bluuebunny/crossref_metadata_2025 huggingface dataset:
Establishment of Fibroblast Cultures
Authors: Takashima Akira
Published: June 2004

Fibroblasts are fairly easily isolated from a variety of tissues, in this case skin. The cells are fast growing and can be rapidly expanded from small samples. They are suitable for behavioral, functional, biochemical, and genomic studies. Skin fibroblast cultures are started from explants in which the epidermis is enzymatically removed to prevent contamination of the fibroblasts with epidermal cells. Alternatively, the dermis can be enzymatically dissociated to provide a suspension of fibroblasts.
Preparation of Endothelial Cells
Authors: Kleinman Hynda K. , Cid Maria C.
Published: June 2004

Endothelial cells, which line blood vessels, can be prepared from a variety of tissues. They are frequently prepared from the umbilical vein, which is relatively easy to obtain. The procedure is clearly described and provides a large population of highly purified endothelial cells. There are also methods for obtaining endothelial cells from other tissues such as fat, skin, and mucosa. These methods require special care and generate smaller populations of cells.
Generation of Continuously Growing B Cell Lines by Epstein‐Barr Virus Transformation
Authors: Biddison William E.
Published: June 2004

Primary cultures of cells tend to have a limited life span, which in turn limits the availability of a consistent population of cells to study. In this unit, Epstein‐Barr virus produced by a marmoset cell line is used to transform B cells into a continuously growing cell line that produces no virus. These cell lines can be used as a constant source of cells for further study.
Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient Centrifugation
Authors: Graham John M.
Published: June 2004

This unit covers the use of Percoll, Nycodenz, and iodixanol for purification of lysosomes from a light mitochondrial pellet or postnuclear supernatant. The first protocol describes isolation of lysosomes from rat liver using a Percoll gradient; it includes some comments on using brain and kidney tissue as source material. Alternatively, this protocol can be modified to enhance the separation of lysosomes by organelle density perturbation. Another alternative uses a discontinuous gradient of Nycodenz to purify lysosomes from a rat liver light mitochondrial pellet. A continuous iodixanol gradient, which can be used to purify other organelles in the light mitochondrial fraction, is yet another alternative for purifying lysosomes. Lysosomes can also be isolated from some of the more commonly used cultured cells. The unit also includes assays for common lysosome marker enzymes.
Proper Alignment and Adjustment of the Light Microscope
Authors: Salmon Edward D. , Canman Julie C.
Published: June 2004

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications (e.g., brightfield, phase‐contrast, differential interference contrast, and fluorescence epi‐illumination), and how it should be maintained as required to obtain maximum image‐forming capacity and resolution. The principles of microscopy and step‐by‐step alignment and adjustment procedures are described in this unit.
Fluorescence Microscopy
Authors: Herman Brian
Published: June 2004

Absorption of energy as light by some molecules and emission of energy as fluorescence can occur only at certain wavelengths, which are characteristic for a given molecule (fluorophore). Fluorescence microscopes are equipped to observe the fluorescence of one or more specific fluorophores to localize specific molecules and analyzed cellular structures. This unit discusses the optics and other components of the fluorescence microscope, their functions and adjustments, and the digital darkroom.
Immunofluorescence Staining
Authors: Donaldson Julie G.
Published: June 2004

This unit provides a protocol for indirect immunofluorescence, which is a method that provides information about the locations of specific molecules and the structure of the cell. Antibody molecules for a specific target molecule are exposed to the cell or tissue being investigated. The binding of these molecules is detected by incubating the sample with a secondary antibody specific for immunoglobulin molecules and conjugated to fluorophore. This provides both a visible signal and amplification of the signal and the results are observed with a fluorescent microscope.
Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria
Authors: Terasaki Mark , Loew Leslie , Lippincott‐Schwartz Jennifer , Zaal Kristien
Published: June 2004

The ability to distinguish and identify specific subcellular compartments is essential to understanding organelle function, biogenesis, and maintenance within cells and to defining protein trafficking pathways. Fluorescent dyes and/or fluorescently labeled lipid derivatives can be used to identify ER, Golgi complex, and mitochondria. Specific conditions for labeling each of these compartments are described.
Cryo‐Immunogold Electron Microscopy
Authors: Peters Peter J.
Published: June 2004

This unit describes subcellular localization of proteins/antigens using high‐resolution cryo‐immunogold electron microscopy, which allows study of topological biochemistry at the ultrastructural level. This is the most sensitive procedure for immunodetection of antigens on ultrathin sections prepared from chemically fixed cells or tissues, since aldehyde fixation is the only denaturation step. The omission of harsh organic solvents (such as those used for plastic embedding) ensures better preservation of protein antigenicity. Support protocols describe how to embed fixed material in gelatin, cryosection, and mount the sections on Formvar‐coated grids.
Polarization Microscopy
Authors: Inoué Shinya
Published: June 2004

