bluuebunny/crossref_metadata_2025 huggingface dataset:
Measurement of Interleukin 3 and Other Hematopoietic Cytokines, such as GM‐CSF, G‐CSF, M‐CSF, Erythropoietin, Steel Factor, and Flt‐3 Ligand
Authors: Cooper Scott H. , Broxmeyer Hal E.
Published: June 2004

Detection and quantitation of IL‐3 and other hematopoietic cytokines in serum samples and culture supernatants has played an important role in understanding their function and pleiotropic properties during the process of hematopoiesis. Several methods for detecting and quantitating cytokines—such as the colony‐stimulating factors granulocyte/macrophage colony‐stimulating factor (GM‐CSF), granulocyte colony‐stimulating factor (G‐CSF), macrophage colony‐stimulating factor (M‐CSF, also called CSF‐1), interleukin 3 (IL‐3), and erythropoietin (Epo), as well as two potent costimulating cytokines steel factor (SLF) and Flt3/Flk‐2 ligand (Flt3‐L)—are described in this unit.
Measurement of Mouse and Human Interferon γ
Authors: Schreiber Robert D.
Published: June 2004

This unit describes two protocols that can be used to quantitate interferon γ (IFN‐γ), an important modulatory lymphokine that regulates natural, cell‐mediated, and humoral immunity by eliciting a number of biological responses in many different cell types. Depending upon the specificity of the reagents used, the assays will detect either human or murine IFN‐γ. The is an enzyme‐linked immunosorbent assay (ELISA) based on IFN‐γ's unique antigenic structure. The is a functional activity assay based on IFN‐γ's ability to induce major histocompatibility (MHC) class II antigens (also termed Ia antigens) on responsive cells. The advantage of the ELISA protocol is its specificity, while the advantage of the MHC protocol is its sensitivity.
Isolation of Whole Mononuclear Cells from Peripheral Blood and Cord Blood
Authors: Kanof Marjorie E. , Smith Phillip D. , Zola Heddy
Published: June 2004

Peripheral blood is the primary source of lymphoid cells for investigations of the human immune system. Its use is facilitated by the Ficoll‐Hypaque density gradient centrifugation method described here. It is a simple and rapid method of purifying peripheral blood mononuclear cells (PBMC) that takes advantage of the density differences between mononuclear cells and other elements found in the blood sample. The mononuclear cell sample can be purified from monocytes by adherence or by exposure to L‐leucine methyl ester; methods are described for both procedures. Cord blood and peripheral blood from infants contain immature cells, including nucleated red cells, that can result in significant contamination of the mononuclear cell layer, and removal of these cells requires additional steps that are described. The isolation procedures presented here can also be applied to cell populations derived from tissues.
Purification of T Cell Subpopulations
Authors: Kanof Marjorie E.
Published: June 2004

This unit describes a procedure for isolating T cell populations or subpopulations using the method of indirect panning. In this method, cells are selected by their capacity to bind to antibody‐coated plates on the basis of particular cell‐surface markers. It is superior to the antibody/complement lysis method (also presented) because the nonselected cell population can be retrieved.
Isolation of Mononuclear Cells From Tonsillar Tissue
Authors: Ryon Judith J.
Published: June 2004

This unit describes a protocol for the derivation of lymphoid cell populations from tonsillar tissue. The procedure can also be applied with little or no modification to spleen and lymph node tissue. Human tonsils are the most readily available lymphoid organs and are often used as a source of large numbers of cells characteristic of local lymphoid tissue. Mononuclear cells are isolated by mechanical disruption of tonsil tissue followed by Ficoll‐Hypaque gradient centrifugation. Cells may then be separated into subpopulations according to general protocols used for peripheral blood lymphocytes.
Measurement of Immunoglobulin Synthesis Using the ELISPOT Assay
Authors: Lycke Nils Y. , Coico Richard
Published: June 2004

The enzyme‐linked immunospot (ELISPOT) assay for detection of single antibody‐secreting cells has become the best alternative method to the conventional plaque‐forming cell (PFC) assays. Among its several advantages are better antigen stability and specificity as well as fewer limitations in the diversity of antigens that can be used in the assay. In addition, the ELISPOT assay can be used to detect two antigenically different secreted antibodies simultaneously by two‐color analysis and offers the unique possibility of quantifying the number of antibody molecules secreted per cell. Finally, the assay can be used to detect single antibody‐secreting cells in tissues that usually confront the immunologist with difficulties, e.g., gut lamina propria from humans or mice. This unit presents the ELISPOT assay in three steps: coating of antigen to a solid phase, incubation of antibody‐producing cells in appropriate dilution, and detection of the antigen‐antibody complex formed at the site of the active antibody‐secreting cell. The assay can be performed using polystyrene plates or nitrocellulose membrane in microtiter plates or using nitrocellulose membrane in a blotting manifold.
Measurement of Cellular Adhesion Under Static Conditions
Authors: Mobley James L. , Shimizu Yoji
Published: June 2004

This unit describes procedures for measuring the adhesion of lymphoid cells to immobilized ligands or adherent cell monolayers. The basic protocol, which is optimized for human T cells, can be easily modified to study almost any cell type in suspension if the corresponding adhesive ligand is available. Alternate protocols describe modifications to the basic protocol that allow analysis of cell adhesion in the presence of stimulators of lymphocyte activation or monoclonal antibodies (MAbs) that react with adhesion molecules or their ligands, or analysis of cell adhesion to adherent cell monolayers. A support protocol is provided for determining the optimal concentration of adhesion ligands and choosing the appropriate type of microtiter plate for static adhesion assays.
Isolation of Human Eosinophils
Authors: Lim Kaiser G. , Weller Peter F.
Published: June 2004

