine and flow cytometry. The kinetic parameters were derived from the relative movement method at varying sampling times (time between injection of bromodeoxyuridine and tumor excision). Ts and Tpot were close to the expected (extrapolated) values of the two parameters with sampling time within the range 60–100% of the expected Ts. With short sampling time, Ts and Tpot were considerably over‐ or underestimated. By use of the original method by (Begg et al., Cytometry 6:620–626, 1985), the Tpot came out with a twofold overestimation with a short sampling time. Generally, modified methods for calculation of Ts did not improve the results. When Tpot is measured in human tumors, there is no a priori knowledge of the kinetic parameters. Consequently, the sampling time must be based on the general experience of tumor cell kinetics in the specific tumor type measured. A relatively long sampling time should be aimed for when measuring Tpot in human tumors. With proper sampling time, Tpot assessed by flow cytometry was found to be a precise and reproducible parameter. © 1994 Wiley‐Liss, Inc.
Enhanced assessment of DNA/proliferative index by depletion of tumor infiltrating leukocytes prior to monoclonal antibody gated analysis of tumor cell DNA
Authors: Kenyon Norma S. , Schmittling Robert J. , Siiman Olavi , Burshteyn Alexander , Bolton Wade E.
Published: June 2005

Flow cytometry has become an important tool for the analysis of breast tumors, and assessment of S phase fraction and DNA ploidy are potential indicators of tumor aggression. Due to masking or dilution of infrequent tumor cell events, the presence of normal cell types, such as inflammatory cells and fibroblasts, can interfere with accurate DNA analysis of solid tumor samples. MDA‐MB‐175‐VII human breast carcinoma cells, WI‐38 human lung fibroblast cells, and peripheral blood leukocytes were mixed, in varying proportions, in order to represent human breast tumor samples. The cells were subsequently treated with CD45 conjugated magnetic microspheres to deplete tumor infiltrating leukocytes, thus enriching for tumor cells. The tumor cell mixtures were then stained with a pancytokeratin specific monoclonal antibody or with a monoclonal antibody that reacts with breast epithelial membrane antigen (EMA). When used in combination with monoclonal antibody gating, utilization of this bead‐based technology resulted in enhanced precision of DNA analysis. © 1994 Wiley‐Liss, Inc.
Time‐resolved flow cytometry for the measurement of lanthanide chelate fluorescence: I. Concept and theoretical evaluation
Authors: Condrau Marc A. , Schwendener Reto A. , Niederer Peter , Anliker Max
Published: June 2005

The concept of a flow cytometer suited for the time‐resolved measurement of lanthaide chelate luminescence with a decay time on the order of 10 μs to 2 ms is presented and evaluated. The instrument proposed encompasses a continuouswave laser for fluorescence excitation and an optical switch for the elimination of cellular autofluorescence decaying within 1 ns to 1 μs during the luminescence detection period. The slowly decaying fluorescence is to be quantified by a photon‐counting system, whereas light scatter and prompt fluorescence parameters are acquired by a conventional detection system.

The detection limit of the method, in terms of the smallest detectable number of fluorescing chelates per cell, is examined. It was found to be nearly 30,000 complexes of a europium chelate with a decay time of 1.6 ms and a quantum efficiency of 17%, independent of fast decaying cellular autofluorescence or prompt dye emission intensity. The probability of cells passing through the instrument without being detected while the laser beam is turned off was estimated, and the implications for cell throughput and sorting performance of the instrument were assessed. At typical fluorescence detection intervals of 500 μs to 1 ms, cell flow rates of 100–200 particles per second lead to detection probabilities of more than 90% and sorting purities comparable to those found in conventional fluorescence‐activated cell sorting. © 1994 Wiley‐Liss, Inc.