This unit provides an introduction to polarization microscopy, the optics involved, and practical considerations for observing submicroscopic structures. The unit also includes specific examples for visualization of microtubules in the mitotic spindle, chromatin within maturing spermatids, and the biocrystalline skeletal spicules in larval echinoderms.
Two‐Photon Excitation Microscopy for the Study of Living Cells and Tissues
Authors: Rocheleau Jonathan V. , Piston David W.
Published: June 2004

Two‐photon excitation microscopy is an alternative to confocal microscopy that provides advantages in three‐dimensional and deep tissue imaging. This unit will describe the basic physical principles of two‐photon excitation and discuss the advantages and limitations of its use in laser‐scanning microscopy. The advantages of two‐photon microscopy are reduced phototoxicity, increased imaging depth, and the ability to initiate localized photochemistry.
Two‐Dimensional Gel Electrophoresis
Authors: Harper Sandra , Mozdzanowski Jacek , Speicher David
Published: June 2004

While one‐dimensional SDS‐PAGE separates proteins on the basis of size, two‐dimensional gel electrophoresis separates proteins first on the basis of isoelectric point, then on the basis of size. This method is capable of resolving 1000 to 2000 separate proteins when combined with sensitive detection methods. This unit describes methods for characterizing cell lysates by two‐dimensional gel electrophoresis, including modifications for acidic and basic proteins, the use of immobilized pH gradients, and nonreducing/reducing electrophoretic separations. In addition there are support protocols for determining pH profiles of gels, casting Immobiline gels, preparing cell and tissue samples for isoelectric focusing, preparing molecular weight standards, and using two‐dimensional protein databases.
Staining Proteins in Gels
Authors: Dell'Angelica Esteban C. , Bonifacino Juan S.
Published: June 2004

Once proteins are separated by gel electrophoresis, staining can be used to visualize the proteins. This unit presents protocols for numerous staining methods. The most common method is staining with Coomassie blue, which after washing gives blue bands on a clear background. This technique can also be applied to isoelectric focusing gels. A second, more sensitive but also more technically challenging method is silver staining. Here the proteins are seen as dark brown to black bands on a clear background. If the gel is incubated with SYPRO Ruby, a fluorescent compound that interacts specifically with proteins, the bands fluoresce when illuminated on a standard transilluminator. Finally, proteins can be reversibly stained with zinc, which precipitates the SDS from the gel leaving protein bands as clear spots against an opaque white background.
Fluorescence Detection of Glycoproteins in Gels and on Electroblots
Authors: Patton Wayne F.
Published: June 2004

Glycosylation of proteins is a dynamic post‐translational modification commonly found in eukaryotes, changing with development, differentiation, and disease state. Identification of glycoproteins is important for completely characterizing the population of proteins found at a given point in time. This unit contains protocols for identifying glycoproteins in general using a fluorescence based periodate/Schiff base method and those containing specific carbohydrates using a lectin‐based fluorescent method.
Metabolic Labeling with Amino Acids
Authors: Bonifacino Juan S.
Published: June 2004

Metabolic labeling techniques are used to study the biosynthesis, processing, intracellular transport, secretion, degradation, and physical‐chemical properties of proteins. This unit provides protocols for metabolic labeling of cells with [35S]‐labeled amino acids to provide material suitable for analysis by immunoprecipitation, for characterization of cellular proteins, for analysis of protein trafficking, and for one‐ and two‐dimensional gel electrophoresis. Three procedures are described for suspension and adherent cells: pulse‐labeling, pulse‐chase labeling, or continuous long‐term labeling. A support protocol describes TCA precipitation to measure the extent of labeling.
Metabolic Labeling with Sulfate
Authors: Tooze Sharon A.
Published: June 2004

Post‐translational modifications of proteins make it possible to determine where a protein normally resides or to follow its transport through the cell. One such modification is addition of sulfate either to tyrosine residues or to carbohydrate side chains. Labeling studies with [35S] sulfate can be done as continuous or pulse‐chase experiments, as described here.
Metabolic Labeling with Fatty Acids
Authors: Jackson Caroline S. , Magee Anthony I.
Published: June 2004

Covalent attachment of radiolabeled fatty acids (e.g., [3H]myristate or palmitate) is an alternative method for labeling proteins. This unit contains methods for biosynthetic labeling with fatty acids, analysis of the fatty acid linkage with protein, analysis of total protein‐bound fatty acid level in cell extracts, and analysis of the identity of the bound fatty acid.
Cell‐Substrate Adhesion Assays
Authors: Humphries Martin J.
Published: June 2004

In the intact organism, cells adhere to a variety of substrates. It is
possible to investigate the nature of the molecules to which cells adhere in
two different assays. In the first protocol presented in this unit, physical
adhesion of a cell is assessed by determining the extent to which the cell
spreads on a defined substrate‐‐the plate is coated with the
test substance, cells are added and allowed time to attach and spread, and
the extent of spreading is assessed using phase contrast microscopy. In a
second assay, an aliquot of cells is added to the well of a microtiter plate
coated with a test adhesion molecule and the cells are allowed to attach.
Nonattached cells are removed, and the percent of added cells attached to the
substrate is quantified by crystal violet staining.
Quantitative Measurement of Cell Adhesion Using Centrifugal Force
Authors: McClay David R. , Hertzler Philip L.
Published: June 2004