Highly purified eosinophils can be isolated from peripheral blood by negative selection using anti‐CD16 immunomagnetic beads. The basic protocol describes a sequential fractionation of peripheral blood in which eosinophils are separated first from mononuclear cells and then from CD16+ neutrophils. This technique is easy to use, fast, and highly reproducible. A support protocol describes a staining method that can be used to evaluate the purity of eosinphils.
Immunoaffinity Chromatography
Authors: Springer Timothy A.
Published: June 2004

This unit describes the isolation of soluble or membrane‐bound protein antigens from cells or homogenized tissue by immunoaffinity chromatography. The technique involves the elution of a single protein from an immunoaffinity column after prior elution of nonspecifically adsorbed proteins. To elute the bound antigen from the immunoaffinity matrix, the antibody‐antigen interaction is destabilized by brief exposure to high‐pH or low‐pH buffer. The use of batch purification of antigens is an alternate procedure and results in shorter column loading times. The detergent octyl β‐D‐glucoside can be used instead of Triton X‐100 for elution. Because octyl β‐D‐glucoside has a high critical micelle concentration (CMC), it can be readily removed by dialysis, as described.
Electroelution of Proteins from Stained Gels
Authors: Smith John A.
Published: June 2004

In this procedure an SDS‐polyacrylamide gel containing electrophoresed proteins is stained with Coomassie brilliant blue, the gel band of interest is excised, and the stained protein is electrophoretically eluted from the minced gel. After removal of most of the salt and sodium dodecyl sulfate (SDS) by dialysis, the protein is precipitated to remove remaining detergent and salt and then analyzed.
Phage Display Selection and Analysis of Ab‐Binding Epitopes
Authors: Enshell‐Seijffers David , Gershoni Jonathan M.
Published: June 2004

The identification and characterization of B cell epitopes by combinatorial phage display peptide analyses is based on the principle that unique peptides can be affinity‐purified from an enormous collection of random peptides. Moreover, once selected, the peptide sequence can be elucidated; filamentous bacteriophages have been genetically engineered to incorporate the DNA template corresponding to the peptide displayed on its surface. This unit begins with a discussion of some of the factors that distinguish available libraries. Protocols are then provided for affinity selection of antibody‐specific phages, determination of phage titer, confirmation and amplification of positive phages, phage characterization, and construction of custom‐tailored phages. The selection protocol in this unit is specific and designed for libraries that are used in the authors' laboratory and are based on the fth1 or fd‐tet derived vectors. However, information is included for adapting these protocols to the specific requirements of other phage display libraries.
Preparation of Genomic DNA from Mammalian Tissue
Authors: Strauss William.M.
Published: June 2004

There are a number of different procedures for the preparation of genomic DNA. They all start with some form of cell lysis, followed by deproteinization and recovery of DNA. The main differences between various approaches lie in the extent of deproteinization and in molecular weight of the DNA produced. The isolation procedure described here is relatively brief and relies on the powerful proteolytic activity of proteinase K combined with the denaturing ability of the ionic detergent SDS.
Reagents and Radioisotopes Used to Manipulate Nucleic Acids
Authors: Struhl Kevin
Published: June 2004

The application of molecular biological techniques to immunological studies is based in large part on the manipulation of nucleic acids‐‐RNA, DNA, and oligonucleotides. This unit offers very basic procedures that are used in a number of protocols later in the chapter. In particular, preparation of nucleoside triphosphates‐‐the substrates for almost all synthetic reactions using nucleic acids‐‐is described; the relevant radioisotopes, 32P, 35S, and 3H are discussed; and methods for quantitating radioisotope and biotin incorporation into nucleic acids are presented. Finally, column chromatographic (gel filtration) procedures for separating labeled DNA from synthetic precursors are described.
Isolation and Use of Rafts
Authors: Brown Deborah A.
Published: June 2004

This unit describes methods for isolating and analyzing rafts by detergent insolubility. To distinguish these rafts from raft‐like membranes isolated by other methods, they are referred to here as detergent‐resistant membranes (DRMs). DRMs can be isolated by flotation on sucrose density gradients or by pelleting after detergent extraction. DRM proteins can be analyzed by SDS‐PAGE and immunoblotting. Additionally, radiolabeled DRM proteins can be analyzed, and lipids can be quantitated by high‐performance thin layer chromatography. Support protocols needed for the lipid analysis are also provided. Finally, protocols for raft disruption by cholesterol removal and measuring the kinetics of such removal are included together with a method that reverses the cholesterol removal (cholesterol repletion).
Measurement of Fcγ Receptor–Mediated Binding and Phagocytosis
Authors: Politis Alexander D. , Vogel Stefanie N.
Published: June 2004

Macrophages are perhaps best recognized for their ability to phagocytose foreign particles. Phagocytosis can be nonspecific, but is often dramatically augmented with the aid of receptors that recognize the Fc portion of antibodies bound to the intruder. Macrophages utilize receptors that recognize IgG (FcγR), which is a major opsonic mediator of phagocytosis. This unit describes two functional assays that quantify macrophage FcγR surface expression. In each assay, IgG‐opsonized sheep red blood cells (SRBC) labeled with Na251CrO4 are used to detect interaction with macrophages. The is used to measure phagocytosis of target cells. The is used to measure FcγR‐mediated binding of target cells to macrophages. A choice of opsonin in both methods allows for some discrimination between subclasses of FcγRs that are responsible for the observed results.
Measurement of Interferon‐Mediated Antiviral Activity of Macrophages
Authors: Falk Lydia A.
Published: June 2004