In order to study the biophysical forces involved in
cell‐substrate (or cell‐cell) adhesion, it is necessary to
measure the strength of adhesion. Two questions can be addressed using the
centrifugal cell adhesion assay provided in this unit: what is the
ligand‐receptor affinity for cells adhering at 4oC and what
is the strength of the ATP‐dependent processes that strengthen
adhesion at 37oC. In both cases, the strength of adhesion is
measured as the resistance to disruption in the presence of a measured
centrifugal force.
Analyzing Integrin‐Dependent Adhesion
Authors: Mould A. Paul
Published: June 2004

This unit describes methods for the analysis of integrin‐ligand
binding in both cell‐based assays and solid‐phase assays. A
major application of cell adhesion assays is in investigating whether a
certain cell type can adhere to a specific adhesive substrate, and, if so,
which receptors are involved. Particularly if the substrate is a matrix
component (e.g., fibronectin), members of the integrin family are likely to
play a dominant role in adhesion. Procedures are described here for assessing
which integrins are involved in this process. A detailed analysis of ligand
recognition by individual integrins can be performed using a
solid‐phase receptor‐ligand binding assay. The unit also
contains support protocols for integrin purification and coupling of
antibodies to Sepharose for use in this purification, as well as for
biotinylation of integrin ligands to be used in the solid‐phase
assay.
Measurement of Adhesion Under Flow Conditions
Authors: Kucik Dennis F.
Published: June 2004

This unit describes the analysis of dynamic cell adhesion using a flow chamber assay. The flow chamber enables the researcher to reconstruct cell systems in the presence of shear stress to assay adhesion under well&defined forces. These assays are most commonly used to study leukocyte adhesion, either to cultured endothelial cell monolayers or to purified substrates, simulating physiological interactions of leukocytes with endothelial cells. This assay can be also be used to characterize transient adhesive events or adhesion strengthening even for cells that do not normally experience shear stress, because contact time between cells and substrates and anti&adhesive forces can be closely regulated by stopping and starting the flow. Flow chamber assays are also useful for measuring bacterial adhesion under flow.
Purification of Vitronectin
Authors: Akiyama Steven K.
Published: June 2004

This unit describes the purification of extracellular vitronectin from plasma by a series of affinity chromatography steps. First plasma is depleted of fibronectin and other heparin‐binding proteins; the sample is then treated with urea to activate the heparin‐binding activity of vitronectin. The resulting vitronectin is ∼98% pure.
Proteoglycan Isolation and Analysis
Authors: Woods Anne , Couchman John R.
Published: June 2004

Proteoglycans can be difficult molecules to isolate and analyze due to large mass, charge, and tendency to aggregate or form macromolecular complexes. This unit describes detailed methods for purification of matrix, cell surface, and cytoskeleton‐linked proteoglycans. Methods for analysis of glycoaminoglycan size and type and of core protein species are described.
Matrix Metalloproteinases
Authors: Birkedal‐Hansen Henning , Yamada Susan , Windsor Jack , Poulsen Anne Havemose , Lyons Guy , Stetler‐Stevenson William , Birkedal‐Hansen Bente
Published: June 2004

Matrix metalloproteinases are a class of enzymes that play an important role in the remodeling of the extracellular matrix in development and cancer metastasis. This unit describes a set of methods‐cell‐mediated dissolution of type I collagen fibrils, direct and reverse zymography, enzyme capture based on a‐2 macroglubulin and TIMP‐1 and ‐2, and demonstration of crytic thiol groups in metalloproteinase precursors‐that are used to characterize the functions of matrix metalloproteinases and their inhibitors.
Purification and Analysis of Thrombospondin‐1
Authors: Yee Karen O. , Duquette Mark , Ludlow Anna , Lawler Jack
Published: June 2004

Thromboapondin 1 (TSP‐1) is a trimeric matricellular protein that is expressed by many cells. It contains several different domains that allow it to participate in cell adhesion, cell migration, and cell signaling. Recently TSP‐1 has been shown to activate transforming growth factor β (TGF‐β) and to inhibit both angiogenesis and tumor growth. This unit contains protocols for the purification of TSP‐1 from platelet‐rich plasma and the purification of TSP‐1 proteolytic fragments.
In Vitro Analysis of Endoplasmic‐Reticulum‐to‐Golgi Transport in Mammalian Cells
Authors: Allan Bernard B. , Balch William E.
Published: June 2004

A temperature‐sensitive mutant of vesicular stomatitis G protein is used to follow the movement of that protein from the endoplasmic reticulum to transport vesicles to cis‐Golgi and finally medial/trans‐Golgi by assessing the maturation of two asparagine‐linked oligosaccharides. These assays can be used to identify the factors that are required for and regulate protein trafficking through these compartments.
In Vitro Analysis of SV40 DNA Replication
Authors: Yin Yi‐Ling , Dutta Anindya
Published: June 2004