Macrophages have been shown to produce interferons (IFNs) that exert an antiviral effect‐‐resistance to the cytopathic effect (CPE) of viral infection‐‐on sensitive cell types such as fibroblasts. In some cases, IFN levels in macrophage supernatants may be below the limits of detection but still may be sufficient to confer antiviral activity on macrophages. This unit provides an assay for the qualitative demonstration of the presence or absence of endogenous IFN production in murine macrophages by measuring the resistance of macrophages to the CPE of infection with vesicular stomatitis virus (VSV).
Animal Models of Airway Sensitization
Authors: Hamelmann Eckard , Gelfand Erwin W.
Published: June 2004

Airway inflammation and hyperresponsiveness (AHR) are hallmarks and distinguishing features of bronchial asthma. This unit contains protocols that employ a mouse model for the study of allergen‐induced AHR. It includes methods for sensitizing mice to allergens via the airways and assessing tracheal smooth muscle reactivity in vitro and in vivo. Protocols to measure and evaluate immunological mechanisms that contribute to the development to allergen‐induced asthma are also provided.
Presenting Exogenous Antigen to T Cells
Authors: Harding Clifford V.
Published: June 2004

Antigen‐processing experiments can be performed with a wide variety of antigen‐presenting cells (APCs). An important technical distinction for some types of experiments is whether the APCs are adherent or nonadherent, since this dictates the procedures that must be used to wash the cells as the medium is changed. This unit describes approaches that can be used for adherent cells (e.g., macrophages or fibroblasts) or nonadherent cells (e.g., B lymphoma cell lines), or both, using similar cell numbers. The protocols utilize T hybridomas to detect expression of peptide‐MHC complexes, since these cells provide the most convenient, consistent, and flexible T cell readout systems for these purposes. If desired, antigen‐specific T cell clones can be used in lieu of T hybridoma cells, but T cell clones often give poorer responses than T hybridomas to fixed APCs due to fixation‐induced loss of costimulator function.
Measurement of Peptide‐MHC Interactions in Solution Using the Spin Column Filtration Assay
Authors: Buus Søren , Lise Lauemøller Sanne , Stryhn Anette , Østergaard Pedersen Lars
Published: June 2004

This unit describes how peptide‐MHC complexes can be generated in vitro using affinity‐purified MHC and synthetic peptide. The unit first describes how the interaction between peptide and MHC interaction can be measured in an accurate, quantitative biochemical assay. This procedure has been optimized for efficient separation of free peptide and MHC‐bound peptide through a novel principle, termed “gradient centrifugation.” The first two support protocols describe how to set up a biochemical fluid‐phase binding reaction between peptide and MHC class I and class II, respectively. Also, an alternative procedure for setting up a biochemical fluid phase binding reaction between β2m and MHC class I is included. Finally a more versatile inhibition assay is described. The assay is simple and robust, and has several advantages compared to the classical gel‐filtration assay, including increased sensitivity and throughput. It also demands fewer resources both in terms of unique reagents and labor, and it generates less hazardous waste. Thus, the spin column gel‐filtration assay is ideal for routine work.
Animal Models for the Analysis of Immune Responses to Leishmaniasis
Authors: Sacks David L. , Melby Peter C.
Published: June 2004

This unit focuses on the murine model of cutaneous leishmaniasis and models of visceral leishmaniasis in mice and hamsters. Each basic protocol describes the methods used to inoculate parasites and to evaluate infections with regard to lesion progression and visceralization, and quantification of parasite load. Five support protocols are provided; two for the growth and isolation of metacyclic promastigotes from in vitro culture, one for isolation of tissue amastigotes from infected animals, one for cryopreservation of parasites, and one for the preparation of blood agar plates for quantitation of parasite numbers in infected tissue.
Animal Model for Infection with Listeria monocytogenes
Authors: Busch Dirk H. , Vijh Sujata , Pamer Eric G.
Published: June 2004

This unit describes methods for infecting mice with L. monocytogenes. Optimal media for growth and methods to maintain bacterial virulence by passage through mice are included. Methods for determining the severity of splenic and hepatic infection are detailed, with strategies for distinguishing innate from specific immune responses following L. monocytogenes infection. This infection induces MHC class I‐restricted CD8+ cytolytic T lymphocytes that clear infection and provide long‐term immunity. This unit describes methods that can be used for in vitro expansion of L. monocytogenes‐specific T cells.
Animal Model of Nippostrongylus brasiliensis and Heligmosomoides polygyrus
Authors: Camberis Mali , Le Gros Graham , Urban Joseph
Published: June 2004

Animal models of Nippostrongylus brasiliensis and Heligmosomoides polygyrus infection are powerful tools for the investigation of the basic biology of immune responses and protective immunity. In particular, they model the induction and maintenance of Th2 type immune responses and exhibit all the requisite hallmarks of CD4 T cell–dependent IgE production, eosinophilia, mastocytosis, and mucus production. This chapter describes simple, cost‐effective techniques for using and maintaining these easy‐to‐work‐with parasites in the context of a modern laboratory.
The P815 Mastocytoma Tumor Model
Authors: Gajewski Thomas F. , Markiewicz Mary A. , Uyttenhove Catherine
Published: June 2004