This unit describes a method for studying cellular factors that affect eukaryotic replication by using a plasmid that contains an SV40 origin of replication and supplying the viral helicase T antigen. Replication factors are supplied by S100 extracts of 293 cells. The unit also contains protocols for expression and purification of T antigen and analysis of the products of replication.
In Vitro Transcription
Authors: Kamakaka Rohinton T. , Kraus W. Lee
Published: June 2004

In vitro analysis of transcription and the factors that play a role in transcription require preparation of an extract that faithfully reproduces in vivo transcription. This unit describes protocols for generating extracts of mammalian cells and Drosophila embryos that are competent for in vitro transcription reactions using naked DNA or chromatin templates and for primer extension analysis of transcripts.
Analysis of RNA Export Using Xenopus Oocytes
Authors: Duricka Deborah , Ullman Katharine S.
Published: June 2004

This unit describes a procedure for monitoring RNA export in Xenopus oocytes. The technique involves synthesizing labeled RNA in vitro and microinjecting the RNA into oocyte nuclei. Following incubation the oocytes are dissected into nuclear and cytoplasmic fractions. These samples are then processed for RNA analysis, allowing the extent of export to be quantitatively assessed.
Cell Wound Assays
Authors: McNeil Paul L. , Clarke Mark F.S. , Miyake Katsuya
Published: June 2004

Many mammalian and invertebrate cells that reside in mechanically active tissue environments normally and frequently experience disruptions in plasma membrane integrity which are rapidly repaired and do not lead to cellular death. This unit describes methods for identifying “wounded” cells, often present as a minority in culture or tissues. The use of these methods can provide information about the frequency and extent of cellular wounding.
Optical Microscopy–Based Migration Assay for Human Neutrophils
Authors: Pierini Lynda M. , Maxfield Frederick R.
Published: June 2004

This unit describes an in vitro microscopy assay for examining the migration of human neutrophils in two dimensions to identify the underlying cause of a migration defect and to evaluate a variety of migration parameters that cannot be studied using migration through a porous filter. Freshly isolated human neutrophils a placed in the chamber, stimulated, and images are collected at various time points. The data can be used to determine the effects of a pharmacological treatment on the migration of individual cells.
In Vitro Motility Assay to Study Translocation of Actin by Myosin
Authors: Sellers James R.
Published: June 2004

A basic property of myosin is its ability to interact with and translocate actin. This unit describes an in vitro motility assay that can be used to study the translocation, or sliding, of actin filaments by myosin bound to a coverslip. The assay makes use of the ability to image single F‐actin filaments labeled with rhodamine phalloidin, a high‐affinity fluorescent ligand using fluorescence microscopy. The system is fast, easy to set up and maintain, uses only small amounts of protein, and yields quantitative results.
Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP
Authors: Hawes Chris , Brandizzil Federica , Batoko Henri , Moore Ian
Published: June 2004

By the addition of plant or mammalian targeting sequences, green fluorescent protein (GFP) can be directed to the endoplasmic reticulum, the Golgi apparatus, or both organelles in plant cells. This unit describes the application of rapid Agrobacterium and virus‐mediated transient expression systems in leaf tissue to permit visualization of ER and Golgi dynamics in vivo by epifluorescence or confocal microscopy.
Overview of Protein Trafficking in the Secretory and Endocytic Pathways
Authors: Enns Caroline
Published: June 2004

Proteins migrate from the site of their synthesis to intracellular, surface, or extracellular sites depending on their function. This well‐illustrated overview provides an introduction to the pathways of protein trafficking. It includes descriptions of the relevant cellular compartments, modifications associated with the movement of proteins through these compartments, signals used for targeting and translocation, mechanisms for transit between compartments, and methods for sorting proteins.
Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum
Authors: Francis Edwin , Daniels Robert , Hebert Daniel N.
Published: June 2004

Proteins that travel through the secretory pathway undergo post‐translational folding and oxidation steps that lead to correct conformation of the final protein. This unit focuses on methods for the analysis of folding and oxidation events and the factors responsible for their proper execution. Alkylation and nonreducing SDS‐PAGE is use to analyze disulfide bond formation in ER‐derived microsomes. If proteins are synthesized under reducing conditions, it is possible to initiate folding by addition of oxidizing agents, thus allowing analysis of factors necessary for the folding process. As folding progresses, the protein of interest shows a change in sensitivity to proteolysis. Co‐immunoprecipitation or crosslinking and denaturing immunoprecipitation are used to explore the role of molecular chaperones and other factors. Conformation‐specific antibodies can be used to probe folding. In addition, folding can be analyzed in intact or semi‐permeabilized adherent or suspension cells.
Analysis of Caspase Activation During Apoptosis
Authors: Kaufmann Scott H. , Kottke Timothy J. , Martins L. Miguel , Henzing Alexander J. , Earnshaw William C.
Published: June 2004