This unit presents an experimental tumor model which has led to pivotal advances in tumor immunology culminating in the preclinical development of human cancer vaccines for melanoma. The model employs the use of the P815 mastocytoma cell line. Although the P815 cell line belongs to the mast cell lineage, it offers several advantages for in vivo experimentation of the tumor‐host relationship. It grows progressively in the majority of syngeneic DBA/2 mice and can be implanted either intraperitoneally or subcutaneously. Moreover, immunogeneic variants have been created yielding tumors that are spontaneously rejected BB a behavior that has provided a context in which to study the immunologically relevant molecules and cells that dictate a successful anti‐tumor response.
The TRAMP Mouse as a Model for Prostate Cancer
Authors: Hurwitz Arthur A. , Foster Barbara A. , Allison James P. , Greenberg Norman M. , Kwon Eugene D.
Published: June 2004

The transgenic adenocarcinoma of the mouse prostate (TRAMP) model closely mirrors the pathogenesis of human prostate cancer. Male TRAMP mice uniformly and spontaneously develop autochthonous (orthotopic) prostate tumors following the onset of puberty. Prostate cancer occurs consequent to the expression of SV40 T antigen. The versatility of the TRAMP model has been extended by establishment of several TRAMP‐derived prostate tumor cells lines that can be injected into syngeneic male nontransgenic C57BL/6 hosts to induce ectopic prostate tumorigenesis. Subcutaneous tumor induction using the TRAMP‐C cell lines has provided the basis for two additional murine models, the first of which can be used for rapid screening of experimental therapies for the treatment of primary prostate tumors and the second for testing the effectiveness of adjunctive therapies targeting prostate cancer metastases. Detailed descriptions for the harvesting and microdissection of TRAMP prostates and tumors, and the evaluation and scoring of tumors are also provided.
Peptide Binding Motifs for MHC Class I and II Molecules
Authors: Biddison William E. , Martin Roland
Published: June 2004

This overview discusses the use of peptide‐bindnig motifs to predict interaction with a specific MHC class I or II allele, and gives examples for the use of MHC binding motifs to predict T‐cell recognition.
Nomenclature of the Human Immunoglobulin Genes
Authors: Lefranc Marie‐Paule
Published: June 2004

The human immunoglobulins (Ig) are the products of three unlinked sets of genes: the immunoglobulin heavy (IGH), the immunoglobulin κ (IGK), and the immunoglobulin λ (IGL) genes, localized on chromosome 14 (14q32.33), 2 (2p12), and 22 (22q11.2), respectively. This appendix presents tabulated lists of the human immunoglobulin heavy, κ, and λ genes named in accordance with the International ImMunoGeneTics database (IMGT) and approved by the Human Genome Organization (HUGO) Nomenclature Committee in 1999. Three additional tables list corresponding nomenclatures for these genes.
Detection and Quantitation of Radiolabeled Proteins and DNA in Gels and Blots
Authors: Voytas Daniel , Ke Ning
Published: June 2004

This appendix presents procedures for visualizing and quantitating radiolabeled proteins or DNA separated by polyacrylamide gel electrophoresis or affixed to filter membranes. Autoradiography is the most common method by which this is accomplished; X‐ray film is the traditional recording medium. Film images can be analyzed by densitometry as described to obtain a quantitative measure of the radioactivity in the sample. The use of X‐ray films for autoradiography, however, suffers from lack of sensitivity and limited linear range. Protocols are provided for overcoming these limitations using fluorography and intensifying screens, both of which enhance the radioactive signal. Sensitivity and linear ranges of measurement can be greatly extended by using a phosphor imaging system, which also makes it much faster and easier to quantify radioactive samples.
Monoclonal Antibodies to Human Cell Surface Antigens
Authors: Stockinger Hannes , Majdic Otto , Knapp Walter , Swart Bernadette , Nicholson Ian , Zola Heddy
Published: June 2004

This appendix lists the cluster of differentiation (CD) designations for the nearly 300 different leukocyte surface molecules that have been assigned and approved by the nomenclature committee of the International Union of Immunological societies (IUIS).
Pulsed‐Field Gel Electrophoresis for Long‐Range Restriction Mapping
Authors: Gemmill Robert M. , Bolin Richard , Albertsen (LiDS prep) Hans , Tomkins Jeff P. , Wing Rod A.
Published: June 2004

This unit describes procedures for generating long‐range restriction maps of genomic DNA and for analysis of large insert clones. The basic protocol details restriction digestion of agarose‐embedded DNA, PFGE separation, Southern transfer, and hybridization. Support protocols describe the preparation of high‐molecular‐weight genomic DNA samples in agarose blocks and in agarose microbeads, respectively. Additional support protocols describe the preparation of DNA size standards from l phage and two yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe. An alternative method of preparing S. cerevisiae size standards using lithium dodecyl sulfate (LiDS) solubilization is provided. The final protocol details the preparation of BAC DNA suitable for digestion, mapping, and sequencing.
Gene Identification: Methods and Considerations
Authors: Baxevanis Andreas D.
Published: June 2004

This unit introduces readers to some of the more commonly used techniques for gene identification. The author discusses the general problem behind accurately predicting genes in both prefinished and finished sequence data, provides a handson description of programs available in the public domain, and suggests strategies for how to best tackle the prediction problem at various stages of data generation and assembly.This unit introduces readers to some of the more commonly used techniques for gene identification.
Sequence Similarity Searching Using the BLAST Family of Programs
Authors: Wolfsberg Tyra G. , Madden Thomas L.
Published: June 2004

Database sequence similarity searching is carried out thousands of times each day by researchers worldwide. This unit describes the BLAST (Basic Local Alignment Search Tool) family of sequence similarity search programs. It compares basic and advanced searches, discusses the BLAST algorithm, and presents the variety of databases that can be searched. Examples of various BLAST searches (e.g., BLASTP, BLASTX, BLASTN) are included. The unit also includes a discussion of different search strategies, such as filtering repeats and low‐complexity regions, and tips for analyzing genomic DNA, mRNA, and short sequences. Two unit appendices detail the BLAST parameters and sequence identifier syntax.
Accessing the Human Genome
Authors: Church Deanna , Pruitt Kim D.
Published: June 2004

The majority of the sequence for the human genome is now available. Regardless of the researcher's area of interest, it is quite likely that they will want to use some aspect of this data. This unit helps researchers achieve that goal. It presents the gene models available at NCBI, the UCSC Genome Browser, and Ensembl. It reviews the features and options available from the three web sites to query, display, and download the data. In addition, the unit illustrates how to query each of the databases in order to identify information, such as the genomic location of a novel cDNA, a BAC clone that contains a particular gene, and homologous human genes to a particular protein sequence from a different organism.
Preparation, Culture, and Analysis of Amniotic Fluid Samples
Authors: Meron Patricia Minehart
Published: June 2004

The describes an in situ method for the preparation, culture, harvest, and analysis of amniotic fluid samples. In this procedure, cells are grown and harvested for metaphase spreads on glass coverslips. The metaphase spreads are analyzed without being removed from the coverslips. describes a harvesting technique that yields higher‐resolution banding. describes a method for growing more cells by passaging cells from a backup monolayer culture. An describes a flask method for the preparation, culture, harvest, and analysis of amniotic fluid samples that can be used instead of the in situ method. Accompanying is that describes subculturing cells from flasks.
Multiplex PCR for Identifying Dystrophin Gene Deletions
Authors: Beggs Alan H.
Published: June 2004

The identification of dystrophin as the defective protein in patients with Duchenne and Becker muscular dystrophies (DMD and BMD) has allowed the development of sensitive and specific tests to establish a diagnosis and to aid in genetic counseling and prenatal diagnosis. The describes three complementary multiplex PCR assays that detect 26 dystrophin gene exons. The describes preparation and storage of stock PCR reaction mixes with primers for each of the three diagnostic assays. The is a modification of the for radioactive detection of duplications in males and deletions in carrier females.The identification of dystrophin as the defective protein in patients with Duchenne and Becker muscular dystrophies
Metaphase Harvest and Cytogenetic Analysis of Malignant Hematological Specimens
Authors: Cin Paola Dal
Published: June 2004

Cytogenetic analysis of malignant hematological disease is an important methodology used by clinicians and researchers, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance. The describes the culture, harvest, and preparation of chromosome spreads from bone marrow aspirates. Five alternate protocols describe adaptations of the for preparation of specimens from chronic lymphocytic leukemia (CLL) bone marrow, leukemic peripheral blood, CLL peripheral blood, post‐bone marrow transplant (post‐BMT) peripheral blood, and aplastic anemia bone marrow and peripheral blood. The modifications involve slightly different culture and harvest methods that are necessary to assure optimal results.
Oligonucleotide Arrays for Expression Monitoring
Authors: Byrne Michael C. , Whitley Maryann Z. , Follettie Maximillian T.
Published: June 2004

This unit provides protocols for the amplification and labeling of mRNA (and the necessary controls) for hybridization to oligonucleotide arrays. It also describes methods for processing and normalizing the raw gene expression data in preparation for clustering and further analysis.
Profiling Human Gene Expression with cDNA Microarrays
Authors: Jiang Yuan , Lueders John , Glatfelter Arthur , Gooden Chris , Bittner Michael
Published: June 2004

As a result of genomics initiatives worldwide, it has become increasingly easy to obtain cDNA clones representing the 3 ends of many human genes. This unit describes methods that allow these clones to be used as hybridization detectors in a highly parallel assay of gene expression. Protocols are provided for preparing cDNA microarrays, extracting RNA from cells of interest and preparing fluorescently labeled cDNA representations of the message pools, and hybridizing the labeled cDNAs to the microarrays.
Semliki Forest Virus and Sindbis Virus Vectors
Authors: Ehrengruber Markus U. , Lundstrom Kenneth
Published: June 2004

Semliki Forest virus (SFV) and Sindbis virus (SIN) are two, positive‐strand RNA viruses of the alphavirus genus. Vectors for both have been developed to express high levels of foreign genes in vitro and in vivo. describes the preparation of packaged SFV and SIN replicons by co‐electroporation of helper and vector RNA into baby hamster kidney (BHK)‐21 cells. describes the activation of packaged SFV replicons with a‐chymotrypsin. provides a method for the infection of hippocampal slices. is a technique for the infection of primary cultures of dispersed neurons with infectious SFV and SIN replicons. The describes a method for the cotransfection of in vitro‐transcribed vector and helper RNA into BHK‐21 cells. describes determining the titers of infectious SFV and SIN replicon stocks, and for metabolic labeling of infected cells.
Production of Retroviral Vectors
Authors: Miller A. Dusty
Published: June 2004