This unit describes three methods for the detection of caspase activation as cells undergo apoptosis. Simple and relatively quantitative enzymatic assays are provided using suitable substrates. Because the various low‐molecular‐weight substrates available for these assays are not selective, however, the assays do not accurately distinguish between various caspases. Immunoblotting is described for following the activation of specific caspases. When coupled with subcellular fractionation, this method can provide large amounts of temporal and spatial information about caspase activation. Finally, affinity labeling protocols are provided for detecting active caspases in whole‐cell lysates or subcellular fractions.
Analysis of Mitochondrial Dysfunction During Cell Death
Authors: Gogvadze Vladimir , Orrenius Sten , Zhivotovsky Boris
Published: June 2004

Attempts to identify a common underlying step in the apoptotic program in response to various cytotoxic stimuli have focused on the role of mitochondria in this form of cell death. This unit contains a family of protocols that can be used to assess mitochondrial functions during apoptotic responses. Protocols are included for the collection and analysis of released proteins, for detection of the mitochondrial permeability transition, for measurement of mitochondrial membrane potential, and for preparation of mitochondria from different tissue sources.
Tail Vein Assay of Cancer Metastasis
Authors: Elkin Michael , Vlodavsky Israel
Published: June 2004

The most damaging change during cancer progression is the switch from a locally growing tumor to a metastatic killer. This switch involves numerous alterations that allow tumor cells to complete the complex series of events needed for metastasis. In considering steps required for successful metastasis, extravasation from blood vessels in target organs is regarded as a critical process. Circulating tumor cells arrested in the capillary beds of different organs must invade the endothelial cell lining of blood vessels and degrade its underlying basement membrane in order to escape into the extravascular tissue where they establish metastasis. This unit describes the most common assay applied to evaluate the metastatic potential of blood‐borne tumor cells. The protocol is often called “experimental metastasis”, distinct from “spontaneous metastasis”, where the tumor cells are first allowed to form a primary tumor in the site of injection and then escape into lymphatic or blood circulation. Cultured tumor cells are injected into the tail vein and allowed to circulate. After 12 to 20 days the recipients are euthanized and the lungs are evaluated for the presence of metastatic tumors.
SAGE Analysis from 1 µg of Total RNA
Authors: Cai Jerry , Ash David , Jabs Ethylin Wang
Published: June 2004

Serial analysis of gene expression (SAGE) is a powerful transcription‐profiling method that allows simultaneous expression analysis of thousands of transcripts, provides absolute digital readout of the expression level, and identifies new genes. A disadvantage of SAGE is the relatively high amount of input RNA required. Consequently, several techniques have been developed to overcome this limitation, so that SAGE can be applied to very limited amounts of starting material, e.g., small biological samples such as tissue biopsies or microdissected materials. Here we describe a modified version of the original microSAGE protocol, which requires only 1 to 2 ug total RNA. This method avoids PCR amplification of cDNA and reamplification of ditags, procedures that potentially compromise the quantitative nature of this technique.
The Importance of Biological Databases in Biological Discovery
Authors: Baxevanis Andreas D.
Published: June 2004

Biological databases play a central role in bioinformatics. They offer scientists the opportunity to access sequence and structure data for tens of thousands of sequences from a broad range of organisms. This unit provides a brief overview of some existing sequence databases, such as GenBank, UCSC Genome Browser, and Ensembl. It also discusses non‐sequence centric databases, such as OMIM.
Using the NCBI Map Viewer to Browse Genomic Sequence Data
Authors: Wolfsberg Tyra G.
Published: June 2004

This unit includes an introduction to the Map Viewer, which describes how to perform a simple text‐based search of the genome to view the genomic context of a gene, navigate along the chromosome, zoom in and out, and change the displayed maps to hide and show information. It also describes some of NCBI's sequence‐analysis tools, which are provided as links from the Map Viewer. The Alternate Protocols describe different ways to query the genome sequence, and also illustrate additional features of the Map Viewer. shows how to perform and interpret the results of a BLAST search against the human genome. demonstrates how to retrieve a list of all genes between two STS markers. Finally, shows how to find all annotated members of a gene family.
Using the Blocks Database to Recognize Functional Domains
Authors: Henikoff Jorja G. , Greene Elizabeth A. , Taylor Nick , Henikoff Steven , Pietrokovski Shmuel
Published: June 2004

Blocks are ungapped multiple alignments of related protein sequence segments that correspond to the most conserved regions of the proteins. The Blocks Database is a collection of blocks representing known protein families that can be used to compare a protein or DNA sequence with documented families of proteins. Protocols in this unit describe the analysis of proteins and families using Blocks‐based tools, including searching, exploring relationships with trees, making new blocks, and designing PCR primers from blocks for isolating homologous sequences.
Identifying Protein Domains with the Pfam Database
Authors: Finn Robert , Griffiths‐Jones Sam , Bateman Alex
Published: June 2004

Pfam is a database of such protein domain families, with each family represented by multiple sequence alignments and profile hidden Markov models (HMMs). In addition, each family has associated annotation, literature references and links to other databases. The entries in Pfam are available via the worldwide web and in flatfile format. This unit contains detailed information on how to access and utilise the information present in the Pfam database, namely the families, multiple alignments and annotation. Details on running Pfam, both remotely and locally are presented.
Finding Homologs to Nucleic Acid or Protein Sequences Using the Framesearch Program
Authors: Healy Matthew
Published: June 2004

The Framesearch algorithm includes the possibility of a frameshift error in its alignment algorithm, and therefore can find alignments that span different reading frames. Protocols in this unit describe the use of Framesearch to search a protein sequence database for sequences that are similar to a query nucleotide sequence, and to search a nucleotide sequence database for sequences that are similar to a query protein sequence. Three alternate protocols describe ways to improve the speed of Framesearch and thus make it practical for routine use. Framesearch is especially appropriate for low‐quality single‐read nucleotide sequence data, such as ESTs (expressed sequence tags) or early drafts of genomic sequences; it does not offer any significant advantage over less CPU‐intensive algorithms for relatively high‐quality nucleotide sequences without many single‐nucleotide insertion or deletion errors.
Finding Homologs in Amino Acid Sequences Using Network BLAST Searches
Authors: Ladunga Istvan
Published: June 2004

BLAST, Basic Local Alignment Search Tool is used more frequently than any other biosequence database search program. The purpose of this unit is not only to show how to run searches on the Web, but also to demonstrate how to fine‐tune arguments for a specific research project. It also offers guidance for interpreting results, handling statistical significance and biological relevance issues, and selecting complementary analyses. This unit covers three classes of the BLAST program: standard protein‐to‐protein searches, translated searches when either the query or the database consists of nucleotide sequences translated into proteins, and finally programs for comparing two sequences (as opposed to searching one sequence against a database of sequences).
Selecting the Right Protein‐Scoring Matrix
Authors: Wheeler David
Published: June 2004

Every program for searching protein sequences against a database includes a choice of a protein weight matrix, also called a scoring matrix. Weight matrices add sensitivity to the search, while statistical significance adds selectivity. Virtually every user chooses the default, typically PAM 250 or BLOSUM62. Despite the fact that the choice of matrix can strongly influence the outcome of the analysis, most users do not know why a particular matrix should be used. In general, scoring matrices implicitly represent a particular theory of protein sequence evolution. Understanding the assumptions underlying the PAM and BLOSUM scoring matrices can aid in making the proper choice. The purpose of this unit is to guide the choice of a scoring matrix. It covers the selection of PAM matrices, BLOSUM matrices and provides a brief overview of the wide variety of specialized scoring matrices.
An Overview of Multiple Sequence Alignment
Authors: Simossis Victor , Kleinjung Jens , Heringa Jaap
Published: June 2004

Multiple sequence alignment is perhaps the most commonly applied bioinformatics technique. It often leads to fundamental biological insight into sequence‐structure‐function relationships of nucleotide or protein sequence families. In this unit, an overview of multiple sequence alignment techniques is presented, covering a history of nearly 30 years from the early pioneering methods to the current state‐of‐the‐art techniques. Methodological and biological issues and end‐user considerations, as well as alignment evaluation issues, are discussed.
Using TWINSCAN to Predict Gene Structures in Genomic DNA Sequences
Authors: Hu Ping , Brent Michael R.
Published: June 2004

TWINSCAN is a gene‐prediction system that combines the methods of ab initio predictors like GENSCAN with information derived from genome comparison. This unit describes the use of TWINSCAN to identify gene structures in eukaryotic genomic sequences. Protocols for using TWINSCAN through its Web interface and from the command line in a Linux environment are provided. Detailed discussion about the appropriate parameter settings, input sequence processing, and choice of genome for comparison are included.
Modeling Protein Structure from its Sequence
Authors: Marti‐Renom Marc A. , Madhusudhan M.S. , Eswar Narayanan , Pieper Ursula , Shen Min‐yi , Sali Andrej , Fiser Andras , Mirkovic Nebojsa , John Bino , Stuart Ashley
Published: June 2004

Functional characterization of a protein sequence is one of the most frequent problems in biology. This task is usually facilitated by accurate three‐dimensional (3‐D) structure of the studied protein. In the absence of an experimentally determined structure, comparative or homology modeling can sometimes provide a useful 3‐D model for a protein that is related to at least one known protein structure. Comparative modeling predicts the 3‐D structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). The prediction process consists of fold assignment, target‐template alignment, model building, and model evaluation. This unit describes generic considerations in all four steps of comparative modeling, typical modeling errors, and applications of comparative protein structure models. Finally, it illustrates these considerations in practice by discussing in detail one application of our program MODELLER.
FAMS and FAMSBASE for Protein Structure
Authors: Umeyama Hideaki , Iwadate Mitsuo
Published: June 2004