In this unit, the basic protocol generates stable cell lines, which produce retroviral vectors that carry selectable markers. applies when the retroviral vector does not carry a selectable marker. presents rapidly generating retroviral vector preparations by transient transfection. describes construction of the retrovirus vectors. The methods for generating virus from retroviral vector plasmids rely on the use of packaging cells that synthesize all of the retroviral proteins but do not produce replication‐competent virus. One packaging cell line is transiently transfected to produce virus carrying the vector RNA that can be used to infect a second packaging cell line. Virus that carries a selectable marker can be titered and viral stocks can be tested for the presence of helper virus, as described. Vectors that contain sequence for alkaline phosphatase can be assayed histochemically.In this unit, the basic protocol generates stable cell lines, which produce retroviral vectors that carry selectable markers.
Particle‐Mediated Gene Delivery In Vivo and In Vitro
Authors: Yang Ning‐Sun , Burkholder Joseph , McCabe Dennis , Neumann Veronica , Fuller Deb
Published: June 2004

Particle‐mediated or “gene gun” technology has been developed as a nonviral method for gene transfer into various mammalian tissues. Gene delivery is achieved by physical force: a strong shock wave is generated that accelerates DNA‐coated gold particles to high speeds, providing them with the momentum needed to penetrate the targeted cells. This unit describes general procedures for in vivo and in vitro DNA and RNA transfections by particle‐mediated delivery. The and an alternate protocol address in vivo delivery to mouse skin. In vitro delivery to cryopreserved and adherent cells is also described.
DNA Vaccination
Authors: Ulmer Jeffrey B. , Rappuoli Rino
Published: June 2004

This unit details some of the key methods for setting up and testing DNA vaccines in animal models. The basic procedures are discussed, as well as alternative methods that have been developed over the past several years. The gives step‐by‐step instructions for administering the DNA vaccine via intramuscular injection of the quadriceps muscle, while an alternate procedure details injection of the anterior tibialis.
Methods for Cancer Gene Therapy
Authors: Kwong Yok‐Lam , Chen Shu‐Hsia , Woo Savio L.C.
Published: June 2004

First, the testing of therapeutic gene vectors in vitro is described. This is followed by a discussion of the administration of therapeutic vectors in vivo. Two methods for assessing the development of anti‐tumor immunity after cytokine gene therapy are provided. In addition, two methods for the generation of murine tumor models in syngeneic hostsone subcutaneous and one orthotopicare also included.
Laboratory Safety
Authors: Meisenhelder Jill , Semba Kentaro , Lunn George , Strober Warren
Published: June 2004

Persons carrying out the protocols in this manual may encounter the following hazardous or potentially hazardous materials: (1) radioactive substances; (2) toxic chemicals and carcinogenic, mutagenic, or teratogenic reagents; and (3) pathogenic and infectious biological agents. Most governments regulate the use of these materials; it is essential that they be used in strict accordance with local and national regulations. Cautionary notes are included in many instances throughout the manual, and some specific guidelines are also provided in this appendix. However, users must proceed with the prudence and precautions associated with good laboratory practice, under the supervision of personnel responsible for implementing laboratory safety programs at their institutions and in compliance with designated guidelines of federal, state, and local officials.
Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy
Authors: Gallagher Sean R.
Published: June 2004

Reliable quantitation of nanogram and microgram amounts of DNA and RNA in solution is essential to researchers in molecular biology. In addition to the traditional absorbance measurements at 260 nm, two more sensitive fluorescence techniques using Hoechst 33258 and ethidium bromide are presented in this appendix. These three procedures cover a range from 5 to 10 ng/ml DNA to 50 µg/ml DNA.
Internet Basics for Biologists
Authors: Baxevanis Andreas D. , Ouellette B.F. Francis
Published: June 2004

With the explosion of sequence and structural information available to researchers, the field of bioinformatics is playing an increasingly large role in the study of fundamental biomedical problems. The challenge facing computational biologists will be to aid in gene discovery and in the design of molecular modeling, site‐directed mutagenesis, and experiments of other types that can potentially reveal previously unknown relationships with respect to the structure and function of genes and proteins. This appendix begins with a review of the Internet and its terminology, also discussing major classes of Internet protocols, without becoming overly engaged in the engineering minutiae underlying these protocols. This appendix also discusses matters of connectivity, ranging from simple modem connections to digital subscriber lines (DSL). Finally, one of the most common problems that has arisen with the proliferation of Web pages throughout the worldWith the explosion of sequence and structural information available to researchers, the field of bioinformatics is playing.
Genetic Linkage Reference Maps: Access to Internet‐Based Resources
Authors: Haines Jonathan L.
Published: June 2004

The goal of this appendix is to introduce the reader to some of the most useful reference map resources now available through the World Wide Web (WWW). The maps presented in earlier versions of this appendix have been eliminated, since they are no longer updated and the most common methods of access and retrieval of up‐to‐date reference maps are now through the WWW. It should be noted that the very nature of the WWW is dynamic change. New sites appear and old ones disappear with somewhat disruptive regularity. The sites mentioned here are the most likely to remain stable, and thus to remain useful for the human gene mapper.
Double child burial from sunghir (Russia): Pathology and inferences for upper paleolithic funerary practices
Authors: Formicola Vincenzo , Buzhilova Alexandra P.
Published: June 2004