The computer program FAMS (Full Automatic Modeling System) performs homology modeling of protein structures by means of an algorithm consisting of database searches and simulated annealing. FAMS produces a model in which the torsion angles of the backbone and sidechains are highly accurate. The operations of the FAMS algorithm are fully automated, and, therefore, special knowledge, techniques or experience are not required in order to obtain a biologically worthwhile protein structure.
Modeling Membrane Proteins Utilizing Information from Silent Amino Acid Substitutions
Authors: Kochva Uzi , Leonov Hadas , Arkin Isaiah T. , Adams Paul D.
Published: June 2004

This unit describes predicting the structure of simple transmembrane α‐helical bundles. The protocol is based on a global molecular dynamics search (GMDS) of the configuration space of the helical bundle, yielding several candidates structures. The correct structure amongst these candidates is selected using information from silent amino acid substitutions, employing the following premise: Only the correct structure must (by definition) accept all of the silent amino acid substitutions. Thus, the correct structure is found by repeating the GMDS for several close homologues and selecting the structure that persists in all of the trials.
Inferring Evolutionary Trees with PAUP*
Authors: Wilgenbusch James C. , Swofford David
Published: June 2004

This unit provides a general description of reconstructing evolutionary trees using PAUP* 4.0. The protocol takes users through an example analysis of mitochondrial DNA sequence data using the parsimony and the likelihood criteria to infer optimal trees. The protocol also discusses searching options available in PAUP* and demonstrates how to import non‐NEXUS formats. Finally, a general discussion is given regarding the pros and cons of the “model‐free” and “model‐based” methods used throughout the protocol.
Using MODELTEST and PAUP* to Select a Model of Nucleotide Substitution
Authors: Posada David
Published: June 2004

Models of nucleotide substitution are commonly used in the analysis of DNA sequences. This unit describes the use of the program MODELTEST (coupled with PAUP*) to find the best‐fit model of substitution for the sequence alignment at hand. An example data file is analyzed and the interpretation of the results is discussed. Some background theory on model selection and a discussion of the relevance of models is included at the end of the unit.
Analyzing Molecular Interactions
Authors: Petsko Gregory A.
Published: June 2004

This unit offers an overview of issues that must be considered when analyzing molecular interactions. The importance of bound solvent, binding affinity, and electrostatic interactions is discussed.
Introduction to Comparing Large Sequence Sets
Authors: Page Roderic D.M.
Published: June 2004

Comparisons of whole genomes can yield important insights into the evolution of genome structure, such as the role of inversions in bacterial evolution and the identification of large‐scale duplications in the human genome. This unit briefly compares two tools for aligning whole genome sequences: MUMmer and PipMaker. These tools differ in both the underlying algorithms used, and in the interface they present to the user.
PipMaker: A World Wide Web Server for Genomic Sequence Alignments
Authors: Elnitski Laura , Riemer Cathy , Schwartz Scott , Hardison Ross , Miller Webb
Published: June 2004

PipMaker is a World‐Wide Web site used to compare two long genomic sequences and identify conserved segments between them. This unit describes the use of the PipMaker server and explains the resulting output files. PipMaker provides an efficient method of aligning genomic sequences and returns a compact, but easy‐to‐interpret form of output, the percent identity plot (pip). For each aligning segment between two sequences the pip shows both the position relative to the first sequence and the degree of similarity. Optional annotations on the pip provide additional information to assist in the interpretation of the alignment. The default parameters of the underlying blastz alignment program are tuned for human‐mouse alignments.
Using MUMmer to Identify Similar Regions in Large Sequence Sets
Authors: Delcher Arthur L. , Salzberg Steven L. , Phillippy Adam M.
Published: June 2004

The MUMmer sequence alignment package is a suite of computer programs designed to detect regions of homology in long biological sequences. Version 2.1 makes several improvements to the package, including: increased speed and reduced memory requirements; the ability to handle both protein and DNA sequences; the ability to handle multiple sequence fragments; and new algorithms for clustering together basic matches. The system is particularly efficient at comparing highly similar sequences, such as alternative versions of fragment assemblies or closely related strains of the same bacterium.
Large Scale Sequencing
Authors: Stein Lincoln
Published: June 2004

DNA sequencing and assembly is the Philosopher's Stone of bioinformatics, where the raw material of wet lab enzymatic chemistry is transformed into the bits and bytes of sequence information. The sequence information becomes the foundation for extensive downstream analyses. This unit provides a brief overview of the sequencing process. It describes several software programs typically used for base calling, assembling, and finishing sequence data such as STADEN, Phred, Phrap, and Consed.
An Overview of RNA Structure Prediction
Authors: Stormo Gary D.
Published: June 2004

This unit briefly describes the two fundamentally different methods for predicting RNA structures. The first is to find that structure with the minimum free energy of folding, as predicted by various thermodynamic parameters related to base‐pair stacking, loop lengths, and other features. If one has only a single sequence, this thermodynamic approach is the best available method. The second fundamental approach to RNA structure prediction is to use multiple, homologous sequences for which one can infer a common structure, and then try and predict a structure common to all of the sequences. Such an approach is referred to as a comparative method or phylogenetic method of RNA structure prediction.
IUPAC/IUB Single‐Letter Codes Within Nucleic Acid and Amino Acid Sequences
Authors: Leonard Shonda A.
Published: June 2004

The International Union of Pure and Applied Chemistry (IUPAC) and the International Union of Biochemistry and Molecular Biology (IUBMB) have established standards for representing nucleic acids and amino acids with single capital letters. The tables provided in this appendix summarize the codes for bases in nucleic acid sequences and individual amino acid residues within protein sequences.
Optimal sensor placement for state reconstruction of distributed process systems
Authors: Alonso Antonio A. , Frouzakis Christos E. , Kevrekidis Ioannis G.
Published: June 2004

In this contribution we propose a systematic approach to field reconstruction of distributed process systems from a limited and usually reduced number of measurements. The method exploits the time scale separation property of dissipative processes and concepts derived from principal angles between subspaces, to optimally placing a given number of sensors in the spatial domain. Basic ingredients of the approach include the identification of a low‐dimensional subspace capturing most of the relevant dynamic features of the distributed system, and the solution of a max–min optimization problem through a guided search technique. The low‐dimensional subspace can be defined either through a spectral basis (eigenfunctions of a linear or linearized part of the operator) or through a semiempirical expansion known in the engineering literature as the Proper Orthogonal Decomposition (POD) or Karhunen–Loeve expansion. For both cases, the optimal sensor placement problem will be solved by taking advantage of the underlying algebraic structure of the low‐dimensional subspace. The implications of this approach for dynamic observer design will be discussed together with examples illustrating the proposed methodology. © 2004 American Institute of Chemical Engineers AIChE J, 50: 1438–1452, 2004
A cluster of male breast cancer in office workers
Authors: Milham Samuel
Published: June 2004

Three cases of male breast cancer were diagnosed among a small group of men who worked in a basement office of a multi‐story office building. This office was adjacent to an electrical switchgear room which generated high magnetic fields in their work space. The risk of male breast cancer in this group was increased about 100‐fold (observe three cases, expect 0.03 cases; P < 0.00001). Since 1991, 15 epidemiologic studies have associated male breast cancer with exposure to electromagnetic field. Am. J. Ind. Med. 46:86–87, 2004. © 2004 Wiley‐Liss, Inc.
Good practices in managing work‐related indoor air problems: A psychosocial perspective
Authors: Lahtinen Marjaana , Huuhtanen Pekka , Vähämäki Kari , Kähkönen Erkki , Mussalo‐Rauhamaa Helena , Reijula Kari
Published: June 2004

Background

Indoor air problems at workplaces are often exceedingly complex. Technical questions are interrelated with the dynamics of the work community, and the cooperation and interaction skills of the parties involved in the problem solving process are also put to the test. The objective of our study was to analyze the process of managing and solving indoor air problems from a psychosocial perspective.

Methods

This collective case study was based on data from questionnaires, interviews and various documentary materials. Technical inspections of the buildings and indoor air measurements were also carried out.

Results

The following four factors best differentiated successful cases from impeded cases: extensive multiprofessional collaboration and participative action, systematic action and perseverance, investment in information and communication, and process thinking and learning. The study also proposed a theoretical model for the role of the psychosocial work environment in indoor air problems.

Conclusions

The expertise related to social and human aspects of problem solving plays a significant role in solving indoor air problems. Failures to properly handle these aspects may lead to resources being wasted and result in a problematic situation becoming stagnant or worse. Am. J. Ind. Med. 46:71–85, 2004. © 2004 Wiley‐Liss, Inc.
Impacts of health and safety education: Comparison of worker activities before and after training
Authors: Becker Paul , Morawetz John
Published: June 2004

Background

The International Chemical Workers Union Council (ICWUC) Center for Worker Health and Safety Education in Cincinnati, Ohio, trains workers to protect themselves from hazards due to chemical spills and other chemical exposures. We evaluated whether the ICWUC Hazardous Waste Worker Training Program affects the attitudes and post‐training activities, of trained union workers.

Methods

Detailed survey questionnaires were administered to 55 workers prior to and 14–18 months following training. Surveys queried trainees' interest and involvement in safety and health, use of information resources, training activities at their worksite, and their attempts and successes at making worksite improvements.

Results

Post‐training, the study population showed an increase in training of other workers, use of resources, attempts at improvements, success rates for those attempting change, and overall success at making improvements. Self‐reported interest decreased, and self reported involvement in health and safety did not significantly change.

Conclusion

The study demonstrates that workers are more willing to attempt to change worksite conditions following training, and that their efficacy at making changes is substantially greater than before they were trained. The study confirms earlier work and strengthens these conclusions by using statistically tested comparisons of impact measures pre‐ and post‐training. Am. J. Ind. Med. 46:63–70, 2004. © 2004 Wiley‐Liss, Inc.