The double child burial from Sunghir (Russia) is a spectacular Mid Upper Palaeolithic funerary example dated to about 24,000 BP. A boy (Sunghir 2) and a girl (Sunghir 3), about 12–13 and 9–10 years old, respectively, were buried at the same time, head to head, covered by red ocher and ornamented with extraordinarily rich grave goods. Examination of the two skeletons reveals that the Sunghir 3 femora are short and exhibit marked antero‐posterior bowing. The two femora do not show any asymmetry in the degree of shortening and bowing. Bowing affects the whole diaphysis and shows a regularly incurved profile, with the highest point at midshaft. Pathology is confined to the femora, and no other part of this well‐preserved specimen shows abnormality. The isolated nature of the Sunghir 3 anomalies points to cases reported in the medical literature under the label of “congenital bowing of long bones” (CBLB). These are a group of rare conditions exhibiting localized, sometimes bilateral, bowing and shortening which are nonspecific and may result from different causes, including abnormalities of the primary cartilaginous anlage (i.e., the aggregation of cells representing the first trace of an organ). Localized ossification disturbances, possibly linked to a diabetic maternal condition, might explain the shortening and the coincidence of maximum midshaft curvature with the position of the primary ossification center, as well as the lack of involvement of other skeletal parts. This scenario, rather than other possibilities (early bilateral midshaft fracture, acute plastic bowing deformities, or faulty fetal posture), provides the most likely explanation for the Sunghir 3 femoral deformities. The intriguing combination of a pathological condition apparent since birth with a spectacular burial of unusually positioned young individuals of different sexes recalls significant aspects of the triple burial from the contemporary site of Dolní Věstonice (Moravia), evoking a patterned relationship between physical abnormality and extraordinary Upper Paleolithic funerary behavior. Am J Phys Anthropol, 2003. © 2003 Wiley‐Liss, Inc.
Titelbild: Al50C120H180: Eine Pseudofullerenhülle aus 60 Kohlenstoffatomen und 60 Methylgruppen schützt einen Clusterkern aus 50 Aluminiumatomen (Angew. Chem. 24/2004)
Authors: Vollet Jean , Hartig Jens R. , Schnöckel Hansgeorg
Published: June 2004

Ein metalloider Al50‐Cluster ist Bestandteil der größten bisher bekannten molekularen metallorganischen Verbindung, die ausschließlich aus Metall, Kohlenstoff und Wasserstoff besteht. Eine zentrale Al8‐Einheit (im Bild blau) ist von 42 Al‐Atomen (silber) umgeben, von denen 12 einen Pentamethylcyclopentadienyl(Cp*)‐Rest tragen. Die 60 Methylgruppen der Cp*‐Liganden (rote Fünfecke) ergeben eine an einen Fußball erinnernde Oberflächentopologie. Näheres zu diesem Al50Cp${{{\ast \hfill \atop 12\hfill}}}$‐Cluster finden Sie in der Zuschrift von H. Schnöckel et al. auf S. 3248 ff.
Effects of high static magnetic field exposure on different DNAs
Authors: Potenza Lucia , Cucchiarini Luigi , Piatti Elena , Angelini Umberta , Dachà Marina
Published: June 2004

The effects of magnetic fields produced by permanent magnets on different DNA sources were investigated in vivo and in vitro. Escherichia coli DNA, plasmid, and amplification products of different lengths were used as the magnetic field target. The in vivo assays did not reveal any DNA alterations following exposure, demonstrating the presence of cell dependent mechanisms, such as the repair system and the buffering action of the heat shock proteins DNA K/J (Hsp 70/40). The in vitro assays displayed interactions between the magnetic field and DNA, revealing principally that magnetic field exposure induces DNA alterations in terms of point mutations. We speculate that the magnetic field can perturb DNA stability interacting with DNA directly or potentiating the activity of oxidant radicals. This genotoxic effect of the magnetic field, however, is minimized in living organisms due to the presence of protective cellular responses. Bioelectromagnetics 25:352–355, 2004. © 2004 Wiley‐Liss, Inc.
0.2 T magnetic field inhibits angiogenesis in chick embryo chorioallantoic membrane
Authors: Ruggiero Marco , Bottaro Donald P. , Liguri Gianfranco , Gulisano Massimo , Peruzzi Benedetta , Pacini Stefania
Published: June 2004

Inhibition of angiogenesis is a major target in the fight against cancer and other diseases. Although the effects of static magnetic fields on cancer development and cell growth have been investigated, effects on angiogenesis have received no attention so far. In this study we report the effects on angiogenesis of exposure to 0.2 T static magnetic field. Angiogenesis was analyzed using the chick embryo chorioallantoic membrane assay. Exposure to 0.2 T static magnetic field was achieved by placing the eggs for 3 hr in the isocentre of the magnet of a sectorial magnetic resonance tomograph used in clinical practice. In sham exposed specimens treated with phosphate buffered saline (negative control), no significant vascular reaction was detectable; 3 hr exposure to 0.2 T static magnetic field did not affect the basal pattern of vascularization or chick embryo viability. Prostaglandin E1 and fetal calf serum elicited a strong angiogenic response in sham exposed eggs. This angiogenic response was significantly inhibited by 3 hr exposure to 0.2 T static magnetic field. These findings point to possible use of static magnetic field in inhibiting angiogenesis; this effect could be exploited for treatment of cancer and other diseases where excessive angiogenesis is involved. Bioelectromagnetics 25:390–396, 2004. © 2004 Wiley‐Liss, Inc.
A Series of Redox Active, Tetrathiafulvalene‐Based Amidopyridines and Bipyridines Ligands: Syntheses, Crystal Structures, a Radical Cation Salt and Group 10 Transition‐Metal Complexes
Authors: Devic Thomas , Avarvari Narcis , Batail Patrick
Published: June 2004

Amidopyridine and −2,2′‐bipyridine derivatives of EDT‐TTF and BTM‐TTF (EDT=ethylenedithio, BTM=bis(thiomethyl), TTF=tetrathiafulvalene) have been synthesized and crystallographically characterized. In the solid state, the different supramolecular organization of all these donors results from the competition between the intermolecular interactions, that is, van der Waals, hydrogen‐bonding, π–π stacking, and donor–acceptor interactions. The electron‐donating properties of the new donors have been investigated by cyclic voltammetry measurements. A radical cation salt, formulated [EDT‐TTF‐CONH‐m‐Py].+[PF6]−, has been prepared by electrocrystallization and its crystal structure determined by X‐ray analysis. Square planar dicationic complexes with the same donor and MIIL2 fragments (M=Pd, Pt, L2=bis(diphenylphosphino)propane (dppp) or bis(diphenylphosphino)ethane (dppe)) have been synthesized and one of them, containing the Pd(dppp) unit, has been structurally characterized. The conformation of the complex in the crystalline state is anti, with the coexistence of the dl racemic pair of enantiomers.
Ruthenium Complexes of Easily Accessible Tridentate Ligands Based on the 2‐Aryl‐4,6‐bis(2‐pyridyl)‐s‐triazine Motif: Absorption Spectra, Luminescence Properties, and Redox Behavior
Authors: Polson Matthew I. J. , Medlycott Elaine A. , Hanan Garry S. , Mikelsons Larisa , Taylor Nick J. , Watanabe Masashi , Tanaka Yasutaka , Loiseau Frédérique , Passalacqua Rosalba , Campagna Sebastiano
Published: June 2004

A family of tridendate ligands 1 a–e, based on the 2‐aryl‐4,6‐di(2‐pyridyl)‐s‐triazine motif, was prepared along with their hetero‐ and homoleptic RuII complexes 2 a–e ([Ru(tpy)(1 a–e)]2+; tpy=2,2′:6′,2′′‐terpyridine) and 3 a–e ([(Ru(1 a–e)2]2+), respectively. The ligands and their complexes were characterized by 1H NMR spectroscopy, ES‐MS, and elemental analysis. Single‐crystal X‐ray analysis of 2 a and 2 e demonstrated that the triazine core is nearly coplanar with the non‐coordinating ring, with dihedral angles of 1.2 and 18.6°, respectively. The redox behavior and electronic absorption and luminescence properties (both at room temperature in liquid acetonitrile and at 77 K in butyronitrile rigid matrix) were investigated. Each species undergoes one oxidation process centered on the metal ion, and several (three for 2 a–e and four for 3 a–e) reduction processes centered on the ligand orbitals. All compounds exhibit intense absorption bands in the UV region, assigned to spin‐allowed ligand‐centered (LC) transitions, and moderately intense spin‐allowed metal‐to‐ligand charge‐transfer (MLCT) absorption bands in the visible region. The compounds exhibit relatively intense emissions, originating from triplet MLCT levels, both at 77 K and at room temperature. The incorporation of triazine rings and the near planarity of the noncoordinating ring increase the luminescence lifetimes of the complexes by lowering the energy of the 3MLCT state and creating a large energy gap to the dd state.
Preparation of Tripeptide‐Bridged Dicatechol Ligands and Their Macrocyclic Molybdenum(VI) Complexes: Fixation of the RGD Sequence and the WKY Sequence of Urotensin II in a Cyclic Conformation
Authors: Albrecht Markus , Stortz Patrick , Runsink Jan , Weis Patrick
Published: June 2004

Dicatechol ligands were prepared with caprylic acid (6‐H4) or the naturally occurring RGD (23‐H4) or WKY sequences (32‐H4) as spacers. 6‐H4 was prepared by solution‐phase amide coupling chemistry, while 16, the precursor of 23‐H4, was obtained by solution‐phase and solid‐phase preparation. In the latter case, a polystyrene resin with a hydrazine benzoate linker was used as the solid support. The last coupling step was performed simultaneously with cleavage of the peptide from the resin. The protecting groups of 16 were all removed in one step to yield the free ligand 23‐H4. The WKY‐bridged derivative 32‐H4 was obtained by a similar solid‐phase synthesis followed by deprotection. The reaction of all three ligands with dioxomolybdenum(VI) bis(acetylacetonate) afforded 19‐membered metallamacrocycles in which the short peptides are conformationally fixed in a turn‐type structure. Hereby, the side‐chain functionalities of the peptides do not interfere in the metal complexation.
Cover Picture: Self‐Assembled Monolayers of Dendritic Polyglycerol Derivatives on Gold That Resist the Adsorption of Proteins (Chem. Eur. J. 11/2004)
Authors: Siegers Conrad , Biesalski Markus , Haag Rainer
Published: June 2004

The cover picture shows the formation of a self‐assembled monolayer of dendritic polyglycerol on a semitransparent gold‐film and its high protein resistant properties. The polyglycerol derivative is readily obtained by ester coupling with thioctic acid, which provides a stable covalent linker to the gold surface. Several dendritic polyglycerol derivatives as well as the monomer equivalent (thioglycerol) were screened for their protein resistant properties with a fibrinogen solution by surface plasmon resonance spectroscopy (SPR). The excellent protein resistant properties and the high thermal stability of this new material, which classifies for the coating of biomedical devices, is reported in detail by R. Haag et al. on p. 2831 ff.
GaAs5 and InAs5: Quantum‐Chemical and Experimental Investigations of Their Stability and Structure.
Authors: Koeppe Ralf , Schnoeckel Hansgeorg
Published: June 2004

For Abstract see ChemInform Abstract in Full Text.
Unusual Electronic and Bonding Properties of the Zintl Phase Ca5Ge3 and Related Compounds. A Theoretical Analysis
Authors: Mudring Anja‐Verena , Corbett John D.
Published: June 2004

For Abstract see ChemInform Abstract in Full Text.